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Environmental DNA (eDNA) methods are increasingly applied in the marine environment to identify species and community structure. To establish widely applicable eDNA techniques for elasmobranchs, we used the Critically Endangered largetooth sawfish (Pristis pristis Linnaeus, 1758) as a model species for: (1) assessing eDNA particle size distribution; (2) assessing the efficiency of long‐term preservation of water samples; and (3) comparing the efficiency and detection sensitivity of filtration and precipitation methods. Water samples (1 L) collected from a tank containing one largetooth sawfish specimen were sequentially filtered through five filter membranes of decreasing pore size (20, 10, 5, 1.2, and 0.45 μm). The proportion of sawfish eDNA retained within each size class was determined through quantitative real‐time PCR (qPCR) using a species‐specific TaqMan probe assay. A linear mixed‐effects model (lme) showed that the 1.2 and 20 µm filters captured most of the eDNA particles present in the sampled water. Additionally, whole water samples (0.375 L) were preserved in Longmire's buffer, stored at tropical ambient temperatures (26.3°C ± 3.0 SD) and extracted at five time points: immediately, one, two, and three months after collection, as well as frozen and extracted three months later, to assess the preservation efficiency of Longmire's buffer via qPCR analysis. A linear mixed‐effects model showed that samples maintained maximal eDNA yield for at least three months after collection at ambient storage. Lastly, when comparing the filtration and precipitation methods, filtration using 0.45 µm pore size was more sensitive to capture of largetooth sawfish eDNA than filtration with 20 µm filter or water precipitation. However, water precipitation was more efficient when accounting for volume of water processed. These results provide options for best capture and preservation of elasmobranch eDNA. Widely applicable techniques for the study of highly threatened elasmobranchs (sharks and rays) using eDNA are warranted if we are to mitigate their risk of extinction. We used the Critically Endangered largetooth sawfish as a model species to assess, for the first time of any shark or ray, eDNA particle size distribution, long‐term preservation efficiency, and detection sensitivity of filtration and precipitation methods. The results presented here can aid researchers, agencies, and conservation practitioners on best eDNA capture and preservation methods for the study of sharks and rays.

Environmental DNA (eDNA)‐based methods are increasingly used by government agencies to detect pests and threatened species, and for broader biodiversity monitoring. Given rapid technological advances and a growing number of commercial service providers, there is a need to standardize methods for quality assurance and to maintain confidence in eDNA‐based results. Here, we introduce two documents to provide best‐practice guidelines for Australian and New Zealand eDNA researchers and end‐users (available from https://sednasociety.com/publications): the Environmental DNA protocol development guide for biomonitoring provides minimum standard considerations for eDNA and environmental RNA projects across the complete workflow, from ethical considerations and experimental design to interpreting and communicating results. The Environmental DNA test validation guidelines outline key steps to be used in assay development and validation for species‐specific testing and metabarcoding. Both guidelines were developed as an initiative of the Australian Government Department of Agriculture, Fisheries and Forestry and led by the Southern eDNA Society in a collaborative process including multiple consultation rounds with eDNA experts, end‐users, and stakeholders to adapt the guidelines to Australian and New Zealand needs. The aim of these guidelines is not to be prescriptive, but to set minimum standards to support a consistent and best‐practice approach to eDNA testing. We anticipate that the guidelines will be reviewed and regularly updated as required. Our aspiration is that these best‐practice guidelines will ensure environmental managers are provided with robust scientific evidence to support decision‐making. We introduce two documents to provide best‐practice guidelines for Australian and New Zealand eDNA researchers and end users: the “Environmental DNA protocol development guide for biomonitoring” and the “Environmental DNA test validation guidelines.” Both guidelines were developed in a collaborative process including multiple consultation rounds with eDNA experts, end‐users, and stakeholders to adapt the guidelines to Australian and New Zealand needs. The aim of these guidelines is to set minimum standards to support a consistent and best‐practice approach to eDNA testing.

Environmental conditions experienced during the larval dispersal of marine organisms can determine the size‐at‐settlement of recruits. It is, therefore, not uncommon that larvae undergoing different dispersal histories would exhibit phenotypic variability at recruitment. Here, we investigated morphological differences in recently settled southern rock lobster (Jasus edwardsii) recruits, known as pueruli, along a latitudinal and temporal gradient on the east coast of Tasmania, Australia. We further explored whether natural selection could be driving morphological variation. We used double digest restriction site‐associated DNA sequencing (ddRADseq) to assess differences in the genetic structure of recently settled recruits on the east coast of Tasmania over 3 months of peak settlement during 2012 (August–October). Phenotypic differences in pueruli between sites and months of settlement were observed, with significantly smaller individuals found at the northernmost site. Also, there was a lack of overall genetic divergence; however, significant differences in pairwise FST values between settlement months were observed at the southernmost study site, located at an area of confluence of ocean currents. Specifically, individuals settling into the southernmost earlier in the season were genetically different from those settling later. The lack of overall genetic divergence in the presence of phenotypic variation indicates that larval environmental history during the dispersal of J. edwardsii could be a possible driver of the resulting phenotype of settlers. We found that Jasus edwardsii settlers exhibit phenotypic variability across a geographical and temporal gradient; however, this is not correlated with genetic divergence. Given the protracted pelagic larval duration of the species, we hypothesize that dispersal history drives phenotypic variability in this species.

Environmental DNA (eDNA) monitoring has revolutionized the way biodiversity is surveyed and has been proposed as a reliable method to inform management decisions. Recognition of eDNA techniques as reliable tools to inform management and biosecurity require stringent standards to assess sample quality and reliability of results. Laboratories can have their workflows assessed and certified through accreditation and be involved in proficiency testing schemes provided to test the accuracy and precision of molecular methods. Currently, there is only one eDNA‐based proficiency testing scheme designed to test competency of laboratories in amplifying eDNA from the Great Crested Newt, Triturus cristatus (Laurenti, 1768) in water samples. This test, however, is a closed scheme currently run by invitation only to laboratories in the United Kingdom. Given the paucity of eDNA‐based proficiency schemes to ensure high‐quality services, this commentary discusses how future proficiency testing schemes could be designed to assess eDNA sample quality and reliability on detection results for environmental management and biosecurity applications. We discuss the use of tissue‐derived and synthetic oligonucleotides as reference materials, the need for proficiency testing schemes to assess the capacity of analytical facilities to determine sample quality as well as accurately detecting trace eDNA in blind samples and discuss the context in which fit for purpose eDNA testing schemes could be designed. To complement the future development of eDNA proficiency testing schemes, we provide firsthand accounts and lessons learned while developing the current Great Crested Newt eDNA proficiency scheme. Lastly, we highlight current limitations in standardizing rapidly improving eDNA‐based techniques and discuss disadvantages to accreditation and standardization from an Australian‐centered perspective as a means to promote an active debate on the topic of future eDNA accreditation and proficiency testing. This commentary provides an Australian‐based perspective for the development of future eDNA proficiency testing schemes.

Eradicating invasive species is difficult, but success is more likely when populations are small after arrival. The cane toad, Rhinella marina, is an invasive pest species that threatens native fauna worldwide. Increasingly, environmental DNA (eDNA) is used as a technique to monitor the presence of invasive species given its power to detect low numbers of individuals. We aimed to investigate eDNA persistence in freshwater at three different temperatures (25, 30 and 35°C) and eDNA detection thresholds for R. marina using controlled experiments in aquaria. For the latter, two water volumes and two cane toad exposure times were used (800 or 200 L volume with 5 or 30 min exposure). A 15‐ml water sample was collected from each replicated aquaria and preserved with 5 ml Longmire's buffer. Environmental DNA was extracted and four technical quantitative PCR replicates were analyzed targeting the cane toad 16S rDNA mitochondrial gene. Environmental DNA decayed rapidly in water and was reliably detected for up to 3 days after cane toad removal, regardless of the temperature treatment. Also, cane toad eDNA was detected in the water after a 5‐min initial exposure of a single individual in 800 L of water. Under the physical parameters tested here, a positive detection means that a cane toad has been in contact with the water body between 1 and 3 days prior to the sampling event. The results of the present study show the importance of eDNA for determining the presence of a species that occurs at low abundance in a small water body, such as at the onset of a cane toad invasion. This study aimed to investigate cane toad (Rhinella marina) eDNA persistence in freshwater and eDNA detection thresholds. Environmental DNA proved to be a sensitive tool for detecting low abundances of cane toads in small experimental water bodies from the first day of exposure. Environmental DNA methods can be successfully incorporated into monitoring programs aiming to detect new incursions of cane toads.

Population outbreaks of the corallivorous crown-of-thorns seastar (CoTS; Acanthaster spp.) are significant threats to the Indo-Pacific reefs. Although recent research demonstrated that environmental DNA (eDNA) techniques could improve CoTS monitoring, the interpretation of surveillance results has been limited by uncertainties about eDNA dynamics. Here, we conducted aquarium experiments to identify biotic and abiotic factors affecting the shedding and degradation rates of CoTS eDNA. In the first experiment, we investigated the effect of two temperatures (24 and 28 °C) and three feeding treatments (no coral, coral accessible and coral inaccessible) on eDNA shedding rate of laboratory-raised 8-month-old juvenile CoTS. In the second experiment, we quantified CoTS eDNA degradation rate under three temperatures (24, 26 and 28 °C). We found that eDNA shedding rate was affected by feeding treatment (p < 0.0001) but not temperature. Specifically, the shedding rate under coral accessible treatment was about seven times higher than that of coral inaccessible treatment (p < 0.0001), whereas the presence of coral reduced the shedding rate by half (coral inaccessible vs no coral, p = 0.0249). Degradation of CoTS eDNA was rapid (half-life = 14 h) and not affected by temperature. Our results demonstrated that feeding activity increased eDNA release, but some of the released DNA was lost, potentially due to binding to coral surface mucus layer or skeleton. The rapid degradation rate indicated that results of eDNA surveillance likely reflects recent and local occurrence of CoTS. Although further testing is needed, this study provided support for using eDNA as a novel detection tool for early life stages of CoTS on coral reefs.

Globally, amphibian species have suffered drastic population declines over the past 40 years. Hundreds of species are now listed as Critically Endangered, with many of these considered \"possibly extinct\". Most of these species are stream-dwelling frogs inhabiting remote, montane areas, where remnant populations are hard to find using traditional surveys. Environmental DNA (eDNA) could revolutionize surveys for 'missing' and endangered amphibian populations by screening water samples from downstream sections to assess presence in the upstream catchments. However, the utility of this survey technique is dependent on quantifying downstream detection probability and distances. Here we tested downstream detection distances in two endangered stream frogs ( and ) that co-occur in a remote stream catchment in north-east Australia, and for which we know precise downstream distributional limits from traditional surveys. Importantly, the two last populations of persist in this catchment: one small (~1,000 frogs) and one very small (~100 frogs). We conducted eDNA screening at a series of sites kilometers downstream from the populations using precipitation from two fixed water volumes (15 and 100 mL) and water filtering (mean 1,480 L). We detected and the small population (~1,000 frogs) at most sampling sites, including 22.8 km downstream. The filtration method was highly effective for far-downstream detection, as was precipitation from 100 mL water samples, which also resulted in consistent detections at the far-downstream sites (including to 22.8 km). In contrast, we had limited downstream detection success for the very small population (~100 frogs). The ecological aspects of our study system, coupled with thorough traditional surveys, enabled us to measure downstream eDNA detection distances with accuracy. We demonstrate that eDNA from a small population of approximately 1,000 frogs can be detected as far as 22.8 km downstream from the population. Water filtration is considered best for eDNA detection of rare aquatic species-indeed it was effective in this study-but we also achieved far-downstream detections when precipitating eDNA from 100 mL water samples. Collecting small water volumes for subsequent precipitation in the lab is more practical than filtration when surveying remote areas. Our downstream detection distances (>20 km) suggest eDNA is a valuable tool for detecting rare stream amphibians. We provide recommendations on optimal survey methods.

Background Approximately 50% of freshwater turtles worldwide are currently threatened by habitat loss, rural development and altered stream flows. Paradoxically, reptiles are understudied organisms, with many species lacking basic geographic distribution and abundance data. The iconic Irwin’s turtle, Elseya irwini , belongs to a unique group of Australian endemic freshwater turtles capable of cloacal respiration. Water resource development, increased presence of saltwater crocodiles and its cryptic behaviour, have made sampling for Irwin’s turtle in parts of its range problematic, resulting in no confirmed detections across much of its known range for > 25 years. Here, we used environmental DNA (eDNA) analysis for E. irwini detection along its historical and contemporary distribution in the Burdekin, Bowen and Broken River catchments and tributaries. Five replicate water samples were collected at 37 sites across those three river catchments. Environmental DNA was extracted using a glycogen-aided precipitation method and screened for the presence of E. irwini through an eDNA assay targeting a 127 base pair-long fragment of the NADH dehydrogenase 4 (ND4) mitochondrial gene. Results Elseya irwini eDNA was detected at sites within its historic distribution in the lower Burdekin River, where the species had not been formally recorded for > 25 years, indicating the species still inhabits the lower Burdekin area. We also found higher levels of E. iriwni eDNA within its contemporary distribution in the Bowen and Broken Rivers, matching the prevailing scientific view that these areas host larger populations of E. irwini . Conclusions This study constitutes the first scientific evidence of E. irwini presence in the lower Burdekin since the original type specimens were collected as part of its formal description, shortly after the construction of the Burdekin Falls Dam. From the higher percentage of positive detections in the upper reaches of the Broken River (Urannah Creek), we conclude that this area constitutes the core habitat area for the species. Our field protocol comprises a user-friendly, time-effective sampling method. Finally, due to safety risks associated with traditional turtle sampling methods in the Burdekin River (e.g., estuarine crocodiles) we propose eDNA sampling as the most pragmatic detection method available for E. irwini .

Halophytes, such as seagrasses, predominantly form habitats in coastal and estuarine areas. These habitats can be seasonally exposed to hypo-salinity events during watershed runoff exposing them to dramatic salinity shifts and osmotic shock. The manifestation of this osmotic shock on seagrass morphology and phenology was tested in three Indo-Pacific seagrass species, Halophila ovalis, Halodule uninervis and Zostera muelleri, to hypo-salinity ranging from 3 to 36 PSU at 3 PSU increments for 10 weeks. All three species had broad salinity tolerance but demonstrated a moderate hypo-salinity stress response--analogous to a stress induced morphometric response (SIMR). Shoot proliferation occurred at salinities <30 PSU, with the largest increases, up to 400% increase in shoot density, occurring at the sub-lethal salinities <15 PSU, with the specific salinity associated with peak shoot density being variable among species. Resources were not diverted away from leaf growth or shoot development to support the new shoot production. However, at sub-lethal salinities where shoots proliferated, flowering was severely reduced for H. ovalis, the only species to flower during this experiment, demonstrating a diversion of resources away from sexual reproduction to support the investment in new shoots. This SIMR response preceded mortality, which occurred at 3 PSU for H. ovalis and 6 PSU for H. uninervis, while complete mortality was not reached for Z. muelleri. This is the first study to identify a SIMR in seagrasses, being detectable due to the fine resolution of salinity treatments tested. The detection of SIMR demonstrates the need for caution in interpreting in-situ changes in shoot density as shoot proliferation could be interpreted as a healthy or positive plant response to environmental conditions, when in fact it could signal pre-mortality stress.

The success of environmental DNA (eDNA) approaches for species detection has revolutionized biodiversity monitoring and distribution mapping. Targeted eDNA amplification approaches, such as quantitative PCR, have improved our understanding of species distribution, and metabarcoding‐based approaches have enabled biodiversity assessment at unprecedented scales and taxonomic resolution. eDNA datasets, however, are often scattered across repositories with inconsistent formats, varying access restrictions, and inadequate metadata; this limits their interoperation, reuse, and overall impact. Adopting FAIR (Findable, Accessible, Interoperable, and Reusable) data practices with eDNA data can transform the monitoring of biodiversity and individual species and support data‐driven biodiversity management across broad scales. FAIR practices remain underdeveloped in the eDNA community, partly due to gaps in adapting existing vocabularies, such as Darwin Core (DwC) and Minimum Information about any (x) Sequence (MIxS), to eDNA‐specific needs and workflows. To address these challenges, we propose a comprehensive FAIR eDNA (FAIRe) Metadata Checklist, which integrates existing data standards and introduces new terms tailored to eDNA workflows. Metadata are systematically linked to both raw data (e.g., metabarcoding sequences, Ct/Cq values of targeted qPCR assays) and derived biological observations (e.g., Amplicon Sequence Variant (ASV)/Operational Taxonomic Unit (OTU) tables, species presence/absence). Along with formatting guidelines, tools, templates, and example datasets, we introduce a standardized, ready‐to‐use approach for FAIR eDNA practices. Through broad collaboration, we seek to integrate these guidelines into established biodiversity and molecular data standards, promote journal data policies, and foster user‐driven improvements and uptake of FAIR practices among eDNA data producers. In proposing this standardized approach and developing a long‐term plan with key databases and data standard organizations, the goal is to enhance accessibility, maximize reuse, and elevate the scientific impact of these valuable biodiversity data resources. Environmental DNA (eDNA) has emerged as a transformative tool for biodiversity monitoring and species detection, yet inconsistent metadata practices hinder data interoperability and reuse. To address these challenges, we present the comprehensive FAIR eDNA (FAIRe) guidelines and metadata checklist, supported by practical tools and multi‐disciplinary collaborations, to advance the adoption of FAIR (Findable, Accessible, Interoperable, and Reusable) principles in eDNA workflows.