Fluorescence light microscopy provided convincing evidence for the domain organization of plant plasma membrane (PM) proteins. Both peripheral and integral PM proteins show an inhomogeneous distribution within the PM. However, the size of PM nanodomains and protein clusters is too small to accurately determine their dimensions and nano-organization using routine confocal fluorescence microscopy and super-resolution methods. To overcome this limitation, we have developed a novel correlative light electron microscopy method (CLEM) using total internal reflection fluorescence microscopy (TIRFM) and advanced environmental scanning electron microscopy (A-ESEM). Using this technique, we determined the number of auxin efflux carriers from the PINFORMED (PIN) family (NtPIN3b-GFP) within PM nanodomains of tobacco cell PM ghosts. Protoplasts were attached to coverslips and immunostained with anti-GFP primary antibody and secondary antibody conjugated to fluorochrome and gold nanoparticles. After imaging the nanodomains within the PM with TIRFM, the samples were imaged with A-ESEM without further processing, and quantification of the average number of molecules within the nanodomain was performed. Without requiring any post-fixation and coating procedures, this method allows to study details of the organization of auxin carriers and other plant PM proteins.

The widespread species Parmotrema crinitum (Ach.) M. Choisy and Parmotrema perlatum (Huds.) M. Choisy are mainly distinguished by their reproductive strategies. While P. crinitum propagates by isidia, P. perlatum produces soredia. In this study, we aim to evaluate the phylogenetic relationship between both species and to critically examine their species boundaries. To this purpose, 46 samples belonging to P. crinitum and P. perlatum were used in our analysis, including 22 for which we studied the morphology and chemistry, before extracting their DNA. We used 35 sequences of the internal transcribed spacer region of nuclear ribosomal DNA (ITS) of Parmotrema perlatum from Europe and Africa (20 of which were newly generated), and 11 of Parmotrema crinitum from Europe, North America and North Africa (two newly generated). Additionally, 28 sequences of several species from Parmotrema were included in the ITS dataset. The ITS data matrix was analyzed using different approaches, such as traditional phylogeny (maximum likelihood and Bayesian analyses), genetic distances, automatic barcode gap discovery (ABGD) and the coalescent-based method poisson tree processes (PTP), in order to test congruence among results. Our results indicate that all samples referred to P. crinitum and P. perlatum nested in a well-supported monophyletic clade, but phylogenetic relationships among them remain unresolved. Delimitations inferred from PTP, ABGD and genetic distance analyses were comparable and suggested that P. crinitum and P. perlatum belong to the same lineage. Interestingly, two samples of P. perlatum separate in a different monophyletic clade, which is supported as a different lineage by all the analyses.