The phylogenetic position of Vincetoxicum pannonicum (Borhidi) holub supports the species’ allopolyploid hybrid origin

The Pannonian endemic species Vincetoxicum pannonicum was described from specimens collected in Hungary and occurs at only few locations. It is considered “vulnerable” according to the International Red List. The chromosome set was reported to be tetraploid, and the species was hypothesized to be an allotetraploid hybrid of the Balkan species V. fuscatum and the Adriatic species V. hirundinaria subsp. adriaticum. We investigated the origin of V. pannonicum using molecular phylogenetic methods by separately analyzing the multicopy nuclear ribosomal internal transcribed spacer (nrITS) and the plastid-encoded trnH-psbA DNA regions and by evaluating discrepancies between the produced gene trees. Paralogs in the nrITS region clustered in two main groups, one of which was closest to V. fuscatum, and the other included V. hirundinaria subsp. adriaticum. According to trnH-psbA sequences, V. pannonicum and V. hirundinaria subsp. adriaticum formed a single group. Our results show that V. pannonicum diversified because of hybrid speciation, in which V. fuscatum was the pollen donor. We discovered a similar placement of V. maeoticum, which suggests a further hybridization event between V. fuscatum and a species of the V. hirundinaria group. Our genome-size estimate indicates almost sixfold larger genome size in V. pannonicum compared to the maternal diploid parent, suggesting hexaploidy; however, V. pannonicum is tetraploid. This may suggest cytological diploidization in the allopolyploid V. pannonicum. We observed substantial genetic distance between V. hirundinaria subsp. adriaticum and all other subspecies of V. hirundinaria, and we therefore propose that V. adriaticum should be regarded as a separate species.