Insights into the phylogeny of systematically controversial haptorian ciliates (Ciliophora, Litostomatea) based on multigene analyses

Abstract

The ciliate subclass Haptoria is a diverse taxon that includes most of the free-living predators in the class Litostomatea. Phylogenetic study of this group was initially conducted using a single molecular marker small-subunit ribosomal RNA (SSU rRNA genes). Multi-gene analysis has been limited because very few other sequences were available. We performed phylogenetic analyses of Haptoria incorporating new SSU rRNA gene sequences from several debated members of the taxon, in particular, the first molecular data from Cyclotrichium. We also provided nine large-subunit ribosomal RNA (LSU rRNA) gene sequences and 10 alpha-tubulin sequences from diverse haptorians, and two possible relatives of controversial haptorians (Plagiopylea, Prostomatea). Phylogenies inferred from the different molecules showed the following: (i) Cyclotrichium and Paraspathidium were clearly separated from the haptorids and even from class Litostomatea, rejecting their high-level taxonomic assignments based on morphology. Both genera branch instead with the classes Plagiopylea, Prostomatea and Oligohymenophora. This raises the possibility that the well-known but phylogenetically problematic cyclotrichiids Mesodinium and Myrionecta may also have affinities here, rather than with litostomes; (ii) the transfer of Trachelotractus to Litostomatea is supported, especially by the analyses of SSU rRNA and LSU rRNA genes, however, Trachelotractus and Chaenea (more uncertainly) generally form the two deepest lineages within litostomes; and (iii) phylogenies of the new molecular markers are consistent with SSU rRNA gene information in recovering order Pleurostomatida as monophyletic. However, Pleurostomatida branches cladistically within order Haptorida, as does subclass Trichostomatia (on the basis of SSU rRNA phylogenies). Our results suggest that the class-level taxonomy of ciliates is still not resolved, and also that a systematic revision of litostomes is required, beginning at high taxonomic levels (taxa currently ranked as subclasses and orders).

Figure 1.

Morphology and infraciliature of nine haptorian ciliates in vivo and after silver impregnation (two images (m,n) are from Pan et al. [15], others are from Lin et al. [16]). (a,b) Amphileptus marinus, (c,d)  Chaenea teres, (e,f) Phialina salinarum, (g,h) Paraspathidium apofuscum, (i,j) Trachelotractus entzi, (k,l)  Chaenea vorax, (m,n) Epiphyllum shenzhenense, (o,p) Loxophyllum jini, (q,r) Cyclotrichium cyclokaryon.

Figure 2.

Models of the secondary structure of variable region 4 (V4) of the SSU rRNA molecule, and the phylogenetic tree of 15 taxa based on V4 region primary sequences and secondary structure combined. Models of the secondary structure comprising helices E23-1,(2), 4, 7, 8, 9, 10, 11, 12, 13, 14 and two pseudoknots formed by helices E23-9 to 12, helices E23-13 and 14 are shown. The number of nucleotides in Helix E23-1 and 2 (or only E23-1) for each species is given above the long arrow. Note the Helix E23-7 in Paraspathidium–Cyclotrichium and the classes Plagiopylea and Prostomatea (arrows).

Figure 3.

(a) Maximum-likelihood (ML) tree of 80 SSU rRNA gene sequences including all ciliate classes, with emphasis on the class Litostomatea. New sequences are shown in bold text. GenBank accession numbers are given after names of species. Numbers at nodes show bootstrap values from ML, Bayesian inference (BI) posterior probabilities, and bootstrap values from maximum parsimony (MP) in this order; instances in which a particular node was not recovered in the estimated BI or MP tree are designated by an asterisk (*). Nodes receiving maximal support (100% ML, 1.00 BI, 100% MP) are indicated by solid circles. (b) The subtree representing clade X from the ML tree of supplementary analysis ‘set 3’, showing the position of the Askenasia spp. clade. For clarity, only backbone support values are shown (ML bootstrap values, BI posterior probabilities). For both trees, the scale bars correspond to 0.05 expected substitutions per site. Certain groups were simplified as solid triangles to reduce the size of the figure; width and length indicates the number of sequences and the average branch length in this group.

Figure 4.

ML tree estimated from the LSU rRNA sequences of 20 ciliate taxa, with myzozoan alveolates as outgroups. New sequences are shown in bold text. Numbers at nodes are the following: ML bootstrap values followed by BI posterior probability and MP bootstrap values. Nodes not present in the estimated MP tree are designated by asterisks (*). Nodes receiving maximal support (100% ML, 1.00 BI, 100% MP) are indicated by solid circles. The scale bar corresponds to 0.1 expected substitutions per site.

Figure 5.

ML tree for the 54-taxon alpha-tubulin dataset (amino acid-level analysis). Newly sequenced species are shown in bold text. Numbers at nodes are ML bootstrap values followed by BI posterior probabilities. Nodes not recovered by BI are designated by asterisks (*). The scale bar corresponds to 0.02 expected substitutions per site.