AbstractThe ciliate genus Spirostomum comprises eight morphospecies, inhabiting diverse aquatic environments worldwide, where they can be used as water quality indicators. Although Spirostomum species are relatively easily identified using morphological methods, the previous nuclear rDNA-based phylogenies indicated several conflicts in morphospecies delineation. Moreover, the single locus phylogenies and previous analytical approaches could not unambiguously resolve phylogenetic relationships among Spirostomum morphospecies. Here, we attempt to investigate species boundaries and evolutionary history of Spirostomum taxa, using 166 new sequences from multiple populations employing one mitochondrial locus (CO1 gene) and two nuclear loci (rRNA operon and alpha-tubulin gene). In accordance with previous studies, relationships among the eight Spirostomum morphospecies were poorly supported statistically in individual gene trees. To overcome this problem, we utilised for the first time in ciliates the Bayesian coalescent approach, which accounts for ancestral polymorphisms, incomplete lineage sorting, and recombination. This strategy enabled us to robustly resolve deep relationships between Spirostomum species and to support the hypothesis that taxa with compact macronucleus and taxa with moniliform macronucleus each form a distinct lineage. Bayesian coalescent-based delimitation analyses strongly statistically supported the traditional morphospecies concept but also indicated that there are two S. minus-like cryptic species and S. teres is non-monophyletic. Spirostomum teres was very likely defined by a set of ancestral features of lineages that also gave rise to S. yagiui and S. dharwarensis. However, molecular data from type populations of the morphospecies S. minus and S. teres are required to unambiguously resolve the taxonomic problems.