Abstract

Genetic crosses between the dioecious Bryonia dioica Jacq. (Cucurbitaceae) and the monoecious B. alba L. in 1903 provided the first clear evidence for Mendelian inheritance of dioecy and made B. dioica the classic case of XY sex determination in plants. We use chloroplast (cp) and nuclear (nr) DNA sequences from 129 individuals representing all morphological species to study species relationships and distribution, sexual system evolution, and association of ploidy-level with dioecy in Bryonia. Chloroplast and nuclear trees mostly fit morphological species concepts; there are seven dioecious and three monoecious species, together ranging from the Canary Islands to Central Asia. Bryonia verrucosa, the morphologically most differing species from the Canary Islands is sister to all other species. Our data argue for the inclusion of the narrowly endemic Central Asian species B. lappifolia and B. melanocarpa in B. monoica. Conflicts between cp and nr topologies imply that the dioecious hexaploid B. cretica arose from hybridization(s) involving the diploid species B. dioica, B. syriaca, and/or B. multiflora. The tetraploid B. marmorata likely originated via autopolyploidy. The nr phylogeny implies at least two transitions between dioecy and monoecy, but no correlation between change in sexual system and ploidy level. Fossil-calibrated molecular clocks using family-wide rbcL data with a Bryonia-centered sampling suggest that the deepest divergence in Bryonia occurred ca. ten million years ago and that monoecious and dioecious species crossed in the classic studies are separated by several million years of evolution. Traits, such as annual regrowth from a tuberous rootstock and other adaptations to a seasonal climate, as well as species and haplotype abundance, point to an origin of Bryonia in the Middle East. Species and haplotype poverty north of the Alps together suggest recolonization there after the last glacial maximum. Most species of Bryonia have 10 chromosomes (as confirmed by my own counts), and there appears to be no morphologically distinct pair that would represent the sex chromosomes. However, we know from the crossings carried out by Correns and others that in B. dioica, sex shows monofactorial dominant inheritance, setting up the hypothesis that B. dioica may have a pair of chromosomes on which key sex-determining gene(s) and sexlinked genes have accumulated. To gain insight into the possible presence of such a pair of sex chromosomes in B. dioica, it is necessary to sequence a fairly long sex-linked region to study its substitution behavior and to eventually visualize its physical placement using FISH. As a first step towards this goal, I developed a sex-linked SCAR marker for B. dioica from AFLP bands and sequenced it for individuals representing the full distribution range of the species from Scotland to North Africa. The region north of the Alps harbours distinct Y and X alleles that differ in a 197-bp indel, with the Y allele being perfectly linked to the male sex. In southern Europe, however, the XY system appears to break down (to an extent that is not clear), and there are signs of recombination between the Y and X homologues. Population genetic analyses suggest that the sex-linked region I amplified (i.e., the SCAR marker) experienced different evolutionary pressures in northern and southern Europe. These findings fit the evidence from my phylogenetic and phylogeographic analyses that the XY system in Bryonia is evolutionarily labile. Overall, my work suggests that Bryonia may be a good, but very complex, system in which to study the early steps of plant sex chromosome evolution.