Supplementary Methods - Download Word file (43 KB ) - Nature

websterhissBiotechnology

Oct 1, 2013 (3 years and 8 months ago)

79 views


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).