The phylogeny of extant starfish (Asteroidea: Echinodermata) including Xyloplax, based on comparative transcriptomics.

Multi-locus phylogenetic studies of echinoderms based on Sanger and RNA-seq technologies and the fossil record have provided evidence for the Asterozoa-Echinozoa hypothesis. This hypothesis posits a sister relationship between asterozoan classes (Asteroidea and Ophiuroidea) and a similar relationship between echinozoan classes (Echinoidea and Holothuroidea). Despite this consensus around Asterozoa-Echinozoa, phylogenetic relationships within the class Asteroidea (sea stars or starfish) have been controversial for over a century. Open questions include relationships within asteroids and the status of the enigmatic taxon Xyloplax. Xyloplax is thought by some to represent a newly discovered sixth class of echinoderms - and by others to be an asteroid. To address these questions, we applied a novel workflow to a large RNA-seq dataset that encompassed a broad taxonomic and genomic sample. This study included 15 species sampled from all extant orders and 13 families, plus four ophiuroid species as an outgroup. To expand the taxonomic coverage, the study also incorporated five previously published transcriptomes and one previously published expressed sequence tags (EST) dataset. We developed and applied methods that used a range of alignment parameters with increasing permissiveness in terms of gap characters present within an alignment. This procedure facilitated the selection of phylogenomic data subsets from large amounts of transcriptome data. The results included 19 nested data subsets that ranged from 37 to 4,281loci. Tree searches on all data subsets reconstructed Xyloplax as a velatid asteroid rather than a new class. This result implies that asteroid morphology remains labile well beyond the establishment of the body plan of the group. In the phylogenetic tree with the highest average asteroid nodal support several monophyletic groups were recovered. In this tree, Forcipulatida and Velatida are monophyletic and form a clade that includes Brisingida as sister to Forcipulatida. Xyloplax is consistently recovered as sister to Pteraster. Paxillosida and Spinulosida are each monophyletic, with Notomyotida as sister to the Paxillosida. Valvatida is recovered as paraphyletic. The results from other data subsets are largely consistent with these results. Our results support the hypothesis that the earliest divergence event among extant asteroids separated Velatida and Forcipulatacea from Valvatacea and Spinulosida.

EchinoDB, an application for comparative transcriptomics of deeply-sampled clades of echinoderms.

BACKGROUND: One of our goals for the echinoderm tree of life project (http://echinotol.org) is to identify orthologs suitable for phylogenetic analysis from next-generation transcriptome data. The current dataset is the largest assembled for echinoderm phylogeny and transcriptomics. We used RNA-Seq to profile adult tissues from 42 echinoderm specimens from 24 orders and 37 families. In order to achieve sampling members of clades that span key evolutionary divergence, many of our exemplars were collected from deep and polar seas. DESCRIPTION: A small fraction of the transcriptome data we produced is being used for phylogenetic reconstruction. Thus to make a larger dataset available to researchers with a wide variety of interests, we made a web-based application, EchinoDB (http://echinodb.uncc.edu). EchinoDB is a repository of orthologous transcripts from echinoderms that is searchable via keywords and sequence similarity. CONCLUSIONS: From transcripts we identified 749,397 clusters of orthologous loci. We have developed the information technology to manage and search the loci their annotations with respect to the Sea Urchin (Strongylocentrotus purpuratus) genome. Several users have already taken advantage of these data for spin-off projects in developmental biology, gene family studies, and neuroscience. We hope others will search EchinoDB to discover datasets relevant to a variety of additional questions in comparative biology.

Large scale patterns of genetic variation and differentiation in sugar maple from tropical Central America to temperate North America.

BACKGROUND: Geological events in the latter Cenozoic have influenced the distribution, abundance and genetic structure of tree populations in temperate and tropical North America. The biogeographical history of temperate vegetation that spans large ranges of latitude is complex, involving multiple latitudinal shifts that might have occurred via different migration routes. We determined the regional structuring of genetic variation of sugar maple (Acer saccharum subsp. saccharum) and its only subspecies in tropical America (Acer saccharum subsp. skutchii) using nuclear and chloroplast data. The studied populations span a geographic range from Maine, USA (46°N), to El Progreso, Guatemala (15°N). We examined genetic subdivisions, explored the locations of ancestral haplotypes, analyzed genetic data to explore the presence of a single or multiple glacial refugia, and tested whether genetic lineages are temporally consistent with a Pleistocene or older divergence. RESULTS: Nuclear and chloroplast data indicated that populations in midwestern USA and western Mexico were highly differentiated from populations in the rest of the sites. The time of the most recent common ancestor of the western Mexico haplotype lineage was dated to the Pliocene (5.9 Ma, 95% HPD: 4.3-7.3 Ma). Splits during the Pleistocene separated the rest of the phylogroups. The most frequent and widespread haplotype occurred in half of the sites (Guatemala, eastern Mexico, southeastern USA, and Ohio). Our data also suggested that multiple Pleistocene refugia (tropics-southeastern USA, midwestern, and northeastern USA), but not western Mexico (Jalisco), contributed to post-glacial northward expansion of ranges. Current southern Mexican and Guatemalan populations have reduced population sizes, genetic bottlenecks and tend toward homozygosity, as indicated using nuclear and chloroplast markers. CONCLUSIONS: The divergence of western Mexican populations from the rest of the sugar maples likely resulted from orographic and volcanic barriers to gene flow. Past connectivity among populations in the southeastern USA and eastern Mexico and Guatemala possible occurred through gene flow during the Pleistocene. The time to the most common ancestor values revealed that populations from the Midwest and Northeast USA represented different haplotype lineages, indicating major divergence of haplotypes lineages before the Last Glacial Maximum and suggesting the existence of multiple glacial refugia.

New taxa and taxonomic revisions to the Poraniidae (Valvatacea; Asteroidea) with comments on feeding biology.

New molecular phylogenetic data and new specimens provide the basis for a revision of the family Poraniidae. We present molecular phylogenetic data for five out of 11 genera in the Poraniidae including a newly discovered taxon from the North Pacific. Bathyporania ascendens nov. gen., nov. sp., is described from Davidson Seamount (35º43'N, 122º43'W). Another newly discovered poraniid taxon, Clavaporania fitchorum nov. gen., nov. sp. is described from south of Macquarie Island (56º21'S, 158º 28'E) but was not included in the analysis. Revision of the Poraniidae has been undertaken. We present two new genera and reinstate the previously synonymized genus Glabraster and return Culcitopsis to genus level. The genus Porania sensu Clark (1993) and Clark and Downey (1992) is not monophyletic. Porania, Poraniomorpha and Poraniopsis are revised. In situ feeding observations of Bathyporania are described and compared with other poraniid feeding accounts.

Difference in larval type explains patterns of nonsynonymous substitutions in two ancient paralogs of the histone H3 gene in sea stars.

Paralogous genes frequently show differences in patterns and rates of substitution that are typically attributed to different selection regimes, mutation rates, or local recombination rates. Here, two anciently diverged paralogous copies of the histone H3 gene in sea stars, the tandem-repetitive early-stage gene and a newly isolated gene with lower copy number that was termed the "putative late-stage histone H3 gene" were analyzed in 69 species with varying mode of larval development. The two genes showed differences in relative copy number, overall substitution rates, nucleotide composition, and codon usage, but similar patterns of relative nonsynonymous substitution rates, when analyzed by the d(N)/d(S) ratio. Sea stars with a nonpelagic and nonfeeding larval type (i.e., brooding lineages) were observed to have d(N)/d(S) ratios that were larger than for nonbrooders but equal between the two paralogs. This finding suggested that demographic differences between brooding and nonbrooding lineages were responsible for the elevated d(N)/d(S) ratios observed for brooders and refuted a suggestion from a previous analysis of the early-stage gene that the excess nonsynonymous substitutions were due to either (1) gene expression differences at the larval stage between brooders and nonbrooders or (2) the highly repetitive structure of the early-stage histone H3 gene.

Recent relaxation of purifying selection on the tandem-repetitive early-stage histone H3 gene in brooding sea stars.

Patterns of nucleotide substitution differ between marine species that have a pelagic feeding (planktotrophic) larval stage and related species that lack such a stage, for both adaptive and non-adaptive reasons. Here, patterns of nucleotide and inferred amino acid substitution are analyzed for the tandem-repetitive early-stage histone H3 gene in 36 sea star species of the order Forcipulatida with documented larval habitat. The relative rate of nonsynonymous substitution (expressed as ω=d(N)/d(S)) was significantly higher in lineages with a brooded non-feeding (lecithotrophic) larval form than in lineages with a planktotrophic larval form. There was also a significant excess of conservative over radical substitutions. The increase in ω for brooders as compared to non-brooders was much greater than for previously analyzed mitochondrial sequences in echinoderms. These data are consistent with the hypothesis that purifying selection on this gene has been relaxed in brooding lineages compared to non-brooding lineages. The hypotheses of adaptive or neutral evolution are less plausible, although recent pseudogenization following a period of relaxed purifying selection could also explain the results.

An ancient repeat sequence in the ATP synthase beta-subunit gene of forcipulate sea stars.

A novel repeat sequence with a conserved secondary structure is described from two nonadjacent introns of the ATP synthase beta-subunit gene in sea stars of the order Forcipulatida (Echinodermata: Asteroidea). The repeat is present in both introns of all forcipulate sea stars examined, which suggests that it is an ancient feature of this gene (with an approximate age of 200 Mya). Both stem and loop regions show high levels of sequence constraint when compared to flanking nonrepetitive intronic regions. The repeat was also detected in (1) the family Pterasteridae, order Velatida and (2) the family Korethrasteridae, order Velatida. The repeat was not detected in (1) the family Echinasteridae, order Spinulosida, (2) the family Astropectinidae, order Paxillosida, (3) the family Solasteridae, order Velatida, or (4) the family Goniasteridae, order Valvatida. The repeat lacks similarity to published sequences in unrestricted GenBank searches, and there are no significant open reading frames in the repeat or in the flanking intron sequences. Comparison via parametric bootstrapping to a published phylogeny based on 4.2 kb of nuclear and mitochondrial sequence for a subset of these species allowed the null hypothesis of a congruent phylogeny to be rejected for each repeat, when compared separately to the published phylogeny. In contrast, the flanking nonrepetitive sequences in each intron yielded separate phylogenies that were each congruent with the published phylogeny. In four species, the repeat in one or both introns has apparently experienced gene conversion. The two introns also show a correlated pattern of nucleotide substitutions, even after excluding the putative cases of gene conversion.

Apparent selection intensity for the cytochrome oxidase subunit I gene varies with mode of reproduction in echinoderms.

When most amino acid substitutions in protein-coding genes are slightly deleterious rather than selectively neutral, life history differences can potentially modify the effective population size or the selective regime, resulting in altered ratios of non-synonymous to synonymous substitutions among taxa. We studied substitution patterns for the mitochondrial cytochrome oxidase subunit I (COI) gene in a sea star genus (Leptasterias spp.) with an obligate brood-protecting mode of reproduction and small-scale population genetic subdivision, and compared the results to available COI sequences in nine other genera of echinoderms with pelagic larvae: three sea stars, five sea urchins and one brittle star. We predicted that this life history difference would be associated with differences in the ratio of non-synonymous (dN) to synonymous (dS) substitution rates. Leptasterias had a significantly greater dN/dS ratio (both between species and within species), a significantly smaller transition/transversion rate ratio, and a significantly lower average nucleotide diversity within species, than did the non-brooding genera. Other explanations for the results, such as altered mutation rates or selective sweeps, were not supported by the data analysis. These findings highlight the potential influence of reproductive traits and other life history factors on patterns of nucleotide substitution within and between species.

Differential tolerance among cryptic species: a potential cause of pollutant-related reductions in genetic diversity.

Differential mortality of cryptic species (i.e., morphologically similar but genetically distinct sibling species) may contribute to observed reductions in genetic diversity at contaminated sites if the members of a complex of cryptic species exhibit differential responses to the contaminants that are present. We conducted toxicity bioassays with both polynuclear aromatic hydrocarbon and metal contamination on Cletocamptus fourchensis and C. stimpsoni from two intensively sampled locations. Previous molecular and detailed morphological analyses segregated these as cryptic species from the cosmopolitan C. deitersi. We found that these species occur together at two field sites and that they exhibit unique toxic responses to heavy metals, suggesting differential tolerances at contaminated sites. These findings suggest that reported losses of genetic diversity at contaminated sites may represent a reduction in species diversity rather than a loss of the presumed less-tolerant genotypes within a species. They also suggest that members of a cryptic species complex should not be used in laboratory toxicity tests unless populations are genetically characterized. Future studies using genetic diversity as a marker of contaminant effects should consider the possibility of undetected cryptic species.

Invertebrate species with nonpelagic larvae have elevated levels of nonsynonymous substitutions and reduced nucleotide diversities.

Under a nearly neutral model in which most amino acid substitutions are slightly deleterious, variation in demography, population structure, and other ecological factors among closely related species can potentially modify the effective population size or the selective regime, leading to differences in the rate of nonsynonymous substitution. Ratios of nonsynonymous to synonymous substitutions (d(N)/d(S)) between species were analyzed in a sea star genus (Patiriella) and a molluscan genus (Littorina), each with diverse modes of reproduction, including multiple lineages with pelagic and nonpelagic larvae. In both genera, lineages with nonpelagic larvae had significantly higher d(N)/d(S) ratios than lineages with pelagic larvae. The hypothesis that the elevated d(N)/d(S) ratios in species with nonpelagic larvae was due to reduced effective population size was tested by comparing nucleotide diversities in three genera of gastropod mollusks (Littorina, Crepidula, and Hydrobia), each with several modes of reproduction. Overall, there was a significant (p < 0.05) reduction in nucleotide diversity in species with nonpelagic larvae compared to species with pelagic larvae.

DO BLACK-TAILED PRAIRIE DOGS MINIMIZE INBREEDING?

Considerable controversy surrounds the importance of inbreeding in natural populations. The rate of natural inbreeding and the influences of behavioral mechanisms that serve to promote or minimize inbreeding (e.g., philopatry vs. dispersal) are poorly understood. We studied inbreeding and social structuring of a population of black-tailed prairie dogs (Cynomys ludovicianus) to assess the influence of dispersal and mating behavior on patterns of genetic variation. We examined 15 years of data on prairie dogs, including survival and reproduction, social behavior, pedigrees, and allozyme alleles. Pedigrees revealed mean inbreeding coefficients (F) of 1-2%. A breeding-group model that incorporated details of prairie dog behavior and demography was used to estimate values of fixation indices (F-statistics). Model predictions were consistent with the minimization of inbreeding within breeding groups ("coteries," asymptotic FIL = -0.18) and random mating within the subpopulation ("colony," asymptotic FIS = 0.00). Estimates from pedigrees (mean FIL = -0.23, mean FIS = 0.00) and allozyme data (mean FIL = -0.21, mean FIS = -0.01) were consistent with predictions of the model. The breeding-group model, pedigrees, and allozyme data showed remarkably congruent results, and indicated strong genetic structuring within the colony (FLS = 0.16, 0.19, and 0.17, respectively). We concluded that although inbreeding occurred in the colony, the rate of inbreeding was strongly minimized at the level of breeding groups, but not at the subpopulation level. The behavioral mechanisms most important to the minimization of inbreeding appeared to be patterns of male-biased dispersal of both subadults and adults, associated with strong philopatry of females. Incest avoidance also occurred, associated with recognition of close kin via direct social learning within the breeding groups.