DROSOPHILA PAULISTORUM GROUP1. The Rockefeller Uniuersity, New York City. Received March 17, 1971

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1 MECHANISMS OF MALE STERILITY IN HYBRIDS OF THE DROSOPHILA PAULISTORUM GROUP1 SANTIAGO PEREZ-SALAS2 AND LEE EHRMAN3 The Rockefeller Uniuersity, New York City Received March 17, 1971 ROSOPHILA PAULISTORUM is a superspecies composed of five semispecies (the Centroamerican, Amazonian, Andean, Orinocan, and Interior), and a heterogeneous group of Transitional populations (DOBZHANSKY, PAV- LOVSKY and EHRMAN 1969; SPASSKY et al ). External morphological, physiological, and ecological differences between the semispecies are too small to be of use in distinguishing the semispecies (PASTEUR 1970). The semispecies can be diagnosed by examination of the gene arrangements in their chromosomes (KASTRITSIS 1969). By far the easiest way to identify the semispecies is, however, by outcrossing the strains to be tested to tester strains known to belong to the respective semispecies. As a rule, fertile progeny are obtained only in crosses to a single tester strain. The gene flow between the semispecies is limited or prevented, presumably in nature as well as in laboratory experiments, by ethological isolation and by sterility of male hybrids. Hybrid females are always vigorous as well as fertile. Male sterility is of at least two kinds, that of the F, hybrid males which is due to their own genetic constitutions, and that of the backcross males, which apparently is induced by the genetic constitution of their mothers. The F, hybrid males have chromosome sets of two different semispecies, and the cytoplasm of one of them. The sterility is genic, since chromolsome pairing at meiosis is usually normal, while degenerative changes take place in the spermatocytes and spermatids before and after meiosis. ( DOBZHANSKY, unpublished observations). The sterility of males from the backcross progenies between the Centroamerican, Amazonian. and Andean semispecies was found by EHRMAN (1960) to be due not to the genetic constitution of these males themselves but to that of their mothers. What seems to be involved here is a maternal effect: sons of hybrid mothers are sterile even if they have chromosomes of only a single semispecies, and are in this sense nonhybrids. As shown by EHRMAN (1962), the sterility of the F, and backcross males is due to different causes. A few crosses even give fertile F, but sterile backcross males, and others sterile F, but fertile males in the backcrosses. Furthermore, one instance of hybrid sterility in Drosophila paulistorum, i.e. between the Santa Marta and Mesitas strains, is due to an incompatibility of the Y chromosome with the cytoplasms of the alternate strain. For ' Work supported under contract No. AT-(30-1)-3096, U. S. Atomic Energy Commission. Permanent address: Escuela de Biologia, Universidad Central, Apartado 10098, Caracas, Venezuela United States Public Health Service Career Award, 5K03 IID Genetics 69: Septemk, 1971

2 64 S. PEREZ-SALAS AND L. EHRMAN several backcross generations, if the Y chromosome and the cytoplasm come from the same strain, the male is fertile, while if their sources are different, the male is usually sterile (EHRMAN 1963). More recently, DOBZHANSKY, PAVLOVSKY and EHRMAN (1969) observed that the F, hybrids of Transitional with Centroamerican strains are sterile, while at least some males in the backcrosses are fertile. The Interior semispecies is more closely related to the Orinocan than is any other pair of semispecies ( PEREZ-SALAS et al. 1970). One of us ( PEREZ-SALAS) found, while testing the hybrids of Interior with Orinocan, Centroamerican and Transitional populations, that the backcross males are, contrary to the rule, fertile. This necessitates a reexamination of the whole problem of male sterility in the backcrosses. The results of such a reexamination are reported in the present article. MATERIALS AND METHODS The strains of D. paulistorum used in these experiments are listed below. Old strains, collected before 1967 and kept since then in laboratory cultures are marked with asterisks*. The geographic position of the localities Number 1-16 are shown on the map, Figure 1. Numbers 17 and 18 are off the map, to the south. 1. Lancetilla, Honduras, Centroamerican. 2. Turbo, Colombia, Orinocan. 3a. Santa Marta-I, Colombia, Transitional*. 3b. Santa Marta-2, Colombia, Orinocan. 3c. Santa Marta-4, Colombia, Transitional. 4a. SarareA, Venezuela. Transitional. 4b. Sarare-C, Venezuela, Orinocan. 5. Valparaiso, Caqueta, Colombia, Interior. 6. Palmira, Colombia, Andean*. 7. Fusagasuga, Colombia, Andean. 8. New Llanos, south of Villavicencio, Colombia*. (The properties of this strain have been described by DOBZHANSKY and PAVLOVSKY (1967).) 9. Puerto Ayacucho, Amazonas, Venezuela, Interior. IO. Ocamo, Amazonas, Venezuela, Interior. 11. Mitxi, Vaupes, Colombia, Interior. 12. Leticia-Marco, Amazonas, Colombia, tk Brasil, Interior. 13. Georgetown, Guyana, Orinman*. 14. Belem, Brazil, Amazonian*. 15. Manaus, Amazonas, Brazil, Interior. 16. Tapuruquara, Amazonas, Brazil, Interior. 17. Tingo Maria, Peru, Andean. 18. Angra, Rio, Brazil, Andean*. The crosses were made by placing about ten virgin females of one strain with about the same number of males of another strain and transferring the parents to fresh culture bottles a week later. All cultures were kept at 25 C. The F, hybrids were also placed for a week in one culture bottle, and then transferred to a fresh bottle. If no larvae appeared in either culture, the F, hybrid males were recorded as sterile. The females were then backcrossed to one or to both parental strains, and the fertility or sterility of the backcross males was tested similarly, in two successive cultures. When the backcross males were found to be sterile, it was desirable to reconfirm this with a greater number of males. Since D. paulistorum has three pairs of chromosomes, one-eighth of the backcross hybrids should have the chromosomes of a single semispecies. Recombination within the chromosomes is reduced owing to the presence of numerous inversions. If 100 backcross males

3 MALE STERILITY IN D. paulistorum 04 R n FIGURE 1.-The in this study. geographic origin of some of the Drosophila paulistorum strains employed are tested, about a dozen of them should be effectively nonhybrids as far as their chromosomal genes are concerned. Their sterility can then be ascribed to their descent from hybrid mothers. The flies in the backcross test cultures were transferred to fresh culture bottles at three day intervals for a total of three weeks. If no larvae appeared in any of the cultures (though eggs were abundantly deposited, since the females are fertile), the males were judged to be sterile. RESULTS The intercrosses of Interior strains (the first six strains from the top and on the left of Figure 2) succeed easily and yield fertile F, hybrids of both sexes. The New Llanos strain behaves as though it belonged to the Interior semispecies. The 24 crosses of Interior to Orinocan (Sarare-C, Georgetown, Turbo) and to Centroamerican (Lancetilla) produce hybrid progenies, but, without exception, the F, males are sterile. Backcrosses were then made of the F, hybrid females separately to males of both parental semispecies. Of the 48 backcrosses, 41 produced enough progeny to make possible tests of the fertility of the backcross males. With a single exception, fertility was observed in all tests. This proves that some, though not necessarily all, males were fertile. The exception is the cross New Llanos? x Sarare-C8 ; the F, hybrid females were backcrossed to New Llanos as well as to Sarare-C males; the males from the backcross to New Llanos were sterile, and those from that a Sarare-C were fertile. The two Transitional strains from Santa Marta behave very differently. S. Marta-I crosses easily to Interior, Centroamerican and Orinocan, the PI hybrid males are sterile (except with Centroamerican), but in the backcrosses fertile

4 66 S. PEREZ-SALAS AND L. EHRMAN (8) New Llanos (9) Pto. Ayacucho (IO) Ocamo (11) Mi+; (12)Leticia-Marco (5) Val para ;so (4) Sarare C (13) Georqetown (2) Turbo A (I) Lancetilla (3) Sta. Marta I SF SF SF! FIGURE 2.-Fertility or sterility of the F, and BC, hybrid males produced by crosses between strains of the Drosophila paulistorum semispecies. SF sterile F, but some fertile BC, males; SS = sterile F, and sterile BC, males; F = fertile F, males, no backcross progeny tested; S = sterile F, males, no backcross progeny tested. males do appear (Figure 2). S. Marta-4 is refractory to crossing with Interior and with Orinocan. The third Transitional strain tested, Sarare-A, behaves like S. Marta-4. Interior, Transitional, and Orinocan strains are difficult to cross to Andean or to Amazonian strains. Out of 24 crosses attempted, only two yielded progeny. The successful crosses were Fusagasuga (Andean) 0 x S. Marta-4 (Transitional) 8, and Sarare-A (Transitional) 0 x Palmira (Andean) 8. The F, hybrid males were, of course, sterile. The F, females from the first cross were backcrossed to S. Marta-4 males; the male backcross products were sterile. The backcross of the F, females from the second cross to Palmira males produced fertile sons.

5 MALE STERILITY IN D. paulistorum 67 Backcross male fertility is, thus, a much more common phenomenon than heretofore known. Interior, the most recently discovered semispecies, is particularly capable of crossing with Orinocan, Centroamerican, and Transitional strains, and of producing at least some fertile males in the progeny of the first backcrosses. Tests of sperm motility, made in physiological insect saline, on backcross males show low percentage of males with motile spermatozoa, as follows (the fractions represent the number of males displaying motile sperm out of the number dissected) : (New Llanos, Interior 0 x Sarare C Orinocan 8 ) F, P X Sarare C (Puerto Ayacucho, Interior 0 x Georgetown, Orinocan 8 ) F, x Georgetown 8. (Ocamu, Interior 0 x Sarare C, Orinocan 8 ) F, 0 X Sarare C (Turbo, Orinocan 0 x Leticia-Marco, Interior 8 ) F, 0 X Turbo (Turbo, Onnocan? x Lancetilla, Centroamencan$) F, P x Lancetilla (Puerto Ayacucho, Interior 0 x Santa Marta, Transitional 8 ) F, 0 ~Puert~ Ayacuchoa (Santa Marta, Transitional 0 x New Llanos, Interior 8 ) F, 0 x New Llanos 8.. (Santa Marta, Transitional 0 x Sarare C, Orino'can 8 ) F, 0 x Sarare C (Lancetilla, Centroamerican 0 x Mith, Interior 8 ) F, 0 x Mith (Sarare A, Transitional 0 x Palmira, Andean 8 ) F, 0 X Palmira /20=30% 4/16=25 % 4/19=21% 1/12=8% 3/25=12% 3/30=10% 1/10=10% 1/10=10'% 5/26=19% 5/21=24% EHRMAN'S ( 1960) all-or-none hypothesis concerning the sterility of backcross males must evidently be modified. That it holds for some crosses between the semispecies has been confirmed by the following crossing experiments: 1. Belem (Amazonian) 0 x Lancetilla (Centroamerican) 8 2. Belem (Amazonian) 0 X S. Marta-2 (Orinocan) 8 3. Belem (Amazonian) 0 x Fusagasuga (Andean) 8 4. Lancetilla (Centroamerican) 0 x Angra (Andean) 8 5. Lancetilla (Centroamerican) 0 x Tingo Maria (Andean) 8 6. Lancetilla (Centroamerican) 0 x Fusagasuga (Andean) 8 7. Tingo Maria (Andean) P X Belem (Amazonian) 8 8. Tingo Maria (Andean) 0 x S. Marta-2 (Orinocan) 8 9. Tingo Maria (Andean) 0 x Tapuruquara (Interior) Angra (Andean) 0 x Belem (Amazonian) Fusagasuga (Andean) 0 X Lancetilla (Centroamerican) Tapuruquara (Interior) 0 x Belem (Amazonian) S. Marta-2 (Orinocan) 0 x Belem (Amazonian) 8 Experiments No. 4, 5, 6, and 10 were most satisfactory; backcrosses of the F, hybrid females to males of both parental semispecies gave sufficient progeny to test for fertility; more than 100 males from each backcross. All males were sterile. Similar sterility was observed in the experiments No. 1, 2, 7, and 11, but fewer than 100 males per backcross were available for testing. In No. 3 and 13, progeny was obtained in only a single backcross, and a relatively small number of males were tested for fertility (48 in No. 3, 50 in No. 13). All were sterile. In the experiments No. 9 and 12, backcrosses to one parental semispecies yielded only sterile males, while in the offspring of the other backcrosses at least some males were fertile. The backcrosses (Tingo Maria 0 x Tapuruquara 8) 0 x Tingo Maria 8 and (Tapuruquara P x Belem 8) 0 x Belem 8 gave sterile males,

6 68 S. PEREZ-SALAS AND L. EHRMAN while the backcrosses to Tapuruquara males gave fertile progeny. Finally, experiment No. 8 gave fertile males in the progeny of the two reciprocal backcrosses. The nature of the difficulty of securing enough males for fertility tests in some of the crosses deserves comment. Ethological isolation between some strains belonging to different subspecies is so strong that F, hybrids are hard to obtain. However, if a cross is repeated again and again, F, hybrids between probably any two strains of D. paulistorum can eventually be obtained. Some backcrosses succeed without difficulty, because the F, hybrid females easily accept males of both parental semispecies. Other F, females easily accept males of one but not the other parental semispecies, while some refuse to mate with either parental form (EHRMAN 1960). DISCUSSION The data presented in this article show a remarkable variety of fertility and sterility conditions among males obtained in the first backcrosses between semispecies of D. paulistorum. This variety contrasts sharply with the uniform sterility of the F, hybrids. Excepting only some Transitional strains, the F, hybrids between strains belonging to different semispecies are invariably completely sterile. The backcross males may also be completely sterile, their testes being in some crosses even more abnormal than those of the F, male hybrids. Yet the males in some other backcrosses seem to be fully fertile and anatomically normal. In still other backcrosses intermediate conditions are observed. Although no systematic study of the cytology of hybrid males has been made, we have found ostensibly normal motile spermatozoa in backcross males which were sterile, and also grossly disturbed spermatogenesis in males from other backcrosses, All backcross males tested were sterile in the following experiments: Amazonian x Andean (5 combinations of strains) Centroamerican x Andean (4 combinations) Amazonian x Orinocan (2 combinations) Andean x Orinocan (1 combination) Amazonian x Centroamerican (1 combination) Transitional x Interior (1 combination) In the following experiments both reciprocal backcrosses yielded at least some fertile males: Interior x Orinocan (14 combinations of strains) Interior x Transitional (5 combinations) Interior x Centroamerican (4 combinations) Transitional x Orinocan (2 combinations) Andean x Orinocan (1 Combination) Orinocan x Centroamerican (2 combinations) Transitional x Centroamerican (1 combination). In two experiments one of the reciprocal backcrosses gave some fertile males, while the other backcross produced only sterile ones: Interior x Andean (1 combination) Interior x Amazonian (1 combination).

7 MALE STERILITY IN D. paulistorum 69 It can be seen that most experiments involving strains of the Amazonian semispecies produce only sterilemales in the backcrosses. Amazonian is the most distinctive among the semispecies in its cytological characteristics ( KASTRITSIS 1967), in low crossability to the other semispecies (CARMODY et al. 1962), in its song (BENNET-CLARK and EWING 1970), but not in its morphology (PASTEUR 1970). Transitional and Interior seem, on the contrary, most apt to give fertile backcross males when crossed to each other or to the other semispecies (they do not, however, cross easily to Amazonian). Orinocan and Andean are intermediate. Orinocan crosses most easily to Interior, and gives fertile backcross males. In crosses of Orinocan to Andean either sterile or fertile backcross males can be obtained, depending on the strains used. Most interesting are the crosses in which all first backcross hybrid males are sterile, even though some of them must have chromosomes of a single semispecies. This condition can be explained most readily on the assumption that the hybrid chromosome complement present in the F, mothers so modifies the egg cytoplasm that a male developing from this egg is sterile regardless of its own chromosomal complement. Professor J. PREER and Dr. R. RICHMOND of the University of Indiana have kindly called our attention to the fact that the maternal effect cannot be explained by a conflict between the chromosome set of one semispecies and the cytoplasm of the other. Indeed, in several experiments the males were sterile in backcrosses to both parental semispecies, although in one of the backcrosses the source of the cytoplasm (obtained from the parental generation females) was the same as the chromosome set in the sterile males. The maternal effect is consequently induced by the chromosome set present during the oogenesis or even at earlier stages of the germ line development. Whether or not the partial sterility and fertility of backcross males in other experiments is due to a similar but less extreme maternal effect is now an open problem. SUMMARY Crosses between the different Drosophila paulistorum semispecies (Centroamerican, Amazonian, Andean, Orinocan, and Interior) produce wholly sterile F, male hybrids, but in the first backcross generation, a variety of situations of complete and partial male fertility and sterility are observed. The F, sterility is due to the genotype of the males themselves; that of the backcross males however, is sometimes due to the genotype of their mother and, in one instance, to an interaction between the cytoplasm and a foreign Y chromosome. Most surprisingly, as shown by the data in this present report, sons derived from backcrosses of hybrid females to males from the maternal strain, may still be sterile despite apparent homozygosity for maternal genes. The mode and magnitude of the maternal effects producing these assorted degrees of hybrid male sterility await clarification. Most backcrosses involving strains of the Amazonian semispecies produce only sterile males. The opposite is true of the Interior semispecies, while Centroamerican, Andean, and Orinocan semispecies are intermediate.

8 70 S. PEREZ-SALAS AND L. EHRMAN LITERATURE CITED BENNET-CLARK, H. and A. EWING, 1970 The love song of the fruit fly. Scientific American 223 : CARMODY, G., A. DIAZ C~LLAZO, TH. DOBZHANSKY, LEE EHRMAN, I. S. JAFFREY, S. KIMBALL, S. OBREBSKI, S. SILAGI, T. TIDWELL and R. ULLRICH, 1962 Mating preferences and sexual isolation within and between the incipient species of Drosophila paulistorum. Amer. Midland Naturalist 6$: DOBZHANSKY, TH. and 0. PAVLOVSKY, 1967 Experiments on the incipient species of the Drosophila paulistorum complex. Genetics 55 : DOBZHANSKY, TH., 0. PAVLOVSKY and LEE EHRMAN, 1969 Transitional populations of Drosophila paulistorum. Evolution 23 : EHRMAN, LEE, 1960 A genetic constitution frustrating the sexual drive in Drosophila paulistorum. Science 131: , 1962 Hybrid sterility as an isolating mechanism in the genus Drosophila. Quarterly Rev. of Biology 37: , 1963 Apparent cytoplasmic sterility in Drosophila paulistorum. Proc. Natl. Acad. Sci. U.S. 49: KASTRITSIS, C. D., 1967 A comparative study of the chromosomal polymorphs in the incipient species of the Drosophila paulistorum complex. Chromosoma 23: , 1969 A cytological study of some recently collected strains of Drosophila paulistorum. Evolution 23: PASTEUR, G., 1970 Biometrical data on the semispecies of the Drosophila paulistorum complex. Evolution 24: PEREZ-SALAS, S., R. C. RICHMOND, C. D. KASTRITSIS, L. EHRMAN and TH. DOBZHANSKY, 1970 The Interior semispecies of Drosophila paulistorum. Evolution 24: SPASSKY, B., R. C. RICHMOND, S. PEREZ-SALAS, 0. PAVLOVSKY, C. A. MOURAO, A. S. HUNTER, H. HOENIGSBERG, TH. DOBZHANSKY and F. J. AYALA, 1971 Geography of the sibling species related to Drosophila willistoni, and of the semispecies of the Drosophila paulistorum complex. Evolution 25: