Rigidohymena inquieta (Stokes, 1887) Berger, 2011

Yang, Caiting, Liu, An, Xu, Yusen, Xu, Yuan, Fan, Xinpeng, Al-Farraj, Saleh A., Ni, Bing & Gu, Fukang, 2015, Phylogenetic positions of four hypotrichous ciliates (Protista, Ciliophora) based on SSU rRNA gene, with notes on their morphological characters, Zootaxa 4000 (4), pp. 451-463 : 454-455

Rigidohymena inquieta (Stokes, 1887) Berger, 2011

( Figure 1 A–L; Table 1)

This species has been long considered as a member of Cyrtohymena , until Berger (2011) transferred it to the genus Rigidohymena because of its rigid body without cortical granules. Since previous reports contained some misobservations and conflicts between populations, redescription and an improved diagnosis are needed.

Improved diagnosis: Body 95–130 × 30–50 µm in vivo. Pellicle rigid without cortical granules. Two macronuclear nodules. One contractile vacuole located in anterior 1/2 of body length near left margin. Adoral zone occupies 40%–50% of body length and composed of 37–50 membranelles. Marginal rows do not confluent posteriorly containing 13–24 on left and 18–26 on right. Eight fronto-ventral and five transverse cirri. Four or five ventral cirri. Six dorsal kineties and three caudal cirri. Morphogenesis is typical Oxytricha- pattern.

Deposition of voucher slides: One voucher slide (with registry number TWJ–20140427) was deposited in the Laboratory of Protozoology, East China Normal University, Shanghai, China.

Morphological description of Chinese population: In vivo 110–130 µm × 30–50 µm, body elliptical in outline, with anterior end broadly rounded, length: width ratio approximately 2.5: 1 ( Fig. 1 A, F–H). Buccal field about 40% of body length ( Fig. 1 A, G). Collar part of adoral zone of membranelles extends to dorsal side ( Fig. 1 A, J). Pellicle firm without cortical granules. Cytoplasm contains numerous crystals of different size ( Fig. 1 A, H). Two ellipsoidal macronuclear nodules, about 45 × 25 µm in size, located at the left of cell mid-line, with spherical nucleoli scattered in. 2–4 micronucleus located around macronuclear nodules. Single contractile vacuole about 13 µm across, located at the left of cell mid-line, slightly in the front of mid-body, pulsing at about 14 s intervals ( Fig. 1 G). Body grayish in appearance when observed at low magnifications, usually containing small lipid droplets and cytoplasmic granules 1–5 µm in diameter ( Fig. 1 A, G, H). Cysts, spherical, about 50 µm across, with contractile vacuole contracting as usual. ( Fig. 1 K). Locomotion by swimming and rotating about its longitudinal axis moderately fast, or slowly crawling on bottom of Petri dishes or on debris.

Paroral membrane distinctly curved in anterior half ( Fig. 1 B, L). Three frontal cirri in life about 12–19 µm long. One buccal cirrus, located at the inflexion of paroral membrane. Three postoral ventral cirri located behind buccal vertex. Five or six transverse cirri, about 20 µm in life, projecting beyond posterior body margin by 2/3 of their length. Right marginal cirri began at the level of penultimate fronto-ventral cirri. Both two marginal rows ended terminally and almost confluent posteriorly ( Fig. 1 B, L). Invariably three caudal cirri are about 17 µm long in life. Six dorsal kineties (DK), DK1–3 bipolar, DK4 commencing posterior, DK5 stretching to posterior 1/2 of cell and DK 6 short ( Fig. 1 C). A late stage of reorganization observed: new dorsal kineties formed in typical Oxytricha -pattern, i. e., DKA 1–3 developed intrakinetally within parental structure, while DK4 formed by fragmentation of DKA3; three caudal cirri formed at the end of DK 1, 2 and 4; two dorsomarginal kineties (DK5 and 6) formed by two anlagen (DMA) that develop de novo at anterior right of right marginal row ( Fig. 1 D, E).

Remarks: Chinese population corresponds well with previous studies in terms of living morphology, especially the body size and rigid pellicle without cortical granules. However, the infraciliature shows some conflicts between populations. Both original reports of Stokes (1886) and Grolière (1975) draw four ventral cirri, which Berger (1999) considered as a typical character of this species. While the organism reported by Gu and Zhang (1992), a highly possible synonym of C. inquieta considered also by Berger (1999), as well as present population, has constantly five ventral cirri. Thus, we inclined to accept the variation in number of ventral cirri as population dependent.

Depending on our observations and the work of Gu and Zhang (1992), there should be six dorsal kineties and three caudal cirri following typical Oxytricha- pattern during ontogenesis.

Çapar (2007) also reported a population of C. inquieta from Anatolia with only three ventral cirri, but the infraciliature is possibly unreliable for the reason that the typical Cyrtohymena -like paroral membrane was not shown in his drawing.

R. inquieta thus share the same infraciliature with its congener R. candens and differs from the latter mainly in smaller body length (<150 µm vs.>150 µm). Although body size is not a good diagnostic character, the discrepancy of their SSU rRNA gene sequences is strong support to separate the two species. Accordingly, Cyrtohymena candens var. depressa (Gellért, 1942) Foissner, 1989 is synonymy of R. inquieta , since tts rigid body and Cyrtohymena -like undulating membrane demonstrate it as a member of Rigidohymena , and the small body size (100–120 µm) indicates it is a population of R. inquieta rather than R. candens ( Berger 1999; Chen et al. 2013).

Characters Species Min Max Mean SD CV n

Body length Rig. 91 120 100.6 8.3 8.3 16 Pat. 297 480 349.8 53.2 15.2 13 Not. 113 179 143.4 19.6 13.6 18 Cyr. 195 318 250.5 40.0 16.0 10

Body width Rig. 40 73 51.3 7.6 14.9 16 Pat. 151 270 200.6 35.6 17.8 13 Not. 37 81 53.1 12.3 23.1 18 Cyr. 90 150 120.5 20.2 16.8 10

Buccal field length Rig. 34 53 40.8 5.1 12.5 16 Pat. 123 197 158.0 23.5 14.9 10 Not. 39 61 47.8 7.0 14.7 16 Cyr. 65 110 87.0 14.6 16.8 10

DE-Value Rig. 15 35 22.9 5.6 24.6 14 Pat. 38 65 50.3 7.6 15.1 10 Not. 10 20 15.7 2.5 15.8 15 Cyr. 23 40 30.0 5.4 18.2 10

Membranelles width Rig. 7 11 9.2 1.2 13.0 16 Pat. 32 41 36.1 3.7 10.2 10 Not. 6 11 8.4 1.1 13.7 16 Cyr. 13 20 16.0 2.6 16.6 10

Adoral membranelles, Rig. 30 45 36.8 4.9 13.4 17

number Pat. 44 78 63.7 9.2 14.4 12 Not. 36 44 41.2 2.7 6.6 15 Cyr. 44 60 51.5 5.5 10.6 10

Macronuclei length Rig. 11 20 15.9 3.2 20.3 19 Pat. 15 25 19.8 3.2 16.2 10 Not. 21 37 29.7 3.8 12.9 18 Cyr. 14 22 19.3 0.8 12.3 10

Macronuclei, number Rig. 2 2 2.0 0 0 16 Pat. 21 35 27.4 3.8 13.7 17 Not. 2 3 2.1 0.3 13.0 14 Cyr. 2 2 2.0 0 0 10

Left marginal cirri, Rig. 17 24 20.0 2.1 10.9 15

number Pat. 13 37 29.2 6.1 20.7 12 Not. 33 48 40.8 4.5 10.9 13 Cyr. 32 47 40.3 4.0 10.0 10

Right marginal cirri, Rig. 18 26 20.6 1.9 9.1 15 ......continued on the next page Characters Species Min Max Mean SD CV n number Pat. 14 36 29.6 5.6 18.8 12 Not. 35 46 41.7 3.2 7.7 15 Cyr. 28 41 36.5 3.9 10.6 10 Frontal cirri, number Rig. 3 3 3.0 0 0 15 Pat. 6 9 7.5 0.8 11.3 10 Not. 3 3 3.0 0 0 15 Cyr. 3 3 3.0 0 0 10 Ventral cirri, number Rig. 10 10 10.0 0 0 15 Pat. 18 27 21.3 3.0 14.0 10 Not. 10 10 10.0 0 0 15 Cyr. 9 10 9.9 0.1 3.2 10 Transverse cirri, Rig. 5 6 5.1 0.4 6.9 15 number Pat. 8 11 10.3 0.9 8.3 13 Not. 5 5 5.0 0 0 15 Cyr. 5 6 5.1 0.3 6.2 10 Dorsal kineties, Rig. 6 6 - - - 2 number Pat. 14 15 14.9 0.3 2.1 10 Not. 6 6 6.0 0 0 15 Cyr. 6 12 7.5 1.0 13.0 10

Figures

FIGURE 1. Rigidohymena inquieta from life (A, F – K) and after staining with protargol (D, E, L). A. Ventral view of living cells; B, C, L. Ventral (B, L) and dorsal (C) view of infraciliature; D, E. Ventral and dorsal view during later stage of reorganization; F, G. Ventral view of two typical individuals, arrow in G marks the contractile vacuole; G. Showing the crystals in cytoplasm; H. Showing the membranelles of adoral zone (arrowhead) and the transverse cirri (arrow); I. Details of oral structure, arrowhead marks the anteriorly curved Cyrtohymena - like paroral membrane; J. The collar part of adoral zone of membrane; K. Cyst; FC, frontal cirri; LMR, left marginal row; Ma, macronuclei; Mi, micronuclei; PM, paroral membrane; RMR, right marginal row; VC, ventral cirri; TC, transverse cirri. Scale bars = 50 Μm.