The green algal genus Microthamnion (Microthamniales, Trebouxiophyceae) has been
extensively studied, but the question of species delimitation remained a matter of
controversy. The morphological traits used to discriminate species in Microthamnion
have shown to be quite polymorphic, and the restriction enzyme analysis performed in
a first molecular attempt does no longer meet modern standards.
The present study used a detailed molecular approach and combined several methods
to clarify the matter of species delimitation on a molecular level. A multi-gene
alignment comprising the nuclear-encoded 18S, and 28S rRNA genes, and the ITS2
molecule, as well as the plastid-encoded rbcL gene was assembled and used for
concatenated phylogenetic analyses. The 74 Microthamnion strains investigated in this
study fell into four monophyletic clades (one with a distinct subdivision) and nine longbranched
lineages, which are assumed to correspond to species level. An apomorphy
analysis was performed in order to find non-homoplasious synapomorphies (NHSs),
and thus unique molecular signatures, for the clades and lineages inferred from the
phylogeny. In a novel approach that interpreted molecular data in an alternative way,
these NHSs and other ‘phenotypic molecular characters’ were compiled in a data
matrix and used for a parsimony tree reconstruction. The clade boundaries and
lineages inferred from the Microthamnion phylogeny were confirmed by both, unique
molecular signatures and the tree based on all phenotypic molecular characters,
resulting in 14 putative species delineated by molecular methods
