Mechanisms of speciation in bryophytesevolutionary studies in the moss Funaria hygrometrica along environmental gradients

Résumé

The general objective of this Doctoral Thesis was to contribute to the knowledge on the importance of geography and climate in shaping genetic variation and their role as speciation mechanisms in bryophytes that exhibit wide distributions. The cosmopolitan moss Funaria hygrometrica was used as a study model. A multidisciplinary study was carried out in which a ploidy analysis, the study of genetic variability using various genetic markers and methodologies, landscape genetics, transcriptome analysis, quantitative morphometrics and biogeographic approaches were combined. Samples from four geographical areas of Spain with different climatic regimes were used (Asturias, Leon and Murcia, and the Sierra Nevada Mountains), in addition to a sample from Chile and other species of the genus Funaria (F. aequidens, F. arctica, F. flavicans, and F. polaris). Four cytotypes were detected by flow cytometry. Only one of them was haploid and was present in 67.4% of the individuals analysed. The rest of the individuals had one of the other three cytotypes, which were diploid, but with different genome sizes. One possible mechanism to explain these differences within diploids is the variable number of accessory chromosomes, which are known to be present in certain moss species. To describe the levels of genetic differentiation among F. hygrometrica populations in the four studied areas, all collected samples were cultivated in vitro and genotyped using the Genotyping by sequencing (GBS) genomic technique. Sanger sequencing of the three genomic compartments (nuclear, mitochondrial, and chloroplastic) was also carried out in some individuals from the Sierra Nevada Mountains. Based on all these analyses, the presence of two clearly differentiated genotypes was confirmed: one restricted to the highlands of the Sierra Nevada Mountains (above 1600 m a.s.l.) and a few individuals from the rest of the studied areas; and another generalist genotype, present in the entire altitudinal gradient of the Sierra Nevada Mountains and preponderant in the rest of the studied territory. A greater phylogenetic and gene expression proximity was observed between the restricted distribution genotype and the sample of F. hygrometrica from Chile, which shows its high long-distance dispersal capacity, while the generalist genotype was more related to the other studied Funaria species. Hybridization between both lineages could not be confirmed in the Sierra Nevada Mountains, not even in localities where they coexist, suggesting reproductive isolation. Sexual reproduction by self-fertilization in both genomic groups might explain the absence of recombination between them. Landscape genetics to understand the importance of geography (isolation by distance, IBD) versus ecology (isolation by environment, IBE) in order to explain genetic differentiation patterns revealed that the genetic structure does not align with an environmental barriers in populations of Asturias, Leon, and Murcia, while along the altitudinal gradient of the Sierra Nevada Mountains, the climatic variable isothermality constituted the model that best explains the observed genetic structure. This suggests that a potential barrier, although not absolute, might restrict dispersal or the effective establishment, ultimately promoting ecological isolation. To compare gene expression between samples subjected to heat stress and control conditions was possible using the RNAseq technique. Transcriptomic data pointed to the direction that there might be differences in heat stress tolerance between the two genetic lineages since the genes differentially expressed under thermal stress conditions belonged to the gene ontology category "Unfolded protein binding", which consists mainly of heat shock proteins. A greater difference in intra-specific than inter-specific gene expression was observed. The biometric study of individuals from Sierra Nevada Mountains did not show significant morphological differentiation between individual groupings classified according to their genetic cluster, altitudinal distribution, and ploidy level. On the contrary, the species F. aequidens, F. arctica, F. flavicans, and F. polaris were morphologically distinguished from F. hygrometrica even though the genetic data showed greater proximity to the individuals with a wide distribution genotype from the Sierra Nevada Mountains. This lack of morphological variation within F. hygrometrica suggests that the two genotypes could be cryptic species within a morphospecies, which seem to have a faster evolution or divergence at the molecular level than at the morphological one. A simulation of the distribution patterns of the two possible cryptic species showed that they could share the same habitat, even if they compete strongly for the same resources and one of them has a certain selective advantage in that habitat. The condition is that a sufficient number of migrants arrive in this habitat from other localities, where the selective advantage is inverted. This work emphasizes the importance of considering multiple sources of empirical evidence to infer evolutionary processes and apply taxonomic criteria in broadly distributed bryophyte species.