Please cite this article in press as: J. Pérez-Yépez, et al., Evaluation of seven housekeeping genes for multilocus sequence analy- sis of the genus Mesorhizobium: Resolving the taxonomic affiliation of the Cicer canariense rhizobia, Syst. Appl. Microbiol. (2014), http://dx.doi.org/10.1016/j.syapm.2014.10.003 ARTICLE IN PRESS G Model SYAPM-25652; No. of Pages 7 Systematic and Applied Microbiology xxx (2014) xxx–xxx Contents lists available at ScienceDirect Systematic and Applied Microbiology j ourna l h omepage: www.elsevier.de/syapm Evaluation of seven housekeeping genes for multilocus sequence analysis of the genus Mesorhizobium: Resolving the taxonomic affiliation of the Cicer canariense rhizobia  Juan Pérez-Yépez a , Natalia Armas-Capote a , Encarna Velázquez b , Ricardo Pérez-Galdona a , Raúl Rivas b , Milagros León-Barrios a,* a Departamento de Bioquímica, Microbiología, Biología Celular y Genética, Universidad de La Laguna, 38071 La Laguna, Tenerife, Spain b Departamento de Microbiología y Genética, Universidad de Salamanca, 37007 Salamanca, Spain a r t i c l e i n f o Article history: Received 23 July 2014 Received in revised form 2 October 2014 Accepted 8 October 2014 Keywords: Cicer canariense Mesorhizobium Housekeeping genes MLSA (multilocus sequence analysis) ANI (average nucleotide identity) Species delineation a b s t r a c t Cicer canariense is a wild chickpea that can be nodulated by Mesorhizobium strains belonging to nine different genomic groups or genospecies. In this study, multilocus sequence analysis (MLSA) of seven protein-coding genes, recA, glnII, dnaK, rpoB, gyrB, truA and thrA, was used to resolve the phylogenetic relationships and taxonomic affiliation of 27 representative strains from all the genotypes. Individual phylogenies were mostly congruent, although there were a few discrepancies. Some genes were more discriminative than others, but concatenation of the seven genes produced a robust phylogeny of the genus Mesorhizobium. MLSA gave good support for the taxonomic affiliations of most of the genomic groups to previously recognized species and delineated several potential new species. Five genospecies found in C. canariense nodules showed average nucleotide identity values for seven genes (ANIg7) of >96% and they could be assigned to previously described Mesorhizobium species. Two large closely related genomic groups had M. caraganae as the closest species and they shared ANIg7 values in the 94–95% range, suggesting that they could be different subspecies or sister species. The predominant genospecies represented a novel monophyletic lineage that was well resolved from all currently recognized species of Mesorhizobium, with the highest ANIg7 below 92%. Other single strains represented potential new species. © 2014 Elsevier GmbH. All rights reserved. Introduction The taxonomy of bacteria nodulating leguminous plants has been elucidated much further in the last ten years. The strains involved are classified within five “classic” rhizobia genera: Rhizo- bium, Sinorhizobium (Ensifer), Mesorhizobium, Bradyrhizobium, and Azorhizobium, which currently contain more than 100 species. A polyphasic approach using phenotypic and genetic methods is mandatory for species recognition, and percentage genomic DNA–DNA hybridization (DDH) is the gold standard technique for species delineation. However, DDH has several drawbacks, since it  Accession numbers for the sequences generated in this study are: LM654027–LM654053 for dnaK, LM654109–LM654137 for gyrB, LM654054–LM654079 for rpoB, LM654080–LM654108 for thrA and LM654138–LM654167 for truA. * Corresponding author. Tel.: +34922318481; fax: +34922318666. E-mail address: mileonba@ull.es (M. León-Barrios). is laborious, scarcely reproducible, inapplicable to non-culturable strains and, above all, it lacks databases to compare or assign new taxa. As methods have improved, whole genome sequencing has become feasible and the average nucleotide identity (ANI) between two genomes is now recognized as a robust test for comparing the relationships between bacteria. Comparing whole genomes from the analysis of conserved core genes of several groups of organisms has shown that the classical 70% DDH cut-off for species delineation corresponds to an ANI cut-off of approximately 95–96% [17,18,29]. Consequently, it has been proposed that ANIs calculated from sequencing approximately 20% of the genomes could replace DDH [29]. Nevertheless, sequence similarity values from concatenation of six to eight randomly selected genes should also allow an accu- rate estimation of the total genome ANI value and give a significant prediction of whole-genome relatedness [18,29]. By combining different housekeeping genes distributed along the chromosome, multilocus sequence analysis (MLSA) can provide more precise phylogenetic relationships that reveal good correlations with DDH http://dx.doi.org/10.1016/j.syapm.2014.10.003 0723-2020/© 2014 Elsevier GmbH. All rights reserved.