Linking multi-gene and morphological data in the subclass Scuticociliatia (Protista, Ciliophora) with establishment of the new family Homalogastridae fam. nov

Abstract

Scuticociliatia is one of the most species-rich subclasses in the phylum Ciliophora. The evolutionary relationships among Scuticociliatia groups have long been very unclear due to the homogeneity of morphology and insufficiency of molecular data. With morphological and multi-gene-based molecular data presented here, the evolutionary phylogeny of several Scuticociliatia taxa that were hitherto especially poorly defined is analyzed and discussed. The results indicate: (1) all scuticociliates cluster into two well supported and one poorly supported group, representing three order-level taxa; (2) with the support of both morphological and molecular data, a new family Homalogastridae fam. nov. is proposed in the order Philasterida; (3) Parauronema is formally transferred to Uronematidae and Potomacus is treated as incertae sedis in the order Philasterida, therefore Parauronematidae is proposed to be a junior synonym of Uronematidae; (4) the genus Madsenia and the species Parauronema longum and Pseudocyclidium longum are treated as incertae sedis, while the genus Protophyra should be maintained in the family Ancistridae. In addition, the putative secondary structure of internal transcribed spacer 2 (ITS2) of representative taxa from the three orders of Scuticociliatia are analyzed, and consensus structures and nucleotide composition in each order are exhibited.

Supplementary information: The online version contains supplementary material available at 10.1007/s42995-024-00264-8.

Keywords: Ciliated protists; Homalogastridae fam. nov.; Phylogeny; Scuticociliates; rRNA secondary structure.

Fig. 1

Morphological schematic illustration of a representative of the subclass Scuticociliatia (A) and the phylogenetic position of the class Oligohymenophorea in the phylum Ciliophora according to Gao et al. (2016) (B)

Fig. 2

Source distribution of SSU rRNA gene sequences of three Scuticociliatia clades (A), numbers indicate the number of sequences; comparison of maximum likelihood (ML) tree bootstrap values before and after adding environmental sequences (B); and comparison of G + C content percentage of SSU rRNA gene sequences from three Scuticociliatia orders (C). Wilcoxon test was used for comparison. *p < 0.05; **p < 0.01; ***p < 0.001; ns not significant

Fig. 3

Maximum likelihood (ML) tree based on SSU rRNA gene sequence data, showing the phylogenetic positions of the taxa of subclass Scuticociliatia. Numbers at nodes denote ML bootstrap values/Bayesian inference (BI) posterior probabilities. Asterisks (*) indicate a mismatch in topologies between ML and BI analyses. Fully supported (100%/1.00) nodes are marked with solid circles. The scale bar corresponds to 10 substitutions per 100 nucleotide positions. All branches are drawn to scale. The systematic classification mainly follows Gao et al. (2016). New assignments proposed in the present study are marked with the letter “N” in the circles. Sequences with question marks indicate the identity or taxonomic assignment of these sequences needs to be confirmed. Family names with question marks indicate families that are polyphyletic and need further investigation in future studies. The number of sequences from each of the three orders of Scuticociliatia is provided in the pie chart

Fig. 4

Maximum likelihood (ML) tree based on LSU rRNA gene (A) and COI gene sequence data (B). Numbers at nodes denote ML bootstrap values/Bayesian inference (BI) posterior probabilities. Asterisks (*) indicate a mismatch in topologies between ML and BI analyses. Fully supported (100%/1.00) nodes are marked with solid circles. The scale bar corresponds to 10 substitutions per 100 nucleotide positions. All branches are drawn to scale except for the basal branch in the LSU rRNA gene tree. The systematic classification mainly follows Gao et al. (2016). New taxonomic assignments proposed in the present study are marked with letter “N” in the circles. Sequences with question marks indicate the identity or taxonomic assignment of these sequences should be confirmed, while family names with question marks indicate families that are polyphyletic and need further investigation in future studies

Fig. 5

Maximum likelihood (ML) tree based on concatenated gene sequence data. In the three-gene tree, the sequences are concatenated in the order: SSU rRNA gene, LSU rRNA gene and ITS1-5.8S-ITS2 gene region; in the four-gene tree, the order is: COI gene, SSU rRNA gene, LSU rRNA gene and ITS1-5.8S-ITS2 gene region. The SSU rRNA gene sequence is available for all the taxa whereas the COI gene, LSU rRNA gene and ITS1-5.8S-ITS2 gene region are available for only a subset of these taxa (Supplementary Table S2). Numbers at nodes denote ML bootstrap values/Bayesian inference (BI) posterior probabilities. Asterisks (*) indicate a mismatch in topologies between ML and BI analyses. Fully supported (100%/1.00) nodes are marked with solid circles. The scale bar corresponds to 10 substitutions per 100 nucleotide positions. All branches are drawn to scale. The systematic classification mainly follows Gao et al. (2016). New assignments proposed in the present study are marked with the letter “N” in the circles. Family names with question marks indicate families that are polyphyletic and need further investigation in future studies

Fig. 6

Maximum likelihood (ML) tree based on SSU rRNA gene sequence data of Scuticociliatia (in colored blocks), other subclass (with no blocks) and related environmental sequences (in grey blocks). ML bootstrap values and G + C content (%) are represented by different colors. Source and location information are obtained from the NCBI database or related references. The scale bar corresponds to 10 substitutions per 100 nucleotide positions. All branches are drawn to scale. The systematic classification mainly follows Gao et al. (2016). Family names with question marks indicate the identity of the sequences or family assignment of the species should be confirmed. Straight lines after family names and environmental sequences indicate those environmental sequences are possibly members of the corresponding families. For detailed sequence information, see Supplymentary Table S3

Fig. 7

Collapsed maximum likelihood tree of Scuticociliatia based on SSU rRNA gene sequence data with schematic oral structure diagrams of some representative members of the three Scuticociliatia orders. The colored clades represent family assignment, and the colored lines at the outer side of the clades represent order assignment. The scale bar corresponds to 10 substitutions per 100 nucleotide positions. All branches are drawn to scale. The systematic classification mainly follows Gao et al. (2016). M1–3, membranelle 1–3; PM, paroral membrane; Sc, scutico-vestige; SK, somatic kinety

Fig. 8

Comparison of putative ITS2 region secondary structure of the taxa from Uronematidae, Homalogastridae, and Glauconematidae of the order Philasterida (A) and the consensus secondary structure based on these selected sequences, with nucleotides composition in different helixes (B). Colored circles in A indicate bulges with at least two nucleotides. Yellow circles represent bulges with the same number of nucleotides at both sides, green circles represent bulges with more nucleotides at 5’ ends than 3’ ends of ITS2 region, and pink circles represent bulges with more nucleotides at 3’ ends than 5’ ends of ITS2 region. Small arrows in B indicate pyrimidine-pyrimidine mismatch of Helix II, box indicates the highly conserved motif in Helix III, dash lines indicate terminal bulges in helixes, and solid lines indicate bulges in helixes with more than one nucleotide. Fully conserved positions of the consensus structure are marked with specific nucleotides