Stronger evidence for genetic ancestry than environmental conditions in shaping the evolution of a complex signalling trait during biological invasion.

Introductions of invasive species to new environments often result in rapid rates of trait evolution. While in some cases these evolutionary transitions are adaptive and driven by natural selection, they can also result from patterns of genetic and phenotypic variation associated with the invasion history. Here, we examined the brown anole (Anolis sagrei), a widespread invasive lizard for which genetic data have helped trace the sources of non-native populations. We focused on the dewlap, a complex signalling trait known to be subject to multiple selective pressures. We measured dewlap reflectance, pattern and size in 30 non-native populations across the southeastern United States. As well, we quantified environmental variables known to influence dewlap signal effectiveness, such as canopy openness. Further, we used genome-wide data to estimate genetic ancestry, perform association mapping and test for signatures of selection. We found that among-population variation in dewlap characteristics was best explained by genetic ancestry. This result was supported by genome-wide association mapping, which identified several ancestry-specific loci associated with dewlap traits. Despite the strong imprint of this aspect of the invasion history on dewlap variation, we also detected significant relationships between dewlap traits and local environmental conditions. However, we found limited evidence that dewlap-associated genetic variants have been subject to selection. Our study emphasizes the importance of genetic ancestry and admixture in shaping phenotypes during biological invasion, while leaving the role of selection unresolved, likely due to the polygenic genetic architecture of dewlaps and selection acting on many genes of small effect.

Signal size allometry in Anolis lizard dewlaps.

Positive allometry of signalling traits has often been taken as evidence for sexual selection. However, few studies have explored interspecific differences in allometric scaling relationships among closely related species that vary in their degree of ecological similarity. Anolis lizards possess an elaborate retractable throat fan called a dewlap that is used for visual communication and differs greatly in size and colour among species. We observed that Anolis dewlaps demonstrate positive allometry: relative dewlap size increases with body size. We also observed that coexisting species are divergent in signal size allometries, while convergent species-similar in other aspects of ecology, morphology and behaviour-typically share similar dewlap allometric scaling relationships. These patterns suggest that dewlap scaling relationships may follow the same pattern as other traits in the anole radiation, where ecologically different sympatric species have evolved a suite of divergent traits.

Signal detection shapes ornament allometry in functionally convergent Caribbean Anolis and Southeast Asian Draco lizards.

Visual ornaments have long been assumed to evolve hyper-allometry as an outcome of sexual selection. Yet growing evidence suggests many sexually selected morphologies can exhibit other scaling patterns with body size, including hypo-allometry. The large conspicuous throat fan, or dewlap, of arboreal Caribbean Anolis lizards was one ornament previously thought to conform to the classical expectation of hyper-allometry. We re-evaluated this classic example alongside a second arboreal group of lizards that has also independently evolved a functionally equivalent dewlap, the Southeast Asian Draco lizards. Across multiple closely related species in both genera, the Anolis and Draco dewlaps were either isometric or had hypo-allometric scaling patterns. In the case of the Anolis dewlap, variation in dewlap allometry was predicted by the distance of conspecifics and the light environment in which the dewlap was typically viewed. Signal efficacy, therefore, appears to have driven the evolution of hypo-allometry in what was originally thought to be a sexually selected ornament with hyper-allometry. Our findings suggest that other elaborate morphological structures used in social communication might similarly exhibit isometric or hypo-allometric scaling patterns because of environmental constraints on signal detection.

Evolution of hormone-phenotype couplings and hormone-genome interactions.

When selection favors a new relationship between a cue and a hormonally mediated response, adaptation can proceed by altering the hormonal signal that is produced or by altering the phenotypic response to the hormonal signal. The field of evolutionary endocrinology has made considerable progress toward understanding the evolution of hormonal signals, but we know much less about the evolution of hormone-phenotype couplings, particularly at the hormone-genome interface. We briefly review and classify the mechanisms through which these hormone-phenotype couplings likely evolve, using androgens and their receptors and genomic response elements to illustrate our view. We then present two empirical studies of hormone-phenotype couplings, one rooted in evolutionary quantitative genetics and another in comparative transcriptomics, each focused on the regulation of sexually dimorphic phenotypes by testosterone (T) in the brown anole lizard (Anolis sagrei). First, we illustrate the potential for hormone-phenotype couplings to evolve by showing that coloration of the dewlap (an ornament used in behavioral displays) exhibits significant heritability in its responsiveness to T, implying that anoles harbor genetic variance in the architecture of hormonal pleiotropy. Second, we combine T manipulations with analyses of the liver transcriptome to ask whether and how statistical methods for characterizing modules of co-expressed genes and in silico techniques for identifying androgen response elements (AREs) can improve our understanding of hormone-genome interactions. We conclude by emphasizing important avenues for future work at the hormone-genome interface, particularly those conducted in a comparative evolutionary framework.

Visual acuity and signal color pattern in an Anolis lizard.

Anolis lizards communicate with colorful dewlaps that often include detailed patterns. We measured the visual acuity of Anolis sagrei. Lizards viewed a checkerboard pattern of red and yellow-green squares that were too small to resolve, and thus appeared uniform in color. We quickly replaced the center portion of the display with a pattern of larger squares. If the new pattern could be resolved, the lizards perceived a change in color and reflexively shifted their gaze toward the target. The acuity threshold was 1.21 cycles deg-1 We also calculated acuity based on published anatomical data for Anolis carolinensis It was similar to that of A.sagrei for the visual periphery. Foveal acuity was 10 times greater. We approximated the effects of viewing conditions on the visibility of fine details of a conspecific's dewlap. For peripheral vision, no detailed patterns were visible at ≥0.5 m. For foveal vision, color-pattern details were visible at 1.0 m.

Gliding lizards use the position of the sun to enhance social display.

Effective communication requires animal signals to be readily detected by receivers in the environments in which they are typically given. Certain light conditions enhance the visibility of colour signals and these conditions can vary depending on the orientation of the sun and the position of the signaller. We tested whether Draco sumatranus gliding lizards modified their position relative to the sun to enhance the conspicuousness of their throat-fan (dewlap) during social display to conspecifics. The dewlap was translucent, and we found that lizards were significantly more likely to orient themselves perpendicular to the sun when displaying. This increases the dewlap's radiance, and likely, its conspicuousness, by increasing the amount of light transmitted through the ornament. This is a rare example of a behavioural adaptation for enhancing the visibility of an ornament to distant receivers.

Adaptive signal coloration maintained in the face of gene flow in a Hispaniolan Anolis Lizard.

BACKGROUND: Studies of geographic variation can provide insight into the evolutionary processes involved in the early stages of biological diversification. In particular, multiple, replicated cases of geographic trait divergence present a powerful approach to study how patterns of introgression and adaptive divergence can vary with geographic space and time. In this study, we conduct replicated, fine-scaled molecular genetic analyses of striking geographic dewlap color variation of a Hispaniolan Anolis lizard, Anolis distichus, to investigate whether adaptive trait divergence is consistently associated with speciation, whereby genetic divergence is observed with neutral markers, or whether locally adapted traits are maintained in the face of continued gene flow. RESULTS: We find instances where shifts in adaptive dewlap coloration across short geographic distances are associated with reproductive isolation as well as maintained in the face of gene flow, suggesting the importance of both processes in maintaining geographic dewlap variation. CONCLUSION: Our study suggests that adaptive dewlap color differences are maintained under strong divergent natural selection, but this divergence does not necessarily lead to anole speciation.

Multilocus phylogenetic analyses of Hispaniolan and Bahamian trunk anoles (distichus species group).

The distichus species group includes six species and 21 subspecies of trunk ecomorph anoles distributed across Hispaniola and its satellite islands as well as the northern Bahamas. Although this group has long served as a model system for studies of reproductive character displacement, adaptation, behavior and speciation, it has never been the subject of a comprehensive phylogenetic analysis. Our goal here is to generate a multilocus phylogenetic dataset (one mitochondrial and seven nuclear loci) and to use this dataset to infer phylogenetic relationships among the majority of the taxa assigned to the distichus species group. We use these phylogenetic trees to address three topics about the group's evolution. First, we consider longstanding taxonomic controversies about the status of several species and subspecies assigned to the distichus species group. Second, we investigate the biogeographic history of the group and specifically test the hypotheses that historical division of Hispaniola into two paleo-islands contributed to the group's diversification and that Bahamian and Hispaniolan satellite island populations are derived from colonists from the main Hispaniolan landmass. Finally, third, we use comparative phylogenetic analyses to test the hypothesis that divergence between pale yellow and darkly pigmented orange or red dewlap coloration has occurred repeatedly across the distichus species group.

The role of heritable and dietary factors in the sexual signal of a Hispaniolan Anolis lizard, Anolis distichus.

The diversity of sexual signals is astounding, and divergence in these traits is believed to be associated with the early stages of speciation. An increasing number of studies also suggest a role for natural selection in driving signal divergence for effective transmission in heterogeneous environments. Both speciation and adaptive divergence, however, are contingent on the sexual signal being heritable, yet this often remains assumed and untested. It is particularly critical that the heritability of carotenoid-based sexual signals is investigated because such traits may instead be phenotypically plastic indicators of an individual's quality that exhibit no or little heritable variation. We present the first study to investigate the relative contribution of genetic and environmental factors to the striking diversity of dewlap color and pattern in Anolis lizards. Using a breeding experiment with Anolis distichus populations exhibiting different dewlap phenotypes, we raise F1 offspring in a common garden experiment to assess whether dewlap color is inherited. We follow this with carotenoid supplementation to investigate the influence of dietary pigments to dewlap color variation. We find significant differences in several aspects of dewlap color and pattern to persist to the F1 generation (fathers: N = 19; F1 males: N = 50; P < 0.01) with no change in dewlap phenotype with carotenoid supplementation (N = 52; P > 0.05). These results strongly support that genetic differences underlie dewlap color variation, thereby satisfying a key requirement of natural selection. Our findings provide an important stepping-stone to understanding the evolution of an incredibly diverse signal important for sexual selection and species recognition.

Genetic differentiation among populations of a Hispaniolan trunk anole that exhibit geographical variation in dewlap colour.

Long neglected by classic island biogeographical theory, speciation within and among islands is increasingly recognized as a major contributor to insular diversity. Although the factors responsible for island speciation remain poorly understood, this process appears critically dependent on geographical variation and speciation in allopatry or parapatry. Here, we investigate geographical variation and speciation in a complex of Hispaniolan trunk anoles (Anolis distichus), where populations with strikingly distinct dewlap colours and patterns correspond with deeply divergent mtDNA structure. Using a multilocus, population-level analysis, we investigate whether these phenotypically and mitochondrially distinct populations exhibit the type of nuclear differentiation expected among species or incipient species. Along a transect that extends across a recently recessed marine barrier, our results are consistent with the persistence of an abrupt phenotypic and mitochondrial transition following secondary contact, in spite of little or no evidence for a reduction in nuclear gene flow. Along a second transect extending across a steep environmental gradient, our phenotypic and microsatellite data suggest a sharp genetic break with little or no admixture, whereas mtDNA recovers a signature of extensive unidirectional introgression. Together, these results are consistent with previous studies of Lesser Antillean anoles, suggesting that allopatric divergence alone is insufficient for speciation, whereas reduced gene flow and partial reproductive isolation may accumulate in the presence of ecological gradients.

Evolutionary drivers of sexual signal variation in Amazon Slender Anoles.

Phenotypic variation among populations, as seen in the signaling traits of many species, provides an opportunity to test whether similar factors generate repeated phenotypic patterns in different parts of a species' range. We investigated whether genetic divergence, abiotic gradients, and sympatry with closely related species explain variation in the dewlap colors of Amazon Slender Anoles, Anolis fuscoauratus. To this aim, we characterized dewlap diversity in the field with respect to population genetic structure and evolutionary relationships, assessed whether dewlap phenotypes are associated with climate or landscape variables, and tested for nonrandom associations in the distributions of A. fuscoauratus phenotypes and sympatric Anolis species. We found that dewlap colors vary among but not within sites in A. fuscoauratus. Regional genetic clusters included multiple phenotypes, while populations with similar dewlaps were often distantly related. Phenotypes did not segregate in environmental space, providing no support for optimized signal transmission at a local scale. Instead, we found a negative association between certain phenotypes and sympatric Anolis species with similar dewlap color attributes, suggesting that interactions with closely related species promoted dewlap divergence among A. fuscoauratus populations. Amazon Slender Anoles emerge as a promising system to address questions about parallel trait evolution and the contribution of signaling traits to speciation.

Transcriptomic Analysis of Skin Color in Anole Lizards.

Color and color pattern are critical for animal camouflage, reproduction, and defense. Few studies, however, have attempted to identify candidate genes for color and color pattern in squamate reptiles, a colorful group with over 10,000 species. We used comparative transcriptomic analyses between white, orange, and yellow skin in a color-polymorphic species of anole lizard to 1) identify candidate color and color-pattern genes in squamates and 2) assess if squamates share an underlying genetic basis for color and color pattern variation with other vertebrates. Squamates have three types of chromatophores that determine color pattern: guanine-filled iridophores, carotenoid- or pteridine-filled xanthophores/erythrophores, and melanin-filled melanophores. We identified 13 best candidate squamate color and color-pattern genes shared with other vertebrates: six genes linked to pigment synthesis pathways, and seven genes linked to chromatophore development and maintenance. In comparisons of expression profiles between pigment-rich and white skin, pigment-rich skin upregulated the pteridine pathway as well as xanthophore/erythrophore development and maintenance genes; in comparisons between orange and yellow skin, orange skin upregulated the pteridine and carotenoid pathways as well as melanophore maintenance genes. Our results corroborate the predictions that squamates can produce similar colors using distinct color-reflecting molecules, and that both color and color-pattern genes are likely conserved across vertebrates. Furthermore, this study provides a concise list of candidate genes for future functional verification, representing a first step in determining the genetic basis of color and color pattern in anoles.

Does breeding season variation affect evolution of a sexual signaling trait in a tropical lizard clade?

Sexually selected traits can be expected to increase in importance when the period of sexual behavior is constrained, such as in seasonally restricted breeders. Anolis lizard male dewlaps are classic examples of multifaceted signaling traits, with demonstrated intraspecific reproductive function reflected in courtship behavior. Fitch and Hillis found a correlation between dewlap size and seasonality in mainland Anolis using traditional statistical methods and suggested that seasonally restricted breeding seasons enhanced the differentiation of this signaling trait. Here, we present two tests of the Fitch-Hillis Hypothesis using new phylogenetic and morphological data sets for 44 species of Mexican Anolis. A significant relationship between dewlap size and seasonality is evident in phylogenetically uncorrected analyses but erodes once phylogeny is accounted for. This loss of strong statistical support for a relationship between a key aspect of dewlap morphology and seasonality also occurs within a species complex (A. sericeus group) that inhabits seasonal and aseasonal environments. Our results fail to support seasonality as a strong driver of evolution of Anolis dewlap size. We discuss the implications of our results and the difficulty of disentangling the strength of single mechanisms on trait evolution when multiple selection pressures are likely at play.

The brown anole dewlap revisited: do predation pressure, sexual selection, and species recognition shape among-population signal diversity?

Animal signalling structures are amongst the most variable characteristics, as they are subjected to a diversity of selection pressures. A well-known example of a diverse signalling system in the animal kingdom is the dewlap of Anolis lizards. Dewlap characteristics can vary remarkably among and within species, and also between sexes. Although a considerable amount of studies have attempted to disentangle the functional significance of the staggering dewlap diversity in Anolis, the underlying evolutionary processes remain elusive. In this study, we focus on the contribution of biotic selective pressures in shaping geographic variation in dewlap design (size, colour, and pattern) and dewlap display behaviour at the intraspecific level. Notably, we have tried to replicate and extend previously reported results hereof in both sexes of the brown anole lizard (Anolis sagrei). To do this, we assembled a dataset consisting of 17 A. sagrei heterogeneous island populations from the Caribbean and specifically tested whether predation pressure, sexual selection, or species recognition could explain interpopulational variation in an array of dewlap characteristics. Our findings show that in neither males nor females estimates of predation pressure (island size, tail break frequency, model attack rate, presence of predatory Leiocephalus lizards) or sexual selection (sexual size dimorphism) could explain variation in dewlap design. We did find that A. sagrei males from larger islands showed higher dewlap display intensities than males from smaller islands, but the direct connection with predation pressure remains ambiguous and demands further investigation. Last, we could show indirect support for species recognition only in males, as they are more likely to have a 'spotted' dewlap pattern when co-occurring with a higher number of syntopic Anolis species. In conclusion, we found overall limited support for the idea that the extensive interpopulational variability in dewlap design and use in A. sagrei is mediated by variation in their biotic environment. We propose a variety of conceptual and methodological explanations for this unexpected finding.

The effect of recent competition between the native Anolis oculatus and the invasive A. cristatellus on display behavior.

Invasive species are a global threat to biodiversity. Cases where the invasion has been tracked since its beginning are rare, however, such that the first interactions between invasive and native species remain poorly understood. Communication behavior is an integral part of species identity and is subject to selection. Consequently, resource use and direct interference competition between native and invasive species may drive its evolution. Here, we tested the role of interactions between the recently introduced invasive lizard Anolis cristatellus and the native Anolis oculatus on variation in behavior and communication in Calibishie (Dominica). From May to June 2016, we filmed 122 adult males of both species displaying in banana farms under two contexts (allopatry and sympatry). We then recorded (i) the proportion of time spent displaying and (ii) the relative frequency of dewlap vs. push-up displays. To control for habitat variation, we measured and compared the habitat characteristics (canopy openness and habitat openness) of 228 males in allopatry and sympatry. While the habitat characteristics and total display-time did not differ between the contexts for the two species, the proportion of display-time spent dewlapping by A. cristatellus decreased in sympatry. The display of A. oculatus did not differ between the contexts, however. Shifts in microhabitat use, predation pressure, or interspecific interference are potential factors which might explain the behavioral changes in display observed in A. cristatellus. This study highlights the role of behavioral traits as a first response of an invasive species to recent competition with a closely related native species.

A genomic assessment of species boundaries and hybridization in a group of highly polymorphic anoles (distichus species complex).

Delimiting young species is one of the great challenges of systematic biology, particularly when the species in question exhibit little morphological divergence. Anolis distichus, a trunk anole with more than a dozen subspecies that are defined primarily by dewlap color, may actually represent several independent evolutionary lineages. To test this, we utilized amplified fragment length polymorphisms (AFLP) genome scans and genetic clustering analyses in conjunction with a coalescent-based species delimitation method. We examined a geographically widespread set of samples and two heavily sampled hybrid zones. We find that genetic divergence is associated with a major biogeographic barrier, the Hispaniolan paleo-island boundary, but not with dewlap color. Additionally, we find support for hypotheses regarding colonization of two Hispaniolan satellite islands and the Bahamas from mainland Hispaniola. Our results show that A. distichus is composed of seven distinct evolutionary lineages still experiencing a limited degree of gene flow. We suggest that A. distichus merits taxonomic revision, but that dewlap color cannot be relied upon as the primary diagnostic character.

Resurrection of Anolis ustus Cope, 1864 from synonymy with Anolis sericeus Hallowell, 1856 (Squamata, Dactyloidae).

In this study, based on a morphological analysis, the resurrection of the name Anolis ustus Cope 1864, is proposed for populations from the Yucatán Peninsula (Campeche, Yucatán, and Quintana Roo, Mexico, and Belize), formerly referred as Anolis sericeus Hallowell, 1856. Anolis ustus differs from Anolis sericeus by its mean snout-vent length and number of gorgetal scales in males, in tibia length and head width in females, and dorsal and ventral scales for both sexes. In addition, Anolis ustus has a small dewlap of similar size between males and females, whereas in Anolis sericeus males have a dewlap much larger than that of the females. These characteristics allow Anolis ustus to be identified within the Anolis sericeus complex. In this study, a description of the characteristics of the hemipenis is also provided, and its importance in the taxonomy of Anolis is discussed.

Intersexual chemo-sensation in a "visually-oriented" lizard, Anolis sagrei.

While the conspicuous visual displays of anoles have been studied in great depth, the possibility that these lizards may also interact through chemical signalling has received hardly any consideration. In this study, we observed the behaviour of male brown anoles (Anolis sagrei) when introduced into an environment previously inhabited by female conspecifics, and compared it to when they were introduced into an untreated environment. The males in our tests exhibited significantly more elaborate display behaviour (i.e., greater number of dewlap extensions and head-nods) and a significantly greater number of tongue extrusions while in the cage formerly occupied by females than when placed in the untreated, control cage. The absolute numbers of tongue extrusions, however, were relatively low in comparison to average tongue-flick rates of 'true' chemically-oriented lizards. Our results strongly suggest that the males were capable of detecting chemical cues left behind by the females. These observations provide the first evidence of intersexual chemo-sensation in an anole lizard.

The incredible shrinking dewlap: signal size, skin elasticity, and mechanical design in the green anole lizard (Anolis carolinensis).

The expression of male secondary sexual traits can be dynamic, changing size, shape, color, or structure over the course of different seasons. However, the factors underlying such changes are poorly understood. In male Anolis carolinensis lizards, a morphological secondary sexual signal called the dewlap changes size seasonally within individuals. Here, we test the hypothesis that seasonal changes in male dewlap size are driven by increased use and extension of the dewlap in spring and summer, when males are breeding, relative to the winter and fall. We captured male green anole lizards prior to the onset of breeding and constrained the dewlap in half of them such that it could not be extended. We then measured dewlap area in the spring, summer, and winter, and dewlap skin and belly skin elasticity in summer and winter. Dewlaps in unconstrained males increase in area from spring to summer and then shrink in the winter, whereas the dewlaps of constrained males consistently shrink from spring to winter. Dewlap skin is significantly more elastic than belly skin, and skin overall is more elastic in the summer relative to winter. These results show that seasonal changes in dewlap size are a function of skin elasticity and display frequency, and suggest that the mechanical properties of signaling structures can have important implications for signal evolution and design.