A small system--high-resolution study of metabolic adaptation in the central metabolic pathway to temperate climates in Drosophila melanogaster.

In this article, we couple the geographic variation in 127 single-nucleotide polymorphism (SNP) frequencies in genes of 46 enzymes of central metabolism with their associated cis-expression variation to predict latitudinal or climatic-driven gene expression changes in the metabolic architecture of Drosophila melanogaster. Forty-two percent of the SNPs in 65% of the genes show statistically significant clines in frequency with latitude across the 20 local population samples collected from southern Florida to Ontario. A number of SNPs in the screened genes are also associated with significant expression variation within the Raleigh population from North Carolina. A principal component analysis of the full variance-covariance matrix of latitudinal changes in SNP-associated standardized gene expression allows us to identify those major genes in the pathway and its associated branches that are likely targets of natural selection. When embedded in a central metabolic context, we show that these apparent targets are concentrated in the genes of the upper glycolytic pathway and pentose shunt, those controlling glycerol shuttle activity, and finally those enzymes associated with the utilization of glutamate and pyruvate. These metabolites possess high connectivity and thus may be the points where flux balance can be best shifted. We also propose that these points are conserved points associated with coupling energy homeostasis and energy sensing in mammals. We speculate that the modulation of gene expression at specific points in central metabolism that are associated with shifting flux balance or possibly energy-state sensing plays a role in adaptation to climatic variation.

Parapatric divergence of sympatric morphs in a salamander: incipient speciation on Long Island?

Speciation is often categorized based on geographic modes (allopatric, parapatric or sympatric). Although it is widely accepted that species can arise in allopatry and then later become sympatrically or parapatrically distributed, patterns in the opposite direction are also theoretically possible (e.g. sympatric lineages or ecotypes becoming parapatric), but such patterns have not been shown at a macrogeographic scale. Here, we analyse genetic, climatic, ecological and morphological data and show that two typically sympatric colour morphs of the salamander Plethodon cinereus (redback and leadback) appear to have become parapatrically distributed on Long Island, New York, with pure-redback populations in the west and pure-leadback populations in the east (and polymorphic populations in between and on the mainland). In addition, the pure-leadback populations in eastern Long Island are genetically, ecologically and morphologically divergent from both mainland and other Long Island populations, suggesting the possibility of incipient speciation. This parapatric separation seems to be related to the different ecological preferences of the two morphs, preferences which are present on the mainland and across Long Island. These results potentially support the idea that spatial segregation of sympatric ecotypes may sometimes play an important part in parapatric speciation.

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.

The phylogeny of advanced snakes (Colubroidea), with discovery of a new subfamily and comparison of support methods for likelihood trees.

The superfamily Colubroidea (> 2500 species) includes the majority of snake species and is one of the most conspicuous and well-known radiations of terrestrial vertebrates. However, many aspects of the phylogeny of the group remain contentious, and dozens of genera have yet to be included in molecular phylogenetic analyses. We present a new, large-scale, likelihood-based phylogeny for the colubroids, including 761 species sampled for up to five genes: cytochrome b (93% of 761 species sampled), ND4 (69%), ND2 (28%), c-mos (54%), and RAG-1 (13%), totaling up to 5814bp per species. We also compare likelihood bootstrapping and a recently proposed ultra-fast measure of branch support (Shimodaira-Hasegawa-like [SHL] approximate likelihood ratio), and find that the SHL test shows strong support for several clades that were weakly-supported by bootstrapping in this or previous analyses (e.g., Dipsadinae, Lamprophiidae). We find that SHL values are positively related to branch lengths, but show stronger support for shorter branches than bootstrapping. Despite extensive missing data for many taxa (mean=67% per species), neither bootstrap nor SHL support values for terminal species are related to their incompleteness, and that most highly incomplete taxa are placed in the expected families from previous taxonomy, typically with very strong support. The phylogeny indicates that the Neotropical colubrine genus Scaphiodontophis represents an unexpectedly ancient lineage within Colubridae. We present a revised higher-level classification of Colubroidea, which includes a new subfamily for Scaphiodontophis (Scaphiodontophiinae). Our study provides the most comprehensive phylogeny of Colubroidea to date, and suggests that SHL values may provide a useful complement to bootstrapping for estimating support on likelihood-based trees.

Combining phylogenomics and fossils in higher-level squamate reptile phylogeny: molecular data change the placement of fossil taxa.

Molecular data offer great potential to resolve the phylogeny of living taxa but can molecular data improve our understanding of relationships of fossil taxa? Simulations suggest that this is possible, but few empirical examples have demonstrated the ability of molecular data to change the placement of fossil taxa. We offer such an example here. We analyze the placement of snakes among squamate reptiles, combining published morphological data (363 characters) and new DNA sequence data (15,794 characters, 22 nuclear loci) for 45 living and 19 fossil taxa. We find several intriguing results. First, some fossil taxa undergo major changes in their phylogenetic position when molecular data are added. Second, most fossil taxa are placed with strong support in the expected clades by the combined data Bayesian analyses, despite each having >98% missing cells and despite recent suggestions that extensive missing data are problematic for Bayesian phylogenetics. Third, morphological data can change the placement of living taxa in combined analyses, even when there is an overwhelming majority of molecular characters. Finally, we find strong but apparently misleading signal in the morphological data, seemingly associated with a burrowing lifestyle in snakes, amphisbaenians, and dibamids. Overall, our results suggest promise for an integrated and comprehensive Tree of Life by combining molecular and morphological data for living and fossil taxa.

Quantifying interactions within the NADP(H) enzyme network in Drosophila melanogaster.

In this report, we use synthetic, activity-variant alleles in Drosophila melanogaster to quantify interactions across the enzyme network that reduces nicotinamide adenine dinucleotide phosphate (NADP) to NADPH. We examine the effects of large-scale variation in isocitrate dehydrogenase (IDH) or glucose-6-phosphate dehydrogenase (G6PD) activity in a single genetic background and of smaller-scale variation in IDH, G6PD, and malic enzyme across 10 different genetic backgrounds. We find significant interactions among all three enzymes in adults; changes in the activity of any one source of a reduced cofactor generally result in changes in the other two, although the magnitude and directionality of change differs depending on the gene and the genetic background. Observed interactions are presumably through cellular mechanisms that maintain a homeostatic balance of NADPH/NADP, and the magnitude of change in response to modification of one source of reduced cofactor likely reflects the relative contribution of that enzyme to the cofactor pool. Our results suggest that malic enzyme makes the largest single contribution to the NADPH pool, consistent with the results from earlier experiments in larval D. melanogaster using naturally occurring alleles. The interactions between all three enzymes indicate functional interdependence and underscore the importance of examining enzymes as components of a network.

Phylogenetic relationships of phrynosomatid lizards based on nuclear and mitochondrial data, and a revised phylogeny for Sceloporus.

Phrynosomatid lizards are among the most common and diverse groups of reptiles in western North America, Mexico, and Central America. Phrynosomatidae includes 136 species in 10 genera. Phrynosomatids are used as model systems in many research programs in evolution and ecology, and much of this research has been undertaken in a comparative phylogenetic framework. However, relationships among many phrynosomatid genera are poorly supported and in conflict between recent studies. Further, previous studies based on mitochondrial DNA sequences suggested that the most species-rich genus (Sceloporus) is possibly paraphyletic with respect to as many as four other genera (Petrosaurus, Sator, Urosaurus, and Uta). Here, we collect new sequence data from five nuclear genes and combine them with published data from one additional nuclear gene and five mitochondrial gene regions. We compare trees from nuclear and mitochondrial data from 37 phrynosomatid taxa, including a "species tree" (from BEST) for the nuclear data. We also present a phylogeny for 122 phrynosomatid species based on maximum likelihood analysis of the combined data, which provides a strongly-supported hypothesis for relationships among most phrynosomatid genera and includes most phrynosomatid species. Our results strongly support the monophyly of Sceloporus (including Sator) and many of the relationships within it. We present a new classification for phrynosomatid lizards and the genus Sceloporus, and offer a new tree with branch lengths for use in comparative studies.

An expanded phylogeny of treefrogs (Hylidae) based on nuclear and mitochondrial sequence data.

The treefrogs (Hylidae) make up one of the most species-rich families of amphibians. With 885 species currently described, they contain >13% of all amphibian species. In recent years, there has been considerable progress in resolving hylid phylogeny. However, the most comprehensive phylogeny to date (Wiens et al., 2006) included only 292 species, was based only on parsimony, provided only poor support for most higher-level relationships, and conflicted with previous hypotheses in several parts (including the monophyly and relationships of major clades of Hylinae). Here, we present an expanded phylogeny for hylid frogs, including data for 362 hylid taxa for up to 11 genes (4 mitochondrial, 7 nuclear), including 70 additional taxa and >270 sequences not included in the previously most comprehensive analysis. The new tree from maximum likelihood analysis is more well-resolved, strongly supported, and concordant with previous hypotheses, and provides a framework for future systematic, biogeographic, ecological, and evolutionary studies.

Loss and re-evolution of complex life cycles in marsupial frogs: does ancestral trait reconstruction mislead?

Using phylogeny-based methods to identify evolutionary transitions has become an integral part of evolutionary biology. Here, we demonstrate the potential for these methods to give statistically well-supported but misleading inferences about character evolution. We also show how inferences of character evolution can be informed using GIS-based methods to reconstruct ancestral environmental regimes. We reconstruct a phylogeny for marsupial frogs (Hemiphractidae) using nuclear and mitochondrial DNA sequences and estimate patterns of life-history evolution across the resulting tree. We find that Gastrotheca species with complex life cycles (i.e., egg, tadpole, and adult stages) are phylogenetically nested among species and genera with direct development (i.e., egg and adult stages only). Assuming a single rate for gains and losses in likelihood reconstructions, there is strong statistical support for the hypothesis that the tadpole stage was lost early in the phylogeny but reappeared within Gastrotheca. Assuming different rates of gain and loss, the model with significantly higher statistical support, the tadpole stage seems to have been lost multiple times but never regained. Given that both hypotheses cannot be correct, at least one reconstruction model must be giving well-supported but misleading results. Several lines of evidence (including GIS-based reconstructions of the ancestral climatic regime) suggest that the former hypothesis is correct, and that the tadpole stage has evolved from direct development within Gastrotheca, the only known case of such a reversal in frogs.

The intensity of selection acting on the couch potato gene--spatial-temporal variation in a diapause cline.

Cosmopolitan populations of Drosophila melanogaster have co-opted a form of reproductive diapause to overwinter in northern populations. Polymorphism in the couch potato gene has been implicated in genetic variation for this diapause trait. Using a collection of 20 populations from Florida to Canada and 11 collections from 3 years in a Pennsylvania orchard, we estimated the allele frequencies for 15 single nucleotide polymorphisms (SNPs) in the couch potato gene. These include the specific polymorphism associated with diapause inducability. We find that the SNP polymorphism, 48034(A/T), is correlated with latitude and its frequencies are predicted by the incidence of diapause trait. We find that the clinal patterns for cpo SNPs sampled in 1997 are similar to the same SNPs sampled in 2009-2010. SNPs that show apparent associations with cpo expression are also clinal with the low-expression allele increasing in frequency, as would be predicted from functional knockout studies of cpo. Finally, we see a significant pattern where the frequency of the diapause-causing allele drops in frequency during the summer season, consistent with the drop in the incidence of the diapause trait. The selection required to drive this response is large, roughly 24% to 59% per generation depending on the degree of dominance.

Branch lengths, support, and congruence: testing the phylogenomic approach with 20 nuclear loci in snakes.

Many authors have claimed that short branches in the Tree of Life will be very difficult to resolve with strong support, even with the large multilocus data sets now made possible by genomic resources. Short branches may be especially problematic because the underlying gene trees are expected to have discordant phylogenetic histories when the time between branching events is very short. Although there are many examples of short branches that are difficult to resolve, surprisingly, no empirical studies have systematically examined the relationships between branch lengths, branch support, and congruence among genes. Here, we examine these fundamental relationships quantitatively using a data set of 20 nuclear loci for 50 species of snakes (representing most traditionally recognized families). A combined maximum likelihood analysis of the 20 loci gives strong support for 69% of the nodes, but many remain weakly supported, with bootstrap values for 20% ranging from 21% to 66%. For the combined-data tree, we find significant correlations between the length of a branch, levels of bootstrap support, and the proportion of genes that are congruent with that branch in the separate analyses of each gene. We also find that strongly supported conflicts between gene trees over the resolution of individual branches are common (roughly 35% of clades), especially for shorter branches. Overall, our results support the hypothesis that short branches may be very difficult to confidently resolve, even with large, multilocus data sets. Nevertheless, our study provides strong support for many clades, including several that were controversial or poorly resolved in previous studies of snake phylogeny.