Computational protocol: Sea star Henricia spiculifera (Clark, 1901) in the northwestern Pacific: one species or three?

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Protocol publication

[…] Observations and sample collections were taken by SCUBA-diving in 2014 through 2017 in Rudnaya Bay of the Sea of Japan and in the Gulf of Avacha (Avacha Bay and Starichkov Is.), Kamchatka (). Underwater images were taken with a Nikon D810 camera equipped with Nikkor 105/2.8G lens and appropriate Sea & Sea underwater housing. The other images were taken with a Nikon D810 or D7000 cameras and a Nikkor 60/2.8 lens. The specimens are in 96% ethanol and deposited in the Museum of National Scientific Center of Marine Biology, Russian Academy of Sciences, Vladivostok. Skeletal plates and spines were denuded using 5–15% sodium hypochlorite solution. Scanning electron images of the spines were obtained using Zeiss Sigma and Zeiss Evo electron microscopes after carbon coating. Other studied specimens are preserved in the collections of Zoological Institute of Russian Academy of Sciences, St. Petersburg, Russia (ZIN), and National Science Center of Marine Biology, Russian Academy of Sciences (MIMB), and Hokkaido University, Sapporo, Japan.DNA was extracted using the Diatom™ DNA Prep 100 kit (Isogene Lab, Moscow, Russia) according to manufacturer’s protocol. Partial sequence of mitochondrial 16S rRNA gene (16S) was used in this study. The author successfully used this marker earlier for delimitation of Henricia species (). The primers used to amplify that fragment () were used here to amplify the region of interest. The master mix for each sample was prepared using 34.75 µL H2O, 5.00 µL PCR Buffer (Evrogen, Moscow), 5.00 µL 25 mM MgCl2, 1.00 µL 40 mM dNTPs, 1.00 µL 10 mM primer 1, 1.00 µL primer 2, 0.25 µL 5 mg/mL Taq, and 1.00 µL extracted DNA. Reaction conditions were an initial denaturation for 3 min at 95 °C, 39 cycles of (1) denaturation for 45 s at 94 °C, (2) annealing for 45 s at 50 °C, and (3) elongation for 2 min at 72 °C, and a final elongation for 10 min at 72 °C. PCR products yielding bands of ca. 600 b.p. were purified using ethanol precipitation. Sequencing was conducted by Sanger ddNTP termination method using BrightDye reagent (Nimagen) and ABI 3130 Genetic Analyser (Applied Biosystems) at Far Eastern Federal University, Vladivostok. The sequences were assembled and edited using BioEdit (). BioEdit was also used to extract the consensus sequences. The sequences used in this study, including those mined from GenBank, are listed in the .Two methods for species delimitation and identification were used: comparing tree topologies, and Automatic Barcode Gap Discovery (ABGD). The p-distances (i.e., the proportion of variable positions) and Neighbor-Joining (NJ) () and Maximum Likelihood (ML) gene trees were calculated using MEGA 7 software (). Hasegawa-Kishino-Yano (HKY  + Γ + I) () evolutionary model was suggested by -lnL value found using Model Selection analysis implemented in MEGA. ABGD method () is based on pairwise distances, detecting the breaks in the distribution referred to as the “barcode gap” () without any prior species hypothesis. The ABGD program is available at http://wwwabi.snv.jussieu.fr/public/abgd/abgdweb.html. I analyzed 16S alignment using either uncorrected p-distance or Kimura-2-Parameter (K2P) () and Jukes-Cantor (JC) () distances. X (relative gap width) was set to 1.4, the other settings remained as default. Single pure (SPu) character attributes, i.e., species-specific barcoding positions (; ; ), were detected manually because of low number of variable sites in H. cf. spiculifera group alignment.The electronic version of this article in Portable Document Format (PDF) will represent a published work according to the International Commission on Zoological Nomenclature (ICZN), and hence the new names contained in the electronic version are effectively published under that Code from the electronic edition alone. This published work and the nomenclatural acts it contains have been registered in ZooBank, the online registration system for the ICZN. The ZooBank LSIDs (Life Science Identifiers) can be resolved and the associated information viewed through any standard web browser by appending the LSID to the prefix http://zoobank.org/. The LSID for this publication is: urn:lsid:zoobank.org:pub:398553F6-F96E-4B82-A6C5-366D7200AF0D. The online version of this work is archived and available from the following digital repositories: PeerJ, PubMed Central and CLOCKSS. […]

Pipeline specifications

Software tools BioEdit, ABGD, MEGA
Application Population genetic analysis