Mitochondrial haplotype variation and phylogeography of Iberian brown trout populations

The biogeographical distribution of brown trout mitochondrial DNA haplotypes throughout the Iberian Peninsula was established by polymerase chain reaction-restriction fragment polymorphism analysis. The study of 507 specimens from 58 localities representing eight widely separated Atlantic-slope (north and west Iberian coasts) and six Mediterranean drainage systems served to identify five main groups of mitochondrial haplotypes: (i) haplotypes corresponding to non-native, hatchery-reared brown trout that were widely distributed but also found in wild populations of northern Spain (Cantabrian slope); (ii) a widespread Atlantic haplotype group; (iii) a haplotype restricted to the Duero Basin; (iv) a haplotype shown by southern Iberian populations; and (v) a Mediterranean haplotype. The Iberian distribution of these haplotypes reflects both the current fishery management policy of introducing non-native brown trout, and Messinian palaeobiogeography. Our findings complement and extend previous allozyme studies on Iberian brown trout and improve present knowledge of glacial refugia and postglacial movement of brown trout lineages.     

Mitochondrial DNA differentiation between two forms of trout Salmo letnica, endemic to the Balkan Lake Ohrid, reflects their reproductive isolation

Mitochondrial haplotype diversity in sympatric populations of Ohrid trout, Salmo letnica was investigated by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the mtDNA control region and ND1, ND3/4, ND5/6 segments. A 310 bp fragment at the 5' end, and a 340-572 bp fragment at the 3' end of the control region were sequenced from representatives of the populations studied. Based on pairwise comparison of the sequences, five new haplotypes were identified plus one identical with the brown trout Andalusian haplotype from the southern Iberian Peninsula. The combination of both RFLP and sequence data sets yielded a total of 10 composite haplotypes. A high degree of genetic subdivision between S. letnica typicus and S. letnica aestivalis populations was observed. The notion of a sympatric origin for the two morphs is discussed. Length variation of the mtDNA control region due to the presence of an 82 bp unit, tandemly repeated one to four times, in the region between the conserved sequence block-3 (CSB-3) and the gene for phenylalanine tRNA is reported. Further, we demonstrate that a single duplication of the approximately 82 bp repeat unit is a common element of the salmonid mitochondrial control region. The unique genetic structure of Ohrid trout represents a highly valuable genetic resource that deserves appropriate management and conservation.     

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.     

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     

Diversity of brown trout <Emphasis Type="Italic">Salmo</Emphasis> cf. <Emphasis Type="Italic">trutta</Emphasis> in the River Danube basin of Western Balkans as assessed from the structure of their mitochondrial Control Region haplotypes

The part of the River Danube basin in the Western Balkans region (11 sampled localities and 57 in total with using of published data) revealed the remarkable diversity of brown trout assessed using the Control Region (CR) of mitochondrial DNA (mtDNA) as a molecular marker. The greatest number of brown trout populations holds brown trout of the Da1 haplotype. Particular distinct haplotypes are limited to isolated brown trout stocks. There are haplotypes of Danubian (Da), and those of Atlantic (At), Adriatic (Ad) and marmoratus (MA) lineages introduced there. Phylogenetic relationships inferred between them implicate the plesiomorhic character of Da*Vr, Da*D? and Da-s6 haplotypes that were resolved as ancestral clades, with the intermediate position between clades holding haplotypes of the non-Danubian lineage and more advanced haplotypes of the Danubian lineage. A uniform rate of evolution was found for all clades. The recent widespread distribution and exclusivity of Da1 haplotype imply its ancestral character among advanced Danubian haplotypes. Populations in the Sava, Drava, Una and Drina revealed an expansion, whereas those in the Kupa and Zapadna Morava catchments revealed both stability in size and great differentiation. Gene flow between stocks was found to be negligible.     

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.     

Population differentiation in the redshank (<Emphasis Type="Italic">Tringa totanus</Emphasis>) as revealed by mitochondrial DNA and amplified fragment length polymorphism markers

The redshank (Tringa totanus) is declining throughout Europe and to implement efficient conservation measures, it is important to obtain information about the population genetic structure. The aim of the present study was two-fold. First, we analysed the genetic variation within and between populations in the Baltic region in southern Scandinavia. Evidence of genetic structure would suggest that different populations might require separate management strategies. Second, in an attempt to study large-scale genetic structure we compared the Baltic populations with redshanks from northern Scandinavia and Iceland. This analysis could reveal insights into phylogeography and long-term population history. DNA samples were collected from six breeding sites in Scandinavia presumed to include two subspecies (totanus and britannica) and a further sample from Iceland (subspecies robusta). Two methods were used to study the population genetic structure. Domain II and III of the mitochondrial control region was analysed by DNA sequencing and nuclear DNA was analysed by screening amplified fragment length polymorphism (AFLP) markers. Mitochondrial DNA showed no variation between individuals in domain II. When analysing an 481 bp fragment of domain III seven haplotypes were found among birds. On the basis of mtDNA sequences, redshanks showed some evidence of a recent expansion from a bottlenecked refugial population. Bayesian analyses of AFLP data revealed a significant genetic differentiation between suggested subspecies but not between populations within the Baltic region. Our results indicate that populations of redshanks in Europe constitute at least three separate management units corresponding to the recognised subspecies.     

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     

Mitochondrial haplotype variation in wild trout populations (Teleostei: Salmonidae) from northwestern Mexico

The variation and composition of Mexican wild trout mitochondrial DNA haplotypes throughout northwestern Mexico was determined by means of polymerase chain reaction–restriction fragment polymorphism analysis (PCR–RFLP), of one region of mitochondrial DNA between cytochrome b and the D-loop. This analysis was based on 261 specimens taken in 12 basins and four hatcheries from northwestern Mexico. From 23 haplotypes, 15 wild trout haplotypes were identified and classified in four groups: (1) one restricted to Nelson’s trout (Oncorhynchus mykiss nelsoni), (2) four restricted to Río Mayo and RíoYaqui trout (O. mykiss sspp.), (3) six to Mexican golden trout (O. chrysogaster) with two subgroups, and (4) one exclusive to Río Piaxtla trout. Distributions of native haplotypes broadly overlap the distribution of non-native hatchery rainbow trout reflecting the historical management of introductions of exotic rainbow trout and the artificial transference of these trout among basins.     

Genetic diversity of Garra rufa Heckel, 1843 (Teleostei: Cyprinidae) in Anatolia

In the present study, mitochondrial DNA polymerase chain reaction-restriction-fragment length polymorphism (PCR-RFLP) and nuclear DNA inter-simple sequence repeat (ISSR) assays were used to assess the phylogenetic and phylogeographic relationships among Garra rufa samples from Anatolia. The complete mtDNA NADH 3/4 dehydrogenase (ND-3/4) gene amplified by PCR was digested with eight restriction enzymes. These enzymes produced 20 composite haplotypes for G. rufa populations. All the mtDNA haplotypes detected were highly diverged from each other and each lineage had a unique genetic profile. The evaluation of mtDNA PCR-RFLP data coupled with geological history of Anatolia indicated a deep genetic divergence among the mtDNA haplotypes of G. rufa populations from drainages of the Mediterranean and Persian Gulf, suggesting an early isolation of Tigris-Euphrates with Orontes river and other rivers draining into the Mediterranean Sea. In general, data from both mtDNA and nDNA were congruent.     

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.

     

Assessment of the stocked or wild origin of anadromous brown trout (Salmo trutta L.) in a Danish river system, using mitochondrial DNA RFLP analysis

Declines in the number of anadromous brown trout in the Karup River in Denmark, due to environmental degradation, led to the stocking of large numbers of hatchery trout during the 1980s. This practice was gradually replaced by stocking with the offspring of electrofished local trout The genetic contribution of the hatchery fish to the current population of anadromous trout in the river was estimated by restriction fragment length polymorphism analysis of mitochondrial DNA, using seven restriction endonucleases. Fish from the hatchery strain as well as from five locations in the river system, and from a further unstocked river were screened. Eight haplotypes were observed. The distribution and frequencies of the observed haplotypes revealed little genetic differentiation among stocked populations. The hatchery strain differed significantly from the stocked populations. One haplotype which was found at a high frequency in the hatchery strain was almost absent from the stocked populations. This suggests that the genetic contribution of the hatchery trout to the current population is much less than would be expected from the number of stocked fish. The possible reasons for the failure of the hatchery trout to contribute to the gene pool, and also the implications for conservation biology, are discussed.     

Genetic structure of brown trout (Salmo trutta, L.) populations from south-western France: data from mitochondrial control region variability

The genetic relationships between several French Atlantic populations of brown trout were analysed using polymerase chain reaction-single-strand conformation polymorphism and sequencing of two parts of the mitochondrial control region. Ten mitochondrial haplotypes were identified, separated by a small number of mutations. The distribution of these haplotypes confirmed our hypothesis of the existence of two genetically well-differentiated groups of populations in this area, which has already been suggested using nuclear markers. Nuclear and mitochondrial markers, however, show different patterns of genetic differentiation: some populations which belong to one group according to allozyme results appear to be part of the other when using mitochondrial DNA. The origin of these different forms is discussed; they seem to belong to the Atlantic grouping. Nevertheless, the hypothesis of an ancestral and a modern form suggested for northern populations does not appear valid in this particular case.     

Mitochondrial DNA restriction map for the mediterranean fruit fly,Ceratitis capitata

Molecular genetic research on the Mediterranean fruit fly,Ceratitis capitata, will provide tools to permit determination of source populations for new pest infestations. Restriction fragment length polymorphism (RFLP) of mitochondrial DNA provides some interpopulation discrimination. A restriction map, including the informative variableEcoRV andXbaI restriction sites, is constructed for the Mediterranean fruit fly, and several restriction sites are associated with specific gene regions based on polymerase chain reaction-RFLP and sequence analyses. A partial sequence of the mitochondrial 16S ribosomal RNA gene is reported.     

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.     

Mitochondrial DNA sequence variation and phylogeography among Salmo trutta L. (Greek brown trout) populations

To investigate the phylogenetic relationships and geographical structure among brown trout S. trutta L. populations from the South Adriatic-Ionian and Aegean sea basins, mitochondrial DNA nucleotide sequence comparisons were used. A 310-base-pair (bp) segment of the control region (D-loop), and an additional 280-bp segment of the cytochrome b gene were sequenced from representatives of 13 brown trout populations. Phylogenetic analyses, conducted after combining the data presented with published data from other Eurasian brown trout, revealed four major phylogenetic groups, three of which were found widely distributed within the southern Balkan region. The phylogeographical patterns revealed by mtDNA represent one of the few cases where phylogenetic discontinuity in a gene tree exists without obvious geographical localization within a species' range and has most likely resulted from the differentiation of the major mtDNA clades during Messinian or early Pleistocene times. Finally, the genetic relationships among the populations suggested by mtDNA were generally not in accordance with either allozyme or morphological data.     

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.     

Mitochondrial DNA Diversity of the Asian Moon Scallop, <Emphasis Type="Italic">Amusium pleuronectes</Emphasis> (Pectinidae), in Thailand

Sequence variation of the mitochondrial DNA 16S rRNA region of the Asian moon scallop, Amusium pleuronectes, was surveyed in seven populations along the coast of Thailand. A total of 16 unique haplotypes were detected among 174 individuals with a total 27 variable sites out of 534 bp sequenced. The mitochondrial haplotypes grouped into two distinct arrays (estimated to differ by about 2.62% to 2.99% nucleotide divergence) that characterized samples collected from the Gulf of Thailand versus the Andaman Sea. Low levels of intrapopulation variation were observed, while in contrast, significant divergence was observed between populations from the Gulf of Thailand and Andaman Sea. Results of AMOVA reveal a high F _ST value (0.765) and showed that the majority of the total genetic variance (76.03%) occurred among groups (i.e., Andaman Sea and the Gulf of Thailand) and little among populations within the group (0.52%) and within populations (23.45%). The genetic differentiation between the populations recorded in the present study is similar to that observed in a variety of marine species in the Indo-Pacific. The implications of the findings for management of A. pleuronectes genetic resources in Thailand are discussed.     

Phylogeographic inferences from the mtDNA variation of the three-toed skink, Chalcides chalcides (Reptilia: Scincidae)

Genetic diversity was analyzed in Chalcides chalcides populations from peninsular Italy, Sardinia, Sicily and Tunisia by sequencing 400 bp at the 5' end of the mitochondrial gene encoding cytochrome b (cyt b) and by restriction fragment length polymorphism (RFLP) analysis of two mitochondrial DNA segments (ND-1/2 and ND-3/4). The results of the phylogenetic analysis highlighted the presence of three main clades corresponding with three of the four main geographical areas (Tunisia, Sicily and the Italian peninsula), while Sardinia proved to be closely related to Tunisian haplotypes suggesting a colonization of this island from North Africa by human agency in historical times. On the contrary, the splitting times estimated on the basis of cyt b sequence data seem to indicate a more ancient colonization of Sicily and the Italian Peninsula, as a consequence of tectonic and climatic events that affected the Mediterranean Basin during the Pleistocene. Finally, the analysis of the genetic variability of C. chalcides populations showed a remarkable genetic homogeneity in Italian populations when compared to the Tunisian ones. This condition could be explained by a rapid post-glacial expansion from refugial populations that implied serial bottlenecking with progressive loss of haplotypes, resulting in a low genetic diversity in the populations inhabiting the more recently colonized areas.     

The Balkans and the colonization of Europe: the post‐glacial range expansion of the wild boar,Sus scrofa

Aim We focus on the biogeographical role of the Balkan Peninsula as a glacial refugium and source of northward post‐glacial dispersal for many European taxa. Specifically, we analysed the genetic structure and variation of wild boar (Sus scrofa) samples primarily from Greece, a region that has repeatedly served as a glacial refugium within the Balkan Peninsula. Location Continental Greece, the Aegean island of Samos and Bulgaria. Methods We analysed wild boar samples from 18 localities. Samples from common domestic breeds were also examined to take into account interactions between wild and domesticated animals. Phylogenetic analyses were carried out on a 637‐bp fragment of the mitochondrial DNA control region in 200 wild boar and 27 domestic pigs. The sequences were also compared with 791 Eurasian wild boar and domestic pig D‐loop sequences obtained from GenBank. Results Ninety‐four haplotypes were identified in the European wild boar data set, of which 68 were found in the Balkan samples and assigned to two previously described clades: the E1 European and Near Eastern clades. All of the continental samples clustered in the E1 clade and the samples from Samos fell into the Near Eastern clade, consistent with the island’s proximity to Asia Minor. Intriguingly, 62 novel haplotypes were identified and are found exclusively in the Balkans. Only six haplotypes were shared between wild boar and domestic pigs. Main conclusions Our data reveal numerous novel and geographically restricted haplotypes in wild boar populations, suggesting the presence of separate refugia in the Balkans. Our analyses support the hypothesis of a post‐glacial wild boar expansion consistent with the leading edge model, north and west from modern day Greece, and suggest little maternal introgression of Near Eastern and domestic haplotypes into wild Balkan populations.