*Département de Biologie, Université du Québec à Rimouski, Rimouski, Québec, Canada;
Département de Biologie, GIROQ, Pavillon Alexandre-Vachon, Université Laval, Sainte-Foy, Québec, Canada
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Evidence of interspecific mitochondrial genome introgression has been accumulating, especially in recent years. Complete mitochondrial genome (mtDNA) replacement in whole populations has been documented for various animal groups, including mammals, amphibians, insects, and fish (see Avise 2000
). Most authors typically attribute the occurrence of introgression to historical demographic events, whereas relatively few consider this occurrence in light of the possible selective advantages in natural populations (Ballard and Kreitman 1994
, 1995
).
Patterns of nonneutral evolution in different mtDNA coding regions have been reported in humans, rats, and Drosophila (Nachman et al. 1996
; Kennedy and Nachman 1998
; Wise, Sraml, and Easteal 1998
; Ballard 2000a
, 2000b
). Furthermore, in a recent study on mammals, Schmidt et al. (2001)
observed that the mtDNA-encoded residues of cytochrome c oxidase that are in close physical proximity to nuclear-encoded residues evolve more rapidly than the other mitochondrial-encoded residues, which suggests positive selection. Therefore, the tacit neutral assumption of interspecific mtDNA introgression appears paradoxical when considering the evidence for nonneutrality as well as the functional importance of the 13 mtDNA-encoded genes involved in the mitochondrial respiratory chain leading to energy production. Given the high sensitivity of mitochondrial metabolism to temperature changes (Blier and Lemieux 2001
) and the potential impact of single amino acid substitution on the functional properties and thermal sensitivity of enzymes (Holland, McFall-Ngai, and Somero 1997
), it is conceivable that the mitochondrial genome may evolve such that animals living in different thermal regimes are able to compensate for the thermal constraints of their environment.
The recent discovery of seven allopatric populations of wild brook charr, Salvelinus fontinalis (Pisces, Salmonidae), in eastern Québec (Canada) that are completely introgressed with mtDNA from arctic charr, S. alpinus, in the apparent absence of nuclear introgression provides a unique model for investigating the possible selective importance of mtDNA introgression (Bernatchez et al. 1995
; Glémet, Blier, and Bernatchez 1998
). The evidence at hand suggests that the respiratory enzymes of the introgressed populations of brook charr are encoded by their own nuclear DNA and by arctic charr mtDNA (Glémet, Blier, and Bernatchez 1998
). Originally, these two species evolved in distinct thermal environments: the brook charr has a more southern distribution and is typically associated with the warmer waters of lacustrine littoral zones, whereas the arctic charr is typically associated with cold water in arctic environments or deep lakes (or both) (Scott and Crossman 1973
; Baroudy and Elliot 1994
). Because introgressed populations of brook charr are found in high-altitude lakes, we could assume that the introgression of a mitochondrial haplotype that partly evolved in a cold environment could give a selective advantage to brook charr populations living at the northern limit of their distribution. The overall mtDNA nucleotide divergence between the two species has been estimated at 3% (Grewe, Billington, and Hebert 1990
), a value comparable to the intraspecific level of divergence reported for most freshwater fishes with a more southern distribution (Bernatchez and Wilson 1998
). Both species show highly reduced mtDNA diversity, with a single haplotype being found at a frequency of 98% (brook charr) and 95% (arctic charr) in populations surveyed in northeastern Canada, where the introgressed brook charr populations are found (Wilson et al. 1996
; Danzmann et al. 1998
).
In this study we perform a comparative sequence analysis of the whole mitochondrial genomes of both brook and arctic charr with the primary objective of localizing the distribution of mutational differences and determining which mtDNA-encoded peptide(s) would be likely to demonstrate specific adaptations to the thermal environment. We then performed a cladistic approach with both S. alpinus and S. fontinalis and two other salmonids, the Atlantic salmon (Salmo salar) and the rainbow trout (Oncorhynchus mykiss), to assign the derived state of the amino acid substitution observed in Salvelinus. We also compare the brook and arctic charr mitochondrial genomes with that of the rainbow trout to further document the relative mtDNA mutation patterns between the two Salvelinus species. Although the analysis of the complete mtDNA variation (mitogenomics) is becoming more common in studies of deep phylogenies (e.g., Curole and Kocher 1999
), few studies, at least on animals, have focused on comparisons at lower levels of divergence, such as within-family. Thus, we quantify the extent of mutations across genes and species. This should be of particular use for future phylogenetic studies in salmonid fishes as well as for our understanding of the pattern of substitution on mtDNA evolution at relatively low levels of divergence.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
PCR Amplification
The entire mitochondrial genomes of brook and arctic charr were amplified using the same primers, except for the D-loop region of arctic charr, which needed different primers. PCR amplifications were performed in 50 µl total volume, with approximately 100 ng of total DNA, 0.5 µl Taq DNA polymerase, 15 mM MgCl2, 100 mM Tris HCl buffer, 1% Triton, 500 mM KCl, 0.2 mM of each dNTP, and 0.8 µM of each primer. The thermal cycles were 3 min at 95°C followed by 32 cycles: denaturation for 1 min at 94°C, annealing for 1 min at 4350°C (depending on the primers), and extension for 12.5 min (depending on fragment size) at 72°C. The last cycle was ended by a termination step of 6 min at 72°C. The first segments amplified encompassed the cox1, cox2, nad/5-6, nad1, and nad2 subunits (total of 9,072 bp). We used the following primerscox1: TyrCOI (5' CTG TTT ATG GAG CTA CAA TC 3') and SerCOI (5' GTG GCA GAG TGG TTA TG 3'); cox2: AspCOII (5' GGT CAA GGC AAA ATT GTG 3') and LysCOII (5' GCT TAA AAG GCT AAC GCT 3'), designed from the mtDNA sequence of rainbow trout (Zardoya, Garrido-Pertierra, and Baustita 1995
); nad5/6: C-leu and C-glu (Park et al. 1993
); nad1: NDI5 and NDI3, unnamed primers described in Hall and Narowrocki (1995)
; and nad2: ND2B-L (5' AAG CTT TCG GGC CCA TAC CC 3') and ND2E-H (5' CCG TTT AGA GCT TTG AAG GC 3'), modified from T. Dowling (Department of Biology, Arizona State University, Tempe, Ariz.).
Direct Sequencing of PCR Products
The double-stranded PCR products were gel-purified in 1% ultra-pure low-melting agarose (GIBCO-BRL), excised from gel under UV light, and purified with a QIAquick Gel Extraction Kit (QIAGEN). The quantities of double-stranded PCR products used for the cycle sequencing reactions were determined according to the Big Dye Terminator Cycle Sequencing Ready Reaction Kit protocol (Applied Biosystems). The resulting cycle-sequencing products were purified with 3 M sodium acetate solution (pH 4.6) in 95% ethanol, followed by two consecutive 70% ethanol washings, and then run on an ABI 377 automated DNA sequencer. Sequencing of the two mtDNA molecules were completed by using the walking primer method with specific primers designed for the sequences obtained. All PCR and cycle-sequencing reactions were performed with the same primers and are available upon request.
Sequence Analysis
Sequence alignment and translation were done with Sequence Navigator (1.0.1) (Applied Biosystems). The sequence comparisons, including nucleotide differences, synonymous substitutions, and total amino acid differences, were carried out with MEGA, version 1.01 (Kumar, Tamura, and Nei 1993
, 130 p.). We also quantified nonconservative amino acids substitution, i.e., the replacement of one amino acid by another belonging to a different amino acid class. These classes are based on the properties of the lateral chain of the amino acid (Lehninger, Nelson, and Cox 1993
, pp. 112116).
The nucleotide, synonymous, and nonsynonymous substitution rates were estimated for the mitochondrial protein-coding genes by comparison with the rainbow trout according to Kimura's (1980)
two-parameter method (Kimura-2p) and by using the Oncorhynchus-Salvelinus divergence time (12%16% sequence divergence/MYr) estimated from IgM gene introns (Andersson et al. 1995
). The comparison of the mitochondrial genomes between each species, which included brook or arctic charr, rainbow trout, and Atlantic salmon, was based on the number of synonymous (Ks) and nonsynonymous (Ka) differences per site calculated with the DNAsp 3.50 program (Rozas and Rozas 2000
) for protein-coding genes. The complete mtDNA sequences of brook and arctic charr have been deposited in the GenBank data library under accession numbers AF154850 and AF154851, respectively.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Divergence Between Brook and Arctic Charr Mitochondrial Genomes: Coding Regions
The nucleotide frequency of the light strand was equivalent between species and had the following percentages (for brook and arctic charr, respectively): A = 28.2% and 28.1%; T = 26.5% and 26.5%; C = 28.3% and 28.5%; G = 16.9% and 16.9%. The overall nucleotide divergence between the two complete mtDNA genomes was 4.6%, a value substantially higher than the 3% estimated by Grewe, Billington, and Hebert (1990)
using RFLP analyses. The alignment between the two complete mtDNA sequences revealed 13 indels. One of these was found within the noncoding space between the nad2 and tRNA-Trp genes, whereas others occurred within the control region. Table 1
presents the comparison of the 13 protein-coding genes between brook and arctic charr. The total nucleotide differences ranged from 3.0% in the atp8 gene to 7.5% in the nad6 gene, whereas the synonymous substitutions ranged from 2.4% in the atp8 gene to 7.1% in the nad6 gene. The transition to transversion ratio was highly variable among genes, ranging from 2.3 in the nad4L gene to 21.5 in the atp6 gene.
|
On the basis of established phylogenetic relationships within the salmonid family, with Salvelinus and Salmo genera being closer than Oncorhynchus (Crane, Seeb, and Seeb 1994
; Lee et al. 1998
; Oakley and Phillips 1999
; Osinov and Lebedev 2000
), we assigned the derived state of all replacement substitutions observed between brook and arctic charr with a cladistic approach using rainbow trout and Atlantic salmon as out-groups. Thus, amino acids observed in charr species were considered as derived character states if they differed from the amino acid at a given gene position that was shared by both out-groups. This exercise indicated that 27 of the 47 diverged amino acids observed between arctic and brook charr are autapomorphic characters in one of the two charr species (table 2
). Seventeen of these 27 substitutions were observed only in brook charr, with the other three salmonids having the same amino acid at these positions; the other 10 substitutions are found only in arctic charr, whereas brook charr, rainbow trout, and Atlantic salmon have the same amino acid at that position. A statistical comparison of the ratios of specific substitutions for both charr species for these 27 substitutions (10/27 and 17/27, for arctic and brook charr, respectively) did not indicate any significant differences between brook charr and arctic charr (chi-square on the scaled overlap of the confidence intervals: P = 0.257; chi-square on frequency table: P = 0.420). Hence, neither brook nor arctic charr demonstrated any bias in the rate of accumulation of the amino substitutions.
|
|
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The occurrence of mutations in mitochondrial-encoded peptides, which are closely associated with the nuclear-encoded genes (complexes I and IV in particular), could indicate where natural selection acts to maintain the physiological function of enzyme complexes and to favor the evolutionary coadaptation of the interaction between mitochondrial peptides (nuclear and mitochondrial encoded, see Schmidt et al. 2001
). Some studies have demonstrated that amino acid substitutions may have an impact on nuclear-mitochondrial interactions and on the mechanisms regulating mitochondrial functioning (reviewed in Blier, Dufresne, and Burton 2001
). Thus, future comparative analyses will be required to determine the extent of nuclear amino acid substitution divergence between the two charr to understand better the potential consequence of mitochondrial introgression between brook and arctic charr on mitochondrial functions.
Evolution of Mitochondrial Genes in Salmonids
Even when considering genes separately, our data demonstrated a highly significant level of correlation (fig. 1
) and a relatively constant mutation rate (table 4
) when we compared the two charr or either charr and rainbow trout (for nucleotide, amino acid, and synonymous substitution comparisons). Comparative studies of mitochondrial gene evolution have shown important differences in mutational accumulation, patterns, and rates of amino acid substitutions among mitochondrial genes and their consequences for deep phylogeny reconstruction (e.g., Mindell and Thacker 1996
; Russo, Takezaki, and Nei 1996
). Our results clearly show that despite the absence of recombination and the haploid nature of mitochondrial DNA, a comparison of closely related species also reveals individual patterns of evolution of the different mitochondrial genes; these patterns seem relatively independent of the phylogenetic distance of species. Therefore, the relatively constant mutational differences observed for each gene between rainbow trout and both charr species suggest that comparable estimates of relative divergence would be obtained regardless of the selected mitochondrial gene segments used for phylogenetic reconstruction among salmonid species.
Given that synonymous substitutions appear to accumulate relatively rapidly in the atp6, cox1, and cob genes (see tables 1
and 4
), it was surprising to find no amino acid substitutions in these genes between brook and arctic charr. Likewise, the highest synonymous differences were not observed in genes with higher numbers of amino acid substitutions. The patterns of divergence (nucleotide and amino acid) of each gene and the relative substitution rates between genes, established during the independent evolution of both charr species, are relatively similar to those observed during the independent evolution of the Oncorhynchus and Salvelinus genera (fig. 1
). Consequently, the substitution rate does not appear to be established randomly. Furthermore, it appears that substitution patterns among different mitochondrial genes were not determined by synonymous substitution rates. Our data (fig. 2 ) suggest a relaxation of purifying selection or the presence of a positive selection in the nad2, nad3, and nad5 genes compared with the more conservative genes such as cox1, cox2, and nad4L. In fact, several studies have reported the possible interaction of functional constraints in the evolution rate of mitochondrial genes (Ballard 2000a,
2000b
). In addition, Rand and Kann (1996)
mention that opposing evolutionary pressures may act on different regions of the mitochondrial genes and genomes. Accordingly, the absence of parallelism in our results between the accumulation rate of amino acids and synonymous substitutions suggests that functional constraints on the substitution rate of the different mitochondrial genes are maintained in different species. Surprisingly, 10 substitutions are inferred to be homoplasious, and eight of these 10 involve only I, V, L, and M (six of these substitutions involve V and I [table 2
]). These amino acids have methylene and methyl groups, and their most important property is that their side chain is hydrophobic. This suggests important structural and functional constraints in the evolution of these sites. Because they constitute more than 20% of the amino acid substitutions observed between brook and arctic charr, these constraints could be highly significant.
There is increasing evidence that selection acts on silent sites in Drosophila mtDNA (Akashi and Schaeffer 1997
; Moriyama and Powell 1997
; Kennedy and Nachmann 1998
). Although closely related species should not be affected by multiple substitutions and should provide a better representation of evolutionary events and substitution dynamics (Meyer 1993
), our results suggest a mutational saturation with increasing gene divergence among closely related species (fig. 2
). The data also indicate that whereas synonymous substitutions accumulate similarly in both species for a given gene, there may be a possibility of important mutational constraints at silent sites among genes, particularly in the atp8 gene, that show a lower synonymous substitution rate than do the other genes (table 4
). Akashi (1994)
supports the possibility that selective constraints act on synonymous codon usage through the translation efficiency.
![]() |
Conclusions |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Acknowledgements |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Footnotes |
---|
Keywords: mitochondria
charr
mtDNA
comparative sequence analysis
Salvelinus
Oncorhynchus
Address for correspondence and reprints: Pierre Blier, Département de Biologie, Université du Québec à Rimouski, 300 allée des Ursulines, Rimouski, Québec, Canada G5L 3A1. pierre_blier{at}uqar.qc.ca
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Akashi H., 1994 Synonymous codon usage in Drosophila melanogaster: natural selection and translational accuracy Genetics 136:927-935
Akashi H., S. W. Schaeffer, 1997 Natural selection and the frequency distribution of "silent" DNA polymorphism in Drosophila Genetics 146:295-307
Andersson E., B. Peixoto, V. Törmänen, T. Matsunaga, 1995 Evolution of the immunoglobulin M constant region genes of salmonid fish, rainbow trout (Oncorhynchus mykiss) and Arctic charr (Salvelinus alpinus): implications concerning divergence time of species Immunogenetics 41:312-315[ISI][Medline]
Arnold M. L., 1992 Natural hybridization as an evolutionary process Annu. Rev. Ecol. Syst 23:237-261[ISI]
Arnold M. L., 1997 Natural hybridization and evolution Oxford University Press, Oxford, N.Y
Avise J. C., 2000 Phylogeography: the history and formation of species Harvard University Press, Cambridge, Mass
Ballard J. W. O., 2000a. Comparative genomics of mitochondrial DNA in members of the Drosophila melanogaster subgroup J. Mol. Evol 51:48-63[ISI][Medline]
. 2000b. Comparative genomics of mitochondrial DNA in Drosophila simulans J. Mol. Evol 51:64-75[ISI][Medline]
Ballard J. W. O., M. Kreitman, 1994 Unraveling selection in the mitochondrial genome of Drosophila Genetics 138:757-772
Ballard J. W. O., M. Kreitman, 1995 Is mitochondrial DNA a stricly neutral marker? TREE 10:485-488
Baroudy E., J. M. Elliott, 1994 The critical thermal limits for juvenile Arctic charr Salvelinus alpinus J. Fish Biol 45:1041-1053[ISI]
Bernatchez L., H. Glémet, C. C. Wilson, R. G. Danzmann, 1995 Introgression and fixation of arctic char (Salvelinus alpinus) mitochondrial genome in an allopatric population of brook trout (Salvelinus fontinalis) Can. J. Fish. Aquat. Sci 52:179-185[ISI]
Bernatchez L., C. C. Wilson, 1998 Comparative phylogeography of nearctic and palearctic fishes Mol. Ecol 7:431-452[ISI]
Blier P. U., F. Dufresne, R. Burton, 2001 Natural selection and the evolution of mtDNA-encoded peptides: insights from studies of protein function and cytonuclear coadaptation Trends Genet 17:400-406[ISI][Medline]
Blier P. U., H. Lemieux, 2001 The impact of the thermal sensitivity of cytochrome oxidase on the respiration rate of Arctic charr red muscle mitochondria J. Comp. Physiol. Biol 171:247-253
Crane P. A., L. W. Seeb, J. E. Seeb, 1994 Genetic relationships among Salvelinus species inferred from allozyme data Can. J. Fish. Aquat. Sci 51: (Suppl. 1) 182-197[ISI]
Curole J. P., T. D. Kocher, 1999 Mitogenomics: digging deeper with complete mitochondrial genomes TREE 14:394-398[Medline]
Danzmann R. G., R. P. Morgan II,, M. W. Jones, L. Bernatchez, P. E. Ihssen, 1998 A major sextet of mitochondrial DNA phylogenetic assemblages extant in eastern North American brook trout (Salvelinus fontinalis): distribution and postglacial dispersal patterns Can. J. Zool 76:1300-1318[ISI]
Glémet H., P. Blier, L. Bernatchez, 1998 Geographical extent of arctic char (Salvelinus alpinus) mtDNA introgression in brook char populations (S. fontinalis) from eastern Québec, Canada Mol. Ecol 7:1655-1662[ISI]
Grewe P. M., N. Billington, P. D. Hebert, 1990 Phylogenetic relationship among members of Salvelinus inferred from mitochondrial DNA divergence Can. J. Fish. Aquat. Sci 47:984-991[ISI]
Hall H. J., L. W. Narowrocki, 1995 A rapid method for detecting mitochondrial DNA variation in the brown trout, Salmo trutta L J. Fish Biol 46:360-364[ISI]
Heiser C. B. Jr., 1973 Introgression re-examined Bot. Rev 15:645-687
Holland L. Z., M. McFall-Ngai, G. N. Somero, 1997 Evolution of lactate dehydrogenase-A homologs of barracuda fishes (genus Sphyraena) from different thermal environments: differences in kinetic properties and thermal stability are due to amino acid substitutions outside the active site Biochemistry 36:3207-3215[ISI][Medline]
Jones D. T., W. R. Taylor, J. M. Thornton, 1994 A mutation data matrix for transmembrane proteins FEBS Lett 339:269-275[ISI][Medline]
Kennedy P., M. W. Nachman, 1998 Deleterious mutations at the mitochondrial ND3 gene in South American marsh rats (Holochilus) Genetics 150:359-368
Kimura M., 1980 A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences J. Mol. Evol 16:111-120[ISI][Medline]
Kumar S., K. Tamura, M. Nei, 1993 MEGA: molecular evolutionary genetics analysis Version 1.01. Pennsylvania State University, University Park
Lee J.-Y., T. Tada, I. Hirono, T. Aoki, 1998 Molecular cloning and evolution of transferrin cDNAs in salmonids Mol. Mar. Biol. Biotechnol 7:287-293[ISI][Medline]
Lehninger A. L., D. L. Nelson, M. M. Cox, 1993 Principles of biochemistry. 2nd edition Worth Publishers, New York
Meyer A., 1993 Evolution of mitochondrial DNA in fishes Pp. 138 in P. W. Hochachka and T. P. Mommsen, eds. Biochemistry and molecular biology of fishes, Vol. 2. Elsevier, Amsterdam
Mindell D. P., C. E. Thacker, 1996 Rates of molecular evolution: phylogenetic issues and applications Annu. Rev. Ecol. Syst 27:279-303[ISI]
Moriyama E. N., J. R. Powell, 1997 Synonymous substitution rates in Drosophila: mitochondrial versus nuclear genes J. Mol. Evol 45:378-391[ISI][Medline]
Nachman M. W., W. M. Brown, M. Stoneking, F. Aquadro, 1996 Nonneutral mitochondrial DNA variation in humans and chimpanzees Genetics 142:953-963
Oakley T. H., R. B. Phillips, 1999 Phylogeny of Salmonine fishes based on growth hormone introns: Atlantic (Salmo) and Pacific (Oncorhynchus) salmon are not sister taxa Mol. Phylogenet. Evol 11:381-393[ISI][Medline]
Osinov A. G., V. S. Lebedev, 2000 Genetic divergence and phylogeny of the Salmoninae based on allozyme data J. Fish Biol 57:354-381[ISI]
Park L. K., M. A. Brainard, D. A. Dightman, G. A. Winans, 1993 Low levels of intraspecific variation in the mitochondrial DNA of chum salmon (Oncorhynchus keta) Mol. Mar. Biol. Biotechnol 2:362-370[Medline]
Pesole G., C. Gissi, A. De Chirico, C. Saccone, 1999 Nucleotide substitution rate of mammalian mitochondrial genomes J. Mol. Evol 48:427-434[ISI][Medline]
Rand D. M., L. M. Kann, 1996 Excess amino acid polymorphism in mitochondrial DNA: contrasts among genes from Drosophila, mice, and human Mol. Biol. Evol 13:735-748[Abstract]
Rozas J., R. Rozas, 2000 DNA sequence polymorphism. Version 3.5 Departament de Genètica, Universitat de Barcelona, Barcelona
Russo C. M., N. Takezaki, M. Nei, 1996 Efficiencies of different gene and different tree-building methods in recovering a known vertebrate phylogeny Mol. Biol. Evol 13:525-536[Abstract]
Schmidt T. R., W. Wu, M. Goodman, L. I. Grossman, 2001 Evolution of nuclear- and mitochondrial-encoded subunit interaction in cytochrome c oxidase Mol. Biol. Evol 18:563-569
Scott W. B., E. J. Crossman, 1973 Freshwater fishes of Canada Bull. Fish. Res. Board Can 184:966.
Wilson C. C., P. D. N. Hebert, J. D. Reist, J. B. Dempson, 1996 Phylogeography and postglacial dispersal of artic charr Salvelinus alpinus in North America Mol. Ecol 5:187-197[ISI]
Wise C. A., M. Sraml, S. Easteal, 1998 Departure from neutrality at the mitochondrial NADH dehydrogenase subunit 2 gene in humans, but not in chimpanzees Genetics 148:409-421
Zardoya R., A. Garrido-Pertierra, J. M. Baustita, 1995 The complete nucleotide sequence of the mitochondrial DNA genome of the rainbow trout, Oncorhynchus mykiss J. Mol. Evol 41:942-951[ISI][Medline]