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

Molecular systematics of Salmonidae: combined nuclear data yields a robust phylogeny
ARTICLE IN PRESS
MOLECULAR
PHYLOGENETICS
AND
EVOLUTION
Molecular Phylogenetics and Evolution xxx (2003) xxx–xxx
www.elsevier.com/locate/ympev
Molecular systematics of Salmonidae: combined nuclear data yields
a robust phylogeny
Bernard J. Crespi* and Michael J. Fulton
Behavioural Ecology Research Group, Department of Biosciences, Simon Fraser University, 8888 University Drive, Burnaby, BC, Canada V5A 1S6
Received 12 May 2003; revised 15 August 2003
Abstract
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 sistergroup 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.
Ó 2003 Published by Elsevier Inc.
Keywords: Salmonidae; Phylogeny; Total evidence; Anadromy
1. Introduction
The family Salmonidae comprises three subfamilies,
Coregoninae (whitefish and ciscoes), Thymallinae
(grayling), and Salmoninae (char, trout, and salmon).
The most speciose of these, Salmoninae, includes five
genera distributed throughout the Northern Hemisphere, Brachymystax (lenok), Hucho (huchen and taimen), Oncorhynchus (Pacific trout and salmon), Salmo
(Atlantic salmon and brown trout), and Salvelinus
(char) (Hart, 1973; Hendry and Stearns, 2003; Scott and
Crossman, 1973). Salmonid fishes have long been of
great interest due to the commercial and recreational
value of some species, and they are becoming increasingly important as model systems for addressing a wide
range of evolutionary and ecological questions (Elliot,
*
Corresponding author. Fax: +604-291-3496.
E-mail address: [email protected] (B.J. Crespi).
1055-7903/$ - see front matter Ó 2003 Published by Elsevier Inc.
doi:10.1016/j.ympev.2003.08.012
1994; Groot and Margolis, 1991; Hendry and Stearns,
2003). Inference of a robust phylogeny for this group is
important for comparative analyses of salmonid adaptations (e.g., Crespi and Teo, 2002; Fleming, 1998),
comparative genomics (e.g., Woram et al., 2003), studies
involving inference of ancestral states (e.g., McDowall,
1997; McLennan, 1994; Stearley, 1992), and evaluation
of conservation priorities (Crandall et al., 2000).
Despite the importance of salmonids to humans, and
to terrestrial and marine ecosystems, their evolutionary
history has remained a matter of considerable dispute
for many years (e.g., Domanico et al., 1997; McKay et
al., 1996; McPhail, 1997; Norden, 1961; Oakley and
Phillips, 1999; Phillips and Oakley, 1997; Phillips and
Pleyte, 1991; Regan, 1914; Utter et al., 1973; Utter and
Allendorf, 1994). Previous species-level and genus-level
phylogenetic research on salmonid fishes have provided
insights into some relationships, but numerous questions remain, most notably the among-genus diver-
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gences, and species-level relationships within Oncorhynchus and Salvelinus.
The lack of a comprehensive, well-resolved and wellsupported phylogeny for Salmonidae can be largely attributed to previous studies using relatively small subsets
of extant salmonid diversity, and only one or at most
several genes or other character sets (e.g., morphology
or karyology). To overcome these limitations, we have
assembled and analyzed all available DNA-sequence
data for the species in this family. The main goals of our
study are twofold: (1) to use these data to infer the best
tree for the family as a whole, and for particular lineages; and (2) to assess what additional data (i.e., sequence from which genes) are needed to achieve a
species-level tree for the entire group.
2. Methods
2.1. Data set
We compiled all of the available sequence data for
salmonid fishes and one outgroup (Plecoglossus altivelis)
(Salmoniformes: Osmeridae), which comprised 269 sequences of mitochondrial DNA (from 16 genes) and
nuclear DNA (from 9 genes) for 31 species (Table 1).
The bulk of these data were from Genbank, with several
additional sequences graciously provided to us by T.
Oakley and R. Phillips. Some species for which very
little data were available (e.g., only one or several genes)
were not included. However, some species with substantial amounts of missing data were included, as inclusion of such taxa has been shown to increase
phylogenetic accuracy and is not expected to produce
misleading results (Wiens and Reeder, 1995; Wiens,
1998a). Complete mitochondrial DNAs were available
for C. lavaretus, O. mykiss, O. tshawytscha, P. altivelis,
S. salar, Sv. alpinus, and Sv. fontinalis. The MHC genes
used were chosen randomly, one for each species, from
the larger sample of alleles in Genbank.
The sequences were aligned gene by gene using
Clustal X (Thompson et al., 1997) and by eye, and
regions with ambiguous alignments (e.g., parts of the
DLOOP) were excluded. The full data set had 27,593
base pairs, and it is available as a NEXUS file from
BC.
2.2. Phylogenetic analyses
We used maximum parsimony and maximum likelihood in PAUP (Swofford, 2002) and Bayesian analysis in MrBayes (http://brahms.biology.rochester.edu/
software.html) (Alfaro et al., 2003; Hall, 2001; Huelsenbeck et al., 2001; Rannala and Yang, 1996; Yang
and Rannala, 1997) for our analyses. We analyzed each
gene separately (for genes with at least 13 taxa repre-
sented, and for vitellogenin and MHC), the full mitochondrial data set, the full nuclear-gene data set, and
all of the data combined. For most of the analyses of
mitochondrial data, P. altivelis was used as the outgroup. However, because data from this species were
not available for nuclear genes, C. lavaretus, B. lenok,
or H. perryi were also used as outgroups, depending on
which was available and closest to the ingroup based
on previous studies. MrMODELTEST (http://
www.ebc.uu.se/systzoo/staff/nylander.html) was used to
choose the most appropriate models of molecular
evolution for the likelihood analyses of each separate
gene, and for the combined data sets (Posada and
Crandall, 1998, 2001; Sullivan and Swofford, 2001). To
assess the robustness of the inferred trees, we used
bootstrapping with 500 replicates under maximum
parsimony and bootstrapping with 200 replicates for
maximum likelihood. Maximum likelihood bootstrapping was not computationally feasible for the combined data sets. For the Bayesian analyses, we used at
least 5000 trees after stabilization of the likelihoods to
compute the a posteriori probabilities, which can be
interpreted as the probabilities that particular clades
are correct. These probabilities tend to be less conservative than maximum-likelihood bootstrap values
(Alfaro et al., 2003; Douady et al., 2003). Although
they tend to identify more correct monophyletic groups
than do parsimony or likelihood bootstrapping in
simulations, Bayesian support values may also overestimate the degree of clade support, especially for
lineages descending from short internodes (Alfaro et
al., 2003; Douady et al., 2003).
We have taken a Ôconditional combinationÕ approach (Bull et al., 1993; De Queiroz et al., 1995;
Huelsenbeck et al., 1996) to analyze data derived from
multiple genes and loci. This approach involves an
assessment of congruence, using various means, prior
to a decision to combine data sets or analyze them
separately. We follow this approach for two reasons.
First, some genes or loci may produce inconguent and
incorrect results, due to such processes as sampling
error, hybridization, natural selection, rate variation
among lineages, variation in base composition, or a
high degree of saturation (Sanderson and Schaffer,
2002; Slowinksi and Page, 1999). Second, given only
minor effects from such processes in a combined data
set, a total evidence analysis should yield the best results, because different genes should provide resolution
and support in different regions of the tree (Bull et al.,
1993; Huelsenbeck et al., 1996).
Our analysis of congruence was constrained by the
variable sets of taxa for which data were available for
each gene, which precluded direct comparisons of genes
on a pairwise basis with ML tests (e.g., Huelsenbeck and
Bull, 1996) or other methods (see Barker and Lutzoni,
2002). As a result, we evaluated the degree of congru-
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Table 1
Genbank sequences used in the analyses
B. lenok
C. artedi
C. autumnalis
C. clupeaformis
C. kiyi
C. lavaretus
H. hucho
H. perryi
O. clarki
O. gorbuscha
O. keta
O. kisutch
O. masou
O. mykiss
O. nerka
O. rhodurus
O. tshawytscha
P. altivelis
P. coulteri
P. williamsoni
S. orhidana
S. salar
S. trutta
Sv. alpinus
Sv. confluentus
Sv. fontinalis
Sv. leucomaenis
Sv. malma
Sv. namaycush
T. arcticus
T. thymallus
DLOOP
12S
16S
AF125519
AF246932
AJ250996
AF239253
U95191
AB034824
AF125513
AF125513
ND2
CO1
AB034824
AB034824
L29771
L29771
L29771
AF246933
AB034824
AF254863
AB034824
AB034824
AF296347
AF296345
AF296344
AF296342
AF125510
L29771
AF296343
AF254865
AB039901
AF318037
AF429780
L29771
U59926
AF113119
AF392054
AB047553
AY008713
AY008696
AF392054
AB047553
AF392054
AB047553
AF392054
AB047553
AF392054
AB047553
AF392054
AB047553
AF133701
U62286
AF154851
AF133701
AF133701
AF133701
AF154851
AF126004
AF154850
AF154851
AF133701
AF117718
AF154851
AF154851
AF133701
M64917
AF154851
AF154850
AF060445
AF154850
AF154850
AF154850
AF076906
AF036381
AF076908
AF036381
Atp6
CO3
ND3
ND4L
AF154850
AF297988
AF298043
AF297989
AF329990
AF329989
CO2
B. lenok
C. artedi
C. autumnalis
C. clupeaformis
C. kiyi
C. lavaretus
H. hucho
H. perryi
O. clarki
O. gorbuscha
O. keta
O. kisutch
O. masou
O. mykiss
O. nerka
O. rhodurus
O. tshawytscha
P. altivelis
P. coulteri
P. williamsoni
S. orhidana
S. salar
S. trutta
Sv. alpinus
Sv. confluentus
Sv. fontinalis
Sv. leucomaenis
Sv. malma
Sv. namaycush
ND1
AF113117
L29771
Atp8
AF246934
AJ133367
AB034824
AB034824
AB034824
D84147
L29771
L29771
D63336
L29771
AF392054
AB047553
AF392054
AB047553
AF392054
AB047553
AB034824
AB034824
AF294830
AF294831
D84147
AF294829
D63336
L29771
AF294832
AF312575
AF055090
AF055089
AF055092
U28364
L29771
AF055091
U28363
AF392054
AB047553
AF392054
AB047553
AB034824
D84147
D63336
L29771
AF392054
AB047553
AJ133369
AF133701
AF133701
AF133701
AF154851
AF133701
X76247
AF154851
AF154851
AF154850
AF133701
AF154851
AF133701
U61181
AF154851
AF154850
AF154850
AF154850
AF154850
AF154850
U61182
AF154851
3
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Table 1 (continued)
CO2
Atp8
Atp6
CO3
ND3
ND4L
ND4
ND5
ND6
Cyt b
GH1c
GH2c
AF125052
AF125213
AF005919
AF005917
AF005920
AF005924
AF005926
AF005927
AF005925
AB001865
AF005907
AF005908
AF005913
AF075572
L04688
U04931
AF005923
U14551
J03797
U14535
Oakley
AF005914
T. arcticus
T. thymallus
B. lenok
C. artedi
C. autumnalis
C. clupeaformis
C. kiyi
C. lavaretus
H. hucho
H. perryi
O. clarki
O. gorbuscha
O. keta
O. kisutch
O. masou
O. mykiss
O. nerka
O. rhodurus
O. tshawytscha
P. altivelis
P. coulteri
P. williamsoni
S. orhidana
S. salar
S. trutta
Sv. alpinus
Sv. confluentus
Sv. fontinalis
Sv. leucomaenis
Sv. malma
Sv. namaycush
T. arcticus
T. thymallus
AJ251592
AB034824
AB034824
AY032633
U66039
AY032633
U66039
AF125051
L29771
L29771
AF125050
L29771
AF392054
AB047553
AF392054
AB047553
AF392054
AB047553
AF133701
AF133701
AF133701
AF154851
AF154851
AF154851
AF154850
AF154850
AF154850
ITS1
AB034824
AF172397
D58396
AY032633
AF165077
AF165078
AF165079
D58403
L29771
AJ314568
AF392054
AB047553
AY008700
AY008701
AF202033
AF133701
X77526
AF154851
ITS2
Oakley
AF005921
AF005915
M21573
AF005912
AF005909
AF005911
AF154850
D58397
AF319544
AF270689
AF270858
GH2d
B. lenok
C. artedi
C. autumnalis
C. clupeaformis
C. kiyi
C. lavaretus
H. hucho
H. perryi
O. clarki
O. gorbuscha
O. keta
O. kisutch
O. masou
O. mykiss
O. nerka
O. rhodurus
O. tshawytscha
P. altivelis
P. coulteri
P. williamsoni
S. orhidana
S. salar
S. trutta
Sv. alpinus
Sv. confluentus
Sv. fontinalis
AB034824
18s
AF005922
Oakley
AF005910
GH1d
VIT
MHC
AF243426
AB001865
L04688
J03797
U14535
AF454745
M94900
AF174612
AF170535
Oakley
Oakley
AF170536
AF170533
Oakley
AF170539
AF170538
AF170540
AF170542
AF170543
AF170537
AF170534
AF170541
AF030250
AF243427
AF243428
AF454747
AJ011689
U14551
AF454748
U34717
U34703
U34692
U34697
U34715
U34711
U34719
AY008709
AY008695
M21573
AF201313
AF201312
AF072862
AF059899
M94902
M94903
AF174609
AF174613
AF174611
AJ427629
X98839
AF469620
Phillips
Phillips
Phillips
Phillips
Phillips
X70166
AF454750
AF454751
AF454752
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Table 1 (continued)
CO2
Sv. leucomaenis
Sv. malma
Sv. namaycush
T. arcticus
T. thymallus
Atp8
Atp6
M94904
M94905
M94906
AF174607
AF174608
AF174610
CO3
ND3
ND4L
Phillips
Phillips
Phillips
AF454753
Fig. 1. Bootstrap majority-rule phylogenies inferred from individual-gene DNA data sets. MP ¼ maximum parsimony, ML ¼ maximum likelihood,
BAY ¼ Bayesian analysis. GH ¼ growth hormone, ITS ¼ internal transcribed spacer, VIT ¼ vitellogenin, MHC ¼ major histocompatibility complex.
ML bootstraps were not computationally feasible for the DLOOP data set.
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Fig. 1. (continued)
ence between the results from analyses of separate genes
via inspection of bootstrap or a posteriori probability
values, to determine how many different genes and loci
supported a given monophyletic group and to what
extent, and to identify any strongly supported nodes
that differed among genes (De Queiroz et al., 1995).
Given potential incongruence in one or more parts of
a phylogeny, we agree with Wiens (1998b) that combining data may still represent the best strategy for inferring the most-accurate tree, subject to the caveat that
the gene or genes involved in possible incongruence
should be considered questionable and may require removal from a combined analysis. In addition to analyzing each gene separately, we also compared the
degrees of resolution and support obtained from analyses of the combined mitochondrial data set, the combined nuclear data set, the combined nuclear data set
without the MHC data (since salmonid MHC is known
to be under strong balancing selection, Miller and
Withler, 1996), all of the data except MHC, and all of
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Fig. 1. continued)
the data combined. Cunningham (1997) has shown that
congruence and phylogenetic accuracy tend to be positively correlated, such that strongly supported combined-data trees are likely to reflect congruence among
trees from the individual data sets. Finally, alternative a
priori hypotheses for the placement of particular species
and sets of species were also evaluated using the SH test
(Shimodaira and Hasegawa, 1999) and the Templeton
test (Templeton, 1983), implemented in PAUP* as described below.
3. Results
3.1. Phylogenetic analyses of individual and combined
data
Bootstrap majority-rule consensus trees from analyses using maximum parsimony and maximum likelihood, and a posteriori clade support values from
Bayesian analysis, are shown in Fig. 1 for each of the
individual genes, and Fig. 2 shows maximum-parsimony
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Fig. 1. (continued)
bootstraps and Bayesian support for the combined data
sets. The individual-gene trees differ considerably in the
species included and the degree of support for various
relationships. We assessed the degree of support for the
main phylogenetic hypothesis among Salmonidae by
collating the bootstrap and Bayesian a posteriori values
relevant to each putative monophyletic group of interest
(Table 2). This analysis allows us to assess the degree to
which the results from the different data sets are con-
gruent, reinforce one another, or provide conflicting
signal.
3.2. Oncorhynchus
The monophyly of Oncorhynchus, which is strongly
supported by virtually all previous studies (Murata et
al., 1993, 1996, 1998; Oakley and Phillips, 1999; Oleinik,
1997; Phillips and Oakley, 1997 and references therein),
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Fig. 2. Bootstrap majority-rule and Bayesian phylogenies for the combined data sets: (A) all mtDNA, (B) all nuclear DNA excluding MHC, (C) all
nuclear DNA, (D) all nuclear DNA and mtDNA excluding MHC, (E) all data.
was also supported in our analyses by all of the individual genes and by the combined data sets (Table 2).
However, the strength of support varied considerably
across data sets, with the nuclear genes tending to provide higher bootstrap and Bayesian a posteriori values
than the mitochondrial genes, and the combined
mtDNA data set (Fig. 2A) returning the weakest support.
The monophyly of Oncorhynchus excluding O. clarki,
O. masou, and O. mykiss was strongly supported here by
GH1C and by the combined nuclear DNA. By contrast,
this group was not supported by analyses of ITS1, CO3,
ND3, and GH2C, the other genes that provided any
degree of bootstrap majority-rule resolution for this
clade. However, the monophyly of this group was not
strongly contradicted by these analyses, as the relevant
bootstrap values were low. The reduced support for
this clade, compared to the genus as a whole, is due
primarily to the presence of O. mykiss, O. clarki or
O. masou among the other Oncorhynchus in some of the
trees.
The clades (O. nerka, O. gorbuscha, O. keta) and
(O. gorbuscha, O. keta) have been inferred in most
previous studies of Oncorhynchus (Domanico and Phillips, 1995; Kitano et al., 1997; McKay et al., 1996;
Murata et al., 1993, 1996, 1998; Oakley and Phillips,
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Table 2
Bootstrap values from maximum likelihood (200 replicates, in plain text) or Bayesian analyses (majority-rule of at least 5000 trees, in italics), and
maximum parsimony (500 replicates, in parentheses) analyses, for relevant clades in Figs. 1 and 2
All Oncorhynchus (Pacific salmon and Pacific trout)
(O. tshawytscha, O. kisutch, O. nerka, O. gorbuscha, O. keta, O. mykiss, O. clarki, O. masou)
GH1C
100 100 (100)
ITS1
100 100 (100)
ITS2
100 100 (100)
GH2C
96 100 (93)
CYTB
70 93 (98)
CO3
71 84 (96)
ND3
86 52 (81)
16S
82 97 (59)
MHC
(62)
All mt DNA
76 (61)
All nuclear DNA (excl. MHC)
100 (100)
All nuclear DNA
100 (99)
All nuclear DNA (excl. MHC) + mtDNA
100 (100)
All nuclear + mt DNA
100 (100)
Pacific salmon excluding O. masou
(O. tshawytscha, O. kisutch, O. nerka, O. gorbuscha, O. keta)
GH1C
98 100 (98)
ITS2
55
GH2C
Not monophyletic
CO3
Not monophyletic
ITS1
Not monophyletic
CYTB
Not monophyletic
ND3
Not monophyletic
All nuclear DNA (excl. MHC)
100 (94)
All nuclear DNA
100 (92)
All nuclear DNA (excl. MHC) + mtDNA
Not monophyletic
54 93 (66)
60 99
73 95 (56)
79
100
(includes O. masou, 99)
(O. nerka, O. gorbuscha, O. keta)
GH1C
GH2C
MHC
ITS2
CYTB
ITS1
ND3
CO3
16S
All mt DNA
All nuclear DNA (excl. MHC)
All nuclear DNA
All nuclear DNA (excl. MHC) + mtDNA
All nuclear + mt DNA
100 100 (100)
98 100 (93)
69 99 (74)
62 86 (75)
72
65 84 includes O. kisutch
87 100 (92) includes O. kisutch
Not monophyletic 60 99 (monophyletic, 56)
Not monophyletic O. nerka with O. tshawytscha, O. kisutch 55,63 77,88 (55,71)
92 (85)
100 (100)
100 (100)
100 (100)
100 (100)
(O. gorbuscha, O. keta)
CYTB
ITS2
GH1C
GH2C
MHC
ITS1
ND3
CO3
All mt DNA
All nuclear DNA (excl. MHC)
All nuclear DNA
All nuclear DNA (excl. MHC) + mtDNA
All nuclear + mt DNA
94 98 (99)
69 91
58 95 (69)
54 53
52 86
50 (51)
(Not monophyletic O. gorbuscha with O. nerka, O. kisutch: 57)
Not monophyletic 60 99 (monophyletic, 66)
97 (94)
100 (92)
100 (90)
100 (100)
100 (100)
(O. tshawytscha, O. kisutch)
MHC
VIT
100 100 (99)
99 100 (99)
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Table 2 (continued)
ITS2
CYTB
GH1C
CO3
16S
ITS1
GH2C
ND3
All mt DNA
All nuclear DNA (excl. MHC)
All nuclear DNA
All nuclear DNA (excl. MHC) + mtDNA
All nuclear + mt DNA
90 100 (88)
91 99 (95)
62 85 (73)
Not monophyletic 60 99
Not monophyletic, includes O. nerka: 55,63 77,88 (55,71)
Not monophyletic 73 95 (56)
Not monophyletic 78 100 (76)
Not monophyletic 87 100 (92)
Not monophyletic 88 (75)
100 (72)
100 (99)
Not monophyletic 100 (90)
100 (59)
Pacific trout and O. masou
(O. mykiss, O. masou)
ITS2
CO3
ITS1
ND3
MHC
DLOOP
CYTB
All mt DNA
All nuclear DNA
All nuclear DNA (excl. MHC) + mtDNA
All nuclear + mtDNA
76 80 (90)
78
54 76 (56, includes O. tshawytscha)
Not monophyletic 52
Not monophyletic 81 95 (62)
Not monophyletic 83
Not monophyletic 98
Not monophyletic 98 (90) (O. mykiss with O. clarki)
Not monophyletic 100 (O. mykiss with O. clarki)
Not monophyletic 100 (100) (O. mykiss with O. clarki)
Not monophyletic 100 (100) (O. mykiss with O. clarki)
(O. mykiss, O. clarki)
GH1C
CYTB
GH2C
DLOOP
ND3
CO3
All mt DNA
All nuclear DNA (excl. MHC) + mtDNA
All nuclear DNA
All nuclear + mt DNA
100 100 (100)
84 98 (95)
84 100 (87)
83
Not monophyletic 50 50
Not monophyletic 60 99
98 (90)
100 (100)
100
100 (100)
(O. mykiss, O. clarki, O. masou)
ND3
CO3
CYTB
ND3
All
All
All
All
nuclear DNA (excl. MHC)
nuclear DNA
nuclear DNA (excl. MHC) + mtDNA
nuclear + mt DNA
O. masou as sister-taxon to all other Oncorhynchus; Not monophyletic 52
Not monophyletic 60 99
ML, MP: O. masou basal but unresolved in Oncorhynchus
ML, MP: O. masou basal but unresolved in Oncorhynchus
Not monophyletic 79
Not monophyletic 50; O. masou basal in Oncorhynchus; (MP: O. masou sister to
other Oncorhynchus, 71)
100 (95)
100 (79)
Not monophyletic 99 (71), O. masou with O. tshawytscha
96, Not monophyletic (51), O. masou basal in Pacific salmon
Salvelinus
(Sv. alpinus, Sv. malma, Sv. confluentus, Sv. leucomaenis, Sv. fontinalis, Sv. namaycush)
GH1C
100 100 (100)
ITS1
100 100 (100)
ITS2
100 100 (100)
GH2C
92 100 (94)
GH2D
57 69 (55)
All nuclear DNA (excl. MHC)
100 (100)
All nuclear DNA
100 (100)
All nuclear DNA (excl. MHC) + mtDNA
100 (100)
All nuclear + mt DNA
100 (100)
11
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Table 2 (continued)
(Sv. alpinus, Sv. malma, Sv. confluentus, Sv. leucomaenis)
ITS2
ITS1
GH1C
GH2D
GH2C
All mtDNA
All nuclear DNA (excl. MHC)
All nuclear DNA
All nuclear DNA (excl. MHC) + mtDNA
All nuclear + mt DNA
91 99 (97)
59 84 (54)
Not monophyletic
Not monophyletic
Not monophyletic
Not monophyletic
99 (75)
100 (73)
99
Not monophyletic
(Sv. alpinus, Sv. malma)
ITS1
GH2C
GH2D
DLOOP
GH1C
ITS2
All mt DNA
All nuclear DNA (excl. MHC)
All nuclear DNA
All nuclear DNA (excl. MHC) + mtDNA
All nuclear + mt DNA
96 100 (99)
87 100 (94)
88 92 (79)
(63)
52
Not monophyletic 96 100 (96)
87 (52)
100 (100)
100 (100)
100 (100)
100 (100)
(Sv. confluentus, Sv. leucomaenis)
ITS1
GH1C
GH2C
ITS2
GH2D
All mt DNA
All nuclear DNA (excl. MHC)
All nuclear DNA
All nuclear DNA (excl. MHC) + mtDNA
All nuclear + mt DNA
98 100 (96)
58 97 (71)
60 82 (55)
Not monophyletic 96 100 (97)
Not monophyletic 68 79 (62)
Not monophyletic 87 (75)
100 (72)
100 (73)
100 (74)
100 (73)
(Sv. fontinalis, Sv. namaycush)
ITS2
GH1C
ITS1
GH2D
GH2C
ND3
All mtDNA
All nuclear DNA (excl. MHC)
All nuclear DNA
All nuclear DNA (excl. MHC) + mtDNA
All nuclear + mt DNA
100 100 (100)
66 86 (52)
(52)
Not monophyletic
Not monophyletic
Not monophyletic
Not monophyletic
100 (98)
100 (99)
100 (75)
100 (77)
Sister-group to Oncorhynchus
(Oncorhynchus, Salvelinus)
GH1C
VIT
ND3
DLOOP
CO3
CYTB
All nuclear DNA (excl. MHC)
All nuclear DNA
All nuclear DNA (excl. MHC) + mtDNA
All nuclear + mt DNA
100 100 (100)
81 100 (92)
85 100 (81)
78 (includes P. coulteri, B. lenok, Oncorhynchus paraphyletic)
51 54 (67)
58 (53) (Salvelinus lineage includes Hucho and B. lenok)
100 (94)
100 (93)
64, Salvelinus includes Hucho and B. lenok (61)
91 (57)
(Oncorhynchus, Salmo)
ITS1
96 100 (96)
50
68
76
87
(monophyletic: 52)
79 (62)
100 (82)
(75)
51 (50)
68 79 (62)
76 100 (82)
83 (89)
87
Results from genes for which relationships remain unresolved in the 50% majority-rule consensus trees are not shown. For clades that are not
monophyletic, the highest bootstrap or a posteriori Bayesian value supporting the lack of monophyly is listed. Genes are listed from top to bottom in
order of the degree to which they tend to support (top) or contradict (bottom) the presence of the monophyletic group shown.
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1999; Oohara et al., 1997, 1999; Osinov, 1999; Phillips
and Oakley, 1997; Shedlock et al., 1992 and references
therein) and have yet to be strongly contradicted. In our
analyses, we found support for (O. nerka, O. gorbuscha,
O. keta) from the nuclear genes GH1C, GH2C, MHC,
and ITS2, as well as from each of the combined data
sets. The results that were incongruent with (O. nerka,
O. gorbuscha, O. keta) tended to involve relatively low
bootstrap and Bayesian values, excepting those from
ND3 and GH2D. The group (O. gorbuscha, O. keta) was
accorded bootstrap and Bayesian support from CYTB,
ITS2, GH1C, GH2C, ITS1, and MHC though most of
these support values were rather low. However, the
contradictory results from ND3 and CO3 were weak,
and the combined data sets provided very strong support for this clade.
The sister-taxon status of O. tshawytscha with O.
kisutch is supported by considerable previous phylogenetic work, and has yet to be contradicted strongly (Du
et al., 1993; Kitano et al., 1997; McKay et al., 1996;
Murata et al., 1993, 1996, 1998; Oakley and Phillips,
1999; Oohara et al., 1997, 1999; Osinov, 1999; Phillips
and Oakley, 1997 and references therein). This clade was
supported in our analyses by MHC, VIT, ITS2, CYTB,
GH1C, the combined nuclear data sets, and the complete data set. By contrast, analyses of CO3, 16S, ITS1,
GH2C, ND3, the mtDNA data, and the complete data
set without MHC, yielded results contradictory to the
presence of this clade. However, the bootstrap and
Bayesian values for these genes tended to be relatively
low compared to the clade support values from the
genes that supported it. Moreover, the phylogenetic
positions of these two species were inconsistent across
CO3, 16S, ITS1, GH2C, and ND3 (Fig. 1), such that
there was no evidence for one or more specific alternative placements. Considered together, these results are
consistent with the sister-taxon status of O. tshawytscha
13
and O. kisutch, although support for this grouping is not
entirely unambiguous.
The phylogenetic placement of O. masou, in relation
to the other Oncorhynchus, has not received strong
support from previous analyses (e.g., Kitano et al., 1997;
McKay et al., 1996; Murata et al., 1996; Oleinik, 2000;
Oohara et al., 1997, 1999). To elucidate the relationships
of O. mykiss, O. clarki and O. masou among themselves
and to the other Oncorhynchus, we jointly evaluated the
degrees of support for the clades (O. mykiss, O. clarki),
(O. mykiss, O. masou), and (O. mykiss, O. clarki, O.
masou) (Table 2). The sister-taxon relationship of O.
mykiss with O. clarki was clearly upheld by the analyses
of GH1C, CYTB, GH2C, the mtDNA data, and the full
combined data set, and it was relatively weakly incongruent with results from ND3 and CO3. In addition, a
relationship of O. mykiss with O. masou was inferred
from ITS2 and (marginally) from ITS1. Considering
all three species together, we note that by most singlegene analyses, O. masou appears relatively basal in
Oncorhynchus but its position is unresolved, as are the
positions of O. mykiss, O. clarki, or both in the analyses
of CO3, 16S, and ND3. A basal but weakly resolved
position for O. masou is also apparent in analyses of the
combined mtDNA data (Fig. 2A), the full data set with
MHC excluded (Fig. 2D), and the the full data set
(Fig. 2E) using maximum parsimony.
The clearest results for the position of O. masou come
from analyses of the nuclear DNA data set, with or
without MHC (Fig. 2B and C). These analyses provided
strong bootstrap support, Bayesian support, or both for
the monophyly of (O. mykiss, O. clarki, O. masou). The
Bayesian analyses also strongly supported sister-taxon
status of O. mykiss and O. clarki. These results indicate
that the various individual genes provide no support
or weak to moderate support for the clade (O. masou,
(O. mykiss, O. clarki)), and that no evidence strongly
Table 3
Results from Templeton and SH tests that in each case compare the best (unconstrained) tree to an alternative, constraint tree: (a) best unconstrained
tree vs. best tree showing O. masou not with O. mykiss and O. clarki, (b) best unconstrained tree vs. best tree with Salmo as sister-group to
Oncorhynchus, (c) best unconstrained tree vs. best tree where O. kisutch and O. tshawytscha are not sister-taxa
Constraint
Data set
Nuclear data without MHC
MP
ML
All nuclear data
MP
ML
(a) Not (O. masou,
O. mykiss, O. clarki)
(b) (Salmo,
Oncorhynchus)
(c) Not (O. kisutch,
O. tshawytscha)
1703 vs. 1709
P ¼ 0:058
22712.21 vs. 22729.14
P ¼ 0:030
1703 vs. 1718
P ¼ 0:022
22712.21 vs. 22733.51
P ¼ 0:046
1700 vs. 1702
P > 0:40
22712.21 vs. 22718.89
P > 0:10
1911 vs. 1914
P > 0:40
25687.05 vs. 25696.96
P ¼ 0:087
1911 vs. 1926
P ¼ 0:022
25687.05 vs. 25708.15
P ¼ 0:050
1908 vs. 1919
P ¼ 0:012
25687.05 vs. 25717.39
P ¼ 0:018
For Templeton tests (MP), tree lengths are shown, and for SH tests (ML), likelihoods are shown. For the MP analysis of (c), H. hucho was
excluded due to apparent long-branch attraction into Oncorhynchus in the constraint tree. Qualitatively similar results were obtained using the
constraint tree ‘‘Not (Oncorhynchus, Salvelinus)’’.
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contradicts these relationships. However, taken together, the nuclear ones reinforce and complement one
another sufficiently to provide strong evidence that O.
masou belongs with the Pacific trout.
3.3. Salvelinus
The monophyly of Salvelinus was supported by all of
the genes for which data on this genus was available,
though support from GH2D, and from the combined
mitochondrial DNA data set, was relatively weak (Table
2). Some of the relationships within Salvelinus were
reasonably clear, but for others different genes yielded
incongruent results. Thus, the monophyly of (Sv. alpinus, Sv. malma, Sv. confluentus, Sv. leucomaenis) was
supported strongly by ITS2, moderately supported by
the complete nuclear DNA data set, and weakly supported by ITS1 and GH1C. However, the differing results from analyses of GH2D and GH2C, the combined
mtDNA data set, and the full data set, preclude unambiguous interpretation of this result. Similarly, the
groups (Sv. alpinus, Sv. malma), (Sv. confluentus, Sv.
leucomaenis), and (Sv. fontinalis, Sv. namaycush) were
each upheld, often with high bootstrap and Bayesian
support values, for one or more genes, but analyses of
one or more other genes yielded notably incongruent
results. For each of these cases, the combined data sets,
especially the nuclear data, lent strong or moderate
support to the group, but this support tended to stem
from only one or two genes that may or may not be
indicating the correct phylogeny.
The simplest interpretation of these heterogeneous
results is that one or more of the genes does not reflect
the species tree for Salvelinus. In particular, the data
from ITS2 disrupts the monophyly of both (Sv. alpinus,
Sv. malma) and (Sv. confluentus, Sv. leucomaenis), because it strongly groups Sv. confluentus with Sv. alpinus
and Sv. malma, and the data from GH2C, GH2D, and
ND3 prevents (Sv. alpinus, Sv. malma, Sv. confluentus,
Sv. leucomaenis) and (Sv. fontinalis, Sv. namaycush)
from each being monophyletic, because they position
Sv. namaycush strongly with Sv. alpinus and Sv. malma.
As discussed below, these findings are consistent with
the hypothesis that extensive hybridization has obfuscated relationships among species of Salvelinus as inferred from DNA-sequence data.
3.4. Relationship of Oncorhynchus to Salvelinus and
Salmo
A sister-taxon relationship between Oncorhynchus
and Salvelinus is strongly supported by GH1C, VIT,
ND3, and the combined nuclear data sets, and weakly
supported by CO3 and CYTB (Table 2). By contrast,
the ITS1 data groups Oncorhynchus and Salmo
with such a high degree of confidence that is it clearly
incongruent with the results from these other data sets.
This analysis of ITS1 appears problematic, however,
because our analyses of other genes have shown that
H. perryi may not be an appropriate outgroup for an
analysis of intergeneric relationships between Oncorhynchus, Salmo, and Salvelinus. CYTB groups
H. perryi with Salvelinus with 57% bootstrap confidence
in the parsimony analysis, and GH1C and the complete
nuclear DNA data set group H. perryi with Salmo, with
90–92% and 66% confidence respectively. Thus, given
that H. perryi may belong in the ingroup, the analysis of
ITS1 cannot be used to address relationships between
Oncorhynchus, Salmo, and Salvelinus. The fish species
that are closest to this set of taxa, but definitely not in
the ingroup, are Cichlidae, which are highly divergent
in ITS1 (i.e., on the order of 50% or more divergent in
nucleotide sequence). Use of Neochromis nigricans
(Genbank U67338) as an outgroup, aligned to the taxa
in the ITS1 data set using Clustal X, yielded a phylogeny
with Oncorhynchus and Salmo as sister-taxa but with
only 58% bootstrap support from maximum parsimony
analysis (500 replicates).
3.5. Statistical tests of alternative hypotheses
We used SH tests and Templeton tests to evaluate
alternative hypotheses for three important questions in
salmonid phylogenetics: (1) the phylogenetic position of
O. masou, (2) the sister-group to Oncorhynchus, and (3)
the monophyly of (O. tshawytscha, O. kisutch) (Table 3).
These tests used the two combined nuclear-gene data
sets, which, as described below, provide what we believe
is the best estimate of salmonid phylogeny.
The monophyly of (O. masou, O. mykiss, O. clarki)
was statistically supported by the SH test of the nuclear
data without MHC, and support was marginally nonsignificant (0:05 < P < 0:10) for the Templeton test of
this data set and the SH test of the full nuclear data set
(Table 3). These results are consistent with erosion of
bootstrap and Bayesian support for (O. masou, O. mykiss, O. clarki) with the addition of the MHC data
(Fig. 2).
A sister-taxon relationship between Oncorhynchus
and Salmo was statistically rejected at the 0.05 level by
all four of the analyses (Table 3). These results concur
with the strong bootstrap and Bayesian values for
(Oncorhynchus, Salvelinus) shown in Fig. 2, and they
show that the apparently incongruent results from ITS1
do not substantially disrupt the monophyly of these two
genera.
The relationship (O. tshawytscha, O. kisutch) was
statistically supported by both the SH test and the
Templeton test using the full nuclear data set (Table 3).
By contrast, the SH test on the data set excluding MHC
gave a result that was non-significant (P ¼ 0:14), and the
Templeton test result provided no support for the
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monophyly of this pair of species. Overall, these findings
are consistent with the moderate (72%) maximum-parsimony bootstrap support for (O. tshawytscha, O. kisutch) in the nuclear data set excluding MHC (Fig. 2D),
the strong Bayesian support for this group in this data
set, and the 100% bootstrap and Bayesian support for
this group from the combined nuclear data set (Fig. 2C).
4. Discussion
This is the first study of salmonid phylogenetics that
uses virtually all of the DNA sequence data currently
available. Our analyses of the data from each gene
separately, followed by combined analyses of the mitochondrial data, the nuclear data, and the full combined data set, showed that the mitochondrial data
yielded levels of resolution and support that were
substantially lower than the nuclear data, and that the
nuclear data showed higher levels of resolution and
support than did the nuclear and mitochondrial data
combined (Fig. 2). These findings indicate that although some of the individual mitochondrial genes
provide good evidence for some salmonid relationships
(Table 2), the mitochondrial data taken together reduced the strength of the phylogenetic signal. The high
noise to signal ratio of the mitochondrial data is
probably due to saturation, effects of hybridization,
selection (Bernatchez et al., 1995; Wilson and Bernatchez, 1998) or some combination of these processes,
and it was not alleviated by removal of third-codon
positions for protein-coding genes (results not shown).
Such a lack of clear, strong signal in mitochondrial
data has probably been responsible for much of the
ongoing uncertainty regarding the molecular phylogenetics of Salmonidae.
In the combined nuclear DNA data sets, the different
genes reinforced and complemented one another to a
considerable degree, yielding generally well-resolved and
well-supported trees (Fig. 2B and C). These trees agree
with the results of most previous studies, but also help to
resolve some long-standing uncertainties regarding the
placement of O. masou, the phylogeny of the Pacific
salmon, relationships within Salvelinus, and the sistertaxon to Oncorhynchus.
4.1. Oncorhynchus masou
By our combined nuclear phylogenies, Oncorhynchus
masou forms a monophyletic group with O. mykiss and
O. clarki, and these three taxa comprise the sister-group
to the five other Oncorhynchus species. This result appears to provide a striking case of data from different
genes complementing and reinforcing one another.
Thus, none of the genes analyzed separately provides
information on the monophyly of this group of three
15
species, but GH1C, GH2C, ITS1, ITS2, and CYTB each
supported the monophyly of (O. mykiss, O. clarki) or
(O. mykiss, O. masou). Taken together, the nuclear
DNA indicated good support for this clade from maximum-parsimony bootstraps (95% without the MHC
data, and 79% with MHC) and Bayesian support values
(100% for other data sets). Moreover, the Bayesian
analysis of the full nuclear data set also provided 100%
support for (O. mykiss, O. clarki), which is consistent
with numerous previous studies (e.g., Kitano et al.,
1997; McKay et al., 1996; Oakley and Phillips, 1999;
Oleinik, 1997; Oohara et al., 1997, 1999; Phillips and
Rab, 2001). Results of the SH and Templeton tests
(Table 3) are also consistent with the group (O. masou,
O. mykiss, O. clarki), although only the SH test on the
data set excluding the questionable MHC data achieved
statistical significance.
Previous studies have generally considered O. masou
to be basal within the Pacific salmon or within Oncorhynchus as a whole (Kitano et al., 1997; McKay et al.,
1996; Murata et al., 1996; Oohara et al., 1997, 1999; see
also Oleinik, 2000). Our findings provide the first firm
evidence for its phylogenetic position within the clade of
Pacific trout. This inference is consistent with diverse
additional forms of evidence from allozymes, morphology, behavior, biogeography, and life history (Table 4),
and it should motivate more-detailed evaluation of the
evolution of phenotypic traits within this lineage.
4.2. Pacific salmon
Our analyses provide a fully resolved and well-supported multi-gene phylogeny for Oncorhynchus excluding O. masou, O. mykiss, and O. clarki. The
relationships among O. tshawytscha, O. kisutch, O.
nerka, O. gorbuscha, and O. keta shown in Fig. 2B and C
have been believed for some time from a variety of
morphological, genetic, and other data (Domanico and
Phillips, 1995; Domanico et al., 1997; Kitano et al.,
1997; McKay et al., 1996; Murata et al., 1993, 1996,
1998; Oakley and Phillips, 1999; Oleinik, 1997; Oohara
et al., 1997, 1999; Osinov, 1999; Phillips and Oakley,
1997; Phillips and Pleyte, 1991; ShedÕko et al., 1996;
Shedlock et al., 1992; Smith and Stearley, 1989; Takasaki et al., 1994; Thomas and Beckenbach, 1989; Thomas et al., 1986; Utter et al., 1973; Utter and Allendorf,
1994). However, previous analyses have lacked unambiguous or strong support for at least one of the nodes,
usually many more.
We suspect that the prior lack of conclusive results
for the phylogeny of Pacific salmon has been due to a
combination of saturation of mitochondrial DNA (e.g.,
McKay et al., 1996), such that it provides little evidence for more-basal nodes, possible selection on
mtDNA (e.g., Bernatchez et al., 1995; Wilson and
Bernatchez, 1998), and potential hybridization of O.
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Table 4
Evidence from previous studies that (O. masou, O. mykiss, O. clarki) represents a monophyletic group
Evidence
References
(1) Chromosome number of O. masou (2n ¼ 66) is most similar to that of O. mykiss
(58–64) and O. clarki (64–68)
(2) O. masou similar to O. mykiss in muscle proteins and to O. mykiss and
O. clarki in allozymes
(3) O. masou is Ômost troutlikeÕ of Oncorhynchus in morphology and behavior
Phillips and Rab (2001)
Tsuyuki and Roberts (1966), Utter et al. (1973)
Neave (1958), Yoshiyasu (1973),
Stearley (1992)
Tanaka (1965), Tsuyuki and Roberts (1966),
Kato (1991), Healey et al. (2001)
Chevassus (1979)
(4) Some O. masou males and females are iteroparous
(5) Male O. masou interbreed best with female O. mykiss, compared to crosses
with other salmonids (i.e., low levels of post-zygotic isolation in laboratory studies)
(6) O. mykiss, O. clarki, and O. masou have similar life histories, with freshwater
residence times 1–2+ years, freshwater populations common
(7) O. masou feed and mature during freshwater spawning migration, like
O. mykiss and O. clarki but unlike other Pacific salmon
(8) Distribution of O. masou is precisely parapatric to that of O. mykiss,
with line of demarkation near Amur River, Sea of Othotsk
tshawytscha or O. kisutch with one or more of the
other three species. Indeed, O. tshawytscha and O.
kisutch show a curious tendency to group with O. keta,
O. gorbuscha, and O. nerka in analyses of the GH2C,
16S, and ND3 data sets. Given that the fertility of
some of the crosses between (O. tshawytscha or O.
kisutch) and (O. keta, O. gorbuscha, or O. nerka) is
currently high (Chevassus, 1979), it should have been
even higher in the past, and hybridization events could
have led to the moderate degree of discordance between gene trees observed here (see also Rosenfield et
al., 2000). Regardless of such apparent incongruities,
the clade (O. tshawytscha, O. kisutch) is strongly supported (99–100% bootstrap or Bayesian support values)
by all of the analyses of the full nuclear data set, by the
Bayesian analysis of the nuclear data set with MHC
excluded, and by the SH and Templeton tests for the
full nuclear data set.
Rounesfell (1958), Willson (1997)
Miller and Brannon (1981),
Groot and Margolis (1991)
Lee et al. (1980), Kato (1991)
4.3. Salvelinus
Our combined nuclear DNA data sets provide a wellresolved and generally well-supported phylogeny for
within the genus Salvelinus. This phylogeny is generally
concordant with the results of most previous moleculargenetic studies (reviewed in Phillips and Oakley, 1997;
Westrich et al., 2002), and also helps in diagnosing some
incongruent findings from single-gene studies. A close
relationship between Sv. alpinus and Sv. malma is well
supported by morphology (Behnke, 1984; Cavender,
1980), karyotypes (Cavender, 1984; Phillips et al., 1989),
allozymes (Crane et al., 1994), and all studies using
DNA sequence. Sv. confluentus and Sv. leucomaenis are
also usually grouped together by morphology and allozymes (Table 6). However, Sv. confluentus groups
strongly with Sv. alpinus and Sv. malma by analyses of
mtDNA restriction sites, ND3, ITS2, and satellite DNA
Table 5
Summary of evidence from previous studies for a sister-taxon relationship between Oncorhynchus and Salvelinus (rather than Salmo and Oncorhynchus)
Evidence
References
(1) Vitellogenin gene organization groups Oncorhynchus and Salvelinus together
(2) Microsatellite gene structure groups Oncorhynchus and Salvelinus together
(3) The number of chromosome arms in the karyotype is the same (100) in basal
Salvelinus, O. masou, and the Pacific salmon; inferred to have changed to 104 in
branch leading to (O. mykiss, O. clarki).
(4) Some Salvelinus, O. mykiss, O. clarki, and O. masou have similar life histories, with
freshwater residence times 1–2+ years, freshwater populations common
(5) Some morphological traits that link Salmo and Oncorhynchus are related to large
size and breeding competition, and thus may be convergent
(6) Oncorynchus and Salvelinus have diversified mainly in the Pacific and Nearctic
respectively, whereas Salmo is in the Palearctic and Atlantic
(7) Oncorhynchus and Salvelinus both diversified over the same general time period
(roughly 6–15 mya), from fossil and molecular-clock evidence
Buisine et al. (2002)
Angers and Bernatchez (1997)
Phillips and Rab (2001)
(8) Independent evolution of highly developed anadromy inOncorhynchus and Salmo
in different ocean basins is not unexpected on ecological grounds
Kato (1991), Groot and Margolis (1991),
Stearley (1992), Willson (1997)
Stearley and Smith (1993) (as reinterpreted here)
Angers and Bernatchez (1997)
Cavender and Miller (1972), Cavender (1980),
Smith et al. (1982), Shedlock et al. (1992),
McKay et al. (1996), Oohara et al. (1997)
Northcote (1978), Stearley (1992),
Hansen and Quinn (1998)
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17
Table 6
Evidence from sources other than DNA sequence for relationships among species of Salvelinus, compared to total evidence nuclear-DNA tree inferred here
Evidence and reference
Phylogeny
Morphology (Behnke, 1984)
Morphology (Stearley, 1992)
Morphology and karyology (Cavender and Kimura, 1989)
Satellite DNA (Hartley and Davidson, 1994)
Allozymes (Crane et al., 1994)
Karyology (Phillips et al., 1989, 2002)
This study (Fig. 2B and C)
fontinalis, namaycush, ((confluentus, leucomaenis), (alpinus, malma))
((leucomaenis, fontinalis, (confluentus, namaycush)), (alpinus, malma)
(fontinalis, namaycush), ((confluentus, leucomaenis), (alpinus, malma))
leucomaenis, (fontinalis, (namaycush, (alpinus, malma, confluentus)))
fontinalis, (namaycush, ((confluentus, leucomaenis), (alpinus, malma)))
fontinalis, namaycush, ((confluentus, leucomaenis), (alpinus, malma))
(fontinalis, namaycush), ((confluentus, leucomaenis), (alpinus, malma))
Our nuclear DNA trees were compatible with the results of Phillips et al. (1989, 2002), and identical to the results of Cavender and Kimura (1989).
(Grewe et al., 1990; Hartley and Davidson, 1994; Phillips et al., 1994, 1995), but it groups with Sv. leucomaenis
by analysis of ITS1 and allozymes (Crane et al., 1994;
Phillips et al., 1994). Sv. fontinalis, Sv. namaycush, and
Sv. leucomaenis have the same chromosome number,
which appears to be primitive within the genus (Phillips
et al., 1994); in Sv. fontinalis and Sv. namaycush this
karyotype comprises 104 chromosome arms, while Sv.
leucomaenis and Sv. confluentus have 100 arms and Sv.
malma and Sv. alpina have 98. Sv. fontinalis and Sv.
namaycush are basal to the other four species in most
previous DNA studies (and allozymes: Crane et al.,
1994), though they form strongly supported sister taxa
only by the ITS1 analysis of Pleyte et al. (1992). Taken
together, DNA-sequence studies of relationships within
Salvelinus have yielded strikingly incongruent results,
especially with regard to the positions of Sv. confluentus,
Sv. fontinalis, and Sv. namaycush.
The discordance among phylogenetic studies of
Salvelinus appears to be the result of hybridization
(Phillips et al., 1994, 1995; Westrich et al., 2002). The
main evidence for this hypothesis comes from the many
examples of ancient and current hybridization between
Salvelinus species. Ancient introgression of mtDNA has
been demonstrated for Sv. alpinus and Sv. fontinalis
(Bernatchez et al., 1995; Glemet et al., 1998), and for Sv.
alpinus and Sv. namaycush (Wilson and Bernatchez,
1998), and ongoing hybridization and introgression
have been reported for Sv. alpinus and Sv. namaycush
(Wilson and Hebert, 1993), Sv. malma and Sv. confluentus (Baxter et al., 1997), and Sv. confluentus and Sv.
fontinalis (Kanda et al., 2002; Redenbach and Taylor,
2002; Spruell et al., 2001). Moreover, most laboratory
crosses between Sv. alpinus, Sv. malma, Sv. fontinalis,
and Sv. namaycush and other Salvelinus result in fertile
offspring (Chevassus, 1979). The extent of hybridization
in Salvelinus appears to be higher than within Oncorhynchus (e.g., Allendorf and Leary, 1988; Campton
and Utter, 1985; Chevassus, 1979; Dangel et al., 1973;
Smith, 1992; Taylor, 2003), though it may be comparable to levels in the coregonids (Ferguson et al., 1978).
Introgression of mtDNA in Salvelinus may also in some
cases be driven by selection (Bernatchez et al., 1995;
Wilson and Bernatchez, 1998), which would tend to
amplify its effects.
Substantial levels of hybridization throughout the
evolutionary history of a group make phylogenetic inference problematic (Arnold, 1992). Indeed, given extensive ongoing hybridization, even the geographic
location of Salvelinus samples used for DNA sequencing
could substantially affect the inferences. Our combined
nuclear data phylogenies, especially those inferred from
Bayesian analyses, provide very good resolution and
support overall. However, support for the grouping of
(Sv. alpinus, Sv. malma, Sv. confluentus, Sv. leucomaenis)
appears to derive predominantly from a single gene
(ITS2). Similar considerations apply to the support for
(Sv. confluentus, Sv. leucomaenis), mainly from ITS1, and
(Sv. fontinalis, Sv. namaycush), with support mainly from
ITS2. Indeed, when the ITS1 and ITS2 data are excluded,
the only group within Salvelinus that is accorded maximum-parsimony bootstrap support over 70% is (Sv. alpinus, Sv. malma) (93%). We believe that the best strategy
in such cases is the gathering of DNA-sequence data from
as many independently evolving nuclear DNA loci as
possible, as well as liberal use of other types of character,
such as genome structure, karyotypes and allozymes.
Taken together, previous phylogenetic inferences from
the use of morphology, allozymes and karyological
characters (Table 6) are consistent with our phylogeny,
although levels of support for the topologies from such
sources of data are difficult to ascertain. Given this concordance among diverse data types, we believe that our
DNA phylogeny of Salvelinus (Fig. 2B and C) is very
likely to be correct. However, additional data from nuclear genes are needed to rigorously test this hypothesis.
4.4. Sister-group to Oncorhynchus
The sister-group to Oncorhynchus has long been believed to be Salmo (e.g., Murata et al., 1996; Phillips and
Oakley, 1997; Phillips and Pleyte, 1991; Stearley and
Smith, 1993), although analysis of data from the GH1C
gene by Oakley and Phillips (1999) provided evidence
that Salmo and Oncorhynchus are not sister taxa.
Our analyses concur with this result of Oakley and
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Phillips, and show that a sister-taxon relationship between Oncorhynchus and Salvelinus is well supported by
data from three genes (GH1C, VIT, and ND3), by the
combined data sets that exclude mitochondrial DNA,
and by SH and Templeton tests using these combined
data sets. Evidence for a sister-taxon relationship between Oncorhynchus and Salvelinus also comes from
data on gene organization, karyology, life history,
morphology, biogeography, and ecology (Table 5). The
primary molecular-genetic evidence against (Oncorhynchus, Salvelinus) is the results from analysis of ITS1,
which show strong bootstrap support for a sister-taxon
relationship between Oncorhynchus and Salmo (Fig. 1D).
However, we note that this analysis is compromised by
use of an outgroup (H. perryi) that may belong among
the ingroup taxa; when the closest available fish species
outside of these genera is used (a cichlid), support for
(Oncorhynchus, Salmo) is substantially reduced. Considered together, our analyses, and data from previous
studies, constitute strong evidence for a sister-taxon relationship between Oncorhynchus and Salvelinus. These
results should compel further evaluation of the data
from morphology (e.g., position of the vomerine teeth)
that has been used to support a sister-taxon relationship
between Oncorhynchus and Salmo.
4.5. Implications for the evolution of salmonid fishes
Our results present a number of interesting implications for understanding the evolution of salmonid life
history, behavior, and diversification. First, the finding
that Salvelinus forms the sister-group to Oncorhynchus
indicates that anadromy, in the form of long ocean migrations followed by a return to the natal stream, migration tightly linked to reproduction, and semelparity or a
very low degree of iteroparity, has evolved at least twice,
once in Salmo and once in Oncorhynchus (Oakley and
Phillips, 1999; Stearley, 1992). This parallel evolution of
life history also involves large body size for age in both
sexes, due to extensive feeding at sea, and strong male–
male competition, probably a result of high breeding
densities (Crespi and Teo, 2002; Stearley, 1992). Such
parallel changes in behavior and life history are ultimately
a consequence of the parallel ecological opportunities
favoring anadromy in the north Pacific and north Atlantic
oceans (Dodson, 1997; Gross et al., 1988; Hansen and
Quinn, 1998; McDowall, 1988; Northcote, 1978).
Second, the sister-taxa Oncorhynchus and Salvelinus
have apparently diversified in parallel on a large scale:
each genus has given rise to exclusively freshwater species (reproductively isolated kokanee forms of O. nerka,
Sv. namaycush), forms with interior (freshwater) and
sea-run populations (e.g., in O. mykiss and Sv. malma),
and exclusively Asian species (O. masou, Sv. leucomaenis) (Rounesfell, 1958; Stearley, 1992; Willson, 1997).
These similarities are consistent with the parallel radia-
tion of the two genera from a common ancestor, subject
to relatively similar selective pressures, opportunities for
dispersal, and vicariant events.
Third, given that O.masou groups with O. mykiss and
O. clarki rather than with the other so-called Pacific
salmon, semelparity has apparently evolved twice in
Oncorhynchus, once in the lineage leading to O. masou,
and once in the lineage leading to (O. tshawytscha, O.
kisutch, O. nerka, O. gorbuscha, O. keta). Alternatively,
O. masou may be less strictly semelparous than is currently believed, as females of some populations of
landlocked O. masou exhibit a small degree of iteroparity (Healey et al., 2001), and one of the main forms
of evidence in the literature for semelparity in O. masou
appears to have been its presumed phylogenetic position
among the Pacific salmon (Kato, 1991).
4.6. Congruence, total evidence, and clade support
Our analyses raise a number of issues regarding the
use of multiple data sets and criteria for evaluating
congruence. First, our results provide good examples of
both the strengths and limitations of a conditional
combining approach to phylogenetic congruence.
Overall, the data sets from the nuclear genes complemented and reinforced one another to yield a robust
tree, which is what one hopes that combining of data
will achieve (Bull et al., 1993; Cunningham, 1997). By
contrast, the results from mtDNA genes tended to
contradict the results from nuclear genes, and in the
combined data sets the inclusion of mtDNA reduced the
degree of resolution and bootstrap or Bayesian support.
But because the individual data sets overlapped only
partially in the taxa that they included, it was not possible to apply statistically based congruence tests (e.g.,
Huelsenbeck and Bull, 1996) to our data sets, which
could more-objectively justify the exclusion of mtDNA
or other data sets such as MHC.
Second, the use of bootstrap or Bayesian a posteriori
support values to evaluate congruence is subject to important caveats. Majority-rule bootstrap trees may differ
from best or strict consensus trees, or the bootstrap
profiles (sets of bootstrapped trees) from analyses of
different genes may exhibit little or no overlap (Page,
1996; Sanderson, 1989). Moreover, low bootstraps
across a clade can be due to only one or few ÔrogueÕ
species whose position is especially uncertain due to
long-branch attraction, a paucity of data, or other
processes (Page, 1996; Sanderson and Schaffer, 2002).
For our separate and combined data sets, the bootstrap
majority-rule consensus trees were almost always the
same as the best or strict consensus trees, subject to the
lack of resolution shown in many of the bootstrap trees;
these findings suggest that rogue species are not unduly
influencing our results. Such limitations do not apply to
Bayesian a posteriori probability values, which appear
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to provide a more accurate metric of support for the true
tree due to their lack of bias and higher sensitivity to
phylogenetic signal (Alfaro et al., 2003). However, such
Bayesian probability values are less conservative than
bootstraps, and given the vagaries of such factors as
sampling error and imprecise model specification,
Bayesian probability values may in some cases imply
strong support for relationships that are accorded only
weak support by bootstraps (Alfaro et al., 2003; Douady
et al., 2003) or by other metrics such as tree length. In
our view, these considerations imply that high Bayesian
clade support values should be interpreted cautiously,
and that they should be accorded high confidence only
in conjunction with high likelihood or parsimony
bootstraps, or results from SH or Templeton tests.
Third, our analyses of the combined nuclear data sets
show that in some cases, adding data from an additional
gene can substantially increase maximum-parsimony
bootstrap support for some nodes while notably decreasing support for others. Thus, the exclusion of the
MHC data from our combined nuclear data set yields a
tree with very strong maximum-parsimony bootstrap
support for (O. mykiss, O. masou, O. clarki, 95% maximum-parsimony bootstrap) but only moderate support
for (O. tshawytscha, O. kisutch, 72%), while its inclusion
gives the reverse: weaker support for the former clade
(79%), but very high support for the latter (99%). Because MHC is known to be under strong selection in
salmonids (Miller and Withler, 1996), we hesitate to
include it without reservations, even if when analyzed
separately these data provide a tree that does not appear
to be unequivocally incongruent with others.
In contrast to these parsimony results, the Bayesian a
posteriori probabilities for (O. mykiss, O. masou, O.
clarki) and for (O. tshawytscha, O. kisutch) remained
high (100%) whether or not the MHC data was included
(Fig. 2). These results appear to reflect the higher sensitivity of Bayesian analysis (vs. maximum parsimony
bootstraps) to phylogenetic signal, its increased accuracy in recovering monophyletic groups, and the high
susceptibility of maximum parsimony analysis to longbranch attraction, which can erode bootstrap support
for the affected clades (Alfaro et al., 2003). Overall, we
find it difficult to argue against the monophyly of both
(O. mykiss, O. masou, O. clarki) and (O. tshawytscha, O.
kisutch), as there is considerable support for each clade
and no notably conflicting results.
4.7. Optimizing future studies
One of the most important results of this study is its
role in mapping the best route for future molecular-phylogenetic studies of salmonids, with the ultimate goal of a
robust tree for all species and subspecies in the family. In
our study, the most-informative genes were GH1C,
GH2C, VIT, CYTB, ITS1, ITS2, and MHC. Of these
19
genes, evidence for incongruence was observed with the
results from ITS1, ITS2, and MHC. Since the apparent
cause of the incongruence can be surmised in each case
(i.e., hybridization and selection respectively), we believe
that the data from these genes should be treated with
reservations in combined analyses. Despite such cautions,
in each of these cases the apparent incongruence involved
the placement of only a few species, the data from the
other genes appeared to supercede the problematic effects,
and the inclusion of the data from these genes thus increased the robustness of the combined nuclear data tree
overall. These results imply that the sequencing of some or
all of the genes GH1C, GH2C, VIT, ITS1, ITS2, and
possibly CYTB and MHC, for an enlarged set of salmonid
species, is likely to provide the best estimate for the phylogeny of Salmonidae, until additional nuclear markers
are developed. Indeed, a combined data set that includes
only these genes provides almost as well-resolved and
supported a phylogeny as the full nuclear data set.
When data from the same large suite of taxa are
available for a collection of genes, statistically based
methods for the analysis of congruence can also be applied, to better assess the extent to which different data
partitions agree on one true species tree. Such a data set
should also allow robust inference of the nature and
timing of major events in salmonid diversification, using
data from fossils (Behnke, 1992; Cavender, 1980; Cavender and Miller, 1972; McPhail, 1997; Smith and
Stearley, 1989; Stearley and Smith, 1993), paleoclimatology (Pearcy, 1992), geology (Montgomery, 2000),
paleobiogeography (McPhail, 1997; Minckley et al., 1986;
Neave, 1958), and the biogeography and ecology of the
Esociformes, a freshwater group that has recently been
shown to be the sister taxon to salmonids (Ishiguro et al.,
2003). In conjunction with recent methods for use of
DNA sequence to infer divergence times (Arbogast et al.,
2002; Sanderson, 2002), such data will provide a comprehensive, interdisciplinary picture of the adaptive radiation of Salmonidae.
Acknowledgments
We are grateful to Robert Behnke, Ian Fleming,
Andrew Hendry, Todd Oakley, Ruth Phillips, Jerry
Smith, Eric Taylor, John Taylor, Kyle Young, and
two anonymous reviewers for helpful comments and
discussion, and the Natural Sciences and Engineering
Research Council of Canada for financial support.
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