Evolutionary genetics and biogeographic structure of Rhizobium gallicum sensu lato, a widely distributed bacterial symbiont of diverse legumes

We used phylogenetic and population genetics approaches to evaluate the importance of the evolutionary forces on shaping the genetic structure of Rhizobium gallicum and related species. We analysed 54 strains from several populations distributed in the Northern Hemisphere, using nucleotide sequences of three ‘core’ chromosomal genes (rrs, glnII and atpD) and two ‘auxiliary’ symbiotic genes (nifH and nodB) to elucidate the biogeographic history of the species and symbiotic ecotypes (biovarieties) within species. The analyses revealed that strains classified as Rhizobium mongolense and Rhizobium yanglingense belong to the chromosomal evolutionary lineage of R. gallicum and harbour symbiotic genes corresponding to a new biovar; we propose their reclassification as R. gallicum bv. orientale. The comparison of the chromosomal and symbiotic genes revealed evidence of lateral transfer of symbiotic information within and across species. Genetic differentiation analyses based on the chromosomal protein‐coding genes revealed a biogeographic pattern with three main populations, whereas the 16S rDNA sequences did not resolve that biogeographic pattern. Both the phylogenetic and population genetic analyses showed evidence of recombination at the rrs locus. We discuss our results in the light of the contrasting views of bacterial species expressed by microbial taxonomist and evolutionary biologists.

[1]  T. Whittam,et al.  Analysis of genetic structure in soil populations of Rhizobium leguminosarum recovered from the USA and the UK , 1995 .

[2]  A. Willems,et al.  Bradyrhizobium canariense sp. nov., an acid-tolerant endosymbiont that nodulates endemic genistoid legumes (Papilionoideae: Genisteae) from the Canary Islands, along with Bradyrhizobium japonicum bv. genistearum, Bradyrhizobium genospecies alpha and Bradyrhizobium genospecies beta. , 2005, International journal of systematic and evolutionary microbiology.

[3]  T. Whittam,et al.  Species limits in Rhizobium populations that nodulate the common bean (Phaseolus vulgaris) , 1995, Applied and environmental microbiology.

[4]  Ych-chu Wang Molecular ecology , 1992, Journal of Northeast Forestry University.

[5]  J. Caballero-Mellado,et al.  Rhizobium Phylogenies and Bacterial Genetic Diversity , 1996 .

[6]  L. Excoffier,et al.  Analysis of molecular variance inferred from metric distances among DNA haplotypes: application to human mitochondrial DNA restriction data. , 1992, Genetics.

[7]  J. Lawrence,et al.  Lateral gene transfer: when will adolescence end? , 2003, Molecular microbiology.

[8]  H. Spaink Root nodulation and infection factors produced by rhizobial bacteria. , 2000, Annual review of microbiology.

[9]  D. Prévost,et al.  Genetic diversity of rhizobial symbionts isolated from legume species within the genera Astragalus, Oxytropis, and Onobrychis , 1997, Applied and environmental microbiology.

[10]  I. Pepper,et al.  Competitiveness and effectiveness of strains of Rhizobium phaseoli isolated from the sonoran desert , 1984 .

[11]  Michael P. Cummings,et al.  PAUP* [Phylogenetic Analysis Using Parsimony (and Other Methods)] , 2004 .

[12]  D. Griffin,et al.  The Global Transport of Dust , 2002, American Scientist.

[13]  N. Requena,et al.  At least five rhizobial species nodulate Phaseolus vulgaris in a Spanish soil , 1999 .

[14]  J. Hamrick,et al.  A hierarchical analysis of population genetic structure in Rhizobium leguminosarum bv. trifolii , 1996, Molecular ecology.

[15]  P. Vinuesa,et al.  Species Delineation and Biogeography of Symbiotic Bacteria Associated with Cultivated and Wild Legumes , 2004 .

[16]  L. Segovia,et al.  Rhizobium tropici, a novel species nodulating Phaseolus vulgaris L. beans and Leucaena sp. trees. , 1991, International journal of systematic bacteriology.

[17]  M. Riley,et al.  Rhizobium gone native: unexpected plasmid stability of indigenous Rhizobium leguminosarum. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[18]  D. Werner,et al.  Population genetics and phylogenetic inference in bacterial molecular systematics: the roles of migration and recombination in Bradyrhizobium species cohesion and delineation. , 2005, Molecular phylogenetics and evolution.

[19]  J. M. Smith,et al.  How clonal are bacteria? , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[20]  R. Hudson,et al.  A statistical test for detecting geographic subdivision. , 1992, Molecular biology and evolution.

[21]  D. Griffin,et al.  The Global Transport of Dust An intercontinental river of dust , microorganisms and toxic chemicals flows through the Earth ’ s atmosphere , 2003 .

[22]  D. M. Ward A natural species concept for prokaryotes. , 1998, Current opinion in microbiology.

[23]  O. M. Aguilar,et al.  Analysis of Rhizobium etli and of its symbiosis with wild Phaseolus vulgaris supports coevolution in centers of host diversification. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[24]  R. Sokal,et al.  Multiple regression and correlation extensions of the mantel test of matrix correspondence , 1986 .

[25]  W. D. de Vos,et al.  Classification of Austrian rhizobia and the Mexican isolate FL27 obtained from Phaseolus vulgaris L. as Rhizobium gallicum. , 1997, International journal of systematic bacteriology.

[26]  C. Istock,et al.  Bacterial species and evolution: Theoretical and practical perspectives , 1996, Journal of Industrial Microbiology.

[27]  F. Blattner,et al.  Extensive mosaic structure revealed by the complete genome sequence of uropathogenic Escherichia coli , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[28]  R. Hudson,et al.  Estimating the recombination parameter of a finite population model without selection. , 1987, Genetical research.

[29]  W. Modell Population, Species and Evolution , 1971 .

[30]  F. -. Wang,et al.  Corresponding 16S rRNA gene segments in Rhizobiaceae and Aeromonas yield discordant phylogenies , 1996, Plant and Soil.

[31]  O. Gascuel,et al.  A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. , 2003, Systematic biology.

[32]  D. N. Rodriguez-Navarroa,et al.  Characterization of Rhizobium spp . bean isolates from South-West Spain , 2022 .

[33]  Y. Fu,et al.  Statistical tests of neutrality of mutations against population growth, hitchhiking and background selection. , 1997, Genetics.

[34]  D. R. Zeigler,et al.  Gene sequences useful for predicting relatedness of whole genomes in bacteria. , 2003, International journal of systematic and evolutionary microbiology.

[35]  D. Romero,et al.  The Symbiotic Plasmids of the Rhizobiaceae , 2004 .

[36]  J. Huelsenbeck,et al.  Bayesian phylogenetic analysis of combined data. , 2004, Systematic biology.

[37]  T. A. Hall,et al.  BIOEDIT: A USER-FRIENDLY BIOLOGICAL SEQUENCE ALIGNMENT EDITOR AND ANALYSIS PROGRAM FOR WINDOWS 95/98/ NT , 1999 .

[38]  E. Bedmar,et al.  Molecular systematics of rhizobia based on maximum likelihood and Bayesian phylogenies inferred from rrs, atpD, recA and nifH sequences, and their use in the classification of Sesbania microsymbionts from Venezuelan wetlands. , 2005, Systematic and applied microbiology.

[39]  D. Piñero,et al.  Genetic Structure of Rhizobium etli biovar phaseoli Associated with Wild and Cultivated Bean Plants (Phaseolus vulgaris and Phaseolus coccineus) in Morelos, Mexico , 1994, Applied and environmental microbiology.

[40]  Sudhir Kumar,et al.  MEGA2: molecular evolutionary genetics analysis software , 2001, Bioinform..

[41]  L. Segovia,et al.  Reclassification of American Rhizobium leguminosarum biovar phaseoli type I strains as Rhizobium etli sp. nov. , 1993, International journal of systematic bacteriology.

[42]  David Posada,et al.  MODELTEST: testing the model of DNA substitution , 1998, Bioinform..

[43]  John P. Huelsenbeck,et al.  MrBayes 3: Bayesian phylogenetic inference under mixed models , 2003, Bioinform..

[44]  P. Normand,et al.  Evidence that two genomic species of Rhizobium are associated with Medicago truncatula , 1996, Archives of Microbiology.

[45]  J. Young,et al.  The evolution of specificity in the legume-rhizobium symbiosis. , 1989, Trends in ecology & evolution.

[46]  R. Selander,et al.  Genetic diversity and relationships among isolates of Rhizobium leguminosarum biovar phaseoli , 1988, Applied and environmental microbiology.

[47]  Zhenshui Zhang,et al.  Distinct Types of rRNA Operons Exist in the Genome of the Actinomycete Thermomonospora chromogena and Evidence for Horizontal Transfer of an Entire rRNA Operon , 1999, Journal of bacteriology.

[48]  W. Whitman,et al.  Report of the ad hoc committee for the re-evaluation of the species definition in bacteriology. , 2002, International journal of systematic and evolutionary microbiology.

[49]  J. Young,et al.  Phylogenies of atpD and recA support the small subunit rRNA-based classification of rhizobia. , 2001, International journal of systematic and evolutionary microbiology.

[50]  F. Cohan What are bacterial species? , 2002, Annual review of microbiology.

[51]  E. Wang,et al.  Rhizobium yanglingense sp. nov., isolated from arid and semi-arid regions in China. , 2001, International journal of systematic and evolutionary microbiology.

[52]  J. Thompson,et al.  The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. , 1997, Nucleic acids research.

[53]  P. Drouin,et al.  Classification of rhizobia based on nodC and nifH gene analysis reveals a close phylogenetic relationship among Phaseolus vulgaris symbionts. , 2001, Microbiology.

[54]  M. Slatkin,et al.  Estimation of levels of gene flow from DNA sequence data. , 1992, Genetics.

[55]  C. Silva,et al.  Reticulated and epidemic population genetic structure of Rhizobium etli biovar phaseoli in a traditionally managed locality in Mexico , 1999 .

[56]  J. Young,et al.  Proposed Minimal Standards for the Description of New Genera and Species of Root- and Stem-Nodulating Bacteria , 1991 .

[57]  J. Rozas,et al.  Statistical properties of new neutrality tests against population growth. , 2002, Molecular biology and evolution.

[58]  P. Reeves,et al.  Intraspecies variation in bacterial genomes: the need for a species genome concept. , 2000, Trends in microbiology.

[59]  T. A. Campbell,et al.  Rhizobium mongolense sp. nov. is one of three rhizobial genotypes identified which nodulate and form nitrogen-fixing symbioses with Medicago ruthenica [(L.) Ledebour]. , 1998, International journal of systematic bacteriology.

[60]  C. Rodríguez,et al.  Multiple recombination events maintain sequence identity among members of the nitrogenase multigene family in Rhizobium etli. , 1998, Genetics.

[61]  U. Priefer Genes involved in lipopolysaccharide production and symbiosis are clustered on the chromosome of Rhizobium leguminosarum biovar viciae VF39 , 1989, Journal of bacteriology.

[62]  E. Martínez-Romero Diversity of Rhizobium-Phaseolus vulgaris symbiosis: overview and perspectives , 2003, Plant and Soil.

[63]  M. Riley,et al.  Comparison of the evolutionary dynamics of symbiotic and housekeeping loci: a case for the genetic coherence of rhizobial lineages. , 1999, Molecular biology and evolution.

[64]  J. Thies,et al.  Structure and diversity among rhizobial strains, populations and communities : a review , 2004 .

[65]  V. Souza,et al.  Rhizobium etli and Rhizobium gallicum Nodulate Common Bean (Phaseolus vulgaris) in a Traditionally Managed Milpa Plot in Mexico: Population Genetics and Biogeographic Implications , 2003, Applied and Environmental Microbiology.

[66]  Julio Rozas,et al.  DnaSP version 3: an integrated program for molecular population genetics and molecular evolution analysis , 1999, Bioinform..

[67]  E. Wang,et al.  Seeds of Phaseolus vulgaris bean carry Rhizobium etli , 1998 .

[68]  D. Swofford PAUP*: Phylogenetic analysis using parsimony (*and other methods), Version 4.0b10 , 2002 .

[69]  J. Young,et al.  The glutamine synthetases of rhizobia: phylogenetics and evolutionary implications. , 2000, Molecular biology and evolution.

[70]  C. Fraser,et al.  Fuzzy species among recombinogenic bacteria , 2005, BMC Biology.

[71]  E. Wang,et al.  Rhizobium etli bv. mimosae, a novel biovar isolated from Mimosa affinis. , 1999, International journal of systematic bacteriology.

[72]  F. Cohan,et al.  Discovery and classification of ecological diversity in the bacterial world: the role of DNA sequence data. , 1997, International journal of systematic bacteriology.

[73]  J. Olivares,et al.  Conservation of Plasmid-Encoded Traits among Bean-Nodulating Rhizobium Species , 2002, Applied and Environmental Microbiology.

[74]  John W. Taylor,et al.  Geographic Barriers Isolate Endemic Populations of Hyperthermophilic Archaea , 2003, Science.

[75]  R. Mhamdi,et al.  Different species and symbiotic genotypes of field rhizobia can nodulate Phaseolus vulgaris in Tunisian soils. , 2002, FEMS microbiology ecology.

[76]  G. Laguerre,et al.  Rhizobium gallicum sp. nov. and Rhizobium giardinii sp. nov., from Phaseolus vulgaris nodules. , 1997, International journal of systematic bacteriology.

[77]  W. D. de Vos,et al.  Characterization of Rhizobium etli and other Rhizobium spp. that nodulate Phaseolus vulgaris L. in an Austrian soil , 1997 .

[78]  P. Reeves,et al.  When does a clone deserve a name? A perspective on bacterial species based on population genetics. , 2001, Trends in microbiology.

[79]  F. Ayala Molecular systematics , 2004, Journal of Molecular Evolution.

[80]  F. Tajima Statistical method for testing the neutral mutation hypothesis by DNA polymorphism. , 1989, Genetics.

[81]  B. Rannala,et al.  Phylogenetic methods come of age: testing hypotheses in an evolutionary context. , 1997, Science.

[82]  L. Kuykendall,et al.  Changing concepts in the systematics of bacterial nitrogen-fixing legume symbionts. , 2003, The Journal of general and applied microbiology.

[83]  J. Wakeley,et al.  A coalescent estimator of the population recombination rate. , 1997, Genetics.

[84]  E. Bedmar,et al.  Identification of fast-growing rhizobia nodulating tropical legumes from Puerto Rico as Rhizobium gallicum and Rhizobium tropici. , 2004, Systematic and applied microbiology.

[85]  J. Burdon,et al.  Conflicting phylogeographic patterns in rRNA and nifD indicate regionally restricted gene transfer in Bradyrhizobium. , 2002, Microbiology.

[86]  R. Mhamdi,et al.  Genotypic diversity and symbiotic effectiveness of rhizobia isolated from root nodules of Phaseolus vulgaris L. grown in Tunisian soils , 1999, Biology and Fertility of Soils.

[87]  Leo M. Schouls,et al.  Horizontal Transfer of Segments of the 16S rRNA Genesbetween Species of the Streptococcus anginosusGroup , 2003, Journal of bacteriology.

[88]  Lars Paulin,et al.  Discordant Phylogenies within the rrn Loci of Rhizobia , 2003, Journal of bacteriology.

[89]  Duckchul Park,et al.  Diversity of 16S rDNA sequences of Rhizobium spp. implications for species determinations. , 2004, FEMS microbiology letters.

[90]  Michael P. Cummings,et al.  MEGA (Molecular Evolutionary Genetics Analysis) , 2004 .

[91]  P. Vandamme,et al.  Legume Symbiotic Nitrogen Fixation byβ-Proteobacteria Is Widespread inNature , 2003, Journal of bacteriology.

[92]  D. Romero,et al.  Rhizobium plasmids in bacteria-legume interactions , 1996, World journal of microbiology & biotechnology.

[93]  X. Xia,et al.  DAMBE: software package for data analysis in molecular biology and evolution. , 2001, The Journal of heredity.