Morphology and Phylogeny of a New Species of Anaerobic Ciliate, Trimyema finlayi n. sp., with Endosymbiotic Methanogens - PubMed (original) (raw)

Morphology and Phylogeny of a New Species of Anaerobic Ciliate, Trimyema finlayi n. sp., with Endosymbiotic Methanogens

William H Lewis et al. Front Microbiol. 2018.

Abstract

Many anaerobic ciliated protozoa contain organelles of mitochondrial ancestry called hydrogenosomes. These organelles generate molecular hydrogen that is consumed by methanogenic Archaea, living in endosymbiosis within many of these ciliates. Here we describe a new species of anaerobic ciliate, Trimyema finlayi n. sp., by using silver impregnation and microscopy to conduct a detailed morphometric analysis. Comparisons with previously published morphological data for this species, as well as the closely related species, Trimyema compressum, demonstrated that despite them being similar, both the mean cell size and the mean number of somatic kineties are lower for T. finlayi than for T. compressum, which suggests that they are distinct species. This was also supported by analysis of the 18S rRNA genes from these ciliates, the sequences of which are 97.5% identical (6 substitutions, 1479 compared bases), and in phylogenetic analyses these sequences grouped with other 18S rRNA genes sequenced from previous isolates of the same respective species. Together these data provide strong evidence that T. finlayi is a novel species of Trimyema, within the class Plagiopylea. Various microscopic techniques demonstrated that T. finlayi n. sp. contains polymorphic endosymbiotic methanogens, and analysis of the endosymbionts' 16S rRNA gene showed that they belong to the genus Methanocorpusculum, which was confirmed using fluorescence in situ hybridization with specific probes. Despite the degree of similarity and close relationship between these ciliates, T. compressum contains endosymbiotic methanogens from a different genus, Methanobrevibacter. In phylogenetic analyses of 16S rRNA genes, the Methanocorpusculum endosymbiont of T. finlayi n. sp. grouped with sequences from Methanomicrobia, including the endosymbiont of an earlier isolate of the same species, 'Trimyema sp.,' which was sampled approximately 22 years earlier, at a distant (∼400 km) geographical location. Identification of the same endosymbiont species in the two separate isolates of T. finlayi n. sp. provides evidence for spatial and temporal stability of the Methanocorpusculum-T. finlayi n. sp. endosymbiosis. T. finlayi n. sp. and T. compressum provide an example of two closely related anaerobic ciliates that have endosymbionts from different methanogen genera, suggesting that the endosymbionts have not co-speciated with their hosts.

Keywords: Methanocorpusculum; Trimyema; anaerobic; ciliate; endosymbiont; methanogen; phylogeny.

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Figures

FIGURE 1

FIGURE 1

A schematic drawing of the (A) ventral and (B) dorsal sides of a Trimyema finlayi n. sp. cell. CCK, caudal cilium kinety; G1, first ciliary girdle; G2, second ciliary girdle; G3, third ciliary girdle; G4, fourth ciliary girdle; Ma, macronucleus; mn, micronucleus; OK, oral kineties.

FIGURE 2

FIGURE 2

Microscopic imaging of T. finlayi n. sp. whole cells. DIC images of silver carbonate impregnated cells, (A,B) and (C,D) show two sides of the same cells, (E) squashed cell showing oral kineties, (F) squashed cell showing cytoproct. (G,H) Maximum intensity projection of a Z-stack of confocal images across a single T. finlayi cell double-labeled with two FISH probes. (G) _Methanocorpusculum_-specific probe (SYM5) dual-labeled with 6-FAM. (H) Archaea-specific probe (ARCH915) dual-labeled with Cy3, white arrows indicate extracellular Archaea that were not labeled by the probe SYM5 (G). (I) F420 auto-fluorescence. (J–L) In vivo DIC images. CCK, caudal cilium kinety; CP, cytoproct; FV, food vacuole; G1, first ciliary girdle; G2, second ciliary girdle; G3, third ciliary girdle; G4, fourth ciliary girdle; MA, macronucleus; NK, N-kineties; OC, oral cavity; OK, oral kineties. Scale bars = 10 μm.

FIGURE 3

FIGURE 3

Transmission electron microscopy (TEM) images of T. finlayi n. sp. showing polymorphic methanogenic endosymbionts and hydrogenosomes (red arrowheads). Disc-shaped (blue arrowheads) and stellate form (yellow arrowheads) morphotypes are shown, as well as intermediate stages (green arrowheads). FV, food vacuole. Scale bars (A) = 5 μm, (B) = 1 μm.

FIGURE 4

FIGURE 4

Bayesian phylogeny inferred from 1640 nucleotide alignment of 18S rRNA genes of Plagiopylea species using the GTR+Γ+I model. Support values represent maximum likelihood bootstrap support/Bayesian posterior probabilities. Scale bar represents the number of substitutions per site.

FIGURE 5

FIGURE 5

Bayesian phylogeny inferred from a 1372 nucleotide alignment of methanogenic Archaea 16S rRNA genes using the GTR+Γ+I model. Support values represent maximum likelihood bootstrap support/Bayesian posterior probabilities. Scale bar represents the number of substitutions per site.

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