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Laboratorium voor Microbiologie (WE10), Universiteit Gent, KL Ledeganckstraat 35, B-9000 Gent, Belgium
Correspondence
Anne Willems
Anne.Willems{at}UGent.be
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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. Int J Syst Evol Microbiol 53, 2107–2110]. Data obtained previously and these new data indicate that Ensifer adhaerens and ‘Sinorhizobium morelense’ are not heterotypic synonyms, but represent separate species. However, transfer to the genus Ensifer is not possible at present because the species name is the subject of a pending Request for an Opinion, which would affect whether a novel species in the genus Ensifer or a new combination based on a basonym would be created.
The GenBank/EMBL/DDBJ accession numbers of new sequence data for dnaK, glnA, gltA, recA, thrC and 16S rRNA genes are given in Table 1
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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; Zakhia & de Lajudie, 2001; Broughton, 2003; Sawada et al., 2003). The current classification of rhizobia, as for other bacterial groups, is strongly influenced by polyphasic classification, taking into account phenotypic characteristics as well as genetic characteristics (Vandamme et al., 1996), with 16S rRNA gene phylogeny providing the backbone of the classification (Willems & Collins, 1993; Young & Haukka, 1996; Yanagi & Yamasato, 1993; Young et al., 2001).
The rhizobia that belong to the Alphaproteobacteria include the genera Allorhizobium, Azorhizobium, Bradyrhizobium, Rhizobium, Mesorhizobium and Sinorhizobium (de Lajudie et al., 1994, 1998; Dreyfus et al., 1988; Jordan, 1982; Jarvis et al., 1997). In 16S rRNA gene-based phylogeny, Agrobacterium and Allorhizobium species are phylogenetically dispersed between Rhizobium species (Willems & Collins, 1993; de Lajudie et al., 1998; Terefework et al., 1998; van Berkum et al., 2003). This observation, and the absence of clear phenotypic distinctions, has led to a proposal to merge these three genera into a single genus, Rhizobium (Young et al., 2001). However, this scheme has been challenged and is not generally accepted (Farrand et al., 2003). It rests largely on the 16S rRNA gene phylogeny and the resulting genus Rhizobium would have the phylogenetic breadth of a bacterial family when neighbouring groups in the alphaproteobacterial 16S rRNA gene tree are considered (see Fig. 1 of Young et al., 2001). There is no single phylogenetic breadth for ranks above species (Brenner et al., 2001); however, as pointed out by Konstantinidis & Tiedje (2005), there is great comparative value in having taxonomic ranks that are comparable among lineages. Although this may not be achievable over the whole of prokaryote diversity, we can endeavour to establish broadly comparable groups within this part of the alphaproteobacterial tree, taking into account several genes and ultimately genomes (Konstantinidis & Tiedje, 2005). We agree with Young et al. (2001) that Agrobacterium rhizogenes is best regarded as a member of Rhizobium, as it is always found to be closely related to the type species Rhizobium leguminosarum. However, at present we consider that uniting Agrobacterium, Allorhizobium and Rhizobium in a single genus (Young et al., 2001) is not a better solution. It is hoped that the taxonomy of these organisms will be resolved in the near future on the basis of a comprehensive analysis of additional markers (sequence and non-sequence). In this paper we use the Agrobacterium nomenclature, except for Rhizobium rhizogenes.
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). In recent years, several studies have shown that the rRNA genes of rhizobia may occasionally undergo lateral transfer and genetic recombination, resulting in sequence mosaicism (Eardly et al., 1996, 2005; Sullivan et al., 1996; van Berkum et al., 2003; Vinuesa et al., 2005). These observations imply that the rhizobial rRNA gene phylogeny may not always accurately reflect prokaryotic phylogeny. A further disadvantage of 16S rRNA gene phylogeny is that closely related species cannot always be distinguished because of the high level of sequence conservation. To overcome these limitations, protein-encoding genes, with a higher level of sequence divergence than rRNA genes, but sufficiently conserved to retain phylogenetic signal and to allow primers to be designed, have been suggested as alternative phylogenetic markers (Stackebrandt et al., 2002). Ideally, these would be single copy chromosomal genes that are universally distributed among prokaryotes. Examples of genes that are used frequently as molecular markers in various bacterial groups include recA (Eisen, 1995), gyrB (Yamamoto & Harayama, 1995), rpoB (Mollet et al., 1998), rpoD (Yamamoto et al., 2000), groEL (Viale et al., 1994) and atpD (Ronner et al., 1991). Several housekeeping genes have already been used as alternative phylogenetic markers within rhizobia: for example, atpD and recA (Gaunt et al., 2001), glnA, glnB (Turner & Young, 2000; Wernegreen & Riley, 1999), dnaK (Stepkowski et al., 2003) and gltA (Hernández-Lucas et al., 2004). Their phylogenies are not always in agreement with 16S rRNA gene phylogeny. Indeed, trees based on single genes do not always reflect organismal phylogeny because of possible horizontal gene transfer or recombination, variable mutation rates and simple stochastic variation (Gevers et al., 2005; Coenye et al., 2005).
The ad hoc committee for re-evaluation of the species definition suggested that ‘species should be identifiable by readily available methods (phenotypic, genomic)’ and that one promising approach towards this end was the determination of a minimum of five housekeeping genes (Stackebrandt et al., 2002). Zeigler (2003) even suggested that sequence analysis of less than five suitable housekeeping genes might be sufficient for a reliable classification. Multilocus sequence analysis (MLSA), using the sequences of multiple protein-coding genes for genotypic characterization of a group of prokaryotes (Gevers et al., 2005), is used increasingly to study and elucidate taxonomic relationships between species (Wertz et al., 2003; Adékambi & Drancourt, 2004; Christensen et al., 2004; Holmes et al., 2004; Naser et al., 2005; Thompson et al., 2005). MLSA is distinct from multilocus sequence typing (Maiden et al., 1998), a method that is used mostly in epidemiology to characterize strains at an infraspecific level by comparing allelic mismatches in housekeeping genes.
In this study we used Sinorhizobium (now renamed Ensifer, see below) as a model system to assess the use of MLSA for taxonomic purposes. This genus contains eleven species, most of which have been described in polyphasic studies: Sinorhizobium fredii, Sinorhizobium xinjiangense (Chen et al., 1988), Sinorhizobium meliloti, Sinorhizobium saheli, Sinorhizobium terangae (de Lajudie et al., 1994), Sinorhizobium medicae (Rome et al., 1996), Sinorhizobium arboris, Sinorhizobium kostiense (Nick et al., 1999), Sinorhizobium kummerowiae (Wei et al., 2002), ‘Sinorhizobium morelense’ (Wang et al., 2002) and Sinorhizobium americanum (Toledo et al., 2003). From 16S rRNA gene comparisons, it is apparent that the monotypic genus Ensifer is highly related to Sinorhizobium (Balkwill, 2005). This organism comprises predatory soil bacteria that were originally described in a separate genus as Ensifer adhaerens, based mainly on morphological and phenotypic characteristics (Casida, 1982). Phylogenetic analyses of 16S rRNA and recA genes (Balkwill, 2005; Willems et al., 2003) indicate that Ensifer forms a well-supported cluster together with Sinorhizobium. From a polyphasic study, Willems et al. (2003) concluded that the genera Ensifer and Sinorhizobium can be considered as a single taxon and therefore should be merged. According to Rule 38 of the Bacteriological Code (Lapage et al., 1992), the oldest legitimate name, Ensifer Casida 1982, has priority over Sinorhizobium Chen et al. 1988. However, after discussion in the Subcommittee on the taxonomy of Rhizobium and Agrobacterium (Lindström & Martínez-Romero, 2002) and in view of several arguments against abandoning the name Sinorhizobium, Willems et al. (2003) in a Request for an Opinion proposed that an exception might be made to Rule 38, allowing the transfer of Ensifer adhaerens to Sinorhizobium as Sinorhizobium adhaerens comb. nov. In a separate Request for an Opinion, Young (2003) proposed that the combination Sinorhizobium adhaerens is illegitimate because it contravenes Rules 23 and 24 of the Code and consequently proposed to transfer all Sinorhizobium species to the genus Ensifer. Recently, the Judicial Commission of the International Committee on Systematics of Prokaryotes ruled that the case for granting an exception to the Rules is too weak (P. De Vos, personal communication). We have therefore adopted the Ensifer nomenclature (Young, 2003).
In the present study, an MLSA approach was used to investigate the relationships of these bacteria by determining the phylogeny of the housekeeping genes recA (recombinase A), glnA (glutamine synthetase type I), gltA (citrate synthase I) dnaK (heat-shock protein Hsp70) and thrC (threonine synthase). Their phylogenies were examined and compared to that of the 16S rRNA gene. In addition, several combined analyses were performed. The results and their possible taxonomic implications are discussed below.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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): 26 strains representing all known former Sinorhizobium species (except for S. kummerowiae, for which we could not obtain a bona fide strain, and S. americanum, for which we could not obtain the complete set of genes) and seven strains representing the three genomovars (A, B and C) of E. adhaerens. In addition, 12 rhizobial strains were included as reference strains. These represent the different groups of the 16S rRNA gene phylogram of rhizobia and belong to the genera Bradyrhizobium, Rhizobium, Mesorhizobium and Agrobacterium. All bacterial strains were grown on yeast extract mannitol agar at 28 °C.
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).
Primers for amplification and sequencing.
The following genes were studied: recA, glnA, 16S rRNA, gltA, dnaK and thrC. To design primers for PCR amplification and sequencing of these genes, we used corresponding sequences derived from the genome sequences of related bacteria: Agrobacterium tumefaciens C58 (Wood et al., 2001; Goodner et al., 2001), S. meliloti 1021 (Galibert et al., 2001), Mesorhizobium loti MAFF 303099 (Kaneko et al., 2000), Brucella melitensis 16MT (DelVecchio et al., 2002) and Bradyrhizobium japonicum USDA 110 (Kaneko et al., 2002). The dnaK, glnA, gltA, recA and thrC sequences were compared using the program BioNumerics (Applied Maths) to identify conserved regions. The primers designed are listed in Table 2. Primers for the amplification of recA and dnaK were obtained from the literature (Gaunt et al., 2001; Stepkowski et al., 2003).
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. The PCR products and their concentration were verified by electrophoresis of 3 µl product on a 1 % agarose gel and staining with ethidium bromide. A molecular mass marker (Smartladder-Eurogentec) was included to estimate the length of the amplicons. The amplified products were purified using a Qiaquick PCR purification kit (Qiagen). The purified DNA was sequenced using the dideoxynucleotide chain-termination method with fluorescent ddNTPs (Applied Biosystems) on an ABI Prism 3100 capillary sequencer, according to the manufacturer's instructions (Applied Biosystems). Consensus sequences were constructed using the AutoAssembler software (Applied Biosystems).
Phylogenetic data analysis.
Nucleotide sequence alignments were made using CLUSTAL_X (Thompson et al., 1997), taking into account the corresponding amino acid alignments for protein-coding genes. To assess the influence of noise due to saturation of the third codon position, we performed incongruence-length difference (ILD) tests (Farris et al., 1994), as implemented in PAUP* version 4.0b10 (Swofford, 2002), using the different codon positions as separate partitions in 1250 replications. As a further test to assess saturation, we used the index of substitution saturation described by Xia et al. (2003) as implemented in DAMBE version 4.2.13 (Xia & Xie, 2001). The same set of strains was used for all genes and sequence data for Caulobacter crescentus CB15, extracted from the complete genome sequence (Nierman et al., 2001), were used as an outgroup. Neighbour-joining (NJ), maximum-parsimony (MP) and maximum-likelihood (ML) analyses were performed with PAUP*. NJ analyses were performed using the Kimura-2 correction and 1000 bootstrap replications; MP analyses were performed using the heuristic search option. For the 16S rRNA gene, this resulted in 46 best trees, which were reduced to five by repeating the analysis using the Goloboff-fit criterion (GK=2; Goloboff, 1993). For ML analyses, the optimal models of nucleotide substitution were estimated using the program MODELTEST 3.7 (Posada & Crandall, 1998), using both hierarchical likelihood ratio tests and the Akaike Information Criterion (AIC). Using both options, the same model was obtained in all cases, except for the 16S rRNA gene and dnaK, where models obtained with AIC were used (Posada & Buckley, 2004). The MP trees were used as starting trees for the heuristic search procedure. Bootstrap analyses were performed using 1000 replications of heuristic searches for MP and 100 replications for ML. The ILD test implemented in PAUP* and using 1250 replicates was used to assess incongruence between datasets. To assess particular hypotheses, the Shimodaira–Hasegawa test (Shimodaira & Hasegawa, 1999) as implemented in PAUP* was used. The significance of differences in the likelihood scores was assessed by a bootstrap test with 1000 replications.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). We also included corresponding sequences retrieved from the complete genome sequences of Agrobacterium tumefaciens C58, Brucella melitensis 16MT, Brucella suis 1330T, C. crescentus CB15, Rhodopseudomonas palustris CGA009 and Bradyrhizobium japonicum USDA 110, available in GenBank/EMBL/DDBJ. The choice of genes was guided by the following requirements: single copy occurrence, relatively high degree of conservation (as established from the literature) and separate location on the main chromosome (as assessed from the complete genome of S. meliloti). Sequence alignments were inspected visually to identify positions with uncertain alignment to be omitted from further analyses. The lengths of the alignments used for further analysis were: 498 bp for recA, 975 bp for glnA, 639 bp for gltA, 285 bp for dnaK, 636 bp for thrC and 1419 for the 16S rRNA gene. No parts of the sequences were omitted, except in the case of the 16S rRNA gene for the type strain of Rhizobium leguminosarum where, in two different positions, a single C residue was omitted because it was absent from all other taxa included in the alignment.
Individual gene analyses
For each Ensifer species (or genomovar) studied, sequences of the same gene for the various strains included (two to four per species) were highly similar (ranging from 98.0–100 % for gltA to 98.7–100 % for glnA; values for 16S rRNA genes were 99.6–100 %). Initial NJ trees that included all strains revealed that, in all cases, strains belonging to a single species clustered very closely together. For this reason, the type strains only were selected for further analysis in order to reduce computing times (except for ‘S. morelense’ where both strains were included, E. adhaerens where the three genomovars were represented and B. japonicum where subgroups I and Ia were represented). Among Ensifer species, the sequence similarities of the same housekeeping genes were clearly lower, ranging from 79.5–94.2 % for thrC to 87.5–96.9 % for glnA (for the 16S rRNA genes values were in the range 97.9–99.8 %). For the calculation of these inter-species sequence similarity ranges, Ensifer xinjiangensis (two strains) was excluded because, in all comparisons, it was identical or very similar to Ensifer fredii [four strains; 99–100 % sequence similarity for all genes except gltA (98.3–100 %)], strongly suggesting that the two species are in fact synonymous. The clear gap between within-species and between-species sequence similarity values (Fig. 1) demonstrates the greater potential for species discrimination using protein-encoding housekeeping genes compared with 16S rRNA genes, where there is in fact no gap. With any of the housekeeping genes used here, discrimination between the existing species and genomovars (except E. fredii and E. xinjiangensis) is clear and thus, for species identification purposes in Ensifer, these genes are superior to the 16S rRNA gene.
According to ILD tests using first, second or third codon positions as separate partitions in pairwise comparisons, potential third codon saturation was found not to cause conflicting phylogenetic signals for most genes. For thrC, the ILD test gave a P value of 0.868 for the datasets containing only the first and the second positions, respectively, and 0.0376 for the datasets containing the first and third codon positions, respectively. However, based on the P value of 0.5816 for a comparison of the partitions containing the second and the third codon positions, respectively, we decided to include the third position in further analyses. Furthermore, the indices of substitution saturation (Xia et al., 2003) did not indicate any significant saturation for any of the genes (data not shown).
Sequence characteristics are provided in Table 3 and demonstrate that the housekeeping genes are more informative than 16S rRNA genes. glnA was the largest gene fragment sequenced (975 bp) and contained the greatest number of parsimony-informative characters (388), representing 40 % of sequenced positions. By comparison, thrC was one-third shorter in length, contained 60 % informative characters and was the most information-rich of the genes studied. dnaK was the shortest fragment but, of its 285 positions, 51 % were still parsimony informative.
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. Significant groupings in the different phylogenies are discussed below; however, it is clear that the different genes yield phylogenies that can differ to varying extents. This may be due to lateral gene transfer events, but also to the size of the dataset (in particular, the dnaK sequence is relatively short) and the taxa included. Bootstrap support for the observed groups was not always high in the ML trees, which generally have lower bootstrap values than corresponding NJ and MP trees (see below and Table 4).
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Concatenated sequence analyses
One approach to combined data analysis is to combine only congruent data and to exclude data partitions with a significant level of incongruence, as they can introduce errors that may obscure reliable data and lead to erroneous topologies (Bull et al., 1993; Miyamoto & Fitch, 1995).
The congruence of the different gene phylogenies was assessed using pairwise ILD tests. This revealed that dnaK, thrC and glnA had compatible phylogenetic signals (P values of 0.112–0.0936) and recA also showed significant congruence with glnA (P=0.0640). For dnaK this was surprising as visual comparison of gene trees (Fig. 2) shows a rather aberrant grouping. We assume that the tree represents a reduced picture of the information content and in this case highlights particular relationships that do not have significant bootstrap support. This may be related to the relatively short sequence used for this gene so that a strong spurious signal can dominate the information content. In the ILD test, this effect appeared to be levelled out to reveal the underlying signal, which in this case appears congruent with thrC and glnA.
Surprisingly, none of the housekeeping genes showed significant congruence with the 16S rRNA gene (P<0.01). This may be due to intragenic mosaicism, which has been reported previously to occur in these organisms (Eardly et al., 2005; Vinuesa et al., 2005). We divided the alignment into three partitions (1–357, 358–744 and 745–1419) that were reported previously to have different phylogenetic affiliations in Rhizobium galegae (Eardly et al., 2005). ILD tests were repeated for each of these partitions against each of the five housekeeping genes. No significant congruence was observed (P<0.05 in all combinations), indicating that mosaicism may be present in these partitions. To establish this with certainty, more detailed analyses of the alignments will be needed.
As a further test to detect aberrant phylogenetic signals, we performed an ILD test for each gene versus a partition comprising the remaining five genes (Table 3, last column). This test gives an indication of how well the phylogenetic signal in one gene is in-line with the phylogenetic signal of the other five genes taken together. Again, glnA, dnaK and thrC gave P values of greater than 0.05, whereas recA yielded a P value of 0.0320 and 16S rRNA and gltA had P values of less than 0.01. The values for the 16S rRNA gene and gltA could not be improved by eliminating one or two taxa potentially responsible for incongruence as deduced by visual inspection of the trees (data not shown). However, in the case of recA, exclusion of Rhizobium huautlense, Rhizobium galegae or both yielded P values of 0.3024, 0.0944 and 0.1064, respectively, indicating that these species contribute strongly to the incongruence of the recA dataset. In view of the ILD test results, we concatenated the aligned sequences for dnaK, thrC and glnA only and obtained an alignment of 1896 nucleotides, comprising 782 invariable sites, 198 variable but parsimony-uninformative sites and 916 (48 %) parsimony-informative sites. MODELTEST was used to select the optimal substitution model for ML analysis (Table 3). NJ, MP and ML trees were now very similar and bootstrap support was generally higher (Fig. 3).
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) and will contribute more strongly to the branching patterns near the tips, whereas in the deeper parts of the tree their phylogenetic signal may be less clear because of noise from the higher number of substitutions. However, the effect of noise from individual partitions may be reduced in the larger complete dataset (Barrett et al., 1991; Kluge, 1989). The concatenated alignment of the six genes comprised 4452 positions, 2464 and 1613 (36 %) of which were constant and parsimony informative, respectively. MODELTEST was used to select the most suitable substitution model for ML analysis. Again, NJ, MP and ML trees were very similar and bootstrap support was high (Fig. 4). There was excellent agreement with the trees obtained based on the concatenation of dnaK, thrC and glnA. In this case, the combination of six genes with varying levels of congruence appeared to level out aberrant signals that may be present in partly incongruent partitions, to yield trees that are highly similar, both in topology and bootstrap support, to those obtained by concatenating only congruent partitions (Fig. 3).
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presents a simple but effective way of assessing the most strongly supported groups in all 24 NJ, MP and ML analyses. As was expected, the two reference strains of Brucella and those of Mesorhizobium each grouped together always. Similarly, Rhodopseudomonas palustris always grouped with Bradyrhizobium species, peripheral to the Bradyrhizobium cluster in the recA, gltA, thrC and glnA phylogenies and inside the Bradyrhizobium cluster according to 16S rRNA and dnaK gene phylogenies. In the case of dnaK, Rhodopseudomonas palustris grouped closer to Bradyrhizobium elkanii whereas, according to the 16S rRNA gene phylogeny, the same species is closer to Bradyrhizobium japonicum groups I and Ia. Agrobacterium radiobacter, Agrobacterium tumefaciens and Agrobacterium rubi were grouped together with high bootstrap support in all analyses except the dnaK phylogeny. Rhizobium galegae and Rhizobium huautlense grouped together with high bootstrap support in the 16S rRNA gene and in the two combined phylogenies, whereas Rhizobium gallicum, Rhizobium leguminosarum and Rhizobium rhizogenes grouped together with significant support only in the combined analyses. In Ensifer, several groupings recurred, but not always with strong bootstrap support. A close relationship between E. meliloti, E. arboris and E. medicae was strongly supported. E. adhaerens grouped particularly with ‘S. morelense’; bootstrap support for this relationship was strong in the glnA and thrC phylogenies and the combined analyses. For ‘S. morelense’ we included the type strain and strain LMG 20571. The latter strain was previously assigned as representing ‘S. morelense’ on the basis of phenotypic and protein profile similarities (Willems et al., 2003). Our present data suggest that it may actually represent a separate but closely related genospecies. Our data do not support the proposal (Young, 2003) that ‘S. morelense’ is a later heterotypic synonym of E. adhaerens, but confirm that it represents a separate species, as has been established previously (Willems et al., 2003). The name of the species ‘S. morelense’ is considered to not be validly published because, at the time of publication, the type strain was not deposited in two publicly accessible culture collections in different countries (Euzéby & Tindall, 2004). The type strain is now available from the BCCM/LMG (Belgium, http://bccm.belspo.be/about/lmg.php) and NBRC (Japan, http://www.nbrc.nite.go.jp/e/index.html) culture collections and thus fulfils the criteria for validation. However, because the name is the subject of a pending Request for an Opinion (Euzéby & Tindall, 2004), ‘S. morelense’ cannot be transferred to the genus Ensifer before a ruling is published. The name of the species will only be established when an opinion from the Judicial Commission is approved and published. Therefore, at present we cannot adopt the Ensifer nomenclature for this species and the species name ‘S. morelense’ is used. It is evident from most trees that Ensifer adhaerens groups with the former Sinorhizobium species, although the bootstrap support for the whole group is strong only in the thrC phylogeny and in the two combined analyses of three and six genes. Our data thus support the proposal to unite the genera Ensifer and Sinorhizobium. The topologies of some trees suggest the possibility that E. adhaerens and ‘S. morelense’ together form a separate group that may be regarded as a sister taxon to the rest of Ensifer (the former Sinorhizobium). However, bootstrap support is high only in the thrC phylogeny and in the two combined analyses. We tested this hypothesis by using the Shimodaira–Hasegawa test (Shimodaira & Hasegawa, 1999): for each gene, new ML trees (using the substitution models selected in MODELTEST) were constructed with the constraint that E. adhaerens and ‘S. morelense’ should form a group, the remaining Ensifer species should form a second group and that the two groups form a single cluster separate from the other taxa included. The likelihood of the constrained tree was compared with the likelihood of the original unconstrained ML tree using the Shimodaira–Hasegawa test. The test showed significant support for the hypothesis in all five housekeeping genes and for the 16S rRNA gene (Table 5). Running the test for the concatenated dataset of dnaK, glnA and thrC and for the concatenation of the six genes yielded P values of 1 and 0.1454, respectively.
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; van Berkum et al., 2003; Vinuesa et al., 2005). Taken together, these observations indicate that, at least in this group, 16S rRNA gene data should be interpreted with caution and should always be compared with data from other genes so as to minimize the impact of possible recombination events when assessing phylogenetic relationships for taxonomic purposes.
The new data presented here confirm the close relationship between Ensifer and the former genus Sinorhizobium and provide supporting evidence for uniting the two genera, as proposed by Young (2003). An alternative scenario in which Ensifer would be restricted to E. adhaerens and ‘S. morelense’ and the genus Sinorhizobium maintained for the other Ensifer species had some support as apparent from the Shimodaira–Hasegawa test and analyses of concatenated sequences, but little support from the individual gene analyses. Furthermore, redefining genera in this group would require additional biological data. We conclude that, from the present evidence of six genes, classification as a single genus Ensifer is most appropriate.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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