Location: East Pacific Rise 9 N 104 W depth 2500m

Collection at the seafloor:

Sampling was conducted near 9°47’N on the East Pacific Rise (EPR) on dives with the Human Occupied Vehicle (HOV) Alvin during R/V Atlantis cruise AT50-06 (cruise DOI: 10.7284/909880); this dataset includes collections from HOV Alvin Dives 5134 and 5135 at Lucky’s Mound on 18 and 19 December 2022 and from Dive 5142 at Sentry Spire on 27 December 2022. This dataset also includes one sampling event from the Remotely Operated Vehicle (ROV) Jason II Dive 1311 at Sentry Spire on 9 April 2021 during R/V Revelle cruise RR2102 (cruise DOI: 10.7284/909120). Sampling events are specified in the dataset as “rock grab” or “slurp.” For rock grabs - the manipulators were used to break off a rock then place it into an individual compartment of a closable, vehicle-mounted container (biobox). For slurps - the substratum (but not the rock targeted for sampling) was vacuumed with a five-chambered hydraulic slurp sampler. The sampling event log was associated by time to renavigated vehicle data including latitude, longitude, and depth (for AT50-06 used renavigated vehicle data version June 2023). We thank the captain, crew, Alvin and Jason teams, and scientists on-board both cruises for enabling sample collection.

Shipboard sample processing:

After recovery on deck, animals were either rinsed or gently scraped off the surfaces of sulfides or siphoned out of the biobox compartment for the collected rock. Siphoned biobox water and slurps were poured over a sieve (60 micrometer on AT50-06). The animal samples were placed into 4°C filtered seawater, sorted in petri dishes to high rank taxonomic groups (e.g., gastropods), and placed into 2.5-mL cryotubes. On cruise AT50-06 gastropods were either preserved in 80% ethanol and stored at -20°C or fixed in 10% buffered formalin; the formalin material was transferred to 80% ethanol after 1-2 days, refreshed with 80% ethanol after 1 day, and again after 1 day, then accidentally placed in a -80°C freezer on-board the research vessel. On cruise RR2102 gastropods were preserved in 95% ethanol and stored at room temperature.

Laboratory sorting and morphological identification:

Cryotubes containing gastropod specimens from a total of 8 rock grabs and 2 slurps from cruise AT50-06 were thawed and poured into a dish for sorting by morphotype. A subset of sorted specimens was photographed and placed into new 1-mL tubes [recorded as “samples and images” metadata tables for the sorted tubes (1 for ethanol, 1 for formalin)]. The new tubes were shipped at room temperature from WHOI to JAMSTEC.

Specimens were identified morphologically, recorded into ID tables for cruise AT50-06 (1 for ethanol, 1 for formalin); specimens from 1 rock grab also were identified for cruise RR2102. Gastropod specimens were gently cleaned using small brushes, and observed under a stereo dissecting microscope (Olympus SZX9). To obtain the operculum and radula for scanning electron microscopy, dissections were done under the microscope using fine forceps and tungsten needles. As listed in the compiled data table column otherCatalogNumbers, a subset of specimens was deposited in Senckenberg Natural History Museum, Frankfurt (SMF), National Museum of Nature and Science, Tsukuba (NSMT), or Muséum National d’Histoire Naturelle, Paris (MNHN). For more information about gastropod morphological identification, please see Chen et al. (2024).

Genetic evidence for identification:

DNA was extracted from one whole specimen of each of the three Melanodrymia species using the DNeasy Blood & Tissue kit (Qiagen) following the manufacturer’s protocols. The same specimen's DNA extraction was used for both mitochondrial COI and mitogenome sequencing, with sequences deposited in National Center for Biotechnology Information (NCBI) GenBank under accession numbers OR828527-OR828529 (Chen et al. 2024) and under BioProject number PRJNA1048888 (Zhang et al. 2024), respectively.