S9
1.
P
hylogenetic
analyses of the Homeobox domain of Cnidarians
Datasets.
Phylogenetic analyses have
therefore
been conducted on alignment
s
of 60 amino
acid sites of the
complete
H
omeobox domain
using
six
different datasets.
1.
HomBil82:
82
H
ox and
Pa
raHox
sequences
from slow evolving bilaterians with
three
protostom
ian
s
Drosophila melanogaster
(Dm)
,
Nereis virens
(Nev),
and
Cupiennius salei
(Cs)
and three deuterostom
ians
Mus musculus
(Mm)
,
Branchiostoma floridae
(Bf),
Ptychodera flava
(Pf)
.
2. HomBilN
v
92
:
T
he 82 Hox and
ParaHox
bilaterian sequences plus 10 Hox

like genes from
the
anthozoan
Nematostella vectensis
(Nv)
.
3. HomBilHm89:
T
he 82 Hox and
ParaHox
bilaterian sequences plus 7 Hox

like genes
from
the hydrozoan
Hydra magnipapillata
(Hm)
.
4. HomBil
Cx
87
:
The 82 Hox and
ParaHox
bilaterian sequences plus 5 Hox

like genes from
the scyphozoan
Cassiope
a xamachana
(Cx)
.
5. HomBilEd85:
The 82 Hox and
ParaHox
bilaterian sequences plus 3 Hox

like genes from
the hydrozoan
Eleutheria dichotoma
(Ed)
.
6. HomBilCn
id107:
The 82 Hox and
ParaHox
bilaterian sequences plus the 2
5
Hox

like genes
from the four cnidarian species.
Model selection
.
The best fitting model of protein sequence evolution was selected using
P
ROT
T
EST
1.2.7 (Abascal et al. 2005) among a set of 40
candidate models constituted by
all
the combination
s
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
alignment
s
.
Phylogenetic analyses.
Distance

based phylogenetic
trees
were
inferred
by applying
the
BioNJ algorithm (Gascuel 1997) in
S
PLITS
T
REE
4.2 (Huson
and
Bryant 2006) on
ML JTT+
8
distances
computed
using
the ML
P
ROT
T
EST
.
Neighbor

Net
networks
(
Bryant
and Moulton 2004
)
were constructed from
the same dista
nce
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
T
REE
F
INDER
(
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
S
EQ
B
OOT
of the
P
HYLIP
package
(
Felsenstein 2001
)
.
Bayesian phylogenetic
analyses
w
ere
conducted
using MrBayes
3
.
1
.2
(Ronquist
and
Huelsenbeck 200
3
).
Two independent runs of
f
our
incrementally heated
Metropolis

coupled
Markov chain Monte Carlo (MCMCMC) chains were simultaneously run for
2
,
5
00,000
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 value
s
of the marginal likelihood
,
the
rat
e heterogeneity parameter (
)
, and
clade posterior probabi
lities
through generations
using the AWTY web server (
Wilgenbusch
et al
.
2004
)
. Bayesian
clade
Posterior Probabilities (PP) w
ere obtained
from the 50% majority
rule consensus of
1
2
,
5
00 trees sampled every
10
0 generations
on both independent runs
after
remo
ving the
12
,
5
00
fi
rst 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
T
REE

P
UZZLE
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
C
ONSEL
0.1i
(Shimodaira and
Has
egawa 2001) using a multiple bootstrap procedure with 1
,
000
,
000 replicates.
R
EFERENCES
Abascal, F., R. Zardoya, and D. Posada. 2005. ProtTest: selection of best

fit models of protein
evolution. Bioinformatics 21:2104

210
5.
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
mixe
d models. Bioinformatics 19:1572

157
4.
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

PUZZL
E:
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 i
nference.
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. A
ll cDNA clones were confirmed by
sequencing.
To produce a BAC library from
Nematostella
,
approximately 5 x 10
5
primary polyps were
harvested, dissociated into single cell suspension by PronaseE (Sigma) digestion and 6.7 x 10
7
cells were embedded in an ag
arose 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 t
hen 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 r
eproduction 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).
Comments 0
Log in to post a comment