Symbioses between cool-season grasses (Subfamily Pooideae) and endophytic fungi in the genera Epichlo and Neotyphodium straddle a continuum of interactions from antagonistic to highly mutualistic. Although these two genera of endophytes are closely related, Neotyphodium endophytes are strictly seed-transmitted and provide many physiological and defensive benefits to their hosts, while Epichlo spp. have an obligately sexual contagious stage wherein host inflorescences are replaced by fungal sexual structures (stromata), effectively sterilizing the plant. Between these two extremes of interactions are Epichlo spp. with a mixed strategy, where some grass tillers are sterilized while others develop normally and yield healthy endophyte-infected seeds. These symbioses offer a unique opportunity to dissect evolutionary mechanisms that may drive movement along this continuum. The research presented characterizes distinct hybridization processes in endophytes and grasses that result in the generation of astounding genetic diversity for the symbiosis. Interspecific hybridization via hyphal anatomosis is a common feature of Neotyphodium endophytes, and may promote mutualism by combining suites of defensive alkaloid genes and ameliorating the adverse evolutionary effects of an asexual lifestyle. My results demonstrate that several genetically distinct hybrid endophytes infect grass species in tribe Poeae. Further, I show that a highly mutualistic asexual endophyte infecting tall fescue (=Festuca arundinaceum Schreb.), Neotyphodium coenophialum, also infects two closely related and interfertile relatives of this host. My findings suggest that this seed-borne endophyte may have been introgressed into these grasses through sexual grass hybridization events. These findings highlight interspecific hybridization as a means of generating tremendous genetic variability in both endophytes and their hosts, thus magnifying the adaptive evolutionary potential of these symbioses. Further, I establish a phylogenetic framework for grasses naturally harboring Epichlo and Neotyphodium endophytes. I show that patterns of genetic divergence among grass lineages are emulated by those of their fungal symbionts. These results suggest that endophytes have co-evolved with grasses in subfamily Pooideae, and may have played a critical role in the evolutionary success and radiation of this group of grasses.

Doctor of Philosophy / Department of Biology / Carolyn J. Ferguson / Morus L. (Moraceae) is a temperate and subtropical genus of ten to 15 species distributed in Asia, Africa, Europe, North, Central and South America. Despite its broad distribution and economic importance, it has received little attention from systematic botanists. Two species of this genus, the native M. rubra and the exotic M. alba, co-occur in eastern North America including the Flint Hills region of the Central Plains. In my dissertation research, I have conducted both species level and population level studies to obtain insights into the diversification of Morus. At the species level, my objectives were to re-evaluate the taxonomy and reconstruct the phylogeny of Morus. Based on herbarium and literature study as well as some field study, I recognize 13 species: eight species occurring in Asia, one in Africa and four in the New World. I used sequence data from the internal transcribed spacer (ITS) region of the nuclear ribosomal DNA and the trnL-trnF region of the chloroplast DNA to reconstruct the evolutionary history of Morus. The phylogenies were congruent and indicate a) a monophyletic core group of Morus with two well-supported geographical clades (one containing Asian taxa and one of New World taxa); and b) that Morus, as currently circumscribed, is non-monophyletic. At the population level, I studied sex expression pattern variation between the Morus native-exotic pair in the Flint Hills region, and assessed hybridization between these species at Konza Prairie Biological Station (KPBS). Both species are subdioecious, and Flint Hills populations exhibit significantly male-biased sex ratios, with sex expression being size independent. Approximately 10% of individuals of each species changed sex annually. In the population study at KPBS, I applied randomly amplified polymorphic DNA (RAPD) markers and microsatellites. The Morus species were moderately ([Theta]II = 0.079; RAPD data) to highly differentiated genetically (F[subscript]ST = 0.233; microsatellite data). Analysis of genetic structure suggested interspecific gene flow and indicated the presence of later generation hybrids. The presence of the exotic congener may affect the existence and genetic integrity of the native species. Overall, these studies contribute to our understanding of diversity in this interesting plant study system.

The thesis deals with phenotypic, ploidy and genetic variation of two Campanula species occurring in the Krkonoše Mts., namely the endemic C. bohemica and widespread C. rotundifolia subsp. rotundifolia. For comparative purposes subspecies sudetica of the latter species was also included. The main aim was to get insight into population structure and assess the threat of interspecific hybridization to the survival of the endemic bluebell. Flow cytometry, distance-based morphometrics and molecular analysis were used to address these questions. Three distinct groups of fluorescence intensities were revealed by flow cytometry, corresponding to DNA diploids, tetraploids and pentaploids. While diploids morphologically matched the nominate subspecies of C. rotundifolia, tetraploids corresponded either to C. rotundifolia subsp. sudetica or C. bohemica. Most populations from the Krkonoše Mts. were uniform although a sympatric growth of diploids and tetraploids was encountered in 12 populations. Only two pentaploids individuals, most likely of hybrid origin, were found, which indicated that interspecific hybridization is much less common than previously assumed. The two tetraploid taxa were distinguished by molecular markers. A combination of flow cytometry and molecular analyses thus allowed reliable...

Canyon shaped reservoirs are characteristic by specific environmental horizontal gradients, so they enable existence of several species of the Daphnia longispina complex in one water body. Due to preference of distinct environmental conditions Daphnia species occurre in the different localities. The aim of my thesis was to analyze detail taxonomical and clonal structure of Daphnia longispina group by ten microsatellite markers on longitudinal gradient and compare it between two consecutive seasons. Simultaneously I received newly discovered divergent mitochondrial lineage from Želivka reservoir. It was confirmed, that the distribution of species and their hybrids in water reservoir was non-concidental and the taxonomic spatial distribution is in two consecutive seasons relatively constant. On the contrary the spatial and temporal distribution of clones was very heterogeneous. Clonal diversity in the interspecific hybrids was lower than in the coexisting parental species. This finding supports the hypothesis, that there exist reproductive barriers between parental genomes. Most of the clones were substantively variable, but several clones that occurred in both seasons in similar localities were found. It is possible that some clones are able of overwintering in hypolimnion and in the spring...

Hybridizing species of the Daphnia longispina complex are key taxa in plankton communities of many European lakes. In several of these lakes, it has been documented that the taxonomic structure of the complex during the 20th century has substantially changed following human-mediated environmental changes, particularly eutrophication and re-oligotrophication and fish stock changes. We characterize these changes in Lago Maggiore (Italy/Switzerland), a southern pre-alpine lake, which also passed through the human-mediated environmental changes. Lago Maggiore is one of best and longest studied European lakes so a large number of historical Daphnia samples from regular monitoring are available. Because local Daphnia do not form dormant egg banks suitable for genetic analysis (being able to overwinter in the water column), we used a combination of morphology and geometric morphometrics (elliptic Fourier analysis) to evaluate the taxonomic and phenotypic changes in the Daphnia longispina complex in Lago Maggiore since the mid-20th century (1948-2012), and attampted to characterize the impact of environmental changes over the respective period on Daphnia body shape and size. Examination of Daphnia phenotype indicates indeed the prevailing presence of D. longispina (hyalina morph) in the 1940s, dominance of...

This study investigates the hybridization of two species of cattails, Typha latifolia (Common Cattail) and T. angustifolia (Narrow-leaved Cattail) in the Czech Republic. The aim of this study was to determine, how often T. latifolia and T. angustifolia hybridize, whether hybridization is allowed by overlapping flowering time of these species and whether it is possible these species controlled cross in a culture. For detection of hybrid individuals were used microsatellite DNA markers, which allow to detect hybridization events and differentiate hybrids from the parental species. Molecular analysis revealed that hybridization of T. latifolia and T. angustifolia occurs in the Czech Republic, but it is not frequent. Of the 267 analyzed individuals, 130 individuals were pure species T. latifolia, 108 individuals pure species T. angustifolia and 29 individuals were hybrids. Of the hybrids, 23 were advanced hybrids, 5 were backcrosses and only one individual was F1 hybrid. Flowering time of T. latifolia and T. angustifolia overlaps, which allows hybridization, and flowering time to not act as a prezygotic reproductive isolation barrier and gametes T. latifolia and T. angustifolia can blend together. In controlled crosses the female spikes T. latifolia and T. angustifolia created seeds, but these were...

Šiame moksliniame darbe nagrinėjama paprastojo (Quercus robur L.) ir bekočio (Q. petraea [Matt.] Liebl.) ąžuolų hibridizacija Trako miške (Alytaus raj.), bei su ja susiję ąžuolų pomiškio plitimo, lapijos nuokritų biocheminės sudėties bei įtakos dirvožemio mikrobiotai procesai. Šis darbas nuo kitų Europoje atliktų tyrimų išsiskiria savo specifika, kadangi tyrimų objektas apima bekočio ąžuolo arealo pakraštį. Nustatyta, kad tarprūšiniai hibridai Trako miške plito greičiau nei bekočiai. Trako miško motinmedžių ir jų palikuonių palyginamoji taksonominė analizė parodė, kad tarprūšinių paprastojo ir bekočio ąžuolų hibridų morfologinių lapų požymių variacija juvenaliniame amžiuje buvo didesnė nei vienos kurios tėvinių rūšių. Molekulinė-genetinė analizė patvirtino rūšinės priklausomybės nustatymo pagal morfologinius lapų požymius tinkamumą ir efektyvumą. Įrodyta, kad hibridizacijos procesams būdingas asimetrinio kryžminimosi pobūdis ir introgresijos procese dalyvauja ribotas individų skaičius. Vyrauja grįžtamasis kryžminimasis su viena iš tėvinių rūšių, šiuo atveju su bekočiu ąžuolu. Didesnė lignino koncentracija paprastojo ąžuolo lapuose sąlygojo mažesnį bakterijų ir mikroorganizmų, ypač mikromicetų, aktyvumą šio ąžuolo rizosferoje. Palyginus su paprastuoju ąžuolu, bekočio ir hibridinių ąžuolų lapų nuokritos ir rizosferos organinės medžiagos buvo intensyviau skaidomos. Todėl šių ąžuolų aplinkoje sąlygos plisti pomiškiui yra palankesnės negu po paprastaisiais ąžuolais. / This study analyzes hybridization of pedunculate (Quercus robur L.) and sessile (Q. petraea [Matt.] Liebl.) oak in forest Trakas (Alytus district), including the following processes related to hybridization: the prevalence of oak undergrowth, leaf litter fall and its biochemical composition, impact on soil microbiota. This study differs from other studies carried out in Europe by its specifics, since the research object is situated at the edge of the natural distribution of sessile oak. It was found the increase of the number of hybrid oaks in the undergrowth. A comparative taxonomic analysis of the parental trees of the Trakas forest showed that the variation of morphological traits of leaves of interspecific hybrids of pedunculate and sessile oaks was higher at juvenile age if compared with any paternal species. Molecular-genetic analysis confirmed suitability and effectiveness of the use of leaf morphological traits for species discrimination. Studies confirmed that hybridization process had a character of asymmetric crossings, a limited number of individuals participate in the introgression process. Backcross with one of the parental species – sessile oak prevails. The leaves litter fall of pedunculate oak was distinguished for a higher content of lignin, which determines decreased activeness of bacteria and, especially, micromicetes in the rhizosphere. The decomposition rate of leaves litter fall and organic compounds in rhizosphere under sessile and hybrid oaks were... [to full text]

Les écosystèmes lacustres sont de plus en plus soumis à de multiples perturbations mais leur réponse est encore trop rarement considérée de façon globale. En effet, au travers d'un jeu d'interactions complexes, chaque perturbation peut moduler l'intensité de l'impact des autres sur les lacs, et de ce fait, leur réponse. Dans ce contexte, ce travail doctoral est axé sur l'approfondissement de notre compréhension de la réponse des lacs à un contexte de perturbations multiples, sur le long terme. Pour cela, trois grands lacs profonds (le lac Léman, du Bourget et d'Annecy), soumis à une perturbation régionale similaire (i.e., le réchauffement climatique) et à deux perturbations locales (i.e., les changements dans le statut trophique et dans les pratiques de gestion piscicole) d'intensités différentes, ont été choisis. La réponse de ces lacs à ces changements a été abordée au travers d'une approche paléolimnologique, basée sur les cladocères. En effet, la capacité de ceux-ci à intégrer les changements temporels dans le réseau trophique pélagique en réponse aux trois perturbations environnementales en fait un modèle adéquat pour reconstruire les changements à l'échelle de la communauté pélagique. Plus précisément, nous avons abordé (i) les changements dans les communautés de cladocères, au cours du siècle dernier, en réponse aux trois perturbations environnementales et (ii) l'impact de ces perturbations sur les stratégies de reproduction des Daphnies. Cependant, le manque de connaissance dans les grands lacs profonds sur la représentativité des communautés sources de cladocères par les assemblages de restes archivés dans les sédiments a tout d'abord conduit à faire un point méthodologique. Ce dernier a abouti à la mise en évidence de différences dans le niveau de représentation de communautés sources de cladocères selon le type de restes utilisés. La composition taxonomique et la structure en taille des restes parthénogénétiques (i.e., exosquelettes) reflètent relativement bien celle des communautés sources. Quant aux éphippies (œufs de dormance), elles se révèlent des indicateurs fiables pour reconstruire les comportements de reproduction. Suite à ces résultats, les reconstructions paléolimnologiques des communautés de cladocères montrent que leur trajectoire écologique était contrôlée par des interactions complexes entre les trois pertubations différentes, selon les périodes de temps et entre les lacs. De plus, la réponse des communautés de cladocères au climat était différente entre les trois lacs comme une fonction de l'intensité des forçages locaux. Ces observations suggèrent qu'il est important d'inclure le rôle des perturbations locales dans la prédiction de l'impact futur des changements climatiques sur les lacs. L'impact des perturbations environnementales sur le comportement de reproduction des Daphnies est tel que des évènements d'hybridation interspécifique ont été observés. Cependant, ces évènements d'hybridation sont issus de sensibilité aux trois perturbations environnementales différentes entre les espèces de Daphnies. / Lake ecosystems are increasingly subjected to multiple perturbations but their response is too rarely considered in overall. But, though a complex set of interactions, each perturbation can modulate the intensity of the impact of each other on lakes, and thus their response. In this context, this work is focused on deepening our understanding of the response of lakes in a context of multiples perturbations, over the long-term. We studied this response performing a paleolimnological approach (based on cladoceran) on three large, deep lakes (Lakes Geneva, Bourget and Annecy) submitted to a similar regional perturbation (i.e., climate warming) and varying intensities of two local perturbations (i.e., changes in nutrient inputs and fisheries managment practices). The ability of cladoceran to integrate the temporal changes in the pelagic food web in response to three environmental perturbations makes them a suitable model to reconstruct changes in the whole pelagic community. Specifically, we addressed (i) changes in cladoceran communities, during the last century, in response to three environmental perturbations and (ii) the impact of these perturbations on reproductive behavior of Daphnia. However, the lack of knowledge in large, deep lakes on the representativity of source cladoceran communitites by fossil assemblages in sediments firstly led to a methodological calibration. This last showed that the represention level of source communities was different according to the type of remain. Parthenogenetic remain assemblages (i.e., exoskeleton) reflect accurately the taxonomical and size structure of source communities in contrast to ephippia which are reliable proxy to reconstruct reproductive behavior. Following these results, the paleolimnological reconstructions of cladoceran communities showed that their ecological trajectories are controlled by complex interactions between the three perturbations, according to time periods and lakes. Moreover, the response of cladoceran communties to climate was different between the three lakes as a function of the intensity of local forcings. These observations suggest that it is important to include the role of local perturbations in predicting the future impact of climate change on lakes. The impact of environmental perturbations on the reproductive behavior of Daphnia is such that interspecific hybridization events were observed. However, these hybridization events were the result of different sensitivity to perturbations among Daphnia species.

La polyploïdie est un processus important et un moteur de la diversification et de l'évolution des plantes. Peu de polyploïdes naturels ont été décrits chez Juniperus, un genre de conifère représenté par 75 espèces d'arbres ou arbustes à feuilles persistantes, largement réparties dans l'hémisphère nord. Dans ce travail de recherche, l’implication de la polyploïdie dans l'évolution de Juniperus et l’élucidation des mécanismes sous-jacents à ces événements de polyploïdisation sont explorées. La taille du génome (TG) et le niveau de ploïdie ont été évalués chez 111/115 taxons en utilisant la cytométrie en flux et les comptages chromosomiques. Le taux de polyploïdie chez les genévriers s’est avéré être exceptionnellement élevé : 15 taxons sont des tétraploïdes et un seul taxon (J. foetidissima) est hexaploïde. Juniperus foetidissima représente le seul conifère hexaploïde découvert à ce jour à part Sequoia sempervirens. Nous avons également utilisé des approches de modélisation phylogénétique pour déterminer la TG ancestrale dans les trois clades de Juniperus et pour reconstruire le processus évolutif de la polyploïdisation chez ce genre. Au moins 10 événements de polyploïdisation ont eu lieu au cours de l'évolution et de la diversification de Juniperus. Nous avons ensuite exploré l’origine de la polyploïdie chez certaines espèces méditerranéennes. La variation de la TG et le niveau de ploïdie de deux variétés de J. sabina ont été estimés : Les populations échantillonnées de J. sabina var. sabina se sont avérées être diploïdes, tandis que les populations de J. sabina var. balkanensis étaient toutes tétraploïdes. Ces derniers auraient été issus d'une ancienne hybridation entre le tétraploïde J. thurifera et le diploïde J. sabina. Dans les Alpes françaises, où J. sabina var. sabina et J. thurifera sont en sympatrie, des individus présentant des morphologies intermédiaires entre ces deux espèces sont observés. Suite à des estimations des TG, de séquençage des ITS et de régions chloroplastiques, ces individus sont considérés comme des hybrides triploïdes. Enfin, l’utilisation des marqueurs AFLP pour déchiffrer les relations phylogénétiques entre des espèces méditerranéenne a montré que plusieurs pools génétiques contribuent à la diversité de Juniperus. Aussi ces marqueurs ont contribué à la découverte des contributions de ces pools génétiques aux taxons polyploïdes. Alors que les populations libanaises de l'hexaploïde J. foetidissima sont issues d'une lignée ancestrale unique, la population grecque semble résulter d'un mélange inégal de deux lignées anciennes. Ces deux lignées contribuent également au tétraploïde J. thurifera. Cette analyse a également montré que l’espèce méditerranéenne J. excelsa et l’espèce africaine J. procera partagent la même lignée ancestrale. Cependant, des analyses supplémentaires sont nécessaires pour une interprétation plus complète des données. L'importance de l'hybridation interspécifique et de la polyploïdie dans l'évolution des espèces de Juniperus nécessite d’amples recherches visant à comprendre le lien entre ces mécanismes et l'adaptation de ces espèces à un large spectre d'habitats extrêmes. Ces recherches futures devraient aussi contribuer à découvrir comment les espèces de conifères peuvent s’adapter aux changements climatiques. / Polyploidy is considered as an important phenomenon and a key driving force for plant diversification and evolution. Few natural polyploid species have been described in Juniperus, a coniferous genus represented by 75 species of evergreen trees or shrubs widely distributed in the North Hemisphere. The occurrence of polyploidy in the evolution of this genus as well as a more comprehensive view of pathways that were involved in these polyploidization events are explored in this research work. Genome size (GS) and ploidy level assessments were conducted on 111/115 taxa using flow-cytometry and chromosome counts. Juniperus holds an exceptionally high rate of polyploidy, 15 taxa being tetraploids and just one (J. foetidissima) being hexaploid. It represents the only hexaploid conifer discovered to date after Sequoia sempervirens. We also used phylogenetically-informed trait evolution modelling approaches to determine ancestral GS in the three clades of Juniperus and to reconstruct the evolutionary process of polyploidization in Juniperus. At least 10 polyploidization events have occurred during Juniperus evolution and diversification. We then explored the origin of polyploidy in selected Mediterranean species. The GS variation and the ploidy level of two J. sabina varieties were estimated: J. sabina var. sabina sampled populations were shown to be diploid, while J. sabina var. balkanensis populations were all tetraploid. The latter has been postulated to have arisen from an ancient hybridization between the tetraploid J. thurifera and the diploid J. sabina. In the French Alps, where J. sabina var. sabina and J. thurifera occur in sympatry, individuals with intermediate morphologies between these two species are observed. Evidences based on GS assessments, ITS and chloroplastic sequences demonstrated these individuals as triploid hybrids. Finally, the use of AFLP markers to decipher phylogenetic relationships between Mediterranean and Eastern Mediterranean species showed that multiple lineages contributes to Juniperus diversity and shed light on some polyploid taxa origins. While the Lebanese populations of the hexaploid J. foetidissima are issued from a unique ancestral lineage, the Greek population seems to be the result of an unequal admixture of two ancient lineages. These two lineages contribute also to the tetraploid J. thurifera. This analysis showed also that the Mediteranean J. excelsa and the African taxa J. procera shares the same ancestral lineage. However, further analyses are needed for a more complete interpretation of the data. The importance of interspecific hybridization and of polyploidization in the evolution of Juniperus species argues in favor of the development of researches aiming at understanding the link between these mechanisms and the adaptation of those species to a wide range of extreme habitats. Such future researches should contribute to predict how conifer species may adapt to dramatic changes in the Earth’s climate.

Hybridization is an important phenomenon in plant evolution because it is one of the sources of new genetic variability. Hybridization is the merging of genomes of formerly isolated evolutionary lineages. In many taxonomic groups, the detection of plants of hybrid origin is challenging. A wide spectrum of methods for their detection has been employed since the beginning of botanical research. The introduction of genomic in situ hybridization has had a great impact on the study plants of hybrid origin. This molecular cytogenetic approach allows to reveal the genomic contributions of particular parental species to hybrid taxa. The tribe Triticeae is a prime example of a group whose present-day diversity has been strongly influenced by hybridization (together with polyploidy). The majority of its species are allopolyploids resulting from frequent interspecific and intergeneric hybridization. The structure of relationships within the tribe is therefore highly reticulate. This thesis includes three papers dealing with the hybrid complex of Elytrigia repens - E. ×mucronata - E. intermedia: (1) The representatives of this hybrid complex are morphologically poorly differentiated, and only two morphological characters are used to their distinguishing. Among anatomical characters on the leaf blade, some...