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 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 the 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 80–125 μm long and 30–55 μm wide in vivo in fully extended, 65–119 μm long and 31–58 μm wide protargol preparations (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 long and 5–9 μm wide; 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. Movement by irregular swimming with rotation about main cell axis.
Somatic ciliary pattern complex, that is, ventral kinety, dorsal kinety, posterior kinety, right ciliary field, left ciliary field, and lateral ciliary field present (Figures 1C–E, 2F–I). Ventral kinety begins anteriorly to the second kinety of right ciliary field, about 4–8 μm below the anterior end of cell, goes around right ciliary field from left side before parallel to kineties of ciliary field posteriorly; 39–66 μm long, with 41–61 monokinetids, composed 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 consists of 9–11 kineties, kineties about 2–5 μm away from their neighbors; each kinety has 5 to 18 monokinetids and one anterior dikinetid; kinetids of first kinety more densely arranged than those in the remaining kineties; all kineties commence at the same level (about 9 μm below the anterior end of cell), except for the first kinety that starts about 2 μm posteriorly to other kineties (Figures 1C, E, 2F). Left ciliary field with 9–12 kineties, begins about 9 μm below the anterior end of cell, kineties about 2–5 μm away from their neighbors, composed of one anterior dikinetid and 2–13 monokinetids each; the leftmost two or three kineties always shorter, each only including three to five kinetids (Figures 1C–E, 2G–I). The anterior basal bodies of dikinetids in left and right ciliary fields bear elongated cilia, about 20 μm long from life and 10 μm long after protargol staining while the cilia on posterior basal bodies are similar to ones on monokinetid in length, about 3 μm long after protargol staining (Figures 1A, B, 2E, I). Lateral ciliary field commences about 9 μm posteriorly to the anterior end of cell, comprises 11–20, relatively densely arranged monokinetidal kineties; 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 silver staining (Figures 1D, E, 2G, H). Dorsal kinety about 66–97 μm in length and consisting of 35–56 dikinetids, commences about 5 μm posteriorly to anterior cell end, about 5 and 10 μm away from right and left ciliary field, respectively; only the posterior basal body bearing a cilium about 8–10 µm long after protargol staining (Figures 1D, E, 2G, I). Posterior kinety 37–58 μm long and consisting of 11–22 dikinetids, commences posteriorly to right portion of left ciliary field, with 29–41 μm away from the anterior cell end and curves rightwards; only the posterior basal body bearing a cilium about 8–10 µm long after protargol staining (Figures 1D, E, 2G–I).
Adoral zone of membranelles composed of 20–22 collar membranelles with, four or five of which extend into buccal cavity with longest bases about 30 μm; cilia in collar membranelles about 25–35 μm in length; polykinetid structures could not be recognized (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 consisting 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 about 60–95 μm long and 35–50 μm wide from life when it is fully extended, 46–65 μm long and 38–64 μm wide in protargol preparation. 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, contractile vacuole, cytopyge, and capsules not observed. Locomotion by rotation about main cell axis.
Somatic ciliary pattern complex, that is, ventral kinety, dorsal kinety, posterior kinety, right ciliary field, left ciliary field, and lateral ciliary field present (Figures 3B, C, E, 4H–K). Ventral kinety 22–37 μm long, commences anteriorly to third or fourth kinety of right ciliary field and about 4 μm below the anterior cell end, 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 consists of 10–13 kineties about 2–5 μm away from each other, 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 consists of 9–11 kineties 2–5 μm away from each other, each kinety commences 6–13 μm posteriorly to the anterior cell end and comprises of two to eight monokinetids and one anterior dikinetid; the number of kinetids of leftmost kinety always minimum (i.e., three or four). The anterior basal bodies of dikinetids in left and right ciliary field bear elongated cilia, about 15 μm long from life and 5 μm long after protargol staining while the cilia on posterior basal bodies with similar length to monokinetid-based ones, about 1 μm long after protargol staining (Figures 3A, D, 4C, H–J). Lateral ciliary field comprises 13–19 monokinetidal kineties of similar length, each apart 6–13 μm from the anterior cell end, 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, apart about 4 μm from the anterior cell end, about 5 μm from right ciliary field and 13 μm from left ciliary field (Figures 3C, E, 4J, K). Posterior kinety 21–29 μm long, consists of 11–18 dikinetids, commences posteriorly to lateral ciliary field, with 28–49 μm away from the anterior cell end (Figures 3C, E, 4J). Cilia of dorsal and posterior kinety are insufficiently stained.
Adoral zone of membranelles comprises 16–18 collar membranelles with cilia about 25–35 μm long, three of which extend into buccal cavity; the longest bases about 30 μm; 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 comprised 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). Wall of lorica heterogeneously agglutinated with mineral particles (Figures 5A, 6A–C).
Cell proper about 25–50 μm long and 20–35 μm wide from life when fully extended, 25–56 μm long and 18–28 μm wide 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, contractile vacuole, cytopyge, and capsules not observed. Locomotion by rotation about main cell axis.
Ventral kinety 14–35 μm long with 17–28 monokinetids, commences anteriorly to first kinety of right ciliary field, about 2 μm posteriorly to the anterior cell end, goes around right ciliary field from the left side and extending parallel to kineties of ciliary field posteriorly (Figures 5B, D, 6E). Right ciliary field consists of 7–8 kineties, 1–3 μm apart; all kineties commenceabout 2 μm below the anterior cell end, except for the first kinety that commences about 1 μm posteriorly to remaining kineties; the second kinety always shorter with only 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 consists of 6–8 kineties about 2 μm away from the anterior cell end, and is composed of one anterior dikinetid and 1–7 monokinetids, with decreasing length from right to left (Figures 5C, D, 6F, G). The anterior basal bodies of dikinetids in left and right ciliary field bear elongated cilia, about 5 μm long in both live and protargol-stained specimens while the cilia on posterior basal bodies are similar to ones on monokinetids, about 1 μm long after protargol staining (Figures 5A, 6E–J). Lateral ciliary field begins about 2 μm posteriorly to 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 21–41 μm long, and consisting of 17–29 dikinetids, begins about 2 μm posteriorly to anterior cell end, about 2 and 3 μm away from right and left ciliary fields, respectively; only the posterior basal body bearing a cilium about 3–5 μm long after protargol staining (Figures 5C, D, 6F, G). Posterior kinety begins posterior to the middle kinety of the left ciliary field and 12–21 μm apart from the anterior end of cell; 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).
Adoral zone of membranelles consists of 18 or 19 collar membranelles with about 20–25 μm long cilia, three or four of which extend into buccal cavity; the longest bases about 10 μm; polykinetid structures could not be recognized (Figures 5A–D, 6A, D, H). Single buccal membranelle, 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) the deposited type materials are unknown; (ii) only lorica features are reported in the original description, 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 nearby 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 Maximum Likelihood (ML) and Bayesian Inference (BI) trees were similar and therefore only the ML trees are shown (Figures 7, 8, 9, S1). Tintinnopsis hemispiralis forms a fully-supported clade with T. subacuta (EU399541; [53]) based on SSU rDNA; 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].