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As the most diverse taxonomic groups in freshwater ecosystems, benthic invertebrates are widely employed as bioindicators for aquatic monitoring and health assessment. In this study, environmental DNA (eDNA) metabarcoding was used to reveal the diversity and spatiotemporal variations in benthic invertebrates in the Hun River Basin (the middle and upper reaches). Sampling was conducted in spring, summer, and autumn, with sites arranged to cover both anthropogenic activities and watershed environmental conditions. A total of 3 phyla, 7 classes, 18 orders, 56 families, 114 genera, and 161 species were detected. Insecta was the predominant class, and the combined EPT taxa (Ephemeroptera, Plecoptera, and Trichoptera) along with Diptera accounted for 64.71%, 74.18%, and 68.75% of the total taxa in spring, summer, and autumn. Community composition exhibited variations across both seasonal and spatial scales, as reflected by differences in OTU richness, dominant taxa and their relative sequence abundances, and alpha diversity indices. The top 10 dominant genera were all regionally common taxa, among which seven belonged to Insecta (including four from Chironomidae), whereas Sulcospira was the most spatially widespread dominant genus. Alpha and beta diversity analyses revealed significant seasonal differentiation but no significant spatial structuring. The SIMPER analysis identified the key genera that contributed substantially to observed community dissimilarity among sampling groups. This study provides fundamental data for freshwater biodiversity conservation and validates the potential applicability of eDNA metabarcoding for benthic invertebrate monitoring, while also revealing the current limitations of this technique.<\/jats:p>","DOI":"10.3390\/biology15181597","type":"journal-article","created":{"date-parts":[[2026,9,10]],"date-time":"2026-09-10T15:54:19Z","timestamp":1789055659000},"page":"1597","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Environmental DNA Metabarcoding Reveals Diversity and Spatiotemporal Variations of Benthic Invertebrates in Hun River Basin, China"],"prefix":"10.3390","volume":"15","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-3964-0211","authenticated-orcid":false,"given":"Peng","family":"Hou","sequence":"first","affiliation":[{"name":"College of Life Science and Bioengineering, Shenyang University, Shenyang 110044, China"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"conceptualization","vocabulary":"credit"},{"role":"methodology","vocabulary":"credit"},{"role":"software","vocabulary":"credit"},{"role":"formal-analysis","vocabulary":"credit"},{"role":"investigation","vocabulary":"credit"},{"role":"writing-original-draft","vocabulary":"credit"}]},{"ORCID":"https:\/\/orcid.org\/0009-0006-3181-3954","authenticated-orcid":false,"given":"Xin","family":"Ma","sequence":"additional","affiliation":[{"name":"College of Life Science and Bioengineering, Shenyang University, Shenyang 110044, China"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"software","vocabulary":"credit"},{"role":"formal-analysis","vocabulary":"credit"},{"role":"resources","vocabulary":"credit"},{"role":"data-curation","vocabulary":"credit"},{"role":"writing-original-draft","vocabulary":"credit"},{"role":"visualization","vocabulary":"credit"}]},{"given":"Li","family":"Liu","sequence":"additional","affiliation":[{"name":"College of Life Science and Bioengineering, Shenyang University, Shenyang 110044, China"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"validation","vocabulary":"credit"},{"role":"investigation","vocabulary":"credit"},{"role":"visualization","vocabulary":"credit"}]},{"given":"Ying","family":"Men","sequence":"additional","affiliation":[{"name":"College of Life Science and Bioengineering, Shenyang University, Shenyang 110044, China"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"software","vocabulary":"credit"},{"role":"validation","vocabulary":"credit"},{"role":"data-curation","vocabulary":"credit"},{"role":"visualization","vocabulary":"credit"}]},{"given":"Jianfeng","family":"Wang","sequence":"additional","affiliation":[{"name":"College of Life Science and Bioengineering, Shenyang University, Shenyang 110044, China"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"validation","vocabulary":"credit"},{"role":"supervision","vocabulary":"credit"}]},{"given":"Ding","family":"Yang","sequence":"additional","affiliation":[{"name":"Department of Entomology, College of Plant Protection, China Agricultural University, Beijing 110193, China"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"conceptualization","vocabulary":"credit"},{"role":"methodology","vocabulary":"credit"},{"role":"writing-review-editing","vocabulary":"credit"},{"role":"supervision","vocabulary":"credit"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9514-0502","authenticated-orcid":false,"given":"Chuntian","family":"Zhang","sequence":"additional","affiliation":[{"name":"College of Life Science, Shenyang Normal University, Shenyang 110034, China"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"conceptualization","vocabulary":"credit"},{"role":"methodology","vocabulary":"credit"},{"role":"writing-review-editing","vocabulary":"credit"},{"role":"funding-acquisition","vocabulary":"credit"},{"role":"supervision","vocabulary":"credit"}]}],"member":"1968","published-online":{"date-parts":[[2026,9,10]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"eabf2675","DOI":"10.1126\/sciadv.abf2675","article-title":"Erosion of global functional diversity across the tree of life","volume":"7","author":"Carmona","year":"2021","journal-title":"Sci. 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