Advances in Genetics Volume 9

ADVANCES IN GENETICS VOLUME IX Edited by M. DEMEREC Carnegie Institution, Cold Spring Harbor, N. Y. Editorial Board ...

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ADVANCES IN GENETICS VOLUME IX

Edited by

M. DEMEREC Carnegie Institution, Cold Spring Harbor, N.

Y.

Editorial Board G. W. BEADLE

JAY L. LUSH

WILLIAM C. BOYD

ALFRED MIRSKY

TH. DOBZHANSKY

J. T. PATTERSON

1. C. DUNN

M. M. RHOADES

MERLE T. JENKINS

CURT STERN

1958 ACADEMIC PRESS INC

PUBLISHERS

NEW YORK

COPYRIQHT @ 1958 BY

ACADEMIC PRESSINC. 111 FIFTHAVENUE NEW YORK3, N. Y.

All Rights Reserved No part of this book m y be reproduced i n any form by photostat, microfilm, or any other means, without written permission from the publishers.

Library of Congress Catalog Card Number 47-30313

PRINTED IN THE UNITED STATES O F AMERICA

CONTRIBUTORS TO VOLUME IX HAMPTON L. CARSON,Department of Zoology, Washington University, St. Louis, Missouri ETTAKAFER,Department of Genetics, The University, Glasgow, Scotland* HAIGP. PAPAZIAN, 169 Cold Spring Street, New Haven 11, Connecticut G. PONTECORVO, Department of Genetics, The University, Glasgow, Scotland G. LEDYARD STEBBINS,University of California, Davis, California M. WESTERGAARD, Institute of Genetics, University of Copenhagen, Denmark

*

Prerent name and addreer: Etta Kdfer Boothroyd, Department of Genetics, McGill Uniueraity, Montreal, Canada.

V

THE POPULATION GENETICS OF Drosophila robusta Hampton L. Carson Department of Zoology, Washington University, St. Louis, Missouri

Page I. Introduction.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Taxonomy and Distribution. . . . . . . . . . . 111. Ecology.. . . . IV. Cytological S ............................................... 15 1. Metaphase Chromosomes.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 2. Salivary Gland Chromosomes. . . . . . . . . . . . . . . . . . . . . . . . . 3. Paracentric Inversions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 4. Pericentric Inversions. . . . V. Distribution of Gene Arra 1. Geographical Distribut .............................. 20 2. Altitudinal and Region .................................. 26 3. Seasonal and Perennial Studies of Single Localities.. . . . . . . . . . . . . . . . . 28 VI. Morphological Variability. . . ........ 31 VII. Combinations of Inversions i .................... 32 32 1. The Effect of Inversions on Crossing-Over.. ....................... 2. Linkage and Positional Relationships within the Structural Karyotype 33 3. Marginal Homozygosity for Gene Arrangement.. . . . . . . . . . . . . . . . . . . . 34 VIII. Experimental Laboratory Populations. ........................ IX. Conclusion: The Genetic Nature of Drosophila robusta as a Specie X. Acknowledgments.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 XI. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38

I. INTRODUCTION Drosophila robusta is a large, dark-colored fly which is one of the commonest species of the genus in the deciduous forest of the eastern United States. The present paper is a review of approximately ten years of work on the genetics and ecology of natural and experimental populations of this form. Interest in such a review has been expressed by various nongenetical investigators of the biota of the eastern United Statestaxonomists, ecologists, evolutionists, and students of speciation and population dynamics. Drosophila work provides fundamental information of a rather unique sort on the genetical nature of the species. The interpretative value of such information may be of wide application and it is in a way unfortunate for those interested in the eastern flora and fauna that the pioneer quantitative work on Drosophila was done on a western species group, Drosophila pseudoobscura and its relatives. The present paper is intended to serve the general audience referred to above. 1

2

HAMPTON L. CARSON

The writer has also used this opportunity to include a considerable amount of previously unpublished data on the geographical, local, and seasonal variations in frequencies of the gene arrangements of this species. A new type of cytological map has been prepared.

11. TAXONOMY AND DISTRIBUTION Patterson and Stone (1952) include five species in the robusta group of the subgenus Drosophila. D. robusta Sturtevant and D. colorata Walker

FIG. 1. Geographical distribution of Drosophila robusta (closed circles) and D. colorata (open circles). Each point represents a verified record given in Tables 1and 2.

are found in the United States and southern Canada (Fig. 1). D . cheda Tan, Hsu, and Sheng, D. pullata Tan, Hsu, and Sheng are described from China; D. sordidula Kikkawa and Peng is found in Japan. The Asiatic members of the group are not well known, although their close phylogenetic relationship to D . robusta and D . colorata is clear and unequivocal. Furthermore, there is apparently at least one additional undescribed species of this group known from Japan (Makino et al., 1955; Momma, 1954). Figure 1 shows the distribution of D. robusta (closed circles) and D. colorata (open circles) in North America. Each dot on the map represents one or more collection records each of which is based on either a record in the literature or on an unpublished record of various workers; the precise data for each collection are given in Tables 1 and 2.

POPULATION GENETICS O F DROSOPHILA ROBUSTA

3

TABLE 1 Collection Records of Drosophila robusta State

County

Locality

Reference

Alabama

Mobile Morgan Tuscaloosa Shelby Chilton Pike Pike Dale Henry Sumter Dekalb Sumter Winston Marshall

Kushla Lacon University Pelham Verbena Orion Troy Ozark Abbeville Epes Fort Payne Demopolis Haleyville Guntersville

Sturtevant, 1916 C and S, 1947* C and S, 1947 C and S, 1947 C and S, 1947 C and S, 1947 C and S, 1947 C and S, 1947 C and S, 1947 P and W, 19431 P and W, 1943 UTLS UTL UTL

Arkansas

Conway Carroll Hempstead Phillips

Morrilton Eureka Springs Hope Helena

P and W, 1943 P and W, 1943 P and W, 1943

Connecticut

New Haven Windham

Wallingford Putnam

UTL UTL

District of Columbia

District of Columbia

Florida

Orange Oceola Jefferson Suwanee Taylor Citrus Putnam Gadsden Clay Okaloosa Citrus

Orlando UTL Lake Wales UTL Mont icello C and S, 1947 Wellborn C and S, 1947 Perry P and W, 1943 Lake Tsala Apopka P and W, 1943 Palatka P and W, 1943 Tallahassee UTL Keystone Heights UTL Crestview UTL Floral City UTL

Georgia

Cobb Cobb Calhoun

Acworth Kcnnesaw Edison

* Carson and

Sturtevant, 1916

Malloch and McAtee, 1924

UTL C and S, 1947 C and S, 1947

Stalker (194713).

t Patterson and Wagner (1943).

1 Unpublished records of the University of Texas Laboratory.

4

HAMPTON L. CARSON

TABLE 1. (Continued) State

Locality

County

Reference

C and S, 1947 C and S, 1947 C and S, 1947 P and W, 1943 P and W, 1943 UTL UTL UTL UTL

Ware Carroll

Leary Albany Thomasville Bolton Marietta Indian Springs Trenton Savannah Crooked River State Park Waycross Villa Rica

Illinois

Cook Macoupin

Glen Ellyn Carlinville

C and S, 1947 UTL

Indiana

Vanderburg Vigo La Porte Madison Montgomery Clark Knox Posey

Evansville Terre Haute Smith Station Anderson Crawfordsville Jeff ersonville Vincennes New Harmony

H. D. Stalker, unpublished H. D. Stalker, unpublished A. Sokoloff , unpublished P and W, 1943 H. D. Stalker, unpublished UTL H. D. Stalker, unpublished H. D.-Stalker, unpublished

Iowa

Linn Page Lee Johnson

Mount Vernon Clarinda Keokuk Iowa City

C and S, 1947 M. E. Annan, unpublished H. D. Stalker, unpublished H. D. Stalker, unpublished

Kansas

Sedgwick Rooks

Wichita Stockton

P and W, 1943 H. L. Carson, unpublished

Kentucky

Breathitt

Noble

Fleming

Muses Mills

Washington

Tatham Springs

Hickman Jefferson Union Christian Fayette

Clinton Louisville Sturgis Hopkinsville Spears

J. M. Carpenter, unpublished J. M. Carpenter, unpublished J. M. Carpenter, unpublished C and S, 1947 P and W, 1943 P and W, 1943 H. D. Stalker, unpublished J. M. Carpenter, unpublished

Georgia

Calhoun Dougherty Thomas Fulton Cobb Butts Dade Chatham Camden

UTL UTL

POPULATION GENETICS O F DROSOPHILA ROBUSTA

TABLE 1. (Continued) State Kentucky

County

Locality

Reference

Jessamine

Nicholasville

J. M. Carpenter, unpub-

Mercer

Harrodsburg

J. M. Carpenter, unpub-

Whitley

Cumberland Falls State Park

J. M. Carpenter, unpub-

lished lished

lished

Louisiana

Caddo Ascension St. James St. Charles St. John Orleans St. Landry

Cross Lake Sorrento Gramercy La Place Garyville New Orleans Krotz Springs

P and W, 1943 H. D. Stalker, unpublished H. D. Stalker, unpublished H. D. Stalker, unpublished H. D. Stalker, unpublished P and W, 1943 UTL

Maine

Hancock Aroostook Oxford

Mt. Desert Island Guerette Bethel

UTL UTL UTL

Maryland

Montgomery

Cabin John

Sturtevant, 1916

Massachusetts

Barnstable

Woods Hole

Hampshire Hampshire Suff olk Norfolk Middlesex Essex

Pelham Amherst Boston Braintree Cambridge Ipswich

Sturtevant, 1916;C and S, 1947 C and S, 1947 C and S, 1947 Spiess, 1949 Spiess, 1949 Spiess, 1949 UTL

Allegan Emmet Dickinson Menominee Mackinac Cheboygan Jackson Roscommon Wayne

Plainwell Petoskey Iron Mountain Menominee St. Ignace Cheboygan Jackson Houghton Lake Grosse Point

H. D. H. D. H. D. UTL UTL D. D. UTL UTL H. D.

Carlton Stearns

Cloquet Big Fish Lake

Clearwater Morrison

Itasca State Park Little Falls

UTL C. P. Oliver, unpublished; C and S, 1947 H. T. Speith, unpublished H. D. Stalker, unpublished

Michigan

Minnesota

Stalker, unpublished Stalker, unpublished Stalker, unpublished Miller, unpublished Stalker, unpublished

5

6

HAMPTON L. CARSON

TABLE 1. (Continued) State

Minnesota

County Murray Winona St. Louis Beltrami Ramsey

Mississippi

Lee Alcorn Lowndes Warren Claiborne Jefferson Yazoo Washington Harrison

Locality

Reference

Lake Shetek State Park Winona Ely Lake Bemidji Stillwater

UTL

Tombigbee State Park Corinth Columbus Vicksburg Port Gibson

UTL

UTL UTL UTL UTL

Yazoo City Hollandale Gulfport

H. D. Stalker, unpublished P and W, 1943 H. D. Stalker, unpublished H. D. Stalker, unpublished H. D. Stalker, unpublished P and W, 1943 UTL UTL

Missouri

Stoddard Shannon Boone St. Louis St. Louis St. Louis St. Louis St. Louis St. Louis St. Louis Franklin Franklin Jefferson Jefferson Crawford St. Louis Dent St. Louis Greene

Dexter Eminence Columbia St. Louis Creve Coeur Lake Olivette University City Manchester Pond Sappington Gray Summit St. Clair Oermann House Springs Steelville Eureka Montauk Fenton Springfield

UTL H. L. Carson, unpublished P and W, 1943 H. L. Carson, unpublished H. L. Carson, unpublished Carson and Stalker, 1949 Carson and Stalker, 1949 C and S, 1947 C and S, 1947 C and S, 1947 C and S, 1947 C and S, 1947 C and S, 1947 H. D. Stalker, unpublished H. L. Carson, unpublished H. L. Carson, unpublished C and S, 1947 H. L. Carson, unpublished P and W, 1943

Montana

Roosevelt Blaine

Poplar Chinook

UTL UTL

Nebraska

Nemaha Richardson Dawes Lancaster

Peru Falls City Chadron State Park Lincoln

Williams and Williams and Williams and Williams and

Miller, Miller, Miller, Miller,

1952 1952 1952 1952

POPULATION GENETICS O F DROSOPHILA ROBUSTA

7

TABLE 1. (Continued) State

County

Locality

Reference

Platte Cass Saline Seward Thomas Buffalo Knox Merrick Antelope Adams Richardson Dixon

Halsey Ravenna Niobrara Silver Crcck Oakdale Hastings Humboldt Ponca State Park

Williams and Miller, 1952 Williams and Miller, 1952 Williams and Miller, 1952 Williams and Miller, 1952 H. L. Carson, unpublished H. L. Carson, unpublished D. D. Miller, unpublished UTL UTL UTL D. Williamson, unpublished D . D. Miller, unpublished

New Hampshire

Grafton Rockingham

Hanover Ham pt on

Sturtevant, 1921 UTL

New Jersey

Bergen Cape May Bergen Mercer

Tenafly Cape May Point Englewood Cliffs Princeton

C and S, 1947 C and S, 1947 Levitan, 1951a C. S. Pittendrigh, unpublished

New York

Cattaraugus

UTL

Suffolk Monroe Tompkins Chautauqua Tompkins Richmond Bronx Westchester Richmond Ulster Hamilton Steuben Wayne

Alleghany State Park Cold Spring Harbor Rochester Varna Chautauqua Ithaca Staten Island Van Cortlandt Park Tibbets Brook Park Mariner's Harbor Kerhonkson Blue Mountain Lake Jasper Webster

Sturtevant, 1916 C and S, 1947 C and S, 1947 C and S, 1947 Sturtevant, 1916 Sturtevant, 1921 Levitan, 1951a Levitan, 1951a Levitan, 1951a UTL UTL UTL H . D. Stalker, unpublished

Bladen Columbus Wake Stanley Martin Guilford Macon

White Lake Chadbourn Raleigh Albemarle Williamston Jamestown Highlands

R. C. Lewontin, unpublished R. C. Lewontin, unpublished R. C. Lewontin, unpublished UTL UTL UTL R. C. Lewontin, unpublished

Nebraska

North Carolina

Monroe

8

HAMPTON L. CARSON

TABLE 1. (Continued) Slate

County

Locality

Reference

Chester Oxford Akron Circleville Ghent Ironton Mansfield Piqua West Chester Celina Buckeye Lake Cantwell Cliffs State Park Independence State Park Galion

Marion

Th. Roosevelt Reserve Prospect

Ashland

Loudonville

Mercer

Skeels

Crawford

Galion

Ashland

Mohicanville

Wayne Cuyah oga Butler

Wooster Chagrin Falls College Corner

C and S, 1947 C and S, 1947 P and W, 1943 P and W, 1943 P and W, 1943 P and W, 1943 P and W, 1943 P a n d W, 1943 P and W, 1943 P and W, 1943 P and W, 1943 H. D. Stalker and W. P. Spencer, unpublished H. D. Stalker and W. P. Spencer, unpublished H. D. Stalker and W. P. Spencer, unpublished H. D. Stalker and W. P. Spencer, unpublished H. D. Stalker and W. P. Spencer, unpublished H. D. Stalker and W. P. Spencer, unpublished H. D. Stalker and W. P. Spencer, unpublished H. D. Stalker and W. P. Spencer, unpublished H. D. Stalker and W. P. Spencer, unpublished P and W, 1943 H. D. Stalker, unpublished UTL

Oklahoma

Tulsa Delaware Mayes Latimer

Tulsa Kansas Salina Wilburton

P and P and P and P and

Pennsylvania

Dauphin Philadelphia Lawrence Montgomery

Millersburg Philadelphia New Wilmington Abington

UTL C and S, 1947 C and S, 1947 D. A. Hungerford, unpublished

Rhode Island

Providence

Chepachet

UTL

Ohio

Meigs Butler Summit Pickaway Summit Lawrence Richland Miami Butler Mercer Licking Hocking Defiance Crawford Scioto

W, W, W, W,

1943 1943 1943 1943

POPULATION GENETICS O F DROSOPHILA ROBUSTA

TABLE 1. (Continued) State

County

Locality

Reference

South Carolina

Georgetown Spartanburg

Georgetown Cross Anchor

South Dakota

Custer Custer

Wind Cave National H. L. Carson, unpublished Park Bluebell H. L. Carson, unpublished

Blount

Townsend

Tennessee

Sevier Bedford Gibson Weakley Cum berlan d Benton Shelby Polk Hamilton Shelby Texas

UTL UTL

J. M. Carpenter, unpublished Gatlinburg C and S, 194815 Shelbyville C and S, 1947 Milan C and S, 1947 Greenfield C and S, 1947 Crossville P and W, 1943 Eva P and W, 1943 Memphis P and W, 1943 Reliance P and W, 1943 Chattanooga P and W, 1943 Shelby Forest State P and W, 1943 Park

Liberty Liberty San Augusti ne San Augustine Caddo Lake State Harrison Park Cherokee Rusk Travis Austin Milam Milano Cass Atlanta Brazos Burleson Shelby Center Liberty Cleveland Fayette Robertson Franklin Galveston Williamson Georgetown Sahine Hemphill Rusk Henderson Houston Walker Huntsville Cherokee Jacksonville Leon Newton Anderson Palestine

P and W, 1943 P and W, 1943 P and W, 1943 P and W, P and W, P and W, P and W, P and W, P and W, P and W, P and W, P and W, P and W, P and W, P and W, P and W, P and W, P and W, P and W, P and W, P and W, P and W, P and W,

1943 1943 1943 1943 1943 1943 1943 1943 1943 1943 1943 1943 1943 1943 1943 1943 1943 1943 1943 1943

9

10

HAMPTON L. CARSON

TABLE 1. (Continued) State

County

Locality

Reference

Texas

Hardin Gonzales Orange

Saratoga P and W, 1943 Palmetto State Park UTL Orange UTL

Vermont

Orange Windham Grand Isle

Randolph South Hero

C and S, 1947 Spiess, 1949 UTL

Virginia

Arlington Montgomery Henrico Fairfax

Falls Church Blacksburg Richmond Fairfax

Sturtevant, 1916 Levitan, 1951b UTL UTL

West Virginia

Preston

Coopers Rock State Dorsey and Carson, 1956 Forest

Wisconsin

Dane Ashland Eau Claire Rock Bay field

Madison Mellen Augusta Beloit Iron River

C and S, 1947 H. L. Carson, unpublished C. J. Bennett, unpublished UTL H. D . Stalker, unpublished

Ontario (Canada)

Oakville

H. D. Stalker, unpublished

Quebec (Canada)

Ste. Anne de Bellevue Gatineau Park Ile Perrot

D. D. Miller, unpublished D. D. Miller, unpublished D. D. Miller, Unpublished

The distribution of Drosophila robusta appears to be approximately coextensive with the eastern deciduous forest of the United States and the known facts about the breeding sites and other ecological relationships of this species support this conclusion. Thus, the species appears to be absent from the coniferous forests of the Canadian Zones of the northeast and north central portions of the United States and southern Canada and the Appalachian uplift. The southwestern distribution follows the deciduous forest into central Texas; the species then extends northwestward from eastern Oklahoma following the slightly more mesic areas of the central plains to northeastern Montana. I n the northwest and north central, the species is confined to riparian locations in sheltered stream valleys which carry elements of the eastern forest into the great plains. These marginal populations have been given special attention recently (Carson, 1955a,b, 1956a).

POPULATION GENETICS OF DROSOPHILA ROBUSTA

11

TABLE 2 Collection Records of Drosophila colorata State

County

Locality

Reference

District of Columbia

Malloch and McAtee, 1924 Sturtevant, 1921

Georgia

Sturtevant, 19 16 Patterson and Wagner, 1943

Idaho

Adams

New Meadows

UTL *

Maine

Aroostook

Guerette

UTL

Maryland

Montgomery

Cabin John

Sturtevant, 1916

Massachusetts

Hampden

Chester

Sturtevant, 1916

Michigan

Dickinson Emmet

Iron Mountain Petosky

H. D. Stalker, unpublished H. D. Stalker, unpublished

Minnesota

Clearwater Lake

Itasca State Park Little Marais

H. T. Speith, unpublished H. L. Carson, unpublished

Mississippi

Warren

Vicksburg

H. D. Stalker, unpublished

Missouri

St. Louis Crawford St. Louis

Olivette Steelville Eureka

Carson and Stalker, 1951 H. L. Carson, unpublished H. L. Carson, unpublished

New Hampshire

coos Grafton Grafton

Bretton Woods Franconia Hanover

Sturtevant, 1916 Sturtevant, 1916 Sturtevant, 1916

New York

Chautauqua Tompkins Suffolk

Chautauqua W. P. Spencer, unpublished Ithaca Sturtevant, 1921 Cold Spring Harbor D. D. Miller, unpublished

Ohio

Wayne

Wooster

Cuyahoga

H. D. Stalker and W. P. Spencer, unpublished Cantwell Cliffs State H. D. Stalker and W. P. Park Spencer, unpublished Buckeye Lake H. D. Stalker and W. P. Spencer, unpublished Chagrin Falls UTL

Pennsylvania

Montgomery

Pottstown

Sturtevant, 1916

South Carolina

Georgetown

Georgetown

UTL

Hocking Licking

HAMPTON L. CARSON

TABLE 2. (Continued) State

County

Locality

Reference

Grainger

H. L. Carbon and H. D. Stalker, unpublished Unaka Mountain Stevenson, 1952 Great Smoky Moun- Patterson, 1943 tain National Park M. R. Wheeler, unpublished Rutledge

Vermont

Windsor

Bridgewater

H. L. Carson, unpublished

Virginia

Alexandria

Glencarlyn

Sturtevant, 1916

West Virginia

Preston

Coopers Rock St. Forest

Dorsey and Carson, 1956

Washington

Chelan

Wenatchee

UTL

Wiaconsin

Ashland

Mellen

H. L. Carson, unpublished

Ontario (Canada)

Algonquin Park Ottawa

D. D. Miller, unpublished Sturtevant, 1916

Manitoba (Canada)

Selkirk

H. L. Carson, unpublished

Quebec (Canada)

Laurentides Park

D. D. Miller, unpublished

Tennessee

Sevier Unicoi Sevier

Gatlinburg

* Unpublished records of the University of Texas Laboratory. Drosophila colorata is much less well known. The largest collections of this species have been made by Dorsey and Carson (1956) in West Virginia (188 out of 15,081 Drosophilidae collected). I n these collections, however, D. robusta was abundant (944 individuals, thus greatly outnumbering D . colorata). This is close to the ratio of colorata to robusta found by the writer a t Mellen, Wisconsin in July 1952 (26: 112 out of 2,388 drosophilids captured). Almost without exception, all other records of D. colorata are represented by a few specimens only. One might be tempted to regard D. colorata as a “rare” species were it not for the fact that there is really no way of knowing how efficiently any Drosophila species is being attracted to the baits used. Indeed, Dorsey and Carson (1956) have presented evidence that D. colorata is less well attracted to a vinegar-molasses-water mixture than are most other common species of Drosophila. I n several instances, however, very extensive collections have been made within the known range of the species with only meager results. Levitan (1954~)did not record the species among more than 8,000

POPULATION GENETICS O F DROSOPHILA ROBUSTA

13

specimens collected in northern New Jersey and southeastern New York. I n the unpublished records of Dr. H. D. Stalker and the writer, of collections made a t Olivette, St. Louis County, Missouri, only two specimens of this species were captured in approximately 48,000 individuals attracted to baits. It is probably significant that the species has never been recorded from Texas, in view of the exhaustive work of Patterson and his co-workers a t Aldrich Farm, Austin and elsewhere. Williams and Miller (1952), furthermore, have not recorded the species in extensive collections from Nebraska, although i t has been taken in the far northwest (Idaho and Washington). 111. ECOLOGY Drosophila robusta may be caught in large numbers during the warm months of the year by placing lures consisting of fermenting fruit in low, relatively damp and dense, deciduous woods. The species is less abundant on oak-hickory hillsides, in suburban areas, and in orchards. Association of this species with the deciduous forest appears to be well explained by the facts concerning its natural breeding sites. Carson and Stalker (1950, 1951) have detailed the breeding of the species on the sap exudations of 14 species of deciduous trees, one species of woody vine (Vitis) and one fleshy fungus. The writer has recently been able to add Salix wardii as a host for the species. Despite this wide latitude of natural breeding sites, there appears to be a distinct tendency for the American Elm (Ulmus americana) to serve as the principal host tree. I n fact, this tree has been successfully used as a clue to good collecting places for the species and has served in this capacity even a t the margin of the species range in extreme northwest Nebraska (Carson, 195Ga). In fact, the geographical distribution of D. robusta (Fig. 1) superimposes strikingly on that of U. americana (see Carson, 195513). Spieth (see Patterson and Stone, 1952, p. 94) and Williams and Miller (1952) have both confirmed elm fluxes as breeding sites for Drosophila robusta. The breeding site of D. colorata is unknown. The adults of D. robusta show great opportunism with respect to feeding sites. The species has been actually observed to feed on a large variety of materials on which it has never been observed to oviposit. This includes a great variety of fungus, exudates from Black Locust, persimmon, apples, and peaches. On occasion, especially in wooded suburban areas, it is attracted to garbage but it is generally not characteristic of domestic habitats. I n Minnesota, Dr. H. D. Stalker observed a great number of this species feeding on a large fermenting mass of compost. Carson and Stalker (1951), Carson (1951) and Carson et al. (1956)

14

HAMPTON L. CARSON

have cited observations which led them to conclude that breeding and feeding sites are largely separate for most wild species of Drosophila. In the latitude of St. Louis, females of D. robusta caught after approximately September 15, show a striking reproductive diapause (Carson and Stalker, 1948a). Thus, 95% of the females a t this time are uninseminated, have extremely small ovaries and very large quantities of body fat. It is inferred that these changes represent a biological adjustment of the species to overwintering as adult flies. That D. robusta overwinters as an adult is also borne out by the fact that collections of flies made on the very first warm days of spring yield adults of this species. Such adults are invariably dark-colored, with occasional missing bristles and somewhat torn wings. In short, they are very unlike young adults which have recently emerged from the pupal case. The latter are lightly pigmented and darken rather slowly over a period of 6 days at 25.5"C. (Galey, 1949). It is thus possible to recognize a young fly of this species with considerable ease. Young flies of D. robusta do not appear in collections in the St. Louis area, in most years, until about the second week in June. Such flies apparently result from eggs laid by overwintering adults. Oviposition has been observed as early as the third week in April. In any event, characteristically large populations have built up by the third week in June. Oviposition continues throughout July and August ;maximum abundance of the species is reached in July and diapause becomes pronounced again by the 15th of September. Such a cycle as described above is in accord with the observations of Patterson (1943) who found the species rare or absent from collections at Austin, Texas except during April and May. Austin may be considered a marginal area for the species in the southwest, with the population peak occurring early, or approximately two months prior to that observed a t St. Louis. Population peaks in more northern areas are apparently reached somewhat later in the year, e.g., in Nebraska (Williams and Miller, 1952; Carson, 1956a) and in New Jersey (Levitan, 1954~). Drosophila robusta, although it may be reared on most of the standard laboratory media for the genus, is nevertheless partial to rather soft laboratory food. This is most easily achieved by reducing the amount of agar in the standard formulae. A medium which has proved very successful in this laboratory consists of: agar, 75 g. ; Karo (maize) syrup, 210 cc. ; yellow cornmeal (maize), 683 g.; 10% solution of Tegosept in 70% alcohol, 62 cc. ; water, 6,950 cc. Dissolve agar in 4,450 cc. water and bring to a boil. Add syrup. Mix remainder of cool water with cornmeal and add. Bring again to a boil with constant stirring. Cover and boil 7 min-

POPULATION GENETICS OF DROSOPHILA ROBUSTA

15

Utes. Add Tegosept, stir well, and pour. This recipe will make 120-130 half -pint bottles. When fully fed with excess fresh yeast suspended in water at 25.5"C., the time for development from egg to adult for this species is about 15 days for females and 16 days for males. Under conditions where the food supply is limited, as in experimental populations (e.g., Carson, 1957), this time is greatly extended and even at 25°C. some normal individuals will hatch from eggs laid 8 weeks previously. It is also likely that the generation time in nature is much greater than under ideal conditions in the laboratory. Both males and females have maturation periods as adults before they will reproduce. Under optimal conditions, this is about 4 days after emergence for females and about 8 days for males.

IV. CYTOLOGICAL SYNOPSIS 1. Melaphase Chromosomes

Metaphase cliromosomes of Drosophila robusta have been studied in aceto-orcein smears of the larval brain or the ovaries of young adults. The chromosome group apparently universally consists of three pairs of V-shaped chromosomes of descending sizes and a pair of dot-like chromosomes. The deswiptioir by Carson and Stalker (1947b) of metaphases in which oiie or both of the medium-sized V's are replaced by J's in the presence of gene arrarigenierit 21,-3 cannot now be confirmed and appears to be in error.+ The original strain from which this condition was described is still being maintained in the laboratory and has beeii repeatedly checked. This strain (Big Fish Lake, Minnesota) is still, as originally, homoeygoris for gene arrangement 2L-3 but does not show J-shaped chromosomes a t metaphase. Apparently a prominent secondary constriction was mistaken for the primary constriction. In any event, although gene arrangement 2L-3 is paracentric, two pericentric inversions are definitely known in the species (2L-R and 3L-R, see below and Fig. 2). Both of these inversions, however, extend about equal distances on each side of the centromere so that their presence does not detectably alter the metaphase shape of the chromosome involved. 2. Salivary Gland Chromosomes

Figure 2 is a new type of photographic chromosome map or idiogram of the salivary gland chromosome group of Drosophila robusta. The photographs chosen for the map are of chromosomes, stained with aceto-orcein,

* The error was exclusively that of the senior author.

16 HAMPTON L. CARSON

POPULATION GENETICS O F DROSOPHILA ROBUSTA

17

in which only the minimum amount of stretching and distortion have occurred. There are six major euchromatic arms in addition to the fourth chromosome. These are designated XL, XR, 2L, 2R, 3L, and 3R and represent the left and right arms, respectively, of the X, second, and third chromosomes. I n each case, the banding pattern shown is th a t of the sequence which has been arbitrarily designated as Standard. Distal ends (e.g., XLD,etc.) are shown a t the left (except chromosome 4). As in previous diagrams of the chromosomes of this species (Carson and Stalker, 1947b; Carson, 1953) the genome has been divided into 34 basic regions. The ‘ I break” points of the inversions have wherever possible been used to delimit these regions from one another. The same regions used in former maps have been retained in the present map. Newly discovered break points within the confines of one of the original regions have resulted in the subdivision of that region into new subregions, e.g., Fig. 2, a t the proximal part of 2L (2L’); old region 23 is now subdivided into 23A, 23B, 23C, and 23D. The inversion break points are indicated by ink lines connected to numbers above the photographs. These numbers give both the name of the arrangement and its extent. Thus, for example, the distal and proximal breaks of the inversion by which gene arrangement XL-1 differs from Standard XL may be told by looking for 1” (distal break of gene arrangement XL-1) and l p(proximal break of gene arrangement XL-1). The approximate position of the centromere in each of the V-shaped chromosomes is indicated by “C.” Unless specially mentioned in the text of this article, all inversions are one-step derivatives of their respective standard arrangements. The following shorthand method of writing the structural karyotype of an individual female of Drosophila robusta has been adopted by Carson (1953) and Levitan (1955). The conditions in each chromosome are written as three sets of symbols, from left to right, following the order X, 2, and 3. The capital letter “S”is used to refer to the Standard arrangement in each case and numerals are used to refer to the various alternative gene orders in th at particular arm. Thus the formula for a n individual female homozygous for all standard arrangements, a s in Fig. 2, would be written : X 2 3

s s s s

~-

s s

m

s s

s-s

FIG.2 . Photographic map of the salivary gland chromosomes of Drosophila robusta. Distal ends of the Standard arrangements (XLD, XRD, etc.) are to the left. Lines connected to numbers indicate break points of inversions (e.g., 2D and 2 ’ in XL indicate distal and proximal breaks of the inversion by which XL-2 differs from Standard XL). Smaller numbers (1-34) indicate chromosome regions.

18

HAMPTON L. CARSON

The centromeres are inferred as being between the two “S’”S in each symbol. An individual heterozygous, for example, for the three inversions XL/XL-l, 2R/2R-11 and 3R/3R-1 would be:

X

2

s s __ s s 1 s s 1

3

s s s 1

or, as it will be written in the text of this article: SS/lS, SS/Sl, SS/S1.

3. Paracentric Inversions The majority of the inversions shown in Fig. 2 are naturally-occurring and are widespread in natural populations (see Fig. 3 and Tables 3 and 4). Certain of them, however, have peculiarities which deserve special mention. Gene arrangement XL-3 is known only from a single strain collected at Lake Tsala Apopka, Florida over ten years ago. This strain is still extant in the laboratory. XR-1 and 2R-1, both widespread inversions, are cytologically terminal in the salivary gland chromosome. 2L-2, 2L-3, and 2L-6 are unique in that, as in the case of one inversion in D. nebulosa (Pavan, 1946), one of the two breaks is in, or very close to the heterochromatin. The precise location of these breaks cannot be determined by examination of the salivary gland chromosomes. All three inversions, however, are apparently paracentric. 2L-6, which is known only from a single wild female collected at Lake Itasca, Minnesota, arose by inversion of gene arrangement 2L-3. As can be determined from a study of Fig. 2, it is essentially a reinversion of that arrangement. This female, when originally isolated, produced no off spring and had thus presumably exhausted sperm. She was subsequently mated to a Standard male, She proved to be carrying two other peculiarities, both of which were also in chromosome 2. Connected to the same centromere as 2L-6 and showing no crossing-over among 20 larvae examined, was a new independent gene arrangement, designated as 2R-2. The other second chromosome transmitted by this female was 2L-3:2B1 but the small ragged terminal “band” of region 27 appeared to be duplicated. This was designated 2R-dp. In 2R/2R-dp heterozygotes, the 2R-dp homolog is slightly longer than 2R. Both of these peculiar chromosomes was shown to be viable in the homozygous condition to the late larval stage, a t least. Gene arrangements 2L-4 and 2L-7 are derived by inversion from 2L-1. The former is known only from an old laboratory strain collected at Kushla, Alabama; 2L-7 was found in two wild individuals collected in 1956 at Olivette Woods, St. Louis County, Missouri (Table 6). Inasmuch as this area had been continually sampled in a detailed fashion over a nine-year period prior to this time (see Table 6) it appears likely that this

POPULATION GENETICS O F DROSOPHILA ROBUSTA

19

may represent a case where a newly arising gene arrangement has been transmitted to a t least two individuals in a natural population. Gene arrangement 2L-5 is an interesting case. It was discovered by Levitan (1951~)a t Blacksburg, Virginia in the offspring of a single wild female collected in October, 1950. A single female captured near Fenton, St. Louis County, Missouri, in June, 1955, was heterozygous for 2L-5, a fact which was proved by crossing F, individuals from the wild fly to stock flies of 2L-5 kindly supplied by Dr. Levitan. No explanation of such a disjunct distribution in two places over 600 miles and 5 years apart can be given at the present time. Chromosome 3 is normally a short V-shaped element a t metaphase and in the salivary gland chromosomes the two arms commonly break out of the chromocenter in association with one another. Levitan (1952a) discovered a single wild female to be carrying in heterozygous condition an extraordinary modification of this; the two distal ends of one of the homologs were seen to be joined end to end in the salivary gland chromosome. Metaphase figures of such heterozygotes show one closed ring chromosome and one normal V-shaped chromosome. The evidence indicates that this aberration arose in t h 6 germ line of the female in question. By selection, Levitan (in litt.) has produced a stock homozygous for the ring chromosome. In the third generation of study of the ring chromosome Levitan (1952~)discovered a new gene arrangement in 3R, designated 3R-2. Because of the intensive study of Fl’s and Fz’sfrom the wild, without recovering this arrangement, this appears to be a clear instance of the spontaneous occurrence of an inversion in the laboratory in this species. Levitan (in litt.) has also found a 4-band deletion involving the distal three large bands in region 26 and the first large band of region 27 of 2R. This clearly also occurred in the laboratory; it was found once in one larva, but appeared in all salivary gland cells of this individual.

4. Pericentric Inversions In all of the inversions mentioned in the previous section, both break points are found in one arm only. I n two instances, however, inversions have been found which are pericentric. These are designated as 3L-R and 2L-R, in chromosomes 3 and 2, respectively. I n Fig. 2, the left-arm breaks are designated “L-RL” and the right-arm breaks “L-RR.” 3L-R is a very common inversion in natural populations of the north central United States; in some localities it reaches approximately 50% (Table 3, Fig. 3) and homozygotes are fully viable. 2L-R was carried by a wild female collected at Eureka, Missouri in October, 1953. Individuals homozygous for this arrangement are viable and although the laboratory stock was not rendered homozygous for this arrangement, the inversion was still

20

HAMPTON L. CARSON

abundant in the stock when last checked (May, 1955) indicating that it has heterotic effects.

V. DISTRIBUTION OF GENE ARRANGEMENTS IN SPACEI AND TIME 1. Geographical Distribution

Tables 3 and 4 give the frequencies of the gene arrangements of D. robusta as observed in 33 population samples from different geographical areas within the species range. The data for No. 11, Table 3 are from Levitan (1951a) and for No. 19, Table 4 from Levitan (1951b); the remainder are either from Carson and Stalker (1947b, 1949), Stalker and Carson (1948a)) or are previously unpublished chromosomal data obtained by the writer. Figure 3 gives data for 22 of the larger samples in an abbreviated graphic form. I n this figure, each locality is represented by a histogram, placed in its approximate geographical location. The histograms follow the pattern of the enlarged, self-explanatory histogram in the lower lefthand corner of Fig. 3. Those gene arrangements having primarily northern distributions are diagrammed in black (including 3L-R, which is indicated by white spots on a black background). Conversely, gene arrangements tending to be southern are left open. Hatched and stippled histogram sections represent arrangements without easily discerned geographical tendencies. Although the fact that there are distinct north-south tendencies may be observed a t a glance, it is perhaps more illuminating to observe the clinal change in individual chromosome arms by taking visual transects of the histograms of Fig. 3 from north to south. Thus, for example, it may be observed that XL-1, 2L-3, and 3R (including 3L-R) decline as one goes south. The clines in these three arrangements are perhaps the most striking in the species. These clines furthermore are gradual over long distances. Abrupt “steps” in the clines which might tempt one to erect taxonomic entities are generally lacking. An occasional such step may be found in one or two arms; for instance, the abrupt change in 2L and 3R between localities 17 and 20 may be cited. I n this case, however, 2L and 2R do not change concomitantly. Even in some areas where pronounced racial tendencies appear, as in Minnesota-Wisconsin and Georgia-Florida, inspection reveals that adjacent populations are wholly intermediate, indicating genetic continuity. The north-south differentiation of the genetic material of the species seems to be more pronounced than its east-west differentiation. The most extensive east-west transect (Fig. 3) is that beginning at Chadron, Nebraska (No. 1) going east through Nos. 2, 3, 4, 14, 10 to 11 (New

D

u i FIG. 3. Geographical distribution of the gene arrangements in four chromosome arms of Drosophila robusta. The histograms, which are explained in the enlarged symbol at the lower left carry numbers which correspond to the quantitative data given in Tables 3 and 4. Sections with a black background indicate arrangements with northern distributions; the open background indicates southern distributions; the remainder generally show no such relation.

TABLE 3 Frequencies (in per cent) of the Various Gene Arrangements in Population Samples of Drosophila r o h t a from Localities in the Northern Part of the Species Range (The numbers of the localities correspond to those given on the map (Fig. 3). Nx = total number of X-chromosomes examined; Na = total number of autosomes examined.)

-

-

Fig. 3, no: State: Locality: county:

1 Nebraska Chadron Dawes

2 Nebraska Ravenna BuEalo

3 Nebraska Lincoln Lancaster

4 Iowa Mt. Vernon Linn

Iowa Keokuk Lee

5 Minnesota Itasca Clearwater

Minnesota Little Falls Morrison

6 Wisconsin Mellen Ashland

Date:

Aug., 1955

Jul., 1955

Jun., 1955

Aug., 1946

Jun., 1956

Jul., 1952

Aug., 1956

Jul., 1952

XL XI.-1 XI.-2

100.0 -

57.1 42.9

-

62.9 37.1 -

52.3 47.7

-

83.3 8.3 8.3

100.0

-

100.0 -

100.0

XR XR-1 XR-2 XR-3

99.4 0.6

14.7 85.3

7.4 92.6

4.6 93.1 2.3

16.7 75.0 8.3

35.7

62.5 37.5

-

65.3

20.8 17.7 0.7 60.8

2L 2L-1

-

31.9 13.1

55.3 23.7

-

-

-

-

55.0

-

50.0 33.3

100.0

21.0

56.2 9.2 3.8 30.8

12.5

2L-2 2L-3

-

16.7

94.5:

87.5

96.0

2R

100.0

94.0

86.8

90.0

100.0

94.5:

100.0

93.6

3R 3R-1

100.0

97.8 2.2

89.5 10.5

91.5 8.5

94.4 5.6

53.0

87.5

-

50.9

-

-

47.0

12.5

49.1

Nx

337

170

27

130

12

14

8

130

N'

398

229

38

130

18

17

8

173

3L-R

-

-

-

-

-

-

-

-

-

* T w o new arrangements: 2G6; 2R-2 and 2R-dp. See Cytological Synopsis.

-

-

-

-

4.0

-

-

-

-

-

Fig. 3, no: State: Wisconsin Locality: Iron River County: Bayfield

7 Wisconsin Augusta Eau Claire

Wisconsin Madison Dane

Michigan Iron Mt. Dickinson

8 Michigan Petoskey Emmet

Michigan Cheboygan Cheboygan

9 Michigan Plainwell Megan

10 Ohio Wooster Wayne

11 New Jersey Englewood Bergen

Aug., 1954

Jul., 1952

Aug., 1956

Aug., 1956

Aug., 1956

Jul., 1954

Aug., 1956

Aug., 1946

1948-1949

6.7 93.3

33.3 66.7

-

-

-

100.0

-

100.0

-

100.0

44.9 55.1

40.2 59.8

-

39.8 59.4 0.2

9.0 45.5

29.2 68.8

8.3 91.7

45.5

2.0

21.2 76.3 2.5

40.0 36.0 26.0

88.0 0.3 11.8

7.5

80.0 3.0 5.9 11.0

42.0 28.3 4.8 25.0

Date:

XL

XL-1 XL-2

XR XR-1 XR-2 XR-3

100.0

-

66.7

-

33.3

-

-

-

13.3 86.7

-

-

-

-

-

-

-

-

-

25.0

-

-

7.1 14.3

100.0

85.0

75.0

92.5

78.6

100.0

57.6 5.9 8.5 28.0

2R

90.0

100.0

100.0

100.0

96.4

100.0

89.8

89.0

98.2

3R 3R-1 3L-R

70.0 30.0

85.0

100.0

58.3

91.7

41.7

87.5 12.5

94.1 4.2 1.7

85.0 15.0 -

97.6 2.4

2L 2G1 2L-2 2L-3

15.0

-

-

15.0

-

-

-

-

-

-

8.3

-

N=

9

15

6

11

48

12

118

102

399

N.

10

20

8

12

56

12

118

102

545

cd

8 F-

2

80 M

2

2 d W

r

a

Ld

8

8 !A 0

m

s s

TABLE 4 Frequencies (in per cent) of the Various Gene Arrangements in Population Samples of Drosophila robusta from Localities in the Central and Southern Part of the Species Range (The numbers of the localities correspond to those given on the map (Fig. 3). Nx = total number of X-chromosomes examined; N* = total number of autosomes examined.) Fig. 3, no: State: Locality: County: Date:

12 Missouri Missouri Olivette University City St. Louis St. Louis

1946-1948

Sept., 1946

13 Missouri Steelville Crawford

Missouri Montauk Dent

Aug., 1955 Jun., 1946

14 15 Indiana Indiana Crawfordsville Terre Haute Montgomery Vigo

Indiana Evansville Vanderburg

Indiana New Harmony Posey

Jun., 1956

Oct., 1956

Oct., 1956

Oct., 1956 75.0

XL xG1 xG2

97.8 1.3 0.9

98.1 0.6 1.3

78.5 15.2 6.3

76.5 14.7 8.8

76.9 21.5 1.6

90.7 7.7 1.6

100.0 -

-

25.0

XR XR1 XR-2 XR-3

43.1 54.8 2.1

-

35.8 61.4 2.8

-

25.1 46.5 28.4 -

17.7 52.9 29.4

24.6 72.3 3.1

-

36.1 57.7 6.2 -

33.3 50.0 16.7 -

41.7 41.7 16.6 -

2L 2L-1 2L-2 2L-3

50.0 39.5 8.6 0.9

63.0 30.1 6.3 0.6

68.3 24.7 5.0 2.0

82.4 14.7 2.9 -

72.4 15.3 9.2 3.1

76.3 13.7 9.2 0.8

60.0 30.0 10.0

-

55.0 35.0 10.0 -

2R

83.2

78.5

78.2

67.6

88.8

80.3

70.0

70.0

3R 3R-1 3L-R

70.6 29.4 -

68.4 31.6

59.7 40.3 -

55.9 44.1 -

78.6 21.4

67.9 32.1

50.0 50.0

65.0 35.0

-

-

-

-

-

-

-

Nx

3862

316

303

34

65

194

6

12

N.

4221

316

303

34

98

249

10

20

Fig.3, no:

18 Tennessee Gatlinburg Sevier

19 Virginia Blacksburg Montgomery

20 Alabama

21 Alabama Henry

22 Georgia Calhoun Dougherty

Apr., 1946

Apr., 1946

Apr., 1946

72.0

85.3 14.7

100.0

16 Kentucky Hopkinsville Christian

17 Tennessee Weakley Gibson

Date:

Jun., 1956

Apr., 1946

XL XL- 1

63.3 1.7 35.0

92.0 8.0

33.5 31.8 34.7

42.1 48.5 9.4

XR XR-1 XR-2 XR-3

35.0 13.3 51.7

28.0 56.0 16.0

26. 1 73. 9 -

22.2 77.8 -

95.0 -

2L 2L-1 2L-2 2L-3

55. 9 30. 4 13. 7 -

48.5 33.3 18.2 -

18.6 23.6 7.0 50.8

27.0 21.8 17.7 33.3

15.4 53.8 30.8 -

7.9 77.8 14.3 -

18.3 65.8 15.9

2R

72.5

60.6

88.7

90.5

65.4

69.8

53.7

3R 3R-1

31.4 68.6

-

36.4 63.6 -

19.9 80.1

53.4 46.6 -

100.0 -

100.0 -

100.0 -

60

25

245

617

20

50

68

102

33

301

843

26

63

82

State: Locality : County:

XL-2

3L-R

Chilton

Jul., 1947 Jun.-Oct., 1950 Apr., 1946

-

30.0 70.0 5.0

-

{ gf,",

-

28.0

100.0

2'd C

7.4

-

92.6 -

100.0

-

10

0

q

26

EUMPTON L. CARSON

Jersey). Although slight differences occur along this transect, it is the similarities which are extraordinary. Obviously the species can exist over a broad range of ecological conditions without extensive variation of the karyotypic condition. This might lead one to suggest that direct sensitivity to local ecological conditions is not the sole determining factor in the striking north-south clines. Certain gene arrangements appear to have the center of their distributions in the central part of the United States. Most striking in this regard are the X R arrangements (Tables 3 and 4;XR is not included in Fig. 3). XR-1 is a fraction of 1%a t Chadron, Nebraska (No. 1); it rises t o a peak in Iowa and Missouri and then declines eastward where it again occurs as a fraction of a per cent near Gatlinburg, Tennessee (see Stalker and Carson, 1948a). 2. Altitudinal and Regional Studies Stalker and Carson (1948a) described sharp clines of gene arrangement frequencies at 6 collecting localities extending from 4,000 to 1,000 feet in the Great Smoky Mountains near Gatlinburg, Tennessee. The clearly northern gene arrangements XL-1 and 2L-3 declined precipitously with altitude (with, of course, complementary increases in other gene arrangements). Of the 14 gene arrangements, 11 showed some change. Again, the abrupt changes in ecological conditions are not reflected by changes in every chromosome arm; some, in fact, like 2R, show almost no change with altitude. The extent of revision of the genotype over this transect, approximately 18 linear miles, is nonetheless very great when compared with what is found between areas of comparable geographical closeness but without striking ecological change. Carson and Stalker (1949) sampled two woods, three miles apart, a t Olivette and University City, Missouri, simultaneously. The data for these two samples are given in Table 6, Column 1 and Table 4,Column 2, respectively. They found that although there were satistically significant differences in the gene frequencies of several of the arrangements, these differences are small when compared to samples taken 50 or more miles apart (e.g., cf. No. 12, Table 4 with University City and then with No. 14). Similar results were obtained by Levitan (1954a, 1955) for two woods, three miles apart, near Blacksburg, Virginia. Differences in the latter case are numerous and seasonal variations occur in one woods which do not occur in the other. Table 5 presents hitherto unpublished data pertaining to this subject. From 1953 to 1955 a number of samples of D.robusta were taken a t three collecting stations on the Meramec River near St. Louis. These places are: (1) Fenton (Highway 21 bridge), St. Louis County, Missouri, which

27

POPULATION GENETICS O F DROSOPHILA ROBUSTA

is about 13 linear miles southeast of the Olivette, Missouri location (see No. 12, Table 4); (2) Eureka, St. Louis County, Missouri, 20 river miles upstream from Fenton; and (3) Steelville (Steelville Ford on the Meramec River), Crawford County, 127 river miles from Eureka. A detailed account of these stations will be found in Blight (1955). The TABLE 5 Gene Arrangement Frequencies (in per cent) over Three Years a t Three Locations along the Meramec River, Missouri* Locality Gene arrangement

Fenton St. Louis County

Steelville Crawford County

1954

1955

1953

1954

1955

1953

1954

1955

XL-1 XL-2

100.0 98.8 0.0 0.0 0.0 1 . 2

99.3 0.0 0.7

95.8 4.2 0.0

93.7 5.3 1.0

96.4 1.7 1.9

68.8 21.9 9.3

77.1 14.3 8.6

78.5 15.2 6.3

XR XR-1 XR-2

70.7 50.0 26.8 48.8 2.5 1.2

45.4 52.6 2.0

37.5 62.5 0.0

43.5 47.4 9.1

40.8 51.9 7.3

34.4 40.6 25.0

23.6 49.3 27.1

25.1 46.5 28.4

2L 2L-1 2L-2 2L-3

5 4 . 0 63.0 4 4 . 0 28.3 2.0 5.4 0.0 3 . 3

68.4 25.0 6.3 0.0

73.3 16.7 6.7 3.3

68.2 25.7 5.1 1.0

74.0 19.6 6.1 0.3

67.4 25.6 4.7 2.3

70.4 17.4 10.2 2.0

68.3 24.7 5.0 2.0

2R

88.0 85.9

80.9

60.0

73.7

78.0

79.1

75.5

78.2

3R

70.0 69.6

64.1

56.7

61.8

59.8

60.5

62.2

59.7

N=

41

86

304

24

285

358

32

140

303

N"

50

92

304

30

296

358

43

196

303

XL

~~

1953

Eureka St. Louis County

* Nx and Na mean the same as in Tables 1 and 2.

general ecology of this riparian habitat changes gradually from that of Mississippi River floodplain a t Fenton, where the Meramec is silt-laden and sluggish, to the conditions found a t Steelville where the river is fastrunning and clear and the surrounding floral conditions are those characteristic of the Ozark uplift. The change in altitude upriver is only about 400 feet. A station-to-station comparison of this transect is best made using the extensive data obtained from all three stations during the early

28

HAMPTON L. CARSON

summer of 1955 (Table 5). There is a striking increase in the frequency of a number of the chromosome inversions as one proceeds towards the headwaters of the Meramec River. XR-2 rises from 2% to 28%, XL-1 from 0 to 15% and XL-2 from less than 1% to 6%. Some tendencies are observable in certain other arrangements, for example, 2R shows about a 10% decrease from Fenton to Eureka and is thereafter unchanged despite the fact that the biggest changes in the X-chromosome arrangements occur between Eureka and Steelville. The Fenton frequencies are most similar to those found a t Olivette Woods (Table 6). Again, both distance and ecological difference are associated with karyotypic differences. It is suggestive that the ecological change must be quite obviously great before a sharp change in the inversion frequencies is encountered. It is difficult to account for the rise of XL-1 on the basis of its “northern” and “high altitude” properties. 3. Seasonal and Perennial Studies of Single Localities

Carson and Stalker (1949) followed the frequencies of the gene arrangements over a three-year period at the Olivette, Missouri station. Samples were taken every month for one of the years and spring and fall for two years thereafter (1946-1948). June samples appear in the first three columns of Table 6. I n Table 6 are also to be found previously unpublished data of the writer which complete a ten-year period of observation on this woods. Carson and Stalker (1949) found little evidence of any seasonal change in the frequencies of the gene arrangements. A number of slight changes in frequency occurred. These were not correlated with season and most were sooner or later reversed. The sample obtained at Olivette Woods in 1949 is of some interest in that the adults on which the sample is based were caught as they fed on a sap exudation of a White Oak (Quercus alba) tree near the usual collecting site. Thus no banana or other artificial bait was used. These data are strikingly similar to those obtained in former years, especially in view of the fact that minor fluctuations in frequency have been detected in banana trap collections alone. Of special interest is the fact that gene arrangement 2R, which has never fluctuated more than 2 or 3 percentage points either within or between years at this location is very close to its usual level. The conclusion is inescapable that the bleeding oak attracted flies from a population similar to that which was sampled in former years by banana traps. Banana traps would thus appear to be vindicated from the charge that they result in a highly unnatural sample of the population. The Olivette collecting area was originally an unspoiled 25-acre area of rich elm lowland with a hillside of large oak and hickory trees above it (upper photograph, Fig. 4, area to the right of the dirt road). A small

29

POPULATION GENETICS OF DROSOPHILA ROBUSTA

stream ran thxough it, and the whole area was rather remote from human habitation. In the fall of 1953 the entire wooded area was removed and graded for a residential subdivision, houses were built, and a large shopping center erected nearby (lower photograph, Fig. 4). Although a few original trees were left standing, most of these died and were removed TABLE 6 Gene Arrangement Frequencies (in per cent) at Olivette Woods, St. Louis County, Missouri over a Ten-Tear Period* Gene arranyenient

Date of sample June, 1946

June, 1947

June, 1948

June, 1949

June, 1953

June, 1954

June, 1956

XL m-1 XL-2

98.7 0.4 0.9

96.8 1.6 1.6

95.8 2.7 1.5

98.2 0.9 0.9

96.9 2.5 0.6

98.9 0.5 0.5

99.1 0.3 0.6

XR XR-1 XR-2

42.5 56.2 1.3

44.4 51.5 4.1

45.4 51.5 3.1

-13.5 53.5 3. 3

39.3 56.2 4.5

47.9 50.0 2.1

39.8 58.6 1.6

2L 2%-1 2L-2 2L-3 2L-7

52.3 38.6 8.7 0.4 -

57.3 30.3 11.3 0.9 -

50.8 39.1 9.2 0.9 -

48.2 43.7 7.8 0.3 -

58.2 34.5 7.3 0.0 -

57.3 31.3 10.4 1.0 -

48.3 43.1 8.0 0.2 0.4

2R.

83.5

84.1

83.3

83.5

81.4

87.5

88.1

3R

74.9

T1.6

68.0

74.8

67.2

67.7

73.1

N=

459

320

480

216

354

188

319

Na

474

320

588

309

354

192

453

* Nx and N* mean the same as in Tables 1 and

2.

prior to 1956. It would be difficult to experinientally accomplish a more complete alteration of the ecological conditions. In 1954, a small area of trees was left standing approximately onequarter mile from the site of the original woods. sample of Drosophila robusta was obtained from this area; the gene frequencies in this sample were essentially unchanged over the levels of previous years. In June, 1956, two and one-half years from the beginning of the destruction of the habitat another sample was obtained from the same general area. As the clump of trees used in 1954 had been removed, a narrow ravine a few

30

HAMPTON L. CARSON

FIG.4. A: Olivette Woods, St. Louie County, collecting area in June, 1953,before subdivision. Traps were always placed within the woods to the right of the dirt road. B: The same view in October, 1956, after subdivision. Note fence for orientation; the fireplug was moved to the left. The clumps of trees in which the 1954 and 1956 collections were made are off the picture to the left.

POPULATION GENETICS OF DROSOPHILA ROBUSTA

31

hundred yards to the north of this location was chosen. This contained a moderate number of oak and elm trees. Again, it may be observed that there was virtually no major change of frequencies. As previously, certain frequencies may shift significantly as compared with the previous year, but in every case the observed frequencies approximate some previously observed level. Essentially what has been observed here is equilibrium over a 10-year period. Shifts in this extraordinary stability are slight, despite drastic change in gross ecology of the region. The major adaptive properties of this chromosomal polymorphism thus do not appear to be related to gross environmental features, such as, for example, frequencies of certain types of host trees or the density of their stands. Levitan (1951b; 1951d; 1957) has described well-documented seasonal changes in the frequencies of two chromosome arms particularly (2L and XL) near Blacksburg, Virginia. Such a phenomenon is striking in view of the absence of anything comparable in the St. Louis data or in data from New Jersey (Levitan, 1951a). It is perhaps significant that the seasonal changes are such that gene arrangements 2L-3 and XL-1 are high in overwintering flies and decline as spring and summer progress. Both of these arrangements are especially frequent in northern and high-altitude areas; thus their connection with low temperature is inescapable. It is of particular interest, however, that Levitan has found that the cyclic changes described are more characteristic of the adult males than of the adult females. This is true in both the X-chromosome and in chromosome 2. Levitan (1951a, 195413) has furthermore shown that in experimental populations of this species there is a great preponderance of females among the adult flies. A study of the sex ratios of eclosing flies, moreover, shows that this high excess of females is a result of differential mortality favoring the females during the adult period. These studies, together with those t o be described in section VII below, open up a whole new series of interpretations of how balanced polymorphism is maintained in natural populations. VI. MORPHOLOGICAL VARIABILITY Stalker and Carson have made three detailed studies of the morphological variability in Drosophila robusta, namely, geographically (1947), altitudinally (19484, and seasonally (1949). Geographically, 45 strains from 22 localities throughout the range of the species were studied and the flies reared in such a way as t o minimize phenotypic variability. North-south clines were found for thorax length, head width, femur length, and wing length. The first two dimensions were greatest in the

32

HAMPTON L. CARSON

south and the last two greatest in the north. Thus one may speak of a “southern” and “northern” morphology. The morphological clines parallel in a general way the clines in frequencies of gene arrangements. The same morphological characteristics were studied in parallel fashion with the gene arrangements on flies collected along an altitudinal gradient in the Great Smoky Mountains, near Gatlinburg, Tennessee (Stalker and Carson, 1948a). Four morphological characters, thorax length, femur length, wing length, and wing width, showed significant decrease with decreasing altitude. Thus there is a general correlation with the geographical studies, although some changes (e.g., decrease in thorax length) are the opposite of what might be expected on geographical grounds. The areas of most rapid change in morphology are not the same as those in which the gene arrangements are changing fastest. Finally, a closely parallel seasonal study of morphology and gene arrangements was carried out at a single woods near St. Louis (Stalker and Carson, 1949; Carson and Stalker, 1949). Five hundred Fz progenies were reared under uniform laboratory conditions. The population underwent a regular, highly significant shift toward a southern morphology during the summer months June through August. This was proved to be a genetic change, and it is inferred that it is a cyclic seasonal one, although this has not been directly observed. This seasonal change is accomplished in the absence of any seasonal changes in the frequencies of the gene arrangements.

VII. COMBINATIONS OF INVERSIONS IN NATURAL POPULATIONS 1. The E$ect of Inversions on Crossing-Over The three major chromosome pairs of Drosophila robusta are metacentric, and paracentric inversions occur in all chromosome arms except for the left arm of the third chromosome. Considerable attention (Nelson, 1951; Carson, 1953; Levitan, 1953a, 1953b), has been given to the study of the recombination pattern imposed upon the species by the presence of these inversions. I n a female which has an inversion present in each arm of chromosome 2 (for example: Sl/lS), one might expect crossing-over to produce second chromosomes which were SS and 11. Such recombinations are very rare (far less than 1%) unless inversions are present in the other chromosomes. In general, however, the more inversions present in other chromosomes the more crossing-over occurs in the central portion X 2 of the second chromosome. In a female of the karyotype 55/22, S1/1S, 3 S1/SS, recombinations between the arms of chromosome 2 rise to about 34% .The relationship is not a simple one, however, and some X-chromo-

POPULATION GENETICS O F DROSOPHILA ROBUSTA

33

some combinations have greater interchromosomal effects than others. The X-chromosome is rather refractory to boosting effects. Differences in the results under similar conditions of heterozygosity indicate that other factors are also involved. In any event, the pattern of recombination in this species under natural conditions is far from a simple one of randomly distributed cross-overs. Wallace (1953) has presented the hypothesis that three overlapping inversions cannot become coadapted in pairs in the same population. This is because it would be expected that certain sections of the arrangements would undergo breakdown through crossing-over. Three such arrangements should thus rarely be found together. This ingenious idea is supported by the nearly complete absence of populations of D. pseudoobscura in which all three members of such triads are present in high frequencies. Wallace’s extension of this idea to D. robusta has been questioned by Levitan et al. (1954). Not only are there quite a few populations in which such triads coexist in high frequencies but most of the data on the effects of these inversion combinations on crossing-over point to a strong intrachromosomal suppression of crossing-over adjacent to the configurations (Carson, 1953). Secondly, most of the inversions in D. robusta overlap one another broadly (Fig. 2), so that the size of the segments that would be involved in the Wallace effect, if it applies in this species, would be generally quite small. One can only again emphasize that the evidence indicates a strongly canalized recombination pattern in this species. 2. Linkage and Positional Relationships within the Structural Karyotype Levitan (1954a, 1955), using a method which involves effective de-sperming of wild females, or use of naturally uninseminated fall diapause females, and mating to laboratory males of known composition has been able to extend complete karyotype analysis to females as well as males. Such an analysis makes it possible to observe the actual “linkage types” that are carried by wild flies in nature. The term linkage type has been employed by Levitan to distinguish between individuals having identical karyotypes but which have the inversions in different spacial arrangements or linkages. For example a female which is heterozygous for 2L/2L-3 and 2R/2R-1 may be either of two linkage types, to wit:

s 1

3 3

or

s s

-

3

1

The linkage types are therefore similar to cis and trans phases, and involve certain combinations of left- and right-arm inversions. Levitan has analyzed the linkage types in chromosome 2 in 2,160 adult

34

HAMPTON L. CARSON

flies collected over a 4-year period. The data, particularly in crucial cases of flies heterozygous for arrangement in both arms, give evidence that certain linkage types are above (and others correspondingly below) expectation on the hypothesis that their occurrence is at random. I n males, for instance, S1/3S individuals greatly outnumber SS/31, although both have the same structural karyotype. Levitan points out that the data indicate that the discrepancy cannot be due to a relic of the mode of origin of the arrangements or to simple interaction of genes in a leftarm arrangement with genes in a right-arm arrangement. He argues convincingly that the only adequate explanation for the results is the hypothesis that positional differences of genes in the arrangements leads to adaptively different phenotypes, at least among the males. Levitan (1957), in studying the seasonal change in the composition of the X-chromosome in his Blacksburg, Virginia populations has shown that each year XL is scarce in the spring (overwintering flies) but increases in the summer; XL-1 has a reciprocal cycle. Not all combinations of the arrangements participate in the seasonal changes. For instance, XL-1 X R - 2 chromosomes consistently show seasonal fluctuation while XL-1 X R do not. It is not entirely clear whether this is due to selection between phenotypes determined by interaction of genetic materials on both arms of the X-chromosome (Levitan, 1957) or differences between positional linkage types, as apparently is the case in chromosome 2 (Levitan, 1954d). An instance of the former has been described by Carson and Stalker (194713) for gene arrangement XR-2. This arrangement is virtually never found except in the combination XL-2 XR-2. Positional, or organizational, effects may play an important role in the maintenance of sectional heterozygosity in natural populations. Persistence of inversions in natural populations depends, in the last analysis, on the adaptive superiority of the inversion heterozygote. Levitan points out that selection may favor a certain kind of heterozygote, possibly a very complex one, not necessarily any heterozygote. The data for the second chromosome of D. robusta appears to provide a striking case of just such a situation, and further analysis is awaited with interest. 3. Marginal Homozygosity for Gene Arrangement

Scrutiny of Tables 3 and 4 and Fig. 2 reveals a rather pronounced tendency for inversion frequencies to be very high in the center of the species range (e.g., Missouri; Tennessee) while at the periphery or margin of the range (Wisconsin-Minnesota; Western Nebraska; GeorgiaAlabama) the species tends towards homozygosity for gene arrangement. Carson (1953, 1955a) has measured the lengths of the inversions and

POPULATION GENETICS OF DROSOPHILA ROBUSTA

35

determined their gross effects on recombination. From these facts and their frequencies in various parts of the range, he has calculated a n “index of free recombination” for a number of the populations of this species. This index is determined by finding out how much of the euchromatin of the population is free to undergo recombination and how much is blocked by inversion heterozygosity. The more marginal the geographical area, the freer the recombination potential becomes ; the most striking and extreme case yet found is among flies collected a t Chadron, Nebraska, a t the terminus of the eastern flora along a tributary of the White River in the northwestern corner of the state (Carson, 1956a). This population proved to be virtually homozygous for gene arrangement (see No. 1, Table 3), an unprecedented situation in D. robusta. These facts are interpreted to mean th a t central populations are genetically more specialized, many genes being effectively tied up in nonrecombining coadapted groups. Marginal populations, on the other hand, tend t o be structurally homozygous and have a greater free recombination. The latter are thus more flexible in the sense of capacity for adjustment t o a major change in new conditions. The formation of major adaptive novelties, therefore, would be most easily accomplished in species or populations which have a high index of free crossing-over (Carson, 195513).

VIII. EXPERIMENTAL LABORATORY

POPULATIONS

Levitan (1951a) performed twenty-one population cage experiments involving Drosophila robusta. These were made up of flies collected in the New York City area which were carrying known inversions in the left arm of chromosome 2. Mixtures were prepared in various proportions of two of the left-arm arrangements. Most of the populations underwent pronounced directional changes in arrangement frequencies which were the result of natural selection. The frequencies of the gene arrangements in the populations in almost every case appeared to attain a stable equilibrium in which the competing gene arrangements were present in constant frequencies. I n no case was one arrangement eliminated by another, and in a number of instances replicate experiments did not result in the same equilibrium between the arrangements. Levitan concludes that these results indicate that the frequencies of the gene arrangements in nature are determined by natural selection in a similar manner as in cage experiments, probably acting through the adaptive superiority of inversion heterozygotes. Carson and Stalker (unpublished data; Table 7) established four replicate population cages in April, 1949, two a t 25OC. and two at 17°C. Gene arrangements introduced were XL; XR,XR-1 ; 2L,2L-1,2L-3;

36

HAMPTON L. CARSON

2R,2R-1; 3R,3R-1. The strains used came from Fall, 1948 collections at Olivette Woods, St. Louis County, Missouri (see Table 6 ) . The arrangements were introduced in approximately the equilibrium frequencies for this locality, except that 2L-3 was high (Tables 6 and 7). I n all cages there appears to have been an immediate equilibrium shift in 2L, with standard 2L decreasing a t the expense of 2L-1. Although certain other slight shifts are apparent (e.g., in 3R) there is no tendency to major TABLE 7 Gene Arrangement Frequencies (in per cent) in Four Population Cages Prepared with Flies from Olivette Woods, St. Louis County, Missouri* Initial % for all Gene cages arrange- May, ment 1949

Cage 111, 17°C.

Cage V, 17°C.

Aug., 1950

Nov., 1949

Nov., 1949

Cage 11, 25°C.

Cage IV, 25°C.

Sept., 1949

Aug., 1950

Sept., 1949

~

_

_

XR (XR-1)

50.0

52.7

55.3

54.7

62.3

45.7

47.3

2L 2L-1 2L-3

56.7 39.5 3.8

39.3 55.0 5.7

38.9 53.5 7.7

38.3 53.7 8.0

38.7 54.7 6.6

43.3 53.0 3.7

45.3 46.0 8.7

2R (2R-1)

82.4

86.0

81.9

83.3

94.3

72.0

84.0

3R (3R-1)

76.1

77.7

62.0

72.7

52.0

69.3

74.0

N=

238

300

291

300

300

300

300

Nu

238

300

326

300

300

300

300

* Nx and Na mean the same as in Tables 1 and 2. directional change and no correlation with temperature. The basic result is obvious: frequency equilibria similar to those found in nature in the population from which the flies came are essentially maintained. Such a result can only mean that the chromosomal polymorphism within a given population, although it may have been originally molded by the environment in nature, is not easily moved from its optimal population level by a drastic change in ecological conditions, such as removal to the laboratory. This result appears to be in accord with what has been observed after destruction of Olivette Woods (see above). I n this regard, it seems noteworthy that it has been the repeated observation of the

_

POPULATION GENETICS OF DROSOPHILA ROBUSTA

37

writer that ordinary laboratory strains of D. robusta are highly tenacious of the gene arrangements they have a t the beginning of laboratory culture. I n most cases i t does not appear to be necessary to render a strain homozygous in order to keep a naturally occurring arrangement in culture.

IX. CONCLUSION : THE GENETICNATUREOF DrosophiEa robusta AS A

SPECIES

Drosophila robusta, with few relatives, appears to be an old, conservative and clearly isolated number of the fauna of the eastern deciduous forest of the United States. It breeds on a characteristic tree of this forest, the American Elm. The three major pairs of V-shaped chromosomes occur in a number of alternate gene sequences, differing from one another by large inversions. Seventeen such arrangements are known from two or more localities; most are very widespread; one is a pericentric inversion. Geographically, the species shows clines in external morphology and in gene arrangements; they are roughly parallel. The gene arrangements are sensitive to geographical change but the relation to specific ecological components is not always clear. Both morphological and cytological clines are steep with altitude and nowhere are steps in the clines pronounced. Marginally, the species shades into homozygosity for gene arrangement. I n some areas, seasonal changes are sharp; their nature is complex and in some instances selection appears to favor complex positional arrangements of certain inversions. The inversions profoundly canalize crossing-over. Conditions in the species support the view that the chromosomally highly heterozygous central portions are in a sense genetically specialized. Thus the extensive chromosomal heterosis found in this species is tenacious in natural and artificial populations. Its presence reduces recombination throughout the genome. Central populations would thus appear to be less capable of the synthesis by recombination of true evolutionary novelties. In this sense, a t least, marginal populations would be expected to respond more quickly to major evolutionary challenges. X. ACKNOWLEDGMENTS I wish to express in particular my great appreciation to Dr. Max Levitan for stimulating letters and discussions and for his generosity in making available to me much unpublished data. Dr. H. D. Stalker has likewise allowed use of unpublished data, has personally collected many of the samples, and has continually been a source of valuable ideas. Dr. A. H. Sturtevant was kind enough to allow full use of his unpublished data. The following persons have supplied unpublished material for

38

HAMPTON 1;. CARSON

Tables 1 and 2 :Dr. J. T. Patterson, Dr. W. S. Stone and Dr. M. R. Wheeler, in particular, who have generously allowed inclusion of much valuable data of the University of Texas Laboratory, Drs. A. Sokoloff, M. E. Annan, J. M. Carpenter, D. D. Miller, C. P. Oliver, H. T. Speith, W. P. Spencer, C. S. Pittendrigh, D. Williamson, R. C. Lewontin, D. A. Hungerford, W. C. Blight, A. Romano, and C. J. Bennett. To all of these persons who have-contributed so much to this paper, I am most grateful. Much of the recent preparation of slides was done by Miss Eleanor Suchland, Miss Bozena Kallus and Miss Barbara Fretwell. My wife, Meredith Carson, has not only been a tolerant companion on many a grueling collecting trip but has caught many flies as well as prepared most of the figures for this paper. These studies have been supported by grants from The Rockefeller Foundation, The Office of Naval Research and The National Science Foundation.

XI. REFERENCES Blight, W. C., 1955. A cytological study of linear populations of Drosophila americana near St. Louis, Missouri. Doctor’s Thesis; Washington University, St. Louis, Missouri. Carson, H. L., 1951. Breeding sites of Drosophila pseudoobscura and Drosophila persimilis in the Transition zone of the Sierra Nevada. Evolution 6, 91-96. Carson, H.L., 1952. Contrasting types of population structure in Drosophila. Am. Naturalist 86, 239-248. Carson, H. L., 1953. The effects of inversions on crossing over in Drosophila robusta. Genetics 38, 168-186. Carson, H. L., 1955a. Variation in genetic recombination in natural populations. J . Cellular Comp. Phusiol. 46, 221-236. Carson, H.L., 1955b. The genetic characteristics of marginal populations of Drosophila. Cold Spring Harbor Symposia Quant. Biol. 20, 276-287. Carson, H. L., 1956a. Marginal homozygosity for gene arrangement in Drosophila robusta. Science 123, 630-631. Carson, H. L., 1956b. Response of Drosophila robusta to selection for motility. Genetics 41, 636-637. Carson, H.L., 1957. Production of biomass as a measure of fitness of experimental populations of Drosophila. Records Genet. Soc. Am. 26, 636-637. Carson, H.L., and Stalker, H. D., 1946a. Chromosome studies on Drosophila robusta. Genetics 31, 213. Carson. H.L., and Stalker, H. D., 1947a. A seasonal study of gene arrangement frequencies and morphology in Drosophila robusta. Genetics 32, 81. Carson, H.L., and Stalker, H. D., 1947b. Gene arrangements in natural populations of Drosophila robusta Sturtevant. Evolution 1, 113-133. Carson, H. L., and Stalker, H. D., 1948a. Reproductive diapause in Drosophila robusta. Proc. Natl. Acad. Sci. U.S. 34, 124-129. Carson, H. L., and Stalker, H. D., 1948b. An altitudinal transect of gene arrangement frequencies in Drosophila robusta. Genetics 33, 100. Carson, H. L., and Stalker, H. D., 1949. Seasonal variation in gene arrangement frequencies over a three-year period in Drosophila robusta Sturtevant. Evolution 3, 322-329.

POPULATION GENETICS O F DROSOPHILA ROBUSTA

39

Carson, H. L., and Stalker, H. D., 1950. Natural breeding sites for Drosophila robusta. Genetics 36, 100. Carson, H. L., and Stalker, H. D., 1951. Natural breeding sites for some wild species of Drosophila in the eastern United States. Ecology 32, 317-330. Carson, H. L., Knapp, E. P., and Phaff, H. J., 1956. Studies on the ecology of Drosophila in the Yosemite region of California. 111. The yeast flora of the natural breeding sites of some species of Drosophila. Ecology 37, 538-544. Dorsey, C. K., and Carson, H. L., 1956. Selective responses of wild Drosophilidae to natural and artificial attrahents. Ann. Entomol. SOC.Am. 49, 177-181. Frolova, S. L., 1936a. Struktur der Kerne in den Speicheldrusen von Drosophila sulcata. Stert. Byull. Eksptl. Biol. Med. 2, 93-96. Frolova, S. L., 1936b. Several spontaneous chromosome aberrations in Drosophila. Nature 138, 204-205. Galey, M. M., 1949. The rate of maturation of the imago in Drosophila robusta. Master's Thesis. Washington University, St. Louis, Missouri. Kikkawa, H., and Peng, F. T., 1938. Drosophila species of Japan and adjacent localities. Japan. J . 2001.7, 507-552. Levitan, M., 1950. Retention of a pericentric inversion in populations of Drosophila robusta. Genetics 36, 674. Levitan, M., 1951a. Experiments on chromosomal variability in Drosophila robusta. Genetics 36, 285-305. Levitan, M., 1951b. Selective differences between males and females in Drosophila robusta. Am, Naturalist 86, 385-388. Levitan, M., 1951c. 2L-5: (a new gene arrangement). Drosophila Inform. Serv. 26, 94. Levitan, M., 1951d. Response of the chromosomal variability in Drosophila robusta to seasonal factors in a southwest Virginia woods. Genetics 36, 561-562. Levitan, M., 1951e. Locus of natural selection in an experimental population. Virginia J. Sci. 2, 35. Levitan, M., 1952a. A ring-chromosome in wild-caught Drosophila. Genetics 37, 600. Levitan, M., 195213. Drosophilidae in a southwest Virginia woods. Virginia J . Sn'. 3, 298. Levitan, M., 1952c. 3R-2: (a new gene arrangement). Drosophila Inform. Serv. 26, 87. Levitan, M., 1953a. Interchromosomal influences on recombination of chromosomal segments in Drosophila robusta. J . Tenn. Acad. Sci. 28, 183. Levitan, M., 1953b. Crossing over adjacent to overlapping and included inversions. Drosophila Inform. Serv. 27, 97-98. Levitan, M., 1954a. Position effects in natural populations. Am. Naturalist 88,419-423. Levitan, M., 1954b. Sex factors and selection in experimental populations, with a note on selection and the sex ratio. Virginia J . Sci. 6, 131-143. Levitan, M., 19540. Drosophilidae in New York and New Jersey. Am. Midland Naturalist 62, 453-459. Levitan, M., 1954d. Additional evidence of position effects in natural populations. Genetics 39, 979. Levitan, M., 1955. Studies of linkage in populations. I. Associations of second chromosome inversions in Drosophila robusta. Evolution 9, 62-74. Levitan, M., 1957. Natural selection for linked gene arrangements. Anat. Rec. 127, 430. Levitan, M., Carson, H. L., and Stalker, H. D., 1954. Triads of overlapping inversions in Drosophila robusta. Am. Naturalist 88, 113-114. Makino, S., Momma, E., Takada, H., and Wakahama, K., 1955. Drosophilidae collected in Hokkaido. Drosophila Inform. Serv. 29, 134-135. Malloch, J. R., and McAtee, W. L., 1924. Flies of the family Drosophilidae of the

40

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L. CARSON

District of Columbia region, with keys to genera and other notes of broader application. Proc. Biot. SOC.Wash. 37, 25-42. Momma, E., 1954. New Karyotype found in the genus Drosophila. Drosophila Inform. Serv. 28, 137. Nelson, S. E., 1951. The influence of inversions on crossing over in the X-chromosome of Drosophila robusta. Master’s Thesis. Washington University, St. Louis, Missouri. Patterson, J. T., 1943. The Drosophilidae of the Southwest. Univ. Tezas Publ. NO. 4313,7-216. Patterson, J. T., and Stone, W. S., 1952. “Evolution in the Genus Drosophila.” Macmillan, New York. Patterson, J. T., and Wagner, R. P., 1943. Geographical distribution of species of the genus Drosophila in the United States and Mexico. Univ. Texas Publ. No. 4313, 217-281. Pavan, C. 1946. Chromosomal variation in Drosophila nebulosa. Genetics 31, 546-557. Spiess, E. B. 1949. Drosophila in New England. J . N . Y . Entomol. Soc. 67, 117-137. Stalker, H. D., and Carson, H. L., 1946. Geographical variation in the morphology of Drosophila robusta. Genetics 31, 231. Stalker, H. D., and Carson, H. L., 1947. Morphological variation in natural populations of Drosophila robusta Sturtevant. Evolution 1, 237-248. Stalker, H. D., and Carson, H. L., 1948a. An altitudinal transect of Drosophila robusta Sturtevant. Evolution 2, 295-305. Stalker, H. D., and Carson, H. L., 1948b. Seasonal changes in gene arrangement frequencies and morphology of Drosophila robusta. Genetics 33, 629-630. Stalker, H. D., and Carson, H. L., 1949. Seasonal variation in morphology of Drosophila robusta Sturtevant. Evolution 3, 330-343. Stevenson, R. 1952. Altitudinal distribution of species of the genus Drosophila (Diptera) on Unaka Mountain, Tennessee-North Carolina. J . Tenn. Acad. Sci. 27, 97-103. Sturtevant, A. H., 1916. Notes on North American Drosophilidae with descriptions of twenty-three new species. Ann. Entomol. SOC.Am. 9, 323-343. Sturtevant, A. H., 1921. The North American species of Drosophila. Carnegie Inst. Wash. Publ. No. 301. Tan, C. C., Hsu, T. C., and Sheng, T. C., 1949. Known Drosophila species in China with descriptions of twelve new species. Univ. Texas Publ. No. 4920, 196-206. Wallace, B., 1953. On coadaptation in Drosophila. Am. Naturalist 87, 343-358. Williams, D. D., and Miller, D. D., 1952. A report on the Drosophila collections in Nebraska. Bull. Univ. Nebraska State Museum 3, 1-19.

THE GENETICS OF BASIDIOMYCETES Haig

P. Papazian*

169 Cold Spring Street, New Haven 1 1 , Conn.

Page 41 42 43 43

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Mating System.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ....... 1. Comparison with Flowering Plants.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Di-mon Matings and Relative Sexuality.. . ................. 111. Heterokaryons. . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Dikaryon.. . . . . .... ............................... 2. “Flat” Mycelia.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Mutation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Induced Mutants.. . . . . . . . . . . . . . . . . .................... V.

2. Undetermined Mutagens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Incompatibility Factors. . . . . . . ................................ Segregation.. . . . . . . . . . . . . . . . . . . ................. 1. Somatic Recombination. .................... 2. Tetrads . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ............. 3. Recombination between Incompatibility Genes 4. Labile Heterozygosity for One Incompatibility Factor. . . . . . . . . . . . . . . 5. 3: 1 Tetrad Segregation., ................................ 6. Spontaneous Dikaryotization.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cytoplasmic Autonomy. . . . . . . . . . . . . . . . . . . . . ........... 1. The Harder Operation.. . . . . . . . . . . . 2. Affinity of Mating Type.. . . . . . . . . . ........................ 3. Dwarf Mycelia.. . . . . . . . . . . . . . . . . . 4. Sectoring Variants.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nucleocytoplasmic Relations. . . . . . . . . . . . . . . . . . . . . . .......... References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

.

VI.

VII. VIII.

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49 52 57 58

59 60

62 64 65 67

I. INTRODUCTION I n the recent history of genetics fungi hold a respectable place. Interest in these organisms has followed, on the whole, phylogenetic sequence. I n the first quarter of this century the work of Blakeslee, Burgeff, and others focused attention on the Phycomycetes. I n the next quarter century pioneering work of Winge and of Lindegren on yeast and the intensive work on the biochemical genetics of Neurospora brought the Ascomycetes into prominence. During this period the Basidiomycetes received the attention of such eminent geneticists as H. Kniep and A. Quintanilha but their work was not continued by other workers a t

* Original work on Coprinus reported here was supported by N.S.F. 41

grant #1031.

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the time. May this review nourish the thought that the genetics of Basidiomycetes will flower in the present third quarter of this century. Basidiomycetes have several interesting characteristics. I n a sense they bridge the gap between microorganisms and higher organisms. On the one hand they are haploid organisms in which biochemical characters can be easily studied and in which tetrads can be isolated; on the other hand they are cellular organisms developing large fruiting bodies with a complex morphology and highly differentiated cells and tissues. A unique feature of the Basidiomycetes is the dikaryon mycelium which is open to wide exploitation. Basidiomycetes have been the subject of investigations in many different areas of genetics and these will be described in various sections. The rusts and smuts, though taxonomically Basidiomycetes, are genetically rather distinct owing to their mating system and the special techniques required for their culture. I have not worked with them and for these reasons the extensive genetic work on both rusts and smuts will not be included here. 11. MATINGSYSTEM The mating system of Basidiomycetes resembles in some aspects that of the flowering plants (Lewis, 1954). I n a population there are a large number (Whitehouse, 1949) of incompatibility alleles, or factors, such that two plants having different factors are interfertile but two plants having similar factors are incompatible. The word factor will be used rather than allele for the operational unit since their genetic control is a subject for discussion. There are no sexual organs and plasmogamy occurs between vegetative hyphae or between an oidium and a vegetative hypha. A complication arises in the case of the majority of Basidiomycetes and in particular in Schizophyllum and Coprinus lagopus. I n these so-called “ tetrapolar ” species there is not one but two series of incompatibility factors, the “ A ” and the “ B ” series. There are many factors in each series and a fertile mating only occurs when both the A and the B factors are different in the two mates. Sexual reproduction is normally bi-parental so that in a tetrapolar species only two factors of each series goes into and comes out of a fruiting body giving, in combination, spores of four different mating types from any fruiting body. Hence the misleading term “ tetrapolar.” Mycelia derived from a single basidiospore have typically 1 nucleus per cell in heterothallic species and are called monokaryons. After hyphal fusion between two compatible monokaryons a dikaryon mycelium, having 2 nuclei per cell grows and produces, in due course, the fruiting body. I n some Coprinus species fruiting bodies have been seen to arise from a single dikaryon cell (Brefeld, 1877) and this may well be true of

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all Basidiomycetes. Karyognmy occurs in the basidium immediately succeeded by meiosis. No technique has yet been developed to identify mating types other than by making crosses with appropriate tester strains. The dikaryon in compatible matings can be recognized b y the presence of clamp connections between all adjacent cells. 1. Comparison with Flowering Plants

There are two important differences between the sexuality of Basidiomycetes and the flowering plants which should be borne in mind while speculating on the mode of action of these multi-factor systems. First, one of the units concerned in the flowering plants, the style, is diploid. Secondly, the incompatibility reaction in flowering plants is extracellular, growth or no growth of a pollen tube in the tissue of a style; in the Basidiomycetes i t is intracellular, migration or no migration of a nucleus into cytoplasm. The fact that hyphal fusions occur in Coprinus lagopus and other species regardless of mating type shows th a t the incompatibility reaction does not occur a t cell contact or cell fusion. 2. Di-mon Mntings and Relative Sexuality

The extensive dikaryon stage in Basidiomycetes allows matings between dikaryon and monoksryon mycelia to be made. Such di-mon matings were first described by Buller (1931) (the “Buller phenomenon”). Three kinds can be distinguished: compatible di-mons where the monokaryon, e.g. A’B’, is compatible with both components of the dikaryon, e.g., A2B2 A3B3,hemi-compatible di-mons where it is compatible with one but not the other, e.g. A’B’ X (A’B’ A2B2),and noncompatible where i t is compatible with neither, e.g. AIB1 X (A1B2 A2B1). In Schixophyllum, in Coprinus lagopus, and in some other species, the monokaryon is converted into a dikaryon in all three cases. The mechanism involved in noncompatible di-mons will be discussed in the section “Segregation.” It will be seen that in compatible di-mons the monokaryon can be dikaryotized by either nucleus of the dikaryon and the one elected will presumably be the one with the greatest affinity if there be any such. A series of compatible di-mons was made with Schizophyllum (Papazian, 1950) using four factors of the A and of the B series in 20 different combinations and although the same nucleus dikaryotized the monokaryon in any particular cross, no order or law could be seen in the results taken as a whole. This provides evidence against the very general views of Hartmann (1943, 1955) on relative sexuality as they are applied t o the Basidiomycetes. It is true th at in a somewhat similar but less extensive experiment Quintanilha (1939) was able to assign degrees of strength to

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four incompatibility factors such that the dikaryon was always formed between nuclei having the greatest difference but the probability of this being possible with random data is too high to warrant giving much significance to this interpretation. There remains the fact that in both Quintanilha’s and my experiments the elected nucleus was not random but constant for a particular combination; these experiments, however, do not indicate that the incompatibility factors themselves are the determinants and it is likely that the rest of the genome contributes the factors responsible for these secondary affinities (see below, “ Cytoplasmic Autonomy”). One general characteristic of the mating system in Basidiomycetes is perplexing. I n contrast to the Phycomycetes and Ascomycetes, the Basidiomycetes are notoriously sterile in interspecific or even intervarietal crosses. What relation this bears to the multi-factorial mating system is a matter for speculation. Each species might have a completely different set of incompatibility factors or largely the same incompatibility factors with an overriding sterility factor. The question is whether the fertility of similarity and infertility of difference exemplified in interspecific matings is due to the same factors that cause infertility of similarity and fertility of difference in intertype matings within a species. Greater sterility of interspecific and intervarietal crosses could be merely a reflection of the taxonomists’ judgment in the group but it is unlikely that the Basidiomycetes represent a “lumpers ” market. I n one of the best taxonomic studies of a section of Coprinus (Lange, 1952) the general conclusion was that “. . intersterility barriers divide the studied strains into groups corresponding to, or in some cases more narrow than, the species defined according to morphological characters . . .”

.

111. HETEROKARYONS 1. Dikar yon

In a tetrapolar species when two mycelia which have different incompatibility factors in both A and the B series meet, a heterokaryotic mycelium, the dikaryon, is established. This dikaryon has, in most species, exactly two nuclei in all cells, forms a clamp-connection between all cells, and is very stable. By inoculating a mycelium near the periphery of a compatible mycelium already grown to an appropriate diameter Buller (1931) obtained an estimate of the rate of migration of the invading nuclei of about 1 mm. per hour. I n more precise experiments with Coprinus macrorhixus f. microsporus Kimura (1954a) followed the course of migration by cutting out rows of small samples from the large mycelium and obtained rates of migration of 3.2 mm. per hour. The rates of growth

GENETICS OF BASIDIOMYCETES

45

for monokaryon and dikaryon mycelia in this species were 0.15 and 0.3 mm. per hour, respectively. The most acceptable interpretation of these experiments is that invading nuclei migrate through the cells of a compatible mycelium with few divisions and set up dikaryon cells with clamp connections when they reach the terminal cells. The subject is still an open one, however, for it is difficult to be sure whether invading hyphae may not grow throughout the older mycelium and fuse only when they reach the periphery. The association of clamp connections with the dikaryon is normally very reliable but a dikaryon is not sufficient cause for the formation of clamps. Some species do not form clamps in their dikaryon (Brunswik, 1924) and occasionally clamps will be absent in a species normally forming them. Furthermore, Harder (1927) obtained monokaryons, derived from dikaryons, in which “ pseudo-clamps” were produced for a considerable number of cell generations of the monokaryon.

2. “Flat” Mycelia In matings between mycelia having different and therefore compatible factors in one series but not the other, peculiar relations exist. In Schixophyllum when the named A factors are similar and the B factors different a mycelium, “Flat,” with special properties is formed (Papazian, 1950). A somewhat similar mycelium, but less stable, is formed between mycelia of Schizophyllum having different A but similar B factors and in this species it is responsible for the so-called “ barrage phenomenon.” There are indications that similar relations hold in some other species (see Papazian, 1950; Raper and San Antonio, 1954), but I have failed to demonstrate such a phenomenon in several stocks of Coprinus Zagopus. There is no evidence that the “barrage phenomenon” in Lenxites (Brodie, 1936) is a similar phenomenon to the barrage in Schizophyllum. Flat mycelia in Schizophyllum are stable when cultured from large inocula, have a distinct morphology (Papazian, 1950), and show distinct growth characteristics (Raper, 1954). They exhibit in addition the property of unilateral dikaryotization, acting as donors or males but not as acceptors in matings with normal mycelia of mating type compatible with that of either of the Flat components. These findings indicate that Flat mycelia are heterokaryotic. Yet when inocula about 1 mm. square or single hyphae are cultured, normal mycelia having single specific mating types are obtained in the vast of majority of cases. This indicates contrariwise that Flat mycelia are heterohyphal rather than heterokaryotic. The distinction is difficult, technically, to evaluate. There is no indication from microscopic observation that hyphae are bunched together more than in normal monokaryons; in fact the hyphae are more

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sparse in Flat mycelia. If the characteristics of Flat are a result of loose proximity of hyphae of the two different components then the same characteristics should be exhibited when the two components are grown either side of a permeable membrane. I have failed to demonstrate any interaction between mycelia of Schizophyllum of various mating types grown either side of a permeable membrane. Contact and almost certainly plasmogamy is therefore necessary t o produce a Flat mycelium, yet heterokaryotic cells are rare, presumably they do not persist, or a t least grow, for long. Experiments in which hyphae of Flat mycelia were made to cross an air gap (Raper and San Antonio, 1954) have been interpreted as showing that one of the component nuclei can migrate for some distance through hyphae of the other component. This is a point of interest and conflicts with the general view of Flat mycelia which will be discussed again in a later section. I n Raper’s experiments a disk of complete nutrient agar was placed in the center of a Petri dish separated by an air gap 1-mm. wide from minimal medium which filled the rest of the disk. The Flat mycelia used consisted of a uracil deficiency mutant for one component and a morphological mutant for the other and were inoculated onto the center disk. The first hyphae t o become established on the minimal medium across the air gap stopped growing after 1-3 mm. indicating that they were the uracilless component. Then, after a day to a week’s delay, hyphae on the minimal medium became “increasingly gnarled and densely and irregularly branched.” From some of these branches but “rarely from the tips of the original hyphae” the morphological mutant component grew characteristically. The conclusion drawn from these experiments was that the morphological mutant nuclei crossed the air gap through the hyphae of the uracilless component. The crucial question is whether any hyphae of the morphological mutant crossed the air gap and the significance of the conclusions depends upon the likelihood that such hyphae would be detected. Confusion is introduced when the characteristics of Flat in Schizophyllum are compared to ((Blotchy” mycelia in Cyathus stercoreus (Fulton, 1950). Blotchy mycelia are also produced in matings between two monokaryons having similar incompatibility factors in one series but not in the other. Cytologically they have two nuclei per cell in contrast to Flat which has one, none, or perhaps occasionally two. More curious is the fact that Blotchy dikaryotizes unilaterally but as a n acceptor not as a donor. This reversed behavior can be generalized further. I n Cyathus all aberrant mycelia which dikaryotize unilaterally act as acceptors and have more than 1 nucleus per cell. I n Schizophyllum several unilaterally dikaryotizing mutants are known and they invariably act as donors and have, in the cases examined, 1 nucleus per cell.

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47

IV. MUTATION 1. Induced Mutants There have been few studies of spontaneous or induced mutants in the higher Basidiomycetes so that the following brief account is fairly complete. Mycelia of Coprinus macrorhizus growing on malt agar have been irradiated with some 11,000 r. by Dickson (193613) and then cut up into squares and subcultured. I n this seemingly inefficient way several morphological variants were obtained but none were shown to be single gene effects. One fluffy variant appeared repeatedly in a particular mating type but there is no evidence that this was correlated with the mating type rather than with some other characteristic of that particular culture. The possibility that this fluffy saltant was not nuclear will be discussed in the section on “Cytoplasmic Autonomy.” Basidiospores of Coprinus lagopus have been irradiated with X-rays a t 50,000 r. by Mittwoch (1951). The number of spores involved is not given but 4 morphological variants are described all of which segregate approximately 1 : l . One of them appeared to be linked to one of the incompatibility factors. Lisbeth Fries (1948) has obtained more quantitative data with Coprinus Jimetarius which may be equivalent to C. lagopus. Ultraviolet irradiation had no significant effect on mutation or viability of basidiospores. Nitrogen mustard at 0.25% and 0.5% for 30 minutes reduced germination by a factor of about 10 and 16, respectively. Among 916 mycelia from mustard-treated spores many morphological variants were seen and 5 biochemical variants were obtained. Two of the latter required hypoxanthine or adenine, 2 required methionine or cysteine, and 1 required an undetermined amino acid. In Schizophyllum, Raper (1953) reports that a large number of biochemical and morphological mutants were obtained by ultraviolet irradiation of monokaryotic mycelia, he has characterized one ultraviolet mutant isolated by the present author as uracil deficient. More recently D. Lewis (unpublished) has obtained 4 adenineless, 1 riboflavineless which also grew on inositol, 1 nicotinamideless, and several morphological mutants by ultraviolet irradiation of oidia of Coprinus species and enrichment by filtration. 2. Undetermined Nutagens

The first of a series of papers on “Heterokaryotic mutagenesis in Hymenomycetes” by Raper and San Antonio (1954) threatens to be a major contribution to our understanding of mutation. The title refers to the fact that in Flat cultures of Schizophyllum morphological variants can frequently be seen (Papazian, 1950). When isolated and crossed with

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wild type these variants show typical Mendelian segregation. I n addition to the original “streak” and dwarf “streak” (Papaaian, 1950-1951), 6 more distinguishable types have been repeatedly isolated (Raper, 1953). The interest in this phenomenon centers around the fact that these 8 or more types obtained by “ heterokaryotic mutagenesis” are not ones that are readily obtained from the same stocks by irradiating monokaryotic mycelia with ultraviolet. Conversely biochemical mutants are not readily isolated following “ heterokaryotic mutagenesis.” That the heterokaryotic condition is determining a preferential increase in mutation rate at these few loci can only be well established by reliable quantitative data and for this Schizophyllum, having no asexual spores or uninucleate stage in its life cycle besides the basidiospore, is poorly equipped. Where several sectors of variant growth are picked up on a Petri dish already covered with mycelium it cannot be assumed that each sector originated from an independent event. One event could give rise to several apparently unconnected sectors by migration of nuclei or by growth of small undetected strands of hyphae. Apart from this it is increasingly evident that in microbial genetics one should never underestimate the power of selection. Almost any mutant can be isolated in large numbers with appropriate selection procedures and the possibility of a selective mechanism operating in the case of “heterokaryotic mutagenesis” is obvious. Furthermore, the most prominent characteristic of the mutant types gives a clue to the way in which selection might act. The mutant types are distinguished by dikaryotizing unilaterally as donors. Now whatever the extent of heterokaryosis in Flat mycelia may be, it does appear established that plasmogamy and a t least local heterokaryosis takes place and that this contributes to the poor, stunted growth of Flat mycelia. A variant which refused to accept foreign nuclei would then be free from this handicap. If selection plays a major role in the regular appearance of these mutant types in Flat mycelia it is not unlikely that there is a general random increase of mutation rates in old Flat mycelia. Chemical mutagens are now known to be very numerous and it would not be surprising if some were produced by aging Flat mycelia. It would not even be surprising if such mutagens had a slight differential effect on some loci (see Kolmark and Westergaard, 1953). A mutagenic process affecting some 8 loci to the almost complete exclusion of other loci would represent quite a different order of specificity and would suggest some mutagenic process much more intimately connected with a gene than is probably the case with the known chemical mutagens. Evidence for an unorthodox and important phenomenon of this sort needs to be all the more critical.

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49

One property of “streak,” the first mutant obtained from Flat mycelia, is of physiological interest. Streak which is controlled by a single gene linked to the A incompatibility locus always exhibits the property of elasticotropism (Papazian, 1950). When grown on agar which is under mechanical stress it grows very precisely along the lines of stress. This is not true of wild type monokaryons. A curiously high proportion of variants has been found in monokaryons which have been obtained by microsurgical operations (see below, “Cytoplasmic Autonomy”) from dikaryotic hyphae. Both biochemical and physiological variants have been obtained (Ashan, 1952; Fries and Ashan, 1952), and some of the morphological variants in Schizophyllum show Mendelian segregation (Papazian, 1955) and must be regarded as gene mutants. 3. Incompatibility Factors

Mutation of the incompatibility factors has deservedly received considerable attention. Most workers have noted a great stability of these factors to spontaneous change during vegetative growth (Kniep, 1929). In attempts to induce such mutations mycelia from two monokaryons of Schizophyllum differingin only one factor (e.g. AIB1and A1B2)were cut up into short hyphae in a Waring blender, plated out and irradiated. Any mutation of the factor that was common (e.g., A]) would provide a nucleus compatible with half the hyphae on the plate. Dikaryotic mycelia might then be expected in large sectors of the periphery of the growing inoculum. None such were found. An objection to this technique was realized when it was discovered that for a day or two after irradiation with ultraviolet monokaryons of Schizophyllum were abnormal in their mating reactions, particularly in their ability to accept nuclei in a cross. Further experiments were made with C. lagopus using mixtures of oidia and of short hyphae of appropriate mating type and treating with acriflavine, camphor, and epichlorohydrin. The results were uniformly negative. Some 108 nuclei in hyphae and lo9 nuclei in oidia were treated but the low germination (about 0.1%) of oidia would make the effective number with oidia smaller. The various data certainly indicate that the incompatibility factors in Basidiomycetes, like those in flowering plants, are unexpectedly stable as regards spontaneous and induced mutation, but the possibility that physiological effects prevent the detection of new factors in these observations must not be overlooked. In contrast to the stability of the incompatibility factors in vegetative mycelia or spores Kniep (1928-1930), Brunswik (1924), and others noted the frequent appearance of unexpected factors in mycelia arising from

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basidiospores. Since these variants can be interpreted as arising through crossing-over and other more complex phenomena, they will be described in later sections.

V. SEGREGATION

Few clearly segregating factors are known in the higher Basidiomycetes. Besides the mutants obtained by Mittwoch and by Lisbeth Fries described above, Zattler (1924) found that the fruit character Knauel-Frucht-korper ” in Schizophyllurn was recessive to wild type and segregated about 1:1 independently of either of the incompatibility factors. He also concluded that brown color versus white in Collybia velutipes was controlled by two genes and brown was dominant in the dikaryon mycelium. It should be emphasized that deformed fruiting bodies and brown mycelia are extremely variable characters in many Basidiomycetes. They can be due to environmental, genic, and unknown causes which makes tetrad analysis very desirable in establishing Mendelian segregations. In a series of three articles Dickson (1934, 1935, 1936a) has described very extensive studies on growth rates of Coprinus sphaerocarpus. Strains were obtained from the mud bricks in the Lahun pyramid and were believed to be 4,000 years old! Over 3,500 growth rates were measured and the results appear to fit with polygenic determination. Extranuclear factors were probably not important since dikaryons derived from either mate in a cross gave similar results. An interesting character “luminosity” in P a n u s stypticus was studied by Macrae (1942). Mycelia as well as fruiting bodies of North American species shine yellowgreen in the dark. European species do not shine. I n crosses, luminescence is completely dominant both in the dikaryon and in the fruiting body. The f l progeny gave 11 shining and 9 nonshining monokaryonsdistributed randomly among the 4 mating types. As these few examples indicate, the higher Basidiomycetes do exhibit orthodox behavior as regards mutation and dominance, and show linkage as often as expected of organisms with about 4 (Olive, 1953) chromosomes. Studies on the segregation of the incompatibility factors show on the other hand many deviations from that expected from classical genetic theory and invite interesting comparisons with more recently discovered phenomena in other organisms. In tetrapolar species the incompatibility factors represent two independent loci or complexes (vide infra) with multiple alternatives. These ready-made genetic markers are to be found in any stock isolated and have no effect on viability. Their identification is, however, somewhat laborious and can be misleading. Many factors both genic and nongenic undoubtedly affect mating reactions and peculiarities of the incompatibility factors can be sufficiently established only by isolating basidio-

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spores in tetrads, mating unknown mycelia with numerous reliable tester strains, and carrying a factor of particular interest through several filial generations. 1. Somatic Recombination I n both Schizophyllum and Coprinus lagopus it has been reasonably demonstrated that a dikaryon having a constitution such as AIB1 A2B2 can, at least under the conditions of a noncompatible di-mon mating, produce a nucleus of constitution A1B2or A2B1.When a dikaryon AIB1 A2B2is grown next to a noncompatible monokaryon A1B2 the monokaryon is dikaryotized (Fig. 1). In some cases the new dikaryon has the same constitution as the inoculated dikaryon, AIB1 A2B2,but in other cases it has the constitution A1B2 A2B1. The distinction cannot be made easily since both types of dikaryon will give identical progeny from their fruit. The distinction has been made by several methods, however, involving further di-mon matings with the unknown dikaryon to extract

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FIG.1. The formation of a new nucleus, AZA', in a n incompatible di-mon mating.

and determine the constitution of its nuclei (see Papazian, 1954). Where the constitution of the newly formed dikaryon proves to be A1B2 A2B1 it is claimed that the A2B1 nucleus was derived from the two nuclei AIB1 A2B2of the inoculated dikaryon. One particular di-mon mating analyzed was (A2B2M1 AIBIS) X A1B2+ (A2B1M1 A1B2),where S (streak) is loosely linked to A and M 1 (uracilless) is probably independent of A and B. The fact that M 1is recovered in the newly formed dikaryon rules out the possibility that mutations of both incompatibility factors are induced in some nuclei of the monokaryon, but does not eliminate the possibility of a directed mutation of the B factor in the A2B2M1 nucleus of the dikaryon. Sufficient data are not yet available to discriminate between possible mechanisms operative in this phenomenon. It resembles parasexual recombination " (Pontecorvo and Sermonti, 1954) in the circumstance that two haploid nuclei can eventually produce a new haploid nucleus but no intervening diploid nucleus has been detected here and the nature of the dikaryon mycelium makes it unlikely that diploid nuclei ever become frequent. Whether karyogamy followed by

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meiosis or a form of parasexual recombination occurs, or whether single whole chromosomes are exchanged between the synchronously dividing nuclei of the dikaryon, is an open question. The very limited data available (Papazian, 1954) show no evidence for crossing-over. A similar phenomenon is probably operative in the production of triparental fruiting bodies of Coprinus jimetarius reported by Brunswik (1924). He grew three haploid strains together, e.g., A'B', A2B2,A3B3 and obtained two fruits which produced spores of four types such as A1B2,A1B3,A2B2,A2B3.Only two factors in each series were obtained so that only two haploid nuclei contributed to these fruits yet they contain factors derived from all three inoculated mycelia. Two successive recombination events must have taken place and since two fruiting generations would certainly have been detected, some form of vegetative recombination is required. A dikaryon AIB1 A3B3might have been formed which produced a new nucleus, A1B3, which then dikaryotized some A2B2 mycelium which was still monokaryotic. This dikaryon, AIBa A2B2, would produce a fruiting body with spores of the types found.

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2. Tetrads

A large number of tetrads of tetrapolar Basidiomycetes were analyzed and discussed by various workers before the simple explanation of the kinds of tetrads obtained was clearly set down by Whitehouse (1949). Whitehouse concludes that there is good evidence in the tetrapolar Basidiomycetes generally that the A and the B factors segregate independently and that one or both are situated far from their centromeres. This conclusion agrees with the data (but not the reasoning) from Newton (1926) in which the spatial position of the four spores of a basidium was taken into account. If we assume that the spindles of the second meiotic division are parallel and that nuclei move into the spores with their relative positions unchanged then diagonal spores never contain sister nuclei. If we further assume that a gene goes randomly to one or the other pole at the second meiotic division independently of its position in the first meiotic division and of the direction its allelomorph in the other second meiotic division goes, then a locus for which the second division is equational will give a tetrad in which diagonal spores are dissimilar, and for second division segregation, diagonal spores will have an equal chance of being similar or dissimilar (Fig. 2). Hence the frequency with which diagonals are similar represents half the frequency of second division segregation and provides an estimate, with certain reservations (Papazian, 1952), of map distance from the centromere. Data provided by the 31 tetrads Newton analyzed give map distances from the centromere of 16 and 29 for the A and B factors, respectively.

GENETICS O F BASIDIOMYCETES

53

Newton was unfortunately unaware of the relation between cross-overs and segregation so his conclusions are different and irrelevant. ?+meiotic division

2nd meiotic

d i vision

FIG.2. Position of basidiospores in species where second meiotic division spindles lie parallel and in a plane a t right angles to the basidium. Above: 1st division segregation-diagonal spores unlike. Below: 2d division segregation-diagonal spores alike in 50 % of tetrads.

3. Recombination between Incompatibility Genes I n contrast to the stability of incompatibility factors during vegetative growth numerous aberrant mycelia have been obtained from basidiospores by various workers. The appearance of unexpected incompatibility factors is of considerable interest since they do not seem to be due to ordinary spontaneous mutation. In Schixophyllum there is evidence (Papazian, 1951) th at the incompatibility factors are controlled b y two or more genes between which crossing-over can occur. I n a cross of A2B1 X A1B2San aberrant tetrad was obtained in which three spores were obtained: they were A2B2,A7B1, and A8B2S. The two mycelia containing the unexpected factors A7 and A8 were crossed and two more aberrant tetrads were obtained. I n these both of the grandparental factors were recovered, the constitution of one tetrad was AIBIS, A2B2,

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ASB'S and A7B2.The proposed origin of new factors by crossing-over is shown diagrammatically in Fig. 3. The phenomena can be described in the language of classical genetics b y saying th a t two genes, defined as cross-over units, interact to produce the phenotype known as A' or A2. A change in either gene will produce a different functional factor, thus A7 is compatible with A', A2, and A8 but not with another ,Q7. They are not pseudo-alleles unless th at term is to include any fairly closely linked genes which have related functions. Although each gene cannot be distinguished by its phenotype this may only be a matter of technique; it is

r i -

+

A2

S

A'

FIG.3. Production of new incompatibility factors by crossing-over. Above: new factors A7, A* from a cross of A' X A2. Below: recovery of A' and A2 from a cross of A7 X A8. conceivable that antigens could be prepared that would react to a factor controlled by each separate gene. The frequency of crossing-over is of the order of 1% based on the assumption th a t factors identical to A7 and AS arising from some 300 random spores were produced in the same way. Making the further assumption that all new incompatibility factors arise by crossing-over Raper (1953) was led to the idea that each factor is controlled by 4-10 closely linked genes and therefore each gene need only have two alternatives to account for the estimated number of different factors in a population. Against this assumption we must allow, however, from evolutionary considerations, that new alleles do arise by mutation. They may also arise by other, as yet undetected mechanisms. Some experiments of Kniep (1930) give information on the number of genes operating. He inoculated medium suitable for fruiting with a large

GENETICS OF BASIDIOMYCETES

55

number of basidiospores from a stock containing mating types A1A2B1B2 (my nomenclature). When this culture fruited he again took a large number of basidiospores from this F1 generation to produce a n F2 generation. Twenty-four generations were thus produced by mass inocula and the mating-type factors present in fruits from various generations were determined. Two new A factors, A3 and A4 and 2 new B factors, B3 and B4 appeared sporadically in the first eight generations. How frequently they appeared in the first eight generations can be estimated very roughly. About 70 spores were analyzed from fruits of the first eight generations and among these 10 supernumerary A factors appeared and 1 supernumerary B factor. Supernumerary means here that more than 2 A and B factors were found in a single fruit, the factor occurring a t lowest frequency is called supernumerary and is presumed to have arisen by crossing-over. The frequencies of recombination are thus of the order of 10% and 1%for the A and the B factors, respectively. The data fit well with the hypothesis that these new factors arose by crossingover and that there is only one cross-over region. Two and only two new factors appeared. If the two genes representing factors A' and A2 are alal and a2a2,respectively, the two new factors produced by recombination would be a1a2and a2a1.If there were more than one cross-over region more than two new factors would be expected. From the 8 th to the 24th generation no supernumerary factors were produced and the stable pair of A factors was A2A3or genotypes a2a2,a1a2.The fact th a t one locus is now homozygous means that recombination of A2 X A3 will not produce any new factors unless there are other cross-over regions. Since no new factors were found the indication is that there were only two loci concerned. If there are, in any species, n loci determining the A incompatibility factors the array of individuals th at are heterozygous a t 1, 2, 3 . . . n loci will be given by the lst, 2nd, 3rd . . . n th coefficient of the binomial expansion (a b)n. If there are 7 loci the probability of getting not more than 2 heterozygous loci, which represents Kniep's case, would be 29/128 = 0.3. The frequencies of recombination cannot be regarded as very low and it may well be that although there is only one cross-over region with a frequency of 1% or more, more intense study would show other cross-over possibilities with a lower order of frequency. Comparison with pseudoalleles in other organisms is difficult because the phenotype observed is not the presence or absence of an enzyme or anatomical structure but a specificity. The specificity of the two genes controlling a factor works in such a way t ha t a change in one or in both genes produces a new factor specificity (see Table 1). This contrasts with the relations between A and B factors for here a change in one factor does not produce a new functional

+

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HAIQ P. PAPAZIAN

specificity for the organism, both the A and the B factors must be different for two monokaryons to be compatible (see Table 2 ) . The production of different incompatibility factors by recombination has an evolutionary significance. Multiple-factor incompatibility is an outbreeding mechanism. In a tetrapolar species with 100 different A and B factors random spore pairs taken from a population are compatible in about 99% of cases but taken from the same fruiting body only 25% are compatible. The many factors in the population will be kept at about TABLE 1 Reactions between Genotypes Differing at the Two Loci Controlling the A Incompatibility Factor in Schizophylluin; the B Factors Are Omitted*

*

+ + +

-

alal a2aa alaa a2a1

+ + +

+ = clamps.

-

+ + +

+ + + -

TABLE 2 Reactions between Monokaryons of the Four Mating Types from a Single Fruiting Body in Schizophyllum*

A1B1 AaBa A1B2 A2B1

*

+ = clamps;

karyon).

0

+

0

+

0 B F

F

B = overlap;

F

=

F B 0

+

B F

+

0

flat (heterokaryon); B = barrage (hetero-

the same frequency by a feed-back mechanism. Factors that are rare will be at a selective advantage over those a t the equilibrium frequency while those that are too common will be a t a selective disadvantage. Thus deviations in either direction actuate their own correction. The effect of recombination will be twofold. In the first place it will increase the frequency of compatibility of random spore pairs taken from the same fruiting body, it will increase inbreeding. This effect will be slight for with a frequency of recombination of 1% the increase will be to <25.5%. ( F = (1 2m - m 2 ) / 4 where m = frequency of new factors, all different) (Papazian, 1951). A more important effect will be to diminish the chances of loss of factors by random sampling. The number of factors

+

GENETICS OF BASIDIOMYCETES

57

maintained in a population depends upon the rate of appearance of new factors by mutation and the rate of random loss. The rate of loss is determined by population size and the selective factors in a way similar to the case of sterility factors in flowering plants analyzed by Wright (1939). If we imagine the extreme case where there are 7 loci each, with only 2 alternatives determining 128 different factors then, if the population size is 1,000, each allele will have an equilibrium number of 500. The chances of loosing 1 allele out of 14 each with 500 duplicates is less than loosing 1 allele out of 100 each with 10 duplicates. It must be admitted that loss of 1 allele in the case where 7 loci determine a factor is more serious since all recombinants or 64 factors are eliminated but without precise calculation it would appear th at random loss of factors would be less where 2 or more loci determined each factor. Another aspect is that of small populations. A population size of 127 or less cannot possibly contain 128 different factors where 1 locus is determining but where 7 loci are determining, a population as small as 2 can rapidly generate 128 different factors by recombination, granting that the 2 have different alleles a t all 7 loci. A number of other irregular segregations of the incompatibility factors have been reported in the literature. Some of these are difficult to evaluate and others are ambiguous. Three such types of irregular segregation will now be described which I have encountered recently under fairly reliable conditions.

4. Labile Heteroz ygosity for One Incompatibility Factor

In C. lagopus, when matings are made between monokaryons having one incompatibility factor in common, nothing resembling the Flat mycelium in Schixophyllum is produced. Instead, a dikaryon is formed fairly frequently. Such mycelia have been referred t o as arising through ‘‘ Durchbrechungskopulationen ” and as having not true clamp connections but “Pseudoschnallen” (Kniep, 1928). Among crosses between 400 tetrad and 50 random spores, 11 matings having common A factors gave rise to mycelia having normal clamps. Forms resembling “Pseudoschnallen” were seen but did not appear to be particularly frequent in common A matings. This frequency, 2.5%, should be considered a maximum since not all were analyzed sufficiently. I n one typical case (Papazian, 1955) a monokaryon which should have been A2B6 according t o its parentage and tetrad sibs, formed a dikaryon not only with A6B2 but also with A2B2. The latter dikaryon ?(A2B6 A2B2) fruited and gave rise to f l spores of 4 types A2B2,A6B6,A2B6,and A6B2.All spores were isolated as tetrads and were mated with 8 or with 10 tester strains representing appropriate incompatibility factors in different genotypes.

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HAIG P. PAPAZIAN

It is thus reasonably certain that no artifact in the testing of factors was involved. The appearance of A6 in the f l means that the parent monokaryon which was assumed to be A2B6must have carried the A6 factor as well. It was heterozygous a t the A locus. When crossed, the compatible pairs of factors appeared in the progeny in their normal haploid state: A2(6)B6 X A2B2+ A2B2, A6B6, A2B6, A6B2. After a few weeks’ growth the A2(6)B6 monokaryon no longer formed a dikaryon with tester A2B2; it had lost the supernumerary A6 factor. Many inocula from various parts of the original A2(6)B6mycelium were taken and tested, no A6B6 sector was isolated but small sectors would have been missed. One can think of a chromosome section controlling A6 duplicating twice during meiosis in the grandparent, which was A2B2 A6B6, and being stuck loosely onto an -42 strand. The whole nucleus was not diploid since the B factors showed regular segregation.

+

5. 3 :1 Tetrad Segregation

Another type of irregular segregation is exemplified by a cross of A2B6 X A6B201,in which O1 represents a gene mutant producing abundant oidia. A tetrad from this cross was obtained having the following constitution A2B2, A6B601, A6B2, A6B601. This represents 3 : 1 tetrad segregation for the A factors and could be due to nothing more than the duplication of an A6B601nucleus following meiosis with loss of the A2B602 nucleus. Two other cases, however, have been obtained, one involving an A and the other a B factor in which duplication of a whole nucleus is unlikely. I n one of these a marker, brown color, was different in the two nuclei with similar incompatibility factors and in the other the difference in growth rate and general morphology indicated that they were not of identical genotype. Color is a rather unreliable character in Coprinus and it would be very desirable to obtain further 3 :1 segregations with good markers. If whole nuclei are not duplicated then whole chromosomes may be. The regular and normal behavior of progeny from all four spores of these tetrads preclude the simple occurrence of nondisjunction but there may be an extra duplication of a whole chromatid and loss or nonduplication of another some time during meiosis. If neither whole nuclei or whole chromosomes are over-duplicated then the word conversion” is perhaps a better description of the phenomenon than errors of duplication and loss of a gene. Comparison with instances of “conversion” in yeast (Lindegren, 1953) and Neurospora (Mitchell, 1955) shows that, whereas in the latter cases the loci involved have alleles which determine the functioning or nonfunctioning of an enzyme or enzymes, in the incompatibility instance all the alleles concerned determine factors which are equally functional and differ only in specificity. No evidence for tem((

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G E N E T I C S O F BASIDIOMYCETES

porary heterozygosity a t the A or B loci was detected in these cases but mating from the first few branches of the germinated spore might have revealed it. It is conceivable th at the same mechanism is operative here as in labile heterozygosis but the expected allele is displaced on the axis by the supernumerary allele and is lost in mitotic divisions (Fig. 4). Two 3 :1 tetrads for the A factors were found in the series of 400 tetrad spores. This represents a frequency per strand of 0.25% for axial dislodgement as compared to 2.5% for temporary fellow traveling. I n 100 tetrads whose mating reactions were published by Brunswik (1924) 4 % gave 3 : 1 segregation for the A or the B factor. None showed a double mating reaction. Both phenomena bring to mind the limited unilateral

-4[-1.

__t____

R

P

:

___9____

FIG.4. Aberrant segregations in Coprinus lagopus. Top : temporary heterozygosity (center diagram) for an incompatibility factor with eventual loss of supernumerary factor. Below: formation of tetrads with 3: 1 segregation for a n incompatibility factor. These diagrams are of mnemonic rather than affirmative value.

genetic substitution of bacterial transductions. I n both Salmonella (Lederberg, 1955) and Escherichia coli K12 (Morse et al., 1956) a small segment of a chromosome or linkage row may apparently be diploid and heterozygous following transduction. I n Salmonella the induced factor is sometimes passed on without duplication at each division to one daughter bacterium while the indigenous factor which goes to the other daughter can duplicate. I n K12 both transduced and indigenous factors commonly duplicate forming colonies with sectors.

6. Spontaneous Dikaryotization I n C. lagopus I have isolated 3-monkaryons in 3 separate tetrads and 2 monokaryons from the same tetrad which have become spontaneously dikaryon, a frequency of some 1%. When fruited they gave rise to progeny of all four incompatibility factors of the stock. This is similar t o the appearance of spontaneous dikaryons in Schizophyllum

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HAIG P. PAPAZIAN

(Papazian, 1951 ; Kniep, 1928-1929). These mycelia, which first behave like normal haploid monokaryons, might be carrying extra A and extra B factors which later segregate out into a separate nucleus and produce a dikaryon, but they would have to be carried without their influencing the incompatibility phenotype which is incongruous. We must accept that dikaryons arise spontaneously from monokaryons and that they are heterozygous for both factors but the event does not fit into any hypothesis which covers other aberrant segregations. VI. CYTOPLASMIC AUTONOMY Under this title several very different phenomena will be included. Non-Mendelian or cytoplasmic inheritance is a poorly defined genetic category. No general laws comparable to Mendelian laws have been established and there is no generally accepted technique for recognizing cytoplasmic determinants; each of the few well-established cytoplasmic determinants is distinct theoretically and technically. It is useful t o distinguish between those characters that are controlled by genes in linkage groups and those that are controlled by autonomous determinants duplicating in the cytoplasm or, to be precise, outside the mitotic apparatus. This raises the question as to how autonomous they might be. Through the ontogeny of a single cell? Through several or very many cell divisions? Or through the life cycle of the organism? All differences between differentiated cells of an organism may belong in the first two categories as well as the differences between sporophyte and gametophyte in the mosses and other diplobiontic organisms. I n fact wherever the difference between two cells can be shown to be due to their past history and not to their contemporary environment and where there is also evidence that the difference is not determined by a difference in Mendelian genes then the character can be said to be determined by cytoplasmic determinants. It will be seen below that it is necessary to consider whether “cytoplasmic ” includes regions within the nucleus but not located on chromosomes. 1. The Harder Operation

A direct way to distinguish between nuclear and cytoplasmic determinants would be to obtain a nucleus from one cell in the cytoplasm of another, different cell. This can and has been done in the Basidiomycetes, first by Harder (1927) and later by Ashan (1952), Fries and Ashan (1952), and Papazian (1955). The technique is to kill, by microsurgery, the terminal cell and the clamp connection cell of a dikaryon soon after nuclear division and to recover the hyphae growing out of the subterminal cell (Fig. 5 ) . The mycelium thus obtained is often monokaryotic with

G E N E T I C S O F BASIDIOMYCETES

GI

the genotype, a t least as regards mating type, of one of the components of the dikaryon from which i t was derived. The argument is that in these monokaryons the two nuclei have been completely separated and only one type retained whereas nothing in the cytoplasm has been separated. The resulting monokaryon is thus uniformly dicytoplasmic. Let us consider now what kind of cytoplasmic factor might be detected with this technique. The experiment is in fact being performed each time an organism reproduces sexually. The four basidiospores in a tetrad contain each a different nucleus yet the cytoplasm of all four is derived from the same heterozygous cytoplasm of the basidium. It can sensibly be maintained, however, th at the formation of a sexual spore is a

n’B’ DI-CYTOPLASMIC

FIG.5. The “Harder operation” which produces a di-cytoplasmic monokaryon. Cells of the dikaryon AIB1 AZB2marked with a double arrow are killed by microsurgery.

+

very special stage in the life history of an organism and th a t many factors, autonomous during vegetative growth, are destroyed and created de novo by nuclear determinants at the sexual spore stage. Harder’s technique might detect these. An important consideration is whether the cytoplasm of the dikaryon is truly dicytoplasmic. During the formation of a dikaryon, nuclei from each monokaryon are commonly supposed to migrate through the opposite mycelium and set up a dikaryon at the periphery. It can hardly be said th at the first dikaryon cells will be composed of equal parts of the two cytoplasms. The migrating nuclei may carry some cytoplasm with them but surely not as much a s a whole cell volume. A growing dikaryon, then, is only di-cytoplasmic as regards those cytoplasmic factors which are produced de novo during vegetative growth and determined by the nucleus. The cytoplasmic factors detected by Harder’s technique are therefore likely t o be ones formed de novo in the dikaryon unless the monokaryons investigated contain nuclei opposite in type t o t ha t of the side of the mating from which the dikaryon came.

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HAIG P. PAPAZIAN

I n the published accounts the region of the original mating from which the dikaryon was taken is usually not identified. I n the experiments of Ashan, Fries and Ashan, and of Papazian, the di-cytoplasmic monokaryons exhibited new characters not present in either of the component monokaryons or in the dikaryon. Some (Papazian) were gene mutants; others (Fries and Ashan) may have been phenotypic. The high frequency of variants following Harder’s operation is impressive and whether the variants are phenotypic or are gene mutants the cause may be an incompatibility of nucleus and cytoplasm as Ashan and Fries have suggested or it may be due to trauma. One way of deciding this is by obtaining similar di-cytoplasmic monokaryons without causing any trauma. This can be done in Coprinus by using hair cells or cystidial cells from the fruiting body (Papazian, 1956), if hyphae growing out of these cells are subcultured before much growth they will produce monokaryotic mycelia. Mycelia obtained in this way do not exhibit the high frequency of variants that di-cytoplasmic monokaryons obtained by Harder’s operation do. This indicates that trauma is responsible for the variants but comparable experiments have not been performed, however, on the same species. I n Harder’s experiments, on the other hand, besides some rather ill-defined new morphological characters, two characters typical of the dikaryon were obtained. In Schizophyllum Harder found that the di-cytoplasmic monokaryons produced clamp connections or pseudo clamp connections during considerable vegetative growth. I n Pholiota mutabilis the character examined was the production of brown pigment when in contact with various other Pholiota mycelia and more clearly with an unknown mycelia designated ( ( Y ”which was obtained as a contaminant. Pholiota dikaryons produced pigment when in contact with ((Y”whereas neither component monokaryon did. The di-cytoplasmic monokaryons behaved like the dikaryon and produced pigment when in contact with (‘Y.” I n both cases the character was ephemeral, the pigment production of Pholiota disappeared after 246-6 months’ vegetative growth, and the clamps after a few dozen cell divisions. 2. Ajinity of Mating Type Another series of experiments (Kimura, 1954b) which are claimed to demonstrate non-Mendelian determinants also depend on the di-cytoplasmic nature of dikaryon mycelia. Kimura made a series of compatible di-mon matings in Caprinus macrorhizus (see above ‘(mating systems”). He was interested in discovering what factors influence the choice between dikaryotizing nuclei under conditions where the origin of the dikaryon was known. When a dikaryon, made up of components from two different stocks c and C in Table 3, was mated with a monokaryon

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GENETICS OF BASIDIOMYCETES

from one of these stocks, C, the dikaryotizing nucleus was always th a t from the other stock, c. A dikaryon so formed was allowed to fruit and the f l mycelia of various mating types were assumed to have cytoplasm made up equally of C and c type. Another compatible di-mon was then arranged such t h at a monokaryon of mixed cytoplasm (>dC) could be dikaryotized by either a pure C or a X C nucleus. The pure C or most different nucleus was always chosen. This rule held (Table 3) to the point where a constant distinction was made between a 3 i C and a %C mycelium. It will have been noticed that in order to describe this experiment I have been obliged to use the terms pure C (for cytoplasmic) nuclei and $$C nuclei. This reveals an important point. The object th a t is chosen TABLE 3 Compatible di-mon matings of Kimura (1954b) to demonstrate autonomous cytoplasmic determinants influencing mating reactions. I n the second and subsequent lines the strain indicated by the vertical arrow was the same as that used in the previous di-mon but the two other strains came from the sexual progeny (fl, f p , and f a ) of the dikaryon in the line above. Nomenclature is not Kimura's. A'B'C

X (A'BzC

+ A'B'C)

+

(A'B'C

+ A3B3c)

-+ f l

I A'B'35C X (A2B2C + A3B3>4C) (A'B1>4C + A2B2C) fz I AIB'XC X (A2B23iC + A3B3jBC) + (A'B'XC + A3B3>$C)+ I AIBIBBC X (AZBz%C + A3B3%C) (A'B'XC + A2B2%C) +

-+

f3

-+

by the monokaryon is a nucleus and in order to maintain th a t the experiment demonstrates a cytoplasmic factor, one would have to suppose th a t the nuclei of the dikaryon are each surrounded by their own distinctive cytoplasm which is the determining factor in choice of nuclei. This is not particularly shocking but the imagination is unbearably strained when you consider the precise mixing of these bits of cytoplasm surrounding the nuclei t o XC and 4gC through successive sexual cycles. Kimura avoids this by proposing th at the determinants are in the caryoplasm which mix in equivalent proportions when the nuclei fuse in the basidium. This is getting rather close to Mendelian genes. The only consideration which leads us to believe th at a number of genes is not determining is that the blending appears very precise with no sign of segregation. Whether the determining factor lies in a number of Mendelian genes, or in cytoplasm held within the nuclear membrane or in cytoplasm surrounding the nuclear membrane, these experiments demonstrate another item: the secondary incompatibility factors determining election do not

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HAIG P . PAPAZIAN

correspond to the A and B incompatibility factors themselves. This in contradiction to Hartmann’s theory of relative sex as applied to Basidiomycetes (Hartmann, 1955).

3. Dwarf Mycelia That characters which do not segregate at meiosis indicate nonMendelian or cytoplasmic inheritance is the argument implied in Quintanilha and Balle’s (1940) work with dwarf mycelia of Coprinus jimetarius. Dwarf mycelia have been obtained by many workers from single basidiospore cultures in the Basidiomycetes. Their frequency may be as high as 20% or 30% and they appear in the progeny of normal parents. I have regularly observed a difference in variability between single spore cultures of Schizophyllum and Coprinus lagopus, the uniformity of the former and the great morphological variability of the latter is striking. The variability of C. lagopus is not environmental since oidia produce very uniform mycelia. Quintanilha found that the dwarf mycelia he obtained could be put into two categories according to their progeny in a cross with a normal mycelium. Some of them segregated giving two dwarf and two normal mycelia in each tetrad, others when crossed with normals gave tetrads in which all four spores produced normal mycelia. The latter were considered to be dwarf because of cytoplasmic determinants. A very similar situation is found in more recent studies with yeast (Ephrussi, 1953) where “ segregational ” and cytoplasmic dwarfs are found. The dwarf character in C . lagopus is not as sharply defined as in yeast and whether segregation of numerous genes was actually occurring can only be judged by the degree to which Quintanilha’s progeny resembled normal. The possibility of lethality of certain types can be discounted because Quintanilha obtained mycelia from all four basidiospores in the tetrads he examined. An indication that the “fluffy” variant obtained by Dickson (1935) in Coprinus macrorhizus (see above “mutation ”) was cytoplasmically determined is that the fluffiness spread to other mycelia in contact with it. If heterokaryon formation in C. macrorhizus is similar to that in Schizophyllum, C. lagopus, and other species of Basidiomycete, it is unlikely that this effect is due to nuclear migration. On the other hand, it is not clear from the published account how likely it is that some extracellular substance, produced by fluffy, might be responsible.

4. Sectoring Variants The recognition of variation caused by a non-Mendelian determinant

is often, unfortunately, a matter of judging the weight of various different indications. This is well-illustrated by the study of sectoring strains of Schizophyllum (Papazian, 1955). These strains were obtained as follows:

GENETICS OF BASIDIOMYCETES

65

+

Harder operations were performed on a dikaryon of constitution A'B2 A2B1swhere s represents the mutant streak. Among the resulting monokaryons a dwarf was obtained which had, most probably, the constitution A2B1sd4where d4 is a new mutant. I n crosses with wild type A2B'sd4 produced the tetrads expected from this genotype except th a t the mycelia which were expected to be streak appeared as semi-dwarf mycelia which sectored into streak. The rate of sectoring was irreversible and of a frequency amenable to quantitative study. Mutagenic agents did not affect the rate of sectoring; neither did changes in the medium. The only environmental factor which did have an effect was temperature, a 12°C. rise increasing the rate of sectoring 16 times. When p u t through a sexual cross or a Flat heterokaryon, only streak mycelia could be extracted but the spontaneous rate of change to streak made the significance of this data doubtful. If we permit ourselves to regard the circular mycelium growing in a Petri dish as an individual these sectoring strains represent a nice example of differentiation. Each individual consists, morphologically, of two parts, the semi-dwarf part and the streak sector in rather constant proportions. The cause of this differentiation is undetermined but the difficulties in accepting the idea of a regular gene mutation to the semi-dwarf and then a back mutation to streak in the sectors inclines one t o a nongenic hypothesis. Further, the idea of a cytoplasmic particle being lost during irreversible change from semi-dwarf to streak necessitates the de now appearance of the particle in these aberrant strains. By elimination, the favored hypothesis is th at the sectoring is due to a change from one metabolic pathway to another more stable pathway. This would involve autonomy of a process rather than a particle. A very limited, but nevertheless interesting, cytoplasmic autonomy is exemplified by the Flat mycelia of Schizophyllum. It was argued in a n earlier section t hat these mycelia consist mainly of monokaryotic hyphae with heterokaryotic cells being continuously formed by hyphal fusions but immediately breaking down again. Flat mycelia donate but will not accept nuclei in a cross. Now if monokaryotic cells and short hyphae containing compatible nuclei are present, this refusal to accept a nucleus must involve cytoplasmic autonomy. There is no reason to doubt th a t a dikaryon, once formed in a Flat mycelium, would grow so as t o be detected. A genic determinant is incongruous because hyphae isolated from Flat and grown into a mycelium do accept a compatible nucleus. The effect is, in fact, not permanent enough to be considered a gene change. VII. NUCLEOCYTOPLASMIC RELATIONS The last topic introduces a promising aspect of Basidiomycete genetics. As was mentioned in the first section Basidiomycetes are

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HAIG P. PAPAZIAN

amenable to microbiological techniques but a t the same time they are cellular organisms. The very remarkable constancy with which one nucleus is to be found in each cell of the higher plants and animals is worthy of greater respect and attention than it commonly receives. Bensaude (1918) first described in the Basidiomycete, C . lagopus, what happens to a second nucleus if it gets into a cell. In fusions between a monokaryon and a dikaryon either nucleus of the monokaryon or the two nuclei of the dikaryon were lysed. It would seem that a strict ratio between nucleus and volume of cytoplasm is maintained; 1: 1 in monokaryons and 2 : l in dikaryons where nuclei of compatible types are present. Extra nuclei which would upset the ratio are lysed. The inability of Basidiomycetes to form heterokaryons of the same mating type is in contrast to the behavior of the Ascomycetes and Phycomycetes and is related perhaps to the cellular structure of Basidiomycetes. I n Flat mycelia lysis of one nucleus may constantly follow hyphal fusion. If the combination of one kind of A factor and two different B factors determines a 1:1 ratio the association (A’B’ A1B2)in the same cell would lead to lysis. When this combination of factors is present in the same nucleus as is the case of the exceptional Coprinus mycelia (A2(6)B6) the 1 :1 ratio obtains and there is no lysis or associated metabolic disturbance. I n Flat mycelia let us argue that the presence of different B factors causes hyphal fusions or perhaps migration of invading nuclei to be particularly persistent, lysis under these conditions may involve more profound metabolic disturbances and these may persist for a time even after the 1 :1 ratio is established by lysis. Hence the limited “cytoplasmic autonomy” in the inability to accept nuclei, of the monokaryotic hyphae of Flat. The ephemeral heterokaryotic cells of Flat would not be expected to accept a compatible nucleus since three nuclei per cell would be inconsistent with a 1: 1 or a 2 : 1 ratio. It must be admitted that fusions between hyphae of similar mating type, for instance between hyphae of the same mycelium, should lead to lysis and might be expected to cause the same characters that are associated with Flat. There may be a qualitative difference, however, between fusions of noncompatible hyphae (e.g., from the same mycelium) and common-A hyphae of Flat. Hyphal fusions in monokaryotic mycelia of Schizlphyllum are in any case rare. I n Coprinus A2(6)B6mycelia can accept as well as donate nuclei in compatible crosses and form a dikaryon. This is expected for the presence of compatible pairs of both A and B factors would determine a 2: 1 ratio even if one of the factors is present in double dose A2c6)B6 A6B2. These ideas can be usefully applied to homothallic species. Coprinus sterquilinus is homothallic and its basidiospores have two nuclei (Harder, 1926). This fact would suggest that it was secondarily homothallic, each

+

+

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67

basidiospore containing nuclei th at were genetically diff erent and compatible. Harder performed his operation on dikaryons of C. sterquilinus and found t ha t hyphae which he isolated containing one nucleus per cell soon reverted to a typical dikaryon with two nuclei per cell which had clamps and fruited. This must mean that the two nuclei are not genetically complementary but are similar as regards mating behavior. C . sterquilinus is then truely homothallic, not secondarily homothallic. One can speculate as to whether the two nuclei of the dikaryon are phenotypically differentiated into some sort of complementarity, but in the present context the phenomenon can be interpreted as meaning th a t the genotype of a single nucleus of C. sterquilinus determines a 2 :1 ratio. Perhaps because each nucleus contains two compatible factors of the related heterothallic species. A final phenomenon found recently in C. lagopus points in the same direction. If hair cells or cystidial cells are isolated from a fruiting body and placed on nutrient agar, several hyphae will grow out of each cell. If these hyphae are isolated singly and subcultured they will, in about half the cases, grow into monokaryons with a normal mating type of one or the other components of the fruiting body. The characteristic of these cells is that they are very large compared to mycelial cells. We might suppose, then, that in these cells the nuclear cytoplasmic ratio is so disturbed t ha t the dikaryotic association breaks down and each nucleus established a 1: 1 ratio independently. The value of these speculations depends to a disturbing degree on Bensaude’s description of nuclear lysis in Coprinus which depends in turn, I believe, on one lucky slide preparation. It is greatly t o be hoped that techniques will be developed whereby her findings can be confirmed.

VIII. REFERENCES Ashan, K., 1952. Studies on dediploidization mycelia of the Basidiomycete Collybia velutipes. Svensk Botan. Tidskr. 46, 366-392. Bensaude, M., 1918. Recherches sur le cycle Evolutif et la sexualit6 chez les Basidiomyc8tes. ThBse. Univ. Paris, Nemours, France. Brefeld, O., 1877. ‘ I Untersuchungen uber Schimmelpilze,” Vol. 3, 226 pp. Arthur Felix, Leipzig. Brodie, H. J., 1936. The Barrage phenomenon in Lenzites betulina. Genet. 18, 61-73. Brunswik, H., 1924. “ Untersuchungen uber die Geschlechts-und Kernverhaltnisse bei der Hymenomyzetengattung Coprinus,” 152 pp. Gustav Fischer, Jena, Germany. Buller, A. H. R., 1931. “Researches on Fungi,” Vol. IV, 329 pp. Longmans, Green, London. Dickson, H., 1934. Studies in Coprinus sphaerocarpus I. Ann. Botan. 48, 527-547. Dickson, H., 1935. Studies in Coprinus sphaerocarpus 11. Ann. Botan. 49, 181-204. Dickson, H., 1936a. Studies in Coprinus sphaerocarpus 111. Ann. Botan. 60, 219-246.

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Dickson, H., 1936b. Observations of inheritance in Coprinus macrorhizus (Pers) Rea. Ann. Botan. 60, 719-733. Ephrussi, B., 1953. “Nucleo-cytoplasmic relations in microorganisms,” 127 pp. Oxford Univ. Press, London and New York. Fries, L., 1948. Mutations induced in Coprinus jimetarius (L) by nitrogen mustard. Nature 162, 846-847. Fries, N., and Ashan, K., 1952. The physiological heterogeneity of the dikaryotic mycelium of Polyporus abietinus investigated with the aid of micrurgical technique. Svensk Botan. Tidskr. 46, 429-445. Fulton, I. W., 1950. Unilateral nuclear migration and the interactions haploid mycelia in the fungus Cyathus stercoreus. Proc. Natl. Acad. Sci. U.S. 36, 306-312. Harder, R., 1926. Mikrochirurgische Untersuchungen uber die geschlechtliche Tendenz der Paarkerne des homothallischen Coprinus sterquilinus Fries. Planta 2, 446-453. Harder, R., 1927. Zur Frage nach der Rolle von Kern and Protoplasma im Zellgeschehen und bei der ttbertragung von Eigenschaften. 2.Botan. 19, 337-407. Hartmann, M., 1943. “Die Sexualitat,” 427 pp. Gustav Fischer, Jena, Germany. Hartmann, M., 1955. Sex problems in algae, fungi and protozoa. Am. Naturalist 89, 321-346. Kimura, K., 1954a. Diploidization in the Hymenomycetes. I. Preliminary experiments. Biol. J . Okayama Univ. 4 , 226-233. Kimura, K., 1954b. On the diploidization by the doubly compatible diploid mycelium in the Hymenomycetes. Botan. Mag. (Tokyo) 67, 238-242. Kniep, H., 1928. “Die Sexualitat der niederen Pflanzen,” 544 pp. Gustav Fischer, Jena, Germany. Kniep, H., 1.929. Vererbungserscheinungen bei Pilzen. Bibliotheca Genet. 6, 371-475. ‘Kniep, H., 1930. tfber Selektionswirkungen in fortlaufenden Massenaussaaten, usw. 2.Botan. 23, 510-536. Kolmark, G., and Westergaard, M., 1953. Further studies on chemically induced reversions a t the adenine locus of Neurospora. Hereditas 39, 209-224. Lange, M., 1952. Species concept in the genus Coprinus. Dansk. Botan. Arkiv 14,l-164. Lederberg, J., 1955. Recombination mechanisms in bacteria. J. Cellular and Comp. Physiol. 46, 108 (suppl. 2). Lewis, D., 1954. Comparative incompatibility in angiosperms and fungi. Advances i n Genet. 6, 235-285. Lindegren, C . C . , 1953. Gene conversion in Saccharomyces. J . Genet. 61, 625-637. Macrae, R., 1942. Interfertility studies and inheritance of luminosity in Panus stypticus. Can. J. Research 20, 411-434. Mitchell, M. B., 1955. Aberrant recombination of pyridoxine mutants of Neurospora. Proc. Natl. Acad. Sci. U.S. 41, 215-220. Mittwoch, U., 1951. Studies in the genetics of some x-ray-induced morphological mutants in Coprinus lagopus. J . Genet. 60, 202-205. Morse, M. L., Lederberg, E. M., and Lederberg, J., 1956. Transduction in Eschen’chia coli K-12. Genetics 41, 142-156. Newton, D. E., 1926. The distribution spores of diverse sex on the Hymenium of Coprinus lagopus. Ann. Botan. 40, 891-917. Olive, L., 1953. The structure and behavior of fungus nuclei. Botan. Rev. 19, 439-586. Papazian, H. P., 1950. Physiology of the incompatibility factors in Schizophyllum commune. Botan. Gaz. 112, 143-163. Paparian, H. P., 1951. The incompatibility factors and a related gene in SchizophyZlum commune. Genetics 36, 441-459.

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Papasian, H. P., 1952. The analysis of tetrad data. Genetics 37, 175-188. Papazian, H. P., 1954. Exchange of incompatibility factors between the nuclei of a dikaryon. Science 119, 691-693. Papazian, H. P., 1955. Sectoring variants in Schizophyllum.Am. J. Botany 42,394-400. Papazian, H. P., 1956. Sex and cytoplasm in the fungi. Trans. N . Y . Acad. Sci. 18, 388-397. Pontecorvo, G., and Sermonti, G., 1954. Parasexual recombination in Penicillium chrysogenum. J . Gen. Microbiol. 11, 94-104. Pontecorvo, G., Gloor, E. T., and Forbes, E., 1954. Analysis of mitotic recombination in Aspergillus nidulans. J. Genet. 62, 226-237. Quintanilha, A., 1939. Etude g6n6tiqe du phenorn6ne de Buller. Boll. SOC.Broteriana 13, 425-486. Quintanilha, A., and Balle, S., 1940. Etude gEn6tique des phhnomhes de nanisme ches les Hymbnomycetes. Boll. SOC. Proteriana 14, 17-46. Raper, J. R., 1953. Tetrapolar sexuality. Quart.Rev. Biol. 28, 233-259. Raper, J. R., and San Antonio, J. P., 1954. Heterokaryotic mutagenesis in Hymenomycetes. I. Heterokaryosis in Schiiophyllum commune. Am. J. Botany 41, 69-86. Whitehouse, H. L. K., 1949. Multiple-allelomorph heterothallism in the fungi. New Phytologist 48, 212-244. Wright, S., 1939. The distribution of self-sterility alleles in populations. Genetics 24, 538-552. Zattler, F., 1924. Vererbungsstudien an Hutpilzen. Z. Botan. 16, 433-499.

GENETIC ANALYSIS BASED ON MITOTIC RECOMBINATION* G. Pontecorvo and Etta Kafer Department of Genetics, The University, Glargow, Scotland

Page I. Introduction. .............................. 71 11. Principles of ................................... 72 1. Location of Markers into Linkage Groups by Means of Haploidization . . . 72 2. Determination of the Sequence of Linked Markers.. . . . . . . . . . . . . . . . . . . 73 3. Location of Centromeres.. . . . . . . . . . . 4. Frequency and Distribution of Mitotic Crossing-Over: Mitotic Chromosome Maps . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74 5. Selection of Mitotic Recombinants.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75 111. Methods and Examples of Application in Aspergillus nidulans. . . . . . . . . . . . 76 1. Methods of Isolation and Classification of Segregants.. .

3. Diploids Y and Z.. 4. Determination of the Positions of the Centromeres.. . . . . . . . . . . . . . . . . . 96 5. Mitotic Chromosome Maps.. . . . . . . . . . ......................... 98 IV. Conclusion V. References

........................ ........................

I. INTRODUCTION Classical genetic analysis is based on the two fundamental features of meiosis in sexually reproducing organisms : reassortment of nonhomologous centromeres and crossing-over. By means of these two processes we resolve the genome into linkage groups and each linkage group into a series of linearly arranged loci. Until recently, apart from a few minor additions of limited application, e.g., the use of polysomics, of chromosome rearrangements, etc., this was the only way to do it. Two substantially different methods of genetic analysis have become available in recent years. One is based on linked transduction (including transformation) in bacteria (Stocker et al., 1953 ; Hotchkiss, 1954; Ephrussi-Taylor, 1951; Demerec et al., 1956), a process, virus-mediated or not, not likely to be confined to bacteria. The other (the object of the present paper) is based on mitotic recombination, a process which we believe to be of widespread, perhaps

* The research reported in this paper has been supported by a grant from the Nuffield Foundation. 71

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G . PONTECORVO AND E. KAFER

general, occurrence in diploid cells, and which may replace meiotic recombination in the genetic systems of asexual fungi (Pontecorvo, 1954, 1956). The classic work of Stern (1936) on somatic crossing-over in Drosophila could have made possible a method of mapping based on it twenty years ago. This, however, was not attempted; in Drosophila, it would have been far less efficient and of more limited scope than mapping through sexual recombination. Furthermore, Stern’s masterly analysis showed that a second process occurring side by side with somatic crossingover in fungi, a process of somatic reduction (“haploidization” as we call it for short), is usually not detectable in Drosophila and may only occur as a rare accident or lead to the formation of cells too inviable to form detectable patches. Mitotic recombination in Aspergillus nidulans and other fungi is the consequence of two distinct processes: one is mitotic crossing-over, with modalities as deduced by Stern in Drosophila; the other is “haploidization,” with modalities as yet not well established. As will be seen later, the occurrence, though rare, of nonmeiotic haploidization in Aspergillus nidulans has been one of the two most important elements in the use of mitotic recombination for mapping in this species. Another element is the possibility, in this species as in other microorganisms, of ascertaining the genotypes of the segregant nuclei arisen through either mitotic crossing-over or haploidization. It would be as if it were possible to isolate cells from somatic mosaic spots in Drosophila, grow them into complete flies, breed these flies, and deduce the genotypes of the cells of any one spot. Though the present chapter will give examples of genetic analysis via mitotic recombination exclusively in Aspergillus nidulans, the techniques described here have already been applied to at least five other species of filamentous fungi: Aspergillus niger (Pontecorvo et al., 1953a), Penicillium chrysogenum (Pontecorvo and Sermonti, 1954), Fusarium oxysporum forma pisi (Buxton, 1956), and Aspergillus oryzae and Aspergillus sojae (Ishitani et al., 1956). In all five of these species, unlike Aspergillus nidulans, a sexual stage is unknown. Genetic analysis via mitotic recombination in asexual organisms is no longer a mere possibility: the Ifirst steps have already been made.

11. PRINCIPLES OF MITOTICANALYSIS 1. Location of Markers into Linkage Groups by Means of Haploidization Early work with Aspergillus nidulans (Pontecorvo el al., 1954) had revealed that, in haploidization, whole chromosomes segregate and reassort; i.e., linked markers segregate en bloc but they recombine freely

GENETIC ANALYSIS BASED ON MITOTIC RECOMBINATION

73

with markers on other chromosomes. This makes it possible, as with the Drosophila inversion technique, to identify immediately to which chromosome a new marker belongs, if a tester strain is available with a t least one marker in each chromosome (even if only few meiotically unlinked markers of a chromosome are known, they can be identified as belonging to the same mitotic linkage group). For instance, we can synthesize a diploid between such a strain and another strain carrying a nonlocated marker 2. The diploid will give haploid segregants showing free recombination between x and all the markers located on chromosome pairs other than that carrying x, and no recombination with those located on the homolog of the chromosome carrying x. (A large number of markers have been located in this way in Aspergillus nidulans and 8 mitotic linkage groups have been established; Kafer, 1958.) 2. Determination of the Sequence of Linked Markers

Diploid segregants arising via mitotic crossing-over can be used to determine the sequence of markers which are on the same chromosome arm. If three markers have the sequence: centromere, a, 6 , c, a mitotic exchange between c and the centromere in a diploid heterozygous for the three markers in coupling, if followed by segregation of one cross-over with one noncross-over strand, will lead to homozygosis for c, but only in part of the cases for b or a and b as well. Crossing over between b and c will lead to homozygosis of c only, crossing over between a and b will lead to homozygosis of b and c and crossing-over between the centromere and a will lead to homozygosis of a, b, and c. Thus i t is possible t o deduce the order of the mutants on one chromosome arm and determine in each case in which interval the mitotic exchange occurred. As mitotic crossing-over is rare, multiple exchanges can be neglected for the crude approximation required to determine the sequence of linked markers. This cannot, however, be extrapolated to “distances” so small that negative interference, which seems to be even stronger in mitotic than meiotic crossing-over, begins t o be effective and frequencies are almost of the order of magnitude of mutation rates (e.g., Pritchard, 1955). 3. Location of Centrorneres

Analysis by means of mitotic crossing-over resolves the genome into chromosome arms. This analysis does not distinguish between two arms of one chromosome and two arms one of each of two nonhomologous chromosomes. As said above, however, in haploidization the markers on one chromosome, even if on different arms, segregate en bloc. Haploidiza-

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G. PONTECORVO AND E. KAFER

tion, thus, permits t o link as the two arms of one chromosome two linkage groups which mitotic crossing-over identifies as separate. Mitotic location of centromeres consists of the following. Suppose that mitotic crossing-over had detected the sequence: centromere, a, b, c for one linkage group and centromere, d , e, f for another one. If haploidization revealed that a, b, c and d , e, f segregate as a single group the position of the centromere could only be between a and d : in other words, the two centromeres are identified as one. Examples of location of centromeres are given further on for Aspergillus nidulans. These locationsdetermined by mitotic analysis-were later confirmed by tetrad analysis by Dr. Strickland in this Department. 4. Frequency and Distribution of Mitotic Crossing-Over: Mitotic Chromosome Maps The absolute incidence of crossing-over is small for any one marker and varies in different diploids. For a few markers tested (Pontecorvo et al., 1954) the incidence of segregant conidia among all conidia was measured as about 1 per thousand and it may vary from more than 1 in 500 to less than 1in 2000 (Kafer, unpublished) partly depending on the position of the marker and its distance from the centromere. These values would about correspond to the total incidence of crossing-over in one chromosome arm, if the position of the marker tested were distal. The total incidence of mitotic crossing-over in the whole genome must then be fairly large; taking into account each arm of the eight visible chromosomes (at least six of them fairly large), a n estimate would fall above one in a hundred. The rarity and clonal distribution of mitotic crossing-over makes it impractical to measure its absolute incidence in any one ‘ I segment )’of a linkage group in the way it is done with meiotic crossing-over, i.e., as the proportion of nuclei in which exchanges have occurred out of a random sample of nuclei. A further complication arises from the use of different selective systems for each arm (most of which would scarcely permit t o estimate the total number of nuclei contained in the random sample). This would make it necessary to measure separately the absolute incidence of segregation for each selective marker (“selector”) used and to apply treatments for clonal distributions similar to those used for mutation rates in microorganisms. Applied to the clonal distribution of 4 selectors, or even worse to all major types of diploid segregants (22 types in Table 4), this would be too laborious. A good deal of information, however, can be gathered from much cruder estimates: those of what proportion of crossing-over in a whole arm falls into various marked intervals. These estimates of “distance” will be based on units different

GENETIC ANALYSIS BASED ON MITOTIC RECOMBINATION

75

for each arm but will permit interesting intra-arm comparisons all the same. Comparisons of this releatiu incidence of mitotic crossing-over measured from various diploids in the intervals of the same chromosome arm can indicate whether a characteristic distribution of mitotic crossingover exists. A possible source of variation from diploid to diploid may be differential multiplication of segregants. When recessive mutants, which show reduced viability on the media used, are in coupling with the selector, part of the segregants become homozygous for these mutants and may show reduced multiplication rates. T o test and correct for such possible distortions of a regular distribution, in each experiment a set of diploids carrying the same relevant markers in coupling or repulsion with the selector have been analyzed and compared- (for details see pp. 82-84 and Table 2). The results compared from different diploids were rather more homogeneous than expected. A possible explanation is th a t segregant nuclei in the parental cytoplasm (as in a heterokaryon) may be less a t a disadvantage than conidia or ascospores of a corresponding phenotype in competitive platings, and in these conditions multiply a t almost normal rate. This is certainly true for aneuploid segregants (as has been shown in recent experiments). On the other hand results can deviate extremely from expected ratios (e.g., the 1:l ratio of two types of haploids, see p. 86) when conditions are not optimal. Methods of isolation, which make use of systems with strong selection pressure and allow little heterokaryotic growth (of segregants with the parental strain), will tend to reinforce effects of differential viability (as is for example the case in acriflavine selection, where a n exceptionally low fraction of haploids is isolated, because the haploids are all auxotroph, while the diploids are prototroph; see Table 3). The findings of a very consistent distribution of mitotic crossing-over makes the construction of mitotic chromosome maps possible which can serve in organisms without sexual reproduction in the place of meiotic maps. If both are available, comparison for each arm independently is possible if the same measure of relative incidence is used for both types of crossing-over. 5. Selection of Mitotic Recombinants T o select rare mitotic recombinants suitable markers, which lend themselves t o selection, must be used. T o give information on the position of a n exchange such selectors must be distal to?other markers on the same chromosome arm. A proximal selector, i.e., located between the

76

G. PONTECORVO AND E. KAFER

centromere and other linked markers, can only permit to confirm that these others are linked and distal to it, but does not tell us anything about their order. The reason is, that a single mitotic exchange leads t o segregation (i.e., homozygosis) for all markers distal t o the position of exchange (Table 2). As it will be shown, the fortunate distal position of three available selectors which would approach this ideal requirement has been the key t o efficient mitotic mapping in Aspergillus nidulans. Five types of selection of segregants have been applied so far: (a) selection for visible characters; (b) selection for “suppressors” of nutritional requirements; (c) selection for resistance t o toxic substances; (d) selection for multiple nutritional requirements under conditions of starvation; (e) selection of “cis” heterozygotes from “trans” heterozygotes for a nutritional marker (Roper and Pritchard, 1955). Clearly, many more types of selection could be designed: any design for selection of mutants is potentially applicable to the selection of mitotic segregants: e.g., filtration for auxotrophic mutants (Fries, 1948; Catcheside, 1954) ; differential survival to fungicidal agents, under conditions of differential germination of conidia, as in the Lederberg-Davis penicillin technique for bacteria (Forbes, 1952) ; selective survival to antisera; recessive or incompletely dominant resistance to poisons, viruses, or other pathogens, etc.

111. METHODS AND EXAMPLES OF APPLICATION IN Aspergillus nidulans* The main purpose of the experiments presented in this paper was the analysis of the distribution of mitotic crossing-over in Aspergillus nidulans and the comparison with meiotic maps. Strains with suitable markers were built up and diploids synthesized in the usual way (Roper, 1952). A first set of 3 diploids had 1 chromosome arm well marked; a second set consists of 2 diploids with 3 fully marked chromosome arms. 1. Methods of Isolation and Classijication of Segregants

The procedure of analysis was the following: segregants from these diploids were selected and isolated in various ways and care was taken to avoid repeated isolation of the same segregant clone. The segregants were then classified as to phenotype (color, requirement, and resistance) and sorted out into haploids and diploids. If necessary the genotype of the diploid segregants was analyzed by further segregation, to identify for each diploid segregant the position of mitotic crossing-over. I n detail *For techniques and symbols in use see: Kafer (1958) and Pontecorvo et al. (195313).

GENETIC ANALYSIS BASED ON MITOTIC RECOMBINATION

77

the techniques used for the work exemplified in this paper were as follows: a. Selective Systems. (i) Selection for visible characters. The nonlinked markers (Table 4) y/y+ (yellow/green) and w/w+ (white/colored, with w epistatic t o the former pair) which determine cell-localized differences in color of the conidia have been used extensively. A diploid heterozygous y/y+ W/W+ has the wild type color of conidia (green). Single mitotic crossing-over gives origin either to y / y w/w+ (yellow) or y/y+ w/w (white) segregants which can be identified by inspection under the dissecting microscope as small spots or single heads. Haploidization also gives origin t o yellow and white spots. Conidia of the heterozygous diploid were plated on complete medium and yellow and white segregants were isolated from individual colonies (one yellow and/or white segregant from each colony). (ii) Selection for “suppressed ” nutritional requirements. A mutant ad20 requires adenine for full growth, but can grow without i t as a thin mycelium, not forming any conidia. A recessive suppressor of ad20 (sulad20) restores normal growth even on medium without adenine (Pritchard, 1955). I n the absence of adenine, a diploid homozygous ad20/ad20 and heterozygous sul ad20/+ grows without forming conidia a t the somewhat reduced rate characteristic of ad20. A diploid homozygous ad20/ad20 sulad20/sul ad20 or the corresponding haploid grow like wild type on adenineless medium. The diploid adgO/ad20 sulad20/+ grows without conidia a t the mentioned reduced rate and gives off vigorous, well-sporulating sectors which are either haploid ad20 sul ad20 or diploid ad20/ad20 sul ad%’O/sulad2O. This selection was carried out as follows: about 30 conidia per plate of the heterozygous diploids were embedded in a medium supplemented with all growth factors for which the diploids were heterozygous b u t not with adenine (Pritchard, 1955, p. 355), which the diploids required for full growth. After 3-4 days almost each of the poorly growing colonies showed well-sporulating segregant sectors of “suppressed ” phenotype. It was found that some of these segregant sectors were partly heterokaryotic with the original diploid strain, but pure isolates were obtained by picking small &mounts of conidia. (iii) Selection for resistance to toxic substances. A spontaneous mutant ( A c r l ) confers resistance to acriflavine (Roper and Kiifer, 1957). On concentrations of acriflavine (25ylml.) on which the sensitive haploid, or a homozygous sensitive diploid, does not grow a t all, the heterozygote forms stunted colonies, and the resistant haploid or the homozygous resistant diploid grow almost as well as in the absence of acriflavine. Colonies of heteroxygous diploids on acriflavine produce vigorous sectors which turn out to be either haploid or diploid homozygous resistant.

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G . PONTECORVO AND E. KAFER

Conidia of the heterozygous ( A w l / + ) diploid were plated on complete medium plus acriflavine to give about 15 colonies per plate; after 4 to 5 days segregant sectors, which were marked as early a s possible from the reverse to avoid unconscious selection of colors, were isolated, not more than 1 per colony. (iv) Selection for multiple auxotrophy following starvation. (For details of this technique and its implications see: Macdonald and Pontecorvo, 1953.) Diploids b i l l b i l a d l 4 / + require biotin for growth and their conidia die off a t a fast rate on agar medium devoid of it. Diploids b i l l b i l a d l 4 / a d l 4 or haploids bil ad14 require both biotin and adenine for growth, but their conidia die off more slowly on a medium devoid of both. Hence if conidia of a strain b i l l b i i a d i d / + are plated on such a medium and among them there is a small proportion of constitution . bil ad14 (haploid) or b i l l b i l a d l ~ $ / a d l Q(diploid) these will be enriched after “starvation” for a number of days. (v) Selection for lLcisJ’heterozygotes f r o m “trans” helerozygotes. It is well known that a double heterozygote in “trans” for two recessive alleles of the same gene-originated by independent mutation-has usually the recessive phenotype. A heterozygote in “cis” (with the same two recessives but both in one chromosome and with nonmutant homolog) has usually the dominant phenotype. Mitotic crossing-over, just like meiotic crossing-over, in a heterozygote in “trans” can produce, though with extremely low frequency, a strand carrying the two recessives in “cis” and the reciprocal nonmutant strand (Roper and Pritchard, 1955; Pritchard, 1955). I n the case of nutritional mutants, the nonmutant strand determines independence from the relevant growth factor requirement. The cross-overs can therefore be selected by plating on deficient medium. Among the cross-overs there will be two classes: those in which the nonmutant strand has segregated with the complementary double mutant strand; and those in which it has segregated with a noncross-over strand. The latter segregants only are relevant for mapping. The information they provide is only as to whether any one or more linked markers are located proximally or distally relative to the nutritional marker used for selection. Examples of this kind of selection are not given in the present paper (but see: Kafer, 1958). Because of the ease with which nutritional markers can be obtained, it has very wide scope and some interesting possibilities. The high incidence of further exchanges within distances as minute as those involved in these cases, however, makes the analysis somewhat more complicated. We cannot take it, as we can for exchanges between markers located far apart, that segregation for the selected

G E N E T I C ANALYSIS BAS E D ON MITOTIC R EC O M B I N A TI O N

79

marker usually goes with segregation of all others distally located on the same member of a chromosome pair. b. Classification of Segregants inio Haploids or Diploids. Segregants isolated by any one of the selective methods mentioned above are usually pure enough for further tests; otherwise they are restreaked for purification. To sort out haploids and diploids various independent criteria are used. Ploidy can be determined by measurement of the conidial diameter; pure haploid or diploid segregants are easy t o distinguish b y this measurement (but in aneuploid segregants, which are found a s unstable products in the course of haploidization and are all listed here as haploids, conidium size is too variable to permit reliable measurement). There are several other devices for distinguishing haploids from diploids: they are mainly based on the fact th a t the products of haploidization have phenotypes showing all possible recombinations between unlinked markers but very seldom showing any recombination between markers on the same chromosome, while diploid segregants as products of mitotic crossing-over seldom show any recombination between unlinked markers, For example, in all diploids used both homologous BI-chromosomes ( = linkage group I) were marked with various mutants determining requirements; consequently white or acriflavine-resistant segregants (w and A c r l are markers on the W-chromosome = linkage group 11) were always requirer when haploid, carrying either of the two homologous chromosomes of linkage group I, while diploids were prototroph (no mutants linked t o w or A c r l were used) ; on minimal medium only diploid segregants would grow. Independent additional markers also help in recognizing haploids; e.g., with two additional markers on different chromosomes 3d of the haploids will show either or both, in contrast with much less than 1% of diploids; two such markers on homologous chromosomes in repulsion automatically mark all haploids. By simple and automatic devices like these, combined with the measurement of conidia the classification into haploids and diploids is unequivocal in most cases. The diploids alone are then used for the construction of mitotic chromosome maps and classified according to the position of crossing-over. c. Analysis of the Genotype of Diploid Recombinants. Whether the position of mitotic crossing-over is directly evident from the phenotype of a diploid segregant, depends on the position of the relevant markers with respect t o the selector used. If recessive mutants are in coupling with the selector of the same chromosome arm, the position of crossing-

00

0

TABLE 1 Phenotypes and Genotypes of Yellow and White Mitotic Segregants from Diploids ws prol bil I - ad14*+ pabal Y 11 -*pro1 ad14 pabal bil WS ad14 bii IV - -0 prol pabal y

+

+ +

+ + ws+ + + + +1! + + + + + + +

From diploid I Phenotype *

YELLOW Diploid prototrophs

Genotype

ad pabat pro pabat

Phenotype

No.

No.

+ prol + y + + ad14 + pabal Y + w3 + prol pabal y + or[7 ad14 + pabal y + y + + ++ pabal ad14 pabal Y +

32 w3 -

o’\t$

WS ad14

l T wS

I T -

119

+

From diploid IV

From diploid I1

+

pabal y

?

prol pabal y ?

+

+

YELLOW

Diploid

No.

+ prol + y + + ad14 + pabal Y + + pro1 + Y + +ad14 + + Y +

ws

32

78

,

11

palm t

1

119

Phenotype

Genotype

132

NO. NO. YELLOW Diploid 3o prototrophs

51

WS

WS

paba pro paba

.+

-t pro1 pabal y ad14 pabal y

++

Genotype

1

82

21: /all

3 371

TABLE 1. (Continued) From diploid II

From diploid I

z

0

From diploid ZV

mc3

U

d

Phenotype*

YELLOW Haploid ad paba pabat

WHITE

Diploid prototrophs Haploid pro bi ad paba ( y )

Genotype

No. 19 -1 20

56 25 25 50

* Under the heading “Phenotype”

Phenotype

YELLOW Haploid Pro

Genotype

No. 9

Phenotype

YELLOW Haploid propaba

Genotype

No. 183

L4

B

td

k

M U 0

WHITE Diploid prototrophs Haploid pro ( Y ) ad paba bi

5

1:

79

10 9 19

the symbols of the alleles indicate the phenotypes. “Genotype” indicates only that part of the genotype which could not be deduced from the phenotype but which was ascertained by tests of further mitotic or meiotic segregat,ion. The question mark is used for those cases in which this further analysis did not distinguish between diploid homozygosis and monosomy. t Exceptional types, see discussion in text.

5r3

0 r3 d

w

?d

id

2 z

82

G. PONTECORVO AND E. KAFER

over can be determined from the phenotype: all mutants distal to the exchange will be homozygous in the segregant and become expressed. But segregants from diploids carrying recessive markers in repulsion with the selector must be analyzed further to identify the position of exchange (see p. 84 and Table 2). Various procedures are available for further analysis of diploid segregants. Essentially it is a matter of recovering intact, i.e., without crossingover and in haploid condition, from a segregant either or both chromosomes of one or more relevant pairs. Since mitotic crossing-over in one arm leaves usually unchanged the rest of the genotype, diploid segregants are still capable of segregation in respect of those selective markers which have not been used in the isolation of the segregant. For instance, if the original diploid is heterozygous for four selective markers, say y, w, Acrl , and sul ad20, any segregant isolated by means of one selective method will usually still be heterozygous for the three other selectors : y/y segregants will still be capable of segregation for w, A w l , and su; sulsu segregants will still be capable of segregation for y, w, and Acrl, etc. These residual selectors can then be used to select haploids which reveal the genotype of the diploid segregants. If they are in suitable positions, e.g., one selector on the chromosome strand to be analyzed and one on another chromosome, a simple system of “double selection” can be devised, which automatically yields only haploids of the desired type. Such second-order segregants, selected a t the same time for two mutants on different chromosomes, e.g., y Acrl or w sulad20 are in general haploid, because coincidence of mitotic crossing-over in two different chromosomes is very rare.

2. Diploids I , 11, and IV These three diploids, synthesized in the usual way (Roper, 1952) were heterozygous for the same six markers on two chromosomes in different arrangements. One chromosome arm (the right arm of linkage group I) with y a s selector was fully marked to analyze the distribution of mitotic crossing-over (Table 1). Yellow and white (only from diploids I and 11) segregants were isolated and classified as to phenotype in respect of the segregating markers, and as t o ploidy by measurement of the conidial diameters. The results are given in Table 1. T o determine the position of crossingover most of the yellow segregants from diploids I and 11, each of which had one relevant marker in repulsion with y, were analyzed by further segregation, mainly by isolating and classifying white haploids from them. The bulk of the yellow diploid segregants (117 out of 119 from diploid I; 131 out of 132 from diploid 11; and all 371 from diploid IV) fall into the three classes expected as a result of a single mitotic crossing over (either between pabal and y or between pro1 and pabal, or between

GENETIC ANALYSIS BASED O N MITOTIC RECOMBINATION

83

the centromere and p r o l ) followed by segregation of one cross-over and one noncross-over strand to the same pole. All the 135 white diploid segregants from diploid I and I1 (no whites were isolated from diploid IV) were prototrophs, indicating that mitotic crossing-over in one chromosome is independent of crossing-over in another chromosome. The full genotypes were ascertained for the three yellow segregants of unexpected types (marked with an asterisk in Table 1). The one from diploid I1 turned out to be a “second-order segregant”: the genotype of the colony from which it arose was not that of diploid I1 but must have w3 prol bil This genotype would result from th a t been ad14 paba1 Y of diploid I1 as a consequence of crossing-over between pabal and y followed by segregation of the two complementary products to the same pole. As, however, this genotype is the same as th a t of diploid I which had been used shortly before, contamination cannot be excluded. Of the other two unexpected yellow types (from diploid I) one had all the markers of one parental chromosome: it could be a n example of “nondisjunction’’ as will be discussed later. The other would have required the successive occurrence of crossing-over and nondisjunction. Neither of these two exceptional types could have originated by contamination, as no strains of these genotypes existed.

+

+

+

+ + +

3. Diploids Y and Z Diploids I, 11, and IV were analyzable only for 1 arm of 1 chromosome. The results reported above prompted the synthesis of 2 further diploids (Y and Z) analyzable in 3 chromosome arms, the 2 arms of linkage group I and the “left” arm of linkage group 11. I n addition these diploids had one marker on each of two more chromosomes (pyr04 of linkage group IV and cho of linkage group VII). Diploid Y

+ + + + ad20 b i l

+

Acrl w.2 puro4 cho prol pabai 1~ ad20 Oadl Diploid Z riboi an1 a d i 4 . Y add0 Acrl pyr04 cho sulad20 p r o i pabal 4- ad20 b i l w2.f

s u i a d 2 0 riboi ani

+

+

+ + +

+ + +

+ +

+ + +

I

+

I

+ I

+ +

77

+ +

centromeres loci used for selection Symbols of mutant alleles: (a) Requirements: riboi = riboflavine; ani = aneurin; p r o i = proline; pabai = p-aminobenzoate; bil = biotin; ad1 = adenine (absolute requirement) ; ad14 = adenine (absolute requirement) ; add0 = adenine (partial requirement, distinguishable from ad1 or ad14) ; sulad20 = suppressor of ad20 (specific for this ad allele); pyro4 = pyridoxine; cho = choline. (b) Conidial colors: y = yellow; ur2 = colorless (white), epistatic; w’y’ = green, wild type. (c) Resistance: Acrl = resistance to acriflavine, semi-dominant.

T

=

=

84

a. PONTECORVO

AND E. KAFER

T o permit the detection of differences in the recovery of the various segregant types (homozygous either for mutants or their wild type alleles), diploid Y had all relevant markers in coupling with the selectors; diploid Z had them all in repulsion. Table 2 gives an example of the different consequences of mitotic crossing-over in the two diploids. I n the case of diploid Y (“coupling”) an exchange proximal to a marker other than the selector gives a segregant homozygous for the recessive allele of that marker, i.e., distinguishable phenotypically from diploid Y in respect of that marker. I n the case of diploid Z (“repulsion”) fin exchange in the same position gives a segregant homozygous for the wild type allele of that marker, i.e., not distinguishable phenotypically from diploid 2 in respect of that marker (except in the case of the semi-dominant A c r l ) , though different genotypically. Analysis by means of further segregation was necessary for all diploid segregants from diploid Z in order to ascertain the position of the exchange producing each segregant. Three types of selection were possible, as diploids Y and 2 were both homozygous for add0 and heterozygous for visible markers, for a recessive “suppressor” of add0 and a partially dominant marker ( A c r l ) conferring resistance to acriflavine: (A) visual selection of yellow or white spots; (B) selection of sectors homozygous su/su or haploid su, i.e., not requiring adenine, and (C) selection of sectors homozygous A c r l l A c r l or haploid Awl, i.e., more resistant to acriflavine than the heterozygous parent. Again the segregants from each of these selections (A), (B), and (C) were classified as t o phenotypes (color, requirements, and resistance), sorted out into haploids and diploids by at least two independent criteria and the diploid segregants from diploid 8, the parent with repulsion arrangement, further analyzed as to genotypes. a. Haploid Segregants. Let us consider first the haploid segregants derived from the three selections applied t o the two diploids (Table 3). Selection ( A )for yellow and white heads. Yellow haploids (selection A) from diploid Y carried, with 1 exception out of 127, all the markers of linkage group I in coupling with y; the exception (y pyro.4 cho) is unexplainable, and not carrying add0, for which diploid Y was homozygous, is most likely a contaminant. Of the remaining 126, all were acriflavine sensitive (Acrl+) and all but one carried a d l , showing again the rarity of coincidence of crossing-over with haploidization in respect of both multiply marked chromosome pairs. The single segregant which carried adl+ probably represents a case of crossing-over between wd and a d l : meiotically this segment is over 100 units long. I n respect of the

TABLE 2 The Consequences of Mitotic Crossing-Over in the Same Interval in Diploid Y (“Coupling”) as Compared with Diploid Z (“Repulsion”) 1 2

“Coupling” heterozygote ( Y ) pabal y ad20 pabal y ad20

+ + + + prol + + + + ()pro1 s u ribol an1 su ribol an1

+ +

I

+ I + + ad20 bil + + ad20 bil () +

“Repulsion” heterozygote ( Z ) y ad20 y ad20

+ ribol an1 ad14 + + ribol an1 ad14 () + 2 su + + + prol su + + + () prol

+ +

pabal pabal

Segregant

+ +

+ + + +

3

+ +

(homosygous p a b a l )

+ +

+ ad20 bil ’+ ad20 bil

- p r o 1 pabal y ad20 pabal y ad20 y ad20

+ + Phenotype: pabal

su ribol an1

+ ribol an1 adl4. + + + + + prol +

su

3

Phenotype: y ad20 (homozygous pabal+)

Y ad20 y ad20

+ +

86

G. PONTECORVO AND E. KAFER

independent markers the 126 yellow haploids were as follows: 24 pyro4 cho; 27 pyr0.4; 40 cho; 35 Yellow haploids (selection A) from diploid Z were all (27) acriflavine resistant ( A c r l ) and again carried all the markers of linkage group I in coupling with y . They were distributed as follows in respect to the independent markers: pyr04 cho 4 ; pyr04 3; cho 7; 13. White haploids (selection A) from diploid Y were all (112) acriflavine resistant; they were distributed between the 4 possible classes in respect

++.

++

TABLE 3 Summary of the Genotypes of 449 Haploid Segregants from Diploids Y and Z Selection A (for color of conidia) Yellow

_

From diploid

Y

_

Z

Noncross-over * 125 27 0 Cross-over 1 0 1 Unexplained - Total haploids: 127 27 Total diploids including “nondisjunctionals” 163 (Table 4) : - 147 Total segregants in sample 290 174 Total segregants: 1831

White

_

Selection B (for “suppressed” addo)

~

Selection C (for fullacriJlavine resistance)

z

Y

z

Y

111 25 1 0 0 _ -0 112 25

80 0 3 83

36 0 0 36

31 0 0 31

_75 _54

281 -

160 -

363 -

139 -

187 79

364

196

394

147

Y

Z

~~

~~~

~~

* Noncross-over means carrying all the markers of the selected chromosome and

any combination of the markers of other chromosomes not requiring crossing-over.

to pyro4 and cho (19 pyro4 cho; 16 pyro4; 41 cho; 36 ++), and between the 2 in respect to the chromosome pair of linkage group I (103 prol pabal ad20 and 8 ribol an1 b i l ) . The gross departure from 1: 1 ratios of the 2 members of chromosome pair I was later found to be due to insufficient riboflavine in the complete medium in this experiment. Only 1 haploid segregant showed a constitution incompatible with segregation of whole linkage groups, i.e., requiring crossing-over in linkage group I (ribol an1 prol pabal). White haploids (selection A) from diploid Z were all (25) acriflavine sensitive, again distributed among 4 classes in respect to the markers of

GENETIC ANALYSIS BASED O N MITOTIC RECOMBINATION

87

linkage groups IV (pyro4) and VII (cho): (7 pyro4 cho; 5 pyro4; 8 cho and 5 + + , and among the two classes in respect to chromosome pair I: 15 prol pabal b i l ; 10 ribol an1 a d l 4 ) . Selection ( B )for “suppressed” ad2O. From diploid Y, haploid segregants hemizygous su (and therefore not showing the adenine requirement determined by addO) were all (83) white and acriflavine resistant. Haploid segregants with the homologous chromosome of linkage group I1 were in this diploid eliminated by this selection, because they would carry a d l . Of these 83, all but three carried all the markers of chromosome pair I in coupling with su, i.e., ribol an1 b i l . Three (Acrl w 2 pyro4 cho) were unexplainable on any simple scheme (as they were discarded without further analysis it is impossible to decide whether they were contaminants). I n respect of the independent markers the 80 were: 13 pyro4 cho; 30 pyro4; 19 cho; 18 From diploid Z, the haploid segregants hemizygous su (36) carried all the markers of linkage group I in coupling with su, i.e., prol pabal b i l l and they were distributed as follows in respect to the other markers: 19 acriflavine-sensitive white, of which 3 pyr04 cho; 3 pyro4; 7 cho; 6 17 acriflavine-resistant green, of which 3 pyro4 cho; 4 pyro4; 6 cho; 4 Again the haploids obtained by means of selection (B) show independent recombination between different chromosome pairs with no connection between mitotic crossing-over and haploidization. Selection ( C ) for full resistance to acri$avine. Haploids Acrl from diploid Y were all (31) white and distributed as follows in respect t o the other chromosomes: prol pabal ad20, 19 (of which 8 pyro4 cho; 3 pyro4; 5 cho; 3 ++);ribol an1 b i l l 12 (of which 4 pyro4 cho; 2 pyro4; 3 cho; 3 ++I. Haploids Acrl from diploid Z were all (8) colored: 2 green prol pabal b i l (of which one pyro4 cho and one pyrod), and 6 yellow ribol an1 ad14 (of which 2 pyro4 cho; 2 pyro4; 2 cho). All haploids not requiring adenine are presumably su ad2O; but the genotype of haploid segregants was not analyzed. I n summary (Table 3) of the 449 haploid segregants from the 2 diploids Y and Z, isolated by means of 3 different selections, all b u t 6 were examples of haploidization with recombination between nonhomologous chromosomes but without crossing-over. Of the six exceptions two were examples of coincidence of mitotic crossing-over and haploidization in one clone though not necessarily in one nucleus; one was probably a contaminant; the other three were not sufficiently analyzed. b. Mechanisms of Haploidization. A working hypothesis proposed by Pontecorvo et al. (1954) for the process of haploidization was that a rare

++.

++;

++.

88

G . PONTECORVO AND E. KAFER

failure of regular chromosome separation at mitosis would lead to nuclei with a random distribution of the four chromatids of each chromosome pair and random recombination between chromosomes. The minor proportion of nuclei from such a process with a balanced haploid set would be the recovered haploids. This hypothesis had a testable consequence, i.e., that diploid segregants homozygous for all the markers in more than one pair should also arise with frequencies predictable from those of the haploids. This prediction has not come true (Table 4).What we call “nondisjunctional” diploids are all (50) homozygous only for the selected chromosome of linkage group I, while the hypothesis predicts that one-sixth of them should also be homozygous for a t least one relevant member of the three other marked chromosomes. A possible new approach is suggested by the following observations. A considerable proportion of segregants when first isolated grow very poorly but throw out vigorous sectors which soon overgrow the poorly growing inoculum and go on growing vigorously after repeated transfers. Reisolation from the center of such a newly isolated segregant usually repeats the same behavior. The conidial size from vigorously growing sectors is unmistakably haploid, that from the center is variable and ranges from almost diploid to haploid. Seven segregants of this type (3 from diploid Y and 4 from diploid Z) were tested by plating conidia from the center and classifying for phenotypes about 30 sectors. All these segregants turned out to segregate for p y r o 4 / + , as in each case about half of the sectors were pyr04, the others pyro4+. The provisional conclusion is that they were aneuploid, i.e., disomic for at least linkage group IV (carrying pyrod), and that they produced, by elimination of either the pyroQ or the pyro4+ member of the pair, fully haploid sectors selectively a t a n advantage. The question then arises of whether most or all haploids originate as aneuploids and gradually settle down t o haploidy by eliminating the extra chromosomes. This question is a t present under investigation by one of us (E.K.). c. Diploid Segregants. After having sorted out the haploids, which constituted anything from less than 5% t o over 50% of all the segregants in a sample (Table 3), the remainder of the segregants isolated turned out t o be diploid. The analysis of the distribution of mitotic crossing-over was based on these diploids. Apart from the fact that diploid Y was heterozygous for ad1 of linkage group 11, and diploid Z for ad14 of linkage group I, the two diploids both homozygous for ad20 differed only in the arrangement of the same eleven markers for which they were heterozygous. The arms and their

GENETIC ANALYSIS BASED ON MITOTIC RECOMBINATION

89

segments, analyzable as t o mitotic crossing-over, were (Table 4): (a) “left ” arm of linkage group I : segment between centromere and sul addo; three intervals analyzable in diploid Y, four in diploid Z (I, 11, 111, IV); total meiotic length 85 units, the most distal marker sulad.20 used for selection; (b) “right” arm of linkage group I: segment between centromere and y; three intervals analyzable (V, VI, VII); total meiotic length 44 units, y used for selection; (c) “left” arm of linkage group pair 11: segment between centromere and A c r l ;two intervals analyzable (aand b) ; total meiotic length 46 units. I n respect of each of the three distal markers used in selection (y, su, and A w l ) the proximal markers were all in coupling in diploid Y and all in repulsion in diploid Z. Thus, as shown above (p. 84 and Table 2)) the phenotype of a segregant would indicate where the exchange giving origin t o that segregant had occurred in the case of diploid Y but not in the case of diploid Z. Consequently all diploid segregants from diploid Z have been analyzed by haploidization to recover the relevant chromosome strand (except in the case of selection (C) where heterozygosis for white could be determined simply by inspection of a colony area sufficiently large to contain about 10-20 yellow spots which showed a similar number of white spots only if the segregant carried the w2 allele in heterozygous condition). “Double selection” (see p. 82), giving automatically haploids carrying the relevant chromosome strands, was possible for selections (A) (selection of su wd haploids) and (B) (selection of y A c r l haploids). For selection (A) e.g., these haploids were isolated in the following way: each yellow diploid segregant from diploid Z was transferred to adenineless medium in order to select suppressed ” ad20 second-order segregants. Among these a proportion were white and haploid as expected, because their origin requires simultaneous segregation for two nonhomologous chromosomes. The su allele selected the relevant chromosome of linkage group I. Classification of requirement for p-aminobenzoic acid, for p-aminobenzoic acid and proline or for neither identified the position of the exchange which had given origin t o the yellow segregant. Table 4 classifies all the diploid segregants obtained from the diploids Y and Z by means of the three selections. (i) Nondisjunctional segregants. I n every selection where they were identifiable, there were about of5 yo segregants (referred t o as “nondisjunctional”) with the characteristics already mentioned in the case of certain segregants from diploid I (Table 1) : they carried all the markers on both arms of one chromosome in homo- or hemizygous condition: it has not yet been established with certainty which of these two alternatives is correct. The first (homozygosis) is the more probable one, because

Phenotypes

ad20 y pabal ad90 y pabal ad20 pyro4 y pro1 pabal ad20

Genotypes as f a r as ascertained

VII

+ + + Y ad20 (?) su prol pabal y ad20 + + +

154

m

y ad20

-4 0

Genotypes Crossinga s f a r as over ascertained intervals Phenotypes su ad20

1/

0

From diploid (2)

From diploid ( Y )

ad20 ad20 ad20 ad20 (?) Y ad20 yad.20 (1) y ad20 y ad20 (?) y ad20 yad20

+ Y su prol pabal y (?) y su prol y

+ +

+

+ (?)+ +

su

+ + + + + su prol +

su prol

Crossing-over intervals

+.

bzl (?)

+ (?I + (?) Acrl + + (?) (?) -~ cho A w l + + (?) + w2

3

+

$4

30 VII 1 VII and VIII or IX

98 VI 7

v

1 VI and b

1 VI

c: 4

’ H

+ +

+ + + +

1 su ribol an1 ad14 y ad20 A~crl 1 VI and I adz’“ 139 su pro1 y ad20 w 2 139 With the exception of the y ad20 cho segregant, all were ascertained as heterozygotes for a t least either pyro4 or cho. The analysis was based on 2-10 haploids from each segregant. In these small samples, 97 already yielded both mutants. WHITE su ad20 b lw2 Awl+ ad20 54 ad20

+ + +

+

+

+

74 su ad20 b ad20 1 -s‘ b and I w2 Awl ad20c - su ad20 75 I A proportion of these segregants could be of nondisjunctional origin in respect to linkage group 11. w2 A c r l ad20

~

1

-~~

y Acrl w2

+ + +

y+

58--

wb Awl

1 ’

w2+

w2f

wd+ y+

w2

(?I

y A c r l w2

20 -

~

1 ++

ribol an1 adid y A c r l

+

+ ++v an1 ad14 y A c r l + (?) + + + Txi + + ad14 y A c r l + (?I (?I + + 2-

I I and b

I1 + ++ y A c r l w2 cho I1 ribol cho 1 -~ + + +(?I y A c r l wd ribol an1 181 - ___ -26+1 + + 111 or IV

y+ ribol

w2+ y+

Selection (B) : “Suppressed” ad20

w2+ y+

+ A +c r l w2+

+ ++ ++ pro1 + + bil + + pabal Y

+

32

I

9

I1

9

I11

91

1v

1 I V andVI

ribol an1 ad14 y A c r l + 1 I and a ++Acrlfw2 ribol an1 ad14 y A c r l pyro4 w2+ y+ pyro4 1 1 I w.2 (9 w2+ y 2 not analyzed __ w2 Awl+ 1 144 147 See remarks under Selection (A) as to heterozygosis for pyro4 and cho w2+

y+

Awl+

1

+ +

+ + + + +

+

TABLE 4. (Continued) Selection ( C ) : Full Resistance to Acriflavine From diploid ( Y )

Genotypes as f a r as ascertained

Phenotypes Awl w2+ y+ ad20

From diploid (2)

+ ad20 Acrlwd + ad20 Aerl + s u ad20 A c rl w2 (311 ___ + ad20 (?) su

48 -~

?I

~

Acrl w2 add0

A e r l w2 ribol an1 ad2O+

Phenot ypas

+ ad20 A c r l w 2 + y ad20 Awl +

su

a

+y

b

b and I, I1

Crosszngover intervals

Genotypes as f a r as ascertained

ad20

(?)

26 139

I11 or IV su ribol an1 ad20 bil A c r l w2 b and I11 01 su ribol an1 ad20 (?) IV

+

Acrl w 2 ad20 cho Acrl w2 ad20 pyrod

Crossingover intervals

b su ad20 A c r l w 2 pyro4 - ad20 (?) (?) 363

+

b

A proportion of the Awl w 2 segregants from diploid ( Y ) and of the nondisjunctional origin in respect to linkage group 11.

~

(?I

-k segregants from diploid (2)could have been of

"

Nondisjunctiond '' Segregants

SELECTION (A): YELLOW From diploid Y y prol pabal ad20

9"

From diploid Z y ribol an1 ad14

8*

SELECTION (B): SUPPRESSED From diploid Y w2+ y+ ribol a n 1 b i l

20"

From diploid Z w2+ y+ prol pabal b i l

1O d

su+prol pabal y ad20

(?I

ribol an1 ad14 y

('0

ribol an1 y+ b i l

(9

+ + + prol

y+ prol

3" 50

A c r l w2 pyro4

& o

+ + + + + A c r l + pyro4 @ + + + w 2 ~~

A c r l wB pyro4 @ ~-

+ + + +

pabal yf b i l

Acrl

+

py04

cho

+ + pyr04 cho Acri + _ + + + pro1 + y + ~ _ + + + p r o l pa6al + 021 + W B + + (?) \

wB+

+

I

+ w B

_

5 analyzed. 2 analyzed. 13 analyzed. d 4 analyzed. 0 All 3 analyzed; coincidence of "nondisjunction" with crossing-over in VI. 0

b

CD

w

94

G. PONTECORVO

AND E. KAFER

these strains do not show obvious abnormalities of the kinds usually associated with aneuploidy, and they seem to have a diploid diameter of conidia (no measurements accurate enough t o detect the small deviations expected from loss of one chromosome have been made). Thus, it is likely that the relevant chromosome was actually represented in diploid condition. I n those segregants which were fully analyzed (27 out of 50) the rest of the chromosome set was certainly diploid and still heterozygous like in the parent strains, a t least in respect to the marked chromosome pairs. For the origin of these (‘nondisjunctional ” diploid segregants there are two conceivable mechanisms: one is nondisjunction, i.e., migration of both sister chromatids of one chromosome to the same pole a t mitosis; the other is the occurrence of two-strand double mitotic exchanges, one on each side of the centromere, in frequencies much higher than those expected from coincidence. There is as yet no operation which would clearly distinguish between these two processes. But in view of recent results it seems likely th a t a t least some “nondisjunctional” diploids originate in connection with the formation of aneuploids in haploidization, independent of crossing-over and we shall exclude them from calculations on the distribution of mitotic crossing-over. Either way, their numbers are not such as to make a substantial difference. (ii) Coincidence of mitotic crossing-over in diferent chromosomes. Diploid segregants for one chromosome are usually still heterozygous for the markers on other chromosomes. Excluding “nondisjunctional” segregants, the bulk of which we assume to originate by a process other than mitotic crossing-over, all but 1 of 293 yellow segregants were still heterozygous A c r l l S and w 2 l - k ; all but 1 of 129 white segregants were still heterozygous su/+; all but 3 of 408 sulsu segregants were still heterozygous A w l / + and w2/+; all but 2 of 502 Acrl segregants were still heterozygous su/+. Altogether out of 1332 diploids selected for segregation (other than nondisjunction) of markers of either linkage group I or 11, only 7 were also segregants for markers on the other one. As t o the other 2 chromosomes (linkage groups IV and VII), marked with pyro4 and cho, respectively, the same trend is apparent: out of the said 1332 diploids segregant for markers of linkage groups I or 11, only 3 were also segregants for pyro4 and 3 for cho. I n all, diploid segregants for 1chromosome which were also identifiable as segregant for an additional chromosome out of the 4 marked chromosomes constituted in this sample 13/1332, or about 1 %. (iii) Coincidence of mitotic crossing-over in the two arms of one chromosome. Excluding again ‘(nondisjunctional)’segregants, diploid segregants

GENETIC ANALYSIS BASED ON MITOTIC RECOMBINATION

95

for markers on one arm of one chromosome are usually still heterozygous for markers on the other arm of the same chromosome. This conclusion is based almost entirely on linkage group I . Out of 293 yellow diploid segregants all but one were still heterozygous su/+ : the exception was a case of three-strand double in intervals I and VI. Out of 408 su/su diploid segregants only 3 were cases of 2 exchanges, 1 in each arm. I n 1 of the 3 the 2 exchanges were in intervals IV and VI, i.e., in those (Table 5) with the maximum relative incidence of mitotic crossing-over. I n the other 2 not analyzed, the exchanges were in undetermined intervals. Altogether, excluding the “nondisjunctional ’’ segregants (which could well all originate from two-strand double exchanges astride the centromere) we have 4 cases of double cross-overs, with 1 exchange in each arm of linkage group I, out of 701 relevant segregants. (iv) Coincidence of mitotic crossing-over in one arm. Double crossingover in one arm is very uncommon. We have found only 1 case though certain types of double cross-overs would have been detectable in both diploids Y and Z, if the second exchange fell in interval IX in selection for y/y, and in interval a in selection for wblwd. The total sample in which only 1 double was found is made up of 293 yellow and 129 white segregants. The case found (Table 4, diploid Z, yellow segregants) could have been the consequence of either a two-strand or a three-strand double in intervals VII and VIII or IX. It could also have been the consequence of a very improbable coincidence: one exchange in VII followed, in the segregant clone arising from it, by mutation from bi+ t o bi. Finally, as in most other cases of doubles, it could have involved two successive exchanges in a clone: the first in interval IX (or less likely in the very short interval VIII) with segregation together of the two complementary products of the exchange, and the second in interval VII. The scarcity of examples of coincidence of two exchanges in one arm, when intervals are wide apart or both large, makes even more interesting Pritchard’s (1955) finding of their very high frequency in close proximity of one another. (v) Coincidence of crossing-over with nondisjunction and with haploidization. Table 3 (and the discussion of it, pp. 84-87) shows coincidence of haploidization with crossing-over in linkage group I1 in 1 out of 154 yellow haploids, and with crossing-over in linkage group I in 1 out of 137 white haploids. Table 4 shows only three (identical) segregants which could be examples of coincidence of crossing-over (in linkage group I, interval VI) with nondisjunction, i.e., segregation of a cross-over chromatid t o the same pole as its sister noncross-over chromatid (selection B for su/su,

96

Q. PONTECORVO AND

E. KAFER

diploid Z). We have no cases of coincidence of nondisjunction in more than one chromosome. The question arises of whether or not any one or more of the kinds of coincidence listed above occur with frequencies too large to be explained by chance coincidence of two events in one nucleus. The impression is that a t least some of them do. For instance, a second crossing-over has occurred in about 1%of the segregants selected because of one crossingover, rather than about 1 in 300-500, which would be the expected coincidence when each marked chromosome arm has a crossing-over incidence of about one per thousand (measured as the incidence of segregant conidia among all conidia; Pontecorvo et al., 1954). This is no more than an impression, for the following reasons. First, the proportion of segregant conidia was not measured in the case of diploids Y and Z, and it is known to vary from one diploid to another; nor is it likely to be the same for all chromosome arms marked in these Gploids. Second, segregants are clonally distributed and the measurement of the incidence of each event giving origin to a segregant clone (i.e. crossing-over, or haploidization or “nondisjunction”) requires the experimental design and the tedious measurements appropriate to clonal distribution. Because of clonal distribution we do not know what proportion of segregants which require two events originated from coincidence of the two events at one nuclear division and what proportion from successive occurrences. We know for certain that in some cases the “double” segregant was the result of two successive events, as shown by the fact that the mother colony from which it was isolated turned out t o have not the genotype of the diploid used in the experiment but a genotype resulting already from a first crossing-over (e.g., p. 83). In summary, and with the qualifications just mentioned, the cases of coincidence were ; Two cross-overs in two different chromosomes Two cross-overs in two arms Two cross-overs in one arm One cross-over and haploidization ’ One cross-over and nondisjunction

13 out 4out 1 out 2 out 3 out

of of of of of

1332 701 422 449 701

4. Determination of the Positions of the Centvomeres

We shall use the data of Tables 1 and 4 as an example of how centromeres could be located by means of mitotic analysis. In actual fact the centromeres of linkage groups I and I1 were located by means of other diploids. In the first place it is clear from an inspection of the haploids in Table 1 that adid, p r o l , pabai, y and bU are linked. It is also clear from

GENETIC ANALYSIS BASED ON MITOTIC RECOMBINATION

97

diploid I V t ha t prol, pabal, and y are in this order, with pro1 nearest the centromere. I n the second place, the position of b i l is established unequivocally as distal t o y by those yellow diploid segregants from diploids I and I1 which were analyzed completely as to genotypes: every one of the 119 82 = 201 such segregants was homo- or hemizygous for the b i l + allele in coupling with y. I n the third place, the same completely analyzed segregants show that, with two exceptions, the 119 yellow segregants from diploid I (in which ad14 was in coupling with y) were still heterozygous ad14 and so were all of the 82 from diploid I1 (in which ad14 was in repulsion with y). This shows that ad14 cannot be distal to y, and is either on the same arm but closer t o the centromere than prol, or on the other arm. We can thus establish mitotically the order of all five of these linked markers as ad14, prol, pabal, y, b i l with the centromere certainly left of prol but either left or right of adl4. I n the present example this order was, of course, already known from meiotic mapping, but we have shown here how it could have been deduced purely from mitotic mapping. We are left only with the alternatives of a centromere location between ad14 and pro1 or beyond adl4. Diploids Y and Z (Table 4) can decide between these two alternatives. The 92 su/su diploid segregants from diploid Z classified as cross-overs in interval IV, were still heterozygous a t the y locus. This establishes the whole sequence of linked loci as su, ribol , an1, adld, prol , pabal , y, b i l and the position of the centromere between ad14 and prol. The data on the su/su segregants from diploid Y-which was not marked with adl4-support this conclusion, though to the cruder approximation th at the centromere must be between an1 and prol ; all of the 181 diploid segregants homozygous for ribol an1 were still heterozygous a t the y locus. T ha t the centromere of chromosome I lies between ad14 and prol was confirmed by Dr. Strickland using the standard method of tetrad analysis for unordered tetrads, i.e., by means of the joint segregation of three markers unlinked to each other. His estimates of the map distances are adl4-centromere 0.20 f 0.03, and centromere-pro1 0.20 k 0.02. The location of the centromere of linkage group I1 is apparent from the kinds of diploid segregants in respect t o this chromosome produced by diploids Y and Z (Table 4). All the white segregants from diploid Y (coupling) were homozygous Acrl /Awl , all those from diploid Z (repulsion) were homozygous Acrl+/Acrl+. On the other hand, of the A w l / Acrl segregants from diploid Y about 87 % were w2/w2 and 13 % w2/+, and of those from diploid Z about 82% were w2+/wd+ and 18% w2/+. Clearly both Acr1 and w 2 are on the same arm and the order is centro-

+

98

G . PONTECORVO AND E. KAFER

mere-w2--Acrl. This order was again confirmed by Dr. Strickland by means of tetrad analysis; the map distance between the centromere and w2 (which is allelic to w3 used in diploids I, 11, and IV) is 0.21 & 0.04. It may be mentioned th at the first attempt a t locating centromeres mitotically was made (Pontecorvo and Kafer, 1956) by selecting ad1 4/ ad14 segregants from a diploid with ad14 and y in coupling: they turned out t o be heterozygous y/y+. As it was already known, both from meiotic and from mitotic analysis, that the loci of a d l 4 , p r o l , pabal, y, and bil were in this order, that prol was proximal relative to pabal , y, and b i l l and t ha t ad14 was beyond p r o l , these results placed the centromere unmistakably between ad14 and p r o l . Should the centromere have been “left” of a d l 4 , all ad14/adl4 segregants would have been also homozygous y/y in a diploid with these two markers in coupling (for two further cases of centromere location see Kafer, 1958). 5. Mitotic Chromosome M a p s The preceding examples show th at the identification of linkage groups and of the order of loci and centromeres can be done mitotically in three steps. The first step makes use of haploidization, in which linked loci segregate e n bloc. The second step determines the sequence of loci in each arm of a mitotic linkage group by making use of mitotic crossingover and of the rule that an exchange followed by segregation together of one cross-over and one noncross-over chromatid leads practically always to homozygosis for the markers distal to it, but not for those proximal or on the other arm. The third step determines the position of the centromere as located between the two adjacent markers which do not become homozygous simultaneously. From the distribution of mitotic crossing-over mitotic map distances can then be estimated; they are based (as mentioned above p. 75) on the relative incidence of crossing-over which falls into the various intervals, measured as per cent, where the total crossing-over selected in each arm equals 100%. Table 5 gives the summary of all the cross-overs in the different intervals of chromosomes I and I1 collected from Table 4. I n this summary the “nondisjunctional” diploids have been left out, and of course each double cross-over, either in two chromosomes or in two arms or in one arm, is entered only once, i.e., as cross-over for the interval for which the selection detecting it was appropriate. Table 5 shows that the relative incidences of crossing-over in each interval are homogeneous between diploid Y (coupling) and Z (repulsion). It will be remembered (Table 2) th at in respect to the proximal markers the segregants were homozygous for recessive alleles in the former case

99

GENETIC ANALYSIS BASED ON MITOTIC RECOMBINATION

TABLE 5 Summary of Cross-Overs in Each Interval, from the Data of Table 4 (Excluding “Nondisjunctional”) * Linkage group I , “left” a r m Selection ( B )

Diploid Y Diploid Z Both

Intervals

I 59 34 93 23 .O

No. No. No. %

I1

I11 IV

2 1.

I) 30 7.4

181 9 92 282 69.6 ~

Total for a r m 261 144 405 100

= 0.89 P = 0.60

X2

Linkage group 1, “right” a r m Selection ( A )

Diploid Y Diploid Z Both

-

Intervals

v

9

No.

No. No.

7 -

16 5.5

%

VII

VI 110 101 211 72 .O

35 31 66 22.5

Total for a r m 154 139 293 100

x2 = 0 . 1 2 P = 0.94

Linkage group 11, “left” arm Selection ( C )

Diploid Y Diploid Z Both

Intervals

a 48 26 74 14. 7

No. No. No. % ~

b 315 113 428 85.3

Total for arm 363 139 502 100

= 2.42 P = 0.12

~2

~~

* The data from the two diploids are homogeneous: hence the incidence of crossing

over in each interval, a8 a percentage of the total in all the intervals of one arm, has been calculated by pooling the data from both diploids.

and for wild type dominant alleles in the latter. I n spite of this difference the unexpected homogeneity of results (also found with diploids I, 11, and IV, xz = 3.1,P = 0.5 based on results given in Table 1) shows th a t there was no appreciable differential recovery of the different segregant types under the conditions used. We can now build mitotic chromosome maps for each of the three arms analyzed as follows (Table 5 ) : Linkage group I, “left” arm: su-23.0--ribol-7.4-anl-6.2-adl~-G3.4-centromere Linkage group I, “right” arm:

100

G. PONTECORVO AND E. KAFER

Centromere-5.5-prol-72.0-pabai-22.5Linkage group 11, “left” arm: Acr1-14.7-~2-85.3-centromere

y

It should be stressed again that the units used represent for each interval the proportion ( %) of segregants resulting from crossing-over in that interval out of all the segregants for that arm: they are not units comparable between arms. For the “right” arm of linkage group I the corresponding values can also be computed from the numbers of each cross-over type found in the diploids I, 11, and IV (Table 1). These values are given in Table 6. For the calculation of weighted mean and standard error (after angular TABLE 6 Relative Incidence (Per Cent) of Mitotic Crossing-Over in All Diploids (“Right” arm of linkage group I, intervals V, VI, and VII) Intervals Diploid Diploid Diploid Diploid Diploid Diploid

Mean

I

I1 IV Y Z

V

VI

VII

6 8* 8*

6 5

67 59 66 72 73

27 33 26 22 22

6.9 f 0 . 5

67.1 k 1 . 9

25.7 f 1 . 6

* Rare “nondisjunctionals”

Total number of analyzed Total recombinants 100 100 100 100 100 99.7

117 131t 371 154 139 912

included.

t Only % of cross-overs in interval VI or VII completely analyzed. transformation) the frequencies from diploids Y and Z are included. The difference between the two sets of experiments, which is evident from these figures, is not statistically significant (but x2 = 4.8 with two degrees of freedom is fairly large, P = 0.1). Such a difference may be due t o the difference in genetic background caused by the crossing in of the additional markers which are present in diploids Y and Z but not in diploids I, 11, and IV. 6. Comparison of Mitotic with Meiotic Maps

We can now ask the question of how this relative incidence of mitotic crossing-over compares with the relative incidence of meiotic crossingover in the same intervals. The meiotic maps based on the most recent estimates (Kiifer, 1958) are given in Table 4. If we take the total meiotic map lengths of the arms, centromere-su, centromere-y, and centromere

GENETIC ANALYSIS BASED ON MITOTIC RECOMBINATION

101

-Acrl, divide by them the lengths of the various intervals in each arm and multiply by 100 we shall have expressed the distribution of meiotic crossing-over in each arm in relative units comparable to those of mitotic crossing-over. We can then compare the relative distributions of crossingover at meiosis and a t mitosis. Linkage group I, “left” arm (85 units): Intervals

mitotic: meiotic:

I

I1

I11

IV

Total

23.0 45.8

7.4 22.4

6.2 8.2

63.4 23.6

100 100

Linkage group I, “right” arm (44 units): Intervals

V

VI

VII

Total

5.5 46.1

72.0 18.0

22.5 35.8

100 100

~

mitotic: meiotic :

Linkage group 11, “left” arm (46 units): Intervals

mitotic : meiotic:

a

b

Total

14.7 54.3

85.3 45.7

100 100

The significance of the differences between the distribution of mitotic and meiotic crossing-over may be estimated for the “right” arm of chromosome I, using the pooled values from all diploids (Table 6) and the corresponding accurate meiotic frequencies (meiotic units for the three intervals: V: 20.2 f 2.3; VI: 7.9 f 0.4; VII: 15.7 1.0):

+

Intervals

mitotic: meiotic:

V

VI

VII

Total

6 . 9 k 0.5 46.1 f 5.3

67.1 1.9 18.0 k 0.9

25.7 f 1 . 6 35.8 k 2 . 3

99.7 100

102

G . PONTECORVO AND

E.

KAFER

These results show (Pontecorvo and Kafer, 1956) that mitotic crossing-over, besides having in Aspergillus nidulans an absolute incidence per mitotic nucleus over 1000 times smaller than th a t of meiotic crossingover per meiotic nucleus, has also a different relative distribution along the chromosomes. The most striking differences are in the three intervals (IV, V, and b, Table 4) nearest to the centromeres, all 3 of meiotic lengths of about 20 units: intervals I V and b show a relative incidence of mitotic crossing-over about double that of meiotic crossing-over ; interval V shows a relative incidence of mitotic crossing-over almost ten times smaller than t hat of meiotic crossing-over. I n this arm the maximum relative incidence of mitotic crossing-over-over 3 times that a t meiosisfalls in the next interval (VI) which has a meiotic length of 8 units and starts 20 units away from the centromere. Another region in which the distributions a t meiosis and a t mitosis diverge substantially is interval 11. We cannot draw any conclusion as to trends in the differences between relative distributions of crossing-over a t mitosis and meiosis, except that they exist and they are substantial. The maximum concentration of mitotic crossing-over occurs, in all three arms examined, within 28 (meiotic) units from the centromere. But the “right arm” of linkage group I shows that this concentration is not in the immediate vicinity of the centromere but in a small region 20 units away. Only the use of additional markers within 30 units of the centromere could tell whether or not this is true of all 3 arms; but such markers are not yet available. From the analysis of further chromosome arms i t will be possible to judge how general these characteristics of the distribution of mitotic crossing-over are. Preliminary results from a diploid well marked on the right arm of linkage group 11, which is meiotically at least 150 units long, show an extreme preponderance of mitotic crossing-over in the interval of about 30 units between the centromere and the most proximal marker (Kafer, 1958).

IV. CONCLUSIONS The work summarized here shows th at formal genetic analysis by means of mitotic recombination is perfectly feasible. It leads to the identification of linkage groups, of the order of genes, and of their “distances,” though in units different from those of meiotic analysis. I n Aspergillus nidulans, which has a sexual cycle and in which the results of genetic analysis based on meiosis are available, mitotic mapping has been compared with, and helped by, meiotic mapping. But in other similar organisms, such as Aspergillus niger, in which a sexual cycle is unknown, mitotic recombination is the only means available so far

GENETIC ANALYSIS BASED ON MITOTIC RECOMBINATION

103

towards formal genetic analysis (Pontecorvo et al., 1953a) and towards (‘breeding’’ for scientific or applied purposes. T he possibility of applying mitotic analysis to somatic tissues of higher organisms, better if in tissue cultures, is clearly a t hand. Even if somatic crossing-over were not of widespread occurrence, the analysis could at least identify linkage groups making use of breakdowns of mitosis, similar to the “haploidization ” described here, which are known to occur as rare accidents in higher organisms. The principles of mitotic analysis expounded here are clearly adaptable, with minor modifications, to a wide variety of special cases.

V. REFERENCES Buxton, E. W., 1956. Hetenkaryosis and parasexual recombination in pathogenic strains of Fusarium oxysporum. J. Gen. Microbiol. 16, 133-139. Catcheside, D. G., 1954. Isolation of nutritional mutants of Neurospora crassa by filtration enrichment. J. Gen. Microbiol. 11, 34-36. Demerec, M., and Demerec, Z. E., 1956. Analysis of linkage relationships in Salmonella by transduction techniques. Brookhaven Symposia in Biol. 8, 75-87. Ephrussi-Taylor, H., 1951. Genetic aspects of transformations of Pneumococci. Cold Spring Harbor Symposia Quant. Biol. 16, 445-455. Forbes, E., 1952. The use of SO2 for selecting auxotrophs in filamentous fungi. Microbial Genetics Bull. 6, 26-28. Fries, N., 1948. Viability and resistance of spontaneous mutations in Ophiostoma representing different degrees of auxotrophy. Physiol. Plantarum 1, 330-341. Hotchkiss, R. D., 1954. Bacterial transformation. J. Cellular Comp. Physiol. 46, 1-22. Ishitani, C., Ikeda, Y., and Sakaguchi, K., 1956. Hereditary variation and genetic recombination in Koji-molds (Aspergillus oryzae and A s p . sojae). VI. Genetic recombination in heterozygous diploids. J . Gen. A p p l . Microbiol. ( J a p a n ) 2, 40 1-430. Kafer, E., 1958. An 8-chromosome map of Aspergillus nidulans. Advances in Genet. 9, 1 05-145. Macdonald, K. D., and Pontecorvo, G., 1953. “Starvation” technique. Advances in Genet. 6, 159-170. Pontecorvo, G., 1954. Mitotic recombination in the genetic systems of filamentous fungi. Caryologia 6 (supp.), 192-200. Pontecorvo, G., 1956. The parasexual cycle in fungi. Ann. Rev. Microbiol. 10, 393-400. Pontecorvo, G., and Kafer, E., 1956. Mapping the chromosomes by means of mitotic recombination. Proc. Roy. Phys. SOC.Edinburgh 26, 16-20. Pontecorvo, G., and Sermonti, G., 1954. Parasexual recombination in Penicillium chrysogenum. J. Gen. Microbiol. 11, 94-104. Pontecorvo, G., Roper, J. A., and Forbes, E., 1953a. Genetic recombination without sexual reproduction in Aspergillus niger. J. Gen. Microbiol. 8, 198-210. Pontecorvo, G., Roper, J. A., Hemmons, L. M., Macdonald, K. D., and Bufton, A. W. J., 195313. The genetics of Aspergillus nidulans. Advances in Genet. 6, 141-238. Pontecorvo, G., Tam Gloor, E., and Forbes, E., 1954. Analysis of mitotic recombination in Aspergillus nidulans. J . Genet. 62, 226-237.

104

G . PONTECORVO AND E. KAFER

Pritchard, R. H., 1955. The linear arrangement of a series of alleles of Aspergillus nidulans. Heredity 9, 343-371. Roper, J. A., 1952. Production of heterozygous diploids in filamentous fungi. Experientia 8, 14-15. Roper, J. A., and Klifer, E., 1957. Acriflavine resistant mutants of Aspergillus nidulans. J . Gen. Microbiol. 16, 660-667. Roper, J. A., and Pritchard, R. H., 1955. Recovery of the complementary products of mitotic crossing over. Nature 176, 639. Stern, C., 1936. Somatic crossing over and segregation in Drosophila rnelanogaster. Genetics 21, 625-730. Stocker, B. A., Zinder, N. D., and Lederberg, J., 1953. Transduction of flagellar characters in Salmonella. J. Gen. Microbiol. 9, 410-433.

AN 8-CHROMOSOME M A P OF Aspergillus nidulans Etta Kijfer* Department of Genetics, The university, Glargow, Scotland

Page I. Introduction.. . . . . . . . . . . . . . . . . . .. 11. Material and Methods. . . . . . . . . . ...................... 106 111. Establishment of Eight Linkage Groups by Means of Mitotic Haploidiza................................... 107 rkers of Linkage Groups I-IV.. . . . . . . . 114 2. Establishment of Linkage Groups V-VIII.. . . . . IV. Determination of the Sequence of Markers and Location of Centromeres . 123 V. Meiotic Linkage Maps of Linkage Groups I and VI. Mitotic Linkage Maps and Comparison with Me VII. Summary.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140 VIII. References .................................................. 144

I. INTRODUCTION In species with a sexual cycle, location of markers and establishment of linkage groups is carried out by means of meiotic analysis. Meiotic mapping is laborious, however, when a species has a large number of chromosomes or when, as in Aspergillus nidulans, most chromosomes have long maps. This is especially true a t the beginning of the analysis. I n the first crosses of A. nidulans, for example, about 20 markers were used but only 2 small linkage groups were found (Pontecorvo, 1953). Mitotic segregation in diploids of Aspergillus nidulans (Roper, 1952) offers the possibility of mapping by means of mitotic recombination. Two processes of mitotic segregation have been found (Pontecorvo et al., 1954): mitotic crossing-over, as analyzed by Stern (1936) in Drosophila, and “haploidization,” a process of somatic reduction. In asexual species of filamentous fungi mitotic mapping is the only method available (Pontecorvo 1954, 1956), but even in species with a sexual cycle mitotic analysis may facilitate mapping. Mitotic haploidization, in which no crossing-over is found but whole chromosomes reassort at random, gives results similar to those obtained by the inversion technique in Drosophila. The linkage groups to which the various markers belong can, therefore, be determined easily by means of haploidization. This method is especially advantageous if, as in

* Present address: Department of Genetics, McGill University, Montreal, Canada. 105

106

ETTA KAFER

A. nidulans, markers of the same chromosome often show free recombination in crosses, whereas it is less useful in species (e.g., Neurospora) in which chromosome maps are short and meiotic linkage detection is relatively efficient (Perkins, unpublished). Mitotic crossing-over permits the determination of the sequence of markers for each chromosome arm. Mitotic analysis of both arms of a chromosome locates the centromere. This method is more efficient than tetrad analysis in A. nidulans, in which the spores are not linearly arranged and have to be dissected by micromanipulation. I n addition, because the maps are large, few markers are found meiotically linked t o their centromeres. For the various steps of mapping in A . nidulans, both mitotic and meiotic analysis are used. Extensive mapping has been carried out with the aim of locating a t least two markers in each linkage group. Marking of all chromosomes was necessary for the analysis of the process of haploidization, which seems to result from a gradual loss of chromosomes (details will be published elsewhere). Besides, it became desirable to determine the total number of linkage groups when a fifth linkage group was established with certainty by mitotic haploidization, while cytological studies had led to the conclusion th at the number of chromosome pairs in the zygote was four (Pontecorvo, 1953; Elliott, 1956). This conclusion has now become doubtful even on cytological grounds (Elliott, unpublished). Thus the aim of the present work was to determine the number of linkage groups as well as to map them. 11. MATERIALAND METHODS Strains. Table 1 gives a list of all mutants located up to March, 1957. All strains used in the work with Aspergillus nidulans in the Department of Genetics in Glasgow are descendants of one wild type strain, from which strains carrying various new mutants were isolated at different times (for details see Pontecorvo, 1953, p. 150). All multiply-marked strains are descendants of crosses between the various mutant strains, except the l y s l strain used in cross 59 (Table 14), which was obtained as a haploid recombinant from diploid J. Of the mutants used in this work two morphological ones (wS and co) and all mutants increasing resistance to acriflavine (Acrl, AcrS and acrd) and suppressors ( d a d 20, Sulpro and Su4pro) arose spontaneously. Most other mutants have been induced by ultraviolet irradiation, but a few early X-ray induced ones have also been used. Many mutants were isolated by means of the starvation technique, which makes use of the fact that bil spores die more quickly than double mutants (Macdonald and Pontecorvo, 1953).

CHROMOSOMfi MAPPING OF ASPERGILLUS NIDULANS

107

Techniques. For the work carried out in Glasgow, standard media were used (Pontecorvo, 1953). At Cold Spring Harbor certain changes in the media were necessary to obtain comparable results. To the minimal medium trace elements were added (as used for Chlamydomonas, Eversole, 1956). I n the complete medium Difco yeast extract and N-Z-case (Sheffield Farms Co., New York) were used in place of “Yeastrel” and hydrolyzed casein. The yeast nucleic acid hydrolyzate was prepared from sodium yeast nucleate (Schwartz Laboratories, New York). Standard methods were used with respect to incubation temperature, plating techniques, etc. (Pontecorvo, 1953). For the collection of meiotic linkage data a random sample of spores from a single crossed perithecium was analyzed (perithecium analysis, Hemmons et al., 1953). The techniques of mitotic analysis are described in the preceding chapter (Pontecorvo and Kafer, 1957).

111. ESTABLISHMENT OF EIGHT LINKAGEGROUPSBY MEANS O F MITOTICHAPLOIDIZBTION Mitotic haploidization in diploids was first found in A . nidulans by Pontecorvo et al. (1954), when mitotic segregation was investigated in two diploids heterozygous for the same five markers in different arrangements. Haploid segregants could be recognized on the basis of their conidial diameters; diploid conidia show twice the volume of haploid ones (Roper, 1952). It was found that the three meiotically linked mutants pabal, y , and bil showed complete linkage in haploids, while w3 and ad1 segregated independently from them, but were completely linked t o each other. Results from mitotic crossing-over supported the conclusion t ha t the two groups of completely linked markers were located on different chromosomes. They indicated th a t pabal, y , and bil are on the same chromosome arm, while w3 and ad1 are on opposite arms of another chromosome. It was concluded th at the three markers pabal, y, and bil belong to the “bi”-linkage group (linkage group I), while w3 and ad1 are markers of a second linkage group (11). The suspected meiotic linkage between all these markers (Pontecorvo, 1953) was explained as due to inclusion of rare diploid products of meiosis in the early calculations of linkage. The conclusion th at haploid segregants arise b y a process which results in random reassortment of chromosomes without crossing-over has been confirmed by extensive later investigations (Pontecorvo and Kafer, 1957). Only 1 % of exceptional types have been found in over 1000 tested haploids from diploids heterozygous for many markers of linkage groups I and 11. Exceptional types result from rare coincidence or consecutive occurrence of mitotic crossing-over and haploidization. Results

s2

F

TABLE 1

Located Mutants of Asper@us nidulans Origin of mutant Symbol *

w l (wa) Y

Bw

W

sm

Designation

VISrnLE white 1 yellow brown

compact small

RESISTANT Am1 acriflavine 1 UCR) act8

acriflavine 2

Phenotype

Linkage Alblic group mutants*

Colorless conidia, 11 L epistatic to y Yellow conidia IR Small brown conidial heads, ? epistatic to y and w, dominant Compact colony VIII Small colony I11 Resistant to acri- I1 L flavine and malachite green, semi-dominant Resistant to acri- I1 R tlavine and malachite green, recessive

wNw) wS(w,)

-

Strain

Mode

Year

wild type

Spontaneous X-rays Spontaneous X-rays Spontaneous

1936 1946 1951 1946 1954

Y

pabal bil wild type +pro1 Y ad14 pabal bil

+

+

+

+ +

Referemet

d

P

w

P:

kJ

-

m m

pabal bil bil; lys6

Spontaneous 1952 Spontaneous 1950 or UV

pabal y; co

Spontaneous 1955

ACTS b i l ; adl; SO; pyro4 (ACRS) ad16pabal y

Spontaneous 1955

!

Spontaneous 1956

Roper and Kiifer, 1957

ad1 ad3 ad8

NUTRITIONAL adenine 1 adenine 3 adenine 8

Growth response to Adenine I1 R Adenine I1 R Adenine I R

-

-

Y

X-rays X-rays

ad10

y; thil bil bil

uv

ad11

bil

UV, starva-

ad12

bil

ad16

bil

ad19

bil

ad20

bil

ad81

bil

ad22

bil

UV, starva-

1946 1948 1950 1950

tion

ad9

adenine 9

Adenine

I R

bil ad13

bil

ad15

bil

ad17

bil

ad32

bil ; w3

tion UV, starvation UV, starvation UV, starvation UV, starvation UV, starvation UV, starvation UV, starvation UV, starvation UV, starvation UV, starvation UV, starvation

1950

1951 1950 1950

0

W

c

TABLE 1. (Continued)

I-.

0

Origin of mutant Symbol*

ad14

Designation

NUTRITIONAL adenine 14

Phenotype

Linkage Allelic group mutants*

Growth response to Adenine IL

Strain

Mode

bil

UV, starva-

bil

UV, starvation UV, starva-

tion

ad2S

adenine 23

Adenine

I1 L

bil

an1

aneurin 1

Aneurin

IL

bil

bil

biotin 1

Biotin

IR

cho (chol) cysd

choline cystine 2

Choline Cystine

VII VIII

wild type y; thil y ; thil bil b i l ; wS

lysl lys6

lysine 1 lysine 5

Lysine Lysine

VI V

wl bil

methl (met)

methionine 1

Methionine

IV L

bil

ni&

niclO

nicotinic 2 nicotinic 10

Nicotinic acid/ V anthranilic acid V I

wild type b i l ; Acrl wS

nie8

nicotinic 8

Nicotinic acid/ VII anthranilic acid/ tryptophan

bil

tion UV, starvation X-rays X-rays X-rays

uv

UV, starvation X-rays UV, starvation UV, starvation X-rays

uv

UV, starvation

Year

Referencei

1g50)F'ritchard, 1950 unpublished 1950

roper,

M

1951

1948 1950 1954 1947 1950

1951 1947 1956 1950

e

p" 1950

+: ?4 q

M

a

niS

nitrite 3

orn$

ornithine 4

pabal

Nitrite

Arginine/ ornithine p-aminobeneoic 1 p-Aminobenzoic acid

panto phen2

pantothenic phenylalanine 2

pro1

proline 1

Pantothenic acid Phenylalanine/ phenylpyruvic acid Arginine/proline

I1 R

bil ; wS

IV R

bil

IR

bil paba2

bil

pabas

bil bil

paba6 paba6

bil bil y; thil

I11 111

bil

IR

bil pro2

bil

pro6

bil

pro6

bil

pro7

bii

pro8

bil

UV, starvation UV, starvation X-rays, starvation UV, starvation

1954 1950 d

1950

$

uv

0

X-rays, Roper, 19531 starvation X-rays UV, starva- 1950 tion X-rays 1948 UV, starva- 1950 tion UV, starvation UV, starva-' tion uv, starvation UV, starvation UV, starvation UV, starvation

5:

E g 5z 4

0

0 q

%v

19501

B La n

1950

L= F

1950 1950 1950 1950 F

c . ' F

TABLE 1. (Continued)

c-' CL

c 4

Origin of mutant Symbol *

pros

Designation

NUTRITIONAL proline 3

Phenotype

Linkage Allelic group mutants*

Growth response to Arginine/proline I R

Strain

bil bil

pu (putr)

putrescine

pyro4

pyridoxine 4

Putrescinel spermidine Pyridoxine

I1 R

-

IV R

b i l ; w3 bil bil bil bil

bil bil bil PYl-09

Sulfite

I11

pyrol0

s7

b i l ; Acrl W S b i l ; Acrl w3 wild type b i l ; WS

Mode

UV, starvation UV, starvation UV, starva-' tion UV, starvation X-rays, starvation X-rays, starvation UV, starvation UV, starvation UV, starvation UV, starvation

uv

W

Nitrogen mustard W, starvation

Year

Referencei

lg50 Forbes, 1956 and 1950 unpublished 1954 1950 1949 1950 1950

I

lg50\

lg50 C. Martin1951 unpublished 19561 1956 1948 1954

M d G

* ?? *: kl

E

sl

SS

thi4

sulfite 1

sulfite 3

thiazole 4

SUPPRESSORS sufaa0 suppressor 1 (SuodZO-I) adenine 20 Sufpro suppressor 1 ( S u p Z) proline Su4pro suppressor 4 ( S u p ZV) p r o l i e

Sulfite

Sulfite

I11

prol-.& pro?'

I11

prol-6

I11

1950

Y

X-rays

1946

ad2O; pyro4

Spontaneous 1954

pro1 pabal y X pro?' bil pro5 bil

Spontaneous

S8

bil

84

bil

95

bil; wS

s6

bil : WS

s8

bif; wS

s9

bil; wS

VI

Aneurin/ I1 R 4methyl5-hydroxy ethylthiasole Suppressed mutants ad20 IL

UV, starvation UV, starvation UV, starvation UV, starvation UV, starvation UV, starvation UV, starvation UV, starvation UV, starvation UV, starvation UV, starvation

bil

bil s f0

bil; W S

sll

bil; wS bil

Spontaneous

* Earlier symbols in brackets. 7 Information on several mutants already published (Pontecorvo, 1953); some details revised.

1950 1950 1954 1954 1954 1954 1950 1954

0 q

1954 1951

Forbes,

114

ETTA RAFER

from recent investigations indicate that mitotic haploidization may be a stepwise breakdown of the diploids as a result of repeated loss of one or a few chromosomes. Poorly growing aneuploids are found, disomic for some chromosomes. They break down into more vigorous haploids and are outgrown. Markers located on the same chromosome are therefore lost or retained together and show complete linkage. The number of chromosomes should correspond to the number of groups of totally linked markers found in haploidization. The process of haploidization seems to occur independently of mitotic crossing-over and chance coincidence is low, as both processes are very rare (Pontecorvo et al., 1954). The low frequency of mitotic segregation makes selection for mitotic recombinants necessary. Several systems of selection have been worked out (summarized by Pontecorvo and Kafer, 1957). The following markers are generally used: y and w (visual selection), suladdO (selection of suppressed segregants from diploids homozygous for ad.20) and A c r l or acrd (selection of segregants fully resistant to acriflavine). Diploids are produced (Roper, 1952) heterozygous for one or several of the selective markers (“ selectors”). Segregants, either diploid homozygous for the selector or haploid carrying it, can be isolated on the appropriate media. Automatic isolation of haploids is possible when two such markers located on different chromosomes are selected for a t the same time. Because coincidence of mitotic crossing-over in two different chromosomes is very rare, such “double selection” yields haploids in most cases.

1. Linkage Detection f o r N e w Markers of Linkage Groups I-IV Early results from haploidization indicated th a t two further linkage groups existed in addition to linkage groups I and 11. Three markers, SO,methl, and pyro4 were found to segregate independently from markers of the two established linkage groups, but methl and pyro4 showed complete linkage with each other (Elliott, unpublished). It was assumed th a t SO would be located on a third chromosome, while methl and pyro4 formed linkage group IV. a. Linkage Group I . Linkage of the markers of linkage group I was only determined by mitotic haploidization in a few cases. Most mutants show meiotic linkage to pabal , y , and b i l l markers generally used in the first cross with a new mutant. The following markers were therefore located meiotically: ad8, ad9, p r o l , pro3, and ad14 (for allelic mutants and references see Table 1). The markers an1 and ribol were located mitotically by Elliott (unpublished) and found completely linked to each other and t o the markers of linkage group I in haploids from a heterozygous diploid. The recessive suppressor suladd0 is specific for add0, a n allele of ad8,

115

CHROMOSOME MAPPING O F ASPERGILLUS NIDULANS

and has been isolated by Pritchard (1955). This marker has been extensively used for the selection of mitotic recombinants, and double selection for sulad2O and w or y is the most convenient method for isolation of haploids. Complete linkage has been found in haploids between sul ad20 and all markers of linkage group I (Pontecorvo and Kafer, 1957). b. Linkage Group I I . Most of the markers of linkage group 11, which contains w (3 alleles, w l , w2, w3; Table 1) and adl, have been located by means of haploidization, but the close linkage of ad3 with ad1 was found meiotically (Pontecorvo, 1952). The three acriflavine-resistant mutants Acrl , Acr3, and acr2 all showed complete linkage with w or ad1 in haploids from various diploids (Roper and Kafer, 1957). They are used for selection of mitotic recombinants on acriflavine media and haploids are often isolated by double selection for y and Awl. The markers thi4, pu, and ni3 were all located mitotically by haploidization and found completely linked with markers of linkage group I1 (pu was obtained and first tested for segregation by Sneath, 1955: cross 30, Table 9). The results obtained from various diploids are given in Table 2; only relevant markers are indicated. The complete genotypes of all diploids are listed in Table 13. I n Table 3 the genotypes of all haploids from diploid B1 are given in detail. They show the complete linkage found between markers of the same linkage group and the independent segregation of markers on different chromosomes. Haploids from diploids D1 and Dz (heterozygous t h i l / + , Table 13) showed complete linkage of thil with markers of linkage group 11. It seemed probable that thil would be allelic t o thi4 located in this linkage group. This was confirmed by diploid El which was isolated from a heterokaryon between a thil and a thi4 strain. Both heterokaryon and diploid were thiazole requiring. ad23 was found t o be nonallelic t o all located adenine-requiring mutants (allelism tests in heterokaryons by Forbes, unpublished). Linkage with markers of linkage group I1 was found in diploid F

("+""

):'.

A:l 16 Awl haploids were isolated and all found ad23+, while all w3-haploids carried add3. c. Linkage Group I I I . The two allelic mutants s l and s2 have been found meiotically linked to SO (v. Arkl, unpublished). Sulpro showed loose meiotic linkage with SO and linkage of Su4pro with SO was determined by means of mitotic haploidization (recent results b y Forbes, unpublished). Meiotic linkage of about 10 units was found between sm and phend in the first cross between these two mutants. They were therefore used together, in coupling, for mitotic mapping in the diploids A, B1 (Table 3),

116

ETTA KAFER

TABLE 2 Linkage Detection of New Markers in Haploids Haploids Linkage New group markers

I1

thi4

Diploid

++ +

thi4 A -~

+

an1 y Acrl

+

PU

w3 p u ad1 Bi Y + + +

+

PU -

Bz Y

+y

su

ni5

I11 p h e n d s m A

+

+

ad20

Selected phenotype

Number

anl+y+ A c r l

13

+ + + + +

y Awl sm phend SO -

y +smphend +w5+

+

Y

12 p u 11

+

y

Acrl w5

Y y Acrl

y (w9)

panto

y

W5

+

smphend

+ + v + phen2 + D i -+ - Acrl + panto

Bz

+

y Acrl

16 smphend 7 + + 8 smphed 11

+ +

7 phend panto+

++ ++

2 panto 1* 1 phend

+

sulad2Owd

14 phen2 panto+ 3 + panto

s-u ad2Ow2 orn4 add0 pyro4

sulad2Owb

+ad20

A

++ ++ 1.+ + + SO?

11 panto+ SO

+ +

+

--+

+

+

SO

E su -ad20 w 2 phend orn4

4 12 12 3*

smphends0 + + + s m phen2 not tested for so

suladd0 wd

+

s u ad20 wd

IV

9 9

+ w+d n+i 5

Awl+ w 3 ni5 Acrl

+ + +

Acrl

DZ--+

40 27 28

6 smphend 17 SO

Y

so

+++

19 w S p u a d l 23

Acrl

Bi --____

A c r l thi.4

WS Y (wS+)

w3 ni5 suladdO wd

+ addOAcr1 + +

Genotype

panto

orn4

+

Y Acrl P Y ~ O ~

+

+ + + + + + orn4 R1 7methl pyro4 + * Exceptional recombinants.

Y

+ + orn.4 methl pyro4 orn4+ + + om4

PYr04 8

4

117

CHROMOSOME MAPPING OF ASPERGILLUS NIDULANS

and Bz. Free recombination of sm and phend with markers of linkage groups I and I1 (as well as IV and V) is shown in Table 4.I n the haploids from all three diploids complete linkage of sm and phend with SO was found (Table 2). These results established the third linkage group. The mutant panto was found completely linked to markers of linkage group 111, when haploids were isolated and tested from diploids DI, Dz, and E (Table 2). TABLE 3 Yellow and White Haploids from Diploid BI L i n k a g e groups

I

I1

I11

IV

V

+ bil

+++

sm phend

w3 p u a d i

+ +

PYT04

+

lys6 -

Y + Y + Y +

w3 p u a d i w3 p u ad1 w3 p u ad1

sm phend sm phend sm phend

PY7.04

+ : 2 lys6: 2 3

Y + Y + Y +

wS p u ad1 wS p u ad1 wS p u ad1

+ + + +

++

$-

Y f Y Y Y Y

+ + + +

Y + Y + Y +

+ bil

wS p u a d i

+ + + + + + +

+ + + + + + +

+ + + + + + +

wS p u ad1

+ + + +

sm phend sm phend sm phend

sm phend

+ + + + + +

sm phend

+

+:

lys6: 1

+:a

lys6: 3

+:5

PYT04 PYTOC

+ +

lys6: 3

+ : 7

lys6: 3 f : 3 lys6: 2 + : 3 lys6: 2

PYT04

+ :-1 42

d. Linkage Group I V . Linkage of or@ with methl and pyro4 (linkage group IV) was found when haploids were isolated from several diploids (diploids A, El and R1,Table 2). 2. Establishment of Linkage Groups V-VIII

In the course of the location of various markers by means of haploidization, several markers were found which failed to show linkage with markers of any of the four established linkage groups. Various diploids were formed and analyzed in detail by selection of a large number of

118

ETTA KAFER

haploids. It was found that ten markers, which showed no linkage with markers of chromosomes I-IV, fell into four further linkage groups. a. Linkage Group V . The marker lys5 was the first shown with certainty not to be located on any of the established linkage groups I-IV (see Table 4, results from diploids A, B1, and Bz, heteroaygous for Zys5 and markers of linkage groups I-IV). nicd also segregated independently of linkage groups I-IV, but showed complete linkage with Zys5 (out of 19 haploids from diploid G heteroaygous for lys5 and nicd in repulsion, 10 were lys5 nicd+ and 9 lys5+ nicd; no recombinant types were found). Table 4 shows the number of recombinants with respect to lys5 (or nicd) and any of the markers of linkage TABLE 4 Mitotic Recombination between lys6 (and nicd) and Markers of Linkage Groups I-IV in Haploids Recombinants between lys6 and markers of linkage groups Diploid

I

I1

I11

IV

Total

A Bi

24 25 12 7 68

24 19 15 10 68

21 24 16 14 75

21 17 12 14 64

40 42 24 19 125

Bz

- G Total

groups I-IV among the haploids isolated from diploids A, B1, Bz, and G. These results establish a fifth linkage group containing lys6 and nic.2. b. Linkage Group V I . The mutant sS shows no linkage with markers of linkage groups I-V. It was first tested for mitotic linkage in the haploids from diploids H (Table 5), which carries sd (linkage group 111) (18) were found sulfite requirand sS in repulsion. Of the 25 haploids ing, as expected if the two markers are located on different chromosomes. These haploids had to be classified for sd or s3 by cross-feeding tests. They showed independent segregation of s3 from markers of the four linkage groups I-IV. Independent segregation of s3 from markers of linkage group V was found in the haploids isolated from diploid G (Table 5). lysl was recognized in diploid J (Table S), which had been used for the location of AcrS, as a mutant possibly not located on the same chromosome as markers of linkage groups I-IV. The genotypes of all haploids isolated from this diploid are given in detail because this is the only case

119

CHROMOSOME MAPPING OF A S P E R G I L L U S N I D U L A N S

TABLE 5 Recombination between s3 (lysl and n i c l 0 ) and Markers of Linkage Groups I-V in Haploids Recombinants between s3 or lysl and markers of linkage groups ~

V

Total

11

19 25 39 14 20 27

Diploid

I

I1

I11

IV

G H J K Mi

9 13 24 9 10 14 79 144

10 12 15 11 8 13 69 144

6 15 15 10 14 60 130

9 9 19 7 12 18 74 144

7 18 36 66

I11

IV

VI

so

pl/ro4

Mz

Recombinant Total

-

TABLE 6 Haploids from Diploid J Linkage groups

I

I1

+ + bil

pabal y

pabal Y pabal Y pabal Y pabal Y pabal y pabal Y pabal Y pabal Y

+ + + ++ +

+ ++ + ++ + ++

+ bil + bil + bil + bil + bil + bil

AcrS

+

AcrS Acr3 Acr3

AcrS Acr3 Acr3

++

Acr3 Acr3

++ ++

+ ad1

w3

+

++ ad1 ad1 + ad1 + + + + ++ w3 w3

++

++ ad1 ad1

w3 w3 w3

w3

++ ++

+

so so so

so

+

+

1ysl

lysl: 2 + : 3 + : 5

+ : 5

so

lysl: 6

so

lysl: 2 1

so

lysl: 1 1

so so

lysl: 2 + : 2 lysl: 2 + :2 39

so so so

so

so

f :5

+:

+:

120

ETTA KAFER

where a high frequency of irregular segregation was found. A large number of haploid segregants show crossing-over between wS and a d l , two markers of linkage group I1 (discussed later, p. 122). Besides, all haploids carry the mutant allele SO,instead of a ratio of about 1 :1 for SO :SO+; irregularities with respect to segregation of SO have been found in other diploids as well, but no plausible explanation is possible on the basis of the available data. Results from diploids J and K

(2

___ 1:1)

show independent segrega-

tion of lysl with respect to markers of linkage groups I-IV (Table 5 ) and complete linkage between s3 and lysl (nine haploids from diploid K were sS lysl and five sS+ and lysl+). Independent segregation of lysl from

'7')

lys6 (linkage group V) was shown in diploid L (l;5 -- which had been synthesized to test for allelism between the two lysine mutants. The diploid was prototroph and of the haploids were nonlysine requiring (5 out of 17). Thus the sixth linkage group was established. niclO was found to be complementary to nic2 and nic8 in heterokaryon tests. In the two diploids Ml and Mz, both heterozygous for sS and n i c l 0 in repulsion, practically complete linkage of n i c l 0 with s3 (1 cross-over out of 47) and independent segregation of niclO from markers of linkage groups I-V were found. Table 5 summarizes the results obtained from the various diploids which established the independent segregation of the three markers s3, l y s l , and n i c l 0 of linkage group VI with respect to linkage groups I-V. c. Linkage Group V I I . nic8 showed free recombination with markers of linkage groups I-VI in the haploids from several diploids (e.g., diploids C and N1, Table 13). In diploid Nz nic8 was shown t o be complementary to, and to segregate independently from, nic2. It was postulated t o be located on a seventh chromosome. The original cho strain was found to carry a translocation. The segregation of cho was therefore analyzed in a large number of diploids and results will be published elsewhere. Haploids from several diploids showed complete linkage of cho with nic8 in all cases regardless of the presence of the translocation, which indicated that these markers form a seventh linkage group. Independent segregation of nic8 with respect to linkage groups I-VI and VIII is shown in Table 7, which contains results from two diploids heterozygous for nic8. d. Linkage Group V I I I . co was found as a spontaneous mutant in strain pabal b i l . In early attempts to locate it, haploids from diploid O1 showed independent segregation of co from all other markers (linkage groups I-IV). Lately it has been tested for mitotic linkage with chromo-

121

CHROMOSOME MAPPING O F ASPERGILLUS NIDULANS

somes I-VII in diploid 02; 27 haploids were isolated and tested. None of the markers showed complete linkage with co, indicating that co does not belong t o any of the established linkage groups I-VII (Table 7). ribod was a second marker tested at the same time as co, which showed no linkage with markers of any of the seven established linkage groups (37 haploids selected as yellow or white “suppressed” sectors from diploid P1are included in results shown in Table 7). I n diploid P2heterozygous in repulsion for co and ribod, these two markers were tested for mitotic linkage with each other. Of the 42 haploids which were selected, 16 were found to be co ribodf and 25 co+ ribo2. TABLE 7 Mitotic Recombination (per cent) in Haploids between Markers of Different Linkage Groups Linkage groups VIII VII VI V IV I11

I1

I 59.4 40.6 34.8 40.6 46.9 45.3 32.4*

I1

I11

IV

V

VI

VII

50.0 53.1 37.5 46.9 46.9 39.1

57.8 64.1 54.7 51.6 51.6

50.0 50.0 53.1 43.8

50.0 43.8 56.3

43.8 40.6

56.3

* Not reliable, because markers of linkage groups I and I1 used for selection. One exceptional haploid was wild type with respect t o both markers and may either have been disomic and heterozygous

c(:;

~

r , 2 ) l or a case of

coincidence or consecutive occurrence of mitotic crossing-over and haploidization. This complete linkage between co and rib02 established the eighth linkage group. The mutant cysd was tested for mitotic linkage against markers of linkage groups I-VII in diploid Q1. No linkage was found among the 34 haploids which were tested. Diploid Q z was formed, heterozygous for cysd and rib02 in repulsion. Among 19 haploids, 17 showed complete linkage of these two markers, but two were found recombinant. It is provisionally concluded that cysd belongs t o linkage group VIII. Table 7 gives the percentage of recombination found between the markers assigned to the eight linkage groups as observed in 64 haploids from diploids O2 and PI. Statistical tests indicate complete fit with random segregation for any pair of markers from different linkage groups. Only 1 out of 27 values deviates significantly from 50% (xz = 5.3,

122

ETTA KAFER

P = 0.02) and one such case is expected out of 20-50 of these ratios. These results show that free recombination is found in mitotic haploidization between markers of the eight postulated linkage groups. The complete linkage between markers of any one of the eight linkage groups can be analyzed in 37 cases, where segregation of chromosomes with two or more markers can be followed. The mean number of haploids tested in each case is about 25. Only in 7 out of the 37 cases have a few haploids of exceptional types been found. A clonal distribution of these cross-over haploids (among all haploids) is found, because of the usually consecutive occurrence of the two events (first mitotic crossing-over and haploidization later after a number of normal mitotic divisions). A very large clone of cross-overs is found among the haploids from diploid J (Table 6), where 17 out of 38 haploids showed crossing over between w3 and a d l , which are two markers of linkage group 11.I n four further cases two or three exceptional haploids showed the same type of crossing-over (e.g., two cases from diploid A, Table 2). Considering the extremely small probability that among 25 haploids 2 cross-overs of the same type have independently occurred, it seems likely that these cases also represent small clones from just one event. Correcting for such clonal distribution, the frequency of cross-over types found among all haploids is less than 1% (as had been found in diploids Y and Z ; Pontecorvo and Kiifer, 1957). It is about 2.5% if each exceptional haploid is counted separately, as 26 cross-overs have been found in nearly 1,000 cases, in which crossing-over could have been detected. The frequency of such second order segregation is increased if diploids are subcultured, as was the case with diploid J. Diploids are therefore better resynthesized than transferred, if large amounts of conidia are required a t various times. These results show that in Aspergillus nidulans it was possible t o establish eight linkage groups by means of mitotic haploidization, even though only a small number of markers was available in most of these linkage groups. Complete mapping of a large number of markers and the centromeres, by means of mitotic as well as meiotic crossing-over, has only been carried out for linkage groups I and 11. But the results from this analysis show that free recombination found between markers in haploidization is a reliable indication that these markers are located on different chromosomes. It is usually safe to conclude that pairs of completely linked markers, or even single new markers, which do not show linkage with markers of established linkage groups, are located on a n additional, nonmarked chromosome. The opposite conclusion, that completely linked markers belong t o the same linkage group, does not always hold because aberrations can

CHROMOSOME MAPPING OF ASPERGILLUS N I D U L A N S

123

produce results which show complete linkage between markers of different chromosomes. For the standard procedure of mitotic mapping of a new mutant by means of haploidization a strain carrying s u l a d 2 0 pabal y ad20 of linkage group I and markers of all other linkage groups is now available. This yellow strain is used when the new marker x has been induced in a white strain (usually carrying only the mutants w 3 and b i l ) . The diploid as selected from the heterokaryon between these two strains will show the following genotype (for example assuming z to belong to linkage group V) :

+

+

+

~.

sulad2O pabal y ad20 Awl phen2 s3 __ nic8 rib02 _ _ pyro4 - lys5 . + + + b i l + w S + + x + +

+

~

+

~

+

This diploid will be green and prototroph. By picking and testing a few yellow segregants from this diploid, the following yellow, adenine-requiring strain (homozygous for y ad20 and bii+ as a consequence of mitotic crossing-over between pabal and y) is easily isolated :

+

+

+

sulad20 pabal y ad20 A ~c r l ~phen2 pyro4 ~ lys5~ s3 nic8 _ rib02_ yaddO++ w3 x

+ -+

+ + +

+ + +

If this diploid is grown on minimal medium supplemented with all growth factors except adenine, s u l a d 2 0 w 3 haploids can be isolated as white sectors. These haploids will show complete linkage of x with the marker on the homologous chromosome (in this example complete repulsion with l y s 5 ) , and free recombination with all others. Such results usually determine without doubt the linkage group or chromosome t o which the mutant x belongs. On the basis of genetic analysis alone we can conclude th a t there are at least eight chromosomes in Aspergillus nidulans, but the possibility that there might be more than this cannot be excluded. However, the genetic evidence of eight chromosomes has recently been supported also by results from cytological analysis (Elliott, unpublished). This evidence of eight pairs of chromosomes observed in meiotic stages of A. nidulans makes it probable th at the number of chromosomes is eight and th a t we are not likely t o find any more linkage groups. IV. DETERMINATION OF THE AND

SEQUENCE OF

MARKERS

LOCATION OF CENTROMERES

While in A . nidulans linkage detection is more efficient mitotically by means of haploidization than meiotically, the sequence of markers on any one chromosome is usually determined most easily by crossing. But when meiotic distances between available markers are long, so t h a t

_

124

ETTA

KXFER

their order cannot be established with certainty, mitotic crossing-over can supply the required information, locating the centromere at the same time. Mitotic crossing-over occurs a t the four-strand stage (Stern, 1936). It leads to homozygosis for all markers distal to a single exchange, when the two cross-over strands move to opposite poles, i.e., segregate together with the noncross-over strands. As mitotic crossing-over is very rare, recombinants are detected only if crossing-over occurs in a diploid heterozygous for a marker which, in the homozygous condition, produces a phenotype distinguishable from the heterozygous parent. In these homozygous recombinants crossing-over has occurred somewhere between the selective marker and its centromere. If markers of the same arm are used in coupling with the selective marker, such segregants will always be homozygous for any marker distal to the “selector.” But they will only be homozygous for proximal markers part of the time, depending on the position of the exchange. The sequence of the markers between the centromere and the selector can therefore be deduced from the phenotypes of these segregants. The details of the procedure for selection of mitotic recombinants and for analysis of segregants, when markers are used in repulsion to the selector, are described for A . nidulans by Pontecorvo and Kafer (1957). If the sequence of the markers on both arms of a chromosome has been determined by means of mitotic crossing-over the position of the centromere can be deduced as being between the two most proximal markers. As such mapping by means of mitotic crossing-over is only possible in chromosome arms on which a distal marker can be used for selection, it has only been applied in linkage groups I, 11, and IV. a. Linkage Group I . The sequence of most markers of linkage group I (Table 8) has been determined meiotically. Results from mitotic crossingover in two well-marked diploids confirmed the sequence of the following markers :

suladdO-ribol

-ad-adl4-centromere-pro1

-pabai- y-bil (Pontecorvo and Kafer, 1957).

Also the position of ad8, ad9 (Pritchard, 1955 and unpublished) and pro3 (Forbes, 1956) have been determined meiotically and confirmed by mitotic analysis: ad8 distal but closely linked to y, ad9 proximal closely linked to pabal and pro3 proximal closely linked to prol. Extensive analysis has been carried out and a large number of further alleles (Table 1) have been located for the following mutants: bil and pabal (Roper, 1950, 1953), ad8 and ad9 (Pritchard, 1955 and unpublished and Calef, 1957), prol and pro3 (Forbes, 1956).

CHROMOSOME MAPPING O F ASPERGILLUS N I D U L A N S

125

b. Linkage Group I I . Left arm. Acrl and Acr3 have been located mitotically distal to w (Roper and Kafer, 1957). They both showed approximately the same meiotic linkage with w. The mean distance of 24.8 k 0.8 units between Acrl and w was calculated from results obtained in eleven crosses. Colonies from about 250 ascospores are usually tested from each cross (listed in Table 14). For the calculation of means and standard errors angular transformation and weighting with the total number of analyzed colonies has been used in all cases. The mean distance Acr3-w was found t o be 22.0 k-0.3 units in crosses 36 and 37. The two mutants Acrl and Acr3 are probably allelic, but as they are semidominant, tests for allelism in diploids were not conclusive. I n crosses between the two mutants (crosses 38 and 39 with a total of about 3,000 colonies tested) a few sensitive (Acr+) recombinants were found. If these have arisen by crossing-over, it would suggest a distance of about 0.3 units (0.34 k 0.06) between the two mutants, with Acrl distal to Acr3. The position of the mutant ad23 distal to Acrl was determined from diploid F

(adLS 4+ Acrl

+

w3) and the corresponding cross 27. All wS/wS

diploid segregants selected from diploid F were adenine requiring, ad23/ ad23 placing ad23 on the same arm as w 3 and probably distal to it. I n cross 27 slight linkage of ad23 with Acrl was found (about 30% recombination), but none with w3 indicating that the sequence of these markers is adWS-Acrl-w3--centromere. Right arm. The meiotic distances between most of the markers of this chromosome arm are very large. Only after extensive crossing with all markers could the following sequence be established (crosses 20-33, Table 9) : thi,J-pu-ni3-adl-ad3-acr2. This sequence was confirmed by results from mitotic analysis, using acr2 for selection of diploid recomy .thi4 ni3 acr2 binants. Diploids S1

+ a + ++

wS.thi.4 p u ni3 ad1 acr2 I / + + + + + + were used for this analysis. Diploid segregants homozygous for acr2lacrd were selected from diploid S1 in three platings. All were green, but heterozygous y/+ and w/+. The following phenotypes were found: lhi4 ni3 acr2 (the majority of segregants), ni3 acr2 (only 2 segregants, each from a different plating), acr2 (several in each plating).

+ + +

Diploid acrd/acrd segregants were selected from Sz in 24 platings. All

126

ETTA KAFER

segregants which were checked were found to be heterozygous y/+ and w/+. The various phenotypes were found to be:

thi4 p u ni3 ad1 p u ni3 ad1 ni3 ad1 ad1

+ + + + + + + + + +

acr2, acr2, acrd, acr2, acr2,

as a consequence of as a consequence of as a consequence of as a consequence of as a consequence of

crossing-over in interval CZ, crossing-over in interval d, crossing-over in interval e, crossing-over in interval f, crossing-over in interval g.

I n Table 12 the numbers of the segregants of each type which were found in all 24 platings are added, but the numbers of platings in which they occurred are indicated. The relative frequency of mitotic crossing-over in each interval was calculated, applying correction for clonal distribution (as discussed later, p. 139). From these data the position of the centromere between w and thi4 can be determined. The sequence of markers as found in meiotic analysis is confirmed and they are established as markers of the right arm of linkage group I1 as follows: centromerethi4-pu-ni3-adl -ad3-acr2. c. Linkage Group I I I . The various mutants of linkage group 111have been mapped in a number of crosses, but no analysis by means of mitotic crossing-over has been attempted. The position of the centromere of linkage group I11 has therefore not been determined and it is impossible t o assign the various mutants to the two chromosome arms. Their sequence can only be concluded tentatively from the available meiotic data. The sequence sm-phen2-Su4pro was established by results from crosses 4 4 4 1 (sm-phen2: 10.7 i- 1.0; phen2-Su4pro: 21.7 f 1.5; and sm-SuQpro: 29.2 i- 2.2, that is, about the sum of the other two distances). All three markers show practically free recombination with SO. The pooled values from crosses 44-50 are: sm-so: 47.4 i- 1.8; phen2-sO: 45.4 i- 1.7; Su4pro-sO: 45.1 t- 1.9. It is tentatively concluded from these data that the longest distance is between s m and SO and that SO is located to the “right” of Su4pro. The mutants s l and s2 have been analyzed in crosses and diploids by v. Ark1 (unpublished). Both showed close linkage to SO (crosses 40 and 4 1 ) . A diploid heterozygous with the two mutants in repulsion showed the mutant phenotype (requiring sulfite) ; no cross-feeding was found between them. They are therefore considered to be allelic. Cross-€ceding tests between 12 sulfite-requiring mutants (s0-sll) were made in all possible combinations. Three groups of mutants could be distinguished on the basis of this test (as had been found for SO and other sulfite-requiring mutants of Aspergillus nidulans by Hockenhull, 1949). That lack of cross-feeding represented a valid test for allelelism

CHROMOSOME MAPPING O F ASPERGILLUS N I D U L A N S

127

was checked in some cases by crossing. Practically no crossing-over was found between mutants inside each group. I n other cases heterokaryons and the corresponding diploids were formed, and these were always sulfite requiring when two mutants which had shown no cross-feeding were used in repulsion. The mutants s4, s5, s6, s8, and s9 were found to be allelic to s l (and s2), s7 was found to be allelic to SO, and s3, s10, and s l l formed the third group of allelic mutants (Table 1). The distance between s4 and SO was determined in crosses 42 and 43; the mean distance between SO and mutants of the sl group ( s l , s2, and s4) was calculated t o be 2.9 & 0.4 units. The position of panto with respect to other markers of linkage group I11 was established provisionally in crosses 4.2 and 52. I n cross 42

182 colonies were tested from a plating of ascospores on (complete + +medium. s41 Two were found nonsulfite requiring (presumably so +

recombinants SO+ s4+), and both these recombinants carried the wild type allele panto+, which had been in coupling with SO+. This may indi(s4 is an cate that the sequence of the three markers is: panto-so-sl allele of the sl group). I n cross 52 the sequence phen2-panto-SO was indicated by the following results : recombination between phen2 and panto: 30.8%; between panto and SO: 10.8%, and between phend and SO: 36.8 % (176 recombinants tested). Linkage of S u l p r o with SO was measured by Forbes in two crosses which showed about 35% recombination between the two mutants (almost 1,500 segregants tested). No detectable linkage of S u l p r o with s m was found in crosses 43 and 53, nor with panto in cross 42. This would indicate that S u l p r o is located far out to the “right” of SO. The sequence of all markers of the linkage group I11 Lwould then be the following: sm-p hen2-8u4propanto-so-sl--Xu1 pro. d. Linkage Group I V . It was not possible to determine meiotically the sequence of the three markers of linkage group IV. Meiotic data only showed that pyro4 and orn4 are closely linked (0.42 k 0.25% recombination in crosses 34 and 56 with about 1,000 segregants tested), b u t both segregate practically independently of methl . (i) pyro4 Alleles. The inability to establish heterokaryons between pyro4 and all other pyro mutants on minimal medium indicated th a t these mutants are all allelic. For pyrol allelism with pyro4 was confirmed in cross 67: selective platings on medium lacking pyridoxine showed no recombinants among several thousand ascospores tested. Diploids TI (pyrod/pyro4) and T, ( pyro5/pyro4) are both pyridoxine requiring, which confirms allelism of pyrod with pyro2 and pyro5 (for other pyr04 alleles see Table 1).

128

ETTA KAFER

(ii) Sequence of orn4~yro4-Centromere. Determination of this sequence was attempted by means of mitotic crossing-over analyzed in the two pyridoxine-requiring diploids

+).

sulad2O y ad20 pyro5 + andTI( + pyro4 orn4 The diploids were either plated at high density (106-107 conidia per plate) on minimal medium (supplemented with ornithine), or a t low density on minimal medium (plus ornithine) to which a limiting amount of pyridoxine was added. Addition of x o o of the normal amount of pyridoxine allows adaptive growth of a pyridoxine-requiring strain as a thin mycelium without formation of conidia. A few colonies using the former

+ +

+

Prototroph segregants

“Cis” heterozygote, segregation of cross-over strands together

“Trans” heterozygote, requiring pyrodoxine

2 1 =

pyro2

+

pyro2

+

*3 ~*- --_--xpyro4

+

pyro2

_Prr!_4_--0!!!!

3 -+---- -+------

+

+

Segregation of cross-over with noncross-over strand

-____________ orn4 + pyro4 orn4 4 ....................... J

4

=

+

3

pyre 2

+

+

-*----_--___--

+

+

FIG.1. Selection of “cis” hetercmygotes from “trans” heterozygotes to determine sequence of linked markers on chromosome IV. Segregants resulting from single crossing-over between pyro mutants.

method and rare sectors using the latter were selected and found to be prototroph. No ornithine-requiring recombinants were found among about 50 tested. Nonpyridoxine-requiring segregants, which resulted from single mitotic crossing-over between the two pyro mutants, would be selected by this method, as had been shown possible for other allelic mutants (Roper and Pritchard, 1955: selection of “cis” heterozygotes from “trans” heterozygotes). They are expected to be either heterozygous for the two mutants in (‘cis” arrangement, or homozygous for the wild type allele of the more distal pyro mutant and all other genes distal to pyro (Fig. l), depending on the way in which the various strands segregated. The types of segregants obtained from diploids TIand Tz were

CHROMOSOME MAPPING OF ASPERGILLUS NIDULANS

129

similar and the sequence of the three markers is therefore assumed to be the same in both. Thirteen prototroph segregants were completely or partially analyzed suladdO y ad20 by means of haploidization. I n several cases a recombin-

+

y

add0

ant was isolated from the prototroph recombinant and this yellow segregant further analyzed by selection of “suppressed” haploids. Distinction between the various pyro mutants was not possible. From each of seven prototroph recombinants two types of haploids were obtained and the following genotypes deduced:

+

pyro

pyro

+ -++

0rn4 (possibly single crossing-over and segregation of the

+ two cross-over strands together; “cis” arrangement of the two pyro mutants?) + (possibly single crossing-over and segregation of cross+ over with noncross-over strand) orn4 (not explainable on the basis of single crossing-over or + mutation).

From each of six prototroph recombinants only one type of haploid was obtained (in one case a single haploid carrying pyro o r n p and in five cases several haploids carrying all wild type alleles). Some difficulties in testing as well as in interpretation reduce the reliability of these data. If either of the two orn4+/orn4+ segregants resulted from a single crossingover, orn4 must be distal to pyro and the mutant pyro4 would be distal to pyrod and pyro5. (iii) Location of the Centromere. Two diploids R1 and R2, carrying methl and pyro4 in coupling and homozygous for b i l l were formed. It was planned to select segregants showing double requirements which would be at an advantage compared to the original biotin-requiring strain under conditions of starvation (Macdonald and Pontecorvo, 1953). Assuming that methl is closer to the centromere than pyro4 (as indicated by tetrad analysis; N. S. Strickland, unpublished) the recombinant diploids homozygous for methl or pyro4 or both should reveal the position of the centromere: (1) if the centromere is between methl and pyro4, only segregants homozygous for either methl or pyr0.4, but not for both, are expected (except for nondisjunctional diploids) ; (2) if the centromere is to the ‘(left” of methl and pyro.4, segregants homozygous either for pyro4 only or for both mutants are expected, but none homozygous for methl only (except double cross-overs) . The following pyridoxine and/or methionine requiring diploid segregants were obtained in six platings from diploid R1 and two platings from diploid R2:

130

ETTA KAFER

R1: 4 meth (from three platings)

3 pyro (from one plating) 8 meth pyro (from five platings) Rz: 1 meth 9 pyro (all from one plating).

It seems obvious that these results cannot be explained on the basis of single mitotic crossing-over alone. As “ nondisjunctionals” were found with much higher frequency than double cross-overs in diploids with markers a t similarly large distances (Pontecorvo and Kafer, 1957), the most likely interpretation would be that methl and pyro4 are on different arms. The segregants requiring either methionine or pyridoxine only would then represent single cross-overs and the segregants requiring both methionine and pyridoxine nondisjunctionals. The sequence of the markers of linkage group I V would be deduced as follows: methl-centromere-pyro4-orn4. e. Linkage Groups V-VIII. As these linkage groups have been established very recently only few markers are available in each one and the sequence of these markers has not yet been determined. Results from a few crosses (58-60) are indicated in Fig. 2. The mutant Bw has not yet been located. Results from the diploid, in which the mutation occurred, indicated th a t it does not belong to linkage groups I or 11; in crosses 37 and 61 no meiotic linkage was found with the markers SO, phen2, pyro4, lys6, s3, and nic8. Efficient mapping by means of mitotic crossing-over is restricted t o those chromosome arms which carry a distal selective marker (the four arms of linkage groups I and 11). The results from mitotic mapping in linkage group I V show that the two methods which are applicable more generally t o many nutritional markers present difficulties in interpretation. Recombinants are selected and isolated which cannot be interpreted as resulting from a single mitotic crossing-over. I n the case of starvation an unusually high frequency of nondisjunction was assumed to explain the results obtained. Whether this was a peculiarity of the two diploids (which is very unlikely, as they showed normal results when yellow segregants were selected), or whether i t was rather the system of selection used in this analysis which caused the increase in the frequency of “nondisjunctionals,” is still to be determined. If there is any connection between nondisjunction and the formation of “aneuploids” (as indicated by recent results obtained in the analysis of haploidixation), results from treatment with chelating agents may point to the latter interpretation. After extreme starvation by addition of E D T A (ethylenediaminetetraacetic acid) to the medium, a large

CHROMOSOME MAPPING O F ASPERGILLUS NIDULANS

131

number of segregants, almost all aneuploids of various kinds, was isolated. I n the case of “cis-Lrans” selection with allelic pyro mutants one out of the seven completely analyzed segregants requires for explanation multiple crossing-over in the extremely small regions between pyro5, pyro.4, and orn4. Similar cases, not explainable by a single mutation or single crossing-over, have been found by Pritchard (1955) in the mitotic analysis of the ad8 region, indicating high negative interference in mitotic crossing-over in small regions. Besides presenting difficulties of interpretation, these two methods are also very laborious, so that only small samples of segregants are usually classified. The analysis of the sequence of centromere and markers on further chromosome arms by means of mitotic crossing-over will therefore depend mostly on the location of new selective mutants.

v. MEIOTICLINKAGEMAPS O F LINKAGE

GROUPS1 AND 11 Table 8 shows the meiotic distances for markers of linkage group I, which were calculated from data obtained in crosses 1-19, each involving at least four of the markers (Table 14; not included are markers pro3, ad9, and ad8, Table 1 and Fig. 2). I n Table 9 the corresponding data for linkage group 11, from crosses 20-39, are given in detail (no distinction is made between the position of ad1 and ad3, which are closely linked). The arrangement of the markers of linkage group I1 is shown for each cross. As no consistent differences have been found between the coupling and repulsion arrangement of markers, data from all crosses have been pooled. Only distances between adjacent markers in each cross are given, because the sequence of these markers is well established and has been ascertained meiotically and mitotically. The mean values and standard errors are calculated for the various intervals, using angular transformation and weighting with the total number of segregants analyzed in each cross. They are listed a t the bottom of the table together with the number of crosses and the total numbers of analyzed segregants on which the measurement of each distance is based. These values are reliable in most cases, as the genotype of each segregant can be classified with certainty from the available tests. This is less the case for the adl-ucr2 interval, because testing for acr2 is very difficult in crosses between these two markers. Addition of adenine decreases the inhibitory effect of acriflavine (Roper and Kafer, 1957), so that ad1 recombinants, which use up the supplemented adenine, become more sensitive regardless of their genotype with respect to acr2. The small standard error found in practically all cases indicates th a t the data from the various crosses are mostly homogeneous. This has been

TABLE 8 Meiotic Recombination (per cent) in Linkage Group I Located mutants Intervals

suald2@-

a

ribol

Mean distance 39.4 f 2 . 3 Number of crosses 5 Total number of segregants 1020

-

ani

-

ad14

b

C

19.4 k 1 . 0 6 1446

6.7 kO.5 4 1022

-+- pro1 d 29.8 k 1 . 2 10 2747

-

pabal

-

M

y ad20

e

f

7.9 k 1.2 14 3938

15.7 k 1 . 0 17 4228

- bil g

5.7 k 0 . 4

17

4098

e l

?

$: 2 a

4

0"

& 20

21 22 23 24

25 26 27 28 29 30 31

32 33

-

Parental geotypes

TABLE 9 Meiotic Recombination (per cent) in Linkage GrouD I1 Located mutants Tested Ac +- thi4 - PU segre- ad23I I gants 'ntersals: a b d C 23.4 235 47.7 37.9

+ niS + ad3 { + + + + ad1 + niS + + acr: { W+S thi4 + p u + ad1 + + + ad1+ ++ acr:+ { W+S thi4 + p+u ni3 { A c+r l w+3 thi4 ++ + + p u + ad1 { + w3 thi4 + niS + + A c r l + + + + + ad3 + niS + + + { w3 ++ thi4 + + + + + acr: thi4 + + I wS + + niS ad23 + w3 + Acrl + Acrl + + + + + + w3 + p u + ad1 + ++ p+u ++ ad1 + { w3 I+T:+ 7+ :dl ++ { W S + p u + ad1 $ +f& a& w S + + + + + + { + + + + + + acr: A c r l w3 thi4

PU

-

ni3 I

e 19.6

-

ad1 Id3 -acr%

f 33.5

g

222

46.4

31.1

15.3

34.2

26.1

425

50.8

33.8

20.9

27.3

30.8

30.7

231

27.2

45.8

233

27.9

43.8

258

43.4

205

41.9

130 132

30.0

27.9 31.8

'

42.4

3

0

41.2

29.2

34.5

4: 4

i 4

34,.8

4

153

47.2

150

44.6

47.3

233

49.7

35.2

30.7

396 (a1 w3) 125

Wean distance Vumber of crosses Total of tksted segregants * Results from 6 further crosses included.

30.3 55.2

I

1

1

30.0 k 4.C 4 . 8 f 0.1 4 . 9 f 0 . 8 3 3 . 5 ?I 1.2119.2 5 1 . 2 3 0 . 6 rt 1 . 3 2 9 . 2 f 1 . 6 1 11* 7 4 3 2 2914 130 1384 1113 882 1278 647

I

F

w w

134

ETTA KAFER

confirmed by homogeneity tests. Only in the case of the distance pabal-y (interval f of linkage group I, Table 8) a certain amount of heterogeneity was found, due mostly to crosses 3 and 17, which showed exceptionally high values. But even for this interval the standard error is fairly small, because the number of crosses giving information is especially large. The homogeneity of most data on crossing-over in A . nidulans reflects the genetic homogeneity of all strains used in this type of work. This is largely due to the fact that all these strains of Aspergillus nidulans are descendants of the same wild type strain, which was originally grown from a single uninucleate spore. This may explain why these results are much more consistent than those obtained in crosses of Neurospora, where distances between two markers often differ significantly in crosses TABLE 10 Recombinants Classified According to Number and Position of Crossing-Over Observed Numbers of the Two Reciprocal Types Single cross-overs i n intervals Cross ~

Total _

1 2

3

Selected number

_

With special genotype

Nonforg

crossovers

b

/ c i

d

lei-

~

_

250 413

208 205

w+

122 105

w+

227

W

W

-

68+57

13+9 3+3 16+16 4+3 3+0 2 + 2

55 +36 51 +44

4 f l l 3 $3 21 +13 1 +O 7+5 1 +2 54-3 7 + 8 2+118+224+0

16+23 lS+19

0 4+5 4+101+0

9+8 2 + 3 4 + 4 1 + 5 9+4 0+1 7+5

Double cross-overs in intervals Cross

bc

bd

b (for g) be _____ cd bf bg

ce

_

0 3+62+01+2

_

~~~

1

3+03+5

_

_

c (for g) ~

de

d (f or g) e (f or g)

-

cf

~

cg

0

0

0 1+42+21+21+2

-

-

df

_

dg

_

ef

~

eg

~

fg ~

0 0+2

0 1+16+31+10+1 0 4+l 1+20+20+10+10+12+0 0 1+2 1+1 0 1+1 2+3 2[3 Of2 0 4+f l+1

0 ?

0 0+1 0 1 + 4 2 + l 0 0 + 1 0 + 1 0 1 + 1 7 + 3 1 + 2 0 + 1 0 0+2 1+1 1+4 0 1+0 1+0 4 + 3 1+0 ? 0 3+2 1+0

~

~

_

I

Quadruple cross-overs i n intervals

Triple cross-overs i n intervals

bd (for g)

Cross bcd

bcf

be (for g)

bde

cd (f or g) bfg

bdf

bdg

bef

O + l

0

beg

bcd (for g) bde (for g)

de (for g) bcde

cde cdf

cdg

def

~~

3

0

1+0

0 0

0 0

0

0

l + O l + l 0 0 2+3

0 0+1 0 1+1(1+0 1+0 0 O + l 0

* Probably some diploids included.

l + O l + O

0 0 0+1 0 I 0 O + l

0 ?

0

0

1+00+1 ? Of1

0

0

0+10+10+6*1+0 0 0 0+1 1 +O

0

0

l + O O + l

4*+00+1 0 1+0 0

0 I 0 0

bdfg bcdf bcdg bdef bdeg ~ -

deg

l + O

0

0

0

0 0

0

0

I

0

0

0

~

O + l

0

0 ?

1+0/ 0 0

0 ?

0

0 4

G

d

-

U

TABLE 11 Recombinants Observed and Expected in the Various Cross-Over Classes

A. LINKAGEGROUPI

w

Q,

Single cross-overs in intervals Crosses

Total

Noncross-overs

1+2+3

890

obs. 387 exp.442.2

4

259

obs. 120 exp. 118

73 68

5 (w+ only)

389

obs. 229 exp. 227

78 82

b

c

d

f,g

Total

Doubles

Triples

56 79.4

301 370.5

157 147.1

35* 27.5

16 16

30 33

119 117

17 22

3 2

12 11

34 39

124 132

34 28

2 2

56 82.9

301 364.8

157 137.9

32 24.2

Total

Doubles

Triples

e

75 16 136 18 83.223.8153.530.6

Figures corrected b y exclusion of possible diploids obs. 381 75 16 +3 881 exp.351 82.8 20.2

136 18 151.8 27.1

Quadruples Quintuples

10* 2.7

-

E,

4 2.2

B. LINKAGEGROUPI1 Single cross-overs in intervals Crosses

Total

Noncross-overs

b

c

d

e

f

20

235

obs. 33 exp. 31

10 9

25 26

16 18

8 9

17 12

+ 22

647

obs. 104 exp. 88

81 86

45 44

20 21

21

* Probably some diploids included.

g

30 29 37 37

Quadruples Quintuples

76 74

82 90

35 28

12

9

-

205 225

212 214

100 97

23 21

3 2

CHROMOSOME MAPPING O F ASPERGILLUS NIDULANS

137

with different strains (Barratt et al., 1954; Stadler, 1956). Besides, care was taken whenever possible to use only strains with mutants which were induced by ultraviolet irradiation or arose spontaneously. These would be less likely to carry rearrangements than X-ray-treated strains. Most crosses were analyzed for interference, but generally no significant deviation from random distribution of crossing-over was found. Crosses 1-5 and 20-22, which give information on the distribution of crossing-over in a large number of adjacent intervals, have been analyzed in detail. (All recombinants from crosses 1-3, classified with respect to number and position of crossing-over, are given in Table 10.) The expected numbers of recombinants of each cross-over type were calculated on the assumption of random distribution of crossing-over in chromosome strands. The expected values were calculated on the basis of the total amount of recombination which had been observed for each interval in these crosses (as shown in Table 9 for crosses 20-22). The various values were calculated separately for each of the crosses 4, 6, and 20, but combined for the similar crosses 1, 2, and 3 of linkage group I and crosses 21 and 22 of linkage group 11. Table 11 summarizes the numbers which were expected for all cross-over types with the same number of cross-overs and shows the corresponding observed values for comparison. A very good fit with random distribution of crossing-over is found for crosses 4, 5, and 20. The pooled values from crosses 1 , 2, and 3, on the other hand, show a significant deviation. An excess of multiple crossovers and noncross-overs and a corresponding shortage of single crossover-that is negative interference-is observed (e.g., for the difference found in single cross-overs x2 = 16, P = 0.01). The same is found to a small extent in the combined values of crosses 21 and 22, but in this case no significant differences are found for any one class of recombinants. It seemed possible th at the inclusion of diploid ascospores in the analysis of these crosses might simulate negative interference. Diploid ascospores are formed with a frequency of 0.1-1 %, and are predominantly wild type, as they arc probably formed by fusion of nuclei during or after meiosis (Pritchard, 1954). I n these crosses involving large numbers of markers, diploids would be less likely t o be detected and would get classified as multiple cross-overs. For example in cross 2 (Table 10) four triple (intervals cde) and six quadruple (intervals bcde) cross-overs were most likely diploids. When nine of these are excluded as probable diploids the differences between expected and observed values become somewhat smaller, but not substantially so (e.g., the shortage of single cross-overs is still very large, x2 = 12, P = 0.01). This shows, th a t a few diploids included by mistake in these data cannot simulate the observed deviation from random distribution. Why negative interference is found in some

138

ETTA a F E R

crosses and not in others, and what the basis is for the apparent negative interference in crosses involving a large number of markers cannot be decided on the incidental data available. The problem of negative interference which was found to a much higher degree in the analysis of small regions in Aspergillus nidulans by Pritchard (1955) and Calef (1957) will therefore not be discussed here.

VI. MITOTICLINKAGEMAPS A N D COMPARISON WITH MEIOTICMAPS The distribution of mitotic crossing-over was found t o be very consistent in various diploids and mitotic linkage maps were calculated for Linkage group I dad20 ribol an1 ad14 30 pro3 pro1 ad9 pabal

Meiotic units : Mitotic frequencies:

I I 0.3

30

I

11

I

22

I

w

1

.

401 69.0

45

9

I I 3

1

18’

I

0.4

Linkage group VZ 93 lysl niclO

I

I

I

34 4.5

:

i

ad8 b i l

I

ni3

ad1 ad3

19 31 : 4.5 : 7.9

0.1

I

I

I I :

I

acrd

29 14.1

I

I

Sulpro

panto SO sl

I

I

I

18 0.5 8 0.3 16 0.1 6 6.9 : 67.1 : 25.71 pu

I

I

I I

thi4

Linkage group ZV methl 44 pyro4 orn4

I

I

45

25 20 14.7 : 85.31

Linkage group 111 s m phenB Su4pro

I

I

39 19 7 20 23.0 : 7.4 : 6.2: 63.4

I

Linkage group 11 ad23 Acrl Acr3

1

I

I

I

35

Linkage group V lys6 nicd

I

I

3

I

Linkage group V Z I nicd cho

I

I

Linkage group V I I I co rib02 cys2

I

I

-__-

I

35 1 43 45 1 FIG.2. Linkage maps of Aspergillus nidulans based on mitotic and meiotic analysis.

the three analyzed chromosome arms (Pontecorvo and Kafer, 1957). As the selective methods, which were used for the isolation of mitotic segregants, did not permit the determination of absolute crossing-over frequencies, only the percentage of crossing-over (the relative frequency) occurring in each interval of a chromosome arm could be calculated. The values for the two arms of linkage group I and the “left” arm of linkage group I1 are shown in Fig. 2. The construction of a corresponding mitotic map became possible for the “right” arm of linkage group I1 when acrd was located distal on

139

CHROMOSOME MAPPING O F ASPERGILLUS NIDULANS

that chromosome arm. The mitotic map distances could be calculated on the basis of the results obtained in diploid Sz (see above, p. 126). Because the plating method (Roper and Kafer, 1957) had been used for the selection of acrdlacrd recombinants, correction for clonal distribution had to be applied (Luria and Delbruck, 1943). As the total number of conidia from which the segregants were selected had not been determined, it was also impossible in this case to calculate the absolute crossing-over frequencies. The relative frequency of mitotic crossing-over in each interval could be determined, however, and is shown in Table 12 as TABLE 12 Linkage Group 11, Mitotic Crossing-Over in “Right” Arm Genotype

thi4

w3 Diploid Sz --e Intervals

+

Number of acrdlacrd segregants Number of platings Relative frequency of mitotic crossing-over Relative meiotic frequency

GI

c2

+

ni3

PU

d

+

e

+

adi

f

+

acrd

g

4 3

3 3

5

7

15 7

69.0

4.5

4.5

7.9

14.1

26.2

22.0

12.5

20.1

19.2

205 24

+

percentage of the crossing-over observed in the whole arm (centromereacrd). For comparison on a corresponding scale the relative frequencies of meiotic crossing-over are also calculated; the map distance of each interval (Table 9) is divided by the length of the whole arm (centromereacrd = 152.5 units) and multiplied by 100 (Table 12). The comparison of these values for mitotic and meiotic distribution of crossing-over shows that mitotic crossing-over is relatively much more frequent in the interval between the centromere and the most proximal marker (thi4): about 70% of the mitotic crossing-over occurs in this interval, which meiotically is only about of the total length. A similar concentration of mitotic crossing-over near the centromere had been found for the two meiotically much shorter “left” arms of linkage groups I and I1 (Fig. 2). As the meiotic distance between the centromere and thi4 is estimated to be about 40 units, no information is gained on

140

ETTA

KXFER

whether mitotic crossing-over increases towards the centromere or is concentrated in a small preferential region a t a certain distance from the centromere, as is indicated by the distribution of mitotic crossing-over found in the “right” arm of linkage group I (Fig. 2).

VII. SUMMARY Techniques for mapping by means of mitotic recombination have been used to locate new markers and establish linkage groups in the filamentous fungus Aspergillus nidulans. Results from mitotic mapping are comparable to meiotic data. Mitotic mapping proved especially useful in A. nidulans because the meiotic linkage maps are very long. (1) By means of mitotic haploidization about 40 markers are assigned to 8 linkage groups. (2) Eleven of these markers fall into linkage group I on which research in A. nidulans has been concentrated, and nine into linkage group 11, which seems to have the longest map. For linkage group I1 the sequence of markers on the right arm and the position of the centromere were determined by mitotic crossing-over. Mitotic mapping for all known markers and the centromeres of linkage groups I and I1 was thereby completed. Agreement with results from meiotic mapping was found in all cases. (3) The sequence of markers could only be tentatively concluded from meiotic results for linkage group I11 and from mitotic data for linkage group IV. Two to three markers were located in linkage groups VVIII by mitotic haploidization, but meiotic linkage was found only in a few cases. (4) Meiotic distances were calculated from a large number of crosses for linkage groups I and 11. Data from the various crosses were found to be homogeneous and have been pooled for the construction of meiotic linkage maps. Random distribution of crossing-over is found in most crosses. (5) The comparison of meiotic and mitotic “map distances” in the “right” arm of linkage group I1 indicates that mitotic crossing-over differs in its distribution from meiotic crossing-over and seems to be relatively more frequent in the region near the centromere. This agrees with similar results obtained earlier in the other three arms of linkage groups I and 11. The combined evidence from all these methods of analysis, together with the most recent cytological information, makes it most probable that all linkage groups have been established and that in Aspergillus nidulans the haploid chromosome number is eight.

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CHROMOSOME MAPPING OF ASPERGILLUS NIDULANS

TABLE 13 List of Dioloids Symbol

A B, B2 C

DI D2

E F G

H J K L

MI M2

Ni N2 0 1

0 2

P1 P2

Qi

Q2

Genotype

+ ad14 + b i l + thi4 + + + + orn4 + + an1 + y + A c r l + s m p h e n d s 0 pyro4 + lys6 y + + + + smphend + lys5 + bil w 3 p u a d l pllr04 f ribol ad14 y + + sm phend + + lys5 ++ +pabal +bil@ + + sOpYro$+ y add0 + + w 3 n i S + + + s u l a d d 0 + + add0 b i l A c r l + + SO pyro4 nic8 ribol y + + + wd + phend + + + addO bil A c r l + l h i l + panto suladdO ribol an1 addO bil A c r l wd + + SO + cho + + + ad20 bil A c r l + t h i l panto + pyro4 + + add0 + A c r l + t h i l + panto pyr04 + suladdOaddObi1 + w d t h i l phend + + ornL + +' + prol pabal y + a d d s + b3 + + + + bil + A c r l + phend lys5 s3 nic8 prol y + A c r l + + pyro4 + nicd + + + b i l + wd phend + lvs5 + s3 cho T(VI1,I) ribol y + wd + _+ _ s3 + + b i l -+_s2_pyr04 + cho+ (VII) + + hi1 AcrS + ad1 SO pyr04 + pabal ?i + + ?US + 77 l?/sl -y + A c r l + + + + cho + bil AcrS ad1 pyro4 s3 l y s l + + bil A c r S a d l + + SO pyr04 +lysl -~ -Y + + + sinphend + + lvs5 + s u l a d 2 O p a b a l y add0 _ A c r l + _+ _ - + ic10 _ -+ - f n~ + + + add0 A c r l_wd phend pyro4 lys5 sS + suladdOpaba1 add0 _ Acrl + + + _+_+-niclO _ ~ -+ + + + ad20 A c r l_wd _phend_ pyrod 1ys5 s3 + nic8 ribol y + + + + + nic8

Location of

ribol

'

thi4,sm,phend orn4, 1ys5 p u , sm, phen.2

n i S , nic8

t hi1, panto ornL

add3 lys6, nicd, s3 s3 AcrS, lysl sS, l y s l , cho

1y s l

1 1

+

niclo

+ b i l ~ p h e n d l ~ ~ f nic8 ad80 hi1 A c r l SO pyro4 nic8 sKadd0 a n 1 prol y addO Acrl nicd bil w3 SO pyr04 an1 y Acrl ads co suladdO addO A c r l wd phend lys5 s J n i c 8 co a n 1 pabal y ad20 b i l A c r l + pyro4 co s u l a d d o p a b a i y addObil A c r l ribod --- - - -. addO A c r l w 2 phend pyro4 lysci sS nic8 ribod suladd0 pabal y add0 hi1 nic8 ribod an1 y a d d O b i l w b pyro4 co s u l a d d 0 p a b a l y ad20 cysd addO A c r l wd p h e n l pyro4 2ys5 s3 nic8 sulad20pabal yaddO cysd y add0 A c r l w 3 ribod

+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + ++ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + ~

1 1

142

ETTA K ~ F E R

TABLE 13. (Continued) Symbol ribol

+ + +

+ + + + + + + +

bil thi4 orn4 prol pabal y bil methl pyro4 prol p a b a l y b i l methl pyro4 f Rz bil ad23 A c r l w 3 nic8 ribol y thi4niSacr2 _ s1 an1 bil w 3 SO pyro4 bil w 3 thi4 p u niS ad1 acr2 & methl sz ribodadl4pabal y sulad20pabal y ad20 Acrl pyro2 T1 bil Acrl thi4 pyro4orn4 sulad20pabal y ad20 Acrl pyrod Tz bil A c r l thi4 pyro4orn4

R1

Location of

Genotype

+

+ + +

7

+ ++ + + + + + + + + + + + ++ ++ + + + + + + + + + + + + + + + + + + + + +

1 1

07-4

sequence of

sequence of

TABLE 14 List of All Crosses

No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

Genotype of one parental strain Allele+: mutant carried by other parent; allele* 1 mutant carried by both parents

ribol an1 ad14 prof pabal y bil+; Acrl+; methi pyro4* ribol an1 adl4+ prol pabal+ y+ b i l ; w8; methl+ pyro4 sulad20 ribol an1 ad14 prol+ pabal+ y addO* bil+; A c r l w2+; pyro4; cho ad14 prol pabal y bil+; w3; methl+ ad14 prol pabal y ; w 3 ribol a n l + ad14 prol+ pabal y bil+; Awl+ wd; methi ribol a n l * ad14 prol pabal+ y+ ad20+ b i l ; Awl+ wd; SO+; pyro4+; cho+ ribol ad14 prol pabal y bil+; phend+ ribol ad14 prol pabal y bil+; ni&+ ribol ad14 prol pabal y bil+; orn4+ sul ad30 ribol+ prol + pabal+ y f ad80 bil+; pyro4 suladdO ribol a n l + pabal+ y+ addo* b i l ; Acrl wd+; SO+; pyro4; cho ribol a n l + prol pabal+ y+ ad2O+ b i l ; w2+; pyro4+ an1 prol+ pabal+ y* ad80 b i l ; w3+ adl+ sulad80 ribol prol* p a h a l f y+ ad20 b i l ; wSf; methl+ sulad20 ribol prol y+ ad20 b i i ; Acrl+; pyro4; cho ribol ad14 prol* pabal y bil+ an1 pabal+ y* ad2O+ bil+; w2; pyro4 suladdO* ribol prol+ pabal+ ad3O* b i l ; A c r l wd; SO; pyro4 ribol b i l ; A c r l w3 thi4 pu+ ni3 adl+ ad3 ribol y bil+; w3+ thi4 pu+ ni3 a d l + acrd ribol y+ b i l ; wS+ thi4 pu+ niS+ a d l + a d i ; orn4 ribol y+ b i l ; A c r l f wS+ thi4 pu+ adi+; cho+ ribol prol+ b i l * ; A c r l f w3 thi4 niS adsi ribol ad16+ pabal+ y+ b i l ; w 3 thi4 ni3 a c r P ribol b i l * ; wSi thi4 ni3+; o m 4

CHROMOSOME MAPPING O F ASPERGILLUS NIDULANS

143

TABLE 14. (Continued) 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52

53 54 55 56 57 58 59 60

61

pro1 pabal y b i l f ; add3 Awl+ w3; phenB+; lys5+; sS+; nic8+ y bil+; A c r l w3+ pu+ a d l + ; cho y bil+; w3+ p u + adl+; SO; pyro4 bil ; w3 p u a d l + prol y bil+; wS+ pu+ a d l + prol* y bil+; A c r l + w3 niP ad3+; SO; m e t h l ; nicd prol ad16+ pabal+ y+ b i l ; w3 acrd+ ribol addO+ b i l ; A w l f wd+ thi4; p h e n d f ; pyr04' orn4 pabal y bil+; A c r l wS+ ads+; co ribol prol pabal add0 b i l ; AcrS+ w3 a d l + ; so;i pyro4+ prol y addo+ bil+; Ac& w3; so+; p y r o d f ; Bw pabal y bil+; A c r l A c r P ; co ribol y+ b i l ; A c r l + AcrS wd ad3+ y bil+; wd+ a d l ; SO s l + S u l p r o ; pyrodf y bil+; wd+ a d l ; SO sl+;pyro4+ prol y+ b i l ; panto+ SO+ s4 S u l p r o s u l a d d 0 prodf pabal y add0 b i l + ; sm+ SO+ 94 S u l p r o + ad14 prol * pabal y bil+; s m phend+ Su4pro SO+ prol * pabal y * ; Awl+; sm+ phen2+ S u e p r o SO+ prol* pabal y bil+; Awl+; s m phen%+ SO+ ribol ad14 y bil+; smf phend; lys6+ anl prol y* ad2O; A w l ; sm+ phend+ SO; pyro4; lys5+ ad14 prol* pabal+ y+ b i l ; sm+ phend* Su4pro+ SO ad14 prol* pabal y b i l f ; wS+ n i 9 ; s m Sudpro SO+; methl+; nit%+ ribol ad14 p r o l f pabal+ y+ b i l ; sm S u 4 p r o f s u l a d d 0 adl4+ prol+ pabal+ y+ add0 b i l ; A c r l + sm+ phend+ Su4pro+ panto so+; pyro4 prol prod+ pabal y bil+; Acrl+; sm phen2+ SO+ S u l p r o + ribol pabal y bil+; thi4; methl pyro4 o r n p prol pabal y b i l ; A c r l wS+;methl pyro4+ pabal y ad20 bil+; A c r l * w3 thi4+; pyro4+ orn4+; ribod pabal y bil+; pyr04 pyrol+ prol y bil+; A c r l wd+; phen2+; pyro4; lys5+ nicb; sS+ prol y bil+; AcrS+adl+; SO+; pyro4+; nic2+; s3 lysl+ pabal y bil+; A c r l * wS+;ribod+ co add0 bil+; A c r l + wd;phend+; pyro4; lys5+; s3+; nic8+; Bw

ACKNOWLEDGMENTS This work was part of a program supported by the Nuffield Foundation, and most of the investigations were undertaken while the author held a fellowship in the Department of Genetics a t the University of Glasgow. Thanks are due to Prof. G. Pontecorvo, head of the Department, for his stimulating criticism, and to various associates for permission t o use unpublished data. The assistance of Mr. J. Hutchinson with some of the experiments is also acknowledged.

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The study was continued while the writer was a guest of the Long Island Biological Association a t Cold Spring Harbor and completed a t the Department of Genetics, McGill University, Montreal. The provision of facilities at these two institutions by Dr. M. Demerec and Prof. J. W. Boyes is gratefully acknowledged. VIII. REFERENCES Barratt, R. W., Newmeyer, D., Perkins, D. D., and Garnjobst, L., 1954. Map construction in Neurospora crassa. Advances in Genet. 6, 1-93. Calef, E., 1957. Effect on linkage maps of selection of cross-overs between closely linked markers. Heredity 11, 265-279. Elliott, C. G., 1956. Chromosomes in micro-organisms. Symposium SOC.Gen. Microbiol. 6, 279-295. Eversole, R. A., 1956. Biochemical mutants of Chlamydomonas reinhardi. Am. J. Botany 43, 404-407. Forbes, E. C., 1956. Recombination in the pro region in Aspergillus nidulans. Microbial Genetics Bull. No. 13, 9-11. Hemmons, L. M., Pontecorvo, G., and Bufton, A. W. J., 1953. Perithecium analysis and relative heterothallism. Advances in Genet. 6, 194-201. Hockenhull, D. J. D., 1949. The sulfur metabolism of mould fungi: The use of “biochemical mutant” strains of Aspergillus nidulans in elucidating the biosynthesis of cystine. Biochim. el Biophys. Acta 3, 326-335. Luria, S. E., and Delbriick, M., 1943. Mutations from virus sensitivity to virus resistance. Genetics 28, 491-511. Macdonald, K. D., and Pontecorvo, G., 1953. “Starvation” technique. Advances in Genet. 6, 159-170. Pontecorvo, G., 1952. Genetical analysis of cell organization. Symposia SOC.Ezptl. Biol. 6, 218-229. Pontecorvo, G., 1953. The genetics of Aspergillus nidulans. Advances in Genel. 6, 141-238. Pontecorvo, G., 1954. Mitotic recombination in the genetic systems of filamentous fungi. Caryologia Suppl. 6, 192-200. Pontecorvo, G., 1956. The parasexual cycle in fungi. Ann. Rev. Microbiol. 10, 393-400. Pontecorvo, G., and Klifer, E., 1956. Mapping the chromosomes by means of mitotic recombination. Proc. Roy. phys. SOC.(Edinburgh) 26, 16-20. Pontecorvo, G., and Kafer, E., 1957. Genetic analysis based on mitotic recombination. Advances in Genet. 9, 71-104. Pontecorvo, G., Tarr-Gloor, E., and Forbes, E. C., 1954. Analysis of mitotic recombination in Aspergillus nidulans. J. Genet. 62, 226-237. Pritchard, R. H., 1954. Ascospores with diploid nuclei in Aspergillus nidulans. Caryologia Suppl. 6, 1117. Pritchard, R. H., 1955. The linear arrangement of a series of alleles of Aspergillus nidulans. Heredity 9, 343-371. &per, J. A., 1950. Search for linkage between genes determining a vitamin requirement. Nature 166, 956-957. Roper, J. A., 1952. Production of heterozygous diploids in filamentous fungi. Ezperientia 8, 14-18. Roper, J. A., 1953. Pseudo-allelism. Advances in Genet. 6, 208-215.

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Roper, J. A., and Kkfer, E., 1957. Acriflavine resistant mutants in Aspergillus. J. Gen. Microbiol. 16, 660-667. Roper, J. A., and Pritchard, R. H., 1955. The recovery of the complementary products of mitotic crossing over. Nature 176, 639. Sneath, P. H. A., 1955. Putrescine as an essential growth factor for a mutant of Aspergillus nidulans. Nature 176, 818. Stadler, D. R., 1956. A map of linkage group VI of Neurospora crassa. Genetics 41, 528-543. Stern, C . , 1936. Somatic crossing-over and segregation in Drosophila melanogaster. Genetics 21, 625-730.

THE INVIABILITY, WEAKNESS, AND STERILITY O F INTERSPECIFIC HYBRIDS G. Ledyard Stebbins University of California, Davis, California

Page ........................................... ty and Weakness.. . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Incompatibility between Parental Chromosomes and Genes.. . . . . . . . . 149

IV. V. VI. VII. VIII. IX. X. XI. XII.

1. Diplontic Genic Sterility.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Diplontic Sterility Due to Disharmony between Genome and Cytoplasm 3. Haplontic Sterility.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Hybrid Breakdown .............................. Sterility, and Sex.. . . . . . . . . . . . . . Relation between Inc enetic Basis of Weakness and Sterility Segregation for Fer Relation between Morphological Species Differences and Interspecific Barriers of Reproductive Isolation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Isolating Barriers and the Systematic Position of the Species.. . . . . . . The Occurrence of Inviability and Sterility Barriers within Species.. . . . . . The Origin of Hybrid Inviability, Weakness, and Sterility.. . . . . . . . . . . . . Conclusion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ............ References ................................................

168 173 174 183 184 188 190 203 203

I. INTRODUCTION The problems connected with the genetics of interspecific hybrids have received increasing attention in recent years, particularly by students investigating the dynamic phase of organic evolution. Foremost among these problems are the ones connected with the development of barriers of reproductive isolation, which prevent or restrict gene exchange between Mendelian populations. With the widespread acceptance of this process as “ t h e essence of the process of speciation” (Dobzhansky, 1951, p. 263), both the species as a biological unit and the nature of these barriers have acquired increased significance. When two groups of organisms have developed a certain degree of reproductive isolation from each other, they can thereafter exist side by side in the same region, exploiting their environment in different ways. This makes possible and even stimulates continuing divergent evolution, and is responsible for most of the diversity of organic life which is the outcome of evolution. The develop147

148

Q. LEDYARD STEBBINS

ment of reproductive isolation is, therefore, one of the most important if not the most important step in evolution. Reproductive isolation may be achieved by a great variety of different mechanisms, acting a t various stages in the life histories of the organisms concerned. The most concise classification of these phenomena is that of Dobzhansky (1951, p. 181).The present review is restricted to that group of interrelated mechanisms designated by Dobzhansky as Hybrid Inviability, Hybrid Sterility, and Hybrid Breakdown. This includes all mechanisms which act after the parental gametes have united in fertilization, i.e., during the development, growth, and reproduction of the F1 hybrids and their progeny of later generations. It does not include barriers which prevent the uniting of the parental gametes, such as Sexual and Mechanical Isolation. the various barriers to the uniting of sperm and egg nuclei in animals, and the growth of pollen tubes and the penetration of pollen tube nuclei in plants. Although the subject of hybrid inviability and sterility has been treated in a number of review papers in recent years, most of these have confined their attention to particular groups of animals or plants, or at least to only one of the two major kingdoms of organisms. Important reviews of the phenomena in animals are those of Hertwig (1936) and Muller (1942), while the evidence from plants has been reviewed by Itenner (1929) and the present writer (Stebbins, 1942, 1945, 1950). The most recent comprehensive review covering both kingdoms is that of Dobzhansky (1951). The purpose of the present review is to present all of the available evidence bearing on theories concerning the way in which these isolating mechanisms originate. For this reason, the descriptive account of the different types of barriers is followed by a discussion of their genetic basis and their relationship to other differences between species. The final section, dealing with theories concerning the origin of species, is based chiefly on the factual information reviewed in the previous sections.

11. HYBRIDINVIABILITY AND WEAKNESS Under this heading are included all of those mechanisms which prevent or retard the development of hybrids from the first division of the zygote up to the final differentiation of the reproductive organs and the gametes or spores which they produce. These mechanisms act a t various stages of development and to varying degrees. Growth and development may ‘be totally inhibited, so that the hybrid dies before reaching adulthood, or the hybrid may be variously retarded or weakened, so that it can be raised to the adult condition under artificial culture, but would surely perish before reaching maturity under natural conditions. The latter

HYBRID INVIABILITY, WEAKNESS, AND STERILITY

149

condition provides as effective a barrier to gene exchange under natural conditions as the former, and so is equally important. The immediate causes of hybrid weakness or degeneration may be grouped into three categories, as follows. First, the disharmony may reside entirely or chiefly in the chromosomes and genes of the two parental species, as they are combined in the hybrid nuclei. Second, there may be a disharmonious interaction between the chromosomes or genes of one species and the cytoplasm of the other, as it has been contributed by the egg of the maternal parent. Third, the hybrid embryo may be perfectly capable of developing so far as its own constitution is concerned, but may be inhibited by the action of the maternal tissue which surrounds it, or in higher plants by the endosperm which nourishes it. These three situations will be discussed in turn.

1. Incompatibility between Parental Chromosomes and Genes Inviability of hybrids due t o incompatibility between parental chromosomes and genes is probably the commonest and the most significant toward a n understanding of evolutionary divergence. If this type of incompatibility is present, reciprocal hybrids between two parents are equally weak or inviable, and the removal of the embryo from its maternal surroundings does not help it to develop. For most examples of incompatibility of this nature, the genetic basis is not known, but there are a few examples of specific genes which cause inviability or abnormality in interspecific hybrids. a. Incompatibility of a General Nature. Most examples of hybrid inviability and weakness which belong in this category have been attributed t o disharmonious interactions between the parental chromosomes. This disharmony is most clearly evident in the first cleavage divisions of eggs of animal species which have been fertilized by sperm belonging to widely different genera. The extensive literature on this subject, which deals particularly with echinoderms and fishes, has been reviewed by Hertwig (1936). I n these forms, elimination of chromosomes commonly occurs during the cleavage mitoses, and further development is arrested. I n extreme examples, such as Parechinus X Arbacia, all of the paternal chromosomes are eliminated, so that no effective union of gametes occurs. I n hybrids between more closely related forms, no abnormalities are evident in the earliest mitoses of the zygote and young embryo, but they occur a t various later stages in development. I n both animals and plants, the time of degeneration of the embryo most often coincides with some critical or maximal period of differentiation of its tissues. I n animals, the most extensive evidence for this generalization has been obtained from amphibia. I n these forms, interspecific or intergeneric hybrids often

150

G. LEDYARD STEBBINS

degenerate a t the time of gastrulation, and the degeneration is most evident in that part of the gastrula in which the differentiation of the primary tissue layers is taking place. This has been shown for Triton cristatus X T . palmatus by Pariser (1932), for T . palmatus X Salamandra maculosa by Schonmann (1938), and for Rana pipiens X R. sylvatica by Moore (1947). I n recording the results of 41 hybrid combinations between different North American species of Rana, Moore (1949) reported 7 combinations in which no cleavage occurred, 23 which failed at the beginning of gastrulation, 3 which failed during the neurula and tailbud stages, and 8 which developed normally to adults. I n the fish hybrid Coregonus baeri X Salmo fario, Rubaschev (1935) found th a t abnormalities which lead to degeneration begin in the tissues which are ‘differentiating at the time of gastrulation, although most of the embryos develop considerably beyond this stage. I n plants, the relatively few studies which have been made of embryology in inviable hybrids point in the same direction. Brieger (1928), studying hybrids of Nicotiana, found th at breakdown of the embryo occurs most often a t the stage when the vegetative growing point is being differentiated. McCray (1933), also working with Nicotiana hybrids, recognized three different stages at which the embryo broke down. These were (1) the 4-%celled stages of the early embryo (in hr.rustica X glauca and N . rustica X longiflora), (2) the stage of differentiation of the vegetative growing point (in N . rustica X “rusbyi” and N . rustica X palmeri) and (3) the stage of seed germination (in N . nudicaulis X tabacum, N . suaveolens X tabacum, N . tabacum X glauca, and N . paniculata X glauca). Each of these three stages is a critical one in the development of the embryo. To pass through the first, the chromosomes of the hybrids must be able t o work together in the process of mitosis and in the synthesis of new cytoplasmic and nuclear components. Passage through the second depends upon the ability of the cells to form a n apical meristem with its precisely arranged and integrated growth centers, upon the integration of which depends the entire vegetative growth and organization of the plant. Successful development of the mature embryo into the seedling and the consequent germination of the seed depends upon the ability of the chromosomes and genes present in the hybrid to work together and construct a n efficient mechanism for photosynthesis and for the elaboration of protoplasm from the immediate products of photosynthesis. Observations and experiments with embryos of amphibians, chiefly the work of Baltzer (1940a,b) and his students, have shown th a t the degeneration of the embryo at a critical stage is due not to the complete inability of the hybrid nuclei to progress beyond this stage, but to their inability t o carry through certain particular processes of differentiation.

HYBRID INVIABILITY, WEAKNESS, AND STERILITY

151

Two types of hybrids have been used in these studies; sexual hybrids resulting from normal fertilization, and merogonic hybrids, in which the egg nucleus has been destroyed and replaced by a haploid nucleus derived from the sperm. The results of both types of experiments are in general similar, although the merogonic hybrids develop more poorly than the diploid ones. The experiments with merogonic hybrids were accompanied by control experiments in which the egg nucleus was destroyed and replaced by a sperm nucleus belonging to its own species. Since such intraspecific merogonic embryos develop normally, the abnormalities of the interspecific merogonic hybrids can be attributed to the absence of nuclear material belong to the maternal species. I n the merogonic Triturus (“Triton”) palmatus ( 9 ) X cristatus ( 3 )whole embryos can never be raise‘d past the stage of gastrulation. But Hadorn (1937) and De Roche (1937, cf. Baltzer, 1940a) have shown that tissue from such embryos, excised before degeneration sets in, will develop further when cultured in an artificial medium, and even better when implanted to the developing embryo of a pure species, either T. palmatus or T . alpestris. The most important fact is th at implants of different organs or regions of the merogonic embryo have very different capabilities for development. The heart, notochord, digestive tube, and pronephros develop normally as implants; the head mesoderm does not develop at all. Such structures as the gills and neural tube develop to some extent, and fail later on either because they fail to receive a necessary stimulus from the inductive region of the head mesoderm, or because of other difficulties which affect their later development. Baltzer (1940a) concluded that the development of the successful organs cannot be explained on the basis of plasmatic inheritance, and th at the explanation of predetermination through the action of substances already present in the egg is very unlikely. Most probably, therefore, the organs which develop successfully are those in which the genes of T . cristatus can successfully replace those of T . palmatus in activating and organizing the cytoplasm of T. palmatus, and in the development of the unsuccessful organs the genes of T . cristatus act in such different ways from those of T . palmatus th a t they are ineffective in the cytoplasm of the latter species. I n the case of these interspecific combinations of Triturus, the diploid hybrid, containing a hybrid nucleus in the cytoplasm of T. palmatus, develops normally at least to the late larval stage. For a n understanding of hybrid inviability, therefore, it is very important to compare them with the intergeneric combination Triton palmatus 9 X Salamandra maculosa 3,of which the diploid hybrid fails to develop beyond the gastrula stage (Schonmann, 1938). These hybrid embryos, moreover, are characterized by marked cytological disturbances, particularly

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stickiness, lagging, and fragmentation of the chromosomes. I n the merogonic hybrid, these abnormalities are even more marked, and the embryo fails to develop beyond the blastula stage (Boehringer, 1938). Successful implantations of merogonic tissues have not been made, but Luthi (1938) has made very significant observations on the behavior of tissues and organs of the diploid hybrid implanted into embryos of T. palmatus. All of those implanted, including epidermis from various parts of the body, brain tissue, pericardium, notochord, head mesenchyme, and muscle fiber, developed normally in the host. At the time of death of the whole embryo, many apparently healthy cells were observed. Whether further development was confined to these cells, or whether some of the partly degenerating cells recovered through the influence of the host, was not certain. But a t any rate, the results seem to indicate that the hybrid nucleus is capable of carrying through a large number of the developmental processes necessary for producing an adult animal, and that the early death of the embryo is due to inability to perform certain specific functions concerned with late blastula development and gastrulation. Based on these results, Baltzer (1940) has suggested that we must lay aside the general concept of lethality. I n his opinion, lethality affects primarily only separate organs or embryonic regions, while the remaining regions degenerate only secondarily, or not a t all. This he considers to be true of both the action of incompatible nuclei or nucleoplasmatic relationships in hybrids and the action of homozygous lethal genes within a species. Hadorn (1951) has recently reviewed the evidence from lethal genes in Drosophila which points toward phase-and perhaps functionspecificity for most if not all of them. Judging from the similarity between the developmental-physiological studies of homozygous lethals and those of inviable hybrids, a plausible hypothesis is that lethality due to the action of homozygous genes or deficiencies and hybrid inviability are essentially similar phenomena from the developmental-physiological point of view. Both appear to be due to the inability of certain genes, chromosomal deficiencies, or gene combinations to carry out specific metabolic processes or chemical syntheses necessary for development. This hypothesis is supported by additional evidence from several different hybrids. Moore (1947-1948) studied the competence of the gastrula ectoderm and the dorsal lip region of Rana pipiens X Rana syluatica hybrids by means of transplant studies. This hybrid normally dies at or before gastrulation. Tissues of the above mentioned regions, transplanted to R. palustris embryos, showed much less competence than did similar transplants taken from normal embryos of R. pipiens. Since haploid embryos, containing only one set of R. pipiens chromosomes in

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cytoplasm of this species, develop normally, Moore attributes the failure of hybrid development to inhibitory action of genes from R. sylvatica. Specifically, he suggests that the foreign (sylvatica) genes compete with the pipiens genes for the utilization of some substrate, combining with this substrate t o form analogous substances which do not result in full competence of the tissue. Pariser (1935, 1936) and Hamburger (1935, 1936), studying hybrids of Triton, noted specific stages of larval development at which mortality is particularly high, and at which abnormalities occur. The most conspicuous of these is at the formation of the hind limbs, particularly the toes, and abnormalities of these structures are particularly common in the hybrids. Bystinski-Salz (1933) found th a t females of the Lepidopteran hybrid Celerio gallii 8 X euphorbiae 0 fail to develop, but th a t implants of wing or ovary primordia develop normally in parental hosts, just as do implants of inviable amphibian hybrids. b. Inviability Due to the Action of SpeciJic Genes and Chromosomes. The examples of hybrid inviability cited in the previous section cannot be analyzed genetically, but are probably due to the interaction of many different factors. There are, however, a few examples of inviability or weakness due t o the action of single genes. The best known of these is the interspecific lethal described by Hollingshead (1930) in Crepis tectorum, producing lethality in hybrids with C. capillaris, but similar examples have been described b y Brieger (1929) in Nicotiana 1ongiJEora X sanderae, by Melchers (1939) in Hutchinsia alpina X brevicaulis, by Hutchinson (1932), Silow (1941), and Gerstel (1954) in Gossypium, by Lein (1943) in wheat, affecting crossability with rye, by Sears (1944) in Triticum monococcum, affecting crossability with Aegilops and Haynaldia, and by Saunders (1952) in Vigna. Although the mode of action of the gene is not known in any of these examples, Sears has suggested that the factors in T . monococcum act through the failure to provide the embryo with some substance essential for growth. I n Crepis, Nicotiana, Gossypium, and Triticum the species concerned are well isolated from each other by barriers other than the Mendelian factors analyzed. I n Hutchinsia and Vigna the inviability factors occur only in a few strains of the species concerned, and do not form part of an interspecific barrier. I n no instance, therefore, are specific genes identified which have contributed to a n effectively operating barrier of hybrid inviability. An unusual but effective type of isolation barrier resulting from gene interaction is the formation of tumors in hybrids. The best known example is the gene X p in Platypoecilus maculatus (Kosswig, 1929; Gordon, 1931, 1941; Gordon and Smith, 1938). I n the genic background of its own species, the X p gene of P. maculatus gives a characteristic pattern

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of spotting due to the development of macromelanophores in specific regions. This pattern is essentially similar in homozygotes and in S p s p heterozygotes. I n crosses with Xiphophorus helleri, however, the F1 hybrids, with the constitution Spsp, form melanic tumors in place of spots. These tumors, though they never cause death, interfere seriously with the activity of the fish, and often cause such abnormalities as complete absence of the tail fins. Backcrosses to Xiphophorus result in complex segregation for intensity of the tumor development, with some individuals having more strongly developed tumors than the PIhybrids. This indicates that X. h.elleri possesses a series of genes modifying the action of S p so as to produce the abnormalities. In hybrids between P. maculatus and other “species” of Platypoecilus the tumor formation occurs in varying degrees, the greatest development being in hybrids between forms of which the geographic range is farthest removed from that of P. maculatus (Gordon and Smith, 1938). Tumor formation also occurs in hybrids of Nicotiana (Whitaker, 1934; Brieger and Forster, 1942; Kostoff, 1943; Kehr, 1951; Kehr and Smith, 1954). They appear after the end of meristematic activity, and may affect all parts of the plant, including the roots. They are not associated with any pathogenic organisms, or with conspicuous disturbances of mitosis, and early reports of reciprocal differences in their occurrence, indicating an influence of the cytoplasm, were not confirmed by later work (Kehr, 1951). In N . langsdorfii X glauca, where they are particularly strongly developed, Kehr and Smith (1954) were unable to locate any one gene or chromosome derived from N . glauca and for their occurrence, but their evidence from progeny of the sesquidiploid hybrid (langsdorfiiX glauca 4n) X langsdorfii (LAG), which develops tumors, suggests that a certain number of glauca chromosomes added to the genotype of langsdorfii may induce tumor formation. Kehr (1951) suggested that the tumors are caused by abnormal phytohormone relationships, a hypothesis apparently supported by the higher content of free auxin in the N . glaucalangsdorfii amphidiploid as compared to the parental species (Kehr and Smith, 1954). I n the genus Gossypium, Stephens (1946, 1950b) has demonstrated the existence of three different gene complexes which in certain heterozygous combinations give phenotypes with conspicuous growth disturbances. Two of these, “corky” and “crinkled,” are on a single chromosome in the D genome of the tetraploid New World species G. barbadense and G. hirsutum, and may be pseudoallelic. They act as an effective isolating mechanism between certain strains of these species which are sometimes cultivated together. The third complex, that of “crumpled” in the A genome of the diploid Asiatic cottons, has not developed into an isolat-

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ing mechanism. I n view of the recent results on Nicotiana, the suggestion tha t the action of these genes causes an abnormal unbalance of the hormonal system of the plant seems reasonable. I n Datura, Cole (1956) found that the ability of D. stramonium to form hybrids with other species, particularly D. ceratocaula, was greatly influenced by the trisomic condition for various chromosomes and chromosome segments. I n certain individual characteristics affecting hybrid formation, such as the percentage of pollen germination on the stigma, the rate of pollen tube growth through the styles, and the percentage of seeds and embryos produced, several of the trisomics were superior to the diploid controls when pollinated with one or the other of two different strains of D. ceratocaula. On the other hand, superiority in one of these characteristics was not usually associated with superiority in the others; none of the trisomics exceeded the controls in all of the characteristics contributing to hybrid formation. Furthermore, the trisomics reacted differently to two different strains of D. ceratocaula used as pollen parents. I n addition a strong environmental effect on crossability was noted. The secondary trisomic 17.17 (‘Ldwarfll),which was the most successful of the trisomics in forming embrxos with D. ceratocaula pollen, gave better results when the plants were growing poorly in a pot bound condition. These results show that successful pollination, fertilization, and embryo development in crosses within a species depend upon a delicate balance between the action of many different genes. The upset of this balance which results from interspecific pollinations can be corrected in certain respects by changing the chromosomal and genic balance of one of the parental species, but the establishment of a new balanced condition is very difficult. 2. Incompatibility Involving Cytoplasmic and Plastid Diflerences

I n some crosses between plant species, there are marked reciprocal differences in the success of the F1 hybrid. The most intensively studied examples of this type are in the genus Epilobium, and are thoroughly reviewed by Michaelis (1954) in a recent volume of Advances in Genetics. If the cross Epilobium hirsutum is made with E. luteum as the female parent, the F1 hybrid is vigorous, normal in all of its parts, and partly fertile, while the F1 of the reciprocal cross is abnormal in development, and has abortive, completely sterile flowers. By repeated backcrossing of the vigorous E. luteum X hirsutum F1 with E . hirsutum, Michaelis produced several true breeding lines which had all of the morphological characteristics of E . hirsutum, combined with the crossing behavior, and therefore the cytoplasmic characteristics of E. luteum. Since the cytoplasm retained the characteristics of E . luteum even after 24 generations

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of backcrossing with E. hirsutum, Michaelis concluded that these characteristics are determined by self-reproducing genes residing in the cytoplasm. The genetic behavior of any strain of Epilobium is determined by interaction between its nuclear genes and its cytoplasmic genes. These cytoplasmic inhibitory effects, though obviously of great interest in connection with the problem of cytoplasmic inheritance, are not very effective as interspecific isolating mechanisms, since gene exchange can take place via the reciprocal hybrid unless other barriers are present. In Epilobium, furthermore, the phenomenon is only partly correlated with the pattern of speciation in the genus. The cytoplasmic inhibitory effect does not get progressively greater as the species are more distantly related. Rather, it is the property of a particular group of species belonging to the European section Eriophora. Furthermore, there is considerable differentiation of the cytoplasm within the species E. parvijlorum and E. hirsutum. Not only do different strains of the same species differ in the degree of inhibition of their hybrids with E. Euteum and other species, but in addition typical dwarf, sterile plants result from crosses between certain races of E. hirsutum. The type of abnormality is apparently specific to the race used as the female parent, but the degree.of its expression varies greatly with the male parent used. I n addition to these abnormalities of growth, pollen sterility exists to a greater or lesser degree in these hybrids, and is effective whichever way the cross is made. This nuclear determined pollen sterility, with an accompanying and similarly determined seed sterility, may have a greater general significance as a reproductive isolating mechanism in the genus Epilobium than the more spectacular abnormalities determined by the cytoplasm. I n the genus Oenothera, reciprocal differences have long been known to be associated with the failure of the chloroplasts to develop normally. This subject has been well reviewed by Renner (1929), and no results have been obtained since then which would alter his conclusions. This type of barrier, also, is restricted to a relatively small group of species; it does not appear to have played an important role in speciation in other sections of the genus (Hecht, 1950). Reciprocal differences in the vigor of F1 hybrids of a less conspicuous nature have been noted by Grant (1956) in certain interspecific hybridizations of Gilia, but these contribute relatively little to the isolating barriers in this genus. Reciprocal differences in the degree of inhibition or abnormality of animal hybrids appear to be due principally to interaction between chromosomes from the sperm and the large amount of cytoplasm in the eggs. Differences of this sort are common in Echinoderm crosses (see review by Hertwig, 1936). I n many of these, and more strikingly in the am-

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phibian cross Bufo viridis X vulgaris (Montalenti, 1933), the development of the hybrid is normal when the egg belongs to a species with a slow developmental rhythm, and the sperm to a species with a relatively rapid rhythm, and abnormal in the reverse situation. There are, however, exceptions to this rule, as Hertwig (1936) and others have pointed out. I n Culex, Laven (1953, 1956) has demonstrated a clear example of hybrid inviability due to plasmagenes. When a strain of C. pipiens from southern Germany is used as male parent in crosses with strains from northern Germany and various parts of western Europe, viable hybrids are obtained, but if the South German strain is the female parent, the cross fails. Laven backcrossed the (North Q X South 3 )F1 hybrid with South males, and repeated the backcross for 44 generations, after which time he assumed that the nucleus was entirely of the South German type in genetic constitution. Nevertheless, when males of this backcross strain were used to fertilize South German females, the same inviability was found as in typical (South Q x North 3 ) hybridizations. The only explanation of this result is that the cytoplasm of the backcross flies retained plasmagenes derived from the cytoplasm of the original North German female, and that these plasmagenes influenced the behavior of their spermatoza. Since reciprocal differences in crossability have been found several times in mosquitoes (Downs and Baker, 1949; Bonnet, 1950; Perry, 1950) plasmagenes of this sort may be widespread in the group.

3. Incompatibility between the Embryo and the Surrounding Tissue I n the higher plants, hybrid inviability is often due to causes other than incompatibility between the parental chromosomes as they affect the development of the embryo itself. This fact was discovered long ago by Laibach (1925), who found that in the cross between Linum austriacum and L. perenne, viable seeds are never formed, but hybrid plants can be produced by dissecting the embryos from the seeds and culturing them in vitro. The hybrids thus produced were found by Laibach to be both vigorous and fertile. The production of hybrids by embryo culture from otherwise incompatible matings has now been carried out in a number of genera. The most extensive work in this field has been done by Blakeslee and his associates (Blakeslee, 1945; Blakeslee and Satina, 1944), in the genus Datura. They found that young embryos, as small as 0.1 mm. in length could be cultured by adding malt extract to the medium. Furthermore certain embryos produced seedlings containing shoots but no roots, and these could be induced to grow by grafting them on to young, vigorous plants of D. stramonium. Using these methods, McLean (1946) was able to obtain seedlings from 12 out of 18 interspecific hybrid com-

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binations involving D . ceratocaula, whereas previously no hybrids involving this species had been produced. Since all of these F1 hybrids were highly sterile, the species concerned are separated by several different reproductive isolating mechanisms, as is usually the case with distantly related species of plants. Attempts to analyze the causes of this type of hybrid inviability have been carried out principally along two different lines; first, a comparison of the different degree of success found in reciprocal matings, and second, studies of fixed and stained preparations showing the development of embryo, endosperm, and ovular tissue following interspecific pollination, as compared t o the results of selfing or intraspecific pollinations. I n hybrids between different diploid species, there often is no marked difference in compatibility whichever way the cross is made. I n other instances, however, there are marked reciprocal differences. One of the most complete series of crosses involving such differences are those in Datura. McLean (1946) found that in combinations between D. ceratocaula and nine other species, eight gave better results when D . ceratocaula was the male parent, and only one, D. ceratocaula X metel, gave possibly, though not markedly better results with D. ceratocaula a s the female parent. I n six combinations, hybrids were obtained by embryo dissection with D . ceratocaula as the pollen parent, while the reciprocal combination gave no embryos a t all. Of the opposite type is D.inozia, which is more successful as the female parent in crosses with D . stramonium, D. discolor, D. metel, and D . ceratocaula, the only combinations recorded for it. The other species used in hybridization, D . stramonium, D. discolor, D . metel, and D. meteloides, are sometimes more successful as the male and sometimes as the female parent. Valentine (1953-1955) found a similar crossing behavior in three species of Primula, P. elatior, P. vulgaris, and P. veris. When P. elatior is the female parent, endosperm production is scanty and no seeds contain embryos, but with P. veris as the seed parent, up to 80% of seeds have endosperm, and up to 30% contain embryos. Primula vulgaris is more successful with P. elatior as a female parent, but with P. veris as a male parent; it crosses more easily with either P. elatior or P. veris than these species cross with each other. Valentine interpreted these results by assigning a numerical “genetic value” to each species. These values, 1 for P. elatior, 1.3 for P. vulgaris, and 2 for P. veris were so assigned th a t the ratio endosperm :embryo in the most compatible crosses approached the value 1.5 which exists in intraspecific pollinations. Later Valentine (1955) suggested that the interaction between endosperm and maternal tissues is more important than that between endosperm and embryo. More recent work on hybrids of Datura (Satina et al., 1950; Rappa-

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port et al., 1950) has established the presence of a substance in the proliferating endothelium of the incompatible crosses which inhibits the growth in vitro of selfed embryos of the parental species. This substance is water soluble, heat stable, and capable of self-duplication to some extent. The chemical nature of this substance, and the reason for its accumulation in the “tumorous ” endothelial tissue of the maternal parent, are not known. I n still other hybridizations of Datura (Sachet, 1948; Sanders, 1948), as also in Melilotus (Greenshields, 1954) the results are very irregular, and the causes for failure are obscure. These observations emphasize the need for more exact knowledge of the physiological causes of hybrid inviability. I n the more extensive series of hybridizations in Nicotiana involving species having the same chromosome number, as summarized by Kostoff (1943), reciprocal differences are often present, but in no instance is a particular species consistently more successful either as a male or a female parent. This inconsistency seems t o be the most common situation in crosses between species having the same chromosome number. Recent studies of chromosome behavior in hybrid endosperms have pointed toward one cause of their breakdown. Brock (1954, 1955) has found that chromosome breakage during mitosis is a prelude to degeneration, both in hybrids between Lilium species having the same chromosome number, and in hybrids between garden hyacinths which have different numbers, constituting an aneuploid series. I n the latter material, the presence of particular extra chromosomes had a greater effect than quantitative chromosomal unbalance per se. Rutishauser (1954) found tha t failure of seed development in hybrids between pseudogamous apomicts of the Ranunculus auricomus complex, as well as in crosses between these apomicts and a diploid, sexual form, was due to unbalanced conditions in the endosperm itself, since in these forms the embryo, which is produced by a diploid, unfertilized egg cell, has the same balanced chromosomal constitution possessed by the maternal tissue. This failure was accompanied by chromosome breakage in the endosperm. I n hybrids between Trillium and Paris (Rutishauser, 1955 ; Rutishauser and LaCour, 1956), chromosome breakage is found when Paris is the female parent, but not in the reciprocal hybrid. This breakage is confined to certain chromosomes of the Trillium complement, and is particularly frequent in distal regions of these chromosomes. The most logical explanation of this situation is th at either the hybrid cytoplasm or some conditions of metabolism in the hybrid cells exert a strong effect on Trillium chromosomes, but have little or no effect on those of Paris. Crosses between diploids and autotetraploids of the same species are

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usually difficult to make, and are often more successful when the tetraploid is the female parent. I n many instances, such as Campanula persicifolia, Primula sinensis, Solanum nigrum (Thompson, 1930), and Datura stramonium (Sansome et al., 1942), the tetraploid usually fails as a pollen parent because its pollen tubes burst in the styles of the diploid, but even when fertilization is successful in both directions, embryo and endosperm development of the hybrid may be better when in maternal tissue with the tetraploid chromosome number. Seven such examples of reciprocal hybridizations have been subjected to embryological studies, namely Galium mollugo (Fagerlind, 1937), Datura stramonium (Sansome et al., 1942), Lycopersicon pimpinellifoliurn (Cooper and Brink, 1945), Secale cereale (Hhkansson and Ellerstrom, 1950), Zea mays (Cooper, 195 1), Galeopsis pubescens (Hhkansson, 1952), and Hordeum uulgare (Hhkansson, 1953). I n Galium mollugo, endosperm development in the 2n X 4 n cross is very irregular, and in most ovules the endosperm eventually degenerates, after having produced nuclei with varying chromosome numbers. Cell wall formation is usually retarded. Embryo formation is also somewhat irregular, but in ovules with well-developed endosperm the embryo also develops nearly or quite normally, and triploid F1 hybrids can be produced from this cross. I n the 4 n X 2n cross, the endosperm grows more slowly than in 4 n X 4n, and always degenerates. Cell wall formation is precocious in the early stages of development. The embryo develops slowly but otherwise normally until the endosperm is exhausted, then degenerates. Hybrids never resulted from the cross 4 n X 2 n Galium mollugo. In Datura stramonium, pollen tube growth is normal in the 4 n X 2 n cross, and can be induced in the 2n X 4 n cross by using a female parent which is a periclinal chimera with an epidermal layer consisting of 4 n cells. Under these conditions, fertilization usually occurs in both crosses, and the proembryo grows to the 4-6 or sometimes the 8-celled stage, after which both endosperm and embryo usually degenerate. There is no noticeable difference in the success of the two reciprocal crosses, and the phenomena of degeneration resemble closely those found in incompatible crosses between different species having the same chromosome number. In Lycopersicon pimpinellifolium, there is likewise no difference in the degree of success of the 4 n X 2n as compared to the 2n X 4 n cross, and the mode of degeneration is the same in both instances. This begins with the failure of the endosperm nuclei to divide and develop normally, and is characterized by excessive growth or hyperplasia of the endothelial tissues, just as in the incompatible crosses in Datura. A noteworthy fact is that, in hybridizations with the distantly related diploid species L.

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peruvianum, the tetraploid L. pimpinellifolium functions better than the diploid of that species. The incompatibility due to differences in chromosome number within L. pimpinellifolium and that due to genic differences between this species and L. peruvianum are similar to each other in their effect on development, but are sufficiently different in their physiological action so that one effect can partly counteract the other. I n Secale cereale, endosperm nuclei in the 2n X 4n cross divide at first very rapidly, but abnormal mitoses soon appear. Cell walls do not form between the nuclei, and the endosperm finally degenerates, followed by degeneration of the embryo. I n the 4n X 2n cross, endosperm development is very slow, but otherwise more nearly normal than in 2n X 4n, and cell wall formation is precocious. The endosperm and embryo both degenerate eventually in most of the ovules, but survive in some instances. I n Zea mays, the endosperm in the 2n X 4n cross develops rapidly and precociously, but contrary to the situation in Secale, no abnormal mitoses occur, and cell wall formation is normal. The embryo begins growth normally, but is finally crowded out by the endosperm tissue, which continues to proliferate excessively. I n the 4n X 2n cross, the endosperm develops abnormally slowly, but development of the embryo is nearly normal, until it ceases, apparently because it has used up all of the food material provided by the endosperm. I n Galeopsis pubescens the rare fertilizations in the 2n X 4n crosses produce a vigorous initial development of endosperm, followed by degeneration, as in Secale and Hordeum, while the 4n X 2n fertilizations develop further. The endospermal haustorium which develops at the micropylar end of the embryo sac in normal 2n X 2n crosses becomes well developed in 2n X 4n, but not in the reciprocal. I n this example, also, the cause of seed abortion is probably the changed chromosome relations between endosperm and maternal tissue. A cross which belongs in this category, since i t is between an autotetraploid and a related and possibly ancestral diploid, is that between tetraploid Medicago sativa and diploid M . falcata (Ledingham, 1940). This combination resembles those of Galium, Secale, Hordeum, and Galeopsis in that endosperm development is precocious and abnormal in the 2n X 4n cross. The development of endosperm in the 4n X 2n cross was not described, but degeneration of both endosperm and embryo apparently occur as quickly and regularly as they do in the 2n X 4n cross. Of these six combinations between diploids and autotetraploids two, Lycopersicon and Datura, show no differences either in the degree or the nature of development, once fertilization has been accomplished, regardless of whether the diploid or the tetraploid is the female parent. I n all of the other combinations, the endosperm develops precociously in the

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2n X 4n cross, and is retarded in 4n X 2n. I n Galium, Secale, and Medicago, precocious endosperm development is accompanied by mitotic abnormalities and by retardation or failure of cell wall formation. I n every example, early development of the embryo is nearly or quite normal, and its later degeneration appears to depend upon failure of the endosperm. Less complete data on additional examples of crosses between diploids and autotetraploids of the same species agree with the descriptions given above. I n Gossypium arboreum, a predominantly self-fertilized species, Stephens (1942)found no significant difference in behavior of the 4n X 2n and 2n X 4n crosses; both were completely unsuccessful. I n the crossfertilized species Brassica oleracea Howard (1939)found that the 4n X 2n cross gives small but viable seeds, while its reciprocal gives only empty seeds. Nishiyama (1952; Nishiyama and Inamori, 1953) obtained the same result in both Brassica and Raphanus. I n the cross-fertilized species Populus tremula Johnsson (1945) found an unusually high degree of success in the 2n X 4n cross. The reciprocal cross was not available. Relatively few data are available on hybrids between allopolyploids and one of their parental diploids. One example is that of Lamium intermedium and its diploid ancestors, L. purpureum and L. amplexicaule (Bernstrom, 195313). In this case the 4n X 2n and 2n X 4n crosses were almost equally unsuccessful. On the other hand, the artificially produced autotetraploid of L. purpureum produced hybrids rather easily as the female parent with diploid L. amplexicaule, as a male parent with the allopolyploid L. intermedium, and in both directions with the allopolyploid L. hybridum, of which it is also one of the diploid ancestors. The autotetraploid form of L. amplexicaule produced many hybrids when crossed in both directions with L. intermedium. On the other hand the cross between L. amplexicaule and L. hybridum, which have no genomes in common with each other, succeeds in both directions when the diploid L. amplexicaule is used, i.e., when the forms differ in chromosome number, but fails if tetraploid L. amplexicaule is combined with the natural tetraploid L. hybridum. This series of crosses shows in striking fashion that success in crossing depends upon both quantitative and qualitative adjustment in the dosage of chromosomes and genes present. A similar example is that of hybrids between the amphiploid Gossypium hirsutum and its diploid ancestors (Weaver, 1955). The combination of G. arboreum with G. hirsutum is somewhat more successful when G. hirsutum is the female parent. I n both directions, however, endosperm growth is superior to that of the embryo, and in occasional ovules of G. hirsutum X arboreum in which no embryo is formed the endosperm grows continuously and vigorously until it fills the ovular cavity. In this example, therefore, there is no disharmonious interaction between the paren-

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tal chromosomes in the endosperm nuclei, or between endosperm and maternal tissue. The failure of embryo development is probably due either to incompatibility between parental genomes in the embryo itself, or to interaction between embryo and endosperm. Furthermore, if mixed pollinations are made so that some ovules are fertilized by pollen nuclei of G . hirsutum and others by G . arboreum, a larger percentage of hybrids develops than in pure cross-pollinations. This suggests th a t embryo and endosperm growth stimulate the development of maternal tissue. Crosses between related allopolyploids having different degrees of polyploidy have been extensively discussed (Watkins, 1927, 1932; Thompson, 1930; Muntzing, 1930, 1933; Kihara and Nishiyama, 1932; Boyes and Thompson, 1937; Walker, 1955; Beamish, 1955). I n these examples, endosperm and embryo always develop better when the higher polyploid is the ovulate parent. Rosa is not a n exception to this rule (Fagerlind, 1944, 1948) since 4n X 2n crosses in this genus fail because of lack of fertilization. The only extensive study of embryo and endosperm development in crosses of this type is that of Boyes and Thompson in Triticum. I n this genus, the behavior is similar t o that in most of the crosses between diploids and autotetraploids of cross-fertilized species. I n 6n X 4n and 4n X 2n crosses, endosperm development is retarded, but otherwise not very abnormal. I n the reciprocal combinations, early development of the endosperm is very rapid, but the mitoses are abnormal. The fact that this type of behavior is found in intraspecific 2n X 4n crosses of cross-fertilized species, and in interspecific crosses of polyploids of self-fertilized species, but apparently not in crosses between diploids and synthetic autotetraploids of self-fertilized species suggests that, as Watkins (1927) and Thompson (1930) have suggested, the reciprocal differences are due principally to differences in dosages of genes and genomes in the endosperm, and not merely to abnormal chromosome number relationships between embryo, endosperm, and maternal tissue. Further evidence in favor of the gene dosage hypothesis was obtained b y Nishiyama and Iizuka (1952) who secured two Fl hybrids by pollinating the diploid Avena strigosa with X-rayed pollen of the tetraploid A . barbata and the hexaploid A . sativa. The most plausible explanation of this result, which has never been obtained with normal pollen of the species concerned, is that the pollen grains which produced the viable F1 hybrids had suffered loss mutations of certain genes which contributed most strongly to the dosage unbalance in hybrids made with nonirradiated pollen. Finally, there is a series of crosses between species possessing different degrees of polyploidy, but distantly related t o each other, so th a t they have no genomes in common. The best known of these are in Nicotiana (East, 1935; Brink and Cooper, 1941; Kostoff, 1943; Poddubnaja-Arnoldi

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and Lodkina, 1945) and in certain cereals and related grasses. I n Nicotiana, crosses usually are more successful when the higher polyploid is the maternal parent, but there are several exceptions, particularly those involving N . paniculata. This species can function as a maternal parent in crosses with tetraploids, even though it is itself a diploid species. I n Dianthus Buell (1953) found that D. chinensis (an = 60) X D. plumarius ( 2 n = 90) is slightly less abnormal than its reciprocal, although neither combination produced hybrids. In Hordeum jubatum X Secale cereale (Cooper and Brink, 1944; Brink and Cooper, 1944; Brink et al., 1944), a tetraploid X diploid cross, the endosperm develops abnormally as in many 2n X 4n crosses, with much chromosome fragmentation. Similar abnormalities occur also in the antipodals. Since these cells carry only the maternal haploid genome, their abnormalities cannot be ascribed to genic interaction within the tissue itself. It is possible, however, that as in Datura the disharmonious interaction of genes in the endosperm, or between this and maternal tissue in some way generates substances with an inhibitory effect, and that these are directly responsible for the abnormalities. Embryo development is much more nearly normal in this cross, and hybrids can be obtained by embryo culture in vitro. I n Elymus virginicus X Agropyron repens, an incompatible 4n X 6n cross from which no hybrids have been obtained (Beaudry, 1951), the antipodals are likewise abnormal, and this abnormality is considered by Beaudry to be the most probable cause for the failure of the cross. I n the Gramineae and some other angiosperms) the antipodal cells multiply considerably before fertilization, and their nuclei become considerably enlarged immediately after this stage. They apparently aid in nutrition of the very young endosperm and embryo. Since this function of the antipodals is not found in most groups of higher plants, abnormalities of the antipodals cannot be considered as a general cause for failure of embryo development in crosses. I n grasses, abnormal functioning of the antipodals may be the immediate cause of breakdown, but these abnormalities are probably the result of genic disharmony of a more fundamental nature, which is similar to that in other plants, but differently expressed. The results of all of these studies of pseudo-incompatibility (Fagerlind, 1948) may be summarized as follows. I n higher plants, the successful production of an interspecific hybrid depends not only upon the ability of the genes of the parental species to work together harmoniously in embryo development, but also upon their ability to produce an endosperm capable of nourishing the young embryo, and on harmonious interaction between three tissues: embryo, endosperm, and maternal tissue. I n most crosses, the development of the embryo is more nearly normal than that

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of the endosperm, and failure of successful development after fertilization usually is the result of abnormalities in the endosperm, the maternal tissues, or the antipodals. Some of these abnormalities are probably due to disharmonious interactions between these three tissues, or more often between endosperm and maternal tissue alone, a phenomenon termed " somatoplastic sterility" by Cooper and Brink (1940), but others are very likely due to genic disharmony within the endosperm itself. The presence of genic disharmony in the endosperm but not in the embryo may be due largely to the fact that this tissue contains two sets of chromosomes derived from the maternal gamete, and only one from the paternal gamete. I n interspecific crosses, this inequality may cause an unbalance in the dosage of various alleles, which is not compensated for, as i t is in crosses between individuals of the same species. Reciprocal differences in the degree of pseudo-incompatibility may be due either to genic differences between the species concerned, or to differences in chromosome number. I n the latter case, the cross is usually more successful if the female parent has the higher chromosome number, and in crosses in which the female parent has the lower chromosome number, abnormalities of endosperm development are particularly prevalent. These abnormalities may be due in part t o disturbance of the quantitative relationship between the chromosome numbers of endosperm, maternal tissue, and embryo, but in most instances unbalance of gene dosage is probably responsible for some or all of the abnormality.

111. HYBRID STERILITY The fact t ha t species hybrids are usually sterile has been recognized from the beginnings of the scientific concepts of species. This sterility may occasionally be associated with the constitutional weakness of the F1hybrid, but more often it is not (Dobzhansky, 1951, p. 212). The exact basic causes of hybrid sterility are as yet only partly known, and are probably numerous, but all may be considered as special examples of the same kind of genetic unbalance which is responsible for hybrid inviability. If this unbalance affects primarily the metabolic and synthetic processes which take place in the early development of the zygote, embryo, and young organism, hybrid inviability results; but if the primary effect of unbalance is upon the gonads, the gametes, or the gametophytes, the result is hybrid sterility. Two sorts of classifications of the hybrid sterility phenomena have been proposed. Renner (1929) and Miintzing (1930) distinguished between haplontic or gametic sterility, which acts on the gametes or gametophytes; and diplontic sterility, which affects diploid tissue, either in the F1before or a t meiosis, or in certain zygotes and early embryos of the

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segregating Fz generation. These distinctions are, therefore, based on the stage at which genetic disharmony acts to produce the sterility. Dobzhansky (1951), on the other hand, separates genic from chromosomal sterility, based upon the type of genetic disharmony present. Genic sterility is typically due to the genetic constitution of the organism (Dobzhansky, 1951) and so is diplontic. It is possible, however, that disharmonious combinations of genes may be segregated to the gametes, and so produce haplontic genic sterility, as has been suggested by Oka (1952a,b) for intervarietal hybrids in the rice plant. This latter type of genic sterility is very difficult to distinguish from chromosomal sterility, and the possibility of its existence weakens both the practicality and the theoretical significance of the distinction made by Dobzhansky. As has been pointed out by Darlington (1937, pp. 198-199), Dobzhansky (1951, pp. 146-149) and the present author (Stebbins, 1950, pp. 218227), the only practical criterion for distinguishing between genic and chromosomal sterility is provided by the effects of doubling the chromosome number. Chromosomal sterility results from structural differences between chromosomes, which reduce their degree of homology and consequently their pairing, and which cause deficiency-duplication and other disharmonious combinations of chromosome segments to be distributed to the gametes. Doubling of the chromosome number, particularly in somatic tissue, results in the presence a t meiosis of a complete homolog for every chromosome in the organism. Pairing and segregation are therefore usually regular, and no chromosomal unbalance results. If the sterility is genic and diplontic, the unbalance in the somatic tissue of the diploid is retained in the corresponding tissue of the tetraploid, and the sterility persists. If, however, the sterility is genic and haplontic in action, or if it results from inviability of Fz zygotes, then the changed segregation ratios which take place in the tetraploid will automatically cause a rise in fertility. This point may be illustrated by a hypothetical example based upon the mechanism suggested by Oka (1952a) to explain intraspecific hybrid sterility in rice. He suggests that if two strains 1 and 2 differ from each other in respect to two pairs of complementary genes in such a way that strain 1 is A d l a z a z and strain 2 is alalA2A2, then their F1 heterozygote, AlalAzaz, can produce two types of gametes AIAz and a1a2,which are not formed by either parent. The results of certain intervarietal crosses made by Oka are best explained by assuming that gametes must carry either A1 or A2 t o be viable, so that the inviability of the u1u2gametes results in abortion of 25% of the pollen and embryo sacs. Although one set of complementary gene pairs of this nature has far too small an effect to form an effective interspecific barrier, a number of

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such sets, acting independently or reinforcing each other, could lower the fertility of a n F1 hybrid to a point which would effectively reduce gene exchange. I n an organism heterozygous for two such sets, AlalA2a2BlblB2b2, with the presence of A1 or A2 and B 1 or B2 necessary to produce a functional gamete, the percentage of viable gametes becomes (%)2, or 56.3 %. Each additional independently acting set (C1cIC2c2,etc.) lowers the percentage of viable gametes by the same amount, so that with n such sets the percentage of viable gametes becomes (N)”.For n = 10, 15, and 20 the values are respectively 5.6%, 1.3%, and 0.56%, which are not unusual figures for the fertility of F1hybrids between closely related species. Doubling the chromosome number in such a n F1 hybrid will greatly complicate segregation, which will follow the tetrasomic pattern for each group of four alleles, derived from a single allele pair in the diploid. If the same assumption is made in the tetraploid as in the diploid, namely tha t a gamete to be viable must contain a t least one dominant allele a t each locus, then the fertility of a hybrid heterozygous for one set of complementary alleles, i.e., A1AlalalA2A2aza2, becomes 35/36, or 97.3%, and for n sets of such factors the fertility is (35/36)”. With n = 10, n = 15, and n = 20 these figures are, respectively, 75.5%, 65.6%, and 56.9%, so tha t the genic sterility has been partly eliminated by the doubling process. As has recently been shown by Hall (1955), strong preferential pairing can have an even greater effect on fertility, but the fact remains th a t if hybrid sterility is haplontic in nature, the effects of chromosome doubling provide a relatively poor criterion of whether the sterility is genic or chromosomal. A further difficulty arises due to the fact that in organisms for which a large number of genetic markers is not available small chromosomal differences will apparently segregate in the same manner as genic differences. If, for instance, we postulate th at two genotypes, I and 11, differ by a single small nonreciprocal translocation so th a t a single particular vital chromosome segment, x, is located on chromosome pair A-A in genotype I, and on pair B-B in genotype 11, then we get the following result. Type I (A,A,BB) X Type I1 (AAB,B,) gives an FI hybrid of the condition A,A, B,B. Three of the four possible types of gametes produced by this hybrid (A,B,, A,B, AB,), contain x, and therefore are viable, while the fourth, AB, is inviable because of a deficiency for x. The effect of such a system is, therefore, exactly the same as that of the genetic factor scheme proposed by Oka, and there is no way of distinguishing between the two except through careful cytological analysis. Stadler (1954) in his recent discussion of the nature of the gene emphasized the importance of defining a phenomenon “in terms of the actual operations t ha t may be applied in dealing with it.” I n this sense, the

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separation of diplontic from haplontic sterility is clearly operational, while within the category of haplontic sterility, the distinction between a genic and a chromosomal basis cannot be made by means of any techniques now available for most hybrids. I n the present discussion, therefore, hybrid sterility of the diplontic type will be discussed separately from haplontic sterility. The former category contains most of the known examples of genic sterility, while the great majority of examples of haplontic sterility are probably chromosomal. Evidence for the latter conclusion will be presented below. 1. Diplontic Genic Sterility

This type of sterility is by far the commonest one in animals. The overwhelming majority of the barriers of reproductive isolation which separate closely related animal species consist of sexual isolation, genic sterility, or both of these phenomena. Genic sterility also occurs in many hybrids of plants, although here it is usually less common than chromosomal sterility, at least in hybrids between closely related species. When viewed from the standpoint of development, genic sterility is seen to be produced by a variety of genes with different types of action. They may interfere with the development of the reproductive parts a t any point, from the earliest differentiation of the gonads to the final stages of meiosis. By far the most frequent abnormalities, however, occur either during the differentiation of the various cells of the gonads or during the process of meiosis itself. I n the higher animals, breakdown during cell differentiation has been observed most frequently in the testes of hybrid males. Examples may be cited from various groups of vertebrates, such as Aphyosemion bivittatum X splendopleum's (Bozkurt, 1945) and hybrids of Coregonus (Shupakov and Kharchevko, 1954 ; Lemanov, 1955) among fishes; Triton cm'status X vulgaris (Benazzi and Lepori, 1949) and Hynobius nebulosus x naevius (Kawamura, 1952) among Amphibia; various combinations of Anatidae (Poll, 1910), Galliformes (Poll, 1910; Ghigi, 1934; Levi, 1937; Owen, 1941), and Ploceidae (Yamashina, 1940) among birds; Bos X Bison (Iwanoff, 1911) and Bos X Poephagus (Zuitin, 1930; Zawadowsky, 1931) among mammals. I n all instances in which the histological development has been described, the accessory cells of the gonads, such as Sertoli and nurse cells, develop normally, and abnormalities are confined to the seminiferous tubules themselves, particularly to the spermatogonia. Judging from the frequencies of abnormalities observed, the differentiation of spermatocytes from spermatogonia and of oocytes from oogonia must involve a series of critical metabolic processes which can easily be 'upset by genic disharmony. A significant fact is that the examples in amphibians

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and birds represent hybrid combinations which are difficult t o obtain. In some of them, such as Triton cristatus X vulgaris and Gallus domesticus X Numida meleagris (Owen, 1941), the majority of the embryos have been shown to degenerate a t an early age, and even the beginning of gonad differentiation takes place only in the few most successful embryos. This suggests that some of the same genic disharmonies which cause the failure of early embryonic differentiation may also contribute to the failure of differentiation of the cells of the gonads. Hybrid inviability and genic sterility should by no means be regarded as entirely separate phenomena; they may both depend on disturbances of the same metabolic processes. Breakdown of meiosis may be due to either genic or chromosomal sterility. When genic sterility is involved, the chromosomes often pair normally a t the early stages of meiosis, as in Pavo cristatus X Numida meleagris (Poll, 1920), Cairina moschata X A n a s platyryncha (according to Crew and Koller, 1936, but see Yamashina, 1941, for conflicting observations), various intergeneric hybrids of Phasianidue (Sandnes, 1954), Peromyscus truei X nasutus (Moree, 1946, 1948), in a subspecies hybrid of Drosophila pallidipennis (Patterson and Dobzhansky, 1945), and in males made t o develop testes by artificial hypophyseal injections in Triton cristatus X vulgaris (Benazzi and Lepori, 1949). Degeneration may set in immediately after pachytene, as in Pavo X Numida, Triton, and Peromyscus, or may follow after more or less normal first metaphase, a s in Cairina X Anus, the Phasianidae, Drosophila pallidipennis, and certain hybrids of Cobitis (Minamori, 1950, 1951). I n either case, the breakdown is most probably due partly to disharmonies in timing, involving the meiotic cycles of the chromosomes, spindle mechanism, and cytoplasm, and partly to the failure of the genes to promote the successful operation of metabolic processes necessary for meiosis. I n other instances, the breakdown may be manifest through reduced and irregular pairing of the chromosomes, particularly a t first metaphase. When this is the case, the decision as to whether the sterility is due t o the disharmonious interaction of genes or to structural differences in the chromosomes may be difficult to make. Additional evidence may be available, however, from various other phenomena. In Drosophila pseudoobscura X persimilis (Dobzhansky, 1934) reduced pairing is associated with abortion of the testes, and there is a direct correlation between the size of the testis as a whole and the degree to which meiosis approaches normality. Furthermore, occasional spermatocytes with the tetraploid complement of chromosomes showed no more pairing than did diploid spermatocytes, indicating th at pairing is prevented even when two sets of structurally identical chromosomes are present. I n interracial hybrids of Triturus (Callan and Spurway, 1951), a somewhat irregular meiosis is

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followed by a complete degeneration of spermatids, which is probably not a direct result of chromosomal unbalance in these cells. I n the higher plants, diplontic sterility is less common than haplontic sterility, and is nearly always associated with the latter phenomenon. Several examples are known of the abortion of anthers, the gynaecium or of all of the floral parts before meiosis is completed, as in Paeonia albiflora X anomala veitchii (Saunders and Stebbins, 1938) , Elymus condensatus X glaucus (Stebbins and Walters, 1949), some hybrids between races or microspecies of Elymus glaucus (Snyder, 1950), and certain interspecific hybrids of Geum (Gajewski, 1953b), and of the Luzula campestris-multiflora complex (Nordenskiold, 1956). These examples suggest that in plants as in animals the early developmental stages of the reproductive organs may be more easily upset by genic disharmony than are the farmative stages of other organs, but this difference is apparently less pronounced in plants. In plants as in animals, the male organs are more easily upset than the female ones. This is not clearly evident from the interspecific hybrids mentioned above, but is the only explanation of the fact that in several species of higher plants genes which cause male sterility are well known and not uncommon (Rick, 1948; Jones, 1954, 1956), while the writer knows of no good example of female sterility combined with male fertility except in dioecious species such as Melandrium, where this condition can be obtained by partial sex reversal. The best known example of genically determined female sterility is in Nicotiana sylvestris X tomentosiformis and related hybrids (Greenleaf, 1942; Ar-Rushdi, 1956), where it is combined with a high development of chromosomal sterility, so that it becomes evident only in artificial amphidiploids. A similar but less pronounced female sterility of this type was found by Davis (1955) in Allium cepa X Jistulosum. Boyes and Walker (1954) found that most of the sterility existing in Triticum-Agropyronamphiploids is associated with " fertilization-stasis," a term applied t o various abnormalities occurring a t the time of fertilization and immediately afterward. These are due to disharmonies existing between the genomes of the parental species, and undoubtedly contributed largely to the sterility of the undoubled Triticum X Agropyron hybrids from which the amphiploids were obtained. Diplontic sterility which is manifest through disturbances of meiosis is well known in plants as in animals, although it appears to be less strongly developed in the plant kingdom. The meiotic disturbances are of three types: failure of pairing a t diakinesis or I metaphase, i.e., asynapsis or desynapsis; lack of synchronization a t I anaphase or a t later stages of meiosis; and chromosome fragmentation. Failure of chromosomes to pair a t meiosis may be caused either by

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lack of structural homology, by lack of synchronization of the various metabolic processes which take place a t the early stages of meiosis, or by both of these conditions existing together. I n species hybrids of higher plants, lack of synchronization is rarely found in hybrids between species which are closely enough related so th at their chromosomes are completely homologous with each other. Consequently, genically conditioned synapsis is usually associated with partial lack of homology, and cannot be recognized by observation of meiosis in a diploid F1hybrid. If, however, an allopolyploid is produced from such a hybrid b y somatic doubling, two sets of completely homologous chromosomes are obtained, and failure of these chromosomes to pair must be genically determined. This condition was demonstrated by Sears (1941) and Bell and Sachs (1953) in amphidiploids of Aegilops species as well as of Aegilops X Triticum and Aegilops X Haynaldia, and by Pope and Love (1952), Bell and Sachs (1953), and Gaul (1954) in Triticum x Agropyron. Genically determined asynapsis can also be detected by comparing related F1 hybrids. I n Populus alba X grandidentutu, Pet0 (1938) obtained sister F1 plants having mean numbers of 0, 0.4, 1.6, 3.7, 16.2, and 34.4 univalents. Such differences could not possibly be due to variation in chromosome homology, and must have resulted from the fact th a t one or both parents were heterozygous for genes affecting meiosis in the interspecific hybrid. I n Geum, Gajewski (1953a) found th at partial asynapsis exists in the F1 hybrid G. rivale X macrophyllum, although both of the parental species form hybrids which have nearly complete chromosome pairing when crossed with either G. aleppicum or G. canadense. This indicates th a t the chromosomes of all four species are a t least partly homologous with each other. The situation in Geum is, however, complicated by the fact th a t in the amphidiploid G. macrophyllum-rivale meiosis is nearly regular and complete pairing of the 42 bivalents occurs in some cells. Gajewski explains this result b y assuming th at genic-physiological unbalance in timing of the meiotic prophase caused the asynapsis in the diploid, and that the effect of polyploidy on developmental processes was to restore their normal balance. The best evidence in favor of this hypothesis is that (1) nearly all F1hybrids involving G. macrophyllum and other species of Geum are asynaptic so th at the condition may be due to genetic factors exsiting in this species; (2) some of these hybrids have reduced vitality, disturbances in development, and abnormalities in their flowers; and (3) in FBand Fq generations from the Fz amphidiploid there was considerable segregation for fertility, with the more fertile segregates showing a higher degree of synapsis than in Fz. On the other hand, if the G . macrophyllum and G. rivale sets were completely homologous with

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each other, one would expect a t least occasionally the formation of multivalents in the amphidiploid, and these were apparently not observed. An alternate explanation would be to assume that G. macrophyllum is actually more distantly related to other species of Geum than they are to each other, and that part of the failure of pairing in its hybrids is due to structural differences in the chromosomes, producing partial homology. The genes affecting synapsis have a relatively large influence on such partly homologous chromosomes, but relatively little influence on pairing of the completely homologous chromosomes found in the amphidiploid. This example illustrates very well the point of view expressed later by Gajewski (1954), that failure of pairing in plant hybrids is often due to the combined effects of structural differences and genic unbalance. In the amphiploid from Crepis multijlora X zacintha (Bilquez, 1955) diplontic sterility is due to nonsynchronization of the parental chromosomes in the meiotic cycle. The chromosomes of C . zacintha contract more rapidly, reaching the diplotene condition when those of C . multijlora are still a t pachytene. The chromosomes of C . multijlora, on the other hand, are more rapid in anaphase separation. Genically determined asynapsis can also be detected by varying the environment of an F1 hybrid. Grant (1952a) found that F1 hybrids of Gilia millejoliata X achilleaefolia had a low degree of pairing when grown in sand under poor nutritional conditions, but much better synapsis when grown in fertile soil. The same conditions prevailed in the allopolyploid. Disturbances a t later stages of meiosis, not associated with previous genically controlled asynapsis, are uncommon in plants, but have been recorded in Agropyron trachycaulum x Hordeum brachyantherum ( I ' A . paucijorum X H . nodosum," Stebbins et al., 1946b), and in Lilium hybrids (Brock, 1954). Spontaneous chromosome breakage has been carefully studied in F1 hybrids of Bromus by Walters (1950, 1952). Since it has been found only in hybrids between distantly related species, it is associated with lack of chromosomal homology, and so is only a contributing factor to the sterility. I n the only allopolyploid obtained from a hybrid showing chromosome breakage in the F1, a normal meiotic balance was apparently restored, and no breakage was found. Although much of the failure of pairing not due to lack of homology is genically conditioned, the influence of the cytoplasm must not be overlooked. I n such genera as Epilobium (Michaelis, 1954) and Zea (Jones, 1951) cytoplasmically determined male sterility is known both in plants belonging to a species and in intervarietal or interspecific hybrids. I n F d progeny from the hybrid Paraixeris denticulata X Crepidiastrum platyphyllum, Ono (1951) found that some sterile individuals underwent a

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partial restoration of fertility due to cytoplasmic changes. The detection of cytoplasmically conditioned sterility depends chiefly on observations of reciprocal F, hybrids, which are not available in many instances. The effect of the cytoplasm on hybrid sterility needs further exploration. The careful and complete review by Gaul (1954) on genically controlled asynapsis has emphasized the fact that the physiologic-developmental unbalance which produces this phenomenon can be determined by the action of single genes or by multiple factors existing in one species, a s well as by the interaction of genes in an interspecific hybrid. I n both instances all degrees of development of the phenomenon exist, from slight reduction of the chiasma frequency to complete failure of pairing. Failure of metaphase pairing is often, though not always, preceded by more complete pairing a t pachytene, so t.hat the genes may sometimes act to suppress chiasma formation. I n most instances, however, they apparently disturb the timing relationships of the meiotic process. Although various hypotheses exist concerning the nature of this disturbance, which bring up such ooncepts as “precocity,” “retardation,” “effective splitting” of the chromosomes, and “ despiralization,” the actual fact is th a t we know far too little about the causes of the initiation of the meiotic or mitotic prophase, or of chromosome pairing, to judge the validity of these or any other hypotheses.

2. Diplontic Sterility Due to Disharmony between Genome and Cytoplasm Recent studies have shown th at diplontic sterility may in some instances be due to disharmonious interaction between the chromosomes of one parent and the cytoplasm of the other. Clayton (1950), by crossing Nicotiana debneyi X tabacum and making successive backcrosses of the hybrid with tabacum pollen, found increasingly greater pollen abortion as the proportion of tabacum genes in debneyi cytoplasm was increased. When these sterile backcross plants were pollinated by debneyi, the resulting offspring were completely fertile. Apparently the cytoplasm of N . debneyi inhibits the action of genes from N . tabacum controlling pollen production. A similar relationship exists between N . megalosiphon and N . tabacum. Kihara (1951) obtained similar results in crosses between Aegilops longissima and A . aucheri, as well as between Aegilops caudata and Triticum vulgare. I n addition, numerous examples are now known of cytoplasmic male sterility in races of a single species (Jones, 1951, 1954, 1956; Stephens and Holland, 1954). Several of these were obtained by crossing widely different varieties of the species. Jones has demonstrated the existence in maize of cytoplasmic determiners of male sterility, of fertility-restoring genes which counteract the effects of cytoplasmic male sterility, and of

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still other, completely independent genes which determine male sterility in the homozygous condition. Such a system of genic and cytoplasmic determiners of sterility could, by segregation of appropriate combinations in a single population, cause such a population to become partially isolated reproductively from other populations of its species, and so initiate speciation.

3. Haplontic Sterility Sterility which becomes apparent only a t the time of the formation of gametes or gametophytes, and is not associated with any disturbances of development or with any irregularities in meiosis other than those due to structural differences between the pairing chromosomes, is rare in animals, but very common in the higher plants. Among the few animal hybrids in which abnormalities of meiosis due to structural differences in the chromosomes may be the principal disturbances found are Orygia thyellina X antiqua (Lepidoptera, Cretschmar, 1928) , Drosophila mercatorum pararepleta X paranaensis (Dreyfus and De Barros, 1948) and certain hybrids between subspecies of Triturus (Spurway and Callan, 1950; Spurway, 1953). I n these hybrids of Triturus, however, the chromosomal irregularities a t meiosis do not lead to sterility, and in the truly sterile hybrids between valid species of Triturus chromosomal disturbances of this sort are accompanied by various abnormalities typical of gene-controlled diplontic sterility (White, 1946 ; Benazzi and Lepori, 1949; Lantz and Callan, 1954). The opinion of White (1954, p. 265), that structural changes in the chromosomes have been of little importance in building up barriers of reproductive isolation between species, is apparently valid for animals, but comes as a distinct surprise to anyone who has paid attention chiefly to interspecific hybrids in plants. The fact t hat differences in structural pattern between the parental chromosomes is the decisive factor in determining the sterility of F1 hybrids between many plant species of all degrees of relationship has long been recognized. The clearest evidence for this fact comes from the long list of sterile hybrids which have been rendered fertile by artificial doubling of the chromosome number, and the still longer list of natural allopolyploids which have most probably been derived from sterile interspecific hybrids. This subject has been thoroughly reviewed by the writer elsewhere (Stebbins, 1950, pp. 218-227), so th a t the present discussion will consider only the more recently published examples. Hybrids in which the sterility is clearly connected with structural differences in the chromosomes are Helianthus annuus X bolanderi (Heiser, 1949), H . annuus X argophyllus (Heiser, 1951), Hordeum compressum X californicum (Covas and Schnack, 1951), Tragopogon dubius X porrifolius,

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T . porrifolius X pratensis, and T . dubius X pratensis (Ownbey, 1950), Holocarpha virgata X heermannii, H . virgata X macradenia, and other combinations in Holocarpha (Clausen, 1951), several combinations in the genera Lamium (Bernstrom, 1952, 1953a), Lilium (Brock, 1954), and in Gilia (Grant, 1952a,b; 1954a-c; Grant and Grant, 1954). Among the fungi, Emerson and Wilson (1954) have described an interesting example in Allomyces arbuscula X javanicus. I n all of these examples lack of structural homology between the parental chromosomes is indicated not only by a reduced amount of pairing, but also by the occasional presence of rings or chains, indicating heterozygosity for interchanges or translocations, and of bridge-fragment configurations, which as a rule suggest heterozygosity for inversions, or indicate differences between the parental chromosomes in the position of the centromere. I n Holocarpha additional evidence is provided by the morphology of the somatic chromosomes, while in Tragopogon and in Gilia achilleaefolia X millifoliata (Grant, 1954b) the demonstration that natural amphiploids exist which have been derived from these hybrids has shown th a t the sterility of the F, is due entirely to these chromosomal differences. Species hybrids with essentially normal meiosis but with consequent sterility are Primula elatior X veris (Valentine, 1952), Glandularia laciniata X peruviana (Schnack and Solbrig, 1953), Agropyron caespitosum X spicatum (Stebbins and Pun, l953), and Geum urbanum X molle (Gajewski, 1954). Similar conditions were found by Snyder (1951) in hybrids between “strains” of Elymus glaucus by Morley et al. (1956) in hybrids between varieties of Trifolium subterraneum, and have long been known to exist in hybrids between cultivated varieties of rice, Oryza sativa (Terao and Midusima, 1939; Cua, 1951, 1952; Oka, 1952a,b, 1953-1955a; Oka et al., 1954). I n the examples of Primula and Agropyron there is no direct evidence to indicate whether the sterility is genic or chromosomal, but the presence of demonstrated chromosomal sterility in hybrids involving other species of these same genera (Stebbins, 1950) suggests th a t in these examples also the sterility may be chromosomal. I n Glandularia and Geum amphiploids were produced. Although these were more fertile than the diploid hybrids, the increase of fertility in both examples was not greater than that which could be accounted for by changed segregation ratios, as explained above. O n the other hand, multivalent configurations in both of these amphiploids were much less frequent than is usual in autopolyploids, indicating the presence of preferential pairing, and therefore of a reduced homology between the chromosomes of the parental species. If reduced homology is always due to structural differences, then the assumption must be made that the sterility in both of the above examples is chromosomal, and is due to cryptic structural

176

G. LEDYARD STEBBINS

hybridity for small chromosomal segments, as the authors of both papers concluded. The strongest evidence for the existence of haplontic sterility controlled by gene interaction exists in Oryza. I n cultivated rice, hybrids between different varieties, particularly those belonging to subspecies japonica crossed with subspecies indica, show varying degrees of sterility. The FI pollen and seed fertility varies from nearly 100% ’ to less than 1%, depending upon the cross, and the,degree of pollen and of seed fertility in a single plant may differ considerably. The original exploratory work of Terao and Midusima on this problem has now been followed up by more careful cytogenetic analyses on the part of Cua, Oka, and Mashima and Uchiyamada (1955). They have shown that autotetraploids produced from pure varieties are consistently lower in fertility than the diploid types. This depression in fertility varies greatly from one variety to another, and those autopolyploids which show a greater degree of vegetative vigor are usually the most fertile. On the other hand, tetraploids produced from intervarietal hybrids are often more fertile than their diploid progenitors, and this increase in fertility is greatest in hybrids of which one parent is of the upland or insular type (subspecies japonica) and the other parent belongs to the mainland series (subspecies indica). These intervarietal tetraploids, whether produced through artificial doubling of diploid hybrids or through crossing autotetraploids, have a smaller number of quadrivalents and a higher frequency of bivalents than do autotetraploids from a single variety. Oka (195513) has pointed out the following characteristics about these rice hybrids: (1) crossing between all varieties is easy, regardless of the morphological differences between the parents or the fertility of the FI; (2) the FI plants show no disturbances in chromosome pairing; (3) microand macrospores begin to deteriorate a t a definite stage of development after meiosis; (4) the percentage of good pollen depends upon the parentage of the F1, and is affected little by environmental conditions; (5) the percentage of good pollen is generally similar to the percentage of seed setting; ( 6 ) there is no significant difference between reciprocal crosses. I n addition, Oka has shown that segregation ratios for certain genes, particularly those for glutinous pollen and for colored apiculus, are altered in just the manner which would be expected if they were linked with genes affecting gametophyte development. The critical hybrids are those which involve three strains A, B, and C, related in such a way that A forms fertile hybrids with both B and C, but the hybrid B X C is sterile. If the F1 (A X B) is crossed to C, then a bimodal curve for fertility is often found in the progeny, and the homozygotes for a particular marker are more frequent among the high fertility class. From these data,

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Oka has concluded th at there exists a series of gametophyte development (GD) genes in rice, which act in complementary fashion in such a way tha t the presence of two recessive alleles (x1x2)in one micro- or megaspore inhibits gametophyte development. The parental strains are always homozygous for one dominant and one recessive allele (XIXlx2x2) or (ZlZ1X2X2).

The chromosome number of several of these partly sterile hybrids was doubled, and the resulting polyploids were as a rule more fertile than the diploid hybrids from which they arose. Furthermore, the results agreed with expectation on the basis of the assumption th a t the sterility of these hybrids was chromosomal in nature; i.e., the more sterile the initial hybrid, the greater was the increase in fertility produced by doubling the chromosome number. I n regard t o the fertility of these tetraploids in later generations, the results are conflicting. Oka (1955a) recorded a decline in fertility from the Fz to the Fg generation, in spite of selection for high fertility, while Mashima and Uchiyamada (1955) reported t h a t selection in tetraploid hybrids brought about an increase in fertility of about 10% t o 25% from the Fz to the Fs generation. These conflicting results, however, do not affect directly the question of genic versus chromosomal differences, since without selection a decline in fertility would be expected on both hypotheses, and differences between the effectiveness of selection on chromosomal as compared to genic differences are not readily apparent. The principal argument in favor of interpreting the sterility in these hybrids as chromosomal is the lower frequency of multivalents in hybrid tetraploids as compared to strict autotetraploids. This phenomenon is best explained as preferential autosyndesis between chromosomes derived from the same parent. Oka (1955) has considered this explanation improbable, since he has observed F2 segregation ratios in several characters which are intermediate between the 35: 1 and 20.8: 1 ratios which would be expected in a n autotetraploid. These segregation ratios are not, however, definite evidence against preferential pairing, since the amount of increased autosyndesis required to reduce the multivalent frequency from a mean of about 9 per nucleus, as observed in strict autotetraploids, to about 6 per nucleus, which is typical of the hybrid tetraploids, is not great, and a large amount of allosyndesis undoubtedly takes place in all of the hybrid tetraploids. One would expect, therefore, on the basis of the preferential pairing hypothesis that many gene loci would show typical tetrasomic ratios, while others, perhaps a minority of the total, would show a n excess of heterozygotes. Oka (1952a,b, 1955a) has suggested the following reasons for assuming that the sterility in the diploid hybrids of Oryza is due to gene

178

G. LEDYARD STEBBINS

combinations which are lethal to the gametes. First, particular sterility factors have been identified, which are linked to genes for morphological characters. Second, seed fertility is greatest in tetraploid hybrids which show hybrid vigor, suggesting a connection between the sterility factors and the genetic mechanism which controls hybrid vigor. Finally, if small chromosomal rearrangements were sufficiently frequent to produce the observed sterility, one might expect that occasional interstrain hybrids would be heterozygous for rearrangements large enough to produce visible disturbances of meiosis. These have never been found in natural hybrids, although they are easily induced by x-radiation (Oka et al., 1953). The first and last of these points are by no means incompatible with the idea that most of the sterility in the hybrids is chromosomal. Small chromosomal rearrangements could be expected to segregate in the same way as genes, and to show similar linkage relationships. As pointed out by Stebbins and Vaarama (1954), several lines of evidence, particularly that of McClintock on the activator and dissociator genes in maize, and of Lima de Faria on pachytene pairing in rye, suggest that small chromosome rearrangements may be far more common than had been hitherto supposed, so that we need not assume they would always exist side by side with large differences. For this reason, the present reviewer is inclined to agree with Mashima and Uchiyamada (1955) that structural differences in the chromosomes are largely responsible for the sterility in hybrids between the japonica and indica types of rice, and that the “gametophyte development ” genes of Oka may represent rearrangements of chromosomal segments which are so small that they contain perhaps only one to five genes. This detailed study of a particular example serves to emphasize the difficulty of distinguishing between the genic and the chromosomal basis of hybrid sterility, and to indicate further that both the disharmonious effects of gene recombination as well as the results of deficiencies and duplications for chromosomal segments may be operating to produce any particular series of examples of hybrid sterility which, as in Oryza, are obviously conditioned by many genetic factors. IV. HYBRIDBREAKDOWN I n many hybrid combinations among both plants and animals, the F1 individuals are apparently fully fertile, but segregates in the Fz and later generations are either weak or sterile. Among animals, this situation has often been recorded in hybrids of Drosophila (Dobzhansky, 1951 ;Lamy, 1948; Patterson and Stone, 1952), and is known in some interspecific as well as intergeneric hybrids among birds, such as the Anatidae and

HYBRID INVIABILITY, WEAKNESS, AND STERILITY

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Phasianidae (Phillips, 1915, 1921), Gallus (Steiner, 1945), and Columbidae (Staple-Brown 1923; Cole and Hollaender, 1950), a s well a s Amphibia (Steiner, 1945; Spurway, 1953) and fishes (Minamori, 1951). Among plants, the significance of hybrid breakdown as a n isolating mechanism was first recognized by Clausen et al. (1939), and examples have been found in Zauschneria (Clausen et al., 1940), Layia (Clausen et al., 1941; Clausen, 1951), Hemizonia (Clausen, 1951), Solidago (Goodwin, 1937), Solanum (Propach, 1940), Larix (Syrach Larsen, 1937),Populus (Johnson, 1947), P i s u m (Lutkov, 1930), Gossypium (Hutchinson, 1932; Silow, 1941; Stephens, 1949, 1950a; Menzel and Brown, 1955), Sambucus (Winge, 1944), Aster (Avers, 1953), and Cichorium (Rick, 1953). A probable example of hybrid breakdown is in Solanum rybinii x chacoense, in which Koopmans (1952, 1954, 1955) found many floral abnormalities among the F f individuals, which he attributed to plasmatic effects. The cause of hybrid breakdown has usually been attributed to disharmonious interaction between combinations of the genes of the parental species, but Stephens (1949, 1950a) has produced evidence th a t in Gossypium the genetic unbalance may be due to deficiencies and duplications for small chromosomal segments, or cryptic structural hybridity. It thus seems likely that hybrid breakdown, like haplontic sterility of F1 hybrids, may be due to differences between the parents in chromosome structure as well as in gene content. If this hypothesis is correct, then the principal difference between haplontic sterility and hybrid breakdown is th a t in some organisms chromosomal deficiencies and duplications nearly always prevent the gametes or gametophytes from developing and functioning, while in other organisms these structures may remain viable in spite of chromosomal unbalance, may transmit this unbalance to their off spring, and consequently produce a lethal effect during various critical stages of development . Genes promoting hybrid breakdown have been identified by Dobzhansky (1941, 1951) in Drosophila pseudoobscura X persimilis. Using various marked stocks of the parental species, he showed th a t the viability of backcross segregates from this cross depended not so much upon the degree to which the parental chromosomes were mixed or belonged chiefly to one or other of the parental species, but rather upon the presence or absence of certain specific genes. The sex-linked recessive genes beaded, ( b d ) , yellow ( y ) , and short ( s ) from D. pseudoobscura and the dominant gene Bare, on the second chromosome of this species, were particularly active in reducing the survival of backcross segregates, although they have no depressing effect a t all on D. pseudoobscura itself. Minamori (1951, 1955), has shown th at in backcrosses of hybrids between “races” of the spinous loach (Cobitis taenia striata) hybrid

180

G. LEDYARD STEBBINS

breakdown is associated with various abnormalities of embryo development, including the failure to develop the usual head-tail gradient in metabolic activity, and is probably associated with a general lowering of the metabolic rate. I n these hybrids the developmental and physiological manifestation of hybrid breakdown is very similar to that of hybrid inviability, and is probably due to similar causes. Similar phenomena exist in embryos produced by backcrossing the hybrid (salmon X brown trout) as males to females of the brown trout (Svardson, 1945; Winge and Ditlevsen, 1948). Svardson emphasizes the presence of mitotic irregularities in these backcross embryos. H e found similar irregularities in embryos of another species of Salmonidae, Coregonus lavaretus, and they have long been known t o exist in the embryos of many F1hybrids between distantly related species of animals (Hertwig, 1936). This serves to emphasize further the essential similarity between breakdown of Fz progeny and the initial inviability of interspecific hybrids. Muller (1940) has pointed out that the unbalance of complementary gene systems might be expected to be greater in certain Fz individuals than in any of the F1hybrids. I n the case of all pairs of autosomal alleles with incomplete dominance or additive effects, the F1 will have one allele from each parent, but certain Fz segregates will be homozygous for alleles derived from one species which require for their proper functioning the presence of other alleles derived from this same species. If the organism possesses instead of these requisite complementary alleles the corresponding noncomplementary alleles from the second species in the homozygous condition, the effects of unbalance will be evident in terms of inferior viability or greater sterility. As an example, we might suppose that in one species the gene A is balanced by the nonallelic gene B , while the corresponding genes a and b are likewise complementary in a second species; but b is not complementary to A nor is a to B. Thus the parental combinations A A B B and aabb are both balanced and viable, while AaBb, found in the F1, is likewise balanced. On the other hand the Fzsegregates will include genotypes like aaBB and AAbb, in which the genes are noncomplementary, and produce the effects of unbalance. From this model, it is easy to see why hybrid breakdown in the Fz generation would be expected more frequently than haplontic sterility of the F1 in animals, but less frequently in plants. Many experiments with irradiation have shown that the gametes of animals, which are the only haploid cells and which do not have to undergo haploid mitoses, can withstand the presence of deficiencies or lethal genes, which they transmit to the zygote. I n plants, on the other hand, the haploid spores must undergo a few or several haploid mitoses, in which the effects of genic

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unbalance could easily cause abnormal growth or degeneration, so that mature gametophytes and gametes containing these unbalanced combinations would never be formed.

V. RELATIONBETWEEN INCOMPATIBILITY, STERILITY,A N D SEX Many years ago Iilaldane (1922) pointed out that in species hybrids of animals individuals of the heterozygous sex are usually less viable, less fertile, or both, than those of the homosygous sex. Since then, the greater inviability or sterility of the male has been demonstrated in a large number of hybrids of Diptera and mammals, and in the smaller number of additional examples among birds and Lepidoptera a greater disturbance among the females has usually existed. The situation in Amphibia and fishes is not completely clear. Due to the fact that the nature of sex determination varies from one group of fishes to another, and is known in only a relatively small number of species, no clear-cut examples are known in fishes which would either favor or go against Haldane’s law. In salamanders of the genus Triton, F1 interspecific hybrids are mostly females (Pariser, 1936; Benazzi, 1946; Spurway, 1953), and this has led to the belief, following Haldane’s law, that the male is the heterosygous sex in this genus. Actual data on this point, however, are not available. In interspecific hybrids of Rana, on the other hand, two separate studies (Diirken, 1935; Kawamura, 1949) have shown the predominance of males among the sterile F, animals. As has been clearly shown by Witschi (1929), the male is the heterozygous sex in this genus, so that Rana constitutes an exception to Haldane’s law. At least four other exceptions exist to Haldane’s law. One of these is in the Lepidopteran hybrid, Pygaera curtula X pigra (Federley, 1931), in which the sex ratio of the F1adults is normal, but in which the males (the homosygous sex) are more sterile than the females. The other three are all in Drosophila. Sturtevant (1920) long ago reported that the cross D. melanogaster 0 X simulans 8’produces only females among the F1 progeny with normal chromosome complements, but that its reciprocal produces almost entirely males. He believed that this was due to an incompatibility between the egg cytoplasm of simulans and the X chromosome of melanogaster. Icing (1947) found that the F1 females of guarzi X subbadia produced chiefly males when outcrossed to guarani, and supposed that this was due to a disharmonious interaction between the X chromosome of guarani and that of either guarQ or subbadia. In the F1 of D . montana 0 X texana 3 , 80 to 100% of the progeny are males (Patterson and Griffen, 1944; Patterson and Stone, 1952, pp. 469-471). These exceptions fit well with the explanations of Haldane’s law of-

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G. LEDYARD STEBBINS

fered by Haldane (1922), Dobzhansky (1937, 1951, pp. 195-196), Craft (1938) and particularly Muller (1940). The latter author pointed out that a hybrid of the homozygous sex (2A XX) contains alleles belonging t o both parental species in its sex chromosomes as well as in its autoXY or 2A XO) somes, while a hybrid of the heterozygous sex (2A contains sex chromosome alleles belonging t o only one species. Hence if any recessive or semi-dominant gene exists in one species which produces lethal or sterility effects in combination with autosomal genes of the other species, these effects will be covered in the homozygous sex by the corresponding dominant allele in the second X chromosome, but will be expressed in the heterozygous sex, where the dominant allele is absent. A comparable genic unbalance will exist in examples in which a particular gene locus exists on an autosome in one species but has been translocated to the sex chromosome in the other. The exception of D. simulans 9 X melanogaster 3 is only in part a n exception, since the reciprocal hybrid follows the rule, so th a t this is clearly a special case. The guar4-subbadia-guarani example is likewise a special case, since the qua& X subbadia hybrid follows Haldane’s rule, and guarani cannot be crossed directly with either of the parental species. The exception in D. montana X texana is due to a somewhat different combination of complementary factors from that suggested by Muller to explain Haldane’s law, There is apparently a dominant gene on the X chromosome of texana which is lethal in combination with recessive autosoma1 genes from montana. This explanation was verified by the results of reciprocal matings, showing th at the sex ratio is normal in D. texana 9 X montana 3,and by various combinations of triple hybrids which showed that any hybrid male containing an X chromosome from texana produces only male offspring when mated to female montana, but th a t F, females of any bybrid between montana and another species give normal sex ratios when mated t o texana. Since gene combinations of this sort would rarely be expected, and since other hybrids between species of the virilis group give either normal sex ratios or an excess of females, this exception is entirely in accord with the genetic explanation of Haldane’s law. The exception in Pygaera has not been given a genetic explanation, but it is of a somewhat different nature from th a t of Drosophila montana x tesana, since it involves sterility rather than inviability of the F1 individuals. It is probably due to the fact tha t in many organisms the development of the male sex organs is more sensitive to genetic disturbances than is t hat of the female structures. I n hermaphroditic plants, for instance, male sterility is in general much more common than female sterility (Soost, 1951). The numerous partial exceptions t o Haldane’s

+

+

+

HYBRID INVIABILITY, WEAKNESS, AND STERILITY

183

law, those in which the hybrids of the heterozygous sex are just as common and as fertile or sterile as the hybrids of the homozygous sex, can be accounted for on the basis of Muller’s explanation by assuming th a t in these cases no important interactions exist between genes on the sex chromosomes and those on the autosomes.

VI. SEGREGATION FOR FERTILITY AND THE GENETIC BASISOF WEAKNESS AND STERILITY I n a n earlier section of this review, many examples were given of increasing weakness or sterility of Fz progeny as compared to the F1 of interspecific hybrids. The reverse phenomenon of increase in fertility in later generations has been observed in many genera of plants such as Viola, Galeopsis, Nicotiana, Tragopogon, Triticum, Clarkia (“Godetia ”), Bromus, and Elymus. These examples have already been reviewed by the author elsewhere (Stebbins, 1950, pp. 286-287). Additional examples among plants are in Melilotus (Webster, 1950) and Helianthus (Stebbins et al., unpublished). The establishment of fertile, true-breeding lines from the progeny of partly sterile interspecific hybrids without change in the chromosome number has been accomplished in several genera of plants, and there is every reason to believe th a t it has occurred repeatedly as a natural phenomenon in plant evolution. So far as this writer is aware, this phenomenon has not been observed among animals, although in numerous instances backcross segregates have proved t o be more fertile than their F1 progenitors. The fertile segregates which appear among the progeny in later generations of partly sterile hybrids have apparently recovered a genic balance or harmony which was lacking in the F1. The question immediately arises, therefore, as to whether this balanced combination of genes corresponds to the balance which existed initially in one of the parents of the hybrid, or whether i t represents a new balance. I n the latter case, hybrids between the fertile derivative and its original parents would be partly sterile, and this derivative could be considered a s the initial stock of a new species. The writer knows of only one recorded instance of backcrossing the fertile homoploid derivative of a species hybrid with its original parents, i.e., Elymus glaucus X Sitanion jubatum (Stebbins, unpublished). I n this instance, the derived line produced highly sterile hybrids when crossed t o its original parents, and showed that it had acquired a new genetic or chromosomal balance. The writer has elsewhere (Stebbins, 1950, pp. 279-287) discussed the high degree of probability of such an event, if the initial barrier consists of many genetic factors which segregate independently of each other. All of the data on species hybrids reviewed in this and the preceding

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0. LEDYARD STEBBINS

sections point to the same general conclusion regarding the genetic nature of the interspecific barriers which cause hybrid inviability and sterility. Any effective barrier which separates valid species always consists of many different genetic factors. In some instances these factors are all differences at particular gene loci, while in others the barrier consists entirely of structural differences in the chromosomes. I n the majority of examples, however, the interspecific barrier consists of both genic and chromosomal differences. As effective isolating mechanisms, gene differences are far more important than chromosomal rearrangements in the higher animals, while in the higher plants chromosomal rearrangements are equally important or more so than strictly genic differences. The generalizations made in the above paragraph are the first to emerge from the data reviewed which have a definite bearing upon theories concerning the origin of isolating mechanisms. Before considering these theories, however, the writer wishes to establish three other sets of generalizations. These deal with the relation between morphological species differences and interspecific barriers of reproductive isolation, with the development of isolating barriers in relation to the systematic position of the species, and with the occurrence of partial isolating barriers within some species.

VII. RELATION BETWEEN MORPHOLOGICAL SPECIESDIFFERENCES AND INTERSPECIFIC BARRIERS OF REPRODUCTIVE ISOLATION Since species are usually separated from each other on the basis of visible morphological differences, the relationship between such differences and those reproductive isolating mechanisms which act after fertilization, i.e., hybrid inviability or sterility, must be an important factor to be considered in the formulation of any theories concerning the origin of these mechanisms. Equally important in this connection, though harder to analyze, are the genetic factors which determine the habitat preferences of different species. Three different types of relationship exist between the genetic factors determining these morphological-physiological species differences and the factors responsible for hybrid inviability and sterility. In some instances, genes with pleiotropic effects may contribute to visible differences and also to the genetic isolating barriers. In others, factors responsible for the visible differences may be genetically linked to those promoting the barriers. Finally, genetic differences, particularly of the chromosomal type, may contribute to the isolating barrier without having any effect on visible differences or adaptive preferences; in such instances the two types of genetic factors segregate independently of each other in progeny of interspecific hybrids.

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185

Examples of species-isolating genes which have pleiotropic effects on morphological characteristics are very hard t o establish. They can be recognized only in organisms such as Drosophila, which are well known genetically, and even in these organisms the sterility of F1hybrids between valid species plus the reduced crossing-over due to incomplete pairing makes the distinction between linked genes and genes with pleiotropic effects very hard to establish. Nevertheless, the probable existence of such genes can be inferred from the fact th a t in Drosophila pseudoobscura and probably other species mutations with conspicuous morphological effects can contribute to the inviability of Fz segregates from a n interspecific cross (Dobzhansky, 1951, p. 198). Linkage between factors promoting sterility in species hybrids and those responsible for visible morphological differences between the species is best shown by skewed genetic ratios in backcrosses of species hybrids to their parents, or by the increased difficulty or inability to transfer a gene from one species to another by means of hybridization and backcrossing. The earliest example known t o the writer is th a t of the gene for position of petal spot in “Godetia amoena” and “G. whitneyi” (now correctly named, respectively, Clarkia rubicunda and C. amoena, see Lewis and Lewis, 1955), which is discussed in detail elsewhere (Stebbins, 1950, p. 253). I n Gossypium Stephens (1950a) has reported examples of skewed backcross ratios, particularly in G. barbadense X hirsutum, two closely related species. The best example is provided by the transfer of the crinkle gene from barbadense t o hirsutum, as shown by the d a ta of Harland. I n the first backcross generation to hirsutum, there was a statistically significant deficiency of 25 % in the appearance of the crinkle gene, but in the second to the fourth backcross generations this deficiency was slight and statistically insignificant or absent. Stephens concluded th a t the data on Gossypium are best explained by assuming the existence of small nonhomologous chromosomal segments near the genes involved, but the possibility of linkage between crinkle and a specific gene causing inviability of the gametes is not excluded. I n view of the fact th a t most morphological differences between species involve many genes, and th a t the sterility barriers are also multifactorial, some linkage between these two types of factors is to be expected in the progeny of almost any interspecific hybrid. I n the higher plants, the best known and clearest examples of segregation in the progeny of interspecific hybrids show independence between segregation for morphological differences and for the sterility factors. This subject has already been discussed by the author with pertinent examples (Stebbins, 1950, pp. 230-232) and no good examples are known to him from the more recent literature. I n animals the fact th a t most

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G. LEDYARD STEBBINS

hybrids are either highly fertile or completely sterile a t least in one sex makes the gathering of comparable data much more difficult, and there appear to be no good examples of simultaneous segregation of sterility factors and genes governing the morphological differences between species. It is likely, however, that such data would not show the high degree of independence between the two sets of factors which is observed in plants.

VIII. ISOLATING BARRIERS AND THE SYSTEMATIC POSITION OF SPECIES

'

THE

Systematists who are acquainted with many groups of animals and plants recognize the fact that the degree of difference between species varies greatly from one group to another. Mayr (1942, 1948) states that sibling species, which are so much alike that they often can be told apart only by means of special techniques, are common in Diptera and ants, while in birds they occur in some families, but are rare or absent in others. Furthermore, there are some groups of birds and mammals (Phasianidae, Canidae) within which hybrids between strikingly different forms, some of them ordinarily placed in separate genera, are a t least partly fertile (Mayr, 1942, p. 259). I n the higher plants the present author (Stebbins, 1950, pp. 234-236) has noted that in some groups barriers of inviability and sterility are weakly developed, so that partly fertile hybrids can be formed between species which are morphologically very distinct from each other, while in other groups there exist strong barriers of sterility between very similar species, or even between certain populations within a species, as will be mentioned below. Furthermore, the type of speciation pattern shown by a group is related to certain other characteristics. Detailed evidence supporting this generalization will be presented elsewhere, in a review of the known facts about species hybrids in individual genera of angiosperms, but a condensation of the data compiled is presented in (Table 1). In this table genera of seed plants are divided into six groups: (1) Hybrids possible between species widely separated on morphological grounds, these have good chromosome pairing and are more or less fertile. (2) As in (1) but hybrids more or less sterile. (3) As in (1) but hybrids sterile and with a low degree of chromosome pairing and/or conspicuous irregularities of meiosis. (4) Hybrids possible only between morphologically similar species are more or less fertile and have good pairing. (5) As in (4)) but hybrids partly or completely sterile. ( G ) As in (4) but hybrids sterile and with irregular meiosis. Some of the genera had to be classified into two of these groups; these were assigned $5 to each group. They were also classified according to growth habit, and according to

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HYBRID INVIABILITY, WEAKNESS, AND STERILITY

primitiveness or specialization in respect to three conventional characteristics of the flower; polypetaly versus sympetaly, actinomorphic (regular) versus zygomorphic (irregular) flowers, and superior versus inferior ovary. The results of Table 1 show that among woody plants barriers of hybrid inviability and sterility are more poorly developed than among herbs, while in the latter group, annuals possess more pronounced interspecific barriers than perennials. This situation can sometimes be noted TABLE 1 Relationship between Hybrid Behavior, Growth Habit, and Systematic Position in Genera of Angiosperms Hybridization group*

Total number of genera in group Number of genera which are: (a) Woody (b) Perennial herbs (c) Annual herbs

1

2

3

24

9

23

18 6

335

0

5

6

Total

11 10

14

91

1

1 4 9

33

4

4

1

5j5 11 0 8

5 5

736

1M

2836 2935

Number of genera which have: (a) Polypetalous, dichalamydous perianth (b) Monochlamydous perianth (c) Sympetalous, dichlamydous perianth

1035 6 8 1

10 4

3 0

3 1

3)5

36

2

16

535 2

9

8

6

835

39

Number of genera which have: (a) Actinomorphic corollas (b) Zygomorphic corollas

19

7

1234

7

2

1035

4

7 3

5

5

9

5735 3335

* For

explanation, see text.

within particular genera. Menzel (1951) found that among the perennial species of Physalis natural hybrids occur between some species placed in different sections (heterophyllae and viscosae), and that these hybrids are fertile enough to produce hybrid swarms. On the other hand, no natural hybrids were found between annual species, even closely related ones belonging to the same section, and with few exceptions artificial hybrids could not be obtained between such species. Among the perennial species of the grass tribe Hordeae, hybrids between species placed in different genera, such as Agropyron, Elymus, and Sitanion, may have very nearly normal meiosis (Stebbins et al., 1946a), while within the

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genus of annuals, Aegilops, strong chromosomal differentiation is found between relatively closely related species (Kihara, 1940). Heiser (1947, 1949, 1951) has shown that F1 hybrids between annual species of Helianthus have many chromosomal irregularities at meiosis, but between the more numerous and diverse perennial species all hybrids which can be obtained have essentially normal meiosis (Long, 1955). I n the higher plants, therefore, the progression from woody plants t o perennial herbs to annual herbs has often been accompanied by the strengthening of genetic barriers between species, and by the building up of various types of genetic isolating mechanisms between populations which are increasingly similar to each other in external morphology as well as in their ecological adaptations. Table 1 shows another trend. The percentage of groups with sympetalous corollas is about twice as great among the types (No. 4-6) which do not form hybrids between widely different species as among those (No. 1-3) which do. There is also a slightly higher percentage of genera with zygomorphic flowers in groups 4-6. Part of this difference may be a reflection of the fact that there are more annuals in these genera with more highly specialized flowers. Nevertheless, if the comparison is confined to perennial herbs, there is still a greater frequency of genera with sympetalous corollas in groups 4-6. This suggests that the evolution of certain, though by no means all types of floral specialization has been accompanied by a tendency for interspecific barriers of hybrid incompatibility and sterility to become stronger. This tendency is probably associated with increasing specialization and diversity of the floral parts in response to selection by pollinating animals. Grant (1949) has shown that, in groups possessing flowers specialized for cross-pollination by Hymenoptera, Lepidoptera, and birds, a relatively large proportion of the characteristics which diff erentiate species are found in the structure of the flower. I n these groups the structure of the mature flower, and consequently its developmental pattern has been repeatedly modified in various ways. One would expect, therefore, that combining gene systems which determine different ones of these floral developmental patterns would relatively often lead to disharmonies, and consequent abnormalities in the development of the reproductive organs as well as of the gametes and gametophytes which they produce.

IX. THEOCCUERENCE OF INVIABILITY AND STERILITY BARRIERS WITHIN

SPECIES

The fact has long been recognized that the different populations of a species are by no means all alike and homogeneous in respect to the

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factors which are responsible for hybrid inviability and sterility. Hollingshead (1930) long ago noted the presence in varying frequency in different populations of Crepis tectorum of a gene which causes hybrids with C. capillaris to die in the early seedling stage. Strains of C. tectorum not possessing this gene form vigorous but highly sterile and meiotically irregular hybrids with C. capillaris. Population systems belonging to a single taxonomic species may possess three types of diversity with respect to the genic and chromosomal differences which produce hybrid inviability and sterility. I n some instances the “species” is broken up into a series of subunits, the individuals of which form viable and fertile hybrids with each other, but inviable or sterile hybrids when crossed with members of other subdivisions of the same “species.” Examples are known in many groups of insects (Drosophila, Anopheles, Culex Trichogramma, Calandra, termites) , and in Protozoa (Paramecium, Euplotes). These have been reviewed by Dobzhansky (1951, pp. 268-269), who rightly maintains that these subdivisions should be regarded as species, even though they are difficult for taxonomists to recognize. Examples of such “sibling species” in plants are in Holocarpha (Clausen, 1951) and in Elymus glaucus (Snyder, 1951). Since there is no reason for believing that the origin of these clusters of “sibling species” is materially different from the origin of the more familiar types of species possessing well-marked morphological characteristics, they do not have any particular significance in relation to our understanding of the origin of isolating mechanisms. Individuals and populations of a given species may also differ from each other in respect to the ease of crossing and sterility of hybrids with another related species. The example of Crepis tectorum and C. capillaris has already been cited, and similar examples exist in Gossypium, Drosophila, Platypoecilus, and other genera (see review in Dobzhansky, 1951, pp. 199-202). The genetic heterogeneity of a species in respect to factors affecting its hybrids with related species is probably a general phenomenon, since it has been found in all examples known to the writer in which several different strains of a species have been crossed with a different species. Its significance for an understanding of the origin of interspecific barriers lies principally in those examples in which the differences exhibit a regular pattern, as in Gossypium (Stephens, 1946). These patterns will be discussed further below. The final and most interesting type of intraspecific diversity with respect to isolating mechanisms is the presence of inviability or sterility barriers between two populations which are linked together by other populations compatible with both of them. For example, hybrids between populations A and B are inviable or sterile, but both A and B can form

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partly or wholly viable and fertile hybrids with a third population, C, or with still other populations. This situation has been studied most extensively in Rana pipiens (Moore, 1946, 1950; Volpe, 1954; Rubial, 1955), where it consists entirely of hybrid inviability between races which are widely separated from each other geographically or are adapted to very different climates. I n this species the disharmony which produces the death of the F1 embryos is directly correlated with and may result from differences in the rate of developmental processes in the parental races. A similar condition in the beetle Epilachna chrysomelina has been described by Zimmermann (1936) and Strausburger (1936). Similarly, Minamori (1953) has found that the percentage of inviable hybrids resulting from the cross between the mud loach (Misgurnus anguillicaudatus) and various races of the spinous loach (Cobitis taenia stm'ata) depends upon certain characteristics of the Cobitis race which is employed. Those races of Cobitis which differ the most widely from Misgurnus in cell size and in reaction to temperature during development also give the highest percentage of abnormal interspecific hybrids. I n the higher plants several examples have been described of the sporadic or frequent occurrence of incompatibility or F1sterility between otherwise interconnected populations of valid species. The example of Oryxa sativa has been known for some time (Terao and Midusima, 1939) and more recently examples have been described in Holocarpha obconica, Layia glandulosa (Clausen, 1951), in Clarkia dejlexa and probably other species of Clarkia (Lewis, 1953), and Gilia achilleaefolia (Grant, 1954b), and in the Luxula campestris-multiflora complex (Nordenskiold, 1956). I n Oryza, as well as in a similar example found by the writer in the complex of Bromus carinalus (Stebbins and Tobgy, 1944; Stebbins, unpublished), the most sterile hybrids occur between forms native to regions widely separated from each other geographically, but in the other examples, particularly Clarkia and Gilia,this does not seem to be the case.

X. THE ORIGINOF HYBRIDINVIABILITY, WEAKNESS,AND STERILITY The problem of the origin of the barriers of hybrid inviability, weakness, and sterility which separate most species has recently been discussed by several authors, particularly Goldschmidt (1940, 1952), Lamprecht (1941,1945,1948), Muller (1942,1950), Dobzhansky (1951, pp. 206-211), and the present author (Stebbins, 1950, pp. 236-250). The viewpoints expressed by these authors are of two widely divergent sorts. Goldschmidt and Lamprecht maintain that a single mutation can and usually does establish both the morphological distinctness and the reproductive isolation of a newly arisen species, while the other authors believe that speciation, like the formation of races, is a gradual process, guided at least in

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part by natural selection, and distinguished chiefly by the establishment in isolated populations of genetic changes which cause internal disharmony in combination with the genotypes of all other populations. Goldschmidt’s hypothesis is based upon his belief that species differences are of a different nature from racial differences, both morphologically and physiologically, and that no true intermediate conditions exist between races and well-isolated species. Neither of these beliefs is supported by the wealth of evidence on interspecific barriers which is now available. In Drosophila one can find in the compilation of Patterson and Stone (1952) a complete progression from interfertile but chromosomally differentiated subspecies (D. americana americana and D. a. texana) through subspecies which hybridize to form highly sterile F1 male but completely fertile F1 female off spring (D. pallidipennis pallidipennis and D. p . centralis) and species which may hybridize frequently but whose F1 hybrids are completely sterile as males and partly sterile as females (D. pseudoobscura and D. persimilis), to species which are fully isolated from each other through the complete sterility of their FI hybrids in both sexes (D. melanogaster and D . simulans). The nature of the barriers separating the fully isolated species cannot, of course, be tested, but in the example of D. pseudoobscura and D. persimilis the multiple factor basis of the hybrid sterility is well known (Dobzhansky, 1951, pp. 222-223) and except in the case of certain subspecies the available evidence indicates that sterility barriers in Drosophila are generally multifactorial. Unfortunately, detailed genetic analysis of hybrid sterility is not available in any other genus of animals. I n plants, however, many analyses of hybrid sterility barriers have been made, and several of them have been mentioned in preceding sections of this article. With few exceptions, they indicate that sterility barriers are multifactorial, and often segregate independently of the factors controlling the morphological differences between species. One exception, that described by Lamprecht in Phaseolus vulgaris X coccineus, has already been reviewed in detail elsewhere (Stebbins, 1950, pp. 232-234) and no new facts have been obtained to alter the opinion previously expressed, that the apparent species-determining character of the genes in Phaseolus for the nature of the stigma and for the type of germination of the seedlings is due to linkage between these gene loci and chromosomal differences which produce disharmonious combinations when segregated to the gametes of the hybrids. A second exception is Clarlcia lingulata, the relationships of which have recently been worked out in elegant fashion by Lewis and Roberts (1956). Clarlcia lingulata differs from its nearest relative, C. biloba australis, in only two characteristics, the petal shape and the chromosome number, since C. lingulata has 9 pairs of chromosomes and C . biloba only 8 pairs.

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The F1hybrid is highly sterile, but produces some functional pollen and seed. Segregation in Fz and backcross progeny shows that the genetic factor or factors determining petal shape are located on the extra chromosome of C. lingulata. The sterility of the F1hybrid between C. lingulata and C. biloba australis can be explained on the basis of heterozygosity for two major translocations. On the other hand, the sterility of the hybrid between C. lingulata and another subspecies of C. biloba, C. b. brandegeae, though this hybrid is somewhat less sterile than C. lingulata X australis, is apparently determined by many genetic factors. The hypothesis of Lewis and Roberts, that C. lingulata arose from C. b. australis recently and by a rapid process involving few steps is amply supported by their data. There is, however, good reason to suspect that this is not merely an example of concomitant mutation and chromosome rearrangement. The authors point out that in another species, C. unguicuZata, many types with extra chromosomes are known, but none of these differ in external morphology from the normal diploid type. I n Clarkia, as in other genera, there are also many examples of single factor differences with a conspicuous effect on external morphology, but which do not alter the chromosomes or affect the fertility of the hybrid. On the other hand, the association of two chromosomal changes with the alteration of a single morphological characteristic is not known elsewhere in Clarkia or any other genus. A direct causal connection between these two events is, therefore, hard to postulate. If they are not causally connected then the association of chromosomal and morphological changes in the origin of C. lingulata could be explained only by either chance, natural selection, or some common cause for both changes. The chance association of the two types of alterations in this particular instance is highly improbable. If this were so, we should expect to find established by chance many more populations of Clarkia which differ from their ancestors by only one of these types of changes; i.e., there should be %paired races morphologically indistinguishable from %paired ones, and local populations which differ from each other only by a single monofactorial morphological difference. No such examples are known, in spite of the rather thorough investigation of Clarkia which has been made by Lewis and his associates. The association by natural selection is also hard to imagine, since australis and lingulata occupy very similar habitats, and no selective value can be recognized for either the morphological or the chromosomal change. There remains the third possibility, that both the chromosomal and the morphological changes have a common cause. Lewis and Roberts point out that the petal shape of C. lingulata is found in another closely related species, C. modesta, which grows near it. Although the formation

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of hybrids between C. australis and C.modesta has not been possible in the garden, their natural occurrence is not excluded. Since most species of Clarkia differ from each other in respect to chromosomal rearrangements] this is probably true of C. biloba and C. modesta. Furthermore, the establishment of stabilized, fertile types from sterile species hybrids is well known and has been discussed earlier in this review. I n this writer's opinion, therefore, the hypothesis suggested but not favored by Lewis and Roberts, t ha t C. lingulata is a product of hybridization and introgression of C. modesta genes and chromosome segments into C. biloba, is the most plausible one. If this is true, then the generalization still holds th a t the rapid origin of new reproductively isolated populations is known or reasonably inferred only as a result of interspecific hybridization and introgression. A t any rate, the bulk of the available factual evidence on segregation in species hybrids supports the more widespread hypothesis th a t species barriers are multifactorial in nature. Since no mechanism is known by which multifactorial differences can arise between two populations except through natural selection, the available data on species hybrids lead us inevitably t o the hypothesis th at selection plays a n important role in the origin of the inviability and sterility barriers which separate species, as i t probably does in most other phases of evolution. The methods suggested by Muller, Dobzhansky, and the present author for the origin of these genetic barriers can be summarized as follows. First, the populations which are to be separated b y such barriers must be geographically isolated from each other. Dobzhansky has, however, emphasized the important fact that this isolation need not be great. In many sedentary species populations isolated from each other by a few hundred yards interbreed so little th at separate isolating mechanisms can arise in them. Given .an initial geographic isolation, natural selection can promote the origin of genetic isolating mechanisms in the following three ways. First, if the two isolated pobulations are subjected to divergent environmental changes, they will often respond by evolving divergent ecological adaptations, different developmental rates, or both. I n most instances such divergences will lead t o external isolating barriers, such as ecological or seasonal isolation. I n addition, interaction between rate genes established in the divergent populations will often lead to internal disharmony in the development of their hybrids. The internal disharmony is in this case produced as a byproduct of divergent adaptation through the pleiotropic action of certain genes. A good example of possible early stages in this type of divergence is the embryo abortion in hybrids between geographically and ecologically separated races of Rana pipiens (Moore,

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1946, 1950, etc.). One can easily imagine how the extreme races of this species could become distinct species merely through the extinction of intermediate populations. Second, natural selection can promote divergence in chromosome structure through the selective advantage of polygenic blocks, which are tied together by linkage and are protected from crossing-over by means of inversions or translocations. The existence and selective value within populations of this type of chromosomal diversity has now been amply demonstrated in Drosophila, and a similar explanation of the adaptive value of chromosomal diversity in plant populations, such as Paris, Trillium, and Paeonia, has now been made more probable by the discovery that certain chromosomal arrangements have regular geographical distributions (J. L. Walters, 1952; Haga and Kurabayashi, 1954). As yet, no examples are available to show how chromosomal diversity within populations can evolve through the guidance of natural selection to reach the stage of chromosomal divergence between populations. Nevertheless, as suggested earlier by the present author (1950, p. 246), this type of evolution could occur if a population system containing many different chromosomal types became broken up into several isolated populations, which diverged from each other in respect to gene combinations held together on chromosomal segments. Third, if two populations have evolved phrtial barriers of reproductive isolation from each other while geographically isolated, and if they then become sympatric, natural selection can reinforce these initial barriers because of the selective disadvantage of the reproductive wastage caused by the production of many sterile or partly sterile hybrids. Dobzhansky (1951, pp. 208-210) has emphasized the importance of this reinforcement of reproductive isolation in Drosophila, and has cited experimental evidence in favor of it. I n Gossypium Stephens (1946) found that the “corky” gene complex of G. hirsutum, which produces gross abnormalities and weakness in F1 hybrids with G . barbadense, is found chiefly in those regions where the two species are sympatric. Since the vigorous F1 plants which result from crossing between G. barbadense and strains of G . hirsutum not containing “corky” give a large number of weak and degenerate Fz progeny, selection against this source of deterioration in the cotton crop is believed to have resulted in the establishment of the (‘corky’’ complex in regions where the damaging hybrids could be formed. The fact must be emphasized, however, that these species of cotton are cultivated plants, and hence the selection involved was not natural selection, but a type of indirect artificial selection practiced by the human cultivators. Natural selection against reproductive wastage has undoubtedly been

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largely responsible for the development of sexual or ethological isolation in animals (Dobzhansky, 1951, pp. 208-209), and probably has contributed largely to differences in floral structure between plant species with highly specialized flowers, such as Scrophulariaceae, Orchidaceae, and Asclepiadaceae (Grant, 1949; Straw, 1956). On the other hand, the distribution within species of genetic mechanisms causing inviability or sterility of interspecific hybrids does not favor the hypothesis th a t this type of natural selection has played a large role in building up barriers of hybrid inviability and sterility. I n examples of two species with overlapping ranges of distribution, the available d a ta do not indicate th a t those strains of the two species which occur sympatrically are more likely to form inviable or sterile hybrids than strains of the same two species which occur in different regions. Examples are the distribution of the “strong” and (‘weak” races of Drosophila pseudoobscura (Dobzhansky, 1941, pp. 313-314), the behavior in interspecific crosses of different races of Bujo americanus and B. fowleri (Volpe, 1955)) “interracial” hybrids of Cobitis (Minamori, 1955), the behavior of different microspecies of the Elymus glaucus complex when crossed with each other (Snyder, 1950, 1951) and with Sitanion jubatum or S. hystrix (Stebbins and Vaarama, 1954), and the closely related species Bromus carinatus and B. marginatus (Stebbins, unpublished). I n all of these examples, allopatric populations are equally or more strongly isolated from each other than are sympatric ones. On the other hand, Kawamura (1953) found th a t the evidence from interspecific hybrids in the salamander genus Hynobius supports Dobzhansky’s hypothesis. Reproductive isolation through hybrid inviability is in general greater between sympatric populations of this genus than between allopatric ones, and this condition is particularly noticeable in comparing strains of the two species H . nebulosus and H . nigrescens. The point must be emphasized th at these methods of action of natural selection are complementary to each other, and th a t different methods probably assume major importance in different groups of organisms. I n the higher animals, with their closed system of growth and the high degree of integration of their circulatory, muscular, nervous, and endocrine systems, we might expect th at alteration of some primary growth process would have far reaching effects on these various systems, and that pleiotropic end effects of genic differences would be particularly common. I n the higher plants, on the other hand, the open system of growth, with greater independence in growth of individual parts and a lower degree of integration of the adult system, would lead us to expect tha t pleiotropic effects should be less common. This difference between animals and plants in the nature of growth and integration is probably responsible for the fact that hybrid inviability

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and diplontic genic sterility are among the most common methods of reproductive isolation in animals, while they are relatively uncommon in plants. As has been pointed out in earlier sections of this review, these barriers are intimately connected with disturbances of internal developmental processes, and so are most likely to result as pleiotropic effects of genes which are altering the rates and interaction of developmental processes. The earlier sections of this paper dealing with hybrid inviability, diplontic genic sterility, and hybrid breakdown in the Fz generation have pointed out the similarity between these types of barriers. They all are due to the failure of certain gene-controlled processes to be carried through in a normal fashion. The processes most strongly affected, moreover, are those such as gastrulation, neurulation, and gonad development, which involve intense cellular activity, with numerous mitotic divisions occurring either simultaneously or in close succession. For this activity, an increased rate of cellular respiration, i.e. glycolysis and oxygen consumption, is necessary, and is found to exist in normal animals a t these developmental stages (Barth, 1946; Gregg, 1948; Brachet, 1950). Furthermore, the same authors have noted that in interspecific hybrids increased oxygen consumption and breakdown of glycogens does not occur a t these critical stages, or does so to only a limited degree, and this faulty activity is associated with the hybrid breakdown. Brachet, moreover, (1950, pp. 484-485) has suggested that these observed deficiencies in hybrid metabolism are secondary effects of a deficient nucleoprotein metabolism, particularly the synthesis of ribonucleoproteins and ribonucleic acid. Since this type of synthesis is usually regarded as directly associated with genic activity, its deficiency may be a direct result of the genic disharmony in the hybrid nucleus. Muller (1950) has cited convincing evidence of the very precise balance of the developmental processes which lead to the formation and arrangement of bristles in species of Drosophila. Sturtevant (1929) found that although the related species D. melanogaster and D. simulans have an identical pattern of bristles, the F1 hybrids between them show much disturbance of this pattern, and various bristles are often missing. Muller concludes from these facts that the common ancestor of the two species, which must have existed millions of generations ago, probably had the same bristle pattern as the two modern species. This adult pattern, which he assumes to have a high selective value, appears to have remained constant in the two evolutionary lines which led to the modern D. melanogaster and D. simulans. The gene-controlled developmental processes which influence the pattern have nevertheless diverged to such an extent that they interact disharmoniously in the hybrid to produce the observed

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abnormalities of its adult pattern. The most likely way in which such alteration could have occurred is through the establishment of gene combinations which caused these divergences of development along with other changes t hat had a sufficiently great adaptive value so th a t they could have been established by natural selection. One can easily imagine that divergence in respect to gene combinations having pleiotropic effects of a similar nature but more pronounced could lead to the typical examples of hybrid inviability and sterility reviewed in earlier sections of this paper. It is perhaps a not insignificant fact th at in the higher plants, in spite of the very large number of hybrids which have been made, very few instances are known of the disturbance of the morphological pattern in a hybrid between two species so closely related to each other th a t they are alike in respect to the morphological structures in question. The polycarpy found in certain hybrids of Paeonia (Saunders and Stebbins, 1938) is a possible example, but in the large number of hybrids in the grass family known to the writer, no comparable example of developmental disturbance exists. This is further evidence for the belief th a t pleiotropic gene action has been less important in plants than in animals as a cause of genetic disharmony in species hybrids. On the other hand, much evidence now exists in favor of the belief tha t differentiation in gross chromosomal structure has been far more important in plants than in animals as a source of hybrid sterility. White (1954, p. 265) has stated th at “there is very little evidence for chromosomal sterility in animal hybrids . . . ,” a conclusion with which this writer must agree. Nevertheless, such evidence as exists, together with evidence of chromosomal repatterning from comparative morphology of the salivary chromosomes in Drosophila and other Diptera, suggests th a t alterations of chromosomal structure have occurred in animal evolution nearly or quite as frequently as in plants, but th a t their role in species isolation has been subordinate because of the much greater role of gene changes with pleiotropic effects. Evidence for the importance of structural differentiation of the chromosomes as a source of hybrid sterility in plants is provided both by the existence in many species hybrids of the type of meiotic disturbances which are caused by structural hybridity, and by the fact that fertile allopolyploids derived from wide species crosses have arisen frequently, both through artificial doubling of the chromosomes of known hybrids and through spontaneous doubling of natural hybrids which have occurred throughout the geologic ages during which the vascular plants have existed. Successful allopolyploids can be produced from sterile species hybrids only if the sterility of the diploid hybrid is caused exclu-

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sively by structural hybridity for chromosomal segments. This is apparently true of a large proportion of species hybrids in vascular plants, but is relatively uncommon in animals. Among the higher plants, however, both the evidence from chromosome behavior in species hybrids and that from the occurrence of allopolyploids indicates that chromosomal sterility is far more frequent and highly developed in some groups than in others. Furthermore, as mentioned in a n earlier section, most of the groups with poorly developed chromosomal sterility in species hybrids resemble each other in being trees or shrubs, while those with many conspicuous chromosomal differences between closely related species are for the most part either perennial or particularly annual herbs. This relationship between growth habit and chromosomal differentiation is probably connected with differences in population structure between the two types of plants. The writer has pointed out earlier (Stebbins, 1950, pp. 248-249) the fact th a t woody plants, because they tend to dominate their habitats, usually form relatively large populations of a constant size, and evolve slowly in response to major climatic changes. Many of the shorter-lived plants, on the other hand, are subject to great fluctuations in population size, either because they are more sensitive to climatic differences from year to year, or because they occupy pioneer habitats or other temporary ecological niches, and so must be continually building up new populations. When populations are at a minimum, new chromosomal arrangements can become established in them in two ways. If the large ancestral population contained many different chromosomal types, occasional isolates from i t could become homogeneous for different ones of these through the action of random fixation, as suggested by Wright (1940). Secondly, the small isolates would probably be under considerable environmental stress, and selection pressures would tend to be very strong in them. Under such conditions, individuals which happened to possess a group of linked genes with a particularly high adaptive value, associated with a certain chromosomal arrangement, might come to dominate a particular isolate, and so to make that isolate homogeneous for the arrangement concerned. Another isolate might in the same manner become homogeneous for a completely different chromosomal arrangement, since the ancestral population would have contained several potentially adaptive gene combinations linked together in different ways on the various chromosomal types for which it was heterogeneous. I n this manner natural selection is believed to act through the medium of linkage to establish the types of chromosomal divergence which lead to the formation of separate species isolated from each other by chromosomal sterility.

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The third way in which natural selection promotes the development of reproductive isolation, through the prevention of reproductive waste by strengthening initial barriers, is likewise very effective in some groups but ineffective in others. Its importance in Drosophila is undoubtedly great, as attested by both observational and experimental evidence (Dobzhansky, 1951, pp. 208-210). On the other hand, this process is relatively ineffective in many groups of higher plants, in which species adapted t o very different ecological conditions and possessing strongly divergent rhythms of growth can often form hybrid swarms consisting of large numbers of highly vigorous, fertile or sterile individuals. Differences of this sort are easily explained by the very different modes of life of the organisms concerned. I n Drosophila, the short life of the individual and its complete lack of defense mechanisms means th a t populations depend for survival almost exclusively on maintaining a high reproductive rate. Reproductive efficiency, therefore, has an exceptionally high selective value, and natural selection tends t o build up effective systems of reproductive isolation between differently adapted population systems. I n many groups of higher plants, on the other hand, individuals live for hundreds of years, and only an infinitesimally small part of the progeny of each individual can possibly grow to maturity and form a part of the population. I n addition, the importance of sexual reproduction is in many instances much lessened by the possession of highly effective means of asexual reproduction. Gustafsson (1946, p. 52) cites the example of the hybrid between Circaea alpina and C . lutetiana, which, though completely sterile, is a widely distributed, highly successful plant because of its asexual reproduction through subterranean runners. T h e writer is familiar with a similar example, Elymus condensatus x triticoides, in California. Since the parental species also reproduce by vegetative means, sexual reproduction in these species has been relegated to a minor role in the maintenance of the population, and is important chiefly as a means of providing new genetic variability. Under such conditions, the advantage of securing new variability through the development even very rarely of backcross progeny from a highly sterile hybrid, with the consequent introgression which these progeny make possible, may equal or outweigh the disadvantage caused by loss of reproductive potential through the sterility of the hybrid. Selective pressure favoring reinforcement of the isolating mechanism may therefore be initially weak, and in addition may actually be countered by pressure in the reverse direction. We should expect, therefore, th at in groups which have a high reproductive potential, species would be separated from each other by strongly developed barriers consisting of several different types of reproductive isolation, while these barriers would be progressively weaker and com-

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posed of fewer elements in organisms depending for survival less and less on reproductive potential and more on other means of defense. This may be responsible for the fact that sibling species are apparently particularly common in such organisms as Diptera and Protozoa, while examples of hybrids between widely different species of animals are best known among forms with relatively slow reproduction, such as gallinaceous birds, the larger carnivores, and particularly some types of fishes (Hubbs, 1955). A final way in which new barriers of hybrid sterility can arise is a s a result of segregation from interspecific hybrids. As has already been pointed out in earlier publications (Stebbins, 1942, 1950) and a n earlier section of this paper as well as by Anderson (1953), independent assortment of the numerous genic and chromosomal differences which make up any barrier of hybrid sterility can cause occasional progeny of highly but not completely sterile interspecific hybrids to be fertile in themselves but isolated from both parental species because they possess a new combination of the sterility factors concerned. This possibility has been experimentally demonstrated in the progeny of Elymus glaucus X Sitanion jubatum, and indirect evidence suggests that it occurs rather frequently in some groups of plants. The most favorable conditions for its occurrence are in perennial herbs which are self-compatible. I n such groups, a sterile hybrid may live for many years, and produce many thousands or millions of meiotic cells and gametes. If a rare viable pollen grain can germinate on the stigma of the same plant, it can fertilize the occasional egg cell produced by equally rare female gametophyte, so that offspring can be produced even though the percentage of viable gametes is very low. Furthermore, if a single Fz individual arises which is partly fertile but isolated from both of the parental species because it possesses a new harmonious combination of chromosomal rearrangements, it can perpetuate itself by self-fertilization, and so become the single progenitor of a new “sibling species.” The end result of this process is the variation pattern which Grant (1953) has termed the homogamic hybrid complex. Its characteristics can be summarized as follows. All of its species usually have the same chromosome number, although complexes of species with basic numbers differing in aneuploid fashion, such as n = 7 and n = 8, may exist. Nevertheless, on the basis of morphological, distributional, and cytogenetic studies the species can be grouped into two general categories, original species and hybrid derivatives. The original species, although admittedly hard t o recognize, usually have the following characteristics. They represent extremes of the morphological variation pattern in the complex, and often occur on the edges of its range of distribution, either geographically or ecologically. They are to be expected in habitats which are relatively

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stable and old geologically. Intraspecific variability is either low, or forms a regular pattern of geographic subspecies, with relatively little genetic variability present in a single population. The derived entities are of two sorts. Some of them, derived by introgression and possessing essentially the same chromosomal arrangement and combination of fertility genes as one of the original species, are still connected to th a t original species by cross-compatibility and the presence of intermediate forms. These are properly designated subspecies; Grant cites the example of Gilia capitata paciJica and the Santa Ana Canyon population of G. c. abrotanifolia. Other derived entities are intermediate between two of the original species, but are reproductively isolated from both of them. Their derived nature is evident from the fact th at they occupy chiefly recently available habitats, from the high degree of intrapopulational variability, and from the irregular distribution of variation between populations. Gilia achilleaefolia (Grant, 195413) is a fine example of such a species. Furthermore, plants resembling G. achilleaefolia occur naturally together with G. angelensis and G. capitata abrotanifolia, and in this locality appear t o be of hybrid origin between these two species. Although new barriers of hybrid inviability and sterility are most likely to originate from hybridization in self-compatible species, this sequence of events is by no means impossible in cross-fertilizing species. Gilia achilleaefolia, though entirely self-compatible, contains some populations which are normally cross-pollinated because of their floral structure, and produce weakened offspring if they are inbred. One feature of these populations is that some of them are separated from each other by barriers of hybrid inviability which are somewhat irregularly distributed, but which divide the species into three compatibility groups which are partially isolated from each other. Grant has suggested th a t these incompatibility barriers have a relatively simple genetic basis, and that they are derived by segregation from the more complex barriers which separate the two putative ancestral species, G. capitata and G. angelensis. On the other hand, if the three compatibility groups of G. achilleaefolia became more strongly isolated from each other geographically, and if some of their populations became extinct, these compatibility groups could evolve into three separate species. The situation in Gilia achilleaefolia is not unique, but is approached by two or three other groups of plants. Clarkia dejlexa occurs in almost the same geographic area and in similar habitats (Lewis and Lewis, 1955; Lewis, 1953). Furthermore, i t has much irregularly distributed variation between populations, and barriers of hybrid incompatibility and sterility separating many of them. It is intermediate morphologically between two sections of the genus, and a hybrid origin is suggested. A similar pattern of

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variation, both as to external morphology and the existence of incompatibility and sterility barriers within the species, exists in Layia glandulosa and the species of Holocarpha, and Clausen (1951, pp. 178-180), without mentioning specific examples, has suggested that occasional interspecific hybridization has played a role in the evolution of these species. In Lolium, Jenkin (1954) found that the two species L. remotum and L. temulentum form an almost completely sterile FI hybrid, which nevertheless can produce occasional backcross progeny. I n addition there is a fertile, stable garden line of unknown origin (“L. canadense”) which is intermediate between these two species, forms partly fertile hybrids with both, and resembles the artificial hybrid, The hybrid origin of this line is suspected. I n Nicotiana, Goodspeed (1954, p. 285) has suggested that hybridization without chromosome doubling has played a prominent role in the origin of species. These examples are now numerous enough and well enough established so that interspecific hybridization must be considered as one source of hybrid incompatibility and sterility barriers in the higher plants. A small initial number of such barriers can be greatly multiplied by the hybridization process. The objection might be raised that if new barriers are formed through segregation of the factors responsible for preexisting barriers, the new ones would inevitably consist of fewer genic or chromosomal differences than the old ones, and in this way species would tend to fuse rather than diverge from each other. The validity of this objection is, however, weakened by the existence of evidence indicating that incomplete barriers of hybrid inviability or sterility can be strengthened during the course of evolution. As has already been mentioned, one way in which this can be done is through natural selection against reproductive wastage. Although this process would be less likely to occur in plants than in animals, it nevertheless might be expected in many groups of annuals, which depend for their existence upon a high reproductive potential. I n addition, new chromosomal rearrangements can arise in species hybrids through spontaneous breakage. Walters (1950, 1952) has found extensive breakage of chromosomes in certain hybrids of Bromus. Although these hybrids are far too sterile ever to give progeny except through amphiploidy, the same phenomenon, which is apparently produced by some type of genetic disharmony between the parental species, may very well exist t o a much lesser degree in partly fertile species hybrids. I n that case, it could be responsible for new chromosomal arrangements, which could occasionally become established in the homozygous condition in hybrid derivatives, and serve t o reinforce the sterility barriers separating them from the parental species. Hybridization, therefore, not only can produce new barriers by means of segregation, but it

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can also provide a genetic environment favorable to further divergence by means of chromosomal change and selection.

XI. CONCLUSION While all of these considerations are as yet more or less speculative, and definite generalizations are as yet premature, the present review of available data clearly points to the conclusion that the causes underlying the erection of barriers of reproductive isolation, and therefore of the origin of species, differ considerably from one group of organisms to another. Furthermore, these differences are related to other differences between groups of organisms, such as size and structure of populations, rates of reproduction, and methods of adaptation. I n the future, therefore, the study of the origin of species should not be a search for any more general causes, which might be considered as responsible for speciation in all groups of organisms, but a Series of special and comparative investigations, by means of which the interrelationships mentioned above, as well as others as yet unknown, can be further explored and more firmly established. XII. REFERENCES Anderson, E., 1953. Introgressive hybridization. Biol. Revs. Cambridge Phil. SOC.28, 280-307. Ar-Rushdi, A. H., 1956. Inheritance in Nicotiana tabacum. XXVI. Sterility genes from Tomentosae species. J . Genet. 64, 9-22. Avers, C. J., 1953. Biosystematic studies in Aster, 11: Isolating mechanisms and some phylogenetic considerations. Evolution 7, 317-327. Baltzer, F., 1940a. u b e r erbliche lethale Entwiclclung und Austauschbarkeit artverschiedener Kerne bei Bastardcn. Naturwissenschaften 28(12), 177-187; (13), 196-206. Baltzer, F., 1940b. Das Problcm der Teilidentitaet artverschiedmer I k n e im Lichte der Entwicklung embryonaler Bastardtransplantate. Boll. SOC. ital. biol. s p w i m . 16, 39-47. Barth, L. G., 1946. Nature of block to gastrulation in the hybrid egg. Anat. Record 94, 401. Beamish, K. I., 1955. Seed failure following hybridization between the hexaploid Solanum demissum and four diploid Solanunt species. Am. J . Botan. 43, 297-304. Beaudry, J. R., 1951. Seed development following the mating Elymus virginicus L. X Agropyron repens (L.) Beauv. Genetics 36, 109-133. Bell, G. D. H., and Sachs, L., 1953. Investigations in the Triticinae. 11. The cytology and fertility of intergeneric and interspecific F1 hybrids and their derived amphidiploids. J . Agr. Sci. 43, 105-115. Benazzi, M., 1946. I1 sesso degli ibridi Triton cristatus 0 X T . vulgaris 3. Monit. zool. ital. 66, 132-137. Benazzi, M., and Lepori, N. G., 1949. Le gonadi nei maschi dell’ibrido Triton cristatus 0 X Triton vulgaris 3. Sci. Genet. 3, 113-130. Bernstrom, P., 1952. Cytogenetic intraspecific studies in Lamium. I. Heredilas 38, 163-220.

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THE MECHANISM OF SEX DETERMINATION IN DlOEClOUS FLOWERING PLANTS M. Westergaard Institute of Genetics, University of Copenhagen, Denmark

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I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Terminology.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. The Genetical Theory of Sex Determination.. . IV. The Heterogametic Sex.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Heteromorphic Sex Chromosomes in Plants. . . . . . . . . . . . . . . . . . . . . . . . 2. Crosses between Dioecious and Bisexual .................... .. .. ... 3. Investigations of Subdioecious Species.. . V. The Localization of the Sex-Deciding Genes .................... 1. Evidence from Crosses between Dioecious and Monoecious Species.. .. 2. Evidence from Subdioecious Species.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Evidence from Polyploids.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. The Evolution of Dioecism.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII. Cytoplasmic Factors in Sex Determination. . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII. The Physiology of Sex Determination.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..... IX. Polyploidy and Dioecism. . X. Summary and Conclusions ..... XI. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

220 221 227 235 247 247 249 251 260 268 269 273

I. INTRODUCTION

It is one of the most striking examples of evolutionary specialization that sexual dioecism, which has become a well-established outbreeding mechanism all through the animal kingdom, has been more or less of a failure among the higher plants. According t o Yampolsky and Yampolsky (1922) only 5 % of the genera of the higher plants are wholly dioecious (but 75% of the families have some dioecious species). Lewis (1942) gives figures for the British flora shown in Table 1. In most cross-fertilizing (allogamous) plants, outbreeding has, however, been secured by other mechanisms, like the S-mechanism, or by heterostyly, protandry, protogeny, etc. Besides, many plants have no mechanism for the prevention of inbreeding and seem t o propagate almost exclusively by self-fertilization (autogamous species). There is no doubt that dioecism has arisen independently in different plant families and plant genera. In the majority of cases the evolution of 217

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M. WESTERGAARD

dioecism has apparently taken place on the species level; in some cases on the subgeneric level (Rumex) or generic level (Humulus). Few families (Salicaceae) comprise only dioecious species. Dioecious plants offer in many cases better tools for studying the genetics of sex determination than dioecious animals. First, the fact that dioecious plants have arisen independently gives an opportunity to study the different ways in which dioecism may become established. Second, in plants the step from dioecism to bisexuality is often a short one and in most cases dioecism is not clear-cut. I n many dioecious species (e.g., Mercurialis) bisexual types are found in nature often with a rather high frequency. Such bisexual individuals of normally dioecious plant species are almost always fertile and can be studied genetically whereas similar TABLE 1 The Distribution of Sex Forms among Species of the British Flora*

Sex form Completely hermaphrodite Completely monoecious Completely dioecious Hermaphrodite monoecious Hermaphrodite dioecious Monoecious dioecious Hermaphrodite monoecious dioecious

+ + + + +

Total

Families

Genera

63 6 6 4 7 2 3

468 28 15 1 9 2

2080 122 54

91

523

2256 (approx.)

-

Species

-

92.0% 5.4% 2.0% -

-

-

* From Lewis (1942). bisexual animals are sterile intersexes. Finally, in plants it is possible to follow both the evolution of dioecism from hermaphroditism (or monoecism) and the reverse process. As pointed out by Allen (1940), most mutations in the sex genes of hermaphroditic species will tend towards unisexuality, whereas most mutations in dioecious species will tend towards hermaphroditism (or monoecism), e.g., phylogenetically backwards.’ During the last decades these possibilities have been explored by many authors and valuable information has been added to the classical work on Bryonia, Rumex, Melandrium, etc. These investigations are the subject of the present review. For earlier reviews see Correns (1928), Allen (1940), Kuhn (1942), and Lewis (1942). 11. TERMINOLOGY I n order to avoid unnecessary confusion, the terminology t o be used is summarized as follows:

SEX DETERMINATION I N PLANTS

219

A species is unisexual or dioecious when the male ( 3 ,staminate) and female ( 9 , pistillate) flowers occur on different individuals. A species is bisexual if the male and female flowers occur on the same plant. Bisexual plants may be hermaphroditic ( g ) , when the two sexes are found in the same flower, or monoecious [ 9 31 when they are restricted t o separate flowers on the same plant. A species is subdioecious if some bisexual plants occur regularly in nature besides the strict male and female plants (Mercurialis and others). The term intersex (Goldschmidt) describes an individual (belonging to an otherwise dioecious species) which is more or less bisexual, but sterile. 111. THE GENETICAL THEORY OF S m DETERMINATION The formal genetics of the inheritance of sexual dioecism was worked out by Correns in the beginning of this century. By crossing dioecious and monoecious species of Bryonia he showed that sexual dimorphism was inherited after the scheme of a Mendelian backcross, one sex (the heterogametic sex) producing two types of gametes (male determining and female determining) in equal proportions, whereas the other (homogametic sex) produces only one type of gametes. The simultaneous discovery of heteromorphic sex chromosomes (McClung) provided the cytological (chromosomal) vehicle for the segregating sex genes. It was soon realized that the genetics of sex determination is more complicated than would appear from this simple scheme of formal inheritance. The classical works of Correns, Hartmann, and Goldschmidt led t o the formulations of the basic theory of sex determination, expressed by Hartmann as “The law of the bisexual potentiality of both sexes” (“ Das Gesetz der allgemeinen bisexuellen Potenz ”) e.g., each sex has the potentiality to develop the sex organs of the opposite sex. Moreover, sex expression is a quantitative phenomenon with maleness and femaleness expressed to varying degrees, and the main problem in sex genetics has been to understand how the inheritance of a potentially quantitative system can follow a clear-cut qualitative scheme. The key to the understanding of this problem is the realization of two types of sex genes, the sex-deciding genes which are confined t o the sex chromosomes (X or Y or both) which through interaction with “basic sex genes” in the autosomes (and sex chromosomes) determine which sex shall be expressed. This dualism was already realized by Correns, and i t has perhaps been most clearly stated by Muller (1932). This concept is one of the basic approaches for the understanding of genetics of sexual dimorphism. Thanks t o the work of Bridges on Drosophila and many Japanese investigators on Rumex, i t soon became possible to show th a t the sex-

220

M. WESTERGAARD

deciding genes in these two species are located in the X-chromosome, which interacts with autosomal sex genes, whereas the Y-chromosome does not contain any sex-deciding genes. A different type, in which the Y-chromosome contains sex-deciding genes was found in Lebistes by Winge, probably also in Lymantria (Winge, 1937) and in many plants, the most conclusive evidence in the latter case comes from the work on polyploid Melandrium (references later). Another important step in the formulation of the genetical theory of sex determination was the introduction of the concept of the structural hybridity of the heterogametic sex (Darlington, 1931, 1932, 1934). As pointed out by Darlington, crossing-over must be suppressed in the segments of the sex chromosomes which contain the sex-deciding genes if the mechanism is to work with precision. These segments are called the diferential segments. Hence chiasma formation cannot take place within these segments and (according to the chiasmatype theory of crossing-over and of chromosome pairing) these segments cannot be associated during the first meiotic prophase and metaphase. On the other hand, normal crossing-over and chiasma formation may take place between segments of the sex chromosomes which do not contain sexdeciding genes, these segments are called the homologous segments. This structural differentiation of sex chromosomes postulates three types of sex-linked inheritance : strictly X-linked or Y-linked inheritance of genes in the differential segments of the X- and Y-chromosome, respectively, and X-Y-linked (incomplete sex-linked) inheritance of genes in the homologous segments. As will appear from the above summary, there are three consecutive steps in the analysis of the sexual genetics of a given species. The first step is t o establish which sex is the heterogametic one. The second is to localize the sex-deciding gene complexes in the sex chromosomes, and to understand their interaction with the autosomal genes. The third step is t o break down these gene complexes and to explain the end-result in terms of individual gene action. The third step will sooner or later lead into problems of biochemical and physiological genetics. Only when these three problems are solved does i t become possible to discuss the final problem: the evolution of dioecism.

IV. THEHETEROGAMETIC SEX Correns (1928) mentions four ways by which the heterogametic sex may be identified : (a) Cytologically, through the demonstration of heteromorphic sex chromosomes (Section IV, 1). (b) Through sex-linked inheritance. X-linked, Y-linked, and X-Y-

SEX DETERMINATION IN PLANTS

221

linked inheritance in plants has been found only in Melandrium (for summary see Winge, 1931). X-Y-linked inheritance was found in two cases in Carica papaya (Hofmeyr, 1939a). One was a gene Y / y for flower color, the second pair M / m controls stem color. The crossing-over value between the sex-deciding gene(s) and Y is 23.7%, and the distance between Y and M is 17.3%. I n both species the inheritance follows the scheme for male heterogamety. (c) By competition (certation) experiments. Male heterogamety was first established in Melandrium by Correns by means of competition experiments. Plants pollinated with excess of pollen gave a n excess of female progeny. Plants pollinated with few pollen gave equal proportions of the two sexes. Failure to change the sex ratio in Fragaria by similar experiments led Correns to assume female heterogamety in this species (summary in Correns, 1928). (d) From crossings between dioecious and bisexual species (the “ Bryonia-method”) (Section IV, 2).

To these methods may be added: (e) Self-fertilization (or intercrossing) of bisexual individuals from normally dioecious or subdioecious species (Kuhn, 1939 ; Gabe, 1939, see Section IV, 3). (f) Crossings between experimentally produced tetraploids and diploids (Warmke, 1942, see Section IV, 3). 1. Heteromorphic Sex Chromosomes in Plants

Sex chromosomes in higher plants were first described by Blackburn (1923), and Winge (1923) in il!lelandrium, Humulus, and other species; by Kihara and Ono (1923) in Rumex acetosa and by Santos (1923) in Elodea. Heteromorphic sex chromosomes have now been claimed to occur in many dioecious plants, but in most cases it is difficult t o accept the evidence as fully convincing. I n most cases a heteromorphic pair of chromosomes is observed in one sex during metaphase I. In the present author’s opinion this is not enough. Heteromorphic bivalents have been described in many cases also in bisexual species, and they may have nothing to do with sex determination. Besides, most dioecious plants have unfortunately very small chromosomes and the drawings and photomicrographs of heteromorphic sex chromosomes are not always too convincing. It might be pointed out that there is no a priori reason to assume that heteromorphic sex chromosomes should be of widespread occurrence among dioecious plants, where dioecism is in most cases of recent origin. It may be well to recall th at in the animal kingdom, where dioecism is

222

M. WESTERGAARD

much older and much more firmly established, heteromorphic sex chromosomes have never been convincingly demonstrated among reptiles, fishes, or birds (cf. Matthey, 1949, 1951). In order to be convincingly established, claims of heteromorphic sex chromosomes should be based on the following criteria: (a) Demonstration on an unequal XY-pair during the meiotic cycle of the heterogametic sex. (b) The absence of such an unequal pair in the homogametic sex. (c) The identification of the sex chromosomes in the somatic cells of both sexes. Species where heteromorphic sex chromosomes have been convincingly demonstrated on the basis of these strict criteria are listed in Table 2. The references given in the table are not always the earliest ones, but those which give the most complete idiogram of both sexes. Cannabis sativa is included in this list with some hesitation. The sex chromosomes were first described by Hirata (1924, 1929) whereas McPhee (1924) failed to recognize them. The photomicrographs published by Hoffmann (1952) are not too convincing, but Yamada (1943) has identified the sex chromosomes in somatic plates of both sexes, the Y-chromosome is the largest, and both X and Y are larger than any of the autosomes. The sex chromosomes of Humulus lupulus were first observed by Winge (1923). Recently Dark (1952) failed to find heteromorphic sex chromosomes in European hops. However, Jacobsen’s investigation leaves no doubt as to the existence of a heteromorphic XY-pair. The Y-chromosome is smaller than X, contrary to Winge’s original inference. The tetrapartite sex chromosomes of the wild Japanese hops (var. cordifolius) have undoubtedly originated through a reciprocal translocation between X Y and a pair of autosomes (cf. Sinoto, 1929a; Darlington, 1932, and Ono, 1937). Jacobsen has recently shown that the two Y-chromosomes of Humulus japonicus are heterochromatic. This is the first published demonstration of heterochromatic sex chromosomes in plants. The original interpretation that the male Humulus japonicus had three X-chromosomes (Winge, 1923, 1929) has been abandoned in favor of Kihara’s interpretation (XY, Yz). Translocations between sex chromosomes and autosomes have also been found in H . japonicus (Kihara, 1929b). Rumex. Although Meurman (1925) and Kihara (1925, 1927) failed to recognize heteromorphic sex chromosomes in hexaploid Rumex acetosella, A. Love’s (1943) investigation of diploid and tetraploid types must be considered convincing. The sex chromosomes of the octoploid Rumex graminifolius have not been studied in detail; the sex chromosome constitution is inferred from the species with lower numbers. I n R. pauci-

TABLE 2 Well-Established Cases of Heteromorphic Sex Chromosomes in Plants

Moraceae Cannabis sativa 18 Humulus lupulus 18 Humulus lupulus var. cordifolius 16 Humulus japonicus 14 Polygonaceae Rumex subgenus acetosella Rumex angiocarpus Rumex tenuijolius Rumex acetosella s. str. Rumex graminifolius Rumex subgenus acetosa Rumex hastatulus Rumex acetosa and related species Rumex paucifolius Caryophyllaceae 22 Melandrium album Melandrium rubrum

2n 3

2n 0

Sprcies

m

+ XX + XX + X1XIXzX2 + XX

+ xx 22 + xx

References

Yamada (1943) Jacobsen (1957) Ono (1937) Kihara (1929a);Kihara and Hirayoshi (1932); Jacobsen (1957) 12 24 36 48 6 12 24

+ XY A. Love (1943) A. Love (1943) + (XX)XY + (XXXX)XY A. Love (1943) + ( X X x X X X ) x Y A.Love (1943)

+ XYiYz + XYlY2

+ (XX)XY 22 + XY 22 + XY

Smith (1955) On0 (1930, 1935); Yamamoto (1938) A. Love and Sarkar (1956)

8 u

M

e M m

5

2

2

0

z

Q 'd L+

* 5 vl

Ono (193913); Westergaard (1940); Warmke (1946a-c)

D. Love (1940, 1944) f3

f3

w

224

M. WESTERGAARD

folius, A. Love and Sarkar (1956) have so far only identified the sex chromosomes in somatic plates, meiosis having not yet been studied. Melandrium. The heteromorphic sex chromosomes of Melandrium were discovered independently in 1923 by Winge and by Blackburn. According to Blackburn the Y-chromosome was the largest member of the pair whereas Winge held the opposite view. Winge’s interpretation was supported by Meurman (1925), BBlai; (1925), and others, but the argument was finally settled in favor of Blackburn’s interpretation by Ono (1939b) , Warmke and Blakeslee (1940), and by Westergaard (1940). Table 3 gives a list of less well-established cases, in which further data are needed (for an earlier list see Allen (1940)). Coccinea indica should perhaps have been included in Table 2. Kumar et al. have shown that the males have a large Y-chromosome in the somatic cells, but the X-chromosome has not been identified and the meiotic divisions have not been studied. All the other examples listed in Table 3 are based upon the demonstration of a heteromorphic bivalent in metaphase I. I n a number of cases (Fragaria, Hydrilla, and others) the somatic chromosomes have been studied but the heteromorphic chromosomes could not be identified. I n the present author’s opinion Kihara’s investigation of the female Fragaria, and Santos’ old investigation of Elodea are among the most convincing. An investigation of the dioecious, tetraploid strawberries (F. orientalis), where the female sex is likewise heterogametic, is much needed (cf. Staudt, 1952). It would also be highly desirable to have Elodea reexamined by more up-to-date cytological methods. This American species has been introduced into Europe, but strangely enough only the female plants occur (although there are a few recordings in the literature of isolated populations of male plants). Elodea is also interesting because the pollen grains remain in tetrads and (according to Santos) two grains of the tetrads are larger than the two others. Strasburger’s old attempt to establish a relationship between this pollen-dimorphismus and sex determination was never completed (cf. Correns, 1928). Least convincing are the strange observations and interpretations by Billings (1932-1934) with respect to the sex chromosomes of Loranthaceae and Atriplex. The recent report of heteromorphic sex chromosomes in cultivated spinach by Zoschke (1956) disagrees with most other observations. A reinvestigation of Urtica dioica and Valeriana dioica is much needed. I n the Salicaceae and several other genera the chromosomes are so small that it will probably be impossible to reach a t an unbiased decision as to the occurrence of a heteromorphic pair. In Dioscoreaceae the demonstration of an XO-type cannot be

225

SEX DETERMINATION IN PLANTS

TABLE 3 Questionable or Insufficiently Established Cases of Heteromorphic Sex Chromosomes in Plants

Species Hydrocharitaceae Elodea canadensis Elodea gigantea Hydrilla verticillata Palmae Trachycarpus excelsus Trachycarpus fortunei Liliaceae Smilax species Dioscoreaceae Dioscorea species Salicaceae Salix Populus Toisusu

Moraceae Cudrania triloba Morus bombycis Urticaceae Urtica dioica Santalaceae Buckleya joan Loranthaceae Phoradendron jlavescens Phoradendron villosum Chenopodiaceae Atriplex hymenelytra

Spinacia tetrandra Spinacia oleracea Caryophyllaceae Silene otites Silene densijlora Cercidiphyllaceae Cercidiphyllum japonicum

References

Santos (1924) Santos (1923) Sinoto and Kiyohara (1928); Sinoto (1929b) Sinoto (1929b) Sinoto (1929b) Present: Nakajima (1937, 1942) Absent: Lindsay (1930) Nakajima (1937, 1942); Smith (1937) ( 0 :XX, 8 : XY or XO) Present: Blackburn and Harrison (1924) ; Meurman (1925); Harrison (1926); Blackburn (1929); Erlanson and Hermann (1927); Sinoto (1929b); Nakajima (1937, 1942); Suto (1944) Absent: Pet0 (1938); Hdkansson (1938); Muntzing (1936); Johnsson (1940); Wilkinsson (1944) Sinoto (1 92913) Sinoto (1929b) Meurman (1925) (doubtful, cf. 1942)

A.

and D. Love,

Nakajima (1937) Billings (1932, 1933) Billings (1932, 1933) (

' *: : 2A1

+

2A +

Billings (1934) ( 0 : 2A X1X1X2X2, d:2A XiXzY) Araratjan (1939) Present: Zoschke (1956) Absent: Haga (1935); Araratjan (1939); Bemis and Wilson (1953)

+

Favarger (1946) Favarger (1946) Nakajima (1942)

226

M. WESTERGAARD

TABLE 3. (Continued) Species Menispermaceae Coccolus trilobus Rosaceae Fragaria elatim Rutaceae Zanthoxylum piperitum Daphniphyllaceae Daphniphyllum macropodum Aquifoliaceae Ilex serrata var. Aceraceae Acer negundo Simarubeceae Picrassima quassioides Dilleniaceae Actinidia kolomicta Actinidia polyganaa Theaceae Eurreya japonica Datiscaceae Datisca cannabina Valerianaceae . Valeriana dioica Cucurbitaceae Trichosanthes cucumeroides Trichosanthes japonica Trichosanthes dioica Trichosanthes multiloba Coccinea indica

References

Nakajima (1937) Iiihara (1930)( 0 : XY, 3 : XX) Sinoto (192913);Nakajima (1937) Sinoto (192913) Nakajima (1942) Present: Sinoto (192913) Absent: Foster (1933) Nakajima (1942) Nakajima (1942) Nakajima (1942) Nakajima (1942)

Sinoto (1929b) Meurman (1925) Nakajima (1937) Nakajima (1937) Pate1 (1952) Kurita (1939) Kumar et al. (1952)

considered final although the evidence is suggestive (cf. Smith, 1937). We have not included references to H. V. Jensen’s papers (see 1940a, 1940b) in the above bibliography. This author denies the existence of sex chromosomes in all dioecious plants. I n some cases his criticism may be valid, but he also denies the existence of such chromosomes in species where they have been convincingly demonstrated both cytologically and genetically. We have refrained from giving a list of dioecious plants with no heteromorphic sex chromosomes. Such a list is given by Allen (1940, p. 262). To this list should be added Sedum (Rhodiola) rosea. The earlier claim of a heteromorphic pair by Levan (1933) has been refuted (Uhl,

S E X DETERMINATION I N PLANTS

227

1952). The same is the case with respect t o Empetrum nigrum (Hagerup, 1927). A reexamination of Hagerup’s slides by the present author (Westergaard, 1940) did not confirm Hagerup’s observations. This also invalidates Hagerup’s interpretation of the “sex chromosomes” of hermaphroditic Empetrum hermaphroditum. The list of dioecious plants in which heteromorphic sex chromosomes are absent includes a number of species which have been intensively studied genetically, and which will be discussed in the following paragraphs, e.g. , Asparagus, Bryonia, Mercurialis, Thalictrum. I n Carica papaya, Kumar et al. (1945) have observed a pair of chromosomes which shows precocious separation in anaphase I . They identify this pair as the sex bivalent; this interpretation is, however, not accepted by Storey (1953). 2. Crosses between Dioecious and Bisexual Species

a. Dioecious X Monoecious Species. Bryonia. Correns’ classical Bryonia studies have recently been repeated and extended b y Heilbronn and his collaborators. The results differ from those of Correns in one important respect: Correns’ F1 hybrids were all sterile whereas Heilbronn’s dioica X alba hybrid could be backcrossed to Bryonia alba. Furthermore, in the recent Bryonia experiments two new species were included, the monoecious B. macrostylis, and the dioecious B. multijlora. The results of these crosses are summarized in Table 4. As appears from the table, all the backcrosses were highly sterile and gave only very few offspring. These were males or females except in the fourth backcross generation where two monoecious types appeared. The results of intercrossing the two dioecious species, and the two monoecious species are perhaps most interesting. The cross dioica 9 X multijlora 3 gave monoecious off spring only, whereas the reciprocal cross which was done by Arsan and only briefly reported by Heilbronn, gave a n equal proportion of males and females. This unexpected result of crossing two dioecious species is matched by Bilge’s cross between monoecious B. alba as female, and monoecious B. macrostylis as male. This cross gave one male and one female. Hence the main results of the Bryonia crosses may be summarized as follows : Those between dioecious and monoecious species give males and females, in ratios which are in conformity with male heterogamety of the dioecious species. Crosses between two dioecious species may give monoecious off spring, and crosses between two monoecious species may give dioecious off spring. Amaranthaceae. By far the most extensive data on crosses between dioecious and monoecious species are provided by Murray (1940a). He

228

M. WESTERGAARD

crossed dioecious Acnida species with monoecious Amaranthus species, and his material comprises more than 50,000 plants. Some of these data are summarized in Table 5. The monoecious species used in these crosses are of two types, Amaranthus retrojlexus, hybridus, caudatus, and powelii TABLE 4 Crossings between Dioecious and Monoecious Bryonia Species*

1. Bryonia dioica 0 X Bryonia alba. (da) 2. Bryonia alba X Bryonia dioica 8.(ad) 3. Bryonia dioica 0 X Bryonia macrostylis 4. Bryonia macrostylis X Bryonia dioica 3 5. Bryonia multijlora 9 X Bryonia macrostylis 6. Bryonia dioica 0 X Bryonia multiflora 3 7. Bryonia multiflora 0 X Bryonia dioica 8 8. Bryonia macrostylis X Bryonia alba 9. Bryonia alba X Bryonia macrostylis 10. F1(dioica X alba) X alba = daz 11. daz X a = dar 12. da, X a = dad 13. daz X dat 14. d X daz 15. a X daz

N

9 9

?

100%

? ?

33 [ 0 8 1 0

50% 50%

0 Heilbronn (1948) Bilge (1955)

1

1

0 1 Bilge (1955)

l 7 Heilbronn (1953)

l7 1

0 Heilbronnand Bagarman (1942)

loo’%

50% 50% 3

References

50% 50%

l2

0

l2 Bilge (1955)

2

1

1

31 24 2 17 30 12

1st 241 0 6 13 50

13 0 0 11 17 75

0 0 0 2 Heilbronn and 0 Bagarman (1942); 0 Heilbronn (1948) 0

* From Heilbronn and BaBarman (1942); Heilbronn (1948, 1953); Bilge (1955). t Eleven sterile. $ Twelve sterile. 8 All sterile.

are called ‘(first type” species whereas Amaranthus spinosus is a “second type” monoecious species. I n the “first type” species, the first flower in each flower cluster is male, all the rest are females. Hence there is only one male flower in each flower cluster. I n the “second type” species all the flowers in one cluster are of the same sex, but the clusters of staminate flowers are placed terminally on the main axes and on the lateral branches,

229

S E X DETERMINATION I N PLANTS

whereas the female flowers develop only on the axes of the branches and on the base of the terminal influorescence. Hybrids between Acnida species and “first type” monoecious species require little comment; they are in conformity with the assumption th a t TABLE 5 Crossings between Dioecious Acnida Species (Ac.) and Monoecious Amaranthus Species (Am.)*

Ac. cuspidata 9 X

8

(Am. retrollexus Am. hybridus

\Am . powellii Am. retrollexus\ Am. hybridus X Ac. cuspidata 3 Am. caudatus Am. powellii Am. retroflexus Ac. tuberculata O X Am. hybridus Am. caudatus Am. retrojlexus X Ac. tuberculata 3 Am. caudatus (Am. retroflexus Am. hybridus Ac. tamariscina O X

I

{

Am. retroflexus Am. hybridus Am. caudatus Am. powellii Ac. tamariscina 9

* From

I

x

\Am. powellii 713

x Ac. tamariscina 3 3543 927

1

Am. spinosus (1) Am. spinosus (2) Am. spinosus (3) Ac. tamariscina Ac. tamariscina Ac. cuspidata Ac. tuberculata

8 8 8 3

350 254 3 22 190 6 59

8 0 100% 0 100% 0 100% 0 50% 50% 50% 50% 50% 50% 100%

0

50% 50% 50% 50% 50% 50% 100% 0 100% 0 100% 0 100% 0

0 0 0 0 0 0 0 0

339 374 1536 2007 517 410

0 0 0

347 207 3 17 186 3 52

3 41 0

0 6 0 1 0 0 0

4 4

3 7

+ “neuter” plants (see text)

+ “neuter” plants 100 % germination 50%germination O%germination k O%germination 100 % germination lOO%germination 100% germination

+

Murray (1940a).

the dioecious species has male heterogamety. The only complication appears in crosses where Acnida tamariscina is one of the parents. I n such crosses sex ratios deviating from the expected are found. I n addition a varying proportion of nonflowering, sexless neuter” plants are pro-

230

M. WESTERGAARD

duced. This segregation of neuters could be traced back to a recessive gene for the “neuter” character which was present in certain strains of Acnida tamariscina. Some strains were found to be homozygous, other heterozygous for the “neuter” gene (in some strains it was absent). The interesting thing is that this gene manifests itself only when the dioecious species is outcrossed to monoecious “first type” species, whereas it is never expressed in intrageneric crossings between Acnida species. The most interesting results came from crosses between Acnida species and the “ second type ” Amaranthus species ( A . spinosus). This cross produced almost 100% male plants, and in addition a few slightly TABLE 6 Backcross Ratios from Acnida (Ac.) X “First type” Amaranthus (Am.) Hybrids*

F1 (Ac. tamariscina X Am. hybridus) X Ac. tamariscina F1 (Ac. tamariscina X Am. retrojlexus) X Ac. tamariscina F, (Am. hybridus X Ac. tamariscina) X Am. hybridus F1 (Ac. tamariscina X Am. hybridus) X Am. hybridus F1 (Ac. tamariscina X Am. retrojlexus) X Am. retrojlexus F1 (Am. retrojlexus X Ac. tamariscina) X Am. retrojlexus Fl (Am. retrojlexus X Ac. cuspidata) X Am. retrojlexus

1029

488

520

6

15

94

37

52

0

15

288

255

14

16

3

2032 1588

328

82

14

60

54

0

6

0

268

263

0

4

1

6

6

0

0

0

* From Murray (1940a). monoecious plants. Seed germination was very poor in some crosses, but the segregation with respect to sex was independent of the germination percentage. This can only be explained by assuming that, unlike in Bryonia, the male sex genes of the monoecious parent are epistatic to the female sex genes of the dioecious species, whereas the male sex genes of the dioecious species are epistatic to the female genes of the monoecious parent (as in Bryonia). We shall see a third variation of this type of interaction in the Ecballium crosses to be described in the following paragraph. Some of the hybrids between Acnida and “first type” Amaranthus species were partly fertile and could be backcrossed to both parental species. The results are summarized in Table 6 . It will be seen that a small proportion of monoecious plants is pro-

231

SEX DETERMINATION I N PLANTS

duced irrespective of whether the backcross is made to the monoecious, or to the dioecious parental species. Ecballium. Two varieties of Ecballium elaterium (Cucurbitaceae) are found in Spain. In northern Spain the populations consist of monoecious species only (var. monoicum) whereas the populations of southern Spain (var. dioicum) are dioecious. When intracrossed, the monoecious type produces monoecious off spring only, and the dioecious species gives males and females in equal proportions. The results of extensive intercrosses have been published by GalAn (1950, 1951; cf. Mather, 1949). The results are summarized in Table 7. The F1hybrids differ from those described earlier by being completely fertile. As a consequence it has been possible in Ecballium t o raise large Fz and backcross families. These data are also grouped in Table 7. As TABLE 7 Crosses between Dioecious Ecballium elaterium var. dioicum and var. monoicum*

1. 2. 3. 4. 5. 6. 7.

Ecballium dioicum 0 X E. monoicum. (dm) Ecballium monoicum X E . dioicum 8 . (md) dm X md md X md md X d m X md dm X m

881 805 50 214 123 1033 59

0 41. 16 7t 35 3t lt

0 423 21. 105 48 483 0

881 378 32 102 30 547 58

* From GalBn (1951).

t Should be added to the monoecious group, see text. appears from the table, Ecballium shows a third type of interaction between the sex genes in bisexual and unisexual species: When a female gamete from the dioecious species is fertilized by a gamete from the monoecious species, a bisexual plant results. In Bryonia and in “first type” Acnida X Amaranthus crosses such a zygote produced a female. I n “second type” Acnida X Amaranthus crosses it gave rise to a male. Certain female (and male plants) marked with an asterisk in Table 7, should probably be classified with the bisexual group since it has been observed that some truly monoecious plants were unisexual the first year and exhibited their bisexual character only in the second year. The significance of these interesting crosses together with GalAn’s interpretation will be discussed later (Section V, see also Table 16). b. Dioecious x Hermaphroditic Species. Among the more recent experiments of this type, the following two are of special interest: Thalictrum. Kuhn (1930a) crossed the dioecious (or rather subdioe-

232

M. WESTERGAARD

cious) T. fendleri with three hermaphroditic species (Table 8). All the crosses were sterile, and the numbers are rather small but in conformity with the assumption that T . fendleri females are homogametic, a hypothesis which was confirmed by other experiments to be described later. Each intercross produces sterile hermaphrodites in which the male organs were normally developed but in which the gynoecia were to a greater or lesser extent reduced; the number of carpels per flower being fewer than in the parental species (9-11 in T . fendleri, 7-8 in T. foetidum and delawayi, 9-12 in aquilegifolium). When aquilegifolium was the male parent, the hybrids were almost pure males, the average number of carpels per flower being 0.5; thus this hybrid resembles the cross between Acnida and “second type” Amaranthus species which were described above. TABLE 8 Crossings between Dioecious Thalictrum fendleri and Hermaphroditic Species*

Thalictrum fendleri 9 X T . foetidum Thalictrum fendleri 0 X T. delawayi Thaliclrum fendleri 0 X T . aquilegifolium

N

0 0

$3

0P

No. of carpels perfiwer

10 14 3

0 0 0

0 0 0

10 14 3

3 . 7 f 0.11 3 . 7 f 0.05 0 . 5 f 0.07

* From Kuhn (1930a). Fragaria. Table 9 summarizes some of the many crosses which have been carried out with different species and varieties of strawberries. All the data are consistent with female heterogamety. This idea was first proposed by Morgan, Sturtevant, and Bridges (1915, quoted from Correns, 1928), on the bases of Richardsson’s data; it was confirmed independently by Valleau (1923) and by Correns (cf. Correns, 1928). Petrov and Tukan (1937) crossed diploid F . vesca with tetraploid F. orientalis and came to the conclusion that the male sex was heterogametic in the tetraploid. species. This conclusion has, however, been invalidated through the recent careful investigation by Staudt (1952, 1955) who has repeated the cross made by the Russian authors. Curiously enough Hartmann (1956) still accepts the old interpretation. The distribution of sex forms in the genus Fragaria is a very interesting one. The species form a polyploid series with 2n = 14, 28, 42, and 56. All the diploids are hermaphrodites. All the polyploid, wild species are dioecious, but many hermaphroditic garden varieties have been produced within the hexaploids and octoploids. Fragaria grandijlora (2n = 56) is thus probably a hybrid between F . chiloensis 0 x F . virginiana 8 (see Kubn, 1930b). Occasional unisexual forms (female-sterile males) are

Fragaria virginiana 0 X grandiflora $' Fragaria grandi8ora 0 X grandijlora g Fragaria grandiflora 0 X oirginiana g Fragaria grandijlora Q X chiloensis 3 Fragaria virginiana 0 X elatior g Fragaria grandijlora Q X elatior 3 Fragaria grandijlora 0 X hagenbachiana Fragaria elatior 0 X nipponiea Fragaria elatior 0 X collina Q Fragaria vesca X moscata 8 Fragaria vesca $7 X orientalis 8 Fragaria viridis ST X orientalis 3 Fragaria orientalis 0 X viridis $ Fragaria vesca $ X vesca 3 62 X 2s X 22 x 22 x 42 x 22 x

22 62 42 42 22 22

82 X 82 8s X 82 82 X 82 82 X 82 82 X 6s 82 X 6s 82 X 22 62 X 2x

Combination

118

6

47 35 67 6

? ?

338 139

34

N 20 280 169 77 half 0 25 15 38 0 0 0 3 0

00

118

4

all 0 19 29 1(42) 0 0

0

0

0

0

0

88

0 5 (52) 100 % 100 % 0 0

1

14 276 169 62 half 0 22

PP

~~

References

Lilienfeld (1933, 1936) Schiemann (1037) Schiemann (1943) Staudt (1952) Staudt (1952) Staudt (1952) Schiemann (1937)

Richardson (1914) Valleau (1923) Schiemann (1931) Schiemann (1931) Mangelsdorf and East (1927) Correns (1928) Schiemann (1931)

TABLE 9 Crossings between Dioecious and Hermaphroditic Species and Varieties of Fragaria

m

z

F

v

2

$

E2,

M 0

e

U M

u,

F!

234

M. WESTERGAARD

found among the diploid species. Schiemann (1931) crossed such a male plant of F . vesca to a normal, hermaphroditic vesca type and obtained 118 male plants in the offspring. These male plants were of the same “sex” as the male parent, but in some of them female sterility was not complete and a small Fz family could be raised. This gave 16 3 3 and 27 8 c7 with the female organs developed to a varying degree. The great difficulty with all the work on sex determination in Fragaria is apparently the proper classification of the sexes. Staudt (1952) has recently made a careful investigation of the males and females of the wild, tetraploid, dioecious F . orientalis. Both sexes show rudiments of the opposite sex. I n the young female buds, anthers develop so far that PMC’s are formed but they degenerate before meiosis takes place. I n the young male buds, a gynoecium is formed, EMC’s develop, and may go through reduction division, but then the gynoecium degenerates. Obviously this sex pattern in the normal, dioecious species makes it practically impossible to classify the sexes of the sterile interspecific crosses properly. Both Schiemann and Lilienfeld have done a most careful investigation of their hybrids and Fz families but even so the classification of the hybrids with respect to sex is somewhat arbitrary and must be accepted with some reservation. In many of the older works where the classification has not been supported by histological examinations, it must be considered quite arbitrary. This does not interfere with the main issue, which is the proof of female heterogamety in all species of the genus, but it makes it somewhat difficult to accept some of the more elaborate genetical hypotheses which various authors have built up in order to explain their results. Some of the intraspecific crosses have been partly fertile, and Fz and backcross families have been raised both by Schiemann and by Lilienfeld. Lilienfeld’s data are summarized in Table 10. F2

TABLE 10 and Backcross Ratios from the Cross 62 Fragaria elalior 8 X Zx Fragaria nipponica Q *

Fragaria elatior X nipponica Fz (FI X Fd Fa (Fz 9 X Fz 8) F3 (Fz9 X FZ Q ) Fragaria elatior 0 X Fz Q Fz Q selfed Fz Q X Fa 3

* From Lilienfeld (1936).

N

9 9

35 283

15 77 146 65 52

444

233 188 102 153

0 0

ST Q 20 79 91 14 49 8 14

0

28 69 34 1 42 28

NotJtowered 99 (35%) 138 (31%) 120 (51%) 86 (45%) 52 (50%) 111 (73%)

SEX DETERMINATION IN PLANTS

235

The reservation stated above with regard to a n unbiased classification of the sex of a given plant is of course also relevant to these data. A further complication comes from the phenomenon of “sex reversal’’ described both by Schiemann and by Lilienfeld. The sex expression in these hybrid plants is very much influenced by the environment, and the plant may be classified as a male one year and as a hermaphrodite in the following year. Despite these reservations, the work on Fragaria has convincingly shown that the genus takes up a rather unique place among dioecists: (1) The female sex is heterogametic. If Kihara’s demonstration of heteromorphic sex chromosomes is accepted, this is the only case where the evolution of female heterogamety has been accompanied by a differentiation of heteromorphic sex chromosomes. According to Matthey (1949) such heteromorphic sex chromosomes are absent in all animals displaying female heterogamety. (2) The diploid species are hermaphrodites whereas the polyploid species are dioecious. In a later section (Section IX ) this problem will be discussed in some detail. (3) The sex ratio does not always follow “Haldane’s rule.” According to Haldane (1922) i t is the heterogametic sex which is likely to be in deficit if the sex ratio deviates from the expected 1 : 1 ratio. Staudt (1952) has recently found that in the tetraploid F. orientalis, where female heterogamety is now convincingly established, the sex ratio is 253 0 9 : 158 3 8 , e.g., a great deficit of the homogametic sex. (4) It is the homogametic sex, the males, which seem t o display the greatest variation in sex expression contrary to other dioecious species where i t is the heterogametic sex (likewise the male) which shows the greatest variation.

3. Investigations of Xubdioecious Species I n subdioecious species, occasional bisexual plants may occur in both sexes, or in one sex only. A male plant with occasional female or hermaphroditic flowers is called subandroecious; a female with occasional male or bisexual flowers is called subgynoecious. The sex expression in such plants may be strongly influenced by the environment, and examples of such sex reversals will be given below. The earlier work on the genetics of subdioecious species is summarized by Correns (1928) who himself contributed most of the earlier investigations. I n the earlier literature it is most often stated that subandroecious plants, when selfed or intercrossed, give male off spring whereas subgynoecious plants give female off spring. These observations, which were

236

M. WESTERGAARD

always difficult t o explain, are no longer valid. I n 1939, Kuhn in Germany and Gabe in USSR did the crucial experiments, which have provided the clue to our understanding of the sexual genetics of subdioecious species. Table 11 summarizes their data and those of later investigators. Thalictrum. Subandroecious plants give females and more or less subandroecious males in the proportion 1 Q : 3 3 3. The males are of two different types, as can be shown when they are crossed to females: N of the males give Q 9 and 3 d in equal proportion when crossed to normal females, of the males, however, give unisexual families consisting only of male plants. This observation is easily explained by assuming that the male sex is heterogametic and that both males and subandroecious hermaphrodites have the constitution XY. When selfed, XY-plants will segregate 1 XX, 2 XY, 1 YY. Both the last two types being males, the proportion will be 1 9 :3 3 3. The YY-males when crossed to XXfemales will give 100 % XY males. Subgynoecious plants (constitution XX) should give female or subgynoecious plants only, as was actually found. The same mechanism works in Asparagus (Table l l ) , where it has proved to be of some practical importance as a method for producing unisexual male strains. The male plants of Asparagus give a slightly higher yield than the female plants (Rick and Hanna, 1943). Mercurialis. In Thalictrum and Asparagus the XY- and YY-males are phenotypically alike and can only be identified when crossed to females. I n Mercurialis, however, both Gabe and Kuhn found that the YY-males differ from the normal XY-males by producing very little pollen. All such males, which Gabe calls “abnormal” and Kuhn “ pollenarme” give 100 % normal XY-males when crossed to females. The subgynoecious plants never produce male or subandroecious off spring, in accordance with expectation (Table 11). Such 1 :3 ratios of 9 9 : 3 3 proves that (1) the male sex is heterogametic, (2) YY-types are viable. It should be remembered that Humphrey (1945) used a similar method for demonstrating female heterogamety in the axolotl. He was able to change females into males, and crosses between such modified females and normal males produced 3 9 Q : 1 3 . Some of these females gave 100% female offspring when crossed to normal males. Silene otites. I n view of these facts, the inheritance of sex forms in Silene otiites becomes very interesting. This subdioecious species differs from most other dioecious species by its sex ratio. Correns (1928) states a ratio of 38% 9 Q :62% d d,e.g., a surplus of males which is rarely found among dioecious species. The bisexual forms are subandroecious according t o Correns, and he states that they produce (after selfing)

237

SEX DETERMINATION IN PLANTS

TABLE 11 Offspring (in per cent) of Hermaphroditic Plants of Subdioecious Species (Selfed or Intercrossed) Zejerences

1. Thalictrum fendleri selfed

Male ~

2. 52 individual males from (1) X females 3. Thalictrum dasycarpum Q selfed 4. Thalictrum dasycarpum Q selfed 5. Thalictrum polyganum Q selfed 6. Thalictrum polyganum Q selfed

72 ~

_

50 100

Female

?

100

-

Male

?

75

25

Female

?

100

-

Male

?

75

25

12 5

50 0

50 100

198

78

22

50

50 100

Male

17

8

Q

Female

Q

Male

12. Normal males from (11) X females 13. Abnormal males from (11) X females

-

-

22 _

50 0

9. 25 individual males from (8) X females 10. Mercurialis a n n u a selfed 11. Mercurialis a n n u a selfed

~

36 16

7. 17 individual males from (6) X females

8. Asparagus o f i i n a l e Q selfed

224

6369

100

-

602

29

50

1074

47

53

672

-

100

170 79

0 0

_

_

_ ~

_

~

~

-

Kuhn (1939)

Rick and Hanna (1943)

21

Kuhn (1939) 2abe (1939)

~~

14. Silene otites 15. (I" X d

Q selfed

See text

100*

loot

Sansome (1938)

~

~

~

238

M. WESTERGAARD

males or subandroecious males only, but never females. These observations were extended by Sansome (1938) (cf. Table 11). Sansome explains these data by assuming female heterogamety for Silene otites. However, he also assumes that the bisexual plants are modified females, but this assumption is quite inconsistent with his data, and contrary to Correns’ classification of this type. As far as the evidence goes, the inheritance of sex forms in Silene otites investigated by Correns and Sansome can be explained by assuming: (1) female heterogamety, and (2) that the bisexual plants are subandroecious and homogametic. The same may be true for Silene roemeri (Correns, 1928) in which no female off spring appear when subandroecious plants are selfed or intercrossed. These data must, however, be considered preliminary, and i t should be remembered that, also, in the older investigations of Mercurialis and Thalictrum, subandroecious males were reported to produce male offspring only. According to Sansome (1938) the closely related S. pseudotites has male heterogamety. Warmke (1942) has investigated the problem of the heterogametic sex in S. otites by means of his ingenious polyploidy method: Artificially raised tetraploids will be of two types: 4A XXXX and 4A XXYY. The latter type will produce a great surplus of XY gametes in addition to a small proportion of XX and YY gametes (because in metaphase I the association X-X and Y-Y is much more frequent than the associations X-Y, X-Y). I n case of male heterogamety the cross 4n Q X 2 n 3 (XXXX X XY) will give an equal proportion of XXX females and XXY males (or hermaphrodites). I n case of female heterogamety, the cross XXYY p X XX 8 will give a high proportion of XXY plants in addition to a small proportion of XXX and XYY plants, e.g., the cross will give almost unisexual off spring. Colchicine-induced tetraploid females of S. otites were crossed to diploid males and the offspring consisted of 50% 0 0 and 50% c? 8,showing that the male sex is heterogametic. This is consistent with Favarger’s cytological observations (cf. Table 3). Nevertheless, Sansome’s results are difficult to explain without assuming female heterogamety in his strains. Further investigations are certainly needed before these problems can be finally settled, but the present evidence suggests that the genus Silene may consist of some species with female heterogamety and closely related types with male heterogamety, a pattern which has been found in several cases in fishes (cf. Gordon, 1947; Winge, 1934). Valerianu dioica. I n this species also, subandroecious plants are claimed to give only male offspring (Correns, 1928), but the actual figures are not given. According to Correns there is also a preponderance of males in natural populations. The problem of the sex ratio has been

+

+

239

S E X DETERMINATION I N PLANTS

more closely investigated b y Moewus (1950) who showed th a t the ratio may vary in different populations and in different crosses. Moewus has experimented with pollen germination in various strains and found a great variation in the germination percentage. He connects this observation with the different sex ratios, assuming male heterogamety. Again further investigations are needed. Vitis. I n grapes, very extensive investigations on sex determination have been published. Most wild species of grapes are probably dioecious, but human selection has produced hermaphroditic strains in most cultivated grapes, where pure males are obviously undesirable from the point TABLE 12 Sex Combinations in Vitis*

1. 2. 3.

g q

SF

selfed or selfed or selfed or

4 . o x q 5. o x q

q X q x g X q

-

All

25% 3

75% 9

50%

50%

4

All

* Condensed from Breider and Scheu (1938); Oberle (1038); Negrul (1936); Loomis et al. (1954, crossing no. 3). of view of production. The extensive crossings between dioecious and hermaphroditic grapes are summarized in Table 12. Evidently there are a t least three types of hermaphrodites: Those which breed true to type, others segregating females and hermaphrodites in the proportion 3: 1, and still others which give 9 :3 :4 ratios. The further discussion of these d ata will be reserved to a later paragraph. Cannabis. Probably the most complicated sex pattern found in any dioecious species is encountered in the cultivated hemp. T o a certain extent the pattern found in Vitis is repeated. The wild Cannabis is dioecious, but from the point of view of fiber production, the normal males are undesirable, because they die off shortly after flowering, much earlier than the females. Therefore human selection has attempted to produce hemp varieties which are either monoecious (or hermaphroditic), or varieties where both sexes mature a t the same time, the latter type will be referred to as “uniformly ripening” hemp. Such varieties have

240

M. WESTERGAARD

been produced in many countries, but most of the recent work is due to Hoffmann (1947, 1952), Sengbusch (1952), Huhnke et al. (1950), and Neuer and Sengbusch (1943) in Germany. Another method to increase the yield and quality of hemp has been the production of colchicineinduced tetraploid strains. These will be discussed in a later paragraph. A great variety of sex forms exists in the cultivated dioecious strains and the sex ratio may vary very considerably from one strain to another, but mostly with a great preponderance of females. Moreover, the sex expression is very much influenced by the environment. I n dioecious hemp the two sexes differ rather strikingly in growth form, there is a “feminine” growth type and a “masculine” growth type. Especially among the females a great variation in sex expression is found, but it is strongly influenced by the condition of growth, time of sowing, day length, etc. Much less variation is found among the male types, and here the sex expression is far less influenced by the environment (Sengbusch, 1952; Huhnke et al. 1950; for a description of the various sex types in dioecious hemp, see also Maekawa, 1929, Hirata, 1927). Hoffmann distinguishes between the following sex types in cultivated uniform and monoecious hemp : (1) Plants of “feminine” growth type with female flowers only, corresponding to the female type of wild hemp. (2) Plants with “feminine” growth type and a greater or smaller proportion of male flowers and hermaphroditic flowers (monoecious types). Sengbusch classifies this group into 5 subgroups called “1-, 2-, 3-, 4-, 5-cross monoecious plants.” A “ l-cross” plant is almost male with a few female flowers. A “5-cross” is a female with only a few male flowers. Pure females which are cut back and have the female flowers removed, may later produce a number of male flowers. (3) Plants of “feminine” growth type, but with male flowers only. The reading of Hoffmann’s and Sengbusch’s papers is complicated by the fact that Hoffmann refers to this type as a “feminized male,” whereas Sengbusch calls it a “masculinized female.” This is inhuman. Hoffmann claims that a similar variation is found among plants of masculine growth type namely: (4) Males with masculine growth type and male flowers only, corresponding to the males of the wild dioecious species. (5) Plants of masculine growth type but with flowers of both sexes (monoecious types). This type is very rare. (6) Plants of masculine growth type, but with female flowers (very rare). According to Hoffmann all 6 types are found among the monoecious and the “uniform” varieties, although 5 and 6 are very rare. Sengbusch,

241

S E X DETERMINATION I N PLANTS

however, claims th at types 4,5, 6 never occur among monoecious strains. He explains their presence in the cultures as due to : (1) contamination from dioecious species and (2) mutation of a gene “locker” which, when present in plants with feminine growth type, changes its phenotype so that it resembles the more open growth which is characteristic of the dioecious males. According t o Hoffmann (1952) the distribution of sex types within the two varieties of hemp is as shown in Table 13. It is a standard practice in the breeding work to remove the plants marked with a n asterisk in the table. The various crosses which have been carried out by Hoffmann and Sengbusch are summarized in Table 14. Although the results of the two authors agree to a great extent, their interpretations of the data are different. According t o Sengbusch, TABLE 13 Sex Types (in per cent) in Monoecious and Uniformly Ripening Cannabis* Feminine growth

cj‘q

9 9 -~

~~~

~

“Uniform,” Muncheberg Monoecious, Sengbusch

* From

33

Masculine growth 9 9

$B

83

N

~~~

38.00 28.95 4.47 94.71

31.02t 0.63t

0.02 0.01 2.00t 0 0.19t 0

9258 1587

Hoffmann (1952). before flowering.

t Discarded

all the normal male plants in crosses f, 10, 17, I8 are due t o contamination. This explanation is not accepted by Hoffmann but his case is not too strong. It should be remembered that all this work was more or less of a sideline, as part of a large-scale breeding program. Hemp is windpollinated and all Sengbusch’s crosses and most of Hoffmann’s have been isolated by distance. True, some of Hoffmann’s crosses were made in greenhouses, but even this cannot be considered as a sufficient precaution against contamination in a wind-pollinated species. Hoff mann claims that the typical male plants which he finds in the above crosses are different from normal males of dioecious species, and he believes th a t this interpretation is supported b y crosses il-13 in which “masculine monoecious males’’ were the pollen parents. He claims th a t the proportion of male plants in these crosses is much lower than when dioecious males are pollen parents. This conclusion is, however, not supported by his data, because both in cross 6 and Bc, in which normal dioecious males are used, the proportion of males is just as low as in crosses 11-13. According to both Sengbusch and Hoffmann, dioecious hemp has the constitution XX Q and XY 6 , e.g., normal male heterogamety. Accord-

242

M. WESTERGAARD

TABLE 14 Crosses between Various Sex Forms of Cannabis satiua* This table is condensed from Hoffmann (1947, 1952) (H.) and Sengbusch (1952) (S.). The number in the references refer to the table numbers. The following strains were used in the crosses: Monoecious hemp from Sengbusch (rnon.) Uniformly ripening hemp from Muncheberg (unif.) Dioecious hemp strain from Schurig (Sch.) Dioecious French strain (Fr.) Dioecious “Hellstenglich” strain (He.) Dioecious “ Max-Planck” strain (MP.) Feminine growth

Masculine

type

growth type

9 9

Q Q dd

1. dioecious 9 X mon. Q a. Sch. 9 X mon. Q b. Sch. 0 X mon. Q c. Sch. 9 X mon. Q d. Fr. 9 X mon. Q e. He. 9 X mon. Q f. MP. 9 X mon. $7‘ g. MP. 9 X unif. Q

5000 99.8 820 94.9 121 89.1 146 87.0 442 98.0 1296 88.7 1750 95.5

0.2 4.0 0 10.9 - 8.9

2. mon. 9 Xmon. Q

1144 47.6 52.4

N

3. dioecious 0 (Sch.) X dioecious 8 (Sch.) 4. monoecious 9 X dioecious d (Sch.) 5. unif. 9 X dioecious d 6a. mon. Q X dioecious 3 (Sch.) b. mon. Q X dioecious d (Sch.) c. mon. Q X dioecious d (He.) 7. Sch. 9 X d from 6a 8. mon. 0 X 8 from 6a 9. mon. Q X 3 from 6a 10. mon. Q selfed

4000 54.0

-

11.2 3.1

9 9

Q Q d8 -

-

-

-

- S.

- - - -

2184 52.7 - 690 66.7 1 . 7 1.4

-

- S. la, 1 1.1 H . 3 b - H. 3b

4.1 2.0 0.1 1.4

-

-

References

- - -

H.3b H. 3b H. 1952/2 H. 1952/2 la, 2

46.0 S. lb, 1 47.3 5. lb, 2 30.2 H. 6

-

-

- 47.9 S. l b , 3

-

-

-

50 32.0 30.0 4.0

-

- 34.0 H. 4a

-

-

- 47.8 S. Ic, 1 49.4 s. l c , 2 46.3 S. Ic, 3

1221 52.1

49 49.0 6.1

6768 50.9 1.3 5605 36.0 14.6 2207 11.4 42.3

44.9 H.4b

- - - - -

2054 18.0 65.0 11.0

- -

6.0 H. 11,l-3

243

S E X DETERMINATION IN PLANTS

TABLE 14. (Continued) Feminine growth type

N 11. dioecious 9 (Sch.) X mon. 3 (masculine type) 12. mon. 0 X mon. 3 (masculine type) 13. mon. $? X unif. 3 (masculine type) 14. unif. X unif. 3 (masculine type) 15. unif. 9 (masculine type) X unif. 16. unif. 9 and $? (masculine type) X dioec. 3 (Sch.) 17a. b. 18a. b. c. d.

unif. 9 X mon. 3 same unif. 9 X mon. 3 same same same

9 0

Masculine growth type

Q Q 33 0

9

gQ

C? CT References

1055 69.1 2.6 0.01 -

- 28.2 H. 5a

932 55.5 2.4 3.4 -

- 36.6 H. 5b

2.4 -

-

112 35.7 56.2 3.6 -

-

379 33.0 17.7 20.8 -

- 11.3 H. 14

244 37.7

3 . 3 4.9

-

-

51.0 31.5 55.9 32.9 48.2 76.6

25.8 19.7 19.5 48.8 19.8 10.7 14.6 45.2 25.3 20.2 16.7 6.7

-

-

85 55.2

964 215 383 82 559 30

7.1

-

35.3 H.12 4 . 5 H. 15

34.8 H. 7

3.5 0.5 - 13.0 - 4.9 - 6.3 - -

H. H. H. H. H.

9/la 9/lb 9/2a 9/2b 9/3 H. 9/4

* Some of the figures from Hoffmann’s table do not add up to 100 % because types classified to growth type only, not to sex, are omitted. ing to Sengbusch, all plants belonging to the monoecious or “uniform” varieties have the constitution XX, the variation in sex expression being due t o heterozygosity of sex genes in the X-chromosomes and in the autosomes, and variation in growth type being due t o the “locker” gene. According to Hoffmann, the monoecious and “uniform” strains may comprise both XX-, XY-, and YY-types of all sex types. The sex chromosomes are of minor importance in sex determination in these strains, their function having been taken over by the autosomes. This explanation has a close resemblance with Winge’s interpretation of his results in Lebistes (Winge, 1934). Hoffmann has also made cytological observations; he claims to have demonstrated heteromorphic XY-bivalents in both males and monoecious plants of both varieties. He is, however, unable to identify the XX- or YY-types by studying meiosis only. Unfortunately Hoffmann has not studied the somatic chromosomes of the various sex types where i t should be possible to settle the problem definitely, provided Yamada’s observations are correct (see Table 2). Sengbusch has not made any cytological investigations.

244

M. WESTERGAARD

Both authors agree th at the data are insufficient to settle their argument, and a further discussion here must likewise be considered premature. I n order to resolve this very interesting sex pattern in cultivated hemp, the following precautions must be taken: (1) The strains must be homogeneous, and raised under conditions such that contamination is excluded. (2) The crosses must likewise be carried out with better precautions against contamination. (3) The plants must be grown under uniform conditions during the whole growth period. (4) The proper classification of the male and female growth types in relationship t o the “locker” type must be cleared up. TABLE 15 The Offspring of Different Types of Hermaphroditic Spinacia*

All 9 3 1

0 3 0 1

0

1 0

1 1

2

* From Bemis and Williams

0 4 1

0 1 1 1

(1953).

(5) The somatic chromosomes of the various sex types must be studied in order to identify the sex chromosomes in the postulated XX and YY plants. (6) It will probably be necessary to carry out parallel experiments with wild strains which have not been exposed to human selection. It is fully realized th at it will be impossible to fulfill most of these requirements if the work is done as a part of a practical breeding program. None the less, from a theoretical point of view it seems worth the effort. Spinacia. Recent work on sex determination in spinach is due to Bemis and Wilson (1953), Zoschke (1956), and Sugimoto (1948, the paper unavailable to the present author). Unfortunately the American authors have given only a very brief summary of their data (Table 15) and only ratios for the offspring of p q . The table certainly has a strong resemblance t o that of Vitis (Table 13). Assuming male heterogamety in both species, the data suggest th at in Spinacia also, YY-types are viable.

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SEX DETERMINATION I N PLANTS

Zoschke’s work is concerned with the production of a monoecious strain of spinach, because, as in Cannabis, the male plants are of less practical value than the females. The selection work is in many ways a repetition of the similar program in Cannabis, but the genetical data are preliminary and a discussion must be considered premature. Carica papaya. The papaya breeding work, which has been done on a large scale during the last decades, has been closely related to problems of sex genetics. Thanks to the work of Hofmeyr in South Africa, Storey on the Hawaiian Islands, and other workers, rather precise information about the inheritance of sex types has become available. There are three main sex forms in the papayas: males and females of dioecious strains, and hermaphrodites. The females are very stable and show little variation in sex expression whereas the male trees sometimes produce female TABLE 16 Crosses between the Different Sex Forms of Carica papaya*

9 x 3 O X $ “ 3 ” x “ 8” or selfedt ST X or selfed Q X 3 or reciprocal

* Condensed

1 1 1 1 1

0 1 0 2 1

1 0 2 0 1

from Hofmeyr (1938, 1939b); Storey (1953).

t Subandroecious males.

or hermaphroditic flowers during part of the year (subandroecious plants). Also the hermaphrodites may be female-sterile during part of the year, and the difference between males and hermaphrodites is according to Storey (1953) mainly in the shape of the inflorescence. The results of crosses between the three main sex forms are summarized in Table 16. The crosses show a very interesting deviation from the pattern which has become familiar from the foregoing tables. The off spring of hermaphrodites or of subandroecious males give 1 :2 ratios, not 1:3 ratios. In such crosses 25% loss of fertility is invariably found. This means that the YY-combination is lethal, or-expressed in a different way-plants without an X-chromosome are inviable. As mentioned in the above paragraph (Section IV, l),heteromorphic sex chromosomes have not been found in Carica. Both Hofmeyr (1938, 1939a, 1939b, 1953) and Storey (1953) have proposed various formulae to explain the segregations shown in Table 16. As in the preceding cases the discussion of these hypotheses will be post-

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M. WESTERGAARD

poned to a later paragraph, here we only intend to state the actual experimental facts. Such 1 :2 segregations, indicating that the YY-combination is inviable, is probably also characteristic of all dioecious species with heteromorphic sex chromosomes, like Melandrium (Winge, 1931). Plants without at least one X-chromosome have never been described in Humulus, Rumex, and other species with more complex sex chromosome structure. Hence we may consider the viability of the YY-combination in Mercurialis, Thalictrum, etc., as a primitive trait, indicating that the dioecious species is not very far removed from its bisexual ancestors. The significance of this will be discussed in more detail in Section VI. I n Antennaria dioica, Ubisch (1936) has studied the progeny of subandroecious males. The data (71 9 9 :51 $ $ :99 g g ) suggest a 1:2 ratio of 9 9 : 0” d’ g g (71: 140). No data are given for seed fertility in such plants. It would be interesting to have this work followed up. The evidence as to the heterogametic sex in plants, as it appears from the data presented in this chapter may be summarized as follows: (1) There is well-established cytological evidence for male heterogamety of all the species listed in Table 2. Compound sex chromosomes have been convincingly demonstrated in Humulus and Rumex only. (2) The cytological evidence for male (or in the case of Fragaria) female heterogamety of the species listed in Table 3 is insufficient to be accepted without reservation. (3) The occurrence of females constituted XX and males XO has not been demonstrated convincingly in plants. (4) Male heterogamety has been convincingly demonstrated genetically through interspecific crossings in Bryonia, Acnida, and Ecballium. ( 5 ) By the same method female heterogamety has been established convincingly in all dioecious species of Fragaria (42, 6z, 8 x species). (6) Male heterogamety has been convincingly established in the following subdioecious species: Mercurialis, Thalictrum, Asparagus, Vitis, Cannabis, Spinacia, and Carica. (7) Incomplete data suggest female heterogamety in some strains of Silene otites. Silene roemeri, Valeriana dioica, and Circium arvense (Correns, 1928) need a reinvestigation, before any definite conclusions can be drawn. ( 8 ) The older genetical data suggest female heterogamety in many other species (Saliz, Populus, and others, see Allen, 1940 for older references, and Seita, 1953, 1954 for more recent work). Preliminary investigations on Morus alba are rather confusing (Schaffner, 1925, 1929, 1936) because both subgynoecious and subandroecious plants give 9 9 , d’3 , and g g ; no recent investigations are known to the present author.

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S E X DETERMINATION I N PLANTS

V. THELOCALIZATION OF

THE

SEX-DECIDING GENES

I n bisexual plants both the sexual potentialities (maleness and femaleness) of the zygote are released. I n dioecious plants, one of the potentialities is suppressed (the male potentiality in the female and vice versa). I n species with a genetic sex-determining mechanism this suppression is brought about through the sex-deciding trigger mechanism. We shall now try to analyze the genetical trigger mechanism somewhat in detail. The first problem is to localize the sex-deciding genes in one or both sex chromosomes. The next step is to find out, how these genes may interact with autosomal sex genes, with cytoplasmic factors, and possibly, with the environment as well. The third step is to try to explain the functioning of the sex-determining mechanism in terms of gene action. It should always be kept in mind, that there are two aspects to these problems, a qualitative aspect and a quantitative one. I n diploid dioecious species the information obtained is principally on the qualitative level. In order to study the problems on the quantitative level, it is in most cases necessary to draw upon information obtained from polyploid material. Therefore the material for such an analysis of the sex-determining mechanism is provided partly from the material presented in the preceding chapter, and partly from the study of natural and experimentally produced polyploids to be described in this chapter. The fact that the heterogametic sex has been identified by cytological methods does not per se tell anything about the localization of the sexdeciding genes except if one sex is of the XO-type in which case the sexdeciding genes must obviously be in the X-chromosome-with the possibility of cytoplasmic factors as well. Since it is still doubtful whether this type occurs in plants, we shall restrict the discussion to the genetical data.

1. Evidence from Crosses between Dioecious and Monoecious Species As shown in Section IV, 2, crosses of dioecious males and females with monoecious species show a very polymorphic pattern: Let x represent the female gamete from the dioecious species (A X) ; y the male gamete (A Y), and z the gamete from the bisexual species, then the sex expression in the combinations xz and yz in the various crossings may be summarized as shown in Table 17. Two striking facts appear from this table. One is the consistency in sex expression of the yz combination, which always gives a male plant, the second one is the variation in sex expression in the xz combination: I n Bryonia and “first type” Amaranthus crosses, such plants are females.

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M. WESTERGAARD

I n “second type” Amaranthus crossings it is a male and in Ecballium it is a bisexual plant. Thalictrum probably takes up an intermediate position between Ecballium and “second type ” Amaranthus hybrids. On the basis of this pattern, it is tempting to infer that the Y-chromosome in all these dioecious species contain sex-deciding genes which in some way or another prevent the manifestation of the female sex potentials. The same line of reasoning suggests that the X-chromosome of Bryonia likewise contains sex-deciding genes which prevent the formation of the male organs, and that the Ecballium X-chromosome plays no role in sex determination. There is, however, a serious objection to such an inference: Conclusions about the interaction of two gametes in intraspecific crosses are drawn from their interactions in crosses to another species. What we TABLE 17 Sex Expression in Crosses between Dioecious and Monoecious or Hermaphroditic Species

Acnida X 1-type ” Amaranthus

Bryonia dioica X alba

Acnida X “&type” Amaranthus

Ecballium dioicum X monoicum

+

Thalictrum fendlen X hermaphrodites

want to know, is how the combination xx (2A XX) results in femaleness and xy (2A XY) in maleness, and this we deduce from the separate interaction of x and y with the “neutral” z-gamete. The validity of this reservation is amply demonstrated from the behavior of the Acnida tamariscina x-gamete when it combines with a z-gamete from a “first type” Amaranthus species, and with a 2;-gamete of a “second type” Amaranthus. I n the first case the xz-combination results in a female, in the second case it is a male. This is a beautiful example of what is called “relative sexuality” in Hartmann’s terminology. It is at the same time a warning against deducing from the “external balance” (xz and yz), to the “internal balance” xx and xy. The various pitfalls of this method are also apparent from Heilbronn’s crosses between two dioecious Bryonia species which in some combinations gave bisexual off spring, and from the crosses between two monoecious species which may give unisexual offspring (cf. Table 4). As long as such interspecific crosses result in sterile F1 families it is, moreover, difficult to decide how important a role cytoplasmic factors and interaction between autosomal genes and sex chromosome localized

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SEX DETERMINATION IN PLANTS

249

genes actually play in sex determinination. Even when the F1 hybrids are slightly fertile as in Bryonia and in some Acnida-Amaranthus crosses it is difficult to accept too elaborate factorial explanations for such crosses, because the high degree of sterility makes a strong gametic selection possible. Both Heilbronn and Murray have proposed various formulae to explain the results of their F2 and backcross families, but one can only agree with Hartmann (1956) that such formulae go far beyond what can be safely deduced from the very scanty data [Heilbronn’s material comprises only 25 plants representing 6 backcross and F2 generations (Table 4)J. GalBn’s very unique Ecballium material is obviously not open to the same criticism, because the F1 hybrids are fully fertile so that very large F2 and backcross families could be raised. Galftn has proposed a multiple allelic scheme to explain the results of his crossings. He postulates three multiple alleles a D , a+ and ad. The first one is dominant over a+ and ad, whereas a+ is dominant:over ad. The monoecious Ecballium has the formula a+a+, a male plant of the dioecious variety has the formula a d a D whereas a female is adad.This hypothesis gives a satisfactory explanation of all the crosses (see Table 7). It is obviously a “Y-chromosome” mechanism, because the dominant sex-deciding allele is carried in the Y-chromosome. In the present author’s opinion it is, however, doubtful, whether such a simple multiple allelic scheme-although formally satisfactory-is sufficient to explain the evolution of a dioecious species from a monoecious one. This problem will be discussed in the following chapter (see also Table 20). 2. Evidence from Subdioecious Species

There is an interesting possibility for obtaining some information about the localization of the sex-deciding genes from the investigations of a number of subdioecious species. In Thalictrum, Asparagus, Mercurialis, and others, two types of males have been identified, XY and YY (cf. Table 11). In such species a plant is apparently a male irrespectively of the presence or absence of an X-chromosome, and irrespective of whether the Y-chromosome autosome ratio is 1:2 or 2:2. I n Bridges’ classical work on sex determination in Drosophila and from Ono’s and Yamamoto’s work on Rumex acetosa it could be shown that the sex was independent of the presence or absence of the Y-chromosome; the sexdeciding genes are in the X-chromosome, and the sex depends upon the X/autosome ratio. Here we have the opposite pattern, and we can make the opposite deduction namely the sex-deciding genes are in the Y-chromosome. Apparently the presence of a Y-chromosome makes a male and its absence makes a female.

250

M. WESTERQAARD

There is an interesting difference between Mercurialis on the one hand, and Thalictrum-Asparagus on the other hand (Table 11). I n the latter species the XY- and YY-males are identical both with respect to morphology and sex. In Mercurialis they are morphologically identical, but the YY-males have a much reduced pollen fertility. This suggests the interesting possibility that in this species some of the genes necessary for full male fertility are provided by the normal X-chromosome. It may of course also be due to the altered Y/autosome ratio ( 2 : 2 against normally 1 :2). The segregation ratios found in Vitis (Table 12) also suggest that the YY-combination is viable (cross 2 ) . I n Spinacia (Table 15)) the ratio 1 3 : 3 8 8 or Q Q has not been observed, but some hermaphrodites give a ratio of 3 Q Q : 1 8 . It seems premature t o comment upon the interesting segregation ratios found in Spinacia, until a more detailed publication is available. Both in Vitis and in Spinacia, certain types of hermaphrodites segregate 9 : 3 : 4 ratios when selfed (Tables 12 and 15). Evidently, at least one pair of autosomal sex genes must interact with the sex chromosome genes in such types. It should be remembered that we are here dealing with secondary hermaphrodites in species which have for a long time been exposed to human selection, which has aimed at stabilizing more or less bisexual strains of an original dioecious species. It will probably not be possible to understand the sex-determining mechanism in such species unless the sex-determination in the wild, dioecious ancestors of the cultivated strains is also investigated. To sum up: We may deduce from the 1 9 : 3 8 c? ratios in subdioecious plants that the Y-chromosome must contain the sex-deciding genes. However, this is primarily a qualitative statement and no material has so far become available which allows us to study the mechanism on a quantitative level. I n Carica papaya (and maybe also in Antennaria dioica) the 1 :3 ratio is changed into an 1:2 ratio, and this change has been accompanied by a 25% reduction of fertility, e.g., the YY-type is inviable. I n Thalictrum, etc., the Y-chromosome apparently differ from the X-chromosome in the sex-deciding genes only, and it can replace the X-chromosome in all respects except in sex determination. In Curica, X and Y have become differentiated to such an extent that vitality genes present in the X-chromosome have been lost in the Y-chromosome. I n such plants, with " 1 :2 ratios," it is not possible to locate the sex-deciding genes without further data. Hofmeyr and Storey have, however, from the Fz ratios and from crosses between hermaphroditic and dioecious types (Table 16) proposed a factorial explanation of the sex-determining mechanism which strongly

SEX DETERMINATION I N PLANTS

251

resembles the multiple allelic formula which Galhn has introduced to explain the Ecballium data. According t o these formulae a female Carica has the constitution mm, a male is nl,m and a hermaphrodite M 2 m , the three genes M I , M z , and m being allelic. The combination M I M 1 ,M2Ad2, and M I M z are inviable. Again such a hypothesis gives a fully satisfactory explanation of all the observed segregations. However, as realized by Storey (1953), the formulae may be too simple to give a satisfactory explanation of how such a dioecious system has originated from bisexual ancestors during the course of evolution. This will be discussed in the next chapter. There are obvious limitations to the information which can be obtained from crosses on the diploid level simply because the number of combinations between sex chromosomes and autosomes are too limited to allow an analysis on the quantitative level. The best way to break up the internal balance between sex chromosomes and autosomes is obviously through polyploidy, the method which has been so successfully applied to Drosophila and Rumex acetosa. The possibilitics for this " polyploidy method" are of course much better in plants than in animals. It is surprising that i t has so far been extended to so few species. 3. Evidence from Polyploids

Many spontaneous triploid, tetraploid, and aneuploid mutants of dioecious plants have been described in Rumex acelosa. An investigation of the sex expression of such aberrant types by Yamamoto, Ono, and other Japanese workers show convincingly th a t sex determination in this species follows the Drosophila scheme, e.g., sex is determined by an X-chromosome/autosome balaiice, the Y-chromosome playing no role in sex determination. This excellent work still stands as a classical demonstration of the Drosophila type of balance in plants (for summary see Ono, 1935 arid Yamamoto, 1938). I n Populus tremula, spontaneous triploids were discovered in 1936 by Nilsson-Ehle. These triploids have now been crossed to diploids, and preliminary investigations of the sex-determining mechanism has been reported by Johnsson (1940, 1942, 1945). The data suggest a sex-determining mechanism different from th at found in Rumex, and more like the one found in Melandrium (to be described below). However, the data must be considered preliminary. Seitz (1953) has studied triploid hermaphrodites originating from a diploid, hermaphroditic Populus type. Triploid Salix have been reported, among others by Hskansson, 1938. I n Coccinea indica, Kumar and Vishveshwaralah (1952) found a triploid plant (3A XXY) which was of male sex.

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M. WESTERGAARD

Experimentally produced polyploid strains of dioecious species have been reported in many cases, e.g., Humulus japonicus, Humulus lupulus var. cordifolius, Cannabis, Spinacia, Rumex acetosa, acetosella, and hastatus, Fragaria elatior, and Asparagus (Ono, 1939a). Polyploid Humulus lupulus strains have been produced by Dark (1952), Skovsted (1953), and Neve and Farvar (1954). Tetraploid Carica papaya have been produced by Hofmeyr and van Elden (1942), Hofmeyr (1945) ; tetraploid grapes (hermaphroditic strains) have been produced in several countries (see Olmo, 1952). Warmke and Blakeslee (1939a) report the production of tetraploid spinach, hemp, and Silene otites, in addition to Melandrium. Tetraploid plants of Acnida have been made by Murray (1940b). Tetraploid strains of Rumex angiocarpus, etc., have been briefly reported by A. Love and Sarkar (1956). By far the most extensive work has, however, been done on tetraploid Melandrium (Lychnis). The first successful results of chromosome doubling in this species was published by Warmke and Blakeslee (1939a), Westergaard (1938) and Ono (193913). More detailed accounts were given by Warmke and Blakeslee (1939b, 1940), Warmke (1946), Westergaard (1940, 1946, 1948), and summarized by Westergaard (1953) (see also Ono, 1940b; Rizet, 1945; Nygren, 1955). Except in the case of Melandrium and Acnida, only preliminary results have been published. Melandrium. It is not a coincidence that so many authors choose Melandrium for studying sex determination in experimental polyploids. It is one of the classical objects in sex genetics, Correns, Baur, G. and P. Hertwig, Shull, and Winge had already made comprehensive studies on sex determination in the natural diploid strains (for summaries see Correns, 1928; Winge, 1931; Allen, 1940). The heterogametic sex had been identified, different types of sex-linked inheritance were discovered, and the inheritance of different types of diploid hermaphrodites was investigated. The diploid hermaphrodites in Melandrium show a great variation in sex expression. Most of them are “ androhermaphrodites” in which only the first flowers are bisexual, the later flowers being pure males. Others are ‘(euhermaphrodites” (this terminology was proposed by Correns) with all the flowers bisexual. Some of the older hermaphrodites originated in the inbred off spring of crosses between Melandrium album and rubrum. Others came in inbred strains of the pure species. Some must have arisen by spontaneous mutations. Finally Correns recovered a very peculiar type of hermaphrodite after pollinating normal females with stored pollen (30-day-old pollen) from normal males. Some of the diploid hermaphrodites give offspring consisting of females, hermaphrodites, and some males. Others give exclusively females and hermaphrodites, and Winge found a type which bred true,

SEX DETERMINATION IN PLANTS

253

giving hermaphroditic offspring only. This is explained as being due to a balanced lethal system. All the diploid hermaphrodites must he considered to be heteroganietic, modified, or mutated XY-males. Correns’ hermaphrodites which originated from pollination with old pollen behaved differently because they gave mostly female offspring with a few hermaphrodites, but no males. The genetics of this type of hermaphrodite can still not be considered satisfactorily explained. I n polyploid Melandrium plants which originated by colchicine treatment or temperature shock from diploid strains, very comprehensive material is now available. When the data provided by the various authors are pooled, it is possible to get a rather clear-cut picture of: (1) The role of the Y-chromosome in sex-determination; (2) The role of the X-chromosomes; (3) The role of the autosomes. The material can conveniently be grouped in three parts: (a) Plants with euploid autosomes sets, but with aneuploid numbers of sex chromosomes. (b) Plants with a fragmented Y-chromosome (and in one case also a fragmented X-chromosome). (c) Triploid offspring with various combinations of sex chromosomes and autosomes. (a) Sex expression in plants with euploid autosome sets. These data are summarized in Table 18 together with the corresponding typesof Drosophila, and a thorough description of this material is given by Warmke (1946a). The table shows conclusively that the sex-deciding mechanism in Melandrium is quite different from that found in D.rosophila (and in R u m e x acetosa). I n the euploid Melandrium plants, a plant is a male if the Y-chromosome is present and a female if it is absent. I n Drosophila the sex expression is independent of the presence or absence of the Y-chromosome. In Melandrium the sex-determining influence of one Y-chromosome is so strong that it can suppress the female potentials of three X-chromosomes and four sets of autosomes. However, when this ratio is shifted from Y/XXX to Y/XXXX the female potentialities of the X-chromosomes become expressed, since such plants are bisexual in most flowers (in Warmke’s strains; they are mostly pure males or only slightly hermaphroditic in Westergaard’s strains). Warmke also found that plants with 2A XXY, 3A XXY, 4A XXY, where the X/Y chromosome ratio is 2/1 have some hermaphroditic flowers. However, as pointed out by Warmke, the sex expression in the series having XXY plus 2, 3, or 4 sets of autosomes is not influenced by the number of autosomal sets. It is therefore not possible to show any influence of the autosomes on sex determination in the euploid strains. The main conclusion from this material is that the Y-chromosome must play a decisive part in the sex-determining mechanism in Melandrium and this of course raises the question: How does the mechanism

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254

M. WESTERQAARD

operate? It has been possible to get important information on this point from plants with fragmented Y-chromosomes. (b) Plants with fragmented Y-chromosomes. Such plants have arisen both in Warmke's and in Westergaard's material but only very brief reports are available on the American material (Warmke, 1946a-c) ; a TABLE 18 The Relation between Chromosome Constitution and Sex in Melandrium and Drosophila*

Chromosome constitution

+ XX + XXX +X + XX + XXX + XX + XXX + XXXX + XXXXX 10. 2A + XY 11. 2A + XYY 12. 2A + XXY 13. 2A + XXYY 14. 3A + XY 15. 3A + XXY 16. 3A + XXXY 17. 4A + XY 18. 4 A + X X Y 19. 4A + XXYY 20. 4A + XXXY 21. 4A + XXXYY 22. 4A + XXXXY 23. 4A + XXXXYY 1. 2A 2. 2A 3. 3A 4. 3A 5. 3A 6. 4A 7. 4A 8. 4A 9.4A

Melandrium

Drosophila

0 0

0 0 d

Q

0 d

Q

0

8

d d d

d 0 0

d d d d d d d d

-

Q

0

-

g

$4

8

-

-

* From Westergaard (1953); see also Warmke (1946a), and Bridges (1939). more detailed account is given by the present author (Westergaard, 1946). Plants with deficient Y-chromosomes may also occur in diploid strains. Wkerlund (1927) has described the only case of a Melandrium hermaphrodite with the constitution AA XX. However, as pointed out by Westergaard (1946) a fragmented Y-chromosome has the same size as a normal X-chromosome and may be classified as such. I n Melandrium two normal X-chromosomes are associated by two terminal chiasmata in most cells during metaphase I (Westergaard, 1940). However, Akerlund's " X X "-chromosomes never showed this expected

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association, they were always associated in one arm only, as would be the case if he actually had a plant with a normal X-chromosome and a fragmented Y-chromosome. There are two main types of fragmented Y-chromosomes. In the first (Y1, see Fig. 1) the distal part of the differential segment is absent. Such plants are euhermaphrodites, e.g., both male and female sex organs are normally developed and all the flowers are bisexual. When crossed to normal females or selfed, such plants give females and hermaphrodites, but never males; when pollinated by normal males, they give females

FIG.1. Schematic drawing of the sex chromosomes of Melandrium. 1-11-111: The differential segment of Y. IV: The homologous segments. V: Differential segment of X. The Y *-chromosome has lost segment I. Segments 111-IV are absent in the Y 3-chromosome. Segment I contains the female suppressor gene(s) SUF. Segment I1 contains the gene(s) M I which initiates anther development. Segment I11 controls the last stages in anther development ( M , ) . See text and Fig. 2 (from Westergaard, 1953).

with normal X-chromosomes, hermaphrodites with the fragmented Y-chromosome plus one o r more X-chromosomes, and normal males which in addition to a normal Y-chromosome and one or more X-chromosomes may have a fragmented Y-chromosome (the inheritance of these types were mainly followed in polyploid material). Evidently, the genetic function of the segment of the normal Y-chromosome which is lost in the Y1-type is to suppress the formation of the female sex organs. Since the absence of this segment does not affect the normal development of the male organs or of the vegetative characters of the plants, it is unlikely that other genes are located in this region. A second type of aberrant Y-chromosome, designated Y3, lacks the pairing segment and part of the differential segment (Fig. 1). Plants with Y3, instead of a normal Y-chromosome, are male-sterile; PMC’s

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M. WESTERGAARD

are formed in the anthers and they go through a normal meiosis, but then they degenerate and viable pollen is never formed. Fortunately some of these male-sterile plants, which were found among triploids, were slightly hermaphroditic and could be crossed t o normal males. Plants with one or more fragmented Y3-chromosomes are always malesterile, but the loss of this section of the Y-chromosome does not release the female potency of the zygote. Plants with a normal Y-chromosome Ya are normal males. By means of such fragmentations, it has thus been possible to identify three different regions of the Y-chromosome, each with a separate function in sex determination. If the distal part is absent (the Y1-type), a normal bisexual plant results. Therefore the function of this segment must be to suppress the formation of the female sex organs. If part of the other arm is lost (the Y3-type) a sterile male results. Hence this segment must include genes (or a gene) which controls the last stages in anther development. If the whole Y-chromosome is absent (in XXplants), a female plant is the result. Hence the middle region of the Y-chromosome must include genes (or a gene) which control the initiation of anther formation. These results give a very clear picture of how the Y-chromosome in M e l a n d r i u m determines the sex: Through complete linkage between genes (or a gene), which suppresses female development and genes which initiate and complete anther development. I have previously (1953) compared M e l a n d r i u m to a machine which has 2 potential functions, male production and female production. A machine can be stopped in two different ways, either by applying the brakes or by removing some essential wheels without which the machine cannot work. The Y-chromosome serves both purposes. When it is present the brakes are pulled on the female-producing part of the engine, and the essential wheels are in place in the male-producing part. The result is a male plant. When the Y-chromosome is absent the brake is released from the femaleproducing part, but at the same time, some essential wheels are removed from the male-producing part, the result is a female. The efficiency of this principle is due to the complete linkage between the “female brake” and some essential “male wheels.” We may carry this analogy one step further. If the dimensions of the female machinery are increased (by increasing, for instance, the number of X-chromosomes) the brake is no longer strong enough to stop the female engine from running, and a bisexual plant is produced. It is, however, much more difficult to compensate for the loss of some essential wheels by increasing the dimensions of the other wheels, and this may be why bisexual plants without a Y-chromosome or a fragment of a Y-chromosome have never been con-

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vincingly established in Melandrium. The only exception to this rule is that female plants infected with the smut-fungus Ustilago violacea develop anthers. Evidently the fungus can do the same trick as the Y-chromosome. Is i t because it provides some essential sex hormone, the production of which is normally controlled b y the anther-initiating gene in the Y-chromosome? If this question could be answered we might perhaps have the clue to the biochemical and physiological aspect of the sexdetermination mechanism (Westergaard, 1953). Warmke (1946a) has also obtained plants in which the differential arm of the X-chromosome is absent, and he also has shown th a t the female potencies in the X-chromosome must be located in this arm as would be expected. (c) Sex expression in ogspring of triploid plants. Neither the euploid series, nor the plants with aberrant sex chromosomes give any reason to believe that the autosomes play any role in sex determination, and this is also Warmke’s conclusion. This is further supported by the fact th a t he gets only a few hermaphroditic plants among the offspring of triploid plants ( 4 out of 200). Moreover no bisexual plant appeared among 36 primary trisomic females ( 2 A a XX). This, however, would hardly be expected in view of what we now know about the role of the Y-chromosome; unfortunately no comparable data are available on trisomic males (2A a XY). The present author’s triploid material behaved strikingly different from that of the American investigators (Westergaard, 1948). Among 46 plants from the cross 3n Q ( 3 A X X X ) X 3n 3 (3A XXY) 10 plants were more or less hermaphroditic, 21 were females, 15 males. The off spring of these hermaphrodites was studied through several generations and the results, which are grouped in Table 19 can only be explained by assuming that both the X-chromosomes and certain autosome combinations may, under special conditions, counterbalance the female suppressor in the Y-chromosome. The role of the X-chromosome is evident from the fact that when the number of X-chromosomes in the triploid material increases from 1 t o 4, the number of hermaphrodites increases from 0-100%. The role of the autosomes is evident from the variation in sex expression in plants with the same sex chromosome constitution but with different autosome combinations. Among 205 aneuploid XXXY-plants, 72 were males and 133 hermaphrodites. This variation can only be ascribed to the single variable factor, the autosomes. The hermaphrodites which appeared among these triploid families were different from the euhermaphrodites originating through fragmentation of the Y-chromosomes. The former were mostly “androhermaphrodites” with only the first flower being bisexual. However, i t was

+ +

+ +

+

+

258

M. WESTERGAARD

possible through selection to shift the sex expression towards that of the euhermaphroditic phenotype. However, all such plants, when crossed to females or selfed, gave both males, females, and hermaphrodites in the progeny, whereas the euhermaphrodites originating through loss of the female suppressor, never gave male off spring. We can now summarize the sex-deciding mechanism in Melandrium as follows: The Y-chromosome plays a decisive part in sex determination. The trigger mechanism is built up by an absolute linkage between a female suppressor region and a t least 2 blocks of essential male sex genes. This trigger mechanism interacts with the X-chromosomes and the TABLE 19 Chromosome Constitution and Sex in Triploid Offspring Plants of MeZandrium*

Sex chromosome constitution

Number of X-chromosomes

$3

QQ

% 3d

Total ~~

XY XXY XXYY XXYYY

~

15

100

89

25 37 1

3 5 0

28 42 1

XXXY XXXYY

3

72 7

133 5

205 12

36

XXXXY XXXXYY

4

0 0

8 1

8 1

0

157

155

312

Total

* From Westergaard (1948). autosomes, the X-chromosome has female potencies, and so have certain autosomes. This quantitative interaction can, however, only be demonstrated when the normal balance is broken up through polyploidy and aneuploidy. Whether the female potentials of the X-chromosome and certain autosomes are controlled by major female sex genes or whether they contain blocks of modifying genes is unknown. The latter possibility appeals most to the present author. Acnida. Murray (1940b) produced tetraploids in Acnidu tamariscina by colchicine treatment. Crosses between tetraploid 4 X 0 X 4n XXYY 8 gave 131 9 9 : 1633 8 8 . This agrees with the findings in Melandrium, and is explained by the fact that X X X Y plants are males. XXXY-plants are always produced in excess in the cross XXXX 9 X X X Y Y 8

259

SEX DETERMINATION I N PLANTS

because a preponderance of X Y gametes are formed b y the X X Y Y X X X X Q X 4A X X X Y 8’ gave equal proplants. The cross 4A portions of males and females. As in Melandrium polyploidy is no hindrance for the establishment of a tetraploid, dioecious strain. The sex expression in triploids is in accordance with the assumption th a t the sex determination follows the Melandrium scheme, i.e., the Y-chromosome plays a decisive role in sex determination. Humulus. Both in Humulus lupulus and in H . japonicus, the d a ta are preliminary. Triploid strains of commercial diploid varieties of hops include many bisexual types, but such types were also present in some of the diploid parental strains. There is no report known to the present author of the cross 4n 9 X 4n c? (cf. Neve and Farrar, 1954). Ono (1940~)states that triploid Humulus japonicus, derived from crosses between diploid and colchicine-induced tetraploids, comprise females with 22 chromosomes and monoecious forms with 25 chromosomes. The preliminary evidence suggests th at sex determination in H . japonicus follows the Drosophila-Rumex acetosa scheme. Cannabis. Tetraploid families has been raised in hemp b y Warmke and Davidsson (1944), by Nishiyama et al. (1947); see also Takenaka (1953). Unlike in Melandrium and Acnida the offspring of the cross 4n Q X 4n 8’ results in a great excess of females and female hermaQP; phrodites, Warmke and Davidsson (1944) : 11 c? 8 :83 9 9 Both authors conNishiyama et al. (1947): 87% 9 9 and 13% 88’). clude that the XXXY-plants in Cannabis must be females or female hermaphrodites, not males as in Melandrium and Acnida. Whether we are here dealing with a “Drosophila-Rumex acetosa type” of balance, or with a third type of sex balance, remains to be seen. Silene otites. Warmke (1942) crossed tetraploid males and females and obtained a ratio of 5 8’ 8’ to 1 9.When tetraploid females were crossed to diploid males an equal proportion of males and females resulted. These data can only be explained by assuming male heterogamety in this strain of Silene otites and a “Melandrium type” of sex determination where the XXXY-plants are males. As pointed out previously, the possibility cannot be excluded that other strains of Silene have female heterogamety. Rumex acetosella. I n their paper on R. paucifolius, A. Love and Sarkar (1956) refer to a number of unpublished results with experimentally produced autotetraploids of the diploid Rumex angiocarpus. Apparently also the experimental polyploids of Rumex subgenus acetosella follow the “ Melandrium scheme.” Carica papaya. Hofmeyr (1945) crossed colchicine-induced tetraploid females and males and obtained a ratio of 3 8’c?:l 9 (51:21). The

+

+

+

260

M. WESTERGAARD

X X X Y plants are thus males but more female-like than the XXYYplants. Both types are subandroecious, however, and can be selfed. The XXYY-plants gave 12 3 3 and 2 Q Q , no data are given for the offspring of XXXY-plants or of triploids. Among the other species which were mentioned in the beginning of this section no detailed investigations on sex determination is known to the present author. It has only been stated th a t colchicine-induced tetraploids have been produced in one or both sexes. Summing up: An analysis of the sex-determining mechanism in diploid and polyploid plants have clearly shown the existence of a t least two types of trigger mechanisms: One type has a n “active Y-chromosome” which plays a decisive role in sex determination (Thalictrum, Mercurialis, Vitis, Asparagus, and other subdioecious species, Melandrium, Acnida, Carica, Rumex subgenus acetosella). I n others the X-chromosome/autosome ratio is a decisive factor in sex determination and the Y-chromosome is inactive (Rumex acetosa, H . japonicus) and perhaps also some cultivated strains of Cannabis sativa. It is, however, most likely that Cannabis may represent a new type, but i t might be expected that different commercial strains may behave differently and th a t i t will be necessary t o obtain tetraploids of the wild species, in order to get to the bottom of this problem. VI. THE EVOLUTION OF DIOECISM There is today sufficient information about the mechanism of sex determination in plants to show a t least two ways in which dioecism may be established. We shall take it for granted that, in the higher plants a t least, the bisexual state is the original one, and th a t dioecious species have originated from hermaphrodites or monoecious species through mutation and natural selection. The unisexual species differ from the bisexual ones only by having developed a trigger mechanism which suppresses the potentialities of the opposite sex in males and females. The trigger is balanced in such a way that the two sexes occur in nature in approximately equal proportions. As pointed out by Allen (1940) it is possible to study this interesting evolutionary process from two angles, namely, (1) the evolution from the bisexual to the unisexual state, and (2) the “ backwards evolution” from dioecism to bisexuality. The best information of the first type comes from the artificial synthesis of dioecious strains of maize (Emerson, 1932; Jones, 1932, 1934). The best information from the second “backward” step probably comes from the Melandrium material where the trigger mechanism can be analyzed by means of spontaneous chromosome mutations.

261

SEX DETERMINATION IN PLANTS

We shall try to fit this information into a more general pattern by approaching the problem in a somewhat roundabout way. We want to explain the evolution of dioecism from bisexuality in terms of specific mutations and gene function and for this purpose i t may be worthwhile to start from the field of genetics where most is known about gene function, namely the field of biochemical-physiological genetics. The work on Neurospora, Aspergillus, bacteria, etc. has shown convincingly th a t the synthesis of essential amino acids and vitamins goes through a series of intermediate steps, each step is catalyzed by a specific enzyme, the activity of which may be under controI of one specific gene. Let M be one such end-product, the synthesis of which may go through say ten steps, each under control of ten different genes (M1, M 2 . . . Mlo). Let F represent another end-product which in order to be synthesized, requires the presence of ten different genes (Fl, F 2 . . . Flo).For the synthesis of both M and F, all F- and M-genes must be present. If the expression of just one M-gene is blocked in some way, the end-product (M) cannot be synthesized even though all the other normal M-genes are present. If, for instance, MT does not function the chain of reactions activated by genes M I t o Me are possible, and the precursor, the synthesis of which is activated by M6, may pile up. We also know th at the normal function of a gene may be blocked in two different ways, namely by-mostly recessive-loss mutations or by dominant, epistatic suppressor mutations (Su). How(M1 --+ ml), ever, in each case, the expression of the mutant gene will of course depend upon the general genetical background. Hence both a system of the constitution ml, M 2 , M a . . . Mloor M 1 , M 2 , M a . . Mlo,XU* will be unable t o synthesize the end-product M. Let us now turn from this pattern which is so familiar in biochemical genetics (although it certainly has many more variations than stated here), t o the genetics of sexuality. Let M now stand for the normally developed and functional male sex, and F for the normal female sex. I n order t o make both the end-products M and F, all normal F-genes and M-genes must be functioning. This will make a bisexual plant. If M 1 and F1 represents the earliest, and Mloand F l o the latest steps in the development of the functional sex organs then a mutation in (or suppression of) the M1-gene will result in a plant which is purely female. A plant where, for instance ME,is blocked will be a female with rudiments of the opposite sex (staminodia) whereas, changes in the expression of the last genes (Ms-Mlo) may result in male sterility or imperfect male fertility. As pointed out above, there are two types of mutations which may interfere with the normal sex expression, namely recessive, loss mutations, and dominant, epistatic, suppressor mutations. A plant of the constitution :

.

262

M. WESTERGAARD

FIFl . . . FIOFIO,mlmlMzMz . . . MloMlowill be a female, but so will a plant of the constitution FlFl . . . F 1 ~ lMlMl ~ , . . . M I O M I OSU*. , There may be several ways in which such recessive and dominant mutations can be combined to build up a functional trigger mechanism, where one sex is homogametic and the other heterogametic. We shall, however, only consider two such possible combinations here, namely, the one which has been established in dioecious maize and the one which is found in Melandrium, because they are the only two for which there is good experimental evidence. I n maize, dioecious strains have been synthesized experimentally by combining two recessive mutations which occurred in normal monoecious corn (Emerson, 1932; Jones, 1932, 1934). One of Jones’ strains had the constitution: 9 : slc/sk, tsz/tsz and 3 : sk/sk, Tsz/tsz. sk is a mutation, “silkless” which in homozygous condition gives a male plant. ts, “tassel seed,” changes the male flowers into carpillate flowers, homozygous “ tassel seed ” plants are therefore females. Using the general F-M symbols proposed above, Sk would be a gene in the F-series and T s a gene in the M-series. The mutant genes, sk and ts may for instance be designated f l and ml, and we may write the formula for the dioecious maize as follows: 9 : mlmlflfl, 3 : M l m f z f l . I n Melandrium the trigger is of a different type, namely a combination of a recessive and a dominant mutation. The formula is 9:

xx

XY

where SUF is a female suppressor and SUF its normal allele. SuF and M I are absolutely linked. This is the essential or “primitive” Melandrium

trigger, which will be considered first. It is actually more complex, as will be shown later. Let us compare these two triggers in some details: (1) I n both cases, the dioecious species is two mutation steps away from the bisexual species. (2) I n maize, there is a one-gene difference between the two sexes. I n the Melandrium type there is a two-gene difference. (3) Both may be called ‘ I Y ”-mechanisms, since the dominant genes are carried on the Y-chromosome. Maize has, however, essentially an Y/autosome mechanism, since the sex is decided through interaction between the Y-chromosome and recessive autosomal genes. Melandrium has a n X/Y-mechanism. (4) The efficiency of the maize trigger is independent of whether the

SEX DETERMINATION I N PLANTS

263

genes are linked or not (in the actual case sk and t s happened to be on different chromosomes) because there is a one-gene difference between the X-and the Y-chromosome; suppression of crossing-over is therefore not required to make the trigger work, i.e., there is no need for a n evolution of a “differential segment.” The Melandrium trigger is only effective if the two genes are linked. Therefore a “differential segment” must necessarily be built up. (Crossing-over would of course be suppressed simultaneously with the mutation suF -+ SuF, if the mutation was accompanied by a chromosome inversion.) ( 5 ) The maize trigger depends upon a very special type of interaction between two recessive mutations, namely the mlmlffl plant being a female. This is probably not a very common type of interaction. I n Rubus similar mutations have been studied by Crane and Lawrence (1931). Here the mlmlflflplants were asexual “neuter” plants, as might a priori be expected, and they consequently failed to construct a dioecious strain in this species (cf. Lewis, 1942). It is probably only possible to build up a trigger mechanism from two recessive mutations when the double recessive is a female, in which case a dioecious system with male heterogamety may become established, or if it is a male plant where a system with female heterogamety may result. Emerson (1932) combined the two mutations ba (barren stalk) and a different tassel-seed mutation (ts3). Here the balba, t s 3 / t s 3 plant is a male and a strain with female heterogamety was constructed, the two sexes having the following constitution: 9 : balba, Ts3/ts3;3 : balba, ts3/ts3. ( 6 ) It is possible to predict the sex of offspring from crosses between dioecious species and the bisexual ‘(wild type” forms. I n maize such crosses ( 9 X g or B X 3 ) will always produce 100% bisexual plants (SklSk, T s / T s X s k / s k , Ts/ts or slc/sk, t s l t s ; all F1 plants have Sk and Ts and will be bisexual). I n the case of Melandrium, the situation is different. The (hypothetical) bisexual ancestor must have the constitution M1-suF. The cross 9 (ml-suF) X (M1-suF) will give bisexual plants. The cross g X 3 (M1-suF X M1-SuF and ml-suF) will give equal proportions of males and bisexual plants. Table 20 shows all the possible F1, Fz and backcross ratios expected with a trigger of the Melandrium type. No cases are known where crosses between dioecious and bisexual species give all bisexual offspring (cf. Table 17). This may indicate th a t the maize type of trigger, which does not require a differential segment, but which depends upon a very special type of interaction between two recessive mutations, is not of widespread occurrence in nature. The Melandrium trigger, however, which is a more conventional” combination of a recessive and a dominant mutation, will explain all the ratios ((

TABLE 20 The expected F1, F2, and backcross ratios from crossings between bisexual strains and dioecious strains with a “Melandriumtype” of trigger, which is built up through linkage between a dominant female suppressor (Sup)and a male-promoting gene M I in the Y-chromosome and the two recessive genes in the X-chromosome (sup-ml). The corresponding “wild type” chromosome of the bisexual strain, which we will call the Z-chromosome has the constit,ution (suF-MI).

N

$

Ratios found in Ecballium (Table 7)

0 0

100% (881) 50%

(378)

0 50% (423)

1

53.

2

!d

0

0

25 %

25 %

0

0 0

50 % 0

0

0

50 %

25 %

25 %

25% (16) 0

75% (34)

50%

(109)

0 50% (105)

Not made Not made 0 100% 0 (58) 0 50% 50% (550) (483) 25% 25% 50% (35) (30) (48)

0 P P

s U

265

S E X DETERMINATION I N PLANTS

which have been observed in the Ecballium crosses (Table 20, cf. Table 7). True enough, GalBn’s multiple allelic hypothesis will also explain the observed ratios, but it seems rather unlikely (although not impossible), that the difference between a dioecious male and female and a bisexual type should depend upon such a simple relationship, where a mutation from the bisexual a+ allele t o the dominant a” and to the recessive ad should be able to produce a stable dioecious state. Therefore we would like t o propose the primitive “Melandrium trigger” as a more likely explanation of the sex-determining mechanism in Ecballium. G alBn’s Carrca papaya m p ml V 0 ’ ” mp m1

Ecballium dioicum Ecballium monoicum MI

SUF

Mi

SUP

. ‘

0

z

ml

SUF

ml

‘ X SUF

:

.

,

Ml

SUF a

’

X

Melandrium

m7 f + ml V sup

0

m p ml

$ ml

SUF

SURX

x

2

d

v

’

:

mp M1

V

suF

u

SUE’

~:

X

X

d

SlP

m p ml V 6 ’ ” Mp M 1

SUF ’

-:

. x

’ X SUP

m7 f + ml

v

m7 f + ml ’ MI Mi

v suF . x

‘ Y u SUF

X

; .SUFY

FIG.2. Tentative chromosome maps of the differential segments of the sex chromosomes of Ecballium, Carica, and Melandrium. The Z-chromosome of Ecballium monoicum represents the bisexual “wild type ” state. Crossing-over is inhibited between the X- and the Y-chromosomes and between the X- and the Z-chromosomes of the secondary hermaphroditic Carica papaya. The gene symbols are explained in the text.

formulae do not require a differential segment where crossing-over is suppressed. If complete X- or Y-linked inheritance was found in Ecballium, this would disprove GalBn’s hypothesis. The results of the Carica crosses (Table 16) cannot be explained by a maize trigger, but can again by a modified Melandrium trigger. A multiple allelic system as proposed by Hofmeyr will of course always explain 1: 1, 1 :3, and 1:2 : 1 ratios in a formally satisfactory way, and it is difficult t o disprove. However, as pointed out also by Storey it is again most unlikely that dioecism can be established by a system which depends upon a one-gene difference between males, females, and hermaphrodites. I n order to explain the Carica crosses it is only necessary to make one more assumption, namely that the X-chromosome carries a vitality gene ( V ) which is absent in the Y-chromosome (v), the combination vv being therefore lethal. The X- and Y-chromosomes may therefore have the structure shown in Fig. 2.

266

M. WESTERGAARD

(7) The possible ways in which the two trigger mechanisms may break down may also be considered. The maize trigger can only break down through mutation and such mutations will tend to restore the stable bisexual state. It should be pointed out that a maize plant heterozygous both in Sk and Ts will give a ratio of 9 p p : 3 3 3,and 4 Q Q ; 1 out of 9 q p would breed true (such ratios were actually found by Jones). This is, however, not the ratio (9 p p : 3 9 9 :4 3 3)which has been observed in Vitis and Spinacia (Tables 11 and 15); hence it is unlikely that this is the actual trigger found in these species. The Melandrium trigger may break down through mutation or through occasional crossing-over in the differential segment. In the latter case we shall have (ml-SuF) on one chromosome and (M1-suF) on the other. If plants with gametes carrying such (M1-suF) chromosomes, whether they have originated from crossing-over or from a back-mutation in the suppressor locus, are crossed to normal females, bisexual plants of the constitution (mrsuF/MrsuF) will appear. If such bisexual plants are selfed, they will give a ratio of 1 Q :3 r$ p provided the YY-combination is viable, or else a 1:2 ratio. In the first case, of the bisexual plants will be homozygous both in M1 and in SUF, e.g., they will breed true. Hence, one back-mutation, or an occasional breakdown of the crossingover inhibition is enough to restore a constant bisexual state in such primitive, dioecious species where YY-plants are viable. Such constant hermaphroditic strains have actually been produced in Spinacia and Vitis.If the YY-constitution is inviable, as in Carica and in Melandrium, it is more difficult to reconstitute a true breeding bisexual strain. The only example of such a reconstitution is Winge’s constant hermaphroditic Melandrium strain which has been stabilized through an intricate balanced lethal system, where both the XX- and YY-combination is inviable. Figure 2 shows (‘chromosome maps” of the differential segments in Ecballium, Carica, and Melandrium, all derived from the Melandrium trigger ”-the reality of which is experimentally demonstrated in this species only. Ecballium would be the most primitive type, and it is probably also the dioecious wild species which is most closely related to its bisexual ancestor (var. monoicum) since the F, hybrids are fully fertile. Hence it is assumed that X and Y differ only in the sex-deciding genes. Carica represents a more advanced state, where some vitality genes, present in the X-chromosome, have been lost in the Y-chromosome. As pointed out by Storey, the Y1-chromosome of the secondary hermaphroditic Carica strains differs from the Y-chromosome of the male plants in a gene which controls the shape of the inflorescence. According to Storey this may actually be the only difference between the hermaph((

SEX DETERMINATION IN PLANTS

267

roditic and male plants of Carica. I n Melandrium the differential arm of the Y-chromosome also contains a gene (here arbitrarily called Mr) which controls the later stages in anther development. When it is absent (in plants with the fragmented Y3-chromosome) the plants are malesterile. I n addition, it is known th at the differential arm of the X-chromosome contains genes which, when present in sufficient quantity, counterbalance the female suppressor in Y. We have designated these genes by the symbol f+ (female modifiers) which enhance the expression of the F-genes. The results of the triploid Melandrium crosses, which show that the autosomes also interact with the sex chromosomes, can be explained by assuming th at some autosomes also contain ff-modifiers, others m+-modifiers. All the three species shown in Fig. 2 have active Y-chromosomes, Ecballium representing the most primitive, and Melandrium the most advanced type. Such a system, once established through mutations and crossing-over restriction, probably carries with it its own doom. The Y-chromosome, as the only “constant haploid chromosome’’ in a n otherwise diploid system, is very susceptible to loss mutations, and it will gradually become inert (cf. Darlington, 1932). This degeneration is already apparent in Carica and Melandrium where Y lacks certain vitality genes. Natural selection would therefore favor the evolution of a secondary trigger mechanism, where the Y-chromosome becomes outmaneuvered. This will have to be a n X-chromosome/autosome mechanism, because when the Y-chromosome is left out, the X-chromosome/ autosome balance is the only difference left between a male zygote (2A X) and a female zygote (2A XX)-still with the possibility of cytoplasmic interaction t o be kept in mind. This is the mechanism in Drosophila and certainly in all animals without Y-chromosomes. I n plants this balance has so far been demonstrated only in Rumex acetosa and perhaps also in Humulus japonicus. Both species also show other indications of representing an evolutionarily more advanced state which is much further removed from the bisexual ancestors. They both have compound sex chromosomes, and in H . japonicus at least, the Y-chromosomes are heterochromatic. There are several indications th a t Melandrium represents a transitional stage from a primitive Y-type to a secondary X/autosome-type. The Y-chromosome is partly inert, and the differential arms fail to pair in most PMC’s in polyploid XXYY-plants (Westergaard, 1940). Besides, modifying genes are present both in the sex chromosomes and in autosomes (ff-modifiers in the X-chromosome and in certain autosomes and m+-modifiers probably in other autosomes). Such modifiers may be regarded as the raw material from which a secondary X/autosome balance system may be built up through natural

+

+

268

M. WESTERGAARD

selection. As is well known the presence of f+-modifiers in the differential segment of the Drosophila X-chromosome has been demonstrated by Dobzhansky and Schultz (1934). We have gone through this process of filling out the differential segments of the sex chromosomes in some detail in order to show that there is no problem in sex determination which cannot be dealt with within the normal framework of genetic systems (recessive and dominant genes, modifiers, linkage, crossing-over, and mutations) and by using the conventional genetic symbols. Such a system describes fully satisfactorily the bisexual potentiality of both sexes which is the basic concept in sex genetics, and it takes into account both the quantitative and the qualitative aspect of the mechanisms. Nothing is therefore gained and much is lost by trying to build up a special symbol system in sex genetics as is still advocated by Hartmann (1956) and Goldschmidt (1955). As pointed out by Allen (1940): “A difficulty that has prevailed in the consideration of this problem (sex determinination) has been a persistance of a traditional conception of sexual differences as a category distinct from other individual differences” (see also Muller, 1932 ;Winge, 1937; Westergaard, 1948). One difficulty with the AGMF symbol system of Hartmann and the M / F balance system of Goldschmidt is that they were established at a time when Darlington’s concept of the structural hybridity of the heterogametic sex was not realized, and they failed to build this concept into the formula. When the preconceived symbol systems of Goldschmidt and Hartmann are applied to sex problems in dioecious plants, their inadequacy becomes quite evident. They become a strait jacket which limits instead of stimulates an unbiased analysis of the actual facts. To quote again from Allen’s paper: “Under the influence of a similar conception, the analysis of size inheritance would be even more difficult than it is now.”

VII. CYTOPLASMIC FACTORS IN SEX DETERMINATION There is probably no well-established case where sex determination in a strictly dioecious plant species is the function of the interaction between nuclear genes and the cytoplasm. I n the so-called gynodioecious species, however, which comprise females and hermaphrodites in approximately equal proportions, an outbreeding mechanism has become established in several species, which depends upon a cytoplasmic/genic interaction. The genetics of gynodioecious species like Cirsium oleraceum, Satureia hortensis and others were first studied by Correns (see Correns, 1928). Females (which of course must be pollinated by hermaphrodites) give only female off spring, and hermaphrodites ‘ (selfed or intercrossed) give hermaphroditic off spring. I n other gynodioecious species, the picture

SEX DETERMINATION IN PLANTS

269

is not as clear-cut, but cannot be explained by simple Mendelian segregations (see Lewis, 1942). The Cirsium-Satureia type is explained by cytoplasmic conditioned male sterility; the cytoplasm of the female suppresses anther development. This hypothesis which was first proposed by v. Wettstein is now generally accepted. Lewis (1941, 1942) has analyzed the adaptive value of the cytoplasmic conditioned outbreeding mechanism in gynodioecious species and he showed that such a cytoplasmic system is superior to a system where male sterility is controlled by nuclear genes, because if the system is genic conditioned, the maintenance of equilibrium between the two sexes in natural populations cannot be obtained unless the females have a much higher degree of fitness than the hermaphrodites. As Lewis also pointed out, gynodioecism is probably not a transitional state which may lead to the evolution of true dioecism. It is a n outbreeding system of its own merit which in plants may have certain advantages over the conventional dioecious outbreeding system (see also Mather, 1940). Recently Lewis and Crowe (1956) have studied the outbreeding mechanism in Origanum vulgare (Labiatae), a gynodioecious species which was formerly assumed to have cytoplasmic conditioned malesterility. However, they found th at the outbreeding mechanism is due to the interaction of two dominant genes; one gene, F , causes anther abortion,the other, H , suppresses the expression of the first one. Hermaphrodites have both dominant genes or H alone and may be FfHh, FFHH or $Hh. Females are Ffhh or PFhh. The double recessive type $hh is lethal. The equilibrium between the two sex types in natural populations is maintained through a slightly better fitness of the females over the hermaphrodites and the lethality of the $hh genotype. The discovery of this interesting outbreeding system may be a warning against accepting all cases of gynodioecism as being established through a cytoplasmic/ genic interaction. Cytoplasmic conditioned male sterility has been described in maize (see Rhoades, 1952). It is a phenomenon frequently encountered in interspecific crosses for instance in Epilobium hybrids (see Michaelis, 1951) and in Streptocarpus (see Oehlkers, 1941). Especially the beautiful Streptocarpus work of Oehlkers shows various types of quantitative interaction between the cytoplasm and the nuclear sex genes. Readers are referred t o Caspari’s earlier review of this work in the present journal (Caspari, 1948). VIII. THE PHYSIOLOGY OF SEX DETERMINATION One of the most unsatisfactory aspects of the work on genetics of sex determination is its remoteness from any attempt to bring the prob-

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lems down to the biochemical and physiological level. Unfortunately, in this respect the work on plants and animals share the same fate. Several plant physiologists have studied the influence of the environment on flower formation and sex expression in hermaphrodites or monoecious species (see Nitsch et al., 1952; Resende, 1950; Resende and Kana, 1952). Nitsch and his colleagues studied the effect of temperature and day length on the development on male and female flowers on the acorn squash (Cucurbita pepo). High temperature and long days promote the formation of male flowers, whereas low temperature and short days speeds up the development of the female flowers. As the ultimate result of this feminization, parthenocarpic fruits were produced. Both Nitsch et al. and Resende (the latter working on Bryophyllum and Kalanchoe) points t o the possible relationship between auxins and sex expression and Resende emphasizes especially the auxin-antiauxin balance. See also the recent review by Heslop Harrison (1957), and the paper by the same author on auxin and sexuality in Cannabis (Heslop Harrison, 1956). Experiments to influence the sex by grafting females on males or vice versa are negative (see Kuhn, 1941) although some recent Russian experiments of the “Lysenko type” claim positive results. A. and D. Love (1940, 1945) claim to have changed the sex expression in some dioecious plants by applying animal sex hormones. The evidence is not too convincing, and the work has been strongly criticized by Kuhn (1941). It should be repeated with proper controls before it can be accepted without reservation. Basarman (1946) has made physiological studies of both sexes of Mercurialis, but this is the only investigation known t o the present author on the physiology of subdioecious plants. This interesting group seems to have been badly neglected by plant physiologists. There are a number of facts from the genetics of sexuality in plants which suggest that there may be a competition for a common substrate (precursor) a t some stage during the process of differentiation of male and female sex organs and it is tempting again to think of the auxins in this connection, but no satisfactory experiments are yet available. As pointed out previously (see also Westergaard, 1953), the stimulation of anther development in Melandrium females infected by Ustilago violacea may one day open up the unknown territory of the physiological genetics of sexuality in plants. A second clue may come from the observation that certain plant hormones like maleic hydrazide induce male sterility. This interesting chemical which also cuts down plant respiration (and induces chromosome breakages in the heterochromatin) has been shown to induce male sterility in several species (Rehm, 1952; Wittwer and Hillyer, 1954; see also Zukel, 1952). It might finally be pointed out that the genetic interaction between

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27 1

sex-promoting genes ( M - and F-genes) and sex-inhibiting suppressor genes from which a trigger mechanism may be built up is very close to the physiological concept of promotion and inhibition, as i t has recently been discussed by Thimann (1956).

IX. POLYPLOIDY AND DIOECISM There are two striking differences in the evolutionary pattern of plants and animals, namely the scarcity of dioecious species and the widespread occurrence of polyploidy in plants, against the scarcity of polyploids in animals where most of the species are dioecious. It is obviously tempting t o link these two phenomena together and explain the absence of polyploidy among animals through their dioecious outbreeding system. This was pointed out by Muller (1925) and his argumentation was strongly supported by the Drosophila type of sex determination. I n Drosophila tetraploids would be X X X X 9 and X X Y Y $. Crossings between such tetraploids would give a preponderance of X X X Y types which in Drosophila are sterile intersexes and become a barrier which will prevent the establishment of a tetraploid, dioecious strain. Such a barrier is, however, not established if tetraploid X X X Y individuals are males as in Melandrium, Acnida, Rumex acetosella, etc. On the contrary: From the crossings between a X X X X P and a X X X Y 3,a tetraploid dioecious strain with equal proportions of males and females will result, where the females are 4A X X X X , and the males 4A XXXY. Such constant, tetraploid and dioecious strains have been constructed both in Melandrium, Acnida, and Silene otites without any difficulties. It is possible to make two predictions from Muller’s hypothesis: (1) dioecism should be confined to the diploid plant species and be rare among polyploids, and (2) sexual polyploids would be expected to occur among hermaphroditic animals. However, none of these two predictions have been confirmed. Table 21 gives a list of polyploid, dioecious plants. The most interesting cases are those of Fragaria and Rumex. I n strawberries, dioecism has apparently been established on the tetraploid level and from there on is maintained in hexaploids and octoploids. I n Rumex subgenus acetosa, the recent interesting work of Smith (1955) leads to the conclusion that Rumex acetosa must be a tetraploid; diploids being represented by the American species Rumex hastatulus. Although Rumex acetosa has a Drosophila type of sex balance, it nevertheless seems to be of a polyploid origin! The genus Rumex certainly offers unique possibilities for studying the evolution of different outbreeding mechanism, as pointed out by A. Love a t several occasions, but until more d a ta are available especially on experimentally produced polypoids of the diploid

+

+

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species, there is no point in discussing this interesting Rumex pattern any further. Muller’s hypothesis is also weakened by the scarcity of sexual polyploids among hermaphroditic animals. When polyploidy occurs in animals, the polyploids are almost always parthenogenetic, irrespective of whether the diploids are hermaphroditic or dioecious. The recent investigations of polyploidy among, for instance, Oligochaetes (Muldal, 1952 for earthworms; and Christensen and Nielsen, 1955 for Enchytraes) show quite clearly that polyploidy always causes a breakdown of sexual reproduction even in such strictly hermaphroditic families. Further TABLE 21 Polyploid, Dioecious Plant Genera and Families

References Vallisneria spiralis (2n = 20); Vallisneria gigantea (2n = 40) Dioscoreaceae 2n = 20 - 40 - 60 - 80 ca. 140 Salicaceae 2n = 38 - 76 - 114 152 (19-series) 2n = 44 - 88 - 176 (22-series) Aucuba chinensis (2n = 16); Aucuba japonica (2n = 32)

-

-

Jorgensen (1927) Smith (1937) See Darlington and Wylie (1955) Darlington and Wylie (1955)

Rumex subgenus acetosella Rumes angiocarpus (2n = 14), Rumex tenuijolius (2n = 28) See Table 2 Rumex acetosella (2n = 42), Rumex graminijolius (2n = 56) Rumex subgenus acetosa Rumes hastatus (2s), Rumex acetosa (4s), Rumex paucifolius See Table 2 (62) Fragaria, 42 - 6s - 8x species (diploids hermaphroditic) See Darlington and Wylie (1955); Staudt (1951)

evidence is summarized by White (1954). One of the few cases of polyploidy in dioecious, sexual species of higher animals may be th a t of the hamster Cricetus cricetus (Sachs, 1952). It therefore seems more difficult than ever t o explain the absence of polyploidy in the animal kingdom by Muller’s hypothesis. See also the discussion by Vandel (1937).

X. SUMMARY AND CONCLUSIONS It seems possible to recognize among dioecious plant species a series of types, which represent different degrees of remoteness from the bisexual ancestors, and which thus show different stages in the evolution from bisexuality towards dioecism. (1) The most primitive type is characterized by the viability of the 2A YY type. (Y differs from X in the sex-deciding genes only.) This

+

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type may be not more (but probably also not less) than two mutational steps removed from the bisexual ancestor. Dioecism is not firmly established and sex expression may be modified b y the environment. Bisexual plants (both subandroecious and subgynoecious) may be produced through modifications. Constant, bisexual strains may easily be reconstituted by means of mutations or crossing-over. (2) X and Y differ to such a degree that 2A YY plants are inviable, but the Y-chromosome still plays a decisive role in sex determination. The sex expression is less influenced by the environment and the homogametic sex is more stable than the heterogametic sex. Bisexual types may arise through mutations rather than through modifications. Reconstitution of constant, bisexual strains is difficult. (3) The Y-chromosome takes no part in sex determination and may be inert. Sex is determined through the X/autosome balance. Bisexual types originates through mutations and sex expression is hardly influenced by the environment. Reconstitution of constant, bisexual types is difficult or impossible. Ecballium may represent the most primitive type, but Mercurialis, Thalictrum, and other subdioecious species also belong to type 1. Type 2 is represented by Carica and Melandrium. Carica probably being somewhat more primitive than Melandrium. Type 3 is represented by Rumex acetosa and probably also Humulus japonicus. It is not yet quite clear into which category Humulus lupulus and Rumex subgenus acetosella belongs, but i t is most likely that the latter belongs to type 2, and the former to type 3.

+

XI. REFERENCES Akerlund, E., 1927. Ein Melandrium-Hermaphrodit mit weiblichem Chromosomenbestand. Hereditas 10,153-159. Allen, C. E., 1940. The genotypic basis of sex-expression in angiosperms. Botan. Rev. 6, 227-300. Araratjan, A. G., 1939. Heterochromosomes in the wild spinach. Compt. rend. acad. sci. U.R.S.S. 24, 56. Batarman, M., 1946. La transpiration, facteur influent, dans la proportion des sexes de Mercurialis annua L. Rev. jac. sci. univ. Istanbul Sir. B11, 31-60. Bklaf, K.,1925. Der Chromosomenbestand der Melandrium Zwitter. Z. Induktive Abstammungs-u. Vererbungslehre 39, 184-190. Bemis, W. P., and Wilson, G. B., 1953. A new hypothesis explaining t h e genetics of sex determination in Spinacia oleracea L. J . Heredity 44, 91-95. Bilge, E., 1955. Recherches morphologiques, anatomique et genetique sur Bryonia macrostylis Heilb. et Bilge. Rev. fac. sci. univ. Istanbul Sdr. B20, 121-146. Billings, F.H., 1932. Microsporogenesis in Phoradendron. Ann. Botan. 46, 979-992. Billings, F. H., 1933. Development of the embryo-sac in Phoradendron. Ann. Botan. 47, 261-278.

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Resende, F., and Viana, M. J., 1952. Some effects of synthetic auxins and antiauxins on the floral state. Bol. SOC.Port. Cienc. Nat. 4, 74-88. Rhoades, M., 1952. Origin of cytoplasmic male sterility in corn. Sizth hybrid cornindustry conference, 7-10. Richardson, C. W., 1914. A preliminary note on the genetics of Fragaria. J. Genet. 3, 171-177. Rick, C. M., and Hanna, G. C., 1943. Determination of sex in Asparagus ojicinale L. Am. J. Botany 30, 711-714. Rizet, G., 1945. Sur le comportement des h6t6rochromosomes 8. la meiose chez les m&lestbtraploides de Melandrium album. Compt. rend. 220. Sachs, L., 1952. Polyploid evolution and mammalian chromosomes. Heredity 6, 357-364. Sansome, F. W., 1938. Sex determination in Silene otites and related species. J. Genet. 36, 387-396. Santos, J. K., 1923. Differentiation among chromosomes in Elodea. Botan. Gaz. 76, 42-59. Santos, J. K., 1924. Determination of sex in Elodea. Botan. Gaz. 77, 353-376. Schaffner, J. H., 1925. Experiments with various plants to produce change of sex in the individual. Bull. Torrey Botan. Club 62, 35-47. Schaff ner, J. H., 1929. Progeny resulting from self-pollination of staminate plants of Morus alba showing sex reversal. Botan. Gaz. 87, 653-659. Schaffner, J. H., 1936. Offspring of a self-pollinated reversed carpillate plant of Morus alba. Botan. Gaz. 98,425-428. Schiemann, E., 1931. Geschlechts- und Artkreuzungsfragen bei Fragaria. Goebel Botan. Abh. 18,5-112. Schiemann, E., 1937. Artkreuzungen bei Fragaria (11).2. Induktive Abstammungs-u. Vererbungslehre 73,375-390. Schiemann, E., 1943. Artkreuzungen bei Fragaria 111. Flora (Jena) [N.F.] 37, 166-192. Seitz, F. W., 1953. tfber anomale Zwitterbluten eines Klones der Gattung Populus, Section Leuce. 2.Forstgenet. u. Forstpflanzenziicht. 2, 77-90. Seitz, F. W., 1954. Uber das Auftreten von Triploiden nach der Selbstung anomaler Zwitterbluten einer Graupappelform. 2. Forstgenet. u. ForstpflanzenzJcht. 3, 1-6. Sengbusch, R. von, 1952. Ein weiterer Beitrag zur Vererbung des Geschlechts bei Hanf als Grundlage fur die Zuchtung eines monozischen Hanfes. 2.PfEanzenzJcht. 31, 319-338. Sinoto, Y., 1929a. On the tetrapartite chromosome in Humulus lupulus. Proc. I m p . Acad. (Tokyo) 6, 46-47. Sinoto, Y., 1929b. Chromosome studies in some dioecious plants, with special reference to the allosomes. Cytologia 1, 109-191. Sinoto, Y., and Kiyohara, K., 1928. A preliminary note on the chromosomes of Hydrilla verticillata Presl. Botan. Mag. (Tokyo) 42, 82-85. Skovsted, A., 1953. Some colchicine experiments with hops. Hereditas 39, 156-160. Smith, B. W., 1937. Notes on the cytology and distribution of the Dioscoreaceae. Bull. Torrey Botan. Club 64, 189-197. Smith, B. W., 1955. Sex chromosomes and natural polyploidy in dioecious Rumex. J . Heredity 46, 226-232. Staudt, G., 1951. tfber Fragen der phylogenetischen Entwicklung einiger Arten der Gattung Fragaria. Zuchter 21, 222-232. Staudt, G., 1952. Genetische Untersuchungen an Fragaria orientalis Los. und ihre Bedeutung fur Artbildung und Geschlechtsdifferenzierung in der Gattung Fragaria L. 2.Induktive Abstammungs-u. Vererbungslehre 84, 361-416.

280

M. WESTERGAARD

Staudt, G., 1955. Die Vererbung des Geschlechts bei der tetraploiden, diosischen Fragaria orientalis Los. Ber. deut. botan. Ges. 67, 385-387. Storey, W. B., 1953. Genetics of the Papaya. J. Heredity 44, 70-78. Suto, T., 1944. Meiotic chromosome behaviour in Populus nigra L. and Toisusu cardiophylla Kimura. J . Fac. Sci. Hokkaido Univ. 6 , 249-262. Takenaka, Y., 1953. Cytogenetic studies on sex in Cannabis sativa L. 11. Ann. Rept. Natl. Inst. Genet. Japan 3, 48-49. Thimann, K. V., 1956. Promotion and inhibition. Twin themes in physiology. A m . Naturalist 90, 145-162. Ubiach, G. von, 1936. Genetic studies on the nature of hermaphroditic plants in Antennaria dioica (L.) Gaertn. Genetics 21, 282-294. Uhl, C. H., 1952. Heteroploidy in Sedum rosea (L.) Scop. Evolution 6 , 81-86. Valleau, W. D., 1923. The inheritance of flower types and fertility in the strawberry. Am. J . Botany 10, 259-274. Vandel, A., 1937. Chromosome number, polyploidy and sex in the animal kingdom. PTOC.ZOO^. SOC.SeT. A107, 519-542. Warmke, H. E., 1942. A new method for determining the sex heterosygote in species with morphologically undifferentiated sex chromosomes, and its application to Silene otites. Genetics 27, 174. Warmke, H. E., 1946a. Sex determination and sex balance in Melandrium. Am. J . Botany 33,648-660. Warmke, H. E., 1946b. An analysis of male development in Melandrium by means of Y chromosome deficiencies. Genetics 31, 234-235. Warmke, H. E., 1946c. A study of spontaneous breakage of the Y chromosome in Melandrium. Am. J. Botany 33, 224. Warmke, H. E., and Blakeslee, A. F., 1939a. Effect of polyploidy upon the sex mechanism in dioecious plants. Genetics 24, 88. Warmke, H. E., and Blakeslee, A. F., 193913. Sex mechanism in polyploids of Melandrium. Science 89, 391-392. Warmke, H. E., and Blakeslee, A. F., 1940. The establishment of a 4n dioecious race in Melandrium. Am. J. Botany 27, 751-762. Warmke, H. E., and Davidson, H., 1944. Polyploidy investigations. Carnegie Inst. Wash. Yearbook 43, 135-139. Westergaard, M., 1940. Studies on cytology and sex determination in polyploid forms of Melandrium album. Dansk botan. Arkiv 10, 1-131. Westergaard, M., 1946. Aberrant Y chromosomes and sex expression in Melandrium album. Hereditas 32, 419-443. Westergaard, M., 1948. The relation between chromosome constitution and sex in the offspring of triploid Melandrium. Hereditas 34, 257-279. Westergaard, M., 1953. Vber den Mechanismus der Geschlechtsbestimmung bei Melandrium album. Naturwissenschaften 40, 253-260. White, M. J. D., 1954. ‘‘Animal Cytology and Evolution,’’ 2nd ed. Cambridge Univ. Press, New York. Wilkinson, J., 1944. The cytology of Saliz in relation to its taxonomy. Ann. Botany [N.S.] 8, 269-284. Winge, 1923. On sex chromosomes, sex determination and preponderance of females in some dioecious plants. Compt. rend. trav. lab. Carlsberg 16, 1-26. Winge, 1929. On the nature of the sex chromosomes in Humulus. Hereditas 12, 53-63. Winge, g., 1931. X- and Y-linked inheritance in Melandrium. Hereditas 16, 127-165.

om,

o.,

SEX DETERMINATION IN PLANTS

o.,

28 1

U’inge, 1934. The experimental alteration of sex chromosomes into autosomes and vice versa, as illustrated by Lebistes. Compt. rend. trav. lab. Carlsberg 21, 1-49. Winge, g., 1937. Goldschmidt’s theory of sex determination in Lynzantria. J. Genet. 34,81-89. Wittwer, S. H., and Hillyer, I. G., 1954. Chemical induction of male sterility in cucurbits. Science 120, 893-894. Yaniada, I., 1943. The sex-chromosomes of Cannabis safiva L. Rept. Kihara Inst. B i d . Iiesearch 2, 64-68. Ynmanioto, Y., 1938. Karyologische Untersuchungen bei der Gattung Ricmez 6. Alem. Coll. Agr. Kyoto Univ. 43, 1-59. Yampolsky, C., and Yampolsky, H., 1922. Distribution of sex forms in the phanerogamic flora. Bibliotheca Genet. 3, 1-62. Zoschke, U., 1956. Untersuchungen uber die Bestimmung des Geschlechts beim Spinat (Spinacia oleracea L.) unter besoriderer Beriicksichtigung der Zuchtung eines monozischen oder gleichzeitig schossenden Spinates. 2. Pflanzenziicht. 36, 257-296. Zukel, J. W., 1952. Literature sumniary on maleic hydrazide. U.S. Rubber Co. MHIS 6, 1-28.

AUTHOR INDEX A Abraham, A., 224,226, 227, 276 k e r l u n d , E., 254, 273 Allen, C. E., 217, 224, 226, 252, 260, 268, 273 Anderson, E., 203 Annan, M. E., 4, 67, 68 Araratjan, A. G., 225, 273 Ar-Rushdi, A. H., 170, 203 Ashan, K., 49, 60, 62, 67, 68 Ausherman, L. E., 164, 204 Avers, C. J., 179, 203

B

Brachet, J., 196, 204 Brefeld, O., 42, 67 Breider, H., 239, 274 Bridges, C. B., 219, 254, 274 Brieger, F., 150, 153, 154, 204 Brink, R. A., 160, 163-165, 204, 205 Brock, R. D., 159, 172, 175,204,210 Brodie, H. J., 45, 67 Brown, M. S., 179, 210 Brunswik, H., 45, 49, 52, 59, 67 Buell, K. M., 164, 204 Bufton, A. W. J., 76, 103, 107, 144 Buller, A. H. R., 43, 44, 67 Buxton, E. W., 72, 103 Bytinski-Salz. H., 153, 204

Baker, R. H., 157, 205 C Balle, S., 64, 69 Calef, E., 109, 124, 138, 144 Baltzer, F., 150-152, 203 Callan, H. G., 169, 174, 204, 209, 213 Barratt, R. W., 137, 144 Carpenter, J. M., 4, 5, 9 Barth, L. G., 196, 203 Carson, H. L., 1, 3-15, 17, 20, 26, 28, Bagarman, M., 228, 270, 273, 275 31-35 Beamish, K. I., 163, 203 Caspari, E., 269, 274 Beaudry, J. R., 164, 203 Catcheside, D. G., 76, 103 BBlaf, K., 224, 273 Chang, T. T., 178, 211 Bell, G. D. H., 171, 203 Christensen, B., 272, 274 Bemis, W. P., 225, 244, 273 Clausen, J., 175, 179, 189, 190, 202, 204, Benazzi, M., 168, 169, 174, 181, 203 205 Bennett, C. J., 10 Clayton, E. E., 173, 205 Bensaude, M., 66, 67 Cole, K., 155, 179, 205 Bernstrom, P., 162, 175, 203, 204 Cooper, D. C., 160, 163-165, 204, 205 Bilge, E., 227, 228, 273 Correns, C., 217, 219-221, 224, 227, 232, Billings, F. H., 224, 225, 273, 274 233,235, 236, 238, 246,252,253.268, Bilquez, M. A., 172, 204 274 Blackburn, K. B., 221, 224, 225, 274 Blakeslee, A. F., 157-160, 204, 212, 224, Covas, G., 174, 205 Craft, W. A., 182, 205 252, 274 Crane, M. B., 263, 274 Blight, W. C., 27, 38 Cretschmar, M., 174, 205 Boehringer, F., 152, 204 Crew, F. A. E., 169, 205 Bonnet, D. A., 157, 204 Crowe, K., 269, 277 Boyes, J. W., 163, 170, 204 Cua, L. D., 175, 176, 205 Bozekurt, B., 168, 204 283

284

AUTHOR INDEX

D

G

Dark, S. 0. S., 222, 252, 274 Darlington, C. D., 166,205,220,222,267, 268, 272, 274 Davern, C. I., 175, 210 Davidson, H., 259, 280 Davis, E. W., 170, 205 De Barros, R., 174, 206 Delbruck, M., 139, 144 Demerec, M., 71, 103 Demerec, Z. E., 71, 103 Detlevsen, E., 180, 215 Dickson, H., 47, 50, 64, 67, 68 Dobshansky, T., 147, 148, 165, 166, 169, 178,179, 182, 185,189-191, 193-195, 199, 205, 211, 268, 274 Dorsey, C. K., 10, 12, 39 Downs, W. G., 157, 205 Dreyfus, A., 174, 206 Diirken, B., 181, 206

Gabe, D. R., 221, 236, 237, 274 Gajewski, W., 170-172, 175, 206 GalBn, F., 231, 249, 251, 265, 274 Galey, M. M., 14, 39 Garnjobst, L., 137, 144 Gaul, H., 171, 173, 206 Gerstel, D. U., 153, 206 Ghigi, A., 168, 206 Gloor, E. T., 69 Goldschmidt, R., 190, 191, 206, 219, 268, 274 Goodspeed, T. H., 202, 206 Goodwin, R. H., 179, 206 Gordon, M., 153, 154, 206, 238, 274 Grant, Alva, 175, 207 Grant, V., 156, 172, 175, 188, 190, 195, 200, 206, 207 Greenleaf, W. H., 170, 207 Greenshields, J. E. R., 159, 207 Gregg, J., 196, 207 Griffen, A. B., 181, 211 Gustafsson, A., 199, 207

E East, E. M., 163, 206, 233, 277 Ellerstrom, S., 160, 207 Elliott, C. G., 106, 114, 122, 144 Emerson, R., 175, 206 Emerson, R. A., 260, 262, 263, 274 Ephrussi, B., 64, 68 Ephrussi-Taylor, H., 71, 103 Erlanson, E. W., 225, 274 Eversole, R. A., 144

F Fagerlind, F., 163, 164, 206 Farrar, R. F., 252, 259, 278 Favarger, C., 225, 238, 274 Federley, H., 181, 206 Forbes, E., 69, 72, 74, 76, 87, 96, 103 Forbes, E. C., 107, 111-115, 124, 144 Forster, R., 154, 204 Foster, R., 226, 274 Fries, L., 47, 50, 68 Fries, N., 60, 62, 68, 103 Frolova, S. L., 39 Fulton, I. W., 46, 68

H Hadorn, E., 151, 152, 207 Haga, T., 194, 207, 225, 275 Hagerup, O., 227, 275 HBkansson, A., 160, 207, 225, 251, 274 Haldane, J. B. S., 181, 182, 207, 235, 275 Hall, B. M., 167, 207 Hamburger, V., 207 Hanna, G. C., 236, 237, 279 Harder, R., 45, 60-62, 65-68 Harrison, J. W. Heslop, 225, 274, 275 Hartmann, M., 43, 64, 68, 219, 232, 248, 249, 268, 275 Hecht, A., 156, 207 Heilbronn, A., 227, 228, 248, 249, 275 Heiser, C. B., Jr., 174, 188, 207, 208 Hemmons, L. M., 76, 103, 107, 144 Hermann, F. J., 225, 274 Hertwig, P., 148, 149, 156, 157, 180, 208 Heslop-Harrison, J., 270, 275 Hiesey, W. M., 179, 205 Hillyer, I. G., 270, 281 Hirata, K., 222, 240, 275 Hirayoshi, I., 223, 276

285

AUTHOR INDEX

Hockenhull, D. J. D., 126, 144 Hoffmann, W., 222, 240-243, 275 Hofmeyer, J. D. J., 221, 245, 250, 252, 259, 265, 275 Holland, R. F., 173, 214 Hollander, W. F., 179, 205 Hollingshead, L., 153, 189, 208 Hong, M. S., 178, 211 Hotchkiss, R. D., 71, 103 Howard, H. W., 162, 208 Hshek, S. C., 175, 211 Hsu, T. C., 40 Huang, T. S., 175, 211 Hubbs, C. L., 200, 208 Huhnke, W., 240, 275 Humphrey, R. R., 236, 275 Hungerford, D. A., 8 Hutchinson, J. B., 153, 179, 208

I Iizuka, M., 163, 211 Ikada, Y., 72, 103 Inamori, Y., 162, 211 Ishitani, C., 72, 103 Iwanoff, E. J., 168, 208

J Jacobsen, P., 222, 223, 275 Jenkin, T. J., 202, 208 Jensen, H. W., 226, 275 Johnson, L. P. V., 162, 208 Johnsson, H., 225, 251, 276 Jones, D. F., 170, 172, 173, 179, 208, 260, 262, 266, 276 Jordan, C., 240, 275 Jergensen, C. A,, 272, 276

K Kafer, E., 71, 73, 74, 76-78, 98, 100, 102, 103-108,114,115,122,124,125,130, 131, 138, 139, 144, 145

Kawamura, T., 168, 181, 195, 208 Keck, D. D., 179, 205 Kehr, A. E., 154, a08 Kharchevko, I. I., 168, 213 Kihara, H., 163, 173, 188, 208, 221, 222, 224, 226, 234, 276

Kikkawa, H., 39 Kimura, K., 44, 62, 63, 68 King, J. C., 181, 208 Kiyohara, K., 225, 279 Knapp, E. P., 13, 39 Kniep, H., 41, 49, 54, 55, 57, 60, 68 Koller, P. C., 169, 205 Kolmark, G., 48, 68 Koopmans, A., 179, 208, 209 Kosswig, C., 153, 209 Kostoff, D., 154, 159, 163, 209 Kuhn, E., 217, 221, 231, 232, 236, 237, 270, 276 Kumar, L. S. S., 224, 226, 227, 251, 276 Kurabayashi, M., 194, 207 Kurita, M., 226, 276 Kyrtz, E. B., 270, 278

L La Cour, L. F., 159, 212 Laibach, F., 157, 209 Lamprecht, H., 190, 191, 209 Lamy, R., 178, 209 Lange, M., 44, 68 Lantz, L. A., 174, 209 Laven, H., 157, 209 Lawrence, W. J. C., 263, 274 Lederberg, E. M., 59, 68 Lederberg, J., 59, 68, 71, 104 Ledingham, G. F., 161, 209 Lein, A., 153, 209 Lemanov, N. A., 168, 209 Lepori, N. G., 168, 169, 174, 203 Levan, A., 226, 276 Levi, G., 168, 209 Levitan, M., 7, 10, 12, 14, 17, 19, 20, 26, 31-35, 39 Lewis, D., 42, 47, 68, 217, 218, 263, 269, 276, 277 Lewis, H., 185, 190-193, 201, 209 Lewis, M. E., 185, 201, 209 Lewontin, R. C., 7 Lilienfeld, F. A., 233-235, 277 Lindegren, C. C., 58, 68 Lindsay, R. H., 225, 277 Liverman, J. L., 270, 278 Lodkina, M. M., 164, 212 Long, R. W., Jr., 188, 209 Loomis, N. H., 239, 277

286

AUTHOR INDEX

Liive, A., 222-225,252,259,270,271,277 Liive, D., 223, 270,277 Love, R. M., 171, 212 Luria, S. E., 139, 144 Lutkov, A. N., 179, 209

M McAtee, W. L., 3, 11, 39 McCray, F. A., 150, 209 Macdonald, K. D., 76, 78, 103, 106, 129, 144 McLean, S. W., 157, 158, 210 McPhee, H. C., 222, 277 Macrae, R., 50, 68 Maekawa, T., 240, 277 Makino, S., 2, 39 Malloch, J. R., 3, 11, 39 Mangelsdorf, A. J., 233, 277 Martin-Smith, C., 112 Mashima, I., 176, 177, 178, 209 Mather, K., 231, 269, 277 Matthey, R., 222, 234, 277 Mayr, E., 186, 209 Melchers, G., 153, 210 Menrel, M. Y., 179, 187, 210 Meurman, O., 222, 224-226, 277 Mezaki, M., 259, 278 Michaelis, P., 155, 156, 172, 210, 269, 277 Midusima, U., 175, 176, 190, 214 Miller, D. D., 5-7, 10-14, 40 Minamori, S., 169, 179, 190, 195, d10 Mitchell, M. B., 58, 68 Mittwoch, U., 47, 50, 68 Moewus, F., 239, 277 Momma, E., 2, 39, 40 Montalenti, G., 157, 210 Moore, J., 150-153, 190, 193, 210 Moree, R., 169, 210 Morley, F. H. W., 175, 210 Morse, M. L., 59, 68 Muldal, S., 272, 277 Muller, H. J., 148, 180,182, 190, 193,196, 210, 219, 268, 271, 272, 277 Miintzing, A., 163, 165, 210, 225, 277 Murphy, M. M., 239, 277 Murray, M. J., 227, 229, 230, 249, 252, 277

N

Negrul, A. M., 239, 278 Nelson, S. E., 32, 40 Neuer, H., 240, 275, 278 Neve, R. A., 252, 259, 278 Newmeyer, D., 137, 144 Newton, D. E., 52, 53, 68 Newton, W. C. F., 24 Nielsen, C. O., 272, 274 Nilsson-Ehle, H., 278 Nishiyama, I., 162, 163,208,211,259,278 Nitsch, J. P., 270, 278 Nordenskiold, H., 170, 190, 211 Nygren, A., 252, 278

0 Oberle, G. D., 239, 278 Oehlkers, F., 269, 278 Oka, H., 166, 175-178, 211 Olive, L., 50, 68 Oliver, C. P., 5 Olmo, H. P., 252, 278 Ono, H., 172, 211 Ono, T., 221-224, 249, 251, 276, 278 Owen, R. D., 168, 169, 211 Ownbey, M., 175, 211

P Papaaian, H. P., 41, 43,45, 47-49, 51-53, 56, 57, 60, 62, 64, 68, 69 Parker, K., 150, 153, 181, 211 Patel, G. I., 226, 278 Patterson, J. T., 2-6, 8-14, 40, 169, 178, 181, 191, 211, 212 Pavan, C., 17, 40 Pellew, C., 211 Peng, F. T., 39 Perkins, D. D., 106, 137, 144 Perry, W. J., 157, 212 Peto, F. H., 171, 212, 225, 278 Petrov, D. F., 232, 278 Phaff, H. J., 13, 39 Phillips, J. C., 179, 212 Pittendrigh, C. S., 7 Poddubnaja-Amoldi, V. A., 163, 212 Poll, H., 168, 169

AUTHOR INDEX

Pontecorvo, G., 51, 69, 71, 72, 74, 76, 78, 87, 96, 98, 102, 103, 105-107, 113115, 122, 124, 129, 130, 138, 144 Pope, W. K., 171, 212 Pritchard, R. H., 73, 76-78, 95, 104, 109, 110, 115, 124, 128, 131, 137, 144, 145 Propach, H., 179, 212 Pun, F. T., 175, 213

Q Quintanilha, A., 41, 43, 44, 64, 69

R Raper, J. R., 4547, 54, 69 Rappaport, J., 158, 159, 212 Rehm, S., 270, 278 Renner, O., 148, 156, 165, 212 Resende, F., 270, 278, 279 Rhoades, M., 269, 279 Richardson, C. W., 232, 233, 279 Rick, C. M., 170, 179, 212,236, 237, 279 Rizet, G., 252, 279 Roberts, M. R., 191-193, 209 Roper, J. A., 72,76-78,103-105, 107, 108, 110, 111, 114, 115, 124, 125, 128, 131, 139, 144, 145 Rubaschev, S. J., 150, 212 Ruibal, R., 190, 212 Rutishauser, A., 159, 212

S Sachet, M.-H., 159, 212 Sachs, L., 171, 203, 272, 279 Sakaguchi, K.,' 72, 103 San Antonio, J. P., 45-47, 69 Sanders, G. C., 169, 212 Sanders, M. E., 159, 212 Sansome, E., 160, 212 Sansome, F. W., 237, 238, 279 Santos, J. K., 221, 224, 225, 279 Sarkar, Nina, 223, 224, 252, 259, 277 Satina, S., 157-160, 204, 212 Saunders, A. P., 170, 197, 212 Saunders, A. R., 153, 212 Schaffner, J. H., 246, 279 Scheu, H., 239, 274 Schiemann, E., 233, 234, 279

287

Schnack, B., 174, 175, 205, 213 Schonmann, W., 150, 151, 213 Schultz, J., 268, 274 Sears, E. R., 153, 171, 213 Seita, F. W., 246, 251, 279 Sengbusch, R. von, 240-243,275,278,279 Sermonti, G., 51, 69, 72, 103 Sheng, T. C., 40 Shupakov, I. G., 168, 213 Silow, R. A., 153, 179, 213 Sinoto, Y., 222, 225, 226, 279 Skovsted, A., 252, 279 Smith, B. W., 223,225,226,271,272,279 Smith, G. M., 153, 154, 206 Smith, H. H., 154, 208 Sneath, P. H. A., 115, 145 Snyder, L. A., 170, 175, 189, 195, 213 Sokoloff, A., 4 Solbrig, 0. T., 175, 213 Soost, R. K., 182, 213 Speith, H. T., 5, 11, 13 Spencer, W. P., 8, 11 Spiess, E. B., 5, 10, 40 Spurway, H., 169, 174, 179, 181, 204, 213 Srinivasan, V. K., 224, 226, 237, 276 Stadler, D. R., 137, 145 Stadler, L. J., 167, 213 Stalker, H. D., 3-15, 17, 20, 26, 28, 3135, 38-40 Staple-Browne, M. A,, 179, 213 Staudt, G., 224, 232-235, 272, 279, 280 Stebbins, G. L., Jr., 147, 148, 166, 170, 172,174,175,178,183,185-187,190, 191, 194, 195, 197, 198,200,212, 213 Steiner, H., 179, 213 Stephens, J. C., 173, 214 Stephens, S. G., 154, 162, 179, 185, 189, 194, 214 Stern, C., 72, 104, 105, 124, 145 Stevenson, R., 12, 40 Stocker, B. A., 71, 104 Stone, W. S., 2, 13, 40, 178, 181, 191, 212 Storey, W. B., 227,245,250,251,265,280 Strasburger, E. H., 190, 214 Straw, R. M., 195, 214 Strickland, N. S., 129 Sturtevant, A. H., 3, 5, 7, 10-12, 40, 214 Suto, T., 225, 280 Sviirdson, G., 180, 214 Syrach-Laraen, C., 179, 214

288

AUTHOR INDEX

T Takada, H., 2, 39 Takenaka, Y., 259, 280 Tan, C. C., 40 Tarr-Gloor, E., 72, 74, 87, 96, 103, 107, 114, 144 Terao, H., 175, 176, 190, 214 Texas University Laboratory, 3-12 Thimann, K. V., 271, 280 Thompson, W. P., 160, 163, 204, 214 Tobgy, H. A., 190, 213 Tukan, D. A., 232, 278

U Ubisch, G. von, 246, 280 Uchiyamada, H., 176-178, 209 Uhl, C. H., 226, 280

V Valtrama, A., 178, 195, 213 Valencia, J. I., 172, 183, 187, 213 Valencia, R. M., 172, 183, 187, 213 Valentine, D. H., 158, 175, 214 Valleau, W. D., 233, 280 Vandel, A., 272, 280 Van Elden, H., 252, 275 Viana, M. J., 270, 279 Vishveshwaralah, S., 251, 276 Volpe, E. P., 190, 195, 214

W Wagner, R. P., 3-6, 8-11, 40 Wakahama, K., 2, 39 Walker, G. W. R., 204 Walker, R. I., 163, 170, 214 Wallace, B., 33, 40 Walters, J. L., 194, 202, 214 Walters, M. S., 170, 172, 213, 215

Warmke, H. E., 221-224, 238, 252-254, 257, 259, 274, 280 Watkins, A. E., 163, 215 Weaver, J. B., Jr., 162, 215 Webster, G. T., 183, 215 Went, F. W., 270, 278 Westergaard, M., 48, 68, 217, 223, 224, 227,252-255,257,258,267,268,270, 280 Wheeler, M. R., 12 White, M. J. D., 174, 197, 215, 272, 280 Whitehouse, H. L. K., 42, 52, 69 Wilkinson, J., 225, 280 Williams, C. F., 239, 244, 277 Williams, D. D., 6, 7, 13, 14, 40 Williamson, D., 7 Wilson, C. M., 175, 206 Wilson, G. B., 225, 244, 273 Winge, g., 179, 180, 215, 220-222, 224, 238,243, 246,252,266, 268, 280, 281 Witschi, E., 181, 215 Wittwer, S. H., 270, 281 Wright, S., 57, 69, 215 Wylie, A. P., 272, 274

Y Yamada, I., 222, 223, 243, 259, 278, 281 Yamamoto, Y., 223, 249, 251, 276, 281 Yamashina, M. Y., 168, 169, 215 Yampolsky, C., 217, 281 Yampolsky, H., 217, 281

2 Zattler, F., 50, 69 Zawadowsky, M. M., 168, 215 Zimmermann, K., 190,215 Zinder, N. D., 71, 104 Zoschke, U., 224, 225, 244, 245, 281 Zuitin, A., 168, 215 Zukel, J. W., 270, 281

SUBJECT INDEX A

B

Abnormalities, in development of animal Bacteria, linked transduction in, 71 hybrids, 156, 157 Basidiomycetes, “ Buller Phenomenon ” reproductive, in interspecific hybrids, in, 43 147-2 15 characteristics of, 42 Amphibian hybrids, time of degeneration “cytoplasmic autonomy” in, 44, 47, in embryo, 149-153 49, 60, 65 Animal hybrids, interspecific, genic sterildikaryon in, 43-45, 51, 52, 57, 58, 62 ity in, 168, 169 dwarf mycelia in, 64, 66 Ascomycetes, in history of genetics, 41 genetics of, 41-69 Aspergillus nidulans, chromosome segreHarder’s technique in, 60-62, 65, 67 gation in, 72 heterokaryosis in, 44, 45, 48, 66 incompatibility factors in, 42, 44, 49, classification and isolation of segregants in, 76-95, 97 50, 53, 54, 56, 57, 59, 63, 64 eight-chromosome map of, 105-145 induced mutants in, 47, 49 haploidization in, 105-107, 114, 115, mating system in, 42, 44 123, 129, 130, 140 mycelia in, 42, 44, 45, 62, 65, 66 induced and spontaneous mutants in, nucleo-cytoplasmic relations in, 65 production of incompatible factors by 106-113, 117, 120, 126, 127, 129, crossing-over, in, 52, 54, 55 131, 133, 137 linkage groups in, 105-108, 110-127, recombination between incompatibility genes in, 53, 55-57 130, 131, 136-138, 140 list of crosses in, 142, 143 segregation in, 50, 53, 57, 58, 60 of diploids in, 141, 142 sexuality in, 43 location of centromeres in, 106, 123, somatic recombination in, 51, 52 124, 126, 129, 130 spontaneous dikaryolization in, 59, 60, 63 of markers in, 105-107,114-123,127, 131, 140 tetrad segregation in, 58, 59 meiotic analysis in, 105, 106, 124 tetrads in, 52, 53, 64, 65 meiotic linkage maps of, 13 undetermined mutagens in, 47, 48 meiotic recombination in, 132, 133 Bisexuality in plants, 218, 219 mitotic analysis in, 106 “Blotchy” mycelia in Cyanthus stermitotic crossing-over in, 76, 78, 81-86, carcus, 46 89, 94-96, 98-102, 105-107, 114, Breakdown in interspecific hybrids, 147, 122-126, 130, 131, 138-140 148, 178-181 mitotic linkage maps of, 138 Bryonia, crossing between dioecious and monoecious species of, 228, 230, 246, mitotic recombination in, 105, 114, 115, 247, 248 121, 140 genetics of sex determination in, 218, mitotic segregation in, 105, 107, 114, 128, 129, 131 219, 228, 230 sequence of markers in, 123-129, 140 heteromorphic sex chromosomes in, 227 289

290

SUBJECT INDEX

“ Buller

Phenomenon I’ in Basidiomycetes, 43

C Cannabis saliva, crosses between sex forms of, 242, 243 Centromeres, location of, in Aspergillus rtidulans, 106, 123, 124, 126, 129, 130 non-homologous, in genetic analysis, 71 Chromosome constitution and sex in Drosophila, 254 in Melundrium, 254-256, 258 Chromosome maps, mitotic, 98-101 Chromosomes and genes, parental, incompatibility between, in interspecific hybrids, 149-155 Chromosomes at metaphase, in Drosophila robusta, 15, 19 Chromosome segregation in Aspergillus nidulans, 72 Coprinus lagopus, “flat” mycelia in, 45, 47, 66 incompatibility factors in, 42, 43, 47 monokaryon in, 43, 67 mutation of incompatibility factors in, 49, 51 Crosses between dioecious and hermaphroditic species of Fragaria, 232-235, 246 between dioecious and monoecious species of Bryonia, 228, 230, 246248 between sex forms of Cannabis saliva, 242, 243 dioecious and bisexual, in plant species, 227-233, 247, 248 list of, in Aspergillus nidulans, 142, 143 Crossing-over in Basidiomycetes, production of incompatibility factors by, 52, 54, 55 in sexual genetics of plant species, 220 mitotic, frequency and distribution of, 74, 75, 84 mitotic, in Aspergillus nidulans, 76, 78, 81,82-86,89, 94-96, 98-102, 105107, 122-126, 130, 131, 138-140 mitotic, in genetic analysis, 71-74 somatic, in Drosophilu, 72

Cyanthus stercorcus, l 1 blotchy” mycelia in, 46 Cytology in Drosophila robusla, 15, 37 Cytoplasm and sex-determination in plants, 268, 269 “ Cytoplasmic autonomy ” in Basidiomycetes, 44, 47, 49, 60, 65

D Dalura hybrids, incompatibility between embryo and surrounding tissue in, 157-159 Degeneration in embryo and endosperm in interspecific plant hybrids, causes of, 159-165 time of, in interspecific hybrids, 149, 150 Dikaryoliration, spontaneous, in Basidiomycetes, 59, 60, 63 Dikaryon in Basidiomycetes, 43-45, 51, 52, 57, 58, 62 Dioecious and bisexual crosses in plant species, 227-233, 247, 248 Dioecious flowering plants, mechanism of sex-determination in, 217-281 Dioecious plants, polyploids in, 251-259, 271,272 Dioecious (unisexual) species, 218, 219 Dioecism, evolution of, in plants, 260-268 Diploids, list of, in Aspergillus nidulans, 141, 142 Drosophila, chromosome constitution and sex in, 254 colorata, distribution of, 12 genetics of sex-determination in, 219, 271 lethal genes in, 152 robusta, chromosomes a t metaphase in, 15, 19 cytology of, 15, 37 distribution of, 2-13 ecology of, 13, 14 gene arrangement, homorygosity in, 34, 35, 37 gene arrangement frequencies in altitude, in, 26, 28, 31 in laboratory populations of, 35-37 seasonal and perennial in, 28-32, 34,37

29 1

SUBJECT INDEX

gene arrangements in, 18, 19 geographical distribution in, 20-25, 27 inversions, effect of, on crossing-over in, 32, 33, 37 paracentric, in, 18, 32 pericentric, in, 19, 20, 37 linkage and positional relationship in, 33, 34 media for, 14, 15 morphological variations in, 31, 32, 37 oviposition in, 14 population genetics of, 1-40 salivary gland chromosomes in, 1519 taxonomy of, 2 somatic crossing-over in, 72 Dwarf mycelia in Basidiomycetes, 64, 66

E Eight-chromosome map of Aspergilhs nidulans, 105-145 Ecology of Drosophila robusta, 13, 14 Epilobium, cytoplasmic incompatibility in hybrids of, 155, 156 Evolution of dioecism in plants, 260-268

F Fish, tumors in interspecific hybrids of, 153, 154 “Flat ” mycelia in Coprinus lagopus, 45, 47, 66 in Schizophyllum, 45, 46, 48, 49, 57, 65 Fragaria, crosses between dioecious and hermaphroditic species of, 232-235, 246 Fungi, haploidization in, 72-74, 77, 79, 84, 87, 95, 98, 103

G Gene arrangement frequencies in altitude, in Drosophila robwrta, 26,28,’31 in laboratory populations of Drosophila robusta, 35-37 seasonal and perennial, in Drosophila robwrta, 28-32, 34, 37

Gene arrangement, in Drosophila robusta, 18, 19 geographical distribution, in Drosophila robusta, 20-25, 27 homozygosity in Drosophila robusta, 34, 35,37 Genes, lethal, in Drosophila, 152 in plant hybrids, 153 Genes, sex-determining, in plants, localization of, 247-249, 250 Genetic analysis, linkage groups in, 71, 73, 74, 79, 83, 88, 89, 91, 92, 95, 96, 98, 100-103 location of centromeres in, 73, 74, 96, 97,98 mitotic crossing-over in, 71-74 non-homologous centromeres in, 71 sequence of linked markers in, 73 Genetics, history of, in Ascomycetes, 41 in Phycomycetes, 41 in yeast, 41, 64 Genetics, of Basidiomycetes, 41-69 of Neurospora, 41 of sex-determination in dioecious plants, 218, 219 in Drosophila, 219, 271 in Melandrium, 218, 220, 251, 252, 254, 262-266, 271 in Rumex, 218, 219, 246, 249 of subdioecious species in plants, 235245, 249, 250

H Haldane’s Law, 181-183, 235 Haploidization i n Aspergillus nidulans, 105-107, 114, 115, 123, 129, 130, 140 in fungi, 72-74, 77, 79, 84, 87, 95, 98, 103 Harder’s technique in Basidiomycetes, 60-62, 65, 67 Heterogametic sex in plants, identification of, 220, 236-239, 246 Heterokaryosis in Basidiomycetes, 44,45, 48, 66 Heteromorphic sex chromosomes in Bryonia, 227 in Melandrium, 221-224 in plants, 221-227, 234, 235 in Rumex, 221-223

292

SUBJECT INDEX

Hybrids, of animals, abnormality in development of, 156, 157 genic sterility in, 168, 169 breakdown in, 147, 148, 178-181 of Datura, incompatibility between embryo and surrounding tissue in, 157-159

of Epilobium, cytoplasmic incompatibility in, 155, 156 of fish, tumors in, 153, 154 incompatibility between parental chromosomes and genes in, 149155

interspecific, genetics of, 147-203 inviability in, 147-165 mechanism of, 148, 149, 183, 184, 190-203

origin of, 190-203 natural selection vs. inviability and sterility in, 190-199 of Nicotiana, growth disturbances in, 154, 155, 185

lethal genes in, 153 time of degenerationinembryo of, 150 tumors in, 154 of plants, cause of degeneration in embryo and endosperm development in, 159-165 cytoplasmic incompatibility in, 156,

I Incompatibility, cytoplasmic, in interspecific plant hybrids, 156, 157 factors in Basidiomycetes, 42, 44, 49, 50, 53, 54, 56, 57, 59, 63, 64 in Coprinus lagopus, 42, 43, 47 mutation of, in Coprinus lagopus, 49, 51

in Schizophyllum, 49, 51, 56 production of, by crossing-over in Basidiomycetes, 52, 54, 55 in Schizophyllum, 42, 49, 53, 57 genes, recombination between, in Basidiomycetes, 53, 55-57 Inversions, effect of, on crossing-over in Drosophila robusta, 32, 33, 37 paracentric, in DTosophila robusta, 18,32 pericentric, in Drosophila robusta, 19, 20,37

Inviability, in interspecific hybrids, 147165

mechanism of, in interspecific hybrids, 148, 149, 183, 184, 190-203

origin of, in interspecific hybrids, 190203

1

157

Linkage groups in Aspergillus nidulans,

193, 200-202

Linkage maps, meiotic, in Aspergillus nidulans, 131 mitotic, in Aspergillus nidulans, 138 Linkage, positional relationship in Drosophila robusta, 33, 34 Linked markers, sequence in genetic analysis, 73 Location of centromeres in genetic analysis, 73, 74, 96-98 of markers, in fungi, 72-74

genic sterility in, 170-173 lethal genes in, 153 reproduction of, abnormalities in, 147ff. segregation for fertility in, 183, 184, sterility in, 147, 148, 165-178 classification of, 165, 166 diplontic, causes of, 173, 174 genic, of animals, 168, 169 in Drosophila, 185 of plants, 170-173 haplontic, 174-178 intraspecific diversity in species, 188-1 90

mechanism of, 190-203 relation between incompatibility and sex in, 181-183 time of degeneration in, 149, 150 in amphibian embryos, 149-153 in embryos of Nicotiana, 150

105-108, 110-127, 130,131,136-138, 140 in genetic analysis, 71, 73, 74, 79, 83, 88, 89, 91, 92, 95, 96, 98, 1 W 1 0 3

M Markers, location of, in Aspergillus nidulans, 105-107, 114-123, 127. 131, 140

293

SUBJECT INDEX

location of, in fungi, 72-74 sequence of, in Aspergillus nidulans, 123-129, 140 Mating system, in Basidiomycetes, 42, 44 Media for Drosophila robusta, 14, 15 Meiotic analysis in Aspergillus nidulans, 105, 106, 124 Meiotic linkage maps in Aspergillus nidulans, 131 Meiotic recombination in Aspergillus nidulans, 132, 133 Melandrium, chromosomes, constitution and sex in, 254-256, 258 genetics of sex-determination in, 218, 220, 251, 252, 254, 262-266, 271 heteromorphic sex chromosomes in, 221-224 Mitotic analysis in Aspergillus nidulans, I06 in fungi, 72 Mitotic chromosome maps, 98-101 Mitotic crossing-over, frequency and distribution of, 74, 75, 84 in Aspergillus nidulans, 76, 78, 81-86, 89, 94-96, 98-102, 105-107, 114, 122-126, 130, 131, 138-140 in genetic analysis, 71-74 Mitotic linkage maps in Aspergillus nidulans, 138 Mitotic recombination in Aspergillus nidulans, 105, 114, 115, 121, 140 in fungi, 72, 102 in genetic analysis, 71-104 Mitotic segregation in Aspergillus nidulans, 105, 107, 114, 128, 129, 131 Monokaryon in Coprinus lagopus, 43, 67 in SchizophyElum, 43, 49, 56-58, 62 Morphological variations in Drosophila robusta, 31, 32, 37 Mutagens, undetermined, in Basidiomycetes, 47, 48 Mutants, in Aspergillus nidulans, induced and spontaneous, 106-113, 117, 120, 126, 127, 129, 131, 133, 137 induced, in Basidiomycetes, 47, 49 Mutation of incompatibility factors in coprinus lagopus, 49, 51 in Schizophyllum, 49, 51, 56 Mycelia in Basidiomycetes, 42, 44, 45, 62, 65, 66

N Natural selection vs. inviability and sterility in interspecific hybrids, 190199 Neurospora, genetics, 41 Nicotiana hybrids, growth disturbances in, 154, 155, 185 lethal genes in, 153 time of degeneration in embryo of, 150 tumors in, 154 Nucleocytoplasmic relations in Basidiomycetes, 65

0 Outbreeding in plants, 217 Oviposition in Drosophila robusta, 14

P Phycomycetes in history of genetics, 41 Physiology of sex determination in plants, 269-271 Plants, bisexuality in, 218, 219 Polyploids in dioecious plants, 251-259 271, 272 Population genetics of Drosophila robusta, 1-40

R Recombination between incompatibility genes, in Basidiomycetes, 53, 55-57 meiotic, in Aspergillus nidulans, 132, 133 mitotic, in Aspergillus nidulans, 105, 114, 115, 121, 140 in fungi, 72, 102 in genetic analysis, 71-104 somatic, in Basidiomycetes, 51, 52 Rumex, genetics of sex-determination in, 218, 219, 246, 249 heteromorphic 5ex chromosomes in, 221-223

S Salivary gland chromosomes in Drosophila robusta, 15-19

294

SUBJECT INDEX

Schizophyllum, “flat ” mycelia in, 45, 46, 48, 49, 57, 65 incompatibility factors in, 42,49,53, 57 monokaryon in, 43, 49,56-58, 62 mutation of incompatibility factors in, 49, 51, 56 Segregants, classification and isolation in Aspergillus nidulans, 76-95, 97 Segregation for fertility in interspecific hybrids, 183, 184, 193, 200-202 Segregation in Basidiomycetes,50,53,57, 58, 60 mitotic, in Aspergillus nidulans, 105, 107, 114, 128, 129, 131 tetrad, in Basidiomycetes, 58, 59 Sex chromosomes, heteromorphic, in plants, 221-227, 234, 235 Sex determination and cytoplasm in plants, 268, 269 genetics of, in Bryonia, 218, 219, 221, 228, 230 in dioecious plants, genetics of, 218, 219 mechanism of, in dioecious flowering plants, 217-281 physiology of, in plants, 269, 270, 271 Sex-determininggenes in plants, localization of, 247-250 Sexual dioecism in plants, 217 Sexual genetics in plant species, crossingover in, 220 Sexuality in Basidiomycetes, 43 Sterility, classification of, in interspecific hybrids, 165, 166 diplontic, causes of, in interspecific hybrids, 173, 174

genic, in interspecific animal hybrids, 168, 169 in interspecific hybrids of Drosophila, 185 in interspecificplant hybrids, 170-173 haplontic, in interspecific hybrids, 174178 incompatibility and sex, relation between, in interspecific hybrids, 181-183 in interspecific hybrids, 147, 148, 165178 in hybrids, and intraspecific diveksity in species, 188-190 in hybrids, and systematic position of species, 186-188 mechanism of, in interspecific hybrids, 190-203 origin of, in interspecific hybrids, 190203 Subdioecious species in plants, genetics of, 235-245, 249, 250 Systematic position of species and sterility in hybrids, 186-188

T Taxonomy of Drosophila robustu, 2 Tetrad segregation in Basidiomycetes, 58, 59 Tetrads in Basidiomycetes, 52, 53, 64,65 Transduction, linked, in bacteria, 71

Y Yeast, in history of genetics, 41, 44