A genetic discontinuity in root-nodulating bacteria of cultivated pea in the Indian trans-Himalayas PRAVEEN RAHI,* RITI KAPOOR,* J. P. W. YOUNG† and ARVIND GULATI* *Plant Pathology and Microbiology Laboratory, CSIR-Institute of Himalayan Bioresource Technology, PO Box 6, Palampur-176061 (HP), India, †Department of Biology, University of York, York YO10 5DD, UK Abstract Evolutionary relationships of 120 root-nodulating bacteria isolated from the nodules of Pisum sativum cultivated at 22 different locations of the trans-Himalayan valleys of Lahaul and Spiti in the state of Himachal Pradesh of India were studied using 16S rRNA gene PCR-RFLP, ERIC-PCR, sequencing of 16S rRNA, atpD, recA, nodC and nifH genes, carbon-source utilization pattern (BIOLOGÔ), and whole-cell fatty acid profiling. The results demonstrated that all isolates belonged to Rhizobium leguminosarum symbiovar viciae (Rlv). Isolates from the two valleys were clearly separated on the basis of ERIC fingerprints, carbon-source utilization pattern, and whole-cell fatty acid methyl esters. Phylogenetic analysis of atpD, recA, nodC and nifH genes revealed a common Rlv sublineage in Spiti valley. Lahaul valley isolates were represented by three sequence types of atpD and recA genes, and four sequence types of nodC and nifH genes. Genotypes from the two valleys were completely distinct, except for two Lahaul isolates that shared nodC and nifH sequences with Spiti isolates but were otherwise more similar to other Lahaul isolates. Isolates from the two highest Spiti valley sites (above 4000 m) had a distinctive whole-cell fatty acid profile. Spiti valley isolates are closely related to Rlv sublineages from Xinjiang and Shanxi provinces in China, while Lahaul valley isolates resemble cosmopolitan strains of the western world. The high mountain pass between these valleys represents a boundary between two distinct microbial populations. Keywords: biogeography, evolutionary relationships, Indian trans-Himalayas, Rhizobium leguminosarum symbiovar viciae Received 7 June 2011; revision received 21 September 2011; accepted 10 October 2011 Introduction The trans-Himalayan region of India consists of the cold deserts of Ladakh and Kargil in Jammu and Kashmir and Lahaul and Spiti in Himachal Pradesh. The Lahaul and Spiti district of Himachal Pradesh is located between elevations of 3048–4572 m spread over an area of 13 693 km 2 . The sparsely inhabited district presents extreme climatic conditions because of the winter tem- perature plunging much below freezing, wide diurnal fluctuations in temperature, scanty rainfall, low air pressure and strong solar radiation. Differences in alti- tude, topography and geographical location create great variation in climate with Spiti valley, in particular, becoming a harsh cold desert during winter. Humidity remains low throughout the year, with scanty rainfall in Spiti in comparison with Lahaul. The low productivity in the fragile ecosystem with treacherous mountainous terrain is limiting to the development of agriculture. Grains like buckwheat (Fagopyrum esculentum) and bar- ley (Hordeum vulgare) were the major crops cultivated in the two valleys, with black pea (Pisum sativum var. arvense) also cultivated as a local crop in Spiti valley. Lathyrus emodii, Lathyrus sativus and Lens culinaris are also reported in sporadic cultivation and are often met with as escapes in Lahaul and Spiti (Aswal & Mehrotra 1994). In the past three decades, the scenario changed with the introduction and replacement of traditional crops by pea (Pisum sativum) and potato (Solanum Correspondence: Arvind Gulati, Fax: +911894230433; E-mails: gal_arvind@yahoo.co.in; arvindgulati@ihbt.res.in Ó 2011 Blackwell Publishing Ltd Molecular Ecology (2011) doi: 10.1111/j.1365-294X.2011.05368.x