Rhizobia supply legumes with fixed nitrogen using a set of symbiosis genes. These can cross rhizobium species boundaries, but it is unclear how many other genes show similar mobility. Here, we investigate inter-species introgression using de novo assembly of 196 Rhizobium leguminosarum sv. trifolii genomes. The 196 strains constituted a five-species complex, and we calculated introgression scores based on gene-tree traversal to identify 171 genes that frequently cross species boundaries. Rather than relying on the gene order of a single reference strain, we clustered the introgressing genes into four blocks based on population structure-corrected linkage disequilibrium patterns. The two largest blocks comprised 125 genes and included the symbiosis genes, a smaller block contained 43 mainly chromosomal genes, and the last block consisted of three genes with variable genomic location. All introgression events were likely mediated by conjugation, but only the genes in the symbiosis linkage blocks displayed overrepresentation of distinct, high-frequency haplotypes. The three genes in the last block were core genes essential for symbiosis that had, in some cases, been mobilized on symbiosis plasmids. Inter-species introgression is thus not limited to symbiosis genes and plasmids, but other cases are infrequent and show distinct selection signatures.

Bacteria currently included in Rhizobium leguminosarum are too diverse to be considered a single species, so we can refer to this as a species complex (the Rlc). We have found 429 publicly available genome sequences that fall within the Rlc and these show that the Rlc is a distinct entity, well separated from other species in the genus. Its sister taxon is R. anhuiense. We constructed a phylogeny based on concatenated sequences of 120 universal (core) genes, and calculated pairwise average nucleotide identity (ANI) between all genomes. From these analyses, we concluded that the Rlc includes 18 distinct genospecies, plus 7 unique strains that are not placed in these genospecies. Each genospecies is separated by a distinct gap in ANI values, usually at approximately 96% ANI, implying that it is a ‘natural’ unit. Five of the genospecies include the type strains of named species: R. laguerreae, R. sophorae, R. ruizarguesonis, “R. indicum” and R. leguminosarum itself. The 16S ribosomal RNA sequence is remarkably diverse within the Rlc, but does not distinguish the genospecies. Partial sequences of housekeeping genes, which have frequently been used to characterize isolate collections, can mostly be assigned unambiguously to a genospecies, but alleles within a genospecies do not always form a clade, so single genes are not a reliable guide to the true phylogeny of the strains. We conclude that access to a large number of genome sequences is a powerful tool for characterizing the diversity of bacteria, and that taxonomic conclusions should be based on all available genome sequences, not just those of type strains.

Image-based phenotype data with high temporal resolution offers advantages over end-point measurements in plant quantitative genetics experiments, because growth dynamics can be assessed and analysed for genotype-phenotype association. Recently, network-based camera systems have been deployed as customizable, low-cost phenotyping solutions. Here, we implemented a large, automated image-capture system based on distributed computing using 180 networked Raspberry Pi units that could simultaneously monitor 1,800 white clover ( Trifolium repens ) plants. The camera system proved stable with an average uptime of 96% across all 180 cameras. For analysis of the captured images, we developed the Greenotyper image analysis pipeline. It detected the location of the plants with a bounding box accuracy of 97.98%, and the U-net-based plant segmentation had an intersection over union accuracy of 0.84 and a pixel accuracy of 0.95. We used Greenotyper to analyze a total of 355,027 images, which required 24–36 h. Automated phenotyping using a large number of static cameras and plants thus proved a cost-effective alternative to systems relying on conveyor belts or mobile cameras.

Sequencing and PCR errors are a major challenge when characterizing genetic diversity using high‐throughput amplicon sequencing (HTAS). We have developed a multiplexed HTAS method, MAUI‐seq, which uses unique molecular identifiers (UMIs) to improve error correction by exploiting variation among sequences associated with a single UMI. Erroneous sequences are recognized because, across the data set, they are over‐represented among the minor sequences associated with UMIs. We show that two main advantages of this approach are efficient elimination of chimeric and other erroneous reads, outperforming dada2 and unoise3, and the ability to confidently recognize genuine alleles that are present at low abundance or resemble chimeras. The method provides sensitive and flexible profiling of diversity and is readily adaptable to most HTAS applications, including microbial 16S rRNA profiling and metabarcoding of environmental DNA.

BackgroundGene transfer between bacterial species is an important mechanism for adaptation. For example, sets of genes that confer the ability to form nitrogen-fixing root nodules on host plants have frequently moved between Rhizobium species. It is not clear, though, whether such transfer is exceptional, or if frequent inter-species introgression is typical. To address this, we sequenced the genomes of 196 isolates of the Rhizobium leguminosarum species complex obtained from root nodules of white clover (Trifolium repens).ResultsCore gene phylogeny placed the isolates into five distinct genospecies that show high intra-genospecies recombination rates and remarkably different demographic histories. Most gene phylogenies were largely concordant with the genospecies, indicating that recent gene transfer between genospecies was rare. In contrast, very similar symbiosis gene sequences were found in two or more genospecies, suggesting recent horizontal transfer. The replication and conjugative transfer genes of the plasmids carrying the symbiosis genes showed a similar pattern, implying that introgression occurred by conjugative plasmid transfer. The only other regions that showed strong phylogenetic discordance with the genospecies classification were two small chromosomal clusters, one neighbouring a conjugative transfer system. Phage-related sequences were observed in the genomes, but appeared to have very limited impact on introgression.ConclusionsIntrogression among these closely-related species has been very limited, confined to the symbiosis plasmids and a few chromosomal islands. Both introgress through conjugative transfer, but have been subject to different types of selective forces.

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1 Background: Sequencing and PCR errors are a major challenge when characterising genetic 2 diversity using high-throughput amplicon sequencing (HTAS). 3 4 Results: We have developed a multiplexed HTAS method, MAUI-seq, which uses unique 5 molecular identifiers (UMIs) to improve error correction by exploiting variation among 6 sequences associated with a single UMI. We show that two main advantages of this approach 7 are efficient elimination of chimeric and other erroneous reads, outperforming DADA2 and 8 UNOISE3, and the ability to confidently recognise genuine alleles that are present at low 9 abundance or resemble chimeras. 10 11 Conclusions:The method provides sensitive and flexible profiling of diversity and is readily 12 adaptable to most HTAS applications, including microbial 16S rRNA profiling and 13 metabarcoding of environmental DNA. 14

Temperate phages play important roles in bacterial communities but have been largely overlooked, particularly in non-pathogenic bacteria. In rhizobia the presence of temperate phages has the potential to have significant ecological impacts but few examples have been described. Here we characterize a novel group of 5 Rhizobium leguminosarum prophages, capable of sustaining infections across a broad host range within their host genus. Genome comparisons identified further putative prophages infecting multiple Rhizobium species isolated globally, revealing a wider family of 10 temperate phages including one previously described lytic phage, RHEph01, which appears to have lost the ability to form lysogens. Phylogenetic discordance between prophage and host phylogenies suggests a history of active mobilization between Rhizobium lineages. Genome comparisons revealed conservation of gene content and order, with the notable exception of an approximately 5 kb region of hypervariability, containing almost exclusively hypothetical genes. Additionally, several horizontally acquired genes are present across the group, including a putative antirepressor present only in the RHEph01 genome, which may explain its apparent inability to form lysogens. In summary, both phenotypic and genomic comparisons between members of this group of phages reveals a clade of viruses with a long history of mobilization within and between Rhizobium species.