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 parapanesthiae ( Kidder, 1937 ) Figs 9–11 Description of Thai I population Size in vivo about 70–110 × 40–65 μm, usually 90 × 50 μm, as calculated from some in vivo measurements and morphometric data; length:width ratio very stable, i.e., ranging from 1.7:1 to 1.9: 1 in protargol preparations ( Table 4 ). Body broadly spade-shaped, widest slightly above posterior third of body, i.e., just above level of contractile vacuole.Anterior end bluntly pointed; posterior body portion differentiated into a conspicuous, moderately long peristomial projection; left margin distinctly notched at base of peristomial projection and recurved posteriorly toward peristomial projection, forming a distinct lobe; dorsoventrally flattened 1.3:1 ( Figs 9 A–N, 11C–D). Macronucleus located in second and third fourth of body, close to right body margin; tear-shaped with a length:width ratio of 2.1–3.4: 1 in protargol preparations; anterior end broadly rounded and near cell’s midline, posterior end acute and in parallel with right body margin; 27–47 × 12–17 μm in size after protargol impregnation; filled with numerous globular structures (presumably nucleoli) 0.6–1.6 μm in diameter after protargol impregnation, well observable in vivo and after protargol impregnation. Karyophore attached to right body margin near base of peristomial projection ( Table 4 ; Figs 9 A–L, 11C–D). Micronucleus not observed. Contractile vacuole in lobe of left body side, i.e., at level of base of peristomial projection ( Figs 9A, L , 11C ). Cortex flexible, no cortical granules recognizable. Cytoplasm colorless; finely granulated; numerous refractile bodies scattered throughout cytoplasm; some (symbiotic?) bacteria and/or archaea freely scattered throughout main body portion; few food vacuoles, about 2.5–5.2 μm across and containing prey prokaryotes ( Fig. 9A ). Swims slowly; dies quickly on microscope slides, possibly due to presence of oxygen; body shape changes in dying and strongly squeezed cells, i.e., lobe of left side becomes less noticeable. Fig. 9. Clevelandella parapanesthiae ( Kidder, 1937 ) . Thai I specimens isolated from Panesthia angustipennis angustipennis (Illiger, 1801) from life (A) and after protargol impregnation (B–N). A . Ventral view of a representative specimen, length 90 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. 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). Scale bars = 30 μm. Fig. 10. Clevelandella parapanesthiae ( Kidder, 1937 ) . Vietnamese specimens isolated from Panesthia angustipennis cognata Bey-Bienko, 1969 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 = 30 μm. Table 4. Morphometric data on Clevelandella parapanesthiae ( Kidder, 1937 ) from Thailand and Vietnam.

Characteristica	Pop	Mean	M	SD	SE	CO	Min	Max	n
Body, length	TH	78.8	79.5	10.1	3.2	12.8	60.0	93.0	10
VN	70.3	68.5	5.8	1.8	8.2	63.0	79.0	10
Body, maximum width	TH	44.2	44.0	6.1	1.9	13.8	34.0	54.0	10
VN	35.3	35.0	3.1	1.0	8.7	31.0	40.0	10
Body, length:width ratio	TH	1.8	1.8	0.1	0.0	3.3	1.7	1.9	10
VN	2.0	2.0	0.1	0.0	6.1	1.8	2.1	10
Peristomial projection, length	TH	19.0	19.0	2.5	0.8	13.4	15.0	23.0	10
VN	16.4	16.5	1.1	0.3	6.6	14.0	18.0	10
Peristomial projection, width	TH	10.6	10.0	0.8	0.3	8.0	10.0	12.0	10
VN	12.0	12.0	0.9	0.3	7.9	11.0	14.0	10
Peristomial projection, % of body length	TH	24.2	24.5	1.8	0.6	7.6	21.3	28.0	10
VN	23.4	23.5	1.4	0.4	5.9	21.3	25.4	10
Peristomial opening, length	TH	16.2	16.0	2.1	0.7	13.3	13.0	20.0	10
VN	15.5	15.0	1.6	0.5	10.6	13.0	18.0	10
Peristomial opening, width	TH	7.6	7.5	0.7	0.2	9.2	7.0	9.0	10
VN	8.6	9.0	1.3	0.4	14.7	6.0	10.0	10
Peristomial opening, % of body length	TH	20.6	20.4	1.4	0.4	6.6	18.0	23.2	10
VN	22.0	22.4	1.0	0.3	4.7	20.0	23.1	10
Adoral zone of membranelles, length	TH	37.1	37.0	2.4	0.8	6.5	34.0	41.0	10
VN	34.4	34.5	2.7	0.8	7.8	30.0	39.0	10
Adoral zone of membranelles, % of body length	TH	47.7	46.7	5.7	1.8	12.0	41.2	56.7	10
VN	49.1	48.8	4.8	1.5	9.8	43.6	58.2	10
Anterior body end to macronucleus, distance	TH	18.5	18.0	3.8	1.2	20.4	10.0	24.0	10
VN	14.5	13.5	2.2	0.7	15.0	12.0	18.0	10
Posterior body end to macronucleus, distance	TH	25.1	26.0	3.4	1.1	13.5	21.0	30.0	10
VN	30.3	30.5	3.6	1.1	11.9	24.0	37.0	10
Macronucleus, length	TH	39.5	40.5	5.6	1.8	14.1	27.0	47.0	10
VN	30.9	31.0	3.0	0.9	9.6	26.0	35.0	10
Macronucleus, width	TH	14.4	15.0	2.0	0.6	14.0	12.0	17.0	10
VN	15.0	14.5	1.4	0.4	9.4	13.0	17.0	10
Macronucleus, length:width ratio	TH	2.8	2.7	0.4	0.1	12.9	2.1	3.4	10
VN	2.1	2.0	0.2	0.0	7.4	1.9	2.3	10
Macronucleus, number	TH	1.0	1.0	0.0	0.0	0.0	1.0	1.0	10
VN	1.0	1.0	0.0	0.0	0.0	1.0	1.0	10
Adoral membranelles, number	TH	37.6	39.0	3.0	0.9	7.9	30.0	40.0	10
VN	36.0	36.5	2.8	0.9	7.9	30.0	39.0	10

a Data based on mounted, protargol-impregnated, and randomly selected specimens. Measurements in µm. Fig. 11. Clevelandella parapanesthiae ( Kidder, 1937 ) . Vietnamese specimens (A–B, E–G) isolated from Panesthia angustipennis cognata Bey-Bienko, 1969 and Thai I specimens (C–D) isolated from Panesthia angustipennis angustipennis (Illiger, 1801) from life (A, E–G) and after protargol impregnation (B–D). A–D . Ventral views of specimens with well-preserved body shape. Black arrowheads mark the proximal end of the adoral zone of membranelles. E–G . A strongly squeezed specimen by pressure of the cover slip, causing the body to become markedly wider and the notch at the base of the peristomial projection to be lost. The general body organization is shown in (E), the ciliary pattern of ventral and dorsal sides is shown in (F) and (G). Asterisks mark the position of the ciliary whorl (posterior suture), white arrowhead denotes the karyophore attaching to right body margin, white double arrowhead denotes the right suture. Scale bars = 30 μm. Somatic ciliature holotrichous; cilia about 4.5–6.0 μm long in vivo and very narrowly arranged. About 90 ciliary rows narrowly spaced over entire body surface and about 25 ciliary rows running onto peristomial projection. Peristomial ciliary rows in a form of strongly oblique lines in ventral view, while in a form of shallow arcs in dorsal view ( Fig. 9 M–N). Almost all body ciliary rows begin from a whorl (posterior suture) on left body side slightly above base of peristomial projection, i.e., near location of contractile vacuole ( Fig. 9 M–N, arrowheads) to radiate over ventral and dorsal sides toward right body margin; some kineties shortened anteriorly or posteriorly. Right suture extends from base of peristomial projection to anterior body end; formed by obliquely abutting ventral and dorsal ciliary rows ( Fig. 9M , arrow). Peristomial projection conspicuous because it occupies on average 24% of body length and measures 15–23 × 10–12 μm in protargol preparations. Peristomial opening situated on left side of peristomial projection, roughly triangular and slender, i.e., about 21% of body length and 13–20 × 7–9 μm in size after protargol impregnation ( Figs 9 A–M, 11C–D). Peristomial funnel about 37 μm long in protargol preparations. Adoral zone extends slightly obliquely from distal end of peristomial projection across right side of peristomial funnel to terminate about in half of body length; occupies 41% to 57% of body length; composed of on average 38 membranelles; cilia of distalmost membranelles about 8 μm long in vivo and projecting out of peristomial funnel ( Table 4 ; Figs 9A, L , 11 C–D). 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 about at level of proximal end of peristomial opening and terminates near cytostome at proximal end of peristomial funnel ( Fig. 9L ). Pharyngeal fibres originate from proximal end of adoral zone and paroral membrane, run transversely leftwards forming a conical funnel about 25–30 μm long in vivo. Notes on Vietnamese population The Vietnamese population exhibits similar morphological features as the Thai population. However, the Vietnamese population shows a slightly narrower range in the body length (70–90 μm vs 70–110 μm) and is slightly more slender (length:width ratio 1.8–2.1:1 vs 1.7–1.9:1) than the Thai population. The macronucleus is shorter in some Vietnamese specimens, but its variability completely falls within the range of the Thai population (length 26–35 μm vs 27–47 μm). The variability of body size and shape of the Vietnamese population is summarized in Table 4 and shown in Figs 10 A–J, 11A–B, E–G.