Comparative genomics of Japanese encephalitis virus shows low rates of recombination and a small subset of codon positions under episodic diversifying selection.

Orthoflavivirus japonicum (JEV) is the dominant cause of viral encephalitis in the Asian region with 100,000 cases and 25,000 deaths reported annually. The genome is comprised of a single polyprotein that encodes three structural and seven non-structural proteins. We collated a dataset of 349 complete genomes from a number of public databases, and analysed the data for recombination, evolutionary selection and phylogenetic structure. There are low rates of recombination in JEV, subsequently recombination is not a major evolutionary force shaping JEV. We found a strong overall signal of purifying selection in the genome, which is the main force affecting the evolutionary dynamics in JEV. There are also a small number of genomic sites under episodic diversifying selection, especially in the envelope protein and non-structural proteins 3 and 5. Overall, these results support previous analyses of JEV evolutionary genomics and provide additional insight into the evolutionary processes shaping the distribution and adaptation of this important pathogenic arbovirus.

Environmental correlates of phenotypic evolution in ecologically diverse Liolaemus lizards.

Evolutionary correlations between phenotypic and environmental traits characterize adaptive radiations. However, the lizard genus Liolaemus, one of the most ecologically diverse terrestrial vertebrate radiations on earth, has so far shown limited or mixed evidence of adaptive diversification in phenotype. Restricted use of comprehensive environmental data, incomplete taxonomic representation and not considering phylogenetic uncertainty may have led to contradictory evidence. We compiled a 26-taxon dataset for the Liolaemus gracilis species group, representing much of the ecological diversity represented within Liolaemus and used environmental data to characterize how environments occupied by species' relate to phenotypic evolution. Our analyses, explicitly accounting for phylogenetic uncertainty, suggest diversification in phenotypic traits toward the present, with body shape evolution rapidly evolving in this group. Body shape evolution correlates with the occupation of different structural habitats indicated by vegetation axes suggesting species have adapted for maximal locomotory performance in these habitats. Our results also imply that the effects of phylogenetic uncertainty and model misspecification may be more extensive on univariate, relative to multivariate analyses of evolutionary correlations, which is an important consideration in analyzing data from rapidly radiating adaptive radiations.

A new lineage of Galapagos giant tortoises identified from museum samples.

The Galapagos Archipelago is recognized as a natural laboratory for studying evolutionary processes. San Cristóbal was one of the first islands colonized by tortoises, which radiated from there across the archipelago to inhabit 10 islands. Here, we sequenced the mitochondrial control region from six historical giant tortoises from San Cristóbal (five long deceased individuals found in a cave and one found alive during an expedition in 1906) and discovered that the five from the cave are from a clade that is distinct among known Galapagos giant tortoises but closely related to the species from Española and Pinta Islands. The haplotype of the individual collected alive in 1906 is in the same clade as the haplotype in the contemporary population. To search for traces of a second lineage in the contemporary population on San Cristóbal, we closely examined the population by sequencing the mitochondrial control region for 129 individuals and genotyping 70 of these for both 21 microsatellite loci and >12,000 genome-wide single nucleotide polymorphisms [SNPs]. Only a single mitochondrial haplotype was found, with no evidence to suggest substructure based on the nuclear markers. Given the geographic and temporal proximity of the two deeply divergent mitochondrial lineages in the historical samples, they were likely sympatric, raising the possibility that the lineages coexisted. Without the museum samples, this important discovery of an additional lineage of Galapagos giant tortoise would not have been possible, underscoring the value of such collections and providing insights into the early evolution of this iconic radiation.

A return-on-investment approach for prioritization of rigorous taxonomic research needed to inform responses to the biodiversity crisis.

Global biodiversity loss is a profound consequence of human activity. Disturbingly, biodiversity loss is greater than realized because of the unknown number of undocumented species. Conservation fundamentally relies on taxonomic recognition of species, but only a fraction of biodiversity is described. Here, we provide a new quantitative approach for prioritizing rigorous taxonomic research for conservation. We implement this approach in a highly diverse vertebrate group-Australian lizards and snakes. Of 870 species assessed, we identified 282 (32.4%) with taxonomic uncertainty, of which 17.6% likely comprise undescribed species of conservation concern. We identify 24 species in need of immediate taxonomic attention to facilitate conservation. Using a broadly applicable return-on-investment framework, we demonstrate the importance of prioritizing the fundamental work of identifying species before they are lost.

Color Polymorphism is a Driver of Diversification in the Lizard Family Lacertidae.

Color polymorphism-two or more heritable color phenotypes maintained within a single breeding population-is an extreme type of intraspecific diversity widespread across the tree of life. Color polymorphism is hypothesized to be an engine for speciation, where morph loss or divergence between distinct color morphs within a species results in the rapid evolution of new lineages, and thus, color polymorphic lineages are expected to display elevated diversification rates. Multiple species in the lizard family Lacertidae are color polymorphic, making them an ideal group to investigate the evolutionary history of this trait and its influence on macroevolution. Here, we produce a comprehensive species-level phylogeny of the lizard family Lacertidae to reconstruct the evolutionary history of color polymorphism and test if color polymorphism has been a driver of diversification. Accounting for phylogenetic uncertainty with multiple phylogenies and simulation studies, we estimate an ancient origin of color polymorphism (111 Ma) within the Lacertini tribe (subfamily Lacertinae). Color polymorphism most likely evolved few times in the Lacertidae and has been lost at a much faster rate than gained. Evolutionary transitions to color polymorphism are associated with shifts in increased net diversification rate in this family of lizards. Taken together, our empirical results support long-standing theoretical expectations that color polymorphism is a driver of diversification.[Color polymorphism; Lacertidae; state-dependent speciation extinction models; trait-dependent diversification.].

Environmental drivers of sexual dimorphism in a lizard with alternative mating strategies.

Understanding the relative importance of sexual and natural selection in shaping morphological traits is a long-standing goal of evolutionary ecology. Male-biased sexual size dimorphism (SSD) is typically associated with male-male competition. Similarly, male polymorphisms are considered a consequence of competitive social interactions. This classic paradigm overlooks the fact that environmental factors mediate social interactions and can lead to ecological adaptations. Common side-blotched lizards, Uta stansburiana, are a model system for this paradigm due to well-known rock-paper-scissors social dynamics between male morphs. SSD in this species has been considered primarily a consequence of social interactions, with male size resulting from the number of morphs in each population and female size being constrained through fecundity benefits. We test if the environment explains intraspecific variation in SSD and number of male morphs in U. stansburiana. By compiling data from 49 populations, we show that environmental variables are stronger predictors of SSD than the number of male morphs. Similarly, we show that the environment mediates SSD and potentially contributes to morph loss in colder environments. We propose that the environment favours smaller males in areas of high seasonality. Our results demonstrate the importance of the environment as a mediator of SSD.

Trait differences among discrete morphs of a color polymorphic lizard, Podarcis erhardii.

Color polymorphism defies evolutionary expectations as striking phenotypic variation is maintained within a single species. Color and other traits mediate social interactions, and stable polymorphism within a population is hypothesized to be related to correlational selection of other phenotypic traits among color morphs. Here, we report on a previously unknown throat color polymorphism in the Aegean Wall Lizard (Podarcis erhardii) and examine morph-correlated differences in traits important to social behavior and communication: maximum bite force capacity and chemical signal profile. We find that both sexes of P. erhardii have three color morphs: orange, yellow, and white. Moreover, orange males are significantly larger and tend to bite harder than yellow and white males. Although the established color polymorphism only partially matches the observed intraspecific variation in chemical signal signatures, the chemical profile of the secretions of orange males is significantly divergent from that of white males. Our findings suggest that morph colors are related to differences in traits that are crucial for social interactions and competitive ability, illustrating the need to look beyond color when studying polymorphism evolution.

Giant tortoise genomes provide insights into longevity and age-related disease.

Giant tortoises are among the longest-lived vertebrate animals and, as such, provide an excellent model to study traits like longevity and age-related diseases. However, genomic and molecular evolutionary information on giant tortoises is scarce. Here, we describe a global analysis of the genomes of Lonesome George-the iconic last member of Chelonoidis abingdonii-and the Aldabra giant tortoise (Aldabrachelys gigantea). Comparison of these genomes with those of related species, using both unsupervised and supervised analyses, led us to detect lineage-specific variants affecting DNA repair genes, inflammatory mediators and genes related to cancer development. Our study also hints at specific evolutionary strategies linked to increased lifespan, and expands our understanding of the genomic determinants of ageing. These new genome sequences also provide important resources to help the efforts for restoration of giant tortoise populations.

Population genomics through time provides insights into the consequences of decline and rapid demographic recovery through head-starting in a Galapagos giant tortoise.

Population genetic theory related to the consequences of rapid population decline is well-developed, but there are very few empirical studies where sampling was conducted before and after a known bottleneck event. Such knowledge is of particular importance for species restoration, given links between genetic diversity and the probability of long-term persistence. To directly evaluate the relationship between current genetic diversity and past demographic events, we collected genome-wide single nucleotide polymorphism data from prebottleneck historical (c.1906) and postbottleneck contemporary (c.2014) samples of Pinzón giant tortoises (Chelonoidis duncanensis; n = 25 and 149 individuals, respectively) endemic to a single island in the Galapagos. Pinzón giant tortoises had a historically large population size that was reduced to just 150-200 individuals in the mid 20th century. Since then, Pinzón's tortoise population has recovered through an ex situ head-start programme in which eggs or pre-emergent individuals were collected from natural nests on the island, reared ex situ in captivity until they were 4-5 years old and subsequently repatriated. We found that the extent and distribution of genetic variation in the historical and contemporary samples were very similar, with the latter group not exhibiting the characteristic genetic patterns of recent population decline. No population structure was detected either spatially or temporally. We estimated an effective population size (N e) of 58 (95% CI = 50-69) for the postbottleneck population; no prebottleneck N e point estimate was attainable (95% CI = 39-infinity) likely due to the sample size being lower than the true N e. Overall, the historical sample provided a valuable benchmark for evaluating the head-start captive breeding programme, revealing high retention of genetic variation and no skew in representation despite the documented bottleneck event. Moreover, this work demonstrates the effectiveness of head-starting in rescuing the Pinzón giant tortoise from almost certain extinction.

Theory, practice, and conservation in the age of genomics: The Galápagos giant tortoise as a case study.

High-throughput DNA sequencing allows efficient discovery of thousands of single nucleotide polymorphisms (SNPs) in nonmodel species. Population genetic theory predicts that this large number of independent markers should provide detailed insights into population structure, even when only a few individuals are sampled. Still, sampling design can have a strong impact on such inferences. Here, we use simulations and empirical SNP data to investigate the impacts of sampling design on estimating genetic differentiation among populations that represent three species of Galápagos giant tortoises (Chelonoidis spp.). Though microsatellite and mitochondrial DNA analyses have supported the distinctiveness of these species, a recent study called into question how well these markers matched with data from genomic SNPs, thereby questioning decades of studies in nonmodel organisms. Using >20,000 genomewide SNPs from 30 individuals from three Galápagos giant tortoise species, we find distinct structure that matches the relationships described by the traditional genetic markers. Furthermore, we confirm that accurate estimates of genetic differentiation in highly structured natural populations can be obtained using thousands of SNPs and 2-5 individuals, or hundreds of SNPs and 10 individuals, but only if the units of analysis are delineated in a way that is consistent with evolutionary history. We show that the lack of structure in the recent SNP-based study was likely due to unnatural grouping of individuals and erroneous genotype filtering. Our study demonstrates that genomic data enable patterns of genetic differentiation among populations to be elucidated even with few samples per population, and underscores the importance of sampling design. These results have specific implications for studies of population structure in endangered species and subsequent management decisions.

Genome-Wide Assessment of Diversity and Divergence Among Extant Galapagos Giant Tortoise Species.

Genome-wide assessments allow for fuller characterization of genetic diversity, finer-scale population delineation, and better detection of demographically significant units to guide conservation compared with those based on "traditional" markers. Galapagos giant tortoises (Chelonoidis spp.) have long provided a case study for how evolutionary genetics may be applied to advance species conservation. Ongoing efforts to bolster tortoise populations, which have declined by 90%, have been informed by analyses of mitochondrial DNA sequence and microsatellite genotypic data, but could benefit from genome-wide markers. Taking this next step, we used double-digest restriction-site associated DNA sequencing to collect genotypic data at >26000 single nucleotide polymorphisms (SNPs) for 117 individuals representing all recognized extant Galapagos giant tortoise species. We then quantified genetic diversity, population structure, and compared results to estimates from mitochondrial DNA and microsatellite loci. Our analyses detected 12 genetic lineages concordant with the 11 named species as well as previously described structure within one species, C. becki. Furthermore, the SNPs provided increased resolution, detecting admixture in 4 individuals. SNP-based estimates of diversity and differentiation were significantly correlated with those derived from nuclear microsatellite loci and mitochondrial DNA sequences. The SNP toolkit presented here will serve as a resource for advancing efforts to understand tortoise evolution, species radiations, and aid conservation of the Galapagos tortoise species complex.

Temporal Mitogenomics of the Galapagos Giant Tortoise from Pinzón Reveals Potential Biases in Population Genetic Inference.

Empirical population genetic studies generally rely on sampling subsets of the population(s) of interest and of the nuclear or organellar genome targeted, assuming each is representative of the whole. Violations of these assumptions may impact population-level parameter estimation and lead to spurious inferences. Here, we used targeted capture to sequence the full mitochondrial genome from 123 individuals of the Galapagos giant tortoise endemic to Pinzón Island (Chelonoidis duncanensis) sampled at 2 time points pre- and postbottleneck (circa 1906 and 2014) to explicitly assess differences in diversity estimates and demographic reconstructions based on subsets of the mitochondrial genome versus the full sequences and to evaluate potential biases associated with diversity estimates and demographic reconstructions from postbottlenecked samples alone. Haplotypic diversities were equal between the temporal samples based on the full mitochondrial genome, but single gene estimates suggested either decreases or increases in diversity depending upon the region. Demographic reconstructions based on the full sequence were more similar between the temporal samples than those based on the control region alone, or a subset of 3 regions, where the trends in population size changes shifted in magnitude and direction between the temporal samples. In all cases, the estimated coalescent point was more distant for the historical than contemporary sample. In summary, our results empirically demonstrate the influence of sampling bias when interpreting population genetic patterns and punctuate the need for careful consideration of potentially conflicting evolutionary signal across the mitochondrial genome.

Author Correction: Identification of Genetically Important Individuals of the Rediscovered Floreana Galápagos Giant Tortoise (Chelonoidis elephantopus) Provides Founders for Species Restoration Program.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Identification of Genetically Important Individuals of the Rediscovered Floreana Galápagos Giant Tortoise (Chelonoidis elephantopus) Provide Founders for Species Restoration Program.

Species are being lost at an unprecedented rate due to human-driven environmental changes. The cases in which species declared extinct can be revived are rare. However, here we report that a remote volcano in the Galápagos Islands hosts many giant tortoises with high ancestry from a species previously declared as extinct: Chelonoidis elephantopus or the Floreana tortoise. Of 150 individuals with distinctive morphology sampled from the volcano, genetic analyses revealed that 65 had C. elephantopus ancestry and thirty-two were translocated from the volcano's slopes to a captive breeding center. A genetically informed captive breeding program now being initiated will, over the next decades, return C. elephantopus tortoises to Floreana Island to serve as engineers of the island's ecosystems. Ironically, it was the haphazard translocations by mariners killing tortoises for food centuries ago that created the unique opportunity to revive this "lost" species today.

Evolutionary and natural history of the turtle frog, Myobatrachus gouldii, a bizarre myobatrachid frog in the southwestern Australian biodiversity hotspot.

Southwest Australia (SWA) is a global biodiversity hotspot and a centre of diversity and endemism for the Australo-Papuan myobatrachid frogs. Myobatrachus gouldii (the turtle frog) has a highly derived morphology associated with its forward burrowing behaviour, largely subterranean habit, and unusual mode of reproduction. Its sister genera Metacrinia and Arenophryne have restricted distributions in Western Australia with significant phylogeographic structure, leading to the recent description of a new species in the latter. In contrast, Myobatrachus is distributed widely throughout SWA over multiple climatic zones, but little is known of its population structure, geographic variation in morphology, or reproduction. We generated molecular and morphological data to test for genetic and morphological variation, and to assess whether substrate specialisation in this species may have led to phylogeographic structuring similar to that of other plant and animal taxa in SWA. We assembled sequence data for one mitochondrial and four nuclear DNA loci (3628 base pairs) for 42 turtle frogs sampled throughout their range. Likelihood phylogenetic analyses revealed shallow phylogeographic structure in the mtDNA locus (up to 3.3% genetic distance) and little variation in three of the four nDNA loci. The mtDNA haplotype network suggests five geographically allopatric groups, with no shared haplotypes between regions. These geographic patterns are congruent with several other SWA species, with genetic groups restricted to major hydrological divisions, the Swan Coastal Plain, and the Darling Scarp. The geographically structured genetic groups showed no evidence of significant morphological differentiation (242 individuals), and there was little sexual size dimorphism, but subtle differences in reproductive traits suggest more opportunistic breeding in lower rainfall zones. Call data were compared to sister genera Metacrinia and Arenophryne and found to be highly conservative across the three genera. Like many taxa in SWA, topographic variation and Plio-Pleistocene arid fluctuations likely were historic drivers of diversification in M. gouldii.

Ecological Divergence, Adaptive Diversification, and the Evolution of Social Signaling Traits: An Empirical Study in Arid Australian Lizards.

Species diversification often results from divergent evolution of ecological or social signaling traits. Theoretically, a combination of the two may promote speciation, however, empirical examples studying how social signal and ecological divergence might be involved in diversification are rare in general and typically do not consider range overlap as a contributing factor. We show that ecologically distinct lineages within the Australian sand dragon species complex (including Ctenophorus maculatus, Ctenophorus fordi, and Ctenophorus femoralis) have diversified recently, diverging in ecologically relevant and social signaling phenotypic traits as arid habitats expanded and differentiated. Diversification has resulted in repeated and independent invasion of distinct habitat types, driving convergent evolution of similar phenotypes. Our results suggest that parapatry facilitates diversification in visual signals through reinforcement as a hybridization-avoidance mechanism. We show that particularly striking variation in visual social signaling traits is better explained by the extent of lineage parapatry relative to ecological or phylogenetic divergence, suggesting that these traits reinforce divergence among lineages initiated by ecologically adaptive evolution. This study provides a rare empirical example of a repeated, intricate relationship between ecological and social signal evolution during diversification driven by ecological divergence and the evolution of new habitats, thereby supporting emergent theories regarding the importance of both ecological and social trait evolution throughout speciation.

Description of a New Galapagos Giant Tortoise Species (Chelonoidis; Testudines: Testudinidae) from Cerro Fatal on Santa Cruz Island.

The taxonomy of giant Galapagos tortoises (Chelonoidis spp.) is currently based primarily on morphological characters and island of origin. Over the last decade, compelling genetic evidence has accumulated for multiple independent evolutionary lineages, spurring the need for taxonomic revision. On the island of Santa Cruz there is currently a single named species, C. porteri. Recent genetic and morphological studies have shown that, within this taxon, there are two evolutionarily and spatially distinct lineages on the western and eastern sectors of the island, known as the Reserva and Cerro Fatal populations, respectively. Analyses of DNA from natural populations and museum specimens, including the type specimen for C. porteri, confirm the genetic distinctiveness of these two lineages and support elevation of the Cerro Fatal tortoises to the rank of species. In this paper, we identify DNA characters that define this new species, and infer evolutionary relationships relative to other species of Galapagos tortoises.

Biogeography and speciation of terrestrial fauna in the south-western Australian biodiversity hotspot.

The south-western land division of Western Australia (SWWA), bordering the temperate Southern and Indian Oceans, is the only global biodiversity hotspot recognised in Australia. Renowned for its extraordinary diversity of endemic plants, and for some of the largest and most botanically significant temperate heathlands and woodlands on Earth, SWWA has long fascinated biogeographers. Its flat, highly weathered topography and the apparent absence of major geographic factors usually implicated in biotic diversification have challenged attempts to explain patterns of biogeography and mechanisms of speciation in the region. Botanical studies have always been central to understanding the biodiversity values of SWWA, although surprisingly few quantitative botanical analyses have allowed for an understanding of historical biogeographic processes in both space and time. Faunistic studies, by contrast, have played little or no role in defining hotspot concepts, despite several decades of accumulating quantitative research on the phylogeny and phylogeography of multiple lineages. In this review we critically analyse datasets with explicit supporting phylogenetic data and estimates of the time since divergence for all available elements of the terrestrial fauna, and compare these datasets to those available for plants. In situ speciation has played more of a role in shaping the south-western Australian fauna than has long been supposed, and has occurred in numerous endemic lineages of freshwater fish, frogs, reptiles, snails and less-vagile arthropods. By contrast, relatively low levels of endemism are found in birds, mammals and highly dispersive insects, and in situ speciation has played a negligible role in generating local endemism in birds and mammals. Quantitative studies provide evidence for at least four mechanisms driving patterns of endemism in south-western Australian animals, including: (i) relictualism of ancient Gondwanan or Pangaean taxa in the High Rainfall Province; (ii) vicariant isolation of lineages west of the Nullarbor divide; (iii) in situ speciation; and (iv) recent population subdivision. From dated quantitative studies we derive four testable models of historical biogeography for animal taxa in SWWA, each explicit in providing a spatial, temporal and topological perspective on patterns of speciation or divergence. For each model we also propose candidate lineages that may be worthy of further study, given what we know of their taxonomy, distributions or relationships. These models formalise four of the strongest patterns seen in many animal taxa from SWWA, although other models are clearly required to explain particular, idiosyncratic patterns. Generating numerous new datasets for suites of co-occurring lineages in SWWA will help refine our understanding of the historical biogeography of the region, highlight gaps in our knowledge, and allow us to derive general postulates from quantitative (rather than qualitative) results. For animals, this process has now begun in earnest, as has the process of taxonomically documenting many of the more diverse invertebrate lineages. The latter remains central to any attempt to appreciate holistically biogeographic patterns and processes in SWWA, and molecular phylogenetic studies should - where possible - also lead to tangible taxonomic outcomes.

Lineage fusion in Galápagos giant tortoises.

Although many classic radiations on islands are thought to be the result of repeated lineage splitting, the role of past fusion is rarely known because during these events, purebreds are rapidly replaced by a swarm of admixed individuals. Here, we capture lineage fusion in action in a Galápagos giant tortoise species, Chelonoidis becki, from Wolf Volcano (Isabela Island). The long generation time of Galápagos tortoises and dense sampling (841 individuals) of genetic and demographic data were integral in detecting and characterizing this phenomenon. In C. becki, we identified two genetically distinct, morphologically cryptic lineages. Historical reconstructions show that they colonized Wolf Volcano from Santiago Island in two temporally separated events, the first estimated to have occurred ~199 000 years ago. Following arrival of the second wave of colonists, both lineages coexisted for approximately ~53 000 years. Within that time, they began fusing back together, as microsatellite data reveal widespread introgressive hybridization. Interestingly, greater mate selectivity seems to be exhibited by purebred females of one of the lineages. Forward-in-time simulations predict rapid extinction of the early arriving lineage. This study provides a rare example of reticulate evolution in action and underscores the power of population genetics for understanding the past, present and future consequences of evolutionary phenomena associated with lineage fusion.

Species detection and individual assignment in species delimitation: can integrative data increase efficacy?

Statistical species delimitation usually relies on singular data, primarily genetic, for detecting putative species and individual assignment to putative species. Given the variety of speciation mechanisms, singular data may not adequately represent the genetic, morphological and ecological diversity relevant to species delimitation. We describe a methodological framework combining multivariate and clustering techniques that uses genetic, morphological and ecological data to detect and assign individuals to putative species. Our approach recovers a similar number of species recognized using traditional, qualitative taxonomic approaches that are not detected when using purely genetic methods. Furthermore, our approach detects groupings that traditional, qualitative taxonomic approaches do not. This empirical test suggests that our approach to detecting and assigning individuals to putative species could be useful in species delimitation despite varying levels of differentiation across genetic, phenotypic and ecological axes. This work highlights a critical, and often overlooked, aspect of the process of statistical species delimitation-species detection and individual assignment. Irrespective of the species delimitation approach used, all downstream processing relies on how individuals are initially assigned, and the practices and statistical issues surrounding individual assignment warrant careful consideration.