Discussion
Rapid adaptation is a common feature of biological invasions (e.g.,
Colautti & Barrett, 2013; Stern & Lee, 2020; Battlay et al., 2023).
Counter to results for many invasions, previous studies of the brown
anole in the southeastern United States have revealed a lack of evidence
for adaptation to the novel environmental conditions experienced there
(Kolbe et al., 2007a, Bock et al., 2021, 2023, Baeckens et al., 2023).
This is surprising given that the colonization of the non-native range
in this species likely coincided with substantial changes in ecological
interactions, as well as a shift from a tropical to a temperate climate.
Consistent with previous studies of phenotypic evolution in the brown
anole invasion, our results show that variation in dewlap
characteristics among non-native populations is primarily explained by
genetic ancestry. Admixture of multiple native-range lineages strongly
influences the current genetic composition of non-native populations
(Kolbe et al., 2004, 2007b, 2008, Bock et al., 2021). GWAS results
corroborated these findings and indicated that most dewlap traits appear
to have a complex genetic architecture. While we find that multiple
aspects of the dewlap are related to local environmental conditions,
genomic analyses did not indicate that dewlap-associated SNPs retain a
signature consistent with local adaptation. Whether natural selection
plays a role in signal divergence during the brown anole invasion thus
requires further investigation. This study provides insight into the
ongoing evolutionary processes occurring in biological invasions,
highlighting the importance of genetic ancestry in brown anole dewlap
variation among populations. Below, we put our findings in the context
of previous studies of the brown anoles to better understand how
populations have evolved during this invasion.
Genetic ancestry and its role in shaping dewlap
phenotypes
Genetic clustering analyses revealed that genetic ancestry of invasive
brown anoles can be summarized at a coarse level as a combination of two
genetically distinct native lineages: Western Cuba and Central-eastern
Cuba. Genetic ancestry had strong effects on several aspects of the
dewlap; lizards with increasing Western Cuba ancestry have larger,
brighter red dewlaps with lower UV reflectance and less yellow (Table 1,
Figure 1b, S8a-k). As well, the probability of lizards having a solid
dewlap increased with the frequency of Western Cuba ancestry (Figure
S9c). This result is consistent with findings from the native range
which, while based on more limited sampling, also find that Western Cuba
populations of A. sagrei have a greater proportion of solid
dewlaps, whereas Central-eastern Cuba populations have a greater
proportion of spotted dewlaps (Driessens et al., 2017). These results
point to an important role of genetic ancestry in shaping multiple
dimensions of dewlap variation across invasive populations in this
system.
Our genome-wide association analyses further explored the effect of
genetic ancestry on dewlap variation. We identified several SNPs
significantly associated with red coloration and brightness of dewlaps,
and dozens of SNPs with suggestive associations with all dewlap traits
(Figure 3, Table S2). Our finding that genetic ancestry has strong and
widespread effects on dewlap differentiation among non-native
populations is consistent with previous studies of invasive species
(e.g., Silene vulgaris , Keller & Taylor, 2010; sticklebacks,
Lucek et al., 2010).
We previously used the same samples to identify a large-effect locus
significantly associated with limb length in A. sagrei (Bock et
al., 2021). Our results here indicate that the genetic architecture of
the dewlap is likely different and more complex than that characterizing
limb length, at least for A. sagrei of Cuban ancestry. This
interpretation is further supported by among-trait correlations. While
all limb length traits show strong and significant pairwise correlations
(Bock et al., 2021), dewlap traits are characterized by more moderate
among-trait correlations that are occasionally non-significant (Figure
S11).
The associations identified here between SNPs and dewlap traits should
be interpreted with caution for two reasons. First, aside from
associations reported for red composition and total and mean dewlap
brightness, the strength of the association signal is reduced for any
one SNP. Second, in-depth analyses of standard and ancestry-specific
associations (Figures 3 and 4) revealed that genotype classes with the
largest difference in trait values are also the ones with the lowest
sample size (e.g., genotype GG for Western Cuba ancestry, at Chr2:
166.587 Mb; Figure 4). Therefore, some of these results could be due to
uneven sample sizes among genotype classes. With these caveats in mind,
we note that the direction of the ancestry-specific effects reported
here is consistent with our observations of the effect of Western Cuba
ancestry on dewlap trait variation. Specifically, linear mixed effects
models of dewlap traits based on genetic ancestry as well as the
ancestry-specific GWAS indicated that Western Cuba ancestry is
associated with increased brightness of dewlaps.
In many organisms, carotenoid pigments have been shown to be a source of
variation in red, orange, and yellow ornamental coloration within and
between species (reviewed in Toews et al., 2017). In most animal
species, carotenoids cannot be synthesized and must be ingested. The
extent to which dewlap color variation is influenced by nutritional
condition has so far been investigated in a few anole species, A.
distichus (Ng et al., 2013b) and A. sagrei (Steffen et al.,
2010). Both studies found no difference in color between carotenoid and
control treatments and dewlap coloration was heritable in A.
distichus lizards. Moreover, biochemical investigations of dewlap
pigment composition in several Anolis species have found that
pterin pigments are important sources of coloration, although
carotenoids were detected as well (Ortiz, 1962; Macedonia et al., 2000,
Steffen & McGraw, 2007, 2009). In contrast to carotenoids, pterins are
produced endogenously (reviewed in Andrade & Carneiro, 2021). Even so,
identifying genes involved in pterin synthesis has been challenging
because of the complexity of underlying biochemical pathways. For
example, studies contrasting skin patches of different color in other
reptiles have identified tens to hundreds of genes that are
differentially expressed, and that are likely involved in the production
of these pigments (e.g., McLean et al., 2017). Our finding that dewlap
phenotypes are correlated with genome-wide estimates of genetic ancestry
across non-native populations is in line with these previous studies and
suggests a complex genetic architecture for dewlap traits.
Potential adaptation to light environments
We tested the hypothesis that dewlap phenotypes are correlated with
environmental variation, which is expected under local adaptation.
Supporting this prediction, we found significant relationships between
multiple environmental variables and dewlap characteristics. Our
strongest results show that dewlaps tend to be darker with relatively
high UV reflectance as canopy openness (i.e., habitat light) increases.
Also, we found that brown anoles occurring in habitats with greater
precipitation had more UV reflectance. These findings are consistent
with those obtained by previous studies in the native range of A.
sagrei . Driessens et al. (2017) found that lizards occupying open
forest habitats had higher UV reflectance than populations inhabiting
environments with little light exposure.
Studies in other Anolis species show different correlations
between UV reflectance and the environment, suggesting the relationship
between aspects of the dewlap and the local environmental conditions
might be species specific. For example, Ng et al. (2013a) found no
difference in UV reflectance among A. distichus populations
inhabiting environments that differed in light characteristics. Also,
Leal and Fleishman (2002, 2004) reported that A. cristatelluspopulations in Puerto Rico occupying closed forest habitats with little
light penetration exhibit highly reflective and transmissive dewlaps
with more UV reflectance. As light exposure increased (i.e., greater
canopy openness), dark dewlaps with low UV reflectance were favored
(Leal & Fleishman, 2004).
Several studies on local adaptation of Anolis dewlaps have
contrasted populations in xeric and mesic habitats. These habitats
differ in characteristics such as habitat light, temperature, or
precipitation. We sampled brown anole populations across a latitudinal
gradient throughout Florida and into southern Georgia. The southeast
region of the United States is known to have relatively uniform climatic
conditions within seasons. As expected, we found less variation in
annual mean temperature and precipitation patterns among our sampled
populations (Figure S3b,c) as compared to previous studies of tropical
mesic and xeric habitats in Puerto Rico and the Dominican Republic (Leal
& Fleishman, 2004, Ng et al., 2013a). Despite this reduced variation in
temperature and precipitation, we found that lizards inhabiting cooler
environments tend to have dewlaps with greater orange composition, which
is consistent with previous studies of A. distichus (Ng et al.,
2013a). While this may indicate convergent evolution of dewlap orange
composition in relation to temperature in A. distichus andA. sagrei , the underlying mechanism is unknown.
In contrast to analyses at the trait level discussed above, analyses at
the SNP level did not provide evidence of adaptation. Specifically, SNPs
associated with dewlap traits had similar F STvalues as random genome-wide SNPs (Figure S12a). As well,
dewlap-associated SNPs were rarely classified asF ST outliers (Figure S12b). Moreover, the small
number of dewlap-associated SNPs that do have extremeF ST values are highly genetically differentiated
in one or two population pairs at most (Figure S12b). These results
therefore indicate that, if some of the dewlap-associated SNPs reported
here are involved in local adaptation, their contribution is likely
specific to a small number of populations, with different SNPs being
recruited by natural selection in different populations. The
genotype-environment association analyses further supported these
results, indicating that dewlap-associated SNPs are not also associated
with the environmental variables predicted to shape dewlap traits under
local adaptation (Figure S13). Given evidence presented here that points
to a complex genetic architecture for dewlaps and considering that
invasive brown anole populations are highly genetically diverse, it is
possible that adaptation occurs via small changes in allele
frequency at a large number of loci. Identifying the genetic signature
of natural selection at loci involved in the control of dewlap is needed
to confirm the occurrence of adaptation, as inferred from
trait-environment correlations. Achieving this task will be challenging,
and will require much denser genome-wide data, in line with observations
for other highly polygenic traits (Lowry et al., 2017).
In conclusion, the brown anole invasion allowed us to study the
evolution of a complex signaling phenotype during a biological invasion,
revealing how genetic ancestry strongly influences among-population
variation in dewlaps in the non-native range. Our study supports the
importance of invasion history and admixture in determining patterns of
phenotypic divergence during biological invasions (Kolbe et al., 2004;
Keller & Taylor, 2010). Although we found some evidence that aspects of
the dewlap are correlated with environmental variation among non-native
populations, which is consistent with previous studies and suggestive of
local adaptation, the loci underlying these dewlap characteristics did
not show a genetic signature consistent with the action of natural
selection. Future studies should consider denser sampling of SNPs along
the genome, which may allow the signature of natural selection to be
recovered for these polygenic traits. Additionally, to better understand
dewlap evolution in non-native brown anole populations, future studies
should consider other potential sources of selection, including species
recognition (Losos, 1985; Vanhooydonck et al., 2009; Baeckens et al.,
2018a), sexual selection (Vanhooydonck et al., 2009), and intrasexual
selection (i.e., male-male competition) (Vanhooydonck et al., 2005;
Lailvaux & Irschick 2007; Baeckens et al., 2018b).