JEZ 0816

JOURNAL OF EXPERIMENTAL ZOOLOGY (MOL DEV EVOL) 288:105–119 (2000)

Phylogenetic Analysis of the Wnt Gene Family and Discovery of an Wnt-10 Orthologue

1 2 E.L. JOCKUSCH * AND K.A. OBER 1Department of Molecular and Cellular Biology, University of Arizona, Tucson, Arizona 85721 2Interdisciplinary Program in 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 his- tory and molecular evolution of this gene family. We have identified a novel Wnt gene in a diver- sity of 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 consis- tently 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 (reviewed in Nusse and Varmus, ’92). To date, Wnt class of genes that has undergone a great deal of genes have only been identified in metazoans. No expansion, presumably through gene duplication Wnt homologues were found in the complete yeast and divergence, within the Metazoa (Holland and genome (Goffeau et al., ’96; Chervitz et al., ’98), García-Fernández ’96; Chervitz et al., ’98). This but Wnt family members are known from deu- has been hypothesized to be essential for the mor- terostomes (van Ooyen and Nusse, ’84; Sidow, ’92; phological diversification of the Metazoa, and par- Ferkowicz et al., ’98; Sasakura et al., ’98), arthro- ticularly of the jawed vertebrates, whose origin pods (e.g., Rijsewijk et al., ’87; Nulsen and Nagy, was accompanied by duplication of many regula- ’99), a nematode (Shackleford et al., ’93; Herman tory genes (Holland and García-Fernández ’96; Val- et al., ’95; Thorpe et al., ’97; Maloof et al., ’99; The entine et al., ’96; Chervitz et al., ’98). Early models C. elegans sequencing consortium, ’98), an annelid of the fate of duplicated genes predicted that loss (Kostriken and Weisblat, ’92), and a brachiopod of function of one copy should be a much more com- (Holland et al., ’91). They are characterized by the mon event than functional diversification (Allendorf, conservation of 22 cysteines and the spacing be- ’79; Li, ’80; Waterson, ’83). The evidence in favor of tween some of these cysteines (Nusse and Varmus, functional diversification in many gene families (e.g. ’92). While at most four Wnt family members have Nadeau and Sankoff, ’97), including the Wnt fam- been identified in any arthropod (in D. melano- ily, presents an empirical challenge to these early gaster: Rijsewijk et al., ’87; Eisenberg et al., ’92; theoretical models. Understanding the history of Russell et al., ’92; Graba et al., ’95), at least 17 duplication, divergence, and loss in individual gene have been identified in the mouse Mus musculus. families will contribute to evaluating hypotheses about the role that these play in morphological and functional evolution. Grant sponsor: National Science Foundation; Grant number: DEB- One family of developmental regulators that ap- 9420219; Grant sponsor: Sloan Foundation; Grant number: 94-4-3ME; Grant sponsor: University of Arizona Research Training Grant in the pears to be a metazoan invention is the Wnt gene Analysis of Biological Diversification; Grant number: DIR-9113362. family. Wnt genes encode secreted signaling pro- *Correspondence to: E.L. Jockusch, Department of Ecology and Evolutionary Biology, 75 N. Eagleville Rd., University of Connecti- teins that have diverse functions during develop- cut, Storrs, CT 06269. E-mail: [email protected] ment in both deuterostomes and protostomes Received 13 August 1999; Accepted 15 December 1999 © 2000 WILEY-LISS, INC. 106 E.L. JOCKUSCH AND K.A. OBER Previous phylogenetic analysis suggested that were amplified using degenerate primers coding at least seven Wnt genes (orthologues of verte- for the conserved amino acid sequences 5′ECKCH- brate Wnt-1 to Wnt-7) were present in the com- GMSG and complementary to the amino acid se- mon ancestor of arthropods and vertebrates, and quence 5′CTFHWCCAV. Products were sized on an that an additional round of duplication occurred agarose gel, and the band of the expected size was at the base of the jawed vertebrates (Sidow, ’92). purified from the gel, then sequenced directly us- Sidow (’92) also found that the rate of amino acid ing dye-labeled terminators. Genomic DNA from substitutions in many Wnt genes decreased four- Thermobia and Triops was amplified using degen- fold at the base of the jawed vertebrates, despite erate primers coding for the sequence 5′QECKCHG the duplication of many of these genes along the and complementary to the sequence 5′HWCC(A/V)V same internode. This is surprising in light of theo- (Sidow, ’92). Products in the expected size range retical predictions that following gene duplication, were ligated into TOPO vector 2.0 (Invitrogen), genes should be under relaxed selection because cloned, and sequenced using dye-labeled termina- of functional redundancy and therefore experience tors. All of the above primers were expected to am- increased rates of molecular evolution (Li and plify Wnt-1 orthologues; however, we also obtained Gojobori, ’83; Li, ’85). representatives of what appeared to be a novel ar- Within arthropods, Wnt-1 (wingless) orthologues thropod Wnt gene. To try to obtain an orthologue have been identified in many groups (e.g. Rijsewijk of this novel Wnt sequence from D. melanogaster, et al., ’87; Nagy and Carroll, ’94; Nulsen and Nagy, representatives of all size classes amplified from ’99), and have been used to infer the phylogenetic its genomic DNA with the primers sets described history of several insect groups (Baker and De- above were cloned and sequenced. No new Wnt Salle, ’97; Brower and DeSalle, ’98). To date, no genes were obtained in this way. Therefore, new other Wnt family members have been reported primers were designed to favor the novel gene over from any arthropod other than D. melanogaster. other Wnt family members by including at the 3′ We have identified a Wnt family member from in- end of each primer nucleotides coding for a Wnt- sects and crustaceans that does not appear to be 10 specific amino acid (primer sequences coding an orthologue of any of the D. melanogaster Wnt for 5′TCWK(S/A)APD and complementary to genes. Phylogenetic analyses of the entire Wnt 5′RFHWCC(A/V)V, with underscoring indicating family identify this gene as an arthropod ortho- amino acids specific to our target). These primers logue of chordate Wnt-10. These analyses concur were used both directly on D. melanogaster ge- with Sidow (’92) that the complement of Wnt genes nomic DNA and in nested reactions following am- present in the common ancestor of protostomes plification with the primers described above. and deuterostomes was greater than the number that has been isolated from any protostome group, Taxon sampling and alignment for indicating that gene loss may have occurred in phylogenetic analysis protostomes. In addition, we used an expanded In order to identify the arthropod Wnt gene frag- sampling of arthropod Wnt-1 and Wnt-10 genes ments obtained, we included them in phylogenetic to compare rates of molecular evolution in proto- analyses of a representative set of Wnt genes. stome and deuterostome lineages and find no evi- Analyses were done both on a data set containing dence of a rate decrease at the base of the jawed primarily sequences of the complete coding region, vertebrates. and on a larger sampling of taxa with sequence restricted to the region between our PCR prim- MATERIALS AND METHODS ers. Use of the complete coding sequence increases the number of characters in the analysis, but data DNA extraction, amplification, and are not available for many taxa. The complete se- sequencing quence data set consisted of the Wnt gene frag- Genomic DNA was extracted following standard ments isolated from Triops (Wnt-1 gene reported protocols (Maddison et al., ’99, for the carabid by Nulsen and Nagy, ’99) and Thermobia (both Desera sp. and lecontei and with sites outside the PCR fragment coded as simuliid black flies [Prosimulium formosum and missing data) plus 21 complete coding sequences: Metacnephia sommermanae]; genomic tip kit the four known D. melanogaster Wnt genes, 15 [Qiagen] for the thysanuran insect Thermobia mouse genes selected to represent the previously domestica and the branchiopod crustacean Triops identified Wnt family members and when possible longicaudatus). and blackfly Wnt genes to span the deepest split in each group, and two WNT GENE FAMILY EVOLUTION 107 additional vertebrate sequences–chick Wnt-14, to points to possible causes of the disagreement. represent a group for which the complete mouse Therefore, we have used maximum likelihood, par- sequence was not yet known, and Brachydanio simony, and distance methods to analyze the rerio (zebrafish) Wnt-8, to sample as deeply di- nucleotide data and parsimony to analyze the pro- verged representatives of the Wnt-8 group as pos- tein data. All trees are unrooted, but the root is sible relative to the mouse sequence. assumed not to separate deuterostome and arthro- Wnt genes for the PCR fragment data set were pod sequences that are nearest neighbors, which selected to include representatives of all Wnt fam- allows us to determine sister group relationships ily members identified to date, and within those between arthropod and deuterostome genes. All groups to span the deepest possible split within analyses were conducted in PAUP* (versions d64, the deuterostomes in order to minimize the prob- d65, b1 and b2; Swofford, ’99). Details of the analy- lems of long branch attraction (Felsenstein, ’78). ses are summarized in Appendix B. In most cases, this meant including sequence from Protein sequences were analyzed using parsi- a jawed vertebrate and an echinoderm or non- mony. Two different character weighting schemes jawed vertebrate. Although numerous additional were used, equal weights, unordered, and a step vertebrate sequences are available for this gene matrix in which the distance between amino ac- region, we limited the number used in order to ids was calculated as the minimum number of reduce the computational time of the phylogenetic nucleotide changes required to convert a codon for analyses. Because we have spanned the deepest one amino acid into a codon for the other. All possible splits within deuterostome groups, and analyses were conducted with gaps coded as an generally recovered the expected relationships of extra state (in the step matrix, a change from an proposed deuterostome orthologues, the effects of amino acid to a gap was treated as requiring three omission of additional taxa from our analyses steps) and with gaps coded as missing. Heuristic should be minimal. For arthropods, our new se- searches consisting of at least 100 random addi- quences and representatives of all other arthro- tion replicates with starting trees obtained by pod Wnts were included (for Drosophila Wnt-2, 3, stepwise addition were used for each analysis. To and 4, the D. melanogaster sequences are the only test the effects of inclusion of regions of uncer- available sequences; for Wnt-1, representatives tain alignment, all analyses were also carried out from throughout the , and from one addi- with and without regions of uncertain alignment. tional crustacean were used; note that the Droso- This produced a total of eight analyses per data phila and vertebrate numbers do not always set (2 gap treatments × 2 character weighting identify expected orthologues). Two versions of the schemes × 2 character inclusion sets) for both the PCR fragment data set, including and excluding complete and PCR fragment data sets (analyses the five C. elegans Wnt sequences, were used in 1 to 16 in Appendix B). In order to estimate rela- phylogenetic analyses of the amino acid data. C. tive support for all clades, bootstrapping was done elegans sequences were excluded from all analy- (100 bootstrap replicates with 10 to 20 random ses of the nucleotide sequences. Genes of C. addition replicates/bootstrap replicate) for all elegans have been shown to evolve extremely rap- analyses (Felsenstein, ’85). idly (Aguinaldo et al., ’97), which may negatively We used likelihood, parsimony, and minimum affect phylogeny reconstruction (Felsenstein, ’78). evolution to analyze the nucleotide data set. Trees The GenBank accession numbers of all sequences of highest likelihood were found by a single heu- used are listed in Appendix A. For both data sets, ristic search with TBR branch-swapping. The initial protein sequence alignment was done in model of character evolution chosen for the search Clustal W (Thompson et al., ’96), and extensively (GTR + codon position rates) fit the data signifi- adjusted by eye in MacClade (Maddison and cantly better than other models explored. TBR Maddison, ’99). A nucleotide alignment corre- branch swapping was performed on a starting tree sponding to the PCR fragment data set was made of high likelihood to search for the tree of highest using the protein alignment as a template. likelihood. Minimum length parsimony trees were sought Phylogenetic analyses by conducting 600 random addition sequence Different phylogenetic methods are sensitive to searches for equal weights parsimony and 700 different kinds of bias in the data. The use of mul- random addition sequence replicates for weighted tiple inference methods gives increased confidence parsimony, with each search beginning from trees when results are in agreement and otherwise acquired by stepwise addition. For equal weights 108 E.L. JOCKUSCH AND K.A. OBER parsimony analyses, characters were treated as VERGE program in the Wisconsin Genetics Group unordered and with equal weights and gaps were GCG package, the number of nonsynonomous coded as missing data. For the weighted parsi- (amino acid) substitutions per site, the number of mony searches, codon positions were weighted by nondegenerate and two-fold degenerate sites, and the reciprocal of the maximum likelihood estimate the standard deviation were calculated between of their relative rates of change (position 1 = 3, each arthropod and vertebrate Wnt-1 and Wnt-10 position 2 = 4, and position 3 = 1) and gaps were and the appropriate outgroup using a one-param- coded both as missing data and as an extra state. eter model of nucleotide substitution. Substitution Minimum evolution distance trees were found rates were considered different if the difference using distances calculated according to the HKY85 between the two ingroup-outgroup rates was (Hasegawa et al., ’85) and LogDet (Lockhart et greater than twice the standard deviation. Ta- al., ’94) model of nucleotide evolution. Starting jima’s (’93) test used a similar, simpler calcula- trees were found by neighbor joining (Saitou and tion to compare the rates of Wnt amino acid Nei, ’87). One minimum evolution distance analy- evolution. A chi-square test determined if the rates sis was also performed using the model of nucle- of amino acid substitution were significantly dif- otide change estimated from the ML analysis with ferent from each other. 800 random addition sequence searches. Bootstrap values for weighted parsimony, HKY85 RESULTS and LogDet minimum evolution distance analyses We obtained orthologues of a novel arthropod were calculated using heuristic searches with ten Wnt gene from two blackflies (Metacnephia and random addition sequence replicates with starting Prosimulium ), a carabid beetle (Desera), a thysa- trees built by stepwise addition for each of 365 to nuran insect (Thermobia), and a branchiopod crus- 800 bootstrap replicates. Decay indices for weighted tacean (Triops) (Fig. 1). We were unable to obtain parsimony clades were calculated by constraining an orthologue of this gene from D. melanogaster, 100 random addition sequence replicate PAUP* and it is not presently represented in the Droso- searches to find the most parsimonious trees with- phila genome sequencing project databases. The out that clade. new sequences were clearly identifiable as Wnt genes based on the conservation of eight cysteines Relative rate analyses in the amplified region. Amino acid identity among To determine if there was any difference in the these sequences ranges from a high of 85% be- rate of molecular evolution between deuterostome tween the two blackfly species to a low of 45% and arthropod Wnt genes, two different relative between Triops and a blackfly. On average, these rate tests were performed on all the available Wnt- sequences are 45% identical to D. melanogaster 1 and Wnt-10 orthologues, the only two orthologue Wnt-1, 40% to D. melanogaster Wnt-2, 37% to D. groups in which arthropods and deuterostomes are melanogaster Wnt-3, and 35% to D. melanogaster both represented by diverse taxa. The GenBank Wnt-4. Initial BLASTx searches suggested that we accession numbers of sequences used are listed had obtained orthologues of chordate Wnt-10. On in Appendix A. Relative rate tests compare the average, our inferred amino acid sequences are sequences of the two lineages of interest (ingroups) 48% identical to Mus Wnt-10a and Wnt-10b. Wnt- to an outgroup sequence to test whether one 1 orthologues were obtained from Thermobia, ingroup sequence has diverged significantly more Triops (Nulsen and Nagy, ’99), and a carabid beetle from the outgroup than has the other ingroup se- (Galerita) (Fig. 1). The inferred amino acid se- quence. Other closely related Wnt gene family quences differed from conspecific Wnt-10 se- members were used as outgroup sequences (Mus quences by 55% (Thermobia) and 48% (Triops) Wnt-6 and Mus Wnt-7b) when comparisons were respectively. The carabid beetle Wnt-1 and Wnt- made between deuterostomes and arthropods, as 10 amino acid sequences differed by 56%. Wnt-1 and Wnt-10 orthologues are not known from The two blackfly sequences appeared to contain other metazoans outside the clade containing an intron. The intron was initially posited based arthropods and vertebrates. For rate comparisons on much greater divergence between the blackfly within the arthropods a Mus orthologue was used sequences in that region, which could not be as the outgroup and for rate comparisons within aligned with any of the other taxa, and a shift in the deuterostomes the Thermobia orthologue was reading frame that would have resulted in loss of used. We compared results obtained using two similarity more 3′ in the inferred amino acid se- similar tests (Tajima, ’93; Li, ’97). Using the DI- quence. The hypothesized boundaries had se- WNT GENE FAMILY EVOLUTION 109

Fig. 1. Alignment of novel arthropod Wnt genes, with ver- Wnt-10 orthologues. Arrow indicates site of an amino acid tebrate Wnt-10 and all D. melanogaster Wnt genes included insertion [not shown] in D. melanogaster Wnt-1. for comparison. * indicates amino acids conserved across all 110 E.L. JOCKUSCH AND K.A. OBER quences highly similar to consensus splice se- slightly more recent Mus/Brachydanio Wnt-8 quences for invertebrates (Shapiro and Senapathy, pair was also supported in 100% of analyses). In ’87). This region (58 bp in Prosimulium and 59 bp analyses of the PCR fragment, support for many in Metacnephia) was removed from all analyses. of the previously suggested deuterostome relation- In order to test the identity of these novel ar- ships was also present (in both amino acid and thropod genes as Wnt-10 orthologues, we undertook nucleotide analyses). Clades that consistently oc- phylogenetic analysis of selected Wnt sequences rep- curred and received bootstrap support >50% were resenting all known clades from arthropods and mouse Wnt-2b/Branchiostoma Wnt-2; Mus Wnt-4/ deuterostomes. In general, relationships within deu- Strongylogentrotus Wnt-4; Mus Wnt-7a/Bran- terostome orthologue groups, which reflect relatively chiostoma Wnt-7; Eptatretus Wnt-8/Branchio- recent divergences, were recovered in all analyses. stoma Wnt-8; and Mus Wnt-10a/Mus Wnt-10b/ Slightly deeper relationships, such as relationships Eptatretus Wnt-10. between proposed arthropod and deuterostome One additional, previously unidentified deu- orthologues, also received some support. However, terostome group was present in every analysis and more ancient divergences (reflecting duplication received high bootstrap support (analysis 15 was events in ancestral metazoans, as well as some of the only one with bootstrap support <80%). This the presumed deuterostome-protostome relation- was a clade containing mouse Wnt-15, human ships) were neither consistently obtained nor well- Wnt-14, and shark (Alopias) and hagfish (Epta- supported in any of the analyses. In analyses of tretus) Wnt-9 genes. Mouse Wnt-15 and human the PCR fragment, no deeper divergences ever Wnt-14 were never sister taxa, and a human Wnt- yielded bootstrap support above 50%. Use of the 15 orthologue has been identified (Bergstein et al., complete coding sequence yielded some deep nodes ’97) making it clear that these belong to different with bootstrap support greater than 50%. This in- orthology groups. It is possible that mammalian crease in support for deeper nodes would be ex- Wnt-14 and Wnt-15 resulted from the same du- pected if lack of resolution is simply a result of plication event at the base of the jawed verte- too few characters in the PCR fragment data set. brates that created the vertebrate a/b pairs. However, the supported nodes were highly sensi- However, the frequent grouping of shark and hag- tive to the method of analysis used, with trees fish Wnt-9 genes with different mammalian se- produced by different methods having conflicting quences raises the possibility that these genes branches with bootstrap support above 50%. Thus, result from a duplication event earlier in deuteros- increasing the number of characters used did not tome history and that the shark and hagfish Wnt- increase confidence in the deeper level phylogeny. 9 genes may not be orthologous. If the Wnt-9 genes Inclusion of the C. elegans genes does not affect are not orthologous, then it is likely that the these general conclusions, although it did affect Alopias Wnt-9 is more closely related to mouse the topology of individual analyses and usually Wnt-15, a relationship recovered in 10/16 analy- resulted in a large increase in the number of most ses with bootstrap support of 54% to 95%. A sis- parsimonious trees. Detailed discussion of the ter group relationship between Eptatretus Wnt-9 orthology between arthropod and chordate genes and human Wnt-14 was recovered in 11/16 analy- is therefore based on the analyses in which C. ses with bootstrap support of 53% to 83%. In the elegans was excluded. It appears that the phylo- analyses of complete coding data, this clade was genetic information retained by Wnt paralogues represented by a single sequence, chick Wnt-14. may be insufficient to recover the early history of At an intermediate level of divergence, repre- this gene family. senting relationships between presumed proto- Although Wnt genes do not appear to be useful stome and deuterostome orthologues, consistency for recovering deeper phylogeny, many more re- of results and level of support varied across analy- cent relationships were consistently and strongly supported (Fig. 2). In all analyses of the complete coding sequence, mouse a/b paralogue relation- Fig. 2. Results of phylogenetic analysis. Tree A is the con- ships, believed to result from gene duplication at sensus of 8 trees resulting from parsimony analysis 11 with the base of the jawed vertebrates (Sidow, ’92), were amino acid data from the PCR fragment. Tree B is a consen- present in the strict consensus of most parsimo- sus of 2 trees from parsimony analysis 3 of the complete cod- ing sequence. The numbers on the branches in A and B nious trees, and had high bootstrap support (100% indicate bootstrap support. Tree C is the tree of highest like- for Mus Wnt-2/2b, Wnt-3/3a, Wnt-5a/5b, and Wnt- lihood for the nucleotide data from the PCR fragment (analy- 7a/7b pairs, 81% to 100% for Mus Wnt-10a/b; the sis 19). WNT GENE FAMILY EVOLUTION 111

Figure 2. 112 E.L. JOCKUSCH AND K.A. OBER ses. For each analysis, the inferred sister group terostome Wnt-10. These are discussed in more of each arthropod clade and the support for that detail below. Relationships of the other arthropod clade are shown in Table 1. The best supported Wnt genes, D. melanogaster Wnt-2, 3, and 4, are relationships were between arthropod and deu- not as clear. DWnt-2 has been previously sug- terostome Wnt-1 and between arthropod and deu- gested 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 Wnt-10 DWnt-2 DWnt-3 DWnt-4

Amino acids Complete coding sequence 1 M1,D1,(Td1,Tl1)-98%2 M10a,b (M7a,b) ((M5a,b)(M2,2b)) D4 D3 2 M1-91% large poly large poly D4 D3 3 M1-93% large poly M7a,b-62% large poly large poly 4 M1-96% large poly (M7a,b)((M5a,b)(M2,2b)) D4 D32 5 M1-97% Tl10(Td10 M7a,b-64% D4 D3 (M10a,b))-55%4 6 M1-96% M3,3a M7a,b-64% M5a,b-62% G14-66% 7 M1-89% G14 M8,Z8-76% M5a,b-57% All Wnt1-54% 8 M1-98% M10a,b M7a,b-94% M5a,b-79% G14-99% PCR fragment 9 M1 deut10,Td10, large poly large poly large poly Tl10,I102 10 M1,M6,B15 deut10-74% M3 (M2,Z2),(D4, deut9/14/15 deut9/14/15) 11 M1,B15 deut10,Tl10,I1 M3 large poly deut9/14/15- 0-60%2 76% 12 M1,M6,B15 deut10-73% M3 (m2,b2)(D4, deut9/14/15- deut9/14/15) 73% 13 (M1,B1)(Wnt-10 I10(Tl10,deut10)- E3 S5 M7,S7 (D2,E3),M3) 63%4 14 M1,B1 deut10,Tl10, E3 S5 M7,S7 I10-74% 15 large poly6 deut10 D4,deut9/14/15-77% S5-71% deut9/14/15- 97% 16 M1,B1 I10(Tl10,deut10)- E3-62% S5 deut9/14/15- 75% 92% Nucleotides PCR fragment 17 M1 M3 (E3,deut10)(D4((m2,b2) S5 (M2,Z2), (deut9/14/15)) (deut9/14/15) 18 M1 deut10-59% D4,E3 S5 E3 Decay index 17 9 3 4 5 19 M1,M6 deut10 D4 S5 D2 20 (M1,M6)B1 deut10 D4(M7,S7) deut9/14/15 M7,S7 21 M1,B1 deut10-61% D4(M7,S7) deut9/14/15 M7,S7 22 (M1,M6)B1 deut10-72% M3 deut9/14/15 M7,S7 23 (M1,M6)B1 deut10-53% D4(M7,S7) deut9/14/15 M7,S7 24 M1-57% deut10 (D4,deut9/14/15),E32 S5-60% deut9/14/15- 98% 1Details 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). 2Polytomy; bootstrap support, if above 50%, is given for the arthropod + deuterostome clade. 367% bootstrap support for sister group relationship with chick Wnt-14. 4Arthropod Wnt-10 not monophyletic; bootstrap support is for all Wnt-10. 5Arthropod Wnt-1 forms a polytomy with the clades indicated. 657% bootstrap support for clade containing all Wnt-1 genes. WNT GENE FAMILY EVOLUTION 113 (Sidow, ’92), DWnt-3 a deuterostome Wnt-5 homo- cover the expected relationships among these ar- logue (Sidow, ’92), and DWnt-4 possibly a verte- thropod genes. In particular, Coleoptera was not brate Wnt-9 homologue (Graba et al., ’95). As monophyletic and the association between crusta- shown in Table 1, these are the only relationships cean and Thermobia Wnt-1 found in analyses in- that received bootstrap support >50% in multiple cluding the complete coding sequences persisted. analyses; however, other relationships were found Furthermore, in analyses of the nucleotide data but in the best trees from different analyses. Because not of the amino acid data, this latter clade usually numerous equally parsimonious trees were found nested within the rest of the insects. in analyses including C. elegans Wnt sequences, The deuterostome Wnt-1 group was represented their strict consensus trees typically had very little by two sequences, whose relationship to each other resolution. Relationships between most C. elegans and to arthropod Wnt-1 and mouse Wnt-6 was sen- Wnt genes and deuterostome orthologues could not sitive to the weighting scheme and method of be confidently identified. Bootstrap support rang- analysis used. Deuterostome Wnt-1 monophyly ing from 35% to 91% identified a possible sister was only recovered in four analyses (13, 14, 16, group relationship between C. elegans mom-2 and 21) and was present in some but not all of the Drosophila Wnt-4. A sister group relationship was most parsimonious trees in four other analyses also consistently inferred between C. elegans Wnt- (10 to 12, 15). In all analyses but two (13, 15), 1 and deuterostome Wnt-4, though with low boot- the sister group of arthropod Wnt-1 was either strap support (36% to 62%). mouse Wnt-1 only, mouse + Branchiostoma Wnt- 1, or a clade containing mouse Wnt-1, mouse Wnt- Wnt-1 relationships 6 and Branchiostoma Wnt-1 (Table 1). However, In the analysis of complete coding sequences, in no case except analysis 24 did bootstrap sup- only a single chordate (mouse Wnt-1) and three port for the node leading to arthropod Wnt-1 and arthropod Wnt-1 sequences (complete Drosophila its sister group exceed 50% (Table 1). Because the coding sequence and partial coding sequences from relationship between mouse Wnt-1 and Wnt-6 dis- Triops and Thermobia) were included. The group- appeared in analyses of complete sequences and ing of these four sequences was strongly supported because improved taxon sampling in the PCR frag- in all analyses (89% to 98% bootstrap support). ment analyses did not lead to any evidence An arthropod Wnt-1 clade was present in the con- strongly contradicting the well-supported arthro- sensus of most parsimonious trees in all analyses pod-deuterostome Wnt-1 relationship found in except one, in which a strict consensus of most analyses of the complete coding sequences, we con- parsimonious trees resulted in a trichotomy of the clude that arthropod and deuterostome Wnt-1 mouse, Drosophila and (Thermobia + Triops) Wnt- genes are orthologous. 1 sequences. Support for the arthropod Wnt-1 clade ranged from 48% (analysis 1) to 100% (in Wnt-10 relationships analysis 7). In only one analysis (6) were the ex- A monophyletic group containing the three chor- pected relationships among the three arthropod date and five arthropod Wnt-10 sequences was re- sequences [(Triops (Thermobia, Drosophila))] re- covered in all analyses of the PCR fragment but covered. The rapid evolution of Drosophila Wnt-1 one (analysis 17). Even though bootstrap support (see below) may provide part of the explanation was low to moderate (42% to 75%), the monophyly for this incongruity. of this group was insensitive to the method of In analyses of the PCR fragment, support for analysis, which increases our confidence in it. Wnt- Wnt-1 monophyly decreased, likely because of the 10 monophyly was recovered in only three analy- decrease in number of informative sites. These ses (1, 5, 8) of the complete coding sequence; analyses included one additional deuterostome Wnt- however, in two of the five remaining analyses, 1 (Branchiostoma) and six additional arthropod Wnt-10 monophyly was one of the equally most Wnt-1 sequences. Arthropod Wnt-1 monophyly was parsimonious solutions, and in one of the remain- recovered in all analyses, but both deuterostome ing cases (6), Wnt-10 monophyly receives higher Wnt-1 monophyly and arthropod + deuterostome bootstrap support than the alternate relationship Wnt-1 monophyly were not. Bootstrap support for present in the most parsimonious tree. Since no the arthropod Wnt-1 clade ranged from 69% (analy- complete coding sequences for arthropod Wnt-10 sis 10) to 100% (analysis 15). Despite the increased genes have been isolated, it is not surprising that taxon sampling relative to the complete coding se- these analyses did not lead to increased support quence analyses, these analyses also failed to re- for an arthropod-deuterostome Wnt-10 clade. 114 E.L. JOCKUSCH AND K.A. OBER Within the Wnt-10 clade, chordate Wnt-10 genes 10 clade than they do for the generally accepted form a monophyletic group in all analyses. Two chordate-arthropod Wnt-1 clade. Although in some chordate Wnt-10 genes, mouse Wnt-10a and Wnt- analyses the crustacean Wnt-10 appears more 10b, were included in the analyses of complete closely related to the deuterostome Wnt-10 clade sequences, and a hagfish (Eptatretus) Wnt-10 se- than to the insect Wnt-10 clade, it seems more quence was added for the analyses of the PCR reasonably to conclude that the Triops sequence fragment. Bootstrap support for chordate Wnt-10 is misplaced in these analyses than to argue that ranged from 63% to 75% in all analyses of the there was an ancestral gene duplication event pro- PCR fragment except 15 where support dropped ducing two copies of Wnt-10, with one copy hav- to 39%. Also, in all analyses except 15, the ex- ing been lost independently in the lineages leading pected relationship [(Eptatretus (mouse Wnt-10a, to crustaceans and chordates while the other copy Wnt-10b))] was recovered. The mouse Wnt-10a, was lost in the lineage leading to insects. Wnt-10b clade received bootstrap support rang- ing from 71% to 100%. Relative rates analyses A monophyletic arthropod Wnt-10 group was Using the Li relative rate tests, arthropod Wnt- also found in most analyses, though usually with 1 and Wnt-10 genes were found to have slightly lower bootstrap support than was found for the but not significantly higher rates of amino acid chordate Wnt-10 clade. The two partial sequences substitution than their vertebrate orthologues included were inferred to be most closely related (Table 2). The Tajima test results were concordant, (bootstrap support 49% to 87%) to each other in but with one exception: D. melanogaster Wnt-1 had all analyses using complete coding sequences ex- significantly more amino acid substitutions than cept number 5. In 5, Thermobia appeared more all deuterostomes relative to the outgroup, pro- closely related to the mouse Wnt-10 clade, with viding evidence for a rate increase in the Droso- Triops the sister to this larger group. Additional phila lineage (Table 2). The closest relatives of D. Wnt-10 sequences from a beetle (Desera) and two melanogaster included in this analysis were the blackflies (Metacnephia and Prosimulium) were lepidopterans Manduca, Bombyx, and Junonia, included in analyses of the PCR fragment. The which showed no evidence of a rate increase. Thus, five arthropod Wnt-10 sequences formed a clade the rate increase occurred in the lineage leading in all analyses of the nucleotide data (17 to 24), to D. melanogaster sometime following the diver- with bootstrap support ranging from 79% to 98%. gence of flies from butterflies and moths. However, an arthropod Wnt-10 clade was present We also investigated rate variation within the in only three of eight analyses (10, 12, 15) of the arthropods, using Mus Wnt-1 or Wnt-10 sequences amino acid data. In the other five analyses, ar- as the outgroup. No significant difference in the thropod Wnt-10 monophyly is not supported be- rate of amino acid substitutions was found in any cause it is either equally parsimonious (analyses of the arthropod comparisons using the Li rela- 9, 11, and 14) or more parsimonious (analyses 13, tive rate test. However, results from the Tajima 16) to place Triops as the sister to the deuteros- test indicated that Drosophila and Artemia Wnt- tome sequences. Bootstrap support for a monophyl- 1 sequences had significantly higher rates of etic arthropod Wnt-10 is always low in analyses 9 amino acid substitutions in almost all compari- to 16, exceeding 50% only in analyses using equal sons to other arthropods. Substitution rates were weights (range of support 51% to 62%). Our confi- not significantly different between Drosophila and dence in some of the nucleotide analyses (18, 19, Tribolium or between Artemia and Manduca. 24) was increased by recovery of the expected rela- There was no significant rate difference in any of tionships among arthropod Wnt-10 genes [(Triops the arthropod Wnt-10 comparisons. No significant (Thermobia (Desera (Metacnephia, Prosimulium))))]. differences were found in the rate of evolution of In other analyses, Desera was either misplaced chordate Wnt-1 or Wnt-10 genes. within the arthropod Wnt-10 clade, or its position was not fully resolved. DISCUSSION The consistency with which monophyly of all Wnt genes are a family of developmental regu- Wnt-10 sequences and all deuterostome Wnt-10 latory proteins that underwent extensive diversi- sequences were recovered provides the strongest fication prior to the divergence of the major evidence that these genes are orthologous. Indeed, metazoan lineages. We have identified a novel ar- analyses based on the PCR fragment provide thropod Wnt gene that appears to be an orthologue stronger support for the chordate-arthropod Wnt- 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 Arthropod Arthropod Deuterostome Deuterostome taxa substitution taxa substitution Significance Li test sampled rate sampled rate s.d. × 2 level

Wnt-1 9 0.171–0.351 11 0.123–0.283 0.249–0.298 NS Wnt-10 5 0.189–0.334 11 0.191–0.389 0.277–0.353 NS Arthropod Deuterostome taxa Arthropod taxa Deuterostome Significance Tajima test sampled substitutions sampled substitutions Chi-square level Wnt-1 8 5–12 11 3–10 0–1.67 NS Drosophila Wnt-1 1 14–16 11 3–6 3.86–8.89 P < 0.05 Wnt-10 5 5–13 11 6–12 0–2.88 NS Within deuterostome

Jawed Jawed Other Other vertebrate vertebrate deuterostome deuterostome taxa substitution taxa substitution Significance Li test sampled rate sampled rate s.d. × 2 level

Wnt-1 10 0.101–0.249 1 0.152–0.216 0.199–0.213 NS Wnt-10 10 0.146–0.171 1 0.269–0.340 0.312–0.322 NS Jawed Other vertebrate Jawed deuterostome Other taxa vertebrate taxa deuterostome Significance Tajima test sampled substitutions sampled substitution Chi-square level

Wnt-1 10 6–11 1 6–10 0–0.60 NS Wnt-10 10 9–14 1 8–13 0.037–0.222 NS 1The 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. me- early history of Wnt gene duplication prior to the lanogaster (Rijsewijk et al., ’87; Eisenberg et al., divergence of the major metazoan groups, mak- ’92; Russell et al., ’92; Graba et al., ’95), this brings ing the reconstruction of early gene duplication to five the minimum number of Wnt genes present events inconclusive. Nevertheless, most trees in- in a common arthropod ancestor. Five Wnt genes dicate that the number of genes present in the were also found in the complete genome sequence common ancestor of deuterostomes and proto- of the nematode C. elegans; however, they do not stomes was closer to the eleven inferred for a deu- appear to represent the same groups as found in terostome ancestor than the five known from arthropods. Wnt genes are more numerous in ver- arthropods or nematodes (Fig. 2). Sidow (’92) tebrates: at least eleven were present in the most reached a similar conclusion, that orthologues of recent common ancestor of chordates (orthologues vertebrate Wnt-1 through 7 were most likely present of Wnt-1 through 8, 10, and 11, and 9/14/15), many in the common ancestor of arthropods and deuteros- of which have undergone an additional round of du- tomes, when considering a smaller set of Wnt genes. plication within the vertebrates (Sidow, ’92). Pres- Our analyses support the generally accepted ortho- ently, the greatest number of Wnt genes known from logy between deuterostome and arthropod Wnt-1 a single species is 17 in Mus musculus. The diver- and identify for the first time a Wnt-10 orthologue sity of Wnt genes remains poorly known in most outside of deuterostomes. Sidow (’92) also found sup- major groups of metazoans. Surveys of Wnt genes port for grouping Drosophila Wnt-2 with vertebrate in other taxa will help fill in our knowledge of the Wnt-7 and Drosophila Wnt-3 with vertebrate Wnt- evolutionary history of this gene family. 5, relationships that appeared in some of our analy- Our phylogenetic analyses failed to resolve the ses of the complete coding sequence but were not 116 E.L. JOCKUSCH AND K.A. OBER consistently supported. Other candidates for proto- velopmental regulatory genes could be the result stome-deuterostome orthology are Drosophila Wnt- of increased selection caused by an increase in the 4 with chordate Wnt-9, 14 and 15, C. elegans mom-2 number of developmental functions. While this with this same chordate clade, and C. elegans Wnt- model remains plausible, we suggest that it does 1 with deuterostome Wnt-4. The clade containing not apply in this case, particularly in the case of chordate Wnt-9, 14, and 15, DWnt-4, and Ce mom- Wnt-10 in which a gene duplication event occurred 2 is the only orthologue group with evidence of rep- along the same internode as the postulated rate resentatives that have persisted in arthropods, decrease. Under many circumstances, duplications nematodes and deuterostomes. are expected to lead to at least transient increases The reduced compliment of Wnt genes in both in the non-synonymous substitution rate because arthropods (where sampling is still incomplete) and selection on each copy should be relaxed in the C. elegans indicates that gene loss may have played presence of an initially identical, redundant sec- a greater role in Wnt family evolution in protostome ond copy (Li and Gojobori, ’83; Li, ’85). We found lineages, while gene duplication in this family has no evidence of such a predicted rate increase in clearly been important in vertebrates. Our failure Wnt-10, which could be because functional redun- and the failure of others using both PCR and low dancy was never present, or only present briefly. stringency library screening (Russell et al., ’92) to A transient rate increase would likely not have isolate a Wnt-10 orthologue from D. melanogaster left a sufficient signal to be detected hundreds of and its absence from the genome sequencing data- millions of years later using the relative rates base suggest that this may be an instance of recent tests (which have fairly low power). gene loss. A Wnt-10 orthologue was obtained from Our phylogenetic analyses highlight the fact the closest relatives of D. melanogaster that we sur- that the most recent common ancestor of deuteros- veyed, two simuliid blackflies. tomes and protostomes already possessed a large Sidow (’92) argued that the vertebrate Wnt complement of Wnt genes. Similar results have genes had undergone a fourfold decrease in the been obtained for other developmental regulatory rate of protein evolution relative to D. melano- gene families such as the Hox genes (de Rosa et gaster and non-jawed vertebrate deuterostomes. al., ’99). Understanding the evolutionary history The divergence dates were based on maximum of these gene families provides a necessary first likelihood estimates of non-synonymous substitu- step in evaluating their evolutionary significance. tion rates in combination with divergence dates estimated from the fossil record. Using relative ACKNOWLEDGMENTS rate tests, which calibrate rates of molecular evo- lution using an outgroup sequence rather than di- We thank J.K. Moulton for the blackfly data and vergence dates, we found no evidence of a decreased L. Nagy for the Triops data. L. Nagy and two anony- substitution rate for two Wnt genes in jawed verte- mous reviewers provided helpful comments on a brates when compared to arthropods or other deu- draft of this paper. E.L.J. was supported by an NIH terostomes. The only significant rate difference was NRSA postdoctoral fellowship. We are grateful to between D. melanogaster and deuterostome Wnt-1 D. Maddison and L. Nagy for laboratory space and orthologues and the D. melanogaster rate was also research funding (NSF DEB-9420219 to D.M., Sloan significantly higher than the substitution rates of 96-4-3ME to L.N.) during this project. nearly all other arthropod Wnt-1 genes, indicating that this rate increase occurred in a relatively re- LITERATURE CITED cent Drosophila ancestor. None of the other eight Aguinaldo AM, Turbeville JM, Linford LS, Rivera MC, Garey arthropod Wnt-1 orthologues showed evidence of JR, Raff RA, Lake JA. 1997. 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APPENDIX A. Genbank accession numbers for taxa included in phylogenetic analysis or relative rates analysis

Taxon Genbank no. Analyses1

Chordates Mus musculus Wnt-1 M11943 PCR, RR M. musculus Wnt-2b/Wnt-13 AF038384/AF070988 CC, PCR M. musculus Wnt-3 P17553 CC M. musculus Wnt-3a X56842 CC, PCR M. musculus Wnt-4 M89797 CC, PCR M. musculus Wnt-5a M89798 CC, PCR M. musculus Wnt-5b P22726 CC M. musculus Wnt-6 M89800 CC, PCR, RR M. musculus Wnt-7a M89801 CC, PCR M. musculus Wnt-7b P28047 CC, RR M. musculus Wnt-8 Q64527 CC M. musculus Wnt-10a U61969 CC, PCR, RR M. musculus Wnt-10b U61971 CC, PCR, RR M. musculus Wnt-11 X70800 CC, PCR M. musculus Wnt-15 AF031169 PCR Homo sapiens Wnt-1 X03072 RR H. sapiens Wnt-10b AF028700 RR H. sapiens Wnt-14 AF028702 PCR Brachydanio rerio Wnt-2 U51266 PCR B. rerio Wnt-8 U10869 CC, PCR B. rerio Wnt-10a U02544 RR Alopias vulpinus Wnt-1 M91250 RR A. vulpinus Wnt-9 M91258 PCR A. vulpinus Wnt-10a M91251 RR Eptatretus stoutii Wnt-9 M91271 PCR E. stoutii Wnt-10 M91263 RR Branchiostoma floridae Wnt-1 AF061974 PCR, RR Oncorhynchus sp. Wnt-1 M91287 RR Fugu rubripes Wnt-1 AF056116 RR Eumeces skltonianus Wnt-1 M91277 RR Sceloperus occidentalis Wnt-1 M91928 RR Pituophis melanoleucus Wnt-1 M91296 RR Ambystoma mexicanum Wnt-1 X55270 RR Xenopus laevis Wnt-1 X13138 RR X. laevis Wnt-10 L07530 RR Plethodon jordani Wnt-10a M91288 RR P. jordani Wnt-10b M91289 RR Pleurodeles waltl Wnt-10a U65428 RR Tetraodon fluviatilis Wnt-10b U56642 RR Gallus gallus Wnt-14 AF031168 CC Echinoderms Evasterias troschelii Wnt-3 M91273 PCR E. troschelii Wnt-8 M91276 PCR Strongylocentrotus purpuratus Wnt-2 M91303 PCR S. purpuratus Wnt-4 M95840+M95841 PCR S. purpuratus Wnt-5 U58982 PCR S. purpuratus Wnt-6 M91304 PCR S. purpuratus Wnt-7 M91305 Brachiopods Terebratulina retusa Wnt-7 X62687 PCR2 (continued) APPENDIX A. (continued)

TaxonWNT GENE FAMILY Genbank EVOLUTION no. Analyses 1191

Arthropods Drosophila melanogaster Wnt-1 M17230 CC, PCR, RR D. melanogaster Wnt-2 X64753 CC, PCR D. melanogaster Wnt-3 X64736 CC, PCR D. melanogaster Wnt-4 L25316 CC, PCR Junonia coenia Wnt-1 L42142 PCR, RR Bombyx mori Wnt-1 D14169 PCR, RR Manduca sexta Wnt-1 Z30280 PCR, RR Galerita lecontei Wnt-1 AF214031 PCR, RR Tribolium castaneum Wnt-1 S41156 PCR, RR Thermobia domestica Wnt-1 AF214035 CC, PCR, RR Artemia franciscana Wnt-1 PCR, RR Triops longicaudatus Wnt-1b AF082219 CC, PCR, RR Desera sp. Wnt-10 AF214032 PCR, RR Metacnephia sommermanae Wnt-10 AF214033 PCR, RR Prosimulium formosum Wnt-10 AF214034 PCR, RR Thermobia domestica Wnt-10 AF214036 CC, PCR, RR Triops longicaudatus Wnt-10 AF214037 CC, PCR, RR Nematodes WO1B6-1 CAA92624 PCR3 W08D2-1 CAA94237 PCR3 mom-2 AAC47749 PCR3 CeWnt-1 P34888 PCR3 lin-44 A57234 PCR3 1CC 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. 2Only included in amino acid analyses in which C. elegans sequences were also included. 3Nematode 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 Analysis Gaps treated Trees no.1 Data set, weighting, distance, or model,2 sites included method as found

Amino acid analyses 1 complete coding, equal weights, all parsimony missing data 4 2 complete coding, equal weights, conf. aligned3 parsimony missing data 3 3 complete coding, equal weights, all parsimony extra state 2 4 complete coding, equal weights, conf. aligned parsimony extra state 2 5 complete coding, weighted, all parsimony missing data 3 6 complete coding, weighted, conf. aligned parsimony missing data 1 7 complete coding, weighted, all parsimony extra state 1 8 complete coding, weighted, conf. aligned parsimony extra state 2 9 PCR fragment, equal weights, all parsimony missing data 20 10 PCR fragment, equal weights, conf. aligned parsimony missing data 12 11 PCR fragment, equal weights, all parsimony extra state 8 12 PCR fragment, equal weights, conf. aligned parsimony extra state 12 13 PCR fragment, weights, all parsimony missing data 2 14 PCR fragment, weights, conf. aligned parsimony missing data 4 15 PCR fragment, weighted, all parsimony extra state 13 16 PCR fragment, weighted, conf. aligned parsimony extra state 2 Nucleotide analyses 17 PCR fragment, equal weights, all parsimony missing data 2 18 PCR fragment, weighted, all parsimony missing data 1 19 PCR fragment, GTR + codonpos. rates,4 all max. like. — 1 20 PCR fragment, MLE distance, all min. evol. — 1 21 PCR fragment, LogDet distance, all min. evol. — 1 22 PCR fragment, HKY85 distance, all nei. join. — 1 23 PCR fragment, HKY85 distance, all min. evol. — 1 24 PCR fragment, weighted, all parsimony extra state 3 1Used to identify analyses in Table 1. 2Details in text. 3Conf. 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. 4The 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.