Shifting evolutionary sands: transcriptome characterization of the Aptostichus atomarius species complex.

BACKGROUND: Mygalomorph spiders represent a diverse, yet understudied lineage for which genomic level data has only recently become accessible through high-throughput genomic and transcriptomic sequencing methods. The Aptostichus atomarius species complex (family Euctenizidae) includes two coastal dune endemic members, each with inland sister species - affording exploration of dune adaptation associated patterns at the transcriptomic level. We apply an RNAseq approach to examine gene family conservation across the species complex and test for patterns of positive selection along branches leading to dune endemic species. RESULTS: An average of ~ 44,000 contigs were assembled for eight spiders representing dune (n = 2), inland (n = 4), and atomarius species complex outgroup taxa (n = 2). Transcriptomes were estimated to be 64% complete on average with 77 spider reference orthologs missing from all taxa. Over 18,000 orthologous gene clusters were identified within the atomarius complex members, > 5000 were detected in all species, and ~ 4700 were shared between species complex members and outgroup Aptostichus species. Gene family analysis with the FUSTr pipeline identified 47 gene families appearing to be under selection in the atomarius ingroup; four of the five top clusters include sequences strongly resembling other arthropod venom peptides. The COATS pipeline identified six gene clusters under positive selection on branches leading to dune species, three of which reflected the preferred species tree. Genes under selection were identified as Cytochrome P450 2c15 (also recovered in the FUSTr analysis), Niemann 2 Pick C1-like, and Kainate 2 isoform X1. CONCLUSIONS: We have generated eight draft transcriptomes for a closely related and ecologically diverse group of trapdoor spiders, identifying venom gene families potentially under selection across the Aptostichus atomarius complex and chemosensory-associated gene families under selection in dune endemic lineages.

Host and geography together drive early adaptive radiation of Hawaiian planthoppers.

The interactions between insects and their plant host have been implicated in driving diversification of both players. Early arguments highlighted the role of ecological opportunity, with the idea that insects "escape and radiate" on new hosts, with subsequent hypotheses focusing on the interplay between host shifting and host tracking, coupled with isolation and fusion, in generating diversity. Because it is rarely possible to capture the initial stages of diversification, it is particularly difficult to ascertain the relative roles of geographic isolation versus host shifts in initiating the process. The current study examines genetic diversity between populations and hosts within a single species of endemic Hawaiian planthopper, Nesosydne umbratica (Hemiptera, Delphacidae). Given that the species was known as a host generalist occupying unrelated hosts, Clermontia (Campanulaceae) and Pipturus (Urticaceae), we set out to determine the relative importance of geography and host in structuring populations in the early stages of differentiation on the youngest islands of the Hawaiian chain. Results from extensive exon capture data showed that N. umbratica is highly structured, both by geography, with discrete populations on each volcano, and by host plant, with parallel radiations on Clermontia and Pipturus leading to extensive co-occurrence. The marked genetic structure suggests that populations can readily become established on novel hosts provided opportunity; subsequent adaptation allows monopolization of the new host. The results support the role of geographic isolation in structuring populations and with host shifts occurring as discrete events that facilitate subsequent parallel geographic range expansion.

Co-occurrence of ecologically similar species of Hawaiian spiders reveals critical early phase of adaptive radiation.

BACKGROUND: The processes through which populations originate and diversify ecologically in the initial stages of adaptive radiation are little understood because we lack information on critical steps of early divergence. A key question is, at what point do closely related species interact, setting the stage for competition and ecological specialization? The Hawaiian Islands provide an ideal system to explore the early stages of adaptive radiation because the islands span ages from 0.5-5 Mya. Hawaiian spiders in the genus Tetragnatha have undergone adaptive radiation, with one lineage ("spiny legs") showing four different ecomorphs (green, maroon, large brown, small brown); one representative of each ecomorph is generally found at any site on the older islands. Given that the early stages of adaptive radiation are characterized by allopatric divergence between populations of the same ecomorph, the question is, what are the steps towards subsequent co-occurrence of different ecomorphs? Using a transcriptome-based exon capture approach, we focus on early divergence among close relatives of the green ecomorph to understand processes associated with co-occurrence within the same ecomorph at the early stages of adaptive radiation. RESULTS: The major outcomes from the current study are first that closely related species within the same green ecomorph of spiny leg Tetragnatha co-occur on the same single volcano on East Maui, and second that there is no evidence of genetic admixture between these ecologically equivalent species. Further, that multiple genetic lineages exist on a single volcano on Maui suggests that there are no inherent dispersal barriers and that the observed limited distribution of taxa reflects competitive exclusion. CONCLUSIONS: The observation of co-occurrence of ecologically equivalent species on the young volcano of Maui provides a missing link in the process of adaptive radiation between the point when recently divergent species of the same ecomorph occur in allopatry, to the point where different ecomorphs co-occur at a site, as found throughout the older islands. More importantly, the ability of close relatives of the same ecomorph to interact, without admixture, may provide the conditions necessary for ecological divergence and independent evolution of ecomorphs associated with adaptive radiation.

Transcriptomic signatures for ovulation in vertebrates.

The central roles of luteinizing hormone (LH), progestin and their receptors for initiating ovulation have been well established. However, signaling pathways and downstream targets such as proteases that are essential for the rupture of follicular cells are still unclear. Recently, we found anovulation in nuclear progestin receptor (Pgr) knockout (Pgr-KO) zebrafish, which offers a new model for examining genes and pathways that are important for ovulation and fertility. In this study, we examined expression of all transcripts using RNA-Seq in preovulatory follicular cells collected following the final oocyte maturation, but prior to ovulation, from wild-type (WT) or Pgr-KO fish. Differential expression analysis revealed 3567 genes significantly differentially expressed between WT and Pgr-KO fish (fold change⩾2, p<0.05). Among those, 1543 gene transcripts were significantly more expressed, while 2024 genes were significantly less expressed, in WT than those in Pgr-KO. We then retrieved and compared transcriptional data from online databases and further identified 661 conserved genes in fish, mice, and humans that showed similar levels of high (283 genes) or low (387) expression in animals that were ovulating compared to those with no ovulation. For the first time, ovulatory genes and their involved biological processes and pathways were also visualized using Enrichment Map and Cytoscape. Intriguingly, enrichment analysis indicated that the genes with higher expression were involved in multiple ovulatory pathways and processes such as inflammatory response, angiogenesis, cytokine production, cell migration, chemotaxis, MAPK, focal adhesion, and cytoskeleton reorganization. In contrast, the genes with lower expression were mainly involved in DNA replication, DNA repair, DNA methylation, RNA processing, telomere maintenance, spindle assembling, nuclear acid transport, catabolic processes, and nuclear and cell division. Our results indicate that a large set of genes (>3000) is differentially regulated in the follicular cells in zebrafish prior to ovulation, terminating programs such as growth and proliferation, and beginning processes including the inflammatory response and apoptosis. Further studies are required to establish relationships among these genes and an ovulatory circuit in the zebrafish model.

An evaluation of sampling effects on multiple DNA barcoding methods leads to an integrative approach for delimiting species: a case study of the North American tarantula genus Aphonopelma (Araneae, Mygalomorphae, Theraphosidae).

The North American tarantula genus Aphonopelma provides one of the greatest challenges to species delimitation and downstream identification in spiders because traditional morphological characters appear ineffective for evaluating limits of intra- and interspecific variation in the group. We evaluated the efficacy of numerous molecular-based approaches to species delimitation within Aphonopelma based upon the most extensive sampling of theraphosids to date, while also investigating the sensitivity of randomized taxon sampling on the reproducibility of species boundaries. Mitochondrial DNA (cytochrome c oxidase subunit I) sequences were sampled from 682 specimens spanning the genetic, taxonomic, and geographic breadth of the genus within the United States. The effects of random taxon sampling compared traditional Neighbor-Joining with three modern quantitative species delimitation approaches (ABGD, P ID(Liberal), and GMYC). Our findings reveal remarkable consistency and congruence across various approaches and sampling regimes, while highlighting highly divergent outcomes in GMYC. Our investigation allowed us to integrate methodologies into an efficient, consistent, and more effective general methodological workflow for estimating species boundaries within the mygalomorph spider genus Aphonopelma. Taken alone, these approaches are not particularly useful - especially in the absence of prior knowledge of the focal taxa. Only through the incorporation of multiple lines of evidence, employed in a hypothesis-testing framework, can the identification and delimitation of confident species boundaries be determined. A key point in studying closely related species, and perhaps one of the most important aspects of DNA barcoding, is to combine a sampling strategy that broadly identifies the extent of genetic diversity across the distributions of the species of interest and incorporates previous knowledge into the "species equation" (morphology, molecules, and natural history).

Comparative transcriptomics of maturity-associated color change in Hawaiian spiders.

Discontinuous variation within individuals is increasingly recognized as playing a role in diversification and ecological speciation. This study is part of an effort to investigate the molecular genetic underpinnings of adaptive radiation in Hawaiian spiders (genus Tetragnatha). This radiation is found throughout the Hawaiian Islands, showing a common pattern of evolutionary progression from older to younger islands. Moreover, the species are characterized by repeated evolution of similar ecomorphs that can be recognized on the basis of color--Green, Maroon, Large Brown, and Small Brown. However, 2 species (including T. kauaiensis from Kauai) are polyphenic, changing from 1 ecomorph (Green) to another (Maroon) at a specific developmental period. The current study focuses on the age-associated color change in the early stages of the radiation to determine whether this ancestral flexibility in phenotype may have translated into diversification of more derived taxa. We conducted a comparative analysis of transcriptome data (expressed genes) from the Maroon morph of T. kauaiensis and T. perreirai (Oahu), which exhibits a single fixed ecomorph (Maroon). Over 70 million sequence reads were generated using Illumina sequencing of messenger RNA. Using reciprocal best hit BLAST searches, 9027 orthologous genes were identified, of which 32 showed signatures of positive selection between the 2 taxa and may be involved in the loss of the ancestral developmental polyphenism and associated switch to separate monophenic ecomorphs. These results provide critical groundwork that will allow us to advance our understanding of the genomic elements associated with adaptive radiations.

De novo characterization of the gene-rich transcriptomes of two color-polymorphic spiders, Theridion grallator and T. californicum (Araneae: Theridiidae), with special reference to pigment genes.

BACKGROUND: A number of spider species within the family Theridiidae exhibit a dramatic abdominal (opisthosomal) color polymorphism. The polymorphism is inherited in a broadly Mendelian fashion and in some species consists of dozens of discrete morphs that are convergent across taxa and populations. Few genomic resources exist for spiders. Here, as a first necessary step towards identifying the genetic basis for this trait we present the near complete transcriptomes of two species: the Hawaiian happy-face spider Theridion grallator and Theridion californicum. We mined the gene complement for pigment-pathway genes and examined differential expression (DE) between morphs that are unpatterned (plain yellow) and patterned (yellow with superimposed patches of red, white or very dark brown). RESULTS: By deep sequencing both RNA-seq and normalized cDNA libraries from pooled specimens of each species we were able to assemble a comprehensive gene set for both species that we estimate to be 98-99% complete. It is likely that these species express more than 20,000 protein-coding genes, perhaps 4.5% (ca. 870) of which might be unique to spiders. Mining for pigment-associated Drosophila melanogaster genes indicated the presence of all ommochrome pathway genes and most pteridine pathway genes and DE analyses further indicate a possible role for the pteridine pathway in theridiid color patterning. CONCLUSIONS: Based upon our estimates, T. grallator and T. californicum express a large inventory of protein-coding genes. Our comprehensive assembly illustrates the continuing value of sequencing normalized cDNA libraries in addition to RNA-seq in order to generate a reference transcriptome for non-model species. The identification of pteridine-related genes and their possible involvement in color patterning is a novel finding in spiders and one that suggests a biochemical link between guanine deposits and the pigments exhibited by these species.

Killer Knots: Molecular Evolution of Inhibitor Cystine Knot Toxins in Wandering Spiders (Araneae: Ctenidae).

Venom expressed by the nearly 50,000 species of spiders on Earth largely remains an untapped reservoir of a diverse array of biomolecules with potential for pharmacological and agricultural applications. A large fraction of the noxious components of spider venoms are a functionally diverse family of structurally related polypeptides with an inhibitor cystine knot (ICK) motif. The cysteine-rich nature of these toxins makes structural elucidation difficult, and most studies have focused on venom components from the small handful of medically relevant spider species such as the highly aggressive Brazilian wandering spider Phoneutria nigriventer. To alleviate difficulties associated with the study of ICK toxins in spiders, we devised a comprehensive approach to explore the evolutionary patterns that have shaped ICK functional diversification using venom gland transcriptomes and proteomes from phylogenetically distinct lineages of wandering spiders and their close relatives. We identified 626 unique ICK toxins belonging to seven topological elaborations. Phylogenetic tests of episodic diversification revealed distinct regions between cysteine residues that demonstrated differential evidence of positive or negative selection, which may have structural implications towards the specificity and efficacy of these toxins. Increased taxon sampling and whole genome sequencing will provide invaluable insights to further understand the evolutionary processes that have given rise to this diverse class of toxins.

The Effects of Social Experience on Host Gut Microbiome in Male Zebrafish (Danio rerio).

AbstractAlthough the gut and the brain vastly differ in physiological function, they have been interlinked in a variety of different neurological and behavioral disorders. The bacteria that comprise the gut microbiome communicate and influence the function of various physiological processes within the body, including nervous system function. However, the effects of social experience in the context of dominance and social stress on gut microbiome remain poorly understood. Here, we examined whether social experience impacts the host zebrafish (Danio rerio) gut microbiome. We studied how social dominance during the first 2 weeks of social interactions changed the composition of zebrafish gut microbiome by comparing gut bacterial composition, diversity, and relative abundance between socially dominant, submissive, social isolates and control group-housed communal fish. Using amplicon sequencing of the 16S rRNA gene, we report that social dominance significantly affects host gut bacterial community composition but not bacterial diversity. At the genus level, Aeromonas and unclassified Enterobacteriaceae relative abundance decreased in dominant individuals while commensal bacteria (e.g., Exiguobacterium and Cetobacterium) increased in relative abundance. Conversely, the relative abundance of Psychrobacter and Acinetobacter was increased in subordinates, isolates, and communal fish compared to dominant fish. The shift in commensal and pathogenic bacteria highlights the impact of social experience and the accompanying stress on gut microbiome, with potentially similar effects in other social organisms.

Phylogenetics of the millipede genus Brachycybe Wood, 1864 (Diplopoda: Platydesmida: Andrognathidae): patterns of deep evolutionary history and recent speciation.

The genus Brachycybe Wood is a little known group of millipedes comprising eight nominal species distributed throughout North America, Japan, South Korea, Taiwan, and China. The group's species are relatively morphologically homogenous and have been described primarily on the basis of differences in somatic morphology largely ignoring the often-used characters in millipede taxonomy and systematics - male genitalia (the gonopods). The objectives of this study were to survey male gonopods with the aim of evaluating inter-specific variation, assess existing species boundaries and phylogeny using molecular characters, examine the historical biogeography of the genus, and estimate the timing of lineage divergence using a molecular clock. We surveyed two mitochondrial genes (cytochrome c oxidase I and cytochrome b) and one nuclear protein-coding gene (glutamyl and prolyl-tRNA synthetase) each comprising 952, 746, and 555 aligned base pairs respectively. Phylogenetic inference coupled with an analysis of species delimitation using a generalized mixed Yule coalescent model recovered eight species, two of which were considered to be cryptic. Molecular dating analyses coupled with ancestral range reconstructions indicate that the group is quite ancient (age>50 million years) and its origins are likely traced back geographically to the mountains of California's Sierra Nevada and Coastal Ranges. The genus Brachycybe appears to have expanded its range at least twice out of present day California into eastern North America and at least once into Asia. This study highlights the need for integrative approaches to describe biodiversity and furthers the evidence for cryptic diversity even in groups where genitalia are generally thought to be rapidly evolving, diagnostic features.

Shifts in morphology, gene expression, and selection underlie web loss in Hawaiian Tetragnatha spiders.

BACKGROUND: A striking aspect of evolution is that it often converges on similar trajectories. Evolutionary convergence can occur in deep time or over short time scales, and is associated with the imposition of similar selective pressures. Repeated convergent events provide a framework to infer the genetic basis of adaptive traits. The current study examines the genetic basis of secondary web loss within web-building spiders (Araneoidea). Specifically, we use a lineage of spiders in the genus Tetragnatha (Tetragnathidae) that has diverged into two clades associated with the relatively recent (5 mya) colonization of, and subsequent adaptive radiation within, the Hawaiian Islands. One clade has adopted a cursorial lifestyle, and the other has retained the ancestral behavior of capturing prey with sticky orb webs. We explore how these behavioral phenotypes are reflected in the morphology of the spinning apparatus and internal silk glands, and the expression of silk genes. Several sister families to the Tetragnathidae have undergone similar web loss, so we also ask whether convergent patterns of selection can be detected in these lineages. RESULTS: The cursorial clade has lost spigots associated with the sticky spiral of the orb web. This appears to have been accompanied by loss of silk glands themselves. We generated phylogenies of silk proteins (spidroins), which showed that the transcriptomes of cursorial Tetragnatha contain all major spidroins except for flagelliform. We also found an uncharacterized spidroin that has higher expression in cursorial species. We found evidence for convergent selection acting on this spidroin, as well as genes involved in protein metabolism, in the cursorial Tetragnatha and divergent cursorial lineages in the families Malkaridae and Mimetidae. CONCLUSIONS: Our results provide strong evidence that independent web loss events and the associated adoption of a cursorial lifestyle are based on similar genetic mechanisms. Many genes we identified as having evolved convergently are associated with protein synthesis, degradation, and processing, which are processes that play important roles in silk production. This study demonstrates, in the case of independent evolution of web loss, that similar selective pressures act on many of the same genes to produce the same phenotypes and behaviors.

DNA methylation analyses identify an intronic ZDHHC6 locus associated with time to recurrent stroke in the Vitamin Intervention for Stroke Prevention (VISP) clinical trial.

Aberrant DNA methylation profiles have been implicated in numerous cardiovascular diseases; however, few studies have investigated how these epigenetic modifications contribute to stroke recurrence. The aim of this study was to identify methylation loci associated with the time to recurrent cerebro- and cardiovascular events in individuals of European and African descent. DNA methylation profiles were generated for 180 individuals from the Vitamin Intervention for Stroke Prevention clinical trial using Illumina HumanMethylation 450K BeadChip microarrays, resulting in beta values for 470,871 autosomal CpG sites. Ethnicity-stratified survival analyses were performed using Cox Proportional Hazards regression models for associations between each methylation locus and the time to recurrent stroke or composite vascular event. Results were validated in the Vall d'Hebron University Hospital cohort from Barcelona, Spain. Network analyses of the methylation loci were generated using weighted gene coexpression network analysis. Primary analysis identified four significant loci, cg04059318, ch.2.81927627R, cg03584380, and cg24875416, associated with time to recurrent stroke. Secondary analysis identified three loci, cg00076998, cg16758041, and cg02365967, associated with time to composite vascular endpoint. Locus cg03584380, which is located in an intron of ZDHHC6, was replicated in the Vall d'Hebron University Hospital cohort. The results from this study implicate the degree of methylation at cg03584380 is associated with the time of recurrence for stroke or composite vascular events across two ethnically diverse groups. Furthermore, modules of loci were associated with clinical traits and blood biomarkers including previous number of strokes, prothrombin fragments 1 + 2, thrombomodulin, thrombin-antithrombin complex, triglyceride levels, and tissue plasminogen activator. Ultimately, these loci could serve as potential epigenetic biomarkers that could identify at-risk individuals in recurrence-prone populations.

Multi-omic analysis of stroke recurrence in African Americans from the Vitamin Intervention for Stroke Prevention (VISP) clinical trial.

African Americans endure a nearly two-fold greater risk of suffering a stroke and are 2-3 times more likely to die from stroke compared to those of European ancestry. African Americans also have a greater risk of recurrent stroke and vascular events, which are deadlier and more disabling than incident stroke. Stroke is a multifactorial disease with both heritable and environmental risk factors. We conducted an integrative, multi-omic study on 922 plasma metabolites, 473,864 DNA methylation loci, and 556 variants from 50 African American participants of the Vitamin Intervention for Stroke Prevention clinical trial to help elucidate biomarkers contributing to recurrent stroke rates in this high risk population. Sixteen metabolites, including cotinine, N-delta-acetylornithine, and sphingomyelin (d17:1/24:1) were identified in t-tests of recurrent stroke outcome or baseline smoking status. Serum tricosanoyl sphingomyelin (d18:1/23:0) levels were significantly associated with recurrent stroke after adjusting for covariates in Cox Proportional Hazards models. Weighted Gene Co-expression Network Analysis identified moderate correlations between sphingolipid markers and clinical traits including days to recurrent stroke. Integrative analyses between genetic variants in sphingolipid pathway genes identified 29 nominal associations with metabolite levels in a one-way analysis of variance, while epigenomic analyses identified xenobiotics, predominately smoking-associated metabolites and pharmaceutical drugs, associated with methylation profiles. Taken together, our results suggest that metabolites, specifically those associated with sphingolipid metabolism, are potential plasma biomarkers for stroke recurrence in African Americans. Furthermore, genetic variation and DNA methylation may play a role in the regulation of these metabolites.

Pick Your Poison: Molecular Evolution of Venom Proteins in Asilidae (Insecta: Diptera).

Robber flies are an understudied family of venomous, predatory Diptera. With the recent characterization of venom from three asilid species, it is possible, for the first time, to study the molecular evolution of venom genes in this unique lineage. To accomplish this, a novel whole-body transcriptome of Eudioctria media was combined with 10 other publicly available asiloid thoracic or salivary gland transcriptomes to identify putative venom gene families and assess evidence of pervasive positive selection. A total of 348 gene families of sufficient size were analyzed, and 33 of these were predicted to contain venom genes. We recovered 151 families containing homologs to previously described venom proteins, and 40 of these were uniquely gained in Asilidae. Our gene family clustering suggests that many asilidin venom gene families are not natural groupings, as delimited by previous authors, but instead form multiple discrete gene families. Additionally, robber fly venoms have relatively few sites under positive selection, consistent with the hypothesis that the venoms of older lineages are dominated by negative selection acting to maintain toxic function.

Natural history of the social millipede Brachycybe lecontii Wood, 1864.

The millipede Brachycybe lecontii Wood, 1864 is a fungivorous social millipede known for paternal care of eggs and forming multi-generational aggregations. We investigated the life history, paternal care, chemical defence, feeding and social behaviour of B. lecontii and provided morphological and anatomical descriptions, using light and scanning electron microscopy. Based on observations of B. lecontii from 13 locations throughout its distribution, we report the following natural history aspects. The oviposition period of B. lecontii lasted from mid-April to late June and the incubation period lasted 3-4 weeks. Only males cared for the eggs and subsequent care of juveniles was not observed. In one case, the clutches of two males became combined and they were later cared for by only one of the males. The defensive compound of B. lecontii is stored in large glands occupying a third of the paranotal volume and were observed only in stadia II millipedes and older. We observed B. lecontii feeding on fungi of the order Polyporales and describe a cuticular structure on the tip of the labrum that may relate to fungivory. We found that their stellate-shaped aggregations (pinwheels) do not form in the absence of fungus and suggest the aggregation is associated with feeding. We describe and illustrate a previously undescribed comb-like structure on the tibia and tarsi of the six anterior-most leg-pairs and measure the colour and spectral reflectance of the B. lecontii exoskeleton.

TOXIFY: a deep learning approach to classify animal venom proteins.

In the era of Next-Generation Sequencing and shotgun proteomics, the sequences of animal toxigenic proteins are being generated at rates exceeding the pace of traditional means for empirical toxicity verification. To facilitate the automation of toxin identification from protein sequences, we trained Recurrent Neural Networks with Gated Recurrent Units on publicly available datasets. The resulting models are available via the novel software package TOXIFY, allowing users to infer the probability of a given protein sequence being a venom protein. TOXIFY is more than 20X faster and uses over an order of magnitude less memory than previously published methods. Additionally, TOXIFY is more accurate, precise, and sensitive at classifying venom proteins.

Diversity and function of fungi associated with the fungivorous millipede, Brachycybe lecontii.

Fungivorous millipedes (subterclass Colobognatha) likely represent some of the earliest known mycophagous terrestrial arthropods, yet their fungal partners remain elusive. Here we describe relationships between fungi and the fungivorous millipede, Brachycybe lecontii. Their fungal community is surprisingly diverse, including 176 genera, 39 orders, four phyla, and several undescribed species. Of particular interest are twelve genera conserved across wood substrates and millipede clades that comprise the core fungal community of B. lecontii. Wood decay fungi, long speculated to serve as the primary food source for Brachycybe species, were absent from this core assemblage and proved lethal to millipedes in pathogenicity assays while entomopathogenic Hypocreales were more common in the core but had little effect on millipede health. This study represents the first survey of fungal communities associated with any colobognath millipede, and these results offer a glimpse into the complexity of millipede fungal communities.

FUSTr: a tool to find gene families under selection in transcriptomes.

BACKGROUND: The recent proliferation of large amounts of biodiversity transcriptomic data has resulted in an ever-expanding need for scalable and user-friendly tools capable of answering large scale molecular evolution questions. FUSTr identifies gene families involved in the process of adaptation. This is a tool that finds genes in transcriptomic datasets under strong positive selection that automatically detects isoform designation patterns in transcriptome assemblies to maximize phylogenetic independence in downstream analysis. RESULTS: When applied to previously studied spider transcriptomic data as well as simulated data, FUSTr successfully grouped coding sequences into proper gene families as well as correctly identified those under strong positive selection in relatively little time. CONCLUSIONS: FUSTr provides a useful tool for novice bioinformaticians to characterize the molecular evolution of organisms throughout the tree of life using large transcriptomic biodiversity datasets and can utilize multi-processor high-performance computational facilities.

Repeated Diversification of Ecomorphs in Hawaiian Stick Spiders.

Insular adaptive radiations in which repeated bouts of diversification lead to phenotypically similar sets of taxa serve to highlight predictability in the evolutionary process [1]. However, examples of such replicated events are rare. Cross-clade comparisons of adaptive radiations are much needed to determine whether similar ecological opportunities can lead to the same outcomes. Here, we report a heretofore uncovered adaptive radiation of Hawaiian stick spiders (Theridiidae, Ariamnes) in which different species exhibit a set of discrete ecomorphs associated with different microhabitats. The three primary ecomorphs (gold, dark, and matte white) generally co-occur in native forest habitats. Phylogenetic reconstruction mapped onto the well-known chronosequence of the Hawaiian Islands shows both that this lineage colonized the islands only once and relatively recently (2-3 mya, when Kauai and Oahu were the only high islands in the archipelago) and that the distinct ecomorphs evolved independently multiple times following colonization of new islands. This parallel evolution of ecomorphs matches that of "spiny-leg" long-jawed spiders (Tetragnathidae, Tetragnatha), also in Hawaii [2]. Both lineages are free living, and both have related lineages in the Hawaiian Islands that show quite different patterns of diversification with no evidence of deterministic evolution. We argue that repeated evolution of ecomorphs results from a rugged adaptive landscape, with the few peaks associated with camouflage for these free-living taxa against the markedly low diversity of predators on isolated islands. These features, coupled with a limited genetic toolbox and reduced dispersal between islands, appear to be common to situations of repeated evolution of ecomorphs.

A biogeographical profile of the sand cockroach Arenivaga floridensis and its bearing on origin hypotheses for Florida scrub biota.

Florida scrub is a xeric ecosystem associated with the peninsula's sand ridges, whose intermittent Pliocene-Pleistocene isolation is considered key to scrub endemism. One scrub origin hypothesis posits endemics were sourced by the Pliocene dispersal of arid-adapted taxa from southwestern North America; a second invokes Pleistocene migration within eastern North America. Only one study to date has explicitly tested these competing hypotheses, supporting an eastern origin for certain scrub angiosperms. For further perspective, we conducted a genetic analysis of an endemic arthropod, the Florida sand cockroach (Arenivaga floridensis), with two aims: (1) to reconstruct the peninsular colonization and residence history of A. floridensis and (2) determine whether its biogeographic profile favors either origin hypothesis. We sequenced the cox2 mitochondrial gene for 237 specimens (65 populations) as well as additional loci (cox1, nuclear H3) for a subset of Florida roaches and congeners. Using Network and Bayesian inference methods, we identified three major lineages whose genetic differentiation and phylogeographical structure correspond with late Pliocene peninsula insularization, indicating Arenivaga was present and broadly distributed in Florida at that time. Stem and crown divergence estimates (6.36 Ma; 2.78 Ma) between A. floridensis and western sister taxa span a period of extensive dispersal by western biota along an arid Gulf Coast corridor. These phylogeographical and phylogenetic results yield a biogeographic profile consistent with the western origin hypothesis. Moreover, age estimates for the roach's peninsular residence complement those of several other endemics, favoring a Pliocene (or earlier) inception of the scrub ecosystem. We argue that eastern versus western hypotheses are not mutually exclusive; rather, a composite history of colonization involving disparate biotas better explains the diverse endemism of Florida scrub.