Optimizing collection methods for noninvasive genetic sampling of neotropical felids

Abstract

Field-sampling methods for molecular scatology studies must be optimized, especially when working on elusive species in challenging tropical environments where rates of DNA degradation are elevated because of hot and humid weather conditions. To maximize polymerase chain reaction (PCR) amplification success and genotyping accuracy rates and to minimize genotyping error rates for fecal DNA samples of jaguars (Panthera onca) and co-occurring Neotropical felids collected in Belize, Central America, we evaluated the performance of two fecal DNA storage techniques (dimethyl sulfoxide saline solution [DETs buffer] and 95% ethanol [EtOH]) suitable for long-term preservation at remote tropical sites. Additionally, we tested fecal samples collected from 4 different locations on the scat (top, side, bottom, inside) at 2 different tropical forest types (tropical broadleaf and tropical pine forests). DETs buffer was the superior fecal DNA preservation method, with 44% higher PCR amplification success (P = 0.009) and 17% higher genotyping accuracy (P = 0.021) than 95% EtOH-stored samples. Polymerase chain reaction amplification success of fecal DNA collected at the more open, pine-forest (Pinus sp.) site differed significantly across locations on the scat, with highest mean success rates obtained from the top (85% ± 6.5% SD), followed by the side (79% ± 9.4% SD), bottom (76% ± 11.9% SD), and inside (69% ± 10.3% SD) of scat samples. Scat samples collected at the more closed-canopy broadleaf site did not show any significant differences in amplification success rates across scat locations. We recommend that researchers optimize field-sampling methods, including collection and storage protocols, by conducting a pilot study prior to their molecular scatology research efforts.