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.

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.

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.

Influence of light/dark cycle and orexins on breathing control in green iguanas (Iguana iguana).

Light/dark cycle affects the physiology of vertebrates and hypothalamic orexin neurons (ORX) are involved in this function. The breathing pattern of the green iguana changes from continuous to episodic across the light/dark phases. Since the stimulatory actions of ORX on breathing are most important during arousal, we hypothesized that ORX regulates changes of breathing pattern in iguanas. Thus, we: (1) Localized ORX neurons with immunohistochemistry; (2) Quantified cyclic changes in plasma orexin-A levels by ELISA; (3) Compared breathing pattern at rest and during hypoxia and hypercarbia; (4) Evaluated the participation of the ORX receptors in ventilation with intracerebroventricular microinjections of ORX antagonists during light and dark phases. We show that the ORX neurons of I. iguana are located in the periventricular hypothalamic nucleus. Orexin-A peaks during the light/active phase and breathing parallels these cyclic changes: ventilation is higher during the light phase than during the dark phase. However, inactivation of ORX-receptors does not affect the breathing pattern. Iguanas increase ventilation during hypoxia only during the light phase. Conversely, CO2 promotes post-hypercarbic hyperpnea during both phases. We conclude that ORXs potentiate the post-hypercarbic (but not the hypoxic)-drive to breathe and are not involved in light/dark changes in the breathing pattern.

Defining intraspecific conservation units in the endemic Cuban Rock Iguanas (Cyclura nubila nubila).

Defining conservation units is an important step in species management and requires interpretation of the genetic diversity and ecological function of the taxon being considered. We used the endemic Cuban Rock Iguanas (Cyclura nubila nubila) as a model to highlight this challenge and examined patterns of its intraspecific genetic diversity across Cuba. We evaluated nuclear (microsatellite loci) and mitochondrial diversity across eight populations from the island and its off-shore cays, and applied the population genetics results for assignment of Management Unit (MU) status and Evolutionary Significant Units (ESUs) based on phylogeographic and time of divergence information. We identified at least six distinct Cuban Rock Iguana MUs, encompassing demographically isolated and genetically differentiated populations across Cuba, most with low effective population size, declining populations, and with high risk of inbreeding and genetic drift. Hence, each MU should be considered of urgent conservation priority. Given the key ecological seed dispersal role of C. n. nubila, the disappearance of any MU could trigger the loss of local ecological functional diversity and major negative impacts on their ecosystems. Two divergent ESUs were also identified, exhibiting an historical east-west geographic separation on Cuba. Based on a Caribbean phylogeographic assessment, our findings strengthen the conclusion that all geographically and evolutionarily differentiated Cyclura species and subspecies across the archipelago warrant ESU distinction.

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.

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.

ZW Sex Chromosomes in Australian Dragon Lizards (Agamidae) Originated from a Combination of Duplication and Translocation in the Nucleolar Organising Region.

Sex chromosomes in some reptiles share synteny with distantly related amniotes in regions orthologous to squamate chromosome 2. The latter finding suggests that chromosome 2 was formerly part of a larger ancestral (amniote) super-sex chromosome and raises questions about how sex chromosomes are formed and modified in reptiles. Australian dragon lizards (Agamidae) are emerging as an excellent model for studying these processes. In particular, they exhibit both genotypic (GSD) and temperature-dependent (TSD) sex determination, show evidence of transitions between the two modes and have evolved non-homologous ZW sex microchromosomes even within the same evolutionary lineage. They therefore represent an excellent group to probe further the idea of a shared ancestral super-sex chromosome and to investigate mechanisms for transition between different sex chromosome forms. Here, we compare sex chromosome homology among eight dragon lizard species from five genera to identify key cytological differences and the mechanisms that may be driving sex chromosome evolution in this group. We performed fluorescence in situ hybridisation to physically map bacterial artificial chromosome (BAC) clones from the bearded dragon, Pogona vitticeps' ZW sex chromosomes and a nucleolar organising region (NOR) probe in males and females of eight Agamid species exhibiting either GSD or TSD. We show that the sex chromosome derived BAC clone hybridises near the telomere of chromosome 2q in all eight species examined. This clone also hybridises to the sex microchromosomes of three species (P vitticeps, P. barbata and Diporiphora nobbi) and a pair of microchromosomes in three others (Ctenophorus pictus, Amphibolurus norrisi and Amphibolurus muricatus). No other chromosomes are marked by the probe in two species from the closely related genus Physignathus. A probe bearing nucleolar organising region (NOR) sequences maps close to the telomere of chromosome 2q in all eight species, and to the ZW pair in P. vitticeps and P. barbata, the W microchromosome in D. nobbi, and several microchromosomes in P. cocincinus. Our findings provide evidence of sequence homology between chromosome 2 and the sex chromosomes of multiple agamids. These data support the hypothesis that there was an ancestral sex chromosome in amniotes that gave rise to squamate chromosome 2 and raises the prospect that some particular property of this chromosome has favoured its role as a sex chromosome in amniotes. It is likely that the amplification of repetitive sequences associated with this region has driven the high level of heterochromatinisation of the sex-specific chromosomes in three species of agamid. Our data suggest a possible mechanism for chromosome rearrangement, including inversion and duplication near the telomeric regions of the ancestral chromosome 2 and subsequent translocation to the ZW sex microchromosomes in three agamid species. It is plausible that these chromosome rearrangements involving sex chromosomes also drove speciation in this group.

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.

All iguana families with the exception of basilisks share sex chromosomes.

Once believed to be restricted only to endotherms (mammals and birds), several poikilothermic amniote lineages have recently been documented to possess long-term evolutionary stability in their sex chromosomes. However, many important lineages were not included in these tests. Previously, based on molecular evidence, we documented the homology of well-differentiated sex chromosomes among seven families of iguanas (Pleurodonta), with basilisks (Corytophanidae) being the only exception, as the tested genes linked to X, but missing on the Y chromosome, in other iguanas were autosomal or pseudoautosomal in basilisks. In this study, we test the homology of sex chromosomes in the remaining, previously unstudied iguana families (Hoplocercidae, Leiosauridae, Liolaemidae, Polychrotidae) and in the basilisk genus Corytophanes. Our results show that 12 currently recognized families of iguanas share X-specific gene content conserved from the common ancestor living in the Cretaceous period. However, the results in the genus Corytophanes indicate the loss of the ancestral differentiated sex chromosomes from the ancestor of basilisks. Our new data further confirm the extensive stability of sex chromosomes in iguanas, thus enabling molecular sexing based on the comparison of the number of X-specific genes by quantitative PCR (qPCR) in all but one family of this widely diversified clade.

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.

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.

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.

Predictability in evolution: Adaptation of the Bonaire anole (Anolis bonairensis) to an extreme environment.

The extent to which evolution is deterministic (predictable), or random, is a fundamental question in evolution. This case study attempts to determine the extent to which interspecific divergence can be predicted from intraspecific trends related species. The mountainous Lesser Antilles are occupied by one or two anole species with very substantial intraspecific differences in the quantitative traits between xeric and rainforest habitats. These ecologically determined differences tend to be in parallel in each island species. A related species (Anolis bonairensis) lives on the far more xeric island of Bonaire, and this study tests the extent to which its interspecific divergence in hue and pattern traits can be predicted from the parallel intraspecific variation exhibited in Lesser Antillean anoles. Regression against a multivariate climate variable suggests that the hue and pattern of the Bonaire anole are consistently predicted from the ecologically determined intraspecific variation of its Lesser Antillean relatives. However, this predictability may be less consistent with other character systems, for example, scalation.

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.

The complete mitochondrial genomes of the Galápagos iguanas, Amblyrhynchus cristatus and Conolophus subcristatus.

The Galápagos iguanas are among the oldest vertebrate lineages on the Galápagos archipelago, and the evolutionary history of this clade is of great interest to biologists. We describe here the complete mitochondrial genomes of the marine iguana, Amblyrhynchus cristatus (Genbank accession number: KT277937) and the land iguana Conolophus subcristatus (Genbank accession number: KT277936). The genomes contain 13 protein-coding genes, 22 transfer RNAs, and two ribosomal RNAs genes, as well as a control region (CR). Both species have an identical gene order, which matches that of Iguana iguana. The CR of both Galápagos iguanas features similar tandem repeats units, which are absent in I. iguana. We present a phylogeny of the Iguanidae based on complete mitochondrial genomes, which confirms the sister-group relationship of Galápagos iguanas. These new mitochondrial genomes constitute an important data source for future exploration of the phylogenetic relationships and evolutionary history of the Galápagos iguanas.

MIG-seq: an effective PCR-based method for genome-wide single-nucleotide polymorphism genotyping using the next-generation sequencing platform.

Restriction-enzyme (RE)-based next-generation sequencing methods have revolutionized marker-assisted genetic studies; however, the use of REs has limited their widespread adoption, especially in field samples with low-quality DNA and/or small quantities of DNA. Here, we developed a PCR-based procedure to construct reduced representation libraries without RE digestion steps, representing de novo single-nucleotide polymorphism discovery, and its genotyping using next-generation sequencing. Using multiplexed inter-simple sequence repeat (ISSR) primers, thousands of genome-wide regions were amplified effectively from a wide variety of genomes, without prior genetic information. We demonstrated: 1) Mendelian gametic segregation of the discovered variants; 2) reproducibility of genotyping by checking its applicability for individual identification; and 3) applicability in a wide variety of species by checking standard population genetic analysis. This approach, called multiplexed ISSR genotyping by sequencing, should be applicable to many marker-assisted genetic studies with a wide range of DNA qualities and quantities.

Mitochondrial Involvement in Vertebrate Speciation? The Case of Mito-nuclear Genetic Divergence in Chameleons.

Compatibility between the nuclear (nDNA) and mitochondrial (mtDNA) genomes is important for organismal health. However, its significance for major evolutionary processes such as speciation is unclear, especially in vertebrates. We previously identified a sharp mtDNA-specific sequence divergence between morphologically indistinguishable chameleon populations (Chamaeleo chamaeleon recticrista) across an ancient Israeli marine barrier (Jezreel Valley). Because mtDNA introgression and gender-based dispersal were ruled out, we hypothesized that mtDNA spatial division was maintained by mito-nuclear functional compensation. Here, we studied RNA-seq generated from each of ten chameleons representing the north and south populations and identified candidate nonsynonymous substitutions (NSSs) matching the mtDNA spatial distribution. The most prominent NSS occurred in 14 nDNA-encoded mitochondrial proteins. Increased chameleon sample size (N = 70) confirmed the geographic differentiation in POLRMT, NDUFA5, ACO1, LYRM4, MARS2, and ACAD9. Structural and functionality evaluation of these NSSs revealed high functionality. Mathematical modeling suggested that this mito-nuclear spatial divergence is consistent with hybrid breakdown. We conclude that our presented evidence and mathematical model underline mito-nuclear interactions as a likely role player in incipient speciation in vertebrates.

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.