2.3 Pollen DNA metabarcoding and plant-pollinator networks
The taxonomic composition of the pollen carried on insects’ bodies was
used to retrieve information about their interactions with plants
following a procedure similar to Tommasi et al (2021 a). Briefly, pollen
was recovered from insects by vortexing the tubes containing insects and
ethanol for at least 10 s. Insects were removed from the ethanol and
tubes were centrifuged at 14000 rpm for 10 minutes. Afterwards, the
ethanol was removed through evaporation in a chemical hood. Pollen was
grinded through a Tissue Lyser ® (Qiagen) after being frozen in liquid
nitrogen, then DNA was extracted following DNeasy Plant mini kit ®
(Qiagen) according to manufacturer’s protocol. Negative controls were
also produced for each day of laboratory activity during the DNA
isolation phase. Primers S2F and S3R (Chen et al., 2010) were used with
the addiction of the Illumina overhang sequence adapters to amplify the
internal transcribed spacer 2 (ITS2) region. Illumina standard protocol
was used for Library preparation and sequencing was performed through
Illumina Miseq 600 V3 (2 × 300-bp paired-end sequencing). Raw sequencing
reads were paired using QIIME2 (ver. 2019.4; https://qiime2.org/)
(Bolyen et al., 2019). After primer trimming, removal of chimera and
low-quality reads, a 0.97 clustering was performed, keeping only
features between 200 and 500bp (Torbjørn et al., 2016)
DNA reference sequences of the plant species identified at the study
sites but not available in NCBI GenBank, were obtained by collecting
leaf samples and sequencing the ITS2 region as described in Tommasi et
al. (2021 a) (details on the produced DNA sequences are available in
Supporting information, Table S3). Reads taxonomic assignments were
carried out using the BLAST algorithm (Camacho et al., 2009) on the NCBI
nucleotide database and on the local database including the reference
sequences we produced. Only assignments with a max identity and a query
coverage ≥ 98% were accepted. Species occurrences were filtered by
removing implausible matches (i.e. plants found not to be present in the
study area), and by removing those represented by a reads count lower
than the maximum number of reads (i.e 24 reads) produced by negative
controls (Tommasi et al., 2021 b).
The taxonomic identification of pollen samples was used to retrieve
interactions between plants and pollinators. First, the interaction
matrix between pollinator insects and plants obtained from DNA
metabarcoding was used to calculate the network index of Connectance,
through the R package Bipartite (Dormann, Gruber, & Fründ,
2008). This, calculated as the number of actually observed
interactions divided by the number of the possible interactions (Biella,
Ollerton, Barcella & Assini, 2017), provides an overall estimation of
network specialization and complexity. Furthermore, to evaluate
intraspecific changes in the foraging strategies in response to habitat
fragmentation, we selected three pollinator species belonging to three
different body size classes (estimated measuring inter tegular (IT)
distance of thirty individuals per species) and that were uniformly
distributed across islands: Braunsapis picitarsis . (small, mean
IT 1.3 mm), Lasioglossum albescens (intermediate, mean 1.6 mm),
and Xylocopa fenestrata (large, mean 7.1 mm). Specifically, the
number of plant taxa found in pollen samples retrieved from each
individual pollinator (individual degree) was calculated.