The Complete Protist Symbiont Communities of Coptotermes formosanus and Coptotermes gestroi: Morphological and Molecular Characterization of Five New Species.

Coptotermes formosanus Shiraki and Coptotermes gestroi (Wasmann) (Blattoidea: Rhinotermitidae) are invasive subterranean termite pest species with a major global economic impact. However, the descriptions of the mutualistic protist communities harbored in their respective hindguts remain fragmentary. The C. formosanus hindgut has long been considered to harbor three protist species, Pseudotrichonympha grassii (Trichonymphida), Holomastigotoides hartmanni, and Cononympha (Spirotrichonympha) leidyi (Spirotrichonymphida), but molecular data have suggested that the diversity may be higher. Meanwhile, the C. gestroi community remains undescribed except for Pseudotrichonympha leei. To complete the characterization of these communities, hindguts of workers from both termite species were investigated using single-cell PCR, microscopy, cell counts, and 18S rRNA amplicon sequencing. The two hosts were found to harbor intriguingly parallel protist communities, each consisting of one Pseudotrichonympha species, two Holomastigotoides species, and two Cononympha species. All protist species were unique to their respective hosts, which last shared a common ancestor ~18 MYA. The relative abundances of protist species in each hindgut differed remarkably between cell count data and 18S rRNA profiles, calling for caution in interpreting species abundances from amplicon data. This study will enable future research in C. formosanus and C. gestroi hybrids, which provide a unique opportunity to study protist community inheritance, compatibility, and potential contribution to hybrid vigor.

Molecular Phylogenetic Position of Hoplonympha natator (Trichonymphea, Parabasalia): Horizontal Symbiont Transfer or Differential Loss?

Hoplonympha natator is an obligate symbiont of Paraneotermes simplicicornis (Kalotermitidae), from southwestern North America. Another Hoplonympha species inhabits Hodotermopsis sjostedti (Archotermopsidae), from montane Southeast Asia. The large phylogenetic and geographical distance between the hosts makes the distribution of Hoplonympha puzzling. Here, we report the phylogenetic position of H. natator from P. simplicicornis through maximum likelihood and Bayesian analysis of 18S rRNA genes. The two Hoplonympha species form a clade with a deep node, making a recent symbiont transfer unlikely. The distribution of Hoplonympha may be due to an ancient transfer or strict vertical inheritance with differential loss from other hosts.

Molecular Identity of Holomastigotes (Spirotrichonymphea, Parabasalia) with Descriptions of Holomastigotes flavipes n. sp. and Holomastigotes tibialis n. sp.

Holomastigotes is a protist genus (Parabasalia: Spirotrichonymphea) that resides in the hindguts of "lower" termites. It can be distinguished from other parabasalids by spiral flagellar bands that run along the entire length of the cell, an anterior nucleus, a reduced or absent axostyle, the presence of spherical vesicles inside the cells, and the absence of ingested wood particles. Eight species have been described based on their morphology so far, although no molecular data were available prior to this study. We determined the 18S rRNA gene sequences of Holomastigotes from the hindguts of Hodotermopsis sjostedti, Reticulitermes flavipes, Reticulitermes lucifugus, and Reticulitermes tibialis. Phylogenetic analyses placed all sequences in an exclusive and well-supported clade with the type species, Holomastigotes elongatum from R. lucifugus. However, the phylogenetic position of Holomastigotes within the Spirotrichonymphea was not resolved. We describe two new species, Holomastigotes flavipes n. sp. and Holomastigotes tibialis n. sp., inhabiting the hindguts of R. flavipes and R. tibialis, respectively.

Diversity, Roles, and Biotechnological Applications of Symbiotic Microorganisms in the Gut of Termite.

Termites are global pests and can cause serious damage to buildings, crops, and plantation forests. The symbiotic intestinal flora plays an important role in the digestion of cellulose and nitrogen in the life of termites. Termites and their symbiotic microbes in the gut form a synergistic system. These organism work together to digest lignocellulose to make the termites grow on nitrogen deficient food. In this paper, the diversity of symbiotic microorganisms in the gut of termites, including protozoan, spirochetes, actinomycetes, fungus and bacteria, and their role in the digestion of lignocellulose and also the biotechnological applications of these symbiotic microorganisms are discussed. The high efficiency lignocellulose degradation systems of symbiotic microbes in termite gut not only provided a new way of biological energy development, but also has immense prospect in the application of cellulase enzymes. In addition, the study on the symbiotic microorganisms in the gut of termites will also provide a new method for the biological control of termites by the endophytic bacteria in the gut of termites.

Exclusive Gut Flagellates of Serritermitidae Suggest a Major Transfaunation Event in Lower Termites: Description of Heliconympha glossotermitis gen. nov. spec. nov.

The guts of lower termites are inhabited by host-specific consortia of cellulose-digesting flagellate protists. In this first investigation of the symbionts of the family Serritermitidae, we found that Glossotermes oculatus and Serritermes serrifer each harbor similar parabasalid morphotypes: large Pseudotrichonympha-like cells, medium-sized Leptospironympha-like cells with spiraled bands of flagella, and small Hexamastix-like cells; oxymonadid flagellates were absent. Despite their morphological resemblance to Pseudotrichonympha and Leptospironympha, a SSU rRNA-based phylogenetic analysis identified the two larger, trichonymphid flagellates as deep-branching sister groups of Teranymphidae, with Leptospironympha sp. (the only spirotrichosomid with sequence data) in a moderately supported basal position. Only the Hexamastix-like flagellates are closely related to trichomonadid flagellates from Rhinotermitidae. The presence of two deep-branching lineages of trichonymphid flagellates in Serritermitidae and the absence of all taxa characteristic of the ancestral rhinotermitids underscores that the flagellate assemblages in the hindguts of lower termites were shaped not only by a progressive loss of flagellates during vertical inheritance but also by occasional transfaunation events, where flagellates were transferred horizontally between members of different termite families. In addition to the molecular phylogenetic analyses, we present a detailed morphological characterization of the new spirotrichosomid genus Heliconympha using light and electron microscopy.

New Species of Spirotrichonympha from Reticulitermes and the Relationships Among Genera in Spirotrichonymphea (Parabasalia).

Spirotrichonymphea is a class of hypermastigote parabasalids defined by their spiral rows of many flagella. They are obligate hindgut symbionts of lower termites. Despite more than 100 yr of morphological and ultrastructural study, the group remains poorly characterised by molecular data and the phylogenetic positions and taxonomic validity of most genera remain in question. The genus Spirotrichonympha has been reported to inhabit several termite genera, including Reticulitermes, Coptotermes, and Hodotermopsis. The type species for this genus, Spirotrichonympha flagellata, was described from Reticulitermes lucifugus but no molecular data are yet available for this species. In this study, three new Spirotrichonympha species are described from three species of Reticulitermes. Their molecular phylogenetic position indicates that the genus is not monophyletic, as Spirotrichonympha species from Coptotermes, Paraneotermes, and Hodotermopsis branch separately. In contrast, the genus Holomastigotoides is monophyletic, as demonstrated using new sequences from Holomastigotoides species. The presence of Holomastigotoides in Prorhinotermes and the distinct phylogenetic positions of Spirotrichonympha from Reticulitermes and Coptotermes are consistent with a previously proposed symbiont fauna replacement in the ancestor of Reticulitermes.

The role of host phylogeny varies in shaping microbial diversity in the hindguts of lower termites.

The hindguts of lower termites and Cryptocercus cockroaches are home to a distinct community of archaea, bacteria, and protists (primarily parabasalids and some oxymonads). Within a host species, the composition of these hindgut communities appears relatively stable, but the evolutionary and ecological factors structuring community composition and stability are poorly understood, as are differential impacts of these factors on protists, bacteria, and archaea. We analyzed the microbial composition of parabasalids and bacteria in the hindguts of Cryptocercus punctulatus and 23 species spanning 4 families of lower termites by pyrosequencing variable regions of the small-subunit rRNA gene. Especially for the parabasalids, these data revealed undiscovered taxa and provided a phylogenetic basis for a more accurate understanding of diversity, diversification, and community composition. The composition of the parabasalid communities was found to be strongly structured by the phylogeny of their hosts, indicating the importance of historical effects, although exceptions were also identified. Particularly, spirotrichonymphids and trichonymphids likely were transferred between host lineages. In contrast, host phylogeny was not sufficient to explain the majority of bacterial community composition, but the compositions of the Bacteroidetes, Elusimicrobia, Tenericutes, Spirochaetes, and Synergistes were structured by host phylogeny perhaps due to their symbiotic associations with protists. All together, historical effects probably resulting from vertical inheritance have had a prominent role in structuring the hindgut communities, especially of the parabasalids, but dispersal and environmental acquisition have played a larger role in community composition than previously expected.

The phylogenetic position of Kofoidia loriculata (Parabasalia) and its implications for the evolution of the Cristamonadea.

Kofoidia loriculata is a parabasalid symbiont inhabiting the hindgut of the lower termite Paraneotermes simplicicornis. It was initially described as a lophomonad due to its apical tuft of multiple flagella that disintegrate during cell division, but its phylogenetic relationships have not been investigated using molecular evidence. From single cell isolations, we sequenced the small subunit rRNA gene and determined that K. loriculata falls within the Cristamonadea, but is unrelated to other lophomonads. This analysis further demonstrates the polyphyly of the lophomonads and the necessity to re-assess the morphological and cellular evolution of the Cristamonadea.

Molecular Evidence for the Polyphyly of Macrotrichomonas (Parabasalia: Cristamonadea) and a Proposal for Macrotrichomonoides n. gen.

Macrotrichomonas (Cristamonadea: Parabasalia) is an anaerobic, amitochondriate flagellate symbiont of termite hindguts. It is noteworthy for being large but not structurally complex compared with other large parabasalians, and for retaining a structure similar in appearance to the undulating membrane (UM) of small flagellates closely related to cristamonads, e.g. Tritrichomonas. Here, we have characterised the SSU rDNA from two species described as Macrotrichomonas: M. restis Kirby 1942 from Neotermes jouteli and M. lighti Connell 1932 from Paraneotermes simplicicornis. These species do not form a clade: M. lighti branches with previously characterised Macrotrichomonas sequences from Glyptotermes, while M. restis branches with the genus Metadevescovina. We examined the M. restis UM by light microscopy, scanning electron microscopy, and transmission electron microscopy, and we find common characteristics with the proximal portion of the robust recurrent flagellum of devescovinids. Altogether, we show the genus Macrotrichomonas to be polyphyletic and propose transferring M. restis to a new genus, Macrotrichomonoides. We also hypothesise that the macrotrichomonad body plan represents the ancestral state of cristamonads, from which other major forms evolved.

Identification criteria of the rare multi-flagellate Lophomonas blattarum: comparison of different staining techniques.

Bronchopulmonary lophomoniasis (BPL) is an emerging disease of potential importance. BPL is presented by non-specific clinical picture and is usually accompanied by immunosuppression. Culture of Lophomonas blattarum is difficult and its molecular diagnosis has not yet been developed. Therefore, microscopic examination of respiratory samples, e.g., bronchoalveolar lavage (BAL) or sputum, is the mainstay of BPL diagnosis. Creola bodies and ciliocytophthoria are two forms of bronchial cells which occur in chest diseases with non-specific clinical picture like that of BPL. Both forms could be misrecognized as multi-flagellates because of their motile cilia in the wet mounts and due to shape variability of L. blattarum in stained smears. The aim of the study is to compare different staining techniques for visualizing L. blattarum to improve the recognition and diagnosis of BPL, to distinguish respiratory epithelial cells from L. blattarum and to decide which stain is recommended in suspected cases of BPL. BAL samples from patients which contain L. blattarum, creola bodies, and ciliocytophthoria were collected then wet mounts were examined. The BAL samples were also stained by Papanicolaou (PAP), Giemsa, hematoxylin and eosin (H & E), trichrome, Gram, and Diff-Quik (DQ) stains. The different staining techniques were compared regarding the stain quality. In wet mounts, the ciliary movement was coordinate and synchronous while the flagellar movement was wavy and leaded to active swimming of L. blattarum. In stained slides, bronchial cells were characterized by the presence of basal nucleus and the terminal bar from which the cilia arise. Trichrome was the best stain in demonstration of cellular details of L. blattarum. H & E, PAP, and Giemsa stains showed good quality of stains. Gram and DQ stains showed only pale hues of L. blattarum. We recommended adding Wheatley's trichrome staining to the differential diagnosis workup of cases of non-specific chest infections, especially when BPL is suspected, to avoid overdiagnosis or underdiagnosis of it.

Molecular characterization of parabasalian symbionts Coronympha clevelandii and Trichonympha subquasilla from the Hawaiian lowland tree termite Incisitermes immigrans.

An important and undervalued challenge in characterizing symbiotic protists is the accurate identification of their host species. Here, we use DNA barcoding to resolve one confusing case involving parabasalian symbionts in the hindgut of the Hawaiian lowland tree termite, Incisitermes immigrans, which is host to several parabasalians, including the type species for the genus Coronympha, C. clevelandii. We collected I. immigrans from its type locality (Hawaii), confirmed its identity by DNA barcoding, and characterized the phylogenetic position of two symbionts, C. clevelandii and Trichonympha subquasilla. These data show that previous molecular surveys of "I. immigrans" are, in fact, mainly derived from the Caribbean termite I. schwarzi, and perhaps also another related species. These results emphasize the need for host barcoding, clarify the relationship between morphologically distinct Coronympha species, and also suggest some interesting distribution patterns of nonendemic termite species and their symbionts.

Morphology and molecular phylogeny of Staurojoenina mulleri sp. nov. (Trichonymphida, Parabasalia) from the hindgut of the kalotermitid Neotermes jouteli.

Staurojoenina is a large and structurally complex genus of hypermastigont parabasalians found in the hindgut of lower termites. Although several species of Staurojoenina have been described worldwide, all Staurojoenina observed to date in different species of North American termites have been treated as the same species, S. assimilis. Here, we characterize Staurojoenina from the North American termite Neotermes jouteli using light microscopy, scanning electron microscopy, and phylogenetic analysis of small subunit ribosomal RNA, and compare it with S. assimilis from its type host, Incisitermes minor. The basic morphological characteristics of the N. jouteli symbiont, including its abundant bacterial epibionts, are similar as far as they may be compared with existing data from S. assimilis, although not consistently identical. In contrast, we find that they are extremely distantly related at the molecular level, sharing a pairwise similarity of SSU rRNA genes comparable to that seen between different genera or even families of other parabasalians. Based on their evolutionary distance and habitat in different termite genera, we consider the N. jouteli Staurojoenina to be distinct from S. assimilis, and describe a new species, Staurojoenina mulleri, in honor of the pioneering parabasalian researcher, Miklos Muller.

Trichomonosis in free-ranging Eurasian collared doves (Streptopelia decaocto) and African collared dove hybrids (Streptopelia risoria) in the Caribbean and description of ITS-1 region genotypes.

We report the first documented occurrence of an outbreak of trichomonosis in a free-ranging small flock of Eurasian collared doves (Streptopelia decaocto) and African collared dove hybrids (Streptopelia risoria) in the Caribbean. In total, 18 birds were examined, including six African collared dove x Eurasian collared dove hybrids and 12 Eurasian collared doves. The affected age class consisted of adults. Sex distribution was equal. With a flock population size of 200 birds, mortality rate for the outbreak was estimated at 15-20%. Living birds were weak, showing evidence of mucus-stained beaks and open-mouth breathing. Caseous ulcerative yellow lesions were restricted to the upper gastrointestinal tract, with the exception of one bird, which had lesions in the upper gastrointestinal tract and in the liver. Ninety-four percent (17/18) of the affected birds had multiple extensive lesions. Lesions located on the roof of the oral cavity extended in 33% (6/18) into the orbit and in 11% (2/18) into the braincase. Using wet-mount microscopy, we were able to confirm Trichomonas gallinae in 22% (4/18) of the sampled animals. Fifteen samples submitted for PCR analysis tested positive. Sequence analysis of the internal transcribed spacer 1 (ITS-1) region of the ribosomal RNA (rRNA) revealed two distinct genotypes of Trichomonas. One sequence had 100% identity to the prototype T. gallinae isolate, whereas the other sequences had 98-100% identity to recently described Trichomonas-like parabasalid. On the basis of gross and histologic findings, along with the sequence results from the columbids in this report, it is likely that this Trichomonas-like parabasalid is pathogenic.

Molecular and morphological analysis of the family Calonymphidae with a description of Calonympha chia sp. nov., Snyderella kirbyi sp. nov., Snyderella swezyae sp. nov. and Snyderella yamini sp. nov.

Calonymphids are a group of multinucleate, multiflagellate protists belonging to the order Cristamonadida (Parabasalia) that are found exclusively in the hindgut of termites from the family Kalotermitidae. Despite their impressive morphological complexity and diversity, few species have been formally described and fewer still have been characterized at the molecular level. In this study, four novel species of calonymphids were isolated and characterized: Calonympha chia and Snyderella yamini spp. nov., from Neotermes castaneus and Calcaritermes nearcticus from Florida, USA, and Snyderella kirbyi and Snyderella swezyae, spp. nov., from Calcaritermes nigriceps and Cryptotermes cylindroceps from Colombia. Each of these species was distinguished from its congeners by residing in a distinct host and by differences at the molecular level. Phylogenetic analyses of small subunit (SSU) rDNA indicated that the genera Calonympha and Stephanonympha were probably not monophyletic, though the genus Snyderella, previously only represented by one sequence in molecular analyses, appeared with these new data to be monophyletic. This was in keeping with the traditional evolutionary view of the group in which the morphology of the genus Snyderella is considered to be derived, while that of the genus Stephanonympha is ancestral and therefore probably plesiomorphic.

Symbiosis, morphology, and phylogeny of Hoplonymphidae (Parabasalia) of the wood-feeding roach Cryptocercus punctulatus.

Anaerobic cellulolytic flagellate protists of the hindguts of lower termites and the wood-feeding cockroach Cryptocercus are essential to their host's ability to digest lignocellulose. Many have bacteria associated with their surfaces and within cytoplasmic vesicles-likely important symbioses as suggested by molecular and other data. Some of the most striking examples of these symbioses are in the parabasalid family Hoplonymphidae, but little or no data exist on the structural aspects of their symbioses, their relationships with bacteria through different life-cycle stages, or their diversity and phylogenetic relationships in Cryptocercus. We investigated these areas in the hoplonymphid genera Barbulanympha and Urinympha from Cryptocercus punctulatus using light and electron microscopy, and analysis of small subunit rRNA. Microscopy reveals variation in density of bacterial surface symbionts related to life-cycle stage, a glyococalyx possibly important in bacterial adhesion and/or metabolite exchange, and putative viruses associated with bacterial surface symbionts. Patterning of surface bacteria suggests protists emerging from the resistant (dormant) stage are colonized by a small population of bacterial cells, which then divide to cover their surface. Additionally, cytoplasmic protrusions from the protist are covered by bacteria. Phylogenetic analysis rejects the monophyly of Hoplonymphidae, suggesting multiple origins or losses of these bacterial symbioses.

Morphology and molecular phylogeny of Pseudotrichonympha hertwigi and Pseudotrichonympha paulistana (Trichonymphea, parabasalia) from neotropical rhinotermitids.

Pseudotrichonympha is a large hypermastigote parabasalian found in the hindgut of several species of rhinotermitid termites. The genus was discovered more than 100 years ago, and although over a dozen species have since been described, this represents only a small fraction of its likely diversity: the termite genera from which Pseudotrichonympha is known are all species rich, and in most cases their hindgut symbionts have not been examined. Even formally described species are mostly lacking in detailed microscopic data and/or sequence data. Using small subunit ribosomal RNA gene sequences and light and scanning electron microscopy we describe here the morphology and molecular phylogenetic position of two Pseudotrichonympha species: the type species for the genus, Pseudotrichonympha hertwigi from Coptotermes testaceus (described previously in line drawing only), and Pseudotrichonympha paulistana from Heterotermes tenuis (described previously based on light microscopy only).

Characterization of new cristamonad species from kalotermitid termites including a novel genus, Runanympha.

Cristamonadea is a large class of parabasalian protists that reside in the hindguts of wood-feeding insects, where they play an essential role in the digestion of lignocellulose. This group of symbionts boasts an impressive array of complex morphological characteristics, many of which have evolved multiple times independently. However, their diversity is understudied and molecular data remain scarce. Here we describe seven new species of cristamonad symbionts from Comatermes, Calcaritermes, and Rugitermes termites from Peru and Ecuador. To classify these new species, we examined cells by light and scanning electron microscopy, sequenced the symbiont small subunit ribosomal RNA (rRNA) genes, and carried out barcoding of the mitochondrial large subunit rRNA gene of the hosts to confirm host identification. Based on these data, five of the symbionts characterized here represent new species within described genera: Devescovina sapara n. sp., Devescovina aymara n. sp., Macrotrichomonas ashaninka n. sp., Macrotrichomonas secoya n. sp., and Macrotrichomonas yanesha n. sp. Additionally, two symbionts with overall morphological characteristics similar to the poorly-studied and probably polyphyletic 'joeniid' Parabasalia are classified in a new genus Runanympha n. gen.: Runanympha illapa n. sp., and Runanympha pacha n. sp.

Strict cospeciation of devescovinid flagellates and Bacteroidales ectosymbionts in the gut of dry-wood termites (Kalotermitidae).

The surface of many termite gut flagellates is colonized with a dense layer of bacteria, yet little is known about the evolutionary relationships of such ectosymbionts and their hosts. Here we investigated the molecular phylogenies of devescovinid flagellates (Devescovina spp.) and their symbionts from a wide range of dry-wood termites (Kalotermitidae). From species-pure flagellate suspensions isolated with micropipettes, we obtained SSU rRNA gene sequences of symbionts and host. Phylogenetic analysis showed that the Devescovina spp. present in many species of Kalotermitidae form a monophyletic group, which includes also the unique devescovinid flagellate Caduceia versatilis. All members of this group were consistently associated with a distinct lineage of Bacteroidales, whose location on the cell surface was confirmed by fluorescence in situ hybridization. The well-supported congruence of the phylogenies of devescovinids and their ectosymbionts documents a strict cospeciation. In contrast, the endosymbionts of the same flagellates ('Endomicrobia') were clearly polyphyletic and must have been acquired independently by horizontal transfer from other flagellate lineages. Also the Bacteroidales ectosymbionts of Oxymonas flagellates present in several Kalotermitidae belonged to several distantly related lines of descent, underscoring the general perception that the evolutionary history of flagellate-bacteria symbioses in the termite gut is complex.

Identification and localization of the multiple bacterial symbionts of the termite gut flagellate Joenia annectens.

The hindgut of wood-feeding lower termites is densely colonized by a multitude of symbiotic micro-organisms. While it is well established that the eukaryotic flagellates play a major role in the degradation of lignocellulose, much less is known about the identity and function of the prokaryotic symbionts associated with the flagellates. Our ultrastructural investigations of the gut flagellate Joenia annectens (from the termite Kalotermes flavicollis) revealed a dense colonization of this flagellate by diverse ecto- and endosymbiotic bacteria. Phylogenetic analysis of the small-subunit rRNA gene sequences combined with fluorescence in situ hybridization allowed us to identify and localize the different morphotypes. Furthermore, we could show that K. flavicollis harbours two phylotypes of J. annectens that could be distinguished not only by their small-subunit rRNA gene sequences, but also by differences in their assemblages of bacterial symbionts. Each of the flagellate populations hosted phylogenetically distinct ectosymbionts from the phylum Bacteroidetes, one of them closely related to the ectosymbionts of other termite gut flagellates. A single phylotype of 'Endomicrobia' was consistently associated with only one of the host phylotypes, although not all individuals were colonized, corroborating that 'Endomicrobia' symbionts do not always cospeciate with their host lineages. Flagellates from both populations were loosely associated with a single phylotype of Spirochaetales attached to their cell surface in varying abundance. Current evidence for the involvement of Bacteroidales and 'Endomicrobia' symbionts in the nitrogen metabolism of the host flagellate is discussed.

Cryptic diversity of free-living parabasalids, Pseudotrichomonas keilini and Lacusteria cypriaca n. g., n. sp., as inferred from small subunit rDNA sequences.

Ultrastructural and molecular phylogenetic evidence indicate that the Parabasalia consists of seven main subgroups: the Trichomonadida, Honigbergiellida, Hypotrichomonadida, Tritrichomonadida, Cristamonadida, Spirotrichonymphida, and Trichonymphida. Only five species of free-living parabasalids are known: Monotrichomonas carabina, Ditrichomonas honigbergii, Honigbergiella sp., Tetratrichomonas undula, and Pseudotrichomonas keilini. Phylogenetic analyses show that free-living species do not form a clade and instead branch in several different positions within the context of their parasitic relatives. Because the diversity of free-living parabasalids is poorly understood, the systematics of these lineages is in a significant state of disarray. In order to better understand the phylogenetic distribution of free-living parabasalids, we sequenced the small subunit rDNA from three different strains reminiscent of P. keilini; the strains were isolated from different geographical locations: (1) mangrove sediments in Japan and (2) sediments in Cyprus. These data demonstrated that the free-living parabasalids P. keilini and Lacusteria cypriaca n. g., n. sp., form a paraphyletic assemblage near the origin of a clade consisting mostly of parasitic trichomonadids (e.g. Trichomonas vaginalis). This paraphyletic distribution of similar morphotypes indicates that free-living trichomonadids represent a compelling example of morphostasis that provides insight into the suite of features present in the most recent free-living ancestor of their parasitic relatives.