Holospora spp. and “Candidatus Gortzia infectiva”, known as Holospora-like bacteria (HLB), are commonly found as nuclear endosymbionts of ciliates, especially the Paramecium genus. HLB are related by phylogenetic relationships, morphological features, and life-cycles, which involve two alternating morphotypes: reproductive and infectious forms (RF, IF). In this paper we describe a novel species belonging to the “Ca. Gortzia” genus, detected in P. multimicronucleatum, a ciliate for which infection by an HLB has not been reported, discovered in India. This novel endosymbiont shows unusual and surprising features with respect to other HLB, such as large variations in IF morphology and the occasional ability to reproduce in the host cytoplasm. We propose the name of “Candidatus Gortzia shahrazadis” for this novel HLB. Moreover, we report two additional species of HLB from Indian Paramecium populations: “Ca. Gortzia infectiva” (from P. jenningsi), and H. obtusa (from P. caudatum); the latter is the first record of Holospora from a tropical country. Although tropical, we retrieved H. obtusa at an elevation of 706 m corresponding to a moderate climate not unlike conditions where Holospora are normally found, suggesting the genus Holospora does exist in tropical countries, but restricted to higher elevations.

The subclass Peniculia (Oligohymenophorea, Intramacronucleata) is one of the most known groups of the phylum Ciliophora, being composed by very notorious representatives, such as Paramecium and Frontonia. Nevertheless, phylogenetic relationships among genera within this subclass are still far from being resolved. Moreover, for several members of the group the characterization by molecular markers is still lacking, such as for Wenrichia, Clathrostoma, Paraclathrostoma, Didieria and, to date, also for Neobursaridium. The finding of one strain of Neobursaridium gigas from India led to the first molecular characterization of this uncommonly sampled ciliate. The 18S rDNA sequence and the COI sequence were obtained and used for phylogenetic analyses. Moreover, the partial mitochondrial genome of N. gigas was sequenced, annotated and employed for phylogenomics analysis. To increase the sampling effort for the Paramecium clade, several newly obtained 18S rDNA sequences of parameciids are herein presented. Unexpectedly, the inclusion of N. gigas's molecular data in phylogenetics/phylogenomics analyses did not help to solve the complex evolution relationships inside Peniculia. Conversely, it raised new and intriguing questions about Paramecium phylogeny, since N. gigas clustered inside Paramecium clade as sister species of Paramecium bursaria in all the performed analyses. A critical revision of past and present data led to rename N. gigas as Paramecium gigas (Balech, 1941) comb. nov., and triggered the revision of genus Paramecium, with the proposal of the new subgenus Gigaparamecium subgen. nov. Hypotheses on the evolution of giant morphologies in ciliates are also discussed.

Symbioses between bacteria and eukaryotes are widespread and may have significant impact on the evolutionary history of symbiotic partners. The order Rickettsiales is a lineage of intracellular Alphaproteobacteria characterized by an obligate association with a wide range of eukaryotic hosts, including several unicellular organisms, such as ciliates and amoebas. In this work, we characterized the Rickettsiales symbionts associated with two different genotypes of the freshwater ciliate Paramecium caudatum originated from freshwater environments in distant geographical areas. Phylogenetic analyses based on 16S rRNA gene showed that the two symbionts are closely related to each other (99.4% identity), belong to the family Rickettsiaceae, but are far-related with respect to previously characterized Rickettsiales. Consequently, they were assigned to a new species of a novel genus, namely "Candidatus Spectririckettsia obscura." Screening on a database of short reads from 16S rRNA gene amplicon-based profiling studies confirmed that bacterial sequences related to the new symbiont are preferentially retrieved from freshwater environments, apparently with extremely scarce occurrence (< 0.1% positive samples). The present work provides new information on the still under-explored biodiversity of Rickettsiales, in particular those associated to ciliate host cells.