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RevisedphylogenyofRhizobiaceae:ProposalofthedelineationofPararhizobiumgen.nov.,and13newspeciescombinations

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Systematic and Applied Microbiology

j ourna l h omepage: www.elsev ier .de /syapm

evised phylogeny of Rhizobiaceae: Proposal of the delineation ofararhizobium gen. nov., and 13 new species combinations

eyed Abdollah Mousavia,e,∗, Anne Willemsb, Xavier Nesmec, Philippe de Lajudied,ristina Lindströme

University of Helsinki, Division of Microbiology and Biotechnology, Department of Food and Environmental Sciences, Biocentre 1, Viikinkaari 9, P.O. Box6, Helsinki FIN-00014, FinlandLaboratory of Microbiology, Department of Biochemistry and Microbiology, Faculty of Science, Ghent University, K. L. Ledeganckstraat 35, B-9000 Gent,elgiumUniversity of Lyon; Université Lyon1; Ecologie Microbienne, UMR CNRS 5557/USC INRA 1364, 16 rue R. Dubois, F-69622 Villeurbanne cedex, FranceIRD, Laboratoire des Symbioses Tropicales et Méditerranéennes, Campus de Baillarguet TA A-82/J, 34398 Montpellier Cédex 5, FranceUniversity of Helsinki, Department of Environmental Sciences, Viikinkaari 2a, P.O. Box 65, Helsinki FIN-00014, Finland

r t i c l e i n f o

rticle history:eceived 23 September 2014eceived in revised form 7 December 2014ccepted 12 December 2014

eywords:LSAousekeeping genes

a b s t r a c t

The family Rhizobiaceae accommodates the seven genera Rhizobium, Neorhizobium, Allorhizobium,Agrobacterium, Ensifer (syn. Sinorhizobium), Shinella and Ciceribacter. However, several so-called Rhizo-bium species do not exhibit robust phylogenetic positions. Rhizobium is extremely heterogeneous and isin need of major revision. Therefore, a phylogenetic examination of the family Rhizobiaceae by multilocussequence analysis (MLSA) of four housekeeping genes among 100 strains of the family was undertaken.Based on the results we propose the delineation of the new genus Pararhizobium in the Rhizobiaceae fam-ily, and 13 new species combinations: Agrobacterium nepotum comb. nov., Agrobacterium pusense comb.

hizobiaceae nov., Agrobacterium skierniewicense comb. nov., Allorhizobium vitis comb. nov., Allorhizobium taibaisha-nense comb. nov., Allorhizobium paknamense comb. nov., Allorhizobium oryzae comb. nov., Allorhizobiumpseudoryzae comb. nov., Allorhizobium borbori comb. nov., Pararhizobium giardinii comb. nov., Pararhizo-bium capsulatum comb. nov., Pararhizobium herbae comb. nov., and Pararhizobium sphaerophysae comb.nov.

© 2015 Published by Elsevier GmbH.

ntroduction

The family Rhizobiaceae Conn 1938, belongs to the order Rhizo-iales in the class Alphaproteobacteria. The Rhizobiaceae comprisesix genera harboring plant-associated species, Rhizobium, Neorhi-obium, Allorhizobium, Agrobacterium, Ensifer (syn. Sinorhizobium),hinella, and the genus Ciceribacter described for strains isolatedrom chickpea rhizosphere soil and representing one species. Thistudy mainly focuses on the plant-associated (either pathogenic

Please cite this article in press as: S.A. Mousavi, et al., Revised phylogegen. nov., and 13 new species combinations, Syst. Appl. Microbiol. (20

r nitrogen-fixing that were isolated from plant tissues) bacterialembers of the Rhizobiaceae family.

∗ Corresponding author at: Department of Environmental Sciences, Viikinkaaria, P.O. Box 65, University of Helsinki, Helsinki FIN-00014, Finland.el.: +358 44 3527991.

E-mail addresses: s.abdollah.mousavi@gmail.com, seyed.mousavi@helsinki.fiS.A. Mousavi).

ttp://dx.doi.org/10.1016/j.syapm.2014.12.003723-2020/© 2015 Published by Elsevier GmbH.

The current classification in this family is mostly based on phe-notypic features, DNA–DNA hybridizations, and 16S rRNA genesequences. Since the genomes of rhizobia may lose or gain plasmidsor genomic islands bearing genes governing catabolic capacities,performing phenotypic tests, mostly for utilization of carbon andnitrogen sources, may not be that informative for rhizobial tax-onomy [8,24]. DNA:DNA hybridization (DDH) is a recommendedmethod to delineate novel species; nevertheless, in practice theobtained results of the DDH are different because of variationbetween laboratories and methods [10,18]. The 16S rRNA gene isconsidered as a suitable molecular marker for phylogenetic analy-ses. However several studies pointed out that the 16S rRNA genephylogeny may not provide a perfect snapshot of evolution ofprokaryotes, since the gene may be subject to horizontal genetransfer and genetic recombination at high frequency [40]. More-

ny of Rhizobiaceae: Proposal of the delineation of Pararhizobium15), http://dx.doi.org/10.1016/j.syapm.2014.12.003

over, a phylogeny based on 16S rRNA genes does generally notallow resolution of closely related species due to the high degree ofconservation of the gene. Because of the drawbacks of the abovesystematics methods, MLSA of housekeeping genes has become

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ore favored and acceptable method to establish taxonomy ofrokaryotes. It is now widely assumed that phylogeny based oneveral well-chosen housekeeping genes, exhibiting sufficient con-ervation and distribution in bacterial genomes, accurately reflectshe phylogeny of the bacteria [20,21].

Some long standing taxonomic uncertainties in the family Rhi-obiaceae have been clarified by MLSA results. For instance, thencertain phylogenetic position of the former R. galegae and threeelated species was resolved by transferring the four species, R. gale-ae, R. vignae, R. alkalisoli, and R. huautlense to Neorhizobium gen.ov. [22]. One of the most controversal genera in the Rhizobiaceae

s Agrobacterium, a group of occasionally pathogenic bacteria phy-ogenetically close to Rhizobium. On the basis of 16S rRNA genehylogeny, Young et al. [47] transferred the Agrobacterium specieso Rhizobium, which introduced a controversy about the validityf the genus Agrobacterium [9,47]. However, several studies con-luded that Agrobacterium must be retained as a genus name forhe species A. radiobacter, A. rubi, “A. fabrum”, and agrobacterialenomovars (G1–G13) that have not yet received a Latin bino-ial [4,19]. More species currently classified in Rhizobium, i.e. R.

kierniewicense and R. nepotum, are candidates to join Agrobac-erium [30], while the former Agrobacterium rhizogenes is nowefinitively designated Rhizobium rhizogenes [19]. Rhizobium is

heterogeneous genus accommodating two major sub-clusters,round R. tropici and R. leguminosarum respectively. The genusame Rhizobium encompasses 56 species; however, it is not aroper name for all, since some of them are phylogenetically

nterspersed among members of other genera in Rhizobiaceae. Fornstance, R. giardinii and R. oryzae did not group with Rhizobium

embers in several MLSA studies [20,22]. The genus name Allorhi-obium was designated for one species, Al. undicola [5]; furthertudies showed that Agrobacterium vitis and R. taibaishanense clus-ered with A. undicola [22]. Another controversial group in thehizobiaceae family is the genus Sinorhizobium (syn. Ensifer). Theenus Ensifer was created for the species E. adhaerens by Casida [2];owever, Willems et al. [42] proposed that the species E. adhaerens

s phylogenetically related to the larger group of Sinorhizobium spp.he genus Sinorhizobium was created by Chen et al. [3] and con-ained 11 species by 2007. The phylogenetic relationships of 16SRNA gene sequences of Ensifer adhaerens and Sinorhizobium spp.ere so close that it caused nomenclatural controversies [42,46].owever, according to the rules of Bacteriological Code giving pri-rity to older name, the genus name Ensifer was adopted for bothinorhizobium and Ensifer taxa (Opinion 84 of the Judicial Com-ission). The genus Ensifer now contains 15 species. Although the

umber of new species of the family Rhizobiaceae has been increas-ng dramatically, the phylogeny of all species of the family has noteen simultaneously studied recently. Including more taxa of theamily could be considered as a solution to estimate the phylogenyf the family more robustly.

In order to solve the taxonomic uncertainties concerning thelant-associated members of the Rhizobiaceae family, we per-ormed MLSA of 100 strains of the family Rhizobiaceae and 16hizobial strains from other rhizobial families, using four house-eeping genes namely 16S rRNA, atpD (ATP synthase F1, betaubunit), recA (recombinase A), and rpoB (RNA polymerase, betaubunit).

aterial and methods

acterial samples, DNA preparation

Please cite this article in press as: S.A. Mousavi, et al., Revised phyloggen. nov., and 13 new species combinations, Syst. Appl. Microbiol. (20

We studied a total of 100 strains representing 81 species of theamily Rhizobiaceae that were described and validated by January014 in the present work (Table S1). Strains for which the sequences

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were not available in GenBank were obtained from LMG and HAMBIculture collections. The strains received from culture collectionswere grown in 5 ml Tryptone-Yeast Extract (TY) broth at 28 ◦C for48 h, and were then cultured on yeast mannitol agar (YMA) mediumat 28 ◦C for 2–3 days [34]. Single colonies of the bacteria were grownin 5 ml TY broth for preservation in 20% glycerol–YEM broth at−80 ◦C and DNA isolation. The UltraClean Microbial DNA IsolationKit (MO BIO Laboratories, Inc.) was used for DNA extraction fromthe samples. The DNA samples were kept at −20 ◦C.

PCR amplification and gene sequencing

For the MLSA approach, four housekeeping genes, 16S rRNA,atpD, recA, and rpoB, were used in this study. Most of thesequences were obtained from GenBank (http://www.ncbi.nlm.nih.gov/genbank). PCR amplification and sequencing were per-formed according to Mousavi et al. [22]. The sequences werechecked and edited using GAP4 [35], and blasted (http://blast.ncbi.nlm.nih.gov/Blast.cgi?CMD=Web&PAGE TYPE=BlastHome). Theaccession numbers of the sequences of the studied housekeepinggenes are listed in Table S1.

Sequence analyses

Newly obtained nucleotide sequences, together with sequencesobtained from GenBank (http://www.ncbi.nlm.nih.gov/genbank),were aligned applying MUSCLE [7] software at EML-EBI [11] for 16SrRNA, and ClustalW [17] in BioEdit version 7.0.5.3 [12] for atpD,recA, and rpoB genes. The alignments were edited manually. Thebest-fit nucleotide substitution models were selected accordingto Akaike information criterion (AIC) for each locus using MEGA5[36]. The maximum likelihood phylogenetic trees based on the 16SrRNA sequences were constructed with 1000 bootstrap (BS) repli-cates; and the mean distance between the groups was computedby MEGA5. Bayesian analyses of the concatenated four housekeep-ing genes were implemented by running the algorithm Metropoliscoupled Markov chain Monte Carlo (MCMCMC) for 20 million gen-erations twice with MrBayes 3.2 [32]. The program Tracer v1.5was used to analyze the trace files generated by Bayesian MCM-CMC runs, and the Bayesian trees were visualized by FigTree v1.4.0(http://tree.bio.ed.ac.uk/software/figtree/).

Results

Analyses of 16S rRNA gene sequences and individualprotein-coding housekeeping genes

The general time reversible plus gamma distribution plus invari-able sites (GTR+G+I) model was selected as the best-fit modelof phylogenetic analysis of the sequences of the 16S rRNA genedataset. The 16S rRNA gene sequences (1279 bp) of 160 rhizobialstrains (listed in Table S1), including 81 species of the family Rhi-zobiaceae, were analyzed by maximum likelihood phylogeny (Fig.S1). The taxa belonging to the genera Agrobacterium, Allorhizo-bium, Ciceribacter, Ensifer, Neorhizobium, Ochrobactrum, Rhizobium,and Shinella stand in a moderately supported clade (BS = 71%). Thegeneric clade accommodating A. radiobacter, “A. fabrum”, A. rubi,A. larrymoorei, and the genomovars that have not received a Latinbinomial, is strongly supported (BS = 84%). The Rhizobium-affiliatedspecies do not form a monophyletic group, with several of themrepresenting separate lineages. Thirty-one plant-isolated Rhizo-

eny of Rhizobiaceae: Proposal of the delineation of Pararhizobium15), http://dx.doi.org/10.1016/j.syapm.2014.12.003

bium species group together and form the Rhizobium clade in the16S rRNA tree, with two groups, the so-called R. tropici group, con-sisting of 11 species and the so-called R. leguminosarum sub-cluster,with low confidence branching (<70%). The Ensifer cluster forms a

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ighly supported clade (BS = 87%). Two member species of the fam-ly Brucellaeceae, Ochrobactrum lupini and O. cytisi, are placed insidehe Rhizobiaceae clade.

The GTR+G+I model was chosen as the best-fit nucleotide sub-titution model for atpD, recA, and rpoB genes based on Akaikenformation criterion, corrected (AICc). The individual trees, fortpD (489 bp), recA (459 bp), and rpoB (583 bp) loci, based onayesian inference were constructed performing two runs of theCMCMC algorithms for 10 million generations. The individual

rees constructed for each of the three studied protein-codingousekeeping genes, atpD, recA and rpoB, showed disparate branch-

ng patterns. Among the family Rhizobiaceae, only the genuseorhizobium formed a robust monophyletic lineage with highosterior probability (0.97 < PP ≤ 1.00) in the individual gene treesdata not shown).

oncatenated sequence analyses (phylogenetic groups)

A combined gene phylogenetic tree was constructed usingayesian approach. The GTR+G+I model was selected as the best-fitodel of phylogenetic analysis of the partitions of the com-

ined dataset, rrs–atpD–recA–rpoB. The MCMCMC algorithms wereun twice for 10 million generations, and the trees were sam-led every 1000. Sequences of the four housekeeping genes, 77lant-associated strains of the family Rhizobiaceae along with 23on-plant-associated related Rhizobium strains and 16 rhizobialtrains from other rhizobial families were applied in the concate-ated gene tree (Table S1). Eleven lineages with high posteriorrobabilities (≥0.95) can be distinguished in the combined generee (A1–G in Fig. 1), from which nine lineages (clades A1–4,, D, E, and G) accommodate the so-called Rhizobium species.

n addition five species R. tarimense, R. cellulosilyticum, R. soli, R.etrolearium, and R. phenanthrenilyticum clustered with the genuseorhizobium; however, at a low posterior probability (PP = 0.86).he species R. halophytocola, and R. subbaraonis were separateFig. 1). The genus Neorhizobium including N. galegae, N. alkalisoli,nd N. huautlense forms clade B with high posterior probability1.00). Two pathogenic species of so-called Rhizobium, R. nepotumnd R. skierniewicense, grouped with A. radiobacter, “A. fabrum”,. rubi, A. larrymoorei, and the well-delineated unnamed Agrobac-erium genomovars (clades C1 and C2).

Three aquatic species, Agrobacterium albertimagni, R. roset-iformans and R. aggregatum, along with R. naphthalenivorans,. selenitireducens, and R. daejeonense formed sister lineages ofgrobacterium in the concatenated gene tree including non-plant-

solated species (Fig. 1). Furthermore, the species A. vitis, R.aibaishanense, Al. undicola, R. paknamense, R. oryzae, R. pseudoryzae,nd “R. qilianshanense” formed a monophyletic lineage, which had. borbori as an outlier. The aligned atpD gene sequences showedhat the members of the clade E harbor two sites (9 bp and 6 bp),hich are unique to this group in the family Rhizobiaceae. All Ensifer

pecies clustered together (PP = 1); however, E. adhaerens appeareds an outlier of this genus. Surprisingly, the species Blastobacterapsulatus grouped with R. giardinii, R. herbae, “R. helanshanense”,nd R. sphaerophysae with the posterior probability of 1.00 (clade); however, the clade was located close to the Ensifer clade and

ar from the genus Rhizobium. The species Shinella kummerowiae iseparate in Rhizobiaceae. At a higher taxonomic level, the generagrobacterium, Allorhizobium, Ciceribacter, Ensifer, Neorhizobium,hizobium, and Shinella were grouped together with high posteriorrobability (1.00), and formed the Rhizobiaceae lineage distantlyrom other rhizobia.

Please cite this article in press as: S.A. Mousavi, et al., Revised phylogegen. nov., and 13 new species combinations, Syst. Appl. Microbiol. (20

The mean distances between groups A–G of the concate-ated gene tree (Fig. 1) were computed based on 16S rRNA geneequences (Fig. S1, panel B). The distance between the clades

and D is lower than 0.05. Excluding missing data from the

PRESSd Microbiology xxx (2015) xxx–xxx 3

datasaet showed that applying taxa which are missing sequencesdid not affect the topoly of the conctaneated gene tree (data notshown).

Discussion

The present MLSA of four housekeeping genes of 100 strainsrepresenting 81 species in the Rhizobiaceae family and 16 strainsbelonging to other bacterial families that contain some rhizobia,clarified several taxonomic uncertainties in this family.

Though the 16S rRNA gene is a widely recommended phyloge-netic marker to study rhizobial taxonomy, it has reached its limitsdue to the issues of horizontal gene transfer and strong sequenceconservation. Vinuesa et al. [41] reported some of the imperfec-tions of the 16S rRNA inferred phylogeny specifically in the caseof rhizobia; for instance, mosacism of 16S rRNA sequences, andvariations of 16S rRNA phylogenetic representation depending onincluded strains. We included a wide selection of taxa representingother rhizobial families (160 taxa in total) in our 16S rRNA phy-logenetic studies to optimize the topology. The Rhizobium speciesform a polyphyletic clade in the 16S rRNA gene based tree and thecombined gene tree of this study. The species Blastobacter capsula-tus, R. giardinii, R. herbae, R. sphaerophysae, and “R. helanshanense”form a monophyletic group with high posterior probability distantfrom the genus Rhizobium in the combined gene tree. Four so-calledRhizobium species, R. giardinii, R. herbae, R. sphaerophysae, and “R.helanshanense” grouped either with Neorhizobium (formerly Rhi-zobium) or in Agrobacterium in their original papers [1,29,31,43].These results are consistent with the previous observation thatR. giardinii does not belong to the Rhizobium genus [22]. Thegenus Blastobacter was described by Zavarzin [48] and representsa heterogeneous group in Alphaproteobacteria. Hugenholz et al.[14] proposed that, with the exception of Bl. capsulatus, the otherdescribed species should be reclassified. Two former Blastobac-ter species were transferred to Bradyrhizobiaceae and Rhizobiaceae,respectively as Bradyrhizobium denitrificans and Rhizobium aggrega-tum [15,39]. Bl. capsulatus is now the only remaining species in thegenus Blastobacter for which strains are available as no cultures areavailable of the type species Bl. henricii (http://www.bacterio.net)and its phylogenetic position therefore is uncertain. Consideringthe results of the MLSA, we propose to transfer the species Bl. capsu-latus, R. giardinii, R. herbae, “R. helanshanense”, and R. sphaerophysaeto a novel genus in the Rhizobiaceae family. The name Pararhizo-bium gen. nov. (Pa.ra.rhi.zo’bi.um), which means adjacent (Para)Rhizobium is proposed for this group of the family Rhizobiaceae.Moreover, the species A. vitis, R. taibaishanense, R. paknamense, R.oryzae, R. pseudoryzae, “R. qilianshanense” and R. borbori formeda monophyletic clade with high posterior probability (1.00) withAl. undicola, distant from the genera Rhizobium and Agrobacterium.Grouping of Allorhizobium undicola and Agrobacterium vitis waspointed out in previous studies by Costechareyre et al. [4] andMousavi et al. [22]. Thus, Costechareyre et al. proposed the transferof Allorhizobium to Agrobacterium; in contrast, Mousavi et al. pro-posed to transfer Agrobacterium vitis to Allorhizobium. The results ofthe current research support the latter proposal along with the fur-ther transfer of Rhizobium taibanshenase, R. paknamense, R. oryzae,R. pseudoryzae, “R. qilianshanense” and R. borbori to Allorhizobium.The genus Allorhizobium exhibits two unique sequence motifs inthe atpD locus that differentiate this genus from other genera inthe family Rhizobiaceae. Including the related non-plant-associatedspecies R. borbori in the combined gene dataset showed that, R. sub-

ny of Rhizobiaceae: Proposal of the delineation of Pararhizobium15), http://dx.doi.org/10.1016/j.syapm.2014.12.003

baraonis and R. haophytocola are separate from the genus Rhizobiumand from the other genera in the Rhizobiaceae family.

In addition, three Rhizobium species, R. nepotum, R. pusense,and R. skierniewicense, grouped in the Agrobacterium clade. Thus

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Fig. 1. Phylogenetic tree of four concatenated housekeeping genes (rrs–atpD–recA–rpoB) of 116 rhizobial and agrobacterial strains, constructed based on a Bayesian inferencea re shoA type s

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nalysis. Only the family Rhizobiaceae clade and the posterior probabilities ≥0.95 allorhizobium, E., Ensifer, N., Neorhizobium, P., Pararhizobium, and R., Rhizobium. The

he genus name Agrobacterium seems the proper genus nameor these three species. The placement of R. nepotum, R. pusense,nd R. skierniewicense in the Agrobacterium clade was previouslyuggested [27,28,30]; however, due to current taxonomic classifi-ation (transfer of all Agrobacterium species to Rhizobium), the threepecies were described as Rhizobium. The species Agrobacteriumlbertimagni, R. rosettiformans, R. aggregatum, R. naphthalenivo-ans, R. selenitireducens, and R. daejeonense were placed in a sisterlade of Agrobacterium (the “R. aggregatum complex”). Neverthe-ess, members of this sister clade of Agrobacterium have neither ainear chromid nor the protelomerase encoding gene telA (unpub-ished results) that are the specific characteristic of this genus30]. Considering the results of MLSA and the lack of Agrobac-erium specific traits, the species of the “R. aggregatum complex”ould be considered as a separate genus, though the sequencesf 16S rRNA gene of this group are over 96% identical with the

Please cite this article in press as: S.A. Mousavi, et al., Revised phyloggen. nov., and 13 new species combinations, Syst. Appl. Microbiol. (20

grobacterium clade. Seven Rhizobium species including R. soli,. tarimense, R. cellulosilyticum, R. petrolearium, R. phenanthreni-

yticum, R. halophytocola, and R. subbaraonis do not group withny of the well-delineated clades of the genera Agrobacterium,

wn in the tree. The genus names are abbreviated as follows: A., Agrobacterium, Al.,trains are shown by a “T” at the end of each strain code.

Allorhizobium, Pararhizobium, Ciceribacter, Ensifer, Neorhizobium,Rhizobium, and Shinella.

In conclusion, based on the results of this MLSA study of theplant-associated species of the family Rhizobiaceae and severalspecies of the family that were not isolated from plants, we proposenew species combinations for 13 species. In addition, potentiallytwo more genera could be described in the family Rhizobiaceaein the future; nonetheless, further phylogenomic analyses arerequired to establish a robust phylogenetic representation of thisfamily.

Description of Pararhizobium gen. nov.

Pararhizobium gen. nov. (Pa.ra.rhi.zo’bi.um. Gr. prep. para,beside, alongside of; N.L. neut. n. Rhizobium a bacterial genericname; N.L. neut. n. Pararhizobium, a genus adjacent to Rhizobium).

eny of Rhizobiaceae: Proposal of the delineation of Pararhizobium15), http://dx.doi.org/10.1016/j.syapm.2014.12.003

Aerobic, Gram-negative, non-spore-forming rods that are0.3–0.9 �m wide by 1.2–2.5 �m long. Colonies are circular, con-vex, white or cream color, translucent to opaque, with a diameterof 1–4 mm within 2–4 days on YMA at 28 ◦C. The maximum growth

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emperature is 35–40 ◦C. Pronounced turbidity develops after 2–3ays in agitated broth media. The type species of the genus Pararhi-obium is P. giardinii (Rhizobium giardinii, Amarger et al. 1997).

The genus Pararhizobium includes four validated species, P.iardinii, P. capsulatum, P. herbae, and P. sphaerophysae, the G+Content of which varies from 57.6 mol% to 63.5 mol%. The mem-ers of the genus are dispersed worldwide, and were isolated from:haseolus vulgaris, Astragalus membranaceus, Oxytropis cashmiriana,aragana sinica, Albizia kalkora, Kummerowia stipulacea, Astragalusanicus, Sphaerophysa salsula, and fresh water. The fatty acid pro-les of the novel genus showed that Pararhizobium species possess14:0, C16:0, C16:0 3OH, C18:0, summed feature 3 (C16:1 iso I/C14:0OH), summed feature 4 (C15:0 iso 2OH/C16:1�7c), and summedeature 7 (C18:1�7c/C18:1�9t/C18:1�12t) fatty acids in common.istinctive features of the species now transferred to Pararhizo-ium, and discriminative features between P. giardinii and otherelated genera were tested comprehensively in previous studies1,5,6,13,29,31,33,37,43], and summarized in Table S2.

escription of Pararhizobium giardinii (Amarger et al., 1997)omb. nov.

The description of the species was provided by Amarger et al.1], Ren et al. [31], Qin et al. [29], Xu et al. [43] and Tighe et al. [37].n addition the species can be differentiated from other Pararhizo-ium species at molecular level by the sequences of 16S rRNA, atpD,lnA, glnII, recA, rpoB, and thrC genes. The whole genome shotgunequence is available for Pararhizobium giardinii H152T in GenBankARBG00000000.1).

Basonym: Rhizobium giardinii by Amarger et al. 1997.The type strain is H152T (=HAMBI 2323T).

escription of Pararhizobium herbae (Ren et al., 2011) comb. nov.

The description of the species is provided by Ren et al. [31]. Inddition the species can be differentiated from other Pararhizobiumpecies at molecular level by G+C content (57.6–59.1 mol%), and theequences of 16S rRNA, atpD, glnII, recA, rpoB, and thrA genes.

Basonym: Rhizobium herbae Ren et al. 2011.The type strain is CCBAU 83011T (=HAMBI 3117T).

escription of Pararhizobium sphaerophysae (Xu et al., 2012)omb. nov.

The description of the species is provided by Xu et al. [43] andin et al. [29]. In addition the species can be differentiated fromther Pararhizobium species at molecular level by G+C content62.9–63.5 mol%), and the sequences of 16S rRNA, atpD, recA andpoB genes.

Basonym: Rhizobium sphaerophysae Xu et al. 2011.The type strain is CCNWGS0238T (=HAMBI 3074T).

escription of Pararhizobium capsulatum (Hirch and Müller,986) comb. nov.

The description of the species is provided by Hirch and Müller13] and Sittig and Hirsch [33]. In addition the species can be dif-erentiated from other Pararhizobium species at molecular level by

Please cite this article in press as: S.A. Mousavi, et al., Revised phylogegen. nov., and 13 new species combinations, Syst. Appl. Microbiol. (20

+C content (58.9–60.2 mol%), and the sequences of 16S rRNA, atpD,ecA and rpoB genes.

Basonym: Blastobacter capsulatus Hirch and Müller 1985.The type strain is IFAM 1004T (=DSM1112T).

PRESSd Microbiology xxx (2015) xxx–xxx 5

Description of Agrobacterium nepotum (Puławska et al., 2012)comb. nov.

The description of the species is provided by Puławska et al. [27].In addition the species can be differentiated from other Agrobac-terium species at molecular level by the sequences of 16S rRNA,atpD, glnA, gyrB, recA, and rpoB genes.

Basonym: Rhizobium nepotum Puławska et al. 2012.The type strain is 39/7T (=LMG 26435T).

Description of Agrobacterium pusense (Panday et al., 2011) comb.nov.

The description of the species is provided by Panday et al. [25]. Inaddition the species can be differentiated from other Agrobacteriumspecies at molecular level by the sequences of 16S rRNA, atpD, andrpoB genes.

Basonym: Rhizobium pusense Panday et al. 2011.The type strain is NRCPB10T (=LMG 25623T).

Description of Agrobacterium skierniewicense (Puławska et al.,2012) comb. nov.

The description of the species is provided by Puławska et al. [28].In addition the species can be differentiated from other Agrobac-terium species at molecular level by the sequences of 16S rRNA,atpD, glnA, gyrB, recA, and rpoB genes.

Basonym: Rhizobium skierniewicense Puławska et al. 2012.The type strain is Ch11T (=LMG 26191T).

Description of Allorhizobium vitis (Ophel and Kerr et al., 1990)comb. nov.

The description of the species is provided by Ophel and Kerr [23],de Lajudie et al. [5,6], and Tighe et al. [37]. In addition the speciescan be differentiated from other Allorhizobium species at molecularlevel by the sequences of 16S rRNA, atpD, glnA, glnII, recA, rpoB, andthrC genes. The sequence of chromosomes 1 and 2 of Al. vitis S4 isavailable in GenBank (BioProject: PRJNA58249).

Basonym: Agrobacterium vitis Ophel and Kerr 1990.The type strain is NCPPB 3554T (=HAMBI 1817T).

Description of Allorhizobium taibaishanense (Yao et al., 2012)comb. nov.

The description of the species is provided by Yao et al. [45]. Inaddition the species can be differentiated from other Allorhizobiumspecies at molecular level by the sequences of 16S rRNA, atpD, glnA,glnII, recA, rpoB, and thrC genes.

Basonym: Rhizobium taibaishanense Yao et al. 2012.The type strain is CCNWSX 0483T (=HAMBI 3214T).

Description of Allorhizobium paknamense (Kittiwongwattana andThawai, 2013) comb. nov.

The description of the species is provided by Kittiwongwattanaand Thawai [16]. In addition the species can be differentiated fromother Allorhizobium species at molecular level by the sequences of

ny of Rhizobiaceae: Proposal of the delineation of Pararhizobium15), http://dx.doi.org/10.1016/j.syapm.2014.12.003

16S rRNA, atpD, glnII, gyrB, recA genes.Basonym: Rhizobium paknamense Kittiwongwattana and

Thawai 2013.The type strain is L6-8T (=NBRC 109338T).

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ARTICLEYAPM-25672; No. of Pages 7

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escription of Allorhizobium oryzae (Peng et al., 2008) comb. nov.

The description of the species is provided by Peng et al. [26], Xut al. [44], Zhang et al. [50], and Kittiwongwattana and Thawai [16].n addition the species can be differentiated from other Allorhizo-ium species at molecular level by the sequences of 16S rRNA, atpD,lnA, glnII, gyrB, recA, rpoB, and thrC genes.

Basonym: Rhizobium oryzae Peng et al. 2008.The type strain is Alt 505T (=LMG 24253T).

escription of Allorhizobium pseudoryzae (Zhang et al., 2011)omb. nov.

The description of the species is provided by Zhang et al. [50],u et al. [44], and Kittiwongwattana and Thawai [16]. In addition

he species can be differentiated from other Allorhizobium speciest molecular level by the sequences of 16S rRNA, atpD, glnII, gyrB,nd recA genes.

Basonym: Rhizobium pseudoryzae Zhang et al. 2011.The type strain is J3-A127T (=ACCC 10380T).

escription of Allorhizobium borbori (Zhang et al., 2011) comb.ov.

The description of the species is provided by Zhang et al. [49] andittiwongwattana and Thawai [16]. In addition the species can beifferentiated from other Allorhizobium species at molecular levely the sequences of 16S rRNA, atpD, glnA, recA, rpoB, and thrC genes.

Basonym: Rhizobium borbori Zhang et al. 2011.The type strain is DN316T (=LMG 23925T).

cknowledgments

We would like to thank Riitta Saastamoinen, Els Vercoutere andlaudine Vereecke for providing us the bacterial strains used inhis study. We thank Janina Österman for technical assistance inene amplification and sequencing. This research was funded by thecademy of Finland (grant number: 132544), Societas Scientiarumennica, and Oskar Öflund Foundation.

ppendix A. Supplementary data

Supplementary data associated with this article can beound, in the online version, at http://dx.doi.org/10.1016/j.syapm.014.12.003.

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