Abyssal fauna of the UK-1 polymetallic nodule exploration claim, Clarion-Clipperton Zone, central Pacific Ocean: Echinodermata

Abstract We present data from a DNA taxonomy register of the abyssal benthic Echinodermata collected as part of the Abyssal Baseline (ABYSSLINE) environmental survey cruise ‘AB01’ to the UK Seabed Resources Ltd (UKSRL) polymetallic-nodule exploration claim ‘UK-1’ in the eastern Clarion-Clipperton Zone (CCZ), central Pacific Ocean abyssal plain. Morphological and genetic data are presented for 17 species (4 Asteroidea, 4 Crinoidea, 2 Holothuroidea and 7 Ophiuroidea) identified by a combination of morphological and genetic data. No taxa matched previously published genetic sequences, but 8 taxa could be assigned to previously-described species based on morphology, although here we have used a precautionary approach in taxon assignments to avoid over-estimating species ranges. The Clarion-Clipperton Zone is a region undergoing intense exploration for potential deep-sea mineral extraction. We present these data to facilitate future taxonomic and environmental impact study by making both data and voucher materials available through curated and accessible biological collections.


Introduction
We present data from a DNA taxonomy register of the abyssal benthic Echinodermata collected as part of the Abyssal Baseline (ABYSSLINE) environmental survey cruise 'AB01' to the UK Seabed Resources Ltd (UKSRL) polymetallic-nodule exploration claim 'UK-1' in the eastern Clarion-Clipperton Zone (CCZ), central Pacific Ocean .
This paper is the start of an iterative approach to providing regional taxonomic synthesis for a region that is undergoing intense deep-sea mineral exploration for high-grade polymetallic nodules regulated by Sponsoring States (here the United Kingdom Government) and the International Seabed Authority (ISA 2014b, Smith 2003, Wedding et al. 2015). Our study is not yet a comprehensive faunal guide to the region, but a data paper that will be updated with new additions following future collections and analyses. New versions will contain all the data contained in the previous version, plus additional descriptions and records from future research cruises.
The abyssal zone of the world's oceans has been defined as the seafloor between depths of 3000m and 6000m, a bathymetric zone that encompasses 54% of the geographic surface of the planet (Smith et al. 2008). Echinoderms form a characteristic and abundant group in this region. Current online data sources list 698 echinoderm species recorded at abyssal depths from between 3000m and 6000m (OBIS 2015) out of a total of 3,272 echinoderm species recorded from depths greater than 500m (Glover et al. 2015).
The Clarion-Clipperton Zone (hereafter, CCZ) is so called as it lies between the Clarion and Clipperton Fracture Zones, topographical highs that extend longitudinally across almost the entire eastern Pacific. There is no strict definition of the region, but it has come to be regarded as the area between these fracture zones that lies within international waters and encompasses the main areas of commercial interest for polymetallic-nodule mining. Areas licensed for mining by the International Seabed Authority (ISA), as well as mining reserve areas and areas protected from mining by the ISA (ISA 2014a, Wedding et al. 2013 extend from 115°W (the easternmost extent of the UK-1 claim) to approximately 158°W, and from 22°N to 2.5°S (Fig. 1). This is an area of 6 million sq km, approximately 1.7% of the ocean's surface.
Within the 6 million sq km CCZ, as defined above, current online data sources prior to this publication list only 50 known species of echinoderms from 290 records (OBIS 2015). This is obviously the result of lack of sampling and/or taxonomy given that an abundant and diverse echinoderm fauna is suspected in the region from photographic and video survey (e.g. Foell and Pawson 1986). The goal of the DNA taxonomy part of the ABYSSLINE program is to start to rectify these gaps in our knowledge and make data publically available that will eventually allow for a complete taxonomic synthesis of the CCZ supported by openly-available molecular and morphological data. Here we provide version 1.0 of the Echinodermata taxonomic synthesis from the ABYSSLINE program, consisting of taxon records, high-resolution imagery, genetic data from multiple markers and phylogenetic analysis from the first research cruise (AB01) aboard the RV Melville in October 2013. This open data publication is intended to be supported by equivalent similar data publications on the Annelida, Mollusca, Bryozoa, Cnidaria, Porifera and other taxa forming a suite of taxonomic syntheses of biodiversity in the region, supported by a contract between the company UK Seabed Resources Ltd and the Natural History Museum, London and Uni Research, Bergen, and the University of Hawaii at Manoa.

Materials and methods
It is widely accepted that knowledge of baseline biodiversity and biogeography in the CCZ is severely hampered by a lack of modern DNA-supported taxonomic studies (ISA 2014b). With this in mind, four fundamental principles underpin our methodological pipeline: (1) A sampling design pipeline with consideration to the spatial scale of the required data, the differing biases in sampling gear and the requirement for at-sea taxonomic study, (2) A field pipeline with consideration to the successful collection of high-quality specimens using The Clarion-Clipperton Zone, central Pacific Ocean (purple box) is a 6 milllion km region at the time of writing containing only 290 online-databased records of echinoderm species (OBIS 2015). The UK Seabed Resources Ltd 'UK-1' polymetallic nodule exploration claim area is highlighted (yellow box).
live-sorting in a 'cold-chain' from depths of 4000-5000m in the central tropical Pacific, (3) A laboratory pipeline with consideration to the needs to collect both DNA sequences and morphological data in a timely and cost-effective manner suited to the immediate needs of the science community and (4) A data and sample management pipeline that includes the publication of results with consideration to the accessibility of data and materials. Our complete methodology for DNA taxonomy in the CCZ, including deployment protocols for the various sampling gears, methods for live-sorting and microscope photography at sea and details of sample and data curation are provided in a separate open-access publication (Glover et al. 2016).

Field pipeline
The ABYSSLINE environmental baseline survey includes three 30x30km survey boxes (strata), distributed across the UK-1 claim area, and an additional reference sites outside of the UK-1 claim area (Smith et al. 2013b, Glover et al. 2016. Within each survey stratum, sample sites for a variety of benthic sampling gears are selected randomly -a randomized, stratified sampling design that assumes no a priori knowledge of the benthic environment (Smith et al. 2013b). The UK-1 strata are being sampled in a series of oceanographic cruises during the course of the project, which commenced in July 2013, with the first cruise (AB01) taking place in October 2013 aboard the RV Melville. During this cruise, the first stratum was comprehensively mapped with multibeam bathymetry and sampled for a range of biological, environmental and geophysical parameters (Fig. 2, Smith et al. 2013). 'UK-1 Stratum A' ABYSSLINE biological baseline survey box sited within the UK-1 polymetallic nodule exploration claim. Stratum A is a 30x30km survey box in the northern sector of the 58,000 km claim area. Echinoderm sample localities are indicated by green circles from the AB01 RV Melville survey cruise, October 2013. Inset map A: the site location within the central Pacific, inset map B: all the echinoderm sampling locations (including site 'ROV7' to the west). Both inset maps use GEBCO 2014 bathymetry (global 30 arc-second interval grid data set). Seafloor bathymetry from the RV Melville ABYSSLINE cruise is shown in the main map. 2 A comprehensive description of our DNA taxonomy pipeline is provided in Glover et al. 2016. In summary, deep-sea benthic specimens from the UK-1 Stratum A were collected using a range of oceanographic sampling gears including box core (BC), epibenthic sledge (EBS), remotely operated vehicle (ROV) and megacore (MC) (Fig. 2, Fig. 3). Geographic data from sampling activities were recorded on a central GIS database. Live-sorting of sediment and specimen samples was carried out aboard the RV Melville under the 'coldchain' pipeline, in which material was immediately transferred and maintained in chilled, filtered seawater held at 2-4°C (Fig. 3). Specimens were preliminarily identified at sea and imaged live using stereomicroscopes with attached digital cameras and strobe lighting. The specimens were then transferred to individual microtube vials containing an aqueous solution of 80% non-denatured ethanol, numbered and barcoded into a database and kept chilled until return to the Natural History Museum (NHM), London. Larger, megafaunalsized, animals were sub-sampled for DNA (with the tissue and DNA sample being taken to NHM, London) with the remaining intact specimen preserved in 10% formalin solution and taken to the University of Hawaii, Honolulu, USA for further study. ABYSSLINE UK-1 polymetallic nodule exploration claim field pipeline for DNA taxonomy. ABYSSLINE AB01 cruise sampling aboard RV Melville in October 2013. (a) Preparing Box Core (BC) for deployment, (b) BC entering the water, (c) Megacore entering the water, (d-f) Epibenthic Sledge shown on recovery in water and cod-end where samples are taken, (g) controlling BC deployment on seafloor, (h) echosounder trace showing BC approaching seabed reflection, (i) successful BC surface after recovery, 50cm x 50cm, (j) carefully sifting mud in chilled filtered seawater (approx. temp 5-7°C) to remove live animals in undamaged state, (k) live-sorting aboard ship, taking samples for DNA and photomicrographs of specimens down to <1mm in size. All images by Glover, Dahlgren & Wiklund. A more comprehensive description of our methods is provided in Glover et al. 2016.

Laboratory pipeline
Extraction of DNA was done with DNeasy Blood and Tissue Kit (Qiagen) using a Hamilton Microlab STAR Robotic Workstation. About 1800 bp of 18S, 450 bp of 16S, and 650 bp of cytochrome c oxidase subunit I (COI) were amplified using primers listed in Table 1. PCR mixtures contained 1 µl of each primer (10µM), 2 µl template DNA and 21 µl of Red Taq DNA Polymerase 1.1X MasterMix (VWR) in a mixture of total 25 µl. The PCR amplification profile consisted of initial denaturation at 95°C for 5 min, 35 cycles of denaturation at 94°C for 45 s, annealing at 55°C for 45 s, extension at 72°C for 2 min, and a final extension at 72°C for 10 min. PCR products were purified using Millipore Multiscreen 96-well PCR Purification System, and sequencing was performed on an ABI 3730XL DNA Analyser (Applied Biosystems) at The Natural History Museum Sequencing Facility, using the same primers as in the PCR reactions plus two internal primers for 18S (Table 1).

Data pipeline
The field and laboratory pipelines created a series of databases and sample sets that were then integrated into a data-management pipeline (Fig. 4). This includes the transfer and management of data and samples between a central collections database, a molecular collections database, an online scratchpad (website for faunal data) and external repositories (e.g GenBank, WoRMS, OBIS, GBIF) through a DarwinCore archive. This provides a robust data framework to support DNA taxonomy, in which openly-available data and voucher material is key to quality data standards. A further elaboration of the data pipeline is published in Glover et al. 2016

Taxonomic assignments
All future studies of biogeographic and bathymetric ranges, gene-flow, extinction risks, natural history, reproductive ecology, functional ecology and geochemical interactions of CCZ species are dependent on accurate identifications faciliated by taxonomy. This taxonomy, presented here, is itself dependent on a sound theoretical underpinning -a species concept -coupled with the availability of both raw data and voucher samples. Here we use a phylogenetic species concept, sensu Donoghue 1985 with species determined by DNA-based phylogenetic analysis and the recognition of distinct monophyletic groups as species. For those taxa where the typical morphological data that allows determination of species are missing, we provide the lowest-level taxonomic name possible, but Data and sample management workflow on the ABYSSLINE DNA taxonomy project. Processes relating to a) physical samples are shown in grey, b) institution level data in dark green and c) externally-available data in blue.
determination to species with genetic data. For species similar to a morphologically well defined species name where we lack comparable genetic data from type material or from the type locality, or when genetic data previously published in Genbank is incompatible with ours, we use the open nomenclature expression "cf.". Material (including archived frozen tissue) and genetic data are accessible through the Natural History Museum, London, together with the morphological data presented in this paper, original specimens for some larger (megafaunal) taxa remain in the collection of Craig R. Smith, University of Hawaii -these specimens are indicated in the taxon treatments below. As such our species hypotheses are easily open to further evaluation and iterative improvement, e.g. full descriptions for new taxa with improved data from future cruises. A localised identification field guide to the CCZ fauna will be the subject of a future publication as more species are described, but for the present we recommend DNA-based identification (barcoding) of our species coupled with morphological comparisons made possible through this publication.

Data resources
The following sections detail the phylogenetic analysis and data resources that underpins the species hypotheses presented in the taxon treatments. A full list of all taxa including Natural History Museum Accession Numbers, NHM Molecular Collection Facility (NHM-MCF) FreezerPro numbers and NCBI GenBank Accession numbers is provided in Table 2.

Phylogenetic analysis of the Asteroidea
Phylogenetic analysis of the Asteroidea (Fig. 5) reveals the presence of 4 distinct lineages of ABYSSLINE specimens which we interpret as the 4 species described below based on their genetic data.

Phylogenetic analysis of the Crinoidea
Phylogenetic analysis of the Crinoidea (Fig. 6) reveals the presence of 4 distinct lineages of ABYSSLINE specimens which we interpret as the 4 species described below based on their genetic data. Phylogenetic analysis of the Asteroidea. 50% majority rule consensus tree from the Bayesian analyses, combining the three genes 18S, 16S and COI and using in total 60 taxa. Some of the clades are collapsed in order to make the tree smaller and easier to read.

Phylogenetic analysis of the Holothuroidea
Phylogenetic analysis of the Holothuroidea (Fig. 7) reveals the presence of 2 distinct lineages of ABYSSLINE specimens which we interpret as the 2 species described below based on their genetic data. Phylogenetic analysis of the Crinoidea. 50% majority rule consensus tree from the Bayesian analyses, combining the three genes 18S, 16S and COI and using in total 113 taxa. Some of the clades are collapsed in order to make the tree smaller and easier to read.

Phylogenetic analysis of the Ophiuroidea
Phylogenetic analysis of the Ophiuroidea (Fig. 8) the presence of 7 distinct lineages of ABYSSLINE specimens which we interpret as the 7 species described below based on their genetic data. Phylogenetic analysis of the Holothuroidea. 50% majority rule consensus tree from the Bayesian analyses, using 16S and in total 115 taxa. Some of the clades are collapsed in order to make the tree smaller and easier to read.  Phylogenetic analysis of the Ophiuroidea. 50% majority rule consensus tree from the Bayesian analyses, combining the three genes 18S, 16S and COI and using in total 79 taxa. Some of the clades are collapsed in order to make the tree smaller and easier to read.
Genetic data for this taxa with new GenBank accession numbers are provided in Table  2 Diagnosis Morphologically and genetically close to Eremicaster sp (Fig. 5) Description 15cm long arm fragments of a Freyellidae recovered from ROV biobox. Identified by DNA and morphological examination (Fig. 10). Morphological identification suggests Freyastera benthophila detailed in Sladen 1889).
Genetic data for this taxa with new GenBank accession numbers are provided in Table  2.
Genetic data for this taxa with new GenBank accession numbers are provided in Table  2.
Genetic data for this taxa with new GenBank accession numbers are provided in Table  2.

Diagnosis
Morphologically matches diagnosis of Styracaster paucispinus based on descriptions in Madsen 1961, Ludwig 1905

Description
Calyx 1.5mm long and 1.4mm wide with arms possibly incomplete. Arms present with 0.24mm in width, 0.95mm in length. Total length of calyx and distal part of stalk preserved 6.5mm. Stalk 0.32mm in width, stalk columnals approx 1mm in length. (Fig.  13) Genetic data for this taxa with new GenBank accession numbers are provided in Table  2.

Diagnosis
Morphologically close to Hyocrinus foelli Roux and Pawson 1999 but incomplete specimen prevents full identification. Forms a unique monophyletic clade distinct from other AB01 specimens. No genetic matches on GenBank or Barcode of Life Database.

Ecology
Specimen observed live on a small potato-sized polymetallic nodule from the eastern CCZ abyssal plain.

Description
Specimen including stalk and crown, calyx with arms, 8mm in total length, 5 arms, 0.31mm in width, as present in original specimen, prior to DNA sampling, with length 1.3mm from distal portion of calyx. Distally, pinnules observed on arms (Fig. 14).
Genetic data for this taxa with new GenBank accession numbers are provided in Table  2.

Diagnosis
Forms a unique monophyletic clade distinct from other AB01 specimens. No genetic matches on GenBank or Barcode of Life Database.

Ecology
Found living on polymetallic nodule.

Description
Specimen including stalk and crown, calyx with proximal arms only, 5mm in total length. Calyx 0.62mm in width, including proximal arms 0.86mm in length. Distally, pinnules observed on arms arising laterally from arms (Fig. 15).
Genetic data for this taxa with new GenBank accession numbers are provided in Table  2.

Diagnosis
Forms a unique monophyletic clade distinct from other AB01 specimens. No genetic matches on GenBank or Barcode of Life Database.
Genetic data for this taxa with new GenBank accession numbers are provided in Table  2.

Diagnosis
Forms a unique monophyletic clade distinct from other AB01 specimens. No genetic matches on GenBank or Barcode of Life Database. Lacks crown and calyx.

Ecology
Found on polymetallic nodule.
Genetic data for this taxa with new GenBank accession numbers are provided in Table  2.

Diagnosis
Forms a unique monophyletic clade distinct from other AB01 specimens and no match (16S) to any GenBank or BOLD databases. Morphologically consistent with Benthodytes sanguinolenta or B. typica. The type locality of B. sanguinolenta is in the Pacific ocean (34°7'S; 73°56'W, 4000m depth) while type locality of B. typica is Atlantic (35°47'N; 8°23'W, 2000m depth) (Théel 1882). We assign the tentative name Benthodytes cf. sanguinolent to this material until we have a better understanding of genetic variation within the species B. sanguinolenta and B. typica including data from the type localities.
Genetic data for this taxa with new GenBank accession numbers are provided in Table  2.

Diagnosis
Forms a unique monophyletic clade distinct from other AB01 specimens and no match to any GenBank or BOLD databases. Morphologically consistent with Psychropotes semperiana Théel, 1882. The type locality of Psychropotes semperiana is Atlantic (5°4 8'N; 14°20'W, 4500m depth) and we use the tentative name Psychropotes cf. semperiana for this material until we have a better understanding of genetic variation within the species including data from the type locality.

Ecology
Observed moving on the seabed amongst polymetallic nodules.

Description
Voucher material recovered from megacore sample, specimen with disc of 1cm diameter (Fig. 19). Additional material including juveniles recovered from box core and epibenthic sledge. Agrees with Amphioplus (Unioplus) daleus as detailed in (Lyman 1879).
Genetic data for this taxa with new GenBank accession numbers are provided in Table  2.

Diagnosis
Forms a unique monophyletic clade distinct from other AB01 specimens.
Morphologically consistent with Amphioplus (Unioplus) daleus Lyman 1879. No genetic data for this species yet on GenBank. The type locality of A. daleus is Atlantic (36°4 4'S; 46°16'W; 4800m depth) and we use the tentative name Amphioplus cf. daleus for this material until we have a better understanding of genetic variation within the species including data from the type locality.

Ecology
Recovered from a range of sampling gears, NHM_447 recovered alive in top of multiple core tube.

Description
Voucher material, NHM_329, disc approx 20mm in diameter. Additional voucher material (12 specimens) ranges in size from 2mm to 20mm in diameter (Fig. 20). Range of polymorphs observed characterised by pattern of disc dorsal coloration (Fig.  20). Juveniles observed and identified from DNA data.
Genetic data for this taxa with new GenBank accession numbers are provided in Table  2.

Ecology
The most abundant brittle-star in the UK-1 exploration claim survey box UK-1 Stratum A, frequently observed by the ROV on the sediment surface and on nodules.

Description
Voucher material, consisting of a series of fragments of arms and one partial disc. All material specimens form a monophyletic clade based on DNA. Arm processes (parasols) suggestive of Ophiotholia sp affinity. In NHM_076, arms are 0.31mm wide, with parasol-shaped processes of 0.19mm in length, parasols, 0.048mm in width (Fig.  21).
Genetic data for this taxa with new GenBank accession numbers are provided in Table  2.

Diagnosis
Forms a unique monophyletic clade distinct from other AB01 specimens. Morphologically perhaps close to Ophiotholia but requires further sampling.

Ecology
Specimens recovered from two box cores, two specimens from each. Specimens from the same box cores genetically identical, so could be fragments of the same species.

Description
Small disc fragments found in several samples, distinct petal arrangement visible ventrally (Fig. 22). Genetic data for this taxa with new GenBank accession numbers are provided in Table  2.

Diagnosis
Forms a unique monophyletic clade distinct from other AB01 specimens.

Description
Small fragment consisting of orange-coloured disc, arms absent or missing (Fig. 22).
Genetic data for this taxa with new GenBank accession numbers are provided in Table  2.

Diagnosis
Forms a unique monophyletic clade distinct from other AB01 specimens.
Morphologically not recognisable. Genetic data for this taxa with new GenBank accession numbers are provided in Table  2.

Diagnosis
Forms a unique monophyletic clade distinct from other AB01 specimens. Morphologically agrees with Perlophiura profundissima but no genetic data available from type locality or any location for this taxon but type locality appears to be North Pacific at abyssal depths (Belyaev and Litvinova 1972).

Discussion
Within the entire 6 million sq km Clarion Clipperton Zone, the best current online databases (OBIS 2015) list only 290 echinoderm records from 50 species. In this study, we report 48 new records from 17 species. This is an increase of ~25% for echinoderm species records from just a single 25-day cruise to a 30x30km location, with a relatively modest number of samples. All of our data are publically available through the Darwin Core outputs on this manuscript which are automatically fed into data aggregators such as GBIF and OBIS . All of our species determinations are supported by molecular DNA sequences, the data made available on GenBank and the voucher materials deposited in the Molecular Collections Facility of the Natural History Museum, London where they are available for future study by research visit or loan.
It is noteworthy that there was not a single 100% match for any of our sequences obtained with data on either NCBI GenBank or BOLD databases (BOLD 2015). This observation reinforces the point that there are very few taxonomic or genetic data available on the benthic biology of this region, an area undergoing intense exploration for mineral resources within the framework of the International Seabed Authority regulatory system (Wedding et al. 2015). The ISA has recently recognised the need for urgent action to make taxonomic data for the CCZ available from the large number of research cruises that are taking place supported by Sponsoring States (national governments) or private contractors (ISA 2014a). It is interesting to note that in the first 6 months of 2015 alone, there have been 3 largescale benthic biology cruises to the eastern end of the CCZ, supported by the ABYSSLINE project (funded by UK Seabed Resources Ltd, cruise AB02), the German Government (the EU JPI cruise aboard the RV Sonne) and the EU 'MIDAS' programme (cruise JC120, partially funded by the Natural Environment Research Council, UK). As an example, from the ABYSSLINE cruise AB02 in March 2015 alone, we have an additional 289 echinoderm samples that are currently being identified and analysed for DNA; these results will be reported in future data papers over the course of the ABYSSLINE project.
The lack of comparative genetic data also has implications for our understanding of species ranges. We know that cryptic diversity is common in the deep sea (Knowlton 2000, Havermans et al. 2013, Jennings et al. 2013. With the routine use of molecular data in taxonomy, we will be better at detecting sibling species and defining species ranges, including for species considered to have distributions across multiple ocean basins and over very wide bathymetric ranges. To avoid exaggerated species ranges, we here follow a precautionary principle and have therefore avoided the use of taxon names based on morphological similarity alone unless the identity also is corroborated by a justifyable bathymetric and geographic proximity to type locality of the species name (i.e. abyssal Pacific).
The increased activity in terms of research cruises and sample collection in the CCZ make it more important than ever to provide taxonomic data quickly for an iterative building of baseline biodiversity knowledge in the CCZ region. Making these data available through rapid publication in open-access journals that support data-aggregator online systems is a key first step in this process.