Species of cyanolichens from Pseudocyphellaria with indistinguishable ITS sequences have different photobionts

•  Cyanobacteria were isolated from bipartite cyanolichen species of Pseudocyphellaria and the identity of the major photobionts established. The specificity of the cyanobacterial-fungal association was also examined. •  Comparison of 16S rRNA gene sequences distinguished cyanobacterial and green algal isolates, and both 16S rRNA gene and tRNA^Leu  (UAA) intron sequences of isolates and lichen thalli identified candidate photobionts. In addition, the genetic diversity of both the cyanobiont and mycobiont was investigated using the comparison of tRNA^Leu  (UAA) intron sequences and ITS sequences, respectively. •  The 16S rRNA gene sequences identified two species-specific photobionts with similar sequences; however, the tRNA^Leu  (UAA) intron sequences unambiguously discriminated between the two symbiotic cyanobacterial strains. Moreover, the fungal ITS sequences of the two corresponding lichens, Pseudocyphellaria crocata and Pseudocyphellaria neglecta, showed little variation. •  The cyanobacterial-fungal associations of P. crocata and P. neglecta were specific for all samples. However, the similarity of the ITS sequences raised the possibility that they represent the same species and that their different morphology is influenced by the cyanobacterial symbiont.