1
Appendix S6: AANAT Polymorphism and Phylogeny
2
Phylogenetic reconstructions
3
Because the only AANAT crystal structure available is from O. aries (Hickman
4
et al. 1999a, 1999b) we chose this species as an outgroup for all performed
5
phylogenies. Genbank and Ensembl were searched for all Teleost AANAT2 cDNA
6
sequences available (Table S3). Non AANAT2 isoforms were excluded thanks to
7
nomenclature verification using Blast and preliminary phylogenetic reconstructions.
8
Analyses were restricted to the full AANAT2 ORF ranging from 627 to 693 bp length,
9
depending on the species.
10
Two mitochondrial genes were searched in Genbank for the 15 species
11
investigated: the Cytochrome b (Cytb) and Cytochrome C Oxidase subunit 1 (COX1)
12
(Table S3). The genes were cloned for the catfish O. sifontesi because they were not
13
available in the data bases. For this purpose, total RNA was extracted from catfish
14
skin using Trizol. The cDNA library generation and the Cytb or COX1 amplification
15
was performed using the same protocols as mentioned earlier on for AANAT2
16
cloning. The majority of O. sifontesi cytb and COX1 genes were obtained
17
(respectively 1,005 and 1,385bp).
18
Each set of sequences was aligned using MUSCLE codon algorithm (Edgar
19
2004) displayed in MEGA 5 (Tamura et al. 2011). Nucleotide or amino acids
20
alignments were then imported in Geneious Pro 5.4.6 (Drummond et al. 2010).
21
Amino acid matrices are displayed for the three genes in Figure S2. The nucleotide
22
AANAT2 alignment was analyzed by maximum likelihood (ML). The model of
23
nucleotide substitution was selected in Modeltest v 3.8 (Posada 2006) using the
24
Akaike Information Criterion (AIC) for each position of the codon. The more complex
25
model was found at the first position (GTR + G), and was applied to all tree positions
26
in the ML reconstructions. A ML heuristic search, using a starting tree obtained by
27
Maximum Parsimony, was then conducted using PAUP 4.0b10 package (Swofford
28
2003). Node support was assessed with the bootstrap technique, using 100
29
replicates. Bayesian phylogenetic analyses were also performed on nucleotide
30
sequences using MrBayes 3.0 (Ronquist & Huelsenbeck 2003) implemented in
31
Geneious. The Bayesian analysis was performed with the Metropolis-coupled Markov
32
chain Monte Carlo algorithm using a Codon Model. The tree-space was explored by
33
using four chains run during 2 million generations and saving every 100th tree. The
34
first 25,000 trees were discarded (Burnin’) based on preliminary runs that allowed
35
empirical checking of the point when chains reached apparent stationarity. A
36
consensus was built with the remaining trees. Bayesian probabilities were used to
37
evaluate branch support.
38
Amino acid AANAT2 sequences were also analyzed by ML. The model of
39
nucleotide substitution was selected in Prottest webserver (Abascal et al. 2005). The
40
best model based on the AIC was JTT+G (alpha = 0.73). It was implemented in
41
PhyML displayed in Geneious for further ML reconstructions (Guindon & Gascuel
42
2003). Node support was assessed with the bootstrap technique, using 1000
43
replicates. Bayesian phylogenetic analyses were also performed on this amino acid
44
matrix using MrBayes 3.0 (Ronquist & Huelsenbeck 2003) implemented in Geneious.
45
The Bayesian analysis was performed with the Metropolis-coupled Markov chain
46
Monte Carlo algorithm using a Mixed model. The tree-space was explored by using
47
four chains run during 4 million generations and saving every 100 th tree. The first
48
25,000 trees were discarded (Burnin’) using the same strategy as previously
49
mentioned for nucleotide sequences. A consensus was built with the remaining trees.
50
Bayesian probabilities were used to evaluate branch support.
51
Phylogenies based on the two mitochondrial genes were performed using the
52
same techniques as presented before for the AANAT2 gene. For nucleotide and
53
amino acid Bayesian reconstructions, the same parameters as the one employed for
54
AANAT2 were used. The more complex model was found at the second and first
55
position of the codon (GTR + I + G) for, respectively, Cytb and COX1, and was
56
applied to all tree positions in the ML reconstructions performed in PAUP. Amino acid
57
sequences of the two genes were concatenated and this new matrix was also used
58
as an input for further analyses. The best substitution models provided by Prottest
59
webserver (Abascal et al. 2005) were MtArt+I+G+F for COX1, and MtMam+I+G for
60
cytb and MtArt+I+G+F for the concatenation. They were used as previously
61
mentioned as inputs for the analysis in PhyML. Bayesian reconstructions were run
62
each time for 2 million generations.
63
Substitution rate and natural selection
64
AANAT2 polymorphism was assessed using DNAspV5 (Librado & Rozas
65
2009). We searched for signatures of positive or negative selection at every codon of
66
Teleost AANAT2 and O. aries AANAT using the HyPhy package (available through
67
the Datamonkey webserver (Delport et al. 2010; Kosakovsky Pond & Frost 2005).
68
Putative recombination within sequences was assessed with the GARD algorithm
69
that partitioned sequences in 4 non recombinant fragments. These fragments were
70
analyzed separately with the SLAC codon-based maximum likelihood method. The
71
best time-reversible model was detected and substitution rate parameters were
72
estimated. These values along with branch length (NJ tree) allowed obtaining a
73
global non synonymous / synonymous substitutions (dN/dS) ratio. Ancestral
74
sequences were then inferred site by site by ML providing the level of significance of
75
positive or negative selection at each codon position.
76
77
78
79
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