J. Phycol.

52, 793805 (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

o mez2
Fernando G

Carmen Campos Panisse 3, E-11500, Puerto de Santa Mar
a, 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 Universit
aria, 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
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
1
Received 14 December 2015. Accepted 19 May 2016.
2
Author for correspondence: e-mail fernando.gomez@fitoplancton.
com.
Editorial Responsibility: R. Wetherbee (Associate Editor)
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).
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 athe-
cate (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 con-
tents of alcohol-preserved salps. This method only

793
794
F E R N A N D O G OM
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. cari-
natus, and he described P. inflatus Pavillard from a
single individual that showed a swollen morphology
when compared to the flattened P. carinatus. Pavil-
lard also considered the smaller half of the cell with
the carina as the hyposome. Schiller (1937)
reported these three taxa as valid species of Pty-
chodiscus F. Stein and also included the Murray and
Whittings misidentification in P. noctiluca. Schiller
maintained the orientation with the carina in the
hyposome as reported by Kofoid and Pavillard. Paul-
sen (1949) found P. inflatus near the Faroe Islands.
He pointed out that previous illustrations in ven-
trodorsal 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. noc-
tiluca, 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 ner-
itic 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,

EZ ET AL.
Balech 1962, G
omez 2007, Kim et al. 2013). Stei-
dinger (1979) transferred the red-tide species Gymno-
dinium breve C.C. Davis (now Karenia brevis [C.C.
Davis] Gert Hansen et Moestrup) into the genus Pty-
chodiscus. 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 draw-
ings of dividing cells reported by Cleve (1901).
Dodge (1984) placed Ptychodiscus within the family
of Gymnodinium F. Stein. Sournia (1986) and Chr
eti-
ennot-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 Pty-
chodiscales including Balechina Loeblich et A.R. Loe-
blich, Brachidinium F.J.R. Taylor, Ceratoperidinium
Margalef, and Kolkwitziella among other genera. Stei-
dinger and Tangen (1997) also placed Herdmania
J.D. Dodge within the Ptychodiscaceae. Recently, Adl
et al. (2012) placed Balechina, Ptychodiscus, and Sclero-
dinium J.D. Dodge within the Ptychodiscales.
While there are molecular data for most of the
above-mentioned genera (Henrichs et al. 2011, Yam-
aguchi et al. 2011, Re~ n
e et al. 2013, G
omez et al.
2015, Mertens et al. 2015), the lack of molecular
data of Ptychodiscus, the type of the order Ptychodis-
cales, renders it impossible to clarify the phyloge-
netic relationships within the Ptychodiscales. In
addition, P. noctiluca possesses morphological fea-
tures such as a cell covering with intermediate char-
acteristics 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 col-
lected from the Mediterranean Sea by slowly filtering surface
seawater taken from the pier of the Station Marine
dEndoume at Marseille, France (43160 48.05 N, 5200
56.22 E, bottom depth 3 m) from October 2007 to Septem-
ber 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 (42280 50 N, 3080 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 (43410 10 N, 7190
00 E, water column depth ~80 m). Water column samples
(080 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 micro-
scope (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
(39270 38.13 N, 0190 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-oligo-
trophic Mediterranean) cruise on board R/V LAtalante from
the south of France to the south of Cyprus (20 June18 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 Lugols iodine
solution and stored at 5C. Samples of 500 mL were concen-
trated via sedimentation in glass cylinders. The top 450 mL
of sample was slowly siphoned off with small-bore tubing dur-
ing 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 (23500 4.05 S, 45240
28.82 W), and from December 2013 to December 2015 off
Ubatuba (23320 20.15 S, 4550 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 microscopes eyepiece. For further morpho-
logical studies using an epifluorescence microscope, live spec-
imens 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 (Olym-
pus 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 con-
centration; Sigma, St. Louis, MO, USA) and Calcofluor White
M2R (Fluorescent Brightener 28; Sigma) and examined with
the same microscope.
795
Incubations. Specimens of P. noctiluca from Brazil were iso-
lated 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. Sev-
eral variations of this protocol were tested. In all the cases,
the culture plates were placed in an incubator used for
microalgae culturing, at 23C, 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. noc-
tiluca 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 speci-
mens 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).
Lugols 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 con-
tainer, the bottom of which was formed by a Nuclepore filter
(Whatman, Brentford, UK) clamped into place. After collect-
ing 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-pal-
ladium in a Polaron cool sputter coater. Finally, the specimens
were examined in a JEOL JSM 25S scanning electron micro-
scope (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 cen-
trifuged 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 55C. DNA extraction and purification followed a previ-
ously 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 denatur-
ing step of 94C for 4 min; 35 cycles of 94C for 30 s, 56C for
30 s, 72C for 45 s, and a final extension step of 72C, 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 per-
formed. The first was based on nearly complete SSU rDNA
796
F E R N A N D O G OM
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 spe-
cies classified within the Ptychodiscales (Balechina, Brachi-
dinium, Kolkwitziella, Herdmania, Ceratoperidinium,
Cucumeridinium F. G
omez, P. L
opez-Garc
a, H. Takayama et
D. Moreira), and a wide selection of dinokaryotes represent-
ing the major orders including Peridiniales and Gymno-
diniales. 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 Pty-
chodiscus was aligned with sequences of dinokaryotic dinoflag-
ellates 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 align-
ment of 1,402 characters in both cases that was used to con-
struct 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 consen-
sus. 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 Lugols iodine fixed
samples from the open Mediterranean Sea, the
records of P. noctiluca were restricted to two speci-
mens from a sample collected at 75 m depth
between Menorca and Sardinia. During the observa-
tions of live samples in the coastal NW Mediter-
ranean Sea (Marseille, Banyuls sur Mer, Villefranche
sur Mer, and Valencia), the occurrence of the spe-
cies 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 non-
trained observers.
There were no apparent morphological differ-
ences between the specimens of the Mediterranean
Sea (Fig. 1) and Brazil (Fig. 2). The specimens
showed chloroplasts in recently collected live sam-
ples (Figs. 1, an; 2, be). The live specimens of
P. noctiluca tended to remain immobile settling in
the apicalantapical plane, and occasionally they
swam slowly (see Video S1 in the Supporting

EZ ET AL.
Information). The cells of the most common mor-
photype, P. noctiluca/P. carinatus, were anteroposte-
riorly compressed with a length/width ratio ~1:4
and formed by two disk-shaped halves joined by the
well-marked cingulum (Fig. 1, gh). The cell dimen-
sions 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 epi-
some was ~85% that of the hyposome (Figs. 1, ab,
ef; 2, ef). 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, gi) with a semicircular shape
in lateral view (Figs. 1h and 2x). The carina was not
exactly located along the ventrodorsal axis, but dis-
placed toward the left side of the cell (Figs. 1, a, e;
2, bd). 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 cin-
gulum, 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, ab; 2b). The sul-
cus formed a groove with a rounded posterior end
and extended for about 1/4 the hyposome diame-
ter. The sulcus was not exactly located along the
dorsoventral axis, but was displaced toward the right
side of the cell (Figs. 1b; 2, de, r). The posterior
end of the sulcus harbored the relatively short
(~30 lm long), longitudinal flagellum (Fig. 1, cd,
see Video S1).
During the observations with an inverted micro-
scope, 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 bot-
tom 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, jm; 2, gh).
The chloroplasts had a yellowish-green pigmenta-
tion (Figs. 1, ef; 2, be). The nucleus was hardly
visible using light microscopy (Fig. 2z). Under
 MORPHOLOGY AND PHYLOGENY OF PTYCHODISCUS 797

FIG. 1. Light micrographs of Ptychodiscus noctiluca from the NW Mediterranean Sea. (an) Live cells. (oq) Ethanol-preserved cells.
(ab) 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. (ei) Several views of the same individual. (e, f) Apical view. (g, h)
Lateral view. (i) Oblique apical view. (jm) 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. (oq) 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-
taraldehyde 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 micro-
scopy 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, jk). The
cell covering resisted treatment with the most com-
mon preservatives (formaldehyde, glutaraldehyde,
ethanol, and Lugols iodine). While the plates of
thecate dinoflagellates decompose after treatment
with sodium hypochlorite, the cell covering of Pty-
chodiscus 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
Lugols iodine, the cell covering was slightly stained
when compared to the cell contents (Fig. 2m). Pty-
chodiscus 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, oq).
798
EZ ET AL.
F E R N A N D O G OM

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. (bd) 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) Lugols 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. (pr) Dead individual of the form P. inflatus. Note the rigid cell covering. (tab) Division of three different individuals.
(tx) 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. (zab) 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. infla-
tus 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, pr). 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, uw) in the
form of P. inflatus or in the dorsal side of the hypo-
some 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 speci-
mens of Ptychodiscus showed numerous chloroplasts
(Figs. 1, a and b; 2, e and h). There was no evi-
dence of food vacuoles that could suggest a hetero-
trophic nutrition. Specimens were isolated with the
aim to establish permanent cultures. However, the
attempts were unsuccessful and the cells did not sur-
vive 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 smal-
ler swollen stage (P. inflatus; Fig. 2a). The yellowish-
green chloroplasts progressively disappeared, and
dark granules appeared in the cell center (Fig. 2, a
and f). The f/2 medium successfully used for peri-
dinin-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 bor-
ders of the cingulum as folds (Fig. 3, ab). 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, ac). 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
799
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, fh). 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 con-
tained a variety of dinoflagellate sequences, espe-
cially sequences of species classified within
Ptychodiscales, as well as Gymnodiniales and Peri-
diniales. A first tree built to include all the available
sequences of ptychodiscaceans showed that the
sequences of the ptychodiscaceans Balechina, Brachi-
dinium, Ceratoperidinium, Cucumeridinium, Kolk-
witziella, 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 short-
branching sequences of the dinokaryotic core as a
lineage between the clades of BrachidiniumKarenia
and KarlodiniumTakayama, without support (Fig. 4).
DISCUSSION
Ptychodiscus noctiluca appears to be relatively eury-
thermal, tolerating a range from tropical waters
(Kofoid 1907, Rampi 1950, this study) to cold waters
of the Atlantic Ocean (Paulsen 1949) or sub-Antarc-
tic 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 liv-
ing in the euphotic zone.
Our observations of Ptychodiscus from natural sam-
ples and the incubation experiments agree with
Boalch (1969) who concluded that P. noctiluca,
P. carinatus, P. inflatus, and D. antarctica were mor-
photypes of the same species. All subsequent reports
agreed with Boalch, with the exception of Balech
(1988), who considered P. carinatus as an indepen-
dent species based on formaldehyde-preserved sam-
ples. Boalch (1969) considered the swollen stage,
P. inflatus, as a preservation artifact. In fact, the pre-
served cells tended to resemble P. inflatus, and for
that reason, most of the records in the literature
800
EZ ET AL.
F E R N A N D O G OM

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. (ei) 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 (ah, j), 1 lm (i, k).

corresponded to that form (Paulsen 1949, Gaarder
1954, Balech 1962, 1967, 1988, Taylor 1976). How-
ever, 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 speci-
mens (Fig. 2, pr). In the attempts to culture P. noc-
tiluca, 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 yel-
lowish-green pigmentation (Fig. 2a).
The description by von Stein (1883) was quite
precise, although restricted to the apicalantapical
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 mark-
ings in the species description. However, his illustra-
tions 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 formalde-
hyde-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 anteriorposteriorly compressed cell of
 MORPHOLOGY AND PHYLOGENY OF PTYCHODISCUS
Hematodinium perezi EF065718
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.
P. noctiluca. These ridges were not evident in our
LM observations, although the form P. inflatus
showed irregular surface ridges under SEM observa-
tions (Fig. 3j).
The cell covering of Ptychodiscus is able to resist
the preservation with formaldehyde and even etha-
nol (Fig. 1, oq). The appearance of the ethanol-
preserved cells of Ptychodiscus is identical to
D. antarctica (Figs. 1, pr; 5b). Fensome et al. (1993,
p. 54) defined the ptychodiscaceans as cells with cel-
lulose as the principal component of the pellicle.
When fixed with Lugols 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 mate-
rial 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 the-
cal plates in the cell covering after staining with Cal-
cofluor White. The cell covering is tough and
flexible (Fig. 2, jk). Using high magnification, the
801
Syndinium turbo DQ146404
0.60 Brachidinium capitatum HM066998
Karenia bidigitata HM067002
Karenia mikimotoi EF492505
1
Karenia mikimotoi AF009131
0.99 Karenia mikimotoi FR865627
1
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
1
Karlodinium veneficum JN986577
1 Takayama acrotrocha HM067010
Takayama cf. pulchellum AY800130
1 1 Dinophysis acuta AJ506973
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
0.86 Peridinium willei EF375879
Peridinium cf. centenniale EF058237
1 Parvodinium umbonatum GU001637
1 Peridinium cinctum EF058243
Peridinium willei EF375879
1 Durinskia capensis AB271107
1 Durinskia baltica AF231803
0.97
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
Ptychodiscus
Pty
tychodidiscus nocti
noctiluca
tiluca KU640194
cell covering showed a polygonal surface reticulum
(Fig. 3i). This pattern of amphiesmal vesicles can be
found in gymnodinioid dinoflagellates such as Kare-
nia (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 classifica-
tions, 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 the-
cate dinoflagellate closely related to Protoperidinium
excentricum (Paulsen) Balech (Mertens et al. 2015,
Fig. S1). Steidinger and Tangen (1997) placed Herd-
mania within the Ptychodiscaceae. However, Herdma-
nia is a thecate dinoflagellate closely related to
other thecate dinoflagellates such as Archaeperi-
dinium Jrgensen (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
802
EZ ET AL.
F E R N A N D O G OM

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). (ko) 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~ n
e et al. 2013). The molecu-
lar data have revealed that Ceratoperidinium is a close
relative of the species formerly known as Gyrodinium
falcatum Kofoid et Swezy and several species classi-
fied as Cochlodinium F. Sch utt (Re~n
e 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 BrachidiniumKarenia. The transfer
of Gymnodinium breve (now Karenia brevis) into Pty-
chodiscus as P. brevis (C.C. Davis) Steidinger was not
accepted further (Steidinger 1979). Fensome et al.
(1993) did not place Gymnodinium breve within Pty-
chodiscales. 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 formalde-
hyde 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). Fur-
ther studies have revealed that Brachidinium is clo-
sely 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 unar-
mored dinoflagellates whose major carotenoid is
fucoxanthin, nucleus without a capsule and cells
with a straight apical groove. The chloroplasts of Pty-
chodiscus show a distinctive yellowish-green pigmen-
tation that could be an indicator of the presence of
fucoxanthin (Fig. 1, ef). In this study, we show a
straight apical groove that bisects the carina of Pty-
chodiscus (Fig. 3, cd). The apical groove of P. noc-
tiluca 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 spe-
cies 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 Daug-
bjerg et al. (2000) fit with Ptychodiscus.
The cingulum of Karenia and Ptychodiscus is
descending and displaced 11.59 the cingular width
(Fig. 3e; Haywood et al. 2004). Both genera showed
concavities in the cell surface. The species of Astero-
dinium 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 Pty-
chodiscus (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 spe-
cies 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
803
1982, Sournia 1986) illustrated the cingulum of
P. noctiluca with vertically oriented cingular ridges.
Species such as K. brevis are characterized by verti-
cally 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 com-
pressed (Fig. 5, ko), its life stage P. inflatus is glob-
ular or slightly compressed (Figs. 2n; 5, ef). 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). Unfor-
tunately, attempts to establish a permanent culture
of Ptychodiscus have so far failed. The paucity of
material renders it difficult to conduct ultrastruc-
tural 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 BrachidiniumKarenia and Karlodinium
Takayama (Fig. S1 and Fig. 4). However, the general
tree topology is poorly resolved, rendering uncer-
tain the relationship between Ptychodiscus and these
fucoxanthin-containing genera.
The Ptychodiscales have been established for unar-
mored 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, Herdma-
nia), naked dinoflagellates with thick cell covering
(Balechina, Cucumeridinium), and other unarmored
dinoflagellates with typical cell coverings (Brachi-
dinium, Ceratoperidinium). Other genera lacking
molecular data have been placed within Ptychodis-
cales, 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 Cient
fico e Tecnol
ogico (grant numbers
BJT 370646/201314 to F.G., and 402759/20125 and
311936/20130 to R.M.L.) and the United States National
Science Foundation (grant number EF0629624 to S.L.).
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Supporting Information
Additional Supporting Information may be
found in the online version of this article at the
publishers 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 805

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.