Evaluation of fish biodiversity in estuaries using environmental DNA metabarcoding

single day sample, the number and list of species from obtain results reflected the ecology of each fish and environmental conditions, such as eutrophication and temperature, in each river. We believe further development of the eDNA technique will offer an alternative method for accurate and non-invasive monitoring of aquatic life. Supporting information S1 Text. Entire procedure of library preparation, sequencing, data preprocessing and taxo- nomic assignment. (PDF) S1 Table. Summary of data preprocessing steps and subsequent taxon assignment using pipeline analysis (MiFish ver. 2.3). Numerals are read numbers and those in parentheses of “Data preprocessing” are percentages against the raw read numbers, while those in “Taxon assignment” are percentages against the denoised read numbers. (PDF) S2 Table. List of species detected at the sampling stations. Plus (+) represents occurrence. HT: river mouth at high tide; LT: river mouth at low tide; L1: left 500 m; L2: left 1 km; R1: right 500 m; R2: right 1 km. †: endangered species according to the IUCN (https://www. iucnredlist.org). ‡: endangered species according to the Ministry of the Environment of Japan (http://ikilog.biodic.go.jp/Rdb/env). †‡: endangered species according to both classifications. (PDF) Acknowledgments We are grateful to Dr. Komei Kadowaki and Mr. Shingo Takada for their help with fieldwork, Dr. Aya Yamazaki and Dr. Natsuko Kondo for guidance during molecular experiments and genetic analysis, Dr. Yoshiaki Kai and Dr. Yumi Henmi for confirming the species list, and

Ms. Yuka Hayakawa for help with data analysis and arrangement. Author Contributions Conceptualization: Yoh Yamashita, Akihide Kasai. Data curation: Hyojin Ahn, Manabu Kume, Yuki Terashima, Feng Ye, Satoshi Kameyama, Akihide Kasai. Formal analysis: Hyojin Ahn, Manabu Kume, Masaki Miya. Funding acquisition: Yoh Yamashita. Investigation: Feng Ye, Satoshi Kameyama. Validation: Hyojin Ahn. Visualization: Hyojin Ahn. Writing – original draft: Hyojin Ahn, Manabu Kume, Satoshi Kameyama, Masaki Miya, Aki- hide Kasai. Writing – review & editing: Hyojin Ahn. References 1. Ginsburg RN. Proceedings of the colloquium on global aspects of coral reefs: Health, Hazards and His- tory (Ed.) Miami: University of Miami; 1994. PLOS ONE Biodiversity of five estuaries PLOS ONE | https://doi.org/10.1371/journal.pone.0231127 October 6, 2020 12 / 15