Integrative taxonomy and molecular phylogeny of three poorly known tintinnine ciliates, with the establishment of a new genus (Protista; Ciliophora; Oligotrichea)

The taxonomic classification of the suborder Tintinnina Kofoid & Campbell, 1929, a species-rich group of planktonic ciliated protistans with a characteristic lorica, has long been ambiguous largely due to the lack of cytological and molecular data for most species. Tintinnopsis is the largest, most widespread, and most taxonomically complex genus within this group with about 170 species occurring in nearshore waters. Previous molecular phylogenetic studies have revealed that Tintinnopsis is polyphyletic. Here we document the live morphology, infraciliature, gene sequences, and habitat characteristics of three poorly known tintinnine species, viz. Tintinnopsis karajacensis Brandt, 1896, Tintinnopsis gracilis Kofoid & Campbell, 1929, and Tintinnopsis tocantinensis Kofoid & Campbell, 1929, isolated from the coastal waters of China. Based on a unique cytological feature (i.e., an elongated ciliary tuft with densely arranged kinetids) in the former two species, Antetintinnopsis gen. nov. is erected with Antetintinnopsis hemispiralis (Yin, 1956) comb. nov. (original combination: Tintinnopsis hemispiralis Yin, 1956) designated as the type species. Moreover, A. karajacensis (Brandt, 1896) comb. nov. (original combination: Tintinnopsis karajacensis Brandt, 1896) and A. gracilis (Kofoid & Campbell, 1929) comb. nov. (original combination: Tintinnopsis gracilis Kofoid & Campbell, 1929) are placed in a highly supported clade that branches separately from Tintinnopsis clades in phylogenetic trees based on SSU rDNA and LSU rDNA sequence data, thus supporting the establishment of the new genus. One other species is assigned to Antetintinnopsis gen. nov., namely A. subacuta (Jörgensen, 1899) comb. nov. (original combination Tintinnopsis subacuta Jörgensen, 1899). The findings of the phylogenetic analyses support the assertion that cytological characters are taxonomically informative for tintinnines. This study also contributes to the broadening of our understanding of the tintinnine biodiversity and evolution.

and oceanic water bodies. Furthermore, they are of great interest to protozoologists working in the field of planktonic ecology for the following reasons: i. they are ideal models for examining the diversity and biogeography of protistans [10,11]; ii. they are bioindicators of water quality status and hydrological circulation [12][13][14][15][16]; iii. they are consumers of algae (primary producers) and prey for medium-sized metazoans (e.g. fish larvae), and thus play an important role in the transfer of matter and energy between the microbial loop and classical food chain [17][18][19].
Tintinnines are characterized by the possession of a lorica, which may be simple (tube-or vase-shaped) or complex (irregular-shaped) [20]. Since the first tintinnine was originally described by Müller [21] under the name of Trichoda inquilinus, more than 1000 nominal species have been described based almost exclusively on their lorica characters [22][23][24][25][26][27][28]. Nevertheless, lorica-based approaches alone are now thought to be inadequate for species identification and discrimination, since the lorica could be polymorphic owing to the influence of environmental conditions or physiological state of the cell itself [23,28]. Recently, a few tintinnine species were studied using integrative techniques (live observation, silver staining, electron microscopy, and gene sequencing), which resulted in new insights into their systematics [29][30][31]. There is increasing evidence of lorica plasticity and cryptic species diversity among tintinnines [24,25,[32][33][34]. Using tintinnines as a model, Santoferrara et al. [35] updated procedures for species identification of loricate protists based on integrative studies of their morphology (cellular and lorica), gene sequence data, and ecology.
Tintinnopsis was established by Stein [36] with T. beroidea Stein, 1867 as type species and was later revised by Jörgensen [37] and Kofoid & Campbell [26]. Currently, there are about 170 nominal species of Tintinnopsis which renders it the largest genus within Tintinnina [38]. The genus is characterized by the possession of an agglutinated and hard lorica. However, species delimitation remains difficult because many species overlap in terms of lorica size and shape. To date, cellular features have been investigated in detail for only 17 species of Tintinnopsis and SSU rDNA sequences are available for only 22 species [39][40][41][42][43][44][45][46][47][48][49][50]. Furthermore, combined data of cytological features and SSU rDNA sequences are available for only ten of these species. Previous molecular phylogenetic studies have revealed that Tintinnopsis is polyphyletic [51,52], most of its members being distributed among at least eleven lineages with some species clustering with taxa that have either a sparsely agglomerated lorica (Leprotintinnus, Rhizodomus, Stylicauda) or a particle-free lorica (Climacocylis, Helicostomella) [52]. These stable and well-supported clades indicated that these unique and ambiguous Tintinnopsis-like species may belong to several distinct genera and families as indicated by their divergent cell features, lorica ultrastructure, and, especially, synapomorphies of the somatic ciliary pattern, which have been confirmed as key diagnostic trait at genus level in other well-known genera [29,52].
In the current study, we redescribed three tintinnine ciliates, viz. Tintinnopsis karajacensis Brandt, 1896, T. gracilis Kofoid & Campbell, 1929, and T. tocantinensis Kofoid & Campbell, 1929, from coastal waters of China. The lorica and cellular morphology of the former two species were investigated based on live and protargolstained specimens for the first time. Based on its unique somatic ciliary pattern, Antetintinnopsis gen. nov. is established for the former two species as well T. hemispiralis Yin, 1956 and T. subacuta Jörgensen, 1899. Furthermore, the SSU rDNA and LSU rDNA sequences of the three species were characterized and analyzed to determine their phylogenetic positions within Tintinnina.
Etymology. The genus name Antetintinnopsis is a composite of the Latin prefix ante-("before in place or time") and the genus name Tintinnopsis, indicating that the new genus is similar to Tintinnopsis in lorica features but differs in somatic ciliary pattern. Feminine gender. (Brandt, 1896) (Figs. 1B, 3D). Micronuclei insufficiently stained to be observed. Tentaculoids slender pin-shaped, about 4-8 μm long, located in outer portions of intermenmbranellar ridges (Figs. 1A, E, 2F, I). Accessory combs, contractile vacuole, cytopyge, and capsules not observed in either living or stained specimens. Cytoplasm colorless, with several food vacuoles up to 7 μm across containing ingested yellow microalgae (Fig. 2I, M). Locomotion by swimming slowly while rotating about main cell axis (speed not measured), cell retracting quickly into lorica with its contractile peduncle when disturbed ( Fig. 2A).

Antetintinnopsis karajacensis
Somatic ciliary pattern consisting of a ventral, dorsal, and posterior kinety as well as a right, left, and lateral ciliary field (Figs. 1C, F, G, 3A-J). Ventral kinety commencing about 3 μm posteriorly to collar membranelles and 4 μm anteriorly to firstly kinety of right ciliary field, which is comprised of three portions: i. anterior portion which is composed of about 14 monokinetids about 1 μm apart, each bearing a cilium that is 1 μm long after protargol staining; anterior of this part curving slightly rightwards at same level as commencement of first kinety of right ciliary field; ii. middle portion which extends longitudinally, consisting of extremely densely spaced kinetids each bearing a long cilium, that is no less than the cell length forming the ciliary tuft; iii. posterior portion which terminates at mid-body, composed of about eight widely spaced monokinetids 2-3 μm apart, each bearing a cilium that is 2-4 μm long in stained specimens (Figs. 1C, F, 2C, I, M, N, 3F, J). Right ciliary field comprising 10-13 kineties each of which commences about 6 μm posteriorly to collar membranelles except for the first kinety which starts about 7 μm posteriorly to collar membranelles; each kinety separated by about 3 μm from its neighboring kineties; length of kineties and number of kinetids in each kinety highly variable; each kinety monokinetidal with one anterior dikinetid, except for the three leftmost kineties each of which has two or three anterior dikinetids; cilia in right field about 5 μm long in vivo and 3-5 μm long after protargol staining, except for cilia of anterior dikinetids which are about 15-20 μm in vivo and about 15 μm after protargol staining (Figs. 1C, E, F, 2I, 3A, E, F). Dorsal kinety dikinetidal, commencing about 6 μm posteriorly to collar membranelles and separated from right and left ciliary fields by conspicuously broad unciliated stripes (about 7 μm wide); anterior and posterior portions curving rightward; length ranging from 31 μm to 73 μm and the number of dikinetids from 16 to 43; only posterior basal body of each dikinetid bears a cilium that is about 3 μm long after protargol staining (Figs. 1C, F, G, 3E). Left ciliary field separated from collar membranelles by about 4 μm long unciliated stripe; consisting of about 10-14 kineties that slightly decrease in length in clockwise direction (when viewed from apical aspect); each kinety with 6-12 widely spaced monokinetids and one anterior dikinetid; each basal body bears a cilium about 3 μm long except for the anterior basal body of each dikinetid, which has a cilium about 15 μm in vivo and 12 μm after protargol staining (Figs. 1C, F, G, 3F, G). Lateral ciliary field commencing about 5 μm posteriorly to collar membranelles, composed of on average 16 monokinetidal kineties, which generally increase in length in clockwise direction (when viewed from apical aspect), each separated from its neighboring kineties by a 1 μm gap. Posterior kinety usually commencing about 25 μm posteriorly to collar membranelles, curving rightwards with posterior portion parallel to that of dorsal kinety, about 19 μm long and composed of on average 15 dikinetids; only posterior basal body of each dikinetid bears a cilium that is about 3-5 μm long after protargol staining (Figs. 1C, F, G, 3B, F, G).
Deposition of neotype and voucher specimens. A protargol slide including the neotype specimen (Fig. 5E, G) was deposited in the Laboratory of Protozoology, Ocean University of China (registration number: WR2017101603-1). One additional protargol slide with      (Fig. 4D). Ellipsoidal or irregular-shaped macronuclear nodules scattered in cytoplasm, seven to 12 in number, 9 µm in diameter/cross section each, and with several spherical nucleoli (Figs. 4B, C, 5E-G). Contractile vacuole, cytopyge, striae, accessory combs, tentaculoids, and capsules not recognizable. Cytoplasm colorless; food vacuoles about 3-6 μm across containing microalgae (e.g. diatoms). Locomotion usually by swimming while rotating about main cell axis, but rapidly reversing on contact with obstacles. When disturbed, cell retracts quickly into lorica.
Somatic ciliature composed a ventral, dorsal, and posterior kinety as well as a right, left, and lateral ciliary field (Figs. 4B, C, E, 5D-G). Kinetids of each ciliary row ostensibly connected by argyrophilic fibers (Fig. 5G). Ventral kinety commencing about 3 μm posteriorly to collar membranelles and 2 μm anteriorly to first kinety of right ciliary field, extending parallel to last kinety of left ciliary field with three portions: i. anterior portion that is about 5 μm long, usually composed of six monokinetids each of which bears a cilium that is 1 μm long cilium; kinetids spaced ca. 1 μm apart; ii. middle portion that is about 20 μm long, consisting of extremely densely arranged of kinetids (kinetids' number not quantified) with long cilia that form the ciliary tuft; whose length of ciliary tuft could not be determined in vivo because it was always lying within the lorica, however, it appears to be at least as long as the cell length. iii. posterior portion which terminates in mid-region of cell; composed of about 15 widely spaced monokinetids spaced 1-2 μm apart, each bearing a 2 μm long cilium (Figs. 4B, E, 5G). Right ciliary field separated from collar membranelles by a 5 μm gap, composed of 10-13 kineties that are highly variable in length; neighboring kineties about 2-3 μm apart; each kinety consisting of about five to 15 monokinetids and one anterior dikinetid; cilia in right field about 2 μm long except for the anterior one in each dikinetid which measure about 7 μm after protargol staining (Figs. 4B, E, 5G). Dorsal kinety commencing about 9 μm posteriorly to collar membranelles, separated from right and left ciliary fields by conspicuously broad, unciliated stripes that are about 7 μm wide; curving slightly rightward curvature, and terminating posteriorly below rightmost kinety of right ciliary field; about 72 μm long and   Table 1) Remarks. An improved diagnosis based on a Shenzhen population was provided by Jiang et al. [48]. The current population corresponds well with the Shenzhen specimens from the South China Sea. Hence, we only provided a redescription based on a new population from the East China Sea at Taizhou.
Cylindrical part as wide as aperture, 40-80 μm long. Bulbous part ovoid, on average 48 μm wide. Posterior portion tapered, stout, about 32 μm in length, usually open at rear end (Fig. 6A, E, G, H). Ratio of bowl length to lorica length about 0.28; ratio of posterior projection length to lorica length about 0.4. Lorica wall densely agglutinated with mineral particles (Fig. 6H).
Cell proper 45-80 × 17-33 μm in fully extended living specimens and 42-75 × 20-35 μm after protargol staining. Posterior end narrowed and always forming a peduncle (up to 55 μm long), which attaches to tapered portion of lorica (Fig. 6A, E, G, H). Invariably two globular macronuclear nodules in mid-region of cell, about 8-23 × 7-19 μm in size after protargol staining (Fig. 6B, C, K). Striae, accessory combs, contractile vacuole, cytopyge, and capsules not recognized in either living or stained specimens. Tentaculoids between membranelles recognizable in vivo, about 15 μm long (Fig. 6A, H). Cytoplasm colorless, containing food vacuoles up to 7 μm across with diatoms and microalgae (Fig. 6H). Locomotion by swimming while rotating about main cell axis, rapidly reversing on contact with obstacles. Cell retracts through quickly into lorica when disturbed; on cessation of disturbance, cell slowly extends through lorica aperture spreading its collar membranelles almost perpendicularly to main cell axis and resumes swimming and feeding.
Somatic ciliature consisting of a ventral, dorsal, and posterior kinety as well as a right, left, and lateral ciliary field ( Fig. 6B-D, I-K). Ventral kinety commencing 4 μm posteriorly to collar membranelles, curving slightly leftwards before extending posteriorly in parallel to kineties of lateral ciliary field and terminating at mid-portion of cell proper; composed of densely spaced monokinetids in anterior portion but more widely ones in posterior portion; on average 28 μm long with about 28 monokinetids; cilia about 3 μm long after protargol staining (Fig. 6B, D, K). Right ciliary field composed of seven kineties, each separated from collar membranelles by a 7 μm gap except for first kinety which starts about 1 μm posteriorly to other kineties; each kinety consisting of six widely spaced monokinetids and one anterior dikinetid expect for the first kinety which has nine monokinetids; cilia of right field about 3-5 μm long except for elongated cilium of each dikinetid which is about 7-10 μm after protargol staining (Fig. 6B, D, I). Dorsal kinety dikinetidal, commencing about 5 μm posterior to collar membranelles, separated from right and left ciliary fields by conspicuously broad, unciliated stripes that are about 5 μm and 10 μm wide, respectively; anterior and posterior portions extending rightward and leftward, respectively; consisting of 31 dikinetids on average, posterior basal body of each kinetid bearing a cilium that is about 3 μm long after protargol staining (Fig. 6C, D, I). Left ciliary field separated from collar membranelles by a 6 μm unciliated stripe; consisting of nine kineties that slightly increase in length in clockwise direction (when viewed from apical aspect); each kinety consisting of monokinetids and one anterior dikinetid; cilia of left field about 2 μm long, except for elongated anterior cilia of dikinetids measuring about 6 μm after protargol staining (Fig. 6B-D, J, K). Lateral ciliary field with on average nine monokinetidal kineties, commencing about 5 μm posterior to collar membranelles except for first kinety which commences about 1 μm anteriorly to second kinety of right field; almost parallel to the ventral kinety; monokinetids more densely spaced in right portion than in left portion of field (Fig. 6B, D, J, K). Posterior kinety composed of 11 dikinetids; usually commencing below ventral kinety, about 2 μm posteriorly to posteriormost kinetosome of ventral kinety; 18 μm long on average; only posterior basal body of each dikinetid bears a cilium that is 2 μm long (Fig. 6B, D, K).
Adoral zone of membranelles closed, lying orthogonal to main cell axis; consists of 20-24 collar membranelles with cilia up to 25-30 μm long, including three that are significantly prolonged, polykinetids of which terminating 6-12 μm posteriorly to apical cell end. Single buccal membranelle (Fig. 6A-E). Endoral membrane insufficiently stained to be observed.

Gene sequences and phylogenetic placement (Figs. 7, 8; Table 2; Additional file 4)
All new sequences were deposited in the NCBI database, with the accession numbers, lengths, and GC contents of each as shown in Table 2. For both gene marker, the topologies of the BI and ML trees are basically congruent with variable support value, therefore only the ML tree topology (with nodal support from both methods) is shown for each (Figs. 7, 8). In the phylogenetic analysis of SSU rDNA sequence data, Tintinnopsis-like species clusters into 14 Fig. 7). In this clade, A. karajacensis comb. nov. first clusters with A. hemispiralis comb. nov. (MT435073) with moderate to high support (74% ML, 0.95 BI) and then with A. subacuta comb. nov. with strong support (99% ML, 1.00 BI). Antetintinnopsis gracilis comb. nov. occupies the basal position in this clade. Additionally, the Haikou population of Tintinnopsis tentaculata (MK036423) was found to be most closely to two species of Stenosemella (94%ML/0.92BI); Tintinnopsis orientalis (MK036422) is sister to the clade formed by species of Cyttarocylididae with maximum support.
In terms of the LSU rDNA sequences, Antetintinnopsis karajacensis comb. nov. differs from Antetintinnopsis gracilis comb. nov. by 33 bp, from Antetintinnopsis hemispiralis comb. nov. (MT435076) by 78 bp, and from Tintinnopsis spp. by 81-210 bp. In phylogenetic trees based on analyses of LSU rDNA sequence data, eight clades for Tintinnopsis-like species, including the Antetintinnopsis gen. nov. cluster (clade 8), were recovered (Fig. 8), which basically corresponds with the SSU rDNA analyses. Furthermore, the evolutionary relationships within the Antetintinnopsis gen. nov. clade are also consistent with those in the SSU rDNA tree. The newly sequenced Taizhou population of T. tocantinensis (MT764262) clusters with the Long Island population (JN831921) with full support forming a group that is sister to the Tintinnopsis cylindrica (JN831901) + T. levigata (KM982847) + T. uruguayensis (JN831923 and MT435078) clade with maximum support.

Establishment of the new genus Antetintinnopsis gen. nov.
Historically, lorica features have been the main diagnostic character used to delineate genera in Tintinnina owing to their ease of collection and observation. In recent decades, however, the application both of cytological characters based on live and stained specimens and phylogenetic analyses based on sequence data has led to the traditional lorica-based classification being challenged [e.g., 29,30,53]. Hence, detailed investigations of cytological features of cell are necessary in order to distinguish among forms with a similar lorica. In the present study we revealed that the complex ciliary pattern in T. karajacensis and T. gracilis, is sufficiently distinct to distinguish these from most other tintinnines as follows: i. kinetids in the ventral kinety conspicuously densely arranged in the middle portion as compared with anterior and posterior portions (vs. basically even distributed); ii. kinetids in the middle portion of the ventral kinety bear extremely long cilia that form a ciliary tuft (vs. absent) [e.g., [47][48][49][50][51][52][53][54]. However, two species of Tintinnopsis (T. subacuta and T. hemispiralis) cannot be separated from T. karajacensis and T. gracilis based on these characters. Tintinnopsis subacuta, for example, was described as having both of these two distinctive features [49,55]. Most recently, the elongated ciliary tuft was also recognized in T. hemispiralis [45]. Consequently, we here establish Antetintinnopsis gen. nov. for the four species of Tintinnopsis with these two synapomorphies, and assign them to the new genus as follows: Antetintinnopsis gracilis (Kofoid & Campbell, 1929) comb. nov., Antetintinnopsis hemispiralis (Yin, 1956) comb. nov., Antetintinnopsis karajacensis (Brandt, 1896) comb. nov., and Antetintinnopsis subacuta (Jörgensen, 1899) comb. nov. Furthermore, phylogenetic analyses based on sequence data from two nuclear ribosomal loci support the validity of the new genus.

Type designation within the new genus
Antetintinnopsis hemispiralis (Yin, 1956) comb. nov. is selected as type species because its description is based on not only silver staining but also live observations, and type materials are available following the designation of a neotype specimen by Bai et al. [45]. Furthermore, its ribosomal gene sequence data were first available for a member of this genus [45]. (Brandt, 1896) comb. nov.

Comments on Antetintinnopsis karajacensis
An adequate sample size avoids the establishment of new combination based on possibly atypical specimens and allows rough estimates of the intraspecific variability [35]. In this sense, it is also important to study several populations wherever possible. In the present study, specimens with congruent lorica morphologies and cell features were collected in Haikou and Beihai which are within a comparatively short distance of each other (no more than 200 km apart) along the southern Chinese coast of the South China Sea. However, specimens of a third population collected from Zhoushan on the East China Sea coast, about 1900 km away from Haikou, show smaller dimensions of both lorica and cell when compared with the two other populations. Nevertheless, all the specimens agree well in terms of the diameter of the lorica opening and the presence of an elongated ciliary tuft, plus they have identical SSU rDNA sequences. Hence, we believe that the size differences are very likely environment-and/or population-dependent.
Antetintinnopsis karajacensis comb. nov. was originally isolated by Brandt from Karajak-Fjord in Davis Strait and was described under the name Tintinnopsis karajacensis based exclusively on lorica features (Fig. 1D) [57]. We identified our three populations as being conspecific with this taxon because all of them correspond well with the type population in the following features: i. lorica structure (hard and entirely agglomerated) and shape (cylindrical and rounded at the aboral end); ii. opening diameter of lorica (Zhoushan population: 35-40 μm, Beihai population: 35-40 μm, Haikou population: 40-45 μm, type population: about 40 μm).
To date, more than ten populations have been described worldwide under the name Tintinnopsis karajacensis by having a cylindrical agglutinated lorica, which is a dominant form shared by different tintinnine species in the pelagial of marine and brackish coastal waters. Moreover, they also present a considerable variability in lorica size [38]. Accordingly, based on these specimens there must be some misidentifications in the literature. Considering that the diameter and characteristics of the lorica opening are widely accepted as taxonomic characters, we recognize only eight of these populations of Tintinnopsis karajacensis as being congruent with the original population (Additional files 2, 5) [22,27,[56][57][58][59][60][61].

Comments on Antetintinnopsis gracilis (Kofoid & Campbell, 1929) comb. nov.
Tintinnopsis gracilis was established by Kofoid & Campbell [26] to describe a morphotype previously recorded by Brandt [62] as Tintinnopsis karajacensis var. α from Schott off the West Coast of Borneo. Since the cell features of the Schott population are unknown, species identification is only based on lorica morphology. The Qingdao specimens match the original illustrations of Brandt (1907) [62]. Hence, the Qingdao population is considered to be conspecific with T. gracilis.
However, for all previously reported populations, the descriptions are cursory and data both for cell features and gene sequences are unavailable. Therefore, a detailed comparison with conspecific populations is impossible.
Antetintinnopsis hemispiralis (Yin, 1956) comb. nov., A. subacuta (Jörgensen, 1899) comb. nov., and Tintinnopsis tubulosoides resemble our specimens of A. gracilis comb. nov. in terms of the overall shape of the lorica and/or cell features. However, A. subacuta comb. nov. differs from A. gracilis comb. nov. by having a subspherical aboral end (vs. obconical aboral end) and fewer kineties in the left ciliary field (on average 11  vs. 14) [49,55]. Antetintinnopsis hemispiralis comb. nov. can be separated from our specimens by the presence of 3-5 spiral striations in the collar of the lorica (vs. spiral striations absent), the commencing position of the ventral kinety (i.e., anteriorly to the second kinety vs. first kinety) of right ciliary field, and the length of the ventral kinety (extending to posterior one-quarter to one-third of cell proper vs. terminating at the middle portion of cell proper in A. gracilis comb. nov.) [45]. Furthermore, the SSU rDNA sequences of A. subacuta comb. nov. (EU399541) and A. hemispiralis comb. nov. (MT435073) has a dissimilarity of 2.8% (46 bp) and 2.9% (48 bp), respectively, compared with our new sequence of A. gracilis comb. nov., which represents an interspecies-level divergence in tintinnine ciliates [51]. Tintinnopsis tubulosoides can be separated from Qingdao population of A. gracilis comb. nov. by the presence (vs. absence) of spiral striations in the collar portion of the lorica [26].

Comments on Tintinnopsis tocantinensis Kofoid & Campbell, 1929
Tintinnopsis tocantinensis can be recognized by its unique lorica appearance, i.e., lorica tripartite, composed of a cylindrical portion, a bulbous part, and a tapered portion. It was first established by Kofoid & Campbell [26] and reported described by Brandt [57] as T. aperta var. α (Fig. 6F). We identified Taizhou population as T. tocantinensis because it matches the original population very well in the lorica shape, although it is considerably longer (110-160 μm vs. 85 μm). Considering the variability of the lorica length in tintinnines [35], we believe that this variation is a population-dependent difference. The infraciliature of T. tocantinensis has been previously reported on a population from Shenzhen, China [48]. Our Taizhou population resembles the Shenzhen population in both lorica features and ciliary pattern, which supports the conspecificity of both forms [48].
In terms of lorica shape, only Tintinnopsis aperta Brandt, 1906 resembles T. tocantinensis in having a cylindrical portion, a bulbous part, and a tapered portion. However, the latter can be distinguished from the former by its shorter (16-42 μm vs. 45-105 μm) and stouter tapered portion [57].

Neotypification
Considering the requirement of Article 75.3.6 of the International Code of Zoological Nomenclature [68], we neotypify A. karajacensis comb. nov. and A. gracilis comb. nov. with two Chinese populations for the following reasons: i. no type specimens are available for either species; ii. the existing descriptions are too incomplete, e.g., they lack detail cytological and molecular information, to allow accurate identification; iii. neotype slides are of a good quality allowing the specific features to be clearly recognized. Unfortunately, both neotypes do not come from near the original type locality (Chinese coast water vs. Karajak-Fjord in Davis Strait, western Greenland and Schott off the west coast of Borneo, respectively). However, according to the rather wide distribution of both species, it seems justified to designate neotypes from different sites, especially as both sites are marine habitats that are connected by oceanic currents. Thus, this point should not be overinterpreted (for a thorough discussion of this problem, see Foissner et al. [69]).

Phylogenetic analyses
Currently, phylogenetic relationships within Tintinnina are uncertain and the systematic positions of most Tintinnopsis species remain unresolved because of limited data [e.g., 29,52]. Nevertheless, members of Tintinnopsis and related species were generally well grouped in the SSU rDNA and LSU rDNA trees, which corresponds with previous phylogenetic studies [52]. One well-supported monophyletic lineage should be concerned, which is marked as tintinnine 8 in Figs. 7 and 8, and consists of all Antetintinnopsis species, viz. A. gracilis comb. nov., A. hemispiralis comb. nov., A. karajacensis comb. nov., and A. subacuta comb. nov. These four species share some morphological synapomorphies, i.e., configuration of the ventral kinety, presence of an elongated ventral ciliary tuft, and multiple macronuclear nodules. These data provided strong evidence that the genus Tintinnopsis should be revised pending the congruence between morphological and molecular data, however, this taxonomic action should be carried out after most Tintinnopsis-like species are cytologically and genetically studied.

Conclusions
The findings of the integrative morphological and phylogeny investigations on two poorly known Tintinnopsis species support the establishment of Antetintinnopsis gen. nov., and the assignment of two additional Tintinnopsis species to the new genus, based on their distinct somatic ciliary pattern and their clustering patterns in gene trees. Moreover, the results of our phylogenetic study based on sequences of two nuclear ribosomal loci provides compelling evidence that Tintinnopsis comprises several distinct evolutionary lineages, necessitating a detailed morphological review of these organisms. The taxonomic novelties reported in this study highlight the importance of integrative studies, that is, the combination of morphological and molecular characters of various populations, in resolving the systematics of tintinnines. Fig. 9; Additional file 1)

Sample collection and environmental factors (
All the tintinnine ciliates redescribed herein were collected from surface water (0-2 m depth) by horizontal towing of a 25-μm meshed plankton net at different sites on the coast of China. The Haikou population of Antetintinnopsis karajacensis comb. nov. was isolated from coastal waters of the South China Sea at Haikou Bay, Hainan Province (20°02′32″N, 110°17′2″E) in November 2017. The Zhoushan population of A. karajacensis comb. nov. was collected from coastal waters of the East China Sea at an inlet in Zhoushan, Zhejiang Province (29°57′2"N, 122°22′29"E) in October 2018. The Beihai population of A. karajacensis comb. nov. was collected from a port in the Beibu Gulf, Guangxi Province (21°24′29"N, 109°9′31"E) in July 2018. Antetintinnopsis gracilis comb. nov. was collected on 16 October 2017 from coastal waters of the Yellow Sea at a wharf in Qingdao, Shandong Province (35°44′31″N, 120°00′52″E). Tintinnopsis tocantinensis was isolated on 15 June 2018 from coastal waters off the East China Sea in Taizhou, Zhejiang Province (29°5′59″N, 121°39′27″E). Water temperature, salinity, and pH were measured in situ using a water quality-measuring instrument (YSI Professional Plus, America).

Taxonomy studies
The samples were transferred into Petri dishes (5 cm across) and the ciliates were immediately isolated under a dissecting microscope (Guiguang XTL-200, China) for further study. Attempts to establish pure cultures were unsuccessful. Swimming motion and cell flexibility and contractility were observed in undisturbed specimens under a dissecting microscope. The morphology of living cells and lorica were investigated using bright-field and differential interference contrast microscopy (Olympus BX 51, Tokyo, Japan) at magnifications of 100-1000 × . Protargol staining was applied to reveal the infraciliature and nuclear apparatus [70]. The protargol reagent was made according to Pan et al. [71]. The cells were stained after removal of their lorica. Drawings of living cells were based on photomicrographs and those protargol-stained cells were made with the help of a camera lucida. In vivo measurements were performed at magnifications of 40-1000 × . Counts and measurements of stained specimens were performed at 400-1000 × magnifications. Terminology is according to Agatha & Riedel-Lorjé [39].

DNA extraction, amplification and sequencing
Although clonal cultures were not established, we are confident that all morphological and molecular studies of each isolate dealt with a single species because i) all the five populations investigated here can be easily recognized by their lorica features and no cryptic species were present in the subsamples, and ii) no other tintinnine morphotypes were present in the protargol preparations. For each population, a single specimen was observed in detail at high magnification (up to 1000 ×) by careful evaluation of morphological features of the lorica and the cell, washed four or five times using sterilized, filtered in situ water to exclude potential contamination, and then transferred to a 1.5 mL microfuge tube with a minimum volume of water. The genomic DNA was extracted using the DNeasy Blood & Tissue kit (Qiagen CA) following the manufacturer's instructions. The amplification of the SSU rDNA sequences was performed with the primers 18 s-F (5′-AAC CTG GTT GAT CCT GCC AGT-3′) and 18 s-R (5′-TGA TCC TTC TGC AGG TTC ACC TAC-3′) [72]. Amplification of the LSU rDNA sequences was performed with the primers F3 (5ʹ-ACC/C CGC TGA/G AT/CT TAA GCA T-3ʹ) and R2 (5ʹ-AAC CTT GGA GAC CTG AT-3ʹ) [73]. PCR amplifications were performed according to the following protocol: 98 °C for 30 s, followed by 18 cycles of 98 °C for 10 s, 69 °C for 40 s with the remaining cycles stepping down by 1 °C for each cycle; then 72 °C for 90 s and 18 cycles of 98 °C for 10 s, 51 °C for 40 s, 72 °C for 90 s; and a final extension at 72 °C for 4 min. Q5 ® Hot Start High-Fidelity DNA Polymerase (New England BioLabs, USA) was used to minimize the possibility of PCR amplification errors. The PCR products were sequenced bidirectionally by the Tsingke Biological Technology Company (Beijing, China).

Molecular phylogeny
In addition to the nine newly obtained sequences, other sequences used in the present analyses were downloaded from the NCBI database, including six SSU rDNA and four LSU rDNA sequences of hypotrichs and Halteria grandinella as the outgroup taxa, respectively.
All sequences were aligned using the MUSCLE program package on the European Bioinformatics Institute web server). The resulting alignments were then edited manually with trimming both ends included 1705 sites of SSU rDNA (107 taxa) and 1292 sites of LSU rDNA (73 taxa). ML analyses of both SSU rDNA and LSU rDNA sequences were performed on the CIPRES Science Gateway (URL: http:// www. phylo. org/ sub_ secti ons/ portal) [74], with RAxML-HPC2 on XSEDE using the GTR-GAMMA + I model as selected by Modeltest v. 3.4 [75]. Searches for the best tree were conducted starting from 100 random trees, and 1000 nonparametric bootstrap replicates were done to assess the reliability of the internal branches. BI analyses were performed on the CIPRES