Phylogeny of a new ciliate family Clampidae fam. nov. (Protista: Ciliophora), with notes on morphology and morphogenesis Author Xu, Wenxin Author Ma, Jiyang Author Li, Yuan Author Bourland, William A. Author Petroni, Giulio Author Luo, Xiaotian Author Song, Weibo text Zoological Journal of the Linnean Society 2022 2022-01-25 196 88 104 https://doi.org/10.1093/zoolinnean/zlab102 journal article 10.1093/zoolinnean/zlab102 0024-4082 7034929 C7008A79-ABF6-403A-9366-2C68AE7C405C CLAMPIA SINICA SP. NOV. Z o o b a n k r e g i s t r a t i o n : u r n: l s i d: z o o b a n k. org:act: 894A94B9-FD7C-4E85-AB08-EC470D2DE 78F . Diagnosis: 100–145 × 30–50 µm in vivo , slender elliptical in shape; cortical granules colourless, spherical and 0.8–1.0 µm across, mainly distributed along cirral rows and dorsal kineties; about 33 adoral membranelles; invariably three frontoventral rows, with the anteriormost cirrus slightly enlarged; generally three short midventral rows, left and right long midventral row composed of about 28 and 40 cirri, respectively; on average six buccal and seven transverse cirri; left and right marginal row composed of about 35 and 37 cirri respectively; limnetic habitat. Etymology: Specific epithet is the feminine Latin adjective sinica , Chinese , refers to the country where this species was discovered. Type locality: Freshwater fishpond in Honghu ( 29°57′57.85″N ; 113°46′35.17″E ), central China (for details, see section ‘Sample collection, observation and terminology’) . Type material: One protargol-impregnated slide (no. XWX201904Y401–01) with the holotype specimen ( Figs 3F , 4A , marked with ink circle on the slide) and five paratype slides (no. XWX201904Y401–02, 03, 04, 05, 06) were deposited in the Laboratory of Protozoology , Ocean University of China , Qingdao, China . MORPHOLOGICAL DESCRIPTION OF CLAMPIA SINICA ( FIGS 3A–S , 4A–D; TABLE 1) Size 100–145 × 30–50 µm in vivo , length to width ratio 3.0–4.5: 1 in vivo ( Fig. 3H–M ) and 1.7–2.8:1 after protargol staining. Elongated body, elliptical in shape, both ends broadly rounded, posterior end slightly narrowed, body flexible but not contractile ( Fig. 3A, B, H–M ). Single contractile vacuole, about 17 µm in diameter at end-diastole, located at left body margin slightly behind buccal vertex ( Fig. 3E, M , arrow). Cortical granules colourless, spherical, about 0.8– 1.0 µm across, often arranged in small groups near cirri, grouped or scattered along and between cirral rows and dorsal kineties ( Fig. 3C, D, N–P ). Cytoplasm colourless, usually packed with many lipid droplets (about 1–2 µm across), spheroidal granules (about 1–4 µm across), a few irregularly shaped crystals (about 2–3 × 0.5 µm), other undefined inclusions and several food vacuoles sometimes containing small-sized ciliates ( Figs 3R , 4D , arrows). Macronuclear nodules 50–120, scattered throughout cell body ( Figs 3G , 4D , arrowhead). Locomotion by swimming while rotating about the main body axis or slow to moderately fast crawling on substrates with body folding or twisting. Adoral zone question mark-shaped, with 29–36 membranelles, occupies 32–42% of cell length in vivo . Undulating membranes more or less in Oxytricha pattern, paroral and endoral membranes almost equal in length, slightly curved and optically intersected, both diplostichomonad, the paroral membrane begins slightly anterior to the endoral membrane ( Figs 3F , 4A, B ). Ventral ciliature as follows ( Figs 3F , 4A ): frontoventral cirri arranged in three short rows, each row usually composed of four or five cirri, with the anteriormost cirrus slightly enlarged, with cilia 10–12 µm long in vivo . On average, six buccal cirri arranged longitudinally to the right of the paroral membrane ( Figs 3F , 4A, B ). Generally, three short and invariably two long midventral rows (MV). MV n–1 (i.e. the left long midventral row) composed of 22–34 cirri, begins at the level of the posterior end of or slightly above buccal cirral row and terminates at about 85% of cell length. MV n (i.e. the right long midventral row) composed of 28–48 cirri, commences near the distal end of the adoral zone and terminates at the rightmost transverse cirrus. Transverse cirri four to nine, arranged in an oblique pseudorow, with cilia about 16 µm long in vivo , protruding beyond the posterior end of the cell ( Figs 3A, B, F , 4A ). One left and one right marginal row, extending towards the posterior end of cell, separated by a distinct gap, with cilia about 9–11 µm long in vivo ; left marginal row composed of 24–42 cirri and J-shaped, right marginal row with 26–44 cirri ( Figs 3F , 4A ). Figure 3. Morphology of Clampia sinica gen. et sp. nov. from life (A–E), after protargol staining (F, G) and photomicrographs of Clampia sinica in vivo (H–K, Q–S, bright field; L–P, differential interference contrast). A, B, ventral views of representative specimens. C, D, ventral (C) and dorsal (D) view showing the distribution of cortical granules. E, ventral views of individuals, showing different body shapes, location of contractile vacuoles. F, G, ventral (F) and dorsal (G) view of the holotype specimen showing the infraciliature and nuclear apparatus. H–M, ventral views of typical individuals, showing the flexibility of body and different body shapes, arrow marks the contractile vacuole. N–P, ventral (N) and dorsal (O, P) views, showing cortical granules (arrowheads). Q, ventral view of the anterior portion, to show adoral zone of membranelles and frontoventral cirri. R, posterior part of cell, showing irregular granules of cytoplasm and a food vacuole (arrow). S, macronuclear nodules (arrowheads). Abbreviations: AZM, adoral zone of membranelles; BC, buccal cirri; e, endoral membrane; FVR1–3, frontoventral rows; LMR, left marginal row; Ma, macronuclear nodules; MV n and n–1, long midventral rows; p, paroral membrane; RMR, right marginal row; TC, transverse cirri; DK1–3, dorsal kineties; 1–3, short midventral rows. Scale bars: 50 µm. Figure 4. Photomicrographs of Clampia sinica gen. et sp. nov. after protargol staining. A, ventral view of the infraciliature of the holotype specimen. B, ventral view of the anterior portion, to show frontoventral rows, short midventral rows, long midventral rows, and undulating membranes. Cirri are linked according to anlagen. C, dorsal view of the anterior portion, to show the dorsal kineties. D, ventral view of the middle portion, showing macronuclear nodules (arrowhead) and an ingested small-sized ciliate (arrow). E, ventral view of an early divider, arrows show the basal body patches forming the oral primordium of the opisthe. F, ventral view of an early-middle divider, arrows show anlage n and n–1, arrowheads mark marginal anlagen. G, ventral view of a middle divider. At this stage the macronuclear nodules have fused into a single mass, arrows show the newly formed undulating membranes, double arrowheads mark the anteriormost cirrus of the first frontoventral row generated from the undulating membranes anlage. H, a middle-late divider, showing the fission of macronuclear nodules and micronuclei (arrows). I, J, ventral (I) and dorsal (J) view of a middle-late divider to show one transverse cirrus formed at the rear end of each frontoventral-transverse cirral anlage and the newly formed dorsal kineties (arrowheads). K, dorsal view of a late divider, arrow marks two extra bristles at the anterior end of the right marginal anlage. L, ventral view of a late divider, arrows show two long midventral rows, arrowheads show transverse cirri. Abbreviations: AZM, adoral zone of membranelles; BC, buccal cirri; DK1–3, dorsal kineties; e, endoral membrane; LMR, left marginal row; Ma, macronuclear nodules; MV n and n–1, long midventral rows; p, paroral membrane; RMR, right marginal row; TC, transverse cirri. Scale bars: 50 µm (A), 30 µm (B–L). Invariably three dorsal kineties (DK), four out of 21 specimens investigated have two or three extra dorsal bristles to the right of the anterior end of DK3, DK2 and DK3 nearly equal to the whole body length, DK1 slightly shortened anterior end ( Figs 3G , 4C ). The length of dorsal bristles is about 4 µm in vivo . Caudal cirri are absent. MORPHOGENESIS OF CLAMPIA SINICA ( FIGS 4E–L , 5A–H ) Seven morphogenetic specimens were observed: one at early stage ( Figs 4E , 5A ); two at early-middle stage ( Figs 4F , 5B–D ); one at middle stage ( Figs 4G , 5E, F ); one at middle-late stage ( Fig. 4H–J ); and two at late stage ( Figs 4K, L , 5G, H ). As some key stages, such as early stages between Figure 5A and Figure 5B , were not observed, the origins of some anlagen are not clear. Table 1. Morphometric characterization of the new species Clampia sinica gen. et sp. nov.

Character *	Min	Max	Mean	M	SD	CV	N
Body, length	94	156	126.4	125	17.1	13.5	21
Body, width	40	89	61.4	65	13.0	21.2	21
Body length: width, ratio	1.7	2.8	2.1	2	0.3	15.4	21
Adoral zone, length	40	61	49.8	50	5.2	10.5	21
Adoral membranelles, number	29	36	33.0	33	2.0	6.1	21
Buccal cirri, number	4	7	5.7	6	0.7	11.6	21
Frontoventral cirri, number	10	17	14.5	15	1.8	12.6	21
Frontoventral rows, number	3	3	3.0	3	0.0	0.0	21
Cirri in frontoventral row 1, number	3	6	4.1	4	0.7	17.5	21
Cirri in frontoventral row 2, number	3	7	5.0	5	1.0	19.3	21
Cirri in frontoventral row 3, number	3	7	5.3	5	0.9	17.1	21
Short midvenral rows, number	2	4	3.0	3	0.5	18.3	21
Cirri in short midventral row 1, number	3	6	4.3	4	0.7	16.9	21
Cirri in short midventral row 2, number	3	5	4.3	4	0.6	13.3	21
Cirri in short midventral row 3, number	3	7	4.7	5	1.0	20.8	18
Cirri in short midventral row 4, number	5	5	5.0	5	0.0	0.0	3
Long midventral rows, number	2	2	2.0	2	0.0	0.0	21
Cirri in left long midventral row, number	22	34	27.6	28	3.1	11.3	21
Cirri in right long midventral row, number	28	48	39.8	40	5.2	13.0	21
Transverse cirri, number	4	9	6.9	7	1.0	14.4	21
Left marginal cirri, number	24	42	34.7	34	4.4	12.7	21
Right marginal cirri, number	26	44	37.4	39	5.4	14.3	21
Dorsal kineties, number	3	3	3.0	3	0.0	0.0	21
Extra dorsal bristles, number	0	3	0.5	0	1.0	216.4	21
Dorsal kinety 1, number of bristles	13	22	17.0	17	2.5	14.7	15
Dorsal kinety 2, number of bristles	16	25	20.0	20	2.3	11.5	21
Dorsal kinety 3, number of bristles	15	25	21.4	22	2.2	10.5	19
Macronuclear nodules, number	54	113	85.0	85	16.3	19.2	21
Macronuclear nodule, length†	6	19	10.4	10	3.8	36.7	21
Macronuclear nodule, width†	2	8	4.5	4	1.8	39.9	21

*All data are based on protargol-stained specimens. Measurements in µm. †Macronucleus nodules were selected randomly in each individual. Abbreviations: CV, coefficient of variation in %; M, median; Max, maximum; Mean, arithmetic mean; Min, minimum; N , number of specimens measured; SD, standard deviation. Stomatogenesis: In the earliest divider observed, several groups of closely spaced basal body patches forming the oral primordium of the opisthe are developed near the rightmost short midventral row and by dedifferentiation of some posterior cirri of MV n–1 ( Figs 4E , 5A , arrows). In the early-middle stage, the parental undulating membranes disorganize and develop into the undulating membranes anlage (UMA) in the proter, while the parental adoral zone of membranelles remains unchanged. Simultaneously, in the opisthe, the oral primordium gives rise to new adoral membranelles, to the right of which appears the streak-like UMA ( Fig. 5B ). Subsequently, the proter’s oral primordium appears near the posterior end of UMA ( Fig. 5C , double arrowheads). At the middle stage, in both proter and opisthe, UMA splits longitudinally to form the paroral and endoral membranes ( Figs 4G , 5E , arrows), with differentiation commencing at the anterior end to form the leftmost frontoventral row ( Figs 4G , 5E , double arrowheads). Simultaneously, the oral primordium of the proter moves to the proximal end of parental adoral zone of membranelles ( Fig. 5E , arrowhead). From middle to late stage, the posterior portion of parental adoral zone of membranelles is partly renewed ( Fig. 5G , arrow). At the same time, the new adoral zone of membranelles of the opisthe continues to differentiate posteriad and anterior part of the adoral zone bends towards the right ( Figs 4I, L , 5E, G ). Frontoventral-transverse cirri: At the early-middle stages, seven to nine streak-like frontoventraltransverse cirral anlagen (FVTA) are formed in both proter and opisthe, of which the two rightmost anlagen are separated from the others ( Figs 4F , 5B, C ). Posterior portion of the short parental frontoventral rows is likely to take part in the formation of proter’s FVTA. In both proter and opisthe, parental cirri in MV n disaggregate to develop the FVTA n–1 ( Fig. 5B, C , arrows), which forms the new MV n–1. The origin of FVTA n ( Fig. 5B, C , arrowheads) is not clear and it may develop de novo or, alternatively, it may split off from the FVTA n–1 anlage at an early stage. Subsequently, the streaks broaden, break apart and migrate to their final positions during cytokinesis. Cirri develop as follows: FVTA I (= UMA) forms the leftmost frontoventral row and the new undulating membranes; FVTA II forms the middle frontoventral row, four to seven buccal cirri, and the leftmost transverse cirrus; FVTA III forms the rightmost frontoventral row and one transverse cirrus; FVTA IV to FVTA n–2 each forms one short midventral row and one transverse cirrus, respectively; FVTA n–1 forms MV n–1 and the penultimate transverse cirrus; FVTA n forms MV n and the rightmost transverse cirrus. The absorption of old cirri occurs during the morphogenetic process. Figure 5. Morphogenesis of Clampia sinica gen. et sp. nov. after protargol staining. A, portion of ventral view of an early divider, arrows show the basal body patches forming the oral primordium of the opisthe. B, ventral view of an earlymiddle divider to show the frontoventral-transverse cirral anlagen, arrows denote the anlage n–1 which forms the left long midventral row, arrowheads denote the anlage n that develops into the right long midventral row. C, D, ventral (C) and dorsal (D) view of an early-middle divider, showing marginal anlagen and dorsal kineties anlagen developed intrakinetally, arrows and arrowheads mark the anlage n–1 and anlage n respectively, double arrowheads denote the proter’s oral primordium. E, F, ventral (E) and dorsal (F) view of a middle divider to show the differentiated frontoventral-transverse cirral anlagen, the macronuclear nodules fused into a single mass, the separation of undulating membranes (arrows), the proter’s oral primordium (arrowhead in E), and dorsal kineties anlagen (arrowheads in F), double arrowheads mark the leftmost frontoventral row generated from the undulating membranes anlagen, dashed areas denote buccal cirral rows. G, H, ventral (G) and dorsal (H) view of a late divider, showing all the cirri almost in their final positions, which are linked according to anlagen. Arrow shows the partly renewed adoral zone of membranelles of proter, arrowheads mark extra dorsal bristles. Abbreviations: AZM, adoral zone of membranelles; DKA1–3, dorsal kineties anlagen; e, endoral membrane; LMA, left marginal anlagen; LMR, left marginal row; Ma, macronuclear nodules; MV n and n–1, long midventral rows; OP, oral primordia; p, paroral membrane; RMA, right marginal anlagen; RMR, right marginal row; TC, transverse cirri; I–VI, frontoventral-transverse cirral anlagen. Scale bars: 50 µm. Marginal rows: Two marginal anlagen develop intrakinetally within each parental marginal row ( Figs 4G , 5E ). These commence asynchronously, i.e. one of the anlagen appears first ( Figs 4F , arrowheads, 5C). These anlagen then increase in size and develop into new cirri that eventually replace the old ones in both proter and opisthe ( Figs 4G, I, L , 5E, G, H ). Dorsal kineties: Two anlagen develop intrakinetally in each parental row, which then stretch towards both ends of the cell and develop into new structures along with the incorporation or absorption of parental structures ( Figs 4J , 5D, F, H ). In some dividers, there are two or three extra dorsal bristles ( Fig. 5H , arrowheads) near the anterior end of RMA. No caudal cirri were formed during ontogenesis. Nuclear apparatus: Macronuclear nodules fuse into many masses in the early-middle stage of morphogenesis ( Fig. 5D ), and then fuse into a single mass ( Fig. 5F ), which subsequently divides several times to form new structures for both proter and opisthe ( Figs 4H , 5H ).