Computational protocol: Impatiens pandurata (Balsaminaceae), a new species from Yunnan, China

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Protocol publication

[…] DNA sequences of 151 species of Impatiens were used and three species, Hydrocera triflora (L.) Wight & Arn. (Balsaminaceae), and Marcgravia umbellata L. and Norantea guianensis Aubl. (Marcgraviaceae), were included as outgroups, based on the results of Yuan et al. (), Janssens et al. () and Yu et al. (). All sequences were downloaded from GenBank, except those of the new species, I. pandurata, which were newly generated for this study (Genbank accession numbers XXXX, XXXX for ITS, XXXX, XXXX for atpB-rbcL and XXXX, XXXX for trnL-F). Vouchers and GenBank accession numbers are listed in Additional file : Table S1.Three molecular markers were used: ITS, atpB-rbcL and trnL-F. Total genomic DNA was extracted from silica gel-dried leaves using a modified CTAB protocol from Doyle and Doyle (). Primers and PCR protocols for ITS, atpB-rbcL and trnL-F are derived from White et al. (), Janssens et al. () and Taberlet et al. (), respectively. PCR products were purified using a GFX™PCR DNA and Gel Band Purification Kit (Amersham Pharmacia Biotech, Piscataway, NJ, USA). Sequencing reactions were carried out using an ABI Prism Bigdye Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA). Products were analyzed on an ABI3730xl automated DNA sequencer. Sequences were aligned using the default parameters in Clustal X v.1.83 (Thompson et al. ) and further adjusted manually in BioEdit v.7.0 (Hall ). Four difficult-to-align regions in trnL-F (encompassing 73 sites) and one difficult-to-align region in atpB-rbcL (encompassing 42 sites) were excluded from the analyses.Maximum parsimony (MP) and Bayesian inference (BI) were used to analyze the ITS and plastid data sets. The MP analyses were carried out in PAUP* v.4.0b10 (Swofford ). Heuristic searches were conducted with 1000 replicates of random addition, one tree held at each step during stepwise addition, tree-bisection-reconnection (TBR) branch swapping, MulTrees in effect, and steepest descent off. Bootstrapping was conducted with 1000 replicates with 10 random taxon additions and heuristic research options. The BI analyses were carried out in MrBayes v.3.0b4 (Ronquist and Huelsenbeck ). Each of the three regions (ITS, atpB-rbcL, and trnL-F) was assigned its own model of nucleotide substitution, as determined by the Akaike information criterion (AIC) in Modeltest v.3.06 (Posada and Crandall ). […]

Pipeline specifications

Software tools Clustal W, BioEdit, PAUP*, MrBayes, ModelTest-NG
Application Phylogenetics