High epizoochorous specialization and low DNA sequence variation in Mediterranean Cynoglossum (Boraginaceae): evidence from fruit traits and ITS region

Fruit morpho-anatomy and DNA sequence diversity in Euro-Mediterranean taxa of Cynoglossum and the closelyrelated genera Solenanthus and Pardoglossum were analysed to assess the structural traits that promote dispersal throughtransport via the fur of mammals, and to evaluate the phylogenetic value of carpological variation in the group. Electron andlight microscopy showed striking epizoochorous adaptations in characters of the pluricellular projections of the epicarp (glochids),such as multiple apical hooks, conical shape, and finely tuberculate surface, as well as the heavy mineralization of thecell walls with especially silicon and calcium revealed by X-ray microanalysis. The attachment potential of nutlets to sheepfleece, estimated through a General Linear Model, was species-specific and relatively high in the taxa with small and lightdiaspores (> 60%). ITS sequences from 29 specific and infraspecific accessions were poorly variable, with pairwise geneticdistances ranging from 0.002 to 0.097 (mean 0.044). A comparative analysis of ITS sequence diversity in relation to the differentdispersal strategies in the four main Boraginaceae tribes revealed substantially higher levels of variation and interspecificgenetic distances in the non-epizoochorous groups, including the Cynoglosseae genera Myosotis and Omphalodes. Bayesian andmaximum parsimony tree construction suggested paraphyly of Cynoglossum due to the nested position of Solenanthus apenninusand Pardoglossum. Species-level relationships remained largely unresolved, preventing an estimation of the phylogeneticsignificance of fruit characters. Rapid spread across the Mediterranean region via epizoochory by wild mammals is possiblythe primary cause for the lack of genetic divergence among species. During the Holocene, nomadic and transhumant pastureby domesticated herbivores has likely contributed to further spreading and mixing of previously isolated taxa, possibly causingevents of homoploid hybridization and introgression. More variable molecular markers should be tested to better understandthe impact of these processes in the evolutionary history of Cynoglossum and to solve relationships within Cynoglosseae.