Order Choreotrichida Small and Lynn, 1985
Suborder Tintinnina Kofoid and Campbell, 1929
Genus Tintinnopsis Stein, 1867
Tintinnopsis hemispiralis Yin, 1956 (Figures 1A–E, 2A–J; Table 1)
Terminology
Tintinnopsis hemispiralis possesses a cluster of extremely long cilia originating from the ventral kinety that has only been reported for Tintinnopsis subacuta [50]. This character is here defined as follows.
Ciliary tuft. An extraordinary long tuft of cilia originated from densely-arranged kinetids in the middle portion of the ventral kinety.
Improved diagnosis (based on the type and neotype populations)
Lorica 88–182 μm long, comprising a cylindrical, spiraled collar and an obconical bowl. Opening 34–59 μm in diameter. Cell proper elongate, obconical when fully extended, size in vivo 80–125 × 30–55 μm. Seven to 11 moniliform macronuclear nodules. On average 21 collar membranelles, of which four or five elongate into buccal cavity; one buccal membranelle. Ventral kinety composed of about 53 monokinetids, commences anteriorly to second kinety of right ciliary field. Ciliary tuft about 150–250 μm long. Right and left ciliary fields consist of about ten kineties each. Lateral ciliary field comprises on average 15 kineties. Dorsal kinety composed of about 47 dikinetids. Posterior kinety with about 17 dikinetids, positioned below left ciliary field.
Deposition of neotype and other voucher materials
A protargol slide including the neotype (Figures 2F, G) was deposited in the Laboratory of Protozoology, Institute of Evolution and Marine Biodiversity, Ocean University of China (registration number: BY201805280101). One additional protargol slide was deposited in the same collection (registration number: BY201805280102).
Redescription based on the Ningde population
Lorica 143–182 μm long, comprises a cylindrical, truncated collar and an obconical bowl (Figures 1A, 2A–D). Opening 45–59 μm across. Ratio of lorica length to opening diameter 2.9–3.2:1. Collar 64–97 μm long, with three to five inconspicuous spiraled striations (Figures 1A, 2B). Bowl often slightly wider than opening (49–66 μm in diameter), about 68–88 μm long, with a posterior angle of 45° (Figures 1A, 2B, C, D). Wall of lorica heterogeneously agglutinated with mineral particles: collar slightly less agglutinated than bowl because adhered particles sparser and thinner (Figures 1A, 2B, C, D).
Cell proper elongate, obconical when fully extended, size 80–125 × 30–55 μm in vivo, and 65–119 × 31–58 μm after protargol staining (Figures 1B, 2E). Posterior portion of cell proper narrows gradually forming a peduncle with a branched posterior end, which is about 60–110 μm long and attaches to bottom of lorica (Figures 1B, 2D, E). Seven to 11 moniliform macronuclear nodules, each about 5–10 × 5–9 μm in size; anterior nodule 17–21 μm posterior to the anterior cell end in protargol-stained specimens (Figures 1C, D, 2F, I, J). Micronuclei, striae, tentaculoids, accessory combs, a contractile vacuole, a cytopyge, and capsules not observed. Cytoplasm colourless, with food vacuoles of various sizes containing ingested ovoidal microalgae (Figures 1B, 2E). Locomotion by rotation about main cell axis. Disturbed individuals retract into lorica with motionless membranelles bent to centre of peristomial field.
Somatic ciliary pattern composed of a ventral, a dorsal, and a posterior kinety and right, left, and lateral ciliary fields (Figures 1C–E, 2F–I). Kinetids of each ciliary row ostensibly connected by argyrophilic fibers (Figure 2F, G). Ventral kinety commences between collar membranelles and second kinety of right ciliary field (about 4–8 μm below the anterior end of cell) and curves leftwards before extending, parallel to kineties of lateral ciliary field posteriorly; 39–66 μm long, with 41–61 monokinetids, consisting of three portions: (1) anterior portion comprised of eight to 14 kinetids about 0.5–1 μm apart; (2) middle portion consisting of 16–24 more densely arranged kinetids (with no measurable gap) with long cilia and forming the ciliary tuft, about 150–250 μm long in vivo; (3) posterior portion containing sparsely arranged monokinetids (more than 1 μm apart), extending posteriorly and terminating at about two thirds to three fourths of cell (Figures 1A–C, E, 2C, F, G). Right ciliary field comprises 9–11 kineties, neighboring kineties about 2–5 μm apart, each composed of about 5–18 widely spaced monokinetids and one anterior dikinetid; kinetids of first kinety more densely arranged; all kineties commence at the same level (about 9 μm below the anterior end of cell), except for the first kinety which commences about 2 μm below the level of remaining kineties (Figures 1C, E, 2F). Left ciliary field comprises 9–12 kineties, neighboring kineties about 2–5 μm apart; each kinety commences about 9 μm below the anterior end of cell and is composed of one anterior dikinetid and 2–13 widely spaced monokinetids; the leftmost two or three kineties always shorter, only including three to five kinetids (Figures 1C–E, 2G–I). Each basal body in left and right ciliary fields bears a cilium, the anterior one in each dikinetid being about 20 μm long in vivo and 10 μm long after protargol staining; the other cilia (on posterior kinety of dikinetids and on monokinetids) are about 3 μm long after protargol staining (Figures 1A, B, 2E, I). Lateral ciliary field consists of 11–20, relatively densely arranged monokinetidal kineties, each of which commences about 9 μm below the anterior end of cell; kineties in middle region always shorter than those at both ends of field (i.e., including only half the number of kineties); cilia about 3 μm long after protargol staining (Figures 1D, E, 2G, H). Dorsal kinety commences about 5 μm below the anterior end of cell, about 5 and 10 μm apart from right and left ciliary field, respectively; curves towards the left-posterior portion; 66–97 μm long and composed of 35–56 dikinetids, with only the posterior basal body bearing a cilium about 8–10 µm long after protargol staining (Figures 1D, E, 2G, I). Posterior kinety usually commences below right portion of left ciliary field (29–41 μm below the anterior end of cell) and curves rightwards, terminating near posterior end of cell proper; 37–58 μm long and with 11–22 dikinetids; only the posterior basal body bearing a cilium about 8–10 µm long after protargol staining (Figures 1D, E, 2G–I).
Oral apparatus occupies anterior portion of cell. Adoral zone of membranelles closed; composed of 20–22 collar membranelles, four or five of which extend into buccal cavity; bases about 30 μm of longest membranelles; polykinetid structures could not be recognized; cilia of membranelles about 25–35 μm long (Figures 1A–E, 2A–E, J). Single buccal membranelle within buccal cavity, with polykinetid about 40 μm long (Figure 1C, E). Argyrophilic fibers originate in the proximal portions of the elongated collar membranelles and the buccal membranelle, and extend posteriorly; three thick fibers commencing from the middle of cell below right ciliary field and extending towards anterior part of cell; ends not observed due to insufficient staining (Figure 2F). Endoral membrane composed of a single row of basal bodies, extends in a semicircle across the peristomial field and right wall of buccal cavity (Figure 1C). An early divider was observed with the oral primordium posterior to the ventral kinety and lateral ciliary field (Figure 2H).
Tintinnopsis kiaochowensis Yin, 1956 (Figures 3A–E, 4A–K; Table 1)
Improved diagnosis (based on the type and neotype populations)
Lorica 79–112 μm in length, composed of an irregular collar and an ellipsoidal bowl with a rounded posterior end, both separated by a constriction. Opening 30–71 μm in diameter. Cell proper obconical when fully extended, size in vivo about 60–95 × 35–50 μm. Two ellipsoidal macronuclear nodules. On average 16 collar membranelles, three of which extend into buccal cavity; one buccal membranelle. Ventral kinety with an average of 49 densely arranged monokinetids. Right, left, and lateral ciliary fields include, on average, 11, 10, and 16 kineties, respectively. Dorsal kinety composed of about 31 dikinetids. Posterior kinety composed of about 15 dikinetids, positioned below lateral ciliary field.
Deposition of neotype and other voucher materials
A protargol slide including the neotype (Figure 4H, I) was deposited in the Laboratory of Protozoology, Institute of Evolution and Marine Biodiversity, Ocean University of China (registration number: BY201805280201). One additional protargol slide was deposited in the same collection (registration number: BY201805280202).
Redescription based on the Ningde population
Lorica 79–112 μm in length, composed of an irregular collar and an ellipsoidal bowl (Figures 3A, 4A–C). Opening 44–71 μm in diameter; rim irregular. Ratio of lorica length to opening diameter 1.4–2:1. Collar 32–46 μm high, not flaring at the opening margin, occasionally slightly layered because of agglutinated particles arranged in horizontal rows (Figures 3A, 4A–C). Region between collar and bowl constricted, about 38–63 μm in diameter (Figures 3A, 4A, B). Bowl about 43–70 μm long and 57–83 μm across. Posterior end usually rounded to bluntly tapered (Figures 3A, 4A, B).
Cell proper elongate, obconical when fully extended, about 60–95 × 35–50 μm in vivo, and 46–65 × 38–64 μm after protargol stained. Posterior cell portion narrows successively forming a peduncle about 25 μm long and attached to the bottom of lorica (Figures 3A, D, 4B). Two ellipsoidal macronuclear nodules, each about 15–22 × 12–17 μm in protargol-stained specimens; anterior nodule 11–24 μm from the anterior cell end (Figure 3C). Micronuclei, striae, tentaculoids, accessory combs, a contractile vacuole, a cytopyge, and capsules not observed. Cytoplasm colourless, usually with food vacuoles containing diatoms and ovoidal microalgae (Figures 3D, 4B). Locomotion by rotation about main cell axis.
Somatic ciliary pattern complex, with a ventral, a dorsal, and a posterior kinety, and right, left, and lateral ciliary fields (Figures 3B, C, E, 4H–K). Ventral kinety 22–37 μm long, commences between collar membranelles and third or fourth kinety of right ciliary field (about 4 μm below the anterior end of cell), anterior third curves leftwards before extending parallel to kineties of lateral ciliary field posteriorly; 43–56 densely arranged monokinetids (Figures 3B, E, 4H). Right ciliary field comprising 10–13 kineties about 2–5 μm apart, the space between the leftmost five to six kineties wider than others; all kineties commence 6–13 μm below the anterior end of cell; composed of 5–14 widely spaced monokinetids and one anterior dikinetid, except for: (i) the first kinety almost parallel to ventral kinety, with two or three anterior dikinetids and eight to 12 monokinetids, more densely arranged than other kineties in right ciliary field; and (ii) the second kinety parallel to rest of kineties, with an angle of about 20° with the first kinety, including four or five monokinetids and two anterior dikinetids (Figures 3B, E, 4H). Left ciliary field comprises 9–11 kineties about 2–5 μm apart, each kinety commencing 6–13 μm below the anterior end of cell and comprised of two to eight widely spaced monokinetids and one anterior dikinetid; the number of kinetids of leftmost kinety always minimum (i.e., three or four). Each basal body in left and right ciliary fields bears a cilium, with the cilium on anterior basal body of each dikinetid being about 15 μm long in vivo and 5 μm long after protargol staining; other cilia (on posterior basal body of each dikinetid and on monokinetids) are about 1 μm long after protargol staining (Figures 3A, D, 4C, H–J). Lateral ciliary field with 13–19 monokinetidal kineties of similar length, each of which commences 6–13 μm below the anterior end of cell, except for the rightmost kinety that commences anteriorly to the second or third kinety of right ciliary field, about 4 μm below the anterior cell end, with the anterior portion curving rightwards before extending towards posterior part; cilia about 2 μm long after protargol staining (Figures 3B, C, E, 4H, I). Dorsal kinety 29–53 μm long, comprises 25–37 dikinetids, commences about 4 μm below the anterior end of cell, about 5 μm from right ciliary field and 13 μm from left ciliary field apart, and extends to the left before curving towards posterior end of cell (Figures 3C, E, 4J, K). Posterior kinety 21–29 μm long, consists of 11–18 dikinetids, commences below lateral ciliary field (28–49 μm below the anterior end of cell) and extends to posterior end of cell (Figures 3C, E, 4J). Cilia of dorsal and posterior kinety are insufficiently stained.
Oral apparatus occupies anterior portion of cell. Adoral zone of membranelles closed, composed of 16–18 collar membranelles with cilia about 25–35 μm long, three of which extend into buccal cavity; bases about 30 μm of longest membranelles; kinetal structures of membranelles could not be recognized (Figures 3A–E, 4E–G, H). Single buccal membranelle in buccal cavity, with polykinetid about 20 μm long (Figures 3B, E, 4E). Endoral membrane composed of a single row of basal bodies, extends in a semicircle across the peristomial field and right wall of buccal cavity (Figures 3B, C, 4E, F). Argyrophilic fibers associated with oral apparatus insufficiently impregnated to be observed.
Tintinnopsis uruguayensis Balech, 1948 (Figures 5A–D, 6A–J; Table 1)
Improved diagnosis (based on the type and present populations)
Lorica 50–73 μm long, bullet-like with a flared collar and a posterior process about 8–10 μm long. Opening 22–42 μm in diameter, with an irregular rim. Cell proper obconical when fully extended, size in vivo about 25–50 μm × 20–30 μm. Two macronuclear nodules. On average 18 collar membranelles, of which three or four extend into buccal cavity; one buccal membranelle. Ventral kinety composed of about 20 monokinetids. Right and left ciliary fields consist of about seven kineties each. Lateral ciliary field comprises on average 12 kineties. Dorsal kinety with about 21 dikinetids. Posterior kinety with about eight dikinetids, posterior to lateral ciliary field.
Deposition of voucher materials
Two protargol slides with voucher specimens were deposited in the Laboratory of Protozoology, Institute of Evolution and Marine Biodiversity, Ocean University of China (registration numbers: BY201811120101 and BY201811120102).
Redescription based on the Qingdao population
Lorica 50–73 μm long, composed of a flared collar about 15 μm long with a jagged rim, and an ovoidal bowl about 32–52 μm long and 25–41 μm wide (Figures 5A, 6A–C). Opening diameter 24–42 μm. Region between collar and bowl narrowed, about 17–29 μm in diameter (Figures 5A, 6A–C). Posterior end projected, about 10 μm long (Figures 5A, 6A). Lorica wall densely agglutinated with mineral particles (Figures 5A, 6A–C).
Cell proper obconical when fully extended, about 25–50 μm × 20–35 μm in size in vivo, and 25–56 × 18–28 μm in size after protargol staining. Posterior end of cell proper becomes spherical when escaped from lorica (Figure 6D). Two ellipsoidal (occasionally elongated) macronuclear nodules, 6–14 × 4–10 μm in size after protargol staining; anterior nodule 4–9 μm posteriorly to the anterior cell end after protargol staining (Figures 5A–C, 6E, H). Micronuclei, striae, tentaculoids, accessory combs, a contractile vacuole, a cytopyge, and capsules not observed. Cytoplasm colourless, with food vacuoles of various sizes containing ingested microalgae (Figure 6D). Locomotion by rotation about main cell axis. Disturbed individuals retract into lorica with motionless membranelles bent to centre of peristomial field.
Somatic ciliary pattern composed of a ventral, a dorsal, and a posterior kinety, and right, left, and lateral ciliary fields (Figures 5B–D, 6E–I). Ventral kinety 14–35 μm long with 17–28 monokinetids, commences between collar membranelles and first kinety of right ciliary field, about 2 μm below the anterior end of cell, curves leftwards before extending parallel to kineties of lateral ciliary field posteriorly (Figures 5B, D, 6E). Right ciliary field comprises 7–8 kineties, 1–3 μm apart; all kineties commence at the same level (about 2 μm below the anterior end of cell), except for the first kinety that commences about 1 μm below the level of remaining kineties; the second kinety always shorter, i.e. only comprising two or three kinetids; others composed of 6–7 widely spaced monokinetids and one anterior dikinetid, except first kinety comprised of two to four monokinetids and two or three anterior dikinetids; first kinety usually commences below anterior portion of ventral kinety (Figures 5B, D, 6E, I). Left ciliary field comprises 6–8 kineties; each kinety commences about 2 μm below the anterior end of cell and is composed of one anterior dikinetid and 1–7 widely spaced monokinetids, with decreasing length from right to left (Figures 5C, D, 6F, G). Each basal body in left and right ciliary fields bears a cilium, with the cilium on anterior basal body of each dikinetid being about 5 μm long in vivo and after protargol staining; other cilia (on posterior basal body of each dikinetid and on monokinetids) are about 1 μm long after protargol staining (Figures 5A, 6E–J). Lateral ciliary field commences about 2 μm below the anterior end of cell, with 9–16 monokinetidal kineties of similar length, with cilia about 1 μm long after protargol staining (Figures 5B, D, 6E). Dorsal kinety commences about 2 μm below anterior cell end, about 2 and 3 μm apart from right and left ciliary fields, respectively, curves towards the left-posterior portion of cell; 21–41 μm long, consists of 17–29 dikinetids, with only the posterior basal body bearing a cilium about 3–5 μm long after protargol staining (Figures 5C, D, 6F, G). Posterior kinety usually commences below the middle kinety of the left ciliary field (12–21 μm below the anterior end of cell) and curves leftwards; 11–22 μm long, consists of 7–9 dikinetids, with only the posterior basal body bearing a cilium about 3–5 µm long after protargol staining (Figures 5C, D, 6F).
Oral apparatus occupies anterior portion of cell. Adoral zone of membranelles closed; composed of 18 or 19 collar membranelles with cilia up to about 20–25 μm long, three or four of which extend into buccal cavity; bases about 10 μm of longest membranelles; polykinetid structures could not be recognized (Figures 5A–D, 6A, D, H). Single buccal membranelle in buccal cavity, with polykinetid about 8 μm long (Figures 5B, D, 6H). Argyrophilic fibers insufficiently impregnated to be observed. Endoral membrane not recognized. One middle divider was observed with the oral primordium located left of ventral kinety and posterior to the lateral ciliary field (Figure 6J).
Neotypification
The neotypes of Tintinnopsis hemispiralis and T. kiaochowensis are designated because (i) no type materials are known to be deposited; (ii) the original descriptions are restricted to lorica features, while the present redescriptions include also cytological and molecular analyses; and (iii) the type locality of the original populations is Qingdao, East China, with no further details [44]. The type location of the two species is near by the collection site of the present populations (Meng Bay, Ningde, East China; detailed information provided in ‘Materials and Methods’), thus meeting the requirement of Article 75.3.6 of the International Code of Zoological Nomenclature [51]. Protargol slides containing the neotype specimens were deposited (see ‘Deposition of neotype and other vouched materials’), thus meeting the requirements of Article 75.3.7 of the Code [51]. A neotype is not established for T. uruguayensis because the type location corresponds to a different ocean basin [52].
Sequence comparison and phylogenetic analyses
For the three species investigated, the length, G+C content and GenBank accession numbers of the SSU rDNA, ITS1-5.8S rDNA-ITS2 and LSU rDNA sequences are compiled in Table 2. For each of the three loci and concatenated sequences, the topologies of the ML and BI trees were similar and therefore only the ML trees are shown (Figures 7, 8, 9, S1). Based on SSU rDNA, T. hemispiralis forms a fully-supported clade with T. subacuta (EU399541; [53]); both sequences are 99.3% similar. Based on ITS1-5.8S-ITS2, a sequence previously obtained for this species in Qingdao, China (KU715813; [48]) groups with our sequence, and both are 96.2% similar. Tintinnopsis kiaochowensis forms a fully-supported clade with T. everta (MG461220; [33]) based on SSU rDNA, and both sequences are 99.0% similar. The newly sequenced population of T. uruguayensis forms a fully-supported clade with the North-Atlantic population of the same species, based on both SSU rDNA and LSU rDNA (JN831838 and JN831923; [25]); the two populations are 100% identical in both markers. The concatenated tree (Figure S1, Table S1) shows similar relationships than SSU rDNA, except that Tintinnina were inferred as non-monophyletic. This inference is probably artifactual given the well-known monophyly of this suborder [22][39].