Abstract:Myrmecia israeliensis has been traditionally considered as a green coccoid free-living microalga. This microalga was previously suggested as the primary phycobiont in the lichens Placidium spp., Heteroplacidium spp., and Psora decipiens. However, due to the absence of ITS rDNA sequences (barcode information) published along with these investigations, the symbiotic nature of M. israeliensis might be confirmed by using the DNA barcoding and different microscopic examinations both in the symbiotic state and in culture. The aim of this study was to settle the presence of M. israeliensis as the primary microalga in squamulose lichens growing in terricolous communities (Psora spp., Placidium spp. and Claviscidium spp.) in 32 localities within European and Canary Island ecosystems by using both molecular and ultrastructural techniques. The lichen-forming fungi were identified using ITS rDNA as a barcode, and in the case of P. decipiens specimens, the mycobiont analyses showed an unexpected variability. Phycobiont phylogenetic analyses were made using both chloroplast (LSU rDNA) and nuclear (ITS rDNA) molecular markers. Our results proved that M. israeliensis is the primary symbiotic microalga in all the chosen and analyzed lichens. In addition, fluorescence microscopy, transmission electron microscopy and scanning electron techniques were used to characterize M. israeliensis. Finally, the presence of this microalga in lichen thalli was verified using different microscopic observations. A combination of different techniques, both molecular and microscopic, allowed for the accurate identification of this symbiotic microalga, beforehand mainly known as free living. Here, we suggest the combination of these techniques to prevent incorrect identification in microalgal lichen studies.