We observed that amino acid sequences inferred from coding—but rarely from non-coding—DNA sequences accumulated alignments in low-stringency BLAST searches, suggesting that this alignments accumulation could be used to highlight coding regions in sequenced DNA. To investigate this possibility, we developed a computer program (AnABlast) that generates profiles of accumulated alignments in query amino acid sequences using a low-stringency BLAST strategy. To validate this approach, all six-frame translations of DNA sequences between every two annotated exons of the fission yeast genome were analysed with AnABlast. AnABlast-generated profiles identified three new copies of known genes, and four new genes supported by experimental evidence. New pseudogenes, ancestral carboxyl- and amino-terminal subtractions, complex gene rearrangements, and ancient fragments of mitDNA and of bacterial origin, were also inferred. Thus, this novel in silico approach provides a powerful tool to uncover new genes, as well as fossil-coding sequences, thus providing insight into the evolutionary history of annotated genomes.Genome annotation, assisted by computer programs, is one of the great advances in modern biology. Nevertheless, the  In addition to the extraordinary support given by experimental approaches, computational analysis is an essential task for protein-coding gene identification in present genome biology.10 The fission yeast Schizosaccharomyces pombe is one of these model organisms.11 The 12.5 Mb S. pombe genome was among the first genomes that were entirely sequenced. Based on standard computational prediction, together with experimental data, 4,824 genes and 31 pseudogenes were initially annotated.12 However, while >95% of the genome is transcribed,15 only 55% of the genome corresponds to annotated coding sequences,16 raising the possibility that more protein-coding genes await to be discovered. Accordingly, high-throughput cDNA sequencing (RNA-Seq) and mass spectrometry-based proteomic approaches19 increased this initial number to the 5,123 protein-coding genes that are currently annotated in PomBase (http://www.pombase.org), the S. pombe database.20 Some pseudogenes were also reassigned as genes, leaving 29 pseudogenes annotated at present. More recently, the use of ribosome profiling (Ribo-Seq) to investigate the translational landscape of meiosis added further 46 new protein-coding genes to the fission yeast genome.18 Most of these new findings correspond to genes coding for small polypeptides and/or coding sequences lacking significant alignments in databases, highlighting that the in silico identification of this set of coding sequences is still challenging in genome annotation.Recent experimental data from ‘omic-technologies’ generated in model organisms are creating the possibility to accurately update published genome annotations.22 Since protein-coding genes mostly arise from previous ones during evolution, even highly divergent coding sequences may harbour ancient footprints of common ancestral proteins to be found among the millions of proteins available in databases. These footprints are not present in non-coding DNA sequences.22 Thus, alignments accumulated in predicted amino acid sequences might provide a method to discern coding from non-coding DNA, a new strategy that may overcome limitations of current in silico algorithms.The identification of similar proteins through BLAST analysis is one of the most useful strategies in annotation tasks. Finding significant alignments facilitates assignment of putative functions to query amino acid sequences through the identification of related proteins in databases. Interestingly, non-significant alignments (below the significant threshold) are often found in conventional BLAST searches. In polypeptides derived from the electronic translation of non-coding DNA, such alignments occur by chance. In polypeptides from coding DNA, however, in addition to random matches, non-significant alignments may also underlie small amino acid patterns representing random footprints of common ancestors.18 Overall, several new genes and ancient coding sequences were efficiently identified by using AnABlast in the extremely well annotated S. pombe genome. The strategy was also successfully applied in a genomic region of the nematode Caenorhabditis elegans, suggesting that this novel tool may be applicable to the in silico analysis of any other sequenced genome.To use this strategy for the identification of coding regions at genome level, we developed a computer program, named AnABlast, which generates profiles of accumulated alignments in query amino acid sequences using a low-stringency BLAST strategy. To test its potential use as a gene-finder algorithm, a genome-wide search of coding sequences was carried out in fission yeast, and Ribo-Seq data used to validate protein-coding regions were highlighted by AnABlast profiles.2.2.1.Ancestral-patterns search through A BLAST-based strategy), to search for putative coding sequences in genomic regions. Briefly, to search for new coding regions in a genome, all inter-exon DNA sequences of the genome (intergenic and intronic regions) were translated in all six frames, masking stop codons with ‘X’ characters; in this way, the analysis become independent of traditional ORF signals. The algorithm then executes a BLASTP search with the six resultant amino acid sequences  and the UniRef50 database (2014_02 version). The UniProt Reference Clusters (UniRef) provide clustered sets of sequences from virtually all the published protein sequences,23 being the clusters from UniRef50 built with amino acid sequences having at least 50% sequence identity. AnABlast only uses the representative member from each UniRef50 cluster, and thus, it only considers non-similar sequences at 50% identity threshold (avoiding biases coming from sequence redundancy).We have designed a computational workflow, here named AnABlast . Using the appropriate scoring matrix during the BLAST search (among available BLOSUM and PAM standard matrices) can improve AnABlast sensitivity. In this study, the BLOSUM90 matrix was selected to favour the search of high identity sequences in very short alignments, enough to be considered protomotifs21 or ancient footprints of protein sequences.22 The low-complexity filter SEG is also used to avoid sequence repeats, thereby improving specificity. Sequence alignments were trained with coding sequences from the analysed genome (data not shown), and a bit score threshold (given by the score matrix and the search space size) that better highlighted coding regions was used. In the S. pombe genome, a threshold of 30 in the score alignment was optimal in our search strategy, corresponding to an E-value of ∼12,000 . Finally, up to a maximum of 10,000 alignments by query is allowed.BLAST analysis efficiently identifies homologous sequences through highly significant alignments.nce Fig. . To this22 Under the above parameters, randomized and non-coding sequences yielded AnABlast profiles with peaks rarely reaching a maximum of 70 accumulated sequences. In this study, a cut-off threshold of 70 accumulated sequences was applied to optimize the identification of putative present and fossil-coding regions. The AnABlast algorithm is summarized in Fig. http://www.bioinfocabd.upo.es/ab.In a second step, AnABlast takes the positions from the acquired hits and counts the number of alignments (belonging to different clusters) that matches each position. As a result of this analysis, AnABlast yields a profile where discrete peaks highlight specific regions in the genome. Independently of the level of significance of each particular alignment, the high number of sequence alignments accumulated at a specific strand and reading frame reveals a significant region in terms of biological function and/or evolution.2.2.S. pombe annotations, protein and DNA sequences, and RNA expression data available from PomBase (http://www.pombase.org/) and C. elegans (https://www.wormbase.org/) on 1 February 2014 were used. The amino acid sequence delimited by each AnABlast peak was further studied manually by using BLAST3 and Pfam5 from their web applications . To determine expression levels in AnABlast domains, genome-wide transcriptome Ribo-Seq data from proliferating and sporulating cells.18 Ribo-Seq and AnABlast data were visualized with the Integrated Genome Viewer.24 Genome-wide RNA-Seq data from cells subjected to temperature or nutritional stress19 were also used (available at http://www.pombase.org/).For all analysis, 3.3.1.S. pombe genome interval containing the universal protein kinase Cdc2 (encoded in the forward strand) and its flanking genes SPBC11B10.08 (forward strand), and pht1 (reverse strand) was used to this purpose. All six-frame translations of this DNA interval were obtained. To allow the complete analysis of the corresponding amino acid sequence independently of existing ORFs, stop codons were ignored (masked with ‘X’). As expected, AnABlast analysis produced a profile of discrete peaks that matched regions of annotated exons and RNA expression patterns . Thus, on average, >85% of the selected AnABlast peaks are expected to identify true-positive coding sequences.To perform a genome-wide search of new coding regions in fission yeast, we analysed all nucleotide sequences between every two adjacent annotated exons, accounting for a total of 5,870 intergenic regions and 4,782 introns. AnABlast profiles were generated for each of the six-frame translation sequences of these inter-exon regions. Among them, 158 regions contained at least one significant peak and were selected for further analysis. As a negative control, we randomized the corresponding DNA sequence in each of the analysed inter-exon regions. With this data set, only 19 significant peaks were obtained  indicate that the sequence identified by AnABlast in this annotated intron is in fact a coding sequence of the snu23 gene . In addition, AnABlast peaks uncovered a new pseudogene . A second new pseudogene uncovered within rearranged DNA regions will be described later.Annotated transposons and pseudogenes were not excluded for intergenic regions analysis. Accordingly, a number of transposons and all 29 annotated pseudogenes were detected in AnABlast profiles . Another correction is suggested in the two annotated exons of the SPAPB24D3.05c pseudogene. AnABlast also identified two regions separated by a single frameshift, although region intervals and the reading frame for the first region differ from those in the annotated exons . Aside corrections, AnABlast peaks matching Ribo-Seq data profiles in pseudogenes suggest that at least two of them could be reassigned as coding genes .Detailed analysis of AnABlast profiles generated in annotated pseudogenes suggested a number of corrections to their genomic annotations. Two additional coding domains (in different reading frames) flanking the SPBPB10D8.03 pseudogene suggest carboxyl- and amino-terminal expansions in the annotated region . Importantly, four other ORF-containing AnABlast peaks identified putative new protein-coding genes, experimentally validated by Ribo-Seq expression patterns18 .Pfam and BLAST searches found that three of the selected ORFs uncovered new copies of annotated genes -bearing non-coding RNA SPNCRNA.31. Both the AnABlast ORF (42 amino acid length) and the annotated non-coding RNA are found in the forward strand, with the Ribo-Seq profile confirming the translation of the ORF Fig. A.26 Polypeptides encoded by these two new genes lack significant homologs or motifs in databases.The second new gene contains a short 28 residues ORF that is highly expressed Fig. B. The coSchizosaccharomyces octosporus and eight in Schizosaccharomyces cryophilus, ranging in length from 102 to 190 residues (Supplementary Table S1B). RNA expression profile  likely resulted from premature nonsense mutations, generating a fossil carboxyl-terminal sequence that in the remaining related proteins is still active.The other two AnABlast ORFs, in addition to being translated, encode polypeptides that share significant similarity to known proteins. A main AnABlast peak, flanked by two others in different reading frames, identifies one of these new genes . In fact, RNA-Seq data suggest the existence of at least two exons in this region  along the entire sequence of the WP_002758301.1 protein from Supplementary Fig. S4).We cannot rule out the existence of new genes in the remaining ORFs found by AnABlast search. However, neither significant translation levels nor sequence/motif conservation support their actual gene status .The 3.5.Supplementary Table S1C).As shown above, our coding sequences search strategy can both find new genes and uncover their associated evolutionary history, revealing fossil-coding sequences that have being subtracted by changes in stop positions Figs B and 3C 29 and the stop codons.26 Our findings indicate that AnABlast may be useful to identify subtractions occurred during evolution. Remarkably, AnABlast can identify long terminal changes which, according to previous studies,29 represent rare events among terminal subtractions and expansions. Since evolutionary expansions are part of present genes, AnABlast analysis of intergenic regions carried out in this study cannot uncover these evolutionary events.Evolutionary changes in coding sequence length seem to be dynamic processes via shifts in the position of the start3.6.Supplementary Table S1D). This finding casts light on how random combinatorial protein domains, including exogenous DNA, may contribute to the emergence of new traits during evolution.The vast majority of the selected AnABlast profiles identified single peak regions without putative ORFs . Since AnABlast identifies coding sequences independently of ORFs, most of these peaks could represent gene fragments arisen from mutations and/or genome rearrangements. Accordingly, careful BLAST and Pfam analysis of predicted amino acid sequences in some of these peaks revealed fragments of tf1-retrotransposon Fig. A, of a Mtlh1/tlh2 genes) provide good examples of rearranged genes. As shown by AnABlast profiles, at least two regions containing tlh1/tlh2 DNA fragments located in subtelomeric regions of Chromosomes I and II, respectively, harbour identical rearranged structures  Fig. D, or up see Fig. E and F. see Fig. , albeit Interestingly, the identical genomic gene fragments pattern revealed by AnABlast profiles in both subtelomeric intervals suggests that evolutionary events yielding such fragments likely occurred earlier than their translocations to the subtelomeric region. However, we cannot rule out that occasionally, these AnaBlast results could point to possible genome assembly errors rather than rearranged fragments and/or missing annotations.3.7.30 transferred from other genomes.As described above, BLAST analysis of a particular AnABlast peak identified a possible fossil transposase sequence, perhaps of bacterial origin , indicating that this genome fragment has a mitochondrial origin Fig. A. Furtheied Fig. D.Figure3.8.in silico strategy (AnABlast) to reveal present and ancient coding regions in sequenced DNA. We have shown that the identification of rearranged and fossil sequences by AnABlast may be helpful to better understand the evolutionary history of the S. pombe genome. This property, together with the ability to uncover novel genes and pseudogenes, suggests that AnABlast provides a new strategy of general interest for the genome-wide search of present and fossil sequences in any genome.Here we report that low-stringency alignments accumulation in predicted amino acid sequences may discern coding from non-coding DNA, thereby providing a new J.J. and A.P.P. conceived the study and designed the experiments. A.P.P. developed the computer programme. A.P.P. and M.G. generated AnABlast profiles. C.D.S.D. and J.M. performed Ribo-Seq experiments. J.J. did the analyses of AnABlast peaks and wrote the manuscript. J.J., C.D.S.D., M.G., J.M., and A.P.P. critically read and approved the manuscript.Supplementary data are available at www.dnaresearch.oxfordjournals.org.Ministry of Economy and Competitiveness of the Spanish Government grant BFU2013-46923-P (J.J.) and by Biotechnology and Biological Sciences Research Council (UK) research grant BB/J007153/1 (J.M.). Authors declare no competing interest. Funding to pay the Open Access publication charges for this article was provided by the Ministry of Economy and Competitiveness of the Spanish Government.This research was supported by the


Staphylococcus aureus arises from a complex interplay between host and pathogen. Genetic variation can result in increased susceptibility to infection within the host and an increased capacity for virulence within the pathogen, resulting in a wide array of clinical syndromes. This review presents evidence for the role of bacterial and human genetic variation in influencing the clinical outcome of S. aureus infections.The clinical diversity of syndromes caused by S. aureus isolates [S. aureus isolates of the same genetic background, or clonal complex (CC). Staphylococcal toxic shock syndrome, a disease of unchecked inflammatory cascade induced by superantigen toxic shock syndrome toxin-1 (TSST-1), provides a classic example of the potential impact of MGE on clinical virulence of S. aureus. The gene that encodes for TSST-1, tst, is located on the MGE SaPI1 and is spread horizontally in distinct CCs of clinical S. aureus isolates [Genetic variation that encodes for virulence, antibiotic resistance, and host adaptation can be introduced through horizontal transfer of mobile genetic elements (MGE)—including bacteriophages, pathogenicity islands (SaPI), plasmids, transposons, and cassette chromosomes—between isolates . MGE tenisolates ,3.S. aureus CCs may be associated with an increased risk for other types of S. aureus infection. S. aureus from CC5 and CC30 genotypes were significantly associated with hematogenous complications, including left-sided native valve infective endocarditis (IE), when compared with clinical S. aureus isolates of other genotypes from the same referral area [S. aureus isolates from patients with either left-sided IE or soft tissue infection [S. aureus isolates belonging to the USA200, a genotype defined by pulsed-field gel electrophoresis that approximately corresponds to CC30 in the multi-locus sequence typing strategy, were significantly more likely to cause IE but less likely to cause lethal sepsis than isolates from either USA300 (CC8) or USA400 (CC1) genetic backgrounds [Asymmetric clustering of adhesins and toxins within specific ral area . The asskgrounds . The attkgrounds –9.hla, the gene in S. aureus that encodes for the potent virulence factor alpha toxin [S. aureus differed from isolates of other lineages by significantly higher expression of protein A (spa), putative membrane proteins , and exported proteins , as well as significantly lower expression levels of genes within the pyrimidine biosynthesis pathway , iron repressible ABC transport , and an azoreductase (acpD) [What makes the CC30 clonotype distinct? The reduced sepsis virulence of CC30 is at least partially explained by a stop codon mutation in ha toxin . A numbee (acpD) . Which oS. aureus. For example, fibronectin-binding protein A (FNBPA), encoded by fnbA, is thought to play a critical role in the initiation of IE [fnbA of S. aureus bloodstream isolates would be associated with an increased risk of cardiac device infection (CDI) in patients who developed S. aureus bacteremia [fnbA of bloodstream S. aureus isolates, were significantly associated with an increased risk of CDI in the source patient. Using atomic force microscopy (AFM), isolates containing these SNPs exhibited significantly higher frequency and strength of binding to fibronectin. Synthesized peptides containing two of the three polymorphisms (H782Q and K786N double mutant) exhibited 34% higher binding activity than the wild type by AFM. In silico molecular dynamics simulations demonstrated that residues of each of the three polymorphisms in FNBPA formed extra hydrogen bonds with fibronectin, providing a potential explanation for this observation of higher binding affinity. The association between specific fnbA SNPs and an increased risk of CDI was recently validated in a cohort of German patients with cardiac devices and S. aureus bacteremia [S. aureus bacteremia [fnbA SNPs and infection type may be due in part to the fact that arthroplasties lack a fibrin sheath, the fibronectin-rich coating present on endovascular leads of cardiac devices [Bacterial genetic variation can also occur on the level of polymorphisms within specific genes that contribute to the virulence of on of IE . FNBPA bcteremia . Three ncteremia . Interescteremia . The app devices .S. aureus infections have been observed in genetically distinct ethnic populations [S. aureus infection. Finally, different strains of sheep [S. aureus infection, sepsis, and death.Host genetic characteristics can also influence the host–pathogen interaction . Higher ulations –18. Patiulations , Hyper-Iulations , IRAK-4 ulations , MyD88 dulations , and chrulations  also exhof sheep , cattle of sheep , and micof sheep  have difS. aureus colonization and infection. In a study of adult Danish twins, investigators report S. aureus nasal carriage in 26.3% of the 617 twin pairs studied, with a concordance rate among monozygotic twins only slightly greater than the overall prevalence [Despite this indirect evidence, none of the handful of studies published to date have confirmed the role of human genetic variation in evalence . No signS. aureus infection. Nelson et al. used a GWAS approach to compare 361 Caucasian patients with healthcare-associated S. aureus bacteremia (SAB) to 699 hospitalized controls without S. aureus infection [p < 9.2x10-8). However, upon excluding the interaction between host SNP and bacterial CC, the investigators did note that rs2043436, an SNP located on the candidate gene CDON, which encodes a cell surface receptor that is a member of the immunoglobulin family, was associated with severity of infection at the level of p = 1.64x10-6. Ye et al. (2014) used GWAS to compare 309 cases with S. aureus infection to 2,925 uninfected adult Northern European control subjects. Again, none of the SNPs identified met genome-wide significance (p < 5x10-8). Four SNPs approached significance at a level of p < 10−5. Genes associated with these SNPs were PDE4B (rs2455012), involved in bacterial-induced inflammation; TXNRD2 (rs3804047), involved in the maintenance of thioredoxin in a reduced state; VRK1, which encodes a serine and/or threonine kinase; BCL11B (rs7152530), which encodes a repressor involved in T cell development; and PNPLA5 (rs470093), involved in autophagosome function.Three genome-wide association studies (GWAS) have looked at potential associations between common genetic variants and human susceptibility to nfection . No genoS. aureus infection. The investigators genotyped a Caucasian population of 4,701 cases of S. aureus infection and 45,344 matched controls [p = 1.3 x 10−10 and rs35079132: p = 3.8 x 10−8) achieved genome-wide significance, and one adjacent genotyped SNP was nearly significant genome-wide (rs4321864: p = 8.8 x 10−8). These polymorphisms were located near HLA-DRA and HLA-DRB1 genes on chromosome 6 in the HLA class II region. Significant evidence supports the possibility that HLA class II haplotypes may influence human susceptibility to S. aureus infection. First, specific HLA haplotypes (HLA II DR14 and/or DQ5) are associated with susceptibility to invasive Streptococcus pyogenes infection in patients [S. pyogenes [S. aureus [S. aureus superantigens, including TSST-1, bind to the HLA II DR1 molecule [S. aureus bacteremia and endocarditis [S. aureus is associated with the HLA-DR3 and HLA-DR7 class II serotypes [S. aureus infection.Most recently, DeLorenze et al. provide the first GWAS evidence of human genetic susceptibility to controls . Two imppatients  and detepyogenes  and S. amolecule  and are erotypes , an inflS. aureus infection will identify high-risk populations in whom expensive and invasive diagnostic and therapeutic strategies can be invested in an increasingly cost-conscious healthcare environment. Achieving these potential advances, however, will require overcoming a number of scientific and practical limitations. First, virulence in S. aureus is noteworthy for its redundancy, with many proteins exhibiting overlapping function. For example, at least four S. aureus proteins have the capacity to bind fibrinogen: FNBPA, clumping factor A, clumping factor B, and bone sialoprotein-binding protein [S. aureus, including multiple mutations in the same genes [Studying the impact of bacterial genetic variation on infection severity in patients will improve our understanding of pathogenesis and will ultimately inform vaccine development and future therapeutic targets. Similarly, insights into the role of human genetic variation on invasive  protein . At leasme genes . This wiS. aureus. Variation introduced by the inclusion of multiple infection types  can be reduced by focusing on a single, carefully defined clinical syndrome. For example, focusing on complementary bacterial receptors and host ligands, such as S. aureus FNBPA and human fibronectin in patients with hematogenous cardiac device-associated infections, reduces the possibility of a false negative result by minimizing the number of host–pathogen interactions at play. Finally, human genetic variation may be minimized by limiting study populations to single ethnic backgrounds when conducting human genotyping studies.Given these multiple sources of potential confusion, translational investigations focused on staphylococcal pathogenesis should strictly minimize sources of study variation. Bacterial genetic variation could be reduced by limiting studies to infections caused by a specific genotype of S. aureus infection exists. Within the pathogen, evidence is found at the clonal, gene, and SNP levels. Observational studies of genetically distinct ethnic populations and inbred animals also suggest the importance of host genetic variation on the initiation and severity of S. aureus infection. More translational studies investigating the role of host genetic variability in S. aureus infection are warranted. The confounding impact of heterogeneity introduced into genetic association studies in S. aureus can be minimized by limiting study populations by infection type, pathogen genotype, and host ethnicity.In conclusion, substantial evidence for the impact of genetic variation on susceptibility to


Carotenoids are a class of diverse pigments with important biological roles such as light capture and antioxidative activities. Many novel carotenoids have been isolated from marine organisms to date and have shown various utilizations as nutraceuticals and pharmaceuticals. In this review, we summarize the pathways and enzymes of carotenoid synthesis and discuss various modifications of marine carotenoids. The advances in metabolic engineering and synthetic biology for carotenoid production are also reviewed, in hopes that this review will promote the exploration of marine carotenoid for their utilizations. Carotenoids are a class of naturally occurring pigments originated in the chloroplasts and chromoplasts of plants, algae and some photosynthetic microorganisms ,2,3,4. AThe ocean is a complex aquatic ecosystem covering about 71% of the Earth’s surface, which is around 300 times larger than the habitable volume of the terrestrial habitats on Earth. A large proportion of all life on Earth lives in the ocean. Ecologically distinct from the terrestrial ecosystem, the ocean constitutes a unique reservoir of marine biodiversity and provides a vast resource of foodstuffs, medicines, and other useful materials. As such, more than 250 novel carotenoids have originated from marine species , many ofMany carotenoids have been reported from a wide range of marine species. The advances in current technologies facilitate the elucidation of the carotenoid biosynthetic pathways and relevant enzymes from marine species, which would enable the production of important carotenoids from marine organisms.C-methyl-d-erythritol 4-phospahte (MEP) pathway and the mevalonic acid (MVA) pathway, are responsible for the synthesis of IPP and DMAPP. These two pathways have been reviewed in detail elsewhere [Escherichiacoli and CrtE of Pantoeaagglomerans) [Biosynthetic routes to carotenoids begin with the basic building blocks isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP), although carotenoids are very diverse in chemical structure. Two distinct pathways, the 2-lsewhere ,20,21,22omerans) ,24. As somerans) ,26.Enzymes involved in the biosynthesis of carotenoids have been mainly investigated in carotenogenic cyanobacteria and land plants ,28. They Acaryochlorismarina and Prochlorococcusmarinus are clustered with green algae and land plant tomato  to red , which benefits carotenogenic gene mining and engineering upon carotenoid synthesis pathway. To date, many carotenoid biosynthetic genes have been cloned from plants, bacteria, and fungi based on their abilities to render different colors to the host ,69,70. TSaccharomycescerevisiae and Candidautilis by the expression of carotenogenic enzymes from Pantoeaananatis [Corynebacterium glutamicum is a native producer of decaprenoxanthin and its glucosides, and it has been engineered to synthesize C50 carotenoids C.P.450 and sarcinaxanthin [E. coli by the expression of carotenogenic enzymes from P. agglomerans (27 mg/L) than from P. ananatis (12 mg/L) [P. agglomeras and P. ananatis in E. coli [Agrobacteriumaurantiacum extends the zeaxanthin β-d-diglucoside pathway from P. ananatis, and synthesizes novel astaxanthin β-d-diglucoside and adonixanthin β-d-diglucoside [d-xylulose-5-phosphate synthase and reductoisomerase led to a 3.6-fold increase in lycopene production in E. coli when compared with the native MEP pathway for IPP and DMAPP supply [Xanthophyllomycesdendrorhous significantly increased β-carotene production in S. cerevisiae [Streptococcuspneumonia and Enterococcusfaecalis into E. coli, which enables the recombinant host to produce 465 mg/L of β-carotene [Pseudomonas putida and Bacillussubtilis have also been developed as platform hosts for carotenoid production [Carotenoid biosynthesis emerges from the central isoprenoid pathway, either the MEP pathway or the MVA pathway, existing in all organisms ,22. The ananatis ,76. Coryaxanthin . To date12 mg/L) . Metabol12 mg/L) ,79,80. β E. coli . In anotlucoside . GeneralP supply . Overexprevisiae . A greatcarotene . With mooduction ,85.E. coli to minimize diffusion limitation and achieve a 77-fold increase in mevalonic acid production [Citrobacterfreundii has been heterologously recasted in E. coli [E. coli promoters that respond to the toxic FPP have been successfully used to dynamically regulate the amorphadiene synthesis pathway and improve the production by two-fold over common inducible promoters and constitutive promoters [Carotenoids synthesis involves multiple enzymes . The expoduction . The pro E. coli . Some in E. coli . To avoiromoters . The Ntrromoters .denovo synthesis of carotenoids is initiated from acetyl-coA by the MVA pathway or glycolytic metabolites pyruvate and glycraldehyde-3-phosphate (G3P) by the MEP pathway. The direct efforts are focused on the modification of associated genes to these pathways. Deletion of pyruvate kinases PykFA can balance the availability of pyruvate and G3P for the MEP pathway, and increase lycopene production by 2.8-fold in E. coli [For the most efficient carotenoid production, the biological system of the host organism also needs to be optimized, by, for example, redirecting cellular carbon flux to the carotenoid synthesis pathway. The  E. coli . The del E. coli . Deletio E. coli . Replace E. coli .S. cerevisiae with periodic hydrogen peroxide shocking, resulting in a three-fold increasee of β-carotene production. Subsequent transcriptome analysis indicates that some genes related with lipid biosynthesis and MVA pathways are up-regulated in the adopted strains [ROX1, YJL064W, and YJL062W shows an almost four-fold increase in total carotenoid production [hnr, yjfP, and yjiD related to the improvement of lycopene production have been identified from a global transposon E. coli mutant library [ gdhA, cyoA, ppc, gpmA, gpmB, eno, glyA, aceE, talB, and fdhF have been insilico identified using a stoichiometric model [gdhA, aceE and fdhF was validated to increase lycopene production by nearly 40% in E. coli over the engineered parental strain. A similar set of gene deletions dhA, cyoA, gpmA, gpmB, icdA, and eno have been also in silico identified using different metabolic network models [E. coli [E. coli [E. coli genes in an a priori knowledge-independent way [A heterologous pathway is not just an independent entity. It communicates with the native cellular metabolism and is therefore governed by the global regulation of the host organisms. Adaptive laboratory evolution is a traditional route for strain engineering to achieve desirable industrially relevant phenotypes. Owing to the antioxidant properties of carotenoids, adaptive evolution has been successfully applied to an engineered  strains . It alsooduction . Gene de library . Other gic model . The trik models . Overexp[E. coli . With a [E. coli ,114. ATP[E. coli . The adv[E. coli . The moddent way .E. coli strain has been developed to produce lycopene and astaxanthin [recA gene. With this approach, lycopene production has been increased by 60% from single copy integrated strain. The ClChE strategy has been further modified to eliminate antibiotic marker for environmental safety and health issue after the evolution of the recombinant host strain [Metabolic engineering for the production of valuable compounds often heavily relies on plasmid-based expression of the synthesis pathway in a heterologous host. Although plasmids are easily manipulated and allow strong expression of targeted enzymes, the plasmid-based systems suffer from genetic instability such as plasmid loss, an additional antibiotic cost, and a potential risk of antibiotic marker spreading to other organisms . Accordiaxanthin . The expaxanthin . ClChE at strain .Sphingomonassp. [ Paracoccussp. result in the improvement if its specificity of to synthesize astaxanthin [Staphylococcusaureus dehydrosqualene (C30) synthase has evolved to synthesize lycopene by mutation F26L or F26S [P. agglomerans and P. ananatis results in the isolation of a variant favoring the production of fully conjugated tetradehydrolycopene [insilico simulation and the prediction using a priori enzyme information, which greatly liberates biologists from onerous screening task. This strategy requires adequate information to predict specific targeted amino acid mutations, which can confer desired enzyme traits [Pathway engineering for efficient production of desired chemicals is often challenged by limitations associated with the pathway enzymes themselves, such as low turnover numbers and promiscuities generating unwanted by-products . Proteinonassp. . Three maxanthin . Staphyl or F26S . DNA shulycopene . Rationae traits . UnfortuTsugacanadensis GPPS has been screened using the lycopene synthesis pathway as a colorimetric reporter. The GPPS variant (S239C and G295D) is created to increase levopimaradiene production with a 1.7-fold increase over the wild type in E. coli [As aforementioned, carotenoids are derived from the central isoprenoid pathway, which is also employed to synthesize several essential and secondary metabolites in nature. The carotenoid-based colorimetric screening has been developed for evolution of other isoprenoid pathway enzymes. Mutations of GGPP synthase are hypothesized to affect the binding efficiency of the magnesium ions needed for substrate anchoring and improve its catalysis. An error-prone PCR library of  E. coli . Augment E. coli .i.e., seaweed) and unicellular microalgae. Microalgae have recently garnered interest for production of valuable chemicals including carotenoids [i.e., D.salina for β-carotene and H. pluvialis for astaxanthin). The carotenoid production from microalgae is closely related to culture conditions such as illumination, pH, temperature, nitrogen availability and source, salinity, the oxidant substances, and growth rate [D. salina is a model species of green microalgae which is widely cultivated outdoors for β-carotene production [D.salina [Chlamydomonasreinhardtii is the first and the best studied transformed Chlorophyte, and the nuclear genetic manipulation is easy and well established. It has been engineered with β-carotene ketolase from H. pluvialis to synthesize ketolutein (Hb1H'a1, a1H'a2, Algae are a diverse group of aquatic, photosynthetic organisms, generally categorized as macroalgae (otenoids ,131, becotenoids . Some miwth rate ,133,134..salina . The abi.salina , in a si.salina . The freHb1H'a1,  and adonThe vast and mysterious ocean breeds diverse marine lives and provides unexhausted foodstuffs, nutriment, and drugs for humans. Diverse carotenoids are found from marine species and show broad utilities as colorant fragrance cosmetics and pharmaceuticals. The synthetic pathway of several carotenoids has been illuminated from marine species, which could benefit engineering processes in several host organisms for the production of carotenoids such as β-carotene, astaxanthin, and lutein. On the other hand, carotenoids such as β-carotene often undergo a series of modifications in the miraculous marine ecosphere. And indeed, several novel carotenoids have been isolated during the exploration of the marine ecosphere, while their pharmaceutical potentials remain to be examined due to the limited amount of extracts. Metabolic engineering and synthetic biology allow the assembly of such a chimeric pathway in a tractable organism for the mass production of rare carotenoids and also exhibit the potential to extend the catalogs of carotenoids to non-natural carotenoids, which could accelerate the exploration of novel carotenoids. It is noted that decoded carotenoid pathways and enzymes are still limited to a few marine organisms, although the J. Craig Venter Institute with worldwide collaboration had sequenced and annotated the genomes of 177 marine microbes up until 2010. However, we believe that the developed and developing technologies will allow us to search for novel marine carotenoid pathways in the future.


E. coli, K. pneumoniae, P. aeruginosa and A. baumannii were assessed. Overall prevalences of E. coli, K. pneumoniae, P. aeruginosa, and A. baumannii were 94%, 16%, 12%, and 2%, respectively. Bacterial recovery rates were independent from the sampling-timepoint during hospital stay. Polyurethane-cellular-foam or nylon-flocked swabs recovered significantly more bacteria as compared to rayon swabs. Intraanal swabbing resulted in significantly higher bacterial quantities as compared to perianal swabbing. In contrast, for the detection of A. baumannii, perianal swabbing seems more suitable than intraanal swabbing. Gender-related differences in bacterial recovery could be detected from perianal but not from intraanal swabs.Screening recommendations for multidrug-resistant Gram-negative bacteria comprise microbiological analyses from rectal swabs. However, essential specifications of the preanalytic steps of such screenings, i.e. the sampling technique, sampling devices and sampling site, are lacking. For standardized and optimum screening conditions these parameters are indispensable. Here, the optimum parameters were examined irrespective of the antibiotic resistance patterns of the target bacteria in order to establish a general basis for this type of screening. Swabs with rayon, polyurethane-cellular-foam and nylon-flocked tips were tested. Different sampling locations were evaluated, i.e. perianal, intraanal and deep intraanal. Subjects were swabbed and quantities of  E. coli, K. pneumoniae, P. aeruginosa, and A. baumannii are among the most frequently isolated bacteria from nosocomial infections and revealed increasing resistance rates in the last years5678910Infections caused by multidrug-resistant Gram-negative organisms (MDR-GNO) are associated with higher mortality rates, longer hospitalization and higher costs as compared to infections by corresponding non-resistant isolates123Recommendations on active screening are discordant. Some health authorities suggest an active screening, i.e. in Germany12in vitro and in vivo performance data with a broad variation of results concerning the bacterial yield1718192021In microbiological diagnostics swab-based sample acquisition is commonly used due to its performance ease and swiftness, both from screening sites and sites of infection. Combinations of different swabs and transport systems have been evaluated for E. coli, K. pneumoniae, P. aeruginosa and A. baumannii from three defined rectal swabbing locations, i.e. perianal, intraanal and deep intraanal, is systematically assessed. Aim of the study was to define the optimum preanalytic conditions for rectal screening in terms of recovering the highest quantities for each of the four bacterial species.The present study investigates the screening performance of three widely used swab-tip materials, i.e. rayon, polyurethane cellular foam or nylon-flocked fibers. Quantitative recovery of E. coli, K. pneumoniae, P. aeruginosa and A. baumannii were quantitatively assessed. Datasets from three different swabbing sites were included.To investigate the optimum swab-tip material, by means of recovering the highest quantities of bacteria, subjects were swabbed with rayon, PU-foam and nylon-flocked swabs. CFUs of Highest quantities were recovered when utilizing swabs with PU-foam and nylon-flocked tips. Mean quantities of recovered bacteria varied significantly between swabs with rayon and PU-foam tips (p < 0.001), as well as between swabs with rayon and nylon-flocked tips (p < 0.001). Swabs with tips made of PU-foam and nylon-flocked fibers performed similarly with no statistically significant differences. In detail, mean recovered bacterial quantities were 1,188 CFU, 50,063 CFU and 77,015 CFU for swabs with rayon, PU-foam and nylon-flocked tips, respectively .To analyze the impact of the sampling site on the bacterial recovery rates, subjects were swabbed at three different locations, i.e. perianally, as well as 1 cm and 5 cm intraanally. Datasets from the three different swab-types were included.Highest bacterial quantities were recovered when swabbing deep intraanally. Mean quantities of recovered bacteria varied significantly between perianally and 1 cm intraanally swabbing (p < 0.001), as well as between perianally and 5 cm intraanally swabbing (p < 0.001). No statistically significant differences were observed between the two intraanal settings (p > 0.05). In detail, mean recovered bacterial quantities were 16,041 CFU, 42,039 CFU and 70,187 CFU from swabs taken perianally, 1 cm and 5 cm intraanally, respectively . AnalyseE. coli, K. pneumoniae, P. aeruginosa and A. baumannii were assessed for each sampling site. Only datasets from subjects colonized with the corresponding bacterium were included in the statistical analysis.With this study, we intended to define the optimum sampling site when in search for a specific bacterial species - a test condition that resembles the clinical situation of screening a transferred patient to verify a previously reported colonization with multidrug-resistant Gram-negative rods. Therefore, the recovered bacterial quantities for E. coli (p < 0.001), K. pneumoniae (p < 0.01) and P. aeruginosa (p < 0.01). For A. baumannii greater quantities were found perianally as compared to 1 cm or 5 cm intraanal swabbing. Due to the small number of Acinetobacter-colonized patients, these differences were not statistically significant . Perianally, statistically significant less bacterial quantities could be recovered from screening female subjects as compared to male subjects (p < 0.001) .To investigate the potential influence of exposure to the hospital environment on screenings results, data was evaluated for screenings performed within the first 2 days after hospital admission and compared to screenings performed thereafter. Datasets from the three different swab-types were included.Performing screenings at early or late timepoints during the hospital stay revealed no statistically significant differences concerning recovered bacterial quantities (p > 0.05) .E. coli, K. pneumoniae, P. aeruginosa and A. baumannii, prevalences of these species were calculated  risk factors for carriage, (ii) appropriate transport conditions and duration, (iii) (easy accessible and well defined) screening site or body region, respectively, (iv) optimum sampling device. While risk factors for the carriage of MDR-GNO are at least partly defined23242526272829For MSSA or MRSA the predominant colonization habitat has been identified as the anterior part of the nasal cavity32Obviously, different tip materials have different physical and chemical characteristics. This in turn influences the process of specimen collection and subsequent release. Therefore, concerning MDR-GNO screening, it seems prudent to first define standards, such as the optimum sampling location and sampling device, before providing general recommendations on screening.By the present study it was demonstrated that swabs with PU-foam or nylon-flocked tips are superior in a rectal screening situation as compared to conventional rayon swabs. The better performance of these materials as compared to conventional rayon swabs is in line with findings from various clinical analyses34353637E. coli and K. pneumoniae, i.e. the colon and rectum. Also, P. aeruginosa prefers moist habitats3940Klebsiella sp. resulted in a loss of bacteria within 150 minutes43A. baumannii still highly controversial but analyses indicate a potential location rather on the skin than in the colon2545464748A. baumannii recovered by swabbing from the perianal skin than from intraanal sites. Especially in an outbreak scenario or when screening transferred patients with a pre-reported MDR-GNO colonization status these colonization differences should be considered and consecutively might help to better detect a specific species by a targeted screening.Furthermore, the present study revealed that deep intraanal swabbing leads to higher bacterial quantities when culturing the extracted material on solid media. This is explainable by the natural colonization habitat of the enterobacteriaceae E. coli in subjects. It could also be appropriate when trying to detect K. pneumoniae and P. aeruginosa, even though the prevalences of the two species are rather low. With a prevalence of 2% for A. baumannii rectal swabbing seems to be not suitable for a reliant detection by screening in this anatomical region. Of note, within our study group, bacterial prevalences and recovered bacterial quantities were marginally affected by the length of hospital stay allowing for comparable screening results independent from sampling-timepoints.Differences between male and female subjects regarding in the bacterial quantities recovered by intraanal swabbing are not present, generally allowing for the same screening procedures for male and female patients. Differences in recovered bacterial quantities by perianal swabbing were observed, the reason remains in the range of speculation since personal hygiene habits were not examined. Regarding the investigated species-specific prevalences one could infer that rectal screening is definitely suitable for the detection of However, differences in recovered bacterial quantities due to swab-tip materials or swabbed body regions might be bypassed when utilizing feces instead of swabs. Here, standardized amounts could be processed in the laboratory. For example, analyses for Vancomycin-resistant enterococci carriage displayed higher detection rates when analyzing stool samples compared to swab-based screenings50Additional loss of time could be due to using the wrong sampling device or swabbing at the wrong sampling site, which in turn leads to now clearly demonstrated, statistically significant lower bacterial recovery rates. As a consequence, subcultures or broth enrichments steps could be necessary, resulting in a delay to definite diagnosis of at least one day. As patients in general benefit from fast diagnostics9525354Finally, the precise bacterial quantities detected by optimum sampling devices and locations could increase the awareness of the medical staff towards the sites most important for the transmission of the bacteria and thus, should stimulate correct hygienic behavior.A. baumannii by rectal swabs remains questionable. Screening recommendations must comprise precise information on screening localization, sampling devices and sampling techniques as well as transport conditions.This study outlines the enormous effects of swabbing devices and testing of defined body regions on the recovered bacterial quantities. Specific suggestions for optimum sampling are given. In search for MDR-GNO, screening should be performed intraanally by swabs with PU-foam or nylon-flocked tips. With this approach, gender-specific differences can be neglected. Reliable detection of 50 subjects were included . Inclusion criteria were age >18 years and no antibiotic treatment in the last 4 weeks. Exclusion criteria were pregnancy and inability for a written consent to this study. Patients were recruited from the following units of the Rostock University Hospital: pneumology, gastroenterology, neurology, orthopedics, urology, and surgery. Patients were examined between the first and seventh day of hospital admission (mean 2.82 d). For analyses regarding the influence of the duration of the hospital stay and the timepoint when the screening was performed, patients were separated in two groups, i.e. early screening  and late screening .Rayon tip: Sarstedt, Nuembrecht, Germany, neutral swab, via Copan, Brescia, Italy, cat. no. 80.1301.Polyurethane cellular foam tip: MWE medical wire, Corsham Wiltshire England, Sigma Dry Swab Tubed, Σ-Swab, ref. MW941.Nylon-flocked fiber tip: Copan, Brescia, Italy, FLOQSwabs, ref. 552C.The following swabs were tested:Swabs were obtained from three different locations: perianal, intraanal (1 cm), and deep intraanal (5 cm).The screening was performed by the same investigator at all instances. Each screening location was swabbed by circulating while simultaneously rotating the swab for 360° and exerting gentle pressure. Each of the subjects was swabbed with each swab-type and at each anatomical site, resulting in nine swabs in total (i.e. three swab-types at three different locations). The order of the utilized swab-types was blinded/randomly chosen to avoid bias due to cyclic order. The swabbed location had to be accurately targeted and therefore one sided blinding was no option.2/5% CO2 atmosphere for 48 h, followed by counting of macroscopically visible colonies (CFU).All swabs were placed into the corresponding transport tube and were subjected to microbiological analysis within 60 minutes after rectal swabbing. Quantitative detection was performed according to CLSI quantitative swab elution methodRecovered microorganisms were identified by matrix-assisted laser-desorption-ionization time-of-flight mass spectrometry (MALDI-TOF-MS) using a Shimadzu “AXIMA Assurance” MALDI-TOF mass spectrometer  as described elsewhereAll data were stored and analyzed using GraphPad Prism Version 6 . All p-values resulted from two-tailed, nonparametric Wilcoxon-Mann-Whitney U-test of not normal-distributed variables. Marginal-distributions between the investigated parameters were analyzed. p-values < 0.05 were considered as significant . In repeated measurements analyses adjustments of alpha levels were carried out using the Bonferroni correction, i.e. the level of significance was lowered to 0.05/3 = 0.017 (for each of the three pairwise comparisons). Raw data is provided in the E.coli between swabs (averaged over locations)) three means could be calculated: 4,705 , 199,000 (1 cm), 307,000 (5 cm). Hence, the average of means was 170,000, the variance of means (V = Σ(μi − μ)2/3) was about 1.56*1010, standard deviations were 27,000 , 765,000 (1 cm), 1,360,000 (5 cm) and therefore average was 717,333. Spearman’s rho correlation between Rayon and PU foam was 0.664, between Rayon and Nylon-flocked was 0.640 and between PU foam and Nylon-flocked was 0.826, resulting in an overall average of 0.71. By using this information, sample size calculation with the nQuery, Vers. 7.0, modul MOT2-1 software and the “Univariate one-way repeated measures analysis of variance (constant correlation)” yielded n = 33 for each swab-type at a test significance level α = 0.05 and a power of 80%. The patient number was n = 42 for a power of 90%. Thus, the chosen sample size of n = 50 was well in the range of both power calculations.Since expected differences and standard deviations of results from different swab-types and sampling locations were initially unknown, sample size calculation could not be performed in advance. But by measuring the primary parameter  of University Medicine Rostock  in line with national and ICH-GCP guidelines. All participants provided written informed consent.How to cite this article: Warnke, P. et al. Screening for Gram-negative bacteria: Impact of preanalytical parameters. Sci. Rep.6, 30427; doi: 10.1038/srep30427 (2016).


Magnaporthe oryzae (anamorph Pyricularia oryzae) is the causal agent of blast disease of Poaceae crops and their wild relatives. To understand the genetic mechanisms that drive host specialization of M. oryzae, we carried out whole genome resequencing of four M. oryzae isolates from rice (Oryza sativa), one from foxtail millet , three from wild foxtail millet S. viridis, and one isolate each from finger millet (Eleusine coracana), wheat (Triticum aestivum) and oat (Avena sativa), in addition to an isolate of a sister species M. grisea, that infects the wild grass Digitaria sanguinalis.M. oryzae Oryza and Setaria isolates form a monophyletic and close to another monophyletic group consisting of isolates from Triticum and Avena. This supports previous phylogenetic analysis based on a small number of genes and molecular markers. When comparing the host specific subgroups, 1.2–3.5 % of genes showed presence/absence polymorphisms and 0–6.5 % showed an excess of non-synonymous substitutions. Most of these genes encoded proteins whose functional domains are present in multiple copies in each genome. Therefore, the deleterious effects of these mutations could potentially be compensated by functional redundancy. Unlike the accumulation of nonsynonymous nucleotide substitutions, gene loss appeared to be independent of divergence time. Interestingly, the loss and gain of genes in pathogens from the Oryza and Setaria infecting lineages occurred more frequently when compared to those infecting Triticum and Avena even though the genetic distance between Oryza and Setaria lineages was smaller than that between Triticum and Avena lineages. In addition, genes showing gain/loss and nucleotide polymorphisms are linked to transposable elements highlighting the relationship between genome position and gene evolution in this pathogen species.Whole genome sequence comparison confirmed that M. oryzae isolates revealed gain and loss of genes as a major evolutionary mechanism driving specialization to Oryza and Setaria. Transposable elements appear to facilitate gene evolution possibly by enhancing chromosomal rearrangements and other forms of genetic variation.Our comparative genomics analyses of host-specific The online version of this article (doi:10.1186/s12864-016-2690-6) contains supplementary material, which is available to authorized users. Magnaporthe oryzae causes disease on an array of important crops such as rice, wheat, barley, finger millet, foxtail millet, and wild grasses [M. oryzae has received considerable attention as the causal agent of rice blast disease [M oryzae is categorized into several host-specific subgroups that are pathogenic on a variety of plants that include Oryza spp., Setaria spp., Triticum spp., Avena spp., Eleusine spp. etc. [M. oryzae reproduction is predominantly asexual. When environmental conditions are conducive, M. oryzae generates an abundance of asexual clones that rapidly colonize its host. The rapid spread of such asexual pathogens poses a significant threat to global food security, human health and biodiversity [ grasses . M. oryz disease , which l disease . M oryzapp. etc. , 4, 5. Aiversity , 7.M. oryzae is limited to specific geographic regions such as Yunnan, China and India [Sexual reproduction for nd India –10. The nd India . For insnd India . In fungnd India –16. Effend India , 17.M. oryzae, whole-genome comparative analysis between two Oryza isolates P131 (from Japan) and Y34 (from China) revealed frequent chromosomal rearrangements, resulting in structural variation such as gain and loss of genes [AVR-Pita was more variable among asexual rice infecting isolates compared with other cereal infecting isolates, suggesting that selection pressures acting from its cognate resistance gene Pita of rice may trigger the diversity in the genome location of AVR-Pita [AVR-Pik that differ in just a few amino acids interact with different specificities with alleles of the resistance gene Pik [For of genes . The genAVR-Pita . This obAVR-Pita , 20. Forgene Pik , 22.M. oryzae to specific hosts remain unclear. It has been proposed that Oryza-specific isolates of M. oryzae are most closely related to those identified on Setaria [M. oryzae is considered to have happened less than 10,000 years ago following rice domestication [M. oryzae on Triticum was only discovered in Brazil in the late 20th century [M. oryzae sub-populations. Almost all Oryza isolates apart from the center of origin propagate asexually and are only found on cultivated rice (O. sativa) [Oryza isolates cannot infect any other host plant. In contrast, M. oryzae isolates that are adapted to wild Setaria spp. have the same host range as isolates from cultivated foxtail millet S. italica [Setaria isolates can escape the artificial selection of breeding by shifting to wild host species. This difference between Oryza and Setaria pathogens could influence their genetic variation within these host-adapted M. oryzae populations.To date, the evolutionary mechanisms that drive adaptation of  Setaria . Whereas century . This ti sativa) . This ma italica , suggestM. oryzae host adaptation, we reconstructed the genome sequences of representative isolates from M. oryzae specifically infecting Oryza, Setaria, Eleusine, Avena and Triticum plant species and its sister species M. grisea. Host species specificity in M. oryzae has been studied by genetic analyses of these representatives of M. oryzae [Oryza and Setaria pathogens to examine genetic polymorphisms within population of these pathogens. Recently, Chiapello et al. 2015 [M. oryzae host-specific subgroups. They revealed that most genes were conserved among M. oryzae host-specific subgroups, and only a small number of gene families was associated with host specificity [Oryza-Setaria vs. Avena-Triticum pathogens) using different approaches. We discovered that genes showing gain/loss and excess of nonsynonymous substitutions usually encoded proteins whose functional domains are present in multiple copies in each genome. We identified a higher degree of presence/absence polymorphisms for Oryza and Setaria infecting M. oryzae isolates when compared to isolates from Triticum and Avena even though the genetic distance between Oryza and Setaria pathogens was smaller than that between isolates from Triticum and Avena. We also found that the lysin motif (LysM) domain effector gene Secreted LysM protein 1 (Slp1) was lost in the tested Setaria isolates. These observations suggest that gain and loss of genes in M. oryzae may act as a major evolutionary mechanism driving specialization to Oryza and Setaria host species. We could also link genes showing presence/absence and nucleotide polymorphisms to transposable elements (TEs), suggesting that TEs may trigger chromosomal rearrangements and mutation.To investigate the evolutionary mechanisms that drive . oryzae –28. In aal. 2015  studied cificity . CompareM. oryzae Oryza isolates, four Setaria isolates, five host-specific subgroup species and its sister species M. grisea, a pathogen of Digitaria species for full genome sequencing  and M. grisea Dig41  [M. oryzae 70-15 strain [M. oryzae host-specific isolates excluding Br48 was consistent with that of M. oryzae 70-15 and P131. However, the M. oryzae Br48 and M. grisea Dig41 isolates showed a lower percentage of CEGs in Group1 and Group3 compared with the controls although these percentages were still comparable to those reported for other published fungal genome sequences [We selected four ae 70-15 , but wer–14,781) . To furt–14,781) . We used5 strain  for comp5 strain . Group1 equences .Table 1SM. oryzae and M. grisea isolates, we performed OrthoMCL analysis [M. oryzae isolates and M. grisea Dig41 in addition to the published gene models of 70-15 strain, Y34 isolate and P131 isolate. We obtained 13,274 orthogroups, 7845 of which are single copy orthologs in our tested isolates. The number of single copy orthologs is larger than that (6976) in Chiapello et al. 2015 [M. oryzae and M. grisea, we generated a phylogenetic tree based on the third codon position of the single orthologous gene sets using a Maximum likelihood model  model  from the reference genomes of Setaria, Eleusine, Triticum and Avena isolates.For ultivars , 20. HowM. oryzae. We assigned loss and gain of genes into three categories: (1) genes that were lost once in the evolutionary history of M. oryzae, (2) genes that were subjected to multiple deletion events from different processes such as horizontal gene transfer and recombination, and (3) genes that uniquely existed in one host-specific subgroup. The number on each branch of the tree indicates the number of genes belonging to the first category  as compared to those of Triticum and Avena (8–18) although the genetic distance per synonymous site between the Oryza and Setaria isolates (0.0027 ± 0.0001) was smaller than that between the Triticum and Avena isolates (0.0053 ± 0.0000). This tendency was more prominent when considering genes encoding putative secreted proteins , which is commonly used as a parameter of positive selection  and Avena (Br58) isolates, a higher proportion of genes showed outliers of dN/dS. When the Triticum isolate Br48 and Avena isolate Br58 were compared to Oryza (Ina168) and Setaria (GFSI1-7-2) isolates, a higher proportion of genes showed outliers of dN/dS.Nucleotide substitutions can contribute to host specialization . To dete AVR-Pik , 22, gen AVR-Pik . Given tM. oryzae isolates, but the accumulation of nonsynonymous substitutions was dependent on divergence time.To examine how this compared to the presence/absence analysis, we plotted the number of genes with outlier values of dN/dS when genes with dN/dS = 99 were included or excluded or number of genes identified as absent in the reference genome against the genetic distance at synonymous sites (dS), which is proportional to time from separation for the two pathogen isolates Fig. . The numOryza isolate Ina168 shared 119 genes with Setaria isolate GFSI1-7-2, but only 39 genes with Triticum isolate Br48 in the Z2-1 gene model  analysis against the Pfam database  were shared between proteins independently lost for each lineage in the host-specific subgroups Fig. . SimilarM. oryzae, which are absent from at least one of the subgroups, we sorted genes based on the 70-15 strain published gene models [To test how many genes encoding proteins shown to be involved in various stages of appressorium development and infection showed loss among host-specific subgroups of e models  and genee models   in ps Table . The isoM. oryzae Oryza and Setaria subgroups, the synonymous/nonsynonymous ratio of DNA polymorphisms levels did not conform to that of divergence  test  to the oce Table . In partM. oryzae, effector genes displaying presence/absence polymorphisms are often located next to transposable elements (TEs) [Slp1 of Oryza isolate Ina168 resides between two multiple mapping read-rich regions including partial sequences of long terminal repeat (LTR) retrotransposons  , 18, 19.ons Fig. . It is ived Fig. , suggestM. oryzae Oryza and Setaria specific subgroups, respectively, genetic distance (Da) between Oryza and Setaria specific subgroups for Supercontigs 8.1 to 8.7  for 8.7 Fig. . The valM. oryzae genes according to their distance to the closest TE according to the same criteria of the analysis of presence/absence polymorphisms  was examined . From these results, we conclude that presence of transposable elements may increase genetic diversity and divergence of genes located nearby.To further investigate the relationships between the transposable elements and DNA polymorphisms of the linked genes, we classified sms Fig. . For eacM. oryzae with only a small fraction (1.2–3.5 % of the genes) dynamically gained and lost, as reported in Chiapello et al. 2015 [M. oryzae genes. Interestingly, we observed that in comparisons between host-specific subgroups, the isolate infecting rice showed the largest number of genes lost from the genome (345–441 genes), followed by the isolate from Setaria spp. (269–389 genes) and the isolate from Eleusine spp. (300–355 genes), respectively. The number of genes lost from the genomes of Triticum and Avena pathogens when compared to all other host-specific subgroups was relatively small (ranging from 151 to 243)  Fig. . These ohe 1980s , suggestM. oryzae when genes with dN/dS = 99 were included. Genes with outlier values of dN/dS could be classified into two groups. One group was potentially under positive selection. Positive selection was common among the effector genes AVR-Pizt, Slp1, PWL3, AVR-Pita3 and BAS2 . Ofisolates , 37.Another group of genes with outlier values of dN/dS had a few nonsynonymous substitutions and zero synonymous substitutions. Small synonymous divergence is not enough to give statistical power to detect positive selection . The numM. oryzae [M. oryzae.Recently, de Guillen et al. 2015  reported. oryzae  is consiBAS1 and AVR-Pik are observed in the Oryza-specific subgroup and not in the Setaria-specific subgroup. These effectors have been shown to be specifically induced during the biotrophic phase of M. oryzae infection on rice [M. oryzae subgroup to rice plants. On the other hand, the LysM domain effector gene Slp1 was missing in two Oryza isolates (TH-3 and P2) and in the Setaria subgroup. The Slp1 is an apoplastic effector that binds Chitin and contributes to the avoidance of pathogen-associated molecular pattern (PAMP) triggered immunity [M. oryzae on rice plants [Oryza isolate Ina168 had one additional putative secreted protein containing a LysM domain. Furthermore, the Setaria-specific subgroup lost Slp1 but harbor an extra LysM domain-containing protein, which potentially could compensate for the lack of Slp1. The loss of Slp1 from the Setaria-specific subgroup could be due to (1) functional redundancy driven by genetic drift, or (2) Slp1 being deleterious for adapting Setaria plants driving loss by purifying selection. As all other crop-infecting M. oryzae and also M. grisea Dig41 maintain Slp1, this suggests an essential role for the effector, with its loss in Setaria-specific subgroup likely a result of host specialization. However, we only examined a limited number of Setaria isolates. To challenge this hypothesis, it is necessary to survey a larger number of Setaria isolates to estimate the effector frequency in population of Setaria isolates.The effector  on rice , 59. Thiimmunity . Furthere plants . The 70-M. oryzae that predominantly propagates asexually, genetic recombination by sexual crosses is limited. Asexual propagation limits genetic diversity that is needed for adaptation to new environments and enhances the accumulation of deleterious mutations (Muller’s ratchet) reducing the mean fitness of the species (population). We hypothesize that TEs may substitute the function of sexual recombination to contribute to maintaining genetic diversity. For instance, genes closer to TEs could potentially be inadvertently mutagenized during imperfect TE-insertion mediated mismatch repair, thereby enhancing genetic diversity during prolonged periods of exclusively asexual reproduction.TEs are known to contribute to insertion, deletion and duplication of DNA segments in a genome. We observed that genes tightly linked to TEs show a higher incidence of presence/absence polymorphisms Fig. . In addiOryza-specific subgroup. Since there is no wild rice species in Japan, Japanese Oryza isolates survive only on cultivated rice. In contrast, Setaria isolates can survive on wild Setaria species, which are commonly distributed across Japan. Therefore, Oryza M. oryzae isolates, are more vulnerable to artificial selection of rice breeding. These different selection pressures could generate more frequent presence/absence polymorphisms in the Oryza-specific subgroup even though the level of nucleotide diversity in Oryza isolates was smaller than that in Setaria isolates. This excess of nonsynonymous polymorphisms in the Orzya subgroup could be explained by two general mechanisms. One is positive selection on protein coding genes in the Oryza-specific subgroup. For example, AVR-Pik seems to be engaged in an arms race with its cognate resistance gene Pik of rice [Oryza spp. may be caused by relaxation of purifying selection by its smaller population effective size. In either mechanism, the relatively higher level of nonsynonymous DNA polymorphisms in the Oryza-specific subgroup may provide the genetic and phenotypic diversity that becomes favorable when the population encounters abrupt changes of environment, e.g. changes in host cultivars. Such high level of intraspecific genetic plasticity should facilitate the persistence of this pathogen lineage despite the constantly changing host environment.We identified an excess of nonsynonymous DNA polymorphisms as compared to synonymous ones and presence/absence polymorphisms in the  of rice , 22. TheM. oryzae specialization to host plant species. We investigated gain and loss of genes and nucleotide substitutions between host-specific subgroups of M. oryzae. We found that redundancy of functional domains in the genome may have enabled a high degree of intra-specific gain and loss of genes and accumulation of nonsynonymous substitutions. Interestingly, these two mechanisms of genetic variations showed contrasting patterns in the comparisons of the host-specific subgroups (Oryza-Setaria vs. Avena-Triticum pathogens). The number of presence/absence of genes was not correlated with genetic divergence between the host specific subgroups, while the number of genes with outlier values of dN/dS correlated well with genetic distance. Though the genetic distance between the isolates infecting Oryza and Setaria was smaller than that between those infecting Triticum and Avena, the loss and gain of genes in Oryza and Setaria isolates were more frequently observed than those in Triticum and Avena isolates. Considering that Oryza and Setaria specific subgroups of M. oryzae have a longer evolutionary history of host specificity when compared to Triticum specific subgroup, this observation suggests that the differences are not explained by the elapsed time of genetic isolation but rather by coevolution with the specific host. In addition, the study of variations in Oryza- and Setaria-specific subgroups of M. oryzae revealed that the isolates pathogenic on Oryza had higher number of nonsynonymous polymorphisms, despite having smaller nucleotide diversity than the isolates pathogenic on Setaria. Differences in hosts and frequency in abrupt changes of environments may have shaped the contrasting patterns of genetic variations between these host-specific subgroups of M. oryzae.This comparative genomics study sheds light on the evolutionary mechanisms underpinning M. oryzae and M. grisea, which are maintained in Laboratory of Plant pathology, Kobe University, Iwate Biotechnology Research Center and National Institute of Agrobiological Sciences Genbank, using the DNeasy Plant Maxi kit (QIAGEN). Five μg of the DNA was used for constructing the paired-end libraries following manufacturer's instructions. The libraries were used for cluster generation on a flow cell and sequenced for 76 cycles on an Illumina Genome Analyzer IIx. Base calling and filtering of low quality bases were performed using sequence control software (SCS) real-time analysis, BCL converter and the GERALD module .We extracted DNA from mycelia of http://compbio.brc.iop.kcl.ac.uk/software/index.php). We conducted De novo assembly using a software ABySS [ab-initio and empirical methods. We estimated gene structure based on the published transcript sequences version 8 of M. oryzae 70-15 strain (Broad institute) and known M. oryza effectors [M. oryzae and M. grisea isolates were predicted using a software AUGUSTUS version 2.7 [Adapter sequences were removed from short reads adapter using a software cutadapt . Unpairere ABySS  accordinre ABySS . We evalre ABySS . We predffectors , and theffectors . Genes osion 2.7  after thsion 2.7 . Functiosion 2.7 . BLASTN sion 2.7  was run sion 2.7  as inputsion 2.7 . We usedMagnaporthe species, paired-end short reads from each species were aligned with BWA [M. oryzae isolates. Alignment files were converted to SAM files using SAMtools version 0.1.8 [Magnaporthe species. Most of heterozygous SNPs could be caused by misalignments or reads that are aligned on multiple genomic regions.To identify SNPs and indels among with BWA  to the 7on 0.1.8 . To remoon 0.1.8 . This piM. oryzae isolates, Venn diagram was created using an online diagram software Cleately .To detect loss of genes in tested isolates from the reference genome, we estimate breadth coverage over a gene based on the alignments according to Raffaele et al. 2010 . When shM. oryzae and M. grisea isolates were estimated based on the consensus sequences using yn00 [rd quartile of dN/dS in each pairwise comparison between isolates. If a threshold value was < 1.5, 1.5 was used as the threshold value.The maximum likelihood trees were constructed using the RAxML software . Consensing yn00 , which iOryza isolates and Setaria isolates, we calculated nucleotide diversity π [Oryza isolates and Setaria isolates by using the Bioperl libraries. Sliding window analysis of π, and Da was performed with the 500 Kbp widow size and 50 Kbp step size. R, a programming language for statistical computing, was used for visualization of the sliding window analysis. To test the neutral hypothesis of molecular evolution, McDonald and Kreitman test [To examine level and pattern of nucleotide polymorphisms within ersity π  and the ersity π  as a parman test  was carrman test .M. oryzae transposable TEs  and genetic variations, positions of TEs were identified. We performed BLASTN  using knThis article does not contain any studies with human participants or animals performed by any of the authors.Not applicable.M. oryzae isolates Ina168, GFSI1-7-2, Br48, Br58 and Z2-1 and M. grisea isolate Dig41 have been deposited to DDBJ Sequenced Read Archive under the accession numbers DRZ007458, DRZ007459, DRZ007460, DRZ007461, DRZ007462, and DRZ007468, respectively. The annotation files of M. oryzae isolates Ina168, GFSI1-7-2, Br48, Br58 and Z2-1 and M. grisea isolate Dig41 are available in the DDBJ Sequenced Read Archive under the accession numbers DRZ007463, DRZ007464, DRZ007465, DRZ007466, DRZ007467 and DRZ007469, respectively.Nucleotide sequence data reported are available in the DDBJ Sequenced Read Archive under the accession numbers DRR059884-DRR059895. The assembled genome sequences of


Linnaea has a circumboreal to north temperate distribution. In this study, we conducted phylogenetic and biogeographic analyses for Linnaeoideae and its close relatives based on sequences of the nuclear ribosomal ITS and nine plastid  markers. Our results support that Linnaeoideae is monophyletic, consisting of four eastern Asian lineages , the Mexican Vesalea, and Linnaea. The Mexican Vesalea was formerly placed in Abelia, but it did not form a clade with the eastern Asian Abelia; instead Vesalea and Linnaea are sisters. The divergence time between the eastern Asian lineages and the Mexican Vesalea plus the Linnaea clade was dated to be 50.86 Ma, with a 95% highest posterior density of 42.8 Ma (middle Eocene) to 60.19 Ma  using the Bayesian relaxed clock estimation. Reconstructed ancestral areas indicated that the common ancestor of Linnaea plus Vesalea may have been widespread in eastern Asia and Mexico or originated in eastern Asia during the Eocene and likely migrated across continents in the Northern Hemisphere via the North Atlantic Land Bridges or the Bering Land Bridge. The Qinling Mountains of eastern Asia are the modern-day center of diversity of Kolkwitzia-Dipelta-Diabelia clade. The Diabeliaclade became highly diversified in Japan and eastern China. Populations of Diabelia serrata in Japan and eastern China were found to be genetically identical in this study, suggesting a recent disjunction across the East China Sea, following the last glacial event.Linnaeoideae is a small subfamily of erect or creeping shrubs to small trees in Caprifoliaceae that exhibits a wide disjunct distribution in Eurasia, North America and Mexico. Most taxa of the subfamily occur in eastern Asia and Mexico but the monospecific genus  Intercontinental disjunct distributions of plants have fascinated botanists for centuries. The East Asian—North American plant disjunctions have attracted much attention in the last two decades –9. The dZabelia) [Linnaeoideae (Dumortier) P.F. Stevens is a subfamily of Caprifoliaceae s.l.  ,95. ThisZabelia) –22. The Zabelia) ,28,38,73rbcL, ITS sequences and morphological data from 45 samples , Donoghue et al. [ndhF and trnL-F from 26 samples , Zhang et al. [ndhF, trnL-F, trnL, matK and rbcL-atpB regions from 30 samples , Bell et al.[trnK, matK, atpB-rbcL and trnL-F) from 51 samples , Jacobs et al. [Abelia, Dipelta, Kolkwitzia, Linnaea as well as Vesalea. They also questioned the position of Zabelia (Rehder) Makino and a sister relationship with Morinoideae or Valerianoideae was proposed but with low support [rbcL, ndhF, trnL intron, trnL-F spacer and matK) from 31 samples , Landrein et al. [Diabelia.Improvements from successive molecular studies have provided important insights into the phylogeny of Linnaeoideae. (1) Based on e et al.  found thg et al.  supporteg et al. . (3) Basll et al. supportes et al.  were abl support . (5) Usin et al.  identifiLinnaea L. to include all members of Linnaeoideae except Zabelia. Major morphological differences within Linnaeoideae as well as a need to keep the number of nomenclatural changes to a minimum would direct to keep the monophyletic clades distinct. The redefined Linnaeoideae here includes six genera: Abelia R.Br., Diabelia Landrein, Dipelta Maxim., Kolkwitzia Graebn., Linnaea Dumortier and Vesalea M. Martens & Galeotti , Northeast China, Central Asia, the Caucasus, the Alps, the Appalachian and the Rocky Mountains  are endof China ,32. Vesag and Li ).rbcL, trnS-G, matK, trnL-F, ndhA, trnD-psbM, petB-D, trnL-rpl32 and trnH-psbA) with a nearly complete taxon sampling, including all genera, and most species, except Dipelta wenxianensis. Our objectives are to: (1) reconstruct the phylogeny of Linnaeoideae; (2) estimate the divergence times of the major lineages of Linnaeoideae using a fossil-calibrated molecular clock method; and (3) explore the evolution of the unique biogeographic distributions in Linnaeoideae, emphasizing on the intercontinental disjunctions in Eurasia, Mexico, and North America.The phylogeny and biogeography of Linnaeoideae remain poorly understood, and the unique disjunct distribution in East Asia and Mexico deserve far more attention. We thus conducted a phylogenetic study on Linnaeoideae using one nuclear marker (ITS) and nine chloroplast markers . A total of 32 accessions of Linnaeoideae representing seven species of Abelia, five species of Vesalea [Dipelta, three subspecies of Linnaea and the only species of Kolkwitzia were collected from China, Finland, Japan, and Mexico; 12 accessions representing ten outgroup genera were also added . The thermal cycling conditions were 3 min at 94°C, followed by 35 cycles of 30 s at 94°C, 40 s at 52°C and 1.5 min at 72°C, with a final extension of 10 min at 72°C. The obtained PCR products were purified with PEG8000 and sequenced using ABI Prism BigDye Terminator Cycle Sequencing Kits v. 3.1 on an ABI 3730xl DNA Analyzer  following the manufacturer’s instructions.Total DNA was extracted from silica gel-dried leaf tissue using the modified Cetyltrimethyl Ammonium Bromide (mCTAB) method . Approxin et al.  for matKt et al.  for trnLw et al.  for trnSn et al.  for ITS.g et al. . Each poThe sequences were edited and assembled using Sequencher v. 4.7 . The resulting sequences were combined with those downloaded from GenBank, aligned using Clustal W implemented in Mega version 6.0 software  and manuPhylogenetic analyses were performed using PAUP* v4b10  for maxihttp://www.phylo.org/portal2/) [MrModeltest 3.7  was run ortal2/) .http://www.phylo.org/portal2/) [A partitioned Bayesian analysis of the plastid dataset was also implemented by applying the previously determined models to each data partition . For BI ortal2/) .Abelia-like fruit fossils were reported from Late Oligocene to Middle Eocene [Abelia fossils is the shape of fruits. In the extant Abelia, the typical fruit is an oblong achene crowned with 2~5 persistent sepals. Crane [A. trialata, A. quadrialata, A. quinquealata and one Abelia sp. were probably correctly determined, while three additional Abelia-like fossils were wrongly identified. Fruits of A. quadrialata and A. trialata have been found to have hypogynous rather than epigynous fruits [Abelia-like fossils are correctly determined, it is difficult to identify which genera they belong to. Due to this uncertainty, we did not use the Abelia-like fossils. Manchester and Donoghue [Abelia-like fossils for their study.Seven e Eocene . The moss. Crane  thought s fruits . MoreoveDonoghue  also disWeigela are known from the Miocene and Pliocene in Poland [Weigela and its sister Diervilla at 23 Ma with mean = 0, SD = 1.0, offset = 23 Ma.Our tree was calibrated with three points. First, seeds of n Poland , the Mion Poland ,103, andn Poland . TherefoDiplodipelta from the late Eocene Florissant flora of Colorado (36–35 Ma), and from the Ruby, and Mormon Creek floras of Montana. The infructescence is made of two achene-like fruits of similar size enclosed by three bracts; two are wing-like, fused to the peduncle at base and the third one is hypothesized to be folded transversely and enveloping the two achenes. In order to interpret the Diplodipelta fossils we present two theoretical morphologies that could correspond to extant inflorescences in Linnaeoideae [Diabelia  TheDiabelia . (2) Thelkwitzia .In both cases the epicalyx is theoretically formed of six prophylls of different origins . If Diplodipelta is related to the genus Dipelta, it is hard to explain why the bracts have reduced in size whereas the bracteole-1 became wing-like. The fact that the bracts of Diplodipelta are fused at the base to the peduncle could form an explanation. We thus differ from Manchester and Donoghue’s [D. floribunda is often either peltate or cordate).As shown by Manchester and Donoghue , it seem species , and a fnoghue’s  reconstrDiplodipelta infructescences could not be dissociated from Linnaeoideae but the fossil genus could also be sister to Diabelia as well as Dipelta. The stratigraphic record of Diplodipelta, together with the occurrence of genuine Dipelta fruits in the late Eocene of England and Mississippi [Diplodipelta, Dipelta and Diabelia fossils at about 36–35 Ma and set the stem of Dipelta with lognormal mean = 0, SD = 1.0, offset = 36 Ma.When taking into account new phylogenetic results and inflorescence ontogenetic data, sissippi , indicatsissippi . We therThird, Caprifoliaceae is a family within eudicots, the oldest fossils of eudicots were recorded at about 125 Ma with their distinctive tricolpate pollen ,105–107.The estimation of divergence times was obtained using a Yule process speciation prior and an uncorrelated lognormal (UCLN) model of rate change with a relaxed clock . The anaAs with many other genera endemic to China , the spehttp://www.reelab.net/lagrange/configurator). The MCC tree from BEAST analysis was used as the input tree. The probability of dispersal between areas was modeled as equal, and all values in the dispersal constraint matrix were set to 1.The distribution of each species was assigned to at least one of these regions. The ancestral distributions were inferred using a likelihood approach under the dispersal-extinction-cladogenesis (DEC) model implemented in Lagrange . Python Dipelta and Diplodipelta fossil taxa.Many recent studies have incorporated fossils into biogeographic reconstruction e.g., –67. The (i)Dipelta europaea was found in southern England in the late Eocene to early Oligocene [D. europaea in Europe into the BEAST tree.The fossil of ligocene . We inco(ii)Dipelta sp. was reported from the Eocene of Mississippi [Dipelta sp. in North America was incorporated into the BEAST tree.The fossil sissippi . The age(iii)Diplodipelta fossil was described by Manchester and Donoghue [Diplodipelta as sister to the Diabelia—Dipelta clade in the BEAST tree.Donoghue  from theDiplodipelta may be sister to Diabelia or Dipelta, we made two alternative estimations: (1) sister relationship between Diplodipelta and Dipelta in the BEAST tree, and (2) sister relationship between Diplodipelta and Diabelia in the BEAST tree.Given that the fossil of trnL-rpl32 is the most variable fragment with a π (nucleotide diversity) value of 0.020 35, while psbM-trnH is the least variable fragment with a π value of 0.005 4. The concatenated length of the nine chloroplast regions reached 7641 bp with 230 parsimony-informative characters. ITS is also very variable in Linnaeoideae with π = 0.019 2 and 40 parsimony-informative characters.Length of aligned matrices, number of constant characters and potentially parsimony-informative characters, as well as consistency and retention indices of the nine chloroplast regions and the nuclear ribosomal ITS are summarized in trnL-F which showed sistership between Zabelia and Morina + Cryptothladia, while other markers suggested a more basal position of Zabelia. ILD tests showed that ITS is incongruent with the nine plastid markers at a significant level (p = 0.01). Thus, we only concatenated all nine plastid markers to build better resolved phylogenetic trees using MP, ML and BI methods  Ma in the middle Eocene, and Kolkwitzia split from Dipelta at 40.18 (95% HPD 36.85–44.68) Ma. The crown group of Abelia was dated at 23.76 (95% HPD 12.99–35.66) Ma, and the divergence of Abelia chinensis with other Abelia species was dated at 19.47 (95% HPD 10.63–29.94) Ma. The crown groups of Vesalea, Dipelta and Linnaea were estimated at 11.04 (95% HPD 4.23–19.95) Ma, 9.92 (95% HPD 1.83–20.85) Ma and 7.42 (95% HPD 2.23–14.07) Ma, respectively  and Diabelia Landrein (= Abelia ser. Serrata Graebner) left Abelia as a monophyletic genus.When phyletic ,38. The Abelia, Diabelia, Dipelta, Kolkwitzia, Linnaea, and Vesalea are well supported in this study and the recently established genera Diabelia and Vesalea are also supported [Diabelia and Vesalea were formerly treated as part of the genus Abelia [Abelia, Diabelia and Vesalea are probably due to convergent evolution, a common phenomenon among the mesic forest elements of the eastern Asian—North American disjunct plants [Abelia, Diabelia and Vesalea share three synapomorphies: accrescent calyx, reduced episepals (except in V. floribunda var. foliacea) and ovaries flattened dorsi-ventrally. Kolkwitzia and Dipelta also have ovaries flattened dorsi-ventrally but they have a well developed epicalyx and reduced calyx but this is clearly linked to dispersal mechanisms  racDivergent characters could be explained by an adaptation to their different environments. The two genera vicariously occupy the Rocky Mountains and the Sierra Madre Oriental, with extant populations being only separated by a few hundred kilometers.Abelia and Vesalea. The 5-sepaled species of Abelia  are well resolved, but the 2-sepaled species  are poorly delimited. Yang and Landrein [A. uniflora species complex. This study suggests that most of the 2-sepaled taxa of the genus are very closely related. Similarly to the 2-sepaled Abelia species, the species in Vesalea, which are 5-sepaled, are poorly resolved. Five species, V. floribunda, V. coriacea, V. grandifolia, V. mexicana and V. occidentalis, have been considered to occur in Mexico [Vesalea are also difficult to separate morphologically.All six genera have distinct morphological characters that have been described in detail by some taxonomists ,72. HoweLandrein  treated n Mexico . Many spAbelia, Diabelia, Dipelta and Vesalea. Achenes of Diabelia serrata and Abelia uniflora only possess two sepals instead of five. In Dipelta the wings originate from the epicalyx and two large bracts are present allowing for wind dispersal. In Kolkwitzia the spiny achenes are surrounded by corky episepals [Linnaea the calyx is deciduous in fruit but two of the episepals are covered by large and numerous sticky glandular hairs. Fruit dispersal adaptations, number of sepals and episepals do not seem good indicators of systematic relationships and this could be due to convergence.Linnaeoideae is a group of shrubby or small tree species producing achenes; these fruits are adapted for wind dispersal in the genera pisepals  and the and Diplodipelta distributionDipelta : The crown group for Diabelia, Dipelta and Kolkwitzia was dated at 40.18 (95% HPD 36.85–44.68) Ma in the middle Eocene  in the Eocene , with occurrences in Europe and North America, although the genus is restricted to western China today. The Tertiary disjunct distribution of Dipelta between Europe and North America may be explained by extinctions in large parts of its former ranges. Extinction events could have extirpated the old stem relatives that diverged prior to the extant crown radiation, leaving a phylogeny that includes only extant taxa with long stems and species-rich crowns [Dipelta stem and the beginning of the extant radiation in the early Miocene  and without the fossils showed the same ancestral area for Linnaeoideae   and the Linnaea borealis is divided into three subspecies, subsp. borealis in Europe, Asia and Alaska; subsp. longiflora (Torr.) Piper & Beattie along the Pacific coast of western North America from Alaska to California, and subsp. americana (J. Forbes) Hultén in the rest of Canada and USA as well as Greenland [Linnaea most likely represents an example of migration from Eurasia to North America via Beringia. Linnaea borealis is the only Linnaeoideae showing a continuous intercontinental extant distribution with populations along the Bering Strait islands, Chukotka and the Alaska Peninsula. Linnaea borealis is clearly the most cold-resistant species in Linnaeoideae and could have survived the conditions in the Bering Land Bridge area in the late Tertiary. Smith [Weigela middendorffiana and the North American Diervilla [reenland ,34. The y. Smith  concludeiervilla .Linnaea-Vesalea clade originated in central and western China (B) and Mexico (E) at 41.03 (95% HPD 24.07–55.19) Ma in the middle Eocene .First, since the genera of Linnaeoideae originated in the Eocene, migration through BLB seems likely. Tiffney and Manchester  argued tVesalea or its common ancestor. Vesalea species were presumably more commonly distributed than its present range prior to the Pleistocene glaciations, and its perennial growth habit may have allowed it to survive the subsequent millennia locally within this former range in various high-elevation or otherwise cool and moist habitats in the highlands, like a few other Northern Hemisphere disjunct plants, such as Aralia L. [Liquidambar L. [Platanus L. [Toxicodendron Mill. [Second, the NALB might be a more likely route for the migration of the Mexican ralia L. , Liquidaambar L. , Platanutanus L. , and Toxon Mill. . Many thon Mill. ,13,93 inon Mill. ,13,41,93Diabelia is a widespread genus in Japan and is only recorded in one locality of Zhejiang province of East China [Kolkwitzia, Dipelta and Diabelia form a well-supported clade (81/99/1)  and the same species collected in Japan could not be differentiated with the molecular markers we employed (100/100/1). It is therefore likely that Diabelia in China represents a relatively recent refugium following the last glacial event.st China ,32. The st China . Kolkwit81/99/1) ; their sin Japan . KolkwitCercidiphyllum japonicum [Kalopanax septemlobus [Platycrater arguta [Kirengeshoma palmata [Diabelia spathulata also growing in Zhejiang. Further phylogeographic studies using population genetic data may allow discovering whether a possible admixture, isolation or ‘filter’ event occurred in this species.During glaciations in the Quaternary, the East China Sea level was lowered and a continuous belt of forests connected the now disjunct populations of East China, South Japan and Korea . The Easaponicum  and Kalotemlobus  have beer arguta  and Kire palmata . DespiteThe following new combinations are made in light of the phylogenetic results;Vesalea occidentalis  H.F. Wang & Landrein, comb. nov.Abelia occidentalis Villarreal, Brittonia 49 (1), 84. 1997Basionym,—Villarreal-Quintanilla, J.A.  8180 (MEXU).Holotype,—Mexico. DURANGO, Mpio. Suchil, Reserva de la Michilia, Cienega Los Caballos, Vesalea grandifolia  H.F. Wang & Landrein, comb. nov.Abelia grandifolia Villarreal, Brittonia 52(2), 174. 2000Basionym,—Servin, B. 1101 (CAS).Holotype,—Mexico. QUERÉTARO, Mpio. de Jalpan, Cerro Grande, 13 June 1991, Vesalea mexicana  H.F. Wang & Landrein, comb. nov.Abelia mexicana Villarreal, Brittonia 52 (2), 172. 2000Basionym,—Calzada, J.I. 21100 (MEXU).Holotype,—Mexico. OAXACA, Mpio. San Sebastian Tecomaxtlahuaca, Vesalea coriacea Hemsl. var. subcoriacea  H.F. Wang & Landrein, comb. nov.Abelia coriacea Hemsl. var. subcoriacea Villarreal, Acta Bot. Mex. 102, 115, 2013Basionym,—Stewart, R.M. 1081 (MEXU).Holotype,—Mexico. COAHUILA, Mpio. Sierra Mojada, Sierra Mojada, Near Esmeralda, above San Salvador Mine, S1 DatasetThe aligned sequence data as presented in nexus format.(DOC)Click here for additional data file.S1 Fig(PDF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S3 Fig(PDF)Click here for additional data file.S4 Fig(PDF)Click here for additional data file.


Oysters have important ecological functions in their natural environment, acting as global carbon sinks and improving water quality by removing excess nutrients from the water column. During their life-time oysters are exposed to a variety of pathogens that can cause severe mortality in a range of oyster species. Environmental stressors encountered in their habitat can increase the susceptibility of oysters to these pathogens and in general have been shown to impact on oyster immunity, making immune parameters expressed in these marine animals an important research topic.S. glomerata tissues exposed to different environmental stressors resulted in a total of 484,121,702 paired-end reads. When reads and assembled transcripts were compared to the C. gigas genome, an overall low level of similarity at the nucleotide level, but a relatively high similarity at the protein level was observed. Examination of the tissue expression pattern showed that some transcripts coding for cathepsins, heat shock proteins and antioxidant proteins were exclusively expressed in the haemolymph of S. glomerata, suggesting a role in innate immunity. Furthermore, analysis of the S. glomerata ORFs showed a wide range of genes potentially involved in innate immunity, from pattern recognition receptors, components of the Toll-like signalling and apoptosis pathways to a complex antioxidant defence mechanism.Paired-end Illumina high throughput sequencing of six S. glomerata, showing the complex network of innate immune components that exist in this species. The results confirmed that many of the innate immune system components observed in mammals are also conserved in oysters; however, some, such as the TLR adaptors MAL, TRIF and TRAM are either missing or have been modified significantly. The components identified in this study could help explain the oysters’ natural resilience against pathogenic microorganisms encountered in their natural environment.This is the first large scale RNA-Seq study carried out in  Saccostrea glomerata) also have a substantial economic value, having contributed about AU$30 million to the New South Wales  economy in 2012/2013 -binding kinase 1). The complex then phosphorylates IRF3 (IFN-regulatory factor 3), which leads to IRF3 dimer formation and its localisation into the nucleus. There, IRF3 triggers the expression of type I IFN genes with the aid of p300 and CBP (cAMP-responsive-element-binding protein [CREB]-binding protein) , 60. TypS. glomerata transcriptomes. These components were TBK1, TRAF, IRF, p300/CBP, RIP1, JAK and STAT  have been found in our study, as well as in other molluscs, it is possible that a TRIF-related pathway exists in molluscs that can be triggered either independently of the adaptor protein TRIF or through the actions of an as yet unknown adaptor protein  through its C-terminal region , 60. Altand STAT . TBK was. edulis , 26, 54,incialis . Further protein .S. glomerata transcriptome   and M. galloprovincialis [GenBank:AHI17287].Alignment of (DOCX)Click here for additional data file.S5 FigS. glomerata big defensin transcripts  with big defensins from C. gigas , A. irradians [GenBank:DQ334340] and V. philippinarum [GenBank:HM562672].Alignment of (DOCX)Click here for additional data file.S6 FigS. glomerata hydramacin transcript (c328639.graph_c0_seq1|m.16116) with V. philippinarum hydramacin [GenBank:AGM14601].Alignment of (DOCX)Click here for additional data file.S1 File(DOCX)Click here for additional data file.S1 TableCLC Genomics Workbench (version 7.5) mapping statistics of individual tissue reads for determination of transcript distribution across the six tissues.(DOCX)Click here for additional data file.