We evaluated the efficacy of sample collection approaches and DNA metabarcoding to identify plants utilized by nectivorous bats, Leptonycteris yerbabuenae and Choeronycteris mexicana. Samples included guano collected from beneath bat roosts and pollen-swabs from bat fur. Plant-specific markers amplified were rbcL, TrnL, and psbA. Sequencing was conducted on a Roche 454 GS FLX DNA sequencer using titanium chemistry at the University of Floridas Interdisciplinary Center for Biotechnology Research.
DNA was extracted from each guano (N = 130) and pollen-swab (N = 152) sample individually using a modified CTAB extraction protocol. Genomic DNA concentrations (ng/l) were determined with the NanoDrop ND-1000 UV-Vis Spectrophotometer (Nanodrop Technologies), then equimolar concentrations were pooled according to groups (see https://doi.org/10.1139/gen-2018-0102). Library preparation followed the 454 Sequencing System Guidelines for Amplicon Experimental Design. We employed three plant chloroplast DNA barcode regions, rbcL, psbA-trnH, and trnL-F. To enable pooling of samples and markers, we ordered a unique set of the three plant chloroplast primers for each group. To enable sequencing on a Roche 454 GS FLX DNA sequencer, the 5' end of each forward and reverse PCR primer was tagged with the A and B 454 adapter (A: CGTATCGCCTCCCTCGCGCCATCAG, B: CTATGCGCCTTGCCAGCCCGCTCAG), respectively. PCR reactions were conducted in 25uL volumes consisting of 2 U of Platinum Taq DNA polymerase (Invitrogen), 1 TaqDNA Polymerase PCR Buffer without magnesium [containing Tris HCl (pH 8.4), and KCl], 2.0 mM MgCl2, 0.2 mM of each dNTP, 1 uM of both forward and reverse primers, and nuclease-free water. PCR temperature cycling conditions were as follows: (I) 3 min at 95 C, (II) denaturation for 30 s at 95 C, (III) annealing for 30 s at 55 C, (IV) extension for 30 s at 72 C, (V) eight repetitions of steps 24, and (VI) a final elongation at 72 C for 5 min. PCR products were quantified, pooled in equimolar ratios, and submitted for next-generation DNA sequencing using a -plate run with on a Roche 454 GS FLX DNA sequencer using titanium chemistry at the University of Floridas Interdisciplinary Center for Biotechnology Research.