Multiple lines of evidence supports the two varieties of Halenia elliptica (Gentianaceae) as two species

Abstract

Delimiting species requires multiple sources of evidence. Here, we delimited two varieties of <i>Halenia elliptica</i> (Gentianaceae) using several lines of evidence, including morphological traits and mating system in a sympatric population, phylogenetic relationships based on nrITS and cpDNA (rpl16) data, and complete chloroplast genome sequences. Comparative analysis of 21 morphological traits clearly separates the two varieties of <i>H. elliptica</i>. Examination of the flowering process and pollination treatments indicate that <i>H. elliptica</i> var. <i>grandiflora</i> produces seeds via outcrossing, whereas <i>H. elliptica</i> var. <i>elliptica</i> produces seeds via mixed mating. Furthermore, hand-pollinated hybridization of the two varieties produced no seeds. Observations of pollinators showed that when bees began a pollination bout on <i>H. elliptica</i> var. <i>grandiflora</i> they preferred to continue pollinating this variety; however, when they began a pollination bout on <i>H. elliptica</i> var. <i>elliptica</i>, they showed no preference for either variety. Phylogenetic analysis confirmed the monophyly of <i>H. elliptica</i>, which was further divided into two monophyletic clades corresponding to the two varieties. A large number of variants from the chloroplast genomes reflected remarkable genetic dissimilarities between the two varieties of <i>H. elliptica</i>. We recommend that the two varieties of <i>H. elliptica</i> should be revised as two species (<i>H. elliptica</i> and <i>H. grandiflora</i>). Our findings indicate that <i>H. elliptica</i> varieties may have split into two separate species due to a shift in mating system, changes in flowering phenology and/or post-pollination reproductive isolation.