Methods to maximise recovery of environmental DNA from water samples

<div><p>The environmental DNA (eDNA) method is a detection technique that is rapidly gaining credibility as a sensitive tool useful in the surveillance and monitoring of invasive and threatened species. Because eDNA analysis often deals with small quantities of short and degraded DNA fragments, methods that maximize eDNA recovery are required to increase detectability. In this study, we performed experiments at different stages of the eDNA analysis to show which combinations of methods give the best recovery rate for eDNA. Using Oriental weatherloach (<i>Misgurnus anguillicaudatus</i>) as a study species, we show that various combinations of DNA capture, preservation and extraction methods can significantly affect DNA yield. Filtration using cellulose nitrate filter paper preserved in ethanol or stored in a -20°C freezer and extracted with the Qiagen DNeasy kit outperformed other combinations in terms of cost and efficiency of DNA recovery. Our results support the recommendation to filter water samples within 24hours but if this is not possible, our results suggest that refrigeration may be a better option than freezing for short-term storage (i.e., 3–5 days). This information is useful in designing eDNA detection of low-density invasive or threatened species, where small variations in DNA recovery can signify the difference between detection success or failure.</p></div

Improving the detection of rare native fish species in environmental DNA metabarcoding surveys

The presence of threatened or endangered species often strongly influences management and conservation decisions. Within the Murray–Darling Basin (MDB), Australia, the presence of threatened native fish affects the management and allocation of water resources. These decisions are currently based on traditional fisheries data and a predictive MaxEnt model. However, it is important to verify the model's predictive power given the implication it may have, but this requires methods with a high detection sensitivity for rare species. Although the use of environmental DNA (eDNA) metabarcoding achieves a higher detection sensitivity compared with traditional methods, earlier surveys in the MDB have shown that the highly abundant and invasive common carp (Cyprinus carpio) can reduce detection probabilities for rare species. Consequently, a polymerase chain reaction (PCR) blocking primer designed to block the amplification of carp eDNA could increase the detection probabilities for rare native species while simultaneously reducing the required sampling effort and survey costs. Although PCR blocking primers are often used in ancient DNA and dietary studies, no aquatic eDNA metabarcoding study to date has evaluated the potential benefits of using PCR blocking primers. A laboratory and field‐based pilot study was used to address this knowledge gap and assess the impact of a blocking primer on the detection probabilities of native species and the minimum sampling effort required. Results showed that the inclusion of the blocking primer increased the detection probabilities for native species by 10–20% and reduced the minimum required sampling effort by 25–50%. These findings provide important insights into possible methods for optimizing eDNA metabarcoding surveys for the detection of rare aquatic species

Does mesocosm validation of environmental DNA methods translate to natural environment monitoring applications? A case study detecting a high-profile invader; the red eared slider turtle, Trachemys scripta elegans, in Australia

Environmental DNA (eDNA) surveys have gained popularity as a highly sensitive detection tool that generally outperform traditional detection techniques. eDNA surveys can provide a cost-effective means to identify species’ distributions and recent incursions, informing the control or containment of invasive species. The red-eared slider turtle, Trachemys scripta elegans, is one of the world’s most invasive species and is listed as a priority pest species for management in Australia. In this study, we validate two eDNA assays to detect this invasive turtle in Australia. We demonstrate high sensitivity in a laboratory setting and perfect detection rates in mesocosms for one of these eDNA assays but show that this does not translate to high detection rates in urban waterbodies at sites of known occupancy. In fact, our results suggest eDNA surveys provide sub-optimal performance compared to traditional detection methods for T.s. elegans. We suggest the capacity for eDNA surveys to provide a highly sensitive detection tool must be evaluated in natural environments on a species-by-species basis to understand any limitations and to avoid high error rates from eDNA surveys leading to wasted resources or inappropriate management decisions. For management of T.s. elegans in Australia, clearly defining the utility of certain eDNA based approaches to detect T.s. elegans and their incursions is vital for effective management of this pest species.</p

Food from faeces:Evaluating the efficacy of scat DNA metabarcoding in dietary analyses

Scat DNA metabarcoding is increasingly being used to track the feeding ecology of elusive wildlife species. This approach has greatly increased the resolution and detection success of prey items contained in scats when compared with other classical methods. However, there have been few studies that have systematically tested the applicability and reliability of this approach to study the diet of large felids species in the wild. Here we assessed the effectiveness of this approach in the cheetah Acinonyx jubatus. We tested how scat degradation, meal size, prey species consumed and feeding day (the day a particular prey was consumed) influenced prey DNA detection success in captive cheetahs. We demonstrated that it is possible to obtain diet information from 60-day old scats using genetic approaches, but the efficiency decreased over time. Probability of species-identification was highest for food items consumed one day prior to scat collection and the probability of being able to identify the species consumed increased with the proportion of the prey consumed. Detection success varied among prey species but not by individual cheetah. Identification of prey species using DNA detection methods from a single consumption event worked for samples collected between 8 and 72 hours post-feeding. Our approach confirms the utility of genetic approaches to identify prey species in scats and highlight the need to account for the systematic bias in results to control for possible scat degradation, feeding day, meal size and prey species consumed especially in the wild-collected scats

Reliable Discrimination of 10 Ungulate Species Using High Resolution Melting Analysis of Faecal DNA

Identifying species occupying an area is essential for many ecological and conservation studies. Faecal DNA is a potentially powerful method for identifying cryptic mammalian species. In New Zealand, 10 species of ungulate (Order: Artiodactyla) have established wild populations and are managed as pests because of their impacts on native ecosystems. However, identifying the ungulate species present within a management area based on pellet morphology is unreliable. We present a method that enables reliable identification of 10 ungulate species (red deer, sika deer, rusa deer, fallow deer, sambar deer, white-tailed deer, Himalayan tahr, Alpine chamois, feral sheep, and feral goat) from swabs of faecal pellets. A high resolution melting (HRM) assay, targeting a fragment of the 12S rRNA gene, was developed. Species-specific primers were designed and combined in a multiplex PCR resulting in fragments of different length and therefore different melting behaviour for each species. The method was developed using tissue from each of the 10 species, and was validated in blind trials. Our protocol enabled species to be determined for 94% of faecal pellet swabs collected during routine monitoring by the New Zealand Department of Conservation. Our HRM method enables high-throughput and cost-effective species identification from low DNA template samples, and could readily be adapted to discriminate other mammalian species from faecal DNA

Phylogeography of the Endangered Otago Skink, Oligosoma otagense: Population Structure, Hybridisation and Genetic Diversity in Captive Populations

Climatic cooling and substantial tectonic activity since the late Miocene have had a pronounced influence on the evolutionary history of the fauna of New Zealand's South Island. However, many species have recently experienced dramatic range reductions due to habitat fragmentation and the introduction of mammalian predators and competitors. These anthropogenic impacts have been particularly severe in the tussock grasslands of the Otago region. The Otago skink (Oligosoma otagense), endemic to the region, is one of the most critically endangered vertebrates in New Zealand. We use mitochondrial DNA sequence data to investigate the evolutionary history of the Otago skink, examine its population genetic structure, and assess the level of genetic diversity in the individuals in the captive breeding program. Our data indicate that the Otago skink diverged from its closest relatives in the Miocene, consistent with the commencement of tectonic uplift of the Southern Alps. However, there is evidence for past introgression with the scree skink (O. waimatense) in the northern Otago-southern Canterbury region. The remnant populations in eastern Otago and western Otago are estimated to have diverged in the mid-Pliocene, with no haplotypes shared between these two regions. This divergence accounts for 95% of the genetic diversity in the species. Within both regions there is strong genetic structure among populations, although shared haplotypes are generally evident between adjacent localities. Although substantial genetic diversity is present in the captive population, all individuals originate from the eastern region and the majority had haplotypes that were not evident in the intensively managed populations at Macraes Flat. Our data indicate that eastern and western populations should continue to be regarded as separate management units. Knowledge of the genetic diversity of the breeding stock will act to inform the captive management of the Otago skink and contribute to a key recovery action for the species