Multilocus phylogeny, species age and biogeography of the Lesser Antillean anoles.

Lesser Antillean anoles provide classic examples of island radiations. A detailed knowledge of their phylogeny and biogeography, in particular how the age of species relate to the ages of their respective islands and the age of their radiation, is essential to elucidate the tempo and mechanisms of these radiations. We conduct a large-scale phylogenetic and phylogeographic investigation of the Lesser Antillean anoles using multiple genetic markers and comprehensive geographic sampling of most species. The multilocus phylogeny gives the first well-supported reconstruction of the interspecific relationships, and the densely sampled phylogeography reveals a highly dynamic system, driven by overseas dispersal, with several alternative post-dispersal colonisation trajectories. These radiations currently occupy both the outer-older (Eocene to Miocene), and the inner-younger (<8mybp), Lesser Antillean arcs. The origin of these radiations corresponds with the age of the ancient outer arc. However, the ages of extant species (compatible with the age of other small terrestrial amniotes) are much younger, about the age of the emergence of the younger arc, or less. The difference between the age of the radiation and the age of the extant species suggests substantial species turnover on older arc islands, most likely through competitive replacement. Although extant anoles are extremely speciose, this may represent only a fraction of their biodiversity over time. While paraphyly enables us to infer several recent colonization events, the absence of the younger arc islands and extant species at the earlier and middle stages of the radiation, does not allow the earlier inter-island colonization to be reliably inferred. Reproductive isolation in allopatry takes a very considerable time (in excess of 8my) and sympatry appears to occur only late in the radiation. The resolved multilocus phylogeny, and relative species age, raise difficulties for some earlier hypotheses regarding size evolution, and provide no evidence for within-island speciation.

Widespread parallel population adaptation to climate variation across a radiation: implications for adaptation to climate change.

Global warming will impact species in a number of ways, and it is important to know the extent to which natural populations can adapt to anthropogenic climate change by natural selection. Parallel microevolution within separate species can demonstrate natural selection, but several studies of homoplasy have not yet revealed examples of widespread parallel evolution in a generic radiation. Taking into account primary phylogeographic divisions, we investigate numerous quantitative traits (size, shape, scalation, colour pattern and hue) in anole radiations from the mountainous Lesser Antillean islands. Adaptation to climatic differences can lead to very pronounced differences between spatially close populations with all studied traits showing some evidence of parallel evolution. Traits from shape, scalation, pattern and hue (particularly the latter) show widespread evolutionary parallels within these species in response to altitudinal climate variation greater than extreme anthropogenic climate change predicted for 2080. This gives strong evidence of the ability to adapt to climate variation by natural selection throughout this radiation. As anoles can evolve very rapidly, it suggests anthropogenic climate change is likely to be less of a conservation threat than other factors, such as habitat loss and invasive species, in this, Lesser Antillean, biodiversity hot spot.

Convergence of multiple markers and analysis methods defines the genetic distinctiveness of cryptic pitvipers.

Using multiple markers and multiple analytical approaches is critical for establishing species boundaries reliably, especially so in the case of cryptic species. Despite development of new and powerful analytical methods, most studies continue to adopt a few, with the choice often being subjective. One such example is routine analysis of Amplified Fragment Length Polymorphism (AFLP) data using population genetic models despite disparity between method assumptions and data properties. The application of newly developed methods for analyzing this dominant marker may not be entirely clear in the context of species delimitation. In this study, we use AFLPs and mtDNA to investigate cryptic speciation in the Trimeresurus macrops complex that belongs to a taxonomically difficult lineage of Asian pitvipers. We analyze AFLPs using population genetic, phylogenetic, multivariate statistical, and Bayes Factor Delimitation methods. A gene tree from three mtDNA markers provided additional evidence. Our results show that the inferences about species boundaries that can be derived from population genetic analysis of AFLPs have certain limitations. In contrast, four multivariate statistical analyses produced clear clusters that are consistent with each other, as well as with Bayes Factor Delimitation results, and with mtDNA and total evidence phylogenies. Furthermore, our results concur with allopatric distributions and patterns of variation in individual morphological characters previously identified in the three proposed species: T. macrops sensu stricto, T. cardamomensis, and T. rubeus. Our study provides evidence for reproductive isolation and genetic distinctiveness that define these taxa as full species. In addition, we re-emphasize the importance of examining congruence of results from multiple methods of AFLP analysis for inferring species diversity.

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.

Genetic tests for ecological and allopatric speciation in anoles on an island archipelago.

From Darwin's study of the Galapagos and Wallace's study of Indonesia, islands have played an important role in evolutionary investigations, and radiations within archipelagos are readily interpreted as supporting the conventional view of allopatric speciation. Even during the ongoing paradigm shift towards other modes of speciation, island radiations, such as the Lesser Antillean anoles, are thought to exemplify this process. Geological and molecular phylogenetic evidence show that, in this archipelago, Martinique anoles provide several examples of secondary contact of island species. Four precursor island species, with up to 8 mybp divergence, met when their islands coalesced to form the current island of Martinique. Moreover, adjacent anole populations also show marked adaptation to distinct habitat zonation, allowing both allopatric and ecological speciation to be tested in this system. We take advantage of this opportunity of replicated island coalescence and independent ecological adaptation to carry out an extensive population genetic study of hypervariable neutral nuclear markers to show that even after these very substantial periods of spatial isolation these putative allospecies show less reproductive isolation than conspecific populations in adjacent habitats in all three cases of subsequent island coalescence. The degree of genetic interchange shows that while there is always a significant genetic signature of past allopatry, and this may be quite strong if the selection regime allows, there is no case of complete allopatric speciation, in spite of the strong primae facie case for it. Importantly there is greater genetic isolation across the xeric/rainforest ecotone than is associated with any secondary contact. This rejects the development of reproductive isolation in allopatric divergence, but supports the potential for ecological speciation, even though full speciation has not been achieved in this case. It also explains the paucity of anole species in the Lesser Antilles compared to the Greater Antilles.

Quantitative traits and mode of speciation in Martinique anoles.

We investigate extensive quantitative trait variation (dewlap hue, colour pattern, dorsum hue, body proportions and scalation) in the Martinique anole across eight transects representing nascent parapatric ecological speciation, nascent allopatric speciation and allopatric divergence without sufficient genetic structure to suggest speciation. Quantitative trait divergence can be extremely large between adjacent sets of populations, but with one exception that this is associated with difference in habitat rather than past allopatry. Nascent ecological speciation shows the greatest level of quantitative trait divergence across all character sets including those implicated in natural, as well as sexual selection. The sole example of nascent allopatric speciation is associated with fairly strong quantitative trait divergence among most character sets, but not the set most implicated in natural (rather than sexual) selection. The role of sexual selection in ecological speciation is discussed, both in terms of female choice with assortative mating and male-male competition with condition-dependant sexual signals.

Phylogenetic structure and species boundaries in the mountain pitviper Ovophis monticola (Serpentes: Viperidae: Crotalinae) in Asia.

We investigated phylogenetic structure and morphological variation in Asian mountain pitvipers of the genus Ovophis (comprising 3-4 species some of which are considered polytypic) by sequencing four mitochondrial markers (cytochrome b, NADH dehydrogenase subunit 4, 12S and 16S rRNA) from 72 specimens, and analysed them in a Bayesian framework together with another 26 sequences from closely related genera. We reconstructed the region of origin and direction of dispersal of the major clades, and of Ovophis as a whole, using likelihood framework analysis. We also defined morphogroups from 280 specimens from across the range of Ovophis to allow the geographic extent of the major clades to be determined, as well as to allow inclusion of specimens lacking sequence data. Phylogenetic analyses confirmed the monophyly of Ovophis as currently defined, and revealed that it contains two major lineages, eastern (mainly Chinese) and western, with both occurring in southwestern China, central and northern Viet Nam. The most likely origin of the genus, and of individual lineages, coincides with the northeastern boundary of the Indomalayan hotspot. Major diversification in this species group likely corresponded to major climatic changes arising from the uplift of the Tibetan Plateau in the early to mid Miocene. With reference to the defined morphogroups, we suggest that at least five species are present and provide appropriate names. With a few exceptions, the newly defined species boundaries do not correspond to the existing taxonomy.

Inclusion of nuclear intron sequence data helps to identify the Asian sister group of New World pitvipers.

Despite much effort towards resolving the molecular phylogenetic tree for pitvipers, some aspects remain unresolved. In particular, the sister group of the diverse New World radiation has remained impossible to identify with any certainty. In this study, which for the first time includes nuclear intron data from all major groups of Asian pitvipers as well as representatives of the New World radiation, Bayesian inference allows Gloydius to be identified as the most likely sister group to the New World radiation and sheds light on other ambiguous relationships among the Old World pitvipers. The sister group relationship of "Ovophis"okinavensis and "Trimeresurus"gracilis is confirmed by the addition of nuclear genes, and we hypothesise that they form a sister group to the Gloydius+New World clade, best supported when the phylogenetic signal from gaps is included in the form of a simple-coded matrix.

Microsatellite data show evidence for male-biased dispersal in the Caribbean lizard Anolis roquet.

Dispersal is a key component of an organism's life history and differences in dispersal between sexes appear to be widespread in vertebrates. However, most predictions of sex-biased dispersal have been based on observations of social structure in birds and mammals and more data are needed on other taxa to test whether these predictions apply in other organisms. Caribbean anole lizards are important model organisms in various biological disciplines, including evolutionary biology. However, very little is known about their dispersal strategies despite the importance of dispersal for population structure and dynamics. Here we use nine microsatellite markers to assess signatures of sex-biased dispersal on two spatial sampling scales in Anolis roquet, an anole endemic to the island of Martinique. Significantly higher gene diversity (H(S)) and lower mean assignment value (mAIC) was found in males on the larger spatial sampling scale. Significant heterozygote deficit (F(IS)), lower population differentiation (F(ST)), mAIC and variance of assignment index (vAIC) was found in males on the smaller spatial scale. The observation of male biased dispersal conform with expectations based on the polygynous mating system of Anolis roquet, and contributes to an explanation of the contrasting patterns of genetic structure between maternal and biparental markers that have been reported previously in this, and other anoline, species.

The roles of allopatric divergence and natural selection in quantitative trait variation across a secondary contact zone in the lizard Anolis roquet.

Populations of the Caribbean lizard, Anolis roquet, are thought to have experienced long periods of allopatry before recent secondary contact. To elucidate the effects of past allopatry on population divergence in A. roquet, we surveyed parallel transects across a secondary contact zone in northeastern Martinique. We used diagnostic molecular mitochondrial DNA markers to test fine-scale association of mitochondrial DNA lineage and geological region, multivariate statistical techniques to explore quantitative trait pattern, and cline fitting techniques to model trait variation across the zone of secondary contact. We found that lineages were strongly associated with geological regions along both transects, but quantitative trait patterns were remarkably different. Patterns of morphological and mitochondrial DNA variation were consistent with a strong barrier to gene flow on the coast, whereas there were no indications of barriers to gene flow in the transitional forest. Hence, the coastal populations behaved as would be predicted by an allopatric model of divergence in this complex, while those in the transitional forest did not, despite the close proximity of the transects and their shared geological history. Patterns of geographical variation in this species complex, together with environmental data, suggest that on balance, selection regimes on either side of the secondary contact zone in the transitional forest may be more convergent, while those either side of the secondary contact zone on the coast are more divergent. Hence, the evolutionary consequences of allopatry may be strongly influenced by local natural selection regimes.

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.

Common garden and natural selection experiments support ecotypic differentiation in the Dominican anole (Anolis oculatus).

The theory behind ecotypic differentiation and ecological speciation assumes a predominant role for natural selection working on characteristics with genetic variance, but experimental support for these assumptions is limited. Lesser Antillean anoles show marked ecotypic variation within islands and the potential for ecological speciation. Common garden rearing experiments on the Dominican anole (Anolis oculatus) suggest that the characters showing geographic variation have genetic variance and are not primarily determined by environmental plasticity. Replicated natural selection experiments using large-scale enclosures show that translocated montane samples experience significant (multivariate) directional selection in both wet and dry seasons in both males and females. The targets of selection appear to be spread among the various character systems. An experiment on 12 geographically segregated populations along a coastal xeric-montane rainforest gradient (four replicate enclosures) clearly showed that the magnitude of the directional selection intensity is positively related to the position along this gradient. The results of the common garden and natural selection experiments support the interpretation that the geographic differentiation is primarily driven by natural selection and are compatible with the potential for ecological speciation in this system.

The dynamics of genetic and morphological variation on volcanic islands.

Oceanic archipelagos of volcanic origin have been important in the study of evolution because they provide repeated natural experiments allowing rigorous tests of evolutionary hypotheses. Ongoing volcanism on these islands may, however, affect the evolutionary diversification of species. Analysis of population structure and phylogeographic patterns in island populations can provide insight into evolutionary dynamics on volcanic islands. We analysed genetic and morphological variation in the gecko Tarentola boettgeri on the island of Gran Canaria and compared it with Tarentola delalandii on Tenerife, a neighbouring volcanic island of similar age but distinctly different geological past. Intraspecific divergence of mitochondrial haplotypes indicates long-term persistence of Tarentola on each island, with a phylogeographic signal left by older volcanic events. More recent volcanic eruptions (approximately 0.2 million years ago on Tenerife, approximately 2.2 million years ago on Gran Canaria) have left a signature of population expansion in the population genetic structure, the strength of which depends on the time since the last major volcanic eruption on each island. While these stochastic events have left traces in morphological variation in Tenerife, in Gran Canaria geographical variation was solely associated with environmental variables. This suggests that historically caused patterns in morphology may be overwritten by natural selection within 2 million years.

Targeting optimal introns for phylogenetic analyses in non-model taxa: experimental results in Asian pitvipers.

Nuclear introns are increasingly used as phylogenetic markers. Here, we present a multidisciplinary approach towards optimal locus selection and amplification using Asian pitvipers as an example of a non-model taxon, and raise the profile of length variant heterozygotes (LVHs) in intron loci. Taxon-specific primers were identified using a bioinformatic approach, and also designed from existing exon primed, intron crossing (EPIC) primer amplifications. Eleven further universal EPIC primer pairs were assayed using a range of PCR optimization strategies. Taxon-specific primers yielded the most consistent amplifications, but assaying a large number of universal EPIC primers yielded another appropriate locus for phylogenetic purposes. Modified Taq DNA polymerases such as JumpStart™Taq either significantly improved the specificity and yield of EPIC PCR amplifications (of low copy number nuclear targets), or resulted in amplifications that were not significantly worse than those derived from a generic Taq DNA polymerase. Finally, LVHs were detected in all loci that were sequenced suggesting that they are relatively common in introns. This study provides an efficient and cost effective template for the successful identification of intron markers for molecular systematics which is universally applicable to other non-model taxon groups.

The importance of being genomic: Non-coding and coding sequences suggest different models of toxin multi-gene family evolution.

Studies of multi-gene protein families, including many toxins, are crucial for understanding the role of gene duplication in generating protein diversity in general. However, many evolutionary analyses of gene families are based on coding sequences, and do not take into account many potentially confounding evolutionary factors, such as recombination and convergence due to selection. We illustrate this using snake venom gene sequences from the Phospholipase A2 (PLA2) subfamily. Novel gene sequences from 20 species of understudied Asian pitvipers were analyzed alongside available genomic PLA2 sequences from another four crotaline and several viperine species. In contrast to previous analyses of this toxin family based on cDNA sequences, we find that duplication events are concentrated at the tips of the tree, suggesting that major functions such as presynaptic neurotoxicity have evolved convergently multiple times in pitvipers. We provide evidence that this discrepancy is due to differing evolutionary patterns between introns and exons. The effects of several well-known sources of bias on the phylogeny were small, compared to the effect of analyses based on different partitions of the gene (whole gene sequence, non-coding regions, cDNA sequence). Switches of function were found to be largely associated with strong selection, and with duplication events. Use of coding sequences for phylogeny estimation potentially produces incorrect inferences about the action of selection on individual lineages and sites. Our results have major implications for phylogenomic methods of functional inference as well as for our understanding of the evolution of multigene families.

Offshore insular variation in the diet of the Taiwanese bamboo viper Trimeresurus stejnegeri (Schmidt).

Dietary data were ascertained for 229 T. stejnegeri (snout vent length >300mm) from 36 localities throughout the main island of Taiwan and the outlying Orchid (Lanyu) and Green (Ludau) Islands. Twenty nine percent of the snakes were devoid of any prey, and of the snakes containing prey, 43% of the cases were unidentifiable. This relatively large proportion of unidentifiable prey items (observed in the hindgut) may reflect either rapid digestion of amphibian prey and/or rapid venting of feces as an evolutionary adaptation to arboreal life. Trimeresurus stejnegeri appears euryphagous, taking primarily amphibians, but additionally reptilian, mammalian and insect prey. There was no discrepancy in prey composition based on comparisons of where the prey item was recorded in the digestive tract. No sexual variation in diet composition was evident, although males were more likely to contain prey than females, indicating the utilisation of different foraging strategies on similar sympatric prey items. Variation in diet composition was observed between mainland Taiwan and offshore islands, which is most likely the result of differences in prey availability.

Predicting function from sequence in a large multifunctional toxin family.

Venoms contain active substances with highly specific physiological effects and are increasingly being used as sources of novel diagnostic, research and treatment tools for human disease. Experimental characterisation of individual toxin activities is a severe rate-limiting step in the discovery process, and in-silico tools which allow function to be predicted from sequence information are essential. Toxins are typically members of large multifunctional families of structurally similar proteins that can have different biological activities, and minor sequence divergence can have significant consequences. Thus, existing predictive tools tend to have low accuracy. We investigated a classification model based on physico-chemical attributes that can easily be calculated from amino-acid sequences, using over 250 (mostly novel) viperid phospholipase A2 toxins. We also clustered proteins by sequence profiles, and carried out in-vitro tests for four major activities on a selection of isolated novel toxins, or crude venoms known to contain them. The majority of detected activities were consistent with predictions, in contrast to poor performance of a number of tested existing predictive methods. Our results provide a framework for comparison of active sites among different functional sub-groups of toxins that will allow a more targeted approach for identification of potential drug leads in the future.

Estimating genetic variability in non-model taxa: a general procedure for discriminating sequence errors from actual variation.

Genetic variation is the driving force of evolution and as such is of central interest for biologists. However, inadequate discrimination of errors from true genetic variation could lead to incorrect estimates of gene copy number, population genetic parameters, phylogenetic relationships and the deposition of gene and protein sequences in databases that are not actually present in any organism. Misincorporation errors in multi-template PCR cloning methods, still commonly used for obtaining novel gene sequences in non-model species, are difficult to detect, as no previous information may be available about the number of expected copies of genes belonging to multi-gene families. However, studies employing these techniques rarely describe in any great detail how errors arising in the amplification process were detected and accounted for. Here, we estimated the rate of base misincorporation of a widely-used PCR-cloning method, using a single copy mitochondrial gene from a single individual to minimise variation in the template DNA, as 1.62×10(-3) errors per site, or 9.26×10(-5) per site per duplication. The distribution of errors among sequences closely matched that predicted by a binomial distribution function. The empirically estimated error rate was applied to data, obtained using the same methods, from the Phospholipase A(2) toxin family from the pitviper Ovophis monticola. The distribution of differences detected closely matched the expected distribution of errors and we conclude that, when undertaking gene discovery or assessment of genetic diversity using this error-prone method, it will be informative to empirically determine the rate of base misincorporation.

Genetic data from 28 STR loci for forensic individual identification and parentage analyses in 6 bird of prey species.

Twenty-eight STR loci were screened in wild populations of six bird of prey species providing allele frequencies and population genetic parameters necessary for the application of STRs in wildlife forensic genetic casework. Individual STR loci were validated according to forensic recommendations in specimens of golden eagle (Aquila chrysaetos), goshawk (Accipiter gentilis), merlin (Falco columbarius), peregrine falcon (Falco peregrinus), gyr falcon (Falco rusticolus) and saker falcon (Falco cherrug). Deviations from Hardy-Weinberg expectations and linkage disequilibrium between locus pairs were examined. The average probability of identity (PI(ave)) and power of exclusion (PE) suggest the profiling systems of golden eagle, goshawk, merlin and peregrine falcons are capable of providing robust and highly discriminatory forensic evidence for legal proceedings. Due to low sample numbers the allele frequency data for gyr and saker falcons is not currently capable of providing an effective probability of identity. Further work should focus on increasing the size of these data sets.