From Harder et al (2016):

Sample collection
Samples were collected in 2006 and 2012-2013 aboard the RVIB Nathaniel B. Palmer and the ASRV Laurence M. Gould. Pycnogonids were obtained using a Blake trawl or epibenthic sled, and subsequently identified to genus level prior to preservation and shipment in ~95 % ethanol. Individuals were identified to species level according to standard pycnogonid taxonomic procedures (Child 1995; Weis and Melzer 2012b; Weis et al. 2014). In total, 64 specimens belonging to the genus Pallenopsis were collected and identified from 21 sampling sites, ranging from 53 16'S to 76 59'S and from 140 26'E to 37 26'W (Fig. 1). Sampling site locality information, including depth (when available), and GenBank accession numbers for all sequences generated for use in our analyses are provided in Table 1.

Molecular techniques
A tissue sample was taken from each individual as a 1-cm piece from the first tibial segment, and DNA was extracted from these using a Qiagen DNeasy Blood & Tissue Kit (Qiagen Inc., Valencia, CA) per manufacturer’s instructions. A ~650 bp portion of the cytochrome c oxidase subunit I (COI) gene was amplified using LCO-1490 (Folmer et al. 1994) and HCOoutout (Prendini et al. 2005). Each PCR mixture consisted of 1x PCR buffer, 0.75 U Taq DNA polymerase (5 PRIME Inc., Gaithersburg, MD), 2.5 mM Mg^+2, 10 nmol of each dNTP, 1 ul of template DNA, 0.5 uM of each primer, and water to 25 ul. The PCR cycling program began with an incubation at 94C for 2 min, followed by 38 cycles of 94C for 20 s, 46C for 30 s, and 65 C for 80 s, and concluded with a final extension at 65C for 7 min. Successful amplification was confirmed by visualizing PCR products on a 1% agarose gel stained with ethidium bromide. Target DNA was gel extracted and purified using a Qiagen QIAquick Gel Extraction Kit (Qiagen Inc., Valencia, CA) according to the manufacturer’s recommendations. Bidirectional Sanger sequencing of amplicons was performed atHigh Throughput Genomics Center (Seattle, WA). Obtained sequences were assembled and screened using Sequencher version 5.29 sequence analysis software (Gene Codes Corporation, Ann Arbor, MI), and aligned using Clustal W v1.8 (Thompson et al. 1994) in BioEdit v7.2.5 (Hall 1999). COI sequences were translated into amino acid sequences to further screen for sequencing error, including checking for frameshift mutations and stop codons.

Harder_2016_T1_sites.csv