Azolla (Azollaceae) is a genus of aquatic, leptosporangiate, heterosporous ferns. Currently, the seven extant species are placed within two sections (Azolla and Rhizosperma). The genus has, however, been subject to a long and complex taxonomic history, especially within sect. Azolla, where species boundaries have been debated. Previous studies have employed morphological, cytological, and molecular data in an attempt to circumscribe the seven extant species, but none have been entirely successful. We employed cladistic analysis of DNA sequence data from three non-coding regions (ITS, atpB-rbcL IGS, and trnL-trnF region) to reconstruct phylogenetic relationships among the species of Azolla. Our results largely uphold the current taxonomic system, suggesting that (1) A. pinnata and A. nilotica are sister taxa in a monophyletic sect. Rhizosperma; (2) the five species of sect. Azolla form a monophyletic group; (3) A. rubra is distinct from A. filiculoides; and (4) A. caroliniana is distinct from A. microphylla and A. mexicana. Although A. microphylla and A. mexicana form a single evolutionary lineage, they do not appear to represent two distinct species. Rather, the multiple accessions sampled from these putative species were interdigitated within this clade, a result that can be explained by several possible hypotheses. In light of geographic distributions and morphology, we suggest the Azolla microphylla – mexicana lineage arose through hybridization and homoploid speciation involving A. caroliniana and A. filiculoides.

Key words: Azolla, homoploid speciation, molecular systematics