Carrhotus xanthogramma, Philodromus cespitum, Clubiona spp., Ebrechtella tricuspidata, Xysticus spp. and ‘Other salticids’) exhibit different levels of predation on different prey groups and the trophic web’s structure changes depending on the time of year. Hunting spiders show a high functional redundancy in their predation, but contrary to their polyphagous nature, the examined spider taxa showed differences in their natural diet, exhibited a certain degree of prey specialization and selected prey by size and taxonomic identity. Guilds  did not consistently predict either prey composition or predation selectivity of arboreal hunting spider species. From the economic standpoint, Ph. cespitum and Clubiona spp. were found to be the most effective natural enemies of apple pests, especially of aphids. Finally, the trophic niche width of C. xanthogramma and Ph. cespitum increased during ontogeny, resulting in a shift in their predation. These results demonstrate how specific generalist predators can differ from each other in aspects of their predation ecology even within a relatively narrow taxonomic group.Spiders (Araneae) form abundant and diverse assemblages in agroecosystems such as fruit orchards, and thus might have an important role as natural enemies of orchard pests. Although spiders are polyphagous and opportunistic predators in general, limited information exists on their natural prey at both species and community levels. Thus, the aim of this study was to assess the natural prey  of arboreal hunting spiders, their role in trophic webs and their biological control potential with direct observation of predation events in apple orchards. Hunting spiders with prey in their chelicerae were collected in the canopy of apple trees in organic apple orchards in Hungary during the growing seasons between 2013 and 2019 and both spiders and their prey were identified and measured. Among others, the composition of the actual (captured by spiders) and the potential (available in the canopy) prey was compared, trophic niche and food web metrics were calculated, and some morphological, dimensional data of the spider-prey pairs were analyzed. Species-specific differences in prey composition or pest control ability were also discussed. By analyzing a total of 878 prey items captured by spiders, we concluded that arboreal hunting spiders forage selectively and consume a large number of apple pests; however, spiders’ beneficial effects are greatly reduced by their high levels of intraguild predation and by a propensity to switch from pests to alternative prey. In this study, arboreal hunting spiders showed negative selectivity for pests, no selectivity for natural enemies and positive selectivity for neutral species. In the trophic web, the dominant hunting spider taxa/groups ( AlthougIt is hard to assess what spiders’ diets consist of or what role various species play in food webs or trophic cascades. In laboratory experiments, spiders might accept more prey types than in their natural environment  and thusSpiders form abundant and diverse assemblages in apple orchards and can contribute to the suppression of various apple pests , althougN = 788, almost 90% of the data) came from one organic apple orchard located at Újfehértó , in Szabolcs-Szatmár-Bereg County, eastern Hungary. A further 37, 31 and 22 observations on the hunting spiders’ natural prey  were collected in apple orchards of the Szent István University in the vicinity of Újfehértó (Szabolcs-Szatmár-Bereg County), in Pest County and Bács-Kiskun County, respectively. The orchard located in Újfehértó  was planted on flat land, on a fine sandy soil in autumn 2002 and contained the cultivars ‘Florina’, ‘Prima’, ‘Rajka’, ‘Releika’, ‘Rewena’, ‘Rubinola’ and ‘Topaz’ on ‘M9’ and ‘Remo’ and ‘Resi’ on ‘M26’ rootstocks. It had 32 rows, each consisting of ~90–135 trees. Rows were spaced 5 m apart and apple trees were spaced 1.5 and 2.25 m apart within rows. The orchard was surrounded by other orchards  as well as other agricultural areas.Data on the natural diet  of the arboreal hunting spider assemblage was collected between 2013 and 2019 in apple orchards in Hungary. For this, apple trees were visually inspected regularly in organic orchards, and hunting spiders with prey in their chelicerae were collected during the growing season (from the beginning of April to the end of October). The vast majority of the observations (Philodromus species), spiders were raised to the adult stage (on Drosophila hydei Sturtevant) in the laboratory. Spiders were identified after Our in situ observations were conducted both day and night (approximate ratio 7:3) to get information not only on the prey of the diurnal hunting spiders but also on the nocturnal ones. Apple trees were examined mainly between 9:00 and 12:00, between 14:00 and 18:00 and between 20:00 and 23:00 (after sunset). Spiders with prey in their chelicerae were collected  and the prey was taken from the spiders to prevent any further degradation. In some cases, just the prey was collected because the spider escaped or because we did not want to influence other trials conducted in the orchard. After collecting the spiders with their prey, the material was taken to the laboratory of the Department of Entomology, Szent István University , and both the spider and the prey were identified  to the lowest taxonomic level possible. Moreover, in spiders, the width of the prosoma and in case of the preys  the width of the thorax were measured with 0.1 mm accuracy using an ocular micrometer calibrated with a stage micrometer. In juvenile spiders where the species-level identification was not possible . On each sampling date, five samples were taken. Each sample consisted of suction samples collected from one (left or right) side of the canopy of four randomly selected apple trees in a randomly selected row. For the samplings, a ~25 cm long, tapering gauze bag (mesh < 0.5 mm) was inserted into the 12 cm diameter intake nozzle of the D-VAC sampler. Suction sampling was carried out during dry weather, approximately between 9:00 and 14:00. The collected material was sorted and identified  in the laboratory.Carrhotus xanthogramma (Latreille), (2) Other salticids, (3) Philodromus cespitum , (4) Ebrechtella tricuspidata (Fabricius), (5) Xysticus spp. s. lat. and (6) Clubiona spp. The main criterion for group formation was that the number of records in a particular group should exceed 5% of the total sample  at the lowest possible taxonomic level. Thus, while C. xanthogramma, Ph. cespitum and E. tricuspidata were collected in sufficiently large numbers (44 < n) for analyses, the other species had to be placed in genus- (Xysticus spp. and Clubiona spp.) or family-level  groups. The group ‘Other salticids’ comprises the data on other spider species belonging to the family Salticidae, mainly three species: (1) Heliophanus auratus C. L. Koch, (2) H. cupreus , (3) Salticus scenicus (Clerck), but not including C. xanthogramma. The group ‘Xysticus spp. s. lat.’ (hereafter Xysticus spp.) comprises the following seven species: (1) Xysticus acerbus Thorell, (2) X. cristatus (Clerck), (3) X. kochi Thorell, (4) X. lanio C. L. Koch, (5) X. striatipes L. Koch  and (7) unidentified juveniles of Xysticus spp. Finally, the group ‘Clubiona spp.’ consists of C. frutetorum L. Koch and unidentified juveniles of Clubiona spp. , (2) ambushers  and (3) foliage runners (Clubiona spp.). Based on a more recent guild classification by E. tricuspidata and Xysticus spp.) and other hunters .For most analyses, the spiders were classified into six groups: (1) ona spp.  and S2. ona spp. . AccordiUsing the slightly modified prey classification system of To evaluate the biological control potential of the hunting spiders, the prey items were categorized according to their economic status in apple orchards in Central Europe as pests, natural enemies and neutral arthropod groups. A prey species was considered to be a pest if at least one of its life stages is known to feed on any parts of the apple tree. The pest category included some beetles (mainly weevils), some moths , some leafhoppers and planthoppers, lace bugs and all aphid and psyllid (Sternorrhyncha) species. Natural enemies are defined as species that can feed (at least in one of their life stages) on any stage of arthropods that were previously categorized as pests. This category includes red velvet mites (Trombidiidae), spiders, predatory beetles , parasitoid wasps, hoverflies, zoophagous bugs  and lacewings. Finally, the neutral category was comprised of other  prey species. For prey that could be identified only to suborder, such as Nematocera, the classification was made according to the dominant characteristics of the taxon .Dasineura mali (Keifer), Resseliella oculiperda (Rübsaamen) (Nematocera) and Phytomyza heringiana Hendel (Brachycera)) and they are of minor importance (Drosophila suzukii (Matsumura)  can breed exclusively on overripe, bruised and rotten apples, and usually infests fallen fruits. Therefore, in apple orchards, this species considered to be as a decomposer  statistical environment . For allGeneralised Linear Mixed Models with binomial error structure (GLMM-b)  were useTo assess the degree of selectivity shown by spiders, Ivlev’s electivity indices (IE) were computed based on the relative abundances of the actual and potential prey categories (collected in Újfehértó 2016–2017). To filter out the effect of year, first, the index values were calculated for each year and prey category (taxonomic or economic), and then mean index values were calculated. For economic categories, the abundances were calculated as sums of all prey items belonging to the given category. The IE values range between −1 and +1, where negative and positive values indicate negative and positive prey selection relative to prey availability in the environment, respectively .The accuracy of our hand sampling method was evaluated and the selectivity in spider predation was examined from another perspective as well. For this, a Mantel test based on Morisita dissimilarity distance was performed to calculate the correlation between the matrices of monthly abundance of the six spider groups in hand-collected versus suction samples  was calculated using “H2fun” function, while species-level specialization (d′) was calculated for the six spider taxa using the “dfun” function of the R package “bipartite” (B) , trophic niche width, and degree of niche overlap were calculated based on the taxonomic composition of the spiders’ prey   was calcite” (B) . Niche oite” (B) . Bonferrite” (B)  using “mite” (B) . Levins’ overlap . As preyThe “fourth-corner analysis” is an adequate multivariate technique for testing relationships between abundance data and species or other environmental trait matrices . The anaC. xanthogramma and Ph. cespitum), given both species were collected in large numbers. Within both species, the individuals were grouped into two life-stages, juveniles and adults . In adults, the data of different sexes were pooled for both species, because there were no sex-specific differences in the investigated morphological traits . The following model structure was run in both species, separately: the log-transformed thorax-prosoma ratio was the response variable, while the Life-stage , Prey taxa and Season  were the predictor variables (as factors). For testing the post-hoc contrasts, Welch’s t-test with the Holm’s correction (to avoid the errors of multiple comparisons) was used.To analyze the spider-prey size relationship, GLMs with gamma error structure and log-link (GLM-g) were used due to the Gamma distribution of the prey thorax widths . As a neC. xanthogramma and Ph. cespitum, the variances (S2) in prey–predator size ratios were computed ) the key pest of apple in Europe. Two salticid individuals (one each of C. xanthogramma and Heliophanus sp.) were observed to feed on lacewing eggs. For the monthly abundance count of spider and prey groups see F1, 647 = 235.74, P < 0.001, R2 = 0.23), with the average prey thorax width and spider prosoma width being 1.13 and 1.72 mm, respectively, while the average prey–predator size ratio was 0.67 (SD: 0.34)  see .r = 0.605, P = 0.004), which showed that our hand-collected sample represented well the total hunting spider assemblage in the canopy. However, the Mantel test showed no correlation between the seasonal composition of actual (held in the chelicerae) and potential (suction-sampled) prey groups  , demonstrating that hunting spiders, as a community, showed selectivity in their diet. Brachycera and Nematocera were captured significantly more , while Coleoptera was captured significantly less  frequently than their abundance would suggest , but not among years .In the apple orchard located in Újfehértó in 2016 and 2017 the seasonal composition of hand-collected spiders (with prey) and suction-sampled spiders correlated , as compared with the relative abundance of potential prey  without difference between years .Considering the economic status of prey species, the proportions of the categories in the actual prey differed significantly from the potential prey  interaction: LRTial prey . Naturalial prey . The dieial prey . Based oial prey . The pror = 0.515, P = 0.003) with those from other sites or years  was the highest in spring and the lowest in summer (d′) was low  and their trophic niche breadth (B) was wide. Xysticus spp., followed by Ph. cespitum, was the most specialized (most stenophagous) group and, in accordance with this, had the narrowest niche breadth  , except for arisons) . The higalticids . Spider 12, 5 = 148.95, P = 0.01) and Season  significantly contributed to the prey selection by spiders. Furthermore, the interaction between Spider groups and Season also explained a significant amount of variance in prey abundance . Coefficients for the significant predictors of prey abundance are depicted in C. xanthogramma was positively associated (PA) with the prey groups Formicidae  and Coleoptera and negatively associated (NA) with Nematocera, Lepidoptera and Heteroptera. Similar selectivity was observed in other spider groups as well: Other salticids , Ph. cespitum , E. tricuspidata , Xysticus spp. , Clubiona spp.   . The coeeoptera) .Ph. cespitum vs. Xysticus spp. and E. tricuspidata vs. Xysticus spp. ) and high interguild similarities  were found in the composition (taxonomic or taxonomic + size) of natural prey (Ph. cespitum vs. E. tricuspidata vs. Xysticus spp.), Ph. cespitum vs. Clubiona spp., C. xanthogramma vs. Ph. cespitum, C. xanthogramma vs. Clubiona spp. and E. tricuspidata vs. Xysticus spp. , seasons , and also between prey taxa . On average, C. xanthogramma and Xysticus spp. had the widest, while Clubiona spp. had the narrowest prosoma , seasons  and between prey taxa . Xysticus spp. had prey with the widest, while Clubiona spp. had prey with the narrowest thorax , among the seasons  and among the different prey groups . Compared to their own size, Ph. cespitum and C. xanthogramma caught the smallest whereas Xysticus and Clubiona spp. caught the relatively largest prey items and the difference between the first two species and Clubiona spp. was significant  and decreased in fall  , with Brachycera being the largest prey caught by a same-sized spider, indicating that the taxonomic identity of the prey influenced the prey–predator ratio  ; Fig. S5= 0.020) . Howeveror ratio .C. xanthogramma and Ph. cespitum were collected in numbers high enough to analyze their prey in more detail. Comparing the prey of the spider life stages, the following results were obtained: C. xanthogramma adults had the widest trophic niche breadth (B = 6.76), followed by C. xanthogramma juveniles (B = 5.85), Ph. cespitum adults (B = 4.32) and Ph. cespitum juveniles (B = 2.51)  and across niche space incorporating these two functional traits , indicating an ontogenetic niche shift  . Conside = 2.51) , these fhogramma . When C.he shift . Taking C. xanthogramma: F1, 260 = 2.814, P = 0.095; Ph. cespitum: F1, 91 = 1.288, P = 0.259; C. xanthogramma: F10, 260 = 24.133, P < 0.001; Ph. cespitum: F8, 91 = 6.338, P < 0.001). The three most abundant prey groups  differed from each other: (1) C. xanthogramma: Brachycera vs. Nematocera (P < 0.001), Brachycera vs. Sternorrhyncha (P < 0.001), Nematocera vs. Sternorrhyncha (P = 0.001) and (2) Ph. cespitum: Brachycera vs. Nematocera (P < 0.001), Brachycera vs. Sternorrhyncha (P < 0.001), Nematocera vs. Sternorrhyncha (P = 0.019) in relation to thorax-prosoma ratio, that is, a spider of a given size mostly caught larger Brachycera prey items than those of Nematocera or Sternorrhyncha  but not for Ph. cespitum .Although prey size was significantly related to spider size in both species , differerrhyncha . Among sC. xanthogramma adults had numerically lower size variance, while Ph. cespitum adults had significantly greater variance (S2) in their size (prosoma width) compared to that in juveniles . Adults of both species had significantly greater variance in the size of their prey (thorax width) than juveniles . However, the variance of thorax-prosoma ratio (niche width with respect to prey size) was not different between the life stages . For detailed data see We found that Based on 878 predator-prey records, we analyzed the prey composition, biological control potential, and trophic interactions of arboreal hunting spiders in apple orchards. Although they were found to be polyphagous predators in general, hunting spiders selectively preyed upon canopy arthropods and the different spider species/groups differed from each other either in their diet composition or their prey size selection.Dysaphis plantaginea (Passerini) and Aphis pomi De Geer). Although aphids are regarded as a low-quality food (Lasius niger (L.)) made up almost 8% of the prey of hunting spiders and comprised of both ant workers and winged males/queens. The fifth most frequent prey taxon was Araneae , which comprised 6.5% of the total prey , Metcalfa pruinosa (Say) (Auchenorrhyncha: Flatidae), and psyllids, (M. pruinosa). The rest of the prey (15% of the diet) was made up of natural enemies such as spiders, zoophagous bugs, parasitic wasps, hoverflies and lacewings (Hunting spiders preyed mostly (54% of the diet) on arthropods irrelevant to pest management in apple orchards in Central Europe such as Brachycera (excluding hoverflies), Nematocera and Formicidae . They alsyllids,  althoughacewings .The hunting spider assemblage showed the highest selectivity (positive preference) for neutral prey species, and spiders preyed on pests less than would be expected based on their availability in the canopy of apple trees –4. NaturWe found that different spider species exerted different levels of predation on a given prey taxon . AlthougPh. cespitum and Xysticus spp. showed the highest level of stenophagy and in accordance with that the narrowest niche breadth  than by the other spiders (60–88%)  . In conte pepper . Based oPh. cespitum but preferred by C. xanthogramma and Xysticus spp. Similar to our results, Philodromus species they studied rejected these two types of prey. The formicid prey of C. xanthogramma consisted almost exclusively of winged males as salticids usually refuse dangerous ant workers as prey , compared to the number of natural enemies it consumed  exerted considerable predation pressure on aphids, especially in autumn  or particular pest-consuming functional groups might be more effective in biological control rather than enhancing predator biodiversity, as the effect of increased diversity is highly context-dependent  when choosing prey . In a web-builder spider, Argiope amoena L. Koch, Ontogenetic niche shifts are common in the animal kingdom . Such shuveniles . Howevere adults  and 11. e stages  and 11. e stages . Howeveruveniles . Bartos lckenaer . In connPh. cespitum and Clubiona spp. were found to be the most effective natural enemies because of their high level of aphid (Ph. cespitum and Clubiona spp.) and Lepidoptera (Clubiona spp.) consumption and low level of intraguild predation. (8) The trophic niche width of C. xanthogramma and Ph. cespitum increased during ontogeny where adults prey upon a wider taxonomic and size range of arthropods than juveniles. Ph. cespitum exhibited an ontogenetic shift in prey type, whereas no such pattern was observed for C. xanthogramma.By analyzing a total of 878 hunting spider prey items collected from the canopy of apple trees in apple orchards in Hungary we concluded that (1) although highly polyphagous, arboreal hunting spiders forage selectively and therefore cannot be considered as entirely opportunistic predators. We found that more Brachycera, Nematocera (and possibly Other (non-formicid) Hymenoptera, Lepidoptera and Sternorrhyncha) and less Coleoptera (and possibly Auchenorrhyncha) were consumed by the hunting spider assemblage than would be expected from their abundance in the canopy of apple trees. (2) Hunting spider assemblages consume a large number of pests. However, this beneficial effect is strongly constrained by the high predation levels on natural enemies (intraguild predation) and on neutral insects . In this study, the hunting spider assemblage showed positive selection for neutral prey, neutral selection for natural enemies and negative selection for pests. (3) In trophic webs, different hunting spider taxa/groups mediate different strengths of trophic effects on different prey taxa, and the web structure changes considerably with the season. (4) The natural prey of hunting spider species is highly overlapped, showing functional redundancy in their predation. (5) Nevertheless, hunting spider species show different trophic niche occupancy, also exhibit a certain level of stenophagy (species-specific prey preference) and select prey by its taxonomic identity and size. (6) The guilds do not determine the preferred or rejected prey types consistently, thus the diet of hunting spiders classified into the same guild can be considerably different. (7) From an economic point of view, 10.7717/peerj.9334/supp-1Supplemental Information 1For detailed information see the Info sheet of the file.Click here for additional data file.10.7717/peerj.9334/supp-2Supplemental Information 2For detailed information see the Info sheet of the file.Click here for additional data file.10.7717/peerj.9334/supp-3Supplemental Information 3Click here for additional data file.10.7717/peerj.9334/supp-4Supplemental Information 4Click here for additional data file.


Phalaris genera that distribute almost all over the temperate regions of the world. Among them, reed canary grass  and hardinggrass  have been long cultivated as forage grass and have received attention as bio-energy materials in recent years. We aimed to facilitate inter-species/ploidies comparisons, and to illuminate the degree of sequence variation within existing gene pools, chloroplast (cp) genomes of three Phalaris cytotypes  were sequenced and assembled. The result indicated that certain sequence variations existed between the cp genomes of P. arundinacea and P. aquatica. Several hotspot regions  were found, and variable genes  were detected. SNPs (single nucleotide polymorphisms) and/or indels (insertions and deletions) were confirmed by the high Ka/Ks and Pi value. Furthermore, distribution and presence of cp simple sequence repeats (cpSSRs) were identified in the three Phalaris cp genomes, although little difference was found between hexaploid and tetraploid P. arundinacea, and no rearrangement was detected among the three Phalaris cp genomes. The evolutionary relationship and divergent time among these species were discussed. The RNA-seq revealed several differentially expressed genes (DEGs), among which psaA, psaB, and psbB related to leaf color were further verified by leaf color differences.There are 22 species in the  Phalaris species, it is necessary to know the information coded in their nuclear genome and the accompanying organelle genomic (mitochondria and/or chloroplast) sequence [To fully understand the evolutionary paths of sequence . Given tsequence . Mutatiosequence . Especiasequence ,2,3. A tsequence ,4. Recensequence ,6.Phalaris L. (Poaceae) wildly distributes in temperate regions of both hemispheres and in the mountains of tropical Africa and South America. At least 22 Phalaris species have been identified [Phalaris aquatica and P. arundinacea both originated in the Mediterranean region and are closely allied with each other in the lineage with x = 7 basic chromosomes. The P. arundinacea (6x) was reported genetically closer to P. aquatica (4x) than to P. arundinacea (4x) because of a higher hybridization rate between P. arundinacea (6x) and P. aquatica (4x) [P. arundinacea (6x) was the outcome of the hybridization of P. aquatica (4x) and P. arundinacea (4x). However, more genetic and fossil evidence is needed to support this hypothesis [entified ,8,9,10. pothesis .Phalaris species are not only very important for grazing, but also receive close attention as a biomass and bio-energy crop which showed great yield potential in consideration of both dry matter and crude protein via seed dispersal or extensive rhizome systems [Phalaris species possess adaptation to cold, drought, and flooding, as well as all kinds of soils; therefore, they are mainly planted on marginal land that is unsuitable for other agricultural uses [Phalaris have evident variation in morphological traits, plus the vital role of polyploidy in their evolution , their potential value in terms of speciation, chromosomal evolution, and biogeography in Poaceae were manifested [P. arundinacea is related to the moisture stress and nitrogen application, as well as clipping and light intensity, which react as a response to the external environmental changes and work as stimulating or regulating factors in plant growth, metabolism, and reproduction [P. arundinacea was reported to be accumulated mainly in the leaf blades with a percentage of more than 59% of the concentration [Phalaris or resulted from the interaction of nuclear and plastids genes. In this case, cp genome modification of Phalaris is feasible because engineering the cp genome for biotechnology applications was reported to succeed in many other species [ systems ,12. Moreral uses ,10. Addinifested . The pronifested . Accordioduction ,14,15. Mntration . A previntration . Hence, Identification of genomic variation provides an opportunity for the uncovering of correlation between genotype and phenotypes . Given tPhalaris species were sequenced and compared. We revealed sequence synteny and rearrangements, gene loss/pseudogenization, and IR expansions and contractions among these species. Divergence times of the sequenced Phalaris species from other complete sequenced Pooideae grass were estimated. Our analyses of the three Phalaris cp genomes (P. arundinacea (tetraploid and hexaploid) and P. aquatica (tetraploid)) provide detailed genetic information distinguishing different Phalaris ploidies and facilitate breeding programs and species identification. In addition, RNA-seq was carried out to detect the differentially expressed genes (DEGs) and to explore their corresponding functions.In this study, cp genomes of two P. arundinacea (4x), P. arundinacea (6x), and P. aquatica (4x) were 135,898 base pairs (bp), 135,910 bp, and 136,249 bp, respectively  and P. arundinacea (6x) both had 28 tRNAs, 4 rRNAs, and 72 mRNA genes  and 44 (P. aquatica) unique photosynthesis-related genes, and five other genes: matK, clpP, cemA, ycf1, and ycf2   .Phalaris cp genomes using mafft software [P. aquatica (4x) vs. P. arundinacea (6x), and P. aquatica (4x) vs. P. arundinacea (4x), among which 6 indels were observed in the coding sequences. There were 14 indels between P. arundinacea (6x) and P. arundinacea (4x) existing in the noncoding sequence (P. aquatica vs. P. arundinacea (6x)  and P. aquatica vs. P. arundinacea (4x) . It is worth noting that both Tv and Tn were dominantly located in the intergenic region, and more Tvs were found than Tns in both the genic and intergenic regions  and SNPs  and Tn (transition)) were identified among the three software  , there were no SNPs/indels in the genic region , indicating high divergence.SNPs and indels were also counted in the quadripartite structure  and P. arundinacea (4x). In addition, the identified non-synonymous substitutions between P. aquatica and P. arundinacea were involved in 14 (6x) and 15 (4x) genes  and synonymous (Ks) rates and the Ka/Ks ratio . The resx) genes .Phalaris cp genomes were compared with each other. As shown in ndhF-ndhH and rpl22-rps19-psbA were located at the junctions of IR and single copy regions. The ndhH gene spanned the SSC/IRA region with 181 bp located at the IRB region. Spacers ranged from 36 (P. arundinacea) to 38 bp (P. aquatica), separating the rps19 gene from the LSC/IRA border and from the LSC/IRB border. In addition, a 94 bp spacer separated the ndhF gene from the SSC/IRB border in all three Phalaris species.The shrinkage and expansion of IR regions in the three Phalaris cp genomes was evaluated by the calculation of nucleotide diversity (Pi) of 107 common genes unique in every quadripartite region using DnaSP v5.0 software [Phalaris cp genomes was 0.01628, showing a high level of intergeneric differentiation. The SSC region exhibited the highest nucleotide diversity in view of its average Pi value (PiSSC = 0.04606), while the IR regions had the lowest level of Pi (PiIR = 0.00012), suggesting a much higher conservation of the IR region  were located in the LSC region.Genetic distance among software . The meaR region . The comP. aquatica, P. arundinacea (6x), and P. arundinacea (4x), respectively. Most SSRs were found to be at the LSC region within its intergenic areas, while there were no SSRs found in intron and intergenic portion within SSC region. We found that the percentage of SSRs was higher at the LSC or SSC region, but lower at the IR regions in P. aquatica than those of the others  in total in the cp genomes of e others A. Among ploidies B.P. aquatica), 185 (P. arundinacea/6x), and 179 (P. arundinacea/4x) pairs of repeats (15 bp or longer) were detected using the program REPuter [Phalaris cp genomes  cp genomes , 2098 (10.872%), and 2125 (10.862%) in P. arundinacea (4x), P. arundinacea (6x), and P. aquatica, respectively. The least abundant amino acid was cysteine, which possessed 211 (1.079%), 208 (1.078%), and 208 (1.078%) codons in each genome, respectively  was UGG, which encoded tryptophan (The codon usage frequency and relative synonymous codon usage (RSCU) were thought to be a combination of natural selection, mutation, and genetic drift. Here, the RSCU values were calculated based on 72 protein-coding genes of the three ectively . Codon uyptophan .Phalaris species along with other 12 Poaceae species were estimated based on their cp genomes. Obviously, the phylogeny of these species was in consistence with the classical botanical classification , and P. arundinacea (4x) clustered together in the Phalaris clade. The divergence time between P. aquatica and P. arundinacea was about 3.3055 million years (Mya), and P. arundinacea (6x) and P. arundinacea (4x) were separated from each other about 0.1446 Mya ago.Evolutionary divergence of the two fication . As expepsbM gene showed a much higher level of expression in all cp genomes, followed by psaC, psbD, and psbA (P. arundinacea (6x) vs. P. arundinacea (4x) and P. arundinacea (6x) vs. P. aquatica were tRNA-/rRNA- genes, especially trnfM-CAU with a high FC value. In addition, three genes of psaA, psaB, and psbB, which were annotated with a gene ontology (GO) molecular function of chlorophyll binding and photosynthesis, were found up-regulated in P. aquatica, compared to P. arundinacea (6x) and P. arundinacea (4x) ; especially the and psbA . The intcea (4x) . This reaquatica B. The rendinacea A.Phalaris cp genome applying RNA-seq dataset, among which RNA editing (RE) was dominant. With a cut-off value of at least 5% edited reads, 407, 76, and 83 RE sites were identified in total in the cp genomes of P. aquatica, P. arundinacea (4x), and P. arundinacea (6x), respectively, among which 214 (52.58%), 54 (71.05%), and 56 (67.47%) were located in the genic region  showed high Pi value would have the potential to be good markers for DNA barcoding and phylogenetic analyses, and to expand the cytoplasmic gene pool related to yield enhancement, seed retention, alkaloid reduction, and so on. It is remarkable that the infA gene was found in all the three Phalaris cp genomes, as it is commonly lost in angiosperm groups due to its hydrophilicity [Phalaris or Poaceae species [Phalaris cp genomes, while the extension of IRs/SC boundaries neighboring ndhH and rps19 in the IR regions was observed. Although expansions and contractions of the IRs/SC junctions in angiosperm cp genomes are well investigated, the ones in the Pooideae are unique and still useful [Several protein-coding genes  or P. arundinacea (4x) were roughly similar to each other, while those between two P. arundinacea cp genomes showed a different pattern. No SNPs or indels were found in the genic or IRs regions between the two P. arundinacea cp genomes, which indicated that IR regions were the most conservative quadripartite structure in the cp genome, and the detailed reason is still unknown [The Ka/Ks value is an important indication of natural selection and molecular sequence evolution, which is a result of both frameshift and missense mutations . Compari unknown . Phalaris species. The RSCU is thought to be influenced by a combination of natural selection, mutation, and genetic drift [Phalaris cp genomes, and our result indicated that the most and least abundant amino acids were leucine and cysteine, respectively, which is consistent with the previous study in the angiosperm cp genomes [One of the most valuable tools in population genetics is cpSSRs marker, which has unique characters such as, non-recombination, haploidy, and so on . Severalic drift . In this genomes . Besides genomes .Phalaris cp genomes and other Pooideae species, and the phylogenetic clades in Bayesian tree were in consistence with the classical botanical taxonomy. The divergence of P. aquatica and P. arundinacea was estimated to be approximately 3.3055 million years (Mya) ago with the arrival of quaternary glacial period, which may partly explain why the tetraploid P. arundinacea is more cold-resistant [P. arundinacea to tetraploidy P. arundinacea than to tetraploidy P. aquatica, though a closer kinship on the production traits between P. arundinacea (6x) and P. aquatica was reported [We conducted a phylogenetic analysis based on the three whole esistant ,35. Furtreported , which wPhalaris cp genomes [Phalaris species may be inherited from their ancestors or caused by the nonconservative replication of enzymes. In view of the cp genes expression pattern revealed by RNA-seq, the down-regulated DEGs were almost related to replication and transcription function in P. aquatica compared to P. arundinacea (4x) and P. arundinacea (6x), while the up-regulated DEGs were mostly enriched in photosynthesis, energy metabolism, and chlorophyll synthesis. In addition, the RE pattern of P. arundinacea (6x) and P. arundinacea (4x) were more alike, as the cp DNA of P. arundinacea (6x) may be inherited from P. arundinacea (4x) in maternal inheritance way [Phalaris cultivars was observed different, and the related genes psaA, psaB, and psbB are worth to be studied with regard to their significantly increased expression in P. aquatica.RE sites were considered as mismatches between RNA-seq reads and the cp genome sequence, that would account for the majority of within-genome polymorphism and indicated multi-haplotypes in each of the three sequenced  genomes . The RE  genomes . Given tance way . The leaPhalaris cultivars, Phalaris arundinacea L. (including tetraploid and hexaploid) and Phalaris aquatica L. (tetraploid), were all NPGS  collections  [Phalaris arundinacea  cp sequence [Phalaris was drawn with the Organellar Genome DRAW 1.3.1  [The three studied  Russia)  and Gapf Russia)  softwaresequence  from NCBsequence . The Gapsequence  pipelinesequence  softwaresequence  softwarePhalaris cp genomes, using the cp genome of NPGS Phalaris arundinacea (NC027481.1) as a reference. Synteny and rearrangement analysis of above-mentioned species were performed based on Mauve [Phalaris cp genomes, and DNAsp 5.0  [The mVISTA   program on Mauve  pipelineon Mauve  online son Mauve  software, Spain)  was usedMISA v1.0 (Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) Gatersleben, Corrensstr. 3, 06466 Seeland, Germany)  softwareBecause of the degeneracy of codons, each amino acid was coded by one to six codons. The utilization rate of genomic codon varies greatly among different species and organisms. This unequal use of synonymous codons is known as Relative Synonymous Codon Usage (RSCU), which was thought to be influenced by natural selection, mutation, and genetic drift. The RSCU analysis was performed using CodonW 1.4.4  software .Phalaris cp genomes, and the Ka/Ks rates of these common coding genes were calculated using the KaKs Calculator v2.0  [When base variation results in changes in amino acids, it is non-synonymous substitution (Ka), or else, synonymous substitution (Ks). Non-synonymous mutations are generally affected by natural selection. The ratio of non-synonymous mutation rate to synonymous mutation rate (Ka/Ks) indicates the selection effect: Ka/Ks ratio > 1, indicated positive selection effect; Ka/Ks ratio < 1, indicated purification selection effect; and Ka/Ks ratio = 1, mean neutral evolution. Here, mafft 7.037 software  was used Arabia) .Phalaris cp genomes sequenced in this study were collected to conduct the phylogenetic analysis, with Bambusa multiplex (NC024668.1) and Panicum virgatum (HQ731441.1) as outgroups [Twelve published Poaceae genomes in NCBI  and threx NC02466.1 and Pautgroups . The MCMutgroups  was perfutgroups .For transcriptome analysis, fresh young plant leaves of each accessions were cut and used to construct cDNA libraries, respectively. Total RNA was extracted using Trizol Reagent and treated with RNase-free DNase I , and then a NanoDrop ND1000 spectrophotometer  along with 1% agarose gel electrophoresis were used for quality examination of the total RNA. The sequencing of RNA samples was performed in Illumina Novaseq PE150 platform by Biomarker Technologies . FPKM values for cp genes of each accession were used for transcript abundance calculation by mapped the RNA-seq reads to their own cp genome.Phalaris accession were chosen in vegetative growth stage which was the same to that of RNA-seq sampling. In each plant, ten leaves from the apex of different tillers were randomly selected to measure at base of leaf blade. Significant differences for the relative chlorophyll average values were determined based on the least significant difference (LSD) test at p ≤ 0.05. The relative chlorophyll (chl) content of the leaves was recorded with a chl content colorimeter CL-01 . The instrument determines the relative chl content on the basis of dual wavelengths of the spectral absorbance at 620 and 940 nm . Three cPhalaris species P. aquatica and P. arundinacea (including tetraploid and hexaploid) were firstly sequenced and annotated in this study, which was beneficial to the evolutionary study of Phalaris genus. Though gene content were highly conserved and no arrangement among those three Phalaris cp genomes, some hotspot regions and variable genes were identified. Furthermore, the divergence time and evolutionary relationship among these three Phalaris cp genomes along with other Gramineae species was discussed. The DEGs obtained by RNA-seq might be one of the explanations for the differences in leaf color among those three accessions.The cp genomes of two


Helicobacter pylori(H. pylori)-uninfected patients and its typical histological feature is signet ring cell carcinoma (SRCC) within the mucosal layer. However, the biological characteristics of SRCC remain unclear. We aimed to clarify the pathological and genetic features of SRCC in H. pylori-uninfected patients.Gastric cancer develops even in H. pylori-uninfected patients with mucosal SRCCs were enrolled and their clinicopathological characteristics were compared with those of H. pylori-infected patients with mucosal SRCCs. Seven SRCCs without H. pylori-infected, including two invasive SRCCs, and seven H. pylori-infected SRCCs were subjected to a genetic analysis using next-generation sequencing.Seventeen H. pylori-uninfected patients with mucosal SRCCs revealed male dominancy and a significantly higher prevalence of smokers among them as compared with the H. pylori-infected patients with SRCC. A CDH1 mutation (frame shift indel) was detected in one H. pylori-uninfected cancer not only in the mucosal SRCC but also in the invasive portion. A TP53 mutation was detected in one SRCC without H. pylori-infected. In the control group, ARID1A and TP53 mutations were detected in one SRCC each. The C to A mutation, which is a characteristic smoking-induced mutation, was not found in any of the samples.H. pylori-uninfected patients may have a malignant potential similar to that of SRCCs in H. pylori-infected patients. Smoking may not be the main carcinogenic factor for the development of SRCCs among the H. pylori-uninfected patients.Some SRCCs in  Helicobacter pylori (H. pylori)-infected is the main cause of gastric cancer development [H. pylori-infected [H. pylori-uninfected patients and its typical histological feature is signet ring cell carcinoma (SRCC) within the mucosal layer [It is well recognized that elopment . In Japainfected , and anoinfected . Howeveral layer . Endoscoal layer .H. pylori-uninfected patients show a lower proliferative activity, few extensive spread, and slower progression, compared with SRCCs with H. pylori-infected patients [H. pylori-uninfected patients resemble those in patients with hereditary diffuse gastric cancer (HDGC), which is caused by a germline mutation of CDH1 [H. pylori-uninfected patients can invade into the submucosal layer. In addition, the pathogenesis of SRCCs in H. pylori-uninfected patients remains uncertain. Recently, Horiuchi et al. reported the implication of smoking in the disease pathogenesis of these patients, noting that their findings should be verified by other studies [Previous reports have suggested that SRCCs in patients . The fea of CDH1 . In pati studies .H. pylori-infected. Moreover, we investigated the genomic characteristics of these lesions to identify their biological characteristics and assess whether smoking may be involved in their pathogenesis.In the present study, we compared the clinical features of SRCCs between patients with and without H. pylori-infected who were diagnosed in Hiroshima University Hospital from 1998 to 2017. All the patients received endoscopic or surgical resection. The entire tumor was cut into parallel 2–4 mm-thick sections for investigation. Histological evaluation was performed according to the Japanese gastric cancer treatment guidelines [H. pylori-uninfected subjects was judged by our criteria as described in a previous publication [We enrolled 19 consecutive patients with SRCC  without H. pylori-infected SRCC diagnosed in the same period were enrolled as controls. We compared the clinical characteristics  between the two groups . Furthermore, we randomly selected seven patients who were habitual smokers among both the H. pylori-uninfected and -infected SRCCs, for next-generation sequencing DNA analysis. This retrospective study was approved by the institutional review board and the ethics committees of Hiroshima University (No. E-1096) and was performed in accordance with the Helsinki Declaration and its later amendments.A total of 34 patients with H. pylori-uninfected SRCCs.In the experiments with next-generation sequencing, we randomly selected seven patients who were habitual smokers among the H. pylori-infected (Case 5 and 6).In addition, two DNA samples  were extracted from two cases with invasive SRCC without We examined 10 diffuse-type gastric cancer associated genes  referring to previous paper [H. pylori-infected. Therefore, in total, nine and seven DNA samples were extracted from the H. pylori-uninfected and -infected patients, respectively.In the control group, seven samples were collected from only mucosal SRCC with Pathologic tumor tissues were dissected from ten 10-μm-thick slides made from formalin-fixed paraffin embedded (FFPE) specimens, which were deparaffinized, stained, and dehydrated using the Arcturus® Paradise® PLUS Reagent System . Dissections were performed using a laser capture microdissection system , in accordance with the pathogenesis diagnosis. The DNA was extracted from these tissues using the GeneRead DNA FFPE kit , and concentrations were determined using a Qubit® 1.0 fluorometer . The quantity and quality of the FFPE-derived DNA samples were checked by calculating the normalized DNA integrity scores (ΔΔCq) via quantitative polymerase chain reaction (PCR) analysis using the Agilent NGS FFPE QC kit .CDH1, TP53, ARID1A, KRAS, PIK3CA, ERBB3, FBXW7, TGFBR1, RHOA, and MAK2K1) were enriched using the SureSelectXT HS Custom panel (Agilent). The resulting pooled libraries were quality control-checked via the High Sensitivity D1000 ScreenTape system using the 2200 TapeStation instrument (Agilent). Sequencing was performed with paired-end reads via the HiSeq 2500 platform .DNA extracted from tumors was fragmented into 150–200 bp portions by sonication using an S2 sonicator  and used for library construction according to the manufacturer’s instructions. In all cases, 10 ng of DNA was prepared for sequencing. The exons of ten oncogenes (https://togovar.biosciencedbc.jp) databases. The remaining mutations in cancerous tissues were considered to be candidate cancer-specific mutations. To reduce the false positive rate, we set the cutoff values for somatic mutation in cancerous tissues as follows: variant score > 0.3; minimum quality for base > 30; variant call quality > 100; allele frequency > 0.1; and number of reads supporting the variant allele > 3 . PCR duplicates were removed and low-frequency mutations in template DNA molecules were detected using the molecular barcode system . To ident test or Wilcoxon rank-sum test to compare continuous data. A p-value of < 0.05 was considered significant. All statistical analyses were performed using JMP® software .All clinicopathological features were analyzed using the Chi-square test or Fisher’s exact test to compare categorical data and Student’s H. pylori-uninfected and -infected patients with SRCCs located within the mucosal layer. As shown in Table H. pylori-uninfected patients with mucosal SRCCs revealed male dominancy and a slightly younger age than the H. pylori-infected patients. The SRCCs in the H. pylori-uninfected patients tended to be located in the lower third of the stomach and its tumor size were significantly smaller than those in the H. pylori-infected patients. Although no difference was detected in blood type, family history of gastric cancer, or alcohol consumption.First, we compared the clinicopathological features between 17 DNA analysis by next-generation sequencing was performed in 16 samples from 14 patients (seven patients from each group) as shown in Table A single gene mutation was detected in each of five samples (Table CDH1 mutation (frame shift indel) was detected only in one H. pylori-uninfected cancer lesion (case 6) . In the H. pylori-infected control group, mutations in ARID1A (case 11) and TP53 (case 12) were detected in SRCCs  which associated somatic genomic alterations associated with the unique characteristics of sporadic diffuse gastric cancers [es Table , Fig. 1. 6) Fig.  and 3. A cancers . . Although, the Brinkman index was not different between the two groups. Moreover, the C to A mutation, which is a characteristic smoking-induced mutation, was not found in any of the 17 H. pylori-uninfected mucosal SRCC.As a result of comparison for smoking status in H. pylori-infected. SRCCs in patients without H. pylori-infected showed different features compared with those in patients with H. pylori-infected. Clinically, the H. pylori-uninfected patients with mucosal SRCCs showed male dominance and the tumor tissue tended to be located in the lower third of the stomach. These observations imply that H. pylori-uninfected SRCCs have distinct biological characteristics and probably have a different carcinogenetic pathway as compared with H. pylori-infected SRCCs.In the present study, we examined the clinical and genetic characteristics of SRCCs in patients without H. pylori-infected. Generally, diffuse-type cancers including SRCC are classified into the “genomically stable group” according to the four-subtype classification [CDH1 have frequently been detected in SRCCs in patients with HDGC [CDH1 gene, regulates signaling pathways leads to increase in cell proliferation, decrease in cell apoptosis followed by gastric cancer development [CDH1 was extremely low in SRCCs without H. pylori- infected. This suggests that the carcinogenic pathway of SRCCs without H. pylori- infected differs from that of SRCCs in patients with HDGC. The SRCC lesions found in H. pylori-uninfected patients seem to reflect not only a defect in cell-cell adhesion but also other neoplastic alterations.Particular focus should be placed on the genetic alterations detected in SRCCs without fication . Under tith HDGC . Moreoveelopment . HoweverTP53, which are known as a representative driver mutation in gastric cancer carcinogenesis, were detected in SRCCs without H. pylori-infected as well as those with H. pylori-infected. This also strongly suggests that SRCCs in H. pylori-uninfected patients may be true neoplastic lesions. Previous reports have demonstrated that SRCC tissue gains an invasive ability after the mutation of TP53 followed by conversion to PDA [H. pylori-uninfected SRCC is a true neoplasm, and clinical features of SRCC without H. pylori-infected may be different from that in HDGC. In the present study, we demonstrated the presence of similar genetic alterations between mucosal SRCC and invasive PDA in a H. pylori-uninfected patient, suggesting the progression of SRCC to invasive PDA. Indeed, we have diagnosed two cases with invasive SRCC without H. pylori-infected (paper under submission). SRCC without H. pylori-infected may have a malignant potential to invade into the submucosal layer and should be treated as a diffuse-type gastric cancer as described in the guidelines for treatment [Mutations in n to PDA . Accordireatment .H. pylori-uninfected patients with SRCCs than among the H. pylori-infected patients. This finding is compatible with the previous report by Horiuchi et al. [H. pylori-infected.Notably, it should be emphasized that the prevalence of smokers was higher among the i et al. . Howeveri et al. , 18. SmoH. pylori-infected [H. pylori-infected is essential for EB virus-induced carcinogenesis [H. pylori-infected remains unclear and should be clarified in future research.In addition to genetic mutations, gastric carcinogenesis may also be influenced by epigenomic alterations including gene methylation. Epstein–Barr virus (EB virus) infection, which induces several epigenetic alterations in host genes, may be another carcinogenetic factor besides infected . Howeverogenesis . The patH. pylori-infected are diagnosed when the lesions measure about 10 mm in diameter; therefore, it is difficult to extract high quality DNA from SRCC tissue. The second limitation is that DNA samples from control lymphocytes were not analyzed. Therefore, we excluded disease-specific germline mutations and Japanese healthy germline mutations when searching the dbSNP 137 and TogoVar databases.This study has several limitations. The first limitation is the limited number of patients. To accurately evaluate the prevalence of any given genetic mutation, a large number of cases should be examined. In addition, we examined only ten genes in our cancer panel and we could not cover their complete exon sequences. In clinical practice, SRCCs without H. pylori should be treated as having a malignant potential similar to that for SRCCs occurring in the presence of H. pylori-infected. Smoking may not be the main carcinogenic factor for the development of SRCCs among the H. pylori-uninfected patients.Our findings indicate that SRCCs without Additional file 1.


TERT) is essential for the maintenance of telomeres. Previously we described the presence of three TERT paralogs in the allotetraploid plant Nicotiana tabacum, while a single TERT copy was identified in the paleopolyploid model plant Arabidopsis thaliana. Here we examine the presence, origin and functional status of TERT variants in allotetraploid Nicotiana species of diverse evolutionary ages and their parental genome donors, as well as in other diploid and polyploid plant species. A combination of experimental and in silico bottom-up analyses of TERT gene copies in Nicotiana polyploids revealed various patterns of retention or loss of parental TERT variants and divergence in their functions. RT–qPCR results confirmed the expression of all the identified TERT variants. In representative plant and green algal genomes, our synteny analyses show that their TERT genes were located in a conserved locus that became advantageous after the divergence of eudicots, and the gene was later translocated in several plant groups. In various diploid and polyploid species, translocation of TERT became fixed in target loci that show ancient synapomorphy.The gene coding for the telomerase reverse transcriptase ( Flowering plants (angiosperms) are important for the existence of many terrestrial organisms, including humans, and a long history of plant breeding has taught us that polyploidization can be advantageous in terms of quantitative traits of crops. Gains and losses of paralogs, their neofunctionalization and sub-functionalization, have all been associated with the generation of duplicate gene copies, e.g., by whole-genome duplications (WGDs) and further rounds of genome duplication/reduction, resulting in genetic diversity upon which the fittest combinations thrived in a competitive environment ,2,3,4. AAllium  and Cestrum  [Nicotiana tabacum possesses three sequence variants of the TERT gene [Nicotiana make the genus an ideal experimental model system to study the long-term evolution of TERT following natural gene duplication. The increasing number of publicly available assembled plant genomes enables the exploration of TERT genomic loci, gene copy numbers and gene synteny in diverse plant species for comparisons with the data from Nicotiana polyploids and the diploid species most closely related to their progenitors . The Nicotiana genus [N. tabacum (section Nicotianae), N. rustica (sect. Rusticae), N. arentsii (sect. Undulatae) that formed approx. 0.4–0.6 million years ago, (ii) N. clevelandii and N. quadrivalvis , (iii) four species from the 4–5 million years old section Repandae , and (iv) ~35 species including the model N. benthamiana from the oldest section Suaveolentes formed about 6 million years ago [N. tabacum, they share an ancient genome donor, N. sylvestris, and these speciation events happened at different times. In N. tabacum, two TERT variants originated from the maternal N. sylvestris genome  and one from the N. tomentosiformis paternal genome (TERT_Ct). Variants TERT_Cs and TERT_Ct code for a full-length functional protein, while the TERT_D variant is truncated and contains several indels resulting in premature stop codons, suggesting that it is a pseudogene [TERT variants [Telomerase reverse transcriptase (TERT) is involved in the maintenance of telomeres, nucleoprotein structures that are essential for genome stability ,12,13. Tlanales) ,16,17,18lanales)  and not lanales) ). Moreovlanales) ,21,22. Ilanales) ,24. Planlanales) ,27,28,29ERT gene . Variousna genus ,33,34,35ears ago . Among teudogene . All thrvariants ,36.TERT variants in N. tabacum, we explored the fate of TERT paralogs in other Nicotiana polyploids to determine whether both parental TERT genes are conserved in allotetraploid genomes, whether they are transcribed, present in syntenic, collinear arrays with their progenitor diploids, and whether any relationship exists between telomere lengths in polyploids and their progenitor diploids. Of particular interest in this study was to clarify the origin of the presumed pseudogene variant TERT_D in N. sylvestris, a diploid genome donor of N. tabacum, as well as of even older species from sections Repandae and Suaveolentes. In addition, we investigated in silico whether diploid and polyploid plants outside of the family Solanaceae sustained TERT paralogs/pseudogenes in their genomes, and we explored syntenic relationships of genes adjacent to TERT to interpret the evolutionary success of TERT copies after translocation.Based on previously described Nicotiana allopolyploids and their parents. To characterize experimentally the number, identity and origin of TERT copies in genomes of polyploid Nicotiana species and representatives of their diploid progenitors, we employed several primer combinations derived from conserved TERT regions of the evolutionarily distant relatives N. sylvestris and N. tomentosiformis  and/or indels evidencing the presence of multiple TERT variants. Primer positions were with respect to Nicotiana TERT gene structure with 13 exons  in N. sylvestris [N. attenuata and N. obtusifolia, species representing parents of polyploid sections Polydicliae and Repandae, we further confirmed our results by in silico analysis using genome assemblies . To complete the set of representative parental species, we assembled available transcriptomic SRA data of N. noctiflora (GenBank accession: SRR2106514) and identified one TERT variant. In conclusion, our results show the presence of more than one TERT variant in diploid N. sylvestris [Nicotiana polyploids.At the beginning of this project, there was limited genomic sequence data available for the majority of siformis , designevariants ,36. Thes13 exons A, which s  and three variants were identified in N. nudicaulis  and (ii) analysis of raw genomic NGS reads [N. benthamiana TERT and representative parental TERT sequences (N. benthamiana TERT sequence (accession number NbS00010427g0116.1) was more similar to N. sylvestris TERT_C variant than to the TERT sequence cloned from N. alata (GenBank accession MG242421) or deduced from N. noctiflora SRA data  [N. sylvestris  and N. obtusifolia , and it comprises four species—N. nudicaulis, sister to and distinct  from the remaining three species—N. repanda, N. nesophila and N. stocktonii [TERT sequences in cloned PCR products from N. repanda, N. nesophila and N. stocktonii and three TERT sequences in N. nudicaulis  differed between N. nudicaulis and three other species. The TERT _O variants from N. repanda, N. nesophila and N. stocktonii shared a striking 102 nt-long in-frame deletion within exon 9  showed a nucleotide transition from G to A that would change the essential residue Asp (D) to Asn (N) within the motif C , all other 2x = 24) ,32,35. Tocktonii . We captdicaulis . Using Tproducts A–C, we cs origin . Moreove primers , we deten exon 9 A,B that n exon 9 . The TERN. tabacum, which originated from the most recent polyploidization event [TERT variants  were characterized in detail experimentally [TERT_D variant that is transcriptionally active and developmentally regulated in N. sylvestris and N. tabacum [N. sylvestris genome assembly (TW136) in silico. BLAST search identified three contigs (TERT_C variant (NW_009540950) and (ii) TERT_D variant (NW_009367114). A comparison of (i) and (ii) TERT variants revealed a TERT_D similarity with TERT_Cs, starting from the repetitive sequence region within intron 7 and ending within exon 12. (iii) In addition, we found a 92 nt region homologous to the 3′ end of exon 12 and also a putative 3′UTR region of TERT within contig NW_009526057 that apparently represents a part of the TERT sequence (TERT_12exD) that is missing in the TERT_D variant within the contig (ii). A BLAST search for the TERT_12D sequence in N. tabacum cv. TN90 genome assembly identified an unplaced genomic scaffold NW_015807891 with a sequence similar to TERT_12exD, thus indicating that N. tabacum retained all TERT-like sequences of N. sylvestris origin. To verify the number of TERT-like copies in silico, we analyzed raw data from SRA archives of N. tabacum, N. tomentosiformis and N. sylvestris genome sequencing projects. Quantification of a number of mapped SRA reads corresponding to respective TERT variants revealed equal occurrences of TERT_Cs:TERT_Ct:TERT_D in N. tabacum, and TERT_C:TERT_D variants in N. sylvestris, and a single TERT gene copy in the N. tomentosiformis  and N. rustica (Rusticae)  [TERT variants in N. arentsii by sequencing of cloned PCR products and assigned them to their parental origins  and N. undulata  in the N. rustica genome (Two independent and relatively recent polyploidization events gave rise to usticae) . N. arendulatae) ,40. Expe origins . Nicotiaa genome .TERT gene were transcribed  and N. obtusifolia-like (TERT_O), were transcribed at a comparable level in N. repanda seedlings. Using TERT-variant-specific primers in N. nudicaulis, RT–qPCR analysis revealed a higher transcript level of TERT_O in comparison with the TERT_Cs variant, while TERT_D transcripts were detectable but heavily under-represented. In N. rustica (Rusticae) seedlings, our RT–qPCR analyses showed comparable transcript levels of both TERT variants, TERT_P of maternal origin and TERT_U of paternal origin.We further focused on the question of whether multiple variants of the  primers  spanninganalysis D revealeN. sylvestris-like TERT_Cs and TERT_D variants in N. tabacum [N. nudicaulis experimentally  showed that the Ogre-SD1_I sequence was associated with both TERT_D and TERT_12exD sequences. RepeatMasker [TERT_12exD and TERT_D originated from the same ancient TERT locus. We presume that either retrotransposition- or transposon-facilitated ectopic recombination shattered the TERT_12exD and TERT_D sequences and transferred TERT_D to a completely new genomic locus. Another possibility is that the change within the ancestral TERT locus was mediated by a 1.3 kb long repeated sequence that is interspersed all over the N. sylvestris genome, including all three TERT contigs   copy is a pseudogene. MtATPO is usually a single-copy gene, and it is often associated with TERT within land plants, including N. tomentosiformis  . A copy ined see .TERT copies within Nicotiana polyploid genomes directed our attention to the question of the genomic arrangement of the TERT locus. TERT-containing contigs in N. sylvestris are short and/or gene-free  of TERT neighboring genes across representative plant genomes  and species representing basal clades—Vitis vinifera , and Nelumbo nucifera , but not Amborella (early diverging angiosperm lineage) or green algae, suggesting that the detected TERT microsynteny may have originated in eudicots subsequent to the divergence of the Amborella lineage. Interestingly, 16 of 18 genes neighboring TERT in S. penellii were almost co-linear within the Vitis TERT locus, and 14 of 18 genes were shared in the Nelumbo TERT locus. Thus, with the exception of the carbohydrate esterase gene that is exclusive to the TERT loci in Solanales, these genes occurred within the TERT locus in species grouped by ancestral eudicot-like microsynteny  in eudicots  and Amborella, but not in monocots or representatives of other basal clades . Moreover, several genomes showed the occurrence of more loci with eudicot-like microsynteny, either complete . For example, the syntelog derived from the TERT locus of Citrus sinensis (Citrus-like) occurred in Amborella, Nelumbo, Vitis, Eucalyptus and Fragaria genomes. Within the Citrus sinensis genome, loci with the eudicot-like (represented by Vitis-like query) and Arabidopsis-like synteny were present. Interestingly, Theobroma cacao  that harbors the species-specific TERT locus contained loci with eudicot-like, Ricinus-like, Eucalyptus-like and Arabidopsis-like synteny, but loci with Theobroma-like synteny did not occur in the other genomes investigated.A reciprocal search was then carried out using syntenic genes neighboring TERT locus and number of gene copies were asked about polyploid model species Glycine max, Gossypium hirsutum, Brassica napus, Camelina sativa, Mimulus luteus and Actinidia chinensis  is a paleotetraploid derived from an ancestor with x = 14 [N. benthamiana speciation, with changes in chromosome number in the section Suaveolentes. One TERT locus and three additional loci with eudicot-like synteny were present in the A. chinensis genome  is a paleopolyploid with a highly duplicated genome, where nearly 75% of the genes are present in multiple copies [TERT gene is no exception, and we identified two loci on chromosomes 15 and 8  that contain functional copies of TERT. The TERT locus on chromosome 15 shares a full eudicot-like synteny, while the arrangement of the TERT locus on chromosome 8 was similar to the eudicot-like synteny only in the region upstream of syntenic TERT  resembles Glycine max with two TERT loci (S and Sd) and two additional loci without a TERT gene  and a truncated TERT-like sequence (LOC18443854) covering 208 amino acids from the N-terminal part of the TERT protein. However, the origin of the truncated TERT-like sequence is unclear  associated with high-copy repetitive sequences, and the MtATPO gene, within a novel genomic locus in the N. sylvestris genome  originated as a result of gene/segment duplication of the TERT_C (plus MtATPO) locus or vice versa  A large-scale segment/genome duplication event had created an additional TERT locus, presumably encoding a TERT pseudogene on chromosome 1 in Populus, (ii) two TERT copies placed on the same scaffold in Mimulus. (iii) A completely different arrangement comprising an additional TERT variant of Vigna radiata that is formed by two adjacent inverted copies of exon 9 of TERT, and this TERT-like sequence was annotated as ncRNA  and adopted the TERT gene later in the ancestral parent of Nelumbo. The original Amborella TERT locus was probably fragmented. Another translocation of the TERT gene into novel loci grouped in all investigated malvids and Malphigiales (in fabids), and further translocations to other loci, took place later on. Interestingly, in several cases, we detected a translocation into loci that had already existed in ancestral genomes for a long time, e.g., the locus with the Citrus-like synteny originated in early eudicots, as assumed from the locus synapomorphy. The first TERT translocation from a locus with eudicot-type synteny was not caused by locus fragmentation because these loci occur in current eudicots , N. obtusifolia, N. nesophila and N. stocktonii were kind gifts from Prof. Marie-Angèle Grandbastien . N. sylvestris (accession A04750326), N. repanda, N. nudicaulis, N. paniculata, N. undulata, N. rustica, N. clevelandii, N. attenuata and N. alata were gifts from Prof. Andrew Leitch . N. sylvestris , N. quadrivalvis, N. wigandoides and N. arentsii were purchased from Imperial Tobacco Bergerac (France). Plants were grown in growth chambers under conditions of 16 h light, 22 °C and 8 h dark, 19 °C, illumination 150 µmol·m−2·s−1. DNA for qPCR experiments was isolated from plant leaves, according to Dellaporta et al. [® RNA kit . RNA was purified by DNaseI treatment , and its integrity was checked by electrophoresis on a 1% (w/v) agarose gel. RNA concentration was measured using a spectrophotometer (Nanodrop).a et al. . Total RTERT sequences in a single PCR reaction (25 µL), we used 200 ng of genomic DNA from various Nicotiana species as a template, KAPA Taq DNA Polymerase  and primer combinations listed in w/v) agarose gel. For sequencing , we purified PCR products using a QUIaquick PCR purification kit . In the case of N. repanda, N. nesophila and N. stocktonii, PCR products were purified from an agarose gel using a QUIaquick gel purification kit). Alternatively, PCR products were cloned into the pCRIITOPO vector using a TOPO TA cloning kit  and sequenced to analyze individual TERT variants. Representative sequences were submitted to GenBank  in a Rotorgene 6000 cycler . For comparative quantification of paralogous TERT transcripts in N. repanda and N. rustica, we optimized qPCR conditions to reach the same efficiency for TERT variant-specific reactions  in a Rotorgene 6000 cycler under the following conditions: 3 min at 95 °C, 45 cycles of 5 s at 95 °C, 20 s at 61 °C, 15 s at 72 °C, final extension 3 min at 72 °C. The proportion of TERT transcripts in allopolyploids were calculated by the delta Ct method [For RT–qPCR, we prepared cDNA from 2 μg of total RNA according to the manufacturer’s protocol using M-MuLV reverse transcriptase  and random nonamers . Quantification of the relative transcription levels of the eactions . Optimizt method  and normTERT gene sequences were constructed in silico using genome assembly data from N. attenuata and N. obtusifolia  and transcriptomic SRA data from N. noctiflora (SRR2106514) and N. clevelandii (SRX3866257).TERT variants in N. benthamiana, we carried out a BLAST (BLASTn) search in genome assembly  as a query. Subsequently, we analyzed raw whole-genome NGS data : two PE libraries comprising 4 files, 2 × 100 nt; 500 bp insert size; 16 Gb compressed file size/each corresponding to ≥30× genome coverage. We built a nucleotide BLAST database on MetaCentrum using BLAST+ command-line applications. TERT-like reads were identified by BLAST and mapped back to the query using Geneious software . As a proof of concept, this approach was tested on publicly available genomic SRA data from N. tabacum, N. tomentosiformis and N. sylvestris . Identification of similarities among TERT contigs from N. sylvestris and repetitive elements was performed using GEvo with/without masking of non-CDS regions and validated manually. To determine the parental origin of TERT variants in polyploids, cloned fragments and/or corresponding sequences reconstructed from genomic/transcriptomic databases were aligned with TERT sequences from their progenitor diploids. Alignments were generated, and pairwise % identities  or BLAST (www.ncbi.nlm.nih.gov) for subsequent syntenic analysis. The syntenic analysis of the TERT loci (TERT genomic region from one species against genomes (CoGe) of other Angiosperms. SynFind identified syntenic regions using a TERT locus query from one genome against any set of genomes. Then TERT genomic regions from species that shared gene synteny were grouped and analyzed using the GEvo tool in CoGe [TERT loci) are listed in TERT neighboring genes, but not the TERT gene in other species, was performed using SynFind . We used representative TERT loci for each synteny type (We selected 49 representative species  across tERT loci  was perf in CoGe . GEvo seeny type  as a que


Moschus moschiferus, is an Endangered species in South Korea due to its decreasing population size caused by illegal hunting and habitat destruction. In this study, the complete mitochondrial genome of M. moschiferus was determined using next-generation sequencing. Total length of its mitogenome is 16,356 bp in length, encoding 13 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes, and one control region. Its AT contents are 62.4%, which are higher than its GC contents (37.7%) . Phylogenetic relationship of genus Moschus showed topologies similar to those reported in previous studies. Sequence comparison between two M. moschiferus from South Korea indicated high sequence variations with 122 nucleotide differences. These results provide useful information necessary for further phylogenetic studies of Moschus species.The Siberian musk deer,  Moschus moschiferus is a species in genus Moschus, small deer-like ruminants have primitive features  to provide basic genetic information about this species.Siberian musk deer Tsendjav . This spTsendjav . Woo . It was deposited at National Institute of Biological Resources at Incheon, South Korea. Total genomic DNA was isolated from a muscle tissue sample using Genome Wizard Kit  according to the manufacture’s instruction. Genomic DNA isolated from the sample was then sequenced using NGS . A tandem repeat was not found in the D-loop region. Overall base compositions for A, C, G, and T in this mitogenome were 34.1, 24.9, 12.8, and 28.3%, respectively, with AT contents of 62.4%. Similar to typical vertebrate mitogenome, all genes in mitogenome of M. moschiferus are distributed in the H-strand except ND6 subunit gene and eight tRNAs that are encoded on the L-strand. The mitogenome of M. moschiferus generated in this study was deposited in GenBank under accession number KT337321.A specimen (IN156) of Moschus, 13 protein-coding genes from seven complete mitogenomes were retrieved from GenBank  in this study was remotely related to other sequences of M. moschiferus (FJ469675 and JN632662). A total of 122 and 102 of nucleotide differences were observed between KT337321 and other two haplotypes of M. moschiferus, FJ469675 and JN632662, respectively. This sequence comparison indicated relatively larger genetic polymorphism within M. moschiferus than that within other Moschus species (32 for M. chrysogaster and 18 for M. anhuiensis). These results will contribute to improve our understanding of phylogenetic relationships of genus Moschus species and subspecies of M. moschiferus.For phylogenetic analyses of genus


E. coli to reduce its tumble bias and hence increase its run duration during swarming is expected to maintain and promote side-by-side alignment and cohesion within the bacterial packs. In this study, we observed a similar low tumble bias in five different bacterial species, both Gram positive and Gram negative, each inhabiting a unique habitat and posing unique problems to our health. The unanimous display of an altered run-tumble bias in swarms of all species examined in this investigation suggests that this behavioral adaptation is crucial for swarming.Bacteria within a swarm move characteristically in packs, displaying an intricate swirling motion in which hundreds of dynamic rafts continuously form and dissociate as the swarm colonizes an increasing expanse of territory. The demonstrated property of  Escherichia coli, wherein the chemotaxis pathway is remodeled to decrease tumble bias (increase run durations), with running speeds increased as well. We show here that the modification of motility parameters during swarming is not unique to E. coli but is shared by a diverse group of bacteria we examined—Proteus mirabilis, Serratia marcescens, Salmonella enterica, Bacillus subtilis, and Pseudomonas aeruginosa—suggesting that increasing run durations and speeds are a cornerstone of swarming.Many bacteria use flagellum-driven motility to swarm or move collectively over a surface terrain. Bacterial adaptations for swarming can include cell elongation, hyperflagellation, recruitment of special stator proteins, and surfactant secretion, among others. We recently demonstrated another swarming adaptation in  Escherichia coli, the mechanics of flagella are similar during both swimming and swarming in that peritrichous flagella driven by bidirectional rotary motors switch between counterclockwise (CCW) and clockwise (CW) directions. However, while CCW rotation promotes formation of a coherent flagellar bundle that propels the cell forward (run) during both swimming and swarming, a transient switch in rotational direction (CW) causes the cell to tumble while swimming but reverse direction while swarming  on hard agar (1.5% and above)   but not lagellum , while tard agar  and B. s numbers , 18, whi numbers . S. enteE. coli , unchanged from that observed in liquid . Preliminary tracking experiments with S. marcescens cells taken from liquid showed large circular trajectories (E. coli and Caulobacter crescentus swimming close to a glass surface (S. marcescens (S. marcescens mutant deficient in serrawettin production abolished the circular motion (B. subtilis makes a surfactant similar to serrawettin (srfA mutant deficient in surfactin synthesis. P. aeruginosa surfactant has a different structure (rhamnolipid)  –25.10.1128/mBio.01189-20.1FIG S1Serratia marcescens and RH1041, a serrawettin-deficient mutant  ~ Treatment + 1|Replicate) with a Gaussian distribution link function. All the statistical analyses were done by sampling of the respective mixed-effect generalized linear models using the RSTAN  were calculated by sampling the posterior probability distributions. Download Table S1, DOCX file, 0.02 MB.Mean posterior probabilities for the median tumble biases and swimming speeds and comparisons of he RSTAN  and BRMShe RSTAN  in R ; this physiological adaptation is different from the adaptation of the chemotaxis pathway through methylation  for a majority of the bacterial species increased significantly between liquid and swarm as follows: P. mirabilis, 9.01 to 13.3; S. enterica, 23.1 to 30.7; B. subtilis, 18.6 to 31; and P. aeruginosa, 21.9 to 41.6 (statistics in E. coli were 21 μm/s in liquid and 25 μm/s in swarmers (As observed for swarmers .E. coli (E. coli, elevation or stabilization of the chemotaxis component CheZ is responsible for the low TB (S. marcescens, surfactants themselves contribute to lowering TB (In summary, keeping swarming conditions the same, we demonstrated that despite different natural habitats and widely different swarming adaptations discovered in the laboratory, the swarmers studied all modify their TB, and a majority modify run speeds during swarming, similar to what was reported for E. coli . This ape low TB . Other cering TB , althougering TB , 27. FutCell culture, swarm setup, tracking experiments, and analysis were largely carried out as described previously .


Corynebacterium diphtheriae, the agent of diphtheria, is a genetically diverse bacterial species. Although antimicrobial resistance has emerged against several drugs including first-line penicillin, the genomic determinants and population dynamics of resistance are largely unknown for this neglected human pathogen.C. diphtheriae. We used 247 strains collected over several decades in multiple world regions, including the 163 clinical isolates collected prospectively from 2008 to 2017 in France mainland and overseas territories.Here, we analyzed the associations of antimicrobial susceptibility phenotypes, diphtheria toxin production, and genomic features in pbp2m is widespread in other Corynebacterium species, and its expression in C. glutamicum demonstrated its effect against several beta-lactams. A novel 73-kb C. diphtheriae multiresistance plasmid was discovered.Phylogenetic analysis revealed multiple deep-branching sublineages, grouped into a Mitis lineage strongly associated with diphtheria toxin production and a largely toxin gene-negative Gravis lineage with few toxin-producing isolates including the 1990s ex-Soviet Union outbreak strain. The distribution of susceptibility phenotypes allowed proposing ecological cutoffs for most of the 19 agents tested, thereby defining acquired antimicrobial resistance. Penicillin resistance was found in 17.2% of prospective isolates. Seventeen (10.4%) prospective isolates were multidrug-resistant , including four isolates resistant to penicillin and macrolides. Homologous recombination was frequent (r/m = 5), and horizontal gene transfer contributed to the emergence of antimicrobial resistance in multiple sublineages. Genome-wide association mapping uncovered genetic factors of resistance, including an accessory penicillin-binding protein (PBP2m) located in diverse genomic contexts. Gene C. diphtheriae in the context of phylogenetic structure, biovar, and diphtheria toxin production and provides a blueprint to analyze re-emerging diphtheria.This work uncovers the dynamics of antimicrobial resistance in Supplementary information accompanies this paper at 10.1186/s13073-020-00805-7. Diphtheria, if untreated, is one of the most severe bacterial infections of humans. It typically affects the upper respiratory tract causing pseudomembrane formation, sometimes leading to suffocation and death. The infection can be complicated by systemic symptoms, caused by the diphtheria toxin. Other forms of disease are skin and invasive infections, including endocarditis , 2.Corynebacterium diphtheriae, a member of the phylum Actinomycetes , and also ermX, dfrA16, dfrA15b, dfrA1, and sul1 genes. We observed a strong correlation between the presence of ARGs and the expected resistance phenotypes , sul1 , and aph (3′)-Ia . In strains FRC0137 and FRC0375, this latter gene was linked to aph (3′)-Ib (strA), aph (6)-Id (strB), and ermX on a Tn5432-like genomic region with an IS1249 insertion sequence [ermX was present either in proximity to gene pbp2m (see below) or in a fragmented insertion-sequence rich accessory region. Gene dfrA16 was associated with sul1 on a reported [C. diphtheriae . This approach led to the detection of 12 ARGs  was strongly associated with penicillin resistance. This gene is described in more detail below.GWAS analysis of accessory genes demonstrated significant associations with phenotypic resistance. Associated genes included those mentioned above for erythromycin, tetracycline, kanamycin, sulfonamide, and trimethoprim  ranging from 0.19 to 1.5 mg/L. Two of these isolates were also resistant to amoxicillin and one was in addition resistant to oxacillin -like (71%) tetracycline efflux gene. To define the genomic context of resistance genes, a complete genome sequence was obtained. The assembly revealed a chromosome of 2,397,465 bp and a circular plasmid of 73,763 bp .Strain FRC0402, a (Z)-like 1% tetracpbp2m gene was located on the large plasmid in a region comprising three other genes, a blaB beta-lactamase family gene, a LysR family regulator gene, and the ermX gene, flanked by two insertion sequences (IS1628) of the IS6 family  with a fragment identified in pLRPD  or epidemiological typing value.Historically, ociation , our phyene spuA . AlthougC. diphtheriae pathogenicity is the diphtheria toxin. Despite early realization that it is encoded on a prophage [tox gene in C. diphtheriae [tox-positive strains mainly belong to the Mitis lineage and to early-diverging branches of the Gravis lineage. The distribution of tox-positive isolates into multiple Mitis sublineages is strongly indicative of independent acquisitions of the toxin gene. Alternately, this pattern might result from initial acquisition of the tox gene, followed by secondary loss in multiple sublineages. The phylogenetic pattern is also consistent with an ancestral presence of the tox-bearing phage in the Gravis lineage, with subsequent loss of the tox gene in the branch leading to the ancestor of most Gravis isolates. Future work should investigate the dynamics of the lysogenic corynephages and molecular determinants of their sublineage distribution. One important open question is the likelihood of tox-negative strains acquiring the tox gene during colonization, infection, or short-term epidemiological timeframes [The most important factor of prophage , few stuhtheriae , 9. Heremeframes .tox gene. This sublineage happens to correspond to the largest outbreak in recent times, which occurred in Newly Independent States of the ex-Soviet Union in the 1990s [tox-positive lineages, which belong to the Mitis lineage. Hence, its antigenic structures or other pathogenicity properties may have diverged from those of more common tox-positive isolates, which might have contributed to its exceptional transmission in the 1990s, in addition to the decline in vaccine coverage [C. diphtheriae immunogens [C. diphtheriae populations, and possible variations of cross-protection as a function of strain diversity, is currently undefined. This work provides a framework onto which future studies can build to address this important question.Remarkably, except for early-diverging sublineages, only one Gravis sublineage was found to carry the he 1990s , 14, 38.coverage , 69. Recmunogens . The impC. diphtheriae strains have been reported on numerous occasions [C. diphtheriae is largely fragmented and suffers from the lack of harmonization. Breakpoints used to define resistance vary according to world region and have changed over time within single countries [Although antimicrobial-resistant ccasions , 32, knoountries . The lacC. diphtheriae. This approach should in the future be extended to MIC values [C. diphtheriae strains, regardless of lineage, biovar, or toxigenic status. This study further suggests that acquired resistance to penicillin, the first-line therapy against diphtheria, is far from being rare, affecting > 15% of C. diphtheriae isolates collected in the last decade in France and its overseas territories. Besides, 10.4% of these isolates were multidrug-resistant. The high prevalence of resistance to penicillin, tetracycline, and trimethoprim/sulfamethoxazole found here is consistent with susceptibility surveys of recent C. diphtheriae isolates in Algeria [C. diphtheriae.We aimed to define biologically meaningful cutoffs  based onC values  and shou Algeria , Indones Algeria , 72, and Algeria . Many hi Algeria , as thisC. diphtheriae [Corynebacterium species [C. diphtheriae. Here, we defined the prevalence and phylogenetic distribution of previously reported and newly identified resistance determinants in C. diphtheriae. We demonstrate the co-occurrence of resistance phenotypes and genes, suggesting a causative link in multiple instances. We further show that resistance genes have been acquired independently in multiple sublineages, demonstrating a dynamic resistome in C. diphtheriae. In addition, we demonstrate an association between alterations in chromosomally encoded targets and phenotypic resistance for quinolone, trimethoprim, and rifampicin. Fluoroquinolone resistance was previously linked to mutations in the gyrA gene in C. amycolatum [C. striatum [C. belfantii [C. diphtheriae. Finally, we demonstrate the co-occurrence within some strains of multiple resistance determinants and uncover a previously undescribed large resistance plasmid in C. diphtheriae. The mechanism of genetic transfer of this plasmid remains to be investigated. This work provides the first overview of the C. diphtheriae resistome and will facilitate further studies into the evolutionary emergence of multiresistant C. diphtheriae strains.Knowledge of the genetic mechanisms of antimicrobial resistance is critical for defining appropriate treatments, refining diagnostics, and conducting epidemiological studies of antimicrobial resistance. Resistance genes to several antimicrobial classes have been described in htheriae , 37, 75, species  might alycolatum , C. stristriatum , and C. elfantii  but seemCorynebacterium species, clearly demonstrates its horizontal transfer, and we revealed a multiplicity of genomic contexts in which it is found within Corynebacterium. PBP2m is a putative low-affinity PBP, which would explain why it is less affected by beta-lactam antibiotics. Very recently, Forde et al. [C. diphtheriae strain BQ11 and have attributed this phenotype to a penicillin-binding protein located on transposon Tn3503. We found that the pbp2m-containing unit is 99.92% similar to Tn3503, which additionally harbors a helicase  that was hypothesized to confer high-level penicillin resistance [pbp2m in its variable genetic backgrounds are required to understand the observed difference in penicillin resistance levels. The location of pbp2m on a mobile element and its association with beta-lactam and carbapenem resistance represents a public health threat.Here, we discovered an accessory PBP (PBP2m) and experimentally showed it to confer resistance to penicillin and other beta-lactam antimicrobial agents. Its distribution in multiple sublineages, and its presence in other e et al.  have repsistance . FurtherC. diphtheriae (including the newly annotated PBP4b) were investigated to identify amino acid sequence polymorphisms associated with penicillin resistance. Although several alterations were significantly associated, none was directly implicated with the catalytic residues of the transpeptidase or transglycosylase domains. The association with resistance may not be directly linked to these catalytic residues but could be due to secondary sites that are thought to interfere with beta-lactam ligand binding. While biochemical and structural studies are necessary to understand how these mutations affect penicillin susceptibility, we postulate that some changes in these domains could lead to allosteric effects ultimately resulting in beta-lactam resistance, as described for Staphylococcus aureus PBP2a [Streptococcus pneumoniae PBP2x [The seven chromosomal PBPs of us PBP2a , 82 or Sae PBP2x . Other Sae PBP2x .C. diphtheriae is highly diverse and comprises multiple sublineages that are grouped into two major lineages, characterized by distinctive associations with biovars Mitis and Gravis and with the diphtheria toxin gene. Antimicrobial resistance remains rare in France and its overseas territories, but multidrug-resistant strains were found and have a wide phylogenetic distribution. Analyses of the genetic bases of antimicrobial resistance defined the contribution of previously described mechanisms of resistance and uncovered a penicillin-binding protein, PBP2m, associated at the population level with penicillin resistance and shown experimentally to confer resistance to several beta-lactam antimicrobial agents. PBP2m is encoded in diverse genetic contexts, including on a large putatively conjugative resistance plasmid. This study reveals how biovars, diphtheria toxin gene, and antimicrobial resistance are associated with the phylogenetic diversity of C. diphtheriae strains, and provides novel data into the evolutionary dynamics of these medically important features. This framework will facilitate future studies investigating the emergence of sublineages of C. diphtheriae involved in diphtheria outbreaks, antimicrobial resistant infections, and the patterns and drivers of global spread of C. diphtheriae sublineages.Our phylogenetic analyses showed that Additional file 1:Table S1. Strains used in this study and their characteristics.Additional file 2:Table S2. Antimicrobial agents and zone diameters interpretative breakpoints used.Additional file 3:Table S3. Susceptibility data and interpretative categories based on current EUCAST recommendations (SIR) and proposed ecological cutoffs (ECOFF).Additional file 4. Contains Supplementary Figures S1 to S14.Additional file 5:Table S4. Molecular explanations for non-toxigenic tox gene-bearing (NTTB) strains.Additional file 6:Table S5. Associations with resistance phenotypes based on treeWAS results for accessory genes, SNPs in core genes and whole-genome SNPs; or based on Kleborate or direct analysis of potential target sequences.Additional file 7:Table S6. Susceptibility phenotypes of transformed C. glutamicum ATCC 13032 strains.Additional file 8:Table S7. Detection of the pbp2m gene in other Corynebacterium genomes.