Delineation of Paraburkholderia tuberum sensu stricto and description of Paraburkholderia podalyriae sp. nov. nodulating the South African legume Podalyria calyptrata.

Since the discovery of Paraburkholderia tuberum, an indigenous South African species and one of the first beta-rhizobia described, several other South African rhizobial Paraburkholderia species have been recognized. Here, we investigate the taxonomic status of 31 rhizobial isolates from the root nodules of diverse South African legume hosts in the Core Cape Subregion, which were initially identified as P. tuberum. These isolates originate from the root nodules of genera in the Papilionoideae as well as Vachellia karroo, from the subfamily Caesalpinioideae. Genealogical concordance analysis of five loci allowed delineation of the isolates into two putative species clusters (A and B). Cluster A included P. tuberum STM678T, suggesting that this monophyletic group represents P. tuberum sensu stricto. Cluster B grouped sister to P. tuberum and included isolates from the Paarl Rock Nature Reserve in the Western Cape Province. Average Nucleotide Identity (ANI) analysis further confirmed that isolates of Cluster A shared high genome similarity with P. tuberum STM678T compared to Cluster B and other Paraburkholderia species. The members of Cluster B associated with a single species of Podalyria, P. calyptrata. For this new taxon we accordingly propose the name Paraburkholderia podalyriae sp. nov., with the type strain WC7.3bT (= LMG 31413T; SARCC 750T). Based on our nodA and nifH phylogenies, P. podalyriae sp. nov. and strains of P. tuberum sensu stricto (including one from V. karroo) belong to symbiovar africana, the symbiotic loci of which have a separate evolutionary origin to those of Central and South American Paraburkholderia strains.

Bradyrhizobium altum sp. nov., Bradyrhizobium oropedii sp. nov. and Bradyrhizobium acaciae sp. nov. from South Africa show locally restricted and pantropical nodA phylogeographic patterns.

Africa is known for its rich legume diversity with a significant number of endemic species originating in South Africa. Many of these legumes associate with rhizobial symbionts of the genus Bradyrhizobium, of which most represent new species. Yet, none of the Bradyrhizobium species from South Africa have been described. In this study, phylogenetic analysis of 16S rRNA gene sequences of fourteen strains isolated in southern Africa from root nodules of diverse legumes (i.e., from the tribes Crotalarieae, Acacieae, Genisteae, Phaseoleae and Cassieae) revealed that they belong to the Bradyrhizobium elkanii supergroup. The taxonomic position and possible novelty of these strains were further interrogated using genealogical concordance of five housekeeping genes (atpD, dnaK, glnII, gyrB and rpoB). These phylogenies consistently recovered four monophyletic groups and one singleton within Bradyrhizobium. Of these groups, two were conspecific with Bradyrhizobium brasilense UFLA 03-321T and Bradyrhizobium ivorense CI-1BT, while the remaining three represented novel taxa. Their existence was further supported with genome data, as well as metabolic and physiological traits. Analysis of nodA gene sequences further showed that the evolution of these bacteria likely involved adapting to local legume hosts and environmental conditions through the acquisition, via horizontal gene transfer, of optimal symbiotic loci. We accordingly propose the following names Bradyrhizobium acaciae sp. nov. 10BBT (SARCC 730T = LMG 31409T), Bradyrhizobium oropedii sp. nov. Pear76T (SARCC 731T = LMG 31408T), and Bradyrhizobium altum sp. nov. Pear77T (SARCC 754T = LMG 31407T) to accommodate three novel species, all of which are symbionts of legumes in South Africa.

Paraburkholderia youngii sp. nov. and 'Paraburkholderia atlantica' - Brazilian and Mexican Mimosa-associated rhizobia that were previously known as Paraburkholderia tuberum sv. mimosae.

Previous studies have recognized South and Central/Latin American mimosoid legumes in the genera Mimosa, Piptadenia and Calliandra as hosts for various nodulating Paraburkholderia species. Several of these species have been validly named in the last two decades, e.g., P. nodosa, P. phymatum, P. diazotrophica, P. piptadeniae, P. ribeironis, P. sabiae and P. mimosarum. There are still, however, a number of diverse Paraburkholderia strains associated with these legumes that have an unclear taxonomic status. In this study, we focus on 30 of these strains which originate from the root nodules of Brazilian and Mexican Mimosa species. They were initially identified as P. tuberum and subsequently placed into a symbiovar (sv. mimosae) based on their host preferences. A polyphasic approach for the delineation of these strains was used, consisting of genealogical concordance analysis (using atpD, gyrB, acnA, pab and 16S rRNA gene sequences), together with comparisons of Average Nucleotide Identity (ANI), DNA G+C content ratios and phenotypic characteristics with those of the type strains of validly named Paraburkholderia species. Accordingly, these 30 strains were delineated into two distinct groups, of which one is conspecific with 'P. atlantica' CNPSo 3155T and the other new to Science. We propose the name Paraburkholderia youngii sp. nov. with type strain JPY169T (= LMG 31411T; SARCC751T) for this novel species.

The synergistic effect of concatenation in phylogenomics: the case in Pantoea.

With the increased availability of genome sequences for bacteria, it has become routine practice to construct genome-based phylogenies. These phylogenies have formed the basis for various taxonomic decisions, especially for resolving problematic relationships between taxa. Despite the popularity of concatenating shared genes to obtain well-supported phylogenies, various issues regarding this combined-evidence approach have been raised. These include the introduction of phylogenetic error into datasets, as well as incongruence due to organism-level evolutionary processes, particularly horizontal gene transfer and incomplete lineage sorting. Because of the huge effect that this could have on phylogenies, we evaluated the impact of phylogenetic conflict caused by organism-level evolutionary processes on the established species phylogeny for Pantoea, a member of the Enterobacterales. We explored the presence and distribution of phylogenetic conflict at the gene partition and nucleotide levels, by identifying putative inter-lineage recombination events that might have contributed to such conflict. Furthermore, we determined whether smaller, randomly constructed datasets had sufficient signal to reconstruct the current species tree hypothesis or if they would be overshadowed by phylogenetic incongruence. We found that no individual gene tree was fully congruent with the species phylogeny of Pantoea, although many of the expected nodes were supported by various individual genes across the genome. Evidence of recombination was found across all lineages within Pantoea, and provides support for organism-level evolutionary processes as a potential source of phylogenetic conflict. The phylogenetic signal from at least 70 random genes recovered robust, well-supported phylogenies for the backbone and most species relationships of Pantoea, and was unaffected by phylogenetic conflict within the dataset. Furthermore, despite providing limited resolution among taxa at the level of single gene trees, concatenated analyses of genes that were identified as having no signal resulted in a phylogeny that resembled the species phylogeny of Pantoea. This distribution of signal and noise across the genome presents the ideal situation for phylogenetic inference, as the topology from a ≥70-gene concatenated species phylogeny is not driven by single genes, and our data suggests that this finding may also hold true for smaller datasets. We thus argue that, by using a concatenation-based approach in phylogenomics, one can obtain robust phylogenies due to the synergistic effect of the combined signal obtained from multiple genes.

Genome-informed Bradyrhizobium taxonomy: where to from here?

Bradyrhizobium is thought to be the largest and most diverse rhizobial genus, but this is not reflected in the number of described species. Although it was one of the first rhizobial genera recognised, its taxonomy remains complex. Various contemporary studies are showing that genome sequence information may simplify taxonomic decisions. Therefore, the growing availability of genomes for Bradyrhizobium will likely aid in the delineation and characterization of new species. In this study, we addressed two aims: first, we reviewed the availability and quality of available genomic resources for Bradyrhizobium. This was achieved by comparing genome sequences in terms of sequencing technologies used and estimated level of completeness for inclusion in genome-based phylogenetic analyses. Secondly, we utilized these genomes to investigate the taxonomic standing of Bradyrhizobium in light of its diverse lifestyles. Although genome sequences differed in terms of their quality and completeness, our data indicate that the use of these genome sequences is adequate for taxonomic purposes. By using these resources, we inferred a fully resolved, well-supported phylogeny. It separated Bradyrhizobium into seven lineages, three of which corresponded to the so-called supergroups known for the genus. Wide distribution of key lifestyle traits such as nodulation, nitrogen fixation and photosynthesis revealed that these traits have complicated evolutionary histories. We present the first robust Bradyrhizobium species phylogeny based on genome sequence information for investigating the evolution of this important assemblage of bacteria. Furthermore, this study provides the basis for using genome sequence information as a resource to make important taxonomic decisions, particularly at the species and genus levels.

Corrigendum: Genome Data Provides High Support for Generic Boundaries in Burkholderia Sensu Lato.

[This corrects the article on p. 1154 in vol. 8, PMID: 28694797.].

Mixta gen. nov., a new genus in the Erwiniaceae.

The Erwiniaceae contain many species of agricultural and clinical importance. Although relationships among most of the genera in this family are relatively well resolved, the phylogenetic placement of several taxa remains ambiguous. In this study, we aimed to address these uncertainties by using a combination of phylogenetic and genomic approaches. Our multilocus sequence analysis and genome-based maximum-likelihood phylogenies revealed that the arsenate-reducing strain IMH and plant-associated strain ATCC 700886, both previously presumptively identified as members of Pantoea, represent novel species of Erwinia. Our data also showed that the taxonomy of Erwinia teleogrylli requires revision as it is clearly excluded from Erwinia and the other genera of the family. Most strikingly, however, five species of Pantoea formed a distinct clade within the Erwiniaceae, where it had a sister group relationship with the Pantoea + Tatumella clade. By making use of gene content comparisons, this new clade is further predicted to encode a range of characters that it shares with or distinguishes it from related genera. We thus propose recognition of this clade as a distinct genus and suggest the name Mixta in reference to the diverse habitats from which its species were obtained, including plants, humans and food products. Accordingly, a description for Mixta gen. nov. is provided to accommodate the four species Mixta calida comb. nov., M. gaviniae comb. nov., M. intestinalis comb. nov. and M. theicola comb. nov., with M. calida as the type species for the genus.

Genome Data Provides High Support for Generic Boundaries in Burkholderia Sensu Lato.

Although the taxonomy of Burkholderia has been extensively scrutinized, significant uncertainty remains regarding the generic boundaries and composition of this large and heterogeneous taxon. Here we used the amino acid and nucleotide sequences of 106 conserved proteins from 92 species to infer robust maximum likelihood phylogenies with which to investigate the generic structure of Burkholderia sensu lato. These data unambiguously supported five distinct lineages, of which four correspond to Burkholderia sensu stricto and the newly introduced genera Paraburkholderia, Caballeronia, and Robbsia. The fifth lineage was represented by P. rhizoxinica. Based on these findings, we propose 13 new combinations for those species previously described as members of Burkholderia but that form part of Caballeronia. These findings also suggest revision of the taxonomic status of P. rhizoxinica as it is does not form part of any of the genera currently recognized in Burkholderia sensu lato. From a phylogenetic point of view, Burkholderia sensu stricto has a sister relationship with the Caballeronia+Paraburkholderia clade. Also, the lineages represented by P. rhizoxinica and R. andropogonis, respectively, emerged prior to the radiation of the Burkholderia sensu stricto+Caballeronia+Paraburkholderia clade. Our findings therefore constitute a solid framework, not only for supporting current and future taxonomic decisions, but also for studying the evolution of this assemblage of medically, industrially and agriculturally important species.

Burkholderia kirstenboschensis sp. nov. nodulates papilionoid legumes indigenous to South Africa.

Despite the diversity of Burkholderia species known to nodulate legumes in introduced and native regions, relatively few taxa have been formally described. For example, the Cape Floristic Region of South Africa is thought to represent one of the major centres of diversity for the rhizobial members of Burkholderia, yet only five species have been described from legumes occurring in this region and numerous are still awaiting taxonomic treatment. Here, we investigated the taxonomic status of 12 South African root-nodulating Burkholderia isolates from native papilionoid legumes (Hypocalyptus coluteoides, H. oxalidifolius, H. sophoroides and Virgilia oroboides). Analysis of four gene regions (16S rRNA, recA, atpD and rpoB) revealed that the isolates represent a genealogically unique and exclusive assemblage within the genus. Its distinctness was supported by all other aspects of the polyphasic approach utilized, including the genome-based criteria DNA-DNA hybridization (≥70.9%) and average nucleotide identities (≥96%). We accordingly propose the name B. kirstenboschensis sp. nov. for this taxon with isolate Kb15(T) (=LMG 28727(T); =SARC 695(T)) as its type strain. Our data showed that intraspecific genome size differences (≥0.81 Mb) and the occurrence of large DNA regions that are apparently unique to single individuals (16-23% of an isolate's genome) can significantly limit the value of data obtained from DNA-DNA hybridization experiments. Substitution of DNA-DNA hybridization with whole genome sequencing as a prerequisite for the description of Burkholderia species will undoubtedly speed up the pace at which their diversity are documented, especially in hyperdiverse regions such as the Cape Floristic Region.