Variation in mitochondrial DNA and post-glacial colonization of north western Europe by brown trout

A purified mitochondrial DNA (mtDNA) probe was used to examine restriction fragment length polymorphisms produced by six restriction enzymes ( Xba I, Eco RV, Ava II, Hinf I, Hae III, Mbo I) in 915 brown trout from western Europe. A total of 20 composite haplotypes were found with one to seven haplotypes in individual populations. Icelandic trout samples from north, south, east, and west coast drainages showed only a single common haplotype in contrast to the high level of polymorphism found in Irish and Scottish populations. The phylogeny of mtDNA haplotypes and the pattern of haplotype distribution suggests that post-glacial colonization of brown trout in NW Europe was more complex than the dual colonization model which has been proposed on the basis of differential LDH-5* allele distribution. For example, Lough Melvin (Ireland) appears to have been independently     

Genetic differentiation among the sympatric brown trout (Salmo trutta) populations of Lough Melvin, Ireland

Three morphotypes of brown trout have been described from Lough Melvin in north-west Ireland: gillaroo; sonaghen; ferox. The extensive genetic differences among the three types indicate that they are reproductively isolated, i.e. separate stocks of trout. For example, only gillaroo possess the Ldh -1(n) allele. Ferox show a high frequency of the Ldh -5(100) allele and this allele does not exceed a frequency of 0.02 in the other two types. Sonaghen are characterized by relatively much higher frequencies for Ck -2(115) and Gpi- 2(135) alleles. Sampling over a period of 7 years and experiments with artificial stocks of gillaroo and sonaghen have demonstrated that these differences are temporally stable. On the basis of observation of mature adults and the genetic composition of fry samples, it has been shown that the three types maintain their genetic integrity as the result of their distinctive spawning habits with gillaroo spawning in the lake and outflowing river, sonaghen in the smaller inflowing rivers and ferox in the deep downstream section of the largest inflowing river. The ferox is a relict population of an early post-glacial trout colonist while gillaroo and sonaghen are representative of a more recent independent colonization. However, it cannot be determined at present whether or not gillaroo and sonaghen were already differentiated prior to colonizing Melvin although the weight of evidence favours a sympatric split. It is proposed that the three types of trout be designated as separate subspecies to highlight the need for independent management and urgent requirement for conservation action.     

Kinetic studies on the lactate dehydrogenase (LDH-5) isozymes of brown trout, Salmo trutta L

The LDH-5 isozymes of brown trout, Salmo trutta L., were purified and subjected to enzyme kinetic analysis. A hierarchy of magnitude existed for both Km(pyr) and Km(lac) such that LDH-5(105/105) greater than (100/105) greater than (100/100). The results suggest that the 105 allele has been replaced by 100 at Ldh-5 under the action of natural selection.     

Historical biogeography of Mediterranean trout

Complete sequencing of the mitochondrial control region was used to describe phylogenetic relationships of brown trout populations (Salmo trutta) in the Mediterranean river basins of Iberia and to review the historical biogeography of trout from the Mediterranean regions. Phylogenetic relationships among trout lineages suggested that the Danubian one is the most ancestral, in accordance with the eastern origin of most of the European freshwater fish species. Nested-clade and mismatch analyses suggested that the present distribution of haplotypes of the Adriatic and Mediterranean lineages resulted from population expansions originated, respectively, from central and western Europe, which favoured extensive secondary contacts between lineages. Reduced diversity detected within 50% of the analysed populations and large intrabasin differentiation indicated restricted gene flow in post-glacial periods.     

Allozyme diversity in brown trout (Salmo trutta) from Central Spain: Genetic consequences of restocking

1.  The brown trout ( Salmo trutta ) represents one of the main freshwater resources in Spain, but habitat alterations and overharvesting have contributed to the decline or disappearance of numerous natural populations. In addition, reinforcement programs of wild populations based on releases of hatchery reared fish of exogenous origin compromise the conservation of remnant native trout resources.
2.  We present allozymic data from Central Spain trout populations including stocked and unstocked populations. Although the levels of genetic variation observed were low and affected by hatchery releases (p = 18.23%, Ho= 3.39%), they were within the range observed in other European areas.
3.  The effective introduction of hatchery reared fish is genetically homogenising the populations in the studied area and disturbing the ancestral pattern of genetic variation that distinguishes the Tajo and Duero basins. Within the eight natural populations analysed, seven had alleles assigned to the foreign trout. The introgression in these populations, following the LDH-5 * 90 allele frequency, ranged between 2% and 29.4%, but those values are not in concordance with the respective stocking effort undertaken in each population. Moreover, the release of hatchery-reared fish does not solve the problems related to the reduced size of wild populations and their recruitment instability.     

Genetic diversity of brown trout in central Italy

Genetic diversity was analysed in brown trout Salmo trutta populations living in an area of central Italy using RFLP analysis of two mtDNA segments and of the nuclear locus LDH‐C1 *. The data indicated a genetic structure profoundly altered by repeated stockings with allochthonous material of Atlantic origin. In fact, four and 11 of the haplotypes detected were, respectively, identical or genetically very close to haplotypes found in Danish populations, the putative source of stocked brown trout. Furthermore, the LDH‐C1 * 90 allele, typical of north‐western Europe, was widespread among the samples studied. Nonetheless, four populations are characterized by a high frequency of both putative autochthonous haplotypes and the LDH‐C1 * 100 allele, common in the Mediterranean basin. These populations, sampled in areas where S. trutta is documented historically, might represent a remnant of the species' indigenous biodiversity, showing the scope for improving the management of brown trout in central Italy.     

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.     

Silencing of duplicate genes: a null allele polymorphism for lactate dehydrogenase in brown trout (Salmo trutta)

A previously described isozyme polymorphism at one of two skeletal muscle LdhA loci in brown trout is due to a null allele, Ldh1(n), producing no detectable catalytic activity. Homozygotes for this allele have approximately only 56% of the LDH activity in skeletal muscle relative to homozygotes for the active allele. The remaining activity results from enzyme subunits produced by other LDH loci. The Ldh1(n) allele is common and widespread throughout brown trout populations in Sweden and is also found in populations from Ireland. The persistence of duplicate gene expression for the LdhA loci in almost all salmonid species is best explained by natural selection against individuals containing null alleles. However, there is no indication of natural selection against brown trout with the Ldh1(n) allele: We suggest that the selection against individuals containing null alleles that is apparently responsible for the persistence of duplicate LdhA loci in salmonids occurs only under certain environmental conditions.      

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

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

Partial nucleotide sequences, and routine typing by polymerase chain reaction-restriction fragment length polymorphism, of the brown trout (Salmo trutta) lactate dehydrogenase, LDH-C1*90 and *100 alleles

The cDNA nucleotide sequences of the lactate dehydrogenase alleles LDH-C1*90 and *100 of brown trout (Salmo trutta) were found to differ at position 308 where an A is present in the *100 allele but a G is present in the *90 allele. This base substitution results in an amino acid change from aspartic acid at position 82 in the LDH-C1 100 allozyme to a glycine in the 90 allozyme. Since aspartic acid has a net negative charge whilst glycine is uncharged, this is consistent with the electrophoretic observation that the LDH-C1 100 allozyme has a more anodal mobility relative to the LDH-C1 90 allozyme. Based on alignment of the cDNA sequence with the mouse genomic sequence, a local primer set was designed, incorporating the variable position, and was found to give very good amplification with brown trout genomic DNA. Sequencing of this fragment confirmed the difference in both homozygous and heterozygous individuals. Digestion of the polymerase chain reaction products with BslI, a restriction enzyme specific for the site difference, gave one, two and three fragments for the two homozygotes and the heterozygote, respectively, following electrophoretic separation. This provides a DNA-based means of routine screening of the highly informative LDH-C1* polymorphism in brown trout population genetic studies. Primer sets presented could be used to sequence cDNA of other LDH* genes of brown trout and other species.     

Body pigmentation pattern to assess introgression by hatchery stocks in native Salmo trutta from Mediterranean streams

Five qualitative and seven quantitative colouration and spotting pattern features were measured in 23 brown trout Salmo trutta populations and two hatchery stocks. Simultaneously, the LDH‐C1 *, a diagnostic locus fixed for *90 and *100 alleles in stocking and native populations from southern Europe, respectively, was analysed to classify the brown trout studied according to their origin: native, hatchery stock and hybrids. The three genotypes showed significant differences in the colouration and spotting features and a discriminant function analysis could correctly identify 79% of the individuals. The most discriminating variables were dorsal fin margin colour, number of opercular spots, presence of the preopercular mark and diameter of black spots. Given the low cost, ease and possibility of field identification of native fish, the results indicate great opportunities for the application of morphological‐based classification models on the conservation and management of native brown trout stocks.     

Failure of a stocking policy, of hatchery reared brown trout, Salmo trutta L., in Asturias, Spain, detected using LDH-5* as a genetic marker

A useful genetic marker exists through the apparent fixation of the LDH-5 * 100 allele in wild populations of brown trout in rivers from Asturias, Spain, contrasted with the near fixation of the LDH-5 * 90 allele in hatchery populations used to stock these rivers. In sampling locations where natural reproduction occurred, the * 100 allele was found exclusively in all areas having no record of hatchery stocking. The * 100 allele also predominated in three stocked areas having natural reproduction; in two of these areas a few individuals of the 0 + age class were homozygous for the * 90 allele. These data indicated that all catchable and reproductive fish originated from indigenous populations and thus the policy of hatchery supplementation was a failure in these areas.     

Allozymic evidence of parapatric differentiation of brown trout (Salmo trutta L.) within an Atlantic river basin of the Iberian Peninsula

The genetic variation of brown trout from Duero, one of the main Atlantic Iberian river basins, was assessed at 34 enzymatic loci in 62 native populations. A strong intrabasin differentiation was detected (G(ST) = 0.46; range D: 0-0.066), mainly attributable to the existence of two divergent groups of populations within Duero: southern and northern groups. This divergence was mainly a consequence of the unequal distribution of *75 and *100 alleles at sMDH-B1,2* isoloci, which were correlated with substantial differences in genetic diversity among regions. The Lower Course region (nearly fixed for the *100 allele) and Pisuerga River (nearly fixed for the *75 allele) showed lower heterozygosities (H approximately 0.8%) in contrast with adjacent areas, which evidenced intermediate frequencies for both alleles and higher heterozygosities (H: 2.2-3.1%). Vicariance appeared as the more probable explanation for the significant positive correlation detected between genetic and geographical distances in Duero Basin. Genetic relationships with adjacent Iberian drainages indicate a close similarity between the southern group and Cantabric trout, whereas the northern group constitutes an ancient form from this basin. This study confirmed complex genetic relationships in brown trout from northwest Iberia, reasserting the existence of clines at several loci and for genetic diversity. The interaction between Cantabric and Duero trout, as well as the location of the limit of the anadromous form around the 42 degrees N parallel, are both required to understand the genetic characteristics of brown trout from this area.     

Genetic structure of two Turkish brown trout populations

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

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.     

Natural and artificial secondary contact in brown trout (Salmo trutta,L.) in the French western Pyrenees assessed by allozymes and microsatellites

Analysis of allozyme polymorphism in brown trout (Salmo trutta) populations from south-western France shows that two genetically differentiated wild forms (characterised by the LDH-C1*100 and 90 alleles) introgress in this area. As allozymes could not evaluate the impact of stocking in the Atlantic basin, microsatellites have been necessary to detect the influence of hatchery fish and to confirm that the observed structure was natural. Microsatellites confirm the distinctness of the two wild forms based on allozyme loci. This situation provides a new example of secondary contact for this species in the Atlantic basin, with various levels of mixing being seen between the two population groups. The origin of these forms is discussed in the light of previous studies concerning modern and ancestral Atlantic trout (Hamilton et al, 1989) and lineages stemming from different glacial refuges (Garcia Marin et al, 1999; Weiss et al, 2000). This local analysis provides new insights in defining the evolutionary history of this species and confirms the important role of glaciation events in this history.     

Alleles of sAAT-1,2* isoloci in brown trout: potential diagnostic marker for tracking routes of post-glacial colonization in northern Europe

Distribution alleles of sAAT-1,2 * isoloci in brown trout Salmo trutta populations of northern Europe revealed two glacial refugia, 'Baltic' and 'Atlantic'.     

Mitochondrial haplotype diversity among Portuguese brown trout Salmo trutta L. populations: relevance to the post-pleistocene recolonization of northern Europe

Mitochondrial haplotype diversity in seven Portuguese populations of brown trout, Salmo trutta L., was investigated by sequencing the 5' end of the mitochondrial DNA (mtDNA) control region. Five new haplotypes were described for this species, each two to three mutational steps distant from the common north Atlantic haplotype. Significant population subdivision of mtDNA haplotypes was also apparent. Based on these results, as well as on published data describing the distribution of both mtDNA haplotypes and allozyme alleles throughout Europe, the postglacial recolonization of northern Europe was re-evaluated. It is argued that the available data do not support the contribution of two major glacial refugia (southwest Atlantic and Ponto-Caspian Basin) to this postglacial recolonization, as proposed in a recently published model. The unique genetic architecture of Portuguese brown trout within the Atlantic-basin clade of this species represents a highly valuable genetic resource that should be protected from introgression with nonendemic strains of hatchery fish.     

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.     

Phylogenetic origin of <Emphasis Type="Italic">Salmo trutta</Emphasis> L 1758 from Sicily,based on mitochondrial and nuclear DNA analyses

The phylogenetic status of brown trout Salmo trutta L 1758 in Sicily is uncertain as some reports describe these trout as S. macrostigma or S. cettii on one hand while other, contradictory reports imply a hatchery origin on the other. In order to clarify this situation, we performed sequence analysis of the mtDNA control region and restriction fragment analysis of the nuclear lactate dehydrogenase (LDH-C1*) gene. A single mitochondrial haplotype (At-s6) found previously in brown trout in Morocco, and two alleles at LDH-C1* (the ancestral*100, at a high frequency, and *90) were revealed. Our results suggest that Sicilian brown trout are native and that they probably colonized Sicily from west to east in an expansion, from the Atlantic Ocean basin, along the North-West African coast. Handling editor: C. Sturmbauer