Selection Shapes the Genomic Landscape of Introgressed Ancestry in a Pair of Sympatric Sea Urchin Species.

A growing number of recent studies have demonstrated that introgression is common across the tree of life. However, we still have a limited understanding of the fate and fitness consequence of introgressed variation at the whole-genome scale across diverse taxonomic groups. Here, we implemented a phylogenetic hidden Markov model to identify and characterize introgressed genomic regions in a pair of well-diverged, nonsister sea urchin species: Strongylocentrotus pallidus and Strongylocentrotus droebachiensis. Despite the old age of introgression, a sizable fraction of the genome (1% to 5%) exhibited introgressed ancestry, including numerous genes showing signals of historical positive selection that may represent cases of adaptive introgression. One striking result was the overrepresentation of hyalin genes in the identified introgressed regions despite observing considerable overall evidence of selection against introgression. There was a negative correlation between introgression and chromosome gene density, and two chromosomes were observed with considerably reduced introgression. Relative to the nonintrogressed genome-wide background, introgressed regions had significantly reduced nucleotide divergence (dXY) and overlapped fewer protein-coding genes, coding bases, and genes with a history of positive selection. Additionally, genes residing within introgressed regions showed slower rates of evolution (dN, dS, dN/dS) than random samples of genes without introgressed ancestry. Overall, our findings are consistent with widespread selection against introgressed ancestry across the genome and suggest that slowly evolving, low-divergence genomic regions are more likely to move between species and avoid negative selection following hybridization and introgression.

Extensive introgression among strongylocentrotid sea urchins revealed by phylogenomics.

Gametic isolation is thought to play an important role in the evolution of reproductive isolation in broadcast-spawning marine invertebrates. However, it is unclear whether gametic isolation commonly evolves early in the speciation process or only accumulates after other reproductive barriers are already in place. It is also unknown whether gametic isolation is an effective barrier to introgression following speciation. Here, we used whole-genome sequencing data and multiple complementary phylogenomic approaches to test whether the well-documented gametic incompatibilities among the strongylocentrotid sea urchins have limited introgression. We quantified phylogenetic discordance, inferred reticulate phylogenetic networks, and applied the Δ statistic using gene tree topologies reconstructed from multiple sequence alignments of protein-coding single-copy orthologs. In addition, we conducted ABBA-BABA tests on genome-wide single nucleotide variants and reconstructed a phylogeny of mitochondrial genomes. Our results revealed strong mito-nuclear discordance and considerable nonrandom gene tree discordance that cannot be explained by incomplete lineage sorting alone. Eight of the nine species examined demonstrated a history of introgression with at least one other species or ancestral lineage, indicating that introgression was common during the diversification of the strongylocentrotid urchins. There was strong support for introgression between four extant species pairs (Strongylocentrotus pallidus ⇔ S. droebachiensis, S. intermedius ⇔ S. pallidus, S. purpuratus ⇔ S. fragilis, and Mesocentrotus franciscanus ⇔ Pseudocentrotus depressus) and additional evidence for introgression on internal branches of the phylogeny. Our results suggest that the existing gametic incompatibilities among the strongylocentrotid urchin species have not been a complete barrier to hybridization and introgression following speciation. Their continued divergence in the face of widespread introgression indicates that other reproductive isolating barriers likely exist and may have been more critical in establishing reproductive isolation early in speciation.

Ancestral sequences from an elite neutralizer proximal to the development of neutralization resistance as a potential source of HIV vaccine immunogens.

A major challenge in HIV vaccine development is the identification of immunogens able to elicit broadly neutralizing antibodies (bNAbs). While remarkable progress has been made in the isolation and characterization of bNAbs, the epitopes they recognize appear to be poorly immunogenic. Thus, none of the candidate vaccines developed to date has induced satisfactory levels of neutralizing antibodies to the HIV envelope protein (Env). One approach to the problem of poor immunogenicity is to build vaccines based on envelope (env) genes retrieved from rare individuals termed elite neutralizers (ENs) who at one time possessed specific sequences that stimulated the formation of bNAbs. Env proteins selected from these individuals could possess uncommon, yet to be defined, structural features that enhance the immunogenicity of epitopes recognized by bNAbs. Here we describe the recovery of envs from an EN that developed unusually broad and potent bNAbs. As longitudinal specimens were not available, we combined plasma and provirus sequences acquired from a single time-point to infer a phylogenetic tree. Combining ancestral reconstruction data with virus neutralization data allowed us to sift through the myriad of virus quasi-species that evolved in this individual to identify envelope sequences from the nodes that appeared to define the transition from neutralization sensitive envs to the neutralization resistant envs that occur in EN plasma. Synthetic genes from these nodes were functional in infectivity assays and sensitive to neutralization by bNAbs, and may provide a novel source of immunogens for HIV vaccine development.

Sequence-Based Analysis of Thermal Adaptation and Protein Energy Landscapes in an Invasive Blue Mussel (Mytilus galloprovincialis).

Adaptive responses to thermal stress in poikilotherms plays an important role in determining competitive ability and species distributions. Amino acid substitutions that affect protein stability and modify the thermal optima of orthologous proteins may be particularly important in this context. Here, we examine a set of 2,770 protein-coding genes to determine if proteins in a highly invasive heat tolerant blue mussel (Mytilus galloprovincialis) contain signals of adaptive increases in protein stability relative to orthologs in a more cold tolerant M. trossulus. Such thermal adaptations might help to explain, mechanistically, the success with which the invasive marine mussel M. galloprovincialis has displaced native species in contact zones in the eastern (California) and western (Japan) Pacific. We tested for stabilizing amino acid substitutions in warm tolerant M. galloprovincialis relative to cold tolerant M. trossulus with a generalized linear model that compares in silico estimates of recent changes in protein stability among closely related congeners. Fixed substitutions in M. galloprovincialis were 3,180.0 calories per mol per substitution more stabilizing at genes with both elevated dN/dS ratios and transcriptional responses to heat stress, and 705.8 calories per mol per substitution more stabilizing across all 2,770 loci investigated. Amino acid substitutions concentrated in a small number of genes were more stabilizing in M. galloprovincialis compared with cold tolerant M. trossulus. We also tested for, but did not find, enrichment of a priori GO terms in genes with elevated dN/dS ratios in M. galloprovincialis. This might indicate that selection for thermodynamic stability is generic across all lineages, and suggests that the high change in estimated protein stability that we observed in M. galloprovincialis is driven by selection for extra stabilizing substitutions, rather than by higher incidence of selection in a greater number of genes in this lineage. Nonetheless, our finding of more stabilizing amino acid changes in the warm adapted lineage is important because it suggests that adaption for thermal stability has contributed to M. galloprovincialis' superior tolerance to heat stress, and that pairing tests for positive selection and tests for transcriptional response to heat stress can identify candidates of protein stability adaptation.

Genome-wide signals of positive selection in strongylocentrotid sea urchins.

BACKGROUND: Comparative genomics studies investigating the signals of positive selection among groups of closely related species are still rare and limited in taxonomic breadth. Such studies show great promise in advancing our knowledge about the proportion and the identity of genes experiencing diversifying selection. However, methodological challenges have led to high levels of false positives in past studies. Here, we use the well-annotated genome of the purple sea urchin, Strongylocentrotus purpuratus, as a reference to investigate the signals of positive selection at 6520 single-copy orthologs from nine sea urchin species belonging to the family Strongylocentrotidae paying careful attention to minimizing false positives. RESULTS: We identified 1008 (15.5%) candidate positive selection genes (PSGs). Tests for positive selection along the nine terminal branches of the phylogeny identified 824 genes that showed lineage-specific adaptive diversification (1.67% of branch-sites tests performed). Positively selected codons were not enriched at exon borders or near regions containing missing data, suggesting a limited contribution of false positives caused by alignment or annotation errors. Alignments were validated at 10 loci with re-sequencing using Sanger methods. No differences were observed in the rates of synonymous substitution (d S), GC content, and codon bias between the candidate PSGs and those not showing positive selection. However, the candidate PSGs had 68% higher rates of nonsynonymous substitution (d N) and 33% lower levels of heterozygosity, consistent with selective sweeps and opposite to that expected by a relaxation of selective constraint. Although positive selection was identified at reproductive proteins and innate immunity genes, the strongest signals of adaptive diversification were observed at extracellular matrix proteins, cell adhesion molecules, membrane receptors, and ion channels. Many candidate PSGs have been widely implicated as targets of pathogen binding, inactivation, mimicry, or exploitation in other groups (notably mammals). CONCLUSIONS: Our study confirmed the widespread action of positive selection across sea urchin genomes and allowed us to reject the possibility that annotation and alignment errors (including paralogs) were responsible for creating false signals of adaptive molecular divergence. The candidate PSGs identified in our study represent promising targets for future research into the selective agents responsible for their adaptive diversification and their contribution to speciation.

Studying the genetic basis of speciation in high gene flow marine invertebrates.

A growing number of genes responsible for reproductive incompatibilities between species (barrier loci) exhibit the signals of positive selection. However, the possibility that genes experiencing positive selection diverge early in speciation and commonly cause reproductive incompatibilities has not been systematically investigated on a genome-wide scale. Here, I outline a research program for studying the genetic basis of speciation in broadcast spawning marine invertebrates that uses a priori genome-wide information on a large, unbiased sample of genes tested for positive selection. A targeted sequence capture approach is proposed that scores single-nucleotide polymorphisms (SNPs) in widely separated species populations at an early stage of allopatric divergence. The targeted capture of both coding and non-coding sequences enables SNPs to be characterized at known locations across the genome and at genes with known selective or neutral histories. The neutral coding and non-coding SNPs provide robust background distributions for identifying FST-outliers within genes that can, in principle, identify specific mutations experiencing diversifying selection. If natural hybridization occurs between species, the neutral coding and non-coding SNPs can provide a neutral admixture model for genomic clines analyses aimed at finding genes exhibiting strong blocks to introgression. Strongylocentrotid sea urchins are used as a model system to outline the approach but it can be used for any group that has a complete reference genome available.

Introgression between invasive and native blue mussels (genus Mytilus) in the central California hybrid zone.

The ecological and genetic factors determining the extent of introgression between species in secondary contact zones remain poorly understood. Here, we investigate the relative importance of isolating barriers and the demographic expansion of invasive Mytilus galloprovincialis on the magnitude and the direction of introgression with the native Mytilus trossulus in a hybrid zone in central California. We use double-digest restriction-site-associated DNA sequencing (ddRADseq) to genotype 1337 randomly selected single nucleotide polymorphisms and accurately distinguish early and advanced generation hybrids for the first time in the central California Mytilus spp. hybrid zone. Weak levels of introgression were observed in both directions but were slightly more prevalent from the native M. trossulus into the invasive M. galloprovincialis. Few early and advanced backcrossed individuals were observed across the hybrid zone confirming the presence of strong barriers to interbreeding. Heterogeneous patterns of admixture across the zone of contact were consistent with the colonization history of M. galloprovincialis with more extensive introgression in northern localities furthest away from the putative site of introduction in southern California. These observations reinforce the importance of dynamic spatial and demographic expansions in determining patterns of introgression between close congeners, even in those with high dispersal potential and well-developed reproductive barriers. Our results suggest that the threat posed by invasive M. galloprovincialis is more ecological than genetic as it has displaced, and continues to displace the native M. trossulus from much of central and southern California.

Genome-wide patterns of codon bias are shaped by natural selection in the purple sea urchin, Strongylocentrotus purpuratus.

Codon usage bias has been documented in a wide diversity of species, but the relative contributions of mutational bias and various forms of natural selection remain unclear. Here, we describe for the first time genome-wide patterns of codon bias at 4623 genes in the purple sea urchin, Strongylocentrotus purpuratus. Preferred codons were identified at 18 amino acids that exclusively used G or C at third positions, which contrasted with the strong AT bias of the genome (overall GC content is 36.9%). The GC content of third positions and coding regions exhibited significant correlations with the magnitude of codon bias. In contrast, the GC content of introns and flanking regions was indistinguishable from the genome-wide background, which suggested a limited contribution of mutational bias to synonymous codon usage. Five distinct clusters of genes were identified that had significantly different synonymous codon usage patterns. A significant correlation was observed between codon bias and mRNA expression supporting translational selection, but this relationship was driven by only one highly biased cluster that represented only 8.6% of all genes. In all five clusters preferred codons were evolutionarily conserved to a similar degree despite differences in their synonymous codon usage distributions and magnitude of codon bias. The third positions of preferred codons in two codon usage groups also paired significantly more often in stems than in loops of mRNA secondary structure predictions, which suggested that codon bias might also affect mRNA stability. Our results suggest that mutational bias has played a minor role in determining codon bias in S. purpuratus and that preferred codon usage may be heterogeneous across different genes and subject to different forms of natural selection.

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.

Range-wide genetic homogeneity in the California sea mussel (Mytilus californianus): a comparison of allozymes, nuclear DNA markers, and mitochondrial DNA sequences.

We tested for genetic differentiation among six populations of California sea mussels (Mytilus californianus) sampled across 4000 km of its geographical range by comparing patterns of variation at four independent types of genetic markers: allozymes, single-copy nuclear DNA markers, and DNA sequences from the male and female mitochondrial genomes. Despite our extensive sampling and genotyping efforts, we detected no significant differences among localities and no signal of isolation by distance suggesting that M. californianus is genetically homogeneous throughout its range. This concordance differs from similar studies on other mytilids, especially in the role of postsettlement selection generating differences between exposed coastal and estuarine habitats. To assess if this homogeneity was due to M. californianus not inhabiting estuarine environments, we reviewed studies comparing allozymes with other classes of nuclear DNA markers. Although both types of markers gave broadly consistent results, there was a bias favouring studies in which allozymes were more divergent than DNA markers (nine to three) and a disproportionate number of these cases involved marine taxa (seven). Furthermore, allozymes were significantly more heterogeneous than DNA markers in three of the four studies that sampled coastal and estuarine habitats. We conclude that the genetic uniformity exhibited by M. californianus may result from a combination of extensive gene flow and the lack of exposure to strong selective gradients across its range.

Ice-age survival of Atlantic cod: agreement between palaeoecology models and genetics.

Scant scientific attention has been given to the abundance and distribution of marine biota in the face of the lower sea level, and steeper latitudinal gradient in climate, during the ice-age conditions that have dominated the past million years. Here we examine the glacial persistence of Atlantic cod (Gadus morhua) populations using two ecological-niche-models (ENM) and the first broad synthesis of multi-locus gene sequence data for this species. One ENM uses a maximum entropy approach (Maxent ); the other is a new ENM for Atlantic cod, using ecophysiological parameters based on observed reproductive events rather than adult distribution. Both the ENMs were tested for present-day conditions, then used to hindcast ranges at the last glacial maximum (LGM) ca 21kyr ago, employing climate model data. Although the LGM range of Atlantic cod was much smaller, and fragmented, both the ENMs agreed that populations should have been able to persist in suitable habitat on both sides of the Atlantic. The genetic results showed a degree of trans-Atlantic divergence consistent with genealogically continuous populations on both sides of the North Atlantic since long before the LGM, confirming the ENM results. In contrast, both the ENMs and the genetic data suggest that the Greenland G. morhua population post-dates the LGM.

Molecular population genetics of the male and female mitochondrial DNA molecules of the California sea mussel, Mytilus californianus.

The presence of two gender-associated mitochondrial genomes in marine mussels provides a unique opportunity to investigate the dynamics of mtDNA evolution without complications inherent in interspecific comparisons. Here, we assess the relative importance of selection, mutation, and differential constraint in shaping the patterns of polymorphism within and divergence between the male (M) and female (F) mitochondrial genomes of the California sea mussel, Mytilus californianus. Partial sequences were obtained from homologous regions of four genes (nad2, cox1, atp6, and nad5) totaling 2307 bp in length. The M and F mtDNA molecules of M. californianus exhibited extensive levels of nucleotide polymorphism and were more highly diverged than observed in other mytilids (overall Tamura-Nei distances >40%). Consistent with previous studies, the M molecule had significantly higher levels of silent and replacement polymorphism relative to F. Both genomes possessed large numbers of singleton and low-frequency mutations that gave rise to significantly negative Tajima's D values. Mutation-rate scalars estimated for silent and replacement mutations were elevated in the M genome but were not sufficient to account for its higher level of polymorphism. McDonald-Kreitman tests were highly significant at all loci due to excess numbers of fixed replacement mutations between molecules. Strong purifying selection was evident in both genomes in keeping the majority of replacement mutations at low population frequencies but appeared to be slightly relaxed in M. Our results suggest that a reduction in selective constraint acting on the M genome remains the best explanation for its greater levels of polymorphism and faster rate of evolution.

Positive Darwinian selection at the pantophysin (Pan I) locus in marine gadid fishes.

Maximum-likelihood models of codon substitution were used to test for positive Darwinian selection at the vesicle protein pantophysin in two allelic lineages segregating in the Atlantic cod Gadus morhua and in 18 related species of marine gadid fishes. Positive selection was detected in the two intravesicular loops of the integral membrane protein but not in four membrane-spanning regions or the 3' cytoplasmic tail. The proportion of positively selected sites (24.9%) and the mean nonsynonymous/synonymous rate ratio (omega = d(N)/d(S) = 5.35) were both greater in the first intravesicular (IV1) domain compared with the second intravesicular (IV2) domain (11.0% positively selected sites with mean omega = 3.76). Likelihood ratio tests comparing models that assume identical omega ratios along all branches of the phylogeny to those that allow omega ratios to vary among lineages were not significant for either the IV1 or IV2 domains, indicating that the selective pressures favoring amino acid replacements have operated consistently in both regions during the diversification of the group. Positive selection was observed in the IV1 domain in both G. morhua allelic lineages, and, although three of the four codons that differ between alleles were targets of positive selection in the broader group, no similar polymorphisms were detected in other taxa. The two G. morhua Pan I alleles appeared to have evolved before the speciation event separating it from its sister taxon, Theragra chalcogramma, and on the basis of a standard mtDNA clock are estimated to be at least 2 Myr old. Although the function of pantophysin remains unknown, the strong signal of positive selection at specific sites in the IV1 and IV2 domains may help clarify its role in cellular trafficking pathways.

Natural selection and the genetic differentiation of coastal and Arctic populations of the Atlantic cod in northern Norway: a test involving nucleotide sequence variation at the pantophysin (PanI) locus.

To examine the role of contemporary selection in maintaining significant allele frequency differences at the pantophysin (PanI) locus among populations of the Atlantic cod, Gadus morhua, in northern Norway, we sequenced 127 PanIA alleles sampled from six coastal and two Barents Sea populations. The distributions of variable sites segregating within the PanIA allelic class were then compared among the populations. Significant differences were detected in the overall frequencies of PanIA alleles among populations within coastal and Arctic regions that was similar in magnitude to heterogeneity in the distributions of polymorphic sites segregating within the PanIA allelic class. The differentiation observed at silent sites in the PanIA allelic class contradicts the predicted effects of widescale gene flow and suggests that postsettlement selection acting on cohorts cannot be responsible for the genetic differences described between coastal and Arctic populations. Our results suggest that the marked differences observed between coastal and Arctic populations of G. morhua in northern Norway at the PanI locus reflect the action of recent diversifying selection and that populations throughout the region may be more independent than suggested by previous studies.

DNA HETEROZYGOSITY AND GROWTH RATE IN THE ATLANTIC COD GADUS MORHUA (L).

Relationships between growth rate and the degree of individual heterozygosity at ten nuclear RFLP loci were examined in two populations of the Atlantic cod, Gadus morhua, from northern Norway. A highly significant positive correlation was observed between growth rate and DNA heterozygosity in one population (Balsfjord) but not in the other (Barents Sea). Our results provide support for an important prediction of the associative overdominance hypothesis that heterozygosity-fitness correlations can be detected at neutral genetic markers and suggest that environmental conditions might play a dominant role in the manifestation of the correlation.