Comparative Analysis of Benthic Invertebrates and Fish Communities in Three Korean Streams using Environmental DNA Metabarcoding

Abstract

Aquatic organisms have been considered as the useful bioindicators to understand the environmental characteristics, which would be useful to assess ecological health and biodiversity of freshwater ecosystems. By parallel sequencing, environmental DNA (eDNA) metabarcoding is currently one of the most promising tools to monitor the fish diversity in freshwater ecosystem due to numerous benefits, including low costs and labors for the analysis, high sensitivity and accuracy in identification, the least harmful impacts on habitats during collecting water samples, and revealing several ecological phenomena in the communities. In this study, invertebrate and fish communities were assessed by DNA metabarcoding from three Korean streams (Seom, Gilan, and Oshipcheon). In the first the section, we aim to evaluate if the novel metabarcoding analysis can be one of the useful tools to analyze the benthic invertebrate community by examined the pipeline using the unsorted sediment samples collected from the three streams. As the result of metabarcoding of samples collected from three streams using 18Sv9 rRNA, a total of 3,056,351 merged reads and 275 Operational Taxonomic Units (OTUs) were respectively obtained, which covered 114 invertebrate families. Invertebrate communities significantly varied by spatiotemporal differences not only among three streams but also within a stream. Statistical analysis showed that both OTUs and its families exhibited significant spatio-temporal difference, even among mesohabitats, suggesting the community study at the family level was sufficient to distinguish the benthic invertebrate assemblages in each sample. The taxa presenting ecological characteristics of each sample site by the metabarcoding were in accordance to the previous studies using traditional survey methods. Ephemeridae, Chironomidae, and Ephemerellidae showed seasonal variability for their aquatic-terrestrial life cycle, while abundance of many aquatic insects (Simuliidae, Hydropsychidae, Baetidae, Ephemerellidae, Tipulidae, Leptophlebiidae, and Heptageniidae), two molluscans (Cyrenidae and Semisulcospiridae), an annelid (Naididae), and a platyhelminthes (Dugesiidae) was significantly different by the mesohabitat characteristics. Naididae was the most abundant taxa in Seom and Gilan, while Dugesiidae was the most abundant taxa in Oshipcheon, in which salinity and temperature mainly contributed to the difference in Oshipcheon. Among the mesohabitats, riffle showed the highest correlations (r = 0.721 to 0.884) to overall communities suggesting that metabarcoding analysis of fast-flowing habitat would be the most representative site to monitor each stream. In the second section, we here examined the additional potential applications of eDNA metabarcoding to uncovers regional characteristics of freshwater ecosystem, haplotype variations and even metaphylogeographical profiles in Korea. Using MiFish eDNA pipeline, we analyzed fish diversity in those three small streams, each of which was located in three main biogeographical subdistricts in Korean peninsula. Total 107 amplicon sequence variants (ASVs) were identified from nine samples sites in two seasons, which covered 20 families, 46 genera and 76 species. Regardless of sample collection sites, we failed to identify any difference in fish assemblage among three mesohabitat types, riffle, run, and pool. Changes in species numbers and proportions between March and August suggested that eDNA metabarcoding would be useful to monitor the seasonal/reproductive migration within or outside river system. Of the 76 detected species, 29 were endemic freshwater species in South Korea, and they played a significant role in structuring fish communities among surveyed streams. The relative abundance and presence-absence data of fish communities generated comparable β-diversity metrics corresponding to spatiotemporal variation. The fish communities among streams exhibited clearly segregated groups, presenting Gilan was closely related to Seom rather than to Oshipcheon and demonstrating well-known allopatric studies of freshwater fish in the Korean Peninsula. We also identified the possible sources of six translocated species to the eastern region by identifying their ASVs. By eliminating those possible fish translocations, no endemic fish was simultaneous coincidentally detected among three streams and phylogeography of endemic fish Odontobutis platycephala, Coreoperca herzi, Microphysogobio yaluensis, Coreoleuciscus splendidus, Nipponocypris koreanus, and Koreocobitis rotundicaudata were clearly observed among three biogeographical regions. In addition, the relative recovered species among mesohabitat types did not significantly differ in wadable stream. Our results proposed the potential of water-derived eDNA metabarcoding for detecting endemic fishes, disentangling the biogeography and phylogeography of allopatric populations of freshwater fishes, and providing genetic forensic to estimate the original sources of translocated species.