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.     

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.     

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.     

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.     

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.     

Juxtaposed microsatellite systems as diagnostic markers for admixture: an empirical evaluation with brown trout (Salmo trutta) as model organism

A juxtaposed microsatellite system (JMS) is composed of two microsatellite repeat arrays separated by a sequence of less than 200 bp and more than 20 bp. This paper presents the first empirical evaluation of JMSs for the study of genetic admixture induced by man, with brown trout (Salmo trutta) as model organism. Two distinct admixture situations were studied: native populations from streams of the Atlantic basin and of the Mediterranean basin, respectively, all stocked with domestic strains originating from the Atlantic basin. For these two situations, we first evaluated by simulation the ability of JMSs to differentiate between alien alleles and naturally shared homoplasious or ancestral alleles, and thus to behave as diagnostic markers for admixture. Simulations indicated that JMSs are expected to be reliable diagnostic markers in most divergent (i.e. Mediterranean) populations and nonreliable diagnostic markers in most closely related (i.e. Atlantic) populations. Three JMSs were genotyped in domestic strains as well as in nonstocked and stocked populations of brown trout sampled in different rivers of the Mediterranean and Atlantic basins. The observed distributions of JMS haplotypes were consistent with simulation predictions confirming that JMSs were reliable diagnostic markers only over a given proportion of the species range, i.e. in substantially divergent populations. JMSs also reinforced the diagnostic character of three microsatellite sites for the studied Mediterranean populations. This last result is consistent with our simulation results which showed that, although much less frequently than at JMSs, diagnostic markers are likely to be found at single site microsatellites provided that the native Mediterranean population has a sufficiently small effective population size. For each population of the Mediterranean basin admixture coefficients did not differ significantly across JMSs and mean admixture coefficients sometimes differ among populations. The interpretation of the origin of JMS haplotypes based on the allele length variants was supported by nucleotide sequence analysis.     

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.     

Genetic Structure of Brown Trout (Salmo trutta) Populations from Turkey Based on Microsatellite Data

This present study investigated micro- and macro-geographic microsatellite DNA variations using five polymorphic microsatellite loci from 27 brown trout populations in Turkey. Average number of alleles and average observed heterozygosity were 7.4 and 0.254, respectively. Even populations from the same sea basin and river system (the so called micro-geographic regions) had unique alleles. Genetic variation among the populations from macro-geographic regions (different sea basins and river systems) was 45.78%. The mtDNA lineages of brown trout that have previously been identified by mtDNA analyses were supported by the analysis of the microsatellite DNA data in general. The Çatak population, which belongs to the Tigris lineage, was clustered together with the Euphrates populations within the Adriatic mtDNA lineage, based on microsatellite data. Both mitochondrial and microsatellite DNA analyses have made it possible to determine a secondary contact between Adriatic and Tigris lineages.     

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.     

Genetic introgression between wild and stocked brown trout in the Douro River basin, Spain

The genetic diversity of Spanish brown trout is currently threatened by stocking with exogenous brown trout from Central and Northern Europe. In the Douro River basin 25% of the analysed populations in the present study showed introgression by genes of hatchery origin. The mean introgression estimated by the single locus approach ( S ) varied from 0 to 22% among populations, with a mean value of 3%. The hatchery allele markers were absent in populations where stocking ceased in 1993. However, the introgression effect was observed in all populations stocked until 1998. It seems that cessation of stocking is a good measure for restoring native populations. A thorough review of published and present data of genetic interactions between wild and stocked brown trout in Spanish rivers indicates different levels of introgression between basins. The absence of a clear geographical pattern in the introgression level suggests that ecological interactions and local stocking programmes may play an important role in stocking success. Finally, several guidelines are provided for conservation and management of native brown trout populations in Spanish rivers.     

Genetic structure of important resident brown trout breeding lines in Poland

The history of brown trout Salmo trutta L. stocking has long tradition in the European Union and other countries. Hundreds of hatchery facilities on continent have artificial broodstocks used for enhancement of neighbouring and also geographically far river basins. These practices have substantial effect on wild brown trout populations. To illuminate this phenomenon, eleven hatchery stocks and wild populations from northern Poland and Carpathian region were analysed using 13 microsatellite markers. Obtained results revealed high genetic diversity between studied stocks and clear differentiation between northern and southern populations and hybridization between these two major clads. As a recommendation, the principle of treating regions as metapopulations should be applied, which, in the case of Poland, means using the division of the northern and southern genetic lines that were revealed in the present study.     

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.     

Comparative analysis of introgression at three marker classes: a case study in a stocked population of brown trout

Comparative analysis of protein loci, microsatellite and mtDNA markers revealed generally comparable estimates for introgression and apparent admixture rates in stocked brown trout populations at two sites in the River Doubs (Rhône dainage, Switzerland), which are 10 km apart and which belong to the same management unit. At one site, a significant deviation between mtDNA and nuclear markers could be explained by stocking of F1 hybrids originating from crosses between hatchery females and males from the local population. Substantial differences between diagnostic protein loci and protein loci having non-fixed private alleles indicated that caution must be exercised when using genetic markers not strictly diagnostic for the distinction of the populations under investigation. Congruent estimates of introgression and apparent admixture rates between diagnostic protein loci and presumed diagnostic microsatellite loci suggest that the latter can be regarded as reliable genetic markers for the estimation of introgression in Mediterranean brown trout populations stocked with trout of Atlantic origin. Significant differences in introgression and apparent admixture rates between the two sites and between age-classes of one study site were observed. Introgression is suggested to depend on environmental factors. Significantly lower introgression rates in age-class 2+ years as compared to juvenile trout might further indicate that introduced Atlantic brown trout and hybrids decrease in proportion between age-classes 1+ and 2+ years.     

Reticulate evolution: ancient introgression of the Adriatic brown trout mtDNA in softmouth trout Salmo obtusirostris (Teleostei: Salmonidae)

Two populations of softmouth trout ( Salmo obtusirostris ) from the rivers Neretva (Bosnia and Herzegovina) and Jadro (Croatia), along with two neighbouring populations of brown trout ( Salmo trutta ) were analysed with a suite of genetic markers (two mtDNA genes, two nuclear genes, and nine microsatellites) as well as morphological characters. The Jadro softmouth trout were fixed for a brown trout mtDNA haplotype of the Adriatic lineage, which is 1.7% divergent from a previously described haplotype characteristic for the Neretva softmouth trout. All other genetic markers, as well as morphological analysis, supported the clear distinction of softmouth trout from the rivers Neretva and Jadro from brown trout in neighbouring populations, and thus a mtDNA capture event is assumed. Population specific microsatellite allele profiles, as well as a high number of private alleles for both populations of softmouth trout, support the hybridization between brown trout and the Jadro softmouth trout most likely being of ancient origin, thus leading to a reticulate evolutionary pattern of mtDNA in this taxon.  © 2007 The Linnean Society of London, Biological Journal of the Linnean Society , 2007, 90 , 139–152.     

Microsatellite DNA data point to extensive but incomplete admixture in a marble and brown trout hybridisation zone

Marble trout, endemic to the Adriatic drainage basin, is severely threatened by hybridisation with non-native brown trout. In the present study, we analysed 12 microsatellite DNA loci to assess genetic population structure and differentiation between sympatric phenotypic marble and brown trout at nine sampling sites in the upper Etsch/Adige River system. F _ST and AMOVA analyses revealed significant genetic differentiation between marble and brown trout samples. Thus, admixture between brown and marble trout appears to be incomplete. However, factorial correspondence analysis depicted marble trout, Atlantic brown trout and intermediate genotypes. Bayesian-based individual assignment tests identified indigenous marble trout at five sampling sites. In four other samples no ‘pure’ marble trout were detected. Bidirectional, first-generation hybridisation, involving both sexes of both parental species was observed. In locations where ‘pure’ marble trout still exist, post-F1 hybridisation appears to be directed towards brown trout. This has likely slowed the rate of hybridisation between the two trout species and the decline of relic marble trout populations. Based on these results, restoration management actions are proposed, such as the abandonment of brown trout stocking activities, sharper angling policies, establishment of indigenous marble trout breeding strains and the elaboration of a conservation priority list.     

Balancing selection on MHC class I in wild brown trout Salmo trutta

Evidence is reported for balancing selection acting on variation at major histocompatibility complex (MHC) in wild populations of brown trout Salmo trutta. First, variation at an MHC class I (satr-uba)-linked microsatellite locus (mhc1) is retained in small S. trutta populations isolated above waterfalls although variation is lost at neutral microsatellite markers. Second, populations across several catchments are less differentiated at mhc1 than at neutral markers, as predicted by theory. The population structure of these fish was also elucidated.     

Characterization of 13 microsatellites for Lahontan cutthroat trout (Oncorhynchus clarki henshawi) and cross-amplification in six other salmonids

Thirteen newly developed tri- and tetranucleotide repeat microsatellite markers were developed for Lahontan cutthroat trout (Oncorhynchus clarki henshawi), a threatened subspecies endemic to the Lahontan hydrographic basin in the western USA. These loci are highly polymorphic with five to 30 alleles per locus and observed heterozygosities ranging from 0.4 to 0.7. Cross-species amplification of these markers was most successful in the closely related rainbow trout, Oncorhynchus mykiss, with only three loci amplifying in brown trout, Salmo trutta. Nonoverlapping allelic distributions for many of these loci among the six salmonid species screened suggest these markers may be useful for hybrid determination.     

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.     

Phylogeography,genetic structure,and conservation of the endangered Caspian brown trout, <Emphasis Type="Italic">Salmo trutta caspius</Emphasis> (Kessler, 1877), from Iran

The Caspian Sea, the largest inland closed water body in the world, has numerous endemic species. The Caspian brown trout (Salmo trutta caspius) is considered as endangered according to IUCN criteria. Information on phylogeography and genetic structure is crucial for appropriate management of genetic resources. In spite of the huge number of studies carried out in the Salmo trutta species complex across its distribution range, very few data are available on these issues for S. trutta within the Caspian Sea. Mitochondrial (mtDNA control region) and nuclear (major ribosomal DNA internal transcribed spacer 1, ITS-1, and ten microsatellite loci) molecular markers were used to study the phylogeography, genetic structure, and current captive breeding strategies for reinforcement of Caspian trout in North Iranian rivers. Our results confirmed the presence of Salmo trutta caspius in this region. Phylogenetic analysis demonstrated its membership to the brown trout Danubian (DA) lineage. Genetic diversity of Caspian brown trout in Iranian Rivers is comparable to the levels usually observed in sustainable anadromous European brown trout populations. Microsatellite data suggested two main clusters connected by gene flow among river basins likely by anadromous fish. No genetic differences were detected between the hatchery sample and the remaining wild populations. While the current hatchery program has not produced detectable genetic changes in the wild populations, conservation strategies prioritizing habitat improvement and recovering natural spawning areas for enhancing wild populations are emphasized.