J. Phycol. 52, 793–805 (2016) © 2016 Phycological Society of America DOI: 10.1111/jpy.12438
MORPHOLOGICAL AND MOLECULAR CHARACTERIZATION OF PTYCHODISCUS NOCTILUCA REVEALED THE POLYPHYLETIC NATURE OF THE ORDER PTYCHODISCALES (DINOPHYCEAE)1 Fernando G o mez2 Carmen Campos Panisse 3, E-11500, Puerto de Santa Marıa, Spain
Dajun Qiu CAS Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Science, Guangzhou, China
John D. Dodge The Old Farmhouse, Ashton under Hill, Evesham WR11 7SW, UK
Rubens M. Lopes Laboratory of Plankton Systems, Oceanographic Institute, University of S~ao Paulo, Cidade Universitaria, Butant~a, S~ao Paulo 05508-900, Brazil
and Senjie Lin Department of Marine Sciences, University of Connecticut, Groton, Connecticut, USA
within the short-branching dinokaryotic dinoflagellates as an independent lineage with affinity to Brachidinium/Karenia and Karlodinium/ Takayama in a generally poorly resolved clade. Our results indicated that the order Ptychodiscales, established for unarmored dinoflagellates with a strongly developed pellicle, has artificially grouped thecate dinoflagellates (Kolkwitziella, Herdmania), naked dinoflagellates with thick cell covering (Balechina, Cucumeridinium) and other insufficiently known unarmored genera with typical cell coverings (Brachidinium, Ceratoperidinium).
The planktonic dinoflagellate Ptychodiscus noctiluca combined distinctive morphological features such as a disk-shaped anteroposteriorly compressed cell body and an apical carina, together with a flexible and tough cell covering, suggesting intermediate characteristics between thecate and naked dinoflagellates. Ptychodiscus noctiluca was examined by light, epifluorescence, and scanning electron microscopy from specimens collected in the Mediterranean Sea and the North and South Atlantic Ocean. Ptychodiscus noctiluca showed a straight apical groove that bisected the carina, a cell covering with a polygonal surface reticulum, nucleus without capsule, sulcal intrusion in the episome, sulcal ventral flange, and yellowish-green chloroplasts that are shared characters with Brachidinium/Karenia. The cell division was the typical binary fission of gymnodinioid dinoflagellates, although exceptionally in an oblique transversal axis. We examined the intraspecific variability during incubation experiments. In the fattened cells, termed as Ptychodiscus carinatus, chloroplasts transformed into dark granules, and the cell acquired the swollen and smaller stage, termed as P. inflatus. Ptychodiscus carinatus, P. inflatus, and Diplocystis antarctica are synonyms of P. noctiluca. Molecular phylogeny based on the SSU rDNA sequence revealed that Ptychodiscus branched
Key index words: alveolates; amphiesma; Atlantic Ocean; Dinophyta; Gymnodiniales; Mediterranean Sea; Peridiniales; phytoplankton; ptychodiscacean; thick pellicle; unarmored Dinoflagellata Abbreviations: BI, Bayesian inference; bp, base pairs Two major groups of dinoflagellates can be distinguished based on their cell coverings. Thecate (armored) dinoflagellates with large amphiesmal vesicles filled with cellulosic material, and the athecate (unarmored or “naked”) dinoflagellates contain hundreds of alveoli lacking cellulosic material (Dodge 1971). The naked dinoflagellates are usually fragile and delicate, and lyse under poor fixation conditions. However, the separation between armored and unarmored species is not clear cut, and the first example was Ptychodiscus noctiluca F. Stein. von Stein (1883) examined the stomach contents of alcohol-preserved salps. This method only
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Received 14 December 2015. Accepted 19 May 2016. Author for correspondence: e-mail fernando.gomez@fitoplancton.
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com. Editorial Responsibility: R. Wetherbee (Associate Editor)
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preserved the dinoflagellates with thecal plates. However, P. noctiluca F. Stein was preserved despite the apparent absence of thecal plates. von Stein illustrated P. noctiluca in apical and antapical views as a cell consisting of anterior and posterior circular disks connected by a well-marked cingulum. He reported that the episome was the smaller half with an apical lamella-like structure, while the hyposome was larger showing a notch that corresponded to the sulcus. Murray and Whitting (1899) reported P. noctiluca and proposed a new variety. However, their illustrations corresponded to a thecate dinoflagellate (Diplopsalis-like or a flattened cell of Protoperidinium Bergh, see Paulsen 1949). Cleve (1901) described Diplocystis antarctica Cleve as a cyst from alcohol-preserved samples collected in the southern Atlantic and Indian Oceans. Kofoid (1907) described Ptychodiscus carinatus Kofoid from the equatorial Pacific Ocean. He did not compare the new species with P. noctiluca and reported that the episome was the larger half of the cell, while the hyposome was smaller and showed the carina (= keel sensu Kofoid). In the NW Mediterranean Sea, Pavillard (1916) observed one individual of P. carinatus, and he described P. inflatus Pavillard from a single individual that showed a swollen morphology when compared to the flattened P. carinatus. Pavillard also considered the smaller half of the cell with the carina as the hyposome. Schiller (1937) reported these three taxa as valid species of Ptychodiscus F. Stein and also included the Murray and Whitting’s misidentification in P. noctiluca. Schiller maintained the orientation with the carina in the hyposome as reported by Kofoid and Pavillard. Paulsen (1949) found P. inflatus near the Faroe Islands. He pointed out that previous illustrations in ventrodorsal view by Kofoid and Pavillard were upside down, and he considered the smaller half with the carina as the episome. Gaarder (1954) reported P. inflatus in several locations of the temperate Atlantic Ocean. She agreed with the orientation proposed by Paulsen (1949) and considered D. antarctica as a synonym of P. inflatus. Boalch (1969) provided the first micrographs and made the first attempts to culture P. noctiluca from live cells collected in the English Channel. He confirmed that the smaller half with the carina corresponded unequivocally to the episome. Boalch observed the cell covering was flexible, without surface markings, and the dimensions and cell shape changed when preserved. Boalch (1969) concluded that P. noctiluca, P. carinatus, P. inflatus, and D. antarctica were different morphotypes of the same species, and P. inflatus was a preservation artifact. Ptychodiscus noctiluca is widely distributed from neritic to oceanic waters and from tropical to cold water areas such as the Mediterranean Sea (G omez 2003), the Atlantic Ocean (Balech 1967, 1988, Steidinger and Williams 1970, Dodge 1993), the Indian Ocean (Taylor 1976), and the Pacific Ocean (Rampi 1950,
Balech 1962, G omez 2007, Kim et al. 2013). Steidinger (1979) transferred the red-tide species Gymnodinium breve C.C. Davis (now Karenia brevis [C.C. Davis] Gert Hansen et Moestrup) into the genus Ptychodiscus. Her proposal, based on the resemblance of the apical lamella with the carina of K. brevis, received no further acceptance in the literature. In earlier classifications, Ptychodiscus was placed as the only genus of its own family. Lindemann (1928) placed Ptychodiscus in the family Kolkwitziellaceae within the Peridiniales. Schiller (1937) also placed Ptychodiscus within the Peridiniales and included Kolkwitziella Er. Lindemann, Berghiella Kofoid et J.R. Michener, and Lophodinium Lemmermann within the Ptychodiscaceae. Gaarder (1954) suggested an affinity with Gymnodiniales based on the line drawings of dividing cells reported by Cleve (1901). Dodge (1984) placed Ptychodiscus within the family of Gymnodinium F. Stein. Sournia (1986) and Chr etiennot-Dinet et al. (1993) also placed Ptychodiscus within the Gymnodiniales. In contrast, Taylor (1987) placed Ptychodiscus within the order Kolkwitziellales, family Kolkwitziellaceae as a thecate dinoflagellate. Fensome et al. (1993) proposed the new order Ptychodiscales including Balechina Loeblich et A.R. Loeblich, Brachidinium F.J.R. Taylor, Ceratoperidinium Margalef, and Kolkwitziella among other genera. Steidinger and Tangen (1997) also placed Herdmania J.D. Dodge within the Ptychodiscaceae. Recently, Adl et al. (2012) placed Balechina, Ptychodiscus, and Sclerodinium J.D. Dodge within the Ptychodiscales. While there are molecular data for most of the above-mentioned genera (Henrichs et al. 2011, Yamaguchi et al. 2011, Re~ ne et al. 2013, G omez et al. 2015, Mertens et al. 2015), the lack of molecular data of Ptychodiscus, the type of the order Ptychodiscales, renders it impossible to clarify the phylogenetic relationships within the Ptychodiscales. In addition, P. noctiluca possesses morphological features such as a cell covering with intermediate characteristics between naked and thecate dinoflagellates that are important for understanding dinoflagellate evolution. In this article, we describe the morphology and intraspecific variability of P. noctiluca by light, epifluorescence, and scanning electron microscopy and present the first molecular phylogenetic data. MATERIALS AND METHODS
Sampling, isolation, and light microscopy. Specimens were collected from the Mediterranean Sea by slowly filtering surface seawater taken from the pier of the Station Marine d’Endoume at Marseille, France (43°160 48.05″ N, 5°200 56.22″ E, bottom depth 3 m) from October 2007 to September 2008. A strainer of 20, 40, or 60-lm mesh size was used to collect planktonic organisms from water volumes ranging between 10 and 100 L, depending on particle concentration. The plankton concentrate was scanned in settling chambers at 9100 magnification with an inverted microscope (Nikon Eclipse TE200; Nikon Inc., Tokyo, Japan). Cells were
MORPHOLOGY AND PHYLOGENY OF PTYCHODISCUS
photographed alive at 9200 or 9400 magnification with a Nikon Coolpix E995 digital camera. Further specimens were collected using the same method from October 2008 to August 2009 in the surface waters (depth of 2 m) of the port of Banyuls sur Mer, France (42°280 50″ N, 3°080 09″ E). The concentrated sample was examined in Uterm€ ohl chambers with an inverted epifluorescence microscope (Olympus IX51; Olympus Inc., Tokyo, Japan) and photographed with an Olympus DP71 digital camera. Sampling continued from September 2009 to February 2010 in the Bay of Villefranche sur Mer, France. For this location, sampling was performed at the long-term monitoring site Point B (43°410 10″ N, 7°190 00″ E, water column depth ~80 m). Water column samples (0–80 m) were obtained using a phytoplankton net (53 lm mesh size, 54 cm diameter, 280 cm length). Samples were prepared according to the same procedure as described above, and specimens were observed with an inverted microscope (Olympus IX51; Olympus Inc.) and photographed with an Olympus DP71 digital camera. Sampling continued from May 2012 to February 2013 in the port of Valencia, Spain (39°270 38.13″ N, 0°190 21.29″ W, water column depth of 4 m). Specimens were obtained using a phytoplankton net (20 lm mesh size). Samples were prepared according to the same procedure as described above, and specimens were observed with an inverted microscope (Nikon Eclipse T2000; Nikon Inc.) and photographed with an Olympus DP71 digital camera. In addition, samples were collected during the BOUM (Biogeochemistry from the Oligotrophic to the Ultra-oligotrophic Mediterranean) cruise on board R/V L’Atalante from the south of France to the south of Cyprus (20 June–18 July 2008). Seawater samples were collected with Niskin bottles from 30 stations. At each station, 6 depths were sampled between 5 and 125 m, with an additional sample at 250 m depth. These samples were preserved with acid Lugol’s iodine solution and stored at 5°C. Samples of 500 mL were concentrated via sedimentation in glass cylinders. The top 450 mL of sample was slowly siphoned off with small-bore tubing during 6 d. The remaining 50 mL of concentrate, representing 500 mL whole water, was then settled in composite settling chambers. The sample was examined in Uterm€ ohl chambers at 9100 magnification with a Nikon inverted microscope (Nikon Eclipse TE200; Nikon Inc.), and the specimens were photographed with a digital camera (Nikon Coolpix E995; Nikon Inc.). In the South Atlantic Ocean, sampling continued after March 2013 in S~ao Sebasti~ao Channel (23°500 4.05″ S, 45°240 28.82″ W), and from December 2013 to December 2015 off Ubatuba (23°320 20.15″ S, 45°50 58.94″ W). The Brazilian specimens were obtained using a phytoplankton net (20 lm mesh size) in surface waters. The living concentrated samples were examined in Uterm€ ohl chambers at magnification of 9200 with inverted microscopes (Diaphot-300; Nikon Inc. at S~ao Sebasti~ao, and Eclipse TS-100; Nikon Inc. and Olympus IX73; Olympus Inc. at Ubatuba), and photographed with a digital camera (Cyber-shot DSC-W300; Sony, Tokyo, Japan) mounted on the microscope’s eyepiece. For further morphological studies using an epifluorescence microscope, live specimens were transported to the University campus (~250 km far away from the coastal laboratory). The stressed live cells were examined using an Olympus BX51 epifluorescence microscope equipped with an Olympus DP72 camera (Olympus Inc.). In order to determinate the position and shape of the nucleus and presence of thecal plates, the cells fixed with glutaraldehyde (5% final concentration) were stained with DAPI (40 ,60 diamino-2-phenylindole, 0.1 lg mL1 final concentration; Sigma, St. Louis, MO, USA) and Calcofluor White M2R (Fluorescent Brightener 28; Sigma) and examined with the same microscope.
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Incubations. Specimens of P. noctiluca from Brazil were isolated with the aim to establish cultures. Cells were isolated using a micropipette and placed in 12-well tissue culture plate with 0.2 lm-filtered seawater collected that day from the same locality, and they were supplemented with f/2 medium without silicates. Two days later, the healthy specimens were re-isolated and placed into a 6-well tissue culture plate with f/2 medium made with filtered and sterilized seawater. Several variations of this protocol were tested. In all the cases, the culture plates were placed in an incubator used for microalgae culturing, at 23°C, 100 lmol photons m2 s1 from cool-white tubes and photoperiod 12:12 L:D. As the attempts to establish long-term cultures of P. noctiluca were unsuccessful, the specimens used for molecular analysis were isolated from natural samples. The specimens were micropipetted individually with a fine capillary into a clean chamber and washed several times in a series of drops of 0.2 lm-filtered and sterilized seawater. Finally, 50 specimens collected in S~ao Sebasti~ao Channel on April 22, 2013, were placed in a 0.2 mL tube filled with absolute ethanol. The sample was kept at room temperature and in darkness until the molecular analysis could be performed. Scanning electron microscopy. Samples were collected from the North-East Atlantic Ocean as reported in Dodge (1993). Lugol’s iodine and formaldehyde-preserved samples were examined by light microscopy at low magnification. Cells of Ptychodiscus were picked out of the drop of sample by using a micro-pipette. The cells were placed in the well of a brass container, the bottom of which was formed by a Nuclepore filter (Whatman, Brentford, UK) clamped into place. After collecting a number of cells, an acetone series was flushed through to dehydrate the cells and the chambers was closed with another filter at the top. The container was then placed in a critical point apparatus (Polaron, Watford, UK), and drying was carried out using liquid carbon oxide. The filters were mounted on 13 mm diameter stubs and coated with gold-palladium in a Polaron cool sputter coater. Finally, the specimens were examined in a JEOL JSM 25S scanning electron microscope (JEOL Ltd., Tokyo, Japan), usually run at 15 kV. DNA extraction, PCR amplification of small subunit rRNA gene, and sequencing. Prior to DNA extraction, the tube was centrifuged for 10 min at 500 g to settle the Ptychodiscus cells, and the ethanol was aspirated. One hundred microliters of DNA lysis buffer (0.1 M ethylenediaminetetraacetic acid pH 8.0, 1% Sodium Dodecyl Sulfate (SDS), 200 lg mL1 proteinase K) was used to re-suspend the cell pellet; the resuspension was transferred into a 1.5 mL tube, and the process was repeated for five times. Then, the resultant 0.5 mL was incubated for 48 h at 55°C. DNA extraction and purification followed a previously reported protocol (Qiu et al. 2011). At the end of the extraction process, DNA of Ptychodiscus was eluted in 50 lL Tris-HCl solution. Next, 1 lL of the extracted DNA was PCR amplified. The small subunit (SSU) rDNA, ~1,800 base pairs, was amplified using the primers Dino18SF1 and 18ScomR1 (Qiu et al. 2013). The PCR amplifications were carried out in 25 lL reaction volumes containing 0.125 lL of TaKaRa Ex Taq HS in the PCR master mix (TaKaRa Bio, Dalian, China), both forward and reverse primers (final concentration 0.2 lM) and template DNA. Thermocycling conditions included a denaturing step of 94°C for 4 min; 35 cycles of 94°C for 30 s, 56°C for 30 s, 72°C for 45 s, and a final extension step of 72°C, 10 min. PCR products were resolved by agarose gel electrophoresis with the DL2000 DNA ladder (TaKaRa Bio), and the bands with expected sizes were excised to remove primer dimers. DNA was purified and directly sequenced as previously reported (Qiu et al. 2011). Phylogenetic analyses. Two phylogenetic analyses were performed. The first was based on nearly complete SSU rDNA
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sequences, comprising dinokaryotic sequences most similar to P. noctiluca, as identified through BLAST search (http:// blast.ncbi.nlm.nih.gov/Blast.cgi), complete sequences of species classified within the Ptychodiscales (Balechina, Brachidinium, Kolkwitziella, Herdmania, Ceratoperidinium, Cucumeridinium F. G omez, P. L opez-Garcıa, H. Takayama et D. Moreira), and a wide selection of dinokaryotes representing the major orders including Peridiniales and Gymnodiniales. To improve the resolution of trees and clarify the relationships between Ptychodiscus and the other species, the second analysis included additional sequences from Karenia and Karlodinium and removed sequences from Kolkwitziella and Cucumeridinium. The new SSU rDNA sequence of Ptychodiscus was aligned with sequences of dinokaryotic dinoflagellates collected from GenBank database in ClustalX, using default parameters (Larkin et al. 2007). Ambiguously aligned regions and gaps were removed manually from the initial alignment using Bioedit 7.0 (Hall 1999), leaving a final alignment of 1,402 characters in both cases that was used to construct phylogenetic trees. The GTR + I + G evolutionary model was selected by Modeltest v3.7 (Posada and Crandall 1998) and used for Bayesian inference (BI) analysis. The BI analysis was conducted using primarily MrBayes 3.1.2. Four simultaneous chains were run for 8,500,000 generations, with sampling every 100 generations, and the first 21,250 (25%) trees discarded as burn-in. The remaining trees were used to calculate posterior probabilities using a majority rule consensus. Our sequence was deposited in DDBJ/EMBL/GenBank under accession number KU640194. RESULTS
Morphology. Ptychodiscus noctiluca was occasionally detected in the Mediterranean Sea and the Atlantic Ocean. After the analysis of 212 Lugol’s iodine fixed samples from the open Mediterranean Sea, the records of P. noctiluca were restricted to two specimens from a sample collected at 75 m depth between Menorca and Sardinia. During the observations of live samples in the coastal NW Mediterranean Sea (Marseille, Banyuls sur Mer, Villefranche sur Mer, and Valencia), the occurrence of the species was occasional and restricted to a few specimens per day. Consequently, no clear temporal pattern was found although the occurrence appeared to be more frequent in winter season. In the South Atlantic Ocean at the coasts of S~ao Paulo State, P. noctiluca also appeared occasionally in any period of the year. Some days several tens of specimens appeared in the samples, while later the species was absent for several months. It should be noted that under low magnification, the usually immobile cells of Ptychodiscus, a flattened cell with a circular contour with a yellowish-green chloroplasts, could be mistaken for drum-shaped diatoms by nontrained observers. There were no apparent morphological differences between the specimens of the Mediterranean Sea (Fig. 1) and Brazil (Fig. 2). The specimens showed chloroplasts in recently collected live samples (Figs. 1, a–n; 2, b–e). The live specimens of P. noctiluca tended to remain immobile settling in the apical–antapical plane, and occasionally they swam slowly (see Video S1 in the Supporting
Information). The cells of the most common morphotype, P. noctiluca/P. carinatus, were anteroposteriorly compressed with a length/width ratio ~1:4 and formed by two disk-shaped halves joined by the well-marked cingulum (Fig. 1, g–h). The cell dimensions typically ranged from 50 to 90 lm width (transdiameter), and sporadically specimens of less than 50 lm wide were observed co-existing with the typical specimens (Fig. 2a). The disk-shaped halves, episome and hyposome, were not circular. The depth was ~90% smaller than the width giving a slightly ellipsoidal shape with a ventrodorsal compression. The diameter of the episome was ~85% that of the hyposome (Figs. 1, a–b, e–f; 2, e–f). The episome was flattened with a slight concave anterior face and a carina that extended radially along the 2/3 of the episome toward the ventral side (Fig. 1, g–i) with a semicircular shape in lateral view (Figs. 1h and 2x). The carina was not exactly located along the ventrodorsal axis, but displaced toward the left side of the cell (Figs. 1, a, e; 2, b–d). The cingulum was descending, wide and deep, except in the ventral side at the sulcus level (Figs. 1p; 2, n and p). The typical dinoflagellate transverse flagellum encircled the cell along the cingulum, and the flagellar pore was located in the anterior end of the sulcus (Figs. 1d and 2f; see Video S1). In addition to the carina, the most distinctive structure was the sulcus. In apical or antapical views, the sulcus formed a notch or indentation in the contour of the hyposome (Figs. 1, a–b; 2b). The sulcus formed a groove with a rounded posterior end and extended for about 1/4 the hyposome diameter. The sulcus was not exactly located along the dorsoventral axis, but was displaced toward the right side of the cell (Figs. 1b; 2, d–e, r). The posterior end of the sulcus harbored the relatively short (~30 lm long), longitudinal flagellum (Fig. 1, c–d, see Video S1). During the observations with an inverted microscope, the specimens settled into two positions: (i) Antapical position, with the flattened hyposome entirely touching the bottom glass. The hyposome can be observed in the frontal focus (Fig. 1b), while the episome with the carina appeared in the rear focus of the cell (Fig. 1a); and (ii) Apical position, the main cell body was inclined with the carina and the dorsal side of the hyposome touching the bottom glass. The carina and the dorsal hyposome were observed in the frontal focus (Fig. 1e), while the ventral side of the cell and sulcus in the rear focus (Fig. 1f). The cell of recently collected specimens showed a flattened morphology and numerous small rounded or slightly ellipsoidal chloroplasts, as revealed by epifluorescence microscopy (Figs. 1, j–m; 2, g–h). The chloroplasts had a yellowish-green pigmentation (Figs. 1, e–f; 2, b–e). The nucleus was hardly visible using light microscopy (Fig. 2z). Under
MORPHOLOGY AND PHYLOGENY OF PTYCHODISCUS
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FIG. 1. Light micrographs of Ptychodiscus noctiluca from the NW Mediterranean Sea. (a–n) Live cells. (o–q) Ethanol-preserved cells. (a–b) Antapical view of one individual. Note the yellowish-green chloroplasts. (a) Frontal focus. (b) Rear focus. (c, d) Swimming individual in dorsal view. Note the longitudinal and transversal flagella. (e–i) Several views of the same individual. (e, f) Apical view. (g, h) Lateral view. (i) Oblique apical view. (j–m) Individual in antapical and lateral views. (j, k) Light and epifluorescence antapical view, respectively, of the form P. inflatus. (l, m) Light and epifluorescence in lateral view, respectively. Note the fluorescence of the chloroplasts. (n) Dividing cell. (o–q) Two individuals preserved in ethanol. (o) Antapical view. (p) Dorsal view of the same individual. (q) Ventroantapical view of other individual with the form Diplocystis antarctica. ca, carina; ci, cingulum; lf, longitudinal flagellum; tf, transversal flagellum; su, sulcus; scale bars, 20 lm.
epifluorescence microscopy of cells fixed with glutaraldehyde and stained with DAPI, the nucleus was ellipsoidal and located in the left dorsal side of the cell (Fig. 2i). Other glutaraldehyde-fixed specimens were stained with Calcofluor White in other to test the presence of thecal plates. No thecal plates were observed and the cell covering using light microscopy appeared as a continuous layer. Individuals were squashed, and the cell covering was tough and flexible, and changed its dimension and shape. The cell covering resembled a plastic bag, and the carina and cingulum appeared as creases (Fig. 2, j–k). The cell covering resisted treatment with the most common preservatives (formaldehyde, glutaraldehyde,
ethanol, and Lugol’s iodine). While the plates of thecate dinoflagellates decompose after treatment with sodium hypochlorite, the cell covering of Ptychodiscus also resisted the treatment with sodium hypochlorite, although the cell strongly deformed and acquired an unrecognizable globular shape (Fig. 2l). When the specimens were fixed with Lugol’s iodine, the cell covering was slightly stained when compared to the cell contents (Fig. 2m). Ptychodiscus did not show the dark brown color of the cellulosic thecal plates of armored dinoflagellates. In ethanol-preserved samples, the cell acquired a swollen shape that corresponded to P. inflatus or more precisely to D. antarctica (Fig. 1, o–q).
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FIG. 2. Light micrographs of Ptychodiscus noctiluca from the South Atlantic Ocean. (a) Note the different size of three individuals. A large depigmented individual on the left, a pigmented individual in the center, and on the right a small depigmented individual of the form P. inflatus after several days in culture conditions. (b–d) Different focuses of one individual. (b) Rear focus. (c) Middle focus. (d) Frontal focus. (e) Antapical view in frontal focus of a live individual. Note the yellowish-green chloroplasts. (f) Antapical view in frontal focus of other live individual after 2 d under culture conditions. Note the reduction in the number of chloroplasts and the numerous dark granules. (g, h) Stressed cell observed with an upright epifluorescence microscope. (h) Epifluorescence image of the chloroplasts. (i) Glutaraldehyde-fixed cell. Note the nucleus stained with DAPI. (j, k) Individual squashed by gently pressing the coverslip over it. Note the shape of the flexible cell covering. (l) Two individuals treated with sodium hypochlorite. Note the tough cell covering. (m) Lugol’s iodine fixed individual. (n) Ventral view of a live individual formerly termed P. inflatus. Note the concave hyposome. (o) Lateral view of a live individual of the form P. inflatus. (p–r) Dead individual of the form P. inflatus. Note the rigid cell covering. (t–ab) Division of three different individuals. (t–x) Another individual of the form P. inflatus. (t) Apical view. (u) Antapical view. The arrow head points a notch, tentatively the beginning of the cytokinesis. (v) Ventral view. (w) Dorsal view. (x) Lateral view. (y) Individual in first division stage. The arrow head points a notch in the dorsal side. (z–ab) Other dividing cell. (z) Frontal focus. Note the nuclei and transversal flagellum. (aa) Middle focus. Note the sulci. (ab) Rear focus. Note the carina. ca, carina; ci, cingulum; n, nucleus; tf, transversal flagellum; su, sulcus; scale bars, 20 lm.
MORPHOLOGY AND PHYLOGENY OF PTYCHODISCUS
The dominant morphotype was the flattened P. noctiluca/P. carinatus, while the form described as P. inflatus was rarer. Consequently, the form P. inflatus was not only an artifact of sample preservation, as live healthy specimens with the morphology of the form P. inflatus appeared in natural samples (Fig. 2o). The P. inflatus morphology was also found in dead specimens devoid of cell contents (Fig. 2, p–r). This suggested that under unfavorable conditions prior to the cell death or due to the preservation, the flattened individuals transformed into the morphology known as P. inflatus. The form P. inflatus was usually smaller in width and larger in length (Fig. 2, a and o). Cell division. The cytokinesis began as notch that appeared in the left dorsal side (Fig. 2, u–w) in the form of P. inflatus or in the dorsal side of the hyposome in the form of P. carinatus (Fig. 2y). The cytokinesis extended toward the right ventral side. Cell division was desmoschitic and oblique along the transversal axis. One daughter cell received the carina (Fig. 2ab) and the other one the sulcus (Fig. 2aa). The two daughter cells remained joined in the right ventral side of the daughter cell that received the carina (Figs. 1o; 2z). Both daughter cells showed their transversal flagella while joined (see Video S1). Incubations and intraspecific variability. The specimens of Ptychodiscus showed numerous chloroplasts (Figs. 1, a and b; 2, e and h). There was no evidence of food vacuoles that could suggest a heterotrophic nutrition. Specimens were isolated with the aim to establish permanent cultures. However, the attempts were unsuccessful and the cells did not survive more than 10 d. Some cells divided within the first days (with filtered seawater supplemented with f/2 medium). The larger and flattened specimens (P. noctiluca/P. carinatus) transformed into the smaller swollen stage (P. inflatus; Fig. 2a). The yellowishgreen chloroplasts progressively disappeared, and dark granules appeared in the cell center (Fig. 2, a and f). The f/2 medium successfully used for peridinin-containing dinoflagellates (Heterocapsa F. Stein, Coolia Meunier, Prorocentrum Ehrenberg) failed with P. noctiluca. Scanning electron microscopy. The cell covering of P. noctiluca appeared as a deflated layer and the borders of the cingulum as folds (Fig. 3, a–b). Some vertical folds appeared in the cingulum. However, it was difficult to discern whether they were a result of the cell deflating or corresponded to truly vertical ridges in the cingulum (Fig. 3, a and b). The carina appeared as a radial semicircular crest oriented along the ventrodorsal axis (Fig. 3, a–c). The carina emerged from the right side of the sulcal intrusion and was bisected by a groove (Fig. 3c). The straight apical groove showed rolled margins (Fig. 3d). The episome showed scattered pores (Fig. 3a) and groups of pores in the antapex (Fig. 3, e and g). The cingulum was descending and displaced about
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one cingular width (Fig. 3e). The sulcus showed a closed intrusion into the episome (Fig. 3e) and was oriented toward the right hyposome (Fig. 3, f–h). A ventral ridge or flange appeared in the sulcus between the ends of the cingulum (Fig. 3f). The cell surface was smooth with hexagonal amphiesmal vesicles and scattered pores in depressed areas (Fig. 3i). One specimen with the morphology of the form P. inflatus was observed. The cell surface showed irregular longitudinal furrows (Fig. 3j). The deep and wide cingulum conserved the transversal flagellum (Fig. 3k). Molecular phylogeny. Initial BLAST comparisons showed that the closest identified relatives in the database were dinokaryotes, such as the thecate dinoflagellates Pentapharsodinium tyrrhenicum (Balech) Montresor, Zingone et D. Marino (AF022201) and Pentapharsodinium sp. (AF274270). We studied the phylogenetic position of P. noctiluca in two SSU rDNA phylogenetic trees. The trees contained a variety of dinoflagellate sequences, especially sequences of species classified within Ptychodiscales, as well as Gymnodiniales and Peridiniales. A first tree built to include all the available sequences of ptychodiscaceans showed that the sequences of the ptychodiscaceans Balechina, Brachidinium, Ceratoperidinium, Cucumeridinium, Kolkwitziella, or Herdmania were not related to Ptychodiscus or even to each other (Fig. S1 in the Supporting Information). A second tree was built using almost complete sequences excluding the long-branch sequence of Kolkwitziella (Fig. 4). The sequence of P. noctiluca branched within the shortbranching sequences of the dinokaryotic core as a lineage between the clades of Brachidinium–Karenia and Karlodinium–Takayama, without support (Fig. 4). DISCUSSION
Ptychodiscus noctiluca appears to be relatively eurythermal, tolerating a range from tropical waters (Kofoid 1907, Rampi 1950, this study) to cold waters of the Atlantic Ocean (Paulsen 1949) or sub-Antarctic waters (Cleve 1901). Ptychodiscus noctiluca has never been observed to reach high abundances in the natural marine environments. Its records are episodic despite it being a photosynthetic species living in the euphotic zone. Our observations of Ptychodiscus from natural samples and the incubation experiments agree with Boalch (1969) who concluded that P. noctiluca, P. carinatus, P. inflatus, and D. antarctica were morphotypes of the same species. All subsequent reports agreed with Boalch, with the exception of Balech (1988), who considered P. carinatus as an independent species based on formaldehyde-preserved samples. Boalch (1969) considered the swollen stage, P. inflatus, as a preservation artifact. In fact, the preserved cells tended to resemble P. inflatus, and for that reason, most of the records in the literature
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FIG. 3. Scanning electron micrographs of Ptychodiscus noctiluca from the North-east Atlantic Ocean. (a) Dorsoapical view. See the dorsal end of the carina. The arrow heads point the pores. (b) Ventroapical view. (c) Detail of carina. Note the straight apical groove. (d) Detail of the apical groove with rolled margins. (e–i) Another individual of P. noctiluca. (e) Ventro-antapical view. (f) Detail of the sulcus and the ventral carina. Note the sulcal ventral flange or ridge. (g) Antapical view. The arrow head points a group of pores. (h) Detail of the sulcus. (i) Detail of the cell surface. Note the pores and the pattern of amphiesmal vesicles as a polygonal reticulum. (j, k) Individual of the form P. inflatus. (j) Dorsal view. Note the furrows in the cell surface. (k) Detail of the transversal flagellum. ag, apical groove; ca, carina; ci, cingulum; fu, furrow; si, sulcal intrusion; su, sulcus; tf, transversal flagellum; vf, ventral flange; scale bars, 10 lm (a–h, j), 1 lm (i, k).
corresponded to that form (Paulsen 1949, Gaarder 1954, Balech 1962, 1967, 1988, Taylor 1976). However, healthy cells with the morphology of the form P. inflatus can be found in natural samples (Fig. 2o); although in some cases, such swollen stage is an indicator of stressed or moribund specimens (Fig. 2, p–r). In the attempts to culture P. noctiluca, we have observed that the healthy and chloroplast-rich flattened specimens of P. noctiluca/ P. carinatus converted themselves into smaller and swollen cells of the form P. inflatus lacking the yellowish-green pigmentation (Fig. 2a). The description by von Stein (1883) was quite precise, although restricted to the apical–antapical view (Fig. 5a). Cleve (1901) described Ptychodiscus (as D. antarctica) as a cyst. However, he illustrated the cyst under binary division (Fig. 5b). The description of P. carinatus by Kofoid (1907) reported that the ventral edge of the carina acts as
the sulcus with the longitudinal flagellum that runs along it. His sulcal groove is in fact the apical groove (Fig. 3c) and no flagellum runs along it (see Video S1). Kofoid (1907) did not cite surface markings in the species description. However, his illustrations showed longitudinal striae or ridges in the cingulum and the carina (Fig. 5c). Pavillard (1916) described P. inflatus from a single specimen (Fig. 5d). It is possible that he examined formaldehyde-preserved material because the morphology coincided with that described by authors examining formaldehyde-preserved samples such as Gaarder (fig. 5f), Taylor (1976) (fig. 5g), and Balech (1988) (fig. 5j). Following Kofoid (1907), other authors have illustrated P. noctiluca with longitudinal striae in the cingulum (Paulsen 1949, fig. 5e; Taylor 1976, fig. 5g; Dodge 1982, fig. 5h; Sournia 1986, fig. 5i). It is difficult to observe the surface of the cingulum in the anterior–posteriorly compressed cell of
MORPHOLOGY AND PHYLOGENY OF PTYCHODISCUS
FIG. 4. Bayesian phylogenetic tree of dinoflagellate SSU rDNA sequences, based on 1,402 aligned positions. Name in bold represents sequence obtained in this study. Numbers at nodes are posterior probabilities. Accession numbers are provided between brackets. The scale bar represents the number of substitutions for a unit branch length.
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Syndinium turbo DQ146404 Hematodinium perezi EF065718 Brachidinium capitatum HM066998 0.60 Karenia bidigitata HM067002 Karenia mikimotoi EF492505 1 Karenia mikimotoi AF009131 0.99 Karenia mikimotoi FR865627 1 Karenia selliformis HM067007 Karenia brevis FJ587219 Karenia brevis EF492503 0.94 Karenia papilionacea HM067005 0.80 Karlodinium veneficum EF036540 Karlodinium veneficum AY245692 1 Karlodinium veneficum AF172712 Karlodinium veneficum EF492506 1 Karlodinium veneficum JN986577 1 Takayama acrotrocha HM067010 Takayama cf. pulchellum AY800130 1 Dinophysis acuta AJ506973 1 Phalacroma rotundatum AJ506975 0.52 1 Gyrodinium fusiforme AB120002 Gyrodinium spirale AB120001 Duboscquodinium collinii HM483398 1 Scrippsiella sweeneyae AF274276 0.83 Scrippsiella trochoidea EF492513 0.99 Peridinium aciculiferum EF417314 Peridinium willei EF375879 0.86 Peridinium cf. centenniale EF058237 Parvodinium umbonatum GU001637 1 Peridinium cinctum EF058243 1 Peridinium willei EF375879 Durinskia capensis AB271107 1 1 Durinskia baltica AF231803 0.97 Galeidinium rugatum AB195668 1 0 .7 4 Kryptoperidinium foliaceum EF492508 Gymnodinium fuscum AF022194 1 Polykrikos geminatum JX967270 Herdmania litoralis AB564305 0.86 1 Amphidiniopsis dragescoi AY238479 Archaeperidinium minutum GQ227501 1 Heterocapsa circularisquama LC054932 Heterocapsa niei EF492499 1 0.55 Heterocapsa rotundata AF274267 Heterocapsa triquetra GU594638 1 Polarella glacialis EF434275 Symbiodinium microadriaticum M88521 0.99 Prorocentrum mexicanum EF492510 1 Prorocentrum micans AY585526 0.1 Prorocentrum minimum DQ336072 Ptychodiscus Pty tychodi discus nocti noctiluca tiluca KU640194
P. noctiluca. These ridges were not evident in our LM observations, although the form P. inflatus showed irregular surface ridges under SEM observations (Fig. 3j). The cell covering of Ptychodiscus is able to resist the preservation with formaldehyde and even ethanol (Fig. 1, o–q). The appearance of the ethanolpreserved cells of Ptychodiscus is identical to D. antarctica (Figs. 1, p–r; 5b). Fensome et al. (1993, p. 54) defined the ptychodiscaceans as cells with cellulose as the principal component of the pellicle. When fixed with Lugol’s iodine, the cell covering did not show the dark brown color of thecate dinoflagellate plates (Fig. 2m, G omez 2007), which suggests the absence or scarcity of cellulosic material in the cell covering. Gaarder (1954) reported that the cell covering of Ptychodiscus could be entirely dissolved by sodium hypochlorite. However, we have observed that after the addition of sodium hypochlorite, the cell covering remained, although strongly deformed (Fig. 2l). We did not observe thecal plates in the cell covering after staining with Calcofluor White. The cell covering is tough and flexible (Fig. 2, j–k). Using high magnification, the
cell covering showed a polygonal surface reticulum (Fig. 3i). This pattern of amphiesmal vesicles can be found in gymnodinioid dinoflagellates such as Karenia (de Salas et al. 2004). As consequence of the unusual cell covering, specimens of Ptychodiscus appeared in samples fixed with formaldehyde or ethanol in which most of the naked dinoflagellates were lost. In the classifications, Ptychodiscus has moved between Peridiniales and Gymnodiniales. Fensome et al. (1993) erected the order Ptychodiscales within the unarmored dinoflagellates. Fensome et al. placed Kolkwitziella within Ptychodiscales. However, Kolkwitziella is a thecate dinoflagellate closely related to Protoperidinium excentricum (Paulsen) Balech (Mertens et al. 2015, Fig. S1). Steidinger and Tangen (1997) placed Herdmania within the Ptychodiscaceae. However, Herdmania is a thecate dinoflagellate closely related to other thecate dinoflagellates such as Archaeperidinium Jørgensen (Yamaguchi et al. 2011; Fig. S1). Fensome et al. (1993) and Adl et al. (2012) also placed the unarmored genus Balechina within the Ptychodiscales. The species of Balechina have been recently split into two genera, Balechina with thick
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FIG. 5. Line drawings of Ptychodiscus noctiluca. (a) P. noctiluca redrawn from von Stein (1883). (b) Diplocystis antarctica redrawn from Cleve (1901). (c) P. carinatus redrawn from Kofoid (1907). (d) P. inflatus redrawn from Pavillard (1916). (e) P. inflatus redrawn from Paulsen (1949). (f) P. inflatus from Gaarder (1954). (g) P. noctiluca redrawn from Taylor (1976). (h) P. noctiluca redrawn from Dodge (1982). (i) P. noctiluca redrawn from Sournia (1986). (j) P. noctiluca redrawn from Balech (1988). (k–o) P. noctiluca based on this study. Apical view. (l) Antapical view. (m) Ventral view. (n) Right lateral view. (o) Dividing cells in antapical view. ca, carina; n, nucleus.
double-layer cell covering with bossed surface and Cucumeridinium with well-marked longitudinal ridges (G omez et al. 2015). Balechina, Cucumeridinium, and Ptychodiscus are distantly related among each other in the SSU rDNA molecular phylogeny (Fig. S1 and Fig. 4). The placement of Brachidinium and Ceratoperidinium within the Ptychodiscales was due to insufficient information on these genera. In the case of Ceratoperidinium, the etymology of the name is confusing because it refers to thecate
dinoflagellates (Cerato = horn, usually associated with a rigid structure, and “Peridinium”). Loeblich (1982) classified Ceratoperidinium as a thecate dinoflagellate within the Peridiniales. However, Ceratoperidinium is an unarmored dinoflagellate (G omez et al. 2004, Re~ ne et al. 2013). The molecular data have revealed that Ceratoperidinium is a close relative of the species formerly known as Gyrodinium falcatum Kofoid et Swezy and several species classified as Cochlodinium F. Sch€ utt (Re~ ne et al. 2013).
MORPHOLOGY AND PHYLOGENY OF PTYCHODISCUS
These taxa do not possess the thick pellicle that defines the ptychodiscaceans. The morphology suggests a relationship between Ptychodiscus and Brachidinium–Karenia. The transfer of Gymnodinium breve (now Karenia brevis) into Ptychodiscus as P. brevis (C.C. Davis) Steidinger was not accepted further (Steidinger 1979). Fensome et al. (1993) did not place Gymnodinium breve within Ptychodiscales. However, they placed Brachidinium within the Ptychodiscales. Brachidinium was first described from a formaldehyde-preserved sample (Taylor 1963), and consequently, it was assumed that the cell possesses a thick pellicle. Unarmored dinoflagellates may occasionally resist the formaldehyde preservation, but the cell shape is strongly deformed and the lack of distinctive features makes the identification impossible even at the genus level. In the case of Brachidinium, it was recognized due to its distinctive body extensions (Taylor 1963). Further studies have revealed that Brachidinium is closely related, if not a synonym, of the unarmored genus Karenia (G omez et al. 2005, Henrichs et al. 2011, fig. 4). However, Karenia and Brachidinium do not possess a thick pellicle, and the cell covering does not differ from that of other typical naked dinoflagellates (de Salas et al. 2004). Daugbjerg et al. (2000) defined Karenia as unarmored dinoflagellates whose major carotenoid is fucoxanthin, nucleus without a capsule and cells with a straight apical groove. The chloroplasts of Ptychodiscus show a distinctive yellowish-green pigmentation that could be an indicator of the presence of fucoxanthin (Fig. 1, e–f). In this study, we show a straight apical groove that bisects the carina of Ptychodiscus (Fig. 3, c–d). The apical groove of P. noctiluca showed rolled margins as in K. papilionacea A.J. Haywood et Steidinger (Fig. 3d; Haywood et al. 2004). The ventral surface of the apical groove meets the extension of the sulcal intrusion as in species of Karenia (Fig. 3c; Haywood et al. 2004). In Ptychodiscus and Karenia, the nucleus is spherical to slightly oval, without capsule, and located in the left side of the cell (Fig. 2i; Haywood et al. 2004). The diagnostic characters of Karenia as defined by Daugbjerg et al. (2000) fit with Ptychodiscus. The cingulum of Karenia and Ptychodiscus is descending and displaced 1–1.59 the cingular width (Fig. 3e; Haywood et al. 2004). Both genera showed concavities in the cell surface. The species of Asterodinium Sournia, Karenia, and Karlodinium showed a sulcal structure traversing the proximal and distal ends of the cingulum (Haywood et al. 2004, G omez et al. 2005). We found a similar structure in Ptychodiscus (Fig. 3f). The cell surface of Karenia and the flattened form of P. noctiluca are smooth with polygonal amphiesmal vesicles (Fig. 3i, de Salas et al. 2004). Groups of pores can be found in species such as K. brevis (Haywood et al. 2004) as in the case of Ptychodiscus (Fig. 3g). Kofoid (1907) and other authors (Paulsen 1949, Taylor 1976, Dodge
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1982, Sournia 1986) illustrated the cingulum of P. noctiluca with vertically oriented cingular ridges. Species such as K. brevis are characterized by vertically oriented cingular ridges (Haywood et al. 2004). Other species such as K. umbella de Salas, Bolch et Hallegraeff showed furrows in the episome (de Salas et al. 2004), which can be also observed in the form P. inflatus (Fig. 3j). The anteroposteriorly flattening of Ptychodiscus is unusual, rarely found in unarmored dinoflagellates (i.e., Gymnodinium opressum W. Conrad). While the form P. noctiluca is highly anteroposteriorly compressed (Fig. 5, k–o), its life stage P. inflatus is globular or slightly compressed (Figs. 2n; 5, e–f). The degree of dorsoventrally compression is variable between the species of Karenia (Haywood et al. 2004). During incubation experiments, the small cells of the form P. inflatus appeared as a life stage of P. noctiluca. The cultures of Karenia also showed small and large cells (Haywood et al. 2004). Unfortunately, attempts to establish a permanent culture of Ptychodiscus have so far failed. The paucity of material renders it difficult to conduct ultrastructural analyses on the nature of the chloroplasts, and especially on the cell covering. In the SSU rDNA molecular phylogeny, P. noctiluca branched between clades of Brachidinium–Karenia and Karlodinium– Takayama (Fig. S1 and Fig. 4). However, the general tree topology is poorly resolved, rendering uncertain the relationship between Ptychodiscus and these fucoxanthin-containing genera. The Ptychodiscales have been established for unarmored dinoflagellates with a thick pellicle (Fensome et al. 1993, Adl et al. 2012). The molecular phylogeny reveals that the order Ptychodiscales has artificially grouped thecate dinoflagellates (Kolkwitziella, Herdmania), naked dinoflagellates with thick cell covering (Balechina, Cucumeridinium), and other unarmored dinoflagellates with typical cell coverings (Brachidinium, Ceratoperidinium). Other genera lacking molecular data have been placed within Ptychodiscales, such as Amphilothus Kofoid ex Poche, Achradina Lohmann, Berghiella, Lophodinium, or Sclerodinium. However, the morphology of these insufficiently known genera is distantly related to Ptychodiscus. This research is supported by the Brazilian Conselho Nacional de Desenvolvimento Cientıfico e Tecnol ogico (grant numbers BJT 370646/2013–14 to F.G., and 402759/2012–5 and 311936/2013–0 to R.M.L.) and the United States National Science Foundation (grant number EF–0629624 to S.L.). Adl, S. M., Simpson, A. G. B., Lane, C. E., Lukes, J., Bass, D., Bowser, S. S., Brown, M.et al. 2012. The revised classification of eukaryotes. J. Eukaryot. Microbiol. 59:429–514. Balech, E. 1962. Tintinnoinea y Dinoflagellata del Pacıfico seg un material de las expediciones Norpac y Downwind del Instituto Scripps de Oceanografıa. Rev. Mus. Arg. C. Nat. ‘B. Rivadavia’ Zool. 7:1–253. Balech, E. 1967. Dinoflagelados nuevos o interesantes del Golfo de Mexico y Caribe. Rev. Mus. Arg. C. Nat. ‘B. Rivadavia’ Hidrobiologia 2:77–126.
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Supporting Information Additional Supporting Information may be found in the online version of this article at the publisher’s web site: Figure S1. Bayesian phylogenetic tree of dinoflagellate SSU rDNA sequences, based on 1,402 aligned positions. Name in bold represents
MORPHOLOGY AND PHYLOGENY OF PTYCHODISCUS
sequence obtained in this study. Numbers at nodes are posterior probabilities. Accession numbers are provided between brackets. The scale bar represents the number of substitutions for a unit branch length.
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Video S1. Morphology of Ptychodiscus noctiluca, https://youtu.be/drhTBaauTvw.
