The use of low-copy nuclear genes in the radiation of the Macaronesian Crassulaceae Sempervivoideae – Phylogeny and evolutionary processes Dissertation Korinna Esfeld Born in Lutherstadt Wittenberg 2009 Dissertation submitted to the Combined Faculties for the Natural Sciences and for Mathematics of the Ruperto-Carola University of Heidelberg, Germany for the degree of Doctor of Natural Sciences presented by Diplom-Biologin Korinna Esfeld born in: Lutherstadt Wittenberg Oral-examination: ....................................... i The use of low-copy nuclear genes in the radiation of the Macaronesian Crassulaceae Sempervivoideae – Phylogeny and evolutionary processes Referees: Prof. Dr. Marcus Koch Prof. Dr. Claudia Erbar ii ... für meine Familie und meine Freunde, die geduldig daran geglaubt haben! Am Ende ist alles gut und wenn es nicht gut ist, dann ist das nicht das Ende.... iii Contents Contents Contents................................................................................................................................iv Figures ...................................................................................................................................v Tables .................................................................................................................................. vii 0.1. Summary...................................................................................................................... viii 0.2. Zusammenfassung.........................................................................................................ix 1. Introduction ....................................................................................................................... 1 2. Materials and Methods .....................................................................................................21 2.1. Study species ............................................................................................................21 2.2. Molecular methods ....................................................................................................26 2.2.1. RNA material ......................................................................................................26 2.2.2. DNA material ......................................................................................................26 2.2.3. Amplification of the low-copy nuclear genes........................................................27 2.2.4. Cloning of the low-copy nuclear genes................................................................30 2.2.5. Colony PCR........................................................................................................31 2.2.6. Amplification of nrITS and cpDNA regions ..........................................................32 2.2.7. Sequencing.........................................................................................................32 2.2.8. Sequence analysis..............................................................................................32 2.2.9. Definition of the gene regions .............................................................................33 2.2.10. Improvements of the datasets...........................................................................33 2.2.11. Partition Homogeneity Tests .............................................................................34 2.2.12. Phylogenetic reconstructions ............................................................................34 2.2.13. Blast analyses...................................................................................................35 2.2.14. Neighbor-joining reconstructions for homologs of PEPC , AP1 , and AP3 ...........35 2.2.15. Species phylogeny............................................................................................36 2.2.16. Nucleotide differences, replacements, and amino acid substitutions.................36 2.2.17. Relative Rate Tests...........................................................................................37 2.2.18. Selection pressure ............................................................................................37 3. Results .............................................................................................................................39 3.1. Datasets ....................................................................................................................39 3.2. Phylogenetic reconstructions .....................................................................................45 3.3. Blast and Neighbor-joining analyses..........................................................................64 3.4. Species phylogeny based on nrITS ...........................................................................66 3.5. Gene duplications......................................................................................................67 3.6. Nucleotide differences, replacements, and amino acid substitutions..........................69 3.7. Relative Rate Tests ...................................................................................................73 3.8. Ka/Ks-values .............................................................................................................74 3.9. Selection pressure.....................................................................................................76 4. Discussion........................................................................................................................78 4.1. Phylogenetic reconstructions .....................................................................................78 4.2. Gene duplications......................................................................................................86 4.3. Selection pressure.....................................................................................................97 4.4. Regulatory versus structural genes..........................................................................103 5. Summary........................................................................................................................108 Literature............................................................................................................................110 Abbreviations .....................................................................................................................125 Overview of scientific contributions.....................................................................................127 Appendices ........................................................................................................................128 Acknowledgment................................................................................................................150 iv Figures Figures Fig. 1: Map of Macaronesia and the Canary Islands after Mort et al. (2002). Taken from the website http://www.eiu.edu/~bio_data/posters/2002/poster_016.htm............................... 2 Fig. 2: Maximum parsimony phylogram of the MCS species based on cpDNA/nrITS data (from Mort et al. 2002)........................................................................................................... 9 Fig. 3: MIKC-like structure of the plant MADS-box proteins (adapted after Purugganan et al. 1995). ..........................................................................................................................15 Fig. 4: The extended ABCDE model adapted after Erbar (2007)..........................................17 Fig. 5: Schematic exon and intron structure of the MCS_PEPC gene sequences. Exons are shown as boxes and introns as lines. The relative length of the respective parts is given in table 4. ....................................................................................................................40 Fig. 6: Schematic exon and intron structure of the MCS_AP1 gene sequences. Exons are shown as boxes and introns as lines. The relative length of the respective parts is given in table 6. ....................................................................................................................42 Fig. 7: Schematic exon and intron structure of the MCS_AP3 gene sequences. Exons are shown as boxes and introns as lines. After the last exon box the 3´-UTR is indicated as line. The relative length of the respective parts is given in table 8....................................44 Fig. 8: BI phylogram based on the full-length MCS_PEPC data. Posterior probabilities are given at the nodes..........................................................................................................47 Fig. 9: BI phylogram based on the MCS_PEPC exon data. Posterior probabilities are given at the nodes................................................................................................................49 Fig. 10: ML phylogram based on the MCS_PEPC intron data. Bootstrap support is given at the nodes. ........................................................................................................................51 Fig. 11: ML phylogram based on the MCS_AP1 full-length data. Bootstrap support is given at the nodes................................................................................................................53 Fig. 12: ML phylogram based on the MCS_AP1 exon data. Bootstrap support is given at the nodes. ........................................................................................................................55 Fig.