Genetics of Reproductive Isolation in the Drosophila simulans Clade : DNA Marker - Aisisted Mapping and Characterization of a Hybrid - Male

In this study, we address the question of whether there exist major genes that cause complete male sterility in the interspecific hybrids of Drosophila and, if they do, how these genes may be characterized at the molecular level. Our approach is to introgress small segments of the X chromosome from Drosophila mauritzana (or Drosophila sechellia) into Drosophila simulans by repeated backcrosses for more than 20 generations. The introgressions are monitored by both visible mutations and a series of DNA markers. We compare the extent of introgressions that cause male sterility with those that do not. If a major sterility factor exists, there should be a sharp boundary between these two classes of introgressions and their breakpoints should demarcate such a gene. Furthermore, if male sterility is the only major fitness effect associated with the introgression, recombination analysis should yield a pattern predicted by the classical three-point cross. Both the genetic and molecular analyses suggest the presence of a major sterility factor from D. mauritiana, which we named Odysseus (Ods), in the cytological interval of 16D. We thus formalize three criteria for inferring the existence of a major gene within an introgression: (1) complete penetrance of sterility, (2) complementarity in recombination analysis, and (3) physical demarcation. Introgressions of Ods from D. sechellia do not cause sterility. Twenty-two introgressions in our collection have breakpoints in this interval of about 500 kb, making it possible to delineate Ods more precisely for molecular identification. The recombination analysis also reveals the complexity of the introgressed segments-even relatively short ones may contain a second male sterility factor and partial viability genes and may also interfere with crossovers. The spermatogenic defects associated with Ods and/or a second factor were characterized by phasecontrast microscopy. T HE evolution of reproductive isolation is undoubtedly one of the central issues in evolutionary biology (DARWIN 1859; DOBZHANSKY 1970). Our understanding of the genetic basis of this important phenomenon, unfortunately, has remained primitive in an era when large strides have already been made on many difficult biological questions, such as morphogenesis and sex determination (e .g . , HODGKIN 1990). In this report, we wish to provide a framework of analysis that attempts genetic fine-mapping and characterization of genes involved in reproductive isolation by means of DNA markers. We are optimistic that this approach, in conjunction with others (WATANABE 1979; PANTAZIDIS and ZOUROS 1988; HUTTER, ROOTE and ASHBURNER 1990; JEAN-FRANCOIS 199 1 ; PALUMBI 1992; ORR 1992; SAWAMURA, TAIRA and WATANABE 1993), may eventually lead to an understanding of reproductive isolation at the molecular level. Among the different aspects of reproductive isolation, hybrid male sterility is of particular interest for ’ To whom correspondence should be addressed. Genetics 133: 261-275 (May, 1993) several reasons. First, in animal species whose males are heterogametic (such as mammals and Drosophila), hybrid male sterility appears very quickly after species divergence as is evidenced by a large number of interspecific crosses (Wu 1992). In fact, the rapid appearance of hybrid male sterility accounts for the majority of cases in Drosophila and mammals that follow Haldane’s rule ( 1 922), which states that if only one of the two sexes in the F1 hybrids is inviable or sterile, it is the heterogametic sex. Inviability in hybrid males, in contrast, is relatively infrequent between incipient species despite a much greater mutagenic potential for inviability than for sterility (WU and DAVIS 1993). Second, hybrid male sterility represents a well-defined developmental system for genetic analysis, namely spermatogenesis. Studies have shown that hybrid sterility in Drosophila is germ-cell autonomous (DOBZHANSKY and BEADLE 1936) and that some hybrid sterility factors may have no detectable effects on viability (JOHNSON and WU 1993). It is possible to view hybrid male sterility as a pure developmental genetic question where the spermatogenic “mutants” are evolutionarily successful variants of another species. De262 D. E. Perez et al. spite their crucial role in postmating reproductive isolation, spermatogenic defects have been characterized cytologically in only a few hybridization studies (e.g., SCHAEFER 1978; NAVEIRA and FONTDEVILA 1991; PANTAZIDIS et al. 1993). Third, traits of postmating reproductive isolation per se (hybrid inviability and sterility) are apparently "maladaptive." Thus, the evolution of these traits is a perplexing problem. A central question about the genetic basis of hybrid sterility is whether there are a small number of discrete factors, each with a complete (or at least major) effect on male fertility or, alternatively, a large number of genes, each having only minor effects. An understanding of reproductive isolation at the molecular level is immensely more achievable if the former is true. Although many publications have already suggested the existence of major effect genes (e.g., ZOUROS 1981; Wu and BECKENBACH 1983; COYNE and CHARLESWORTH 1986), in almost all cases the observations are also compatible with the alternative interpretation that many genes of minor effect are responsible. Some authors indeed consistently favor the latter view (NAVEIRA and FONTDEVILA 1986, 1991; NAVEIRA 1992). The contrasting interpretations of essentially the same type of data clearly point out a need for more rigorous criteria. In this report, we propose three criteria for inferring the existence of a major effect gene. First, under the major gene hypothesis, penetrance of sterility for any genotype should be (nearly) complete. It is important to note that sterility or fertility is the property of an introgression genotype and this property should be deduced from a population of genotypically identical individuals. Second, recombination analysis by markers flanking the putative sterility factor should map the factor to the same location from both ends (complementarity). Third, the putative major factor should be assignable to an ever more refined interval demarcated by a series of DNA markers. In DISCUSSION, we will review briefly the quest to identify major genes in light of these criteria. Physical demarcation of hybrid sterility genes can now be attempted thanks to many recent developments in DNA technology. In theory, if two species have diverged by 1 %, one expects two chromosomes to have one base pair (bp) different out of 100 bp, which is the theoretical limit of marker density. In practice, the resolution of mapping depends on the number of recombinant lines that can be generated and analyzed for their DNA markers in stage I1 of Figure 1. Thus, the practical limit is determined by the biology of the species chosen (such as their chromosomal constitution and the genetic tools available in the species), the crossing scheme employed and the available molecular techniques for detecting nucleotide differences at specific chromosomal locations. + + + \ I. / I" "7 " x