Environmental DNA (eDNA) metabarcoding and fish visual census reveals the first record of Doboatherina magnidentata in the Philippines

Simple SummarySeafood, especially the traditional one in Taiwan, is rarely sourced from a fixed species and routinely from similar species depending on their availability. Hence, species diversity in seafood could be potentially complicated. While a DNA-based approach has been extensively utilized for species identification, a large scale of seafood species identification in fish markets and nearby seafood restaurants could be challenging (e.g., elevated cost and time-consuming only for a limited number of species identification). Environmental DNA (eDNA) metabarcoding has emerged as a promising tool for the simultaneous identification of multiple species in the environments. In this work, we aimed to identify the majority of fish species potentially consumed in fish markets and nearby seafood restaurants using this novel approach. A total of 153 fish species have been identified. Specifically, 22 chondrichthyan fish, 14 Anguilliformes species, and 15 Serranidae species were potentially linked with smoked sharks, braised moray eels, and grouper fish soups, respectively. This is the first study to examine the feasibility of a large scale of seafood identification using eDNA metabarcoding. Our findings also further imply the species diversity in traditional seafood might be seriously underestimated and crucial for the conservation and management of marine resources.Seafood, especially the traditional one in Taiwan, is rarely sourced from a fixed species and routinely from similar species depending on their availability. Hence, the species composition of seafood can be complicated. While a DNA-based approach has been routinely utilized for species identification, a large scale of seafood identification in fish markets and restaurants could be challenging (e.g., elevated cost and time-consuming only for a limited number of species identification). In the present study, we aimed to identify the majority of fish species potentially consumed in fish markets and nearby seafood restaurants using environmental DNA (eDNA) metabarcoding. Four eDNA samplings from a local fish market and nearby seafood restaurants were conducted using Sterivex cartridges. Nineteen universal primers previously validated for fish species identification were utilized to amplify the fragments of mitochondrial DNA (12S, COI, ND5) of species in eDNA samples and sequenced with NovaSeq 6000 sequencing. A total of 153 fish species have been identified based on 417 fish related operational taxonomic units (OTUs) generated from 50,534,995 reads. Principal Coordinate Analysis (PCoA) further showed the differences in fish species between the sampling times and sampling sites. Of these fish species, 22 chondrichthyan fish, 14 Anguilliformes species, and 15 Serranidae species were respectively associated with smoked sharks, braised moray eels, and grouper fish soups. To our best knowledge, this work represents the first study to demonstrate the feasibility of a large scale of seafood identification using eDNA metabarcoding approach. Our findings also imply the species diversity in traditional seafood might be seriously underestimated and crucial for the conservation and management of marine resources.

Surveys of fish diversity in complex heterogeneous environments are highly challenging to perform using traditional survey methods. Although environmental DNA (eDNA) metabarcoding has been effectively used to evaluate fish diversity, studies exploring the spatial and temporal variability of fish communities in mosaic habitats and their connection to water quality after ecological project implementation are still scarce. Here, we evaluated the changes in water quality and fish assembles using the traditional method and environmental DNA (eDNA) metabarcoding after Ecological water replenishment (EWR) and the links between fish communities and water quality were established in the Baiyangdian (BYD) ecosystem in the North China Plain. All water quality variables including TN, NH3-N, CODMn and TP showed a conspicuous improvement pattern, and the number of fish species increased notably after EWR. In addition, 6 more introduced fish species were recorded when compared with the historical data before the implementation of the EWR project. Furthermore, the species richness showed a highly significant difference among the four habitats in the summer 2020 and spring 2021 (spring: P = 0.000; summer: P = 0.002), and obvious discrimination of fish communities across two seasons was observed (P = 0.001) with eDNA metabarcoding. The water quality variables driving the changes of fish communities during the same period varied significantly across different habitats, while not all showed noticeable discrepancy in driving cross-seasonal fish community changes. Thus, our study highlights that the continuous EWR improves the water quality and fish richness but potential ecological issues associated with introduced species should be carefully considered after EWR. Our results also confirm that eDNA is a reliable tool for assessing fish diversity and distinguishing spatiotemporal variability of fish communities in mosaic habitat ecosystems.

Abstract. Roesma DI, Tjong DH, Syaifullah, Nofrita, Janra MN, Prawira FDL,Salis VM, Aidil DR. 2023.The importance of DNA barcode reference libraries and selection primer pair in monitoring fish diversity using environmental DNA metabarcoding. Biodiversitas 24: 2251-2260. Environmental DNA (eDNA) metabarcoding has become an alternative method used for biodiversity monitoring of an ecosystem. The eDNA metabarcoding has advantages compared to the conventional method because it is non-invasive, quick, and requires less cost. However, the effectiveness of the eDNA method is highly dependent on the coverage of the DNA barcode reference and primerpair. A study using the eDNA method was conducted for fish biodiversity monitoring in Singkarak Lake. Two-liter water samples were collected using sterile bottle samples at each sampling site (five sites). The universal primers (Fish FI and Fish R1) used for Next-generation sequencing (GRIDION, Nanopore, Oxford Technologies). The study detected 152 fish species using eDNA metabarcoding. Ten species out of the 30 originally reported in Singkarak Lake were detected using eDNA metabarcoding. The low percentage of fish detected is thought to be due to several factors; incomplete/unavailability of freshwater fish DNA barcodes in Indonesia registered in the database repository, inappropriate primer pair selection, low DNA quality, and the absence of target species DNA in collected water samples. The results demonstrated the significance of correctly registering DNA barcodes to the database and appropriate primer pair selection to identify eDNA metabarcoding. This study provides recommendations using eDNA metabarcoding for monitoring in future work.

Environmental DNA (eDNA) metabarcoding has emerged as a potentially powerful tool to monitor invasive fish species. As an alternative (or complementary) tool for biodiversity monitoring, eDNA metabarcoding had been used to detect species in aquariums, which represents an important transit avenue for introducing non-indigenous species with high population densities. In this study, eDNA metabarcoding as well as morphological characterization were used to reveal the diversity of non-indigenous species in a large aquarium at Qingdao Underwater World. Environmental DNA metabarcoding of 14 water samples at five locations from the Big Water Tank detected 24 non-indigenous species and four putative non-indigenous operational taxonomic units (OTUs). In contrast, only 20 non-indigenous species were observed by morphological characterization. Some species undetected by morphological characterization, such as Oreochromis niloticus (Linnaeus, 1758), are highly adaptable to various environments and/or have invaded preferred regions where they threaten native aquatic species. eDNA metabarcoding also detected seven local fishes that were not identified by morphological characterization. However, analysis of OTU diversity among stations and sample replications revealed that eDNA varied within and/or between stations. Increasing sampling effort as well as negative controls are required to increase the detection rate of species and to eliminate false-positive OTUs.

Fish in the Yong River basin have been significantly impacted by pollution, habitat modification and overfishing. In order to facilitate the recovery of freshwater biodiversity, a fishing ban has been implemented in the Yong River basin since 2022. However, the high level of human activity along the waterways of the Yong River basin poses a significant challenge to the monitoring of fish biodiversity using established methods. It is essential, therefore, to have effective monitoring tools to assess the effectiveness of the fishing ban policy. In this study, environmental DNA (eDNA) metabarcoding was employed to assess fish species biodiversity across five river sections of the Yong River basin in October 2022. Additionally, we checked the results against fishing gear (drift gillnets and ground cages) surveys and historical records. A total of twenty-two fish species were found from eDNA metabarcoding, fourteen species were found via fishing, and only two species were found from both methods. The most dominant fish species identified by eDNA was Alburnus mossulensis, present in all river sections. However, a decline in species biodiversity was observed in the whole Yong River basin, with a significant difference in community composition across the Upper Yao River (UYAR) and Yong River (YOR). Moreover, eDNA detected fish and potentially invasive species consistent with their geographic distribution. Overall, the results of this study provide a snapshot of the current fish biodiversity in the Yong River basin, offering baseline data for future evaluations of fishing ban policies in this ecosystem.

The use of environmental DNA (eDNA) metabarcoding to analyze fish species diversity across different aquatic ecosystems is well documented. Nonetheless, there is a gap in validating eDNA metabarcoding studies on the diversity and structure of fish communities in coastal ecosystems, particularly in comparing these findings with bottom trawl catch data. In this study, we employed eDNA metabarcoding to explore species composition and relative abundance in fish communities, taxonomic-level diversity variations, and the interplay between community structures and environmental factors in the Yellow Sea and compared these results with those obtained from bottom trawl catches. In addition, we compared the various methods used to estimate the distributions of taxonomic, phylogenetic, and functional diversity factors. We found that eDNA metabarcoding detected a greater number of species (86 vs. 41), genera (73 vs. 37), and families (42 vs. 25) than bottom trawl results at each sampling station. eDNA metabarcoding provided higher Shannon, Simpson, and Chao1 alpha diversity indices than the bottom trawl results. The PCoA results showed that eDNA metabarcoding samples could be more clearly separated at the sampling sites in the Zhuanghe (ZH) and Lianyungang (LYG) areas than bottom trawling samples. The RDA analysis indicated that temperature, along with NO3- and NH4+ concentrations, were pivotal in shaping the geographical patterns of fish communities, as identified through eDNA metabarcoding, echoing findings from bottom trawling studies. Furthermore, our findings suggest that eDNA barcoding surpasses bottom trawling in detecting taxonomic and phylogenetic diversity, as well as in uncovering greater functional diversity at the local level. Conclusively, eDNA metabarcoding emerges as a valuable complement to bottom trawling, offering a multifaceted approach to biodiversity monitoring that not only boosts efficiency but also reduces environmental impact on coastal ecosystems.

To understand the status of fish diversity in the middle reaches of the Yangtze River’s Yuanshui River basin during the ten-year fishing ban, this study employed environmental DNA (eDNA) metabarcoding technology. Fish resources were surveyed and diversity analyzed across 38 sampling points within seven river sections of the Yuanshui River basin in September 2023. The results were compared with data obtained using conventional fishing gear methods from four identical sections (Changde, Yuanling, Chenxi, Hongjiang) to explore the application of eDNA technology for fish resource monitoring in the Yangtze River basin and to investigate the similarities and differences in monitoring outcomes between the two methods. The eDNA metabarcoding approach identified a total of 94 fish species, belonging to 10 orders, 17 families, and 57 genera. Cypriniformes was the most abundant order (65 species, 69.15%), and Cyprinidae dominated (54 species, 57.45%) within families. Among the 94 species, 12 were endemic to the Yangtze River basin, 11were key protected wildlife species in Hunan Province, and 3 were non-native species. The predominant ecological traits of the fish assemblage were characterized by lentic-resident habits (56.38%), omnivorous feeding (63.83%), and demersal habitat utilization (45.74%). Diadromous migrants, herbivorous feeders, and lower water column species were relatively scarce. A comparative study in four identical sections of the Yuanshui River utilized environmental DNA (eDNA) metabarcoding and conventional fishing gear to assess fish diversity. Across all sections, eDNA detected a total of 89 fish species while conventional methods detected 93 species, with 62 species co-detected by both approaches. Site-specific comparisons revealed that within the Changde section, eDNA identified 70 species compared to 55 species captured by conventional gear, resulting in 40 shared species. In the Yuanling section, eDNA detected 71 species against 53 species from conventional sampling, yielding 35 co-detected species. The Chenxi section showed eDNA detection of 44 species and conventional gear detection of 38 species, with 20 species found by both methods. Similarly, in the Hongjiang section, eDNA recorded 49 species and conventional methods 47 species, also sharing 20 species. Both the total number of fish species and the number of shared species detected in the Changde and Yuanling sections were significantly higher than those in the Chenxi and Hongjiang sections. Within the same river section, the Shannon diversity index and Pielou evenness index calculated based on the conventional fishing gear data were consistently greater than those derived from the eDNA analysis. This study demonstrates the high credibility of environmental DNA (eDNA) technology for monitoring fish stocks within the Yangtze River Basin. Combining eDNA methods with conventional fishing gear surveys provides a more comprehensive assessment of fish diversity throughout the basin. These findings offer valuable references for evaluating the effectiveness of fishing bans, protecting fish resources, and informing fisheries management strategies in the Yuanshui River and broader Yangtze Basin.

Human‐induced global warming has triggered a persistent decline in the health of marine ecosystems, particularly coral reefs, which are experiencing increasingly frequent and severe bleaching and mortality events. Refining cost‐effective and precise monitoring tools, such as environmental DNA (eDNA) metabarcoding, is essential to supplement future coral reef monitoring programs, with ongoing efforts focused on improving methods, validating results, and understanding limitations. Although eDNA has been widely used in aquatic ecosystem studies, its application to corals (Anthozoa) remains underexplored. Here, we investigate the use of eDNA metabarcoding with molecular markers targeting the ITS2 region of Anthozoa for monitoring coral communities in a remote and relatively undisturbed atoll system. We integrate three mainstream taxonomic assignment approaches (IDTAXA, BLAST Top Hits, and BLAST LCA), retaining only consensus identifications across methods for downstream analyses. This conservative strategy ensures highly robust and reliable taxonomic resolution, with over 90% of the sequences classified within Anthozoa, encompassing 18 genera and 15 genera of hard corals (Scleractinia). A considerable overlap in coral identification is observed between eDNA and traditional benthic transect surveys, giving support to the ability of eDNA to identify the community composition of Anthozoan taxa. Importantly, cryptic genera, such as Cycloseris , Cyphastrea , Merulina , Oxypora , and Turbinaria were identified by the eDNA approach but not the traditional surveys. Conversely, genera such as Alveopora , Astreopora , Caulastrea , Fungia , Galaxea , Halomitra , Herpolitha , Leptastrea , Platygyra , Plerogyra , and Stylophora were identified by the traditional surveys but not the eDNA approach, likely due to primer bias, taxonomic resolution or incomplete reference databases, supporting the complementary use of both methods. We also observe that the eDNA metabarcoding may capture differences in coral community structure between our lagoonal and seaward reef habitat types and point to potential characteristic taxa. This study underscores the utility of eDNA metabarcoding as a noninvasive, cost‐effective tool for coral biodiversity monitoring and provides insights into how to improve eDNA techniques for use as a coral biodiversity monitoring tool.

An environmental DNA (eDNA) metabarcoding approach has been widely used for biodiversity monitoring of fishes, although it has rarely been applied to tropical and subtropical aquatic ecosystems, where species diversity is remarkably high. This study examined the extent to which species richness can be estimated in a small coral reef lagoon (1,500 × 900 m) near Okinawa Island, southern Japan, where the surrounding waters are likely to harbor more than 1,500 species of fish. During 2015–2017, a total of 16 capture‐based surveys were conducted to create a faunal list of fish species, followed by eDNA metabarcoding based on seawater samples taken from 11 sites in the lagoon on a day in May 2019. We also tested whether eDNA metabarcoding could detect differences between adjacent fish communities inhabiting the offshore reef edge and shore‐side seagrass beds within the lagoon. A total of 217 fish species were confirmed by the capture‐based samplings, while 291 fish species were detected by eDNA metabarcoding, identifying a total of 410 species distributed across 119 families and 193 genera. Of these 410 species, only 96 (24% of the total) were commonly identified by both methods, indicating that capture‐based surveys failed to collect a number of species detected by eDNA metabarcoding. Interestingly, two different approaches to estimate species richness based on eDNA data yielded values close to the 410 species, including one that suggested an additional three or more eDNA surveys from 11 sites (36 samples) would detect 90% of the 410 species. In addition, nonmetric multidimensional scaling for fish assemblages clearly distinguished between the fish communities of the offshore reef edge and those of the shore‐side seagrass beds. This study demonstrates that an eDNA metabarcoding approach is useful for estimating species richness and detection of habitat segregation even in ecosystems with remarkably high species diversity.

Global biodiversity in freshwater and the oceans is declining at high rates. Reliable tools for assessing and monitoring aquatic biodiversity, especially for rare and secretive species, are important for efficient and timely management. Recent advances in DNA sequencing have provided a new tool for species detection from DNA present in the environment. In this study, we tested whether an environmental DNA (eDNA) metabarcoding approach, using water samples, can be used for addressing significant questions in ecology and conservation. Two key aquatic vertebrate groups were targeted: amphibians and bony fish. The reliability of this method was cautiously validated in silico, invitro and insitu. When compared with traditional surveys or historical data, eDNA metabarcoding showed a much better detection probability overall. For amphibians, the detection probability with eDNA metabarcoding was 0.97 (CI=0.90-0.99) vs. 0.58 (CI=0.50-0.63) for traditional surveys. For fish, in 89% of the studied sites, the number of taxa detected using the eDNA metabarcoding approach was higher or identical to the number detected using traditional methods. We argue that the proposed DNA-based approach has the potential to become the next-generation tool for ecological studies and standardized biodiversity monitoring in a wide range of aquatic ecosystems.

Abstract. Andriyono S, Jobaidul Alam Md, Kim HW. 2019. Environmental DNA (eDNA) metabarcoding: Diversity study around the Pondok Dadap fish landing station, Malang, Indonesia. Biodiversitas 20: 3772-3781. Molecular identification of species is now fast growing and currently widely applied method in the diversity estimation of aquatic biota; even though morphological identification is still carried out. The molecular approach is beneficial complementing on regular surveys, e.g. use of nets, traps, fishing rods, and even with poisons. In this study, the eDNA metabarcoding was applied to water samples around the Pondok Dadap fish landing station, Indonesia to determine the diversity of fish around the waters and also to identify marine fish landed in this area. Molecular identification was carried out on fish samples obtained from the fish market improved GenBank database on COI and ITS. While, seawater samples were carried out by using the next-generation sequencing (NGS) platform to obtain the eDNA metabarcoding data for the first time. Molecular identification obtained 34 species (68 sequences of COI and ITS regions) belonging to 28 genera, 18 families, 4 orders, while the eDNA metabarcoding approach identified 53 marine fish species by using the MiFish pipeline representing 38 genera, 27 families, and 7 orders. From the present study, we can able to estimated fish diversity by eDNA metabarcoding, and this finding will be helpful for baseline data preparation for future effective management of resources in this area.

The karst aquifer of the Yucatán Peninsula (YP) in southeastern Mexico is a unique ecosystem in which water-filled sinkholes, locally known as cenotes, connect subterranean waters with the surface. This system is home to around 20 species of freshwater fishes, including several that are endemic and/or threatened. Studies on this unique ichthyofauna have been partially hampered by the technical difficulties associated with sampling these habitats, particularly submerged caves. In this proof-of-concept study, we use environmental DNA (eDNA) metabarcoding to survey the diversity of freshwater fishes associated with the YP karst aquifer by sampling six cenotes from across the Ring of Cenotes region in northwestern Yucatán, a 180-km-diameter semicircular band of abundant karst sinkholes. Through a combination of conventional sampling (direct observation, fishing) and eDNA metabarcoding, we detected eight species of freshwater fishes across the six sampled cenotes. Overall, our eDNA metabarcoding approach was effective at detecting the presence of fishes from cenote water samples, including one of the two endemic cave-dwelling fish species restricted to the subterranean section of the aquifer. Although our study was focused on detecting fishes via eDNA, we also recovered DNA from several other vertebrate groups, particularly bats. These results suggest that the eDNA metabarcoding approach represents a promising and largely noninvasive method to assay aquatic biodiversity in these vulnerable habitats, allowing more effective, frequent, and wide-ranging surveys. Our detection of DNA from aerial and terrestrial vertebrate fauna implies that eDNA from cenotes, besides being a means to survey aquatic fauna, may also offer an effective way to quickly survey non-aquatic biodiversity associated with these persistent water bodies.

In recent years, biodiversity loss has become one of the most serious environmental issues worldwide, especially in aquatic ecosystems. To avoid diversity loss, it is necessary to monitor biological communities, and environmental DNA (eDNA) metabarcoding has been developed as a rapid, noninvasive, and cost-effective method for aquatic biodiversity monitoring. Although this method has been applied to various environments and taxa, a detailed assessment of the efficient sampling methods for monitoring is still required. In this study, we explored eDNA metabarcoding sampling methods for fish at a single site to maximize the number of detected species using realistic effort in a natural, small river. We considered the following three parameters: sample type (water or sediment), sample position at a site (right and left shore and center of the river), and water volume (10–4000 mL). The results suggested that the number of detected species from sedimentary eDNA was equivalent to that from aqueous eDNA, although the species composition was different. The number of detected species could be saturated by collecting a 1000 mL water sample, regardless of sampling position within a survey site. However, sedimentary eDNA showed a spatially heterogeneous species composition between sampling positions within a survey site despite the short distance (5 m) between positions, without apparent differences in physical properties such as velocity and sediment particle distribution. By completing eDNA biodiversity monitoring of fish with 1000 mL water samples across the whole river, we detected more fish species than in previous traditional surveys conducted at the same sites. Thus, the aqueous eDNA metabarcoding method is as efficient as traditional surveys, while sedimentary eDNA metabarcoding could complement the results of aqueous eDNA metabarcoding.

The future of biodiversity monitoring and conservation utilizing environmental DNA

Context The ability to accurately assess biodiversity is a critical first step towards effective conservation and management. However, assessment of biodiversity using conventional monitoring programs is often constrained by high cost and a lack of taxonomic expertise. Environmental DNA (eDNA) metabarcoding may be a useful tool to efficiently catalogue biodiversity in areas that cannot be easily assessed using other methods. Aims Here, we evaluated the potential of eDNA metabarcoding for assessing fish biodiversity and distribution in the Kumbe River, Papua Province, Indonesia. Methods We selected four sampling locations and collected seven eDNA samples from each location. We used eDNA metabarcoding of the Cytochrome-b gene to characterise the fish community. Key results A total of 23 species were detected, three of which comprised 92% of sequence reads detected: Melanotaenia goldiei (32%), Craterocephalus randi (31%), and the invasive tilapia Oreochromis niloticus (29%). Only five species that were previously detected using conventional methods were detected by metabarcoding: M. goldiei, Craterocephalus stercusmuscarum, O. niloticus, Neoarius graeffei, and Arius arius. We detected 18 species (70% native) that have never been recorded from the Kumbe River. Conclusions This work has demonstrated that fish biodiversity is substantially underestimated in the Kumbe River. Environmental DNA metabarcoding is a promising rapid, non-invasive and cost-effective method for assessing fish biodiversity in Papua. Implications The findings support future investment in eDNA metabarcoding to characterise the fish biodiversity in Papua. This will assist in allocating the limited resources for conservation and management to areas most at risk from anthropogenic impacts.