Computational protocol: Historical Isolation versus Recent Long-Distance Connections between Europe and Africa in Bifid Toadflaxes (Linaria sect. Versicolores)

Similar protocols

Protocol publication

[…] We sampled a total of 57 populations of Linaria sect. Versicolores (one individual per population), including representatives of 25 species and subspecies (, ). We failed to sample L. dissita. This is a poorly known African taxon of minor relevance for our objectives, because it seems to be closely related to other African species, with which it could even be con-specific . We made special emphasis in the sampling of multiple populations of morphologically variable, widely distributed, and intercontinental species in order to test biogeographic hypotheses. To test the monophyly of section Versicolores, we also sampled nine additional species representing the remaining six sections of Linaria. One species of Chaenorhinum and one of Antirrhinum were included as the outgroup on the basis of a previous phylogeny of the tribe Antirrhineae . Plant material was collected in the field and dried in silica gel or obtained from herbarium collections (RNG, MA, ATH, UPOS, SALA; ).Total genomic DNA was extracted using DNeasy Plant Mini Kit (QIAGEN Inc., California). A pilot study using 6 samples of different species was performed to find the most variable sequences among 14 plastid DNA regions previously used in phylogenetic and phylogeographic analyses , . DNA regions were amplified in an Eppendorf Mastercycler Epgradient S (Westbury, NY) or a MJ Research PTC-200 (Massachusetts) thermal cycler. After 1 min pretreatment at 95°C, PCR conditions were: 30 cycles of 1 min at 94°C, 1–2 min at 48–55°C and 1–2 min at 72°C. In certain reactions, a volume of 1 µL of bovine serum albumine (BSA) at 1 mg ml−1 was included in each 25 mL reaction to improve the efficiency of amplification. Amplified products were treated with ExoSAP-IT (USB Corporation, Ohio) and submitted to Macrogen Inc. (Seoul, South Korea) for sequencing. Resulting sequence data were assembled and edited using Geneious Pro v5 . We identified two highly variable cpDNA regions: rpl32-trnLUAG and trnK-matK and then the sequencing of these regions was extended to every sampled individual. In order to facilitate amplification and sequencing from partially degraded DNA obtained from herbarium specimens, we designed the following internal primers for both regions and used them in combination with the standard primers: rpl32-trnL_intF (5′-CATTTCCAAGGTGGGGAGTCT-3′), rpl32-trnL_intR (5′-AGAAATAGGTTGATGGGGA-3′), trnK-matK_intF1 (5′-ACCTGTCTCCGAGGTATCTA-3′), trnK-matK_intF2 (5′-GGGGTTTGCATTTATTGTGG-3′), trnK-matK_intR1 (5′-CACGATCATGAGCAAACGCA-3′), and trnK-matK_intR2 (5′-CCACAATAAATGCAAACCCC-3′). We also designed a reverse primer specific to Linaria sect. Versicolores for trnK-matK: 1470R_Lvers (5′- AAGATGTTGATCGTAAATCC-3′). All sequences were submitted to GenBank (see for accession numbers). [...] Sequences of each cpDNA region (rpl32-trnLUAG and trnK-matK) were aligned using MAFFT 6 with default parameters, and further adjustments were made by visual inspection. The two regions were combined in a single matrix, and phylogenetic relationships were assessed using maximum parsimony (MP), maximum likelihood (ML) and Bayesian inference (BI). The MP analysis was performed in TNT 1.1 using a heuristic search with 10,000 replicates saving two most-parsimonious trees per replicate, followed by a second heuristic search retaining all best trees and using the trees obtained in the previous 10,000 replicates as the starting ones. Bootstrap support (MP-BS) of clades was assessed using 1000 standard replicates. For ML and BI analyses, the simplest model of sequence evolution that best fits the sequence data (GTR for trnK-matK and GTR+G for rpl32-trnLUAG and the combined dataset) was determined under the Akaike Information Criterion (AIC) in jModelTest 0.1.1 . ML was implemented in PhyML 3.0 with 500 non-parametric bootstrap replicates (ML-BS). BI was performed in MrBayes v3.1.2 using two searches with 10 million generations each and a sample frequency of 1000. The two regions were partitioned and unlinked. Chain convergence was assessed with Tracer 1.4 , and a 50% majority rule consensus tree with Bayesian posterior probabilities (PP) of clades was calculated to obtain the Bayesian estimate of phylogeny after removing the first 10% generations as burn-in. [...] To estimate divergence times among Linaria sect. Versicolores lineages, we implemented a relaxed molecular-clock approach in BEAST v.1.6.0 , , a software that simultaneously estimates tree topology and node ages. Identical sequences of the rpl32-trnLUAG/trnK-matK matrix were removed from the analysis. Gaps were treated as missing data. Since no reliable fossils of Linaria are known to date, only molecular estimates were available for temporal calibration of the tree. The divergence time between Chaenorhinum and Linaria was modelled as a normal distribution with mean = 29 Ma and standard deviation = 4.6, on the basis of an estimate obtained in a relaxed molecular-clock analysis of tribe Antirrhineae (P. Vargas et al., unpublished). This analysis incorporates a calibration of 97 Ma for the crown-age of Lamiales and minimum stem-age constraints for Lamiales families and tribes based on five fossils: Fraxinus wilcoxiana (Oleaceae, Middle Eocene) , Catalpa rugosa (Bignoniaceae, Early-Middle Oligocene) , Ajuginucula smithii (Lamiaceae, Early-Middle Oligocene) , Gratiola tertiaria (Gratioleae, Miocene) and Plantaginacearumpollis (Plantaginaceae s.str., Middle Miocene) . All these fossils have been considered reliable and proposed as calibration points for molecular dating in previous studies , , . The substitution rate variation was modelled using an uncorrelated lognormal distribution, and a Birth-Death process was employed as tree prior. Two MCMC analyses were run for 10 million generations, with a sample frequency of 1000. Both chains were combined using LogCombiner 1.4.8, after discarding the first 10% of sampled generations as burn-in. Parameter analysis in Tracer 1.4 confirmed adequate sample size, with ESS values above 650 and plots showing equilibrium. Trees were summarized in a maximum clade credibility (MCC) tree obtained in TreeAnotator 1.4.8 and visualized in FigTree 1.1.2. […]

Pipeline specifications

Software tools Geneious, MAFFT, jModelTest, PhyML, MrBayes, BEAST, FigTree
Application Phylogenetics