Computational protocol: Origin and evolution of Petrocosmea (Gesneriaceae) inferred from both DNA sequence and novel findings in morphology with a test of morphology-based hypotheses

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[…] Sequences were aligned using Clustal X1.83 [] and adjusted manually using BioEdit5.0.9 []. All combined DNA data were analyzed with maximum parsimony (MP), maximum likelihood (ML) and Bayesian inference (BI) methods, which were implemented in PAUP**4.0b10 [], RAxML 8.1.11 [], and MRBAYES version 3.0b4 [], respectively.For MP analysis, all characters were given equal weight and character states were unordered. Heuristic searches were performed 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. Bootstrap support [] for each clade was estimated from 1000 heuristic search replicates as described above.For ML analysis, the optimal model and parameters were determined under the Akaike information criterion (AIC) in Modeltest 3.06 []. A BIONJ tree was employed as a starting point []. Statistical support for the node on the ML tree was estimated by 1000 replicates of bootstrap analyses.In the BI analysis, the model choice of nucleotide substitution was the same as described in ML analysis. Four chains of the Markov Chain Monte Carlo were run each for 10,000,000 generations and were sampled every 10,000 generations. For each run, the first 200 samples were discarded as burn-in to ensure that the chains reached stationary. In the majority rule consensus from Bayesian analysis, posterior probability (PP) was used to estimate robustness.For combined sequence data, the incongruence length difference (ILD) test [] as implemented in PAUP* 4.0b10 [] was performed to assess character congruence between cpDNA data and nDNA data, with 1000 replicates, each with 100 random additions with TBR branch swapping. The p value was used to determine whether the two data sets contained significant incongruence (0.05). [...] The morphological dataset is based on 41 characters, of these, 25 are floral and important traits previously used for subgeneric classification within Petrocosmea (see Additional file : Appendix S1). The morphological data and the combined matrix of DNA plus morphological data were analyzed with MP and BI methods. The Mk1 model was used for the morphological characters in BI. Characters are equally weighted and the states were unordered.The evolution of twelve diagnostic characters (for detail see Results) was analyzed on the posterior set of trees from the combined molecular MP analysis. The analysis was performed using unordered maximum parsimony as implemented in Mesquite ver. 3.02 (available from http://mesquiteproject.org). The results are summarized on the majority rule consensus tree of the posterior set of trees. [...] To reconstruct the possible ancestral ranges of Petrocosmea, we conducted an S-DIVA analysis [] using the software package RASP []. By utilizing the bootstrap distribution of trees resulting from a MP analysis and generating credibility support values for alternative phylogenetic relationships, the S-DIVA method can minimizes the phylogenetic uncertainties [, , ].We used the most parsimonious tree generated from analysis of combined cpDNA and nDNA data as a final representative tree. Four geographic regions were coded: Region A, the border region of China, Thailand, India, and Myanmar, lying east and southeast of Himalaya-Tibetan Plateau; Region B, the Hengduan Mountain-Yunnan Plateau region in southwestern China; Region C, The central China; Region D, the north-central China. By loading the representative tree file and the distribution file based on the geographic region codes as mentioned above, the statistical Dispersal-Vicariance Analysis (S-DIVA) was executed in the software package RASP. Ancestral areas were reconstructed with the “max areas” constrained to three because most species occur in fewer than three areas.The geographical distribution was generated by ARCGIS 10.2(ESRI,US). Locations of Petrocosmea distribution were obtained from collection records and herbarium. The transition from location to longitude and latitude was carried out online (www.gpsspg.com). [...] All the phylogenetic data used in this study have been deposited to the Treebase (http://purl.org/phylo/treebase/phylows/study/TB2:S17643). […]

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