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.