Biological replicates were treated independently (N = 5) for each of the 9 time points (week one–six, eight, ten, 16 and the day 0 outgroup for a total of 46 data points). Total extracted nucleic acids were RNAse A treated in triplicate. The ribosomal 16S rRNA gene V4 region was targeted with primers, 515f-Y GTGYCAGCMGCCGCGGTAA (5′–3′) [44] and 806R GGACTACNVGGGTWTCTAAT (5′–3′) [45]. The internal transcribed region 2 (ITS2) region was targeted with primers, fITS7 GTGARTCATCGAATCTTTG (5′–3′) [46] and ITS4ngs TCCTSCGCTTATTGATATGC (5′–3′) [47]. Cluster identification was enhanced with a random dodecamer sequence NNNHNNNWNNN (5′–3′) prepended to the forward primer [48].

16S rRNA gene polymerase chain reactions (PCR) were performed in 25 μL volumes containing 200 μM dNTPs, 0.5 μM 515fY-MN, 0.5 μM 806rMN, 50 ng gDNA, 0.5 U Phusion HF polymerase (#M0530) and 1x Phusion HF Buffer. Thermocycling conditions included an initial denaturation at 98 °C for 30 s, followed by 28 cycles of 98 °C for 10 s, 53 °C for 30 s and 72 °C for 15 s and 72 °C for 10 min. ITS2 PCR were performed as above with thermocycling conditions including an initial denaturation at 98 °C for 30 s, followed by 34 cycles of 98 °C for 10 s, 57 °C for 30 s and 72 °C for 20 s, with final extension at 72 °C for 5 min. Indexing was performed using the Nextera XT™ library preparation kit (Illumina FC-131-1001). The libraries were pooled in equimolar concentrations to 4 nM, spiked to 1% PhiX and run on a MiSeq 250 bp × 2 cartridge (MiSeq Reagent Kit v2 (500 cycles) MS-102-2003, Illumina).

The generated 16S rRNA genes libraries averaged 54,929 sequences. Fastq merging was performed with Vsearch version 1.11.1 [49]. The generated ITS2 libraries averaged 50,843 sequences and were processed with ITSx [50] to filter non-fungi sequences. The resulting fungi only ITS2 libraries averaged 28,972 sequences. The primer sequences were trimmed using Cutadapt (version 1.11.). Sequences were trimmed to global lengths of 250 bp using Usearch (version 9, -fastx_truncate) [51]. Amplicon profiles were dereplicated, purged of singletons, assigned abundance and sorted by size using Usearch (version 7, -derep_fulllength) [51]. Clustering was performed using the UPARSE algorithm [52], with concurrent de novo chimaera detection using Usearch (version 9, -cluster_otus) with a 97% identity threshold resulting in 5122 non-chimeric operational taxonomic units (OTUs) that were taken forward for analysis. Representative sequences for each OTU were then mapped to original sequences using Usearch (version 7, -usearch_global). Taxonomy was assigned with QIIME [53] (version 1.9, assign_taxonomy.py) using SILVA 132 [35] for the 16S rRNA libraries and UNITE v7.1 [54]. Rarefaction analysis [53] displayed curves that begin to reach asymptotic levels, indicating sufficient depth for analysis but not complete diversity coverage (Additional file 1: Figure S2). The taxonomy of any unassigned OTUs (N = 610), using UNITE in the ITS2 libraries were further classified using BLASTn against the GenBank non-redundant nucleotide database. Non-fungal OTUs were discarded and missing taxonomies of on target OTU sequences were manually curated (N = 393) resulting in a total of 920 fungal OTUs which were subsequently analysed. Fungal OTUs were classified into functional guilds using FUNGuild [55] which assigned a functional guild to 419 OTUs from 724 matches of the original 920, this represented 51.4 ± 2.12% mean OTU abundance across all time points and was taken forward for analysis. All commands for the analysis pipeline are available in Additional file 1: Table S3.

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