Snake venom gene expression is coordinated by novel regulatory architecture and the integration of multiple co-opted vertebrate pathways.

Understanding how regulatory mechanisms evolve is critical for understanding the processes that give rise to novel phenotypes. Snake venom systems represent a valuable and tractable model for testing hypotheses related to the evolution of novel regulatory networks, yet the regulatory mechanisms underlying venom production remain poorly understood. Here, we use functional genomics approaches to investigate venom regulatory architecture in the prairie rattlesnake and identify cis-regulatory sequences (enhancers and promoters), trans-regulatory transcription factors, and integrated signaling cascades involved in the regulation of snake venom genes. We find evidence that two conserved vertebrate pathways, the extracellular signal-regulated kinase and unfolded protein response pathways, were co-opted to regulate snake venom. In one large venom gene family (snake venom serine proteases), this co-option was likely facilitated by the activity of transposable elements. Patterns of snake venom gene enhancer conservation, in some cases spanning 50 million yr of lineage divergence, highlight early origins and subsequent lineage-specific adaptations that have accompanied the evolution of venom regulatory architecture. We also identify features of chromatin structure involved in venom regulation, including topologically associated domains and CTCF loops that underscore the potential importance of novel chromatin structure to coevolve when duplicated genes evolve new regulatory control. Our findings provide a model for understanding how novel regulatory systems may evolve through a combination of genomic processes, including tandem duplication of genes and regulatory sequences, cis-regulatory sequence seeding by transposable elements, and diverse transcriptional regulatory proteins controlled by a co-opted regulatory cascade.

Patterns of genetic diversity in the polymorphic ground snake (Sonora semiannulata).

We evaluated the genetic diversity of a snake species with color polymorphism to understand the evolutionary processes that drive genetic structure across a large geographic region. Specifically, we analyzed genetic structure of the highly polymorphic ground snake, Sonora semiannulata, (1) among populations, (2) among color morphs (3) at regional and local spatial scales, using an amplified fragment length polymorphism dataset and multiple population genetic analyses, including FST-based and clustering analytical techniques. Based upon these methods, we found that there was moderate to low genetic structure among populations. However, this diversity was not associated with geographic locality at either spatial scale. Similarly, we found no evidence for genetic divergence among color morphs at either spatial scale. These results suggest that despite dramatic color polymorphism, this phenotypic diversity is not a major driver of genetic diversity within or among populations of ground snakes. We suggest that there are two mechanisms that could explain existing genetic diversity in ground snakes: recent range expansion from a genetically diverse founder population and current or recent gene flow among populations. Our findings have further implications for the types of color polymorphism that may generate genetic diversity in snakes.

Evolution of paedomorphosis in plethodontid salamanders: ecological correlates and re-evolution of metamorphosis.

Life-history modes can profoundly impact the biology of a species, and a classic example is the dichotomy between metamorphic (biphasic) and paedomorphic (permanently aquatic) life-history strategies in salamanders. However, despite centuries of research on this system, several basic questions about the evolution of paedomorphosis in salamanders have not been addressed. Here, we use a nearly comprehensive, time-calibrated phylogeny of spelerpine plethodontids to reconstruct the evolution of paedomorphosis and to test if paedomorphosis is (1) reversible; (2) associated with living in caves; (3) associated with relatively dry climatic conditions on the surface; and (4) correlated with limited range size and geographic dispersal. We find that paedomorphosis arose multiple times in spelerpines. We also find evidence for re-evolution of metamorphosis after several million years of paedomorphosis in a lineage of Eurycea from the Edwards Plateau region of Texas. We also show for the first time using phylogenetic comparative methods that paedomorphosis is highly correlated with cave-dwelling, arid surface environments, and small geographic range sizes, providing insights into both the causes and consequences of this major life history transition.

Biogeography, phylogeny, and morphological evolution of central Texas cave and spring salamanders.

BACKGROUND: Subterranean faunal radiations can result in complex patterns of morphological divergence involving both convergent or parallel phenotypic evolution and cryptic species diversity. Salamanders of the genus Eurycea in central Texas provide a particularly challenging example with respect to phylogeny reconstruction, biogeography and taxonomy. These predominantly aquatic species inhabit karst limestone aquifers and spring outflows, and exhibit a wide range of morphological and genetic variation. We extensively sampled spring and cave populations of six Eurycea species within this group (eastern Blepsimolge clade), to reconstruct their phylogenetic and biogeographic history using mtDNA and examine patterns and origins of cave- and surface-associated morphological variation. RESULTS: Genetic divergence is generally low, and many populations share ancestral haplotypes and/or show evidence of introgression. This pattern likely indicates a recent radiation coupled with a complex history of intermittent connections within the aquatic karst system. Cave populations that exhibit the most extreme troglobitic morphologies show no or very low divergence from surface populations and are geographically interspersed among them, suggesting multiple instances of rapid, parallel phenotypic evolution. Morphological variation is diffuse among cave populations; this is in contrast to surface populations, which form a tight cluster in morphospace. Unexpectedly, our analyses reveal two distinct and previously unrecognized morphological groups encompassing multiple species that are not correlated with spring or cave habitat, phylogeny or geography, and may be due to developmental plasticity. CONCLUSIONS: The evolutionary history of this group of spring- and cave-dwelling salamanders reflects patterns of intermittent isolation and gene flow influenced by complex hydrogeologic dynamics that are characteristic of karst regions. Shallow genetic divergences among several species, evidence of genetic exchange, and nested relationships across morphologically disparate cave and spring forms suggests that cave invasion was recent and many troglobitic morphologies arose independently. These patterns are consistent with an adaptive-shift hypothesis of divergence, which has been proposed to explain diversification in other karst fauna. While cave and surface forms often do not appear to be genetically isolated, morphological diversity within and among populations may be maintained by developmental plasticity, selection, or a combination thereof.

Sequence variation in the Mc1r gene for a group of polymorphic snakes.

Studying the genetic factors underlying phenotypic traits can provide insight into dynamics of selection and molecular basis of adaptation, but this goal can be difficult for non-model organisms without extensive genomic resources. However, sequencing candidate genes for the trait of interest can facilitate the study of evolutionary genetics in natural populations. We sequenced the melanocortin-1 receptor (Mc1r) to study the genetic basis of color polymorphism in a group of snake species with variable black banding, the genera Sonora, Chilomeniscus, and Chionactis. Mc1r is an important gene in the melanin synthesis pathway and is associated with ecologically important variation in color pattern in birds, mammals, and other squamate reptiles. We found that Mc1r nucleotide sequence was variable and that within our focal Sonora species, there are both fixed and heterozygous nucleotide substitutions that result in an amino acid change and selection analyses indicated that Mc1r sequence was likely under purifying selection. However, we did not detect any statistical association with the presence or absence of black bands. Our results agree with other studies that have found no role for sequence variation in Mc1r and highlight the importance of comparative data for studying the phenotypic associations of candidate genes.

Nuclear-mitochondrial discordance and gene flow in a recent radiation of toads.

Natural hybridization among recently diverged species has traditionally been viewed as a homogenizing force, but recent research has revealed a possible role for interspecific gene flow in facilitating species radiations. Natural hybridization can actually contribute to radiations by introducing novel genes or reshuffling existing genetic variation among diverging species. Species that have been affected by natural hybridization often demonstrate patterns of discordance between phylogenies generated using nuclear and mitochondrial markers. We used Amplified Fragment Length Polymorphism (AFLP) data in conjunction with mitochondrial DNA in order to examine patterns of gene flow and nuclear-mitochondrial discordance in the Anaxyrus americanus group, a recent radiation of North American toads. We found high levels of gene flow between putative species, particularly in species pairs sharing similar male advertisement calls that occur in close geographic proximity, suggesting that prezygotic reproductive isolating mechanisms and isolation by distance are the primary determinants of gene flow and genetic differentiation among these species. Additionally, phylogenies generated using AFLP and mitochondrial data were markedly discordant, likely due to recent and/or ongoing natural hybridization events between sympatric populations. Our results indicate that the putative species in the A. americanus group have experienced high levels of gene flow, and suggest that their North American radiation could have been facilitated by the introduction of beneficial genetic variation from admixture between divergent populations coming into secondary contact after glacial retreats.

Analyzing the relationship between sequence divergence and nodal support using Bayesian phylogenetic analyses.

Determining the appropriate gene for phylogeny reconstruction can be a difficult process. Rapidly evolving genes tend to resolve recent relationships, but suffer from alignment issues and increased homoplasy among distantly related species. Conversely, slowly evolving genes generally perform best for deeper relationships, but lack sufficient variation to resolve recent relationships. We determine the relationship between sequence divergence and Bayesian phylogenetic reconstruction ability using both natural and simulated datasets. The natural data are based on 28 well-supported relationships within the subphylum Vertebrata. Sequences of 12 genes were acquired and Bayesian analyses were used to determine phylogenetic support for correct relationships. Simulated datasets were designed to determine whether an optimal range of sequence divergence exists across extreme phylogenetic conditions. Across all genes we found that an optimal range of divergence for resolving the correct relationships does exist, although this level of divergence expectedly depends on the distance metric. Simulated datasets show that an optimal range of sequence divergence exists across diverse topologies and models of evolution. We determine that a simple to measure property of genetic sequences (genetic distance) is related to phylogenic reconstruction ability in Bayesian analyses. This information should be useful for selecting the most informative gene to resolve any relationships, especially those that are difficult to resolve, as well as minimizing both cost and confounding information during project design.

Phylogeographic analysis and environmental niche modeling of the plain-bellied watersnake (Nerodia erythrogaster) reveals low levels of genetic and ecological differentiation.

Species that exhibit geographically defined phenotypic variation traditionally have been divided into subspecies. Subspecies based on phenotypic features may not comprise monophyletic groups due to selection, gene flow, and/or convergent evolution. In many taxonomic groups the number of species once designated as widespread is dwindling rapidly, and many workers reject the concept of subspecies altogether. We tested whether currently recognized subspecies in the plain-bellied watersnake Nerodia erythrogaster are concordant with relationships based on mitochondrial markers, and whether it represents a single widespread species. The range of this taxon spans multiple potential biogeographic barriers (especially the Mississippi and Apalachicola Rivers) that correspond with lineage breaks in many species, including other snakes. We sequenced three mitochondrial genes (NADH-II, Cyt-b, Cox-I) from 156 geo-referenced specimens and developed ecological niche models using Maxent and spatially explicit climate data to examine historical and ecological factors affecting variation in N. erythrogaster across its range. Overall, we found little support for the recognized subspecies as either independent evolutionary lineages or geographically circumscribed units and conclude that although some genetic and niche differentiation has occurred, most populations assigned to N. erythrogaster appear to represent a single, widespread species. However, additional sampling and application of nuclear markers are necessary to clarify the status of the easternmost populations.

Evolution of the Schlafen genes, a gene family associated with embryonic lethality, meiotic drive, immune processes and orthopoxvirus virulence.

Genes of the Schlafen family, first discovered in mouse, are expressed in hematopoietic cells and are involved in immune processes. Previous results showed that they are candidate genes for two major phenomena: meiotic drive and embryonic lethality (DDK syndrome). However, these genes remain poorly understood, mostly due to the limitations imposed by their similarity, close location and the potential functional redundancy of the gene family members. Here we use genomic and phylogenetic studies to investigate the evolution and role of this family of genes. Our results show that the Schlafen family is widely distributed in mammals, where we recognize four major clades that experienced lineage-specific expansions or contractions in various orders, including primates and rodents. In addition, we identified members of the Schlafen family in Chondrichthyes and Amphibia, indicating an ancient origin of these genes. We find evidence that positive selection has acted on many Schlafen genes. Moreover, our analyses indicate that a member of the Schlafen family was horizontally transferred from murine rodents to orthopoxviruses, where it is hypothesized to play a role in allowing the virus to survive host immune defense mechanisms. The functional relevance of the viral Schlafen sequences is further underscored by our finding that they are evolving under purifying selection. This is of particular importance, since orthopoxviruses infect mammals and include variola, the causative agent of smallpox, and monkeypox, an emerging virus of great concern for human health.

Evolution of gigantism in amphiumid salamanders.

The Amphiumidae contains three species of elongate, permanently aquatic salamanders with four diminutive limbs that append one, two, or three toes. Two of the species, Amphiuma means and A. tridactylum, are among the largest salamanders in the world, reaching lengths of more than one meter, whereas the third species (A. pholeter), extinct amphiumids, and closely related salamander families are relatively small. Amphiuma means and A. tridactylum are widespread species and live in a wide range of lowland aquatic habitats on the Coastal Plain of the southeastern United States, whereas A. pholeter is restricted to very specialized organic muck habitats and is syntopic with A. means. Here we present analyses of sequences of mitochondrial and nuclear loci from across the distribution of the three taxa to assess lineage diversity, relationships, and relative timing of divergence in amphiumid salamanders. In addition we analyze the evolution of gigantism in the clade. Our analyses indicate three lineages that have diverged since the late Miocene, that correspond to the three currently recognized species, but the two gigantic species are not each other's closest relatives. Given that the most closely related salamander families and fossil amphiumids from the Upper Cretaceous and Paleocene are relatively small, our results suggest at least two extreme changes in body size within the Amphuimidae. Gigantic body size either evolved once as the ancestral condition of modern amphiumas, with a subsequent strong size reduction in A. pholeter, or gigantism independently evolved twice in the modern species, A. means and A. tridactylum. These patterns are concordant with differences in habitat breadth and range size among lineages, and have implications for reproductive isolation and diversification of amphiumid salamanders.

Vicariant origin of malagasy reptiles supports late cretaceous antarctic land bridge.

Since the acceptance of Wegener's theory of plate tectonics in the 1960s, continental drift vicariance has been proposed as an explanation for pan-Gondwanan faunal distributions. Given the recognition of historical connections among continents, it no longer was necessary to invoke hypotheses of dispersal across nearly insurmountable barriers. The application of continental drift vicariance theory to Gondwanan floral and faunal distributions provided reasonable explanations for such unusual distributions as that of the southern beech (Nothofagus) and chameleons. However, recent studies have demonstrated a significant, if not dominant, role for dispersal in the present-day distributions of these and numerous other "Gondwanan" taxa. The evolutionary histories of three Malagasy groups (boid snakes, podocnemid turtles, and iguanid lizards) commonly have been interpreted as reflecting vicariance because of continental drift associated with the breakup of Gondwana. Bayesian analyses of divergence ages suggest that this pattern is the result of vicariance coincident with the isolation of Madagascar in the Late Cretaceous (approximately 80 million years ago). This represents the first temporal evidence linking the vicariant origin of extant Malagasy vertebrates to a single geologic event. Specifically, our data provide strong, independently corroborated evidence for a contiguous Late Cretaceous Gondwana, exclusive of Africa and connected via Antarctica.

Dispersal and vicariance: the complex evolutionary history of boid snakes.

Since the early 1970s, boine snakes (Boidae: Boinae) have served as a prime example of a group whose current distribution was shaped by vicariant events associated with the fragmentation of the supercontinent Gondwana. Early phylogenetic treatments of this group, and what were thought to be closely related groups (Erycinae and Pythoninae) based on morphological features, produced a relatively stable view of relationships that has strongly influenced subsequent molecular-based work. We examined 4307 base pairs (bp) of nucleotide sequence data obtained from five nuclear loci (c-mos, NT3, BDNF, RAG1, and ODC) and one mitochondrial locus (cyt b) for all genera of erycines and boines, plus representatives of other groups, including those previously thought to be closely allied with boines (Ungaliophiidae, Loxocemidae, Xenopeltidae, and Pythoninae). Our results suggest that the Boidae is not monophyletic, and its current division into three subfamilies (Erycinae, Boinae, and Pythoninae) does not accurately reflect evolutionary history. We find that the evolutionary relationships are better reflected by current geographic distributions and tectonic history than by the morphological characters that have long served as the foundation of boid phylogeny. Divergence time estimates suggest that this strong congruence between geography and phylogeny is the result of several vicariant and dispersal events in the Late Cretaceous and Paleocene associated with the fragmentation of the Gondwanan supercontinent. Our results demonstrate the importance of both vicariance and dispersal in shaping the global distributions of terrestrial organisms.

Streambed microstructure predicts evolution of development and life history mode in the plethodontid salamander Eurycea tynerensis.

BACKGROUND: Habitat variation strongly influences the evolution of developmentally flexible traits, and may drive speciation and diversification. The plethodontid salamander Eurycea tynerensis is endemic to the geologically diverse Ozark Plateau of south-central North America, and comprises both strictly aquatic paedomorphic populations (achieving reproductive maturity while remaining in the larval form) and more terrestrial metamorphic populations. The switch between developmental modes has occurred many times, but populations typically exhibit a single life history mode. This unique system offers an opportunity to study the specific ecological circumstances under which alternate developmental and life history modes evolve. We use phylogenetic independent contrasts to test for relationships between a key microhabitat feature (streambed sediment) and this major life history polymorphism. RESULTS: We find streambed microstructure (sediment particle size, type and degree of sorting) to be highly correlated with life-history mode. Eurycea tynerensis is paedomorphic in streams containing large chert gravel, but metamorphoses in nearby streams containing poorly sorted, clastic material such as sandstone or siltstone. CONCLUSION: Deposits of large chert gravel create loosely associated streambeds, which provide access to subsurface water during dry summer months. Conversely, streambeds composed of more densely packed sandstone and siltstone sediments leave no subterranean refuge when surface water dries, presumably necessitating metamorphosis and use of terrestrial habitats. This represents a clear example of the relationship between microhabitat structure and evolution of a major developmental and life history trait, and has broad implications for the role of localized ecological conditions on larger-scale evolutionary processes.

Rapid diversification, incomplete isolation, and the "speciation clock" in North American salamanders (Genus Plethodon): testing the hybrid swarm hypothesis of rapid radiation.

The history of life has been marked by several spectacular radiations, in which many lineages arise over a short period of time. A possible consequence of such rapid splitting in the recent past is that the intrinsic barriers that prevent gene flow between many species may have too little time to develop fully, leading to extensive hybridization among recently evolved lineages. The salamander genus Plethodon in eastern North America has been proposed as a possible example of this scenario, but without explicit statistical tests. In this paper, we present a nearly comprehensive phylogeny for the 45 extant species of eastern Plethodon, based on DNA sequences of mitochondrial (two genes, 1335 base pairs) and nuclear genes (two genes, up to 3481 base pairs). We then use this phylogeny to examine rates and patterns of diversification and hybridization. We find significantly rapid diversification within the glutinosus species group. Examining patterns of natural hybridization in light of the phylogeny shows considerable hybridization within this clade, including introgression between species that are morphologically distinct and distantly related. Reproductive isolation increases over time and may be very weak among the most recently diverged species. These results suggest that the origin of species and the evolution of intrinsic reproductive isolating mechanisms, rather than being synonymous, may be decoupled in some cases (i.e., rapid origin of lineages outstrips the "speciation clock"). In contrast to the conclusions of a recent review of adaptive radiation and hybridization, we suggest that extensive hybridization sometimes may be a consequence, rather than a cause, of rapid diversification.

Phylogenetic evidence for a major reversal of life-history evolution in plethodontid salamanders.

The transition from aquatic to terrestrial eggs is a key evolutionary change that has allowed vertebrates to successfully colonize and exploit the land. Although most amphibians retain the primitive biphasic life cycle (eggs deposited in water that hatch into free-living aquatic larvae), direct development of terrestrial eggs has evolved repeatedly and may have been critical to the evolutionary success of several amphibian groups. We provide the first conclusive evidence for evolutionary reversal of direct development in vertebrates. The family Plethodontidae (lungless salamanders) contains the majority of salamander species, including major radiations of direct developers. We reconstruct the higher level phylogenetic relationships of plethodontid salamanders using molecular and morphological data and use this phylogeny to examine the evolution of direct development. We show that the predominantly biphasic desmognathines, previously considered the sister group of other plethodontids, are nested inside a group of direct-developing species (Plethodontini) and have re-evolved the aquatic larval stage. Rather than being an evolutionary dead end, the reversal from direct developing to biphasic life history may have helped communities in eastern North America to achieve the highest local diversity of salamander species in the world.

When are phylogenetic analyses misled by convergence? A case study in Texas cave salamanders.

Convergence, i.e., similarity between organisms that is not the direct result of shared phylogenetic history (and that may instead result from independent adaptations to similar environments), is a fundamental issue that lies at the interface of systematics and evolutionary biology. Although convergence is often cited as an important problem in morphological phylogenetics, there have been few well-documented examples of strongly supported and misleading phylogenetic estimates that result from adaptive convergence in morphology. In this article, we propose criteria that can be used to infer whether or not a phylogenetic analysis has been misled by convergence. We then apply these criteria in a study of central Texas cave salamanders (genus Eurycea). Morphological characters (apparently related to cave-dwelling habitat use) support a clade uniting the species E. rathbuni and E. tridentifera, whereas mitochondrial DNA sequences and allozyme data show that these two species are not closely related. We suggest that a likely explanation for the paucity of examples of strongly misleading morphological convergence is that the conditions under which adaptive convergence is most likely to produce strongly misleading results are limited. Specifically, convergence is most likely to be problematic in groups (such as the central Texas Eurycea) in which most species are morphologically very similar and some of the species have invaded and adapted to a novel selective environment.

PATTERNS OF POSTZYGOTIC ISOLATION IN FROGS.

From literature data on 116 taxa crosses involving 46 species of frogs, we found a positive correlation between degree of divergence (measured as Nei's genetic distance, D) and degree of postzygotic isolation. In anurans, hybrid sterility appears to evolve more quickly than inviability, which is consistent with the conclusions of other studies that involved Drosophila species. The lower threshold of D = 0.30 for evolution of hybrid inviability that we found is similar to that observed for Drosophila. This consistency suggests that there may be a general pattern in the acquisition of reproductive isolation in animals.