Swinging back from the brink? Polygamous mating strategies revealed for an iconic threatened freshwater fish.

Catastrophic fish death events are increasing in frequency and severity globally. A series of major recent fish deaths in the semi-arid lower Darling-Baaka river system (LDBR) of Australia are emblematic of these issues with tens of millions of native fish perishing. In 2018-2019 there was a major death event for Australia's largest freshwater fish, Murray cod (Maccullochella peelii). To aid the recovery and guide restoration activities of local Murray cod populations, it is essential to gather information on the mating strategies and effective population size following the fish death event. After the fish deaths, we collected larvae during the 2020 and 2021 breeding seasons and used single nucleotide polymorphisms (SNPs) to provide insight mating strategies and to estimate effective population size. Larvae were detected in both years along the entire length of the LDBR. Sixteen percent of the inferred breeding individuals were found to contribute to multiple pairings, confirming a complex and polygamous mating system. A high frequency of polygamy was evident both within and between years with 100 % polygamy identified among parents that produced offspring in both 2020 and 2021 and 95 % polygamy identified among parents involved in multiple spawning events within years. Post-larval Murray cod samples collected between 2016 and 2021 were co-analysed to further inform kinship patterns. Again, monogamy was rare with no confirmed cases of the same male-female pair contributing to multiple breeding events within or between seasons. Effective population size based on Murray cod collected after the fish death event was estimated at 721.6 (CI 471-1486), though this has likely declined following a subsequent catastrophic fish death event in the LDBR in March 2023. Our data provide insight into the variability of Murray cod mating strategies, and we anticipate that this knowledge will assist in planning conservation actions to ultimately help recover a species in crisis.

Identifying error and accurately interpreting environmental DNA metabarcoding results: A case study to detect vertebrates at arid zone waterholes.

Environmental DNA (eDNA) metabarcoding surveys enable rapid, noninvasive identification of taxa from trace samples with wide-ranging applications from characterizing local biodiversity to identifying food-web interactions. However, the technique is prone to error from two major sources: (a) contamination through foreign DNA entering the workflow, and (b) misidentification of DNA within the workflow. Both types of error have the potential to obscure true taxon presence or to increase taxonomic richness by incorrectly identifying taxa as present at sample sites, but multiple error sources can remain unaccounted for in metabarcoding studies. Here, we use data from an eDNA metabarcoding study designed to detect vertebrate species at waterholes in Australia's arid zone to illustrate where and how in the workflow errors can arise, and how to mitigate those errors. We detected the DNA of 36 taxa spanning 34 families, 19 orders and five vertebrate classes in water samples from waterholes, demonstrating the potential for eDNA metabarcoding surveys to provide rapid, noninvasive detection in remote locations, and to widely sample taxonomic diversity from aquatic through to terrestrial taxa. However, we initially identified 152 taxa in the samples, meaning there were many false positive detections. We identified the sources of these errors, allowing us to design a stepwise process to detect and remove error, and provide a template to minimize similar errors that are likely to arise in other metabarcoding studies. Our findings suggest eDNA metabarcoding surveys need to be carefully conducted and screened for errors to ensure their accuracy.

Does Size Matter? An Experimental Evaluation of the Relative Abundance and Decay Rates of Aquatic Environmental DNA.

Environmental DNA (eDNA) is increasingly used to monitor aquatic macrofauna. Typically, short mitochondrial DNA fragments are targeted because these should be relatively more abundant in the environment as longer fragments will break into smaller fragments over time. However, longer fragments may permit more flexible primer design and increase taxonomic resolution for eDNA metabarcoding analyses, and recent studies have shown that long mitochondrial eDNA fragments can be extracted from environmental water samples. Nuclear eDNA fragments have also been proposed as targets, but little is known about their persistence in the aquatic environment. Here we measure the abundance of mitochondrial eDNA fragments of different lengths and of short nuclear eDNA fragments, originating from captive fish in experimental tanks, and we test whether longer mitochondrial and short nuclear fragments decay faster than short mitochondrial fragments following fish removal. We show that when fish are present, shorter mitochondrial fragments are more abundant in water samples than both longer mitochondrial fragments and short nuclear eDNA fragments. However, the rate of decay following fish removal was similar for all fragment types, suggesting that the differences in abundance resulted from differences in the rates at which different fragment types were produced rather than differences in their decay rates.

A framework for estimating the sensitivity of eDNA surveys.

Imperfect sensitivity, or imperfect detection, is a feature of all survey methods that needs to be accounted for when interpreting survey results. Detection of environmental DNA (eDNA) is increasingly being used to infer species distributions, yet the sensitivity of the technique has not been fully evaluated. Sensitivity, or the probability of detecting target DNA given it is present at a site, will depend on both the survey method and the concentration and dispersion of target DNA molecules at a site. We present a model to estimate target DNA concentration and dispersion at survey sites and to estimate the sensitivity of an eDNA survey method. We fitted this model to data from a species-specific eDNA survey for Oriental weatherloach, Misgurnus anguillicaudatus, at three sites sampled in both autumn and spring. The concentration of target DNA molecules was similar at all three sites in autumn but much higher at two sites in spring. Our analysis showed the survey method had ≥95% sensitivity at sites where target DNA concentrations were ≥11 molecules per litre. We show how these data can be used to compare sampling schemes that differ in the number of field samples collected per site and number of PCR replicates per sample to achieve ≥95% sensitivity at a given target DNA concentration. These models allow researchers to quantify the sensitivity of eDNA survey methods to optimize the probability of detecting target species, and to compare DNA concentrations spatially and temporarily.