Morphological versus molecular delimitation of ciliate species: a case study of the family Clevelandellidae (Protista, Ciliophora, Armophorea) Author Pecina, Lukáš 8B83834D-884C-448A-A0CD-5A1FB9A3BB8C Department of Zoology, Comenius University in Bratislava, 842 15 Bratislava, Slovakia. pecinalukas1@gmail.com Author Vďačný, Peter 47A28E80-E04F-40C4-93A3-F7F685C9533A Department of Zoology, Comenius University in Bratislava, 842 15 Bratislava, Slovakia. peter.vdacny@uniba.sk text European Journal of Taxonomy 2020 2020-08-06 697 1 46 journal article 21346 10.5852/ejt.2020.697 58523d91-e809-474b-b5c8-a9c1d64ad17c 3978463 8962B6E6-B278-4EF5-9E62-3E858726E2F2 Clevelandella constricta ( Kidder, 1937 ) Figs 1–3 Description of Vietnamese population Size in vivo about 100–155 × 25–50 μm, usually 120 × 35 μm, as calculated from some in vivo measurements and morphometric data; length:width ratio ranging from 3:1 to 4.2:1 after protargol impregnation ( Table 1 ). Body spindle-shaped, more or less distinctly constricted in anterior third, usually widest at mid-portion, i.e., about at level of proximal end of adoral zone of membranelles; dorsoventrally flattened 1.4:1. Anterior end bluntly pointed; posterior body portion differentiated into a short, inconspicuous peristomial projection, distinctly constricted at its base; left and right body margins slightly concave at level of macronucleus ( Figs 1 A–N, 3A, E–G). Macronucleus located in anterior second fifth of body length; ellipsoidal to almost spherical, with a length:width ratio of 1.1– 1.8: 1 in protargol preparations; 20–28 × 15–20 μm in size after protargol impregnation; filled with numerous globular structures (very likely nucleoli) 0.7–1.8 μm across after protargol impregnation, well recognizable in vivo and sometimes in lightly impregnated specimens. Karyophore attached to right and left cell’s margins in middle third of body, usually at level of macronucleus, rarely in midbody. Micronucleus attached to anterior side of macronucleus, i.e., near the place where longitudinal cell axis crosses macronucleus or on its left side; globular and about 4.1–4.3 × 4.5–4.7 μm in size in vivo ( Table 1 ; Figs 1 A–L, 3E). Contractile vacuole near left body margin in posterior third of cell, i.e., close to canal leading to cytopyge ( Fig. 1A, L ). Cortex flexible, no cortical granules recognizable. Cytoplasm colorless; finely granulated; divided by karyophore into an anterior and a posterior part; cytoplasm anterior to macronucleus contains a frontal lamina transversely stretching slightly posterior to anterior body end and densely packed, oval, refractile bodies (probably paraglycogen platelets) observable in vivo and after protargol impregnation; compartment posterior to macronucleus contains some (symbiotic?) bacteria and/or archaea freely scattered throughout the main body portion and food a Data based on mounted, protargol-impregnated, and randomly selected specimens. Measurements in µm. Fig. 1. Clevelandella constricta ( Kidder, 1937 ) . Vietnamese specimens isolated from Panesthia angustipennis cognata Bey-Bienko, 1969 from life (A) and after protargol impregnation (B–N). A . Ventral view of a representative specimen, length 120 μm. B–K . Variability of body shape and size as well as of the nuclear (shaded grey) and oral (shaded yellow) apparatus. L . Semi-schematic diagram, showing the general body organization. Black double arrowhead marks densely packed, oval, refractile bodies (probably paraglycogen platelets). M–N . Ciliary pattern of ventral and dorsal sides. Arrow marks the right suture, black arrowheads indicate the position of the ciliary whorl (posterior suture). O . Prokaryotes freely scattered throughout the cytoplasm posterior to the macronucleus. P . Detail of oval, refractile bodies (probably paraglycogen platelets) anterior to the macronucleus. Scale bars = 50 μm. Table 1 (continued on next page). Morphometric data on Clevelandella constricta ( Kidder, 1937 ) from Vietnam and Thailand.

Characteristica	Pop	Mean	M	SD	SE	CO	Min	Max	n
Body, length	VN	105.2	105.0	12.6	4.0	12.0	87.0	133.0	10
TH	116.2	115.0	21.5	6.8	18.5	81.0	150.0	10
Body, maximum width	VN	28.9	27.5	5.6	1.8	19.3	25.0	44.0	10
TH	27.8	29.0	4.9	1.5	17.5	22.0	34.0	10
Body, length:width ratio	VN	3.7	3.7	0.4	0.1	9.5	3.0	4.2	10
TH	4.2	4.2	0.5	0.2	12.2	3.4	5.0	10
Peristomial projection, length	VN	16.8	16.5	3.4	1.1	20.0	12.0	23.0	10
TH	21.7	22.5	5.0	1.6	23.2	13.0	28.0	10
Peristomial projection, width	VN	18.3	18.5	1.8	0.6	9.7	15.0	20.0	10
TH	15.2	15.5	4.4	1.4	29.1	10.0	24.0	10
Peristomial projection, % of body length	VN	16.0	15.5	3.0	0.9	18.4	12.6	20.9	10
TH	18.7	18.7	2.9	0.9	15.8	14.9	23.3	10
Peristomial opening, length	VN	11.9	11.0	1.6	0.5	13.4	10.0	15.0	10
TH	14.6	15.0	1.6	0.5	10.8	12.0	17.0	10
Peristomial opening, width	VN	15.9	16.5	1.6	0.5	10.0	13.0	18.0	10
TH	17.5	17.0	2.7	0.9	15.5	14.0	22.0	10
Peristomial opening, % of body length	VN	11.4	11.2	1.2	0.4	10.6	9.9	13.8	10
TH	12.8	12.4	1.5	0.5	11.6	11.1	14.8	10
Adoral zone of membranelles, length	VN	50.5	49.5	4.5	1.4	8.9	46.0	60.0	10
TH	59.2	58.0	10.0	3.2	16.9	38.0	73.0	10
Adoral zone of membranelles, % of body length	VN	48.3	48.0	3.9	1.2	8.1	41.4	54.5	10
TH	51.1	50.0	3.7	1.2	7.2	46.9	58.9	10
Anterior body end to macronucleus, distance	VN	24.9	24.5	4.1	1.3	16.6	19.0	34.0	10
TH	28.5	27.5	6.8	2.2	23.9	20.0	40.0	10
Posterior body end to macronucleus, distance	VN	63.4	61.5	9.2	2.9	14.5	53.0	83.0	10
Macronucleus, length	TH VN	73.8 22.4	73.0 21.5	13.2 2.5	4.2 0.8	17.8 11.0	51.0 20.0	94.0 28.0	10 10
TH	18.0	18.5	1.8	0.6	9.8	15.0	20.0	10

Table 1 (continued). Morphometric data on Clevelandella constricta ( Kidder, 1937 ) from Vietnam and Thailand.

Characteristica	Pop	Mean	M	SD	SE	CO	Min	Max	n
Macronucleus, width	VN	16.6	16.0	2.0	0.6	12.1	15.0	20.0	10
TH	14.7	15.0	1.6	0.5	10.7	11.0	16.0	10
Macronucleus, length:width ratio	VN	1.4	1.3	0.2	0.1	13.3	1.1	1.8	10
TH	1.2	1.2	0.1	0.0	9.9	1.1	1.4	10
Macronucleus, number	VN	1.0	1.0	0.0	0.0	0.0	1.0	1.0	10
TH	1.0	1.0	0.0	0.0	0.0	1.0	1.0	10
Adoral membranelles, number	VN	55.0	55.5	3.2	1.0	5.7	50.0	59.0	10
TH	52.7	53.5	6.4	2.0	12.2	37.0	60.0	10

vacuoles about 2.0–5.2 μm across and encompassing prey prokaryotes ( Figs 1A, L , O–P, 3E). Swims slowly; dies quickly on microscope slides, possibly due to presence of oxygen. Somatic ciliature holotrichous; cilia about 4.5–6.5 μm long in vivo and narrowly arranged.Approximately 80 ciliary rows narrowly spaced over entire body surface and about 10 ciliary rows encroaching onto peristomial projection. Almost all ciliary rows commence from a whorl (posterior suture) on left side near contractile vacuole to radiate over ventral and dorsal sides toward right body margin; some kineties shortened anteriorly or posteriorly ( Figs 1 M–N, 3F–G). Right suture extends from base of peristomial Fig. 2. Clevelandella constricta ( Kidder, 1937 ) . Thai I specimens isolated from Panesthia angustipennis angustipennis (Illiger, 1801) after protargol impregnation. A–J . Variability of body shape and size as well as of the nuclear (shaded grey) and oral (shaded yellow) apparatus. Scale bar = 50 μm. Fig. 3. Clevelandella constricta ( Kidder, 1937 ) . Vietnamese (A, E–G) and Cambodian (D) specimens isolated from Panesthia angustipennis cognata Bey-Bienko, 1969 , as well as Thai I specimens (B– C) isolated from Panesthia angustipennis angustipennis (Illiger, 1801) from life (A, D–G) and after protargol impregnation (B–C). A–C . Ventral view of specimens with well-preserved body shape. D–E . Ventral view, showing the general body organization. Arrows mark oval, refractile bodies anterior to the macronucleus, black arrowheads mark the proximal end of the adoral zone of membranelles, white arrowheads denote the karyophore attached to the right and left body margins and black double arrowhead marks the canal leading from the contractile vacuole to the cytopyge. F–G . Ciliary pattern of ventral and dorsal sides. Asterisks mark the position of the ciliary whorl (posterior suture), white double arrowhead denotes the right suture. Scale bars: A–C, E–G = 50 μm; D = 20 μm. projection to anterior body end; formed by obliquely abutting ventral and dorsal ciliary rows ( Figs 1N , arrow, 3G, white double arrowhead). Peristomial projection occupies on average 16% of body length and measures 12–23 × 15–20 μm in protargol preparations. Peristomial opening situated on ventral side of peristomial projection, triangular and short, i.e., about 11% of body length and 10–15 × 13–18 μm in size after protargol impregnation ( Figs 1 A–M, 3A, E). Peristomial funnel approximately 50 μm long after protargol impregnation. Adoral zone extends obliquely from distal end of peristomial projection along left side of peristomial funnel to terminate about in mid-portion of cell; occupies 41% to 55% of body length; composed of on average 55 membranelles; cilia of distalmost membranelles about 9 μm long in vivo and projecting out of peristomial opening ( Table 1 ; Figs 1A, L , 3E ). Paroral membrane diplostichomonad, i.e., composed of two rows of basal bodies; extends in parallel with adoral zone but on opposite side of peristomial funnel; commences at level of proximal end of peristomial opening and terminates near cytostome at proximal end of peristomial funnel ( Fig. 1L ). Pharyngeal fibres spread from proximal end of adoral zone and paroral membrane, run transversely leftwards forming a conical funnel about 20 μm long in vivo. Notes on Thai I population The Thai population matches very well the Vietnamese population. However, the Thai population shows a slightly wider range in the body length (95–175 μm vs 100–155 μm) and is slightly more slender than the Vietnamese population (length:width ratio 3.4–5.0:1 vs 3.0– 4.2:1). The variability of body size and shape of the Thai population is summarized in Table 1 and shown in Figs 2 A–J, 3B–C.