Speciation genes in free-spawning marine invertebrates

Research on speciation of marine organisms has lagged behind that of terrestrial ones, but the study of the evolution of molecules involved in the adhesion of gametes in free-spawning invertebrates is an exception. Here I review the function, species-specificity, and molecular variation of loci coding for bindin in sea urchins, lysin in abalone and their egg receptors, in an effort to assess the degree to which they contribute to the emergence of reproductive isolation during the speciation process. Bindin is a protein that mediates binding of the sperm to the vitelline envelope (VE) of the egg and the fusion of the gametes' membranes, whereas lysin is a protein involved only in binding to the VE. Both of these molecules are important in species recognition by the gametes, but they rarely constitute absolute blocks to interspecific hybridization. Intraspecific polymorphism is high in bindin, but low in lysin. Polymorphism in bindin is maintained by frequency-dependent selection due to sexual conflict arising from the danger of polyspermy under high densities of sperm. Monomorphism in lysin is the result of purifying selection arising from the need for species recognition. Interspecific divergence in lysin is due to strong positive selection, and the same is true for bindin of four out of seven genera of sea urchins studied to date. The differences between the sea urchin genera in the strength of selection can only partially be explained by the hypothesis of reinforcement. The egg receptor for lysin (VERL) is a glycoprotein with 22 repeats, 20 of which have evolved neutrally and homogenized by concerted evolution, whereas the first two repeats are under positive selection. Selection on lysin has been generated by the need to track changes in VERL, permitted by the redundant structure of this molecule. Both lysin and bindin are important in reproductive isolation, probably had a role in speciation, but it is hard to determine whether they meet the strictest criteria of "speciation loci," defined as genes whose differentiation has caused speciation.     

Duplication and selection on abalone sperm lysin in an allopatric population

While gene duplication is a major source of evolutionary novelty, the importance of this process in reproductive protein evolution has not been widely investigated. Here, we report the first known case of gene duplication of abalone sperm lysin in an allopatric subspecies found in the Eastern Atlantic, Haliotis tuberculata coccinea. Mass spectrometry identified both copies of the lysin protein in testis tissue, and 3-dimensional structural modeling suggests that both proteins remain functional. We also detected positive selection acting on both paralogs after duplication and found evidence of a recent selective sweep. Because H. t. coccinea occurs in geographic isolation from other abalone species, these findings suggest that the evolution of lysin is not driven to create reproductive barriers to unfit hybrid formation with an overlapping species. Instead, sexual selection or sexual conflict acting during abalone fertilization could be responsible for the recent positive selection on this protein. The presence of multiple, rapidly evolving lysin genes in H. tuberculata presents an opportunity to study the early stages of diversification of a protein whose function is well understood.     

Detection and characterization of gamete-specific molecules in Mytilus edulis using selective antibody production

The mussel Mytilus edulis can be used as model to study the molecular basis of reproductive isolation because this species maintains its species integrity, despite of hybridizing in zones of contact with the closely related species M. trossulus or M. galloprovincialis. This study uses selective antibody production by means of hybridoma technology to identify molecules which are involved in sperm function of M. edulis. Fragmented sperm were injected into mice and 25 hybridoma cell clones were established to obtain monoclonal antibodies (mAb). Five clones were identified producing mAb targeting molecules putatively involved in sperm function based on enzyme immunoassays, dot and Western blotting as well as immunostaining of tissue sections. Specific localization of these mAb targets on sperm and partly also in somatic tissue suggests that all five antibodies bind to different molecules. The targets of the mAb obtained from clone G26-AG8 were identified using mass spectrometry (nano-LC-ESI-MS/MS) as M6 and M7 lysin. These acrosomal proteins have egg vitelline lyses function and are highly similar (76%) which explains the cross reactivity of mAb G26-AG8. Furthermore, M7 lysin was recently shown to be under strong positive selection suggesting a role in interspecific reproductive isolation. This study shows that M6 and M7 lysin are not only found in the sperm acrosome but also in male somatic tissue of the mantle and the posterior adductor muscle, while being completely absent in females. The monoclonal antibody G26-AG8 described here will allow elucidating M7/M6 lysin function in somatic and gonad tissue of adult and developing animals.     

Polymorphism in abalone fertilization proteins is consistent with the neutral evolution of the egg's receptor for lysin (VERL) and positive darwinian selection of sperm lysin

The evolution of species-specific fertilization in free-spawning marine invertebrates is important for reproductive isolation and may contribute to speciation. The biochemistry and evolution of proteins mediating species-specific fertilization have been extensively studied in the abalone (genus Haliotis). The nonenzymatic sperm protein lysin creates a hole in the egg vitelline envelope by species-specifically binding to its egg receptor, VERL. The divergence of lysin is promoted by positive Darwinian selection. In contrast, the evolution of VERL does not depart from neutrality. Here, we cloned a novel nonrepetitive region of VERL and performed an intraspecific polymorphism survey for red (Haliotis rufescens) and pink (Haliotis corrugata) abalones to explore the evolutionary forces affecting VERL. Six statistical tests showed that the evolution of VERL did not depart from neutrality. Interestingly, there was a subdivision in the VERL sequences in the pink abalone and a lack of heterozygous individuals between groups, suggesting that the evolution of assortative mating may be in progress. These results are consistent with a model which posits that egg VERL is neutrally evolving, perhaps due to its repetitive structure, while sperm lysin is subjected to positive Darwinian selection to maintain efficient interaction of the two proteins during sperm competition.     

Positive selection is a general phenomenon in the evolution of abalone sperm lysin

Lysin is a 16kDa acrosomal protein used by abalone sperm to create a hole in the egg vitelline envelope (VE). The interaction of lysin with the VE is species-selective and is one step in the multistep fertilization process that restricts heterospecific (cross-species) fertilization. For this reason, the evolution of lysin could play a role in establishing prezygotic reproductive isolation between species. Previously, we sequenced sperm lysin cDNAs from seven California abalone species and showed that positive Darwinian selection promotes their divergence. In this paper an additional 13 lysin sequences are presented representing species from Japan, Taiwan, Australia, New Zealand, South Africa, and Europe. The total of 20 sequences represents the most extensive analysis of a fertilization protein to date. The phylogenetic analysis divides the sequences into two major clades, one composed of species from the northern Pacific (California and Japan) and the other composed of species from other parts of the world. Analysis of nucleotide substitution demonstrates that positive selection is a general process in the evolution of this fertilization protein. Analysis of nucleotide and codon usage bias shows that neither parameter can account for the robust data supporting positive selection. The selection pressure responsible for the positive selection on lysin remains unknown.      

The crystal structure of a fusagenic sperm protein reveals extreme surface properties

Sp18 is an 18 kDa protein that is released from abalone sperm during the acrosome reaction. It coats the acrosomal process where it is thought to mediate fusion between sperm and egg cell membranes. Sp18 is evolutionarily related to lysin, a 16 kDa abalone sperm protein that dissolves the vitelline envelope surrounding the egg. The two proteins were generated by gene duplication followed by rapid divergence by positive selection. Here, we present the crystal structure of green abalone sp18 resolved to 1.86 A. Sp18 is composed of a bundle of five alpha-helices with surface clusters of basic and hydrophobic residues, giving it a large dipole moment and making it extremely amphipathic. The large clusters of hydrophobic surface residues and domains of high positive electrostatic surface charge explain sp18's ability as a potent fusagen of liposomes. The overall fold of sp18 is similar to that of green abalone lysin; however, the surface features of the proteins are quite different, accounting for their different roles in fertilization. This is the first crystal structure of a protein implicated in sperm-egg fusion during animal fertilization.     

Purification and immunocytochemical localization of the vitelline coat lysin of abalone spermatozoa

Spermatozoa of the abalone Haliotis discus were treated with high-calcium seawater to induce the acrosome reaction. The soluble components released from the sperm acrosomal vesicles showed potent lytic activity on the egg vitelline coat. A vitelline coat lysin was purified by salting-in, preparative polyacrylamide gel electrophoresis, and high-performance liquid chromatography. Its molecular weight was 15,500 and its isoelectric point 9.6. These properties were similar to those of other molluskan vitelline coat lysins. The lysin was immunocytochemically localized using a protein A-gold technique, in the posterior half of the acrosomal vesicle.     

鲍配子识别蛋白的研究

配子相互作用的生化机制对于进一步阐明生殖过程具有重要作用,它是深入了解细胞内识别的理想体系。精卵细胞相互作用包括一系列的步骤,开始于精子与卵细胞外被的接触,终止于两性细胞的融合及精子核进入卵细胞质中,而精卵细胞的识别具有建立于各自性细胞表面成分基础上的种的特异性,鲍则是研究精卵识别的好材料。鲍精子在发生顶体反应时释放出两种蛋白质——细胞溶素(1ysin)和18ku糖蛋白(spl8),其中的细胞溶素与其卵黄膜上的受体紧密结合,并利用非酶反应在卵黄膜上穿一个小孔,整个精子则从此孔穿过卵黄膜与卵细胞融合;spl8释放后则覆盖到精子细胞膜表面,起到溶解卵细胞脂质体的作用,即spl8介导精、卵细胞膜的融合。鲍卵细胞膜上存在细胞溶素受体,它是大的不分支的糖蛋白分子,占据了卵黄膜30％的组分,可以专一性地与细胞溶素相结合。这些配子识别蛋白共同进化且速度很快,其中细胞溶素和18ku糖蛋白通过正向选择进化,而细胞溶素受体进行协同进化。     

Positive selection on an acrosomal sperm protein,M7 lysin,in three species of the mussel genus Mytilus

Marine invertebrate sperm proteins are particularly interesting because they are characterized by positive selection and are likely to be involved in prezyogotic isolation and, thus, speciation. Here, we present the first survey of interspecific and intraspecific variation of a bivalve sperm protein among a group of species that regularly hybridize in nature. M7 lysin is found in sperm acrosomes of mussels and dissolves the egg vitelline coat, permitting fertilization. We sequenced multiple alleles of the mature protein-coding region of M7 lysin from allopatric populations of mussels in the Mytilus edulis species group (M. edulis, M. galloprovincialis, and M. trossulus). A significant McDonald-Kreitman test showed an excess of fixed amino acid replacing substitutions between species, consistent with positive selection. In addition, Kolmogorov-Smirnov tests showed significant heterogeneity in polymorphism to divergence ratios for both synonymous variation and combined synonymous and nonsynonymous variation within M. galloprovincialis. These results indicate that there has been adaptive evolution at M7 lysin and, furthermore, show that positive selection on sperm proteins can occur even when postzygotic reproductive isolation is incomplete.     

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.     

The high resolution crystal structure of green abalone sperm lysin: implications for species-specific binding of the egg receptor

Abalone sperm lysin is a 16 kDa acrosomal protein used by sperm to create a hole in the egg vitelline envelope. Lysins from seven California abalone exhibit species-specificity in binding to their egg receptor, and range in sequence identity from 63 % to 90 %. The crystal structure of the sperm lysin dimer from Haliotis fulgens (green abalone) has been determined to 1.71 A by multiple isomorphous replacement. Comparisons with the structure of the lysin dimer from Haliotis rufescens (red abalone) reveal a similar overall fold and conservation of features contributing to lysin's amphipathic character. The two structures do, however, exhibit differences in surface residues and electrostatics. A large clustering of non-conserved surface residues around the waist and clefts of the dimer, and differences in charged residues around these regions, indicate areas of the molecule which may be involved in species-specific egg recognition.     

Coevolution of Interacting Fertilization Proteins

Reproductive proteins are among the fastest evolving in the proteome, often due to the consequences of positive selection, and their rapid evolution is frequently attributed to a coevolutionary process between interacting female and male proteins. Such a process could leave characteristic signatures at coevolving genes. One signature of coevolution, predicted by sexual selection theory, is an association of alleles between the two genes. Another predicted signature is a correlation of evolutionary rates during divergence due to compensatory evolution. We studied female–male coevolution in the abalone by resequencing sperm lysin and its interacting egg coat protein, VERL, in populations of two species. As predicted, we found intergenic linkage disequilibrium between lysin and VERL, despite our demonstration that they are not physically linked. This finding supports a central prediction of sexual selection using actual genotypes, that of an association between a male trait and its female preference locus. We also created a novel likelihood method to show that lysin and VERL have experienced correlated rates of evolution. These two signatures of coevolution can provide statistical rigor to hypotheses of coevolution and could be exploited for identifying coevolving proteins a priori. We also present polymorphism-based evidence for positive selection and implicate recent selective events at the specific structural regions of lysin and VERL responsible for their species-specific interaction. Finally, we observed deep subdivision between VERL alleles in one species, which matches a theoretical prediction of sexual conflict. Thus, abalone fertilization proteins illustrate how coevolution can lead to reproductive barriers and potentially drive 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.     

Crystal structure and subunit dynamics of the abalone sperm lysin dimer: egg envelopes dissociate dimers, the monomer is the active species

Lysin is a 16-kD acrosomal protein used by abalone spermatozoa to create a hole in the egg vitelline envelope (VE) by a nonenzymatic mechanism. The crystal structure of the lysin monomer is known at 1.9 A resolution. The surface of the molecule reveals two tracks of basic residues running the length of one surface of the molecule and a patch of solvent-exposed hydrophobic residues on the opposite surface. Here we report that lysin dimerizes via interaction of the hydrophobic patches of monomers. Triton X-100 dissociates the dimer. The crystal structure of the dimer is described at 2.75 A resolution. Fluorescence energy transfer experiments show that the dimer has an approximate KD of 1 microM and that monomers exchange rapidly between dimers. Addition of isolated egg VE dissociates dimers, implicating monomers as the active species in the dissolution reaction. This work represents the first step in the elucidation of the mechanism by which lysin enables abalone spermatozoa to create a hole in the egg envelope during fertilization.     

Forensic genetic identification of abalone (<Emphasis Type="Italic">Haliotis</Emphasis> spp.) of the northeastern Pacific Ocean

International trade in abalone meats, exclusive of their shells, is a lucrative business based upon both legally and illegally harvested abalone from many jurisdictions. The inability to visually identify abalone meat to species in the absence of the shell impedes enforcement efforts to reduce the illegal exploitation of the world’s abalone resources. We describe species-specific DNA sequences for the gamete recognition proteins, sperm lysin and vitelline egg receptor for lysin, and their use in forensic species identification among abalone of the northeastern Pacific Ocean. Some commercially relevant abalone species from the Northern and Southern hemispheres can be differentiated on the basis of the length of the second intron of the sperm lysin gene. The seven North American species of abalone that occupy the waters of Mexico, the USA and Canada can be distinguished based on sequence differentiation in the first three repeats of the vitelline receptor gene.     

Bindin from a sea star

SUMMARY The genetic basis for the evolution of development includes genes that encode proteins expressed on the surfaces of sperm and eggs. Previous studies of the sperm acrosomal protein bindin have helped to characterize the adaptive evolution of gamete compatibility and speciation in sea urchins. The absence of evidence for bindin expression in taxa other than the Echinoidea has limited such studies to sea urchins, and led to the suggestion that bindin might be a sea urchin-specific molecule. Here we characterize the gene that encodes bindin in a broadcast-spawning asterinid sea star ( Patiria miniata ). We describe the sequence and domain structure of a full-length bindin cDNA and its single intron. In comparison with sea urchins, P. miniata bindin is larger but the two molecules share several general features of their domain structure and some sequence features of two domains. Our results extend the known evolutionary history of bindin from the Mesozoic (among the crown group sea urchins) into the early Paleozoic (and the common ancestor of eleutherozoans), and present new opportunities for understanding the role of bindin molecular evolution in sexual selection, life history evolution, and speciation among sea stars.     

Inbreeding shapes the evolution of marine invertebrates

Inbreeding is a potent evolutionary force shaping the distribution of genetic variation within and among populations of plants and animals. Yet, our understanding of the forces shaping the expression and evolution of nonrandom mating in general, and inbreeding in particular, remains remarkably incomplete. Most research on plant mating systems focuses on self-fertilization and its consequences for automatic selection, inbreeding depression, purging, and reproductive assurance, whereas studies of animal mating systems have often assumed that inbreeding is rare, and that natural selection favors traits that promote outbreeding. Given that many sessile and sedentary marine invertebrates and marine macroalgae share key life history features with seed plants (e.g., low mobility, modular construction, and the release of gametes into the environment), their mating systems may be similar. Here, we show that published estimates of inbreeding coefficients (F_IS) for sessile and sedentary marine organisms are similar and at least as high as noted in terrestrial seed plants. We also found that variation in F_IS within invertebrates is related to the potential to self-fertilize, disperse, and choose mates. The similarity of F_IS for these organismal groups suggests that inbreeding could play a larger role in the evolution of sessile and sedentary marine organisms than is currently recognized. Specifically, associations between traits of marine invertebrates and F_IS suggest that inbreeding could drive evolutionary transitions between hermaphroditism and separate sexes, direct development and multiphasic life cycles, and external and internal fertilization.     

Structural features of the abalone egg extracellular matrix and its role in gamete interaction during fertilization

Abalone eggs are surrounded by a complex extracellular coat that contains three distinct elements: the jelly layer, the vitelline envelope, and the egg surface coat. In this study we used light and electron microscopy to describe these three elements in the red abalone (Haliotis rufescens) and ascribe function to each based on their interactions with sperm. The jelly coat is a spongy matrix that lies at the outermost margin of the egg and consists of variably sized fibers. Sperm pass through this layer with their acrosomes intact and then go on to bind to the vitelline envelope. The vitelline envelope is a multilamellar fibrous layer that appears to trigger the acrosome reaction after sperm binding. Next, sperm release lysin from their acrosomal granules, a nonenzymatic protein that dissolves a hole in the vitelline envelope through which the sperm swims. Sperm then contact the egg surface coat, a network of uniformly sized filaments lying directly above the egg plasma membrane. This layer mediates attachment of sperm, via their acrosomal process, to the egg surface. © 1995 Wiley-Liss, Inc.     

The Tegula tango: a coevolutionary dance of interacting, positively selected sperm and egg proteins

Reproductive proteins commonly show signs of rapid divergence driven by positive selection. The mechanisms driving these changes have remained ambiguous in part because interacting male and female proteins have rarely been examined. We isolate an egg protein the vitelline envelope receptor for lysin (VERL) from Tegula, a genus of free-spawning marine snails. Like VERL from abalone, Tegula VERL is a major component of the VE surrounding the egg, includes a conserved zona pellucida (ZP) domain at its C-terminus, and possesses a unique, negatively charged domain of about 150 amino acids implicated in interactions with the positively charged lysin. Unlike for abalone VERL, where this unique VERL domain occurs in a tandem array of 22 repeats, Tegula VERL has just one such domain. Interspecific comparisons show that both lysin and the VERL domain diverge via positive selection, whereas the ZP domain evolves neutrally. Rates of nonsynonymous substitution are correlated between lysin and the VERL domain, consistent with sexual antagonism, although lineage-specific effects, perhaps owing to different ecologies, may alter the relative evolutionary rates of sperm- and egg-borne proteins.