DNA metabarcoding for diet analysis of an endangered endemic bitterling, Acheilognathus majusculus (Teleostei; Cyprinidae)

The bitterling species, Acheilognathus majusculus , is a freshwater fish, endemic to the Nakdong and Seomjin rivers in South Korea and listed as an endangered species by the Ministry of Environment in South Korea due to its biological, ecological, and genetic importance. Dietary information is crucial for understanding the role of A. majusculus within an ecosystem and planning its restoration strategy. In this study, we employed a non-invasive DNA metabarcoding approach to characterize the diet using 34 A. majusculus fecal samples collected from the Nakdong and Seomjin rivers. Analysis of 1642037 clean reads generated by applying the next generation sequencing yielded 210 amplicon sequence variants (ASVs). Taxonomic assignment successfully identified 10 phyla, 16 classes, 23 orders, 29 families, 29 genera, and 26 species. Green algae (Chlorophyta) and diatoms (Bacillariophyta) were predominantly detected in all samples, with percentages ranging from 5.56 to 43.59% and 30.00 to 66.67%, respectively. Smaller percentages of other taxa such as parasitic protists of Ichthyosporea (Opisthokonta incertae sedis), ciliates (Ciliophora), mostly eustigmatophytes (Ochrophyta), fungi (Ascomycota), vertebrates (Chordata), flatworms (Platyhelminthes), and green plants (Streptophyta) were also detected. The dietary composition of A. majusculus remained consistent irrespective of body size or sex. However, there was pronounced seasonal variation between summer samples and those from spring and fall, as demonstrated by beta-diversity analyses. These findings provide valuable insights into the dietary composition and seasonal variations of A. majusculus and highlight the need to protect and restore aquatic habitats and regulate water flow from weirs to ensure the consistent availability of essential food resources for effective habitat management strategies of this endangered species.