The genetic basis of prezygotic reproductive isolation between Drosophila santomea and D. yakuba due to mating preference

Sexual isolating mechanisms that act before fertilization are often considered the most important genetic barriers leading to speciation in animals. While progress has been made toward understanding the genetic basis of the postzygotic isolating mechanisms of hybrid sterility and inviability, little is known about the genetic basis of prezygotic sexual isolation. Here, we map quantitative trait loci (QTL) contributing to prezygotic reproductive isolation between the sibling species Drosophila santomea and D. yakuba. We mapped at least three QTL affecting discrimination of D. santomea females against D. yakuba males: one X-linked and one autosomal QTL affected the likelihood of copulation, and a second X chromosome QTL affected copulation latency. Three autosomal QTL also affected mating success of D. yakuba males with D. santomea. No epistasis was detected between QTL affecting sexual isolation. The QTL do not overlap between males and females and are not disproportionately concentrated on the X chromosome. There was some overlap in map locations of QTL affecting sexual isolation between D. santomea and D. yakuba with QTL affecting sexual isolation between D. simulans and D. mauritiana and with QTL affecting differences in pigmentation between D. santomea and D. yakuba. Future high-resolution mapping and, ultimately, positional cloning, will reveal whether these traits do indeed have a common genetic basis.     

Mechanisms of conspecific sperm precedence in Drosophila

The postmating, prezygotic isolating mechanism known as conspecific sperm precedence (CSP) may play an important role in speciation, and understanding the mechanism of CSP is important in reconstructing its evolution. When a Drosophila simulans female mates with both a D. simulans male and a D. mauritiana male, the vast majority of her progeny are fathered by D. simulans, regardless of the order of mating. The dearth of hybrid progeny does not result from inviability of eggs fertilized by heterospecific sperm or from the relative inviability of heterospecific larvae. Instead, CSP apparently results from a prefertilization obstacle to heterospecific sperm. We identified two independent barriers to heterospecific fertilization, sperm displacement and incapacitation, whose action depends on the order of mating. When a D. simulans female mates first with a conspecific male, the seminal fluid from this mating incapacitates heterospecific sperm transferred two days later. This sperm incapacitation occurs with no change in the retention of stored sperm over time, but does not occur when the conspecific mating lasts for only 5 min. When the order of matings is reversed, the seminal fluid from the second mating physically displaces heterospecific sperm from storage, even if the conspecific copulation lasts only 5 min. Conspecific sperm are not susceptible to displacement by a second conspecific copulation, but are susceptible to interference by heterospecific sperm if the conspecific copulation is interrupted after 12 min. Curing the D. mauritiana males of their infection with the endosymbiont Wolbachia had no effect on CSP. Sperm displacement and incapacitation involve the same basic mechanisms seen in second-male sperm precedence within species, supporting the hypothesis that CSP is an evolutionary by-product of adaptations affecting sperm competition within species.     

Males,but not females,contribute to sexual isolation between two sympatric species of <Emphasis Type="Italic">Gambusia</Emphasis>

Reproductive isolation restricts genetic exchange between species. Various pre- and post-mating barriers, such as behavior, physiology and gametic incompatibility, have been shown to evolve in sympatry. In certain scenarios, isolation can be asymmetrical, where species differentially prefer conspecifics. We examined sexual isolation via conspecific mate preference between Gambusia affinis and G. geiseri in both sexes. To investigate male contribution to sexual isolation, we compared the number of mating attempts (gonopodial thrusts) directed at either a conspecific or a heterospecific female, in both species. We also examined sperm priming and expenditure in males in the presence of conspecific or heterospecific females. We then measured female preference for either a conspecific or heterospecific male, in both species. We found that males of both species preferred to mate with conspecific females, but showed no difference in sperm production or expenditure between conspecific and heterospecific females. Females of both species did not prefer conspecific over heterospecific males. Our results suggest that sexual isolation might be mediated by male mate choice in this system and not female choice, suggesting that there is asymmetrical reproductive isolation between the sexes in G. affinis and G. geiseri, but symmetrical species isolation.     

Strong cryptic prezygotic isolation despite lack of behavioral isolation between sympatric host races of the leaf beetle Lochmaea capreae

One of the major goals in speciation research is to understand which isolation mechanisms form the first barriers to gene flow. This requires examining lineages that are still in the process of divergence or incipient species. Here, we investigate the presence of behavioral and several cryptic barriers between the sympatric willow and birch host races of Lochmaea capreae. Behavioral isolation did not have any profound effect on preventing gene flow. Yet despite pairs mating indiscriminately, no offspring were produced from the heterospecific matings between birch females and willow males due to the inability of males to transfer sperm to females. We found evidence for differences in genital morphology that may contribute to failed insemination attempts during copulation. The heterospecific matings between willow females and birch males resulted in viable offspring. Yet fecundity and hatchability was remarkably reduced, which is likely the result of lower efficiency in sperm transportation and storage and lower survival of sperm in the foreign reproductive tract. Our results provide evidence for the contribution of several postmating‐prezygotic barriers that predate behavioral isolation and act as primary inhibitors of gene flow in this system. This is a surprising, yet perhaps often overlooked feature of barriers acting early in sympatric speciation process.     

Faster fertilization rate in conspecific versus heterospecific matings in house mice

Barriers to gene flow can arise at any stage in the reproductive sequence. Most studies of reproductive isolation focus on premating or postzygotic phenotypes, leaving the importance of differences in fertilization rate overlooked. Two closely related species of house mice, Mus domesticus and M. musculus , form a narrow hybrid zone in Europe, suggesting that one or more isolating factors operate in the face of ongoing gene flow. Here, we test for differences in fertilization rate using laboratory matings as well as in vitro sperm competition assays. In noncompetitive matings, we show that fertilization occurs significantly faster in conspecific versus heterospecific matings and that this difference arises after mating and before zygotes form. To further explore the mechanisms underlying this conspecific advantage, we used competitive in vitro assays to isolate gamete interactions. Surprisingly, we discovered that M. musculus sperm consistently outcompeted M. domesticus sperm regardless of which species donated ova. These results suggest that in vivo fertilization rate is mediated by interactions between sperm, the internal female environment, and/or contributions from male seminal fluid. We discuss the implications of faster conspecific fertilization in terms of reproductive isolation among these two naturally hybridizing species.     

INTRINSIC REPRODUCTIVE ISOLATION BETWEEN TWO SISTER SPECIES OF DROSOPHILA

Drosophila santomea and D. yakuba are sister species that live on the volcanic African island of São Tomé. Previous work has revealed several barriers to gene flow, including sexual isolation, hybrid sterility, and “extrinsic” ecological isolation based on differential adaptation to and preference for temperature. Here, we describe several new “intrinsic” barriers to gene flow—barriers that do not depend on the species’ ecology. These include reduced egg number, reduced egg hatchability, and faster depletion of sperm in interspecific compared to intraspecific matings. Further, hatching interval and egg‐to‐adult development time are significantly longer in interspecific than intraspecific crosses. If a female of either species is initially mated to a heterospecific male, she lays fewer and less‐fertile eggs than if she is first mated to a conspecific male, so that heterospecific matings permanently reduce female fertility. Finally, D. santomea females mated to D. yakuba males do not live as long as virgin or conspecifically mated females. The “poisoning” effects of heterospecific ejaculates may be byproducts of antagonistic sexual selection. Although these species diverged relatively recently, they are clearly separated by many isolating barriers that act both before and after mating.     

Cryptic reproductive isolation in the Drosophila simulans species complex

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.     

Divergence in Multiple Courtship Song Traits between Drosophila santomea and D. yakuba

The courtship song of Drosophila is useful for species recognition and sexual selection. A new species of the melanogaster group of Drosophila , D. santomea , has recently been described from the island of São Tomé in the Gulf of Guinea. We describe the courtship song of D. santomea and compare it with that of its sibling species D. yakuba . Both species have a relatively unusual song pattern for melanogaster-group species, in that they have two types of pulse song but no sine song. There are large differences in the inter-pulse interval of both types of song, but no major differences in pulse shape or intrapulse frequency between the species. The song of D. yakuba is similar in lines from the African mainland (allopatric to D. santomea ) and from São Tomé (sympatric). We test if song pattern might influence sexual isolation by examining the mating success of wingless males with homo- and hetero-specific females. We show that song pattern contributes to sexual stimulation, but the differences in song patterns alone are unlikely to explain patterns of sexual isolation such as the asymmetrical isolation seen between species.     

Genetic studies of two sister species in the Drosophila melanogaster subgroup, D. yakuba and D. santomea

We performed genetic analysis of hybrid sterility and of one morphological difference (sex-comb tooth number) on D. yakuba and D. santomea, the former species widespread in Africa and the latter endemic to the oceanic island of S?o Tomé, on which there is a hybrid zone. The sterility of hybrid males is due to at least three genes on the X chromosome and at least one on the Y, with the cytoplasm and large sections of the autosomes having no effect. F1 hybrid females carrying two X chromosomes from either species are perfectly fertile despite their genetic similarity to completely sterile F1 hybrid males. This implies that the appearance of Haldane's rule in this cross is at least partially due to the faster accumulation of genes causing male than female sterility. The larger effects of the X and Y chromosomes than of the autosomes, however, also suggest that the genes causing male sterility are recessive in hybrids. Some female sterility is also seen in interspecific crosses, but this does not occur between all strains. This is seen in pure-species females inseminated by heterospecific males (probably reflecting incompatibility between the sperm of one species and the female reproductive tract of the other) as well as in inseminated F1 and backcross females, probably reflecting genetically based incompatibilities in hybrids that affect the reproductive system. The latter 'innate' sterility appears to involve deleterious interactions between D. santomea chromosomes and D. yakuba cytoplasm. The difference in male sex-comb tooth number appears to involve fairly large effects of the X chromosome. We discuss the striking evolutionary parallels in the genetic basis of sterility, in the nature of sexual isolation, and in morphological differences between the D. santomea/D. yakuba divergence and two other speciation events in the D. melanogaster subgroup involving island colonization.     

The effects of heterospecific mating frequency on the strength of cryptic reproductive barriers

Heterospecific mating frequency is critical to hybrid zone dynamics and can directly impact the strength of reproductive barriers and patterns of introgression. The effectiveness of post‐mating prezygotic (PMPZ) reproductive barriers, which include reduced fecundity via heterospecific matings and conspecific sperm precedence, may depend on the number, identity and order of mates. Studies of PMPZ barriers suggest that they may be important in many systems, but whether these barriers are effective at realistic heterospecific mating frequencies has not been tested. Here, we evaluate the strength of cryptic reproductive isolation in two leaf beetles (Chrysochus auratus and C. cobaltinus) in the context of a range of heterospecific mating frequencies observed in natural populations. We found both species benefited from multiple matings, but the benefits were greater in C. cobaltinus and extended to heterospecific matings. We found that PMPZ barriers greatly limited hybrid production by C. auratus females with moderate heterospecific mating frequencies, but that their effectiveness diminished at higher heterospecific mating frequencies. In contrast, there was no evidence for PMPZ barriers in C. cobaltinus females at any heterospecific mating frequency. We show that integrating realistic estimates of cryptic isolation with information on relative abundance and heterospecific mating frequency in the field substantially improves our understanding of the strong directional bias in F1 production previously documented in the Chrysochus hybrid zone. Our results demonstrate that heterospecific mating frequency is critical to understanding the impact of cryptic post‐copulatory barriers on hybrid zone structure and dynamics, and that future studies of such barriers should incorporate field‐relevant heterospecific mating frequencies.     

The magnitude of behavioral isolation is affected by characteristics of the mating community

Gene exchange between species occurs in areas of secondary contact, where two species have the opportunity to hybridize. If heterospecific males are more common than conspecific males, females will experience more encounters with males of other species. These encounters might increase the likelihood of heterospecific matings, and lead to the production of hybrid progeny. I studied the mating behavior of two pairs of sibling species endemic to Africa: Drosophila yakuba/Drosophila santomea and Drosophila simulans/Drosophila sechellia. Drosophila yakuba and D. simulans are cosmopolitan species widely distributed in the African continent, while D. santomea and D. sechellia are island endemics. These pairs of species hybridize in nature and have the potential to exchange genes in natural conditions. I used these two pairs of Drosophila species, and constructed mating communities of different size and different heterospecific:conspecific composition. I found that both the total number of potential mates and the relative frequency of conspecific versus heterospecific males affect female mating decisions in the cosmopolitan species but not in the island endemics. These results suggest that the population characteristics, in which mating occurs, may affect the magnitude of premating isolation. Community composition might thus facilitate, or impair, gene flow between species.     

Heterospecific sperm reduction in interspecific crosses between two closely related phytophagous ladybird beetles,Henosepilachna vigintioctomaculata and H. pustulosa (Coleoptera: Coccinellidae)

In interspecific crosses, a mismatch in internal physiological conditions between two species can reduce sperm viability in the interval from insemination to fertilization, leading to gametic isolation. Two closely related Japanese phytophagous ladybird beetles, Henosepilachna vigintioctomaculata and H. pustulosa, show several isolating barriers, including reduction in the number of heterospecific sperm in the female reproductive tract and low egg‐hatching rates in interspecific matings. However, the mechanisms of these two potential isolating barriers and the association between them are unknown. Here we investigated temporal changes in the number of sperm stored in the female reproductive tract and egg‐hatching rates in inter‐ and intraspecific crosses between these species. Although the number of sperm decreased after both inter‐ and intraspecific crosses, the reduction was more drastic in inter‐ than in intraspecific crosses for females of both species. Most of the sperm reduction occurred early on, during sperm transfer from the bursa copulatrix to the paired ampullae of the common oviduct (the sperm storage organs). These two species also demonstrated stably low egg‐hatching rates in interspecific crosses. Since the degree and timing of the sperm reduction did not correlate with egg‐hatching rates, the reduction in heterospecific sperm in interspecific crosses may not directly cause the low hatching rates. These two isolating barriers could be different expressions of the physiological mismatch and/or genetic incompatibility between gametes of these species.     

Cryptic gametic interactions confer both conspecific and heterospecific advantages in the Chrysochus (Coleoptera: Chrysomelidae) hybrid zone

Most species pairs are isolated through the collective action of a suite of barriers. Recent work has shown that cryptic barriers such as conspecific sperm precedence can be quite strong, suggesting that they evolve quickly. However, because the strength of multiple barriers has been formally quantified in very few systems, the relative speed with which conspecific sperm precedence evolves remains unclear. Here, we measure the strength of both conspecific sperm precedence and cryptic non-competitive isolation between the hybridizing sister species, Chrysochus auratus and C. cobaltinus (Coleoptera: Chrysomelidae), and compare the strength of those barriers to the strength of other known reproductive barriers in this system. Overall, cryptic barriers in this system are weaker than other barriers, indicating that they have not evolved rapidly. Furthermore, their evolution has been asymmetric. Non-competitive barriers substantially reduce the production of hybrid offspring by C. auratus females but not by C. cobaltinus females. In multiply-mated C. cobaltinus females, heterospecific sperm outcompete conspecific sperm, as evidenced by the fact that heterospecific males sired disproportionately more offspring than predicted from the results for singly-mated females. In C. auratus females, neither sperm type has a competitive advantage. Such asymmetries explain why nearly all F1 hybrids in the field are from crosses between C. cobaltinus females and C. auratus males. We discuss these findings in terms of understanding the cost of mating 'mistakes' in the Chrysochus hybrid zone. In addition, our discovery that 95% confidence intervals for commonly-used isolation statistics can be very wide has important implications for speciation research. Specifically, to avoid biases in the interpretation of such isolation metrics, we suggest that studies should routinely include error estimates in their analyses of reproductive isolation.     

CONTRIBUTION OF MULTIPLE ISOLATING BARRIERS TO REPRODUCTIVE ISOLATION BETWEEN A PAIR OF PHYTOPHAGOUS LADYBIRD BEETLES

Reproductive isolation between species may often be attained by multiple isolating barriers, but the components are rarely studied in animal taxa. To elucidate the nature of multiple isolating barriers, we quantified the strength of three premating barriers, including ecologically based ones (seasonal, habitat, and sexual), two postmating–prehatching barriers (reduced egg hatchability and conspecific sperm precedence [CSP]), and one posthatching barrier, including four components of F₁ hybrid reduced fitness, between two phytophagous ladybird beetles, Henosepilachna vigintioctomaculata and H. pustulosa . We detected five positive barriers (habitat isolation, sexual isolation, reduced egg hatchability, CSP, and reduced egg hatchability in backcrosses of F₁ hybrids). None of these barriers entirely prevents gene exchange when it acts alone, but jointly they generate nearly complete reproductive isolation even between sympatric populations. Host fidelity contributed most strongly to reproductive isolation by reducing interspecific hybridization through several important types of ecological isolation, including microspatial, habitat, and seasonal isolation. The existence of multiple isolating barriers likely helps keep reproductive isolation stable and robust, by complementing changes in the strength of leaky barriers. This complementarity of multiple isolating barriers yields the concept of robustness of reproductive isolation, which is important when considering the long-term maintenance of species boundaries in coexisting species pairs.     

Remating responses are consistent with male postcopulatory manipulation but not reinforcement in D. pseudoobscura

Reinforcement occurs when hybridization between closely related lineages produces low‐fitness offspring, prompting selection for elevated reproductive isolation specifically in areas of sympatry. Both premating and postmating prezygotic behaviors have been shown to be the target of reinforcing selection, but it remains unclear whether remating behaviors experience reinforcement, although they can also influence offspring identity and limit formation of hybrids. Here, we evaluated evidence for reinforcing selection on remating behaviors in Drosophila pseudoobscura, by comparing remating traits in females from populations historically allopatric and sympatric with Drosophila persimilis. We found that the propensity to remate was not higher in sympatric females, compared to allopatric females, regardless of whether the first mated male was heterospecific or conspecific. Moreover, remating behavior did not contribute to interspecific reproductive isolation among any population; that is, females showed no higher propensity to remate following a heterospecific first mating than following a conspecific first mating. Instead, we found that females are less likely to remate after initial matings with unfamiliar males, regardless of species identity. This is consistent with one scenario of postmating sexual conflict in which females are poorly defended against postcopulatory manipulation by males with whom they have not coevolved. Our results are generally inconsistent with reinforcement on remating traits and suggest that this behavior might be more strongly shaped by the consequences of local antagonistic male–female interactions than interactions with heterospecifics.     

Asymmetric reproductive isolation between two sympatric annual killifish with extremely short lifespans

Background

Interspecific reproductive isolation is typically achieved by a combination of intrinsic and extrinsic barriers. Behavioural isolating barriers between sympatric, closely related species are often of primary importance and frequently aided by extrinsic factors causing spatial and temporal interspecific separation. Study systems with a severely limited role of extrinsic factors on reproductive isolation may provide valuable insights into how reproductive isolation between sympatric species is maintained. We used no-choice experimental set-up to study reproductive barriers between two closely related sympatric African killifish species, Nothobranchius furzeri and Nothobranchius orthonotus. These fish live in small temporary savannah pools and have complete spatial and temporal overlap in reproductive activities and share a similar ecology.

Principal Findings

We found that the two species display largely incomplete and asymmetric reproductive isolation. Mating between N. furzeri males and N. orthonotus females was absent under standard experimental conditions and eggs were not viable when fish were forced to mate in a modified experimental setup. In contrast, male N. orthonotus indiscriminately mated with N. furzeri females, the eggs were viable, and offspring successfully hatched. Most spawnings, however, were achieved by male coercion and egg production and embryo survival were low. Behavioural asymmetry was likely facilitated by mating coercion from larger males of N. orthonotus and at relatively low cost to females. Interestingly, the direction of asymmetry was positively associated with asymmetry in post-mating reproductive barriers.

Significance

We showed that, in fish species with a promiscuous mating system and multiple matings each day, selection for strong mate preferences was relaxed. This effect was likely due to the small proportion of resources allocated to each single mating and the high potential cost to females from mating refusal. We highlight and discuss the fact that males of rarer species may often coercively mate with females of a related, more abundant species.     

Reinforcement shapes clines in female mate discrimination in Drosophila subquinaria

Reinforcement of species boundaries may alter mate recognition in a way that also affects patterns of mate preference among conspecific populations. In the fly Drosophila subquinaria, females sympatric with the closely related species D. recens reject mating with heterospecific males as well as with conspecific males from allopatric populations. Here, we assess geographic variation in behavioral isolation within and among populations of D. subquinaria and use cline theory to understand patterns of selection on reinforced discrimination and its consequences for sexual isolation within species. We find that selection has fixed rejection of D. recens males in sympatry, while significant genetic variation in this behavior occurs within allopatric populations. In conspecific matings sexual isolation is also asymmetric and stronger in populations that are sympatric with D. recens. The clines in behavioral discrimination within and between species are similar in shape and are maintained by strong selection in the face of gene flow, and we show that some of their genetic basis may be either shared or linked. Thus, while reinforcement can drive extremely strong phenotypic divergence, the long‐term consequences for incipient speciation depend on gene flow, genetic linkage of discrimination traits, and the cost of these behaviors in allopatry.     

Conspecific sperm precedence in Callosobruchus subinnotatus (Coleoptera: Bruchidae): mechanisms and consequences

Conspecific sperm precedence (CSP) has been identified as an important post-copulatory, pre-zygotic mechanism that can act to reduce gene flow between populations. The evolution of CSP is thought to have arisen as a by-product of male and female coevolution in response to intraspecific post-copulatory sexual selection. However, little is known about the mechanisms that generate CSP. When Callosobruchus subinnotatus females copulate with both C. subinnotatus and Callosobruchus maculatus males, regardless of mating order, the majority of eggs are fertilized by conspecific sperm. The low number of heterospecific fertilizations does not result from general differences in the viability of sperm in the female reproductive tract, as heterospecific sperm fertilized equivalent numbers of eggs as conspecific sperm in the absence of sperm competition. Instead, CSP results from disadvantages to heterospecific sperm that are manifest only when in competition with conspecific sperm. CSP in C. subinnotatus appears to result from two, not mutually exclusive, mechanisms. First, conspecific sperm are better able to displace heterospecific sperm from female storage. Second, conspecific sperm achieve disproportionately higher numbers of fertilizations relative to their proportional representation in the fertilization set. Thus, we provide evidence of differential sperm use from the female spermatheca.     

Asymmetrical reinforcement and Wolbachia infection in Drosophila

Reinforcement refers to the evolution of increased mating discrimination against heterospecific individuals in zones of geographic overlap and can be considered a final stage in the speciation process. One the factors that may affect reinforcement is the degree to which hybrid matings result in the permanent loss of genes from a species' gene pool. Matings between females of Drosophila subquinaria and males of D. recens result in high levels of offspring mortality, due to interspecific cytoplasmic incompatibility caused by Wolbachia infection of D. recens. Such hybrid inviability is not manifested in matings between D. recens females and D. subquinaria males. Here we ask whether the asymmetrical hybrid inviability is associated with a corresponding asymmetry in the level of reinforcement. The geographic ranges of D. recens and D. subquinaria were found to overlap across a broad belt of boreal forest in central Canada. Females of D. subquinaria from the zone of sympatry exhibit much stronger levels of discrimination against males of D. recens than do females from allopatric populations. In contrast, such reproductive character displacement is not evident in D. recens, consistent with the expected effects of unidirectional cytoplasmic incompatibility. Furthermore, there is substantial behavioral isolation within D. subquinaria, because females from populations sympatric with D. recens discriminate against allopatric conspecific males, whereas females from populations allopatric with D. recens show no discrimination against any conspecific males. Patterns of general genetic differentiation among populations are not consistent with patterns of behavioral discrimination, which suggests that the behavioral isolation within D. subquinaria results from selection against mating with Wolbachia-infected D. recens. Interspecific cytoplasmic incompatibility may contribute not only to post-mating isolation, an effect already widely recognized, but also to reinforcement, particularly in the uninfected species. The resulting reproductive character displacement not only increases behavioral isolation from the Wolbachia-infected species, but may also lead to behavioral isolation between populations of the uninfected species. Given the widespread occurrence of Wolbachia among insects, it thus appears that there are multiple ways by which these endosymbionts may directly and indirectly contribute to reproductive isolation and speciation.     

Genetic detection of sperm from forced copulations between sympatric populations of Poecilia reticulata and Poecilia picta

The reproductive success of the sneaky mating tactic is difficult to determine in fish with internal fertilization. We approached this problem by developing an assay that allows the recovery of sperm DNA from the reproductive tract of females. This assay was used to test whether sperm transfer occurs between sympatric populations of the guppy, Poecilia reticulata , and its putative sister species, Poecilia picta . Six hundred females of both species from two sympatric sites in Trinidad were collected, and then the contents of the females' reproductive tracts were analysed using reciprocally diagnostic microsatellite markers. Conspecific sperm occurred in approximately 86% of females, whereas heterospecific sperm were found in only 4% of females. Because females of either species do not mate willingly with heterospecific males, the results indicate that sneaky mating results in the transfer of sperm. The data are consistent with the idea that sexual conflict can result in differences in the strength of behavioural isolation between the sexes, and they suggest that behavioural isolation is unlikely to have driven speciation between the guppy and P. picta .  © 2006 The Linnean Society of London, Biological Journal of the Linnean Society , 2006, 88 , 397–402.