Hidden diversity of marine zooplankton under sea ice in the Okhotsk Sea: Insights from community and population analyses based on DNA metabarcoding

The Okhotsk Sea is a semi-closed marine area characterized by unique environments, including seasonal sea ice during winter, and is expected to harbor distinct zooplankton diversity. However, zooplankton beneath sea ice remain inadequately researched, primarily because of sampling and methodological challenges. In this study, we used the Okhotsk Tower and icebreakers Garinko II/III, typically used for sightseeing, to collect zooplankton samples during the sea-ice season off the coast of Mombetsu, Hokkaido, Japan. We used DNA metabarcoding of mitochondrial cytochrome oxidase subunit I (COI) and nuclear small ribosomal subunit (18S) genes to assess species- and population-level diversity. Metabarcoding analyses of both COI and 18S revealed clear shifts in zooplankton community composition based on operational taxonomic units (OTUs), which aligned with environmental differences in water temperature and chlorophyll concentration. BLAST searches indicated that 37.6% of COI and 21.5% of 18S OTUs shared <97% identity with known sequences, indicating potentially hidden species diversity or inadequate reference data in public databases. The dominant COI-identified taxa included Clione okhotensis, Neocalanus flemingeri, Pseudocalanus acuspes, and P. newmani, which were described or known to have distinct diversity in the Okhotsk Sea. For the other dominant species, namely Acartia longiremis, Eucalanus bungii, and Hybocodon prolifer, dominant haplotypes were not shared with other geographic regions based on amplicon sequence variants. Although diversity patterns are influenced by the ecological traits of zooplankton species, our metabarcoding approach effectively captured both species- and population-level diversity, thereby highlighting the presence of previously unrecognized zooplankton diversity beneath the sea ice of the Okhotsk Sea.