Please cite this article in press as: Z. Ji, et al., Genetic divergence among Bradyrhizobium strains nodulating wild and cultivated Kum- merowia spp. in China, Syst. Appl. Microbiol. (2018), https://doi.org/10.1016/j.syapm.2018.10.003 ARTICLE IN PRESS G Model SYAPM-25950; No. of Pages 9 Systematic and Applied Microbiology xxx (2018) xxx–xxx Contents lists available at ScienceDirect Systematic and Applied Microbiology journal homepage: www.elsevier.de/syapm Genetic divergence among Bradyrhizobium strains nodulating wild and cultivated Kummerowia spp. in China Zhaojun Ji a,b,c , Tianyan Liu b,c , Jixing Zhang a , Hui Yan b,c , Entao Wang d , Qingguo Cui e , Wenxin Chen b,c , Wenfeng Chen b,c,∗ a College of Life Science and Horqin Plant Stress Biology Research Institute, Inner Mongolia University for the Nationalities, Tongliao 028042, Inner Mongolia, China b State Key Laboratory of Agrobiotechnology, Beijing 100193, China c College of Biological Sciences and Rhizobium Research Center, China Agricultural University, Beijing 100193, China d Departamento de Microbiología, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, México D. F. 11340, México e State Key Laboratory of Vegetation and Environmental Change, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China a r t i c l e i n f o Article history: Received 9 August 2018 Received in revised form 7 October 2018 Accepted 9 October 2018 Keywords: Bradyrhizobium Kummerowia Microevolution Phylogeny Recombination Urban segregation a b s t r a c t Distribution of rhizobial species is affected by geographical isolation and selected by leguminous hosts, however, little is known about the molecular evolution of rhizobia nodulating the same legume in dif- ferent eco-environments. In present study, the microevolution of Bradyrhizobium associated with the leguminous grass Kummerowia grown in exurban areas and cultivated in urban areas in China was inves- tigated. Total 14 genospecies, including seven new groups, were identified based on a concatenated sequence analysis of taxonomic markers (SMc00019, truA and thrA) for 94 representative strains. Results demonstrated that lower levels of nucleotide diversity were found in the strains isolated from urban areas compared with those isolated from exurban areas, based on the evolutional analyses of three house- keeping genes (atpD, glnII and recA), two symbiosis-related genes (nodC and nifH), and the taxonomic markers. Moreover, compared with urban areas, gene exchange and recombination occurred more fre- quently among the genospecies isolated from exurban areas, regardless of the geographical distribution. Finally, the evolutionary lineage of Bradyrhizobium strains isolated from urban areas was independent of that of the strains isolated from exurban areas. In summary, the evolutionary history of Kummerowia bradyrhizobia may have been gradually segregated to different evolutionary lineages, irrespective of distinct biogeography. © 2018 Elsevier GmbH. All rights reserved. Introduction Kummerowia stipulacea (Korean clover) and Kummerowia stri- ata (Japanese clover) are legume grasses widely distributed along the roadside, hillside and lakeside as well as in other exurban areas habitats in China and other countries [22]. Because of its high oestrogenic activity and production of flavonoids, which are used for menopausal dysfunction treatment, anti-HIV and antitu- mor medicine, herbal K. striata has been deliberately cultivated [34,39]. Furthermore, Kummerowia has been grown as a flowering grass in university campuses in urban areas. Symbiotic nitrogen fixation of Kummerowia with rhizobia plays an important role in the healthy growth of the plant without the requirement of ∗ Corresponding author. E-mail address: chenwf@cau.edu.cn (W. Chen). fertilisers. Rhizobial strains of Kummerowia have been recorded previously [19], and slow-growing strains, such as Bradyrhizobium spp., are the predominant microsymbionts in China. Some fast- growing strains grown in the Loess Plateau have been identified as Sinorhizobium kummerowiae [35,38], whereas some of those grown in campus lawns have been identified as Rhizobium cauense [20]. Furthermore, 14 reference strains (Bradyrhizobium diazo- efficiens, Bradyrhizobium canariense, Bradyrhizobium japonicum, Bradyrhizobium betae, Bradyrhizobium huanghuaihaiense, Bradyrhi- zobium daqingense, Bradyrhizobium liaoningense, Bradyrhizobium yuanmingense, Bradyrhizobium iriomotense, Bradyrhizobium elkanii, Bradyrhizobium pachyrhizi, Bradyrhizobium jicamae and Bradyrhi- zobium lablabi and an outgroup genospecies S. kummerowiae) have been employed here to identify the phylogenetic relationship of these rhizobial strains isolated from nodules on the roots of Kum- merowia distributed in exurban and urban areas [19]. https://doi.org/10.1016/j.syapm.2018.10.003 0723-2020/© 2018 Elsevier GmbH. All rights reserved.