PhD Abstracts

Jaroslav Zahradníček	PhD	"Phylogeny and evolutionary trends in subtribe Hieraciinae (Asteraceae)"
Abstract: Subtribe Hieraciinae includes the taxonomically intricate polyploid and mostly apomictic genera Pilosella and Hieracium as well as the diploid sexual genus Andryala. It offers a unique possibility to compare evolutionary trends and processes in closely related genera with contrasting frequency of polyploids, modes of reproduction, and geographical distrubution. The thesis is focused on Hieracium s.str. and Andryala; the genus Pilosella was studied by other authors. Genome size of so-called 'basic' Hieracium s. str. (Hieracium subgen. Hieracium) was estimated and correlated with results of phylogenetic analysis based on the nuclear DNA marker ETS, ploidy level, breeding system and ecogeographic features. Inter- and intraspecific variability in genome size was also analyzed. Genome size variation corresponded with results of a molecular phylogeny that separated three main clades reflecting geographical distribution in Europe ('western', 'eastern', and hybridogenous). The monoploid genome size in the 'western' species was significantly lower than in the 'eastern' ones. Intraspecific variability was generally low. Genome size downsizing was confirmed in a comparison of monoploid C values among diploid and polyploid cytotypes. Correlation of genome size with longitude was apparent for the whole data, correlations with latitude and altitude were not significant. Evolutionary history and genome size pattern and evolution were explored in Andryala, a medium-size genus distributed mainly in the Mediterranean Basin and Macaronesia. To reconstruct the relationships within the genus we used three nuclear markers (ETS, ITS and single-copy gene SQS) and two chloroplast markers (trnT-trnL and trnV-ndhC). While cpDNA analysis confirmed a previously inferred chloroplast capture event with the sister genus Pilosella, nuclear markers supported the monophyletic origin of Andryala. None of phylogenetic analyses resulted in sufficient resolution, due to very low levels of nucleotide divergence of two nuclear and two chloroplast markers and a high degree of homoplasy and incomplete lineage sorting in the variable SQS marker. Only two well-supported basal lineages corresponding to relict species A. laevitomentosa and A. agardhii were separated. The rest of Andryala species collapsed to a well-supported large group named here 'Major Radiation Group'. Relationships inside this group are largely unresolved. Regarding the genome size, highest C values were detected in basal relict species (A. laevitomentosa, A. agardii) and in two populations of A. ragusina. Another two populations of A. ragusina have distinctly lower C values. Higher intraspecific variation of genome size in a few species might be explained by allopatric differentiation including island populations. In addition, special attention was also payed to phylogeography and cytotype structure of Hieracium intybaceum, the sister member of all Hieraciinae genera forming a transitional and still clearly unexplored lineage among the four Hieraciinae genera. 43 populations collected across the distribution range in the Alps and the Vosges Mts were explored using flow cytometry and AFLP molecular markers. We detected two ploidy levels, diploid and tetraploid, with contrasting modes of reproduction (sexuality in diploids, apomixis in tetraploids). Diploids were found all across the Alps, while tetraploids only in the westernmost Alps and the Vosges Mts. Genetic variation was very low. Bayesian clustering identified four clusters/genetic groups, which are partly congruent with the ploidy pattern and geographical distribution. We suppose that diploids colonized the deglaciated areas from source populations most likely located in the southern part of the recent distribution range and in the western Alps.
Karol Krak	PhD	"Molecular phylogeny and evolutionary trends in Hieracium (Asteraceae, Lactuceae)"
Abstract: 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.
Pavel Trávníček	PhD	"Microevolutionary processes and inter-cytotype interactions in mixed-ploidy populations"
Abstract: This thesis is aimed at a better understanding of cytotype co-existence in mixed-ploidy populations with an emphasis on microevolutionary processes behind it. Our past knowledge was based on a few thoroughly investigated model taxa like Chamerion angustifolium and Heuchera grossulariifolia, but some generalizations seem to be premature in the light of new findings. A detailed research of other taxa included in the thesis showed that polyploid complexes can vary dramatically in their ability to cope with the co-existence of cytotypes in mixed-ploidy populations. Whereas mixed-ploidy populations are virtually lacking in some species (an example being Vicia cracca), ploidy-heterogeneous populations are very common in others, maintained by free mating interactions and the absence of reproductive isolation among cytotypes (e.g. Pilosella echioides). The strength and cumulative effect of various breeding barriers (both pre- or postzygotic) govern the position of a particular multi-ploidy complex between these two extremes and co-determine the type of cytotype co-existence in its mixed-ploidy populations. Despite the fact that the number of studies revealing cytotype co-existence has been increasing rapidly, evolutionary background and consequences of such coexistence are usually unknown. To answer these questions, a detailed and time consuming research involving long-term field observations and experiments is needed. Results presented in this thesis are mostly based on pilot investigation, which is essential to reveal the status quo, but is insufficient for the understanding of the evolutionary processes behind it. Therefore, an ongoing thorough research on selected plant groups (namely the Gymnadenia conopsea agg. and Pilosella echioides) is being conducted to gather as much information as possible from various points of view. This research is focused on: (i) the determination of the rate of inter-ploidy crosses in field conditions, (ii) the understanding of the further fate of inter-ploidy hybrids under natural selection, their ability to backcross and produce viable progeny, and finally (iii) the detection of the role of such hybrids in the further evolution of mixed-ploidy populations. Although the papers included in this thesis hopefully broaden our horizon and shed some light on possible ways of cytotype co-existence, further thorough investigation is needed to cover the spectrum of evolutionary mechanisms shaping the population structure in species with ploidy heterogeneity. The value of such research lies in our understanding of processes behind polyploid evolution and the evolution of plant biota in general.
Tomáš Urfus	PhD	"Evolutionary mechanisms and relationships among taxa of genus Pilosella"
Abstract: Several case studies at diffrent levels (population, regional, comparative etc.) were carried out to evaluate the importance of different sources of variation and to follow microevolutionary traits in Pilosella (Asteraceae). The gradient of different spatial levels and comparative studies of closely related taxa group (characterised by partly different type of ongoing microevolution - genus Picris) revealed some common processes while at the same time highlighted the uniqueness of genus Pilosella. Microevolutionary potential was studied at the population level using morphometric approach (hybridization tendencies of highly complex hybrid swarm in Prague - Praha Vysočany). Cytogeography of Pilosella officinarum was then investigated at the regional scale (Central Europe). Tetraploid cytotype was detected almost exclusively in Bohemia region and further to the west whereas pentaploid and hexaploid cytotypes prevailed in Moravia and Slovakia and further to southeast. Cytogeography was followed by morphometric evaluation of three most abundant ploidy levels (4x, 5x and 6x) of P. officinarum combined with the analyses of reproduction mode. Results confirmed the possibility to distinguish the particular cytotypes (intermediate pentaploids partly overlapping with the tetra- and hexaploids). Different morphological traits of sexual vs. apomictic hexaploids were uncovered. Finally the microevolutionary processes of Pilosella were compared to those observed in Picris hieracioides group. The existence of two distinct morphological groups of P. hieracioides was confirmed by differences in their absolute genome size. Genome size and morphology of Balkan endemic Picris hispidissima was found to be significantly different compared to the closely related taxon to P. hieracioides. The comparative case studies of Picris revealed highly distinct microevolutionary pattern compared to genus Pilosella.
Tomáš Peckert	PhD	"A taxonomical study of Hieracium sect. Echinina in Central Europe"
Abstract: The present study bringst the first detailed information about polyploidy and agamospermy in Hieracium echioides. The species was used as a model one to contribute to the solution of general questions about evolutionary processes in polyploid plant species. In addition, genetic variation, chromosome number and breeding system were investigated in agamospermous populations of H. echioides subsp. echioidesvar. tauscheri in the Danube Basin region. Experimental crossing among diploids, triploids and tetraploids of Hieracium echioides was made to examine mating interactions. Specifically, cytotype diversity in progeny from experimental crossing, intercytotype pollen competition as a reproductive barrier among diploids and tetraploids, differences in seed-set, pollen size and beginning of flowering periode and differences in genome size among cytotypes were studied. Only diploids were found in progeny from 2x × 2x crosses. The other types of crosses yielded more than one cytotype in progeny, however, one cytotype predominated in each cross type: triploids (93%) in reciprocal crosses between diploids and tetraploids, diploids (92%) in 2x × 3x crosses, tetraploids (88%) in 3x × 2x, tetraploids (60%) in 3x × 3x crosses, pentaploids (56%) in 3x × 4x crosses, triploids (80%) in 4x × 3x crosses and tetraploids (88%) in 4x × 4x crosses. No aneuploids were detected among karyologically analysed plants. Unreduced egg cells production was detected in triploids and tetraploids, on the contrary, formation of unreduced pollen was recorded only in two cases in triploids. Triploid plants produced x, 2x and 3x gametes, however, in male gametes predominated x (92%) gametes whereas in female gametes predominated 3x (88%) gametes. No statistically significant difference in seed-set obtained from intra- and intercytotype crosses between diploids and tetraploids were observed suggesting absence of post-zygotic reproductive barriers among cytotypes. Cytotype diversity of progeny from crossing where diploids and tetraploids were pollinated by mixture of pollen from diploid and tetraploid plants suggested intercytotype pollen competition to serve as a prezygotic reproductive barrier. There was no significant difference in the seed-set of diploids and tetraploids, but they differed significantly from seed-sets of triploids and pentaploids. No difference among cytotypes in beginning of flowering periode was found. Values of pollen size were associated with ploidy level. Genome size of diploids, triploids and tetraploids was found to be in principle identical. Only pentaploids had lower values of genome size than the other cytotypes. The origin of polyploidy in H. echioides is discussed based on the knowledge of genome size pattern in the subgenus. Detailed flow cytometric analysis of population of co-existing diploid, triploid, tetraploid and pentaploid cytotypes of Hieracium echioides was carried out at the model locality (Havranické vřesoviště heathland, Czech Republic, south-west Moravia). The analysed population is formed by a system of in size restricted subpopulations. In total, 392 plants from twelve subpopulations were analysed. Random selection of 25 plants per subpopulation was carried out in nine subpopulations. Three subpopulationswere analysed in detail (all plants were analysed). Analysis of 225 randomly selected plants from nine subpopulations revealed 24 (10.67%) diploids, 154 (68.44%) triploids, 42 (18.67%) tetraploids and 5 (2.22%) pentaploids. Detailed analysis of three subpopulations revealed almost only diploid plants. Analysed subpopulations differed in cytotype structure. One cytotype predominated in each subpopulation. The subpopulations were divided into three categories according to the dominant cytotype. In most subpopulations, triploids predominated. In one subpopulation, the most common cytotype was represented by tetraploi one. Finally, in four subpopulations (ncluding three in detail analysed ones), diploids prevailed. Generally, the pattern of cytotype distribution has striking clump-like character. The dynamic of the population is discussed with respect to results obtained during previous hybridization experiments. Six populations of Hieracium echioides subsp. echioides var. tauscheri from the Danube Basin between Bratislava and Budapest (locations: Balinka, Čenkov, Devín, Dorog, Győr, Pilis) were analysed using allozyme and karyological analysis. Five allozyme systems (EST, LAP, 6PGDH, PGM, and SKDH) were used to analyse the genetic structure of the examined populations. Analyses revealed low genetic variation both within- and among populations. Four multilocus allozyme phenotypes were detected; three populations (Čenkov, Devín, and Győr) possessed phenotype I exclusively, while phenotype II was found only in the Balinka and Dorog populations. Two different phenotypes were found in the population of Pilis (phenotypes III and IV). However, due to the complex banding patterns generated for EST, allelic interpretation was not possible, and the Balinka and Dorog populations appeared to possess different phenotypes. All populations proved to be tetraploid (2n = 36) and agamospermous. The geographic distribution pattern of the analysed populations (one allozyme phenotype at several isolated localities) may reflect a more common occurrence of the taxon in the past. Landscape changes, caused by changes in human management of the country, may have resulted in a loss of suitable localities, mainly open sandy habitats. These changes may have caused the reduction and fragmentation of H. tauscheri habitat.
Zita Ferreira	PhD	"Biosystematics of the genus Andryala L. (Asteraceae)"
Abstract: Andryala (Asteraceae: Cichorieae) is a little-known Mediterranean-Macaronesian genus whose taxonomy is much in need of revision. The aim of the present biosystematic study was to elucidate species relationships within this genus based on morphological and molecular data. In this study, several taxa are recognised: 17 species, 14 subspecies, and 3 hybrids. Among these, 5 species are Macaronesian endemics (A. glandulosa, A. sparsiflora, A. crithmifolia, A. pinnatifida, and A. perezii), 4 species are Northwest African endemics (A. mogadorensis, A. maroccana, A. chevallieri, and A. nigricans), and one species is endemic to Romania (A. laevitomentosa). The historical background regarding the taxonomic delimitation in the genus is addressed from Linnaean to present day concepts, as well as the origin of the name Andryala. The origin of Asteraceae and the systematic position of Andryala is shortly summarised. The morphological study was based on a bibliographic review and the revision of 1066 specimens of 13 herbaria as well as additional material collected during fieldwork. The variability of the morphological characters of the genus, including both vegetative taxonomic characters (root, stem, leaf and indumentum characters) and reproductive ones (inflorescence, floret, fruit and pappus characters), was assessed. Numerical analysis of the morphological data was performed using different similarity or dissimilarity measures and coefficients, as well as ordination and clustering methods. Results support the segregation of the recognised taxa and the congruence of several analyses in the separation of the recognised taxa (using quanititative, binary, or multi-state characters). The proposed taxonomy for Andryala includes a new infra-generic classification, new taxa, and new combinations and ranks, typifications and diagnostic keys (one for the species and several for subspecies). For each taxon, a list of synonyms, typification comments and a detailed description are provided, also comments on taxonomy and nomenclature, and a brief discussion on karyology. Additionally, information on ecology and conservation status as well as on distribution, and a list of the material studied are also presented. Phylogenetic analyses based on different nuclear and chloroplast DNA markers, using Bayesian and maximum parsimony methods of inference, were performed. Results support three main lineages: separate ones for the relict species A. agardhii and A. laevitomentosa and a third including the majority of the Andryala species that underwent a relatively rapid and recent speciation. They also suggest a single colonization event of each, Madeira and the Canary Islands, from the Mediterraneaen region, followed by insular speciation. Biogeography and speciation within the genus are briefly discussed, including a proposal for the centre of origin of the genus and possible dispersal routes.