Target enrichment improves phylogenetic resolution in the genusZanthoxylum(Rutaceae) and indicates both incomplete lineage sorting and hybridization events

Abstract

Background and aims: Zanthoxylum L. is the only pantropical genus within Rutaceae, with a few species native to temperate eastern Asia and North America. Efforts using Sanger sequencing failed to resolve the backbone phylogeny of Zanthoxylum. In this study, we employed target enrichment high-throughput sequencing to improve resolution. Gene trees were examined for concordance and sectional classifications of Zanthoxylum were evaluated. Off-target reads were investigated to identify putative single-copy markers for bait refinement, and low-copy markers for evidence of putative hybridization events. Methods: We developed a custom bait set for target enrichment of 745 exons in Zanthoxylum and applied it to 45 Zanthoxylum species and one Tetradium species as the outgroup. Illumina reads were processed via the HybPhyloMaker pipeline. Phylogenetic inferences were conducted using coalescent and concatenated methods. Concordance was assessed using quartet sampling. Off-target reads were assembled and putative single- and low-copy genes were extracted. Additional phylogenetic analyses were performed based on these alignments. Key results: Four major clades are supported within Zanthoxylum: the African clade, the Z. asiaticum clade, the Asian-Pacific-Australian clade, and the American-eastern Asian clade. While overall support has improved, regions of conflict are similar to those previously observed. Gene tree discordances indicate a hybridization event in the ancestor of the Hawaiian lineage, and incomplete lineage sorting for the American backbone. Off-target putative single-copy genes largely confirm on-target results, and putative low-copy genes provide additional evidence for hybridization in the Hawaiian lineage. Only two of the five sections of Zanthoxylum are resolved as monophyletic. Conclusion: Target enrichment is suitable to assess phylogenetic relationships in Zanthoxylum. Our phylogenetic analyses reveal that current sectional classifications need revision. Quartet tree concordance indicates several instances of reticulate evolution. Off-target reads are proven useful to identify additional phylogenetically informative regions for bait refinement or gene tree based approaches.

The Euscaphis japonica genome and the evolution of malvids

“…The branching order for E. japonica , fabids, and malvids displayed a high level of discordance among gene trees, with two nearly equally supported (and one slightly less supported) topologies in both nucleotide and amino acid sequence‐based analyses (Figure S7 b,d). Incomplete lineage sorting may play a role in confounding the resolution of diverging branches within angiosperms (Mirarab et al., 2014 ; Reichelt et al., 2021 ). Therefore, we speculate that the incomplete lineage sorting during the diversification of the ancestral population may have led to the unstable phylogenetic position of E. japonica .…”