Campylobacter spp.; however, little is known about the genotypes and their role in human infections. We studied intestinal content from small wild mammals collected in their natural habitats in Finland in 2010–2017, and in close proximity to 40 pig or cattle farms in 2017. The animals were trapped using traditional Finnish metal snap traps. Campylobacter spp. were isolated from the intestinal content using direct plating on mCCDA. A total of 19% of the captured wild animals (n = 577) and 41% of the pooled farm samples (n = 227) were positive for C. jejuni, which was the only Campylobacter species identified. The highest prevalence occurred in yellow-necked mice (Apodemus flavicollis) and bank voles (Myodes glareolus) which carried Campylobacter spp. in 66.3 and 63.9% of the farm samples and 41.5 and 24.4% of individual animals trapped from natural habitats, respectively. Interestingly, all house mouse (Mus musculus) and shrew (Sorex spp.) samples were negative for Campylobacter spp. C. jejuni isolates (n = 145) were further characterized by whole-genome sequencing. Core genome multilocus sequence typing (cgMLST) clustering showed that mouse and vole strains were separated from the rest of the C. jejuni population . Very little or no alleles were shared with C. jejuni genomes described earlier from livestock or human isolates. FastANI results further indicated that C. jejuni strains from voles are likely to represent a new previously undescribed species or subspecies of Campylobacter. Core-genome phylogeny showed that there was no difference between isolates originating from the farm and wild captured animals. Instead, the phylogeny followed the host species-association. There was some evidence (one strain each) of livestock-associated C. jejuni occurring in a farm-caught A. flavicollis and a brown rat (Rattus norvegicus), indicating that although small mammals may not be the original reservoir of Campylobacter colonizing livestock, they may sporadically carry C. jejuni strains occurring mainly in livestock and be associated with disease in humans.Small mammals are known to carry  Campylobacter spp., especially C. jejuni and C. coli, are common causes of gastroenteritis in humans globally, campylobacteriosis being the most frequently reported zoonosis in the EU area , yellow-necked mouse (Apodemus flavicollis), house mouse (Mus musculus), and brown rat (Rattus norvegicus) have been shown to carry Campylobacter species in their intestinal tracts  R. norvegicus from organic swine farms campylobacter-positive, but no shared genotypes with those from pig manure were detected with AFLP typing. No other rodent species or shrews were found to carry campylobacters  for the presence of Campylobacter spp. both in woodland habitats and on cattle farms in the United Kingdom. They found 41 and 18% of M. glareolus from the woodlands and on farms to carry Campylobacter spp., respectively, while only 1% of the A. sylvaticus, and only on the farms, were positive. Furthermore, all isolates from M. glareolus represented a novel C. jejuni clone, multilocus sequence type (ST) 3704, which was also identified from a calf. This clone was also detected in three of the six positive A. sylvaticus while the other positive A. sylvaticus harbored isolates with ST 61 and ST 583, which were also identified from cattle.Little research exists on the prevalence and genotypes of obacters . In contobacters . In Swedpothesis . WilliamCampylobacter isolates from various sources, more powerful typing methods based on whole-genome sequencing (WGS) give us considerably higher discriminatory power for phylogenetic analyses and epidemiologic and source attribution studies , based on seven house-keeping genes, has been widely used for the typing of  studies . Also, a species . Only a  mammals . Their rCampylobacter spp. isolates from small mammals, mainly rodents and shrews, caught from 40 Finnish beef, dairy, and swine farms, and from natural habitats throughout Finland, (ii) to compare them to isolates collected from other sources, and (iii) to estimate their role as vectors for Campylobacter spp. for Finnish livestock and humans.Our aims were (i) to explore the occurrence, genotypes, and resistance markers of n = 442) were caught by Natural Resources Institute Finland. The trapping was done over two consecutive nights at every farm and the traps were checked in each day. These were all included as farm samples in the successive analyses.During October and November 2017, rodent traps were set on 40 farms in western and south-western Finland. Of these, 18 were beef cattle farms, two were dairy farms and 20 were swine farms. Both live and snap traps (traps that instantly kill the rodent) – 100 traps in total – were set at and near production buildings and at the edges of the farmyard areas. The small mammals (n = 128), and in May–June and September–November 2017 (n = 384). The trapping sites were located throughout Finland in forest and field habitats  of A. flavicollis trapped inside office and storage buildings in southern Finland in 2010–2015 was also included for comparison.A sample  with cut-off at 2.0. The colonies identified as C. jejuni with a score lower than 2.0 were further confirmed with microscopy. After identification, the isolates were stored in media containing brain heart infusion broth and 15% glycerol at −80°C.Sample preparation was performed following the ISO-6887/6 (2013): The intestines and stomachs were first cut into small pieces with a sterile scalpel. Then, the subsamples were pooled so that one pool contained subsamples from a maximum of ten individuals of the same species, caught from a single farm on the same night. After pooling, 1:1 (w/w) buffered peptone water was added to the pooled sample. The sample was cultivated following the ISO-10272-1 (2017) standard Detection procedure C. The colonies were confirmed as 2, 10% ± 3% CO2, ≤10% H2, balanced with N2; Anoxomat System, Mart Microbiology, Netherlands or ThermoForma Series 2 Water Jacketed Incubator (HEPA filter) Model 3131, Thermo Fisher Scientific, United States) at 41.5 ± 1.0°C for 48–72 h. One typical colony was confirmed as C. jejuni by the lack of aerobic growth on blood agar at 25°C, microscopy, and using species-specific PCR  and all 102 isolates from the wild animal samples were subjected to WGS . The sequencing of the wild animal isolates was performed by Istituto Zooprofilattico Sperimentale Dell’Abruzzo e Molise, Teramo, Italy under a Memorandum of Intent between the faculty and the institution. Sequencing libraries were prepared with a Nextera XT Library Prep kit  and sequenced on the Illumina NovaSeq6000 or NextSeq platform , with a 2 × 100-bp paired-ended protocol according to the manufacturer’s instructions, aiming at >100X coverage.1. In brief, after calculating if the sample raw data fulfils the expected coverage (min. 15x), INNUca checks read quality using FastQC2, and processes the reads using Trimmomatic  alleles between two groups.To analyze allele segregation according to source, the cgMLST profiles of a total of 4,323 strains for which source information was available were analyzed using proCompare.py  availablC. jejuni genomes, all-against-all pairwise average nucleotide identity (ANI) values for the mouse and vole strains were calculated using FastANI (v1.0) with default parameters (C. jejuni subsp. jejuni NCTC 11168 (GenBank Accession number: NC_002163.1), C. jejuni subsp. jejuni RM1221 (NC_003912.7), C. jejuni subsp. doylei 269.97 (CP000768.1), C. coli 76339 (NC_022132.1), and C. coli OR12 (NZ_CP019977.1).To evaluate the taxonomical position of the newly described rameters . In addiC. jejuni strains selected among the 6,830 genomes, as described above, excluding the samples isolated from humans and those without information on source  .The antimicrobial resistance determinants of the isolates were searched from the quality-controlled assemblies using default settings in ResFinder 3.2 , accesseCampylobacter prevalence between different sampling times and locations were tested using the Chi-square or Fisher’s Exact tests in Microsoft Excel 2016. Statistically significant differences were considered for p-values below 0.05.Statistical significance of the observed differences in A. flavicollis, M. musculus, Micromys minutus (harvest mouse), and R. norvegicus were almost exclusively caught on-farm (year 2017) or inside buildings  (Myodes rutilus), tundra/root voles (Alexandromys oeconomus), and gray-sided voles (Craseomys rufocanus) are limited to northern parts of Finland and accordingly, these species were not caught on farms in south-western or western Finland. M. glareolus, Sorex araneus (common shrew), and Microtus agrestis (field vole) were common among both in-farm caught samples and those collected from natural habitats throughout Finland.lection) . The disA. flavicollis) of the individual animals captured from office and storage buildings were positive for C. jejuni  oeconomus (tundra/root vole), and M. musculus samples were negative for Campylobacter species.A total of 41% (93/227) of the pooled samples from farms, and 19%  compared to forests (18%) (p < 0.0001) in 2017. The samples from fields were comparable in Campylobacter prevalence with those from farms. In natural habitats, M. glareolus was significantly more often Campylobacter-positive in southern Finland (42%) compared to Lapland (19%) in northern Finland (p < 0.0001) in 2017. In Lapland, Campylobacter prevalence in M. glareolus was significantly higher in 2017 (19%) compared to 2015 (7%) (p = 0.0001). For other parts of the country (excluding Lapland), we had samples only from 2017.C. jejuni isolates from mice, rats, or voles matched a previously defined 7-gene MLST profile, most of which were associated with a clonal complex (CC): ST 1304 , ST 2219 , ST 3704 (n = 1), ST 4791 , or ST 8562 (n = 1) (n = 5) were from rodents captured from farms. ST-45 CC was isolated from A. flavicollis from a pig farm (ST 2219) and from R. norvegicus from a cattle farm (ST 4791). ST 1304 was isolated from A. flavicollis from a cattle farm and from two M. glareolus from pig and cattle farms. Two other previously identified STs, ST 3704 and ST 8562, were both isolated from M. glareolus from a forest in central Finland and from northern Finland, respectively.Only seven out of the 147  (n = 1) . For the (n = 1) . All theM. glareolus, M. agrestis and M. rutilus, and ST 9477 only identified in A. flavicollis. ST 9473 was identified among C. jejuni isolates from M. glareolus from both on-farm (pig and cattle farms) and natural habitats in both 2015 and 2017. ST 9477 was identified among C. jejuni isolates from A. flavicollis from both on-farm (pig and cattle farms) and other buildings (office and storage buildings) in southern Finland from 2010 to 2017. In addition, ST 9470 was isolated from M. minutus and ST 9467 from M. agrestis caught both on-farm and from natural habitats. Furthermore, the same STs occurred in C. jejuni isolates from both M. agrestis and M. glareolus.Novel STs that were most prominent in the dataset included ST 9473 from C. jejuni strains isolated from voles and mice with previously undefined ST designation, the cgMLST based on 678 loci yielded a total of 105 unique profiles. This revealed that the C. jejuni strains isolated from these animals form distinct populations that diverge largely from the ones described so far. Changes in the composition of clusters was investigated at all goeBURST thresholds. At the threshold of 671 allelic differences (99.0% of the core loci), the C. jejuni strains from voles formed two separate clusters from the rest of the strains. At 636 allelic differences (93.8% of core genes), the C. jejuni strains from mice separated from the larger cluster , which was split further into two sub-clusters at 531 allelic differences (78.3% of core genes). One contained strains isolated exclusively from A. flavicollis and one was composed of strains isolated exclusively from M. minutus  and 53 (max 141) with voles and other taxa, respectively. Similar results were obtained for strains isolated from M. minutus. Very few alleles were shared between strains isolated from voles and any other taxa, 18 being the maximum number of shared alleles detected. Overall, this analysis confirmed the divergent nature of the C. jejuni population circulating both in mice and voles.Considering the large diversity in the cgMLST profiles observed between mouse and vole strains and the rest of the C. jejuni isolated from voles and other taxa is an indication that these strains might form a different Campylobacter species or subspecies. To verify this hypothesis, we calculated the ANI percentage between mouse and vole strains and reference strains from C. jejuni subspecies and C. coli . The mouse strains had >95% ANI versus C. jejuni subspecies and <87% ANI versus C. coli. On the contrary, the vole strains had on average 90.9% ANI versus C. jejuni subspecies and <84% ANI versus C. coli. Both Campylobacter strains from voles and mice formed cohesive groups with ANI average at 99% and min. 96%. This data confirmed the hypothesis that Campylobacter strains from voles might form a different taxonomic group. The maximum likelihood phylogenetic tree based on the alignment of 864 core genes supports the ANI results . For the mouse lineages, 225 recombination events were detected, 32 on terminal branches and the rest on internal nodes, and the imported sizes ranged between 2 and 6,597 bp (median 712). The posterior mean calculated for the ratio of recombination events compared to mutation (rho/theta) was 0.342058 (posterior variance 1.82762 × 10–05) for the vole and 0.111041 (posterior variance 2.78479 × 10–05) for the mice. This analysis showed that mutation was approximately 3 and 9 times more frequent than recombination in the vole and mouse lineages, respectively.For investigating the presence of phylogeographical or temporal signals and to see whether there were differences between strains isolated from animals captured in the proximity of the farm or not, we reconstructed the genealogy of the mouse and vole specific lineages based on Campylobacter strains did not segregate clearly according to space or time, and no major differences were observed between animals captured within farms and those captured in the wild. However, the smaller cluster of Campylobacter strains from voles, which diverged significantly from the main population (M. rutilus (orange taxon) and few isolates from M. glareolus (yellow taxon). Furthermore, for the Campylobacter isolates from mice, the smaller cluster consisted of strains from M. minutus (orange taxon) and the larger one from A. flavicollis (purple taxon)  . The CamC. jejuni isolates . Only one isolate had a beta-lactamase gene with 100% nucleotide identity to that previously deposited to Genbank. Not surprisingly, this isolate, derived from A. flavicollis trapped on a swine farm, had a previously recognized ST . All isolates that had the same ST carried identical resistance markers. Both the gyrA mutation and beta-lactamase genes were never present in the same isolates, and only one isolate [from R. norvegicus trapped from a cattle farm having ST 4791 (ST-45 CC)] had neither.We found a nucleotide substitution resulting in P104S amino acid change in GyrA in 62.6% (92/147) of the isolates . No otheCampylobacter jejuni occurred in nearly all animal species sampled, even those collected from the less populated northern parts of Finland . The prevalence of Campylobacter was slightly higher among animals trapped on farms compared to those caught in their natural habitats. However, this is likely due to the fact that the farm samples were pooled from 1–10 individual animals compared to the animals from natural habitats which were studied individually. A. flavicollis were almost exclusively (except for one) caught near or inside buildings and mainly in the fall, and showed the highest prevalence  of Campylobacter. Previously, C. jejuni has rarely been isolated from A. flavicollis , from human dwellings during the cold season in rural areas around Uppsala, Sweden (C. jejuni was isolated from 5/18 (28%) and C. upsaliensis from 2/18 (11%) A. flavicollis, respectively, from pig farms, chicken farms, and non-farm locations in Sweden . Another common mammal that is widespread in Europe, M. agrestis, was less often  positive for Campylobacter than M. glareolus. Previous studies have reported contradictory results concerning the occurrence of campylobacter in M. glareolus. While research from the United Kingdom and Sweden showed a prevalence of 18–77%  and we emphasize that this comparison was made between similar samples, i.e., breeding bank voles in early summer. Even if the exact reason for the great difference between 2 years is not known, it is important to realize generally in epidemiological sampling that temporal differences can be pronounced.In Lapland , nce 1970 . ConsequM. musculus and S. araneus were consistently Campylobacter negative despite being quite common among the studied samples. A similar finding concerning M. musculus was presented recently from South Korea  C. coli, and three (20%) C. jejuni strains were isolated from 15 M. musculus caught on a Dutch organic pig farm  and Campylobacter spp. from 1/30 (3.3%) of M. musculus, respectively.th Korea  involvinectively . The autig farms . In anotpig farm . CampyloS. araneus, our results were in line with a previous study in which all shrews (10 S. araneus common shrews and 119 Crocidura russula greater white-toothed shrews), were negative for Campylobacter species (C. jejuni from the spleens of one water (Neomys fodiens) and one common shrew (S. araneus), even though they were not isolated from the gut, in the United Kingdom  of the pooled R. norvegicus samples from farms. Previous studies have also shown that R. norvegicus may be carriers of Campylobacter. C. jejuni was isolated from three and C. coli from nine percent of R. norvegicus, respectively, collected from pig farms, chicken farms, and non-farm locations in Sweden  of these were isolated from on-farm pooled samples. These STs have previously been isolated from, e.g., wild birds in Sweden and New Zealand , chicken offal or meat in Denmark , and various sources including chicken, cattle, dog, and human gastroenteritis worldwide . ST 2219 was isolated from A. flavicollis (one strain isolated from a pig farm sample), and the other STs from R. norvegicus, M. glareolus, and A. flavicollis from on-farm samples. Our findings are in agreement with previous studies finding also similar genotypes from small mammals and livestock , further suggesting C. jejuni lineages specific to M. glareolus.To our knowledge, MLST or WGS has only previously been used for typing e fields , and in le farms . Our isoR. norvegicus, the Dutch study identified C. coli from 1/8 (12.5%) R. norvegicus caught on a pig farm in Netherlands . The majority of the cgMLST alleles were segregated according to species (A. flavicollis and M. minutus) or group (vole). Very few or no alleles were shared with livestock or human Campylobacter strains. Core-genome phylogeny of the animal-associated strains confirmed the separation of the lineages as observed in cgMLST. The strains from voles formed a distinct clade that was clearly separate from C. jejuni, while the strains isolated from mice grouped monophyletically within the diversity of the C. jejuni population. Moreover, there was no difference between Campylobacter strains from farm versus wild captured animals, whose phylogeny followed the animal species-association indicating adaptation to different host species. The only major exceptions to this were the clustering of different Campylobacter strains from M. glareolus, M. agrestis, and M. mystacinus (syn. M. levis) isolated from different locations throughout Finland , and M. glareolus and M. rutilus isolated from northern Finland. The overlap in habitat selection among vole species and the commonness of M. glareolus could explain the spread of Campylobacter from M. glareolus to other sympatric vole species. The larger cluster of vole strains also contained the bank vole having ST 3704, the genotype that was previously also identified in the United Kingdom  that are almost as separate as different species. The ANI value for C. coli clade 3 versus clade 1, which included most of the human and farm animal isolates (Campylobacter strains from voles showed 90 to 91% ANI with C. jejuni and <85% ANI with C. coli (same with C. hepaticus), while strains from mice shared ≥95% ANI with both C. jejuni subspecies, clearly indicating that the mice strains belong to C. jejuni, more specifically C. jejuni subsp. jejuni. The isolates from voles tested hippurate positive in the phenotypic hippurate hydrolysis test. However, the majority of them were only weakly identified as C. jejuni using MALDI-TOF-MS, further suggesting they may represent a novel sub-species.FastANI further indicated that the vole strains might form a different species or sub-species of ≥95% ANI . Howeverisolates , for exagyrA  database and is publicly available under the project accession number Animal trapping was carried out according to stipulations of national animal welfare and environmental legislature (permits from Ministry of Agriculture and Forestry of Finland and Ministry of the Environment of Finland). The Animal Ethics Council of the State Provincial Office of Southern Finland has prior to this study ruled that terminal snap trapping of small rodents does not constitute an animal experiment and therefore does not require an animal experimentation permit. No human studies are presented in this manuscript. No potentially identifiable human images or data is presented in this study.RK, MR, MH, PT, MS, OH, SO, JN, and HH contributed to the conception and design of the study. JN, OH, and HH collected and prepared all the animal samples. MR, JT, MS, AJ, SO, and RK analyzed the data. MR performed the phylogenomic analyses. MR and RK interpreted the results. RK performed the statistical analysis. MR wrote the first draft of the manuscript. RK, SO, and MS wrote sections of the manuscript. All authors made substantial intellectual contributions, revised the manuscript critically, and approved the final version of the manuscript.http://www.efsa.europa.eu.MR is employed with the European Food Safety Authority (EFSA) in the Unit BIOCONTAM that provides scientific and administrative support to EFSA’s Scientific Activities in the area of Rapid Outbreak Assessment and molecular typing of foodborne pathogens. However, the present article is published under the sole responsibility of MR and may not be considered as an EFSA scientific output. The positions and opinions presented in this article are those of the author/s alone and are not intended to represent the views/any official position or scientific works of EFSA. To know about the views or scientific outputs of EFSA, please consult its website under The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Solenopsis invicta is an invasive ant that has quickly spread worldwide causing significant ecological and economic impacts. Its virome has begun to be characterized pertaining to potential use of viruses as natural enemies. Although the S. invicta virome is the best characterized among ants, most studies have been performed in its native range, with less information from invaded areas.Advances in sequencing and analysis tools have facilitated discovery of many new viruses from invertebrates, including ants. S. invicta, in two introduced areas, U.S and Taiwan. The data set used here was obtained from different stages  of S. invicta life cycle. Publicly available RNA sequences from GenBank’s Sequence Read Archive were downloaded and de novo assembled using CLC Genomics Workbench 20.0.1. Contigs were compared against the non-redundant protein sequences and those showing similarity to viral sequences were further analyzed.Using a metatranscriptome approach, we further identified and molecularly characterized virus sequences associated with S. invicta transcriptomes. Sequence comparisons revealed extensive divergence across ORFs and genomic regions with most of them sharing less than 40% amino acid identity with those closest homologous sequences previously characterized. The first negative-sense single-stranded RNA virus genomic sequences included in the orders Bunyavirales and Mononegavirales are reported. In addition, two positive single-strand virus genome sequences and one single strand DNA virus genome sequence were also identified. While the presence of a putative tenuivirus associated with S. invicta was previously suggested to be a contamination, here we characterized and present strong evidence that Solenopsis invicta virus 14 (SINV-14) is a tenui-like virus that has a long-term association with the ant. Furthermore, based on virus sequence abundance compared to housekeeping genes, phylogenetic relationships, and completeness of viral coding sequences, our results suggest that four of five virus sequences reported, those being SINV-14, SINV-15, SINV-16 and SINV-17, may be associated to viruses actively replicating in the ant S. invicta.We characterized five putative new viruses associated with S. invicta in introduced areas with potential to be used as biological control agents, which will require further biological characterization.The present study expands our knowledge about viral diversity associated with  Insects are the most abundant and diverse group of animals on earth . High thSolenopsis invicta, is an invasive pest causing significant ecological impact and economic loss in invaded areas [S. invicta was accidentally introduced into the southern region of the United States (U.S.) almost a century ago, becoming a serious problem [S. invicta in introduced areas [S. invicta populations may reach sizes even greater than those observed in its native range, making control difficult and even more costly [S. invicta populations in the U.S. have facilitated its dispersal across the world, contributing to repeated introduction events in several countries, such as China, Taiwan, and Australia [S. invicta infestation. Highly competitive ability, generalist feeding habits and high populations densities make S. invicta a successful invasive species causing huge disturbance in biodiversity by displacing native ants and other arthropods in introduced regions [S. invicta [The red imported fire ant, ed areas , 16. Ori problem . Since t problem . Limiteded areas –21. There costly . High deustralia . Morrisoustralia  demonstr regions . Current invicta . Low effS. invicta virome pertaining to potential use in biological control [S. invicta virome is composed of mainly positive-sense single-strand RNA (+ ssRNA) viruses in the order Picornavirales. These include eleven viruses from families Dicistroviridae, Polycipiviridae, Iflaviridae, Soliniviridae and two unclassified viruses [Totiviridae and Parvoviridae, respectively, have been characterized [S. invicta in its native range, only the species Solenopsis invicta virus 1, Solenopsis invicta virus 2, Solenopsis invicta virus 3, Solenopsis invicta virus 6 and Nylanderia fulva virus 1 have been reported in the United States [S. invicta virome, the effect of these viruses and their potential use in biological control will require additional investigation. Interactions among S. invicta and Solenopsis invicta virus 1 (SINV-1), SINV-2 and SINV-3, have been the only ones previously characterized [S. invicta colonies, with greatest potential as a biological control agent [Classical biological control has been one strategy used in an attempt to control this pest in the U.S., with viruses considered a promising resource to be used as biopesticides –26. Over control , 27–30.  viruses , 31. Addcterized , 32. Whid States , 31. Altcterized , 33, 34.ol agent , 35.S. invicta in two introduced areas, U.S and Taiwan. These investigations utilized existing publicly available RNA sequences deposited in NCBI GenBank as Sequence Read Archive (SRA) data files  virus sequences included in the orders Bunyavirales and Mononegavirales reported.In addition, two + ssRNA viruses sequences, included in the family Iflaviridae and an unassigned species, and a partial ssDNA virus sequence, were also characterized.Here using a metatranscriptome approach, we identified and molecularly characterized five new virus genome sequences associated with es Table . Five neS. invicta collected in Mississippi, U.S. [S. invicta, six libraries [−2 were downloaded and further analyzed. The final data set analyzed here consisted of 13 libraries, all from S. invicta transcriptomes. Detailed information on the samples are presented in Table Contigs corresponding to putative new virus genome sequences were initially obtained from a transcriptome project studying differential gene expression between larvae and pupae stages of pi, U.S. . To furtibraries  were dowS. invicta were first filtered by mapping them back to the S. invicta genome (GenBank accession: GCA_000188075.2) and only unmapped reads were used for de novo assembling . Multiple sequence alignments of deduced amino acid sequences were prepared using the MUSCLE option in MEGA7 . Alignmecox1), that presented high transcriptional levels, ribosomal protein L18 (rpl18) and translation elongation factor 1 (eif1-beta), both with lower transcriptional levels. Viral and internal genes abundance were calculated as percentage of viral or housekeeping reads per total number of reads in each library  and dinucleotide composition bias has been used to infer putative host-virus association and taxonomic placement for a given virus sequence , 44. To  package  in R sof package , both imS. invicta transcriptomes collected from three geographic locations in the U.S. and one location from Taiwan, representing different stages of the S. invicta life cycle , the only species within the genus Horwuvirus , suggests that they are putative new members of this genus within the family Nyamiviridae.The SINV-15 genome comprises a linear negative-sense, single strand RNA varying from 10,113 to 10,135 nt  including RdRp, helicase, and the canonical picornavirus capsid protein domain . These results indicate that SINV-16 is a new iflavirus reported from S. invicta.SINV-16 has a positive sense single-stranded RNA genome varying from 10,355 to 10,369 nt , isolated from a pool of marine crustaceans within the genus ia fulva .Parvoviridae , predicted to encode three ORFs on the positive strand, with typical organization of viruses within the family dae Fig. F. The ORrus Fig. D. While cox1, rpl18 and eif1-beta  will be challenging with invasive ant systems.S. invicta. Curiously, while a great diversity of positive and negative sense viruses have been reported from ants [S. invicta virome has been composed mainly of   ssRNA viruses in the order Picornavirales, with no negative sense RNA viruses previously reported. One factor that could be responsible for this bias is the selection of polyadenylated RNA for library preparation in previous studies [Bunyavirales [Mononegavirales associated with S. invicta. Although we did not perform any amplification step of viruses genomes characterized here, the presence of untruncated ORFs carrying intact functional domains , associated with a pool of horseflies (family Tabanidae), and proposed that it may represent a transitional form between plant-infecting virus and arthropod-specific viruses. In addition, a partial genome sequence of another tenui-like, FCTenV1, has been reported associated to Culex annulirostris [S. invicta transcriptome (Order Hymenoptera). Interestingly, in contrast with WhHV, which lacks an ambisense coding strategy, the SINV-14 genome sequence exactly mirrors the genomic structure of typical plant tenuivirus, predicted to encode proteins using ambisense strategy, and also has the conserved sequences at the ends of all four segments identical to those found in tenuivirus genomes  related to plant tenuiviruses. Further identity analysis showed that sequence is 99.8% identical to SINV14 RNA4. They suggested that the sequence would be likely a contamination due to the ant diet feeding either plant or infected insect. Tenuiviruses are typical plant viruses that replicate in the insect vector [S. invicta. Furthermore, the highest virus abundance was found in a sample prepared from a dissected ant brain, which rules out the possibility of contamination due to feeding on plants or association with insect vectors infected with a tenuivirus. The Maize stripe virus (MSpV), a typical plant tenuivirus, was detected in the brain of its vector Peregrinus maidis, providing evidence of replication of tenuiviruses in this tissue [Valles et al.  using ant vector , 54, ands tissue . Tenuivis tissue , 57; this tissue . Althougs tissue , our resS. invicta, suggest an intimate and long-term codivergence process between virus-host.While SINV-14 is evolutionarily closely related to insect tenui-like viruses and plant tenuivirus, the synonymous codon usage and dinucleotide analyses demonstrate a distinct compositional bias compared to FCTenV1 and WhHV, and all other viruses analyzed here, indicating that the virus may be actively replicating in ants rather than plants and other insects. The active replication may have driven the virus genome to distinct compositional bias at the nucleotide level, while maintaining protein integrity at the amino acid level and close relationship with those of other tenui-like viruses, as observed through phylogenetic analysis. The fact that most sequences examined here are probably from viruses that replicate in plant or vertebrate hosts and also in the insect vector could be the reason driving such difference between SINV-14, most likely associated only with ant, compared to other viruses. Furthermore, phylogenetic congruence across different segments that mirror the genetic structure of invasive We presented strong evidence that the sequences from SINV-14 may be from a virus that has been associated long-term and may actively replicate in ants. However, the possibility of this virus, and other tenui-like viruses, replicating in plants is unknown. The presence of the protein carrying an NS4 domain, that is related to cell-to-cell and long-distance movement in plants  in insecS. invicta. Our results suggest fluid shifts in the virome of this invasive species. Further research describing this virome in native and invasive regions and ecosystems could provide insight on virus evolution and invasion mechanics.Altogether, based on virus abundance compared to housekeeping genes, abundant viruses with viral reads higher than 0.01%, phylogenetic relationship, complete viral coding sequence regions recovered, and compositional bias for SINV-14, our results suggest that four out five viruses reported here, those being SINV-14, SINV-15, SINV-16 and SINV-17 are truly replicating in S. invicta in introduced areas. In addition to revealing new virus-host interactions, contributing to better understanding of viral evolutionary history and emergence, the discovery of new viruses expands the range of agents with potential to be used in biocontrol programs, which will require further biological characterization. By understanding these interactions, we may be better equipped to cope with ongoing global changes and introductions of invasive organisms and their associated viruses.The present study expands our knowledge about viral diversity associated with Additional file 1.  Table S1: Descriptive statistics of fire ant transcriptome datasets analyzed in this study.Additional file 2.  Table S2: Open reading frames (ORFs) characterization from putative new viruses based on ORFinder followed by BLASTp analyses, as implemented at National Center for Biotechnology Information Search database (NCBI).Additional file 3.  Table S3: Viral conserved domains detected in ORFs from putative new viruses based on Conserved Domains Database (CDD).Additional file 4.  Table S4: Data set used in the compositional analyses.Additional file 5. Figure S1: Neighbor-Joining phylogenetic tree (1000 bootstrap replications) showing the clustering of the SINV-14 segments. Isolates are highlighted according to sampled place as indicated at the bottom of the figure.Additional file 6.  Figure S2: Alignment of non-structural protein 4 (NS4) amino acid sequence of SINV-14 with those from typical plant tenuiviruses. Positions with single conserved amino acid are highlighted in black, groups of similar properties are highlighted in light gray and positions in SINV-14 that shares the same amino acid with at least one plant tenuivirus sequence, are highlighted in dark gray. Sites containing residues essentials for cell-to-cell movement and long-distance movement are indicated by asterisk and filled circle, respectively. These sites were mapped using Maize stripe virus (MSpV) as a model (59). Matrix of pairwise identity of NS4 amino acid sequence are shown at the bottom of the figure. Multiple sequence alignment and pairwise identity were generated in Clustal O v. 1.2.4. European wheat striate mosaic virus (EWSMV), MN044345; Rice hoja blanca virus (RHBV), MG566077; Echinochloa hoja blanca virus (EHBV), L48441; Rice stripe virus (RSV), D10979; Maize stripe virus (MSpV), L13438.


Phaseolus vulgaris (common bean) microsymbionts belonging to the bacterial genera Rhizobium, Bradyrhizobium, and Ensifer (Sinorhizobium) have been isolated across the globe. Individual symbiosis genes  of these rhizobia can be different within each genus and among distinct genera. Little information is available about the symbiotic structure of indigenous Rhizobium strains nodulating introduced bean plants or the emergence of a symbiotic ability to associate with bean plants in Bradyrhizobium and Ensifer strains. Here, we sequenced the genomes of 29 representative bean microsymbionts  and compared them with closely related reference strains to estimate the origins of symbiosis genes among these Chinese bean microsymbionts.Rhizobium strains. Analysis of vertically transferred genes uncovered 191 (out of the 2654) single-copy core genes with phylogenies strictly consistent with the taxonomic status of bacterial species, but none were found on symbiosis plasmids. A common symbiotic region was wholly conserved within the Rhizobium genus yet different from those of the other two genera. A single strain of Ensifer and two Bradyrhizobium strains shared similar gene content with soybean microsymbionts in both chromosomes and symbiotic regions.Comparative genomics demonstrated horizontal gene transfer exclusively at the plasmid level, leading to expanded diversity of bean-nodulating Rhizobium microsymbionts were assigned to four defined species and six putative novel species. The symbiosis genes of R. phaseoli, R. sophoriradicis, and R. esperanzae strains that originated from Mexican bean-nodulating strains were possibly introduced alongside bean seeds. R. anhuiense strains displayed distinct host ranges, indicating transition into bean microsymbionts. Among the six putative novel species exclusive to China, horizontal transfer of symbiosis genes suggested symbiosis with other indigenous legumes and loss of originally symbiotic regions or non-symbionts before the introduction of common bean into China. Genome data for Ensifer and Bradyrhizobium strains indicated symbiotic compatibility between microsymbionts of common bean and other hosts such as soybean.The 19 native bean  Phaseolus vulgaris L. (common bean) is an important leguminous food crop cultivated worldwide in a broad range of cropping systems and environments. This species was domesticated from a wild-growing vine around 7000 years ago, in two primary centers of origin located in Mexico/Central America and the southern Andes  [Rhizobium species have been isolated from common bean root nodules. In addition, the symbiovar (sv.) mediterranense in Ensifer (Sinorhizobium) meliloti [E. fredii [E. americanum [Most legumes can establish mutualistic symbiosis with certain Alphaproteobacteria or Betaproteobacteria known as rhizobia . Such sygentina) , 4. Likegentina) –7. To dameliloti , E. fred. fredii , and E. ericanum  can noduericanum .Rhizobium, including six defined species and eight novel genospecies, while the remaining strains were Bradyrhizobium (2.4%) and Ensifer (8.4%) [R. anhuiense [E. fredii [Bradyrhizobium [Ensifer [Vicia faba (fava bean) and Trifolium spp. (clover) in China [Rhizobium, Bradyrhizobium, and Ensifer) evolved into microsymbionts of common bean after its introduction into China is very interesting.Common bean is believed to have been introduced into China directly from Latin America around 400 years ago , and Chir (8.4%) . To datenhuiense , E. fred. fredii , and somhizobium  and Ensi[Ensifer  have onlin China . How sucRhizobium species [Mesorhizobium huakuii, M. amorphae [Ensifer species [Bradyrhizobium species [M. loti [M. ciceri [Sesbania rostrata secrete flavonoids that induce nodulation in the rhizobium-legume mutualistic symbiosis while enhancing the transfer of Azorhizobium caulinodans symbiosis islands [Glycine max L. (soybean), both members of the Phaseoleae family, diverged 19 million years ago [Ensifer and Bradyrhizobium strains) have been reported in various ecoregions of China [Ensifer and Bradyrhizobium strains that nodulate common bean and soybean in China are of interest.Concerning the location of symbiosis genes (on plasmids or chromosomes),  species , Mesorhiamorphae , 18, and species  differ f species , 21, M. [M. loti , and M. . ciceri . Growing. ciceri , 25. Hos islands . Howeverears ago . Soybeanof China , 29. TheMedicago sativa L. ; (2) those stringently selected on symbiosis gene background only, such as common bean, Robinia pseudoacacia L. (black locust) [Aspalathus carnosa [Sophora flavescens [R. etli CFN42T and R. esperanzae CNPSo668T) with different chromosomal backgrounds share nodC gene similarities of 97.3–100% [nod, nol, and noe) [nif, fix, and fdx) [Nodule-forming leguminous plants have been divided into three categories based on symbiotic specificities , 31: (1) locust) , and Asp carnosa ; (3) tho carnosa  and Sophavescens . Approxi7.3–100% . In cont7.3–100% , 34, 35.and noe)  and nitrand fdx)  in rhizoand fdx) . Howeverand fdx) .nodC, could provide insight into the phylogenetic relationships [nod, nif, nol, fix, exo, and lps) [To clarify how different rhizobial species were recruited as symbionts of common bean in China, comparison of individual symbiosis genes, such as ionships . So far,and lps) , 42. Anaand lps) . In partRhizobium, two Ensifer, and four Bradyrhizobium), and four from Mexico (two Rhizobium and two Ensifer), for comparative genomics analysis with reference strains. The genome analyses could shed light on different genetic associations while fully explaining the genetic structure of rhizobial strains. The goals of this study were (i) to estimate the origins of symbiosis genes among the common bean-nodulating rhizobial strains belonging to Rhizobium, Ensifer, and Bradyrhizobium, and (ii) to gain genomic insight into different symbiovars among the rhizobia investigated.Herein, we chose 25 representatives of common bean -nodulating rhizobia from China  at the 95% average nucleotide identity (ANI) threshold for species delineation; this is consistent with the grouping results of digital DNA:DNA hybridization (dDDH) estimation and multilocus sequence analysis of housekeeping genes [To analyze genomic features among strains in the ng genes .Rhizobium strains, we clustered 315,181 coding sequences (CDSs) obtained from the 50 available genomes. This yielded a pan-genome containing 30,767 homologous gene families in the genus Rhizobium, with 2777 homologous genes in the core genome and 14,243 genes in the dispensable genome. The core genome represented 39.93 to 47.59% of the repertoire of protein-coding genes in each strain. Moreover, 13,747 genes belonging to the unique genome represented only one strain of Rhizobium  species-related genes were located on chromosomes. With universal distribution and strictly vertical transfer among the 29 rhizobial genomes, 16 highly-conserved genes encode hypothetical proteins. These 16 genes may perform essential biological functions in the survival of rhizobial strains. In general, 65 genes were related to metabolism , 24 genes were linked to translation and biogenesis , 18 genes were associated with transcription , and eight genes were involved in defense mechanisms and signal transduction mechanisms  to verify the symbiotic specificity. The rhizobium-legume symbiosis was highly specific, such that each rhizobial genus/species/strain could nodulate only a specific group of legume, and vice versa [Rhizobium strains from clusters (species) containing the corresponding symbiovars. The results confirmed that all representative strains could nodulate their original host only  were found to be related to the host of origin , which comprised a chromosome (4.57 Mb) with a G + C content of 61.5% and four plasmids (0.67–0.85 Mb) with a G + C content of 58.4–61.1%. Most of the nod, nif, and fix symbiosis genes were clustered in a 0.1 Mb region on symbiosis plasmid pRapFH23a  of HGT genes  with the ANI values between the 447 species pairs. The results indicate that phylogenetic distance was not a significant barrier for recent HGT events among Rhizobium species.To explore the effects of HGT among the 35 nod/nif/fix) and mobile elements, encoding plasmid proteins with key functions such as (1) replication  and (2) conjugation . Moreover, we found that all inferred HGT events occurred within the Rhizobium species isolated from P. vulgaris root nodules only, and ANI values of some species pairs from different host plants were considerably smaller . In summary, symbiosis plasmids in R2-JJW1, R11-L43, and R18-FH23 were substantially different from other symbiosis plasmids with regard to P. vulgaris-Rhizobium symbiosis.It is worth noting that two different HGT groups were identified based on the occurrence of HGT events among the 35 ris Fig. c, and thies Fig. d. We theEnsifer and four Bradyrhizobium microsymbionts of common bean, we obtained six and 13 related reference genomes from Genbank, respectively  displayed close relationships with soybean microsymbionts, while the other two Bradyrhizobium strains (C9 and Y36) exhibited unique genome content  in the eight common bean microsymbionts. Among the four Ensifer strains, Ensifer sp. III FG01 and NG07B, isolated from root nodules of common bean in Mexico, shared almost identical nodulation- and nitrogen fixation-related genes, and these genes differed from those in Ensifer sp. III CCBAU05631 isolated from nodules of soybean. Despite their highly similar genomic background, these three strains exhibited differences in their symbiosis gene content, which could reflect host characteristics. Thus, these two symbiovars may be indicative of Ensifer sp. III. Nodulation genes of FG01/NG07B were highly similar to those of Acacia farnesiana and P. vulgaris microsymbionts, and most similar to strains with different hosts  [P < 0.001; Additional file We identified significant differences in both mean genome size and mean G + C content among rhizobial strains in the three distinct genera  . The resstrains) . High coRhizobium strains from different regions with diverse environmental conditions and host plants. The 50 Rhizobium strains were divided into 19 taxonomic clusters, with a shared core genome that represented less than half of the repertoire of protein-coding genes in each strain. Additionally, a high frequency of gene exchange with other taxa was evidenced by their large number of homologous gene in the dispensable and unique genomes. Of note, strains in the same cluster, despite isolated from diverse environments (countries or hosts), were more inclined to recruit lineage-specific shell genes under direct or indirect control through the speciation process, based on hierarchical clustering of the pan-genome. Concordance between pan-genome phylogenetic tree and core genome tree was also found for Aeromonas hydrophila [Bradyrhizobium, Ensifer [Streptococcus [Pan-genome analysis is an efficient tool for revealing the diversity and composition of the gene repertoire . Herein,drophila , but notdrophila . In addi Ensifer , and Strtococcus .Rhizobium species [nod genes and ribosomal/housekeeping genes for microsymbionts of Acacia mearnsii, indicating that microsymbionts of different host plants possess distinct evolutionary histories [Genetic material can be acquired by bacteria via gene transfer. In vertical gene transfer, genetic information is passed from parents to offspring by asexual splitting; in the case of HGT, a donor organism transfers genes to a recipient organism that is not a descendant mainly by transformation, conjugation, and transduction . Here we species . Althoug species , 54. Ana species . Howeveristories .Rhizobium strains, we assigned the 19 sequenced Chinese common bean microsymbionts to four defined species  and six putative novel species [R. phaseoli, R. sophoriradicis, and R. esperanzae were found to be closely related to Mexican strains, not only in terms of chromosomal background, but also in symbiosis genes. Such close phylogenetic relationships indicate that the three Rhizobium species share the same origin. R. anhuiense may be the dominant common bean microsymbiont in China, despite the reference strain of R. anhuiense WYCCWR10015 has been isolated from root nodules of Trifolium repens [R. anhuiense type strain CCBAU23252T is from Vicia faba [R. anhuiense (R9) were related to their host of origin. Furthermore, three symbiovars  have been identified in this species [R. anhuiense possess different repABC operons, suggesting their independent origins. It appears that R. anhuiense is more flexible in terms of acquiring symbiosis plasmids. Thus, common bean microsymbionts might be microsymbionts of other indigenous legumes before the introduction of common bean into China. In addition to R. anhuiense, another six putative novel species were exclusive to China, although they share many recently transferred symbiosis genes with R. etli CFN42T. Therefore, these strains might have transitioned from microsymbionts of other legumes or soil saprophytes to bean endosymbionts. Herein, we present strong evidence that indigenous Rhizobium strains likely acquired their common bean-specific symbiosis genes from other Rhizobium species that were introduced alongside bean crops.Based on comparison of the genomes of 50  species . Strainsm repens , and thecia faba . In the R. etli to other species in introduced regions appears to be a common phenomenon [R. tropici, which is thought to be indigenous to Brazil, and which has been successfully applied for bean inoculation in Brazil [Rhizobium genus. Despite the high plasticity of symbiosis plasmids, they appear to have been transferred in an interspecies manner within the Rhizobium genus only. Our results suggest that dispersion of key symbiosis genes is rare between rhizobial generaa; however, expansion within the same genus is frequent, which could explain the emergence of multi-symbiovars [Ensifer and Bradyrhizobium strains possess diverse symbiotic regions compared with Rhizobium strains. Comparative genomic analysis and cross-nodulation tests of Ensifer and Bradyrhizobium strains indicated symbiotic compatibility between soybean microsymbionts and common bean microsymbionts. Nodulation genes of Ensifer strains FG01/NG07B/BJ1 and Bradyrhizobium strain L2 are heterogeneous, as they are diverse when compared with similar strains interacting with a different host.The transfer of symbiosis genes mediated by symbiosis plasmids from enomenon . Herein,n Brazil . Nonethembiovars . In addiRhizobium genus belong to a phylogenetically compact group, although common bean can be nodulated by rhizobia from distinct genera; this result indicates that Rhizobium species exhibit a restricted host range. Overall, our findings provide insight into various aspects of rhizobial evolution, but experiments are needed to clarify the frequency of HGT of symbiosis genes among Rhizobium lineages, and the effects of host plants on transfer of these genes. HGT of specific symbiosis genes within rhizobial genera is an important mechanism that allows legumes to form symbioses with rhizobia adapted to particular soils. It is also critical to understand the strategies taken by different legume species to select particular partners. Such knowledge will facilitate the application of nitrogen fixation mechanisms to non-leguminous crops and thereby contribute to sustainable agriculture.Rhizobia can live as saprophytes in soil or establish symbiotic relationships with legumes, and they can change their host characteristics through HGT of symbiosis plasmids . HoweverR. phaseoli, R. sophoriradicis, and R. esperanzae may have been introduced together with bean seeds from Mexico. Strains of R. anhuiense and six putative novel Rhizobium species are exclusive to China, and they appear to have evolved into common bean microsymbionts via HGT events with Mexican rhizobia after being introduced into China. Common bean-nodulating Ensifer and Bradyrhizobium strains possess symbiotic regions distinct from those in Rhizobium strains, indicating symbiotic compatibility between microsymbionts of soybean and common bean. These findings provide insight into symbiotic and housekeeping genes during rhizobial evolution.Among the 19 sequenced Chinese common bean microsymbionts, strains of Rhizobium, four Ensifer, and four Bradyrhizobium), including 25 from China and four from Mexico. Other reference genomes used in the study included 29 Rhizobium strains , six Ensifer strains , and 13 Bradyrhizobium strains  following the manufacture’s instructions. The complete genome of R. acidisoli FH23 was sequenced using the PacBio RS II platform at Novogene. Low-quality reads were filtered using SMRT Analysis v2.3.0 with default parameters [R. acidisoli FH23 have been deposited in the GenBank database under project accession no. PRJNA403813.The draft genomes of 29 common bean microsymbionts were obtained as previously described  using thrameters , and therameters . The drap-values among gene functional categories and genome size were obtained using the corr.test function in the R package ‘psych’ and the obtained correlation matrix was visualized using the R package ‘corrplot’.A whole-genome BLAST search  , Mimosa pudica (mimosa), P. vulgaris (common bean), Leucaena leucocephala (white popinac), and Glycine max (soybean). More than 50 alternative strains to be selected for genome sequencing were subjected to reinfection test with common bean. In addition, cross-nodulation tests were performed using 15 representative strains, including seven sequenced Rhizobium strains , three Ensifer strains , and five Bradyrhizobium strains .Five leguminous plant species were selected for reinfection tests, namely 2 for 3–5 min. After rinsing and imbibition, the seeds were germinated on 0.8% water-agar plates in the dark [Briefly, leguminous plant seeds were washed with sterilized distilled water two to four times, followed by 95% ethanol (v/v) for 1–3 min. Then the seeds were surface-sterilized with 0.2% HgClthe dark . Once ththe dark . ControlThe RecentHGT automatic pipeline  was implP. vulgaris . A bipartite ‘gene families-plasmids’ network was constructed based on the 13 proteomes using AccNet v1.2. Specifically, all homologous proteins sharing > 40% sequence similarity were clustered with kClust parameters -s = 1.73, −c = 0.8, and -e = 1e-10. The bipartite graph was hierarchically clustered with a practical similarity threshold of 85%. The resulting network files including nodes, edges, and clusters were then imported into Gephi v0.92 to visualize the bipartite graph [Symbiosis plasmids were identified in 13 completely sequenced rhizobial strains nodulating Ensifer strains and 17 Bradyrhizobium strains were calculated using JspeciesWS with the MUMmer algorithm [All pairwise ANI values among the 10 lgorithm , and corlgorithm . Multipllgorithm  to identlgorithm .Additional file 1: Table S1. Genomes used in this study. Table S2. Core genes for which phylogenies are strictly consistent with the species tree. Table S3. Recent horizontal gene transfer (HGT) predictions among 50 Rhizobium strains. Table S4. Average nucleotide identity  and digital DNA:DNA hybridization  values of 10 Ensifer strains and 17 Bradyrhizobium strains based on whole-genome alignments. Table S5. Recent HGT predictions among 10 Ensifer strains and 17 Bradyrhizobium strains. Table S6. Identities (%) of nodulation- and nitrogen fixation-related genes between sequenced strains and their most closely related strains.Additional file 2: Figure S1. Neighbor-joining species tree based on the 2110 single-copy core genes shared by 50 rhizobial genomes used in this study. The name of each strain is preceded by the cluster number indicated in Additional file Rhizobium strains in bold font were determined in this study. Figure S2. Cross-nodulation tests on Trifolium pretense, Mimosa pudica, Phaseolus vulgaris, and Leucaena leucocephala with seven representative Rhizobium strains. * n = 3 cultivation bags per treatment. ** bold italic letters before strain names indicate the original host. P, Phaseolus vulgaris; T, Trifolium pretense. Figure S3. Core genes of 29 representative Rhizobium strains related to hosts. Neighbor-joining trees of 12 critical symbiosis genes were constructed using 1000 bootstrap replicates (only bootstrap support > 60% is shown). Bar = 5% sequence divergence. Symbiotic characteristics are indicated by different colored solid lines . Figure S4. Graphical circular map of the complete genome of R. acidisoli FH23. The outermost circle represents the coordinates of the genome sequence. Circles from outside to inside indicate protein-coding genes, genes BLAST searched against COG, KEGG, and GO databases, non-coding RNA, deviations in G + C content (green/red), and G-C skew (light green/pink). Outermost to innermost positions indicate genes in forward and reverse orientations. Figure S5. Distribution of homologous genes based on COG assignment of unique genes within the symbiosis plasmid of R18-FH23 (R. acidisoli). Figure S6. Prediction of recent HGT genes among strains isolated from different legumes. a Ensifer strains. b Bradyrhizobium strains. The width of links is proportional to the number of predicted recent HGT genes. The darker the link color, the larger the predicted magnitude of the recent HGT event. Figure S7. Correlogram showing correlations between genome size, protein-coding sequences, and COG assignments of the 29 rhizobial strains in three distinct genera. Positive correlations are indicated by cells with lower triangles or circles in upper triangles colored blue from lower left to upper right. Negative correlations are represented by red coloring from the upper left to the lower right. The darker and more saturated the color, the greater the magnitude of the correlation. Weak correlations (near zero) are almost colorlessAdditional file 3. Comparison of genomic features among three distinct rhizobial genera.