Xanthium strumarium

Manzo, Eleonora & Tomasello, Salvatore, 2025, Recent Long-Distance-Dispersal Explains the Range Disjunction of the Old-Word Cockleburs (Xanthium strumarium), Journal of Biogeography (e 15104) 52 (5), pp. 1-16 : 8

2.7 | Phylogeny of the Xanthium strumarium Species Complex

A total of 48 X. strumarium samples and six outgroups ( Table 1) were employed. Data processing and assembly was done in HybPhyloMaker using the aforementioned 2150 sequences as reference. Read mapping was achieved using BWA v0.7.16a ( Li and Durbin 2009), with the addition of a mismatch penalty of 8 to make reads more stringently map to the reference. Consensus sequences were generated in ConsensusFixer as it was done for the dating. BLAT was utilised for the alignment of the mapped reads with the targeted exons to generate PSLX files. The minimum sequence identity between the probe and the sample (termed ‘minident’) was set to 99. The subsequent steps were carried out as for the age estimation analyses. Further putative paralog sequences were excluded by employing the “HybPhyloMaker4a2_selectNonHet.sh” script and designating a maximum of 5 heterozygous sites per locus (“maxhet” in the HybPhyloMaker settings file). Missing data filtering was again performed as for the age estimation analyses.

A total of 744 loci ( Table S1b) were retrieved, concatenated, and used to generate an ultrametric Bayesian phylogenetic tree. The analyses were conducted using the Beast2 software, employing the “Optimized Relaxed Clock”, the GTR + G as substitution model, and the “Birth-Death Model” tree model. To obtain absolute divergence times, the crown age of X. strumarium was calibrated using the age obtained in the above analysis (i.e., it was fixed to the estimate obtained in the age estimation analyses). Two independent analyses were run for 100,000,000 generations, sampling every 10,000 iterations. Convergence and effective sample size (ESS) were checked in Tracer v.1.7. The tree files from the two independent runs were combined using LogCombiner v.2.7 ( Bouckaert et al. 2019), and a maximum clade credibility tree was calculated in TreeAnnotator v.2.6 applying 10% burn-in, a posterior probability limit of 0.5, and “Mean Heights” for node heights.