Systematics, speciation processes, reproductive strategies and genetic structure in Ruppia

Resumen

Aquatic plants of the genus Ruppia inhabit coastal lagoons, saltmarshes, wetlands and inner saline waters, playing several key ecological roles. These systems are characterized by extreme variations of environmental conditions such as salinity, temperature and inundation. The complex evolutionary history of the genus Ruppia has resulted in uncertainty regarding the delimitation of species in the genus. These species exhibit a wide diversity of biological strategies to survive in these extreme environments, such as annual and perennial life-cycles, sexual reproduction and vegetative propagation, epihydrophily and hydroautogamy as strategies of pollination, and self- and cross-fertilisation. In addition, there are different vectors that can facilitate gene flow between populations, including sea-currents, waterbirds and fishes. These biological features of Ruppia have an important influence on its patterns of speciation, its genotypic and genetic diversity, and its population structure. Thus, genetic analyses provide important information to delimit species and taxa within this genus, evaluate diversity, and identify processes and fluxes acting at various temporal and spatial scales. The main objectives of this thesis are: to infer the evolutionary and biological processes of speciation and diversification, and to assess the prevalence and population structure, of European Ruppia species. This thesis is focused mainly on coastal locations from the Iberian Peninsula, due to the high diversity and sympatric occurrence of Ruppia species registered in this geographical area. To achieve these objectives, in Chapter I, ten new polymorphic molecular markers (i.e. microsatellites) were developed and validated for Ruppia cirrhosa (Petagna) Grande. Aditionally cross-amplification of two further microsatellites previously described for R. maritima were acheived. These molecular tools are important for studying clonal plants and therefore they have been used together with nuclear and chloroplast sequences in the following chapters. In Chapter II, the systematics of the genus Ruppia in Iberia was studied using morphology, highly polymorphic microsatellite markers and sequences of nuclear (ITS) and chloroplast (psbA-trnH) markers. Based on the different mutation rates and the inheritance mechanisms of these markers, the important role of hybridization and introgression in the evolutionary history of this genus was emphasized. R. drepanensis Tineo ex Guss and R. cirrhosa are in the same phylogenetic clade for both nuclear and chloroplast markers, and they can therefore be considered sister species. R. maritima Linnaeus is included in a more distant phylogenetic clade supported by both markers. In addition, two new genetic entities, R. cf. maritima and "R. hybrid", were identified. This entities showed some incongruences between nuclear and chloroplast gene trees, as well as a combination of microsatellite alleles suggesting hybridization and/or introgression effects. In Chapter III, by studying microsatellites in different populations of R. cirrhosa from the Iberian Peninsula and Sicily, a strong population genetic structure was detected. In general terms a low level of gene flow was recorded, ocurring mainly between geographically close populations or those populations located in the same hydrological body. In addition, different hypotheses were evaluated to explain the connectivity between the populations through correlations between geographic and genetic distances, the results suggesting that the most likely vector of dispersion among R. cirrhosa populations in the Iberian Peninsula are aquatic birds. By compiling results from Chapter II and Chapter III, the effects of different reproductive strategies on genotypic and genetic diversity of Ruppia were evaluated. All genetic entities showed high rates of sexual reproduction. In R. cirrhosa, the highest rates of sexual reproduction were detected from the most hydrologically unstable habitats. This could promote seed germination and establishment by a low intraspecific competition for space, light and other resources existing in less dense meadows. Higher levels of genetic diversity were detected in the epihydrophilous R. drepanensis, R. cirrhosa and probably "Ruppia hybrid" (this has not been confirmed) than in the hydroautogamous Ruppia cf. maritima, suggesting a strong influence of the pollination model on patterns of genetic diversity. In Chapter IV, R. maritima was for the first time identified in Cape Verde (Santiago Island) based on morphological and phylogenetic analyses. This information extends the geographic range distribution of this species to West Africa. The results obtained in this thesis provide basic information on the evolutionary processes linked with speciation and diversification of Ruppia. In addition, the biological strategies of adaptation and population connectivity of Ruppia taxa inhabiting shallow waters are identified. Finally, this thesis provides recommendations to improve the conservation of the genus Ruppia which inhabit one of the most threatened ecosystems in the world.