S9
1. Phylogenetic analyses of the Homeobox domain of Cnidarians
Datasets. Phylogenetic analyses have therefore been conducted on alignments of 60 amino
acid sites of the complete Homeobox domain using six different datasets.
1. HomBil82: 82 Hox and ParaHox sequences from slow evolving bilaterians with three
protostomians Drosophila melanogaster (Dm), Nereis virens (Nev), and Cupiennius salei
(Cs) and three deuterostomians Mus musculus (Mm), Branchiostoma floridae (Bf),
Ptychodera flava (Pf).
2. HomBilNv92: The 82 Hox and ParaHox bilaterian sequences plus 10 Hox-like genes from
the anthozoan Nematostella vectensis (Nv).
3. HomBilHm89: The 82 Hox and ParaHox bilaterian sequences plus 7 Hox-like genes from
the hydrozoan Hydra magnipapillata (Hm).
4. HomBilCx87: The 82 Hox and ParaHox bilaterian sequences plus 5 Hox-like genes from
the scyphozoan Cassiopea xamachana (Cx).
5. HomBilEd85: The 82 Hox and ParaHox bilaterian sequences plus 3 Hox-like genes from
the hydrozoan Eleutheria dichotoma (Ed).
6. HomBilCnid107: The 82 Hox and ParaHox bilaterian sequences plus the 25 Hox-like genes
from the four cnidarian species.
Model selection. The best fitting model of protein sequence evolution was selected using
PROTTEST 1.2.7 (Abascal et al. 2005) among a set of 40 candidate models constituted by all
the combinations of the Dayhoff, Blosum62, JTT, WAG, and VT empirical matrices of amino
acid substitution with a gamma distribution with eight categories (+8) and a proportion of
invariable sites (+I). All statistical criteria unanimously selected the JJT+8 model as the best
fitting model for all six sequence alignments.
Phylogenetic analyses. Distance-based phylogenetic trees were inferred by applying the
BioNJ algorithm (Gascuel 1997) in SPLITSTREE 4.2 (Huson and Bryant 2006) on ML JTT+8
distances computed using the ML 
PROTTEST.
Neighbor-Net networks (Bryant and Moulton 2004) were constructed from the same distance
estimates. Bootstrap proportions were also obtained from 100 replicates using the same
distance correction. Bootstrap networks were then constructed from all splits that occurred in
any of the 100 bootstrap replicates.
Maximum Likelihood (ML) analyses were performed using TREEFINDER (Jobb et al. 2004)
under the JJT+8 model. ML bootstrap proportions were obtained from the 50% majority rule
consensus of the 100 ML tree inferred using the same model from pseudo-replicates generated
with the program SEQBOOT of the PHYLIP package (Felsenstein 2001).
Bayesian phylogenetic analyses were conducted using MrBayes 3.1.2 (Ronquist and
Huelsenbeck 2003). Two independent runs of four incrementally heated Metropolis-coupled
Markov chain Monte Carlo (MCMCMC) chains were simultaneously run for 2,500,000
generations under the JTT+8
model using the program default priors as starting values for
model parameters and branch-lengths. The convergence of MCMCMC was monitored by
examining the values of the marginal likelihood, the rate heterogeneity parameter (), and
clade posterior probabilities through generations using the AWTY web server (Wilgenbusch
et al. 2004). Bayesian clade Posterior Probabilities (PP) were obtained from the 50% majority
rule consensus of 12,500 trees sampled every 100 generations on both independent runs after
removing the 12,500 first trees as a conservative "burn-in".
Statistical tests of alternative topologies. Likelihood-based tests of alternative topologies were
performed in two steps. First, ML branch lengths and site-wise log-likelihood values of
alternative topologies were computed assuming the JTT+8
model using TREE-PUZZLE 5.2
(Schmidt et al. 2002). Second, p-values of the SH (Shimodaira and Hasegawa 1999) and AU
(Shimodaira 2002) likelihood-based tests were calculated with CONSEL 0.1i (Shimodaira and
Hasegawa 2001) using a multiple bootstrap procedure with 1,000,000 replicates.
REFERENCES
Abascal, F., R. Zardoya, and D. Posada. 2005. ProtTest: selection of best-fit models of protein
evolution. Bioinformatics 21:2104-2105.
Bryant, D., and V. Moulton. 2004. Neighbor-Net: An agglomerative method for the
construction of phylogenetic networks. Mol. Biol. Evol. 21:255-265.
Felsenstein, J. 2001. PHYLIP (PHYLogeny Inference Package), version Version 3.06b.
Department of Genome Sciences, University of Washington.
Gascuel, O. 1997. BIONJ: an improved version of the NJ algorithm based on a simple model
of sequence data. Mol. Biol. Evol. 14:685-95.
Huson, D. H., and D. Bryant. 2006. Application of phylogenetic networks in evolutionary
studies. Mol. Biol. Evol. 23:254-257.
Jobb, G., A. von Haeseler, and K. Strimmer. 2004. TREEFINDER: a powerful graphical
analysis environment for molecular phylogenetics. BMC Evol. Biol. 4:18.
Ronquist, F., and J. P. Huelsenbeck. 2003. MrBayes 3: Bayesian phylogenetic inference under
mixed models. Bioinformatics 19:1572-1574.
Shimodaira, H., and M. Hasegawa. 1999. Multiple comparisons of log-likelihoods with
applications to phylogenetic inference. Mol. Biol. Evol. 16, 1114-6.
Shimodaira, H., and M. Hasegawa. 2001. CONSEL: for assessing the confidence of
phylogenetic tree selection. Bioinformatics. 17:1246-7.
Shimodaira, H. 2002. An approximately unbiased test of phylogenetic tree selection. Syst.
Biol. 51, 492-508
Schmidt, H.A., K. Strimmer, M. Vingron, and A. von Haeseler. 2002. TREE-PUZZLE:
maximum likelihood phylogenetic analysis using quartets and parallel computing.
Bioinformatics. 18:502-4.
Wilgenbusch, J. C., D. L. Warren, and D. L. Swofford. 2004. AWTY: A system for graphical
exploration of MCMC convergence in Bayesian phylogenetic inference.
http://ceb.csit.fsu.edu/awty.
2. Other methods
Culture of Nematostella polyps and induction of gametogenesis was carried out as described
(Hand and Uhlinger, 1992; Fritzenwanker and Technau, 2002). cDNA clones of the Hox
genes were isolated by PCR by gene specific primers using first strand cDNA from mixed
embryonic stages or adult polyps. In selected cases genomic DNA was used as template in
PCR reactions to confirm the bioinformatic predictions. All cDNA clones were confirmed by
sequencing.
To produce a BAC library from Nematostella, approximately 5 x 105 primary polyps were
harvested, dissociated into single cell suspension by PronaseE (Sigma) digestion and 6.7 x 107
cells were embedded in an agarose block. The generation of the library was carried out as
described (Osoegawa et al., 1998). Approximately 27,000 clones with an average insert size
of 168 kb representing a 14x genome coverage have been arrayed into 72 384-well microtiter
dishes and then gridded onto nylon filters for screening by probe hybridization. Filter
hybridisation of the BAC library with Digoxigenin-labeled cDNA probes was carried out
using standard protocols.
Hand, C. & Uhlinger, K. (1992). The culture, sexual and asexual reproduction and growth of
the sea anemone Nematostella vectensis. Biol. Bull. 182, 169-176.
Fritzenwanker, J.H & Technau, U. Induction of gametogenesis in the basal cnidarian
Nematostella vectensis (Anthozoa). Dev Genes Evol. 212, 99-103 (2002).
Osoegawa, K., Woon, P.Y,, Zhao, B., Frengen, E., Tateno, M., Catanese, J.J. & de Jong, P.J.
An improved approach for construction of bacterial artificial chromosome libraries.
Genomics 52, 1-8 (1998).