eDNA-based occurrence dataset on fungi of fine woody debris studied in Northwestern Siberia

Study Extent

To investigate the structure and dynamics of the small woody debris fungal community, we employed a method involving standard substrates and total DNA extraction (metabarcoding) from wood (pine, larch, and birch) in the middle taiga zone of Western Siberia. Plots were established where the substrates were buried in the upper layer of forest litter in four habitat types: coniferous and deciduous forest, swampy forest, and raised bog. In total, around 200 wooden pegs were installed in July 2022 in the vicinity of the "Mukhrino" and "Shapsha" research stations of the Yugra State University (Khanty-Mansiysk): 5 plots, with 20 chronosequence points in each, and 2 pegs of three wood types at each point. A total of 189 wood dowels were extracted by the end of the second season. The same number of samples of environmental DNA, extracted from the wood, were analysed using ITS2 region (Illumina MiSeq platform). Between 13 and 15 chronosequence points were extracted throughth 2 year period of decomposition from each habitat.

Sampling

All field operations were made wearing gloves and the instruments (knife, scissors and tweezers when necessary) were sterilised between samples with bleach and alcohol according to recommendations (Tedersoo et al. 2022). After extraction, wooden dowels were wrapped in paper bags, dried in a drying cabinet at 40°C for 24 hours and stored in a dry stage before extraction according to recommendations (Shumskaya et al., 2023).

Method steps

1. To study the total DNA of the dead wood community, we used an approach of standardised substrates (Shumskaya et al. 2023) developed to describe the community in the early stages of wood decay. Sterilised wooden dowels of three tree species (pine, larch and birch) were buried in the upper peat surface in peatland and forest habitats and were extracted at two-week intervals throughout two years.
2. The collected dowels were wrapped in sterile bags and dried at 40°C for a day.
3. The homogenisation of wood substrates was done according to the following: the interior of each dowel was drilled by a 2 mm fire-sterilised drill bit and the sawdust was collected into sterile plastic centrifuge tubes.
4. Further extraction was done according to the instructions of the SileksMagNA kit, by addition of 40 µl of lysis buffer, soaking and homogenising with glass beads.
5. The samples of extracted DNA were outsourced for processing by an independent company (Evrogen, Moscow). The quality of the obtained metagenomic DNA was checked by electrophoresis on an agarose gel. Quantification was carried out by measuring the concentration of DNA by Qubit 2, using the dsDNA HS reagent kit (ThermoFisher Scientific). The preparation of libraries for sequencing was carried out in accordance with the protocol described in 16S Metagenomic Sequencing Library Preparation (Part #15044223 Rev. B; Illumina).
6. Amplification of ITS variable regions was carried out using primers: fITS7: 5'-GTGARTCATCGAATCTTTG-3' and ITS4: 5'-TCCTCCGCTTATTGATATGC-3' (White et al. 1990, Ihrmark et al. 2012). After obtaining the amplicons, the libraries were purified and pooled equimolarly with the SequalPrep™ Normalization Plate Kit (ThermoFisher, Cat #A10510-01). Quality control of the libraries was carried out using the Fragment Analyzer and quantitative analysis was carried out with qPCR.
7. The library was sequenced on Illumina MiSeq (length of reads – 300 bp on both side fragments) using MiSeq Reagent Kit v.3 (600 cycles). FASTQ files were obtained using bcl2fastq v.2.17.1.14 Conversion Software (Illumina). The PhiX phage library was used to control sequencing parameters. Most of the readings related to phage DNA were removed during demultiplexing.
8. The obtained sequences were processed using QIIME2 (Quantitative Insights Into Microbial Ecology 2, version 2023.9).
9. The pipeline of sequence analysis is applied as follows: 1) Indexes were removed using trim-paired (QIIME cutadapt trim-paired); 2) Forward and backward reads were merged using merge-pairs (QIIME vsearch merge-pairs); 3) Quality filtering done using q-score (QIIME quality-filter q-score); 4) Dereplication made using dereplicate-sequences (QIIME vsearch dereplicate-sequences); 5) Internal de-novo clustering with an identity parameter of 99% (QIIME vsearch cluster-features-de-novo); 6) Clustering based on the UNITE database (version 9.0 16 October 2022) using cluster-features-closed-reference with 97% identity 7) parameter (QIIME vsearch cluster-features-closed-reference); 7) Chimeras removed using uchime-ref (QIIME vsearch uchime-ref); 8) Classification classify-sklearn (QIIME feature-classifier classify-sklearn) on a classifier that was trained using the naive Bayes classifier algorithm (QIIME feature-classifier fit-classifier-naive-bayes).

The sequence analysis revealed a total of 1642 SHs classified into 745 species, 502 genera, 241 families, 94 orders, 33 classes, eight phyla and one kingdom at a 99% similarity level. About 45% of taxa were identified at the species level, 30% at the genus level and the rest at higher taxonomic levels.

1. Leucosporidiales
   rank: order
2. Phaeomoniellales
   rank: order
3. Mortierellomycota
   rank: phylum
4. Filobasidiales
   rank: order
5. Agaricostilbales
   rank: order
6. Rhizophydiomycetes
   rank: class
7. Trichosphaeriales
   rank: order
8. Wallemiales
   rank: order
9. Auriculariales
   rank: order
10. Phaeothecales
    rank: order
11. Sordariales
    rank: order
12. Pucciniales
    rank: order
13. Myriangiales
    rank: order
14. Wallemiomycetes
    rank: class
15. Mytilinidiales
    rank: order
16. Tremellomycetes
    rank: class
17. Pisorisporiales
    rank: order
18. Thyridiales
    rank: order
19. Orbiliales
    rank: order
20. Onygenales
    rank: order
21. Spizellomycetes
    rank: class
22. Atractiellomycetes
    rank: class
23. Mucoromycota
    rank: phylum
24. Malasseziales
    rank: order
25. Tremellodendropsidales
    rank: order
26. Microascales
    rank: order
27. Mortierellomycetes
    rank: class
28. Cystofilobasidiales
    rank: order
29. Ustilaginales
    rank: order
30. Togniniales
    rank: order
31. Trichosporonales
    rank: order
32. Sebacinales
    rank: order
33. Microbotryomycetes
    rank: class
34. Erythrobasidiales
    rank: order
35. ITS
    rank: phylum
36. Lichinales
    rank: order
37. Corticiales
    rank: order
38. Geoglossales
    rank: order
39. Erysiphales
    rank: order
40. Mortierellales
    rank: order
41. Rhytismatales
    rank: order
42. Microstromatales
    rank: order
43. Helotiales
    rank: order
44. Glomeromycetes
    rank: class
45. Malasseziomycetes
    rank: class
46. Chaetosphaeriales
    rank: order
47. Sareales
    rank: order
48. Agaricales
    rank: order
49. Atheliales
    rank: order
50. Trechisporales
    rank: order
51. Boliniales
    rank: order
52. ITS2
    rank: class
53. Dothideomycetes
    rank: class
54. Lauriomycetales
    rank: order
55. Jobellisiales
    rank: order
56. Glomeromycota
    rank: phylum
57. Candelariomycetes
    rank: class
58. Ostropales
    rank: order
59. Geastrales
    rank: order
60. Candelariales
    rank: order
61. Pleosporales
    rank: order
62. Umbelopsidales
    rank: order
63. Eurotiomycetes
    rank: class
64. Lichinomycetes
    rank: class
65. Pezizales
    rank: order
66. Chytridiales
    rank: order
67. Glomerellales
    rank: order
68. GS21
    rank: order
69. Kriegeriales
    rank: order
70. Abrothallales
    rank: order
71. Lecanoromycetes
    rank: class
72. GS18
    rank: order
73. Spizellomycetales
    rank: order
74. Polyporales
    rank: order
75. Glomerales
    rank: order
76. Umbelopsidomycetes
    rank: class
77. Sporidiobolales
    rank: order
78. Taphrinales
    rank: order
79. Chytridiomycetes
    rank: class
80. Mycosphaerellales
    rank: order
81. Eurotiales
    rank: order
82. Capnodiales
    rank: order
83. Exobasidiomycetes
    rank: class
84. Olpidiomycota
    rank: phylum
85. Saccharomycetes
    rank: class
86. Sordariomycetes
    rank: class
87. Ascomycota
    rank: phylum
88. Hymenochaetales
    rank: order
89. Xylariales
    rank: order
90. Geoglossomycetes
    rank: class
91. Lecanorales
    rank: order
92. Russulales
    rank: order
93. Hypocreales
    rank: order
94. Atractiellales
    rank: order
95. Pucciniomycetes
    rank: class
96. Botryosphaeriales
    rank: order
97. Trapeliales
    rank: order
98. Tremellales
    rank: order
99. Chaetothyriales
    rank: order
100. Venturiales
     rank: order
101. Archaeorhizomycetes
     rank: class
102. Exobasidiales
     rank: order
103. Calosphaeriales
     rank: order
104. Agaricomycetes
     rank: class
105. Cystobasidiomycetes
     rank: class
106. Thelebolales
     rank: order
107. Mytilinidales
     rank: order
108. Tubeufiales
     rank: order
109. Rozellomycota
     rank: phylum
110. Ustilaginomycetes
     rank: class
111. Rosettozymales
     rank: order
112. Endogonomycetes
     rank: class
113. Thelephorales
     rank: order
114. Rhizophydiales
     rank: order
115. Taphrinomycetes
     rank: class
116. Leotiales
     rank: order
117. Sareomycetes
     rank: class
118. Basidiomycota
     rank: phylum
119. Saccharomycetales
     rank: order
120. Boletales
     rank: order
121. Pezizomycetes
     rank: class
122. Archaeorhizomycetales
     rank: order
123. Coniochaetales
     rank: order
124. Amylocorticiales
     rank: order
125. Cantharellales
     rank: order
126. Chytridiomycota
     rank: phylum
127. Illumina MiSeq
     rank: order
128. Cystobasidiales
     rank: order
129. Phacidiales
     rank: order
130. Xylonomycetes
     rank: class
131. Leotiomycetes
     rank: class
132. Orbiliomycetes
     rank: class
133. Dothideales
     rank: order
134. Symbiotaphrinales
     rank: order
135. Diaporthales
     rank: order
136. Agaricostilbomycetes
     rank: class
137. Amphisphaeriales
     rank: order
138. Xylonales
     rank: order

text text text text text text text text text text text text