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Abundance of fungal species in slug feces collected in a temperate forest


Tuno, Nobuko (2022), Abundance of fungal species in slug feces collected in a temperate forest, Dryad, Dataset,


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.


For species identification of fungal spores in slug’s feces, DNA barcoding analysis was performed. Eight M. fruhstorferi 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.