The planktonic freshwater ciliate Balanion planctonicum (Ciliophora, Prostomatea): A cryptic species complex or a "complex species"?

The globally distributed ciliate Balanion planctonicum is a primary consumer of phytoplankton spring blooms. Due to its small size (~20 μm), identification and quantification by molecular tools is preferable as an alternative to the laborious counting of specimen in quantitative protargol stains. However, previous sequencing of the 18S rDNA V9 region of B. planctonicum from Lake Zurich (Switzerland) and subsequent quantification by fluorescence in situ hybridization yielded significantly lower cell numbers than using morphotype counting. This raised the question of whether B. planctonicum shows a cryptic diversity or whether it is just a 'complex species' with intra-clonal polymorphisms. Over three years, we established numerous monoclonal cultures, and long-read sequencing of rDNA operons revealed four distinct dominant haplotypes (BpHs 1-4). The gene sequences of BpHs 1 and 3 differed by 6% and did not share intra-clonal polymorphisms, providing evidence for two distinct clades. Furthermore, phylogenetic analyses corroborate the sister relationship between Balanion and Askenasia (plus Hexasterias and Radiosperma). Morphologically, the two Balanion clades are nearly indistinguishable with small differences in macronucleus size and in the cell length to width ratio. CARD-FISH analyses indicated that the diversity of B. planctonicum is even more extensive with still unidentified clades.