CHAPTER 1: Reconstructing ancient Mediterranean crossroads in Deronectes diving beetles Aim: To reconstruct the evolutionary history of a genus of freshwater beetle with a pan- Mediterranean distribution, to test classic hypotheses which proposed a Miocene origin for groups with high biodiversity in the Iberian and Anatolian peninsulas. Location: Mediterranean basin. Methods: We sequenced four mitochondrial and one nuclear gene from 51 specimens of 30 of the c. 60 extant species of Deronectes (Dytiscidae), all typical of mid-mountain streams from North Africa and Iberia over most of Europe to the Middle East. We used maximum likelihood, Bayesian probabilities with an a priori evolutionary rate and a dispersal–extinction–cladogenesis model to reconstruct their biogeographical history. Results: Deronectes has two major lineages which originated in the mid Miocene; one including mostly eastern and another mainly western and central Mediterranean species. From these two areas, range expansions, mainly at the end of the Miocene and beginning of the Pliocene, resulted in the many species groups and some of the extant species of the genus. Most of the current diversity and distributions are, however, of Plio-Pleistocene origin, particularly in widespread European species. Main conclusions: In line with traditional hypotheses, we found an ancient division between eastern and western Mediterranean lineages of Deronectes, likely resulting from the isolation of Europe west of the Alps from the Balkans and Anatolia during the early-middle Miocene. The history of the genus was strongly influenced by major geological and climatic events, with successive cycles of fragmentation and subsequent eastward and westward range expansions, resulting in a steady accumulation of species across the basin. Most of these range movements took place through the north side of the Mediterranean, with only local displacements in the south during the Messinian salinity crisis and a recent (Pleistocene) colonization of the Italian Peninsula, which remained largely submerged through most of the genus’ evolutionary history. CHAPTER 2: The origin of widespread species in a poor dispersing lineage (diving beetle genus Deronectes) In most lineages most species have restricted geographic ranges, with only few reaching widespread distributions. How these widespread species reached their current ranges is an intriguing biogeographic and evolutionary question, especially in groups known to be poor dispersers. We reconstructed the biogeographic and temporal origin of the widespread species in a lineage with particularly poor dispersal capabilities, the diving beetle genus Deronectes (Dytiscidae). Most of the ca. 60 described species of Deronectes have narrow ranges in the Mediterranean area, with only four species with widespread European distributions. We sequenced four mitochondrial and two nuclear genes of 297 specimens of 109 different populations of the four lineages of Deronectes including widespread species, together with their closest narrow range relatives, covering their entire distributions. Using Bayesian probabilities with an a priori evolutionary rate we performed (1) a global phylogeny/phylogeography to estimate the relationships of the main lineages within each group and root them, and (2) demographic analyses of the best population coalescent model for each species group, including a reconstruction of the geographical history estimated from the distribution of the sampled localities. We also selected 56 specimens to test for the presence of Wolbachia, a maternally transmitted parasite that can alter the patterns of mtDNA variability. All species of the four studied groups originated in the southern Mediterranean peninsulas and were estimated to be of Pleistocene origin. In three of the four widespread species the central and northern European populations were nested within those in the northern areas of the Anatolian, Balkan and Iberian peninsulas respectively, suggesting a range expansion at the edge of the southern refugia. In the Mediterranean peninsulas the widespread European species were replaced by vicariant taxa of recent origin. The fourth species (D. moestus) was proven to be a composite of unrecognised lineages with more restricted distributions around the Western and Central Mediterranean. The analysis of Wolbachia showed a high prevalence of infection among Deronectes, especially in the D. aubei group, where all sequenced populations were infected with the only exception of the Cantabrian Mountains, the westernmost area of distribution of the lineage. In this group there is a phylogenetic incongruence between the mitochondrial and the nuclear sequence, although not clear pattern links this discordance to the Wolbachia infection. Our results suggest that in different glacial cycles populations that happened to be at the edge of the newly deglaciated areas took advantage of the optimal ecological conditions to expand their ranges to central and northern Europe. Once this favourable ecological window ended populations become isolated, resulting in the presence of closely related but distinct species in the Mediterranean peninsulas. CHAPTER 3: Pleistocene range expansions in Western Palaearctic aquatic Coleoptera The Quaternary glacial cycles lead to large changes in the size and location of the geographic distribution of many species. During glacial maxima large areas of central and northern Europe become inhospitable to temperate species, which are generally assumed to have been recolonized by range expansions from Mediterranean refugia in the interglacials. An alternative possibility is that the recolonization was from non-Mediterranean sources, from glacial refugia in central Europe or western Asia, but data are still scarce concerning the origin of the central and north European species with very large geographic distributions, especially for insects. In this work we studied some species of three wide distributed lineages of freshwater beetles (Platambus maculatus complex, H. gracilis complex, and the genus Oreodytes), all typical of running waters and including both narrowly distributed southern endemics and widespread European species, some with distributions spanning the whole Palearctic. The main goal was to understand the role of the Pleistocene glaciations in the diversification and current distribution of these species. For this we sequenced four mitochondrial and two nuclear genes of 212 specimens of 107 different populations of the different lineages and used Bayesian probabilities and Maximum Likelihood to reconstruct the phylogenetic relationships, age and geographical origin of the western Palaearctic species of these three widely distributed lineages. All species of the three studied groups were estimated to be of Pleistocene origin. In the H. gracilis complex, a secondary fast range expansion from northern Anatolian populations to western and northern Europe resulted in the current widespread European H. gracilis. However, in the other two groups we found widespread central and northern European species the origin of which was not the Mediterranean area but central Asia, but with peripheral isolated forms in the southern Mediterranean peninsulas. These peripheral forms could have been confined as remnants of earlier diversification cycles or be result of incipient isolation of the most recent post-glacial expansion. The accumulation of narrow endemics of these lineages in the Mediterranean may thus be the result of successive cycles of range expansions with subsequent speciation (and local extinction in glaciated areas) through the multiple Pleistocene glacial cycles. CHAPTER 4: Range expansion and ancestral niche reconstruction in the Mediterranean diving beetle genus Meladema (Coleoptera, Dytiscidae) Species of the genus Meladema (Dytiscidae, Colymbetinae) are some of the largest macroinvertebrates in the western Palearctic region, being top predators in fishless streams. Two of the three described species, M. imbricata (Wollaston, 1871) and M. lanio (Fabricius, 1775) are Macaronesian endemics from the Canary Islands and Madeira respectively, while the third, M. coriacea Laporte, 1835, is widely distributed from Morocco and the Iberian Peninsula to Turkey, including the Canary Islands. Previous phylogenetic analysis using only mitochondrial markers revealed the existence of two cryptic lineages within M. coriacea, one restricted to Corsica and the other including the rest of sampled populations. We reconstruct here the evolutionary history of the species of Meladema using a more comprehensive sampling covering its whole geographical range, adding nuclear markers and Bayesian molecular dating. Using environmental niche modelling we test for possible differences in climatic preferences among lineages, and reconstruct their ancestral climatic niche. Our results strongly supported the existence of four monophyletic lineages represented by the three recognised species plus a fourth cryptic lineage with populations of M. coriacea from the Tyrrhenian islands (Corsica, Sardinia and Montecristo). This pattern is not likely to be the result of mitochondrial artefacts due to Wolbachia infection, as all 11 tested individuals were negative for this parasite. Dating analysis placed the origin of Meladema in the Middle Miocene although diversification among extant Meladema lineages started in the early Pleistocene and took place in a relatively short time period. Phylogeographic analysis inferred a continental origin of Meladema, with an independent colonization of the Macaronesian and Mediterranean islands. From the southwestern Mediterranean region the continental M. coriacea expanded its range up to Turkey in the northern basin, and to Tunisia in the southern. Results of niche modelling showed that seasonality is the critical factor in shaping the current distribution of Meladema. Island lineages (M. imbricata, M. lanio and the Thyrrenian lineage of M. coriacea) occur in sites with low seasonality, within the range of the reconstructed ancestral climatic niche of the genus. On the contrary, continental M. coriacea expanded its range to localities outside the ancestral climatic range of the genus, with a higher seasonality and aridity., Una pregunta básica sobre los rangos geográficos es comprender por qué unas especies están ampliamente distribuidas mientras otras, estrechamente emparentadas tienen distribuciones restringidas. En este estudio pretendo comprender los factores que llevaron a algunas especies a expandir su rango original y sugerir posibles hipótesis que expliquen las enormes diferencias de rango entre especies emparentadas. Para ello, reconstruyo la historia evolutiva de varios linajes de coleópteros acuáticos del Paleártico Occidental bajo el contexto de la compleja historia geológica y climática de este área, con el fin de aclarar los procesos que han contribuido a la actual distribución de especies en estos grupos. Estos linajes son típicos de aguas corrientes (es decir, con baja capacidad dispersiva) e incluyen tanto endemismos distribuidos en pequeñas áreas, como especies de amplia distribución, mostrando su potencial de expansión de rango. Las reconstrucciones biogeográficas llevadas a cabo sugieren la existencia de varios eventos de expansión de rango entre la zona oriental y occidental de la Cuenca Mediterránea bajo condiciones favorables, seguidos de fragmentación cuando las condiciones empeoraban. Estas expansiones debieron producirse principalmente a lo largo de la costa norte del Mediterráneo, ya que por la sur hay ausencia de especies al este de Túnez. A pesar de que no es posible establecer un origen temporal común para los grupos estudiados, el Mioceno con sus múltiples conexiones entre el Mediterráneo occidental y oriental seguidas de aislamiento, parece estar implicado en el origen y disyunción este-oeste en varios grupos. Sin embargo, la mayor parte de las especies actuales se originaron durante el Pleistoceno, incluyendo aquellas que han alcanzado amplias distribuciones, siendo por tanto la época mas influyente en la distribución actual de especies. Las sucesivas rondas de expansión y fragmentación de rango resultaron principalmente en la acumulación de especies restringidas en el área Mediterránea, especialmente en Anatolia y la Península Ibérica. A pesar de la evidencia en muchos casos de especiación con estabilidad de rango dentro del área Mediterránea, algunas especies lograron expandir su rango original hasta alcanzar amplias distribuciones en áreas mas al norte, probablemente aprovechando una posición geográfica ventajosa cuando las condiciones ambientales fueron ventajosas. No obstante, diferencias en tolerancia fisiológica y/o ecológica también pudieron favorecer las expansiones de rango en unas especies y limitarlo en otras, aunque futuras investigaciones experimentales se requieren para poder testar esta hipótesis.