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Appendix S1
Data sets
Data Set A: Anolis marmoratus Basse Terre Guadeloupe. A defined (Malhotra
1992) subset of traits found in Data set B recorded on adult males only.
Data Set B: Anolis oculatus Dominica. A defined (Malhotra 1992) set of traits
recorded from both males and females, subject to several studies of natural selection
and associated fields (Malhotra 1992; Malhotra & Thorpe 1991a,b, 1992, 1997, 1999;
Thorpe & Malhotra 1992; Thorpe et al 2004, 2005).
Data set C: Anolis roquet complex northwest and central Martinique. A
defined (Thorpe et al 2008) set of traits recorded on adult males only, from along
specific transects (Thorpe et al 2008, 2010, 1012; Surget-Groba et al. 2012) and the
subject of studies of phylogeography, adaptation and speciation (Johansson et al
2008; Ogden & Thorpe 2002; Surget-Groba et al 2012; Thorpe 2005; Thorpe et al
2003, 2008, 2010,). In addition to the traits used in Fig. 4 and Table S1a, the colour
pattern traits chevron intensity, A mark on neck black reticulations on trunk, and nonUV white spots on trunk were also recorded (Thorpe et al 2008) (Table S1d).
The data set C Martinique sites (Fig 1) are from the publications (Thorpe et al 20101;
Surget-Groba 20122) in the table below.
Label
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Citation
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Transect
site
Habitat
NW1
III
1
xeric coastal
NW2
1
III
5
montane rainforest
NW3
1
III
6
montane rainforest
NW4
1
IV
1
xeric coastal
NW5
1
IV
2
xeric coastal
NW6
1
IV
5
montane rainforest
NW7
1
IV
6
montane rainforest
NW8
2
combined
1
xeric coastal
C1
1
III
7
montane rainforest
C2
1
IV
7
montane rainforest
C3
1
VIII
6
xeric coastal
C4
1
VIII
7
xeric coastal
Data set D. Anolis roquet complex northwest and central Martinique. A defined
(Malhotra 1992) set of traits recorded on adult males and females from scattered
localities across all Martinique (Gianassi 1997). Sites used are figured below.
Martinique sample sites for data set D (Thorpe & Stenson 2003).
Northwest
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19
33
47
8
12
54
38
7
Central
59
37
Southwest
South
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Data set E. Anolis luciae St Lucia. A defined (Malhotra 1992) set of traits recorded
on adult males and females from scattered localities across all St Lucia (Gianassi
1997).
Data set F. Anolis trinitatis St Vincent. A set of traits (hue, colour pattern, body
dimensions, body length, scalation) recorded from males and females from specific
localities in St Vincent. The characters were recorded from live specimens under
anaesthetic or from high-resolution digital macrophotographs. Hue (proportion of
red, green, blue) was recorded from the lateral trunk using Adobe Photoshop.
Achromaticity (greyness) was computed from this as conformity/departure from equal
RGB scores. For colour pattern, the intensity of chevrons was recorded on an ordinal
scale (0-5) using benchmarks. Both hue and colour pattern follow data set C (Thorpe
et al 2008). Scalation characters were 1 ventral scales, 2 circum-trunk scales, 3 dorsal
scales along body recorded from trunk and 4 neck 5 giving overall number along body
as figured below.
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Body proportions were 1 snout-vent length, 2 jaw (mouth) length, 3 head length, 4
head depth, 5 head width, 6 upper leg length, 7 lower leg length, 8 toe length and 9
dewlap length as figured below.
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Where covariates were significant (SVL) characters were adjusted by ANCOVAR
(Thorpe et al 2008).
Data set G. Anolis richardii Grenada. A set of traits (hue, colour pattern, body
dimensions, body length, scalation) recorded from males and females from specific
localities in Grenada. Traits were recorded and treated as for data set F.
Data set H. Anolis aeneus Grenada. A set of traits (hue, colour pattern, body
dimensions, body length, scalation) recorded from males and females from specific
localities in Grenada. Traits were recorded and treated as for data set F.
Comparisons are also made with the invasive A. cristatellus and dry island species A.
bonairensis and A. blanquillanus. For the Anolis cristatellus (Dominica invasive),
two scalation traits were recorded from specific localities in Dominica (Eales et al.
2008, 2010; Malhotra et al. 2007).
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Molecular and phylogenetic methods.
Novel DNA sequences were generated by extracting total DNA from anole tail tips
using both commercial kits (Qiagen, cat. no. 69506) and previously published
methods (Surget Groba et al. 2001). Mitochondrial fragments were then PCR
amplified using the primers MTA-S (5'-ATCTCAGCATGATGAAACTTCG-3') and
MTF-S (5'-TTTGGTTTACAAGACCAATG-3') (Thorpe et al. 2008) for MT-CYB,
and LCO1490 (5'- GGTCAACAAATCATAAAGATATTGG-3') and HCO2198 (5'TAAACTTCAGGGTGACCAAAAAATCA-3') for MT-CO1 (Folmer et. al. 1994).
Sanger sequencing was performed by Macrogen Inc. (www.macrogen.com) using
both forward and reverse primers. Forward and reverse sequence chromatograms were
proof-read and merged, and the resulting sequences aligned and translated to check
for unexpected stop codons or frameshift mutations, using the software Codoncode
Aligner version 3.5.6 (www.codoncode.com).
Sequence alignments for each species (details in table below) were analysed
individually using a Bayesian phylogenetic approach in BEAST v.1.7.4 (Drummond
et al. 2012). Gene sequences were partitioned into first, second and third codon
positions and separate, unlinked substitution models specified for each. jModelTest
(Guindon & Gascuel 2003: Posada 2008) was used to select optimal substitution
models according to the corrected Akaike Information Criterion (see table below).
Several molecular clock models and coalescent tree priors (suitable for dense
intraspecific sampling) were tested in order to determine the most appropriate model
specifications. Preliminary BEAST runs using uncorrelated lognormal relaxed clocks
were conducted and the posterior distributions of parameters describing variation in
evolutionary rate examined. These posterior distributions were found to abut zero for
all species, thus failing to reject a constant rate of evolution, and so a strict molecular
clock model was employed for all species. A uniform prior of 0.5–1% per million
years was applied to the per-lineage substitution rate for the complete MT-CYB,
sequence (as opposed to codon positions), based on published rates from other
squamate lineages (Surget-Groba & Thorpe 2013). Using a similar procedure, we then
tested a coalescent tree prior with exponential population growth, and found the
posterior distributions of parameters describing variation in population size to exclude
zero for all species, thus rejecting constant population sizes over time. The
exponential population growth model was therefore retained for all species. Two
independent BEAST runs of 2x107 generations were conducted, sampling the MCMC
chain every 2000 generations. The maximum clade credibility (MCC) tree topology
was selected from the posterior sample of trees and annotated with posterior clade
probabilities and node heights equal to the median value from the posterior sample
using TreeAnnotator. For MrBayes analyses, a consensus tree was constructed from
the post burn-in trees using all compatible groups. The final trees were visualised in
FigTree.
BEAST trees for A. luciae and A. richardii (which lacked a well-supported
primary dichotomy) were compared to trees from an alternative Bayesian approach in
MrBayes 3 (Ronquist and Heulsenbeck 2003). A representative of the bimaculatus
Series (A. oculatus) was used as outgroup in both cases. MrBayes analyses involved
three independent runs each using one cold and three heated chains, and ran for 13x107 generations sampling the MCMC chain every 1000 generations. Tracer v1.5
(http://beast.bio.ed.ac.uk/Tracer) was used to verify convergence and adequate
sampling of all parameters, and determine appropriate burn-in (10% in all cases) for
each Bayesian analysis. A consensus tree was constructed from the post burn-in trees
using all compatible groups. The final trees were visualised in FigTree and exported
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for graphics from TreeView. All sequence alignments and tree files have been
uploaded to Dryad and novel DNA sequences have been addition uploaded to
GenBank.
Details of DNA sequence alignments
species
individuals gene
marmoratus 35
MT-CYB
bp
1008
CP1
TrNef+I
CP2
CP3
HKY+I+G TrN+G
downstream
-
oculatus
luciae
37
93
MT-CYB
MT-CYB
1068
1001
TrNef+I
HKY
TrN+I
HKY+G
GTR+G
K80+I
HKY
-
trinitatis
23
MT-CYB
987
TrNef+G
HKY+G
GTR+G
-
richardii
45
MT-CYB
1008
HKY/GTR HKY/HKY TN93/GTR
-
aeneus
38
MT-CYB
966
K80+G
HKY
-
aeneus
38
MT-CO1
657
K80
luciae
93
MT-CYB
1001
HKY/F81
HKY
TrN
TrN+G/HK
Y+G
K80+I/K80+I -
TrN
Legend for above table. “individuals” shows number of individuals sequenced; “gene”
indicates the sequenced gene; “bp” shows the length of the sequence alignment
analysed, in base-pairs; “CP1”, “CP2”, “CP3” show the substituion models assigned
to each codon position for phylogenetic analysis (where both BEAST and MrBayes
analyses were performed, the substitution models used in each are shown before and
after the forward-slash, respectively); “downstream” shows the substitution model
assigned to a short section of sequence downstream of MT-CYB that was analysed in
A. oculatus. Note that the Anolis roquet tree is taken from Surget-Groba & Thorpe
(2013).
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