Morphology and molecular basis of cell-cell recognition in Euplotes songi sp. nov., a phylogenetically early-emerging new species of Euplotes (Ciliophora, Spirotrichea).

Euplotes songi sp. nov. is described as a new morphospecies of Euplotes, isolated from shallow interstitial waters off the southern Italian coast of the Adriatic Sea. Its small (49×33 µm) body has a slightly asymmetric elliptical shape, contains a relatively large hook-shaped macronucleus consistently associated with a single micronucleus and harbours γ-proteobacteria as endosymbionts. Formation of resting stages was not observed, either as a result of culture ageing or in response to conditions commonly inducing encystation. The dorsal surface features a silver-line system ('dargyrome') of the double type, with two longitudinal inter-kinety rows of argentophilic polygons that are markedly unequal in width. Four prominent longitudinal ridges, each bearing one kinety of 9-12 dikinetids, run along the dorsal body side, while two shorter kineties extend along the lateral cell margins. The ventral surface bears 10 fronto-ventral, five transverse, two stiff and elongated caudal cirri and a single short, highly motile marginal cirrus. The buccal field extends nearly four-fifths of the body length and is bordered by 28-31 adoral membranelles. In the Euplotes SSU rRNA gene-based phylogenetic tree, E. songi clusters with Euplotes huizhouensis, Euplotes petzi and Euplotes sinicus, forming the earliest emerging clade. Consistent with other Euplotes species, E. songi cells grow and mate under the control of a family of waterborne, cell-type-specific protein pheromones. Native pheromones, purified from genetically distinct cultures, are active at micromolar concentrations and consist of short 33-aa sequences, which include eight cysteine residues and fold into a bundle of three antiparallel helical segments. This bundle likely represents an evolutionary forerunner of the more complex helical bundles characteristic of pheromone families synthesized by phylogenetically derived Euplotes species. A simple organization is similarly exhibited by the macronuclear pheromone-coding genes, having full-length sequences of only 728 or 732 bp and an intron-free ORF.