The parabasalian symbionts of lower termite hindgut communities are well-known for their large size and structural complexity. The most complex forms evolved multiple times independently from smaller and simpler flagellates, but we know little of the diversity of these small flagellates or their phylogenetic relationships to more complex lineages. To understand the true diversity of Parabasalia and how their unique cellular complexity arose, more data from smaller and simpler flagellates are needed. Here, we describe two new genera of small-to-intermediate size and complexity, represented by the type species Cthulhu macrofasciculumque and Cthylla microfasciculumque from Prorhinotermes simplex and Reticulitermes virginicus, respectively (both hosts confirmed by DNA barcoding). Both genera have a single anterior nucleus embeded in a robust protruding axostyle, and an anterior bundle flagella (and likely a single posterior flagellum) that emerge slightly subanteriorly and have a distinctive beat pattern. Cthulhu is relatively large and has a distinctive bundle of over 20 flagella whereas Cthylla is smaller, has only 5 anterior flagella and closely resembles several other parababsalian genera. Molecular phylogenies based on small subunit ribosomal RNA (SSU rRNA) show both genera are related to previously unidentified environmental sequences from other termites (possibly from members of the Tricercomitidae), which all branch as sisters to the Hexamastigitae. Altogether, Cthulhu likely represents another independent origin of relatively high cellular complexity within parabasalia, and points to the need for molecular characterization of other key taxa, such as Tricercomitus.

Competing interests: The authors have the following interests: Specimens of P. simplex were collected as part of a Caribbean Basin survey of termite diversity funded by Terminix International. There are no patents, products in development or marketed products to declare. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors.

Funding: This work was supported by grants from the Natural Sciences and Engineering Research Council of Canada (227301) and a grant from the Tula Foundation to the Centre for Microbial Diversity and Evolution. Specimens of P. simplex were collected as part of a Caribbean Basin survey of termite diversity funded by Terminix International. PJK is a Fellow of the Canadian Institute for Advanced Research. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2013 James et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Here we describe Cthulhu macrofasciculumque found in Prorhinotermes simplex (Hagan), and Cthylla microfasciculumque, found in Reticulitermes virginicus (Banks), newly described genera and type species of relatively small parabasalian termite symbionts that address both these issues. These two similar but morphologically distinct genera are the first identified members of a clade of unidentified environmental sequences derived from several distantly related rhinotermitids and kalotermitids. Altogether, these new genera help provide a morphological framework to a previously uncharacterized lineage of environmental sequences, and Cthulhu in particular represents a newly identified morphological type for Parabasalia, as well as a new case of accelerating size and complexity.

In order to fully understand the evolution of the large, complex, and conspicuous parabasalians, it is necessary to pay greater attention to the smaller forms because the phylogeny of parabasalians makes it clear that large size and structural complexity have evolved more than once [2] , [9] – [13] . The basic cytoskeletal unit of parabasalian body plans is the karyomastigont, a suite of connected structures including the nucleus, basal bodies and flagella, and accessory cytoskeletal and endomembrane structures [2] , [14] . In some cases, cell size has increased but the karyomastigont complexity does not [11] , [15] . In other cases, cellular complexity has increased by massive duplication of the entire karyomastigont system resulting in huge multinucleate cells [13] , [16] – [18] . In still other cases only part of the karyomastigont system is massively duplicated, resulting in mononucleate cells with huge numbers of flagella and complex cytoskeletons [4] , [6] , [7] , [19] , [20] . Each of these types evolved independently, and each has probably evolved more than once from distinct and simpler ancestors. However with our incomplete knowledge about the simpler forms, together with an overabundance of clades comprised entirely of “unidentified environmental sequences”, it is difficult to reconstruct these events.

The large and complex hypermastigotes are most conspicuous and have therefore attracted the most attention in studies of the diversity and taxonomy of termite symbionts. Indeed, after over 100 years of study, it is arguable that most of the major types (i.e., genera) of large and complex hypermastigotes have probably been observed and described (although it is also increasingly clear that most species, even of the large parabasalian genera, remain to be described). The likelihood that many, if not most, of the major types of large hypermastigtes have been described is also supported by phylogenetic analysis of molecular surveys of hindgut diversity: there are many lineages in the tree of parabasalians that are made up entirely of “unidentified environmental sequences” from termite hindguts [9] , but only a few of these are likely to represent organisms of large size and substantial complexity. Thus, current data suggest that most of the unexplored diversity of parabasalians will correspond to relatively small and simple flagellates, which will make up the bulk of the molecular diversity of the group.

The hindgut of lower termites has long been known for the diversity of its community of symbiotic protists [1] . The community is dominated by Parabasalia, which are not only of interest due to the critical ecological role they play in the breakdown of lignocellulose, but also because these symbionts have evolved a remarkable range of sizes and structural complexities [2] . The largest and most complex so-called hypermastigotes can reach hundreds of microns in length (visible to the naked eye) and can be covered by as many as 50,000 flagella, underpinned by a highly organized suite of cytoskeletal elements [2] – [7] . At the other end of the extreme are many other species of small, simple flagellates, typically with three to six flagella, and most likely similar to the common ancestor of the parabasalians as a whole [2] , [8] .

Results and Discussion

Barcode identification of termite hosts The misidentification of host termites has often proved to be a complicating factor in the study of parabasalian symbionts. To avoid this issue, we first acquired a positive identification of all termite species by both examining their morphology and by characterizing mitochondrial 16S rRNA (mtLSU rRNA), an established DNA barcode marker for lower termites. Barcode sequences from field identified termites confirmed the identification of R. virginicus (Figure 1A), which shared 98.9% identity with a large number of R. virginicus barcodes (R. virginicus may be polyphyletic, but our isolate branches with the main group of R. virginicus barcodes). No comparable barcode sequence for P. simplex is available in public databases, so we also barcoded eight other isolates from across the Caribbean basin (including Central American records for P. simplex from Guatemala, Honduras, and Belize, all of which represent new ranges for this species). Comparing these and our P. simplex isolate confirmed its identification with a 99.7% shared identity. (Figure 1B). We also analyzed environmental sequences from three other termites, Calcaritermes nearcticus (Snyder), Cryptotermes cylindroceps (Scheffrahn & Krecek), and Heterotermes tenuis (Hagan), the identities of which were all confirmed by barcoding that was reported previously [21], [22]. PPT PowerPoint slide

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larger image TIFF original image Download: Figure 1. Barcode identification of termite hosts based on mitochondrial 16S (LSU) rRNA. At the top are all available barcodes from the genus Prorhinotermes and below are representative barcodes from all available species of Reticulitermes including represenative barcodes from R. virginicus (22 of 33 available sequences). Isolates used in this study are indicated by black boxes. Since no P. simplex barcodes were available, they were generated here from samples independently isolated from Florida, Puerto Rico, Bahamas, Belize, Guatemala, and Honduras. https://doi.org/10.1371/journal.pone.0058509.g001

Molecular phylogeny of cthulhumonads and hexamastigids To determine the phylogenetic position of Cthulhu and Cthylla, we characterized the SSU rRNA from both new species. For Cthulhu, SSU rRNA was sequenced from 5 individual manually isolated cells and from two pools of 5 cells and a single pool of 12cells, and from whole P. simplex gut contents. Altogether 10 individual clones were sequenced from manually isolated cells, and found to share 99% identity. Two sequences from whole hindgut material were also found to share 99% identity with those from isolated cells, altogether suggesting the cells matching the morphology of Cthu. macrofasciculumque were a single coherent species. From Cthylla, SSU rRNA was sequenced from 7 individual manually isolated cells and from whole R. virginicus gut contents. Four sequences from whole hindgut material were also found to share 98% identity with those from isolated cells, altogether suggesting the cells matching the morphology of Cthy. microfasciculumque were also a single coherent species. We also sequenced SSU rRNA from whole gut contents of three other termites, Cryptotermes cylindroceps, Calcaritermes nearcticus, and Heterotermes tenuis, which yielded environmental sequences that were closely related to Cthulhu and Cthylla. In SSU rRNA phylogenies including a broad representation of parabasalian diversity, Cthulhu and Cthylla sequences branch together with the genus Hexamastix and a group of formerly unidentified environmental sequences from other termite hindguts (Figure S1). A detailed analysis of this subgroup of parabasalia and closely related trichomonads used as an outgroup (Figure 5), shows that Cthulhu and Cthylla belong (with high/moderate support) to a group of environmental sequences from H. tenuis (JX975351: this study) and Reticulitermes chinensis (310871926), respectively. This suggests flagellates with similar morphology may be found in these termites. We have neither species available to investigate, but we did review video footage from the same isolate of H. tenuis that our environmental sequence was derived, and identified two small flagellates from which the Cthulhu-related sequences might plausibly be derived (Figure 4F–G). Unfortunately the footage was targeted at larger hypermastigotes and was therefore at too low a magnification to make out details, but one can see 10 µm flagellates with a bundle of anterior flagella and a protruding axostyle. PPT PowerPoint slide

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larger image TIFF original image Download: Figure 5. Phylogenetic relationships between Cthulhu, Cthylla, environmental sequences, and closely related parabasalians. Bayesian tree with posterior probabilities (upper) and maximum likelihood bootstraps (lower) indicated for each node, and major identified groups named to the right. Cthulhu and Cthylla are both related to unidentified environmental sequences from Heterotermes tenuis and Reticulitermes chinensis, respectively. They are all closely related to a large clade of unidentified environmental sequences from Cryptotermes, Glyptotermes, and Calcaritermes, which has previously been hypothesized to represent Tricercomitus. If true, then Cthulhu and Cthylla would be best considered members of the Tricercomitidae, thought this will depend on molecular characterization for this group. https://doi.org/10.1371/journal.pone.0058509.g005 Cthulhu, Cthylla, and their environmental relatives are in turn related to a cluster made up entirely of environmental sequences from a diverse group of kalotermitids (Cryptotermes, Glyptotermes, and Calcaritermes), and more distantly to the genus Hexamastix and several more unidentified sequences from termites, including some we identified from termite whole hindgut preparations: unidentified symbionts from Cryptotermes cylindroceps (JX975352) and Cryptotermes nearcticus (JX975353) (Figure 5). According to the recent classification of Čepička et al. (2010), this would place the cthulhumonads within the Honigbergiellida, which primarily includes the genera Hexamastix and Tricercomitus [9]. Interestingly, Čepička et al. singled out the clade of unidentified sequences that we now find are closely related to Cthulhu and Cthylla and hypothesize based on its distribution in termites and phylogenetic position that this lineage corresponds to the genus Tricercomitus. Both Cthulhu and Cthylla share certain features in common with the morphology of Tricercomitus and Hexamastix, which are about the same size as Cthylla, and also have robust, protruding axostyles ending in a point and lacking undulating membranes: [2]. Hexamasitx has five anterior flagella and a short recurrent one, like Cthylla, but Tricercomitus has only three anterior flagella and a very long recurrent one, unlike either Cthulhu or Cthylla. Taking morphology and phylogeny together, we speculate that both Hexamastix and Tricercomitus as currently described will emerge as polyphyletic genera in need of revision due to the difficulty in identifying and describing these small taxa without molecular data. If the unidentified clade related to Cthulhu and Cthylla does indeed turn out to correspond to the flagellates called Tricercomitus in the kalotermitids, then it will be important to characterise molecular data from Tricercomitus termopsidis (Kirby) in Zootermopsis angusticollis (Hagan) since this is the type host for the type species [23], and it is important to eventually test these hypotheses with additional genes, which are presently too sparsely sampled to allow such tests.