Computational protocol: Differentiation of three common deep-water hermit crabs (Crustacea, Decapoda, Anomura, Parapaguridae) from the South African demersal abundance surveys, including the description of a new species of Paragiopagurus Lemaitre, 1996

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[…] Since 2011, targeted invertebrate specimens retained in the research trawl nets were collected during the DAFF demersal research abundance surveys, using a German otter trawl design with various configurations, and a 75 mm mesh cod-end fitted with a 35 mm mesh liner. Trawls were deployed for 30 minutes (bottom time) over all feasible habitats on the South African shelf (for detailed methods see ). During the 2015 research surveys, hermit crabs were pre-sorted on board by scientific staff, and all specimens of S. dimorphus and P. bouvieri were separated. Three male specimens with “green eyes” were obtained during the 2015 surveys. During the 2016 research surveys, a subsample of approximately 100 hermit crab specimens from each trawl were separated and frozen for further identification at the University of Cape Town (UCT). Three additional males and 23 females with “green eyes” were obtained from trawls at two West Coast stations during the 2016 surveys. All specimens with “green eyes” were found to be the new species of Paragiopagurus herein described. No specimens with “green eyes” were found in trawls from the South Coast. Live images of S. dimorphus and P. bouvieri were taken in the laboratory at the University of Cape Town, and in a photographic tank on board of the RS Africana during an additional South Coast spring survey in September/October of 2016.The µCT scan of the holotype of the new species of Paragiopagurus was performed at the CT Scanner Facility at Stellenbosch University, South Africa, using a General Electric Phoenix V|Tome|X L240 with NF180 option (du ). The specimen was defrosted and placed on top of a plastic rod with dense polystyrene foam as a platform, and consecutively scanned at an X-ray voltage of 100 kV and 100 µA, and a resolution of 35 µm. Images were recorded in 3200 steps in one full rotation of the sample averaging two image acquisitions at every step. Using a detector shift function between images reduced ring artifacts. The projection images were reconstructed using the system-supplied General Electric Datos reconstruction software, which were subsequently utilized for the visualization of the right cheliped using Volume Graphics VGStudioMax 3.1. (Heidelberg, Germany).Illustrations were drawn using a Wild stereomicroscope equipped with a camera lucida, and digitally traced in Inkscape 0.91 (www.inkscape.com). Colour photographs were processed in Gimp 2.8 (www.gimp.com).Specimens examined in this report are deposited in the Iziko South African Museum, Cape Town, South Africa (SAMC), the National Museum of Natural History, Smithsonian Institution, Washington DC (USNM), as well as in the Lee Kong Chian Natural History Museum, Singapore (ZRC). Morphological terminology for parapagurids is that used by . Measurements of specimens, in millimeters (mm), listed in the material examined sections are for shield length (SL), taken from the tip of the rostrum to the midpoint of the posterior margin of the shield. Other abbreviations used are: ovig: ovigerous; SCDSA: South Coast Demersal Survey Autumn; SCDSS: South Coast Demersal Survey Spring; WCDSS: West Coast Demersal Survey Summer; sta: station; and in the material examined sections, months are abbreviated by the first three letters.Muscular tissue, usually from the merus of the right cheliped, was extracted from freshly frozen specimens, placed in 96% ethanol, and sent to the South African Institute for Aquatic Biodiversity (SAIAB). At SAIAB, DNA extractions were carried out using a standard “salting out – ethanol precipitation” protocol (), followed by the amplification of the ‘barcoding’ () fragment of the cytochrome c oxidase subunit I (CO1) gene for each sample by Polymerase Chain Reaction (PCR), using the universal invertebrate primers (LCOI-1490 and HCOI-2198) of , or their degenerate variants (dgLCO1490 and dgHCO2198; ). PCR recipes and conditions followed and , with annealing performed at 48 °C for the latter. Successful amplification was determined by visualising products under UV light, following electrophoresis in 1% agarose gels, stained with ethidium bromide, in a TBE buffer. PCR products were purified with an Exonuclease I – Shrimp Alkaline Phosphate (Exo/SAP, ThermoFisher Scientific) protocol (), sequenced in both the forward and reverse directions using BigDye v3.1 (Applied Biosystems, Austin, Texas) terminator chemistry and analyzed on an ABI-Hitachi 3500 Genetic Analyser (Applied Biosystems) at SAIAB. The resulting sequences were checked against their chromatograms for misreads and sequencing errors using ChromasLITE (Technylesium). Sequences were aligned, edited and the consensus DNA barcode compiled using Lasergene SeqMan Pro 9 (DNASTAR, Madison, Wisconsin). Barcodes were uploaded to the SeaKeys (SEAKY) project on BOLD (www.boldsystems.org; Ratnasingham and Hebert 2007) and were submitted to GenBank. For a number of specimens, tissues were submitted to the Canadian Centre for DNA Barcoding, Biodiversity Institute of Ontario, University of Guelph, for barcoding. These data were also uploaded to SEAKY on BOLD. For future reference and studies, the database gene codes are included under each species. […]

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