Environmental DNA Metabarcoding Reveals Spatiotemporal Variation in Fish Communities of the Middle Raohe River, China

Environmental DNA (eDNA) metabarcoding can provide fish-community inventories across river networks. We surveyed ten sites in the Jingdezhen section of the Raohe River system on 20-23 October 2023 and 2-5 June 2024, covering the Donghe River, Chang River, and Le'an River. Three technical process replicates were pooled per site and campaign to produce 20 community profiles. The two surveys detected 96 and 94 taxa, respectively, including 100 fish taxa overall, of which 90 were shared. Richness, Shannon diversity, Simpson diversity, and Pielou evenness did not differ significantly between paired campaigns. Community composition differed between campaigns (Bray-Curtis PERMANOVA: R² = 0.293; Jaccard: R² = 0.185; both adjusted p = 0.002). PERMDISP did not detect unequal dispersion. River-group differentiation was clearer in 2023, whereas neither distance metric was significant in 2024 after multiple-testing correction. A reduced environmental partial RDA accounting for site and campaign effects was not significant (F = 0.971, p = 0.420). These findings provide a two-campaign molecular inventory for the ten surveyed sites and identify between-campaign compositional differences, without demonstrating seasonal dynamics or independent environmental drivers.