A standardized workflow combining coarse pre-filtration, taxa-specific primers (e.g., MiFish for fish), and manual validation against expanded local databases to strengthen eDNA-based biodiversity assessments in Indonesia is recommended.
Abstract
ARTICLE HIGLIGHTS- Environmental DNA studies in Indonesian freshwaters remain uneven.- Multi-step filtering improves species detection and reduces false findings in tropical condition.- Taxa-specific primers detect native species better than broad-range primers.- Local databases and manual plausibility checks reduce wrong species matches.ABSTRACTIndonesian freshwater ecosystems harbor immense biodiversity yet remain understudied due to logistical constraints of conventional methods. Environmental DNA (eDNA) metabarcoding offers a noninvasive, high-resolution alternative for biodiversity monitoring, but its uptake in Indonesia is still nascent and methodologically heterogeneous. We reviewed 106 peer-reviewed studies (of which 28 studies were eligible) published between 2015 and 2025 across Google Scholar, PubMed, DOAJ, and GARUDA, charting sampling designs, molecular workflows, and bioinformatics pipelines. Studies were heavily skewed toward Java and West Sumatra (86%) and overwhelmingly employed filtration-based sampling. Broad-range COI and 12S markers dominated (39% and 36% of studies, respectively), whereas fish-specific MiFish-U primers, demonstrating superior sensitivity and specificity, were only used in 14% of cases. Correspondingly, non-specific primer in shotgun metagenomics proven imprudent. Studies using 0.22 µm filters collected a lot of species as much as non-target amplification, while 0.45 µm filters performed inconsistently. Bioinformatics approaches (QIIME2, DADA2, mBRAVE) differed widely but showed no clear impact on detection outcomes. Key limitations included geographic and taxonomic biases, poor reference library coverage, and a lack of expert-driven plausibility checks. We recommend a standardized workflow combining coarse pre-filtration, taxa-specific primers (e.g., MiFish for fish), and manual validation against expanded local databases to strengthen eDNA-based biodiversity assessments in Indonesia.
Environmental DNA (eDNA) metabarcoding has emerged as a powerful molecular tool for biodiversity monitoring, particularly in remote and logistically challenging environments such as Antarctic marine ecosystems, where traditional surveys are often limited. Despite the ecological significance of the Ross Sea, comprehensive molecular datasets for this region remain scarce. Here, we present a two-year eDNA metabarcoding survey in the Ross Sea region Marine Protected Area (RSR MPA) during 2024 and 2025. Seawater samples were collected from 20 stations (n = 61) in 2024 and 12 stations (n = 40) in 2025. Eukaryotic communities were characterized across multiple taxonomic levels and assigned to functional groups (autotrophs, heterotrophs, mixotrophs, and other protist-related groups) to assess the associated ecological roles. Annual comparison revealed that 92 species were consistently detected across survey years, indicating ecological stability within core community components. Species-level taxonomic assignments revealed that foundational components, including dinoflagellate and Antarctic diatom species, remained present despite environmental variability. Environmental variables, particularly thermal stratification and water mass properties, emerged as the primary drivers structuring eukaryotic communities in the Ross Sea, generating distinct vertical and horizontal stratification patterns and effectively explaining spatial separations among sample groups. The dataset generated om this study contributes to expanding molecular reference resources for polar marine ecosystems and provides a foundation for future integrative analyses that combine eDNA monitoring with environmental and oceanographic datasets, with broad applicability to the conservation and management of the RSR MPA.
Soyun Choi, E. Choi, Inseo Kim et al.· Marine Environmental Researc...· 0 citations
Groundwater is among the planet's most essential natural resources and a crucial part of the global freshwater cycle. It also harbours a specialized aquatic fauna, the stygofauna, which plays a pivotal role in subterranean ecosystem functioning. DNA metabarcoding provides a powerful tool to better uncover the previously largely hidden aquifer communities. It does so by circumventing taxonomic impediments and uncovering cryptic species, simultaneously enabling the assessment of the impacts of pollution and climate change. However, the application of (e)DNA metabarcoding for groundwater metazoans is hindered by two major obstacles: the scarcity of validated primer pairs and the limited taxonomic coverage of reference databases. In this study, we sought to strengthen the molecular toolbox for groundwater biodiversity research by tackling these two challenges. We demonstrate that the primer pair fwhF2 / fwhR2n, targeting the mitochondrial COI region, provides the most effective amplification of central European groundwater invertebrates, particularly crustaceans. In addition, we contributed targeted updates to public databases (for this study assembled in the Groundwater invertebrate Database; GwinD), particularly for 30 Copepoda, five Bathynellacea, and five Ostracoda species. The toolbox was applied in a case study, demonstrating its utility for (e)DNA metabarcoding of central European groundwater communities while highlighting persisting limitations for several non-crustacean groups.
Tobias Hoeschel, Hannah Rau, Melanie Sinn et al.· Ecotoxicology and Environmen...· 0 citations
ABSTRACT The sustainability of Southeast Asian fisheries hinges on high‐throughput tools for monitoring early life‐stage fish biodiversity. However, applying DNA metabarcoding to hyper‐diverse tropical ichthyoplankton requires rigorous calibration to ensure quantitative reliability. We systematically evaluated the metabarcoding workflow using controlled mock communities, revealing that taxonomic recovery is governed by a stochastic limit of detection at a normalised proxy biomass threshold of ≤ 0.05. Quantitative analysis confirmed a significant linear relationship between specimen size and read abundance (R 2 up to 0.817), demonstrating that biomass‐driven template competition induces frequent false negatives for low‐biomass taxa, a phenomenon exacerbated by increasing community complexity (ANOVA: p < 0.001). To mitigate these systemic biases, we applied a size‐stratified specimen‐balancing strategy intended to increase the representation of small‐bodied components in natural bulk samples. Applying this optimised workflow to field samples from Khanh Hoa, Vietnam, we identified 139 species and unmasked a North–South biogeographic dichotomy (PERMANOVA: R 2 = 53%, p = 0.001) driven by transect‐scale environmental gradients and local hydrography. Notably, we identified diversity hotspots requiring > 200,000 reads for saturation, suggesting these sites act as critical larval retention zones. The contrast between functional management zones was highly significant (p = 0.002), with the conservation area (Zone B) exhibiting higher alpha richness and a nine‐fold increase in unique indicator species compared to high‐activity areas (18 vs. 2). Our work demonstrates that comprehensive validation is vital for accurate metabarcoding, offering a robust framework to understand how ecological gradients and localised human pressures shape Vietnam's critical marine spawning sites and nursery grounds.
Cam Hong Van, L. Nguyen, O. Truong et al.· Molecular Ecology Resources· 0 citations
The rapid expansion of environmental DNA (eDNA)-based biodiversity monitoring has heightened the need for accurate and well-curated taxonomic reference databases. Although mitochondrial cytochrome oxidase I (COI) is widely used, the mitochondrial 12S ribosomal RNA gene (12S rRNA) has increasingly demonstrated strong taxonomic assignment success in chondrichthyan metabarcoding applications. The South African coastline supports one of the most diverse chondrichthyan faunas globally. However, as in many regions, coverage of 12S rRNA reference databases has remained limited. Well-represented reference libraries are critical for improving the accuracy of eDNA-based species detection. Here, we aimed to expand the regional 12S rRNA reference database for South African chondrichthyans, thereby supporting both local and global eDNA applications for widely distributed species. A total of 36 chondrichthyan species, represented by 69 individuals, were sequenced to expand the 12S rRNA reference library for sharks, rays and chimaeras from South Africa’s Indian and Atlantic Ocean coastlines. A newly developed primer set (V05F_898E / teleoR_Elas), designed to overlap with the commonly used Elas02 region, achieved consistent amplification across diverse lineages. The new data increased regional reference coverage from 50.3% to 69.1% and added 18 species globally. Sequence authenticity was confirmed through comparison with GenBank, and Bayesian phylogenetic analyses showed that 97.2% of sequences clustered within their expected family-level lineages, although some uncertainties remained in groups with low mitochondrial divergence or complex taxonomic histories. Percent identity analyses revealed high intraspecific similarity and clear interspecific separation across taxa. However, comparative in silico analyses of the shorter Elas02 fragment demonstrated reduced phylogenetic resolution and less consistent clustering among closely related taxa. While the Elas02 region is suitable for accurate amplicon sequence variant (ASV) matching in eDNA metabarcoding, the longer 12S rRNA fragment generated here provides improved phylogenetic resolution and may aid in the identification of unknown or ambiguous ASVs. The expanded 12S rRNA reference library and associated phylogenetic analyses expanded genetic resources for South African chondrichthyans and support global applications. The newly developed marker can be incorporated in future eDNA studies dealing with elasmobranchs, since it can have the added benefit of providing more phylogenetic resolution than the short Elas02 fragment.
Kristina Loosen, Sara Andreotti, Conrad Matthee· BMC Zoology· 0 citations
Freshwater mussels (family Unionidae) are among the most endangered aquatic species worldwide, making effective monitoring vital for their conservation. Traditional surveys are laborious, time-consuming, and often miss rare or low-density populations. Environmental DNA (eDNA) is a highly sensitive, non-invasive approach for freshwater mussel detection and monitoring; however, uncertainties in ecological interpretation continue to limit its broader application in freshwater biodiversity monitoring. We conducted a systematic review of 49 peer-reviewed studies, identified through an extensive literature search, to assess temporal research trends, methodological advancements, ecological contexts, and regulatory readiness of freshwater mussel eDNA monitoring. Findings show that freshwater mussel eDNA research has surged since 2020, particularly in the United States, which accounts for the largest share of published studies (38.8%). Quantitative polymerase chain reaction (qPCR) was the most frequently applied detection method (46%), followed by metabarcoding (32%), indicating a shift from species-specific detection toward community-level biodiversity assessment. Water was the principal sampling matrix, while freezing and silica-based extraction kits were the most commonly reported preservation and DNA-extraction approaches. The review demonstrates that eDNA greatly enhances the detection of freshwater mussels, particularly rare and endangered species, and supports large-scale biodiversity assessments and conservation planning. However, significant variation exists in sampling strategies, laboratory workflows, molecular markers, bioinformatics pipelines, and quality control practices. Ecological interpretation remains complicated by factors such as hydrological transport, seasonal biological variation, eDNA degradation, PCR inhibition, incomplete reference databases, and unclear relationships between eDNA levels and population size. Overall, this review highlights that eDNA has complemented traditional surveys rather than replacing them. To facilitate regulatory adoption and conservation, ongoing efforts should focus on standardizing protocols, expanding reference databases, improving quantitative analyses, across laboratories, and integrating ecological and hydrological data.
Mizanur Rahman, Md Saydur Rahman, Richard J. Kline· Frontiers in Conservation Sc...· 0 citations
Marker choice is a critical methodological factor in DNA metabarcoding, as it directly influences taxonomic composition and abundance structure of communities. In diatoms, metabarcoding is increasingly evaluated as an alternative to microscopy-based assessments, particularly for ecological status evaluation under the Water Framework Directive (WFD).
Here, we compared two widely used
rbc
L markers (263 bp and 331 bp long) using identical benthic diatom samples to evaluate their suitability for ecological assessment.
Both markers produced highly consistent community patterns and IPS-based ecological classifications. Differences between datasets were mainly restricted to low-abundance taxa, while dominant taxa—driving most of the IPS signal—were consistently detected. Decomposition of IPS differences showed that variation was primarily driven by changes in the relative abundance of shared taxa and inconsistencies in species-level assignment, whereas marker-specific taxa had negligible influence.
Overall, the 331-bp marker provided higher taxonomic resolution for abundant taxa, and its lower detection of rare taxa did not compromise ecological assessment. It is therefore well suited for routine biomonitoring under the WFD. Although marker-specific calibration may further improve quantitative accuracy, our results indicate that the 331-bp marker can be reliably applied without major methodological adjustments.
P. Urbánková, Jan Štěpka· Metabarcoding and Metagenomi...· 0 citations