Palacký University Olomouc Faculty of Science Department of Botany Microevolutionary processes in apomictic genus Taraxacum. Ph.D. Thesis RNDr. Ľuboš Majeský Supervisor: RNDr. Radim Jan Vašut, Ph.D. Olomouc 2013 2 BIBLIOGRAFICKÁ IDENTIFIKACE Meno a priezvisko autora: RNDr. Ľuboš Majeský Názov práce: Mikroevoluční procesy v apomiktickém rodě Taraxacum. Typ práce: doktorská Pracovisko: Katedra botaniky – UP Olomouc Štúdijný program: Biologie Štúdijný odbor: Botanika Školiteľ: RNDr. Radim Jan Vašut, Ph.D. Rok obhajoby: 2013 ABSTRAKT Rod Taraxacum představuje druhově velmi početnou a morfologicky variabilní skupinu vytrvalých rostlin se složitými evolučními vztahy. Tato komplexnost je způsobena retikulární evolucí zahrnující diploidní (sexuální) a polyploidní (apomiktické) druhy. V rodě převažuje obligátní asexuální rozmnožování, ačkoli některé sexuální druhy mají rovněž široký areál rozšíření. Ze své povahy mají apomiktické druhy (jedinci) jen velice omezené možnosti k získaní variability. V předložené práci jsem se zabýval studiem genetické variability apomiktických a sexuálních druhů s cílem popsat procesy probíhající v populacích apomiktických klonů. Za populaci apomiktického klonu je považována morfologicky homogenní jednotka rozlišována v taxonomii tohoto rodu na úrovni tzv. apomiktického mikrodruhu. V práci byly použity jednak v součastnosti známe (vědecky popsané druhy), a součastně výrazné (morfologicky stabilní) morfotypy, které dosud validně popsány nebyly, a to ze dvou sekcí T. sect. Taraxacum a T. sect. Erythrosperma. Pro získání co největšího množství informací pro vysvětlení procesů, které mohly vést k pozorované strukture klonů, byly použity tři typy molekulárních markerů – SSR, AFLP, cpDNA. Výsledky mé práce ukazují, že apomiktické klony pampelišek jsou geneticky vysoce homogenní, přestože nejsou výlučně tvořeny pouze jediným klonem. Populace apomiktických klonů jsou tvořeny i) klonálními genotypy, ii) odvozenými klonálními genotypy (lišící se nahromaděnými somatickými mutacemi) a iii) klonálními liniemi. Klonální linie pocházejí z hybridizace mezi asexuálními a sexuálními jedinci. Klony apomiktických mikrodruhů jsou geneticky dobře charakterizovatelné a navzájem vyhraněné. V oblastech, kde apomiktičtí a sexuální jedinci tvoří smíšené populace, dochází k hybridizaci a tím ke vzniku nových apomiktických klonů. Opakovaná hybridizace nově vzniklých apomiktických klonů může následně vést k vytvoření nového apomiktického mikrodruhu, pokud se klon dokáže rozšířit na větší vzdálenosti. Hybridizace je zde interpretována a chápána jako proces zvyšující variabilitu v populacích apomiktů, který umožňuje získat nové vlastnosti a tak přispět k pozitivní selekci daných taxonů. S přispěním experimentálních dat z této dizertace byla potvrzena genetická homogenita jednoho dříve rozlišovaného a vzácnějšího morfotypu – Taraxacum pudicum, který byl proto popsán jako nový druh pro vědu. Klíčová slova: Taraxacum, apomixie, klon, variabilita, mutace, hybridizace Počet stran: 142 Jazyk: anglický 3 4 BIBLIOGRAPHIC IDENTIFICATION Author’s first name and surname: RNDr. Ľuboš Majeský Title of the Thesis: Microevolutionary processes in apomictic genus Taraxacum. Type of Thesis: Ph.D. Thesis Department: Department of Botany – UP Olomouc Study program: Biology Field of Study: Botany Supervisor: RNDr. Radim Jan Vašut, Ph.D. Year of defence: 2013 ABSTRACT Taraxacum represents abundant and widespread genus of perennial herbs well known for its high evolutional and morphological complexity. This is caused by reticulate evolution including diploid (sexual) and polyploid (apomictic) species. Obligate apomixis is prevalent in the genus and sexually reproducing species with wide distribution are known for only some of the groups. Thus in general, apomictic taxa have restricted sources for acquiring of any variability. In this thesis, I have investigated genetic variability of apomictic and also sexual taxa with the aim to describe the processes ongoing on the level of populations of apomictic clones. Population of apomictic clone represents here a morphological unit, traditionally recognized as microspecies in the taxonomy of dandelions. I used scientifically recognized microspecies as well as undescribed morphotypes (stable in their morphology) from two sections: T. sect. Taraxacum and T. sect. Erythrosperma. Three different molecular markers (SSR, AFLP, cpDNA) were used to gain all the possible information on processes leading to the observed genetic structure of the clones. Results of my work showed that apomictic clones of dandelions are genetically highly homogenous, although not always uniclonal. Populations of apomictic clones consist of i) clonal genotypes, ii) clonal mates with accumulated somatic mutations and iii) clonal lineages resulting from hybridisation events among apomicts and sexuals. Apomictic clones— microspecies—are genetically well separated from each other with specific fingerprint. In regions with sympatric occurrence of apomicts and sexuals, new apomictic clones are formed in hybridisation process. Recurrent hybridisation of neo-apomictic clones may lead to formation of new apomictic microspecies, which after successful spreading may become widespread. Hybridisation is seen also as process for increasing the variability and possibility to gain a new positively selected trait. Results of this thesis contributed also to scientific description of one new apomictic microspecies of dandelions – Taraxacum pudicum, which was found to be genetically uniform and thus of a clonal origin. Key words: Taraxacum, apomixis, clone, variability, mutation, hybridisation Number of pages: 142 Language: English 5 6 DECLARATION I hereby declare that this thesis has been worked out by myself together with listed coauthors. All literary sources cited in this thesis are listed in the References section. AUTHOR CONTRIBUTIONS CHAPTER 1 Introduction ĽM wrote the text. CHAPTER 2 The pattern of genetic variability in apomictic clones of Taraxacum officinale indicates the alternation of asexual and sexual histories of apomicts Published in PLoS ONE. RJV and ĽM conceived and designed the experiments. ĽM performed the experiments and analyzed the data. ĽM, BT, MK and RJV contributed reagents/material/analysis tools. ĽM and RJV wrote the paper. BT and MK edited manuscript. BT phenotyped the plant material. MK – contributed to optimalization of AFLP. CHAPTER 3 Genotypic variability of obligate apomicts is enriched by the gene pool of sexuals in contact zones between sexual-apomictic dandelions (Taraxacum sect. Erythrosperma) BMC Evolutionary biology (submitted). RJV conceived the idea of the study, and RJV and ĽM designed the study. Plant material was collected by RJV and ĽM or was kindly provided by colleagues, RJV phenotyped the plant material. ĽM did the molecular work and performed the statistical analyses. MK contributed to AFLP analyses and helped with the lab work. ĽM wrote the first draft, and MK and RJV improved subsequent versions of the manuscript. All authors read and approved the final manuscript. CHAPTER 4 Taraxacum pudicum, a new apomictic microspecies of the section Erythrosperma from Central Europe Manuscript prepared to be submitted to Preslia. RJV discovered the described species. RJV did comparative cultivation, flow- cytometric analyses, counted chromosome numbers, tested microsatellites for the species and made scientific description. ĽM cultivated F1 offspring of the species and studied genetic diversity of the species, its F1 offspring and its relatives. 7 CHAPTER 5 Where is the place for apomictic taxa in taxonomic hierarchy of apomictic genera? Let's take a look on apomictic dandelions Manuscript prepared to be submitted to Taxon. ĽM and RJV conceived the idea of the review. ĽM wrote the first draft and RJV improved the subsequent version. 8 ACKNOWLEDGEMENTS This thesis could not be done without interplay of coincidences, which led me to come to Olomouc to Palacký University and without help of many people to, which I am very grateful. I would like to express my thanks to my supervisor Dr. Radim J. Vašut, to provide me the opportunity to do my research at the Department of Botany UP Olomouc. I am thankfull for his supervision, for the time we spent in the field collecting of dandelions necessary for present thesis, for his help and critical comments about writing of scientific papers. I thank to Dr. Miloslav Kitner (Department of Botany, UP Olomouc) for his lab assistance, especially with AFLP technique and for valuable comments. I am thankfull also to Associate professor Bohumil Trávniček (Department of Botany, UP Olomouc), for his expert knowledge of dandelions, for providing seeds and herbarium vouchers of plants necessary for my research and for his valued suggestions. I thanks to Tim Sharbel (IPK Gatersleben) for allowing me to spent valued time in his work group and his lab and to perform some experiments and also for his improving suggestions that help me in my research. I thank to Dr. Martin Dančák (Department of Ecology UP Olomouc) for his friendship and for his great passion in plants. I express my thanks also to Dr. Petr Nádvorník (Department of Cell biology and Genetics, UP Olomouc) for initial help with SSR analyses, to Petra Šarhanová and Petra Macháčková for assistance with flow cytometry and to Tereza Pěnkavová for taking care of my plants. My special thanks go to my grandfather, who showed me the beauty of nature and learned me to love and respect the nature, to my grandmother who was always helpful whenever