Cyrtohymena seorakensis, Omar & Jung, 2020

Omar, Atef & Jung, Jae-Ho, 2020, Morphology and Molecular Phylogeny of the Soil Ciliate Cyrtohymena seorakensis sp. n. (Ciliophora, Hypotricha, Oxytrichidae), Zootaxa 4758 (3), pp. 561-572 : 562-564

Cyrtohymena seorakensis sp. n.

( Figs. 1–5 ; Table 1)

urn:lsid:zoobank.org:act:0F24868A-99CC-4F0C-9F83-139BC5482E9C

Diagnosis. Size in vivo 100–150 × 25–35 μm; body slenderly ellipsoid with posterior end narrower than anterior; cytoplasm colorless, cell appear brownish under low magnification due to many crystals. Contractile vacuole at left margin of mid-body, with two inconspicuous collecting canals. Cortical granules spherical, randomly scattered, yellowish, and 0.5–0.8 μm across. Two macronuclear nodules with 2 or 3 micronuclei. Adoral zone occupies about 32% of body length, consists of an average of 28 membranelles. 17 frontal-ventral-transverse cirri of which only four transverse cirri. 13–19 right and 17–21 left marginal cirri. Six dorsal kineties of which kinety 4 commences posterior to mid-body and kineties 5 and 6 are dorsomarginal.

Zoobank registration of Cyrtohymena seorakensis sp. n.: urn:lsid:zoobank.org:act:0F24868A-99CC-4F0C-9F83-139BC5482E9C

Type locality. Terrestrial moss from Seorak Mountain, South Korea (N38° 11′ 7″, E128° 28′ 39″) .

Type material. The holotype ( NIBRPR 0000110249) and two paratype slides ( NIBRPR 0000110219 and NI- BRPR 0000110220) with protargol-impregnated specimens have been deposited in the National Institute of Biological Resources ( NIBR), South Korea. Ten other paratype slides with protargol-impregnated specimens have been kept in our laboratory.

Etymology. Named after the Mountain in which it was discovered.

Morphological description. Size in vivo 100–150 × 25–35 μm; on average 97 × 27 μm after protargol prepa- rations ( Table 1). Body flexible and slightly contractile; ellipsoid with both ends rounded, posterior end distinctly narrower than anterior, right margin usually slightly concave, left slightly convex; ventral surface flat to slightly convex, dorsal surface distinctly convex ( Figs 1A, C , 2A, B, F, G, H ). Nuclear apparatus left of midline, in middle half of cell, composed of two ellipsoid macronuclear nodules separated with 2–14 μm wide distance in protargol preparations, and 2 or 3 globular micronuclei near or attached to macronuclear nodules ( Figs 1A, E , 2F, G ). Contractile vacuole at left margin of mid-body, with two inconspicuous collecting canals ( Figs 1A , 2A ). Cortical granules yellowish, randomly scattered, and 0.5–0.8 μm across ( Figs 1C , 2C, D ). Cytoplasm colorless, contains few to many 1–3 μm across lipid droplets, up to 10 μm-sized food vacuoles with fungi, starch grains, and algae, and few to many crystals making cells appear brownish under low magnification ( Figs 1A , 2 A–D). Usually crawls slowly forward and backward on bottom of Petri dish culture, rarely swims.

Adoral zone of membranelles (AZM) occupies about 32% of body length and composed of 28 membranelles on average, cilia up to 18 μm long in vivo and about 14 μm in SEM preparations ( Figs 1A, D , 2 F–I). Undulating membranes in typical Cyrtohymena pattern; paroral membrane commences at about 7.5% of body length and about 1 or 2 μm ahead of endoral, paroral cilia about 8 μm in SEM preparations, attached to each other and covering buc- cal cavity; pharyngeal fibers extend to mid-body ( Figs 1D , 2 F–I).

Cirral pattern oxytrichid, invariably 17 frontal-ventral-transverse cirri in ~50 morphostatic specimens studied (the opisthe of a single late divider specimen observed with only 14 cirri). Three slightly enlarged frontal cirri about 15 μm in SEM preparations, right cirrus posterior to distal-most adoral membranelle, middle cirrus anterior to buc- cal cirrus, left cirrus anterior to curved paroral membrane. One buccal cirrus right to middle portion of undulating membranes. Invariably four frontoventral cirri in two oblique pairs (V-shaped); anterior pair right of the buccal cirrus and in line with right marginal row; posterior pair slightly anterior and left to right marginal row. Three postoral cirri in anterior half of cell, cirri IV/2 and V/4 close to buccal vertex, distance between V/4 and V/3 about 8 μm on average. Two obliquely arranged pretransverse cirri, with cilia about 13 μm in SEM preparations. Invariably four, slightly thickened, transverse cirri at posterior cell end about 18 μm in vivo. Right marginal cirral row composed of about 16 cirri, commences at about 22% of body length and ends at level of first pretransverse cirrus. Left marginal cirral row composed of an average of 19 cirri, commences left of posterior portion of AZM at about 27% of body length and curved posteriorly to right of cell midline at posterior cell margin ( Figs 1A, D, E , 2 F–J; Table 1).

Six dorsal kineties (DK) including two dorsomarginal kineties (DK5 and DK6) composed of a total of 62 bristles on average, kinety 1 slightly shortened anteriorly, kineties 2 and 3 bipolar, kinety 3 fragmented posteriorly, kinety 4 begins posterior to mid-body, kinety 5 ends posterior to mid-body, kinety 6 ends in anterior third of body and composed of 3–6 dikinetids. Dorsal bristles 3–4 μm long on average in vivo. Three caudal cirri slightly right of cell’s midline at end of kineties 1, 2, and 4, about 12 μm in SEM preparations ( Figs 1E , 2G ; Table 1).

Resting cyst. Cysts in vivo spherical, 18–20 μm in diameter. Cyst wall about 1 μm thick, structureless in light microscope. Macronuclear nodules fused to a central mass. Cytoplasm hyaline packed with minute lipid droplets up to 2 μm across ( Fig. 1B , 2E ).

Notes on ontogenesis. Only three cells in the middle and late stages of division were observed. The postoral cirrus V/3 was not observed during the late division stages indicating that it was involved in the primordia formation as in other Cyrtohymena species ( Singh & Kamra 2015; Voss 1991) ( Figs 3A, C, E , 4 A–C). The dorsal morphogenesis is of typical Oxytricha -pattern, that is, parental kineties develop intrakinetally to form kineties 1–3 in both the proter and opisthe. Kinety 3 fragments posteriorly and forms fourth anlage (kinety 4). The parental kineties 4–6 dedifferentiate, and the new kineties 5 and 6 develop de novo on the right side of the cells as dorsomarginal kineties. The three caudal cirri are formed at the posterior end of kineties 1, 2, and 4 of both the proter and opisthe ( Figs 3B, D, F , 4D ).

Phylogeny. The SSU rDNA sequence of Cyrtohymena seorakensis sp. n. is 1,539 base pairs long and has a GC content of 45.44% and available under GenBank accession number MT103333 . Cyrtohymena seorakensis sp. n. shows a similarity of 99.9% (one nt difference) with the Indian population of C. citrina and cluster together in the phylogenetic trees ( Fig. 5 ). This clade is a sister to a clade containing C. muscorum , which shows 99.6% similarity with C. seorakensis sp. n., and two urosomoidid species. The Namibian population of C. citrina nests at the base of the clade containing these three Cyrtohymena sequences and shows a similarity of 99.3% (10 nt difference) to C. seorakensis sp. n. The other two sequences of C. citrina are identical to each other (possibly from the same population) and almost identical (0.1% dissimilarity = 1 nt difference) to Australocirrus shii . They cluster together with three neokeronopsid species distant from other Cyrtohymena species.

Figures

FIGURE 1. Line drawings of Cyrtohymena seorakensis sp. n. from life (A–C) and after protargol impregnation (D, E). A. A representative specimen showing the body shape and the arrangement of the ventral ciliature. B. Optical section of a resting cyst showing the thin, smooth wall (opposite arrowheads), the lipid droplets in the cytoplasm, and the fused macronuclear nodules. C. Dorsal view showing the randomly arranged cortical granules. D, E. Ventral and dorsal view of the holotype specimen showing the infraciliature and the nuclear apparatus. Dotted line in (E) connects dikinetids of dorsal kinety 4. AZM—adoral zone of membranelles; BC—buccal cirrus; CC—caudal cirri; CG—cortical granules; DK1–6—dorsal kineties; EM—endoral membrane; FC—frontal cirri; LD—lipid droplets; LMR—left marginal row; MA—macronucleus; MI—micronucleus; PF—pharyngeal fibers; PM—paroral membrane; PTC—pretransverse cirri; RMR—right marginal row; TC—transverse cirri. Scale bars = 30 μm (A, D, E), 10 μm (B).

FIGURE 2. Photomicrographs of Cyrtohymena seorakensis sp. n. from life (A–E), after protargol impregnation (F, G), and in the scanning electron microscope (H–J). A, B. Ventral and lateral view of a free swimming specimen under low magnification, showing the body outline, the distinctly convex dorsal surface (arrows in B), and the brownish color due to the crystals in the cytoplasm. C, D. Dorsal views showing the yellow, randomly scattered cortical granules (arrows). E. Optical section of a cyst showing the thin wall (opposite arrowheads) and the fused macronuclear nodules. F, G. Ventral and dorsal view of the holotype specimen showing the infraciliature and nuclear apparatus. H–J. Ventral views showing the arrangement of frontal-ventraltransverse cirri (H), the shape of the paroral membrane (arrows in H and I), and the invariably four transverse cirri (arrowheads in J). AZM—adoral zone of membranelles; BC—buccal cirrus; CC—caudal cirri; CV—contractile vacuole; DK1–6—dorsal kineties; FC—frontal cirri; LD—lipid droplets; LMR—left marginal row; MA—macronucleus; MI—micronucleus; PF—pharyngeal fibers; PM—paroral membrane; PTC—pretransverse cirri; RMR—right marginal row. Scale bars: 30 μm (A–C, F–H), 20 μm (D, I, J), 10 μm (E).

FIGURE 3. Line drawings of protargol-impregnated dividing specimens of Cyrtohymena seorakensis sp. n. Parental cirri and dorsal bristles drawn as outlines, newly formed structure are in black. A, B. Ventral and dorsal view of a mid-divider specimen, showing the frontal-ventral-transverse anlagen (arrows), the undulating membranes anlagen (arrowheads), and the dorsal kineties 1–3 anlagen. Note the fusion of the macronuclear nodules. C, D. Ventral and dorsal view of late divider, showing the frontal-ventral-transverse cirri migrate to their original locations, the dorsomarginal kineties (kineties 5 and 6) anlagen originate very close to the right marginal cirral row, and the new caudal cirri. Note the opisthe has 14 frontal-ventral-transverse cirri differing from all other cells in the preparations (~50 non-dividing cells). E, F. Ventral and dorsal view of very late divider, showing the frontal-ventral-transverse cirri already in their specific locations, the fragmentation of kinety 3 producing kinety 4 (arrows) posteriorly. CC—caudal cirri; DK1–6—dorsal kineties; DM—dorsomarginal kineties; LMR—left marginal row; MA—macronucleus; MI—micronucleus; OP—oral primordium; RMR—right marginal row; TC—transverse cirri. Scale bars: 30 μm.

FIGURE 4. photomicrographs of protargol-impregnated dividing specimens of Cyrtohymena seorakensis sp. n. A. Ventral view of a mid-divider specimen, showing the frontal-ventral-transverse anlagen (arrows) and the undulating membranes anlagen (arrowheads). B. Ventral view of late divider, showing the frontal-ventral-transverse cirri migrate to their original locations. Note the opisthe is the only specimen observed with less number (only 14) of frontal-ventral-transverse cirri in all preparations. C, D. Ventral and dorsal view of very late divider, showing the frontal-ventral-transverse cirri already in their specific locations, and the newly formed caudal cirri (arrowheads) at the end of kineties 1, 2, and 4. CC—caudal cirri; DK1–6—dorsal kineties; DM—dorsomarginal kineties; LMR—left marginal anlage; MA—macronucleus; MI—micronucleus; OP—oral primordium; PTC—pretransverse cirri; RMR—right marginal anlage; TC—transverse cirri. Scale bars: 30 μm.

FIGURE 5. A maximum likelihood (ML) tree based on 18S rRNA genes, showing the phylogenetic position of Cyrtohymena seorakensis sp. n. Numbers at the nodes represent the maximum likelihood (ML) bootstrap values and the Bayesian inference (BI) posterior probabilities. Newly obtained sequence is in bold. Sequences belong to the genus Cyrtohymena are shaded in grey. GenBank accession numbers follow species names. Bootstrap values lower than 50% and posterior probabilities in BI lower than 0.5 were not shown. The scale bar represents two nucleotide substitutions per 100 nt.