DNA extraction, PCR and sequencing
For this study, we focused on nectar and fruit eating species, which
produced faecal samples consisting of either seeds or digested fruit
pulp. For the DNA extraction, PCR and sequencing of the samples we
followed standard protocols for plants and all works was conducted by
the Canadian Centre for DNA barcoding (CCDB) following these procedures
(Ivanova, Kuzmina, & Fazekas, 2011). In brief, dried plant material
from faeces (fruit pulp or seed) was placed in a sterile strip-tube with
pre-aliquoted sterile stainless steel beads and the tissue was ground
using a Tissue Lyser (Qiagen, USA). The ground material was incubated
with 2x CTAB buffer at 65°C for 1 hour and DNA extraction was performed
using a semi-automated glass fiber filtration method (Ivanova, Fazekas,
& Hebert, 2008; Fazekas et al. 2012). Following established methods, we
amplified a 552 bp fragment of the 5’ end of the large subunit of
RuBisCO (rbc L) and the ~350 bp second nuclear
encoded internal transcribed spacer (ITS2) flanking by the partial 5.6S
and 26S ribosomal genes. Sanger sequencing was performed using a ABI
3730xl capillary sequencer (Ivanova, DeWaard, Hajibabaei, & Hebert,
2005; Ivanova & Grainger, 2006; Kuzmina & Ivanova, 2011a; Kuzmina &
Ivanova, 2011b; Fazekas et al. 2012). Although plant DNA barcoding
yields lower species resolution compared to fungi and animals
(Hollingsworth, Graham, & Little, 2011), generally it provides robust
results for identification of vascular plants at the genus level (Kress
et al. 2009; Parmentier et al. 2013; Braukmann, Kuzmina, Sills,
Zakharov, & Hebert, 2017). For the samples from the normal year, plant
specimens collected on the sites were identified using rbcL andmatK and the supplementary non-coding plastid regiontrnH-psbA (see Clare et al. 2019 for full methods).