RAPD analysis of Anabaena-free Azolla and its application in the study of interspecific relationships within section Azolla

Azolla Lam. is a small genus of free_floating ferns symbiotic with cyanobacteria. The heterogeneity of Azolla samples has been usually ignored when DNA was extracted from intact Azolla_Anabaena complex for Azolla phylogeny analysis based on RAPD. In this study, the Anabaena_free Azolla was induced from Azolla_Anabaena complex to compare the RAPD banding patterns amplified by different primers with intact Azolla as well as isolated Anabaena azollae. The banding patterns of Azolla with Anabaena were detected to be affected by two symbiotic partners and their contributions to PCR products varied with the tested Azolla sample and the used primer, which indicates the importance of using Anabaena_free Azolla for RAPD analysis of Azolla species. For a better understanding of the interspecific relationships within the section Azolla, 11 accessions classified as five species in this section were identified by RAPD marker with their Anabaena_free Azolla. A total of 127 polymorphic bands amplified from nine primers were selected to generate Jaccard's similarity coefficients and to construct UPGMA dendrogram. The results have shown that three accessions referred to as A. filiculoides were grouped together with intraspecific similarity coefficients from 0.456 to 0.702; two accessions of A. mexicana (similarity coefficients 0.587) were grouped with two accessions of A. microphylla (similarity coefficient 0.203) and two accessions of A. caroliniana were grouped together with similarity coefficient 0.368; but two accessions referred to as A. rubra were distinctly separated with similarity coefficient 0.190 and the accession collected from Japan clustered with A. filiculoides group. Coupled with other evidence such as isozyme and sexual hybridization among sect. Azolla, the following problems have been discussed: (1) the rationality to refer Azolla accessions from both Japan and Oceania to A. rubra; (2) the affinity among A. filiculoides, A. microphylla and A. mexicana; and (3) the taxonomic treatment of A. caroliniana. It is considered that (1) A. japonica and A. rubra should be treated as two independent species, (2) A. filiculoides is an independent species while A. microphylla and A. mexicana cannot be treated as two separate species because of their very close genetic relationship, and (3) the taxonomic status of A. caroliniana cannot yet be determined with certainty.