Abstracts of Presentations

Bräutigam et al.	2000	Hybridogenous complex of Hieracium floribundum/H. pilosella in the low mountains of Central Europe: a complex view.
Abstract: Hieracium (hawkweeds) is one of the world's most diverse and species-rich genera (1000-10000 species, depending on species concept). Three clearly separated subgenera exist (also treated as independent genera by some authors), which clearly differ in their mode of reproduction. Apomixis of different types is common in subgen. Hieracium (diplospory) and subgen. Pilosella (apospory), the latter exhibiting an enormous variability in respect to ploidy levels (ranging from diploid to octoploid in the field). Facultative and obligate apomicts as well as sexuals occur, leading to extremely complex reticulate patterns of populations and species. In the present study, we investigated four species of a hybridogenous series of Hieracium subgen. Pilosella - H. floribundum Wimm. & Grab and H. pilosella L. as putative parents and H. iseranum Uechtr. and H. piloselliflorum N. & P. s.l. [including H. apatelium N. & P.] as hybridogenous types - by different approaches. Morphologically, H. iseranum is more similar to H. floribundum while H. piloselliflorum is closer to H. pilosella. The two first mentioned species show only low morphological variability while great variability is observed in the last two. With the exception of widespread H. pilosella, the main distribution of the species within Central Europe lies in the eastern mountain regions. Our study is constrained to an area spreading from the Erzgebirge Mts (N slopes in Saxony) to the Krkonoše (West Sudeten, S slopes in the Czech Republic), i.e., the borderland of Germany, Poland and the Czech Republic. With rare exceptions, H. floribundum, H. iseranum and H. pilosella are tetraploid; for H. piloselliflorum, tetra-, penta- and hexaploid plants were found (rarely, all cytotypes were found to occur together). H. floribundum and H. iseranum individuals of the Krkonoše exhibit a single, unusually large "marker" chromosome, which occurs only very rarely in H. piloselliflorum and which is completely lacking in H. pilosella. Haploid DNA content determined by flow cytometry is intermediate for H. piloselliflorum with respect to H. floribundum/H. iseranum and H. pilosella indicating a presumed hybrid origin. Isoenzyme analysis and Southern hybridisation by mini- and microsatellite probes reveal the existence of different clones within populations of (supposedly apomictic) H. iseranum and H. piloselliflorum. PCR-RFLP analyses of non-coding chloroplast DNA (trnT-trnF) indicate an identical haplotype for H. pilosella and H. piloselliflorum. The H. iseranum haplotype differs from those of the other species investigated. Experimental crosses between H. pilosella (as mother plant) and H. iseranum or H. piloselliflorum lead to variable offspring being similar to H. piloselliflorum. While natural populations contain only euploid plants, artificially produced offspring is partly aneuploid in heteroploid crosses. Evidence from the different approaches taken together with observations under field conditions lead to the hypothesis, that H. iseranum might be a stabilised species of ancient hybridogenous origin. In contrast, H. piloselliflorum is most likely a complex of early generation hybrids with H. pilosella as maternal parent.
Chrtek	2000	The genus Hieracium s.str. in the Carpathians - once more to the question of apomictic 'microspecies'.
Abstract: The Carpathians are extremely rich in montane Hieracium taxa. The richest areas are situated in the highest parts of the Western Carpathians (Slovakia, S Poland) and in the south part of the arc (the Retyezat Mts.). Sytematic studies on selected species groups have revealed important differences between the Western and Eastern Carpathians. In the Western Carpathians, diploid sexual species are most likely lacking completely nowadays; in the Eastern Carpathians, besides apomicts, sexual species occur. While many triploid and most probably obligate agamospermous taxa have been found in the Western Carpathians, in the eastern part, triploids are extremely rare, and most of the agamospermous taxa are pollen-bearing tetraploids. Consequently, recent gene exchange between sexual diploids as well as between pollen-bearing tetraploid apomicts and sexual diploids (or their hybrids) in the Eastern Carpathians is highly expected (experimental studies are now in progress, experimental crosses between H. alpinum and H. transsilvanicum resulting in progeny morphologically similar to H. krasani have been performed). Isozyme patterns revealed higher genetic variation of Eastern Carpathian tetraploid apomicts compared to apomicts from the Western Carpathians (considerably high genetic variation was only found in H. alpinum s.str.). Low genetic variation of selected taxa was also confirmed by RAPD analysis. Recent processes of differentiation and consequently patterns of morphological variation in the Eastern Carpathians cause serious problems in taxonomic treatment. In contrast, fixed and often clearly separated agamospermous taxa in the Western Carpathians can be evaluated at the species level.
Fitze & Fehrer	2000	PCR-RFLP studies of non-coding chloroplast DNA in European Hieracium subgen. Pilosella.
Abstract: As part of an ongoing project about the biodiversity, genetic structure and differentiation of Hieracium, subgenus Pilosella, the relationships of the species occurring in the borderland of Saxony (Germany), the Czech Republic and Poland have been investigated by PCR-RFLP analysis of the chloroplast trnT-trnF spacer region. As outgroups, species of Hieracium subgen. Hieracium and Chionoracium as well as of the closely related genus Andryala were included. The target region was PCR-amplified using the universal primers described by TABERLET et al. (1991. Plant Mol. Biol. 17: 1105-1109). Amplification products were submitted to restriction enzyme digestions. Out of 15 enzymes tested, only AluI, MboI, BclI, BglII and EcoRI yielded differences between the species and were therefore used for further analysis. No changes in restriction sites, but only length variations were observed. Fragment sizes were determined. Identical haplotypes were observed for representatives of the following species: H. cymosum/H. glomeratum, H. pilosella/H. piloselliflorum, H. bauhini/H. leptophyton, H. lactucella/H. floribundum/H. aurantiacum, the three subspecies of H. caespitosum (madarum, caespitosum and colliniforme), H. piloselloides ssp. obscurum/H. echioides ; H. sabaudum/H. umbellatum, H. schneiderianum/H. alpinum. In three cases (H. glomeratum, H. piloselliflorum, H. leptophyton), the species have previously been considered as presumed early generation hybrids based on their morphology, occurrence, DNA content and ploidy levels. Because of the maternal inheritance of the chloroplast DNA, evidence for the seed parent is now suggested. The result is in accordance with the assumed modes of reproduction. H. floribundum - although being a "main" species in the sense of NÄGELI & PETER (1885. Die Hieracien Mittel-Europas, Piloselloiden. München) - was suggested to be a hybrid between H. lactucella and H. caespitosum (KRAHULCOVÁ & KRAHULEC, 1999. Preslia 71: 217-234). Usually, the few sexual species (H. pilosella - tetraploid, H. cymosum and H. lactucella - diploid) served as the seed parent. In the case of H. bauhini, however, an unreduced chromosome set of this pentaploid apomict might have been pollinated by diploid (reduced) pollen of H. pilosella, yielding the heptaploid hybrid H. leptophyton. For the remaining haplotypes, no direct evidence from other sources is as yet available to explain the patterns. More data are needed to elucidate these relationships and homology of the length variations has to be established by DNA sequencing. Generally, the Hieracium species of all subgenera as well as of closely related Andryala showed only little sequence variation as judged by the missing RFLPs. Insertions/deletions seem to account for most variation as has also been shown recently for other Asteraceae of the Lactuca tribe (VIJVERBERG & BACHMANN, 1999. Amer. J. Bot. 86: 1448-1463). However, most variation was found between trnT-trnL in our case, but not in the trnL-trnF part of the amplified fragment, as indicated by a restriction site map and partial DNA sequencing.
Krahulcová & Krahulec	2000	Offspring diversity in Hieracium subgen. Pilosella: new cytotypes obtained from hybridization experiments.
Abstract: We studied within-species patterns of Hieracium subg. Pilosella occurring in the Krkonoše Mts (Riesengebirge), a range with high diversity of different types. They differ in ploidy level and reproductive systems. To understand their morphological patterns found in the field, a series of experimental crosses was carried out, in which the following species of the Hieracium subgen. Pilosella were used as parents: diploid H. lactucella and tetraploid H. pilosella (both sexuals, used predominantly as seed parents), tetraploid H. caespitosum, tetraploid H. aurantiacum, pentaploid H. glomeratum and pentaploid H. piloselliflorum (all at least facultative apomicts, used predominantly as pollen donors). The chromosome numbers within the F1 progeny of crosses were examined. In addition, the karyological diversity in the offspring of some of the selected open pollinated F1 hybrids was studied, as well as in the F1 progeny of open pollinated hexaploid hybridogenous species H. rubrum. Some of the individual hybrid seed parents hybridised easily being open pollinated, which led to considerable karyological diversity within their progeny: it is indicative for their (at least) facultative sexuality. Furthermore, both the haploid parthenogenesis and the fertilisation of the unreduced egg cell contributed to offspring diversity. These phenomena were especially manifested in H. rubrum. The haploid partheno-genesis in an aneuploid hybrid individual obtained from experimental cross was recorded as a new observation. There is a discrepancy between the rare occurrence of aneuploids in nature in Europe and the abundant viable aneuploid progeny that can be easily obtained from experimental crosses. A similar case concerns triploids: their sparse occurrence in the field is in contrast to the common rise of viable triploids from the crosses between diploids and tetraploids. The reasons for these disproportions are discussed, expecting the reduced competitive ability of polyhaploids in the field, but, on the other hand, the more common participation of unreduced egg cells in the origin of natural polyploids.
Mráz et al.	2000	Genetic variability in selected taxa of Hieracium sect. Alpina.
Abstract: Two taxa included in the present study belong to the Hieracium rohacsense group. H. rohacsense KIT. is considered to be endemic to the high mountain ranges of the W Carpathians. However, many Central European authors have treated not only W Carpathian populations under the name H. rohacsense s.str., on which the name is based, but also those from the other mountains of Central Europe. Pollen production in cultivated plants in experimental fields was found to be completely disturbed, although a small amount of pollen of heterogeneous size with normal exine has been observed in two studied plants. The male sterility phenomenon underlines the genetic isolation of H. rohacsense. The population of the probably still unnamed taxon closely related to H. rohacsense was found during the expedition to the Ukrainian E Carpathians on Mt. Pop Ivan (Marmarosh Mts.) in 1996. Plants from Mt. Pop Ivan have less grey and darker involucral bracts than H. rohacsense because of less numerous stellate and clothing trichomes and more abundant glandular ones. The plants from this population produce considerable quantities of heterogeneous-sized pollen grains with normal exine in the field and in cultivation. H. rohacsense as well as plants from Mt. Pop Ivan are tetraploid (2n=36) with an apomictic mode of reproduction proved by emasculation experiments. The study was aimed at the evaluation of the genetic variation within and between populations of H. rohacsense. We investigated whether isozymes can be useful as discriminatory taxonomic markers between H. rohacsense s.str. and the closely related population from Mt. Pop Ivan. Five isozyme systems (AAT, ADH, LAP, PGM, SKD) were studied. No genetic intra- and inter-population variability was detected in H. rohacsense, which is in accordance with its narrow morphological variation. In contrast, the population from Mt. Pop Ivan is represented by three genotypes. The plants on this locality vary in density of the indumentum on involucral bracts, but this variation does not deserve taxonomic recognition. Presence of diploid sexual taxa in the Ukrainian E Carpathians (H. alpinum L., H. conicum ARV.-TOUV. and H. transsilvanicum HEUFF.) may contribute to the increase of genetic variability. H. rohacsense and plants from Mt. Pop Ivan can be separated by morphological characters and also on the base of their isozyme patterns. Some other tetraploid taxa studied in less detail (H. stygium UECHTR., H. chlorocephalum s.l. and H. nigritum s.l.) have unique isozyme phenotypes within each taxon. The name H. rohacsense KIT. has also been used in a wide sense for a long time for what we now understand as H. rauzense MURR from the E Alps. One electrophoretically analysed plant of H. rauzense differs from H. rohacsense in morphology, ploidy level (the former one is triploid) and by its isozyme banding pattern. The isozyme data provide evidence of heterozygosity, as has been shown in other agamic genera.
Peckert & Chrtek	2000	The Hieracium echioides group in Central Europe.
Abstract: Two species, i.e. Hieracium echioides Lumn. and H. rothianum Wallr. are included in the present study. Hieracium echioides is distributed in Central and Eastern Europe and in the steppe region of Asia. Westwards it reaches central Germany, the Czech Republic and eastern Austria. H. rothianum is an intermediate type linking H. echioides and H. pilosella, more closely resembling H. echioides. Its distribution range, at least in some regions, overlaps with that of H. echioides. The objectives of our study are: (i) to prepare a list of localities of both target species in Central Europe, (ii) to ascertain chromosome numbers and mode of reproduction in selected European populations - and if there is a geographical pattern of the distrubution of particular cytotypes (several cytotypes have been reported in literature, i.e., diploids, triploids and tetraploids), (iii) to test whether karyological differentiation is congruent with patterns of morphological variation, and (iv) to elucidate the origin of H. rothianum populations - whether they are of polytopic hybrid origin or if the recent geographical distribution is a result of migration from one genetic centre. The preliminary results show that all previously reported cytotypes of H. echioides occur within the territory of the Czech Republic and Austria. Two or three cytotypes were found to co-occur at many localities, except of localities with relic character, where only diploids were found (e.g., Mohelno in western Moravia). Only diploids were found in Hungary. Morphologically, populations from the Czech Republic and Austria are rather invariable. Distinct types represent populations from north-eastern Germany (Brandenburg) with densely-haired inflorescence and upper part of stems. Diploids and tetraploids of H. echioides were found to be sexual and allogamous as shown by emasculation and isolation experiments with free pollination. Triploids after isolation of inflorescences did not produce viable achenes. No comparable results from free pollination are available yet. Therefore, no decision between allogamy and sterility can be made. H. rothianum is morphologically variable, e.g., some populations from the Czech Republic differ markedly from type material (Germany). It seems to be tetraploid only (2n=36); all studied plants from the Czech Republic and Slovakia were proved to be agamospermous.
Štorchová et al.	2000	Assessment of relationships between diploid and triploid plants of H. alpinum on the basis of RAPD data.
Abstract: The study of hawkweeds in the mountains of Central Europe and Ukraina represents a good model for microevolution of higher plants. The members of sect. Alpina (Fr.) F.N.Williams inhabit the highest vegetation belts and their stations form an "island" system. H. alpinum L. s.str. differs from other representants of this section from the Tatry Mts. by the considerable level of within-population genetic variability revealed by RAPD (Štorchová et al., submitted). Populations of H. alpinum L. s.str.from Tatry Mts. (Slovakia) together with populations of this microspecies from the Krkonoše and Jeseníky Mts. (Czech Republic) and from the Western Carpathians (Ukraina) were screened with an additional 20 primers. The aim of this study was to find population-specific RAPD primers that distinguish between various populations, but which should not be able to detect within-population variability. Two primers - ABA 07 and ABA 19 - provided reproducible results and divided the populations of triploid plants from the Czech Republic and Slovakia into two groups, roughly corresponding to their geographical distribution. However, the same primers revealed within-population variation in the populations of diploid H. alpinum from Ukraina. This finding is in agreement with the supposition about the contribution of sexuality to the reproduction of diploid plants. The RAPD phenotype typical for the populations of triploid H. alpinum was identical to some individual RAPD phenotypes in populations of Ukrainian hawkweeds. More primers will be checked to compare the population-specific RAPD patterns of triploid H.alpinum to the individual-specific patterns of diploid Ukrainian hawkweed.
Šimek et al.	2001	Study of the species Hieracium cymosum L. in the Czech and Slovak Republic.
Abstract: Hieracium cymosum (H.c.) is a subatlantic / subcontinental species with a distrib-ution from SE France to W Siberia and from the Balkan to Scandinavia. The sparse knowledge about its biology available from literature was additionally confounded by unclear conception of the nature of the species. Therefore, a comprehensive analysis of this species was performed in order to study its geographic distribution and ecology, karyological differentiation, reproductive systems and genetic variability. The investigation area chosen was the territory of former Czechoslovakia. A checklist and distribution map was produced by screening of the literature, investigation of herbarium specimens and by personal field work. Approximately 50 localities were confirmed whereas at 15 described earlier, no H.c. was detected. Localities were concentrated on warm regions with occurrence of relic habitats as pine woods, thermophilous oak forests on rocks, edges of forests and steppe grasslands. In Slovakia, where it showed a scattered distribution, it reached altitudes of about 1000 m. In Czech Republic, three regions of higher abundance were found: Bohemian Highlands (České Středohoří Mts.), canyons of central Bohemian rivers and SW Moravia. For 39 populations, karyological studies were performed. Usually, chromosomes were counted for at least three plants per population. 32 populations consisted of diploid (2n = 18) plants only. In one, a mixture of diploid and tetraploid plants, which could not be distinguished by their morphology was found. According to isolation and emasculation experiments, these individuals were sexual. The same held for another population of tetraploid sexuals only. In a further population, a single triploid plant was detected, which was of a weak condition. At four further localities, morpho-logically different plants ("cf. cymosum") occurred, one consisting of tetraploid plants, two of pentaploids and one of a mixture of 4x, 5x and 6x individuals. All of them were apomictic. Genetic variability within and between populations was assessed by isozyme analysis. Seven sexual populations (five plants each) were investigated with three enzyme systems (AAT, PGM and SHDH). All of them were variable. Between-population variability did not exceed within-population differences, i.e., no sub-structuring of H.c. was detected. The mixed 2x/4x sexual population revealed the highest variability with many additional alleles compared to the mere diploid ones. Additionally, multilocus fingerprinting with a human minisatellite probe (33.15) was used for the identification of apomictic lineages. Of the 76 plants analysed, most showed individual-specific patterns. In some cases, two diploid H.c. had identical fingerprints, which was likely due to vegetative propagation via stolons. In contrast, (nearly) identical fingerprints were obtained for the two pentaploid populations. The diploid and tetraploid sexuals as well as the single triploid plant likely correspond to ZAHN'S H.c. subsp. cymosum and are suggested to be considered as H.c. s.str. The apomictic types (summarised here as "cf. cymosum"), which were morphologically characterised by a higher number of glandular hairs and fewer simple eglandular hairs, differ from each other and may correspond to different subspecies described in the literature. However, the database is not yet sufficient to draw firm conclusions.
Fehrer et al.	2002	Clonal distribution of apomictic Hieracium subgen. Pilosella species revealed by mini- and microsatellite fingerprinting.
Abstract: The geographic distribution of five apomictic species of Hieracium subgen. Pilosella which differ by their assumed origin and species status has been analysed in a transsect from the Erzgebirge to the Krkonoše (Riesengebirge) where their distribution areas overlap. 83 populations (239 plants) were analysed, in detail (populations/plants): H. caespitosum (21/63), H. glomeratum (17/51), H. iseranum (21/69), H. floribundum (20/45), and H. aurantiacum (4/11). In Southern Hybridisations, a human minisatellite probe (33.15) in combination with TaqI-digested genomic DNA produced highly variable individual-specific patterns which allowed to discriminate between single or multiple origin. Although some of the so-defined apomictic clones showed slight variation (more clearly demonstrated by a (GATA)5 microsatellite probe) attributed to somatic mutations, this intraclonal variation could be neglected in comparison to the large differences between the assumed clones (near-isogenic lineages) of single origin. The following distributions of apomictic clones were found: H. caespitosum: Unexpectedly, this species was represented by two clones only, a tetraploid (abundant in the whole region) and a pentaploid one (restricted to the Erzgebirge). H. glomeratum: Most plants formed a single pentaploid clone speading from the Oberlausitz to the Riesengebirge. A single tetraploid population/clone was detected in the Riesengebirge and another pentaploid one in the western Erzgebirge (the only glomeratum population which we found in that mountain range). H. iseranum: This species also consisted of a widespread clone with similar distribution as the abundant glomeratum clone, but six further clones were found (among them subsp. confinium), two even within the same population. All plants were tetraploid. H. floribundum: This species showed most lineages; two of the clones were somewhat more widespread (one in the Erzgebirge and one in the Riesengebirge), the others were local lineages. Material from the type locality did not correspond to any one of the other clones. H. aurantiacum: Only one tetraploid clone was found in the whole region. The so-called "Hauptarten" (caespitosum and aurantiacum) showed the least variability, comprising of 1-2 clones only in the whole investigation area. The other species ("Zwischenarten") form stabilised clones covering broad areas and also local populations indicating polytopic origin on different scale (e.g., few in glomeratum, many in floribundum). A certain geographic isolation between the mountain ranges (Erzgebirge / others) was present. In some cases, the fingerprinting detected erroneously determined plants which could properly be assigned by morphological re-examination. The almost complete corres-pondence between genotypes and morphological findings - not all clones could be distinguished morphologically as can be expected - make the DNA fingerprinting a valuable tool for the detection of apomictic lineages in Hieracium.
Fehrer et al.	2002	Evolution of the Hieracium/Andryala complex based on trnT-trnL sequences.
Abstract: Relationships of the three subgenera of Hieracium s.l. (Pilosella, Hieracium s.str. and Chionoracium), which differ in their mode of reproduction as well as in their distribution range, were studied. Several species of the closely related genus Andryala and some outgroup taxa were also included. Subgenus Pilosella was investigated in more detail. Based on a broader survey of PCR-RFLPs, the trnT-trnL intergenic spacer of cpDNA was chosen for sequencing. Two distinct groups of haplotypes were found in subgenus Pilosella, each represented by about half of the species examined. One of these Pilosella haplotype groups clustered with Andryala while the other clustered with Chionoracium and Hieracium s.str. Basal relationships among the subgenera were not resolved, however, each of the two Pilosella clusters seemed to be derived as well as monophyletic (one of them including Andryala). Fruit morphology, being a valuable trait in Lactuceae taxonomy, as well as other morphological features support a close relationship of Andryala and Pilosella. Also, their DNA content is similar (2.9-3.5 pg) in contrast to Hieracium s.str. (7.0-7.5 pg). As a preliminary conclusion, duplication/halfing of genome size, probably correlated with the diplosporous/aposporous mode of apomixis should have arisen at least two times independently.
Fehrer et al.	2002	Evolution, hybridisation, and clonal distribution of apo- and amphimictic Pilosella species in a Central European mountain range.
Abstract: Pilosella hawkweeds (Asteraceae, Lactuceae) are known for their difficult taxonomic structure which is caused by their mode of reproduction (facultative apomixis of the aposporous type) in combination with frequent hybridisations. In an area where many of these species occur together, i.e., the three-border land of Germany, Poland and Czechia, the genetic structure of several species and their presumed relationships was analysed by PCR-RFLPs and sequencing of cpDNA intergenic spacers and by Southern hybridisations with mini- and microsatellite probes. Additional information was obtained from morphological and cytological analyses (including ploidy level determination), experimental hybridisations and reproduction studies. At different geographic scales, the apomicts differed in their variability: from a single clone across the whole area via more widespread clones, but several origins, to multiple origins, even within the same locality. As a tendency, such apomicts considered as main species were less variable, followed by old fixed hybrids covering considerable areas whereas other intermediate taxa were of more recent origin or apparently hybridised frequently. Surprisingly, cpDNA haplotypes were usually conserved within species and did not yield any detectable geographic pattern. Furthermore, in several cases the seed parent did not have the haplotype of the sexual crossing partner. Only one single hybridogenous species showed one or the other type indicating that different parental species had acted as seed parents. In a phylogenetic framework based on sequences of the trnT-trnL intergenic spacer, Pilosella was divided into two monophyletic groups which were more divergent from each other than each was from the other hawkweed (sub)genera Hieracium and Chionoracium. One of these groups had some ancestral features (particularities of structural mutations) whereas the other was clearly derived. This suggests an early divergence of haplotypes predating the speciation within Pilosella and their general maintenance whithin species boundaries despite the extremely reticulate species structure.
Krahulcová & Krahulec	2002	Diversity in breeding system in Hieracium subgen. Pilosella: comparison of experimental and field data.
Abstract: The diverse mode of seed production contributes significantly to a complicated pattern of variation in this polyploid agamic complex. Moreover, the different ways giving rise to seed progeny can be combined within one individual female plant. Both the sexual reproduction (allogamy, which can be accompanied by autogamy) and apomixis (i. e., the asexual seed production) are known to operate in subgenus Pilosella. Apomixis is of the aposporous type, but with autonomous development of endosperm. Pollination, therefore, is not necessary for endosperm formation and successful seed production. Since apomixis in Pilosella is often facultative, one female plant can produce seeds in both, sexual and asexual, ways. In addition, two different mechanisms occur there, which give rise to seed progeny without fertilisation: somatic parthenogenesis (the embryo develops from an unreduced egg cell and haploid parthenogenesis (the embryo develops autonomously from a reduced egg cell). While the former way maintains the mother genome (including the ploidy level), the latter is connected with meiosis, possible recombination and reduction of mother ploidy level to a half. The demonstration of the breeding system is impossible without experimental work. All ways of seed production mentioned above were recorded in experiments with Pilosella, and the capability of diverse modes of reproduction was proven in this way. The hybridisation experi-ments resulted in hybrid progeny, which were compared with corresponding types from nature (e.g., Peter 1884, Gadella 1991, 1992). New cytotypes, different from parental ones, were recorded among progeny of experimental crosses (e.g. Gadella 1987, 1988, Chapman & Bicknell 2000, Krahulcová & Krahulec 2001), demonstrating the participation of unreduced gametes on the origin of highly polyploid hybrids. The self-fertilisation, operating under the influence of foreign pollen, was recorded in two sexual species, H. pilosella and H. lactucella (Krahulcová et al. 1999). The facultatively apomictic species (as e. g. H. aurantiacum, H. caespitosum and H. praealtum) hybridise both as male and as female parent (Chapman & Bicknell 2000, Krahulcová & Krahulec 2001, unpubl. data). The variation in asexual seed production, including the formation of polyhaploid progeny via haploid parthenogenesis, was demonstrated in experiments with H. aurantiacum (Skalińska 1971, Bicknell 1997), H. rubrum (Krahulcová & Krahulec 2001) and H. brachiatum (Krahulcová & Krahulec, unpubl. data). In the field, it is more difficult to reveal the origin of particular individuals, the parents of which are unknown. In such populations, where both the putative parents and their hybrids occur together, recent hybridisation events can be studied and documented. A shift to more frequent sexual reproduction (and hybridisation) is evidently important for adaptive radiation in New Zealand populations of H. pilosella (Chapman & Brown 2001). The capability for hybridisation in this facultative apomict was evidenced in field experiments in New Zealand (Houliston & Chapman 2001). Study of the expression of sexual reproduction and its correlation with environmental conditions is now in progress. The first case of a polyhaploid progeny (as a seed) was demonstrated in H. rubrum from nature (Krahulcová & Krahulec, unpubl. data). The feasibility to detect in nature the origin of rarely recorded highly polyploid hybrids, and of the putative polyhaploids, is discussed.
Krahulec et al.	2002	The Sudetic group of Hieracium subgen. Pilosella: a synthesis.
Abstract: Detailed research on the Sudetic group of Hieracium subgen. Pilosella has recently been carried out at Görlitz and Průhonice. It included population and species-level studies based on the following methods: studies of karyology and breeding systems, DNA-based studies (haplotypes, fingerprinting), and isozyme studies. This complex research allowed us to make the following conclusions: - The agamic complex of Sudetic species is composed of the following basic species: Sexual diploids: H. lactucella, H. onegense, H. cymosum subsp. cymosum; sexual tetraploids: H. pilosella; apomictic tetraploids: H. caespitosum, H. aurantiacum. - This complex involves common types which behave as independent apomictic entities, and rare types, probably the results of recent hybridisation events. The common types are: H. glomeratum, H. schultesii, H. floribundum, H. iseranum, H. pilo-selliflorum, H. rubrum, H. blyttianum, H. cymosum subsp. cymigerum. This group represents already established hybridogenous species, some of them produced by recent hybridisation events (H. schultesii, H. piloselliflorum, H. cymosum subsp. cymigerum). The rare ones involve: H. dubium, H. macranthelum, H. macrostolonum, H. tubulascens, H. fuscoatrum, H. stoloniflorum, and a diploid type corresponding to H. floribundum (prob-ably the recent hybrid between H. onegense and H. lactucella). These rare types are usually represented by one population only, or very rare individuals dispersed within populations of other types. - Most of the polyploids are represented by one common and one or two rare cytotypes. - It is evident that most of the hybridogenous species with broader distribution are of poly-topic origin (originated several times). There are big differences in number of types and scale, where this phenomenon is expressed. This phenomenon is documented by different cytotypes, different isozyme- and fingerprinting-based phenotypes. - There is growing evidence that at least some of the hybridogenous species might be of polyphyletic origin. Repeatedly, the same phenotype has been obtained from crossings involving different parent species. The differences in haplotypes of H. piloselliflorum suggests this phenomenon occurs in the field, too. Evidence obtained from hybridisation experiments and haplotype studies allows us to suggest the detailed history of individual members of the agamic complex. For example, hexaploid H. rubrum seems to result from a combination of unreduced female gametes (egg cells) of tetraploid H. aurantiacum and diploid pollen of H. pilosella; pentaploid H. stoloniflorum was probably produced by reduced gametes of H. rubrum and H. pilosella (the mother plant should be determined by haplotype study).
Severa & Chrtek	2002	On Hieracium villosum and some related taxa in the Western Carpathians.
Abstract: Hieracium villosum Jacq. belongs, together with its intermediate taxa, to the most interesting groups of mountain hawkweeds in the Western Carpathians (Slovakia, S Poland). We examined pattern of morphological and genetic (using isozyme analysis) variation, chromosome numbers and modes of reproduction of H. villosum (16 populations), H. scor-zonerifolium Vill. (H. villosum ł H. bupleuroides, 2 populations), H. dentatum Hoppe (H. villosum ł H. bifidum, 2 populations) and in one still unnamed and taxonomically unclear taxon resembling (but not fully identical with) H. valdepilosum Vill. (H. villosum - H. pre-nanthoides, 6 populations). Based on morphology, chromosome numbers, and multilocus isozyme genotypes, 5 types were recognised within H. villosum. Generally, all of them are geographic vicariants; a co-occurrence of two types was observed at only three localities. The most widespread type, corresponding to H. villosum subsp. villosum, proved to be an apomictic triploid (2n=27) with aborted pollen; no genetic variation was discovered. The remaining types are apomictic tetraploids (2n = 36) with viable pollen grains, confined to rather small geographic areas. Both intra- and interpopulation genetic variation was detected in two of them; the remainig two are homogeneous in this respect. One of them shares the same multilocus isozyme genotype with H. villosum subsp. villosum; genotypes of two other differ remarkably from H. villosum subsp. villosum by occurrence of unique alleles. Hieracium dentatum was found to be an apomictic tetraploid with surprisingly the same geno-type as detected in H. villosum subsp. villosum. The same chromosome number and mode of reproduction was found in H. scorzonerifolium; comparison of isozyme genotypes of H. scor-zonerifolum and its putative parent species (H. villosum and H. bupleuroides) supports the hypothesis about its hybrid origin. A geographic pattern of both morphological and genetic variation (more or less different races in particular mountain ranges of the Western Carpathians) was detected in a still unnamed taxon from the vicinity of H. valdepilosum; both triploids and tetraploids were found. In addition, two remarkably distinct isozyme genotypes were found in H. bupleuroides. No relationship between genotypes and morphology was observed; on the other hand, geographic areas of particular genotypes are vicariant. The observed variation pattern of H. villosum resembles generally that of some other "Haupt-arten" of Hieracium. The core taxon (H. villosum subsp. villosum) is surrounded by tetraploid apomictic (but pollen-producing) lineages that show some morphological characters of other Hieracium species (nevertheless, they cannot be treated as "Zwischen-arten", because they are very similar to H. villosum subsp. villosum). Their origin is difficult to trace; further detailed studies can perhaps elucidate particular evolutionary processes.
Fehrer	2003	The role of sexual reproduction in recently established facultative apomicts of Hieracium subgen. Pilosella (Asteraceae).
Abstract: Hieracium subgenus Pilosella is of special interest for the study of asexual reproduction, because (i) many of the species are facultative apomicts of the aposporous type. They can serve as a model for the study of sexual and asexual reproduction in the same organism. Besides vegetative reproduction by stolons, seed production without pollination (apomixis in strict sense) and sexual reproduction can occur together in the same plant, even in a single capitulum at the same time; (ii) they are model organisms for the study of apomixis and the search for apomixis genes; (iii) their apomictic mode of reproduction facilitates their invasiveness in North America and New Zealand. We identified apomictic clones of several species and their geographic distribution by minisatellite fingerprinting. A time frame for the origin of some polyploid taxa could be determined by the evaluation of historical reports on man-made habitats in a Central European mountain range, and different relative ages of the clones were suggested by their range of dispersal. Some polyploid taxa have restricted distribution, others cover areas up to half the size of Europe. In two cases, single clones were identified over very large distances - in one case between Europe and North America - obviously mediated by human influence. Study of natural hybridogenous species by chloroplast DNA analysis revealed in several cases unidirectionality of the crosses. Furthermore, the facultative apomict was repeatedly identified as mother plant instead of the sexual parent indicating a larger role of residual sexuality under natural conditions than assumed before.
Fehrer et al.	2003	A chloroplast DNA phylogeny of Hieracium subgen. Pilosella and its relationship to the other subgenera and to Andryala.
Abstract: Chloroplast DNA (cpDNA) is usually maternally transmitted in angiosperms giving evidence for e.g. seed dispersal and the direction of hybridizations. We confirmed maternal transmission in subgen. Pilosella for selected experimental hybrids from reciprocal crosses whose parental species could be distinguished by their chloroplast DNA. Thus, a cpDNA phylogeny will not in first place yield a phylogeny of the species, but rather one of their maternal lineages. The chloroplast genome consists of a single circular chromosome, is therefore haploid - the type of chloroplast DNA found in a species is therefore referred to as its haplotype - and does not undergo recombination, i.e., it is transmitted unchanged except for mutations accumulating over time and separately from the nuclear genome. This leads to the situation that the cpDNA haplotype can still be recognized after repeated backcrosses even if most of the genome of the species transmitting it is diluted out more or less completely, i.e., leaving no trace of the earlier hybridization in the species' morphology or in its nuclear genome. This outcome is called "chloroplast capture", because a species might harbour the cpDNA of a foreign species without any obvious signs of it in its morphology. Such processes are the reason why the cpDNA often rather shows a geographic distribution than reflecting species' boundaries. Especially in a hybridogenous complex like Pilosella, we therefore expected a mixture of different cpDNA haplotypes within species as well as some geographical pattern in their distribution. Based on previous analyses, a part of the chloroplast genome showing a relatively high variability in Hieracium s.l. - the trnT-trnL intergenic spacer - was chosen for sequencing. We found two distinct species groups in subgen. Pilosella exhibiting one or another major type of cpDNA and rather little variability within each of the two groups. Contrary to expectation, at first, no geographic structure at all was found - species with one or the other haplotype were regularly growing intermingled and evidently hybridized without exchanging their cpDNA in most cases. This was even true for intermediate species that were probably of hybridogenous origin. Even if they originated several times, the majority of those analyzed always showed the same type of cpDNA (e.g., H. glomeratum, H. floribundum, H. iseranum) indicating unidirectional hybridizations the reason of which is unknown. Furthermore, in several cases we found the facultative apomict serving as mother plant in hybridizations and the sexual parent providing pollen rather than vice versa (e.g., in H. glomeratum, H. iseranum, H. piloselliflorum) indicating that residual sexuality in facultative apomicts might play a larger role than previously thought. The two PilosellaHieracium s.str. and Chionoracium taxa. Thus, the separation of Pilosella haplotype groups which did not correspond to any morphological characters must have predated the speciation of the (sub)genus. However, it seemed to reflect some geographical/historic as well as ecological characteristics on a broader geographic scale: One of the haplotype groups (showing ancestral features in their sequences) is more restricted to western Europe and never leaves the original forest areal except at high altitudes. The previously missing geographic pattern can be understood as a consequence of our initial concentration on a small investigation area which lies in the overlap zone of both haplotype groups. Andryala turned out to be an ingroup rather than an outgroup taxon. Its cpDNA was unequivocally derived from the second Pilosella haplotype (with more eastern distribution and derived sequence features) showing an extremely close relationship between Pilosella and Andryala which is also supported by morphological characters (achene structure, flower colour etc.) and a similar DNA content in contrast to both Hieracium s.str. and Chionoracium. The latter two could hardly be distinguished by their cpDNA. Hieracium s.l. and Andryala together formed a monophyletic group which was well separated from any other presumably related genera. Hieracium hololeion MAXIM. proved to belong to the outgroup taxa and should be referred to as Hololeion maximowiczii KITAMURA.
Fehrer et al.	2003	Speciation in facultatively apomictic Hieracium subgen. Pilosella taxa: a bottom-up approach.
Abstract: Facultatively apomictic taxa present special difficulties for the elucidation of speciation processes. A vast number of (micro)species and a virtual continuity of forms make their identification and treatment a challenging task. Therefore, we started at the clonal level by multilocus fingerprinting in order to assist with their proper determination and taxonomic delimitation as a foundation for further studies and to determine the number, origin and geographic distribution of clones present in different apomictic taxa. A mixture of basic species, old fixed intermediate forms and recent hybrids was found. Next, we studied hybridization and introgression phenomena of the confirmed taxa at population and species level using sequences and structural properties of the trnT-trnL intergenic spacer. Despite multiple origins of clones, most of the presumed hybrids obtained their chloroplast DNA from the same parent and often, the facultative apomict even proved to be the maternal parent rather than the sexual species. Finally, a molecular phylogeny of all Pilosella basic species was established based on chloroplast as well as nuclear (ITS) sequences. Discrepancies between them reflected either morphological/cytological or distributional /ecological features. The molecular approaches were accompanied by morphological and cytological analyses as well as by studies of reproduction systems and experimental hybridizations.
Mráz et al.	2003	First record on recent natural hybridization in Hieracium s.str.
Abstract: Hieracium s. str. belongs to the richest plant genera of the world. Most of the karyologically so far analyzed (micro)species are tri- or tetraploid with apomictic formation of seeds. The diploid taxa are very rare and they reproduce exclusively sexually. For some of them strict allogamy was proved. Polyploid taxa, at least most of them, are believed to be allopolyploid. There are some indirect proves supporting the hybridogeneous origin of polyploid taxa: (i) morphology - taxa posses the combined characters from two (or more) putative parent species; (ii) presence of fixed heterozygosity (evidence from allozyme markers); (iii) breakdown of normal chromosome pairing during microsporogenesis (univalents are often recorded), disturbance of microsporogenesis results in heterogeneous sized pollen or even in male sterility; (iv) and finally, the diplosporic apomixis can be characterized as an "escape from sterility", generally, sterility is a frequent phenomenon accompanying hybridization. While natural hybridization is a quite common event at present in the closely related genus of Pilosella, there are no reliable data on recent hybridization in the genus Hieracium s. str. so far. Probably the first recent Hieracium hybrid at all was found during a botanical expedition in the Munţii Rodnei (Romanian Eastern Carpathians) in 2001. The single plant was found by the tourist path from the village Borşa to Mt. Pietrosul Mare at the 1350 m a. s. l. in spruce zone. The plant had an intermediate appearance of the two morphologically very distant species - Hieracium alpinum L. and Hieracium transsilvanicum HEUFF. The hybrid plant was accompanied on the locality by plants of both parent species, but the former one occurred as 3 individual plants only and ca 300 m away from the hybrid site. While H. transsilvanicum is a typical representative of spruce (and fir-beech) forests of the Eastern and Southern Carpathians, H. alpinum normally occurs in the alpine and very rarely in subalpine belts only. The presence of 3 individuals of H. alpinum on the borders of the tourist path may be simply explained by the close proximity of the alpine belt and by the fact that this locality was strongly disturbed (and cleared) by forest machines during cutting. Thus, the biotop was suitable for incidental occurrence of H. alpinum at a very unusual altitude. From the reason of probably being the first discovered hybrid in the genus, this plant was transferred to the Botanical garden of P.J. Šafárik University in Košice for cultivation and further studies. Karyological analysis revealed the diploid chromosome number (2n=18). Both parental species are diploid in the Eastern and Southern Carpathians (Romania and Ukraine; CHRTEK 1996, 1997, MRÁZ 2001 and unpubl.). 3 unopened flowers from one head of the hybrid were tested for pollen production, but no pollen has been found. The hybrid plant was crossed with one plant of the parental species, H. transsilvanicum. All seeds obtained from hybrid plant were completely empty and whitish. So, besides several morphological characters, also the ploidy level and male and female sterility underlines the hybridogeneous origin of this plant. Moreover, the morphological intermediate characters present in hybrid plant from the field were confirmed in hybrid progeny from a series of experimental crosses between H. alpinum and H. transsilvanicum. The progeny from these crosses, however, produced pollen, but the seed production (from futher hybridization, open pollination or from isolation experiments) was nearly 1% (near sterile) (MRÁZ & PAULE, unpubl.). Furthermore, we compared allozyme patterns of hybrid plant and its putative parents (plants of H. alpinum and H. transsilvanicum collected in the vicinity of the hybrid). Four allozyme systems were analyzed, i.e. ADH (Alcoholdehydrogenase), LAP (Leucinaminopeptidase), PGM (Phosphoglucomutase), and SKD (Shikimate dehydrogenase). Only two loci, Skd, and Lap-1, showed allelically interpretable variation, for the others, only allozyme patterns were compared. The results partially support the hypothesis about hybrid origin of the target plant.
Suda et al.	2003	Genome Size Variation In Hieracium Subgenus Pilosella
Abstract: Hieracium subg. Pilosella (hawkweed) is a typical example of a taxonomically intricate polyploid complex whose structure is substantially influenced by hybridization. The group consists of two types of taxa: the "basic" species and the "intermediate" species of hybridogenous origin. A continuous variation connecting the intermediate species with the basic ones is common. Flow cytometry was employed to test whether the genome size reflects the species relationships established on the basis of the morphological traits. The genome size (DNA content of the monoploid chromosome set) of the basic species varied from 1.70 pg in hexaploid H. pilosella to 2.11 pg in diploid H. onegense (about 1.24-fold difference). A tendency towards a reduction of the genome size was detected in higher ploidy levels of multiploid species. Nevertheless, these differences were small with a maximum value of 4.5% between the diploid and tetraploid cytotypes of H. cymosum. A low variation in nuclear DNA content within the cytotypes supports a theory of stable genome size. Preliminary results indicate that the location of genome size of the intermediate taxa is in the range between the values of their putative parents, although closer to the parental species with the lower DNA content.
Chrtek et al.	2004	The Hieracium nigrescens group in the Sudeten Mountains and the Western Carpathians.
Abstract: The Hieracium nigrescens group (H. nigrescens s.l.) includes taxa of morphologically intermediate position between H. alpinum and H. murorum, which are closer to the former (H. alpinum ł H. murorum). They are distributed in higher mountains of the Central Europe and in the North, from Greenland to the Ural Mts. This research aims to propose a new taxonomic treatment of the group in the Western Carpathians (Slovakia, S Poland), and the Sudeten Mts. (N Czech Republic, SW Poland). Based on our own observations and studies of previously recognized taxa, we prepared a preliminary taxonomic treatment. To test it, we examined the morphological variation (total of 32 quantitative and qualitative characters), the genetic structure of species/populations by use of isozyme markers, chromosome numbers and the mode of reproduction (using emasculation/isolation experiments). Both multivariate morphometric and isozyme analyses provided support for recognition of 8 taxa, evaluated here at the species level, i.e. H. apiculatum TAUSCH, H. chrysostyloides (ZAHN) CHRTEK jun., H. decipiens TAUSCH, H. nigrescens TAUSCH (all from the Sudeten Mts.), H. mlynicae (HRUBY & ZAHN) CHRTEK jun., H. vapenicanum (LENGYEL & ZAHN) Chrtek jun. and H. koprovanum (RECH. fil. & ZAHN) CHRTEK jun. & MRÁZ (all from the Western Carpathians). Isozyme analysis revealed no variation within each species. On the other hand, the recognized species clearly differ from each other in their banding patterns. With respect to the ploidy level (x = 9), tetraploids were strongly represented (2n = 36, H. apiculatum, H. decipiens, H. nigrescens, H. mlynicae), H. vapenicanum was triploid (2n = 27), and H. chrysostyloides was pentaploid (2n = 45). Agamospermy was confirmed for all species. While all species are genetically formed by one clone each, they differ in morphological variation. The highest phenotypic plasticity was found in H. mlynicae, which also includes genetically identical but morphologically slightly different population from Mount Babia hora. However, some questions remain still open, such as systematic position of H. vapenicanum (which may be better placed between H. alpinum and H. bifidum).
Fehrer et al.	2004	Hybridization in ancestral Hieracium populations inferred from chloroplast capture events predating the speciation of genera.
Abstract: Molecular phylogenies of Hieracium and related genera (Asteraceae, Lactuceae) based on chloroplast trnT-trnL intergenic spacer and on nuclear ribosomal ITS sequences revealed strong discrepancies in some of the major clades: (i) cpDNA placed subgenus Pilosella into two well distinguished haplotype groups that were at least as divergent from each other than from the other subgenera and (ii) Andryala cpDNA was derived from one of those subgroups. In contrast, ITS data revealed monophyly for both Pilosella and Andryala. Several hypotheses about chloroplast transfer between clades were evaluated by mapping the cpDNA haplotypes on the ITS tree. Detailed analysis of character states and structural features of the trnT-trnL suggested a particular sequence of events as the most parsimonious explanation for all data. It requires a first transfer of cpDNA from the Hieracium s.str./Chionoracium ancestor to the Pilosella lineage and a second transfer from the latter to the Andryala ancestor. Both inferred hybridization events seem to have triggered an acceleration of chloroplast DNA sequence evolution. Both molecular data sets are in accordance about the monophyly of Pilosella, Hieracium, Chionoracium, Andryala and Hispidella, which is supported by morphological evidence (e.g., trichome types), thus excluding Tolpis, Hololeion and Arnoseris from the Hieraciinae sensu Bremer.
Košťálová & Krahulec	2004	Distribution of mixed Hieracium bauhini - H. pilosella populations and hybridization within these populations.
Abstract: Mixed plant populations of Hieracium subgenus Pilosella, composed of H. pilosella, H. bauhini / H. piloselloides and their hybrids H. leptophyton and H. brachiatum were investigated in an urban area (Prague city). The number and distribution of such localities, their respective species ratios, ecological and geographic conditions are described. Morphological comparison of the plants, determination of their ploidy level and reproduction experiments was performed in order to assess the species' variability. These localities and plant features, considered together, shall lead to a better understanding of the factors influencing the initiation of new hybrid localities and their population parameters. In 2003, the whole Prague area was systematically screened for hybrid localities. Among the 34 sites found containing Pilosella species were seven with mixed H. pilosella - H. bauhini / H. piloselloides populations. Two localities involved hybridogenous populations with H. leptophyton or H. brachiatum (and both parental species present). During this study, surprisingly, two further hybrid species between H. pilosella and H. bauhini, namely H. aridum and H. visianii, were retrieved, which had not been reported before in the Czech Republic. Interestingly, a total of seven populations with H. aridum as the only Pilosella species were found. In contrast, H. visianii was detected at a single locality together with hexaploid (2n=54) H. pilosella and pentaploid (2n=45) H. bauhini / H. piloselloides. Both H. aridum and H. visianii seem to be strictly apomictic. The following ploidy levels/chromosome numbers and reproduction modes were found for the plants studied: H. pilosella (2n=36, 54; both sexual), H. bauhini / H. piloselloides (2n=45, apomictic), H. leptophyton (2n=45, 47/48; both apomictic), H. brachiatum (2n=36; sexual), H. aridum (2n=36; apomictic) and H. visianii (2n=36; apomictic). These preliminary results point out that hybrid species such as H. leptophyton and H. brachiatum probably originate de novo at individual localities, because they were never found in absence of their parental species. In contrast, the other hybrid species H. aridum seems to form fixed populations in our study area. This conclusion is supported by similarities in morphological features, ploidy level, apomictic reproduction mode and occurrence in the same kind of habitats (ruderal, synantropic places like the ramparts of highways or railways). Future investigations with molecular methods shall show whether all H. aridum plants are descendants of the same clone or whether they have multiple origin. Definite conclusions of Hieracium visianii are not interpreted because of any comparison with other localities. Studies of ecological features of all localities showed a preference of sunny southern places with a slope gradient of about 30-47 degrees for all taxa investigated.
Krahulec et al.	2004	The Hieracium subg. Pilosella in the Šumava Mts.
Abstract: The Pilosellae population occurring in the Šumava Mts. was analysed during the last three years. It consists of the following four basic species: H. pilosella (2n=36), H. lactucella (2n=18), H. aurantiacum (2n=36, 45), and H. caespitosum (2n=36). Three intermediate species are common in the study area, viz. H. glomeratum (2n=36, 45), H. floribundum (2n=36), and H. scandinavicum (H. glomeratum - H. floribundum, 2n=36). Within the region, the following set of recent hybrids was found: H. aurantiacum > H. pilosella (morphotype as well as chromosome number fully corresponding to H. rubrum, 2n=54); H. aurantiacum - H. pilosella (H. stoloniflorum, 2n=36); H. scandinavicum - H. aurantiacum (2n=36); H. caespitosum - H. aurantiacum (morphotype corresponding to H. fuscoatrum, 2n=36); H. floribundum - H. aurantiacum (2n=36); H. lactucella - H. pilosella (H. schultesii, 2n=27, 36); H. floribundum > H. pilosella (morphologically similar to H. iseranum, but 2n=54); H. floribundum < H. pilosella (H. piloselliflorum, 2n=36); H. glomeratum < H. pilosella (H. macranthelum, 2n=38; morphotype similar to H. schultesii). The basic chloroplast haplotypes are the same as in the Sudeten region: Haplotype group 1, and its modification typical of tetraploid H. aurantiacum ; haplotype group 2 and its modification typical of H. cymosum. The distribution of haplotypes among most of the basic and hybridogenous types is very complex. Several species, both basic and hybridogenous, share more than one of them. This fact reflects several phenomena: (i) rather complex pattern within the basic species, as for H. lactucella (the occurrence of haplotypes 1, 2 and aurantiacum sub-type); (ii) recurrent origin of hybridogenous types, with both parents serving reciprocally as mother plants: H. stoloniflorum, H. schultesii; (iii) common function of apomicts as mother plants; (iv) indication of uncertainty of determination of some rare types, e.g. of that morphologically corresponding to H. macrostolonum (H. caespitosum < H. pilosella). Its aurantiacum-haplotype indicates another explanation, viz. yellow type of hybrid between H. aurantiacum and H. pilosella; (v) possible introgression between several species. The above-mentioned traits suggest common recent hybridisation. Most hybrids were found at recently disturbed sites. As a whole, the pattern of the Pilosellae population within the Šumava Mts. is different from that in the western part of the Sudeten Mts. (the Krkonoše Mts.). Most probably, random events in the past led to differences in qualitative and quantitative composition of populations. Common occurrence of H. scandinavicum, its hybrids with H. aurantiacum, and hybridisation between H. floribundum and H. aurantiacum seem to be typical of the Šumava region, while H. iseranum, H. piloselliflorum, and H. blyttianum (H. aurantiacum - H. lactucella) are typical for the Krkonoše Mts. H. rubrum occurs in the Krkonoše as an old stabilized hybrid with its own distribution area and ecology; in the Šumava region, it is a recent hybrid with low number of localities and specimens. The same particular haplotypes-cytotypes combination of the same species in both mountain regions suggests that they are either old entities or that they have originated in the same way.
Peckert et al.	2004	Genetic variation of agamospermous populations of Hieracium echioides s.l. in the Danube basin.
Abstract: Six agamospermous populations of Hieracium echioides s.l. from Southern Slovakia and Northern Hungaria were analysed. The plants studied differed from H. echioides s.str. in having a lower density of hairs on the stem and leaves, less rigid hairs, narrower and more pointed leaves, and smaller flowering heads. The plants are taller and at the same time more slender than H. echioides. They start flowering earlier, and the flowering period is shorter in comparison with H. echioides s.str. From the morphological point of view the populations are supposed to be a result of introgressive hybridization of H. echioides s.str. with H. piloselloides or H. auriculoides which is distributed in this region. Morphologically, the plants match H. echioides subsp. echioides var. tauscheri NAEGELI & PETER well (ZAHN 1921-23). Allozyme analysis, cytology, and analysis of mode of reproduction were used to evaluate variation within and among populations. All populations were tetraploid (2n = 36) and agamospermous. Allozyme analysis revealed four genotypes: three populations were represented by genotype I, two populations by genotype II, and one population by genotypes III and IV. Within-population diversity was found only in one population (population from the Pilis Mountains) where genotypes III and IV were represented equally (genotypic eveness = 0.96). All the remaining populations were genetically completely uniform. The absence of within-population diversity reflects the agamospermous mode of reproduction. The occurence of sexual plants of H. echioides s.str. with these plants was only recorded from the Pilis Mountains. The geographic distribution pattern (one genotype at several isolated localities) can reflect occurrence of the taxa together in the past. Changes of environment and loss of suitable localities, mainly sandy habitats, could cause the reduction and fragmentation of their habitats. The study of herbarium specimens, mainly in BP, confirmed this hypothesis.
Rosenbaumová & Krahulec	2004	Residual sexuality as a factor influencing genetic structure within the agamic complex of Hieracium subgenus Pilosella.
Abstract: Hieracium subgenus Pilosella belongs to the most taxonomically complicated groups of flowering plants. Apomixis, hybridization and polyploidisation considerably influence the taxonomic structure of this group. In apomictic members of the subgenus, seeds are produced without meiosis and fertilisation. Offspring are genetic clones of the mother plant. Apomixis results in a stabilisation and long-termed fixation of the existing composition of the genome. Apomixis is not obligatory and some level of residual sexuality usually also occurs. Sexual reproduction disrupts stability fixed by apomixis and changes the composition of the genome in a fundamental way. The residual sexuality could thus play an important role during formation of genetic structure within the agamic complex of the subgenus Pilosella. The questions to be answered include: In what way is residual sexuality manifested in natural populations? How often do haploid parthenogenesis and fusion of unreduced gametes occur, and what is their significance? How do the sexually derived progeny influence the evolution of genetic structure of the population? How do they contribute genetically to next generation of progeny? How often do they produce new genotypes? To improve our understanding of these problems, the manifestation of residual sexuality in a model system comprising tetraploid sexual accessions of H. pilosella and hexaploid apomictic accessions of H. bauhini is being studied. Reciprocal hybridization experiments are being carried out between model accessions to quantify the proportion of progeny derived by sexual processes in the model system. Because the main part of experiments are planned to be carried out this and next year, only preliminary results from 2003 are presented here. In experiments employing H. bauhini as the mother plant, only polyhaploids (3x) and BII hybrids (5x) were detected, and no progeny originated by fusion of unreduced gametes were obtained. The products of natural hybridization are also being charcterised. Thirty-six plants from model population from Valov u Podbořan (H. pilosella - 14 individuals, H. bauhini - 6 individuals, and hybrids and hybridogenous plants - 16 individuals) are being analysed with respect to ploidy level, DNA content, mode of reproduction, pollen fertility, and isoenzyme and morphological variation. The aim is to bring to light on family relationships between plants in the population and to compare progeny derived from experiments with natural hybrids. The mode of reproduction (fertility/sterility, apomixis/sexuality) of sexually derived progeny will be evaluated to assess the significance of its contribution in evolution of genetic structure of population.
Fehrer et al.	2005	Evolutionary aspects in Hieracium subg. Pilosella
Abstract: The hawkweed subgenus Pilosella (Lactuceae, Asteraceae) is known for its notoriously complicated taxonomic structure due to ongoing reticulate evolution combined with a facultatively apomictic mode of reproduction. Recently, molecular approaches at clone, population and species level have begun to shed some light onto these processes. Geneflow across ploidy levels is common, and parental species of hybridogenous taxa often include apomicts, even as seed parents. Sexual taxa (diploid or polyploid) usually show high genetic variability. Apomicts vary from near clonality across large geographic distances to multiple origins on a small scale. Pilosella chloroplast haplotypes form two major groups whose separation predated most of the subgenus' speciation. Comparison of chloroplast and nuclear markers suggests two ancient hybridization events predating most of the speciation observed in the subtribe Hieraciinae: one between the Hieracium/Chionoracium subgenera ancestor and partly differentiated Pilosella, and a subsequent one between this introgressed Pilosella lineage and the closely related Andryala genus ancestor.
Fehrer et al.	2005	Hybridization in Hieracium s.l. (Lactuceae, Asteraceae).
Abstract: In Hieracium, reproduction modes vary between subgenera. In subgen. Pilosella, natural hybridizations are common between sexuals and facultative apomicts as well as among apomicts, creating a high diversity of new, usually apomictic taxa many of which are now widely distributed. Surprisingly often, such fixed populations have originated from hybridizations of a facultative apomict as seed (maternal) parent and a sexual pollen donor. Depending on the initial species composition at localities and presumed time frame of habitat availability, speciation status and diversity of the resulting hybridogenous types differ, e.g., between mountain ranges. In subgen. Hieracium, comprising very few sexuals and numerous (obligate) apomicts, natural hybridizations are presently very rare due to several internal and external reproduction barriers, but must have been frequent in the past in order to produce thousands of microspecies worldwide. Detailed comparison of nuclear and chloroplast DNA markers revealed that at least one hybridization event predating the major speciation in these two subgenera - that meanwhile differ in their mode of reproduction, DNA content, morphology and distribution - must have had taken place. There are indications that this event may have accelerated molecular and morphological evolution in the introgressed Pilosella lineage.
Krahulec et al.	2005	The role of apomictic species in agamic complexes of Hieracium subgen. Pilosella.
Abstract:Apomixis is often considered as a "blind alley" in evolution. The quantity of apomictic plants as well as their success suggests a different view. In Hieracium subgen. Pilosella, apomicts are facultative, with some degree of residual sexuality. We estimated the role of apomicts in populations by experimental crosses and comparison of progeny with composition of populations in the field. The reproductive pathways were quantified in two model systems of polyploid species: 1) H. bauhinii (apomictic) crossed with H. pilosella (sexual) and 2) a hybridogenous apomict, H. rubrum, crossed with H. pilosella. The resulting variation and also variation of plants obtained from seeds collected in the field from particular facultatively apomictic mothers is surprisingly higher than progeny from fully sexual mothers. In addition to ordinary apomixis (without meiosis and gamete fusion, polyploid apomictic mothers can also reproduce via special additional pathways (haploid parthenogenesis or fusion of unreduced gametes), which omit one of the otherwise characteristic steps of sexual seed formation. These results were supported by studies of field populations. Chloroplast-DNA haplotypes revealed that facultatively apomictic species often function as maternal parents of polyploid derivatives of hybridization. Many of these hybridogenous types are stabilized and widespread, considered as separate taxonomic entities. Facultatively apomictic mothers also serve as a bridge for gene flow through agamic complex.
Chrtek & Mráz	2006	Hieracium s.str. in the Western Carpathians.
Abstract: In the past decade, taxonomic revisions of Hieracium alpinum, H. fritzei, H. nigrescens, H. piliferum, H. rohacsense, H. sparsum and H. villosum (species in the broad sense, species groups) were finished. In comparison with Zahn´s account, most changes concern the Hieracium nigrescens group (several Zahn´s subspecies were assigned to another species groups). Recently, taxonomic position of plants from the Nízke Tatry Mts., referred by Zahn erroneously to Hieracium rohacsense subsp. glandulosodentatiforme (the name is a taxonomic synonym to H. rohacsense) were examined. The plants show no close affinity to H. rohacsense, but are closely allied (but with sure not conspecific) to Hieracium vagnerii, described from the Eastern Carpathians. Hieracium rostanii was discovered as a new species for Slovakia in the Západné Tatry Mts. (Mt. Sivý vrch). Chromosome numbers were reported for members of 27 species in the broad sense (species groups). Triploids and tetraploids predominate (53% and 45%, respectively), diploids (2n = 18) were only found in Hieracium umbellatum. Previously published diploid counts reported for H. sabaudum from Slovakia were found to be erroneous (misidentification with H. umbellatum). From the karyological point of view, the Western Carpathians belong to the best-explored areas of the Hieracium distribution range. Attention was also paid to the genetic structure of populations/species. Until recently, genetic structure was detected in 10 species (in the narrower sense). Considerable variation was revealed in Hieracium alpinum s.str. (using allozyme and RAPD markers), between-population variation was found in H. pinetophilum, H. crassipedipilum (both the H. fritzei group, using allozyme and RAPD markers), and H. silesiacum (H. sparsum group, allozyme markers). In the remaining species, i.e. Hieracium halleri (H. alpinum group), H. krivanense, H. slovacum (both the H. fritzei group), H. jarzabczynum, H. vapenicanum (both the Hieracium nigrescens group) and H. rohacsense (s.str.), genetic variation was very low or absent (allozymes, in some cases RAPD markers).
Fehrer et al.	2006	Species relationships, biogeography, population structure, and reproduction in an agamic complex: Hieracium subgenus Pilosella (Cichoriae, Asteraceae).
Abstract: The genetics and speciation of apomictic groups belong to the most complicated issues in evolutionary biology. Diversification in Pilosella is currently in full progress, combining facultative sexuality of polyploid apomicts, extremely reticulate population structures and a general lack of reproductive isolation with maintenance and stabilization of particular apomictic genotypes. Two divergent chloroplast haplotype groups and their distribution across parental species might result from their origin in different glacial refugia at a time predating most of the young group’s speciation. An ITS sequence-derived phylogeny of diploids, their taxonomic grouping and ecogeographic distribution suggest several cases of chloroplast capture that occurred after secondary contact in a meanwhile large overlap zone. In different areas of this zone, a similar set of parental species can give rise to different hybridogenous types with respect to ploidy levels, chloroplast haplotypes and reproductive modes. Hybrid progeny of apomicts that had acted as seed donors becomes established more often in nature than its formation frequency in experiments would suggest. Apomictic genotypes that form stable populations are characterized by offspring generated mainly by apomixis whereas local genotypes of the same morphologically defined species additionally have much offspring produced by haploid parthenogenesis and hybridization.
Krahulcová et al.	2006	Comparison of Hieracium subgen. Pilosella in Bulgarian and Czech populations: how is the interspecific hybridization reflected in population structure?
Abstract: Frequent interspecific and intercytotype hybridization, in addition to polyploidy and a diverse reproduction mode, is a main source of variation in the polyploid apomictic complex of Hieracium subgen. Pilosella. Past and recent recurrent hybridizations produce plenty of morphotypes, resulting in their intricate taxonomy. Hybrid swarms between facultatively apomictic H. bauhini and closely related sexual species H. pilosella/H. macranthum, common in the Czech Republic and Bulgaria, were chosen as a model system. Populations at particular localities were evaluated with respect to species (morphotype) and cytotype composition, breeding system (apomictic or sexual) and clonal structure of apomictic biotypes. Effective interspecific hybridization operates in both regions against different background: explicit dominance of hexaploids and rare sexuality recorded in Bulgarian populations are in contrast to more diverse ploidy levels and more frequent sexual biotypes in Czech populations.
Krahulec et al.	2006	Diversity of breeding systems in Hieracium subgen. Pilosella: present state of the knowledge.
Abstract: Breeding systems within Hieracium subgenus Pilosella are very diverse. All types have clonal growth (above or underground stolons, formation of new ramets on the root crown and/or on fine roots). Seed reproduction is sexual and/or apomictic. Sexual types have regular meiosis, and the progeny from the breeding of sexual types is very uniform. On the other hand, apomicts produce diverse progeny. In addition to true apomixis, there are different proportions of other reproductive pathways: combination of reduced and unreduced gametes and haploid parthenogenesis. It seems that stabilized hybridogenous types have usually about 90% of true apomictic progeny. In contrast, local hybridogenous types (and recent hybrids from experiments) have a high degree of true sexuality and/or haploid parthenogenesis (up to 80%). The background of this variation is still unknown.
Krahulec et al.	2006	The role of apomictic species in agamic complexes of Hieracium subg. Pilosella.
Abstract: Apomixis is often considered as a „blind alley“ in evolution. The quantity of apomictic plants as well as their success suggests a different view. In Hieracium subgen. Pilosella, apomicts are facultative, with some degree of residual sexuality. We estimated the role of apomicts in populations by experimental crosses and comparison of progeny with composition of populations in the field. The reproductive pathways were quantified in two model systems of polyploid species: 1) H. bauhini (apomictic) crossed with H. pilosella (sexual) and 2) a hybridogenous apomict, H. rubrum, crossed with H. pilosella. The resulting variation and also variation of plants obtained from seeds collected in the field from particular facultatively apomictic mothers is surprisingly higher than progeny from fully sexual mothers. In addition to ordinary apomixis (without meiosis and gamete fusion), polyploid apomictic mothers can also reproduce via special additional pathways (haploid parthenogenesis or fusion of unreduced gametes), which omit one of the otherwise characteristic steps of asexual seed formation. These results were supported by studies of field populations. Chloroplast-DNA haplotypes revealed that facultatively apomictic species often function as maternal parents of polyploid derivatives of hybridization. Many of these hybridogenous types are stabilized and widespread, considered as separate taxonomic entities. Facultatively apomictic mothers also serve as a bridge for gene flow through the agamic complex.
Krak et al.	2006	Molecular tools for inferring phylogeny in Hieracium.
Abstract: Our main aim is inference of genus Hieracium molecular phylogeny with emphasis on the subgenera Chionoracium and Hieracium s.str. (Zahn’s basic and diploid intermediate species). First results are based on the analyses of the nuclear ribosomal DNA internal transcribed spacer (ITS) and the chloroplast (cp) DNA trnT-trnL intergenic spacer, involving 28 and 38 ingroup species, respectively. Cp DNA is maternally inherited and thus allows to trace back a phylogeny of maternal lineages only and often shows geographic pattern. The ITS evolves faster and better reflects species relationships due to its biparental mode of inheritance. According to our data, both subgenera are polyphyletic, and the groups found in phylogenetic analyses are incongruent with sectional classifications based on morphology. In the subgen. Chionoracium, four North American species (H. scabrum, H. venosum, H. argutum, H. albiflorum) are close to each other, and the chloroplast haplotypes of the North-Western H. albiflorum-H.argutum are derived from those of North-Eastern H.scabrum-H.venosum. Further geographic patterning of cp DNA clusters (i) H. carneum from Arizona with H. guatemalense and (ii) all South American species except H. antarcticum. In Hieracium s.str., both molecular markers identify three groups: (i) H. porrifolium and H. glaucum, (ii) H. alpinum and H. pojoritense and (iii) H. hryniawiense, H. umbellatum and H. eriophorum. While cpDNA places H. intybaceum and H. piliferum to the H. alpinum-H.pojoritense-H. sparsum s.str. group, according to ITS they do not belong to Hieracium s.l. at all. This incongruence suggests at least one ancient hybridization event between the progenitor of H. intybaceum/H. piliferum and the ancestor of the above-mentioned group. The latter served as maternal parent. Triploid H. pictum and surprisingly also the diploid H. tomentosum are of hybrid origin. In the case of H. pictum, H. humile was involved as maternal parent, and some taxon close to the H. murorum-H. transsilvanicum-H. amplexicaule group was the paternal parent. H. tomentosum originated probably from H. amplexicaule (or a close relative) as seed parent and some paternal taxon close to H. humile. In several polyploid taxa only a single ITS sequence type was found. This suggests either that these species are autopolyploids, or homogenization of different parental copies (a particular feature of the multicopy ITS region) deleted any evidence of potential hybridizations. Therefore, we are currently developing a single copy nuclear marker to distinguish between these two possibilities. Furthermore, such a fast evolving marker is supposed to give a better resolution of species relationships in this recently diverged complex.
Šingliarová et al.	2006	Pilosella alpicola subsp. ullepitschii, a diploid endemic taxon of the Carpathians: notes on taxonomy, chorology and ploidy level.
Abstract: Two alpine species, Pilosella candollei and P. alpicola [syn. Hieracium alpicola], are distinguished within the section Alpicolina. Beside P. alpicola subsp. ullepitschii, en endemic taxon of the Carpathians, five other subspecies with more or less restricted areas of distribution have been included in latter species: subsp. furcotae from the Vysoké Tatry Mts, nominate subsp. alpicola from the Alps, and three Balcan subspecies - subsp. rhodopea, subsp. glandulifolia and subsp. micromegas. Distribution of P. alpicola subsp. ullepitschii has a strongly disjunct character. It grows in the Western Carpathians in the Vysoké and Západné Tatry Mts (Slovakia, Poland) and extremly rarely in the Eastern (Nemira Mts) and Southern Carpathians (Buçegi Mts, in both cases in Romania). In the Western Carpathians, P. alpicola subsp. ullepitschii is confined to the primarly alpine or subalpine meadows with acid bedrock in communities with Avenella versicolor. In contrary, in the Nemira Mts it occurrs in man made habitats (secondary formed pastures) in the spruce belt. Similary, in the Buçegi Mts it was found in site not typical for this taxon. This circumstatnces evokes a question if this taxon is native in Romania or not? If the nativeness is the case, how is the genetic differentiation of the populations from both distant areas? Two different ploidy levels (diploid and tetraploid) have been previously published for this taxon. However, all individuals from Vysoké and Západné Tatry Mts and Romanian plants from Buçegi and Nemira Mts analysed in 2000-2005 by flow cytometry and classical counting were proved to be diploid. Genetic structure of six Western Carpathian populations was studied by 7 allozyme loci. We found that genetic variation is high within, but low among populations. Hence, the sexual reproduction and strong gene flow could be suggested. Strict allogamy in this taxon was proved by isolation experiments. In three studied plants from Romania we foud two unique alleles (Lap-1-a, Shdh-1-d), missing in Slovak samples, but present in nominate subspecies alpicola from the Col du Simplon (Suisse).
Urfus et al.	2006	Pilosella officinarum: longitudinal differences in ploidy level distribution in the Czech Republic and Slovakia.
Abstract: P. officinarum is a highly structured species with respect to the ploidy level. Previous scattered data indicated a possible differentiation in frequency of particular ploidy level in the Czech Republic and Slovakia (e.g., Mráz & Šingliarová 2005, Rotreklová et al. 2005). Therefore, a detailed sampling and ploidy level analyses were assessed to reveal a boundary of common occurrence of tetraploids on the one hand and higher ploids on the other hand. Ploidy levels and/or chromosome numbers were determined for 987 plants using flow cytometry and/or classical counting. Samples were collected from 311 localities in the Czech Republic, Slovakia and North-Eastern Hungary. Four ploidy levels with contrasting pattern of distribution were found in the study area. The most widespread cytotype in the Bohemian part of the Czech Republic is tetraploid (4x), while pentaploids and hexaploids (5x and 6x, respectively) clearly prevail in Slovakia and the Moravian part of the Czech Republic. The heptaploid level (7x) was found for the first time in Slovakia at one site. Mixed populations consisting of two different ploidy levels were recorded in nearly 12% of the localities. The boundary between common occurrence of tetraploids and higher ploids is very obvious and reflects the geomorphologic boundary between the Bohemian Massif and the Western Carpathians with an adjacent part of Pannonia. This probably important biogeographical line is obvious also from the distribution of other taxa like, e.g., Campanula rotundifolia s.str., Cirsium heterophyllum, different cytotypes of Pilosella bauhinii (cf. Rotreklová 2004), and the ecological differentiation of cytotypes of Allium oleraceum (Šafářová 2004).
Fehrer et al.	2007	Diploid relict species of Asteraceae subtribe Hieraciinae in evolutionary and phylogeographic context.
Abstract: The Hieraciinae are, according to recent results (Mol. Phylogen. Evol. 42 (2007) 347–361), composed of the genera Hieracium, Pilosella, Hispidella and Andryala. While the monotypic Hispidella, all species of Andryala and the American Hieracium subgen. Chionoracium are only known as diploid, Hieracium subgen. Hieracium and Pilosella are notorious for their complex taxonomic structure due to a mixture of sexual and different apomictic reproductive pathways, ancient or recent hybridization, and diploid or various polyploid cytotypes. The difficulties species identification and delimitation pose for non-specialists, the existence of different taxonomic concepts, and insufficient knowledge about geographical distribution, genetic variability and population dynamics have thwarted their proper treatment in Red Data books and thus the conservation of particular species. Our work is aimed at a better understanding of species relationships and patterns of genetic variability in order to identify refuges that have played a role in the speciation of these groups and to provide a better basis for their taxonomic evaluation and improved treatment in floras and field guides. Pilosella chloroplast haplotypes form two major groups with no correlation to morphology or species relationships while haplotypes of Andryala species are derived from one of the Pilosella haplotypes. This can be explained by two ancient hybridization events predating most of the speciation observed in the Hieraciinae: one between the ancestor of Hieracium and partly differentiated Pilosella, and a subsequent one between this introgressed Pilosella lineage and the ancestor of all Andryala species investigated so far. Distribution areas and species numbers of Pilosella species belonging to one or the other haplotype group as well as the extinction of intermediate haplotypes suggest their differentiation in different glacial refuges. Relict species in Pilosella comprise P. argyrocoma, a rather rare species with disjunct distribution on the Iberian Peninsula, and P. vahlii, an endemic species of the high mountains of central Spain (IUCN Red List of Threatened Plants 1997, both as Hieracium); P. breviscapa, an endemic species of the E Pyrenees; and P. alpicola with an extremely disjunct distribution in the Alps, the Carpathians and the Balkan mountains which is generally indicative of a relict situation. Some populations of P. castellana in the mountains of S Spain may as well have relict character. From an evolutionary point of view, these relict species are occurring in all four major lineages of Pilosella as revealed by ITS sequencing. All of them show derivatives of the original chloroplast haplotype except P. alpicola, whose introgressed chloroplast corresponds to its more eastern distribution. Andryala comprises two relict species, A. levitomentosa (IUCN RL of Threatened Plants 1997, Romanian RL), a highly endangered species occurring as a single population of 200-300 plants in the Romanian Carpathians far apart from the current distribution area of the genus, and A. agardhii (Lista Roja de la Flora Vascular Española 2000) which is confined to the Sierra Nevada (Spain) and Moyen Atlas (Morocco). Both form old lineages separated from other species of this genus which today has its main distribution in North Africa, the Iberian Peninsula and Macaronesia. In Hieracium, several partly diploid species/microspecies that are closely related and most likely of relict character occur at the foot of the Pyrenees, e.g. H. gouani, H. recoderi, and H. cordifolium; the latter appears to be of hybrid origin involving a Pyrenean taxon and a species from a different clade. Hieracium stelligerum is only known from ca 15 localities on limestone rocks in S France and can also be supposed to be a relict species. Probably the most endangered species of relict character occurs in Sicily: H. lucidum (IUCN RL of Threatened Species 2006) is only known from a single population consisting of very few plants on north facing limestone cliffs of Monte Gallo near Palermo. In SE Europe, geographical ranges of diploid hawkweeds coincide well with principal refugial areas. This is the case with the recently described local endemics H. petrovae and H. kittanae from the central Rhodopy Mts. in S Bulgaria and with some populations of H. waldsteinii s.l. from Montenegro. Hieracium pojoritense, a diploid species, is endemic to calcareous mountains in NE Romania. Besides these, some populations of otherwise geographically more widespread species might be considered as relict ones, e.g. widespread diploid populations of the otherwise triploid H. alpinum in the E and S Carpathians, and rather rare diploid populations of H. prenanthoides in the westernmost part of the Alps. While not all diploid relict species of this genus have been studied yet, also some polyploids of relict character may exist.
Krahulcová et al.	2007	The role of facultatively apomictic mothers in the generation of ploidy variation in two model populations of hawkweeds (Hieracium subgen. Pilosella, Asteraceae).
Abstract: The capacity of facultatively apomictic maternal biotypes to generate ploidy variation was compared with the corresponding role of sexual biotypes in two polyploid hybrid swarms comprised of Hieracium pilosella (usually sexual, less commonly apomictic), H. bauhini (apomictic) and their homoploid or heteroploid hybrids (sexual, apomictic or sterile). Two investigated populations differed in abundance of established hybrids and complexity of population structure, which was characterized by co-ocurring morphotypes, cytotypes and their reproductive modes. In the more complex model population, also the clonal and cpDNA haplotype structure were studied. The origin of seed progeny arisen spontaneously in the field was determined for sexual and apomictic mothers from both populations, using either i) a comparison of chromosome number/DNA ploidy level, breeding system and morphology between cultivated seedlings and their mothers, or ii) a flow cytometric screening of seeds of the respective mothers (FCSS method). While the sexual mothers predominantly retained a rather narrow range of ploidy levels/chromosome numbers in their progeny, the facultatively apomictic mothers produced progeny of much more variable ploidy. The maternal breeding system was conserved in the majority of the respective offspring plants. However, the fraction of sexual progeny formed by apomictic mothers yet prevailed over that of apomictic progeny formed by sexual mothers. Heptaploid and octoploid hybrids, originated via fertilization of unreduced egg cells of apomicts, displayed a specific reproductive mode. Although they were able to produce viable seed via parthenogenesis as did true apomicts, most of their progeny were polyhaploids (after emasculation) and hybrids (after open pollination). Their high-polyploid genome seems to be unstable and to decrease its ploidy in the next generation. This population study demonstrates the versatility of reproductive pathways operating in polyploid facultative apomicts in the field, namely the importance of their residual sexuality. Thus, the apomictic biotypes can increase population diversity much more than has been supposed.
Krahulec et al.	2007	Progeny structure and breeding behavior of facultative apomicts of Hieracium subgen. Pilosella and their experimental hybrids.
Abstract: During past research we discovered variation within progeny structure of facultatively apomictic species of hawkweeds (Hieracium subgen. Pilosella) and their hybrids. Our main aim was to specify the pattern of this variation and to explain it. To this purpose we studied the progeny structure of 54 emasculated or experimentally crossed maternal plants. Emasculated plants produced either progeny identical with the maternal parent (true apomictic progeny) or progeny of half chromosome number (derived from haploid parthenogenesis). Experimental crosses were designed so that the origin of particular progeny classes reflecting the respective reproductive pathways could be inferred from distinct ploidy levels in the progeny. When facultative apomicts were used as maternal parent, the progeny was highly variable with respect to their origin, comprising 2n+0 plants (true apomicts), n+0 plants (polyhaploids), n+n, 2n+n, n+2n, and 2n+2n hybrids. In addition, a rare progeny type with the maternal ploidy level, but distinct in morphology, originated from selfing. The frequency of non-maternal (aberrant) progeny was of an order of several percent, i.e., other than apomictic progeny occurred in not negligible amounts. The maximum amount of non-apomictic progeny of maternal biotypes from the field, which can be classified as basic species or as stabilized hybrids, was 10%. In contrast, the frequency of aberrant progeny varied considerably among recent hybrids (natural and experimental ones). Most of these plants had individual-specific progeny structures and produced, in addition to sexually and apomictically derived progeny, different proportions of polyhaploid offspring. Analysis of the breeding system of particular progeny classes that originated from reciprocal experimental hybridizations between apomictic and sexual biotypes revealed that: i) Polyhaploids were either apomictic or sterile. ii) Concerning the breeding system of n+n (“ordinary”) hybrids, significant differences between reciprocal crosses were found: When the apomict was used as the maternal parent, most of the progeny was sexual; when the sexual plant was used as the maternal parent, the ratio of sexually to apomictically reproducing hybrids was about 1:1. iii) The breeding behavior found with 2n + n (“addition”) hybrids was unexpected. In spite of the fact that this progeny contained one complete genome of a good, stabilized apomict (either a tetraploid or a hexaploid), these hybrids produced high proportions of polyhaploids and hybrids besides true apomictic progeny, i.e., their residual sexuality was much higher than that of their maternal parent. Moreover, the proportions of polyhaploid and hybrid progeny varied even among full siblings that differed from each other only by the contribution of the paternal genome. This implies a more complicated manifestation of apomixis than is proposed by present models.
Rosenbaumová et al.	2007	Inheritance of apomixis in Hieracium rubrum (Asteraceae).
Abstract: The aim of our study was to gain deeper insight into the rules of transmission of apomixis in Hieracium rubrum, a member of Hieracium subgenus Pilosella. Subgenus Pilosella represents well established model system for studying aposporous apomixis in dicotyledonous plants, and therefore a subject of intense investigations. Formerly, it was supposed that apomixis is transmitted as a single dominant Mendelian locus in the subgenus, however, recent study on H. caespitosum speak in favour of two independently segregating loci. Nevertheless, because of some post-zygotic lethality and transmission distortions, the inheritance of apomixis does not simply follow the Mendelian rules of inheritance. Thus, the question of apomixis inheritance in subgenus Pilosella remain still not fully resolved. Our study was based on analyses of mode of reproduction in segregating progeny (105 individuals) obtained from reciprocal crosses between facultative apomictic hexaploid (2n = 54) H. rubrum and sexual tetraploid (2n = 36) H. pilosella. Segregating progeny fell into three classes distinct in ploidy level (triploid, pentaploid, and heptaploid), all originated from reduced gametes from hexaploid apomictic H. rubrum but differing in genome contribution from tetraploid sexual H. pilosella (no contribution / reduced gamete / unreduced gamete). Such diversity in segregating progeny provided an opportunity to investigate whether and how the way of progeny origin influences the rate of transmission of apomixis. Emasculation experiments and flow cytometry were used to score plants for mode of reproduction. Analyses of mode of reproduction showed that the rate of transmission of apomixis is strongly dependent on the progeny origin. It appears that the main role play the direction of cross in which progeny have originated - whether apomixis was transmitted through the female or the male gametes. In cross where apomictic H. rubrum served as the maternal parent, gametes conferring apomixis developed preferentially parthenogenetically and gave rise to triploids; all triploids were apomictic or sterile. Gametes lacking apomixis gave rise to pentaploid hybrids requiring fertilization; almost all pentaploids were sexual. Contrariwise when H. rubrum served as the paternal parent, pentaploid hybrids inherited apomixis and sexuality in 1:1 ratio. Only a few heptaploid hybrids (all apomictic) arose in both crosses. The presented study revealed several novel features concerning the inheritance of apomixis in H. rubrum, which could shed new light on the rules of transmission of apomixis not only in Hieracium subgenus Pilosella, but also in other apomictic plants.
Krak et al.	2008	Reticulation, speciation and patterns of molecular evolution in Hieracium (Asteraceae)
Abstract: The genus Hieracium (≈650–8000 species, two subgenera) is notorious for its large morphological variability associated with variation in ploidal level, mode of reproduction and extensive past hybridization. Subgenus Hieracium comprises mostly Eurasian species, diploid sexuals as well as polyploid apomicts. For the exclusively American subgenus Chionoracium, only sexual or autogamic diploids were described. To resolve major phylogenetic relationships and to gain insight into the origin of polyploid taxa, we used sequences of the chloroplast trnT-trnL intergenic spacer and the ETS region of nuclear ribosomal DNA for a representative selection of taxa (80 accessions/ 70 species). The genus is monophyletic, but both subgenera are polyphyletic. Despite relatively low interspecific variation, the ETS dataset revealed the existence of two major groups roughly corresponding to species with a Western or Eastern European origin. All Chionoracium species were derived from the eastern clade, multiple lineages suggesting several introductions to the New World. A few additional subclades were found within each of the major groups. Surprisingly many taxa, including diploid species, turned out to be of hybrid origin: polymorphic sites in the ETS region were additive between the two major clades or among different subclades. Further species were found to have been introgressed by species from other lineages according to chloroplast capture. In ETS, a high level of intraindividual polymorphism occurred in both diploids and polyploids. While such a pattern was expected for allopolyploids where concerted evolution should be slow due to a lack of sexual reproduction, this was also true for diploid taxa. Persisting rDNA variants despite sexual reproduction indicate that concerted evolution does not seem to operate well in this species group. A considerable amount of intraindividual polymorphism occurred that was either independent of or additional to the additive patterns accounted for by hybrid origin. Shared intraindividual polymorphisms among closely related species suggest that this variation occurred prior to speciation events while frequent absence of one of the alternative character states could suggest a contribution of extinct taxa to the Hieracium diversity observed today.
Fehrer et al.	2009	Extensive ancient hybridization in sexual and apomictic hawkweeds (Hieracium, Asteraceae) and evidence for extinct diversity.
Abstract: Hawkweeds are one of the largest (500-8000 spp) and most notorious plant genera comprising few diploid sexuals and many polyploid apomicts. Different ploidy levels can occur in the same species. Extensive reticulation in the past and apomixis have resulted in a continuity of forms affecting species delimitation. We present the first molecular phylogeny of Hieracium based on nearly complete sampling of presumed major evolutionary units: species that show a unique set of characters, lack morphological evidence of hybrid origin, and are rather easy to identify. The nrETS region and the trnT-L spacer of cpDNA were used to resolve relationships. Among 60 accessions of 46 taxa, 29 had unexpected hybrid origin, with 17 different parental combinations. Diploids and polyploids were either ‘pure’ species or had hybrid origin. Some species had multiple origin, and some accessions had more than two parents. Hybrids were identified by character additivity at intra-individual polymorphic sites in ETS and excluded from phylogenetic analysis. A deep split of the genus into two major lineages was found. They corresponded to species with mainly Eastern or Western European distribution and also showed significant genome size differences at the same ploidy levels, but these two groups had never been suggested in any taxonomic treatment. 20 hybrid accessions were composed of members of both major clades. Detailed visual analysis of shared intra-individual polymorphisms that were not additive, and cloning of selected accessions revealed the existence of three additional ribotype lineages that occurred only in hybrids; two of them were geographically widespread. Several hybrid cpDNA haplotypes were unique and rather derived; candidate maternal parents were missing. Also, a much larger number of diploids would be necessary to generate the Hieracium species diversity even if the most conservative estimate of species numbers is applied. However, only 10 (of 18) diploid species did not show signs of hybrid origin, and many diploids have relict character or occur only in known glacial refuge areas. We assume that the major species groups survived in different glacial refugia and hybridized after secondary contact. Hybrid origin even of supposed major evolutionary units, multiple origin of some species, and the presence of widespread or Central European taxa in both major clades had largely obscured species relationships. In addition, missing ancestral variation (more likely extinct than unsampled) preserved only in hybrid genomes may have contributed to the taxonomic confusion. Obviously, hybrid origin cannot be inferred from morphology if at least one parent is extinct.
Mráz et al.	2009	Auto- and allopolyploid speciation within the Pilosella alpicola group with consequences for breeding system and genetic variation.
Abstract: The Pilosella alpicola group comprises several allopatric alpine taxa with a very polydisjunctive range across the high mountains of Central and Southern Europe. Morphological study using multivariate morphometric analyses revealed the existence of four morphologically distinguishable species: P. alpicola s.str. (Alps), P. rhodopea (Balkan Peninsula and Southern Carpathians), P. serbica (Serbia) and P. ullepitschii (Carpathians). Karyological and flow cytometric analyses indicate geographic and taxon specific patterns in cytotype distribution. Pilosella ullepitschii and P. serbica are exclusively diploid and strictly self-incompatible taxa. A complex cytotype pattern, including mixed ploidy populations, was found in P. rhodopea including four different cytotypes, varying from diploids to pentaploids. These cytotypes, reproducing by strict allogamy, are not morphologically differentiated and this fact alongside with nuclear ITS data suggests their autopolyploid origin. Allozyme genetic diversity gradually decreases from diploids to tetraploids. In contrast, isolation, castration and flow cytometric seed screen analyses revealed that tetraploids and pentaploids of P. alpicola s.str. produce seeds apomictically. Apomictic reproduction of this taxon is reflected in significantly reduced genetic diversity when compared to P. rhodopea polyploids. Interestingly, ITS sequences proved that P. alpicola s.str. is in fact an allopolyploid taxon originating from hybridisation with another alpine species, Pilosella glacialis. Allopatric distribution of cytotypes (tetraploids in the Walliser Alps and pentaploids in the Dolomites), different multilocus allozyme patterns, slightly different ITS additive polymorphism, and some morphological differences are evidence for a polytopic origin of P. alpicola s.str. We hypothesize that during suitable climatic conditions, polyploid cytotypes of Balkanian P. rhodopea underwent range expansion in a north-westerly direction, where they subsequently hybridized at least twice with P. glacialis giving rise to hybridogeneous P. alpicola s.str. Interestingly, apomixis has not contributed to the further range expansion, and P. alpicola s.str. currently has a very restricted distribution. Surprisingly, some level of introgression was detected also in diploid Pilosella ullepitschii, either from Balkanian P. rhodopea or other closely related species. Our data show that different speciation processes such as auto- and allopolyploidization within closely related taxa might result in different reproductive pathways which in turn have consequences for genetic variation.
Rosenbaumová et al.	2009	Sexual reproduction as a source of ploidy level variation in the agamic complex of Hieracium subgenus Pilosella (H. pilosella and H. bauhini as model systems).
Abstract: Apomixis is clonal reproduction through seeds. When it is combined with sexual reproduction it can lead to the formation of intricately structured agamic complexes characterized by a huge diversity in morphology, ploidy level, mode of reproduction, and degree of hybridity. Study of recent hybridization in young developing agamic complexes, e.g. Hieracium subgen. Pilosella, can improve our understanding the role of sexual reproduction in the diversification within a complex. A model population from the vicinity of Valov (NW Bohemia, Czech Republic) consisted of 4x sexual H. pilosella and 6x apomictic H. bauhini (parental species), and their 5x, 7x, and 8x hybrids. Crosses between parental species were performed to quantify their potential to produce ploidy level variation. Flow cytometry revealed significant ploidy level variation in the progeny, showing that reduced as well as unreduced gametes of both parents participated in crosses. Progeny from the cross where H. pilosella served as a maternal parent (273 plants) consisted of 4x progeny from autogamy (18.3%) and of 5x hybrids (81.7%). Progeny from the cross where H. bauhini served as a maternal parent (821 plants) consisted of apomictically derived 6x progeny (93.2%), of three types of hybrids (5x - 4.8%, 7x - 0.1%, and 8x - 0.7%), and of 3x parthenogenetic progeny (1.2%). To quantify ploidy level variation actually formed in the field, ploidy levels were estimated in the progeny that was obtained from seeds collected from parental species at the locality. Progeny of H. pilosella (317 plants) consisted of 4x progeny from auto/allogamy (98.7%) and of 5x hybrids (1.3%). Progeny of H. bauhini (486 plants) consisted of apomictically derived 6x progeny (92.4%), of four types of hybrids (5x - 2.7%, 7x - 0.4%, 8x - 2.9%, and 10x - 0.4%), and of 3x parthenogenetic progeny (1.2%). Hybridization between H. pilosella and H. bauhini generated significant variation in ploidy level under both experimental and field conditions. Nevertheless, it appears that only a part of this potential variation can influence ongoing evolution in the population as neither 3x, nor 10x adult plants were detected in the field. Some selective disadvantage of these cytotypes can be suggested. When regarded as maternal parents, sexual H. pilosella gave rise to significantly lower ploidy level variation than apomictic H. bauhini, and the proportion of 5x hybrids produced by H. pilosella was much lower in the field than in the experiments. The apomictic species thus appears to be a better source of ploidy variation within populations, evidently due to its ability to take advantage of diverse modes of reproduction.
Rotreklová & Krahulcová	2009	Ploidy level and reproductive behaviour in the facultatively apomictic high-polyploid Hieracium subgen. Pilosella.
Abstract: The reproductive behaviour and the capacity to generate variation in ploidy level was studied in heptaploid and octoploid hybrid mother-plants of Hieracium subgen. Pilosella. This polyploid agamic complex is characterized by diverse ploidy levels and a combination of sexual and apomictic reproduction in different biotypes. The high-polyploid mother-plants under study originated via 2n + n hybridization in three hybrid swarms; in each of them, Hieracium pilosella (pentaploid or hexaploid, sexual or apomictic) was one parent, hybridizing with either H. bauhini (pentaploid or hexaploid, apomictic) or with H. densiflorum (tetraploid, sexual). Seeds were collected from open pollinated plants in the field as well as from open pollinated/emasculated plants in the experimental garden. Flow cytometric seed screening (FCSS; Matzk et al. Pl. J. 21: 97–108. 2000) and a modified method of FCSS (Krahulcová & Suda Biol. Pl. 50: 457−460. 2006) were used to detect the ploidy level and reproductive origin of embryos within particular maternal arrays; chromosome counts and flow cytometric detection of DNA-ploidy level were used to analyse the variation in ploidy level within the cultivated seedlings. Three pathways shaping the ploidy level variation were found: 1) Mating via unreduced gametes (2n + n, n + 2n) increased the ploidy level in the progeny of heptaploid and octoploid mother-plants even further, up to 9x−12x. 2) Mating via reduced gametes (n + n) or true apomixis (2n + 0) resulted in the cytotypes commonly occurring at the locality (4x–6x), or it conserved the maternal ploidy level (7x, 8x), respectively. 3) Haploid parthenogenesis (n + 0) reduced the ploidy level: 3x or 4x polyhaploid progeny was found. Comparison of the cytotype variation of seeds to that of seedlings, selection against progeny cytotypes with extreme ploidy levels (3x–4x or 10x–12x) was found. However, the high-polyploid hybrids may further increase the total ploidy variation within a population and may produce new biotypes with favourable combinations of characters.
Trávníček et al.	2009	Inter-cytotype interaction in populations of plants with ploidy heterogeneity: Pilosella echioides (Asteraceae) as a model system.
Abstract: The formation and maintenance of polyploids via the development of various reproductive barriers rank among the central questions of research on polyploid evolution. However, most of the recent studies dealt with model plants with quite well established diploid-polyploid pre- or postzygotic barriers and thus low frequency of fruitful reciprocal mating interaction. On the contrary, Pilosella echioides with obviously poor or no barriers among at least four contemporarily known cytotypes represents a unique naturally arisen model system for among-cytotypes mating interaction studies. Detailed study of populations at heathlands in the vicinity of Znojmo (SW Moravia, Czech Republic) revealed the co-occurrence of up to four cytotypes (2x, 3x, 4x and 5x), even on a fine spatial scale (several square centimetres). Cytotype proportions (DNA ploidy estimation by flow cytometry) based on a set of more than 2500 adult plants was as follows: 2x – 5.7%, 3x – 76.9%, 4x – 14.6% and 5x – 2.8%. The huge predominance of the triploid cytotype is a puzzle, especially as sexual reproduction (allogamy with little contribution of autogamy) was confirmed for all cytotypes (even triploids and pentaploids). A study of DNA-ploidy levels of embryo and endosperm of seeds (or more precisely achenes) harvested from mother plants of known ploidy level within subpopulations with known spatial cytotype structure was carried out. Absolutely no mature achenes on pentaploid plants (only numerous undeveloped ones) showed a female sterility of this cytotype. On the other hand, the greatest portion of variability of ploidy levels was found within embryos from achenes of tetraploid mother plants. Out of almost 200 mature achenes, 40.9% of triploid, 47.0% of tetraploid, 4.5% of pentaploid and 7.6% of hexaploid embryos were discovered. Up to three ploidy levels, but also huge numbers of aneuploids were produced by triploid mother plants – ca 150 euploid achenes contained 10% of triploid, 46.7% of tetraploid and 43.3% of pentaploid embryos. Finally, the diploid mother plants produced 78.6% of diploid and 21.4% of triploid embryos (out of 250 euploid achenes). Based on the survey of produced achenes, the theoretical proportion of ploidy levels should be as follows: 4.6% of diploid, 16.5% of triploid, 41.7% of tetraploid, 35.4% of pentaploid and 1.8% of hexaploid plants. The real cytotype proportion under natural conditions showed a considerable shift towards triploids and decreasing numbers of tetraploids and pentaploids.
Urfus et al.	2009	Variation in Pilosella officinarum F. W. Schultz et Sch. Bip. in Central Europe: ploidy and breeding systems and their correlation with morphogy.
Abstract: Pilosella officinarum F. W. Schultz et Sch. Bip. (syn. Hieracium pilosella L.; Asteraceae) belongs to a complex group of partly apomictic plants (i.e., its seeds are of clonal origin). Their enormous variability is caused especially by polyploidization, combination of the sexual and apomictic breeding mode, widespread hybridization, and vegetative reproduction (Krahulcová et al. 2000). DNA ploidy level (Flow Cytometry used) or chromosome numbers were determined for 768 plants of Pilosella officinarum from 216 localities from all over the area. Three ploidy levels were recorded within the area of Central Europe. The most widespread cytotype was tetraploid (2n=36, 65%), the second most common was pentaploid (2n=45, 18%) while the least common was hexaploid (2n=54, 17%). Breeding systems of most of the plants were determinated and showed that tetraploids were sexual and pentaploids were apomictic, but hexaploid plants were separated into two distinct groups (apomicts and sexuals) which were also geographically devided. Morphometric analyses were carried out for individual plants of particular cytotypes (4x, 5x, 6x). Principal Component Analysis, Discriminant Analysis and Nonparametrical Classification Analysis detected that tetraploid and hexaploid plants are morphologically distinct while pentaploids share morphological features of 4x and 6x plants. Nevertheless, the pentaploid cytotype shows special characters that slightly distinguish pentaploid plants from others. Even sexual and apomictic hexaploids could be distinguished on the basis of morphological features. These results indicate that the two groups of hexaploids might be unrelated. Sexual hexaploids are considered to be of relict origin, apomictic hexaploids appear to be connected to a possibly hexaploid cytotype distribution centre in the Carpathian Mts. like the majority of the pentaploid cytotypes. The results possibly reflect a reticulate evolution of the group and may be considered as an alternative to the traditional complicated taxonomic view.
Fehrer et al.	2010	Hieracium s.str. speciation and evolution.
Abstract: The large number of species and the presumed extensive past hybridization combined with an abundant occurrence of polyploidy and apomictic reproduction have presented enormous difficulties for the taxonomy of Hieracium s.str. Thus, the study of their relationships was greatly hampered by species circumscription. Our first molecular analysis of this group that focused on diploids and polyploid basic species revealed that the problems go even deeper than anticipated, but also helped to understand the basic processes involved in Hieracium speciation. Only a small part of the basic species did not have hybrid origin. These formed two groups coinciding with Western or Eastern European origin who probably survived in different glacial refugia. The ‘Western’ group comprised H. humile, H. tomentosum, H. pictum, H. stelligerum, H. lawsonii, H. ramondii, and H. recoderi, which have their main distribution in the Western Alps and in the Pyrenees, but also the widespread or Central European species H. bifidum, H. murorum, and H. schmidtii. The only species not fitting geographically into the ‘Western’ group was H. transylvanicum. Hybrid origin within the ‘Western’ group was found for H. lucidum (NW Sicily) and H. cordifolium, H. gymnocerinthe, H. candidum and H. cerinthoides (Pyrenees). Apart from a distinguishable subgroup of Pyrenean species, almost no genetic diversity was found among ‘Western’ species. To the ‘Eastern’ group belonged (i) all species from the Balkans (H. petrovae, H. kittanae, H. naegelianum, H. pannosum and H. sparsum – the latter had apparently hybrid origin), which were most basal, (ii) H. alpinum with a lineage on its own, (iii) a group consisting of H. porrifolium, H. bupleuroides, H. villosum and H. pilosum, and (iv) a group comprising H. umbellatum, H. eriophorum, H. virosum, and H. canadense. Besides, H. pojoritense and one accession of H. bupleuroides had hybrid origin involving ‘Eastern’ parents. Despite these clearly distinguishable species groups, the low genetic variation also occurring among ‘Eastern’ species suggests a rapid recent divergence of the different lineages. Most taxa with hybrid origin comprised species from both groups and may have mainly formed as a result of secondary contact after the retreat of glaciers. They comprised H. prenanthoides, H. amplexicaule, H. gouani, H. mixtum, H. caesium, H. racemosum, H. sabaudum, H. bracteolatum, H. lachenalii, H. laevigatum, H. heterogynum, H. plumulosum (H. waldsteinii s.l.), H. gymnocephalum, H. olympicum, H. glaucum, and single accessions of H. villosum and H. pilosum. Several genetic lineages occurred only in hybrids, but not in any extant ‘pure’ species. For example, H. bracteolatum, H. racemosum, H. sabaudum, H. lucidum, H. olympicum and one accession of each H. prenanthoides, H. villosum, H. pilosum, and possibly also H. mixtum had hybrid origin involving at least one extinct parent; two extinct lineages contributed to H. gymnocephalum, H. plumulosum and H. heterogynum. In the latter two taxa and in two accessions of H. prenanthoides, contributions from 3-4 different parents were detected. A large number of meanwhile extinct taxa involved in the hybrid origin of basic (even diploid) species helps to better understand the taxonomic difficulties in Hieracium s.str.
Krahulcová et al.	2010	Pilosella aurantiaca and P. officinarum hybridizing in the field: population structure of an exemplary hybrid swarm in North Rhine-Westphalia, Germany.
Abstract: The population is comprised of two tetraploid, morphologically distinct parental species, an introduced facultatively apomictic P. aurantiaca, a native sexual P. officinarum and their recent hybrids, which are tetraploid or hexaploid. The hybrid swarm, first found in 1990, is growing on nutrient-poor fallow land, but the meadow was occassionally mown in the past. A wide spectrum of coexisting hybrid morphotypes has practically been unchanged during twenty years, involving types i) more close to P. aurantiaca (corresponding to P. rubra), ii) intermediate between the parental species (P. stoloniflora), and iii) several different types more or less close to P. officinarum. When comparing the present situation with that twenty years ago, the abundance of some morphotypes as well as the whole population size have changed. Recently, the population structure was studied with respect to ploidy level, genome size, breeding system and chloroplast-DNA haplotypes. Using isozyme phenotypes, the genotype structure of apomictic plants was identified. The hybrid corresponding to P. rubra is hexaploid with a variable reproductive mode, producing a considerable amount of sexual and polyhaploid progeny besides true apomictic progeny. Its seed fertility is reduced. The genotype structure and DNA content in this hexaploid suggest a repeated origin via 2n + n hybridization of P. aurantiaca (maternal parent) and P. officinarum. The other coexisting hybrids are tetraploid and sexual. Two chloroplast DNA haplotypes were found in P. officinarum at this locality, one of them shared with P. aurantiaca. This fact did not allow an unequivocal identification of the maternal parent in the hybrids. The different genome size (DNA content in the monoploid chromosome set) in the putative parental species, P. aurantiaca and P. officinarum, is reflected in their homoploid hybrids which have different proportions of parental genomes. Thus, multistep hybridization (backcross to P. officinarum) suggested by morphological characters of the tetraploid hybrids, was supported by genome size data.
Krahulcová et al.	2010	Structure of selected Pilosella populations in Bulgaria and their comparison with Central Europe.
Abstract: During the last years we paid attention to the structure of Pilosella populations in Bulgaria. Chromosome numbers, breeding systems, and additionally chloroplast haplotypes and clonal structure were studied. In comparison with Central Europe, there are pronounced differences: 1. There are more diploid species, but it seems that they are isolated from the polyploid ones; their hybridization is comparatively very rare. 2. Polyploid sexual types are almost absent (except triploid P. alpicola and tetraploid P. pavichii). 3. Most of the polyploids are hexaploids, more rare are pentaploids, and tetraploids (P. officinarum, P. pavichii) are very rare. 4. We did not find any 2n + n hybrids, probably because of low viability of potential high polyploids. 5. Stabilized hybrids are probably very rare in Bulgaria; as far as we know, only P. guthnickiana is such a case.
Krahulec et al.	2010	Variation in apomictic Pilosella species: do we know how and where it is expressed?
Abstract: The present taxonomic concept of apomictic Pilosella species is a broad one; most of the species are represented by many genotypes. This seems very reasonable with respect to the relatively high level of residual sexuality. The infraspecific variation is expressed at several levels: chromosomal (different ploidy levels), genotype and chloroplast DNA haplotype level. Variation in ploidy levels is the best known, because during more than 50 years considerable knowledge has been collected. But even the most known species as P. officinarum (both sexual and apomictic) were only fragmentarily studied in some parts of the distribution area (in this and most other species, especially in the south-eastern part of Europe). Variation at the genotype level is known extremely unevenly. Whereas in some geographic areas that have been studied extensively in the past, the knowledge is deep, in most areas, nothing is known. Most of the basic species have a low level of variation in some parts of the distribution area, while high variation was recorded in some smaller areas. The most variable seems to be P. piloselloides subsp. bauhinii, which has many genotypes at the locality level. The situation of intermediate species is different. Their variation evidently depends on the frequency of their origin: sometimes variation is expressed at the locality level, sometimes on larger geographic scale. There are differences in particular regions, similar to basic species. We have only fragmentary knowledge about the distribution of chloroplast haplotypes. The existing knowledge allowed us to study the origin of some hybridogenous taxa in particular regions, but phylogeographic studies are not possible so far.
Krak et al.	2010	Three new low-copy nuclear markers for low level systematic studies in the Asteraceae – development and preliminary results of the phylogenetic analysis of Hieracium subgen. Hieracium and Hieracium subgen. Chionoracium.
Abstract: In molecular systematics of plants, low-copy nuclear genes are the tools of choice in cases when cpDNA and nrDNA give only restricted resolution and/or conflicting phylogenetic signals. Such results are relatively frequent in studies at species level (or below), especially when polyploidization, hybridization and introgression are the driving forces of the evolutionary processes. Here we present three newly developed low-copy nuclear markers for the Asteraceae: Gamma-glutamylcysteine synthetase (GSH1), Squalene synthase (SQS) and Glycine hydroxymethyltransferase (SHMT). All three markers amplified successfully in the representatives of 7 out of 8 tested tribes of the family. To assess their potential utility for low taxonomic level phylogenies, the phylogenetic signal and the level of variation for each of these markers was estimated on a small number of accessions from the Lactuceae subtribe Hieraciinae. All three markers have considerably higher variation (1.9–4.5 times) than ITS. However, incongruence in tree topologies was observed among the results of the different phylogenetic analyses. Given that all accessions used for the pilot study were confirmed diploids and that no more than two sequence types per accession were obtained despite the high number of sequenced clones, we consider both intraindividual variability and topological incongruence to be results of allelic variation connected with extensive hybridization processes rather than paralogy caused by gene duplication. Two of these markers, SQS and SHMT were analyzed in 70 species of Hieracium subgen. Hieracium and Hieracium subgen. Chionoracium represented by 80 accessions. The preliminary analyses of these datasets resulted in huge topological incongruences when the low copy nuclear gene phylogenies were compared to each other or to previously published results (based on nrDNA ETS and cpDNA). Likewise in morphology, frequent multi-directional hybridization processes affected the evolutionary pathway of these subgenera into a maze, complex and obscure from the molecular point of view as well.
Křišťálová et al.	2010	Populations of the Pilosella species in ruderal habitats within the city of Prague: chromosome numbers and reproductive mode.
Abstract: Pilosella populations were studied on 49 selected localities situated along highways and railways within the city of Prague and its close vicinity. These habitats are especially suitable for the spread, formation and survival of these species, because competition-free habitats are formed. The study was done during a short period; altogether, four basic species and 12 hybridogenous ones (or hybrids) were recorded. The following species, hybridogenous species or hybrids (with ploidy levels/chromosome numbers and reproductive mode) were found: P. aurantiaca, P. caespitosa (4x, 5x), P. officinarum (2n = 36, sexual; 2n = 54, sexual; 2n = 63), P. piloselloides subsp. bauhinii (2n = 45, 54; both apomictic), P. piloselloides subsp. praealta (5x; apomictic), P. brachiata (4x; sterile), P. densiflora (4x), P. flagellaris, P. floribunda, P. erythrochrista, P. glomerata (4x, 5x; apomictic), P. leptophyton (5x; apomictic), P. rothiana (4x, apomictic), P. setigera (apomictic), P. visiani (4x; apomictic), P. ziziana (apomictic), and a so far undescribed hybridogenous type, P. setigera x P. piloselloides. Pilosella visianii is reported from the Czech Republic for the first time. New habitats formed during highway construction seem to be suitable for Pilosella species. Many types previously known to be rare such as P. rothiana find here open space for further spreading, not only at the local scale, but throughout the whole country.
Rosenbaumová et al.	2010	Sexual reproduction as a source of ploidy level variation in the agamic complex of Hieracium subgenus Pilosella (H. pilosella and H. bauhinii as a model system).
Abstract: Apomixis (clonal reproduction through seeds) is not usually regarded as a source of genetic variation. Nevertheless, exceptional diversity in morphology, ploidy level, mode of reproduction, etc. emerges in agamic complexes, where apomixis coexists with sexual reproduction. A study of recent hybridizations in young agamic complexes, e.g. Hieracium subgen. Pilosella, can improve our understanding of the origin of such diversity. A model population from the vicinity of Valov (NW Bohemia) consisted of 4x sexual H. pilosella and 6x apomictic H. bauhinii (parental species), and their 5x, 7x, and 8x hybrids. Crosses between the parental species were performed to quantify their potential to produce ploidy level variation. Flow cytometry revealed significant ploidy level variation in the progeny, showing that reduced as well as unreduced gametes of both parents participated in crosses. The progeny from the cross where H. pilosella served as a maternal parent (273 plants) consisted of 4x progeny resulting from autogamy (18.3%) and of 5x hybrids (81.7%). The progeny from the cross where H. bauhinii served as a maternal parent (821 plants) consisted of apomictically derived 6x progeny (93.2%), of three types of hybrids (5x ­­­– 4.8%, 7x – 0.1%, and 8x – 0.7%), and of 3x parthenogenetic progeny (1.2%). To quantify the ploidy level variation really formed in the field, the ploidy level was estimated in the progeny that was obtained from seeds collected from the parental species in the model population. The progeny of H. pilosella (317 plants) consisted of 4x progeny from auto/allogamy (98.7%) and of 5x hybrids (1.3%). The progeny of H. bauhinii (486 plants) consisted of apomictically derived 6x progeny (92.4%), of four types of hybrids (5x – 2.7%, 7x – 0.4%, 8x – 2.9%, and 10x – 0.4%), and of 3x parthenogenetic progeny (1.2%). Hybridization between H. pilosella and H. bauhinii generated significant variation in ploidy level under both experimental and field conditions. Nevertheless, it seems that only a part of this variation can influence evolution of the population because neither 3x, nor 10x adults were detected in the field. Some selection disadvantage of these cytotypes can be suggested. When regarded as maternal parents, sexual H. pilosella gave rise to significantly lower ploidy level variation than apomictic H. bauhinii, and the proportion of 5x hybrids produced by H. pilosella was much lower in the field than in the experiment. The apomictic species thus appears to be a better source of ploidy variation in the population.
Rotreklová et al.	2010	Variability in morphology, karyology and reproductive behaviour in a hybrid swarm of hawkweeds from Rašovice village (South Moravia).
Abstract: Hieracium subgen. Pilosella has a high taxonomic diversity including hybridogenous types. Population studies on co-occuring sexual and facultative apomictic taxa are necessary to understand the selective processes during the evolution of new types. In a hybrid swarm situated on a steppe slope, the variability of plants in morphology, ploidy level, reproductive systems and progeny origins was studied. On the locality co-occurred two parental taxa, Hieracium pilosella (6x, facultatively apomictic), H. densiflorum (4x, sexual), and their hybrids (4x, 5x, 6x, and 8x; sexual, apomictic or sterile). Hybrids were classified as H. fallacinum and H. pilosellinum, besides plants closely resembling H. pilosella. We found that: 1. Progeny of sexual H. densiflorum mothers was uniform and formed exclusively sexually, via fusion of reduced gametes (n + n progeny). 2. Progeny of facultatively apomictic H. pilosella mothers originated via four different pathways: apomixis (somatic parthenogenesis, 2n + 0 progeny, the dominant breeding system), sexual mating via fusion of either reduced (n + n progeny) or unreduced gametes (2n + n progeny), and haploid parthenogenesis (n + 0, polyhaploids). 3. The reproductive behavior of the hybrids was more diverse: Pentaploids were sterile or semisterile, poor seed-set was produced exclusively sexually (n + n progeny). Hexaploids were either semisterile or produced seeds sexually (n + n progeny prevailed while that of 2n + n origin was rare). Part of the octoploids (recent 2n + n hybrids derived from a pentaploid mother) were semisterile plants, which formed poor seed-set sexually (frequently n + n progeny, rarely 2n + n progeny). The other octoploids, derived from an octoploid apomictic mother, produced progeny apomictically (2n + 0 progeny), almost half of their progeny were produced via haploid parthenogenesis (n + 0 polyhaploid progeny), and only a small part of their seeds were produced sexually (either n + n or 2n + n progeny). 4. Tetraploid progeny, derived from apomictic octoploid mothers via haploid parthenogenesis, again produced mainly tetraploid progeny via apomixis, and rarely n + n or 2n + n progeny of sexual origin. 5. The maternal breeding system was conserved in the majority of the respective progenies. Our study demonstrates the versatility of reproductive pathways operating in polyploid facultative apomicts in the field and the importance of their residual sexuality as we found in a former study on other hybrid swarms of Hieracium subgen. Pilosella.
Rotreklová et al.	2010	Ploidy level and reproductive behaviour in facultatively apomictic high-polyploid Hieracium subgen. Pilosella.
Abstract: The reproductive behaviour and the capacity to generate variation in ploidy level was studied in hexaploid, heptaploid and octoploid mother plants of Hieracium subgen. Pilosella. They originated in the field from hybridizations between facultatively apomictic and sexual species (via fertilization of unreduced gametes) in eight hybrid swarms: seven in the Czech Republic and one in Germany. In all of them, Hieracium pilosella (tetraploid, pentaploid or hexaploid, sexual or apomictic) was one parent, hybridizing either with H. bauhinii (pentaploid or hexaploid, apomictic), H. aurantiacum (tetraploid, apomictic) or with H. densiflorum (tetraploid, sexual). Seeds were collected both from open-pollinated plants in the field and from open-pollinated/emasculated plants in the garden. The method of Flow Cytometric Seed Screen (FCSS) was used to detect ploidy level and reproductive origin of embryos within particular maternal arrays; chromosome counts and flow cytometric detection of DNA-ploidy level were used to detect the variation in ploidy level within the cultivated seedlings. Three ways in shaping the ploidy level variation were found: 1. Mating via unreduced gametes (2n + n, n + 2n) even further increased the maternal ploidy level in their progeny, reaching up to 8x – 12x. 2. Mating via reduced gametes (n + n) or true apomixis (2n + 0) resulted in the cytotypes commonly occurring at the respective localities (4x – 6x), or it conserved the maternal ploidy level (6x, 7x, 8x), respectively. 3. Haploid parthenogenesis (n + 0) reduced the maternal ploidy level, ranging from 3x to 4x in the polyhaploid progeny. The vigour observed in embryos and seedlings of the same origin was different. As many well-developed seeds had detectable embryo and endosperm using the FCSS method, only a small part of the seed-set collected from the same plants were able to germinate. Comparing the variation within the seeds and seedlings, a lack of progeny cytotypes with extreme ploidy levels (lower than 4x and those ranging between 10x – 12x) was found in seedlings. The extremely high ploidy levels are yet more suppressed in the field, as no plants exceeding the octoploid level were found. Nevertheless, the facultatively apomictic high-polyploid hybrids may increase the total ploidy variation in populations. Due to relevant sexual reproduction, they can produce new biotypes with favourable combinations of characters.
Šingliarová et al.	2010	Evolutionary significance of polyploidy and hybridization in the Pilosella alpicola group – what we know and what we would like to know.
Abstract: According to our biosystematic study, the Pilosella alpicola group comprises four morphologically well-differentiated and geographically vicariant species with contrasting cytotype pattern. Pilosella alpicola s.str. is a polyploid (4x and 5x) taxon distributed in the Swiss and Italian Alps. Pilosella ullepitschii is a diploid, self-incompatible taxon endemic to the Carpathians. Four ploidy levels (2x–5x) with a complex cytogeographic pattern and a high frequency of mixed-ploidy populations were recorded in P. rhodopea. Pilosella serbica is a diploid sexual taxon confirmed only from Serbia and Montenegro. In spite of a clear morphological separation, molecular analyses suggest a rather recent and monophyletic origin of the group, with the exception of P. alpicola s.str. While P. rhodopea polyploids originated by autopolyploidization, P. alpicola s.str. is an allopolyploid species that originated from hybridization between Balkan P. rhodopea and Alpine P. glaciale. The reproductive mode of the polyploids is linked to their origin. All cytotypes of P. rhodopea reproduce sexually, while polyploids of P. alpicola s.str. are apomictic. Allozyme differentiation among morphologically well-defined species was rather low, but reflecting the mode of reproduction and the type of polyploidy. Genetic markers used so far (allozymes, cpDNA and ITS sequences) failed to show evolutionary relationships between the closely related P. rhodopea, P. ullepitchii and P. serbica. Therefore, we plan to use more discriminative markers such as AFLPs. Hybridogeneous origin of P. alpicola s.str. will be studied by FISH and GISH techniques to determine how many copies of each parental genome were involved in the tetra- and pentaploid cytotype of P. alpicola s.str. Our further research will be focused on the fine-spatial cytotype structure and on microhabitat preferences of the cytotypes of P. rhodopea in mixed-ploidy populations, on gene flow between cytotypes and on differences in fitness under natural and experimental conditions. Morphological characters discriminating particular taxa of the P. alpicola group are mainly those considered as important phenotypic traits involved in evapotranspiration (density and colour of indumentum). Similarly, cline variation from North (Carpathians) to South (Balkan) was observed in genome size and some ecophysiological traits related to water-use efficiency. It seems that adaptive vicariant speciation after range fragmentation played an important role in the evolutionary history of the group. Preliminary results based on plants collected in the field should be verified in a common garden experiment. Natural hybridization occurs, when P. rhodopea and P. serbica co-occur with P. macrantha or P. pseudopilosella as evidenced by intermediate morphology and additive patterns of ITS sequences. However, we do not know anything about the frequency and direction of the interspecific gene-flow.
Trávníček et al.	2010	Geographic pattern of cytotype variation in Pilosella echioides (Asteraceae) in Europe.
Abstract: Pilosella echioides is a perennial, predominantly sexual and self-incompatible herbaceous plant distributed in Central and Eastern Europe and in steppe regions of Asia. Six ploidy levels have been previously discovered in Central Europe, namely 2x, 3x, 4x, 5x and 6x, and several mixed-ploidy populations have been found. However, a more general geographical pattern of cytotypes and the frequency of particular cytotypes in mixed-ploidy populations remained a puzzle. Principal objectives of the present study were to elucidate the distribution of particular cytotypes (i) in Europe (large-scale pattern), and (ii) in a model area with previously detected mixed-ploidy populations in SW Moravia and an adjacent part of Lower Austria (small-scale pattern). Diploid populations seem to prevail throughout Europe; mixed ploidy populations with two to four intermingled sexual cytotypes (2x, 3x, 4x, 5x) occur in the Czech Republic and in Lower Austria. Populations consisting exclusively of tetraploid apomictic plants were discovered in northern Hungary and southern Slovakia (Danube Basin), their taxonomic position is still unclear. Besides these, sexual tetraploids were found in NW Hungary. The extremely complicated variation in Hungary is underlined by the occurrence of intermediate types between P. echioides and P. rothiana. A detailed survey of mixed-ploidy populations at the Havraníky heathland (SW Moravia, Czech Republic) and adjacent areas revealed a common co-occurrence of up to four cytotypes (2x, 3x, 4x and 5x) even at a fine spatial scale (several square centimetres). Triploids markedly prevailed (more than 75% of the plants), considerably lower frequencies were detected for diploids, tetraploids, and pentaploids. The predominance of the triploid (sexual) cytotype is with no doubt mysterious, especially if we consider that only sexual (allogamy) reproduction was confirmed for all cytotypes and that crosses between two triploids never resulted in triploid progeny as showed by hand pollination. Elucidation of the spatial pattern at the Havraníky heathland falls in a complex project aimed at the population dynamics of mixed ploidy populations. Detailed analysis of cytotype diversity in the progeny of di-, tri- and tetraploid plants from subpopulations with contrasting frequencies of cytotypes and consequent comparison with spatial pattern of adult plants is in progress.
Trávníček et al.	2010	Free sex as a way to long-term persistence of cytotypes in sympatry: study on Pilosella echioides.
Abstract: Two main scenarios are usually considered as an explanation of sympatric coexistence of different cytotypes of autopolyploid origin: i) a temporal stage based on frequency-dependent mating, where one cytotype will outcompete the other over time (minority cytotype exclusion principle), and ii) a more stable system based on the evolution of assortative mating and reproductive isolation of cytotypes (comprising niche differentiation, flowering-time divergence, etc.). Both scenarios assume strong selection against putative inter-cytotype hybrids because of, for instance, triploid block and effort is made to evolve mechanisms preventing their formation. Despite these theoretical considerations, we found a system of long-term persistence of up to five cytotypes maintained by random and obviously non-assortative mating with high viability and fertility of inter-cytotype hybrids in natural populations of Pilosella echioides. This finding, together with an often observed prevalence of triploids in mixed-ploidy populations, throws new light on our understanding of cytotype co-existence and provides the first evidence of such a system in plants with an exclusively sexual mode of reproduction.
Zahradníček & Chrtek	2010	Phylogeography of Hieracium intybaceum (Asteraceae).
Abstract: Effects of historical climate changes on genetic structure of populations and changes in distribution areas (fragmentation, retreat to lower altitudes and recolonization, differentiation, surviving in refuges) in recent history of alpine species belong to very timely research areas. Hieracium intybaceum (Asteraceae) is a well defined species that grows on silicate rocks in high altitudes in the Alps, Vosges and Schwarzwald Mts. Though traditionally recognised as a part of Hieracium genus (subgenus Hieracium) molecular data suggest an older isolated lineage. Combination of molecular and flow-cytometry methods along with morphometrics and reproduction experiments will help to elucidate the phylogeographical pattern within and among cytotypes and the origin of new polyploid cytotypes. It can also contribute to the knowledge of main refuges and migration routes during the recolonization of high altitudes. Surprisingly, only diploid populations were found during a pilot sampling in the Eastern and Central Alps. Very low genetic variation detected by AFLP contradicts sexuality and self-incompatibility. However, more representative sampling is necessary to draw final conclusions.
Zahradníček et al.	2010	Genome size and evolutionary history of Mediterranean-Macaronesian genus Andryala (Hieraciinae, Asteraceae).
Abstract: Depending on taxonomic concept, the genus Andryala comprises about 25 species distributed in the Mediterranean Basin and Macaronesia with centres of diversity in NW Africa, Spain and the Canary Islands. It includes most likely only diploid sexuals (in contrast to the related genus Hieracium s.l.), and is also interesting for its diversity of growth forms. According to our preliminary results based on ITS nuclear DNA markers, the genus Andryala is monophyletic. Chloroplast DNA markers detected introgression from one Pilosella lineage to Andryala by hybridization that preceded the speciation of recent species in this genus. We also intend to include additional molecular markers to elucidate phylogenetic relationships within the genus. Study of genome size evolution is underway, attention is paid to relationships between genome size and phylogeny, geography (genome size seems to be markedly lower on the Macaronesian islands in comparison with the continent), growth form, breeding system (allogamy vs. autogamy), ecological demands, phenology etc.
Fehrer & Chrtek	2011	Evolution of the American Hieracium subgenus Chionoracium.
Abstract: The rather little explored American Hieracium subgenus Chionoracium is distributed from Alaska to Patagonia and consists of approx. 150 species all of which are diploid sexuals as far as known. We investigated their relationships using two non-coding chloroplast inter­genic spacers (trnT-trnL and trnV-ndhC) and the nuclear ribosomal external transcribed spacer (ETS). Previous analyses that included only a few taxa suggested that Chionoracium nests within Hieracium s.str., but the resolution of the markers (trnT-L, matK and ITS) used in that study was relatively low. TrnV-ndhC was more variable and also longer than trnT-trnL, and combined analyses of both cpDNA regions provided relatively high resolution. These results suggest that subgenus Chionoracium is monophyletic and nests near the base of several Hieracium s.str. lineages, i.e., none of the present-day Hieracium subgen. Hieracium taxa show particular affinities to the American lineage. Monophyly in the cpDNA tree may suggest a single introduction of the ancestral taxon into the New World with subsequent speciation and colonization of the entire continent. The ETS tree revealed that Chionoracium is derived from the ‘Eastern’ clade of Hieracium s.str. species. This fits to the relatively high genome size of three Chionoracium taxa analyzed so far. Interestingly, two Chionoracium lineages were found in the ETS tree, which reflect the disjunct distribution of Chionoracium in North America fairly well. One of these lineages consisted of species endemic to NW America (Alaska to California) whereas the other comprised the rest of the analyzed taxa. The two lineages insert at the base of the ‘Eastern’ clade suggesting early rapid speciation from an ‘Eastern’ Hieracium ancestor, comparable to the lineages of Hieracium s.str. taxa that belong to this clade. The colonization of North America by H. umbellatum / H. canadense occurred independently. Both datasets are partly congruent with respect to species relationships. Almost all South American species are apparently derived from Central American species and from taxa endemic to southern North America, with the exception of H. Antarcticum (Patagonia). This species clusters with H. Gracile and H. Triste (both from Alaska / NW North America) in accordance with taxonomic treatments. One possibility for this disjunct distribution is long-distance dispersal via migratory birds. Particularly close relationships and relatively late speciation events were found for the South American radiation (except H. Antarcticum) and for three, mainly NW American taxa (H. albiflorum, H. bolanderi, and H. Argutum); according to cpDNA, the latter lineage may be derived from species occurring mostly in eastern North America. Some discrepancies between the ETS and cpDNA trees are not readily explained and may reflect a complex speciation history of which maternally and biparentally inherited markers reveal different aspects. A denser taxon sampling and more molecular markers may provide better insight into Chionoracium species relationships and colonization events.
Fehrer et al.	2011	When low copy nuclear genes are unable to produce species trees despite large genetic variation: Lessons from a highly reticulate plant group (Hieracium).
Abstract: A major challenge for the reconstruction of species relationships based on molecular phylogenies is presented by groups with recent speciation, abundant hybridization and the occurrence of polyploidy. Hawkweeds (Hieracium) are among the most complex plant groups in these respects. Besides, the fixation of genotypes by apomixis has a major impact on the maintenance of alleles in taxa of hybrid origin. For the elucidation of species relationships and the origin of hybrid taxa, we used seven molecular markers: two chloroplast DNA regions (trnT-L and trnV-ndhC intergenic spacers), two nuclear ribosomal gene regions (ITS and ETS), and three newly developed low copy nuclear (LCN) genes (glycine hydroxymethyltransferase - shmt, squalene synthase - sqs, and gamma-glutamylcysteine synthetase - gsh1). The three LCN genes provided the highest amount of variation, however, the trees were largely incongruent. The lowest variation was found with ITS and the two chloroplast markers. The best estimate of species relationships was provided by the ETS tree. It revealed two major lineages with Eastern or Western European origin and significantly different genome size which contained several subclades each and showed that many accessions had hybrid origin. The LCN gene trees partly matched ETS subclades with taxa of hybrid origin clustering along with at least one parental species revealed by ETS. On the one hand, the LCN genes divided the ETS subclades into two or more divergent lineages while on the other hand they merged taxa that belonged to different major ETS clades within a single lineage, with little variation among them. However, the number of divergent lineages did not exceed the ploidy level of particular accessions. No evidence for pseudogenes or paralogs was found, and the divergent LCN variants were therefore interpreted as divergent alleles of heterozygous (or hybrid) individuals. Relationships among these lineages remained unclear with all three LCN genes (no support for basal lineages, only for crown groups), i.e., neither was able to produce a species tree. According to LCN genes, the genus behaves like a single large population characterized by a rapid diversification of basal lineages and suffers from lineage sorting with a rather accidental distribution of particular alleles among the species. Interpretations are further complicated by the hybrid origin of many taxa. In addition, ETS, cpDNA and LCN genes all showed evidence for extinct diploid species whose ribotypes, haplotypes, or alleles were maintained in accessions with hybrid origin (including diploids), but were not found in any diploid non-hybrid species (only one diploid Hieracium species was not sampled yet). While nuclear ribosomal DNA seemed to best reflect species relationships, the more variable LCN genes could provide deeper insight into the processes that have lead to the origin of particular species. In any case, much care has to be taken with the interpretation of LCN gene trees. Each might provide a different aspect of speciation in a particular group, but to allow or at least facilitate interpretations, a reasonable species tree based on other evidence (nuclear ribosomal DNA, morphology, geographic distribution etc.) is strongly recommended.
Trávníček et al.	2011	Cytotype and genetic variation of Pilosella echioides (Asteraceae) in Central Europe.
Abstract: Pilosella echioides is a perennial, predominantly sexual and self-incompatible herbaceous plant distributed in Central and Eastern Europe and in steppe regions of Asia. Five ploidy levels have been previously discovered in Central Europe, namely 2x, 3x, 4x, 5x and 6x, and several mixed-ploidy populations have been found. Principal objectives of the present study were to elucidate the distribution of particular cytotypes (i) in Europe (large-scale pattern), and (ii) in a model area with previously detected mixed-ploidy populations in SW Moravia and the adjacent part of Lower Austria (small-scale pattern), and to detect the pattern of genetic variation with respect to geography and ploidy level. Diploid populations seem to prevail throughout Europe; mixed ploidy populations with two to four intermingled sexual cytotypes (2x, 3x, 4x, 5x) occur in the Czech Republic and Lower Austria. Populations consisting exclusively of tetraploid apomictic plants were discovered in northern Hungary and southern Slovakia (Danube Basin); their taxonomic position is still unclear. Besides, sexual tetraploids were found in NW Hungary. A detailed survey of mixed-ploidy populations at the Havraníky heathland (SW Moravia, Czech Republic) and adjacent areas revealed a common co-occurrence of up to four cytotypes (2x, 3x, 4x and 5x) even at a fine spatial scale (several square centimetres). AFLP data for the complete data set (Central Europe) did not show a clear structure with respect to both geographic provenience and ploidy level. However, some geographic structure was found in SW Moravia and the adjacent part of Lower Austria. The low genetic differentiation can be explained by rather recent fragmentation of the former geographic range (it is supposed that P. echioides was a common species in the periods with steppe vegetation in Central Europe) and/or gene flow between populations. A close relationships between di- and polyploids supports the hypothesis of autopolyploid origin of the higher ploidy levels. A detailed study of population dynamics of mixed-ploidy populations in SW Moravia is now in progress.
Zahradníček et al.	2011	Phylogeography of the species Hieracium intybaceum (Asteraceae).
Abstract: Hieracium intybaceum (Asteraceae) is a well-defined species that grows only on silicate rocks in high altitudes in the Alps, Vosges and Schwarzwald Mts. Though traditionally recognised as a part of genus Hieracium (subgenus Hieracium), molecular data suggest an older isolated lineage. We have collected 43 populations throughout the Alps and one population from an outlying locality in the Vosges Mts. Surprisingly, diploid sexual and allogamous plants prevail. Tetraploid populations seem to be confined to the western Alps and the Vosges Mts. This distribution pattern of cytotypes is in conflict with most phylogeographic studies in the Alps. Previously published triploid plants have not been found so far. AFLP molecular data revealed a very low variation among diploid populations and high variation between ploidy levels. The latter might indicate an allopolyploid origin of the tetraploid plants. For final conclusions, more tetraploid populations will have to be sampled.
Fehrer et al.	2012	Patterns and processes of speciation in two closely related agamic complexes of hawkweeds.
Abstract: Agamic complexes present particular challenges for the reconstruction of species relationships. The hawkweed genera Hieracium and Pilosella are characterized by relatively small numbers of diploids and high numbers of polyploids. Different ploidy levels can occur in the same species. The genera are closely related, but differ strongly not only morphologically, but also by contrasting modes of reproduction and cytotype distribution. In Hieracium, apomixis of the diplosporous type occurs; all polyploids appear to be obligatory apomicts. Most species are triploid or tetraploid. Many of the basic evolutionary units (diploids and well-defined polyploids with unique morphology) unexpectedly had ancient hybrid origin; recent hybridization does not play a significant role in this genus. Multi- and single-copy nuclear as well as chloroplast markers showed evidence for extinct diploid lineages. Ancestral polymorphism and incomplete lineage sorting further complicated the inference of species relationships. Survival of ancestral diploids in different glacial refugia, very recent speciation, hybridization as a result of secondary contact, and the emergence of polyploidy and apomixis in this context are currently considered as cornerstones of Hieracium speciation. In Pilosella, apomixis of the aposporous type occurs; polyploids can be sexual or facultatively apomictic. Most taxa are tetraploid or pentaploid. Speciation at the diploid level appears to be more regular than in Hieracium, resulting in several distinct species clades. Recent hybridization with gene flow across ploidy levels is abundant in Pilosella; reproductive barriers are almost completely absent. A high proportion of apomictic hybrids arose from apomictic seed parents and sexual pollen donors indicating an evolutionarily significant role of residual sexuality of apomicts in this genus. Based on multilocus fingerprinting and isozyme analyses, some older polyploid species with unique morphology consist of only a single or a few clones; their origin from diploid progenitors is still unclear. Apomictic taxa with intermediate morphology are formed recurrently from the same or similar parents. Those with a high degree of apomictically produced seeds establish stable populations and can spread over large distances. Those with lower proportions of apomictic progeny produce higher amounts of polyhaploids and/or hybrids; this high genotypic diversity can act as raw material for selection of favorable genotypes.
Ferreira et al.	2012	Phylogenetic relationships of Andryala L. (Asteraceae): emphasis on the Macaronesian taxa.
Abstract: Andryala L. (Cichorieae, Asteraceae) includes 16 species distributed in the Mediterranean basin and Macaronesia, where it is represented by 4 endemic species. Phylogenetic analyses based on nuclear ribosomal (ITS and ETS) and chloroplast (trnT-trnL and trnV-ndhC) markers were conducted using maximum parsimony and Bayesian inference in order to clarify species relationships. All markers clearly revealed the monophyletic origin of Andryala, but all datasets also showed very low genetic variation. Two lineages of glacial relict taxa were strongly supported and occurred in basal positions in all trees. A third potential glacial relict species was also well distinguished from the rest of the taxa whose relationships remained largely unresolved. Among the latter, accessions of the same species did not group together, or only with some markers. However, cpDNA showed some indication of geographic structure: North African A. pinnatifida subspecies can be distinguished from those of the Canary Islands; A. integrifolia seems to be a neophyte on the Canary Islands; European and African species can be distinguished from Macaronesian ones. Species relationships among most Andryala species, including those from Macaronesia, remain unresolved. Each of the four markers usually provides sufficient resolution at species level in plants. That this is not true for most Andryala species can be explained by their very recent speciation and rapid radiation.
Koltunow et al.	2012	Apomixis in polyploid Hieracium species.
Abstract: Subgenera Hieracium and Pilosella of the Asteraceae subtribe Hieraciinae contain species that can reproduce solely by sexual reproduction while others form most of their seeds asexually by apomixis. Apomixis is gametophytic in both subgenera in that embryo sacs form apomeiotically (without meiosis) in ovules. Subsequent embryo and endosperm formation within these embryo sacs is fertilization independent, giving rise to progeny with a maternal genotype. The mode of apomeiosis differs in both subgenera. Subgenus Hieracium species exhibit diplospory and subgenus Pilosella species exhibit apospory. The origin of the cell giving rise to the apomeiotic embryo sac enables discrimination between the two modes. We have used subgenus Pilosella species to examine the genetic and molecular basis of apomixis. In H. praealtum accession R35, the dominant LOSS OF APOMEIOSIS (LOA) locus functions to induce somatic aposporous initial (AI) cell formation near sexually programmed cells in the ovule. The AI cells form aposporous embryo sacs by mitosis, and the adjacent sexually programmed cells die. Independent function of the LOSS OF PARTHENOGENESIS (LOP) locus in the aposporous embryo sacs enables fertilization-independent embryo and endosperm development. Deletion of both loci by irradiation leads to sexual reversion indicating apomixis is not a result of mutational inactivation of the sexual process. Incomplete functional penetrance of both loci is thought to give rise to sexual and other "off-type" progeny with altered ploidy levels that are found in these facultative apomicts. Deletion mapping and chromosome walking has led to the identification of markers linked to both loci. LOA is located on a single chromosome surrounded by complex repeats while the chromosomal location of LOP is currently unknown. Analysis of selected LOA and LOP markers in subgenus Pilosella indicates the LOA locus is conserved in eight apomictic species and is absent in apomictic H. aurantiacum and H. pilosella while an LOP marker is more widely distributed throughout examined species. This suggests independent evolution of apospory loci in subgenus Pilosella. Examined LOA and LOP markers are absent in 22 accessions of displosporus and sexual subgenus Hieracium species indicating the independent evolution of apomixis components in subgenus Hieracium relative to the known loci controlling apomixis in subgenus Pilosella. The talk will highlight our recent understanding of factors activating the LOA locus and influencing its penetrance. Pathways identified from transcriptomic studies in cells undergoing apomixis and cells surrounding and supporting the process in ovules will also be discussed.
Krahulcová et al.	2012	Role of residual sexuality in apomictic plants: detection, rate and manifestation in populations of Pilosella (Asteraceae, Lactuceae).
Abstract: Contribution of facultatively apomictic maternal biotypes to population diversity was evaluated in seven hybridizing populations of Pilosella in Central Europe, where apomictic (P. bauhini or P. aurantiaca) and sexual (P. officinarum) biotypes coexist. The parental species were tetraploid (exclusively P. aurantiaca, commonly P. officinarum, rarely P. bauhini), pentaploid (commonly P. bauhini, rarely P. officinarum) or hexaploid (P. officinarum, P. bauhini). Recent hybrids co-occurred with their parental species in all populations. Plants in the field were studied with respect to their ploidy level, reproductive system, morphological characters, clonal structure and chloroplast DNA haplotypes. Reproductive origins of the progeny were assessed in maternal progeny arrays, using a flow cytometric seed screen and/or comparing DNA ploidy level in progeny embryos/seedlings with the maternal ploidy level. Identification of progeny which had arisen by virtue of residual sexuality in maternal apomicts was also based on morphological and reproductive characters of cultivated progeny that were found to differ from their maternal parents. Such "aberrant" progeny had originated either sexually or via haploid parthenogenesis. The structure of progeny arrays generated in the field by facultatively apomictic versus sexual plants showed that apomictic mothers produced progeny that was more variable in ploidy. This effect was demonstrated at both the embryonic and the seedling stage of progeny development. Apomictic maternal parents formed 2n + n hybrids, which were found in all investigated populations. Such high-polyploid hybrids from an apomictic and a sexual parent had similar reproductive behaviour, producing polyhaploid, sexual and (usually minority) apomictic progeny in variable ratios. Residual sexuality in apomicts proved effective also in experimental crosses, giving rise to progeny parallel to spontaneous hybrids from the field. Repeated hybridizations in the field between parental species and/or multi-step crosses can result in advanced hybrid swarms rich in cytotypes and morphotypes. Variation, recorded in such populations, suggests prevailing introgressive hybridization towards the sexual species P. officinarum.
Krahulec et al.	2012	Facultative apomixis in Pilosella and its role in evolution.
Abstract: The role of facultatively apomictic members of the genus Pilosella (Cichorieae, Asteraceae) was studied on different scales by a complex approach which included direct studies of population composition in the field, studies of the progeny of selected plants which originated in the field, and artificial hybridizations. We used a morphometric approach, studies of reproductive systems by both emasculation and analysis of progeny origins, isozyme analyses to identify the minimum number of genotypes, and chloroplast DNA haplotyping. The main results can be summarized as follows: (1) An analysis of population composition in two mountain ranges (the Krkonoše Mts. and the Šumava Mts., Czech Republic) has shown that facultative apomicts served as mother plants in half of the hybrids and hybridogenous species. (2) A progeny analysis of both apomictic and sexual mothers has shown that apomictic plants produce more variation with respect to ploidy level. (3) This is supported by reciprocal experimental crosses between apomictic and sexual parents. A single hybridization of an apomictic mother plant can produce a broad set of progeny differing in ploidy and chromosome numbers. (4) Apomicts serve as the main force of polyploidization because they produce unreduced female gametes more often than sexual plants. (5) Apomicts as mother plants were at the origin of many hybrid swarms and served as a source of genetic material for sexual plants via introgression. (6) Totally unknown is the role of polyhaploids produced by apomicts. We know they can survive and grow well or even produce progeny with a doubled chromosomal set in the polyploid-polyhaploid-polyploid cycle. A new polyploid differs from the original one in being more homozygous. This process is extremely difficult to recognize in the field because we do not know how to detect polyhaploids when we do not know the mother plant. All these facts suggest that facultative apomicts are active agents of different evolutionary events and not an "evolutionary blind alley", as had been thought in the past.
Krak et al.	2012	Reconstruction of phylogenetic relationships in a highly reticulate group with deep coalescence and recent speciation (Hieracium, Asteraceae).
Abstract: The genus Hieracium s.str. is characterized by extensive past hybridization, polyploidy and apo-mictic reproduction. Diploids are rare; the majority of taxa are triploid apomicts. In comparison with other case studies dealing with polyploid speciation, the genus represents a peculiar system: Previous phylogenetic analyses based on nrDNA (ETS) and cpDNA (trnT-L) revealed unexpected hybrid origin of about half of the investigated accessions independent of ploidy as well as extinct diploid ancestors. Rather low overall variation indicated relatively recent speciation, but the ETS tree also showed a deep split of the genus into two major lineages that corresponded to genome size and geographic distribution of the species. Both major groups are thought to have survived in different glacial refugia and to have hybridized as a result of secondary contact. In order to better resolve relationships within the major clades, we added a second, more variable cpDNA (trnV-ndhC) and a newly developed low-copy nuclear (LCN) marker (squalene synthase, sqs) that is applied here for the first time for phylogeny reconstruction. Sqs sequence divergence largely exceeded the variation of nrDNA and cpDNA. Most acces-sions were heterozygous, but the number of sqs alleles did not exceed the ploidy level, suggesting orthology and single-copy status except for a single, apparently duplicated allele. While crown groups were roughly equivalent, basal relationships were largely incongruent with all markers. The major lineages of the ETS tree were absent. Instead, several rather divergent alleles and massive allele sharing among taxa were observed. This pattern can be explained by a rapid and rather ancient diversification of the alleles with lineage sorting. The resolution of relationships was further complicated by the hybrid origin of many taxa, but alleles of putative hybrids were often shared with their putative parental taxa / lineages. These results demonstrate that species trees in closely related and reticulate groups might be approximated by combining evidence from various sources, including seemingly contradictory information. For Hieracium s.str., we (1) consider the nrDNA phylogeny as a 'backbone' for the interpretation of species origin and relationships because of its congruence with geographic and genomic patterns, (2) use LCN phylogenies to elucidate relationships that are beyond the resol-ution power of nrDNA, and (3) incorporate information from cpDNA to infer maternal lineages.
Šmerda et al.	2012	TY3-gypsy copy number is correlated with genome size in the subtribe Hieraciinae (Lactuceae, Asteraceae).
Abstract: Transposable elements can represent up to 50% of the DNA content in plant genomes, and retrotransposon proliferation or removal has been considered as one of the major factors influencing genome size variation among closely related taxa. The subtribe Hieraciinae is composed of the genera Hieracium s.l. (including subgen. Hieracium and Chionoracium), Pilosella, Andryala, and Hispidella. Phylogenetic studies have revealed the existence of three major phylogenetic groups: (1) Pilosella/Hispidella, (2) Hieracium s.l., and (3) Andryala. These lineages differ from each other in reproductive mode, ploidy level, and number of polyploid taxa included. Some information about genome size variation among as well as within Pilosella and Hieracium was already available whereas for Andryala species, hardly any data had been reported so far. The aims of the present study were (1) to fill the gap in the pattern of genome size variation within the Hieraciinae by providing these data for genus Andryala, and (2) to investigate the relation between genome size variation and abundance of gypsy-like retrotransposons in the subtribe. Genome sizes of 14 species and subspecies of Andryala were estimated using flow cytometry. An estimate of gypsy-like retrotransposon density was performed in 23 diploid taxa with known genome size, representing the major phylogenetic lineages of the Hieraciinae using (1) quantitative PCR with degenerate primers and (2) Southern dot blot analysis. Substantial variation in genome size was found among the three major phylogenetic groups of the Hieraciinae. The 1Cx values varied 1.22-fold (3.51-4.34 pg) in subgen. Hieracium, 1.26-fold (1.72-2.16 pg) in Pilosella and 1.66-fold (1.54-2.56 pg) in Andryala. A positive correlation between monoploid genome size and density of gypsy-like retrotransposons was found in the complete dataset. The total proportion of repetitive DNA represented by gypsy-like retrotransposons in the whole genome varied from 6.9% in Andryala to 31.9% in Hieracium s.str. in particular species. While the genome size ranges in Pilosella and Andryala more or less overlapped, species of both genera consistently showed only about 50% of the DNA content compared to Hieracium s.str. despite the same base chromosome number (x = 9). The evolution of genome size in the subtribe Hieraciinae is to a considerable degree associated with gypsy-like retrotransposon proliferation/removal.
Trávníček et al.	2012	Supremacy of sexual triploids: what drives cytotype coexistence in Pilosella echioides?
Abstract: A unique system of sympatric persistence of up to 5 sexually reproducing cytotypes (2x, 3x, 4x, 5x and 6x) that are maintained by apparently free mating was found in some populations of Pilosella echioides (Asteraceae). A surprising feature of one population (the Havranické vřesoviště heathland, S Moravia, Czech Republic) was the dominance (70%) of triploids. Consequently performed intra- and inter-cytotype experimental crosses revealed high ploidal diversity of the progeny, but only limited prospects for triploid dominance. Knowledge on experimental hybridization was used to model the system behaviour, and the in silico results were compared with empirical cytotype frequencies in the field. The data showed that the field frequencies did not fit either the equilibrium or any transitional state of the model (starting with diploids and tetraploids) if identical biological traits of the cytotypes are assumed. The supremacy of triploids might be explained by different performance (e.g. clonal spread by daughter rosettes) of particular cytotypes, assortative mating resulting from a non-random spatial pattern of cytotypes or lower mortality of triploid seedlings.
Urfus et al.	2012	A morphometric analysis of a Pilosella hybrid swarm.
Abstract: Genus Pilosella Hill (Asteraceae) comprises a complex group of partly apomictic taxa (i.e., its seeds are of clonal origin). Their enormous variability is caused especially by: polyploidization, a combination of the sexual and apomictic breeding mode, widespread hybridization, and vegetative reproduction. Hybridisation processes that occur within a population of Pilosella piloselloides subsp. bauhini and P. officinarum were detected by means of a morphometric analysis (incl. ploidy level and reproductive mode data). We analysed a set of maternal plants that occurred in the field and a set of their progeny that originated in the field. The analysis showed common hybridisation of both parents as well as of their hybrids (P. brachiata and P. leptophyton). All plants with different reproductive modes (sexual, facultative apomictic, and variable) participated in these hybridisations.
Vít et al.	2012	Distribution of apomixis-related markers in sexual and apomictic Hieracium subg. Pilosella accessions.
Abstract: Hieracium subg. Pilosella species can reproduce solely by sexual reproduction while others are facultative for apomixis. In H. praealtum accession R35, the dominant LOSS OF APOMEIOSIS (LOA) locus functions to induce somatic aposporous initial (AI) cell formation near sexually programmed cells in the ovule. The AI cells form aposporous embryo sacs by mitosis, avoiding prior meiotic reduction in a process termed apomeiosis. The adjacent sexually programmed cells die. Independent function of the LOSS OF PARTHENOGENESIS (LOP) locus in the "unreduced" aposporous embryo sacs enables fertilization-independent embryo and endosperm development. Deletion mapping and chromosome walking has led to the identification of markers linked to both loci. Previous analyses in individual accessions of apomictic H. piloselloides, H. praealtum and H. caespitosum has shown significant conservation of LOA linked markers. Furthermore, in situ hybridization has shown the LOA locus is located near the telomeric tip of a single chromosome, surrounded by extensive repeats in these plants. In H. caespitosum and H. praealtum accessions, but not in H. piloselloides, the LOA locus resides on a very long chromosome. These LOA locus markers and linked repeats were not evident in the apomictic H. aurantiacum, and sexual H. pilosella isolates. Here, we surveyed the distribution of 5 LOA linked markers (including four surrounding the core genomic region for apomeiosis) and one marker tightly linked to the LOP locus in 6 sexual and 62 apomictic accessions. These included sexual basic species, basic apomictic species, stabilized hybridogenous species, natural hybrids, and progeny arising from selected crosses between sexual and apomictic plants. The presence or absence of a long chromosome was also examined by root tip squashes in some species. Strong conservation of LOA locus markers was evident in six additional apomictic species H. bauhini, H. pavichii, H. floribundum, H. glomeratum, and H. iseranum, and their derived apomictic hybrids (n = 24). LOA markers were absent in sexual and apomictic H. pilosella and H. aurantiacum accessions and in their apomictic hybrids (n = 31) suggesting the evolution of other apomeiosis loci in these apomictic species. The LOP marker was detected in all of the above mentioned apomictic species but did not display perfect correlation with parthenogenesis in the accessions tested.
Zahradníček et al.	2012	Ploidy level diversity and phylogeography of Hieracium intybaceum (Asteraceae).
Abstract: Hieracium intybaceum is a well-defined species growing on silicate rocks in high altitudes in the Alps and the Vosges Mts. Though traditionally recognised as a part of genus Hieracium (subgenus Hieracium), molecular data (ITS and ETS sequences) suggest an older isolated lineage. We have collected 47 populations throughout the Alps and one population from an outlying locality in the Vosges Mts. Two cytotypes with different mode of reproduction were found, namely sexual diploids and agamospermous tetraploids. While diploids are distributed in nearly all parts of the species geographical range (except of the Vosges Mts.), tetraploids seem to be confined to the western Alps and the Vosges Mts. This distribution pattern of cytotypes is in conflict with repeatedly detected refuges of diploids in the western Alps. Previously published triploid counts and mixed-ploidy populations have not been found so far. Both cytotypes are morphologically well separated, mainly by the density and length of glandular hairs, ligule length and leaf dentation. AFLP molecular data revealed a very low variation among diploid populations and high variation between ploidy levels. By comparing the expected genome size of the tetraploids (i.e., twice the genome size of diploids) with the observed genome size, the tetraploids showed downsizing. Our data support the hypothesis of an allopolyploid origin of tetraploids via introgression of the diploids by hitherto undetected hawkweed species.
Fehrer	2013	Evolutionarily successful asexuals: examples from plants and lichens.
Abstract: Most switches to asexual reproduction are independent, evolutionarily young events that are sometimes connected with environmental change. The performance of a range of organisms representing various forms of asexuality is assessed here. Potamogeton, the largest genus of the pondweed family (Potamogetonaceae), has worldwide distribution. Many hybrid taxa are known; most of them are sterile and propagate vegetatively. Some of these genotypes may date back to post-glacial times when hybrid establishment was facilitated by the colonization of new habitats. Although they are evolutionary dead ends, due to their abundance and longevity, Potamogeton hybrids represent a non-negligible part of aquatic plant biodiversity. Hieracium and Pilosella (Asteraceae), two closely related hawkweed genera with mainly European distribution, are characterized by few diploids and large numbers of polyploids. Different kinds of apomixis emerged in these groups. In Hieracium (diplospory), only diploids are sexual; most are rare and confined to un-glaciated refugia. Polyploids are most likely obligate apomicts; many are widespread. The emergence of polyploidy/apomixis may be connected with abundant hybridization when isolated lineages that survived in different glacial refugia came into secondary contact. Although apomicts do not seem to produce additional variation nowadays, due to their high species numbers and wide distribution, they are mainly responsible for the survival and spread of the genus. In Pilosella (apospory), apomixis is a facultative trait. Apomicts are strongly involved in the recurrent formation of new genotypes as male or female parents and produce an even higher diversity of different progeny than sexuals. Favorable genotypes with a high degree of apomictically produced offspring can spread over large distances. As a result, many apomictic Pilosellas are invasive on other continents. Lepraria (Stereocaulaceae) is a genus of sterile lichens with worldwide distribution. As there are no indications of sexual reproduction, these haploid ascomycetes might represent ancient asexuals. Their speciation was accompanied by large genetic diversification, comparable to the level of variation within (sub)tribes or even families in plants. While it is unclear how such a high diversity can be generated in the apparent absence of recombination, asexual reproduction as such obviously does not diminish the evolutionary success of this group.
Fehrer et al.	2013	Emergence of different forms of apomixis in closely related lineages of hawkweeds.
Abstract: Hawkweeds of genera Pilosella and Hieracium (Lactuceae, Asteraceae) - formerly treated as a single genus Hieracium s.l. - consist of few diploid and many polyploid taxa and are the only example for the emergence of different forms of apomixis (apospory and diplospory) in the same plant subtribe. Apomixis is gametophytic in both genera; embryo sacs form without meiosis in ovules. Embryo and endosperm formation is fertilization independent, giving rise to progeny with a maternal genotype. Pilosella species exhibit apospory and Hieracium (s.str.) species exhibit diplospory; these modes differ by the origin of the cell giving rise to the embryo sac which influences the penetrance of apomixis and the resulting cytotype distributions and population structures. In the mainly European genus Pilosella (ca. 180 species, main ploidy levels 2n = 4x = 36 or 2n = 5x = 45), apomixis is a facultative trait. Diploids are rather rare. Most polyploids are widespread; they can be sexual or facultatively apomictic; the latter show different, genotype dependent degrees of residual sexuality. Apomicts are frequently involved in the recurrent formation of new genotypes, not only as male, but also as female parents. Breeding barriers are almost completely absent so that Pilosella species are characterized by extensive contemporary hybridizations. This mixed sex/asex reproductive system results in very complex population structures. The genetic basis of apomixis in Pilosella is quite well investigated. Two independent loci (LOA, LOSS OF APOMEIOSIS; LOP, LOSS OF PARTHENOGENESIS) are responsible for the expression of the apomictic phenotype. LOA is in some species located on a single hemizygous chromosome surrounded by complex repeats; the chromosomal location of LOP is currently unknown. Deletion of both loci by irradiation leads to restoration of the sexual pathway. Incomplete functional penetrance of both loci is thought to be responsible for sexual and other 'off-type' progeny with altered ploidy levels. Screening of selected LOA and LOP markers indicates that LOA is absent in some apomictic species suggesting independent evolution of apospory loci even within genus Pilosella. In the mainly Eurasian genus Hieracium (ca. 500-8000 species, main ploidy level 2n = 3x = 27), only diploids are sexual; they are rare and confined to un-glaciated refugia. Polyploids are widespread and most probably obligate apomicts. Multi- and single-copy nuclear as well as chloroplast markers indicate that almost the entire genetic variation of the genus is encompassed with the diploids and that these have radiated rapidly and relatively recently. The emergence of polyploidy/apomixis in this genus may be connected with abundant past hybridizations when isolated lineages that survived in different glacial refugia came into secondary contact. Neither the LOA nor the LOP locus occur in apomicts of genus Hieracium (nor in sexual species of both genera). Thus, the genetic mechanisms / loci responsible for the apomictic mode of reproduction in this genus are still unknown.