Ultrastructure and morphogenesis of the marine epibenthic ciliate Epiclintes ambiguus (Epiclintidae, n. fam.; Ciliophora)

Europ.J. Protistol. 26, 182-194 (1990) October 19, 1990

European Journal of

PROTISTOLOGY

Ultrastructure and Morphogenesis of the Marine Epibenthic Ciliate Epiclintes ambiguus (Epiclintidae, n. tam.; Ciliophora) Barry J. Wicklow Department of Biology, Saint Anselm College, Manchester, New Hampshire, U.S.A.

Arthur C. Borror Department of Zoology, University of New Hampshire, Durham, New Hampshire, U.S.A.

SUMMARY Epiclintes ambiguus, a highly contractile marine ciliate, has ventral cirri in oblique and marginal rows, and three rows of dorsal cilia, thus is at the hypotrich structural grade. We use light microscopy of living and protargol stained specimens and both scanning and transmission electron microscopy to describe cortical morphogenesis through cell division and interphase cortical structure of E. ambiguus. Cortical morphogenesis proceeds in 2 oblique developmental zones and is based on 4 kinds of primordia: oral, frontal, somatic, and a specialized ventral primordium. Thirteen to fifteen oblique rows of ventral cirri arise primarily from somatic (within-row) primordia (producing the anterior complement of cirral rows) and the ventral primordia (producing the posterior complement of cirral rows); frontal ciliature is limited to one or two short cirral rows. A longitudinal series of transverse cirri differentiate from posterior kinetosomes in (1) frontal streaks, (2) ventral (within-row) streaks, and (3) streaks that develop from ventral primordia. Stomatogenesis in the opisthe begins near the cell surface, then continues within a cortical pit which lengthens posteriorly. Stomatogenesis in the proter occurs dorsally to the parental buccal membranelles. The ultrastructural organization of membranelles and cirri of Epiclintes is consistent with the general structural pattern within the Spirotricha. Dorsal ciliary complexes are dikinetids with the anterior kinetosome ciliated, the posterior nonciliated; each dorsal cilium protrudes from a cylindrical cortical papilla. Dikinetids have postciliary, transverse, and nematodesmal microtubules as well as a kinetodesmal fiber. Nematodesmal granules (d = 55 nm) lie beside nematodesmal microtubules. A system of multilayered, membrane-like materials lie within the cortex. Stomatogenesis, deployment of somatic ciliature, and general organization of microtubular structures in Epiclintes suggest an evolutionary relationship with Kahliella and other members of the Stichotrichina. Morphogenetic and structural specializations however justify inclusion of Epiclintes in a new family, the Epiclintidae.

Introduction Classifications of half a century ago (e.g. [28]) sufficed by putting all ciliates of hypotrich grade of structural organization into three families of a single order. By the early 1970's, it was evident that the 400 or so species of 0932-4739/90/0026-0182$3.50/0

hypotrichs represented at least 6 families in that order [6]. During the past two decades, evidence regarding the complexity of hypotrichs has led to the generally accepted conclusion that such ciliates represent diversephylogenetic branches representing several high level taxonomic groups. The interpretation of morphogenetic and mor© 1990 by Gustav Fischer Verlag, Stuttgart

Figs. 1-4. Scanning electron micrographs of Epiclintes ambiguus. - Fig. 1. Interphase cell (ventral aspect) showing the distribution of somatic and buccal ciliature. Adoral zone of membranelles (Azm), dorsal cilia (De), transverse cirri (Tc), x 920. - Fig. 2. Anterior cell view showing the position of the zone of membranelles (Azm), paroral cirrus (Pc), paroral membrane (Pm), marginal cirri (Me), and ventral cirri (Vc), x 4000. - Fig. 3. The paroral membrane (Pm) lies in a cortical furrow above the right buccal overture, x 4300.Fig. 4. Posterior cell view showing transverse cirri (Tc), marginal cirri (Me), and dorsal cilia (De) within papillae (P), x 2900.

184 . Wicklow and Borror

phostatic evidence has provided the basis for several, often conflicting, classification schemes [10, 13-15,24,36,40]. Such a trend suggests at a minimum that present taxonomic schemes may be insufficient to encompass all hypotrich phylogenetic diversity. Epiclintes ambiguus (Miiller, 1786) a cosmopolitan epibenthic ciliate, is ubiquitous in most marine habitats including estuaries, sandy beaches, salt marshes, and tide pools [23]. The paucity of papers documenting the biology of the species of Epiclintes [e.g. 9, 11,42] underscores the uncertain systematic position of the genus. Since its discovery Epiclintes has been assigned, at least provisionally or as incertae sedis, to several hypotrich (sensu lato) families (Table 1), based primarily on the arrangement of ventral cirral rows. Becauseciliates sharing a similarly arranged ventral ciliature may have strikingly different ontogenies, classifications based solely on the interphase organization of ciliature may be artificial. Thus the phylogenetic position of Epiclintes has remained ambiguous. Wicklow, 1979, presented preliminary information on the structure and morphogenesis of E. ambiguus. The details suggested that Epiclintes exhibited characteristics of the suborder Stichotrichina but also possessed many unique characters. Our objective in this paper is to provide a detailed analysis of interphase cortical ultrastructure and cortical morphogenesis during cell division in E. ambiguus. We then use this information to help resolve the systematic position of Epiclintes. Material and Methods We isolated E. ambiguus from Chondrus detritus and the surface of intertidal mud of Great Bay Estuary, Adams Point, Durham, N.H. and from intertidal sand at Sea Point Beach, Kittery, Maine between 1976 and 1989. Populations were cultured on mixed populations of diatoms collected from Great Bay then grown on F 2 medium [20] or on single species diatom populations of Bellerochea polymorpha or Phaeodactylum tricornutum in 18-25%0 sea water at 15-16°C, We studied living cells as well as Pereny's fixed, protargolstained specimens [43] to reveal cortical ciliature and infraciliature and cortical development through cell division. Counts and measurements were made at 1000 X; a Nikon Biophot microscope was used for photomicrography. After relaxing cells with 8% MgCI2, we processed them for scanning electron microscopy [43], then viewed them using an AMR 1000 scanning electron microscope. For transmission electron microscopy, we fixed cells in 3% glutaraldehyde in a 0.2 M cacodylate buffer containing a balanced salt solution (30 mg/ml NaCl and 20 mg/ml CaCI2 ) for 45 min followed by 4.5 min washes in a 0.2 M cacodylate buffer containing 30 mg/ml NaCl 2 (first wash) with successive decreases in salt content in subsequent washes [41]. We postfixed cellsin 1% OS04 in 0.2 M cacodylate buffer, 30 min, rinsed 4 X (5 min each) in buffer, once (5 min) in distilled water, dehydrated in a graded ethanol series, transferred to propylene oxide, then embedded in an Epon 812/Araldite 502 mixture. After polymerization we sectioned cells with glassknives on a Reichert OMU 3 ultramicrotome then stained with 2% uranyl acetate (30 min) and lead citrate (15 min). We viewedcellsections usingaJEOL 100stransmission electron microscope.

Results

General Morphology and Behavior E. ambiguus (Miiller, 1786) Biitschli, 1889 is elongate, cephalized, and contractile with a long posterior tail (Figs. 1-5). Cells relaxed with MgCh, then fixed with Pereny's, ranged from 170-265 urn long and 35-54 urn wide (n = 8). Active cells at maximum extension ranged from 236-400 urn long, usually over 300 urn (n = 7). At these times cephalization of the anterior is prominent while the posterior is attenuate. Cells feed on a variety of diatoms by gliding forward, maneuvering the supple, cephalized anterior over the substrate. Undisturbed individuals in culture may remain motionless for periods of 30 s except for active lapel membranelles and brief retractions of the cell's anterior. When moving rapidly forward the anterior may bend right or left to change direction. Cells may also back up to change direction; under severe stimuli such as a change in osmolarity, cells can back up rapidly for a distance of several millimeters. E. ambiguus is multimacronucleate. Macronuclei are irregularly shaped, elongate, and show replication bands (Fig. 19) as in other hypotrichs [32]. A biometric characterization of E. ambiguus is shown in Table 2.

Buccal and Somatic Ciliature and Infraciliature Buccal structures. The zone of membranelles is typical of spirotrichs. It comprises an average of 63 paramembranelles, each consisting of 3 or 4 rows of kinetosomes. The first (posteriormost) and second rows are the longest rows and are of equal length (approximately 11 kinetosomes); the third row is shorter (by 2 kinetosomes) and the fourth row is shortest consisting of only 2 kinetosomes. Transverse microtubules arise from row 4 kinetosomes and those kinetosomes of row 3 not bordered by row 4 kinetosomes. Postciliary microtubules arise from row 1 kinetosomes. The paroral membrane consists of a longitudinal series of kinetosomal pairs (diplostichomonade); it lieswithin a cortical furrow along the right buccal overture (Fig. 3). The single kinetosome row composing the endoral membrane (stichomonade) lies within the buccal cavity. One or two paroral cirri are located at the anterior left ventral surface (Fig. 2).

Ventral cirri. Thirteen to fifteen oblique ventral rows of relatively undifferentiated cirri are located between right and left marginal cirral rows (Figs. 1, 2, 5, 6, 8). Each cirrus, 2 kinetosomes wide and 6-8 kinetosomes long, lies in a cortical indentation at a 60° angle to the long axis of the cell. A fibrillar sheath surrounds each cirrus distally, then descends to encircle the cirrus proximally as the matrix from which 3 microtubular bundles (Mb) extend into the cytoplasm (Fig. 5). A narrow Mb extends anteriorly from the cirrus. A larger Mb extends posteriorly and to the cell's right of each cirrus, overlapping the Mb of the next cirrus posteriorly. In living cells, light microscopy reveals refractile myonemes to the cell's right of each ventral oblique row of cirri, and the right marginal row of cirri. The overlapping Mbs appear to correspond, at least

Ultrastructure and Morphogenesis of Epic/intes ambiguus . 185 Table 1. Classifications of Epiclintes ambiguus Author

Date

Familial placement

Kahl Borror Corliss Jankowski Tuffr au

1932 1972 1979 1979 1979; 1987

Oxytrichidae Urostylidae Keronidae Oxyrrichid ae (Psammomitrinae) Keronid ae

in part, to the myoneme visible in living cells. Th ere are no such myonemes parallel to the transverse cirri or the left marginal row of cirri. The transverse Mb extends to the cell's right and centrally from each ventral cirrus, deep to the posterior Mb. Transverse Mbs become progressively shorter and extend more dorsally in the anterior cirr i of each ventral row. Postciliary micro tubules aris e from the posteriormost kinetosomes of each cirrus th en extend po ster iorly to contribute to the po sterior Mb. Transverse microtubules originate near the anteriormost cirral kine tosomes and extend toward the pellicle (Fig. 6).

Transverse cirri. Th ese are the most ma ssive of the cirri, consisting of 5 row s of 8-10 kinetosomes, located in a longitudinal row in th e posterio r half of the cell, mediad of the left marginal cirri (Figs. 1,4, 5, 7). Each is subtended by three bundles of microtubules. The large anterior Mb extends anteriomedially from the anterior edge of the cirrus base . Th e shorter posterior Mb extends posteriorly and laterally from the posterior edge of the cirrus. Th e transverse Mb extends posteriorly but is deeper and to the right of the po sterior Mb. Fig. 5. Ink drawing of Epic/intes ambiguus (ventral aspect) based on a protargolstained specimen. Oral , frontal, and somatic infraciliary structur es are depicted.

Marginal cirri. There is one row each of right and left marginal cirri. Protargol sta ining and EM reveal thr ee sets of microtubules extending dorsolaterally and anteriorly , medially and posteriorly, and medially from each left marginal cirrus (Figs. 1,2,4,5). Two sets of microtubules extend from each right marginal cirrus: an anteriormed ial

Table 2. Biometric characterization of Epic/intes am biguus (Great Bay, N. H. popul ation) based on protargol-stained specimens (N = 25). Min =:....minimum, Max = maximum, SE = standard error of mean, SD = sta ndard deviation, R = range, V = coefficient of variation in %, X = arithmetic mean Character

X

membranelles dorsal kineties ventral rows buccal cirri ventral cirri transverse cirri If. marginal cirri rt. marginal cirri total cirri

63 3 13.8 1.5 217.1 30.4 77.0 82.3 408.4

SD

SE

V

5.0

1.0

8.0

0.8 0.5 25.2 3.4 7.7 7.2 39.0

0.2 0.1 5.0 0.7 1.5 1.4 7.8

5.9 33.5 11.6 11.3 10.0 8.8 9.5

Min

Max

51 3 13 1 176 18 65 63 345

71 3 15 2 253 34 93 94 460

R 19 2 1 77 16 28 31 115

Figs. 6-10. Transmission -electron micrographs of Epic/intes am biguus. - Fig. 6. Ro w 3 ventra l cirri showing the organi zati on of anterior and po sterior microtubular bundles, po stcili ar y micro tubules, transverse microtubules, and parasornal sac , x 28 700. Fig. 7. Oblique section of a tr an sverse cirrus, x 24 5 00. - Fig. 8. Ventral cirrus base sho wing microtubular and microfibrillar stru ctur es, x 28000. - Fig. 9. Multilayered membran e-lik e materials with in the cortex, x 70000. - Fig. 10. Longitudinally sectioned dorsal dikinetid show ing cilium within cylindrical papill a. Transverse microtubules ar e associated within the anterior, ciliated kinetosome, postciliary microtubules are associated with th e posterior kinetosome, x 3 1700. A = anterior, Amb = anter ior microtubular bundle, Om = o ute r membrane, P = po steri or , Pmb = posterior microtubular bundle, Pmt = postciliary microtub ules, Ps = parasomal sac, Tmb = transverse microtubular bundle, T mt = transverse microtubulcs.

11

Figs. 11-19. Transmission electron micrographs of Epiclintes ambiguus (structures in Figs. 11-17 as seen from within the cell) Fig. 11-12. Grazing sections of the dorsal surface showing the basket-like framework of the distal section of the papillae (Fig. 11) as well as the orientation of dikinetids, X 46700, X 20000. - Figs. 13-17. Structure of the dorsal dikinetid. Transverse microtubules are associated with the anterior, ciliated kinetosome, postciliary micro tubules and a kinetodesmal fiber are associated in the posterior kinetosome. Microtubule-associated electron dense material is located on the posterior and right borders of the posterior kinetosome, X 43000. - Figs. 18a and 18 b. Longitudinally sectioned dorsal cilia. Nematodesmal granules (Fig. 18a) and the basket-likeframework (arrowhead) at the distal region of the papilla are evident, X 18800, X 30000. Kd = kinetodesmal fiber, M = electron-dense material, Ng = nematodesmal granules, Pmt = postciliary microtubules, Tmt = transverse microtubules. - Fig. 19. Macronuclei showing replication bands (arrowheads), x 11000.

188 . Wicklow and Bor ror

Mb and a right Mb that extend anteriorly and dor solaterally to overlap in a "herring bone" pattern with lateral Mb's along the right most dor sal kinety (Figs. 20, 21 ). Dorsal cilia. The dorsal kineties consist of thr ee rows of short cilia that emerge from the tips of cylindrical papillae (Figs. 4, 10). The ciliary unit s of most of the dorsal surface are dikinetids with the anterior kinetosome ciliated and the posterior kinetosome unciliated. Trans verse microtubules arise at the anterior kineto some, whereas postc iliary micro tubules and a kinetodesmal fiber originate from the posterior kinetosom e (Figs. 10, 13-17). Microtubuleassociated electron dense material occurs on the po sterior and right side of the dikin etid (Figs. 13-17). Nematodesmal microtubules descend into the cytoplasm from fibrillar material at the base of the bristle kinetosomes. Electron dense granules (d = 55 nm) are arranged in a linear array at 30 nm intervals along the nematodesmal microtubules (Fig. 18a); adjacent granules are linked by fine connections. Faint protargol-p ositive connections occur betw een some dikinetids of the right dorsal ciliary row and the posterior ends of the lateral Mb. Cell Membrane and Dorsal Papillae

Unusual cortical features include a specialized dor sal bristle complex (Figs. 4, 10-18) and a system of multilayered membrane-like mater ial (Figs. 9, 10) located between an outer perilemma and the plasma membr ane. Each dorsal cilium emerges from a cortical papilla th at is supported distally by a framework of dense materials (Figs. 10-12, 18a, b). Morphogenesis Stomatogenesis. During early cortical morphogenesis the opisthe oral primordium (Op) develops near the cell surface as an elabor ation of kinetosomes associated with the dedifferentiation of the anterior-most transverse cirrus (Figs. 25a, 25b). The Op grows into an oval field with a truncated anterior end within which kinetosomes align into promembranelles (Fig. 22 ). The differentiati on of promembranelles proceeds in an anterio r to posterior maturity gradient with in a hemispherically concave depression which continues to invaginate posteriorl y to subtend the first one or two transverse cirri (Figs. 25 c-25 f). Meanwhile, ant erior promembranelles curve medially to form the presumptive collar membr anelles (Fig. 23). Membranellar cilia emerge onto the cell surface during cytokinesis. The proter Op develops later as a field of kinetosomes just posterior to the parental lapel membranelles. Subsequent differentiation of pr oter promembranelles pro ceeds dor sally to the par ental membranelles (Figs. 25 d-25 f). The parental lapel membranelles and paroral app aratu s then dedifferentiate and resorb in a posterior to anterior direction. Proter promembr anelles emerge onto the cell sur face anteriorly as parental membranelles are resorbed . Anlage of paroral and endora l membrane s differenti ate along the medial edge of the Op. Eventually 1-2 par or al cirri bud from the ant erior end of the paroral membrane

then migrate to lie anteriorly near the front al ciliature (Figs. 23, 24, 25 f-h). Frontal cirri. As opisthe promembranelles begin to differentiate, a dikin etid appears medial to the Op and posterior to the first postcytostomal row of vent ral cirri. The dikinetid may arise de novo or by dedifferentiation of a parental cirrus ; its distance from the Op makes an Op origin unlikely. Initially only the posterior kinetosome of the pair is ciliated. The kinetosomes then prol iferat e to form one or two frontal streaks that give rise to the anterior-most cirral rows (malar and paramalar row s) of the opisthe (Figs. 22, 25c-h). The proter frontal ciliature develops later but in a similar way. Kinetosomes appear medial to the developing paroral membrane, proliferate to form first one then two frontal streaks, from which differentiate two anterior ventral rows. Ventral and transverse cirri. An opisthe ventral primordium (Vp) develops at the anterior end of the 8th postcytostomal vent ral row ; the proter Vp develops 3-4 rows anterior to the opisthe Vp (Figs. 25 c, d). Kinetosomes of each Vp develop from the dedifferentiation of the anterior-most cirru s of the ventral row. Ventr al primordia develop to the left of, but independent from , right marginal cirri. The Vp form s 2-3 ventral cirral streaks plus 2-3 transverse cirral strea ks. Subsequently, the Vp expands anteriorly to form 5- 6 ventral cirra l streaks and posteriorly to form about 16 transverse cirral str eaks (Figs. 22, 23, 25c-g). Whereas each of the anterior 5-6 ventr al cirral streaks gives rise to ent ire ventral cirral row and a large tran sverse cirru s, each of the posterior 16 transverse cirral streaks gives rise to a single large transverse cirrus. Additional ventr al cirri develop by the proliferation of kinetosomes as streaks within parental cirral row s (Figs. 22, 23, 25 c-g). Transverse cirri also differentiate from the posterior ends of ventral (within-row) and frontal streaks (Figs. 24, 25g). Thus, transverse cirri develop from 3 anlagen: the anterior 1-2 from front al streaks, the next 4-6 from ventral streaks, and the remainder from the ventral primordium. Dorsal kin eties and marginal cirri. In the right dorsal kinety a dikin etid appears at each of two zones adjacent to, and to the cell's right of, the original ro w, that subsequently develops into an anteroposterior streak of about 20 kinetosome s. A similar process happens in left and the median dorsal kinetics, except that the initial group of basal bodies app ears to the cell's left of the row. About 70 dikinetids occur in each of the dorsal ciliary rows. The earliest stage shows two monokinetids adjacent to one of the parental dikinetids. Figure 26 a illustrates an example with four basal bodies in the opisthe streak. The streaks elongate (Figs. 26b-d) by kinetosome replication resulting in the interphase number of kinetosomes arranged in dikinetids by the time streaks are as large as shown in Fig. 26d. During streak development, the proter and opisthe streaks of the left-most dorsal kinety invaginate in

Ultrastructure and Morph ogenesis of Epiclintes am biguus . 189

21

Vc

24 Figs. 20-24. Cortical structure durin g interphase (Figs. 20, 21) and cell division morph ogenesis (Figs. 22- 24) of Epiclintes ambiguus. Scanning electron micrograph (Fig. 20) and a prot argol srained cell (Fig. 21) showing rhe herringbon e-like arrangement of cortica l microtubular bundles below rhe right lateral cell surface. Arrowhead s and Db = dorsal bristles, Lmb = later al microrubular bundle, Me = marginal cirrus, Rmb = right microtubular bundle, x 2650; x 850. - Figs. 22-24. Th e paroral and frontal primord ia are to the right of the oral primordium (Op); a parora! cirrus (Pc) differentiates from the par ora! primord ium. Vent ral cirri (Vc) and transverse cirri (Tc) differentiate from "within-row" streaks (arrowheads) as well as from ventral primordia (Vp), x 700, x 700, x 800.

shallow pits so as to lie internal to, and to the right of, their form er position. The left marginal primordium in the pr oter develop from 2- 3 kinetosomes that app ear to the right of the anterior parental mar ginal row near the lapel membr anelles. These kinetosomes proliferate to form a streak of kinetosomal pairs. After about 20 kinetosomes are present, th e streak is independent of its site of origin. A similar pro cess occurs in the left margin prim ordium of the opisthe near the Op and the right margin al primordia (Figs. 25 e-h). In summary, anlagen of dor sal and marginal ciliature develop clockwise from parental rows (as seen from

anterior aspect) except th e right dor sal kinety, where anlagen develop counterclock wise from the parental row . Dor sal and marginal ciliature develop synchronously with the differentiation of the vent ra l ciliature. Discussion Ult rastructure. The general organization of or al and somatic polykinetids is con sistent with the hypot rich pattern as described by Lynn [29]. Cirri of Epi clin tes resemble other hypotri chs as well as some heterotrichs [e.g. 1,15,18,26,29,33, 34,43,47]. Thi s general agreement in

190 . Wicklow and Borror

25

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the arra ngement of hypotrich cirral kinetosomes and associated micro tubular ribbons reflects an orderly and conservative develop mental process: early (primary) developmental events are more highly conserved than subsequent, higher level developmental events. Microtub ular bundles associated with the peripheral cirral mat rix differentiate late in development [26] and appear to be one of the most variable cirral constituents among hypotrichs. Th e size, arrangement, and direction of Mbs varies both betw een different kinds of cirri within a species and between cirri of different species. Stichotrich ventr al cirri usually show prominent anterior and posterior Mbs (presumably functio ning to resist mechanical stress during forwa rd and backward cell movement). An additional left tra nsverse M b occur in Epiclintes as well as Kahliella, Gastrostyla, Paraurostyla, and Stylonychia [14, 17, 26, 33]. The urostyline Pseudokeronopsis possesses only anterior and posterior Mbs [47] whereas additional right and left tra nsverse Mbs occur in the related genus Thigmokeronopsis [43]. Mbs range from one to many in cirri of euplotines such as Euplotes [37J and Certesia [45J. The highest degree of differentiation in Mbs occurs in the discocephaline Discocephalus where, in addition to serving as cytoplasmic supports to resist

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Fig. 25. Line drawings of Epiclintes am biguus depicting interphase morphology and a sequence of cortical morphogenetic stages during cell division. Based on protargol-stained specimens.

f

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26

Fig. 26. Epiclintes ambiguus dorsal aspect, showing interphase cell with two rows of dorsal dikinetids and buccal cavity (dash lines). Insets a-d represent successivestages of early morphogenesis of the median kinery in the opisthe (area enclosed in rectangle), showing cell periphery (solid line), opisthe buccal cavity (dash line), and parental and opisthe kinetosomes . Based on pro targol-stained specimens.

Ultrastructure and Morphogenesis of Epiclintes ambiguus . 191

locomotive stress, Mbs appear to contribute to cell form and integrity [44]. In Epiclintes Mbs appear to facilitate or perhaps contribute to contractility. Microtubule-microtubule sliding between the overlapping posterior Mbs of ventral cirri, anterior Mb of left marginal cirri and between the right Mb of right marginal cirri and the lateral Mbs, may provide, in part, the motive force for contraction. The structure of dorsal dikinetids in Epiclintes conforms to the general hypotrich dikinetid pattern [29]. Close comparisons with dikinetids of other hypotrichs, however, reveal both similarities and differences. The positions of postciliary microtubules associated with anterior kinetosome are highly conservative. The kinetodesmal fiber associated with the posterior kinetosome, however, varies in shape and direction and may be absent in mature dikinetids. As in Epiclintes, anteriorly directed Kd fibers occur in dikinetids of the euplotines Aspidisca, Uronychia, Euplotes, and Certesia [e.g. 15,45] and in the discocephaline Discocephalus [44]. Euplotine and discocephaline Kd fibers, however, angle sharply toward the cell surface. Kd fibers have not been observed in dikinetids of Kahliella or Parastrongylidium [14]. (Unlike most hypotrichs, both kinetosomes of the dikinetids of Parastrongylidium are ciliated). A posteriorly directed Kd fiber occurs only in immature dikinetids in the sporadotrich Oxytricha [19]. A more laterally directed Kd occurs in Paraurostyla [27]. The electron-dense, microtubule-associated material predominant on the right of the dikinetid of Epiclintes occurs similarly but predominantly left in Kahliella [14]. A fibrillar mass is found to the left of dikinetids in Oxytricha and Discocephalus but is absent in Euplotes [18, 35, 44]. The nematodesmal granules associated with dikinetids of Epiclintes are reminiscent of - but, because of location, probably not homologous to - lasiosomes within the dorsal cilia of Euplotes [38] and Certesia [45]. Thus, the organization of dikinetid ancillary structure in Epiclintes is distinct. Although numerous stichotrichines, sporadotrichines, and tintinnines possess an outer membrane, the perilemrna, external to the plasma membrane [2], the multilayered membrane system observed in Epiclintes is absent in most ciliates. Similar layers of membranous materials, however, occur in Engelmanniella [48]. As in Engelmanniella the multi-layered membrane system of Epiclintes appears to be located between the outer perilemma and the plasma membrane - perhaps an elaboration of perilemma external to the plasma membrane. Alveoli of [22, 34, 35, 45] euplotines contain multilayered alveolar plates. Bohm and Hausmann [5] determined that the alveolar plates in Euplotes consist mainly of protein with a fine coating of polysaccharide. Such plates in euplotines appear to contribute to cellrigidity, whereas the supple membranous layers in Epiclintes facilitate change in cell form in this highly contractile ciliate. Thus the cortical system of Epiclintes appears nonhomologous to the alveolar plates of euplotines. Cytochemical studies would be helpful in assessing homology between cortical membrane systems in Epiclintes and Engelmanniella. Although rare in ciliates, a tuberculated dorsal surface occurs in a wide range of taxa including Aspidisca,

Discotricha and Peritromus, in addition to Epiclintes. The collar-like papillae in Aspidisca supported by alveolar plates differ in structure from the cylindrical papillae supported by a basket-like framework in Epiclintes. Morphogenesis. Three kinds of ciliary primordia develop during morphogenesis in most hypotrichs: frontal, oral, and somatic [8]. Membranelles as well as paroral and endoral membranes develop from oral primordia; paroral cirri differentiate from anterior kinetosomes of the developing paroral membrane. Frontal ciliature develops from primordial kinetosomes adjacent to the parental paroral membrane in the proter and kinetosomes to the right of the oral primordium in the opisthe. Somatic ciliature such as dorsal kineties and marginal cirral rows differentiate from primordia within or adjacent to parental rows [8]. In general frontal ciliature is considered as an apomorphic character whereas somatic ciliature is considered as a plesiomorphic character in hypotrichs. Frontal ciliature is only minimally represented in Epiclintes. As in Kahliella [3, 12], Cladotricha [7], and Engelmanniella [48], one or two streaks of frontal cirri develop adjacent to opisthe oral primordium. Marginal cirri develop in pattern similar to that of Oxytricha and have similar Mbs. In contrast to most hypotrichs, however, Epiclintes exploits ventral primordia as a means of forming a major portion of its ventral ciliature. During early development, ventral primordia appear similar to the right ventrallateral primordia in Pseudourostyla [25]; they differ, however, in subsequent arrangement as well as in the kinds of cirral rows produced (lateral full-length, longitudinal rows without differentiation of transverse cirri in Pseudourostyla, oblique posterior median rows that differentiate transverse cirri in Epiclintes). Within-row ventral primordia produce 2 longitudinal ventral rows, each of which differentiates a transverse cirrus in Paraurostyla [46]. Longitudinal ventral rows differentiate from streaks that develop from right marginal cirri primordia in Parakahliella macrostoma [4]. Primordia of the right-most dorsal kineties arise from right marginal cirri primordia in Oxytricha, Stylonychia and Laurentiella [19, 30, 31]. Dorsal dikinetids, caudal cirri, and marginal cirri are considered somatic ciliature [8]. Morphogenesis of dorsal kineties in Epiclintes resembles that of Paraurostyla weissei [27], except for the invagination described above and lack of anlage of caudal cirri. The ventral primordia of Epiclintes can be considered as specialized somatic primordia with possible homologies with lesser developed ventral somatic primordia in other stichotrichs and sporadotrichs [30, 31]. Development of ventral cirri within parental ventral rows resembles the process shown by Foissner, Adam and Foissner [16] for what was later named Pseudokahliella marina [4]. Taxonomy and nomenclature. Carey and Tatchell [9] discussed in detail the confused nomenclature of Epiclintes. Based on their comparisons with the illustrations in Muller's 1786 publication they suggested changing the name from E. ambiguus to E. felis. We appreciate their careful comparisons, but have decided here not to regard

192 . Wicklow and Borror

[ells (a possible nomen oblitum) as a valid senior synonym. Whereas only Epiclintes ambiguus was recognized as valid in Borror's 1972 revision, Carey and Tatchell [9] also included Epiclintes caudatus Bullington, 1940 and Epiclintes radiosa Calkins 1902 as valid additional species in the genus. We now agree that Epiclintes caudatusBullington, 1940 warrants consideration as a member of the genus. In his 1940 paper, Bullington also described species of Trachelocerca, Peritromus, Gruberia, and Gastrocirrhus. Our observations of ciliates in these genera convinces us Bullington was a careful observer, and that the ciliate he named Epiclintes caudatus must have resembled E. ambiguus closely except for the apparent alignment of rows of cirri. Pending rediscovery and description of the ciliature with modern methods, however, we consider it at best a member incertae sedis. We are not including Epiclintes radiosa Calkins 1902, listed as a member of the genus by Carey and Tatchell [9]. We agree with Carey and Tatchell that Calkins' ciliate is probably best considered identical with Oxytricha retractilis Clapared & Lachmann, 1858, as well as several more recently published names including Mitra radiosa Quennerstedt 1867. In 1972, Borror included this ciliate in the Oxytrichidae based on its sporadotrich pattern of frontal ciliature. Pending publication of modern observations, we see no point in considering it as a member of the genus Epiclintes. For a review of the nomenclatural problems surrounding "Oxytricha retractilis" see Borror, 1972. Conflicting classifications of Epiclintes (Table 1) are not surprising: its general anatomy and behavior, even at the light microscope level, display bizarre characters making taxonomic judgements difficult. The specific name ambiguus seems well suited. Ultrastructural and morphogenetic data, however, provide additional means to test the phylogenetic fit of Epiclintes with other hypotrich families. For example, the present study justifies exclusion of Epiclintes from the Urosrylina: midventral cirri are absent (indeed frontal ciliature is limited to 1 or 2 rows of cirri in the opisthe). When compared with Kerona and Oxytricha, Epiclintes exhibits significant ultrastructural and developmental differences. In Kerona [23] most oblique cirral rows differentiate from frontal primordia while the remaining cirral rows arise from primordia which originate within parental rows in a similar manner as occurs in Paraurostyla weissei [46]. This information indicates that the ventral ciliature of Epiclintes and Kerona are nonhomologous. We reject the hypothesis that Epiclintes represents a heterotrich at the hypotrich grade related to Peritromus or Plagiotoma. Although it shares with Peritromus dorsal papillae and contractility and shares with Plagiotoma "within-row" development of the ventral rows of ciliature, greater ultrastructural and morphogenetic similarities to the Stichotrichia argue against this hypothesis. Furthermore, neither Peritromus nor Plagiotoma show evidence of frontal ciliature, and differ in structure of cortex and nuclei. Inclusion of E. ambiguus with contractile species in the

genus Psammomitra as some sort of divergent offshoot from Trachelostyla, is unwarranted based on their morphology and morphogenesis. What little is known of the ciliature of members of the genus Psammomitra suggests presence of five transverse cirri only, in the pattern of the Sporadotrichina. The general pattern of cortical morphogenesis of Epiclintes is different from that of the sporadotrichine Oxytricha [18].

Conclusions. Comparative analysis of ultrastructural and morphogenetic data indicate that Epiclintes is a specialized descendent from Kahliella-like stichotrichines. Also likely to have radiated from this group are Engelmanniella in soils, Psilotricha and Eschaneustyla in mosses, Stichotricha in the periphyton of both freshwater and marine habitats, and freshwater, planktonic forms such as Hypotrichidium and Pseudokahliella. Divergent features of the cortex, morphogenetic pattern, and degree of contractility, however, justify the inclusion of Epiclintes in a new stichotrichine family, the Epiclintidae. Such a placement reflects the extraordinary diversity and radiation of stichotrichine ciliates. Family Epiclintidae (n. fam.) Diagnosis. Ventral ciliature is comprised of numerous oblique rows of cirri, the majority of which differentiate during morphogenesis from parental cirral rows and ventral primordia; frontal cirral rows are minimally represented and differentiate from primordia medial to the oral primordium. A longitudinal row of transverse cirri is located medial to the left margin cirri in the posterior half of the cell. Transverse cirri differentiate from the posterior of frontal streaks, ventral (within-rows) streaks, and streaks that develop within ventral primordia. Dorsal cilia project from cylindrical papillae. A system of multiple, membrane-like material is present within the cortex. One genus; Type-Epiclintes Stein, 1862. E. ambiguus (Muller, 1789) Butschli, 1889 (type by subsequent designation). E. caudatus Bullington, 1940 (incertae sedis). Acknowledgements We gratefully acknowledge the University of New Hampshire for providing use of its electron microscope facility, the Jackson Estuarine Laboratory, and the laboratory of Dr. Charles W. Walker.

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Key words: Epiclintes - Hypotrichida - Ultrastructure - Morphogenesis - Systematics Barry J. Wicklow, Department of Biology, Saint Anselm College, Manchester, New Hampshire, 03102, USA