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     

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.     

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.     

Microsatellite DNA Polymorphism in Intensely Enhanced Populations of Sea Trout (Salmo trutta) in the Southern Baltic

Sea trout (Salmo trutta) is an anadromous form of brown trout, a commercially important salmonid species in Europe. Stocking has been used to compensate for the decrease of natural populations and maintenance of fishery and angling catches. Over 1.5 million smolts and 4.5 million alevins are released to Polish coastal rivers each year. Variation at 7 microsatellite loci (Ssa197, Ssa171, Ssa85, Str15, Str73, Str591, and Str543) was used to study genetic polymorphism of spawners returning to 6 rivers. Application of distance method for comparison of pairs of populations based on number of different alleles (F _ST) revealed significant differences between Vistula and Wieprza, and Parseta as well as between Drweca and Wieprza, and Slupia. The level of heterozygosity was similar between most of the studied sea trout populations; considerable differences were found only for Str591. Differences in frequencies of a few alleles between populations were observed. An exact test of sample differentiation based on allele frequencies confirmed lack of significance of differentiation between the 6 pairs of populations (F _ST and R _ST). No admixture was observed in the studied populations. Possible effects of stocking on the genetic polymorphism of the sea trout populations in wild with implications for biotechnology are discussed.     

Estimation of introgression in cutthroat trout populations using microsatellites

Introgressive hybridization, mediated by anthropogenic activity, poses a threat to numerous and diverse taxa. The management of introgressed individuals or populations within species of conservation concern is currently the subject of scientific and political debate. We investigate the utility of 10 non-diagnostic microsatellite loci for investigating admixture from introduced Yellowstone cutthroat trout (Oncorhynchus clarkii bouvieri) and rainbow trout (O. mykiss) within 25 putative Rio Grande cutthroat trout (O. c. virginalis) populations. We apply five different approaches (correspondence analysis, maximum-likelihood assignment tests, an admixture estimator based on allele frequencies, an admixture estimator based on coalescent theory and an admixture estimator implementing a Bayesian method) and use two alternative O. c. virginalis reference samples. All approaches were capable of identifying one population that consisted entirely of introduced O. c. bouvieri, and three out of five approaches enabled us to discriminate those populations with relatively high levels of non-native introgression from those populations with little or none. Actual estimates of admixture coefficients within a test population, varied, however, with the approach and reference sample used. These results have important implications for policies dividing populations into different management categories according to the estimated proportion of non-native genetic material that they contain.     

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.     

Rapid identification of <Emphasis Type="Italic">Salmo trutta</Emphasis> lineages by multiplex PCR utilizing primers tailored to discriminate single nucleotide polymorphisms (SNPs) of the mitochondrial control region

Analyses of the mtDNA control region sequence variation of the brown trout Salmo trutta through its distribution range have demonstrated five distinct phylogenetic lineages. In this work we report the design and implementation of a rapid and accurate molecular diagnostic procedure which allows the assignment of all major phylogenetic lineages. Our method relies on multiplex PCR that discriminates diagnostic single nucleotide polymorphisms (SNPs) characteristic of each lineage. The method is straightforward and easy to set up, involves a single reaction, yields data easy to interpret and does not pose significant risk for false positive/negative lineage assignment of a given individual. Panagotis K. Apostolou and Andreas Georgiadis contributed equally to this work.     

Effects of species invasion on population dynamics,vital rates and life histories of the native species

Invasions occurring in natural environments provide the opportunity to study how vital rates change and life histories evolve in the presence of a competing species. In this work, we estimate differences in reproductive traits, individual growth trajectories, survival, life histories and population dynamics between a native species living in allopatry and in sympatry with an invasive species of the same taxonomic Family. We used as a model system marble trout Salmo marmoratus (native species) and rainbow trout Oncorhynchus mykiss (non-native) living in the Idrijca River (Slovenia). An impassable waterfall separates the stream into two sectors only a few 100 meters apart: a downstream sector in which marble trout live in sympatry with rainbow trout and an upstream sector in which marble trout live in allopatry. We used an overarching modelling approach that uses tag-recapture and genetic data (>2,500 unique marble and rainbow trout were sampled and genotyped) to reconstruct pedigrees, test for synchrony of population dynamics and model survival and growth, while accounting for individual heterogeneity. The population dynamics of the two marble trout populations and of rainbow trout were synchronous. We found higher prevalence of younger parents, higher mortality and lower population density in marble trout living in sympatry with rainbow trout than in marble trout living in allopatry. There were no differences in the average individual growth trajectories between the two marble trout populations. Faster life histories of marble trout living in sympatry with rainbow trout are consistent with predictions of life history theory.     

Strontium content of scales as a marker for distinguishing between sea trout and brown trout

Strontium was determined in trout scales from a river where it is often difficult to distinguish between sea trout and resident brown trout by coloration or other visual marks. Sr values were compared with values in scales from brown trout caught above the anadromous stretch of the same river and in scales from a river where sea trout coloration is typical. In the first river, the Sr concentration was generally low, and as a mean only 50 ppm higher in scales from individuals classified as sea trout from the anadromous stretch than in brown trout scales from the upper stretch. There was no consistency between fish coloration and Sr concentration in scales from presumed sea trout on the anadromous stretch. Individuals with a typical sea trout coloration could have a lower concentration of Sr than individuals that were classified as uncertain sea trout by coloration. Fish weight did not seem to influence Sr levels. The mean Sr concentration in scales from the typical sea trout colored population in the second river was 2.8 times higher than that of the anadromous part of the first river. The high variability of Sr concentration in sea trout scales may be explained by differences in individual and population life history. The Sr levels reflect differences in saltwater exposure, either expressed by length of stay or concentration of salt in marine habitats. The study has shown that fish coloration is an inadequate mean to distinguish between resident and migratory trout. Nor is Sr determination of scales alone sufficient, because of low inter-group and high intra-group variability in some rivers. However, Sr values can give valuable information on individual and population migration on a large scale.     

A Highly Polymorphic Plasma Protein Locus in Brown Trout (Salmo trutta L.) Populations from Portugal

Genetic polymorphism of an unidentified plasma protein (PX) is described for the first time in Salmo trutta (L.) by means of isoelectric focusing. The analysis of 414 individuals from different geographic origins in Portugal allowed the identification of nine alleles. Heterozygosity in natural populations is generally above 0.60, thus giving similar values to those reported for brown trout microsatellite loci. Substructuring of Portuguese brown trout is evident between northern and southern basins. Genetic affinities between the southernmost rivers and the hatchery stock were detected, suggesting the existence of recent stocking influences.     

Genetic characterization of brown trout (<Emphasis Type="Italic">Salmo trutta</Emphasis>) populations from the Southern Balkans using mtDNA sequencing and RFLP analysis

In the present study, a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay was used to survey variation in a 1450-bp mitochondrial DNA (mtDNA) segment which comprises part of the cytochrome oxidase III (COIII) and ATPase subunit VI genes in 8 brown trout (Salmo trutta L) populations from the southern Balkans. In addition, a 300 bp fragment at the 5′ end of the control region was sequenced from representatives of the populations studied providing the opportunity to assign PCR-RFLP haplotypes into major phylogenetic lineages (i.e. Atlantic, Danubian, marmoratus, Adriatic and Mediterranean). The level of polymorphism found in the 1450 bp segment suggests that this PCR-RFLP assay may be useful for future diagnostic analyses of mitochondrial DNA in brown trout populations. A reduced within-population genetic variability but considerable among-population differentiation was observed. The results are in accordance with previous data on phylogeography of Mediterranean brown trout suggesting that mitochondrial DNA haplotypes are distributed in a mosaic pattern as a consequence of a complex evolutionary history. The present study shows that brown trout populations from the Southern Balkans are highly divergent and possess a unique genetic profile that should be taken into account when establishing conservation management programs. Handling editor: C. Sturmbauer     

Museum samples could help to reconstruct the original distribution of Salmo trutta complex in Italy

Partial D‐loop sequences of museum specimens of brown trout and marble trout (Salmo trutta species complex) collected from Mediterranean rivers in the late 19th century were analysed to help to describe the native distribution of these species. All the individuals studied carried native haplotypes, the geographic distribution of which is consistent with published data. These results indicate that museum specimens from the 19th century could represent an opportunity to get a picture of the original genetic diversity distribution of this species complex.     

Temporal variations in the diet of brown trout (Salmo trutta L.) and rainbow trout (S. gairdneri R.) in Rutland Water

The diet of brown trout (Salmo trutta L.) and rainbow trout (S. gairdneri Richardson) in Rutland Water were compared during the first two fishing seasons (April–October 1977 and 1978).Fortnightly samples of approximately forty stomachs were obtained from boat and bank, rod-and-line caught trout giving a total of 1046 stomachs over the two seasons.During 1977 seasonal changes in the diet were divided into two phases; the first being a period of abundant drowned terrestrial food until June. This was followed by a period of more stable water level from July onwards when chironomid larvae and pupae were consistently the most important food items and the diversity of food also increased.In 1978 the proportion of chironomid pupae and larvae declined and they were replaced in the diet by Gammarus and Asellus.     

Spatial and temporal genetic differentiation and effective population size of brown trout (<Emphasis Type="Italic">Salmo trutta</Emphasis>, L.) in small Danish rivers

The spatial and temporal genetic structure of brown trout populations from three small tributaries of Lake Hald, Denmark, was studied using analysis of variation at eight microsatellite loci. From two of the populations temporal samples were available, separated by up to 13 years (3.7 generations). Significant genetic differentiation was observed among all samples, however, hierarchical analysis of molecular variance (AMOVA) showed that differentiation among populations accounted for a non-significant amount of the genetic differentiation, whereas differentiation among temporal samples within populations was highly significant (0.0244, P<0.001). Estimates of effective population size (N _e) using a maximum-likelihood based implementation of the temporal method, yielded small values (N _e ranging from 33 to 79). When a model was applied that allows for migration among populations, N _e estimates were even lower (24–54), and migration rates were suggested to be high (0.13–0.36). All samples displayed a clear signal of a recent bottleneck, probably stemming from a period of unfavourable conditions due to organic pollution in the 1970–1980’s. By comparison to other estimates of N _e in brown trout, Lake Hald trout represent a system of small populations linked by extensive gene flow, whereas other populations in larger rivers exhibit much higher N _e values and experience lower levels of immigration. We suggest that management considerations for systems like Lake Hald brown trout should focus both on a regional scale and at the level of individual populations, as the future persistence of populations depends both on maintaining individual populations and ensuring sufficient migration links among these populations.     

Admixture analysis of stocked brown trout populations using mapped microsatellite DNA markers: indigenous trout persist in introgressed populations

Admixture between wild and captive populations is an increasing concern in conservation biology. Understanding the extent of admixture and the processes involved requires identification of admixed and non-admixed individuals. This can be achieved by statistical methods employing Bayesian clustering, but resolution is low if genetic differentiation is weak. Here, we analyse stocked brown trout populations represented by historical (1943–1956) and contemporary (2000s) samples, where genetic differentiation between wild populations and stocked trout is weak (pairwise F_ST of 0.047 and 0.053). By analysing a high number of microsatellite DNA markers (50) and making use of linkage map information, we achieve clear identification of admixed and non-admixed trout. Moreover, despite strong population-level admixture by hatchery strain trout in one of the populations (70.8%), non-admixed individuals nevertheless persist (7 out of 53 individuals). These remnants of the indigenous population are characterized by later spawning time than the majority of the admixed individuals. We hypothesize that isolation by time mediated by spawning time differences between wild and hatchery strain trout is a major factor rescuing a part of the indigenous population from introgression.     

Vertical distribution and substrate preference of brown trout in a littoral zone

Synopsis We studied vertical distribution, substrate preference and food choice of brown trout, Salmo trutta, from benthic gillnet catches at four littoral sampling locations in a Norwegian hydroelectric reservoir. The sampling locations had different bottom substrates; at one location the bottom substrate consisted of sand, while at the other three, substrates consisted of stones ranging 2–5 cm, 10–30 cm and 30–150 cm in diameter, respectively. Small-sized (< 160 cm) and intermediate-size (164–269 mm) brown trout were mainly caught close to the bottom (0–0.5 m above). Small-sized brown trout were caught in the highest frequency at the location with substrate consisting of 10–30 cm large stones. Intermediate-sized brown trout were also caught in highest frequency at this location, but were also caught in a high frequency at the location with sandy substrate. In contrast, the catches of large-sized ( 270 mm) brown trout did not vary with distance from the bottom or with substrate coarseness. The most important food items for the brown trout were aquatic insects, surface insects, Eurycercus lamellatus and crustacean zooplankton, mainly Daphnia longispina, Bythotrephes longimanus, and Holopedium gibberum. In accordance with the differences in vertical distribution, benthic food was more important to small than to large brown trout. We argue that small brown trout stayed close to the bottom to reduce aggressive behaviour from larger specimens, and that small brown trout were therefore more dependent on benthic food items. We also argue that the observed differences in substrate preference between the size groups of brown trout is explained by variation in access to shelter, visual isolation between individuals and benthic feeding conditions between locations.     

Use of three types of stream-margin habitat by age-0 brown trout late in the growing season

The use of stream-margin habitat by age-0 salmonids has been studied, but differences in use among various types of habitat along stream margins has not been addressed. We described the nighttime use of habitat features by age-0 brown trout (Salmo trutta) among three types of stream-margin habitat late in the growing season (August–September) and assessed the extent to which use of habitat features within each type differed over the sampling period. Differences in water depths, water velocities, distances from shore, and substrate at the locations of fish along the margins of pools, the margins of riffles, and in backwaters were studied. Variation in habitat use also was observed during the study period as fish increased in length. Our observations are important considerations when developing habitat suitability criteria for assessment of instream-flow needs of age-0 brown trout.     

Interspecific association of brown trout (Salmo trutta) with non‐native brook trout (Salvelinus fontinalis) at the fry stage

The introduction of non‐native brook trout (Salvelinus fontinalis) in Europe has led to displacement and decreasing populations of native brown trout (Salmo trutta). Some studies have found that brown trout shift to a diet niche similar to brook trout when the two species live in sympatry, which conflicts with the competitive exclusion principle. A change in feeding niche may be a sign of early interspecific association and social learning, leading to behavioral changes. As a first step to address this possibility, it is essential to assess the interspecific association between the species during the early ontogenetic life stages. In this study, we therefore assess whether juvenile brown trout associate with non‐native juvenile brook trout to the same extent as with conspecifics by setting up two experiments: (i) a binomial choice test allowing visual and chemical cues to estimate the species specificity of group preference, and (ii) an association test without physical barriers to estimate the degree of association of a focal brown trout with a group of either conspecifics or heterospecifics. In experiment (1), we found that focal juvenile brown trout preferred to associate with the stimuli groups and did not discriminate either against conspecific or heterospecific groups. Furthermore, more active individuals showed stronger preference for the stimuli group than less active ones, regardless of species. In experiment (2), we found that brook trout groups had a tighter group structure than brown trout groups, and that focal brown trout showed stronger association with brook trout than with brown trout. These results indicate that brown trout may associate with brook trout at an early life stage, which would allow for interspecific social learning to occur. Future studies should look closer into causes and consequences of interspecific association and social learning, including potential effects on the phenotype selection in brown trout populations.     

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.     

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.