Methods

Tissues from single, wild-collected individuals were used for this study. Three specimens were obtained from the Western Australian Museum (WAM) Marine Mollusca collection (Western Australian Museum 2023), and four were collected live for this study (CPIC, AMNH, MNHN). Live specimens were collected by hand while SCUBA diving and preserved in 95-100% EtOH for DNA sequencing.

DNA extraction was performed using the Qiagen DNEasy genomic extraction kit using the standard process. Paired-end sequencing libraries were constructed using the Illumina TruSeq kit according to the manufacturer’s instructions. The libraries were sequenced on an Illumina Hi-Seq platform in paired-end, 2 × 150bp format. The resulting fastq files were trimmed of adapter/primer sequences and low-quality regions with Trimmomatic v0.33 (Bolger, Lohse, and Usadel 2014). The trimmed sequence was assembled by SPAdes v3.15.4 (Bankevich et al. 2012) followed by a final assembly finishing step using Zanfona (Kieras, O’Neill, and Pirro 2021).

Results and Data Availability

 
Species Locality Date Collected Accession Specimen_voucher
Bornella anguilla Beagle Bay, Kimberley, WA, Australia 12 March 2008 JBDGEK000000000 WAMS:44513
Lomanotus vermiformis Bocas del Toro, Panama 6 Aug 2004 JBBPDF000000000 CPIC:00556
Melibe bucephala Seringapatam Reef, WA, Australia 27 Sept 2007 JBCJKE000000000 WAMS:31224
Melibe viridis South Mole, Fremantle, WA, Australia 15 April 2019 JBCIKM000000000 WAMS:72630
Plocamopherus ceylonicus Kwinana Grain Terminal Jetty, Rockingham, WA, Australia 21 June 2023 JBCJKF000000000 AMNH:IZC-00385163
Plocamopherus imperialis Koumac, New Caledonia 7 Nov 2018 JBCHJU000000000 MHNH-IM-2019-26525
Plocamopherus tilesii Koumac New Caledonia 21 Nov 2019 JBBPCV000000000 MNHN-IM-2019-26526

Funding

Funding was provided by Iridian Genomes grant# IRGEN_RG_2021-1345: Genomic Studies of Eukaryotic Taxa. Field work in Australia was funded by the American Museum of Natural History, and work in New Caledonia was funded by the Gouvernement de la Nouvelle-Calédonie, Province Nord, Agence Française de la Biodiversité (AFB), the Lounsbery Foundation, and Office des Postes et Télécommunications (OPT).

Acknowledgments

We are grateful for specimens and support from Lisa Kirkendale and Corey Whisson at the Western Australian Museum, and collecting assistance from Henry Carrick for Plocamopherus ceylonicus. We also thank Rachel Colin for supporting field work in Bocas del Toro, Panama and facilitating export and collecting permits and Philippe Bouchet for the invitation to participate in the specimens “Our Planet Reviewed”—New Caledonia expeditions (2018–2019), a joint project of MNHN and Conservatoire d’Espaces Naturels (CEN) de Nouvelle-Calédonie.