Repeated evolution of underwater rebreathing in diving Anolis lizards.

Air-based respiration limits the use of aquatic environments by ancestrally terrestrial animals. To overcome this challenge, diving arthropods have evolved to respire without resurfacing using air held between their cuticle and surrounding water.1-4 Inspired by natural history observations in Haiti (unpublished data) and Costa Rica,5,6 we conducted experiments documenting routine air-based underwater respiration in several distantly related semi-aquatic Anolis lizard species. Semi-aquatic anoles live along neotropical streams and frequently dive for refuge or food,7-12 remaining underwater for up to 18 min. While submerged, these lizards iteratively expire and re-inspire narial air bubbles-underwater "rebreathing." Rebreathed air is used in respiration, as the partial pressure of oxygen in the bubbles decreases with experimental submersion time in living anoles, but not in mechanical controls. Non-aquatic anoles occasionally rebreathe when submerged but exhibit more rudimentary rebreathing behaviors. Anole rebreathing is facilitated by a thin air layer (i.e., a "plastron," sensu Brocher13) supported by the animal's rugose skin upon submergence. We suggest that hydrophobic skin, which we observed in all sampled anoles,14,15 may have been exaptative, facilitating the repeated evolution of specialized rebreathing in species that regularly dive. Phylogenetic analyses strongly suggest that specialized rebreathing is adaptive for semi-aquatic habitat specialists. Air-based rebreathing may enhance dive performance by incorporating dead space air from the buccal cavity or plastron into the lungs, facilitating clearance of carbon dioxide, or allowing uptake of oxygen from surrounding water (i.e., a "physical gill" mechanism4,16).

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.

Improving sustainable use of genetic resources in biodiversity archives.

Tissue sample databases housed in biodiversity archives represent a vast trove of genetic resources, and these tissues are often destructively subsampled and provided to researchers for DNA extractions and subsequent sequencing. While obtaining a sufficient quantity of DNA for downstream applications is vital for these researchers, it is also important to preserve tissue resources for future use given that the original material is destructively and consumptively sampled with each use. It is therefore necessary to develop standardized tissue subsampling and loaning procedures to ensure that tissues are being used efficiently. In this study, we specifically focus on the efficiency of DNA extraction methods by using anuran liver and muscle tissues maintained at a biodiversity archive. We conducted a series of experiments to test whether current practices involving coarse visual assessments of tissue size are effective, how tissue mass correlates with DNA yield and concentration, and whether the amount of DNA recovered is correlated with sample age. We found that tissue samples between 2 and 8 mg resulted in the most efficient extractions, with tissues at the lower end of this range providing more DNA per unit mass and tissues at the higher end of this range providing more total DNA. Additionally, we found no correlation between tissue age and DNA yield. Because we find that even very small tissue subsamples tend to yield far more DNA than is required by researchers for modern sequencing applications (including whole genome shotgun sequencing), we recommend that biodiversity archives consider dramatically improving sustainable use of their archived material by providing researchers with set quantities of extracted DNA rather than with the subsampled tissues themselves.

Phylogeographic and phenotypic outcomes of brown anole colonization across the Caribbean provide insight into the beginning stages of an adaptive radiation.

Some of the most important insights into the ecological and evolutionary processes of diversification and speciation have come from studies of island adaptive radiations, yet relatively little research has examined how these radiations initiate. We suggest that Anolis sagrei is a candidate for understanding the origins of the Caribbean Anolis adaptive radiation and how a colonizing anole species begins to undergo allopatric diversification, phenotypic divergence and, potentially, speciation. We undertook a genomic and morphological analysis of representative populations across the entire native range of A. sagrei, finding that the species originated in the early Pliocene, with the deepest divergence occurring between western and eastern Cuba. Lineages from these two regions subsequently colonized the northern Caribbean. We find that at the broadest scale, populations colonizing areas with fewer closely related competitors tend to evolve larger body size and more lamellae on their toepads. This trend follows expectations for post-colonization divergence from progenitors and convergence in allopatry, whereby populations freed from competition with close relatives evolve towards common morphological and ecological optima. Taken together, our results show a complex history of ancient and recent Cuban diaspora with populations on competitor-poor islands evolving away from their ancestral Cuban populations regardless of their phylogenetic relationships, thus providing insight into the original diversification of colonist anoles at the beginning of the radiation. Our research also supplies an evolutionary framework for the many studies of this increasingly important species in ecological and evolutionary research.

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.

Comparative tests of the role of dewlap size in Anolis lizard speciation.

Phenotypic traits may be linked to speciation in two distinct ways: character values may influence the rate of speciation or diversification in the trait may be associated with speciation events. Traits involved in signal transmission, such as the dewlap of Anolis lizards, are often involved in the speciation process. The dewlap is an important visual signal with roles in species recognition and sexual selection, and dewlaps vary among species in relative size as well as colour and pattern. We compile a dataset of relative dewlap size digitized from photographs of 184 anole species from across the genus' geographical range. We use phylogenetic comparative methods to test two hypotheses: that larger dewlaps are associated with higher speciation rates, and that relative dewlap area diversifies according to a speciational model of evolution. We find no evidence of trait-dependent speciation, indicating that larger signals do not enhance any role the dewlap has in promoting speciation. Instead, we find a signal of mixed speciational and gradual trait evolution, with a particularly strong signal of speciational change in the dewlaps of mainland lineages. This indicates that dewlap size diversifies in association with the speciation process, suggesting that divergent selection may play a role in the macroevolution of this signalling trait.

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.

Discovery of a Giant Chameleon-Like Lizard (Anolis) on Hispaniola and Its Significance to Understanding Replicated Adaptive Radiations.

We report a new chameleon-like Anolis species from Hispaniola that is ecomorphologically similar to congeners found only on Cuba. Lizards from both clades possess short limbs and a short tail and utilize relatively narrow perches, leading us to recognize a novel example of ecomorphological matching among islands in the well-known Greater Antillean anole radiation. This discovery supports the hypothesis that the assembly of island faunas can be substantially deterministic and highlights the continued potential for basic discovery to reveal new insights in well-studied groups. Restricted to a threatened band of midelevation transitional forest near the border of the Dominican Republic and Haiti, this new species appears to be highly endangered.

Clonal reproduction shapes evolution in the lizard malaria parasite Plasmodium floridense.

The preponderant clonal evolution hypothesis (PCE) predicts that frequent clonal reproduction (sex between two clones) in many pathogens capable of sexual recombination results in strong linkage disequilibrium and the presence of discrete genetic subdivisions characterized by occasional gene flow. We expand on the PCE and predict that higher rates of clonal reproduction will result in: (1) morphologically cryptic species that exhibit (2) low within-species variation and (3) recent between-species divergence. We tested these predictions in the Caribbean lizard malaria parasite Plasmodium floridense using 63 single-infection samples in lizards collected from across the parasite's range, and sequenced them at two mitochondrial, one apicoplast, and five nuclear genes. We identified 11 provisionally cryptic species within P. floridense, each of which exhibits low intraspecific variation and recent divergence times between species (some diverged approximately 110,000 years ago). Our results are consistent with the hypothesis that clonal reproduction can profoundly affect diversification of species capable of sexual recombination, and suggest that clonal reproduction may have led to a large number of unrecognized pathogen species. The factors that may influence the rates of clonal reproduction among pathogens are unclear, and we discuss how prevalence and virulence may relate to clonal reproduction.

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.

Anolis sex chromosomes are derived from a single ancestral pair.

To explain the frequency and distribution of heteromorphic sex chromosomes in the lizard genus Anolis, we compared the relative roles of sex chromosome conservation versus turnover of sex-determining mechanisms. We used model-based comparative methods to reconstruct karyotype evolution and the presence of heteromorphic sex chromosomes onto a newly generated Anolis phylogeny. We found that heteromorphic sex chromosomes evolved multiple times in the genus. Fluorescent in situ hybridization (FISH) of repetitive DNA showed variable rates of Y chromosome degeneration among Anolis species and identified previously undetected, homomorphic sex chromosomes in two species. We confirmed homology of sex chromosomes in the genus by performing FISH of an X-linked bacterial artificial chromosome (BAC) and quantitative PCR of X-linked genes in multiple Anolis species sampled across the phylogeny. Taken together, these results are consistent with long-term conservation of sex chromosomes in the group. Our results pave the way to address additional questions related to Anolis sex chromosome evolution and describe a conceptual framework that can be used to evaluate the origins and evolution of heteromorphic sex chromosomes in other clades.

Determinism in the diversification of Hispaniolan trunk-ground anoles (Anolis cybotes species complex).

The evolutionary processes that produce adaptive radiations are enigmatic. They can only be studied after the fact, once a radiation has occurred and been recognized, rather than while the processes are ongoing. One way to connect pattern to process is to study the processes driving divergence today among populations of species that belong to an adaptive radiation, and compare the results to patterns observed at a deeper, macroevolutionary level. We tested whether evolution is a deterministic process with similar outcomes during different stages of the adaptive radiation of Anolis lizards. Using a clade of terrestrial-scansorial lizards in the genus Anolis, we inferred the adaptive basis of spatial variation among contemporary populations and tested whether axes of phenotypic differentiation among them mirror known axes of diversification at deeper levels of the anole radiation. Nonparallel change associated with genetic divergence explains the vast majority of geographic variation. However, we found phenotypic variation to be adaptive as confirmed by convergence in populations occurring in similar habitats in different mountain ranges. Morphological diversification among populations recurs deterministically along two axes of diversification previously identified in the anole radiation, but the characters involved differ from those involved in adaptation at higher levels of anole phylogeny.

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.

Asynchronous evolution of physiology and morphology in Anolis lizards.

Species-rich adaptive radiations typically diversify along several distinct ecological axes, each characterized by morphological, physiological, and behavioral adaptations. We test here whether different types of adaptive traits share similar patterns of evolution within a radiation by investigating patterns of evolution of morphological traits associated with microhabitat specialization and of physiological traits associated with thermal biology in Anolis lizards. Previous studies of anoles suggest that close relatives share the same "structural niche" (i.e., use the same types of perches) and are similar in body size and shape, but live in different "climatic niches" (i.e., use habitats with different insolation and temperature profiles). Because morphology is closely tied to structural niche and field active body temperatures are tied to climatic niches in Anolis, we expected phylogenetic analyses to show that morphology is more evolutionarily conservative than thermal physiology. In support of this hypothesis, we find (1) that thermal biology exhibits more divergence among recently diverged Anolis taxa than does morphology; and (2) diversification of thermal biology among all species often follows diversification in morphology. These conclusions are remarkably consistent with predictions made by anole biologists in the 1960s and 1970s.

Using genomic data to revisit an early example of reproductive character displacement in Haitian Anolis lizards.

The pattern of reproductive character displacement (RCD)-in which traits associated with reproductive isolation are more different where two species occur together than where they occur in isolation-is frequently attributed to reinforcement, a process during which natural selection acting against maladaptive mating events leads to enhanced prezygotic isolation between species or incipient species. One of the first studies of RCD to include molecular genetic data was described 40 years ago in a complex of Haitian trunk anole lizards using a small number of allozyme loci. In this example, Anolis caudalis appears to experience divergence in the color and pattern of an extensible throat fan, or dewlap, in areas of contact with closely related species at the northern and southern limits of its range. However, this case study has been largely overlooked for decades; meanwhile, explanations for geographic variation in dewlap color and pattern have focused primarily on adaptation to local signalling environments. We reinvestigate this example using amplified fragment length polymorphism (AFLP) genome scans, mtDNA sequence data, information on dewlap phenotypes and GIS data on environmental variation to test the hypothesis of RCD generated by reinforcement in Haitian trunk anoles. Together, our phenotypic and genetic results are consistent with RCD at the southern and northern limits of the range of A. caudalis. We evaluate the evidence for reinforcement as the explanation for RCD in Haitian trunk anoles, consider alternative explanations and provide suggestions for future work on the relationship between dewlap variation and speciation in Haitian trunk anoles.

Phylogenetic analyses of novel squamate adenovirus sequences in wild-caught Anolis lizards.

Adenovirus infection has emerged as a serious threat to the health of captive snakes and lizards (i.e., squamates), but we know relatively little about this virus' range of possible hosts, pathogenicity, modes of transmission, and sources from nature. We report the first case of adenovirus infection in the Iguanidae, a diverse family of lizards that is widely-studied and popular in captivity. We report adenovirus infections from two closely-related species of Anolis lizards (anoles) that were recently imported from wild populations in the Dominican Republic to a laboratory colony in the United States. We investigate the evolution of adenoviruses in anoles and other squamates using phylogenetic analyses of adenovirus polymerase gene sequences sampled from Anolis and a range of other vertebrate taxa. These phylogenetic analyses reveal that (1) the sequences detected from each species of Anolis are novel, and (2) adenoviruses are not necessarily host-specific and do not always follow a co-speciation model under which host and virus phylogenies are perfectly concordant. Together with the fact that the Anolis adenovirus sequences reported in our study were detected in animals that became ill and subsequently died shortly after importation while exhibiting clinical signs consistent with acute adenovirus infection, our discoveries suggest the need for renewed attention to biosecurity measures intended to prevent the spread of adenovirus both within and among species of snakes and lizards housed in captivity.

Correlation between Anolis lizard dewlap phenotype and environmental variation indicates adaptive divergence of a signal important to sexual selection and species recognition.

Although the importance of signals involved in species recognition and sexual selection to speciation is widely recognized, the processes that underlie signal divergence are still a matter of debate. Several possible processes have been hypothesized, including genetic drift, arbitrary sexual selection, and adaptation to local signaling environments. We use comparative analyses to investigate whether the remarkable geographic variation of dewlap phenotype in a Hispaniolan trunk Anolis lizard (A. distichus) is a result of adaptive signal divergence to heterogeneous environments. We recover a repeated pattern of divergence in A. distichus dewlap color, pattern, and size with environmental variation across Hispaniola. These results are aligned with ecological models of signal divergence and provide strong evidence for dewlap adaptation to local signaling environments. We also find that A. distichus dewlaps vary with the environment in a different manner to other previously studied anoles, thus expanding upon previous predictions on the direction dewlaps will diverge in perceptual color space in response to the environment.

Quantifying the roles of ecology and geography in spatial genetic divergence.

Investigating the properties of ecological landscapes that influence gene flow among populations can provide key insights into the earliest stages of biological divergence. Both ecological and geographical factors can reduce gene flow, which can lead to population divergence, but we know little of the relative strengths of these phenomena in nature. Here, we use a novel application of structural equation modelling to quantify the contributions of ecological and geographical isolation to spatial genetic divergence in 17 species of Anolis lizards. Our comparative analysis shows that although both processes contributed significantly, geographical isolation explained substantially more genetic divergence than ecological isolation (36.3 vs. 17.9% of variance respectively), suggesting that despite the proposed ubiquity of ecological divergence, non-ecological factors play the dominant role in the evolution of spatial genetic divergence.

Are subspecies of Anolis lizards that differ in dewlap color and pattern also genetically distinct? A mitochondrial analysis.

Subspecies of Anolis lizards are often defined on the basis of geographic variation in the color and pattern of the dewlap, an extensible throat fan considered central to species recognition and sexual selection. Among the most impressive examples of this phenomenon are two species of trunk anoles found across Hispaniola and the Bahamas: Anolis distichus is divided into 16 subspecies with dewlap colors ranging from deep wine red to pale yellow while Anolis brevirostris is divided into three subspecies with dewlaps ranging from pale yellow to orange. Limited sampling of allozyme data indicates some genetic divergence among subspecies and suggests that they may deserve recognition at the species-level. Our goal here is to use more comprehensive geographic sampling of mtDNA haplotypes to test whether the five subspecies of A. distichus and three subspecies of A. brevirostris that occur in the Dominican Republic correspond with genetically distinct populations that may warrant recognition under the general lineage concept. We obtain an aligned dataset of 1462bp comprised of the genes encoding ND2 and adjacent tRNAs from 76 individuals of A. distichus from 28 localities and 12 individuals of A. brevirostris from five localities. We find that haplotypes sampled from each Dominican subspecies of A. distichus form well-supported and deeply divergent clades (>10% uncorrected sequence divergence). Strong concordance between mtDNA haplotype structure and previously diagnosed phenotypic variation in traits central to interspecific communication (i.e., the dewlap) leads us to hypothesize that each of the presently recognized Dominican subspecies of A. distichus and A. brevirostris deserves elevation to full species status under the general lineage concept.

Remarkable new evidence for island radiation in birds.

If island biogeography has a sweet spot, it's where islands generate their own species diversity rather than merely taking on mainland immigrants. In birds and other highly dispersive taxa, however, this 'zone of radiation', may be vanishingly small. Darwin's finches and Hawaiian Honeycreepers are among only a handful of examples of island radiation in birds (Price 2008), suggesting that winged powers of dispersal make sufficient isolation from mainland colonists unlikely, while also hindering speciation within and among isolated islands. Nevertheless, two studies in this issue of Molecular Ecology join a string of other recent analyses suggesting that island radiation in birds remains under-appreciated (see also Moyle et al. 2009; Kisel & Barraclough 2010; Rosindell & Phillimore 2011). Melo et al. (2011) use a phylogenetic analysis of white-eyes on islands in the Gulf of Guinea to identify two previously overlooked island radiations, and reveal replicated adaptive divergence on islands where species occur in pairs. Sly et al. (2011), meanwhile, consider possible explanations for speciation and geographic differentiation within a large island, and find the same type of oceanic barriers that are critical to bird speciation across archipelagos may also contribute to divergence that appears to have occurred within a single island.