Genes Genet. Syst. (1997) 72, p. 25–34

Phylogenetic relationship of the genus
Oncorhynchus species inferred from
nuclear and mitochondrial markers
Takashi Kitano1,2, Norimasa Matsuoka1,*, and Naruya Saitou2
1

Department of Biology, Faculty of Science, Hirosaki University,
3 Bunkyo-cho, Hirosaki, Aomori 036, and2
Laboratory of Evolutionary Genetics, National Institute of Genetics,
1111 Yata, Mishima, Shizuoka 411, Japan(Received 24 October, accepted 12 February 1997)

The phylogenetic relationship among the salmonid fishes of the genus
Oncorhynchus has been analyzed using various kinds of markers for a long time.
However, there are three major disagreements among those studies; (1) the
authenticity of the Pacific salmon group as a monophyletic cluster, (2) thephylogenetic relationship among three Pacific salmons (pink salmon, sockeye
salmon, and chum salmon), and (3) the phylogenetic position of masu salmon. We
used allozyme electrophoresis to clarify the phylogenetic relationship between the
Pacific salmon group and the Pacific trout group. Furthermore, we reanalysed
published mitochondrial DNA D-loop sequences (Shedlock et al., 1992). Allozymic
data and mtDNA data indicated the following consistent results; (1) all Pacificsalmons formed a monophyletic cluster, (2) chum salmon and pink salmon wereclustered within those Pacific salmons, (3) masu salmon formed a cluster with other
Pacific salmons and diverged first in this group.
INTRODUCTION
The salmonid fish (the family Salmonidae) are distributed in a relatively cold water of northern hemisphere andnormally have anadromous life histories, that is, naturally
spawn only in some definite regions in the upper reachesof rivers or streams. These fish are also one of the mostimportant resources as commercial or game fish in Japan
as well as in Canada, North America, and eastern Siberia.
The genus Oncorhynchus belongs to the family
Salmonidae and have been the subjects of numerous phylogenetic studies using morphology (Hikita, 1962; Smithand Stearley, 1989), allozyme electrophoresis (Utter et al.,1973), restriction fragment length polymorphism [RFLP]
of ribosomal RNA genes [rDNA] (Phillips et al., 1992),
RFLP of mitochondrial DNA [mtDNA] (Thomas et al.,1986), HindIII fragment (ATPase 6, CO III, tRNAGLY, ND
3, tRNAARG, and ND 4L) sequences of mtDNA (Thomas andBeckenbach, 1989), D-loop sequences of mtDNA (Shedlock
et al., 1992), ATPase 6 and ND 3 sequences of mtDNA(Domanico and Phillips, 1995), and short interspersed repetitive elements [SINEs] (Kido et al., 1991; Murata et al.,
1993; Takasaki et al., 1996). These studies are in generalagreements that Oncorhynchus had branched into fourgroups; (1) the Pacific trout group that consists of rainbow* Corresponding author.

trout (O. mykiss; nijimasu) and cutthroat trout (O. clarki),(2) coho salmon (O. kisutch; ginzake) and chinook salmon(O. tschawytscha; masunosuke) of the Pacific salmon
group [we call them A-lineage in this paper], (3) pinksalmon (O. gorbuscha; karafutomasu), sockeye salmon (O.
nerka; benizake), and chum salmon (O. keta; sake) of the
Pacific salmon group [we call them B-lineage in this paper], and (4) masu salmon (O. masou; yamame) of the Pacific salmon group (Fig. 1). The Pacific trout group inhabits only western America, while the Pacific salmon group
except masu salmon inhabits the whole area of the northern Pacific Ocean. Masu salmon has a restricted geographical distribution mainly in the Japanese archipelago. However, the phylogenetic relationship among
above four groups differs depending on data.
In this study, we analyzed 19 allozyme loci by using protein electrophoresis for inferring the phylogenetic relationship among the four Oncorhynchus groups: the Pacifictrout group, A- and B-lineages of the Pacific salmon groupand masu salmon. It should be noted that Utter et al.
(1973) used only 12 loci, and only 2 loci are overlapped
among the 19 loci used in the present study. They simply
computed similarities for interspecies comparisons, while
we used the standard genetic distance (D) (Nei, 1972,1978) to construct the phylogenetic tree.
Because mtDNA does not recombine, the same topology
is expected to be obtained among different regions of an

26

T. KITANO et al.

mtDNA genome. However, the result of D-loop sequences (Shedlock et al., 1992) is different from other
mtDNA studies. We thus reexamined the mtDNA D-loop
sequences data and compared with our allozyme data tohelp clarify the phylogenetic relationships among those
Oncorhynchus species.
MATERIALS AND METHODS
Allozyme electrophoresis. Eight rainbow trout, fourpink salmon, and two coho salmon individuals werebought from markets, while ten masu salmon were caught
in Kamikasuo, Tochigi Prefecture. Two Japanese charr(Salvelinus leucomaenis; iwana) caught in Oorakumae
river of Aomori Prefecture were also used as the outgroup.
Muscles, livers, and intestines of those fish were individually homogenized with the 20 mM phosphate buffer,
pH 7.0, containing 0.1 M KCl and 20 mM EDTA under acold temperature. The clear supernatants of homogenates obtained by centrifugation (15,000 rpm, 10 min,4°C) were stored at -40°C before electrophoresis.
Polyacrylamide gel (7.5%) was used for electrophoresis
with the electrode buffer (0.38M Glycine-Tris, pH 8.3).
The following thirteen enzymes were analyzed; αglycerophosphate dehydrogenase (α-GPDH), glucose-6phosphate dehydrogenase (G6PD), octanol dehydrogenase(ODH), nothing dehydrogenase (NDH), formaldehyde
dehydrogenase (FDH), peroxidase (PO), superoxide
dismutase (SOD), aspartate aminotransferase (AAT),
phosphoglucomutase (PGM), esterase (EST), alkaline
phosphatase (ALK), leucine aminopeptidase (LAP), andfumarase (FUM).
To estimate the genetic variability within and between
species, average heterozygosity (H), genetic identity (I),and standard genetic distance (D) described by Nei (1972,1978) were calculated from the allele frequency data.
The phylogenetic trees were constructed by using theneighbor-joining method (Saitou and Nei, 1987) and bootstrap tests with 1000 replications (resampling loci) were
performed. A series of programs developed by N. S. wereused for assisting those phylogenetic analyses.
Nucleotide sequences of mtDNA. The D-loop sequence of rainbow trout was retrieved from the DDBJ/
EMBL/GenBank international nucleotide sequence database under the accession number S68946 (Shedlock et al.,1992). D-loop sequences of the other salmonid species
were obtained from published papers (Shedlock et al.,1992). CLUSTAL W version 1.6 (Thompson et al., 1994)
was used for multiple alignment of the D-loop sequence
data. The phylogenetic trees were constructed by using
the neighbor-joining method (Saitou and Nei, 1987), themaximum likelihood method (Felsenstein, 1981), and the
maximum parsimony method (Fitch, 1977). Split decomposition method (Bandelt and Dress, 1992) was also used.

Bootstrap tests with 1000 replications were performed
for the neighbor-joining analysis. Computer packages
MEGA version 1.0 (Kumar et al., 1993), MOLPHY version
2.2 (Adachi and Hasegawa, 1994), PAUP version 3.1.1 (acommercial product distributed from Illinois Natural History Servey), and SplitsTree version 1.0 (developed by D.
Huson and R. Wetzel) were used for assisting the neighbor-joining, the maximum likelihood, the maximum parsimony, and the split decomposition analyses, respectively.
RESULTS AND DISCUSSION
Allozyme electrophoresis. Table 1 shows allele frequencies for 19 genetic loci in five salmonid species.
Monomorphism was observed at the Po locus for fivesalmonid species and at the Aat locus for four
Oncorhynchus species. Sod-2 and Lap-1 were detected
only in rainbow trout and masu salmon, respectively (datanot shown). Only two loci (Sod-1 and Pgm) overlappedwith previous study (Utter et al., 1973).
Genetic identities (I) and standard genetic distances (D)among the five salmonid species are shown in Table 2.
The I values range from 0.161 ± 0.083 (pink salmon - Japanese charr) to 0.504 ± 0.119 (pink salmon - coho salmon),and the D values range from 0.685 ± 0.236 (pink salmon coho salmon) to 1.829 ± 0.515 (pink salmon - Japanesecharr). The average heterozygosity (H) values are also
shown in Table 2, and they range from 0.035 for coho
salmon to 0.084 for pink salmon and masu salmon.
These values are within the range of those for various
fishes species previously reported (e.g., Gyllensten, 1985).
The phylogenetic tree for the four Oncorhynchus species
was constructed from the matrix of D (Table 2) as shown in
Fig. 2. The tree was rooted by applying the method described by Ishida et al. (1995), where constancy of evolutionary rate was assumed on the specified tree topology.
In this tree, pink salmon and coho salmon formed a cluster
and the phylogenetic position of rainbow trout indicatedthat the species diverged earlier among the Oncorhynchus
species. The bootstrap values of both internal branches
were 100%. The unrooted topology of this tree is thesame as that of Utter et al. (1973), though the location of
the root is different. This inconsistency is probably because they used UPGMA.
The origination time (t) of Oncorhynchus species was estimated by the equation described by Nei (1987) (t = 5 × 106
D) from the tree (Fig. 2). The D value (1.02) of the first
divergence among Oncorhynchus species was estimatedunder the assumption of molecular clock using the method
described by Ishida et al. (1995). The time was thus estimated to be about 5 Ma (million years ago) within the
Pliocene. The Oncorhynchus species is distributed onlyin the northern Pacific Ocean and they probably penetrated from the Atlantic Ocean through the Bering
Seaway. Briggs (1970) suggested that the north PacificPhylogeny of the genus Oncorhynchus fishes
Table 1.

27

Allele frequencies at 19 genetic loci in five salmonid species

species*

species*

Locus

A**

1

2

3

4

5

Locus

A**

1

2

3

4

5

α-gpdh

a
b
c
d

1.00
–
–
–
(16)

1.00
–
–
–
(20)

–
0.88
0.12
–
(8)

–
1.00
–
–
(4)

–
–
–
1.00
(4)

Est-2

a
b
c
d
e

–
1.00
–
–
–
(12)

1.00
–
–
–
–
(2)

–
–
–
1.00
–
(8)

–
–
1.00
–
–
(2)

–
–
–
–
1.00
(4)

G6pd

a
b

1.00
–
(16)

–
1.00
(20)

–
1.00
(8)

–
1.00
(4)

1.00
–
(4)

Est-3

a
b
c

a
b
c

–
1.00
–
(16)

–
1.00
–
(20)

1.00
–
–
(8)

–
1.00
–
(4)

–
–
1.00
(4)

–
1.00
–
(16)

1.00
–
–
(20)

–
1.00
–
(8)

1.00
–
–
(4)

–
–
1.00
(4)

Est-4

a
b

1.00
–
(16)

1.00
–
(20)

1.00
–
(8)

–
1.00
(4)

–
1.00
(4)

a
b
c

1.00
–
–
(16)

–
–
1.00
(20)

–
1.00
–
(8)

–
1.00
–
(4)

–
–
1.00
(4)

Est-5

Fdh

a
b

–
1.00
(16)

1.00
–
(20)

1.00
–
(8)

–
1.00
(4)

–
1.00
(4)

a
b
c
d
e
f

–
–
0.12
0.44
–
0.44
(16)

–
0.80
0.10
–
0.10
–
(20)

–
–
1.00
–
–
–
(8)

–
–
1.00
–
–
–
(4)

1.00
–
–
–
–
–
(4)

Po

a

1.00
(12)

1.00
(20)

1.00
(8)

1.00
(4)

1.00
(4)

Alk

Sod-1

a
b
c
d
e
f
g

–
–
–
–
0.94
0.06
–
(16)

–
–
–
1.00
–
–
–
(20)

0.37
–
0.63
–
–
–
–
(8)

–
1.00
–
–
–
–
–
(4)

–
–
0.50
–
–
–
0.50
(4)

a
b
c
d

–
–
1.00
–
(16)

1.00
–
–
–
(12)

–
0.88
–
0.12
(8)

–
1.00
–
–
(4)

1.00
–
–
–
(2)

Lap-2

a
b
c
d

–
–
1.00
–
(16)

–
1.00
–
–
(20)

–
–
0.50
0.50
(8)

0.50
–
0.50
–
(4)

–
1.00
–
–
(4)

Aat

a
b

–
1.00
(16)

–
1.00
(20)

–
1.00
(8)

–
1.00
(4)

1.00
–
(4)

Lap-3

a
b
c

0.69
–
0.31
(16)

0.89
0.11
–
(18)

–
1.00
–
(4)

1.00
–
–
(2)

–
0.50
0.50
(4)

Pgm

a
b
c
d

–
–
1.00
–
(10)

1.00
–
–
–
(20)

–
–
–
1.00
(8)

–
1.00
–
–
(4)

–
–
1.00
–
(4)

Lap-4

a
b
c

1.00
–
–
(6)

–
0.50
0.50
(4)

1.00
–
–
(8)

1.00
–
–
(4)

1.00
–
–
(4)

a
b

1.00
–
(10)

0.70
0.30
(20)

–
1.00
(6)

–
1.00
(4)

1.00
–
(4)

Fum

a
b

–
1.00
(16)

–
1.00
(20)

1.00
–
(8)

–
1.00
(4)

–
1.00
(4)

Odh

Ndh

Est-1

Numbers of genes sampled (2N) for each locus are in parentheses. A hyphen indicates zero frequency.
* 1: rainbow trout, 2: masu salmon, 3: pink salmon, 4: coho salmon, 5: Japanese charr.
** Allele names. They are alphabetically designated starting from “a” with increasing the electrophoretic mobility.

and Atlantic Oceans were isolated from each other fromthe late Mesozoic (200 Ma) until the late Cenozoic when
two periods of trans-Arctic exchange occurred. The first
period took place about 10-12 Ma during a brief Bering

Seaway connection between the two ocean basins. The
second period occurred during the most recent Bering Seaway opening some 3-5 Ma. The origination time ofOncorhynchus species estimated by allozyme data is con-

28

T. KITANO et al.
Table 2. Genetic identities, standard genetic distances, and average heterozygosities
among five salmonid species based on 19 genetic locirainbow

masu

pink

coho

charr

rainbow

0.061± 0.0380.417± 0.1160.319± 0.1090.405± 0.1130.404± 0.117

masu

0.874
± 0.279

0.084± 0.0420.317± 0.1070.409± 0.1140.329± 0.112

pink

1.141
± 0.3411.148± 0.3390.084± 0.0420.504± 0.1190.161± 0.083

coho

0.904
± 0.280

0.894± 0.279

0.685± 0.2360.035± 0.0350.278± 0.108

charr

0.907
± 0.2901.112± 0.3391.829± 0.5151.281± 0.3880.070± 0.048Note. Genetic identities are above the diagonal, standard genetic distances are below thediagonal, and average heterozygosities (H) are on the diagonal. Numbers with ± arestandard errors.

Fig. 1. The scheme of the relationship for the Oncorhynchus species from various kind of data. The strictly consensus relationship ofOncorhynchus species from morphological, allozyme, rDNA, mtDNA, and SINEs data. Names in parentheses are English common names
and Japanese scientific names.

sistent with the second period. It is speculated that
Oncorhynchus species penetrated and radiated in thenorthern Pacific Ocean during the second trans-Arctic
connection. The fossil record of masu salmons also suggests that Oncorhynchus species existed in the late Cenozoic (Uyeno et al., 1975).

Fig. 2. The neighbor-joining tree for the five salmonid species
constructed from Nei’s standard genetic distances based on 19
genetic loci. The value given on each branch is the bootstrap probability (in %).

Nucleotide sequences of mtDNA. We first realigned
the D-loop sequences for nine salmonid species. To obtain a more optimal alignment, profile alignment menu in
CLUSTAL W program was used. In this procedure, thealignment of sequences for closely related three Pacificsalmons (sockeye salmon, chum salmon, and pink salmon)
was first performed. Next, five sequences of other
Oncorhynchus species were joined to the aligned three sequences and finally the sequence of Atlantic salmon(Salmo salar) was joined. The resultant multiple alignment has the total length much shorter (about 80 sites)
than that described by Shedlock et al. (1992). Shedlock etPhylogeny of the genus Oncorhynchus fishesal. (1992) treated gaps in the alignment as fifth character
states for their maximum parsimony analysis, while weeliminated gaps. Unstable sites (the region immediately
downstream of the tRNAPHE gene, that is after position
957) were eliminated following Shedlock et al. (1992).
The remaining 906 nucleotide sites (913 sites when only
eight Oncorhynchus species were analysed) were used forthe following phylogenetic analyses. Variant sites are
only shown (Table 3).
Because transitional nucleotide substitutions seem to bemuch more frequent than transversional substitutions in
mtDNA (Brown et al., 1982), we first calculated the number of nucleotide substitutions among salmonid species byusing the two-parameter method (Kimura, 1980) and constructed the phylogenetic tree by using the neighborjoining tree. A method described by Ishida et al. (1995)
was then applied to obtain integer-value branch lengths.
However, the total number (255) of nucleotide substitutions inferred from this procedure was smaller than that of
the smallest possible number (258). This underestimation was probably caused by the assumption of uniform
evolutionary rate at every site. We thus used the twoparameter method described by Kimura (1980) with agamma distribution model (Jin and Nei, 1990) to obtain amore reasonable distance matrix. The parameter a(0.723) in the gamma distribution was estimated by themethod described by Tamura and Nei (1993). The mean
(0.2859) and variance (0.3988) of the estimated number ofTable 3.

29

nucleotide substitutions per site were estimated by parsimony analysis of first obtained 9 salmonid species tree.
Resultant pairwise distances were shown in the lower diagonal of Table 4. Those values were considerably larger
than proportions of nucleotide difference (upper diagonalof Table 4). This indicates that some parallel nucleotidechanges, multiple substitutions, or back substitutions exist and needed to use a suitable corrected distance matrix
method.
The resulting tree has the same topology as that obtained previously using the two-parameter method described by Kimura (1980) without gamma distribution. A
total of 277 nucleotide substitutions was estimated by applying this procedure (Fig. 3). In this tree, the genus
Oncorhynchus branched into the following four groups:
the Pacific trout group (rainbow trout and cutthroat
trout), the A-lineage of the Pacific salmon group (cohosalmon and chinook salmon), the B-lineage of the Pacificsalmon group (pink salmon, sockeye salmon, and chumsalmon), and masu salmon. All Pacific salmon species
formed a cluster. The tree indicated the same relationship with that estimated from the present allozyme data
(Fig. 2). However, the bootstrap value of each branch
was not so high.
We also used the maximum likelihood and the maximum parsimony methods. Because Atlantic salmon wasmuch divergent from other Oncorhynchus species, we used
only eight Oncorhynchus species for these analyses. All

Sequence comparison of the Realigned D-loop region of nine salmonid mtDNAs

Data from the nucleic acid sequence data bases and Shedlock et al. (1992). Sequence identity is indicated by a dot, and a gap isindicated by a hyphen. At position 764 of Salmo salar, Y = C or T.

30

T. KITANO et al.
Table 4.
rainbow
rainbow

Sequence divergences of D-loop sequences among nine salmonid species

cutthroatchinook

coho

chum

sockeye

pink

masou

Atlantic salmon

0.02980.03200.04080.04300.03200.0596

0.04300.0751

0.05190.04750.05960.05080.07400.06400.09490.03970.05520.03420.07280.0574

0.09380.06620.04080.08170.0596

0.09600.04300.0651

0.06400.09490.05630.0552

0.09160.0806

0.1170cutthroat

0.0315chinook

0.03420.0575coho

0.04430.05180.0431chum

0.04630.06630.06120.0749sockeye0.03390.05580.03650.0441

0.0464pink

0.06610.08480.0831

0.09500.07300.0620masu

0.04670.07270.06450.06700.07230.0613

0.0934

Atlantic salmon

0.08580.11300.11190.11480.1126

0.10820.14480.10150.1232

Values above diagonal are proportions of nucleotide difference (p-distances) and those below diagonal are gamma distances with
Kimura 2-parameter modelFig. 3. The neighbor-joining tree for the nine salmonid species constructed from D-loop sequence data using gamma distances with
Kimura’s 2-parameter model. The value given to each branch is the bootstrap probability (in %).

the 10,395 possible tree topologies for 8 sequences weresearched by using exhaustive options of MOLPHY and
PAUP, and the results for the top 10 trees with highest
likelihoods are presented in Table 5. The rainbow troutcutthroat trout cluster and the chum salmon-pink salmon
cluster were observed in all the 10 trees. The topology ofthe maximum likelihood tree (tree 1) was identical with
the neighbor-joining tree of Fig. 3. Three equally maximum parsimonious trees (trees 1, 2 and 5) were foundwhen transitions and transversions were not weighted(transitions/transversions ratio is 1.0). One of them had
the same topology with the maximum likelihood tree.
Because the estimated transitions/transversions ratio was

4.07 in the maximum likelihood analysis, we also used ratios 2.0 and 4.0 in the maximum parsimony analysis.
Trees 1 and 2 were again equally parsimonious trees inboth cases, while tree 5 was no longer parsimonious (seeTable 5). The topology of the tree 2 was different only on
the shortest branch (bootstrap value 16 %) from tree 1.
We also constructed the phylogenetic tree of six
Oncorhynchus species (rainbow trout, cutthroat trout,coho salmon, chinook salmon, pink salmon, and sockeye
salmon) by using the neighbor-joining and the maximum
likelihood methods based on HindIII fragment sequences(Thomas and Beckenbach, 1989). The resultant trees
had the identical topology with that of the tree in Fig. 3

Phylogeny of the genus Oncorhynchus fishes
Table 5.

31

The maximum likelihood and the maximum parsimony analyses of various kind of
topologies for eight Oncorhynchus species

Tree

Topology*∆ln L

RNM1.0RNM2.0

RNM4.01((my,cl),((ki,ts),((ke,go),ne)),ma)

0.0

196

263

397

2

(((my,cl),((ke,go),ne)),(ki,ts),ma)

–1.8

196

263

397

3

((my,cl),(ki,ts),(ma,((ke,go),ne)))

–2.6

197

264

398

4

((my,cl),((ki,ts),ne),(ma,(ke,go)))

–4.0

197

264

398

5

((my,cl),(((ki,ts),ne),(ke,go)),ma)

–4.1

196

264

400

6

((my,cl),((ki,ne),ts),(ma,(ke,go)))

–6.1

198

265

399

7

((my,cl),(((ki,ne),ts),(ke,go)),ma)

–6.5

197

265

401

8

(((my,cl),(ke,go)),((ki,ts),ne),ma)

–7.2

197

265

401

9

((my,cl),(ki,(ts,((ke,go),ne))),ma)

–7.5

197

265

401

10

((my,cl),((ki,((ke,go),ne)),ts),ma)

–8.5

199

267

403

∆ln L : The difference in log-likelihood from the ML tree (–2334.6).
RNM : Required number of mutations when the maximum parsimony method was applied. The
number followed RNM means the transition/transversion ratio used. Bold letters are minimal
trees for each case.
*Species abbreviations are my : O. mykiss (rainbow trout), cl : O. clarki (cutthroat trout),
ne : O. nerka (sockeye salmon), ts : O. tschawytscha (chinook salmon), ki : O. kisutch (cohosalmon), ke : O. keta (chum salmon), go : O. gorbuscha (pink salmon), ma : O. masou (masusalmon).

Fig. 4. A split decomposition network for the eight Oncorhynchus species based on proportions of nucleotide difference (Table 4). Edges
were drawn to scale.

and bootstrap values were quite high at all branches (datanot shown). However, two Oncorhynchus species (chumsalmon and masu salmon) and an outgroup species (e.g.,
Atlantic salmon) were not included. Therefore HindIII
fragment sequences were not so useful to resolve the phylogenetic problems in this study.
The split decomposition network of eight Oncorhynchus
species was shown in Fig. 4. In this network, six parallelograms were observed and many tree topologies areembedded in this figure. There are three clusters different from the neighbor-joining tree (Fig. 3) in the network;(I) cutthroat trout and coho salmon, (II) coho salmon,
chinook salmon, and sockeye salmon, and (III) pinksalmon and sockeye salmon. However, these three clusters are supported only by a few nucleotide sites. Cluster
I is supported by 4 sites (177, 224, 227, and 730), cluster II
is supported by 1 site (155), and cluster III is supported by
3 sites (171, 345, and 660) (see Table 3). In this study
sequences of mtDNA were used and the possibility of generecombination is eliminated. Thus those sites probably
appeared by some parallel nucleotide changes, multiple
substitutions, or back substitutions. On the other hand,the position of masu salmon is not definite in this network.
The bootstrap value of that branch in the neighbor-joining
tree (Fig. 3) is also quite low.
We estimated the rate (λ) of nucleotide substitution in32

T. KITANO et al.
salmon, and the pacific trout group) differs depending ondata (Table 6A). Allozyme (Utter et al., 1973) and RFLP
of rDNA (Phillips et al., 1992) data suggest that A- and Blineages of the Pacific salmon group form a cluster andmasu salmon forms a cluster with the Pacific trout group(topology I). Morphology (Smith and Stearley, 1989) and
SINEs (Takasaki et al., 1996) data suggest that masu
salmon forms a cluster with A- and B- lineages of Pacificsalmons and diverged first (topology II). Morphology(Hikita, 1962) data suggest that masu salmon forms acluster with A-lineage of the Pacific salmon group (topology III). D-loop sequences of mtDNA (Shedlock et al.,1992) suggest that masu salmon forms a cluster with the
B-lineage of the Pacific salmon group (topology IV).
RFLP of mtDNA (Thomas et al., 1986), HindIII fragment
sequences of mtDNA (Thomas and Beckenbach, 1989),
and ATPase 6 and ND 3 sequences of mtDNA (Domanicoand Phillips, 1995) do not contain masu salmon, therefore
these results were not shown.
The phylogenetic relationship within the B-lineage ofPacific salmons (pink salmon, sockeye salmon, and chumsalmon) is also not clear (Table 6B). Data based on morphology, allozyme, rDNA, and D-loop sequences of mtDNA
suggest that pink salmon and sockeye salmon are sister
species (topology I), while data on RFLP of mtDNA, ATPase 6 and ND 3 sequences of mtDNA (Domanico andPhillips, 1995), and SINEs suggest that pink salmon andchum salmon are sister species (topology II).
The phylogenetic tree constructed from the mtDNA reanalyses in this study (Fig. 3) indicated the same relation-

the mtDNA D-loop region under the assumptions of
the constant evolutionary rate. The equation d = 2λt wasused, where d is the number of nucleotide substitutions
per site between a pair of sequences, and t is the divergence time. As mentioned above, the last Bering Seaway
opening occurred during some 3-5 Ma and it was suggested that Oncorhynchus species penetrated and radiated into the northern Pacific Ocean at around that period.
Therefore, the time (t) of the root of the tree of Fig. 3
was assumed to be 3-5 Ma. The number of nucleotide
substitutions per site of the first divergence among
Oncorhynchus species (d = 0.028) was estimated by applying the method described by Ishida et al. (1995). Thus
the rate (λ = d/2t) becomes 0.2 - 0.4 × 10–8 per nucleotidesite per year. This value is considerably smaller thanthat of human mtDNA (2.5 - 15 × 10–8 estimated by
Tamura and Nei, 1993) and horse mtDNA (2 - 4 × 10–8 estimated by Ishida et al., 1995). Thomas and Beckenbach(1989) suggested that the evolutionary rate of mtDNA in
salmonid fishes is considerably slower than that inprimates. Martin et al. (1992) suggested that rates of
evolution for mtDNA in sharks may be 5 - 7 times slower
than that for mammals, because of the different metabolic
physiology between poikilotherms and homoiotherms.
The value estimated in this study is consistent with above
two studies.
Comparison of various studies. The phylogenetic relationship among four groups (A-linage of the pacificsalmon group, B-linage of the pacific salmon group, masu

Table 6. Comparisons of various studies
A: The phylogenetic relationship among four groups
Various studies*
1
Topology I
Topology II
Topology III
Topology IV((A,B),(M,P))(((A,B),M),P)(((A,M),B),P)(((B,M),A),P)

2

3

4

◯

◯

6

◯

6'

8

9

◯

◯

◯

◯
◯

B: The phylogenetic relationship within the B-linage of the Pacific salmon group
Various studies#

Topology I
Topology II
Topology III((pink,sockeye),chum)((pink,chum),sockeye)((sockeye,chum),pink)

1

2

3

4

5

◯

◯

◯

◯

◯

6

6'

7

8

◯

◯

◯

◯

A: A-linage of the Pacific salmon group, B: B-linage of the Pacific salmon group, M: masu salmon, P:
The pacific trout group* 1: morphology (Hikita, 1962), 2: morphology (Smith and Stearley, 1989), 3: allozyme electrophoresis (Utter et al., 1973), 4: RFLP of rDNA (Phillips et al., 1992), 5: RFLP of mtDNA (Thomas et al.,1986), 6: D-loop sequences of mtDNA (Shedlock et al., 1992), 6': D-loop sequences of mtDNA
reanalysed (present study), 7: ATPase 6 and ND 3 sequences of mtDNA (Domanico and Phillips,
1995), 8: SINEs (Takasaki et al., 1996), 9: allozyme electrophoresis (present study)Phylogeny of the genus Oncorhynchus fishes
ship with that estimated from the present allozyme data
(Fig. 2). However, there are two differences between theprevious results (Shedlock et al., 1992) and those of thepresent study. (1) Masu salmon is clustered with other
Pacific salmons and is not clustered with only B-lineagesof the Pacific salmon group (tree II). This result is supported by morphology (Smith and Stearley, 1989) and
SINEs (Takasaki et al., 1996) data. (2) Pink salmon andchum salmon are clustered (topology II). This result is
supported by two other mtDNA studies (Thomas et al.,
1986; Domanico and Phillips, 1995), therefore the consistency of all mtDNA data was confirmed. This result isalso supported by SINEs data. SINE insertions appear
to be irreversibly therefore provide an informative markers of evolution (Murata et al., 1993).
In the previous study (Shedlock et al., 1992), they used
all 9 salmonid fish (including Atlantic salmon) and Arctic
grayling (Thymallus arcticus: this belongs to a different
subfamily) to obtain the simultaneous alignment. They
also considered only transitional and transversional substitution ratio to construct maximum parsimony andmaximum likelihood trees. However we used the profilealignment procedure and excluded Arctic grayling to obtain a more optimal alignment. We also used a suitablecorrected distance matrix method to consider transitional
and transversional substitution ratio and the rate of substitution varies from site to site and some parallel nucleotide changes, multiple substitutions, or back substitutions. As a result, a more optimal relationship among
salmonid fishes seemed to be obtained.
In conclusion, the following consistent results were obtained by our electrophoretic data and mtDNA analysis;(1) all the Pacific salmon species form a monophyletic cluster, (2) chum salmon and pink salmon are clustered withinthose Pacific salmons, and (3) masu salmon forms a clusterwith other Pacific salmons and diverged first in this group.
All of these results are consistent with SINEs data (Kidoet al., 1991; Murata et al., 1993; Takasaki et al., 1996).
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