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Environmental DNA (eDNA) has revolutionized our ability to identify the presence and distributions of terrestrial and aquatic organisms. Recent evidence suggests the concentration of eDNA could also provide a rapid, cost‐effective indicator of abundance and/or biomass for fisheries stock assessments. Globally, fisheries resources are under immense pressure, and their sustainable harvest requires accurate information on the sizes of fished stocks. However, in many cases the required information remains elusive because of a reliance on imprecise or costly fishery‐dependent and independent data. Here, we review the literature describing relationships between eDNA concentrations and fish abundance and/or biomass, as well as key influencing factors, as a precursor to determining the broader utility of eDNA for monitoring fish populations. We reviewed 63 studies published between 2012 and 2020 and found 90% identified positive relationships between eDNA concentrations and the abundance and/or biomass of focal species. Key influencing biotic factors included the taxon examined as well as their body size, distribution, reproduction, and migration. Key abiotic factors mostly comprised hydrological processes affecting the dispersal and persistence of eDNA, especially water flow and temperature, although eDNA collection methods were also influential. The cumulative influence of these different factors likely explains the substantial variability observed in eDNA concentrations, both within and among studies. Nevertheless, there is considerable evidence to support using eDNA as an ancillary tool for assessing fish population abundance and/or biomass across discrete spatio‐temporal scales, following preliminary investigations to determine species‐ and context‐specific factors influencing the eDNA abundance/biomass relationship. Advantages of eDNA monitoring relative to other approaches include reduced costs, increased efficiencies, and nonlethal sampling. The sustainable harvest of fisheries resources is contingent on accurate information concerning the size of the fished stocks. Here, we review the literature to critically evaluate the utility and limitations of using eDNA as a method to quantify the abundance and/or biomass of fish in marine and freshwater environments.

The analysis of environmental DNA (eDNA) is a powerful and non-invasive method for monitoring the presence of species in ecosystems. However, ecologists and laboratory staff can find it challenging to use eDNA analysis software effectively due to the unfamiliar command-line interfaces used by many of these packages. Therefore, we developed the eDNA-container app, a free and open-source software package that provides a simple user-friendly interface for eDNA analysis. The application is based on the popular QIIME2 library and is distributed as a Docker image. The use of Docker makes it compatible with a wide range of operating systems and facilitates the reproducible analysis of data across different laboratories. The application includes a point-and-click user interface for selecting sequencing files, configuring parameters, and accessing the results. Key pipeline outputs, such as sequence quality plots, denoising, and ASV generation statistics, are automatically included in a PDF report. This open-source and freely available analysis package should be a valuable tool for scientists using eDNA in biodiversity and biosecurity applications.

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 DNA (eDNA) has been widely used for species surveillance. However, the lack of adequate quality control in many eDNA research projects and applications can lead to false‐negative results, greatly affecting biosecurity surveillance and conservation efforts. Exogenous DNA is routinely added to eDNA samples and used as a positive control, typically after DNA extraction. However, this type of positive control is only able to identify false negatives due to errors at the amplification stage. Therefore, errors in upstream processes, such as sample collection will not be identified by an exogenous control. We designed two independent sets of generic quality control qPCR assays (QCqPCR) targeting abundant endogenous DNA that is obtained during sample collection. Our QCqPCR assays target the chloroplast 16S and 23S ribosomal RNA sequences. In silico analyses indicated these regions were highly conserved among plants, algae and bacteria commonly found in freshwater, marine, or terrestrial environments. These QCqPCR assays were purposely mismatched against the human genome to avoid false positives resulting from human DNA contamination. Both assays remained highly efficient and sensitive under annealing temperatures between 58 and 62°C, allowing them to be multiplexed with most qPCR analyses. We validated our assays by multiplexing with a species‐specific Murray cod (Maccullochella peelii) assay on field‐collected environmental water samples. Potential false‐negative reactions can be identified by the failed or suppressed QCqPCR assay and the negative species‐specific assay. We recommend incorporating either one of the QCqPCR assays in qPCR‐based eDNA analysis to identify potential false negatives and improve the reliability of eDNA surveys. We developed two independent sets of generic quality control qPCR assays (QCqPCR) targeting abundant endogenous plant DNA in the environment. Unlike exogenous controls, our QCqPCR assays enable the monitoring of the entire eDNA workflow. We recommend incorporating either one of the QCqPCR assays into qPCR‐based eDNA analysis to detect potential false negatives and improve the reliability of eDNA surveys.

Non-invasive, low-cost methods for censusing depleted fish populations are being prioritised among many jurisdictions worldwide. Collecting environmental DNA (eDNA) could offer one such option for augmenting fish population assessments. However, candidate species need to be carefully selected because species-specific DNA shedding and decay rates are affected by many biotic and abiotic factors that may influence relative abundance estimates. In this study, we sought to ascertain if the eDNA of a depleted Australian teleost, mulloway, Argyrosomus japonicus, reflects its weight under controlled aquaria conditions. With four experiments, we investigated the relationships between mulloway eDNA concentrations and their weight tank−1 as a function of: (1) time post-tank establishment; (2) water temperatures (within the species’ tolerance range); (3) stocking densities; and (4) among individual, similar-sized fish. The concentrations of eDNA in tanks stabilised after six days, and a positive relationship was found between fish weight and eDNA concentration, despite some variability in shedding rates by similar-sized fish. There was also a positive effect of water temperature on eDNA concentrations, which reinforces the need to control for such abiotic factors. We conclude that there is strong utility in applying eDNA concentrations as an index of relative abundance for mulloway under controlled conditions, which justifies future field-based investigations.

Freshwater eel-tailed catfishes ( Tandanus spp.) have a complicated phylogenetic structure including several undescribed cryptic species in eastern Australia. This naturally complex phylogeny is further complicated by their phylogeography, with the existence of numerous populations reported to have been subject to historic translocations. The Murray–Darling Basin (MDB) population of Tandanus tandanus is listed as endangered and is locally extinct over large parts of its historical range. Conservation actions such as captive breeding for release into the wild, or translocations, will be necessary components of a recovery plan for the MDB population. To further understand the phylogeography of Tandanus populations in south-eastern Australia and to provide additional support for the taxonomic position of previously proposed cryptic species, we sequenced a 408 bp fragment of the mtDNA control region from 240 fish collected from 26 catchments. We identified three distinct clades within New South Wales; two from coastal catchments and a third from the MDB. Phylogeographic structure was clearly affected by previous translocations, with the presence of both entirely non-endemic populations, and endemic populations subjected to introgression with fish from non-endemic source populations. Most coastal catchments possessed unique haplotypes resulting in a high degree of genetic structure among catchments, while few MDB sub-catchments possessed unique haplotypes. The phylogenetic structure was consistent with the greater hydrological connectivity of MDB sub-catchments, allowing much greater gene flow compared to coastal catchments that are hydrologically isolated. The complicated phylogenetic and phylogeographic structure observed in T. tandanus has implications for future stocking or translocations.

The Australian freshwater catfish ( Tandanus tandanus ) has suffered a decline in abundance and distribution, and stocking of wild populations with hatchery-bred fish has been suggested to assist with population recovery. Here we describe the isolation and characterisation of eight microsatellite markers that may be used to assess population structure of T. tandanus in the wild to inform future stocking programs of any major genetic boundaries between populations. We tested the variability of the loci in 28–29 individuals from three populations of T. tandanus , as well in 24 individuals from a population representing an undescribed species. Expected heterozygosity for these loci ranged from 0.034 to 0.920 across the four populations. All loci successfully amplified in the three T. tandanus populations, while in the undescribed species one locus failed to amplify and three loci were monomorphic.

Non-invasive, low-cost methods for censusing depleted fish populations are being prioritised among many jurisdictions worldwide. Collecting environmental DNA (eDNA) could offer one such option for augmenting fish population assessments. However, candidate species need to be carefully selected because species-specific DNA shedding and decay rates are affected by many biotic and abiotic factors that may influence relative abundance estimates. In this study, we sought to ascertain if the eDNA of a depleted Australian teleost, mulloway, Argyrosomus japonicus, reflects its weight under controlled aquaria conditions. With four experiments, we investigated the relationships between mulloway eDNA concentrations and their weight tank[sup.−1] as a function of: (1) time post-tank establishment; (2) water temperatures (within the species’ tolerance range); (3) stocking densities; and (4) among individual, similar-sized fish. The concentrations of eDNA in tanks stabilised after six days, and a positive relationship was found between fish weight and eDNA concentration, despite some variability in shedding rates by similar-sized fish. There was also a positive effect of water temperature on eDNA concentrations, which reinforces the need to control for such abiotic factors. We conclude that there is strong utility in applying eDNA concentrations as an index of relative abundance for mulloway under controlled conditions, which justifies future field-based investigations.

Tandanus bellingerensis, new species, is described based on specimens from four river drainages (Bellinger, Macleay, Hastings, and Manning rivers) of the mid-northern coast of New South Wales, Australia. Previously, three species were recognized in the genus Tandanus: T. tropicanus of the wet tropics region of northeast Queensland, T. tandanus of the Murray-Darling drainage and coastal streams of central-southern Queensland and New South Wales, and T. bostocki of southwestern Western Australia. The new species is distinguished from all congeners by a combination of the following morphologic characters: a high count of rays in the continuous caudodorsal and anal fins (range 153–169, mode 159), a high count of gill rakers on the first arch (range 35–39, mode 36), and strongly recurved posterior serrae of the pectoral-fin spine. Additionally, results from previously conducted genetic studies corroborate morphologic and taxonomic distinctness of the new species.

Tandanus bellingerensis, new species, is described based on specimens from four river drainages (Bellinger, Macleay, Hastings, and Manning rivers) of the mid-northern coast of New South Wales, Australia. Previously, three species were recognized In the genus Tandanus: T. tropicanus of the wet tropics region of northeast Queensland, T. tandanus of the Murray-Darling drainage and coastal streams of central-southern Queensland and New South Wales, and T. bostocki of southwestern Western Australia. The new species is distinguished from all congeners by a combination of the following morphologic characters: a high count of rays In the continuous caudodorsal and anal fins (range 153-169, mode 159), a high count of gill rakers on the first arch (range 35-39, mode 36), and strongly recurved posterior serrae of the pectoral-fin spine. Additionally, results from previously conducted genetic studies corroborate morphologic and taxonomic distinctness of the new species.