An efficient method for simultaneous species, individual, and sex identification via in‐solution single nucleotide polymorphism capture from low‐quality scat samples

Abstract

Understanding predator population dynamics is important for conservation management because of the critical roles predators play within ecosystems. Noninvasive genetic sampling methods are useful for the study of predators like canids that can be difficult to capture or directly observe. Here, we introduce the FAECES* method (Fast and Accurate Enrichment of Canid Excrement for Species* and other analyses) which expands the toolbox for canid researchers and conservationists by using insolution hybridization sequence capture to produce single nucleotide polymorphism (SNP) genotypes for multiple canid species from scat-derived DNA using a single enrichment. We designed a set of hybridization probes to genotype both coyotes (Canis latrans) and kit foxes (Vulpes macrotis) at hundreds of polymorphic SNP loci and we tested the probes on both tissues and field-collected scat samples. We enriched and genotyped by sequencing 52 coyote and 70 kit fox scats collected in and around a conservation easement in the Nevada Mojave Desert. We demonstrate that the FAECES* method produces genotypes capable of differentiating coyotes and kit foxes, identifying individuals and their sex, and estimating genetic diversity and effective population sizes, even using highly degraded, low-quantity DNA extracted from scat. We found that the study area harbours a large and diverse population of kit foxes and a relatively smaller population of coyotes. By replicating our methods in the future, conservationists can assess the impacts of management decisions on canid populations. The method can also be adapted and applied more broadly to enrich and sequence multiple loci from any species of interest using scat or other noninvasive genetic samples.

Genetic analyses are more sensitive than morphological inspection at detecting the presence of threatened Mojave desert tortoise (Gopherus agassizii) remains in canid scat and raven pellets

“…We collected 341 putative canid scat samples and 34 raven pellets in and around the Boulder City Conservation Easement Area (BCCEA) between September 2015 and April 2018 (Parker et al, 2021; Figure 1). The BCCEA is a 34,800‐hectare area of public land south of Boulder City, Nevada, in the northeastern Mojave Desert within the Eldorado Valley.…”

“…Sampling details are described in detail in Parker et al (2021). Briefly, we used stratified random sampling (Ratti & Garton, 1994) to select 84 sample localities that were at least 1 km from any other sampling point.…”

“…We extracted DNA from scats and pellets using a Mag-Bind ® Stool DNA kit in 96-sample plate format (Omega Bio-Tek, Norcross, CA) following the manufacturer's "Standard Protocol" with modifications described in Parker et al (2021). We then performed a species identification assay (Bozarth et al, 2010) by amplifying a small fragment of the control region (CR) of the mitochondrial genome that is a different length in each sympatric species in the study area.…”

“…As part of a previous study (Parker et al, 2021), we used in-solution DNA hybridization capture followed by high-throughput sequencing to determine multi-locus single-nucleotide polymorphism (SNP) canid genotypes using the confirmed kit fox and coyote canid scat samples. We identified individual canids by calculating pairwise identity-by-state (IBS) and identity-by-descent (IBD).…”

“…By using in-solution hybridization capture, we were able to successfully determine multi-locus SNP genotypes for 17 out of 41 qPCR-positive kit fox and coyote scats, all of which were unique individuals (Parker et al, 2021). For the remaining 24 samples, we cannot rule out the possibility that some of these represent multiple scats produced by a single individual after consumption of a single tortoise.…”