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Haplotype-level analysis of environmental DNA metabarcoding revealed the biogeography and phylogeography of freshwater fishes in Korean Peninsula

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Abstract

Environmental DNA (eDNA) metabarcoding is a cost-effective, sensitive, and minimally invasive tool for monitoring fish diversity in freshwater ecosystems. To evaluate its suitability for assessing fish biogeography and phylogeography in Korean streams, we analyzed fish assemblages in three streams—Gilan, Seom, and Oshipcheon—located in different Korean biogeographical subdistricts. Over two sampling campaigns at nine sites, a total of 107 amplicon sequence variants (ASVs) were identified, representing 20 families, 46 genera, and 76 species. Among the 76 detected species, 29 were endemic to freshwater habitats in South Korea and played a significant role in shaping fish communities across the surveyed streams. We identified potential sources of six translocated species to the eastern region by examining their ASVs. After excluding these potential translocations, no endemic fish species were simultaneously detected in all three streams, and the phylogeography of endemic fish species Odontobutis platycephala, Coreoperca herzi, Microphysogobio yaluensis, Coreoleuciscus splendidus, Nipponocypris koreanus, and Koreocobitis rotundicaudata was clearly observed across the three biogeographical regions. Additionally, analysis of relative abundance and presence-absence data of fish communities yielded comparable β-diversity metrics reflecting spatiotemporal variation. The fish communities in the streams exhibited distinct groups, with Gilan showing a closer relationship to Seom than to Oshipcheon, consistent with well-known allopatric studies of freshwater fishes in the Korean Peninsula. Interestingly, we did not find any differences in fish assemblages among the three mesohabitat types (riffle, run, and pool), regardless of the sampling site. Our results highlight the potential of water-derived eDNA metabarcoding for detecting endemic fish, unraveling the biogeography and phylogeography of allopatric populations of freshwater fishes, and providing genetic forensic tools to estimate the original sources of translocated species.

Data availability

The raw sequences of the samples are available on the Sequence Read Archive (SRA) under the name Bioproject PRJNA994282.

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Acknowledgements

We thank members of the Marine Molecular Bioresources Laboratory of Pukyong National University and Molecular Evolution Laboratory of Sangji University for assisting in field sampling and pre-processing the samples, and to the Directorate General of Higher Education, Research, and Technology-Ministry of Education, Culture, Research, and Technology of The Republic of Indonesia, for providing a Ph.D. Scholarship (BPPLN) to Muhammad Hilman Fu’adil Amin. This work was supported by a grant from the National Institute of Biological Resources (NIBR), funded by the Ministry of Environment (MOE) of the Republic of Korea (NIBR201933203), and partially supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (RS-2021-NR060118).

Funding

This work was supported by a grant from the National Institute of Biological Resources (NIBR), funded by the Ministry of Environment (MOE) of the Republic of Korea (NIBR201933203), and partially supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (RS-2021-NR060118).

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Authors and Affiliations

  1. Industry 4.0 Convergence Bionics Engineering, Pukyong National University, Busan, 48513, Republic of Korea

    Muhammad Hilman Fu’adil Amin & Hyun-Woo Kim

  2. Advanced Tropical Biodiversity, Genomics, and Conservation Research Group, Department of Biology, Faculty of Science and Technology, Universitas Airlangga, Surabaya, 60115, Indonesia

    Muhammad Hilman Fu’adil Amin

  3. Marine Integrated Biomedical Technology Center, The National Key Research Institutes in Universities, Pukyong National University, Busan, 48513, Republic of Korea

    Ah Ran Kim & Hyun-Woo Kim

  4. Department of Biological Science, Sangji University, Wonju, 26339, Republic of Korea

    Ji Eun Jang & Hyuk Je Lee

  5. Biodiversity Research Department, National Institute of Biological Resources, Incheon, 22689, Republic of Korea

    Hye-Woo Byun

  6. Department of Marine Biology, Pukyong National University, Busan, 48513, Republic of Korea

    Hyun-Woo Kim

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  1. Muhammad Hilman Fu’adil Amin
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  2. Ah Ran Kim
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  3. Ji Eun Jang
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  4. Hye-Woo Byun
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  5. Hyuk Je Lee
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Contributions

M.H.F.A., A.R.K, H.J.L., and H.W.K. conceptualized the study; H.W.K., M.H.F.A., A.R.K, H.W.B., and J.E.J. performed the research methodology; J.E.J., M.H.F.A., and A.R.K contributed to bioinformatic pipeline for study; M.H.F.A., A.R.K, H.W.B., J.E.J., H.J.L. and H.W.K. conducted formal analysis and data curation; H.W.B., H.J.L. and H.W.K. supervised the study; and M.H.F.A., A.R.K, and H.W.K. wrote the original draft. All authors contributed to the review, writing, and editing of the final draft.

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Correspondence to
Hyun-Woo Kim.

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Amin, M.H.F., Kim, A.R., Jang, J.E. et al. Haplotype-level analysis of environmental DNA metabarcoding revealed the biogeography and phylogeography of freshwater fishes in Korean Peninsula.
Sci Rep (2026). https://doi.org/10.1038/s41598-026-36043-x

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  • DOI: https://doi.org/10.1038/s41598-026-36043-x

Keywords

  • Biogeography
  • Native fishes
  • Environmental DNA
  • Freshwater ecosystem
  • Metaphylogeography
  • Metabarcoding

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