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Environmental DNA reveals the distinct genetic diversity and evolutionary pathways of the Chinese Minnow Rhynchocypris oxycephalus in Korean freshwater systems

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Abstract

Establishing an environmental DNA (eDNA) reference library at regional and local scales is essential not only for accurate biodiversity assessment but also for comprehensive long-term monitoring. To date, genetic diversity studies of the Chinese minnow (Rhynchocypris oxycephalus) have largely been restricted to China, leaving substantial knowledge gaps across its broader distribution, including South Korea. Hence, the present study identified suitable regions for guiding eDNA surveys and non-invasive sampling, based on documented occurrences retrieved from the IUCN Geospatial Conservation Assessment Tool (GeoCAT). The newly designed primer pairs successfully amplified long mitochondrial fragments (~ 1 kb) of the cytochrome b (Cytb) and 16S ribosomal RNA (16S rRNA) genes. The generated sequences revealed 29 haplotypes from 41 Cytb sequences and 13 haplotypes from 21 16S rRNA sequences, corresponding to high intraspecific genetic diversity (5.57% for Cytb and 2.46% for 16S rRNA), thereby indicating potential cryptic diversity of R. oxycephalus in South Korea. The phylogenetic analyses, combined with multiple species delimitation methods, resolved several putative molecular operational taxonomic units and highlighted a distinct genetic clade in the Seomjin River basin, likely driven by microhabitat-specific evolutionary processes. In addition, the shared haplotypes across catchments of different river basins indicate either ongoing gene flow or anthropogenic influences contributing to genetic admixture of R. oxycephalus. The time-calibrated phylogenetic analyses also suggest that historical geographic changes and ancient river networks, from the Early Miocene to the Late Pliocene, likely facilitated the diversification of R. oxycephalus across China, the Korean Peninsula, and Japan. Overall, this study represents the first eDNA-based assessment of R. oxycephalus diversity in South Korea, while also providing new evolutionary insights from a broader geographic context in China and Japan. Given the complexity of multiple river networks in South Korea, further investigations using multiple genetic markers are recommended to enhance understanding of this cyprinid species phylogeography in the region.

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Data availability

The generated sequencing data has been deposited in NCBI GenBank (https://www.ncbi.nlm.nih.gov/) with the following accession numbers PQ330261 to PQ330301 and PQ333020 to PQ333040.

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Acknowledgements

The authors wish to express their profound gratitude to the laboratory colleagues of the Molecular Physiology Laboratory, Department of Marine Biology, Pukyong National University for their support during this research.

Funding

This work was supported by Dongwon Research Foundation in 2024 (202404170001) and the corresponding author (Hyun-Woo Kim) was supported by 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

    Gyurim Bang

  2. Research Center for Marine Integrated Bionics Technology, Pukyong National University, Busan, 48513, Republic of Korea

    Soo Rin Lee, Ah Ran Kim & Hyun-Woo Kim

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

    Soo Rin Lee, Ah Ran Kim & Hyun-Woo Kim

  4. Institute of Marine Life Science, Pukyong National University, Busan, 48513, Republic of Korea

    Hye-Eun Kang

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

    Hsu Htoo & Hyun-Woo Kim

  6. Department of Zoology, Bodoland University, Kokrajhar, 783370, India

    Imon Abedin

  7. Advance Tropical Biodiversity, Genomics, and Conservation Research Group, Department of Biology, Faculty of Science and Technology, Airlangga University, Surabaya, 60115, Republic of Indonesia

    Muhammad Hilman Fu’adil Amin

  8. Major of Biomedical Engineering, Division of Smart Healthcare, Pukyong National University, Busan, Republic of Korea

    Myunggi Yi

  9. Molecular Ecology and Evolution Laboratory, Department of Biological Science, Sangji University, Wonju-si, 26339, Kangwon-State, Republic of Korea

    Hyuk Je Lee

  10. Ocean and Fisheries Development International Cooperation Institute, College of Fisheries Science, Pukyong National University, Busan, 48513, Republic of Korea

    Shantanu Kundu

  11. International Graduate Program of Fisheries Science, Pukyong National University, Busan, 48513, Republic of Korea

    Shantanu Kundu

  12. Advance Tropical Biodiversity, Genomics, and Conservation Research Group, Department of Biology, Faculty of Science and Technology, Airlangga University, Surabaya, 60115, Indonesia

    Hyun-Woo Kim

Authors

  1. Gyurim Bang
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  2. Soo Rin Lee
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  3. Hye-Eun Kang
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  4. Ah Ran Kim
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  5. Hsu Htoo
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  6. Imon Abedin
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  7. Muhammad Hilman Fu’adil Amin
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  8. Myunggi Yi
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  9. Hyuk Je Lee
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  10. Shantanu Kundu
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  11. Hyun-Woo Kim
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Contributions

S.K. and H.-W.K. conceived and supervised the study. G.B., S.R.L., and H.H. performed the experiments. G.B., H.-E.K., and A.R.K. performed the data analyses. M.Y., H.J.K., I.A. and M.H.F.A. contributed to the data analyses. G.B., S.K., and H.-W.K. wrote the manuscript. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to
Shantanu Kundu or Hyun-Woo Kim.

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Competing interests

The authors declare no competing interests.

Ethical accordance for procedures

The study is entirely based on environmental water samples; therefore, no animal samples were collected from the wild, and no special permissions were required for this research.

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Bang, G., Lee, S.R., Kang, HE. et al. Environmental DNA reveals the distinct genetic diversity and evolutionary pathways of the Chinese Minnow Rhynchocypris oxycephalus in Korean freshwater systems.
Sci Rep (2025). https://doi.org/10.1038/s41598-025-32073-z

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  • DOI: https://doi.org/10.1038/s41598-025-32073-z

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