Material and methods
Specimens of Protatlanta used for this study were sourced from a number of collections. Fresh specimens for molecular and morphological analysis were collected from the Atlantic Ocean during the Atlantic Meridional Transects AMT24 (N=404, late September to October 2014) and AMT20 (N=68, October to November 2010). Oblique tows were carried out using a CalBOBL bongo net with an aperture diameter of 0.71 m for AMT24 and a WP2 bongo net with an aperture diameter of 0.57 for AMT20. Both nets had a mesh size of 200 μm. For sample collection during AMT24, a flowmeter (General Oceanics 2030RC) was mounted at the mouth of one of the nets to measure the volume of water sampled for quantitative analysis (Burridge et al., in press). Specimens from AMT24 were fixed and preserved in 96% ethanol and stored at -20°C prior to DNA barcoding. Specimens from AMT20 were fixed and stored in 96% ethanol and stored at room temperature. Storage at room temperature is not optimal for the preservation of DNA, therefore, specimens from AMT20 were not used for DNA barcoding. Station information and sampling parameters for all specimens are given in Table S1.
A total of seven specimens from the recent fossil record (0–10 ka) were used for morphometric analysis. Fossil specimens were collected from a marine sediment core from site CAR-MON 2, offshore of Montserrat, Lesser Antilles (Le Friant et al., 2008; Wall-Palmer et al., 2014). For the biogeography, additional specimens were examined from the Challenger collection and the National Institute of Oceanography Discovery collection (N=66, surface sediment and plankton samples Natural History Museum, London), the ACE-ASIA, KH-11-10, VANC10MV and CANCAP Expeditions collections (N=100, plankton samples and surface sediment, Naturalis Biodiversity Center, Leiden), the Dana and Thor collections (N=337, plankton samples, Zoological Museum, Copenhagen) and the APNAP-I-1986 and G0-Snellius-II collections (N=21, plankton samples, Vrije Universiteit, VU, Amsterdam). Collection information is given in Table S1. All biogeographical data was plotted using the software QGIS (QGIS Development Team, 2016).
Morphometric analysisnext section
Morphometric analysis was performed on 19 adult specimens from AMT20 and AMT24 and from the recent fossil record of site CAR-MON 2 (Le Friant et al., 2008; Wall-Palmer et al., 2014). Images of the apical side of each shell were obtained using light microscopy and scanning electron microscopy. The ImageJ software FIJI (Schindelin et al., 2012) was used to measure the shell diameter perpendicular to the nucleus of the protoconch (Fig. 2) at half whorl intervals throughout each shell.
Fig. 2. Method of whorl counting of Protatlanta shells, following the method of whorl numbering by Seapy (1990) and morphometric shell measurements for P. souleyeti (black circles, black lines) and P. sculpta (white triangles, grey lines) shells. For specimens analysed, see supplementary Table S1. Specimen used for method schematic is P. sculpta from Figure 3k.
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).