Heather D. Bracken-Grissom

Targeted Amplicon Sequencing (TAS): A Scalable Next-Gen Approach to Multilocus, Multitaxa Phylogenetics

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.

A comprehensive and integrative reconstruction of evolutionary history for Anomura (Crustacea: Decapoda).

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.

Using phylogenetically-informed annotation (PIA) to search for light-interacting genes in transcriptomes from non-model organisms

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.

Phylogenetics links monster larva to deep-sea shrimp

Mid-water plankton collections commonly include bizarre and mysterious developmental stages that differ conspicuously from their adult counterparts in morphology and habitat. Unaware of the existence of planktonic larval stages, early zoologists often misidentified these unique morphologies as independent adult lineages. Many such mistakes have since been corrected by collecting larvae, raising them in the lab, and identifying the adult forms. However, challenges arise when the larva is remarkably rare in nature and relatively inaccessible due to its changing habitats over the course of ontogeny. The mid-water marine species Cerataspis monstrosa (Gray 1828) is an armored crustacean larva whose adult identity has remained a mystery for over 180 years. Our phylogenetic analyses, based in part on recent collections from the Gulf of Mexico, provide definitive evidence that the rare, yet broadly distributed larva, C. monstrosa, is an early developmental stage of the globally distributed deepwater aristeid shrimp, Plesiopenaeus armatus. Divergence estimates and phylogenetic relationships across five genes confirm the larva and adult are the same species. Our work demonstrates the diagnostic power of molecular systematics in instances where larval rearing seldom succeeds and morphology and habitat are not indicative of identity. Larval–adult linkages not only aid in our understanding of biodiversity, they provide insights into the life history, distribution, and ecology of an organism.

Phylogenetic and transcriptomic analyses reveal the evolution of bioluminescence and light detection in marine deep-sea shrimps of the family Oplophoridae (Crustacea: Decapoda)

Bioluminescence is essential to the survival of many organisms, particularly in the deep sea where light is limited. Shrimp of the family Oplophoridae exhibit a remarkable mechanism of bioluminescence in the form of a secretion used for predatory defense. Three of the ten genera possess an additional mode of bioluminescence in the form of light-emitting organs called photophores. Phylogenetic analyses can be useful for tracing the evolution of bioluminescence, however, the few studies that have attempted to reconcile the relationships within Oplophoridae have generated trees with low-resolution. We present the most comprehensive phylogeny of Oplophoridae to date, with 90% genera coverage using seven genes (mitochondrial and nuclear) across 30 oplophorid species. We use our resulting topology to trace the evolution of bioluminescence within Oplophoridae. Previous studies have suggested that oplophorid visual systems may be tuned to differentiate the separate modes of bioluminescence. While all oplophorid shrimp possess a visual pigment sensitive to blue-green light, only those bearing photophores have an additional pigment sensitive to near-ultraviolet light. We attempt to characterize opsins, visual pigment proteins essential to light detection, in two photophore-bearing species (Systellaspis debilis and Oplophorus gracilirostris) and make inferences regarding their function and evolutionary significance.

Phylogenetic relationships, character evolution, and taxonomic implications within the slipper lobsters (Crustacea: Decapoda: Scyllaridae).

The slipper lobsters belong to the family Scyllaridae which contains a total of 20 genera and 89 species distributed across four subfamilies (Arctidinae, Ibacinae, Scyllarinae, and Theninae). We have collected nucleotide sequence data from regions of five different genes (16S, 18S, COI, 28S, H3) to estimate phylogenetic relationships among 54 species from the Scyllaridae with a focus on the species rich subfamily Scyllarinae. We have included in our analyses at least one representative from all 20 genera in the Scyllaridae and 35 of the 52 species within the Scyllarinae. Our resulting phylogenetic estimate shows the subfamilies are monophyletic, except for Ibacinae, which has paraphyletic relationships among genera. Many of the genera within the Scyllarinae form non-monophyletic groups, while the genera from all other subfamilies form well supported clades. We discuss the implications of this history on the evolution of morphological characters and ecological transitions (nearshore vs. offshore) within the slipper lobsters. Finally, we identify, through ancestral state character reconstructions, key morphological features diagnostic of the major clades of diversity within the Scyllaridae and relate this character evolution to current taxonomy and classification.

A synthetic phylogeny of freshwater crayfish: insights for conservation.

Phylogenetic systematics is heading for a renaissance where we shift from considering our phylogenetic estimates as a static image in a published paper and taxonomies as a hardcopy checklist to treating both the phylogenetic estimate and dynamic taxonomies as metadata for further analyses. The Open Tree of Life project (opentreeoflife.org) is developing synthesis tools for harnessing the power of phylogenetic inference and robust taxonomy to develop a synthetic tree of life. We capitalize on this approach to estimate a synthesis tree for the freshwater crayfish. The crayfish make an exceptional group to demonstrate the utility of the synthesis approach, as there recently have been a number of phylogenetic studies on the crayfishes along with a robust underlying taxonomic framework. Importantly, the crayfish have also been extensively assessed by an IUCN Red List team and therefore have accurate and up-to-date area and conservation status data available for analysis within a phylogenetic context. Here, we develop a synthesis phylogeny for the worlds freshwater crayfish and examine the phylogenetic distribution of threat. We also estimate a molecular phylogeny based on all available GenBank crayfish sequences and use this tree to estimate divergence times and test for divergence rate variation. Finally, we conduct EDGE and HEDGE analyses and identify a number of species of freshwater crayfish of highest priority in conservation efforts.

ON STABILISING THE NAMES OF THE INFRAORDERS OF THALASSINIDEAN SHRIMPS, AXIIDEA DE SAINT LAURENT, 1979 AND GEBIIDEA DE SAINT LAURENT, 1979 (DECAPODA)

The names Gebiidea and Axiidea, erected by de Saint Laurent (1979), have priority over others for the two infraorders of shrimps previously included in Thalassinidea. Importantly, Thalassinidea are not monophyletic and the name should be replaced. Gebiidea and Axiidea, besides having priority and describing two monophyletic taxa, are now in common use (130 citations) and are more stable than alternative schemes proposed by Sakai (2005 and later). The history of the names of higher taxa applied to these groups is reviewed, and all family-group taxa listed.

The Forest for the Trees: Evaluating Molecular Phylogenies with an Emphasis on Higher-Level Decapoda

Since the late 1800s, several infraordinal relationships have been proposed for Decapoda; however, reaching a consensus among higher-level relationships is proving difficult. Molecular methods were first applied to higher-level decapod phylogenetics in the 1990s and have significantly contributed to our understanding of the group: sampling is becoming more thorough, a greater number of phylogenetically informative characters are being sequenced, and analysis procedures are becoming more consistent between studies. However, relationships among the deep lineages of Decapoda remain unclear. Several phylogenetic hypotheses have been suggested, and while there is some agreement among studies, an ultimate consensus among higher-level relationships has yet to be reached. This is largely the result of differences in sampling effort, marker selection, data-recycling, and analysis. Because most studies have generated conflicting phylogenetic hypotheses, the foundation on which the trees were built (data and analysis procedures) must be considered and evaluated. In this review, we summarize the early morphological decapod studies, address common problems that are causing a lack of consensus in molecular studies, present a means of evaluating molecular trees, offer suggestions for good phylogenetic practice, review the previous molecular studies of infraordinal decapod phylogeny, and discuss the future directions of the field, with special attention paid to next-generation sequencing (NGS) techniques.

Phylogenetic evidence from freshwater crayfishes that cave adaptation is not an evolutionary dead-end

Caves are perceived as isolated, extreme habitats with a uniquely specialized biota, which long ago led to the idea that caves are “evolutionary dead‐ends.” This implies that cave‐adapted taxa may be doomed for extinction before they can diversify or transition to a more stable state. However, this hypothesis has not been explicitly tested in a phylogenetic framework with multiple independently evolved cave‐dwelling groups. Here, we use the freshwater crayfish, a group with dozens of cave‐dwelling species in multiple lineages, as a system to test this hypothesis. We consider historical patterns of lineage diversification and habitat transition as well as current patterns of geographic range size. We find that while cave‐dwelling lineages have small relative range sizes and rarely transition back to the surface, they exhibit remarkably similar diversification patterns to those of other habitat types and appear to be able to maintain a diversity of lineages through time. This suggests that cave adaptation is not a “dead‐end” for freshwater crayfish, which has positive implications for our understanding of biodiversity and conservation in cave habitats.