• ALGAE
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• v.34 no.1
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• pp.57-70
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• 2019

DNA metabarcoding is currently used for large-scale taxonomic identification to understand the community composition in various marine ecosystems. However, before being widely used in this emerging field, this experimental and analytic approach still has several technical challenges to overcome, such as polymerase chain reaction (PCR) bias, and lack of well-established metabarcoding markers, a task which is difficult but not impossible to achieve. In this study, we present an adapted PCR-free small-organelles enriched metagenomics (SoEM) method for marine biodiversity assessment. To avoid PCR bias and random artefacts, we extracted target DNA sequences without PCR amplification from marine environmental samples enriched with small organelles including mitochondria and plastids because their genome sequences provide a valuable source of molecular markers for phylogenetic analysis. To experimentally enrich small organelles, we performed subcellular fractionation using modified differential centrifugation for marine environmental DNA samples. To validate our SoEM method, two marine environmental samples from the coastal waters were tested the taxonomic capturing capacity against that of traditional DNA metabarcoding method. Results showed that, regardless of taxonomic levels, at least 3-fold greater numbers of taxa were identified in our SoEM method, compared to those identified by the conventional multi-locus DNA metabarcoding method. The SoEM method is thus effective and accurate for identifying taxonomic diversity and presents a useful alternative approach for evaluating biodiversity in the marine environment.

• Ocean and Polar Research
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• v.43 no.1
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• pp.45-51
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• 2021

Studies on marine zooplankton diversity and ecology are important for understanding marine ecosystem, as well as environmental conservation and fisheries management. DNA metabarcoding is known as a useful tool to reveal and understand diversity among animals, but a comparative evaluation with classical microscopy is still required in order to properly use it for marine zooplankton research. This study compared crustacean mesozooplankton taxa revealed by morphological analysis and metabarcoding of the cytochrome oxidase I (COI). A total of 17 crustacean species were identified by morphological analysis, and 18 species by metabarcoding. Copepods made up the highest proportion of taxa, accounting for more than 50% of the total number of species delineated by both methods. Cladocerans were not found by morphological analysis, whereas amphipods and mysids were not detected by metabarcoding. Unlike morphological analysis, metabarcoding was able to identify decapods down to the species level. There were some discrepancies in copepod species, which could be due to a lack of genetic database, or biases during DNA extraction, amplification, pooling and bioinformatics. Morphological analysis will be useful for ecological studies as it can classify and quantify the life history stages of marine zooplankton that metabarcoding cannot detect. Metabarcoding can be a powerful tool for determining marine zooplankton diversity, if its methods or database are further supplemented.

• Korean Journal of Ecology and Environment
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• v.55 no.4
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• pp.360-367
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• 2022

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.

• Journal of Ecology and Environment
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• v.46 no.2
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• pp.126-135
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• 2022

Background: Pollinators are important ecological elements due to their role in the maintenance of ecosystem health, wild plant reproduction, crop production and food security. The pollinator-plant interaction supports the preservation of plant and animal populations and it also improves the yield in pollination dependent crops. Having knowledge about the plant-pollinator interaction is necessary for development of pesticide risk assessment of pollinators and conservation of endangering species. Results: Traditional methods to discover the relatedness of insects and plants are based on tracing the visiting pollinators by field observations as well as palynology. These methods are time-consuming and needs expert taxonomists to identify different groups of pollinators such as insects or identify flowering plants through palynology. With pace of technology, using molecular methods become popular in identification and classification of organisms. DNA metabarcoding, which is the combination of DNA barcoding and high throughput sequencing, can be applied as an alternative method in identification of mixed origin environmental samples such as pollen loads attached to the body of insects and has been used in DNA-based discovery of plant-pollinator relationship. Conclusions: DNA metabarcoding is practical for plant-pollinator studies, however, lack of reference sequence in online databases, taxonomic resolution, universality of primers are the most crucial limitations. Using multiple molecular markers is preferable due to the limitations of developed universal primers, which improves taxa richness and taxonomic resolution of the studied community.

• Korean Journal of Ecology and Environment
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• v.55 no.4
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• pp.368-375
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• 2022

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.

• Journal of Ecology and Environment
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• v.48 no.1
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• pp.32-48
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• 2024

Background: Longitudinal connectivity in river systems strongly affects biological components related to ecosystem functioning, thereby playing an important role in shaping local biodiversity and ecosystem health. Environmental DNA (eDNA)-based metabarcoding has an advantage of enabling to sensitively diagnose the presence/absence of species, becoming an efficient/effective approach for studying the community structure of ecosystems. However, little attention has been paid to eDNA-based biomonitoring for river systems, particularly for assessing the river longitudinal connectivity. In this study, by using eDNA we analyzed and compared species diversity and composition among artificial barriers to assess the longitudinal connectivity of the fish community along down-, mid- and upstream in the Hotancheon from the Geum River basin. Moreover, we investigated temporal variation in eDNA fish community structure and species diversity according to season. Results: The results of species detected between eDNA and conventional surveys revealed higher sensitivity for eDNA and 61% of species (23/38) detected in both methods. The results showed that eDNA-based fish community structure differs from down-, mid- and upstream, and species diversity decreased from down to upstream regardless of season. We found that there was generally higher species diversity at the study sites in spring (a total number of species across the sites [n] = 29) than in autumn (n = 27). Nonmetric multidimensional scaling and heatmap analyses further suggest that there was a tendency for community clusters to form in the down-, mid- and upstream, and seasonal variation in the community structure also existed for the sites. Dominant species in the Hotancheon was Rhynchocypris oxycephalus (26.07%) regardless of season, and subdominant species was Nipponocypris koreanus (16.50%) in spring and Odontobutis platycephala (15.73%) in autumn. Artificial barriers appeared to negatively affect the connectivity of some fish species of high mobility. Conclusions: This study attempts to establish a biological monitoring system by highlighting the versatility and power of eDNA metabarcoding in monitoring native fish community and further evaluating the longitudinal connectivity of river ecosystems. The results of this study suggest that eDNA can be applied to identify fish community structure and species diversity in river systems, although some shortcomings remain still need to be resolved.

• Journal of the Korean Society of Environmental Restoration Technology
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• v.24 no.6
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• pp.133-144
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• 2021

This study aims to estimate location of land mammals habitat by analyzing spatial data and investigate how to apply environmental DNA monitoring methodology to lotic system in Yangpyeong-gun, Gyeonggi-do. Environmental DNA sampling points are selected through spatial analysis with QGIS open source program by overlaying Kernel density of wild boar(Sus scrofa), elevation, slope and land-cover map, and 81 samples are collected. After 240 mL of water was filtered in each sample, metabarcoding technique using MiMammal universal primer was applied in order to get a whole list of mammal species whose DNA particles contained in filtered water. 8 and 22 samples showed DNA of wild boar and water deer, respectively. DNA of raccoon dog, Eurasian otter, and Siberian weasel are also detected through metabarcoding analysis. This study is valuable that conducted in outdoor lotic system. The study suggests a new wildlife monitoring methodology integrating overlayed geographic data and environmental DNA.

• Korean Journal of Ecology and Environment
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• v.54 no.3
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• pp.221-228
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• 2021

Recently, environmental DNA (eDNA)-based metabarcoding approaches have been proposed to evaluate the status of freshwater ecosystems owing to various advantages, including fast and easy sampling and minimal habitat disruption from sampling. Therefore, as a case study, we applied eDNA metabarcoding techniques to evaluate the effects of an abandoned mine land located near a headwater stream of Nakdonggang River, South Korea, by examining benthic macroinvertebrate diversity and compared the results with those obtained using the traditional Surber-net sampling method. The number of genera was higher in Surber-net sampling (29) than in the eDNA analysis (20). The genus richness tended to decrease from headwater to downstream in eDNA analysis, whereas richness tended to decrease at sites with acid-sulfated sediment areas using Surber-net sampling. Through cluster analysis and non-metric multidimensional scaling, the sampling sites were differentiated into two parts: acid-sulfated and other sites using Surber-net sampling, whereas they were grouped into the two lowest downstream and other sites using eDNA sampling. To evaluate freshwater ecosystems using eDNA analysis in practical applications, it is necessary to constantly upgrade the methodologies and compare the data with field survey methods.

• Korean Journal of Ecology and Environment
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• v.54 no.3
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• pp.199-208
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• 2021

Environmental DNA (eDNA) metabarcoding is effective method with high detection sensitivity for evaluating fish biodiversity and detecting endangered fish from natural water samples. We compared the richness of operational taxonomic units(OTUs) and composition of freshwater fishes according to filters(cellulose nitrate filter vs. glass fiber filter), extraction kits(DNeasy2^® Blood & Tissue Kit vs. DNeasy2^® PowerWater Kit), primer sets (12S rDNA vs. 16S rDNA), and PCR methods (conventional PCR vs. touchdown PCR) to determine the optimal conditions for metabarcoding analysis of Korean freshwater fish. The glass fiber filter and DNeasy2^® Blood & Tissue Kit combination showed the highest number of freshwater fish OTUs in both 12S and 16S rDNA. Among the four types, the primer sets only showed statistically significant difference in the average number of OTUs in class Actinopterygii (non-parametric Wilcoxon signed ranks test, p=0.005). However, there was no difference in the average number of OTUs in freshwater fish. The species composition also showed significant difference according to primer sets (PERMANOVA, Pseudo-F=6.9489, p=0.006), but no differences were observed in the other three types. The non-metric multidimensional scaling (NMDS) results revealed that species composition clustered together according to primer sets based on similarity of 65%; 16S rDNA primer set was mainly attributed to endangered species such as Microphysogobio koreensis and Pseudogobio brevicorpus. In contrast, the 12S rDNA primer set was mainly attributed to common species such as Zacco platypus and Coreoperca herzi. This study provides essential information on species diversity analysis using metabarcoding for environmental water samples obtained from rivers in Korea.

• Journal of Environmental Impact Assessment
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• v.32 no.2
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• pp.94-111
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• 2023

In order to establish an effective management strategy for invasive species early detection and regular monitoring are required to assess their introduction or dispersal. Environmental DNA (eDNA) is actively applied to evaluate the fauna including the presence of invasive species as it has high detection sensitivity and can detect multiple species simultaneously. In Korea, the applicability evaluation of metabarcoding is being conducted mainly on fish, and research on other taxa is insufficient. Therefore, this study identified the feasibility of detecting invasive species in Korea using eDNA metabarcoding. In addition, to confirm the possibility of detection by taxa, the detection of target species was evaluated using four universal primers (MiFish, MiMammal, Mibird, Amp16S) designed for fish, mammals, birds, and amphibians. As a result, target species (Trachemys scripta, 3 sites; Cervus nippon, 3 sites; Micropterus salmoides, 7 sites; Rana catesbeiana, 4 sites) were detected in 17 of the total 55 sites. Even in the selection of dense sampling sites within the study area, there was a difference in the detection result by reflecting the ecological characteristics of the target species. A comparison of community structures (species richness, abundance and diversity) based on the presence of invasive species focused on M.salmoides and T.scripta, showed higher diversity at the point where invasive species were detected. Also, 1 to 4 more species were detected and abundance was also up to 1.7 times higher. The results of invasive species detection through metabarcoding and the comparison of community structures indicate that the accumulation of large amounts of monitoring data through eDNA can be efficiently utilized for multidimensional ecosystem evaluation. In addition, it suggested that eDNA can be used as major data for evaluation and prediction, such as tracking biological changes caused by artificial and natural factors and environmental impact assessment.