Genetic structure of brown trout, salmo trutta l., at the southern limit of the distribution range of the anadromous form

Genetic variation at 33 protein loci was investigated in 41 wild brown trout populations from four river basins in Galicia (northwest Spain) to analyse the amount and distribution of genetic diversity in a marginal area, located in the distribution limit of the anadromous form of this species. The genetic diversity detected within populations (H between 0 and 6%) lies within the range quoted for this species in previous reports. The Mino, the most southern river basin analysed, showed a significantly lower genetic diversity and the highest genetic differentiation among the river basins studied. The hierarchical gene diversity analysis showed high population differentiation in a restricted area (GST = 27%), mostly due to differences among populations within basins (GSC = 22%). The reduction of GST observed when the isolated samples were excluded from the analysis (GST = 17%) showed the importance of habitat fragmentation on the heterogeneity detected. Gene flow among populations was comparatively evaluated by three indirect methods, which in general revealed low figures of absolute number of migrants per generation, slightly higher than 1. The gene flow among basins reflected a positive relationship with geographical distance. This trend was confirmed by the significant correlation observed between geographical and genetic distances, including all population pairs, which suggests a component of isolation by distance in brown trout genetic structure. Nevertheless, the nonsignificant intrabasin correlation demonstrates the complexity of genetic relationships among populations in this species. The model of genetic structure in brown trout is discussed in the light of the results obtained.     

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.     

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.     

Life history and genetic characterisation of sea trout Salmo trutta in the Adriatic Sea

1. The brown trout Salmo trutta is characterised by both anadromous (sea trout) and resident populations, naturally occurring in Atlantic and Ponto-Caspian rivers. Sea trout are currently considered absent from rivers of the Mediterranean area, probably because of the non-optimal chemical–physical characteristics of the Mediterranean Sea. However, the occasional bycatch of smoltified S. trutta in the Adriatic Sea is well known among fishermen and the biological explanation of this phenomenon is still controversial. The aim of this study was to compare the genetic diversity of freshwater and marine brown trout to try to understand the factors underlying the presence of putative anadromous brown trout in the Adriatic Sea.
2. In this study, we analysed the genetic diversity of: (1) wild brown trout collected from the Esino River (central Italy); (2) a domestic strain of brown trout used for stocking the study area; and (3) a sample of Adriatic sea trout collected near the outlet of the Esino River. Together with genetic analysis, we carried out scale analysis in order to track the freshwater/marine stages of the life cycle in the sea trout samples. The genetic characterisation was carried out by polymerase chain reaction–restriction fragment length polymorphism analysis of the mtDNA fragment ND-5/6 and the nuclear locus LDH-C1* and by genotyping 15 microsatellite loci. The genetic polymorphism obtained was used to investigate intra- and inter-population genetic diversity, rates of genetic introgression between wild and domestic samples and the origin of sea trout specimens by using assignment tests.
3. Our genetic analyses demonstrated that the sea trout analysed in this study are from the domestic strain of Atlantic origin used in central Italy for stocking activities. The level of genetic introgression between native and domestic samples is high in the Esino River. The populations more resilient to introgressive hybridisation appeared to be those living in the portion of the river network dominated by carbonate rocks. Assignment tests (GeneClass) suggest the existence of a link between stocking efforts and the freshwater origin of the sea trout. In addition, data obtained from the analysis of scales, size measurement, and sex determination showed a pattern of smolt age, size, and sex ratio very similar to those observed in other anadromous populations.
4. In conclusion, the present study highlighted that sea trout from the central Adriatic Sea originated from brown trout of Atlantic origin inhabiting the Esino River. Their seaward migratory behaviour could represent a consequence of an active migration instead of a passive displacement by water flow. Our results also showed that traditional stocking practices represent a negative activity for the conservation of the last Mediterranean native S. trutta populations.

     

Extreme genetic differentiation among the remnant populations of marble trout (Salmo marmoratus) in Slovenia

Populations of the marble trout (Salmo marmoratus) have declined critically due to introgression by brown trout (Salmo trutta) strains. In order to define strategies for long-term conservation, we examined the genetic structure of the 8 known pure populations using 15 microsatellite loci. The analyses reveal extraordinarily strong genetic differentiation among populations separated by < 15 km, and extremely low levels of intrapopulation genetic variability. As natural recolonization seems highly unlikely, appropriate management and conservation strategies should comprise the reintroduction of pure populations from mixed stocks (translocation) to avoid further loss of genetic diversity.     

Genetic differences among brown trout, Salmo trutta, stocks and their importance for the conservation and management of the species

SUMMARY. 1. Review of published studies on genetic variation, as shown by electrophoretic studies of protein variation, in natural brown trout ( Salmo trutta L.) populations from Britain and Ireland, Finland, France, Greece, Iceland, Norway, Sweden, U.S.A. and U.S.S.R., revealed abundant geographical variation in gene frequency with individual populations containing only a limited part of the gene diversity of the species.
2. Thirty-eight (54%) of the seventy gene loci examined have been found to be polymorphic in the species with an average population showing polymorphism at 16% of its loci (range 0-34.8%).
3. The brown trout is naturally subdivided into a large number of reproductively isolated and genetically distinct populations within, as well as among, drainages.
4. Two independent post-glacial colonizations, by genetically distinct races, followed by independent evolution in separate drainages over the past 13,000 years is seen as responsible for the genetic diversity of brown trout in north-western Europe.
5. Many genetically unique populations have been lost in the past 100 years and there is an urgent need to identify and conserve the remaining genetic diversity. Genetically unique populations are an irreplaceable resource for rational management in relation to angling and future aquaculture potential.     

Demographic Genetics of Brown Trout (Salmo Trutta) and Estimation of Effective Population Size from Temporal Change of Allele Frequencies

We studied temporal allele frequency shifts over 15 years and estimated the genetically effective size of four natural populations of brown trout (Salmo trutta L.) on the basis of the variation at 14 polymorphic allozyme loci. The allele frequency differences between consecutive cohorts were significant in all four populations. There were no indications of natural selection, and we conclude that random genetic drift is the most likely cause of temporal allele frequency shifts at the loci examined. Effective population sizes were estimated from observed allele frequency shifts among cohorts, taking into consideration the demographic characteristics of each population. The estimated effective sizes of the four populations range from 52 to 480 individuals, and we conclude that the effective size of natural brown trout populations may differ considerably among lakes that are similar in size and other apparent characteristics. In spite of their different effective sizes all four populations have similar levels of genetic variation (average heterozygosity) indicating that excessive loss of genetic variability has been retarded, most likely because of gene flow among neighboring populations.     

Phylogeography of the Turkish brown trout Salmo trutta L.: mitochondrial DNA PCR‐RFLP variation

In the present study, mitochondrial DNA polymerase chain reaction‐restriction fragment length polymorphism (PCR‐RFLP) assay was used to assess the phylogenetic and phylogeographic relationships among 27 brown trout Salmo trutta populations from Turkey. The complete NADH 5/6 region and a second segment comprising the cytochrome b gene and D‐loop of mtDNA amplified by PCR were digested with six and five restriction enzymes, respectively. A total of 27 haplotypes were observed and divided into three major phylogenetic assemblages, namely Danubian (DA), Adriatic (AD) and a newly proposed Tigris (TI) lineage. The timing of the net nucleotide divergence between the major lineages along with the geological history of Turkey suggested pre‐Pleistocene isolation of the Turkish brown trout and provided evidence that Turkey could be considered as a centre of diversification for these lineages. The average haplotype diversity (0·1397) and the nucleotide diversity (0·000416) within populations were low in comparison to the observed interpopulation nucleotide diversity (0·021266). PCR‐RFLP analysis showed that most of the mtDNA sequence variation found in the Turkish brown trout populations was imputable to differences among lineages. On the other hand, there was also an obvious relationship between geographical distribution of the populations and their clustering. The present study showed that brown trout populations from Turkey are highly divergent and mainly have a unique genetic profile that could be used for conservation and management purposes.     

Selection and genetic drift in captive versus wild populations: an assessment of neutral and adaptive (MHC-linked) genetic variation in wild and hatchery brown trout (<Emphasis Type="Italic">Salmo trutta</Emphasis>) populations

Captive bred individuals are often released into natural environments to supplement resident populations. Captive bred salmonid fishes often exhibit lower survival rates than their wild brethren and stocking measures may have a negative influence on the overall fitness of natural populations. Stocked fish often stem from a different evolutionary lineage than the resident population and thus may be maladapted for life in the wild, but this phenomenon has also been linked to genetic changes that occur in captivity. In addition to overall loss of genetic diversity via captive breeding, adaptation to captivity has become a major concern. Altered selection pressure in captivity may favour alleles at adaptive loci like the Major Histocompatibility Complex (MHC) that are maladaptive in natural environments. We investigated neutral and MHC-linked genetic variation in three autochthonous and three hatchery populations of Austrian brown trout (Salmo trutta). We confirm a positive selection pressure acting on the MHC II β locus, whereby the signal for positive selection was stronger in hatchery versus wild populations. Additionally, diversity at the MHC II β locus was higher, and more uniform among hatchery samples compared to wild populations, despite equal levels of diversity at neutral loci. We postulate that this stems from a combination of stronger genetic drift and a weakening of positive selection at this locus in wild populations that already have well adapted alleles for their specific environments.     

Using a reference population yardstick to calibrate and compare genetic diversity reported in different studies: an example from the brown bear

In species with large geographic ranges, genetic diversity of different populations may be well studied, but differences in loci and sample sizes can make the results of different studies difficult to compare. Yet, such comparisons are important for assessing the status of populations of conservation concern. We propose a simple approach of using a single well-studied reference population as a ‘yardstick'' to calibrate results of different studies to the same scale, enabling comparisons. We use a well-studied large carnivore, the brown bear (Ursus arctos), as a case study to demonstrate the approach. As a reference population, we genotyped 513 brown bears from Slovenia using 20 polymorphic microsatellite loci. We used this data set to calibrate and compare heterozygosity and allelic richness for 30 brown bear populations from 10 different studies across the global distribution of the species. The simplicity of the reference population approach makes it useful for other species, enabling comparisons of genetic diversity estimates between previously incompatible studies and improving our understanding of how genetic diversity is distributed throughout a species range.     

Electrophoretic variation in six populations of brown trout (Salmo trutta L.)

Starch gel electrophoretic studies of 16 enzymes encoded by 34 Loci were performed on six brown trout populations. One new polymorphism is described at the Pmi-2 locus. Breeding data were analysed for both single and joint segregation of six loci: Aat-1, Cpk-1, G3p-2, Mdh-2, Mdh-3, and Pmi-2. All the loci are shown to segregate in simple mendelian ratios and one nonrandom joint segregation was observed. The polymorphism level, heterozygosities, and genetic distances were estimated and compared with those reported in other studies on brown trout and closely related salmonid species. The polymorphism level (25%) and average heterozygosity (9%) were high. Significant genetic distances were observed, but the average degree of differentiation between populations appeared to be small (9% of the total heterozygosity).     

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.     

Comparing RADseq and microsatellites for estimating genetic diversity and relatedness — Implications for brown trout conservation

The conservation and management of endangered species requires information on their genetic diversity, relatedness and population structure. The main genetic markers applied for these questions are microsatellites and single nucleotide polymorphisms (SNPs), the latter of which remain the more resource demanding approach in most cases. Here, we compare the performance of two approaches, SNPs obtained by restriction‐site‐associated DNA sequencing (RADseq) and 16 DNA microsatellite loci, for estimating genetic diversity, relatedness and genetic differentiation of three, small, geographically close wild brown trout (Salmo trutta) populations and a regionally used hatchery strain. The genetic differentiation, quantified as F_ST, was similar when measured using 16 microsatellites and 4,876 SNPs. Based on both marker types, each brown trout population represented a distinct gene pool with a low level of interbreeding. Analysis of SNPs identified half‐ and full‐siblings with a higher probability than the analysis based on microsatellites, and SNPs outperformed microsatellites in estimating individual‐level multilocus heterozygosity. Overall, the results indicated that moderately polymorphic microsatellites and SNPs from RADseq agreed on estimates of population genetic structure in moderately diverged, small populations, but RADseq outperformed microsatellites for applications that required individual‐level genotype information, such as quantifying relatedness and individual‐level heterozygosity. The results can be applied to other small populations with low or moderate levels of genetic diversity.     

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.     

Long-term temporal changes of genetic composition in brown trout (Salmo trutta L.) populations inhabiting an unstable environment

The genetic structure of brown trout (Salmo trutta) populations inhabiting rivers on the island of Bornholm in the Baltic Sea was studied on a spatial and temporal scale. Low water levels in the rivers during the summer period are assumed to have a significant impact on the persistence of local populations, possibly resulting in a metapopulation structure. Extinctions may, however, also be buffered by a remnant strategy, whereby juveniles escape to river outlets during periods of drought. We compared polymorphism at seven microsatellite DNA loci in contemporary and past samples collected from 1944 to 1997. A principal component analysis, a hierarchical gene diversity analysis and assignment tests showed that the genetic composition of populations was not temporally stable, and that temporal genetic differentiation was much stronger than spatial differentiation. Genetic variability was high and stable over time. Effective population sizes (Ne) and migration rate (m) were estimated using a maximum-likelihood-based implementation of the temporal method. Ne estimates were low (ranging from 8.3 to 22.9) and estimates of m were high (between 0.23 and 0.99), in contrast to other Danish trout populations inhabiting larger and more environmentally stable rivers (Ne between 39.2 and 289.9 and m between 0.01 and 0.09). Thus, the observed spatio-temporal patterns of genetic differentiation can be explained by drift in small persisting populations, where levels of genetic variation are maintained by strong gene flow. However, observations of rivers devoid of trout suggested that population turnover also takes place. We suggest that Bornholm trout represent a metapopulation where the genetic structure primarily reflects strong drift and gene flow, combined with occasional extinction-recolonization events.     

Genetic structure of brown trout (<Emphasis Type="Italic">Salmo trutta</Emphasis> L.) from the Hardangervidda mountain plateau (Norway) analyzed by microsatellite DNA: a basis for conservation guidelines

The Hardangervidda in southern Norway, the largest mountain plateau in Europe, has thousands of lakes and streams, mainly between 1000 and 1300 m above sea level, where brown trout is the only fish species. To describe the current genetic diversity of brown trout in this area, a total of 863 fish from 20 lakes were genotyped with eleven microsatellites. Most diversity is within lake populations, but diversity among geographical groups and populations within groups was significant, too. Neighbor-joining, principle coordinate analysis and Bayesian clustering show three major geographic groups in accordance with the river systems. Bias was caused by recent stocking in two lakes. Low/no genetic differentiation among some populations indicates that intermixing is common when lakes are well-connected, as was also shown by assignment test. We recommend preserving the genetic diversity of brown trout in this unique area by managing stocking in lake systems according to genetic structure.     

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.     

A low-density single nucleotide polymorphism panel for brown trout (Salmo trutta L.) suitable for exploring genetic diversity at a range of spatial scales

The rivers of southern England and northern France which drain into the English Channel contain several genetically unique groups of trout (Salmo trutta L.) that have suffered dramatic declines in numbers over the past 40 years. Knowledge of levels and patterns of genetic diversity is essential for effective management of these vulnerable populations. Using restriction site-associated DNA sequencing (RADseq) data, we describe the development and characterisation of a panel of 95 single nucleotide polymorphism (SNP) loci for trout from this region and investigate their applicability and variability in both target (i.e., southern English) and non-target trout populations from northern Britain and Ireland. In addition, we present three case studies which demonstrate the utility and resolution of these genetic markers at three levels of spatial separation:(a) between closely related populations in nearby rivers, (b) within a catchment and (c) when determining parentage and familial relationships between fish sampled from a single site, using both empirical and simulated data. The SNP loci will be useful for population genetic and assignment studies on brown trout within the UK and beyond.     

Phylogeographical lineages in brown trout (Salmo trutta): investigating microgeographical differentiation between native populations from Northern Spain

SUMMARY 1. The large microgeographical differentiation revealed by allozyme studies in brown trout ( Salmo trutta) populations is one of the most striking features of this species. Additionally, allozymes showed great genetic differences between Atlantic and Mediterranean populations on a macrogeographical scale.
2. This study was carried out in order to assess whether the great differences observed between Atlantic and Mediterranean populations persisted where the two are geographically close (the 'microgeographical scale'). Sixteen populations of brown trout, S. trutta , were screened for genetic variation at 25 allozyme loci. The sampling sites, which occupied a relatively small geographical area, were distributed across Cantabrian (Atlantic) and Mediterranean drainages in Northern Spain.
3. The neighbour-joining tree, inferred from Nei's genetic distance, showed that brown trout populations clustered into two different groups. These groups corresponded to the Cantabrian and the Mediterranean groups of populations, although no clear geographical pattern emerged within each of the groups. This geographical pattern is basically caused by significant differences in the frequency distribution of the CK-A1 * locus, with a higher frequency of * 115 in Cantabrian samples (0.586 ± 0.091) while allele * 100 was more frequent in Mediterranean samples (0.931 ± 0.038). In addition, this study revealed alleles exclusive to the Mediterranean and Cantabrian populations, agreeing with previous findings.
4. Genetic differentiation between Cantabrian and Mediterranean regions (14.19%) was similar to that estimated in Spain at a larger scale (13%), showing that most of the differences between the regions can be observed even in a small geographical area.     

Complex genetic diversity patterns of cryptic,sympatric brown trout (<Emphasis Type="Italic">Salmo trutta</Emphasis>) populations in tiny mountain lakes

Intraspecific genetic variation can have similar effects as species diversity on ecosystem function; understanding such variation is important, particularly for ecological key species. The brown trout plays central roles in many northern freshwater ecosystems, and several cases of sympatric brown trout populations have been detected in freshwater lakes based on apparent morphological differences. In some rare cases, sympatric, genetically distinct populations lacking visible phenotypic differences have been detected based on genetic data alone. Detecting such “cryptic” sympatric populations without prior grouping of individuals based on phenotypic characteristics is more difficult statistically, though. The aim of the present study is to delineate the spatial connectivity of two cryptic, sympatric genetic clusters of brown trout discovered in two interconnected, tiny subarctic Swedish lakes. The structures were detected using allozyme markers, and have been monitored over time. Here, we confirm their existence for almost three decades and report that these cryptic, sympatric populations exhibit very different connectivity patterns to brown trout of nearby lakes. One of the clusters is relatively isolated while the other one shows high genetic similarity to downstream populations. There are indications of different spawning sites as reflected in genetic structuring among parr from different creeks. We used >3000 SNPs on a subsample and find that the SNPs largely confirm the allozyme pattern but give considerably lower F _ST values, and potentially indicate further structuring within populations. This type of complex genetic substructuring over microgeographical scales might be more common than anticipated and needs to be considered in conservation management.