Computational protocol: Chloroplast and nuclear DNA exchanges among Begonia sect. Baryandra species (Begoniaceae) from Palawan Island, Philippines, and descriptions of five new species

Similar protocols

Protocol publication

[…] Molecular phylogenies were constructed based DNA sequences from the chloroplast genome (four noncoding regions ndhA intron, ndhF-rpl32 spacer, rpl32-trnL spacer, trnC-trnD spacer) and the nuclear genome (nuclear ribosomal internal transcribed spacers ITS1 and 2 with the 5.8S gene). Taxon sampling was based on that of Hughes et al. [], supplemented with 14 new accessions including all the new species described herein. The ingroup, Begonia sect. Baryandra, has been found to be strongly supported as monophyletic [,]; the outgroup sampling for both phylogenies matches that of Hughes et al. []. A total of 91 accessions were sampled for the chloroplast regions, representing 67 taxa including outgroups, and 69 accessions representing 50 taxa (including outgroups) for the nuclear region. Voucher and GenBank accession information is listed in . The DNA extraction, PCR and sequencing were carried out as in Hughes et al. []. The following bases were excluded from the chloroplast alignment because of missing data at the ends of regions, or alignment uncertainty due to long mononucleotide repeats: 1–10, 1390–1540, 2510–3050, 4530–4645, 4746–4910, 4943–4997, 5720–5810, 6970–7125, 8050–8089; and from the nuclear alignment bases 494–548 were excluded. Appropriate models of DNA sequence evolution for the aligned datasets were assessed with the program jModeltest 2.1.3 [] using the corrected Akaike information criterion (AICc) to estimate the model with the closest fit to the data. For both the chloroplast and nuclear alignment, the GRT+G+I model was the most probable (AIC weight 1.00 and 0.66 respectively).Bayesian phylogenetic analyses were carried out separately on the chloroplast and nuclear data sets using the program MrBayes 3.2.6 []. Each data set was treated as a single partition, analysed under the appropriate model of sequence evolution and the default parameters of two runs with four chains each, run for 10 000 000 generations with a sample tree taken every 10 000 generations. The convergence of the MCMC chains of the two runs was assessed by inspection of the trace plots of parameters using Tracer ver. 1.6 []. The effective sample sizes (ESS) of all parameters were > 200, indicating that each parameter was sampled satisfactorily. The first 25% of sampled trees were discarded as burn-in, and the remainder summarized as a maximum clade credibility tree. Trees were visualised using the APE package []. Strict and semi-strict consensus trees combining the two analyses were constructed in PAUP []. [...] Alignments with matching sampling for both chloroplast and nuclear markers were constructed for use in conducting an Incongruence Length Difference test (ILD) [] and measures of internode certainty (IC) []. The ILD test was conducted using PAUP, using 100 replicates based on heuristic searches with TBR branch swapping, each with 10 random addition replicates and Maxtrees set to 100. In order to further examine topological incongruence between the datasets, post burn-in samples of trees resulting from the Bayesian phylogenetic analysis of the chloroplast and ITS data were combined into a single majority rule consensus (MRC) tree with IC values for each node using RaxML []. The IC metric (scaled between 0 and 1) represents the certainty for each internal branch, taking into account the frequency of the most prevalent conflicting bipartition in the population of trees used to make the consensus. For example, if there are two conflicting bipartitions between the chloroplast and ITS derived topologies, each present at 100% frequency in the different datasets, then IC = 0, reflecting the complete incongruence between the genomes. A bipartition with 100% frequency in trees derived from the chloroplast dataset and 50% frequency in the ITS dataset, contradicted by 50% of the remaining trees in the ITS dataset would have an IC = 0.18. Comparing all post burn-in trees allows us to further investigate the full range of topologies produced by the two separate analyses, rather than just comparing a separate consensus of each. […]

Pipeline specifications

Software tools jModelTest, MrBayes, APE, PAUP*, RAxML
Application Phylogenetics