Cysttheca relationship of a new dinoflagellate with a spiny round

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Phycological Research 2015; 63: 110–124
doi: 10.1111/pre.12083
Cyst-theca relationship of a new dinoflagellate with a spiny
round brown cyst, Protoperidinium lewisiae sp. nov., and its
comparison to the cyst of Oblea acanthocysta
Kenneth Neil Mertens,1* Yoshihito Takano,2 Haifeng Gu,3 Aika Yamaguchi,4 Vera Pospelova,5 Marianne Ellegaard6 and
Kazumi Matsuoka2
1
Research Unit for Palaeontology, Gent University, Gent, Belgium, 2Institute for East China Sea Research (ECSER),
Nagasaki University, Nagasaki, 4Research Center for Inland Seas, Kobe University, Kobe, Japan, 3Third Institute of
Oceanography, SOA, Xiamen, China, 5School of Earth and Ocean Sciences, University of Victoria, Victoria, British
Columbia, Canada, and 6Department of Plant and Environmental Sciences, University of Copenhagen, Frederiksberg,
Denmark
SUMMARY
Round spiny brown cysts with apiculocavate processes were
isolated from sediments of Lake Saroma, Japan, Changle
Harbor, East China Sea, China, Jinzhou Harbor, Bohai Sea,
China, and San Pedro Harbor, California, USA. Superficially
similar round spiny brown cysts of the species, Oblea
acanthocysta were, for comparison, restudied through light
microscopy and scanning electron microscopy (SEM) and by
sequencing of small subunit (SSU) and large subunit (LSU)
rDNA obtained through a single cyst from Lake Saroma. These
morphological measurements and SEM observations showed
that the new cysts can be discriminated from O. acanthocysta
by the archeopyle, number of processes, shape of process
bases and its apiculocavate processes. Based on LSU
sequences, the most closely related species was Protoperidinium monovelum, for which no cyst stage has been
described so far. However, the thecal morphology of the specimens found in this study differed from P. monovelum in
details of the sulcal plates and shape of apical pore and 2a
plate. We therefore describe Protoperidinium lewisiae sp.
nov., which can be found in estuarine subtropical to temperate
waters of the Pacific Ocean.
Key words: Bohai Sea, Changle Harbor, East China Sea,
Jinzhou Harbor, Lake Saroma, large subunit rDNA, San Pedro
Harbor, small subunit rDNA.
INTRODUCTION
Free-living marine dinoflagellates form a very large and
diverse group of planktonic organisms, currently encompassing 1555 species (Gómez 2005). About 13 to 16% of living
dinoflagellates form so-called resting cysts as part of their
sexual cycle (Head 1996). These cysts can be linked to their
respective motile stage by incubation experiments (Wall &
Dale 1968) and conversely, motile stages may be induced to
form cysts in culture. Molecular phylogenetic analyses based
on either cultured strains or single-cell polymerase chain
reaction (PCR) have helped to elucidate taxonomic relation-
ships within this group (e.g. Bolch 2001; Matsuoka et al.
2006; Takano & Horiguchi 2006; Matsuoka & Head 2013).
Many new cyst species are still being discovered (e.g.
Verleye et al. 2011; Mertens et al. 2014), which is important as dinoflagellate cysts are frequently used for
paleoclimate reconstructions (e.g. Mertens et al. 2009;
Price et al. 2013).
Some dinoflagellate cysts have a distinct round, spinebearing shape and are brown in color, and are therefore
informally designated as ‘round brown spiny cysts’ (see
review in Radi et al. 2013). The paleontological cyst-based
taxonomy has erected two genera to classify these species.
Cysts that belong to the genus Echinidinium (Zonneveld
1997) open with a theropylic archeopyle (Head et al. 2001),
i.e. an angular slit that follows paraplate boundaries but
without complete release of plates (Matsuoka 1988). The
second genus, Islandinium was erected by Head et al.
(2001) and opens with a saphopylic archeopyle, i.e. having
a free operculum (Matsuoka 1988). At species level, characteristics of processes and wall texture are useful distinguishing features of round brown spiny cysts (e.g. Zonneveld
1997; Head et al. 2001; Mertens et al. 2012b; Radi et al.
2013).
Cyst-motile relationships have been established only for a
few species of ‘round brown spiny cysts’: Protoperidinium
monospinum (Paulsen) Zonneveld et Dale (Zonneveld and
Dale 1994), Oblea acanthacysta Kawami, Iwataki et Matsuoka
(Kawami et al. 2006; =cyst of Diplopelta parva (Abé)
Matsuoka sensu Matsuoka 1988), P. tricingulatum Kawami,
van Wezel, Koeman et Matsuoka (Kawami et al. 2009), different types of Archaeperidinium minutum (Kofoid) Jørgensen
– previously called P. minutum (Kofoid) Loeblich III; the
genus Archaeperidinium was reinstated by Yamaguchi et al.
(2011) – and the closely related species A. saanichi Mertens,
*To whom correspondence should be addressed.
Email: kenneth.mertens@ugent.be
Communicating editor: Mona Hoppenrath
Received 6 June 2014; accepted 4 October 2014.
© 2015 Japanese Society of Phycology
Cyst-theca relation of P. lewisiae
Yamaguchi, Kawami et Matsuoka (both reviewed in Mertens
et al. 2012b), P. haizhouense Liu, Gu et Mertens (Liu et al.
2014), Islandinium minutum (Harland et Reid) Head,
Harland et Matthiessen (Potvin et al. 2013) and P. fukuyoi
Mertens, Head, Pospelova et Matsuoka (Mertens et al. 2013).
With the advent of molecular techniques, it has become
increasingly apparent that cyst characteristics such as the
archeopyle and thecate characteristics such as details of the
sulcal plates and cingular plates are of taxonomic importance
(Matsuoka & Kawami 2013; Mertens et al. 2013; Liu et al.
2014).
These round brown spiny cysts are also of specific interest
for paleoclimatological studies since their distributions are
restricted by temperature, salinity, trophic stage and sea-ice
cover (Radi et al. 2013). Some species, in particular
I. minutum, Echinidinium karaense Head, Harland et
Matthiessen and Islandinium? cezare s.l. (de Vernal et al. ex
de Vernal in Rochon et al., 1999) Head, Harland et
Matthiessen, have distinct cold-water distributions (Head
et al. 2001) and have been associated with sea-ice cover (e.g.
de Vernal et al. 2013; Potvin et al. 2013). Others, e.g.
E. bispiniformum Zonneveld, have a distinct warm-water distribution (Zonneveld 1997).
Here we describe the cyst-theca relationship for a new
round spiny brown cyst, by germinating cysts from surface
sediments from China, Japan and USA. We show that this cyst
is produced by a new species, P. lewisiae, and that the cyst
has a theropylic archeopyle. We restudied the superficially
similar cyst of O. acanthocysta that occurs in some of the
same localities to clarify the morphological differences
between them. In addition, we obtained large subunit (LSU)
ribosomal DNA (rDNA) sequences from single cells of
P. lewisiae and small subunit (SSU) and LSU rDNA sequences
from single cysts of O. acanthocysta.
MATERIAL AND METHODS
Morphological analyses
Sediment samples containing round spiny brown cysts were
collected from Lake Saroma, Hokkaido, Japan, Changle
Harbor, East China Sea, China, Jinzhou Harbor, Bohai Sea,
China and San Pedro Harbor California, USA (Fig. 1, Table 1).
Fig. 1. Map showing sampling locations
for culture experiments and sediment
sampling.
© 2015 Japanese Society of Phycology
111
All samples were stored in plastic bags in a refrigerator at 4°C.
In situ sea surface salinities and sea surface temperatures
were measured during sampling (Table 1).
Approximately 0.5–1.0 cm3 of wet sediment was immersed
in filtered seawater and after 1 min of sonication using an
sonic bath US-2R (As One, Osaka, Japan), the immersion was
rinsed through a 20 μm metallic-meshed calibrated sieve
(Sanpo, Osaka, Japan) using filtered seawater. From this
residue, the cyst fraction was separated using
sodiumpolytungstate (SPT) at a density of 1.3 g cm−1 (Bolch
1997). For samples from China, SPT was not used. Single
cysts were then transferred to 0.5 mL microwells (Thermo
Scientific, Waldham, MA, USA) subjected to an irradiance of
100 μmol photons m−2 s−1 and 24-h light, and filled with
Erd-Schreiber Modified medium (Watanabe et al. 2000) at
temperatures and salinities comparable to the respective
natural environments (Table 1). Cysts were regularly checked
for germination and observations of the cells were performed
under an inverted light microscope (LM) IX70 (Olympus,
Tokyo, Japan). Encysted and excysted cysts and vegetative
stages (stained with calcofluor) were photographed and measured using an Olympus BX51 LM with a digital sight module
DS-1L 1 (Nikon, Tokyo, Japan). For every cyst, the body
diameter and the length of the five longest processes were
measured.
Sediment samples containing cysts of Oblea acanthocysta
were collected using a Tokyo University Fisheries Oceanography Lab (TFO) gravity corer from the type locality, Omura Bay,
Japan (Kawami et al. 2006; Table 1). Palynological techniques were used for processing (Mertens et al. 2012a). The
samples were oven-dried at 40°C and then treated with room
temperature 10% HCl to remove calcium carbonate particles.
Material was rinsed twice with distilled water. To dissolve
siliceous particles, samples were treated with 48–50% roomtemperature hydrofluoric acid for 2 days, and then treated for
10 min with room-temperature HCl (10%) to remove
fluorosilicates. The residue was rinsed twice with distilled
water, ultrasonicated for 30 s and finally collected on a 20 μm
mesh. Aliquots of residue were mounted in glycerine jelly.
Measurements were the same as above.
For scanning electron microscope (SEM) observation,
residue from Lake Saroma and Omura Bay was washed with
distilled water and dehydrated in a graded ethanol series (30
to 100% in six steps), critical-point-dried with CO2 (CPD
24-Aug-12
28-Nov-11
28-Apr-11
25-Aug-11
25-Aug-11
32°
25°
40°
33°
33°
55.50′
50.42′
43.62′
44.07′
44.53′
N
N
N
N
N
129°
119°
121°
118°
118°
51.25′
44.77′
03.00′
16.12′
14.87′
E
E
E
W
W
11.1
13
10
7
5
NA
NA
NA
32.2
32.2
NA
NA
NA
20.2
20.2
TFO corer
Grab
Grab
Petite Ponar Grab
Petite Ponar Grab
Y. Takano &
K. Mertens
A. Morinaga
H. Gu
H.Gu
V. Pospelova
V. Pospelova
TFO corer
17.1
32.2
17.3
143° 49.59′ E
44° 07.53′ N
B
C
D
E
F
Omura Bay St. 1, East China Sea, Japan
Changle Harbor St. T21, East China Sea, China
Jinzhou Harbor St. N16, Bohai Sea, China
San Pedro Harbor St. 1 (SCMI docks, Fish Harbor), CA, USA
San Pedro Harbor St. 2 (Old Sea Plane Anchorage), CA, USA
22-Jul-11
21-Jul −11; 02& 10-Aug-11
NA
10-Oct-12
17-Mar-12
31-Aug-11
1-Sep-11
A
Lake Saroma (lagoon) St. 1, Okhotsk Sea, Japan
Sampling
date
Station
name on
the map
Sampling site
Date of isolation
Latitude
Longitude
Water
depth
(m)
SSS
(psu)
SST
(°C)
Sampling device
Sampled by
K. N. Mertens et al.
Table 1. Site location, name on the map, date of cyst isolation, sediment sampling date, latitude, longitude, water depth (m), sea surface salinity (SSS), sea surface temperature (SST),
sampling device and name of person who did the sampling
112
Bal-Tec 030), glued onto a stub, sputter-coated with
platinum/palladium for 90 s using JFC-2300 HR (JEOL,
Tokyo, Japan) and examined using a SEM JEOL 6330F.
Molecular phylogenetic analyses
Cells were isolated after germination of cysts from Jinzhou
Harbor, Bohai Sea, China. Identified cells were rinsed several
times in sterilized distilled water, broken by squeezing the
coverslip above, and then transferred into a PCR tube. The
single cell was used as the template to amplify about 1430 bp
of the LSU rDNA (D1-D6 domains), using the primers D1R
(Scholin et al. 1994) and 28-1483R (Daugbjerg et al. 2000).
The PCR protocol was identical to the one followed by Liu
et al. (2014).
Surface samples were collected from Lake Saroma, Hokkaido, Japan (TFO core sample, sampled on 22 July 2011)
(Fig. 1). Cysts were isolated from the sediment using heavy
liquid separation as described by Bolch (1997). We used
cysts from Lake Saroma for molecular analyses. Isolated
cysts were sonicated in a 200 μL PCR tube with sterilized
seawater. The cysts and the cells were individually transferred to a glass slide covered with a frame of vinyl tape, and
photographed using an Olympus BX51 microscope with an
Olympus DP71 digital camera. The cell was transferred to an
inverted microscope and crushed with a fine glass needle,
and subsequently transferred into a 200 μL PCR tube containing 3 μL of Milli-Q water. We determined partial
sequences of SSU rDNA and LSU rDNA from two singlecysts. The PCR protocol was identical to the one followed by
Mertens et al. (2012c).
SSU rDNA sequences were aligned manually using Mesquite version 2.75 (Maddison & Maddison 2011) based on
the datasets of Horiguchi et al. (2012). The final alignment of
SSU rDNA dataset consisted of 51 taxa and contained 1588
base pairs (Oblea acanthocysta, Accession number
LC005409). LSU rDNA sequences were aligned based on the
dataset of Mertens et al. (2013). The final alignment of the
dataset consisted of 57 taxa and contained 490 base pairs
(Protoperidinium lewisiae, Accession number LC005410).
The apicomplexan Neospora caninum Dubey et al. was used
as an outgroup species for both datasets. The alignments are
available from the authors upon request. Phylogenetic trees
were constructed using maximum likelihood (ML) and Bayesian analysis. The GTR + I + G substitution model was chosen
by the Akaike information criterion implemented in
jModelTest 2.1.4. (Guindon & Gascuel 2003; Darriba et al.
2012) and used for all the analyses. For ML, the datasets were
analyzed by GARLI version 0.951 (Zwickl 2006). Bootstrap
analyses were carried out for ML with 100 replicates (SSU
rDNA dataset) and 500 replicates (LSU rDNA dataset).
MrBayes version 3.2.2 was used to perform Bayesian analyses
on both datasets (Huelsenbeck & Ronquist 2001; Ronquist &
Huelsenbeck 2003). The program was set to operate four
Monte-Carlo-Markov chains starting from a random tree. A
total of 1 000 000 generations (SSU rDNA) and 2 500 000
generations (LSU rDNA) were calculated with trees sampled
every 100 generations. The first 2500 (SSU) and 6250 (LSU
rDNA) trees in each run were discarded as burn-in. Posterior
probabilities (PP) correspond to the frequency at which a
given node was found in the post-burn-in trees.
© 2015 Japanese Society of Phycology
Cyst-theca relation of P. lewisiae
RESULTS
Results of germination experiments
Spiny round brown cysts were isolated from surface sediment
samples and germinated after one or two days of incubation.
Based on morphological characters of this cyst and the corresponding motile stage, as well as LSU DNA sequencebased phylogenetic analysis a new species, here assigned
to Protoperidinium lewisiae sp. nov. is described below. Cells
never divided and died relatively quickly, a few days after
germination.
Species descriptions
Division DINOFLAGELLATA (Bütschli 1885) Fensome et al.
1993
Class DINOPHYCEAE Pascher 1914
Subclass PERIDINIPHYCIDAE Fensome et al. 1993
Order PERIDINIALES Haeckel 1894
Family PROTOPERIDINIACEAE Balech 1988 nom. cons.
Subfamily PROTOPERIDINIOIDEAE (Autonym)
Genus Protoperidinium Bergh 1881
Protoperidinium lewisiae K.N. Mertens, Y. Takano, A.
Yamaguchi, H. Gu, V. Pospelova, M. Ellegaard et K. Matsuoka
sp. nov. (Figs 2–62,65–66).
Diagnosis
A small to medium sized species of the genus Protoperidinium
with the tabulation formula: Po, X, 4′, 3a, 7′′, 4c, 4 s, 5′′′,
2′′′′. The motile cell is slightly ovoidal with a short apical horn
but no antapical extensions. Plate 1a is pentagonal and 2a
and 3a are hexagonal. 2a is elongate, asymmetrical. S.d.
touches the cingulum and carried an extended list on the left.
Plates are smooth with scattered trichocyst pores. Cysts are
spherical to subspherical, smooth and light brown in color.
Cell content is colorless. Numerous equidistant tapering,
apiculocavate processes with circular to ovoidal bases and
acuminate tips. These processes bear very small elongate
spinules that are only visible through SEM or LM with ×100 oil
immersion objectives. Archeopyle theropylic and corresponds
to a split, possibly along the paracingulum.
Holotype
Figs 2–5.
Type locality
Jinzhou Harbor, Bohai Sea, China.
Etymology
The epithet honors Dr. Jane Lewis for her significant taxonomic research on marine cyst-forming dinoflagellates.
Description of motile stage
The motile cells are slightly ovoidal with a short apical horn
and no antapical extensions (Figs 2,7,18). The smooth thecal
plates carry small, randomly arranged trichocyst pores on the
surface (Figs 2–4,20,21). These pores are also present on the
cingular plates, where they are aligned with the cingular lists.
The plate arrangement of the epitheca is asymmetrical. The
© 2015 Japanese Society of Phycology
113
apical pore plate (Po) is elongate and ellipsoidal and connected to an elongate rectangular canal plate (X)
(Figs 2,29,30). This pore plate is surrounded by a low collar
formed by the raised edges of the second, third and fourth
apical plates (2′, 3′ and 4′). The first apical plate (1′) is
rhombic (ortho-type) and asymmetrical (Figs 2,8). Apical
plate 2′ is subpentagonal, apical plates 3′ and 4′ are
subhexagonal and plate 4′ is the largest (Figs 2,23–24,29–
30). There are three anterior intercalary plates (Figs 2–4,22–
24). The first anterior intercalary plate (1a) is pentagonal and
located at the left side of the epitheca (Figs 2,3,21,30). The
second anterior intercalary plate (2a) is hexagonal, asymmetrical, laterally elongated and touches 2′′, 3 ′′, 4′′, 1a, 3a and
3′ (Figs 3,22,23). The third anterior intercalary plate (3a) is
hexagonal (Figs 4,23,29,31).
The precingular series consists of seven plates (Figs 2–
4,8,9,21,22,24,29,31). The first (1′′), second (2′′), fourth
(4′′) and sixth (6′′) precingular plates are pentagonal. The
third (3′′), fifth (5′′) and seventh (7′′) precingular plates are
quadrangular (Figs 3,4,9,20). The cingulum is slightly righthanded (ascending) (Fig. 8), lined with narrow lists, and consists of four cingular plates. There is no transitional plate (t).
Plate 1c is small and reaches the middle of 1′′ (Figs 8,10,21).
2c is slightly larger and reaches the start of 2′′ and the start
of 2′′′ (Figs 8,10). 3c is the longest plate and reaches the
start of 7′′ and the end of 4′′′ (Figs 24,28). Four sulcal plates
are observed (Figs 15–16,19). The anterior sulcal plate (S.a.)
is relatively elongate and its anterior part intrudes between
plates 1′′ and 6′′ and touches 1′ (Figs 8,10,19). The rightsulcal plate (S.d.) is long and touches the cingulum
(Figs 10,15,25). S.d. carries a small sulcal fin on the left
which partly covers the sulcal area (Figs 10,18). The left
sulcal plate (S.s.) was long and formed a J-shaped curve, and
also touches the cingulum (Figs 15,19,25). S.s. has a narrow
list bordering its margin (Fig. 25). The posterior sulcal plate
(S.p.) is symmetrical and U-shaped (Figs 14,19). The plate
arrangement of hypotheca is symmetrical. The theca has five
postcingular plates. The first, third and fifth postcingular plate
(1′′′, 3′′′, 5′′′) are five-sided, the second and fourth
postcingular plates (2′′′, 4′′′) are four-sided (Figs 11–13,26–
28). The antapical series is composed of two pentagonal
plates (Figs 13,27).
The plate formula is Po, X, 4′, 3a, 7′′, 4c, 4s, 5′′′, 2′′′′, and
the complete tabulation is illustrated in Figs 32–36
(Iconotype).
Dimensions of motile stage
The motile cells have a length of 21.7–34.6 μm (27.2 ± 4.7,
mean ± SD; n = 10) and width of 17.3–28.8 μm (23.9 ± 4.0,
n = 10).
Cyst description. The cysts are spherical to subspherical
and brown colored (Figs 37–62,65,66). The living cysts had
abundant greenish pigments and endospore (Fig. 37). A membrane could remain inside the cyst after germination
(Figs 38,39,47). The wall is thin. The cyst surface is
microgranular (Figs 40,52,66). This surface is covered with
more or less equidistant, short to long, slender, erect (more
often curved when the processes are long), apiculocavate
processes (=spines) which are never fused and terminate in
acuminate tips (Figs 37–62). There are 8–20 processes per
10 × 10 μm2 (12.5 ± 4.2, n = 8). Generally there are more
114
K. N. Mertens et al.
Figs 2–16. 2–5. Protoperidinium lewisiae after germination of cysts from Jinzhou Harbor, isolated from cyst shown in Figs 37–39 and
used for single-cell polymerase chain reaction (PCR). 6–16. Motile cells after germination of cysts from Lake Saroma. 6,7. Motile cell
showing general shape, color and progressive lower focus starting from ventral side (SR7A8). 8–16. Details of tabulation shown on one
cell (KATG1). 10 and 15 show sulcal wing (arrow). Scale bars = 10 μm.
© 2015 Japanese Society of Phycology
Cyst-theca relation of P. lewisiae
115
Figs 17–31. Protoperidinium after germination of cysts from San Pedro Harbor. 17. Live cell showing general shape and color
(SPH1B5). 18. Ventral view showing extensions of left sulcal plate (arrow on the right) and right sulcal plate (arrow on the left)
(SPH1B5).19. Details of sulcal plates (SPH2H1).20–25. Details of tabulation (SPH1B5).26–28. Details of tabulation (SPH2A9).29–
31. Details of tabulation (SPH3C4). Scale bars = 10 μm.
© 2015 Japanese Society of Phycology
116
K. N. Mertens et al.
Figs 32–36. Drawing
of
interpreted
tabulation
of
Protoperidinium lewisiae.
32. Ventral
side. 33. Dorsal
side.
34. Sulcal
plates.
35. Epitheca.
36. Hypotheca. Shaded black zone denotes
the position of the flagellar pore.
processes on specimens with shorter processes. These processes have spherical to ovoidal proximal bases
(Figs 41,42,65), and carry many tiny spinules, which are
difficult to observe with LM, but are well-observed with SEM
(Figs 61,62,66). The archeopyle observed in culture is
theropylic, and showed a split, which we situate along the
cingulum (Figs 39,44,45,54).
Cyst dimensions
Germinated cysts have a central body diameter of 22.6–
30.9 μm (26.7 ± 3.1 μm, n = 10). The length of five processes varies 2.8–8.1 μm (5.5 ± 1.6, n = 10 × 5).
Comparable measurements were obtained from cysts
recovered from surface sediments from Lake Saroma and San
Pedro Harbor, which had a central body diameter of 25.7–
33.6 μm (30.1 ± 3.1, n = 14). The average length of
five processes varied between 3.6–7.2 μm (5.7 ± 1.2,
n = 14 × 5).
Gene sequence
The LSU rDNA of the cell collected from Jinzhou Harbor
(Figs 2–5) with GenBank Accession KM820891.
cysts have abundant colorless pigments, and often showed
paratabulation (Figs 69,70). The wall is thin. The cyst surface
is microgranular (Figs 63,64,69,70). This surface is covered
with more or less equidistant, short to long, slender, erect
(more often curved when the processes are long), solid processes which are never fused and terminate in acuminate tips
(Figs 63,64,67–70). There are 4–7 (5.5 ± 1.3) processes per
10 × 10 μm2 (n = 7). Generally there are more processes on
specimens with shorter processes. These processes have circular bases (rarely rectangular) (Figs 74,75), and carry many
tiny round spinules, which are difficult to discern with LM, but
are well-observed with SEM (Fig. 68). The archeopyle
observed is theropylic and is formed by suturing along predetermined plate boundaries.
Cyst dimensions
Cysts had a central body diameter of 28.1–44.2 μm
(37.1 ± 4.1, n = 14). The length of five processes varied
between 6.9–11.7 μm (8.4 ± 1.4, n = 14 × 5). These dimensions were similar to Kawami et al. (2006, p. 186), which
reported a central body diameter of 30.0–53.0 μm (35.4
n = 14) and process lengths of 1–8 μm (n = 14).
Comments
Cysts of P. lewisiae have been described as Dinoflagellate cyst
type B in Matsuoka (1987, Pl. 18, his figs 5–8). Cysts of
P. lewisiae would fit into the cyst-based genus Echinidinium
because of the archeopyle.
Gene sequence
Cyst of Oblea acanthocysta Kawami, Iwataki et
Matsuoka 2006 emend.
Phylogenetic position of Protoperidinium
lewisiae
Remarks
We obtained 1396 base pairs of LSU rDNA of one cell of
P. lewisiae (Figs 2–5) collected from Jinzhou Harbor
(KM820891), and this sequence was used for the phylogenetic analyses (Fig. 81). P. lewisiae and P. monovelum
These spiny brown cysts observed from Omura Bay were superficially similar to cysts of P. lewisiae, and spherical to
subspherical and pale brown in color (Figs 69–80). The living
The SSU and LSU rDNA of the cyst collected from Saroma
Lake (Fig. 78–80) with GenBank Accession No. LC005409
and LC005410, respectively.
© 2015 Japanese Society of Phycology
Cyst-theca relation of P. lewisiae
117
Figs 37–48. 37–42. Cysts of Protoperidinium lewisiae from China. 37. Living cyst from Jinzhou Harbor, isolated for sequencing. 38,39. Same cyst, after germination. 40–42. Cyst isolated from Qingdao. 43–48. Cysts from Lake Saroma. 43–44. Germinated cyst from culture (KATG1) which resulted in the thecate stage depicted in Figs 8–16, showing typical process bases and theropylic
archeopyle. 45,46. Different orientations of germinated cyst showing archeopyle (SR4H4). 46–48. Germinated cyst, fluorescence of
cyst is shown in Fig. 48 (SR5B3). Scale bars = 10 μm.
© 2015 Japanese Society of Phycology
118
K. N. Mertens et al.
Figs 49–60. Cysts of Protoperidinium lewisiae from San Pedro Harbor. 49–51. Germinated cyst from culture (SPH1B5) which resulted
in the thecate stage depicted in Figs 17–18 and 20–25, showing split archeopyle. 52–54. Different orientations of germinated cyst
showing archeopyle (SPH3C4). 55–60. Germinated cyst, progressive focus (SPH1A3). Scale bars = 10 μm.
formed a strongly supported clade. Herdmania litoralis Dodge
positioned at the base of this clade. The clade comprised
Archaeperidinium species and the clade of P. lewisiae
and P. monovelum showed their sister relationship. Five
Protoperidinium species (P. tricingulatum, P. haizhouense,
P. americanum (Gran et Braarud) Balech, P. parthenopes
Zingone et Montresor and P. fukuyoi) and Islandinium
minutum formed a robust clade. This clade and the
clade comprising Archaeperidinium/P. lewisiae/P. monovelum/
H. litoralis showed a sister relationship.
Phylogenetic position of Oblea acanthocysta
SSU rDNA
We determined 1740 base pairs of SSU rDNA sequences of
two single-cysts (one of these shown in Figs 78–80) collected
from Saroma Lake (LC005409). Comparisons of the obtained
SSU rDNA sequences from the cysts shows that these are
identical, and also identical to Oblea acanthocysta.
O. acanthocysta formed a clade with four species of the
diplopsalid group (Gotoius excentricus (Nie) Sournia,
© 2015 Japanese Society of Phycology
Cyst-theca relation of P. lewisiae
Figs 61–64. 61–62. Scanning
119
electron
microscope (SEM) images of cysts of
Protoperidinium lewisiae
from
Lake
Saroma,
63,64. SEM images of cysts of
Oblea acanthocysta from Omura Bay. Scale
bars = 10 μm.
Figs 65–68. 65–66. Enlarged apiculocavate processes of cysts of Protoperidinium lewisiae
from
Lake
Saroma
(palynological preparation) using light
microscope
(LM)
and
SEM. 67–
68. Enlarged solid processes of cysts of
O. acanthocysta from Omura Bay using LM
and SEM. Scale bars = 1 μm.
Diplopsalopsis bomba (Stein) Dodge et Toriumi, D. lebouriae
(Nie) Balech and O. torta (Abé) Balech ex Sournia) (Fig. 82).
However, the phylogenetic relationship between this clade and
the other two species of diplopsalid group (Preperidinium
meunieri (Pavillard) Elbrächter and D. lenticula Bergh 1881)
was not robust because of low statistical support.
LSU rDNA
We obtained 1183 base pairs of LSU rDNA of the same two
single-cysts (LC005410), and this sequence was used for the
phylogenetic analyses (Fig. 81). O. acanthocysta, O. torta and
D. lebouriae formed a robust clade. This clade and a clade of
G. excentricus and D. bomba were sister groups (Fig. 81). The
phylogenetic positions of the other diplopsalid species (Pre.
© 2015 Japanese Society of Phycology
meunieri and D. lenticula) were not robust because of low
statistical support.
DISCUSSION
Seven species of Protoperidinium bearing three intercalary
plates could be discriminated from P. lewisiae by their morphological characteristics as follows: P. asymmetricum (Abé)
Balech is larger (36 μm long and 34 μm wide) and can be
differentiated by a 2a that is pentagonal and a pronounced
straight suture separating plates 2′′, 1a, 2′, 4′ and 3′′, 2a, 3a,
3′ (Abé 1927). P. bolmonense Chomérat et Couté is smaller
(18–22 μm long and 15–18 μm wide) and different in having
a S.d. that does not touch the cingulum, only three cingular
120
K. N. Mertens et al.
Figs 69–80. Cysts of Oblea acanthocysta isolated from Omura Bay (69–77) and from Lake Saroma (78–80). 69–71. Specimen showing
paratabulation, cell content still inside. 72–74. High focus, intermediate and lower focus of empty specimen showing process
distribution. 75–77. Specimen with few processes. Note microgranular wall. 78–80. Live cysts from Lake Saroma with colored
granules selected for polymerase chain reaction (PCR) analysis, note rectangular bases on processes (KATG2, KC60). Scale bars = 10 μm.
plates and a differently shaped S.p. (Chomérat & Couté 2008).
P. monovelum (Abé) Balech has a more elongated apical pore,
a pentagonal 2a, a V-shaped S.p and a larger number of sulcal
plates (Abé 1936). P. parthenopes is larger (30.0–38.8 μm
long, 26.0–35.0 μm wide), has a differently shaped 2a and a
V-shaped S.p and has a larger number of sulcal plates (Zingone
& Montresor 1988). P. fukuyoi is of similar size but has a
symmetrical 2a, a V-shaped S.p and six sulcal plates (Mertens
et al. 2013). P. haizhouense has a heptagonal 2a, a pentagonal
3a, three cingular plates and six sulcal plates (Liu et al. 2014).
P. vorax Siano et Montresor is most similar to P. lewisiae, but is
smaller (16–26 μm long and 16–25 μm wide), has only three
cingular plates, a S.d. that does not touch the cingulum, and an
S.p. that is differently shaped and intrudes between 1′′′′ and
2′′′′ (Siano & Montresor 2005).
The distinct shape of the apiculocavate processes with
their small elongate spinules in combination with the specific
type of theropylic archeopyle makes this species distinguishable from all other spiny round brown cysts. There are three
other species with apiculocavate processes, which differ in
© 2015 Japanese Society of Phycology
Cyst-theca relation of P. lewisiae
121
Fig. 81. Maximum-likelihood (ML) tree
inferred from large subunit rDNA
sequences. ML bootstrap values over 70%
-/0.95
and Bayesian posterior probabilities over
0.95 are shown at the nodes. Thick
branches indicate maximal support (100/
1.00). The scale bar represents inferred
90/1.0
77/1.0
evolutionary distance in changes/site. The
DNA sequences generated in this study
are indicated by black boxes.
74/0.99
P. denticulatum AB255848
P. abei AB255839
Protoperidinium thulesense AB716929
Protoperidinium conicum AB255844
Protoperidinium leonis AB255856
Protoperidinium excentricum AB255855
Protoperidinium conicoides DQ444227
Protoperidinium elegans AB255853
Protoperidinium
81/0.98
Protoperidinium crassipes AB255845
sensu stricto
Protoperidinium divergens AB255851
96/1.0
Protoperidinium angustum DQ444237
Protoperidinium pallidum AB255589
-/0.99
Protoperidinium pellucidum AB255862
-/0.99
Protoperidinium bipes AB284160
Protoperidinium pentagonum AB255864
Protoperidinium punctulatum AB255866
Protoperidinium depressum AB255850
Section
99/1.0
Protoperidinium steidingerae DQ444231
Oceanica
Protoperidinium oblongum AB255857
Protoperidinium claudicans AB255840
Preperidinium meunieri DQ444232 Diplopsalids
Archaeperidinium minutum GQ227502
Archaeperidinium (Minutum)
99/1.0 Archaeperidinium minutum AB781001
subgroup
Archaeperidinium saanichi AB702990
Protoperidinium lewisiae KM820891
Monovelum subgroup
Protoperidinium monovelum AB716928
89/1.0
Herdmania litoralis AB564306
Islandinium minutum JX627345
Protoperidinium tricingulatum KF651042
Protoperidinium haizhouense KF651019
92/1.0
96/1.0
Americanum subgroup
Protoperidinium americanum KF651012
Protoperidinium parthenopes KF651026
Protoperidinium fukuyoi AB780844
Diplopsalis lenticula EF152794
Gotoius excentricus AB716922
Diplopsalopsis bomba AB716930
Diplopsalis lebouriae AB716921
88/1.0
Oblea torta AB716924
81/0.99
Oblea acanthocysta LC005410
Rhinodinium broomense DQ078782
Peridinium willei EF205012
Peridinium cinctum EF205011
Peridiniopsis borgeii FJ236464
Akashiwo sanguinea AB232670
Gymnodinium aureolum DQ917486
Pfiesteria piscicida AY112746
Diplopsalids
70/1.0
Scrippsiella trochoidea HQ670228
Togula britannica AY455679
Prorocentrum micans M14649
Gonyaulax baltica AF260388
Dinophysis norvegica AY571375
Heterocapsa triquetra AF260401
Polarella glacialis AY571373
Gyrodinium spirale AY571371
Karenia umbella EF469239
Alexandrium pseudogoniaulax AY154958
Neospora caninum AF001946
0.1
other features from the cyst of P. lewisiae. Islandinium
minutum has a more granular wall, a larger number of processes and a saphopylic archeopyle (Head et al. 2001).
Echinidinium sleipnerensis Head et Riding is larger (44–
48 μm), and has a smooth wall and much more processes
(Head et al. 2004). Echinidinium sp. A has longer processes
(10–15 μm) and a smooth cyst wall.
The restudy of the cyst of Oblea acanthocysta shows that it
has solid (rarely apiculocavate) processes, not hollow as stated
by Kawami et al. (2006), and this characteristic distinguishes
the cyst of O. acanthocysta from the cyst of P. lewisiae, which
has apiculocavate processes. Furthermore it should be noted
that the cyst surface of O. acanthocysta is microgranular and
not smooth, which also contrasts with what is stated by Kawami
et al. (2006). There are a number of other species with
© 2015 Japanese Society of Phycology
solid processes that are quite different from the cyst of
O. acanthocysta. I. brevispinosum Pospelova et Head is
smaller and has much more and shorter solid processes and a
saphopylic archeopyle (Pospelova & Head 2002), which is very
similar to the cyst of P. haizhouense (Liu et al. 2014). The cyst
of Diplopelta symmetrica Pavillard has short hair-like processes
(Dale et al. 1993). The cyst of P. fukuyoi has distinctive and
solid processes which cluster into straight or arcuate linear
complexes, and a saphopylic archeopyle (Mertens et al. 2013).
There are two other species with solid processes that look
very similar to the cyst of O. acanthocysta, but these differ
particularly in the shape of the archeopyle. E. transparantum
Zonneveld (her fig. 6, Plate III, figs 6–10; invalid according to
Head 2002 but validated here by mentioning the basionym)
has long processes with rectangular bases and the archeopyle
122
K. N. Mertens et al.
P. abei AB181881
P. denticulatum AB181890
P. conicum AB181884
Protoperidinium punctulatum AB181906
Protoperidinium thulesense AB261519
Protoperidinium excentricum AY443021
Protoperidinium bipes AB284159
Protoperidinium
Protoperidinium pellucidum AB181902
sensu stricto
(1/4)
95/1.0
97/1.0
98/1.0
Fig. 82. ML tree inferred from small
subunit rDNA sequences. The branch
leading to fast-evolving species have been
shortened to 1/4 the original length (indicated by 1/4). Other information is the
same as Fig. 81.
Protoperidinium elegans AB255835
Protoperidinium crassipes AB181888
Archaeperidinium saanichi AB702987 Archaeperidinium (Minutum)
Archaeperidinium minutum GQ227501
subgroup
Herdmania litoralis AB564300
Amphidiniopsis dragescoi AY238479
Amphidiniopsis rotundata AB639343
Protoperidinium monovelum AB716913
Monovelum subgroup
Protoperidinium monovelum AB716914
Protoperidinium parthenopes AB716915
Protoperidinium americanum AB716911
Protoperidinium fukuyoi AB780842
Americanum subgroup
Protoperidinium tricingulatum AB716916
Islandinium minutum AB780843
Protoperidinium depressum AB255834 Section
99/1.0
Oceanica
Protoperidinium claudicans AB255833
Diplopsalopsis bomba AB261513
Gotoius excentricus AB261514
Oblea acanthocysta LC005409
99/1.0
Oblea acanthocysta AB273723
Diplopsalids
Oblea torta AB273724
-/0.97
Diplopsalis lebouriae AB261512
Preperidinium meunieri AB716910
Diplopsalis lenticula AB716909
Durinskia baltica AF231803
Pfiesteria piscicida AF077055
Prorocentrum micans AY585526
Gymnodinium fuscum AF022194
Heterocapsa triquetra AF022198
Pentapharsodinium tyrrhenicum AF022201
Peridinium cinctum AB185114
Peridinium willei AF274280
Dinophysis acuta AJ506973
Alexandrium minutum AJ535380
99/1.0
Pyrocystis noctiluca AF022156
76/1.0
Ceratium hirundinella AY443014
Scrippsiella sweeneyae AF274276
Karenia brevis AF172714
Akashiwo sanguinea AF276818
Polarella glacialis EF434275
Gyrodinium spirale AB120001
Noctiluca scintillans AF022200
Neospora caninum L24380
0.1
is a simple split (figs 6–10 in Zonneveld 1997), and it has
no paratabulation as opposed to cysts of O. acanthocysta.
E. zonneveldiae Head has a theropylic archeopyle which forms
a long straight split and has processes with a rectangular base
(Head 2002).
Several characteristics of the motile stage enable an assignment to the Monovela group. The Monovela group was erected
to accommodate Protoperidinium species with or without an
apical horn, no antapical horns/spines, a flat ventral area (flat
sulcus and a sulcal fin positioned at the left suture of the right
sulcal plate) and a plate formula of 4′, 2a-3a, 7′′, 5′′′, 2′′′′ (Abé
1936; p. 669–670). P. lewisiae has all these features, and
additionally the four cingular plates and the absence of a
transitional plate suggests an affinity to the Monovela group
(Mertens et al. 2013). However, previous studies suggested
that the Monovela group is not monophyletic because the
benthic (sand-dwelling) dinoflagellate Herdmania litoralis was
included in the clade with the species of Monovela group
(Mertens et al. 2013; Liu et al. 2014). The LSU rDNA based
molecular phylogeny confirms that the species in the Monovela
group formed three clades; clades of Archaeperidinium
(Minutum) subgroup, Americanum subgroup and Monovelum
subgroup (Mertens et al. 2013; Liu et al. 2014) (Fig. 81).
P. lewisiae belongs to the Monovelum subgroup and shows
close phylogenetic relationship with H. litoralis.
Both LSU and SSU rDNA phylogenies confirmed the
monophyly of two species of Oblea and also of Gotoius
excentricus and Diplopsalopsis bomba. Furthermore, both
phylogenetic results supported that Diplopsalis lebourae was
positioned at the base of the Oblea clade. However, another
© 2015 Japanese Society of Phycology
Cyst-theca relation of P. lewisiae
Diplopsalis species, D. lenticula, did not show any affinity
with the other taxa included in this study.
Cysts of P. lewisiae were found in the surface sediments
from Changle and Jinzhou Harbors, Lake Saroma (coastal
lagoon) and Omura Bay, and San Pedro Harbor (see Table 1
and Fig. 1). All of these locations can be classified as shallow
(up to 20 m) estuarine systems from subtropical to temperate
biogeographic regions of the northeastern and northwestern
Pacific Ocean. Based on our records of cysts of P. lewisiae, we
believe that this species can be associated with annual seasurface temperature ranging from −2° to 32°C, relatively constant average sea-surface salinities at 30–33 psu, and
possibly high surface primary productivity.
CONCLUSIONS
The clarification of the cyst-theca relationship and LSU and
SSU rDNA phylogenies of Protoperidinium lewisiae, a new
species assigned to the Monovela group of Abé (1936), highlights the large morphological variability in both the motile
and cyst stages of species in the Monovela group. Both
phylogenies placed Oblea acanthocysta in the clade comprised with four other species of the diplopsalid group, confirming its place within the diplopsalid group. The difference
in process structure and archeopyle of cysts of P. lewisiae and
O. acanthocysta underlines the importance of these characteristics in their classification, and the polyphyly of spiny
brown cysts. Due to low-bootstrap support of the backbone of
the LSU and SSU rDNA based phylogenies, more cyst-theca
relationships need to be established in combination with
single-cell PCR, to clarify the phylogenetic and evolutionary
relationships within the genus Protoperidinium and the
diplopsaloideans. The cyst of P. lewisiae can be applied in
paleoecological studies as indicator for estuarine subtropical
to temperate waters.
ACKNOWLEDGMENTS
KNM is a postdoctoral fellow of FWO Belgium, who conducted
this research at the University of Victoria (British Columbia,
Canada) and partly at Nagasaki University and was supported
by a Kakenhi Grant 22-00805. The Natural Science and
Engineering Research Council of Canada (NSERC) is acknowledged for partial funding of this project (VP Discovery Grant
224236). Haifeng Gu was supported by the National Natural
Science Foundation of China (41376170). Hiromi Saitoh and
Kimihiko Maekawa are thanked for assistance during sampling
of Saroma Lake. Aya Morinaga is thanked for sampling Omura
Bay. Carrie Wolfe, Adam Willingham, and Dennis Dunn from
the Southern California Marine Institute, http://www.scmi.net/
are thanked for their help with sampling in San Pedro Harbor.
Martin J. Head is acknowledged for sharing published measurements. Two anonymous reviewers and the editors,
Mitsunobu Kamiya and Mona Hoppenrath, are thanked for
helpful comments that significantly improved the manuscript.
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