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Fig. S1. Phylogenetic analysis of Clytia Wnt sequences. To determine Wnt sequence orthology, phylogenetic analysis was performed using complete genomic repertoires of Wnt proteins retrieved by BLAST from NCBI (Nematostella vectensis, Homo sapiens, Strongylocentrtus purpuratus, Tribolium castaneum), JGI (Capitela sp., Lottia gigantae) and Metazome (Hydra magnipapillata) websites. A primary amino acid alignment was performed using Muscle (Edgar, 2004) with manual adjustments made by eye, and usable positions then selected using Gblock (Castresana, 2000), eliminanting the poorly conserved inter-cysteine regions. The final alignment of 239 positions (available on request) was analysed by maximum likelihood (ML) using Phyml (Guindon and Gascuel, 2003) and Bayesian Monte Carlo Markov chain (MCMC) methods using MrBayes (Huelsenbeck and Ronquist, 2001). ML and Bayesian/MCMC analyses were performed with the WAG (Γ4+I) model. ML clade support was estimated by bootstrapping (500 replicates). Four Markov chains, one of which was heated, were run simultaneously for 1 million generations and trees were sampled every 100 generations after stabilisation of posterior likelihoods. For detailed analysis of orthology, the highly divergent CheWntX3 and two Hydra Wnt sequences were excluded to avoid perturbing the alignment. Only three Clytia sequences could be confidently assigned to previously defined orthology groups. Difficulty in assigning orthology to Wnt sequences from early-diverging metazoans as a result of high evolutionary rates has recently been highlighted by analysis of sponge (Amphimedon queenslandica) Wnt sequences, which show no clear relationships with Wnt sequences from eumetazoans (Adamska et al., 2007). In ML analysis, shown here, all previously described orthology groups were confirmed as monophyletic, except for the WntA group. By MCMC analysis, only the Wnt3 group was split (not shown). In both cases, apparent polyphyly was caused by unstable positioning of Nematostella sequences, indicative of the relative weakness of the phylogenic signal of this gene family. Bootstraps values (top; omitted if <50%) and posterior probabilities (bottom; x if node absent in Bayesian analysis) indicate support for nodes of Clytia Wnt sequences. Hma, Hydra magnipapillata; Che, Clytia hemisphaerica; Nve, Nematostella vectensis. Sequences used (in bold, sequences removed from the final ML analysis):
Homo sapiens (NCBI accession number): Wnt1 (NP_005421), Wnt2 (NP_003382), Wnt2B (NP_004176), Wnt3 (NP_110380), Wnt3A (NP_149122), Wnt4 (NP_110388), Wnt5A (NP_003383), Wnt5B (NP_116031), Wnt6 (NP_006513), Wnt7A (NP_004616), Wnt7B (NP_478679), Wnt8A (NP_490645), Wnt8B (NP_003384), Wnt9A (NP_003386), Wnt9B (NP_003387), Wnt10A (NP_079492), Wnt10B (NP_003385), Wnt11 (NP_004617), Wnt16 (NP_476509);
Strongylocentrotus purpuratus (NCBI accession number): Wnt1 (XP_780754.2), Wnt3 (XP_790595.1), Wnt4 (XP_786346.2), Wnt5 (XP_779946.1), Wnt6 (XP_790077.1), Wnt7 (XP_787051), Wnt8 (NP_999832.1), Wnt9 (XP_780823.2), Wnt10 (XP_781564.1), WntA (XP_797603.1);
Lottia gigantae (JGI identification number): Wnt1 (170942), Wnt2 (139906), Wnt5 (130786), Wnt4 (130946), Wnt6 (136505), Wnt7 (179503), Wnt9 (180028), Wnt10 (136550), Wnt11 (109316), Wnt16 (105620), WntA (152125);
Capitella sp. (JGI identification number): Wnt1 (110406), Wnt2 (96953), Wnt4 (181867), Wnt5 (156046), Wnt6 (182518), Wnt7 (112156), Wnt8 (90169), Wnt9 (222661), Wnt10 (110385), Wnt11 (20087), WntA (216606), Wnt16 (149951);
Tribolium castaneum (NCBI accession number): Wnt1 (XP_967980.1), Wnt5 (XP_974684.1), Wnt6 (XP_968055.1), Wnt7 (XP_973159.1), Wnt8 (XP_971439.1), Wnt10 (XP_968210.1), Wnt11 (XP_969261.1), WntA (XP_972893.1);
Nematostella vectensis (NCBI accession number): Wnt1 (XP_001641494.1), Wnt2 (XP_001633773.1), Wnt3 (XP_001635899.1), Wnt4 (XP_001623100.1), Wnt5 (XP_001630693.1) Wnt6 (XP_001629451.1), Wnt7A (XP_001630717.1), Wnt7B (XP_001630717.1), Wnt8A (XP_001630032.1), Wnt8B (XP_001637576.1), Wnt10 (XP_001629450.1),Wnt11 (XP_001629964.1), Wnt16 (XP_001632885.1), WntA (XP_001637670.1);
Hydra magnipapillata (Metazome identification number): 104740, 111695, 111696, 112397, 119947, 130434, 130653, 131265, 132410, 133639, 135223;
Clytia hemisphaerica: Wnt3, Wnt5, Wnt9, WntX1A, WntX1B, WntX2, WntX3.
References
Castresana, J. (2000). Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis. Mol. Biol. Evol. 17, 540-552.
Edgar, R. C. (2004). MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 32, 1792-1797.
Guindon, S. and Gascuel, O. (2003). A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst. Biol. 52, 696-704.
Huelsenbeck, J. P. and Ronquist, F. (2001). MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics 17, 754-755.
Fig. S2. Expression of the five Clytia Wnt genes that could be detected in embryos or larvae. Unfertilised eggs, mid-blastula, early gastrula and 1-day-old planula stages are shown. Embryos are oriented with the oral pole up.
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