Dataset: JTMD Mytilus population genetics
Deployment: JTMD_2012

Mytilus specimens were collected from Japanese tsunami marine debris (JTMD) from 2012-2014 at coastal sites in Washington, Oregon and Hawaii.

Population level analysis of focal JTMD species. Mytilus spp. were one of the most common organisms on JTMD. Cytochrome c oxidase subunit III was chosen for species identification of mussels because available female-specific primers allowed a simpler approach to species discrimination than the standard COI primers, which amplify both male and female sequences and complicate analyses. The proposed method of pooling tissues for bulk DNA extraction and post-sequencing analysis of haplotype variation was set aside due to presumed sequence error observed when sequencing individual mussel amplicons. Instead, 695 mussels were extracted for DNA and sequenced individually on the Ion Torrent platform. Addition of index nucleotides to PCR products allowed post-sequencing separation of sequences by source mussel. Bins of reads from individual mussels were mapped to reference sequences of female M. galloprovincialis, M. trossulus, and M. coruscus. Known distribution of Mytilus suggested that mussels in Japan would be M. galloprovincialis. A sample of mussels for which Ion Torrent sequencing resulted in identification as M. trossulus or M. coruscus were reanalyzed by preparation of new PCR product and Sanger sequencing.

Metagenomic analysis of JTMD assemblages. Four 15 cm2 samples were scraped from the sides and top of the floating dock "Misawa 1" that beached near Newport Oregon in June 2012. Samples were preserved in ethanol and shipped to Moss Landing Marine Laboratories. Entire samples were decanted of ethanol and homogenized in an IKE Basic Mill. Ten grams of each sample were extracted with the Mo Bio Powersoil Mega DNA extraction kit (Mo Bio, Carlsbad, CA). Cytochrome c oxidase subunit I was PCR amplified, and prepared for sequencing on the Ion Torrent PGM. Resulting reads were pooled, quality and size filtered, and assembled into operational taxonomic units (OTUs) using the Velvet assembler. Gene OTUs were compared to eukaryotic COI sequences culled from Genbank by using variants of the search term for COI (COI, COX1, Cytochrome oxidase subunit I, Cytochrome c oxidase subunit I, etc...) and excluding prokaryota. We also specifically searched for COI sequences associated with of morphologically identified JTMD specie and congeners, as well as con-familials when morphological identification was limited to the family level.