Molecular systematics of Salmonidae: combined nuclear data yields a robust phylogeny

The phylogeny of salmonid fishes has been the focus of intensive study for many years, but some of the most important relationships within this group remain unclear. We used 269 Genbank sequences of mitochondrial DNA (from 16 genes) and nuclear DNA (from nine genes) to infer phylogenies for 30 species of salmonids. We used maximum parsimony and maximum likelihood to analyze each gene separately, the mtDNA data combined, the nuclear data combined, and all of the data together. The phylogeny with the best overall resolution and support from bootstrapping and Bayesian analyses was inferred from the combined nuclear DNA data set, for which the different genes reinforced and complemented one another to a considerable degree. Addition of the mitochondrial DNA degraded the phylogenetic signal, apparently as a result of saturation, hybridization, selection, or some combination of these processes. By the nuclear-DNA phylogeny: (1) (Hucho hucho, Brachymystax lenok) form the sister group to (Salmo, Salvelinus, Oncorhynchus, H. perryi); (2) Salmo is the sister-group to (Oncorhynchus, Salvelinus); (3) Salvelinus is the sister-group to Oncorhynchus; and (4) Oncorhynchus masou forms a monophyletic group with O. mykiss and O. clarki, with these three taxa constituting the sister-group to the five other Oncorhynchus species. Species-level relationships within Oncorhynchus and Salvelinus were well supported by bootstrap levels and Bayesian analyses. These findings have important implications for understanding the evolution of behavior, ecology and life-history in Salmonidae.     

Patterns of Historical Balancing Selection on the Salmonid Major Histocompatibility Complex Class II β Gene

Allelic variation in the major histocompatibility class (MHC) IIB gene of salmonids is analyzed for patterns indicative of natural selection acting at the molecular level. Sequence data for the second exon of this MHC gene were generated for 11 species in three salmonid genera: Oncorhynchus, Salmo, and Salvelinus. Phylogenetic analysis of nucleotide sequences revealed: (1) monophyletic grouping of alleles from each genus, (2) transspecies evolution of alleles within Salmo and Salvelinus, and (3) differential patterns of transspecies evolution within the genus Oncorhynchus. Within Oncorhynchus, five of seven species had alleles that were species-specific or nearly so, while the remaining two, O. mykiss and O. clarkii, retained ancestral polymorphisms. The different patterns in Oncorhynchus and the other two genera could be due to historical demographic effects or functional differences in MHC molecules in the three genera, but the two hypotheses could not be distinguished with the current dataset. An analysis of recombination/gene conversion identified numerous recombinant alleles, which is consistent with what has been found in other vertebrate taxa. However, these gene conversion events could not account for the species-specific allelic lineages observed in five of the Oncorhynchus species. Analyses of the relative rates of nonsynonymous and synonymous substitutions revealed the signature of selection on the class IIB gene in all 11 of the salmonid species for both the ABS and the non-ABS codons. Codon-based analyses of selection identified seven codons that have experienced selection in the majority of the species. More than half of these sites were mammalian ABS codons, but several were not, suggesting subtle functional differences in the mammalian and teleost fish MHC molecules.     

A salmon id phylogeny inferred from mitochondrial cytochrome b gene sequences

Partial mitochondrial cytochrome b gene sequences of eight salmonid species were used in a PAUP analysis to generate a phylogeny of the group. The four genera represented are Salmo, Salvelinus, Oncorhynchus and Thymallus . The inferred phylogenetic tree coincides well with the classically derived one for these genera. The recent reclassification of the rainbow trout as a member of the genus Oncorhynchus is supported. The assignment of grayling as the outgroup is vindicated. The utility of gene sequence data to infer the phylogenetic relationships of the Salmonidae is discussed.     

Isolation of a Loma salmonae variant: biological characteristics and host range

A Salvelinus -infecting variant of Loma salmonae , derived from naturally-infected Chinook salmon Oncorhynchus tshawytscha by serial passage through brook trout Salvelinus fontinalis , has been isolated and amplified. Loma salmonae SV ( Salvelinus -variant) has a high preference for species of Salvelinus (brook trout and Arctic charr S. alpinus ) and low virulence and preference for species of Oncorhynchus (rainbow trout O. mykiss , Chinook salmon, cohoSalmon O. kisutch ) or Salmo (Atlantic salmon Salmo salar ). Although this variant of L. salmonae was different from the original, the differences do not justify describing it as a new species, although definitive determination is pending.     

Phylogenetic relationship 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.     

The complete sequence of the mitochondrial genome of the Chinook salmon, Oncorhynchus tshawytscha

The complete sequence of the mitochondrial genome of Chinook salmon, Oncorhynchus tshawytscha, has been determined. The circular genome consisting of 16,644 base pairs encodes thirteen proteins, the 12S and 16S ribosomal RNAs, and 22 transfer RNAs. These genes are ordered in the same way as most other vertebrates. The nucleotide and amino acid sequences of the ribosomal RNAs and the thirteen protein-coding genes were compared with those of other salmonids such as Oncorhynchus mykiss, Salmo salar, Salvelinus fontinalis, Salvelinus alpinus and Coregonus lavaretus. The sequence features of the control region (D-loop), the origin of L-strand replication and a putative peptide codified by the 16S mitochondrial RNA are described and discussed.     

A comparative mitogenomic analysis of the potential adaptive value of Arctic charr mtDNA introgression in brook charr populations (Salvelinus fontinalis Mitchill)

Wild brook charr populations (Salvelinus fontinalis) completely introgressed with the mitochondrial genome (mtDNA) of arctic charr (Salvelinus alpinus) are found in several lakes of northeastern Québec, Canada. Mitochondrial respiratory enzymes of these populations are thus encoded by their own nuclear DNA and by arctic charr mtDNA. In the present study we performed a comparative sequence analysis of the whole mitochondrial genome of both brook and arctic charr to identify the distribution of mutational differences across these two genomes. This analysis revealed 47 amino acid replacements, 45 of which were confined to subunits of the NADH dehydrogenase complex (Complex I), one in the cox3 gene (Complex IV), and one in the atp8 gene (Complex V). A cladistic approach performed with brook charr, arctic charr, and two other salmonid fishes (rainbow trout [Oncorhynchus mykiss] and Atlantic salmon [Salmo salar]) revealed that only five amino acid replacements were specific to the charr comparison and not shared with the other two salmonids. In addition, five amino acid substitutions localized in the nad2 and nad5 genes denoted negative scores according to the functional properties of amino acids and, therefore, could possibly have an impact on the structure and functional properties of these mitochondrial peptides. The comparison of both brook and arctic charr mtDNA with that of rainbow trout also revealed a relatively constant mutation rate for each specific gene among species, whereas the rate was quite different among genes. This pattern held for both synonymous and nonsynonymous nucleotide positions. These results, therefore, support the hypothesis of selective constraints acting on synonymous codon usage.     

Whole genome duplication: challenges and considerations associated with sequence orthology assignment in Salmoninae

To illustrate some of the challenges and considerations in assigning correct orthology necessary for any comparative genomic investigation among salmonids, sequence data from the non-coding regions of different chromosomes in three members of the subfamily Salmoninae, rainbow trout Oncorhynchus mykiss, Atlantic salmon Salmo salar and Arctic charr Salvelinus alpinus, were compared. By analysing c. 55 distinct loci, corresponding to c. 142 kbp sequence information per species, 18 duplicated patterns representative of the two sequential rounds of teleost-specific whole genome duplications (i.e. 3R and 4R WGD) were identified. Sequence similarities between the 4R paralogues were c. 90%, which was slightly lower than those of the 4R orthologues and c. 60% for the 3R products. Through careful examination of the sequence data, however, only 14 loci could reliably be assigned as true orthologues. Locus-specific trees were constructed through maximum parsimony, maximum likelihood and neighbour-joining methods and were rooted using the information from a close relative, lake whitefish Coregonus clupeaformis. All approaches generated congruent trees supporting the {Coregonus [Salmo (Oncorhynchus, Salvelinus)]} topology. The general phenotypic characteristics of sequences, however, were highly suggestive of the basal position of Oncorhynchus, raising the hypothesis of an accelerated rate of nucleotide evolution in this species.     

Seventy‐five EST‐linked Atlantic salmon (Salmo salar L.) microsatellite markers and their cross‐amplification in five salmonid species

An Atlantic salmon (Salmo salar L.) expressed sequence tag (EST) database consisting of 58 146 ESTs was screened for microsatellite sequences. Subsequent development of 75 polymorphic EST‐associated microsatellite markers in this species is described together with cross‐species amplification results of 133 gene‐associated tandem repeat markers in five salmonid species (Salmo trutta, Oncorhynchus mykiss, Salvelinus aplinus, Thymallus thymallus, Coregonus lavaretus). The number of alleles among EST‐linked microsatellites in Atlantic salmon ranged from two to 41 with an average of 12 alleles per locus. Cross‐species amplification resulted in detection of a total of 111 polymorphic locus‐species combinations (12–32 loci per species).     

Vitellogenin isolation,purification and antigenic cross-reactivity in three teleost species

Vitellogenin (Vtg) was isolated from male greenback flounder (Rhombosolea tapirina), rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar) plasma, following induction by estradiol (E(2)) inoculation. The molecular weight of each native molecule, as determined by gel filtration, was 540, 383 and 557 kDa, respectively. With sodium dodecyl sulphate polyacrylamide gel electrophoresis under reducing conditions, Atlantic salmon and greenback flounder Vtg appeared as three major bands (approximately 159, 117, 86 kDa and 155, 104, 79 kDa, respectively), whereas rainbow trout Vtg appeared as one major band (approximately 154 kDa). Several minor bands were also present in each Vtg isolate. Polyclonal antisera, produced against only the highest molecular weight band from each species following excision from reducing gels, were reactive with all major bands in Western blots. In competition ELISA, parallel binding slopes were demonstrated between purified Vtg and plasma from vitellogenic females of the same species, but there was no reaction with plasma from untreated males. These antisera were highly species-specific and little cross-reactivity was noted, even between the two salmonid species. These data suggest that excision of bands from gels is a simple procedure for the preparation of species-specific antisera, and confirm that cross-species assays give highly variable results.     

Genetc divergence and phylogeny of the Salmoninae based on allozyme data

The data on 31–37 allozyme loci in 21 species of nine salmonid genera are used for phylogenetic analysis by seven distance methods and several variants of cladistic analysis. Monophyletic origin for all genera and three sub-families of the Salmonidae is corroborated. The closest phylogenetic relationships are characteristic of Parasalmo and Oncorhynchus (bootstrap support is 88–99%), Brachymystax and Hucho (68–97%), and the clade ( Brachymystax + Hucho )+ Salmo (up to 85%). The patterns of phylogenetic relationships in the group Salmo-Parasalmo-Oncorhynchus are analogous to those in the group Parahucho-Hucho-Brachymystax. The position of Parahucho in phylogenetic trees of the Salmoninae is extremely unstable, although it is most likely associated with the clade ( Brachymystax + Hucho)+Salmo ) or Salvelinus. When using the out group analysis, Salvelinus appears as the earliest branch of the Salmoninae tree, whereas if the molecular clock is assumed, the basal position is occupied by Oncorhynchus. However, the latter genus is probably characterized by a substantially increased rate of molecular evolution.     

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 genus Parahucho, monophyly of Oncorhynchus, and inclusion of Acantholingua ohridana within Salmo. Suprisingly, GH1 intron C (GH1C) did not support the widely accepted hypothesis that Oncorhynchus (Pacific salmon and trout) and Salmo (Atlantic salmon and trout) are sibling genera; GH2C was ambiguous at this node. Previously published data were also examined for support of Salmo and Oncorhynchus as 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.     

Comparison of GNRH3 genes across salmonid genera

Genomic sequences of gonadotropin-releasing hormone genes were amplified and examined for sequence divergence among members of three different genera of the subfamily Salmoninae: rainbow trout (Oncorhynchus mykiss), Atlantic salmon (Salmo salar), and Arctic charr (Salvelinus alpinus). Sequences of GNRH3A and GNRH3B (formerly known as sGnRH1 and sGnRH2) were 97-99% similar in coding regions and 94-98% similar in non-coding regions among genera, but comparisons within species between GNRH3A and GNRH3B were only 90-92% similar in coding regions and 83-89% similar in non-coding regions. Polymorphisms in the parents of mapping families for each species allowed for linkage mapping of the GNRH3B gene in all three species and the GNRH3A gene in rainbow trout. GNRH3B maps to linkage group 6 in rainbow trout, linkage group 16 in Atlantic salmon and linkage group 25 in Arctic charr. GNRH3A mapped to linkage group 30 in rainbow trout.     

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 thymallins, 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 radiated 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 19-16 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 Corergouns ancestral lineages occurred during the same time interval.     

Redescription of Gyrodactylus teuchis Lautraite,Blanc, Thiery,Daniel & Vigneulle, 1999 (Monogenea: Gyrodactylidae); a species identified by ribosomal RNA sequence

Molecular and morphological features of Gyrodactylus specimens from Oncorhynchus mykiss, Salmo trutta and Salmo salar were examined. Sequences from variable region V4 of the small subunit ribosomal RNA gene and the ribosomal RNA internal transcribed spacers, produced by the FRS Marine Laboratory, revealed that these were not the same as other well-characterised Gyrodactylus that are common on European salmonids and were in fact a distinct species. Initial morphological examination of the opisthaptor indicated that this species very closely resembles G. salaris Malmberg, 1957. More detailed analysis revealed differences in the shape of the marginal hook sickle of these two species and thus Gyrodactylus teuchis Lautraite, Blanc, Thiery, Daniel & Vigneulle, 1999 was erected. Analysis of the ribosomal RNA gene or spacer sequences remains the most reliable method of identifying this species. This is believed to be the first record of a Gyrodactylus species identified first from molecular data and confirmed by morphological examination; previous molecular analyses had provided alternative methods for identifying species that had already been described using morphological characters.     

Comparative structures of the apopolysialoglycoproteins from unfertilized and fertilized eggs of salmonid fishes

The complete amino acid sequence of the major polysialoglycoproteins (PSGPs) from two genera of salmonid fish eggs, Salvelinus and Oncorhynchus, has been determined. The occurrence of tandem repeats of a genus-specific dodeca- and tridecapeptide was found for the apoPSGP of Salvelinus leucomaenis pluvius (Slp) and Oncorhynchus masou ishikawai (Omi), respectively, their amino acid sequences being highly homologous with that of rainbow trout [Salmo gairdneri (Sg)] apoPSGP (*denotes the glycosylation site; mean value of N = approximately 25): H-PSGP(Slp): (Asp-Asp-Ala-Thr*-Ser*-Glu-Ala-Ala-Thr*-Gly-Pro-Ser-)N H-PSGP(Omi): (Asp-Asp-Ala-Thr*-Ser*-Glu-Ala-Ala-Thr*-Gly-Pro-Ser-Ser-)N H-PSGP(Sg): (Asp-Asp-Ala-Thr*-Ser*-Glu-Ala-Ala-Thr*-Gly-Pro-Ser-Gly-)N Within 5-7 min following fertilization H-PSGP is converted to the low-molecular-mass PSGP (L-PSGP) by a specific protease (PSGPase). We have purified L-PSGP from the fertilized eggs of S. leucomaenis pluvius and Oncorhynchus keta (chum salmon) and compared it with rainbow trout egg L-PSGP(Sg) by analysis of their amino acid sequence: L-PSGP(Slp): Asp-Ala-Thr*-Ser*-Glu-Ala-Ala-Thr*-Gly-Pro-Ser-Asp L-PSGP(Ok): Asp-Asp-Ala-Thr*-Ser*-Glu-Ala-Ala-Thr*-Gly-Pro-Ser-Ser L-PSGP(Sg): Asp-Asp-Ala-Thr*-Ser*-Glu-Ala-Ala-Thr*-Gly-Pro-Ser-Gly The data support the conclusion that H-PSGP is degraded in vivo 5-7 min after fertilization to L-PSGP by proteolytic cleavage at the position two residues C-terminally to the Pro residue, i.e., -Pro-Ser-Xaa-Asp-(Xaa = either Gly, Ser, or Asp) by the action of PSGPase.     

Application of recA and rpoB sequence analysis on phylogeny and molecular identification of Geobacillus species

Aims:  Some Geobacillus species have highly similar 16S rRNA gene sequences, making 16S rDNA sequence analysis-based identification problematic. To overcome this limitation, recA and rpoB sequence analysis was evaluated as an alternative for distinguishing Geobacillus species.
Methods and Results:  The phylogram of 16S rRNA gene sequences inferred from the neighbour-joining method showed that nine clusters of Geobacillus species were characterized with bootstrap values >90%. The recA and rpoB sequences of 10 reference strains in clusters V, VIb and VIc were amplified and sequenced using consensus primers. Alignment of recA sequences in clusters V, VIb and VIc revealed three types of recA genes, consistent with the putative amino acid sequences and in vivo recA splicing analysis. The phylogram constructed from rpoB sequences showed more divergence than that constructed from 16S rRNA gene sequences.
Conclusions:  recA and rpoB sequence analysis differentiated closely-related Geobacillus species and provided direct evidence for reclassifying some species dubiously categorized as Geobacilli . Additionally, this study revealed three types of recA genes in the different Geobacillus species.
Significance and Impact of the Study:  This study highlights the advantage of recA and rpoB sequence analysis to supplement 16S rRNA gene sequence analysis for efficient and convenient determination of Geobacillus species.     

Nuclear DNA and salmonid phylogenetics

There are many unresolved problems in salmonid systematics, both at the interspecific and sub-specific levels. Some of the major systematic problems in the subfamily Salmoninae are briefly reviewed along with the available molecular methods for their analysis. Nuclear DNA markers available for use in molecular systematics include localized and dispersed highly repetitive DNA sequences, moderately repetitive sequences such as the ribosomal RNA genes (rDNA), and single copy DNA sequences. Both coding and non-coding sequences can be examined in the rDNA and single copy DNA. The rDNA is especially suitable for use in phylogenetic analysis, since different regions evolve at different rates and can be used for comparisons at different taxonomic levels. Comparison of restriction maps of the entire rDNA repeating unit in 17 salmonid species from Hucho. Sahelinus, Salmo and Oncorhynchus has shown that the transcribed spacer regions are the most informative for interspecific comparisons and that the intergenic spacer has potential for use in intraspecific comparisons. Our current approach is to amplify selected regions from each of these spacers for analysis by DNA sequencing. DNA sequence analysis of the internal transcribed spacers should be very informative in elucidating interspecific relationships in Salvelinus and Oncorhynchus . Analysis of a hypervariable region in the intergenic spacer has potential for identification of geographically separated stocks. The relative utility of different types of nuclear DNA sequences for identification of stocks and subspecies is examined.     

High levels of MHC class II allelic diversity in lake trout from Lake Superior

Sequence variation in a 216 bp portion of the major histocompatibility complex (MHC) II B1 domain was examined in 74 individual lake trout (Salvelinus namaycush) from different locations in Lake Superior. Forty-three alleles were obtained which encoded 71-72 amino acids of the mature protein. These sequences were compared with previous data obtained from five Pacific salmon species and Atlantic salmon using the same primers. Although all of the lake trout alleles clustered together in the neighbor-joining analysis of amino acid sequences, one amino acid allelic lineage was shared with Atlantic salmon (Salmo salar), a species in another genus which probably diverged from Salvelinus more than 10-20 million years ago. As shown previously in other salmonids, the level of nonsynonymous nucleotide substitution (dN) exceeded the level of synonymous substitution (dS). The level of nucleotide diversity at the MHC class II B1 locus was considerably higher in lake trout than in the Pacific salmon (genus Oncorhynchus). These results are consistent with the hypothesis that lake trout colonized Lake Superior from more than one refuge following the Wisconsin glaciation. Recent population bottlenecks may have reduced nucleotide diversity in Pacific salmon populations.     

Experimental and natural host specificity of Loma salmonae (Microsporidia)

The microsporidian Loma salmonae (Putz, Hoffman & Dunbar, 1965) Morrison & Sprague, 1981 has caused significant gill disease in Pacific salmon Oncorhynchus spp. Host specificity of the parasite was examined experimentally by per os challenge of selected salmonids and non-salmonids with infective chinook salmon O. tshawytscha gill material. Pink Oncorhynchus gorbuscha and chum salmon O. keta, brown Salmo trutta and brook trout Salvelinus fontinalis, and chinook salmon (controls) were positive, whereas Atlantic salmon Salmo salar and Arctic char Salvelinus alpinus were negative. In addition, no non-salmonids were susceptible to experimental exposure. Wild Pacific salmon species in British Columbia, Canada, were examined for L. salmonae during their freshwater life history stages (smolts, prespawning, spawning). All stages were infected, although infections in smolts were only detectable using a L. salmonae-specific PCR test. Many previous Loma spp. described from Oncorhychus spp. are likely L. salmonae based on host, parasite morphology, and site of infection.