Non-invasive real-time genomic monitoring of the critically endangered kakapo.

We used non-invasive real-time genomic approaches to monitor one of the last surviving populations of the critically endangered kakapo (Strigops habroptilus). We first established an environmental DNA metabarcoding protocol to identify the distribution of kakapo and other vertebrate species in a highly localized manner using soil samples. Harnessing real-time nanopore sequencing and the high-quality kakapo reference genome, we then extracted species-specific DNA from soil. We combined long read-based haplotype phasing with known individual genomic variation in the kakapo population to identify the presence of individuals, and confirmed these genomically informed predictions through detailed metadata on kakapo distributions. This study shows that individual identification is feasible through nanopore sequencing of environmental DNA, with important implications for future efforts in the application of genomics to the conservation of rare species, potentially expanding the application of real-time environmental DNA research from monitoring species distribution to inferring fitness parameters such as genomic diversity and inbreeding.

Confusion will be my epitaph: genome-scale discordance stifles phylogenetic resolution of Holothuroidea.

Sea cucumbers (Holothuroidea) are a diverse clade of echinoderms found from intertidal waters to the bottom of the deepest oceanic trenches. Their reduced skeletons and limited number of phylogenetically informative traits have long obfuscated morphological classifications. Sanger-sequenced molecular datasets have also failed to constrain the position of major lineages. Noteworthy, topological uncertainty has hindered a resolution for Neoholothuriida, a highly diverse clade of Permo-Triassic age. We perform the first phylogenomic analysis of Holothuroidea, combining existing datasets with 13 novel transcriptomes. Using a highly curated dataset of 1100 orthologues, our efforts recapitulate previous results, struggling to resolve interrelationships among neoholothuriid clades. Three approaches to phylogenetic reconstruction (concatenation under both site-homogeneous and site-heterogeneous models, and coalescent-aware inference) result in alternative resolutions, all of which are recovered with strong support and across a range of datasets filtered for phylogenetic usefulness. We explore this intriguing result using gene-wise log-likelihood scores and attempt to correlate these with a large set of gene properties. While presenting novel ways of exploring and visualizing support for alternative trees, we are unable to discover significant predictors of topological preference, and our efforts fail to favour one topology. Neoholothuriid genomes seem to retain an amalgam of signals derived from multiple phylogenetic histories.

Formalin-fixed paraffin-embedded (FFPE) samples help to investigate transcriptomic responses in wildlife disease.

Infectious diseases impact numerous organisms. Knowledge of host-pathogen interactions and host responses to infection is crucial for conservation and management. Obtaining this knowledge quickly is made increasingly possible by a variety of genomic approaches, yet, for many species the bottleneck to understanding this, remains access to appropriate samples and data. Lack of sample availability has also limited our understanding of how pathogens and the immune responses of hosts change over time. Archival materials may provide a way to explore pathogen emergence and host responses over multiple-possibly hundreds-of years. Here, we tested whether formalin-fixed paraffin-embedded (FFPE) tissue samples could be used to understand an unknown pathology, lamprey reddening syndrome (LRS), affecting pouched lampreys (Geotria australis). Our differential expression analyses of dermal tissues from four unaffected lampreys and eight affected lampreys collected in 2012 alluded to several potential agents associated with LRS. Interestingly, the pathways associated with viral infections were overrepresented in affected versus unaffected lamprey. Gene ontology analyses of the affected and non-affected lampreys also provided new insights into the largely understudied immune responses of pouched lampreys. Our work confirms that FFPE samples can be used to infer information about the transcriptional responses of a wildlife species affected by unknown historical pathologies/syndromes. In addition, the use of FFPE samples for transcriptomics offers many opportunities to investigate the genomic responses of a species to a variety of environmental changes. We conclude with a discussion about how to best sample and utilize these unique archival resources for future wildlife transcriptomic studies.

An improved germline genome assembly for the sea lamprey Petromyzon marinus illuminates the evolution of germline-specific chromosomes.

Programmed DNA loss is a gene silencing mechanism that is employed by several vertebrate and nonvertebrate lineages, including all living jawless vertebrates and songbirds. Reconstructing the evolution of somatically eliminated (germline-specific) sequences in these species has proven challenging due to a high content of repeats and gene duplications in eliminated sequences and a corresponding lack of highly accurate and contiguous assemblies for these regions. Here, we present an improved assembly of the sea lamprey (Petromyzon marinus) genome that was generated using recently standardized methods that increase the contiguity and accuracy of vertebrate genome assemblies. This assembly resolves highly contiguous, somatically retained chromosomes and at least one germline-specific chromosome, permitting new analyses that reconstruct the timing, mode, and repercussions of recruitment of genes to the germline-specific fraction. These analyses reveal major roles of interchromosomal segmental duplication, intrachromosomal duplication, and positive selection for germline functions in the long-term evolution of germline-specific chromosomes.

A new species of the diogenid hermit crab genus Pseudopaguristes McLaughlin, 2002 from the Northern Mariana Islands, Micronesia.

The diogenid hermit crab genus Pseudopaguristes McLaughlin, 2002 is currently represented by 15 species worldwide. In this study, we describe a new species of the genus, P. flavioculus, based on a unique male holotype from off Sarigan, Northern Mariana Islands, Micronesia, at depth of 131133 m. The new species appears similar to the type species of the genus, P. janetkae McLaughlin, 2002, but the lack of conspicuous spines on the dorsal face of the right cheliped carpus, the possession of a median row of tiny spines on the dorsal face of the left cheliped carpus, and different colour pattern of the cephalic appendages and the ambulatory legs distinguish P. flavioculus n. sp. from P. janetkae. Pseudopaguristes has been differentiated from the two allied genera, Paguristes Dana, 1851 and Areopaguristes Rahayu, 2012, by the possession of eight functional pairs of gills (versus 13 pairs in Paguristes and 12 pairs in Areopaguristes), but our examination of the new species and three other congeneric species available for study clarifies that the development of arthrobranchs on the maxillipeds 3 and chelipeds is rather variable in species currently assigned to Pseudopaguristes. Careful observation of the anterior arthrobranchs is recommended to avoid misinterpretation in the counts of gill numbers.

Population Genomics of New Zealand Pouched Lamprey (kanakana; piharau; Geotria australis).

Pouched lamprey (Geotria australis) or kanakana/piharau is a culturally and ecologically significant jawless fish that is distributed throughout Aotearoa New Zealand. Despite its importance, much remains unknown about historical relationships and gene flow between populations of this enigmatic species within New Zealand. To help inform management, we assembled a draft G. australis genome and completed the first comprehensive population genomics analysis of pouched lamprey within New Zealand using targeted gene sequencing (Cyt-b and COI) and restriction site-associated DNA sequencing (RADSeq) methods. Employing 16 000 genome-wide single nucleotide polymorphisms (SNPs) derived from RADSeq (n = 186) and sequence data from Cyt-b (766 bp, n = 94) and COI (589 bp, n = 20), we reveal low levels of structure across 10 sampling locations spanning the species range within New Zealand. F-statistics, outlier analyses, and STRUCTURE suggest a single panmictic population, and Mantel and EEMS tests reveal no significant isolation by distance. This implies either ongoing gene flow among populations or recent shared ancestry among New Zealand pouched lamprey. We can now use the information gained from these genetic tools to assist managers with monitoring effective population size, managing potential diseases, and conservation measures such as artificial propagation programs. We further demonstrate the general utility of these genetic tools for acquiring information about elusive species.

Three new species of pagurid hermit crabs (Decapoda: Anomura: Paguroidea) from the Northern Mariana Islands, Micronesia.

We describe three new species of hermit crab of the pagurid genera, Catapagurus A. Milne-Edwards, 1880, Nematopagurus A. Milne-Edwards Bouvier, 1892 and Pagurixus Melin, 1939, on the basis of material collected during the scientific cruise to the Northern Mariana Islands conducted by the U.S. National Oceanographic and Atmospheric Administration (NOAA) in 2015. Catapagurus tenuilamina n. sp., referred to the C. ensifer Henderson, 1893 species group, appears close to C. granulatus Edmondson, 1951, but differs from the latter in the more slender ultimate article of the antennular peduncle, more slender dactyli of pereopods 2 and 3, and the possession of two dorsal subdistal spines in the meri of the pereopods. Nematopagurus marianicus n. sp. resembles N. gardineri Alcock, 1905 and N. lewinsohni Trkay, 1986, but is distinguished from the latter two by the characteristic dense setation on the upper part of the left cheliped carpus and by the elongate dactyli of pereopods 2 and 3 that lack a row of bristle-like setae on the dorsal margins. Pagurixus eminens n. sp. is referred to the P. boninensis Melin, 1939 species group and appears closest to P. annulus Komai Poupin, 2013, but is differentiated from the latter in the shape and ornamentation of the right coxa of pereopod 5 in the male as well as details of the colour pattern on the chelipeds and ambulatory legs. Partial sequences of the mitochondrial 16S rRNA gene from the three new species were sequenced and deposited in the GenBank database for future genetic and phylogenetic study.

Slippery when wet: cross-species transmission of divergent coronaviruses in bony and jawless fish and the evolutionary history of the Coronaviridae.

The Nidovirales comprise a genetically diverse group of positive-sense single-stranded RNA virus families that infect a range of invertebrate and vertebrate hosts. Recent metagenomic studies have identified nido-like virus sequences, particularly those related to the Coronaviridae, in a range of aquatic hosts including fish, amphibians, and reptiles. We sought to identify additional members of the Coronaviridae in both bony and jawless fish through a combination of total RNA sequencing (meta-transcriptomics) and data mining of published RNA sequencing data and from this reveal more of the long-term patterns and processes of coronavirus evolution. Accordingly, we identified a number of divergent viruses that fell within the Letovirinae subfamily of the Coronaviridae, including those in a jawless fish-the pouched lamprey. By mining fish transcriptome data, we identified additional virus transcripts matching these viruses in bony fish from both marine and freshwater environments. These new viruses retained sequence conservation in the RNA-dependant RNA polymerase across the Coronaviridae but formed a distinct and diverse phylogenetic group. Although there are broad-scale topological similarities between the phylogenies of the major groups of coronaviruses and their vertebrate hosts, the evolutionary relationship of viruses within the Letovirinae does not mirror that of their hosts. For example, the coronavirus found in the pouched lamprey fell within the phylogenetic diversity of bony fish letoviruses, indicative of past host switching events. Hence, despite possessing a phylogenetic history that likely spans the entire history of the vertebrates, coronavirus evolution has been characterised by relatively frequent cross-species transmission, particularly in hosts that reside in aquatic habitats.

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.

Molecular phylogeny of extant Holothuroidea (Echinodermata).

Sea cucumbers (Holothuroidea) are a morphologically diverse, ecologically important, and economically valued clade of echinoderms; however, the understanding of the overall systematics of the group remains controversial. Here, we present a phylogeny of extant Holothuroidea assessed with maximum parsimony, maximum likelihood, and Bayesian approaches using approximately 4.3kb of mt- (COI, 16S, 12S) and nDNA (H3, 18S, 28S) sequences from 82 holothuroid terminals representing 23 of the 27 widely-accepted family-ranked taxa. Currently five holothuroid taxa of ordinal rank are accepted. We find that three of the five orders are non-monophyletic, and we revise the taxonomy of the groups accordingly. Apodida is sister to the rest of Holothuroidea, here considered Actinopoda. Within Actinopoda, Elasipodida in part is sister to the remaining Actinopoda. This latter clade, comprising holothuroids with respiratory trees, is now called Pneumonophora. The traditional Aspidochirotida is paraphyletic, with representatives from three orders (Molpadida, Dendrochirotida, and Elasipodida in part) nested within. Therefore, we discontinue the use of Aspidochirotida and instead erect Holothuriida as the sister group to the remaining Pneumonophora, here termed Neoholothuriida. We found four well-supported major clades in Neoholothuriida: Dendrochirotida, Molpadida and two new clades, Synallactida and Persiculida. The mapping of traditionally-used morphological characters in holothuroid systematics onto the phylogeny revealed marked homoplasy in most characters demonstrating that further taxonomic revision of Holothuroidea is required. Two time-tree analyses, one based on calibrations for uncontroversial crown group dates for Eleutherozoa, Echinozoa and Holothuroidea and another using these calibrations plus four more from within Holothuroidea, showed major discrepancies, suggesting that fossils of Holothuroidea may need reassessment in terms of placing these forms with existing crown clades.

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.