Molecular phylogeny of unculturable Karyorelictea (Alveolata, Ciliophora)

Abstract

Karyorelictea is a class of traditionally unculturable ciliates characterized by a non-dividing macronucleus. Their classification has been recently revised and morphological redescription of many species has been performed as well. On the contrary, molecular data of karyorelictean ciliates are largely underrepresented in public databases. In the present article we resumed and improved a method to characterize 18S rRNA gene sequences through direct amplification and sequencing of single cells. Using this approach, we characterized 12 different karyorelictean molecular operational taxonomic units (MOTUs: nine trachelocercids, one Geleia, one Remanella and one Loxodes), most of which were also photo-recorded. These molecular data were used to reconstruct phylogenetic relationships among the three orders (Protostomatida, Loxodida and Protoheterotrichida) in which the class is traditionally subdivided. The most supported tree topology shows an association between orders Loxodida (Loxodes, Remanella) and Protoheterotrichida (Geleia), in contrast with previous works associating orders Loxodida and Protostomatida (trachelocercids) on a morphological basis.

Rare Freshwater Ciliate Paramecium chlorelligerum Kahl, 1935 and Its Macronuclear Symbiotic Bacterium “Candidatus Holospora parva”

“…The amplification of the host 18S rDNA required a semi-nested PCR approach, as reported in [35], in order to achieve sufficient DNA for direct sequencing. In short, the first PCR was carried out with primers 18S F9 Euk (5’-CTGGTTGATCCTGCCAG-3’ [36]) and 18S R1513Hypo (5’-TGATCCTTCYGCAGGTTC-3’ [37]), then using as template the diluted first PCR product, a second step was performed employing two semi-nested PCRs: 1—using primers 18S F9 and Penic R1280 (5’-CGACACGTCCTAACAAGA-3’ [38]) and 2—with primers Penic F82 (5’-GAAACTGCGAATGGCTC-3’ [39]) and 18S R1513Hypo.…”

Phylogeny and Establishment of a New Ciliate Family, Wilbertomorphidae fam. nov. (Ciliophora, Karyorelictea), a Highly Specialized Taxon Represented by Wilbertomorpha colpoda gen. nov., spec. nov.

“…More recently, molecular data on karyorelicteans have begun to accumulate, in particular small subunit (SSU) rRNA gene sequence data (Andreoli et al. ; Gao et al. ; Mazei et al.…”

Specificity in diversity: single origin of a widespread ciliate-bacteria symbiosis

“…PCR product bands were cut from the gel after electrophoresis, purified with the Qiaquick gel extraction kit (Qiagen) and sequenced with BigDye Terminator v 3.1 Cycle Sequencing Kit (Life Technologies) on a 3130 × 1 Genetic Analyzer (Applied Biosystems), using EukA, EukB and 18SF492karyo (AGGACCCACTGGAGGG, modified from [23]) as sequencing primers. Sequence chromatograms were inspected and assembled in Sequencher 4.6 (Gene Codes), retaining sequences that had more than 95% of bases with a Phred score greater than 20.…”