Adaptive evolution of M3 lysin--a candidate gamete recognition protein in the Mytilus edulis species complex.

Marine invertebrate gamete recognition proteins (GRPs) are classic examples of rapid adaptive evolution of reproductive proteins, and hybridizing Mytilus blue mussels allow us to study the evolution of GRPs during speciation following secondary contact. Even with frequent hybridization, positive selection drives divergence of M7 lysin, one of the three Mytilus egg vitelline envelope (VE) lysins. Mytilus trossulus and M. edulis form a broad hybrid zone in the Canadian Maritimes and eastern Maine, isolated by strong (but partial) gamete incompatibility. M7 lysin, however, is an unlikely GRP controlling this gametic incompativility, as earlier studies showed either weak or no positive selection and extensive introgression between the two species. We used reverse transcriptase-polymerase chain reaction and cloned several alleles of M3 lysin, a potent VE lysin encoded by a nonhomologous gene whose evolution has not been studied. McDonald-Kreitman and HKA tests reveal strong positive selection, which PAML branch-site models detect in 19.7% of the codons. Protein structure predictions show that replacements map exclusively to one face of the carbohydrate recognition domain (CRD) of this C-type lectin, with codons under positive selection localizing to CRD regions known to control ligand specificity. Polymorphism/divergence analyses show that selective sweep has purged M. edulis but not M. trossulus of polymorphism, and unique to M3 is an absence of fixed substitutions and broad haplotype sharing between M. edulis and Mediterranean M. galloprovincialis. Taken together, these results suggest that different lysins serve as GRPs in different Mytilus hybrid zones, with M3 likely co-opted to play this role in the western Atlantic.

A comparison of survey method efficiencies for estimating densities of zebra mussels (Dreissena polymorpha).

Abundance surveys are commonly used to estimate plant or animal densities and frequently require estimating detection probabilities to account for imperfect detection. The estimation of detection probabilities requires additional measurements that take time, potentially reducing the efficiency of the survey when applied to high-density populations. We conducted quadrat, removal, and distance surveys of zebra mussels (Dreissena polymorpha) in three central Minnesota lakes and determined how much survey effort would be required to achieve a pre-specified level of precision for each abundance estimator, allowing us to directly compare survey design efficiencies across a range of conditions. We found that the required sampling effort needed to achieve our precision goal depended on both the survey design and population density. At low densities, survey designs that could cover large areas but with lower detection probabilities, such as distance surveys, were more efficient (i.e., required less sampling effort to achieve the same level of precision). However, at high densities, quadrat surveys, which tend to cover less area but with high detection rates, were more efficient. These results demonstrate that the best survey design is likely to be context-specific, requiring some prior knowledge of the underlying population density and the cost/time needed to collect additional information for estimating detection probabilities.

Connectivity of Caribbean coral populations: complementary insights from empirical and modelled gene flow.

Understanding patterns of connectivity among populations of marine organisms is essential for the development of realistic, spatially explicit models of population dynamics. Two approaches, empirical genetic patterns and oceanographic dispersal modelling, have been used to estimate levels of evolutionary connectivity among marine populations but rarely have their potentially complementary insights been combined. Here, a spatially realistic Lagrangian model of larval dispersal and a theoretical genetic model are integrated with the most extensive study of gene flow in a Caribbean marine organism. The 871 genets collected from 26 sites spread over the wider Caribbean subsampled 45.8% of the 1900 potential unique genets in the model. At a coarse scale, significant consensus between modelled estimates of genetic structure and empirical genetic data for populations of the reef-building coral Montastraea annularis is observed. However, modelled and empirical data differ in their estimates of connectivity among northern Mesoamerican reefs indicating that processes other than dispersal may dominate here. Further, the geographic location and porosity of the previously described east-west barrier to gene flow in the Caribbean is refined. A multi-prong approach, integrating genetic data and spatially realistic models of larval dispersal and genetic projection, provides complementary insights into the processes underpinning population connectivity in marine invertebrates on evolutionary timescales.

The genome of the zebra mussel, Dreissena polymorpha: a resource for comparative genomics, invasion genetics, and biocontrol.

The zebra mussel, Dreissena polymorpha, continues to spread from its native range in Eurasia to Europe and North America, causing billions of dollars in damage and dramatically altering invaded aquatic ecosystems. Despite these impacts, there are few genomic resources for Dreissena or related bivalves. Although the D. polymorpha genome is highly repetitive, we have used a combination of long-read sequencing and Hi-C-based scaffolding to generate a high-quality chromosome-scale genome assembly. Through comparative analysis and transcriptomics experiments, we have gained insights into processes that likely control the invasive success of zebra mussels, including shell formation, synthesis of byssal threads, and thermal tolerance. We identified multiple intact steamer-like elements, a retrotransposon that has been linked to transmissible cancer in marine clams. We also found that D. polymorpha have an unusual 67 kb mitochondrial genome containing numerous tandem repeats, making it the largest observed in Eumetazoa. Together these findings create a rich resource for invasive species research and control efforts.

AFLP fingerprinting shows that a single Prymnesium parvum harmful algal bloom consists of multiple clones.

Due to slow rates of molecular evolution, DNA sequences used to identify and build phylogenies of algal species involved in harmful algal blooms (HABs) are generally invariant at the intraspecific level. This means that it is unknown whether HAB events result from the growth of a single clone, a few dominant clones, or multiple clones. This is true despite the fact that several physiological and demographic traits, as well as toxicity, are known to vary across clones. We generated AFLP fingerprints from a set of 6 clonal isolates, taken from a bloom of Prymnesium parvum at a striped bass mariculture facility. This new haptophyte bloom was recently implicated in fish kills at several sites in the United States. The AFLP fragments were highly reproducible and showed that all isolates were distinguishable due to abundant AFLPs unique to single isolates. These results demonstrate that blooms can be genetically diverse outbreaks and indicate that AFLP can be a powerful molecular tool for characterizing and monitoring this diversity.

Structure, function and parallel evolution of the bivalve byssus, with insights from proteomes and the zebra mussel genome.

The byssus is a structure unique to bivalves. Byssal threads composed of many proteins extend like tendons from muscle cells, ending in adhesive pads that attach underwater. Crucial to settlement and metamorphosis, larvae of virtually all species are byssate. By contrast, in adults, the byssus is scattered throughout bivalves, where it has had profound effects on morphological evolution and been key to adaptive radiations of epifaunal species. I compare byssus structure and proteins in blue mussels (Mytilus), by far the best characterized, to zebra mussels (Dreissena polymorpha), in which several byssal proteins have been isolated and sequenced. By mapping the adult byssus onto a recent phylogenomic tree, I confirm its independent evolution in these and other lineages, likely parallelisms with common origins in development. While the byssus is superficially similar in Dreissena and Mytilus, in finer detail it is not, and byssal proteins are dramatically different. I used the chromosome-scale D. polymorpha genome we recently assembled to search for byssal genes and found 37 byssal loci on 10 of the 16 chromosomes. Most byssal genes are in small families, with several amino acid substitutions between paralogs. Byssal proteins of zebra mussels and related quagga mussels (D. rostriformis) are divergent, suggesting rapid evolution typical of proteins with repetitive low complexity domains. Opportunities abound for proteomic and genomic work to further our understanding of this textbook example of a marine natural material. A priority should be invasive bivalves, given the role of byssal attachment in the spread of, and ecological and economic damage caused by zebra mussels, quagga mussels and others. This article is part of the Theo Murphy meeting issue 'Molluscan genomics: broad insights and future directions for a neglected phylum'.

Extraordinary AFLP fingerprint similarity despite strong assortative mating between reef fish color morphospecies.

Recent theoretical models and empirical studies of fruit flies, birds, and fish indicate that assortative mating may initiate speciation when physical barriers to gene flow are absent, and before postzygotic barriers evolve. These are important results for marine animals like coral reef fish, where ocean currents can carry planktonic larvae over broad ranges, interconnecting populations and slowing genetic divergence. The Caribbean hamlets (genus Hypoplectrus) are a flock of reef fish morphospecies with highly distinct color pattern that mate like with like, but show little mitochondrial or microsatellite DNA differentiation. Here, we broadly screen genomic diversity using amplified fragment length polymorphisms (AFLP) and survey mating pair formation between two morphospecies in the Florida Keys, the butter hamlet (H. unicolor) and the blue hamlet (H. gemma). No AFLP was species-diagnostic (fixed), and neighbor-joining analyses revealed no clustering of individuals consistent with morphospecies boundaries. Assignment tests, however, placed most individuals within their morphospecies of origin. Field surveys showed that > 98% of mating pairs, including those of rare morphospecies, were of like color pattern. Spawning by a single mixed pair adds to earlier observations suggesting that infrequent hybridization may be a genetically homogenizing force in Hypoplectrus. This study provides a clear example of strong assortative mating in a system with limited genetic differentiation.

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.

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.

Across-shelf distribution of blue mussel larvae in the northern Gulf of Maine: consequences for population connectivity and a species range boundary.

Studies of population connectivity have largely focused on along-shelf, as opposed to across-shelf, processes. We hypothesized that a discontinuity in across-shelf mixing caused by the divergence of the Eastern Maine Coastal Current (EMCC) from shore acts as an ecological barrier to the supply of mussel larvae to the coast. Existing data on the relative abundance of two congeneric blue mussels, Mytilus edulis and M. trossulus, were analysed to quantify the association of M. trossulus with the colder temperature signal of the EMCC and generate larval distribution predictions. We then sampled the across-shelf distribution of larvae along two transects during 2011. Larvae were identified using restriction digests of PCR amplicons from the mitochondrial 16S rDNA. Mytilus edulis larvae were consistently abundant on either the inshore and offshore transect ends, but not homogeneously distributed across the shelf, while M. trossulus larvae were less common throughout the study area. The divergence of the EMCC from shore appears to create a break in the connectivity of M. edulis populations by isolating those inshore of the EMCC from upstream larval sources. Across-shelf transport processes can thus produce connectivity patterns that would not be predicted solely on the basis of along-shelf processes.

Quantitative analysis of gametic incompatibility between closely related species of neotropical sea urchins.

Species of the sea urchin genus Echinometra found on the two coasts of Panamá are recently diverged and only partially isolated by incomplete barriers to interspecific fertilization. This study confirms previous work that revealed incompatibility between the eggs of the Atlantic E. lucunter and the sperm of the other two neotropical species, whereas eggs of its sympatric congener E. viridis and allopatric E. vanbrunti are largely compatible with heterospecific sperm. Here we quantify fertilization using a range of sperm dilutions. We demonstrate a much stronger block to cross-species fertilization of E. lucunter eggs than was previously shown at fixed sperm concentrations, and mild incompatibility of the other two species' eggs where previous crosses between species were not distinguishable from within-species controls. Additionally, we present evidence for intraspecific variation in egg receptivity towards heterospecific sperm. Our findings here again discount the "reinforcement model" as a viable explanation for the pattern of prezygotic isolation. Gamete incompatibility in these Echinometra has appeared recently-within the last 1.5 million years-but is weaker in sympatry than in allopatry. Accidents of history may help explain why incompatibility of eggs emerged in one species and not in others. Compensatory sexual selection on sperm in this species could follow, and promote divergence of proteins mediating sperm-egg recognition.

Conspecific sperm precedence is a reproductive barrier between free-spawning marine mussels in the northwest atlantic mytilus hybrid zone.

Reproductive isolation at the gamete stage has become a focus of speciation research because of its potential to evolve rapidly between closely related species. Conspecific sperm precedence (CSP), a type of gametic isolation, has been demonstrated in a number of taxa, both marine and terrestrial, with the potential to play an important role in speciation. Free-spawning marine invertebrates are ideal subjects for the study of CSP because of a likely central role for gametic barriers in reproductive isolation. The western Atlantic Mytilus blue mussel hybrid zone, ranging from the Atlantic Canada to eastern Maine, exhibits characteristics conducive to the study of CSP. Previous studies have shown that gametic incompatibility is incomplete, variable in strength and the genotype distribution is bimodal-dominated by the parental species, with a low frequency of hybrids. We conducted gamete crossing experiments using M. trossulus and M. edulis individuals collected from natural populations during the spring spawning season in order to detect the presence or absence of CSP within this hybrid zone. We detected CSP, defined here as a reduction in heterospecific offspring from competitive fertilizations in vitro compared to that seen in non-competitive fertilizations, in five of the twelve crosses in which conspecific crosses were detectable. This is the first finding of CSP in a naturally hybridizing population of a free-spawning marine invertebrate. Our findings support earlier predictions that CSP can promote assortative fertilization in bimodal hybrid zones, further advancing their hypothesized progression towards full speciation. Despite strong CSP numerous heterospecific fertilizations remain, reinforcing the hypothesis that compatible females are a source of hybrid offspring in mixed natural spawns.

Across-shelf transport of bivalve larvae: can the interface between a coastal current and inshore waters act as an ecological barrier to larval dispersal?

Using an integrated physical and biological approach, we examined across-shelf advection and exchange and the associated transport of bivalve larvae in the presence of a strong coastal current separated from the coast by a stratified inshore environment. We tested the hypothesis that the interface of the coastal current and inshore waters can act as an ecological barrier to across-shelf transport of larvae but can be overcome by wind- or tidally-induced transport. Our study region in the Gulf of Maine encompasses a coastal current that diverges from the coast as it moves downshelf. The region inshore of this current is home to several species that exhibit limited recruitment in spite of extensive upshelf larval sources. Analysis of surface water temperatures and wind velocities revealed episodic decreases in temperature along the coast correlated with alongshelf (but not upwelling) winds, indicating wind-forced onshore movement of the cold coastal current. Such wind-driven onshore migrations are more common along the northern portion of the study region where the coastal current is near the coast, tidal currents are strong, and wind directions are more conducive to onshore migration, but rarer further south where the interface between inshore waters and the coastal current is further offshore and suitable wind events are less common. The distribution of bivalve larvae was consistent with the physical measurements. There was little across-shelf variation in larval abundance where the current abuts the coast, indicating strong across-shelf exchange of larvae, but strong across-shelf variation in larval density where the stratified inshore waters separate the current from the coast, indicating weak across-shelf transport of larvae. Our results suggest that the interface between the coastal current and inshore waters may constitute a major ecological barrier to larval dispersal in the southern part of the region that may only be overcome by rare, strong wind-forced events.

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.

Identifying exoskeleton proteins in the blue crab from an expressed sequence tag (EST) library.

A blue crab (Callinectes sapidus) expressed sequence tag project was designed for multiple purposes including discovery of genes for cuticular (exoskeletal) proteins, some of which may regulate mineralization. One of the expression libraries sequenced was from the hypodermis (the epithelium depositing the cuticle). RNAs used for cDNA synthesis were pooled from arthrodial and mid-dorsal hypodermis at both pre-ecdysis and post-ecdysis. This ensured representation from both calcifying and non-calcifying regions and from layers of cuticle deposited both before and after ecdysis. The EST database was mined for cuticular protein sequences in three ways. First, we searched for sequences coding for known cuticle-specific motifs like the Rebers-Riddiford chitin-binding sequence and a motif known only from proteins extracted from mineralized exoskeletons of other decapods. Second, we checked the associated annotations in the EST project for similarity to known cuticular proteins, often from insects. Third, BLAST was used to search the EST data for significant homology to published cuticular protein sequences from other crustaceans. In all, the database contains at least 73 contigs or singlets representing transcripts of cuticular proteins. Forty-five of these distribute among ten clusters of very similar transcripts, possibly representing alternative splicing or recent gene duplications. The rest share less similarity. We have obtained complete sequences for 25 of the transcripts, have produced phylogenetics trees comparing them with similar proteins from insects and other crustaceans, and have determined expression patterns across the molt in calcifying versus non-calcifying cuticle. The combination of homology analysis and gene expression analysis allows us to infer putative functions in cuticle synthesis and calcification.

SEX ALLOCATION AND MALE FITNESS GAIN IN A COLONIAL, HERMAPHRODITIC MARINE INVERTEBRATE.

While simultaneous hermaphroditism occurs in most animal phyla, theories for its adaptive significance remain untested. Sex allocation theory predicts that combined sexes are favored in sedentary and sessile organisms because localized gamete dispersal and local mate competition (LMC) among gametes promote decelerating fitness "gain curves" that relate male investment to reproductive success. Under this LMC model, males fertilize all locally available eggs at low sperm output, additional output leads to proportionally fewer fertilizations, and combined sexes with female-biased sex allocation are favored. Decelerating male gain curves have been found in hermaphroditic flowering plants, but the present paper reports the first analysis in an animal. The colonial hermaphroditic bryozoan Celleporella hyalina forms unisexual male and female zooids that can be counted to estimate absolute and relative gender allocations. I placed "sperm donor" colonies-each with different numbers of male zooids, and each homozygous for diagnostic allozyme alleles-among target maternal colonies on field mating arrays, and estimated donor fertilization success by scoring allozyme markers in target-colony progeny. Fertilization success increased with numbers of donor male zooids, but linear and not decelerating curves fit the data best. Mean sex allocation was not female biased, consistent with nondecelerating male gain. Sperm donors, moreover, did not monopolize matings as expected under high LMC, but rather shared paternity with rival colonies. Hence localized water-borne gamete dispersal alone may not yield decelerating male gain and favor the maintenance of hermaphroditism; relaxed sperm competition in low density populations might also be required. In free-spawning marine organisms, males cannot control access to fertilizations, intense sperm competition may be commonplace, and high male sex allocation may be selected to enhance siring success under competition.

Adaptive evolution of sperm bindin tracks egg incompatibility in neotropical sea urchins of the genus Echinometra.

Bindin is a gamete recognition protein known to control species-specific sperm-egg adhesion and membrane fusion in sea urchins. Previous analyses have shown that diversifying selection on bindin amino acid sequence is found when gametically incompatible species are compared, but not when species are compatible. The present study analyzes bindin polymorphism and divergence in the three closely related species of Echinometra in Central America: E. lucunter and E. viridis from the Caribbean, and E. vanbrunti from the eastern Pacific. The eggs of E. lucunter have evolved a strong block to fertilization by sperm of its neotropical congeners, whereas those of the other two species have not. As in the Indo-West Pacific (IWP) Echinometra, the neotropical species show high intraspecific bindin polymorphism in the same gene regions as in the IWP species. Maximum likelihood analysis shows that many of the polymorphic codon sites are under mild positive selection. Of the fixed amino acid replacements, most have accumulated along the bindin lineage of E. lucunter. We analyzed the data with maximum likelihood models of variation in positive selection across lineages and codon sites, and with models that consider sites and lineages simultaneously. Our results show that positive selection is concentrated along the E. lucunter bindin lineage, and that codon sites with amino acid replacements fixed in this species show by far the highest signal of positive selection. Lineage-specific positive selection paralleling egg incompatibility provides support that adaptive evolution of sperm proteins acts to maintain recognition of bindin by changing egg receptors. Because both egg incompatibility and bindin divergence are greater between allopatric species than between sympatric species, the hypothesis of selection against hybridization (reinforcement) cannot explain why adaptive evolution has been confined to a single lineage in the American Echinometra. Instead, processes acting to varying degrees within species (e.g., sperm competition, sexual selection, and sexual conflict) are more promising explanations for lineage-specific positive selection on bindin.

Genetic mosaic in a marine species flock.

We used molecular approaches to study the status of speciation in coral reef fishes known as hamlets (Serranidae: Hypoplectrus). Several hamlet morphospecies coexist on Caribbean reefs, and mate assortatively with respect to their strikingly distinct colour patterns. We provide evidence that, genetically, the hamlets display characteristics common in species flocks on land and in freshwaters. Substitutions within two mitochondrial DNA (mtDNA) protein-coding genes place hamlets within a monophyletic group relative to members of two related genera (Serranus and Diplectrum), and establish that the hamlet radiation must have been very recent. mtDNA distances separating hamlet morphospecies were slight (0.6 +/- 0.04%), yielding a coalescent estimate for the age of the hamlet flock of approximately 430 000 years. Morphospecies did not sort into distinct mtDNA haplotype phylogroups, and alleles at five hypervariable microsatellite loci were shared broadly across species boundaries. None the less, molecular variation was not distributed at random. Analyses of mtDNA haplotype frequencies and nested clades in haplotype networks revealed significant genetic differences between geographical regions and among colour morphospecies. We also observed significant microsatellite differentiation between geographical regions and in Puerto Rico, among colour morphospecies; the latter providing evidence for reproductive isolation between colour morphospecies at this locale. In our Panama collection, however, colour morphospecies were mostly genetically indistinguishable. This mosaic pattern of DNA differentiation implies a complex interaction between population history, mating behaviour and geography and suggests that porous boundaries separate species in this flock of brilliantly coloured coral reef fishes.