“Finally, sequences identified at the genus-level were verified not to contain redundant information, i.e., no fully identified sequences of the same genus presented the same TRFLP size.”

This assumption avoids underestimating the resolution power, since many sequences in public databases appear to be insufficiently well identified (Nilsson et al., 2006). However,putative species of genera with low levels of sequence divergence (e.g., Cortinarius, Hebeloma) would be missed too.There are two main reasons why this assumption is finally adopted:

-Insufficiently identifiedredundant species are thought to be more frequent than putative low-divergence species.

-Since this is a conceptual limitation of the TRFLP technique, using a perfectly identified database would slightly reduce every enzyme’s resolution power. The relative results presented here would therefore still hold.

Table 1. Enzymes included in the analysis

Enzyme / Target / Enzyme / Target / Enzyme / Target / Enzyme / Target
AatI / AGG/CCT / BfaI / C/TAG / Eco47III / AGC/GCT / NspI / RCATG/Y
AatII / GACGT/C / BglI / GCCNNNN/NGGC / EcoNI / CCTNN/NNNAGG / NspV / TT/CGAA
AccI / GT/MKAC / BglII / A/GATCT / EcoRI / G/AATTC / PacI / TTAAT/TAA
AccIII / T/CCGGA / BmyI / GDGCH/C / EcoRV / GAT/ATC / PaeR7I / C/TCGAG
Acc65I / G/GTACC / Bpu1102I / GC/TNAGC / Fnu4HI / GC/NGC / PflMI / CCANNNN/NTGG
AcsI / R/AATTY / BsaI / GGTCTCN/ / FseI / GGCCGG/CC / PmeI / GTTT/AAAC
AcyI / GR/CGYC / BsaBI / GATNN/NNATC / FspI / TGC/GCA / PpuMI / RG/GWCCY
AflI / G/GWCC / BsaJI / C/CNNGG / HaeII / RGCGC/Y / Psp1406I / AA/CGTT
AflII / C/TTAAG / BseAI / T/CCGGA / HaeIII / GG/CC / PstI / CTGCA/G
AflIII / A/CRYGT / BsePI / G/CGCGC / HincII / GTY/RAC / PvuI / CGAT/CG
AgeI / A/CCGGT / BsiEI / CGRY/CG / HindIII / A/AGCTT / PvuII / CAG/CTG
AhaIII / TTT/AAA / BsiWI / C/GTACG / HinfI / G/ANTC / RsaI / GT/AC
AluI / AG/CT / BsiYI / CCNNNNN/NNGG / HinPI / G/CGC / RsrII / CG/GWCCG
Alw44I / G/TGCAC / Bsp1407I / T/GTACA / HpaI / GTT/AAC / SacI / GAGCT/C
AlwNI / CAGNNN/CTG / BspDI / AT/CGAT / KasI / G/GCGCC / SalI / G/TCGAC
AocI / CC/TNAGG / BspHI / T/CATGA / KpnI / GGTAC/C / Sau96I / G/GNCC
AosI / TGC/GCA / BspLU11I / A/CATGT / KspI / CCGC/GG / ScaI / AGT/ACT
ApaI / GGGCC/C / BsrFI / R/CCGGY / MaeII / A/CGT / ScrFI / CC/NGG
ApyI / /CCWGG / BssGI / CCANNNNN/NTGG / MaeIII / /GTNAC / SexAI / A/CCWGGT
AscI / GG/CGCGCC / Bst1107I / GTA/TAC / MfeI / C/AATTG / SfcI / C/TRYAG
AseI / AT/TAAT / BstEII / G/GTNACC / MluI / A/CGCGT / SmaI / CCC/GGG
AspI / GACN/NNGTC / BstNI / CC/WGG / MseI / T/TAA / SnaBI / TAC/GTA
Asp700 / GAANN/NNTTC / BstUI / CG/CG / NarI / GG/CGCC / SpeI / A/CTAGT
AspEI / GACNNN/NNGTC / BstXI / CCANNNNN/NTGG / NciI / CC/SGG / SphI / GCATG/C
AspHI / GWGCW/C / BstYI / R/GATCY / NcoI / C/CATGG / SrfI / GCCC/GGGC
AsuII / TT/CGAA / CfoI / GCG/C / NdeI / CA/TATG / Sse8387I / CCTGCA/GG
AvaI / C/YCGRG / CfrI / Y/GGCCR / NgoMI / G/CCGGC / SspI / AAT/ATT
AvrII / C/CTAGG / DdeI / C/TNAG / NheI / G/CTAGC / StyI / C/CWWGG
BalI / TGG/CCA / DpnII / /GATC / NlaIII / CATG/ / SwaI / ATTT/AAAT
BamHI / G/GATCC / DraII / RG/GNCCY / NlaIV / GGN/NCC / TaqI / T/CGA
BanI / G/GYRCC / DraIII / CACNNN/GTG / NotI / GC/GGCCGC / TfiI / G/AWTC
BanII / GRGCY/C / DrdI / GACNNNN/NNGTC / NruI / TCG/CGA / XbaI / T/CTAGA
BbrPI / CAC/GTG / DsaI / C/CRYGG / NsiI / ATGCA/T / XcmI / CCANNNNN/NNNNTGG
BclI / T/GATCA / EagI / C/GGCCG / NspBII / CMG/CKG / XmaI / C/CCGGG

Table 2. Distribution of sequences recovered from INSD among Ascomycete and Basidiomycete clades

Set / Ascomycete / Basidiomycete / Total / % Ascomycete / % Basidiomycete
Sequences / 2419 / 2199 / 4618 / 0.5238 / 0.4761
Species / 850 / 902 / 1752 / 0.4851 / 0.5148
Genera / 271 / 195 / 466 / 0.5815 / 0.4184

No sequences outside the Dicaryomycota clade were recovered because of the entrez string employed.

Table 3. Alternative PEP-sets tested (enzymes ordered)

Set / 1-ENZ / 2-ENZ / 3-ENZ / 4-ENZ / 5-ENZ / 6-ENZ
DIVERSITY-SET / MaeII-14 / MaeII-41 / BfaI-41 / BstNI-14 / RsaI-41 / StyI-41
RICHNESS-SET / DdeI-14 / BfaI-14 / ScrFI-41 / DdeI-41 / BstUI-14 / ApyI-14
6E-SET / MaeII-14 / BfaI-14 / BstNI-14 / RsaI-41 / StyI-41 / DdeI-14

Table 4. Richness results for alternativePEP sets

Set / 2-ENZ / SD / 3-ENZ / SD / 4-ENZ / SD / 5-ENZ / SD / 6-ENZ / SD
DIVERSITY-SET / 1123.3 / 9.476 / 1403.0 / 9.043 / 1514.4 / 8.601 / 1533.9 / 9.527 / 1554.5 / 4.517
RICHNESS-SET / 1373.6 / 9.522 / 1519.4 / 9.854 / 1534.6 / 5.997 / 1556.0 / 9.407 / 1561.6 / 6.965
6E-SET / 1379.4 / 8.287 / 1506.3 / 8.243 / 1539.4 / 4.033 / 1556.4 / 6.706 / 1570 / 7.901

The results in Table 4 show the means and standard deviations of richness for 15 replicate rarefacted databases of the fragments obtained from digesting INSD fungal ITS sequences with each of the PEP sets shown in Table 3. Sequence acquisition and processing followed the methods explained the Material and Methods in the main paper. While ‘diversity’ and ‘6E-SET’ sets are quite similar in composition, their respective average richness values differ substantially for the 2-ENZ and 3-ENZ communities. If more enzymes are used the differences disappear, in accordance with the results shown in the main paper (Fig. 2). Comparing the ‘richness’ and ‘6E-SET’ sets, very similar richness values are obtained, but a generally lower standard deviation is obtained with the ‘6E-SET’ set. This deviation increases dramatically in the ‘6E-SET’ when DdeI-14 (the top-richness PEP) is included (6-ENZ data).

Fig. 1 Dendrogram produced using the UPGMA hierarchical clustering method