Plant–orthoptera network reconstruction
The reconstruction of plant–insect trophic networks from fecal samples
relies on a DNA metabarcoding procedure that uses a two-step DNA
amplification PCR-based approach in which samples are individually
tagged by dual-indexing. A full protocol of the wet-lab procedure, from
DNA extraction to sequencing, is provided in Supporting information,
Appendix S2, section Methods 1. In short, after DNA extraction from the
insect feces, the ITS2 nuclear plant marker (360bp) was amplified in the
amplicon PCR. We selected this marker based on its ease of
amplification, high taxonomic resolution, good coverage of the reference
database and successful application to degraded DNA samples (Li et
al. 2011; García-Robledo et al. 2013). In parallel to DNA
metabarcoding library preparation and sequencing, we compiled an ITS2
reference database by recovering sequences from Genbank (Clark et
al. 2016). The database was filtered using in-silico PCRs that allow
only one mismatch between the primers and the priming sites. We expanded
the reference database with custom sequences generated for 54% of the
plant species (see Appendix S2, section Methods 2). After processing of
the raw sequencing data, OTU calling and taxonomic assignment against
the DNA reference database, the OTU table was streamlined to reconstruct
individual networks using R (R Core Team 2019; see complete descriptions
of the bioinformatic and OTU table cleaning procedures in Appendix S2,
section Methods 3). We discarded OTUs that were non-monophyletic and/or
identified above the family level, summed the OTUs belonging to the same
taxon, used vegetation surveys to filter out the OTUs with low read
numbers, and redistributed the count of OTUs assigned above the species
level. In a final step, we computed the relative read abundance (RRA)
for each sample as an estimate of the interaction intensity (Deagleet al. 2019, Roslin et al. 2019).