Computational protocol: Phylogeny and ecology of the ubiquitous saprobe Cladosporium sphaerospermum, with descriptions of seven new species from hypersaline environments

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Protocol publication

[…] For DNA isolation strains were grown on MEA for 7 d. DNA was extracted according to Gerrits van den Ende & de Hoog () by mechanical lysis of approx. 1 cm2 of mycelium. A fragment of the rDNA including the Internal Transcribed Spacer region 1, 5.8S rDNA and the ITS 2 (ITS) was amplified using the primers V9G () and LS266 (). Sequence reactions were done using primers ITS1 and ITS4 (). For amplification and sequencing of the partial actin gene, primers ACT-512F and ACT-783R were applied according to Carbone & Kohn (). For amplification and sequencing of the β-tubulin gene primers T1 and T22 were used according to O'Donnell & Cigelnik (). A BigDye terminator cycle sequencing kit (Applied Biosystems, Foster City, CA, U.S.A.) was used in sequence reactions. Sequences were obtained with an ABI Prism 3700 DNA Analyzer (Applied Biosystems). They were assembled and edited using SeqMan v. 3.61 (DNAStar, Inc., Madison, U.S.A.). Sequences downloaded from GenBank are indicated in the trees by their GenBank accession numbers; newly generated sequences are indicated by strain numbers (see also ). Sequences were automatically aligned using ClustalX v. 1.81 (Jeanmougin et al. 1998). The alignments were adjusted manually using MEGA3 (). Phylogenetic relationships of the taxa were estimated from aligned sequences by the maximum parsimony criterion as implemented in PAUP v. 4.0b10 (). Data sets of the SSU rDNA, ITS rDNA and the β-tubulin and actin genes are analysed separately. Species of Cladosporium s. str. were compared with various taxa of the Mycosphaerellaceae using SSU rDNA sequences and Fusicladium effusum G. Winter (Venturiaceae) as outgroup. The other data sets focus on Cladosporium s. str., using Cladosporium salinae Zalar, de Hoog & Gunde-Cimerman as an outgroup, because this species was most deviant within Cladosporium in the SSU rDNA analysis (see below). Heuristic searches were performed on all characters, which were unordered and equally weighted. Gaps were treated as missing characters. Starting tree(s) were obtained via stepwise, random, 100 times repeated sequence addition. Other parameters included a “MaxTrees” setting to 9 000, the tree-bisection-reconnection as branch-swapping algorithm, and the “MulTrees” option set to active. Branch robustness was tested in the parsimony analysis by 10 000 search replications, each on bootstrapped data sets using a fast step-wise addition bootstrap analysis. Bootstrap values larger than 60 are noted near their respective branches. Newly generated sequences were deposited in GenBank (www.ncbi.nlm.nih.gov); their accession numbers are listed in . Alignments and trees were deposited in TreeBASE (www.treebase.org). Fig. 1. […]

Pipeline specifications

Software tools Clustal W, MEGA, PAUP*
Databases TreeBASE
Application Phylogenetics
Organisms Corynebacterium halotolerans, Sargassum fusiforme, Homo sapiens