Spathidium bromeliophilum, Foissner & Wolf & Kumar & Quintela-Alonso, 2014

Foissner, Wilhelm, Wolf, Klaus W., Kumar, Santosh & Quintela-Alonso, Kuidong Xu and Pablo, 2014, Five New Spathidiids (Ciliophora: Bromeliads Haptoria) from Caribbean Tank, Acta Protozoologica 53 (2), pp. 159-194 : 171-180

Spathidium bromeliophilum nov. spec. ( Figs 7a–r ,

8a–g , 9a–h , 10a–e , 11a–j , 12a–f , 13a–g , 14a–c , 15a–f ; Tables 4, 5)

Diagnosis: Size about 135 × 35 μm in vivo. Narrowly spatulate with oblique, slightly dumbbell-shaped to cuneate oral bulge about as long as widest trunk region and screwed like a propeller blade (∞-shaped); ventral portion of bulge and circumoral kinety more or less bent laterally in 80% of specimens. On average 30 ellipsoidal, scattered macronuclear nodules and several globular micronuclei. Extrusomes bluntly fusiform and asymmetrical, about 5 µm long. Two size-types of cortical granules. On average 17 ciliary rows, 3 anteriorly differentiated to an isostichad dorsal brush occupying 22% of body length: shortest row 1 composed of 17 dikinetids, rows 2 and 3 of similar length but composed of 23 and 17 dikinetids on average, respectively; monokinetidal tail of brush row 3 extends to mid-body. Type III resting cyst.

Type locality: In tank bromeliads from the “Upper Cedar Valley”, southern slope of the Blue Mountains, Jamaica, 18º2′N 76º34′W .

Type material: 1 holotype and 5 paratype slides with protargol-impregnated specimens have been deposited in the Biologiezentrum of the Oberösterreichische Landesmuseum in Linz ( LI), Austria. Relevant specimens have been marked by black ink circles on the coverslip .

Etymology: Composite of Bromeliaceae , the plant family in whose leaf-tanks it occurs, and philum, from the Greek adjective philos (loving).

Description: Size and length:width ratio highly variable in vivo and in protargol preparations, depending on culture age and conditions: 90–180 × 20–50 μm in vivo, usually about 135 × 35 μm (n = 7); length:width ratio 2.4–6.8:1, frequently 4:1; acontractile but very flexible ( Table 4). Shape narrowly spatulate, mid-body circular in cross-section, hyaline anterior region laterally flattened and more or less set off from trunk by the slightly narrowed neck. Anterior end (oral bulge) ordinarily oblique, posterior portion slightly tapering and evenly rounded ( Figs 7a, f–j, m, n , 8a–c , 9a ). On average 30 macronuclear nodules scattered throughout cytoplasm; individual nodules globular to ellipsoidal, about 6 × 4 μm in size in protargol preparations. Micronuclei 1.5–2 μm across, scattered between macronuclear nodules, exact number difficult to determine due to similar-sized and impregnated lipid droplets ( Figs 7a, n , 8a–c , 15f ; Table 4). Contractile vacuole in posterior body end, with several excretory pores in pole area. Extrusomes bluntly fusiform and asymmetrical, about 5 × 0.5–0.6 μm long in vivo, studded in oral bulge and scattered in cytoplasm; anterior third usually lightly impregnated with the protargol method used ( Figs 7a, d , 9c, g , 13b, e ). Cortex very flexible, contains about 10 rows of granules in two size-types (0.2 and 0.4 μm) between each two kineties ( Figs 7e , 9h ). Cytoplasm colourless, more or less packed with lipid droplets up to 10 μm across and food vacuoles with granular contents, except for flattened and hyaline neck region ( Fig. 7a ). Feeds on medium-sized ciliates ( Colpoda spp. , Vorticella sp. ), flagellates, and naked amoebae, the latter be- ing engulfed within about 30 seconds, whereby the oral bulge centre opens and the prey glides into a bursiform sac soon becoming a globular food vacuole ( Figs 7k, l ). Wriggles in slimy bacterial and protozoan masses, showing great flexibility.

Cilia about 8 µm long in vivo, ordinarily spaced (~ 2.5 µm), arranged in an average of 17 ordinarily spaced, equidistant rows (~ 5.7 µm in protargol preparations); those of right side attached to circumoral kinety in acute angles and with 2–7 closely spaced cilia in anterior region, those of left side abutting at nearly right angles ( Figs 7a, m, n , 8a–c, e–g , 9a, b , 13a, b, e ). Dorsal brush three-rowed, rarely a fourth row occurs, comparatively short because occupying on average only 22% of body length; isostichad, i.e., all rows of similar length; inconspicuous because bristles only up to 2.5 µm long and as thin as ordinary somatic cilia; all rows with an anterior tail of 4–8 densely spaced monokinetids; tail rarely lacking. Shortest brush row 1 about 22 µm long and composed of 17 dikinetids, rows 2 and 3 of very similar length (27 µm vs. 26 µm on average) but different in number and spacing of dikinetids, i.e., 23 ordinarily spaced (0.7–1.5 µm) and 17 widely spaced (≥ 1.5 µm) dikinetids, respectively; row 3 continues to mid-body with a monokinetidal tail of 2–2.5 μm long bristles ( Figs 7a, m, q , 8b–d, f, g ; Table 4).

Oral bulge slightly longer than body width on average, inclined about 45º to ventral side, flat to indistinctly convex with dorsal end slightly higher than ventral, slightly cuneate to dumbbell-shaped with bluntly point- ed ventral end; ventral half more or less curved laterally in 80% of specimens, straight in the rest; bright because packed with extrusomes; obliquely striated due to microtubule bundles originating from circumoral kinety; without temporary cytostome ( Figs 7a–c, f–j, r , 8a, b, e, f , 9a, b , 13a, b, e ; Table 4). Bulge base surrounded by an ∞-shaped circumoral kinety composed of closely spaced dikinetids giving rise to nematodesma bundles forming an ordinary oral basket ( Figs 7n, r , 8a–c, e–g , 9b , 13a, b, e ).

Starvation induces formation of type III resting cysts ( Foissner and Xu 2007). Mature cysts on average 42 μm across in vivo ( Table 4). Cyst wall yellowish, smooth, about 1 μm thick, separated from cytoplasm by a ~ 0.5 μm thick, hyaline sheet (endocyst?) not recognizable in squashed cysts ( Figs 7o , 9e, f ). Cyst plasm packed with four large (postconjugants?) or many smaller macronuclear nodules about 6 × 4 μm in size, granules ≤ 1 μm across, and lipid droplets 1–2 μm in size ( Fig. 7p ). Contractile vacuole still visible after one week, be- comes active when cyst is slightly pressed by coverslip. Several degenerated cysts with clumped cytoplasm and contractile vacuole observed in two encystment trials ( Fig. 9d ).

Ontogenesis ( Figs 8c , 10a–e , 11a–j , 12a–f , 13c, f, g , 14a–c , 15a–f ; Table 5)

Essentially identical to that of Spathidium turgitorum described in detail by Foissner et al. (2002), i.e., it is homothetogenic, holotelokinetal, and occurs in nonencysted (freely motile) condition. Thus, we refer the reader to Table 5 and the many figures. However, one process should be highlighted, viz., the origin of the macronuclear nodules, which develop in post-dividers via a three-dimensional netting of the long macronuclear strand present in the dividers ( Figs 11a–j , 15e, f ).

Occurrence and ecology: As yet found only at type locality. A semipure culture could be established .

Remarks: Two species can be confused with S. bromeliophilum . Spathidium anguilla , as redescribed by Foissner (1984), is much more slender than S. bromeliophilum (~ 8:1 vs. ~ 4:1) and has only 11 (vs. 17) ciliary rows on average. The second species, Arcuospathidium multinucleatum (reviewed in Foissner and Xu 2007), has a more cuneate and longer oral bulge (ratio of oral bulge length:body width 1.5–2.4:1 vs. 1.1:1), a temporary cytostome (lacking in S. bromeliophilum ), and an Arcuospathidium (vs. Spathidium ) ciliary pattern. The last character is not very distinct but sustained by three ontogenetic features ( Table 5): the body is distinctly inflated (vs. not inflated in Arcuospathidium ) in fission area of mid-dividers, the oral kinetofragments are separate (vs. aligned) in very late dividers, and the circumoral kinety develops in the simple (vs. complex) manner. There are four other multinucleate species that are similar to S. bromeliophilum and which have been compared by Foissner et al. (2008).

Main differences between S. bromeliophilum and S. alqasabi (averages, protargol): body length (125 µm vs. 171 µm), ratio body length:length of oral bulge (3.8 vs. 5.7), length of dorsal brush row 2 (27 µm vs. 42 µm), number of dikinetids in dorsal brush row 2 (23 vs. 32), temporary cytostome (absent vs. present).

Main differences between S. bromeliophilum and S. fraterculum (averages, protargol): length of dorsal brush kinety 1 (22 µm vs. 34 µm), length of dorsal brush kinety 2 (27 µm vs. 38 µm), length of dorsal brush kinety 3 (26 µm vs. 36 µm), number of dikinetids in brush row 1 (17 vs. 27), in row 2 (23 vs. 40), and in row 3 (17 vs. 27), number of macronuclear nodules (30 vs. 67), number of ciliary rows (17 vs. 28).

Main differences between S. bromeliophilum and S. foissneri (averages, protargol): dorsal brush isostichad vs. heterostichad, length of brush row 1 (22 µm vs. 32 µm), of row 2 (27 µm vs. 37 µm), and row 3 (26 µm vs. 11 µm), dikinetids in brush row 1 (17 vs. 30), in row 2 (23 vs. 35), and in row 3 (17 vs. 10), number of ciliary rows (17 vs. 23).

Main differences between S. bromeliophilum and S. seppelti (averages, protargol): dorsal brush isostichad vs. heterostichad, number of dikinetids in brush row 3 (17 vs. 8), length of brush row 3 (26 µm vs. 10 µm), number of macronuclear nodules (30 vs.> 100), temporary cytostome (absent vs. present).

Figures

Figs 7a–r. Spathidium bromeliophilum from life (a–e, o–p) and after protargol impregnation (f–n, q, r). a – right side view of a representative specimen, length 135 μm. Arrowhead marks end of bristle tail of brush row 3; b, c – the ∞-shaped oral bulge, which is slightly dumbbell-shaped and/or cuneate, is studded with extrusomes. The oblique fibre bundles originate from the circumoral dikinetids; d – two views of the same extrusome, length 5 µm; e – surface view, showing the two size-types of cortical granules, diameter 0.2 µm and 0.4 µm; f–j – variability of shape of body and oral bulge whose ventral third is often curved laterally; k, l – a specimen engulfing a Colpoda (k) and another that has just engulfed a Vorticella; m, n – ciliary pattern of right and left side and nuclear apparatus of holotype specimen, length 138 μm; o, p – detail and overview of a resting cyst with inactive contractile vacuole, diameter 42 µm; q, r – ciliary pattern of anterior dorsal and ventral side, showing the isostichad dorsal brush and the wide spacing of the dikinetids in row 3. B(1–3) – dorsal brush (rows), BA – oral basket, BU – oral bulge, CK – circumoral kinety, CV – contractile vacuole, E – extrusomes, EP – excretory pores, FV – food vacuole, LD – lipid droplets, MA – macronuclear nodules, MI – micronuclei. Scale bars: 40 μm (a, f–n), 20 μm (q–r) and 2 μm (o).

Figs 8a–g. Spathidium bromeliophilum after protargol impregnation. a, b – right and left side view of the same specimen; c – left side view of a specimen with many small macronuclear nodules; d – anterior dorsal view of the proter from a late divider, showing the isostichad dorsal brush; e–g – anterior right (e) and left (f, g) side views of the specimens shown in Figs (a–c). Arrowheads (f, g) mark nematodesma bundles forming the oral basket. B(1–3) – dorsal brush (rows), BU – oral bulge, CK – circumoral kinety, F – oral bulge fibres, MA – mac- ronucleus, MI – micronuclei. (Without scale bars because from ± squashed specimens.)

Figs 9a–h. Spathidium bromeliophilum in the SEM (a, b) and from life (c–h). a – a slender specimen; b – ventrolateral view, showing the cuneate oral bulge (arrowheads) and circumoral cilia (arrow); c, g – the extrusomes are about 5 μm long and are slightly asymmetrical (c); d–f – type III resting cysts, about 40 μm across. Mature cysts (e) show a thin (~ 1 μm) and smooth wall (opposed arrowheads), separated from the cytoplasm by a ~ 0.5 μm thick hyaline sheet (endocyst?) not recognizable in squashed cysts (f). When degenerated, the cytoplasm detaches from the wall (d); h – surface view showing the two-size types of cortical granules, diameter 0.2 μm and 0.4 μm (arrows and ar- rowheads). BU – oral bulge. Scale bars: 40 μm (a), 25 μm (d, e), 10 μm (b) and 5 μm (f, g).

Figs 10a–e. Spathidium bromeliophilum, ontogenesis of ciliary pattern after protargol impregnation. Corresponding body and nuclear changes are shown in Figs 11a–j. a – very early divider, showing basal body production in the prospective division zone. Asterisk denotes a slight indentation in fission area. Nuclear apparatus as shown in Figs 11a, b; b – early divider where the developing dorsal brush rows (1–3) and the circumoral kinety fragments (placed between arrowheads) of the opisthe are already recognizable. Note blebs in fission area left of the circumoral kinety fragments (arrowheads). Nuclear apparatus as shown in Fig. 11c; c – middle stage, showing blebs (arrowheads) in prospective fission area. Nuclear apparatus as shown in Figs 11d, e; d – late divider with a single macronuclear strand (Fig. 11f) and conspicuous division furrow (arrows); e – very late divider with proter and opisthe about to separate, length 170 μm (Fig. 11g). B(1–3) – dorsal brush rows, BU – oral bulge, CK – circumoral kinety, MA – macronucleus, MI – micronuclei.Scale bars: 20 μm (a–d) and 30 μm (e).

Figs 11a–j. Spathidium bromeliophilum, body and nuclear changes in dividers (a–g) and post-dividers (h–j) after protargol impregnation; drawn to scale. For the ciliary pattern, see Figs 10a–e, 12a–f, 15a–d. a – a morphostatic specimen with many macronuclear nodules scattered throughout the cytoplasm; b – early divider with most macronuclear nodules fused; c, d – middle dividers in which the macronuclear nodules fused to an irregular mass. Note micronuclear division and the slightly inflated fission area; e, f – middle-late and late stage, showing elongation of body and macronucleus, and division furrow. The opisthe is considerably narrower than the proter (f), a rare feature (Table 5); g – very late divider, showing proter and opisthe about to separate while the macronucleus has already divided; h–j – post-dividers develop a three-dimensional macronuclear reticulum (h, i) that breaks into many nodules during the maturation of the cell (j, a). BU – oral bulge, CV – contractile vacuole, MA – macronuclear nodules, MI – micronuclei. Scale bar: 40 μm.

Figs 12a–f. Spathidium bromeliophilum, dividers after protargol impregnation. a–c – overview and details of an early divider. Note body indentation (asterisks), basal bodies production in the division zone of all kineties (arrowhead), and beginning fusion of macronuclear nodules (arrow in c); d–f – a middle divider, showing an inflated fission area (small arrowheads), the developing dorsal brush (1–3), the oral kinetofragments (large arrowheads), and the macronuclear strand that developed by fusion of the macronuclear nodules. B(1–3) – dorsal brush rows, MA – macronucleus, MI – micronuclei. (Without scale bars because from ± squashed specimens.)

Figs 13a–g. Spathidium bromeliophilum after protargol impregnation. a, b, e – anterior body portion of three specimens, showing the oral bulge whose ventral third is curved laterally in 80% of the specimens (b) and straight in the rest (a, e). Note the oblique microtubule bundles originating from the dikinetids of the circumoral kinety (a) and forming the oral basket (e, arrowheads); c – view of the inflated fission area of a middle divider, showing micronuclear fission and the macronuclear strand that developed by fusion of the macronuclear nodules; d – post-conjugant, showing four macronuclear nodules and the slightly convex oral bulge whose dorsal end is higher than the ventral end; f, g – an early divider, showing a slight body indentation (asterisk) and basal body production in the prospective division zone of most kineties. B – dorsal brush, BA – oral basket, BU – oral bulge, CK – circumoral kinety, E – extrusomes, F – oral bulge fibres (microtubule bundles), MA – macronucleus, MI – micronuclei. (Without scale bars because from ± squashed specimens.)

Figs 14a–c. Spathidium bromeliophilum, late divider after protargol impregnation. a–c – overview and details of the fission area, showing the ventral (b) and dorsal (c) side. Arrowheads (b) mark the growing nematodesma bundles (oral basket fibres) originating from the diki- netids of the circumoral kinety fragments. Arrow (c) denotes a bleb left of the circumoral kinety fragments of the opisthe. 1, 2, 3 – dorsal brush rows, BU – oral bulge, CK – circumoral kinety, MA – macronucleus, MI – micronuclei. (Without scale bars because from ± squashed specimens.)

Figs 15a–f. Spathidium bromeliophilum after protargol impregnation. a–d – overview and details of a very late divider, showing the elliptical oral field of the opisthe whose circumoral kinety fragments have not yet aligned ventrally; e, f – post-dividers, showing the threedimensional macronuclear reticulum (e) that breaks into many nodules (f). B(1–3) – dorsal brush (rows), BA – oral basket, BU – oral bulge, CK – circumoral kinety,F – oral bulge fibres, MA – macronucleus,MI – micronuclei.(Without scale bars because from ± squashed specimens.)