The use of low-copy nuclear genes in the radiation of the Macaronesian Crassulaceae Sempervivoideae - Phylogeny and evolutionary processes

Speciation and evolution of species are two of the most exciting topics in biology. Radiations, with their wide morphological and physiological variety, provide a promising tool to understand speciation and diversity of species. Numerous studies have revealed that the high morphological diversity of radiated species is not represented at the molecular level. Neutral markers like rDNA nrITS and chloroplast (cp) DNA evolve slowly compared to speciation in radiations and thus, may not provide enough information to resolve phylogenetic relationships. In contrast, low-copy nuclear genes evolve faster and may help to resolve relationships. This is supported by the hypothesis that accelerated changes in regulatory genes, as opposed to structural genes, can explain the evolution of species. To contribute to this ongoing discussion, the radiation of the Macaronesian Crassulaceae Sempervivoideae (MCS) was studied. The polyploid species of the MCS are mainly distributed on the Canary Islands and comprise more than 70 species in three genera (Aeonium, Aichryson, and Monanthes) that display a huge morphological (e.g., flower color, number of floral organs, growth-form) and physiological (e.g., CAM activity) variety. Two regulatory genes, homologs of the floral homoeotic genes APETALA1 and APETALA3, and the structural gene encoding for phosphoenolpyruvate carboxylase (PEPC) were analyzed with respect to the following aims: 1) to evaluate the use of the low-copy nuclear genes to reconstruct phylogenies and to compare genealogies with the species phylogeny; 2) to estimate the impact of the studied genes in the speciation process and elucidate differences between the roles of regulatory and structural genes; 3) to determine if gene duplications occurred and to distinguish duplicates into orthologs and paralogs, and 4) to calculate the selection pressure (Ka/Ks-values) acting on the respective gene copies. The three analyzed low-copy nuclear genes both support and contradict the phylogenetic relationships inferred by other markers. The selection acting on the studied low-copy genes is in contrast with the neutral evolution of nrITS and cpDNA markers and may explain observed differences. In particular APETALA3 seems to be a promising marker for resolving species relationships. In addition, the studied genes may have had an influence in speciation since individually they exhibit accelerated Ka/Ks-values compared to mean Ka/Ks-values estimated for regulatory and structural genes. Their Ka/Ks-values are also much higher than those obtained for other genes in studies with comparable experimental designs. Accelerated evolutionary rates were estimated for the regulatory genes as opposed to the structural gene PEPC. However, summarizing all observations, the impact of these genes may be limited. Further study is recommended to evaluate their true impact. For all studied genes duplications were observed and emphasize the greatest challenge of working with low-copy nuclear genes – the differentiation of orthologs and paralogs. The observed duplication pattern suggests that the gene duplications are the result of polyploidization, a phenomenon to which the island colonization of the MCS species was connected previously. In addition, all gene copies were under purifying selection pressure, even if the estimated Ka/Ks-values for the respective copies varied. Rate differences were estimated for PEPC and APETALA3; the latter also showed significant differences in the Ka/Ks-values comparing copy A and copy B. For APETALA1 similar evolutionary rates and highest Ka/Ks-values were found. Altogether, this thesis offers a promising approach to study speciation and evolution in the radiation of the MCS and is a valuable basis for further studies