A Huge Diversity of Metopids (Ciliophora, Armophorea) in Soil from the Murray River Floodplain, Australia. II. Morphology and Morphogenesis of Lepidometopus platycephalus nov. gen., nov. spec. Author Vď’Ačný, Peter Author Foissner, Wilhelm text Acta Protozoologica 2017 56 1 39 57 https://www.mendeley.com/catalogue/3f0ad1ad-b26e-3840-b42c-281b8827ae9e/ journal article 299022 10.4467/16890027AP.17.004.6968 cc6f0751-bd79-4ad9-b40e-5d4d4999c978 1689-0027 12555179 Lepidometopus platycephalus nov. spec. Foissner and Vďačný Diagnosis: Size about 45 × 20 µm in vivo . Body broadly to narrowly reniform with a somewhat rhomboid appearance when viewed ventro- or dorsolaterally. Macronucleus between anterior and posterior end of adoral zone, globular to oblong; one globular to broadly ellipsoid micronucleus. Contractile vacuole terminal. Epicortical scales about 1.25 × 0.45 µm in SEM, flat with margin curled up. On average 11 ciliary rows; caudal cilium about 20 µm long. Perizonal stripe composed of five kineties extending approximately 46% of body length and forming about 19 false kineties. Adoral zone extends about 50% of body length, composed of an average of 11 polykinetids. Type locality: Loamy soil and leaf litter from the floodplain of the Murray River near to the town of Albury, Australia ( S36°06' E146°54' ) . Type material: The holotype slide and eight paratype slides with protargol-impregnated specimens have been deposited in the Museum of Natural History (Biologiezentrum) in Linz (LI), Austria . The holotype ( Fig. 2K, L ) and relevant paratype specimens as well as dividers have been marked by black ink circles on the coverslip. Etymology: Derived from the Ancient Greek adjective platús ( πλατύς , flat) and the Ancient Greek noun képhalos ( κέφ ᾰ λΟς [m], head), referring to the strongly flattened preoral dome. The composite name is latinized and treated as a noun in the nominative singular standing in apposition to the generic name [Art. 11.9.1.2 of the International Commission on Zoological Nomenclature (1999)]. Description: Size in vivo 35–50 × 15–30 µm, usually about 45 × 20 µm, as calculated from some in vivo measurements and the morphometric data adding 15% preparation shrinkage ( Table 1 ). Body asymmetric and thus multi-shaped: broadly to narrowly reniform in ventro- and dorsolateral views ( Figs 2A, G, K–V , 3A, B , 4C, D ), broadly crescentic in lateral views ( Figs 2F, H , 4B, E , 5B ), and dumbbell-shaped in ventrocaudal views ( Figs 2I , 5C ); body ends somewhat angular providing cells with a rhomboid or triangular appearance, depending on observation perspective; distal portion of preoral dome strongly flattened and thus hyaline, only 2–3 µm thick in vivo ( Fig. 3A–C , asterisks), projects distinctly in lateral views, forming a right or nearly right angle with main body axis ( Fig. 4B, E , opposed arrowheads); postoral body portion unflattened and usually distinctly vaulted ( Figs 4A–E , 5B, C ). Localization of nuclear apparatus very stable, i.e., between anterior and posterior end of adoral zone and left of cell’s midline. Macronucleus broadly ellipsoid (52.4%), ellipsoid (23.8%), narrowly ellipsoid (9.5%) or globular (14.3%), i.e., length:width ratio 1.0–5.0:1, size about 8–20 × 4–9 µm, usually 12 × 7 µm in protargol preparations; nucleoli 0.5–1 µm across. Micronucleus usually attached to anterior portion of right margin of macronucleus; shape and size rather stable, i.e., globular to broadly ellipsoid and 2–3 µm in diameter after protargol impregnation ( Figs 2A, K, J, M, O–V , 3C, E–H ; Table 1 ). Contractile vacuole in posterior body end, globular to ellipsoid during diastole ( Figs 2A, K, O–Q, S–V , 3A, B, D ). Cortex flexible, covered by epicortical scales (lepidosomes) usually forming a 1–2 µm, rarely an up to 4 µm thick layer with slimy or fibro-granular appearance in vivo ( Fig. 3A, C, D , opposed arrowheads) and flake-like appearance in SEM ( Figs 4H , 5A ), not recognizable in protargol or silver carbonate preparations. Individual lepidosomes with irregular shape, flat with margin curled up, rather variable in size, viz., 0.50–1.65 × 0.20– 0.80 µm, on average 1.25 × 0.45 µm in SEM ( Figs 2E , 4H , 5A ). No cortical granules recognizable. Cytoplasm colourless, contains many 3–6 µm-sized food vacuoles with bacterial spores; symbiotic bacteria neither detected in vivo nor in protargol preparations ( Fig. 3A ). Creeps slowly and ungainly on microscope slides, rotates slowly about main body axis when swimming. Fig. 1. Various views of semi-schematized anterior body portion of Lepidometopus . Opposed arrowheads mark the strongly flattened distal portion of the preoral dome. AZP – adoral zone of polykinetids (black); PS – perizonal stripe (grey); SS – side stripe. Table 1. Morphometric data on Lepidometopus platycephalus nov. gen., nov. spec.

Characteristicsa	Mean	M	SD	SE	CV	Min	Max	n
Body, length	37.2	38.0	3.7	0.8	10.0	31.0	44.0	21
Body, maximum width of preoral dome	19.7	20.0	2.6	0.6	13.2	14.0	24.0	21
Body, width at cytostome	17.3	17.0	2.0	0.4	11.6	14.0	21.0	21
Body, maximum postoral width	22.0	22.0	3.5	0.8	16.0	15.0	28.0	21
Body, length:width ratio	1.8	1.7	0.3	0.1	17.7	1.4	2.8	29
Anterior body end to proximal end of PS, distance	17.1	17.0	2.5	0.5	14.4	12.0	21.0	21
Perizonal stripe, percentage of body length	46.1	47.4	5.9	1.3	12.7	34.1	58.3	21
Anterior body end to distal end of AZP, distance	6.3	6.0	1.0	0.2	15.1	5.0	8.0	21
Anterior body end to proximal end of AZP, distance	18.7	19.0	1.7	0.4	9.0	15.0	21.0	21
Adoral zone of polykinetids, percentage of body length	50.3	50.0	3.4	0.7	6.8	45.7	58.8	21
Anterior body end to distal end of PM, distance	9.9	10.0	1.6	0.4	15.8	7.0	13.0	19
Anterior body end to macronucleus, distance	6.5	5.5	2.8	0.6	42.6	3.0	15.0	21
Macronucleus, length	12.4	12.0	2.6	0.6	21.4	8.5	20.0	21
Macronucleus, width	7.1	7.0	1.4	0.3	20.1	4.0	9.0	21
Macronucleus, length:width ratio	1.9	1.6	0.9	0.2	48.1	1.0	5.0	21
Macronucleus, number	1.0	1.0	0.0	0.0	0.0	1.0	1.0	21
Micronucleus, largest diameter	2.5	2.5	–	–	–	2.0	3.0	21
Micronucleus, number	1.0	1.0	0.0	0.0	0.0	1.0	1.0	21
Somatic ciliary rows, total number	11.1	11.0	1.1	0.2	10.1	9.0	13.0	23
Perizonal ciliary rows, number	5.0	5.0	0.0	0.0	0.0	5.0	5.0	21
False kineties in perizonal stripe, number	18.7	19.0	2.5	0.5	13.1	15.0	23.0	21
Adoral polykinetids, number	11.0	11.0	0.9	0.2	7.8	10.0	12.0	23
Paroral membrane, length	10.3	10.0	1.1	0.2	10.2	9.0	13.0	19

a Data based on mounted, protargol-impregnated, and randomly selected specimens from a non-flooded Petri dish culture. Measurements in µm. AZP – adoral zone of polykinetids; CV – coefficient of variation (%); M – median; Max – maximum; Mean – arithmetic mean; Min – minimum; PM – paroral membrane; PS – perizonal stripe; n – number of individuals investigated; SD – standard deviation; SE – standard error of arithmetic mean. Fig. 2. A–V. Lepidometopus platycephalus from life (A, D, F–I), after silver carbonate (B, C) and protargol (J–V) impregnation, and in the SEM (E). A. Ventrolateral view of a representative specimen, length 45 µm. Arrows denote left side cilia; arrowhead marks the distally tapered caudal cilium. B. The perizonal stripe is composed of five rows: the first three rows are arranged more closely than the two last rows whose dikinetids are slightly shifted, providing the stripe with a staggered appearance. C. The oral ciliature consists of an average of 11 adoral polykinetids and a paroral membrane optically intersecting the adoral zone. D. Optical section showing the epicortical layer. E. Epicortical scales have various shapes and are about 1.25 × 0.45 µm in size. F–I. Body perspectives. J, O–V. Variability of body shape and size as well as of nuclear apparatus. Dashed line delimits the strongly flattened anterior body portion. Drawn to scale. K–N. Dorso- and ventrolateral views of ciliary pattern and nuclear apparatus of the holotype (K, L) and of a paratype (M, N) specimen. AZP – adoral zone of polykinetids; CP – cytopharynx; CV – contractile vacuole; LE – lepidosomes; MA – macronucleus; MI – micronucleus; PD – preoral dome; PM – paroral membrane; PS (1–5) – perizonal stripe (rows); SC – somatic cilium; SK – somatic kineties. Scale bars: 20 µm. Fig. 3. A–H. Lepidometopus platycephalus from life (A–D) and after silver carbonate impregnation (E–H). Asterisks mark the strongly flattened preoral dome (A–C, E). Opposed arrowheads denote cortex and epicortical scale layer (A, C, D). A, B, E. Ventro- (A, B) and dorsolateral (E) overviews, showing general body organization. C. Detail of anterior body portion. D. Detail of posterior body end, showing the single elongated caudal cilium. F. Lateral view, showing somatic ciliary pattern and nuclear apparatus. G, H. Dorsolateral views of anterior body portion, showing the oral ciliary pattern, the perizonal stripe, and the nuclear apparatus. AZP – adoral zone of polykinetids; CC – caudal cilium; CV – contractile vacuole; MA – macronucleus; MI – micronucleus; PM – paroral membrane; PS – perizonal stripe; SK – somatic kineties; SC – somatic cilia. Scale bars: 10 µm (C, G, H) and 20 µm (A, B, D–F). Fig. 4. A–J. Lepidometopus platycephalus in the SEM. A–E. Ventral (A), right side (B), dorsolateral (C, D), and left side (E) overview, showing general body organization. Opposed arrowheads mark the strongly flattened distal portion of the preoral dome (B, E); arrows denote left side cilia (B–D). F. Ventrolateral view, showing the paroral membrane and the perizonal stripe. G. Dorsolateral view, showing five perizonal rows. H . Epicortical scales. I, J. Only a single basal body is ciliated in the postoral dikinetids (I) except for the left side kineties where both basal bodies are ciliated (J). (1–5) – perizonal rows; AZP – adoral zone of polykinetids; PD – preoral dome; PM – paroral membrane; PS – perizonal stripe; SC – somatic cilia. Scale bars: 1 µm (H), 2 µm (G), 3 µm (F), 5 µm (I, J), and 20 µm (A–E). Fig. 5. A–E. Lepidometopus platycephalus in the SEM. A. Epicortical scales (lepidosomes). B. Left side overview, showing the strongly flattened preoral dome (asterisk). C. Oblique posterior polar view, showing the globular postoral portion roofed by the cap-shaped preoral dome. D. Detail of oral area, showing the tongue-like paroral membrane and the adoral zone of polykinetids whose cilia spread backwards. The dome lip is very narrow while the side stripe forms a rather deep channel covered with epicortical scales. E. Ventrolateral view of oral body portion. The arrowhead marks entrance to buccal cavity. This cell lost lepidosomes during the preparation process. AC – adoral cilia; DL – dome lip; PD – preoral dome; PM – paroral membrane; PS – perizonal stripe; SC – somatic cilia; SS – side stripe. Scale bars: 1 µm (A), 5 µm (D, E), and 20 µm (B, C). Somatic ciliature composed of dikinetids, anterior cilium lacking in postoral kinetids ( Fig. 4I ) except for those extending along left body margin, an unusual feature observed in vivo , after protargol impregnation, and confirmed in SEM ( Figs 2A , 3C , 4B–D , arrows, J). Somatic cilia comparatively widely spaced, rather rigid, in vivo 10–12 µm long in mid-body, up to 13 µm on rear body end; a single elongated caudal cilium with filiform distal end, 16–30 µm long, usually about 20 µm long in vivo , fragile and thus usually missing in prepared specimens ( Figs 2A , 3A, D ). On average 11 equidistant and ordinarily spaced ciliary rows, i.e., interkinetal distance about 5–6 µm in protargol preparations, follow body curvature ( Figs 2K–N , 3E, F ; Table 1 ). Perizonal stripe begins at left anterior body margin, extends along whole anterior body end, curves perpendicularly to right anterior margin, and terminates on right margin of dorsal side at or slightly anterior to level of proximal end of adoral zone, i.e., forms a Γ-shaped pattern; extends 46% of body length on average; invariably composed of five rows: first three rows more narrowly spaced than the two last rows with dikinetids slightly shifted, providing stripe with a staggered appearance. Stripe rows segmented into an average of 19 false kineties, each perizonal dikinetid with two cilia 15–18 µm long in vivo and 8–12 µm in SEM; number of perizonal rows often difficult to determine in distal portion of stripe due to their narrow spacing and strong flattening of anterior body portion, but ontogenetic data indicate that there are five rows beginning at almost same level ( Figs 2B, K–N , 3E–H , 4G ; Table 1 ). Type 4 oral area. Adoral zone extends vertically to strongly obliquely and about half of body length, roofed by preoral dome, composed of an average of 11 polykinetids up to 6 µm wide; cilia 5 µm long in vivo , usually spread backwards in SEM ( Fig. 5D ); proximal- and distalmost polykinetids rectangular or somewhat irregular and composed of two to three rows of basal bodies, others L-shaped and usually composed of a short row and three long rows ( Fig. 2C ). Paroral membrane begins about 17 µm posterior to anterior body end, extends along right margin of side stripe, optically intersects adoral zone; composed of narrowly spaced, oblique ellipsoids being dikinetids according to the ontogenetic data, only one basal body ciliated according to SEM observations, cilia 3–6 µm long in SEM, form a tongue-like or fimbriate structure ( Figs 2C, L, N , 3G, H , 4F , 5D, E ; Table 1 ). Cytopharyngeal fibres originate from proximal end of adoral zone and paroral membrane, extend backwards forming a funnel about 15 µm long in protargol preparations ( Fig. 2L, N ). Dome lip inconspicuous because only 0.25–0.50 µm wide in SEM. Side stripe a comparatively deep, 2.3–3.3 µm wide channel in the scanning electron microscope, covered by epicortical scales ( Fig. 5D, E ). Morphogenesis of Lepidometopus platycephalus Division mode: Binary fission is homothetogenic and occurs in freely motile (non-encysted) condition. Body shape changes drastically. Stomatogenesis is pleurotelokinetal. The parental oral structures are reorganized but are not involved in the formation of the daughter oral ciliature. Body changes: Early dividers are 48–55 × 23– 25 µm in size, i.e., they are slightly larger than morphostatic specimens and gradually loose the reniform and rhomboid appearance, becoming Metopus -shaped ( Fig. 6A–H ). The cell portion carrying the prospective adoral polykinetids transforms into a small bulge recognizable in lateral view ( Fig. 6E, F ). On the other hand, the parental oral area is still unchanged, i.e., the preoral dome is strongly flattened and distinctly projects from body proper ( Fig. 6C ). In mid-dividers, the body slightly shortens and conspicuously broadens to 45–50 × 30–38 µm. These cells are thus the stoutest and shortest dividers. Body shape drastically changes: (1) the outline becomes broadly elliptic, (2) the ventral side becomes strongly inflated, and (3) the preoral dome turns into a small, rounded protuberance projecting from the left anterior body margin ( Fig. 7A–F ). Just before separation, the daughter cells are broadly ellipsoid without any sign of a preoral dome ( Fig. 8A, B ). However, fundamental changes in cell shape and size take place after division: (1) the body intensively grows from about 33 × 23 µm to about 55 × 22 µm, (2) the anterior portion of the cell flattens, and (3) the preoral dome pulls out of the cell to roof the adoral zone by twisting leftwards taking along the perizonal stripe which thus obtains the typical Γ-shaped pattern ( Fig. 8C–H ). Nevertheless, late post-dividers are still very dissimilar from morphostatic cells. They are oblong, not or only slightly twisted anteriorly ( Fig. 8G, H ), and longer than morphostatic specimens (on average 55 × 22 µm vs. 38 × 20 µm in protargol preparations). This indicates that their further development must be associated with body shortening, possibly as a consequence of massive remodelling to a reniform/rhomboid shape. Fig. 6. A–J. Lepidometopus platycephalus , ciliary pattern and nuclear apparatus of early dividers (A–H) and of an early mid-divider (I, J) after protargol impregnation. Arrowheads mark the prospective adoral polykinetids formed at the posterior end of dorsal and dorsolateral kineties. Asterisks denote the prospective adoral polykinetids developing at the anterior end of the postoral kineties. Arrow in (I) points to two dorsolateral kineties which migrate towards the growing perizonal stripe to become perizonal rows 4’ and 5’ in the opisthe. BU – bulge; CV – contractile vacuole; MA – macronucleus; MI – dividing micronucleus; OAZP – opisthe’s adoral zone of polykinetids; PAZP – proter’s adoral zone of polykinetids; PM – paroral membrane; PPS – proter’s perizonal stripe. Scale bars: 20 µm. Fig. 7. A–F. Lepidometopus platycephalus , ciliary pattern and nuclear apparatus of mid-dividers after protargol impregnation. Asterisks denote scattered dikinetids of perizonal rows 1 and 2 that migrate along the new adoral zone to assemble the opisthe’s paroral membrane. Arrows point to two dorsolateral kineties which migrate towards the opisthe’s perizonal stripe to become rows 4’ and 5’. Arrowheads mark newly formed ciliary rows left of opisthe’s adoral zone. CH – chromosomes; F – fibres; MA – macronucleus; MI – micronucleus; OAZP – opisthe’s adoral zone; OPM – opisthe’s paroral membrane; PAZP – proter’s adoral zone; PD – preoral dome; PPM – proter’s paroral membrane; PS – perizonal stripe rows. Scale bars: 20 µm. Fig. 8. A–H. Lepidometopus platycephalus , ciliary pattern and nuclear apparatus of a late divider (A, B), early post-dividers (C–F), and a late post-divider (G, H) in protargol preparations. Arrowheads in (A, B) mark barren area that forms at the posterior end of the proter and at the anterior end of the opisthe after the parental somatic ciliary rows split in the middle. Dashed lines in (D, F) delimit the flattened anterior body portion, i.e., the preoral dome. AZP – adoral zone of polykinetids; CP – cytopharynx; CV – contractile vacuole; MA – macronucleus; MI – micronucleus; OAZP – opisthe’s adoral zone of polykinetids; OPM – opisthe’s paroral membrane; OPS – opisthe’s perizonal stripe; PAZP – proter’s adoral zone of polykinetids; PD – preoral dome; PM – paroral membrane; PPM – proter’s paroral membrane; PPS – proter’s perizonal stripe; PS – perizonal stripe; SK – somatic kineties. Scale bars: 20 µm. Development of adoral zone: The formation of the opisthe’s adoral zone is associated with two concomitantly proceeding events taking place in early dividers: (1) proliferation of dikinetids (protopolykinetids) in the posterior portion of about six dorsal and dorsolateral kineties ( Fig. 6A–G , arrowheads) and (2) the production of dikinetids in the anterior portion of about three postoral kineties, i.e., slightly posterior to the proximal end of the perizonal stripe ( Fig. 6A, C, E, G , asterisks). In early mid-dividers, the newly formed dikinetids detach from the somatic ciliary rows and migrate to assemble the opisthe’s adoral zone ( Fig. 6I ). The new adoral polykinetids are thus migrating kinetofragments composed of two long rows of basal bodies. The third long row and the short row making the polykinetids L-shaped are mostly added in post-dividers ( Fig. 8C, G ). Reorganization of the parental adoral zone begins in early mid-dividers. The polykinetids of the proter become smaller and loose the interphase L-shaped pattern, very likely due to the resorption of one long row and the short row of basal bodies ( Fig. 6I ). Taking into account that the adoral zone of proter and opisthe has a similar morphology in mid-dividers and late dividers ( Figs 7A, C, E , 8A ), the proter’s adoral zone must obtain the species-specific pattern also post-divisionally. Development of perizonal stripe and paroral membrane: During the formation of the new adoral polykinetids, an intrakinetal proliferation of kinetids commences in the parental perizonal rows which thus elongate posteriorly into the glabrous area between the opisthe’s adoral zone and the posterior end of the split parental somatic kineties ( Fig. 6E, G, I ). In mid-dividers, the posterior portion of perizonal rows 1 and 2 disorders and the resulting dikinetids migrate along the new adoral zone to assemble the opisthe’s paroral membrane ( Fig. 7A, C, E , asterisks); perizonal rows 3–5 remain ordered and their posterior portion becomes perizonal rows 1’–3’ in the opisthe. Opisthe’s perizonal rows 4’ and 5’ are formed from the posterior half of the first two dorsolateral kineties which migrate towards perizonal rows 1’–3’ in very late mid-dividers and late dividers ( Figs 6I , 7A, C, E , arrows). The loss of these two dorsolateral kineties is compensated by the formation of one or two ciliary rows left of the opisthe’s adoral zone ( Fig. 7A, C, E , arrowheads). In connection with body re-shaping, the parental perizonal stripe looses the Γ-shape and moves from dorsolateral to the right margin of the ventral side; the species-specific pattern is obtained post-divisionally. The parental paroral membrane is entirely disordered in mid-dividers ( Fig. 7A, C, E ) and re-assembled in late dividers ( Fig. 8A ). Development of somatic ciliature: After formation of the prospective adoral polykinetids, an intense proliferation of basal bodies begins in all somatic ciliary rows. First, the basal bodies of the individual dikinetids go apart more or less distinctly. Then a new basal body develops in front of the anterior basal body generating a triad. Subsequently, a fourth basal body forms ahead of the posterior basal body producing a tetrad. Eventually, the tetrad splits into two pairs of dikinetids. The replication of dikinetids finishes in mid-dividers, i.e., when all somatic ciliary rows are arranged meridionally and composed of comparatively narrowly spaced dikinetids ( Fig. 7C–F ). In late dividers, the somatic ciliary rows split in the middle leaving a barren area at the posterior region of the proter and at the anterior region of the opisthe ( Fig. 8A, B , arrowheads). The somatic dikinetids are still narrowly arranged in early post-dividers ( Fig. 8C, D ). Their loose spacing is obtained gradually by growth and patterning of the body ( Fig. 8G, H ). Nuclear division: In early dividers, the macronucleus is as in morphostatic cells, i.e., it is oblong and situated between the anterior and posterior end of the adoral zone ( Fig. 6B, D, F, H ). Later on, the macronucleus becomes larger, rounds up, and migrates to mid-body ( Fig. 6J ). In mid-dividers, the centrally located macronucleus becomes broadly fusiform and begins to divide ( Fig. 7B, D, F ). In late dividers, the macronucleus is dumbbell-shaped with a conspicuous constriction in the fission area ( Fig. 8 B ). After binary fission, the macronucleus is lenticular and pointed ( Fig. 8D ). During post-divisional growth, the macronucleus becomes oblong and moves to the species-specific interphase position ( Fig. 8F, H ). When division commences, the micronucleus increases in size from 2.5 µm to 4.0 µm, showing fibrous structures, possibly prophasic chromosomes ( Fig. 6B ). The micronucleus further enlarges to about 6 µm and impregnates heterogeneously, which is indicative of the formation of spindle microtubules ( Fig. 6D, H, J ). Thus, the division spindle and the metaphase plate made of about eight chromosomes, become distinct already in early dividers ( Fig. 6F ). When the macronucleus becomes globular, the micronucleus begins to divide ( Fig. 7B ). The daughter micronuclei impregnate homogenously and are connected by a fiber bundle that conspicuously elongates in mid-dividers ( Fig. 7D, F ). The bundle disappears and the micronucleus achieves the species-specific size already in late dividers ( Fig. 8B ). During post-divisional patterning, the micronucleus moves to the macronucleus ( Fig. 8D, F, H ).