Brice P. Noonan

Resolving the phylogeny of lizards and snakes (Squamata) with extensive sampling of genes and species

Squamate reptiles (lizards and snakes) are one of the most diverse groups of terrestrial vertebrates. Recent molecular analyses have suggested a very different squamate phylogeny relative to morphological hypotheses, but many aspects remain uncertain from molecular data. Here, we analyse higher-level squamate phylogeny with a molecular dataset of unprecedented size, including 161 squamate species for up to 44 nuclear genes each (33 717 base pairs), using both concatenated and species-tree methods for the first time. Our results strongly resolve most squamate relationships and reveal some surprising results. In contrast to most other recent studies, we find that dibamids and gekkotans are together the sister group to all other squamates. Remarkably, we find that the distinctive scolecophidians (blind snakes) are paraphyletic with respect to other snakes, suggesting that snakes were primitively burrowers and subsequently re-invaded surface habitats. Finally, we find that some clades remain poorly supported, despite our extensive data. Our analyses show that weakly supported clades are associated with relatively short branches for which individual genes often show conflicting relationships. These latter results have important implications for all studies that attempt to resolve phylogenies with large-scale phylogenomic datasets.

Evidence for an ancient adaptive episode of convergent molecular evolution

Documented cases of convergent molecular evolution due to selection are fairly unusual, and examples to date have involved only a few amino acid positions. However, because convergence mimics shared ancestry and is not accommodated by current phylogenetic methods, it can strongly mislead phylogenetic inference when it does occur. Here, we present a case of extensive convergent molecular evolution between snake and agamid lizard mitochondrial genomes that overcomes an otherwise strong phylogenetic signal. Evidence from morphology, nuclear genes, and most sites in the mitochondrial genome support one phylogenetic tree, but a subset of mostly amino acid-altering substitutions (primarily at the first and second codon positions) across multiple mitochondrial genes strongly supports a radically different phylogeny. The relevant sites generally evolved slowly but converged between ancient lineages of snakes and agamids. We estimate that ≈44 of 113 predicted convergent changes distributed across all 13 mitochondrial protein-coding genes are expected to have arisen from nonneutral causes—a remarkably large number. Combined with strong previous evidence for adaptive evolution in snake mitochondrial proteins, it is likely that much of this convergent evolution was driven by adaptation. These results indicate that nonneutral convergent molecular evolution in mitochondria can occur at a scale and intensity far beyond what has been documented previously, and they highlight the vulnerability of standard phylogenetic methods to the presence of nonneutral convergent sequence evolution.

Phylogeny of iguanian lizards inferred from 29 nuclear loci, and a comparison of concatenated and species-tree approaches for an ancient, rapid radiation

Iguanian lizards form a diverse clade whose members have been the focus of many comparative studies of ecology, behavior, and evolution. Despite the importance of phylogeny to such studies, interrelationships among many iguanian clades remain uncertain. Within the Old World clade Acrodonta, Agamidae is sometimes found to be paraphyletic with respect to Chamaeleonidae, and recent molecular studies have produced conflicting results for many major clades. Within the largely New World clade Pleurodonta, relationships among the 12 currently recognized major subclades (mostly ranked as families) have been largely unresolved or poorly supported in previous studies. To clarify iguanian evolutionary history, we first infer phylogenies using concatenated maximum-likelihood (ML) and Bayesian analyses of DNA sequence data from 29 nuclear protein-coding genes for 47 iguanian and 29 outgroup taxa. We then estimate a relaxed-clock Bayesian chronogram for iguanians using BEAST. All three methods produce identical topologies. Within Acrodonta, we find strong support for monophyly of Agamidae with respect to Chamaeleonidae, and for almost all relationships within agamids. Within Pleurodonta, we find strong Bayesian support for almost all relationships, and strong ML support for some interfamilial relationships and for monophyly of almost all families (excepting Polychrotidae). Our phylogenetic results suggest a non-traditional biogeographic scenario in which pleurodonts originated in the Northern Hemisphere and subsequently spread southward into South America. The pleurodont portion of the tree is characterized by several very short, deep branches, raising the possibility of deep coalescences that may confound concatenated analyses. We therefore also use 27 of these genes to implement a coalescent-based species-tree approach for pleurodonts. Although this analysis strongly supports monophyly of the pleurodont families, interfamilial relationships are generally different from those in the concatenated tree, and support is uniformly poor. However, a species-tree analysis using only the seven most variable loci yields higher support and more congruence with the concatenated tree. This suggests that low support in the 27-gene species-tree analysis may be an artifact of the many loci that are uninformative for very short branches. This may be a general problem for the application of species-tree methods to rapid radiations, even with phylogenomic data sets. Finally, we correct the non-monophyly of Polychrotidae by recognizing the pleurodont genus Anolis (sensu lato) as a separate family (Dactyloidae), and we correct the non-monophyly of the agamid genus Physignathus by resurrection of the genus Istiurus for the former Physignathus lesueurii.

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 (∼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.

Integrated analyses resolve conflicts over squamate reptile phylogeny and reveal unexpected placements for fossil taxa.

Squamate reptiles (lizards and snakes) are a pivotal group whose relationships have become increasingly controversial. Squamates include >9000 species, making them the second largest group of terrestrial vertebrates. They are important medicinally and as model systems for ecological and evolutionary research. However, studies of squamate biology are hindered by uncertainty over their relationships, and some consider squamate phylogeny unresolved, given recent conflicts between molecular and morphological results. To resolve these conflicts, we expand existing morphological and molecular datasets for squamates (691 morphological characters and 46 genes, for 161 living and 49 fossil taxa, including a new set of 81 morphological characters and adding two genes from published studies) and perform integrated analyses. Our results resolve higher-level relationships as indicated by molecular analyses, and reveal hidden morphological support for the molecular hypothesis (but not vice-versa). Furthermore, we find that integrating molecular, morphological, and paleontological data leads to surprising placements for two major fossil clades (Mosasauria and Polyglyphanodontia). These results further demonstrate the importance of combining fossil and molecular information, and the potential problems of estimating the placement of fossil taxa from morphological data alone. Thus, our results caution against estimating fossil relationships without considering relevant molecular data, and against placing fossils into molecular trees (e.g. for dating analyses) without considering the possible impact of molecular data on their placement.

The role of predator selection on polymorphic aposematic poison frogs

Demonstrations of interactions between diverse selective forces on bright coloration in defended species are rare. Recent work has suggested that not only do the bright colours of Neotropical poison frogs serve to deter predators, but they also play a role in sexual selection, with females preferring males similar to themselves. These studies report an interaction between the selective forces of mate choice and predation. However, evidence demonstrating phenotypic discrimination by potential predators on these polymorphic species is lacking. The possibility remains that visual (avian) predators possess an inherent avoidance of brightly coloured diurnal anurans and purifying selection against novel phenotypes within populations is due solely to non-random mating. Here, we examine the influence of predation on phenotypic variation in a polymorphic species of poison frog, Dendrobates tinctorius. Using clay models, we demonstrate a purifying role for predator selection, as brightly coloured novel forms are more likely to suffer an attack than both local aposematic and cryptic forms. Additionally, local aposematic forms are attacked, though infrequently, indicating ongoing testing/learning and a lack of innate avoidance. These results demonstrate predator-driven phenotypic purification within populations and suggest colour patterns of poison frogs may truly represent a ‘magic trait’.

Estimating divergence dates and evaluating dating methods using phylogenomic and mitochondrial data in squamate reptiles

Recently, phylogenetics has expanded to routinely include estimation of clade ages in addition to their relationships. Various dating methods have been used, but their relative performance remains understudied. Here, we generate and assemble an extensive phylogenomic data set for squamate reptiles (lizards and snakes) and evaluate two widely used dating methods, penalized likelihood in r8s (r8s-PL) and Bayesian estimation with uncorrelated relaxed rates among lineages (BEAST). We obtained sequence data from 25 nuclear loci (∼500-1,000 bp per gene; 19,020bp total) for 64 squamate species and nine outgroup taxa, estimated the phylogeny, and estimated divergence dates using 14 fossil calibrations. We then evaluated how well each method approximated these dates using random subsets of the nuclear loci (2, 5, 10, 15, and 20; replicated 10 times each), and using ∼1 kb of the mitochondrial ND2 gene. We find that estimates from r8s-PL based on 2, 5, or 10 loci can differ considerably from those based on 25 loci (mean absolute value of differences between 2-locus and 25-locus estimates were 9.0 Myr). Estimates from BEAST are somewhat more consistent given limited sampling of loci (mean absolute value of differences between 2 and 25-locus estimates were 5.0 Myr). Most strikingly, age estimates using r8s-PL for ND2 were ∼68-82 Myr older (mean=73.1) than those using 25 nuclear loci with r8s-PL. These results show that dates from r8s-PL with a limited number of loci (and especially mitochondrial data) can differ considerably from estimates derived from a large number of nuclear loci, whereas estimates from BEAST derived from fewer nuclear loci or mitochondrial data alone can be surprisingly similar to those from many nuclear loci. However, estimates from BEAST using relatively few loci and mitochondrial data could still show substantial deviations from the full data set (>50 Myr), suggesting the benefits of sampling many nuclear loci. Finally, we found that confidence intervals on ages from BEAST were not significantly different when sampling 2 vs. 25 loci, suggesting that adding loci decreased errors but did not increase confidence in those estimates.

Multiple Quaternary Refugia in the Eastern Guiana Shield Revealed by Comparative Phylogeography of 12 Frog Species

The Guiana Shield (GS) is one of the most pristine regions of Amazonia and biologically one of the richest areas on Earth. How and when this massive diversity arose remains the subject of considerable debate. The prevailing hypothesis of Quaternary glacial refugia suggests that a part of the eastern GS, among other areas in Amazonia, served as stable forested refugia during periods of aridity. However, the recently proposed disturbance-vicariance hypothesis proposes that fluctuations in temperature on orbital timescales, with some associated aridity, have driven Neotropical diversification. The expectations of the temporal and spatial organization of biodiversity differ between these two hypotheses. Here, we compare the genetic structure of 12 leaf-litter inhabiting frog species from the GS lowlands using a combination of mitochondrial and nuclear sequences in an integrative analytical approach that includes phylogenetic reconstructions, molecular dating, and Geographic Information System methods. This comparative and integrated approach overcomes the well-known limitations of phylogeographic inference based on single species and single loci. All of the focal species exhibit distinct phylogeographic patterns highlighting taxon-specific historical distributions, ecological tolerances to climatic disturbance, and dispersal abilities. Nevertheless, all but one species exhibit a history of fragmentation/isolation within the eastern GS during the Quaternary with spatial and temporal concordance among species. The signature of isolation in northern French Guiana (FG) during the early Pleistocene is particularly clear. Approximate Bayesian Computation supports the synchrony of the divergence between northern FG and other GS lineages. Substructure observed throughout the GS suggests further Quaternary fragmentation and a role for rivers. Our findings support fragmentation of moist tropical forest in the eastern GS during this period when the refuge hypothesis would have the region serving as a contiguous wet-forest refuge.

Alkylresorcinol Synthases Expressed in Sorghum bicolor Root Hairs Play an Essential Role in the Biosynthesis of the Allelopathic Benzoquinone Sorgoleone

Alkylresorcinol synthases (ARSs) are thought to participate in the biosynthesis of a diversity of defense-related plant phenolic lipids. This work presents the functional characterization of two ARS enzymes involved in the biosynthesis of allelochemicals produced in sorghum root hair cells as well as three enzymes likely dedicated to the biosynthesis of alkylresorcinolic phytoanticipins in rice. Sorghum bicolor is considered to be an allelopathic crop species, producing phytotoxins such as the lipid benzoquinone sorgoleone, which likely accounts for many of the allelopathic properties of Sorghum spp. Current evidence suggests that sorgoleone biosynthesis occurs exclusively in root hair cells and involves the production of an alkylresorcinolic intermediate (5-[(Z,Z)-8′,11′,14′-pentadecatrienyl]resorcinol) derived from an unusual 16:3Δ9,12,15 fatty acyl-CoA starter unit. This led to the suggestion of the involvement of one or more alkylresorcinol synthases (ARSs), type III polyketide synthases (PKSs) that produce 5-alkylresorcinols using medium to long-chain fatty acyl-CoA starter units via iterative condensations with malonyl-CoA. In an effort to characterize the enzymes responsible for the biosynthesis of the pentadecyl resorcinol intermediate, a previously described expressed sequence tag database prepared from isolated S. bicolor (genotype BTx623) root hairs was first mined for all PKS-like sequences. Quantitative real-time RT-PCR analyses revealed that three of these sequences were preferentially expressed in root hairs, two of which (designated ARS1 and ARS2) were found to encode ARS enzymes capable of accepting a variety of fatty acyl-CoA starter units in recombinant enzyme studies. Furthermore, RNA interference experiments directed against ARS1 and ARS2 resulted in the generation of multiple independent transformant events exhibiting dramatically reduced sorgoleone levels. Thus, both ARS1 and ARS2 are likely to participate in the biosynthesis of sorgoleone in planta. The sequences of ARS1 and ARS2 were also used to identify several rice (Oryza sativa) genes encoding ARSs, which are likely involved in the production of defense-related alkylresorcinols.

Genetic effects of habitat fragmentation and population isolation on Etheostoma raneyi (Percidae)

The use of genetic methods to quantify the effects of anthropogenic habitat fragmentation on population structure has become increasingly common. However, in today’s highly fragmented habitats, researchers have sometimes concluded that populations are currently genetically isolated due to habitat fragmentation without testing the possibility that populations were genetically isolated before European settlement. Etheostoma raneyi is a benthic headwater fish restricted to river drainages in northern Mississippi, USA, that has a suite of adaptive traits that correlate with poor dispersal ability. Aquatic habitat within this area has been extensively modified, primarily by flood-control projects, and populations in headwater streams have possibly become genetically isolated from one another. We used microsatellite markers to quantify genetic structure as well as contemporary and historical gene flow across the range of the species. Results indicated that genetically distinct populations exist in each headwater stream analyzed, current gene flow rates are lower than historical rates, most genetic variation is partitioned among populations, and populations in the Yocona River drainage show lower levels of genetic diversity than populations in the Tallahatchie River drainage and other Etheostoma species. All populations have negative FIS scores, of which roughly half are significant relative to Hardy–Weinberg expectations, perhaps due to small population sizes. We conclude that anthropogenic habitat alteration and fragmentation has had a profoundly negative impact on the species by isolating E. raneyi within headwater stream reaches. Further research is needed to inform conservation strategies, but populations in the Yocona River drainage are in dire need of management action. Carefully planned human-mediated dispersal and habitat restoration should be explored as management options across the range of the species.