Escherichia coli and Klebsiella pneumoniae and compare the sequence, expression and plasmid stability function of their variants. 27 putative type II TAS were identified from 1063 plasmids of Klebsiella pneumoniae in GenBank. Among these, ccdAB and pemIK were found to be most common, also occurring in plasmids of E. coli. Comparisons of ccdAB variants, taken from E. coli and K. pneumoniae, revealed sequence differences, while pemIK variants from IncF and IncL/M plasmids were almost identical. Similarly, the expression and plasmid stability functions of ccdAB variants varied according to the host strain and species, whereas the expression and functions of pemIK variants were consistent among host strains. The specialised functions of some TAS may determine the host specificity and epidemiology of major antibiotic resistance plasmids.Toxin-antitoxin systems (TAS) are commonly found on bacterial plasmids and are generally involved in plasmid maintenance. In addition to plasmid maintenance, several plasmid-mediated TAS are also involved in bacterial stress response and virulence. Even though the same TAS are present in a variety of plasmid types and bacterial species, differences in their sequences, expression and functions are not well defined. Here, we aimed to identify commonly occurring plasmid TAS in  Toxin-antitoxin systems (TAS) were originally discovered on bacterial plasmids in the 1980s,  but haveparDE TA system is involved in antibiotic and heat tolerance in Escherichia coli [ccdAB contributes antibiotic persistence in E. coli [mpvAT is involved in virulence by maintaining virulence plasmids in Shigella and Salmonella species [Enterobacteriaceae, TAS are common among conjugative plasmids including antibiotic resistance (AbR) plasmids, and often associated with certain plasmid incompatibility (Inc) types. For example, the type II TAS pemIK and vagCD are usually found on IncF and IncL/M plasmids, and IncF and IncHI2 plasmids respectively [It has long been known that plasmid-mediated TAS play a role in plasmid maintenance through postsegregational killing and/or inhibition of growth of plasmid free cells , 8, 9, whia coli . Some ot E. coli , while m species , 18. Witectively , 20.Escherichia coli is well described [Klebsiella pneumoniae species. Existing studies of a collection of clinically relevant K. pneumoniae genomes (encompassing both plasmids and chromosomes) has provided insights into the distribution of TAS in the chromosomes of this species and the association of some TAS with certain plasmid replicon and antibiotic resistance regions, suggesting possible strain specific specialisation of TAS, with some being localised to one species while others are found across species [vagCD TAS on plasmids located in K. pneumoniae [vagCD is a member of the large TA family vapBC, with the toxin VagD being a PilT N-terminal (PIN) domain containing endoribonuclease that inhibits translation [The distribution of TAS in the plasmids found in escribed –25, but  species , 27. Preeumoniae , with eanslation , 30.ccdAB TAS (ccdF) from pathogenic E. coli O157:H7 is involved in post-segregational killing but its chromosomal counterpart (ccdO157) is unable to mediate PSK [e.g. plasmid maintenance or antibiotic tolerance) for plasmid-mediated TA variants were not investigated.TAS with identical names infer similar roles, regardless of the initial source and sequence variations, but functional differences in TAS from different genetic contexts have previously been noted , 32. ForK. pneumoniae retrieved from GenBank. Variation in DNA sequence, expression and function of two common TAS found in both E. coli and K. pneumoniae plasmids, ccdAB and pemIK, were examined.In this study, we analysed the distribution of type II TAS in 1063 fully sequenced plasmids found in ccdAB is a well-studied, primarily plasmid-associated type II TAS, although copies have also been found on bacterial chromosomes where it appears to be involved in the bacterial stress response [pemIK, another well studied type II TAS that is related to the mazEF system, is an mRNA endoribonuclease that inhibits protein synthesis [response , 34. Theresponse , 36. pemynthesis . Here, wK. pneumoniae species available in GenBank at the time of search (August 2019) were retrieved (https://www.ncbi.nlm.nih.gov/genome/?term=klebsiella+pneumoniae), and the sequences of all plasmids >30 kb examined with TA Finder (http://202.120.12.133/TAfinder/TAfinder.php) [https://cge.cbs.dtu.dk/services/PlasmidFinder/) [The names and complete sequences of all plasmids from der.php)  using thFinder/) .ccdAB and pemIK, were chosen, and the nucleotide and amino acid sequences of the toxin and antitoxin coding regions retrieved from GenBank (https://www.ncbi.nlm.nih.gov/genbank/) using coordinates obtained from TA Finder. To verify that these sequences were in fact representative of their specific variant, each of the sequences were used as queries for nucleotide Basic Local Alignment Search Tool (BLAST) searches (https://blast.ncbi.nlm.nih.gov/Blast.cgi) [http://www.megasoftware.net/mega7/) [Representative examples of the two most common TAS, ast.cgi) . The res/mega7/)  using thhttp://bioinf.cs.ucl.ac.uk/psipred/) [Variants were compared with one another by nucleotide alignment as described above. The amino acid sequences were then used to predict the secondary structures of the proteins using PsiPred (sipred/) .http://www.softberry.com/berry.phtml?topic=bprom&group=programs&subgroup=gfindb) (Softberry) with default parameters, with the input being the 500 bp region upstream of the ATG start codon of the antitoxin gene. The putative promoter sequences were then aligned as described above.TAS promoters were predicted using BPROM . The concentrations of each sample were then standardised to OD580 of 1.0 (~ 8.0 x 108 cfu/mL), as determined using the DensiCHEKTM Plus nephelometer . Fluorescence was analysed using a Victor3 plate reader , with an excitation wavelength of 485 nm and an emission wavelength of 535 nm. Experiments were performed in triplicate, and readings were averaged and corrected for background fluorescence by subtracting the pANT3 (no promoter) reading from each sample. The positive control pANT5, which constitutively expresses GFP from the ptac promoter, is a derivative of pANT3 [Relative strengths of putative TAS promoters were determined from TA promoter-eviously . Brieflyof pANT3 . Both plE. coli and K. pneumoniae were transformed with these constructs. Plasmid stability was assessed as described previously [E. coli or K. pneumoniae bacteria carrying the relevant plasmid was grown in LB broth at 37°C with shaking at 225 rpm for 72 or 96 hours, without antibiotic selection. Bacterial cultures were transferred into fresh LB medium at 1:1000 dilution at 16, 24, 40, 48, 64 and 72 hrs for high copy plasmids. Additional dilutions were performed at 88 and 96 hrs for low copy plasmids, as these are not lost as quickly as the high copy plasmids. Samples were taken before every transfer, diluted in saline and plated on to LB agar without antibiotic and incubated at 37°C for 18 h. From each plate, 120 colonies were replica plated onto LB agar plates with and without the indicator antibiotics to estimate plasmid retention.To assess plasmid stability, the whole TAS (including the putative promoter and ribosome binding site) was cloned into a low and a high copy plasmid (pACYC184 and pBSCK+ respectively) and two strains each of eviously . BrieflyccdAB, pemIK and vagCD most common among them  were common in both, with ccdAB and pemIK most commonly shared. Many, such as MNT-HEPN-like, GNAT-RHH-like, Bro-Abr-like and Bro-ArsR-like TAS appear to be most important in Klebsiella plasmids and have not previously been reported in E. coli plasmids [Twenty-seven different putative type II TAS were identified , with ccong them . We compplasmids . E. coliplasmids , 23.K. pneumoniae species belonged to 11 different Inc types   are rarely reported in E. coli.Plasmids found in nc types , althougnc types , which ieriaceae . ReplicaN and H) , Fig 1 ariaceae,  but someccdAB is somewhat specific to IncF plasmids in K. pneumoniae, while pemIK is common to both IncL/M and IncF plasmids  [K. pneumoniae  and IncR (35 of 76) plasmids in K. pneumoniae but not in E. coli. Most IncX plasmids lacked an easily identifiable TAS, save for a few carrying hicBA, also as previously noted [Other associations are noteworthy. For example, plasmids , in liney noted) , and in eumoniae , Fig 1. ly noted .ccdAB and pemIK were the two most predominant TAS modules across both E. coli and K. pneumoniae plasmids , while variants of pemIK were chosen from two plasmid incompatibility types, IncL/M and IncF (henceforth termed “pemIKLM” and “pemIKF” respectively). These sequences were confirmed as representative of their specific variant by phylogenetic analysis ,,pemIK prplasmids , and theccdAB promoters is generally lower than that of pemIK and showed host species specificity. GFP expression from the E. coli specific ccdAB promoter was higher in E. coli than in K. pneumoniae strains. The expression of GFP from the K. pneumoniae specific ccdAB promoter also varied between strains in each species but was relatively lower than from all other promoters tested here. The association between differences in expression of ccdAB from different genetic contexts with differences in function in E. coli has previously been noted [In contrast, expression of GFP from en noted .ccdAB (by host species) and pemIK (by plasmid type) are also reflected functionally, we assessed plasmid stability in two host strains of each species, in both low and high copy number plasmids. The low copy plasmid data are presented , is non-toxic and unable to provide plasmid maintenance [Vibrio fischeri superintegron could not protect from the toxicity of plasmid mediated CcdBF [ species , 34. Theeviously . The finntenance , suggested CcdBF .ccdAB to confer significant plasmid stability in certain host strains raises the possibility that it is performing another role in these strains. It would be of interest to investigate whether these plasmid-mediated ccdAB variants are involved in other known functions of TAS within these strains, such as persister formation, antibiotic and heat tolerance, and other bacterial stress responses [E. coli, both an IncF plasmid mediated ccdAB and chromosomally located variant have previously been shown to be able to confer some level of protection during antibiotic treatment [The inability of esponses , 55, 56.reatment , and thereatment .ccdAB has previously been noted to influence the spread of certain Salmonella serovars [ccdAB and pemIK may contribute to and influence the epidemiology of the conjugative antibiotic resistance plasmids which carry them. It is hoped that an increased understanding of these effects may be able to aid in the surveillance of these plasmids and the spread of the resistance genes carried by them.Variation in serovars , and we S1 Table(XLSX)Click here for additional data file.S1 FigA) ccdAB from plasmids found in E. coli and K. pneumoniae, and (B) pemIK from IncL/M and IncF plasmids. Sequences were aligned in MEGA7 using the ClustalW algorithm. Non-identical residues are highlighted in black.Nucleotide sequence alignments of the coding region of ((TIF)Click here for additional data file.S2 FigccdAB encoded toxins and antitoxins from plasmids found in E. coli and K. pneumoniae. Arrows represent β-strands, cylinder shapes represent α-helices and lines represent random coils. Pred: predicted secondary structure; AA: amino acids; numbers below each structure represent the amino acid positions within the proteins.The predicted secondary structure of the (TIF)Click here for additional data file.S3 FigpemIK encoded toxins and antitoxins from IncL/M and IncF type plasmids. Arrows represent β-strands, cylinder shapes represent α-helices and lines represent random coils. Pred: predicted secondary structure; AA: amino acids; numbers below each structure represent the amino acid positions within the proteins.The predicted secondary structure of the (TIF)Click here for additional data file.S4 FigE. coli (A: Ec BW25113 and B: Ec WH59) and K. pneumoniae (C: Kp ATCC13883 and D: Kp WH49). Data shown are the means of three replicates, with the error bars indicating one standard deviation from the mean.Stability of a high copy number plasmid with and without TAS over 72 h. Each TAS was cloned into a pBCSK+ backbone, and the percentage of cells retaining the plasmid calculated at each time point. Measurements were done in two species, (TIF)Click here for additional data file.


Rubus represents the second largest genus of the family Rosaceae in Taiwan, with 41 currently recognized species across three subgenera . Despite previous morphological and cytological studies, little is known regarding the overall phylogenetic relationships among the Rubus species in Taiwan, and their relationships to congeneric species in continental China. We characterized eight complete plastomes of Taiwan endemic Rubus species: subg. Idaeobatus  and subg. Malachobatus (R. kawakamii and R. laciniastostipulatus) to determine their phylogenetic relationships. The plastomes were highly conserved and the size of the complete plastome sequences ranged from 155,566 to 156,236 bp. The overall GC content ranged from 37.0 to 37.3%. The frequency of codon usage showed similar patterns among species, and 29 of the 73 common protein-coding genes were positively selected. The comparative phylogenomic analysis identified four highly variable intergenic regions . Phylogenetic analysis of 31 representative complete plastomes within the family Rosaceae revealed three major lineages within Rubus in Taiwan. However, overall phylogenetic relationships among endemic species require broader taxon sampling to gain new insights into infrageneric relationships and their plastome evolution.Genus  In addition, Taiwan provided refugia for northern species that migrated south during the glacial periods. These striking biogeographic events and topographic heterogeneity in the island’s environment resulted in floristic affinity between Taiwan and mainland China, as well as the formation of unique floristic elements in Taiwan. For example, approximately 52% of the ca. 4,000 native vascular plant species are closely related to the mainland China, while ca. 26% of natives  are endemic to Taiwan3. A combination of multiple colonization events from geographically close source areas, followed by subsequent speciation on the island without splitting events , and in situ adaptive and nonadaptive speciation after colonization on island likely explains the high endemism of flora in Taiwan2. In particular, several distinctive vegetation zones provided opportunities for the diversification of several rich groups of endemic species, ranging from tropical and coastal evergreen forests, to subalpine shrubs and alpine tundra6.Taiwan originates from the continental Taiwan-Ryukyu Archipelago, lying on the western rim of the Pacific Ocean approximately 150 km from the southeastern coast of China, separated by the Taiwan Strait. Taiwan was repeatedly connected and disconnected during the glacial and interglacial periods of the Pleistocene glaciation cycles, which provided opportunities for plant colonization and isolation between the islands and the mainlandRubus L., with ca. 700 species, is distributed worldwide and is abundant in the Northern Hemisphere, with very few species occurring in the Southern Hemisphere7. Focke established the widely adopted Rubus infrageneric classification system that recognizes 12 subgenera10, and several attempts have been made to unravel the overall phylogeny within the genus as well as the role of hybridization events16. Approximately 41 species from three subgenera are currently recognized in Taiwan; i.e., Chamaebatus (3 species), Idaoeobatus (27 species), and Malochobatus (11 species). Among these species, approximately 40% (15 species) are considered endemic to Taiwan, while the remaining species occur in mainland China , Japan , and the Philippines 17. Despite previous floristic and cytological studies, as well as reports of new natural hybrid taxa18, very little is known regarding the evolutionary history among Rubus species in Taiwan and their phylogenetic relationships to congeneric species in continental China and adjacent countries.The genus 21. In addition, several chloroplast gene loci  have proven useful as DNA barcode markers for species discrimination that provide valuable information to develop conservation strategies and biodiversity assessments23. Achieving adequate resolutions on the basis of the phylogenetic analyses of several concatenated regions has been difficult, especially for recently diverged plant species, because of limited sequence variations in several coding and noncoding regions of chloroplast DNA26. With the advent of next-generation sequencing (NGS) tools, considerable genome-wide variations in phylogenomics have significantly enhanced our understanding of patterns and processes in plant evolution, especially at lower taxonomic levels32. Since the first report of three partial Rubus plastomes being part of the Rosaceae phylogeny33, several complete chloroplast genomes belonging to different subgenera, i.e., Anoplobatus and Idaeobatus, have been recently reported, and useful hotspot regions for phylogenetic analysis have been suggested39. Our knowledge of plastome structure and evolution was primarily synthesized from the subgenus Idaeobatus from the infrageneric classification system of Rubus by Focke10, with the exception of two species from subg. Anoplobatus and one species from subg. Cylactis, based on species from Korea and China. Thus, we know very little regarding the plastome structure and evolution of other subgenera and also from other geographical regions in East Asia.Chloroplasts are essential organelles in plant cells that serve as metabolic centers in green plants and encode many key proteins that are involved in photosynthesis and other metabolite syntheses. Phylogenetic relationships of major plant groups at various taxonomic levels based on plastid genomes have been greatly elucidated by sequence polymorphisms or by hypervariable microsatellites (simple sequence repeats) as efficient genetic markersRubus species endemic to Taiwan, including six species from subgenus Idaeobatus and two species from subgenus Malacobatus. Although few other endemic species of Rubus belong to subgenus Chamaebatus, we were not able to include any representatives in current study. The comparative analysis of these eight plastomes, alongside the other previously reported plastomes within the genus will allow us to elucidate the genome structure, gene order, and gene contents, eventually providing an opportunity to study plastid evolution across groups. In addition, this study will shed light on the plastome structure and evolution of insular endemic species of the Taiwan-Ryukyu Archipelago. Lastly, the results from this study will aid in the development of useful chloroplast markers from hotspot regions, facilitating increased resolution of phylogenetic relationships among closely related species of Rubus.Therefore, we aimed to determine the complete plastomes of eight Rubus species from Taiwan were characterized: six species from subg. Idaeobatus and two species from subg. Malachobatus. The size of complete plastome sequences ranged from 155,566 bp (R. rubroangustifolius) to 156,236 bp (R. laciniatostipulatus). The plastomes were highly conserved with no structural variations or content rearrangements despite their representations from different subgenera 39, the overall GC content ranged from 37.0%  to 37.3% , summarized in Table ndhA, ndhB, petB, petD, rpl2, rpl16, rpoC1, rps12, rps16, trnA-UGC, trnG-UCC, trnI-GAU, trnK-UUU, trnL-UAA, and trnV-UAC) contained one intron, whereas clpP and ycf3 each contained two introns.For the first time, the complete plastomes of eight endemic ycf1 gene  was located in the IRb/SSC junction region, while the complete ycf1 gene  was located in the IR region at the SSC/IRa junction. The infA gene of the eight Taiwanese endemic Rubus plastomes located in the LSC region became a pseudogene. Interestingly, the highly conserved group II intron of atpF was lost, and a frameshift mutation via ATT deletion of the ndhF gene was identified in R. kawakmii of subg. Malachobatus , and caused early termination of translation  to isoleucine (Ile) and tyrosine (Tyr) to phenylalanine (Phe); the former mutation was in R. glandulosopunctatus, R. rubroangustifolius, R. taiwanicolus  to lysine (Lys)  contained the same consistent distribution of amino acids, sequences  up to 26,093 , but the distribution of codon type was consistent. Excluding the AUC codon usage of trnV-GAU in R. laciniatostipulatus , similar patterns of cp genes and codon usage were detected amongst the eight endemic Rubus species , self-replication genes  and others . No RNA editing sites at ndhF and ndhG genes were found in R. glandulosopunctatus, R. rubroangustifolius, and R. taiwanicolus. In addition, RNA editing sites at the rpoC1 gene was observed in R. kawakamii. Compared with other species, the ndhA gene in R. glandulosopunctatus showed exceptionally higher frequencies  in RNA editing sites. The ndhB gene had the highest number of potential editing sites , followed by the ndhD gene . It showed consistent results from previous reports44, except for R. glandulosopunctatus that had the second highest number of potential editing sites in ndhA (10). All editing sites showed base transition from cytosine (C) to thymine (T), and the most frequent transition serine (Ser) conversion to leucine (Leu)  to the R. taiwanicolus plastome, while R. kawakamii was least similar  to R. taiwanicolus.The eight complete plastome sequences of endemic nce Fig. . The resRubus chloroplast genome  over the entire cp genome was 0.010. The most variable region was the rps16/trnQ intergenic region with a 0.05018 Pi value. Also, highly variable regions included three other intergenic regions: petA/psbJ (Pi = 0.04567), rpl32/trnL-UAG (Pi = 0.04165), and trnT-UGU/trnL-UAA (Pi = 0.04147). Therefore, four highly variable regions with a Pi value greater than 0.04 identified in eight endemic Rubus plastomes in Taiwan can be useful for population genetic and phylogeographic study.The sliding window analysis using DnaSP program revealed highly variable regions in the continental Island endemic taxa of the ome Fig. . As the Rubus chloroplast. The Ka/Ks ratio of > 1 had a total of 29 genes out of 73, suggesting that these genes were positively selected in the eight endemic plastomes in Taiwan. Those genes included one ATP subunit gene (atpI), three photosystem subunit genes , two cytochrome b6/f complex gene (petA and petB), eight NADH oxidoreductase genes , Rubisco gene (rbcL), four encoded DNA dependent RNA polymerase genes , four ribosomal subunit genes , maturase gene (matK), one subunit Acetyl-CoA carboxylase gene (accD), one c-type cytochrome synthesis gene (ccsA), one envelop membrane protein gene (cemA), and two unknown genes (ycf1 and ycf 2). Surprisingly, our results from the Taiwanese endemic Rubus suggested that ca. 40% of the protein-coding genes underwent positive selection pressures.The positive selection analysis using DnaSP program with synonymous and nonsynonymous substitution options revealed positively selected genes Fig. . OverallRubus (100% bootstrap support) and the sister relationship between Rubus and the clade containing Fragaria and Rosa (100% bootstrap support). Based on limited available complete plastome sequences of Rubus from four subgenera, Anoplobatus and Malachobatus appear to be monophyletic, while Idaeobatus is not monophyletic  is sister to Malachobatus , and six species of Idaeobatus  are located basal lineage within genus Rubus clade. The continental progenitor-insular derivative species pair in Japan, R. boninensis and R. trifidus, are sister to the other progenitor-derivative species pair R. crataegifolius and R. takesimensis in Korea. Subgenus Anoplobatus appears to be deeply embedded within Idaeobatus . The other clade of Taiwanese endemics including R. taitoensis, R. parviaraliifolius, and R. incanus, is sister to R. coreanus, while R. laciniatostipulatus and R. kawakamii in subg. Malachobatus is sister to R. fockeanus in subg. Cylactis. These suggest that endemic species of Rubus in Taiwan most likely have evolved at least three times from different lineages  analysis conducted on the best-fit model of “TVM + F + R4” revealed the first preliminary phylogenetic relationships among endemic species in Taiwan Fig. . Phylogetic Fig. . Subgenutus Fig. . Resultsges Fig. , requiriRubus species, including two species from subg. Malachobatus. The size of the complete plastome sequences are highly conserved with the total length ranging from 155,566 to 156,236 bp , the plastomes are highly conserved, with no structural variations or content rearrangements. Interestingly, the highly conserved group II intron of atpF gene was lost in all eight plastomes regardless of their subgeneric assignments, as we demonstrated previously in the case of R. boninensis, R. crataegifolius, R. takesimensis and R. trifidus39. Within the major lineages of the family Rosaceae45, we found the complete atpF gene in members of the newly circumscribed subfamily Amygdaloideae, such as Prunus (KP732472), Pyrus , and Malus . However, loss of the atpF intron was also detected in other members of Rosoideae, such as Fragaria , Hagenia (KX0088604), Potentilla (HG931056), and Rosa . It still remains to be determined whether the loss of the atpF intron, that frequently occurs in Rosa and all subgenera of Rubus, has occurred in other major lineages within the family Rosaceae.For the first time, we characterized the eight complete plastomes of Taiwan endemic ndhF gene; which is known to have frameshift mutations and alterations on transcription termination, as a result of higher substitution rate, a wide range of insertion and deletion (indel) variations, a low homoplasy rate, and a high AT content47. In comparative phylogenomic analysis of genus Rosa sect. Synstylae, Jeon and Kim30 revealed frameshift and point mutations on the 3′ end of the ndhF gene. However, our previous study on the comparative analysis of four Rubus plastomes (two from each subgenus Anoplabatus and Idaeobatus) only showed nucleotide substitutions and transcription alterations without size variations39. A frameshift mutation via ATT deletion that caused early termination and translation was identified in R. kawakamii  from the eight Taiwan endemic Rubus plastomes  to lysine (Lys) and tyrosine (Tyr) to phenylalanine (Phe). We also detected point mutations in other endemic Rubus in Taiwan for the same 2,242 bp sequences, which altered transcripts from tyrosine (Tyr) to phenylalanine (Phe), phenylalanine (Phe) to isoleucine (Ile), tyrosine (Tyr) to phenylalanine (Phe), and asparagine (Asp) to lysine (Lys). Furthermore, three Taiwanese endemic Rubus plastomes in subg. Idaeobatus  showed the same distribution of amino acids and sequence lengths  with the four previously analyzed members of Rubus plastomes (two in subg. Idaeobatus and two in Anoplobatus)39. It appears that these changes are neither subgenus specific nor geographically confined to the East Sea (Ulleung Island), the northwestern Pacific Ocean (Bonin Islands), and the western rim of Pacific Ocean (Taiwan).In this study, we detected mutations in the 3′ region of the mes Fig. . Althoug49. In the case of R. trifidus —R. boninensis (insular derivative) species pairs in subg. Anoplobatus, we found similar patterns in codon usage with some exceptions39. When compared with the pair of R. trifidus-R. boninensis, AUG , UCA (trnS-UGA), UAG (no usage), and CAA codon usage (trnQ-UUG) showed different patterns in R. crataegifolius-R. takesimensis species pairs. When compared with these two species pairs, AUG , CAA (trnQ-UUG), and UCA (trnS-UGA) codon usage of the eight Taiwanese Rubus endemics showed similar patterns to R. cratageifolius, R. takesimensis, and R. trifidus from Korea. The specific codon usage amongst eight endemic Rubus was detected in R. laciniatostipulatus  with AUG codon usage (trnV-GAU). The UAG codon usage (stop codon) was similar only to R. crataegifolius and R. takesimensis on Ulleung Island, and R. boninensis on Bonin Islands. Rubus trifidus showed a different UAG codon usage of trnI-CAT. However, the Bonin Islands endemic R. boninensis showed different patterns of codon usage at AUG (trnfM-CAU and trnI-CAU), CAA (trnK-UUG) and UCA (no usage). Given that R. boninensis occurs on Minamiiwojima Island, which is estimated to be as young as 30,000 years old, it was interesting to notice that the endemic Rubus species on geologically younger islands showed more diverse patterns of codon usage than other insular endemics on Ulleung Island (R. takesimensis) and Taiwan (eight species in this study). The biased patterns toward high RSCU values of U and A, at the third codon usage in eight endemic Rubus plastomes in Taiwan, was similar to other angiosperms and algal lineages41.The codon usage bias, which could be manifested primarily by the balance between mutational bias and natural selective forces, provide crucial information to our understanding of molecular evolution and environmental adaptation50. While purifying selection acts to maintain protein functions, positive selection may come into play in response to environmental changes, novel ecological adaptation, or results from coevolutionary processes51. Previous studies showed that Ka/Ks values are usually less than one because synonymous nucleotide substitutions occur more frequently than nonsynonymous substitutions52. Interestingly, our current results from the eight endemic Rubus species from Taiwan suggested that ca. 40% protein-coding genes experienced positive selection pressures. All but two genes (ndhB and ndhC) of the NADH oxidoreductase genes of the eight Taiwan Rubus endemics showed that they were under positive selection pressure. Under strong light conditions, NADH dehydrogenase can protect plants from photoinhibition or photooxidation stress by stabilizing the NADH complex, and adjusting the photosynthetic rate and growth delay caused by drought54. In addition, all Rubus plastid gene encoding proteins related to transcription and post-transcriptional modification  underwent positive selection. In the same Rosaceae family, Fragaria vesca ssp. vesca showed six positively selected genes: rpoC2, ndhD, ndhF, psbB, ycf1, and ycf455. We can only speculate that the positive selection pressure among eight endemic species of Rubus in Taiwan experienced by many genes was likely a result of their adaptation to subtropical climates in the island of Taiwan. This speculation, however, has to be investigated further.Owing to their important function in plant metabolism, proteins and RNA molecules encoding plastid genes are subject to selective pressuresRubus as a maternally inherited marker. Recently, several hot spot regions including genic and non-coding regions across the entire plastome were reported in several members of Rosaceae, such as Malus56, Prunus57, Pyrus27, and Rosa30. In our current study, we also detected four highly variable regions, including rps16/trnQ (Pi = 0.0518) and petA/psbJ (Pi = 0.0466), as two of the most variable hotspots within the eight Taiwan Rubus endemics, with an average nucleotide diversity (Pi) value of 0.01. When compared with our early study , which included subg. Idaeobatus (four taxa) and subg. Cylactis (one taxon), the two most variable noncoding regions, trnL/trnF and rps16/trnQ, were detected with a Pi value of 0.05 and 0.046, respectively35. Yang et al.39 compared the four taxa of progenitor-derivative species pairs in subg. Idaeobatus (R. crataegifolius-R. takesimensis on Ulleung Island) and subg. Anoplobatus (R. trifidus-R. boninensis on the Bonin Islands), and found that the average Pi value (0.005) was substantially lower than that found between subg. Idaeobatus and subg. Cylactis (Pi = 0.01). The same study also suggested that the trnT/trnL region was the most variable region with a Pi value of 0.027. Thus, considering all previous studies on Rubus for the identification of hotspot regions throughout the complete plastomes, two intergenic regions rsp16/trnQ and trnT/trnL were found to be the most variable hotspot regions within genus Rubus, including members of four subgenera  throughout the central mountains in Taiwan, was treated as a synonym of R. niveus, which occurs widely in South Asia and Southeast Asia3. It was considered that R. incanus (narrow cymose panicles or short thyrses) and R. niveus (umbellate corymbs) are two distinct taxa on the basis of the inflorescence type, and our preliminary data based on the complete plastome sequences support the statement of Huang and Hu17  throughout the island and has 5-foliolate leaves with red fruits at maturity, and its sister species in this phylogeny, R. taitoensis, has simple leaves (not divided or 3-lobed) with orange to yellow fruits, occurring in medium altitudes  in the central mountains. It is necessary to include three species of this clade into a broader phylogenetic framework of Rubus in Taiwan and mainland China to precisely determine species relationships among these taxa.Given the scarcity of available complete plastome sequences of genus tus Fig. 14. RubusIdaeobatus, R. glandulosopunctatus, R. rubroangustifolius, and R. taiwanicolus, which is more closely related to the clade of subg. Anoplobatus (R. trifidus and R. boninensis) and subg. Idaeobatus (R. crataegifolius and R. takesimensis) than other members of the same subgenus  , and no previous hypothesis regarding its relationship to other congeneric species exists. In the case of R. glandulosopunctatus, it is considered a synonym of R. rosifolius, which occurs widely in Asia , Africa, and Australia. As pointed out by Huang and Hu17, this widespread species exhibits tremendous morphological variations, requiring more investigation on this complex taxon. Given that the chloroplast and nuclear combined phylogenies  is closely related to the clade containing R. hirsutus, R. eustephanaus var. glandulinger, and R. tsangii var. linearifoliolus. However, the target capture sequencing phylogeny suggested that R. rosifolius is closely related to R. illecebrosus, R. trifidus, and R. craetaegifolius15. In our current complete plastome-based phylogeny  Fig. . In genesis Fig. 14. Rubuses Figs. 14, R. roMalachobatus, R. laciniatostipulatus, and R. kawakamii form a clade with R. lambertianus var. glaber, another species of the same subgenus. R. fockeanus from subg. Cylactis is sister to the clade of subg. Malachobatus. Based on three concatenated regions of chloroplast DNA among Chinese Rubus species, it was shown that subg. Cylactis is closely related to subg. Malachobatus, including few exceptional species from subg. Idaeobatus  and Dalibardastrum (R. amphidasys and R. tsangorum). Subg. Cylactis has also shown to be highly polyphyletic on the basis of target capture sequencing study15. Rubus foeckeanus typically occurs in high elevation  and has 3-foliolate compound leaves. Rubus laciniatostipulatus occurs widely in southern China and southeastern Asia. In Taiwan, it occurs in forest edges in the northern and central parts of the island at low elevations (20–300 m)17. In the case of R. kawakamii, it is commonly distributed in forests at medium altitudes  throughout the central mountains. This species is very difficult to distinguish from R. swinhoei, which occurs at low altitudes  in northern and central parts, and is often treated as a variety of R. swinhoei58. Nevertheless, both R. laciniatostipulatus and R. kawakamii have simple leaves, shallowly 5–7 lobed or not divided, respectively, and display a close relationship14. Overall, phylogenetic relationships among the endemic species, including infraspecific taxa, require broader taxon sampling from China and Taiwan to gain new insights into infrageneric relationships, as well as their plastome evolution. It is also necessary to assemble plastome sequences from members of subg. Rubus and several other subgenera to fully understand plastome evolution and to reveal the complex evolutionary history of Rubus on a global scale.Lastly, the third clade of two endemic species of subg. Rubus species in Taiwan, representing two subgenera, Idaeobatus  and Malachobatus (R. kawakamii and R. laciniastostipulatus) to assemble plastome sequences. Voucher specimens were collected and deposited at SKK . Fresh leaves were collected from Taiwan and dried with silica gel before DNA extraction. Total DNA was isolated by using the DNeasy Plant Mini Kit  and sequenced with an Illumina HiSeq 4000 , yielding 150 bp paired-end read length, at Macrogen Corporation . The resulting paired-end reads were assembled de novo using Velvet v. 1.2.10 with multiple k-mers59. The tRNAs were confirmed via tRNAscan-SE60. Annotation was conducted using Geneious R1061, and the annotated plastome sequences were submitted to GenBank: R. glandulosopunctatus (MT274118), R. incanus (MT274119), R. kawakamii (MT274120), R. laciniastostipulatus (MT274121), R. parviaraliifolius (MT274122), R. rubroangustifolius (MT274123), R. taitoensis (MT274124), and R. taiwanicolus (MT274125). The annotated GenBank format sequence file was used to draw a circular map with the OrganellarGenomeDRAW (OGDRAW) program v1.3.1.62.We first sampled eight out of the 15 endemic 63 in Shuffle-LAGAN mode64, the eight complete plastomes from the endemic Rubus were compared: subg. Idaeobatus  and subg. Malachobatus (R. kawakamii and R. laciniastostipulatus). The eight endemic Rubus plastome sequences in Taiwan were aligned with MAFFT v. 765 and adjusted manually with Geneious61. Using DnaSP v. 6.10 software66, a sliding window analysis with a step size of 200 bp and a window length of 800 bp was conducted to determine the nucleotide diversity (Pi) of the plastome. The codon usage frequency was calculated using MEGA767 with the relative synonymous codon usage (RSCU) value68, which is a simple measure of non-uniform usage of synonymous codons in a coding sequence. The DNA code used by bacteria, archaea, prokaryotic viruses, and chloroplast proteins was used69. Protein-coding genes were run using the online program predictive RNA editor for plants (PREP) suite70, with 22 genes as reference, based on a cut off value of 0.8 to predict the possible RNA editing sites in eight endemic Rubus from Taiwan. Analyses based on the complete cp genomes and concatenated sequences of 75 common protein-coding genes among the studied species were performed via MAFFT v. 765 using Geneious R1061. Using DnaSP v. 6.10 software66, we calculated the Ka/Ks ratios of the eight endemic Taiwanese Rubus plastomes and compared them with each other.Using mVISTARubus, including R. amabilis (NC047211), R. boninensis (MH734123), R. corchorifolius (KY419958), R. coreanus (NC042715), R. crataegifolius (NC039704), R. fockeanus (KY420018), R. niveus (KY419961), R. takesimensis (NC 037991), R. lambertianus var. glaber (MH99240), R. leucanthus (MK105853), and R. trifidus (MK465682); six species from Fragaria; two species from Rosa; one species from Prunus; two species from Pyrus; and one species from Prinsepia) were aligned with MAFFT v. 765 in Geneious61. Maximum likelihood (ML) analysis based on the best-fit model of TVM + F + R4 was conducted with IQ-TREE v. 1.4.271. Prinsepia utilis was used as an outgroup, and a non-parametric bootstrap analysis was performed with 1000 replicates.For the phylogenetic analysis, the complete plastome sequences of 31 representative species from the family Rosaceae (11 species from Supplementary Tables.


MPO. This encodes myeloperoxidase, an essential component of neutrophil azurophil granules. MPO screening in conditions phenotypically related to GPP uncovered further disease alleles in one subject with acral pustular psoriasis  and in two individuals with acute generalized exanthematous pustulosis . A subsequent analysis of UK Biobank data demonstrated that the c.2031−2A>C and c.1705C>T (p.Arg569Trp) disease alleles were also associated with increased neutrophil abundance in the general population . The same applied to three further deleterious variants that had been genotyped in the cohort, with two alleles  yielding p values < 10−10. Finally, treatment of healthy neutrophils with an MPO inhibitor (4-Aminobenzoic acid hydrazide) increased cell viability and delayed apoptosis, highlighting a mechanism whereby MPO mutations affect granulocyte numbers. These findings identify MPO as a genetic determinant of pustular skin disease and neutrophil abundance. Given the recent interest in the development of MPO antagonists for the treatment of neurodegenerative disease, our results also suggest that the pro-inflammatory effects of these agents should be closely monitored.The identification of disease alleles underlying human autoinflammatory diseases can provide important insights into the mechanisms that maintain neutrophil homeostasis. Here, we focused our attention on generalized pustular psoriasis (GPP), a potentially life-threatening disorder presenting with cutaneous and systemic neutrophilia. Following the whole-exome sequencing of 19 unrelated affected individuals, we identified a subject harboring a homozygous splice-site mutation (c.2031−2A>C) in  A tight regulation of neutrophil numbers is crucial to innate immune homeostasis. As mature granulocytes do not divide, their accumulation depends on the balance between progenitor proliferation, release of differentiated cells into the bloodstream, and clearance of aging cells.614204]), a potentially life-threatening condition presenting with flares of neutrophilic skin inflammation (pustular eruptions), fever, increased production of acute phase reactants, and neutrophilia. While disease alleles have been described in IL36RN, AP1S3, and CARD14, the majority of affected individuals do not carry deleterious changes at these loci.Here we focused our attention on generalized pustular psoriasis  (254600]) (The c.2031−2A>C substitution in _000250) B was sel254600]) , an inheMPO coding region was next examined in a validation sample, including 14 GPP subjects and 109 individuals with acral variants of pustular psoriasis (APP). This uncovered a further study participant harboring a homozygous c.2031−2A>C substitution  . Given ttitution .Table 1FMPO changes were detected . This is a severe cutaneous adverse reaction, which can be triggered by drugs , leading to flares of skin pustulation, fever, and systemic neutrophilia.MPO deletion (c.1552_1565del [p.Met519Profs∗21]). A second study participant had inherited the c.2031−2A>C substitution previously observed in GPP/APP, in conjunction with a damaging c.1705C>T (p.Arg569Trp) change (CADD score: 35.0) (To further investigate the impact of e: 35.0) .,MPO alleles observed in our dataset have a well-established impact on protein function.Of note, homozygous c.1705C>T (p.Arg569Trp) mutations have been repeatedly observed in individuals affected by MPOD.MPO mutations, MPOD, and skin pustulation. We examined 28 articles describing the presentation of MPOD in 217 individuals. This uncovered four case reports where the disease manifested with pustular eruptions and a fifth where it was associated with the severe neutrophilic dermatosis known as pyoderma gangrenosum . We queried the UK Biobank dataset, which includes genotype information and health data for a well-characterized population cohort .−6; −28 . As c.2031−2A>C affects the splicing of the last exon, this is in keeping with the expectation of an escape from nonsense-mediated decay.To explore the pathways underlying the effects of ions see . SpecifiMPO wild type) examined in parallel, indicating that the changes are unlikely to be a secondary effect of inflammation.Conversely, we found that 95 genes were upregulated in the affected individual (FDR < 0.05) . The majPBK and GUCYA2) encode proteins  that can inhibit apoptosis.,MPO mutations may affect neutrophil survival.While the experiment was limited by the modest sample size and the number of differentially expressed genes was too small for pathway enrichment analyses, we noted that two of the five most upregulated loci  stimulation and assessed the effects of ABAH pre-treatment on this process. While PMA caused substantial neutrophil death, we found that ABAH supplementation caused an increase in cell viability  to mimic the effects of iability C and a riability D. Thus, MPO mutations and pustular skin disease. While the disease alleles described here have also been implicated in MPOD, we did not observe any evidence of immune deficiency in the individuals examined in this study. Likewise, pustular skin disease is present in only a fraction of people affected by MPOD. Thus, it is tempting to speculate that the manifestations of MPO mutations may be influenced by background polygenic variation, especially as a similar phenomenon has been documented in rare hematological phenotypes.AP1S3 allele ,Our findings  is an inhibitor of myeloid cell apoptosis,Further studies will be required to dissect the molecular mechanisms whereby MPO deficiency downregulates cell death. Given that GSE123787.The neutrophil RNA-sequencing dataset described in this study may be obtained from the Gene Expression Omnibus using identifier A.D.B. has received funding from Boehringer-Ingelheim. C.H.S. has been a principal (or co-) investigator on commercially sponsored clinical trials and investigator-led studies funded by AbbVie, GaxoSmithKline, Janssen, Novartis, Pfizer, Regeneron, Roche, Sanquin, Celgene, Sanofi, LEO Pharma, Boehringer Ingleheim, and UCB Pharma. F.C. has received funding from Boehringer-Ingelheim and consultancy fees from AnaptysBio. J.N.B. has received funding and fees from Abbvie, Boehringer-Ingelheim, Bristol Myers Squibb, Celegene, Ely Lily, Novartis, Pfizer, Samsung, Sienna, and Sun Pharma. N.J.R. has received research funding for clinical trials from AnaptysBio through Newcastle Hospitals NHS Foundation Trust. P.B. and S.V. are Boehringer-Ingelheim employees. S.W. has received non-financial support (sponsorship to attend dermatology conferences) from Janssen, Abbvie, Novartis, and Almirall.


Gossypium species, they are involved in phenolics and flavonoid pathways. Phenolics defend the cellulose fiber from dreadful external conditions of biotic and abiotic stresses, promoting strength and growth of plant cell wall.O-methyltransferases (OMTs) are an important group of enzymes that catalyze the transfer of a methyl group from S-adenosyl-L-methionine to their acceptor substrates. OMTs are divided into several groups according to their structural features. In OMT gene family, containing a total of 192 members, has been identified and characterized in three main Gossypium species, G. hirsutum, G. arboreum and G. raimondii. Cis-regulatory elements analysis suggested important roles of OMT genes in growth, development, and defense against stresses. Transcriptome data of different fiber developmental stages in Chromosome Substitution Segment Lines (CSSLs), Recombination Inbred Lines (RILs) with excellent fiber quality, and standard genetic cotton cultivar TM-1 demonstrate that up-regulation of OMT genes at different fiber developmental stages, and abiotic stress treatments have some significant correlations with fiber quality formation, and with salt stress response. Quantitative RT-PCR results revealed that GhOMT10_Dt and GhOMT70_At genes had a specific expression in response to salt stress while GhOMT49_At, GhOMT49_Dt, and GhOMT48_At in fiber elongation and secondary cell wall stages.An Gossypium species and that they may contribute to salt tolerance or fiber quality formation in Gossypium.Our results indicate that O-methyltransferase genes have multi-responses to salt stress and fiber development in The online version contains supplementary material available at 10.1186/s12870-020-02786-6. Gossypium Species) has the importance for natural fiber all over the globe. The primary goals of upland cotton (G. hirsutum) perspectives have been always to achieve better quality with higher yield [G. hirsutum bears staple fibers 25–40 mm in length and 15 μm in thickness at their full maturity. Fiber cells must undergo four distinct but partially overlapped developmental stages, including initiation, elongation, secondary cell wall deposition, and maturation. The secondary cell wall of fiber, which is mainly composed of cellulose, is important especially for fiber quality perspective. However, some studies have shown that secondary cell wall of fibers of flax (Linum usitatissimum L.), ramie (Boehmeria nivea L.), and Spanish broom (Spartium junceum L.) also contain phenolics along with cellulose. Their fibers are known for their physical properties such as length and strength and have been used for textile purposes. A thicker secondary cell wall was estimated to contain no less than 70% cellulose content while the cotton fiber contains almost 90% cellulose [OMTs) in lignin biosynthesis [OMTs mediate normal plant growth in the presence of lignin [OMT cDNA was described in 1991 [OMT cDNAs have been cloned from diverse plants species, including Zea mays, Arabidopsis thaliana, Iris hollandica, and Nicotiana tabacum [Cotton , II. N-methyltransferases (NMTs), and III. C-methyltransferases (CMTs). Category I OMTs are further classified into five sub-categories. Sub-category I-a comprises caffeoyl coenzyme A 3-O-methyltransferase (CCoAOMT) and caffeic acid 3-O-methyltransferases (COMTs), which are involved in methylation in phenylpropanoids. Sub-categories I-b, I-c, and I-d act in methylation of hydroxyl in flavonoid, alkaloids, and myoinositol, respectively. The fifth sub-category I-e takes part in methylation of carboxyl of diverse acids. The results of a study discovered the crystal structure of OMTs from Medicago sativa [OMT gene that was cloned and characterized from a medicinal plant Ligusticum chuanxiong and contained higher ferulic acid was named as LcCOMT. The differential expression of LcCOMT gene under chilling stress was more than 6-fold higher than that under controlled conditions, suggesting that ferulic acid may increase plant tolerance to cold stress. BLAST analysis showed that LcCOMP was 23.9–40.2% similar to OMTs of alkaloid, flavonoid, isoflavonoid, and phenylpropanoids [According to substrate classification, plant methyltransferases have three major categories, I. O-methyltransferases  with a molecular weight of 6.642 kDa. While GhOMT33_Dt, which was identified on chromosome Dt02, coded the largest protein of 969 aa with a molecular weight of 108.296 kDa among all OMT members in three Gossypium species.A genome wide analysis was conducted to characterize i Table S. Sheet Ad Table S. Sheet BOMT family genes in Gossypium species, the results revealed that 64, 45 and 47 OMTs in G. hirsutum, G. arboreum and G. raimondii contained Pfam domain Pf00891, and that only 20, 10 and 9 OMTs in G. hirsutum, G. arboreum and G. raimondii contained Pfam domain Pf01596. In A. thaliana and T. cacao, 25, 24 OMTs contained Pf00891 domain, and 8, 2 OMTs contained Pf01596 domain respectively.In domain analysis of OMT genes were positioned on their respective chromosomes, while seven of G. raimondii, one of G. arboreum, and 23 of G. hirsutum were positioned in scaffolds , chr11 was mapped with 13 genes followed by chr08 with nine genes. The minimum number of genes in a chromosome was one in chr2, chr6, and chr10 respectively. There was no OMT family members identified in chr01 and chr07    was higher than in At sub-genome (26 genes). The maximum number of genes in a chromosome was seven in Dt04 and At12, followed by four in Dt10 and At10 chromosomes, respectively. Dt01, Dt05, At01, At06, and At11 only had one OMT gene, and Dt06, Dt07, At03, At08, At09, At13 two OMT genes and Dt02, Dt08, and Dt13 three OMT genes respectively  in Gossypium, A. thaliana, and T. cacao species. The results showed that each clade of OMT genes were symmetrically distributed within Gossypium species  was found 152 times in 43/82 genes in G. hirsutum. ABRE elements was detected 119 times in 29/82 OMT genes in G. hirsutum. The ERE element was detected 113 times in 37/82 and G-Box 97 times in 37/82 G. hirsutum OMT genes. An auxin RR-core and cis-acting regulatory element involved in the MeJA-responsiveness (TGACG-motif) were also observed in Gossypium OMT genes where this element was identified 48 times in 25/82 genes. Some other important cis-regulatory elements including wun-motif 44 times in 26/82, W-box 39 times in 31/82, GATA-motif 32 times in 27/82, O2-site 30 times in 22/82 OMT genes respectively, in G. hirsutum  enrichment, kyoto encyclopedia of genes and genomes (KEGG Pathway), and InterPro analyses. GO term analysis verified their O-methyltransferase activity of all 82 OMT genes, while 62 of the 82 genes were also enriched in methyltransferase activity and 53 of the 82 genes in protein dimerization activity  categorized these 82 OMT genes as functional genes of S-adenosyl-L-methionine-dependent methyltransferase . Recent study testified that modern allotetraploid Gossypium species were developed from a natural hybridization between the ancestors of two diploid species of G. raimondii (D-genome) [G. arboreum (A-genome) [OMT genes in G. hirsutum AtDt genome as compared to the total number of OMT genes in A and D genomes. Possibly 19 OMT genes in At sub-genome and 17 in Dt sub-genome in G. hirsutum   and G. a-genome)  1.7 to 1-genome) . The resionation . Besidesunctions .OMTs within Gossypium species that might support the conservative evolution mode of OMT genes within five phylogenetic clades. Previous study also reported five clades of OMT genes in Catalpa bungei [OMT genes in Gossypium species were under a purifying selection pressure. The purifying selection pressure might suggest the importance of OMT genes in Gossypium species. But noticeable exceptions were also observed in some interspecific homologous pairs, in which their Ka/Ks values were > 1, indicating these homologous pairs were under a positive selection pressure. These homologous pair exceptions included GrOMT52_D-GhOMT52_Scaf and GrOMT29_D-GhOMT29_At in G. raimondii and G. hirsutum, GaOMT30_A-GhOMT30_At in G. arboreum and G. hirsutum, GrOMT63_D-GaOMT64_A and GrOMT29_D-GaOMT29_A in G. raimondii and G. arboreum. These results suggested that the OMT genes might had experience positive selection pressures during the evolution from diploids to tetraploids. Previous studies have evidenced that the positive selection pressure might be associated with the onsets of new functions in genes [OMT genes were lost during the formation and evolution of allotetraploid cotton -reticuline 7-O-methyltransferase. 7OMTs convert reticuline to laudanine in tetrahydrobenzylisoquinoline biosynthesis in the opium poppy Papaver somniferum, however, this enzymatic activity is unknown in most higher plants [OMTs that experienced positive selective pressure be lost or take on some novel functions in G. hirsutum during the processes of its evolution and ancestor formation.Phylogenetic analysis showed high similarity and monophyletic distribution of a bungei . Analysein genes , 48. Conr plants . TherefoG. raimondii (D-genome) and G. arboreum (A-genome) are the closest relatives to the Dt and At sub-genomes of allotetraploids, respectively [t or Dt sub-genomes of G. hirsutum [OMT genes that do not have homologs in their relative At and Dt sub-genomes  in CSSLs lines CCRI36 and MBI8255, diverse genes were found differentially expressed in lignin biosynthesis including CCoAOMT, which can adequately utilize lignin and has been characterized in several previous studies [CCoAOMT was up-regulated in response to Verticillium pathogen in cotton and rendered cotton plants a comparable phenotypic resistance as compared to control plants [CCoAOMT in response to V. dahliae, confirming the effect of this OMT gene in the plant response to V. dahliae in cotton [OMT genes in biotic stresses in cotton.The KEGG pathways enrichment analysis revealed the involvement of nditions –72. The nditions , 74, salnditions , droughtnditions , and temnditions . These pnditions , 78. Samtabolism . Higher tabolism , 81. Impn cotton –84. Duri dahliae . In the  dahliae . In resp studies , 88. Anol plants . A RNA-sn cotton . These rOMT genes have been found specific for salt stress tolerance and fruit development in tomato plant (Solanum lycopersicum) [Ipomoea batatas) in response to salt stress [TaCOMT-3D contributes to stem mechanical support [TaCOMT gene was also observed with constitutive expression in stem along with leaf and root [OMT gene (BdCOMT1) was strongly expressed in stem node and internode but poorly expressed in other tissues in Brachypodium distachyon plant [OMT gene family in the transcriptome data of TM-1 [OMT members GhOMT10_Dt and GhOMT70_At might contribute to salt stress tolerance in G. hirsutum. In the qRT-PCR verifications, GhOMT10_Dt and GhOMT70_At showed different expression profiling from 2 h and 6 h after 200 mM NaCl treatment  . The SAMt stress . In whea support . Anotherand root . The OMTon plant . The exp of TM-1  and verient Fig. . ProbablGhOMT48_At and GhOMT49_At were expressed at elongation stages of a CSSL (CS-B25) and TM-1 respectively [OMT genes were consistently identified across various populations and species including TM-1  in G. hirsutum evidenced the regulation of lignin-like phenolics pathway genes including a COMT and others, during cotton fiber elongation and secondary cell wall synthesis stages. The results demonstrated the roles of these genes in regulating the lignification in developing cotton fibers [OMT gene family which is a forward step in research to uncover the possible functions or to support previous studies in exploration the functions of OMT genes in plant response to salt stress and in cotton fiber development.Lignins-like phenolics are widely studied in response to stress . Recent n fibers . This stOMT contributes to diverse phenolics that are essential for plant growth and serves as protective shield against several kinds of stresses. Various bioinformatics analyses revealed that OMT gene family is a strong growth regulator, which not only provide protection to the plant, but also are involved in fiber elongation and secondary cell wall synthesis stages. Furthermore, expression profiling analysis based on several transcriptome data and qRT-PCR validation inferred that GhOMT10_Dt and GhOMT70_At might be the potential candidates for salt stress tolerance and that GhOMT48_At, GhOMT49_At, and GhOMT49_Dt might have significant influence in fiber development at elongation and secondary cell wall thickness stages of G. hirsutum. This proposed study concludes the important roles of OMT family genes in cotton fiber development and in salt stress tolerance.Methyltransferases are versatile class of enzymes. Gossypium species including G. arboreum (CRI), G. raimondii (JGI), and G. hirsutum (NAU) were downloaded from cotton functional genomic database (https://cottonfgd.org/) [A. thaliana  [T. cacao  [OMT genes. The hidden Markov model profiles (PF00891 and PF01596) were downloaded from Pfam database (https://pfam.xfam.org/). The hmmsearch program of HMMER 3.0 software [Gossypium species with the E-value of 1e− 5. OMT protein sequences of A. thaliana and T. cacao were also retrieved from the Phytozome database  for phylogenetic analysis. The proteins with absence of required domains were manually removed. Other features of OMT genes including protein length (aa), and molecular weight (kDa) were characterized by using cotton functional genomic database (http://www.cottonfgd.org/) [G. hirsutum, G. arboreum, G. raimondii, A. thaliana, and T. cacao encoded by OMT genes were aligned with clustalx2 software  [Genome data of three gd.org/) . Genome aliana#)  and T. cTcacao#)  were alssoftware  was usedgd.org/) . The fulal.org/)  with defal.org/) . The topGossypium species.Nomenclature of these members was based on their chromosomal locations, homology and numbers in each OMT genes, to search the homologous pairs of OMT genes between genomes of the three Gossypium species through protein-protein blast . Circle gene viewer model of TBtools software was used to visualize the results of collinearity between homologous gene pairs [TBtools was used to perform the chromosomal mapping of the given ne pairs .OMT genes was analyzed using the online server of Gene Structure Display  [http://meme-suite.org/) by providing protein sequences of OMT genes [The structure of the .edu.cn) . The conMT genes .G. hirsutum (NAU), G. arboreum (CRI), and G. raimondii (JGI) were assigned to TBtools software to estimate the Ka/Ks ratio to predict selection pressure between the genes of each pair in genomes and sub-genomes [OMT genes were retrieved through cotton functional genomic database (http://www.cottonfgd.org) and were submitted to PlantCARE database [OMT family genes were downloaded from cotton functional genomic database (http://www.cottonfgd.org), then annotation was performed by providing KEGG IDs in kyoto encyclopedia of genes and genomes database (https://www.genome.jp/kegg/) [OMT family genes were downloaded from cotton functional genomic database (http://www.cottonfgd.org) and were submitted to Gene ontology database (http://geneontology.org/) to perform GO analysis [The CDS of homologous gene pairs of -genomes . The upsdatabase  to obtaidatabase , 110. KEp/kegg/) . Gene onanalysis .G. hirsutum  (PRJNA248163) [OMT family genes at different growth stages, under abiotic stress treatment stages, ovule development, and in different fiber development stages of cotton. Briefly, 69,307, 0–153, MBI7747, MBI7561, MBI9915, MBI9749, and Hai1 have high fiber quality traits, while 69,362, MBI7285, sGK9708, CCRI36, and CCRI45 have low fiber quality traits. Detailed information of these referenced materials is presented in Table SThe different sets of RNA sequencing data including TM-1, a genetic standard line of A248163) , 31, 69,A248163) , MBI7747A248163) , 34, andA248163) , 34 wereG. arboreum (PRJNA179447) [G. raimondii (PRJNA79005) [OMT genes.Transcriptome data of A179447) , and G. NA79005)  were alsUpland cotton cultivar 0–153 had elite fiber quality while sGK9708 had high yield potential and wide adaptability. They are successfully used to tag fiber quality and yield QTLs in our previous reports , 114. InOMT genes under salt stress, seeds of sGK9708 cultivar were germinated in wet filter papers for 72 h and then were transferred to hydroponic conditions. The seedlings were treated with 200 mM NaCl at three leaves stage. The true leaves, stems, and roots were sampled at 0 h, 2 h, and 6 h of the treatment. The 0 h of treatment was considered as control sample to compare the expression profiling with treated samples.To examine the expression profiling of Gh-Histone3 gene was used as reference to normalize the relative expression level. Primers pairs of five OMT genes were designed by using Oligo 7 [−ΔΔCt method was used to calculate the gene expressions [Total RNA isolation was performed with the RNAprep Pure Plant Kit by . To eliminate the genomic DNA contamination, the RNA samples were treated with DNase1. RNA concentration and integrity was observed on Nano Drop 2000 spectrophotometer  and 1% agarose gel electrophoresis. cDNAs of the RNA samples that the A260/280 ratio reached 2.00 were synthesized using PrimeScript® RT Reagent Kit . qRT-PCR was performed with ABI 7500 fast Real-Time PCR system , with  Oligo 7  , b: in A genome (G. arboreum), c:in AtDt genomes (G. hirsutum), d: in scaffolds of the three genomes. The genes in boxes in A and D genomes represent that their homologous genes in At and Dt sub-genomes are missing, while the genes in boxes in At and Dt sub-genomes represent that their homologous genes in A and D genomes are missing.Additional file 2: Figure S2. Identification of motifs of OMT genes in three Gossypium species.Additional file 3: Figure S3. Identification of cis-regulatory elements of OMT genes.Additional file 4: Table S1. Detail Information of plant materials used for RNA-seq data acquisition.Additional file 5: Table S2. Sequences of Primer Pairs of five selected OMT family genes for qRT-PCR verification.Additional file 6: Table S3. Basic information and analyses of OMT genes in Gossypium species (Sheet A), and in A.thaliana and T. cacao (Sheet B).Additional file 7: Table S4. Analyses of duplication events and selection pressure. Sheet A: The analysis of duplication events of OMT genes in A genome (G. arboreum), D genome (G. raimondii), and AtDt genomes (G. hirsutum). Sheet B: The comparative analysis of selection pressure (Ka/Ks) of OMT genes in G. raimondii, G. arboreum, and G. hirsutum.Additional file 8: Table S5. Structural features of OMT genes.


Phenacoccus solenopsis (Tinsley) has established close mutual relationship with the ghost ant Tapinoma melanocephalum (Fabricius). The sympatric ants, Paratrechina longicornis (Latreille) and Tetramorium bicarinatum (Nylander) were frequently observed to compete for nutrient honeydew produced by P. solenopsis with T. melanocephalum. Herein, we investigated the effects of interference competition between the ant species on the ant-mealybug interactions. Phenacoccus solenopsis benefited from the tending by T. melanocephalum and P. longicornis. Interference competition between T. melanocephalum and P. longicornis interrupted the mutualism, suppressed the trailing activity of both species, but negligibly influenced the parasitism of Aenasius bambawalei Hayat, a solitary endoparasitoid of P. solenopsis. Harmonia axyridis, a predator of P. solenopsis, showed a significant avoidance when encountering with T. melanocephalum or P. longicornis, but not T. bicarinatum. Ant workers showed higher aggressiveness and lower exploratory activity when T. melanocephalum encountered P. longicornis. However, competition between T. melanocephalum and T. bicarinatum seldom influenced the trailing and exploratory activity of T. melanocephalum. It is concluded that interference competition for mutualism between ant species can mediate ant-mealybug associations and the fitness of mealybug colony. Our results also demonstrate that the effects of interference competition between ant species on ant-mealybug mutualism are varied among ant species.Ant-hemipteran mutualism has been well documented, and many studies have reported the interference competition between ant species for the mutualism. However, little is known on how this interference competition impacts the reciprocally beneficial association. Previous studies demonstrated that the invasive mealybug  Mutualism, which is defined as the reciprocally beneficial interaction between two species, is an important ecological interaction ,2. MutuaLasius niger (Linnaeus) differs among aphid species [Although many studies have elucidated the ant-hemipteran interactions and their ecological outcomes, relatively few studies have explored the various outcomes resulting from the competitive mutualism among the interactional species. It is accepted that competition is an important force in natural communities ,21. Prev species . Ants us species . Subsequ species ,27,28. TSolenopsis invicta Buren and native ants for carbohydrate sources from honeydew-producing hemipterans widely exists in both its native and invasion regions [Solenopsis invicta suppresses native ants by monopolization and competition for mutualism with honeydew-producing hemipterans [Tapinoma nigerrimum (Nylander) notably reduces the likelihood of incipient colony establishment and survival of Argentine ant Linepithema humile Mayr [Pseudococcus citriculus Green, can associate with various mutualistic ant species, including Lasius niger and Pristomyrmex pungens Mayr. The territories of these two ant species are completely separated, and there are occasional territory takeovers of one ant species by the other. Lasius niger is considered as a more effective mutualistic partner than P. pungens because of its higher aggressiveness [In comparison with the competition for ant tending among different hemipterans, the interference competition between ant species for mutualist-provided resources has been less studied. Such competition is particularly acute between invasive and native ant species. Intense interspecific competition between  regions . Solenopipterans ,30,31. Bipterans . Due to ile Mayr . Honeydesiveness . These rsiveness ,29,34. HPhenacoccus solenopsis Tinsley (Hemiptera: Pseudococcidae) is a polyphagous invasive species in China. It has established widely mutualistic relationships with a wide range of ant species, including S. invicta, T. melanocephalum, P. longicornis, T. bicarinatum and Pristomyrmex pungens in ecosystems [T. melanocephalum is one of the most dominant native ant species in the areas invaded by S. invicta [Tapinoma melanocephalum also competes for honeydew produced by mealybugs with other native ant species [T. melanocephalum, P. longicornis and T. bicarinatum, on ant-mealybug interactions. Understanding the response of each partner in the beneficial associations to ant competition is critical to clarifying how resource competition between ant species regulates the ecological interactions. Herein, we conducted a series of laboratory experiments to test the effect of ant interference competition on the colony growth of P. solenopsis and the performance of natural enemies of mealybugs. We also evaluated how interference competition mediates the trailing activity, aggressiveness index and exploratory behavior of these three ant species. We hypothesize that T. melanocephalum competes with P. longicornis and T. bicarinatum for mutualist that provides carbohydrate resources. The interference competition between ant species would affect the outcome of ant-mealybug mutualism. Our results may provide more accurate information for better understanding the mechanism by which interspecific competition among ant species mediates ant-hemipteran mutualism. The mealybug osystems ,36,37,38 invicta . Tapinom species . CurrentPhenacoccus solenopsis colonies were collected from a cotton field in Huazhong Agricultural University and transferred to cotton plants in flowerpots. Sixty 1st instar mealybug nymphs were placed on cotton plants and raised for several generations in the laboratory at the temperature of 26 ± 2 °C and relative humidity of 60–70%. The parasitoid Aenasius bambawalei and predator Harmonia axyridis were also collected from cotton field. Both parasitoid and predator colonies were fed mealybug nymphs in the laboratory at 26 ± 2 °C and under an LD 16:8 h photoperiod. Cotton plants  were grown in soil media  within plastic flowerpots. Each plant used in the experiment was approximately 30–40 cm in height with 15–20 true leaves. Tapinoma melanocephalum, Paratrechina longicornis and Tetramorium bicarinatum were collected from the suburb of Wuhan. Ant colonies were separated from the soil by dripping water into the plastic boxes until the colonies floated [Tenebrio molitor L.; Coleoptera: Tenebrionidae) and 50 mL 10% honey water solution. All ant colonies were reared in the laboratory at 26 ± 2 °C and relative humidity of 60–70%.Sympatric ant colonies of  floated . Ants we floated . Each anT. melanocephalum, P. longicornis, T. bicarinatum, a combination of T. melanocephalum and P. longicornis colonies, and a combination of T. melanocephalum and T. bicarinatum colonies. No ant tending was used as a control. Each treatment was replicated 12 times, and the number of live mealybugs per plant was recorded after six weeks. The colony growth rate of mealybugs was defined as the final population density divided by the initial population density. Each potted cotton plant was held in a plastic tray (42 × 26 × 18 cm) and covered with nylon netting surrounded by a wooden cage (90 × 90 × 100 cm). The potted plants were randomly arranged. Thirty 2nd instar mealybugs were transferred to each caged plant. The ant colony fragment was placed into a small plastic container (22 × 15 × 7 cm), and connected with the plant tray by a silicone tube (0.8 cm in diameter and 10 cm in length) . To prevA. bambawalei were also evaluated by the caged bioassay described above. We transferred the 3rd instar mealybugs to caged plants . The ant colony fragment was then connected with the plant tray. The ants were given two mealworms and 50 mL water every two days. Two fertilized female parasitoids were released on each plant after 24 h. Five treatments of ant-tending and a control were implemented as described in 2.2. Each treatment was replicated 12 times. All surviving mealybugs and mummified mealybugs on each plant were collected and counted after two weeks. The parasitism percentage was defined as the number of mummified mealybugs divided by the total number of mealybugs on each plant. The effects of ant competition on the parasitism of T. melanocephalum; P. longicornis; and a combination colony of T. melanocephalum and P. longicornis. The similar experiments were repeated with T. melanocephalum; T. bicarinatum; and a combination colony of T. melanocephalum and T. bicarinatum. Each treatment was replicated 15 times. The trailing activity of ants was evaluated on the plants by counting the number of trailing ants moving up and down the plant trunk for 5 min after 24 h. The trailing activity of each ant species was determined in laboratory assay. One cotton plant was placed in the plastic tray. The 3rd instar mealybugs were introduced to the cotton plants  and allowed to acclimate and feed for 24 h. The ant colony fragment was deprived of any carbohydrate source for 12 h and then connected with the plant tray. Workers could enter into the plant tray and move toward the plant through the outside of the pot. Ant tending treatments were conducted as follows: T. melanocephalum/no ants; P. longicornis/no ants; T. bicarinatum/no ants; T. melanocephalum-P. longicornis/no ants; and T. melanocephalum-T. bicarinatum /no ants. Each comparison was replicated 12 times. The number of ladybeetles in each arena and the number of eggs laid in the Petri dishes were counted at after 48 h.The effects of ant competition on the performance of lady beetles were tested by using a behavior observation apparatus described previously ,41. LadyT. melanocephalum-P. longicornis interaction and T. melanocephalum-T. bicarinatum interaction. Fifty trials were implemented for each treatment. The aggressiveness index was calculated by the following formula as previously described by Errard and Hefetz [iδ represents the score we evaluated in the attack interaction, it means the duration of each behavioral interaction, n means the number of replications in each treatment and T is the total time for each trial.The aggressiveness between different ant species at individual level was quantified with the protocol described by Errard and Hefetz  and Tsutd Hefetz  (1). TheThe exploratory activity of ant species was assessed using a modified apparatus originally described by Grangier and Lester . Three ciδ represents the score evaluated in the exploratory behavior; in means the number of workers in each foraging arena; T is the total time for each trial; and m means the number of replications for each treatment.A. bambawalei was treated by the arcsine square root-transformation. One-way ANOVA was used to compare the variance in means of mealybug colony growth rate and parasitism among different ant tending treatments. An LSD-test was performed for multiple comparisons of mealybug colony growth rate and parasitism between the ant treatments. For experiment 2.4, an Independent sample t-test was used to analyze the difference in ant trailing activity between single colony and combined colony. For experiment 2.5, a Paired sample t-test was performed to analyze the difference in number of lady beetle adults and eggs between ant included arena and ant excluded arena. For experiment 2.6, an Independent sample t-test was used to determine ant aggressiveness index and aggressiveness level between T. melanocephalum-P. longicornis competition and T. melanocephalum-T. bicarinatum competition. For experiment 2.7, an Independent sample t-test was used to analyze the difference in ant exploratory activity and exploratory level between single colony treatment and combined colony treatment. All statistical analyses were conducted with SPSS, version 19.0 .For experiment 2.2 and 2.3, to satisfy the precondition of variance analysis, colony growth rate of mealybug was treated by log10-transformation. Parasitism by 5, 66 = 9.603, P < 0.001). Compared with no ant tending, mealybug colony growth was significantly improved by tending of T. melanocephalum, P. longicornis and a combination of T. melanocephalum-T. bicarinatum . No positive effects on mealybug colony growth were observed for tending by combination of T. melanocephalum-P. longicornis or T. bicarinatum alone (LSD-test). The results showed that the growth of mealybug colony was significantly different under the tending by different ant species in the presence of predation . Compared with no ant tending, parasitism was largely reduced when mealybugs were tended by T. melanocephalum, P. longicornis and a combination of T. melanocephalum-P. longicornis, and a combination of T. melanocephalum-T. bicarinatum , while tending by T. bicarinatum, had no significant effect on parasitism . Percentage of parasitism varied among ant tending treatments . Furthermore, the trailing activity of T. melanocephalum was almost not influenced by interference competition with T. bicarinatum ; however, that of T. bicarinatum rapidly decreased when they encountered T. melanocephalum colony . Both T. melanocephalum, P. longicornis, T. melanocephalum-P. longicornis,and T. melanocephalum-T. bicarinatum combined colonies . Tending by T. bicarinatum had little effect on the predation of lady beetles . Lady beetles produced fewer eggs in the arenas with workers of T. melanocephalum, P. longicornis and a combination of T. melanocephalum-T. bicarinatum . Oviposition of lady beetles was not significantly influenced by T. bicarinatum or combination of T. melanocephalum- P. longicornis . Lady beetle adults apparently avoided the foraging arena occupied by workers of T. melanocephalum and P. longicornis was definitely higher than that between T. melanocephalum and T. bicarinatum . Aggressive acts in avoidance occurred more frequently in T. melanocephalum-T. bicarinatum interaction, while brief touch occurred more frequently in T. melanocephalum-P. longicornis interaction . The aggressiveness index between T. melanocephalum and P. longicornis workers showed much higher exploratory activities in single colony than in combined colony treatment . Interference competition between T. melanocephalum and T. bicarinatum significantly reduced the exploratory activity of T. bicarinatum but not T. melanocephalum . Both T. melanocephalum possessed higher exploratory level in original arena but lower exploratory level in extended arena when treated by combination of T. melanocephalum and P. longicornis . The same case of P. longicornis was observed when treated by combined colony . No significant difference in exploratory level of T. melanocephalum was found between single and combination of T. melanocephalum- T. bicarinatum in each foraging arena . T. bicarinatum showed lower exploratory level in extended arena when treated by combination of T. melanocephalum and T. bicarinatum . Animals usually experience imbalance of food resources in the ecosystem, which often restricts colony abundance and limit the maximum fitness . HoneydeLasius japonicus and P. pungens differ in aggressiveness towards Aphis spiraecola enemies. Compared with L. japonicas, P. pungens shows lower aggressiveness, and thus, has a lower extent of interference with parasitoid oviposition [P. solenopsis was improved by T. melanocephalum and P. longicornis tending. However, the positive effects of ant tending on mealybug colony growth disappeared when two ant species competed for mutualism, suggesting that the interference competition between two native ant species restrains their mutualistic efficiency. In addition, the tending ant species significantly affected the percentage parasitism by parasitoids, with more mealybug mummies in T. bicarinatum tended colonies, which also suggests that T. melanocephalum and P. longicornis take more aggressive actions against parasitoids than T. bicarinatum. Unexpectedly, we found that T. melanocephalum-P. longicornis and T. melanocephalum-T. bicarinatum competitions did not influence the parasitism of parasitoids. This result may be attributed to the distinctive approach of T. melanocephalum against parasitoids compared with that of other native ants. Workers of T. melanocephalum could effectively utilize their pygidial gland secretions as an alarm-defense system during aggressive confrontations [A. bambawalei showed strong avoidance to pygidial gland secretions of T. melanocephalum [A. bambawalei was also observed even when there was intense competition between T. melanocephalum and P. longicornis because of the presence of T. melanocephalum secretions.Intensity of ant-hemipteran interactions varies with ant species. When tending the same hemipteran species, different ant species may have different levels of aggressiveness against the enemies of hemipterans and thereby different extents of interference with prey consumption by predators and oviposition by parasitoids ,50. Lasiposition . Our resntations . A. bambcephalum . TherefoT. melanocephalum significantly declined when S. invicta was introduced, and the foraging activity of T. melanocephalum increased when fire ants were excluded [Dorymyrmex bureni declined in areas invaded by S. invicta due to the competition for mutualist-provided carbohydrates [T. melanocephalum, P. longicornis and T. bicarinatum for carbohydrates produced by mealybugs. However, the trailing activity of T. melanocephalum was not influenced by T. melanocephalum and T. bicarinatum interaction, suggesting a stronger antagonism action between T. melanocephalum and P. longicornis than between T. melanocephalum and T. bicarinatum. This result was also confirmed by subsequent aggressiveness test, which revealed a great passivity of the T. bicarinatum workers during confrontation with T. melanocephalum workers. Furthermore, the performance of predators was definitely impeded by T. melanocephalum and P. longicornis, but not by T. bicarinatum. Competition between T. melanocephalum and T. bicarinatum did not obviously affect the negative effects of T. melanocephalum on predators, which also suggests that the competitiveness in T. melanocephalum and T. bicarinatum interaction is relatively low and the protection effect on mealybugs depends on ant species. A similar study reported that due to the more effective patrolling of L. humile than Tapinoma sessile on plant surfaces to protect honeydew-producing hemipterans, Aphis gossypii was less negatively affected by enemies when tended by L. humile [T. melanocephalum or P. longicornis, but no noticeable difference was observed in arenas with T. melanocephalum and P. longicornis interaction, suggesting that the negative effects of T. melanocephalum or P. longicornis on predators were weakened by the interference competition. The intensity of interference competition between ant speices can be revealed by the foraging activity of ant workers ,54. Honeexcluded . Prevalehydrates . In our . humile . Differe. humile . PredatoLinepithema humile [Prolasius advenus were increased under carbohydrate scarcity [T. bicarinatum. The number of trailing workers of T. bicarinatum showed a sharp decline in T. melanocephalum and T. bicarinatum combined colony compared with a single colony. These results suggest that competition between ant species for honeydew resource produced by hemipterans may inhibit their protection of honeydew producing insects. Carbohydrates serve as a principal metabolic fuel, and carbohydrate scarcity may hinder the aggressive activity of ants to a greater extent than deficiency of protein or other nutrients essential for their growth. Invasive ants and co-occurring native ants showed significantly different behavior responses under competition for limited food resource . Carbohya humile . Howeverscarcity . Our resT. melanocephalum competes for access to mutualist-provided carbohydrates with P. longicornis and T. bicarinatum and suppresses T. bicarinatum from this critical resource. Additionally, our results provide novel evidence that competition for mutualist-provided carbohydrates between native ant species can enhance the performance of natural enemies, and in turn affecting the outcome of ant-mealybug mutualism, which may impact on the fitness and invasion process of the invasive mealybug. Currently, most conclusions are based on laboratory evidence, more studies conducted in field are needed to confirm this perspective. These findings may promote our understanding of the significance of interference competition in mediating ecological interactions and arthropod community.In conclusion, our results showed different efficiencies of two ant interference competition models on the outcome of mutual interactions. The dominant native ant


Micrococcus luteus ATCC 49442, which was reclassified as Pseudarthrobacter sp. strain ATCC 49442 based on its draft genome sequence. Its genome length is 4.98 Mbp, with 64.81% GC content.We present here the draft genome sequence of a pyridine-degrading bacterium,  Micrococcus luteus ATCC 49442, which was reclassified as Pseudarthrobacter sp. strain ATCC 49442 based on its draft genome sequence. Its genome length is 4.98 Mbp, with 64.81% GC content.We present here the draft genome sequence of a pyridine-degrading bacterium,  Micrococcus luteus ATCC 49442 was isolated from a Chalmers silt loam soil, which had not previously been exposed to pyridine, by enrichment using pyridine as a carbon, nitrogen, and energy source (https://kbase.us/) for assembly and genome annotation .y source , with grnotation . During M. luteus based on its morphological and metabolic characteristics . In recent years, however, there has been considerable reorganization of genera within the Micrococcaceae based on phylogenetic and taxonomic analyses  from NCBI (https://github.com/kbaseapps/SpeciesTreeBuilder/tree/master/data/cogs) against the ATCC 49442 draft genome. The RPS-BLAST alignments of marker genes from our draft genome to 34 closely related RefSeq genomes were concatenated, and the tree was then constructed. The approximately maximum-likelihood tree (Pseudarthrobacter, with maximum homology to Pseudarthrobacter sulfonivorans (Pseudarthrobacter sp. strain ATCC 49442.At the time of isolation, ATCC 49442 was identified as eristics . M. luteanalyses , 8. Signood tree  was builood tree , based onivorans , and we Pseudarthrobacter sp. strain ATCC 49442 has been deposited in GenBank under accession number JAAAXP000000000, and the raw sequencing reads are available in the Sequence Read Archive under accession numbers SRR10874394 and SRR10874393.The draft genome sequence of