INTRODUCTION
Mesodinium is a cosmopolitan ciliate genus that inhabits freshwater and marine ecosystems globally. Because the genus is characterized by an equatorial ciliary belt, cirri, and oral tentacles, cells of this genus can be easily recognized by light microscopy (Bary and Stuckey 1950, Taylor et al. 1971, Hibberd 1977, Lindholm 1985, Tamar 1986, 1992, Garcia-Cuetos et al. 2012). Despite the easily distinguishable morphology and global distribution, the number of species formally assigned to the genus remains limited (Lindholm 1985, Garcia-Cuetos et al. 2012). Although recent taxonomic studies have greatly contributed to the establishment of new species based on ultrastructural and molecular data (Garcia-Cuetos et al. 2012, Moestrup et al. 2012, Nam et al. 2015), species diversity in this genus has been insufficiently explored due to the limited number and plasticity of available morphological characters (Lindholm 1985, Tamar 1986, Garcia-Cuetos et al. 2012).
Since the establishment of the genus Mesodinium by Stein (1862), some species have been described as either heterotrophic or mixotrophic (Lindholm 1985, Garcia-Cuetos et al. 2012, Moestrup et al. 2012, Nam et al. 2015, 2016, Mitra et al. 2016, Herfort et al. 2017, Moeller and Johnson 2018, Kim et al. 2019). Mesodinium rubrum is the best-known species of the genus and has long been recognized as a single bloom-forming species in many different locations (Taylor et al. 1971, see table 1). However, cryptic diversity within the M. rubrum morphospecies was indicated by variations in cell dimension and morphology. For example, Lindholm (1985) recorded four forms of M. rubrum from European material, including a medusa form, and referred to these variants as the M. rubrum species complex. Later, Crawford (1993) recognized six morphological variants of M. rubrum from the Southampton Water estuary in southern England.
The somatic ciliature and oral apparatus are important characters for the identification of Mesodinium species. Ultrastructural studies of two heterotrophic and four mixotrophic congeners have been performed; however, the ultrastructure of the somatic ciliature has been investigated in detail in only two species, namely, M. chamaeleon and M. coatsi (Garcia-Cuetos et al. 2012, Moestrup et al. 2012, Nam et al. 2012, 2015). Nam et al. (2012) displayed the oral apparatus of the Korean M. rubrum strain.
The populations assigned to the M. rubrum species complex have shown high genetic diversity. Herfort et al. (2011) investigated the genetic variability of the M. rubrum species complex in 21 water samples from the coastal margin of the Columbia River in spring and summer, where they found at least five genetic variants of the complex. Thereafter, Garcia-Cuetos et al. (2012) found a further genetic variant and described the variant D as M. major. In samples collected from eight globally dispersed habitats, Johnson et al. (2016) recently discovered two new variants belonging to the complex, thereby increasing the total number of variants to eight.
Given that most variants of the M. rubrum / M. major complex have not yet been formally characterized despite the growing body of data about the high genetic diversity, it seems timely to perform a more detailed investigation of their ciliature. This study describes the Korean variant B of the M. rubrum complex a new species, namely, Mesodinium annulatum sp. nov.
MATERIALS AND METHODS
Collection and clonal culture of the ciliate and its prey
Cultures of M. annulatum were established by single cell isolation from plankton samples collected in the Gomso Bay, Korea (35°40′ N, 126°40′ E) in February 2002. Cultures of the prey algae Teleaulax amphioxeia were established by single cell isolation from plankton samples collected at same location but in May 2001, when salinity and water temperature were approximately 18°C and 31.5 psu. Both species were grown in f/2 medium at 15°C under a continuous illumination of 25 μE m−2 s−1 provided by cool-white fluorescent lamps.
Light microscopy
Living M. annulatum cells and cells impregnated by protargol, using the procedure A of Foissner (2014), were studied by means of a Nikon ECLIPSE Ni-U (Nikon, Tokyo, Japan) equipped with differential interference contrast optics. Images were captured, using a Digital Camera (DS-Ri2; Nikon).
Scanning electron microscopy
Cultures of M. annulatum were preserved in 1% (v/v) glutaraldehyde with 0.1 M cacodylate buffer in seawater for 1 h, rinsed three times with 0.1 M cacodylate buffer in seawater, and then postfixed with 1% (w/v) osmium tetroxide in distilled water for 1 h. The fixed cells were collected on 5-μm polycarbonate filters (Isopore Membrane filters; Milipore Ltd., Billerica, MA, USA) and rinsed three times with distilled water. Filters with specimens were serially dehydrated in an ethanol series (30, 50, 70, 90, and 100%) and dried, using a critical point dryer (HCP-2 critical point dryer; Hitachi Co., Tokyo, Japan). The dried filters were mounted on stubs and coated with gold-palladium. Cells were viewed with a JSM 700F FE-SEM (JEOL Ltd., Tokyo, Japan) at 5–10 kV.
Transmission electron microscopy
For transmission electron microscopy, specimens of cultures of M. annulatum were prefixed in 2.5% (v/v) glutaraldehyde with 0.1 M cacodylate in f/2 medium for 1 h at 4°C, washed three times in cacodylate buffer, and postfixed in 1% (w/v) OsO4 for 1 h at 4°C. The fixed cells were rinsed three times with cacodylate buffer. Dehydration was carried out at 4°C, using a graded ethanol series of 50, 60, 70, 80, and 90% for 10 min each and three 10 min changes of pure ethanol. Pellets were then brought to room temperature and transferred through propylene oxide twice for 20 min each, 50%, and 75% Spurr’s embedding resin (Spurr 1969) in propylene oxide for 1 h each, and 100% overnight. The following day, the pellets were moved to new pure resin and polymerized at 70°C. Blocks were thin-sectioned on a PT-X ultramicrotome (RMC Products; Boeckeler Instruments, Tucson, AZ, USA). Sections of 70 nm thickness were collected on slot copper grids, stained with 3% (w/v) uranyl acetate and Reynold’s lead citrate (Reynolds 1963), and observed and photographed, using a JEM-1010 transmission electron microscope operated at 80 kV (JEOL Ltd.). Images of sections were recorded on Kodak EM Film 4489 (Eastman Kodak Co., Rochester, NY, USA) and scanned to digital format, using EPSON PERFECTION V700 PHOTO (Epson Korea Co., Ltd., Seoul, Korea). The three-dimensional reconstruction was generated via CATIA V5R16 (Dassault-Aviation, Argenteuil, France) based on several hundred transmission electron micrographs.
Terminology followed Lynn (2008) and we adopted the kinetosome triplet numbering conventions of Grain (1969) and De Puytorac (1970).
DNA extraction, polymerase chain reaction amplification, and sequencing
Before DNA extraction, we used an inverted light microscope (ZEISS Axio Vert.A1, Jena, Germany) to confirm that the culture of M. annulatum had completely depleted the available prey. Cells were pelleted from culture samples (approximately 4,000 cells 4 mL−1) by centrifugation (VS-15000 CFN II; Vision Scientific, Gwangju, Korea) at 8,000 ×g for 5 min. The supernatant was carefully removed from the cell pellet. The DNA was extracted from the cell pellet and purified, using an AccuPrep Genomic DNA Extraction Kit (Bioneer, Seoul, Korea) according to the manufacturer’s instructions. Amplification of the entire ITS region was carried out with the primer set UNIDEUK880F (ACTGTAAACTATGCCRACTTGG) (Johnson et al. 2004) and D2C (Scholin et al. 1994) as follows: initial denaturation for 5 min at 94°C, followed by 35 cycles of denaturation for 30 s at 94°C, annealing for 30 s at 52°C, and elongation for 3 min at 72°C. Polymerase chain reaction (PCR) purification and sequencing were conducted as described previously (Park et al. 2013). The sequence has been deposited in GenBank under the accession No. KU047955.
Phylogenetic analyses
Internally transcribed spacer region (ITS) of Mesodinium is known to clearly distinguish the intrageneric groups (Garcia-Cuetos et al. 2012, Johnson et al. 2016, Kim and Park 2019). The new sequence was aligned and edited based on the reference database of all available Mesodinium ITS sequences (164 taxa), using MacGDE version 2.4 (http://macgde.bio.cmich.edu/main.html). A total of 605 unambiguously aligned sites were used for phylogenetic analysis of the partial 18S, the entire ITS, and the partial 28S rRNA sequences, excluding those corresponding to the PCR primer regions.
Maximum likelihood (ML) analysis was performed, using RAxML version 8.0.0 (Stamatakis 2014), with a single general time-reversible plus gamma (GTR + GAMMA) model obtained automatically by the program [rate matrix (0.836016, 1.301959, 0.983546, 0.684371, 1.759670, 1.0), state frequency (0.276742, 0.217093, 0.260866, 0.245299), gamma (heterogeneity)]. We used 1,000 independent tree inferences, selecting the -# option in the program to identify the best tree. ML bootstrap support values were calculated, using 1,000 replicates with the same substitution model.
Bayesian analysis was run, using MrBayes 3.2.5 (Ronquist et al. 2012) with a random starting tree, two simultaneous runs (nruns = 2), and four Metropolis-coupled Markov chain Monte Carlo (MC3) chains for 10,000,000 generations, sampling every 1,000 generations. The molecular data were analyzed with a K80 + G model, and the following parameters were specified: preset tratiopr = fixed (1.7187), statefreqpr = fixed (equal), and shapepr = exponential (0.5313). The first 2,500 trees were discarded, and the remaining trees were used to calculate the posterior probabilities (PPs) of each clade. The tree was visualized, using the FigTree v.1.4.2 program (http://tree.bio.ed.ac.uk/software/figtree/).
RESULTS
Light microscopy
The M. annulatum cells consisted of two portions, as in other Mesodinium species (Fig. 1A). The anterior portion had almost the same diameter as the posterior portion (Fig. 1A). The cirri were stiff and extended in three different directions, whereas the equatorial ciliary belt was flexible and attached to the posterior portion (Fig. 1A–C). Together, the cirri and the equatorial ciliary belt formed a girdle between the two portions. One or two macronuclei were clearly visible in the center of protargol-impregnated cells (Fig. 1D–G). Living cells were 23–34 μm long, 14–22 μm wide in the anterior portion, and 14–21 μm wide in the posterior hemi-cell, whereas protargol-impregnated cells were 13–23 μm long, 9–20 μm wide in the anterior portion and 10–22 μm wide in the posterior portion. The protargol-stained cells were shrunken by about 35% (Table 1).
General ultrastructure
When looking at the scanning electron microscope images (Fig. 2A), the anterior and posterior portions were separated by cirri and an equatorial ciliary belt, which covered both the anterior and posterior portions. The chloroplast-mitochondrial complexes were located in the cell’s periphery (Fig. 2B & C), with each chloroplast having an inwardly projecting pyrenoid surrounded by starch (Fig. 2B & C). Up to 23 chloroplasts were observed. Two types of nuclei of ciliate-origin were observed in the cytoplasm of M. annulatum, namely, invariably one micronucleus and one or two macronuclei (Fig. 2B & D). The nucleus of cryptophyte-origin was also observed in the center of the posterior portion close to the ciliate’s macronucleus / macronuclei (Fig. 2B).
Kinetids and associated structures
Mesodinium annulatum had two types of polykinetids, namely, those being associated with the equatorial ciliary belt and the cirri, respectively. The first polykinetids were located on the posterior side of the girdle and formed the equatorial ciliary belt (Figs 1A, 2A, 2D, 3A & B). There were 34–38 double rows (Figs 2D & 3B), each composed of two rows of 18 alternating kinetosomes linked in various ways by fibrillar structures and electron-dense material (ocm) (Fig. 3C–E). A non-striated homologue to a kinetodesmal fibre (Kd) was associated with triplet No. 7 of each right kinetosome and extended rightwards perpendicular to the double row (Fig. 3E). Additionally, cross-banded, Y-shaped fibrils (cbf) connected three adjacent kinetosomes: triplet No. 3 of a kinetosome in the right row, triplet No. 2 of a more anteriorly positioned kinetosome of the left row, and triplet No. 7 of the following (more posteriorly) kinetosome of the left row. Electron-dense material in a X-shaped pattern connects adjacent right kinetosomes: triplets Nos. 1 and 2 of the one kinetosome and triplets Nos. 5 and 6 of the following (more posteriorly positioned) kinetosome.
There were two types of associated microtubules: the postciliary microtubular ribbons (pr) (Fig. 4) and transverse microtubules (Fig. 5). The postciliary microtubular ribbon consisted of three microtubules and originated from near triplet No. 9 of each kinetosome in the right row (Fig. 4A). They extended posteriorly just underneath the cell membrane and overlapped the postciliary microtubules of every following kinetids, forming a stacked ribbon with about seven pairs of microtubules each on the right of the double rows, while the third microtubule of each ribbon terminated shortly before being joined to the plate-like layer (Fig. 4B–F).
A transverse microtubular ribbon (tm) consisted of five microtubules and extended horizontally leftwards, connecting a kinetosome of the left row with a kinetosome of the right row in the adjacent polykinetid (Fig. 5A & B). The transverse microtubular ribbons originating from the two or three posterior-most kinetosomes consisted of 5–15 microtubules (Fig. 5C). Within each transverse ribbon, the microtubules were connected by thin electron-dense strands (arrows in Fig. 5D).
Basal microtubules (bm) formed ribbons of four or five organelles each (Fig. 5E). These microtubules were associated only with the anterior-most left kinetosomes of each polykinetid in the equatorial belt and extended at a distance of 15 nm on the polykinetids left side (Fig. 4A).
Four unciliated kinetosomes (nbb) arranged in a rhomboidal pattern were between the anterior portions of the polykinetids of the equatorial ciliary belt (Fig. 6A). A single microtubule (1r; arrow in Fig. 6C) was associated with the posterior-most kinetosome (kinetosome 4 in Fig. 6A & C). The microtubular quartet (4r) was near the posterior margin of the cirral kinetosomes in row I (Fig. 6D). From this microtubular ribbon composed of approximately seven microtubules, several microtubules (Rmt) extended to the sixth or seventh kinetosome of the left row (Fig. 6A, B, F & G).
A striated fiber associated with a longitudinal row of nine microtubules (MB) occurred at the anterior margin of the polykinetids (Fig. 7A). This microtubule row was adjacent to the anterior-most kinetosome of the polykinetid row I (Fig. 7A). The polykinetids associated with the cirri consisted of 16 kinetosomes arranged in four longitudinal rows (numbered I to IV from right to left) containing five, five, four, and two kinetosomes, respectively (Fig. 7B). Two other types of polykinetid-associated microtubular elements were observed. The first type consisted of three or four microtubules (PKr) located between the kinetosomes of row I and extended parallel to the kinetosomes (Fig. 7C–F). The second type, a single microtubule, was associated with the posterior-most kinetosomes in rows I and II (Fig. 7G). Only eight of the polykinetid kinetosomes formed with their long cilia of the cirri (asterisks in Fig. 7B): the three posterior-most kinetosomes of row I, the three posterior-most kinetosomes of row II, and two posterior kinetosomes of row III. The remaining eight congeners formed short cilia (Fig. 7H). The eight cilia forming the cirrus were arranged in a 7 + 1 pattern: a central-most cilium and seven peripheral cilia (Fig. 7I). The central cilium of each cirrus had a notably distended cell membrane (Fig. 7I).
Mesodinium annulatum had an obconical cytopharynx terminating in the anterior cell portion (Fig. 8A). Cross-section of its proximal portion reveal about 20 single microtubules to form a ring (Fig. 8B). Towards the proximal end of the cytopharynx, these singletons had additional cytopharyngeal microtubules successively added, eventually forming triplets (Fig. 8C & D). Near the distal end of the cytopharynx, the small microtubular cylinders originated from a fibrous annulus. Immediately anterior to the annulus, the large microtubular cylinders arose adjacent each small cylinder. Each small and large cylinder consisted of 11 and 14 microtubules, respectively (Fig. 8E & F). We provide diagrammatic reconstruction of the somatic ciliature (Fig. 9) and schematic drawing (Fig. 10) of M. annulatum to help the reader comprehend three dimensional of structures.
Phylogeny
The topology of the RAxML tree based on the nuclear partial small subunit rRNA, the entire ITS, and the partial large subunit rRNA sequences of 85 taxa was almost identical to that of the Bayesian tree, except for two positions (see the node marked with ‘*’ in Fig. 11). The genus Mesodinium formed eight major clades (A, B, C, D, E, F, G, and H; we refer to the clade name as matching that of Johnson et al. 2016), in accordance with previously reported phylogenies of Mesodinium (Herfort et al. 2011, Garcia-Cuetos et al. 2012, Johnson et al. 2016, Kim and Park 2019). Mesodinium annulatum (MR-MAL01) falls into clade B with strong support values (ML = 90%, PP = 0.95), forming a polytomy with several unidentified environmental sequences. The clades B was sister to the clades C, D (including M. major), and E with low bootstrap support (ML = 59%) and a high PP (0.99). The ITS sequences from the Antarctic strain (JN412738), Danish strain (JN412736), and American strain (KX783613) of M. rubrum were placed within clades A, F, and G, respectively.
DISCUSSION
Ultrastructural comparison of Mesodinium species
Mesodinium annulatum differs distinctly from M. rubrum in the shape of the posterior cell portion and in the somatic ciliature (Table 2). While M. rubrum has a conical posterior cell portion (Lindholm 1985; A form, Crawford 1993; fig. 3B, Garcia-Cuetos et al. 2012), it is broadly rounded in M. annulatum. Based on serial sections, the number of chloroplasts in M. annulatum was estimated to be 18 or 23 (n = 2). Although M. annulatum is smaller than the Danish M. rubrum, its number of chloroplasts is very similar to that of M. rubrum (Table 3).
In early descriptions of M. rubrum, the number of polykinetids in the equatorial ciliary belt (ECB) varied distinctly within and between populations: 43–106 polykinetids in the Wellington Harbour population, New Zealand (Bary and Stukey 1950), 30–40 in the Isefjord population, Denmark (Fenchel 1968), 36–80 in the Baltic Sea population, 112 in the Southampton estuary population, United Kingdom (Lindholm 1985), and 31–36 in Danish culture, the neotype population of M. rubrum (Garcia-Cuetos et al. 2012). Therefore, the number of polykinetids in the equatorial ciliary belt may be an important character for the discrimination of populations or species. In M. annulatum, the number of polykinetids in the equatorial ciliary belt was 34–38 and thus somewhat higher than that in the neotype of M. rubrum. Likewise, the number of kinetosomes in the polykinetids of equatorial ciliary belt varies: each row consists of about 9 kinetosomes in M. pulex; about 11 in M. pupula, M. chamaeleon, and M. coatsi; about 16 in M. rubrum; about 24 to 25 in M. major; and about 18 in M. annulatum (Garcia-Cuetos et al. 2012, Nam et al. 2015).
Two types of microtubules are associated with the polykinetid of the equatorial ciliary belt: a postciliary microtubular ribbon and a transverse microtubular ribbon. In M. chamaeleon, M. coatsi, and M. annulatum, the postciliary microtubular ribbon consists of three microtubules: data on other species are not available. The number of microtubules in the transverse microtubular ribbons is variable: 5–15 in M. annulatum, 5–6 in M. chamaeleon, and 7 in M. coatsi (Grain et al. 1982, Moestrup et al. 2012, Nam et al. 2015).
All Mesodinium species ultrastructurally studied share four unciliated kinetosomes between the anterior ends of the polykinetids of the equatorial ciliary belt (Garcia-Cuetos et al. 2012, Moestrup et al. 2012, Nam et al. 2015), except for the M. rubrum population from the White Sea which has only two unciliated kinetosomes (Grain et al. 1982). With exception of M. pulex and M. major, the unciliated kinetosomes are associated with microtubules. Mesodinium chamaeleon, a green Mesodinium species, has one microtubule, while M. coatsi, another green Mesodinium species, has two microtubules (Moestrup et al. 2012, Nam et al. 2015). Mesodinium pupula, a heterotrophic species, has two types of microtubular associates and even connecting fibers between its two unciliated kinetosomes (Garcia-Cuetos et al. 2012). In M. rubrum from the White Sea, a three-microtubular ciliary root (= Pcn, postciliaries) is associated with the posterior-most unciliated kinetosome (Grain et al. 1982). Mesodinium annulatum has four unciliated kinetosomes, which are associated with only a single microtubule.
The number and arrangement of the kinetosomes in the cirral polykinetids are important distinguishing features (Garcia-Cuetos et al. 2012, Moestrup et al. 2012, Nam et al. 2015). Each cirral polykinetid of M. annulatum consists of 16 kinetosomes arranged in four rows, with rows I–IV (numbered from right to left) comprising 5, 5, 4, and 2 kinetosomes, respectively; this pattern was similar to that observed in M. rubrum, M. major, and M. pulex (Garcia-Cuetos et al. 2012). The remaining congeners, M. pupula, M. chamaeleon, and M. coatsi, have 17 or 18 kinetosomes and a different arrangement of the kinetosomes: 6, 5, 4, and 3 in M. pupula; 5, 5, 5, and 2 in M. chamaeleon, and 5, 5, 5, and 3 in M. coatsi (Garcia-Cuetos et al. 2012, Moestrup et al. 2012, Nam et al. 2015). Furthermore, M. annulatum has a single microtubule at the ends of rows I and II and an additional microtubular ribbon between each pair of kinetosomes in row I. These two types of microtubular associations were reported in only M. coatsi (Nam et al. 2015).
Based on the same culture material, this and previous studies (Nam et al. 2012) confirmed that M. annulatum has a distinctive oral apparatus, consisted of a cone-shaped cytopharynx, 20 to 22 small cylinders composed of 11 microtubules, 20–22 large cylinders composed of 14 microtubules (= oral tentacles bearing up to five extrusomes at their tips), and a conspicuous fibrous annulus (Fig. 8). Lindholm et al. (1988) reported that M. rubrum from the Baltic Sea had six to eight oral tentacles, which consisted of large cylinders with 14 microtubules (Table 3). Danish M. rubrum cells have 16 small and large cylinders, respectively, and each of the 16 tentacles originating from the large cylinders has four extrusomes at its tips; an annulus could not be detected (Garcia-Cuetos et al. 2012).
Molecular diversity of the Mesodinium rubrum complex
The phylogeny based on nuclear ITS sequences showed that the Mesodiniidae formed five well-supported and distinct clades, represented by M. pulex, M. pupula, M. coatsi, M. chamaeleon, and the highly diverse, not fully resolved M. rubrum / major / annulatum complex (Garcia-Cuetos et al. 2012, Johnson et al. 2016). The present data placed M. annulatum in clade B, and its partial 18S, entire ITS, and partial 28S rRNA sequences were identical to that of HQ227851 (Herfort et al. 2011), i.e., specimens identified as M. rubrum and isolated from the Columbia River estuary. Hence, our new species is apparently widely distributed in coastal region and estuaries, as well as is a causative species of the massive Mesodinium blooms occurred in those environmental regimes (Yih et al. 2013, Herfort et al. 2017). Although M. annulatum is genetically similar to M. major, it has many morphological and ultrastructural features that distinguish both species: cell size, number of polykinetids in the equatorial ciliary belt and cirri, and number of kinetosomes in the polykinetids of the equatorial ciliary belt. All data suggest that the strain studied represents a new Mesodinium species.
Mesodinium annulatum S. W. Nam, W. Shin sp. nov
Description
Cells in vivo on average about 27 μm long, anterior portion on average about 17 μm wide, posterior portion on average about 17 μm wide, and 34–38 two-rowed polykinetids in the equatorial ciliary belt each consisting of about 18 kinetosomes in each row. Two types of microtubules associated with polykinetids in equatorial ciliary belt: postciliary ribbons consisting of three microtubules; transverse ribbons mostly consisting of five microtubules. Four or five basal microtubules. Each cirral polykinetid consists of 16 kinetosomes arranged in four longitudinal rows of 5, 5, 4, and 2 organelles, respectively; eight cilia form a cirrus. Obconical cytopharyngeal basket formed by about 20–22 microtubule triplets. Cytopharyngeal annulus anteriorly anchoring small cylinders composed of 11 microtubules each. Tentacles contain large cylinders of 14 microtubules each and up to 5 extrusomes at their tips. Cells contain red (phycoerythrin) and rarely blue-green (phycocyanin) chloroplasts in field and culture material. Marine plankton.
Holotype
A protargol-stained slide containing the holotype marked with an ink circle is deposited at the Nakdonggang National Institute of Biological Resources, Sangju, Korea (NNIBR), NNIBRPR17357.
Isotype
A protargol-stained slide containing the isotype marked with an ink circle is deposited at the Nakdonggang National Institute of Biological Resources, Sangju, Korea (NNIBR), NNIBRPR17358.
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