Three redescriptions in Tintinnopsis (Protista: Ciliophora: Tintinnina) from coastal waters of China, with cytology and phylogenetic analyses based on ribosomal RNA genes

The taxonomy of tintinnine ciliates is vastly unresolved because it has traditionally been based on the lorica (a secreted shell) and it has only recently incorporated cytological and molecular information. Tintinnopsis, the most speciose tintinnine genus, is also the most problematic: it is known to be non-monophyletic, but it cannot be revised until more of its species are studied with modern methods. Here, T. hemispiralis Yin, 1956, T. kiaochowensis Yin, 1956, and T. uruguayensis Balech, 1948, from coastal waters of China, were studied. Lorica and cell features were morphometrically investigated in living and protargol-stained specimens, and sequences of three ribosomal RNA (rRNA) loci were phylogenetically analyzed. The three species show a complex ciliary pattern (with ventral, dorsal, and posterior kineties and right, left, and lateral ciliary fields), but differ in lorica morphology, details of the somatic ciliature and rRNA gene sequences. Tintinnopsis hemispiralis is further distinguished by a ciliary tuft (a ribbon of very long cilia originated from the middle portion of the ventral kinety and extending out of the lorica) and multiple macronuclear nodules. Both T. kiaochowensis and T. uruguayensis have two macronuclear nodules, but differ in the number of somatic kineties and the position of the posterior kinety. Two neotypes are fixed for T. hemispiralis and T. kiaochowensis to stabilize the species names objectively, mainly because of the previous unavailability of type materials. By phylogenetic analysis and comparison with closely-related species, we infer that the ciliary tuft and details such as the commencement of the rightmost kinety in the lateral ciliary field are synapomorphies that may help clarify the systematics of Tintinnopsis-like taxa. The redescriptions of three poorly known Tintinnopsis species, namely T. hemispiralis, T. kiaochowensis, and T. uruguayensis firstly revealed their ciliary patterns and rRNA sequences. This study expands knowledge and database of tintinnines and helps in identifying potential synapomorphies for future taxonomic rearrangements.


Background
Ciliated protists are among the most diverse and numerically important members of microzooplankton, and act as a trophic link in the microbial food web of aquatic ecosystems [1][2][3][4][5]. In particular, tintinnine ciliates are conspicuous due to the diversity of loricae produced by their cell propers. Tintinnines have been of great interest in the field of protistology because they (i) display distinct patterns of diversity and biogeography [6,7]; (ii) serve as bioindicators of water quality and hydrological circulation [8][9][10][11]; (iii) are prey for fish larvae and other small metazoans [12,13]; and (iv) can leave fossilized loricae that are useful in evolutionary studies [14,15].
There are approximately 1000 extant tintinnine species classified almost entirely based on the shape and size of their loricae [16][17][18][19][20][21]. However, it is widely recognized that lorica features alone have shortcomings for determining taxonomic affiliations in this group of ciliates [22,23]. In some species, laboratory cultures have provided clear evidences that the lorica is polymorphic in response to environmental factors or in different stages of the life cycle [24]. More recently, DNA sequencing of several closely-related species has revealed examples of polymorphic and cryptic species [25,26]. Thus, the current lorica-based taxonomy does not allow estimating tintinnine diversity accurately, and it does not provide a natural classification. Accordingly, several studies have incorporated more informative characters, namely, cytological and/ or molecular data, in tintinnine systematics (e.g., [27][28][29][30][31][32][33][34][35][36]). Still, cell characters and DNA sequences are only known for about 3 and 10% of the described tintinnine morphospecies, respectively (e.g., [22,37]), and considerable efforts are needed to increase the availability of these types of information.
The present study investigates the morphology and molecular phylogeny of three Tintinnopsis species, namely, T. hemispiralis Yin, 1956, T. kiaochowensis Yin, 1956, and T. uruguayensis Balech, 1948, which were collected from coastal waters of China. This work includes observations of specimens in vivo and after protargol staining as well as phylogenetic analyses of ribosomal RNA gene markers based on recommendations for tintinnine taxonomy [23] and common practices for other ciliates [49]. The aims of the present study are to combine lorica, cell proper, and molecular data in three Tintinnopsis species and to compare them with related taxa in order to find potential diagnostic features relevant in this problematic tintinnine taxon.

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 (Fig. 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). Somatic ciliary pattern complex, that is, ventral kinety, dorsal kinety, posterior kinety, right ciliary field, left ciliary field, and lateral ciliary field present (Figs. 1c-e, 2fi). 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 (Figs. 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 (Figs. 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 ( (Fig. 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 (Fig. 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 (Fig. 1c). An early divider was observed with the oral primordium posterior to the ventral kinety and lateral ciliary field (Fig. 2h).

Deposition of neotype and other voucher materials
A protargol slide including the neotype (Fig. 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).
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 (Figs. 3a-e, 4e-g, h). Single buccal membranelle in buccal cavity, with polykinetid about 20 μm long (Figs. 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 (Figs. 3b, c, 4e, f). Argyrophilic fibers associated with oral apparatus insufficiently impregnated to be observed.
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 (Figs. 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 (Figs. 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 (Figs. 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 (Figs. 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 (Figs. 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 (Figs. 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 (Figs. 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 (Figs. 5a-d, 6a, d, h). Single buccal membranelle, with polykinetid about 8 μm long (Figs. 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 (Fig. 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 (Figs. 7, 8, 9, S1). Tintinnopsis hemispiralis forms a fullysupported 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 fullysupported 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].
Regarding cell features, T. hemispiralis resembles Tintinnopsis subacuta Jörgensen, 1899 in having a ventral kinety associated with the extraordinarily long ciliary tuft that extends outside of the lorica [50]. Both species are also similar in having multiple moniliform macronuclear nodules [50], which differ from the common finding of only two macronuclear nodules in other Tintinnopsis species (e.g., [22,27,34]). The two species cluster together based on SSU rDNA (Fig. 7), which suggests that the ciliary tuft and multiple moniliform macronuclear nodules are synapomorphies of this clade and may be important for a future reclassification of Tintinnopsis species. Despite the close relationship between T. hemispiralis and T. subacuta, the latter can be distinguished from our specimens by a lorica with a swollen, ovoid (vs. obconical) bowl in the original description [57] and the micrograph of the sequenced specimen [53]. The SSU rDNA divergence for both species, although small (0.7%), is consistent with interspecific variation in this conserved marker [25].

Tintinnopsis kiaochowensis Comparison with type population
The specimens studied here match Tintinnopsis kiaochowensis in lorica size and shape [44]. The lorica dimensions reported in the original description (length = 95-108 μm, opening diameter = 30-52 μm [44];) overlap with those of our specimens (length = 79-112 μm, opening diameter = 44-71 μm; Table 1). Our specimens also resemble to those originally described in a lorica with a cylindrical collar and an ellipsoidal bowl with a constricted connection. However, our specimens differ from the original population in the rounded posterior end of the lorica (vs. obconical) and in the agglutinated

Comparison with similar species
Tintinnopsis kiaochowensis differs from other Tintinnopsis species by its peculiar lorica shape, i.e. swollen bowl divided from a non-flaring collar by a constriction. Compared to our specimens, the most similar species is Tintinnopsis compressa Daday, 1887. However, T. compressa can be separated from our specimens by having a smaller lorica size (45 vs. 79-112 μm in length; 26 vs. 44-71 μm in opening diameter), a flared lorica collar (vs. not flared), and a less obvious constriction between the lorica collar and bowl [18].
Tintinnopsis kiaochowensis is similar to Tintinnopsis everta Kofoid and Campbell, 1929 based on SSU rDNA (Fig. 7) and cytological characters [33], including: (i) elongated anterior portion of the ventral kinety, which forms a curvature above the third, occasionally the fourth, kinety of the right ciliary filed; (ii) elongated anterior portion of the rightmost kinety of lateral ciliary field, which forms a curvature above the second or third kinety of the right ciliary field (with the ventral kinety in between); and (iii) first four to six kineties of the right ciliary field very widely spaced. However, unique cytological features observed in T. everta (the large distance between the collar membranelles and the somatic ciliary fields as well as the position of the posterior kinety [33];) are not present in T. kiaochowensis. Both species also show a different lorica morphology (campanulate lorica with a funnel-shaped collar vs. ellipsoidal bowl and non-flaring collar, respectively) and interspecies-level divergence in SSU rDNA (1% [25];).

Tintinnopsis uruguayensis Comparison with other populations
This species was first described by Balech [52] based on the lorica features of specimens collected in the Southwest Atlantic Ocean. The lorica dimensions reported in the original description (length = 54-63 μm, opening diameter = 22-27 μm [52];) overlap with those of our specimens (length = 50-73 μm, opening diameter = 24-42 μm; Table 1), and both populations match in the characteristic bullet-like shape with a flared collar and a posterior process. Our population presents no divergence in SSU rDNA and LSU rDNA when compared against Long Island Sound specimens of similar lorica features [25].

Comparison with similar species
In terms of a small, bullet-like lorica, three congeners, namely Tintinnopsis baltica Brandt, 1896, Tintinnopsis fimbriata Meunier, 1919, and Tintinnopsis meunieri Kofoid and Campbell, 1929, can be compared to our population. Tintinnopsis baltica has a similar lorica shape, but can be separated from T. uruguayensis by the absence (vs. presence) of a protruding posterior end [58]. Laval-Peuto & Brownlee [59] provided a diagram of the ciliary pattern of T. baltica, which is similar to our specimens in the number of kineties in the right, left, and lateral ciliary fields and the presence of only 2-3 kinetids in the second kinety of right ciliary field, but differs in a shorter ventral kinety. The distant phylogenetic relationship between T. uruguayensis and T. baltica based on SSU rDNA and LSU rDNA (Figs. 7, 9) also separates both species. Tintinnopsis fimbriata differs from T. uruguayensis by a shorter collar (10 μm vs. up to 20 μm) and a wider bowl (40-50 μm vs. 25-41 μm) [60]. Based on cytological data [27], T. fimbriata also differs from the latter in having less kineties in the left ciliary field (4-6 vs. up to 9) and lateral ciliary field (11-14 vs. up to 17). The SSU rDNA sequence labeled as T. fimbriata in GenBank (Fig. 7) has been considered a misidentification [39] and is thus not considered in this comparison. Tintinnopsis meunieri differs from T. uruguayensis in a larger opening diameter (60 μm vs. 24-42 μm) [19].

Conclusion
Tintinnopsis hemispiralis, T. kiaochowensis and T. uruguayensis show hard, fully agglomerated loricae and the most complex pattern of somatic ciliature known for the genus, i.e. a right, left and lateral ciliary field as well as a ventral, dorsal and posterior kinety [22]. However, the three species show differences in the lorica outline and the number, structure and arrangement of somatic kineties (Figs. 1, 2, 3, 4, 5 and 6; Table 1), and species-level divergence in rRNA genes [25,26]. Their distant position and intertwining with other genera in phylogenetic trees (Figs. 7,8,9) confirm, once again, the nonmonophyly of the genus Tintinnopsis [22,38,39].
Tintinnopsis cannot be revised at present, as its type species and most other tintinnine species have not been studied cytologically or genetically [23]. Our work is important to increase the number of tintinnine species investigated with modern methods, which also helps in identifying potential synapomorphies for future taxonomic rearrangements. Our data show the potential taxonomic relevance of (i) details of the somatic ciliary pattern, including the anterior parts of the ventral kinety and the rightmost kinety of the lateral ciliary field [33]; and (ii) the presence of a ciliary tuft and multiple moniliform macronuclear nodules. Our paper contributes important information on the non-monophyletic Tintinnopsis and it thus helps to fill the gaps in modern tintinnine taxonomy.

DNA extraction, PCR amplification and sequencing
Because most tintinnine species are not amenable to culture, clonal cultures could not be established. Thus, we applied common criteria to verify that field-isolated specimens were not confounded with other species (e.g. as done by Gruber et al. [33]): the three species were distinguished by careful evaluation of their morphological features and lorica size in vivo, and the absence of potentially confounding, co-occurring species was confirmed with further analyses of loricae and protargolstained cells. For each species, a single specimen was isolated at 400× magnification and washed five times with 0.22-μm filtered sample water. DNA extraction, PCR amplification and sequencing were done as detailed by Bai et al. [31], except for some of the primers utilized. PCR amplification of the SSU rDNA was performed with the primers 82F (5′-GAA ACT GCG AAT GGC TC-3′ [63];) and either 5.8 s-R (5′-CTG ATA TGC TTA AGT TCA GCG G-3′ [64];) for Tintinnopsis uruguayensis or 18 s-R (5′-TGA TCC TTC TGC AGG TTC ACC TAC-3′ [65];) for the other two species. A fragment containing the ITS1, 5.8S rDNA and ITS2 regions was amplified with the primers 5.8 s-F (5′-GTA GGT GAA CCT GCG GAA GGA TC-3′) and 5.8 s-R (5′-CTG ATA TGC TTA AGT TCA GCG G-3′) [64].
Sequences were assembled and analysed as reported before [31]. In brief, phylogenetic analyses were done separately for SSU rDNA, ITS1-5.8S rDNA-ITS2 and LSU rDNA, as well as after concatenating the three sequence markers. The analyse incorporated additional ciliate sequences were obtained from GenBank and used Halteria grandinella and hypotrichs as outgroup taxa. Sequences were aligned with Muscle 3.7 [66]. Maximum likelihood analyses were done with RAxML v. 8 [67], using the GTRG AMMA model and 1000 bootstraps. Bayesian Inference analyses were done with MrBayes v.3.2.6 [68], using the GTR + I + Γ model, 6000,000 generations with a sample frequency of 100 generations and a burn-in of 6000 trees. Estimates of sequence similarity were done in MEGA 7.0 [69].