First record of two urotsylid ciliates (Protozoa: Ciliophora: Spirotrichea) from Japan expanding the circumscription of species Author Ghaffar, Abdul Author Bai, Yang Author Hu, Xiaozhong text Journal of Natural History 2023 Oxford, England 2023-06-14 57 9 - 12 724 743 http://dx.doi.org/10.1080/00222933.2023.2206586 journal article 54185 10.1080/00222933.2023.2206586 c1c0e6b1-13f2-44b3-beca-d3c70a856927 1464-5262 8071692 Trichototaxis marina Lu et al., 2014 Description ( Figures 1 , 2 ; Table 1 ) The November and April populations studied here are so similar that conspecificity is beyond reasonable doubt. Consequently, the description below includes data from both populations. Body size highly variable, but shape very similar in the two populations. Size 200– 360 × 40–80 μm, mostly 250 × 50 μm in life, and 178–328 × 53–118 μm for stained specimens ( Table 1 ); ratio of length to width about 4–6: 1 in live specimens; flattened ca. 2:1 dorsoventrally with anterior and posterior portions thin. Body elongate elliptical and often sigmoidal in outline, anterior end wider than posterior one, widest at mid-body ( Figures 1A , 2A–C ), conspicuously reddish in colour at low magnification ( Figure 2B, C, F ). Pellicle very flexible and highly contractile ( Figure 2D, F ) and to some extent thigmotactic. Conspicuous furrows recognisable along cirral rows on ventral side ( Figure 2G ). Cortical granules not observed, but rice-shaped granules (1 × 0.5 μm, mitochondria?) densely arranged along cirral rows ( Figure 2E ). Many brick-reddish, weird pigment patches of variable size densely distributed under pellicle and in cytoplasm, which render cells red ( Figure 2I ), and often decreasing in number after some time at room temperature in raw culture. Endoplasm usually packed with numerous small (<1 μm) spherical or roughly elliptical shining globules ( Figure 2G ), and refractive crystals, 1–3 μm across, usually in posterior region of cell. Several food vacuoles usually present in mid-region of cell, mainly containing diatoms, and rod-shaped bacteria ( Figure 2J ). One contractile vacuole about 10 μm across, positioned dorsally at anterior third, near left margin. Figure 1. Morphology of Trichototaxis marina , (A) from life and (B–E) after protargol staining. (A) Ventral view showing representative body shape; (B) ventral view of a specimen, showing three left marginal rows; (C) ventral view of a specimen having two buccal cirri, cirri from the same anlage are connected by a broken line; (D) ventral view of a cell with three buccal cirri, arrow shows paroral membrane, arrowhead indicates one displaced buccal cirrus; (E) dorsal view of a specimen, showing dorsal kineties, macronuclear nodules, and micronuclei (arrowheads). AZM, adoral zone of membranelles; DK1, the leftmost dorsal kinety; DK2, the second dorsal kinety counting from the left; DKn, the rightmost dorsal kinety; F, fibres; FC, frontal cirri; FT, frontoterminal cirri; LMR1, the innermost left marginal row; LMR2, the exterior left marginal row; LMR3, the outermost left marginal row; MN, macronuclear nodule; MP, midventral pair; PT, pretransverse ventral cirri; RMR, right marginal row; TC, transverse cirri. Scale bars: 50 μm. Buccal field 60–110 μm long, occupying ca . 30% of body length in live cells ( Figures 1A , 2A–C, F, G ), its proximal part covered by a thick buccal lip ( Figure 2H and J , arrow). Adoral zone composed of 45 to 80 (on average 63 and 74 in two populations) membranelles, with distal end extending far onto right ventral side ( Figures 1B–D , 2A, D, G, K, N ); cilia of apical membranelles about 10 μm long. Paroral membrane ( Figures 1D , arrow, and 2K, N, P) about half length of endoral membrane ( Figures 1B , arrow, and 2N, arrowhead), almost parallel to each other, the former dikinetidal and the latter monokinetidal ( Figure 2K, P ). Pharyngeal fibres conspicuous in vivo. Twelve to 23 frontal cirri, cilia of which are 10–15 μm long in vivo, arranged in a conspicuous bicorona ( Figures 1B–D and 2N ). Usually 1 buccal cirrus (rarely 2 or 3) ( Figures 1B–D , 2K, N , arrow,P, arrowhead); cilia ca.10 μm long, located adjacent to right posterior third of paroral membrane. Invariably 2 frontoterminal cirri near distal end of adoral zone ( Figures 1B–D , 2K, N ). Midventral complex composed of 39–75 pairs of cirri (including 2 pretransverse ventral cirri), arranged in a zig-zag pattern, running in a slightly sigmoid pattern along ventral surface and extending to posterior portion of body ( Figures 1B–D , 2K–O ). Cirri about 10 µm long, base of right midventral cirrus (about 2 × 6 basal bodies) conspicuously larger than that of left cirri (2 × 3 basal bodies; Figure 2L ). Usually 6 (sometimes 5 or 7; 8 in two of ( Continued ) 35 specimens ) transverse cirri, cilia of which are ca. 20 μm long, arranged in a J-shaped pseudo-row ( Figures 1B–D , 2K, O ). Total number of fronto–ventral–transverse cirri highly variable, ranging from 94 to 178 for two populations. Ciliary fibres of cirri, especially transverse cirri, conspicuous after protargol staining ( Figure 2K, M ). Figure 2. Micrographs of Trichototaxis marina , (A–J) from life and (K–R) after protargol staining. (A) Ventral view of a representative individual, showing general morphology and grooves along cirral rows (arrows); (B, C, F) different body, showing shape variation; (D) illustrates the flexibility of the cell; (E) cortical granules densely arranged along cirral rows (arrows); (G) ventral view of a cell, showing grooves along cirral rows (arrows); (H) indicates the bases of membranelles and cirri; (I) weird pigment spots (arrows); (J) anterior body portion, showing thick buccal lip (arrow) and the ingested diatoms (arrowheads); (K) ventral view to show ciliature and nuclear apparatus; (L) basal body composition of cirri, arrowheads indicate micronuclei; (M) cirri and associated fibres; (N) ventral view of a specimen, showing oral apparatus and ciliary pattern, arrow and arrowhead show buccal cirri and endoral membrane, respectively; (O) ventral view of the same specimen as in N, showing somatic ciliature in posterior portion of cell; (P) ventral view of part of buccal field, showing buccal lip (arrow), buccal cirrus (arrowhead) and undulating membranes; (Q) middle part of a cell, showing the arrangement of macronuclear nodules and micronuclei (arrowheads); (R) macronuclear nodules, micronuclei (arrow) and adoral membranelles. AZM, adoral zone of membranelles; EM, edoral membrane; FC, frontal cirri; FT, frontoterminal cirri; LMR1, the innermost left marginal row; LMR2, the exterior left marginal row; MN, macronuclear nodule; PM, paroral membrane; PT, pretransverse ventral cirri; RMR, right marginal row; TC, transverse cirri. Scale bars: 50 μm. Table 1. Morphometric characteristics of Trichototaxis marina (Np, November population; Ap, April population; Tp, type population). Data are based on protargol-stained specimens. Measurements in μm.

Character	Pop	Min	Max	Mean	SD	SE	CV	n
Body length	Np Ap	178 198	300 328	228.6 273.5	41.34 35.51	10.33 8.88	18.1 13.0	16 16
Body width	Np Ap	58 53	118 103	82.4 76.3	16.13 12.77	4.16 3.19	19.6 16.7	15 16
Buccal field, length	Np Ap	60 78	100 106	75.6 88.8	11.27 7.43	2.82 1.86	14.9 8.4	16 16
Adoral membranelles, number	Np Ap	45 61	75 80	62.6 73.6	7.17 5.37	1.79 1.39	11.5 7.3	16 15
Buccal cirri, number	Np Ap	1 1	3 2	1.2 1.1	0.52 0.25	0.10 0.06	43.3 22.7	25 15
Frontoterminal cirri, number	Np Ap	2 2	2 2	2 2	0 0	0 0	0 0	16 16
Frontal cirri, number	Np Ap	12 12	17 23	14.4 15.3	1.47 3.20	0.31 0.92	10.2 20.9	23 12
Midventral pairs, number	Np Ap	39 60	73 75	57. 67.5	10.32 4.79	2.76 1.33	18.1 7.1	14 13
PT, number	Np Ap	2 2	2 2	2 2	0 0	0 0	0 0	17 16
Transverse cirri, number	Np Ap	5 5	7 8	6.0 6.4	0.59 0.85	0.13 0.23	9.8 13.3	21 14
Total cirri, number*	Np Ap Tp	94 145 123	166 178 186	133.4 162.7 158.8	21.42 9.97 18.48	5.73 3.32 4.24	16.1 6.1 11.6	14 9 19
Left marginal rows, number	Np Ap	2 2	3 4	2.1 2.9	0.33 0.47	0.07 0.13	15.7 16.2	25 14
Cirri in LMR1, number	Np Ap	46 73	81 102	68.3 89.4	10.61 8.86	2.74 2.37	15.5 9.9	15 14
Cirri in LMR2, number	Np Ap	23 32	52 51	35.6 42.1	7.35 5.40	1.69 1.44	20.6 12.8	19 14
Cirri in LMR3, number	Np Ap	3 13	14 26	- 20.5	- 4.42	- 1.28	- 21.6	2 12
Cirri in RMR, number	Np Ap	46 67	87 104	65.2 86.4	11.19 10.43	2.89 2.89	17.2 12.1	15 13
Macronuclear nodules, number	Np Ap	124 126	179 166	- -	- -	- -	- -	6 3
Micronuclei, number	Np Ap	4 2	14 6	7.9 3.1	2.39 1.17	0.50 0.28	30.3 37.7	23 17
Micronuclei, length	Np Ap	3 5	7 9	- -	- -	- -	- -	7 6
Micronuclei, width	Np Ap	2 3	4 6	- -	- -	- -	- -	7 6
Dorsal kineties, number	Np Ap	6 6	6 8	6 6.7	0 0.83	0 0.22	0 12.4	13 14
Dikinetids in DK1, number	Np Ap Tp	21 23 22	37 50 42	28.3 41.8 30.8	5.39 7.89 5.62	1.80 2.19 1.25	19.0 18.9 18.2	9 13 18
Dikinetids in DK2, number	Np Ap Tp	26 29 21	34 48 42	- 41.5 31.0	- 5.18 5.62	- 1.49 1.32	- 12.5 18.1	5 12 18
Dikinetids in DK3, number	Np Ap Tp	21 25 20	35 42 35	- 35.7 26.8	- 5.60 4.05	- 1.62 0.95	- 15.7 15.1	5 12 18
Dikinetids in DK4, number	Np Ap Tp	20 23 19	28 40 36	- 34.1 26.9	- 5.04 4.96	- 1.45 1.17	- 14.8 18.4	6 12 18
Dikinetids in DK5, number	Np Ap Tp	18 23 17	- 37 34	- 32.9 27.1	- 4.33 5.05	- 1.37 1.19	- 13.2 18.6	6 12 18

Table 1. (Continued).

Character	Pop	Min	Max	Mean	SD	SE	CV	n
Dikinetids in DK6, number	Np	14	25	-	-	-	-	6
Ap	21	40	31.2	5.81	1.84	18.6	10
Tp	15	33	23.9	5.26	1.59	22.0	11

*all cirri derived from frontoventral-transverse cirral anlagen. Abbreviations: CV, coefficient of variation, in %; DK, dorsal kinety; LMR, left marginal row; Max, maximum; Mean, arithmetic mean; Min, minimum; n, number of specimens examined; PT, pretransverse ventral cirri; RMR, right marginal row; SD, standard deviation; SE, standard error of the mean. Invariably 1 right marginal row; on average 2 or 3 left marginal rows for November and April populations, respectively ( Figures 1A–D , 2K ). Cilia of marginal cirri ca. 12 μm long, each cirrus usually containing 2 × 6 basal bodies ( Figure 2L ). The innermost left marginal row composed of about 68 (November population) or 89 (April population) densely packed cirri, commencing more or less at level of mid-region of buccal field, and ending at posterior end of body. The next left marginal row consisting of 23–52 sparsely distributed cirri, starting behind level of the innermost row, and terminating at about posterior two-thirds of body; sometimes one or more cirri in this row resorbed, forming several gaps; most cells of the April population having the third left marginal row, which is comprised of about 21 loosely arranged cirri and located at mid-body. Right marginal row starting adjacent to right of frontoterminal cirri, composed of 46–104 cirri, running along right body margin, and terminating at the rear end of body ( Figures 1B–D , 2K ). Six to 8 bipolar dorsal kineties ( Figure 1D ), with cilia 3 μm long in vivo. Numerous (usually more than 100) macronuclear nodules, scattered throughout body; each spherical to ovoid, varying between 3 and 8 μm in length ( Figures 1E , 2K, L, Q ), having usually 1 central nucleolus (rarely 2 to 5) ( Figure 2R ); micronuclei numbering 2–14, each spherical to ellipsoidal ( Figures 1E , 2L and Q , arrowheads, R, arrow) and 3–9 μm long. Locomotion by crawling moderately without pause on debris or on bottom of Petri dish or by swimming in water when disturbed. Cells usually very thigmotactic to the substrate. Deposition of specimens. A voucher slide (registration number: HX20230418-1) with protargol-stained specimens has been deposited in the Marine Biological Museum, Chinese Academy of Sciences, Qingdao, China . Ecological data. Water temperature 14.2°C and 16.5°C, pH 8.2 and 8.2, salinity 34.5‰ and 34.7‰, DO (Dissolved oxygen) 7.38 mg /L and 7.87 mg /L for November and April populations, respectively.