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European Journal of Protistology 59 (2017) 34–49

Morphological descriptions of five including one new species of Falcicyclidium Xinpeng Fana,b, Yuan Xuc, Jiamei Jiangd, Khaled A.S. Al-Rasheide, Yangang Wanga, Xiaozhong Hua,∗ aInstitute of Evolution & Marine Biodiversity, University of China, Qingdao 266003, China bSchool of Life Sciences, East China Normal University, Shanghai 200241, China cState Key Laboratory of Estuarine and Coastal Research, East China Normal University, Shanghai 200062, China dCollege of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306, China eZoology Department, King Saud University, Riyadh 11451, Saudi Arabia

Received 14 April 2016; received in revised form 7 March 2017; accepted 9 March 2017 Available online 16 March 2017

Abstract

Five scuticociliates, collected from China, were morphologically studied using standard methods One represents a new member of the genus Falcicyclidium, F.citriforme nov. spec., which can be recognised mainly by a combination of the following characters: usually two macronuclear nodules, buccal field about half of body length, ten somatic kineties, about 22 kinetal units in somatic kinety 1 and n, and excretory pore near posterior end of somatic kinety n. A redescription for Biggaria bermudensis was provided to include the feature of scutica and argyrome based on new isolate, and variations between different isolates were also discussed. The new population of Sathrophilus holtae differs from the type population by two postoral kineties and fewer kinetal units in the scutica. Its stomatogenesis belongs to the scuticobuccokinetal type, which shows similarities with Dexiotricha among loxocephalids. Morphometric data and brief descriptions were supplied for another two species, i.e., Protocyclidium citrullus and Cyclidium varibonneti. © 2017 Elsevier GmbH. All rights reserved.

Keywords: Biggaria; Falcicyclidium citriforme nov. spec; Morphology; Sathrophilus; Stomatogenesis

Introduction of aquatic animals (Jones et al. 2010; Lobban et al. 2011; Xu et al. 2015). In the past two decades, an extensive body Scuticociliates are one of the most abundant groups of work on the ciliate fauna in Chinese coastal waters has in various biotopes worldwide, and play a vital role in the revealed extremely high diversity of this group of organ- microbial food web (Buosi et al. 2011; Durán-Ramírez et al. isms (e.g. Fan et al. 2011a,b; Gong and Song 2008; Liu 2015; Foissner et al. 2002, 2003; Rossi et al. 2015; Song et al. 2016; Long et al. 2006, 2007a,b; Miao et al. 2010; Pan et al. 2009). They also exist as symbionts, or even pathogens, et al. 2016, 2011, 2015a,b,c; Song 2000; Song et al. 2003; Wang et al. 2008a,b,c, 2009; Xu et al. 2015). Meanwhile, those species that lack detailed documentation, especially ∗Corresponding author. Fax: + 86 532 8203 1610. pleuronematids and philasterids have been re-investigated E-mail address: [email protected] (X. Hu). http://dx.doi.org/10.1016/j.ejop.2017.03.003 0932-4739/© 2017 Elsevier GmbH. All rights reserved. X. Fan et al. / European Journal of Protistology 59 (2017) 34–49 35 using standard methods in order to circumscribe morphos- differential interference contrast microscopy. The protar- pecies (e.g. Fan et al. 2011b; Song and Wilbert 2000, 2002; gol (Wilbert 1975) and Chatton-Lwoff silver-nitrate staining Song et al. 2002a,b; Wang 2009), and analyses based on mor- methods (Song and Wilbert 1995) were used to reveal the phogenesis and molecular phylogeny have been increasingly infraciliature and argyrome (silverline pattern), respectively. undertaken to answer the systematic questions within this Drawings of stained specimens were made with the help of a subclass (e.g. Gao et al. 2012, 2013, 2014; Ma and Song camera lucida. Measurements were made under 100–1250× 2003; Ma et al. 2003, 2006; Pan et al. 2011; Song et al. 2005; magnification. Terminology and classification mainly follow Zhang et al. 2011). Corliss (1979) and Lynn (2008), respectively. Cyclidium-like genera, e.g., Cyclidium Müller, 1773, Fal- cicyclidium Fan et al., 2011, Protocyclidium Alekperov, 1993, and Acucyclidium Gao et al., 2014, share similar cil- iary pattern, but meanwhile, differences in both oral apparatus Results and Discussion and living morphology are gradually recognised and raised Subclass Scuticociliatia Small, 1967 as species- or genera-dependent as taxonomy and phyloge- Order Fauré-Fremiet in Corliss, 1956 netic work are accumulated (Fan et al. 2011a,b; Foissner Family Ctedoctematidae Small and Lynn, 1985 et al. 2002; Gao et al. 2014). Hence, careful reinvestigation Falcicyclidium Fan et al., 2011 of known species may contribute equally as exploring new Falcicyclidium citriforme nov. spec. (Figs 1 A–H, 2 A–N, taxa in understanding the species diversity of this group (Fan Table 2) et al. 2011b; Foissner et al. 1994; Song 2000). Sathrophilus Corliss, 1960 was redefined by Long et al. Synonyms. Falcicyclidium plouneouri (Dragesco, 1963) (2007b), and contains about 15 nominal species so far. Infra- Gao et al., 2014 (misidentification and incorrect combination ciliature data of nearly half of them are available (Fanetal. with Falcicyclidium; see discussion); Cyclidium plouneouri 2010). The morphogenetic information, however, is limited, Dragesco, 1963 sensu Wang (2009) (misidentification; see because only few dividing stages of S. holtae were docu- discussion).

mented (Long et al. 2007b). This resulted in the confusion Diagnosis. Marine Falcicyclidium, in vivo about related to its systematic position (Zhang et al. 2011). 50 × 30 ␮m, lemon-shaped in ventral view, dorso-ventrally The genus Biggaria was first established by Kahl (1934), flattened about 2:1. Buccal field occupying half body but it was nomen nudum until Aescht (2001) fixed the length. 10 somatic kineties, kinety 1 with about 22 kinetal type species, B. bermudensis. The type species had been units, kinety n with about 22 kinetal units and posteriorly described frequently, but sufficient redescription based on shortened. Approximately nine caudal cilia. Usually two modern method is still lacking (Berger 1964; Biggar and macronuclear nodules in anterior half. Contractile vacuole Wenrich 1932; Lucas 1940; Nie 1934; Powers 1933, 1935). posteriorly positioned on ventral side and with excretory As a part of faunistic surveys in coastal water pore posterior to somatic kinety n. of China, the present study documents five scuticociliates, Etymology. The species-group name citriforme (Latin including one new Falcicyclidium species. Morphological adjective, lemon-shaped) recalls the body shape of this traits from live specimen, morphometric data, and ecological species. features are supplied. Type locality. Sandy beach in Qingdao, China (36◦0318N; 120◦2037E; details see Table 1). Ecological features. This species probably typically Material and Methods inhabits sandy sediments. It was found in all surveyed sandy beaches in Qingdao near the Yellow Sea. It was also collected Specimens of Falcicyclidium citriforme nov. spec. and once from a sandy beach in Daya Bay, Huizhou, southern Sathrophilus holtae were collected from sandy beaches in China. China (Table 1). Fine sand containing seawater was taken Deposition of type slides. The protargol-stained slide directly from the surface of sediments and ciliates were containing the holotype specimen (Figs 1 G, H, 2 F, G; regis- flushed out with the help of a strainer. Protocyclidium citrul- tration number: wyg-20051130-01) and two paratype slides lus and Cyclidium varibonneti were collected from seawater, with silver nitrate-stained specimens (registration numbers: which was taken directly with a 500 ml jar. Ciliates became wyg-20051130-02, wyg-20051130-03) had been deposited abundant several days after rice grains were added to the in the Laboratory of Protozoology, Ocean University of raw culture to enrich bacterial food. Biggaria bermudensis China, Qingdao, China. The holotype has been marked by was isolated from the digestive tracts of sea urchins, Hemi- an ink circle.

centrotus pulcherrimus. The host was dissected and then its Description based on Qingdao population. Living mantle was flooded with filtered, ciliate-free marine water. cells about 40–60 × 20–40 ␮m in size; lemon-shaped when For details of the sampling information see Table 1. viewed ventrally (Figs 1 A, 2 A–C), dorsoventrally flat- Cells were isolated with a fine pipette under a dissec- tened about 2:1; anterior end truncated, posterior end broadly tion microscope and were then observed in vivo using rounded (Figs 1 A, B, 2 D, E). Buccal field prominent, 36 X. Fan et al. / European Journal of Protistology 59 (2017) 34–49

Fig. 1. A–L Falcicyclidium citriforme nov. spec. (A–H) and its closely related species (I–L), in vivo (A, B), after protargol preparation (C, F–I, K) and silver nitrate staining (D, E, J, L). A, B. Left lateral (A) and dorsal view (B) of representative individual, arrowheads indicate the multiple caudal cilia. C. Right lateral view showing basal bodies of caudal cilia and barren area (arrow), arrowhead marks the slightly longer somatic kinety 1. D. Ventral view, showing buccal apparatus, extrusomes and excretory pore. E. Part of dorsal argyrome. F. Detail of buccal ciliature. G, H. Ventral (G) and dorsal (H) ciliature of the holotype specimen. I. Cyclidium plouneouri (from Dragesco 1963). J. Cyclidium plouneouri (from Wilbert 1986). K. Falcicyclidium fangi (from Fan et al. 2011a). L. Cyclidium borrori (from Small and Lynn 1985). CC, caudal cilia; EP, excretory pore; Ex, docking sites of extrusomes; M1–3, membranelles 1, 2 and 3; PM, paroral membrane; Sc, scutica; SK1,

n, somatic kineties 1 and n. Scale bars = 25 ␮m. X. Fan et al. / European Journal of Protistology 59 (2017) 34–49 37

Table 1. Sampling information for the five species.

Species Sampling date Sampling location Habitat and salinity

Falcicyclidium citriforme nov. spec. 30 November, 2005 No. 1 Bathing Beach, Qingdao, Typical sandy sediment; 26‰ China (36◦0318N; 120◦2037E) Protocyclidium citrullus 5 August, 2009 Estuary of Baisha River, Seawater; 21‰ Qingdao, China (36◦1510”N; 120◦1931E) Cyclidium varibonneti 15 December, 2009 Shrimp culture pond, Zhanjiang, Seawater; 25‰ China (20◦5515N; 110◦3054E) Sathrophilus holtae 24 October, 2007 Shilaoren Bathing Beach, Typical sandy sediment; 28‰ Qingdao, China (36◦0552N; 120◦2846E) Biggaria bermudensis 26 April, 2007 Hosts from Qingdao, China Digestive tracts of sea urchin (36◦0546N; 120◦3003E) Hemicentrotus pulcherrimus; 30‰ occupying 47%–67% (54% on average, n = 15) of body length transversely oriented kinetosome rows (Figs 1 F, 2 F). Dis-

in protargol-stained specimen, with triangular protrusion on tance between M3 and bottom end of PM about 9 ␮m, ca. 40% left border (Figs 1 A, 2 C). Pellicle rigid, conspicuously of buccal field length (Fig. 1D, F, G). Scutica arranged in two notched (Figs 1 A, 2 D). Cytoplasm colourless, contain- groups, each usually comprising two kinetosomes (Fig. 1F). ing food granules and crystals, especially in posterior half Argyrome composed of rectangular meshes (Figs 1 D, E of body (Fig. 2A, C, E). Usually two, rarely one or up to four 2 J–L). Posterior part of PM subtended by eight or nine oral macronuclear nodules, in anterior half of body, accompanied ribs (Figs 1 D, 2 K). Docking sites of extrusomes arranged in by single micronucleus (Figs 1 G, H 2 H, I, M). Contractile line between ciliary rows (Figs 1 D, 2 N).

vacuole about 10 ␮m across, near posterior end of ventral SSU-rRNA gene sequence and phylogenetic position. side, pulsating at 2 min intervals (Figs 1 A 2 C). Excretory The sequence was previously published in Gao et al. (2014) pore at posterior end of kinety left of buccal field (Figs 1 D, and was already deposited in GenBank with accession num-

2 J). Somatic cilia about 11 ␮m long, densely spaced anteri- ber FJ868181 (see discussion). For the phylogenetic position orly and loosely posteriorly, invisible in vivo in mid-portion of Falcicyclidium citriforme nov. spec., therefore, see the of body on dorsal side (Figs 1 A, 2 B, D, E). Approximately trees in Gao et al. (2014).

nine caudal cilia, each about 20 ␮m long (Figs 1 A, B, 2 A, B, Discussion. Wang (2009) described this form as Cyclidium D, E). Extrusomes undetectable in vivo, but present because plouneouri. Recently, Gao et al. (2014) sequenced the SSU- docking sites recognisable in silver nitrate preparations (Figs rRNA gene using cells from the same population (noted as 1 D, 2 N). Movement by rotating fast around its main axis. Falcicyclidium plouneouri isolate 1 in their publication) as Most of time, cells keep still on substrate or in water for quite well as another two populations from China (noted as F.plou- a few minutes with paroral membrane expanded and lateral neouri isolates 2 and 3 in their publication) and transferred side up. When disturbed, cells start to swim again. it to the genus Falcicyclidium, based on the description of Consistently 10 somatic kineties (SKs) arranged longi- Wang (2009) and their phylogenetic analyses. After carefully tudinally. Kinety right of buccal field (SK1) composed of reinvestigating specimens of the above-mentioned population about 22 kinetal units, including 7–9 posterior monokinetids of Wang (2009), however, we found that it is quite different (Fig. 1C, G). Kinety left of buccal field (SKn) containing from the original report of Cyclidium plouneouri by Dragesco 20–24 (21.8 on average, n = 12) kinetal units and three or (1963), mainly in the ratio of the buccal field to body length four of which are monokinetids posteriorly; kinety left of (0.54 vs. 0.79), and in having fewer somatic kineties (10 SKn (SKn-1) containing 22–26 (24 on average, n = 12) kine- vs. 14–16). Another minor difference lies in the number of tal units (Figs 1 G, 2 F). Other SKs contained dikinetids in kinetal units in SK1 and SKn (21.8 and 21.9 in the present anterior half of body and about six or seven monokinetids species vs. 17 and 13 in the type population of C. plouneouri; in other half (Figs 1 C, H, 2 G). Kinetosomes near rear end data counted from original figure) (Fig. 1I; Table 3). Thus, of body heavily stained and possibly correspond to caudal both Wang (2009) and Gao et al. (2014) misidentified this cilia in vivo (Fig. 1C). Paroral membrane (PM) distinctively organism and it actually represents a new species, which we curved at posterior part (Fig. 1F), distinctively sail-like when named Falcicyclidium citriforme nov. spec. Considering the cilia extended and motionless (Figs 1 A, 2 B, D). Three oral misidentification, the combination of Cyclidium plouneouri membranelles (M) irregularly shaped: M1 and M2 longitudi- with Falcicyclidium by Gao et al. (2014) was premature, nal, composed of two, and two or three rows of kinetosomes, because the existence of multiple caudal cilia, one of the respectively; M3 containing a few scattered kinetosomes and 38 X. Fan et al. / European Journal of Protistology 59 (2017) 34–49

Fig. 2. A–N Falcicyclidium citriforme nov. spec. in vivo (A–E) and after staining with protargol (F–I, M) and silver nitrate (J–L, N). A–E. Left lateral (A, B, E), ventral (C) and right lateral (D) view of typical individuals, arrowheads mark the caudal cilia, arrow indicates the contractile vacuole, double-arrowhead refers to the triangular protrusion on left border of buccal field. F, G. Ventral (F) and dorsal (G) ciliary pattern of the holotype. H, I, M. Variability of nuclear apparatus. J. Left lateral view, arrowhead shows the excretory pore located near the posteriorly shortened somatic kinety n. K. Ventral view, showing buccal apparatus and oral fibres. L. Dorsal argyrome. N. Apical view showing somatic

kineties and extrusomes. M1–3, membranelles 1, 2 and 3; Ma, macronucleus; Mi, micronucleus. Scale bars = 30 ␮m. diagnostic characters of Falcicyclidium does not apply for 12 vs. 10), fewer monokinetids in the somatic kineties on dor- the type population of C. plouneouri (Dragesco 1963). sal side (3 vs. 6 or 7), and sparsely arranged kinetal units in Wilbert (1986) also reported an isolate of Cyclidium plou- SK1 (16 vs. ca. 22) and SKn (11 vs. ca. 22) (Fig. 1J; Table 3). neouri. Although its paroral membrane has a similar shape The type species of Falcicyclidium, F. fangi Fan et al., to that of our organism, Wilbert’s specimens differ from the 2011, has a similar body shape and size, and an identical new species in having slightly more somatic kineties (11 or number of somatic kineties, and therefore it may easily be X. Fan et al. / European Journal of Protistology 59 (2017) 34–49 39

Table 2. Morphometric characterization of Falcicyclidium citriforme nov. spec. (Fa), Protocyclidium citrullus (Pc), Cyclidium varibonneti (Cv), Sathrophilus holtae (Sh) and Biggaria bermudensis (Bi).

Charactera Species Min Max M Mean SD CV (%) n

Body length Fa 48 64 54 55.0 4.3 7.9 15 Pc 20 25 21.5 22.0 1.8 8.3 21 Cv 16 22 19.5 18.9 1.9 10 20 Sh 80 110 94 93.5 8.7 9.3 25 Bi 127 202 155 157.7 16.7 10.6 15 Body width Fa 38 48 40 41.6 3.4 8.1 15 Pc 12 16 14 14 1.2 8.3 21 Cv 11 19 14 14.2 2.2 15.3 20 Sh 30 50 38 40.2 6.4 16.0 25 Bi 55 85 70 69.8 10.2 14.7 15 Length of buccal field Fa 26 36 28 29.5 3.3 11.1 15 Pc 13 15 14 14.2 0.7 5.3 21 Cv 10 14 11 11.4 1.2 10.8 20 Sh 16 21 19 18.9 1.6 8.2 25 Bi–––– –– – Somatic kineties, number Fa 10 10 10 10 0 0 13 Pc 15 15 15 15 0 0 21 Cv 11 12 11 11.3 0.5 4.2 20 Sh 19 23 20 20.6 1.4 6.6 25 Bi 40 52 44 43 3.5 7.9 15 Kinetal units in somatic kinety 1 Fa 20 25 21 21.6 1.5 6.7 13 Pc 15 17 16 16.3 0.7 4.5 21 Cv 12 15 14 14 0.8 5.4 20 Sh–––– –– – Bi–––– –– – Macronuclear nodules, number Fa 1 4 2 2.5 1.0 39.6 18 Pc1111 00 25 Cv 1 3 2 2.0 0.3 16.2 20 Sh1111 00 25 Length of macronuclear nodule Fa 10 26 16 15.8 4.6 29.2 13 Pc 7 10 8 8.3 0.8 9.6 21 Cv 2.0 7.0 5 5.0 1.0 19.5 20 Sh 15 35 24 24 4.4 18.3 25 Bi 24 34 27 28.1 3.1 11.0 15 Width of macronuclear nodule Fa 8 18 12 11.2 3.0 27.0 13 Pc 4 8 6 5.7 1.0 17.6 21 Cv 3.0 6.0 5 4.4 1.0 22.6 20 Sh 10 28 20 18.9 4.3 22.8 25 Bi 17 38 26 26.3 4.9 18.5 15

Measurements in ␮m. CV, coefficient of variation; M, median; Max, maximum; Mean, arithmetic mean; Min, minimum; n, number of specimens investigated; SD, standard deviation. a Data based on protargol-stained specimens. confused with the new species. Falcicyclidium fangi can be occupying 40% of the buccal field length) (Fan et al. 2011a; distinguished from F. citriforme nov. spec., however, by pos- Fig. 1K; Table 3). Their separation as different species was sessing a longer buccal field (ratio of buccal field length to also indicated by the phylogenetic tree in Gao et al. (2014). body length of 75% vs. 54%) and more kinetal units in SK1 Cyclidium borrori Small and Lynn, 1985; also has an oral (ca. 35 vs. ca. 22). Moreover, the distance from M3 to the infraciliature that is similar to Falcicyclidium, but differs from rear end of the paroral membrane is greater in F. fangi than our new species in consistently having one macronuclear nod-

in the new species (about 15 ␮m measured from the figure, ule (vs. 1–4, 2 on average), more somatic kineties (12 or 13

occupying 50% of the buccal field length vs. about 9 ␮m, vs. 10) and fewer kinetal units in SK1 (17 vs. ca. 22) and 40 X. Fan et al. / European Journal of Protistology 59 (2017) 34–49

Table 3. Comparison of Falcicyclidium citriforme nov. spec. with similar species.

Character F. citriforme F. fangi C. plouneouri C. plouneouri C. borrori Present work Fan et al. (2011a,b) Dragesco (1963) sensu Wilbert (1986) Borror (1965)

Body length, in ␮m 48–64 38–57 30–40 25–37 25–30 Ratio of buccal field to 0.54 0.75 0.79a 0.63a 0.54a body length Somatic kineties, number 10 10 14–16 11 or 12 12 or 13 Kinetal units in somatic 21.8 34.8 17a 16a 17a kinety 1b Kinetal units in somatic 21.9 22a 13a 11a 15a kinety nb Macronucleus nodules, 1–4 1 1–4 1 or 2 1 number Monokinetids in the 6or7 ca.8 – 3a – middle somatic kinety on dorsal side

a Measured from figures. b Dikinetid accounted as single kinetal unit.

SKn (15 vs. ca. 22) (Borror 1965; Fig. 1L; Table 3). Cyclid- somatic kineties on dorsal side containing about six or seven ium borrori and C. plouneouri sensu Wilbert (1986) are also dikinetids in anterior half and five monokinetids in poste- both potential members of Falcicyclidium, but they cannot rior half (Fig. 4B). M1 small, composed of two longitudinal yet be finally transferred to the genus until the existence of rows of kinetosomes; M2 conspicuous, comprising about multiple caudal cilia is confirmed. nine transverse rows, which gradually become longer; M3 Although Acucyclidium atractodes (Fan et al., 2011) Gao oriented obliquely (Figs 3 G, H, 4 A). Scutica composed of et al., 2014; has a similar ciliary pattern, it differs from the three dikinetids at posterior end of SKn and paroral mem- new species in having an armed pellicle and distinct spines brane (Figs 3 G, H, 4 A). One spherical macronucleus near at both ends (Fan et al. 2011a; Gao et al. 2014). fore-end, accompanied by single micronucleus (Figs 3 I, J, 4 Protocyclidium citrullus (Cohn, 1866) Foissner et al., 2002 B). (Figs 3 A–J, 4 A, B, Table 2) Deposition of voucher slide. Discussion. This species was transferred from Cyclid- ium to Protocyclidium Alekperov, 1993 by Foissner et al. The voucher slide containing protargol-stained specimens (2002) because of its multiple horizontally orientated kineties (registration number: FXP-20090805-01) had been deposited in membranelles 2 and 3. So far, many populations have in the Laboratory of Protozoology, Institute of Evolution and been morphologically described from Europe, Asia, Amer- Marine Biodiversity, Ocean University of China, Qingdao, ica, Oceania and Africa, which may suggest this species has China. a global distribution (e.g. Agamaliev 1978; Coats and Clamp

Description of Chinese population. Cells in vivo about 2009; Czapik 1963; Esteban et al. 2000; Song and Wilbert

× ␮ 25 13 m in size, slightly dorsoventrally flattened, lemon- 2002; Song et al. 2009; Wilbert 1995). Our population cor-

shaped in ventral view (Fig. 3A–E). Buccal field occupying responds well with previous descriptions in terms of general ␮

60% of body length and paroral cilia about 10 m in length morphology. We discovered, however, that our form, from the ␮

(Fig. 3B, C). Several food vacuoles (ca. 5 m across), and estuary of the Baisha River, is almost identical with the West- ␮ flat circular granules that could be alveoli (2–3 m across) ern European, Oceanian and African populations, in having

present in cytoplasm (Fig. 3B, F). Contractile vacuole about a few densely arranged monokinetids at the posterior end of ␮

8 m across when fully expanded, sub-terminally positioned SKn-1 (Fig. 4A), a feature that is lacking in the population ␮

(Fig. 3E). Somatic cilia about 14 m long (Fig. 3D). Single from Egypt (Wilbert 1995). This implies the possibility of ␮ caudal cilium 25–30 m long (Fig. 3E). Cells move abruptly, different species or subspecies among these C. citrullus pop- then stop and keep still for a short time with their ventral side ulations. facing up. Consistently 15 somatic kineties (Figs 3 G, H, 4 A, B): Cyclidium varibonneti Song, 2000 (Figs 3 K–P, 4 C, D, SK1 extending for whole body length and composed of Table 2) 10–12 dikinetids in anterior part and about four monokinetids Remarks. This species was well described by Song (2000), in remainder (Fig. 4A); somatic kineties on left of buccal and therefore only a brief description of our population was field short (Fig. 4A); SKn shortened posteriorly, terminat- provided here. ing level with cytostome (Fig. 4A); SKn-1 containing five Deposition of voucher slide. The voucher slide con- closely arranged monokinetids at its posterior end (Fig. 4A); taining protargol-stained specimens (registration number: JJM-2009121504) had been deposited in the Laboratory of X. Fan et al. / European Journal of Protistology 59 (2017) 34–49 41

Fig. 3. A–P Protocyclidium citrullus (A–J) and Cyclidium varibonneti (K–P) in vivo (A–F, K–M) and after protargol staining (G–J, N–P). A–E. Different individuals, arrowheads in (A, E) mark the long caudal cilium, arrows in (B, C) indicate the buccal cilia, arrows in (D, E) refer to the contractile vacuole. F. Showing the flat, circular shaped granules which could be aveoli (arrowheads). G, H, J. Ventral (G, H) and dorsal (J) view of ciliary pattern, arrowhead in (H) marks the densely arranged kinetosomes in the posterior end of somatic kinety n-1. I. Macronucleus (Ma) and micronucleus (arrow). K–M. Three individuals, arrowheads in (K) shows the food granules, arrow in (L) marks the

caudal cilium, arrow in (M) depicts the barren truncated anterior end. N, O. Ventral (N) and dorsal (O) view of ciliary pattern. P. Macronucleus.

Ma, macronucleus; M1–3, membranelles 1, 2 and 3; Sc, scutica. Scale bars = 15 ␮m (A, D, E); 10 ␮m (L).

Protozoology, Institute of Evolution and Marine Biodiversity, in SKn-1 arranged slightly more closely than those in other

Ocean University of China, Qingdao, China. somatic kineties (Figs 3 N, 4 C); kineties on dorsal side com-

Description. Cells in vivo measuring 20–25 × 10–15 ␮m posed of six or seven dikinetids in anterior half, and three or in size, oval outline in ventral view, and not flattened four posterior monokinetids (Figs 3 O, 4 D). Oral apparatus (Fig. 3K–M). Food granules of equal size often observed as described originally; scutica possibly composed of three (Fig. 3K). Macronucleus consisting of two closely attached dikinetids arranged closely beneath posterior end of paroral nodules, in anterior half of body (Fig. 3K, P). Distinct pellicle membrane (Figs 3 N, 4 C). ridges present between somatic kineties (Fig. 3M). Somatic Discussion. So far, reports of this species are limited

cilia slightly longer than extended paroral cilia, about 10 ␮m to China Seas (Song 2000). Compared with the type pop-

long. Caudal cilium about 20 ␮m in length (Fig. 3L). ulation from Yellow Sea in northern China, the current Eleven or 12 somatic kineties: SK1 containing 14 kine- population from South China Sea has identical diagnos- tal units; SKn posteriorly shortened, but extending beyond tic characters including ciliature details, e.g., the SKn was paroral membrane (Figs 3 N, 4 C); posterior five kinetids shorter but extending beyond the paroral membrane, and the 42 X. Fan et al. / European Journal of Protistology 59 (2017) 34–49

Fig. 4. A–D Protocyclidium citrullus (A, B) and Cyclidium varibonneti (C, D) after protargol preparation. A, B. Ventral (A) and dorsal (B) view of ciliary pattern, arrowhead indicates the slightly dense arrangement of kinetosomes in posterior part of somatic kinety n-1. C, D. Ventral (C) and dorsal (D) view of the ciliary pattern, arrow indicates the five densely arranged kinetosomes at the posterior end of the somatic

kinety n-1. M1–3, membranelles 1, 2 and 3; PM, paroral membrane; Sc, scutica; SK1, n, somatic kineties 1 and n. Scale bars = 10 ␮m. monokinetids of SKn-1 were narrowly arranged (but not as anterior (SFa) and posterior (SFp) part, and as they moving tight as those in Protocyclidium citrullus), and thus species backwards, the posterior end of the unchanged paroral mem- identification is not in doubt. brane generates new kinetosomes, possibly to form the new Order Small, 1967 scutica of the proter (Figs 5 K, 6 B); (iii) the proter inher- Family Cinetochilidae Perty, 1852 its the parental membranelles and, meanwhile, the new oral Sathrophilus Corliss, 1960 apparatus for the opisthe is formed, despite the fact that the Sathrophilus holtae Long et al., 2007 (Figs 5 A–M, 6 A–D, Table 2) postoral kineties and other somatic kineties are still dividing Remarks. Long et al. (2007b) has documented both the (Figs 5 L, 6 C); (iv) the paroral membrane is in the form of a living morphology and infraciliature of this species, thus single row and the first row of membranelle 1 in the opisthe only new information based on the new population was is not elongated (Figs 5 M, 6 D). supplied here. Discussion. The new population shows consistency with Deposition of voucher slide. The voucher slide con- the original report in both the living morphology and general taining protargol-stained specimens (registration number: ciliary pattern (Long et al. 2007b). It differs from the latter, FXP-20071024-01) had been deposited in the Laboratory of however, in: (i) having two postoral kineties (vs. one), and (ii) Protozoology, Institute of Evolution and Marine Biodiversity, the scutica having fewer kinetal units (ca. 7 vs. ca. 10). More- Ocean University of China, Qingdao, China. over, based on the newly described stomatogenesis stages,

S. holtae possesses a typical scuticobuccokinetal pattern,

Description. Living cells 65–100 × 25–40 ␮m in size. which contradicts the previous observation that its stomato-

Cytoplasm full of food granules, each about 6 ␮m in diam- eter (Fig. 5A–D). Buccal opening having narrow gap at its genesis basically follows the parakinetal mode (Long et al. anterior left corner to contain membranelle 1 (Fig. 5E). Pelli- 2007b). Among the so-called loxocephalids, Sathrophilus cle ridged with prominent extrusomes underneath (Fig. 5H). shares the scuticobuccokinetal pattern with Dexiotricha, and Somatic cilia in mid-portion of body lying flat against pelli- differs from Paratetrahymena, which has a monoparakinetal cle (Fig. 5I). 19–33 somatic kineties: SK1 containing 11–15 pattern, and also differs from Dexiotrichides and Sphenos- densely arranged monokinetids in anterior portion; SKn com- tomella, whose oral primordia originate from the postoral prising 4–7 dikinetids in its anterior part (Fig. 5F). Scutica kinety, and where the scutica is not involved in the formation composed of about seven kinetal units arranged in two groups of the paroral membrane (Grolière 1973; Li et al. 2010; Peck (Fig. 5F, J). Two postoral kineties commencing from left and 1974; Song et al. 2005). right side of scutica, respectively (Fig. 5J). Family Cryptochilidae Berger in Corliss, 1979 Morphogenesis. Four individuals at different divisional Biggaria Aescht, 2001 stages were observed, from which the following features can Biggaria bermudensis (Biggar and Wenrich, 1932) Aescht, 2001 be deduced: (i) scutica generates the first oral primordium (Figs 7 A–E, 8 A–P, Table 2) field; the parental paroral membrane splits longitudinally to form two lines, while the outer one forms a second pri- Remarks: Berger (1964) redescribed the species and com- mordium field (SF) with a small gap in the middle part (Figs pared a number of populations, despite the fact that several 5 G, 6 A); (ii) SF possibly further divides into two parts, the characters, e.g., the scutica and the argyrome, were still unknown. Moreover, the description of the infraciliature was X. Fan et al. / European Journal of Protistology 59 (2017) 34–49 43

Fig. 5. A–M Sathrophilus holtae in vivo (A–E, H, I) and after protargol staining (F, G, J–M). A–C. Ventral view of different individuals, arrow in (A) and (B) marks the contractile vacuole and the caudal cilium, respectively. D. Lateral view, showing dorsoventrally flattened body. E. Buccal field, arrow marks the pellicular gap containing membranelle 1. F, J. Buccal apparatus (F) and part of ventral ciliary pattern (J) of non-dividing individuals, arrowhead in (F) refers to the dikinetids in the anterior part of somatic kinety n, arrowheads in (J) depict the two postoral kineties. G. Stomatogenesis, arrow marks the first primordium originating from the scutica, arrowhead depicts the second field of primordium generated by paroral membrane splitting. H, I. Details of pellicle, arrows in (H) indicate the extrusomes, arrows in (I) refer to the pellicular concaves. K. Middle phase of division, arrow indicates posterior part of parental paroral membrane generating the primordium which possibly develops to form the new scutica; arrowhead marks the posterior part of secondary field moving backward. L. Late phase of division, the buccal structure of both proter and opisthe already formed, arrowheads indicate newly formed scutica. M. Final phase of division, two new cells are about to separate, arrowhead shows the first row of membranelle 1 of opisthe is not elongated at this time. M1–3,

membranelles 1, 2 and 3; PK1, 2, postoral kineties 1 and 2; Sc, scutica; SFa, the anterior secondary field of primordium. Scale bars = 40 ␮m. 44 X. Fan et al. / European Journal of Protistology 59 (2017) 34–49

Fig. 6. A–D Morphogenesis of Sathrophilus holtae. A. Early phase of stomatogenesis; arrowhead marks the first primordium originating from scutica, arrowhead refers to the second field of the primordium (SF) generated by the paroral membrane splitting. B. Middle phase of division, arrow indicates the posterior part of the parental paroral membrane generating the primordium, which is possibly related with the new scutica, arrowhead marks the posterior part of the secondary field (SFp) moving backwards. C. Late phase of division, the buccal apparatus of the proter and opisthe is already formed, arrowhead and arrow indicate the newly formed scutica of proter and opisthe respectively. D. Final phase of division, arrow shows the M1 of opisthe having equal kinety rows, arrowhead shows that the first kinety row of M1 of proter became longer than the other rows. M1–3, membranelles 1, 2 and 3; PK1, 2, postoral kineties 1 and 2; SFa, the anterior secondary field of the primordium. in an unpublished thesis (Berger 1964). A redescription, ually thickened from front to rear part (Figs 7 A, 8 A, C). based on the new population, is therefore provided here. Buccal margin straight, aboral margin gently curved (Figs Deposition of voucher slides. The voucher slides con- 7 A, 8 A, B). Small beak-like protrusion curved to ventral taining protargol-stained specimens (registration number: side present at anterior end, and bigger one at caudal end FXP-2007042601-1) and silver nitrate-stained specimens (Fig. 8B, C, E). Caudal protrusion hardly detectable in living (registration number: FXP-2007042601-2) were deposited in cell, from ventral or dorsal view, it deriving from left side the Laboratory of Protozoology, Institute of Evolution and and slightly pointed to right side (Fig. 8C). Five caudal cilia

Marine Biodiversity, Ocean University of China, Qingdao, inserted at caudal protrusion, 25 ␮m long (Fig. 8F). Buccal China. field depressed and at about 60% of body length (Fig. 8B, H).

Host. Hemicentrotus pulcherrimus collected from coastal Granules (1.5–2.5 ␮m across) present in cytoplasm, usually

water (salinity 30‰) off Qingdao. clustering in posterior third of body and making cells appear

Description. Living cells about 150–200 × 75–100 ␮min dark. A few food vacuoles near buccal field, about 10–15 ␮m size, leaf-like in outline, highly laterally flattened, and grad- in diameter (Fig. 8A). Single spherical or irregularly shaped X. Fan et al. / European Journal of Protistology 59 (2017) 34–49 45

Fig. 7. A–E Biggaria bermudensis in vivo (A) and after protargol staining (B–E). A. Right lateral view of a representative individual, arrowheads mark the caudal cilia, arrow refers to the pointed protrusion at the anterior end. B, C. Right (B) and left (C) lateral view of infraciliature, arrowheads depict the curved anterior part of the somatic kineties with their densely arranged kinetosomes. D, E. Details of somatic kineties near buccal field, showing the three kineties that anteriorly end at different level and form a seem with other kineties of right side (D), and three kineties composed of more densely arranged kinetosomes on the left side (E). CC, caudal cilia; M, oral membranelle; Ma,

macronucleus; PM, paroral membrane; Sc, scutica. Scale bars = 50 ␮m. macronucleus in centre of cell, about 28 × 26 ␮m (Figs 7 C, 8 Argyrome composed of loose grids between ciliary rows, K, L, P). Pellicle formed strong projections along ciliary rows with each grid including four or five kinetosomes of two on right side (Fig. 8D), and curved ridges in anterior portion related somatic kineties (Fig. 8N). of left side, no ectoplasmic projections existed. Somatic cilia Discussion. The new population shows great similarities

about 17 ␮m long (Fig. 8D). with previously reported ones in terms of general morphology Cells isolated from hosts and placed in Petri dishes slowly and their sea urchin host (Berger 1964; Biggar and Wenrich float in water or move on substrate. 1932; Lucas 1940; Nie 1934; Powers 1933, 1935). Powers

Forty to 52 somatic kineties, 21–27 (23.1 on average) on (1935) reported that the size of this species may vary greatly right side and 18–24 (20.8 on average) on left side (Figs 7 between individuals of an isolate (54–210 × 40–98 ␮m). The

B, C, 8 K, L). SKs on right side straight, containing large average size from previous reports and present study may

percentage of dikinetids, kinetosomes of which are all cil- arrange from 107 × 46 ␮mto187× 99 ␮m; while the num- iated (Figs 7 B, 8 K). Inconspicuous suture of kineties of ber of somatic kineties varies from 39 to 69 (Berger 1964; two sides present at anterior margin (Fig. 7B). Three SKs Biggar and Wenrich 1932; Lucas 1940; Nie 1934; Powers right of PM anteriorly shortened from left to right, forming 1933, 1935). narrow seam with other SKs (Figs 7 D, 8 M). Three SKs Previous studies conformably reported the existence of a left of buccal field containing densely arranged kinetosomes caudal protrusion. However, some of them reported that the (Fig. 7C, E); while other SKs of left side containing loosely protrusion originates from the left side (Biggar and Wenrich arranged monokinetids and arranged obliquely with curved 1932; Nie 1934), while Berger (1964) showed an acute pos- anterior ends in most part (Figs 7 C, 8 L). Buccal apparatus terior elongation of the bulbous posterior third of the body, composed of paroral membrane and single oral membranelle i.e., it was related with both sides. In our isolate the caudal (Figs 7 B, D, 8 O). Oral membranelle triangle shaped, con- protrusion originates from the left side according to live- and taining about 25 obliquely oriented kinety rows (Figs 7 D, protargol-prepared specimens; moreover, the caudal protru- 8 O). Scutica behind buccal cavity and composed of short sion is not that obvious in living cells than that in fixed cells, kinety, which is of different length among individuals (Figs possibly due to its retractability. Also, the number of caudal 7 B, D, 8 I, J, O). cilia in the caudal protrusion can be variable: five revealed in present isolate; while six to eight according to Berger (1964). 46 X. Fan et al. / European Journal of Protistology 59 (2017) 34–49

Fig. 8. A–P Biggaria bermudensis in vivo (A–F, H) and after staining with protargol (G, I–L, O, P) and silver nitrate (M, N). A. Right lateral view of an individual, arrow marks the food vacuoles in the posterior part of the cell. B, C. Left (B) and ventral (C) lateral view, showing the small curved protrusion at anterior end (arrows) and the bigger protrusion at posterior end (arrowheads). D, E. Anterior part, showing the rigid pellicle along ciliary rows (D) and the small beak-like protrusion at anterior end (arrowhead in E). F, G. The caudal protrusion, showing the caudal cilia (arrowhead in F) and their kinetosomes (arrowhead in G). H–J, O. Buccal field, showing the buccal depression of living cell (H), and the details of buccal apparatus (I, J, O), arrow in (J) marks the scutica. K, L. Right (K) and left (L) lateral view of ciliary pattern, arrowhead indicates the somatic kineties near buccal field which are different from other kineties, arrow marks the caudal protrusion. M. Right lateral view, showing the seam formed by the three somatic kineties close to buccal field and other kineties. N. Part of argyrome in left

side. P. Macronucleus. M, oral membranelle; Ma, macronucleus; PM, paroral membrane; Sc, scutica. Scale bars = 50 ␮m.

Berger (1964) and Powers (1935) documented that the in Nie (1934) show no such feature. Such a morphological anterior dorsal surface of this species was characterized by difference alone cannot be considered as evidence of a dis- bearing ectoplasmic projections, which was also shown in the tinct species, and it might be caused by different geographic figure of Biggar and Wenrich (1932). However, the present distribution. isolate from Qingdao and the China population from Xiamen X. Fan et al. / European Journal of Protistology 59 (2017) 34–49 47

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