BackgroundThe infraorder Anomura has long captivated the attention of evolutionary biologists due to its impressive morphological diversity and ecological adaptations. To date, 2500 extant species have been described but phylogenetic relationships at high taxonomic levels remain unresolved. Here, we reconstruct the evolutionary history—phylogeny, divergence times, character evolution and diversification—of this speciose clade. For this purpose, we sequenced two mitochondrial (16S and 12S) and three nuclear (H3, 18S and 28S) markers for 19 of the 20 extant families, using traditional Sanger and next-generation 454 sequencing methods. Molecular data were combined with 156 morphological characters in order to estimate the largest anomuran phylogeny to date. The anomuran fossil record allowed us to incorporate 31 fossils for divergence time analyses.ResultsOur best phylogenetic hypothesis (morphological + molecular data) supports most anomuran superfamilies and families as monophyletic. However, three families and eleven genera are recovered as para- and polyphyletic. Divergence time analysis dates the origin of Anomura to the Late Permian ~259 (224–296) MYA with many of the present day families radiating during the Jurassic and Early Cretaceous. Ancestral state reconstruction suggests that carcinization occurred independently 3 times within the group. The invasion of freshwater and terrestrial environments both occurred between the Late Cretaceous and Tertiary. Diversification analyses found the speciation rate to be low across Anomura, and we identify 2 major changes in the tempo of diversification; the most significant at the base of a clade that includes the squat-lobster family Chirostylidae.ConclusionsOur findings are compared against current classifications and previous hypotheses of anomuran relationships. Many families and genera appear to be poly- or paraphyletic suggesting a need for further taxonomic revisions at these levels. A divergence time analysis provides key insights into the origins of major lineages and events and the timing of morphological (body form) and ecological (habitat) transitions. Living anomuran biodiversity is the product of 2 major changes in the tempo of diversification; our initial insights suggest that the acquisition of a crab-like form did not act as a key innovation.

Next-gen sequencing technologies have revolutionized data collection in genetic studies and advanced genome biology to novel frontiers. However, to date, next-gen technologies have been used principally for whole genome sequencing and transcriptome sequencing. Yet many questions in population genetics and systematics rely on sequencing specific genes of known function or diversity levels. Here, we describe a targeted amplicon sequencing (TAS) approach capitalizing on next-gen capacity to sequence large numbers of targeted gene regions from a large number of samples. Our TAS approach is easily scalable, simple in execution, neither time-nor labor-intensive, relatively inexpensive, and can be applied to a broad diversity of organisms and/or genes. Our TAS approach includes a bioinformatic application, BarcodeCrucher, to take raw next-gen sequence reads and perform quality control checks and convert the data into FASTA format organized by gene and sample, ready for phylogenetic analyses. We demonstrate our approach by sequencing targeted genes of known phylogenetic utility to estimate a phylogeny for the Pancrustacea. We generated data from 44 taxa using 68 different 10-bp multiplexing identifiers. The overall quality of data produced was robust and was informative for phylogeny estimation. The potential for this method to produce copious amounts of data from a single 454 plate (e.g., 325 taxa for 24 loci) significantly reduces sequencing expenses incurred from traditional Sanger sequencing. We further discuss the advantages and disadvantages of this method, while offering suggestions to enhance the approach.

Lobsters are a ubiquitous and economically important group of decapod crustaceans that include the infraorders Polychelida, Glypheidea, Astacidea and Achelata. They include familiar forms such as the spiny, slipper, clawed lobsters and crayfish and unfamiliar forms such as the deep-sea and "living fossil" species. The high degree of morphological diversity among these infraorders has led to a dynamic classification and conflicting hypotheses of evolutionary relationships. In this study, we estimated phylogenetic relationships among the major groups of all lobster families and 94% of the genera using six genes (mitochondrial and nuclear) and 195 morphological characters across 173 species of lobsters for the most comprehensive sampling to date. Lobsters were recovered as a non-monophyletic assemblage in the combined (molecular + morphology) analysis. All families were monophyletic, with the exception of Cambaridae, and 7 of 79 genera were recovered as poly- or paraphyletic. A rich fossil history coupled with dense taxon coverage allowed us to estimate and compare divergence times and origins of major lineages using two drastically different approaches. Age priors were constructed and/or included based on fossil age information or fossil discovery, age, and extant species count data. Results from the two approaches were largely congruent across deep to shallow taxonomic divergences across major lineages. The origin of the first lobster-like decapod (Polychelida) was estimated in the Devonian (∼409-372 Ma) with all infraorders present in the Carboniferous (∼353-318 Ma). Fossil calibration subsampling studies examined the influence of sampling density (number of fossils) and placement (deep, middle, and shallow) on divergence time estimates. Results from our study suggest including at least 1 fossil per 10 operational taxonomic units (OTUs) in divergence dating analyses. [Dating; decapods; divergence; lobsters; molecular; morphology; phylogenetics.].

Comprising over 15 000 living species, decapods (crabs, shrimp and lobsters) are the most instantly recognizable crustaceans, representing a considerable global food source. Although decapod systematics have received much study, limitations of morphological and Sanger sequence data have yet to produce a consensus for higher-level relationships. Here, we introduce a new anchored hybrid enrichment kit for decapod phylogenetics designed from genomic and transcriptomic sequences that we used to capture new high-throughput sequence data from 94 species, including 58 of 179 extant decapod families, and 11 of 12 major lineages. The enrichment kit yields 410 loci (greater than 86 000 bp) conserved across all lineages of Decapoda, more clade-specific molecular data than any prior study. Phylogenomic analyses recover a robust decapod tree of life strongly supporting the monophyly of all infraorders, and monophyly of each of the reptant, ‘lobster’ and ‘crab’ groups, with some results supporting pleocyemate monophyly. We show that crown decapods diverged in the Late Ordovician and most crown lineages diverged in the Triassic–Jurassic, highlighting a cryptic Palaeozoic history, and post-extinction diversification. New insights into decapod relationships provide a phylogenomic window into morphology and behaviour, and a basis to rapidly and cheaply expand sampling in this economically and ecologically significant invertebrate clade.

Comprising over 15,000 living species, decapods (crabs, shrimp, and lobsters) are the most instantly recognizable crustaceans, representing a considerable global food source. Although decapod systematics have received much study, limitations of morphological and Sanger sequence data have yet to produce a consensus for higher-level relationships. Here we introduce a new anchored hybrid enrichment kit for decapod phylogenetics designed from genomic and transcriptomic sequences that we used to capture new high-throughput sequence data from 94 species, including 58 of 179 extant decapod families, and 11 of 12 major lineages. The enrichment kit yields 410 loci (>86,000 bp) conserved across all lineages of Decapoda, eight times more molecular data than any prior study. Phylogenomic analyses recover a robust decapod tree of life strongly supporting the monophyly of all infraorders, and monophyly of each of the reptant, 'lobster', and 'crab' groups, with some results supporting pleocyemate monophyly. We show that crown decapods diverged in the Late Ordovician and most crown lineages diverged in the Triassic-Jurassic, highlighting a cryptic Paleozoic history, and postextinction diversification. New insights into decapod relationships provide a phylogenomic window into morphology and behavior, and a basis to rapidly and cheaply expand sampling in this economically and ecologically significant invertebrate clade. Introduction:Decapod crustaceans, broadly categorized into 'shrimp', 'lobsters', and 'crabs', are embedded in the public consciousness due to their importance as a global food source worth over $24 billion [1]. Several ornamental species are also popular in the pet trade [2,3], and some lobsters and crayfish may be promising models for cancer and aging research [4]. Furthermore, decapods are a major faunal component of a bewildering variety of global habitats, including the open ocean, seafloor vents and seeps, caves, coral reefs, mangroves and estuaries, intertidal mud and sand, freshwater streams and lakes, semi-terrestrial locations, and in symbiosis with other animals (Figure 1). Decapods have diversified over the course of 455 million years resulting in over 15,000 living and 3,000 fossil species recognized in approximately 233 families [5,6]. Despite the economic and ecological significance of the clade, higher-level phylogenetic relationships among decapods have proven recalcitrant.The majority of work is restricted to studies using morphology [7][8][9], up to nine targeted mitochondrial and nuclear genes [6,[10][11][12][13][14][15][16][17][18][19], and more recently complete mitogenomes of 13 genes [20][21][22][23][24]. Mitogenomic data can be problematic for reconstructing ancient nodes [25], and indeed, deeper relationships receive poor support [24]. As part of a larger analysis, decapods were included in a recent transcriptomic study [26], but with limited taxon sampling within the order. This plurality of results, several based on the same underlying data [25], have reported conflicting deep relationshi...

BackgroundTools for high throughput sequencing and de novo assembly make the analysis of transcriptomes (i.e. the suite of genes expressed in a tissue) feasible for almost any organism. Yet a challenge for biologists is that it can be difficult to assign identities to gene sequences, especially from non-model organisms. Phylogenetic analyses are one useful method for assigning identities to these sequences, but such methods tend to be time-consuming because of the need to re-calculate trees for every gene of interest and each time a new data set is analyzed. In response, we employed existing tools for phylogenetic analysis to produce a computationally efficient, tree-based approach for annotating transcriptomes or new genomes that we term Phylogenetically-Informed Annotation (PIA), which places uncharacterized genes into pre-calculated phylogenies of gene families.ResultsWe generated maximum likelihood trees for 109 genes from a Light Interaction Toolkit (LIT), a collection of genes that underlie the function or development of light-interacting structures in metazoans. To do so, we searched protein sequences predicted from 29 fully-sequenced genomes and built trees using tools for phylogenetic analysis in the Osiris package of Galaxy (an open-source workflow management system). Next, to rapidly annotate transcriptomes from organisms that lack sequenced genomes, we repurposed a maximum likelihood-based Evolutionary Placement Algorithm (implemented in RAxML) to place sequences of potential LIT genes on to our pre-calculated gene trees. Finally, we implemented PIA in Galaxy and used it to search for LIT genes in 28 newly-sequenced transcriptomes from the light-interacting tissues of a range of cephalopod mollusks, arthropods, and cubozoan cnidarians. Our new trees for LIT genes are available on the Bitbucket public repository (http://bitbucket.org/osiris_phylogenetics/pia/) and we demonstrate PIA on a publicly-accessible web server (http://galaxy-dev.cnsi.ucsb.edu/pia/).ConclusionsOur new trees for LIT genes will be a valuable resource for researchers studying the evolution of eyes or other light-interacting structures. We also introduce PIA, a high throughput method for using phylogenetic relationships to identify LIT genes in transcriptomes from non-model organisms. With simple modifications, our methods may be used to search for different sets of genes or to annotate data sets from taxa outside of Metazoa.Electronic supplementary materialThe online version of this article (doi:10.1186/s12859-014-0350-x) contains supplementary material, which is available to authorized users.

The emergence of cost-effective and rapid sequencing approaches has resulted in an exponential rise in the number of mitogenomes on public databases in recent years, providing greater opportunity for undertaking large-scale comparative genomic and systematic research. Nonetheless, current datasets predominately come from small and disconnected studies on a limited number of related species, introducing sampling biases and impeding research of broad taxonomic relevance. This study contributes 21 crustacean mitogenomes from several under-represented decapod infraorders including Polychelida and Stenopodidea, which are used in combination with 225 mitogenomes available on NCBI to investigate decapod mitogenome diversity and phylogeny. An overview of mitochondrial gene orders (MGOs) reveals a high level of genomic variability within the Decapoda, with a large number of MGOs deviating from the ancestral arthropod ground pattern and unevenly distributed among infraorders. Despite the substantial morphological and ecological variation among decapods, there was limited evidence for correlations between gene rearrangement events and species ecology or lineage specific nucleotide substitution rates. Within a phylogenetic context, predicted scenarios of rearrangements show some MGOs to be informative synapomorphies for some taxonomic groups providing strong independent support for phylogenetic relationships. Additional comparisons for a range of mitogenomic features including nucleotide composition, strand asymmetry, unassigned regions and codon usage indicate several clade-specific trends that are of evolutionary and ecological interest.

Crayfish can be used as model organisms in phylogeographic and divergence time studies if reliable calibrations are available. This study presents a comprehensive investigation into the phylogeography of the European stone crayfish ( Austropotamobius torrentium ) and includes samples from previously unstudied sites. Two mitochondrial markers were used to reveal evolutionary relationships among haplogroups throughout the species’ distributional range and to estimate the divergence time by employing both substitution rates and geological calibration methods. Our haplotype network reconstruction and phylogenetic analyses revealed the existence of a previously unknown haplogroup distributed in Romania's Apuseni Mountains. This haplogroup is closely related to others that are endemic in the Dinarides, despite their vast geographical separation (~600 km). The separation is best explained by the well‐dated tectonic displacement of the Tisza–Dacia microplate, which started in the Miocene (~16 Ma) and possibly carried part of the A. torrentium population to the current location of the Apuseni Mountains. This population may thus have been isolated from the Dinarides for a period of ca. 11 m.y. by marine and lacustrine phases of the Pannonian Basin. The inclusion of this geological event as a calibration point in divergence time analyses challenges currently accepted crayfish evolutionary time frames for the region, constraining the evolution of this area's crayfish to a much earlier date. We discuss why molecular clock calibrations previously employed to date European crayfish species divergences should therefore be reconsidered.