Comparison of mechanical sorting and DNA metabarcoding for diet analysis with fresh and degraded wolf scats

Abstract

DNA metabarcoding has become a powerful technique for identifying the species present in a bulk environmental sample. The application of DNA metabarcoding to wildlife diet analysis is a particularly promising tool for exploring trophic interactions. The extent to which molecular approaches agree with traditional approaches, and how this varies with the quality of field-collected scats, is unknown. Here, we compare diets from wolf scats profiled using both mechanical sorting and metabarcoding of amplified vertebrate DNA sequences. Our objectives were to (1) compare findings from mechanical sorting and metabarcoding as a method of diet profiling and (2) use results to better understand diets of wolves on Prince of Wales Island, a population of conservation concern. We predicted metabarcoding would reveal both higher diversity of prey and identify rare species that are overlooked with mechanical sorting. We found that there was substantial overlap in the diets revealed using both methods, indicating that deer, beaver, and black bear were the primary prey species, but metabarcoding revealed a more diverse diet with greater occurrence of rare species. However, there was a large discrepancy in the occurrence of beaver in scats (52% and 25% from mechanical sorting and metabarcoding, respectively) explained by the high rate of false positives with mechanical sorting methods. While the number of wolf sequence reads for fresh scats was nearly eight times higher than in degraded scats, neither the number of prey sequence reads nor the quantity of DNA to be sequenced varied between fresh and degraded scats suggesting that metabarcoding is sensitive enough to determine prey assemblages in degraded scats. Even using scats from extremely wet conditions hostile to DNA preservation, we found that metabarcoding was more effective than mechanical sorting in describing diet.

Evaluating and integrating spatial capture–recapture models with data of variable individual identifiability

“…Prior to genotyping, the identity of the defecator for each scat was identified using DNA metabarcoding of ˜100 base pairs of the mitochondrial 12S gene region as part of a separate diet study (primers used in Massey et al. [ 2021 ], Eriksson et al. [ 2020 ], and modified from Riaz et al.…”

Temporal variation in translocated Isle Royale wolf diet reflects optimal foraging.

“…However, scat analysis is prone to observer error, can over-represent small prey, assumes constant scat deposition rates, and biomass calculations rely on strong assumptions of carcass use (Spaulding et al ., 2000;Klareat al. , 2011;Massey et al , 2021). Specifically, in IRNP wolves often scavenge and/or partially consume carcasses (see Vucetichet al , 2011), which could bias our biomass calculations.…”

Western spotted skunks provide important food web linkages in forest of the Pacific Northwest

“…In the lab, we identified the diet of western spotted skunks using DNA metabarcoding (Massey et al 2021; Eriksson et al 2019) and mechanical sorting. For DNA metabarcoding, we extracted DNA in a laboratory dedicated to processing degraded DNA using the DNeasy Blood and Tissue kit (Qiagen, Germantown, Maryland) or the QIAamp Fast DNA Stool Mini Kit (Qiagen, Germantown, Maryland).…”

“…Previously, diets of spotted skunks were difficult to study because scats were often deposited in rest sites (Selko 1937; Lesmeister, Gompper, and Millspaugh 2008), not on trails, and because spotted skunks exhibit an omnivorous diet consisting of insects, small vertebrates, and fruit (Baker and Baker 1975; K. R. Crooks and Van Vuren 1995; Crabb 1941; Howell 1906). These scats were typically collected opportunistically, mechanically sorted, and morphologically identified (Ewins et al 1994; Sándor and Ionescu 2009), but these processes had biases related to digestion that may render prey items unrecognizable (Symondson 2002; Galan, Pagès, and Cosson 2012) and misidentification of rare species (Massey et al 2021). This can be particularly problematic for small omnivorous predators that consume a wide breadth of prey items including plants, animals, and invertebrates because identifiers must have taxonomic expertise.…”

“…Moreover, misidentification of carnivores from scat morphology has been problematic and has potentially led to biased results (Morin et al 2016). Newer genetic approaches such as DNA metabarcoding (Ji et al 2013; Monterroso et al 2019) can increase confidence in correctly identifying the carnivore (Morin et al 2016), increase the number of prey items that can be correctly identified (Massey et al 2021), and increase efficiency of identifying diet for a high volume of samples (Kartzinel et al 2015), especially for omnivorous species (De Barba et al 2014).…”