DOI: 10.5061/dryad.vdncjsxvh
title: Abundance of fungal species in slug feces collected in a temperate forest

description: In the DNA barcoding study, a total of 288,630 OTU sequences were
 recovered from feces of eight field-captured slugs. Major taxa were
 Ascomycota (52.5%), Basidiomycota (46.1%), Mortierellomycota (0.2%),
 Chytridiomycota (0.1%) and Mucoromycota (0.1%) (Table 2). In
 Basidiomycota, 17 orders were detected (Table 2). The dominant order was
 Agaricales (66.1%), followed by Trichosporonales (29.7%) and
 Hymenochaetales (2.9%). In Agaricales, the dominant genera were Armillaria
 (35.7%) and Gymnopilus (29.8%). The dominance of Agaricales spores may be
 due to that the slugs used in this study were collected in September and
 October when fruiting bodies of Agaricales were abundant.
description: For species identification of fungal spores in slug’s feces, DNA
 barcoding analysis was performed. Eight <i>M.</i><i>
 fruhstorferi</i> individuals collected in the census in September
 and October in 2017 were individually placed in plastic cups and kept
 under the same conditions as above to allow excretion. After 24 h, 2 g of
 excreted fecal samples were collected into two 1.5 ml centrifuge tubes and
 stored in a freezer at -30°C.  DNA barcoding by
 amplicon sequencing Fecal samples
 were lyophilized on a VD-250R Freeze Dryer (TAITEC Inc., Saitama, Japan)
 and crushed using Shake Master Neo (BMS Inc., Tokyo, Japan). DNA was
 extracted from samples using the MPure Bacterial DNA Extraction Kit (MP
 Bio Inc., Tokyo, Japan). Library preparation was performed by a two-step
 trail PCR method using ExTaq (Takara Bio Inc., Shiga, Japan); the primers
 for 1st PCR were 1st_ITS1-F_KYO1 and 1st_ITS2_KYO2 (Toju et al. 2012) and
 the primers for 2nd PCR were 2nd-F and 2nd-R. The quality of the prepared
 libraries was then checked using Fragment Analyzer and DNA915 Reagent Kit
 (Advanced Analytical Technologies Inc., Iowa, U.S.A). The amplicon
 sequencing analysis was outsourced to Bioengineering laboratory Inc.
 (Kanagawa, Japan). Sequencing analysis was performed using the MiSeq
 Genome Sequencer (Illumina, CA, USA) under 2x300 bp condition. We
 extracted sequences whose beginning was a perfect match to the primer
 using fastq_barcode_splitter in the Fastx toolkit. We then used SICKLE
 TOOLS to remove sequences with quality values less than 20 and discarded
 sequences that were less than 40 bases long and their paired sequences.
 For lead merging, the paired-end merge script FLASH was used to merge 320
 nucleotides of the post-merge fragment length, 280 nucleotides of the lead
 fragment length, and ten nucleotides of the minimum overlap length. The
 sequences being failed to be merged were extracted, 50 bases on the
 3' side of both strands were deleted and merged again. We performed
 two more rounds of the same work. The reads obtained from the four merging
 operations were joined and analyzed in the following steps. Chimera checks
 were performed by adapting USEARCH's UCHIME algorithm for all
 filtered through sequences. The database was 97% UNITE OTU, and all
 sequences that were not determined to be chimeric were extracted. OTU
 creation and phylogenetic inference were performed using a workflow script
 from Qiime under default conditions. OTUs with less than 10 sequences were
 excluded from the analysis. OTUs were divided into Basidiomycota,
 Ascomycota and other taxa.