Genetic introgression on freshwater fish populations caused by restocking programmes

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

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.     

Identification and conservation of remnant genetic resources of brown trout in relict populations from Western Mediterranean streams

Brown trout is a cold-adapted freshwater species with restricted distribution to headwater streams in rivers of the South European peninsulas, where populations are highly vulnerable because Mediterranean regions are highly sensitive to the global climatic warming. Moreover, these populations are endangered due to the introgressive hybridization with cultured stocks. Individuals from six remnant populations in Western Mediterranean rivers were sequenced for the complete mitochondrial DNA control region and genotyped for 11 nuclear markers. Three different brown trout lineages were present in the studied region. Significant genetic divergence was observed among locations and a strong effect of genetic drift was suggested. An important stocking impact (close to 25%) was detected in the zone. Significant correlations between mitochondrial-based rates of hatchery introgression and water flow variation suggested a higher impact of stocked females in unstable habitats. In spite of hatchery introgression, all populations remained highly differentiated, suggesting that native genetic resources are still abundant. However, climatic predictions indicated that suitable habitats for the species in these rivers will be reduced and hence trout populations are highly endangered and vulnerable. Thus, management policies should take into account these predictions to design upstream refuge areas to protect remnant native trout in the region.     

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.

     

Role of environmental factors in the spread of domestic trout in Mediterranean streams

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Population status and ecology of the Salmo trutta complex in an Italian river basin under multiple anthropogenic pressures

Salmonids inhabiting Mediterranean rivers are of particular concern for biodiversity conservation, as they are threatened by various stressors, including habitat alterations, overfishing, climate change, and introgressive hybridization with alien species. In the Tiber River basin (Central Italy), genetic introgression phenomena of the native Salmo cettii with the non‐native Salmo trutta hinder the separate analysis of the two species, which are both included in the S. trutta complex. Little is known about the factors currently limiting the trout populations in this area, particularly with respect to climate change. With the intention of filling this gap, the aims of the current study were to (a) quantify changes in the climate and (b) analyze the distribution, status, and ecology of trout populations, in the context of changing abiotic conditions over the last decades. Fish stock assessments were carried out by electrofishing during three census periods (1998–2004, 2005–2011, and 2012–2018) at 129 sites. The trend over time of meteorological parameters provided evidence for increased air temperature and decreased rainfall. Multivariate analysis of trout densities and environmental data highlighted the close direct correlation of trout abundance with water quality, altitude, and current speed. Climate‐induced effects observed over time in the sites where trout were sampled have not yet led to local extinctions or distribution shifts, indicating a marked resilience of trout, probably due to the buffering effect of intrinsic population dynamics. Decreasing body conditions over time and unbalanced age structures support the hypothesis that variations in hydraulic regime and water temperature could overcome these compensatory effects, which may lead to a severe decline in trout populations in the near future. In a climate change context, habitat availability plays a key role in the distribution of cold‐water species, which often do not have the possibility to move upstream to reach their thermal optimum because of water scarcity in the upper river stretches.     

Effects of stocking on the genetic diversity of brown trout populations of the Adriatic and Danubian drainages in Switzerland

This study focuses on genetic variation of brown trout Salmo trutta populations of the Adriatic and Danubian drainages in Switzerland. The allozyme and other protein loci data show a major replacement of native stocks from the Adriatic drainages by introduced hatchery trout of Atlantic basin origin. In most samples, diagnostic alleles for the Adriatic form of Salmo trutta f. fario and for the marbled trout Salmo trutta marmoratus are found at very low frequencies (f<0.15). Taking into account previous genetic studies on brown trout of this basin, the Danubian samples are not heavily contaminated with foreign alleles. The results are consistent with records of local stocking activities which account in part for the high introgression rates of Atlantic alleles into local populations of the Adriatic drainages. In addition, introgression is enhanced by a decrease of natural reproduction which is caused by a deterioration of trout habitats through human activities. Furthermore, a third mechanism is proposed that may contribute to the high introgression rates observed: if Atlantic trout are introduced, the reproductive barriers between the two native forms, marbled trout and Adriatic fario respectively, break down. Atlantic trout apparently hybridize with both native forms and generate gene flow between them. In some parts of Adriatic drainages in Switzerland, the patterns of introgression and hybridization are further complicated by introduction of trout from the Danubian system. Alleles of the marbled trout are also found in the samples of the Danubian drainage system. These are due to stocking activities across the watershed.     

Evidence for selective angling of introduced trout and their hybrids in a stocked brown trout population

Comparisons of the genetic composition of brown trout Salmo trutta captured by anglers and by electrofishing based on three diagnostic microsatellite loci provided strong evidence that angling is selective in a stocked brown trout population. At two sites, anglers caught significantly younger trout and proportionally more introduced hatchery trout and hybrids than were observed in electrofishing surveys. Selective angling, in combination with a small legal catch size, may have considerably eliminated introduced trout and hybrids before spawning at the study sites, and thus may have reduced the introgression of alien genes into the local gene pool. Angling can be an important factor influencing the genetic structure of fish populations and should be taken into account in studies of introgressive hybridization in stocked fish populations and their management. In this study, demographic consequences of stocking were not assessed. Thus, even though the genetic consequences of stocking may be minimal or largely reversible through angling, resource competition between native and introduced trout, until they reach legal catch size, is expected to have a negative effect on the productivity of the indigenous trout population.     

Molecular Evidence for Introgression and Loss of Genetic Variability in Salmo (trutta) macrostigma as a Result of Massive Restocking of Apennine Populations (Northern and Central Italy)

We assessed structural gene variation (allozymes and mtDNA) of brown trout to evaluate the genetic variability of Apennine stream populations (Northern and Central Italy) and the possibility of introgression by alien genomes after massive restocking with hatchery strains (Atlantic stocks). Genetic variability within and between Apennine populations was extremely low in our samples. Only two allozyme loci were polymorphic and mean hetero-zygosity was also reduced compared to other brown trout populations. Allelic frequencies determined for both loci were similar to the ones detected in the corresponding hatchery spawners. The reduction or total absence of the Mediterranean nuclear (LDH-5) and mitochondrial (16S rDNA) diagnostic markers suggests the domestic origin of most populations, and the introgression effects carried out by non-native genomes. From a taxonomic point of view, a clear differentiation emerges among basins placed on opposite sides of the Apennine chain (Tyrrhenian and Adriatic regions). In particular, the presence of Mediterranean genotypes and haplotypes characterizing Salmo (trutta) macrostigma is sporadic along the eastern Apennine side, adding additional doubts on the original presence and wide distribution of this salmonid along the Adriatic side of the mountain chain. In spite of conservation programs devoted to preservation of local genetic characteristics of S. t. macrostigma, massive restocking practices with hatchery strains obtained by a few spawners is the major cause of significant `founder effect' and `inbreeding depression' even in Apennine regions.     

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.     

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.     

Hybridization rapidly reduces fitness of a native trout in the wild

Human-mediated hybridization is a leading cause of biodiversity loss worldwide. How hybridization affects fitness and what level of hybridization is permissible pose difficult conservation questions with little empirical information to guide policy and management decisions. This is particularly true for salmonids, where widespread introgression among non-native and native taxa has often created hybrid swarms over extensive geographical areas resulting in genomic extinction. Here, we used parentage analysis with multilocus microsatellite markers to measure how varying levels of genetic introgression with non-native rainbow trout (Oncorhynchus mykiss) affect reproductive success (number of offspring per adult) of native westslope cutthroat trout (Oncorhynchus clarkii lewisi) in the wild. Small amounts of hybridization markedly reduced fitness of male and female trout, with reproductive success sharply declining by approximately 50 per cent, with only 20 per cent admixture. Despite apparent fitness costs, our data suggest that hybridization may spread due to relatively high reproductive success of first-generation hybrids and high reproductive success of a few males with high levels of admixture. This outbreeding depression suggests that even low levels of admixture may have negative effects on fitness in the wild and that policies protecting hybridized populations may need reconsideration.     

Breakdown of the brown trout evolutionary history due to hybridization between native and cultivated fish

Genetic changes in the population structure of brown trout Salmo trutta in the eastern Pyrenees were monitored during the 1990s. Stocking with cultivated exogenous fish has resulted in introgressed populations where those with higher introgression coefficients also have the highest values of heterozygosity. Nevertheless, this increase of local diversity reflects a decrease of genetic differentiation between populations and loss of native alleles. These changes obscure native gene pools and modify evolutionary distinctions among native brown trout populations.     

Effectiveness of alien brown trout Salmo trutta L. removal activities for the native trout conservation in Mediterranean streams

In Italian freshwaters the introgressive hybridization with the alien Atlantic trout represents the principal threat to the native endangered Mediterranean trout. The aim of the research was to test the effectiveness of alien trout removal activities carried out by electrofishing in four streams in Central Italy. The four sites were chosen on the basis of the severe genetic introgression level of the trout populations. The removal activities were carried out from December 2014 to July 2017, for a total of 10 eradication campaigns in each site. During each field activity, five environmental parameters (flow rate, conductivity, average depth, average width, accessibility) were collected, since they could influence both the effectiveness of the electrofishing and the biological characteristics of the fish populations. A total of over 22,000 alien trout accounting for a biomass of over 700 kg were removed from the removal sites. For each site, a progressive drastic decrease over time in density and standing crop values was observed. Removal rates ranged from 94.22% to 100.00% for density, and from 96.57% to 100.00% for standing crop. The correlation analysis showed an inverse significant relationship between catchability and populations abundance, confirming that low density populations are more suitable to removal efforts. The progressive removal of specimens improved the catchability over time, indicating that even the largest populations could be eradicated, providing the necessary number of steps in a fishing season. The improved body condition and the greater growth rates observed during the eradication period, in low abundance conditions, seemed to confirm the key role that intraspecific competition and density-dependent phenomena play in the Apennine trout population dynamics. The results obtained in the present research provided evidence that electrofishing removal could be an effective management tool for the Mediterranean trout conservation, especially in watercourses of modest dimensions in terms of flow rates, width and depth.     

An impact assessment of the alien lizard Plestiodon japonicus (Scincidae, Reptilia) on a threatened island population of the native lizard P. latiscutatus at an early phase of the biological invasion

On the island of Hachijojima, central Japan, a formerly abundant population of native lizard, Plestiodon latiscutatus has become endangered by predation from an alien weasel (Mustela itatsi). Recently, an alien lizard, P. japonicus, became established on the island. The impact of the alien lizard and current distribution of the native lizard were assessed by field surveys and DNA analyses. The native lizard was sparsely distributed in the western part of the island. The alien lizard occurred in the northeastern region of the island. Between these regions, several hybrid populations were identified, including some with non-F1 genotypes, suggesting that introgression is occurring. The distribution of the weasel was examined based on field observations, allowing us to estimate predator impacts on both lizard species. We found evidence that the weasel was present throughout the entire island, suggesting that the alien lizard was able to become established despite the presence of the weasel. This indicates that the alien lizard is likely to expand its habitat range and that introgression may spread. We conclude that P. latiscutatus populations will become increasingly endangered by the synergism of genetic introgression from P. japonicus and predation pressure from the weasel.     

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.     

Early effects of the strategies of creating a genetic refuge and direct translocation for conserving and restoring populations of native brown trout

1. The conservation of salmonid inter‐ and intra‐specific diversity is a well‐known challenge, and general management guidelines and conservation processes are available. However, research demonstrating the outcomes of practical conservation actions is largely lacking. 2. We monitored the spatiotemporal genetic and demographic evolution of a native Mediterranean brown trout population in a river in the French Alps to assess the efficacy and early effects of genetic refuge (i.e. cessation of stocking) and wild trout translocation strategies. We also studied the use of angling as a tool to limit the introgression of the wild standing population. 3. We found that the rate of non‐native alleles in wild populations was age dependent, underpinning the importance of using age profiles in the design of genetic conservation studies. 4. Genetic refuge and direct translocation of wild trout resulted in a rapid and significant decrease in the percentages of non‐native alleles. Moreover, the genetic refuge strategy resulted in a significant reduction in the number of pure non‐native individuals, without changing trout densities, whilst direct translocations resulted in the establishment of dense, self‐sustaining native trout populations. Direct translocations changed the distribution of genotype categories and increased densities up to 55‐fold in 3 years. Our results also showed that angling resulted in a selective pressure on non‐native trout introduced at fry stage, whereas non‐native trout issued from natural recruitment were not affected. 5. Our study provides insights for improving the efficacy of practical conservation policies and can be used in other native freshwater fish conservation plans. Proactive measures such as direct translocation need to be implemented together with passive approaches such as genetic refuge policies. Before implementing such actions, accurate genetic and demographic studies at small geographical scales are essential to ensure that no self‐sustaining population of non‐native fish is present. To obtain rapid colonisation, we recommend introducing fish along whole river sections rather than concentrating on a few river stretches. Angling pressure can be used as an additional tool to improve restoration.     

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.     

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.     

Comparison of competitive ability between native and introduced salmonids: evidence from pairwise contests

Brown trout, Salmo trutta, and rainbow trout, Oncorhynchus mykiss, have been introduced to freshwaters in Hokkaido, Japan. Today, it is recognized that these introduced salmonids have negative impacts on native salmonids such as white-spotted charr, Salvelinus leucomaenis, and masu salmon, O. masou. In particular, interspecific competition may be an important mechanism that could contribute to the exclusion for native salmonids. In this study, experimental pairwise contests were conducted to compare interference competitive ability between native and introduced salmonids. We demonstrated that brown trout were competitively superior to white-spotted charr and masu salmon whereas rainbow trout were superior to white-spotted charr. We suggest that introduced brown trout negatively impact both white-spotted charr and masu salmon, and introduced rainbow trout negatively impact white-spotted charr.