Computational protocol: Monophyly or Paraphyly– The Taxonomy of Holcoglossum (Aeridinae: Orchidaceae)

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Protocol publication

[…] To estimate the variation of the ITS, matK and trnL-F sequences across Holcoglossum and related genera, we calculated the pairwise genetic p-distance for each region using MEGA v. 4 . These distances were initially used to evaluate the interspecific divergence with the Kimura 2-Parameter model (K2P). TrnH-psbA was excluded under this analysis because all of the sequences were from Holcoglossum and there was a paucity of related genera. [...] Clustal X 1.83 was used to obtain an initial alignment of the DNA sequences, followed by manual adjustment using BioEdit .The phylogenetic analyses for each matrix were performed using the maximum parsimony (MP) and Bayesian inference (BI) methods in PAUP v4.0b10 and MrBayes v3.0b4 , respectively.For the MP analyses, heuristic searches were conducted with 1,000 replicates of random addition, with one tree held at each step during the stepwise addition, tree-bisection-reconnection (TBR) branch swapping, MulTrees in effect, and the steepest descent off. All of the characters were unordered and equally weighted, and the gaps were coded as missing data. To access the node support, bootstrap analyses were performed using 1,000 replicates, with 10 random taxon additions and heuristic search options.Prior to the Bayesian analysis, the Akaike Information Criterion (AIC) implemented in ModelTest version 3.7 , was used to select the best-fit model of molecular evolution for each dataset. For the BI analyses, four chains of the Markov Chain Monte Carlo (MCMC) were run, sampling one tree every 1,000 generations for 5,000,000, starting with a random tree. Majority rule (>50%) consensus trees were constructed after removing the “burn-in period” samples (the first 20% of the sampled trees). [...] To identify the synapomorphies that are congruent with each of the major clades of Holcoglossum retrieved in the molecular tree and to assess the value of the characters used in the classifications, 45 morphological characters were selected. We used the exemplar method, scoring the morphological characters in the morphological matrix for the same species as used in the molecular analyses. The complete morphological matrix, coding 45 characters for the 35 taxa, is available in . We also prepared a combined morphological and molecular matrix that included the 32 taxa for character mapping.Character evolution was reconstructed onto a 50% major consensus tree generated in PAUP using the parsimony ancestral state reconstruction in Mesquite v.2.75 . All of the morphological characters were considered unordered and unweighted. [...] We displayed the patrocladistic analysis according to Stussey and König . This cladogram with high support based on Bayesian inference served as a structure for the patristic distance. The cladistic distances were calculated from the selected cladogram, and these values were placed in a new cladistic matrix. The patristic distance was defined as the number of apomorphic step changes separating two taxa on the cladogram. The patristic distance was then added to the cladistic distance to form the combined patrocladistic data matrix (). These combined distances are used as input into UPGMA (packages in MEGA) to construct the patrocladogram. […]

Pipeline specifications

Software tools MEGA-V, Clustal W, BioEdit, PAUP*, MrBayes, ModelTest-NG, Mesquite, PATRISTIC, MEGA
Applications Phylogenetics, GWAS
Diseases Varicose Ulcer