The search finds an end: the morphologically chimeric hysterocinetids belong to the subclass Hymenostomatia (Ciliophora: Oligohymenophorea) Author Obert, Tomáš Author Zhang, Tengyue Author Vďačný, Peter peter.vdacny@uniba.sk text Zoological Journal of the Linnean Society 2023 2023-06-29 199 1 97 123 http://dx.doi.org/10.1093/zoolinnean/zlad023 journal article 267062 10.1093/zoolinnean/zlad023 85827197-9d1f-40fa-919c-683b6170544c 0024-4082 8326247 EC132BF4-4C98-49D5-99D8-01ED24128481 Protoptychostomum simplex ( André, 1915 ) Raabe, 1949 ( Figs 1A–I , 2A–I ; Table 1 ) Synonymy: Anoplophrya simplex André 1915: 102 , fig. 1, host: not specified Lumbricidae . Ptychostomum simplex Heidenreich (1935: 530–531 , fig. 3) transferred A. simplex to Ptychostomum and described the living morphology of a German population, host: ‘ Allolobophora caliginosa ’. Possibly a misidentified Protoptychostomum davidis (Rees, 1962) , whose type host is ‘ A. caliginosa ’. Protoptychostomum simplex . Raabe (1949: 36 , fig. 5) transferred A. simplex to Protoptychostomum , host: ‘ Eiseniella tetraedra f. tipica (Savigny)’. Meier (1954: 234–236 , fig. 15) described the morphology of a German population (Mannhof), host: E. tetraedra . Kaczanowski (1961: 257–272 , figs 1–7, plate I) studied morphology, morphogenesis, and silver line system of a Polish population (Lubkowo), host: E. tetraedra . Raabe (1972: 129–130 , fig. 5A–E) revised the species. Figure 1. Protoptychostomum simplex in vivo (A) and asser protargol impregnation (B–I). A, less-side view of a representative specimen. B, F–I, less-side overviews, showing the variability of body shape and size as well as of the nuclear apparatus (shaded grey) and sucker (shaded yellow). Dashed circles in (I) represent contractile vacuoles. C, oral ciliary paưern. The paroral membrane and both membranelles extend along the whole posterior body end to plunge into the infundibulum where they form a helix-like paưern. Membranelle M1 is made up of two rows of basal bodies and is not segmented. Membranelle M2 runs beside M1, is composed of only a single row of basal bodies and is not segmented. Arrowhead in (C) marks the curved anterior end of membranelles M1 and M2. D, E, ciliary paưern of the less (D) and the right (E) side. CP, cytopharynx; CV, contractile vacuoles; M1–2, membranelle 1 and 2; MA, macronucleus; MI, micronuclei; PM, paroral membrane; S, sucker; SK, somatic kineties. Scale bars = 30 µm (I), 40 µm (A, B, D, E), 80 µm (H), 100 µm (F, G). Figure 2. Protoptychostomum simplex in vivo (A, B) and asser protargol impregnation (C–I). A, detail of the anterior body region, showing densely arranged basal bodies of somatic kineties and the unciliated V-shaped sucker lined by regularly spaced, oblique rows of highly refractive granules. B, detail of the posterior body region, showing the oral ciliature and the vacuolized cytoplasm. C, D, less-side overviews, showing the variability of body shape and size, as well as of the nuclear apparatus. E, F, I, details of the infundibular and peristomial ciliature, as well as of the nuclear apparatus, which is composed of an ellipsoidal macronucleus and two globular micronuclei. Arrowhead in (E, F) marks the curved anterior end of membranelles M1 and M2. G, somatic kineties are composed of narrowly spaced monokinetids. Asterisks mark irregularities in the somatic ciliary paưern. H, surface view, showing cortical granules. CP, cytopharynx; F, fibre; G, granules; M1–2, membranelle 1 and 2; MA, macronucleus; MI, micronuclei; OA, oral apparatus; OC, oral cilia; PM, paroral membrane; S, sucker; SK, somatic kineties. Scale bars = 5 µm (I), 10 µm (F, G), 15 µm (A, B), 40 µm (E), 50 µm (C, D). Table 1. Morphometric data on P. simplex (Ps) and H. bellerophon (Hb) isolated from lumbricid earthworms.

Characteristica	Mean	M	CV	SD	Min	Max	N
Body length	Ps	107.0	103.2	18.7	20.0	71.2	146.2	19
Hb	178.2	162.3	17.7	31.6	140.3	224.1	10
Body maximum width	Ps	61.0	61.4	13.3	8.1	48.4	77.8	19
Hb	96.6	98.9	19.7	19.0	62.9	126.3	10
Body length:width ratio	Ps	1.8	1.7	13.2	0.2	1.4	2.2	19
Hb	1.9	1.8	13.1	0.2	1.6	2.3	10
Distance of anterior body end to proximal end of sucker	Ps	7.1	6.5	19.4	1.4	5.3	9.3	10
Hb	23.9	21.7	27.3	6.5	16.3	34.5	6
Sucker maximum width	Ps	29.5	29.5	21.8	6.4	22.2	40.6	10
Hb	40.0	38.1	27.4	11.0	27.8	57.1	7
Length of ventral sucker arm	Ps	21.2	21.0	18.8	4.0	15.4	28.8	10
Hb	27.9	26.8	37.8	10.5	17.1	46.2	6
Length of dorsal sucker arm	Ps	21.5	22.0	14.1	3.0	17.5	26.3	6
Hb	22.4	21.6	31.2	7.0	15.1	34.8	6
Angle formed by sucker arms	Ps	96.7	97.6	4.2	4.0	86.3	101.7	10
Hb	83.8	84.5	9.3	7.8	75.0	93.0	6
Distance of anterior body end to beginning of macronucleus	Ps	51.9	50.3	18.5	9.6	37.6	76.4	19
Hb	37.8	33.8	30.2	11.4	20.9	55.5	10
Macronucleus length	Ps	30.9	30.6	20.9	6.5	22.1	44.1	19
Hb	103.9	98.5	22.7	23.5	64.0	140.2	10
Macronucleus width	Ps	23.5	23.1	19.3	4.5	14.5	35.3	19
Hb	20.5	21.5	33.7	6.9	10.1	32.4	10
Macronucleus length:width ratio	Ps	1.3	1.3	19.1	0.3	1.0	2.0	19
Hb	5.4	5.2	27.7	1.5	3.8	8.8	10
Number of macronuclear nodules	Ps	1.0	1.0	0.0	0.0	1.0	1.0	19
Hb	1.0	1.0	0.0	0.0	1.0	1.0	10
Micronucleus largest diameter	Ps	2.6	2.6	16.3	0.4	2.2	3.1	4
Number of micronuclei	Ps	2.0	2.0	0.0	0.0	2.0	2.0	4
Number of less ciliary rows	Ps	48.3	49.0	5.5	2.6	43.0	51.0	9
Hb	64.0	62.5	11.6	7.4	57.0	74.0	4
Number of right ciliary rows	Ps	78.5	78.5	75.0	82.0	2
Hb	104.6	103.0	7.2	7.5	95.0	114.0	5
Cytopharynx length	Ps	10.8	10.8	21.2	2.3	8.0	14.3	7
Distance in between ciliary rows	Ps	1.8	1.9	12.9	0.2	1.5	2.0	4
Hb	1.6	1.6	7.3	0.1	1.4	1.7	5
Peristome width	Ps	31.5	33.3	10.9	3.4	24.0	36.0	19
Hb	38.7	42.2	33.1	12.8	19.5	62.7	11
Distance of posterior body end to proximal end of peristome	Ps	18.5	18.6	17.4	3.2	12.2	24.1	16
(peristome length)	Hb	33.1	33.7	33.0	10.9	15.4	48.1	11

a Data based on protargol-impregnated specimens.Measurements in μm. Abbreviations:CV,coefficient of variation (%); M, median;Max,maximum; Mean, arithmetic mean;Min, minimum; N , number of individuals investigated;SD, standard deviation. Improved diagnosis (includes all information known): Body size about 85–200 × 50–90 µm in vivo . Body broadly ovoid to ovoid, with anterior end more narrowly rounded than posterior one. Anterior sucker forms an inverted, widened, V-shaped paưern, sucker arms about 15–28 µm long forming an angle of about 97°. Nuclear apparatus situated below midbody, composed of one macronucleus and two micronuclei. Two contractile vacuoles situated less of cell’s midline, anterior and posterior to macronucleus. On average, 48 less and 78 right meridional ciliary rows. Oral ciliature consists of a paroral membrane and two membranelles; paroral and both membranelles begin near ventral margin of posterior body side, extend along whole posterior body end to plunge into infundibulum, where they describe two complete turns of a spiral. Type locality: Lake Maggiore ( Fr. Lac Majeur ). The lake and its shoreline are divided between the Italian regions of Piedmont and Lombardy and the Swiss canton of Ticino . Type host: Not provided in the original paper ( André 1915 ). All reliable records ( Raabe 1949 , 1972 ; Meier 1954 ; Kaczanowski 1961 ; present study) come only from the semi-aquatic Eiseniella tetraedra (Savigny, 1826) . ND1 and COI sequences of Slovak E. tetraedra specimens, which harboured P. simplex , have been deposited in GenBank under the following accession numbers: OP752211–OP752212 and OP755297–OP755298, respectively. Type material: Deposition of type material not mentioned in the original paper ( André 1915 ) and is thus probaby unavailable. Voucher material: A DNA sample of a voucher specimen (CVsk 190 ET ) has been deposited in the Natural History Museum, Vajanského nábrežie 2, 810 06 Bratislava, Slovakia ( ID Collection Code 01426283) . Paratype slides containing protargol-impregnated specimens (reg. no. 2023/1–4- ZTY ) have been deposited at the Department of Zoology , Comenius University in Bratislava , Ilkovičova 6, 842 15 Bratislava , Slovakia . Gene sequences: The 18S rRNA gene, ITS region-28S rRNA gene, and 16S rRNA gene sequences of the voucher specimen CVsk 190 ET have been deposited in GenBank under the following accession numbers: OP755196, OP755170 and OP755221, respectively. Etymology: The Latin adjective simplex (simple) refers to the simple nuclear and contractile vacuole apparatus. Description: Two populations of P. simplex were found. Their conspecificity was confirmed by 18S, ITS region-28S, and 16S rRNA gene sequences. However, the description is based only on the populations from the riparian zone of the Kráľov potok stream in the village of Plavecké podhradie, Malacky district, Slovakia . Body size about 85–175 × 55‒90 µm, usually 130 × 75 µm, as calculated from some in vivo measurements and the morphometric data adding 20% preparation shrinkage ( Table 1 ). Body broadly ovoid to ovoid, with anterior end more narrowly rounded than posterior one; length:width ratio 1.4–2.2:1, near 2.0:1 both in vivo and in protargol preparations; distinctly laterally flaưened ( Figs 1A, B, D–I , 2C, D ; Table 1 ). Sucker occupies anterior body pole, more or less delimited by inconspicuous concavities on lateral body sides; forms an inverted, widened, V-shaped paưern. Angle formed by sucker arms about 97° on average; both arms of similar length, i.e. about 15–28 µm long asser protargol impregnation ( Figs 1A, B, D, F–I , 2C ; Table 1 ). Sucker unciliated and densely doưed by regularly arranged rows of granules; individual granules sometimes weakly impregnate with the protargol method used ( Fig. 2A, C ). No skeletal fibres developed. Nuclear apparatus situated below midbody, i.e. usually about 50 µm apart from anterior body end asser protargol impregnation; invariably composed of one macronucleus and two micronuclei. Macronucleus located in posterior body half, globular to broadly ellipsoidal, and 22–44 × 14–35 µm in size asser protargol impregnation; nucleoli small and more or less globular, evenly distributed over macronucleus. Micronuclei close to each other; consistently aưached to right side of macronucleus at its midportion; about 2.6 µm in diameter asser protargol impregnation; difficult to recognize in vivo , faintly and unevenly impregnated with the protargol method used ( Figs 1A, B, D, F–I , 2C–E ; Table 1 ). Invariably two contractile vacuoles situated less of the midline of the cell, anterior and posterior to macronucleus; excretory pores not observed either in vivo or asser protargol impregnation ( Fig. 1A, I ). Cortex flexible; not, or only slightly, furrowed by ciliary rows; no specific granules recognizable in vivo but faintly impregnated and irregularly scaưered granules sometimes recognizable in protargol preparations ( Fig. 2H ). Cytoplasm colourless; 5.5 µm-sized food vacuoles together with a vast number of densely spaced granules (about 0.8–1.0 µm in diameter) occupy an irregular area between posterior end of macronucleus and peristome ( Figs 1A , 2B ). Swims moderately fast, rotating about main body axis; dies in about half an hour asser extraction from host. Somatic ciliature holotrichous and composed of monokinetids. Somatic cilia about 9 µm long in vivo , narrowly arranged in an average of 48 less and 78 right meridional rows ( Table 1 ). Less ciliary rows start below posterior margin of sucker, while right ciliary rows begin at anterior margin of sucker. Individual ciliary rows densely spaced, i.e. approximately 2 µm apart from each other; frequently with irregularities on both less and right body side, e.g. some rows shortened anteriorly or posteriorly, and/or with short breaks causing a swap between two adjacent and sometimes even more distant ciliary rows ( Figs 1D, E , 2G ). No secant system recognizable in laterally oriented cells, possibly also due to slightly more irregular arrangement of basal bodies at posterior body pole. Oral apparatus located at posterior body pole and composed of an outer peristomial region and an inner infundibular part. Peristomial region follows the curvature of posterior body end, oriented perpendicularly to anteroposterior body axis, and approximately 32 µm wide asser protargol impregnation ( Table 1 ); outer oral cilia about 7 µm long in vivo . Infundibulum 12.2–24.1 µm long in protargol preparations, describing two complete turns of a spiral; infundibular cilia about 8.5 µm long in vivo , form nice metachronal waves. Oral ciliature consists of a paroral membrane and only two membranelles (M1 and M2). Paroral membrane and both membranelles begin near ventral margin of posterior body side, extend along whole posterior body end to plunge into infundibulum where they form a helix-like paưern. Membranelle M1 located anteriormost, made up of two rows of basal bodies, not segmented. Membranelle M2 runs beside M1 and hence both membranelles appear as a single ciliary structure, composed of only a single row of basal bodies, not segmented. Both membranelles anteriorly bent forming an inverted J-shaped paưern. Paroral membrane located posteriormost, distinctly separated from M1 and M2, consists of two rows of kinetosomes, not segmented and not hook-like curved anteriorly. Cytopharynx originates at proximal end of paroral membrane and both membranelles; runs towards anterior body end almost in parallel with main body axis; forms a slender, 8.0–14.3 µm-long funnel faintly impregnating with the protargol method used ( Figs 1A–D, F–I , 2B–F, I ; Table 1 ).