Computational protocol: High Abundance of the Epibenthic Trachymedusa Ptychogastria polaris Allman, 1878 (Hydrozoa, Trachylina) in Subpolar Fjords along the West Antarctic Peninsula

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Protocol publication

[…] Live individuals of Ptychogastria polaris were collected in Andvord and Flandres Bays (), from the RVIB Nathaniel B. Palmer (cruise NBP10-01) and the ARSV Lawrence M. Gould (cruise LMG11-05) in February 2010 and May 2011, respectively (). These individuals were obtained from the top water of megacores (10 cm diameter) (OSIL Environmental Instruments and Systems) from which formalin-preserved (3) and frozen at -80°C (4) voucher specimens were saved for taxonomic identification. All collections were made in international waters, under the auspices of, and with permission from, the United States Antarctic program (USAP). No endangered or protected species were collected in this study. Megacore-collected trachymedusae were humanely sacrificed by rapid freezing, or by rapid warming to room temperature (which anesthetizes Antarctic marine benthos adapted to living at -1.0°C). Field collections of invertebrates within the USAP do not require IACUC approval.Formalin-preserved trachymedusae () were compared to three voucher specimens of the subboreal P. polaris obtained from JAMSTEC collections: 1 specimen (5% formalin), JAMSTEC No. 045607 (2K1284SS7c), collected from Shiribeshi Seamount, Sea of Japan (43.46' N, 139.54' E), 234 m, 19 July 2001 (Cruise NT01-07 Leg 2, Dive no. 2K#1284) [4.9°C, salinity 34.31, dissolved oxygen 1.8 ml/L]; and 2 specimens (3% formalin), JAMSTEC No. 1120031607, 1120031609 (7K549SS5, 7K549SS6), collected off Okushiri Island, Sea of Japan (42.30' N, 139.47' E), 1062 m, 10 March 2012 (Cruise KR12-07, Dive no. 7K#0549) [0.24°C, salinity 34.01]. All formalin-preserved specimens were examined under a Leica MZ16 dissecting microscope with a Leica KL2500LCD illuminator under transmitted, darkfield and polarized light conditions.For DNA sequencing, tentacle tissue was taken from each of the four frozen specimens and transferred immediately into -20°C molecular-grade (99.5%) EtOH. DNA was extracted from tissues using the Biosprint 96 workstation (Qiagen, Hilden, Germany) in conjunction with the Biosprint 96 DNA Blood Kit (cat. no. 940057) at the Laboratories of Analytical Biology (LAB) of the National Museum of Natural History, Smithsonian Institution (Washington, DC, USA). Specimens were barcoded using cytochrome oxidase I (COI) and additional molecular markers (mitochondrial ribosomal 16S, and nuclear-encoded ribosomal 18S and 28S) generated for phylogenetic reconstruction. The primers and PCR conditions used for obtaining 16S, 18S, and 28S were described in []. For COI barcoding the primers described in [], were used for PCR (94°C for 5 min, 30 cycles of 94°C for 1 min, 50°C for 30 s, 72°C for 2.5 min, followed by a final extension step of 72°C for 5 min) and cycle sequencing.PCRs were performed in 10 μl reactions containing 0.5 units Biolase DNA polymerase (Bioline USA Inc., Taunton, MA), 0.3 mM of each primer, 0.5 mM dNTPs (Bioline), 1.5 mM magnesium chloride, 2.5x Bovine serum albumin (BSA) (New England BioLabs Inc., Ipswich, MA), and 1x Buffer, 1 μl template DNA, and DNAase-free H20 to bring the volume to 10 μl. 3 μl of a 1 in 5 dilution of ExoSAP-IT (Affymetrix, USB Products) was added to each PCR reaction, followed by incubation at 37°C for 30 min followed by 80°C for 20 min. 1 μl of the ExoSAP-IT purified PCR product was used in cycle sequencing reaction with Big Dye Terminator (v. 3.1; ThermoFisher Scientific, Waltham, MA), followed by Sephadex G-50 Fine (GE Healthcare Life Sciences, Pittsburgh, PA) clean-up. Purified sequencing reactions were then analysed on an Applied Biosystems 3130xl Genetic Analyzer or Applied Biosystems 3730xl DNA Analyzer. Sequences were assembled in Geneious (v. 9.05; Biomatters Limited, NZ) and their cnidarian origin verified by BLAST searches against the National Center for Biotechnology Information's GenBank database ( were aligned using MAFFT (v. 7.205; []) with default settings. The edges of the COI alignment were trimmed to remove gaps at the ends of the alignments. Kimura 2-parameter distances were calculated from this alignment with the R package APE (v. 3.5; []) to estimate the genetic differentiation among sampling sites.A concatenated matrix for 16S, 18S, and 28S was constructed in Mesquite (v. 3.1; []) using a broad sampling of trachyline species (), with the aim of inferring the relationship between subboreal and Antarctic P. polaris to each other, and their relationship to the remainder of Trachylina. Positions in the concatenated alignment suitable for phylogenetic analysis were identified using Gblocks [] with the least stringent settings implemented in the alignment viewer Seaview (v. 4; []), allowing for smaller blocks, gap positions, and less strict flanking positions in the final alignment. The most appropriate model of sequence evolution for the aligned genes was inferred using jModelTest (v. 2.1.10; []) with default settings; the best fitting model was chosen using the Akaike Information Criterion (AIC). A Bayesian phylogenetic analysis was performed using MrBayes (v. 3.2.5; []). Here, MrBayes performed 4 separate runs with 8 markov-monte-carlo chains each for a maximum of 10,000,000 generations. Trees were sampled every 1,000 generations, discarding the first third of trees as burn-in. The analysis was stopped automatically when the average standard deviation of split frequencies among runs was < 0.01. […]

Pipeline specifications

Software tools Geneious, MAFFT, APE, Mesquite, Gblocks, SeaView, jModelTest, MrBayes
Application Phylogenetics
Diseases Spasms, Infantile