Multiple barriers to gene exchange in a field cricket hybrid zone

Data on patterns of variation within hybrid zones, combined with studies of life history, mate choice, and hybrid performance, allow estimates of the contribution of different pre-zygotic and post-zygotic barriers to reproductive isolation. We examine the role of behavioural barriers to gene exchange in the maintenance of a hybrid zone between North American field crickets Gryllus firmus and Gryllus pennsylvanicus . We consider these barriers in the context of previous studies that documented temporal and ecological isolation and a one-way post-mating incompatibility (i.e. G. firmus females do not produce offspring when they mate only with heterospecific males). Based on no-choice mating experiments in the laboratory, we demonstrate strong behavioural pre-mating barriers between the two species, but no apparent fecundity or fertility costs for G. firmus females when they mate with both conspecific and heterospecific males. Furthermore, we show that G. firmus females do not discriminate between hybrids and conspecifics, whereas G. pennsylvanicus females do. This observation could explain the asymmetric allele introgression observed in the hybrid zone. We also document a failure of heterospecific males to induce normal oviposition in G. firmus females, which may be due to rapid evolution of accessory gland proteins and may serve as an additional barrier to gene exchange.  © 2009 The Linnean Society of London, Biological Journal of the Linnean Society , 2009, 97 , 390–402.     

Do Wolbachia infections play a role in unidirectional incompatibilities in a field cricket hybrid zone?

Two closely related field crickets, Gryllus firmus and G. pennsylvanicus, hybridize along an extensive north-south zone in the eastern United States. Crosses between G. firmus males and G. pennsylvanicus females produce viable and fertile F1, but the reciprocal cross consistently fails to produce offspring. Wolbachia, a bacterial parasite of arthropods that causes unidirectional incompatibilities in a variety of insect species, has been suggested as the cause of the observed incompatibility between G. pennsylvanicus and G. firmus. We examine the presence/absence of Wolbachia strains, defined by sequencing the ftsZ gene, in four cricket populations from the north-eastern United States. Most G. firmus individuals are infected (100% in Guilford, Connecticut; 65% in Seaside Park, New Jersey) and > 95% of those infected harbour a single strain of Wolbachia. All individuals in G. pennsylvanicus populations (Ithaca, New York; Sharon, Connecticut) are infected; the majority of individuals carry a second strain of Wolbachia, but a significant fraction carry the same strain found commonly in G. firmus. The presence of an apparently identical Wolbachia strain in crickets of both species means that some crosses between G. pennsylvanicus males and G. firmus females should be compatible. We have no evidence of such compatibility. Furthermore, if Wolbachia infections are responsible for the observed incompatibility between species, then incompatibilities must also exist within G. pennsylvanicus, because this species harbours both Wolbachia strains. Although some single pair crosses within G. pennsylvanicus do fail to produce offspring, the proportion is lower than expected if Wolbachia were responsible. Therefore, Wolbachia is unlikely to be involved in reproductive isolation between the two cricket species.     

Wolbachia plays no role in the one-way reproductive incompatibility between the hybridizing field crickets Gryllus firmus and G. pennsylvanicus

Wolbachia are cytoplasmically inherited alpha-proteobacteria that can cause cytoplasmic incompatibility (CI) in insects. This incompatibility between sperm and egg is evident when uninfected females mate with infected males. Wolbachia-driven reproductive incompatibilities are of special interest because they may play a role in speciation. However, the presence of Wolbachia does not always imply incompatibility. The field crickets Gryllus firmus and G. pennsylvanicus exhibit a very clear unidirectional incompatibility and have been cited as a possible example of Wolbachia-induced CI. Here, we conduct curing experiments, intra- and interspecific crosses, cytological examination of Wolbachia in testes and Wolbachia quantifications through real-time PCR. All of our data strongly suggest that Wolbachia are not involved in the reproductive incompatibility between G. firmus and G. pennsylvanicus.     

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.     

Searching for candidate speciation genes using a proteomic approach: seminal proteins in field crickets

In many animals, male seminal proteins influence gamete interactions and fertilization ability and are probably involved in barriers to gene flow between diverging lineages. Here we use a proteomic approach to identify seminal proteins that are transferred to females during copulation and that may be involved in fertilization barriers between two hybridizing field crickets (Gryllus firmus and Gryllus pennsylvanicus). Analyses of patterns of divergence suggest that much of the field cricket genome has remained undifferentiated following the evolution of reproductive isolation. By contrast, seminal protein genes are highly differentiated. Tests of selection reveal that positive selection is likely to be responsible for patterns of differentiation. Together, our observations suggest that some of the loci encoding seminal proteins may indeed play a role in fertilization barriers in field crickets.     

Post-mating reproductive barriers in two unidirectionally hybridizing sunfish (Centrarchidae: Lepomis)

The evolutionary sequence of events in the evolution of reproductive barriers between species is at the core of speciation biology. Where premating barriers fail, post-mating barriers, such as conspecific sperm precedence (CSP), gamete incompatibility (GI) and hybrid inviability (HI) may evolve to prevent the production of (often) costly hybrid offspring with reduced fitness. We tested the role of post-mating mechanisms for the reproductive isolation between two sunfish species [bluegill (BG) Lepomis macrochirus and pumpkinseed (PS) Lepomis gibbosus] and their first-generation hybrids. Performing in vitro sperm competition experiments, we observed asymmetric CSP as main post-mating isolation mechanism when BG and PS sperm were competing for PS eggs, whereas when sperm from both species were competing for BG eggs it was HI. Furthermore, hybrid sperm--although fertile in the absence of competition--were outcompeted by sperm of either parental species. This result may at least partly explain previous observations that natural hybridization in the study system is unidirectional.     

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.     

Measuring polyandry in wild populations: a case study using promiscuous crickets

Mating rate has important implications for patterns of sexual selection and sexual conflict and hence for issues such as speciation and the maintenance of genetic diversity. Knowledge of natural mating rates can provide insights into the factors driving female mating behaviour. We investigated the level of polyandry in a Spanish population of the field cricket Gryllus bimaculatus using microsatellite markers. Two approaches were employed: (i) genotyping the offspring of wild-caught gravid females to determine the number of males siring the brood and (ii) genotyping sperm stored in the spermathecae of females mated in the wild to estimate the number of mating partners. We compared existing methods for inferring the minimum and probable number of fathers and described a novel probabilistic technique estimating the number of mates by genotyping stored sperm. Using the most conservative allele-counting method, 71% of females produced offspring sired by at least two males (a minimum mean of 2.4 fathers per clutch), and all females had mated to at least two males with minimum mean estimates of 2.7-5.1 mates per female. Our study reveals high levels of polyandry in the wild and suggests that females mate with more males than sire their offspring.     

Possible glimpses into early speciation: the effect of ovarian fluid on sperm velocity accords with post‐copulatory isolation between two guppy populations

Identifying mechanisms of reproductive isolation is key to understanding speciation. Among the putative mechanisms underlying reproductive isolation, sperm–female interactions (post‐mating–prezygotic barriers) are arguably the hardest to identify, not least because these are likely to operate at the cellular or molecular level. Yet sperm–female interactions offer great potential to prevent the transfer of genetic information between different populations at the initial stages of speciation. Here, we provide a preliminary test for the presence of a putative post‐mating–prezygotic barrier operating between three populations of Trinidadian guppies (Poecilia reticulata), an internally fertilizing fish that inhabits streams with different levels of connectivity across Trinidad. We experimentally evaluate the effect of female ovarian fluid on sperm velocity (a predictor of competitive fertilization success) according to whether males and females were from the same (native) or different (foreign) populations. Our results reveal the potential for ovarian fluid to act as a post‐mating–prezygotic barrier between two populations from different drainages, but also that the strength of this barrier is different among populations. This result may explain the previous finding that, in some populations, sperm from native males have precedence over foreign sperm, which could eventually lead to reproductive isolation between these populations.     

Premating reproductive barriers between hybridising cricket species differing in their degree of polyandry

Understanding speciation hinges on understanding how reproductive barriers arise between incompletely isolated populations. Despite their crucial role in speciation, prezygotic barriers are relatively poorly understood and hard to predict. We use two closely related cricket species, Gryllus bimaculatus and G. campestris, to experimentally investigate premating barriers during three sequential mate choice steps. Furthermore, we experimentally show a significant difference in polyandry levels between the two species and subsequently test the hypothesis that females of the more polyandrous species, G. bimaculatus, will be less discriminating against heterospecific males and hence hybridise more readily. During close-range mating behaviour experiments, males showed relatively weak species discrimination but females discriminated very strongly. In line with our predictions, this discrimination is asymmetric, with the more polyandrous G. bimaculatus mating heterospecifically and G. campestris females never mating heterospecifically. Our study shows clear differences in the strength of reproductive isolation during the mate choice process depending on sex and species, which may have important consequences for the evolution of reproductive barriers.     

RAPID DIVERSIFICATION OF SPERM PRECEDENCE TRAITS AND PROCESSES AMONG THREE SIBLING DROSOPHILA SPECIES

Postcopulatory sexual selection is credited with driving rapid evolutionary diversification of reproductive traits and the formation of reproductive isolating barriers between species. This judgment, however, has largely been inferred rather than demonstrated due to general lack of knowledge about processes and traits underlying variation in competitive fertilization success. Here, we resolved processes determining sperm fate in twice‐mated females, using transgenic Drosophila simulans and Drosophila mauritiana populations with fluorescently labeled sperm heads. Comparisons among these two species and Drosophila melanogaster revealed a shared motif in the mechanisms of sperm precedence, with postcopulatory sexual selection potentially occurring during any of the three discrete stages: (1) insemination; (2) sperm storage; and (3) sperm use for fertilization, and involving four distinct phenomena: (1) sperm transfer; (2) sperm displacement; (3) sperm ejection; and (4) sperm selection for fertilizations. Yet, underlying the qualitative similarities were significant quantitative differences in nearly every relevant character and process. We evaluate these species differences in light of concurrent investigations of within‐population variation in competitive fertilization success and postmating/prezygotic reproductive isolation in hybrid matings between species to forge an understanding of the relationship between microevolutionary processes and macroevolutionary patterns as pertains to postcopulatory sexual selection in this group.     

Sperm performance in conspecific and heterospecific female fluid

Divergent sexual selection within allopatric populations may result in divergent sexual phenotypes, which can act as reproductive barriers between populations upon secondary contact. This hypothesis has been most tested on traits involved in precopulatory sexual selection, with less work focusing on traits that act after copulation and before fertilization (i.e., postcopulatory prezygotic traits), particularly in internally fertilizing vertebrates. However, postcopulatory sexual selection within species can also drive trait divergence, resulting in reduced performance of heterospecific sperm within the female reproductive tract. Such incompatibilities, arising as a by‐product of divergent postcopulatory sexual selection in allopatry, can represent reproductive barriers, analogous to species‐assortative mating preferences. Here, we tested for postcopulatory prezygotic reproductive barriers between three pairs of taxa with diverged sperm phenotypes and moderate‐to‐high opportunity for postcopulatory sexual selection (barn swallows Hirundo rustica versus sand martins Riparia riparia, two subspecies of bluethroats, Luscinia svecica svecica versus L. s. namnetum, and great tits Parus major versus blue tits Cyanistes caeruleus). We tested sperm swimming performance in fluid from the outer reproductive tract of females, because the greatest reduction in sperm number in birds occurs as sperm swim across the vagina. Contrary to our expectations, sperm swam equally well in fluid from conspecific and heterospecific females, suggesting that postcopulatory prezygotic barriers do not act between these taxon pairs, at this stage between copulation and fertilization. We therefore suggest that divergence in sperm phenotypes in allopatry is insufficient to cause widespread postcopulatory prezygotic barriers in the form of impaired sperm swimming performance in passerine birds.     

Egg-hatching benefits gained by polyandrous female locusts are not due to the fertilization advantage of nonsibling males

Several studies suggest that polyandrous females bias paternity in favor of unrelated males to avoid inbreeding depression. Here we tested whether the migratory locust biases sperm usage toward unrelated males by analyzing the paternity of offspring from females mated with either two siblings, or two nonsiblings, or a sibling and a nonsibling in either order. We found that the eggs of females mated only with siblings had decreased hatching success. When females mated with both a nonsibling and a sibling, egg hatchability was significantly increased. Subsequent paternity analyses found no evidence that females could avoid fertilization by sibling males. Therefore, improvement of the hatchability of eggs sired by siblings suggests that rather than biased fertilization by females toward genetically compatible or superior males, male-induced maternal effects or direct effects of male ejaculates might influence the survival of offspring sired by related males.     

BENEFITS OF MULTIPLE MATES IN THE CRICKET GRYLLUS BIMACULATUS

Despite the importance of polyandry for sexual selection, the reasons why females frequently mate with several males remain poorly understood. A number of genetic benefits have been proposed, based on the idea that by taking multiple mates, females increase the likelihood that their offspring will be sired by genetically more compatible or superior males. If certain males have intrinsically “good genes,” any female mating with them will produce superior offspring. Alternatively, if some males have genetic elements that are incompatible with a particular female, then she may benefit from polyandry if the sperm of such males are less likely to fertilize her eggs. We examined these hypotheses in the field cricket Gryllus bimaculatus (Orthoptera: Gryllidae). By allocating females identical numbers of matings but different numbers of mates we investigated the influence of number of mates on female fecundity, and both short- and long-term offspring fitness. This revealed no effect of number of mates on number of eggs laid. However, hatching success of eggs increased with number of mates. This effect could not be attributed to nongenetic effects such as the possibility that polyandry reduces variance in the quantity or fertilizing ability of sperm females receive, because a control group receiving half the number of copulations showed no drop in hatching success. Offspring did not differ in survival, adult mass, size, or development time with treatment. When males were mated to several different females there were no repeatable differences between individual males in the hatching success of their mate's eggs. This suggests that improved hatching success of polyandrous females is not due to certain males having genes that improve egg viability regardless of their mate. Instead, our results support the hypothesis that certain males are genetically more compatible with certain females, and that this drives polyandry through differential fertilization success of sperm from more compatible males.     

Male sperm donation consequences in single and double matings in Anisopteromalus calandrae

Abstract. The number of spermatozoa that a male transfers to the female during copulation is a main component of its individual fitness, especially under the pressure of sperm competition. This paper presents experimental results on the direct relationship between the male's sperm investment and its paternity in the offspring of dual-mated females. An eye colour mutant (red-eyed) is used to study the differences in the mating and fertilization abilities of males through observation of single and dual matings of females in Anisopteromalus calandrae (Hymenoptera, Chalcidoidea, Pteromalidae). Experimentally, females accept dual matings only in the simultaneous presence of two males. Counts of spermatozoa in the seminal vesicles of virgin males show that red-eyed males have more sperm than wild-eyed ones (approximately 1.46-fold greater). Red- and wild-eyed males do not differ in their mating behaviour and females mate indifferently with both phenotypes. Compared with once-mated females, double-mated females increase neither sperm storage nor lifetime fecundity, and the offspring sex ratio is female-biased. Females mated with two males of different phenotypes produce offspring of both phenotypes throughout their reproductive life, whatever the order of males in the copulation sequence. Any mating pattern appears to produce more red- than wild-eyed offspring (between 1.45- and 1.88-fold greater). Thus, proportions of offspring of each male match the proportions of their sperm potential. With no preference of female for red-eye or wild-eye males being demonstrated at either behavioural or physiological levels, a male's investment in sperm quantity appears to determine its individual reproductive success, at least in these experimental conditions.     

Influence of the Male Ejaculate on Post-Mating Prezygotic Barriers in Field Crickets

Post-copulatory interactions between males and females involve highly coordinated, complex traits that are often rapidly evolving and divergent between species. Failure to produce and deposit eggs may be a common post-mating prezygotic barrier, yet little is known about what prevents the induction of egg-laying between species. The field crickets, Gryllus firmus and G. pennsylvanicus are isolated by a one-way reproductive incompatibility; G. pennsylvanicus males fail to fertilize G. firmus eggs or to induce normal egg-laying in G. firmus females. We use experimental crosses to elucidate the role of accessory gland-derived vs. testis-derived components of the G. firmus male ejaculate on egg-laying in conspecific and heterospecific crosses. Using surgical castrations to create ‘spermless’ males that transfer only seminal fluid proteins (SFPs) we test whether G. firmus male SFPs can induce egg-laying in conspecific crosses and rescue egg-laying in crosses between G. pennsylvanicus males and G. firmus females. We find G. firmus SFPs induce only a small short-term egg-laying response and that SFPs alone cannot explain the normal induction of egg-laying. Gryllus firmus SFPs also do not rescue the heterospecific cross. Testis-derived components, such as sperm or prostaglandins, most likely stimulate egg-laying or act as transporters for SFPs to targets in the female reproductive tract. These results highlight the utility of experimental approaches for investigating the phenotypes that act as barriers between species and suggest that future work on the molecular basis of the one-way incompatibility between G. firmus and G. pennsylvanicus should focus on divergent testis-derived compounds or proteins in addition to SFPs.     

Sperm length influences fertilization success during sperm competition in the snail Viviparus ater

Sperm form and size is tremendously variable within and across species. However, a general explanation for this variation is lacking. It has been suggested that sperm size may influence sperm competition, and there is evidence for this in some taxa but not others. In addition to normal fertilizing sperm, a number of molluscs and insects produce nonfertile sperm that are also extremely morphologically variable, and distinct from fertilizing forms. There is evidence that nonfertile sperm play an indirect role in sperm competition by decreasing female remating propensity in Lepidopterans, but in most taxa the function of parasperm is unknown. We investigated the role of nonfertile (oligopyrene) sperm during sperm competition in the fresh water snail Viviparus ater. Previous studies found that the proportion of oligopyrene sperm increased with the risk of sperm competition, and hence it seems likely that these sperm influence fertilization success during competitive matings. In mating experiments in which females were sequentially housed with males, we examined a range of male characteristics which potentially influence fertilization success. We found that the size of oligopyrene sperm was the best predictor of fertilization success, with males having the longer sperm siring the highest proportion of offspring. Furthermore, we found a positive shell size and sperm concentration effect on paternity, and females with multiply sired families produced more offspring than females mating with only one male. This result suggests polyandry is beneficial for female snails.     

Gamete compatibility and sperm competition affect paternity and hybridization between sympatric Asterias sea stars

Gamete interactions may strongly influence speciation and hybridization in sympatric broadcast-spawning marine invertebrates. We examined the role of gamete compatibility in species integrity using cross-fertilization studies between sympatric Asterias sea stars from a secondary contact zone in the northwest Atlantic. In crosses between single males and single females, gametes of both species were compatible and produced viable, fertile hybrid offspring, but with considerable variation in the receptivity of eggs to heterospecific sperm. Differential compatibility of heterospecific gametes was detected in sperm competition studies in which we used a nuclear DNA marker to assign paternity to larval offspring. Several families showed conspecific sperm precedence in A. forbesi eggs, and one family showed competitive superiority of A. forbesi sperm fertilizing A. rubens eggs. Gametic interactions are an important component of prezygotic reproductive isolation in sympatric Asterias. The interaction between gametes of these closely related sea stars is consistent with the function of gamete recognition systems that are known to mediate fertilization success and speciation in other marine invertebrates.     

Female life span and fertility are increased by the ejaculates of preferred males

In animals with internal fertilization, sperm competition among males can favor the evolution of male ejaculate traits that are detrimental to females. Female mating preferences, in contrast, often favor traits in males that are beneficial to females, yet little is known about the effect of these preferences on the evolution of male ejaculates. A necessary condition for female preferences to affect the evolution of male ejaculate characteristics is that females select mates based on a trait correlated with ejaculate quality. Previous work has shown that females of the variable field cricket, Gryllus lineaticeps, prefer males that produce calling songs containing faster and longer chirps. In this study, we tested the hypothesis that females receive more beneficial ejaculates from preferred males. Females were placed on either a high- or a reduced-nutrition diet then mated twice to a male of known song phenotype. Females received only sperm and seminal fluid from males during these matings. There was no effect of male song phenotype on any fitness component for females on the high-nutrition diet. Reduced-nutrition females mated to males that produced preferred song types, however, lived longer, produced more eggs, produced more fertile eggs, and had a higher proportion of their eggs fertilized than those mated to other males. The life-span benefit was positively associated with male chirp duration, and the reproductive benefits were positively associated with male chirp rate. We explored two possible mechanisms for the life span and reproductive benefits. First, a path analysis suggested that part of the effect of male chirp duration on female life span may have been indirect; females mated to males that produced longer chirps showed delayed oviposition, and females that delayed oviposition lived longer. Males that produce longer chirps may thus transfer fewer or less potent oviposition stimulants to females in their seminal fluid. Second, there was a positive correlation between male chirp rate and the number of sperm transferred to females. The fertility benefit may thus have resulted from females receiving more sperm from males that produce faster chirps. Finally, there was a negative phenotypic correlation between male chirp rate and chirp duration, suggesting that females may have to trade off the life span and reproduction benefits when selecting a mate.     

Male house mice evolving with post-copulatory sexual selection sire embryos with increased viability

Although mating is costly, multiple mating by females is a taxonomically widespread phenomenon. Theory has suggested that polyandry may allow females to gain genetic benefits for their offspring, and thus offset the costs associated with this mating strategy. For example, the good sperm hypothesis posits that females benefit from mating multiply when genetically superior males have increased success in sperm competition and produce high quality offspring. We applied the powerful approach of experimental evolution to explore the potential for polyandry to drive evolutionary increases in female fitness in house mice, Mus domesticus. We maintained polygamously mated and monogamously mated selection lines of house mice for 14 generations, before determining whether selection history could account for divergence in embryo viability. We found that males from lineages evolving with post-copulatory sexual selection sire offspring with increased viability, suggesting that polyandry results in the production of higher quality offspring and thus provides long-term fitness benefits to females.