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.     

Stock Transfers in Spanish Brown Trout Populations: A Long-term Assessment

Synopsis Stocking of fish from other populations has been commonly employed for enhancement of wild brown trout, Salmo trutta, populations in north Spain. Young hatchery reared brown trout of central European origin were introduced into some Asturian rivers every year since 1984. Based on variation at the isozyme locus LDH-C1* and at the microsatellite locus BFRO 002, two genetic markers race-specific in Salmo trutta, we detected introgression of foreign genomes into native gene pools in some Spanish trout populations where only pure native individuals were present 10 years ago. We strongly suggest development of alternative management policies for conservation of Spanish natural brown trout populations without endangering the traditional recreational fisheries. Jorge I. Izquierdo, Ana G. F. Castillo: These two authors contributed equally to the article.     

Genetic structure and demographic history of brown trout (Salmo trutta) populations from the southern Balkans

1. The present study was designed to characterize the genetic structure of brown trout ( Salmo trutta ) populations from the southern Balkans and to assess the spread of non-native strains and their introgression into native trout gene pools. We analysed polymorphism at nine microsatellite loci in seven supposedly non-admixed and three stocked brown trout populations.
2. The analyses confirmed the absence of immigration and extraordinarily strong genetic differentiation among the seven non-introgressed populations in parallel with low levels of intrapopulation genetic variability. In contrast, analyses of the stocked populations revealed that the genetic integrity of the local populations had been substantially changed, and the populations must be characterized as hybrid swarms. The pattern of population differentiation observed at microsatellites contrasted to that depicted previously by mtDNA variation. However, the close relationships between populations from the Danube and Axios river systems proposed solely by microsatellites could be explained by palaeogeographic events.
3. Our research showed that most of the populations examined represent unique gene pools, whose existence is critically compromised. Therefore, appropriate management and conservation strategies should be developed urgently in order to protect the subspecific biodiversity and to reverse currently negative trends.     

Evidence of a genetically distinct population of Vrljika softmouth trout Salmo obtusirostris Heckel evolved by vicariance

Mitochondrial DNA haplotypes (control region, partial cytochrome b and ATPase6 genes) indicate a sister relationship between Vrljika and Neretva softmouth (Adriatic) trout Salmo obtusirostris . This relationship was supported by a tree of individuals based on microsatellite results [allele sharing distances ( D _AS)], which revealed three distinctive clusters, corresponding to Jadro softmouth, Neretva brown trout Salmo trutta and Neretva softmouth trout. Within the latter taxon, Vrljika trout are clearly separated from other trout. The genetic results contradict the synonymy of Jadro with Vrljika softmouth trout, as recently proposed in the Red Book of Freshwater Fish in Croatia. Vrljika softmouth trout appear to have originated from a vicariance that split a common ancestor into large (Neretva) and small (Vrljika) fragmented populations 135 000–270 000 years ago. Vrljika softmouth trout can be distinguished by an array of derived phenotypic and molecular character states. For conservation, this population should be recognized formally at the same taxonomic level as the other geographically separated populations of softmouth trout.     

Patterns of genetic structuring in a brown trout (<Emphasis Type="Italic">Salmo trutta</Emphasis> L.) metapopulation

One main challenge in conservation biology is to preserve genetic variability and adaptive variation within and among populations. However, constant anthropogenic habitat modifications have severe effects on the evolutionary dynamics shaping wild populations and pose a serious threat to the natural evolution of biodiversity. The aim of the present study was to unravel the genetic structuring of brown trout (Salmo trutta) populations in the largest freshwater catchment in Ireland, whose habitats have experienced major human-mediated changes over at least two centuries. A total of 419 juvenile fish were sampled from nine main rivers in the Corrib catchment and were genotyped using 12 microsatellites. Both Bayesian clustering and F _ST-based analyses of genetic variance sorted these populations into five main genetically distinct groups, characterized by different extent of genetic differentiation among populations. These groups were also characterized by some degree of admixture, which can be partly explained by recent gene flow. Overall, the study suggests that the Corrib trout may conform to a metapopulation model with local populations that show different degrees of isolation and are interconnected by various level of gene flow. Results add further insights into metapopulation evolutionary dynamics and provide a useful basis to implement appropriate conservation strategies.     

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     

Plitvice Lakes National park harbors ancient,yet endangered diversity of trout (genus Salmo)

National park Plitvice Lakes is the oldest, the largest and the most visited national park in Croatia, well known for its stunning karstic watersheds that supply Plitvice Lakes and exceptionally valuable biodiversity. Lack of knowledge on the taxonomic status, population structure and viability of trout in the Plitvice Lakes National park, as well as the possibility of existence of cryptic diversity, motivated this study which aimed to determine: taxonomic status and phylogenetic relationship of Plitvice Lakes trout with other Salmo species, genetic structure and diversity of their populations, as well as future viability of populations. 150 samples of trout were obtained from 17 localities. Two mitochondrial gene markers, gene for cytochrome b and mitochondrial control region were used in phylogenetic reconstruction, as well as in population genetic investigation. Intraspecific and intrapopulation genetic diversity was described by calculating several measures of genetic polymorphism, while gene flow among populations was estimated as the migration rates and as the number of immigrants per generation. Phylogenetic trees revealed that Plitvice Lakes watershed is inhabited by two evolutionary independent units of the genus Salmo, representing two species, Salmo labrax Pallas, 1814 and Salmo trutta Linnaeus, 1758. Possibly ‘pure’ populations of both species were found alongside several admixed ones in which both species are present. Our data has shown low intraspecific and intrapopulational diversities for Plitvice Lakes trout and restricted gene flow. Small effective sizes of populations and weak interconnections between them, which are most likely a consequence of fragmentation and habitat degradation, accentuating the need for urgent conservation measures.     

Alternative mating strategies in Atlantic salmon and brown trout

By screening variable number of tandem repeat (VNTR) loci, multiple paternity within clutches has been found in wild populations of southern European Atlantic salmon (Salmo salar) and brown trout (Salmo trutta). For Atlantic salmon, we determined the relative contribution of alternative male phenotypes to the next generation. Individual males that are morphologically juvenile yet sexually mature fertilized a large proportion of eggs, and they thereby contributed to an increase of genetic variability in wild populations via (1) balancing the sex ratio, (2) increasing outbreeding, and (3) enlarging the effective population size, in part a consequence of (1) and (2). In addition, these precocious males ensured that interspecific spawns involving Atlantic salmon females and brown trout males (a fairly common occurrence in southern Europe where the two species are sympatric) resulted mostly in Atlantic salmon progeny. For brown trout, preliminary genetic results indicated that multiple paternity, when present, was not due to alternative mating strategies by males, but rather to successive fertilizations by adult suitors.     

The selection of the ‘wild’: A combined molecular approach for the identification of pure indigenous fish from hybridised populations

The identification of pure indigenous fish from hybridised populations represents a key issue in fisheries management and conservation biology. In the present study an approach for selection of purebred marble trout (Salmo trutta marmoratus C.) individuals out of admixed populations was set up and assessed. In a first step, baseline data sets of pure marble trout and pure brown trout specimens based on twelve microsatellite loci were used to simulate five consecutive generations of admixture. The baseline and the resulting simulation data sets were then combined with data of a ‘real’ hybridised marble trout population to perform a single individual assignment test as implemented in STRUCTURE. By this procedure the assignment approach was calibrated and it was possible to compare admixture coefficients obtained for individuals from different populations. The ranking of individual admixture coefficients on a plot and comparison with simulated data revealed that the test population was composed of pure marble trout individuals, first generation hybrids between marble trout and brown trout, and hybrid backcross specimens between both groups. However, by defining a critical q-value of 0.1 and additionally integrating individual sequence data of the mtDNA control region, it was possible to indicate individuals, which could be selected for the establishment of a pure marble trout strain.     

Genetic diversity of trout (genus Salmo) from its most eastern native range based on mitochondrial DNA and nuclear gene variation

Russia and western Asia harbour trout populations that have been classified as distinct species and subspecies, most often on the basis of morphological and ecological variation. In order to assess their origins and to verify whether traditional taxonomy reflects their evolutionary distinctiveness, we documented their genetic relationships on the basis of mitochondrial DNA (mtDNA) RFLP, mtDNA sequence analysis, and allozyme variation. Both mtDNA and nuclear gene variation defined two ancient phylogenetic assemblages of populations distributed among northern (Baltic, White, Barents), and southern (Black, Caspian, Aral) sea basins, between which gene flow has been possible but limited in postglacial times. These results supported the traditional taxonomic differentiation between populations of these two regions. They provided weak support for the taxonomic distinction of southern brown trout (Salmo trutta) populations based on their basin of origin. They also refuted the hypothesis that L. Sevan trout (Salmo ischchan) diverged from a primitive brown trout ancestor. Nevertheless, all trout populations from southern sea basins possessed private alleles or mtDNA genotypes and were genetically distinct Therefore, they represent unique gene pools that warrant individual recognition for conservation and management.     

Sixty years of anthropogenic pressure: a spatio-temporal genetic analysis of brown trout populations subject to stocking and population declines

Analyses of historical samples can provide invaluable information on changes to the genetic composition of natural populations resulting from human activities. Here, we analyse 21 microsatellite loci in historical (archived scales from 1927 to 1956) and contemporary samples of brown trout ( Salmo trutta ) from six neighbouring rivers in Denmark, to compare the genetic structure of wild populations before and after population declines and stocking with nonlocal strains of hatchery trout. We show that all populations have been strongly affected by stocking, with admixture proportions ranging from 14 to 64%. Historical population genetic structure was characterized by isolation by distance and by positive correlations between historical effective population sizes and habitat area within river systems. Contemporary population genetic structure still showed isolation by distance, but also reflected differences among populations in hatchery trout admixture proportions. Despite significant changes to the genetic composition within populations over time, dispersal rates among populations were roughly similar before and after stocking. We also assessed whether population declines or introgression by hatchery strain trout should be the most significant conservation concern in this system. Based on theoretical considerations, we argue that population declines have had limited negative effects for the persistence of adaptive variation, but admixture with hatchery trout may have resulted in reduced local adaptation. Collectively, our study demonstrates the usefulness of analysing historical samples for identifying the most important consequences of human activities on the genetic structure of wild populations.     

Complex evolutionary history in the brown trout: Insights on the recognition of conservation units

We screened genetic variation in a polytypicorganism, whose populations are oftendistributed into numerous isolated habitats,and integrated the results into a critique ofdefining ``units' of conservation for organismswith highly fragmented populations. Sixteenpopulations of brown trout Salmo truttaL. across 8 Portuguese river basins werescreened for variation at 5 loci (mtDNA andallozymes). Population history based on mtDNArevealed a mosaic pattern driven by pastfragmentation and restricted gene flow withlittle correspondence to major river drainagesor recently proposed OCUs on the IberianPeninsula. Such patterns of variation offer achallenge to conservation strategies that basethemselves on defining units of conservation,particularly if such units intend to reflect ahierarchical evolutionary structure. Wesuggest that geographically mosaic patterns ofevolutionary lineages, as well as adaptivelysignificant traits are common characteristicsof many freshwater organisms. Thus,large-scale units, even if diagnosed by mtDNAclades, are often too heterogeneous to considera ``unit' of conservation. Alternatively, abottom-up perspective that prioritizespopulations or metapopulations is both morepractical and more effective in recognizing andpreserving evolutionary diversity.     

Genetic divergence among native trout Salmo trutta populations from southern Balkans based on mitochondrial DNA and microsatellite variation

The genetic structure and the phylogenetic relationships among five Balkan populations of trout Salmo trutta that have been classified earlier into five different taxa were studied, using microsatellite and mitochondrial DNA (mtDNA) analyses. The pattern of population differentiation observed at microsatellites differed to that depicted by mtDNA variation, yet both methods indicated a very strong partitioning of the genetic variation among sampling locations. Results thus suggest that conservation strategies should be directed towards preserving the genetic integrity and uniqueness of each population.     

Genetic diversity among Balkan trout populations based on RAPD analysis

Random amplified polymorphic DNA (RAPD) markers were used to estimate the population structure and phylogenetic relationships among samples of the Salmo trutta complex that inhabit the Balkan Peninsula. Five random oligodecamers were selected to amplify DNA from 140 fish from seven populations. Using these primers, 55 discernible DNA fragments were generated, of which 50 (90.91%) were polymorphic. The statistical results indicated that there was low genetic diversity within populations (with an average percentage of polymorphic bands (P) of 11.69% and a Nei's genetic diversity index (h) of 0.035), but at the same time high genetic differentiation among populations (F(ST) = 0.89). The distribution of genetic diversity among Balkan trout may result from their evolutionary history and reflects genetic drift coupled with bottleneck phenomena. Overall, RAPDs proved valuable tools for quick and reliable stock discrimination and provided information that might be useful regarding conservation and management of trout.     

Effects of sea trout stocking on the population genetics of landlocked brown trout, Salmo trutta L., in the Conwy River system, North Wales, U.K.

The effect of the introduction of fry of anadromous sea trout, Salmo trutta L., on the genetic integrity of landlocked brown trout populations was evaluated. Samples were taken from six brown trout populations from streams above impassable waterfalls in the Conwy river system (North Wales, U.K.) in 1989 and 1990. Three of these streams had no known stocking history and three had been stocked with sea trout fry from the lower Conwy system over the last few years. Representatives of these sea trout were collected from two streams in the lower Conwy system and from a hatchery. Allele frequencies at 13 loci, six of which were polymorphic, were determined by starch gel electrophoresis.
The stocked populations were intermediate in their allele frequencies between unstocked brown trout and sea trout samples. A principal component analysis suggested significant numbers of hybrids in all of the stocked streams. This shows that some of the introduced sea trout did not migrate down the falls to the sea, but stayed in fresh water and hybridized with the local population. The significance of this finding for the conservation of the genetic resource of brown trout stocks is discussed.     

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.     

Genetic structure of two Turkish brown trout populations

The genetic structure of two brown trout Salmo trutta populations living in Lake Abant in Bolu and Üzüm River in Antalya was determined by examining 15 enzyme coding loci ( AAT, ADH, LDH, MDH, MEP, GPI, PGM and SOD ) using starch gel electrophoresis. Population specific mobilities were observed for the fixed alleles of LDH-B2, mMEP-2 and SOD-1 loci. Polymorphisms in sAA T-4, GPI-B2 loci were observed within the populations. Average heterozygosity of Abant and Antalya populations was 0.0358 and 0.0224 respectively. For LDH-C which is the post glaciation marker locus, the ancestral allele * 105 was found to be fixed in both of the populations. Nei's genetic distance between the two populations was 0.2507 which is the level of genetic distance often found between different species. This difference seems to be due to the presence of unique alleles in the LDH-B2, mMEP-2 and SOD-1 loci of the Abant population, indicating that the conservation of the Abant population and its heterozygosity is of prime importance.     

Structure and genetic diversity of small populations of brown trout Salmo trutta in Kandalaksha Bay,the White Sea

The phenetic variability (types of life strategy, age and sex structure of population, biological peculiarities), the anthropogenic pressure, and the genetic diversity were studied by five microsatellite loci for several minor populations of brown trout Salmo trutta inhabiting the waterbodies of Velikaya Salma Strait watershed area (Kandalaksha Bay, the White Sea). The level of genetic and phenetic diversity in small populations was comparable to those representing large water systems. Significant spatial dissimilarity of the populations located in less than 15-km distance was found. Low allelic and genetic diversity was observed for the studied populations. Variability of some studied loci through the time period was observed for the fish sampled from different populations. We assume that minor populations of brown trout may be treated as the standards of native populations for the monitoring activities on the brown trout populations and its environment both in short-term and long-term perspective.     

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.     

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.