Phylogeny of Diplonema ambulator (Larsen and Patterson)

Europ.]. Protistol, 30, 227-237 (1994) May 27, 1994

Europea n Jou rna l o f

PROTISTOLOGY

Phylogeny of Diplonema ambulator (Larsen and Patterson) 1. Homologies of the Flagellar Apparatus Ann E. Montegut-Felkner and Richard E. Triemer Department of Biological Sciences and Bureau of Biological Research, Rutgers University, Piscataway, NJ, USA

SUMMARY The ultrastructure of the flagellar appara tus of Diplonema ambulator was determined to investigate the phylogenetic placement of this genus. Two flagella insert into a pocket and bear distal and proximal tr ansition al plat es. The basal bodies are linked by a connecting fiber and are associated with three microtubular roots. A three-membered dorsal root attaches to the dorsal basal body and ascends along the flagellar pocket. An intermediate root is composed of five microtubules where it attac hes to the ventral basal body (Vb). As it ascends along the flagellar pocket, two additional microtubules become associated, pro ducing a 6 + 1 configura tion. A four-membered ventral root attaches laterally to the Vb and extends into the cytoplasm. Th e flagellar pocket is lined on one side by an independent band of microtubules which are reminiscent of the bodonid MTR . The presence of three asymmetrically arranged microtubular roots, transitional plates similar to those in the flagella of kinetop lastids, and an MTR-like band furth er support the relatio nship of Diplonema with the Euglenozoa. However, the absence of paraxonemal rods and flagellar hairs as well as the unique mitotic chro mosome behavior and peculiar mitochondrial cristae structure suggest that Diplonema may have undergone substantial independent evolution since its point of divergence.

Introduction Griessmann [17] used light microscopy to describe the genus Diplonema as a colorless marine flagellate with a cylindrical, metabolic body which tapers an teriorly and has two short flagella that arise from a flagellar pocket. Later, Schuster et al. [36] used light and electron microscopy to describe the genus Isonema. Ultrastru ctural observations such as the presence of a large vesicular nucleus with a persistent nucleolus and condensed chro mosomes, an intr anu clear spindle, trichocysts, and two subapically inserted flagella led them to suggest th at Isonema may be related to the Euglenida. Upon further investigation, it was later determined that Isonema was not a separate genus but instead should be regarded as the junior synonym of Diplonema [20, 31]. Th is finding was corro borated in the report of a recently cultivated freshwater species of

Diplonema [46].

© 1994 by Gustav FischerVerlag, Stuttgart

Cavalier-Smith [8] coined the term Euglenozoa as a taxonomic unit designed to merge the euglenid flagellates with the Kinetoplastida, another proti st group which comprises the biflagellate bodonid s and uniflagellate trypanosomes (see also [9]). The esta blishment of the Euglenozoa was based prim arily on similarities in fundamental cell structures such as the organization of the periph eral cytoskeletal microtubules, mitochondrial cristae, and paraxonemal rods. In an in depth literatu re review by Kivic and Wa lne [19], additional structura l evidence was presented that provided furth er suppor t for the validity of Cavalier-Smith's Euglenozoa. In addition, they expanded the group to include Diplonema (= Isonema). Among other charac ters, they homologized the microtubules that lined Diplonema's cytostome with the bodonid MTR [6] indicating that the microtubules lining the phagotroph ic apparat us in both organisms originated as flagellar root s. They also suggested that this feeding apparatus complex 0932-4739/94/0030-0227$3.50/0

228 . A. E. Montegut-Felkner and R. E. Triemer

was homologous to that found in some phagotrophic euglenids. However, because Diplonema was not available for study until recently [28, 46], the observations and subsequent phylogenetic implications of these earlier studies were derived from a few published micrographs. As such, the occurrence of an MTRJpocket as well as a more complex feeding apparatus in Diplonema (i.e., the Type 1 and Type II feeding apparatuses of [44]) were overlooked bringing into question the details of homology suggested for the feeding apparatuses. Based on their assumptions, Kivic and Walne [19] proposed a phylogenetic scenario which inferred that Diplonema arose as an evolutionary offshoot during the development of the euglenids from a bodonid-like ancestor. Following an accumulation of additional ultrastructural data, it was later suggested that the euglenids and bodonids diverged from a common ancestor and that Diplonema arose as a separate branch [44]. Although Diplonema does have some euglenozoan characters, the absence of a paraxonemal rod, flagellar hairs, and a complex euglenid pellicle as well as the presence of a unique form of mitosis [43] cause speculation of the exact placement of Diplonema to be problematic and create doubt as to whether or not this organism is even a member of the euglenozoan assemblage. The present paper describes the first of three studies designed to provide new insights into the phylogenetic placement of Diplonema. The purpose of the paper is to compare the three-dimensional structure of the flagellar apparatus in Diplonema ambulator with the basic pattern found in the Euglenozoa. Subsequent papers will report on the homologies of the feeding apparatus and phylogenetic implications derived from the small subunit rRNA gene of

acetate and lead citrate . Sections were viewed with a JEOl 100CX II TEM equipped with a goniometer stage.

Micrograph Orientation and Terminology Unless otherwise stated , cross-sectional series are oriented such that the cell is situated with its dorsal side towards the bottom of the micrograph and its ventral side tow ards the top such that the right and left sides of the micrographs correspond to the right and left sides of the cell. In addition, these series are arranged in the plates from anterior to posterior and viewed from the anterior of the cell as indicated by the clockwise imbrication of the basal bodies [41]. Due to the relative positions of the microtubular roots, the rotational orientation of each longitudinal series differs in order to resolve the microtubules of the individual roots. The direction of view and plane of section for each series is indicated in the corresponding figure legend. Descriptions of the individual components of the flagellar apparatus are accord ing to the terminology set forth by Andersen et al. [1].

Results

General Orientation Diplonema ambulator has 2 short flagella that insert into the dorso-proximal region of a subapical flagellar pocket (Figs. 1, 2, 30). A complex feeding apparatus is situated to the right of the flagellar pocket (arrowhead, Figs. 1,2) . In addition, a microtubule-reinforced pocket (= MTRJpocket of [46]) originates as part of the rightventral side of the flagellar pocket (MTR, Figs. 2, 7, 10-13, 21-24, 30) then extends well below the

Diplonema ambulator.

Material and Methods

Cultures Pure axenic cultures of Diplonema ambulator [46] were maintained at room temperature on agar plates in Sonneborn's Paramecium medium (ATCC 802) supplemented with 10 % horse serum and the antibiotics gentamicin (100 ug/ml), penicillin (100 U/ml), streptomycin (100 ug/rnl), and ampicillin (100 ug/ml).

*

Electron Microscopy Cells were concentrated onto 10 um millipore filters, fixed with 2 % glutaraldehyde in 0.05 M phosphate buffer for 1 hour at room temperature, washed with phosphate buffer, fixed with 2 % osmium tetroxide in 0.05 M phosphate buffer for 1 hour at room temperature, then washed in distilled water . For scanning electron microscopy (SEM), cells on filters were dehydrated in an ethanol series, critical point dried with a Ladd critical point drier, coated with gold/palladium with a Polaron sputter coater, then viewed with a Hitachi S450 SEM. For transmission electron microscopy (TEM), cells on filters were embedded in agar, dehydrated in a graded ethanol series then infiltrated with Quetol resin (Electron Microscopy Sciences, Ft. Washington, PAl and polymerized at 100 °C for 2 hours . Samples were serially sectioned on a Sorvall MT-2B ultramicrotome using a Diatome diamond knife, floated onto formvar coated slot grids, and post-stained with uran yl

Figs. 1-8. General orientation and flagellumlbasal body struc- ~ ture. - Fig. 1. SEM of cell anterior showing complex feeding apparatus (arrowhead) situated to the right of the flagellar pocket (P). Note subapical emergence of the flagella (f) from P. Bar = 0.5 urn. - Fig. 2. Transverse section near cell anterior indicating relative placement of complex feeding apparatus (arrowhead) , microtubule-reinforced area (MTR) of flagellar pocket (P), and ventral and dorsal basal bodies (Vb, Db). Bar = 0.5 urn, - Fig. 3. longitudinal section through ventral and dorsal flagella (Vf, Df). Note distal transitional plate (arrowhead), transitional fibers (small arrow), near perpendicular arrangement of associated non flagellate ventral and dorsal basal bodies (Vb', Db'), and ventral root (VR). Bar = 0.5 urn. - Fig. 4. Transverse section illustrating striated connecting fiber (arrowheads). Bar = 0.5 urn, - Figs. 5-7. Non-adjacent serial sections (30 tilt) through flagellum (Vf) and basal bodies (Vb, Db) illustrating their cross-sectional design. Note concentric rings in the basal bodies (Db, areas 1-3, Fig. 5), transitional fibers (small arrows, Fig. 5), grazing view of striated connecting fiber (arrowheads, Fig. 6), and near perpendicular position of nonflagellate dorsal and ventral basal bodies (Db', Fig. 6; Vb', Fig. 7) with respect to flagellate basal bodies. MTR = microtubule-reinforced region of flagellar pocket (P). Bar = 0.25 urn. - Fig. 8. longitudinal section through flagellum and basal body. Lines indicate cross-sectional planes (x.s.) as follows: 1 = x.s. ofVf as shown in Fig. 5; 2 = x.s. of Db and Vb as shown in Figs.5 and 6, respectively; 3 = x.s. of Db and Vb as shown in Figs. 6 and 7, respectively. Arrowheads = distal and proximal transitional plates, small arrow = transitional fiber. Bar = 0.5 urn, 0

Diplonema ambulator Flagellar Apparatus . 229

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230 . A. E. Montegut-Felkner and R. E. Triemer

point of basal body insertion (Figs. 14, 30). The rnicrotubules that line the pock et are interconnected by a thin fibrou s sheet and each is embedded in an electron dense mass (Fig. 30; Fig. 10 [46]) . A zone of exclusion is usually seen surrounding the cytopla smic side of this MTRJpocket along its entire length (Figs. 2, 24).

Structure of the Flagella The dor sal and ventral flagella lie approximately parallel to one another (Figs. 1, 3, 30) and, since they are nearl y identical, these flagella could only be identified by their development into the dor sal and ventral basal bodies (see Basal Apparatus section ). The axonemes of the flagellar shafts comprise the typical 9 + 2 configuration. There is no evidence for either a paraxonemal body or a paraxonemal rod embedded in the flagellar matrix which surrounds the axoneme nor is there any evidence for the presence of flagellar hair s or scales. The transitional zone (plane 1, Fig. 8) is approximately 350 nm long and is defined as the area delimited by the distal and proximal trans ition al plate s (arrowheads, Figs. 3, 8, 15, 18; DP and PP, Fig. 30). The central microtubule s of the axoneme extend into this region of the flagellum (Vf, Fig. 5; m, Fig. 30) but terminate before the development of the axonemes into the basal bodies (Db, Fig. 5; Vb, Fig. 6). Thin transitional fibers connect the outer doubl ets of the axon eme in the transitional zone to the surrounding flagellar membrane (small arrows, Fig. 5). These fibers elongate, thicken, and become whorled at the point where the axonemes develop into the basal bodies (small arrow, Figs. 3, 8, 15,27).

and Melkonian [41]. The DR is composed of 3 microtubules and appears to be embedded in an electron dense matr ix near its atta chment to the base of the dor sal basal body (Figs. 21-23). This root depart s from the basal body at a sharp angle toward s the cell's right then ascends along the flagellar pocket (large arrow, Figs. 19-20,26-29, obliqu e section; Fig. 30). Although the DR was followed to the distal portion of the pock et, its precise point of termin ation was not determin ed. The IR is anchored to the right-dorsal side of the ventral basal body at a point between the ventral and dorsal basal bodies (Figs. 19-20; arro whead, Figs. 21- 24, 30). At its point of attachment to the ventral basal body, the IR is composed of 5 microtubules (Figs. 9-11 , 30). Thi s root extends anteriorly along the flagellar pocket after it departs from the ventral basal body at a sharp angle to the cell's left (Figs. 17-18,25-29,30). However, as it ascends along the pock et, 2 additional microtubules are added. On e is added next to the right termin al microtubule of the band while the oth er is added to the dor sal side at a point between the second and third microtubules from the left terminal end of the band . Th is gives the root a 6 + 1 configuration (Figs. 25, 30). As is the case for the DR, the precise termination po int of this microtubular root at its distal end is unknown. The VR is compo sed of 4 microtubules and is laterally anchored to the left-ventr al side of the ventral basal body (Figs. 15-18, 30). Unlike the DR and IR, this root does not ascend along the flagellar pocket; rath er, it by-passes the ventral basal body and extends into the cytoplasm approac hing the right-dorsal and left sides of the cell (Figs. 3, 11-13, 19-20,27-29,30).

Basal Apparatus The ventral and dorsal basal bodies lie approximately parallel to one another (Figs. 6, 15-18,21-23,30) and are joined by a thin striated connecting fiber (SCF) (arrowheads, Figs. 4, 6; SCF, Fig. 30). The SCF takes the form of a flattened , striated lens with terminal fibrou s extensions that attach to the basal body triplets. The basal bodies are distinguished by their association with specific microtubular roots which were identified according to the terminolo gy used to describe the root s in euglenid flagellates. The distal region (plane 2, Fig. 8) of the basal bodies has a fibrous component within the lumen and , in cross section, appears as 3 concentric rings of different electron densities (Db, Fig. 5; Vb, Fig. 6). The out er and inner rings (areas 1 and 3, Fig. 5) app ear less electron dense than the intermediate ring (area 2, Fig. 5). A cartwheel configura tion is seen within the lumen at the proximal end (plane 3, Fig. 8) of each basal body (Db, Fig. 6; Vb, Fig. 7). In addition, the ventral and dorsal basal bodies are each associated with a nonflagellate basal body that is oriented nearly perpendicular to its respective partner (Figs. 3, 6, 7, 10, 13,23,24, 30). These non flagellate basal bodies were found in many of the cells examined. Emanating from the basal bodies are three asymmetrically arranged microtubular roots. These were named the dorsal root (DR), ventral root (VR), and intermediate root (IR) according to Solomon et al, [40] as revised by Surek

Figs. 9- 20. Spatial relation ship of the microtubular roots. - ~ Figs. 9- 14. Non -adjacent serial sections in a longitud inal plane which is slightly oblique (ventral to dorsal) to the central plane of basal apparatus as presented in Fig. 30. Sections are viewed from the ventral side and presented in a dorsal to ventral series. The intermediate root (IR) is composed of 5 microtubules at this level (Fig. 9) and att aches to the right-dor sal side of the ventral basal body (Vb, Fig. 11). Note attachment of the ventra l root (VR) to the left-ventr al side of Vb (Figs. 12, 13), extension of microtu bule-reinforced region (MTR ) of pocket below basal apparatus (Fig. 14), and positions of the non flagellate dorsal and ventral basal bodies (Db', Vb', Figs. 10, 13). Of and Vf = dorsal and ventral flagella, Db = dorsal basal body. Bar = 0.25 urn. Figs. 15-18. Non-ad jacent longitudinal series. Micrographs represent a right to left series viewed from the right-ventral side. The ventral root (VR) is composed of 4 microtubules (Figs. 15- 18) and attaches to the left-ventral side of the ventral basal body (Vb, Fig. 18). Note that the intermediate root (IR) passes between Vb and dorsal basal body and approaches Vb at a sharp angle (Figs. 17, 18). Of and Vf = dorsal and ventral flagella, arrowheads = distal and proximal tran sitional plates, small arrow = transitional fiber. Bar = 0.5 urn, - Figs. 19-20. Adjacent serial sections in a plane displaced appro ximatel y 60° (ventral to dorsal) from the plane that par allels the longitudin al axis of the diagram (Fig. 30) and viewed from the right-ventr al side. The ventral root (VR) by-passes the ventral basal body (Vb) and extends into the left and right-dorsal regions of cytoplasm. Note position of the dorsal (large arrow ) and intermediate (IR) roots. P = flagellar pocket. Bar = 0.5 urn,

Diplonema ambulator Flagellar Apparatus . 231

232 . A. E. Mon tegut-Felkner and R. E. Triemer

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Diplonema ambulator Flagellar Apparatus . 233
flagellar-MTR pocket complex in Diplonema ambulator. The drawing represents a view that has been displaced 600 counterclockwise around the centralaxis of the cell to better illustratethe relative positions of the individual components (see arrows for orientation). The doubletsand tripletsof the axonemes and basal bodiesare represented bycylinders. Doublearrowheads = flagellar membrane; Vf,Vb, and Vb' = ventral flagellum, basal body, and non-flagellate basal body, respectively; Df, Db, and Db' = dorsal flagellum, basalbody,and nonflagellate basalbody, respectively; SCF = striatedconnecting fiber;VR = ventralroot; IR = intermediate root; DR = dorsal root; MTR = microtubule-reinforced region of flagellar pocket (P). Window 1 illustrates flagellar insertion into the dorso-proximal side of the flagellar pocket. Window 2 illustratesthe extensionof the central axonemal microtubules (m) into the transitional zone as well as the distal and proximal transitional plates (DP, PP). Bar = 0.25 urn,

Discussion Over the past 20 years, ultrastructural studies have been instrumental in inferring the phylogenetic relationships of several protistan groups. Some of these studies have led to the speculation that, even though the Euglenida and Kinetoplastida do represent two diverse groups of protists, they may share a common phylogenetic origin (see reviews by [19, 44, 53]). Among other features, it was demonstrated that phenotypic similarity exists in the flagellar apparatus of these organisms which comprises two basal bodies that may be linked by a connecting fiber and three asymmetrically arranged microtubular roots. In addition, the flagella are often decorated with non-tubular flagellar hairs and at least one flagellum typically contains a paraxonemal rod (see [14, 19, 26]). The phenotypic state of each of these components in Diplonema and members of the Euglenozoa (sensu [8]) will be individually addressed.

Flagella

M TR D ORSAL

30

PROXIMAL

Eukaryotic flagella can be subdivided into several components. Of importance to this study are the fine


dorsal root (DR, Figs. 21-23) is composed of 3 microtubules and attaches to the right-dorsal side of the dorsal basal body (Db). The intermediate root (arrowhead,Figs. 21-24) descends alongthe flagellar pocketand attachesto the right-dorsal sideof the ventral basal body (Vb). Note position of the nonflagellate basal bodies (Db', Vb') and narrowing of the right side of flagellar pocket to form an extension supported by microtubules (MTR). Vf = ventralflagellum. Bar = 0.5 urn. - Figs. 25-29. Non-adjacentserialsections in an obliqueplanewhichtraverses a ventro-dorsal angleand isviewed from the left-ventral side. The intermediate root (lR)is composed of 7 microtubules in a 6 + ] pattern in the distal region of the flagellar pocket (P) (Fig. 25). The IR is then seen in oblique longitudinal section (I. s.) as it descends along P and attaches to the right-dorsal sideof the ventral basal body (Vb, Fig. 29). The dorsal root (large arrow, Figs. 26-29) is barelynoticeable in oblique I. s. as it descends along P and approachesthe dorsal basal body (Db). The ventral root (VR) attaches to Vb (Fig. 29) as it by-passes Vb and extends into the cytoplasm in both directions (Figs. 27-29). Small arrow, Fig. 27 = transitional fiber, Df = dorsal flagellum . Bar = 0.5 urn.

234 . A. E. Montegut-Felkner and R. E. Triemer

structural details of the axoneme, paraxonemal components, surface, and transitional zone of the flagellum as defined byAndersen et al. [1]. The axonemein the flagellar shaft region of Diplonema ambulator appears to be identical to that found in Diplonema nigricans (Plate 1, Fig. 1 [36]) and Diplonema papillatum [34] in that it is composed of nine outer doublets surrounding two central microtubules. This is the typical condition found in eukaryotic flagella including those of the Kinetoplastida [49] and the Euglenida [22]. Exceptions include a "9 + 0" configuration in the nonemergent (= ventral) flagellum of Colacium libellae (Figs. 10-12 [54]), Cryptoglena pigra [30], Euglena mutabilis [41], Phacus pleuronectes [12], and Trachelomonas hispida var. coronata [52]. However, this is not a definitive character for the nonemergent euglenid flagellum as illustrated by the 9 + 2 pattern seen in both flagella of Euglena gracilis (Fig. 2 [42]; [35]), Khawkinea quartana [37], Menoidium bibacillatum (Fig. 14 [23]), and Rhabdomonas costata (Fig. 20 [23]). It is possible that the reported difference in the axoneme structure of euglenids with a single emergent flagellum is due to a distal placement of the central tubules in the ventral flagella relative to those of the dorsal flagella, hence, providing the two different configurations in a single sectionthrough the proximal regionof the reservoir [35]. Paralleling the axonemeof most euglenozoan flagella is a proteinaceous paraxonemal rod. When present, the paraxonemal rod of the euglenid ventral flagellum is typically more structurallycomplex than that of the dorsal flagellum [3, 7, 11, 50]. On the basis of structural similarity, it has beensuggested that the rods in the ventral (= trailing) flagellum of euglenids are homologous to the rods in the recurrentflagella of bodonidsand the emergent flagella of trypanosomes [11, 50]. A homology between the rods in these protists is further supported by biochemical and immunological studies which infer that euglenozoan rods are composed of homologous proteins (see review by [7]). Another feature of interest that can be found on the euglenozoan flagellum is non-tubular flagellar hairs [22, 48]. In contrast, neither paraxonemal rods nor non-tubular flagellar hairs have been observed in the three species of Diplonema examined ([34, 36], this paper). It should be noted, however, that the absence of paraxonemal rods and flagellar hairs shouldnot be heavily weighted as a criterion questioning the relationship of Diplonema with the Euglenozoa (e.g. [34,36,45]). In fact, paraxonemal rods are absent in the flagella of some trypanosomes (e.g. Blastocrithidia culicis, Crithidia deanei, Crithidia oncopelti [10,15,16]) and the nonemergent flagellum of some euglenids (e.g. Cryptoglena pigra [30], Euglena gracilis [4, 35], Euglena mutabilis [41], Khawkinea quartana [37], Menoidium bibacillatum [23], Phacus pleuronectes [12], Rhabdomonas costata [23], Trachelomonas hispida var. coronata [52]). It should also be noted that cross-reactivity of a monoclonal antibody raised against a trypanosomeparaxonemal rod protein has been demonstrated in the proximal portion of the flagellum of the rodless trypanosome Crithidia oncopelti [16]; it would beinteresting to seeifantibodiesagainstparaxonemalrods

also recognize an epitope within Diplonema flagella. Finally, flagellar hairs are not present on the emergent flagellum of trypanosomes [48], the flagella of some bodonids [47], or the nonemergent flagella of some euglenids [22]. Theseobservationssuggest 1) that the rods and hairs couldhavebeenindependently lost following the divergence of the Kinetoplastida, Euglenida, and Diplonema from a euglenozoan ancestor which had these structuresor 2) that the euglenozoan ancestorlackedthese structures and they developed independently in the Euglenida and Kinetoplastida. Alternatively, Diplonema may have diverged very early in this assemblage before the development of either structure in the ancestor to the Euglenida and Kinetoplastida. The transitional zone is usually defined as the region between the proximalterminationof the centralaxonemal tubulesand the distalend of the basalbody [33]. However, as Moestrup [26] points out, some transitional structures extend beyond these limits and a less stringent definition should be utilized. We define the transitional zone of Diplonema ambulator as the region delimited by the proximal and distal transitional plates. Unlike that of the typicaleukaryotic axoneme, the central tubules penetrate the distal plate and extend into the transitional zone of Diplonema ambulator. This situation was not noted in Diplonema nigricans [36], Diplonema papillatum [34], most trypanosomes and euglenids, or bodonids [47]. However,one or two of the microtubules havebeenshown to enter the transitional zone of some trypanosomes (reviewed in [19]). Likewise, the "transitional helix" of Entosiphon sulcatum extends into a region which surrounds the proximal portion of the central axonemal tubules [25]. The phylogenetic significance, if any, of the pattern of the central tubules in the transitional zone remains obscure. Other components of the transitional zone that are of interest are the electron dense transitional plates. Kinetoplastids typically have a proximal and distal plate which delimit the transitional zone [19, 47, 49]. There is no definitive situation in the euglenids. Proximal plates have been identified in Entosiphon sulcatum [25] and Ploeotia costata [14]. Distal plates may also occur in Menoidium and Rhabdomonas (Figs. 15, 23 [23J). Diplonema ambulator has both proximal and distaltransitionalplatesmuch like those found in the kinetoplastids. The same situation appears to hold true for Diplonema papillatum (terminal plate plus lower electron dense region in Fig. 28 [34]). Although no longitudinal sections were available for evaluation,one can assume that at leasta distalplate exists in Diplonema nigricans (Fig. lc [36]). The transitional fibers found at the proximal region of the transitional zone in Diplonema ambulator become elongated, thickened, and whorled, closely resembling the fibers found at the point of flagellar insertion in many euglenid genera(e.g. curvedbands [21]; Distigma proteus, connective arms, [13]; Euglena gracilis, Fig. 11 [14]; Euglena mutabilis, Fig. 5 [41]; Colacium libellae, Figs. 4, 5 [54]; Cryptoglena pigra, Fig. 12 [30]; Entosiphon sulcatum, Figs. 4, 9 [40]; Eutreptia pertyi, Fig. 4 [11]; Eutreptiella eupharyngea, Fig.9 [51]; Peranema

Diplonema ambulator Flagellar Apparatus . 235

trichophorum, electron dense arms [18]; Rhabdomonas costata and Menoidium bibacillatum, curved bands [23]). These structures might also exist in Diplonema nigricans (Fig. 16 [36]) and Diplonema papillatum (Figs. 24, 28 [34]).To our knowledge,the transitional fibers do not take on such an elaborate appearance in kinetoplastids at the point of flagellar insertion except perhaps in Cryptobia iubilans (Fig. 11 [29]) and Bodo sp. (Fig. 11 [6]).

Basal Bodies The basal bodies of Diplonema ambulator can be divided into two regions. The distal region of the basal body consists of nine triplets that surround a central granular lumen which, in cross-section, appears to consist of three rings of differing electron densities.This structure is not mentioned nor seen in the micrographs describing Diplonema papillatum [34]. Schuster et al. [36] state that the outer triplets of the basal bodies in Diplonema nigricans enclose a central granular region. However, the only visual reference to this structure is in a line drawing (Fig. lc), which is difficult to equate with the fine structure seen in Diplonema ambulator. To our knowledge, no central structure is apparent in the distal region of other euglenozoan basal bodies except perhaps in Entosiphon applanatum [13] and Trypanosoma lewisi (dense amorphous central core [2]). However, the granular core in Diplonema ambulator is often faint and only clearly detected in basal bodiesviewedin perfect cross-section(i.e. compare Figs. 5-6 to 22-23) suggestingthat a re-evaluation of this region of basal bodies may be warranted. At present, though, the granular core should be considered as a novel character in Diplonema. The proximal region of the basal body of Diplonema ambulator is composed of a cartwheel configuration (e.g. [26, 38]). No reference to this structure was made for either Diplonema nigricans [36] or Diplonema papillatum [34]. However, this region of the basal body is short in Diplonema ambulator (approximately 150 nm) and is only clearly seen in near perfect cross-section (compare Db's, Figs. 6 and 23). Moestrup [26] stated that the cartwheel may be lacking in most euglenids. However, subsequent ultrastructural studies have indicated that this structure is in fact present in the proximal basal body region of several euglenids (e.g. Entosiphon sulcatum, Fig. 8 [40]; Peranema trichophorum [18]; 9 + 1 structure in Dinema sulcatum, Distigma proteus, Entosiphon applanatum, and Ploeotia costata [13]). The cartwheel appears to be universal in the proximal ends of kinetoplastid basal bodies [48]. In the Euglenozoa, substantial variation occurs in the size and appearance of the connecting fiber that links the basal bodies of these organisms. In the Euglenida, a striated form of this fiber appears to be prominent and universal in genera with heterodynamic flagella (Dinema sulcatum, Ploeotia costata [14]; Peranema trichophorum [18]; Entosiphon sulcatum [40]). It was suggestedthat the connecting fiber may be a synapomorphy to heterodynamic phagotrophic euglenids [45]. However, a more inconspicuous form of the fiber can be found in some photosynthetic and osmotrophic euglenid genera (e.g.

Euglena gracilis [13, 32]; Euglena mutabilis [41]; Khawkinea quartana [37]). A striated connecting fiber is also present in many kinetoplastid flagellates [48] and Diplonema ambulator (this study). A connecting fiber was not observed in either Diplonema nigricans [36] or Diplonema papillatum [34] but this structure appears to be very thin in Diplonema ambulator and is not seen in every plane of

section. Taken together, these data suggest that a connecting fiber may in fact be synapomorphic to the Euglenozoa where it has become lost or reduced in some members during the course of evolution. Additional nonflagellate basal bodies were observed in many Diplonema ambulator cells. Schuster et al. [36] did not see additional basal bodies in interphase cellsof Diplonemanigricans and thus suggestedthat they must replicate before mitosis where he observed a four basal body state. Although a light microscopic description of mitosis was performed on Diplonema papillatum, no electron microscopic observations were made thereby providing no account of the nonflagellate basal bodies in this species [34]. However, if one accepts that the nonflagellate basal bodies in Diplonema ambulator give rise to flagella during cell division as was implicated for Diplonema nigricans and proven for several euglenid members [5, 14,24], the variation seen in these two species may only represent variations in the timing of basal body replication and the length of the cell cycle as was previously suggested for members of the Euglenida [14].

Flagellar Roots The present study is the first to describe the relative placement and path of the individual components of the basal apparatus in Diplonema and indicates that Diplonema ambulator has a basal apparatus similar to that described for the Euglenozoa (seereviewsby [44,45]). It is composed of two microtubular roots (the IR and VR) that connect to one basal body (Vb) and a third root (the DR) that attaches to the other basal body (Db). The IR and DR ascend along the flagellar pocket as predicted. However, the VR extends into the right-dorsal and left cytoplasmic regions of the cell after attaching laterally to the Vb. This contrasts Kivic and Walne's [19] interpretation that a flagellar root (the VR) is continuous with the root which supports the cytostome in Diplonema (= Isonema) papillatum. While the observation that a VR gives rise to the cytostornal microtubules may hold true for some speciesof Diplonema, their interpretation was based on a few published micrographs since this organism was not available in culture nor was it readily observed in nature. Subsequent studies have indicated that Diplonema ambulator has two feeding apparatuses. The microtubules which support the cytostome of the complex feeding apparatus (Type II of [44]) are continuous with the microtubular loop which lies adjacent to the flagellar pocket (Type I of [44]) and extends below the basal apparatus (personal observation). The need for the threedimensional analysis of the basal apparatus in other cultivated species of Diplonema [28] is warranted to determine if the path of the VR is consistent in this genus.

236 . A. E. Montegut-Felkner and R. E. Triemer

Conclusions The ultrastructural features of the flagellar apparatus presented in this paper further support the placement of Diplonema in the euglenozoan assemblage. The asymmetric arrangement of the three microtubular roots around two basal bodies is consistent with that found in the bodonids and euglenids, The presence of distal and proximal transitional plates in the transitional zone of Diplonema closely resembles the situation found in the bodonids. Finally, the whorled transitional fibers in the proximal region of the transitional zone of Diplonema closely resemble those of the euglenids. The absence of paraxonemal rods and flagellar hairs, the unique chromosome behavior during mitosis [43], and the unusual appearanceof mitochondrialcristae in Diplonema may be explained by independentevolution following the point of divergence from a commoneuglenozoan ancestor.In fact, this has beeninferred from small subunit rRNA sequence analysis [27] which also suggests that the kinetoplastids and euglenids have had a long and separate evolutionary history since their point of divergence [39]. Acknowledgements Th is research was supported by the National Science Found ation (grant no. BSR 9020325) and the Bureau of Biological Research, Rutgers University. The authors wish to thank Carole L. Lewandowski for her technical assistance and for critically read ing the manu script. The authors also wish to thank Lisa Markov for her ideas concernin g the final presentation of the drawing and two anon ymou s reviewers for their helpful comments which have strengthened 'the pap er. This research will be submitted by A. E. Montegut-Felkner in partial fulfillment of the requirements for the Ph.D. degree, Rutgers University, New Brunswick, N. J.

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Key words: Diplonema - Euglenozoa - Flagellar appa ra tus - Isonema - Phylogeny Ann E. Montegut-Felkner, Department of Biological Sciences, Rutgers University, P. O. Box 1059, Piscataway, N] 08855-1059, USA