Identification of plant DNA sequences from bat faecal samples
We initially filtered all sequences for quality and excluded low quality
sequences where the PHRED score was <30 as indexed on the
Barcode of Life Data Management System (BOLD) (Ratnasingham & Hebert,
2007). We compared the obtained rbc L and ITS2 sequences with the
reference libraries of GenBank and BOLD using the BLAST algorithm with
default search parameters (Altschul, Gish, Miller, Myers, & Limpman,
1990) and the combined BLAST and Hidden Markov Model methods implemented
by the BOLD server (Ratnasingham & Hebert, 2007). For each reference
database (BOLD, GenBank), we assigned query sequences to taxon based on
highest percentage similarity, and considered a threshold of ≥97% to be
a reliable assignment (Lamb, Winsley, Piper, Freidrich, & Siciliano,
2016). When there was an agreement between species-level matches for
both markers (rbc L and ITS2) in both databases, with at least one
match >97%, we assigned to the level of species. In cases
where the query matched with equal similarity to multiple taxa of the
same genus, we assigned the taxon to the level of the genus only, and
similarly we used the same approach to assign query sequences to the
level of the family. Where rbc L and ITS2 sequences matched
different species from different genera, both at >97%, we
concluded that two taxa were present in the sample and therefore
assigned to both genera. Query sequences that did not show significant
similarity to a reference were excluded from the analysis.
To corroborate our species assignments, for each candidate genus match,
we reconstructed a gene phylogeny in which we included our query
sequences together with all available reference sequences from species
of the same genus present in BOLD that are also known to occur in Costa
Rica. Sequences from rbc L and ITS2 of each plant genus were
aligned with ClustalW (Larkin et al. 2007) in BioEdit v7.2.5 (Hall,
1999). For each alignment we ran a model selection test to check which
would be the best method to build the phylogenetic tree based on the
lowest BIC value. We ran model selection and built the phylogenetic
trees using MEGA 6.06 (Tamura, Stecher, Peterson, Filipski, & Kumar,
2013). These phylogenies (not shown) recovered paraphyletic groupings
for some species, perhaps through a lack of reference material, and
therefore such species assignments were considered unreliable. To
address this issue, we took a conservative approach and reduced all data
to genus-level designations and repeated our analyses to check for
consistency of results (see Supplementary material).
The identification of plant DNA sequences from bat faecal samples during
the normal year relied on GenBank and BOLD, with the exception of thetrn H-psb A region which was not searchable within BOLD (see
Clare et al. 2019 for more details) for our purposes we used the
assignments as given in Clare et al. (2019).