Computational protocol: Exploring Canadian Echinoderm Diversity through DNA Barcodes

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Protocol publication

[…] Tissue samples were obtained from 1285 specimens in the collections of the Centre for Biodiversity Genomics (CBG) and the Royal British Columbia Museum (RBCM). These samples included 316 specimens representing 78 species from trawl collections made by the RBCM between 2000 and 2006 at depths > 200m at various localities along the coast of British Columbia (). Another 252 specimens were obtained by SCUBA and dredging between 1995 and 2002 from Queen Charlotte Sound and Nunavut () while 589 specimens were collected by SCUBA, by dredging and by hand from sites near Baffin Island, British Columbia, Labrador, Manitoba, and New Brunswick between 2007 and 2014 (). A final 128 specimens were gathered by trawls at depths < 700m from the Beaufort Sea, Baffin Bay and the Labrador Sea in 2010 as part of the Canadian Healthy Oceans Network (). Scientific names follow the World Register of Marine Species (WoRMS, All RBCM specimens were identified by Philip Lambert, an echinoderm specialist. All CBG specimens were identified using regional taxonomic keys (British Columbia: [–]; Atlantic and Arctic Canada: []). When a species-level identification could not be made, an interim name was assigned that coupled a genus-level identification with a Barcode Index Number (BIN) designation []. All specimens are stored as vouchers in 95% ethanol or frozen at -20°C in collections at the RBCM or the CBG (). Specimen and sequence data are available in the dataset DS-COIECH on BOLD, the Barcode of Life Data Systems []. All sequences have also been deposited in GenBank under the following accession numbers: GU670162-65; GU670167-81; GU670187-94; HM400305-69; HM400539; HM405487; HM405870-914; HM473811-74; HM473876-89; HM473902-57; HM542062-420; HM542908-3073; JF891304-16; JN295388; JN314244; KU495734-918. [...] All sequences were edited manually using Sequencher 4.8 and aligned by eye in MEGA 6.0 []. As most sequences were barcode compliant (N = 987), they received a Barcode Index Number (BIN), aiding in species delimitation []. Maximum-likelihood trees were constructed with 1000 bootstrap replicates and either an HKY+I model in MEGA 6.0 [] or with a GTR+G+I model in RAxML v8 []. Models with the highest AIC values generated by jModelTest v2 [,] were selected as most appropriate. Pairwise estimates of intra- and interspecific sequence divergence were calculated using the K2P distance model [] and the Distance Summary tools on BOLD []. The presence of a barcode gap was analysed by plotting maximum intraspecific divergence against Nearest-Neighbor distance. All species with an intraspecific divergence >2% were flagged for further investigation. Maximum and mean intraspecific divergences were plotted against the number of individuals sampled within a species and regression analysis was carried out in RStudio using the Picante and VEGAN packages to determine the significance of this relationship [,]. […]

Pipeline specifications

Software tools BOLD, Sequencher, MEGA, RAxML, jModelTest, Picante, vegan
Applications Phylogenetics, WGS analysis
Organisms Drosophila melanogaster