Improving taxonomic classification of marine zooplankton by molecular approach: registration of taxonomically verified 18S and 28S rRNA gene sequences

Tsuyoshi Watanabe; Junya Hirai; Sirje Sildever; Kazuaki Tadokoro; Kiyotaka Hidaka; Iwao Tanita; Koh Nishiuchi; Naoki Iguchi; Hiromi Kasai; Noriko Nishi; Seiji Katakura; Yukiko Taniuchi; Taketoshi Kodama; Satokuni Tashiro; Misato Nakae; Yuji Okazaki; Satoshi Kitajima; Sayaka Sogawa; Toru Hasegawa; Tomonori Azumaya; Yutaka Hiroe; Daisuke Ambe; Takashi Setou; Daiki Ito; Akira Kusaka; Takeshi Okunishi; Takahiro Tanaka; Akira Kuwata; Daisuke Hasegawa; Shigeho Kakehi; Yugo Shimizu; Satoshi Nagai

doi:10.7717/peerj.15427

Improving taxonomic classification of marine zooplankton by molecular approach: registration of taxonomically verified 18S and 28S rRNA gene sequences

PeerJ. 2023 Jun 7:11:e15427. doi: 10.7717/peerj.15427. eCollection 2023.

Authors

Tsuyoshi Watanabe¹, Junya Hirai², Sirje Sildever^{3

4}, Kazuaki Tadokoro⁵, Kiyotaka Hidaka³, Iwao Tanita⁶, Koh Nishiuchi⁷, Naoki Iguchi⁸, Hiromi Kasai¹, Noriko Nishi⁹, Seiji Katakura¹⁰, Yukiko Taniuchi¹, Taketoshi Kodama^{3

11}, Satokuni Tashiro⁶, Misato Nakae⁸, Yuji Okazaki⁵, Satoshi Kitajima⁷, Sayaka Sogawa³, Toru Hasegawa⁷, Tomonori Azumaya¹, Yutaka Hiroe⁷, Daisuke Ambe³, Takashi Setou³, Daiki Ito³, Akira Kusaka³, Takeshi Okunishi⁵, Takahiro Tanaka⁵, Akira Kuwata⁵, Daisuke Hasegawa⁵, Shigeho Kakehi⁵, Yugo Shimizu⁵, Satoshi Nagai¹²

Affiliations

¹ Kushiro Field Station, Fisheries Resources Institute, Japan Fisheries Research and Education Agency, Kushiro, Japan.
² Atmosphere and Ocean Research Institute, The University of Tokyo, Kashiwa, Japan.
³ Yokohama Field Station, Fisheries Resources Institute, Japan Fisheries Research and Education Agency, Shiogama, Japan.
⁴ Department of Marine Systems, Tallinn University of Technology, Tallinn, Estonia.
⁵ Shiogama Field Station, Fisheries Resources Institute, Japan Fisheries Research and Education Agency, Shiogama, Japan.
⁶ Yaeyama Field Station, Fisheries Technology Institute, Japan Fisheries Research and Education Agency, Ishigaki, Japan.
⁷ Nagasaki Field Station, Fisheries Resources Institute, Japan Fisheries Research and Education Agency, Nagasaki, Japan.
⁸ Niigata Field Station, Fisheries Resources Institute, Japan Fisheries Research and Education Agency, Niigata, Japan.
⁹ AXIOHELIX Co. Ltd., Tokyo, Japan.
¹⁰ City of Mombetsu, Mombetsu, Japan.
¹¹ Present Address: Graduate School of Agricultural and Life Sciences, The University of Tokyo, Japan.
¹² Yokohama Field Station, Fisheries Technology Institute, Fisheries Research and Education Agency, Yokohama, Japan.

Abstract

Background: Zooplankton plays an important role in the marine ecosystem. A high level of taxonomic expertise is necessary for accurate species identification based on morphological characteristics. As an alternative method to morphological classification, we focused on a molecular approach using 18S and 28S ribosomal RNA (rRNA) gene sequences. This study investigates how the accuracy of species identification by metabarcoding improves when taxonomically verified sequences of dominant zooplankton species are added to the public database. The improvement was tested by using natural zooplankton samples.

Methods: rRNA gene sequences were obtained from dominant zooplankton species from six sea areas around Japan and registered in the public database for improving the accuracy of taxonomic classifications. Two reference databases with and without newly registered sequences were created. Comparison of detected OTUs associated with single species between the two references was done using field-collected zooplankton samples from the Sea of Okhotsk for metabarcoding analysis to verify whether or not the newly registered sequences improved the accuracy of taxonomic classifications.

Results: A total of 166 sequences in 96 species based on the 18S marker and 165 sequences in 95 species based on the 28S marker belonging to Arthropoda (mostly Copepoda) and Chaetognatha were registered in the public database. The newly registered sequences were mainly composed of small non-calanoid copepods, such as species belonging to Oithona and Oncaea. Based on the metabarcoding analysis of field samples, a total of 18 out of 92 OTUs were identified at the species level based on newly registered sequences in the data obtained by the 18S marker. Based on the 28S marker, 42 out of 89 OTUs were classified at the species level based on taxonomically verified sequences. Thanks to the newly registered sequences, the number of OTUs associated with a single species based on the 18S marker increased by 16% in total and by 10% per sample. Based on the 28S marker, the number of OTUs associated with a single species increased by 39% in total and by 15% per sample. The improved accuracy of species identification was confirmed by comparing different sequences obtained from the same species. The newly registered sequences had higher similarity values (mean >0.003) than the pre-existing sequences based on both rRNA genes. These OTUs were identified at the species level based on sequences not only present in the Sea of Okhotsk but also in other areas.

Discussion: The results of the registration of new taxonomically verified sequences and the subsequent comparison of databases based on metabarcoding data of natural zooplankton samples clearly showed an increase in accuracy in species identification. Continuous registration of sequence data covering various environmental conditions is necessary for further improvement of metabarcoding analysis of zooplankton for monitoring marine ecosystems.

Keywords: Japan; Marine biology; Metabarcoding; Pacific Ocean; Plankton; Species identification.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Biodiversity
Ecosystem*
Genes, rRNA
RNA, Ribosomal, 28S / genetics
Zooplankton* / genetics

Substances

RNA, Ribosomal, 28S

Grants and funding

This work was supported by “Establishing a network of environment and fisheries information”, Ministry of Agriculture, Forestry and Fisheries, Japan. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.