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.     

Asymmetric conspecific sperm precedence in relation to spawning times in the Montastraea annularis species complex (Cnidaria: Scleractinia)

In broadcast spawners, prezygotic reproductive isolation depends on differences in the spatial and temporal patterns of gamete release and gametic incompatibility. Typically, gametic incompatibility is measured in no‐choice crosses, but conspecific sperm precedence (CSP) can prevent hybridization in gametes that are compatible in the absence of sperm competition. Broadcast spawning corals in the Montastraea annularis species complex spawn annually on the same few evenings. Montastraea franksi spawns an average of 110 min before M. annularis, with a minimum gap of approximately 40 min. Gametes are compatible in no‐choice heterospecific assays, but it is unknown whether eggs exhibit choice when in competition. Hybridization depends on either M. franksi eggs remaining unfertilized and in proximity to M. annularis when the latter species spawns or M. franksi sperm remaining in sufficient viable concentrations when M. annularis spawns. We found that the eggs of the early spawning M. franksi demonstrate strong CSP, whereas CSP appears to be lacking for M. annularis eggs. This study provides evidence of diverging gamete affinities between these recently separated species and suggests for the first time that selection may favour CSP in earlier spawning species when conspecific sperm is diluted and aged and is otherwise at a numeric and viability disadvantage with heterospecific sperm.     

Assortative fertilization and selection at larval stage in the mussels Mytilus edulis and M. galloprovincialis

Assortative mating (prezygotic isolation) and reduced hybrid fitness (postzygotic isolation) are typically invoked to explain the stability of hybrid zones. In the tension zone model, these factors work in opposition to migration, which promotes genetic homogeneity. Many marine animals migrate over long distances through a planktonic larval stage. Therefore, strong reproductive isolation is needed to maintain stable marine hybrid zones. However, surprisingly little is known about mating preferences and hybrid fitness in marine organisms. Smooth-shelled mussels (Mytilus spp.) form a well-known species complex, with hybridization over extensive areas such as the contact zone of M. edulis and M. galloprovincialis around European Atlantic coasts. This paper reports direct experimental evidence of assortative fertilization, hybrid larval inviability, and early heterosis for growth rate in M. edulis and M. galloprovincialis. Four crosses between pure M. edulis and M. galloprovincialis were analyzed with a new polymerase-chain-reaction-based diagnostic marker. Gamete competition between taxa was allowed in two out of the four crosses. Genotype frequencies observed at an early stage (36 h after fertilization) unambiguously revealed assortative fertilization when gamete competition was allowed. A significant reduction in hybrid viability was subsequently observed during the larval stage. At the same stage an antagonistic effect, heterosis, was observed on growth rate. However, even if heterosis is observed in the F1, it is expected to vanish in subsequent hybrid generations. Although specialization for different habitats and asynchronous spawning have been mentioned as factors contributing to the maintenance of the blue mussel hybrid zone in Europe, we argue that assortative fertilization and reduced hybrid fitness are important factors that also contribute to the stabilization of this zone. These results emphasize that multiple factors may act concomitantly in a barrier to gene flow, especially in complex life cycles. Furthermore, they show that assortative mating through gamete preference, as already demonstrated for sea urchins, may play a role in speciation processes taking place in the sea.     

Investigating Incipient Speciation in Arabidopsis lyrata from Patterns of Transmission Ratio Distortion

Our understanding of the development of intrinsic reproductive isolation is still largely based on theoretical models and thorough empirical studies on a small number of species. Theory suggests that reproductive isolation develops through accumulation of epistatic genic incompatibilities, also known as Bateson–Dobzhansky–Muller (BDM) incompatibilities. We can detect these from marker transmission ratio distortion (TRD) in hybrid progenies of crosses between species or populations, where TRD is expected to result from selection against heterospecific allele combinations in hybrids. TRD may also manifest itself because of intragenomic conflicts or competition between gametes or zygotes. We studied early stage speciation in Arabidopsis lyrata by investigating patterns of TRD across the genome in F₂ progenies of three reciprocal crosses between four natural populations. We found that the degree of TRD increases with genetic distance between crossed populations, but also that reciprocal progenies may differ substantially in their degree of TRD. Chromosomes AL6 and especially AL1 appear to be involved in many single- and two-locus distortions, but the location and source of TRD vary between crosses and between reciprocal progenies. We also found that the majority of single- and two-locus TRD appears to have a gametic, as opposed to zygotic, origin. Thus, while theory on BDM incompatibilities is typically illustrated with derived nuclear alleles proving incompatible in hybrid zygotes, our results suggest a prominent role for distortions emerging before zygote formation.     

Gametic incompatibilities between races of Drosophila melanogaster

Reproductive-isolating mechanisms between nascent species may involve sperm-egg recognition and have been best described in externally fertilizing organisms where such recognition is essential in preventing undesirable fertilizations. However, reproductive barriers in internally fertilizing species differ in significant ways, and a direct role for sperm-egg interactions has yet to be demonstrated. Females of many strains of Drosophila melanogaster from Zimbabwe, Africa, do not mate readily with cosmopolitan males. This polymorphism in mate choice is postulated to represent incipient speciation. We now report that, in one direction, crosses between the above populations produce far fewer offspring than reciprocal crosses due to a lower rate of egg hatch. We established that egg inviability in these crosses was due to defects in fertilization. Thus, even in taxa with internal fertilization, gametic incompatibility may be a mechanism relevant to reproductive isolation during incipient speciation.     

Sexual isolation in two bee-pollinated Costus (Costaceae)

Examining reproductive isolating barriers is essential for understanding processes of speciation. Sexual isolation has been shown to contribute to speciation in many sympatric taxa; however, its role in parapatric taxa with reduced interspecific gene flow is poorly understood. I investigated the extent of sexual isolation in two closely related Neotropical understory species, Costus allenii and C. villosissimus, that occur in adjacent habitats within flight distance of their shared pollinators, euglossine bees. Pollination arrays were used to test whether individual pollinators travel between species, to estimate the proportion of hetero- and conspecific pollen deposited on the stigmas, and to examine the proportion of hybrid progeny. In comparison to C. allenii, C. villosissimus produces flowers with larger labella, longer stamen–labellum distances, and longer styles. Pollinators visited both species but preferred C. villosissimus. This preference caused pollinator isolation in C. villosissimus. In C. allenii, the frequency of heterospecific pollinator transitions was not less common than that of conspecific transitions, but floral mechanical isolation greatly reduced the likelihood of heterospecific pollen deposition. The contribution of gametic isolation was not strong in either species. Based on data for pollinator isolation, floral mechanical isolation, and gametic isolation, it appears that sexual isolation is weak in C. allenii, restricting heterospecific gene flow by 25 %, but moderate in C. villosissimus, where gene flow from C. allenii is reduced by 70 %. Further research will estimate the magnitude of other isolation barriers to determine the relative contribution of sexual isolation to total isolation in this parapatric species pair.     

Evidence of prezygotic isolation,but not assortative mating,between locally adapted populations of Fundulus heteroclitus across a salinity gradient

Selection along environmental gradients can drive reproductive isolation and speciation. Among fishes, salinity is a major factor limiting species distributions, and despite its importance in generating species diversity, speciation events between marine and freshwater are rare. Here, we tested for mechanisms of reproductive isolation between locally adapted freshwater and brackish water-native populations of killifish, Fundulus heteroclitus, from either side of a hybrid zone along a salinity gradient. There was evidence for pre-zygotic endogenous reproductive isolation with reduced fertilization success between crosses of freshwater-native males and brackish water-native females. Exogenous pre-zygotic isolation was also present where females had highest fertilization in their native salinity. We used a replicated mass spawning design to test for mate choice in both brackish and fresh water. After genotyping 187 parents and 2523 offspring at 2347 SNPs across the genome, 85% of offspring were successfully assign to their parents. However, no reinforcing mate choice was observed. These results therefore demonstrate emerging, yet limited, reproductive isolation and incipient speciation across a marine to freshwater salinity gradient and suggest that both endogenous and exogenous mechanisms, but not assortative mating, contribute to divergence.     

HYBRIDIZATION OF SYMPATRIC PATIRIELLA SPECIES (ECHINODERMATA: ASTEROIDEA) IN NEW SOUTH WALES

Three species of the asteroid genus Patiriella occur sympatrically in New South Wales and the possibility for hybridization among them was examined through a series of cross-fertilization experiments. Patiriella calcar and P. gunnii are morphologically distinct as adults but indistinguishable as larvae. Patiriella exigua is morphologically distinct in both its adult and larval morphologies. The gametes of P. calcar and P. gunnii were reciprocally compatible: laboratory crosses between these species produced viable hybrid juveniles. In crosses between female P. calcar and male P. gunnii, most of the juveniles metamorphosed with an arm number intermediate between that of the parents, whereas crosses between female P. gunnii and male P. calcar produced juveniles with an arm number more similar to the maternal phenotype. Heterospecific crosses with P. exigua resulted in low fertilization rates, and viable hybrids were not produced. This species appears capable of self-fertilization. Because hybrids between P. calcar and P. gunnii were viable, neither gametic incompatibility nor hybrid inviability appears to ensure reproductive isolation between these species. Ecological or habitat segregation and temporal separation in breeding may isolate these species in the field. The results demonstrate that if gamete surface recognition molecules are involved in fertilization of P. calcar and P. gunnii, then they are not strongly species specific, at least at the sperm concentrations used in this study. Reproductive isolation between these species has evolved despite their gametic compatibility. In contrast, P. exigua is isolated from its congeners because of gametic incompatibility and several features characteristic of its reproduction and development. The implications of these findings for reproductive isolation and speciation of Patiriella and for the evolution of reproductive isolation in free-spawning marine organisms are discussed.     

Experimental evidence for reduced hybrid viability between dwarf and normal ecotypes of lake whitefish (Coregonus clupeaformis Mitchill)

Forces driving the evolution of reproductive isolation among natural populations, as well as the mechanisms involved to maintain it, are still poorly understood. Because sympatric fish ecotypes mainly differ in phenotypic traits associated with occupying distinct trophic niches, it is generally believed that reproductive isolation is mainly driven by ecological divergent selection, excluding genome incompatibility as a basis for postmating isolation. We did cross experiments between dwarf and normal ecotypes of lake whitefish (Coregonus clupeaformis Mitchill) originating from distinct glacial refugia to test the hypothesis that their geographical isolation during the Pleistocene may have led to sufficient genetic divergence for the development of reproductive isolation between them before their secondary contact. Similar fertilization success in pure and hybrid crosses indicated the absence of gametic incompatibility between the two ecotypes. In contrast, daily embryonic mortality rates were 2.4–4.7 times higher in reciprocal hybrid crosses compared to pure crosses, which supports our working hypothesis. These results, along with previous morphological and population genetic studies, indicate that both genetic and ecological mechanisms may jointly act to promote speciation among northern freshwater fish ecotypes.     

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.     

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 gene genealogies reveal asymmetrical hybridization and introgression among strongylocentrotid sea urchins

The evolution of incompatibilities between eggs and sperm is thought to play important roles in establishing and maintaining reproductive isolation among species of broadcast-spawning marine invertebrates. However, the effectiveness of gametic isolation in initiating the speciation process and/or in limiting the introgression of genes among species at later stages of divergence remains largely unknown. In the present study, we collected DNA sequence data from five loci in four species of Strongylocentrotus sea urchins ( S. droebachiensis , S. pallidus , S. purpuratus , and S. franciscanus ) to test whether the susceptibility of S. droebachiensis eggs to fertilization by heterospecific sperm results in gene flow between species. Despite the potential for introgression, a small but statistically significant signal of introgression was observed only between the youngest pair of sister taxa ( S. pallidus and S. droebachiensis ) that was strongly asymmetrical (from the former into the latter). No significant gene flow was observed for either S. purpuratus or S. franciscanus despite the ability of their sperm to readily fertilize the eggs of S. droebachiensis . Our results demonstrate that asymmetrical gamete compatibilities in strongylocentrotids can give rise to asymmetrical patterns of introgression but suggest that gamete traits alone cannot be responsible for maintaining species integrities. The genetic boundaries between strongylocentrotid urchin species in the northeast Pacific appear to be related to postzygotic isolating mechanisms that scale with divergence times and not intrinsic gametic incompatibilities per se .     

Reproductive isolation between two darter species is enhanced and asymmetric in sympatry

Robust reproductive isolation was found between the rainbow darter Etheostoma caeruleum and the orangethroat darter Etheostoma spectabile, as more offspring were produced when conspecific males and females were crossed as compared with heterospecific crosses. Furthermore, fewer eggs resulted from heterospecific crosses involving sympatric E. spectabile females than those using allopatric E. spectabile females, while a similar pattern was not observed in heterospecific crosses using E. caeruleum females. These results suggest that reinforcement, i.e. selection for pre‐zygotic reproductive barriers driven by reduced hybrid fitness, may have contributed to the evolution and maintenance of reproductive barriers between these potentially hybridizing species in sympatry.     

Pervasive genetic associations between traits causing reproductive isolation in Heliconius butterflies

Ecological speciation proceeds through the accumulation of divergent traits that contribute to reproductive isolation, but in the face of gene flow traits that characterize incipient species may become disassociated through recombination. Heliconius butterflies are well known for bright mimetic warning patterns that are also used in mate recognition and cause both pre- and post-mating isolation between divergent taxa. Sympatric sister taxa representing the final stages of speciation, such as Heliconius cydno and Heliconius melpomene, also differ in ecology and hybrid fertility. We examine mate preference and sterility among offspring of crosses between these species and demonstrate the clustering of Mendelian colour pattern loci and behavioural loci that contribute to reproductive isolation. In particular, male preference for red patterns is associated with the locus responsible for the red forewing band. Two further colour pattern loci are associated, respectively, with female mating outcome and hybrid sterility. This genetic architecture in which ‘speciation genes’ are clustered in the genome can facilitate two controversial models of speciation, namely divergence in the face of gene flow and hybrid speciation.     

Evolutionary consequences of introgression at M7 lysin, a gamete recognition locus, following secondary contact between blue mussel species

Hybridizing populations of blue mussels, Mytilus edulis and Mytilus trossulus, in Cobscook Bay (eastern Maine) have been used by our laboratory to study the evolution of gamete incompatibility and molecular evolution of the vitelline coat lysin proteins expressed in sperm. The M7 lysin locus has been the most studied of the three lysins, but evidence for positive selection necessary to help confirm its role in gamete recognition in western Atlantic hybrid zones is contradictory. We developed an alternative test, based on rates of introgression at M7 lysin. Contrary to expectations, introgression at this locus is much higher (instead of much lower) than is introgression at neutral markers. In this article, we present simulations, constructed using synthetic populations of combinations of admixed genotypes, representing various hybrid offspring categories. Simulations produced variation in introgression across loci, but did not generate the massive introgression at M7 lysin observed in natural populations in Cobscook Bay. We consider these results in the context of selection operating on gamete recognition loci, both within and between species, during the third stage of allopatric speciation in Mytilus.     

Comparison of gamete compatibility between two blue mussel species in sympatry and in allopatry

Recent demonstrations of positive selection on genes controlling gamete compatibility have resulted in a proliferation of hypotheses concerning the sources of selection. We tested a prediction of one prominent hypothesis, selection to avoid hybridization (i.e., reinforcement), by comparing heterospecific gamete compatibility in two Mytilus edulis populations: one population in Cobscook Bay, Maine, in which the close congener, M. trossulus, is abundant (a region of sympatry), and one population in Kittery, Maine, in which M. trossulus is absent (a region of allopatry). Three diagnostic nuclear DNA markers were used to identify mussels to species and to estimate the frequency of both species and their hybrids in the two populations. Controlled crosses were then conducted by combining eggs of M. edulis females with a range of M.edulis and M. trossulus sperm concentrations. Results were not consistent with the reinforcement hypothesis. M. edulis females collected from the region of sympatry were no more incompatible with M. trossulus males than were M. edulis females collected from the region of allopatry. A trend in the opposite direction, toward greater compatibility in sympatry, suggests that introgression of M. trossulus genes that control egg compatibility, such as those encoding receptors for sperm, may influence evolution of gametic isolation in hybridizing populations.     

Long- term effects of previous experience determine nutrient discrimination abilities in birds

Background

The evolution of reproductive traits, such as hybrid incompatibility (postzygotic isolation) and species recognition (prezygotic isolation), have shown their key role in speciation. Theoretical modeling has recently predicted that close linkage between genes controlling pre- and postzygotic reproductive isolation could accelerate the conditions for speciation. Postzygotic isolation could develop during the sympatric speciation process contributing to the divergence of populations. Using hybrid fitness as a measure of postzygotic reproductive isolation, we empirically studied population divergence in perch (Perca fluviatilis L.) from two genetically divergent populations within a lake.

Results

During spawning time of perch we artificially created parental offspring and F₁ hybrids of the two populations and studied fertilization rate and hatching success under laboratory conditions. The combined fitness measure (product of fertilization rate and hatching success) of F₁ hybrids was significantly reduced compared to offspring from within population crosses.

Conclusion

Our results suggest intrinsic genetic incompatibility between the two populations and indicate that population divergence between two populations of perch inhabiting the same lake may indeed be promoted by postzygotic isolation.     

Assortative mating in sympatric ascomycete fungi revealed by experimental fertilizations

Mate recognition mechanisms resulting in assortative mating constitute an effective reproductive barrier that may promote sexual isolation and speciation. While such mechanisms are widely documented for animals and plants, they remain poorly studied in fungi. We used two interfertile species of Epichloë (Clavicipitaceae, Ascomycota), E. typhina and E. clarkii, which are host-specific endophytes of two sympatrically occurring grasses. The life cycle of these obligatory outcrossing fungi entails dispersal of gametes by a fly vector among external fungal structures (stromata). To test for assortative mating, we mimicked the natural fertilization process by applying mixtures of spermatia from both species and examined their reproductive success. Our trials revealed that fertilization is non-random and preferentially takes place between conspecific mating partners, which is indicative of assortative mating. Additionally, the viability of hybrid and non-hybrid ascospore offspring was assessed. Germination rates were lower in E. clarkii than in E. typhina and were reduced in ascospore progeny from treatments with high proportions of heterospecific spermatia. The preferential mating between conspecific genotypes and reduced hybrid viability represent important reproductive barriers that have not been documented before in Epichloë. Insights from fungal systems will deepen our understanding of the evolutionary mechanisms leading to reproductive isolation and speciation.     

Reinforcement of Gametic Isolation in Drosophila

Reinforcement, a process by which natural selection increases reproductive isolation between populations, has been suggested to be an important force in the formation of new species. However, all existing cases of reinforcement involve an increase in mate discrimination between species. Here, I report the first case of reinforcement of postmating prezygotic isolation (i.e., barriers that act after mating but before fertilization) in animals. On the slopes of the African island of São Tomé, Drosophila yakuba and its endemic sister species D. santomea hybridize within a well-demarcated hybrid zone. I find that D. yakuba females from within this zone, but not from outside it, show an increase in gametic isolation from males of D. santomea, an apparent result of natural selection acting to reduce maladaptive hybridization between species. To determine whether such a barrier could evolve under laboratory conditions, I exposed D. yakuba lines derived from allopatric populations to experimental sympatry with D. santomea, and found that both behavioral and gametic isolation become stronger after only four generations. Reinforcement thus appears to be the best explanation for the heightened gametic isolation seen in sympatry. This appears to be the first example in animals in which natural selection has promoted the evolution of stronger interspecific genetic barriers that act after mating but before fertilization. This suggests that many other genetic barriers between species have been increased by natural selection but have been overlooked because they are difficult to study.