An ultrastructural study of a free-living retortamonad, Chilomastix cuspidata (Larsen & Patterson, 1990) n. comb. (Retortamonadida, Protista)

Europ. J. Protistol. 33, 254-265 (1997) August 29, 1997

European Journal of

PROTISTOLOGY

An Ultrastructural Study of a Free-living Retortamonad, Chilomastix cuspidata (Larsen & Patterson, 1990) n. comb. (Retortamonadida, Protista) Catherine Bernard, Alastair G. B. Simpson, and David J. Patterson School of Biological Sciences, Zoology Building AD8, Universityof Sydney, Sydney, Australia

Summary The ultrastructure of a free-living retortamonad, Chilomastix cuspidata, originally described as Percolomonas cuspidata Larsen and Patterson, is reported for the first time. This species has a cosmopolitan distribution and has been reported from marine, brackish and freshwater sites with low levels of oxygen. There are four flagella which are located subapically at the head of a ventral groove. The basal bodies give rise to five roots: two major microtubular roots which support the walls of the ventral groove, two minor microtubular roots and one striated root. The root lying in the right wall of the groove also supports the cytopharyngeal region. An electron-dense "lapel" extends dorsally around the site of flagellar insertion and gives rise to microtubules which support the cell membrane. No mitochondria nor dictyosomes were observed. On the basis of this information, the organism is assigned to Chilomastix. These observations are also discussed in relation to the origin of mitochondria within eukaryotes. Key words: Chilomastix, Retortamonadida, Protista, Protozoa, Flagellate, Percolomonas cuspidata, Anaerobe

Introduction The genus Percolomonas was created by Fenchel and Patterson [17] for P. cosmopolitus, a quadriflagellated protist originally assigned to Tetramitus by Ruinen [32]. Percolomonas was originally distinguished by a suite of largely ultrastructural features and assigned to the Heterolobosea [17]. We concur with this assignation, and also regard Psalteriomonas and Lyromonas (the genus created by Cavalier-Smith [14] for the organism originally described as Psalteriomonas vulgaris) as members of the Heterolobosea (after [4]; cf [14,15]). © 1997 byGustav Fischer Verlag

It is in this sense that Heterolobosea will be used in the rest of the paper. Larsen and Patterson [23] used the following criteria to assign flagellates to Percolomonas on the basis of light microscopical characteristics: free-living flagellates with four flagella, flagella inserting at the head of a well-developed groove, and flagella used to draw bacteria into the groove where the bacteria are ingested. They transferred six nominal species previously assigned to Tetramitus and one species originally described in the genus Choanogaster to Percolomonas. Two new species were added: P. membranifera (since transferred to Carpediemonas [16]) and P. cuspidata [23]. The generic assignment of Percolomonas cuspidata has been the subject of dispute. Brugerolle [8] assigned an organism isolated from a salt marsh by Farmer (unpublished), and indistinguishable from P. cuspidata, to Chilomastix. This is one of the two genera of retortamonads. Patterson and Zolffel [29] also noted a close resemblance between P. cuspidata and Chilomastix caulleryi Alexeief], Electron-microscopical observations of retortamonads have revealed a distinctive cellular architecture but have not shown the presence of mitochondria [5, 6]. The retortamonads have been usually regarded as primitively amitochondriate [10, 15,27,28, 35]. All nominal species of retortamonads have been reported as intestinal endocommensals [22] with the exception of four records of free-living species [8,20,34] none of which we regard as unambiguously of retortamonads (see Discussion). Ultrastructural studies have proven effective in establishing and testing hypothesised affinities of protists [27]. This approach has been adopted here to establish the correct generic assignment of the species described by Larsen and Patterson [23].

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Material and Methods Flagellates studied here were isolated from four sites: (1) sediments of a freshwater billabong of the Murray-Darling Basin (Albury-Wodonga, Australia, 146°09'E - 36°08'S) in December 1995;(2) brackish sediments among mangroves of Quibray Bay (south of Sydney, New South Wales, Australia, 151 °lO'E - 33°55'S) in 1995and 1996;(3) marine sediments in Bowling Green Bay (south of Townsville, Queensland, Australia, 147°28'E - 19°20'S) in spring 1986; and (4) brackish sediments from Nivd Bay (north of Copenhagen, Denmark, 12°32'E- 55°56'N) in summer (northern hemisphere) 1992. The 1995/1996 Australian isolates were cultured under anoxic conditions in 30 ml serum vials with about 10-20 ml of boiled source water or brackish water (15%0) and one boiled wheat grain. One drop of a saturated solution of the redox indicator resazurine was added. The medium was flushed with oxygen-free nitrogen, the bottles closed with silicone stoppers and sealed with aluminium caps. The headspace was flushed with oxygen-free nitrogen and cultures inoculated by syringes through the stopper. Decolouration of the resazurine indicated anoxia. Mixed populations of bacteria were present and served as food for the flagellates. The cultures were kept in the dark at room temperature (20-22 °C). Light microscopy was carried out using Zeiss Standard and Axioplan microscopes equipped for photomicrography modified after Patterson [26]. Cells from the Murray-Darling Basin were fixed for ultrastructural examination in a cocktail of 5% glutaraldehyde, 50 mM cacodylate buffer at pH 7.4, and 0.4% OS04 for about 30 minutes on ice. Fixed cells were washed in buffer, embedded in 2% agar and dehydrated in an ascending series of alcohol. Material was embedded in Spurr's resin, and the blocks stained for 2 hours with saturated uranyl in 50% alcohol. Serialsections were cut with a diamond knife using a Reichert Ultracut S. Sections were mounted on pioloforrn-coated slot grids after the technique of Rowley and Moran [31], carbon coated and stained with lead citrate and saturated uranyl acetate in 50% ethanol. Sectionswere examinedwith a Philips CM12 or a Zeiss 902 electron microscope, both fitted with goniometer stages.

Results The cells of the flagellate under study are luteshaped (Figs. 1, 2a-e). They have a posterior spike (Figs. 1, 2a-f) with which they may temporarily attach to the substrate (Fig. 2e). Attached cells usually appear to be feeding. The spike can be as long as the body. The length of the cell, including the spike, ranges from 14 to 33 pm [14 to 29 pm for the isolate from the MurrayDarling Basin (Figs. 2b, c); 15 to 33 pm for the isolate from Quibray Bay (Fig. 2a), 21 to 25 pm for the isolate from Bowling Green Bay (Fig. 2d) and 16 pm for the isolate from Niva Bay (Fig. 2e)]. Cells are ventrally flattened and dorsally convex (Fig. 2g). The cell has a deep groove (G) on its ventral face extending from near the apex anteriorly to just above the spike posteriorly (Figs. 1, 2a-e). The right

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Fig. 1. Diagrammatic representation of Chilomastix cuspidata nov. comb., seen from the ventral face. N: nucleus, G: . groove, the arrowhead indicates the ridge formed by the lapel, the dotted lines at the end of the groove indicate the onset of the cytopharynx. Scalebar = 3 pm.

wall of the groove (G) is more strongly developed than the left one (Figs. 1, 2a-e, g) and has a cusp about one third along its length (Figs. 1, 2a). The cell bulges outward just anterior to the cusp (Figs. 1, 2a-e). Posteriorly the groove (G) becomes a tube which twists to the left and into the cell (Figs. 1, 2a-d). Food is ingested at the inner end of the groove. Food vacuoles are numerous and scattered throughout the cell (Figs. 2a-g). A ridge projects slightly from the surface of the cell dorsal to the site of flagellar insertion (Figs. 1, 2c). This is supported internally by a band of electron-dense material: the lapel (Figs. 2f, i, j, 3a-e, 7). One end of the lapel is adpressed to the ventral face of the left microtubular root (see below). The other end of the lapel terminates dorsally at the level of the posterior basal bodies (Fig. 7). The lapel is narrowest where it connects to the left microtubular root and widest (ca. 800 nm) dorsally (Fig. 7). The lapel gives rise to subpellicular microtubules from its outer margin (Figs. 2i-k). The microtubules form a continuous cortex under the cell membrane excepting under the groove. They extend to the rear of the cell. They become more widely spaced in the mid region of the cell (Fig. 2g), but cluster again at the rear end of the cell to form the posterior spike

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Fig. 3. Details of the flagellar appa ratus and associated struc tures . a-b. From a series of sections. a. Anterior basal bodies (B anterior right; C - anterior left) with linking striated conn ector. b. Posterior basal bodies (A - posterior right, R - recurrent), with left microtubular root (LR), nuclear connector (arro w), arched fibre (open star) and associated electron-dense materi al (black star); the amorphous material associated with basal bodi es A and R is also visible. c. Oblique section through basal bodies A, B and R, showing th e B-r oot to the side of basal body B (slender arro w), the two recurrent rnicrotubules (arrowhead), the left microtubular root (LR) and the striate d fibre (SF) arising from dense material lying on the pr oximal edge of basal body A, and connec ting to the right microtubular band (RB). d. Section throu gh basal body B and the groove (G) showing the B-root (slender arrow), the arched fibre (open star) and associated electr on -dense material (black star). e. Section thr ou gh basal bodi es A and B showi ng th e recurrent microtubul es (small arrowheads) and the connection between basal body.B'and the lapel (plain arrowhead). T he lapel is also indicated on a-d (plain arrowh ead). f. Section along the recurre nt flagellum showi ng th e striated stru ctur e of the vane. g. Section throu gh recurre nt flagellum showing vane and support struc ture . .a-e. Scale bar = 0.5 pm; f-g. Scale bars = 0.25 pm.

Fig. 2. a-e. Light microgra phs of living cells from: a. Quibray Bay; b, c. Albury -Wodonga; d. Bowling Green Bay - Townsville; e. N iva Bay. The plain arrow indi cates th e cusp on th e right wall of the groove, the open arrow indicates the nucl eus and the arrow head indicates th e lapel. f-1. Electron-m icro graphs showing th e general appearance of Chilomastix cuspidata. Whe re possible, these and other micrograp hs are pr esented with dorsal to top and viewed from the anterior, or with anterior to th e top and viewed from th e ventral face. f. Lon gitudinal section throu gh th e cell with the nucleus (N), groove (G) and lapel (arrow head), the flagellar apparatus is visible as well as some associated roots. g. Transverse section th rou gh th e groove (G) showing recurrent flagellum (RF), associated right microtubular band (RB) and hook-b and (H B) separated by the gutter (g). Note the conti nuo us array of subpellicular microtubules und er the cell membrane apart fro m the groove. D ark bod y is indicated by an arrowhead. h. Sectio n near posterior end of the cell, showi ng the subpellicular microtubules gatherin g into the spike; the cytopharynx is also visible. i-k. Serial sections anterior to the flagellar apparatus. i, j. showing the lapel and associated subpellicular micro tu bules; k, I. showing the subpellicular micro tu bules in grazing and in cross-section respecti vely. a-e. Scale bar = 10 )lm; f. Scale bar = 2 urn; g. Scale bar = 1 )lm; h-I, Scale bar = 0.5 )lm.

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(Figs . 2f, h). The microtubules are linked to each other (Fig. 21). The four flagella insert subapically at the head of the groove (G) (Figs. 1, 2a-f, 7). Three flagella of similar length and width extend antero-laterally from the cell (Figs. 1, 2a-e). Their beating causes the cells to swim in a spiral path. The shortest flagellum is recurrent (RF) and lies in the groove (Figs. 1, 2a-e, g, Sa-e, 7). This flagellum has two opposed vanes (Figs. 2g, 3g, 5b, c, 7). Each vane includes a paraxonemal striated structure (Figs . 3f, g, 4f). These structures arise in association with axonemal doublets shortly after the recurrent flagellum (RF) emerges into the groove (Figs. 4e, f). Initially, the support structures are somewhat curled up but become more flattened and expanded distally (Figs. 3g, 4f, 5b, c, 7). No attachment of the recurrent flagellum to the groove has been observed. Th ere are four basal bodies. They are here coded as follows : posterior right = A, anterior right = B, anterior left =C and recurrent = R. The anterior basal bodies (B and C) are linked at their base by a striated connector (Figs. 3a, 7). The basal bodies are approximately 400 nm long . Each basal body has a transitional plate on which the central pair of the axoneme terminates (Figs. 3b, d, e, 4d). Basal bodies A , Band C are directed antero-ventrally and the basal body of the recurrent flagellum is directed posteriorly (Fig. 7). The flagella arising from basal bodies A and B are directed to the right, that from basal body C, more to the left (Figs. 1,7). Basal bodies A and R are located particularly close to each other (Figs. 3b, c, 4a, 7). The flagellar apparatus is anchored to the lapel by several fibres arising from basal body B (Figs. 3a, e, 7). Some amorphous materi al is assoc iated with the anterior basal bodies, extends between those bodies to the cell surface, extends between the two pairs of basal bodies and extends between basal body R to some

of th e roots (Figs. 3b, 4a-e). A further fibrous structure, the nuclear connector, links R to the nuclens (Fig. 3b). The basal bodies give rise to five discrete roots, four of which are microtubular and one is striated (Fig. 7). The right microtubular root is the most prominent root. The right end of this root arises in contact with the base of basal body A (Figs. 4a, 7) and the left end is attached to basal body R (Figs. 4a-e, 7). In this region the microtubules are rolled into a cylinder and appear almost hook-like in cross-section (Figs. 4a-c, 7). The space enclosed within this cylinder is referred to as the gutter (g) and posteriorly becomes continuous with the groove (G) (Figs. 4f, 7). The two anterior ends of the right microtubular root (RR) are held together by electron-dense material (Figs. 4a, 7). This continues posteriorly as dense material associated with the right microtubular band (RB) and which links the walls of the gutter (g) to the inner edge of the right microtubular band (RB) and to the outer edge of the hook-band (HB, see below) (Figs . 4b-f). There are usually about 30 microtubules (including those of the hook-band) separating these two strands within the groove (G) - although the size of the groove and the spacing among these microtubules may vary from cell to cell (Figs . 2g, Sa-c). The right-most microtubules of the right microtubular root (RR) are referred to as the right microtubular band (RB). This is comprised of about 6 microtubules at its origin, but more posteriorly expands by the addition of microtubules into a flat ribbon of about 40 microtubules (Figs. 4b-f, Sa, 7). The vent rally directed face of the right microtubular band (RB) is coated with a thin layer of dens e material via short struts (F igs. 4a-f, Sa). The arched fibre (see below) links the left microtubular root (LR) and the right microtubular band (RB) via this dense material (Figs. 4b-d, 7). The dense

Fig. 4. Serial sections through the flagellar apparatus showing the ori gins of the right microtubular root (RR) and left microtubular ro ot (LR). a. Section showing the right (hooked) end of the right microtubular root (RR) arising between basal bodie s A and R and pushing into a depre ssion in the nucleus; the dense material associated posteriorl y with the arched fibre is visible (black star), as well as the amorphous material associated with basal bodies A and R; basal bod y C is visible. b. More poster iorl y, the right microtubular band (RB) portion of the right microtubular root starts to expand, two strands of material (small arrowheads) lie alongside th e gutter (g) and can be traced through to where the gutter (g) opens in the groove (G ) (Fig. 4f). The arched fibre (open star) becomes visible along side the dense associated material (black star). The left microtubular band (LR) arises on basal body R, near by basal body C. Some coating material forming a layer of dense material lies of the ventral side of the right microtubular band and can be traced through Figure f. c-d, More posteriorly, the arched fibre (open star) links the extending right microtubular band (RB) and the left microtubular root (LR). There is associated electron-dense materi al (black star) with the arched fibre (open star). The recurrent microtubules (large arrowhead) arise near basal body R and can be traced through to where th ey lie near the membrane of the gro ove (Fig. 4f). e. The section is at the point of emergence of th e recurrent flagellum (RF ) from the cell. The arched fibre (open star) is restri cted to the right side of the gutt er; th e associated dense material is still visible (black star). The left microtubular root (LR) is strengthened with associated electron-dense material; th e hook-band (H B) is now well differentiated. f. At the opening of the groove (G), the jun ction with the gutter (g) is set off by th e strands of mat eri al which appeared from the right microtubular root near its or igin (small arrowheads). The recurrent microtubules (large arrow heads) now lie in contact with the cell membrane, and th e gutter is greatly expanded. Th e hook-band (H B) is at th e left edge of the gut ter. Scale bar = 0.5 pm for all figures.

Ultrastructure of Chilomastix cuspidata nov. comb.

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C. Bernard, A. G. B. Simpson, and D. J. Patterson

material disappears posteriorly to the opening of the groove (G) (Figs. 4e, f, Sa). The right microtubular band (RB) extends posteriorly in the right wall of the groove - initially in the irregular bulge above the cusp (Figs. Sa-c). The number of microtubules increases posteriorly to about 60. As the bulge diminishes posteriorly, the right microtubular band (RB) splits into two parts (Fig. Sd). The right part ends after a short distance (Fig. 5e). The left part consists of fewer than 10 microtubules and decreases posteriorly as the right wall of the groove (G) narrows (Fig. Se). It is residual after the groove turns to enter the cell (Fig. Sf). The left-most 10 microtubules of the left edge of the right microtubular root form a tightly linked group called the hook-band and appear electron-dense. The hook-band (HB) arises in association with basal body R (Figs. 4e, f). This band extends posteriorly in close association with the dorsal side of the groove (G) (Figs. 4e, f, Sa-e). The number of microtubules in the hookband (HB) remains unchanged as the root runs posteriorly along the groove (G) (Figs. Sa-e). Approximately at the point where the groove twists into the cell, an additional group of microtubules associated with a paracrystalline fibre (PF) arises along the left edge of the hook-band (HB) (Figs. Sd, e). The hook-band (HB) and the associated structures continue as the cytopharynx (CP) (Figs. sf, 6c-f). The hookband (HB) splits into two bands of microtubules which encircle the developing food vacuoles (Figs. Sf, 6b-f). The paracrystalline fibre (PF) (Fig. 6f) lies on the dorsal side of the cytopharynx (CP) (Figs. 6c-f). The cytopharynx (CP) twists into the cell and eventually is directed anteriorly (Figs. sf, 6b-e). A ribbon of S-6 microtubules, called cytopharynx associated microtubules, arises from the internal end of the cytopharynx

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,. Fig. 5. Transversal sections through the groove, posterior to those of Figure 4. a-e. The right microtubular band (RB) lies under the right wall of the groove (G). The left microtubular root (LR) lies below the left wall of the groove. The hookband (HI~) lies in close associationwith the dorsal part of the groove. The recurrent flagellum (RF) is visible in the groove through to Figure c. In d. the right microtubular band (RB) splits in two parts. A comparison of a-f. demonstrates the reduction of the right wall of the groove. a. The coating material is still present on the right microtubular band; it is not visible in b. c, d. show the left microtubular root (LR) givingrise to a lip, which disappears more posteriorly (e) before the left microtubular root (LR) expands dorsally (f). e. A paracrystalline fibre (PF) and associated microtubules arise at the left of the hook-band (HB). f. The cytopharynx developsinto the cell from an extension of the microtubules of the hook-band (HB), paracrystalline fibre (PF) and associated microtubules. Scale bar = 1 pm for all figures.

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e Fig. 6. a-e. Transversal sections contiguous to those of Figure 5 showing the cytopharynx and associated structures developing into the cell. a. is the most anterior of the series. The cytopharynx (CP), visible in d, e and f, is formed by the invagination of the microtubules of the hook-band (HB) splitting in 2 bands (e) as indicated in f (black and open arrowheads) and of the paracrystalline fibre (PF) and associated microtubules. d. From the anterior and dorsal part of the cytopharynx arise 5-6 cytopharynx associated rnicrotubules (right arrowhead); they run anteriorly (c, b) for about 2 urn, then twist towards the ventral side of the cell (a), extend ventrally and posteriorly to terminate dorsally anterior to the base of the spike (a-e - left arrowheads). g. Section through the paracrystalline fibre (PF) of the cytopharynx, a-e. Scale bar = 1 pm; f, g. Scale bars = 0.5 pm.

(CP), extends anteriorly for 1-2 pm (Figs. 6b-d), then turns towards the ventral side of the cell, extending posteriorly to terminate close to the base of the spike (Figs. 6a, e). The left microtubular root (LR) arises in close association with basal body R and near by basal body C

(Figs. 3b, c, 4b, 7). It extends posteriorly to lie ventral to the hook-band (HB) in the left wall of the groove (G) (Figs. 3b, c, 4c-f, 7). The 15 or so microtubules of this root are sandwiched in thick osmiophilic layers (Figs. 4c-f, 7). The ventral layer of osmiophilic material disappears before the dorsal one (Fig. 4f). The lapel

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Fig. 7. Diagrammatic reconstruction of the flagellar apparatus and associated roots of Chilomastix cuspidata n. comb., showing: A, B, C and R, posterior right, anterior right, anterior left and recurrent basal bodies, respectively, the gutter (g), the groove (G), the hook-band (HB), the left microtubular root (LR), the right microtubular band (RB) of the right microtubular root (RR), the recurrent flagellum (RF), the striated fibre (SF), the lapel (large arrowheads), the recurrent microtubules (small arrowheads), the connections between basal body B and the lapel (short arrow), the B-root to the side of basal body B (slender arrow) and the arched fibre (open star).

arises at the left edge of this mot from the ventral osmiophilic layer (Fig. 7). Posteriorly, the left microtubular root (LR) continues under the left wall of the groove (Figs. Sa-d). At the level of the cusp, the left microtubular root (LR) gives rise to a lip which ends when the groove (G) twists inwards (Figs. Sc, d). The left microtubular root (LR) subsequently develops additional microtubules and extends dorsally (Figs. sf, 6e). The right part of the arched fibre which links the right microtubular band (RB) via the electron-dense material and the left microtubular root (LR), near their origins, is broader than the left part (Figs. 4b-d, 7). The arched fibre passes across the ventral face of the recurrent flagellum (RF) as it enters to the groove (G), and

extends on the right a short distance posteriorly (Figs. 4d-f, 7). A lump of amorphous material is-located on the ventral face of the arched fibre (Figs. 3b, d, 4e-f). This material has an appearance similar to that of the material surrounding the basal bodies. The remaining two microtubular roots are minor. The first of these, called the B-root, usually consists of two microtubules and runs for a short distance along the side of basal body B (Figs. 3c, d, 7). The second minor root, the recurrent root, also comprised of two microtubules, arises to the left of basal body R (Figs. 3c, e, 4c, d, 7) and runs posteriorly and ventrally to eventually form part of the left wall of the groove (Figs. 4f, 7). The microtubules then lie adjacent to the left wall of the groove (G) and between the hook-band (HB) and the left microtubular root (LR) (Fig. 4f). The distance between the hook-band (HB) and the left microtubular root (LR) increases posteriorly (Figs. Sa-d). Additional microtubules arise posterior to the recurrent root to fill in the intervening space. The fifth root is a striated fibre (SF) which arises from dense material lying on the proximal edge of the basal body A (Figs. 3c, 7). This fibre has a periodicity of about 30 nm, and extends to to the right microtubular band (RB) to which it attaches by means of the dense material (Figs. 2f, 3c, 7). The nucleus (N) is located subapically dorsal and to the left of the flagellar insertion (Figs. 1, 2b, f). A small nucleolus is visible in some cells. Division was not observed. Dark rod-shaped bodies (around 0.2 prn) surrounded by one membrane are encountered in the cell (Fig. 2g). Their nature is unknown. No mitochondria, dictyosomes, extrusomes, scales, other secreted structure, cysts or contractile vacuoles were observed in any isolate. The organization of the flagellar apparatus of this isolate is summarised diagrammatically in Figure 7.

Discussion The organisms isolated from the freshwater and brackish sites in Australia and from the brackish water site in Denmark correspond well with Percolomonas cuspidata as described by Larsen & Patterson [23] and also with the organism isolated from a salt-marsh by Farmer and illustrated by Brugerolle [8]. We believe that all these records refer to one species. The ultrastructural features held to characterise Percolomonas include: mitochondria with paddle-shaped cristae, three major microtubular roots and all 4 basal bodies attached to an electron-dense pad [17]. Despite compliance with the light microscopical characteristics of the genus Percolomonas [23], none of these ultra-

Ultrastructure of Chilomastix cuspidata nov. comb.

structural features were encountered in the organism reported here. We also note that the basal bodies in several genera of Heterolobosea are oriented almost in parallel [2, 4, 17], while those of the organism studied here diverge from each other. On the basis of these differences, we conclude that the organism described as P. cuspidata is incorrectly assigned to the genus Percolomonas and to the Heterolobosea. Known groups of completely amitochondriate flagellates are the pelobionts, diplomonads, oxymonads, parabasalids, and retortamonads [11,27], and some less well studied taxa such as Trimastix (unpublished observations). All of these groups, excepting the pelobionts and hypermastigids, contain quadriflagellated members [7,33]. Trichomonads have well developed dictyosomal systems, hydrogenosomes, and a number of cytoskeletal elements such as the pelta and costa [7], not observed in the organism studied here. Oxymonads have cytoskeletal elements such as the axostyle not observed in the organisms studied here, and diplomonads have separated tetrakinetids and dual nuclei [7]. Among the retortamonads, ultrastructural accounts exist for Chilomastix aulastomi [5] and Retortamonas agilis [6], representing both genera currently admitted to the group. Both organisms have four basal bodies, and have a recurrent flagellum from the base of which arise one or more microtubular roots. The recurrent flagellum bears vanes. The ventral groove is supported by two microtubular roots one of which is reinforced with a paracrystalline fibre. Mitochondria, dictyosomes and microbodies/peroxisomes have never been observed [8]. All of these features are found in the organism studied here and are not found together in any other known organism placed outside the retortamonads. We therefore concur with Brugerolle [8] that this organism is assignable to the retortamonads. Retortamonas agilis differs from both Chilomastix aulastomi and the organism studied here in having only two flagella, basal bodies lying as separated pairs, a more obtuse angle between the anterior basal bodies, an extra vane on the recurrent flagellum and a larger nuclear connector [6, 7,9]. The ultrastructure of the isolate studied here is similar to Chilomastix aulastomi [5] in the number and general orientation of basal bodies and associated roots, presence of nuclear connector, arched fibre and cytopharynx with associated microtubules and paracrystalline material. There are some minor differences between the two ultrastructural accounts. The right microtubular root in the organism studied here divides into three elements: the right microtubular band , the microtubules of the gutter and the hook-band. This distinction was not stated explicitly for C. aulastomi, but the same pattern is apparent from published micrographs [5]. Both species have a paracrystalline fibre as-

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sociated with the cytopharynx. However, in the isolate studied here, the paracrystalline fibre is less extensive than in other retortamonads studied to date and is most closely associated with the hook-band, whereas it is linked to the right microtubular band in C. aulastomi [5]. In C. aulastomi, the left root disappears before the cytopharynx develops, while in the isolate studied here the left microtubular root extends beyond the level of the cytopharynx - a feature shared with R. agilis [6]. The orientation of the arched fibre differs in C. aulastomi and in the isolate studied here. The striated fibre arising on basal body A, the lapel, the recurrent root and the two microtubules along the basal body B have not been described from C. aulastomi, nor has a detailed account of the organization of the cytopharynx been provided. We however regard these differences as being differences of degree, and as far as a comparison is possible, the protist studied here shares most of the ultrastructural characters described in Chilomastix aulastomi. We therefore assign the organism studied here to this genus as Chilomastix cuspidata nov. comb. (basionym Percolomonas cuspidata Larsen and Patterson [23]). Chilomastix cuspidata closely resembles some of the other species of Chilomastix - especially the type species Chilomastix caulleryi [1, 19, 24]. There is no evidence that C. cuspidata can live endobiotically, or that any of the endobiont species in the genus can be free-living under natural conditions. It is possible that the free-living organism referred to here as Chilomastix cuspidata could be one of the species previously recorded as an endobiont. However, in the absence of experimental studies, we do not feel that a case for identity with any endocommensal species can be made. We have been able to find four earlier accounts which purport to refer to free-living retortamonads [8,20,34]. The only named species Chilomastix undulate was described by Skuja [34], and was also referred to by Hamar [20]. The description is of an organism with an undulating membrane and therefore is not likely to be a member of the genus Chilomastix. The second organism described by Brugerolle [8] as a free-living Retortamonas is probably referable to Carpedtemonas [16]. We therefore regard this study as the first unambiguous account of a freeliving retortamonad. Chilomastix cuspidata has been reported from several locations in Australia, from Portaferry (Northern Ireland), from Denmark and from the United States. It therefore appears to be cosmopolitan, and has been encountered in habitats ranging from freshwater to marine . It is usually found in sites with little or no oxygen and/or rich in hydrogen sulphide [18]. We have maintained Chilomastix cuspidata under anoxic conditions for months providing additional evidence that it is a free-living anaerobe.

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Chilomastix shares the condition of tetrakonty (i.e. flagella arranged in a group or groups of four) with the amitochondriate diplomonads and oxymonads, with the mitochondriate Heterolobosea and with the hydrogenosome-bearing parabasalids [12, 13]. Tetrakonty is therefore found in taxa perceived to be primitively amitochondriate and in the (probably) monophyletic group consisting of the mitochondriate eukaryotes and their descendants. It has been suggested that tetrakonty evolved once and that mitochondria where originally acquired by a tetrakont host [12, 13, 35]. Chilomastix has been suggested as the extant group most similar to the immediate ancestor of the mitochondriates [12,35], with the Heterolobosea considered by some to be the most basal, or even representing the stem group for the mitochondriates [12,27,35]. Molecular studies provide limited support for the Heterolobosea being the first mitochondriates. They are invariably"deep branching" in analyses of the 16S-like rRNA of eukaryotes [21], however only in a small minority of studies does the group branch "below" the (mitochondriate) euglenozoa [e.g. 3,14,15] or the trichomonads [14], which may also be mitochondriates [30]. No molecular data are available for the retortamonads. Despite ultrastructural differences (including absence/presence of dictyosomes and mitochondria, and the number of microtubular roots) between the retortamonads and Heterolobosea, there are a number of provocative structural similarities between Chilomastix cuspidata and genera of Heterolobosea [2, 4, 12, 17,35]. In Percolomonas cosmopolitus and in the retortamonads, the right wall of the ventral groove is supported by at least two arrays of microtubules. At least two Heterolobosea, Psalteriomonas lanterna [4] and Percolomonas cosmopolitus [17], have a right microtubular root that is C-shaped in cross section at its origin, and lies near the nucleus - as is the case in C. cuspidata. Percolomonas cosmopolitus, P. lanterna and C. cuspidata have electron-dense material associated with the inner face of the right root. One of the right roots of P. cosmopolitus lies in the groove and acts as the source for other microtubules, and may correspond with the hook-band of C. cuspidata. These similarities provide some additional support for the retortamonads and Heterolobosea branching offlfalling immediately "either side" of the evolution of mitochondria on the eukaryotic tree. There are, however, alternative plausible contemporary hypotheses. It has been suggested that the often tetraflagellated parabasalids [e.g. 27] or the biflagellated, but grooveand vane-bearing jakobids [25] may branch off! fall between Retortamonads and Heterolobosea. Furthermore, studies of free-living anaerobic flagellates suggest that the full diversity of amitochondriate protists has yet to be covered by any modern techniques (Bernard

et al. unpublished obs.; Brugerolle pers. com.). Without a significantly greater understanding of- such organisms, progress in testing or choosing between, phylogenetic hypotheses of the evolution of mitochondria may well be difficult. Acknowledgements: The authors thank Russell Shiel at the Murray-Darling Freshwater Research Centre, the Electron Microscopy Unit of the University of Sydney, and Brett Dicks for darkroom work and expertise. Financial support from ABRS, ARC and EERO is acknowledged.

References Alexeieff A. (1909): Les flagelles parasites de l'intestin des batraciens indigenes. C. R. Soc. Bio!., Paris, 67, 199-201. 2 Balamuth W., Bradbury P. c., and Schuster F. L. (1983): Ultrastructure of the amoeboflagellate Tetramitus rostratus. J. Protozoo!., 30, 445-455. 3 Branke J., Berchtold M., Breunig A., Konig H., and ReimannJ. (1996): 16S-like rDNA sequence and phylogenetic position of the diplomonad Spironucleus muris (Lavier 1936). Europ. J. Protisto!., 32, 227-233. 4 Broers C. A. M., Stumm C. A., Vogels G. D., and Brugerolle G. (1990): Psalteriomonas lanterna gen. nov., sp. nov., a free-living amoeboflagellate isolated from freshwater anaerobic sediments. Europ. J. Protistol., 25, 369-380. 5 Brugerolle G. (1973): Etude ultrastructurale du trophozoite et du kyste chez le genre Chilomastix Alexeieff, 1910 (Zoomastigophorea, Retortamonadida Grasse, 1952). J. Protozoo!., 20, 574-585. 6 Brugerolle G. (1977): Ultrastructure du genre Retortamonas Grassi 1879 (Zoomastigophorea, Retortamonadida Wenrich 1931). Protistologica, 13,233-240. 7 Brugerolle G. (1991a): Flagellar and cytoskeletal systems in amitochondrial flagellates: Archamoeba, Metamonada and Parabasala. Protoplasma, 164,70-90. 8 Brugerolle G. (1991b): Cell organization in free-living amitochondriate heterotrophic flagellates. In: Patterson D. J. and Larsen J. (eds): The biology of free-living heterotrophic flagellates, pp. 138-148. The Systematics Association. Clarendon Press, Oxford. 9 Brugerolle G. and Mignot J.-P. (1990): Phylum Zoomastigina, Class Retortamonadida. In: Margulis L., Corliss J. O. and Melkonian (eds): Handbook of Protoctista, pp. 259-265. Jones & Bartlett, Boston. 10 Cavalier-Smith T. (1983): A 6-kingdom classification and a unified phylogeny. In: Schwemmler W. and Schenk H. E. A. (eds): Endocytobiology II, pp. 1027-1034. De Gruyter, Boston. 11 Cavalier-Smith T. (1987): Eukaryotes with no mitochondria. Nature, 326, 332-333. 12 Cavalier-Smith T. (1991): Cell diversification in heterotrophic flagellates. In: Patterson D. J. and Larsen J. (eds): The biology of free-living heterotrophic flagellates, pp. 113-131. The Systematics Association. Clarendon Press, Oxford. 13 Cavalier-Smith T. (1992): Percolozoa and the symbiotic origin of the metakaryote cel!. In: Sato S., Ishida M. and

Ultrastructure of Chilomastix cuspidata nov. comb .

14 15 16

17

18

19 20 21 22

23 24 25

26

Ishikawa H . (eds): Endocytobiology V, pp. 399-406. University Press, Tiibingen. Cavalier-Smith T. (1993): Kingdom Protozoa and its 18 phyla. Microbiol. Rev., 57, 953-994. Cavalier Smith T. (1995): Zooflagellate phylogeny and classification. Cytology, 37,1010-1029. Ekebom J., Patterson D. J., and Vers N. (1996): Heterotrophic flagellates from co ral reef sediments (Great Barrier Reef, Australia). Arch. Protistenkd., 142, 251-272. Fenchel T and Patterson D . J. (1986): Percolomonas cosmopoluus (Ruinen) n. gen., a new type of filter feeding flagellate from marine plankton. J. Mar. BioI. Ass. U.K., 66, 465-482. Fenchel T., Bernard C, Esteban G ., Finlay B. J., Hansen P.J., and Iversen N. (1995): Microbial diversity and activity in a Danish fjord with anoxic deep water. Ophelia, 43, 4~100. . Grasse P.-P. (1926): Contribution a l'etude des flagelles para sites. Arch . Zool. expogen., 65, 345-601. H amar J. (1979): Some new zooflagellates from Hungary. Tiscia (Szeged), 14, 147-162. Hinkle G. and Sogin M. L. (1993): The evolution of the Vahlkampfiidae as deduced from 16S-like ribosomal RNA analysis . J. Euk . Microbiol., 40, 599-603. Kulda J. and Nohynkova E . (1978): Flagellates of the human intestine and of intestines of other species. In: Kreier J. P. (ed.): Parasitic protozoa, Vol. 2., pp. 1-138. Academic Press, New York. Larsen J. and Patterson D. J. (1990): Some flagellates (Protista) from tropical marine sediments. J. Nat. Hist., 24, 801-937. Ni e D. (1948): Th e structure and division of Chiloma stix int estinalis Kucz ynsk i, with notes on similar forms in man and other vertebrates. J. Morph., 82,287-318. O'Kelly C (1993): The jakobid flagellates: structural features of]akoba, Reclinomonas and Histiona and impl ications for the early diversification of eukaryotes. J. Euk. Micr ob iol., 40, 627-636. Patterson D. J. (1982): Photomicrography using a ded icated electronic flash. Microscopy, 34, 437-442.

265

27 Patterson D. J. (1994): Protozoa: evolution and systematics. In: Hausmann K. and Hiilsmann N. (eds): Progress in Pr otozoology, Proceedings of the IX International Congress of Protozoology, Berlin 1993, pp.l-14. Gustav Fischer Verlag, Stuttgart. 28 Patterson D. J. and Sogin M. L. (1992): Eukaryote origins and protistan diversity. In: Hartman H. and Matsuno K. (eds): The orig in and evolution of the cell, pp . 13-46 . World Scientific , Singapore. 29 Patterson D.J. and Zolffel M. (1991): Heterotrophic flagellates of uncertain taxonomic position. In: Patterson D .J. and Larsen J. (eds): The biology of free-living heterotrophic flagellates, pp . 427-475 . The Systematics Associa tion. Clarendon Press, Oxford. 30 Philippe H. and Adoutte A. (1996): How reliable is our current view of eukaryotic phylogeny? In: Brugerolle G. and Mignot J.-P. (eds): Protistological actualities (Proceedings of the Second European Congress of Protistology Clermont-Ferrand, 1995), pp. 17-33. University Blaise-Pascal , Clermont-Ferrand. 31 Rowle y J. C and Moran D. T. (1975): A simple procedure for mounting wrinkle free sections on formvar coated slot grids . Ultramicroscopy, 1, 151-155 . 32 Ruinen J. (1938): Notizen iiber Salzflagellaten. II. Dber die Verbreitung der Salzflagellaten. Arch. Protistenkd, 90, 210-258. 33 Simpson A., Bernard C, Fenchel T., and Patterson D. J. (1997): The organisation of Mastigamoeba schizophrenia n. sp.: more evidence of ultrastructural idiosyncrasy and simplicity in pelobiont protists. Europ. J. Protistol, 33, 87-98 . 34 Skuja H. (1956): Studien iiber das Phytoplankton Schwedischer Binncngewasser, Nova Acta Regiae. Societat is Scientiarum Upsaliensis, 16, 1-404. 35 Sleigh M. A. (1995): Progress in understanding th e phylogen y of flagellates . C ytology, 37, 985-1009. Address for correspondence: Dav id J. Patterson, School of Biological Sciences, Zoology Building A08, University of Sydney, Sydney, NSW 2006, Australia; fax: 61 2 9351 4119. E-mail: [email protected]