Rapid species identification of eight sympatric northern Australian macropods from faecal-pellet DNA

Abstract

Context Conservation of vulnerable and endangered species requires a comprehensive understanding of their distribution and habitat requirements, so as to implement effective management strategies. Visual scat surveys are a common non-invasive method for monitoring populations. However, morphological similarity of scats among sympatric species presents a problem for accurate identification. Visual misidentifications of scats can have major impacts on the accuracy of abundance and distribution surveys of target species, wasting resources and misdirecting management and conservation actions. DNA identification of scats can overcome this issue, while simultaneously providing a rich source of genetic information for population and dietary studies. Aims We developed a simple and reliable method to identify morphologically similar macropod scats from eight sympatric species in north-eastern Australia, using polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) of a portion of the mtDNA ND2 gene. Methods We identified a short (275-bp) polymorphic region of ND2, which is easily amplifiable from degraded DNA, developed a primer set, and identified a set of three restriction endonucleases (AluI, BstNI and HphI) which, in combination, can discriminate among the eight target species. So as to test the effectiveness of this protocol, we collected 914 macropod scats from 53 sites in the north-eastern Australia. Key results In total, 406 of these scats were extracted, with 398 (98%) containing amplifiable macropod DNA. All 398 scats were subsequently identified to species by using our RFLP protocol. Sequencing of a subset of these samples confirmed the accuracy of the test. Species identification of scats by using DNA identified eight species of macropods, five of which were outside their documented distributions, one of which was ~400 km. Conclusions Our PCR–RFLP method is a simple and efficient means to identify macropod scats to species, eliminating the need for sequencing, which is costly, time-consuming and requires additional laboratory equipment. Implications The method allows for rapid and non-invasive assessment of macropod species and is particularly useful for surveying populations across multiple sites.

Stemming the tide: progress towards resolving the causes of decline and implementing management responses for the disappearing mammal fauna of northern Australia

“…Recent advances in genetic analysis now allow for reliable, rapid and cost-effective identification of prey items in predator faeces, predators from prey remains (Banks et al 2003a), herbivore species from their faeces, including north Australian macropods (Telfer et al 2006;Wadley et al 2013), and individuals from faeces or hair samples, which may enable the monitoring of highly threatened species (Banks et al 2003b). Extraction and analyses of DNA extracted from leeches, carrion flies and mosquitoes, may also provide novel methods for improving monitoring of a range of mammal species (Calvignac-Spencer et al 2013;Schnell et al 2012).…”

Genetic inference as a method for modelling occurrence: A viable alternative to visual surveys

“…The indirect genetic method requires samples to undergo laboratory analysis for identification of species. For this study, samples were processed in-house at the University of Adelaide (Wadley et al 2013). However, for the cost benefit analysis we have used commercial costs from an external source (the Australian Genome Research Facility -AGRF) so as to provide a comparison of surveying cost for those without access to the required laboratory equipment.…”

“…Extractions were performed in a DNA extraction laboratory physically separate from PCR-setup and post-PCR laboratories to prevent cross contamination with previously amplified DNA and all extractions included a negative control. All DNA extracts and negative controls were amplified for a 275-bp mtDNA ND2 fragment using macropod specific PCR primers (M496 5′-GCTTGAATAACAAACCAAT GAACCC-3′/M497 5′-CCTCCTCAGCCTCCGAGYAT-3′), digested with three restriction endonucleases (AluI, HphI and BstNI) and examined by gel electrophoresis to determine species (Wadley et al 2013). …”

“…Note that scats could not be located at all 1 km intervals. Species identification of scats was conducted following Wadley et al (2013), whereby a small amount (2 cm 2 ) of the outer surface of each scat was scraped and removed using a sterile scalpel and 'Bioline fecal ISOLATE' kits (Bioline, London, UK) were used for extracting DNA. Extractions were performed in a DNA extraction laboratory physically separate from PCR-setup and post-PCR laboratories to prevent cross contamination with previously amplified DNA and all extractions included a negative control.…”

Phylogeography of the antilopine wallaroo (Macropus antilopinus) across tropical northern Australia

“…DNA was extracted from bone samples using a Qiagen DNeasy blood and tissue kit with the following modifications; the amount of ATL, AL, and ethanol added were doubled. Bone extracts underwent amplification and sequencing using species identification primers (Wadley et al 2013) to determine the success of the extraction and to confirm the species identification as M. antilopinus before continuing with the PCRs for the three targeted mtDNA regions. Bone extracts were amplified and sequenced with primers designed to create a number of short overlapping fragments (Table S2 in Appendix S1).…”