Computational protocol: Elaphroporiaailaoshanensis gen. et sp. nov. in Polyporales (Basidiomycota)

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[…] Morphological studies. The specimens studied are deposited at the herbarium of Southwest Forestry University (SWFC). Macro-morphological descriptions are based on field notes. Special colour terms follow . Micro-morphological data were obtained from the dried specimens and observed under a light microscope following . The following abbreviations were used: KOH = 5% potassium hydroxide, CB = Cotton Blue, CB– = acyanophilous, IKI = Melzer’s reagent, IKI– = both inamyloid and indextrinoid, IKI+ = amyloid, L = mean spore length (arithmetic average of all spores), W = mean spore width (arithmetic average of all spores), Q = variation in the L/W ratios between the specimens studied, n (a/b) = number of spores (a) measured from given number (b) of specimens. DNA extraction and sequencing. CTAB rapid plant genome extraction kit-DN14 (Aidlab Biotechnologies Co., Ltd, Beijing) was used to obtain genomic DNA from dried specimens, according to the manufacturer’s instructions with the modification that a small piece of dried fungal specimen (about 30 mg) was ground to powder with liquid nitrogen. The powder was transferred to a 1.5 ml centrifuge tube, suspended in 0.4 ml of lysis buffer and incubated in a 65 °C water bath for 60 min. After that, 0.4 ml phenol-chloroform (24:1) was added to each tube and the suspension was shaken vigorously. After centrifugation at 13 000 rpm for 5 min, 0.3 ml supernatant was transferred to a new tube and mixed with 0.45 ml binding buffer. The mixture was then transferred to an adsorbing column (AC) for centrifugation at 13 000 rpm for 0.5 min. Then, 0.5 ml inhibitor removal fluid was added in AC for a centrifugation at 12 000 rpm for 0.5 min. After washing twice with 0.5 ml washing buffer, the AC was transferred to a clean centrifuge tube, and 100 ml elution buffer was added to the middle of the adsorbed film to elute the genome DNA. The ITS region was amplified with primer pairs ITS5 and ITS4 (). The nuclear LSU region was amplified with primer pairs LR0R and LR7 (https://sites.duke.edu/vilgalyslab/rdna_primers_for_fungi/). The PCR procedure for ITS was as follows: initial denaturation at 95 °C for 3 min, followed by 35 cycles at 94 °C for 40 s, 58 °C for 45 s and 72 °C for 1 min, and a final extension of 72 °C for 10 min. The PCR procedure for nLSU was as follows: initial denaturation at 94 °C for 1 min, followed by 35 cycles at 94 °C for 30 s, 48 °C for 1 min and 72 °C for 1.5 min, and a final extension of 72 °C for 10 min. The PCR products were purified and directly sequenced at Kunming Tsingke Biological Technology Limited Company. All newly generated sequences were deposited at GenBank (Table ). Phylogenetic analysis. Sequencher 4.6 (GeneCodes, Ann Arbor, MI, USA) was used to edit the DNA sequence. Sequences were aligned in MAFFT 6 (Katoh and Toh 2008, http://mafft.cbrc.jp/alignment/server/) using the “G-INS-I” strategy and manually adjusted in BioEdit (). The sequence alignment was deposited in TreeBase (submission ID 21778). Sequences of Heterobasidion annosum (Fr.) Bref. and Stereum hirsutum (Willd.) Pers. obtained from GenBank were used as outgroups to root trees following in Figure and Xanthoporus syringae (Parmasto) Audet. obtained from GenBank was used as an outgroup to root trees following in the ITS+nLSU analyses (Fig. ).Maximum parsimony analysis was applied to the ITS+nLSU dataset sequences. Approaches to phylogenetic analysis followed and the tree construction procedure was performed in PAUP* version 4.0b10 (). All characters were equally weighted and gaps were treated as missing data. Trees were inferred using the heuristic search option with TBR branch swapping and 1000 random sequence additions. Max-trees were set to 5000, branches of zero length were collapsed and all parsimonious trees were saved. Clade robustness was assessed using a bootstrap (BT) analysis with 1,000 replicates (). Descriptive tree statistics tree length (TL), consistency index (CI), retention index (RI), rescaled consistency index (RC) and homoplasy index (HI) were calculated for each Maximum Parsimonious Tree (MPT) generated. Sequences were also analysed using Maximum Likelihood (ML) with RAxML-HPC2 through the Cipres Science Gateway (www.phylo.org; ). Branch support for ML analysis was determined by 1000 bootstrap replicates.MrModeltest 2.3 (, ) was used to determine the best-fit evolution model for each data set for Bayesian Inference (BI). Bayesian Inference was calculated with MrBayes 3.1.2 with a general time reversible (GTR) model of DNA substitution and a gamma distribution rate variation across sites (). Four Markov chains were run for 2 runs from random starting trees for 5 million generations (Fig. ), for 3 million generations (Fig. ) and trees were sampled every 100 generations. The first one-fourth generations were discarded as burn-in. A majority rule consensus tree of all remaining trees was calculated. Branches that received bootstrap support for maximum likelihood (BS), maximum parsimony (BP) and Bayesian posterior probabilities (BPP) greater than or equal to 75 % (BP) and 0.95 (BPP) respectively, were considered as significantly supported. […]

Pipeline specifications

Software tools Sequencher, MAFFT, BioEdit, RAxML, CIPRES Science Gateway, MrModelTest, MrBayes
Databases TreeBASE
Application Phylogenetics
Organisms Fungi