JEZ 0816
JOURNAL OF EXPERIMENTAL ZOOLOGY (MOL DEV EVOL) 288:105–119 (2000)
Phylogenetic Analysis of the Wnt Gene Family and
Discovery of an Arthropod Wnt-10 Orthologue
E.L. JOCKUSCH1* AND K.A. OBER2
1
Department of Molecular and Cellular Biology, University of Arizona,
Tucson, Arizona 85721
2
Interdisciplinary Program in Insect Science, University of Arizona, Tucson,
Arizona 85721
ABSTRACT
Wnt genes encode a conserved family of secreted signaling proteins that play many
roles in arthropod and vertebrate development. We have investigated both the phylogenetic history and molecular evolution of this gene family. We have identified a novel Wnt gene in a diversity of arthropods that it is likely an orthologue of the vertebrate Wnt-10 group. Wnt-10 is one of
only two cases in which orthology between protostome and deuterostome genes could be consistently assigned based on our analyses. Despite difficulties in assessing orthologies, all of our trees
suggest that the most recent common ancestor of protostomes and deuterostomes possessed more
than the five Wnt genes known from either arthropods or nematodes. This suggests that Wnt gene
loss has occurred during protostome evolution. In addition, we examined the rate of amino acid
evolution in the two arthropod/deuterostome orthology groups we identified. We found little rate
variation across taxa, with the exception that Drosophila Wnt-1 is evolving more rapidly than all
vertebrate and most arthropod orthologues. J. Exp. Zool. (Mol. Dev. Evol.) 288:105–119, 2000.
© 2000 Wiley-Liss, Inc.
Developmental regulatory genes constitute a
class of genes that has undergone a great deal of
expansion, presumably through gene duplication
and divergence, within the Metazoa (Holland and
García-Fernández ’96; Chervitz et al., ’98). This
has been hypothesized to be essential for the morphological diversification of the Metazoa, and particularly of the jawed vertebrates, whose origin
was accompanied by duplication of many regulatory genes (Holland and García-Fernández ’96; Valentine et al., ’96; Chervitz et al., ’98). Early models
of the fate of duplicated genes predicted that loss
of function of one copy should be a much more common event than functional diversification (Allendorf,
’79; Li, ’80; Waterson, ’83). The evidence in favor of
functional diversification in many gene families (e.g.
Nadeau and Sankoff, ’97), including the Wnt family, presents an empirical challenge to these early
theoretical models. Understanding the history of
duplication, divergence, and loss in individual gene
families will contribute to evaluating hypotheses
about the role that these play in morphological and
functional evolution.
One family of developmental regulators that appears to be a metazoan invention is the Wnt gene
family. Wnt genes encode secreted signaling proteins that have diverse functions during development in both deuterostomes and protostomes
© 2000 WILEY-LISS, INC.
(reviewed in Nusse and Varmus, ’92). To date, Wnt
genes have only been identified in metazoans. No
Wnt homologues were found in the complete yeast
genome (Goffeau et al., ’96; Chervitz et al., ’98),
but Wnt family members are known from deuterostomes (van Ooyen and Nusse, ’84; Sidow, ’92;
Ferkowicz et al., ’98; Sasakura et al., ’98), arthropods (e.g., Rijsewijk et al., ’87; Nulsen and Nagy,
’99), a nematode (Shackleford et al., ’93; Herman
et al., ’95; Thorpe et al., ’97; Maloof et al., ’99; The
C. elegans sequencing consortium, ’98), an annelid
(Kostriken and Weisblat, ’92), and a brachiopod
(Holland et al., ’91). They are characterized by the
conservation of 22 cysteines and the spacing between some of these cysteines (Nusse and Varmus,
’92). While at most four Wnt family members have
been identified in any arthropod (in D. melanogaster: Rijsewijk et al., ’87; Eisenberg et al., ’92;
Russell et al., ’92; Graba et al., ’95), at least 17
have been identified in the mouse Mus musculus.
Grant sponsor: National Science Foundation; Grant number: DEB9420219; Grant sponsor: Sloan Foundation; Grant number: 94-4-3ME;
Grant sponsor: University of Arizona Research Training Grant in the
Analysis of Biological Diversification; Grant number: DIR-9113362.
*Correspondence to: E.L. Jockusch, Department of Ecology and
Evolutionary Biology, 75 N. Eagleville Rd., University of Connecticut, Storrs, CT 06269. E-mail: [email protected]
Received 13 August 1999; Accepted 15 December 1999
106
E.L. JOCKUSCH AND K.A. OBER
Previous phylogenetic analysis suggested that
at least seven Wnt genes (orthologues of vertebrate Wnt-1 to Wnt-7) were present in the common ancestor of arthropods and vertebrates, and
that an additional round of duplication occurred
at the base of the jawed vertebrates (Sidow, ’92).
Sidow (’92) also found that the rate of amino acid
substitutions in many Wnt genes decreased fourfold at the base of the jawed vertebrates, despite
the duplication of many of these genes along the
same internode. This is surprising in light of theoretical predictions that following gene duplication,
genes should be under relaxed selection because
of functional redundancy and therefore experience
increased rates of molecular evolution (Li and
Gojobori, ’83; Li, ’85).
Within arthropods, Wnt-1 (wingless) orthologues
have been identified in many groups (e.g. Rijsewijk
et al., ’87; Nagy and Carroll, ’94; Nulsen and Nagy,
’99), and have been used to infer the phylogenetic
history of several insect groups (Baker and DeSalle, ’97; Brower and DeSalle, ’98). To date, no
other Wnt family members have been reported
from any arthropod other than D. melanogaster.
We have identified a Wnt family member from insects and crustaceans that does not appear to be
an orthologue of any of the D. melanogaster Wnt
genes. Phylogenetic analyses of the entire Wnt
family identify this gene as an arthropod orthologue of chordate Wnt-10. These analyses concur
with Sidow (’92) that the complement of Wnt genes
present in the common ancestor of protostomes
and deuterostomes was greater than the number
that has been isolated from any protostome group,
indicating that gene loss may have occurred in
protostomes. In addition, we used an expanded
sampling of arthropod Wnt-1 and Wnt-10 genes
to compare rates of molecular evolution in protostome and deuterostome lineages and find no evidence of a rate decrease at the base of the jawed
vertebrates.
MATERIALS AND METHODS
DNA extraction, amplification, and
sequencing
Genomic DNA was extracted following standard
protocols (Maddison et al., ’99, for the carabid
beetles Desera sp. and Galerita lecontei and
simuliid black flies [Prosimulium formosum and
Metacnephia sommermanae]; genomic tip kit
[Qiagen] for the thysanuran insect Thermobia
domestica and the branchiopod crustacean Triops
longicaudatus). Beetle and blackfly Wnt genes
were amplified using degenerate primers coding
for the conserved amino acid sequences 5′ECKCHGMSG and complementary to the amino acid sequence 5′CTFHWCCAV. Products were sized on an
agarose gel, and the band of the expected size was
purified from the gel, then sequenced directly using dye-labeled terminators. Genomic DNA from
Thermobia and Triops was amplified using degenerate primers coding for the sequence 5′QECKCHG
and complementary to the sequence 5′HWCC(A/V)V
(Sidow, ’92). Products in the expected size range
were ligated into TOPO vector 2.0 (Invitrogen),
cloned, and sequenced using dye-labeled terminators. All of the above primers were expected to amplify Wnt-1 orthologues; however, we also obtained
representatives of what appeared to be a novel arthropod Wnt gene. To try to obtain an orthologue
of this novel Wnt sequence from D. melanogaster,
representatives of all size classes amplified from
its genomic DNA with the primers sets described
above were cloned and sequenced. No new Wnt
genes were obtained in this way. Therefore, new
primers were designed to favor the novel gene over
other Wnt family members by including at the 3′
end of each primer nucleotides coding for a Wnt10 specific amino acid (primer sequences coding
for 5′TCWK(S/A)APD and complementary to
5′RFHWCC(A/V)V, with underscoring indicating
amino acids specific to our target). These primers
were used both directly on D. melanogaster genomic DNA and in nested reactions following amplification with the primers described above.
Taxon sampling and alignment for
phylogenetic analysis
In order to identify the arthropod Wnt gene fragments obtained, we included them in phylogenetic
analyses of a representative set of Wnt genes.
Analyses were done both on a data set containing
primarily sequences of the complete coding region,
and on a larger sampling of taxa with sequence
restricted to the region between our PCR primers. Use of the complete coding sequence increases
the number of characters in the analysis, but data
are not available for many taxa. The complete sequence data set consisted of the Wnt gene fragments isolated from Triops (Wnt-1 gene reported
by Nulsen and Nagy, ’99) and Thermobia (both
with sites outside the PCR fragment coded as
missing data) plus 21 complete coding sequences:
the four known D. melanogaster Wnt genes, 15
mouse genes selected to represent the previously
identified Wnt family members and when possible
to span the deepest split in each group, and two
WNT GENE FAMILY EVOLUTION
additional vertebrate sequences–chick Wnt-14, to
represent a group for which the complete mouse
sequence was not yet known, and Brachydanio
rerio (zebrafish) Wnt-8, to sample as deeply diverged representatives of the Wnt-8 group as possible relative to the mouse sequence.
Wnt genes for the PCR fragment data set were
selected to include representatives of all Wnt family members identified to date, and within those
groups to span the deepest possible split within
the deuterostomes in order to minimize the problems of long branch attraction (Felsenstein, ’78).
In most cases, this meant including sequence from
a jawed vertebrate and an echinoderm or nonjawed vertebrate. Although numerous additional
vertebrate sequences are available for this gene
region, we limited the number used in order to
reduce the computational time of the phylogenetic
analyses. Because we have spanned the deepest
possible splits within deuterostome groups, and
generally recovered the expected relationships of
proposed deuterostome orthologues, the effects of
omission of additional taxa from our analyses
should be minimal. For arthropods, our new sequences and representatives of all other arthropod Wnts were included (for Drosophila Wnt-2, 3,
and 4, the D. melanogaster sequences are the only
available sequences; for Wnt-1, representatives
from throughout the insects, and from one additional crustacean were used; note that the Drosophila and vertebrate numbers do not always
identify expected orthologues). Two versions of the
PCR fragment data set, including and excluding
the five C. elegans Wnt sequences, were used in
phylogenetic analyses of the amino acid data. C.
elegans sequences were excluded from all analyses of the nucleotide sequences. Genes of C.
elegans have been shown to evolve extremely rapidly (Aguinaldo et al., ’97), which may negatively
affect phylogeny reconstruction (Felsenstein, ’78).
The GenBank accession numbers of all sequences
used are listed in Appendix A. For both data sets,
initial protein sequence alignment was done in
Clustal W (Thompson et al., ’96), and extensively
adjusted by eye in MacClade (Maddison and
Maddison, ’99). A nucleotide alignment corresponding to the PCR fragment data set was made
using the protein alignment as a template.
Phylogenetic analyses
Different phylogenetic methods are sensitive to
different kinds of bias in the data. The use of multiple inference methods gives increased confidence
when results are in agreement and otherwise
107
points to possible causes of the disagreement.
Therefore, we have used maximum likelihood, parsimony, and distance methods to analyze the
nucleotide data and parsimony to analyze the protein data. All trees are unrooted, but the root is
assumed not to separate deuterostome and arthropod sequences that are nearest neighbors, which
allows us to determine sister group relationships
between arthropod and deuterostome genes. All
analyses were conducted in PAUP* (versions d64,
d65, b1 and b2; Swofford, ’99). Details of the analyses are summarized in Appendix B.
Protein sequences were analyzed using parsimony. Two different character weighting schemes
were used, equal weights, unordered, and a step
matrix in which the distance between amino acids was calculated as the minimum number of
nucleotide changes required to convert a codon for
one amino acid into a codon for the other. All
analyses were conducted with gaps coded as an
extra state (in the step matrix, a change from an
amino acid to a gap was treated as requiring three
steps) and with gaps coded as missing. Heuristic
searches consisting of at least 100 random addition replicates with starting trees obtained by
stepwise addition were used for each analysis. To
test the effects of inclusion of regions of uncertain alignment, all analyses were also carried out
with and without regions of uncertain alignment.
This produced a total of eight analyses per data
set (2 gap treatments × 2 character weighting
schemes × 2 character inclusion sets) for both the
complete and PCR fragment data sets (analyses
1 to 16 in Appendix B). In order to estimate relative support for all clades, bootstrapping was done
(100 bootstrap replicates with 10 to 20 random
addition replicates/bootstrap replicate) for all
analyses (Felsenstein, ’85).
We used likelihood, parsimony, and minimum
evolution to analyze the nucleotide data set. Trees
of highest likelihood were found by a single heuristic search with TBR branch-swapping. The
model of character evolution chosen for the search
(GTR + codon position rates) fit the data significantly better than other models explored. TBR
branch swapping was performed on a starting tree
of high likelihood to search for the tree of highest
likelihood.
Minimum length parsimony trees were sought
by conducting 600 random addition sequence
searches for equal weights parsimony and 700
random addition sequence replicates for weighted
parsimony, with each search beginning from trees
acquired by stepwise addition. For equal weights
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E.L. JOCKUSCH AND K.A. OBER
parsimony analyses, characters were treated as
unordered and with equal weights and gaps were
coded as missing data. For the weighted parsimony searches, codon positions were weighted by
the reciprocal of the maximum likelihood estimate
of their relative rates of change (position 1 = 3,
position 2 = 4, and position 3 = 1) and gaps were
coded both as missing data and as an extra state.
Minimum evolution distance trees were found
using distances calculated according to the HKY85
(Hasegawa et al., ’85) and LogDet (Lockhart et
al., ’94) model of nucleotide evolution. Starting
trees were found by neighbor joining (Saitou and
Nei, ’87). One minimum evolution distance analysis was also performed using the model of nucleotide change estimated from the ML analysis with
800 random addition sequence searches.
Bootstrap values for weighted parsimony, HKY85
and LogDet minimum evolution distance analyses
were calculated using heuristic searches with ten
random addition sequence replicates with starting
trees built by stepwise addition for each of 365 to
800 bootstrap replicates. Decay indices for weighted
parsimony clades were calculated by constraining
100 random addition sequence replicate PAUP*
searches to find the most parsimonious trees without that clade.
Relative rate analyses
To determine if there was any difference in the
rate of molecular evolution between deuterostome
and arthropod Wnt genes, two different relative
rate tests were performed on all the available Wnt1 and Wnt-10 orthologues, the only two orthologue
groups in which arthropods and deuterostomes are
both represented by diverse taxa. The GenBank
accession numbers of sequences used are listed
in Appendix A. Relative rate tests compare the
sequences of the two lineages of interest (ingroups)
to an outgroup sequence to test whether one
ingroup sequence has diverged significantly more
from the outgroup than has the other ingroup sequence. Other closely related Wnt gene family
members were used as outgroup sequences (Mus
Wnt-6 and Mus Wnt-7b) when comparisons were
made between deuterostomes and arthropods, as
Wnt-1 and Wnt-10 orthologues are not known from
other metazoans outside the clade containing
arthropods and vertebrates. For rate comparisons
within the arthropods a Mus orthologue was used
as the outgroup and for rate comparisons within
the deuterostomes the Thermobia orthologue was
used. We compared results obtained using two
similar tests (Tajima, ’93; Li, ’97). Using the DI-
VERGE program in the Wisconsin Genetics Group
GCG package, the number of nonsynonomous
(amino acid) substitutions per site, the number of
nondegenerate and two-fold degenerate sites, and
the standard deviation were calculated between
each arthropod and vertebrate Wnt-1 and Wnt-10
and the appropriate outgroup using a one-parameter model of nucleotide substitution. Substitution
rates were considered different if the difference
between the two ingroup-outgroup rates was
greater than twice the standard deviation. Tajima’s (’93) test used a similar, simpler calculation to compare the rates of Wnt amino acid
evolution. A chi-square test determined if the rates
of amino acid substitution were significantly different from each other.
RESULTS
We obtained orthologues of a novel arthropod
Wnt gene from two blackflies (Metacnephia and
Prosimulium ), a carabid beetle (Desera), a thysanuran insect (Thermobia), and a branchiopod crustacean (Triops) (Fig. 1). We were unable to obtain
an orthologue of this gene from D. melanogaster,
and it is not presently represented in the Drosophila genome sequencing project databases. The
new sequences were clearly identifiable as Wnt
genes based on the conservation of eight cysteines
in the amplified region. Amino acid identity among
these sequences ranges from a high of 85% between the two blackfly species to a low of 45%
between Triops and a blackfly. On average, these
sequences are 45% identical to D. melanogaster
Wnt-1, 40% to D. melanogaster Wnt-2, 37% to D.
melanogaster Wnt-3, and 35% to D. melanogaster
Wnt-4. Initial BLASTx searches suggested that we
had obtained orthologues of chordate Wnt-10. On
average, our inferred amino acid sequences are
48% identical to Mus Wnt-10a and Wnt-10b. Wnt1 orthologues were obtained from Thermobia,
Triops (Nulsen and Nagy, ’99), and a carabid beetle
(Galerita) (Fig. 1). The inferred amino acid sequences differed from conspecific Wnt-10 sequences by 55% (Thermobia) and 48% (Triops)
respectively. The carabid beetle Wnt-1 and Wnt10 amino acid sequences differed by 56%.
The two blackfly sequences appeared to contain
an intron. The intron was initially posited based
on much greater divergence between the blackfly
sequences in that region, which could not be
aligned with any of the other taxa, and a shift in
reading frame that would have resulted in loss of
similarity more 3′ in the inferred amino acid sequence. The hypothesized boundaries had se-
WNT GENE FAMILY EVOLUTION
Fig. 1. Alignment of novel arthropod Wnt genes, with vertebrate Wnt-10 and all D. melanogaster Wnt genes included
for comparison. * indicates amino acids conserved across all
109
Wnt-10 orthologues. Arrow indicates site of an amino acid
insertion [not shown] in D. melanogaster Wnt-1.
110
E.L. JOCKUSCH AND K.A. OBER
quences highly similar to consensus splice sequences for invertebrates (Shapiro and Senapathy,
’87). This region (58 bp in Prosimulium and 59 bp
in Metacnephia) was removed from all analyses.
In order to test the identity of these novel arthropod genes as Wnt-10 orthologues, we undertook
phylogenetic analysis of selected Wnt sequences representing all known clades from arthropods and
deuterostomes. In general, relationships within deuterostome orthologue groups, which reflect relatively
recent divergences, were recovered in all analyses.
Slightly deeper relationships, such as relationships
between proposed arthropod and deuterostome
orthologues, also received some support. However,
more ancient divergences (reflecting duplication
events in ancestral metazoans, as well as some of
the presumed deuterostome-protostome relationships) were neither consistently obtained nor wellsupported in any of the analyses. In analyses of
the PCR fragment, no deeper divergences ever
yielded bootstrap support above 50%. Use of the
complete coding sequence yielded some deep nodes
with bootstrap support greater than 50%. This increase in support for deeper nodes would be expected if lack of resolution is simply a result of
too few characters in the PCR fragment data set.
However, the supported nodes were highly sensitive to the method of analysis used, with trees
produced by different methods having conflicting
branches with bootstrap support above 50%. Thus,
increasing the number of characters used did not
increase confidence in the deeper level phylogeny.
Inclusion of the C. elegans genes does not affect
these general conclusions, although it did affect
the topology of individual analyses and usually
resulted in a large increase in the number of most
parsimonious trees. Detailed discussion of the
orthology between arthropod and chordate genes
is therefore based on the analyses in which C.
elegans was excluded. It appears that the phylogenetic information retained by Wnt paralogues
may be insufficient to recover the early history of
this gene family.
Although Wnt genes do not appear to be useful
for recovering deeper phylogeny, many more recent relationships were consistently and strongly
supported (Fig. 2). In all analyses of the complete
coding sequence, mouse a/b paralogue relationships, believed to result from gene duplication at
the base of the jawed vertebrates (Sidow, ’92), were
present in the strict consensus of most parsimonious trees, and had high bootstrap support (100%
for Mus Wnt-2/2b, Wnt-3/3a, Wnt-5a/5b, and Wnt7a/7b pairs, 81% to 100% for Mus Wnt-10a/b; the
slightly more recent Mus/Brachydanio Wnt-8
pair was also supported in 100% of analyses). In
analyses of the PCR fragment, support for many
of the previously suggested deuterostome relationships was also present (in both amino acid and
nucleotide analyses). Clades that consistently occurred and received bootstrap support >50% were
mouse Wnt-2b/Branchiostoma Wnt-2; Mus Wnt-4/
Strongylogentrotus Wnt-4; Mus Wnt-7a/Branchiostoma Wnt-7; Eptatretus Wnt-8/Branchiostoma Wnt-8; and Mus Wnt-10a/Mus Wnt-10b/
Eptatretus Wnt-10.
One additional, previously unidentified deuterostome group was present in every analysis and
received high bootstrap support (analysis 15 was
the only one with bootstrap support <80%). This
was a clade containing mouse Wnt-15, human
Wnt-14, and shark (Alopias) and hagfish (Eptatretus) Wnt-9 genes. Mouse Wnt-15 and human
Wnt-14 were never sister taxa, and a human Wnt15 orthologue has been identified (Bergstein et al.,
’97) making it clear that these belong to different
orthology groups. It is possible that mammalian
Wnt-14 and Wnt-15 resulted from the same duplication event at the base of the jawed vertebrates that created the vertebrate a/b pairs.
However, the frequent grouping of shark and hagfish Wnt-9 genes with different mammalian sequences raises the possibility that these genes
result from a duplication event earlier in deuterostome history and that the shark and hagfish Wnt9 genes may not be orthologous. If the Wnt-9 genes
are not orthologous, then it is likely that the
Alopias Wnt-9 is more closely related to mouse
Wnt-15, a relationship recovered in 10/16 analyses with bootstrap support of 54% to 95%. A sister group relationship between Eptatretus Wnt-9
and human Wnt-14 was recovered in 11/16 analyses with bootstrap support of 53% to 83%. In the
analyses of complete coding data, this clade was
represented by a single sequence, chick Wnt-14.
At an intermediate level of divergence, representing relationships between presumed protostome and deuterostome orthologues, consistency
of results and level of support varied across analy-
Fig. 2. Results of phylogenetic analysis. Tree A is the consensus of 8 trees resulting from parsimony analysis 11 with
amino acid data from the PCR fragment. Tree B is a consensus of 2 trees from parsimony analysis 3 of the complete coding sequence. The numbers on the branches in A and B
indicate bootstrap support. Tree C is the tree of highest likelihood for the nucleotide data from the PCR fragment (analysis 19).
WNT GENE FAMILY EVOLUTION
Figure 2.
111
112
E.L. JOCKUSCH AND K.A. OBER
ses. For each analysis, the inferred sister group
of each arthropod clade and the support for that
clade are shown in Table 1. The best supported
relationships were between arthropod and deuterostome Wnt-1 and between arthropod and deu-
terostome Wnt-10. These are discussed in more
detail below. Relationships of the other arthropod
Wnt genes, D. melanogaster Wnt-2, 3, and 4, are
not as clear. DWnt-2 has been previously suggested to be a deuterostome Wnt-7 homologue
TABLE 1. Summary of phylogenetic relationships between arthropod and deuterstome Wnt genes found
in a variety of analyses1
Arthropod clade
Analysis
Wnt-1
Amino acids
Complete coding sequence
1
M1,D1,(Td1,Tl1)-98%2
2
M1-91%
3
M1-93%
4
M1-96%
5
M1-97%
6
7
8
PCR fragment
9
M1-96%
M1-89%
M1-98%
M1
Wnt-10
M10a,b
large poly
large poly
large poly
Tl10(Td10
(M10a,b))-55%4
M3,3a
G14
M10a,b
DWnt-2
DWnt-3
DWnt-4
(M7a,b) ((M5a,b)(M2,2b))
large poly
M7a,b-62%
(M7a,b)((M5a,b)(M2,2b))
M7a,b-64%
D4
D4
large poly
D4
D4
D3
D3
large poly
D32
D3
M7a,b-64%
M8,Z8-76%
M7a,b-94%
M5a,b-62%
M5a,b-57%
M5a,b-79%
G14-66%
All Wnt1-54%
G14-99%
deut10,Td10,
Tl10,I102
deut10-74%
large poly
large poly
large poly
M3
deut10,Tl10,I1
0-60%2
deut10-73%
M3
I10(Tl10,deut10)63%4
deut10,Tl10,
I10-74%
deut10
E3
(M2,Z2),(D4,
deut9/14/15
deut9/14/15)
large poly
deut9/14/1576%
(m2,b2)(D4,
deut9/14/15deut9/14/15)
73%
S5
M7,S7
E3
S5
M7,S7
D4,deut9/14/15-77%
S5-71%
deut9/14/1597%
deut9/14/1592%
10
M1,M6,B15
11
M1,B15
12
M1,M6,B15
13
14
(M1,B1)(Wnt-10
(D2,E3),M3)
M1,B1
15
large poly6
16
M1,B1
I10(Tl10,deut10)75%
E3-62%
S5
Nucleotides
PCR fragment
17
M1
M3
S5
18
Decay index
19
20
21
22
23
24
M1
17
M1,M6
(M1,M6)B1
M1,B1
(M1,M6)B1
(M1,M6)B1
M1-57%
deut10-59%
9
deut10
deut10
deut10-61%
deut10-72%
deut10-53%
deut10
(E3,deut10)(D4((m2,b2)
(deut9/14/15))
D4,E3
3
D4
D4(M7,S7)
D4(M7,S7)
M3
D4(M7,S7)
(D4,deut9/14/15),E32
M3
S5
4
S5
deut9/14/15
deut9/14/15
deut9/14/15
deut9/14/15
S5-60%
(M2,Z2),
(deut9/14/15)
E3
5
D2
M7,S7
M7,S7
M7,S7
M7,S7
deut9/14/1598%
1
Details of analyses are given in Appendix B. The sister group found in the strict consensus of best trees from each analysis is indicated,
using parenthetical notation to convey the phylogenetic structure within the sister group. Boostrap support is given for arthropod-sister
group nodes when it exceeded 50%. Large poly indicates that the node contained a large polytomy. Gene names are given in abbreviated form
as the first letter of the genus name and the Wnt gene number. B = Branchiostoma, D = Drosophila, deut = deuterostome, E = Evasterias, G =
Gallus, I = insect, M = Mus, S = Strongylocentrotus, Td = Thermobia domestica, Tl = Triops longicaudatus, Z = zebrafish (Brachydanio).
2
Polytomy; bootstrap support, if above 50%, is given for the arthropod + deuterostome clade.
3
67% bootstrap support for sister group relationship with chick Wnt-14.
4
Arthropod Wnt-10 not monophyletic; bootstrap support is for all Wnt-10.
5
Arthropod Wnt-1 forms a polytomy with the clades indicated.
6
57% bootstrap support for clade containing all Wnt-1 genes.
WNT GENE FAMILY EVOLUTION
(Sidow, ’92), DWnt-3 a deuterostome Wnt-5 homologue (Sidow, ’92), and DWnt-4 possibly a vertebrate Wnt-9 homologue (Graba et al., ’95). As
shown in Table 1, these are the only relationships
that received bootstrap support >50% in multiple
analyses; however, other relationships were found
in the best trees from different analyses. Because
numerous equally parsimonious trees were found
in analyses including C. elegans Wnt sequences,
their strict consensus trees typically had very little
resolution. Relationships between most C. elegans
Wnt genes and deuterostome orthologues could not
be confidently identified. Bootstrap support ranging from 35% to 91% identified a possible sister
group relationship between C. elegans mom-2 and
Drosophila Wnt-4. A sister group relationship was
also consistently inferred between C. elegans Wnt1 and deuterostome Wnt-4, though with low bootstrap support (36% to 62%).
Wnt-1 relationships
In the analysis of complete coding sequences,
only a single chordate (mouse Wnt-1) and three
arthropod Wnt-1 sequences (complete Drosophila
coding sequence and partial coding sequences from
Triops and Thermobia) were included. The grouping of these four sequences was strongly supported
in all analyses (89% to 98% bootstrap support).
An arthropod Wnt-1 clade was present in the consensus of most parsimonious trees in all analyses
except one, in which a strict consensus of most
parsimonious trees resulted in a trichotomy of the
mouse, Drosophila and (Thermobia + Triops) Wnt1 sequences. Support for the arthropod Wnt-1
clade ranged from 48% (analysis 1) to 100% (in
analysis 7). In only one analysis (6) were the expected relationships among the three arthropod
sequences [(Triops (Thermobia, Drosophila))] recovered. The rapid evolution of Drosophila Wnt-1
(see below) may provide part of the explanation
for this incongruity.
In analyses of the PCR fragment, support for
Wnt-1 monophyly decreased, likely because of the
decrease in number of informative sites. These
analyses included one additional deuterostome Wnt1 (Branchiostoma) and six additional arthropod
Wnt-1 sequences. Arthropod Wnt-1 monophyly was
recovered in all analyses, but both deuterostome
Wnt-1 monophyly and arthropod + deuterostome
Wnt-1 monophyly were not. Bootstrap support for
the arthropod Wnt-1 clade ranged from 69% (analysis 10) to 100% (analysis 15). Despite the increased
taxon sampling relative to the complete coding sequence analyses, these analyses also failed to re-
113
cover the expected relationships among these arthropod genes. In particular, Coleoptera was not
monophyletic and the association between crustacean and Thermobia Wnt-1 found in analyses including the complete coding sequences persisted.
Furthermore, in analyses of the nucleotide data but
not of the amino acid data, this latter clade usually
nested within the rest of the insects.
The deuterostome Wnt-1 group was represented
by two sequences, whose relationship to each other
and to arthropod Wnt-1 and mouse Wnt-6 was sensitive to the weighting scheme and method of
analysis used. Deuterostome Wnt-1 monophyly
was only recovered in four analyses (13, 14, 16,
21) and was present in some but not all of the
most parsimonious trees in four other analyses
(10 to 12, 15). In all analyses but two (13, 15),
the sister group of arthropod Wnt-1 was either
mouse Wnt-1 only, mouse + Branchiostoma Wnt1, or a clade containing mouse Wnt-1, mouse Wnt6 and Branchiostoma Wnt-1 (Table 1). However,
in no case except analysis 24 did bootstrap support for the node leading to arthropod Wnt-1 and
its sister group exceed 50% (Table 1). Because the
relationship between mouse Wnt-1 and Wnt-6 disappeared in analyses of complete sequences and
because improved taxon sampling in the PCR fragment analyses did not lead to any evidence
strongly contradicting the well-supported arthropod-deuterostome Wnt-1 relationship found in
analyses of the complete coding sequences, we conclude that arthropod and deuterostome Wnt-1
genes are orthologous.
Wnt-10 relationships
A monophyletic group containing the three chordate and five arthropod Wnt-10 sequences was recovered in all analyses of the PCR fragment but
one (analysis 17). Even though bootstrap support
was low to moderate (42% to 75%), the monophyly
of this group was insensitive to the method of
analysis, which increases our confidence in it. Wnt10 monophyly was recovered in only three analyses (1, 5, 8) of the complete coding sequence;
however, in two of the five remaining analyses,
Wnt-10 monophyly was one of the equally most
parsimonious solutions, and in one of the remaining cases (6), Wnt-10 monophyly receives higher
bootstrap support than the alternate relationship
present in the most parsimonious tree. Since no
complete coding sequences for arthropod Wnt-10
genes have been isolated, it is not surprising that
these analyses did not lead to increased support
for an arthropod-deuterostome Wnt-10 clade.
114
E.L. JOCKUSCH AND K.A. OBER
Within the Wnt-10 clade, chordate Wnt-10 genes
form a monophyletic group in all analyses. Two
chordate Wnt-10 genes, mouse Wnt-10a and Wnt10b, were included in the analyses of complete
sequences, and a hagfish (Eptatretus) Wnt-10 sequence was added for the analyses of the PCR
fragment. Bootstrap support for chordate Wnt-10
ranged from 63% to 75% in all analyses of the
PCR fragment except 15 where support dropped
to 39%. Also, in all analyses except 15, the expected relationship [(Eptatretus (mouse Wnt-10a,
Wnt-10b))] was recovered. The mouse Wnt-10a,
Wnt-10b clade received bootstrap support ranging from 71% to 100%.
A monophyletic arthropod Wnt-10 group was
also found in most analyses, though usually with
lower bootstrap support than was found for the
chordate Wnt-10 clade. The two partial sequences
included were inferred to be most closely related
(bootstrap support 49% to 87%) to each other in
all analyses using complete coding sequences except number 5. In 5, Thermobia appeared more
closely related to the mouse Wnt-10 clade, with
Triops the sister to this larger group. Additional
Wnt-10 sequences from a beetle (Desera) and two
blackflies (Metacnephia and Prosimulium) were
included in analyses of the PCR fragment. The
five arthropod Wnt-10 sequences formed a clade
in all analyses of the nucleotide data (17 to 24),
with bootstrap support ranging from 79% to 98%.
However, an arthropod Wnt-10 clade was present
in only three of eight analyses (10, 12, 15) of the
amino acid data. In the other five analyses, arthropod Wnt-10 monophyly is not supported because it is either equally parsimonious (analyses
9, 11, and 14) or more parsimonious (analyses 13,
16) to place Triops as the sister to the deuterostome sequences. Bootstrap support for a monophyletic arthropod Wnt-10 is always low in analyses 9
to 16, exceeding 50% only in analyses using equal
weights (range of support 51% to 62%). Our confidence in some of the nucleotide analyses (18, 19,
24) was increased by recovery of the expected relationships among arthropod Wnt-10 genes [(Triops
(Thermobia (Desera (Metacnephia, Prosimulium))))].
In other analyses, Desera was either misplaced
within the arthropod Wnt-10 clade, or its position
was not fully resolved.
The consistency with which monophyly of all
Wnt-10 sequences and all deuterostome Wnt-10
sequences were recovered provides the strongest
evidence that these genes are orthologous. Indeed,
analyses based on the PCR fragment provide
stronger support for the chordate-arthropod Wnt-
10 clade than they do for the generally accepted
chordate-arthropod Wnt-1 clade. Although in some
analyses the crustacean Wnt-10 appears more
closely related to the deuterostome Wnt-10 clade
than to the insect Wnt-10 clade, it seems more
reasonably to conclude that the Triops sequence
is misplaced in these analyses than to argue that
there was an ancestral gene duplication event producing two copies of Wnt-10, with one copy having been lost independently in the lineages leading
to crustaceans and chordates while the other copy
was lost in the lineage leading to insects.
Relative rates analyses
Using the Li relative rate tests, arthropod Wnt1 and Wnt-10 genes were found to have slightly
but not significantly higher rates of amino acid
substitution than their vertebrate orthologues
(Table 2). The Tajima test results were concordant,
but with one exception: D. melanogaster Wnt-1 had
significantly more amino acid substitutions than
all deuterostomes relative to the outgroup, providing evidence for a rate increase in the Drosophila lineage (Table 2). The closest relatives of D.
melanogaster included in this analysis were the
lepidopterans Manduca, Bombyx, and Junonia,
which showed no evidence of a rate increase. Thus,
the rate increase occurred in the lineage leading
to D. melanogaster sometime following the divergence of flies from butterflies and moths.
We also investigated rate variation within the
arthropods, using Mus Wnt-1 or Wnt-10 sequences
as the outgroup. No significant difference in the
rate of amino acid substitutions was found in any
of the arthropod comparisons using the Li relative rate test. However, results from the Tajima
test indicated that Drosophila and Artemia Wnt1 sequences had significantly higher rates of
amino acid substitutions in almost all comparisons to other arthropods. Substitution rates were
not significantly different between Drosophila and
Tribolium or between Artemia and Manduca.
There was no significant rate difference in any of
the arthropod Wnt-10 comparisons. No significant
differences were found in the rate of evolution of
chordate Wnt-1 or Wnt-10 genes.
DISCUSSION
Wnt genes are a family of developmental regulatory proteins that underwent extensive diversification prior to the divergence of the major
metazoan lineages. We have identified a novel arthropod Wnt gene that appears to be an orthologue
of vertebrate Wnt-10. Together with the four Wnt
WNT GENE FAMILY EVOLUTION
115
TABLE 2. Results of the Li (’97) and Tajima (’93) relative rates tests of molecular evolution of Wnt-1 and Wnt-10
genes in deuterostomes and arthropods1
Arthropods vs. deuterostomes
Li test
Arthropod
taxa
sampled
Arthropod
substitution
rate
Deuterostome
taxa
sampled
Deuterostome
substitution
rate
s.d. × 2
Significance
level
Wnt-1
Wnt-10
9
5
0.171–0.351
0.189–0.334
11
11
0.123–0.283
0.191–0.389
0.249–0.298
0.277–0.353
NS
NS
Arthropod
taxa
sampled
Arthropod
substitutions
Deuterostome
taxa
sampled
Deuterostome
substitutions
Chi-square
Significance
level
8
1
5
5–12
14–16
5–13
11
11
11
3–10
3–6
6–12
0–1.67
3.86–8.89
0–2.88
NS
P < 0.05
NS
Tajima test
Wnt-1
Drosophila Wnt-1
Wnt-10
Within deuterostome
Li test
Jawed
vertebrate
taxa
sampled
Jawed
vertebrate
substitution
rate
Other
deuterostome
taxa
sampled
Other
deuterostome
substitution
rate
s.d. × 2
Significance
level
Wnt-1
Wnt-10
10
10
0.101–0.249
0.146–0.171
1
1
0.152–0.216
0.269–0.340
0.199–0.213
0.312–0.322
NS
NS
Tajima test
Wnt-1
Wnt-10
Jawed
vertebrate
taxa
sampled
Jawed
vertebrate
substitutions
Other
deuterostome
taxa
sampled
Other
deuterostome
substitution
Chi-square
Significance
level
10
10
6–11
9–14
1
1
6–10
8–13
0–0.60
0.037–0.222
NS
NS
1
The chordate and arthropod amino acid substitution rates and standard deviation values (s.d.) or chi-square values are represented by the
lower and upper ends of the range in Wnt-1 and Wnt-10 taxa. NS (not significant) indicates P > 0.05. The Tajima test comparing Wnt-1
Drosophila with 11 deuterostome Wnt-1 genes were all significant. No other comparisons between arthropods and deuterostomes or within
deuterostomes were significantly different.
family members previously identified in D. melanogaster (Rijsewijk et al., ’87; Eisenberg et al.,
’92; Russell et al., ’92; Graba et al., ’95), this brings
to five the minimum number of Wnt genes present
in a common arthropod ancestor. Five Wnt genes
were also found in the complete genome sequence
of the nematode C. elegans; however, they do not
appear to represent the same groups as found in
arthropods. Wnt genes are more numerous in vertebrates: at least eleven were present in the most
recent common ancestor of chordates (orthologues
of Wnt-1 through 8, 10, and 11, and 9/14/15), many
of which have undergone an additional round of duplication within the vertebrates (Sidow, ’92). Presently, the greatest number of Wnt genes known from
a single species is 17 in Mus musculus. The diversity of Wnt genes remains poorly known in most
major groups of metazoans. Surveys of Wnt genes
in other taxa will help fill in our knowledge of the
evolutionary history of this gene family.
Our phylogenetic analyses failed to resolve the
early history of Wnt gene duplication prior to the
divergence of the major metazoan groups, making the reconstruction of early gene duplication
events inconclusive. Nevertheless, most trees indicate that the number of genes present in the
common ancestor of deuterostomes and protostomes was closer to the eleven inferred for a deuterostome ancestor than the five known from
arthropods or nematodes (Fig. 2). Sidow (’92)
reached a similar conclusion, that orthologues of
vertebrate Wnt-1 through 7 were most likely present
in the common ancestor of arthropods and deuterostomes, when considering a smaller set of Wnt genes.
Our analyses support the generally accepted orthology between deuterostome and arthropod Wnt-1
and identify for the first time a Wnt-10 orthologue
outside of deuterostomes. Sidow (’92) also found support for grouping Drosophila Wnt-2 with vertebrate
Wnt-7 and Drosophila Wnt-3 with vertebrate Wnt5, relationships that appeared in some of our analyses of the complete coding sequence but were not
116
E.L. JOCKUSCH AND K.A. OBER
consistently supported. Other candidates for protostome-deuterostome orthology are Drosophila Wnt4 with chordate Wnt-9, 14 and 15, C. elegans mom-2
with this same chordate clade, and C. elegans Wnt1 with deuterostome Wnt-4. The clade containing
chordate Wnt-9, 14, and 15, DWnt-4, and Ce mom2 is the only orthologue group with evidence of representatives that have persisted in arthropods,
nematodes and deuterostomes.
The reduced compliment of Wnt genes in both
arthropods (where sampling is still incomplete) and
C. elegans indicates that gene loss may have played
a greater role in Wnt family evolution in protostome
lineages, while gene duplication in this family has
clearly been important in vertebrates. Our failure
and the failure of others using both PCR and low
stringency library screening (Russell et al., ’92) to
isolate a Wnt-10 orthologue from D. melanogaster
and its absence from the genome sequencing database suggest that this may be an instance of recent
gene loss. A Wnt-10 orthologue was obtained from
the closest relatives of D. melanogaster that we surveyed, two simuliid blackflies.
Sidow (’92) argued that the vertebrate Wnt
genes had undergone a fourfold decrease in the
rate of protein evolution relative to D. melanogaster and non-jawed vertebrate deuterostomes.
The divergence dates were based on maximum
likelihood estimates of non-synonymous substitution rates in combination with divergence dates
estimated from the fossil record. Using relative
rate tests, which calibrate rates of molecular evolution using an outgroup sequence rather than divergence dates, we found no evidence of a decreased
substitution rate for two Wnt genes in jawed vertebrates when compared to arthropods or other deuterostomes. The only significant rate difference was
between D. melanogaster and deuterostome Wnt-1
orthologues and the D. melanogaster rate was also
significantly higher than the substitution rates of
nearly all other arthropod Wnt-1 genes, indicating
that this rate increase occurred in a relatively recent Drosophila ancestor. None of the other eight
arthropod Wnt-1 orthologues showed evidence of
a higher substitution rate relative to deuterostomes. There is also evidence from other genes
that D. melanogaster has an unusually high rate
of molecular evolution (Carmean and Crespi, ’95;
Huelsenbeck, ’97). It is possible that some of
Sidow’s (’92) analyses were affected by this fast
rate in Drosophila, the only arthropod species for
which Wnt sequences were available at the time
of his analyses.
Sidow (’92) suggested that rate decreases in de-
velopmental regulatory genes could be the result
of increased selection caused by an increase in the
number of developmental functions. While this
model remains plausible, we suggest that it does
not apply in this case, particularly in the case of
Wnt-10 in which a gene duplication event occurred
along the same internode as the postulated rate
decrease. Under many circumstances, duplications
are expected to lead to at least transient increases
in the non-synonymous substitution rate because
selection on each copy should be relaxed in the
presence of an initially identical, redundant second copy (Li and Gojobori, ’83; Li, ’85). We found
no evidence of such a predicted rate increase in
Wnt-10, which could be because functional redundancy was never present, or only present briefly.
A transient rate increase would likely not have
left a sufficient signal to be detected hundreds of
millions of years later using the relative rates
tests (which have fairly low power).
Our phylogenetic analyses highlight the fact
that the most recent common ancestor of deuterostomes and protostomes already possessed a large
complement of Wnt genes. Similar results have
been obtained for other developmental regulatory
gene families such as the Hox genes (de Rosa et
al., ’99). Understanding the evolutionary history
of these gene families provides a necessary first
step in evaluating their evolutionary significance.
ACKNOWLEDGMENTS
We thank J.K. Moulton for the blackfly data and
L. Nagy for the Triops data. L. Nagy and two anonymous reviewers provided helpful comments on a
draft of this paper. E.L.J. was supported by an NIH
NRSA postdoctoral fellowship. We are grateful to
D. Maddison and L. Nagy for laboratory space and
research funding (NSF DEB-9420219 to D.M., Sloan
96-4-3ME to L.N.) during this project.
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APPENDIX A. Genbank accession numbers for taxa included in phylogenetic analysis or relative rates analysis
Taxon
Chordates
Mus musculus Wnt-1
M. musculus Wnt-2b/Wnt-13
M. musculus Wnt-3
M. musculus Wnt-3a
M. musculus Wnt-4
M. musculus Wnt-5a
M. musculus Wnt-5b
M. musculus Wnt-6
M. musculus Wnt-7a
M. musculus Wnt-7b
M. musculus Wnt-8
M. musculus Wnt-10a
M. musculus Wnt-10b
M. musculus Wnt-11
M. musculus Wnt-15
Homo sapiens Wnt-1
H. sapiens Wnt-10b
H. sapiens Wnt-14
Brachydanio rerio Wnt-2
B. rerio Wnt-8
B. rerio Wnt-10a
Alopias vulpinus Wnt-1
A. vulpinus Wnt-9
A. vulpinus Wnt-10a
Eptatretus stoutii Wnt-9
E. stoutii Wnt-10
Branchiostoma floridae Wnt-1
Oncorhynchus sp. Wnt-1
Fugu rubripes Wnt-1
Eumeces skltonianus Wnt-1
Sceloperus occidentalis Wnt-1
Pituophis melanoleucus Wnt-1
Ambystoma mexicanum Wnt-1
Xenopus laevis Wnt-1
X. laevis Wnt-10
Plethodon jordani Wnt-10a
P. jordani Wnt-10b
Pleurodeles waltl Wnt-10a
Tetraodon fluviatilis Wnt-10b
Gallus gallus Wnt-14
Echinoderms
Evasterias troschelii Wnt-3
E. troschelii Wnt-8
Strongylocentrotus purpuratus Wnt-2
S. purpuratus Wnt-4
S. purpuratus Wnt-5
S. purpuratus Wnt-6
S. purpuratus Wnt-7
Brachiopods
Terebratulina retusa Wnt-7
Genbank no.
Analyses1
M11943
AF038384/AF070988
P17553
X56842
M89797
M89798
P22726
M89800
M89801
P28047
Q64527
U61969
U61971
X70800
AF031169
X03072
AF028700
AF028702
U51266
U10869
U02544
M91250
M91258
M91251
M91271
M91263
AF061974
M91287
AF056116
M91277
M91928
M91296
X55270
X13138
L07530
M91288
M91289
U65428
U56642
AF031168
PCR, RR
CC, PCR
CC
CC, PCR
CC, PCR
CC, PCR
CC
CC, PCR, RR
CC, PCR
CC, RR
CC
CC, PCR, RR
CC, PCR, RR
CC, PCR
PCR
RR
RR
PCR
PCR
CC, PCR
RR
RR
PCR
RR
PCR
RR
PCR, RR
RR
RR
RR
RR
RR
RR
RR
RR
RR
RR
RR
RR
CC
M91273
M91276
M91303
M95840+M95841
U58982
M91304
M91305
PCR
PCR
PCR
PCR
PCR
PCR
X62687
PCR2
(continued)
APPENDIX A. (continued)
1
Analyses119
WNT GENE FAMILY
GenbankEVOLUTION
no.
Taxon
Arthropods
Drosophila melanogaster Wnt-1
D. melanogaster Wnt-2
D. melanogaster Wnt-3
D. melanogaster Wnt-4
Junonia coenia Wnt-1
Bombyx mori Wnt-1
Manduca sexta Wnt-1
Galerita lecontei Wnt-1
Tribolium castaneum Wnt-1
Thermobia domestica Wnt-1
Artemia franciscana Wnt-1
Triops longicaudatus Wnt-1b
Desera sp. Wnt-10
Metacnephia sommermanae Wnt-10
Prosimulium formosum Wnt-10
Thermobia domestica Wnt-10
Triops longicaudatus Wnt-10
Nematodes
WO1B6-1
W08D2-1
mom-2
CeWnt-1
lin-44
M17230
X64753
X64736
L25316
L42142
D14169
Z30280
AF214031
S41156
AF214035
AF082219
AF214032
AF214033
AF214034
AF214036
AF214037
CC, PCR, RR
CC, PCR
CC, PCR
CC, PCR
PCR, RR
PCR, RR
PCR, RR
PCR, RR
PCR, RR
CC, PCR, RR
PCR, RR
CC, PCR, RR
PCR, RR
PCR, RR
PCR, RR
CC, PCR, RR
CC, PCR, RR
CAA92624
CAA94237
AAC47749
P34888
A57234
PCR3
PCR3
PCR3
PCR3
PCR3
1
CC indicates sequence used in phylogenetic analysis of complete coding region; PCR indicates sequence used in phylogenetic analysis of
PCR fragment region; RR indicates sequence used in relative rates tests.
2
Only included in amino acid analyses in which C. elegans sequences were also included.
3
Nematode sequences were only included in amino acid analyses of the PCR fragment. These analyses were done including and excluding the
C. elegans sequences.
APPENDIX B. Summary of phylogenetic analyses and search results for the Wnt genes
Analysis
no.1
Data set, weighting, distance, or model,2 sites included
Amino acid analyses
1
complete coding, equal weights, all
2
complete coding, equal weights, conf. aligned3
3
complete coding, equal weights, all
4
complete coding, equal weights, conf. aligned
5
complete coding, weighted, all
6
complete coding, weighted, conf. aligned
7
complete coding, weighted, all
8
complete coding, weighted, conf. aligned
9
PCR fragment, equal weights, all
10
PCR fragment, equal weights, conf. aligned
11
PCR fragment, equal weights, all
12
PCR fragment, equal weights, conf. aligned
13
PCR fragment, weights, all
14
PCR fragment, weights, conf. aligned
15
PCR fragment, weighted, all
16
PCR fragment, weighted, conf. aligned
Nucleotide analyses
17
PCR fragment, equal weights, all
18
PCR fragment, weighted, all
19
PCR fragment, GTR + codonpos. rates,4 all
20
PCR fragment, MLE distance, all
21
PCR fragment, LogDet distance, all
22
PCR fragment, HKY85 distance, all
23
PCR fragment, HKY85 distance, all
24
PCR fragment, weighted, all
1
Analysis
method
Gaps treated
as
Trees
found
parsimony
parsimony
parsimony
parsimony
parsimony
parsimony
parsimony
parsimony
parsimony
parsimony
parsimony
parsimony
parsimony
parsimony
parsimony
parsimony
missing data
missing data
extra state
extra state
missing data
missing data
extra state
extra state
missing data
missing data
extra state
extra state
missing data
missing data
extra state
extra state
4
3
2
2
3
1
1
2
20
12
8
12
2
4
13
2
parsimony
parsimony
max. like.
min. evol.
min. evol.
nei. join.
min. evol.
parsimony
missing data
missing data
—
—
—
—
—
extra state
2
1
1
1
1
1
1
3
Used to identify analyses in Table 1.
Details in text.
Conf. aligned indicates that only confidently aligned sites were included. This was 222 (of 317 total) parsimony informative sites in the
complete coding sequence data set and 83 (of 112 total) in the PCR fragment data set.
4
The parameter values of eight models of character evolution were estimated on a tree found by a simple search (lset nst=2 basefreq=empirical
tratio=2; hs swap=nni; save trees; lset nst=2 basefreq=empirical tratio=estimate lscore 1; lset nst=2 basefreq=empirical tratio=previous; hs
start=current swap=spr; savetrees). The most complex model (GTR+codon position rates) fit the data significantly better than any of the
simpler models by a likelihood ratio test (Sullivan and Swofford, ’97). This model’s parameter values were fixed for the full likelihood search.
2
3