Population structure
The final LD-filtered SNP set contained 25,532 markers, of which 24,574
were located on the autosomes, and 958 were located on the X chromosome.
Pairwise IBS values between samples averaged 91.52% (range
88.41%-97.01%). By contrast, IBS values estimated for DNA replicates
approached 100% (average 99.91%; range 99.84%-99.96%), as expected
if the genotyping error rate is low (i.e., below 0.16%, as estimated
using the nine replicates). The STRUCTURE analyses performed using the
24,574 autosomal SNPs confirmed our previous results on genomic
variation in these populations (Bock et al., 2021). The delta K
criterion indicated that two genetic clusters (K = 2) are the
best fit for our data (Figure S5a-c). As expected, all individuals were
assigned ancestry from both genetically distinct clusters in varying
proportions. Based on information from mtDNA sequences (Kolbe et al.,
2004, 2007a) and genome-wide SNP data from the native range (Bock et
al., 2021), these clusters can be interpreted as representative of
ancestry from native lineages in Western and Central-eastern Cuba.
Southern Florida populations have higher frequencies of Western Cuba
ancestry, whereas central to northwestern Florida populations are highly
admixed and contain higher frequencies of Central-eastern Cuba ancestry
(Figure 2). Note, however, that DAPC indicated that a clustering ofK = 3 could be a marginally better fit (Figures S6-S7).
Associations
between the dewlap, genetic ancestry, and the environment
We found significant correlations between dewlap traits, genetic
ancestry, and environmental variables. Dewlap variation as represented
by PC1 and PC2 showed significant positive correlations with Western
Cuba ancestry (P < 0.001, Table 1). As the frequency of
Western Cuba ancestry increased, lizards exhibited brighter and redder
dewlaps (Table 1, Figure 1b, Figure S8a,b). Additionally, PC1 was
negatively correlated with canopy openness (P = 0.001; Table 1). As
canopy openness increased (i.e., less canopy cover and lighter
conditions), lizards exhibited darker dewlaps.
To further investigate the effects of genetic ancestry and environmental
variation on distinct aspects of the dewlap and to follow-up on the
PC-based analyses, we built separate models for each dewlap trait (i.e.,
univariate tests), including UV reflectance, total brightness, hue
(cut-on wavelength), color composition (red, orange, yellow), area, and
perimeter. Spectral colorimetric variables differed across dewlap
positions; therefore, further analyses were also done by position (i.e.,
P1, P2 and P3). UV reflectance was negatively correlated with Western
Cuba ancestry across dewlap positions (P1–edge, P = 0.003; P2–edge, P
= 0.017; P3–center, P = 0.003; Table 1). Center UV reflectance showed
significant positive correlations with canopy openness (P2, P
< 0.001; P3, P = 0.021) and annual mean precipitation (P2, P =
0.032). Total brightness was positively correlated with Western Cuba
ancestry (P1, P2 and P3, P < 0.001), negatively correlated
with canopy openness (P1, P < 0.001; P2, P = 0.028), and
negatively correlated with temperature (P3, P = 0.028). These univariate
results were consistent with PC analyses, such that PC1 can be
interpreted as brightness. Both PC1 (Figure S8a,l) and total brightness
(Figure S8f-h,o,p) resulted in significant positive correlations with
Western Cuba ancestry and negative correlations with canopy openness
across dewlap positions (Table 1). PC2 and yellow composition were
correlated with Western Cuba ancestry (P < 0.01; Figure S8);
lizards with low PC2 values had yellow dewlaps (i.e., greater yellow
composition) and low frequencies of Western Cuba ancestry. As for size,
dewlap area and perimeter resulted in significant positive correlations
with Western Cuba ancestry (Figure S8j-k) and negative correlations with
canopy openness (Figure S8q,r; Table 1). The colorimetric variable hue
(cut-on wavelength) was inconclusive due to a lack of model convergence.
Binomial logistic regression indicated that the probability of a lizard
having a solid dewlap increased with Western Cuba ancestry (P = 0.01;
Figure S9c). Although we observed variation in dewlap pattern across
populations (Figs. S9-S10), this trait was not significantly correlated
with any environmental variable (Table 2). Furthermore, we found no
significant correlation between red color composition, genetic ancestry,
and environmental variables. However, yellow color composition was
negatively correlated with Western Cuba ancestry (P = 0.004, Figure
S8i). These results are consistent with analyses of PC2. Lizards with
low PC2 values had yellow dewlaps (Figure 1b) and low frequencies of
Western Cuba ancestry (Figure 1b; Figure S8b).
Genetic architecture and selection tests for dewlap
traits
Analysis of correlations among traits (Figure S11) revealed strong and
significant correlations between our measurements of dewlap size (i.e.,
area and perimeter; r = 0.91) and between our measurements of
dewlap brightness (i.e., mean and total dewlap brightness; r =
1). Moderate, albeit still significant, positive correlations were
further revealed between dewlap hue, yellow chroma, and brightness, as
well as between dewlap size and dewlap red chroma. Finally, dewlap UV
chroma was negatively correlated with most dewlap traits (Figure S11).
The linear mixed model implemented in GEMMA (i.e., standard GWAS),
identified seven loci distributed on chromosomes 2, 4, 6, 7, and 11 as
significantly associated with the percent of red color (Figure 3, Table
S2) at the Bonferroni-adjusted significance level. These associations
were of moderate effect as estimated using percent variance explained
(PVE; 3.5 to 9.6%; Figure 3). All other analyses using the GEMMA model
did not reveal any associations with dewlap traits at the
Bonferroni-adjusted significance level (Table S2). There were, however,
SNPs that passed the suggestive genome-wide significance threshold.
These suggestive associations were identified for nine of the dewlap
traits, and were distributed on all macrochromosomes except chromosome
3, as well as on microchromosomes 9, 11, and 12 (Figure 3, Table S2).
The ancestry-specific GWAS identified one significant locus at the
Bonferroni-adjusted level (Figure 4, Table S2), located on
macrochromosome 2. This locus was associated with total and mean
brightness with a moderate effect in samples of Western Cuba ancestry
(PVE=3%; Figure 4), and a much smaller effect for samples in the
Central-eastern Cuba ancestry group (PVE = 0.7%; Figure 4). Other
ancestry-specific associations (N = 37 loci) were identified at the
suggestive genome-wide threshold for 10 of the dewlap traits,
distributed on chromosomes 1,2,4,5,6 and 11 (Figure 4; Table S2).
The F ST values estimated for the 45
dewlap-associated SNPs (i.e., loci with both significant and suggestive
associations with dewlap traits; Table S2) ranged from 0 to 0.62
(average 0.04), whereas F ST for the 45 random
SNPs ranged from 0 to 0.43 (average 0.03). The difference inF ST between the dewlap-associated SNPs and the
random SNPs was not, however, significant (P = 0.08; Figure
S12a). As well, most dewlap-associated SNPs (i.e., 89%, or 40/45 SNPs)
were not classified as F ST outliers (Figure
S12b). The remaining five dewlap-associated SNPs wereF ST outliers in one or two population pairs at
most (Figure S12b).
The GEA analysis identified one SNP on macrochromosome 2 at coordinate
22,431,024 that was significantly associated with canopy openness
(q = 0.009; Figure S13a). However, this SNP was not associated
with any dewlap traits (Figure S13b; Table S2), and it was not
classified as an F ST outlier, displayingF ST values that ranged from 0 to 0.33 (average
0.05).