Base Composition Bias Research Articles

The polyphyly of Plasmodium: comprehensive phylogenetic analyses of the malaria parasites (order Haemosporida) reveal widespread taxonomic conflict.

The evolutionary relationships among the apicomplexan blood pathogens known as the malaria parasites (order Haemosporida), some of which infect nearly 200 million humans each year, has remained a vexing phylogenetic problem due to limitations in taxon sampling, character sampling and the extreme nucleotide base composition biases that are characteristic of this clade. Previous phylogenetic work on the malaria parasites has often lacked sufficient representation of the broad taxonomic diversity within the Haemosporida or the multi-locus sequence data needed to resolve deep evolutionary relationships, rendering our understanding of haemosporidian life-history evolution and the origin of the human malaria parasites incomplete. Here we present the most comprehensive phylogenetic analysis of the malaria parasites conducted to date, using samples from a broad diversity of vertebrate hosts that includes numerous enigmatic and poorly known haemosporidian lineages in addition to genome-wide multi-locus sequence data. We find that if base composition differences were corrected for during phylogenetic analysis, we recovered a well-supported topology indicating that the evolutionary history of the malaria parasites was characterized by a complex series of transitions in life-history strategies and host usage. Notably we find that Plasmodium, the malaria parasite genus that includes the species of human medical concern, is polyphyletic with the life-history traits characteristic of this genus having evolved in a dynamic manner across the phylogeny. We find support for multiple instances of gain and loss of asexual proliferation in host blood cells and production of haemozoin pigment, two traits that have been used for taxonomic classification as well as considered to be important factors for parasite virulence and used as drug targets. Lastly, our analysis illustrates the need for a widespread reassessment of malaria parasite taxonomy.

English

The phylogenetic utility of the 16S gene in cichlids is assessed. Eighty-six (86) partial sequences belonging to 37 genera of cichlids from the Genbank was analyzed. The alignment had four hundred and sixty three (463) basepairs with 337 conserved sites and 126 variable sites. Base compositional bias is similar to that found in higher organism with Adenine having the highest average of 30.3%, followed by cytosine, guanine and thiamine with the average values of 26.1, 21.9 and 21.7% respectively. The most suitable evolutionary model is the K2+G+I model as this had the lowest Bayesian Information Criterion. There were 4 major indels at basepair positions 328 which is unique to the Heterotilapia buttikoferi, position 369 unique to Gramatoria lemarii, position 396 which is shared by Tilapia sparrmanii, T. guinasana and T. zilli. The indel at position 373 was found in all tested species except the Oreochromis mossambicus. The Tilapine general is the basal group in Cichlids. The 16S gene separates the Tilapia genera without any ambiguity but there were phylogenetic overlaps in the Sarotherodon and Oreochromis. More finite molecular and statistical methodology may be needed to distinguish the Sarotherodon and Oreochromis. The diversity of cichlids is generally very low due to a common ancestry with little differentiation genetically. The grouping of the Oreochromis and Sarotherodon genera together in the same clade is not unconnected with the preservation of genetic beacons that the group retained as it evolved. Key words: Cichlids, 16S gene, phylogeny, evolutionary model, monophyletic, conserved segment, speciose, indels

Characterization of the complete chloroplast genome of the wild Himalayan pear Pyrus pashia (Rosales: Rosaceae: Maloideae)

Pyrus pashia is a deciduous fruiting tree species of high commercial, scientific, nutritional and therapeutic values. Many of its wild populations are in danger, and need urgent conservation. To contribute to such efforts, its complete chloroplast genome was assembled from high-throughput Illumina sequencing data. The circular genome is 160,153 bp long, and displays a typical quadripartite structure of the large (LSC, 88,129 bp) and small (SSC, 19,252 bp) single-copy regions, separated by a pair of inverted repeat regions (IRs, 26,386 bp each). It harbors 112 gene species, including 78 protein-coding, 30 transfer RNA and 4 ribosomal RNA gene species. Being similar to many other confamilial taxa, it has a biased base composition (31.35% A, 18.63% C, 17.93% G and 32.09% T) with an overall A+T content of 63.44%. Phylogenetic analysis suggested that P. pashia was more closely related to its congeners than to the other confamilial taxa with sequenced chloroplast genomes.

Towards a higher-level Ensifera phylogeny inferred from mitogenome sequences

Although mitogenomes are useful tools for inferring evolutionary history, only a few representative ones can be used for most Ensifera lineages. Thirty-two ensiferan mitogenomes were determined using ABI Sanger sequencing and standard primer walking of 2–3 overlapping Long-PCR fragments, or Illumina® HiSeq2000 for “shotgun” sequenced long-PCR-amplified mitochondrial or total genomic DNA. Six patterns of gene arrangements, including the novel trnR-trnSAGN-trnA-trnN-trnG-nad3 in Lipotactes tripyrga (Lipotactinae), were identified from 59 ensiferan mitogenomes. The results suggest that trnM-trnI-trnQ and trnA-trnR-trnE-trnSAGN-trnN-trnF rearrangements might be a shared derived character in Pseudophyllinae and Gryllidae, respectively. We found base composition biases in our dataset, which potentially complicate the inference of higher-level ensiferan phylogeny. Site-heterogeneous Bayesian inference (BI) and site-homogeneous maximum likelihood (ML) analyses recovered all ensiferan superfamilies as monophyletic. The site-homogeneous BI analysis failed to recover the monophyly of Stenopelmatoidea. As Schizodactyloidea was only represented by Comicus campestris, its monophyly could not be tested. In the Triassic/Jurassic boundary, Ensifera diverged into grylloid and non-grylloid clades. All analyses confirmed Grylloidea and Gryllotalpoidea as sister groups. Site-heterogeneous BI analysis found Schizodactyloidea as the most basal lineage and sister to the clade formed by Grylloidea and Gryllotalpoidea, but the site-homogeneous analyses placed it basally to the non-grylloid clade and recovered a sister relationship between Tettigonioidea and (Hagloidea, Rhaphidophoroidea, Stenopelmatoidea), although this clade had a low support. The site-heterogeneous BI analysis found Tettigonioidea and Hagloidea were sister groups (posterior probability (PP)=0.99), Stenopelmatoidea was sister to (Tettigonioidea, Hagloidea) (PP>0.91), and Rhaphidophoroidea was basal to the non-grylloid clade. At a lower level, all analyses divided Tettigonioidea into Phaneropteridae and Tettigoniidae.

The Evolution of LINE-1 in Vertebrates.

The abundance and diversity of the LINE-1 (L1) retrotransposon differ greatly among vertebrates. Mammalian genomes contain hundreds of thousands L1s that have accumulated since the origin of mammals. A single group of very similar elements is active at a time in mammals, thus a single lineage of active families has evolved in this group. In contrast, non-mammalian genomes (fish, amphibians, reptiles) harbor a large diversity of concurrently transposing families, which are all represented by very small number of recently inserted copies. Why the pattern of diversity and abundance of L1 is so different among vertebrates remains unknown. To address this issue, we performed a detailed analysis of the evolution of active L1 in 14 mammals and in 3 non-mammalian vertebrate model species. We examined the evolution of base composition and codon bias, the general structure, and the evolution of the different domains of L1 (5′UTR, ORF1, ORF2, 3′UTR). L1s differ substantially in length, base composition, and structure among vertebrates. The most variation is found in the 5′UTR, which is longer in amniotes, and in the ORF1, which tend to evolve faster in mammals. The highly divergent L1 families of lizard, frog, and fish share species-specific features suggesting that they are subjected to the same functional constraints imposed by their host. The relative conservation of the 5′UTR and ORF1 in non-mammalian vertebrates suggests that the repression of transposition by the host does not act in a sequence-specific manner and did not result in an arms race, as is observed in mammals.

Abstract 3637: Targeted exome and panel analysis from low input and FFPE DNA using hybridization capture for cancer genome studies

Abstract Introduction Hybridization-based target enrichment techniques coupled with Next Generation Sequencing (NGS) provide a useful and cost-efficient means to study disease specific target regions including whole exomes and gene panels. More than 500 whole exome analyses are behind The Cancer Genome Atlas (TCGA), which is the largest knowledge base for cancer studies including research, prevention, treatment, and care. Clinical samples may be limited in input and of compromised quality due to formalin fixation, however most current NGS library preparation methods require 50-100 ng high quality DNA for such studies. Here we present an efficient method which enables high quality target enrichment and variant calling from inputs as low as 1-25 ng. Method NGS libraries for hybridization capture were made from 1 to 100 ng of Coriell Hapmap samples (NA12878, etc), clinical Formalin Fixed Paraffin Embedded (FFPE) and Horizon Discovery (HDx) reference DNA (HDx 701) using the Accel-NGS® 2S Hyb DNA Library Kit. Amplified libraries were then enriched using specific targeted panels (xGen® Pan-Cancer and xGen® AML) or SeqCap™ EZ MedExome using manufacturer's specifications (IDT™ and Roche NimbleGen™). Enriched libraries were captured using streptavidin beads. Captured libraries were then amplified according to the manufacturer's specifications. Targeted panels were sequenced on an Illumina® MiSeq® using V2 chemistry and MedExome on a HiSeq® using V4 chemistry. Sequence analysis was performed with custom pipelines using BWA for alignment and GATK, Samtools, and Freebayes for variant calling. Results Sequencing analyses yielded a minimum average coverage of 30x and more than thirty fold enrichment. Enriched libraries exhibited significant sequence complexity with minimal duplicates and without any base-composition bias. The percent on-target varied with type of input and ranged from 50-80%. Germline variant calling results had > 99% concordance with the NIST GIAB truth list with sensitivity and precision of > 98%, even from inputs as low as 1 ng of DNA. Somatic variant calling down to 1-5% allele frequency was also evaluated at various DNA input quantities and greater depth of sequencing; results for FFPE and DNA standards will be presented. Conclusions Accel-NGS 2S Hyb technology can be used for whole exome and targeted enrichment studies from low quantity and low quality FFPE clinical samples. High complexity libraries with minimal bias yield high quality sequence data which enables discovery and detection of somatic variants in tumor samples to identify molecular drivers associated with different cancer types. The technique facilitates better understanding of complex cancer genomes and guide precision medicine. Citation Format: Sukhinder K. Sandhu, Cassie Schumacher, Laurie Kurihara, Tim Harkins, Vladimir Makarov. Targeted exome and panel analysis from low input and FFPE DNA using hybridization capture for cancer genome studies. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3637.

Genome-Wide Analysis of Codon Usage Bias in Epichloë festucae.

Analysis of codon usage data has both practical and theoretical applications in understanding the basics of molecular biology. Differences in codon usage patterns among genes reflect variations in local base compositional biases and the intensity of natural selection. Recently, there have been several reports related to codon usage in fungi, but little is known about codon usage bias in Epichloë endophytes. The present study aimed to assess codon usage patterns and biases in 4870 sequences from Epichloë festucae, which may be helpful in revealing the constraint factors such as mutation or selection pressure and improving the bioreactor on the cloning, expression, and characterization of some special genes. The GC content with 56.41% is higher than the AT content (43.59%) in E. festucae. The results of neutrality and effective number of codons plot analyses showed that both mutational bias and natural selection play roles in shaping codon usage in this species. We found that gene length is strongly correlated with codon usage and may contribute to the codon usage patterns observed in genes. Nucleotide composition and gene expression levels also shape codon usage bias in E. festucae. E. festucae exhibits codon usage bias based on the relative synonymous codon usage (RSCU) values of 61 sense codons, with 25 codons showing an RSCU larger than 1. In addition, we identified 27 optimal codons that end in a G or C.

Phylogenomics Controlling for Base Compositional Bias Reveals a Single Origin of Eusociality in Corbiculate Bees.

As increasingly large molecular data sets are collected for phylogenomics, the conflicting phylogenetic signal among gene trees poses challenges to resolve some difficult nodes of the Tree of Life. Among these nodes, the phylogenetic position of the honey bees (Apini) within the corbiculate bee group remains controversial, despite its considerable importance for understanding the emergence and maintenance of eusociality. Here, we show that this controversy stems in part from pervasive phylogenetic conflicts among GC-rich gene trees. GC-rich genes typically have a high nucleotidic heterogeneity among species, which can induce topological conflicts among gene trees. When retaining only the most GC-homogeneous genes or using a nonhomogeneous model of sequence evolution, our analyses reveal a monophyletic group of the three lineages with a eusocial lifestyle (honey bees, bumble bees, and stingless bees). These phylogenetic relationships strongly suggest a single origin of eusociality in the corbiculate bees, with no reversal to solitary living in this group. To accurately reconstruct other important evolutionary steps across the Tree of Life, we suggest removing GC-rich and GC-heterogeneous genes from large phylogenomic data sets. Interpreted as a consequence of genome-wide variations in recombination rates, this GC effect can affect all taxa featuring GC-biased gene conversion, which is common in eukaryotes.

Evolutionary Divergence of Aggregatibacter actinomycetemcomitans

Gram-negative facultative Aggregatibacter actinomycetemcomitans is an oral pathogen associated with periodontitis. The genetic heterogeneity among A. actinomycetemcomitans strains has been long recognized. This study provides a comprehensive genomic analysis of A. actinomycetemcomitans and the closely related nonpathogenic Aggregatibacter aphrophilus. Whole genome sequencing by Illumina MiSeq platform was performed for 31 A. actinomycetemcomitans and 2 A. aphrophilus strains. Sequence similarity analysis shows a total of 3,220 unique genes across the 2 species, where 1,550 are core genes present in all genomes and 1,670 are variable genes (accessory genes) missing in at least 1 genome. Phylogenetic analysis based on 397 concatenated core genes distinguished A. aphrophilus and A. actinomycetemcomitans. The latter was in turn divided into 5 clades: clade b (serotype b), clade c (serotype c), clade e/f (serotypes e and f), clade a/d (serotypes a and d), and clade e′ (serotype e strains). Accessory genes accounted for 14.1% to 23.2% of the A. actinomycetemcomitans genomes, with a majority belonging to the category of poorly characterized by Cluster of Orthologous Groups classification. These accessory genes were often organized into genomic islands (n = 387) with base composition biases, suggesting their acquisitions via horizontal gene transfer. There was a greater degree of similarity in gene content and genomic islands among strains within clades than between clades. Strains of clade e′ isolated from human were found to be missing the genomic island that carries genes encoding cytolethal distending toxins. Taken together, the results suggest a pattern of sequential divergence, starting from the separation of A. aphrophilus and A. actinomycetemcomitans through gain and loss of genes and ending with the divergence of the latter species into distinct clades and serotypes. With differing constellations of genes, the A. actinomycetemcomitans clades may have evolved distinct adaptation strategies to the human oral cavity.

Analysis of synonymous codon usage in spike protein gene of infectious bronchitis virus.

Infectious bronchitis virus (IBV) is responsible for causing respiratory, renal, and urogenital diseases in poultry. IBV infection in poultry leads to high mortality rates in affected flocks and to severe economic losses due to a drop in egg production and a reduced gain in live weight of the broiler birds. IBV-encoded spike protein (S) is the major protective immunogen for the host. Although the functions of the S protein have been well studied, the factors shaping synonymous codon usage bias and nucleotide composition in the S gene have not been reported yet. In the present study, we analyzed the relative synonymous codon usage and effective number of codons (Nc) using the 53 IBV S genes. The major trend in codon usage variation was studied using correspondence analysis. The plot of Nc values against GC3 as well as the correlation between base composition and codon usage bias suggest that mutational pressure rather than natural selection is the main factor that determines the codon usage bias in the S gene. Interestingly, no association of aromaticity, degree of hydrophobicity, and aliphatic index was observed with the codon usage variation in IBV S genes. The study represents a comprehensive analysis of IBV S gene codon usage patterns and provides a basic understanding of the codon usage bias.

Analysis of synonymous codon usage in the VP2 protein gene of infectious bursal disease virus.

Infectious bursal disease virus (IBDV) is an important poultry pathogen. The VP2 protein of IBDV is the major host-protective immunogen. Although the functions of the VP2 protein have been well studied, the factors shaping synonymous codon usage bias and nucleotide composition in the VP2 gene have not yet been reported. In the present study, we have analyzed the relative synonymous codon usage and effective number of codons (ENC) using 69 IBDV VP2 genes. The major trend in codon usage variation was studied using correspondence analysis. The plot of ENC values and GC3s as well as the correlation between base composition and codon usage bias suggests that mutational pressure rather than natural selection is the main factor that determines the codon usage bias in the VP2 gene. In addition, other factors, such as the aromaticity, hydrophobicity and aliphatic index also influence the codon usage variation of the VP2 gene. This study represents a comprehensive analysis of IBDV VP2 gene codon usage patterns and provides a basic understanding of the codon usage bias.

Endosymbiont evolution: predictions from theory and surprises from genomes.

Genome data have created new opportunities to untangle evolutionary processes shaping microbial variation. Among bacteria, long-term mutualists of insects represent the smallest and (typically) most AT-rich genomes. Evolutionary theory provides a context to predict how an endosymbiotic lifestyle may alter fundamental evolutionary processes--mutation, selection, genetic drift, and recombination--and thus contribute to extreme genomic outcomes. These predictions can then be explored by comparing evolutionary rates, genome size and stability, and base compositional biases across endosymbiotic and free-living bacteria. Recent surprises from such comparisons include genome reduction among uncultured, free-living species. Some studies suggest that selection generally drives this streamlining, while drift drives genome reduction in endosymbionts; however, this remains an hypothesis requiring additional data. Unexpected evidence of selection acting on endosymbiont GC content hints that even weak selection may be effective in some long-term mutualists. Moving forward, intraspecific analysis offers a promising approach to distinguish underlying mechanisms, by testing the null hypothesis of neutrality and by quantifying mutational spectra. Such analyses may clarify whether endosymbionts and free-living bacteria occupy distinct evolutionary trajectories or, alternatively, represent varied outcomes of similar underlying forces.

Revisiting robustness and evolvability: evolution in weighted genotype spaces.

Robustness and evolvability are highly intertwined properties of biological systems. The relationship between these properties determines how biological systems are able to withstand mutations and show variation in response to them. Computational studies have explored the relationship between these two properties using neutral networks of RNA sequences (genotype) and their secondary structures (phenotype) as a model system. However, these studies have assumed every mutation to a sequence to be equally likely; the differences in the likelihood of the occurrence of various mutations, and the consequence of probabilistic nature of the mutations in such a system have previously been ignored. Associating probabilities to mutations essentially results in the weighting of genotype space. We here perform a comparative analysis of weighted and unweighted neutral networks of RNA sequences, and subsequently explore the relationship between robustness and evolvability. We show that assuming an equal likelihood for all mutations (as in an unweighted network), underestimates robustness and overestimates evolvability of a system. In spite of discarding this assumption, we observe that a negative correlation between sequence (genotype) robustness and sequence evolvability persists, and also that structure (phenotype) robustness promotes structure evolvability, as observed in earlier studies using unweighted networks. We also study the effects of base composition bias on robustness and evolvability. Particularly, we explore the association between robustness and evolvability in a sequence space that is AU-rich – sequences with an AU content of 80% or higher, compared to a normal (unbiased) sequence space. We find that evolvability of both sequences and structures in an AU-rich space is lesser compared to the normal space, and robustness higher. We also observe that AU-rich populations evolving on neutral networks of phenotypes, can access less phenotypic variation compared to normal populations evolving on neutral networks.

Phylogenetic position of the bee genera Ancyla and Tarsalia (Hymenoptera: Apidae): A remarkable base compositional bias and an early Paleogene geodispersal from North America to the Old World

We address the phylogenetic position of the bee genera Tarsalia and Ancyla (currently forming the tribe Ancylaini) on the basis of morphological, molecular and combined data. We assembled a matrix of 309 morphological characters and 5246 aligned nucleotide positions from six nuclear genes (28S, EF-1a, wingless, POL2, LW-Rhodopsin, NAK). In addition to both constituent genera of Ancylaini, we include all three subtribes of the Eucerini as well as a large number of other tribes from the “eucerine line”. The morphological data suggest Ancyla to be sister to Tarsalia+Eucerini and analyses of the entire molecular dataset suggest Tarsalia to be sister to Ancyla+Eucerini. However, analyses of the combined dataset suggests the Ancylaini to be monophyletic. We address possible bias within the molecular data and show that the base composition of two markers (EF-1a and NAK) is significantly heterogeneous among taxa and that this heterogeneity is strong enough to overcome the phylogenetic signal from the other markers. Analyses of a molecular matrix where the heterogeneous partitions have been RY-recoded yield trees that are better resolved and have higher nodal support values than those recovered in analyses of the non-recoded matrix, and strongly suggest the Ancylaini to be a monophyletic sister group to the Eucerini. A dated phylogeny and ancestral range reconstructions suggest that the common ancestor of the Ancylaini reached the Old World from the New World most probably via the Thulean Land Bridge in a time window between 69 and 47mya, a period that includes the Early Eocene Climatic Optimum. No further exchanges between the New World and the Old World are implied by our data until the period between 22mya and 13.9mya. These more recent faunal exchanges probably involved geodispersal over the Bering Land Bridge by less thermophilic lineages.

Analysis of synonymous codon usage in enterovirus 71.

Enterovirus 71 (EV71) is the major cause of hand-foot-and-mouth disease in children. In our study, using the complete genome sequences of 42 EV71 representing all three genotypes, we analyzed synonymous codon usage and the relative dinucleotide abundance in EV71 genome. The general correlation between base composition and codon usage bias suggests that mutational pressure rather than natural selection is the main factor that determines the codon usage bias in EV71 genome. Furthermore, we observed that the relative abundance of dinucleotides in EV71 is independent of the overall base composition but is still the result of differential mutational pressure, which also shapes codon usage. In addition, other factors, such as hydrophobicity and aromaticity, also influence the codon usage variation among the genomes of EV71. This study represents the most comprehensive analysis of EV71 codon usage patterns and provides a basic understanding of the mechanisms for codon usage bias.

Changes in base composition bias of nuclear and mitochondrial genes in lice (Insecta: Psocodea)

While it is well known that changes in the general processes of molecular evolution have occurred on a variety of timescales, the mechanisms underlying these changes are less well understood. Parasitic lice ("Phthiraptera") and their close relatives (infraorder Nanopsocetae of the insect order Psocodea) are a group of insects well known for their unusual features of molecular evolution. We examined changes in base composition across parasitic lice and bark lice. We identified substantial differences in percent GC content between the clade comprising parasitic lice plus closely related bark lice (=Nanopsocetae) versus all other bark lice. These changes occurred for both nuclear and mitochondrial protein coding and ribosomal RNA genes, often in the same direction. To evaluate whether correlations in base composition change also occurred within lineages, we used phylogenetically controlled comparisons, and in this case few significant correlations were identified. Examining more constrained sites (first/second codon positions and rRNA) revealed that, in comparison to the other bark lice, the GC content of parasitic lice and close relatives tended towards 50 % either up from less than 50 % GC or down from greater than 50 % GC. In contrast, less constrained sites (third codon positions) in both nuclear and mitochondrial genes showed less of a consistent change of base composition in parasitic lice and very close relatives. We conclude that relaxed selection on this group of insects is a potential explanation of the change in base composition for both mitochondrial and nuclear genes, which could lead to nucleotide frequencies closer to random expectation (i.e., 50 % GC) in the absence of any mutation bias. Evidence suggests this relaxed selection arose once in the non-parasitic common ancestor of Phthiraptera + Nanopsocetae and is not directly related to the evolution of the parasitism in lice.

Analysis of synonymous codon usage in Newcastle disease virus hemagglutinin–neuraminidase (HN) gene and fusion protein (F) gene

Newcastle disease virus (NDV) hemagglutinin-neuraminidase (HN) is a multifunctional protein, which possesses both the receptor recognition and neuraminidase activities. The fusion (F) protein is a type I membrane glycoprotein that mediates the merger of the viral envelope to the host cell membrane. Although the functions of the HN and F proteins have been well studied, however, the factors shaping synonymous codon usage bias and nucleotide composition in HN and F genes have been few reported. In our study, we analyzed synonymous codon usage using the 69 NDV HN and F genes, respectively. The general correlation between base composition and codon usage bias suggests that mutational pressure rather than natural selection is the main factor that determines the codon usage bias in HN and F genes. In addition, other factors, such as the aromaticity and hydrophobicity, also influence the codon usage variation among HN and F genes. This study represents the most comprehensive analysis to date of NDV HN and F genes codon usage patterns and provides a basic understanding of the mechanisms for codon usage bias.

The complete chloroplast genome sequence of Mahonia bealei (Berberidaceae) reveals a significant expansion of the inverted repeat and phylogenetic relationship with other angiosperms

Mahonia bealei (Berberidaceae) is a frequently-used traditional Chinese medicinal plant with efficient anti-inflammatory ability. This plant is one of the sources of berberine, a new cholesterol-lowering drug with anti-diabetic activity. We have sequenced the complete nucleotide sequence of the chloroplast (cp) genome of M. bealei. The complete cp genome of M. bealei is 164,792bp in length, and has a typical structure with large (LSC 73,052bp) and small (SSC 18,591bp) single-copy regions separated by a pair of inverted repeats (IRs 36,501bp) of large size. The Mahonia cp genome contains 111 unique genes and 39 genes are duplicated in the IR regions. The gene order and content of M. bealei are almost unarranged which is consistent with the hypothesis that large IRs stabilize cp genome and reduce gene loss-and-gain probabilities during evolutionary process. A large IR expansion of over 12kb has occurred in M. bealei, 15 genes (rps19, rpl22, rps3, rpl16, rpl14, rps8, infA, rpl36, rps11, petD, petB, psbH, psbN, psbT and psbB) have expanded to have an additional copy in the IRs. The IR expansion rearrangement occurred via a double-strand DNA break and subsequence repair, which is different from the ordinary gene conversion mechanism. Repeat analysis identified 39 direct/inverted repeats 30bp or longer with a sequence identity ≥90%. Analysis also revealed 75 simple sequence repeat (SSR) loci and almost all are composed of A or T, contributing to a distinct bias in base composition. Comparison of protein-coding sequences with ESTs reveals 9 putative RNA edits and 5 of them resulted in non-synonymous modifications in rpoC1, rps2, rps19 and ycf1. Phylogenetic analysis using maximum parsimony (MP) and maximum likelihood (ML) was performed on a dataset composed of 65 protein-coding genes from 25 taxa, which yields an identical tree topology as previous plastid-based trees, and provides strong support for the sister relationship between Ranunculaceae and Berberidaceae. Molecular dating analyses suggest that Ranunculaceae and Berberidaceae diverged between 90 and 84mya, which is congruent with the fossil records and with recent estimates of the divergence time of these two taxa.

Addressing Gene Tree Discordance and Non-Stationarity to Resolve a Multi-Locus Phylogeny of the Flatfishes (Teleostei: Pleuronectiformes)

Non-homogeneous processes and, in particular, base compositional non-stationarity have long been recognized as a critical source of systematic error. But only a small fraction of current molecular systematic studies methodically examine and effectively account for the potentially confounding effect of non-stationarity. The problem is especially overlooked in multi-locus or phylogenomic scale analyses, in part because no efficient tools exist to accommodate base composition heterogeneity in large data sets. We present a detailed analysis of a data set with 20 genes and 214 taxa to study the phylogeny of flatfishes (Pleuronectiformes) and their position among percomorphs. Most genes vary significantly in base composition among taxa and fail to resolve flatfish monophyly and other emblematic groups, suggesting that non-stationarity may be causing systematic error. We show a strong association between base compositional bias and topological discordance among individual gene partitions and their inferred trees. Phylogenetic methods applying non-homogeneous models to accommodate non-stationarity have relatively minor effect to reduce gene tree discordance, suggesting that available computer programs applying these methods do not scale up efficiently to the data set of modest size analysed in this study. By comparing phylogenetic trees obtained with species tree (STAR) and concatenation approaches, we show that gene tree discordance in our data set is most likely due to base compositional biases than to incomplete lineage sorting. Multi-locus analyses suggest that the combined phylogenetic signal from all loci in a concatenated data set overcomes systematic biases induced by non-stationarity at each partition. Finally, relationships among flatfishes and their relatives are discussed in the light of these results. We find support for the monophyly of flatfishes and confirm findings from previous molecular phylogenetic studies suggesting their close affinity with several carangimorph groups (i.e., jack and allies, barracuda, archerfish, billfish and swordfish, threadfin, moonfish, beach salmon, and snook and barramundi).

The genome-wide distribution of non-B DNA motifs is shaped by operon structure and suggests the transcriptional importance of non-B DNA structures in Escherichia coli

Although the right-handed double helical B-form DNA is most common under physiological conditions, DNA is dynamic and can adopt a number of alternative structures, such as the four-stranded G-quadruplex, left-handed Z-DNA, cruciform and others. Active transcription necessitates strand separation and can induce such non-canonical forms at susceptible genomic sequences. Therefore, it has been speculated that these non-B DNA motifs can play regulatory roles in gene transcription. Such conjecture has been supported in higher eukaryotes by direct studies of several individual genes, as well as a number of large-scale analyses. However, the role of non-B DNA structures in many lower organisms, in particular proteobacteria, remains poorly understood and incompletely documented. In this study, we performed the first comprehensive study of the occurrence of B DNA–non-B DNA transition-susceptible sites (non-B DNA motifs) within the context of the operon structure of the Escherichia coli genome. We compared the distributions of non-B DNA motifs in the regulatory regions of operons with those from internal regions. We found an enrichment of some non-B DNA motifs in regulatory regions, and we show that this enrichment cannot be simply explained by base composition bias in these regions. We also showed that the distribution of several non-B DNA motifs within intergenic regions separating divergently oriented operons differs from the distribution found between convergent ones. In particular, we found a strong enrichment of cruciforms in the termination region of operons; this enrichment was observed for operons with Rho-dependent, as well as Rho-independent terminators. Finally, a preference for some non-B DNA motifs was observed near transcription factor-binding sites. Overall, the conspicuous enrichment of transition-susceptible sites in these specific regulatory regions suggests that non-B DNA structures may have roles in the transcriptional regulation of specific operons within the E. coli genome.