ALBERT

Description: The predicted effect of effective population size on the distribution of fitness effects and substitution rate is critically dependent on the relationship between sequence and fitness. This highlights the importance of using models that are informed by the molecular biology, biochemistry, and biophysics of the evolving systems. We describe a computational model based on fundamental aspects of biophysics, the requirement for (most) proteins to be thermodynamically stable. Using this model, we find that differences in population size have minimal impact on the distribution of population-scaled fitness effects, as well as on the rate of molecular evolution. This is because larger populations result in selection for more stable proteins that are less affected by mutations. This reduction in the magnitude of the fitness effects almost exactly cancels the greater selective pressure resulting from the larger population size. Conversely, changes in the population size in either direction cause transient increases in the substitution rate. As differences in population size often correspond to changes in population size, this makes comparisons of substitution rates in different lineages difficult to interpret.

Description: Populations of widely distributed species encounter and must adapt to local environmental conditions. However, comprehensive characterization of the genetic basis of adaptation is demanding, requiring genome-wide genotype data, multiple sampled populations, and an understanding of population structure and potential selection pressures. Here, we used single-nucleotide polymorphism genotyping and data on numerous environmental variables to describe the genetic basis of local adaptation in 21 populations of teosinte, the wild ancestor of maize. We found complex hierarchical genetic structure created by altitude, dispersal events, and admixture among subspecies, which complicated identification of locally beneficial alleles. Patterns of linkage disequilibrium revealed four large putative inversion polymorphisms showing clinal patterns of frequency. Population differentiation and environmental correlations suggest that both inversions and intergenic polymorphisms are involved in local adaptation.

Description: Wolbachia , endosymbiotic bacteria of the order Rickettsiales, are widespread in arthropods but also present in nematodes. In arthropods, A and B supergroup Wolbachia are generally associated with distortion of host reproduction. In filarial nematodes, including some human parasites, multiple lines of experimental evidence indicate that C and D supergroup Wolbachia are essential for the survival of the host, and here the symbiotic relationship is considered mutualistic. The origin of this mutualistic endosymbiosis is of interest for both basic and applied reasons: How does a parasite become a mutualist? Could intervention in the mutualism aid in treatment of human disease? Correct rooting and high-quality resolution of Wolbachia relationships are required to resolve this question. However, because of the large genetic distance between Wolbachia and the nearest outgroups, and the limited number of genomes so far available for large-scale analyses, current phylogenies do not provide robust answers. We therefore sequenced the genome of the D supergroup Wolbachia endosymbiont of Litomosoides sigmodontis , revisited the selection of loci for phylogenomic analyses, and performed a phylogenomic analysis including available complete genomes (from isolates in supergroups A, B, C, and D). Using 90 orthologous genes with reliable phylogenetic signals, we obtained a robust phylogenetic reconstruction, including a highly supported root to the Wolbachia phylogeny between a (A + B) clade and a (C + D) clade. Although we currently lack data from several Wolbachia supergroups, notably F, our analysis supports a model wherein the putatively mutualist endosymbiotic relationship between Wolbachia and nematodes originated from a single transition event.

Description: Many insects rely on bacterial symbionts with tiny genomes specialized for provisioning nutrients lacking in host diets. Xylem sap and phloem sap are both deficient as insect diets, but differ dramatically in nutrient content, potentially affecting symbiont genome evolution. For sap-feeding insects, sequenced symbiont genomes are available only for phloem-feeding examples from the suborder Sternorrhyncha and xylem-feeding examples from the suborder Auchenorrhyncha, confounding comparisons. We sequenced genomes of the obligate symbionts, Sulcia muelleri and Nasuia deltocephalinicola , of the phloem-feeding pest insect, Macrosteles quadrilineatus (Auchenorrhyncha: Cicadellidae). Our results reveal that Nasuia- ALF has the smallest bacterial genome yet sequenced (112 kb), and that the Sulcia- ALF genome (190 kb) is smaller than that of Sulcia in other insect lineages. Together, these symbionts retain the capability to synthesize the 10 essential amino acids, as observed for several symbiont pairs from xylem-feeding Auchenorrhyncha. Nasuia retains genes enabling synthesis of two amino acids, DNA replication, transcription, and translation. Both symbionts have lost genes underlying ATP synthesis through oxidative phosphorylation, possibly as a consequence of the enriched sugar content of phloem. Shared genomic features, including reassignment of the UGA codon from Stop to tryptophan, and phylogenetic results suggest that Nasuia -ALF is most closely related to Zinderia , the betaproteobacterial symbiont of spittlebugs. Thus, Nasuia / Zinderia and Sulcia likely represent ancient associates that have co-resided in hosts since the divergence of leafhoppers and spittlebugs 〉200 Ma, and possibly since the origin of the Auchenorrhyncha, 〉260 Ma.

Description: Thanks to the microarray technology, our understanding of transcriptome evolution at the genome level has been considerably advanced in the past decade. Yet, further investigation was challenged by several technical limitations of this technology. Recent innovation of next-generation sequencing, particularly the invention of RNA-seq technology, has shed insightful lights on resolving this problem. Though a number of statistical and computational methods have been developed to analyze RNA-seq data, the analytical framework specifically designed for evolutionary genomics remains an open question. In this article we develop a new method for estimating the genome expression distance from the RNA-seq data, which has explicit interpretations under the model of gene expression evolution. Moreover, this distance measure takes the data overdispersion, gene length variation, and sequencing depth variation into account so that it can be applied to multiple genomes from different species. Using mammalian RNA-seq data as example, we demonstrated that this expression distance is useful in phylogenomic analysis.

Description: The control of RNA splicing is often modulated by exonic motifs near splice sites. Chief among these are exonic splice enhancers (ESEs). Well-described ESEs in mammals are purine rich and cause predictable skews in codon and amino acid usage toward exonic ends. Looking across species, those with relatively abundant intronic sequence are those with the more profound end of exon skews, indicative of exonization of splice site recognition. To date, the only intron-rich species that have been analyzed are mammals, precluding any conclusions about the likely ancestral condition. Here, we examine the patterns of codon and amino acid usage in the vicinity of exon–intron junctions in the brown alga Ectocarpus siliculosus , a species with abundant large introns, known SR proteins, and classical splice sites. We find that amino acids and codons preferred/avoided at both 3' and 5' ends in Ectocarpus , of which there are many, tend, on average, to also be preferred/avoided at the same exon ends in humans. Moreover, the preferences observed at the 5' ends of exons are largely the same as those at the 3' ends, a symmetry trend only previously observed in animals. We predict putative hexameric ESEs in Ectocarpus and show that these are purine rich and that there are many more of these identified as functional ESEs in humans than expected by chance. These results are consistent with deep phylogenetic conservation of SR protein binding motifs. Assuming codons preferred near boundaries are "splice optimal" codons, in Ectocarpus , unlike Drosophila, splice optimal and translationally optimal codons are not mutually exclusive. The exclusivity of translationally optimal and splice optimal codon sets is thus not universal.

Description: Autonomous retrotransposons lacking long terminal repeats (LTR) account for much of the variation in genome size and structure among vertebrates. Mammalian genomes contain hundreds of thousands of non-LTR retrotransposon copies, mostly resulting from the amplification of a single clade known as L1. The genomes of teleost fish and squamate reptiles contain a much more diverse array of non-LTR retrotransposon families, whereas copy number is relatively low. The majority of non-LTR retrotransposon insertions in nonmammalian vertebrates also appear to be very recent, suggesting strong purifying selection limits the accumulation of non-LTR retrotransposon copies. It is however unclear whether this turnover model, originally proposed in Drosophila , applies to nonmammalian vertebrates. Here, we studied the population dynamics of L1 in the green anole lizard ( Anolis carolinensis ). We found that although most L1 elements are recent in this genome, truncated insertions accumulate readily, and many are fixed at both the population and species level. In contrast, full-length L1 insertions are found at lower population frequencies, suggesting that the turnover model only applies to longer L1 elements in Anolis . We also found that full-length L1 inserts are more likely to be fixed in populations of small effective size, suggesting that the strength of purifying selection against deleterious alleles is highly dependent on host demographic history. Similar mechanisms seem to be controlling the fate of non-LTR retrotransposons in both Anolis and teleostean fish, which suggests that mammals have considerably diverged from the ancestral vertebrate in terms of how they interact with their intragenomic parasites.

Description: Increasing evidence from sequence data from various environments, including the human gut, suggests the existence of a previously unknown putative seventh order of methanogens. The first genomic data from members of this lineage, Methanomassiliicoccus luminyensis and " Candidatus Methanomethylophilus alvus," provide insights into its evolutionary history and metabolic features. Phylogenetic analysis of ribosomal proteins robustly indicates a monophyletic group independent of any previously known methanogenic order, which shares ancestry with the Marine Benthic Group D, the Marine Group II, the DHVE2 group, and the Thermoplasmatales. This phylogenetic position, along with the analysis of enzymes involved in core methanogenesis, strengthens a single ancient origin of methanogenesis in the Euryarchaeota and indicates further multiple independent losses of this metabolism in nonmethanogenic lineages than previously suggested. Genomic analysis revealed an unprecedented loss of the genes coding for the first six steps of methanogenesis from H 2 /CO 2 and the oxidative part of methylotrophic methanogenesis, consistent with the fact that M. luminyensis and " Ca. M. alvus" are obligate H 2 -dependent methylotrophic methanogens. Genomic data also suggest that these methanogens may use a large panel of methylated compounds. Phylogenetic analysis including homologs retrieved from environmental samples indicates that methylotrophic methanogenesis (regardless of dependency on H 2 ) is not restricted to gut representatives but may be an ancestral characteristic of the whole order, and possibly also of ancient origin in the Euryarchaeota. 16S rRNA and McrA trees show that this new order of methanogens is very diverse and occupies environments highly relevant for methane production, therefore representing a key lineage to fully understand the diversity and evolution of methanogenesis.

Description: Streptococcus dysgalactiae subsp. equisimilis (SDSE) is an emerging human pathogen that causes life-threatening invasive infections such as streptococcal toxic shock syndrome. Recent epidemiological studies reveal that invasive SDSE infections have been increasing in Asia, Europe, and the United States. Almost all SDSE carry Lancefield group G or C antigen. We have determined the complete genome sequence of a human group C SDSE 167 strain. A comparison of its sequence with that of four SDSE strains, three in Lancefield group G and one in Lancefield group A, showed approximately 90% coverage. Most regions showing little or no homology were located in the prophages. There was no evidence of massive rearrangement in the genome of SDSE 167. Bayesian phylogeny using entire genome sequences showed that the most recent common ancestor of the five SDSE strains appeared 446 years ago. Interestingly, we found that SDSE 167 harbors sugar metabolizing enzymes in a unique region and streptodornase in the phage region, which presumably contribute to the degradation of host tissues and the prompted covRS mutation, respectively. A comparison of these five SDSE strains, which differ in Lancefield group antigens, revealed a gene cluster presumably responsible for the synthesis of the antigenic determinant. These results may provide the basis for molecular epidemiological research of SDSE.

Description: Mitochondria are intracellular organelles where oxidative phosphorylation is carried out to complete ATP synthesis. Mitochondria have their own genome; in metazoans, this is a small, circular molecule encoding 13 electron transport proteins, 22 tRNAs, and 2 rRNAs. In invertebrates, mitochondrial gene rearrangement is common, and it is correlated with increased substitution rates. In vertebrates, mitochondrial gene rearrangement is rare, and its relationship to substitution rate remains unexplored. Mitochondrial genes can also show spatial variation in substitution rates around the genome due to the mechanism of mtDNA replication, which produces a mutation gradient. To date, however, the strength of the mutation gradient and whether movement along the gradient in rearranged (or otherwise modified) genomes impacts genic substitution rates remain unexplored in the majority of vertebrates. Salamanders include both normal mitochondrial genomes and independently derived rearrangements and expansions, providing a rare opportunity to test the effects of large-scale changes to genome architecture on vertebrate mitochondrial gene sequence evolution. We show that: 1) rearranged/expanded genomes have higher substitution rates; 2) most genes in rearranged/expanded genomes maintain their position along the mutation gradient, substitution rates of the genes that do move are unaffected by their new position, and the gradient in salamanders is weak; and 3) genomic rearrangements/expansions occur independent of levels of selective constraint on genes. Together, our results demonstrate that large-scale changes to genome architecture impact mitochondrial gene evolution in predictable ways; however, despite these impacts, the same functional constraints act on mitochondrial protein-coding genes in both modified and normal genomes.

Description: Very little is known about genetic factors that regulate life history transitions during ontogeny. Closely related tiger salamanders ( Ambystoma species complex) show extreme variation in metamorphic timing, with some species foregoing metamorphosis altogether, an adaptive trait called paedomorphosis. Previous studies identified a major effect quantitative trait locus ( met1 ) for metamorphic timing and expression of paedomorphosis in hybrid crosses between the biphasic Eastern tiger salamander ( Ambystoma tigrinum tigrinum ) and the paedomorphic Mexican axolotl ( Ambystoma mexicanum ). We used existing hybrid mapping panels and a newly created hybrid cross to map the met1 genomic region and determine the effect of met1 on larval growth, metamorphic timing, and gene expression in the brain. We show that met1 maps to the position of a urodele-specific chromosome rearrangement on linkage group 2 that uniquely brought functionally associated genes into linkage. Furthermore, we found that more than 200 genes were differentially expressed during larval development as a function of met1 genotype. This list of differentially expressed genes is enriched for proteins that function in the mitochondria, providing evidence of a link between met1 , thyroid hormone signaling, and mitochondrial energetics associated with metamorphosis. Finally, we found that met1 significantly affected metamorphic timing in hybrids, but not early larval growth rate. Collectively, our results show that met1 regulates species and morph-specific patterns of brain transcription and life history variation .

Description: Oxidative phosphorylation (OXPHOS), the major energy-producing pathway in aerobic organisms, includes protein subunits encoded by both mitochondrial (mt) and nuclear (nu) genomes. How these independent genomes have coevolved is a long-standing question in evolutionary biology. Although mt genes evolve faster than most nu genes, maintenance of OXPHOS structural stability and functional efficiency may involve correlated evolution of mt and nu OXPHOS genes. The nu OXPHOS genes might be predicted to exhibit accelerated evolutionary rates to accommodate the elevated substitution rates of mt OXPHOS subunits with which they interact. Evolutionary rates of nu OXPHOS genes should, therefore, be higher than that of nu genes that are not involved in OXPHOS (nu non-OXPHOS). We tested the compensatory evolution hypothesis by comparing the evolutionary rates (synonymous substitution rate d S and nonsynonymous substitution rate d N ) among 13 mt OXPHOS genes, 60 nu OXPHOS genes, and 77 nu non-OXPHOS genes in vertebrates (7 fish and 40 mammal species). The results from a combined analysis of all OXPHOS subunits fit the predictions of the hypothesis. However, results from two OXPHOS complexes did not fit this pattern when analyzed separately. We found that the d N of nu OXPHOS genes for "core" subunits (those involved in the major catalytic activity) was lower than that of "noncore" subunits, whereas there was no significant difference in d N between genes for nu non-OXPHOS and core subunits. This latter finding suggests that compensatory changes play a minor role in the evolution of OXPHOS genes and that the observed accelerated nu substitution rates are due largely to reduced functional constraint on noncore subunits.

Description: Recently, we found dramatic mitochondrial DNA divergence of Israeli Chamaeleo chamaeleon populations into two geographically distinct groups. We aimed to examine whether the same pattern of divergence could be found in nuclear genes. However, no genomic resource is available for any chameleon species. Here we present the first chameleon transcriptome, obtained using deep sequencing (SOLiD). Our analysis identified 164,000 sequence contigs of which 19,000 yielded unique BlastX hits. To test the efficacy of our sequencing effort, we examined whether the chameleon and other available reptilian transcriptomes harbored complete sets of genes comprising known biochemical pathways, focusing on the nDNA-encoded oxidative phosphorylation (OXPHOS) genes as a model. As a reference for the screen, we used the human 86 (including isoforms) known structural nDNA-encoded OXPHOS subunits. Analysis of 34 publicly available vertebrate transcriptomes revealed orthologs for most human OXPHOS genes. However, OXPHOS subunit COX8 (Cytochrome C oxidase subunit 8), including all its known isoforms, was consistently absent in transcriptomes of iguanian lizards, implying loss of this subunit during the radiation of this suborder. The lack of COX8 in the suborder Iguania is intriguing, since it is important for cellular respiration and ATP production. Our sequencing effort added a new resource for comparative genomic studies, and shed new light on the evolutionary dynamics of the OXPHOS system.

Description: Human hemoglobins, the oxygen carriers in the blood, are composed by two α-like and two β-like globin monomers. The β-globin gene cluster located at 11p15.5 comprises one pseudogene and five genes whose expression undergoes two critical switches: the embryonic-to-fetal and fetal-to-adult transition. HBD encodes the -globin chain of the minor adult hemoglobin (HbA 2 ), which is assumed to be physiologically irrelevant. Paradoxically, reduced diversity levels have been reported for this gene. In this study, we sought a detailed portrait of the genetic variation within the β-globin cluster in a large human population panel from different geographic backgrounds. We resequenced the coding and noncoding regions of the two adult β-globin genes ( HBD and HBB ) in European and African populations, and analyzed the data from the β-globin cluster ( HBE , HBG2 , HBG1 , HBBP1 , HBD, and HBB ) in 1,092 individuals representing 14 populations sequenced as part of the 1000 Genomes Project. Additionally, we assessed the diversity levels in nonhuman primates using chimpanzee sequence data provided by the PanMap Project. Comprehensive analyses, based on classic neutrality tests, empirical and haplotype-based studies, revealed that HBD and its neighbor pseudogene HBBP1 have mainly evolved under purifying selection, suggesting that their roles are essential and nonredundant. Moreover, in the light of recent studies on the chromatin conformation of the β-globin cluster, we present evidence sustaining that the strong functional constraints underlying the decreased contemporary diversity at these two regions were not driven by protein function but instead are likely due to a regulatory role in ontogenic switches of gene expression.

Description: A tandem repeat’s (TR) propensity to mutate increases with repeat number, and can become very pronounced beyond a critical boundary, transforming it into a microsatellite (MS). However, a clear understanding of the mutational behavior of different TR classes and motifs and related mechanisms is lacking, as is a consensus on the existence of a boundary separating short TRs (STRs) from MSs. This hinders our understanding of MSs’ mutational properties and their effective use as genetic markers. Using indel calls for 179 individuals from 1000 Genomes Pilot-1 Project, we determined polymorphism incidence for four major TR classes, and formalized its varying relationship with repeat number using segmented regression. We observed a biphasic regime with a transition from a faster to a slower exponential growth at 9, 5, 4, and 4 repeats for mono-, di-, tri-, and tetranucleotide TRs, respectively. We used an in vitro mutagenesis assay to evaluate the contribution of strand slippage errors to mutability. STRs and MSs differ in their absolute polymorphism levels, but more importantly in their rates of mutability growth. Although strand slippage is a major factor driving mononucleotide polymorphism incidence, dinucleotide polymorphism incidence is greater than that expected due to strand slippage alone, indicating that additional cellular factors might be driving dinucleotide mutability in the human genome. Leveraging on hundreds of human genomes, we present the first comprehensive, genome-wide analysis of TR mutational behavior, encompassing several motif sizes and compositions.

Description: Eukaryotic genome sequencing projects often yield bacterial DNA sequences, data typically considered as microbial contamination. However, these sequences may also indicate either symbiont genes or lateral gene transfer (LGT) to host genomes. These bacterial sequences can provide clues about eukaryote–microbe interactions. Here, we used the genome of the primitive animal Trichoplax adhaerens (Metazoa: Placozoa), which is known to harbor an uncharacterized Gram-negative endosymbiont, to search for the presence of bacterial DNA sequences. Bioinformatic and phylogenomic analyses of extracted data from the genome assembly (181 bacterial coding sequences [CDS]) and trace read archive (16S rDNA) revealed a dominant proteobacterial profile strongly skewed to Rickettsiales ( Alphaproteobacteria ) genomes. By way of phylogenetic analysis of 16S rDNA and 113 proteins conserved across proteobacterial genomes, as well as identification of 27 rickettsial signature genes, we propose a Rickettsiales endosymbiont of T. adhaerens (RETA). The majority (93%) of the identified bacterial CDS belongs to small scaffolds containing prokaryotic-like genes; however, 12 CDS were identified on large scaffolds comprised of eukaryotic-like genes, suggesting that T . adhaerens might have recently acquired bacterial genes. These putative LGTs may coincide with the placozoan’s aquatic niche and symbiosis with RETA. This work underscores the rich, and relatively untapped, resource of eukaryotic genome projects for harboring data pertinent to host–microbial interactions. The nature of unknown (or poorly characterized) bacterial species may only emerge via analysis of host genome sequencing projects, particularly if these species are resistant to cell culturing, as are many obligate intracellular microbes. Our work provides methodological insight for such an approach.

Description: Following polyploidy, duplicate genes are often deleted, and if they are not, then duplicate regulatory regions are sometimes lost. By what mechanism is this loss and what is the chance that such a loss removes function? To explore these questions, we followed individual Arabidopsis thaliana–A. thaliana conserved noncoding sequences (CNSs) into the Brassica ancestor, through a paleohexaploidy and into Brassica rapa . Thus, a single Brassicaceae CNS has six potential orthologous positions in B. rapa ; a single Arabidopsis CNS has three potential homeologous positions. We reasoned that a CNS, if present on a singlet Brassica gene, would be unlikely to lose function compared with a more redundant CNS, and this is the case. Redundant CNSs go nondetectable often. Using this logic, each mechanism of CNS loss was assigned a metric of functionality. By definition, proved deletions do not function as sequence. Our results indicated that CNSs that go nondetectable by base substitution or large insertion are almost certainly still functional (redundancy does not matter much to their detectability frequency), whereas those lost by inferred deletion or indels are approximately 75% likely to be nonfunctional. Overall, an average nondetectable, once-redundant CNS more than 30 bp in length has a 72% chance of being nonfunctional, and that makes sense because 97% of them sort to a molecular mechanism with "deletion" in its description, but base substitutions do cause loss. Similarly, proved-functional G-boxes go undetectable by deletion 82% of the time. Fractionation mutagenesis is a procedure that uses polyploidy as a mutagenic agent to genetically alter RNA expression profiles, and then to construct testable hypotheses as to the function of the lost regulatory site. We show fractionation mutagenesis to be a "deletion machine" in the Brassica lineage.

Description: Understanding the molecular basis of within and between species phenotypic variation is one of the main goals of Biology. In Drosophila , most of the work regarding this issue has been performed in D. melanogaster , but other distantly related species must also be studied to verify the generality of the findings obtained for this species. Here, we make the case for D. americana , a species of the virilis group of Drosophila that has been diverging from the model species, D. melanogaster , for approximately 40 Myr. To determine the suitability of this species for such studies, polymorphism and recombination estimates are presented for D. americana based on the largest nucleotide sequence polymorphism data set so far analyzed (more than 100 data sets) for this species. The polymorphism estimates are also compared with those obtained from the comparison of the genome assembly of two D. americana strains (H5 and W11) here reported. As an example of the general utility of these resources, we perform a preliminary study on the molecular basis of lifespan differences in D. americana . First, we show that there are lifespan differences between D. americana populations from different regions of the distribution range. Then, we perform five F2 association experiments using markers for 21 candidate genes previously identified in D. melanogaster . Significant associations are found between polymorphism at two genes ( hep and Lim3 ) and lifespan. For the F2 association study involving the two sequenced strains (H5 and W11), we identify amino acid differences at Lim3 and Hep that could be responsible for the observed changes in lifespan. For both genes, no large gene expression differences were observed between the two strains.

Description: Comparative mitochondrial genomics of arbuscular mycorrhizal fungi (AMF) provide new avenues to overcome long-lasting obstacles that have hampered studies aimed at understanding the community structure, diversity, and evolution of these multinucleated and genetically polymorphic organisms. AMF mitochondrial (mt) genomes are homogeneous within isolates, and their intergenic regions harbor numerous mobile elements that have rapidly diverged, including homing endonuclease genes, small inverted repeats, and plasmid-related DNA polymerase genes ( dpo ), making them suitable targets for the development of reliable strain-specific markers. However, these elements may also lead to genome rearrangements through homologous recombination, although this has never previously been reported in this group of obligate symbiotic fungi. To investigate whether such rearrangements are present and caused by mobile elements in AMF, the mitochondrial genomes from two Glomeraceae members (i.e., Glomus cerebriforme and Glomus sp. ) with substantial mtDNA synteny divergence, were sequenced and compared with available glomeromycotan mitochondrial genomes . We used an extensive nucleotide/protein similarity network-based approach to investigate dpo diversity in AMF as well as in other organisms for which sequences are publicly available. We provide strong evidence of dpo -induced inter-haplotype recombination, leading to a reshuffled mitochondrial genome in Glomus sp. These findings raise questions as to whether AMF single spore cultivations artificially underestimate mtDNA genetic diversity. We assessed potential dpo dispersal mechanisms in AMF and inferred a robust phylogenetic relationship with plant mitochondrial plasmids. Along with other indirect evidence, our analyses indicate that members of the Glomeromycota phylum are potential donors of mitochondrial plasmids to plants.

Description: The study of genetic and phenotypic variation is fundamental for understanding the dynamics of bacterial genome evolution and untangling the evolution and epidemiology of bacterial pathogens. Neisseria meningitidis ( Nm ) is among the most intriguing bacterial pathogens in genomic studies due to its dynamic population structure and complex forms of pathogenicity. Extensive genomic variation within identical clonal complexes (CCs) in Nm has been recently reported and suggested to be the result of homologous recombination, but the extent to which recombination contributes to genomic variation within identical CCs has remained unclear. In this study, we sequenced two Nm strains of identical serogroup (C) and multi-locus sequence type (ST60), and conducted a systematic analysis with an additional 34 Nm genomes. Our results revealed that all gene content variation between the two ST60 genomes was introduced by homologous recombination at the conserved flanking genes, and 94.25% or more of sequence divergence was caused by homologous recombination. Recombination was found in genes associated with virulence factors, antigenic outer membrane proteins, and vaccine targets, suggesting an important role of homologous recombination in rapidly altering the pathogenicity and antigenicity of Nm . Recombination was also evident in genes of the restriction and modification systems, which may undermine barriers to DNA exchange. In conclusion, homologous recombination can drive both gene content variation and sequence divergence in Nm . These findings shed new light on the understanding of the rapid pathoadaptive evolution of Nm and other recombinogenic bacterial pathogens.

Description: Organelle DNA is no stranger to palindromic repeats. But never has a mitochondrial or plastid genome been described in which every coding region is part of a distinct palindromic unit. While sequencing the mitochondrial DNA of the nonphotosynthetic green alga Polytomella magna , we uncovered precisely this type of genic arrangement. The P. magna mitochondrial genome is linear and made up entirely of palindromes, each containing 1–7 unique coding regions. Consequently, every gene in the genome is duplicated and in an inverted orientation relative to its partner. And when these palindromic genes are folded into putative stem-loops, their predicted translational start sites are often positioned in the apex of the loop. Gel electrophoresis results support the linear, 28-kb monomeric conformation of the P. magna mitochondrial genome. Analyses of other Polytomella taxa suggest that palindromic mitochondrial genes were present in the ancestor of the Polytomella lineage and lost or retained to various degrees in extant species. The possible origins and consequences of this bizarre genomic architecture are discussed.

Description: Expression quantitative trait loci (eQTLs) have been found to be enriched in trait-associated single-nucleotide polymorphisms (SNPs). However, whether eQTLs are adaptive to different environmental factors and its relative evolutionary significance compared with nonsynonymous SNPs (NS SNPs) are still elusive. Compiling environmental correlation data from three studies for more than 500,000 SNPs and 42 environmental factors, including climate, subsistence, pathogens, and dietary patterns, we performed a systematic examination of the adaptive patterns of eQTLs to local environment. Compared with intergenic SNPs, eQTLs are significantly enriched in the lower tail of a transformed rank statistic in the environmental correlation analysis, indicating possible adaptation of eQTLs to the majority of 42 environmental factors. The mean enrichment of eQTLs across 42 environmental factors is as great as, if not greater than, that of NS SNPs. The enrichment of eQTLs, although significant across all levels of recombination rate, is inversely correlated with recombination rate, suggesting the presence of selective sweep or background selection. Further pathway enrichment analysis identified a number of pathways with possible environmental adaption from eQTLs. These pathways are mostly related with immune function and metabolism. Our results indicate that eQTLs might have played an important role in recent and ongoing human adaptation and are of special importance for some environmental factors and biological pathways.

Description: Environmental or geological changes can create new niches that drive ecological species divergence without the immediate cessation of gene flow. However, few such cases have been characterized. On a recently formed volcano, Mt. Etna, Senecio aethnensis and S. chrysanthemifolius inhabit contrasting environments of high and low altitude, respectively. They have very distinct phenotypes, despite hybridizing promiscuously, and thus may represent an important example of ecological speciation "in action," possibly as a response to the rapid geological changes that Mt. Etna has recently undergone. To elucidate the species’ evolutionary history, and help establish the species as a study system for speciation genomics, we sequenced the transcriptomes of the two Etnean species, and the outgroup, S. vernalis , using Illumina sequencing. Despite the species’ substantial phenotypic divergence, synonymous divergence between the high- and low-altitude species was low (d S = 0.016 ± 0.017 [SD]). A comparison of species divergence models with and without gene flow provided unequivocal support in favor of the former and demonstrated a recent time of species divergence (153,080 ya ± 11,470 [SE]) that coincides with the growth of Mt. Etna to the altitudes that separate the species today. Analysis of d N /d S revealed wide variation in selective constraint between genes, and evidence that highly expressed genes, more "multifunctional" genes, and those with more paralogs were under elevated purifying selection. Taken together, these results are consistent with a model of ecological speciation, potentially as a response to the emergence of a new, high-altitude niche as the volcano grew.

Description: Admixture mapping has been enormously resourceful in identifying genetic variations linked to phenotypes, adaptation, and diseases. In this study through analysis of copy number variable regions (CNVRs), we report extensive restructuring in the genomes of the recently admixed African-Indian population (OG-W-IP) that inhabits a highly saline environment in Western India. The study included subjects from OG-W-IP (OG), five different Indian and three HapMap populations that were genotyped using Affymetrix version 6.0 arrays. Copy number variations (CNVs) detected using Birdsuite were used to define CNVRs. Population structure with respect to CNVRs was delineated using random forest approach. OG genomes have a surprising excess of CNVs in comparison to other studied populations. Individual ancestry proportions computed using STRUCTURE also reveals a unique genetic component in OGs. Population structure analysis with CNV genotypes indicates OG to be distant from both the African and Indian ancestral populations. Interestingly, it shows genetic proximity with respect to CNVs to only one Indian population IE-W-LP4, which also happens to reside in the same geographical region. We also observe a significant enrichment of molecular processes related to ion binding and receptor activity in genes encompassing OG-specific CNVRs. Our results suggest that retention of CNVRs from ancestral natives and de novo acquisition of CNVRs could accelerate the process of adaptation especially in an extreme environment. Additionally, this population would be enormously useful for dissecting genes and delineating the involvement of CNVs in salt adaptation.

Description: As an important subtype of structural variations, chromosomal translocation is associated with various diseases, especially cancers, by disrupting gene structures and functions. Traditional methods for identifying translocations are time consuming and have limited resolutions. Recently, a few studies have employed next-generation sequencing (NGS) technology for characterizing chromosomal translocations on human genome, obtaining high-throughput results with high resolutions. However, these studies are mainly focused on mechanism-specific or site-specific translocation mapping. In this study, we conducted a comprehensive genome-wide analysis on the characterization of human chromosomal material exchange with regard to the chromosome translocations. Using NGS data of 1,481 subjects from the 1000 Genomes Project, we identified 15,349,092 translocated DNA fragment pairs, ranging from 65 to 1,886 bp and with an average size of approximately 102 bp. On average, each individual genome carried about 10,364 pairs, covering approximately 0.069% of the genome. We identified 16 translocation hot regions, among which two regions did not contain repetitive fragments. Results of our study overlapped with a majority of previous results, containing approximately 79% of approximately 2,340 translocations characterized in three available translocation databases. In addition, our study identified five novel potential recurrent chromosomal material exchange regions with greater than 20% detection rates. Our results will be helpful for an accurate characterization of translocations in human genomes, and contribute as a resource for future studies of the roles of translocations in human disease etiology and mechanisms.

Description: Angiosperm mitochondrial genomes exhibit many unusual properties, including heterogeneous nucleotide composition and exceptionally large and variable genome sizes. Determining the role of nonadaptive mechanisms such as mutation bias in shaping the molecular evolution of these unique genomes has proven challenging because their dynamic structures generally prevent identification of homologous intergenic sequences for comparative analyses. Here, we report an analysis of angiosperm mitochondrial DNA sequences that are derived from inserted plastid DNA ( mtpts ). The availability of numerous completely sequenced plastid genomes allows us to infer the evolutionary history of these insertions, including the specific nucleotide substitutions and indels that have occurred because their incorporation into the mitochondrial genome. Our analysis confirmed that many mtpts have a complex history, including frequent gene conversion and multiple examples of horizontal transfer between divergent angiosperm lineages. Nevertheless, it is clear that the majority of extant mtpt sequence in angiosperms is the product of recent transfer (or gene conversion) and is subject to rapid loss/deterioration, suggesting that most mtpts are evolving relatively free from functional constraint. The evolution of mtpt sequences reveals a pattern of biased mutational input in angiosperm mitochondrial genomes, including an excess of small deletions over insertions and a skew toward nucleotide substitutions that increase AT content. However, these mutation biases are far weaker than have been observed in many other cellular genomes, providing insight into some of the notable features of angiosperm mitochondrial architecture, including the retention of large intergenic regions and the relatively neutral GC content found in these regions.

Description: Neotropical primates (NP) are presently distributed in the New World from Mexico to northern Argentina, comprising three large families, Cebidae, Atelidae, and Pitheciidae, consequently to their diversification following their separation from Old World anthropoids near the Eocene/Oligocene boundary, some 40 Ma. The evolution of NP has been intensively investigated in the last decade by studies focusing on their phylogeny and timescale. However, despite major efforts, the phylogenetic relationship between these three major clades and the age of their last common ancestor are still controversial because these inferences were based on limited numbers of loci and dating analyses that did not consider the evolutionary variation associated with the distribution of gene trees within the proposed phylogenies. We show, by multispecies coalescent analyses of selected genome segments, spanning along 92,496,904 bp that the early diversification of extant NP was marked by a 2-fold increase of their effective population size and that Atelids and Cebids are more closely related respective to Pitheciids. The molecular phylogeny of NP has been difficult to solve because of population-level phenomena at the early evolution of the lineage. The association of evolutionary variation with the distribution of gene trees within proposed phylogenies is crucial for distinguishing the mean genetic divergence between species (the mean coalescent time between loci) from speciation time. This approach, based on extensive genomic data provided by new generation DNA sequencing, provides more accurate reconstructions of phylogenies and timescales for all organisms.

Description: Dogs shared a much closer relationship with humans than any other domesticated animals, probably due to their unique social cognitive capabilities, which were hypothesized to be a by-product of selection for tameness toward humans. Here, we demonstrate that genes involved in glutamate metabolism, which account partially for fear response, indeed show the greatest population differentiation by whole-genome comparison of dogs and wolves. However, the changing direction of their expression supports a role in increasing excitatory synaptic plasticity in dogs rather than reducing fear response. Because synaptic plasticity are widely believed to be cellular correlates of learning and memory, this change may alter the learning and memory abilities of ancient scavenging wolves, weaken the fear reaction toward humans, and prompt the initial interspecific contact.

Description: Hirsutella minnesotensis [Ophiocordycipitaceae (Hypocreales, Ascomycota)] is a dominant endoparasitic fungus by using conidia that adhere to and penetrate the secondary stage juveniles of soybean cyst nematode. Its genome was de novo sequenced and compared with five entomopathogenic fungi in the Hypocreales and three nematode-trapping fungi in the Orbiliales (Ascomycota). The genome of H. minnesotensis is 51.4 Mb and encodes 12,702 genes enriched with transposable elements up to 32%. Phylogenomic analysis revealed that H. minnesotensis was diverged from entomopathogenic fungi in Hypocreales. Genome of H. minnesotensis is similar to those of entomopathogenic fungi to have fewer genes encoding lectins for adhesion and glycoside hydrolases for cellulose degradation, but is different from those of nematode-trapping fungi to possess more genes for protein degradation, signal transduction, and secondary metabolism. Those results indicate that H. minnesotensis has evolved different mechanism for nematode endoparasitism compared with nematode-trapping fungi. Transcriptomics analyses for the time-scale parasitism revealed the upregulations of lectins, secreted proteases and the genes for biosynthesis of secondary metabolites that could be putatively involved in host surface adhesion, cuticle degradation, and host manipulation. Genome and transcriptome analyses provided comprehensive understanding of the evolution and lifestyle of nematode endoparasitism.

Description: Anopheles gambiae is a major mosquito vector of malaria in Africa. Although increased use of insecticide-based vector control tools has decreased malaria transmission, elimination is likely to require novel genetic control strategies. It can be argued that the absence of an A. gambiae inbred line has slowed progress toward genetic vector control . In order to empower genetic studies and enable precise and reproducible experimentation, we set out to create an inbred line of this species. We found that amenability to inbreeding varied between populations of A. gambiae . After full-sib inbreeding for ten generations, we genotyped 112 individuals—56 saved prior to inbreeding and 56 collected after inbreeding—at a genome-wide panel of single nucleotide polymorphisms (SNPs). Although inbreeding dramatically reduced diversity across much of the genome, we discovered numerous, discrete genomic blocks that maintained high heterozygosity. For one large genomic region, we were able to definitively show that high diversity is due to the persistent polymorphism of a chromosomal inversion. Inbred lines in other eukaryotes often exhibit a qualitatively similar retention of polymorphism when typed at a small number of markers. Our whole-genome SNP data provide the first strong, empirical evidence supporting associative overdominance as the mechanism maintaining higher than expected diversity in inbred lines. Although creation of A. gambiae lines devoid of nearly all polymorphism may not be feasible, our results provide critical insights into how more fully isogenic lines can be created.

Description: The kallikrein ( KLK ) gene family comprises the largest uninterrupted locus of serine proteases in the human genome and represents a notable case for studying the evolutionary fate of duplicated genes. In primates, a recent duplication event gave rise to KLK2 and KLK3 , both encoding essential proteins for the cascade of seminal plasma liquefaction. We reconstructed the evolutionary history of KLK2 and KLK3 by comparative analysis of the orthologous sequences from 22 primate species, calculated d N / d S ratios, and addressed the hypothesis of coevolution with their substrates, the semenogelins (SEMG1 and SEMG2). Our findings support the placement of the KLK2–KLK3 duplication in the Catarrhini ancestor and unveil the frequent loss of KLK2 throughout primate evolution by different genomic mechanisms, including unequal crossing-over, deletions, and pseudogenization. We provide evidences for an adaptive evolution of KLK3 toward an expanded enzymatic spectrum, with an effect on the hydrolysis of semen coagulum. Furthermore, we found associations between mating system, the number of SEMG repeat units, and the number of functional KLK2 and KLK3 , suggesting complex evolutionary dynamics shaped by reproductive biology.

Description: The question of Jewish ancestry has been the subject of controversy for over two centuries and has yet to be resolved. The "Rhineland hypothesis" depicts Eastern European Jews as a "population isolate" that emerged from a small group of German Jews who migrated eastward and expanded rapidly. Alternatively, the "Khazarian hypothesis" suggests that Eastern European Jews descended from the Khazars, an amalgam of Turkic clans that settled the Caucasus in the early centuries CE and converted to Judaism in the 8th century. Mesopotamian and Greco–Roman Jews continuously reinforced the Judaized empire until the 13th century. Following the collapse of their empire, the Judeo–Khazars fled to Eastern Europe. The rise of European Jewry is therefore explained by the contribution of the Judeo–Khazars. Thus far, however, the Khazars’ contribution has been estimated only empirically, as the absence of genome-wide data from Caucasus populations precluded testing the Khazarian hypothesis. Recent sequencing of modern Caucasus populations prompted us to revisit the Khazarian hypothesis and compare it with the Rhineland hypothesis. We applied a wide range of population genetic analyses to compare these two hypotheses. Our findings support the Khazarian hypothesis and portray the European Jewish genome as a mosaic of Near Eastern-Caucasus, European, and Semitic ancestries, thereby consolidating previous contradictory reports of Jewish ancestry. We further describe a major difference among Caucasus populations explained by the early presence of Judeans in the Southern and Central Caucasus. Our results have important implications for the demographic forces that shaped the genetic diversity in the Caucasus and for medical studies.

Description: The impact of transposable elements (TEs) on genome structure, plasticity, and evolution is still not well understood. The recent availability of complete genome sequences makes it possible to get new insights on the evolutionary dynamics of TEs from the phylogenetic analysis of their multiple copies in a wide range of species. However, this source of information is not always fully exploited. Here, we show how the history of transposition activity may be qualitatively and quantitatively reconstructed by considering the distribution of transposition events in the phylogenetic tree, along with the tree topology. Using statistical models developed to infer speciation and extinction rates in species phylogenies, we demonstrate that it is possible to estimate the past transposition rate of a TE family, as well as how this rate varies with time. This methodological framework may not only facilitate the interpretation of genomic data, but also serve as a basis to develop new theoretical and statistical models.

Description: We generated a genome-wide replication profile in the genome of Lachancea kluyveri and assessed the relationship between replication and base composition . This species diverged from Saccharomyces cerevisiae before the ancestral whole genome duplication . The genome comprises eight chromosomes among which a chromosomal arm of 1 Mb has a G + C-content much higher than the rest of the genome. We identified 252 active replication origins in L. kluyveri and found considerable divergence in origin location with S. cerevisiae and with Lachancea waltii. Although some global features of S. cerevisiae replication are conserved: Centromeres replicate early, whereas telomeres replicate late, we found that replication origins both in L. kluyveri and L. waltii do not behave as evolutionary fragile sites. In L. kluyveri, replication timing along chromosomes alternates between regions of early and late activating origins, except for the 1 Mb GC-rich chromosomal arm. This chromosomal arm contains an origin consensus motif different from other chromosomes and is replicated early during S-phase. We showed that precocious replication results from the specific absence of late firing origins in this chromosomal arm. In addition, we found a correlation between GC-content and distance from replication origins as well as a lack of replication-associated compositional skew between leading and lagging strands specifically in this GC-rich chromosomal arm. These findings suggest that the unusual base composition in the genome of L. kluyveri could be linked to replication.

Description: The vomeronasal organ (VNO) is an olfactory structure that detects pheromones and environmental cues. It consists of sensory neurons that express evolutionary unrelated groups of transmembrane chemoreceptors. The predominant V1R and V2R receptor repertoires are believed to detect airborne and water-soluble molecules, respectively. It has been suggested that the shift in habitat of early tetrapods from water to land is reflected by an increase in the ratio of V1R/V2R genes. Snakes, which have a very large VNO associated with a sophisticated tongue delivery system, are missing from this analysis. Here, we use RNA-seq and RNA in situ hybridization to study the diversity, evolution, and expression pattern of the corn snake vomeronasal receptor repertoires. Our analyses indicate that snakes and lizards retain an extremely limited number of V1R genes but exhibit a large number of V2R genes, including multiple lineages of reptile-specific and snake-specific expansions. We finally show that the peculiar bigenic pattern of V2R vomeronasal receptor gene transcription observed in mammals is conserved in squamate reptiles, hinting at an important but unknown functional role played by this expression strategy. Our results do not support the hypothesis that the shift to a vomeronasal receptor repertoire dominated by V1Rs in mammals reflects the evolutionary transition of early tetrapods from water to land. This study sheds light on the evolutionary dynamics of the vomeronasal receptor families in vertebrates and reveals how mammals and squamates differentially adapted the same ancestral vomeronasal repertoire to succeed in a terrestrial environment.

Description: Orphan genes are defined as genes that lack detectable similarity to genes in other species and therefore no clear signals of common descent (i.e., homology) can be inferred. Orphans are an enigmatic portion of the genome because their origin and function are mostly unknown and they typically make up 10% to 30% of all genes in a genome. Several case studies demonstrated that orphans can contribute to lineage-specific adaptation. Here, we study orphan genes by comparing 30 arthropod genomes, focusing in particular on seven recently sequenced ant genomes. This setup allows analyzing a major metazoan taxon and a comparison between social Hymenoptera (ants and bees) and nonsocial Diptera (flies and mosquitoes). First, we find that recently split lineages undergo accelerated genomic reorganization, including the rapid gain of many orphan genes. Second, between the two insect orders Hymenoptera and Diptera, orphan genes are more abundant and emerge more rapidly in Hymenoptera, in particular, in leaf-cutter ants. With respect to intragenomic localization, we find that ant orphan genes show little clustering, which suggests that orphan genes in ants are scattered uniformly over the genome and between nonorphan genes. Finally, our results indicate that the genetic mechanisms creating orphan genes—such as gene duplication, frame-shift fixation, creation of overlapping genes, horizontal gene transfer, and exaptation of transposable elements—act at different rates in insects, primates, and plants. In Formicidae, the majority of orphan genes has their origin in intergenic regions, pointing to a high rate of de novo gene formation or generalized gene loss, and support a recently proposed dynamic model of frequent gene birth and death.

Description: The most bacteria-like mitochondrial genome known is that of the jakobid flagellate Reclinomonas americana NZ. This genome also encodes the largest known gene set among mitochondrial DNAs (mtDNAs), including the RNA subunit of RNase P (transfer RNA processing), a reduced form of transfer–messenger RNA (translational control), and a four-subunit bacteria-like RNA polymerase, which in other eukaryotes is substituted by a nucleus-encoded, single-subunit, phage-like enzyme. Further, protein-coding genes are preceded by potential Shine–Dalgarno translation initiation motifs. Whether similarly ancestral mitochondrial characters also exist in relatives of R. americana NZ is unknown. Here, we report a comparative analysis of nine mtDNAs from five distant jakobid genera: Andalucia, Histiona, Jakoba, Reclinomonas , and Seculamonas . We find that Andalucia godoyi has an even larger mtDNA gene complement than R. americana NZ. The extra genes are rpl35 (a large subunit mitoribosomal protein) and cox15 (involved in cytochrome oxidase assembly), which are nucleus encoded throughout other eukaryotes. Andalucia cox15 is strikingly similar to its homolog in the free-living α-proteobacterium Tistrella mobilis . Similarly, a long, highly conserved gene cluster in jakobid mtDNAs, which is a clear vestige of prokaryotic operons, displays a gene order more closely resembling that in free-living α-proteobacteria than in Rickettsiales species. Although jakobid mtDNAs, overall, are characterized by bacteria-like features, they also display a few remarkably divergent characters, such as 3'-tRNA editing in Seculamonas ecuadoriensis and genome linearization in Jakoba libera . Phylogenetic analysis with mtDNA-encoded proteins strongly supports monophyly of jakobids with Andalucia as the deepest divergence. However, it remains unclear which α-proteobacterial group is the closest mitochondrial relative.

Description: Large-scale evolutionary studies often require the automated construction of alignments of a large number of homologous gene families. The majority of eukaryotic genes can produce different transcripts due to alternative splicing or transcription initiation, and many such transcripts encode different protein isoforms. As analyses tend to be gene centered, one single-protein isoform per gene is selected for the alignment, with the de facto approach being to use the longest protein isoform per gene (Longest), presumably to avoid including partial sequences and to maximize sequence information. Here, we show that this approach is problematic because it increases the number of indels in the alignments due to the inclusion of nonhomologous regions, such as those derived from species-specific exons, increasing the number of misaligned positions. With the aim of ameliorating this problem, we have developed a novel heuristic, Protein ALignment Optimizer (PALO), which, for each gene family, selects the combination of protein isoforms that are most similar in length. We examine several evolutionary parameters inferred from alignments in which the only difference is the method used to select the protein isoform combination: Longest, PALO, the combination that results in the highest sequence conservation, and a randomly selected combination. We observe that Longest tends to overestimate both nonsynonymous and synonymous substitution rates when compared with PALO, which is most likely due to an excess of misaligned positions. The estimation of the fraction of genes that have experienced positive selection by maximum likelihood is very sensitive to the method of isoform selection employed, both when alignments are constructed with MAFFT and with Prank +F . Longest performs better than a random combination but still estimates up to 3 times more positively selected genes than the combination showing the highest conservation, indicating the presence of many false positives. We show that PALO can eliminate the majority of such false positives and thus that it is a more appropriate approach for large-scale analyses than Longest. A web server has been set up to facilitate the use of PALO given a user-defined set of gene families; it is available at http://evolutionarygenomics.imim.es/palo .

Description: In vitro studies of the haloarchaeal genus Haloferax have demonstrated their ability to frequently exchange DNA between species, whereas rates of homologous recombination estimated from natural populations in the genus Halorubrum are high enough to maintain random association of alleles between five loci. To quantify the effects of gene transfer and recombination of commonly held (relaxed core) genes during the evolution of the class Halobacteria (haloarchaea), we reconstructed the history of 21 genomes representing all major groups. Using a novel algorithm and a concatenated ribosomal protein phylogeny as a reference, we created a directed horizontal genetic transfer (HGT) network of contemporary and ancestral genomes. Gene order analysis revealed that 90% of testable HGTs were by direct homologous replacement, rather than nonhomologous integration followed by a loss. Network analysis revealed an inverse log-linear relationship between HGT frequency and ribosomal protein evolutionary distance that is maintained across the deepest divergences in Halobacteria. We use this mathematical relationship to estimate the total transfers and amino acid substitutions delivered by HGTs in each genome, providing a measure of chimerism. For the relaxed core genes of each genome, we conservatively estimate that 11–20% of their evolution occurred in other haloarchaea. Our findings are unexpected, because the transfer and homologous recombination of relaxed core genes between members of the class Halobacteria disrupts the coevolution of genes; however, the generation of new combinations of divergent but functionally related genes may lead to adaptive phenotypes not available through cumulative mutations and recombination within a single population.

Description: Telomeres, ubiquitous and essential structures of eukaryotic chromosomes, are known to come in a variety of forms, but knowledge about their actual diversity and evolution across the whole phylogenetic breadth of the eukaryotic life remains fragmentary. To fill this gap, we employed a complex experimental approach to probe telomeric minisatellites in various phylogenetically diverse groups of algae. Our most remarkable results include the following findings: 1) algae of the streptophyte class Klebsormidiophyceae possess the Chlamydomonas -type telomeric repeat (TTTTAGGG) or, in at least one species, a novel TTTTAGG repeat, indicating an evolutionary transition from the Arabidopsis -type repeat (TTTAGGG) ancestral for Chloroplastida; 2) the Arabidopsis -type repeat is also present in telomeres of Xanthophyceae, in contrast to the presence of the human-type repeat (TTAGGG) in other ochrophytes studied, and of the photosynthetic alveolate Chromera velia , consistent with its phylogenetic position close to apicomplexans and dinoflagellates; 3) glaucophytes and haptophytes exhibit the human-type repeat in their telomeres; and 4) ulvophytes and rhodophytes have unusual telomere structures recalcitrant to standard analysis. To obtain additional details on the distribution of different telomere types in eukaryotes, we performed in silico analyses of genomic data from major eukaryotic lineages, utilizing also genome assemblies from our on-going genome projects for representatives of three hitherto unsampled lineages (jakobids, malawimonads, and goniomonads). These analyses confirm the human-type repeat as the most common and possibly ancestral in eukaryotes, but alternative motifs replaced it along the phylogeny of diverse eukaryotic lineages, some of them several times independently.

Description: Domain architectures and catalytic functions of enzymes constitute the centerpieces of a metabolic network. These types of information are formulated as a two-layered network consisting of domains, proteins, and reactions—a domain–protein–reaction (DPR) network. We propose an algorithm to reconstruct the evolutionary history of DPR networks across multiple species and categorize the mechanisms of metabolic systems evolution in terms of network changes. The reconstructed history reveals distinct patterns of evolutionary mechanisms between prokaryotic and eukaryotic networks. Although the evolutionary mechanisms in early ancestors of prokaryotes and eukaryotes are quite similar, more novel and duplicated domain compositions with identical catalytic functions arise along the eukaryotic lineage. In contrast, prokaryotic enzymes become more versatile by catalyzing multiple reactions with similar chemical operations. Moreover, different metabolic pathways are enriched with distinct network evolution mechanisms. For instance, although the pathways of steroid biosynthesis, protein kinases, and glycosaminoglycan biosynthesis all constitute prominent features of animal-specific physiology, their evolution of domain architectures and catalytic functions follows distinct patterns. Steroid biosynthesis is enriched with reaction creations but retains a relatively conserved repertoire of domain compositions and proteins. Protein kinases retain conserved reactions but possess many novel domains and proteins. In contrast, glycosaminoglycan biosynthesis has high rates of reaction/protein creations and domain recruitments. Finally, we elicit and validate two general principles underlying the evolution of DPR networks: 1) duplicated enzyme proteins possess similar catalytic functions and 2) the majority of novel domains arise to catalyze novel reactions. These results shed new lights on the evolution of metabolic systems.

Description: Alternative splicing (AS) is a major mechanism of increasing proteome diversity in complex organisms. Different AS transcript isoforms may be translated into peptide sequences of significantly different lengths and amino acid compositions. One important question, then, is how AS is constrained by protein structural requirements while peptide sequences may be significantly changed in AS events. Here, we address this issue by examining whether the intactness of three-dimensional protein structural units (compact units in protein structures, namely protein units [PUs]) tends to be preserved in AS events in human. We show that PUs tend to occur in constitutively spliced exons and to overlap constitutive exon boundaries. Furthermore, when PUs are located at the boundaries between two alternatively spliced exons (ASEs), these neighboring ASEs tend to co-occur in different transcript isoforms. In addition, such PU-spanned ASE pairs tend to have a higher frequency of being included in transcript isoforms. ASE regions that overlap with PUs also have lower nonsynonymous-to-synonymous substitution rate ratios than those that do not overlap with PUs, indicating stronger negative selection pressure in PU-overlapped ASE regions. Of note, we show that PUs have protein domain- and structural orderness-independent effects on messenger RNA (mRNA) splicing. Overall, our results suggest that fine-scale protein structural requirements have significant influences on the splicing patterns of human mRNAs.

Description: Sequencing of the complete mitochondrial genome of the soft coral Paraminabea aldersladei (Alcyoniidae) revealed a unique gene order, the fifth mt gene arrangement now known within the cnidarian subclass Octocorallia. At 19,886 bp, the mt genome of P. aldersladei is the second largest known for octocorals; its gene content and nucleotide composition are, however, identical to most other octocorals, and the additional length is due to the presence of two large, noncoding intergenic regions. Relative to the presumed ancestral octocoral gene order, in P. aldersladei a block of three protein-coding genes ( nad6–nad3–nad4l ) has been translocated and inverted. Mapping the distribution of mt gene arrangements onto a taxonomically comprehensive phylogeny of Octocorallia suggests that all of the known octocoral gene orders have evolved by successive inversions of one or more evolutionarily conserved blocks of protein-coding genes. This mode of genome evolution is unique among Metazoa, and contrasts strongly with that observed in Hexacorallia, in which extreme gene shuffling has occurred among taxonomic orders. Two of the four conserved gene blocks found in Octocorallia are, however, also conserved in the linear mt genomes of Medusozoa and in one group of Demospongiae. We speculate that the rate and mechanism of gene rearrangement in octocorals may be influenced by the presence in their mt genomes of mtMutS , a putatively active DNA mismatch repair protein that may also play a role in mediating intramolecular recombination.

Description: Gene duplication is an important evolutionary mechanism and no eukaryote has more duplicated gene families than the parasitic protist Trichomonas vaginalis . Iron is an essential nutrient for Trichomonas and plays a pivotal role in the establishment of infection, proliferation, and virulence. To gain insight into the role of iron in T. vaginalis gene expression and genome evolution, we screened iron-regulated genes using an oligonucleotide microarray for T. vaginalis and by comparative EST (expressed sequence tag) sequencing of cDNA libraries derived from trichomonads cultivated under iron-rich (+Fe) and iron-restricted (–Fe) conditions. Among 19,000 ESTs from both libraries, we identified 336 iron-regulated genes, of which 165 were upregulated under +Fe conditions and 171 under –Fe conditions. The microarray analysis revealed that 195 of 4,950 unique genes were differentially expressed. Of these, 117 genes were upregulated under +Fe conditions and 78 were upregulated under –Fe conditions. The results of both methods were congruent concerning the regulatory trends and the representation of gene categories. Under +Fe conditions, the expression of proteins involved in carbohydrate metabolism, particularly in the energy metabolism of hydrogenosomes, and in methionine catabolism was increased. The iron–sulfur cluster assembly machinery and certain cysteine proteases are of particular importance among the proteins upregulated under –Fe conditions. A unique feature of the T. vaginalis genome is the retention during evolution of multiple paralogous copies for a majority of all genes. Although the origins and reasons for this gene expansion remain unclear, the retention of multiple gene copies could provide an opportunity to evolve differential expression during growth in variable environmental conditions. For genes whose expression was affected by iron, we found that iron influenced the expression of only some of the paralogous copies, whereas the expression of the other paralogs was iron independent. This finding indicates a very stringent regulation of the differentially expressed paralogous genes in response to changes in the availability of exogenous nutrients and provides insight into the evolutionary rationale underlying massive paralog retention in the Trichomonas genome.

Description: In evolution of mammals, some of essential genes for placental development are known to be of retroviral origin, as syncytin-1 derived from an envelope ( env ) gene of an endogenous retrovirus (ERV) aids in the cell fusion of placenta in humans. Although the placenta serves the same function in all placental mammals, env -derived genes responsible for trophoblast cell fusion and maternal immune tolerance differ among species and remain largely unidentified in the bovine species. To examine env -derived genes playing a role in the bovine placental development comprehensively, we determined the transcriptomic profiles of bovine conceptuses during three crucial windows of implantation periods using a high-throughput sequencer. The sequence reads were mapped into the bovine genome, in which ERV candidates were annotated using RetroTector © (7,624 and 1,542 for ERV-derived and env -derived genes, respectively). The mapped reads showed that approximately 18% (284 genes) of env -derived genes in the genome were expressed during placenta formation, and approximately 4% (63 genes) were detected for all days examined. We verified three env -derived genes that are expressed in trophoblast cells by polymerase chain reaction. Out of these three, the sequence of env -derived gene with the longest open reading frame (named BERV-P env ) was found to show high expression levels in trophoblast cell lines and to be similar to those of syncytin-Car1 genes found in dogs and cats, despite their disparate origins. These results suggest that placentation depends on various retrovirus-derived genes that could have replaced endogenous predecessors during evolution.

Description: Recent studies suggested a role for the human endogenous retrovirus (HERV) group HERV-K(HML-2) in melanoma because of upregulated transcription and expression of HERV-K(HML-2)-encoded proteins. Very little is known about which HML-2 loci are transcribed in melanoma. We assigned 〉1,400 HML-2 cDNA sequences generated from various melanoma and related samples to genomic HML-2 loci, identifying a total of 23 loci as transcribed. Transcription profiles of loci differed significantly between samples. One locus was found transcribed only in melanoma-derived samples but not in melanocytes and might represent a marker for melanoma. Several of the transcribed loci harbor ORFs for retroviral Gag and/or Env proteins. Env-encoding loci were transcribed only in melanoma. Specific investigation of rec and np9 transcripts indicated transcription of protein encoding loci in melanoma and melanocytes hinting at the relevance of Rec and Np9 in melanoma. UVB irradiation changed transcription profiles of loci and overall transcript levels decreased in melanoma and melanocytes. We further identified transcribed HML-2 loci formed by reverse transcription of spliced HML-2 transcripts by L1 machinery or in a retroviral fashion, with loci potentially encoding HML-2-like proteins. We reveal complex, sample-specific transcription of HML-2 loci in melanoma and related samples. Identified HML-2 loci and proteins encoded by those loci are particularly relevant for further studying the role of HML-2 in melanoma. Transcription of HERVs appears as a complex mechanism requiring specific studies to elucidate which HERV loci are transcribed and how transcribed HERVs may be involved in disease.

Description: Microsatellites (SSRs) are highly susceptible to expansions and contractions. When located in a coding sequence, the insertion or the deletion of a single unit for a mono-, di-, tetra-, or penta(nucleotide)-SSR creates a frameshift. As a consequence, one would expect to find only very few of these SSRs in coding sequences because of their strong deleterious potential. Unexpectedly, genomes contain many coding SSRs of all types. Here, we report on a study of their evolution in a phylogenetic context using the genomes of four primates: human, chimpanzee, orangutan, and macaque. In a set of 5,015 orthologous genes unambiguously aligned among the four species, we show that, except for tri- and hexa-SSRs, for which insertions and deletions are frequently observed, SSRs in coding regions evolve mainly by substitutions. We show that the rate of substitution in all types of coding SSRs is typically two times higher than in the rest of coding sequences. Additionally, we observe that although numerous coding SSRs are created and lost by substitutions in the lineages, their numbers remain constant. This last observation suggests that the coding SSRs have reached equilibrium. We hypothesize that this equilibrium involves a combination of mutation, drift, and selection. We thus estimated the fitness cost of mono-SSRs and show that it increases with the number of units. We finally show that the cost of coding mono-SSRs greatly varies from function to function, suggesting that the strength of the selection that acts against them can be correlated to gene functions.

Description: The cAMP receptor protein (CRP)/fumarate and nitrate reduction regulatory protein (FNR)-type transcription factors (TFs) are members of a well-characterized global TF family in bacteria and have two conserved domains: the N-terminal ligand-binding domain for small molecules (e.g., cAMP, NO, or O 2 ) and the C-terminal DNA-binding domain. Although the CRP/FNR-type TFs recognize very similar consensus DNA target sequences, they can regulate different sets of genes in response to environmental signals. To clarify the evolution of the CRP/FNR-type TFs throughout the bacterial kingdom, we undertook a comprehensive computational analysis of a large number of annotated CRP/FNR-type TFs and the corresponding bacterial genomes. Based on the amino acid sequence similarities among 1,455 annotated CRP/FNR-type TFs, spectral clustering classified the TFs into 12 representative groups, and stepwise clustering allowed us to propose a possible process of protein evolution. Although each cluster mainly consists of functionally distinct members (e.g., CRP, NTC, FNR-like protein, and FixK), FNR-related TFs are found in several groups and are distributed in a wide range of bacterial phyla in the sequence similarity network. This result suggests that the CRP/FNR-type TFs originated from an ancestral FNR protein, involved in nitrogen fixation. Furthermore, a phylogenetic profiling analysis showed that combinations of TFs and their target genes have fluctuated dynamically during bacterial evolution. A genome-wide analysis of TF-binding sites also suggested that the diversity of the transcriptional regulatory system was derived by the stepwise adaptation of TF-binding sites to the evolution of TFs.

Description: Hybridization and abiotic stress are natural agents hypothesized to influence activation and proliferation of transposable elements in wild populations. In this report, we examine the effects of these agents on expression dynamics of both quiescent and transcriptionally active sublineages of long terminal repeat (LTR) retrotransposons in wild sunflower species with a notable history of transposable element proliferation. For annual sunflower species Helianthus annuus and H. petiolaris , neither early generation hybridization nor abiotic stress, alone or in combination, induced transcriptional activation of quiescent sublineages of LTR retrotransposons. These treatments also failed to further induce expression of sublineages that are transcriptionally active; instead, expression of active sublineages in F1 and backcross hybrids was nondistinguishable from, or intermediate relative to, parental lines, and abiotic stress generally decreased normalized expression relative to controls. In contrast to findings for early generation hybridization between H. annuus and H. petiolaris , ancient sunflower hybrid species derived from these same two species and which have undergone massive proliferation events of LTR retrotransposons display 2 x to 6 x higher expression levels of transcriptionally active sublineages relative to parental sunflower species H. annuus and H. petiolaris . Implications and possible explanations for these findings are discussed.

Description: It has been long known that insect-infecting trypanosomatid flagellates from the genera Angomonas and Strigomonas harbor bacterial endosymbionts ( Candidatus Kinetoplastibacterium or TPE [trypanosomatid proteobacterial endosymbiont]) that supplement the host metabolism. Based on previous analyses of other bacterial endosymbiont genomes from other lineages, a stereotypical path of genome evolution in such bacteria over the duration of their association with the eukaryotic host has been characterized. In this work, we sequence and analyze the genomes of five TPEs, perform their metabolic reconstruction, do an extensive phylogenomic analyses with all available Betaproteobacteria, and compare the TPEs with their nearest betaproteobacterial relatives. We also identify a number of housekeeping and central metabolism genes that seem to have undergone positive selection. Our genome structure analyses show total synteny among the five TPEs despite millions of years of divergence, and that this lineage follows the common path of genome evolution observed in other endosymbionts of diverse ancestries. As previously suggested by cell biology and biochemistry experiments, Ca. Kinetoplastibacterium spp. preferentially maintain those genes necessary for the biosynthesis of compounds needed by their hosts. We have also shown that metabolic and informational genes related to the cooperation with the host are overrepresented amongst genes shown to be under positive selection. Finally, our phylogenomic analysis shows that, while being in the Alcaligenaceae family of Betaproteobacteria, the closest relatives of these endosymbionts are not in the genus Bordetella as previously reported, but more likely in the Taylorella genus.

Description: Many insect species have established long-term symbiotic relationships with intracellular bacteria. Symbiosis with bacteria has provided insects with novel ecological capabilities, which have allowed them colonize previously unexplored niches. Despite its importance to the understanding of the emergence of biological complexity, the evolution of symbiotic relationships remains hitherto a mystery in evolutionary biology. In this study, we contribute to the investigation of the evolutionary leaps enabled by mutualistic symbioses by sequencing the genome of Blattabacterium cuenoti , primary endosymbiont of the omnivorous cockroach Blatta orientalis, and one of the most ancient symbiotic associations. We perform comparative analyses between the Blattabacterium cuenoti genome and that of previously sequenced endosymbionts, namely those from the omnivorous hosts the Blattella germanica (Blattelidae) and Periplaneta americana (Blattidae), and the endosymbionts harbored by two wood-feeding hosts, the subsocial cockroach Cryptocercus punctulatus (Cryptocercidae) and the termite Mastotermes darwiniensis (Termitidae). Our study shows a remarkable evolutionary stasis of this symbiotic system throughout the evolutionary history of cockroaches and the deepest branching termite M. darwiniensis , in terms of not only chromosome architecture but also gene content, as revealed by the striking conservation of the Blattabacterium core genome. Importantly, the architecture of central metabolic network inferred from the endosymbiont genomes was established very early in Blattabacterium evolutionary history and could be an outcome of the essential role played by this endosymbiont in the host’s nitrogen economy.

Description: Whole-genome duplications (WGDs) have recurred in the evolution of angiosperms, resulting in many duplicated chromosomal segments. Local gene duplications are also widespread in angiosperms. WGD-derived duplicates, that is, ohnologs, and local duplicates often show contrasting patterns of gene retention and evolution. However, many genes in angiosperms underwent multiple gene duplication events, possibly by different modes, indicating that different modes of gene duplication are not mutually exclusive. In two representative angiosperm genomes, Arabidopsis ( Arabidopsis thaliana ) and rice ( Oryza sativa ), we found that 9.6% and 11.3% of unique ohnologs, corresponding to 15.5% and 17.1% of ohnolog pairs, were also involved in local duplications, respectively. Locally duplicated ohnologs are widely distributed in different duplicated chromosomal segments and functionally biased. Coding sequence divergence between duplicated genes is denoted by nonsynonymous (Ka) and synonymous (Ks) substitution rates. Locally duplicated ohnolog pairs tend to have higher Ka, Ka/Ks, and gene expression divergence than nonlocally duplicated ohnolog pairs. Locally duplicated ohnologs also tend to have higher interspecies sequence divergence. These observations indicate that locally duplicated ohnologs evolve faster than nonlocally duplicated ohnologs. This study highlights the necessity to take local duplications into account when analyzing the evolutionary dynamics of ohnologs.

Description: Evolution of prokaryotes involves extensive loss and gain of genes, which lead to substantial differences in the gene repertoires even among closely related organisms. Through a wide range of phylogenetic depths, gene frequency distributions in prokaryotic pangenomes bear a characteristic, asymmetrical U-shape, with a core of (nearly) universal genes, a "shell" of moderately common genes, and a "cloud" of rare genes. We employ mathematical modeling to investigate evolutionary processes that might underlie this universal pattern. Gene frequency distributions for almost 400 groups of 10 bacterial or archaeal species each over a broad range of evolutionary distances were fit to steady-state, infinite allele models based on the distribution of gene replacement rates and the phylogenetic tree relating the species in each group. The fits of the theoretical frequency distributions to the empirical ones yield model parameters and estimates of the goodness of fit. Using the Akaike Information Criterion, we show that the neutral model of genome evolution, with the same replacement rate for all genes, can be confidently rejected. Of the three tested models with purifying selection, the one in which the distribution of replacement rates is derived from a stochastic population model with additive per-gene fitness yields the best fits to the data. The selection strength estimated from the fits declines with evolutionary divergence while staying well outside the neutral regime. These findings indicate that, unlike some other universal distributions of genomic variables, for example, the distribution of paralogous gene family membership, the gene frequency distribution is substantially affected by selection.

Description: The Drosophila Y chromosome is a degenerated, heterochromatic chromosome with few functional genes. Despite this, natural variation on the Y chromosome in D. melanogaster has substantial trans- acting effects on the regulation of X-linked and autosomal genes. It is not clear, however, whether these genes simply represent a random subset of the genome or whether specific functional properties are associated with susceptibility to regulation by Y-linked variation. Here, we present a meta-analysis of four previously published microarray studies of Y-linked regulatory variation (YRV) in D. melanogaster . We show that YRV genes are far from a random subset of the genome: They are more likely to be in repressive chromatin contexts, be expressed tissue specifically, and vary in expression within and between species than non-YRV genes. Furthermore, YRV genes are more likely to be associated with the nuclear lamina than non-YRV genes and are generally more likely to be close to each other in the nucleus (although not along chromosomes). Taken together, these results suggest that variation on the Y chromosome plays a role in modifying how the genome is distributed across chromatin compartments, either via changes in the distribution of DNA-binding proteins or via changes in the spatial arrangement of the genome in the nucleus.

Description: Molecular phylogenetic studies have not yet reached a consensus on the placement of Ginkgoales, which is represented by the only living species, Ginkgo biloba (common name: ginkgo). At least six discrepant placements of ginkgo have been proposed. This study aimed to use the chloroplast phylogenomic approach to examine possible factors that lead to such disagreeing placements. We found the sequence types used in the analyses as the most critical factor in the conflicting placements of ginkgo. In addition, the placement of ginkgo varied in the trees inferred from nucleotide (NU) sequences, which notably depended on breadth of taxon sampling, tree-building methods, codon positions, positions of Gnetopsida (common name: gnetophytes), and including or excluding gnetophytes in data sets. In contrast, the trees inferred from amino acid (AA) sequences congruently supported the monophyly of a ginkgo and Cycadales (common name: cycads) clade, regardless of which factors were examined. Our site-stripping analysis further revealed that the high substitution saturation of NU sequences mainly derived from the third codon positions and contributed to the variable placements of ginkgo. In summary, the factors we surveyed did not affect results inferred from analyses of AA sequences. Congruent topologies in our AA trees give more confidence in supporting the ginkgo–cycad sister-group hypothesis.

Description: Rieske/cytochrome b (Rieske/cyt b ) complexes are proton pumping quinol oxidases that are present in most bacteria and Archaea. The phylogeny of their subunits follows closely the 16S-rRNA phylogeny, indicating that chemiosmotic coupling was already present in the last universal common ancestor of Archaea and bacteria. Haloarchaea are the only organisms found so far that acquired Rieske/cyt b complexes via interdomain lateral gene transfer. They encode two Rieske/cyt b complexes in their genomes; one of them is found in genetic context with nitrate reductase genes and has its closest relatives among Actinobacteria and the Thermus / Deinococcus group. It is likely to function in nitrate respiration. The second Rieske/cyt b complex of Haloarchaea features a split cytochrome b sequence as do Cyanobacteria, chloroplasts, Heliobacteria, and Bacilli. It seems that Haloarchaea acquired this complex from an ancestor of the above-mentioned phyla. Its involvement in the bioenergetic reaction chains of Haloarchaea is unknown. We present arguments in favor of the hypothesis that the ancestor of Haloarchaea, which relied on a highly specialized bioenergetic metabolism, that is, methanogenesis, and was devoid of quinones and most enzymes of anaerobic or aerobic bioenergetic reaction chains, integrated laterally transferred genes into its genome to respond to a change in environmental conditions that made methanogenesis unfavorable.

Description: The availability of genome sequences of Thermotogales species from across the order allows an examination of the evolutionary origins of phenotypic characteristics in this lineage. Several studies have shown that the Thermotogales have acquired large numbers of genes from distantly related lineages, particularly Firmicutes and Archaea. Here, we report the finding that some Thermotogales acquired the ability to synthesize vitamin B 12 by acquiring the requisite genes from these distant lineages. Thermosipho species, uniquely among the Thermotogales, contain genes that encode the means to synthesize vitamin B 12 de novo from glutamate. These genes are split into two gene clusters: the corrinoid synthesis gene cluster, that is unique to the Thermosipho and the cobinamide salvage gene cluster. The corrinoid synthesis cluster was acquired from the Firmicutes lineage, whereas the salvage pathway is an amalgam of bacteria- and archaea-derived proteins. The cobinamide salvage gene cluster has a patchy distribution among Thermotogales species, and ancestral state reconstruction suggests that this pathway was present in the common Thermotogales ancestor. We show that Thermosipho africanus can grow in the absence of vitamin B 12 , so its de novo pathway is functional. We detected vitamin B 12 in the extracts of T. africanus cells to verify the synthetic pathway. Genes in T. africanus with apparent B 12 riboswitches were found to be down-regulated in the presence of vitamin B 12 consistent with their roles in B 12 synthesis and cobinamide salvage.

Description: Gene sequences are routinely used to determine the topologies of unrooted phylogenetic trees, but many of the most important questions in evolution require knowing both the topologies and the roots of trees. However, general algorithms for calculating rooted trees from gene and genomic sequences in the absence of gene paralogs are few. Using the principles of evolutionary parsimony (EP) (Lake JA. 1987a. A rate-independent technique for analysis of nucleic acid sequences: evolutionary parsimony. Mol Biol Evol. 4:167–181) and its extensions (Cavender, J. 1989. Mechanized derivation of linear invariants. Mol Biol Evol. 6:301–316; Nguyen T, Speed TP. 1992. A derivation of all linear invariants for a nonbalanced transversion model. J Mol Evol. 35:60–76), we explicitly enumerate all linear invariants that solely contain rooting information and derive algorithms for rooting gene trees directly from gene and genomic sequences. These new EP linear rooting invariants allow one to determine rooted trees, even in the complete absence of outgroups and gene paralogs. EP rooting invariants are explicitly derived for three taxon trees, and rules for their extension to four or more taxa are provided. The method is demonstrated using 18S ribosomal DNA to illustrate how the new animal phylogeny (Aguinaldo AMA et al. 1997. Evidence for a clade of nematodes, arthropods, and other moulting animals. Nature 387:489–493; Lake JA. 1990. Origin of the metazoa. Proc Natl Acad Sci USA 87:763–766) may be rooted directly from sequences, even when they are short and paralogs are unavailable. These results are consistent with the current root (Philippe H et al. 2011. Acoelomorph flatworms are deuterostomes related to Xenoturbella . Nature 470:255–260).

Description: Phylogenomics has revealed the existence of fast-evolving animal phyla in which the amino acid substitution rate, averaged across many proteins, is consistently higher than in other lineages. The reasons for such differences in proteome-wide evolutionary rates are still unknown, largely because only a handful of species offer within-species genomic data from which molecular evolutionary processes can be deduced. In this study, we use next-generation sequencing technologies and individual whole-transcriptome sequencing to gather extensive polymorphism sequence data sets from Ciona intestinalis . Ciona is probably the best-characterized member of the fast-evolving Urochordata group (tunicates), which was recently identified as the sister group of the slow-evolving vertebrates. We introduce and validate a maximum-likelihood framework for single-nucleotide polymorphism and genotype calling, based on high-throughput short-read typing. We report that the C. intestinalis proteome is characterized by a high level of within-species diversity, efficient purifying selection, and a substantial percentage of adaptive amino acid substitutions. We conclude that the increased rate of amino acid sequence evolution in tunicates, when compared with vertebrates, is the consequence of both a 2–6 times higher per-year mutation rate and prevalent adaptive evolution.

Description: Mitochondria are essential organelles whose replication, development, and physiology are dependent upon coordinated gene interactions with both the mitochondrial and the nuclear genomes. The evolution of coadapted (CA) nuclear–mitochondrial gene combinations would be facilitated if such nuclear genes were located on the X-chromosome instead of on the autosomes because of the increased probability of cotransmission. Here, we test the prediction of the CA hypothesis by investigating the chromosomal distribution of nuclear genes that interact with mitochondria. Using the online genome database BIOMART, we compared the density of genes that have a mitochondrion cellular component annotation across chromosomes in 16 vertebrates. We find a strong and highly significant genomic pattern against the CA hypothesis: nuclear genes interacting with the mitochondrion are significantly underrepresented on the X-chromosome in mammals but not in birds. We interpret our findings in terms of sexual conflict as a mechanism that may generate the observed pattern. Our finding extends single-gene theory for the evolution of sexually antagonistic genes to nuclear–mitochondrial gene combinations.

Description: Phenotypic regression of morphological, behavioral, or physiological traits can evolve when reduced trait expression has neutral or beneficial effects on overall performance. Studies on the evolution of phenotypic degradation in animals have concentrated mostly on the evaluation of resulting phenotypes, whereas much less research has been dedicated to uncovering the molecular mechanisms that underlie phenotypic regression. The majority of parasitoids (i.e., insects that develop on or inside other arthropods), do not accumulate lipid reserves during their free-living adult life-stage and represent an excellent system to study phenotypic regression in animals. Here, we study transcriptional patterns associated with lack of lipogenesis in the parasitic wasp Nasonia vitripennis . We first confirmed that N. vitripennis does not synthesize lipids by showing a reduction in lipid reserves despite ingestion of dietary sugar, and a lack of incorporation of isotopic labels into lipid reserves when fed deuterated sugar solution. Second, we investigated transcriptional responses of 28 genes involved in lipid and sugar metabolism in short- and long-term sugar-fed females relative to starved females of N. vitripennis. Sugar feeding did not induce transcription of fatty acid synthase ( fas ) or other key genes involved in the lipid biosynthesis pathway. Furthermore, several genes involved in carbohydrate metabolism had a lower transcription in fed than in starved females. Our results reveal that N. vitripennis gene transcription in response to dietary sugar deviates markedly from patterns typically observed in other organisms. This study is the first to identify differential gene transcription associated with lack of lipogenesis in parasitoids and provides new insights into the molecular mechanism that underlies phenotypic regression of this trait.

Description: Splicing regulatory elements (SREs) are sequences bound by proteins that influence splicing of nearby splice sites. Constitutively spliced introns have evolved to utilize many different splicing factors. The evolutionary processes that influenced which splicing factors are used for splicing of individual introns are generally unclear. We demonstrate that in the lineage that gave rise to mammals, many introns lost U-rich sequences and gained G-rich sequences, both of which resemble known SREs. The apparent conversion of U-rich to G-rich SREs suggests that the associated splicing factors are functionally equivalent. In support of this we demonstrated that U-rich and G-rich SREs are both capable of promoting splicing of an SRE-dependent splicing reporter. Furthermore, we demonstrate, using the heterologous MS2 tethering system (bacterial MS2 coat fusion-protein and its RNA stem-loop binding site), that both the U-rich SRE-binding protein (TIA1) and the G-rich SRE-binding protein (HNRNPF) can promote splicing of the same intron. We also observed that gain of G-rich SREs is significantly associated with G/C-rich genomic isochores, suggesting that gain or loss of SREs was driven by the same processes that ultimately resulted in the formation of mammalian genomic isochores. We propose the following model for the gain and loss of mammalian SREs. Ancestral U-rich SREs located in genomic regions that were experiencing high rates of A/T to G/C conversion would have suffered frequent deleterious mutations. However, this same process resulted in increased formation of functionally equivalent G-rich SREs, and acquisition of new G-rich SREs decreased purifying selection on the U-rich SREs, which were then free to decay.

Description: GC-biased gene conversion (gBGC) is a process that tends to increase the GC content of recombining DNA over evolutionary time and is thought to explain the evolution of GC content in mammals and yeasts. Evidence for gBGC outside these two groups is growing but is still limited. Here, we analyzed 36 completely sequenced genomes representing four of the five major groups in eukaryotes (Unikonts, Excavates, Chromalveolates and Plantae). gBGC was investigated by directly comparing GC content and recombination rates in species where recombination data are available, that is, half of them. To study all species of our dataset, we used chromosome size as a proxy for recombination rate and compared it with GC content. Among the 17 species showing a significant relationship between GC content and chromosome size, 15 are consistent with the predictions of the gBGC model. Importantly, the species showing a pattern consistent with gBGC are found in all the four major groups of eukaryotes studied, which suggests that gBGC may be widespread in eukaryotes.

Description: Photosynthetic diatom plastids have long been suggested to have originated by the secondary endosymbiosis of a red alga. However, recent phylogenomic studies report a high number of diatom nuclear genes phylogenetically related to green algal and green plant genes. These were interpreted as endosymbiotic gene transfers (EGT) from a cryptic green algal endosymbiosis. We reanalyzed this issue using a larger set of red algal genomic data. We show that previous studies suffer from a taxonomic sampling bias and point out that a majority of gene phylogenies are either poorly resolved or do not describe EGT events. We finally show that genes having a complete descent from cyanobacteria to diatoms through primary and secondary EGTs have been mostly transferred via a red alga. We conclude that, even if some diatom genes still support a putative green algal origin, these are not sufficient to argue for a cryptic green algal secondary endosymbiosis.

Description: Herbivorous insects are among the most successful radiations of life. However, we know little about the processes underpinning the evolution of herbivory. We examined the evolution of herbivory in the fly, Scaptomyza flava , whose larvae are leaf miners on species of Brassicaceae, including the widely studied reference plant, Arabidopsis thaliana ( Arabidopsis ). Scaptomyza flava is phylogenetically nested within the paraphyletic genus Drosophila , and the whole genome sequences available for 12 species of Drosophila facilitated phylogenetic analysis and assembly of a transcriptome for S. flava . A time-calibrated phylogeny indicated that leaf mining in Scaptomyza evolved between 6 and 16 million years ago. Feeding assays showed that biosynthesis of glucosinolates, the major class of antiherbivore chemical defense compounds in mustard leaves, was upregulated by S. flava larval feeding. The presence of glucosinolates in wild-type (WT) Arabidopsis plants reduced S. flava larval weight gain and increased egg–adult development time relative to flies reared in glucosinolate knockout (GKO) plants. An analysis of gene expression differences in 5-day-old larvae reared on WT versus GKO plants showed a total of 341 transcripts that were differentially regulated by glucosinolate uptake in larval S. flava . Of these, approximately a third corresponded to homologs of Drosophila melanogaster genes associated with starvation, dietary toxin-, heat-, oxidation-, and aging-related stress. The upregulated transcripts exhibited elevated rates of protein evolution compared with unregulated transcripts. The remaining differentially regulated transcripts also contained a higher proportion of novel genes than the unregulated transcripts. Thus, the transition to herbivory in Scaptomyza appears to be coupled with the evolution of novel genes and the co-option of conserved stress-related genes.

Description: Continuing advances in genome sequencing technologies and computational methods for comparative genomics currently allow inferring the evolutionary history of entire plant and animal genomes. Based on the comparison of the plant and animal genome paleohistory, major differences are unveiled in 1) evolutionary mechanisms (i.e ., polyploidization versus diploidization processes), 2) genome conservation (i.e ., coding versus noncoding sequence maintenance), and 3) modern genome architecture (i.e., genome organization including repeats expansion versus contraction phenomena). This article discusses how extant animal and plant genomes are the result of inherently different rates and modes of genome evolution resulting in relatively stable animal and much more dynamic and plastic plant genomes.

Description: The majority of proteins in eukaryotes are composed of multiple domains, and the number and order of these domains is an important determinant of protein function. Although multidomain proteins with a particular domain architecture were initially considered to have a common evolutionary origin, recent comparative studies of protein families or whole genomes have reported that a minority of multidomain proteins could have appeared multiple times independently. Here, we test this scenario in detail for the signaling molecules netrin and secreted frizzled-related proteins (sFRPs), two groups of netrin domain-containing proteins with essential roles in animal development. Our primary phylogenetic analyses suggest that the particular domain architectures of each of these proteins were present in the eumetazoan ancestor and evolved a second time independently within the metazoan lineage from laminin and frizzled proteins, respectively. Using an array of phylogenetic methods, statistical tests, and character sorting analyses, we show that the polyphyly of netrin and sFRP is well supported and cannot be explained by classical phylogenetic reconstruction artifacts. Despite their independent origins, the two groups of netrins and of sFRPs have the same protein interaction partners (Deleted in Colorectal Cancer/neogenin and Unc5 for netrins and Wnts for sFRPs) and similar developmental functions. Thus, these cases of convergent evolution emphasize the importance of domain architecture for protein function by uncoupling shared domain architecture from shared evolutionary history. Therefore, we propose the terms merology to describe the repeated evolution of proteins with similar domain architecture and discuss the potential of merologous proteins to help understanding protein evolution.

Description: Horizontal gene transfer (HGT) has been recognized to be an important mechanism that shaped the evolution and genomes of prokaryotes and unicellular eukaryotes. However, HGT is regarded to be exceedingly rare among eukaryotes. We discovered massive transfers of a DNA transposon, a Tc1 element encoding a transposase, between multiple teleost fishes and lampreys that last shared a common ancestor over 500 Ma. Members of this group of Tc1 elements were found to exhibit a mosaic phylogenetic distribution, yet their sequences were highly similar even between distantly related lineages (95%–99% identity). Our molecular phylogenetic analyses suggested that horizontal transfers of this element happened repeatedly, involving multiple teleost fishes that are phylogenetically only distantly related. Interestingly, almost all the affected teleost lineages are also known to be subject to lamprey parasitism, suggesting that the horizontal transfers between vertebrates might have occurred through parasite–host interaction. The genomes of several northern hemisphere lamprey species, including that of the sea lamprey ( Petromyzon marinus ), were found to contain thousands of copies of the foreign elements. Impact of this event is discussed in relation to other peculiar genomic features of lampreys.

Description: Vertebrates have experienced two rounds of whole-genome duplication (WGD) in the stem lineages of deep nodes within the group and a subsequent duplication event in the stem lineage of the teleosts—a highly diverse group of ray-finned fishes. Here, we present the first full Hox gene sequences for any member of the Acipenseriformes, the American paddlefish, and confirm that an independent WGD occurred in the paddlefish lineage, approximately 42 Ma based on sequences spanning the entire HoxA cluster and eight genes on the HoxD gene cluster. These clusters comprise different HOX loci and maintain conserved synteny relative to bichir, zebrafish, stickleback, and pufferfish, as well as human, mouse, and chick. We also provide a gene genealogy for the duplicated fzd8 gene in paddlefish and present evidence for the first Hox14 gene in any ray-finned fish. Taken together, these data demonstrate that the American paddlefish has an independently duplicated genome. Substitution patterns of the "alpha" paralogs on both the HoxA and HoxD gene clusters suggest transcriptional inactivation consistent with functional diploidization. Further, there are similarities in the pattern of sequence divergence among duplicated Hox genes in paddlefish and teleost lineages, even though they occurred independently approximately 200 Myr apart. We highlight implications on comparative analyses in the study of the "fin-limb transition" as well as gene and genome duplication in bony fishes, which includes all ray-finned fishes as well as the lobe-finned fishes and tetrapod vertebrates.

Description: Microsatellites make up ~3% of the human genome, and there is increasing evidence that some microsatellites can have important functions and can be conserved by selection. To investigate this conservation, we performed a genome-wide analysis of human microsatellites and measured their conservation using a binary character birth--death model on a mammalian phylogeny. Using a maximum likelihood method to estimate birth and death rates for different types of microsatellites, we show that the rates at which microsatellites are gained and lost in mammals depend on their sequence composition, length, and position in the genome. Additionally, we use a mixture model to account for unequal death rates among microsatellites across the human genome. We use this model to assign a probability-based conservation score to each microsatellite. We found that microsatellites near the transcription start sites of genes are often highly conserved, and that distance from a microsatellite to the nearest transcription start site is a good predictor of the microsatellite conservation score. An analysis of gene ontology terms for genes that contain microsatellites near their transcription start site reveals that regulatory genes involved in growth and development are highly enriched with conserved microsatellites.

Description: The transition from endosymbiont to organelle in eukaryotic cells involves the transfer of significant numbers of genes to the host genomes, a process known as endosymbiotic gene transfer (EGT). In the case of plastid organelles, EGTs have been shown to leave a footprint in the nuclear genome that can be indicative of ancient photosynthetic activity in present-day plastid-lacking organisms, or even hint at the existence of cryptic plastids. Here, we evaluated the impact of EGT on eukaryote genomes by reanalyzing the recently published EST dataset for Chromera velia , an interesting test case of a photosynthetic alga closely related to apicomplexan parasites. Previously, 513 genes were reported to originate from red and green algae in a 1:1 ratio. In contrast, by manually inspecting newly generated trees indicating putative algal ancestry, we recovered only 51 genes congruent with EGT, of which 23 and 9 were of red and green algal origin, respectively, whereas 19 were ambiguous regarding the algal provenance. Our approach also uncovered 109 genes that branched within a monocot angiosperm clade, most likely representing a contamination. We emphasize the lack of congruence and the subjectivity resulting from independent phylogenomic screens for EGT, which appear to call for extreme caution when drawing conclusions for major evolutionary events.

Description: Gibbons (Hylobatidae) shared a common ancestor with the other hominoids only 15–18 million years ago. Nevertheless, gibbons show very distinctive features that include heavily rearranged chromosomes. Previous observations indicate that this phenomenon may be linked to the attenuated epigenetic repression of transposable elements (TEs) in gibbon species. Here we describe the massive expansion of a repeat in almost all the centromeres of the eastern hoolock gibbon ( Hoolock leuconedys ). We discovered that this repeat is a new composite TE originating from the combination of portions of three other elements (L1ME5, Alu Sz6, and SVA_A) and thus named it LAVA. We determined that this repeat is found in all the gibbons but does not occur in other hominoids. Detailed investigation of 46 different LAVA elements revealed that the majority of them have target site duplications (TSDs) and a poly-A tail, suggesting that they have been retrotransposing in the gibbon genome. Although we did not find a direct correlation between the emergence of LAVA elements and human–gibbon synteny breakpoints, this new composite transposable element is another mark of the great plasticity of the gibbon genome. Moreover, the centromeric expansion of LAVA insertions in the hoolock closely resembles the massive centromeric expansion of the KERV-1 retroelement reported for wallaby (marsupial) interspecific hybrids. The similarity between the two phenomena is consistent with the hypothesis that evolution of the gibbons is characterized by defects in epigenetic repression of TEs, perhaps triggered by interspecific hybridization.

Description: The transition from endosymbiont to organelle in eukaryotic cells involves the transfer of significant numbers of genes to the host genomes, a process known as endosymbiotic gene transfer (EGT). In the case of plastid organelles, EGTs have been shown to leave a footprint in the nuclear genome that can be indicative of ancient photosynthetic activity in present-day plastid-lacking organisms, or even hint at the existence of cryptic plastids. Here, we evaluated the impact of EGT on eukaryote genomes by reanalyzing the recently published EST dataset for Chromera velia , an interesting test case of a photosynthetic alga closely related to apicomplexan parasites. Previously, 513 genes were reported to originate from red and green algae in a 1:1 ratio. In contrast, by manually inspecting newly generated trees indicating putative algal ancestry, we recovered only 51 genes congruent with EGT, of which 23 and 9 were of red and green algal origin, respectively, whereas 19 were ambiguous regarding the algal provenance. Our approach also uncovered 109 genes that branched within a monocot angiosperm clade, most likely representing a contamination. We emphasize the lack of congruence and the subjectivity resulting from independent phylogenomic screens for EGT, which appear to call for extreme caution when drawing conclusions for major evolutionary events.

Description: It has long been known that many proteins require folding via molecular chaperones for their function. Although it has become apparent that folding imposes constraints on protein sequence evolution, the effects exerted by different chaperone classes are so far unknown. We have analyzed data of protein interaction with the chaperones in Saccharomyces cerevisiae using network methods. The results reveal a distinct community structure within the network that was hitherto undetectable with standard statistical tools. Sixty-four yeast chaperones comprise ten distinct modules that are defined by interaction specificity for their 2,691 interacting proteins. The classes of interacting proteins that are in turn defined by their dedicated chaperone modules are distinguished by various physiochemical protein properties and are characterized by significantly different protein expression levels, codon usage, and amino acid substitution rates. Correlations between substitution rate, codon bias, and gene expression level that have long been known for yeast are apparent at the level of the chaperone-defined modules. This indicates that correlated expression, conservation, and codon bias levels for yeast genes are attributable to previously unrecognized effects of protein folding. Proteome-wide categories of chaperone–substrate specificity uncover novel hubs of functional constraint in protein evolution that are conserved across 20 fungal genomes.

Description: The nature of a species remains a fundamental and controversial question. The era of genome/metagenome sequencing has intensified the debate in prokaryotes because of extensive horizontal gene transfer. In this study, we conducted a genome-wide survey of outcrossing homologous recombination in the highly sexual bacterial species Helicobacter pylori . We conducted multiple genome alignment and analyzed the entire data set of one-to-one orthologous genes for its global strains. We detected mosaic structures due to repeated recombination events and discordant phylogenies throughout the genomes of this species. Most of these genes including the "core" set of genes and horizontally transferred genes showed at least one recombination event. Taking into account the relationship between the nucleotide diversity and the minimum number of recombination events per nucleotide, we evaluated the recombination rate in every gene. The rate appears constant across the genome, but genes with a particularly high or low recombination rate were detected. Interestingly, genes with high recombination included those for DNA transformation and for basic cellular functions, such as biosynthesis and metabolism. Several highly divergent genes with a high recombination rate included those for host interaction, such as outer membrane proteins and lipopolysaccharide synthesis. These results provide a global picture of genome-wide distribution of outcrossing homologous recombination in a bacterial species for the first time, to our knowledge, and illustrate how a species can be shaped by mutual homologous recombination.

Description: Vertebrate genome comparisons revealed that there are highly conserved noncoding sequences (HCNSs) among a wide range of species and many of which contain regulatory elements. However, recently emerged sequences conserved in specific lineages have not been well studied. Toward this end, we identified 8,198 primate and 21,128 specific HCNSs as representative ones among mammals from human–marmoset and mouse–rat comparisons, respectively. Derived allele frequency analysis of primate-specific HCNSs showed that these HCNSs were under purifying selection, indicating that they may harbor important functions. We selected the top 1,000 largest HCNSs and compared the lineage-specific HCNS-flanking genes (LHF genes) with ultraconserved element (UCE)-flanking genes. Interestingly, the majority of LHF genes were different from UCE-flanking genes. This lineage-specific set of LHF genes was more enriched in protein-binding function. Conversely, the number of LHF genes that were also shared by UCEs was small but significantly larger than random expectation, and many of these genes were involved in anatomical development as transcriptional regulators, suggesting that certain groups of genes preferentially recruit new HCNSs in addition to old HCNSs that are conserved among vertebrates. This group of LHF genes might be involved in the various levels of lineage-specific evolution among vertebrates, mammals, primates, and rodents. If so, the emergence of HCNSs in and around these two groups of LHF genes developed lineage-specific characteristics. Our results provide new insight into lineage-specific evolution through interactions between HCNSs and their LHF genes.

Description: The role of adaptation is a fundamental question in molecular evolution. Theory predicts that species with large effective population sizes should undergo a higher rate of adaptive evolution than species with low effective population sizes if adaptation is limited by the supply of mutations. Previous analyses have appeared to support this conjecture because estimates of the proportion of nonsynonymous substitutions fixed by adaptive evolution, α, tend to be higher in species with large N e . However, α is a function of both the number of advantageous and effectively neutral substitutions, either of which might depend on N e . Here, we investigate the relationship between N e and a , the rate of adaptive evolution relative to the rate of neutral evolution, using nucleotide polymorphism and divergence data from 13 independent pairs of eukaryotic species. We find a highly significant positive correlation between a and N e . We also find some evidence that the rate of adaptive evolution varies between groups of organisms for a given N e . The correlation between a and N e does not appear to be an artifact of demographic change or selection on synonymous codon use. Our results suggest that adaptation is to some extent limited by the supply of mutations and that at least some adaptation depends on newly occurring mutations rather than on standing genetic variation. Finally, we show that the proportion of nearly neutral nonadaptive substitutions declines with increasing N e . The low rate of adaptive evolution and the high proportion of effectively neutral substitution in species with small N e are expected to combine to make it difficult to detect adaptive molecular evolution in species with small N e .

Description: Microsatellites make up ~3% of the human genome, and there is increasing evidence that some microsatellites can have important functions and can be conserved by selection. To investigate this conservation, we performed a genome-wide analysis of human microsatellites and measured their conservation using a binary character birth--death model on a mammalian phylogeny. Using a maximum likelihood method to estimate birth and death rates for different types of microsatellites, we show that the rates at which microsatellites are gained and lost in mammals depend on their sequence composition, length, and position in the genome. Additionally, we use a mixture model to account for unequal death rates among microsatellites across the human genome. We use this model to assign a probability-based conservation score to each microsatellite. We found that microsatellites near the transcription start sites of genes are often highly conserved, and that distance from a microsatellite to the nearest transcription start site is a good predictor of the microsatellite conservation score. An analysis of gene ontology terms for genes that contain microsatellites near their transcription start site reveals that regulatory genes involved in growth and development are highly enriched with conserved microsatellites.

Description: Two-pronged bristletails (Diplura) are traditionally classified into three major superfamilies: Campodeoidea, Projapygoidea, and Japygoidea. The interrelationships of these three superfamilies and the monophyly of Diplura have been much debated. Few previous studies included Projapygoidea in their phylogenetic considerations, and its position within Diplura still is a puzzle from both morphological and molecular points of view. Until now, no mitochondrial genome has been sequenced for any projapygoid species. To fill in this gap, we determined and annotated the complete mitochondrial genome of Octostigma sinensis (Octostigmatidae, Projapygoidea), and of three more dipluran species, one each from the Campodeidae, Parajapygidae, and Japygidae. All four newly sequenced dipluran mtDNAs encode the same set of genes in the same gene order as shared by most crustaceans and hexapods. Secondary structure truncations have occurred in trnR , trnC , trnS1 , and trnS2 , and the reduction of transfer RNA D-arms was found to be taxonomically correlated, with Campodeoidea having experienced the most reduction. Partitioned phylogenetic analyses, based on both amino acids and nucleotides of the protein-coding genes plus the ribosomal RNA genes, retrieve significant support for a monophyletic Diplura within Pancrustacea, with Projapygoidea more closely related to Campodeoidea than to Japygoidea. Another key finding is that monophyly of Diplura cannot be recovered unless Projapygoidea is included in the phylogenetic analyses; this explains the dipluran polyphyly found by past mitogenomic studies. Including Projapygoidea increased the sampling density within Diplura and probably helped by breaking up a long-branch-attraction artifact. This finding provides an example of how proper sampling is significant for phylogenetic inference.

Description: Microsporidia are an abundant group of obligate intracellular parasites of other eukaryotes, including immunocompromised humans, but the molecular basis of their intracellular lifestyle and pathobiology are poorly understood. New genomes from a taxonomically broad range of microsporidians, complemented by published expression data, provide an opportunity for comparative analyses to identify conserved and lineage-specific patterns of microsporidian genome evolution that have underpinned this success. In this study, we infer that a dramatic bottleneck in the last common microsporidian ancestor (LCMA) left a small conserved core of genes that was subsequently embellished by gene family expansion driven by gene acquisition in different lineages. Novel expressed protein families represent a substantial fraction of sequenced microsporidian genomes and are significantly enriched for signals consistent with secretion or membrane location. Further evidence of selection is inferred from the gain and reciprocal loss of functional domains between paralogous genes, for example, affecting transport proteins. Gene expansions among transporter families preferentially affect those that are located on the plasma membrane of model organisms, consistent with recruitment to plug conserved gaps in microsporidian biosynthesis and metabolism. Core microsporidian genes shared with other eukaryotes are enriched in orthologs that, in yeast, are highly expressed, highly connected, and often essential, consistent with strong negative selection against further reduction of the conserved gene set since the LCMA. Our study reveals that microsporidian genome evolution is a highly dynamic process that has balanced constraint, reductive evolution, and genome expansion during adaptation to an extraordinarily successful obligate intracellular lifestyle.

Description: It is well known that horizontal gene transfer (HGT) is a major force in the evolution of prokaryotes. During the adaptation of a bacterial population to a new ecological niche, and particularly for intracellular bacteria, selective pressures are shifted and ecological niches reduced, resulting in a lower rate of genetic connectivity. HGT and positive selection are therefore two important evolutionary forces in microbial pathogens that drive adaptation to new hosts. In this study, we use genomic distance analyses, phylogenomic networks, tree topology comparisons, and Bayesian inference methods to investigate to what extent HGT has occurred during the evolution of the genus Rickettsia , the effect of the use of different genomic regions in estimating reticulate evolution and HGT events, and the link of these to host range. We show that ecological specialization restricts recombination occurrence in Rickettsia , but other evolutionary processes and genome architecture are also important for the occurrence of HGT. We found that recombination, genomic rearrangements, and genome conservation all show evidence of network-like evolution at whole-genome scale. We show that reticulation occurred mainly, but not only, during the early Rickettsia radiation, and that core proteome genes of every major functional category have experienced reticulated evolution and possibly HGT. Overall, the evolution of Rickettsia bacteria has been tree-like, with evidence of HGT and reticulated evolution for around 10–25% of the core Rickettsia genome. We present evidence of extensive recombination/incomplete lineage sorting (ILS) during the radiation of the genus, probably linked with the emergence of intracellularity in a wide range of hosts.

Description: Major unresolved questions in evolutionary genetics include determining the contributions of different mutational sources to the total pool of genetic variation in a species, and understanding how these different forms of genetic variation interact with natural selection. Recent work has shown that structural variants (SVs) (insertions, deletions, inversions, and transpositions) are a major source of genetic variation, often outnumbering single nucleotide variants in terms of total bases affected. Despite the near ubiquity of SVs, major questions about their interaction with natural selection remain. For example, how does the allele frequency spectrum of SVs differ when compared with single nucleotide variants? How often do SVs affect genes, and what are the consequences? To begin to address these questions, we have systematically identified and characterized a large set of submicroscopic insertion and deletion (indel) variants (between 1 and 200 kb in length) among ten inbred lines from a single natural population of the plant species Mimulus guttatus . After extensive computational filtering, we focused on a set of 4,142 high-confidence indels that showed an experimental validation rate of 73%. All but one of these indels were less than 200 kb. Although the largest were generally at lower frequencies in the population, a surprising number of large indels are at intermediate frequencies. Although indels overlapping with genes were much rarer than expected by chance, approximately 600 genes were affected by an indel. Nucleotide-binding site leucine-rich repeat (NBS–LRR) defense response genes were the most enriched among the gene families affected. Most indels associated with genes were rare and appeared to be under purifying selection, though we do find four high-frequency derived insertion alleles that show signatures of recent positive selection.

Description: Phylogenetic inference is widely used to investigate the relationships between homologous sequences. RNA molecules have played a key role in these studies because they are present throughout life and tend to evolve slowly. Phylogenetic inference has been shown to be dependent on the substitution model used. A wide range of models have been developed to describe RNA evolution, either with 16 states describing all possible canonical base pairs or with 7 states where the 10 mismatched nucleotides are reduced to a single state. Formal model selection has become a standard practice for choosing an inferential model and works well for comparing models of a specific type, such as comparisons within nucleotide models or within amino acid models. Model selection cannot function across different sized state spaces because the likelihoods are conditioned on different data. Here, we introduce statistical state-space projection methods that allow the direct comparison of likelihoods between nucleotide models and 7-state and 16-state RNA models. To demonstrate the general applicability of our new methods, we extract 287 RNA families from genomic alignments and perform model selection. We find that in 281/287 families, RNA models are selected in preference to nucleotide models, with simple 7-state RNA models selected for more conserved families with shorter stems and more complex 16-state RNA models selected for more divergent families with longer stems. Other factors, such as the function of the RNA molecule or the GC-content, have limited impact on model selection. Our models and model selection methods are freely available in the open-source PHASE 3.0 software.

Description: Symbiotic associations between animals and microbes are ubiquitous in nature, with an estimated 15% of all insect species harboring intracellular bacterial symbionts. Most bacterial symbionts share many genomic features including small genomes, nucleotide composition bias, high coding density, and a paucity of mobile DNA, consistent with long-term host association. In this study, we focus on the early stages of genome degeneration in a recently derived insect-bacterial mutualistic intracellular association. We present the complete genome sequence and annotation of Sitophilus oryzae primary endosymbiont (SOPE). We also present the finished genome sequence and annotation of strain HS, a close free-living relative of SOPE and other insect symbionts of the Sodalis -allied clade, whose gene inventory is expected to closely resemble the putative ancestor of this group. Structural, functional, and evolutionary analyses indicate that SOPE has undergone extensive adaptation toward an insect-associated lifestyle in a very short time period. The genome of SOPE is large in size when compared with many ancient bacterial symbionts; however, almost half of the protein-coding genes in SOPE are pseudogenes. There is also evidence for relaxed selection on the remaining intact protein-coding genes. Comparative analyses of the whole-genome sequence of strain HS and SOPE highlight numerous genomic rearrangements, duplications, and deletions facilitated by a recent expansion of insertions sequence elements, some of which appear to have catalyzed adaptive changes. Functional metabolic predictions suggest that SOPE has lost the ability to synthesize several essential amino acids and vitamins. Analyses of the bacterial cell envelope and genes encoding secretion systems suggest that these structures and elements have become simplified in the transition to a mutualistic association.

Description: Sea ice is a highly dynamic and productive environment that includes a diverse array of psychrophilic prokaryotic and eukaryotic taxa distinct from the underlying water column. Because sea ice has only been extensive on Earth since the mid-Eocene, it has been hypothesized that bacteria highly adapted to inhabit sea ice have traits that have been acquired through horizontal gene transfer (HGT). Here we compared the genomes of the psychrophilic bacterium Psychroflexus torquis ATCC 700755 T , associated with both Antarctic and Arctic sea ice, and its closely related nonpsychrophilic sister species, P. gondwanensis ACAM 44 T . Results show that HGT has occurred much more extensively in P. torquis in comparison to P. gondwanensis . Genetic features that can be linked to the psychrophilic and sea ice-specific lifestyle of P. torquis include genes for exopolysaccharide (EPS) and polyunsaturated fatty acid (PUFA) biosynthesis, numerous specific modes of nutrient acquisition, and proteins putatively associated with ice-binding, light-sensing (bacteriophytochromes), and programmed cell death (metacaspases). Proteomic analysis showed that several genes associated with these traits are highly translated, especially those involved with EPS and PUFA production. Because most of the genes relating to the ability of P. torquis to dwell in sea-ice ecosystems occur on genomic islands that are absent in closely related P. gondwanensis , its adaptation to the sea-ice environment appears driven mainly by HGT. The genomic islands are rich in pseudogenes, insertional elements, and addiction modules, suggesting that gene acquisition is being followed by a process of genome reduction potentially indicative of evolving ecosystem specialism.

Description: Papillomaviruses (PVs) are widespread pathogens. However, the extent of PV infections in bats remains largely unknown. This work represents the first comprehensive study of PVs in Iberian bats. We identified four novel PVs in the mucosa of free-ranging Eptesicus serotinus (EserPV1, EserPV2, and EserPV3) and Rhinolophus ferrumequinum (RferPV1) individuals and analyzed their phylogenetic relationships within the viral family. We further assessed their prevalence in different populations of E. serotinus and its close relative E. isabellinus . Although it is frequent to read that PVs co-evolve with their host, that PVs are highly species-specific, and that PVs do not usually recombine, our results suggest otherwise. First, strict virus–host co-evolution is rejected by the existence of five, distantly related bat PV lineages and by the lack of congruence between bats and bat PVs phylogenies. Second, the ability of EserPV2 and EserPV3 to infect two different bat species ( E. serotinus and E. isabellinus ) argues against strict host specificity. Finally, the description of a second noncoding region in the RferPV1 genome reinforces the view of an increased susceptibility to recombination in the E2-L2 genomic region. These findings prompt the question of whether the prevailing paradigms regarding PVs evolution should be reconsidered.

Description: Ion transporters are fundamental to life. Due to their ancient origin and conservation in sequence, ion transporters are also particularly well suited for comparative genomics of distantly related species. Here, we perform genome-wide ion transporter profiling as a basis for comparative genomics of eukaryotes. From a given predicted proteome, we identify all bona fide ion channels, ion porters, and ion pumps. Concentrating on unicellular eukaryotes ( n = 37), we demonstrate that clustering of species according to their repertoire of ion transporters segregates obligate endoparasites ( n = 23) on the one hand, from free-living species and facultative parasites ( n = 14) on the other hand. This surprising finding indicates strong convergent evolution of the parasites regarding the acquisition and homeostasis of inorganic ions. Random forest classification identifies transporters of ammonia, plus transporters of iron and other transition metals, as the most informative for distinguishing the obligate parasites. Thus, in silico ionomics further underscores the importance of iron in infection biology and suggests access to host sources of nitrogen and transition metals to be selective forces in the evolution of parasitism. This finding is in agreement with the phenomenon of iron withholding as a primordial antimicrobial strategy of infected mammals.

Description: Bidirectional best hits (BBH), which entails identifying the pairs of genes in two different genomes that are more similar to each other than either is to any other gene in the other genome, is a simple and widely used method to infer orthology. A recent study has analyzed the link between BBH and orthology in bacteria and archaea and concluded that, given the very high consistency in BBH they observed among triplets of neighboring genes, a high proportion of BBH are likely to be bona fide orthologs. However, limited by their analysis setup, the previous study could not easily test the reverse question: which proportion of orthologs are BBH? In this follow-up study, we consider this question in theory and answer it based on conceptual arguments, simulated data, and real biological data from all three domains of life. Our analyses corroborate the findings of the previous study, but also show that because of the high rate of gene duplication in plants and animals, as much as 60% of orthologous relations are missed by the BBH criterion.

Description: Enterohemorrhagic Escherichia coli (EHEC) O26:H11/H – is the predominant non-O157 EHEC serotype among patients with diarrhea, bloody diarrhea, and hemolytic uremic syndrome (HUS) worldwide. To elucidate their phylogeny and association between their phylogenetic background and clinical outcome of the infection, we investigated 120 EHEC O26:H11/H – strains isolated between 1965 and 2012 from asymptomatic carriers and patients with diarrhea or HUS. Whole-genome shotgun sequencing (WGS) was applied to ten representative EHEC O26 isolates to determine single nucleotide polymorphism (SNP) localizations within a predefined set of core genes. A multiplex SNP assay, comprising a randomly distributed subset of 48 SNPs, was established to detect SNPs in 110 additional EHEC O26 strains. Within approximately 1 Mb of core genes, WGS resulted in 476 high-quality bi-allelic SNP localizations. Forty-eight of these were subsequently investigated in 110 EHEC O26 and four different SNP clonal complexes (SNP-CC) were identified. SNP-CC2 was significantly associated with the development of HUS. Within the subsequently established evolutionary model of EHEC O26, we dated the emergence of human EHEC O26 to approximately 19,700 years ago and demonstrated a recent evolution within humans into the 4 SNP-CCs over the past 1,650 years. WGS and subsequent SNP typing enabled us to gain new insights into the evolution of EHEC O26 suggesting a common theme in this EHEC group with analogies to EHEC O157. In addition, the SNP-CC analysis may help to assess a risk in infected individuals for the progression to HUS and to implement more specific infection control measures.