Ľubomír Rajter

Ciliates have a long history of being central in evolutionary and ecological studies on eukaryotic microorganisms. Although thousands of species have been discovered, their total diversity still remains unknown. Here, we will discuss two unsolved problems that hinder the further exploration of ciliate diversity at the species level, and potential solutions to these problems are proposed. First, ciliate morphospecies are difficult to identify because the different silver stains are not scalable (they do not represent high-throughput methods) and basic supplies are lacking (e.g., protargol); a solution may be the development of fluorescent staining techniques. Second, ciliate phylogenetic species are difficult to identify because of extensive paralogy in nuclear-protein-coding genes; a solution may be to concentrate on sequencing mitochondrial genomes. These two approaches could be integrated into a high-throughput fluorescent-single-cell sorting and mitochondrial genomes sequencing process that would enable the observation and better understanding of ciliate species on a massive scale.

We investigated interrelationships between four free-living litostomatean lineages, using 18S rRNA gene and ITS region sequences as well as the secondary structure of the ITS2 molecules. Our phylogenetic analyses confirmed the deep split of free-living litostomateans into Rhynchostomatia and Haptoria represented here by Haptorida, Pleurostomatida, and Spathidiida. This bifurcation is also corroborated by the signature of the rhynchostomatian and haptorian ITS2 molecules. Specifically, the consensus stems of helices II and III are longer by one base pair in Rhynchostomatia, while the terminal loops of both helices are longer by one or two nucleotide/-s in Haptoria. A close relationship of Pleurostomatida and Haptorida is favored by quartet likelihood-mapping and supported by a 5’-AG vs. CU-3’ motif in the variable part of helix II and by two morphological apomorphies, i.e., meridionally extending somatic kineties and a non-three-rowed dorsal brush. Although monophyletic origin of Spathidiida is poorly supported in phylogenetic trees, the unique motif 5’-GA vs. UC-3’ present in the consensus helix II stem could be an important molecular synapomorphy of spathidiids, apart from the ancestrally anteriorly curved somatic kineties and the three-rowed dorsal brush. The peculiar family Pseudoholophryidae has very likely found its phylogenetic home among spathidiids, as an early branching lineage.

The morphology and phylogenetic position of a haptorian ciliate, Phialina pupula (Müller, 1773) Foissner, 1983, isolated from microaerobic sandy sediments of the floodplain area of the Boise River, Idaho, U.S.A., were studied using live observation, protargol impregnation, scanning electron microscopy, and the 18S rRNA gene as  well as the ITS region. The Boise population of P. pupula is characterized by a size of about 60–130 × 20–50 μm, an elliptical macronucleus with a single micronucleus, highly refractive dumbbell-shaped inclusions scattered throughout the cytoplasm and concentrated in the anterior body half, a single subterminal/terminal  contractile vacuole, about 10 μm long rod-shaped extrusomes, and an average of 15 ciliary rows. In phylogenetic analyses, the newly obtained sequences from P. pupula and Lacrymaria olor clustered within the family Lacrymariidae with full to moderate statistical support. Neither the genus Phialina nor the genus Lacrymaria was depicted monophyletic both in the single gene and multigene phylogenetic inferences. Specifically, the genus Phialina was shown as a paraphyletic assemblage containing members of the polyphyletic genus Lacrymaria. This indicates that the phialinid bauplan, i.e., an anterior body end differentiated into a head-like structure  directly attached to the trunk, might represent the ground pattern in the family Lacrymariidae. On the other hand, the long highly contractile neck carrying the head-like structure probably evolved later and convergently in multiple Lacrymaria species from Phialina-like ancestors.

Pleurostomatids are raptorial ciliates that form a very distinct group within the Haptoria. Traditionally, the order Pleurostomatida was divided into two families: the Amphileptidae with two perioral kineties and a suture formed by the right side ciliary rows, and the Litonotidae with three perioral kineties and without suture. However, molecular phylogenies depicted the “traditional” Amphileptidae as a paraphyletic assemblage nesting also the Litonotidae. To overcome this problem we have analyzed genealogy of pleurostomatids using morphological data and 18S rRNA gene sequences, including newly sequenced genera Acineria and Kentrophyllum. Specifically, we have combined a morphological and molecular approach and have used also some other phylogenetic tools such as phylogenetic networks, split spectrum analysis, quartet mapping as well as the likelihood method of tracing history of morphological characters. These analyses show that: (1) there are not two but three distinct pleurostomatid lineages – Epiphyllidae fam. nov., Amphileptidae and Litonotidae; (2) epiphyllids (Epiphyllum + Kentrophyllum) represent a basal pleurostomatid group which is defined by two perioral kineties, by the presence of a suture on both the right and the left side of the body, by the loss of the oral bulge extrusomes, and by the extrusome fringe extending all around the body except for the oral region; (3) the families Amphileptidae and Litonotidae are monophyletic each, and represent sister groups; (4) Acineria belongs to the Litonotidae, as already indicated by morphological data; (5) Loxophyllum is a monophyletic and crown genus of the Litonotidae; and (6) Litonotus is paraphyletic, which could be very likely caused by a rapid radiation event that did not allow primary nucleotide homologies to be fixed.