Abstract
Aesculus L. comprises 19 putative species which are horticulturally important trees and shrubs. A recent molecular study using RAD-seq data with 68 samples of Hippocastaneae showed that, in contrast to Billia Peyr. which was supported in previous studies, a sister relationship between Aesculus and Handeliodendron Rehder was strongly supported for the first time. In the current study, we tested the sister genus of Aesculus by evaluating the newly generated trnH-trnK data alongside published chloroplast coding region matK and the entire ITS region (ITS1+5.8S+ITS2), singly and in combination in 80 individuals representing 13 species of the genus Aesculus. We also included the highest number of outgroups to date from the remaining Hippocastanoideae subfamily and from the Xanthoceroideae, Dodonaeoideae and Sapindoideae subfamilies. The results of our phylogenetic analyses with broad and diverse taxon sampling (i.e., up to 82 outgroups) revealed that the sister genus of Aesculus is Billia and not Handeliodendron, as was recently suggested. Therefore, the current study highlights the importance of outgroup sampling and the crucial effect of outgroups on ingroup phylogeny.