Computational protocol: Temperate Pine Barrens and Tropical Rain Forests Are Both Rich in Undescribed Fungi

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Protocol publication

[…] Protocols described in Zhang et al and Luo and Zhang were used for DNA extraction, PCR amplification and sequencing of the internal transcribed spacer (ITS) of the fungal ribosomal RNA genes. The UltraClean Soil DNA Isolation Kit (MoBio, California) was used for DNA isolation following the manufacturer's protocol. Primers ITS1 and ITS4 were used for PCR and sequencing . The ITS sequences were designated to phylotypes by using 97% similarity criterion in Usearch 7 . The ITS sequences of representative phylotypes were aligned in Clustal X V.1.8 and edited in BioEdit 7.0.5 . Separate phylogenetic analyses were performed for three major groups identified from this collection: Sordariomycetes and Leotiomycetes; Dothideomycetes, Eurotiomycetes, and Pezizomycetes; and Basidiomycota and early diverging lineages. A Bayesian inference (BI) analysis was conducted with the Markov Chain Monte Carlo method in MrBayes 3.2.1 under the nucleotide substitution model selected by using Hierarchical Likelihood Ratio Tests (hLRTs) and Akaike Information Criterion (AIC) in MrModeltest 2.3 . The general time reversible with a proportion of invariable sites and gamma distributed rate variation among sites (GTR+I+G) was the selected model for Sordariomycetes and Leotiomycetes; and Dothideomycetes, Eurotiomycetes, and Pezizomycetes. The Hasegawa-Kishino-Yano model with a proportion of invariable sites (HKY+I) was used for Basidiomycota and the early diverging lineages. Trees were sampled every 100 generations from 10 000 000 generations resulting in 100 000 trees. The first 25 000 trees were discarded as the burn-in and the remaining 75 000 trees were chosen to calculate posterior probability values of clades in a consensus tree. Fungal phylotypes were identified by searching with Blastn in GenBank ( and the AFTOL ( databases and morphology (). A number of phylogenetic analyses for each individual species/genus were performed to further confirm fungal identification. A species was categorized as undescribed when it had lower than 97% ITS sequence similarity with any known taxa in GenBank, and morphologically, there was no match with available fungal descriptions in the literature. Collected samples are preserved in both Zhang lab at Rutgers University, USA and Cai lab in China. Described new fungal taxa () were also deposited in CBS-KNAW Fungal Biodiversity Centre (details and accession numbers in , ). Fungal DNA sequences from this study were deposited in GenBank (). […]

Pipeline specifications

Software tools USEARCH, Clustal W, BioEdit, MrBayes, MrModelTest, BLASTN
Application Phylogenetics