Redescriptions of three tintinnine ciliates (Ciliophora: Tintinnina) from coastal waters in China based on lorica features, cell morphology, and rDNA sequence data

Journal Pre-proof Redescriptions of three tintinnine ciliates (Ciliophora: Tintinnina) from coastal waters in China based on lorica features, cell morphology, and rDNA sequence data Yang Bai, Rui Wang, Weiwei Liu, Alan Warren, Yan Zhao, Xiaozhong Hu

PII:

S0932-4739(19)30096-3

DOI:

https://doi.org/10.1016/j.ejop.2019.125659

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EJOP 125659

To appear in:

European Journal of Protistology

Received Date:

6 November 2018

Revised Date:

15 November 2019

Accepted Date:

20 November 2019

Please cite this article as: Bai Y, Wang R, Liu W, Warren A, Zhao Y, Xiaozhong H, Redescriptions of three tintinnine ciliates (Ciliophora: Tintinnina) from coastal waters in China based on lorica features, cell morphology, and rDNA sequence data, European Journal of Protistology (2019), doi: https://doi.org/10.1016/j.ejop.2019.125659

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European Journal of Protistology

Y. Bai et al.

Redescriptions of three tintinnine ciliates (Ciliophora: Tintinnina) from coastal waters in China based on lorica features, cell morphology, and rDNA sequence data Yang Baia, Rui Wanga, Weiwei Liub, Alan Warrenc, Yan Zhaod, *, Xiaozhong Hua, *

a Institute of Evolution and Marine Biodiversity & Key Laboratory of Mariculture, Ministry of

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Education, Ocean University of China, Qingdao 266003, China b Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Science, Guangzhou 510301, China

c Department of Life Sciences, Natural History Museum, London SW7 5BD, United Kingdom

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d College of life Sciences, Capital Normal University, Beijing 100048, China

* Corresponding authors: Yan Zhao (E-mail: [email protected]), Xiaozhong Hu (E-mail:

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Abstract

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[email protected])

Three species of tintinnines, namely Tintinnopsis tentaculata Nie and Cheng, 1947, Tintinnopsis

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orientalis Kofoid and Campbell, 1929, and Eutintinnus lususundae (Entz, 1885) Kofoid and Campbell, 1939, were isolated from coastal waters of China. The morphology of each was investigated based on observations of live and protargol-stained specimens, and their SSU rDNA- and LSU rDNA-based

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phylogenetic relationships were analyzed. The ciliary patterns of these species are revealed for the first time. Based on the original descriptions and data from the present study, an improved diagnosis is

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given for each species. Unlike its congeners, the second dorsal kinety of Eutintinnus lususundae is displaced below the left ciliary field, which may suggest that the second dorsal kinety is evolving into a posterior kinety by a migration process. The ventral kinety in Eutintinnus is redefined. A neotype is fixed for T. tentaculata to stabilize the species name objectively, mainly because of the unavailability of type material.

Keywords: Ciliary pattern; Lorica; Phylogeny; Ribosomal gene; Tintinnines 1

Introduction Planktonic ciliates are common members of the microzooplankton community and play key roles in controlling the transfer of materials and the flux of energy in the microbial loop (Azam et al. 1983; Dolan 2010; Landry and Calbet 2004; Worden et al. 2015; Xu et al. 2017). Consequently, they are increasingly the subject of interest in studies of biodiversity, biogeography, systematics, and ecology (Agatha 2011; Agatha and Strüder-Kypke 2012; Chen et al. 2017; Dolan et al. 2013; Gao et al. 2009; Gao et al. 2016; Liu et al. 2017; Santoferrara et al. 2018; Song et al. 2018; Wang et al. 2018). Tintinnines are an important group of planktonic ciliates that are characterized by the possession of a lorica which may be one of five types: (i) soft, agglomerated with a rounded posterior end; (ii) soft, agglomerated with a subterminal membrane; (iii) hard and entirely agglomerated; (iv) hard, with a hyaline collar and an agglomerated bowl; and (v) entirely hyaline (Agatha and Strüder-Kypke 2012).

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There are approximately 1,000 extant tintinnine species, the classification of which is based almost

entirely on lorica characters (e.g., Agatha and Strüder-Kypke 2012; Kofoid and Campbell 1929, 1939). However, it is now widely recognized that lorica features alone are insufficient for determining

taxonomic affiliations in tintinnines since the lorica may be polymorphic and change in response to

environmental factors or stages in the life cycle (Laval-Peuto 1981; Santoferrara et al. 2017; Zhang et

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al. 2017). Recent studies have revealed that the ciliature and molecular information (e.g., SSU rDNA and LSU rDNA sequences) are valuable characters for tintinnine taxonomy (Agatha 2008, 2010a,

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2010b; Agatha and Tsai 2008; Jiang et al. 2012; Gruber et al. 2018; Santoferrara et al. 2016, 2017; Smith et al. 2018; Xu et al. 2013). Unfortunately, rDNA sequences and ciliature data are available for

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only about 10% and 5% of tintinnine morphospecies, respectively (e.g., Agatha and Strüder-Kypke 2012; Bachy et al. 2012; Foissner and Wilbert 1979; Zhang et al. 2017). The genus Tintinnopsis Stein, 1867 has a hard and entirely agglomerated lorica and is the most

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common tintinnine genus in inshore habitats (Dolan et al. 2013). It comprises about 140 morphospecies, many of which differ only in minute lorica features (e.g., Kofoid and Campbell 1929, 1939). About 50 species have been reported from China Seas, however ciliature data are lacking for

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almost all of these (Hu et al. 2019; Liu 2008; Xu et al. 2001; Xu and Song 2005; Zhang et al. 2012). Eutintinnus Kofoid and Campbell, 1939, the type genus of the family Eutintinnineae, is characterized by a hyaline, tube-shaped lorica with anterior and posterior openings (Dolan et al. 2013;

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Kofoid and Campbell 1939). Although 40 Eutintinnus species have been described (e.g., Hada 1938; Kofoid and Campbell 1929, 1939; Nie and Cheng 1947), the ciliary pattern is known for only three of these (Choi et al. 1992). In the present study, the morphology of three tintinnine species, namely Tintinnopsis tentaculata,

T. orientalis, and Eutintinnus lususundae, collected from coastal waters of China, was investigated using observations of specimens in vivo and after protargol staining. In addition, their molecular phylogeny was inferred based on SSU rDNA and LSU rDNA sequence data. The main aim of the study is to expand knowledge and understanding of the taxonomy and phylogeny of these three 2

species.

Material and Methods Sample collection, observation, and identification Tintinnopsis tentaculata was collected from surface water (0–2 m water depth) in Haikou Bay, Haikou, China (20°02′0.40″N 110°17′47.83″E), on 19 October 2017 using a plankton net (mesh size 25 μm). Tintinnopsis orientalis and Eutintinnus lususundae were collected from Yellow Sea coastal waters of Qingdao, China (36°03′35.66″N 120°18′53.77″E) in September 2017 using the same method (Fig. 1). The samples were transferred into Petri dishes of 20 cm diameter with a water depth of about 5 mm. Samples were maintained at room temperature until processed. The ciliates were isolated by means of micropipettes under a stereo microscope (Olympus SZX2-TR30, Tokyo, Japan) at 45×

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magnification. Live cells were studied with bright-field and differential interference contrast microscopy. Lorica measurements were calculated from photomicrographs taken at low to medium magnifications (100–400×), and approximate values (i.e., accurate to 5 μm) were estimated. The

protargol method of Wilbert (1975) was used to reveal the ciliary pattern and the nuclear apparatus.

After fixation in Bouin’s solution, the cells were removed from their loricae using an eyebrow brush.

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The protargol powder was manually synthesized following the method described by Pan et al. (2013). Measurements of protargol-stained specimens were performed at 1,000× magnification. Drawings

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were made at 1,000× magnification with the aid of a camera lucida. Identifications were based on original species descriptions (Entz 1885; Kofoid and Campbell 1929; Nie and Cheng 1947).

Adl et al. (2019), respectively.

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Terminology follows Agatha and Riedel-Lorjé (2006) and Gruber (2018), and classification follows

DNA extraction, PCR amplification and sequencing

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Although clonal cultures were not established, the three tintinnine species investigated here could easily be distinguished by their lorica features as no similar tintinnine morphotypes were detected in any of the the protargol preparations. Hence, the morphological and molecular studies of each very

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likely dealt with a single species. For each species, the identity of a single cell isolated for DNAbarcoding was checked by inspection in vivo at 400× magnification. It was then washed five times

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with sterilized (0.22 μm filtered) in situ water. Total genomic DNA was extracted from the washed cell using the DNeasy Blood & Tissue kit (Qiagen). PCR amplification of the SSU rDNA was performed with the primers 82F (5’-GAA ACT GCG AAT GGCTC-3’) (Jerome et al. 1996) and 18s-R (5’-TGA TCC TTC TGC AGG TTC ACC TAC-3’) (Medlin et al. 1988). PCR amplification of the LSU rDNA was performed with the primers F3 (5’-ACS CGC TGR AYT TAA GCA-3’) and R2 (5’-AAC CTT GGA GAC CTG AT-3’) (Moreira et al. 2007). Q5® Hot Start High-Fidelity DNA Polymerase (New England BioLabs, USA) was used to minimize the possibility of PCR amplification errors. PCR amplifications were performed according to the following protocol: 98 °C for 30 s, followed by 18 3

cycles of 98 °C for 10 s, 69 °C for 40 s with the remaining cycles decreasing in temperature by 1 °C for each cycle; then 72 °C for 90 s and 18 cycles at 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. The PCR products were purified using the EasyPure®Quik Gel Extraction Kit (TransGen, EG101, Beijing). Each purified PCR product (about 1,800 kb) was cloned into the pEASY®-T1 cloning kit (TransGen, CT101, Beijing) according to the manufacturer's instructions, and then sequenced bidirectionally on an ABI 3700 sequencer (GENEWIZ Biotechnology Co., Ltd., Beijing, China). All new SSU and LSU sequences were deposited in the GenBank database under accession numbers MK036421–MK036423 and MK809531–MK809533, respectively (supplementary Tables S1 and S2).

Phylogenetic analyses

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Besides the six newly obtained sequences (i.e., SSU rDNA and LSU rDNA for each of the three tintinnines), further sequences used in the present phylogenetic analyses were downloaded from

GenBank. Only sequences of definitively identified species were used, including: (1) SSU rDNA

sequences of 86 tintinnines, three aloricate choreotrichids, four oligotrichids, Halteria grandinella, and three hypotrichs; (2) LSU rDNA sequences of 45 tintinnines, one aloricate choreotrichid, one

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oligotrichid, and three hypotrichs. Halteria grandinella, Oxytricha granulifera, Neokeronopsis

asiatica, and Urostyla grandis were used as the outgroup taxa in the SSU rDNA phylogenetic tree, and

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Oxytricha granulifera, Hemiurosomoida longa, and Stylonychia mytilus were used as the outgroup taxa in the LSU rDNA phylogenetic tree (for details of these sequences, see figures 8 and 9 and supplementary Tables S1 and S2).

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The sequences were aligned with Muscle 3.7 implemented in phyml (http://phylogeny.lirmm.fr/phylo_cgi/index.cgi) (Edgar 2004). The alignments were manually adjusted using the program BioEdit 7.0 (Hall 1999).

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Maximum likelihood (ML) analyses were performed in RAxML via the CIPRES Science Gateway (http://www.phylo.org) using the GTRGAMMA model provided by RAxML-HPC2 v.8.2.10 on XSEDE (Stamatakis 2014). The branch support values were assessed using 1,000 bootstrap

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replicates. Bayesian inference (BI) analyses were executed with MrBayes v.3.2.6 on XSEDE (Ronquist et al. 2012), using the best-fit model GTR + I + Γ selected by the Akaike Information

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Criterion in MrModeltest 2.2 (Nylander 2004). Approximations of these distributions were obtained using Markov chain Monte Carlo (MCMC) simulations by running 6,000,000 generations with a sample frequency of 100 generations and a burn-in of 6,000 trees (10%). All remaining trees were used to calculate the posterior probabilities using a 50% majority rule consensus. MEGA 7.0 (Kumar et al. 2016) was employed to visualize the tree topologies.

ZooBank registration ZooBank registration number of present work: urn:lsid:zoobank.org:pub:5A67BC28-89AB4

4AAD-8115-C58323511B25.

Results Oder Choreotrichida Small and Lynn, 1985 Suborder Tintinnina Kofoid and Campbell, 1929

Genus Tintinnopsis Stein, 1867 Tintinnopsis tentaculata Nie and Cheng, 1947 (Figs. 2A–H, 3A–N; Table 1) Improved diagnosis (based on original description and this study). Lorica globular with stout posterior process or pointed end, about 45–65 μm long and 35–60 μm wide. Opening 20–45 μm in diameter, with a short, cylindroidal inner collar and an outer collar with five or six crescent-shaped

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projections, each 10–15 μm long. Cell proper sub-obconical and slightly contractile, with a peduncle nearly 8 μm long. Two macronuclear nodules. On average 19 collar membranelles, of which four or five extend into buccal cavity; one buccal membranelle. Ventral kinety composed of on average 34

monokinetids, commences anteriorly to first kinety of right field. Right and left ciliary fields consist of

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about nine and 12 kineties, respectively. Lateral ciliary field comprises on average 19 kineties. Dorsal kinety with about 50 dikinetids. Posterior kinety with on average 15 dikinetids, located posterior of

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first kinety of lateral ciliary field.

Deposition of neotype material. A protargol slide containing the neotype specimen (Fig. 2D, E)

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was deposited in the Natural History Museum, London, UK, with the registration number NHMUK 2019.4.18.1. A voucher slide was deposited in the Laboratory of Protozoology, Institute of Evolution

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and Marine Biodiversity, Ocean University of China, with the registration number BY201710190201.

Description of Haikou population

Lorica globular, about 45–65 μm × 20–45 μm in size, with a short cylindroidal inner collar

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approximately 3 μm high and an outer collar with five or six anteriorly directed, crescent-shaped projections, each 10–15 μm long (Figs. 2A–C, 3A–C). Lorica opening about 20–45 μm in diameter. Posterior end pointed or with a stout process, rarely rounded (Figs. 2A–C, 3A, F). Lorica wall densely

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agglomerated with mineral particles of various sizes causing an irregular outline (Figs. 2A, C, 3A–C). Cell proper obconical, about 30–55 × 20–45 μm in size when extended, merging gradually into a

peduncle that is about 10 μm long and attached to base of lorica (Fig. 2B). Two ellipsoidal macronuclear nodules, 15–25 × 14–20 μm in size; anterior nodule located 12–17 μm from the collar membranelles after protargol staining (Fig. 2E). Micronuclei not recognizable because they were insufficiently stained with protargol. Cytoplasm colourless, with food vacuoles of various sizes containing ingested ovoidal microalgae (Figs. 2B, 3E). Tentaculoids pin-shaped, inserted between membranelles, about 10 × 1 μm in size (Fig. 3D, E). Accessory combs, striae, contractile vacuole, 5

cytopyge, and extrusomes not recognized. Locomotion by rotation about main cell axis. Disturbed individuals retract into lorica with motionless membranelles bent to centre of peristomial field. Somatic ciliary pattern complex with ventral, dorsal, and posterior kineties and right, left, and lateral ciliary fields (Figs. 2D–F, 3G–J, M, N). Length and number of kineties variable, especially in lateral ciliary field. Ventral kinety commences between collar membranelles and first kinety of right ciliary field and curves leftwards before extending posteriad, parallel to kineties of lateral ciliary field; 14–26 μm long, composed of 29–41 densely spaced monokinetids (Figs. 2D, F, 3H). Right ciliary field comprises 7–10 kineties, neighboring kineties about 3–5 μm apart; all kineties commence at same level (about 5 μm below membranellar zone) except for first kinety which commences about 5 µm below level of remaining kineties; each kinety composed of about 12 widely spaced monokinetids and one anterior dikinetid (Figs. 2D–F, 3I). Left ciliary field comprises 10–14 kineties, neighboring

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kineties about 4–5 μm apart; each kinety commences about 5 μm below collar membranelles and is composed of 6–14 widely spaced monokinetids and one anterior dikinetid (Figs. 2D–F, 3G, H). Each basal body in left and right ciliary fields bears a cilium: in protargol-stained cells, the anterior cilium of each dikinetid is about 8 μm long, the others (i.e., cilia of monokinetids and posterior cilium of each dikinetid) are about 3 μm long (Fig. 3G). Lateral ciliary field consists of 16–22 monokinetidal kineties

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each of which commences about 5 μm below collar membranelles; kinetids and kineties more densely spaced in right portion than in left portion; cilia about 3 μm long after protargol staining (Figs. 2D, F,

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3G, H). Dorsal kinety commences about 5 μm below collar membranelles, about 5 μm from right ciliary field and 20 μm from left ciliary field, curves towards left-posterior but not reaching posterior end of cell proper as it is distinctly shortened; about 42–60 μm long and composed of 39–60

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dikinetids; only posterior basal body of each dikinetid bears a cilium that is about 8 µm long after protargol staining (Figs. 2E, F, 3I, M). Posterior kinety usually commences below first kinety of lateral ciliary field (14–25 μm below collar membranelles) and curves rightwards, terminating near posterior

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end of cell proper; 12–25 μm long and composed of 11–22 dikinetids; only posterior basal body of each dikinetid bears a cilium that is about 8 µm long after protargol staining (Figs. 2D–F, 3J). Oral apparatus occupies anterior portion of cell. Adoral zone of membranelles closed, orthogonal

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to the main cell axis when contracted, composed of 17–22 collar membranelles, of which four or five extend into buccal cavity; bases of membranelles (polykinetids) about 10–20 μm long, structure of

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polykinetids not recognizable. Cilia of membranelles up to 20–30 μm long (Figs. 2A, B, 3A, I, K). Single buccal membranelle in buccal cavity, base about 15 μm long (Figs. 2D–F, 3I). Argyrophilic fibres associated with adoral zone of membranelles were insufficiently stained to be clearly observed in protargol preparations. Endoral membrane composed of a single row of basal bodies, commences on dorsal side of peristomial field and extends parallel to collar membranelles before plunging into buccal cavity (Figs. 2D, 3L). One early divider was observed; oral primordium was left of ventral kinety and posterior to lateral ciliary field (Fig. 3N).

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Neotypification. A neotype is designated because (i) no type material is known to be deposited; (ii) the original description is restricted to lorica features; (iii) the taxonomy of Tintinnopsis species is problematic, and; (iv) the type locality of the original population was given only as Ching-lan, Hainan Island (Nie and Cheng 1947). The present population was collected from the same geographical region as the original population, i.e., Haikou Bay, Hainan Island, and therefore meets the requirement of Article 75.3.6 of the International Code of Zoological Nomenclature, i.e., that “the neotype came as nearly as practicable from the original type locality” (ICZN 1999). A detailed description of the new type locality, that is the sample site of the neotype population, is given above. A protargol slide containing the neotype specimen was deposited in the Natural History Museum, London (for details, see ‘Deposition of Neotype Material’) thus meeting the requirements of Article 75.3.7 of the Code (ICZN 1999). A protargol slide with voucher specimens was deposited in the Ocean

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University of China. The SSU rDNA and LSU rDNA sequences were deposited in the GenBank database (for details see ‘Sequence Comparison and Phylogenetic Analysis’).

Tintinnopsis orientalis Kofoid and Campbell, 1929 (Figs. 4A–E, 5A–J; Table 1)

Improved diagnosis (based on original description and this study). Lorica campanulate, about

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90–140 μm long and 61–90 µm wide, with slightly flared collar that is 20–35 µm high, bowl

ellipsoidal and posteriorly rounded, lorica opening about 70–100 μm in diameter. About 22–41

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macronuclear nodules. On average 16 collar membranelles of which three or four extend into buccal cavity; one buccal membranelle. Ventral kinety commences anteriorly to first kinety of right ciliary field, comprises on average 41 monokinetids. Right and left ciliary fields composed of on average 11

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and 14 kineties, respectively. Lateral ciliary field comprises on average 30 kineties. Dorsal kinety with on average 47 dikinetids. Posterior kinety located below left half of left ciliary field, with on average

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23 dikinetids.

Deposition of voucher material. A protargol slide with voucher specimens was deposited in the Natural History Museum, London, UK, with the registration number NHMUK 2019.4.18.2. Two

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further protargol slides were deposited in the Laboratory of Protozoology, Institute of Evolution and Marine Biodiversity, Ocean University of China, with the registration numbers BY201709190102 and

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BY201709190103.

Description of Qingdao population Lorica campanulate, about 115–140 μm long and 75–90 µm wide, with an opening diameter of

85–100 μm (Figs. 4A, 5A–C). Collar slightly flared with irregular rim, 20–35 µm high (Figs. 4A, 5A– C). Bowl ellipsoidal, with maximum width in posterior half, posterior end rounded to bluntly pointed; narrowed portion between bowl and collar approximately 60–80 μm across. Lorica wall densely agglutinated with mineral particles of various sizes (Figs. 4A, 5A–C); no spiraled or annulated 7

structures observed. Cell proper elongate-obconical when fully extended, about 95–110 × 55–70 μm in vivo, merging gradually into a peduncle that is about 40 μm long and attached to base of lorica (Fig. 4E). About 22– 41 macronuclear nodules, 5–11 × 4–8 μm in size after protargol staining, scattered in the cytoplasm (Figs. 4C, 5J). Micronuclei difficult to recognize because they were insufficiently stained with protargol. Tentaculoids inserted between collar membranelles, pin-shaped, about 35 × 2 μm in size (Fig. 5C). Striae, accessory comb, cytopyge, contractile vacuole, and capsules not recognized. Cytoplasm colourless, usually with food vacuoles containing microalgae (Fig. 4E). Locomotion by rotation about main cell axis. Disturbed individuals retract into lorica with motionless membranelles bent to centre of peristomial field. Somatic ciliary pattern comprises ventral, dorsal, and posterior kineties and right, left, and lateral

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ciliary fields (Figs. 4B–D, 5D–F, H, J). Kinetids of each ciliary row ostensibly connected by argyrophilic fibres. Ventral kinety 48–64 μm long, commences anteriorly to first kinety of right ciliary field, composed of 37–44 monokinetids, densely spaced in anterior portion and gradually more widely spaced in posterior portion (Figs. 4B, D, 5D, E). Right ciliary field comprises 9–12 kineties,

neighboring kineties about 4 μm apart, commences about 5 μm posteriorly to collar membranelles,

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each composed of 4–14 widely spaced monokinetids and one anterior dikinetid, except for first kinety which starts with two dikinetids and commences about 1 μm below level of remaining right kineties

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(Figs. 4B–D, 5D, F). Left ciliary field comprises 13–15 kineties, neighboring kineties about 5 μm apart, commences about 5 μm posteriorly to collar membranelles; kineties in left portion always obviously shorter than remaining kineties; each kinety composed of a single anterior dikinetid and

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widely spaced monokinetids (9–11 in longest kinety, 3–7 in shortest kinety) (Figs. 4B–D, 5H). Each basal body in left and right ciliary fields bears a cilium: in protargol-stained cells, the anterior cilium of each dikinetid is about 8–10 μm long, the others (i.e., cilia of monokinetids and posterior cilium of

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each dikinetid) are about 2 μm long (Fig. 5F, J). Lateral ciliary field with 24–32 monokinetidal kineties of similar length, each commencing about 5 μm posteriorly to collar membranelles, kineties in right portion usually more closely spaced than those of left portion; each monokinetid bears a cilium

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about 2 μm long after protargol staining (Figs. 4B, D, 5D, E). Dorsal kinety 49–92 μm long, commences about 5 μm posteriorly to collar membranelles, about 15–20 μm away from left and right

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ciliary fields respectively, curves leftwards and terminates near posterior end of cell proper; comprises 43–52 dikinetids, only posterior basal body of each dikinetid bears a cilium about 8–10 µm long after protargol staining (Figs. 4C, D, 5F). Posterior kinety 30–45 μm long, commences posteriorly to fourth or fifth kinety of left ciliary field (25–37 μm below collar membranelles) and extends to posterior end of cell proper; comprises 19–25 dikinetids, only posterior basal body of each dikinetid bears a cilium that is about 8–10 µm long after protargol staining (Figs. 4C, D, 5F). Adoral zone of membranelles closed, composed of 14–17 collar membranelles, of which three or four extend into the buccal cavity; cilia of membranelles up to 35–50 μm long, bases of short 8

membranelles about 20 μm long, structure of polykinetids not recognized; one buccal membranelle (Figs. 4B–D, 5I). Two bundles of argyrophilic fibres associated with distal end of each collar membranelle, about 5–6 μm long, extend rightwards and leftwards and merge into neighbouring fibres underneath membranellar zone (Fig. 5G). Endoral membrane composed of a single row of basal bodies, commences in dorsal portion of peristomial field and extends to buccal cavity (Fig. 5I). An early divider was observed; oral primordium left of ventral kinety and posterior to lateral ciliary field (Fig. 5J).

Family Eutintinnidae Bachy et al., 2012 Genus Eutintinnus Kofoid and Campbell, 1939 Eutintinnus lususundae (Entz, 1885) Kofoid and Campbell, 1939 (Figs. 6A–E, 7A–N; Table 1)

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Improved diagnosis (based on original description and present study). Lorica cylindroidal, about 180–230 μm long, anterior opening 40–55 μm across, posterior opening about 20–30 μm across. Macronucleus moniliform, composed of four or five nodules. On average 17 collar membranelles, of which three or four extend into buccal cavity; one buccal membranelle. Ventral kinety comprises on

average 19 monokinetids, commences more posteriorly than other kineties. Right and left ciliary fields

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comprise on average 13 and 17 kineties, respectively. Lateral ciliary field comprises on average six short kineties. Two dorsal kineties; right and left dorsal kineties comprise on average 50 and 34

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kinetids, respectively.

Deposition of voucher material. A protargol slide with voucher specimens was deposited in the

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Natural History Museum, London, UK, with the registration number NHMUK 2019.4.18.3. Two further protargol slides were deposited in the Laboratory of Protozoology, Institute of Evolution and

BY201709190203.

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Marine Biodiversity, Ocean University of China, with the registration numbers BY201709190202 and

Description of Qingdao population

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Lorica cylindroidal to slightly obconical, about 180–205 μm long, anterior opening about 40–50 μm across with trumpet-shaped rim, posterior opening not flared, about 20–30 μm across (Figs. 6A, B,

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7A–D). Cell proper elongate-obconical when fully extended, 80–95 × 35–55 μm in vivo, narrowing posteriorly to form a peduncle (usually bifurcated) that is attached posteriorly to inner lorica wall (Figs. 6A, B, 7A–D). Macronucleus moniliform, composed of four or five nodules, each about 11–25 × 7–14 μm in size, anterior nodule located 11–24 μm away from collar membranelles after protargol staining (Figs. 6D, 7G, J, N). Micronuclei not recognized probably because they were insufficiently stained with protargol. Tentaculoids pin-shaped, located between collar membranelles, about 15 × 2 μm in size (Figs. 6A, 7E). Striae, accessory combs, contractile vacuole, cytopyge, and extrusomes not observed. Cytoplasm colorless, usually with food vacuoles containing green algae and diatoms (Figs. 9

6A, B, 7F). Locomotion by swimming slowly while rotating about main cell axis. Disturbed individuals retract into lorica with motionless membranelles bent to centre of peristomial field (Fig. 6B) Somatic ciliature comprises ventral kinety, two dorsal kineties and left, right, and lateral ciliary fields (Figs. 6C–E, 7H–N). Argyrophilic fibres connect kinetids within each kinety. Ventral kinety 19– 27 μm long, composed of 12–24 monokinetids, commences about 5 µm more posteriorly than kineties of ciliary fields, terminates in posterior half of cell proper (Figs. 6C, E, 7I). Right ciliary field with 11– 15 kineties, commences about 5 μm posteriorly to collar membranelles, progressively decreasing in length towards both sides of the field; first kinety commences about 1 μm below remaining right kineties; each kinety composed of widely spaced monokinetids and one anterior dikinetid (Figs. 6C–E, 7H, I). Left ciliary field with 15–21 kineties of various lengths that commence about 5 μm posteriorly

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to collar membranelles, each composed of widely spaced monokinetids and one anterior dikinetid (Figs. 6C–E, 7J). Each basal body in right and left ciliary fields bears a cilium: in protargol-stained

cells, the anterior cilium of each dikinetid is about 10 μm long, the others (i.e., cilia of monokinetids and posterior cilium of each dikinetid) are about 2 μm long (Fig. 7H). Lateral ciliary field with 5–7 kineties that commence about 5 μm posteriorly to collar membranelles, each composed of

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monokinetids and commences about 1 μm more anteriorly than kineties of left ciliary field, with

anterior portions oblique to main axis; anteriormost 4–6 monokinetids densely spaced, others widely

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spaced; each monokinetid bears a cilium about 2 μm long after protargol staining (Figs. 6C, E, 7I). Two apparently monokinetidal, obliquely oriented dorsal kineties with distinct leftwards curvature in posterior portion of cell proper; left dorsal kinety 40–54 μm long, comprises 27–44 basal bodies and

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commences posteriorly to leftmost kinety of left ciliary field and about 20 µm below collar membranelles; right dorsal kinety about 50–61 μm long, commences about 5 μm posteriorly to collar membranelles and located about 6 μm and 2 μm from right and left ciliary fields, respectively;

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comprises 43–69 basal bodies and commences beside leftmost kinety of left ciliary field; each monokinetid bears a cilium about 5 μm long (Figs. 6D, E, 7J, K). Posterior kinety absent. Adoral zone of membranelles closed, composed of 17 or 18 collar membranelles, of which three

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or four extend into buccal cavity, oriented orthogonal to main cell axis in extended cells while obliquely orientated with a slight ventralisation in contracted cells; cilia of membranelles up to 30–40

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μm long, polykinetids about 15 μm long, structure of polykinetids not recognizable; one buccal membranelle (Figs. 6C–E, 7H–J, M). Argyrophilic fibres associated with membranellar zone insufficiently stained to be observed after protargol staining. Endoral membrane composed of a single row of basal bodies, commences on dorsal portion of peristomial field and extends parallel to membranellar zone, terminating near buccal cavity (Figs. 6C, 7M). Two middle dividers were observed in which the oral primordium is located posteriorly to the ventral kinety (Fig. 7L); left and right ciliary fields show division furrows; a replication band traverses each macronuclear nodule (Fig. 7N). 10

Sequences comparison and phylogenetic analyses Phylogeny based on SSU rDNA sequences The length, GenBank accession number, and G+C content of the SSU rDNA of each of the three newly sequenced species are as follows: Tintinnopsis tentaculata 1,618 bp, MK036423, 45.34%; T. orientalis 1,644 bp, MK036422, 47.57%; Eutintinnus lususundae 1,578 bp, MK036421, 49.30%. The topologies of the ML and BI trees are similar, therefore only the ML tree is shown (Fig. 8). Tintinnine clades 1, 2, and 3 each comprises a maximally supported (100/1.00) cluster of Tintinnopsis species (Fig. 8; Table. S1). The phylogenetic analyses indicate that Tintinnopsis is non-monophyletic, whereas Eutintinnus is paraphyletic. Tintinnopsis tentaculata is sister to the cluster formed by T. fimbriata (AY143560) and two Stenosemella species (S. steini KT792927 and S. ventricosa EU399538)

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(94/1.00), whereas T. orientalis becomes the adelphotaxon to the cluster formed by Cyttarocylis acutiformis (KY290316) and Petalotricha ampulla (KY290317) (100/1.00). The newly sequenced

population of E. lususundae groups with the Jangmok Bay population (JX101858) (100/1.00) which together cluster with E. tenuis (JN871721) (100/1.00).

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Phylogeny based on LSU rDNA sequences

The length, GenBank accession number, and G+C content of the LSU rDNA of the three newly

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sequenced species are as follows: Tintinnopsis tentaculata 1,699 bp, MK809531, 51.15%; T. orientalis 1,725 bp, MK809532, 48.75%; Eutintinnus lususundae 1,699 bp, MK809533, 50.91%. The topologies of the ML and BI trees are similar, therefore only the ML tree is shown for each

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gene (Figs. 8, 9). The topology of the LSU rDNA trees is similar to that of the SSU rDNA tree (Figs. 8, 9). The main difference between them is that, in the LSU rDNA tree, the T. brasiliensis + T. urnula + T. ventricosoides clade is sister to the Dictyocysta sp. + Stenosemella ventricosa clade, whereas in

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the SSU rDNA tree the former clade clusters with other Tintinnopsis species (tintinnine clades 2 and 3). Support for these groupings is, however, weak in both trees (29/* in LSU rDNA tree and 28/* in SSU rDNA tree). Tintinnopsis tubulosoides is sister to the Stenosemella pacifica (JN831883) +

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Tintinnopsis parva (JN831911) clade (100/0.90). Tintinnopsis orientalis clusters with the clade that comprises Favella campanula (KM222151), Schmidingerella arcuata (JN831867), S. quequenensis

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(KU715786), and Rhabdonella valdestriata (KU715782) with high support (84/1.00). Eutintinnus lususundae groups with E. pectinis (JN831856), which together cluster with Eutintinnus sp. (JN831857) with full support (100/1.00).

Discussion Neotypification The International Code of Zoological Nomenclature (ICZN 1999) and subsequent amendments (see on-line version: http://www.iczn.org/iczn/index.jsp) states that a neotype is validly designated 11

“when there is evidence that the neotype came as nearly as practicable from the original type locality” (Article 75.3.6). For Tintinnopsis tentaculata the present population was collected from the same area as the type locality (Ching-lan, Hainan Island), thereby fulfilling Article 75.3.6. The other two species, however, were collected hundreds or thousands of kilometers from the type locality and, although they have long been assumed that free-living marine ciliates are cosmopolitan, there is recent evidence for endemicity among such organisms (Williams et al. 2018). We therefore do not believe that the conditions of the Code have met for the neotypification of Tintinnopsis orientalis or Eutintinnus lususundae.

Tintinnopsis tentaculata Morphological comparison with original description

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Although T. tentaculata has occasionally been reported (Liu 2008), its morphology and morphometry are known only from the original description which was based exclusively on lorica features (Nie and Cheng 1947). The specimens from Haikou match the original population in the

lorica size (length 45–65 μm vs. 52–63 μm), opening diameter (20–45 μm vs. 35–40 μm), bowl width (35–60 µm vs. 57–70 µm, inferred from the original illustration), and the possession of crescent-

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shaped projections around the outer collar (Fig. 2G, H). The Haikou population differs slightly from

the original population in that its bowl is more slender, which is probably population-dependent, and

Tintinnopsis orientalis

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size and shape of the adhered particles.

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the collar projections are regular (vs. irregular) in shape, which can be attributed to differences in the

Morphological comparison with original and subsequent descriptions Kofoid and Campbell (1929) separated T. orientalis from T. dadayi Kofoid, 1905 by its convex

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(vs. concave), more widely flared collar and narrower posterior end. The dimensions of various lorica features (e.g., the diameter of the lorica opening, the width of the narrowed portion, the collar length, and the bowl width) were not given in the original description but can be inferred from the illustration.

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Accordingly, the opening is 70–85 µm across, the collar 20–25 µm high, the bowl 61–75 µm wide, and the narrowed portion 53–65 µm wide (Fig. 4F). The present population closely matches the

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original description in terms of its convex and widely flared collar. However, it differs from the original population in the diameter of the lorica opening (85–100 µm vs. 70–85 µm), the bowl width (75–90 µm vs. 61–75 µm), the lorica length (115–140 μm vs. 90–110 μm) and the less flattened posterior lorica end. These differences are assumed to reflect intraspecific polymorphism. By contrast, the populations described by Nie (1934) and Sharaf (1995) had a significantly larger ratio of length to opening diameter (i.e., about 3:1 vs. about 1.3:1), so we consider these to be misidentifications. It is noteworthy that the populations of T. schotti Brandt, 1906 (original combination Tintinnopsis dadayi schotti Brandt, 1906) reported by Hada (1938) and Nie and Cheng (1947) from the tropical Pacific and 12

Hainan, respectively, differ significantly from the original description of this species. Specifically the widest part of the bowl is in the posterior-to-mid-region (vs. widest part of bowl at anterior region in the original report, see below; Brandt 1906). Therefore, both are considered as synonyms of T. orientalis (Fig. 4G, H).

Comparisons among morphologically similar species Six congeners, namely T. buetschlii Daday, 1887, T. dadayi Kofoid, 1905, T. ecaudata Kofoid and Campbell, 1929, T. failakkaensis Skryabin and Al-Yamani, 2006, T. loricata Brandt, 1906 (original combination Tintinnopsis dadayi loricata Brandt, 1906), and T. schotti Brandt, 1906, have flared collars and therefore should be compared with the Qingdao population of T. orientalis. Tintinnopsis buetschlii can be distinguished from T. orientalis by the absence (vs. presence) of a

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narrowed lorica portion separating the bowl and collar (Daday 1887). Tintinnopsis dadayi differs from T. orientalis in having a concave (vs. convex) collar and a cylindroidal (vs. narrowed) lorica portion that separates the bowl from the collar (Kofoid 1905). Tintinnopsis ecaudata resembles T. orientalis in lorica size (100–150 µm), but can be

distinguished by its distinctly pointed (vs. rounded or bluntly pointed) posterior end and four spiral

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turns (vs. spiral turns absent) in the anterior half of the lorica (Kofoid and Campbell 1929).

Tintinnopsis failakkaensis differs from T. orientalis in having one or two deeply spiraled grooves

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(vs. no grooves) in the lorica wall (Skryabin and Al-Yamani 2006).

Tintinnopsis loricata can be separated from T. orientalis by having a longer lorica (160–170 μm vs. 115–140 μm) and a thus a higher length: width ratio (about 2:1 vs. about 1.3:1) (Brandt 1906,

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1907).

Tintinnopsis schotti can be distinguished from T. orientalis by the bowl shape, i.e., the widest part

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of bowl is in the anterior region (vs. in the posterior half in T. orientalis) (Brandt 1906).

Eutintinnus lususundae

Comparison with original description

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Eutintinnus lususundae was described by Entz (1885) as “Tintinnus lususundae” and later transferred to the genus Eutintinnus by Kofoid and Campbell (1939). Our population corresponds well

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with the original description in term of lorica shape, i.e., with a slightly flared rim around the anterior opening, posterior opening without a flared rim, and the diameter of the posterior lorica opening (30 vs. 20–30 µm) (Entz 1885). There are slight differences in terms of the lorica length (230 vs. 180–205 μm) and the diameter of the anterior lorica opening (55 vs. 35–50 μm), however these are considered to be population-dependent (Entz 1885). In addition, the genetic distance of the SSU rDNA sequences from our specimen and that of a specimen of E. lususundae collected from Jangmok Bay (JX101858; Xu et al. 2013) is only 0.12%. Hence, we identified our specimens as E. lususundae.

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Comparison of lorica features among congeners We compared our specimens with three Eutintinnus species that have a flared anterior lorica portion and lack an inflated lorica middle portion or a flared posterior portion, namely E. stramentus (Kofoid and Campbell, 1929) Kofoid and Campbell, 1939, E. tenuis (Kofoid and Campbell, 1929) Kofoid and Campbell, 1939, and E. tubulosus (Ostenfeld, 1899) Kofoid and Campbell, 1939. Eutintinnus stramentus can be separated from E. lususundae by its shorter lorica (115–174 μm vs. 180–205 μm long), higher ratio of lorica length to anterior opening diameter (4.4–6:1 vs. 3.8–4.5:1), and a more slender posterior lorica region (about 10 μm vs. 20–30 μm across), although it should be noted that the latter measurement was estimated from the drawing since no data were given in the original description of E. stramentus (Kofoid and Campbell 1929). Eutintinnus tenuis differs from E. lususundae by its larger ratio of anterior opening diameter to

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lorica length (4.3–6.1:1 vs. 3.8–4.5:1) (Kofoid and Campbell 1929). Eutintinnus tubulosus differs from E. lususundae by having a shorter lorica (120–150 μm vs. 180–205 μm) (Ostenfeld 1899).

Comparison of somatic ciliary pattern among congeners

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The dorsal kineties of most tintinnines are composed of dikinetids, the only exceptions

documented to date being the eutintinnine species Eutintinnus angustatus, E. pectinis, E. tenuis and

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Dartintinnus alderae (Choi et al. 1992; Smith et al. 2018). However, it cannot be excluded that dikinetids are present but were not recognized due to insufficient staining of the basal bodies. TEM studies, which are lacking for these species, may be required in order to determine the presence or

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absence of dikinetids. It is noteworthy that Choi et al. (1992) deviate in their application of the term “ventral kinety”: in E. angustatus, it is used for the last monokinetidal oblique row, but in E. tenuis and E. pectinis it is used for the posteriorly shifted monokinetidal row and not for the last oblique row.

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Subsequently, Dolan et al. (2013) noted that: (i) in E. tenuis, the posteriorly shifted monokinetidal row is the first kinety of right ciliary field (K2) and the last monokinetidal oblique row is the ventral kinety, and; (ii) in E. pectinis, the posteriorly shifted monokinetidal fragmental row is the ventral

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kinety because this species lacks a monokinetidal oblique row. However, the kineties of the ventral side vary in the genus Eutintinnus, and the application of the term “ventral kinety” as suggested by

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Dolan et al. (2013) is unsatisfactory. In Agatha and Riedel-Lorjé (2006), the definition of the ventral kinety is as follows: “The ventral kinety is on the left side of the right ciliary field and on the right side of oral primordium. It is the longest entirely monkinetidal ciliary row on the ventral side”. Hence, the ventral kinety of genus Eutintinnus should be the monokinetidal ciliary row beside the leftmost kinety of the right ciliary field, even though it may be shifted posteriorly and/or fragmented. Additionally, compared with other congeners described by Choi et al. (1992), the second dorsal kinety of E. lususundae is below the left ciliary field (vs. to the left of the leftmost kinety of the left ciliary field). Gruber et al. (2018) revealed that the posterior kinety of Tintinnopsis everta is to the left 14

of the leftmost kinety of the left ciliary field and suggested that the posterior kinety evolved from the second dorsal kinety. A similar evolutionary process may have occurred in Eutintinnus, although we tend to identify the ciliary row below the left ciliary field of E. lususundae as a dorsal kinety rather than the posterior kinety because it is very close to the first dorsal kinety and all known Eutintinnus species have two dorsal kineties. Eutintinnus angustatus differs from E. lususundae by the absence (vs. presence) of a posteriorly shifted ventral kinety, the position and length of the two dorsal kineties (both kineties commence almost at the same level and are of similar length vs. left dorsal kinety commences significantly higher, and is significantly shorter, than the right), and in having fewer kineties (1 vs. 5–7) in the lateral ciliary field (Choi et al. 1992). Eutintinnus pectinis differs from E. lususundae in having a fragmented (vs. continuous)

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posteriorly shifted ventral kinety, the absence (vs. presence) of a lateral ciliary field, and the presence (vs. absence) of kinety fragments posterior to the left field (Choi et al. 1992; Dolan et al. 2013). Eutintinnus tenuis closely resembles E. lususundae in ciliary pattern, especially having a

posteriorly shifted ventral kinety. However, the former differs from the latter in having fewer kineties in the lateral ciliary field (3–4 vs. 5–7), and only the rightmost two or three kineties have a dense,

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obliquely oriented anterior portion (vs. all kineties of the lateral ciliary field have an anterior portion

of this type) (Choi et al. 1992). The high morphological similarity of these two species is reflected by

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their SSU rDNA sequence (99.0% similarity).

Dartintinnus alderae differs from E. lususundae in the position of the ventral kinety (commencing at the same level as the kineties of lateral ciliary field vs. significantly posterior to the

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kineties of lateral ciliary field), fewer kineties in the right and left ciliary fields (4–6 vs. 11–15 in right ciliary field, and 6–8 vs. 15–21 in left ciliary field), and in having only one (vs. two) dorsal kinety

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(Smith et al. 2018).

Phylogenetic analyses

The genus Tintinnopsis was found to be non-monophyletic in phylogenetic analyses based on

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both SSU rDNA and LSU rDNA sequence data, which is consistent with previous studies (Agatha and Strüder-Kypke 2007; Santoferrara et al. 2017). In our SSU rDNA phylogenetic trees, T. tentaculata

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was closely related to T. fimbriata, Stenosemella ventricosa, and S. steini. This is also supported by the similarities of the lorica shape in these species, i.e., with a broad bowl and a low collar. The newly sequenced Tintinnopsis orientalis grouped with Cyttarocylis acutiformis (KY290316)

and Petalotricha ampulla (KY290317) in the SSU rDNA tree, whereas it formed an independent lineage in the LSU rDNA tree, probably due to a lack of LSU sequences for the above taxa (Figs. 8, 9). All three species have a campanulate lorica but differ in their lorica structures: the lorica wall of Tintinnopsis is entirely agglutinated, that of Cyttarocylis is uniformly and finely reticulate with small subequal polygons, and the lorica of Petalotricha has rows of fenestrae in the anterior portion of the 15

bowl (Kofoid and Campbell 1929). Therefore, the results so far obtained for tintinnine species might indicate that the lorica shape is more phylogenetically informative than the lorica structure.

Author contributions XH conceived and guided the study. YB and RW conducted sampling and performed laboratory work. XH and YB identified the species. YZ did the phylogenetic analyses and the results interpretation. YB drafted the manuscript, and WL, AW, YZ, and XH made further revisions. AW also provided access to the older literature, and dealt with nomenclatural issues. All authors read and approved the final version of manuscript.

Acknowledgements

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This work was supported by the National Natural Science Foundation of China (project numbers: 41776133, 41576124, 31801955, 31702009). We thank Prof. Weibo Song, Ocean University of China for his comments on the manuscript. We are also grateful to the editor and anonymous reviewers for

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their constructive suggestions.

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Liu, W.W., Jiang, J., Xu, Y., Pan, X.M., Qu, Z.S., Luo, X.T., El-Serehy, H., Warren, A., Ma, H.G., Pan, H.B., 2017. Diversity of free-living marine ciliates (Alveolata, Ciliophora): fauna studies in China seas during the years 2011–2016. Eur. J. Protistol. 61, 424–438.

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Moreira, D., Heyden, S., Bass, D., López-García, P., Chao, E., Cavalier-Smith, T., 2007. Global eukaryote phylogeny: combined small-and large-subunit ribosomal DNA trees support monophyly of Rhizaria, Retaria and Excavata. Mol. Phylogenet. Evol. 44, 255–266.

Nie, D., 1934. Notes on Tintinnoinea from the Bay of Amoy. Rep. mar. biol. Ass. China 3, 71–80. Nie, D., Cheng, P.S., 1947. Tintinnoinea of the Hainan Region. Contr. Biol. Lab. Sci. Soc. China 16, 41–86. Nylander, J.A., 2004. MrModeltest, version 2.2. Uppsala: Evolutionary Biology Centre, Uppsala University. 19

Ostenfeld, C.H., 1899. Über Coccosphaera und einige neue Tintinnineen im Plankton des nördlichen Atlantischen Oceans. Zool. Anz. 22, 433–439. Pan, X.M., Bourland, W., Song, W.B., 2013. Protargol synthesis: an in-house protocol. J. Eukaryot. Microbiol. 60, 609–614. Ronquist, F., Teslenko, M., Van Der Mark, P., Ayres, D.L., Darling, A., Höhna, S., Larget, B., Liu, L., Suchard, M.A., Huelsenbeck, J.P., 2012. MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Syst. Biol. 61, 539–542. Santoferrara, L.F., Bachy, C., Alder, V.A., Gong, J., Kim, Y.O., Saccà, A., Silva-Neto, I.D., StrüderKypke, M.C., Warren, A., Xu, D.P., Yi, Z.Z., Agatha, S., 2016. Updating biodiversity studies in loricate protists: the case of the tintinnines (Alveolata, Ciliophora, Spirotrichea). J. Eukaryot. Microbiol. 63, 651–656.

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Santoferrara, L.F., Alder, V.V., McManus, G.B., 2017. Phylogeny, classification and diversity of Choreotrichia and Oligotrichia (Ciliophora, Spirotrichea). Mol. Phylogenet. Evol. 112, 12–22.

Santoferrara, L.F., Rubin, E., McManus, G.B., 2018. Global and local DNA (meta)barcoding reveal new biogeography patterns in tintinnine ciliates. J. Plankton Res. 40, 209–221.

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Arab Emirates waters. Qatar. Univ. Sci. J. 15, 389–398.

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(Ciliophora, Spirotrichea, Tintinninea) from the Kuwaiti waters of the Arabian Gulf. Russ. J. Mar. Biol. 32, 302–307.

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Santoferrara, L.F., 2018. Dartintinnus alderae n. g., n. sp., a brackish water tintinnine (Ciliophora, Spirotrichea) with dual-ended lorica collapsibility. J. Eukaryot. Microbiol. 65, 400– 411.

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Song, W., Wang, L., Li, L.F., Al-Farraj, S.A., Aleidan, A., Smith, S., Hu, X.Z., 2018. Morphological characterizations of four species of Parallelostrombidium (Ciliophora, Oligotrichia), with a note on the phylogeny of the genus. J. Eukaryot. Microbiol. 65, 679–693

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Stamatakis A., 2014. RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics 30, 1312–1313.

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Wang, R., Song, W., Yi, Z.Z., Lin, X.F., Al-Rasheid, K.A.S., Hu, X.Z., 2018. Morphology and molecular phylogeny of two new species of Spirostrombidium (Ciliophora, Oligotrichia), with a key to species in the genus. Syst. Biodivers. 16, 743–756.

Wilbert, N., 1975. Eine verbesserte Technik der Protargolimpragnation fur Ciliaten. Mikrokosmos. 64, 171–179 (in German). Williams, R.A.J., Owens, H.L., Clamp, J.C., Peterson, A.T., Warren, A., Martin-Cereceda, M., 2018. Endemicity and climatic niche differentiation in three marine ciliated protists. Limnology and Oceanography 27, 27–2736. 20

Worden, A., Follows, M., Giovannoni, S., Wilken, S., Zimmerman, A., Keeling, P., 2015. Rethinking the marine carbon cycle: Factoring in the multifarious lifestyles of microbes. Science 347, 1257594. Xu, D.P., Song, W.B., 2005. Tintinnine ciliates from Qingdao (Protozoa, Ciliophora, Tintinninea). Acta Zootaxonom. Sin. 30, 501–508 (in Chinese with English abstract). Xu, D.P., Sun, P., Warren, A., Noh, J., Choi, D., Shin, M., Kim, Y., 2013. Phylogenetic investigations on ten genera of tintinnine ciliates (Ciliophora: Spirotrichea: Tintinninea), based on small subunit ribosomal RNA gene sequences. J. Eukaryot. Microbiol. 60, 192–202. Xu, D.P., Jiao, N.Z., Ren, R., Warren, A., 2017. Distribution and diversity of microbial eukaryotes in bathypelagic waters of the South China Sea. J. Eukaryot. Microbiol. 64, 370–382. Xu, K.D., Hong, H.S., Song, W.B., Ke, L., Ma, H.G., 2001. Studies on tintinnine ciliates in the

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Zhang, W.C., Feng, M.P., Yu, Y., Zhang, C.X., Xiao, T., 2012. An Illustrated Guide to Contemporary

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Tintinnines in the World. Science Press, Beijing (in Chinese).

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Figures and legends

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Fig. 1. A–E Sampling sites and surrounding areas. (A) Map of China with sampling sites (yellow circles). (B, C) Google map of sampling sites, (B) showing the Haikou Bay, Hainan Province, China, (C) showing Qingdao, Shandong Province, China. (D, E) Photographs of Haikou Bay (D) and coast of

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Qingdao (E).

22

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Fig. 2. A–H Drawings of Tintinnopsis tentaculata in vivo (A–C, G, H) and after protargol staining (D– F). (A) Representative specimen; arrows denote the crescent-shaped projections. (B) Specimen

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showing key cell characters. (C) Lorica with large agglutinated particles on the wall, showing crescent-shaped projections (arrows) and broadly rounded posterior end. (D, E) Ciliary pattern of

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ventral (D) and dorsal (E) sides of the neotype specimen. (F) Kinetal map of a morphostatic specimen. (G, H) Showing variations in lorica shape (from Nie and Cheng 1947). BM, buccal membranelle; CM, collar membranelles; DK, dorsal kinety; LA, lateral ciliary field; LF, left ciliary field; PK, posterior kinety; RF, right ciliary field; T, tentaculoids; VK, ventral kinety. Scale bars: 20 μm (A–E), 25 μm (G, H).

23

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Fig. 3. A–P Photomicrographs of Tintinnopsis tentaculata in vivo (A–F) and after protargol staining (G–P). (A) Specimen with slightly extended cell. (B) Oblique view, showing the crescent-shaped

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projections (arrowheads). (C) Lorica with large agglutinated particles on the collar and crescentshaped projections. (D) Arrowhead marks a pin-shaped tentaculoid. (E) Showing the cell with food

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vacuoles, arrowhead marks an extended tentaculoid. (F) Posterior lorica projection. (G) Lateral and left (arrowhead) ciliary fields. (H) A slightly squashed specimen showing lateral ciliary field, left ciliary field, and ventral kinety. (I) Right ciliary field and anterior end of dorsal kinety of a squashed specimen; arrowhead denotes buccal membranelle. (J) Posterior kinety. (K) Apical view, showing the collar membranelles. (L) Endoral membrane. (M) Anterior portion of dorsal kinety (arrowhead). (N) Posterior ventral cell portion of an early divider, showing the oral primordium. CM, collar membranelles; DK, dorsal kinety; EM, endoral membrane; LA, lateral ciliary field; LF, left ciliary field; OP, oral primordium; PK, posterior kinety; RF, right ciliary field; VK, ventral kinety. Scale bars: 24

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15 μm.

Fig. 4. A–H Tintinnopsis orientalis in vivo (A, E–H) and after protargol staining (B–D). (A) Representative specimen. (B, C) Ciliary pattern of ventral (B) and dorsal (C) sides of the same

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specimen. (D) Kinetal map of a morphostatic specimen. (E) Specimen showing key cell characters. (F) Lorica (from Kofoid and Campbell, 1929). (G) Lorica of T. schotti (from Hada, 1938). (H) Lorica

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of T. schotti (from Nie and Cheng, 1947). BM, buccal membranelle; CM, collar membranelles; DK, dorsal kinety; EM, endoral membrane; LA, lateral ciliary field; LF, left ciliary field; PCM, prolonged collar membranelles; PK, posterior kinety; RF, right ciliary field; T, tentaculoids; VK, ventral kinety. Scale bars: 45 μm (A, E), 30 μm (B, C), 25 μm (F–H).

25

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Fig. 5. A–J Photomicrographs of Tintinnopsis orientalis in vivo (A–C) and after protargol staining (D–

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L). (A) Representative specimen. (B) Lorica. (C) Tentaculoids, arrowheads show the gaps (lacunae) on the collar. (D) Ventral view of anterior cell portion, to show right ciliary field and ventral kinety and lateral ciliary field. (E) Showing ventral kinety and lateral ciliary field. (F) Dorso-lateral side view of cell showing dorsal kinety, posterior kinety and right ciliary field. (G) Anterior portion of cell, showing argyrophilic fibres associated with adoral membranelles (arrowhead). (H) Left ciliary field. (I) Apical view showing endoral membrane and collar membranelles. (J) Antapical view of an early divider, to show oral primordium; arrowheads indicate macronuclear nodules. CM, collar membranelles; DK, dorsal kinety; EM, endoral membrane; LA, lateral ciliary field; LF, left ciliary 26

field; OP, oral primordium; PK, posterior kinety; RF, right ciliary field; T, tentaculoids; VK, ventral

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kinety. Scale bars: 55 μm (A–C), 10 μm (I), 15 μm (J).

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Fig. 6. A–E Drawings of Eutintinnus lususundae in vivo (A, B) and after protargol staining (C–E). (A) Representative specimen; arrow shows bifurcate stalk. (B) Contracted cell in the lorica. (C, D) Ciliary pattern of ventral (C) and dorsal (D) sides of the same specimen. (E) Kinetal map of a morphostatic

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specimen, arrowheads show dorsal kineties. BM, buccal membranelle; CM, collar membranelles; DK, dorsal kinety; EM, endoral membrane; LA, lateral ciliary field; LF, left ciliary field; RF, right ciliary field; T, tentaculoids; VK, ventral kinety. Scale bars: 80 μm (A, B), 30 μm (C, D).

27

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Fig. 7. A–N Photomicrographs of Eutintinnus lususundae in vivo (A–F) and after protargol staining (G–N). (A–D) Lateral views of the same dividing individual in extended (A) and contracted (B-D)

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states. (E) Anterior portion of an extended cell, arrowheads mark tentaculoids. (F) Showing food vacuoles and ingested microalgae. (G) Macronuclear nodules (arrowheads). (H) Showing right ciliary

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field. (I) Showing ciliature of ventral side; arrowheads show the densely spaced anterior kinetids in lateral ciliary rows. (J) Showing left ciliary field and dorsal kineties. (K) The two dorsal kineties. (L) Showing oral primordium of opisthe. (M) Part of buccal cavity, showing the endoral membrane; arrowhead shows the buccal membranelle. (N) Dorso-lateral view of a middle divider, showing kinety fragments of the left ciliary field in proter and opisthe. DK, dorsal kineties; EM, endoral membrane; LF, left ciliary field; OP, oral primordium; RF, right ciliary field; VK, ventral kinety. Scale bars: 65 μm (A–D), 20 μm (E, G, N), 30 μm (J).

28

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Fig. 8. Maximum likelihood (ML) tree inferred from SSU rDNA sequences showing nodal support for ML and BI analyses. Newly sequenced species, i.e., Tintinnopsis tentaculata, T. orientalis, and

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Eutintinnus lususundae, are shown in bold. Clade 1 comprises sequences of Tintinnopsis fistularis (KU715770), Tintinnopsis acuminata (JN831840), Tintinnopsis baltica (JN831805), and Tintinnopsis

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nana (JN831821). Clade 2 includes sequences of Tintinnopsis tocatinensis (AY143561), Tintinnopsis sp. (KU715775), Tintinnopsis uruguayensis (JN831838), Tintinnopsis levigata (KM982811), and Tintinnopsis cylindrical (KU715769). Clade 3 comprises sequences of Tintinnopsis beroidea (EF123709), Tintinnopsis major (JN831818), Tintinnopsis dadayi (AY143562), Tintinnopsis buetschlii (JN831809), and Tintinnopsis tubulosa (AB640683). The Oligotrichida clade comprises sequences of Cyrtostrombidium longisomum (KJ609953), Novistrombidium apsheronicum (FJ876958), Spirotontonia turbinata (FJ422994), and Strombidium sulcatum (FJ377546). Asterisks (*) reflect disagreements in topology between the BI and ML trees. The scale bar corresponds to 0.02 expected substitutions per site. 29

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Fig. 9. Maximum likelihood (ML) tree inferred from LSU rDNA sequences showing nodal support for

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ML and BI analyses. Newly sequenced species, i.e., Tintinnopsis tentaculata, T. orientalis and Eutintinnus lususundae, are shown in bold. Asterisks (*) reflect disagreements in topology between

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the BI and ML trees. The scale bar corresponds to 0.05 expected substitutions per site.

30

Min 45 115 180

Max 65 140 205

Mean 54.3 125.7 190.3

Median 55 125 190

SD 5.9 8.6 7.4

CV 10.9 6.9 3.9

N 15 15 15

Lorica, bowl width

T. tentaculata T. orientalis

35 75

60 90

39.7 80.7

35 80

7.4 4.2

18.7 5.2

15 15

Lorica, collar length

T. tentaculata T. orientalis

2 20

3 35

2.8 28.7

3 30

0.4 5.2

14.8 18.0

15 15

Lorica, anterior opening diameter

T. tentaculata T. orientalis E. lususundae

20 85 40

45 100 50

29.0 94.0 44.7

25 95 45

7.6 5.4 3.0

26.2 5.8 6.6

15 15 15

Lorica, posterior opening diameter

E. lususundae

20

30

25.0

25

3.8

15.1

15

Lorica, total T. tentaculata length : anterior T. orientalis opening diameter, E. lususundae ratio

1.1 1.2 3.8

2.8 1.5 4.5

2.0 1.3 4.2

2.0 1.4 4.2

0.5 0.1 0.2

22.9 6.9 6.0

15 15 15

Lorica, narrowed portion diameterb

T. orientalis

60

80

72.4

70

5.4

7.5

15

T. orientalis

1.6

1.9

1.8

1.8

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0.1

4.9

15

Cell proper, length

T. tentaculata T. orientalis E. lususundae

40 60 71

60 104 91

48.8 79.8 78.7

49 82 79

5.5 13.3 6.7

11.2 16.6 8.6

15 15 15

Cell proper, width

T. tentaculata T. orientalis E. lususundae

34 50 27

52 84 42

41.4 64.0 33.4

40 61 31

5.2 10.7 4.7

12.6 16.8 14.0

15 15 15

T. tentaculata T. orientalis E. lususundae

2 22 4

2 41 5

2.0 33.6 4.4

2 34 4

0.0 12.2 0.5

0.0 36.3 11.4

15 15 20

T. tentaculata T. orientalis E. lususundae

15 5 11

25 11 25

18.9 7.9 20.0

8 8 21

2.6 1.8 4.6

14.0 10.0 22.9

15 15 20

Macronuclear nodules, width

T. tentaculata T. orientalis E. lususundae

14 4 7

20 8 14

14.6 6.2 11.8

15 6 12

1.5 1.0 1.5

10.0 16.3 12.6

15 15 20

Anterior cell end to anterior macronucleus nodule, distance

T. tentaculata T. orientalis E. lususundae

12 11 11

17 22 24

13.9 17.1 17.3

14 17 18

1.6 3.4 3.4

11.8 2.0.0 19.8

15 15 20

Ventral kinety, length

T. tentaculata T. orientalis

14 48

26 64

17.3 57.2

17 57

2.9 1.8

16.6 3.5

15 11

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Macronuclear nodules, number

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Lorica, total length : narrowed portion diameter

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Macronuclear nodules, length

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Species T. tentaculata T. orientalis E. lususundae

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Charactera Lorica, total length

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Table 1. Morphometric data of Tintinnopsis tentaculata, T. orientalis, and Eutintinnus lususundae.

19

27

23.3

24

2.4

10.3

15

Ventral kinety, number of kinetids

T. tentaculata T. orientalis E. lususundae

29 37 12

41 44 24

34.1 41.0 19.3

33 41 21

3.5 2.3 3.3

10.1 5.6 17.2

15 11 15

Ventral kinety, distance to collar membranelles

T. tentaculata T. orientalis E. lususundae

4 5 8

7 7 11

5 5.6 9.4

5 5 9

0.8 0.7 0.8

16.9 13.2 8.8

15 15 15

Dorsal kinety1, length

Dorsal kinety 1, distance to right ciliary field

T. tentaculata T. orientalis E. lususundae T. tentaculata T. orientalis E. lususundae T. tentaculata T. orientalis E. lususundae

42 49 50 39 43 43 4 15 5

60 92 61 60 52 69 7 21 7

52.2 69.3 53.9 50.2 46.5 49.9 4.8 18.1 5.7

54 68 53 51 47 50 5 19 5

4.8 13.1 3.1 6.1 3.1 46.0 0.9 1.8 0.9

9.2 18.9 5.7 12.2 6.6 12.1 18.1 10.0 15.8

15 11 15 15 11 15 15 11 15

Dorsal kinety 1, distance to left ciliary field

T. tentaculata T. orientalis E. lususundae

15 15 2

21 20 3

19.4 16.5 2.3

20 16 2

1.6 1.6 0.5

8.2 9.6 20.2

15 11 15

Dorsal kinety 1, distance to collar membranelles

T. tentaculata T. orientalis E. lususundae

4 5 5

7 7 6

5 5.6 5.3

5 5 5

0.8 0.7 0.7

16.9 13.2 20.5

15 15 15

Dorsal kinety 2, length Dorsal kinety 2, number of kinetids Dorsal kinety 2, distance to collar membranelles

E. lususundae

40

54

44.7

44

9.7

15

E. lususundae

27

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4.3

44

33.9

32

4.4

13.0

15

22

19.1

20

2.1

11.0

15

Posterior kinety, length

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E. lususundae

14

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Dorsal kinety 1, number of kinetids

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E. lususundae

T. tentaculata

12

25

16.7

16

3.7

22.0

15

T. orientalis T. tentaculata T. orientalis

30 11 19

45 22 25

35.5 14.8 22.8

35 14 24

4.0 3.1 2.1

11.3 20.6 9.2

11 15 11

Posterior kinety, distance to collar membranelles

T. tentaculata

14

25

19.3

19

2.6

13.3

15

T. orientalis

25

37

30.8

31

3.5

11.5

11

Right ciliary field, number of kineties

T. tentaculata T. orientalis E. lususundae

7 9 11

10 12 15

9.0 10.5 12.7

9 10 13

1.0 0.8 1.0

11.1 7.9 8.1

13 15 15

Longest kinety in right field, length

T. tentaculata T. orientalis E. lususundae T. tentaculata

21 31 15 10

35 42 22 15

27.0 35.1 17.3 12.7

27 34 17 13

4.1 4.1 2.3 1.4

15.3 11.6 12.9 11.3

13 15 15 13

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Posterior kinety, number of kinetids

Longest kinety in right field,

32

12 13 19 11 6 11 5 4

15 15 31 17 11 13 6 7

13.7 14.3 24.7 13.3 8.1 11.8 5.3 5.3

14 14 25 14 8 12 5 5

1.2 0.6 3.9 2.0 1.2 1.0 0.5 1.1

8.7 4.2 15.6 15.1 12.1 8.9 8.3 20.9

15 15 13 15 15 13 11 15

Shortest kinety of lateral ciliary field, length

T. tentaculata T. orientalis E. lususundae T. tentaculata T. orientalis E. lususundae T. tentaculata T. orientalis E. lususundae

16 24 5 16 24 15 9 21 4

22 32 7 24 32 22 14 33 7

19.2 29.8 6.2 19.7 28.6 17.7 10.8 26.2 5.8

19 30 6 20 29 17 11 26 6

1.9 2.3 2.3 2.4 2.3 2.1 1.5 3.4 0.8

9.8 7.6 37.1 12.0 8.0 11.6 13.6 12.9 13.3

15 15 15 15 15 15 15 15 15

Left ciliary field, number of kineties

T. tentaculata T. orientalis E. lususundae

10 13 15

14 15 21

11.8 13.7 17.2

12 13 16

1.1 0.8 2.3

9.2 6.0 13.4

15 15 15

Longest kinety in left field, length

T. tentaculata T. orientalis E. lususundae

20 27 19

32 36 26

25.9 31.4 22.6

25 31 22

3.9 2.7 2.4

15.0 8.7 10.7

15 15 15

Longest kinety in left field, number of kinetids

T. tentaculata T. orientalis E. lususundae

10 10 14

15 12 17

12.4 10.9 15.5

12 11 15

1.2 0.5 0.8

10.0 4.6 5.2

15 11 15

Shortest kinety in left field, length

T. tentaculata T. orientalis E. lususundae

7 15 8

12 22 14

9.5 18.1 10.8

9 17 11

1.4 2.3 1.6

14.9 13.0 14.5

15 15 15

7 4 9

8.3 6.1 9.7

8 6 10

1.4 1.3 0.7

16.8 21.3 7.2

15 11 15

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Lateral ciliary field, number of kineties Longest kinety of lateral ciliary field, length

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Shortest kinety in right field, number of kinetids

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T. orientalis E. lususundae T. tentaculata T. orientalis E. lususundae T. tentaculata T. orientalis E. lususundae

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number of kinetids Shortest kinety in right field, length

11 8 11

Kineties in ciliary field, distance to collar membranelles

T. tentaculata T. orientalis E. lususundae

4 5 5

7 7 6

5 5.6 5.3

5 5 5

0.8 0.7 0.7

16.9 13.2 20.5

15 15 15

Adoral zone of membranelles, diameter

T. tentaculata T. orientalis E. lususundae

33 50 25

54 84 40

43.8 64.0 32.7

44 61 31

5.3 10.7 4.5

12.2 16.8 13.7

15 15 15

Collar membranelles, number

T. tentaculata T. orientalis E. lususundae

17 14 17

22 17 18

19.3 15.9 17.3

19 16 17

1.6 0.9 0.5

8.2 5.5 2.7

15 15 15

Collar membranelles,

T. tentaculata T. orientalis

4 3

5 4

4.8 3.7

5 4

0.5 0.5

10.0 13.3

12 15

T. tentaculata T. orientalis E. lususundae

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Shortest kinety in left field, number of kinetids

33

number of elongated ones Buccal membranelles, number

E. lususundae

3

4

3.1

3

0.4

11.2

15

T. tentaculata 1 1 1.0 1 0.0 0.0 15 T. orientalis 1 1 1.0 1 0.0 0.0 15 E. lususundae 1 1 1.0 1 0.0 0.0 15 a Lorica data are based on living observations, and others are based on protargol-stained specimens. The lorica measurements were calculated from micrographs taken at low to medium magnifications (100–400×), and they were accurate to 5 μm. Measurements are in μm. Abbreviations: CV = coefficient of variation in %; Max = maximum; Mean = arithmetic mean; Min = minimum; n = number of specimens examined; SD = standard deviation. b

Jo

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The narrowest portion of neck of lorica.

34