ABSTRACT The Hong Kong Grouper ( Epinephelus akaara ) is a commercial fish species that suffered at least 50%–80% population declines in the past 40 years throughout its distribution range due to overexploitation. Yet, limited research on distribution and habitat utilization resulted in a lack of species‐specific management strategies. Here we aim to utilize environmental DNA (eDNA) analysis as a potential tool for species detection and to provide both shedding and decay rate for future application in persistence prediction models. We first develop a novel, species‐specific, sensitive, quantitative PCR (qPCR) assay amplifying 71 bp of the mitochondrial ND2 gene. From a mesocosm experiment, we found the decay rate (0.131 ± 0.0111/h) of E. akaara to be similar to other reported marine fish species. However, the shedding rate of E. akaara (1.94 × 10 4 ± 2.07 × 10 3 copies/h/g) is generally lower than reported values of other species, likely due to the relatively low activity and energy use from solitary and sedentary behavior of groupers. This highlights the importance of empirically determining species or taxon‐specific shedding and decay rates to inform accurate abundance estimates with modeling tools for eDNA concentrations. Out of 88 water filters collected across four sampling seasons and 11 sites in Hong Kong, six samples from four different sites showed positive amplification. No samples were measured at a concentration above limit of detection of the assay, and this rarity is consistent with current observational data. Overall, we demonstrate that eDNA with qPCR assay is a promising tool to fill in knowledge gaps of endangered species with insufficient species management and conservation across its distribution.

ABSTRACT Humans rely heavily on marine ecosystems for essential goods and services. However, the diversity and composition of mesophotic ecosystems (ME) across temporal and spatial scales remain poorly understood. Upper (UME; 20–50 m) and lower (LME; 50–150 m) MEs, despite the diversity they host and the threats they face, remain largely unprotected. At two locations in central Chile, we first contrast the diversity and composition of rocky reef invertebrate communities between the UME and the LME using eDNA from ARMS and second compare the invertebrate diversity and composition collected from water eDNA and ARMS eDNA. Alpha diversity was greater in the LME except at one of the sites; the LME contained the highest number of exclusive families with the greatest taxa turnovers detected between LME and UME; and community composition differences were mainly driven by sponges (Callyspongiidae) and crabs (Pilumnoididae, Porcellanidae) in the UME and habitat formers such as sponges (Halisarcidae, Suberitidae) and hydroids (Bougainvilliidae, Pandeidae), feather duster worms (Sabellidae), squat lobsters and sea urchins (Arbaciidae) in the LME. Regarding the eDNA diversity and composition between water and ARMS, little diversity was shared between media. Most of the families characterizing the water eDNA community encompassed pelagic copepods (e.g., Acartiidae, Claussocalanidae), whereas ARMS eDNA revealed primarily benthic taxa, such as fan worms (Sabellidae), polychaetes (Terebellidae) and flatworms (Notocomplanidae). This study contributes to our knowledge of the spatial distribution of UME and LME taxa in the central Humbolt current system, justifying the richness and uniqueness of these unprotected ecosystems. Furthermore, this study provides a comparison between water and ARMS eDNA in ME, highlighting the need to sample multiple media to obtain more complete diversity estimates in marine systems.

ABSTRACT Environmental DNA (eDNA) metabarcoding is a powerful tool for monitoring aquatic biodiversity in extreme environments like the Qinghai–Tibet Plateau (QTP), where traditional methods face significant challenges. However, key knowledge gaps remain, particularly in selecting optimal sampling substrates for target taxonomic groups and understanding substrate‐specific detection differences. Here, using eDNA metabarcoding with five primer sets (targeting mitochondrial, nuclear, and nucleoid sequences), we systematically compared biodiversity detection between sediment and water samples collected from five sites in the Dongjicuona Lake across the tree of life (encompassing archaea, bacteria, algae, fungi, plants, protozoa, invertebrates, fishes, birds, and mammals). The results showed that sediment eDNA exhibited significantly higher amplicon sequence variant (ASV) richness compared to water eDNA for most taxonomic groups. In contrast, the opposite pattern was observed for fishes, while no significant differences were observed for fungi and birds. Community composition differs significantly between sediment and water samples for most taxonomic groups, except for mammals. Across all taxonomic groups, 18.0% of ASVs were shared between sediments and water, with fishes showing the highest overlap (42.6%) and archaea the lowest (4.5%). Additionally, the percentages of ASVs contributing to major community dissimilarity (cumulatively accounting for 80%) ranged from 6.4% (invertebrates) to 17.4% (protozoa), with most taxa exhibiting distinct habitat preferences. These findings support water sampling as an effective method for monitoring fish diversity, while emphasizing the need for a multi‐substrate approach to comprehensively monitor other taxonomic groups in plateau lakes.

ABSTRACT In recent years, European countries have intensified efforts to control or limit the spread of invasive freshwater crayfish and the crayfish plague pathogen Aphanomyces astaci , while working to conserve native species such as the noble crayfish ( Astacus astacus ). Although crayfish shed relatively low amounts of DNA into their environment, environmental DNA (eDNA) approaches have proven effective for detecting their presence. A range of protocols and equipment is currently used in eDNA‐based monitoring of freshwater crayfish. To evaluate how methodological variation influences detection accuracy, we conducted the first European interlaboratory ring test using eDNA to detect A. astacus , the invasive signal crayfish Pacifastacus leniusculus , a chronic carrier of A. astaci , and the pathogen itself. The aim is to harmonize monitoring methods for crayfish and disease surveillance across laboratories. Eleven teams from thirteen European countries participated, each using its own equipment and protocols to collect and filter water from indoor tanks and outdoor ponds where the presence of A. astacus and P. leniusculus had been experimentally manipulated, as well as from a natural lake containing a P. leniusculus population. The resulting samples were analyzed in each team's laboratory. Despite methodological differences, all teams successfully detected DNA from both crayfish species in indoor tanks (3–10 crayfish/m 3 ). However, detection accuracy declined in outdoor ponds where crayfish density was an order of magnitude lower (0.32 crayfish/m 3 ). Detection was most variable for A. astaci , likely due to its very low prevalence in the host stock. Our study demonstrates the challenges of achieving consistent eDNA results across laboratories and highlights the importance of interlaboratory comparisons. It also underscores the need to identify sources of variability and error, an essential step toward developing robust and standardized protocols. This multinational intercalibration and exchange of knowledge improved methodology and enhanced reliability in crayfish detection.

ABSTRACT Mangrove ecosystems support a diverse array of animal species and also provide pivotal ecosystem services, such as coastal protection, food provisioning, and carbon capture. However, these vital habitats are in decline, leading to coastal degradation in many parts of the globe. To address this, a mangrove restoration project in Demak, Java, Indonesia, introduced the use of semi‐permeable coastal protective barriers made of bamboo pilings to safeguard the shore zone and hinterlands. The introduction of such hard substrate in the marine environment can attract a range of species, and it is important to be able to monitor changes in biodiversity from a restoration point of view. Here, we assessed whether environmental DNA metabarcoding can be applied to monitor fish biodiversity in an understudied area. Our results show slight but significant differences in species richness and fish community composition within a short timeframe of only 4 months, although we cannot disentangle the effects of seasonal variation from those of the introduction of hard substrate. More importantly, this study demonstrates a useful level of temporal resolution of eDNA metabarcoding and establishes a baseline for fish species richness in an understudied mangrove coastal zone in Demak, Java, Indonesia. Our results are of value for informing future restoration efforts and other (metabarcoding) biodiversity studies in the region.

ABSTRACTEffective conservation of threatened species relies on precise detection methods to inform conservation management strategies. Here, we developed a new environmental DNA (eDNA) assay to detect the endangered Booroolong frog (Litoria booroolongensis) in river water samples throughout their range in Australia. The suitability of the assay to detect Booroolong frog eDNA was first validated ex situ from a population of captive‐bred frogs at Taronga Zoo, New South Wales. We then established the detectability of Booroolong frog DNA from river water samples collected in situ. The new assay successfully detected Booroolong frog eDNA in water samples collected from the breeding enclosure, from water samples exposed to adult frogs and frog egg masses. Testing in the field confirmed the effectiveness of the assay at detecting Booroolong frog eDNA in environmental water samples collected in situ from sites with known presence of the species determined from prior nocturnal surveys. Our results demonstrate the efficacy of eDNA for detecting Booroolong frogs from river water samples across its geographic range. Ongoing monitoring utilizing eDNA has the potential to support conservation efforts by enabling more efficient and widespread surveys for this endangered species.

ABSTRACT Tetracapsuloides bryosalmonae, the causative agent of Proliferative Kidney Disease (PKD), affects salmonid populations in the northern hemisphere. In Switzerland, PKD monitoring to date has been conducted irregularly using invasive sampling. However, non‐invasive detection protocols based on environmental DNA (eDNA) have been established, and there is still a lack of knowledge about environmental parameters that may influence detection. In this study, we evaluated which sampling period would be best for eDNA‐based monitoring of T. bryosalmonae and which environmental parameters might influence the outcome by regularly sampling water from six Swiss rivers over an entire year and testing for presence of T. bryosalmonae DNA by droplet digital PCR (ddPCR). The T. bryosalmonae infection status of bryozoans from all investigated rivers was also assessed. Water temperature, precipitation, and air temperature were examined as environmental parameters. We found T. bryosalmonae DNA in all rivers by ddPCR. Positive results were almost exclusively retrieved between late spring and early autumn (mid‐April until end of October). 98.3% of T. bryosalmonae positive water samples were collected when average daily water temperatures were above 8°C, and 71.9% of the positive samples when average daily water temperatures were above 14°C. Occupancy modeling corroborated the influence of water and air temperature on detecting T. bryosalmonae eDNA. Precipitation caused early clogging of filters in some cases and reduced detection by reducing sample volumes, suggesting when sampling for eDNA should be avoided. Finally, recommendations for a future eDNA‐based monitoring of T. bryosalmonae are provided.

ABSTRACT The East Asian–Australasian Flyway (EAAF) is experiencing notable population declines in its migratory waterbird species. Understanding the foraging ecology of these waterbirds, including ducks, is crucial for monitoring and safeguarding their food sources and wetland habitats. Here, we used a DNA metabarcoding approach to analyze fecal DNA from duck species to elucidate their dietary composition during the wintering period in a subtropical East Asian wetland. By employing multiple markers ( 18S , COI , and trnL ) targeting different taxonomic groups and levels, we offered a comprehensive dietary analysis for omnivores that consume both plants and animals. We revealed the dietary compositions of common migratory duck species and their intraspecific and interspecific dietary variations. While ducks are generally known to be omnivorous, Anas crecca (green‐winged teal) had a more specialized diet and was primarily herbivorous throughout winter. In contrast, the sympatric Mareca penelope (Eurasian wigeon) and Spatula clypeata (northern shoveler) exhibited more omnivorous foraging behaviors. Moreover, A. crecca displayed less dietary variation among samples, while samples of M. penelope and S. clypeata were highly variable in their compositions. Comparing our results with those of studies conducted in different regions, we found that the dietary compositions of these duck species varied to different degrees across geographic locations. This variation underscores the flexibility of these duck species in their diets and their adaptable foraging strategies. Our findings also indicate that grasslands rich in herbaceous plants and aquatic environments abundant with small aquatic invertebrates are vital foraging habitats for duck species during their winter period.

ABSTRACTEnvironmental DNA (eDNA) metabarcoding is a powerful tool for uncovering hidden biodiversity and identifying underestimated taxa in aquatic ecosystems. In this study, we applied eDNA metabarcoding to assess fish species in isolated environments of the Tibagi River basin, specifically sinkholes. Water samples were collected from three sinkholes (Sites 1, 2, and 4), one site at Dourada Lake within the Vila Velha State Park (VVSP), and two sites along the main channel of the Tibagi River. A fragment of the mitochondrial 12S rDNA gene was amplified and sequenced, yielding a final dataset of 975,197 reads after filtering. We identified 31 Molecular Operational Taxonomic Units (MOTUs) belonging to Actinopterygii, distributed across four taxonomic orders: Characiformes, Cichliformes, Gymnotiformes, and Siluriformes. No MOTUs were detected in sinkhole 1, indicating local extinction. A single MOTU (Psalidodon fasciatus) was identified in sinkhole 2. In sinkhole 4, two distinct MOTUs were found, corresponding to the family Heptapteridae and the genus Cambeva, but the potential presence of P. fasciatus was not confirmed. As expected, the highest species richness was observed in the Tibagi River. Despite the lack of a local reference database, our findings offer valuable insights into the detection of diverse taxa in isolated aquatic environments using eDNA metabarcoding. This study underscores the potential of eDNA to inform conservation strategies in ecologically unique and underexplored habitats and highlights the need for developing region‐specific genetic reference databases to enhance taxonomic resolution and monitoring efforts in the future.