Host collection and identification

Prorhinotermes simplex and Coptotermes gestroi were collected in Fort Lauderdale, Florida, USA, and their morphological identification was confirmed by DNA barcoding (accessions JX975355 and MF373426, respectively). A second species of Coptotermes was collected in Mount Glorious, Queensland, Australia. It was identified to the genus level by morphology in the field. Ethanol-preserved samples were returned to the lab for DNA barcoding using the mitochondrial large subunit rRNA gene (accession KJ438378), which allowed for comparisons with two of the three species of Coptotermes known from the area. Coptotermes frenchi barcodes shared on average 95.5% identity with the Australian barcode and showed no close affinity in the phylogenetic tree (Fig. 1, Suppl. Figure 1). The identity shared with Coptotermes lacteus barcodes was on average 96.7%, and in phylogenetic trees the Australian barcode branched closer to C. lacteus, but did not branch within the clade of C. lacteus barcode isolates (which share 99–100% identity). Based on this, we conclude that the collection corresponds to neither C. lacteus nor C. frenchi. The only other Coptotermes known in this region is Coptotermes acinaciformis, but unfortunately there is no barcoded vouchered specimen of this species (Coptotermes are notorious difficult to identify). Therefore, we conclude that the probable identity of our isolate is C. acinaciformis, but it could also be a presently unknown species. We will henceforth refer to this specimen as Coptotermes cf. acinaciformis.

Figure 1 Maximum likelihood phylogeny of host barcode sequences (mitochondrial LSU rRNA) from Coptotermes. All available Coptotermes barcodes are included. The new specimen from Florida is confirmed to correspond to C. gestroi, while the new specimen from Australia is a species for which no barcode is currently available. Based on the available barcodes to which it does not correspond and the diversity of Coptotermes from this region of Queensland, we infer this is most likely a specimen of C. acinaciformis, and therefore refer to this as Coptotermes cf. acinaciformis. Conspecific clades are collapsed and represented by triangles. Numbers at nodes correspond to ML bootstrap support over 50% (values for nodes with lower support are not shown for clarity), and the scale bar represents a distance of 0.06 substitutions per site. The complete tree with outgroups can be seen in Suppl. Figure 1. Full size image

Morphology of new Pseudotrichonympha species

In all three termite species we observed and photographed cells matching the description of Pseudotrichonympha (Fig. 2). Specifically, all three symbionts were characterized by an apical cap with an adjacent apical band of short rostral flagella, a distinctive posterior band of very long rostral flagella, and intermediate-length flagella covering the remainder of the post-rostral body. Cells contained a single nucleus central in the sub-rostral area (e.g. see Fig. 2E). The Pseudotrichonympha from Coptotermes cf. acinaciformis (Fig. 2F) was very large (length average: 392 µm, length range: 332–448 µm, width average: 100 µm,width range: 94–108, n = 5) and had a wide ovoid shape and a sharply tapering posterior tip. The Pseudotrichonympha from C. gestroi (Fig. 2A–C) had a more slender body (length average: 303 µm, length range: 280–330 µm, width average: 62 µm,width range: 56–71 µm, n = 5), and was distinguished by the tendency to possess a robust sub-apical shoulder region that frequently had the greatest diameter of the body (Fig. 2B). In stressed cells (e.g. during isolation), this region expanded greatly and produced a distinctive cup-shaped cell with the apex at the centre of the cup (Fig. 2C). The Pseudotrichonympha from P. simplex (Fig. 2D,E) tended to have a wide body around the midline (length average: 262 µm, length range: 200–316 µm, width average: 122 µm, width range: 89–180 µm, n = 12), and was most obviously defined by the presence of a strange intracellular body we dubbed the “rotatosome”.

Figure 2 Light micrographs of new Pseudotrichonympha specimens. (A–C) Differential interference contrast images of Pseudotrichonympha leei from Coptotermes gestroi showing the overall body shape, with the long posterior rostral flagella emerging below the pointed apical cap, and shorter post-rostral flagella (A). (B,C) Show the commonly observed robust collar (B) and cup-shaped anterior of stressed cells (C). (D,E) Differential interference contrast images of Pseudotrichonympha pearti from Prorhinotermes simplex showing the overall body shape, single nucleus, and detail of the rostral region, including the apical cap, rostral tube, and long posterior rostral flagella. (F) Phase contrast images (taken in the field) of Pseudotrichonympha lifesoni from Coptotermes cf. acinaciformis showing the basic body shape and size and the single central nucleus. Bars represent 50 µm. Full size image

The rotatosome (see Fig. 3, and Supplemental video files) is a spherical body on average 13 µm in diameter (n = 3) that was only ever observed in a single copy per cell. It could only be observed in some focal planes, so whether it is in all cells or not is not known. It continuously rotated, causing visible turbulence in the cytoplasm around it. The lumen of the rotatosome was not uniform. At certain observational planes the rotatosome could be seen to include a rod 6-7 µm in length that emerged from one side of the circle (which side depended on the angle from which it was viewed), was stationary with respect to the cytoplasm, and in which the material appears to stream towards the outermost layer of the sphere, where it can then be seen to turn and turn again in the direction of the rotation. We examined the possibility that the rotatosome is an endosymbiont living within Pseudotrichonympha by Hoechst staining. In fixed and stained cells, the nucleus consistently showed a strong signal for the presence of DNA (in the pattern expected for a parabasalian, which have condensed interphase chromosomes), but the rotatosome retained no detectible stain (Fig. 3C,D). Similarly, we manually broke three Pseudotrichonympha cells open under the microscope to see if a rotatosome could be observed swimming freely, but the body consistently disappeared when cells were disrupted (not shown). A spherical structure was previously observed, in 1923, in the cytoplasm of a species identified as P. sphaerophora from a termite identified as Rhinotermes nasutus in British Guiana9. In this case, the sphere was reported to be larger (25 µm), consisting of a non-staining centre covered by a lighter layer in a concentric organization. The structured was reported from a fixed and stained sample, so no rotation was observed, and it was hypothesized at the time to function as a stercoma for storing excretory material. Based on the difference in size, lack of detail in the 1923 description, and the fact this is apparently not the same species of host or symbiont, or the same collection location, it would be premature to conclude these are the same structure. Overall, in either case, many questions remain about the function and the form of the rotatosome that will hopefully be resolved in the fullness of time, but it appears to be a sub-cellular structure rather than an endosymbiont.

Figure 3 Details of the “rotatosome” from Pseudotrichonympha pearti. Differential interference contrast details of the rotating structure in the P. pearti cytoplasm (A,B), with (B) showing the projection into the cytoplasm (this can also be seen in the Supplemental video, which also shows how the structure rotates). (C and D) show a rotatosome next to the P. pearti nucleus in fixed and Hoechst stained cells. In C (the DIC image) both structures are visible, but in D (Hoechst stain fluorescence) only the nucleus can be seen to fluoresce, showing the rotatosome does not contain DNA. Bars represent 10 µm. Full size image

Molecular phylogeny

To determine how these Pseudotrichonympha species relate to other members of the genus, we sequenced the SSU rRNA gene from single isolated cells and small pools of cells. In all cases, sequences from the symbionts of a given host were 98.5–99% identical and one representative sequence per taxon was used for phylogenetic analyses and submitted to GenBank under accessions MF373810 –MF373812. In phylogenetic trees, the Pseudotrichonympha symbionts from Coptotermes hosts branch within a single clade, albeit with no support (Fig. 4). However, within this clade are two well-supported subgroups, one of these consists of three sequences from Southeast Asian specimens of Coptotermes (and includes one known species, P. grassii from C. formosanus), and the other consists of P. hertwigi from its type host, C. testaceus, and the two Coptotermes symbionts described here. The P. simplex symbiont is excluded from this clade, but its position is otherwise unresolved and the real relation to the underlying tree of currently sampled Pseudotrichonympha sequences unclear (Fig. 4).

Figure 4 Maximum likelihood (ML) phylogeny of SSU rRNA genes from Pseudotrichonympha, showing the relationships between Pseudotrichonympha leei, Pseudotrichonympha lifesoni, and Pseudotrichonympha pearti, to other members of the genus. The tree is rooted with the closest known relatives to Pseudotrichonympha, Teranympha and Eucomonypha. Numbers at nodes correspond to ML bootstrap support over 70% (values for nodes with lower support are not shown for clarity), and the scale bar represents a distance of 0.06 substitutions per site. Taxon names include GenBank accession numbers and the name of the termite host, where known. Most of the diversity on the phylogeny is represented by sequences from undescribed species, many from unidentified hosts, emphasizing our dearth of knowledge about the diversity of this lineage. Full size image

Taxonomic considerations

All three specimens of Pseudotrichonympha characterized here represent new species. In the case of C. gestroi, flagellates have been reported10 but none, including members of the genus Pseudotrichonympha, have been formally described. In the case of P. simplex, several spirotrichonymphids and the smaller flagellate, Cthulhu, have been described11, but although Pseudotrichonympha has been reported to be present, it was never formally described10,11,12.

In the case of the Australian Coptotermes cf. acinaciformis, we can conclude the symbiont is undescribed without knowing the host species with absolute certainty. Of the three Coptotermes known to exist in the region, one (C. frenchi) has no described Pseudotrichonympha, and the other two (C. lacteus and C. acinaciformis) are both documented as containing “Pseudotrichonympha hertwigi”10,13. However, this host barcode excludes both C. frenchi and C. lacteus, and moreover the confusing history of P. hertwigi is inconsistent with the presence of this species in any of these hosts (reviewed in detail in ref.7). Since its original description and subsequent re-classificiation to a new genus5,6,14, several authors have assumed that Pseudotrichonympha observed in other Coptotermes hosts correspond to the same species, even in geographically distant Australian and Asian termites10,12,13. But this was invariably based only on light microscopy and limited numbers of distinguishable characters. The identity of the host species is also convoluted. The Coptotermes host of P. hertwigi was named Coptotermes hartmanni 15, but it lacked formal description and is now invalid16. Subsequent surveys of neotropical Coptotermes have shown all Brazilian specimens correspond to Coptotermes testaceus: this host has now been barcoded and the P. hertwigi SSU rRNA from this type host has been characterized7. This species does not exist in Australia, and neither the Pseudotrichonympha from the Australian Coptotermes nor the host barcodes match their Brazilian counterparts, leading to the conclusion that neither Australian C. lacteus nor C. acinaciformis (nor any other Australian termite) contain P. hertwigi, but rather contain undescribed species that have been misidentified. The same is also likely true for Asian termites concluded by light microscopy alone to harbour P. hertwigi. Based on this and the phylogenetic and morphological data, we propose three new species of Pseudotrichonympha as detailed below.