3.2 Metabarcoding
We sequenced a total of 9,487,085 reads as part of the DNA metabarcoding analysis, maintaining 3,196,911 non-chimeric reads after filtering, denoising, and merging reads for all primer combinations and all samples (Table 1, Tables S1-S6). The P6 loop of the chloroplast trnL (UAA) intron marker (Figure 3a,b) yielded an average of 56,507 ± 22,886 (SD) non-chimeric processed reads for each of the dissected sections of the gastrointestinal tract. Consistent with results of the metagenomic analysis, sequences assigned to the family Erythroxylaceae (Erythroxylum novagranatense ) predominated in samples from each section, from the stomach to the C12 portion of the colon (Figure 3a). Sequences of E. novagranatense represent 97.3% of the 886,078 sequences derived from all surveyed sections of the gastrointestinal tract. In addition to representatives of Erythroxylaceae, we detected representatives of Amaryllidaceae (0.4% of the total), Poaceae (0.3%), Pinaceae (0.3%), and Malvaceae (0.1%), and Fabaceae and Juglandaceae, which together accounted for 0.05% of the total (Figure 3b).
The internal transcribed spacer 2 (ITS2) marker yielded an average of 19,952 ± 7,762 non-chimeric reads for each section of the gastrointestinal tract, and Erythroxylaceae accounted for 87.2% of the 279,336 total reads. Sequences from Pinaceae (6.1% of the total), Amaryllidaceae (5.9%), Urticaceae (0.64%), Poaceae (0.15%) were also detected, along with traces of Laureaceae, Fabaceae, Ranunculaceae and Hypericaceae, which together accounted for 0.04% of total reads (Figure 3d).
The third marker that provided information for Streptophyta was domain V of the 23S plastid rRNA gene which yielded 1,596 ± 671 reads on average for all sections of the gastrointestinal tract. Again, Erythroxylaceae predominated, accounting for 98.2% of 22,352 total reads (Figure 3e). In addition to Erythroxylaceae and Amaryllidaceae (1.5% of total reads), the next two most abundant taxa were Cyatheaceae and Brassicaceae, which together accounted for <0.6% of total reads (Figure 3f).
For Fungi, the internal transcribed spacer (ITS) of the nuclear ribosomal DNA marker detected a wide variety of Ascomycota and Basidiomycota orders and families (Figure 4) in the 414,990 read total, with an average of 29,642 ± 12,846 reads for each section of the gastrointestinal tract. For Ascomycota, the most abundant families were Cladosporiacea (28.4% of the total reads for this Phylum), Pleosporaceae (20.2% of the total), Sacharomycetaceae (16.6% of the total) and Nectriaceae (14.1% of the total), while the remaining 24 families represent 20.6% of the total, with Phaeococcomycetaceae and Parmeliaceae (lichen-associated families) accounting for 1.6% of the total. For the Phylum Basidiocomycota, the orders Agaricales (40.7% of total reads for this Phylum) and Polyporales (36.5% of the total) represent the most abundant groups, whereas Psathyrellaceae (18.8% of the total), Polyporaceae (13.6% of the total), Agaricaceae (12.0% of the total), and the families Meripilaceae, Fomitopsidaceae and Hyphodermataceae (together accounting for 16.7% of the total) were the most abundant groups across all sections of the gastrointestinal tract.
For Metazoans the marker cytochrome c oxidase subunit I identified 41 amplicon sequence variants (ASV), all of which were assigned toPhyllotis ; none were derived from vicuña or guanaco. Nine ASV remain uncategorized. Finally, the marker cytochrome c oxidase subunit I specifically developed for arthropods did not detect any ASV for the group.