Bioinformatics
Sequences were sorted according to samples and adapters (identification
tags) were removed in Galaxy (https://main.g2.bx.psu.edu/root).
Primer sequences were removed and poor-quality ends were trimmed based
on a 0.02 error probability limit in Geneious Pro 5.6.1 (BioMatters, New
Zealand). Sequences were filtered with USEARCH v.8.0 (Edgar 2010) with
the following settings: all sequences were truncated to 200 bp and
sequences with expected error > 1 were discarded. For each
sample, identical sequences were collapsed into unique sequence types
while preserving their counts. The quality-filtered sequences from all
samples were grouped into operational taxonomic units (OTUs) at 97%
sequence similarity in USEARCH, and global singletons and putative
chimeric sequences were removed. We assigned sequences to taxonomic
groups based on pairwise similarity searches against the curated
UNITE+INSD fungal ITS sequence database (released August 22, 2016),
containing identified fungal sequences with assignments to Species
Hypothesis groups based on dynamic similarity thresholds (Kõljalget al . 2013). We excluded OTUs with < 80% similarity
or < 150 bp pairwise alignment length to a fungal reference
sequence. To minimize artifactual OTUs that may have been generated
during the molecular work, in subsequent analyses we only included OTUs
that occurred in at least two samples. The final dataset contained 20
620 fungal OTUs, representing a total of 3 962 204 high-quality
sequences.
We assigned fungal OTUs to functional groups in two steps. First, we
assigned putative functional guilds using FunGuild (Nguyen et al .
2015). We recognize the limitations of functional inference based on
partial ITS sequences, and here use these guilds as hypothetical
functional groups. Secondly, for genera that comprise species from
multiple functional guilds (e.g., Amanita , Entoloma ,Ramaria , Sebacina and related genera in the Sebacinales),
we assigned ecological function to each OTU on a case by case basis with
available ecological information for the matching Species Hypothesis in
the UNITE database. Fungi that can grow as saprotrophs as well as animal
or plant pathogens were designated as animal or plant pathogens,
respectively, to acknowledge the ecological distinction from purely
saprotrophic fungi. Similarly, we separated fungi capable of degrading
complex carbohydrates of wood from generalist saprotrophs that generally
can utilize only simple sugars. In total, functional assignments were
made for 13 356 OTUs (ca. 65%). The quality-filtered and rarefied
dataset contained 8720, 7829, and 4498 fungal OTUs in the samples from
Argentina, Borneo, and Panama, respectively, of which 5917, 5078, and
2896 OTUs were assigned to functional groups, respectively. Targeted
Locus Study projects corresponding to the three study regions have been
deposited at DDBJ/EMBL/GenBank under accessions KDPX00000000
(Argentinian Yungas), KDPY00000000 (Malaysian Borneo), and KDPZ00000000
(Panama). The versions described in this paper are the first versions,
i.e. KDPX01000000, KDPY01000000, and KDPZ01000000, respectively.