Dispersal, not vicariance, explains the biogeographic origin of iguanas on Madagascar.

Lizards of the clade Iguanidae (sensu lato) are primarily a New World group. Thus, the remarkable presence of an endemic lineage of iguanas (family Opluridae) on the isolated Indian Ocean island of Madagascar has long been considered a biogeographic anomaly. Previous work attributed this disjunct extant distribution to: (1) vicariance at about 140-165 Ma, caused by the breakup of Gondwana and the separation of South America, Africa, and Madagascar (with subsequent extinction of iguanas on Africa, and potentially other Gondwanan landmasses), (2) vicariance at about 80-90 Ma, caused by the sundering of hypothesized land-bridge connections between South America, Antarctica, India, and Madagascar, or (3) long-distance overwater dispersal from South America to Madagascar. Each hypothesis has been supported with molecular divergence dating analyses, and thus the biogeographic origin of the Opluridae is not yet well resolved. Here we utilize genetic sequences of ultraconserved elements for all Iguania families and the majority of Iguanidae (s.l.) genera, and morphological data for extant and fossil taxa (used for divergence dating analyses), to produce the most comprehensive dataset applied to date to test these origin hypotheses. We find strong support for a sister relationship between the Opluridae (Madagascar) and Leiosauridae (South America). Divergence of the Opluridae from Leiosauridae is dated to between the late Cretaceous and mid-Paleogene, at a time when Madagascar was already an island and was isolated from all other Gondwanan landmasses. Consequently, our results support a hypothesis of long-distance overwater dispersal of the Opluridae lineage, either directly from South America to Madagascar or potentially via Antarctica or Africa, leading to this radiation of iguanas in the Indian Ocean.

Phylogenomics and historical biogeography of West Indian Rock Iguanas (genus Cyclura).

The genus Cyclura includes nine extant species and six subspecies of West Indian Rock Iguanas and is one of the most imperiled genera of squamate reptiles globally. An understanding of species diversity, evolutionary relationships, diversification, and historical biogeography in this group is crucial for implementing sound long-term conservation strategies. We collected DNA samples from 1 to 10 individuals per taxon from all Cyclura taxa (n = 70 ingroup individuals), focusing where possible on incorporating individuals from different populations of each species. We also collected 1-2 individuals from each of seven outgroup species of iguanas (Iguana delicatissima; five Ctenosaura species) and Anolis sagrei (n = 12 total outgroup individuals). We used targeted genomic sequence capture to isolate and to sequence 1,872 loci comprising of 687,308 base pairs (bp) from each of the 82 individuals from across the nuclear genome. We extracted mitochondrial reads and assembled and annotated mitogenomes for all Cyclura taxa plus outgroup species. We present well-supported phylogenomic gene tree/species tree analyses for all extant species of Cyclura using ASTRAL-III, SVDQuartets, and StarBEAST2 methods, and discuss the taxonomic, biogeographic, and conservation implications of these data. We find a most recent common ancestor of the genus 9.91 million years ago. The earliest divergence within Cyclura separates C. pinguis from a clade comprising all other Cyclura. Within the latter group, a clade comprising C. carinata from the southern Lucayan Islands and C. ricordii from Hispaniola is the sister taxon to a clade comprising the other Cyclura. Among the other Cyclura, the species C. cornuta and C. stejnegeri (from Hispaniola and Isla Mona) form the sister taxon to a clade of species from Jamaica (C. collei), Cuba and Cayman Islands (C. nubila and C. lewisi), and the eastern (C. rileyi) and western (C. cychlura) Lucayan Islands. Cyclura cychlura and C. rileyi form a clade whose sister taxa are C. nubila and C. lewisi. Cyclura collei is the sister taxon to these four species combined.

Proteomics of Galápagos Marine Iguanas Links Function of Femoral Gland Proteins to the Immune System.

Communication between individuals via molecules, termed chemosignaling, is widespread among animal and plant species. However, we lack knowledge on the specific functions of the substances involved for most systems. The femoral gland is an organ that secretes a waxy substance involved in chemical communication in lizards. Although the lipids and volatile substances secreted by the femoral glands have been investigated in several biochemical studies, the protein composition and functions of secretions remain completely unknown. Applying a proteomic approach, we provide the first attempt to comprehensively characterize the protein composition of femoral gland secretions from the Galápagos marine iguana. Using samples from several organs, the marine iguana proteome was assembled by next-generation sequencing and MS, resulting in 7513 proteins. Of these, 4305 proteins were present in the femoral gland, including keratins, small serum proteins, and fatty acid-binding proteins. Surprisingly, no proteins with discernible roles in partner recognition or inter-species communication could be identified. However, we did find several proteins with direct associations to the innate immune system, including lysozyme C, antileukoproteinase (ALP), pulmonary surfactant protein (SFTPD), and galectin (LGALS1) suggesting that the femoral glands function as an important barrier to infection. Furthermore, we report several novel anti-microbial peptides from the femoral glands that show similar action against Escherichia coli and Bacillus subtilis such as oncocin, a peptide known for its effectiveness against Gram-negative pathogens. This proteomics data set is a valuable resource for future functional protein analysis and demonstrates that femoral gland secretions also perform functions of the innate immune system.

Ghosts of a Structured Past: Impacts of Ancestral Patterns of Isolation-by-Distance on Divergence-Time Estimation.

Isolation-by-distance is a widespread pattern in nature that describes the reduction of genetic correlation between subpopulations with increased geographic distance. In the population ancestral to modern sister species, this pattern may hypothetically inflate population divergence time estimation due to allele frequency differences in subpopulations at the ends of the ancestral population. In this study, we analyze the relationship between the time to the most recent common ancestor and the population divergence time when the ancestral population model is a linear stepping-stone. Using coalescent simulations, we compare the coalescent time to the population divergence time for various ratios of the divergence time over the population size. Next, we simulate whole genomes to obtain single nucleotide polymorphisms (SNPs), and use the Bayesian coalescent program SNAPP to estimate divergence times. We find that as the rate of migration between neighboring demes decreases, the coalescent time becomes significantly greater than the population divergence time when sampled from end demes. Divergence-time overestimation in SNAPP becomes severe when the divergence-to-population size ratio < 10 and migration is low. Finally, we demonstrate the impact of ancestral isolation-by-distance on divergence-time estimation using an empirical dataset of squamates (Tropidurus) endemic to Brazil. We conclude that studies estimating divergence times should be cognizant of the potential ancestral population structure in an explicitly spatial context or risk dramatically overestimating the timing of population splits.

Heterozygosity-Fitness Correlations Reveal Inbreeding Depression in Neonatal Body Size in a Critically Endangered Rock Iguana.

Inbreeding depression, though challenging to identify in nature, may play an important role in regulating the dynamics of small and isolated populations. Conversely, greater expression of genetic load can enhance opportunities for natural selection. Conditional expression concentrates these opportunities for selection and may lead to failure of detection. This study investigates the possibility for age-dependent expression of inbreeding depression in a critically endangered population of rock iguanas, Cyclura nubila caymanensis. We employ heterozygote-fitness correlations to examine the contributions of individual genetic factors to body size, a fitness-related trait. Nonsignificant reductions in homozygosity (up to 7%) were detected between neonates and individuals surviving past their first year, which may reflect natural absorption of inbreeding effects by this small, fecund population. The majority of variation in neonate body size was attributed to maternal or environmental effects (i.e., clutch identity and incubation length); however, heterozygosity across 22 microsatellite loci also contributed significantly and positively to model predictions. Conversely, effects of heterozygosity on fitness were not detectable when adults were examined, suggesting that inbreeding depression in body size may be age dependent in this taxon. Overall, these findings emphasize the importance of taking holistic, cross-generational approaches to genetic monitoring of endangered populations.

The Lesser Antillean Iguana (Iguana delicatissima) on St. Eustatius: Genetically Depauperate and Threatened by Ongoing Hybridization.

The Lesser Antillean Iguana (Iguana delicatissima) is an endangered species threatened by habitat loss and hybridization with non-native Green Iguanas (Iguana iguana). Iguana delicatissima has been extirpated on several islands, and the Green Iguana has invaded most islands with extant populations. Information is essential to protect this species from extinction. We collected data on 293 iguanas including 17 juveniles from St. Eustasius, one of the few remaining I. delicatissima strongholds. Genetic data were leveraged to test for hybridization presence with the Green Iguana using both mitochondrial and nuclear genes, including 16 microsatellite loci. The microsatellites were also analyzed to estimate genetic diversity, population structure, and effective population size. Using molecular and morphological data, we identified 286 I. delicatissima individuals captured during our first fieldwork effort, and 7 non-native iguanas captured during a second effort, showing hybridization occurs within this population. Comparing homologous microsatellites used in studies on Dominica and Chancel, the I. delicatissima population on St. Eustatius has extremely low genetic diversity (HO = 0.051; HE = 0.057), suggesting this population is genetically depauperate. Furthermore, there is significant evidence for inbreeding (FIS = 0.12) and weak spatial genetic structure (FST = 0.021, P = 0.002) within this population. Besides immediate threats including hybridization, this population's low genetic diversity, presence of physiological abnormalities and low recruitment could indicate presence of inbreeding depression that threatens its long-term survival. We conclude there is a continued region-wide threat to I. delicatissima and highlight the need for immediate conservation action to stop the continuing spread of Green Iguanas and to eliminate hybridization from St. Eustatius.

Characterization and distribution of GHRH, PACAP, TRH, SST and IGF1 mRNAs in the green iguana.

The somatotropic axis (SA) regulates numerous aspects of vertebrate physiology such as development, growth, and metabolism and has influence on several tissues including neural, immune, reproductive and gastric tract. Growth hormone (GH) is a key component of SA, it is synthesized and released mainly by pituitary somatotrophs, although now it is known that virtually all tissues can express GH, which, in addition to its well-described endocrine roles, also has autocrine/paracrine/intracrine actions. In the pituitary, GH expression is regulated by several hypothalamic neuropeptides including GHRH, PACAP, TRH and SST. GH, in turn, regulates IGF1 synthesis in several target tissues, adding complexity to the system since GH effects can be exerted either directly or mediated by IGF1. In reptiles, little is known about the SA components and their functional interactions. The aim of this work was to characterize the mRNAs of the principal SA components in the green iguana and to develop the tools that allow the study of the structural and functional evolution of this system in reptiles. By employing RT-PCR and RACE, the cDNAs encoding for GHRH, PACAP, TRH, SST and IGF1 were amplified and sequenced. Results showed that these cDNAs coded for the corresponding protein precursors of 154, 170, 243, 113, and 131 amino acids, respectively. Of these, GHRH, PACAP, SST and IGF1 precursors exhibited a high structural conservation with respect to its counterparts in other vertebrates. On the other hand, iguana's TRH precursor showed 7 functional copies of mature TRH (pyr-QHP-NH2), as compared to 4 and 6 copies of TRH in avian and mammalian proTRH sequences, respectively. It was found that in addition to its primary production site (brain for GHRH, PACAP, TRH and SST, and liver for IGF1), they were also expressed in other peripheral tissues, i.e. testes and ovaries expressed all the studied mRNAs, whereas TRH and IGF1 mRNAs were observed ubiquitously in all tissues considered. These results show that the main SA components in reptiles of the Squamata Order maintain a good structural conservation among vertebrate phylogeny, and suggest important physiological interactions (endocrine, autocrine and/or paracrine) between them due to their wide peripheral tissue expression.

Never judge an iguana by its spines: Systematics of the Yucatan spiny tailed iguana, Ctenosaura defensor (Cope, 1866).

Spiny tailed iguanas are highly diverse clade of lizards in Mesoamerica, ranging from northern Mexico through Panama. Utilizing 2 regions of mitochondrial DNA (1948bp) and 4 nuclear loci (2232bp) we explored the relationships between these species and the phylogeographic history of the major clades. We discovered that the lineage endemic to the Yucatan Peninsula renders the genus Ctenosaura paraphyletic. To resolve this non-monophyly, we resurrect the taxon Cachryx Cope, 1866, and provide a new diagnosis for the genus. We also find that small body-size and highly spinose tails in the species previously referred to the subgenus Enyaliosaurus, have evolved independently 3 times. Cachryx were recovered as sister to the lineage of iguanines endemic to the Galapagos Islands, and we discuss biogeographic scenarios to explain this relationship as well as those among the primary clades of Ctenosaura in Mesoamerica.

Phylogeography and Ecological Niche Modeling of the Desert Iguana (Dipsosaurus dorsalis, Baird & Girard 1852) in the Baja California Peninsula.

Understanding the factors that explain the patterns of genetic structure or phylogeographic breaks at an intraspecific level is key to inferring the mechanisms of population differentiation in its early stages. These topics have been well studied in the Baja California region, with vicariance and the dispersal ability of individuals being the prevailing hypothesis for phylogeographic breaks. In this study, we evaluated the phylogeographic patterns in the desert iguana (Dipsosaurus dorsalis), a species with a recent history in the region and spatial variation in life history traits. We analyzed a total of 307 individuals collected throughout 19 localities across the Baja California Peninsula with 15 microsatellite DNA markers. Our data reveal the existence of 3 geographically discrete genetic populations with moderate gene flow and an isolation-by-distance pattern presumably produced by the occurrence of a refugium in the Cape region during the Pleistocene Last Glacial Maximum. Bayesian methods and ecological niche modeling were used to assess the relationship between population genetic structure and present and past climatic preferences of the desert iguana. We found that the present climatic heterogeneity of the Baja California Peninsula has a marked influence on the population genetic structure of the species, suggesting that there are alternative explanations besides vicariance. The information obtained in this study provides data allowing a better understanding of how historical population processes in the Baja California Peninsula can be understood from an ecological perspective.

Differential responses of the somatotropic and thyroid axes to environmental temperature changes in the green iguana.

Growth hormone (GH), together with thyroid hormones (TH), regulates growth and development, and has critical effects on vertebrate metabolism. In ectotherms, these physiological processes are strongly influenced by environmental temperature. In reptiles, however, little is known about the direct influences of this factor on the somatotropic and thyroid axes. Therefore, the aim of this study was to describe the effects of both acute (48h) and chronic (2weeks) exposure to sub-optimal temperatures (25 and 18°C) upon somatotropic and thyroid axis function of the green iguana, in comparison to the control temperature (30-35°C). We found a significant increase in GH release (2.0-fold at 25°C and 1.9-fold at 18°C) and GH mRNA expression (up to 3.7-fold), mainly under chronic exposure conditions. The serum concentration of insulin-like growth factor-I (IGF-I) was significantly greater after chronic exposure (18.5±2.3 at 25°C; 15.92±3.4 at 18°C; vs. 9.3±1.21ng/ml at 35°C), while hepatic IGF-I mRNA expression increased up to 6.8-fold. Somatotropic axis may be regulated, under acute conditions, by thyrotropin-releasing hormone (TRH) that significantly increased its hypothalamic concentration (1.45 times) and mRNA expression (0.9-fold above control), respectively; and somatostatin (mRNA expression increased 1.0-1.2 times above control); and under chronic treatment, by pituitary adenylate cyclase-activating peptide (PACAP mRNA expression was increased from 0.4 to 0.6 times). Also, it was shown that, under control conditions, injection of TRH stimulated a significant increase in circulating GH. On the other hand, while there was a significant rise in the hypothalamic content of TRH and its mRNA expression, this hormone did not appear to influence the thyroid axis activity, which showed a severe diminution in all conditions of cold exposure, as indicated by the decreases in thyrotropin (TSH) mRNA expression (up to one-eight of the control), serum T4 (from 11.6±1.09 to 5.3±0.58ng/ml, after 2weeks at 18°C) and T3 (from 0.87±0.09 to 0.05±0.01ng/ml, under chronic conditions at 25°C), and Type-2 deiodinase (D2) activity (from 992.5±224 to 213.6±26.4fmolI(125)T4/mgh). The reduction in thyroid activity correlates with the down-regulation of metabolism as suggested by the decrease in the serum glucose and free fatty acid levels. These changes apparently were independent of a possible stress response, at least under acute exposure to both temperatures and in chronic treatment to 25°C, since serum corticosterone had no significant changes in these conditions, while at chronic 18°C exposure, a slight increase (0.38 times above control) was found. Thus, these data suggest that the reptilian somatotropic and thyroid axes have differential responses to cold exposure, and that GH and TRH may play important roles associated to adaptation mechanisms that support temperature acclimation in the green iguana.

Hybridization masks speciation in the evolutionary history of the Galápagos marine iguana.

The effects of the direct interaction between hybridization and speciation-two major contrasting evolutionary processes--are poorly understood. We present here the evolutionary history of the Galápagos marine iguana (Amblyrhynchus cristatus) and reveal a case of incipient within--island speciation, which is paralleled by between-island hybridization. In-depth genome-wide analyses suggest that Amblyrhynchus diverged from its sister group, the Galápagos land iguanas, around 4.5 million years ago (Ma), but divergence among extant populations is exceedingly young (less than 50,000 years). Despite Amblyrhynchus appearing as a single long-branch species phylogenetically, we find strong population structure between islands, and one case of incipient speciation of sister lineages within the same island--ostensibly initiated by volcanic events. Hybridization between both lineages is exceedingly rare, yet frequent hybridization with migrants from nearby islands is evident. The contemporary snapshot provided by highly variable markers indicates that speciation events may have occurred throughout the evolutionary history of marine iguanas, though these events are not visible in the deeper phylogenetic trees. We hypothesize that the observed interplay of speciation and hybridization might be a mechanism by which local adaptations, generated by incipient speciation, can be absorbed into a common gene pool, thereby enhancing the evolutionary potential of the species as a whole.

Species delimitation using Bayes factors: simulations and application to the Sceloporus scalaris species group (Squamata: Phrynosomatidae).

Current molecular methods of species delimitation are limited by the types of species delimitation models and scenarios that can be tested. Bayes factors allow for more flexibility in testing non-nested species delimitation models and hypotheses of individual assignment to alternative lineages. Here, we examined the efficacy of Bayes factors in delimiting species through simulations and empirical data from the Sceloporus scalaris species group. Marginal-likelihood scores of competing species delimitation models, from which Bayes factor values were compared, were estimated with four different methods: harmonic mean estimation (HME), smoothed harmonic mean estimation (sHME), path-sampling/thermodynamic integration (PS), and stepping-stone (SS) analysis. We also performed model selection using a posterior simulation-based analog of the Akaike information criterion through Markov chain Monte Carlo analysis (AICM). Bayes factor species delimitation results from the empirical data were then compared with results from the reversible-jump MCMC (rjMCMC) coalescent-based species delimitation method Bayesian Phylogenetics and Phylogeography (BP&P). Simulation results show that HME and sHME perform poorly compared with PS and SS marginal-likelihood estimators when identifying the true species delimitation model. Furthermore, Bayes factor delimitation (BFD) of species showed improved performance when species limits are tested by reassigning individuals between species, as opposed to either lumping or splitting lineages. In the empirical data, BFD through PS and SS analyses, as well as the rjMCMC method, each provide support for the recognition of all scalaris group taxa as independent evolutionary lineages. Bayes factor species delimitation and BP&P also support the recognition of three previously undescribed lineages. In both simulated and empirical data sets, harmonic and smoothed harmonic mean marginal-likelihood estimators provided much higher marginal-likelihood estimates than PS and SS estimators. The AICM displayed poor repeatability in both simulated and empirical data sets, and produced inconsistent model rankings across replicate runs with the empirical data. Our results suggest that species delimitation through the use of Bayes factors with marginal-likelihood estimates via PS or SS analyses provide a useful and complementary alternative to existing species delimitation methods.

Phylogeography of the endangered Lesser Antillean iguana, Iguana delicatissima: a recent diaspora in an archipelago known for ancient herpetological endemism.

Iguana delicatissima is an endangered endemic of the Lesser Antilles in the Caribbean. Phylogeographic analyses for many terrestrial vertebrate species in the Caribbean, particularly lizards, suggest ancient divergence times. Often, the closest relatives of species are found on the same island, indicating that colonization rates are so low that speciation on islands is often more likely to generate biodiversity than subsequent colonization events. Mitochondrial sequence analysis of the region spanning ND4 was performed on I. delicatissima individuals from islands across the species' range to estimate genetic divergence among geographically isolated populations. Five unique haplotypes were recovered from 46 individuals. The majority of animals carry a single common haplotype. Two of the haplotypes were only present in individuals classified as hybrids from Îles des Saintes. The final 2 haplotypes, single nucleotide substitutions, were present in animals from Îlet Chancel of Martinique and Saint Barthélemy, respectively. Despite the great distances between islands and habitat heterogeneity within islands, this species is characterized by low haplotype diversity. The low mtDNA variation of I. delicatissima suggests a single colonization coupled with rapid range expansion, potentially hastened by human-mediated dispersal.

Cretaceous park of sex determination: sex chromosomes are conserved across iguanas.

Many poikilothermic vertebrate lineages, especially among amphibians and fishes, possess a rapid turnover of sex chromosomes, while in endotherms there is a notable stability of sex chromosomes. Reptiles in general exhibit variability in sex-determining systems; as typical poikilotherms, they might be expected to have a rapid turnover of sex chromosomes. However, molecular data which would enable the testing of the stability of sex chromosomes are lacking in most lineages. Here, we provide molecular evidence that sex chromosomes are highly conserved across iguanas, one of the most species-rich clade of reptiles. We demonstrate that members of the New World families Iguanidae, Tropiduridae, Leiocephalidae, Phrynosomatidae, Dactyloidae and Crotaphytidae, as well as of the family Opluridae which is restricted to Madagascar, all share homologous sex chromosomes. As our sampling represents the majority of the phylogenetic diversity of iguanas, the origin of iguana sex chromosomes can be traced back in history to the basal splitting of this group which occurred during the Cretaceous period. Iguanas thus show a stability of sex chromosomes comparable to mammals and birds and represent the group with the oldest sex chromosomes currently known among amniotic poikilothermic vertebrates.

Characterization of pituitary growth hormone and its receptor in the green iguana (Iguana iguana).

Pituitary growth hormone (GH) has been studied in most vertebrate groups; however, only a few studies have been carried out in reptiles. Little is known about pituitary hormones in the order Squamata, to which the green iguana (gi) belongs. In this work, we characterized the hypophysis of Iguana iguana morphologically. The somatotrophs (round cells of 7.6-10 µm containing 250- to 300-nm secretory granules where the giGH is stored) were found, by immunohistochemistry and in situ hybridization, exclusively in the caudal lobe of the pars distalis, whereas the lactotrophs were distributed only in the rostral lobe. A pituitary giGH-like protein was obtained by immuno-affinity chromatography employing a heterologous antibody against chicken GH. giGH showed molecular heterogeneity (22, 44, and 88 kDa by SDS-PAGE/Western blot under non-reducing conditions and at least four charge variants (pIs 6.2, 6.5, 6.9, 7.4) by isoelectric focusing. The pituitary giGH cDNA (1016 bp), amplified by PCR and RACE, encodes a pre-hormone of 218 aa, of which 190 aa correspond to the mature protein and 28 aa to the signal peptide. The giGH receptor cDNA was also partially sequenced. Phylogenetic analyses of the amino acid sequences of giGH and giGHR homologs in vertebrates suggest a parallel evolution and functional relationship between the GH and its receptor.

Novel herpesvirus in the critically endangered Galapagos pink land iguana.

Virus surveillance in wildlife is important to understanding ecosystem health, taxonomy, and evolution. Nevertheless, viruses in reptiles, and specifically in squamates, continue to be understudied. Herein, we conducted a health assessment on the critically endangered Galapagos pink land iguana (Conolophus marthae) and the vulnerable Galapagos land iguana (Conolophus subcristatus). We collected oral and/or cloacal swabs from 47 clinically healthy iguanas and tested for adenovirus (cloacal swabs, n = 47) and herpesvirus (oral swabs, n = 45) using broad-spectrum PCRs. Two out of 38 (5.3 %) Galapagos pink land iguanas tested positive for herpesvirus, while no herpesvirus was detected in all Galapagos land iguanas (n = 7). Both herpesviral sequences were identical between them and divergent (61.9 % amino acid identity) when compared to the closest herpesvirus sequences available in GenBank/EMBL/DDBJ. The genetic distance between this and other herpesviruses is consistent with its classification as a novel virus species. All iguanas were negative for adenovirus. This is the first description of a herpesvirus in iguanas of the Galapagos islands, and the first report of a potential pathogen for the iconic Galapagos pink land iguana. Further research is needed to understand the implications of this virus in the conservation and management of one of the most endangered iguana species in the world.

Contrasting effects of landscape features on genetic structure in different geographic regions in the ornate dragon lizard, Ctenophorus ornatus.

Habitat fragmentation can have profound effects on the distribution of genetic variation within and between populations. Previously, we showed that in the ornate dragon lizard, Ctenophorus ornatus, lizards residing on outcrops that are separated by cleared agricultural land are significantly more isolated and hold less genetic variation than lizards residing on neighbouring outcrops connected by undisturbed native vegetation. Here, we extend the fine-scale study to examine the pattern of genetic variation and population structure across the species' range. Using a landscape genetics approach, we test whether land clearing for agricultural purposes has affected the population structure of the ornate dragon lizard. We found significant genetic differentiation between outcrop populations (FST  = 0.12), as well as isolation by distance within each geographic region. In support of our previous study, land clearing was associated with higher genetic divergences between outcrops and lower genetic variation within outcrops, but only in the region that had been exposed to intense agriculture for the longest period of time. No other landscape features influenced population structure in any geographic region. These results show that the effects of landscape features can vary across species' ranges and suggest there may be a temporal lag in response to contemporary changes in land use. These findings therefore highlight the need for caution when assessing the impact of contemporary land use practices on genetic variation and population structure.

Quaternary range and demographic expansion of Liolaemus darwinii (Squamata: Liolaemidae) in the Monte Desert of Central Argentina using Bayesian phylogeography and ecological niche modelling.

Until recently, most phylogeographic approaches have been unable to distinguish between demographic and range expansion processes, making it difficult to test for the possibility of range expansion without population growth and vice versa. In this study, we applied a Bayesian phylogeographic approach to reconstruct both demographic and range expansion in the lizard Liolaemus darwinii of the Monte Desert in Central Argentina, during the Late Quaternary. Based on analysis of 14 anonymous nuclear loci and the cytochrome b mitochondrial DNA gene, we detected signals of demographic expansion starting at ~55 ka based on Bayesian Skyline and Skyride Plots. In contrast, Bayesian relaxed models of spatial diffusion suggested that range expansion occurred only between ~95 and 55 ka, and more recently, diffusion rates were very low during demographic expansion. The possibility of population growth without substantial range expansion could account for the shared patterns of demographic expansion during the Last Glacial Maxima (OIS 2 and 4) in fish, small mammals and other lizards of the Monte Desert. We found substantial variation in diffusion rates over time, and very high rates during the range expansion phase, consistent with a rapidly advancing expansion front towards the southeast shown by palaeo-distribution models. Furthermore, the estimated diffusion rates are congruent with observed dispersal rates of lizards in field conditions and therefore provide additional confidence to the temporal scale of inferred phylogeographic patterns. Our study highlights how the integration of phylogeography with palaeo-distribution models can shed light on both demographic and range expansion processes and their potential causes.

Diversification in the Mexican horned lizard Phrynosoma orbiculare across a dynamic landscape.

The widespread montane Mexican horned lizard Phrynosoma orbiculare (Squamata: Phrynosomatidae) represents an ideal species to investigate the relative impacts of Neogene vicariance and Quaternary climate change on lineage diversification across the Mexican highlands. We used mitochondrial DNA to examine the maternal history of P. orbiculare and estimate the timing and tempo of lineage diversification. Based on our results, we inferred 11 geographically structured, well supported mitochondrial lineages within this species, suggesting P. orbiculare represents a species complex. Six divergences between lineages likely occurred during the Late Miocene and Pliocene, and four splits probably happened during the Pleistocene. Diversification rate appeared relatively constant through time. Spatial and temporal divergences between lineages of P. orbiculare and co-distributed taxa suggest that a distinct period of uplifting of the Transvolcanic Belt around 7.5-3 million years ago broadly impacted diversification in taxa associated with this mountain range. To the north, several river drainages acting as filter barriers differentially subdivided co-distributed highland taxa through time. Diversification patterns observed in P. orbiculare provide additional insight into the mechanisms that impacted differentiation of highland taxa across the complex Mexican highlands.

An overlooked pink species of land iguana in the Galapagos.

Despite the attention given to them, the Galápagos have not yet finished offering evolutionary novelties. When Darwin visited the Galápagos, he observed both marine (Amblyrhynchus) and land (Conolophus) iguanas but did not encounter a rare pink black-striped land iguana (herein referred to as "rosada," meaning "pink" in Spanish), which, surprisingly, remained unseen until 1986. Here, we show that substantial genetic isolation exists between the rosada and syntopic yellow forms and that the rosada is basal to extant taxonomically recognized Galápagos land iguanas. The rosada, whose present distribution is a conundrum, is a relict lineage whose origin dates back to a period when at least some of the present-day islands had not yet formed. So far, this species is the only evidence of ancient diversification along the Galápagos land iguana lineage and documents one of the oldest events of divergence ever recorded in the Galápagos. Conservation efforts are needed to prevent this form, identified by us as a good species, from extinction.