A fecal sequel: Testing the limits of a genetic assay for bat species identification

Abstract

DNA metabarcoding assays are powerful tools for delving into the DNA in wildlife feces, giving unprecedented ability to detect species, understand natural history, and identify pathogens for a range of applications in management, conservation, and research. Next-generation sequencing technology is developing rapidly, which makes it especially important that predictability and reproducibility of DNA metabarcoding assays are explored together with the post-depositional ecology of the target taxon’s fecal DNA. Here, we defined the constraints of an assay called ‘Species from Feces’ used by government agencies, research groups, and non-governmental organizations to identify bat species from guano. We tested assay sensitivity by examining how time and humidity affect the ability to recover and successfully sequence DNA in guano, assessing whether a fecal pellet from a rare bat species could be detected in a background of feces from other bat species, and evaluating the efficacy of Species from Feces as a survey tool for bat roosts in temperate and tropical areas. We found that the assay performs well with feces over two years old in dry, cool environments, and fails by 12 months at 100% relative humidity. We also found that it reliably identifies rare DNA, has great utility for surveying roosts in temperate and tropical regions, and detects more bat species than do visual surveys. We attribute the success of Species from Feces to characteristics of the assay paired with application in taxa that are particularly well-suited for fecal DNA survival. In a time of rapid evolution of DNA metabarcoding approaches and their use with feces, this study illustrates the strengths and limitations of applied assays.

Vampyrum spectrum (Phyllostomidae) movement and prey revealed by radio-telemetry and DNA metabarcoding

“…Both vampire species that we detected fed on blood from domestic birds and cattle [ 53 , 54 ]. Although we did not detect cattle in our samples, Walker et al [ 35 ] genetically identified cattle from D . rotundus feces, so it is not unrealistic to conclude that domestic birds could be either prey of V .…”

“…spectrum [ 2 , 7 , 13 , 17 – 19 ]. The SFF marker, which is optimized for identifying bat species from fecal samples [ 35 , 36 ], followed by the 12S marker [ 33 ], provided the greatest diversity of identifiable vertebrate taxa with 10 species confirmed by both markers. The ANML primer [ 32 ] detected fewer vertebrate taxa; however, it was the only one that detected Megascops cooperi .…”

“…We extracted DNA from homogenized samples using a QIAamp Fast DNA Stool Mini Kit (Qiagen Inc., USA) following manufacturer instructions. We performed initial PCRs using four primers that targeted identification of a range of vertebrates and arthropods: ANML [ 32 ], 12S [ 33 ], 18S [ 34 ] and SFF [ 35 , 36 ]. Each primer set was run on replicates of the 16 samples on its own plate with non-template (NTC) and positive control (PTC) for a total of 72 wells across four plates.…”

Pollen assemblage and environmental DNA changes: A 4300-year-old bat guano deposit from Jamaica

“…Overall, in all fecal DNA metabarcoding studies, the gold standard is to collect fresh feces for maximum DNA recovery. Research carried out by Walker et al (2019) revealed that DNA in bat guano, which can be used for host identification, can be recovered up to at least 2.5 years in dry and cool roosts, but at 100% relative humidity, feces that are younger than 6 months are needed. Moisture, particularly in combination with high temperature, promotes the growth of microbes, and thus further DNA There are two versions of abstract in the manuscript.…”

“…Identification of bats from guano-derived DNA dates back to the early 2000 (Zinck et al, 2004). This approach has been successfully applied to fresh and modern -up to 20 years -samples (e.g., Brown et al, 2017;Walker et al, 2019;Guan et al, 2020). Overall, in all fecal DNA metabarcoding studies, the gold standard is to collect fresh feces for maximum DNA recovery.…”