Plant and Soil 254: 219–227, 2003. © 2003 Kluwer Academic Publishers. Printed in the Netherlands. 219 Evolutionary implications of nucleotide sequence relatedness between Alnus nepalensis and Alnus glutinosa and also between corresponding Frankia microsymbionts Rajani Varghese, Vineeta Singh Chauhan & Arvind Kumar Misra ∗ Molecular Genetics Laboratory, Department of Botany, North-Eastern Hill University, Shillong 793 022, India. 1 Corresponding author ∗ Received 19 July 2002. Accepted in revised form 20 August 2002 Key words: Alnus nepalensis, Frankia, evolution, Phylogeny Abstract Frankia DNAs were isolated directly from root nodules of Alnus nepalensis and Alnus nitida collected from various natural sites in India. For comparison, a nodule sample from Alnus glutinosa was also collected from Tuebingen, Germany. Nucleotide sequence analyses of amplified 16S–23S ITS region revealed that one of the microsymbionts from Alnus nepalensis was closely related to the microsymbiont from Alnus glutinosa. A similar exercise on the host was also carried out. It was found that one sample of Alnus nepalensis was closely related to Alnus glutinosa sequence from Europe. Since both Frankia and the host sequences studied revealed proximity between Alnus glutinosa and Alnus nepalensis, it is hypothesised that the common progenitor of all the alders first entered into an association with Frankia, and the symbiotic association has evolved since. Introduction Alnus, commonly known as alder, belongs to the family Betulaceae comprising of six genera (Alnus, Betula, Carpinus, Corylus, Ostrya and Ostrypsis) (Lawrence, 1967) and about 130 species (Chen et al., 1999). Of these six genera, only Alnus is repor- ted to symbiotically associate with the actinomycete Frankia. Forty-seven species of Alnus have been iden- tified so far (Baker and Schwintzer, 1990) and only two of them (nepalensis and nitida) are found in India. It was hypothesised that if the progenitor of genus Alnus had entered into an association with Frankia prior to diversification, the evolutionary pattern of Frankia and alders should have some similarity. That means that the two species of Alnus showing close proximity to each other should harbour closely related Frankia strains as well. The phylogenetic information generated for alders and Frankia was therefore used to determine if this was true. ∗ E-mail: arvindkmisra@nehu.ac.in Earlier investigations had shown genetic diversity in strains of Frankia isolated from nodules of Alnus nepalensis growing in India (Ganesh et al., 1994). The present work was initiated to investigate the differ- ences at molecular level among some Frankia strains present in the nodules of Indian alders. Further, to establish the relationship between Frankia found in India and that found in Europe, a comparison was also made between Indian frankiae with one strain present in the nodules of Alnus glutinosa from Europe. We isolated Frankia DNAs from field-collected nodules and sequenced amplified ribosomal DNA sequences to establish molecular diversity and phylogenetic re- lationship. To reconstruct phylogenetic relationships between the alder hosts, we used sequences of the 18S–28S Internally Transcribed Spacer (ITS) region, primarily because earlier workers had emphasised the utility of this region for inferring phylogenetic relationships at lower taxonomic levels (Chen et al., 1999; Savard et al., 1993). One hundred trees of Alnus nepalensis were investigated and a lot of variability was detec-