2.4 DNA metabarcoding analysis and primer selection
The stomach, cecum, and 12 consecutive segments of the colon were sent
to MrDNA (www.mrdnalab.com) for DNA
extraction and metabarcoding analysis. We identified and discarded false
positives using extraction blanks and multiple PCR replicates to avoid
noise from spurious amplification (Taberlet et al., 2018, Table 1). We
used the following primer pairs for specific taxonomic groups (Table 1):
for plants, we amplified (i) the P6 loop of the chloroplast trnL (UAA) intron using primers P6-trnLF: 5’-GGG CAA TCC TGA GCC AA-3’ and
p6-trnLR: 5’-CCA TTG AGT CTC TGC ACC TAT C-3’ (Taberlet et al 2007), and
(ii) the internal transcribed spacer 2 (ITS2) of nuclear ribosomal DNA
using primers S2F: 5’- ATGCGATACTTGGTGTGAAT-3’ and S2R: 5’-
GACGCTTCTCCAGACTACAAT-3’ (Chen et al 2010); for eukaryotic algae and
cyanobacteria, we amplified domain V of the 23S plastid rRNA gene using
primers p23SrV_f1 5’-GGA CAG AAA GAC CCT ATG AA-3’ and p23SrV_r1
5’-TCA GCC TGT TAT CCC TAG AG-3’ (Sherwood & Presting, 2007); for fungi
(Ascomycota and Basidiomycota), we amplified the internal transcribed
spacer (ITS) of nuclear ribosomal DNA using primers: ITS1-F 5’-CTT GGT
CAT TTA GAG GAA GTA A-3’ (Gardes & Bruns, 1993) and 5’-GCT GCG TTC TTC
ATC GAT GC-3’ (White et al 1990); for metazoans, we amplified the
cytochrome c oxidase subunit I using primers: mlCOIintF: 5’-GGW ACW GGW
TGA ACW GTW TAY CCY CC-3’ (Leray et al., 2013) and jgHCO2198: 5’-TAI ACY
TCI GGR TGI CCR AAR AAY CA-3’ (Geller et al., 2013); and for
invertebrates, we amplified the cytochrome c oxidase subunit I using
primers: fwhF2: 5’-GGD ACW GGW TGA ACW GTW TAY CCH CC-3’ (Vamos et al.,
2017) and EPTDr2n: 5’-CAA ACA AAT ARD GGT ATT CGD TY-3’ (Leese et al.,
2021).