Ana Ramón-Laca

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.

A simple and effective method for obtaining mammal DNA from faeces

The use of faecal DNA, although a promising tool for the population monitoring of mammals, has not yet become a fully exploited and standard practice, mainly because low target DNA concentration, DNA degradation, and co-purification of inhibitors demand extra laboratory procedures to improve success and reliability. Here we evaluate a simple method that enables sampling of DNA in the field through the collection of the intestinal cells present on the surface of a scat using a swab. The swab is immediately placed in a vial containing a lysis buffer that preserves the DNA for its later extraction. DNA extracts of three species of herbivores (goat, fallow deer and white-tailed deer), two carnivores (Iberian lynx and domestic dog) and one omnivore species (brushtail possum) were characterised in terms of target and total DNA quantity, PCR inhibition and genotyping success. Direct comparison was carried out with duplicate samples preserved in 96% ethanol and extracted via a commonly used commercial DNA extraction kit for faecal material. Results from these comparisons show that swabbing the samples in situ not only simplifies field collection and sample handling in the laboratory, but generally optimises target DNA recovery, minimises co-purification of PCR inhibitors and provides good quality DNA for the species tested, especially for herbivores. This method is also less time-consuming and more cost-effective, thus providing a more convenient and efficient alternative for non-invasive genetic studies.

Identification multiplex assay of 19 terrestrial mammal species present in New Zealand

An identification assay has been developed that allows accurate detection of 19 of the most common terrestrial mammals present in New Zealand (cow, red deer, goat, dog, horse, hedgehog, cat, tammar wallaby, mouse, weasel, ferret, stoat, sheep, rabbit, Pacific rat, Norway rat, ship rat, pig, and brushtail possum). This technique utilizes species‐specific primers that, combined in a multiplex PCR, target small fragments of the mitochondrial cytochrome b gene. Each species, except hedgehog, produces two distinctive species‐specific fragments, making the assay self‐confirmatory and enabling the identification of multiple species simultaneously in DNA mixtures. The multiplex assay detects as little as 100 copies of mitochondrial DNA, which makes it a very reliable tool for degraded and trace samples. Reliability, accuracy, reproducibility, and sensitivity tests to validate the technique were performed. The technique featured here enabled a prompt response in a predation specific event, but can also be useful for wildlife management and conservation, pest incursions detection, forensic, and industrial purposes in a very simple and cost‐effective manner.

Molecular identification of the prey range of the invasive Asian paper wasp

The prey range of the invasive Asian paper wasp, Polistes chinensis antennalis, was studied using molecular diagnostics. Nests of paper wasps were collected from urban residential and salt marsh habitats, larvae were removed and dissected, and DNA in the gut of the paper wasp larvae was amplified and sequenced with cytochrome c oxidase subunit I (COI). Seventy percent of samples (211/299) yielded medium-to high-quality sequences, and prey identification was achieved using BLAST searches in BOLD. A total of 42 taxa were identified from 211 samples. Lepidoptera were the majority of prey, with 39 taxa from 91% of samples. Diptera was a relatively small component of prey (three taxa, 19 samples). Conclusive species-level identification of prey was possible for 67% of samples, and genus-level identification, for another 12% of samples. The composition of prey taken was different between the two habitats, with 2.5× more native prey species being taken in salt marsh compared with urban habitats. The results greatly extend the prey range of this invasive species. The technique is a more effective and efficient approach than relying on the collection of “prey balls”, or morphological identification of prey, for the study of paper wasps.

Extraction of DNA from captive-sourced feces and molted feathers provides a novel method for conservation management of New Zealand kiwi (Apteryx spp.)

Abstract Although some taxa are increasing in number due to active management and predator control, the overall number of kiwi (Apteryx spp.) is declining. Kiwi are cryptic and rare, meaning current monitoring tools, such as call counts, radio telemetry, and surveys using detection dogs are labor‐intensive, yield small datasets, and require substantial resources or provide inaccurate estimates of population sizes. A noninvasive genetic approach could help the conservation effort. We optimized a panel of 23 genetic markers (22 autosomal microsatellite loci and an allosomal marker) to discriminate between all species of kiwi and major lineages within species, while simultaneously determining sex. Markers successfully amplified from both fecal and shed feather DNA samples collected in captivity. We found that DNA extraction was more efficient from shed feathers, but DNA quality was greater with feces, although this was sampling dependent. Our microsatellite panel was able to distinguish between contemporary kiwi populations and lineages and provided PI values in the range of 4.3 × 10−5 to 2.0 × 10−19, which in some cases were sufficient for individualization and mark–recapture studies. As such, we have tested a wide‐reaching, noninvasive molecular approach that will improve conservation management by providing better parameter estimates associated with population ecology and demographics such as abundance, growth rates, and genetic diversity.