Core Genes Evolve Rapidly in the Long-Term Evolution Experiment with Escherichia coli.

Bacteria can evolve rapidly under positive selection owing to their vast numbers, allowing their genes to diversify by adapting to different environments. We asked whether the same genes that evolve rapidly in the long-term evolution experiment (LTEE) with Escherichia coli have also diversified extensively in nature. To make this comparison, we identified ∼2000 core genes shared among 60 E. coli strains. During the LTEE, core genes accumulated significantly more nonsynonymous mutations than flexible (i.e., noncore) genes. Furthermore, core genes under positive selection in the LTEE are more conserved in nature than the average core gene. In some cases, adaptive mutations appear to modify protein functions, rather than merely knocking them out. The LTEE conditions are novel for E. coli, at least in relation to its evolutionary history in nature. The constancy and simplicity of the environment likely favor the complete loss of some unused functions and the fine-tuning of others.

Proteinaceous Pheromone Homologs Identified from the Cloacal Gland Transcriptome of a Male Axolotl, Ambystoma mexicanum.

Pheromones play an important role in modifying vertebrate behavior, especially during courtship and mating. Courtship behavior in urodele amphibians often includes female exposure to secretions from the cloacal gland, as well as other scent glands. The first vertebrate proteinaceous pheromone discovered, the decapeptide sodefrin, is a female attracting pheromone secreted by the cloacal gland of male Cynops pyrrhogaster. Other proteinaceous pheromones in salamanders have been shown to elicit responses from females towards conspecific males. The presence and levels of expression of proteinaceous pheromones have not been identified in the family Ambystomatidae, which includes several important research models. The objective of this research was therefore to identify putative proteinaceous pheromones from male axolotls, Ambystoma mexicanum, as well as their relative expression levels. The results indicate that axolotls possess two different forms of sodefrin precursor-like factor (alpha and beta), as well as a putative ortholog of plethodontid modulating factor. The beta form of sodefrin precursor-like factor was amongst the most highly expressed transcripts within the cloacal gland. The ortholog of plethodontid modulating factor was expressed at a level equivalent to the beta sodefrin precursor-like factor. The results are from a single male axolotl; therefore, we are unable to assess how representative our results may be. Nevertheless, the presence of these highly expressed proteinaceous pheromones suggests that male axolotls use multiple chemical cues to attract female conspecifics. Behavioral assays would indicate whether the putative protein pheromones elicit courtship activity from female axolotls.

An Energy-Independent Pro-longevity Function of Triacylglycerol in Yeast.

Intracellular triacylglycerol (TAG) is a ubiquitous energy storage lipid also involved in lipid homeostasis and signaling. Comparatively, little is known about TAG's role in other cellular functions. Here we show a pro-longevity function of TAG in the budding yeast Saccharomyces cerevisiae. In yeast strains derived from natural and laboratory environments a correlation between high levels of TAG and longer chronological lifespan was observed. Increased TAG abundance through the deletion of TAG lipases prolonged chronological lifespan of laboratory strains, while diminishing TAG biosynthesis shortened lifespan without apparently affecting vegetative growth. TAG-mediated lifespan extension was independent of several other known stress response factors involved in chronological aging. Because both lifespan regulation and TAG metabolism are conserved, this cellular pro-longevity function of TAG may extend to other organisms.

Dynamic evolution of the GnRH receptor gene family in vertebrates.

BACKGROUND: Elucidating the mechanisms underlying coevolution of ligands and receptors is an important challenge in molecular evolutionary biology. Peptide hormones and their receptors are excellent models for such efforts, given the relative ease of examining evolutionary changes in genes encoding for both molecules. Most vertebrates possess multiple genes for both the decapeptide gonadotropin releasing hormone (GnRH) and for the GnRH receptor. The evolutionary history of the receptor family, including ancestral copy number and timing of duplications and deletions, has been the subject of controversy. RESULTS: We report here for the first time sequences of three distinct GnRH receptor genes in salamanders (axolotls, Ambystoma mexicanum), which are orthologous to three GnRH receptors from ranid frogs. To understand the origin of these genes within the larger evolutionary context of the gene family, we performed phylogenetic analyses and probabilistic protein homology searches of GnRH receptor genes in vertebrates and their near relatives. Our analyses revealed four points that alter previous views about the evolution of the GnRH receptor gene family. First, the "mammalian" pituitary type GnRH receptor, which is the sole GnRH receptor in humans and previously presumed to be highly derived because it lacks the cytoplasmic C-terminal domain typical of most G-protein coupled receptors, is actually an ancient gene that originated in the common ancestor of jawed vertebrates (Gnathostomata). Second, unlike previous studies, we classify vertebrate GnRH receptors into five subfamilies. Third, the order of subfamily origins is the inverse of previous proposed models. Fourth, the number of GnRH receptor genes has been dynamic in vertebrates and their ancestors, with multiple duplications and losses. CONCLUSION: Our results provide a novel evolutionary framework for generating hypotheses concerning the functional importance of structural characteristics of vertebrate GnRH receptors. We show that five subfamilies of vertebrate GnRH receptors evolved early in the vertebrate phylogeny, followed by several independent instances of gene loss. Chief among cases of gene loss are humans, best described as degenerate with respect to GnRH receptors because we retain only a single, ancient gene.

Conservation genetics and the implication for recovery of the endangered Mitchell's satyr Butterfly, Neonympha mitchellii mitchellii.

The modern delineation of taxonomic groups is often aided by analyses of molecular data, which can also help inform conservation biology. Two subspecies of the butterfly Neonympha mitchellii are classified as federally endangered in the United States: Neonympha mitchellii mitchellii, the Mitchell's satyr, and Neonympha mitchellii francisi, the Saint Francis's satyr. The recent discovery of additional disjunct populations of N. mitchellii in the southeastern US could have important implications for both legal protection and management decisions. We elucidated the relationships among 48 individuals representing 5 N. mitchellii populations using 6 molecular markers (5 nuclear and 1 mitochondrial) under a variety of analytical frameworks. Phylogenetic analysis resulted in moderately supported clades that corresponded with the geographic region where samples originated. Clustering analyses identified 3 groups, wherein the 2 named subspecies formed separate clusters. Coalescent analyses indicated evolutionary divergence between N. m. mitchellii and all other populations but weakly supported divergence among N. m. francisi and the recently discovered populations. Hence, the 2 currently accepted subspecies were clearly different from one another, but the recently discovered populations could not be completely distinguished from N. m. francisi or each other. We propose that N. m. mitchellii and N. m. francisi continue to be managed as separate endangered species.

The complex evolutionary dynamics of Hsp70s: a genomic and functional perspective.

Hsp70 molecular chaperones are ubiquitous. By preventing aggregation, promoting folding, and regulating degradation, Hsp70s are major factors in the ability of cells to maintain proteostasis. Despite a wealth of functional information, little is understood about the evolutionary dynamics of Hsp70s. We undertook an analysis of Hsp70s in the fungal clade Ascomycota. Using the well-characterized 14 Hsp70s of Saccharomyces cerevisiae, we identified 491 orthologs from 53 genomes. Saccharomyces cerevisiae Hsp70s fall into seven subfamilies: four canonical-type Hsp70 chaperones (SSA, SSB, KAR, and SSC) and three atypical Hsp70s (SSE, SSZ, and LHS) that play regulatory roles, modulating the activity of canonical Hsp70 partners. Each of the 53 surveyed genomes harbored at least one member of each subfamily, and thus establishing these seven Hsp70s as units of function and evolution. Genomes of some species contained only one member of each subfamily that is only seven Hsp70s. Overall, members of each subfamily formed a monophyletic group, suggesting that each diversified from their corresponding ancestral gene present in the common ancestor of all surveyed species. However, the pattern of evolution varied across subfamilies. At one extreme, members of the SSB subfamily evolved under concerted evolution. At the other extreme, SSA and SSC subfamilies exhibited a high degree of copy number dynamics, consistent with a birth-death mode of evolution. KAR, SSE, SSZ, and LHS subfamilies evolved in a simple divergent mode with little copy number dynamics. Together, our data revealed that the evolutionary history of this highly conserved and ubiquitous protein family was surprising complex and dynamic.

Occurrence of cyclic di-GMP-modulating output domains in cyanobacteria: an illuminating perspective.

UNLABELLED: Microorganisms use a variety of metabolites to respond to external stimuli, including second messengers that amplify primary signals and elicit biochemical changes in a cell. Levels of the second messenger cyclic dimeric GMP (c-di-GMP) are regulated by a variety of environmental stimuli and play a critical role in regulating cellular processes such as biofilm formation and cellular motility. Cyclic di-GMP signaling systems have been largely characterized in pathogenic bacteria; however, proteins that can impact the synthesis or degradation of c-di-GMP are prominent in cyanobacterial species and yet remain largely underexplored. In cyanobacteria, many putative c-di-GMP synthesis or degradation domains are found in genes that also harbor light-responsive signal input domains, suggesting that light is an important signal for altering c-di-GMP homeostasis. Indeed, c-di-GMP-associated domains are often the second most common output domain in photoreceptors-outnumbered only by a histidine kinase output domain. Cyanobacteria differ from other bacteria regarding the number and types of photoreceptor domains associated with c-di-GMP domains. Due to the widespread distribution of c-di-GMP domains in cyanobacteria, we investigated the evolutionary origin of a subset of genes. Phylogenetic analyses showed that c-di-GMP signaling systems were present early in cyanobacteria and c-di-GMP genes were both vertically and horizontally inherited during their evolution. Finally, we compared intracellular levels of c-di-GMP in two cyanobacterial species under different light qualities, confirming that light is an important factor for regulating this second messenger in vivo. IMPORTANCE: This study shows that many proteins containing cyclic dimeric GMP (c-di-GMP)-regulatory domains in cyanobacteria are associated with photoreceptor domains. Although the functional roles of c-di-GMP domain-containing proteins in cyanobacteria are only beginning to emerge, the abundance of these multidomain proteins in cyanobacteria that occupy diverse habitats ranging from freshwater to marine to soil environments suggests an important role for the regulation of c-di-GMP in these organisms. Indeed, we showed that light distinctly regulates c-di-GMP levels in Fremyella diplosiphon and Synechocystis sp. strain PCC6803. Our findings are consistent with the occurrence of c-di-GMP domains based on evolutionary origin and as an adaptation to specific habitat characteristics. Phylogenetic analyses of these domains clearly separate two distinctive clades, one composed of domains belonging predominantly to cyanobacteria and the other belonging to a mix of cyanobacteria and other bacteria. We further demonstrate that in cyanobacteria the acquisition of c-di-GMP signaling domains occurred both vertically and horizontally.

A peek into the evolution of the sexual lifestyles of yeast.

Tsong et al. characterize the role of mating type genes in Candida albicans and identify a new regulator of mating type and several mating type target genes. Comparison with Saccharomyces cerevisiae provides an in-depth view into the evolution of a well-characterized genetic regulatory circuit.

Genome-scale approaches to resolving incongruence in molecular phylogenies.

One of the most pervasive challenges in molecular phylogenetics is the incongruence between phylogenies obtained using different data sets, such as individual genes. To systematically investigate the degree of incongruence, and potential methods for resolving it, we screened the genome sequences of eight yeast species and selected 106 widely distributed orthologous genes for phylogenetic analyses, singly and by concatenation. Our results suggest that data sets consisting of single or a small number of concatenated genes have a significant probability of supporting conflicting topologies. By contrast, analyses of the entire data set of concatenated genes yielded a single, fully resolved species tree with maximum support. Comparable results were obtained with a concatenation of a minimum of 20 genes; substantially more genes than commonly used but a small fraction of any genome. These results have important implications for resolving branches of the tree of life.

Production of alpha 1,3-galactosyltransferase-knockout cloned pigs expressing human alpha 1,2-fucosylosyltransferase.

The production of genetically engineered pigs as xenotransplant donors aims to solve the severe shortage of organs for transplantation in humans. The first barrier to successful xenotransplantation is hyperacute rejection (HAR). HAR is a rapid and massive humoral immune response directed against the pig carbohydrate Galalpha 1,3-Gal epitope, which is synthesized by alpha 1,3-galactosyltransferase (alpha1,3-GT). The Galalpha 1,3-Gal antigen also contributes to subsequent acute vascular rejection events. Genetic modifications of donor pigs transgenic for human complement regulatory proteins or different glycosyltransferases to downregulate Galalpha 1,3-Gal expression have been shown to significantly delay xenograft rejection. However, the complete removal of the Galalpha 1,3-Gal antigen is the most attractive option. In this study, the 5' end of the alpha 1,3-GT gene was efficiently targeted with a nonisogenic DNA construct containing predominantly intron sequences and a Kozak translation initiation site to initiate translation of the neomycin resistance reporter gene. We developed two novel polymerase chain reaction screening methods to detect and confirm the targeted G418-resistant clones. This is the first study to use Southern blot analysis to demonstrate the disruption of the alpha 1,3-GT gene in somatic HT-transgenic pig cells before they were used for nuclear transfer. Transgenic male pigs were produced that possess an alpha 1,3-GT knockout allele and express a randomly inserted human alpha 1,2-fucosylosyltransferase (HT) transgene. The generation of homozygous alpha 1,3-GT knockout pigs with the HT-transgenic background is underway and will be unique. This approach intends to combine the alpha 1,3-GT knockout genotype with a ubiquitously expressed fucosyltransferase transgene producing the universally tolerated H antigen. This approach may prove to be more effective than the null phenotype alone in overcoming HAR and delayed xenograft rejection.

Drosophila pigmentation evolution: divergent genotypes underlying convergent phenotypes.

Similar phenotypic changes have evolved independently in many animal taxa. It is unknown whether independent changes involve the same or different developmental and genetic mechanisms. Myriad pigment patterns in the genus Drosophila offer numerous opportunities to address this question. Previous studies identified regulatory and structural genes involved in the development and diversification of pigmentation in selected species. Here, we examine Drosophila americana and Drosophila novamexicana, interfertile species that have evolved dramatic pigmentation differences during the few million years since their divergence. Interspecific genetic analysis was used to investigate the contribution of five specific candidate genes and other genomic regions to phenotypic divergence by testing for associations between molecular markers and pigmentation. At least four distinct genomic regions contributed to pigmentation differences, one of which included the ebony gene. Ebony protein was expressed at higher levels in the more yellow D. novamexicana than the heavily melanized D. americana. Because Ebony promotes yellow pigment formation and suppresses melanization, the expression difference and genetic association suggest that evolution at the ebony locus contributed to pigmentation divergence between D. americana and D. novamexicana. Surprisingly, no genetic association with the yellow locus was detected in this study, and Yellow expression was identical in the two species. Evolution at the yellow locus underlies pigmentation divergence among other Drosophila species; thus, similar pigment patterns have evolved through regulatory changes in different genes in different lineages. These findings bear upon understanding classic models of melanism and mimicry.

Landscape scale genetic effects of habitat fragmentation on a high gene flow species: Speyeria idalia (Nymphalidae).

Detection of the genetic effects of recent habitat fragmentation in natural populations can be a difficult task, especially for high gene flow species. Previous analyses of mitochondrial DNA data from across the current range of Speyeria idalia indicated that the species exhibited high levels of gene flow among populations, with the exception of an isolated population in the eastern portion of its range. However, some populations are found on isolated habitat patches, which were recently separated from one another by large expanses of uninhabitable terrain, in the form of row crop agriculture. The goal of this study was to compare levels of genetic differentiation and diversity among populations found in relatively continuous habitat to populations in both recently and historically isolated habitat. Four microsatellite loci were used to genotype over 300 individuals from five populations in continuous habitat, five populations in recently fragmented habitat, and one historically isolated population. Results from the historically isolated population were concordant with previous analyses and suggest significant differentiation. Also, microsatellite data were consistent with the genetic effects of habitat fragmentation for the recently isolated populations, in the form of increased differentiation and decreased genetic diversity when compared to nonfragmented populations. These results suggest that given the appropriate control populations, microsatellite markers can be used to detect the effects of recent habitat fragmentation in natural populations, even at a large geographical scale in high gene flow species.

Transgenic pigs designed to express human CD59 and H-transferase to avoid humoral xenograft rejection.

Research in pig-to-primate xenotransplantation aims to solve the increasing shortage of organs for human allotransplantation and develop new cell- and tissue-based therapies. Progress towards its clinical application has been hampered by the presence of xenoreactive natural antibodies that bind to the foreign cell surface and activate complement, causing humoral graft rejection. Genetic engineering of donor cells and animals to express human complement inhibitors such as hCD59 significantly prolonged graft survival. Strategies to decrease the deposition of natural antibodies were also developed. Expression of human alpha1,2-fucosyltransferase (H transferase, HT) in pigs modifies the cell-surface carbohydrate phenotype resulting in reduced Galalpha1,3-Gal expression and decreased antibody binding. We have developed transgenic pigs that coexpress hCD59 and HT in various cells and tissues to address both natural antibody binding and complement activation. Functional studies with peripheral blood mononuclear cells and aortic endothelial cells isolated from the double transgenic pigs showed that coexpression of hCD59 and HT markedly increased their resistance to human serum-mediated lysis. This resistance was greater than with cells transgenic for either hCD59 or HT alone. Moreover, transgene expression was enhanced and protection maintained in pig endothelial cells that were exposed for 24 h to pro-inflammatory cytokines. These studies suggest that engineering donor pigs to express multiple molecules that address different humoral components of xenograft rejection represents an important step toward enhancing xenograft survival and improving the prospect of clinical xenotransplantation.

The population genetics of host specificity: genetic differentiation in dove lice (Insecta: Phthiraptera).

Some species of parasites occur on a wide range of hosts while others are restricted to one or a few host species. The host specificity of a parasite species is determined, in part, by its ability to disperse between host species. Dispersal limitations can be studied by exploring the genetic structure of parasite populations both within a single species of host and across multiple host species. In this study we examined the genetic structure in the mitochondrial cytochrome oxidase I (COI) gene of two genera of lice (Insecta: Phthiraptera) occurring on multiple sympatric species of doves in southern North and Central America. One genus, Columbicola, is generally less host-specific than the other, Physconelloides. For both genera we identified substantial genetic differentiation between populations of conspecific lice on different host species, generally 10-20% sequence divergence. This level of divergence is in the range of that often observed between species of these two genera. We used nested clade analysis to explore fine scale genetic structure within species of these feather lice. We found that species of Physconelloides exhibited more genetic structure, both among hosts and among geographical localities, than did species of Columbicola. In many cases, single haplotypes within species of Columbicola are distributed on multiple host species. Thus, the population genetic structure of species of Physconelloides reveals evidence of geographical differentiation on top of high host species specificity. Underlying differences in dispersal biology probably explain the differences in population genetic structure that we observed between Columbicola and Physconelloides.

Conservation Genetics, Extinction, and Taxonomic Status: a Case History of the Regal Fritillary.

I investigated the phylogeography of mitochondrial DNA haplotypes in the regal fritillary ( Speyeria idalia ) to determine the conservation status of isolated populations in light of alternative definitions of species, subspecies, and evolutionarily significant units. A total of 1441 bases of partial cytochrome oxidase I and II genes were sequenced from 115 individuals in 18 populations located throughout the range of the species. A relatively disjunct population was differentiated with five synapomorphies, whereas all other populations had little haplotype variation and no apparent association between geographic and genetic distance. The divergence of the disjunct population may be due to either historical isolation among populations or extinction of intermediate populations within a cline. Genetic divergence due to the extinction of populations in fragmented habitat, termed "anthrovicariance," may be a generalizable phenomenon. Similar cases in insects have been described previously. If more cases can be elucidated, taxonomists will need to consider whether allopatry achieved through a natural vicariant event should be treated differently than that achieved via human-induced habitat loss. Mitochondrial DNA sequence divergence, along with ecological and natural-history data, suggest that the disjunct population of S. idalia is a distinct evolutionary lineage. Although taxonomic designation is not a simple task, the data indicate that the disjunct population is clearly of conservation concern.

RESUMEN: Investigué la fitogeografía de los haplotipos de ADN mitocondrial de la fritilaria real ( Speyeria idalia ) para determinar el estatus de conservación de poblaciones aisladas a la luz de definiciones alternativas de especies, subespecies y unidades evolutivamente significativas. Se secuenció un total de 1441 bases de genes I y II de citocromo oxidasa parcial de 115 individuos en 18 poblaciones localizadas a lo largo del rango de la especie. Se diferenció una población relativamente aislada con cinco sinapomorfias, mientras que las demás poblaciones tenían poca variación de haplotipos y no tenían asociación aparente entre la distancia geográfica y genética. La divergencia de la población aislada puede deberse al aislamiento histórico entre poblaciones o a la extinción de poblaciones intermedias en un clin. La divergencia genética debido a la extinción de poblaciones en hábitat fragmentado, llamada "antropovarianza", puede ser un fenómeno generalizable. Previamente se han descrito casos similares en insectos y si hay más casos, los taxónomos tendrán que considerar si la alopatría obtenida por medio de un evento vicariante natural deberá ser tratada de otra forma que la obtenida por medio de la perdida de hábitat inducida por el hombre. La divergencia de secuencias de ADN mitocondrial, además de datos de la ecología y la historia natural, sugiere que la población aislada de S. idalia es una línea evolutiva diferente. Aunque la designación taxonómica no es una tarea fácil, los datos indican que la población aislada claramente tiene importancia para la conservación.