Background. Age-related loss of muscle mass (sarcopenia) increases the incidence of obesity in the elderly by reducing physical activity. This sarcopenic obesity may become self-perpetuating, increasing the risks for metabolic syndrome, disability, and mortality. We investigated the associations of two sarcopenic indices, the ratio of lower extremity muscle mass to body weight (L/W ratio) and the ratio of lower extremity muscle mass to upper extremity muscle mass (L/U ratio), with metabolic parameters related to obesity in patients with type 2 diabetes and obesity.Methods. Of 148 inpatients with type 2 diabetes treated between October 2013 and April 2014, we recruited 26 with obesity but no physical disability. Daily physical activity was measured by a triaxial accelerometer during a period of hospitalization, and which was also evaluated by our previously reported non-exercise activity thermogenesis questionnaire. We measured body composition by bioelectrical impedance and investigated the correlations of L/W and L/U ratios with body weight, body mass index (BMI), waist circumference (WC), waist-to-hip ratio (WHR), visceral fat area, subcutaneous fat area, serum lipid profile, and daily physical activity.Results. The L/W ratio was significantly and negatively correlated with BMI, WC, WHR, body fat mass, body fat percentage, subcutaneous fat area, and serum free fatty acid concentration, was positively correlated with daily physical activity: the locomotive non-exercise activity thermogenesis score, but was not correlated with visceral fat area. The L/U ratio was significantly and positively correlated with serum high-density lipoprotein cholesterol.Conclusions. High L/W and L/U ratios, indicative of relatively preserved lower extremity muscle mass, were predictive of improved metabolic parameters related to obesity. Preserved muscle fitness in obesity, especially of the lower extremities, may prevent sarcopenic obesity and lower associated risks for metabolic syndrome and early mortality. Excess body weight is a serious public health problem and its prevalence has been increasing steadily since 1980 . RecommeSarcopenia is an age-related condition characterized by loss of skeletal muscle mass and decreased muscle strength . The preTo prevent sarcopenia, it is important to increase muscle mass and strength by resistance training. In elderly people, fat-free mass, especially muscle mass decreased and unfavorable changes in body composition were observed . Therefo2, the World Health Organization (WHO) definition of obesity . BMI was calculated as body weight in kilograms divided by the square of body height in meters. WC was measured at the umbilical level at the end of exhalation , and hipBody composition was analyzed using a bioelectrical impedance analysis device . The method is based on the principle that lean body mass contains higher water and electrolyte content than fat tissue, so these tissues can be distinguished by electrical impedance. Segmental body composition was estimated using a patented 8-point tactile electrode system. The device uses 6 frequencies  and produces 30 impedance values for five body segments: right and left upper extremities, trunk, and right and left lower extremities . PreviouVisceral and subcutaneous fat areas were measured using abdominal computed tomography (CT). This CT method was validated previously . Single-Venous blood samples were taken after a 12-h overnight fast. Fasting plasma glucose was measured using an enzymatic method . Hemoglobin A1c (HbA1c) was measured by high-performance liquid chromatography . Total cholesterol, total triglycerides (TG), and high-density lipoprotein cholesterol (HDL-C) were determined enzymatically using the commercially available kits T-CHOKL , Pureauto S TG-N, and Cholestest N HDL , respectively. Low-density lipoprotein cholesterol (LDL-C) was calculated by the Friedewald formula . Free faPhysical activity measurements were performed by the method which we previously reported . BrieflyDaily physical activity was also assessed using our previously reported non-exercise activity thermogenesis (NEAT) questionnaire . Briefly2 and <35.9 kg/m2). Differences in age, the L/W, L/U and U/W ratios and the locomotive NEAT score between the higher and lower BMI groups were analyzed by the Student’s t-test. A p value less than 0.05 was considered statistically significant.All statistical analysis was performed using SPSS version 19 . All values are expressed as mean ± standard deviation (SD). Sex differences in physiological and biochemical data were analyzed by the Student’s t-test. Correlations of the L/W ratio, L/U ratio and the ratio of upper extremity muscle mass to body weight (U/W ratio) with age, BMI, WC, WHR, TG, HDL-C, LDL-C, FFA, PAL and the locomotive NEAT score were assessed using Pearson’s correlation coefficient. Multiple regression analysis was performed to test the independent correlations of L/W and L/U ratios with physical and biochemical data. Models were adjusted for age. We divided participants into higher and lower BMI groups by the median BMI , WC , serum FFA , subcutaneous fat area , body fat mass  and body fat percentage , and positively associated with the locomotive NEAT score . After adjustment for age, L/U ratio was also negatively associated with weight , BMI , WC  and WHR , and positively associated with serum HDL-C . No significant association was found between the L/U ratio and visceral fat area, subcutaneous fat area, body fat mass, or body fat percentage. In men, the L/W ratio was negatively correlated with BMI, WC, subcutaneous fat area, body fat mass, and body fat percentage. However, in women, the L/W ratio was negatively correlated with only body fat percentage and was positively correlated with the locomotive NEAT score. The L/U ratio was negatively correlated with WC and WHR in men. The L/U ratio was also negatively correlated with BMI and WHR in women. Moreover, the L/U ratio was positively correlated with serum HDL-C. The mean age of subjects was 50.7 ± 13.2 years. The mean BMI of subjects was 36.2 ± 4.7 kg/mlass III . About 72) and lower BMI group (<35.9 kg/m2). Both L/W and L/U ratios were significantly lower in the higher BMI group than in the lower BMI group. Furthermore, the locomotive NEAT score was also significantly lower in the higher BMI group  in patients with higher BMI. On the other hand, higher U/W ratio was associated with unfavorable WHR and serum HDL-C. These results strongly suggest that not upper extremity but adequate lower extremity muscle mass is critical for reducing metabolic risk factors in obese type 2 diabetes patients.Sarcopenia is an age-related condition characterized by loss of skeletal muscle mass . Lower eObese patients tend to have a sedentary lifestyle, which increases body weight and decreases muscle mass and strength in the lower extremities, and these in turn contribute to a continued sedentary lifestyle. The locomotive NEAT score was in fact significantly reduced in the higher BMI group, suggesting that the most obese individuals tended not to walk as often as subjects in the lower BMI group. We also observed a significant and positive correlation between the L/W ratio and the locomotive NEAT score, suggesting that increased locomotive activity is associated with increased muscle mass. While we did not find significant correlations of PAL with the L/W and L/U ratios, PAL was measured during a period of hospitalization and so these findings may not represent the true physical activity of subjects in daily life outside the hospital.Free fatty acid levels are usually elevated in obese patients because the enlarged adipose tissue releases FFA and FFA clearance is reduced . ElevateDyslipidemia in obesity is typically characterized by elevated serum TG and FFA as well as lower HDL-C with HDL dysfunction . HDL metA systematic review and meta-analysis to investigate the independent and combined effects of aerobic exercise and resistance training on visceral fat area showed that aerobic exercise was effective in reducing visceral fat area, while resistance training was not . HoweverIn the present study, we observed more significant correlations of L/W and L/U ratios with metabolic parameters related to obesity in men than in women. On the other hand, L/U ratio was positively correlated with serum HDL-C in women not in men. However, the potential mechanism of sex differences is unknown. Further studies are needed to elucidate the differences in the role of lower extremity muscle mass between men and women.2; however, obesity was defined as a BMI over 25.0 kg/m2 in Japanese population (Limitations to our study include the cross-sectional study design, small sample size, and potential confounding factors. The cross-sectional study design does not allow causal relationships to be deduced, and future prospective studies are therefore needed to confirm our results. We recruited obese patients according to WHO definition of obesity, with BMI over 30.0 kg/mpulation . We shoupulation . Forth, We demonstrated that lower extremity muscle mass is predictive of reduced body weight, BMI, WC, WHR, subcutaneous fat area, body fat mass, body fat percentage, and serum FFA as well as higher serum HDL-C in obese patients with type 2 diabetes. Our results suggest that lower extremity muscle mass is more important than upper extremity muscle mass for improving metabolic risk factors. Resistance training targeting the lower extremities may allow obese patients to increase physical activity, preventing sarcopenic obesity and concomitant risks of morbidity and mortality.10.7717/peerj.942/supp-1Supplemental Information 1Click here for additional data file.


Many young people with childhood-onset diseases, including rheumatic diseases, continue to require medical care into adult life. Although there is an extensive evidence base for the need of transitional care, there is a paucity of robust outcome data and a great variability on the models of transitional care.To develop recommendations on the transition from pediatric care to adult care in patients with chronic inflammatory rheumatic diseases with childhood-onset based on the best evidence and experience.st panel meeting to be discussed and to help define recommendations. A first draft of recommendations was generated and circulated for comments and wording refinements. Focal groups with adolescents, young adults and parents were separately. In a 2nd panel meeting the focus group results along with the input from invited nurses and psychologists were used to established definitive recommendations. Then, a Delphi process (2 rounds) was carried out. A large group of 70 pediatricians and rheumatologists took part. Recommendations were voted from 1  to 10 . We defined agreement if at least 70% voted ≥7. The level of evidence and grade or recommendation was assessed using the Oxford Centre for Evidence-based Medicine Levels of Evidence.Recommendations were generated following nominal group methodology and Delphi technique. A panel of experts was established . A systematic literature review  and a narrative review  were performed and presented to the panel in the 1Transition care was defined as a purposeful, planned process that addresses the medical, psychosocial and educational/vocational needs of adolescents and young adults with chronic inflammatory rheumatic diseases with childhood-onset as they move from child-centred to adult-oriented health care systems. The consensus covers: transition needs, barriers and facilitators, transitional issues , physicians and other health professionals knowledge and skills requirements, models/programs, strategies and guideline for implementation. A total of 19 recommendations were developed.These recommendations are intended to provide pediatricians, rheumatologists, patients, families and other stakeholders with a consensus on the transition process from pediatric care to adult care in patients with chronic inflammatory rheumatic diseases with childhood-onset.I. Calvo: None declared., J. Anton: None declared., S. Bustabad: None declared., M. Camacho: None declared., J. De Inocencio: None declared., M. L. Gamir: None declared., G. Graña: None declared., L. La Cruz: None declared., J. C. López-Robledillo: None declared., M. Medrano: None declared., R. Merino: None declared., C. Modesto: None declared., E. Nuñez: None declared., M. J. Rua: None declared., V. Torrente: None declared., C. Vargas: None declared., E. Loza Grant / Research Support from: Abbvie.


Deinococcus geothermalis (D. geo), Deinococcus radiodurans (D. rad), Kineococcus radiotolerans (K. rad) and Rubrobacter xylanophilus (R. xyl). It is revealed that most of the three reference gene sets, i.e. ribosomal proteins, transcription factors and major chaperones, are generally highly expressed in the four bacteria. Recombinase A (recA), a key enzyme in recombinational repair, is predicted to be highly or marginally highly expressed in the four bacteria. However, most proteins associated with other repair systems show low expression levels. Some genes participating in ‘information storage and processing,’ ‘cellular processes and signaling’ and ‘metabolism’ are among the top twenty predicted highly expressed (PHX) genes in the four genomes. Many antioxidant enzymes and proteases are commonly highly expressed in the four bacteria, indicating that these enzymes play important roles in resisting irradiation. Finally, a number of ‘hypothetical genes’ are among the top twenty PHX genes in each genome, some of them might contribute vitally to resist irradiation. Some of the prediction results are supported by experimental evidence. All the above information not only helps to understand the radiation-resistant mechanisms but also provides clues for identifying new radiation-resistant genes from these bacteria.To investigate the general radiation-resistant mechanisms of bacteria, bioinformatic method was employed to predict highly expressed genes for four radiation-resistant bacteria, i.e.  Deinococcus geothermalis DSM 11300 (D. geo),Deinococcus radiodurans R1 (D. rad),Kineococcus radiotolerans SRS30216 (K. rad)Rubrobacter xylanophilus DSM 9941 (R. xyl),Radiation-resistant bacteria can withstand desiccation and many kinds of radiations such as ultraviolet light (UV), Hg(II)-, U(VI)-, Cr(VI)-ionizing radiations (IR), Co-gamma and UV-gamma radiations.D. geo is composed of a circular chromosome and two megaplasmids, which was originally isolated from a hot spring, and subsequently identified from other extreme environments.D. rad is the first deinobacteria discovered and isolated from canned meat that had spoiled following exposure to X rays, which contains two circular chromosomes and two plasmids.Deinococcus, which can survive acute exposures to IR (10 kGy), UV (1 kJ/m2), and can grow under chronic IR (60 Gy/hour).K. rad contains a 4.76 Mb linear chromosome and two plasmids, which is isolated from a high-level radioactive environment.R. xyl contains a circular chromosome.K. rad and R. xyl belong to Actinobacteria phylum, which can withstand relatively high concentration of metals and alkali cations, as well as exposure to extreme doses of IR close to that of D. rad.The above four radiation-resistant aerobic bacteria survive in an intense dosage of radiation that is lethal to most organisms.es to IR  kGy, UV UV 1 kJ/m, and canUV 1 kJ/m, and canBriefly, the prevailing opinions of radiation-resistant mechanisms are: (i) Genome reassembly and DNA repair are facilitated by numerous repeated sequences and morphology features, extended synthesis-dependent strand annealing (ESDSA) repair, nonhomologous end joining (NHEJ) repair and other DNA repair systems.E(g) indexetc. Based on E(g) index, Karlin and coworkers predicted the gene expression level of D. rad.D. rad when exposed to severe conditions of radiations. However, it remains unclear whether these mechanisms are also shared by other radiation-resistant bacteria, which stimulated our interest to do a comprehensive analysis. Considering the validity of E(g) based gene expression prediction methodology,To further understand the radiation-resistant mechanisms of these bacteria, a viable strategy is to identify the highly expressed genes. Thanks to the rapid progress in bioinformatics, several theoretical indices have been proposed to predict the expression levels of prokaryotic genes, which include codon adaptation index (CAI),E(g) values for the four radiation-resistant and one radiation-sensitive bacteria is listed in Escherichia coli str. K12 (E. coli) is much smaller than the four radiation-resistant bacteria. The highest E(g) value ranges from 1.17 in R. xyl to 1.66 in D. rad. E(g) values in E. coli. The following section will analyze these PHX genes with details.The statistics of PHX genes and their fusA), elongation factor Tu, Ts, Crp/Fnr family transcriptional regulator, RNA polymerase beta and beta’ subunit are generally highly expressed in the four species, especially RNA polymerase beta’ subunit, which is among the top twenty PHX genes in three radiation-resistant bacteria. The principal transcription/translation factors ensure microbes to synthesize proteins rapidly and exactly to prevent radiation damage. Some major chaperone proteins, such as DnaK (HSP70), DnaJ, GroEL (HSP60) and co-chaperonin GroES are among the top twenty PHX genes in the four species. DnaK can cooperate with trigger factor in the de novo protein folding by transiently binding to nascent and newly synthesized polypeptides.GroEL and GroES chaperones consist of a nest with a lid, which provide secluded environments for folding molecules to protect them against intermolecular aggregation and facilitate the actual folding process.DnaK, GroEL and GroES are helpful for preventing protein misfolding and increasing their thermal stability, which might be helpful for resisting irradiation.30It is observed that genes in three reference groups  are generally highly expressed . Many 50recA), GCN5-related N-acetyltransferase and DEAD/DEAH box helicase domain-containing protein are top twenty PHX genes in one or more radiation-resistant bacteria. Among them, recA is a key enzyme in recombination and repair, which is highly expressed in three radiation-resistant bacteria and marginally highly expressed in R. xylanophilus. As a major defense against environmental damage to cells, DNA repair is present in all organisms. It is presumed that other proteins involved in DNA repair processes should also be highly expressed, whereas the prediction result is much different from our speculation. Both Karlin et al’s and our analyses showed that most genes involved in direct repair, base excision repair, apurinic/apyrimidinic (AP) endonuclease, mismatch excision repair, nucleotide excision repair, recombinational repair (except recA) and other repair proteins are not highly expressed (data not shown). Therefore, it is speculated that recA gene makes more contribution for resisting irradiations than other repair proteins. All basal DNA repair genes in radiation-resistant bacteria are subject to positive selection.recA in D. rad is substantially up-regulated at early phase of DNA damage,recA render D. rad as sensitive to ionizing radiation as E. coli.5According to the clusters of orthologous groups of proteins (COGs) category,D. geo and D. rad, whereas the other two species have no similar genes.Some genes in ‘cellular processes and signaling’ are among the top twenty PHX genes in the four genomes. However, most of them are not generally highly expressed  except AacnA) of tricarboxylic acid (TCA) cycle, antioxidant protein thioredoxin, F0F1 ATP synthase and oxidoreductase-like protein are usually highly expressed in the four radiation-resistant bacteria. Whereas, other genes, such as aldo/keto reductase, isocitrate lyase (aceA) in glyoxalate bypass and light-independent protochlorophyllide reductase subunit B, are PHX genes in one or two species. In ‘lipid metabolism’ category, only acyl-CoA dehydrogenase-like protein is highly expressed in three bacteria except K. rad. ABC transporter can transport a wide variety of substrates including sugars, amino acids, metal ions, peptides, proteins, and a large number of hydrophobic compounds and metabolites across extra- and intracellular membranes, which is essential for all living organisms.R. xyl, whereas it is not annotated in the other three bacteria. Some extracellular solute binding proteins are highly expressed in D. geo and D. rad. Furthermore, glycine hydroxymethyltransferase is only highly expressed in K. rad. Nucleoside-diphosphate kinase in ‘nucleotide transport and metabolism’ is highly expressed in three bacteria except R. xyl, and bifunctional 2′, 3′-cyclic nucleotide 2′-phosphodiesterase is highly expressed in D. geo and K. rad.Many genes participating in ‘energy production and conversion’, ‘lipid metabolism’, ‘amino acid transport and metabolism’ and ‘nucleotide transport and metabolism’ are among the top twenty PHX genes and generally highly expressed in the four radiation-resistant bacteria. Aconitate hydratase  are highly expressed in the four radiation-resistant species, and some of them are among the top twenty PHX genes  and Mn superoxide dismutase (sodA) are highly expressed in three radiation-resistant species except K. rad. Many catalase and peroxidase participating in detoxification are generally highly expressed in the four species. Thioredoxin reductase (TrxR) in conjunction with thioredoxin (Trx) is a ubiquitous oxidoreductase system with antioxidant and redox regulatory roles.D. geo, whereas the other three bacteria do not have this enzyme.Because the formation of ROS during irradiation is extremely rapid, the antioxidant enzymes in radiation-resistant bacteria must be highly efficient in order to neutralize and remove free radicals and other toxic substances. Many antioxidant enzymes are highly expressed in the four radiation-resistant bacteria . For exaR. xyl. Most highly expressed pro-teases are ATP-dependent proteases. ATP-dependent proteases control diverse cellular processes by degrading specific regulatory proteins. Especially, ATP-dependent protease La is essential for cellular homeostasis by mediating the degradation of abnormal and damaged polypeptides, as well as short-lived regulatory proteins.D. rad. High expression levels of these proteases make sure that the irradiation injury proteins could be degraded efficiently.Some proteases participating in protein degradation are highly expressed in three radiation-resistant bacteria except E(g) values should be compared with experimental evidence. Although the four radiation-resistant bacteria have been sequenced for several years, only D. rad transcriptional profiling can be obtained in gene expression databases up to now. The experimental gene expression data of D. rad were downloaded from NCBI GEO database (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE9636). Then the correlation between experimental and prediction result is analyzed. E(g) values and experimental transcriptional profiling in wild-type D. rad. R = 0.348, P < 0.0001), and (b) shows the correlation of PHX genes and their experimental expression levels . Although the correlation is weak, it is statistically significant. Further, Joshi et al reported the kinetics of proteomic changes accompanying post-irradiation recovery (PIR) in D. rad following exposure to 6 kGy γ-irradiation.acn), groEL, dnaK and some ‘hypothetical genes’ were degraded during 4 h lag period of PIR and re-synthesized to normal levels during the late phase of PIR.D. rad.in silico prediction can provide some valuable information.To evaluate the prediction reliability, recA is more important than other repair proteins in resisting irradiation. Furthermore, many antioxidant enzymes, e.g. superoxide dismutase, catalase, peroxidase and thiol-based thioredoxin and glutaredoxin, play important roles in scavenging free radicals caused by irradiation, which are commonly or uniquely PHX genes in the four radiation-resistant bacteria. Proteases are important for degrading abnormal and damaged polypeptides, some of them, especially ATP-dependent protease La, are PHX genes in most radiation-resistant bacteria. In addition to the common highly expressed genes, each species has its own unique PHX genes to resist irradiation. Another important issue is that a number of ‘hypothetical genes’ are PHX genes in the four radiation-resistant bacteria, and some are among the top twenty, which might be unexplored genes for resisting irradiation. All the above information is helpful for further understanding the radiation-resistant mechanisms and provides important clues to identifying new radiation-resistant genes.Through analyzing the common PHX genes in four radiation-resistant bacteria, it can be inferred that some common radiation-resistant mechanisms are shared by the four species . Our preD. geo (chromosome: NC_008025), D. rad , K. rad (NC_009664), R. xyl (NC_008148) and one radiation-sensitive model bacterium E. coli (NC_000913) were downloaded from NCBI RefSeq at Aug 6, 2008.The complete sequences and corresponding annotation information of the four radiation-resistant bacteria E values can reflect the levels of common genes’ similarity in codon frequencies to RP, TF, and CH groups and of their deviation from the whole genome.As a well-established formulism, Karlin’s methodology select three sets of genes, i.e. ribosomal proteins (RP), principal transcription/translation factors (TF), and the major chaperones/degradation (CH), as the reference of tacit highly expressed genes. Qualitatively, if a gene’s codon usage is similar to that of the three groups of genes but deviates strongly from the average gene of the genome, it is predicted to be a PHX gene.F and G be two groups of genes, the codon usage difference of F relative to G is calculated by the formulaap (F) are the average amino acid frequencies of the genes in F, and f and g are the average codon frequencies for the codon triplet  of each amino acid codon family for gene groups F and G, respectively. Let B (g|G) indicates the codon usage difference of the gene g relative to the gene group G, and C is the totality of all genes in the genome.Let For the four bacterial genomes, the overall expression measure isE value exceeds 1.00 and at least two of ERP(g), ECH(g), and ETF(g) are more than 1.05.PHX genes must satisfy the following two criteria, the


Nilaparvata lugens harbors yeast-like symbionts (YLSs). In present paper, a genome-wide analysis found 115 genes from Ni. lugens and 90 genes from YLSs that were involved in the metabolic degradation of 20 proteinogenic amino acids. These 205 genes encoded for 77 enzymes. Accordingly, the degradation pathways for the 20 amino acids were manually constructed. It is postulated that Ni. lugens can independently degrade fourteen amino acids . Ni. lugens and YLSs enzymes may work collaboratively to break down tryptophan, cysteine, arginine, isoleucine, methionine and valine. We cloned a lysine-ketoglutarate reductase/saccharopine dehydrogenase gene (Nllkr/sdh) that encoded a bifunctional enzyme catalyzing the first two steps of lysine catabolism. Nllkr/sdh is widely expressed in the first through fifth instar nymphs and adults, and is highly expressed in the fat body, ovary and gut in adults. Ingestion of dsNllkr/sdh by nymphs successfully knocked down the target gene, and caused nymphal/adult mortality, shortened nymphal development stage and reduced adult fresh weight. Moreover, Nllkr/sdh knockdown resulted in three defects: wings were shortened and thickened; cuticles were stretched and thinned; and old nymphal cuticles remained on the tips of legs and abdomen and were not completely shed. These data indicate that impaired lysine degradation negatively affects the survival and development of Ni. lugens. Nilaparvata lugens, upregulated transcription level of a proline-degraded gene was observed due to starvation induced by the antifeedant activity of triazophos, and proline as an alternative energy source was then catabolized [Haemaphysalis longicornis, the Hllkr/sdh transcripts were more abundant in starved individuals than in well-fed and engorged ones [In animals, amino acid degradation is critically important. Firstly, amino acids are one of the three major energy sources for animals. Amino acids as an energy source are especially important for carnivorous animals and for all animals during starvation. For example, in the brown planthopper abolized . Lys-ketged ones . Moreoveged ones . All thoNi. lugens, contains high levels of simple sugars but low levels of nitrogenous organic compounds such as free amino acids. Among those free amino acids in rice phloem saps, only asparagine (Asn), glutamate (Glu), glutamine (Gln), threonine (Thr) and valine  are abundant [Ni. lugens. The insect must degrade the excess amino acids that are not needed for protein synthesis to maintain a balanced amino acid composition in hemolymph.Secondly, herbivores tend to obtain free amino acids from plants. However, many plants contain low levels of some essential amino acids. For example, rice phloem sap, as the food of abundant . MoreoveHa. longicornis, silencing Hllkr/sdh by RNA interference (RNAi) seriously affected the osmotic regulation of water balance and egg development of engorged females [Thirdly, an accumulation of amino acids and/or their breakdown intermediates is harmful to animals. For example, high levels of Lys were toxic to plant and mammalian cells –7. In Ha females . In huma females –13.Ni. lugens is a serious pest in paddy fields throughout Asia [Ni. lugens and its symbionts can independently biosynthesize seven non-essential amino acids of Glu, Gln, aspartate (Asp), Asn, alanine (Ala), serine (Ser) and glycine (Gly). Ni. lugens and symbiont enzymes may work collaboratively to catalyze the biosynthesis of proline (Pro), methionine (Met), Val, leucine (Leu), isoleucine (Ile), phenylalanine (Phe) and tyrosine (Tyr). And the symbionts alone may function in the biosynthesis of Lys, arginine (Arg), tryptophan (Trp), Thr, histidine(His) and cysteine (Cys) [out Asia . It harbout Asia –17, mainne (Cys) . An inteNi. lugens and YLS were released recently [Nllkr/sdh, and showed that knocking down of this gene using double stranded-RNA (dsRNA) caused obvious negative effects. Our results indicate that degradation of Lys is critical for the survival and development of Ni. lugens.The genomes of recently . They arNi. lugens were maintained on rice variety Taichung Native 1 (TN1) for more than 170 generations under controlled temperature (28 ± 1°C), relative humidity (80±10%) and photoperiod (14/10 h light/dark) in China National Rice Research Institute. TN1 seedlings were grown in soil at 30–35°C under a long day photoperiod (14/10 h light/dark) in a growth incubator. Planthoppers were transferred to fresh seedlings every 10–14 days to assure sufficient nutrition. All animal works were conducted according to relevant nation and international guidelines.Drosophila melanogaster, Bombyx mori, Anopheles gambiae, Tribolium castaneum, Apis mellifera and Acyrthosiphon pisum, and from model fungi Schizosaccharomyces pombe, Saccharomyces cerevisiae and Metharhizium robertsii were downloaded from the NCBI. These protein sequences were used for TBLASTN searches of Ni. lugens genome and YLS genome [The annotated proteins involved in amino acid degradations from model insects S genome , respectS genome , augustuS genome , FGENESHS genome  and exonS genome . The prede novo transcriptome assembly. The transcriptome was assembled from the clean reads (accession no. SRX326774) using TRINITY [In order to get transcriptional evidence of the genes, each gene was searched against a  TRINITY . Potenti TRINITY . The annNi. lugens and YLS genes were searched (blastx) against nr databases, respectively. All YLS genes shared the highest identities with those of Metarhizium spp, a closest relative to YLSs [Ni. lugens genes, sequences that had the greatest identity with that from Rhodnius prolixus or Ac. pisum, which are closely related to Ni. lugens, were considered as Ni. lugens (host) origin. The output from the blastx searches against nr databases for each gene was further analyzed with MEGAN v7.7.1 [Ni. lugens sequences exhibited essentially perfect identity with Arsenophonus nilaparvatae genome signaling that they were either DNA contaminants or the results of very recent horizontal gene transfer (HGT) from bacteria. Those sequences were excluded from further analysis because it is beyond the scope of the present study. After manual filtering, the analysis produced a list of genes that encode enzymes involved in amino acid degradation (The identified  to YLSs . Among tN v7.7.1 . After eradation .lkr/sdh was identified and the sequence was confirmed by reverse transcription polymerase chain reaction (RT-PCR) using primers listed in lkr/sdh were amplified according to the manual of SMARTer RACE cDNA amplification kit , using antisense and sense gene-specific primers and universal primers. After obtaining the full-length cDNA, a pair of primers (Nllkr/sdh) was submitted to GenBank (KJ958908).Out of those genes and ESTs, a putative  primers  was desiNlLKR/SDH using ClustalW v. 2.1 [Ixodes scapularis was added as an outgroup.LKR/SDH-like proteins from twenty-two insect species were selected and aligned with the W v. 2.1 . The aliW v. 2.1  to selecW v. 2.1 . The relNllkr/sdh dsRNA production and enhanced green fluorescent protein  were synthesized by PCR, using a 598 bp Nllkr/sdh and a 414 bp egfp fragment, and gene-specific primers (DNA samples for  primers  incorporegfp at the concentration of 0.50 mg/mL (negative control), and the diet containing dsNllkr/sdh at the concentration of 0.50 mg/mL. Twenty Ni. lugens nymphs (three days after hatching) were carefully transferred into each feeding chamber of the different diet treatments. All treatments were replicated 15 times (15 chambers) with a total of 300 nymphs in each treatment . The experiments lasted for the whole nymphal stage. Mortality was recorded daily. Nymphal development duration and fresh weight of surviving adults were recorded.For bioassays, a previously reported dietary dsRNA-delivering procedure ,31 was uNllkr/sdh in each sample was estimated by qRT-PCR  and alpha 2-tubulin (tub) as internal control genes and the corresponding primer pairs reported recently [-ΔΔCT method [rps15 and tub for normalization according to the strategy described previously [Total RNA samples were prepared from whole bodies of the first- through fifth-instar  nymphs and adults, and from ventral ganglion (VG), thorax muscles (TM), epidermis (EP), fat body (FB), gut (GU) and ovary (OV) of normal adults or the adult survivors of the dsRNA bioassay using the SV Total RNA Isolation System Kit (Promega). mRNA abundance of the recently . All expT method , using teviously ,35.The data were given as means ± SE, and were analyzed by one-way ANOVA followed by the Tukey-Kramer test, using SPSS for Windows .Ni. lugens genome that was released recently, 115 amino acid degradation-related genes were obtained by manual annotations  and a missing enzyme for the first step of Asp degradation  . YLS may only independently degrade Gln, Glu and Pro . In contrast, YLS may not independently degrade any of Ile, Val and Met, due to lack of dihydrolipoyllysine-residue (EC 2.3.1.168) and/or EC 5.1.99.1 , and YLS may independently breakdown Thr and Trp.In the pyruvate pathway, ion Figs  and 2A. 13) Figs  and 2B. Pro Figs  and 2C. 9.1 Figs  and 2D. Ni. lugens genome and transcriptome. The full-length cDNA encoding the putative LKR/SDH consisted of 3,314 bp (Nllkr/sdh). The lengths of the 5′- and 3′-untranslated regions (UTRs) are 239 and 285 bp, respectively. The 3′-UTR ends with a 39 bp poly (A) tail begins at 21 bp downstream from AATAAA, the eukaryotic consensus polyadenylation signal. The ORF is 2,793 bp and encodes 930 amino acids with a predicted molecular mass of 103.2 kDa and a theoretical isoelectric point of 6.71  with LKR/SDH-like protein from Rh. prolixus. Similarly, it has 81% similarity with that from Ac. pisum, Tr. castaneum and An. gambiae, 80% similarity with that from Bombus impatiens, Bo. terrestris, Ap. florae, Culex quinquefasciatus and Aedes aegypti, 79%-71% similarity with Ap. mellifera, Dr. melanogaster, Nasonia vitripennis, Atta cephalotes, Bo. mori, Pediculus humanus, Heliconius melpomene and Danaus plexippus. Moreover, it shows 69%-46% similarity with those from Plutella xylostella, Acromyrmex echinatior, Camponotus floridanus, Harpegnathos saltator and Ix. scapularis.The NlLKR/SDH belongs to the Hemiptera clade  Wings were shortened and thickened, and were not well extended during adult ecdysis. (2) Cuticle was stretched and thinned, making it transparent as some of the internal organs were easily visible through the cuticle. And (3) Old nymphal exoskeletons remained on the tips of legs and abdomens and could not completely shed .Ni. lugens harbors several species of YLSs [Ni. lugens and YLS genome that were released recently [ of YLSs –17, mainrecently , we idenNi. lugens some enzymes are encoded by genes originated from YLSs and others are encoded by genes of the hoppers. Firstly, all identified genes were successfully mapped to Ni. lugens or YLS genome, respectively [Ni. lugens and YLS gene has transcriptional evidence with EST support, respectively. Thirdly, each Ni. lugens and YLS gene shared a high identity with that of Rh. prolix and Metarhizium spp, respectively. It is postulated that Ni. lugens can independently catabolize fourteen amino acids . Ni. lugens and symbiont enzymes may work collaboratively to catalyze the degradation of Trp, Cys, Arg, Ilv, Val and Met.In the present paper, we provided lines of evidence that in ectively . SecondlHa. longicornis ticks injected with Lys or saccharopine [Nllkr/sdh in Ni. lugens. The NlLKR/SDH protein is a putative bifunctional enzyme with three distinct regions. The phylogenetic analysis revealed that the LKR/SDH-like proteins from twenty-two insect species formed clades agreed with taxonomy. The NlLKR/SDH belongs to the Hemiptera clade as expected. From insect genome data, one lkr/sdh gene was discovered in Ae. aegypti, Ap. mellifera, An. gambiae, Bo. mori, Cu. quinquefasciatus, Da. plexippus, Dr. melanogaster, Pe. humanus, and Na. vitripennis. Two lkr/sdhgenes were detected in Ha. saltator, Pl. xylostella and Ac.pisum, indicating a gene duplication event within the genome. Out of those genes, one or both lkr/sdh is pseudogenes that result from premature stop codons in Ha. saltator, and Pl. xylostella, whereas two lkr/sdh paralogs are protein-coding genes in Ac. pisum. It appears that all insect lkr/sdhs have evolved from a common ancestral gene, and gene duplication or loss have occurred during the evolution. In addition, LKR/SDHs from several insects, including Ac. pisum are annotated as mitochondrial-like or simply mitochondrial proteins. In mammals, lysine is catabolized via mitochondria through the bifunctional enzyme alpha-aminoadipic semialdehyde synthase that is known as LKR/SDH [NlLKR/SDH is encoded in the nucleus and is probably transported to the mitochondria to function.High levels of Lys were toxic to plant and mammalian cells –7. High-haropine . A Lys c LKR/SDH ,41. It sNllkr/sdh was widely expressed in the first- through fifth-instar nymphs and adults. The expression levels were similar among different development stages. Similarly, Hllkr/sdh was expressed in all developmental stages of Ha. longicornis [gicornis . This unNllkr/sdh successfully knocked down the target gene, and reduced the survivorship of the planthoppers. Similarly, all Ha. longicornis ticks injected with dsHllkr/sdh died 25 days after engorgement due to irreversible pathological changes [Nllkr/sdh indicates its importance in the survival and development of Ni. lugens as Nllkr/sdh was highly expressed in vital organs such as the fat body and gut (important organs in insects), moderately expressed in the ventral ganglion, and least expressed in epidermis and thorax muscles. Similarly, Hllkr/sdh was highly expressed in the midgut, fat body and synganglion in unfed Ha. longicornis ticks [lkr/sdh in fat bodies indicates that the insect LKR/SDH may play similar roles to that in mammals. Furthermore, Lys is an important precursor for synthesis of Glu, the most significant excitatory neurotransmitter in mammalian as well as arthropod central nervous systems [The present work provides three pieces of empirical evidence regarding the importance of LKR/DSH. Firstly, the ingestion of dsis ticks ,42, contis ticks ,37,39,42is ticks . The hig systems . The widNllkr/sdh shortened Ni. lugens nymphal development and decreased adult fresh weight. The DmLKR/SDH in Dr. melanogaster is involved in ecdysone-mediated transcription. DmLKR/SDH binds histone H3 and H4 and suppresses ecdysone-mediated transcription of cell death genes by inhibiting histone H3R17me2. In the absence of DmLKR/SDH, histone methylation occurs prematurely and enhances hormone-regulated gene expression to affect the developmental timing. Homozygous deficiency of Dmlkr/sdh produces viable adult flies that exhibit significantly smaller wing size and reduced overall body weight [Ni. lugens suggest that NlLKR/SDH may also be involved in ecdysone-mediated transcriptions.Secondly, we found that knocking down of y weight . SimilarNllkr/sdh caused the three morphological defects associated with wings and cuticles, a sign of imbalanced osmotic pressure. In arthropods, the Malpighian tubules and guts play a major role in salt and water balance. The Malpighian tubules and rectum of insects form a physiological complex that serves as a functional kidney at the organism level. Urine is secreted in the Malpighian tubules, and transported to the rectum for selectively reabsorbing ions and water. This processes of secretion and reabsorption are responsible for maintaining osmotic balance between the intracellular and extracellular compartments [Ha. longicornis, dsHllkr/sdh injection induced 2–4 times greater volume of hemolymph than the control groups. Pathomorphological examination showed that the midgut, Malpighian tubules and rectal sac were filled with a watery liquid. This high volume of hemolymph created a high hydraulic pressure and stretched the tick cuticle thin and transparent so that all the internal organs were easily visible through the cuticle. In addition, the Gene’s organ protruded with a hernia-like morphology is probably due to the high hydraulic pressure from the large volume of hemolymph. Lower amounts of guanine crystals were observed in the rectal sac and Malpighian tubules [Ha. longicornis showed that Hllkr/sdh was highly expressed in the ovary. RNAi-mediated knockdown of Hllkr/sdh showed a clear influence on egg production and tick reproduction [Nllkr/sdh was highly expressed in the ovary. The potential effect of Nllkr/sdh knock-down on reproduction of Ni. lugens is currently under evaluation in our laboratory. The physiological roles of other amino acid catabolism genes also will be delineated through RNAi knockdown in future.Finally, silencing artments . In Ha.  tubules –49. Othe tubules ,49. In r tubules . FurtherS1 Table1A: Pyruvate degradation family; 1B: Oxaloacetate and fumarate degradation family; 1C: α-ketoglutarate degradation family; 1D: Succinyl-CoA degradation family; 1E, Acetyl-CoA, acetoacetyl-CoA and acetoacetate degradation family.(XLS)Click here for additional data file.S2 Table(XLS)Click here for additional data file.


MHC) in vertebrates. The Atlantic cod is missing genes for both MHC-II and CD4, vital parts of the adaptive immune system. Nevertheless, cod are healthy in their ecological niche, maintaining large populations that support major commercial fisheries. Innate immunity is of interest from an evolutionary perspective, particularly in taxa lacking adaptive immunity. Here, we analyze extensive amino acid and nucleotide polymorphisms of the cathelicidin gene family in Atlantic cod and closely related taxa. There are three major clusters, Cath1, Cath2, and Cath3, that we consider to be paralogous genes. There is extensive nucleotide and amino acid allelic variation between and within clusters. The major feature of the results is that the variation clusters by alleles and not by species in phylogenetic trees and discriminant analysis of principal components. Variation within the three groups shows trans-species polymorphism that is older than speciation and that is suggestive of balancing selection maintaining the variation. Using Bayesian and likelihood methods positive and negative selection is evident at sites in the conserved part of the genes and, to a larger extent, in the active part which also shows episodic diversifying selection, further supporting the argument for balancing selection.Natural selection, the most important force in evolution, comes in three forms. Negative purifying selection removes deleterious variation and maintains adaptations. Positive directional selection fixes beneficial variants, producing new adaptations. Balancing selection maintains variation in a population. Important mechanisms of balancing selection include heterozygote advantage, frequency-dependent advantage of rarity, and local and fluctuating episodic selection. A rare pathogen gains an advantage because host defenses are predominantly effective against prevalent types. Similarly, a rare immune variant gives its host an advantage because the prevalent pathogens cannot escape the host’s apostatic defense. Due to the stochastic nature of evolution, neutral variation may accumulate on genealogical branches, but trans-species polymorphisms are rare under neutrality and are strong evidence for balancing selection. Balanced polymorphism maintains diversity at the major histocompatibility complex ( MHC molecules, cell surface molecules with broad (MHC-I) and specialized (MHC-II) pathogen recognition features  genome reveals the evolutionary loss of MHC-II and CD4, major parts of the adaptive immune system , Greenland cod Gadus ogac (Gog), Walleye pollock Gadus chalcogrammus (Gch), and Polar cod Boreogadus saida (Bsa), which is more distantly related. Pacific cod is considered a speciation from an Atlantic cod invasion into the Pacific (Pac) at approximately 4 mya based on genomic mtDNA data, Greenland cod is a recent re-invasion of Pacific cod into the Arctic and Atlantic oceans, and Walleye pollock is a speciation from an Atlantic cod invasion into the Pacific at 3.8 mya  from throughout the distributional range of the species: two each from Greenland (Gre), Barents Sea (Bar), Celtic Sea (Cel), Baltic Sea , Norway (Nor), Faroe Islands (Far), and Canada (Can) and five from around Iceland (Ice). We randomly sampled the individuals from our large sample collection  containi 3.8 mya  (and see 3.8 mya . Labelinhttp://www.wageningenur.nl/en/Expertise-Services/Research-Institutes/imares/Weblogs/Beam-Trawl-Survey.htm) of the Institute for Marine Resources & Ecosystem Studies (IMARES), Wageningen University, the Netherlands, which is approved by the IMARES Animal Care Committee and IMARES Board of Directors.The Icelandic Committee for Welfare of Experimental Animals, Chief Veterinary Office at the Ministry of Agriculture, Reykjavik, Iceland has determined that the research conducted here is not subject to the laws concerning the Welfare of Experimental Animals.  DNA was isolated from tissue taken from dead fish on board research vessels. Fish were collected during the yearly surveys of the Icelandic Marine Research Institute. All research plans and sampling of fish, including the ones for the current project, have been evaluated and approved by the Marine Research Institute Board of Directors. The Board comprises the Director General, Deputy Directors for Science and Finance and heads of the Marine Environment Section, the Marine Resources Section, and the Fisheries Advisory Section. Samples were also obtained from dead fish from marine research institutes in Norway, the Netherlands, Canada and the US that were similarly approved by the respective ethics boards. The samples from the US used in this study have been described in We extracted genomic DNA using a Chelex/proteinase K extraction method . PCR wasEU707291.1 as template.The primers used for PCR were CodCathF1: 5′-TGTTCAGCACAAAGCCAAACT-3′ from ® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) according to the manufacturer’s protocols for plasmid sequencing except that we used 1/16 of the manufacturer’s recommended amount.Universal M13F and M13R primers were used for sequencing, using the BigDye® 4-TOPO vector (Invitrogen™) and Sanger sequenced using an AB-3500xL Genetic Analyser (Applied Biosystems) (The PCR amplification fragments were gel extracted and cloned with PCRsystems) . All seqsystems)  and had systems) . We sequsystems) .KJ831349–KJ831391.Errors occur during PCR amplification and inevitably will be found, mostly as singletons, in the sequences of the cloned DNA. To remove this source of variation from the data we initially had planned to use the strategy of EY975127.1 , FG312333.1 , and ES786338.1 (Gadus morhua spleen SSH library enriched for genes up-regulated by polyriboinosinic polyribocytidylic acid). We also used GW862872.1 . We used DNAsp  with functions from the adegenet package. We used TexShade (ggtree (https://github.com/GuangchuangYu/ggtree) based on ggplot2 . Compared to the other two groups Cath2 had deletions in intron 3 and exon 4  that had some length differences. The first four clones in the alignment  forms of Cath3 clusters by alleles and not by species. The discriminant analysis of principal components (DAPC) lends further support that the variation clusters by alleles  and aa 48 (K) is identical to that of Cath2 for 152018-3.Gch.Pac. That aa is therefore unique for the Gadus chalcogrammus (Gch) species.For instance, the individual clone 152027-1.Gch.Pac  and 104947-2.Gog.Gre (Gadus ogac)  in our data  was characterized by the similar sequence AYIIN). However, this aa sequence also is found in the EST sequence FG312333.1 from Canada  in exon 4  and non-synonymous (N) changes within each codon and calculated either the ratio Nd/Sd or the difference Nd − Sd. For the codons with significant results, described below, both Nd and Sd were greater than zero. We compared several methods, SLAC, REL, FEL, MEME and FUBAR  higher than the suggestive cutoff 0.8 . MEME (Mixed Effects Model of Evolution p ≤ 0.05 indicative of selection (p < 0.05 by MEME but negatively selected by REL.The SLAC (Single Likelihood Ancestor Counting) program, the most conservative compared with the empirical Bayesian and likelihood approaches, found no evidence of selection. Similarly, FEL (Fixed Effects Likelihood), which is less conservative, found no evidence of selection. However, REL (Random Effects Likelihood) found no positively selected sites but found 11 and four negatively selected sites in exon 4 and exons 1–3, respectively. A REL Bayes factor higher than 10 is strong evidence of selection, giving support to positively selected sites in exons 1–3, as also found by FUBAR. REL is highly sensitive but has a tendency to produce false positives because of an of rates . FUBAR  indicating positive and balancing selection mainly in exon 4, the active part. Both results add support for the inference of balancing selection based on the trans-species nature of the within paralogs variation.Overall, the results of the exploratory codon-based analysis are in line with the results of the summary statistics  of approximately 10.000 in Atlantic cod , Gch.Pac , Gma.Pac , Gog.Gre (Gadus ogac) and Gmo (Gadus morhua) from various locations; Iceland (Gmo.Ice), Greenland (Gmo.Gre), Barents Sea (Gmo.Bar), Celtic Sea (Gmo.Cel), Baltic Sea , Norway (Gmo.Nor), Faeroe Islands (Gmo.Far), Canada (Gmo.Can).Highly polymorphic sites are boxed. The four conserved cysteine residues characterizing cathelicidin are shaded. Up arrows represent positively selected sites and down arrows negatively selected sites from Click here for additional data file.10.7717/peerj.976/supp-3Figure S2Bsa.Gre (Boreogadus saida), Gch.Pac , Gma.Pac , Gog.Gre (Gadus ogac) and Gmo (Gadus morhua) from various locations; Iceland (Gmo.Ice), Greenland (Gmo.Gre), Barents Sea (Gmo.Bar), Celtic Sea (Gmo.Cel), Baltic Sea , Norway (Gmo.Nor), Faeroe Islands (Gmo.Far), Canada (Gmo.Can).Click here for additional data file.10.7717/peerj.976/supp-4Figure S3Bsa.Gre (Boreogadus saida), Gch.Pac , Gma.Pac , Gog.Gre (Gadus ogac) and Gmo (Gadus morhua) from various locations; Iceland (Gmo.Ice), Greenland (Gmo.Gre), Barents Sea (Gmo.Bar), Celtic Sea (Gmo.Cel), Baltic Sea , Norway (Gmo.Nor), Faeroe Islands (Gmo.Far), Canada (Gmo.Can).Phylogenetic tree built on amino acid sequences in exons 1, 2, and 3 combined, the conserved part of cathelicidin, of clones from various individuals of Atlantic cod and four closely related taxa. Click here for additional data file.10.7717/peerj.976/supp-5Figure S4Bsa.Gre (Boreogadus saida), Gch.Pac , Gma.Pac , Gog.Gre (Gadus ogac) and Gmo (Gadus morhua) from various locations; Iceland (Gmo.Ice), Greenland (Gmo.Gre), Barents Sea (Gmo.Bar), Celtic Sea (Gmo.Cel), Baltic Sea , Norway (Gmo.Nor), Faeroe Islands (Gmo.Far), Canada (Gmo.Can).Phylogenetic tree of nucleotide sequences of the cathelicidin gene from 43 representative clones of various individuals of Atlantic cod and four sister taxa. Click here for additional data file.10.7717/peerj.976/supp-6Figure S5Window length was 100 bp with a 25 bp step size.Click here for additional data file.10.7717/peerj.976/supp-7Figure S6Window length was 100 bp with a 25 bp step size.Click here for additional data file.10.7717/peerj.976/supp-8Figure S7Window length was 100 bp with a 25 bp step size.Click here for additional data file.10.7717/peerj.976/supp-9Figure S8Window length was 100 bp with a 25 bp step size.Click here for additional data file.


Guadua angustifolia. This genome is 135,331 bp long and comprises of an 82,839-bp large single-copy (LSC) region, a 12,898-bp small single-copy (SSC) region, and a pair of 19,797-bp inverted repeats (IRs). Comparative analyses revealed marked conservation of gene content and sequence evolutionary rates between neotropical and paleotropical woody bamboos. The neotropical herbaceous bamboo Cryptochloa strictiflora differs from woody bamboos in IR/SSC boundaries in that it exhibits slightly contracted IRs and a faster substitution rate. The G. angustifolia chloroplast genome is similar in size to that of neotropical herbaceous bamboos but is ~3 kb smaller than that of paleotropical woody bamboos. Dissimilarities in genome size are correlated with differences in the lengths of intergenic spacers, which are caused by large-fragment insertion and deletion. Phylogenomic analyses of 62 taxa yielded a tree topology identical to that found in preceding studies. Divergence time estimation suggested that most bamboo genera diverged after the Miocene and that speciation events of extant species occurred during or after the Pliocene.To elucidate chloroplast genome evolution within neotropical-paleotropical bamboos, we fully characterized the chloroplast genome of the woody bamboo  The chloroplast or plastid is a cellular organelle that is derived from a free-living cyanobacterial-like prokaryote; this event occurred by endosymbiosis ~1.5 billion years ago . The chlDaucus carota) [Although these conserved chloroplast genome structures are present in most plants, the chloroplasts of several angiosperms are an exception, having independently undergone substantial alterations. Gene loss and pseudogenization commonly occur during chloroplast evolution in parasitic plants , 6. Such carota)  and five carota) , 15.Because these structural mutations are accompanied by speciation over time, such informative changes are well suited to indicate evolution. The very nature of the chloroplast genome, including its small size, simple and conserved structure, tremendous evolutionary-rate variations among gene loci and abundant copies per cell, have rendered the chloroplast genome more useful in molecular evolutionary analyses and phylogenetic reconstruction than mitochondrial and nuclear genomes. Because the entire chloroplast sequence harbors complete and stronger phylogenetic signals compared with the small number of loci, chloroplast genomes have been extensively utilized for comparative and phylogenetic studies in association with large, closely related and globally distributed plant groups , 17, albGuadua angustifolia Kunth, an economically and ecologically important neotropical bamboo that is found in Colombia, Ecuador and Venezuela. The main purpose of this study is to apply comparative genomics approaches to test the hypothesis that the chloroplast genomes of paleotropical and neotropical species have evolved differently due to differences in the selective forces between continents.Selective pressures lead plants to diversify as they adapt to new terrestrial landscapes. Chloroplast genomes bear the scars of these pressures by exhibiting different patterns of diversity in different environments. The evolution of smaller chloroplast genomes, for instance, tends to be associated with harsh environments . CurrentG. angustifolia were harvested in the Hua’an Bamboo Garden, Fujian Province, China . G. angustifolia is not an endangered or protected species, and the sample collection was approved by the Forestry Bureau of Hua’an County.Leaves of G. angustifolia-specific primers  Swallen and the paleotropical woody bamboo Dendrocalamus latiflorus Munro were compared to that of G. angustifolia (the reference species). Variations in gene contents and orders were then visualized using MultiPipmaker (http://pipmaker.bx.psu.edu/pipmaker/). The relative evolutionary rates of the protein-coding sequences of the three chloroplast genomes were quantified based on nonsynonymous (dN) and synonymous (dS) substitutions and their ratios (ω = dN/dS); Ferrocalamus rimosivaginus was used as an outgroup. dN and dS were computed according to the Nei and Gohobori method as implemented in the PAML package v4.4 [ndhH and ycf68) were not included in the analyses. dN, dS and ω were calculated for (1) individual protein-coding genes; (2) all protein-encoding genes combined; and (3) groups of genes with the same functions, e.g., photosynthesis ATP-synthase genes or NADH-dehydrogenase genes. Tajima’s relative rate test, as implemented in MEGA 5 [The chloroplast genomes of the herbaceous bamboo age v4.4  using thn MEGA 5 , was appG. angustifolia, 61 complete chloroplast genomes, representing 38 genera of Poaceae  to verify that the effective sample size (ESS) statistic exceeded 200 for all estimated parameters. The maximum clade credibility tree was summarized using TreeAnnotator v1.7.3. BEAST analyses were also performed at the CIPRES Science Gateway V.3.3 [A Bayesian dating analysis was performed using the BEAST v 1.7.3 package  to estim–34.5 Ma , and (3)Pliocene , 45. Posay V.3.3 .G. angustifolia chloroplast genome (KM365071) exhibited the conventional chloroplast quadripartite structure of land plants and mapped as a single circular double-stranded DNA molecule of 135,331 bp . Amongst the 130 SNPs detected, 57% corresponded to transitions and 43% to transversions. Of the 41 indels, 27 were mononucleotide repeats, and the remainders were small indels (5–27 bp) that were present in noncoding regions .The architecture, gene content and AT content of the  bamboos . In partT. latifolia indicated that all Poaceae chloroplast genomes had relatively compact sizes (~135–139 kb vs ~161 kb). Additionally, as reported in previous studies, [ycf1, ycf2 and accD genes, as well as loss of the introns of clpP and rpoC1. In addition, three DNA inversions between trnSGCU and trnfMCAU within the LSC region were found in all Poaceae species.These genomic characteristics were quite common even when compared with other grasses . Furtherstudies, , featureG. angustifolia, D. latiflorus and C. strictiflora) were visually quantified using “percent identity plots”the annotation of G. angustifolia was used as a reference  than in coding regions. Mutational hotspot regions in the alignment were also identified and encompassed the trnG-trnT, trnD-psbM, rpl32-ccsA, and ndhF-rpl32 regions. Other evolutionary differences among the three chloroplast genomes were mainly inferred from genome size, IR expansion and contraction, and the pseudogene at the IRb/SSC border.Sequence differences among the three representative tropical bamboos . Nevertheless, coding regions and introns remained largely invariant; the total coding region varied from 75,458 bp (D. latiflorus) to 75,888 bp (G. angustifolia), whereas the total intron length varied from 14,064 bp (G. angustifolia) to 14,135 bp (C. strictiflora).The chloroplast protein synthesis apparatus was almost identical in the three bamboos, except that D. latiflorus, an exceptional IR-located 1,521-bp deletion in the trnI-trnL spacers and a 461-bp deletion in rps12-trnV were found in G. angustifolia. These unusual intergenic deletions clearly led to the decreased genome size of G. angustifolia. When the intergenic spacers of G. angustifolia were contrasted with those of C. strictiflora, 12 intergenic spacers were found to have notable length differences >100 bp, 7 of which were expanded and 5 were diminished. Taken together, these length differences accounted for the slight variations in the total length of the spacing regions of these two chloroplast genomes .The genome size differences between the neotropical and paleotropical bamboos were primarily due to length variations in the intergenic spacers that resulted from insertion/deletion changes. In comparison with rps19 genes resided in the IRs at varying distances to the IRb/LSC junction . Although the IR regions of G. angustifolia  were much shorter than those of D. latiflorus , G. angustifolia and D. latiflorus were quite similar at the IR/SSC boundary. The ndhH gene, which is encoded by 196 bp in D. latiflorus and by 198 bp in G. angustifolia, straddled the IRa/SSC junction and generated an incomplete copy (ΨndhH) in the IRb of both chloroplast genomes.Expansion and contraction of IR regions led to differences at the IR borders among the three complete bamboo chloroplast genomes, as shown in C. strictiflora. The IR region of C. strictiflora had contracted; thus, the junction of IRb/SSC falls into the ndhF gene. This contraction also causes the rps15 and ndhH genes to occur as single-copy genes within the SSC rather than in the IR regions, as observed in the two woody bamboos. Consequently, this phenomenon generated the other pseudogene ΨndhF at the IRa/ SSC boundary region and reduced the total number of C. strictiflora genes to 127.The IR/SSC junction locations were slightly disparate in herbaceous bamboo ycf68 gene that was nested in the trnIGAU intron in the IR regions appeared to be a complete 405-bp open reading frame in the woody bamboo G. angustifolia and D. latiflorus but was much shorter in the herbaceous bamboo C. strictiflora. The 45th codon was identified as a stop codon that disrupted the CDS; thus, ycf68 is likely to be a pseudogene in C. strictiflora.The F. rimosivaginus chloroplast genome as a reference, the dN, dS and ω of 76 protein-coding genes in G. angustifolia, D. latiflorus and C. strictiflora were computed and compared  than in G. angustifolia or D. latiflorus. G. angustifolia and D. latiflorus showed similar dN and dS rates ; the value of ω was slightly higher in D. latiflorus, revealing similar evolutionary rates between the paleotropical and neotropical woody bamboos. Relative rate tests further confirmed that the substitution rates were statistically higher in most of the C. strictiflora genes analyzed (49 out of 76 genes) (P<0.001). Only in C. strictiflora was there a slight correlation between dN and dS across genes .Using the compared . UnsurprcemA matK and ccsA genes exhibited the highest dN and ω values, whereas dS was highest in infA (0.0567) , whereas the mean dS values were uniform in each bamboo examined. Not all gene groups contained sufficient numbers of genes to apply multiple comparison tests, which require at least two data points in each group. Thus, the Student Newman Keuls (S-N-K) multiple comparison tests were performed on the first 9 groups shown in psb, pet and psa presented the smallest mean dN values.After sorting the genes into functional categories, significant differences were revealed among the groups. The (0.0567) . Higher e groups . Among tmatK, and the highest dS value was detected in rpl36. Most genes exhibited dN/dS ratios of less than 0.5, indicating the efficiency of purifying selection. Two genes in D. latiflorus, rpl32 and rpl33, both of which were functionally essential in translation activity, exhibited dN/dS ratios that were significantly greater than unity (1.2009 and 1.3791).A comparison among individual genes in each functional group  showed tG. angustifolia was grouped with its congener G. weberbaueri, and both species share a phylogenetic neighborhood with other neotropical bamboos.A dataset of 113,641 unambiguously aligned nucleotide characters was finally subjected to phylogenomic analysis. MP, ML and BI analyses recovered almost identical and strongly supported relationships among the major clades . The phyInitial divergence dates of the major clades within Poaceae were estimated based on entire chloroplast genome data . The divG. angustifolia chloroplast genome bears a high level of conservation in terms of architecture and linear sequence order with other members of Bambusoideae. The most conspicuous difference appeared in chloroplast genome size when G. angustifolia was compared to its woody relatives. With the smallest IR size known in bamboos , a ~3 kb smaller chloroplast genome was present in G. angustifolia , hinting at a lineage-specific effect on this species. Because C. strictiflora is a herbaceous perennial that flowers annually [C. strictiflora clearly support the general observation that molecular evolutionary rates are negatively scaled with generation time [C. strictiflora , indicating that the selective constraints on the coding genes of these two species were comparable. These bamboos are both giant woody species with long generation times, and both exhibit little or no changes in structural organization of the chloroplast genomes; these observations might partly explain why these species evolved at similarly low rates. Based on our chloroplast-level divergence time estimates Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 TableGuadua angustifolia; CS: Cryptochloa strictiflora; DL: Dendrocalamus latiflorus.Genes that evolve at similar rates are shown in bold. P<0.05 indicates rates that are significantly different. GA: (XLSX)Click here for additional data file.S4 TabledN: nonsynonymous substitution rates; dS: synonymous substitution rates; dN/dS: ratio of nonsynonymous to synonymous substitutions.(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.