Phylogeny of Salmonine Fishes Based on Growth Hormone Introns: Atlantic (Salmo) and Pacific (Oncorhynchus) Salmon Are Not Sister Taxa

Though salmonid fishes are a well-studied group, phylogenetic questions remain, especially with respect to genus-level relationships. These questions were addressed with duplicate growth hormone (GH) introns. Intron sequences from each duplicate gene yielded phylogenetic trees that were not significantly different from each other in topology. Statistical tests supported validity of the controversial monotypic genusParahucho,monophyly ofOncorhynchus,and inclusion ofAcantholingua ohridanawithinSalmo.Suprisingly, GH1 intron C (GH1C) did not support the widely accepted hypothesis thatOncorhynchus(Pacific salmon and trout) andSalmo(Atlantic salmon and trout) are sibling genera; GH2C was ambiguous at this node. Previously published data were also examined for support ofSalmoandOncorhynchusas sister taxa and only morphology showed significant support. If not sister taxa, the independent evolution of anadromy—the migration to sea and return to freshwater for spawning—is most parsimonious. While there was incongruence with and among published data sets, the GH1C intron phylogeny was the best hypothesis, based on currently available molecular data.     

Isolation and characterization of salmonid telomeric and centromeric satellite DNA sequences

Satellite DNA clones with a 37 bp repeat unit were obtained from BglII-digested genomic DNA of Masu salmon (Oncorhynchus masou) and Chum salmon (O. keta). Fluorescence in situ hybridization (FISH) analysis with the isolated clones as a probe showed that these repetitive sequences were localized in the telomeric regions of chromosomes in both species. Southern and dot blot analyses suggested conservation of homologous sequences with similar repeat unit in other salmonids including the species of the genus Oncorhynchus and Salvelinus, but lack or scarcity of such sequences in the genus Hucho and Salmo. Similarly, polymerase chain reaction (PCR)-based cloning of satellite DNA referring to a reported Rainbow trout (O. mykiss) centromeric sequence was successful for the Oncorhynchus, Salvelinus and Hucho species. The obtained satellite DNA clones were localized with FISH in the centromeric regions of chromosomes of the species from these three genera. Although PCR cloning of the centromeric satellite DNA had failed in the Salmo species due to some base changes in the priming sites, dot blot hybridization analysis suggested conservation of homologous satellite DNA in the genus Salmo as in the other three genera. In the neighbor-joining tree of cloned centromeric satellite DNA sequences, the genus Oncorhynchus and Salvelinus formed adjacent clades, and the clade of the genus Hucho included the reported centromeric sequence of the genus Salmo. Conservation pattern and molecular phylogeny of the telomeric and centromeric satellite DNA sequences isolated herein support a close phylogenetic relationship between the genus Oncorhynchus and Salvelinus and between the Salmo and Hucho.     

Fish growth hormone genes: Divergence of coding sequences in salmonid fishes

Comparison of coding nucleotide sequences of the paralogous GH1 and GH2 genes, as well as of the growth hormone amino acid sequences, in the species of closely related salmonid genera Salvelinus, Oncorhynchus, and Salmo was performed. It was demonstrated that, in different groups of salmonids, the amino acid substitution rates were considerably different. In some cases, an obvious discrepancy between the divergence of growth hormone genes and phylogenetic schemes based on other methods and approaches was revealed. These findings suggest that the reason may be multidirectional selection at duplicated genes at different stages of evolution.     

Chromosome relationships in the genus Salmo

Chromosome numbers and polymorphisms in rainbow trout, Atlantic salmon, and brown trout are described. The karyotypes of these three species are compared with each other and with those of other salmonid fish from the genera Salmo, Salvelinus, and Oncorhynchus. Karyotype evolution from a postulated ancestral tetraploid is discussed.     

Chromosome painting supports lack of homology among sex chromosomes in Oncorhynchus, Salmo, and Salvelinus (Salmonidae)

The sex chromosome pair has been identified previously as the largest submetacentric pair in the genome in several species of the genus Salvelinus (eastern trouts and chars) including S. namaycush (lake trout) and as a large subtelocentric/acrocentric pair in several species of the genus Oncorhynchus (Pacific trouts and salmon). Sex chromosomes have not been identified in Salmo (Atlantic salmon and brown trout). Two paint probes, one specific for the short arm (Yp) and the other for the long arm (Yq) of the sex chromosome pair in Salvelinus namaycush were hybridized to chromosomes of Oncorhynchus mykiss (rainbow trout) and O. tshawytscha (chinook salmon) and Salmo salar (Atlantic salmon) and S. trutta (brown trout). The two probes hybridized to two different autosomal pairs in each of the Oncorhynchus species, supporting lack of homology between the sex chromosomes in the two genera. The Yp probe hybridized to interstitial regions on two different chromosome pairs in S. salar and one pair in S. trutta. The Yq probe hybridized to a different pair in both species.     

The Lactate Dehydrogenase Gene <Emphasis Type="Italic">LDH-C1</Emphasis>, a New Molecular Marker for Phylogenetic Analysis of Salmonid Fishes (Salmoniformes: Salmonidae)

We tested the locus of the nuclear lactate dehydrogenase gene (LDH-C1) as a phylogenetic marker in specimens of 11 salmonid genera (Thymallus, Coregonus, Hucho, Brachymystax, Salmo, Salmothymus, Acantholingua, Parahucho, Salvelinus, Parasalmo, and Oncorhynchus). All the sequences were veraciously clustered according to their taxonomic affiliation at the species and genus levels. It is shown that used complex of characters contains a phylogenetic signal that represents specific information about the phylogenesis process. This allows us to recommend the LDH-C1 locus to specify the phylogeny of salmonids in the combined analysis of several independent nuclear genes and mitochondrial DNA.     

Fish growth hormone genes: Divergence of intron sequence in charrs of Salvelinus genus

Sequences of the two large introns (C and D) from two paralogous growth hormone genes, GH1 and GH2, were compared in eight charr species of the Salvelinus genus (Osteichthes, Salmonidae). It was demonstrated that the rates of intron divergence in these two genes were remarkably different. Introns in the GH1 gene appeared to be more conservative, while the rate of intron variations was considerably higher in the GH2 gene. These data suggest that noncoding regions of nuclear genes under the selective pressure. The lower congruence of phylogenetic scheme constructed based on an analysis of the GH1 introns compared to that based on the GH2 data, as well as with the traditional views on the evolution of charr species, also favors the supposition on differences in the forces and directions of the selective pressure on the introns.     

Otoliths as natural tags in the systematics of salmonids

Synopsis We studied otolith shape in two species of Salmo and two species of Salvelinus to quantify the accuracy of intraspecific, interspecific, and intergeneric identification. Otolith shape is chiefly population and species specific. Between genera, only 3% of the specimens were incorrectly grouped using discriminant analysis.     

Genetic mapping of Y-chromosomal DNA markers in Pacific salmon

Sex chromosomes in fish provide an intriguing view of how sex-determination mechanisms evolve in vertebrates. Many fish species with single-factor sex-determination systems do not have cytogenetically-distinguishable sex chromosomes, suggesting that few sex-specific sequences or chromosomal rearrangements are present and that sex-chromosome evolution is thus at an early stage. We describe experiments examining the linkage arrangement of a Y-chromosomal GH pseudogene (GH-Y) sequence in four species of salmon (chum, Oncorhynchus keta; pink, O. gorbuscha; coho, O. kisutch; chinook, O. tshawytscha). Phylogenetic analysis indicates that GH-Y arose early in Oncorhynchus evolution, after this genus had diverged from Salmo and Salvelinus. However, GH-Y has not been detected in some Oncorhynchus species (O. nerka, O. mykiss and O. clarki), consistent with this locus being deleted in some lineages. GH-Y is tightly linked genetically to the sex-determination locus on the Y chromosome and, in chinook salmon, to another Y-linked DNA marker OtY1. GH-Y is derived from an ancestral GH2 gene, but this latter functional GH locus is autosomal or pseudoautosomal. YY chinook salmon are viable and fertile, indicating the Y chromosome is not deficient of vital genetic functions present on the X chromosome, consistent with sex chromosomes that are in an early stage of divergence.     

Spawning behaviour of Sakhalin taimen, <Emphasis Type="Italic">Parahucho perryi</Emphasis>, from northern Hokkaido,Japan

A video camera mounted in an underwater housing and remotely operated was used to monitor the behaviour of five different Sakhalin taimen (Parahucho perryi), females and attendant males spawning in three coastal tributary streams in Northern Hokkaido, Japan. Based on three complete and two incomplete spawnings, we describe in detail for the first time the complete spawning behavioural repertoire of this species. The Sakhalin taimen was originally placed within Hucho, then removed from that genus based on morphological, life history and molecular data. Our study supports that removal—none of the behavioural traits we recorded clustered Parahucho with Hucho uniquely. Similarities between the two genera were all plesiomorphic traits that are widespread throughout the salmonines. The immediate behaviour right after spawning was found to be a major difference between Hucho and Parahucho. Like female Oncorhynchus and Salmo, Sakhalin taimen females cover their eggs by beats of their tails immediately after spawning. This is different from the “rest, then cover” behaviour shown by Siberian taimen (Hucho taimen) as well as lenok (Brachymystax lenok), supporting again that the Sakhalin taimen be removed from Hucho and placed in its own genus.     

The complete mitochondrial genome of the Sichuan taimen (Hucho bleekeri): repetitive sequences in the control region and phylogenetic implications for Salmonidae

The complete mitochondrial DNA genome of the Sichuan taimen (Hucho bleekeri) was determined by the long and accurate polymerase chain reaction (LA-PCR) and primer walking sequence method. The entire mitochondrial genome of this species is 16,997 bp in length, making it the longest among the completely sequenced Salmonidae mitochondrial genomes. It consists of two ribosomal RNA (rRNA) genes, 13 protein-coding genes, 22 transfer RNA (tRNA) genes, and one control region (CR). The gene arrangement, nucleotide composition, and codon usage pattern of the mitochondrial genome are similar to those of other teleosts. A T-type mononucleotide microsatellite and an 82 bp tandem repeat were identified in the control region, which were almost identical among the three H. bleekeri individuals examined. Both phylogenetic analyses based on 12 concatenated protein-coding genes of the heavy strand and on just the control region show that H. bleekeri is a basal species in Salmoninae. In addition, Salmo, Salvelinus and Oncorhynchus all represent monophyletic groups, respectively. All freshwater species occupied basal phylogenetic positions, and also possessed various tandem repeats in their mitochondrial control regions. These results support established phylogenetic relationships among genera in Salmonidae based on morphological and molecular analyses, and are consistent with the hypothesis that Salmonidae evolved from freshwater species.     

Phylogeny of Mitochondrial DNA in Salmonids of the Subfamily Salmoninae: Analysis of the Cytochrome b Gene Sequences

On the basis of comparison of the cytochrome b gene nucleotide sequences from genetic databases, the possible phylogenetic relationships of mitochondrial DNA (mtDNA) among all major lineages of Salmoninae (Brachymystax, Parahucho, Salvelinus, Salmo, Parasalmo, and Oncorhynchus) were examined. Three different phylogenetic methods (UPGMA, NJ, and ML) yielded phylogenetic trees of essentially the same topology: (((Brachymystax, Parahucho), Salvelinus, Salmo), (Parasalmo, Oncorhynchus)). The results obtained using the maximum parsimony method were less clear. Apparently, the divergence of the main salmonid lineages occurred during a relatively short time period; hence, the number of synapomorphs marking the order of their divergence was extremely low. This may account for the relative failure to use the maximum parsimony method of phylogenetic reconstruction. The problem of concordance of mtDNA and species phylogenetic schemes is discussed. Their discrepancy in salmonids may be caused by interspecific introgressive hybridization.     

Molecular phylogeny of Salmo of the western Balkans,based upon multiple nuclear loci

Background

Classification of species within the genus Salmo is still a matter of discussion due to their high level of diversity and to the low power of resolution of mitochondrial (mt)DNA-based phylogeny analyses that have been traditionally used in evolutionary studies of the genus. We apply a new marker system based on nuclear (n)DNA loci to present a novel view of the phylogeny of Salmo representatives and we compare it with the mtDNA-based phylogeny.

Methods

Twenty-two nDNA loci were sequenced for 76 individuals of the brown trout complex: Salmo trutta (Danubian, Atlantic, Adriatic, Mediterranean and Duero mtDNA lineages), Salmo marmoratus (marble trout), Salmo obtusirostris (softmouth trout), and Salmo ohridanus (Ohrid belvica or belushka). Sequences were phylogenetically analyzed using maximum-likelihood and Bayesian Inference methods. The divergence time of the major clades was estimated using the program BEAST.

Results

The existence of five genetic units i.e. S. salar, S. ohridanus, S. obtusirostris, S. marmoratus and the S. trutta complex, including its major phylogenetic lineages was confirmed. Contrary to previous observations, S. obtusirostris was found to be sister to the S. trutta complex and the S. marmoratus clade rather than to the S. ohridanus clade. Reticulate evolution of S. obtusirostris was confirmed and a time for its pre-glacial origin suggested. S. marmoratus was found to be a separate species as S. trutta and S. obtusirostris. Relationships among lineages within the S. trutta complex were weakly supported and remain largely unresolved.

Conclusions

Nuclear DNA-based results showed a fairly good match with the phylogeny of Salmo inferred from mtDNA analyses. The comparison of nDNA and mtDNA data revealed at least four cases of mitochondrial–nuclear DNA discordance observed that were all confined to the Adriatic basin of the Western Balkans. Together with the well-known extensive morphological and genetic variability of Balkan trouts, this observation highlights an interesting and variegated evolutionary history of Salmo in this area.     

Genetic basis of creatine kinase isozymes in skeletal muscle of salmonid fishes

The genetic basis of isozyme phenotypes of creatine kinase (CK) from extracts of skeletal muscle of salmonids has been resolved through breeding data including double heterozygous crosses and backcrosses of rainbow trout (Salmo gairdneri), and backcrosses of coho salmon (Oncorhynchus kisutch). The two-, three-, or four-banded phenotypes of homozygous individuals and all heterozygous and hybrid phenotypes of ten salmonid species are readily explained by the following model: (1) there are no detectable heterodimers either between allelic products at a single locus or between loci; (2) each allele is represented electrophoretically by two bands, presumably a reflection of stable posttranslational modification of a single polypeptide unit; (3) CK of salmonid muscle is encoded by two loci—CK-1 and CK-2. The distance separating the paired bands reflecting each allele provides a basis for two groupings—a broad-spaced group (including all species of Oncorhynchus tested excepting O. masou) and a narrow-spaced group (including all species of Salmo tested and O. masou). The relationships among species suggested by the relative mobilities and spacings of these CK bands are consistent with taxonomic schemes inferred from morphological, cytogenetic, and other isozymic data.     

Metazoan parasites of salmonids and coregonids from coastal Labrador*

Three hundred and twenty-three fish of six species (salmonids and coregonids) from four locations on the eastern coast of Labrador were examined for metazoan parasites, using conventional parasitological techniques. Twenty-four genera of parasites were recovered (3 of Monogenea, 8 of Digenea, 5 of Cestoda, 5 of Nematoda, 1 of Acanthocephala, 2 of parasitic Copepoda). Fifty-one new host records were noted. Fourteen genera of parasites were noted in Salmo salar L., while Salvelinus fontinalis (Mitchill) contained 21 genera; S. namaycush (Walbaum,) 12 genera; S. alpinus (L.), 16 genera: Coregonus clupeaformis (Mitchill), 11 genera; Prosopium cylindraceum (Pallas), 11 genera. It was found that the parasite burden of the various fish species examined was not homogeneous when sample areas and sex of the fish was considered. An increase in the number of parasite species per infected host with age was seen in the case of Salmo salar, Salvelinus fontinalis, S. namaycush, S. alpinus. In Coregonus clupeaformis and Prosopium cylindraceum no such correlation was seen, this difference being related to the ecology of the fish. The parasitofauna of the various salmonid species examined was not homogeneous, significant differences being noted in the number of certain species infected with specific parasites. Significant differences were also noted in the parasite burden of the Salmonidae when compared with the Coregonidae. Food items recovered from the fish autopsied were noted.     

Ecological subspecies and locally temporal populations of anadromous fish

Intraspecific organization and structure of anadromous fish populations (in terms of species of the Oncorhynchus, Salvelinus, and Tribolodon genuses) were investigated. It was shown that the ecological, genetic, and morpho-physiological differences in ecology-temporal (seasonal) races of these fish reached the rank of ecological subspecies. The expediency of more careful and detailed study of intraspecific organization, ecological and locally temporal differentiation of fish, and consideration of the results of these researches was shown in the development of a polymorphic species concept as well as optimization of fish industry—the sustainable organization of fishing, artificial fish breeding, and introduction of fish.     

Isolation and characterization of Brachymystax lenok microsatellite loci and cross‐species amplification in Hucho spp. and Parahucho perryi

We isolated and characterized eight polymorphic microsatellite markers for Brachymystax lenok (Pallas, 1773) from genomic libraries enriched for (GATA)_n, (GACA)_n and (ATG)_n microsatellites. The number of alleles per locus ranged from two to 17. Heterozygosity ranged from 0.2 to 0.95. In addition, cross‐species amplification was successful for seven loci in Hucho hucho, eight in H. taimen and seven in Parahucho perryi.     

Phylogeny of salmonids (Salmoniformes: Salmonidae) and its molecular dating: Analysis of nuclear RAG1 gene

The phylogenetic relationships among 26 species of salmonid fishes (family Salmonidae) were studied using the RAG1 gene as phylogenetic marker. No unambiguous relationships between thymalllins, coregonins, and salmonins it was possible to establish. It seems likely, that divergence of these lineages took place during rather short time interval (about 3 to 4 million years). The thymallins are thought to be the first separated lineage. The genera of the subfamily Salmoninae form two distinct monophyletic groups, represented by (1) Brachymystax and Hucho and (2) Salmo, Parahucho, Salvelinus, Parasalmo and Oncorhynchus. Ancestral forms of these two evolutionary lineages could diverge at the Oligocene-Miocene boundary (about 24 million years ago). It is suggested that diversification of the main lineages within the second group was rather rapid, and took place in middle Miocene (about 19?C16 million years ago). Moreover, the lineages of Salvelinus, Parasalmo and Oncorhynchus were the latest to diverge. It seems likely that divergence of the Prosopium and Coregonus ancestral lineages occurred during the same time interval.     

Interspecific Relationships among Charrs Based on Phylogenetic Analysis of Nuclear Growth Hormone Intron Sequences

DNA samples were obtained from six charr species: Salvelinus alpinus, S. malma, S. confluentus, S. leucomaenis, S. namaycush and S. fontinalis and two outgroups: Salmo salar and S. trutta. The C and D introns from the duplicate growth hormone loci (GH1 and GH2) were amplified using the polymerase chain reaction and sequenced from each of the species. Phylogenetic analyses with and without gaps were performed using the beta version of PAUP. The GH introns are evolving relatively slowly, so the data were combined with sequences from the ITS1 and ITS2 for a phylogenetic analysis of the charr species. The GH intron data and the combined data supported two groups of sister species among charrs: S. alpinus and S. malma, and S. confluentus and S. leucomaenis.     

Phylogeny of Salmonoid Fishes (Salmonoidei) Based on mtDNA <Emphasis Type="Italic">COI</Emphasis> Gene Sequences (Barcoding)

We have analyzed the partial sequences of the mitochondrial COI gene along with the amino acid sequences of cytochrome oxidase subunit I, encoded by this gene region, in representatives of 11 genera of salmonoid fish. For amino acid sequences, two alternative networks are constructed with outgroups represented by either Esocoidei or Osmeroidei as the supposed ancestral groups. This way, Osmeroidei appear to be closer to the salmonoid fish than Esocoidei, and their presence in the network as an outgroup explains the available data on the morphology and karyology of salmonoids much better. A number of the results of this study are fundamentally new. In particular, the slowing down of the molecular evolution of the grayling (Thymallidae) is shown. We conclude that the charr (Salvelinus) is one of the modern genera of salmonoids closest to their ancestor. The hypothesis of the phylogenetic proximity of the genera Brachymystax, Hucho, and Salmo has been confirmed. We also discuss the possibility that it is namely the changes in the amino acid sequence of cytochrome oxidase subunit I that lead to postzygotic reproductive isolation between taxa.