Mitochondrial haplotype variability of brown trout populations from Northwestern Iberian Peninsula,a secondary contact area between lineages

Previous studies using mitochondrial markers and microsatellites revealed a new highly differentiated Atlantic lineage of brown trout in the Iberian Peninsula mainly confined to the Upper Duero Basin. Data suggest that Duero lineage (DU) hybridizes with previously described Atlantic (AT) lineages in that basin. However, nuclear and cytogenetic data suggest another hybrid zone between divergent lineages might be present in the Miño Basin, another Atlantic drainage located to the north. Here, we extend those studies by analyzing the mitochondrial haplotype variability of brown trout populations from northwestern Spain, a poorly studied area, to obtain an accurate picture of the distribution of DU. Results show that the DU haplotype is fixed in several isolated populations from the upper course of the Miño River and its main tributary the Sil River. DU haplotype co-occurs with two related AT haplotypes in the Miño Basin revealing a new secondary contact zone. The identification of both longtime stable populations of DU and several other populations showing introgression with widespread AT lineages will be useful for the management and conservation of such an endemic lineage.     

Allozymic evidence of parapatric differentiation of brown trout (Salmo trutta L.) within an Atlantic river basin of the Iberian Peninsula

The genetic variation of brown trout from Duero, one of the main Atlantic Iberian river basins, was assessed at 34 enzymatic loci in 62 native populations. A strong intrabasin differentiation was detected (G(ST) = 0.46; range D: 0-0.066), mainly attributable to the existence of two divergent groups of populations within Duero: southern and northern groups. This divergence was mainly a consequence of the unequal distribution of *75 and *100 alleles at sMDH-B1,2* isoloci, which were correlated with substantial differences in genetic diversity among regions. The Lower Course region (nearly fixed for the *100 allele) and Pisuerga River (nearly fixed for the *75 allele) showed lower heterozygosities (H approximately 0.8%) in contrast with adjacent areas, which evidenced intermediate frequencies for both alleles and higher heterozygosities (H: 2.2-3.1%). Vicariance appeared as the more probable explanation for the significant positive correlation detected between genetic and geographical distances in Duero Basin. Genetic relationships with adjacent Iberian drainages indicate a close similarity between the southern group and Cantabric trout, whereas the northern group constitutes an ancient form from this basin. This study confirmed complex genetic relationships in brown trout from northwest Iberia, reasserting the existence of clines at several loci and for genetic diversity. The interaction between Cantabric and Duero trout, as well as the location of the limit of the anadromous form around the 42 degrees N parallel, are both required to understand the genetic characteristics of brown trout from this area.     

Morphological variation in a secondary contact between divergent lineages of brown trout (Salmo trutta) from the Iberian Peninsula

The aim of this study was to analyze the morphological variation of brown trout (Salmo trutta) in the Duero basin, an Atlantic river basin in the Iberian Peninsula, where a spatial segregation of two divergent lineages was previously reported, based on isozyme, microsatellite and mtDNA data. In these studies, two divergent pure regions (Pisuerga and Lower-course) and several hybrid populations between them were identified. Morphological variation was evaluated in 11 populations representative of the genetic differentiation previously observed in the Duero basin, using multivariate analysis on 12 morphometric and 4 meristic traits. A large differentiation between populations was observed (interpopulation component of variance: 41.8%), similar to that previously detected with allozymes and microsatellites. Morphometric differentiation was also reflected by the high classification success of pure and hybrid individuals to their respective populations, using multivariate discriminant functions (94.1% and 79.0%, respectively). All multivariate and clustering analyses performed demonstrated a strong differentiation between the pure regions. The hybrid populations, though showing large differentiation among them, evidenced an intermediate position between the pure samples. Head and body shape traits were the most discriminant among the morphometric characters, while pectoral rays and gillrakers were the most discriminant among the meristic traits. These results confirmed the high divergence of the brown trout from the Duero basin and suggest some traits on which selection could be acting to explain the spatial segregation observed.     

High Ag-NOR-site variation associated to a secondary contact in brown trout from the Iberian Peninsula

The analysis of nucleolar organizer regions (NORs) using silver (Ag-) staining and in situ hybridization (ISH) in brown trout (Salmo trutta) from various river basins in the Iberian Peninsula revealed high variation in the number and location of NORs. A total of 17 different Ag-NOR sites were revealed in 10 different chromosome pairs. Three different Ag-NOR patterns clustered by river basins and strongly associated to the internal transcribed spacer 1 (ITS1) variation were detected. The main variability in NOR-sites was found in a secondary contact between two divergent lineages of brown trouts at Duero basin. Our results confirmed the abrupt break in the spatial distribution of genetic variation of brown trout populations previously reported at Duero basin. We hypothesize that NOR-site variation might be a consequence of hybridization between divergent lineages of brown trouts and that NORs could play a major role in the maintenance of a hybrid zone in Duero basin via post-zygotic isolation mechanisms.     

Genetic diversity and phylogenetic origin of brown trout Salmo trutta populations in eastern Balkans

The study focuses on the phylogenetic origin and genetic diversity of brown trout in the eastern part of the Balkan Peninsula. It further aims to reveal the impact of human-mediated transfers and stocking with non-indigenous trout on the populations in this area. For these purposes, mtDNA control region and microsatellite variation of 204 individuals from 16 populations were analysed. The results indicate that mtDNA haplotypes from the lower Danube basin and southern Black Sea basins differ substantially from a subclade of the Danubian lineage consisting of haplotypes found so far in the most of the Danube basin and in the Caspian and Aral Sea basins. Considering also the results of demographic analyses, this study evidences a complex evolutionary history of brown trout in the southern and western parts of the Black Sea basin. In the Aegean Sea basin, a high frequency of the central haplotype of Adriatic mtDNA lineage has been found. The other Adriatic lineage haplotypes found in this basin differ from the central haplotype by one mutational step only, indicating a recent evolution of the Adriatic lineage in the Aegean Sea basin. Substantial genetic differentiation among populations and basins was revealed. The hybridization with Atlantic brown trout was indicated in both sea basins, but especially in the Danube basin. Compared to other European regions, it can be inferred that the introgression of exogenous brown trout in the eastern Balkan populations is rather low.     

Northern and Southern expansions of Atlantic brown trout (Salmo trutta) populations during the Pleistocene

The phylogeography of Atlantic brown trout ( Salmo trutta ) was analysed using mitochondrial DNA control region complete sequences of 774 individuals from 57 locations. Additionally, the available haplotype information from 100 published populations was incorporated in the analysis. Combined information from nested clade analysis, haplotype trees, mismatch distributions, and coalescent simulations was used to characterize population groups in the Atlantic basin. A major clade involved haplotypes assigned to the Atlantic (AT) lineage, but another major clade should be considered as a distinct endemic lineage restricted to the Iberian Peninsula. The phylogeography of the Atlantic populations showed the mixed distribution of several Atlantic clades in glaciated areas of Northern Europe, whereas diverged haplotypes dominated the coastal Iberian rivers. Populations inhabiting the Atlantic rivers of southern France apparently contributed to postglacial colonization of northern basins, but also comprised the source of southern expansions during the Pleistocene.  © 2009 The Linnean Society of London, Biological Journal of the Linnean Society , 2009, 97 , 904–917.     

Management units of brown trout from Galicia (NW: Spain) based on spatial genetic structure analysis

Population genetic structure approaches offer the possibility of defining management units in conservation activities of species. The genetic structure of the brown trout Salmo trutta in Galicia (NW Spain) was investigated by using microsatellites. We determined genetic variation across 10 microsatellite loci of 901 individuals from 30 geographical populations representing 18 river basins. The analysis of the spatial distribution of the genetic variation by using different methods clearly revealed strong genetic differentiation among two groups of populations living in the studied area. This result is concordant with previous work using allozymes and mtDNA markers, and confirms a secondary contact among two highly differentiated evolutionary lineages in Miño Basin. Although both lineages might be locally adapted, results suggest that they hybridize at the middle course of the river. The brown trout from the Upper Miño Basin belongs to the previously described Duero lineage, an Iberian endemism threatened by introgression with other Atlantic forms. The results support the recognition of the Upper Miño Basin as a particular biotic region in Galicia. This study illustrates how a multidisciplinary approach in spatial genetic analysis contributes to the delineation of conservation units as conspecific metapopulations.     

Analysis of a secondary contact between divergent lineages of brown trout Salmo trutta L. from Duero basin using microsatellites and mtDNA RFLPs

The analysis of 12 microsatellite loci in 16 native populations of Salmo trutta from Duero basin evidenced a strong genetic differentiation in accordance with the existence of two divergent Atlantic mtDNA lineages, Atlantic (AT) and Duero (DU). These lineages were observed spatially segregated mainly in the Lower-course and in the inner part of the basin. Unlike previous isozyme information, microsatellite data suggested a more downstream location of the sharpest genetic divergence in Duero basin and a more complex structure in the inward area. Putative hybrid populations evidenced higher Hardy–Weinberg and gametic disequilibria than pure ones (Pisuerga, Lower-course), not explained by mixture due to differential immigration pattern across the basin. Hybridization indexes suggested a bimodal pattern of hybridization and a higher weight of Pisuerga region in the genetic composition of hybrid samples in accordance with mtDNA data. The results suggested a limited introgression between AT and DU lineages. Taking into account the time of divergence between both lineages, selection and/or genetic incompatibility appeared necessary to explain the genetic structure observed and the ancient location of DU lineage restricted to this area.     

Mitochondrial haplotype variation and phylogeography of Iberian brown trout populations

The biogeographical distribution of brown trout mitochondrial DNA haplotypes throughout the Iberian Peninsula was established by polymerase chain reaction-restriction fragment polymorphism analysis. The study of 507 specimens from 58 localities representing eight widely separated Atlantic-slope (north and west Iberian coasts) and six Mediterranean drainage systems served to identify five main groups of mitochondrial haplotypes: (i) haplotypes corresponding to non-native, hatchery-reared brown trout that were widely distributed but also found in wild populations of northern Spain (Cantabrian slope); (ii) a widespread Atlantic haplotype group; (iii) a haplotype restricted to the Duero Basin; (iv) a haplotype shown by southern Iberian populations; and (v) a Mediterranean haplotype. The Iberian distribution of these haplotypes reflects both the current fishery management policy of introducing non-native brown trout, and Messinian palaeobiogeography. Our findings complement and extend previous allozyme studies on Iberian brown trout and improve present knowledge of glacial refugia and postglacial movement of brown trout lineages.     

Genetic introgression on freshwater fish populations caused by restocking programmes

The brown trout (Salmo trutta L.) is one of the best studied native salmonids of Europe. Genetic studies on this species suggest that a large proportion of the evolutionary diversity corresponds to southern European countries, including the Iberian Peninsula, where this study is focused. Stocking activities employing non-indigenous hatchery specimens together with the destruction and fragmentation of natural habitats are major factors causing a decrease of native brown trout populations, mostly in the Mediterranean basins of the Iberian Peninsula. The main aim of the present work is to examine the genetic structure of the brown trout populations of the East Cantabrian region, studying the consequences of the restocking activities with foreign hatchery brown trout specimens into the wild trout populations. We have based our study on the Polymerase Chain Reaction and Restriction Fragment Length Polymorphism technique conducted on a mitochondrial fragment of 2700 base pairs and on the lactate dehydrogenase locus of the nuclear DNA. Our results show higher introgression rates in the Ebro (Mediterranean) basin than in the Cantabrian rivers. This revised version was published online in July 2006 with corrections to the Cover Date.     

Historical demography of brown trout (Salmo trutta) in the Adriatic drainage including the putative S. letnica endemic to Lake Ohrid

We explore the historical demography of the Adriatic lineage of brown trout and more explicitly the colonization and phylogenetic placement of Ohrid trout, based on variation at 12 microsatellite loci and the mtDNA control region. All Adriatic basin haplotypes reside in derived positions in a network that represents the entire lineage. The central presumably most ancestral haplotype in this network is restricted to the Iberian Peninsula, where it is very common, supporting a Western Mediterranean origin for the lineage. The expansion statistic R2, Bayesian based estimates of demographic parameters, and star-like genealogies support expansions on several geographic scales, whereas application of pairwise mismatch analysis was somewhat ambiguous. The estimated time since expansion (155,000 years ago) for the Adriatic lineage was supported by a narrow confidence interval compared to previous studies. Based on microsatellite and mtDNA sequence variation, the endemic Ohrid trout represents a monophyletic lineage isolated from other Adriatic basin populations, but nonetheless most likely evolving from within the Adriatic lineage of brown trout. Our results do not support the existence of population structuring within Lake Ohrid, even though samples included two putative intra-lacustrine forms. In the interests of protecting the unique biodiversity of this ancient ecosystem, we recommend retaining the taxonomic epithet Salmo letnica for the endemic Ohrid trout.     

Phylogeographic structure and demographic patterns of brown trout in North-West Africa

The objectives of the study were to determine the phylogeographic structure of brown trout (Salmo trutta) in Morocco, elucidate their colonization patterns in North-West Africa and identify the mtDNA lineages involved in this process. We also aimed to resolve whether certain brown trout entities are also genetically distinct. Sixty-two brown trout from eleven locations across the Mediterranean and the Atlantic drainages in Morocco were surveyed using sequence analysis of the mtDNA control region and nuclear gene LDH, and by genotyping twelve microsatellite loci. Our study confirms that in Morocco both the Atlantic and Mediterranean basins are populated by Atlantic mtDNA lineage brown trout only, demonstrating that the Atlantic lineage (especially its southern clade) invaded initially not only the western part of the Mediterranean basin in Morocco but also expanded deep into the central area. Atlantic haplotypes identified here sort into three distinct groups suggesting Morocco was colonized in at least three successive waves (1.2, 0.4 and 0.2-0.1 MY ago). This notion becomes more pronounced with the finding of a distinct haplotype in the Dades river system, whose origin appears to coalesce with the nascent stage of the basal mtDNA evolutionary lineages of brown trout. According to our results, Salmo akairos, Salmo pellegrini and "green trout" from Lake Isli do not exhibited any character states that distinctively separate them from the other brown trout populations studied. Therefore, their status as distinct species was not confirmed.     

Evidence for phylogeographically informative sequence variation in the mitochondrial control region of Atlantic brown trout

Complete sequencing of the mitochondrial control region was undertaken among brown trout Salmo trutta from North Atlantic areas where previous studies, based on smaller mtDNA fragments, failed to detect any phylogeographic signal. Comparison of sequences suggests that brown trout in the Iberian Peninsula and Scandinavia belong to largely divergent evolutionary units.     

Trout (<Emphasis Type="Italic">Salmo trutta</Emphasis>) mitochondrial DNA polymorphism in the centre of the marble trout distribution area

The marble trout, a lineage of the Salmo trutta complex, is endemic to the Southern Alpine region. Although it is endangered throughout its entire distribution range, population genetic data were lacking for the central area, including the upper Etsch/Adige River system (South Tyrol, Northern Italy). A total of 672 Salmo trutta specimens, comprising phenotypic marble trout and phenotypic brown trout, from 20 sampling sites throughout South Tyrol were analysed by sequencing the complete mitochondrial DNA control region. Thirteen distinct haplotypes were identified, which clustered within three major genetic lineages: the Marmoratus (MA), the Atlantic (AT) and the Danubian (DA) lineage. 41.7% of the investigated individuals carried haplotypes of the MA lineage, 47.9% of the AT lineage and 10.4% of the DA lineage. It is noticeable that AT haplotypes were present at all sampling sites and no “pure” marble trout population with exclusively MA haplotypes was found. This points to a considerable impact of stocking with allochthonous brown trout, given that there is no evidence for natural colonisation by individuals of the AT lineage. However, our data indicate, for at least four localities, a limited gene flow between the native marble trout and hatchery-reared strains. Future conservation and rehabilitation measures will thus have to concentrate on the identification of remnant pure marble trout individuals from such mixed populations. Handling editor: C. Sturmbauer     

Fine-scale phylogeographic contact zone in Austrian brown trout Salmo trutta reveals multiple waves of post-glacial colonization and a pre-dominance of natural versus anthropogenic admixture

Two major mtDNA lineages of brown trout come into contact in the upper Danube basin of Central Europe. The region is additionally confronted with stock management programs that often use domesticated foreign strains creating confusion as to what constitutes native and non-native populations as well as how to define major management units. In a unique system in north eastern Austria we provide additional support that the so-called Atlantic lineage of brown trout is native to the upper Danube and provide a high resolution microsatellite screening protocol that clearly distinguishes between natural and anthropogenic introgression, with the latter being minimal. Sequencing of 6,000 bp of mtDNA also shows that domesticated stocks primarily stem from a distinct albeit genetically diverse sub-clade of the Atlantic lineage, approximately 50,000–100,000 years divergent from the native Atlantic stocks, thought to have expanded into the region during the Holocene. The futility in defining large-scale management units based on mtDNA lineages for such broadly distributed and highly fragmented species is discussed.     

Influence of environment upon the life history of gudgeon, Gobio gobio (L.): a recent and successful colonizer of the Iberian Peninsula

The growth and the reproductive tactics of gudgeon, Gobio gobio (L.) were assessed in three contrasting streams of the Iberian Peninsula: the Rivers Moros and Ucero, tributaries of the R. Duero, and the R. MatarrańGa, a tributary of the R. Ebro. The R. Duero gudgeon have spread in the last 50 years while their presence in the lower tributaries of the R. Ebro followed the construction of reservoirs in the mid 1960s. The life histories of the Moros and MatarrańnGa gudgeon are very similar; rapid growth in spring, early reproduction (1 -year-old), high reproductive cost, short life-span and trend to semelparity. Both populations are strongly influenced by the severe summer droughts that result in (I) reduction in the condition coefficient, (2) reduction in the growth rate (particularly in the River MatarrańGa), (3) single spawning and (4) no gonad development in the summer. The gudgeon in the Ucero delay maturity, grow slower, are multi-spawners and have a longer life span. All the Duero populations come from the same genetic pool and they are actually connected through the populations living along this river basin, whereas the MatarrańGa population is not genetically linked, we hypothesize that the differences between these populations are environmentally induced.     

Genetic structure of northwestern Spanish brown trout (Salmo trutta L.) populations, differences between microsatellite and allozyme loci

Genetic variation in nine wild brown trout (Salmo trutta L.) populations was studied by means of allozyme and microsatellite markers. All brown trout populations were clearly separated into two clusters that represented the Sil and Duero basins. Although both markers revealed a strong genetic differentiation between basins, microsatellite loci resulted much more accurate when population structure at the intrabasin level was analysed. Also pairwise multilocus FST estimates and assignment tests of individual fish to the set of sampled populations demonstrated a much higher efficiency of microsatellites compared to allozymes. The analysis of both markers provides new insights in defining the conservation units at this local area and confirms the existence of a recognized sub-lineage in the Duero basin. The management implications of these findings are discussed and changes in trout release activity are recommended to avoid mixing of trout gene pools mainly in the Sil basin.     

Post-glacial colonization of Western Europe brown bears from a cryptic Atlantic refugium out of the Iberian Peninsula

The European brown bear (Ursus arctos) shows a particular phylogeography that has been used to illustrate the model for contraction-expansion dynamics related to glacial refugia in Southern European peninsulas. Recent studies, however, have nuanced the once generally accepted paradigm, indicating the existence of cryptic refugia for some species further north. In this paper we collected available data on chronology and mitochondrial haplotypes from Western European brown bears, adding new sequences from present day individuals from the Cantabrian (North Iberia) area, in order to reconstruct the dynamics of the species in the region. Both genetics and chronology show that the Iberian Pleistocene lineages were not the direct ancestors of the Holocene ones, the latter entering the Peninsula belatedly (around 10,000 years BP) with respect to other areas such as the British Isles. We therefore propose the existence of a cryptic refugium in continental Atlantic Europe, from where the bears would expand as the ice receded. The delay in the recolonization of the Iberian Peninsula could be due to the orographic characteristics of the Pyrenean-Cantabrian region and to the abundant presence of humans in the natural entrance to the Peninsula.     

Mitochondrial DNA variation in Pleistocene and modern Atlantic salmon from the Iberian glacial refugium

Current understanding of the postglacial colonization of Nearctic and Palearctic species relies heavily on inferences drawn from the phylogeographic analysis of contemporary generic variants. Modern postglacial populations are supposed to be representative of their Pleistocene ancestors, and their current distribution is assumed to reflect the different colonization success and dispersal patterns of refugial lineages. Yet, testing of phylogeographic models against ancestral genomes from glacial refugia has rarely been possible. Here we compare ND1 mitochondrial DNA variation in late Pleistocene (16,000-40,000 years before present), historical and contemporary Atlantic salmon (Salmo salar) populations from northern Spain and other regions of western Europe. Our study demonstrates the presence of Atlantic salmon in the Iberian glacial refugium during the last 40,000 years and points to the Iberian Peninsula as the likely source of the most common haplotype within the Atlantic lineage in Europe. However, our findings also suggest that there may have been significant changes in the genetic structure of the Iberian refugial stock since the last ice age, and question whether modern populations in refugial areas are representative of ice age populations. A common haplotype that persisted in the Iberian Peninsula during the Pleistocene last glacial maximum is now extremely rare or absent from European rivers, highlighting the need for caution when making phylogeographic inferences about the origin and distribution of modern genetic types.     

Genetic verification of native brown trout from the Persian Gulf (Catak Cay River, Tigris basin)

Analysis of the mtDNA control region in six individual brown trout Salmo trutta from the Catak Cay River (Tigris basin) revealed a single, new, haplotype, 1·0 to 1·5% divergent from five other Da lineage haplotypes analysed, that groups with the Danubian clade with low bootstrap support. This highly divergent haplotype combined with unique phenotypic characteristics underscores the novelty and native status of this population, which has probably been isolated from other brown trout lineages for at least several hundred thousand years.