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15 The genetic diversity of Frankia strains nodulating Alnus glutinosa along the basin of the Tormes River was 16 studied on DNA extracted directly from nodules. Frankia strains inhabiting root nodules at 12 different 17 locations, ranging in altitude from 409 to 1181 m, were characterized. For that, we amplified the whole IGS 18 region between 16S–23S rDNA and performed a restriction fragment length polymorphism (RFLP) 19 analysis with four restriction enzymes. Two different RFLP patterns (termed A and B) were obtained with 20 HaeIII, indicating the existence of two different groups of Frankia strains. Three different nodule extracts 21 from each of the two RFLP groups were selected for further analyses. Sequencing of the 16S–23S rDNA 22 IGS showed a 100% of intragroup homology and also confirmed the difference (98.4% level of similarity) 23 between the Frankia strains in the two nodule extract groups. The phylogenetic analyses based on the two 24 16S–23S rDNA IGS sequences obtained in this study and other previously published sequences indicated 25 that Frankia strains TFAg5 and TFAg23 (chosen as representative of HaeIII–RFLP group A and B, 26 respectively) are quite similar to other strains nodulating plants of A. rhombifolia and A. viridis in Cali27 fornia (pairwise levels of similarity including gaps ranged from 97.8% to 98.6%), together with which they 28 form a single group. To put the Frankia strains representative of eachHaeIII–RFLP group in the context of 29 overall Frankia diversity we amplified and sequenced the 16S rDNA and glnII gene from nodular DNA. An 30 also remarkable fact found in this study was that Frankia strains belonging to the HaeIII–RFLP group A 31 were distributed all along the river course, from the lowest site sampled to the highest, while Frankia strains 32 placed into RFLP group B were restricted to the upper Tormes River, being exclusively found at altitudes 33 of 946 m or higher. 34