Larval collection:
These larvae were collected near hydrothermal vent fields on the East Pacific Rise (EPR 9°50'N vent field), on the Mariana Back-Arc Spreading Center (near Snail or Archaean vent fields), and in Pescadero Basin (Auka vent field) in the Gulf of California. The EPR specimens were from McLane WTS-LV50 plankton pumps deployed on-axis near Tica vent site on LADDER 3 cruise AT15-26 in 2007. The pump sampled for 24 hr over a 63 micron mesh at a rate of 30 L/min at a position about 3 meters above bottom. The Mariana specimens were from the same plankton pumps with same configuration deployed on-site or off-site but within 300 m of active hydrothermal vents on R/V Yokosuka cruise YK10-11 in 2010. The Pescadero specimens were from the suction (slurp) sampler on ROV Hercules on E/V Nautilus cruise NA091 in 2017. The slurp sampled for 10 min over a 63 micron mesh at a rate of ~100 L/min at a position about 1 meter above bottom, while the ROV was parked on-site at an active vent ("intensive sampling station", or ISS). ISS 2 was during ROV dive H1657, and ISS 4 was during ROV dive H1658.

We acknowledge the sample collection permits CONAPESCA PPFE/DGOPA-010/17 and INEGI: Autorización EG0072017 associated to the Diplomatic Note number SRE 17-1087 (CTC/06727/17).

Shipboard sample processing:
Pump and slurp samples were processed within an hour upon recovery on deck, with many specimens alive. The sample was washed off the filter using 95% non-denatured ethanol into a 250 mL jar (brand new or rinsed with ethanol, but was not autoclaved).

Laboratory sorting and morphological identification:
Portions of the sample were extracted with a wide-mouthed pipette into a petri dish for sorting under a dissecting scope at magnifications up to 50X (although most tools were rinsed with ethanol, none were autoclaved). Specimens were sorted into vials by major taxa (i.e., all the gastropod larvae together), in some cases with separate vials for those retained on 300um or 63um sieve. Vials were brand new or rinsed with ethanol, but were not autoclaved. Individuals were identified to morphotypes at lowest taxonomic level. Some morphotypes contain morphologically indistinguishable larvae from multiple species (e.g., polychaete nectochaetes).

Specimens selected for DNA extraction:
We selected specimens in several major taxonomic groups (gastropods, polychaetes, crustaceans, and a bivalve) for DNA extraction. The amount of time the larval specimens had been stored in ethanol at the time of DNA extraction was ~10, ~7, and ~1 yrs, respectively, for EPR, Mariana, and Pescadero specimens. Only the Mariana specimens had been stored at 4 degrees C, while the others were at room temperature. Working in a laminar flow hood, we used sterile techniques to place 25 individuals from EPR and 25 individuals from Mariana into separate 1.5 mL vials with 95% ethanol. We thank Diana Franks for helping with sterile techniques. Pescadero specimens were provided in 2 vials each with 5 individuals of the same morphotype using sterile tools but not in a laminar flow hood.

DNA extraction and amplification are described by Carrier et al. (2021), and 28S rRNA larval genetic sequences and 16S rRNA bacterial genetic sequence data are in the Dryad repository (Carrier et al., 2021, doi.org/10.5061/dryad.sqv9s4n18). The 28S larval sequences were compared to those in the NCBI GenBank database using a BLAST search to assist with matching morphotypes to lowest taxonomic level. Results from the BLAST search were insufficient to provide additional taxonomic resolution; therefore, the 28S genetic results were primarily used as support for the morphological identification. We'd like to thank Bethany Fleming for conducting the BLAST searches.

Additional genetic evidence to assist with larval identification:
We'd like to thank Dr. Hiroka Hidaka, Dr. Shigeaki Kojima, and Dr. Hiromi Watanabe for providing histone 3 and Dr. Florence Pradillon for providing cytochrome oxidase subunit 1 (CO1) sequences for other Mariana larval specimens from the same collections. The Canadian Centre for DNA Barcoding provided CO1 sequences for other Pescadero specimens from the same collections; these results may be viewed in the Barcode of Life Data System (BOLD) Public Data Portal using the project search PESPL.

deep_sea_larvae.csv