Allotetraploid Origin and Putative Ancient Introgression in Plantago hakusanensis (Plantaginaceae)

Plantago hakusanensis (2n = 4x = 24) is an endangered endemic species that occurs in subalpine zones in Japan. To clarify the unresolved phylogenetic position of P. hakusanensis within subgenus Plantago, we constructed a phylogenetic tree based on the nuclear-encoded single-copy gene sucrose-proton symporter 1 (SUC1) using 60 previously reported alleles from 24 taxa in subgenus Plantago. We found that P. hakusanensis was closely related to Plantago asiatica var. densiuscula. The phylogenetic relationships between P. hakusanensis and P. asiatica var. densiuscula were further examined by analyses of the SUC1 nuclear regions and the internal transcribed spacer (ITS) of rDNA, genome-wide single-nucleotide polymorphism genotyping (via multiplexed inter-simple sequence repeat genotyping by sequencing), as well as by additional analyses of three chloroplast (cp) regions (trnL-F, ndhF-rpl32, and rpl32-trnL) in 25 individuals of P. hakusanensis and 53 individuals of P. asiatica var. densiuscula covering the species' geographical distribution. Monophyly of P. hakusanensis was suggested by the nuclear marker analyses, whereas the cp haplotypes of P. hakusanensis were shared with P. asiatica var. densiuscula and P. asiatica in China. The disparity between the nuclear and cp data may be explained by the introgression of the cp genome (cp capture). This research provides a phylogenetic tree showing the position of P. hakusanensis within subgenus Plantago and molecular evidence that implies complicated introgressions between P. hakusanensis and P. asiatica var. densiuscula.