Protocruzia, a highly ambiguous ciliate (Protozoa; Ciliophora): very likely an ancestral form for Heterotrichea, Colpodea or Spirotrichea? With reevaluation of its evolutionary position based on multigene analyses.

The ciliate genus Protocruzia belongs to one of the most ambiguous taxa considering its systematic position, possible as a member of the classes Heterotrichea, Spirotrichea or Karyorelictea, which is tentatively placed into Spirotrichea in Lynn's 2008 system. To test these hypotheses, multigene trees (Bayesian inference, evolutionary distance, maximum parsimony, and maximum likelihood) were constructed using the small subunit rRNA (SSU rRNA) gene, internal transcribed spacer 2 (ITS2) and a protein coding gene (histone H4). All analyses agree that: (1) four morphotypes of Protocruzia from different geographical origins group together and form a monophyletic clade, which cannot be assigned to any of the eleven described ciliate classes; (2) it is invariably positioned on an isolated branch separated from the class Spirotrichea suggesting that this clade should be clearly removed from Spirotrichea; (3) this leads us to hypothesize that this taxon may indeed represent a lineage on a class rank. Based on the fact that it is, both morphologically and in molecular features, closely related to heterotrichs, Colpodea and Oligohymenophorea, Protocruziida might be an ancestral form for the subphylum Intramacronucleata in the evolutionary line from the class Heterotrichea (subphylum Postciliodesmatophora) to higher taxa.