Megafauna of the UKSRL exploration contract area and eastern Clarion-Clipperton Zone in the Pacific Ocean: Echinodermata

Abstract Background There is growing interest in mining polymetallic nodules from the abyssal Clarion-Clipperton Zone (CCZ) in the tropical Pacific Ocean. Despite being the focus of environmental studies for decades, the benthic megafauna of the CCZ remain poorly known. In order to predict and manage the environmental impacts of mining in the CCZ, baseline knowledge of the megafauna is essential. The ABYSSLINE Project has conducted benthic biological baseline surveys in the UK Seabed Resources Ltd polymetallic-nodule exploration contract area (UK-1). Prior to these research cruises in 2013 and 2015, no biological studies had been done in this area of the eastern CCZ. New information Using a Remotely Operated Vehicle and Autonomous Underwater Vehicle, the megafauna within the UKSRL exploration contract area (UK-1) and at a site ~250 km east of the UK-1 area were surveyed, allowing us to make the first estimates of megafaunal morphospecies richness from the imagery collected. Here, we present an atlas of the abyssal echinoderm megafauna observed and collected during the ABYSSLINE cruises to the UK-1 polymetallic-nodule exploration contract area in the CCZ. There appear to be at least 62 distinct morphospecies (13 Asteroidea, 5 Crinoidea, 9 Echinoidea, 29 Holothuroidea and 6 Ophiuroidea) identified mostly by imagery but also using molecular barcoding for a limited number of animals that were collected. This atlas will aid the synthesis of megafaunal presence/absence data collected by contractors, scientists and other stakeholders undertaking work in the CCZ, ultimately helping to decipher the biogeography of the megafauna in this threatened habitat.


Introduction
The Clarion-Clipperton Zone (CCZ) is an abyssal region of the tropical eastern Pacific Ocean where deep-sea mining may take place in the near future (Ramirez-Llodra et al. 2011, Wedding et al. 2013; Fig. 1). High-grade polymetallic nodules, which could provide a commercial source of copper, cobalt, nickel, and manganese (among other metals), are abundant in this six million km region that lies in Areas Beyond National Jurisdiction (ABNJ), and thus falls under the legal mandate of the International Seabed Authority (ISA) (Wedding et al. 2013). Thus far, there have been 16 exploration leases (each up to 75,000 km in area) granted by the International Seabed Authority in the CCZ, with those for exploitation expected to soon follow (https://www.isa.org.jm/).
The ABYSSLINE (ABYSSal BaseLINE) Project was designed to undertake benthic biological baseline studies in accordance with ISA environmental guidelines within the UK Seabed Resources Ltd (UKSRL) exploration contract area (UK-1) (Amon et al. 2016b). The UK-1 exploration contract area is one of the easternmost contract areas in the CCZ and encompasses ~58,000 km of seafloor (Fig. 1). The ABYSSLINE Project was led by scientists from the University of Hawai'i at Mānoa (USA), and included scientists from Hawai'i Pacific University (USA), the Natural History Museum, London (UK), the National Oceanography Centre, Southampton (UK), Senckenberg Gesellschaft für Naturforschung (Germany), Uni Research (Norway), and the International Research Institute of Stavanger (Norway). The ABYSSLINE Project aimed to evaluate baseline conditions of community 2 2 2 structure and biodiversity for megafauna, macrofauna, meiofauna and microbes within the UK-1 contract area and across the CCZ (Amon et al. 2016a, Amon et al. 2016b, Glover et al. 2015, Shulse et al. 2016. No faunal studies had been undertaken in the UK-1 contract area prior to licensing in 2013. It is expected that nodule mining will drastically alter this unique deep-sea habitat with recovery expected to be slow (Amon et al. 2016b, Vanreusel et al. 2016, Ramirez-Llodra et al. 2011, Oebius et al. 2001) and yet, despite increases in technology and the number of expeditions to the area, very little is known about the ecology and biogeography of the fauna inhabiting the region (Amon et al. 2016a, Amon et al. 2016b, Foell and Pawson 1986, Bluhm and Gebruk 1999, Martinez-Arbizu et al. 2013, Pawson 1983, Pawson and Foell 1986, Roux 2004, Roux and Pawson 1999, Shulse et al. 2016, Tilot 2006, Vanreusel et al. 2016. The megafauna constitute an important component of the biodiversity in the abyssal deep sea and play a significant role in deep- Locations of megafaunal surveys during the ABYSSLINE cruises, AB01 and AB02, in the Clarion-Clipperton Zone. (a) The location of the UKSRL exploration contract area (UK-1) in the eastern Pacific Ocean. (b) The locations of the 30x30-km survey areas, UK-1 Stratum A and UK-1 Stratum B, in relation to the UK-1 exploration contract area and the AB01 ROV dive site, EPIRB, which was approximately 250 km east of the UK-1 contract area. (c) The locations of ROV dives within UK-1 Stratum A, indicated by purple tracklines labelled with the dive number (e.g. RV01). Stations where samples were collected with a Brenke epibenthic sled (EB04) and megacorer (MC10) are also indicated. (d) The locations of AUV dives within UK-1 Stratum B, indicated by purple tracklines labelled with the dive number (e.g. AV01). Stations where samples were collected with a Brenke epibenthic sled (EB02), box corer (BC02, BC19) and megacorer (MC17, MC25) are also indicated, as well as where imagery was collected with a baited camera (CA02). All maps were created by Seafloor Investigations Ltd for the ABYSSLINE Project using ArcGIS software (https://www.arcgis.com/features/). sea ecosystem function (Amon et al. 2016a, Amon et al. 2016b, Smith et al. 2008, Vanreusel et al. 2016. It has also been suggested that echinoderms may act as indicators of physical disturbance of the seabed, such as that caused by deep-sea polymetallicnodule mining (Bluhm and Gebruk 1999).  reported that a search of OBIS listed 698 echinoderm species recorded at abyssal depths worldwide between 3000m and 6000m, but only 50 species within the CCZ. Amon et al. 2016b was able to confirm that there were no echinoderm or megafauna records for the UK-1 exploration contract area in OBIS. This is likely the result of lack of sampling, taxonomy and/or ensuring data are publicly available, especially as an abundant and diverse echinoderm fauna is already known from the tropical Pacific Ocean from photographic and video survey (Amon et al. 2016a, Amon et al. 2016b, Foell and Pawson 1986, Martinez-Arbizu et al. 2013, Pawson 1983, Pawson and Foell 1986, Roux and Pawson 1999, Tilot 2006, Vanreusel et al. 2016. In order to predict and manage the environmental impacts of mining in the CCZ and within the UK-1 exploration contract area, baseline knowledge of the megafauna is essential and allows for a complete taxonomic and biogeographic synthesis of the fauna of the CCZ (Wedding et al. 2015).
Here, we present the first section (Echinodermata) of an image atlas of benthic megafauna that inhabit the UK-1 exploration contract area based on ROV and AUV surveys and samples collected during two cruises of the ABYSSLINE project. This section will be supported by the following sections in the near future: 1) Cnidaria, 2) Porifera, and 3) All Other Phyla (Annelida, Arthropoda, Bryozoa, Chordata, Ctenophora, and Mollusca). This atlas was crucial during the ABYSSLINE quantitative megafaunal analyses (Amon et al. 2016b) and we hope that it will facilitate the standardization of the putative morphospecies and be useful to other scientists and stakeholders undertaking research in the CCZ in the future.

Materials and methods
The UKSRL exploration contract area (UK-1) is located in the eastern CCZ in the Pacific Ocean ( Fig. 1). There have been two ABYSSLINE research cruises to the UK-1 exploration contract area: the AB01 or MV1313 cruise on the R/V Melville from 3 to 27 October 2013, and the AB02 or TN319 cruise on the R/V Thompson from 12 February to 25 March 2015. The AB01 cruise focused on a 30x30-km stratum (UK-1 Stratum A) centered at 13°49′ N, 116°36′ W in the northern portion of the UK-1 contract area (Fig. 1). During the AB01 cruise, multibeam bathymetric surveys indicated an abyssal seafloor characterized by ridges and valleys running from NNW to SSE at 3900-4400 m. The commercial Remotely Operated Vehicle (ROV) Remora III, operated by Phoenix International Holdings, performed video surveys and sample collections at four randomly-located sites within UK-1 Stratum A in the UK-1 contract area. Additionally, surveys were done ~250 km to the east of the UK-1 contract area, at a site called 'EPIRB' centered at 13°40′ N, 114°24′ W (Fig. 1). Work at the EPIRB site was dictated by an emergency response to an Emergency Position Indicating Radio Beacon (EPIRB) distress signal and, although unplanned, provided a useful broader context for our study. The ROV was equipped with two manipulators, four The criteria used for selection of megafaunal morphospecies was that individuals were greater than 2 cm in maximum dimension and that there was sufficient detail to identify them to a putative 'species-level' morphotype or morphospecies (Amon et al. 2016b). However, this only applied to imagery from AB01 as the AUV imagery collected during AB02 was of poor resolution resulting in only megafauna above 5 cm in the largest dimension being included in this atlas. Morphospecies that could not be identified to species but appeared morphologically distinct were assigned a unique informal species name (e.g. Echinoidea morphospecies 1). These were identified by taxonomic experts or by using the "Atlas of Abyssal Megafauna Morphotypes of the Clarion-Clipperton Fracture Zone" created for the ISA (http://ccfzatlas.com/) (Martinez-Arbizu et al. 2013), as well as Bluhm and Gebruk 1999, Foell and Pawson 1986, Pawson 1983, Pawson and Foell 1986, Roux 2004, Roux and Pawson 1999, Tilot 2006. Morphospecies from this study that matched morphotypes listed in the "Atlas of Abyssal Megafauna Morphotypes of the Clarion-Clipperton Fracture Zone" have had a section titled "Nomenclature" added to their data, in which the identification from the "Atlas of Abyssal Megafauna Morphotypes of the Clarion-Clipperton Fracture Zone" has been included. This is in an effort to provide coherence between these CCZ atlases. For morphospecies that were morphologically similar to a well-defined species name, we use the open nomenclature expression "cf.", although a precautionary approach was taken. Specimens in this atlas that were collected have undergone (relevant GenBank numbers are included) or are currently undergoing molecular analyses Glover et al., unpublished data).
This process provided an estimate of the number of echinoderm morphospecies in the UK-1 contract area and eastern CCZ, and will aid in delimiting species ranges in the CCZ. However, since the majority of the morphospecies were not collected, it is impossible to confirm species identities in most cases or undertake systematic studies on this fauna (Amon et al. 2016b  cf. Freyella morphospecies observed in the UK-1 exploration contract area and eastern CCZ. Images (a-d) correspond with the relevant data above. Notes: Fig. 3 a b c d Figure 3.
Freyastera cf. benthophila observed in the UK-1 exploration contract area. Images (a-d) correspond with the relevant data above.  cf. Paxillosida morphospecies 1 in situ on seafloor in the UK-1 exploration contract area. Image corresponds with the data above. Scale bar is 10 cm.  cf. Paxillosida morphospecies 2 in situ on seafloor in the UK-1 exploration contract area. Image corresponds with the data above. Scale bar is 10 cm. Image attribution: Woods Hole Oceanographic Institution.

cf. Paxillosida morphospecies 3
Nomenclature: In the "Atlas of Abyssal Megafauna Morphotypes of the Clarion-Clipperton Fracture Zone" created for the ISA (http://ccfzatlas.com/), this morphospecies is listed as "Astropectinidae Dytaster morphotype".  cf. Paxillosida morphospecies 3 observed in situ on sponge on seafloor in the UK-1 exploration contract area. Image corresponds with the data above. Scale bar is 10 cm. Image attribution: Woods Hole Oceanographic Institution. Notes: Fig. 8 a b cf. Porcellanaster morphospecies observed in the UK-1 exploration contract area and eastern CCZ. Images (a-b) correspond with the data above.   Notes: Fig. 10 a b Figure 10.

Material
Styracaster paucispinus after collection from the UK-1 exploration contract area. Images (a) and (b) correspond with the data in (a) above.   Notes: Fig. 15 a b Figure 15.
cf. Comatulida morphospecies 1 observed in the UK-1 exploration contract area and eastern CCZ. Images (a-b) correspond with the data above. Notes: Fig. 16 a b Figure 16.
cf. Comatulida morphospecies 2 observed in the UK-1 exploration contract area. Image (a-b) correspond with the data in (a) above.  cf. Echinoidea morphospecies 2 in situ on seafloor in the UK-1 exploration contract area. Image corresponds with the data above. Scale bar is 10 cm. Image attribution: Woods Hole Oceanographic Institution. Notes: Fig. 22 a b Figure 22.
cf. Aspidodiadematidae morphospecies observed in the UK-1 exploration contract. Images (ab) correspond with the data above.  cf. Holasteroida morphospecies 1 in situ on seafloor in the UK-1 exploration contract area. Image corresponds with the data above. Scale bar is 10 cm. Image attribution: Woods Hole Oceanographic Institution. Notes: Fig. 24 a b Figure 24.
cf. Holasteroida morphospecies 2 observed in the UK-1 exploration contract area. Images (ab) correspond with the data above.   Notes: Fig. 26 a b c Figure 26.
Echinocrepis morphospecies observed in the UK-1 exploration contract area. Images (a-c) correspond with the data above.    In the "Atlas of Abyssal Megafauna Morphotypes of the Clarion-Clipperton Fracture Zone" created for the ISA (http://ccfzatlas.com/), this morphospecies is listed as " Mesothuria morphotype".   cf. Synallactidae morphospecies 1 observed in the UK-1 exploration contract area and eastern CCZ. Images (a-c) correspond with the data above.  cf. Synallactidae morphospecies 2 in situ on seafloor in the UK-1 exploration contract area. Image corresponds with the data above. Scale bar is 10 cm. Image attribution: Woods Hole Oceanographic Institution. In the "Atlas of Abyssal Megafauna Morphotypes of the Clarion-Clipperton Fracture Zone" created for the ISA (http://ccfzatlas.com/), this morphospecies is listed as " Synallactes morphotype "white"".
cf. Synallactes morphospecies 2 observed in the UK-1 exploration contract area. Images (a-b) correspond with the data above.

Materials
cf. Deimatidae morphospecies 1 observed in the UK-1 exploration contract area. Images (a-b) correspond with the data above.
a: cf. Deimatidae morphospecies 1 in situ on seafloor. Scale bar is 10 cm. Image attribution: Woods Hole Oceanographic Institution. b: cf. Deimatidae morphospecies 1 in situ on seafloor. Scale bar is 5 cm. Image attribution: Woods Hole Oceanographic Institution.

cf. Deimatidae morphospecies 2
Nomenclature: In the "Atlas of Abyssal Megafauna Morphotypes of the Clarion-Clipperton Fracture Zone" created for the ISA (http://ccfzatlas.com/), this morphospecies is listed as " Orphnurgus morphotype".  cf. Deimatidae morphospecies 2 observed in the UK-1 exploration contract area. Image corresponds with the data above. Scale bar is 10 cm. Image attribution: Woods Hole Oceanographic Institution.

Nomenclature:
In the "Atlas of Abyssal Megafauna Morphotypes of the Clarion-Clipperton Fracture Zone" created for the ISA (http://ccfzatlas.com/), this morphospecies is listed as " Peniagone morphotype "tulip"".  cf. Peniagone morphospecies 1 in situ on seafloor in the UK-1 exploration contract area. Image corresponds with the data above. Scale bar is 10 cm. Image attribution: Woods Hole Oceanographic Institution. Notes: Fig. 49 a b Figure 49.

Materials
Psychronaetes cf. hanseni observed in the UK-1 exploration contract area. Images (a-b) correspond with the data above.  cf. Enypniastes morphospecies observed in situ on seafloor the UK-1 exploration contract area. Image corresponds with the data above. Image attribution: A Leitner and J Drazen, University of Hawai'i.  cf. Benthodytes morphospecies 1 observed in the UK-1 exploration contract area. Images correspond with the data above.

Notes:
Benthodytes cf. incerta observed in the UK-1 exploration contract area and eastern CCZ. Images correspond with the data above. Amphioplus (Unioplus) daleus observed in the UK-1 exploration contract area. Images (a-b) correspond with the data above.  site east of the contract area. Previous studies in the CCZ have identified 38 echinoderm morphotypes (Foell and Pawson 1986), and 46 morphotypes (Tilot 2006). Bluhm and Gebruk (1999) noted 28 holothurian morphotypes from the DISCOL site in the southeastern Pacific Ocean, which is similar to the numbers observed during this study (29). The quantitative study by (Amon et al. 2016b) which utilised many of the images from AB01 included in this study reported 41 echinoderm morphotypes (the second-most speciose phylum behind Cnidaria -48 morphotypes) from UK-1 Stratum A and EPIRB only. However, we recognise that the comparison of echinoderm species richness aross the region is only valid if sampling effort was similar or standardised (Amon et al. 2016b). This suggests that the Echinodermata are the most conspicuous, and therefore, bestcharacterised of all phyla occuring the CCZ.
These morphospecies represent a range of functional traits: the crinoids are sessile or semi-sessile suspension feeders, reliant on the polymetallic nodules as hard substrate, whereas most of the asteroids (excluding brisingids), echinoids, holothurians, and ophiuroids are mobile deposit feeders (Amon et al. 2016b, Vanreusel et al. 2016. Approximately half of the morphospecies in this atlas have been observed in other contract areas in the CCZ (http://ccfzatlas.com/, Bluhm and Gebruk 1999, Pawson 1983, Pawson and Foell 1986, Tilot 2006, although this may be an overestimate given the presence of cryptic species and the problems identifying megafauna from imagery, as has been experienced during studies in other poorly-explored areas (Amon et al. 2016b, Vrijenhoek 2009, Bickford et al. 2006, Linse et al. 2007). Information like this will likely be crucial to inform the future environmental management of the region.
While this image atlas has expanded the knowledge of benthic fauna in the UK-1 exploration claim area and overall CCZ, there is still a need for further high-quality imagery of fauna, and an even more dire need for physical megafaunal specimens to ground-truth the morphospecies observed in images via detailed morphological and molecular analyses. We expect that a number of the morphospecies included in this atlas may be new to science, new records, or poorly known, but this can only be confirmed when specimens are collected and analysed. Molecular analyses are especially important given the presence of cryptic species. The limited collection of voucher specimens in the CCZ thus far has severely hampered reliable estimation of species richness and species distributions and continues to be an issue. Although the taxonomic identification of preserved material is always necessary, we hope that this atlas will aid scientists by showing what these morphospecies look like in situ in their natural surroundings, as well as by providing some ecological information (e.g. feeding modes, preferred habitat etc.). This information will be important in estimating the human impact on this ecosystem. Furthermore, the appearance of morphospecies captured in situ in images can drastically differ from that of collected or preserved material, especially when relatively rudimentary collection equipment (trawls, dredges etc.) are used. As mentioned in Amon et al. (2016b), there is also a need for those working in the CCZ to make available detailed descriptions of equipment and methods to facilitate data standardization and statistically-rigorous regional comparisons. It is also important that the ISA-sponsored online atlas continues to be updated with new imagery (such as the images in this atlas), and that the morphospecies are properly identified with the help of taxonomists.