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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.
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Anaerobic flagellate protozoa play a critical role in lignocellulose digestion in the hindgut of phylogenetically lower termites (Cleveland, 1923; Hungate, 1955; Breznak and Brune, 1994). The flagellates are associated with a diverse community of ectosymbiotic and endosymbiotic bacteria, which represent several phyla and are typically specific for their respective hosts (for reviews, see Brune and Stingl, 2005; Brune, 2006; Ohkuma, 2008). Neither the flagellates nor the bacterial symbionts are in permanent culture, but phylogenetic analysis of their SSU rRNA genes provided first hints that at least some of these symbiotic associations might have evolved by cospeciation (for review, see Ohkuma, 2008).
Cospeciation, resulting from intimate and long-standing association of host and symbiont, exists if molecular phylogenies of hosts and symbionts are significantly more similar than would be expected due to chance alone, and has been observed in various types of symbioses (e.g. Baumann et al., 1997; Peek et al., 1998; Hosokawa et al., 2006; Hughes et al., 2007). To date, two examples of cospeciation between termite gut flagellates and their bacterial endosymbionts have been described, involving Pseudotrichonympha spp. and Bacteroidales (Noda et al., 2007), and Trichonympha spp. and ‘Endomicrobia’ (Ikeda-Ohtsubo and Brune, 2009).
Endosymbionts located within the cytoplasm of a flagellate are easily transferred to the daughter cells when the host divides (Ikeda-Ohtsubo and Brune, 2009). Such a vertical transmission is less likely in the case of ectosymbionts. If the cells are not firmly attached to the host's surface, they may dissociate, leading to a permanent loss, and reattach to another host, resulting in a horizontal transfer of symbionts between host lineages, as was shown for ectosymbiotic Spirochaetes (Noda et al., 2003). Although such mechanisms would reduce the chances of cospeciation in ectosymbiosis, the close phylogenetic relatedness of Bacteroidales ectosymbionts of three devescovinid flagellates (Devescovina spp. and Caduceia versatilis) suggests that also ectosymbionts of termite gut flagellates can be vertically transmitted (Hongoh et al., 2007).
Devescovinid flagellates are present mainly in the gut of dry-wood termites; they have been classified on the basis of their morphological characteristics and the presence or absence of ectosymbiotic bacteria (Kirby, 1941; 1942; 1945). The highest number of species has been assigned to the genus Devescovina (Kirby, 1941), yet knowledge about their phylogeny is scarce (Gerbod et al., 2002). Each of the 20 Devescovina species carry ectosymbionts (Kirby, 1941; Branke, 1996; Radek et al., 1996; Noda et al., 2006a); these laterally attached filamentous rods may cover the entire surface of the host cell (Fig. 1A and B). The consistent presence of such rods on the surface of all Devescovina species offers an excellent opportunity for studying cospeciation of ectosymbionts with their flagellate host.
Figure 1. Scanning electron micrographs of Devescovina sp. (A) and Oxymonas sp. (C) and their ectosymbiotic bacteria (B, D) in Neotermes castaneus (batch BAM-2), exemplifying the two major groups of flagellates investigated in this study. A. Devescovina sp. has a spindle-shaped body, three anterior flagella (fla), and a thicker, ribbon-shaped, recurrent flagellum (flr) typical of this genus (Kirby, 1941). B. As in other Devescovina spp. (Branke, 1996; Radek et al., 1996; Noda et al., 2006a), the entire flagellate is covered by a layer of uniform, filamentous bacteria; the arrows indicate the two ends of a single cell. C. Oxymonas sp. has a rostellum (ro), typical of this genus (Cleveland, 1950; Brugerolle and König, 1997); as in another Oxymonas sp. (Noda et al., 2006b), the flagellate is colonized by two morphotypes of ectosymbiotic bacteria (D): long, slender rods with tapered ends (white arrows), and short, thick rods with rounded ends (black arrows), sometimes in division (arrowhead). Scale bars: 20 μm (A, C), 3 μm (B, D).
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In the present study, we investigated the molecular phylogenies of Devescovina spp. and their bacterial symbionts. Species-pure suspensions of flagellates from numerous species of dry-wood termites (Table 1) were isolated with micropipettes. The phylogenies of host flagellates and bacterial symbionts were reconstructed and compared based on their SSU rRNA gene sequences. Fluorescence in situ hybridization was carried out to determine the exact location of Bacteroidales. Since Bacteroidales ectosymbionts also occur on Oxymonas sp. in Neotermes koshunensis (Noda et al., 2006b), the ectosymbionts of the Oxymonas spp. (exemplified by Fig. 1C and D) present in the termites used in this study were included in the phylogenetic analysis. Finally, we compared the phylogenies of the devescovinid flagellates with that of their host termites using the cytochrome oxidase II (COII) gene of the termite as a phylogenetic marker.
Table 1. Phylotypes of flagellates and their bacterial symbionts in the clone libraries obtained from capillary-picked suspensions of devescovinid and oxymonadid flagellates from different termites.
|Termitesa||Flagellate suspensions||Phylotypes in clone libraries (abundance)b|
|Neotermes castaneus Cuba FN377805||Devescovina lepida||NcDvL11 (4/8) FN377759c||NcDvLB13 (10/24) FN377751||NcDv-1 (3/24) AB298058d|
|NcDvL20 (4/8) FN377760||NcDvLB23 (9/24) FN377752|
|Devescovina arta||NcDvA25 (10/10) FN377761||NcDvAB05 (9/10) FN377753||NcDvAE10 (1/10) FN377767|
|Oxymonas sp.||NcOxA (3/3) AB326383d||NcOxAB12 (8/26) FN377773||NcOx-1 (2/26) AB298081d|
|NcOxAB26 (6/26) FN377774|
|Cryptotermes longicollis Mexico FN377806||Devescovina sp.e||ClDv14 (4/4) FN377762||ClDvB13 (12/18) FN377754||ClDvE07 (4/18) FN377768|
|Cryptotermes secundus Australia DQ278261d||Devescovina sp.||CsDv11 (8/8) FN377763||CsDvB04 (9/9) FN377755||CsDvE04 (3/3)f FN377769|
|Oxymonas sp.||Not determinedg||CsOxB27 (10/27) FN377775||–h|
|CsOxB40 (4/27) FN377776|
|Cryptotermes brevisi Brazil FN377807||Devescovina sp.||CbDv02 (7/7) FN377764||CbDvB11 (22/30) FN377756||CbDvE07 (1/30) FN377770|
|Cryptotermes dudleyii Kenya FN377808||Devescovina sp.||CdDv09 (4/4) FN377765||CdDvB38 (7/10) FN377757||CdDvE17 (2/10) FN377771|
|Cryptotermes havilandi Ghana FN377809||Devescovina sp.||ChDv22 (5/5) FN377766||ChDvB35 (16/20) FN377758||ChDvE11 (2/20) FN377772|
|Cryptotermes cavifronsj USA FN377810||Caduceia versatilis||CcCv DQ855405||CcCv-03 AB299517||–k|
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This is the first report of cospeciation of an ectosymbiotic bacterium with its protistan host. The molecular phylogenies of ‘Candidatus Armantifilum devescovinae’ (Bacteroidales) and devescovinid flagellates (i.e. Devescovina spp. and C. versatilis) perfectly mirror each other. While these ectosymbionts are highly specific to the host flagellates, there are strong indications that the endosymbionts (‘Endomicrobia’) of Devescovina spp. were acquired by multiple horizontal transfers from other gut flagellates. The strict cospeciation of devescovinids and ectosymbionts is maintained despite the horizontal transfer of flagellates among several species of dry-wood termites (Kalotermitidae), which underlines that cospeciation is not merely caused by a spatial separation of flagellates in their termite hosts, but is the result of an obligate symbiosis, leading to strict vertical transmission of ‘A. devescovinae’ by their flagellate hosts.
The results obtained by SSU rRNA gene analysis of capillary-picked Devescovina flagellate suspensions, followed by in situ localization of the sequences in hindgut contents, convincingly show that the filamentous ectosymbionts of all investigated Devescovina spp. form a monophyletic group among the Bacteroidales. Strict cospeciation of ‘A. devescovinae’ with their devescovinid hosts is documented by the fully supported congruence of their phylogenetic trees, and further corroborated by the highly significant results of cospeciation test and coalescence analysis (Hughes et al., 2007). Regression analysis of coalescence times suggests that the SSU rRNA gene of the symbionts evolved two times faster than that of the hosts (Fig. 6); an intercept close to zero indicates almost synchronous cospeciation (Hughes et al., 2007).
The Bacteroidales ectosymbionts previously obtained from two other Devescovina spp. (Cd3, Nk2; Noda et al., 2006a) also fall into the radiation of ‘A. devescovinae’, and the phylogenetic position of their hypothetical host flagellates (Cd3–CdD3-1 and Nk2–NkD2-1) indicates that they are cospeciating just like the symbiotic pairs included in this study. Evidently, ‘A. devescovinae’ represents the filamentous rods described already by Kirby (1941) as characteristic of the genus Devescovina. This is further supported by the fact that also the filamentous ectosymbiont of C. versatilis (Tamm, 1982; Hongoh et al., 2007), a flagellate that clusters within the Devescovina group (Noël et al., 2007; Fig. 2), is affiliated with the ‘A. devescovinae’ cluster. Since filamentous rods are absent on other devescovinid flagellates (Kirby, 1941; 1942; 1945; Strassert et al., 2009), it is safe to assume that the acquisition of the ancestor of ‘A. devescovinae’, a representative of the Bacteroidales Cluster V specific for termite guts, by the common ancestor of Devescovina species (and C. versatilis) gave rise to the extant symbiotic pairs by cospeciation.
The fact that C. versatilis and its filamentous ectosymbiont are one of these pairs corroborates the finding that the devescovinid flagellate C. versatilis (‘Rubberneckia’) clusters with species of the Devescovina group (Noël et al., 2007; Fig. 2). This raises the question whether C. versatilis belongs to the genus Caduceia (Kirby, 1942) or is a species of Devescovina. As morphological features that allow a separation of Devescovina species and Caduceia species (Duboscq and Grassé, 1927; Kirby, 1941; 1942) are weak, Kirby used the presence of filamentous ectosymbiotic rods as an important characteristic separating species in the genus Devescovina from those in all other devescovinid genera (including Caduceia). Notably, the devescovinid flagellate ‘Rubberneckia’ discovered by Tamm and Tamm (1974) was later described as a species of Caduceia (versatilis) by D'Ambrosio and colleagues (1999) even though it possesses filamentous rods similar to those present on Devescovina species (Tamm, 1982). The phylogenetic position of other Caduceia species must be studied in order to resolve this issue.
The flagellate communities in the gut of dry-wood termites are remarkably similar at the generic level (Kitade, 2004). Nevertheless, a single species of dry-wood termites may simultaneously harbour devescovinid flagellates of several genera, and similar morphotypes occur in different termites (Kirby, 1941; 1942, 1945; Yamin, 1979). This can be explained by a horizontal transfer of devescovinids among the termite species – a hypothesis that is supported also by the complete incongruence between the phylogenies of devescovinid flagellates and their termite hosts (Fig. 7). The fact that ‘A. devescovinae’ strictly cospeciated with its host flagellate, even in those cases where the presence of several devescovinid species in the same termite gut should have provided ample opportunities for an exchange of ectosymbionts, underscores the high specificity of this obligate symbiosis.
To date, only two instances of cospeciation in flagellate–bacterium symbioses have been documented; both occur in the termite gut. One is the symbiosis of Pseudotrichonympha flagellates with ‘Azobacteroides pseudotrichonymphae’ (Noda et al., 2007), the other is the symbiosis of Trichonympha flagellates with ‘Endomicrobium trichonymphae’ (Ikeda-Ohtsubo and Brune, 2009). Since Pseudotrichonympha spp. are cospeciating also with their host termites (Rhinotermitidae; Noda et al., 2007), it cannot be excluded that cospeciation with their bacterial symbionts is merely due to a spatial separation of the flagellate hosts within their respective termite species. However, in the case of Trichonympha spp., which show a horizontal transfer of flagellates among termite species of different families (Ikeda-Ohtsubo and Brune, 2009), strict vertical transmission of the symbionts within their host lineage is a prerequisite for explaining cospeciation of the symbiotic pairs. This is exactly the situation we observed here with Devescovina flagellates and ‘A. devescovinae’ in dry-wood termites.
Cospeciation of bacterial endosymbionts and their hosts is a widespread phenomenon (Clark et al., 2000; Sauer et al., 2000; Thao et al., 2000). However, the only ectosymbiotic bacterium reported to cospeciate with its host is ‘Candidatus Ishikawaella capsulata’ (Gammaproteobacteria), which attaches to the intestinal epithelia of stinkbugs (Hosokawa et al., 2006). Specific attachment sites for ectosymbionts, as reported for the flagellates Mixotricha paradoxa (Cleveland and Grimstone, 1964), Joenia annectens (Radek et al., 1996) and Staurojoenina sp. (Stingl et al., 2004), are absent in Devescovina species (Radek et al., 1996).
The driving force behind such intimate symbioses could be metabolic interactions (for a review, see Brune and Stingl, 2005). The most reasonable assumption is that the bacterial ectosymbionts serve as a source of essential amino acids or cofactors for their flagellate hosts. In Devescovina spp., phagocytosis of ectosymbionts has been evidenced by transmission electron microscopy (Radek et al., 1996; Noda et al., 2006a), indicating that the host flagellate uses ‘A. devescovinae’ as nutrient source. A more specific role in nitrogen metabolism would be in amino acid upgrading, as suggested for ‘E. trichonymphae’, the endosymbiont of the distantly related genus Trichonympha (Hongoh et al., 2008a). Although nitrogen fixation is generally absent in members of the phylum Bacteroidetes, the finding of a nifH gene and its expression in ‘A. pseudotrichonymphae’, the endosymbiont of the distantly related genus Pseudotrichonympha, documented the first case of nitrogen fixation by a symbiont of termite gut flagellate (Hongoh et al., 2008b). Other possible functions of ‘A. devescovinae’ include maintenance of the cytoskeletal structure of their host flagellate (Radek et al., 1996); consumption of hydrogen produced by the flagellates, as shown for ‘A. pseudotrichonymphae’ (Inoue et al., 2007); and protection of the oxygen-sensitive host from oxygen diffusing into the gut (Brune, 1998), as suggested for the Desulfovibrio symbionts of Trichonympha spp. (Sato et al., 2009).
Although strict cospeciation may also occur in other instances, it is not necessarily a rule governing the numerous flagellate–bacteria associations in termite guts. This is illustrated by the polyphyletic nature of the Bacteroidales sequences recovered from the capillary-picked Oxymonas spp., which fall, together with the Bacteroidales ectosymbionts of Oxymonas sp. from Neotermes koshunensis (Noda et al., 2006b), into several termite-gut clusters (Ohkuma et al., 2002; Fig. 3), indicating that Oxymonas flagellates must have acquired their ectosymbionts multiple times from a pool of free-living Bacteroidales. Likewise, the endosymbionts (‘Endomicrobia’) of Devescovina spp. must have been acquired independently by their respective host flagellates. As the closest relatives of the ‘Endomicrobia’ associated with Devescovina spp. are endosymbionts of phylogenetically unrelated flagellates from dry-wood termites, many parabasalid flagellates may have acquired their ‘Endomicrobia’ by horizontal transfer from other flagellates present in the same gut (Ikeda-Ohtsubo et al., 2007; Ohkuma et al., 2007). Given multiple acquisitions and horizontal transfer of other symbionts, the reasons for the strict cospeciation of ‘A. devescovinae’ and its devescovinid host remain enigmatic.
Description of ‘Candidatus Armantifilum devescovinae’
Ar.man.ti.fi′ lum. L. part. adj. armans arming; L. neut. n. filum filament; N.L. neut. n. armantifilum, an arming filament. de.ves.co.vi′ nae. N.L. n. Devescovina, a genus of parabasalid flagellates; N.L. gen. n. devescovinae, of Devescovina (referring to the host genus).
Basis of assignment: filamentous rods, laterally attached on the surface of all Devescovina species, originally referred to as ‘Fusiformis-like rods’ by Kirby (1941). So far uncultured, but form a monophyletic group based on 16S rRNA gene sequence analysis (GenBank accession numbers: FN377751–FN377758, AB194938, AB194939).