Systematic and Applied Microbiology 33 (2010) 282–290 Contents lists available at ScienceDirect Systematic and Applied Microbiology journal homepage: www.elsevier.de/syapm Lotus endemic to the Canary Islands are nodulated by diverse and novel rhizobial species and symbiotypes  M a José Lorite a , Javier Donate-Correa b , Marcelino del Arco-Aguilar c , Ricardo Pérez Galdona b , Juan Sanjuán a , Milagros León-Barrios b,∗ a Departamento de Microbiología del Suelo y Sistemas Simbióticos, Estación Experimental del Zaidín, CSIC, Granada, Spain b Departamento de Microbiología y Biología Celular, Facultad de Farmacia, Universidad de La Laguna, 38071 La Laguna, Tenerife, Spain c Departamento de Biología Vegetal (Botánica), Universidad de La Laguna, 38071 La Laguna, Tenerife, Spain article info Article history: Received 27 January 2010 Keywords: Mesorhizobium Lotus Biodiversity Housekeeping genes Nodulation genes Canary Islands abstract Genetic and symbiotic characterization of 34 isolates from several Lotus species endemic to the Canary Islands showed extraordinary diversity, with bacteria belonging to different species of the genera Mesorhi- zobium (17 isolates), Sinorhizobium (12 isolates) and Rhizobium/Agrobacterium (5 isolates). In a previous report, we showed that the Sinorhizobium isolates mostly belonged to S. meliloti. Here, we focused on the remaining isolates. The Lotus mesorhizobial strains were distributed in the rrs tree within six poorly resolved branches. Partial sequences from atpD and recA genes produced much better resolved phyloge- nies that were, with some exceptions, congruent with the ribosomal phylogeny. Thus, up to six different mesorhizobial species were detected, which matched with or were sister species of M. ciceri, M. alhagi, M. plurifarium or M. caraganae, and two represented new lineages that did not correspond to any of the currently recognized species. Neither M. loti nor Bradyrhizobium sp. (Lotus), recognized as the typi- cal Lotus-symbionts, were identified among the Canarian Lotus isolates, although their nodulation genes were closely related to M. loti. However, several subbranches of mesorhizobia nodulating Lotus spp. could be differentiated in a nodC tree, with the isolates from the islands distributed in two of them (A1 and A3). Subbranch A1 included reference strains of M. loti and a group of isolates with a host range compatible with biovar loti, whereas A3 represented a more divergent exclusive subbranch of isolates with a host range almost restricted to endemic Lotus and it could represent a new biovar among the Lotus rhizobia. © 2010 Elsevier GmbH. All rights reserved. Introduction The diversity of rhizobia nodulating Lotus spp. is starting to be elucidated. Until recently, most isolates had been collected from a small number of Lotus species used as pasture-forage in a few locations of South America and New Zealand [11,16,18,26,38]. Mesorhizobium loti and Bradyrhizobium sp. (Lotus) have been tra- ditionally recognized as the typical Lotus rhizobia [32]. M. loti establish effective symbiosis with some Lotus species, which include L. corniculatus, L. japonicus or L. filicaulis, whereas the bradyrhizobial strains either do not nodulate or form ineffective nodules in these species, although they effectively nodulate species such as L. uliginosus or L. angustissimus, which form ineffective  Accession numbers: Nucleotide sequence data reported are available in the GenBank database under the following accession numbers: 16S rDNA: FN563428–FN563445; atpD: FN563446–FN563453 and FN563969; recA: FN563454–FN563462; and nodC: FN563463–FN563471. ∗ Corresponding author. Tel.: +34 922318481; fax: +34 922318477. E-mail address: mileonba@ull.es (M. León-Barrios). symbioses with M. loti [3,4,32,35]. Broad host range mesorhizo- bia, such as NZP 2037, that are able to nodulate both groups of Lotus effectively [34] are rare. Many strains classified as M. loti have been poorly characterized [6,31] and some are probably misclas- sified, which may explain the wide variability in total DNA–DNA homology [6], the different phylogenies [7,50] and the great dif- ferences in N 2 -fixing effectiveness [3,32] detected among “M. loti” strains. Recently, strain “M. loti MAFF303099” was re-classified as M. huakuii biovar loti [42]. Similarly, the broad host range “M. loti NZP2037” strain needs to be better characterized and probably re- classified. Lotus is a genus of legume plants with 120–180 species of herbs and small shrubs distributed mainly across the Northern Hemi- sphere [36], and most Lotus species remain unexplored from the point of view of their rhizobial partners. Thus, larger diversity than that currently recognized can be expected for the Lotus rhizobia, as new isolates from less well known species are studied and bet- ter characterized genetically. Recent studies are starting to change our knowledge about the genetic diversity of the bacteria able to establish symbiosis with Lotus spp. Characterization of root nod- ule bacteria from Lotus creticus, L. argenteus and L. roudairei in an 0723-2020/$ – see front matter © 2010 Elsevier GmbH. All rights reserved. doi:10.1016/j.syapm.2010.03.006