Contrasting insights provided by single and multispecies data in a regional comparative phylogeographic study

Many single‐species freshwater phylogeographic studies have been carried out in south‐east Queensland; however comparative phylogeography requires multiple lines of evidence to infer deep, significant relationships between landscape and biota. The present study aimed to test conclusions resulting from single taxon studies in a multispecies comparative framework: (1) how influential are river basins in the genetic structure of freshwater species; (2) are there biogeographic frontiers between groups of basins; and (3) could deep intraspecific lineages be explained by a single event? New and existing data from 33 freshwater species (23 fishes and 10 crustaceans) were combined, and both standard single‐species analyses (haplotype networks, genetic distances, Φ_ST) and multispecies methods (hierarchical ABC) were carried out for 1814 sequences from eight basins. More than half of the species displayed a high phylogeographic structure and contained at least two distinct lineages. Almost all of the lineage divergences displayed an element of north/south geographic breaks, with the most influential boundary being between the Mary and Brisbane rivers. Of the 11 basin‐pair multispecies coalescent analyses, four implied a single divergence as being most likely. A regional analysis of deep lineages within 16 taxon‐pairs resulted in a strongly supported inference of a single divergence, probably dating to the Pleistocene. Basin boundaries are a key determinant of phylogeographic patterns for most of these freshwater species, although the specific biogeographic relationship between basins often varies depending on the species. There are a number of influential biogeographic frontiers, with the Brisbane‐Mary being the most important. The finding that a single event may be responsible for multiple deep lineages across the region implies that a highly influential climate change event may have been detected. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 111 , 554–569.     

Natural fragmentation in river networks as a driver of speciation for freshwater fishes

Although habitat fragmentation fosters extinctions, it also increases the probability of speciation by promoting and maintaining divergence among isolated populations. Here we test for the effects of two isolation factors that may reduce population dispersal within river networks as potential drivers of freshwater fish speciation: 1) the position of subdrainages along the longitudinal river gradient, and 2) the level of fragmentation within subdrainages caused by natural waterfalls. The occurrence of native freshwater fish species from 26 subdrainages of the Orinoco drainage basin (South America) was used to identify those species that presumably arose from in‐situ cladogenetic speciation (i.e. neo‐endemic species; two or more endemic species from the same genus) within each subdrainage. We related subdrainages fish diversity (i.e. total, endemic and neo‐endemic species richness) and an index of speciation to our two isolation factors while controlling for subdrainages size and energy availability. The longitudinal position of subdrainages was unrelated to any of our diversity measures, a result potentially explained by the spatial grain we used and/or the contemporary connection between Orinoco and Amazon basins via the upstream Casiquiare region. However, we found higher neo‐endemic species richness and higher speciation index values in highly fragmented subdrainages. These results suggest that habitat fragmentation generated by natural waterfalls drives cladogenetic speciation in fragmented subdrainages. More generally, our results emphasize the role of history and natural waterfalls as biogeographic barriers promoting freshwater biodiversity in river drainage basins.     

Low mitochondrial divergence indicates a rapid expansion across Europe in the weather loach, Misgurnus fossilis (L.)

Several phylogeographic studies using mtDNA sequence data have revealed an expressed geographic structure in nearly every European freshwater fish species studied. The authors present a phylogeographic study of Misgurnus fossilis on the base of 43 specimens from 17 localities across a major part of the known distribution area of M. fossilis . Despite the large geographic distance between the sampling points and their origin from different major European river systems, only eight closely related haplotypes in the sequences of the whole mitochondrial cytochrome b were detected. The most common haplotype I included more than 60% of specimens and occurred in the North Sea basin in northern Germany, in the Danube and Elbe basins in the Czech Republic, in the Nieman basin in Poland and in the Dniester and Vistula basins in the Ukraine. Since the highest number of haplotypes (six out of eight) and the most divergent haplotypes were found in the Danube, the authors tentatively consider the Danube to have acted as a refuge area for Misgurnus during the glaciation maxima in the Pleistocene. From this refuge, the species presumably recolonized Central and Eastern Europe but failed to stretch to Western Europe.     

Genetic divergence among invasive and native populations of Plagioscion squamosissimus (Perciformes, Sciaenidae) in Neotropical regions

The genetic divergence among invasive and native populations of Plagioscion squamosissimus from four Neotropical hydrographic basins was assessed using the hypervariable domain of the mitochondrial DNA (mtDNA) control region. Plagioscion squamosissimus is native to the neighbouring hydrographic basins of the Parnaíba and Amazon Rivers, and the latter includes the Araguaia-Tocantins drainage, but it is invasive in other basins due to introductions. The mtDNA nucleotide polymorphism supported the hypothesis that the Amazon and Parnaíba populations constitute the same species and are separated into two independent evolutionary lineages. Absence of nucleotide polymorphism was observed within and among P. squamosissimus populations invasive to the uppper and middle Paraná River basins. Nucleotide divergence was null or low comparing the Paraná invasive populations with the populations native to the Parnaíba River basin, whereas it was significantly high compared to Tocantins populations. These results ascertain that P. squamosissimus populations invasive to the upper Paraná River basin and to the middle Paraná River basin downstream of the Itaipu dam are derived from the Parnaíba River basin. The genetic data presented are potentially useful to assist further studies on P. squamosissimus taxonomic and geographic distribution, development of ecological guidelines for managing populations invasive to the upper Paraná River basin and for preservation of native fish diversity.     

Molecular ecology of zebra mussel invasions

The invasion of the zebra mussel, Dreissena polymorpha, into North American waters has resulted in profound ecological disturbances and large monetary losses. This study examined the invasion history and patterns of genetic diversity among endemic and invading populations of zebra mussels using DNA sequences from the mitochondrial cytochrome oxidase I (COI) gene. Patterns of haplotype frequency indicate that all invasive populations of zebra mussels from North America and Europe originated from the Ponto-Caspian Sea region. The distribution of haplotypes was consistent with invasive populations arising from the Black Sea drainage, but could not exclude the possibility of an origin from the Caspian Sea drainage. Similar haplotype frequencies among North American populations of D. polymorpha suggest colonization by a single founding population. There was no evidence of invasive populations arising from tectonic lakes in Turkey, while lakes in Greece and Macedonia contained only Dreissena stankovici. Populations in Turkey might be members of a sibling species complex of D. polymorpha. Ponto-Caspian derived populations of D. polymorpha (theta = 0.0011) and Dreissena bugensis (one haplotype) exhibited low levels of genetic diversity at the COI gene, perhaps as a result of repeated population bottlenecks. In contrast, geographically isolated tectonic lake populations exhibited relatively high levels of genetic diversity (theta = 0.0032 to 0.0134). It is possible that the fluctuating environment of the Ponto-Caspian basin facilitated the colonizing habit of invasive populations of D. polymorpha and D. bugensis. Our findings were concordant with the general trend of destructive freshwater invaders in the Great Lakes arising from the Ponto-Caspian Sea basin.     

Haplotype (mtDNA) diversity of brown trout Salmo trutta in tributaries of the Austrian Danube: massive introgression of Atlantic basin fish — by man or nature?

Mitochondrial haplotype diversity in 27 populations of brown trout, Salmo trutta L., in Austria was investigated by sequencing the 5' end of the mitochondrial DNA (mtDNA) control region. Although all populations are within the Danube drainage, 44% of all individuals carried Atlantic basin haplotypes. It is argued that the presence of these haplotypes in Austria primarily reflects introgression stemming from the stocking of hatchery-reared fish. However, several lines of evidence suggest that some natural colonization from Atlantic lineages may have contributed to the present haplotype diversity. Nonetheless, the more diverse Danubian clade is represented by regionally distinct haplotype diversity that should be protected from the continued introduction of domesticated strains of exogenous fish     

Regional population structure of the endangered Bridle Shiner (<Emphasis Type="Italic">Notropis bifrenatus</Emphasis>)

In the last 100 years, the Bridle Shiner has declined over significant parts of its range. We used mitochondrial and nuclear microsatellite markers to investigate population structure of this species. Tissue samples were collected from populations in six drainages in PA, NJ, NY and CT. One predominant haplotype was observed in the Delaware, Housatonic, Passaic, and Raritan specimens. Specimens from the Hudson and St. Lawrence drainages had a separate unique haplotype. Microsatellite loci revealed low within-population genetic diversity and pairwise population comparisons of allelic divergence showed significant genetic differences among most drainages. Population structure analysis of microsatellite loci recover the same population clusters inferred using mtDNA. Within the Delaware drainage, there was significant differentiation among areas separated by 128 km. Several alternate scenarios of population divergence and population bottlenecks were investigated using approximate Bayesian computing. These supported a scenario with a bottleneck occurring in the ancestral population of Bridle Shiner followed by recent divergence of Northern and Southern drainages.     

Unique mitochondrial DNA lineages in Irish stickleback populations: cryptic refugium or rapid recolonization?

Repeated recolonization of freshwater environments following Pleistocene glaciations has played a major role in the evolution and adaptation of anadromous taxa. Located at the western fringe of Europe, Ireland and Britain were likely recolonized rapidly by anadromous fishes from the North Atlantic following the last glacial maximum (LGM). While the presence of unique mitochondrial haplotypes in Ireland suggests that a cryptic northern refugium may have played a role in recolonization, no explicit test of this hypothesis has been conducted. The three‐spined stickleback is native and ubiquitous to aquatic ecosystems throughout Ireland, making it an excellent model species with which to examine the biogeographical history of anadromous fishes in the region. We used mitochondrial and microsatellite markers to examine the presence of divergent evolutionary lineages and to assess broad‐scale patterns of geographical clustering among postglacially isolated populations. Our results confirm that Ireland is a region of secondary contact for divergent mitochondrial lineages and that endemic haplotypes occur in populations in Central and Southern Ireland. To test whether a putative Irish lineage arose from a cryptic Irish refugium, we used approximate Bayesian computation (ABC). However, we found no support for this hypothesis. Instead, the Irish lineage likely diverged from the European lineage as a result of postglacial isolation of freshwater populations by rising sea levels. These findings emphasize the need to rigorously test biogeographical hypothesis and contribute further evidence that postglacial processes may have shaped genetic diversity in temperate fauna.     

Inter‐basin exchange and repeated headwater capture across the Sierra Madre Occidental inferred from the phylogeography of Mexican stonerollers

Aim Geomorphic evolution of river basins can shape the structure and diversity of aquatic communities, but understanding the biological significance of basin evolution can be challenging in semi‐arid regions with ephemeral or endorheic conditions and complex drainage configurations such as the Sierra Madre Occidental (SMO) in North America. In this study, we characterized range‐wide patterns of genetic variation in the Mexican stoneroller (Campostoma ornatum) to infer how orogenic and erosional influences on river basin connectivity have given rise to the diverse and largely endemic freshwater communities across the SMO region. Location Twelve drainage basins across northern Mexico and the south‐western United States, centred on the SMO. Methods  We collected 202 specimens from 98 localities across the range of C. ornatum. We performed phylogenetic analyses of DNA sequences from one mitochondrial (cytochrome b) and one nuclear (intron S7) gene. Phylogenetic trees were estimated for each data set using maximum likelihood and Bayesian inference. Results Phylogenetic analyses consistently resolved a monophyletic C. ornatum composed of multiple evolutionary lineages within two markedly divergent clades that differentiate northern drainages from southern drainages in the SMO region. Within‐clade patterns of divergence corresponded to fine‐scale geographic structure within and among SMO drainage basins. However, the geographic distribution of evolutionary lineages within the northern and southern clades did not always correspond to the geographic configuration of drainage basins. Some subclades encompassed multiple drainages, and individuals from a single drainage were sometimes recovered in multiple subclades. Main conclusions Our findings indicate that a common ancestor of Mexican Campostoma is likely to have entered north‐west Mexico through an ancient Rio Grande system that extended as far south as the Rio Nazas and Rio Aguanaval. The geographic orientation of the two strongly divergent clades recovered within C. ornatum provides evidence of long‐standing isolation of southern basins from northern basins within the ancestral Rio Grande system, possibly due to the combined influence of tectonic events and increasing regional aridity. Geographic patterns of genetic variation also provide evidence of range expansion from Atlantic to Pacific drainages due to drainage evolution and river capture events, as well as further inter‐basin exchange via more recent headwater capture events, hydrological connections and possible anthropogenic introductions.     

Maintenance of an endemic lineage of brown trout (Salmo trutta) within the Duero river basin

The Iberian Peninsula contains diverse populations of freshwater fish, with major river basins comprising differentiated biogeographic units. The Duero River flows through the North‐Western Iberian Peninsula and is one of the most important rivers within the Iberian glacial refuge. Brown trout (Salmo trutta) populate this whole basin, and studies using both allozyme and microsatellite loci have detected a geographically sorted distribution of genetic variation in this species. In this work, sequences of the mitochondrial control region obtained from 299 brown trout from the Duero River were compared with other Iberian and European datasets. Two differentiated haplotype groups were detected inside the Duero River basin. One of them was related to the Atlantic (AT) lineage that is present in Northern European populations, whereas the other comprised an unique group that was restricted to the inner region of the basin. The amount of divergence of this Duero group from the other brown trout populations studied is consistent with a new trout lineage (Duero, DU) that is endemic to this river basin and that diverged from other Atlantic populations during the Pleistocene. The distribution of the DU and AT quaternary lineages in the Duero River was consistent with the ichthyological pattern described in the basin that originated during the Miocene–Pliocene. Evidence of selective processes that favour the haplotypes of the DU lineage may explain this discrepancy.     

Biogeographic patterns and current distribution of molecular-genetic variation among populations of speckled dace, Rhinichthys osculus (Girard)

We examined genetic diversity within- and among-populations of speckled dace (Rhinichthys osculus) in five major drainage systems in the state of Oregon in western North America. Analysis of sequence variation in a 670-bp segment of the mitochondrial cytochrome b gene revealed deep divergence among basins and high genetic diversity within basins. Application of a molecular clock indicated that the divergence time among basins reflects vicariant events during the late Miocene to early Pliocene. The high levels of genetic diversity observed within basins is likely due to large historic population sizes, in particular, within the Klamath Basin. Two highly divergent mtDNA lineages were found to co-occur in populations in the Klamath Basin. This result may be indicative of a complex history of isolation and reconnection in this basin and/or multiple colonization events. Based on the observed level of mtDNA divergence these lineages may represent two reproductively isolated sympatric taxa. We recommend that major basins be regarded as distinct ESUs based on high levels of subdivision, deep divergences, and reciprocal monophyly among basins.     

Habitat islands, genetic diversity, and gene flow in a Patagonian rodent

The effects of terrestrial habitat islands on gene flow and genetic diversity in animal populations have been predicted and discussed in theoretical terms, but empirical data are needed to test these predictions and provide an understanding of the relationships of life-history characteristics to genetics of insular species. We studied saxicolous mice ( Phyllotis xanthopygus ) in Patagonia to explore genetic structure, phylogeography, and gene flow in a species inhabiting natural habitat islands. Phylogeographic analyses based on mtDNA sequences revealed two haplotype clades, which presumably reflect early Pleistocene factors that temporarily separated the mice into two geographically isolated groups. The Río Chubut, which lies within a glacial drainage basin bisecting northern Patagonia, might have affected gene flow in the species. Although we anticipated isolation by distance and founder phenomena associated with habitat islands, in some habitat patches we found evidence of high local genetic diversity. The amount of divergence in the mitochondrial cytochrome b gene (≈ 3.4%) in animals at a single locality could best be explained through a combination of historical factors and metapopulation source–sink theory. Demographic shifts, dispersal, and episodic recolonization are important in the life history and genetic population structure of P. xanthopygus .     

Sea‐level variations have influenced the demographic history of estuarine and freshwater fishes of the coastal plain of Paraná, Brazil

This study surveyed the mitochondrial haplotype diversity of nine freshwater fish species and two estuarine–marine species from the coastal basins and drainages of the highland plateaus of Paraná, Brazil. Portions of the cytochrome b gene or the control region were sequenced. The demographic history of each species was inferred using the Bayesian skyline method, mismatch distribution analysis and statistical neutrality tests. Demographic reconstruction analyses revealed a single pattern of variation in the effective population size (N_e) among species. No dramatic changes in N_e were detected in upland species. By contrast, evidence of population expansion over the past 200 000 years was detected in all coastal plain and estuarine species. These findings correspond to periods of low sea‐level (regressions) followed by a rapid increase in the sea‐level by >100 m. The resulting reconnections and subsequent fragmentation and isolation between the estuarine and freshwater bodies were putatively relevant to the historical demography of the fish species in these areas.     

The effect of landscape processes upon gene flow and genetic diversity in an Australian freshwater fish, Neosilurus hyrtlii

1. Flow regime and riverine architecture are two important landscape characteristics that influence genetic diversity and gene flow in riverine species.
2. Using population genetic markers (mtDNA, microsatellites and allozymes), this study aimed to investigate genetic diversity and gene flow in the freshwater fish, Neosilurus hyrtlii , across two major drainage divisions in northern and central Australia (the Gulf of Carpentaria and Lake Eyre basins). These basins lie adjacent to each other and differ in their hydrological inputs and riverine structure, providing an ideal opportunity to identify the impact of landscape processes upon population dynamics of freshwater fish.
3. Populations were strongly structured among basins, among catchments within basins and were weakly structured within catchments in the Lake Eyre Basin, providing support for the Stream Hierarchy Model.
4. Interestingly, mtDNA and microsatellite diversity was much higher in the Gulf of Carpentaria Basin compared to the Lake Eyre Basin. It was concluded that this difference was due to the extreme hydrological variability in this basin and boom-bust population cycles resulting in smaller effective population sizes in the Lake Eyre Basin.     

Phylogeography of the catfish Hatcheria macraei reveals a negligible role of drainage divides in structuring populations

Southern South America provides a set of unusual geographic features that make it particularly interesting for studying phylogeography. The Andes Mountains run along a north-to-south axis and act as a barrier to gene flow for much of the biota of this region, with southern portions experiencing extensive historical glaciation. Geological data reveal a series of drainage reversals, shifting from Pacific Ocean outlets to Atlantic Ocean outlets because of glacier formation that dammed and reversed rivers. Once glaciers melted around 13 000 years ago, drainages returned to the Pacific Ocean. This geologic history predicts that aquatic organisms in Pacific rivers should have their closest relationships to their counterparts in Atlantic rivers immediately to their east. We tested this prediction in the trichomycterid catfish Hatcheria macraei from 38 locations using the mitochondrial cytochrome b gene. Our results show that most populations found in Pacific rivers were closely related to fish found in the adjacent Atlantic draining Río Chubut. Surprisingly, one documented drainage reversal (from Río Deseado into Río Baker) did not result in movement of H. macraei. Overall, we found the lowest levels of genetic structure between most Pacific rivers that are adjacent to the Atlantic draining Río Chubut. We also found low levels of population structuring among three of four contemporary river basins that drain to the Atlantic Ocean. Our findings suggest that drainage basin boundaries have historically not played an important long-term role in structuring between nine of 11 drainages, an unusual finding in freshwater biogeography.     

Biogeography of a southern hemisphere freshwater fish: how important is marine dispersal?

Galaxias maculatus is one of the world's most widely distributed freshwater fish. This species has a marine-tolerant juvenile phase, and a geographical range extending through much of the southern hemisphere. We conducted phylogeographic analyses of 163 control region haplotypes of G. maculatus, including samples from New Zealand (five locations), Tasmania (one location) and Chile (one location). A lack of genetic structure among New Zealand samples suggests that marine dispersal facilitates considerable gene flow on an intra-continental scale. The discovery of a Tasmanian-like haplotype in one of 144 New Zealand samples indicates that inter-continental marine dispersal occurs but is insufficient to prevent mitochondrial DNA differentiation among continents. The sister relationship of Tasmanian and New Zealand clades implies that marine dispersal is an important biogeographical mechanism for this species. However, a vicariant role in the divergence of eastern and western Pacific G. maculatus cannot be rejected.     

Phylogeography and freshwater basins in central Mexico: recent history as revealed by the fish parasite Rhabdochona lichtenfelsi (Nematoda)

Aim The phylogeography of Rhabdochona lichtenfelsi, a nematode parasite specific to endemic goodeids in Mexico, is used to infer the biogeographical history of fragmentation and recent evolution of the Mesa Central drainages. Geological history of the west‐central region of Mexico suggests that extant freshwater basins are the result of different vicariant events that fragmented ancient watercourses and lakes within the Mesa Central. Location Major freshwater river basins of the Mesa Central, Mexico: Ameca, Cotija, Lerma, Rio Verde, Panuco, and lakes Cuitzeo and Zacapu. Methods Haplotype diversity and phylogeographical structure of 10 populations of R. lichtenfelsi, sampled from the complete range of this species, were analysed with partial sequences of cytochrome c oxidase subunit I (456 bp). Analyses performed included phylogenetic tree estimation methods (neighbour‐joining, maximum parsimony and maximum likelihood), genetic diversity, distance and structure estimates, and nested clade analysis. Results High overall haplotype diversity, unique haplotypes, and strongly structured populations were found in the basins sampled. Three phylogenetically and demographically identifiable clades were recovered. These clades fit an isolation‐by‐distance model. Significant population expansion was observed for two clades and for the entire population. Time of divergence was estimated as 1.0 and 0.84 Ma for the different clades. Main conclusions The distribution of R. lichtenfelsi haplotypes does not correspond to the present distribution of the basins of Mesa Central, but instead reflects the distribution of those basins during a recent geological period (Pleistocene). While our current knowledge of the evolution and geographical relationships of the Mesa Central basins comes from studies of freshwater fish encompassing a more ancient history, our results suggest that, during the past million years, old basins and connections existed where today isolated freshwater bodies stand, thus unravelling a novel biogeographical history for the Mesa Central of Mexico.     

Partitioning global patterns of freshwater fish beta diversity reveals contrasting signatures of past climate changes

Here, we employ an additive partitioning framework to disentangle the contribution of spatial turnover and nestedness to beta diversity patterns in the global freshwater fish fauna. We find that spatial turnover and nestedness differ geographically in their contribution to freshwater fish beta diversity, a pattern that results from contrasting influences of Quaternary climate changes. Differences in fish faunas characterized by nestedness are greater in drainage basins that experienced larger amplitudes of Quaternary climate oscillations. Conversely, higher levels of spatial turnover are found in historically unglaciated drainage basins with high topographic relief, these having experienced greater Quaternary climate stability. Such an historical climate signature is not clearly detected when considering the overall level of beta diversity. Quantifying the relative roles of historical and ecological factors in explaining present-day patterns of beta diversity hence requires considering the different processes generating these patterns and not solely the overall level of beta diversity.     

Clarifying an ambiguous evolutionary history: range‐wide phylogeography of an Australian freshwater fish,the golden perch (Macquaria ambigua)

Aim We conducted a range‐wide phylogeographic study of a common Australian freshwater fish, the golden perch (Macquaria ambigua), to investigate the relationship between environmental processes and evolutionary history in drainage basins. Location Inland [Lake Eyre (LEB), Murray–Darling (MDB) and Bulloo (BULL)] and coastal basins [Fitzroy (FITZ)] of eastern Australia. Methods A total of 590 samples were collected from across the entire species’ distribution and a section of the mitochondrial DNA control region was sequenced. In order to reconstruct the evolutionary history of M. ambigua a comprehensive suite of phylogeographic analyses was conducted, including nested clade phylogeographic analysis, mismatch analysis and isolation‐with‐migration model simulations. Results Three major lineages corresponding to the major drainage basins, FITZ, MDB and LEB/BULL, were identified (Φ_ST = 0.92). Lineages from the coastal basin (FITZ) were highly divergent from those of the inland basins (up to 6%). Levels of genetic diversity in the inland basins were relatively low and our analyses indicate that these populations experienced both demographic and range expansions during the Pleistocene. Main conclusions Investigation of the range‐wide phylogeography of M. ambigua has revealed new insights into the biogeography of the Australian arid zone, particularly with regard to evolutionary events chronologically associated with cyclical moist and dry conditions. We propose that M. ambigua originated on the east coast (FITZ) and crossed a major geographic barrier, the Great Dividing Range (GDR), to colonize the inland basins (MDB, LEB and BULL). We infer a series of demographic and range expansion events for M. ambigua consistent with a scenario of moister Pleistocene conditions and increased connectivity of freshwater environments, both within and among drainage basins. Major lineages then diversified following isolation of freshwater environments under increasingly arid climate conditions. We suggest that management priorities for M. ambigua should include the resolution of taxonomic uncertainties and the maintenance of genetic diversity of both stocked populations in the MDB and native populations of the LEB that may be at risk of further isolation and reduced gene flow due to increased aridification under future climate change scenarios.     

Phylogeography and population structure of thornback rays (Raja clavata L., Rajidae)

The phylogeography of thornback rays (Raja clavata) was assessed from European waters, using five nuclear microsatellite loci and mitochondrial cytochome b sequences. Strong regional differentiation was found between the Mediterranean basin, the Azores and the European continental shelf. Allelic and haplotype diversities were high in Portuguese populations, consistent with the existence of a refugium along the Iberian Peninsula. Unexpectedly, high diversity was also found in the English Channel/North Sea area. The lowest genetic diversity was found in the Black Sea. Populations sampled from the Mediterranean, Adriatic and Black Seas were characterized by a single mitochondrial haplotype. This haplotype was also the most ancestral and widespread outside of the Mediterranean basin except for the Azores. Populations from the Azores were dominated by a second ancestral haplotype which was shared with British populations. Results from multidimensional scaling, amova and nested clade analysis indicate that British waters are a secondary contact zone recolonized from at least two refugia--one around the Iberian Peninsula and one possibly in the Azores. Links to a potential refugium known as the Hurd Deep, between Cornwall and Brittany, are discussed. Finally, a historical demographic analysis indicates that thornback ray populations started to expand between 580,000 and 362,000 years ago, which suggests that the Last Glacial Maximum (20,000 years ago) had mainly affected the distribution of populations rather than population size.