Biology Reference
In-Depth Information
5.2. Multiple sequence alignments and phylogenetic analysis
Multiple sequence alignments were carried out using MUSCLE (
Edgar,
2004
), Clustal Omega (
Sievers et al., 2011
), MAFFT employing the
L-INS-i option (
Katoh, Kuma, Toh, & Miyata, 2005
), ProbCons
(
Do, Mahabhashyam, Brudno, & Batzoglou, 2005
) and T-Coffee
(
Di Tommaso et al., 2011
). To eliminate ambiguous alignments, we used
the online version of Gblocks 0.91b (
Castresana, 2000
) with the 'less strin-
gent selection' parameter set (
www.phylogeny.fr
)
. The quality of the align-
ments was assessed by MUMSA (
Lassmann & Sonnhammer, 2005
).
Maximum likelihood (ML) analyses were carried out using RA
ML
(
Stamatakiis et al., 2008
). Neighbour-joining (NJ) analyses were performed
using MEGA version 5.05 (
Tamura et al., 2011
). Distances were corrected
for superimposed events using the Poisson method. All positions containing
alignment gaps and missing data were eliminated only in pairwise sequence
comparisons (pairwise deletion option). The reliability of the branching
pattern was tested by bootstrap analysis with 1000 replications. Bayesian
inference trees were obtained employing MrBayes version 3.1.2
(
Ronquist & Huelsenbeck, 2003
), assuming the WAG model of amino acid
substitution and a gamma distribution of evolutionary rates, as determined
by the substitution model testing option in MEGA 5.05. Two parallel runs,
each consisting of four chains, were run simultaneously for at least 8
10
6
generations and trees were sampled every 1000 generations generating a
total of at least 8000 trees. The final average standard deviations of split
frequencies were stationary in all analyses and posterior probabilities were
estimated on the final 60-80% trees. The CIPRES web portal was used
for the Bayesian analyses (
Miller, Pfeiffer, & Schwartz, 2010
) and MEGA
version 5.05 was used to visualize radial trees. With the exception of
Fig. 9.3
, we employed as outgroups, two non-haem, globin-like stress
response regulators RsbR from
Bacillus subtilis
and
B. amyloliquofaciens
(NP_388348.1 and YP_00391940.1). Although they have a globin-like
secondary structure, their G and H helices are bent inwards, eliminating
the haem-binding cavity (
Murray, Delumeau, & Lewis, 2005
). Phylogenetic
trees were also constructed employing SSU rRNA sequences (
Guillou et al.,
2013
). In compiling the lists of sequences, we identified each sequence
by the first three letters of the two portions of the binomial, the number
of residues, one or more three-letter abbreviations of the taxon, followed
by the identifier. Subcellular localization was identified using PSORT II
(
Nakai & Horton, 1999
).
