ÚSTAV FYZIKÁLNÍ BIOLOGIE JIHOČESKÁ UNIVERZITA V ČESKÝCH BUDĚJOVICÍCH

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1 ÚSTAV FYZIKÁLNÍ BIOLOGIE JIHOČESKÁ UNIVERZITA V ČESKÝCH BUDĚJOVICÍCH PŘIHLÁŠKA STUDENTSKÉHO PROJEKTU Projekt Název projektu: The use of molecular markers for detection of hybrid speciation in the genus Spergularia. Uchazeč Hlavní řešitel Příjmení, jméno, tituly: Kúr Pavel, Mgr. pavel.kur@prf.jcu.cz Tel.: Studium v doktorském studijním programu zahájeno dne: Pracoviště/místo studia Fakulta (VŠ ústav): Přírodovědecká fakulta Katedra (ústav, pracoviště): Katedra botaniky Školitel: Ing. Milan Štech, PhD Vedoucí projektu Příjmení, jméno, tituly: Ing. Milan Štech, PhD stech@prf.jcu.cz Tel.: Pracoviště Fakulta (VŠ ústav): Přírodovědecká fakulta Katedra (ústav, pracoviště): Katedra botaniky Celkové náklady na řešení projektu Náklady nebo výdaje na pořízení hmotného a nehmotného majetku: Další provozní náklady nebo výdaje: Doplňkové náklady nebo výdaje: Celkem Kč: V Českých Budějovicích dne Podpis uchazeče:...

2 Zdůvodnění návrhu projektu Stav řešení problematiky projektu (max. 2 stránky): Hybridization has been long recognized as one of the major forces driving evolution of angiosperm plants (Stebbins 1950, Grant 1981, Arnold 1997). Current estimates suggest that ca 25% of recent plant species have been involved in hybridization processes (Mallet 2005), with ca 11% of current species being of known hybrid origin (Ellstrand et al. 1996). Moreover, there has been an increasing body of evidence that hybridization is very often accompanied by shifts in ploidy level a phenomenon referred to as allopolyploidy (Ramsey & Schemske 1998, Soltis & Soltis 2009). The increased feasibility of allopolyploid formation and establishment in comparison with homoploid hybrids (i.e. hybrids without change in ploidy level) have been chiefly explained by elimination of problems caused by poor meiotic pairing of incompatible parent genomes and better overall adaptability resulting from fixed heterozygozity (Stebbins 1950, Grant 1981, Riesberg & Willis 2007). However, the knowledge of mechanisms of hybrid speciation is still incomplete, and more experimental studies on these processes are required (Soltis & Soltis 2009). The genus Spergularia (Caryophyllaceae) represents a suitable model group for studying hybridization and polyploidy, as suggested by some earlier works (Ratter , Dvořák 1990). Nowadays, these processes can be studied in a more effective way thanks to the availability of modern molecular techniques, which can significantly expand the knowledge obtained by the past studies. A rare opportunity to research both homoploid and polyploid hybridization within one genus can be found in the species S. rubra. It is a tetraploid species (2n = 4x = 36) and the most widespread representative of the genus, secondarily introduced to various types of human-affected stands nearly all over the world (Friedrich 1979, Hartman & Rabeler 2005). The S. rubra expansion to secondary habitats has been considered to cause hybridization with two other Spergularia species in Central Europe. One taxon presumably involved in polyploid hybridization with S. rubra is S. echinosperma, a very rare species of bare pond bottoms that represents an endemic species to Central Europe (Friedrich 1979, Dvořák 1990). Recent studies revealed existence of two cytotypes within S. echinosperma, diploid (2n = 2x = 18) and tetraploid (2n = 4x = 36), distinguishable as separate species (Kúr 2007, Kúr 2010). Based on morphometric and flow cytometric analyses, the tetraploid cytotype has been speculated to be of allopolyploid origin between the diploid cytotype and S. rubra (most probably via unreduced gametes of one of the parent species, Kúr 2007). In attempt to confirm the hybrid origin of the S. echinosperma tetraploids, we carried out sequencing of the ITS region (nrdna) in a limited set of the both S. echinosperma cytotypes and S. rubra (P. Kúr, unpubl.). While the S. echinosperma diploids and S. rubra provided clearly diverse sequences, intragenomic variation in the ITS region, i.e. presence of divergent ITS paralogs, was found in the S. echinosperma tetraploid. Divergent paralogs found in these accessions of tetraploid S. echinosperma showed additive pattern of variation observed in both putative parents, which advocated its allopolyploid origin. However, further analyses are needed in order to confirm the involvement of the S. echinosperma diploid and S. rubra genomes in the formation of the tetraploids. The other species supposed to be involved in hybridization with S. rubra is S. salina (2n = 4x = 36), a widespread halophyte species of seacoast and inland salt-marsh areas. Whereas the Central European natural habitats of S. salina have been vastly destroyed during the last decades (Grulich 1987, Dvořák 1990), the species was found to have expanded along salted roads in this region some 20 years ago (Hetzel 2006). The road margins are concurrently one of the secondary habitats with frequent occurrence of S. rubra, which has led to intermingling

3 of populations of these two species. Morphological observations and preliminary molecular analyses (P. Kúr, unpubl.) indicated presence of extensive hybridization in such mixed populations. In primary biotopes, the hybridization between S. salina and S. rubra has been observed only very rarely, primarily due to a lack of natural habitats with coexistence of the two species (Dvořák 1990), in combination with partial hybridization barriers discovered during artificial crossing experiments (Ratter ). However, the extensive intermingling of the species on the road verges has likely created an opportunity for overcoming the hybridization barriers and formation of viable hybrid genotype. In summary, these two cases of interspecific crossings within the genus Spergularia represent a suitable opportunity for studying hybrid speciation following human intervention into the landscape. Results of this project will serve as a background for further research concerning microspeciation events in the genus. The knowledge of the mechanisms and extent of the hybridization processes in these species will be also utilizable in further ecological and conservational studies on this group of significant indicator species. Stanovení badatelských cílů, metody a způsob řešení (max. 2 stránky): The aim of this study is to answer following questions: 1) Do selected molecular markers support the presence of either polyploid or homoploid hybridization between S. rubra and S. echinosperma or S. rubra and S. salina? 2) If so, does one of the species constantly act as the maternal one, or does reciprocal crossing occur? The issue will be solved with the use of sequencing of selected DNA regions. The nrdna ITS region will be used for detecting the presence of hybridization between the species. A selected cpdna region will be employed for determining the maternity of the involved species. In the case of S. rubra S. echinosperma hybridization, 15 populations of each of S. echinosperma diploids, S. echinosperma tetraploids, and S. rubra (10 individuals from each population) will be sampled across the whole distribution area of S. echinosperma, mainly in South-West Bohemia and North Germany, where the species is most abundant. The S. rubra individuals will be sampled from the same geographical regions as S. echinosperma, preferably in the closest vicinity, in order to cover all possible genetic similarities. The ploidy level of all S. echinosperma individuals will be ascertained by flow cytometry. From the populations of S. salina, 15 mixed roadside populations with expected occurrence of intercrossing and 5 pure populations from natural habitats for each of the species (10 plants per population) will be sampled in Central Europe (mainly Czech Republic, Germany, and Poland). Although preliminary flow cytometric analyses of plants from mixed populations revealed no occurrence of other ploidy levels than tetraploid (P. Kúr, unpubl.), they were based on a few individuals only, so the presence of polyploidization accompanying some of the hybridization events (for example caused by unreduced gametes) cannot be excluded. Therefore, the ploidy level of all analyzed plants will be ascertained by flow cytometry. The laboratory analyses will be carried out in the Laboratory of Plant Molecular Biology at the Faculty of Science of the University of South Bohemia. From the 10 sampled plants per population, 3 5 will be selected for the sequencing. The total genomic DNA will be extracted using the CTAB method (Foster & Twell 1996). The PCR amplification of the ITS region will be carried out using universal plant primers (Roalson et al. 2001). For the cpdna sequencing, a suitable, adequately variable region will be searched for among noncoding chloroplast sequences. In the case of S. echinosperma tetraploids, as well as presumed S. rubra S. salina hybrids if containing ITS paralogs, the PCR product will be cloned using pgem-t

4 Easy Vector System I (Promega). The vector will be transferred into competent cells cultivated on selective LB plates, and 4 successfully transformed colonies will be then used for final sequencing. All selected PCR products will be sequenced in the Laboratory of Genomics, Biology Centre of the Academy of Sciences. The obtained sequential data will be evaluated by appropriate phylogenetic and statistical analyses, e.g. maximum parsimony (PAUP*4.0b1, Swofford 2001), phylogenetic networks (SplitsTree 4, Huson 1998), or AMOVA (Arlequin 3, Excoffier et al. 2005). Arnold M. (1997): Natural hybridization and evolution. Oxford University Press, Oxford. Dvořák F. (1990): Spergularia kuřinka. In: Hejný S. & Slavík B. (eds.), Květena České republiky [Flora of the Czech Republic] 2: 81 86, Academia, Praha. Ellstrand N. C., Whitkus R. & Rieseberg L. H. (1996): Distribution of spontaneous plant hybrids. P. Natl. Acad. Sci. USA 93: Excoffier L., Laval G. & Schneider S. (2005): Arlequin ver. 3.0: An integrated software package for population genetics data analysis. Evol. Bioinform. Online 1: Foster G. D. & Twell D. [eds.] (1996): Plant gene isolation: Principles and practice. John Wiley & Sons Ltd, New York. Friedrich H. C. (1979): Familie Caryophyllaceae. In: Reichinger K.H. (ed.), Illustrierte Flora von Mitteleuropa III/2: , Verlag, Berlin. Grant V. (1981): Plant speciation, 2nd ed. Columbia University Press, New York. Grulich V. (1987): Slanomilné rostliny na jižní Moravě [Halophilous plants in South Moravia]. Český svaz ochránců přírody, Břeclav. Hartman R. L. & Rabeler R. K. (2005): Spergularia. In: Flora of North America Editorial Committee (eds.), Flora of North America 5: 16 23, Oxford University Press, New York and Oxford. Hetzel G. (2006): Die Neophyten Oberfrankens. Floristik, Standortcharakteristik, Vergesellschaftung, Verbreitung, Dynamik [PhD thesis]. Julius-von-Sachs-Institut für Biowissenschaften, Fakultät für Biologie, Würzburg. Huson D. H. (1998): SplitsTree: analyzing and visualizing evolutionary data. Bioinformatics 14: Kúr P. (2007): Cytologická variabilita a potenciální hybridizace druhů Spergularia echinosperma a S. rubra rešerše problematiky v celém rodě a pilotní studie [Cytological variability and possible hybridization of the species Spergularia echinosperma and S. rubra a review of the issue in the whole genus and a pilot study. Bc. Thesis, in Czech]. Faculty of Biological Sciences, University of South Bohemia, České Budějovice. Kúr P. (2010): A cytological, morphometric, and ecological study of Spergularia echinosperma in the Czech Republic and its comparison with a closely similar species S. rubra. MSc. Thesis, in English. 27 pp., Faculty of Science, University of South Bohemia, České Budějovice. Mallet J. (2005): Hybridization as an invasion of the genome. Trends Ecol. Evol. 20: Ramsey J., Schemske D. W. (1998): Pathways, mechanisms, and rates of polyploid formation in flowering plants. Annu. Rev. Ecol. Syst. 29: Ratter J. A. ( ): Cytogenetic Studies in Spergularia I IX. Not. Roy. Bot. Gar. Edinburgh 25: , 1964; 26: et , 1965; 29: et , 1969; 32 (1): , 1972; 32 (2): et , 1973; 34: , Riesberg L. H. & Willis J. H. (2007): Plant speciation. Science 317: Roalson E. H., Columbus J. T. & Friar E. A. (2001): Phylogenetic relationships in Cariceae (Cyperaceae) based on ITS (nrdna) and trnt-l-f (cpdna) region sequences:

5 Assessment of subgeneric and sectional relationships in Carex with emphasis on section Acrocystis. Syst. Bot. 26: Soltis P. S. & Soltis D. E. (2009): The role of hybridization in plant speciation. Annu. Rev. Plant. Biol. 60: Stebbins G. L. (1950): Variation and evolution in plants. Columbia University Press, New York. Swofford D. L. (2001): PAUP*. Phylogenetic analysis using parsimony (*and other methods) version 4.0b1. Sinauer Associates, Sunderland, Massachusetts. Harmonogram prací: June July 2011: Field sampling August December 2011: Laboratory analyses, preparation of the publication of the results Předpokládaný typ vědeckých výsledků projektu The results will be a part of the PhD thesis of the main investigator and will be published in the form of at least two articles in journals with an impact factor. Hlavní řešitel (maximálně 30 řádek) Research: : BC study at the Faculty of Science, Univ. of S Bohemia, specialization biology. Bc. thesis: Cytological variability and possible hybridization of the species Spergularia echinosperma and S. rubra a review of the issue in the whole genus and a pilot study : MSc study at the Faculty of Science, Univ. of S Bohemia, specialization botany. MSc thesis: A cytological, morphometric, and ecological study of Spergularia echinosperma in the Czech Republic and its comparison with a closely similar species S. rubra. since March 2010: PhD study at the Faculty of Science, Univ. of S Bohemia, specialization botany. Topic: Study of microspeciation processes and biological properties in selected representatives of the genus Spergularia. Publications: Kúr P. (2010): A morphometric study and revision of Spergularia echinosperma and its hybridization with S. rubra. [in prep.] Grants: Cytological variability and potential hybridization of an endemic Central European species Spergularia echinosperma (Mattoni Awards for Studies of Biodiversity and Conservation Biology, 2007) main investigator. Vedoucí projektu (maximálně 30 řádek) Štech Milan, PhD Department of Botany, Faculty of Science, University of South Bohemia, Branisovska 31, České Budějovice, CZ , Czech Republic Phone: stech@prf.jcu.cz Research: Systematics and phylogeography of vascular plants, microspeciation processes and hybridization. Publications:

6 Těšitel J., Říha P., Svobodová Š., Malinová T. & Štech M. (2010): Phylogeny, life history evolution and biogeography of the Rhinanthoid Orobanchaceae. Folia Geobotanica 45: XXX-XXX (in press). Košnar J., Košnar J., Herbstová M., Macek P., Rejmánková E. & Štech M. (2010): Natural hybridization in tropical spikerushes of Eleocharis subgenus Limnochloa (Cyperaceae): Evidence from morphology and DNA markers. American Journal of Botany 97: Loureiro J., Trávníček P., Rauchová J., Urfus T., Vít P., Štech M., Castro S. & Suda J. (2010): The use of flow cytometry in the biosystematics, ecology and population biology of homoploid plants. Preslia 82: Kolář F., Štech M., Trávníček P., Rauchová J., Urfus T., Vít P., Kubešová M. & Suda J. (2009): Towards resolving the Knautia arvensis agg. (Dipsacaceae) puzzle: primary and secondary contact zones and ploidy segregation at landscape and microgeographic scales. Annals of Botany 103: Těšitel J., Malinová T., Štech M. & Herstová M. (2009): Variation in the Melampyrum sylvaticum group in the Carpathian and Hercynian region: two lineages with different evolutionary histories. Preslia 81: 1 22.

7 Požadované finanční prostředky Výdaje a náklady na pořízení hmotného a nehmotného majetku (max Kč) Drobný hmotný a nehmotný majetek: 0 Spotřební materiál: Kč Náklady na publikace (max. 2000Kč): 0 Požadované finanční prostředky celkem: Kč Slovní zdůvodnění a rozpis finančních prostředků Spotřební materiál Rukavice latexové nezaprašované S 5680 Kč Rukavice latexové nezaprašované L 1420 Kč Rukavice latexové nezaprašované M 2840 Kč Invisorb Fragment CleanUp, 250 purifications 9120 Kč 2X Red PCR Master mix; 500 reactions Kč