Charles University in Prague, Faculty of Science Department of Botany Molecular phylogeny and evolutionary trends in Hieracium (Asteraceae, Lactuceae) Molekulární fylogeneze a evoluční trendy v rodě Hieracium (Asteraceae, Lactuceae) Karol Krak Ph.D. thesis Prague, May 2012 Supervised by: Dr. Judith Fehrer Content Declaration.........................................................................................................1 Acknowledgements............................................................................................2 Sumary...............................................................................................................3 Introduction.........................................................................................................4 Aims of the thesis.............................................................................................14 References.......................................................................................................15 Papers included in the thesis 1. Intra-individual polymorphism in diploid and apomictic polyploid.................22 hawkweeds (Hieracium, Lactuceae, Asteraceae): disentangling phylogenetic signal, reticulation, and noise. Fehrer J., Krak K., Chrtek J. BMC Evolutionary Biology (2009) 9: 239 2. Genome size in Hieracium subgenus Hieracium (Asteraceae) is...............45 strongly correlated with major phylogenetic groups. Chrtek J., Zahradníček J., Krak K., Fehrer J. Annals of Botany (2009) 104: 161–178 3. Development of novel low-copy nuclear markers for Hieraciinae................63 (Asteraceae) and their perspective for other tribes. Krak K., Álvarez I., Caklová P., Costa A., Chrtek J., Fehrer J. American Journal of Botany (2012) 99: e74–e77 4. Reticulate evolution and lineage sorting in Hieracium s. str. ......................67 (Asteraceae): evidence from low-copy nuclear gene and cpDNA Krak K., Caklová P., Chrtek J., Fehrer J. Conclusions...........................................................................................................97 Professional Curriculum vitae..............................................................................100 Supplementary Files............................................................................................103 Hereby I declare that I made this thesis independently, using the mentioned references. I have not submitted or presented any part of this thesis for any other degree or diploma. Karol Krak (in Prague 18. 5. 2012) Author contribution statement I declare that I made substantial part of the thesis papers (manuscripts). Contribution to each paper is specified below: 1. Judith Fehrer, Karol Krak, Jindřich Chrtek: Intra-individual polymorphism in diploid and apomictic polyploid hawkweeds (Hieracium, Lactuceae, Asteraceae): disentangling phylogenetic signal, reticulation, and noise. Molecular data acquisition, participation on data analyses and manuscript preparation; Total contribution 40% 2. Jindřich Chrtek, Jaroslav Zahradníček, Karol Krak, Judith Fehrer: Genome size in Hieracium subgenus Hieracium (Asteraceae) is strongly correlated with major phylogenetic groups. Molecular data acquisition and substantial contribution to data analysis; Total contribution: 25% 3. Karol Krak, Ines Álvarez, Petra Caklová, Andrea Costa, Jindřich Chrtek, Judith Fehrer: Development of novel low-copy nuclear markers for Hieraciinae (Asteraceae) and their perspective for other tribes. Study design, lab work, data analyses and manuscript preparation; Total contribution: 80% 4. Karol Krak, Petra Caklová, Jindřich Chrtek, Judith Fehrer: Reticulate evolution and lineage sorting in Hieracium s. str. (Asteraceae): evidence from low-copy nuclear gene and cpDNA Lab work, data analyses and interpretation, manuscript preparation; Total contribution: 80% Jindřich Chrtek .................... Petra Caklová .................... Judith Fehrer .................... 1 Acknowledgements I would like to thank to Judith Fehrer for sharing her knowledge on molecular systematics, fruitful discussions and thorough supervision of my work. Jindřich Chrtek, my consultant, was always around, ready to help inconspicuously. His deep knowledge of Hieracium taxonomy was essential for successful completion of the thesis. Petra Caklová supported me continuously during those long years, not only as a colleague with substantial contribution to the labwork, but as well as a good friend. I thank Jaroslav Zahradníček for sharing his data on genome size, Siegfried Bräutigam and Franz Schuhwerk for their help with determination of plant material. Patrik Mráz and Pavel Trávníček are acknowledged for valuable comments on the fourth manuscript. I won’t be able to submit the thesis without the help of Petr Vít and Tomáš Urfus: they patiently corrected the countless typing errors and provided excellent technical assistance during the finalization. I would like to express my thanks to my family and Martina for their love, patience and encouragements. With them even the worst working day turned to a nice evening. I’m grateful to all those that provided plant material: S. Bräutigam, F. Krahulec, A. Krahulcová, G. Mateo, P. Mráz, M. Niketić, Z. Szeląg, P. Trávníček, T. Tyler, V. Vladimirov, J. Zahradníček. The work was supported by the Czech Science Foundation (Projects No. 206/05/0657 and P506/10/1363) and SYNTHESYS program of the EU (Project No ES-TAF-1365). 2 Summary The hawkweed subgenus Hieracium s. str. is notoriously known for its extreme morphological variability and variation in ploidy levels that is associated with differences in modes of reproduction. Extensive past hybridization is supposed for the subgenus, but recent hybridization was evidenced only in few cases. The subgenus attracts the attention of botanists already for more than a century. Therefore the species diversity is largely examined and the taxonomy of the subgenus is well elaborated, although several contradictory taxonomic concepts exist. However the relationships among the species are unknown and haven’t been studied yet. The investigation of these relationships from a phylogenetic perspective using molecular approaches was the main aim of the presented thesis. Basic species (both diploid and polyploid), representing morphologically unique taxa, that are supposed to be the basic evolutionary units of the subgenus were studied. The sequences of two intergenic spacers of the cpDNA (trnT-trnL and trnV-ndhC) and the external transcribed spacer of the nuclear ribosomal DNA (nrDNA ETS) were analyzed. Moreover, three new low-copy nuclear markers with higher variability than nrDNA and cpDNA markers were developed and their suitability for phylogenetic studies in Hieracium s. str. was evaluated. One of these, a part of the gene coding for squalene synthase (sqs) was included to the study as the second nuclear marker. Based on ETS, two previously unrecognized major evolutionary lineages were found, that correlated well with the geographic origin and distribution of the species. The observed pattern in genome size variation was in high correlation with this phylogenetic pattern. On the other hand, the cpDNA and sqs phylogenies were incongruent with the ETS, in respect of these major groups. However, they reflected well the subgroups identified within the major ETS clades. This pattern together with the low interspecific variability of the ETS indicates that Hieracium s. str. is a group with rather recent speciation. The level of interspecific hybridization found greatly exceeded the previous expectations: almost half of the studied accessions was evidenced to be of hybrid origin. Moreover, hybrid origin was inferred not only for the polyploids, but it concerned diploids as well. Genetic material representing most probably already extinct parental lineages was identified in some of the hybrid accessions. Hybridization in the subgenus is probably even more abundant as described above, but its detection was obscured by population level (genetic drift, incomplete lineage sorting) or intragenomic (concerted evolution) processes. 3 Introduction Driving forces of plant evolution Fundamental sources of variation in living organisms are associated with structural changes in genetic material – mutations. Due to these changes, new alleles related to novel functional traits arise in populations. Hence, mutations together with gene flow (migration), genetic drift and natural selection are acting as engines of speciation, the process of novel species formation. However, in the plant kingdom, speciation is shaped by additional processes more extensively than in other groups of organisms. These factors include hybridization, polyploidization and apomixis. Hybridization Hybridization is defined as crossing between individuals belonging to different species or genetically divergent populations within a species (Soltis & Soltis 2009). Despite recognised already by Linné (e.g. Rieseberg 1997), the role of hybridization in plant evolution was underestimated for a long time (Arnold 1997). Nowadays is already known that hybridization may have various evolutionary consequences including the increase of intraspecific genetic diversity, origin of genetic adaptation as well as origin of new ecotypes or species (Rieseberg 1997). Hybridization in higher plants is quite widespread. About 25% of all plant species are known to hybridize at least with one other species. This number may be underestimated, because hybrid origin is often challenging to prove (Mallet 2007). Interspecific hybridization introduces