In lichen-forming fungi, traditional taxonomical concepts are frequently in conflict with molecular data,
and identifying appropriate taxonomic characters to describe phylogenetic clades remains challenging in
many groups. The selection of suitable markers for the reconstruction of solid phylogenetic hypotheses is
therefore fundamental. The lichen genus Usnea is highly diverse, with more than 350 estimated species,
distributed in polar, temperate and tropical regions. The phylogeny and classification of Usnea have been
a matter of debate, given the lack of phenotypic characters to describe phylogenetic clades and the low
degree of resolution of phylogenetic trees. In this study, we investigated the phylogenetic relationships of
52 Usnea species from across the genus, based on ITS rDNA, nuLSU, and two protein-coding genes RPB1
and MCM7. ITS comprised several highly variable regions, containing substantial genetic signal, but also
susceptible to causing bias in the generation of the alignment. We compared several methods of alignment
of ITS and found that a simultaneous optimization of alignment and phylogeny (using BAli-phy)
improved significantly both the topology and the resolution of the phylogenetic tree. However the resolution
was even better when using protein-coding genes, especially RPB1 although it is less variable. The
phylogeny based on the concatenated dataset revealed that the genus Usnea is subdivided into four
highly-supported clades, corresponding to the traditionally circumscribed subgenera Eumitria, Dolichousnea,
Neuropogon and Usnea. However, characters that have been used to describe these clades are often
homoplasious within the phylogeny and their parallel evolution is suggested. On the other hand, most of
the species were reconstructed as monophyletic, indicating that combinations of phenotypic characters
are suitable discriminators for delimitating species, but are inadequate to describe generic subdivisions.