Molecular diet analysis
Field and lab methods To examine bird diets, between April-June
2016 we caught 103 birds and successfully collected 41 faecal samples at
200m and 2000m elevations at the Bengal sites (Supplementary table S2).
We caught an additional 107 birds and collected 82 faecal samples at
1200m, 2300m, 2700m and 3200m elevations in May 2017. All faecal samples
were stored in 95% ethanol until DNA extraction. We used Qiagen QIAamp
DNA Stool Mini kit to extract DNA with some minor modifications in the
protocol, following Zeale et al. (2011).
To examine weaver ant diet, we collected items of food that the weaver
ants were carrying to their nest between April-June 2016. We collected
food items from 25 different colonies, either for an hour or until we
had collected 10 items, whichever happened first. We extracted DNA from
these samples by taking a small part of each of the prey items, crushing
it and then using the Qiagen DNeasy blood and tissue kit and following
the manufacturer’s protocol for DNA extraction. We carried out a 25μL
PCR (polymerase chain reaction) to amplify a segment of 16S rDNA using
the lns16S-short primer pair (5’-3’ TRRGACGAGAAGACCCTATA;
ACGCTGTTATCCCTAAGGTA) described by Clarke et al. (2014). We used this
primer set because it is invertebrate specific, so successful DNA
amplification indicates presence of prey DNA in the bird faecal DNA
extract. We sent successful PCR products to the Sequencing core at the
University of Illinois at Chicago, Chicago, Illinois, for barcoding,
pooling, size selection and sequencing on an Illumina Miniseq platform.
All PCR products had strong primer dimer bands that were removed at the
sequencing core before sequencing with a procedure that selected for
fragments longer than 150 bp. (see Supplementary methods for further
details)
Data analysis We followed the dada2 pipeline (Callahan et al.
2016) to construct an amplicon sequence variant (ASV) table and then
created a FASTA file with all the unique sequences recovered from our
samples. We used blastn in blastplus to BLAST this FASTA file against
the NCBI nr database and used the NCBI-taxcollector script (Dias et al.
2014) to get detailed taxonomic information for the top hit of our BLAST
results. After filtering out bacteria and other contaminating sequences,
we estimated overlap between the diets of weaver ants and birds at low
and mid-elevations at various taxonomic levels using EcoSimR (Gotelli et
al. 2015) and visualized the overlap using venn diagrams and non-metric
multidimensional scaling (NMDS) in R (R Core Team 2014). EcoSim R is
used to estimate niche overlap between species and compare it to a null
distribution of niche overlap given information on resource utilization
(in columns) by each species (rows). The algorithm randomizes resource
utilization for each species by reshuffling the row values and generates
a null distribution of niche overlap. In our case, we considered birds
at low elevations (N = 18), birds at middle elevations (N = 15) and
weaver ants (N = 25) at low elevations as “species” and used the
frequency of occurrence of different orders or families in diets as the
“resources”. All the analyses were done in the R programming
environment and in the shell using the R packages dada2 (Callahan et al.
2016), DECIPHER (Wright 2016), VennDiagram (Chen and Boutros 2011),
ggplot2 (Wickham 2011), vegan (Oksanen et al. 2018) and EcoSimR (Gotelli
et al. 2015). All scripts and details of the steps are available as
supplements to this paper.