Breaking down scats: degradation of DNA from greater bilby (Macrotis lagotis) faecal pellets

Abstract

Isolating DNA from scats (faeces) of threatened species is a valuable, non-invasive method for identifying individuals. To establish whether genotyping of greater bilby (Macrotis lagotis) individuals from faecal pellets collected in the field can be useful for population monitoring, an understanding of the DNA degradation rates is necessary. To determine the relationship between time and degradation of bilby faecal DNA, and assess whether a two-step elution process during extraction results in better-quality DNA, faecal pellets were collected from captive individuals, maintained under seminatural conditions, then harvested at known periods. DNA was amplified from faecal pellets with a 99% success rate and error rates of less than 5% up to 14 days after deposition. The amplification rate decreases, and the rate of allelic dropout increases with time, but DNA can still be amplified at rates above 60% and error rates below 15% at 90–180 days. We found that a second elution step was unnecessary, with more DNA amplified over a longer period using the first eluate. Viable DNA exists on bilby faecal pellets for a long period after deposition, which is useful for obtaining genetic samples for population monitoring programs and studies on population genetics.

Monitoring the Abundance of Wild and Reintroduced Bilby Populations

“…Bilby scats are well suited to this type of sampling; the high water‐conserving capability of bilbies means their scats are relatively desiccated (Gibson and Hume 2000, Gibson et al 2002), and the dry conditions in areas where bilbies are still present likely assists in the preservation of DNA on bilby scats (Carpenter and Dziminski 2017). Thus, DNA can be reliably extracted from bilby scats for up to 2 weeks after deposition (Carpenter and Dziminski 2017). Because rainfall may promote the decay of scat DNA (Piggott 2004, Brinkman et al 2009), we recommend that sampling of bilby scats should be undertaken during the dry season and >2 weeks after rainfall.…”

“…Thus, we were able to assess the age of these scats by examining the state of decomposition of the associated digging. If the digging was very eroded and weathered, indicating it was created probably >2 weeks prior, then we did not collect the associated scats because the scats were less likely to yield DNA (Carpenter and Dziminski 2017). Initially, we collected some samples in paper envelopes and vials with silica gel beads, but we stored the majority of collected scats in labeled 30‐ml plastic tubes, with approximately 33% filled with silica gel beads and a cotton wool ball, until DNA extraction.…”

“…We used the QIAGEN QIAamp DNA Stool Minikit (QIAGEN, Hilden, Germany) as per the manufacturer's protocol, which included the scraping of 100 mg of material from the outside of each pellet, for extractions completed from 2013 to the beginning of 2014. Further testing of different extraction methods in 2014 resulted in amending the protocol to the one described by Carpenter and Dziminski (2017). After 2014 we employed a single elution using 100 µL of buffer ATE and stored DNA samples at −20°C until we amplified samples using polymerase chain reaction (PCR).…”

“…We undertook PCR amplification using up to 8 bilby‐specific polymorphic microsatellite markers (Moritz et al 1997, Smith et al 2009) amplified across 2 multiplexes with fluorescent‐labeled markers from the G5 filter set: multiplex 1 (B02 [6FAM], B17 [VIC], B56 [PET], and B66 [NED]) and multiplex 2 (B55 [6FAM], B22 [VIC], B41 [PET], and B63 [NED]; Moritz et al 1997, Smith et al 2009). We ran PCRs as described in Carpenter and Dziminski (2017) with 2–4 µL of DNA used in a 12.5‐µL reaction for each replicate. We initially performed a minimum of 2 PCRs for each scat sample.…”

“…Sampling bilby scats, and coupling this with genotyping individuals from the DNA in their scats, may allow an accurate calculation of bilby abundance within a population. Bilby scats are easy to collect and store, and viable DNA can readily be extracted from them (Smith et al 2009, Carpenter and Dziminski 2017).…”