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.     

SIMULTANEOUS POSITIVE AND NEGATIVE FREQUENCY‐DEPENDENT SELECTION ON SPERM BINDIN,A GAMETE RECOGNITION PROTEIN IN THE SEA URCHIN STRONGYLOCENTROTUS PURPURATUS

Gamete‐recognition proteins often, but not always, evolve rapidly. We explored how variation in sperm bindin influences reproductive success of the sea urchin Strongylocentrotus purpuratus during group spawning in the sea. Despite large variation in male and female abundance and neighbor distances, males with common genotypes had higher reproductive success than males with rare genotypes. However, males with a relatively uncommon proline‐for‐serine substitution were the most successful. Females also showed a fitness consequence of sperm‐bindin genotype, suggesting linkage disequilibrium between the sperm‐bindin locus and the egg receptor locus. Females with common genotypes had higher reproductive success than rare genotypes, but females with relatively uncommon insertions were most successful. Overall, these results suggest that rare male proteins are selected against, as supported by molecular evidence of purifying selection and probably caused by poor matches to the female receptor protein. Within the pool of moderately common to common alleles, however, individuals with less‐common functional variants were favored and probably maintained by negative frequency‐dependent selection. These results support the hypothesis that sperm availability and sexual conflict influence the evolution of gamete recognition systems in broadcast spawners and highlight the benefits of combining fitness measures with molecular signatures for estimation of patterns of selection.     

Geographic variation and positive selection on M7 lysin, an acrosomal sperm protein in mussels (Mytilus spp.)

Successful fertilization in free-spawning marine organisms depends on the interactions between genes expressed on the surfaces of eggs and sperm. Positive selection frequently characterizes the molecular evolution of such genes, raising the possibility that some common deterministic process drives the evolution of gamete recognition genes and may even be important for understanding the evolution of prezygotic isolation and speciation in the marine realm. One hypothesis is that gamete recognition genes are subject to selection for prezygotic isolation, namely, reinforcement. In a previous study, positive selection on the gene coding for the acrosomal sperm protein M7 lysin was demonstrated among allopatric populations of mussels in the Mytilus edulis species group (M. edulis, Mytilus galloprovincialis, and Mytilus trossulus). Here, we expand sampling to include M7 lysin haplotypes from populations where mussel species are sympatric and hybridize to determine whether there is a pattern of reproductive character displacement (RCD), which would be consistent with reinforcement driving selection on this gene. We do not detect a strong pattern of RCD; neither are there unique haplotypes in sympatry nor is there consistently greater population structure in comparisons involving sympatric populations. One distinct group of haplotypes, however, is strongly affected by natural selection, and this group of haplotypes is found within M. galloprovincialis populations throughout the Northern Hemisphere concurrent with haplotypes common to M. galloprovincialis and M. edulis. We suggest that balancing selection, perhaps resulting from sexual conflicts between sperm and eggs, maintains old allelic diversity within M. galloprovincialis.     

Natural hybridization in the sea urchin genus Pseudoboletia between species without apparent barriers to gamete recognition

Marine species with high dispersal potential often have huge ranges and minimal population structure. Combined with the paucity of geographic barriers in the oceans, this pattern raises the question as to how speciation occurs in the sea. Over the past 20 years, evidence has accumulated that marine speciation is often linked to the evolution of gamete recognition proteins. Rapid evolution of gamete recognition proteins in gastropods, bivalves, and sea urchins is correlated with gamete incompatibility and contributes to the maintenance of species boundaries between sympatric congeners. Here, we present a counterexample to this general pattern. The sea urchins Pseudoboletia indiana and P. maculata have broad ranges that overlap in the Indian and Pacific oceans. Cytochrome oxidase I sequences indicated that these species are distinct, and their 7.3% divergence suggests that they diverged at least 2 mya. Despite this, we suspected hybridization between them based on the presence of morphologically intermediate individuals in sympatric populations at Sydney, Australia. We assessed the opportunity for hybridization between the two species and found that (1) individuals of the two species occur within a meter of each other in nature, (2) they have overlapping annual reproductive cycles, and (3) their gametes cross-fertilize readily in the laboratory and in the field. We genotyped individuals with intermediate morphology and confirmed that many were hybrids. Hybrids were fertile, and some female hybrids had egg sizes intermediate between the two parental species. Consistent with their high level of gamete compatibility, there is minimal divergence between P. indiana and P. maculata in the gamete recognition protein bindin, with a single fixed amino acid difference between the two species. Pseudoboletia thus provides a well-characterized exception to the idea that broadcast spawning marine species living in sympatry develop and maintain species boundaries through the divergence of gamete recognition proteins and the associated evolution of gamete incompatibility.     

GAMETE INTERACTIONS AND GENETIC DIFFERENTIATION AMONG THREE SYMPATRIC POLYCHAETES

The evolution of gamete incompatibility between free-spawning marine invertebrate species has been explained by three hypotheses: (1) independent divergence at gamete recognition loci; (2) selection against hybrids; and (3) a process of sexual selection involving polymorphic gamete recognition loci (Metz and Palumbi 1996). The first two hypotheses predict that gamete incompatibility appears only after gene flow has been halted for other reasons and the third that gamete incompatibility appears simultanously with blocks to gene flow. Here I show that gametes of three sympatric polychaetes in the genus Arctonoe are compatible in all crosses, over a broad range of gamete concentrations and contact times. Although at least some hybrid crosses produce fertile adults, allozyme and mitochondrial DNA sequence data indicate that the three species do not regularly exchange genes. These data are consistent with predictions of the first two hypotheses for the evolution of gamete incompatibility, but allow rejection of the third hypothesis. Gametes of the three species are compatible despite estimated divergence times of 1–3 M.Y.B.P.; in several other marine invertebrates, divergence times of the same magnitude are associated with asymmetric or complete gamete incompatibility. It appears likely that segregation of symbiotic adults on their respective host species restricts mating opportunities, and thus gene flow, among Arctonoe species.     

Two sexes,one body: intra‐ and intersex covariation of gamete phenotypes in simultaneous hermaphrodites

By harboring male and female functions in the same genome and expressing them in every individual, simultaneous hermaphrodites may incur sexual conflict unless both sex functions can evolve phenotypic optima independently of each other. The first step toward understanding their capacity to do so lies in understanding whether sex functions are phenotypically correlated within individuals, but remarkably few data address this issue. We tested the potential for intra‐ and intersex covariation of gamete phenotypes to mediate sexual conflict in broadcast‐spawning hermaphrodites (the ascidians Ciona intestinalis and Pyura praeputialis), for which sex‐specific selection acts predominantly on sperm–egg interactions in the water column. In both species, gamete phenotypes covaried within and across sex functions, implying that selection may be unable to target them independently because its direct effects on male gametes translate into correlated effects on female gametes and vice versa. This alone does not preclude the evolution of a different phenotypic optimum for each sex function, but imposes the more restrictive requirement that selection – which ultimately sorts among whole individuals, not sex functions – aligns with the direction in which gamete phenotypes covary at this level.     

The rapid evolution of reproductive proteins

Many genes that mediate sexual reproduction, such as those involved in gamete recognition, diverge rapidly, often as a result of adaptive evolution. This widespread phenomenon might have important consequences, such as the establishment of barriers to fertilization that might lead to speciation. Sequence comparisons and functional studies are beginning to show the extent to which the rapid divergence of reproductive proteins is involved in the speciation process.     

Evaluation of Sequence Variation and Selection in the Bindin Locus of the Red Sea Urchin, Strongylocentrotus franciscanus

Recent evidence suggests that gamete recognition proteins may be subjected to directed evolutionary pressure that enhances sequence variability. We evaluated whether diversity enhancing selection is operating on a marine invertebrate fertilization protein by examining the intraspecific DNA sequence variation of a 273-base pair region located at the 5′ end of the sperm bindin locus in 134 adult red sea urchins (Strongylocentrotus franciscanus). Bindin is a sperm recognition protein that mediates species-specific gamete interactions in sea urchins. The region of the bindin locus examined was found to be polymorphic with 14 alleles. Mean pairwise comparison of the 14 alleles indicates moderate sequence diversity (p-distance = 1.06). No evidence of diversity enhancing selection was found. It was not possible to reject the null hypothesis that the sequence variation observed in S. franciscanus bindin is a result of neutral evolution. Statistical evaluation of expected proportions of replacement and silent nucleotide substitutions, observed versus expected proportions of radical replacement substitutions, and conformance to the McDonald and Kreitman test of neutral evolution all indicate that random mutation followed by genetic drift created the polymorphisms observed in bindin. Observed frequencies were also highly similar to results expected for a neutrally evolving locus, suggesting that the polymorphism observed in the 5′ region of S. franciscanus bindin is a result of neutral evolution. Received: 19 June 1998 / Accepted: 2 August 2000     

Sexual selection at the protein level drives the extraordinary divergence of sex-related genes during sympatric speciation

An increasing number of molecular studies are indicating that, in a wide variety of species, genes directly related to fertilization evolve at extraordinarily high rates. We try to gain insight into the dynamics of this rapid evolution and its underlying mechanisms by means of a simple theoretical model. In the model, sexual selection and sympatric speciation act together in order to drive rapid divergence of gamete recognition proteins. In this process, intraspecific competition for fertilizations enlarges male gamete protein variation by means of evolutionary branching, which initiates sympatric speciation. In addition, avoidance of competition for fertilizations between the incipient species drives the rapid evolution of gamete recognition proteins. This mechanism can account for both strong stabilizing selection on gamete recognition proteins within species and rapid divergence between species. Moreover, it can explain the empirical finding that the rate of divergence of fertilization genes is not constant, but highest between closely related species.     

Sea urchin bindin divergence predicts gamete compatibility

Studies on the evolution of reproductive proteins have shown that they tend to evolve more rapidly than other proteins, frequently under positive selection. Progress on understanding the implications of these patterns is possible for marine invertebrates, where molecular evolution can be linked to gamete compatibility. In this study, we surveyed data from the literature from five genera of sea urchins for which there was information on gamete compatibility, divergence of the sperm-egg recognition protein bindin, and mitochondrial divergence. We draw three conclusions: (1) bindin divergence at nonsynonymous sites predicts gamete compatibility, whereas (2) bindin divergence at synonymous sites and mitochondrial DNA divergence do not, and (3) as few as 10 amino acid changes in bindin can lead to complete gamete incompatibility between species. Using mitochondrial divergence as a proxy for time, we find that complete gamete incompatibility can evolve in approximately one and a half million years, whereas sister species can maintain complete gamete compatibility for as long as five million years.     

Gamete types, sex determination and stable equilibria of all-hybrid populations of diploid and triploid edible frogs (Pelophylax esculentus)

Background  

Triploid individuals often play a key role in speciation by hybridization. An understanding of the gamete types (ploidy and genomic content) and stability of hybrid populations with triploid individuals is therefore of importance for exploring the role of hybridization in evolution. The all-hybrid populations of the edible frog, Pelophylax esculentus, are unique in their composition and genetic dynamics: Diploid (genotype LR) and triploid (LLR and LRR) hybrids depend on each other's different gamete contributions for successful reproduction and maintenance of the populations, as the parental genotypes P. lessonae (LL) and P. ridibundus (RR) are absent among adults. This study provides data and interpretations on gamete types and sex determination that are essential for understanding the function, evolutionary potential and threats of this intriguing system.     

MORE THAN BINDIN DIVERGENCE: REPRODUCTIVE ISOLATION BETWEEN SYMPATRIC SUBSPECIES OF A SEA URCHIN BY ASYNCHRONOUS SPAWNING

The evolution of reproductive barriers is crucial to the process of speciation. In the Echinoidea, studies have focused on divergence in the gamete recognition protein, bindin, as the primary isolating mechanism among species. As such, the capacity of alternate mechanisms to be effective reproductive barriers and the phylogenetic context in which they arise is unclear. Here, we examine the evolutionary histories and factors limiting gene exchange between two subspecies of Heliocidaris erythrogramma that occur sympatrically in Western Australia. We found low, but significant differentiation between the subspecies in two mitochondrial genes. Further, coalescent analyses suggest that they diverged in isolation on the east and west coasts of Australia, with a subsequent range expansion of H. e. erythrogramma into Western Australia. Differentiation in bindin was minimal, indicating gamete incompatibility is an unlikely reproductive barrier. We did, however, detect strong asynchrony in spawning seasons; H. e. erythrogramma spawned over summer whereas H. e. armigera spawned in autumn. Taken together, we provide compelling evidence for a recent divergence of these subspecies and their reproductive isolation without gamete incompatibility. Western Australian H. erythrogramma may therefore present an intriguing case of incipient speciation, which depends on long‐term persistence of the factors underlying this spawning asynchrony.     

Morphological evidence that the molecularly determined Ciona intestinalis type A and type B are different species: Ciona robusta and Ciona intestinalis

Ciona intestinalis is considered a widespread and easily recognizable tunicate, the sister group of vertebrates. In recent years, molecular studies suggested that C. intestinalis includes at least two cryptic species, named ‘type A’ and ‘type B’, morphologically indistinguishable. It is dramatic to certify that two different species may be hidden under the name of a species widely used as a model species in biological researches. This raised the problem of identifying diagnostic morphological characters capable of distinguishing these types. We compared the morphology of specimens belonging to the two types and found that only type A specimens possess tunic tubercular prominences, allowing unambiguous discrimination. Remarkably, these structures were already described as distinctive of the Japanese species Ciona robusta, Hoshino and Tokioka, 1967; later synonymized under C. intestinalis (sensu Millar, 1953). In this study, we have confirmed that C. intestinalis type A corresponds to C. robusta. Based on the geographic distribution of C. intestinalis type B, and considering that the original C. intestinalis species was described from North European waters, we determined that C. intestinalis type B corresponds to C. intestinalis as described by Millar in 1953 and possibly to Linnaeus' Ascidia intestinalis L., 1767 for which we have deposited a neotype (from Roscoff, France) and for which we retain the name Ciona intestinalis (Linnaeus, 1767).     

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.     

Characterization of novel β‐1,3‐glucan recognition proteins from a Tibetan Plateau ghost moth Thitarodes pui (Lepidoptera,Hepialidae)

β‐1,3‐glucan recognition proteins (βGRPs) function as pattern recognition receptors in the innate immune response against invading pathogens. In the present study, we obtain full‐length cDNA clones for two novel putative βGRPs: TpβGRPc and TpβGRPd from the ghost moth Thitarodes pui (Lepidoptera: Hepialidae). Phylogenetic analysis shows a small distinct lineage, βGRP clade 4, consisting of T. pui βGRPs including TpβGRPa and TpβGRPb that have been identified previously. TpβGRPc and TpβGRPd, comprising 488 and 229 amino acids, have calculated molecular masses of 52 596 and 24 589 Da, respectively. TpβGRPc is 85.52% identical in sequence to TpβGRPa. TpβGRPb and TpβGRPd share the same deletion start site located at the conserved residue Pro 43, although TpβGRPd exhibits a much larger deletion of up to approximately 270 residues covering both the N‐ and C‐terminal regions. Affinity purification, associated with subsequent peptide sequencing, confirms the constitutive occurrence of TpβGRPa and TpβGRPc of similar size (approximately 65 kDa) in sixth‐instar larval haemolymph. These two βGRPs show clear binding affinities to curdlan, an insoluble β‐1,3‐glucan. A quantitative real‐time polymerase chain reaction analysis reveals the high‐level constitutive expression of TpβGRPc and TpβGRPd in the fat body of mid‐instar larvae, which are found to be susceptible to fungal pathogens in field investigations. Remarkable induction of both TpβGRPs occurs in response to haemocoelic challenge with entomopathogenic fungus Beauveria bassiana. The results of the present study suggest that TpβGRPs may contribute to the detection and control of fungal infections.     

From song dialects to speciation in white‐crowned sparrows

The behavioural signals used in mate selection are a key component in the evolution of premating isolating barriers and, subsequently, the formation of new species. The importance of mating signals has a long tradition of study in songbirds, where many species differ in their song characteristics. In oscine songbirds, individual birds usually learn their songs from a tutor. Mistakes during learning can help generate geographic dialects, akin to those within human language groups. In songbirds, dialect differences can often be substantial and there is an intuitive connection between the evolution of song amongst populations at a small scale, and the more substantive song differences between bird species and presumably used in species recognition. However, studies investigating the concordance between putative genetic and behavioural boundaries have generated mixed results. In many cases, this is possibly a function of the poor resolving power of the genetic markers employed. In this issue of Molecular Ecology, Lipshutz et al. ( 2017 ) combine genomic markers with a robust behavioural assay to address the importance of song variation amongst white‐crowned sparrow (Zonotrichia leucophrys) subspecies.     

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.     

Molecular evolution of the sex peptide network in Drosophila

Successful reproduction depends on interactions between numerous proteins beyond those involved directly in gamete fusion. Although such reproductive proteins evolve in response to sexual selection pressures, how networks of interacting proteins arise and evolve as reproductive phenotypes change remains an open question. Here, we investigated the molecular evolution of the ‘sex peptide network’ of Drosophila melanogaster, a functionally well‐characterized reproductive protein network. In this species, the peptide hormone sex peptide (SP) and its interacting proteins cause major changes in female physiology and behaviour after mating. In contrast, females of more distantly related Drosophila species do not respond to SP. In spite of these phenotypic differences, we detected orthologs of all network proteins across 22 diverse Drosophila species and found evidence that most orthologs likely function in reproduction throughout the genus. Within SP‐responsive species, we detected the recurrent, adaptive evolution of several network proteins, consistent with sexual selection acting to continually refine network function. We also found some evidence for adaptive evolution of several proteins along two specific phylogenetic lineages that correspond with increased expression of the SP receptor in female reproductive tracts or increased sperm length, respectively. Finally, we used gene expression profiling to examine the likely degree of functional conservation of the paralogs of an SP network protein that arose via gene duplication. Our results suggest a dynamic history for the SP network in which network members arose before the onset of robust SP‐mediated responses and then were shaped by both purifying and positive selection.