Abstract
Monophyly is essential prerequisite for species delimitation. In protists, molecular phylogenetic studies revealed, in recent years, many polyphyletic groups at all taxonomic levels, which afterwards led to their revisions for getting closer to natural classification. Above all, species-level polyphyly not only causes revisions of species, but also forces taxonomists to face species problems, especially species delimitation.
Technological advancement in molecular biology represented by next-generation sequencing enables to develop various new methods for species delimitation, which open up a new way of taxonomy. Morphologically indistinct groups such as small flagellates or amoebas have been out of search for intraspecific polyphyly because of inability of delimiting their species boundaries. However, a new approach called "reverse taxonomy" including increased taxon sampling shed light on their species delimitation.
