Evaluation of Cottontail Pellets Collected in Suboptimal Conditions for DNA Analysis

Abstract

Occupancy monitoring of the New England cottontail (Sylvilagus transitionalis) relies on collections of fecal pellets made following a snowfall, and subsequent genetic screening to distinguish New England cottontail pellets from those of the eastern cottontail (S. floridanus) and snowshoe hare (Lepus americanus). In years when snowy conditions are not common, less frequent sampling may result in data gaps at long‐term monitoring sites, and jeopardize ongoing field research projects that rely on genetic data from fecal pellets. Such conditions occurred in the lower Hudson Valley, New York, USA, in the winter of 2015–2016, during which we collected pellets from the ground to evaluate performance of genetic testing relative to pellet quality. Pellets were categorized on overall appearance of degradation, assigned a pellet quality score, and then genetically analyzed to identify species using mitochondrial DNA. The pellets identified as being from New England cottontail were further evaluated targeting nuclear microsatellite DNA to obtain a multilocus genotype to identify individual rabbits. Species identification was possible for 89% of the highest quality pellets from the ground, and 68% in the most degraded pellets. A complete multilocus genotype was obtained in 79% of the least degraded pellets, but only 14% of the most degraded pellets. Certain genetic loci strongly influenced this success rate, with some performing better than others. Pellets collected from snow were still most reliable with 100% identified to species, and >97% of New England cottontail with a complete multilocus genotype. Thus, if researchers are genotyping rabbit pellets for applications such as abundance estimations, only the higher quality pellets from the ground or those collected from snow will likely be useful. For occupancy monitoring, a wider range of pellets may be acceptable for species identification, with the understanding that there is a tradeoff with pellet quality and successful DNA analysis. These results indicate that sampling from the ground and qualitative assessment of pellet quality using our categorization scheme is a viable method for New England cottontail monitoring in years with less snow. © 2020 The Wildlife Society.

Landscape context and spatial attributes matter for New England cottontail occupancy

“…We estimated occupancy and metapopulation parameters for New England and eastern cottontails from presence‐absence data collected as part of the Regional Monitoring Program from range‐wide winter pellet surveys during 2016–2020 (Kovach et al 2003, Brubaker et al 2014, Rittenhouse and Kovach 2020, Whipps et al 2020). Sampling occurred over the 4‐winter sampling period, with 21 sites sampled every winter and 149 sites only in certain winters: 74 sites sampled in 2016–2017, 109 sites sampled in 2017–2018, 104 sites sampled in 2018–2019, and 74 sites sampled in 2019–2020.…”

“…Observers chose the direction of the transects within a site, so direction could differ between sites, but the transect direction was consistent within sites. After collection, observers sent samples to University of Rhode Island and University of New Hampshire laboratories for species identification through fecal mitochondrial DNA analysis (Litvaitis and Litvaitis 1996, Kovach et al 2003, Whipps et al 2020). From the sample location data, we created presence‐absence data sets for both species.…”

“…We incorporated snow depth (cm), air temperature (°C), days since the last high wind event (days wind), and days since the last snowfall (days snow) into the detection process of our models because these factors influence cottontail sample detection and quality (Kovach et al 2003, Brubaker et al 2014, Whipps et al 2020). Snow‐covered ground and air temperature can be important for maintaining the quality of samples, ensuring DNA can be extracted and species can be identified (Kovach et al 2003, Whipps et al 2020). Biologists were able to extract DNA from most samples (~99% of samples), but if DNA was not extracted, we excluded the samples from the analysis.…”

Habitat management to reduce competitive interactions: case study of native and invading cottontails

“…We marked individuals with a uniquely numbered metal ear tag and used a scalpel to collect an approximately 0.3‐cm × 0.3‐cm tissue sample from an ear for species confirmation, which we stored in 100% ethanol for genetic analysis. We extracted DNA for species identification from biopsied tissue using the Qiagen DNeasy Blood and Tissue Kit (Qiagen, Germantown, MD, USA) following the methods described by Whipps et al (2020), and implementing methods from earlier studies (Litvaitis and Litvaitis 1996, Kovach et al 2003, Kilpatrick et al 2013). We conducted confirmation of species identification on a subset of samples and any ambiguous results by DNA sequencing as described by Whipps et al (2020).…”

“…We extracted DNA for species identification from biopsied tissue using the Qiagen DNeasy Blood and Tissue Kit (Qiagen, Germantown, MD, USA) following the methods described by Whipps et al (2020), and implementing methods from earlier studies (Litvaitis and Litvaitis 1996, Kovach et al 2003, Kilpatrick et al 2013). We conducted confirmation of species identification on a subset of samples and any ambiguous results by DNA sequencing as described by Whipps et al (2020).…”