Mori et al. Supporting information S1
1
DNA extraction, PCR amplification, and pyrosequencing
2
Fungal communities in soil samples from 72 subplots collected in late May and late October
3
were analyzed. Whole DNA was extracted from individual soil samples (0.25 g wet weight per
4
sample) using Soil DNA Isolation Kit (Norgen Biotek, Thorold, Ontario, Canada) following
5
manufacturer’s instructions. For direct 454 sequencing of the fungal internal transcribed spacer
6
1 (ITS1), we used a semi-nested PCR protocol. The ITS region has been proposed as the formal
7
fungal barcode (Schoch et al. 2012). First, the entire ITS region and 5’ end region of LSU were
8
amplified using the fungus-specific primers ITS1F (Gardes & Bruns 1993) and LR3 (Vilgalys
9
& Hester 1990). PCR was performed in a 20 μl volume with the buffer system of KOD FX
10
NEO (TOYOBO, Osaka, Japan), which contained 1.6 μl of template DNA, 0.3 μl of KOD FX
11
NEO, 9.0 μl of 2× buffer, 4.0 μl of dNTP, 0.5 μl each of two primers (10 μM), and 4.1 μl of
12
distilled water. The PCR conditions were as follows: an initial step of 5 min at 94°C; followed
13
by 20 cycles of 30 s at 95°C, 30 s at 58°C for annealing, and 90 s at 72°C; and a final extension
14
of 10 min at 72°C. The PCR products were purified using ExoSAP-IT (GE Healthcare, Little
15
Chalfont, Buckinghamshire, U.K.) and diluted by adding 225 μl of sterilized water. The second
16
PCR was then conducted targeting the ITS1 region using the ITS1F fused with the 454 Adaptor
17
A (5'-CCA TCT CAT CCC TGC GTG TCT CCG ACT CAG-3') and the eight base-pair DNA
18
tag (Hamady et al. 2008) for post-sequencing sample identification and the reverse universal
19
primer ITS2 (White et al. 1990) fused with the 454 Adaptor B (5'-CCT ATC CCC TGT GTG
20
CCT TGG CAG TCT CAG-3'). PCR was performed in a 20 μl volume with the buffer system
21
of KOD FX Plus NEO (TOYOBO), which contained 1.0 μl of template DNA, 0.2 μl of KOD
22
Plus NEO (TOYOBO), 2.0 μl of 10× buffer, 2.0 μl of dNTP, 0.8 μl each of the two primer (5
23
μM), and 13.2 μl of distilled water. The PCR conditions were as follows: an initial step of 5
24
min at 94°C, followed by 20 cycles of 30 s at 95°C, 30 s at 60°C, 90 s at 72°C and a final
25
extension of 10 min at 72°C. PCR products were checked with agarose gel electrophoresis,
1
Mori et al. Supporting information S1
26
purified with ExoSAP-IT and quantified with Nanodrop. Amplicons were pooled into two
27
libraries and purified using an Agencourt Ampure XP kit (Beckman Coulter, Brea, CA, USA).
28
The pooled products were sequenced following the manufacturer’s instructions in a sequencing
29
reaction of a GS Junior sequencer (Roche) at the Wildlife Research Center of Kyoto University,
30
Japan.
31
32
Bioinformatics
33
A total of 147,170 reads were obtained from pyrosequencing. The full dataset of the runs was
34
deposited in the Sequence Read Archive of DNA Data Bank of Japan (accession: DRA003024).
35
The pyrosequencing reads were trimmed with a minimum quality value of 27 at the 3' tails
36
(Kunin et al. 2010), and the trimmed reads were sorted into individual samples using the
37
sample-specific tags. The 5'- and 3'-primer sequences and tag sequences were then removed
38
from the sorted reads. Of the sorted reads, those that had sequence lengths shorter than 150 bp
39
were excluded, and those longer than 380 bp were shortened to 380 bp by removing bases from
40
the 3'-end. The remaining 108,508 reads were assembled using Assams assembler
41
v0.1.2013.08.10 (Tanabe 2013), a highly parallelized extension of the Minimus assembly
42
pipeline (Sommers et al. 2007). First, in each sample, reads with 99.5% similarity were
43
assembled and singletons were removed, which potentially represented chimeric sequences and
44
pyrosequencing errors. Potentially chimeric sequences were eliminated using UCHIME v4.2.40
45
(Edgar et al. 2001) with a relatively rigorous chimera check option (the minimum score for
46
chimera reporting was 0.8). Pyrosequencing errors were eliminated using an algorithm in CD-
47
HIT-OTU (Li et al. 2012) with an error reporting value of 0.8 (Assams default value). After
48
these filtering procedures, a total of 62,477 reads remained. The number of sequencing reads
49
per plot ranged from 116 to 912 (437±181, mean±S.D.). All sequences were assembled across
50
the subplots using Assams at a threshold similarity of 97%, a value which is widely used for
2
Mori et al. Supporting information S1
51
fungal ITS region (Osono 2014). The resulting consensus sequences represented molecular
52
operational taxonomic units (OTUs).
53
To systematically annotate the taxonomy of the OTUs, we used Claident
54
v0.1.2013.08.10 (Tanabe & Toju 2013), which was constructed using an automated BLAST-
55
search by means of BLAST+ (Camacho C et al. 2009) and the NCBI taxonomy-based sequence
56
identification engine. Using the reference database for taxonomic assignment, homologous ITS
57
sequences of each query were retrieved, and taxonomic assignment was performed based on
58
the lowest common ancestor algorithm (Huson et al. 2007).
59
In total, we recorded 637 fungal OTUs from 144 soil samples. Of these, 428 OTUs
60
were obtained from soil samples collected in late May. In this study, we used the data of 428
61
OTUs from late May to examine the relationship between fungal diversity and
62
multifunctionality. A full species list in the meta-community is archived in DDBJ Sequence
63
Read Archive (http://trace.ddbj.nig.ac.jp/) as submission number of DRA003036.
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
Reference
1.
Camacho C, Coulouris G, Avagyan V, Ma N, Papadopoulos J, Bealer K et al. (2009). BLAST+:
architecture and applications. BMC Bioinformatics, 10, 421.
2.
Edgar, R., Haas, B., Clemente, J., Quince, C. & Knight, R. (2001). UCHIME improves
sensitivity and speed of chimera detection. Bioinformatics, 27, 2194-2200.
3.
Gardes, M. & Bruns, T. (1993). ITS primer with enhanced specificity for basidiomycetes:
application to the identification of mycorrhizae and rust. Molecular ecology, 21, 113118.
4.
Hamady, M., Walker, J., Harris, J., Gold, N. & Knight, R. (2008). Error-correcting barcoded
primers for pyrosequencing hundreds of samples in multiplex. Nature Method, 5, 235237.
5.
Huson, D., Auch, A., Qi, J. & Schuster, S. (2007). MEGAN analysis of metagenomic data.
Genome Research, 17, 377-386.
6.
Kunin, V., Engelbrektson, A., Ochman, H. & Hugenholtz, P. (2010). Wrinkles in the rare
biosphere: pyrosequencing errors can lead to artificial inflation of diversity estimates.
3
Mori et al. Supporting information S1
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
Environmental Microbiology, 12, 118-123.
7.
Li, W., Fu, L., Niu, B., Wu, S. & Wooley, J. (2012). Ultrafast clustering algorithms for
metagenomic sequence analysis. Briefings in Bioinformatics, 13, 656-668.
8.
Osono, T. (2014). Metagenomic approach yields insights into fungal diversity and functioning.
In: Species Diversity and Community Structure: Novel Patterns and Processes in Plants,
Insects, and Fungi. . Springer, Berlin.
9.
Schoch, C., Seifert, K., Huhndorf, S., Robert, V., Spouge, J., Levesque, C. et al. (2012). Nuclear
ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker
for Fungi. Proc Natl Acad Sci U S A, 109, 6241-6246.
10.
Sommers, D., Delcher, A., Salzberg, S. & Pop, M. (2007). Minimus: a fast, lightweight genome
assembler. BMC Bioinformatics, 8, 64.
11.
Tanabe, A. (2013). Claident v0.1.2013.08.10.
12.
Tanabe, A. & Toju, H. (2013). Two new computational methods for universal DNA barcoding:
A benchmark using barcode sequences of bacteria, archaea, animals, fungi, and land
plants. Plos One, 8, e76910.
13.
Vilgalys, R. & Hester, M. (1990). Rapid genetic identification and mapping of enzymatically
amplified ribosomal DNA from several Cryptococcus species. Journal of Bacteriology,
172, 4238-4246.
14.
White, T., Bruns, T., Lee, S. & Taylor, J. (1990). Amplification and direct sequencing of fungal
ribosomal RNA genes for phylogenetics. In: PCR Protocols: a Guide to Methods and
Applications (eds. Innis, M, Gelfand, D, Sninsky, J & White, T). Academic Press New
York, USA, pp. 315-322.
4