The evolutionary dynamics of sex chromosomes differ from those of autosomes due to their unique patterns of inheritance and regions of hemizygosity in non-recombining areas. However, the study of sex chromosomes and sex-linked gene evolution has been limited by the rarity of truly novel sex chromosome complements in model systems. Recent advances in next-generation sequencing have enabled the identification of neo-sex chromosomes, created by the fission or fusion of autosomes with sex chromosomes, providing a new avenue to investigate the dynamics of sex chromosome evolution. Squamate reptiles, particularly Anolis lizards, are an excellent system for studying the consequences of sex-linkage due to their frequent sex chromosome-autosome fusions. The Hispaniolan Bark anole, Anolis distichus, has experienced two sex chromosome and autosome fusions that led to a multiple sex chromosome system (X1X2Y). We present a high-quality whole-genome assembly and annotation of a male A. distichus (X1X2Y), enabling a detailed analysis of all three of its neo-sex chromosomes. We identified the AnoDisX1, AnoDisX2, and AnoDisY chromosomes from assembly scaffolds using an integrative approach, and estimated their degeneration and selection strength. Our results support long-held theories of differential evolutionary pressures on sex chromosomes, such as the Fast X effect and Y degeneration. Additionally, we observed that chromosome 12 has become sex-linked in two different Anolis species, suggesting that some autosomes may be more likely to become sex-linked. Altogether, our genome adds to the diversity of available taxa sequenced and enables novel comparative analyses in a variety of fields, including speciation, chromosomal synteny, and sex chromosome evolution.

The evolution of reproductive isolation lies at the core of our modern concept of species. Despite this central importance, study of the evolution of reproductive isolation is largely limited to a set of laboratory amenable species. When shallowly divergent populations are associated with different habitats, extrinsic isolation is often assumed to play a major role. Because this type of isolation is environmentally mediated it can be ephemeral, for instance, if habitats change in response to climate. In contrast, intrinsic isolation is heritable and therefore loss of this type of isolation requires genetic changes in one or both populations. We performed a hybridization and backcross experiment to test for and characterize intrinsic reproductive isolation between two recently diverged, ecologically differentiated populations of Anolis lizards. We find evidence of substantial intrinsic isolation that appears to operate at the postmating, prezygotic phase. Our findings suggest that intrinsic isolation may play a substantial role in the maintenance of shallow, yet ecologically divergent, lineages. ### Competing Interest Statement The authors have declared no competing interest. National Science Foundation, https://ror.org/021nxhr62, DEB-0920892, DEB-1457774, DEB-1500761, DEB-1927194

Abstract Cuba is the only landmass with more than one species in the Anolis carolinensis subgroup. We test the hypothesis that three rather than two distinct species occur on Cuba, based on substantial prior evidence of paraphyly. To test this hypothesis, we collected phenotypic data from all described species in the subgroup, including eastern and west-central Cuban populations of A. porcatus, and assessed phenotypic diagnosability using uni- and multivariate analyses. We also examined geographic isolation using all available occurrence records for Cuban lineages. Additionally, we conducted ecological niche modeling and niche overlap analyses, considering only Cuban lineages, to test for ecological differentiation. Finally, we reconstructed phylogenetic trees, incorporating all species from the subgroup for the first time. Our results support the recognition of three species in Cuba: A. allisoni and eastern and west-central A. porcatus as two distinct cryptic species, showing minimal phenotypic differentiation but clear geographic isolation, distinct ecological niches, and deep genetic divergence. We restrict the name A. porcatus to west-central Cuba, with Havana as the type locality, and formally describe the eastern Cuban populations as Anolis torresfundorai sp. nov., designating Baracoa, Guantánamo, as the type locality.

Islands are the recipients of numerous invasive amphibians and reptiles, often resulting in negative impacts on native biodiversity. In the Caribbean, Anolis lizards, a well-studied system for their adaptive radiation, are also commonly introduced outside of their native range, which has resulted in reshaping species-area and species-isolation relationships. We used mitochondrial DNA (mtDNA) sequence data in a phylogeographic framework to reconstruct and compare invasion histories of four non-native Anolis species (A. grahami, A. extremus, A. leachii, and A. sagrei) established in Bermuda over the past 120 years. Our findings support different invasion histories for the four species, including the number of native-range sources, genetic diversity, secondary introductions and opportunities for intraspecific hybridization between previously isolated lineages. The extent of population genetic structure in the native range and the mode of introduction (i.e., intentional vs. unintentional) may influence patterns of the invasion history and genetic diversity for introduced Anolis lizards in Bermuda. These results suggest that human-mediated introductions can create substantial variation in invasion dynamics, which in turn may influence fates of species in new environments.

Environmental variation often drives evolutionary processes like population differentiation, local adaptation and speciation. We used genome-scale data to investigate the contribution of environmental variation to evolution of the North Caribbean bark anole (Anolis distichus), a widespread common lizard that exhibits impressive phenotypic variation across varying habitats on the island of Hispaniola. We obtained new double-digest restriction-associated DNA sequence data (ddRADseq) from nearly 200 individuals and used 53 GIS data layers representing a range of environmental variables. We first asked how environmental variation has contributed to genome-wide differentiation across Hispaniola. We found that Hispaniola's three major mountain ranges contribute to deep genome-wide divergence and patterns of migration, that some deeply genomically divergent populations occupy significantly different environments, and that environmental variation is broadly capable of explaining more range-wide genomic differentiation than geographic distance alone. We then asked whether specific loci exhibit evidence of local adaptation to environmental variation using genotype-environment association (GEA) methods. We initially identified hundreds of loci broadly distributed across the genome that are significantly correlated with one or more environmental variables, but ultimately found that fewer than 100 of these candidate loci are shared across different GEA methods applied to our entire dataset, and that only 10 candidate loci are shared by independent analyses of two regional subsets of our dataset, suggesting parallel evolution is infrequent. Our study shows that abiotic environmental variation has played a critical role in explaining the evolution and diversity of a widespread and phenotypically diverse Caribbean anole species.

The Cuban green anoles, members of the Anolis carolinensis species complex, are among the most successful colonizing lineages of anole lizards in the Caribbean. We use ddRAD and Sanger sequence data to clarify the evolutionary relationships among all members of the A. carolinensis species complex (generally called Cuban green anoles), demarcate species boundaries, and explain the geographic origins of this key group of highly dispersive anoles. This study supports the "Out of Cuba" hypothesis involving six or seven independent colonization events from a Cuban source with one possible island-to-island dispersal event between the Cayman Islands and Navassa. Independent colonization events occurred from three main clades delimited by western, central, and eastern Cuba. West Cuban lineages dispersed twice-to the Cay Sal Bank (Bahamas) and to Florida. Central Cuban lineages dispersed twice-to the Bahamas archipelago and the Bay Islands (Honduras), respectively, with the latter seemingly owing to human assistance. East Cuban lineages colonized the Bahamas archipelago once as well as Little Cayman and Navassa, although it is unclear if all these colonizations occurred directly from Cuba. We recommend continued recognition of all species in the complex except A. fairchildi-which we consider a subspecies of A. porcatus-for a total of 8 species in this group: A. allisoni, A. brunneus, A. carolinensis, A. longiceps, A. maynardii, A. porcatus, A. smaragdinus, and A. torresfundorai.

The ENMTools software package was introduced in 2008 as a platform for making measurements on environmental niche models (ENMs, frequently referred to as species distribution models or SDMs), and for using those measurements in the context of newly developed Monte Carlo tests to evaluate hypotheses regarding niche evolution. Additional functionality was later added for model selection and simulation from ENMs, and the software package has been quite widely used. ENMTools was initially implemented as a Perl script, which was also compiled into an executable file for various platforms. However, the package had a number of significant limitations; it was only designed to fit models using Maxent, it relied on a specific Perl distribution to function, and its internal structure made it difficult to maintain and expand. Subsequently, the R programming language became the platform of choice for most ENM studies, making ENMTools less usable for many practitioners. Here we introduce a new R version of ENMTools that implements much of the functionality of its predecessor as well as numerous additions that simplify the construction, comparison and evaluation of niche models. These additions include new metrics for model fit, methods of measuring ENM overlap, and methods for testing evolutionary hypotheses. The new version of ENMTools is also designed to work within the expanding universe of R tools for ecological biogeography, and as such includes greatly simplified interfaces for analyses from several other R packages.

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 Brocher(13)) supported by the animal's rugose skin upon submergence. Wesuggest 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" mechanism(4,16)).

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.

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.

Cryptic species – genetically distinct species that are morphologically difficult to distinguish – present challenges to systematists. Operationally, cryptic species are very difficult to identify and sole use of genetic data or morphological data can fail to recognize evolutionarily isolated lineages. We use morphometric data to test species boundaries hypothesized with genetic data in the North Caribbean bark anole (Anolis distichus), a suspected species complex. We use univariate and multivariate analyses to test if candidate species based on genetic data can be accurately diagnosed. We also test alternative species delimitation scenarios with a model fitting approach that evaluates normal mixture models capable of identifying morphological clusters. Our analyses reject the hypothesis that the candidate species are diagnosable. Neither uni- nor multivariate morphometric data distinguish candidate species. The best-supported model included two morphological clusters; however, these clusters were uneven and did not align with a plausible species divergence scenario. After removing two related traits driving this result, only one cluster was supported. Despite substantial differentiation revealed by genetic data, we recover no new evidence to delimit species and refrain from taxonomic revision. This study highlights the importance of considering other types of data along with molecular data when delimiting species.

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.

A new species of glassfrog (Centrolenidae) is described from the San Jacinto River, an affluent of the Topo River, on the Amazonian slopes of the Ecuadorian Andes. The new species, Hyalinobatrachium adespinosai sp. nov., can be differentiated from all other centrolenids by the combination of its coloration (transparent peritoneum and pericardium) and vocalization (call duration = 0.38-0.44 s, with 9-13 pulses per call; dominant frequency = 4,645-5,001 Hz). However, H. adespinosai sp. nov. is morphologically cryptic with H. anachoretus, H. esmeralda, and H. pellucidum, from which it differs by call traits (in H. anachoretus: call duration = 0.32-0.37 s, with 5 or 6 pulses per call, dominant frequency = 4,670-4,800 Hz; in H. esmeralda: call duration = 0.218-0.257 s, tonal call, dominant frequency = 4,739-5,580 Hz; in H. pellucidum: call duration = 0.112-0.140 s, tonal, dominant frequency = 5,000-5,710 Hz). Biogeographically, the new species is separated from H. anachoretus by a considerable distance and, also, the Maranon valley. Finally, following IUCN conservation criteria, the status of the new species is considered as Data Deficient.

An integrative taxonomic revision of agamid lizards from the genera Amphibolurus and Lophognathus (Lacertilia: Agamidae).Memoirs of Museum Victoria 77: 41-61

AbstractDelimiting 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.

The divergence of signals used in sexual selection and species recognition is thought to play an important role in speciation. As such, the striking diversity of dewlap color in Anolis lizards may have contributed to the diversification of this species-rich group. Whether the dewlap acts as a reproductive isolating barrier however remains unclear. We tested the prediction that the degree of dewlap divergence between the two closely related and highly polymorphic Hispaniolan trunk anoles Anolis distichus and Anolis brevirostris is correlated with genetic divergence and the frequency of hybridization in nature. We use integrative analyses of dewlap color variation and molecular genetics to investigate two pairs of localities where the species co-occur, including one pair of localities where the two species exhibit dissimilar dewlaps and a second pair of localities where they have similar dewlaps. At one site where species share similar dewlap coloration, we found evidence of hybridization and lower levels of genetic differentiation. At all other localities, however, including another site where species share a similar dewlap color, the species were genetically divergent with little evidence of mitochondrial and nuclear gene flow. Together, our results suggest that dewlap color is not consistently associated with reproductive isolation at the species level. Instead, site-specific factors may influence the dewlap's role in maintaining species boundaries.

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.

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.

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.