J. Phycol.

52, 793–805 (2016)
© 2016 Phycological Society of America
DOI: 10.1111/jpy.12438


o mez2
Fernando G
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

The planktonic dinoflagellate Ptychodiscus noctiluca within the short-branching dinokaryotic
combined distinctive morphological features such as dinoflagellates as an independent lineage with
a disk-shaped anteroposteriorly compressed cell affinity to Brachidinium/Karenia and Karlodinium/
body and an apical carina, together with a flexible Takayama in a generally poorly resolved clade. Our
and tough cell covering, suggesting intermediate results indicated that the order Ptychodiscales,
characteristics between thecate and naked established for unarmored dinoflagellates with a
dinoflagellates. Ptychodiscus noctiluca was examined strongly developed pellicle, has artificially grouped
by light, epifluorescence, and scanning electron thecate dinoflagellates (Kolkwitziella, Herdmania),
microscopy from specimens collected in the naked dinoflagellates with thick cell covering
Mediterranean Sea and the North and South (Balechina, Cucumeridinium) and other insufficiently
Atlantic Ocean. Ptychodiscus noctiluca showed a known unarmored genera with typical cell coverings
straight apical groove that bisected the carina, a cell (Brachidinium, Ceratoperidinium).
covering with a polygonal surface reticulum, nucleus Key index words: alveolates; amphiesma; Atlantic
without capsule, sulcal intrusion in the episome, Ocean; Dinophyta; Gymnodiniales; Mediterranean
sulcal ventral flange, and yellowish-green Sea; Peridiniales; phytoplankton; ptychodiscacean;
chloroplasts that are shared characters with thick pellicle; unarmored Dinoflagellata
Brachidinium/Karenia. The cell division was the
typical binary fission of gymnodinioid Abbreviations: BI, Bayesian inference; bp, base pairs
dinoflagellates, although exceptionally in an oblique
transversal axis. We examined the intraspecific
variability during incubation experiments. In the Two major groups of dinoflagellates can be
fattened cells, termed as Ptychodiscus carinatus, distinguished based on their cell coverings. Thecate
chloroplasts transformed into dark granules, and (armored) dinoflagellates with large amphiesmal
the cell acquired the swollen and smaller stage, vesicles filled with cellulosic material, and the athe-
termed as P. inflatus. Ptychodiscus carinatus, cate (unarmored or “naked”) dinoflagellates contain
P. inflatus, and Diplocystis antarctica are synonyms of hundreds of alveoli lacking cellulosic material
P. noctiluca. Molecular phylogeny based on the SSU (Dodge 1971). The naked dinoflagellates are usually
rDNA sequence revealed that Ptychodiscus branched fragile and delicate, and lyse under poor fixation
conditions. However, the separation between
armored and unarmored species is not clear cut,
Received 14 December 2015. Accepted 19 May 2016.
Author for correspondence: e-mail fernando.gomez@fitoplancton.
and the first example was Ptychodiscus noctiluca F.
com. Stein. von Stein (1883) examined the stomach con-
Editorial Responsibility: R. Wetherbee (Associate Editor) tents of alcohol-preserved salps. This method only

794  EZ ET AL.

preserved the dinoflagellates with thecal plates. Balech 1962, G omez 2007, Kim et al. 2013). Stei-
However, P. noctiluca F. Stein was preserved despite dinger (1979) transferred the red-tide species Gymno-
the apparent absence of thecal plates. von Stein dinium breve C.C. Davis (now Karenia brevis [C.C.
illustrated P. noctiluca in apical and antapical views Davis] Gert Hansen et Moestrup) into the genus Pty-
as a cell consisting of anterior and posterior circular chodiscus. Her proposal, based on the resemblance of
disks connected by a well-marked cingulum. He the apical lamella with the carina of K. brevis,
reported that the episome was the smaller half with received no further acceptance in the literature.
an apical lamella-like structure, while the hyposome In earlier classifications, Ptychodiscus was placed as
was larger showing a notch that corresponded to the only genus of its own family. Lindemann (1928)
the sulcus. Murray and Whitting (1899) reported placed Ptychodiscus in the family Kolkwitziellaceae
P. noctiluca and proposed a new variety. However, within the Peridiniales. Schiller (1937) also placed
their illustrations corresponded to a thecate Ptychodiscus within the Peridiniales and included
dinoflagellate (Diplopsalis-like or a flattened cell of Kolkwitziella Er. Lindemann, Berghiella Kofoid et J.R.
Protoperidinium Bergh, see Paulsen 1949). Cleve Michener, and Lophodinium Lemmermann within
(1901) described Diplocystis antarctica Cleve as a cyst the Ptychodiscaceae. Gaarder (1954) suggested an
from alcohol-preserved samples collected in the affinity with Gymnodiniales based on the line draw-
southern Atlantic and Indian Oceans. Kofoid (1907) ings of dividing cells reported by Cleve (1901).
described Ptychodiscus carinatus Kofoid from the Dodge (1984) placed Ptychodiscus within the family
equatorial Pacific Ocean. He did not compare the of Gymnodinium F. Stein. Sournia (1986) and Chr eti-
new species with P. noctiluca and reported that the ennot-Dinet et al. (1993) also placed Ptychodiscus
episome was the larger half of the cell, while the within the Gymnodiniales. In contrast, Taylor (1987)
hyposome was smaller and showed the carina (= placed Ptychodiscus within the order Kolkwitziellales,
keel sensu Kofoid). In the NW Mediterranean Sea, family Kolkwitziellaceae as a thecate dinoflagellate.
Pavillard (1916) observed one individual of P. cari- Fensome et al. (1993) proposed the new order Pty-
natus, and he described P. inflatus Pavillard from a chodiscales including Balechina Loeblich et A.R. Loe-
single individual that showed a swollen morphology blich, Brachidinium F.J.R. Taylor, Ceratoperidinium
when compared to the flattened P. carinatus. Pavil- Margalef, and Kolkwitziella among other genera. Stei-
lard also considered the smaller half of the cell with dinger and Tangen (1997) also placed Herdmania
the carina as the hyposome. Schiller (1937) J.D. Dodge within the Ptychodiscaceae. Recently, Adl
reported these three taxa as valid species of Pty- et al. (2012) placed Balechina, Ptychodiscus, and Sclero-
chodiscus F. Stein and also included the Murray and dinium J.D. Dodge within the Ptychodiscales.
Whitting’s misidentification in P. noctiluca. Schiller While there are molecular data for most of the
maintained the orientation with the carina in the above-mentioned genera (Henrichs et al. 2011, Yam-
hyposome as reported by Kofoid and Pavillard. Paul- aguchi et al. 2011, Re~ ne et al. 2013, Gomez et al.
sen (1949) found P. inflatus near the Faroe Islands. 2015, Mertens et al. 2015), the lack of molecular
He pointed out that previous illustrations in ven- data of Ptychodiscus, the type of the order Ptychodis-
trodorsal view by Kofoid and Pavillard were upside cales, renders it impossible to clarify the phyloge-
down, and he considered the smaller half with the netic relationships within the Ptychodiscales. In
carina as the episome. Gaarder (1954) reported addition, P. noctiluca possesses morphological fea-
P. inflatus in several locations of the temperate tures such as a cell covering with intermediate char-
Atlantic Ocean. She agreed with the orientation acteristics between naked and thecate
proposed by Paulsen (1949) and considered dinoflagellates that are important for understanding
D. antarctica as a synonym of P. inflatus. Boalch dinoflagellate evolution. In this article, we describe
(1969) provided the first micrographs and made the the morphology and intraspecific variability of
first attempts to culture P. noctiluca from live cells P. noctiluca by light, epifluorescence, and scanning
collected in the English Channel. He confirmed electron microscopy and present the first molecular
that the smaller half with the carina corresponded phylogenetic data.
unequivocally to the episome. Boalch observed the
cell covering was flexible, without surface markings,
and the dimensions and cell shape changed when MATERIALS AND METHODS
preserved. Boalch (1969) concluded that P. noc- Sampling, isolation, and light microscopy. Specimens were col-
tiluca, P. carinatus, P. inflatus, and D. antarctica were lected from the Mediterranean Sea by slowly filtering surface
different morphotypes of the same species, and seawater taken from the pier of the Station Marine
P. inflatus was a preservation artifact. d’Endoume at Marseille, France (43°160 48.05″ N, 5°200
Ptychodiscus noctiluca is widely distributed from ner- 56.22″ E, bottom depth 3 m) from October 2007 to Septem-
itic to oceanic waters and from tropical to cold water ber 2008. A strainer of 20, 40, or 60-lm mesh size was used
to collect planktonic organisms from water volumes ranging
areas such as the Mediterranean Sea (G omez 2003), between 10 and 100 L, depending on particle concentration.
the Atlantic Ocean (Balech 1967, 1988, Steidinger The plankton concentrate was scanned in settling chambers
and Williams 1970, Dodge 1993), the Indian Ocean at 9100 magnification with an inverted microscope (Nikon
(Taylor 1976), and the Pacific Ocean (Rampi 1950, Eclipse TE200; Nikon Inc., Tokyo, Japan). Cells were

photographed alive at 9200 or 9400 magnification with a Incubations. Specimens of P. noctiluca from Brazil were iso-
Nikon Coolpix E995 digital camera. Further specimens were lated with the aim to establish cultures. Cells were isolated
collected using the same method from October 2008 to using a micropipette and placed in 12-well tissue culture plate
August 2009 in the surface waters (depth of 2 m) of the port with 0.2 lm-filtered seawater collected that day from the
of Banyuls sur Mer, France (42°280 50″ N, 3°080 09″ E). The same locality, and they were supplemented with f/2 medium
concentrated sample was examined in Uterm€ ohl chambers without silicates. Two days later, the healthy specimens were
with an inverted epifluorescence microscope (Olympus IX51; re-isolated and placed into a 6-well tissue culture plate with
Olympus Inc., Tokyo, Japan) and photographed with an f/2 medium made with filtered and sterilized seawater. Sev-
Olympus DP71 digital camera. Sampling continued from eral variations of this protocol were tested. In all the cases,
September 2009 to February 2010 in the Bay of Villefranche the culture plates were placed in an incubator used for
sur Mer, France. For this location, sampling was performed at microalgae culturing, at 23°C, 100 lmol photons  m2  s1
the long-term monitoring site Point B (43°410 10″ N, 7°190 from cool-white tubes and photoperiod 12:12 L:D.
00″ E, water column depth ~80 m). Water column samples As the attempts to establish long-term cultures of P. noc-
(0–80 m) were obtained using a phytoplankton net (53 lm tiluca were unsuccessful, the specimens used for molecular
mesh size, 54 cm diameter, 280 cm length). Samples were analysis were isolated from natural samples. The specimens
prepared according to the same procedure as described were micropipetted individually with a fine capillary into a
above, and specimens were observed with an inverted micro- clean chamber and washed several times in a series of drops
scope (Olympus IX51; Olympus Inc.) and photographed with of 0.2 lm-filtered and sterilized seawater. Finally, 50 speci-
an Olympus DP71 digital camera. Sampling continued from mens collected in S~ao Sebasti~ao Channel on April 22, 2013,
May 2012 to February 2013 in the port of Valencia, Spain were placed in a 0.2 mL tube filled with absolute ethanol.
(39°270 38.13″ N, 0°190 21.29″ W, water column depth of The sample was kept at room temperature and in darkness
4 m). Specimens were obtained using a phytoplankton net until the molecular analysis could be performed.
(20 lm mesh size). Samples were prepared according to the Scanning electron microscopy. Samples were collected from
same procedure as described above, and specimens were the North-East Atlantic Ocean as reported in Dodge (1993).
observed with an inverted microscope (Nikon Eclipse T2000; Lugol’s iodine and formaldehyde-preserved samples were
Nikon Inc.) and photographed with an Olympus DP71 digital examined by light microscopy at low magnification. Cells of
camera. Ptychodiscus were picked out of the drop of sample by using a
In addition, samples were collected during the BOUM micro-pipette. The cells were placed in the well of a brass con-
(Biogeochemistry from the Oligotrophic to the Ultra-oligo- tainer, the bottom of which was formed by a Nuclepore filter
trophic Mediterranean) cruise on board R/V L’Atalante from (Whatman, Brentford, UK) clamped into place. After collect-
the south of France to the south of Cyprus (20 June–18 July ing a number of cells, an acetone series was flushed through
2008). Seawater samples were collected with Niskin bottles to dehydrate the cells and the chambers was closed with
from 30 stations. At each station, 6 depths were sampled another filter at the top. The container was then placed in a
between 5 and 125 m, with an additional sample at 250 m critical point apparatus (Polaron, Watford, UK), and drying
depth. These samples were preserved with acid Lugol’s iodine was carried out using liquid carbon oxide. The filters were
solution and stored at 5°C. Samples of 500 mL were concen- mounted on 13 mm diameter stubs and coated with gold-pal-
trated via sedimentation in glass cylinders. The top 450 mL ladium in a Polaron cool sputter coater. Finally, the specimens
of sample was slowly siphoned off with small-bore tubing dur- were examined in a JEOL JSM 25S scanning electron micro-
ing 6 d. The remaining 50 mL of concentrate, representing scope (JEOL Ltd., Tokyo, Japan), usually run at 15 kV.
500 mL whole water, was then settled in composite settling DNA extraction, PCR amplification of small subunit rRNA gene,
chambers. The sample was examined in Uterm€ ohl chambers and sequencing. Prior to DNA extraction, the tube was cen-
at 9100 magnification with a Nikon inverted microscope trifuged for 10 min at 500 g to settle the Ptychodiscus cells, and
(Nikon Eclipse TE200; Nikon Inc.), and the specimens were the ethanol was aspirated. One hundred microliters of DNA
photographed with a digital camera (Nikon Coolpix E995; lysis buffer (0.1 M ethylenediaminetetraacetic acid pH 8.0, 1%
Nikon Inc.). Sodium Dodecyl Sulfate (SDS), 200 lg  mL1 proteinase K)
In the South Atlantic Ocean, sampling continued after was used to re-suspend the cell pellet; the resuspension was
March 2013 in S~ao Sebasti~ao Channel (23°500 4.05″ S, 45°240 transferred into a 1.5 mL tube, and the process was repeated
28.82″ W), and from December 2013 to December 2015 off for five times. Then, the resultant 0.5 mL was incubated for
Ubatuba (23°320 20.15″ S, 45°50 58.94″ W). The Brazilian 48 h at 55°C. DNA extraction and purification followed a previ-
specimens were obtained using a phytoplankton net (20 lm ously reported protocol (Qiu et al. 2011). At the end of the
mesh size) in surface waters. The living concentrated samples extraction process, DNA of Ptychodiscus was eluted in 50 lL
were examined in Uterm€ ohl chambers at magnification of Tris-HCl solution. Next, 1 lL of the extracted DNA was PCR
9200 with inverted microscopes (Diaphot-300; Nikon Inc. at amplified. The small subunit (SSU) rDNA, ~1,800 base pairs,
S~ao Sebasti~ao, and Eclipse TS-100; Nikon Inc. and Olympus was amplified using the primers Dino18SF1 and 18ScomR1
IX73; Olympus Inc. at Ubatuba), and photographed with a (Qiu et al. 2013). The PCR amplifications were carried out in
digital camera (Cyber-shot DSC-W300; Sony, Tokyo, Japan) 25 lL reaction volumes containing 0.125 lL of TaKaRa Ex Taq
mounted on the microscope’s eyepiece. For further morpho- HS in the PCR master mix (TaKaRa Bio, Dalian, China), both
logical studies using an epifluorescence microscope, live spec- forward and reverse primers (final concentration 0.2 lM) and
imens were transported to the University campus (~250 km template DNA. Thermocycling conditions included a denatur-
far away from the coastal laboratory). The stressed live cells ing step of 94°C for 4 min; 35 cycles of 94°C for 30 s, 56°C for
were examined using an Olympus BX51 epifluorescence 30 s, 72°C for 45 s, and a final extension step of 72°C, 10 min.
microscope equipped with an Olympus DP72 camera (Olym- PCR products were resolved by agarose gel electrophoresis with
pus Inc.). In order to determinate the position and shape of the DL2000 DNA ladder (TaKaRa Bio), and the bands with
the nucleus and presence of thecal plates, the cells fixed with expected sizes were excised to remove primer dimers. DNA was
glutaraldehyde (5% final concentration) were stained with purified and directly sequenced as previously reported (Qiu
DAPI (40 ,60 diamino-2-phenylindole, 0.1 lg  mL1 final con- et al. 2011).
centration; Sigma, St. Louis, MO, USA) and Calcofluor White Phylogenetic analyses. Two phylogenetic analyses were per-
M2R (Fluorescent Brightener 28; Sigma) and examined with formed. The first was based on nearly complete SSU rDNA
the same microscope.
796  EZ ET AL.

sequences, comprising dinokaryotic sequences most similar to Information). The cells of the most common mor-
P. noctiluca, as identified through BLAST search (http:// photype, P. noctiluca/P. carinatus, were anteroposte-
blast.ncbi.nlm.nih.gov/Blast.cgi), complete sequences of spe- riorly compressed with a length/width ratio ~1:4
cies classified within the Ptychodiscales (Balechina, Brachi-
dinium, Kolkwitziella, Herdmania, Ceratoperidinium, and formed by two disk-shaped halves joined by the
Cucumeridinium F. G omez, P. Lopez-Garcıa, H. Takayama et well-marked cingulum (Fig. 1, g–h). The cell dimen-
D. Moreira), and a wide selection of dinokaryotes represent- sions typically ranged from 50 to 90 lm width
ing the major orders including Peridiniales and Gymno- (transdiameter), and sporadically specimens of less
diniales. To improve the resolution of trees and clarify the than 50 lm wide were observed co-existing with the
relationships between Ptychodiscus and the other species, the typical specimens (Fig. 2a).
second analysis included additional sequences from Karenia
and Karlodinium and removed sequences from Kolkwitziella
The disk-shaped halves, episome and hyposome,
and Cucumeridinium. The new SSU rDNA sequence of Pty- were not circular. The depth was ~90% smaller than
chodiscus was aligned with sequences of dinokaryotic dinoflag- the width giving a slightly ellipsoidal shape with a
ellates collected from GenBank database in ClustalX, using ventrodorsal compression. The diameter of the epi-
default parameters (Larkin et al. 2007). Ambiguously aligned some was ~85% that of the hyposome (Figs. 1, a–b,
regions and gaps were removed manually from the initial e–f; 2, e–f). The episome was flattened with a slight
alignment using Bioedit 7.0 (Hall 1999), leaving a final align-
ment of 1,402 characters in both cases that was used to con-
concave anterior face and a carina that extended
struct phylogenetic trees. The GTR + I + G evolutionary radially along the 2/3 of the episome toward the
model was selected by Modeltest v3.7 (Posada and Crandall ventral side (Fig. 1, g–i) with a semicircular shape
1998) and used for Bayesian inference (BI) analysis. The BI in lateral view (Figs. 1h and 2x). The carina was not
analysis was conducted using primarily MrBayes 3.1.2. Four exactly located along the ventrodorsal axis, but dis-
simultaneous chains were run for 8,500,000 generations, with placed toward the left side of the cell (Figs. 1, a, e;
sampling every 100 generations, and the first 21,250 (25%) 2, b–d). The cingulum was descending, wide and
trees discarded as burn-in. The remaining trees were used to
calculate posterior probabilities using a majority rule consen- deep, except in the ventral side at the sulcus level
sus. Our sequence was deposited in DDBJ/EMBL/GenBank (Figs. 1p; 2, n and p). The typical dinoflagellate
under accession number KU640194. transverse flagellum encircled the cell along the cin-
gulum, and the flagellar pore was located in the
anterior end of the sulcus (Figs. 1d and 2f; see
Video S1).
Morphology. Ptychodiscus noctiluca was occasionally In addition to the carina, the most distinctive
detected in the Mediterranean Sea and the Atlantic structure was the sulcus. In apical or antapical views,
Ocean. After the analysis of 212 Lugol’s iodine fixed the sulcus formed a notch or indentation in the
samples from the open Mediterranean Sea, the contour of the hyposome (Figs. 1, a–b; 2b). The sul-
records of P. noctiluca were restricted to two speci- cus formed a groove with a rounded posterior end
mens from a sample collected at 75 m depth and extended for about 1/4 the hyposome diame-
between Menorca and Sardinia. During the observa- ter. The sulcus was not exactly located along the
tions of live samples in the coastal NW Mediter- dorsoventral axis, but was displaced toward the right
ranean Sea (Marseille, Banyuls sur Mer, Villefranche side of the cell (Figs. 1b; 2, d–e, r). The posterior
sur Mer, and Valencia), the occurrence of the spe- end of the sulcus harbored the relatively short
cies was occasional and restricted to a few specimens (~30 lm long), longitudinal flagellum (Fig. 1, c–d,
per day. Consequently, no clear temporal pattern see Video S1).
was found although the occurrence appeared to be During the observations with an inverted micro-
more frequent in winter season. scope, the specimens settled into two positions: (i)
In the South Atlantic Ocean at the coasts of S~ao Antapical position, with the flattened hyposome
Paulo State, P. noctiluca also appeared occasionally entirely touching the bottom glass. The hyposome
in any period of the year. Some days several tens of can be observed in the frontal focus (Fig. 1b), while
specimens appeared in the samples, while later the the episome with the carina appeared in the rear
species was absent for several months. It should be focus of the cell (Fig. 1a); and (ii) Apical position,
noted that under low magnification, the usually the main cell body was inclined with the carina and
immobile cells of Ptychodiscus, a flattened cell with a the dorsal side of the hyposome touching the bot-
circular contour with a yellowish-green chloroplasts, tom glass. The carina and the dorsal hyposome were
could be mistaken for drum-shaped diatoms by non- observed in the frontal focus (Fig. 1e), while the
trained observers. ventral side of the cell and sulcus in the rear focus
There were no apparent morphological differ- (Fig. 1f).
ences between the specimens of the Mediterranean The cell of recently collected specimens showed a
Sea (Fig. 1) and Brazil (Fig. 2). The specimens flattened morphology and numerous small rounded
showed chloroplasts in recently collected live sam- or slightly ellipsoidal chloroplasts, as revealed by
ples (Figs. 1, a–n; 2, b–e). The live specimens of epifluorescence microscopy (Figs. 1, j–m; 2, g–h).
P. noctiluca tended to remain immobile settling in The chloroplasts had a yellowish-green pigmenta-
the apical–antapical plane, and occasionally they tion (Figs. 1, e–f; 2, b–e). The nucleus was hardly
swam slowly (see Video S1 in the Supporting visible using light microscopy (Fig. 2z). Under

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, respec-
tively, 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) Ventro-
antapical 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 glu- ethanol, and Lugol’s iodine). While the plates of
taraldehyde and stained with DAPI, the nucleus was thecate dinoflagellates decompose after treatment
ellipsoidal and located in the left dorsal side of the with sodium hypochlorite, the cell covering of Pty-
cell (Fig. 2i). Other glutaraldehyde-fixed specimens chodiscus also resisted the treatment with sodium
were stained with Calcofluor White in other to test hypochlorite, although the cell strongly deformed
the presence of thecal plates. No thecal plates were and acquired an unrecognizable globular shape
observed and the cell covering using light micro- (Fig. 2l). When the specimens were fixed with
scopy appeared as a continuous layer. Individuals Lugol’s iodine, the cell covering was slightly stained
were squashed, and the cell covering was tough and when compared to the cell contents (Fig. 2m). Pty-
flexible, and changed its dimension and shape. The chodiscus did not show the dark brown color of the
cell covering resembled a plastic bag, and the carina cellulosic thecal plates of armored dinoflagellates.
and cingulum appeared as creases (Fig. 2, j–k). The In ethanol-preserved samples, the cell acquired a
cell covering resisted treatment with the most com- swollen shape that corresponded to P. inflatus or
mon preservatives (formaldehyde, glutaraldehyde, more precisely to D. antarctica (Fig. 1, o–q).
798  EZ ET AL.

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) Glutaralde-
hyde-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 indi-
vidual. (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 begin-
ning 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.

The dominant morphotype was the flattened one cingular width (Fig. 3e). The sulcus showed a
P. noctiluca/P. carinatus, while the form described as closed intrusion into the episome (Fig. 3e) and was
P. inflatus was rarer. Consequently, the form P. infla- oriented toward the right hyposome (Fig. 3, f–h). A
tus was not only an artifact of sample preservation, ventral ridge or flange appeared in the sulcus
as live healthy specimens with the morphology of between the ends of the cingulum (Fig. 3f). The
the form P. inflatus appeared in natural samples cell surface was smooth with hexagonal amphiesmal
(Fig. 2o). The P. inflatus morphology was also vesicles and scattered pores in depressed areas
found in dead specimens devoid of cell contents (Fig. 3i). One specimen with the morphology of the
(Fig. 2, p–r). This suggested that under unfavorable form P. inflatus was observed. The cell surface
conditions prior to the cell death or due to the showed irregular longitudinal furrows (Fig. 3j). The
preservation, the flattened individuals transformed deep and wide cingulum conserved the transversal
into the morphology known as P. inflatus. The form flagellum (Fig. 3k).
P. inflatus was usually smaller in width and larger in Molecular phylogeny. Initial BLAST comparisons
length (Fig. 2, a and o). showed that the closest identified relatives in the
Cell division. The cytokinesis began as notch that database were dinokaryotes, such as the thecate
appeared in the left dorsal side (Fig. 2, u–w) in the dinoflagellates Pentapharsodinium tyrrhenicum
form of P. inflatus or in the dorsal side of the hypo- (Balech) Montresor, Zingone et D. Marino
some in the form of P. carinatus (Fig. 2y). The (AF022201) and Pentapharsodinium sp. (AF274270).
cytokinesis extended toward the right ventral side. We studied the phylogenetic position of P. noctiluca
Cell division was desmoschitic and oblique along in two SSU rDNA phylogenetic trees. The trees con-
the transversal axis. One daughter cell received the tained a variety of dinoflagellate sequences, espe-
carina (Fig. 2ab) and the other one the sulcus cially sequences of species classified within
(Fig. 2aa). The two daughter cells remained joined Ptychodiscales, as well as Gymnodiniales and Peri-
in the right ventral side of the daughter cell that diniales. A first tree built to include all the available
received the carina (Figs. 1o; 2z). Both daughter sequences of ptychodiscaceans showed that the
cells showed their transversal flagella while joined sequences of the ptychodiscaceans Balechina, Brachi-
(see Video S1). dinium, Ceratoperidinium, Cucumeridinium, Kolk-
Incubations and intraspecific variability. The speci- witziella, or Herdmania were not related to
mens of Ptychodiscus showed numerous chloroplasts Ptychodiscus or even to each other (Fig. S1 in the
(Figs. 1, a and b; 2, e and h). There was no evi- Supporting Information). A second tree was built
dence of food vacuoles that could suggest a hetero- using almost complete sequences excluding the
trophic nutrition. Specimens were isolated with the long-branch sequence of Kolkwitziella (Fig. 4). The
aim to establish permanent cultures. However, the sequence of P. noctiluca branched within the short-
attempts were unsuccessful and the cells did not sur- branching sequences of the dinokaryotic core as a
vive more than 10 d. Some cells divided within the lineage between the clades of Brachidinium–Karenia
first days (with filtered seawater supplemented with and Karlodinium–Takayama, without support (Fig. 4).
f/2 medium). The larger and flattened specimens
(P. noctiluca/P. carinatus) transformed into the smal-
ler swollen stage (P. inflatus; Fig. 2a). The yellowish-
green chloroplasts progressively disappeared, and Ptychodiscus noctiluca appears to be relatively eury-
dark granules appeared in the cell center (Fig. 2, a thermal, tolerating a range from tropical waters
and f). The f/2 medium successfully used for peri- (Kofoid 1907, Rampi 1950, this study) to cold waters
dinin-containing dinoflagellates (Heterocapsa F. of the Atlantic Ocean (Paulsen 1949) or sub-Antarc-
Stein, Coolia Meunier, Prorocentrum Ehrenberg) tic waters (Cleve 1901). Ptychodiscus noctiluca has
failed with P. noctiluca. never been observed to reach high abundances in
Scanning electron microscopy. The cell covering of the natural marine environments. Its records are
P. noctiluca appeared as a deflated layer and the bor- episodic despite it being a photosynthetic species liv-
ders of the cingulum as folds (Fig. 3, a–b). Some ing in the euphotic zone.
vertical folds appeared in the cingulum. However, it Our observations of Ptychodiscus from natural sam-
was difficult to discern whether they were a result of ples and the incubation experiments agree with
the cell deflating or corresponded to truly vertical Boalch (1969) who concluded that P. noctiluca,
ridges in the cingulum (Fig. 3, a and b). The carina P. carinatus, P. inflatus, and D. antarctica were mor-
appeared as a radial semicircular crest oriented photypes of the same species. All subsequent reports
along the ventrodorsal axis (Fig. 3, a–c). The carina agreed with Boalch, with the exception of Balech
emerged from the right side of the sulcal intrusion (1988), who considered P. carinatus as an indepen-
and was bisected by a groove (Fig. 3c). The straight dent species based on formaldehyde-preserved sam-
apical groove showed rolled margins (Fig. 3d). The ples. Boalch (1969) considered the swollen stage,
episome showed scattered pores (Fig. 3a) and P. inflatus, as a preservation artifact. In fact, the pre-
groups of pores in the antapex (Fig. 3, e and g). served cells tended to resemble P. inflatus, and for
The cingulum was descending and displaced about that reason, most of the records in the literature
800  EZ ET AL.

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 ven-
tral 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. in-
flatus. (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 the sulcus with the longitudinal flagellum that runs
1954, Balech 1962, 1967, 1988, Taylor 1976). How- along it. His sulcal groove is in fact the apical
ever, healthy cells with the morphology of the form groove (Fig. 3c) and no flagellum runs along it (see
P. inflatus can be found in natural samples Video S1). Kofoid (1907) did not cite surface mark-
(Fig. 2o); although in some cases, such swollen ings in the species description. However, his illustra-
stage is an indicator of stressed or moribund speci- tions showed longitudinal striae or ridges in the
mens (Fig. 2, p–r). In the attempts to culture P. noc- cingulum and the carina (Fig. 5c). Pavillard (1916)
tiluca, we have observed that the healthy and described P. inflatus from a single specimen
chloroplast-rich flattened specimens of P. noctiluca/ (Fig. 5d). It is possible that he examined formalde-
P. carinatus converted themselves into smaller and hyde-preserved material because the morphology
swollen cells of the form P. inflatus lacking the yel- coincided with that described by authors examining
lowish-green pigmentation (Fig. 2a). formaldehyde-preserved samples such as Gaarder
The description by von Stein (1883) was quite (fig. 5f), Taylor (1976) (fig. 5g), and Balech (1988)
precise, although restricted to the apical–antapical (fig. 5j). Following Kofoid (1907), other authors
view (Fig. 5a). Cleve (1901) described Ptychodiscus have illustrated P. noctiluca with longitudinal striae
(as D. antarctica) as a cyst. However, he illustrated in the cingulum (Paulsen 1949, fig. 5e; Taylor 1976,
the cyst under binary division (Fig. 5b). The fig. 5g; Dodge 1982, fig. 5h; Sournia 1986, fig. 5i).
description of P. carinatus by Kofoid (1907) It is difficult to observe the surface of the cingulum
reported that the ventral edge of the carina acts as in the anterior–posteriorly compressed cell of

Syndinium turbo DQ146404
Hematodinium perezi EF065718
0.60 Brachidinium capitatum HM066998
Karenia bidigitata HM067002
Karenia mikimotoi EF492505
Karenia mikimotoi AF009131
0.99 Karenia mikimotoi FR865627
Karenia selliformis HM067007
Karenia brevis FJ587219
0.94 Karenia brevis EF492503
Karenia papilionacea HM067005
0.80 Karlodinium veneficum EF036540
Karlodinium veneficum AY245692
1 Karlodinium veneficum AF172712
Karlodinium veneficum EF492506
Karlodinium veneficum JN986577
1 Takayama acrotrocha HM067010
Takayama cf. pulchellum AY800130
1 1 Dinophysis acuta AJ506973
FIG. 4. Bayesian phylogenetic Phalacroma rotundatum AJ506975
tree of dinoflagellate SSU rDNA 1 Gyrodinium fusiforme AB120002
sequences, based on 1,402 Gyrodinium spirale AB120001
Duboscquodinium collinii HM483398
aligned positions. Name in bold 1 Scrippsiella sweeneyae AF274276
represents sequence obtained in 0.83 Scrippsiella trochoidea EF492513
this study. Numbers at nodes are Peridinium aciculiferum EF417314
posterior probabilities. Accession 0.86 Peridinium willei EF375879
Peridinium cf. centenniale EF058237
numbers are provided between 1 Parvodinium umbonatum GU001637
brackets. The scale bar represents 1 Peridinium cinctum EF058243
the number of substitutions for a Peridinium willei EF375879
unit branch length. 1 Durinskia capensis AB271107
1 Durinskia baltica AF231803
0 .7 1 Galeidinium rugatum AB195668
4 Kryptoperidinium foliaceum EF492508
1 Gymnodinium fuscum AF022194
Polykrikos geminatum JX967270
0.86 Herdmania litoralis AB564305
1 Amphidiniopsis dragescoi AY238479
Archaeperidinium minutum GQ227501
1 Heterocapsa circularisquama LC054932
1 Heterocapsa niei EF492499
0.55 Heterocapsa rotundata AF274267
Heterocapsa triquetra GU594638
1 Polarella glacialis EF434275
Symbiodinium microadriaticum M88521
0.99 Prorocentrum mexicanum EF492510
1 Prorocentrum micans AY585526
Prorocentrum minimum DQ336072 0.1
tychodidiscus nocti
tiluca KU640194

P. noctiluca. These ridges were not evident in our cell covering showed a polygonal surface reticulum
LM observations, although the form P. inflatus (Fig. 3i). This pattern of amphiesmal vesicles can be
showed irregular surface ridges under SEM observa- found in gymnodinioid dinoflagellates such as Kare-
tions (Fig. 3j). nia (de Salas et al. 2004).
The cell covering of Ptychodiscus is able to resist As consequence of the unusual cell covering,
the preservation with formaldehyde and even etha- specimens of Ptychodiscus appeared in samples fixed
nol (Fig. 1, o–q). The appearance of the ethanol- with formaldehyde or ethanol in which most of the
preserved cells of Ptychodiscus is identical to naked dinoflagellates were lost. In the classifica-
D. antarctica (Figs. 1, p–r; 5b). Fensome et al. (1993, tions, Ptychodiscus has moved between Peridiniales
p. 54) defined the ptychodiscaceans as cells with cel- and Gymnodiniales. Fensome et al. (1993) erected
lulose as the principal component of the pellicle. the order Ptychodiscales within the unarmored
When fixed with Lugol’s iodine, the cell covering dinoflagellates. Fensome et al. placed Kolkwitziella
did not show the dark brown color of thecate within Ptychodiscales. However, Kolkwitziella is a the-
dinoflagellate plates (Fig. 2m, G omez 2007), which cate dinoflagellate closely related to Protoperidinium
suggests the absence or scarcity of cellulosic mate- excentricum (Paulsen) Balech (Mertens et al. 2015,
rial in the cell covering. Gaarder (1954) reported Fig. S1). Steidinger and Tangen (1997) placed Herd-
that the cell covering of Ptychodiscus could be mania within the Ptychodiscaceae. However, Herdma-
entirely dissolved by sodium hypochlorite. However, nia is a thecate dinoflagellate closely related to
we have observed that after the addition of sodium other thecate dinoflagellates such as Archaeperi-
hypochlorite, the cell covering remained, although dinium Jørgensen (Yamaguchi et al. 2011; Fig. S1).
strongly deformed (Fig. 2l). We did not observe the- Fensome et al. (1993) and Adl et al. (2012) also
cal plates in the cell covering after staining with Cal- placed the unarmored genus Balechina within the
cofluor White. The cell covering is tough and Ptychodiscales. The species of Balechina have been
flexible (Fig. 2, j–k). Using high magnification, the recently split into two genera, Balechina with thick
802  EZ ET AL.

a b

c d e

f g

h i j

k l m o
n n

n ca

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 dinoflagellates (Cerato = horn, usually associated
Cucumeridinium with well-marked longitudinal ridges with a rigid structure, and “Peridinium”). Loeblich
(Gomez et al. 2015). Balechina, Cucumeridinium, and (1982) classified Ceratoperidinium as a thecate
Ptychodiscus are distantly related among each other dinoflagellate within the Peridiniales. However,
in the SSU rDNA molecular phylogeny (Fig. S1 and Ceratoperidinium is an unarmored dinoflagellate
Fig. 4). The placement of Brachidinium and (Gomez et al. 2004, Re~ ne et al. 2013). The molecu-
Ceratoperidinium within the Ptychodiscales was due to lar data have revealed that Ceratoperidinium is a close
insufficient information on these genera. In the relative of the species formerly known as Gyrodinium
case of Ceratoperidinium, the etymology of the name falcatum Kofoid et Swezy and several species classi-
is confusing because it refers to thecate fied as Cochlodinium F. Sch€ utt (Re~ne et al. 2013).

These taxa do not possess the thick pellicle that 1982, Sournia 1986) illustrated the cingulum of
defines the ptychodiscaceans. P. noctiluca with vertically oriented cingular ridges.
The morphology suggests a relationship between Species such as K. brevis are characterized by verti-
Ptychodiscus and Brachidinium–Karenia. The transfer cally oriented cingular ridges (Haywood et al.
of Gymnodinium breve (now Karenia brevis) into Pty- 2004). Other species such as K. umbella de Salas,
chodiscus as P. brevis (C.C. Davis) Steidinger was not Bolch et Hallegraeff showed furrows in the episome
accepted further (Steidinger 1979). Fensome et al. (de Salas et al. 2004), which can be also observed in
(1993) did not place Gymnodinium breve within Pty- the form P. inflatus (Fig. 3j).
chodiscales. However, they placed Brachidinium The anteroposteriorly flattening of Ptychodiscus is
within the Ptychodiscales. Brachidinium was first unusual, rarely found in unarmored dinoflagellates
described from a formaldehyde-preserved sample (i.e., Gymnodinium opressum W. Conrad). While the
(Taylor 1963), and consequently, it was assumed form P. noctiluca is highly anteroposteriorly com-
that the cell possesses a thick pellicle. Unarmored pressed (Fig. 5, k–o), its life stage P. inflatus is glob-
dinoflagellates may occasionally resist the formalde- ular or slightly compressed (Figs. 2n; 5, e–f). The
hyde preservation, but the cell shape is strongly degree of dorsoventrally compression is variable
deformed and the lack of distinctive features makes between the species of Karenia (Haywood et al.
the identification impossible even at the genus level. 2004). During incubation experiments, the small
In the case of Brachidinium, it was recognized due to cells of the form P. inflatus appeared as a life stage
its distinctive body extensions (Taylor 1963). Fur- of P. noctiluca. The cultures of Karenia also showed
ther studies have revealed that Brachidinium is clo- small and large cells (Haywood et al. 2004). Unfor-
sely related, if not a synonym, of the unarmored tunately, attempts to establish a permanent culture
genus Karenia (G omez et al. 2005, Henrichs et al. of Ptychodiscus have so far failed. The paucity of
2011, fig. 4). However, Karenia and Brachidinium do material renders it difficult to conduct ultrastruc-
not possess a thick pellicle, and the cell covering tural analyses on the nature of the chloroplasts, and
does not differ from that of other typical naked especially on the cell covering. In the SSU rDNA
dinoflagellates (de Salas et al. 2004). molecular phylogeny, P. noctiluca branched between
Daugbjerg et al. (2000) defined Karenia as unar- clades of Brachidinium–Karenia and Karlodinium–
mored dinoflagellates whose major carotenoid is Takayama (Fig. S1 and Fig. 4). However, the general
fucoxanthin, nucleus without a capsule and cells tree topology is poorly resolved, rendering uncer-
with a straight apical groove. The chloroplasts of Pty- tain the relationship between Ptychodiscus and these
chodiscus show a distinctive yellowish-green pigmen- fucoxanthin-containing genera.
tation that could be an indicator of the presence of The Ptychodiscales have been established for unar-
fucoxanthin (Fig. 1, e–f). In this study, we show a mored dinoflagellates with a thick pellicle (Fensome
straight apical groove that bisects the carina of Pty- et al. 1993, Adl et al. 2012). The molecular phylogeny
chodiscus (Fig. 3, c–d). The apical groove of P. noc- reveals that the order Ptychodiscales has artificially
tiluca showed rolled margins as in K. papilionacea grouped thecate dinoflagellates (Kolkwitziella, Herdma-
A.J. Haywood et Steidinger (Fig. 3d; Haywood et al. nia), naked dinoflagellates with thick cell covering
2004). The ventral surface of the apical groove (Balechina, Cucumeridinium), and other unarmored
meets the extension of the sulcal intrusion as in spe- dinoflagellates with typical cell coverings (Brachi-
cies of Karenia (Fig. 3c; Haywood et al. 2004). In dinium, Ceratoperidinium). Other genera lacking
Ptychodiscus and Karenia, the nucleus is spherical to molecular data have been placed within Ptychodis-
slightly oval, without capsule, and located in the left cales, such as Amphilothus Kofoid ex Poche, Achradina
side of the cell (Fig. 2i; Haywood et al. 2004). The Lohmann, Berghiella, Lophodinium, or Sclerodinium.
diagnostic characters of Karenia as defined by Daug- However, the morphology of these insufficiently
bjerg et al. (2000) fit with Ptychodiscus. known genera is distantly related to Ptychodiscus.
The cingulum of Karenia and Ptychodiscus is
descending and displaced 1–1.59 the cingular width This research is supported by the Brazilian Conselho Nacional
(Fig. 3e; Haywood et al. 2004). Both genera showed de Desenvolvimento Cientıfico e Tecnol ogico (grant numbers
concavities in the cell surface. The species of Astero- BJT 370646/2013–14 to F.G., and 402759/2012–5 and
dinium Sournia, Karenia, and Karlodinium showed a 311936/2013–0 to R.M.L.) and the United States National
Science Foundation (grant number EF–0629624 to S.L.).
sulcal structure traversing the proximal and distal
ends of the cingulum (Haywood et al. 2004, G omez
Adl, S. M., Simpson, A. G. B., Lane, C. E., Lukes, J., Bass, D., Bow-
et al. 2005). We found a similar structure in Pty- ser, S. S., Brown, M.et al. 2012. The revised classification of
chodiscus (Fig. 3f). The cell surface of Karenia and eukaryotes. J. Eukaryot. Microbiol. 59:429–514.
the flattened form of P. noctiluca are smooth with Balech, E. 1962. Tintinnoinea y Dinoflagellata del Pacıfico seg un
polygonal amphiesmal vesicles (Fig. 3i, de Salas material de las expediciones Norpac y Downwind del Insti-
et al. 2004). Groups of pores can be found in spe- tuto Scripps de Oceanografıa. Rev. Mus. Arg. C. Nat. ‘B. Riva-
davia’ Zool. 7:1–253.
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sequence obtained in this study. Numbers at Video S1. Morphology of Ptychodiscus noctiluca,
nodes are posterior probabilities. Accession num- https://youtu.be/drhTBaauTvw.
bers are provided between brackets. The scale bar
represents the number of substitutions for a unit
branch length.