Species From Feces: Order-Wide Identification of Chiroptera From Guano and Other Non-Invasive Genetic Samples

<div><p>Bat guano is a relatively untapped reservoir of information, having great utility as a DNA source because it is often available at roosts even when bats are not and is an easy type of sample to collect from a difficult-to-study mammalian order. Recent advances from microbial community studies in primer design, sequencing, and analysis enable fast, accurate, and cost-effective species identification. Here, we borrow from this discipline to develop an order-wide DNA mini-barcode assay (Species from Feces) based on a segment of the mitochondrial gene cytochrome c oxidase I (COI). The assay works effectively with fecal DNA and is conveniently transferable to low-cost, high-throughput Illumina MiSeq technology that also allows simultaneous pairing with other markers. Our PCR primers target a region of COI that is highly discriminatory among Chiroptera (92% species-level identification of barcoded species), and are sufficiently degenerate to allow hybridization across diverse bat taxa. We successfully validated our system with 54 bat species across both suborders. Despite abundant arthropod prey DNA in guano, our primers were highly specific to bats; no arthropod DNA was detected in thousands of feces run on Sanger and Illumina platforms. The assay is extendable to fecal pellets of unknown age as well as individual and pooled guano, to allow for individual (using singular fecal pellets) and community (using combined pellets collected from across long-term roost sites) analyses. We developed a searchable database (<a href="http://nau.edu/CEFNS/Forestry/Research/Bats/Search-Tool/" target="_blank">http://nau.edu/CEFNS/Forestry/Research/Bats/Search-Tool/</a>) that allows users to determine the discriminatory capability of our markers for bat species of interest. Our assay has applications worldwide for examining disease impacts on vulnerable species, determining species assemblages within roosts, and assessing the presence of bat species that are vulnerable or facing extinction. The development and analytical pathways are rapid, reliable, and inexpensive, and can be applied to ecology and conservation studies of other taxa.</p></div

Bat COI mini-barcode primers designed and tested in this study.

<p>A) Primer sequences; B) PCR amplicon length (bp) and annealing temperature (Ta) for each primer combination. Forward and reverse primers are indicated by f and r, respectively. After lab testing, we selected primer pair SFF_145f/SFF_351r for this study.</p

Laboratory testing of the Species from Feces mini-barcode assay across sample types of 54 <i>a priori</i> identified bat species.

<p>Laboratory testing of the Species from Feces mini-barcode assay across sample types of 54 <i>a priori</i> identified bat species.</p

Multiple bat species detected in each of eight mines in the U.S. Southwest from next-generation amplicon sequencing of pooled guano.

<p>A single DNA extraction was performed on a pooled sample of roughly 200 fecal pellets that were collected from each mine, and the Species from Feces primer pair SFF_145f/SFF_351r was applied with next generation amplicon sequencing on an Illumina MiSeq. *Mine sample NM_1 also contained small proportions of <i>Corynorhinus townsendii</i> and <i>Myotis thysanodes</i>/<i>evotis</i>.</p

Taxonomic resolution provided by the Species from Feces DNA mini-barcode for bats in each geographic region.

<p>Of the species for which COI barcodes are available, 92% can be identified to species, and the remaining share a barcode with other congeners (5%) or across genera (3%). Pie sizes correspond to number of bat species in each geographic region. The dark grey slice includes species not yet barcoded and species for which barcodes are not publically available.</p

<i>In silico</i> taxonomic coverage by genus, comparing bat-specific COI primer pairs developed in the current study, those previously utilized, and all available sequences.

<p>We used all publically available barcode sequences for Chiroptera in BOLD and dereplicated them at full length to alleviate taxonomic overrepresentation. Bar components labeled Chiroptera denote bat barcodes from BOLD that were not given taxonomic identifiers below the level of Order.</p

Evaluation of taxonomic classification of reference library sequences.

<p>The primer-targeted regions of reference library sequences were classified using the RDP Classifier (in QIIME). We evaluated six datasets: unique amplicon regions against the full reference library (F), and five random subsamples of the reference library (1–5) (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162342#sec002" target="_blank">Methods</a>). Classifications with the RDP Classifier were computed with confidence thresholds of 0.5–1.0, and performance was evaluated at the Genus (A) and Species (B) taxonomic level. The percentage of sequences correctly classified, and percentages of false positives and false negatives are displayed. We chose a confidence threshold of 0.8 for classification of bat sequences as this value was deemed to be an acceptable tradeoff between the number of correctly classified sequences and the risk of false positives.</p

Supplemental Material for Dumont et al., 2018

Supplemental figures and tables associated with manuscript entitled: Evolutionary association of sex chromosome architecture and meiotic synapsis in North American vole