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.     

The accumulation of reproductive barriers during speciation: postmating barriers in two behaviorally isolated species of darters (Percidae: Etheostoma)

Identifying the manner in which reproductive barriers accumulate during lineage divergence is central to establishing general principles of species formation. One outstanding question is which isolating mechanisms form the first complete barrier to gene flow in a given lineage or under a particular set of conditions. To identify these initial reproductive barriers requires examining lineages in very early stages of divergence, before multiple reproductive barriers have evolved to completion. We quantified the strength of three postmating barriers in a pair of darter species and compared these estimates to each other and to the strength of behavioral isolation (BI) reported in a previous study. Results reveal no evidence of gametic incompatibility but intermediate levels of conspecific sperm precedence and hybrid inviability. As BI is nearly complete, our analysis comparing the strength of multiple reproductive barriers implicates the evolution of mate choice as central to both the origin and maintenance of these species. Further examination of ecological isolation and hybrid sterility is necessary to determine the role of these barriers in darter speciation.     

The evolution of conspecific sperm precedence in Drosophila

Conspecific sperm precedence takes place when females inseminated by both conspecific and heterospecific sperm preferentially produce conspecific rather than hybrid offspring. Although many studies have documented conspecific sperm precedence, most have only identified it between taxa that are already considered to be good species. Here, we test for sperm precedence between two Drosophila pseudoobscura subspecies and between two Drosophila melanogaster races to evaluate how early in evolutionary divergence sperm precedence evolves. We found evidence of weak conspecific sperm precedence between the Drosophila subspecies but none between the Drosophila races. These pairs of taxa are already separated by mating discrimination and/or hybrid sterility, so our observation suggests that conspecific sperm precedence does not always evolve before other barriers to gene exchange.     

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.     

CONSPECIFIC SPERM PRECEDENCE IS AN EFFECTIVE BARRIER TO HYBRIDIZATION BETWEEN CLOSELY RELATED SPECIES

Conspecific sperm precedence is widespread in animals, appears to evolve rapidly, and is thought to have the potential to prevent hybridization between closely related species. However, to date no study has tested the isolating potential of such a barrier in mixed populations of two taxa under conditions in which other potential barriers to gene flow are controlled for or are prevented from operating. We tested the isolating potential of conspecific sperm precedence in the ground crickets Allonemobius fasciatus and A. socius in population cage experiments in which the frequency of the two species was varied. Despite the observation of abundant interspecific matings, the proportions of hybrid progeny were low and differed statistically from the proportions expected in the absence of conspecific sperm precedence. The results demonstrate that conspecific sperm precedence can severely limit gene flow between closely related species, even when one species is less abundant than the other.     

RAPID EVOLUTION OF ASYMMETRIC REPRODUCTIVE INCOMPATIBILITIES IN STALK‐EYED FLIES

The steps by which isolated populations acquire reproductive incompatibilities remain poorly understood. One potentially important process is postcopulatory sexual selection because it can generate divergence between populations in traits that influence fertilization success after copulation. Here we present a comprehensive analysis of this form of reproductive isolation by conducting reciprocal crosses between variably diverged populations of stalk‐eyed flies (Teleopsis dalmanni). First, we measure seven types of reproductive incompatibility between copulation and fertilization. We then compare fertilization success to hatching success to quantify hybrid inviability. Finally, we determine if sperm competition acts to reinforce or counteract any incompatibilities. We find evidence for multiple incompatibilities in most crosses, including failure to store sperm after mating, failure of sperm to reach the site of fertilization, failure of sperm to fertilize eggs, and failure of embryos to develop. Local sperm have precedence over foreign sperm, but this effect is due mainly to differences in sperm transfer and reduced hatching success. Crosses between recently diverged populations are asymmetrical with regard to the degree and type of incompatibility. Because sexual conflict in these flies is low, postcopulatory sexual selection, rather than antagonistic coevolution, likely causes incompatibilities due to mismatches between male and female reproductive traits.     

The importance of pre-mating barriers and the local demographic context for contemporary mating patterns in hybrid zones of Eucalyptus aggregata and Eucalyptus rubida

The frequency of hybridization in plants is context dependent and can be influenced by the local mating environment. We used progeny arrays and admixture and pollen dispersal analyses to assess the relative importance of pre‐mating reproductive barriers and the local demographic environment as explanations of variation in hybrid frequency in three mapped hybrid zones of Eucalyptus aggregata and E. rubida. A total of 731 open‐pollinated progeny from 36 E. aggregata maternal parents were genotyped using six microsatellite markers. Admixture analysis identified substantial variation in hybrid frequency among progeny arrays (0–76.9%). In one hybrid zone, hybrid frequency was related to pre‐mating barriers (degree of flowering synchrony) and demographic components of the local mating environment (decreasing population size, closer proximity to E. rubida and hybrid trees). At this site, average pollen dispersal distance was less and almost half (46%) of the hybrid progeny were sired by local E. rubida and hybrid trees. In contrast, at the other two sites, pre‐mating and demographic factors were not related to hybrid frequency. Compared to the first hybrid zone where most of the E. rubida (76%) and all hybrids flowered, in the remaining sites fewer E. rubida (22–41%) and hybrid trees (0–50%) flowered and their reproductive success was lower (sired 0–23% of hybrids). As a result, most hybrids were sired by external E. rubida/hybrids located at least 2–3 km away. These results indicate that although pre‐mating barriers and local demography can influence patterns of hybridization, their importance can depend upon the scale of pollen dispersal.     

The evolution of alternative cryptic female choice strategies in age-structured populations

Abstract.— Cryptic female choice is a potentially important aspect of the sexual selection process. According to the theory of sexual dialectics, postcopulation manipulation of relative male fertilization success can provide an avenue by which females can circumvent attempts by males to control female reproduction. Here I use stochastic models to investigate the evolution of cryptic female choice in populations with and without age structure. In populations without age structure, cryptic female choice will evolve only when (1) precopulatory mate choice by females is inefficient, (2) variation in male fitness is correlated with a trait upon which a female can base her choice of mates, and (3) the cost of multiple mating is not too high. In populations with age structure, similar conditions apply. However, selection sometimes favors females that employ alternative strategies of female choice at different ages. These results help to define the types of biological systems in which we should expect to see the evolution of cryptic female choice. They also illustrate that the evolution of choice strategies in females may be complex and may mirror in some important respects the evolution of alternative mating tactics in males.     

Conspecific sperm precedence in sister species of Drosophila with overlapping ranges

Abstract Barriers to gene flow that act after mating but before fertilization are often overlooked in studies of reproductive isolation. Where species are sympatric, such "cryptic' isolating barriers may be important in maintaining species as distinct entities. Drosophilayakuba and its sister species D. santomea have overlapping ranges on the island of Sao Tome, off the coast of West Africa. Previous studies have shown that the two species are strongly sexually isolated. However, the degree of sexual isolation observed in the laboratory cannot explain the low frequency (–1%) of hybrids observed in nature. This study identifies two "cryptic" isolating barriers that may further reduce gene flow between D. yakuba andD. santomea where they are sympatric. First, noncompetitive gametic isolation has evolved between D. yakuba and D. santomea: heterospecific matings between the two species produce significantly fewer offspring than do conspecific matings. Second, conspecific sperm precedence (CSP) occurs when D. yakuba females mate with conspecific and heterospecific males. However, CSP is asymmetrical: D. santomea females do not show patterns of sperm usage consistent with CSP. Drosophila yakuba and D. santomea females also differ with respect to remating propensity after first mating with conspecific males. These results suggest that noncompetitive and competitive gametic isolating barriers may contribute to reproductive isolation between D. yakuba and D. santomea.     

Multiple post‐mating barriers to hybridization in field crickets

Mechanisms that prevent different species from interbreeding are fundamental to the maintenance of biodiversity. Barriers to interspecific matings, such as failure to recognize a potential mate, are often relatively easy to identify. Those occurring after mating, such as differences in the how successful sperm are in competition for fertilisations, are cryptic and have the potential to create selection on females to mate multiply as a defence against maladaptive hybridization. Cryptic advantages to conspecific sperm may be very widespread and have been identified based on the observations of higher paternity of conspecifics in several species. However, a relationship between the fate of sperm from two species within the female and paternity has never been demonstrated. We use competitive microsatellite PCR to show that in two hybridising cricket species, Gryllus bimaculatus and G. campestris, sequential cryptic reproductive barriers are present. In competition with heterospecifics, more sperm from conspecific males is stored by females. Additionally, sperm from conspecific males has a higher fertilisation probability. This reveals that conspecific sperm precedence can occur through processes fundamentally under the control of females, providing avenues for females to evolve multiple mating as a defence against hybridization, with the counterintuitive outcome that promiscuity reinforces isolation and may promote speciation.     

The relationship between conspecific fertilization success and reproductive isolation among three congeneric sea urchins

Few data are available on the effectiveness of reproductive isolating mechanisms in externally fertilizing taxa. I investigated patterns of conspecific and heterospecific fertilization among three coexisting sea urchin species, Strongylocentrotus droebachiensis, S.franciscanus, and S. purpuratus. In the laboratory, both among and within species, eggs from individual females whose eggs are more easily fertilized by conspecific sperm are also most susceptible to heterospecific fertilization. At one extreme, S. droebachiensis requires an order of magnitude fewer conspecific sperm to fertilize eggs than do the other two species and shows very little distinction between conspecific and heterospecific sperm in no choice experiments. Strongylocentrotus franciscanus has an intermediate susceptibility to fertilization by heterospecific sperm. At the other extreme, S. purpuratus rarely cross-fertilizes. Field observations indicate that S. droebachiensis is often surrounded by heterospecific sea urchins. Genetic analysis of larvae produced during heterospecific spawning events indicate that hybrids are generally produced if male conspecifics are more than 1 m from a spawning female S. droebachiensis. Laboratory cultures indicate that these hybrids suffer high mortality relative to conspecific larvae. Comparisons of reproductive success of S. droebachiensis during single-species and multispecies spawning events indicate that the benefits of producing easily fertilized eggs under conditions of sperm limitation may outweigh the costs of losing some offspring to hybrid fertilization. Patterns of variability in heterospecific fertilization are considered in light of three hypotheses: phylogenetic relatedness, reinforcement selection, and sexual selection.     

Sexual isolation between two newt species, Triturus vulgaris and T. montandoni (Amphibia, Urodela, Salamandridae)

In the present study we investigated sexual isolation between Triturus vulgaris and 77 montandoni in mating experiments run under semi-natural conditions. The two newt species offer a suitable model for studying evolution of reproductive isolation and mating preferences because they arc genetically the most similar species within the genus and readily hybridize in nature. Separate experiments were conducted in which groups of virgin females were placed together (in artificial pools) with groups of homospecific, heterospecific or both types of males. The estimates of reproductive isolation and mating propensity were based on the numbers of females producing hybrids and/or non-hybrid progeny. The levels of reproductive isolation, isolation asymmetry (IA) and propensity asymmetry (PA) were significant only for experiments in which females were given a choice between conspecific and heterospecific males. This implies that mating experiments with no interspecific choice may reduce discrimination and affect patterns of IA and PA. Asymmetry in reproductive isolation was also significant when the analysis was confined to just inseminated females. Differences in habitat preferences and condition of females possibly contributed to the relatively high values of PA.     

Sexual conflict does not drive reproductive isolation in experimental populations of Drosophila pseudoobscura

Sexual conflict has been predicted to drive reproductive isolation by generating arbitrary but rapid coevolutionary changes in reproductive traits among allopatric populations. A testable prediction of this proposal is that allopatric populations experiencing different levels of sexual conflict should exhibit different levels of reproductive isolation. We tested this prediction using experimentally evolved populations of the promiscuous Drosophila pseudoobscura. We manipulated sexual conflict by enforcing either monogamy, maintaining natural levels of promiscuity, or elevating promiscuity. Within each treatment, we carried out sympatric and allopatric crosses using replicated populations and examined pre-zygotic (number of mating pairs, mating speed and copulation duration) and post-zygotic (hybrid inviability and sterility) indicators of reproductive isolation. After 50 generations of selection, none of the measures conformed to predictions of sexual conflict driving reproductive isolation. Our results cannot be explained by lack of genetic variation or weak selection and suggest that sexual conflict may not be a widespread engine of speciation.     

Multiple mating increases cocoon hatching success in the earthworm Eisenia andrei (Oligochaeta: Lumbricidae)

In simultaneous hermaphrodites with reciprocal mating, multiple mating may be a male strategy that conflicts with female interests, and therefore an intra‐individual sexual conflict regarding the number of matings may be expected. The evolutionary outcome of this sexual conflict will depend on the costs and benefits that extra mating entails for each sexual function. In the present study, we investigated the costs and benefits of multiple mating on cocoon number, cocoon mass, and cocoon hatching success in the redworm Eisenia andrei, a simultaneous hermaphrodite with reciprocal insemination, by manipulating the number of matings with different partners. We did not detect any reduction in the female reproductive output (number and mass of cocoons) with increasing number of mating partners. However, we found that multiple mating showed benefits for female reproduction that increased the hatching success of the cocoons. This effect may be a result of increased quantity and/or diversity of sperm in the spermathecae of multiple mated earthworms. Further studies are required to clarify the mechanism underlying the increased cocoon hatching success when redworms engage in multiple matings. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, ?? , ??–??.     

A double role of sperm in scorpions: The mating plug of Euscorpius italicus (Scorpiones: Euscorpiidae) consists of sperm

Mating plugs occluding the female gonopore after mating are a widespread phenomenon. In scorpions, two main types of mating plugs are found: sclerotized mating plugs being parts of the spermatophore that break off during mating, and gel‐like mating plugs being gelatinous fluids that harden in the female genital tract. In this study, the gel‐like mating plug of Euscorpius italicus was investigated with respect to its composition, fine structure, and changes over time. Sperm forms the major component of the mating plug, a phenomenon previously unknown in arachnids. Three parts of the mating plug can be distinguished. The part facing the outside of the female (outer part) contains sperm packages containing inactive spermatozoa. In this state, sperm is transferred. In the median part, the sperm packages get uncoiled to single spermatozoa. In the inner part, free sperm is embedded in a large amount of secretions. Fresh mating plugs are soft gelatinous, later they harden from outside toward inside. This process is completed after 3‐5 days. Sperm from artificially triggered spermatophores could be activated by immersion in insect Ringer's solution indicating that the fluid condition in the females' genital tract or females' secretions causes sperm activation. Because of the male origin of the mating plug, it has likely evolved under sperm competition or sexual conflict. As females refused to remate irrespective of the presence or absence of a mating plug, females may have changed their mating behavior in the course of evolution from polyandry to monandry. J. Morphol., 2010. © 2010 Wiley‐Liss, Inc.     

The evolution of gametic compatibility and compatibility groups in the sea urchin Mesocentrotus franciscanus: An avenue for speciation in the sea

The generation of reproductive incompatibility between groups requires a rare genotype with low compatibility to increase in frequency. We tested the hypothesis that sexual conflict driven by the risk of polyspermy can generate compatibility groups in gamete recognition proteins (GRPs) in the sea urchin Mesocentrotus franciscanus. We examined variation in the sperm (bindin) and egg (EBR1) GRPs, how this variation influences fertilization success and how allele frequencies shift in these GRPs over time. The EBR1 gene is a large, 4595 amino acid protein made up of 27 thrombospondin type 1 domain (TSP) and 20 C1s/C1r, uEGF and bone morphogenic protein subdomain (CUB) repeats. Two TSP and two CUB repeats each demonstrate two common non‐synonymous haplotypes (alleles). Sperm bindin and one of these EBR1 repeats (TSP8) shift allele frequencies from one common to two common types over an approximate 200 year interval associated with the removal of predatory sea otters and rising sea urchin abundances; the egg receptor shifts first, followed by the sperm ligand. Laboratory crosses indicate that the historically common sperm and egg gamete recognition proteins have high compatibility as do the new common proteins, with mismatches having lower compatibility. This process of creating compatibility groups sets the stage for reproductive isolation and speciation.     

Multiple paternity and sperm competition in the sibling species Drosophila buzzatii and Drosophila koepferae

Sperm competition (SC) is a major component of sexual selection that enhances intra‐ and intersexual conflicts and may trigger rapid adaptive evolution of sexual characters. The actual role of SC on rapid evolution, however, is poorly understood. Besides, the relative contribution of distinctive features of the mating system to among species variation in the strength of SC remains unclear. Here, we assessed the strength of SC and mating system factors that may account for it in the closely related species Drosophila buzzatii and Drosophila koepferae. Our analyses reveal higher incidence of multiple paternity and SC risk in D. buzzatii wild‐inseminated females. The estimated number of fathers per brood was 3.57 in D. buzzatii and 1.95 in D. koepferae. In turn, the expected proportion of females inseminated by more than one male was 0.89 in D. buzzatii and 0.58 in D. koepferae. Laboratory experiments show that this pattern may be accounted for by the faster rate of stored sperm usage observed in D. koepferae and by the greater female remating rate exhibited by D. buzzatii. We also found that the male reproductive cost of SC is also higher in D. buzzatii. After a female mated with a second male, first‐mating male fertility was reduced by 71.4% in D. buzzatii and only 33.3% in D. koepferae. Therefore, we may conclude that postmating sexual selection via SC is a stronger evolutionary force in D. buzzatii than in its sibling.     

Relative abundance and the species-specific reinforcement of male mating preference in the Chrysochus (Coleoptera: Chrysomelidae) hybrid zone

Most studies of reinforcement have focused on the evolution of either female choice or male mating cues, following the long-held view in sexual selection theory that mating mistakes are typically more costly for females than for males. However, factors such as conspecific sperm precedence can buffer females against the cost of mating mistakes, suggesting that in some hybrid zones mating mistakes may be more costly for males than for females. Thus, the historical bias in reinforcement research may underestimate its frequency. In this study, we present evidence that reinforcement has driven the evolution of male choice in a hybrid zone between the highly promiscuous leaf beetles Chrysochus cobaltinus and C. auratus, the hybrids of which have extremely low fitness. In addition, there is evidence for male choice in these beetles and that male mating mistakes may be costly, due to reduced opportunities to mate with conspecific females. The present study combines laboratory and field methods to quantify the strength of sexual isolation, test the hypothesis of reproductive character displacement, and assess the link between relative abundance and the strength of selection against hybridization. We document that, while sexual isolation is weak, it is sufficient to produce positive assortative mating. In addition, reproductive character displacement was only detected in the relatively rare species. The strong postzygotic barriers in this system are sufficient to generate the bimodality that characterizes this hybrid zone, but the weak sexual isolation is not, calling into question whether strong prezygotic isolation is necessary for the maintenance of bimodality. Growing evidence that the cost of mating mistakes is sufficient to shape the evolution of male mate choice suggests that the reinforcement of male mate choice may prove to be a widespread occurrence.     

Patterns of sperm precedence are not affected by female mating history in Drosophila melanogaster

In promiscuously mating species, there is strong selection on males to maximize their share of paternity through both defensive and offensive means. This has been most extensively examined using the Drosophila melanogaster model system. In these studies, sperm competition has been examined by mating a virgin female to two consecutive males and then determining the fertilization success of both the first male (defending, P1) and the second male (offending, P2). Recent evidence suggests that male defense may be influenced by female mating history (i.e., virgin versus nonvirgin). Here, by mating females to males with three different genotypes, we show that female mating history does not affect male defensive or offensive abilities in sperm competition. We also show that, although female lifetime fecundity was not correlated with the number of times that she mated, it was reduced by increased exposure to males. These data indicate that measures of P1 and P2 previously reported in D. melanogaster may be robust to the specific mating history of the females used in these studies.     

Precopulatory but not postcopulatory male reproductive traits diverge in response to mating system manipulation in Drosophila melanogaster

Competition between males creates potential for pre‐ and postcopulatory sexual selection and conflict. Theory predicts that males facing risk of sperm competition should evolve traits to secure their reproductive success. If those traits are costly to females, the evolution of such traits may also increase conflict between the sexes. Conversely, under the absence of sperm competition, one expectation is for selection on male competitive traits to relax thereby also relaxing sexual conflict. Experimental evolution studies are a powerful tool to test this expectation. Studies in multiple insect species have yielded mixed and partially conflicting results. In this study, we evaluated male competitive traits and male effects on female costs of mating in Drosophila melanogaster after replicate lines evolved for more than 50 generations either under enforced monogamy or sustained polygamy, thus manipulating the extent of intrasexual competition between males. We found that in a setting where males competed directly with a rival male for access to a female and fertilization of her ova polygamous males had superior reproductive success compared to monogamous males. When comparing reproductive success solely in double mating standard sperm competition assays, however, we found no difference in male sperm defense competitiveness between the different selection regimes. Instead, we found monogamous males to be inferior in precopulatory competition, which indicates that in our system, enforced monogamy relaxed selection on traits important in precopulatory rather than postcopulatory competition. We discuss our findings in the context of findings from previous experimental evolution studies in Drosophila ssp. and other invertebrate species.