A random sample of 18 undamaged adult (N=7) and juvenile (N=11) specimens from the Atlantic Ocean were selected from AMT24 samples for DNA barcoding. A mixture of ornamented and non-ornamented specimens of varying colour (brown and white) were analysed. All specimens were imaged prior to analysis using a Zeiss automated z-stage light microscope. DNA extraction was carried out on whole specimens, using the NucleoMag 96 Tissue kit by Macherey-Nagel on a Thermo Scientific KingFisher Flex magnetic bead extraction robot, with a final elution volume of 75 µl. The standard CO1 barcoding fragment (Hebert et al., 2003) was amplified using primers jgLCO1490 and jgHCO2198 (Geller et al., 2013). Primers were tailed with M13F and M13R for sequencing (Messing, 1983). PCR reactions contained 17.75 µl mQ, 2.5 µl 10x PCR buffer CL, 0.5 µl 25mM MgCl2 , 0.5 µl 100mM BSA, 1.0 µl 10 mM of each primer, 0.5 µl 2.5 mM dNTPs and 0.25 µl 5U Qiagen Taq, with 1.0 µl of template DNA, which was diluted 10 or 100 times for some samples. PCR was performed using an initial denaturation step of 180 s at 94°C, followed by 40 cycles of 15 s at 94°C, 30 s at 50°C and 40 s at 72°C, and finishing with a final extension of 300 s at 72°C and pause at 12°C. Sequencing was carried out by Macrogen, Europe.
Sequences were examined and edited in MEGA 6 (Tamura et al., 2013), aligned using Mafft v7 (http://mafft.cbrc.jp/alignment/server/) and are available from GenBank (Table 1). Additional GenBank CO1 sequences from heteropods identified as Atlanta inclinata, Oxygyrus inflatus, Firoloida desmarestia, Pterotrachea hippocampus and Pterotrachea coronata (Jennings et al., 2010) were used in the analysis to represent the families and genera most closely related to the protatlantids. CO1 sequences of the gastropods Crassitoniella flammea, Pisinna albizona, Bembicium nanum, Echinolittorina riisei, Tectonatica sagraiana, Natica vittata and Ataxocerithium sp. from GenBank (Williams and Reid, 2004; Colgan et al., 2003, 2007; Huelsken et al., 2008; Ayre et al., 2009) were used as outgroups. These all belong to Hypsogastropoda (within Caenogastropoda), supposedly closely related to the heteropods, as suggested by Jennings et al. (2010). A maximum-likelihood tree was constructed based on these data using nucleotide sequences and a General Time Reversible model with different rates at the three codon positions (+CP) model with 1000 bootstraps in RAxML (Stamatakis, 2014). Kimura-2-parameter (K2P) genetic distances were calculated between and within species belonging to the family Atlantidae using MEGA 6 (Tamura et al., 2013).