DNA-based occurrence dataset on peatland fungal communities studied by metabarcoding in Northwestern Siberia

Study Extent

In order to study the fungal community of raised bogs, four major substrates were subjected to metabarcoding analysis: peat (dead parts of sphagnum parts 2-30 cm below the surface), plant litter, wood and mycorrhizal roots. Within each of these groups, the experiment design was planned in order of cover spatial and temporal variability, substrate features and some methodological questions.

Sampling

General considerations during sampling. All field operations were made wearing gloves, the instruments (knife, scissors and tweezers when necessary) were sterilized between samples with bleach. The samples were wrapped in sterilized aluminum bags and labeled by permanent marker. The bags with samples were put in a cooling bag with refrigerant immediately after the sampling, and transported to the laboratory to be frozen at -22˚C within a few hours. All substrates except wood were frozen at -20˚C in a refrigerator to be extracted and processed 2 months later. Wood dowels were wrapped in paper bags and dried in drying cabinet at 40˚C during the day of extraction and stored in dry stage before extraction.

Method steps

1. Sampling of peat. To study the fungal community of dead sphagnum/peat, six plots were located in two habitats: treed bogs and open sphagnum lawns. Each plot was sampled regularly in June, July, August and September. Several shoots of dead sphagnum were collected from 10 points 5 m apart within each plot, to create a composite sample of approximately 5 g field wight. The general sampling depth was about 2-5 cm below the Sphagnum surface. Additional sampling at different depths was made in August, where the samples were collected in two plots at four different depth (0-2 cm, 5-7 cm, 10-15 cm and 25-30 cm below the surface). This experiment design resulted in total 46 samples with the following parameters for analyses: habitat (2 types), species of Sphagnum (6 species), peat depth (4 depths) and seasonal variation (4 months). Additional methodological experiment was made related to sampling approach: a series of samples with minimum extraction weight (0.25 g, several shoots) picked from a single point (as opposite to composite samples 5 g) were collected in June and were later compared with the composite sample approach in the same plot.
2. Sampling of plant litter. The community of fungal saprotrophs of 6 most common bog plants was studied by collecting its leaf litter. The litter was picked randomly from the surface of Sphagnum, in approximate area of 10 m^2 in each plot. The sampling was made in the same plots and the same dates as for peat substrate. The total of 5 g field weight substrate of each plant was collected three times per season (June, July, September), totaling in 28 samples.
3. Sampling of mycorrhizal roots. To study the mycorrhizal community of bog trees, we collected ecto-mycorrhizal roots of two common bog species: Pinus sylvestris and P. sibirica. The roots were collected in two localities («Mukhrino» and «Shapsha» located at about 30 km distance) to cover spatial variability. In each locality, 5 to 10 trees growing about 10 m apart were marked for following root extraction and dendrochronological boring. About 30 g of fine roots were extracted from samples taken about 20-30 cm distance in several opposite sites from the trunk. The samples were additionally cleaned from fine debris in the laboratory, cleaned roots were collected in Eppendorf tubes, then frozen. The sampling was done twice a year in the beginning and at the end of vegetation season (June and September), totaling in 40 root samples.
4. Sampling of wood. To study the total DNA of dead wood community, we used an approach of standardized substrates (Shumskaya et al., 2022) developed to describe the community of early stages of wood decomposition. The sterilized wooden dowels of three tree species (pinus, larch and betula) were buried in the upper peat surface in treed bog, and were extracted in each subsequent two weeks throughout the season. Totally, 30 wood dowels were extracted by the end of the first season. The experiment will continue for two years, here we present the first-year results.
5. DNA extraction. The samples were homogenized using different approached depending on the substrate type. All samples of peat were lyophilized and homogenized manually: 1) using sterilized pestle and mortar for 5 g composite samples, 2) using sterilized micro pestles for single 0.25 g samples. From each composite sample, about 0.25 g of peat powder was transferred to 1.5 ml Eppendorf tube. Finally, all samples were soaked in 400 mkl of lysis buffer for a night, then homogenized using a micro-tube homogenizer with glass beds according to instructions for total DNA extraction soil kit SileksMagNA (https://sileks.com/ru/products/soil-dna-rna-isolation-sileksmagna.html). Homogenization of plant leaf litter was made in a different way in order to break down hard plant materials. All samples were packed in sterile paper bags and dried in drying cabinet at 40˚C. Each sample was then grinded in a coffee grinder (all parts were sterilized between the samples). From each composite sample, about 0.25 g of plant powder was transferred to 1.5 ml Eppendorf tube, soaked and homogenized with lysis buffer as above. The mycorrhizal roots were homogenized by two different approaches to compare their final performance. The first group of samples was lyophilized and then homogenized using micro-tube homogenizer to create dry fine powder. The second group of samples was homogenized directly (without lyophilization), using micro-tube homogenizer and glass beds accordingly. The homogenization of wood substrates was done according to the instructions (Shumskaya et al., 2022): the interior of each pin was drilled by a 2 mm fire-sterilized drill bit and the sawdust was collected into sterile plastic centrifuge tubes. Further extraction was done as above, by addition of 40 mkl of lysis buffer, soaking and homogenizing with glass beds according to instructions of SileksMagNA. Totally 114 samples of environmental DNA extracted from 4 substrates were obtained and stored at -20˚C until further processing in outsource company.
6. DNA detection, library preparation, PCR and sequencing. The samples of extracted DNA were sent for sequencing to outsource company (Evrogen, Moscow, https://evrogen.ru/). The quality of the obtained metagenomic DNA was checked by electrophoresis in agarose gel. Quantification was carried out by measuring the concentration of DNA by Qubit 2, using dsDNA HS reagent kit (ThermoFisher Scientific). 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). Amplification of ITS variable regions was carried out using primers: Next-fITS7_For: 5'-GTGARTCATCGAATCTTTG-3' and Next-ITS4_Rev: 5'-TCCTCCGCTTATTGATATGC-3'. After obtaining the amplicons, the libraries were purified and pooled equimolarly with SequalPrep™ Normalization Plate Kit (ThermoFisher, Cat # A10510-01). Quality control of libraries was carried out using the Fragment Analyzer, and quantitative analysis was carried out with qPCR. The library was sequenced on Illumina MiSeq (length of reads – 300 bp on both sides fragments) using MiSeq Reagent Kit v3 (600 cycles). FASTQ files were obtained using bcl2fastq v2.17.1.14 Conversion Software (Illumina). The PhiX phage library was used to control sequencing parameters. Most of the readings related to phage DNA was removed during demultiplexing.
7. Indexes were removed using trim-paired (QIIME cutadapt trim-paired) Forward and backward reads were merged using merge-pairs (QIIME vsearch merge-pairs) Quality filtering done using q-score (QIIME quality-filter q-score) Dereplication made using dereplicate-sequences (QIIME vsearch dereplicate-sequences) Internal de-novo clustering with an identity parameter of 99% (QIIME vsearch cluster-features-de-novo) Clustering based on the UNITE database (version 9.0 October 16, 2022) using cluster-features-closed-reference with 97% identity parameter (QIIME vsearch cluster-features-closed-reference) Chimeras removed using uchime-ref (QIIME vsearch uchime-ref) 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)
8. Data storage. The raw sequence reads were deposited in SRA: https://www.ncbi.nlm.nih.gov/bioproject/PRJNA1007262.

Metabarcoding of ITS2 region (Illumina MiSeq platform) revealed about 1200 OTUs and about 800 Linnean taxa .

1. Fungi
   rank: kingdom

The area is located in the middle taiga zone of Western Siberia, nearby Khanty-Mansiysk (60.89N, 68.68E) – the Mukhrino field station of Yugra State University. The Mukhrino bog is an ombrotrophic landscape covering about 10 x 15 km, and located in the northern border of the large paludified area “Kondinskaya nizmennost”, on the left terrace of Irtysh river close to its confluence with Ob river. The vegetation of the raised bog represented by typical ombrotrophic or oligo-mesotrophic communities from classes Scheuchzerio-Caricetea nigrae, Oxycocco-Sphagnetea and Vaccinio-Piceetea. Two major vegetation classes described by dominant approach are: pine-dwarfshrubs-Sphagnum bogs (treed bogs, dominated by Pinus sylvestris, Chamaedaphne calyculata, Ledum palustre, Rubus chamaemorus, Sphagnum fuscum) and graminoid-Sphagnum lawns (open bogs, dominated by Scheuchzeria palustris, Carex limosa, Eriophorum russeolum, Oxycoccus palustris, Sphagnum balticum).