CRYPTIC REPRODUCTIVE ISOLATION IN THE DROSOPHILA SIMULANS SPECIES COMPLEX

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1 Evolution, 55(1), 2001, pp CRYPTIC REPRODUCTIVE ISOLATION IN THE DROSOPHILA SIMULANS SPECIES COMPLEX CATHERINE S. C. PRICE, CHRISTINE H. KIM, CARINA J. GRONLUND, AND JERRY A. COYNE 1 Department of Ecology and Evolution, The University of Chicago, 1101 East 57th Street, Chicago, Illinois Abstract. Forms of reproductive isolation that act after copulation but before fertilization are potentially important components of speciation, but are studied only infrequently. We examined postmating, prezygotic reproductive isolation in three hybridizations within the Drosophila simulans species complex. We allowed females to mate only once, observed and timed all copulations, dissected a subset of the females to track the storage and retention of sperm, examined the number and hatchability of eggs laid after insemination, counted all progeny produced, and measured the longevity of mated females. Each of the three hybridizations is characterized by a different set of cryptic barriers to heterospecific fertilization. When D. simulans females mate with D. sechellia males, few heterospecific sperm are transferred, even during long copulations. In contrast, copulations of D. simulans females with D. mauritiana males are often too short to allow sperm transfer. Those that are long enough to allow insemination, however, involve the transfer of many sperm, but only a fraction of these heterospecific sperm are stored by females, who also lay fewer eggs than do D. simulans females mated with conspecific males. Finally, when D. mauritiana females mate with D. simulans males, sperm are transferred and stored in abundance, but are lost rapidly from the reproductive tract and are therefore used inefficiently. These results add considerably to the list of reproductive isolating mechanisms in this well-studied clade and possibly to the list of evolutionary processes that could contribute to their reproductive isolation. Key words. Drosophila, fertilization, hybridization, reproductive isolation, sperm. The enumeration, classification, and analysis of reproductive isolating mechanisms has been a central task in the study of speciation, especially for the many biologists who adhere to the biological species concept (Dobzhansky 1935; Mayr 1942). Although barriers to hybridization may exist at any stage of reproduction, barriers between copulation and fertilization have received much less attention than either sexual isolation or hybrid sterility and inviability. This relative neglect is reflected in the common practice of classifying isolating mechanisms as either premating and postmating or prezygotic and postzygotic. As noted by Markow (1998), barriers that act between mating and fertilization are either ignored or grouped together with very different barriers. Evolutionists have long appreciated that gametic incompatibilities are of primary importance in isolating species of plants (Darwin 1859) and of broadcast-spawning marine invertebrates that lack courtship, copulation, and thus sexual isolation (Dobzhansky 1937). Yet even species with internal fertilization may show substantial genetic isolation through barriers to reproduction that act after copulation but before fertilization. For example, the ground crickets Allonemobius fasciatus and A. socius remain distinct across a large contact zone primarily through conspecific sperm precedence, the competitive superiority of conspecific over heterospecific sperm when both exist together within a single female (Howard et al. 1998a). Most species are probably isolated by the cumulative effects of several mechanisms (Coyne and Orr 1998), and our understanding of speciation in any group is necessarily incomplete until one checks for the presence of postmating, prezygotic isolation (Grimaldi et al. 1992; Katakura 1997). In addition, cryptic barriers to fertilization may be the byproducts of a unique and intriguing set of phenomena. These 1 Corresponding author. j-coyne@uchicago.edu The Society for the Study of Evolution. All rights reserved. Received April 11, Accepted August 1, include the evolution of mechanisms that prevent multiple sperm from fertilizing a single egg (polyspermy; Rice and Holland 1997; Howard et al. 1998b) selection for resistance to sexually transmitted diseases (Sheldon 1993), competition among ejaculates of different conspecific males within a single female (Parker 1970), cryptic female choice of sperm (Eberhard 1996) and other varied forms of intersexual reproductive conflict (Rice and Holland 1997). If these processes shape the evolution of reproduction within geographically isolated populations, their divergence among populations may cause reproductive isolation as an incidental byproduct. Furthermore, in some species such divergence may be directly favored by natural selection when the ranges of previously allopatric taxa overlap secondarily, leading to reinforcement, or selection for increased prezygotic isolation that will avoid the production of sterile or inviable hybrids (Howard and Gregory 1993; Wade et al. 1994; Albuquerque et al. 1996; Vacquier et al. 1997). In animals with internal fertilization, postmating barriers to fertilization cannot be directly observed and so are often overlooked in hybridization studies that involve mass matings (Coyne and Orr 1989; Howard and Gregory 1993). Nonetheless, careful observation of interspecific copulations and/ or dissection of females after copulation show that a variety of barriers can prevent heterospecific inseminations from producing hybrid progeny. For example, matings between females of the dermestid beetle Trogoderma glabrum and males of T. inclusum result in successful transfer of a spermatophore, but there is no fertilization because sperm do not migrate from the spermatophore to the sperm storage organs (Vick 1973). Likewise, copulations between Drosophila buzzatii females and D. arizonae males result in successful sperm transfer, but cause an insemination reaction, the formation of a mass in the vagina that inhibits sperm storage and blocks oviposition (Patterson 1946). In matings between Drosophila pulchrella females and D. suzukii males, the heterospecific

2 82 CATHERINE S. C. PRICE ET AL. seminal fluid fails to stimulate oviposition, thereby impairing the fertilization that normally occurs when eggs move past the sperm storage organs (Fuyama 1983). Here we identify cryptic obstacles to hybridization in the D. simulans clade by comparing patterns of sperm transfer, storage and use after single interspecific versus single intraspecific copulations. Drosophila simulans is a worldwide human commensal, whereas its sibling species, D. mauritiana and D. sechellia, are native only to the eponymous islands of the Indian Ocean. The three species are morphologically indistinguishable except for the shape of the male genital arch. All interspecific matings in this group exhibit some degree of sexual isolation, and all produce fertile female but sterile male hybrids (David et al. 1974; Lemeunier et al. 1986). The genetic basis of reproductive isolation within this clade has been studied more extensively than in any other group of species (see summary in Coyne and Orr 1998). We use the term cryptic reproductive isolation instead of postmating, prezygotic isolation so that we may include problems with sperm transfer during copulation. Although not strictly postmating events, problems with sperm transfer not associated with shortened copulations are cryptic they cannot be inferred from direct observation of matings, but must be seen by dissecting mated females (Grimaldi et al. 1992). We previously documented one form of cryptic isolation among D. simulans, D. sechellia, and D. mauritiana known as conspecific sperm precedence (Price 1997). This process occurs when a female mates with both a conspecific and a heterospecific male and leads to heterospecific sperm fertilizing far fewer eggs than they do after single interspecific matings. In contrast to this phenomenon, which requires multiple mating, the barriers to fertilization we describe here can act as isolating mechanisms regardless of whether females mate with one or with multiple males. MATERIALS AND METHODS Drosophila Stocks Stocks were reared in uncrowded cultures at 24 C with a 12-h light-dark cycle on standard cornmeal-yeast-agar medium. Drosophila simulans flies were taken from the Florida City (FC) line, an isofemale line collected in 1985 in Florida City, Florida and maintained in large populations. Drosophila mauritiana flies were taken from the synthetic (SYN) stock, a wild-type line synthesized by combining six isofemale lines collected on Mauritius by O. Kitagawa in Drosophila sechellia males were taken from a wild-type stock collected on Cousin Island in 1980 and provided by H. Robertson. These lines have all been used extensively in studies of reproductive isolation in this species complex (e.g., Coyne 1989). To determine whether cryptic isolating mechanisms varied among strains of a species, we repeated some of our experiments using alternate strains. For these experiments, D. simulans were from an isofemale strain collected in Ottawa, Ontario, in September 1985; D. sechellia from an isofemale strain (ss77 25X) collected in Praslin, Seychelles, by K. Kimura in July 1987; and D. mauritiana from an isofemale line (no. 197) collected at the same time as the other lines composing the synthetic strain described above. Observations of Copulation and Rearing of Progeny Males and females were collected as virgins under CO 2 anesthesia and stored in 8-dram food vials. All copulations took place on the morning of the fourth day after eclosion, at room temperature (21 23 C). Mating observations began within 1 h after lights came on in the incubators. One female and one to five males were transferred without anesthesia into a fresh food vial for observation, the time was recorded at the beginning and end of each copulation, and males were removed from the vial immediately after copulation ended. Up to 50 vials were observed simultaneously, and observations lasted from 45 min to 5 h depending on the ease with which mating occurred. Females who failed to mate within 5 h were discarded. Copulations between D. simulans females and D. mauritiana males that lasted less than 7 min were excluded unless noted, because these are too short to result in sperm transfer (Coyne 1993; additional data not shown). Three intraspecific crosses (within D. simulans, D. mauritiana, and D. sechellia) and three interspecific crosses (D. simulans females D. mauritiana males; D. mauritiana females D. simulans males; D. simulans females D. sechellia males) were performed. The interspecific matings involving D. simulans females proceed with relative ease, but copulations between D. mauritiana females and D. simulans males occur only rarely (David et al. 1974; Coyne 1989). Of 588 D. mauritiana females presented with D. simulans males, only 91 (15.5%) accepted a copulation within 5 h. Copulations between D. mauritiana and D. sechellia or between D. sechellia females and D. simulans males are even rarer (Lachaise et al. 1986) and thus were not examined. Mated females were transferred individually to a fresh vial 2 days after copulation and thereafter transferred to fresh vials every 3 days until either the females stopped laying fertile eggs or were used for a separate experiment (see below). Progeny were reared to adulthood at 24 C with a 12-h lightdark cycle on standard cornmeal-yeast-agar medium. All progeny were counted, and male progeny from matings between D. simulans and D. mauritiana were all confirmed as hybrids by the shape of the genital arch. Drosophila simulans males have a large, helmet-shaped genital arch, whereas the hybrid arch is much narrower (Coyne 1983). Number and Location of Stored Sperm For each mating type, five to 10 females were dissected per day each day at timed intervals after the end of copulation. Females were etherized and their reproductive tracts removed in a drop of phosphate buffered saline (PBS). The spermathecae, seminal receptacle and uterus were each transferred to a separate drop of PBS to prevent mixing of sperm from different organs, and the sperm from each organ were removed with insect pins. Slides were dried at 60 C for 5 10 min, fixed in 3:1 methanol:glacial acetic acid for 5 min, rinsed three times in PBS, and labeled with 0.5 g/ml DAPI (4, 6- diamidino-2-phenylindole) in glycerol. DAPI binds to the DNA in sperm heads, labeling them fluorescent blue. All sperm heads in each of the three storage organs and the uterus of every female were counted using an epifluorescent microscope.

3 CRYPTIC REPRODUCTIVE ISOLATION IN DROSOPHILA 83 TABLE 1. Summary of the prezygotic consequences of interspecific matings. Barriers to hybridization are indicated with an asterisk. Figures showing the data are given under each potential isolating mechanism. Female Male Copulation duration (Fig. 2) Number of sperm transferred (Fig. 3) simulans mauritiana short* normal (excluding 7 min copulations) mauritiana simulans long abnormally many simulans sechellia intermediate abnormally few* Efficiency of initial sperm storage (Fig. 4) Efficiency of sperm use over time (Fig. 6) Rate of sperm loss (Fig. 7) Rate of oviposition (Fig. 9) low* high normal below normal low high low* high fast* normal normal Oviposition Rate and Egg Hatchability A subset of the mated females was set aside for examination of oviposition and egg hatchability. Immediately after mating, these females were transferred individually without anesthesia to vials containing small plastic spoons filled with grape-juice-tinted medium. The females were allowed to lay eggs on these spoons for 24 h, transferred without anesthesia to fresh spoons to lay eggs for another 24 h, and then discarded. Drosophila mauritiana females mated to D. simulans males were transferred to fresh spoons every 24 h for 8 successive days before being discarded. After the females were removed, spoons were stored at 24 C for 28 h, at which time hatched and unhatched eggs were counted using a dissecting microscope. Brown, unhatched eggs, which indicate zygotes that died early in development, were observed only rarely. Nonetheless, we did not determine whether the unhatched eggs we observed were unfertilized or whether they had been fertilized but died early in development. Longevity of Mated Females Previous work in D. melanogaster shows that female longevity is reduced considerably when they mate repeatedly with males, and that this effect is attributable entirely to effects of the seminal fluid, with the act of copulation or the transfer of sperm having no effect on longevity (Chapman et al. 1993, 1995). This toxicity has been attributed to antagonistic coevolution within a species, so that components of the seminal fluid enhancing the reproduction of males have an opposite effect on females (Chapman et al. 1995; Rice and Holland 1997; Holland and Rice 1999). Therefore, we wanted to determine if interspecific evolutionary divergence in seminal fluid or in female response to seminal fluid might lead to a more severe reduction on female longevity after heterospecific than after conspecific matings. Price (1997) gives evidence that such divergence does exist in these species. Nearly all previous studies have examined longevity reductions in females mated to several males in succession, usually by keeping them perpetually or intermittently confined with males. (The one exception is the study of Chapman et al. [1996], in which singly mated D. melanogaster females from both a mutant stock and a wild-type revertant showed no reduction in longevity compared to virgin control females.) In comparing the effects of intraspecific versus interspecific matings on longevity, one must control the exposure of females to males and observe matings because of possible species differences in the courtship behavior, duration, or intensity that might effect longevity independently of insemination. Three treatments were conducted: D. simulans females D. mauritiana males (experimental); D. simulans females D. simulans males (conspecifically mated control); and unmated D. simulans females (unmated control). Four-day-old flies were placed either in pairs (experimental and mated controls) or singly (unmated control) for an observation period of 1 h. All copulations were observed and timed. (We did not use D. simulans females whose mating with D. mauritiana males lasted less than 7 min; as noted above, such matings are usually too short to transfer sperm, but probably do transfer seminal fluid.) The experiment was conducted using two replicates on successive days, with 25 randomly selected flies from each treatment saved for longevity experiments. Immediately after mating, each female was isolated in a food vial and transferred to a fresh food vial every 2 days, with all laid eggs counted at each transfer (eggs were counted because egg-laying is a metabolic cost that might affect longevity). Females were maintained and transferred in this way until all had died. RESULTS Progeny Production Each of the three hybridizations is characterized by a unique set of cryptic barriers to fertilization, which are summarized in Table 1. Figure 1 shows the net result of these barriers, the mean number of progeny produced per female after single inseminations. Those copulations that failed to produce any progeny are excluded from Figure 1 and from all other analyses unless noted, so that the figure presents the minimum level of cryptic reproductive isolation after single successful inseminations. Fewer than 5% of all conspecific copulations failed to produce offspring. In contrast, about half of the matings between D. simulans females and D. mauritiana males are less than 7 min long and never produce offspring (Coyne 1993; additional data not shown). These abnormally short copulations were excluded from further analysis so that we could examine any additional barriers in this hybridization. A D. simulans female singly inseminated by a conspecific male produces on average about 162 progeny (Fig. 1). In contrast, single matings of D. simulans females and D. mauritiana males that are long enough to result in sperm transfer (Coyne 1993) produce on average only 126 progeny, a significantly lower productivity (P 0.02; Mann-Whitney U- test). When copulations shorter than 7 min are included, fe-

4 84 CATHERINE S. C. PRICE ET AL. FIG. 1. Progeny production after single matings in the Drosophila simulans clade. Mean ( SE) total progeny per female after intraspecific (dark bars) and interspecific (light bars) copulations, excluding those females that produced no progeny (see text). Abbreviations: sim, D. simulans; mau, D. mauritiana; sech, D. sechellia; n, number of females. males produce a mean of 65.6 progeny (SE 9.32, n 103). We cannot conclude from these comparisons alone that a D. mauritiana ejaculate suffers from any unique difficulty within a D. simulans female, because the successful interspecific inseminations actually result in significantly more progeny than single conspecific inseminations in D. mauritiana (P 0.001; Mann-Whitney U-test). In contrast, inseminations of D. mauritiana females by D. simulans males produce a mean of only 32 progeny, significantly fewer than produced by either of the two conspecific copulations (P for both comparisons; Mann-Whitney U-tests). Cryptic barriers to hybridization between D. simulans and D. mauritiana are therefore strongest after those copulations that involve D. mauritiana females. Among single copulations between D. simulans females and D. sechellia males, 55% (28 of 51) produce no progeny at all. Those copulations that do result in progeny produce significantly fewer offspring than those produced by conspecific matings within either D. simulans or D. sechellia (P for both comparisons; Mann-Whitney U-tests). This hybridization is therefore characterized by considerable cryptic reproductive isolation. FIG. 2. Mean ( SE) copulation duration, excluding those copulations between Drosophila simulans females and D. mauritiana males that lasted less than 7 min (see text). Abbreviations same as for Figure 1. Copulation Duration Figure 2 shows the mean copulation duration for successful inseminations of all six types. Visual comparison of Figures 1 and 2 reveals that differences in copulation duration cannot fully explain the differences in progeny production just described. Like Cobb et al. (1988) and Coyne (1993), we found that copulations between D. simulans females and D. mauritiana males are abnormally short, whereas the reciprocal matings are not. When all observed copulations are included, the mean copulation of a D. simulans female with a D. mauritiana male lasts only 8.1 min (SE 0.1, n 356). Even when those copulations too short to result in sperm transfer (less than 7 min long) are excluded from analysis, the average copulation lasts only 11.7 min (SE 0.2, n 181), significantly shorter than either the conspecific D. simulans copulations (about 30 min; P , Mann-Whitney U-test) or the conspecific D. mauritiana copulations (about 17 min; P , Mann-Whitney U-test). As observed by Coyne (1993), we also found a significant relationship between hybrid progeny production and duration of the D. simulans female D. mauritiana male copulation, whether copulations that fail to produce hybrids are included (n 103, r 0.57, P ) or excluded (n 53, r 0.52, P ). Matings shorter than 7 min never produce progeny, whereas those longer than 13 min invariably do. Matings between D. mauritiana females and D. simulans males last about 27 min longer than the within-mauritiana copulations (P , Mann-Whitney U-test) and nearly as long as the within-simulans copulations. Therefore, a behavioral interruption of sperm transfer during copulation cannot explain the low productivity of matings between D. mauritiana females and D. simulans males (see below for additional evidence). Copulations between D. simulans females and D. sechellia males last on average about 16 min significantly shorter than intraspecific copulations in either D. simulans or D. sechellia, both of which average about 30 min (P for both comparisons, Mann-Whitney U-tests). It is thus possible that this short copulation interrupts sperm transfer in a manner similar to the copulation between D. simulans females and D. mauritiana males. This is, however, not a likely explanation for the paucity of hybrids between D. simulans females and D. sechellia males because the (slightly positive) regression of progeny production on the duration of copulation is not significant, whether we include copulations failing to produce progeny (n 51, r 0.22, P 0.1) or exclude them (n 23, r 0.25, P 0.2). Even copulations as long as min often fail to produce hybrids. Sperm Transfer during Copulation To determine whether the paucity of hybrids following interspecific copulations could be explained by a lack of sperm transfer, we dissected a group of females within a few hours of copulation and counted the total number of sperm

5 CRYPTIC REPRODUCTIVE ISOLATION IN DROSOPHILA 85 FIG. 3. Minimum number of sperm transferred during copulation, estimated by the mean ( SE) number of sperm found in the spermathecae, seminal receptacle, and uterus of females dissected within4hofcopulation and before oviposition, excluding copulations that failed to transfer sperm. Abbreviations same as for Figure 1. present in the uterus and the three sperm storage organs (two mushroom-shaped spermathecae and a tubular seminal receptacle). Females who had previously laid one or more eggs were not dissected, because excess unstored sperm is lost from the uterus by the passage of the first egg (Fowler 1973; Gilbert 1981). Females were not dissected immediately after insemination (with the exception of D. simulans females mated with D. sechellia males; see below) because the dense packaging of sperm at this time makes direct counts very difficult (Gilbert 1981). Because it is possible that some sperm were lost from the female by passive means within the first few hours after mating, the mean number of sperm found in mated females within the first few hours provides a minimum estimate of the number transferred during copulation (Fig. 3). Although copulations of D. simulans females with D. mauritiana males that result in sperm transfer are much shorter than conspecific copulations between D. simulans males and females (Fig. 2), the interspecific matings involve the transfer of just as many sperm (Fig. 3; P 0.5, two-tailed t-test). Remarkably, copulations between D. mauritiana females and D. simulans males those that produce an average of only 32.5 progeny often result in the transfer of more than three times as many sperm as the average for either type of intraspecific copulation. Clearly, problems with sperm transfer are not responsible for the cryptic reproductive isolation seen in both reciprocal crosses between D. simulans and D. mauritiana. In contrast, six of 14 (43%) D. simulans females mated to D. sechellia males contained no sperm in the uterus or either storage organ when dissected immediately after mating. The remaining females had a mean of only 59 sperm in their reproductive tracts (Fig. 3; n 8, SE 19.3). This was only a fraction of the number transferred during either intraspecific D. simulans copulations or intraspecific D. sechellia copulations (P for both comparisons; Mann-Whitney U-tests). This severe problem with sperm transfer is the most likely explanation for the paucity of hybrids produced by these interspecific matings. FIG. 4. Initial sperm storage after single copulations, estimated by the mean ( SE) number of sperm found in the spermathecae and seminal receptacle, of females dissected 2 24 h after copulation, excluding females with no sperm. Abbreviations same as for Figure 1; percent of transferred sperm stored is estimated by dividing the mean from Figure 4 by the mean from Figure 3 for each mating type. Initial Sperm Storage Because eggs are fertilized as they pass by the openings to the sperm storage organs, it is unlikely that they can be fertilized by sperm that are not first resident in these organs (Fowler 1973). The production of hybrids will therefore be limited by the efficiency with which heterospecific sperm are stored after insemination. The number of sperm found in the three storage organs of females dissected 2 24 h after mating was used as a measure of initial sperm storage (Fig. 4). This interval may be too long to estimate the maximum number of stored sperm, which occurs 4 7 h after copulation in D. melanogaster (Gilbert 1981), but was chosen to give a conservative estimate of the number of sperm that females can use over their entire reproductive life span. There were no significant differences among mating treatments in mean time between mating and dissection (P 0.17; Kruskal-Wallis test). Also, within each mating treatment there were no significant differences in the mean number of stored sperm between females dissected within 6hofmating and those dissected the following day (combined P 0.25, combining probabilities from Mann-Whitney U-tests for each of the six mating treatments). We therefore conclude that our use of a 22-h interval after copulation has not introduced significant variation in our estimate of initial sperm storage. Although D. mauritiana males transfer just as many sperm to D. simulans females as do D. simulans males (Fig. 3), the females store significantly fewer of these heterospecific sperm (Fig. 4; P 0.01, Mann-Whitney U-test). This inefficient sperm storage is the most likely explanation for the cryptic isolation after these copulations. Drosophila mauritiana females store on average about 54% of the conspecific sperm that they receive during copulation (Fig. 4). In contrast, they store only about 26% of the much larger number of D. simulans sperm transferred during interspecific copulations. Despite this apparent inefficiency in heterospecific sperm storage, these D. mauritiana females

6 86 CATHERINE S. C. PRICE ET AL. FIG. 5. Mean ( SE) fraction of sperm stored in the spermathecae of females dissected 2 24 h after copulation, calculated for each female by dividing the number of spermathecae sperm by the total number of stored sperm. Abbreviations same as for Figure 1. still store an abundance of sperm, significantly more than they store when inseminated by conspecific males (P , Mann-Whitney U-test). In contrast, D. simulans females store over 90% of the very small number of D. sechellia sperm transferred, but this still represents significantly fewer sperm than the number of conspecific sperm stored by D. simulans females (P 0.001; Mann-Whitney U-test). The efficiency of initial sperm storage therefore plays a very small role in the eventual reproductive isolation found after matings between D. mauritiana females and D. simulans males or between D. simulans females and D. sechellia males. Efficiency of Sperm Use The spatial distribution of sperm among storage organs may play a role in the efficiency of sperm use because the opening of the seminal receptacle is thought to be closer to the site of fertilization than are the openings of the spermathecae (Nonidez 1920) and the use of seminal-receptacle sperm in fertilization may be correspondingly more efficient (Gilbert 1981). We describe the spatial pattern of sperm storage by dividing the number of sperm stored in the two spermathecae by the total number of stored sperm in females dissected 2 24 h after copulation (Fig. 5). Only females with more than 10 stored sperm are included in this calculation. There is significant variation among the six types of matings for the spatial pattern of storage (ANOVA using arcsinetransformed proportions: F 1, , P ). An estimate of how efficiently females use their stored sperm can be obtained from the relationship between the number of sperm initially stored by a female and the total number of progeny she ultimately produces. Figure 6 juxtaposes the ranked observations among females of the number of total stored sperm 2 24 h after mating with the ranked total number of progeny produced. The fewer sperm wasted by females during fertilization, the closer together these distributions will fall (Zimmering and Fowler 1968; Price et al. 2000). For D. simulans females mated to conspecific males (Fig. 6A) or to D. sechellia males (Fig. 6F), the ranked distribution of sperm number lies entirely above, but close, to, that of progeny number. This implies that females waste a small but roughly constant fraction of their stored sperm. For D. simulans females mated to D. mauritiana males (Fig. 6B), there is remarkable overlap between the distributions, suggesting that females use these stored heterospecific sperm at least as efficiently as they use conspecific sperm. In contrast, D. mauritiana females mated to D. simulans males show extremely inefficient use of stored sperm (Fig. 6C). All of these females produce relatively few hybrid progeny, whether they store only 100 or more than 800 sperm (Fig. 6). This inefficiency is the most likely explanation for the cryptic reproductive isolation characteristic of this cross, and we investigate its cause below. The negative correlation suggested by Gilbert (1981) between degree of sperm storage in the spermathecae and the efficiency of conspecific sperm use cannot explain the variation in efficiency seen in these species. Both D. simulans females mated to D. sechellia males and D. mauritiana females mated to D. simulans males store a large fraction (about 59%) of their sperm in the spermathecae (Fig. 5), but sperm use is clearly much more efficient in the D. simulans females (Fig. 6C versus 6F). Moreover, conspecifically mated D. simulans and D. sechellia females each store about 32% of their sperm in the spermathecae (Fig. 5), but use of conspecific sperm appears to be more efficient in D. simulans females (Fig. 6A) than in D. sechellia females (Fig. 6E). Retention of Stored Sperm Figures 7 and 8 show the patterns of sperm storage and offspring production over time after single inseminations. Drosophila simulans females begin with more stored D. simulans sperm than stored D. mauritiana sperm after single matings (Fig. 4; Fig. 7A, B), but they lose both types of sperm at gradual and roughly similar rates (Fig. 7A, B). Drosophila simulans females inseminated by D. sechellia males lose their few stored sperm quite gradually (Fig. 7C), corresponding to the production of fewer than 10 offspring per 3-day cohort for up to 16 days (Fig. 8). By 13 days after copulation, all D. simulans females have fewer than 50 sperm in storage, regardless of the species of their mate (Fig. 7A C). The rate at which sperm are released from apparently has little to do with the cryptic reproductive isolation found after either of the hybridizations involving D. simulans females. In contrast, D. mauritiana females lose D. simulans sperm much more rapidly than they lose conspecific sperm. Although they start out with up to four times as many heterospecific as conspecific stored sperm (Figs. 4, 7D), by 5 7 days after mating D. mauritiana females all have fewer than 50 sperm in storage, regardless of the species of their mate (Fig. 7D, E). The more rapid loss of heterospecific sperm is consistent with the observation of a short interval during which hybrid progeny are produced. Although D. mauritiana females do not produce any hybrids beyond 10 days after mating (Fig. 8D), they continue to produce conspecific progeny for up to 19 days after a single insemination (Fig. 8E). Rate of Oviposition To determine whether the dearth of D. simulans D. mauritiana hybrids could be explained in part by insufficient stim-

7 CRYPTIC REPRODUCTIVE ISOLATION IN DROSOPHILA 87 FIG. 6. Efficiency of sperm use after single inseminations. Ranked distributions of total number of progeny produced after mating (solid circles) and number of sperm stored in the spermathecae and seminal receptacle of females dissected 2 24 h after mating (open circles). Abbreviations same as for Figure 1. ulation of oviposition, we counted the eggs laid by females over the first 48 h after mating (Fig. 9). Drosophila simulans females mated to heterospecific males lay significantly fewer eggs over this interval than D. simulans females mated to conspecific males (P 0.05, Mann-Whitney U-test), and this reduction (16%; Fig. 9) probably contributes to the reduction in progeny production after these matings (22%; Fig. 1). There is no difference in the number of eggs laid by D. mauritiana females after heterospecific and conspecific copulations (Fig. 9; P 0.5, Mann-Whitney U-test). Fertilization and Egg Hatchability Of the eggs laid by D. simulans females mated to conspecific males, 96% hatch over the first 48 h, compared to a hatch of only 39% when the same females are mated to a D. mauritiana male (P ; Mann-Whitney U-test). Although this low hatchability might reflect an early mortality of hybrid zygotes, more likely it reflects a high proportion of unfertilized eggs for the following reasons. Compared to conspecifically mated females, D. simulans females mated to D. mauritiana males initially store only a little over half as many sperm after copulation (Fig. 4) and lose sperm gradually from storage (Fig. 7B), but lay only slightly fewer eggs (Fig. 9). These observations suggest that females do not release sperm at a rate sufficient to fertilize all the eggs they lay. In contrast, D. mauritiana females show roughly the same egg hatchability over the first 48 h after either conspecific or heterospecific copulations (Fig. 10; P 0.1, Mann-Whitney U-test). Figure 11 shows that D. mauritiana females mated to D. simulans males continue to lay more than 20 eggs per day for the first week after insemination, but the proportion of fertilized eggs drops precipitously, until all females cease laying fertilized eggs by 7 days after mating. These results

8 88 CATHERINE S. C. PRICE ET AL. FIG. 7. Sperm retention over time after single inseminations. Mean ( SE) number of sperm stored in the spermathecae and seminal receptacle of five females dissected per day, each day after copulation. Data are grouped into intervals of 2 and 3 days to allow comparison with intervals that females occupied successive vials for progeny counts (see Figure 8). Abbreviations same as for Figure 1. corroborate the earlier conclusion that D. mauritiana females inseminated by D. simulans males lose sperm very rapidly. Longevity of Mated Females We abbreviate the three treatments as follows: D. simulans females D. simulans, SS; D. simulans females D. mauritiana males, SM; and unmated D. simulans females, UN. The two replicates of each treatment were homogeneous for female longevity and total eggs, and so the replicates are combined to compare the effect of mating treatment. Both female longevity and total egg number did not differ among these three treatments. Mean longevities were 43.7 days for SS (SE 1.6, n 42), 40.8 days for SM (SE 1.6, n 45), and 42.9 days for UN (SE 1.7, n 42) and the survivorship curves were nearly coincident (not shown). These longevities were not statistically heterogeneous (AN- OVA: F 2, , P 0.42). Likewise, there was no significant difference in total egg number (mean lifetime output , , and , respectively; F 2, , P 0.14), although the SS females produced more eggs during the first 10 days of observation. There is thus no evidence these single intraspecific or interspecific matings reduce longevity beyond that seen in unmated females. A smaller reciprocal experiment (data not shown) was conducted using D. mauritiana females (1) mated to D. simulans males; (2) mated to D. mauritiana males; (3) unmated and unexposed to males; and (4) unmated but exposed to D. simulans males for the average time it took to achieve this difficult copulation (male harassment control). Sample sizes per treatment ranged from 11 to 15 females because of the difficulty of obtaining the interspecific copulation of treatment 1. As in the study described above, the treatments were not significantly heterogeneous in either female longevity or total egg number. Again, we see no obvious effect on longevity of interspecific compared to intraspecific copulations. Experiments Using Alternate Strains Observations of copulations, dissections of sperm storage organs, and progeny counts were repeated using genetically different strains from each of the three species. Because the data are extensive, we present only a brief summary of our results. In the hybridization between D. simulans females from the Ottawa strain and D. mauritiana males from strain 197, the results were qualitatively identical to those outlined above. That is, interspecific copulations are significantly shorter than either intraspecific copulation, and generate significantly fewer progeny than intraspecific copulations, even when copulations shorter than 7 min are excluded from analysis. Nevertheless, interspecific matings longer than 7 min involve the transfer of just as many sperm as D. simulans intraspecific matings and more than do D. mauritiana intraspecific matings. In the reciprocal cross, the results are again qualitatively identical to those described above. Copulations between D. mauritiana females and D. simulans males last as long as do intraspecific copulations, and result in the transfer of abnor-

9 CRYPTIC REPRODUCTIVE ISOLATION IN DROSOPHILA 89 FIG. 8. Progeny production over time after single inseminations. Mean ( SE) number of progeny produced per female in each successive vial. Sample sizes: Drosophila simulans females D. simulans males, n 45; D. simulans females D. mauritiana males, n 53; D. simulans females D. sechellia males, n 23; D. mauritiana females D. simulans males, n 52; D. mauritiana females D. mauritiana males, n 79; D. sechellia females D. sechellia males, n 25. Copulations that failed to produce any progeny are excluded. Abbreviations same as for Figure 1. mally large quantities of sperm. As with the other strains, the number of stored heterospecific sperm drops precipitously with time, however, leading to an overall lifetime fecundity per female that is lower than that of either within-species cross. Copulations between D. simulans females from the Ottawa strain and D. sechellia males from strain ss77 25x did not show one isolating mechanism described in the other strains. Instead of transferring abnormally few sperm during copulation as described above (Figs. 3, 4; Table 1), these D. se- FIG. 9. Mean ( SE) number of eggs laid per female over the first 48 h after insemination. Females that failed to lay any eggs are excluded. Abbreviations same as for Figure 1. FIG. 10. Mean ( SE) hatchability of eggs laid by females over the first 48 h after insemination. Females failing to lay any eggs are excluded. Abbreviations same as for Figure 1.

10 90 CATHERINE S. C. PRICE ET AL. FIG. 11. Number and hatchability over time of eggs laid by Drosophila mauritiana females mated to D. simulans males (n 15), excluding females that failed to lay any eggs (n 8). (A) Mean ( SE) number of eggs laid per female per day. (B) Mean ( SE) egg hatchability per female per day. chellia males transferred nearly as many sperm to heterospecific females as to conspecific females. This shows that some traits involved in cryptic reproductive isolation may differ among strains of the same species. This is not unexpected in light of the prediction that internal reproductive anatomy and physiology can evolve very rapidly between populations (Rice 1996; Price 1997; Parker and Partridge 1998). There is ample evidence that the degree of postzygotic isolation between species depends on genetic variation within a species. These latter studies have included D. aldrichi and D. mulleri (Crow 1942), Crepis tectorum and C. capillaris (Hollingshead 1930), and the cottons Gossypium barbadense and G. hirsutum (Stephens 1950). Additional examples are given by Dobzhansky (1951, pp ) and Orr (1997, pp ). DISCUSSION The effect of interspecific mating on gene flow may often depend on the complex dynamics of sperm transfer, storage, and use. The three hybridizations we studied exemplify this complexity. Drosophila simulans Female Drosophila mauritiana Male It was shown previously that copulations between D. simulans females and D. mauritiana males are abnormally short, interrupting the transfer of sperm to females (Cobb et al. 1988; Coyne 1993). Those females who copulate long enough to receive sperm, however, receive just as many sperm as do D. simulans females who copulate with conspecific males (Fig. 3). This result was unexpected given that the average interspecific copulation that can yield sperm transfer (i.e., longer than 7 min) is still shorter than 12 min, itself far less than the 30 min observed for intraspecific copulations in D. simulans (Fig. 2). We suspect that transfer of D. simulans sperm to conspecific females is nearly complete after about 14 min from the start of copulation, because interrupting copulations after 14 min does not decrease progeny production (Price et al. 2000). It is possible that D. simulans males continue to transfer nonsperm components of the seminal fluid during the second half of copulation, perhaps including some components necessary for proper sperm storage (see review in Wolfner 1997). A difference in the amount or nature of such components transferred by D. mauritiana males might explain why D. simulans females store a smaller fraction of transferred D. mauritiana sperm than of conspecific sperm (Fig. 4). We also found that D. simulans females inseminated by D. mauritiana males lay slightly but significantly fewer eggs over the first 48 h after mating (Fig. 9), suggesting that D. mauritiana males may also differ from D. simulans males in the nature or amount of the seminal fluid components that stimulate oviposition (Fuyama 1983; Wolfner 1997). We conclude, however, that cryptic reproductive isolation after insemination of D. simulans females by D. mauritiana males is due primarily to the smaller fraction of heterospecific sperm that are initially stored. Although subsequent use of this sperm is gradual and efficient, the dearth of sperm produces a low overall rate of fertilization. If female longevity were reduced more by interspecific than by intraspecific matings, and this reduction reduced the number of progeny, this would constitute a form of prezygotic isolation, because the parents and not the offspring would suffer from hybridization. We found, however, no effect of hybridization on female longevity; indeed, there was no effect of single conspecific matings on longevity, as was found by Chapman et al. (1996) in strains of D. melanogaster. We used only single matings because of possible interspecific differences in courtship that might themselves affect female longevity were females left continuously with males. It is clear that if interspecific matings between these species have more toxic effects on female longevity than do intraspecific matings, the effects must be rather small. These observations should be repeated using multiple matings with appropriate controls, although such work would be difficult. We are unaware of any work explicitly testing the possibility of cryptic reproductive isolation occurring via reduced female longevity after hybrid matings. Drosophila mauritiana Female Drosophila simulans Male The paucity of hybrids in the reciprocal cross appears to have a very different explanation. Copulations between D.

11 CRYPTIC REPRODUCTIVE ISOLATION IN DROSOPHILA 91 mauritiana females and D. simulans males are long (Fig. 2) and result in the transfer of abnormally large numbers of sperm (Fig. 3). We have anecdotal evidence to suggest that these sperm masses can interfere with oviposition because about 10% of these females die without laying any eggs. Dissection of these dead females revealed a hardened, yellowed mass in the vagina; a phenomenon never observed in any of the other crosses. The surviving females show no difficulty in storing D. mauritiana sperm initially (Fig. 4), but then lose sperm from the storage organs quite rapidly (Fig. 7). This rapid loss is most likely responsible for the steady drop in the fertilization rate over the week following insemination (Fig. 11), leading to an overall inefficiency in the use of stored sperm (Fig. 6C). Drosophila simulans Female Drosophila sechellia Male Finally, copulations between D. simulans females and D. sechellia males involve the transfer of abnormally small numbers of sperm in one of the two sets of strains used (Fig. 3). Nearly half these females receive no sperm during copulation and the remainder very few. This dearth of sperm is not strongly related to the duration of copulation (Fig. 2) because copulations as long as min often fail to result in sperm transfer. The females store over 90% of the few sperm they receive during copulation (Fig. 4), followed by gradual and fairly efficient use of those stored sperm in fertilization (Figs. 6, 7, 8). Spatial Pattern of Sperm Storage The fraction of sperm initially stored in the spermathecae differs considerably among the six types of matings we examined (Fig. 5). The significance of this finding is unclear, however, because we observed no consistent relationship between the location of stored sperm and the overall efficiency of their use (Figs. 5, 6). In addition, in our strains we find no evidence to support the common assertion that the spermathecae are used for long-term storage of sperm (e.g., Nonidez 1920; Fowler 1973; Gromko et al. 1984; Pitnick et al. 1999). Instead, conspecifically mated D. simulans, D. mauritiana, and D. sechellia females dissected beyond the first week after copulation had on average more sperm in their seminal receptacles than in their spermathecae and females with no sperm in the seminal receptacle almost invariably lacked sperm in their spermathecae. In an independent set of dissections, we found that conspecifically mated D. melanogaster females also deplete their store of spermathecae sperm at a rate equal to or greater than that at which they deplete their seminal receptacle sperm (Price et al. 1999; data not shown). Species and strains may differ in their use of the storage organs (Pitnick et al. 1999), but our results indicate that one should be cautious about assuming that spermathecae in this species group are specialized for long-term storage. Cryptic Reproductive Isolation We suggest that the term cryptic reproductive isolation be used in situations where the line becomes blurred between premating and postmating isolation or between prezygotic and postzygotic isolation. For example, problems with heterospecific sperm transfer that occur during copulation cannot accurately be described as either premating or postmating isolation. In addition, gametic incompatibilities may result in postzygotic isolation (hybrids do not appear after mating), but actually span the division between pre- and postzygotic isolation. For example, species of amphibians have (presumably evolved) mechanisms for avoiding polyspermy of eggs; such polyspermy causes egg mortality. However, divergence in these mechanisms among amphibian species leads to polyspermy in heterospecific crosses and death of hybrid zygotes (Gómez and Cabada 1994). Moreover cryptic reproductive isolation between animal species with internal fertilization may be a byproduct of a unique set of evolutionary forces acting within species, including sperm competition, cryptic female choice of sperm, and intersexual evolutionary conflict. These phenomena are only now beginning to attract attention. Because cryptic isolating mechanisms have received much less attention than either sexual isolation or hybrid sterility and inviability, it is too early to judge their relative importance in preventing gene flow between species. Moreover, no comprehensive genetic analysis of a postinsemination barrier to fertilization has been performed in any species (see summary in Coyne and Orr 1998). One would like to know, for example, whether differences at few or many loci are responsible for the reproductive incompatibilities we found (if intraspecific sexual selection involves continual coevolution between males and females drives divergence, one might expect a polygenic basis), whether cryptic isolation results more from evolutionary change in one sex than in the other, and whether cryptic isolation could result from evolutionary divergence of any of the known Drosophila seminal fluid proteins. Such information is essential for testing hypotheses about the evolutionary forces underlying cryptic reproductive divergence, and the species of the D. simulans clade are ideally suited to such a task. ACKNOWLEDGMENTS We thank M. DeAngelis, K. Dyer, L. French, J. Posluszny, and A. Travelli for help with the experiments. This work was funded by National Institute of Health Training Grant GM07097, a National Science Foundation Doctoral Dissertation Improvement Grant, and a Harper Fellowship to CSCP and by National Institute of Health grant GM58260 to JAC. LITERATURE CITED Albuquerque, G. S., C. A. Tauber, and M. J. Tauber Postmating reproductive isolation between Chrysopa quadripunctata and Chrysopa slossonae: mechanisms and geographic variation. Evolution 50: Chapman, T., J. Hutchings, and L. Partridge No reduction in the cost of mating for Drosophila melanogaster females mating with spermless males. Proc. R. Soc. Lond. B 253: Chapman, T., L. F. Liddle, J. M. Kalb, M. F. Wolfner, and L. Partridge Cost of mating in Drosophila melanogaster females is mediated by male accessory gland products. Nature 373: Chapman, T., Y. Choffat, W. E. Lucas, E. Kubli, and L. Partridge Lack of response to sex-peptide results in increased cost of mating in dunce Drosophila melanogaster females. J. Insect Physiol 42: Cobb, M., B. Burnet, and K. Connolly Sexual isolation and