Hybridization plays a major role in speciation. However, hybridization and reticulate evolution in general are poorly understood in tree species because genetic documentation is often missing. Analyses of biparentally inherited gene regions allow detection of reticulate signals. Multicopy and single-copy nuclear markers may show significant intraindividual variability owing to reticulation processes. Naturally, such processes induce incompatible phylogenetic signal resulting in incongruent genealogies. Data from three nuclear markers, two multicopy nrDNA spacers, and the single-copy 2nd intron of the LEAFY gene, mirror ancient and recent horizontal gene flow in plane trees (Platanus). In addition to previously assembled data from the internal transcribed spacers (ITS) of the 35S ribosomal DNA, we found atypical 5S rDNA intergenic spacer sequences (5S-IGS) causing significant intra- and interindividual polymorphism, and a conspicuous LEAFY intron dimorphism. A detailed framework of reticulate molecular evolution of Platanus can be erected using splits graphs based on distances between cloned sequences or individuals, and competing topologies. Two hundred and sixty-one 5S-IGS sequences and LEAFY genotyping of 71 individuals via sequence analysis and PCR-RFLP support a previous ITS study (including pseudogenous and non-pseudogenous variants) suggesting that the modern North American taxa P. rzedowskii and P. occidentalis var. palmeri are the result of ancient hybridization. Platanus occidentalis var. palmeri requires taxonomic revision and is provisionally treated at species rank.
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