Freshwater bivalves contribute to key ecological functions in lake ecosystems, yet their cryptic and benthic lifestyles often hinder detection through conventional surveys. In this study, we applied environmental DNA (eDNA) metabarcoding to assess the diversity and distribution of unionid bivalves in six lakes across Republic of Korea. Water samples were collected from three sampling strategies-Center Surface, Center Mix, and Waterside Surface-and processed using 16S rDNA-targeted primers followed by high-throughput sequencing. A total of four unionid species (Cristaria plicata, Sinanodonta lauta, Unio (Nodularia) douglasiae, and Anodonta woodiana) were detected across 18 sampling points. Notably, eDNA successfully identified unionid presence in all lakes, even where conventional surveys failed to observe individuals. Among the sampling strategies, Center Mix exhibited the highest values for Shannon and Simpson indices as well as ASV richness. Waterside Surface samples generally showed lower diversity and detection frequency. A Venn diagram of ASV occurrences revealed three ASVs shared across all sampling strategies and one unique ASV found only in Center Mix. These results indicate that sampling location significantly affects detection sensitivity and diversity representation in eDNA-based bivalve monitoring. Combined application of Center Mix and Center Surface strategies may enhance both detection efficiency and species diversity coverage in lentic environments.

Environmental DNA (eDNA) analysis has emerged as a powerful tool for biodiversity monitoring due to its efficiency, standardization potential, and cost-effectiveness. We evaluated the applicability of eDNAbased zooplankton monitoring in Korean lakes by comparing three DNA methods-eDNA, iDNA, and eiDNA-with traditional microscopy. Sampling was conducted in six lakes with varying conditions. eDNA was obtained from lake water, iDNA from unpreserved zooplankton incubated in water, and eiDNA from zooplankton incubated in ethanol. DNA metabarcoding detected more taxa than microscopy, but dominant taxa overlapped, mainly Daphnia. While DNA methods showed higher richness, Simpson and Shannon indices were higher in microscopy, reflecting differences in quantification methods. These discrepancies reflect methodological differences in how taxa are quantified and suggest that DNA-based approaches may overrepresent certain groups in richness estimates. In addition, false negatives were observed for several common rotifer species (e.g., Keratella, Polyarthra), likely due to incomplete reference databases and high intraspecific genetic diversity. Conversely, some taxa detected only by DNA-particularly small-bodied or rare crustaceans-may represent false positives relative to microscopy. These findings emphasize the importance of improving reference libraries and interpreting DNA results with caution, while also supporting the utility of DNA-based methods as complementary tools in zooplankton monitoring and national biodiversity assessments.

Phytoplankton play a vital role as primary producers in freshwater ecosystems, contributing to the nutrient cycle, energy flow, and ecological stability. To accurately assess phytoplankton diversity and community composition, this study compared traditional microscopy and environmental DNA (eDNA) metabarcoding in six small lakes located in the Han, Geum, and Nakdong River basins in Korea. eDNA analysis identified 268 species from 161 genera, approximately 2.4 times higher than microscopy, which detected 113 species from 68 genera. The eDNA data were dominated by picocyanobacteria such as Synechococcus and Cyanobium, while microscopy primarily revealed larger taxa, including Stephanodiscus and Scenedesmus. Nonmetric multidimensional scaling (NMDS) based on Bray-Curtis similarity showed clear separation between the two methods, with average similarity values of 0.0326 (1st survey) and 0.0221 (2nd survey) at the species level. Only 6.8% of the 429 total species were commonly detected by both methods, while overlap at the genus level was 18.8%. Spatial heterogeneity in phytoplankton communities based on eDNA was also evident depending on the sampling location, with the centre of the surface showing the highest species richness and overlap, suggesting its suitability for biodiversity monitoring. These findings demonstrate the high resolution and sensitivity of eDNA metabarcoding in capturing phytoplankton diversity and highlight its complementary role in existing biomonitoring programmes. Further improvements in the quantitative reliability of eDNA-based assessments will require efforts such as copy number normalisation, methodological standardisation, and refinement of reference databases.

This study analyzed the epilithic diatom community and ecological health of freshwater streams using environmental DNA (eDNA)-based metabarcoding technology. eDNA metabarcoding is a method that analyzes biological communities by performing PCR amplification followed by next-generation sequencing (NGS), offering higher sensitivity and faster results compared to traditional microscopic analyses. The study compared the eDNA metabarcoding results of ribulose bisphosphate carboxylase large chain gene (rbcL) targeting epilithic diatoms according to Taq polymerases (SuperFi II, GainBlue, EzPCR, and AccuPower). SuperFi II and GainBlue yielded the highest number of reads and zOTUs, with GainBlue showing particularly uniform read distribution, allowing for more accurate analysis for community diversity of epilithic diatoms. On the other hand, EzPCR and AccuPower exhibited lower number of reads and zOTUs, making them less suitable for the community diversity. In terms of community similarity analysis, SuperFi II and GainBlue produced highly similar results, while EzPCR and AccuPower showed significant differences. This study demonstrates that PCR Taq polymerases significantly influence community diversity and similarity analyses of epilithic diatoms, with GainBlue providing the most stable and accurate results. Our findings serve as a valuable foundation for improving the accuracy of eDNA-based metabarcoding analyses of diatoms.

Tricholoma matsutake is an economically important ectomycorrhizal mushroom, but artificial cultivation remains challenging. While much research has been conducted on the bacteria and fungi associated with T. matsutake, studies on archaea have been limited. This study compared and analyzed the diversity and structural differences of archaeal communities in the fairy ring and non-fairy ring soils using a metabarcoding approach. The alpha diversity analysis revealed that the archaeal community in fairy ring soil had lower diversity compared to non-fairy ring soil, and beta diversity analysis clearly separated the community structures between the two soil types. Among all analyzed soils, Nitrososphaeria (Nitrososphaerota) dominated the archaeal community at class level. In fairy ring soils, Nitrosotalea (Nitrosotaleaceae) was predominant, while in non-fairy ring soils, Nitrosocaldaceae dominated. These results suggest that Nitrosotalea may have a close association with T. matsutake, highlighting the need for further in-depth research on archaea as potential growth promoting microorganism for T. matsutake mycelial growth.

Vespa mandarinia (Vespidae: Hymenoptera) is one of the two largest true hornets known to science. The species is a noted predator of social Hymenoptera and a significant pest of managed honey bees in its native range, but is also known to feed on a wide variety of other species when available. Most of the prey records for V. mandarinia are derived from visual observations in Japan, with sparse observations from other parts of its native range. A population of V. mandarinia was detected in North America in 2019 and five nests were removed between 2019 and 2021. We extracted DNA from larval meconia from four nests collected in Washington State, USA, and amplified the CO1 region to determine the potential prey base. We compared these with sequences generated from three nests in the Republic of Korea, and with prey pellets collected from foraging hornets at several locations in Korea. Results indicate that the prey base was much wider in the ROK than the USA, although social Hymenoptera were the most abundant and common prey items in both regions. Prey range seems to be bound by an intersection of organism size and local biodiversity, with little evidence to suggest that the latter is a limiting factor in colony success.

River estuaries are dynamic and productive ecosystems with high regional biodiversity. Environmental DNA (eDNA) has become a useful approach to assessing biodiversity in aquatic ecosystems. This study was conducted to investigate fish community characteristics and species diversity in two river estuary ecosystems, the Taehwa River and Changwon Stream. We further compared conventional and eDNA metabarcoding analyses of the fish communities. The conventional survey was performed in May, July, and October 2022, while the eDNA analysis was conducted only in May. We observed various fish species with different life histories, including carp, goby, and marine fish. We also found that migratory fish, such as dace Tribolodon hakonensis, sweetfish Plecoglossus altivelis, and eel Auguilla japonica, occurred in the Taehwa River, suggesting high river connectivity. Marine fish species were predominant in the Changwon Stream, as this river is located close to the sea. The diversity indices showed that the Taehwa River generally had higher species richness, evenness, and diversity values than the Changwon Stream. A total of 9-19 species were detected in the conventional survey for the three sites, whereas 11-18 species were found from eDNA analysis. The findings indicate that the sensitivity of eDNA was similar to or higher than that of the conventional method. Our study findings suggest the efficiency and efficacy of eDNA-based fish community monitoring, although with some shortcomings in applying the genetic marker to Korean fish, including no clear-cut distinction for Korean endemic species and/or genetically closely related species groups.

Research on food sources through DNA metabarcoding is being used for various organisms based on high resolution and reproducibility. In the study, we investigated the difference in food sources between pre and post-starving in the three bivalve species (Anemina acaeformis, Anodonta woodiana, and Unio douglasiae) through DNA metabarcoding using 18S rRNA V9 primer. The food source of pre-starving appeared in 87 genera, 71 families, 51 orders, 35 classes, and 22 phyla. The primary food sources were the zoo and phytoplankton, including Chlamydomonadales, Euglenales, Ploima, Sphaeropleales, and Stephanodiscales. However, all zoo and phytoplankton were not observed after starving except Schizopyrenida and Rotifera. In Levin’s niche breadth analysis, the Bi index of A. woodiana is 0.3, which was higher than A. acaeformis (0.14) and U. douglasiae (0.21), indicating that they feed on various food sources. The niche overlap of A. acaeformis was measured as 0.78 in A. woodiana, 0.7 in U. douglasiae showing a relative high value compared to other bivalves. The trophic level of A. acaeformis, A. woodiana, and U. douglasiae based on the food source information were investigated as 2.0, 2.0, and 2.5, respectively. The results of the previous study on the trophic level using stable isotopes showed 1.8 to 2.4 values were similar to the results of this study. These results suggest that DNA metabarcoding can be an effective analyzing tool for the gut content in the bivalves.

Gut contents analysis is essential to predict the impact of organisms on food source changes due to variations of the habitat environment. Previous studies of gut content analysis have been conducted using traditional methods, such as visual observation. However, these studies are limited in analyzing food sources because of the digestive process in gut organ. DNA metabarcoding analysis is a useful method to analyze food sources by supplementing these limitations. We sampled marine fish of Pennahia argentata, Larimichthys polyactis, Crangon affinis, Loligo beka and Sepia officinalis from Gwangyang Bay and Yeosu fisheries market for analyzing gut contents by applying DNA metabarcoding analysis. 18S rRNA v9 primer was used for analyzing food source by DNA metabarcoding. Network and two-way clustering analyses characterized the relationship between organisms and food sources. As a result of comparing metabarcoding of gut contents for P. argentata between sampled from Gwangyang Bay and the fisheries market, fish and Copepoda were analyzed as common food sources. In addition, Decapoda and Copepoda were analyzed as common food sources for L. polyactis and C. affinis, respectively. Copepoda was analyzed as the primary food source for L. beka and S. officinalis. These study results demonstrated that gut contents analysis using DNA metabarcoding reflects diverse and detailed information of biological food sources in the aquatic environment. In addition, it will be possible to provide biological information in the gut to identify key food sources by applying it to the research on the food web in the ecosystem.

메타바코딩을 이용한 환경 DNA 분석은 검출 감도가 높아 어류의 생물다양성 평가 및 멸종위기종의 검출에 유용 한 기술이다. 이번 연구는 메타바코딩을 이용해 우리나라 담수어류를 대상으로 높은 검출 효율을 보일 수 있는 적합 한 분석방법을 확인하기 위해 4가지 분석조건별, 즉 필터 (cellulose nitrate filter, glass fiber filter), 추출 키트 (DNeasy® Blood & Tissue Kit, DNeasy® PowerWater Kit), 프라이머 조합 (12S rDNA, 16S rDNA) 그리고 PCR 방법 (conventional PCR, touchdown PCR)로 나타나는 Operational Taxonomic Units (OTUs) 수와 종 조성을 비교하였다. Glass fiber filter와 DNeasy® Tissue & Blood Kit를 이용해 추출한 시료는 12S rDNA와 16S rDNA 프라이머 조합에서 담수어류 OTUs가 가장 많이 검출되었다. 모든 분석조건 중 프라이머 조합에서만 조기어강 (Class Actinopterygii) 평균 OTUs 수에서 통계적으로 유의한 차이를 보였고 (Non-parametric Wilcoxon Signed Ranks Test, p=0.005), 담수어류 평균 OTUs 수는 유의하지 않았다. 종 조성 비교 결과 역시 프라이머 조합에서 유의한 차이를 보였고 (PERMANOVA, Pseudo-F=6.9489, p=0.006), 나머지 분석조건에서는 유의한 차이를 보이지 않았다. NMDS 분석 결과 종 조성은 유사도 65% 기준에서 프라이머 조합에 따라 묶였고, 16S rDNA 프라이머 세트는 주로 멸종위기종인 모래주사 (Microphysogobio koreensis), 꼬치동자개 (Pseudogobio brevicorpus)가 기여하였고, 12S rDNA 프라이머 세트는 주로 일반종인 피라미 (Zacco platypus), 꺽지 (Coreoperca herzi) 등이 기여한 것으로 나타났다. 본 연구는 국내 하천에서 채취한 시료에 대한 메타바코딩을 이용한 종 다양성 분석의 기초정보를 제공한다.