Table of contents Thesis objectives and structure………………......…………..……... 3 CHAPTER I General introduction…………………………...……………………. 5 Marine population genetic structure 5 o Dispersal potential 7 o Population subdivision 9 o Population history 10 Phylogeography of canopy-forming brown macroalgae 12 The model species: Fucus ceranoides 15 CHAPTER II Surfing the wave on a borrowed board: range expansion and spread of introgressed organellar genomes in the seaweed Fucus ceranoides L……………………………….......……………………... 22 Abstract………………………………….……………………. 23 Introduction…………………………………………………… 24 Material & Methods…………………………………………... 27 Results………………………………………………………… 31 Discussion…………………………………………………….. 35 Acknowledgements…………………………………………… 41 Supplementary material………………………………………. 42 CHAPTER III Drifting fronds and drifting alleles: range dynamics, local dispersal and habitat isolation shape the population structure of the estuarine seaweed Fucus ceranoides L.....…………………… 44 Abstract……………………………………………………….. 45 Introduction…………………………………………………… 46 Material & Methods…………………………………………... 49 Results………………………………………………………… 54 1 Discussion…………………………………………………….. 59 Acknowledgements…………………………………………… 66 Supplementary material………………………………………. 67 CHAPTER IV Fine-scale genetic breaks driven by the colonization past and present density barriers in the estuarine seaweed Fucus ceranoides L. …………………………........................................…… 73 Abstract……………………………………………………….. 74 Introduction…………………………………………………… 75 Material & Methods…………………………………………... 80 Results………………………………………………………… 83 Discussion…………………………………………………….. 92 Acknowledgements…………………………………………… 103 Supplementary material………………………………………. 104 CHAPTER V General conclusions……….......….…..……………………………... 108 General references....……………..…………………………………. 112 2 Thesis objectives and structure The major objective of this thesis was to investigate the historical and recurrent processes shaping the genetic make-up of Fucus ceranoides, an estuarine seaweed lacking planktonic developmental stages. To this end, a range of molecular markers was used to describe the population genetic structure of this seaweed throughout its entire range and at more regional spatial scales. Specifically, the modern patterns of genetic diversity and differentiation of F. ceranoides were used to test the expected structuring effects of its particular life-history (e.g. habitat discontinuity, restricted dispersal), to infer its patterns of population genetic connectivity at large and small spatial scales, to infer the extent of introgression with related species, and to reconstruct its historical biogeography in the Northeast Atlantic. CHAPTER I provides a general introduction to population genetics theory, with special emphasis on the factors that interact to shape the population structure of marine organisms. The specificity of large brown seaweeds in the context of marine phylogeography is discussed, and some interesting findings highlighted. Finally, the biology of the species investigated in this study is presented. In CHAPTER II a combination of sequence markers is employed to resolve the phylogenetic relationships and the patterns of genetic exchange between F. ceranoides and two parapatric congeners. Because introgression was apparent for cytoplasmatic markers, the mtDNA phylogeography of F. ceranoides is also described to further investigate the geographical scope of introgression and its relationship with the species demographic history. In CHAPTER III, the population genetic structure of F. ceranoides is examined throughout its entire distributional range with microsatellite markers. The objective was to reconstruct the historical biogeography of F. ceranoides and to investigate the effects of 3 restricted dispersal and demographic history on the geographical organization of its genetic variation. In CHAPTER IV, the fine-scale phylogeographic structure of F. ceranoides in NW Iberia is described using both microsatellites and mtDNA sequence data. In the previous chapters three distinct phylogroups were found in this very restricted shoreline. Here, the historical and recurrent processes contributing to generate (and maintain) these high levels of regional population subdivision (in the absence of obvious dispersal barriers) are investigated. Finally, CHAPTER V provides a synthesis of the results of the preceding chapters and highlights the main contributions of this work. 4 CHAPTER I. GENERAL INTRODUCTION Marine population genetic structure Most, if not all marine species, exhibit some degree of population genetic structure, i.e., their modern populations differ to some extent in their genetic composition. A complete lack of differentiation across a species range (i.e., all populations having the same allele frequencies) would only be possible if the entire species was constituted by a single, very large group of randomly mating individuals. From a variety of reasons (biological, behavioural, geographical, ecological and geological), even the most mobile species are normally far from being panmictic throughout their entire ranges. As a result, populations inevitably diverge in time and space to some extent. Populations evolve by changes in the frequencies of alleles, the alternative forms of genes that constitute heritable diversity among conspecific individuals. Allele frequency change results from the interaction of distinct evolutionary forces: mutation (or introgression) of genes, which introduces new allelic variants in the populations; genetic drift, the stochastic fluctuation of allele frequencies resulting from the inter-generational sub-sampling of alleles in finite populations; gene flow, the exchange of genes between populations resulting from effective migration; and selection, the deterministic increase (and purge) of gene variants due to fitness effects (increased/reduced survival or reproductive output) (Hellberg et al. 2002). Over sufficiently long periods of time, and in the absence of demographical change, the genetic differentiation of populations will arrive at a state of equilibrium determined by the balance among these opposing forces. Mutation, drift and diversifying selection will cause populations to diverge, whereas migration and stabilizing selection will act to genetically 5 homogenise populations and maintain the genetic cohesion of a biological species (Slatkin 1987). The theoretical effect of gene-flow on allele-frequency divergence is the conceptual link between marine connectivity and population genetics (Hedgecock et al. 2007). If equal- sized populations are in equilibrium (i.e. the introduction on new alleles within populations from gene-flow and mutation is in equilibrium with the loss of alleles via genetic drift), the extent of neutral genetic differentiation between them will reflect their average rate of gene flow. In the Island Model (Wright 1931) at equilibrium, the absolute number of migrants exchanged each generation can be estimated as Nm = (1– FST)/ (4FST), where N is the effective size of the local population and m is the proportion of migrants entering that population each generation. Patterns of genetic differentiation can nevertheless represent poor proxies for modern population connectivity, because in natural populations the equilibrium assumption is often violated [see (Marko & Hart 2011b)]. The conditions that generate any particular distribution and subdivision change over ecological and evolutionary time scales, and it takes time for populations to progressively approach new genetic equilibriums [e.g. (Richmond et al. 2009)]. The number of generations required depends on the effective population sizes and rates of gene-flow (Palumbi 2003; Hedgecock et al. 2007; Hellberg 2009; Marko & Hart 2011a), but can be substantial. For species characterized by relatively large effective population sizes and/or low migration rates it may mean several thousands of generations and thus inferred patterns of connectivity will often be severely confounded by the vestiges of history (Benzie 1999; Hellberg et al. 2002; Poissant et al. 2005; Teske et al. 2011). On the 6 positive side, they typically allow the examination of historical patterns of population stability, vicariance and colonization with finer resolution (Hewitt 2000; Pelc et al. 2009). Marine biologists have long been interested in interpreting the geographic patterns of genetic variation and investigate the importance of several interacting factors that may be potentially responsible for them. Among others (e.g. mating systems, selection, introgression), the most important drivers of population genetic structure are the specie‟s intrinsic dispersal potential, population subdivision and population history. Dispersal potential Dispersal, the moviment of individuals or their propagules across space, has profound ecologic and evolutionary consequences. It allows the connectivity of populations in heterogeneous landscapes, the persistence of regional meta-populations (despite local extinctions), and the tracking of favourable environmental conditions in an ever changing world. As such, the patterns of dispersal have wide-ranging ramifications for the population dynamics, genetic structure, cohesion, resilience, distribution and evolution of species. The spatial scales where dispersal is important may vary considerably depending on the process of interest. When referring to population connectivity, the level of exchange must be sufficient to impact the demographic properties of the local population(s) (Johnson