On the Causes of Evolutionary Transition:Transversion Bias.

A pattern in which nucleotide transitions are favored several fold over transversions is common in molecular evolution. When this pattern occurs among amino acid replacements, explanations often invoke an effect of selection, on the grounds that transitions are more conservative in their effects on proteins. However, the underlying hypothesis of conservative transitions has never been tested directly. Here we assess support for this hypothesis using direct evidence: the fitness effects of mutations in actual proteins measured via individual or paired growth experiments. We assembled data from 8 published studies, ranging in size from 24 to 757 single-nucleotide mutations that change an amino acid. Every study has the statistical power to reveal significant effects of amino acid exchangeability, and most studies have the power to discern a binary conservative-vs-radical distinction. However, only one study suggests that transitions are significantly more conservative than transversions. In the combined set of 1,239 replacements (544 transitions, 695 transversions), the chance that a transition is more conservative than a transversion is 53 % (95 % confidence interval 50 to 56) compared with the null expectation of 50 %. We show that this effect is not large compared with that of most biochemical factors, and is not large enough to explain the several-fold bias observed in evolution. In short, the available data have the power to verify the "conservative transitions" hypothesis if true, but suggest instead that selection on proteins plays at best a minor role in the observed bias.

Ancient DNA supports southern survival of Richardson's collared lemming (Dicrostonyx richardsoni) during the last glacial maximum.

Collared lemmings (genus Dicrostonyx) are circumpolar Arctic arvicoline rodents associated with tundra. However, during the last glacial maximum (LGM), Dicrostonyx lived along the southern ice margin of the Laurentide ice sheet in communities comprising both temperate and boreal species. To better understand these communities and the fate of these southern individuals, we compare mitochondrial cytochrome b sequence data from three LGM-age Dicrostonyx fossils from south of the Laurentide ice sheet to sequences from modern Dicrostonyx sampled from across their present-day range. We test whether the Dicrostonyx populations from LGM-age continental USA became extinct at the Pleistocene-Holocene transition ~11000 years ago or, alternatively, if they belong to an extant species whose habitat preferences can be used to infer the palaeoclimate along the glacial margin. Our results indicate that LGM-age Dicrostonyx from Iowa and South Dakota belong to Dicrostonyx richardsoni, which currently lives in a temperate tundra environment west of Hudson Bay, Canada. This suggests a palaeoclimate south of the Laurentide ice sheet that contains elements similar to the more temperate shrub tundra characteristic of extant D. richardsoni habitat, rather than the very cold, dry tundra of the Northern Arctic. While more data are required to determine whether or not the LGM southern population is ancestral to extant D. richardsoni, it seems most probable that the species survived the LGM in a southern refugium.

Coevolution between cannabinoid receptors and endocannabinoid ligands.

Genes for receptors and ligands must coevolve to maintain coordinated gene expression and binding affinities. Researchers have debated whether anandamide or 2-arachidonyl glycerol (2-AG) is a more "intrinsic" ligand of cannabinoid receptors. We addressed this debate with a coevolutionary analysis, by examining genes for CB1, CB2, and ten genes that encode ligand metabolic enzymes: abhydrolase domain containing 4 protein, cyclooxygenase 2, diacylglycerol lipase paralogs (DAGLalpha, DAGLbeta), fatty acid amide hydrolase paralogs (FAAH1, FAAH2), monoglyceride lipase, N-acylethanolamine acid amidase, NAPE-selective phospholipase D, and protein tyrosine phosphatase non-receptor type 22. Gene trees (cladograms) of CB1, CB2, and ligand enzymes were obtained by searching for orthologs (tBLASTn) in the genomes of nine phylogenetically diverse species, aligning ortholog sequences with ClustalX, and applying Bayesian analysis (MrBayes). Mirrored cladograms provided evidence of coevolution (i.e., parallel cladogenesis). Next we constructed phylograms of CB1, CB2, and the ten enzymes. Phylogram branch lengths were proportional to three sets of maximum likelihood metrics: all-nucleotide-substitutions and NS/SS ratios (using PAUP()), and Ka/Ks ratios (using FUGE). Spurious correlations in all-nucleotide-substitutions trees (due to phylogenetic bias) and in Ka/Ks ratio trees (due to simplistic modeling) were parsed. Branch lengths from equivalent branches in paired trees were correlated by linear regression. Regression analyses, mirrored cladograms, and phylogenetic profiles produced the same results: close associations between cannabinoid receptors and DAGL enzymes. Therefore we propose that cannabinoid receptors initially coevolved with a fatty acid ester ligand (akin to 2-AG) in ancestral metazoans, and affinity for fatty acid ethanolamide ligands (e.g., AEA) evolved thereafter.

Tempo and mode in the endocannaboinoid system.

The best-known endocannabinoid ligands, anandamide and 2-AG, signal at least seven receptors and involve ten metabolic enzymes. Genes for the receptors and enzymes were examined for heterogeneities in tempo (relative rate of evolution, RRE) and mode (selection pressure, Ka/Ks) in six organisms with sequenced genomes. BLAST identified orthologs as reciprocal best hits, and nucleotide alignments were performed with ClustalX and MacClade. Two bioinformatics platforms, LiKaKs (a distance-based LWL85 model) and SNAP (a parsimony-based NG86 model) made pairwise comparisons of orthologs in murids (rat and mouse) and primates (human and macaque). Mean RRE of the 18 endocannabinoid genes was significantly greater in murids than primates, whereas mean Ka/Ks did not differ significantly. Next we used FUGE (tree-based maximum-likelihood model) to compute human lineage-specific Ka/Ks calculations for 18 genes, which ranged from 1.11 to 0.00, in rank order from highest to lowest: PTPN22, NAAA, TRPV1, TRPA1, NAPE-PLD, MAGL, PPARgamma, FAAH1, COX2, FAAH2, ABDH4, CB2, GPR55, DAGLbeta, PPARalpha, TRPV4, CB1, DAGLalpha; differences were significant (p < 0.0001). Rat and mouse presented different rank orders (e.g., GPR55 generated the greatest Ka/Ks ratio). The 18 genes were then tested for recent positive selection (within 10,000 yr) using an extended haplotype homozygosity analysis of SNP data from the HapMap database. Significant evidence (p < 0.05) of a positive "selective sweep" was exhibited by PTPN22, TRPV1, NAPE-PLD, and DAGLalpha. In conclusion, the endocannabinoid system is collectively under strong purifying selection, although some genes show evidence of adaptive evolution.

A shifted repertoire of endocannabinoid genes in the zebrafish (Danio rerio).

The zebrafish has served as a model organism for developmental biology. Sequencing its genome has expanded zebrafish research into physiology and drug-development testing. Several cannabinoid pharmaceuticals are in development, but expression of endocannabinoid receptors and enzymes remains unknown in this species. We conducted a bioinformatics analysis of the zebrafish genome using 17 human endocannabinoid genes as a reference set. Putative zebrafish orthologs were identified in filtered BLAST searches as reciprocal best hits. Orthology was confirmed by three in silico methods: phylogenetic testing, synteny analysis, and functional mapping. Zebrafish expressed orthologs of cannabinoid receptor 1, transient receptor potential channel vanilloid receptor 4, GPR55 receptor, fatty acid amide hydrolase 1, monoacylglycerol lipase, NAPE-selective phospholipase D, abhydrolase domain-containing protein 4, and diacylglycerol lipase alpha and beta; and paired paralogs of cannabinoid receptor 2, fatty acid amide hydrolase 2, peroxisome proliferator-activated receptor alpha, prostaglandin-endoperoxide synthase 2, and transient receptor potential cation channel subtype A1. Functional mapping suggested the orthologs of transient receptor potential vanilloid receptor 1 and peroxisome proliferator-activated receptor gamma lack specific amino acids critical for cannabinoid ligand binding. No orthologs of N-acylethanolamine acid amidase or protein tyrosine phosphatase, non-receptor type 22 were identified. In conclusion, the zebrafish genome expresses a shifted repertoire of endocannabinoid genes. In vitro analyses are warranted before using zebrafish for cannabinoid development testing.

The phylogenetic position of the zokors (Myospalacinae) and comments on the families of muroids (Rodentia).

Recent molecular studies have concluded that the genus Myospalax evolved from within the rodent subfamily Cricetinae. This conclusion was tested using the complete sequences from the mitochondrial 12S rRNA and cytochrome b genes. Based on our analyses, Myospalax appears to be sister to a clade containing the subfamilies Spalacinae and Rhizomyinae, and all three of these lineages appear to be basal to the superfamily Muroidea. Based on the position of these three lineages, we suggest that they be placed in a distinct family, the Spalacidae, rather than subsumed as subfamilies in the family Muridae. Finally, our analyses suggest that the earlier placement of Myospalax as a member of the Cricetinae is the result of a single misidentified specimen, which was not a Myospalax.