Content uploaded by Giacomo R. DiTullio
Author content
All content in this area was uploaded by Giacomo R. DiTullio on Feb 05, 2016
Content may be subject to copyright.
DIFFERENCES IN PIGMENTATION BETWEEN LIFE CYCLE STAGES IN SCRIPPSIELLA
LACHRYMOSA (DINOPHYCEAE)
1
Agneta Persson
2,3
Department of Biological and Environmental Sciences, G€
oteborg University, Box 461, G€
oteborg SE-405 30, Sweden
Barry C. Smith
National Oceanic and Atmospheric Administration, National Marine Fisheries Service, Northeast Fisheries Science Center,
Milford Laboratory, 212 Rogers Avenue, Milford, Connecticut 06460, USA
Tyler Cyronak, Emily Cooper, and Giacomo R. DiTullio
Hollings Marine Laboratory, College of Charleston, 331 Fort Johnson Rd, Charleston, South Carolina 29412, USA
Various life cycle stages of cyst-producing
dinoflagellates often appear differently colored under
the microscope; gametes appear paler while zygotes
are darker in comparison to vegetative cells. To
compare physiological and photochemical competency,
the pigment composition of discrete life cycle stages
was determined for the common resting cyst-producing
dinoflagellate Scrippsiella lachrymosa. Vegetative cells
had the highest cellular pigment content
(25.2 0.5 pg cell
1
), whereas gamete pigment
content was 22% lower. The pigment content of zygotes
was 82% lower than vegetative cells, even though they
appeared darker under the microscope. Zygotes of
S. lachrymosa contained significantly higher cellular
concentrations of b-carotene (0.65 0.15 pg cell
1
)
than all other life stages. Photoprotective pigments and
the de-epoxidation ratio of xanthophylls-cycle pigments
in S. lachrymosa were significantly elevated in zygotes
and cysts compared to other stages. This suggests a role
for accessory pigments in combating intracellular
oxidative stress during sexual reproduction or
encystment. Resting cysts contained some pigments
even though chloroplasts were not visible, suggesting
that the brightly colored accumulation body contained
photosynthetic pigments. The differences in
pigmentation between life stages have implications for
interpretation of pigment data from field samples
when sampled during dinoflagellate blooms.
Key index words: accumulation body; dinoflagellate;
gamete; life stage; pellicle cyst; pigment; resting
cyst; Scrippsiella lachrymosa; zygote
Abbreviations: Chl, chlorophyll; DD, diadinoxanthin;
DT, diatoxanthin; Pp, photoprotective pigment
pool; Ps, photosynthetic pigment pool
Dinoflagellates constitute a diverse group of
phytoplankton that is well known for causing toxic
blooms worldwide (Anderson et al. 2012, Cusick
and Sayler 2013). Most species of dinoflagellates,
however, are harmless and play an important role
in the trophic transfer of energy in lakes and
oceans. Scrippsiella lachrymosa (J. Lewis) is one of
several very similar cosmopolitan neritic species of
the genus Scrippsiella forming resting cysts with an
outer wall covered with calcite crystals (Lewis
1991, Nehring 1994, Montresor et al. 2003, Zinss-
meister et al. 2011). Scrippsiella species are non-
toxic but bloom-forming, with the most commonly
described species of the genus, S. trochidea, form-
ing blooms globally (e.g., Ishikawa and Taniguchi
1996).
Dinoflagellates have complex life cycles. At least
10% of all dinoflagellate species (Dale 1983, Head
1996) and 20%–28% in temperate areas (Dale 1979,
Nehring 1997, Persson et al. 2000) have life cycles
involving a dormant, diploid resting stage called the
resting cyst. The actively growing cells propagate by
binary asexual fission and are in the vast majority of
species haploid (Steidinger and Tangen 1997). For
cyst-forming species, the resting cyst is formed by
fusion of two haploid gametes into a diploid zygote
that encysts (Fig. 1).
The different life cycle stages of dinoflagellates
often appear differently colored when viewed under
the microscope; gametes often appear to be paler
and give the impression to be less pigmented than
vegetative cells, whereas zygotes are observed to
appear very dark in comparison to vegetative cells
(Anderson et al. 1983, Fritz et al. 1989, Persson
et al. 2013). Cells from the same laboratory culture
or the same field sample on the same microscope
slide can thus appear differently colored or pig-
mented depending upon the life stage.
The sexual cycle of many different cyst-producing
dinoflagellate species has been studied in the labo-
ratory. The basic pathways of life-stage transforma-
1
Received 23 October 2014. Accepted 13 October 2015.
2
Present address: Smedjebacksv€
agen 13, SE-771 90 Ludvika, Sweden.
3
Author for correspondence: e-mail:agnetapersson77@gmail.com.
Editorial Responsibility: J. Raven (Associate Editor)
J. Phycol. 52, 64–74 (2016)
©2015 Phycological Society of America
DOI: 10.1111/jpy.12364
64
tions in nature are thought to follow those outlined
in Figure 1, since natural cues are likely to be irre-
versible “seasonal” signals (composed of environ-
mental information). A number of additional routes
can be studied after culture manipulations (e.g.,
Anderson and Wall 1978, Figueroa et al. 2007).
Cyst-producing dinoflagellates in temperate areas
usually spend most of the year as dormant resting
cysts in the sediment, and only a brief period of
time (i.e., weeks to months) as vegetative cells in
the water column (Dale 1983). Each stage has speci-
fic metabolic requirements linked to the role of the
stage in perpetuation of the population. For exam-
ple, vegetative cells need to synthesize organelles
and cellular constituents that make up new vegeta-
tive cells and to stimulate division into two new veg-
etative cells. In contrast, the primary aim of gametes
is not to divide, but rather to fuse with another hap-
loid gamete to form a diploid zygote. The zygote
accumulates energy stores before transforming into
a dormant resting cyst (starch grains and lipid dro-
plets; Dale 1983). The entire resting cyst often is
packed densely with these materials, whereas all
other cellular components have become invisible to
light microscopy, except for a bright red or yellow-
orange “accumulation body” that can be seen to
form as the chloroplasts degrade (Anderson 1980,
Fritz et al. 1989). It is thought that the accumula-
tion body contains the pigments when resting cysts
have been formed (Anderson 1980, Fritz et al. 1989
and references therein).
We hypothesized that the observed color differ-
ences among the different life stages would be
directly associated with pigment concentration, com-
position, and function. Accordingly, we conducted
experiments in which gamete formation and sexual
behavior were induced by the use of “encystment
medium”; f/2 media (Guillard and Ryther 1962)
minus nitrogen, as this is the most common method
for resting cyst production in the laboratory (e.g.,
Anderson et al. 1984, Persson et al. 2008a and refer-
ences therein). Nitrogen deprivation is considered
an important environmental cue leading to changes
in metabolism and reproduction specifically leading
toward a sexual life cycle and gamete formation. A
strain of S. lachrymosa isolated by Kalle Olli as a cyst
from surface sediments of Casco Bay, Gulf of Maine,
was found to have an exceptionally high cyst-form-
ing capability (Olli 2001, Olli and Anderson 2002)
and has been used in numerous studies on cyst
water surface
(n)
(n)
(2n)
(2n)
(2n)
FIG. 1. The life cycle of Scrippsiella lachrymosa in nature. An illustration of the major routes within the life cycle of S. lachrymosa (cells
drawn from outlines of photos). The resting cysts germinate from the sediment (bottom, dotted region) at the onset of the new growth
season and the diploid planomeiozytes emerge. Each of these cells divides into two haploid vegetative cells that continue to divide by bin-
ary fission. Vegetative cells perform daily vertical migrations (vertical arrow). At the end of the bloom/growth season, gametes are formed.
These accumulate in the pycnocline and mate (shaded region). Two gametes fuse into a diploid planozygote that continues to swim while
building up nutrient stores of starch and lipid. The diploid resting cyst formed by the zygote sinks to the sediment where it enters a dor-
mancy period, staying quiescent, awaiting environmental conditions suitable for vegetative growth. A temporary encysted stage called the
“pellicle cyst” can also form when cells are disturbed (horizontal arrow); this stage is a cell that has cast off the flagella and outer cell wall
with its thecal plates. It can reform a swimming cell within 24 h and does not withstand long storage, but does provide short-term protec-
tion against adverse conditions. Additional routes can be studied in the laboratory after culture manipulations.
DINOFLAGELLATE LIFE STAGE PIGMENTS 65
formation and cyst grazing (e.g., Olli 2001, Olli and
Anderson 2002, Kremp et al. 2003, Smith and Pers-
son 2004, 2005, Persson et al. 2008b, Persson and
Smith 2009). Unlike other studied cyst-forming
dinoflagellates, discrete life-history stages in
S. lachrymosa can be produced with high purity
which makes it an ideal model organism for com-
parison of pigmentation between sexual life stages.
In an earlier study comparing toxin content
between life stages of Alexandrium fundyense (Persson
et al. 2012) many additional life stage separation
steps were developed.
The hypothesis was that observed differences in
color between sexual life stages of the dinoflagellate
S. lachrymosa would correspond to real differences
in pigmentation. The objectives were to compare
the pigmentation of discrete life stages, not only as
a total amount that might reflect color as perceived
by a human eye through the microscope, but to
examine the physiological and photochemical com-
petency at various stages in the life cycle. We com-
pared intracellular pigment concentrations that
reflected photosynthetically (Ps) active processes
and photoprotective (Pp) functions between life
stages. The ratio between these, the Pp/(Pp+Ps)
ratio, is indicative of the cell’s physiological func-
tion. One major photoprotective pathway is the
intraconversion (by means of de-epoxidation) of the
photoprotective pigment diadinoxanthin (DD) to
diatoxanthin (DT). The de-epoxidation ratio (DT/
DD+DT) is indicative of xanthophyll cycling pro-
cesses that all chromophytic (i.e., Chl c-containing)
phytoplankton utilize as a means of nonphotochem-
ical quenching to dissipate excess energy (Yama-
moto et al. 1963) and combat oxidative stress.
MATERIALS AND METHODS
Experiments were performed at NOAA/NMFS Milford lab-
oratory July 16 to August 6, 2010 using a homothallic
S. lachrymosa strain, B-10 (same as CCMP 2666). The different
life stages were isolated as outlined below. After isolation,
cells were harvested using vacuum filtration onto GF/F filters
(Whatman International, Maidstone, UK), rinsed with 5 mL
of isotonic ammonium formate (NH
4
HCO
2
), and frozen at
80°C. Pigment samples were shipped frozen to the Hollings
Marine Laboratory, Charleston, South Carolina, for subse-
quent analysis.
The purity of gamete and zygote life-stage samples cannot
be viewed as 100% since there exist no way to mechanically
separate swimming life stages from each other. Gametes and
vegetative cells are very similar in size but have different
swimming behavior (as described in Smith and Persson 2005,
Persson et al. 2013 and Persson and Smith 2013) and small
morphological differences (gametes are rounder and paler).
The biovolume of cells was not determined in this experi-
ment since focus was on cellular pigment content. However,
using images from this and other experiments using the same
species, biovolumes of discrete life stages can be estimated.
Gametes have a volume approximately 10%–40% smaller
than vegetative cells while the volume of a zygote is the same
as that of two gametes and slightly larger than a resting cyst.
Zygotes are larger and easy to identify (enabling separate
counts), but cannot be mechanically separated from other
swimming life stages. Cysts on the other hand are stiff due to
the thick nonelastic wall and can easily be separated from
zygotes and residual gametes and vegetative cells by sieving.
Due to the high cyst-forming ability of the S. lachrymosa strain
used, and combined with thorough microscopy and experi-
ence in life-stage identification, high purity of samples could
be reached, but the gamete and zygote life-stage samples
should still be viewed at a population-level and not as totally
pure. For use of the method on other strains and species we
recommend additional cleaning/sorting steps (as in Persson
et al. 2012) combined with thorough morphological and
behavioral studies.
Vegetative cells.Scrippsiella lachrymosa was grown semi-con-
tinuously in 0.2 lm filtered, Milford Harbor seawater
enriched with f/2 minus silicate nutrients and autoclaved for
sterility. Cultures (1.5 L) were grown in 2.8-L Fernbach flasks
in a light room at 16°C, 220 lmol photons m
2
s
1
PAR
and a 12:12 light:dark cycle. The culture is part of a culture
collection kept in these conditions and has been used in sev-
eral studies on cyst formation and sexual life stages (Smith
and Persson 2004, 2005). To ensure maximal exponential
growth, transfers were made every fourth day, three times in
a row prior to harvesting (250 mL culture with only actively
swimming cells transferred to 1250 mL new f/2 growth med-
ium each time). Microscopy confirmed straight (i.e., non-
erratic) swimming patterns and equally sized, drop-shaped
cells with a short but pronounced apical horn (as described
in Lewis 1991, for swimming pattern interpretation, see Smith
and Persson 2005, Persson et al. 2013 and Persson and Smith
2013). Cells were thus harvested in mid-exponential growth
phase. Three replicate samples from the same culture were
used, at a cell density of 15 910
3
cells mL
1
(Table 1).
Effort was spent on producing cells in truly exponential
growth without the presence of other life stages.
Gametes. Gamete formation was induced by diluting
80 mL of a vegetative cell culture (45 910
3
cells mL
1
)
with 920 mL encystment medium (f/2 minus nitrogen;
Anderson et al. 1984, Smith and Persson 2004) in 2.8-L Fern-
TABLE 1. Summary of samples extracted for pigment analysis.
Scrippsiella lachrymosa life stage Number of containers
Number of samples
per container
Total number
of samples
Volume
filtered (mL)
Number (10
4
)of
cells per sample
Vegetative 1 3 3 35 54 0
Pellicle cysts of vegetative Same as vegetative 3 3 35 54 0
Gamete 3 3 9 51 2364
Pellicle cyst of gamete 3 3 9 10 44 5
Zygote 3 3 9 183 25 107 18
Early resting cysts Same as zygotes 3 3 199 40 16
Resting cysts 3 1 3 67 14 58 20
Extra resting cysts Left-over cultures,
5 added together
334214 54 9
66 AGNETA PERSSON ET AL.
bach flasks (six replicates –three for gametes, three for pelli-
cle cysts of gametes). These cultures were monitored by light
microscopy for morphology and swimming behavior of cells
(Persson and Smith 2013, Persson et al. 2013). Cells were
harvested after 2 d when gametes were plentiful. Gametes of
S. lachrymosa were slightly smaller than vegetative cells and
more rounded without a clear drop-shape (without a pro-
nounced apical horn, Fig. 2A). They also displayed typical
gamete swimming patterns, with circular movements and fre-
quent cell contacts (Smith and Persson 2005). Three samples
from each replicate were harvested and analyzed (Table 1).
Pellicle cysts. To induce pellicle cyst formation, S. lachry-
mosa cells were centrifuged at 1,750gfor 10 min at 16°C. Cells
were resuspended in filtered seawater and confirmed to be
intact and nonmotile using light microscopy. Pellicle cyst
samples were prepared from both exponentially growing veg-
etative cells and from gametes. Three samples from each
replicate were harvested and used for pigment analysis
(Table 1).
Zygotes. Zygotes were produced in 1-L baking dishes fol-
lowing the protocol in Smith and Persson (2004). Baking
dishes were placed in the same light room as above, at
16°C. Encystment medium (f/2 minus N) was used, and
the initial cell density was 3.6 910
3
cells mL
1
in 1 L of
medium per dish (six replicates were made, three used for
zygotes and three for resting cysts; Table 1). The cultures
were monitored microscopically for the different life stages
and harvested when they consisted of zygotes and early rest-
ing cysts.
Zygotes were harvested after 11 d: the contents of each
baking dish were poured through a 20-lm nylon-mesh sieve,
thus separating zygotes from resting cysts already formed.
Resting cysts were trapped on the sieve while zygotes and
residual gametes and vegetative cells passed through the sieve
and were collected in a container below. Zygote samples were
harvested for pigment measurements by filtration onto GF/F
filters as previously described for the other life stages (three
samples per replicate). During counting of preserved sam-
ples, zygotes were counted separately from the residual small
cells (gametes and vegetative cells). Resting cysts separated
from zygotes were rinsed with filtered seawater using a plant
sprayer, harvested, and analyzed as “early resting cysts.”
Resting cysts. Resting cysts were produced in baking dishes
in the same way as zygotes (see above). Three replicates were
used (Table 1). Scrippsiella lachrymosa cysts were harvested
from baking dishes on days 11 (early resting cysts, mentioned
above) and 17 (resting cysts) by rinsing them onto a 20-lm
nylon-mesh sieve with filtered seawater using a plant sprayer.
Clean cysts were resuspended in filtered seawater to enable
cell counts and harvesting by filtration onto GF/F filters as
with the other samples. Extra cysts were harvested on day 17
from leftover cultures in encystment medium (cysts from five
different containers were combined).
Pigment analysis. Chlorophyll and accessory pigment com-
position was analyzed by high performance liquid chro-
matography at the Hollings Marine Laboratory, Charleston,
South Carolina, USA. Samples were stored at 80°C until
analysis. Just prior to analysis, cells were sonicated on
ice (Sonic Dismembrator, Model 100; Fisher Scientific,
Pittsburgh, Pennsylvania, USA) to facilitate the breakdown
of cell walls and extraction of the pigments, which was veri-
fied by microscopy, and the pigments were allowed to
extract overnight in 100% acetone at 20°C. The next day,
cell extracts were centrifuged to remove cell debris. Pig-
ments were separated using a gradient elution method
(DiTullio and Geesey 2002), which is a slight modification
of the Zapata et al. (2000) method. Chromatographic sepa-
ration was performed using a Waters C8 Symmetry column,
and pigments were detected using a photodiode array and
fluorescence detector on an Agillent 1100 HPLC as previ-
ously described by DiTullio and Geesey (2002). The pigment
b-Apo-8-carotenal-trans (Fluka Chemical Corp., Wilmington,
Delaware, USA) was added to the pigment extract as an
internal standard. Pigment concentrations were calibrated
against standards from DHI LABS (Hoersholm, Denmark)
and in-house purifications of noncommercially available pig-
ments. Identification of pigments was performed using rela-
tive retention times as well as pigment action spectra
obtained from our pigment library. Individual pigments were
quantified based upon peak area using Chemstation software
(revision B.03.01; Agilent, Technologies, Santa Clara, Califor-
nia, USA). Coefficient of variation among replicate HPLC
injections of pigment standards on this system were <3%,
and the limit of detection was approximately 1 ng L
1
.
Cell counts. Samples for cell counts were preserved with
iodine crystals (~5mgmL
1
sample) and counted
microscopically in a Sedgwick-Rafter chamber during the
experiment (within 17 d).
FIG. 2. (A) The total pigment concentration per cell in Scrippsiella lachrymosa. (B) Pigment composition. Major pigments are colored
dark for carotenoids (upper part) and light for chls. Error bars are standard deviations.
DINOFLAGELLATE LIFE STAGE PIGMENTS 67
Statistical analysis. One-way ANOVA was done for each
variable (pigment) using StatGraphics Plus 5.1. (Statpoint
Technologies, Inc., Warrenton, VA, USA), and for total pig-
ment content. For each dependent variable, multiple compar-
isons of each of the life stages were made to determine which
means were significantly different from others (P<0.05).
The method used to discriminate between means was Fisher’s
least significant difference procedure. Triplicate samples from
each replicate enabled nested analyses of variance for game-
tes, pellicle cysts of gametes, and zygotes (see Table 1). Thus,
the variance within replicates was compared to the variance
between replicates for each of the life stages. For statistical
comparison of proportions (pigment composition), analysis
of variance was conducted on transformed (x’ =arcsin√x)
values as recommended by Underwood (1997).
RESULTS
Pigment analysis. The total cellular pigment
concentration differed significantly among the life
stages of S. lachrymosa (Fig. 2A). The variance
within replicates was insignificant in the nested
analyses for gametes, pellicle cysts of gametes,
and zygotes, so averages for each replicate
were used. Vegetative cells contained the highest
cellular concentration of total pigments
(25.2 0.5 pg cell
1
), gametes had 22% less total
pigment per cell, pellicle cysts of gametes 54% less,
and zygotes and resting cysts were approximately 4-
to 5-fold lower when compared to vegetative cells
or gametes (4.5 1.2 pg cell
1
and
5.6 2.7 pg cell
1
, respectively). Calculating pig-
ment content expressed as biovolume eliminates
the difference between gametes and vegetative cells
while the difference between these life stages and
zygotes and resting cysts is amplified. The major
pigments observed were Chl a, peridinin, and Chl
c2, and minor pigments included diadinoxanthin,
diatoxanthin, dinoxanthin, and b-carotene
(Fig. 2B). Trace amounts (<1%) of diadinochrome,
phaeophytin, chlorophyllide, MgDVP, and mona-
doxanthin were found as well. Pigment composition
differed significantly among life stages (Fig. 2B).
Vegetative cells and gametes (and their correspond-
ing pellicle cysts) were very similar, whereas zygote
samples had a unique pigment composition com-
pared to preceding stages and to resting cysts of dif-
ferent ages. The pureness of zygote samples was
85 4%. The cysts had a number of small,
unknown peaks which probably were breakdown
products of other pigments. Zygotes contained a
larger amount of b-carotene (>14% of total pig-
ment) compared to only 1% of the total in other
swimming stages. There was a significantly larger
proportion of diatoxanthin relative to Chl ain
zygotes and cysts compared to vegetative and
gamete stages (six times higher in zygotes and nine
times higher in resting cysts; Table 2). There also
was a significantly larger proportion of peridinin
relative to Chl ain zygotes and cysts compared to
vegetative and gamete stages (22% higher in
TABLE 2. Ratio of pigment concentration to chl ain the different life stages of Scrippsiella lachrymosa.
Cell type MgDVP Chl c2 Chlorophyllide Phaeophytin Peridinin Diadinoxanthin Diadinochrome Dinoxanthin Diatoxanthin Monadoxanthin Zeaxanthin b-carotene
Vegetative 0.006 0.298 0.000 0.028 0.511 0.136 0.009 0.030 0.017 0.003 0.000 0.015
Vegetative pellicle 0.005 0.369 0.004 0.021 0.598 0.157 0.013 0.034 0.016 0.000 0.000 0.019
Gamete 0.007 0.330 0.006 0.016 0.521 0.131 0.010 0.030 0.014 0.002 0.000 0.020
Gamete pellicle 0.006 0.308 0.000 0.014 0.518 0.135 0.010 0.024 0.017 0.003 0.000 0.026
Zygote 0.008 0.305 0.000 0.023 0.666 0.188 0.010 0.025 0.066 0.001 0.012 0.390
Early cyst 0.008 0.234 0.005 0.016 0.823 0.192 0.017 0.048 0.135 0.006 0.000 0.031
Cyst 0.007 0.237 0.006 0.017 0.883 0.285 0.014 0.065 0.175 0.009 0.000 0.028
Extra cyst 0.014 0.438 0.000 0.013 0.950 0.350 0.021 0.090 0.208 0.007 0.000 0.132
68 AGNETA PERSSON ET AL.
zygotes and 51%–83% higher in resting cysts;
Table 2). Carotenoids were dominant (>50%) in
zygotes and resting cysts, whereas chls were domi-
nant (>60%) in vegetative cells and gametes.
Pigment concentrations were divided into two
main functional groups. The “Ps” pool consisted of
the main light-harvesting, photosynthetic pigments,
including Chl a,Chlc2, and peridinin. We did not
include the minor concentrations of dinoxanthin or
the other light-accessory pigments as they repre-
sented a relatively small percentage of the Ps pool
(i.e., <2%). The Ps pool of pigments was signifi-
cantly higher (2- to 3-fold) in vegetative and swim-
ming-gamete cells compared to the zygote and cyst
stages (Fig. 3A). Another pigment category was
established, the “Pp” pool, which was calculated by
summing the major photoprotective pigments, dia-
toxanthin (DT), diadinoxanthin (DD), and b-caro-
tene. The Pp pool size was approximately an order
of magnitude lower than the Ps pool size but
revealed a similar trend among the life stages
(Fig. 3B). The ratio of photoprotective pigments to
total pigments (Pp/(Ps+Pp)) revealed that,
although the zygote and cyst life stages had lower
cellular Pp pool size compared to the vegetative and
gamete stages, the photoprotective pigment ratio
was actually 72%–186% higher in the cyst and
zygote stages relative to the vegetative and gamete
stages (Fig. 3C). The photoprotective ability of the
Pp pool was determined by calculating the de-epoxi-
dation ratio (DT/(DT+DD)). The de-epoxidation
ratio in the zygote and cyst stages for S. lachyromsa
was on average 248% higher compared to the vege-
tative and gamete stages (Fig. 3D).
Microscopic observations. Gametes of
Scrippsiella lachrymosa appeared paler under the
microscope compared to vegetative cells and had a
more rounded epitheca (Fig. 4A). Zygotes were lar-
ger, lumpier and appeared darker under the micro-
scope and had a conical epitheca (Fig. 4B). All
FIG. 3. (A) The photosynthetic pigment pool (Ps) of Scrippsiella lachrymosa. (B) The photoprotective pigment pool (Pp). (C) The
photoprotective pigment ratio (Pp/(Pp +Ps)) of different life cycle stages. (D) The de-epoxidation ratio of life cycle stages (the propor-
tion of diatoxanthin (DT) in the xanthophyll (diatoxanthin +diadinoxanthin (DD)) cycle. Error bars show standard deviation.
DINOFLAGELLATE LIFE STAGE PIGMENTS 69
swimming life stages (vegetative cells, gametes, and
zygotes) could form pellicle cysts. The accumulation
body (“red spot”), that is an organelle typically pre-
sent in dinoflagellate hypnozygotes (Dale 1983)
soon started to form near the wall in the hypotheca
when centrifuged cells formed pellicle cysts (Fig. 5).
Early resting cysts appeared somewhat brownish
(still containing small chloroplasts), but late resting
cysts were colorless except for the prominent red
accumulation body.
DISCUSSION
Scrippsiella lachrymosa has a complex life history
with alternating life cycle stages (Fig. 1). The cellu-
lar pigment concentration of vegetative cells aver-
aged 25 pg cell
1
, which is in general agreement
with previous reports for the genus (Zapata et al.
2012), while pigment concentrations in other life
stages were significantly lower (Fig. 3A). Scrippsiella
lachrymosa belongs to dinoflagellates categorized as
“Group 1” following the classification scheme of
Zapata et al. (2012). This group is characterized by
containing the accessory pigments peridinin, dinox-
anthin, and Chl c2. Xanthophyll cycling pigments
also were important contributors to the total pig-
ment inventory. Pigment concentrations and com-
positions varied between the life-history stages. For
instance, there was a significantly higher DT:Chl a
ratio in zygotes and cysts compared to other stages
(Table 2). Latasa and Berdalet (1994) found an
increased amount of DT in a Heterocapsa sp. follow-
ing N or P limitation, which may have been caused
by induction of sexuality. Since nitrogen limitation
leads to induction of the sexual cycle in cyst-produ-
cing dinoflagellates, it is not possible to study the
effects of nitrogen deprivation separate from the
effects of sexual life-stage formation. Future devel-
opment of a reliable life-stage marker that could be
used on field samples would greatly improve
research possibilities.
Zeaxanthin is an important intermediate step in
dinoflagellate carotenoid formation (Swift et al.
1982, Takaichi 2011), and trace amounts of this pig-
ment were found in one of the S. lachrymosa zygote
cultures. b-carotene is the precursor pigment of
other carotenoid pigments (e.g., Takaichi 2011),
and its increased occurrence in zygotes may be
indicative of increased carotenoid synthesis at this
stage. b-carotene, however, also serves as an effective
antioxidant that phytoplankton can use to combat
stressful oxidative conditions, such as under severe
nutrient limitation (e.g., Riseman and DiTullio
2004), and following dilution with N-limiting encyst-
ment media. N-limiting conditions in the field may
also trigger this response. Between gamete mating
and resting-cyst formation, zygotes accumulate
stored energy in the form of starch and lipid
A B
FIG. 4. (A) Scrippsiella
lachrymosa gametes losing the
theca after centrifugation. The
rounded shape of empty theca
can be seen. (B) An S. lachrymosa
zygote showing conical epitheca
and a large nucleus. The cell has
dense content with many
organelles, starch grains, and
lipid droplets.
FIG. 5. The accumulation body (arrow) starts forming quickly
near the cell wall in the hypotheca when pellicle cyst formation is
induced in Scrippsiella lachrymosa.
70 AGNETA PERSSON ET AL.
sinking to the seafloor as resting cysts (Fig. 1). Possi-
bly the increased b-carotene content represents a
physiological mechanism for the cell to scavenge
intracellular reactive oxygen species that are
produced during the major internal changes that
occur between gamete fusion and formation of a
thick-walled, dormant, resting cyst.
The color of cells seen in microscopic observa-
tions does not always correlate with the concentra-
tion of photosynthetic pigments present in the cells.
Gametes of S. lachrymosa appeared paler than the
vegetative cells, which is in accordance with the
pigment measurements presented, as gametes had a
lower cellular pigment concentration than vegetative
cells. However, when the pigment content per unit
volume is estimated, gametes have a similar pigment
concentration per lm
3
to that of vegetative cells –
thus the paler appearance is due to the smaller cell
size, not to a lower pigment concentration within
the cell. The most well-known difference in color
among different life stages is the dark color of
zygotes (Anderson et al. 1983, Fritz et al. 1989).
The darker color so commonly observed has
tempted an interpretation as a larger content of
photosynthetic pigments to harvest light energy to
fuel synthesis of storage compounds. This hypothe-
sis, however, was not confirmed in the present
study. On the contrary, the results show that pig-
ments present in the two fused gametes have “disap-
peared” and the pigment composition changed in
the zygote, showing that degradation and reorgani-
zation processes occur. Even though zygotes
appeared darker under the microscope than other
life stages, S. lachrymosa zygotes had a significantly
lower total cellular pigment concentration than
either vegetative cells or gametes. The color per-
ceived by us when viewing a suspension of
dinoflagellate cells in a water body typically will
not be the same as seen when viewing individual
cells under the microscope or be consistent with
the pigment concentration and/or composition.
Spear et al. (2009) showed that the optical density
and light-scattering properties in cultures of Kare-
nia brevis changed over time. Even though this
dinoflagellate has green chloroplasts (e.g., “Group
2” of Zapata et al. 2012), the optical density
increased over time as cell size, organelle size and
numbers increased, leading to light scattering of
longer wavelengths (red light) in older cultures.
Cells that are larger contain more organelles and
vesicles, have a thicker cell wall, are more optically
dense, and appear darker under the microscope
than smaller cells with fewer organelles and a thin-
ner wall. These large, dense cells also reflect or
scatter longer wavelengths than fast-growing, smal-
ler cells (Spear et al. 2009). Thus, the dark color
of zygotes when viewed under a microscope does
not equate to a higher cellular pigment content,
but rather is a function of how optically dense the
cells are.
The significantly decreased pigment content in
pellicle cysts of gametes may indicate that gametes
are more sensitive, either in general due to physio-
logical and/or morphological differences, or more
specifically to the centrifugation procedure used for
pellicle cyst formation compared to vegetative cells.
Centrifugation is sometimes used as a harvesting
method for pigment analyses, but our results
indicate that careful filtration is preferable.
Light can have a significant effect on cellular
pigment composition and concentration through
photoadaptation and/or photoinhibition processes.
Pigment measurements within the same species can
differ significantly if collected from the field or
grown under different light conditions in the labo-
ratory (Rivkin et al. 1982, Schl€uter et al. 2000, Hen-
riksen et al. 2002). The findings presented here,
that pigmentation can differ among various life
stages when grown under the same light conditions
may have implications for the interpretation of pig-
ment data from field samples, where computed
models often rely upon stable proportions between
pigments in each phytoplankton group. The pro-
gram CHEMTAX uses an iterative process to find
the optimal pigment:Chl aratios and generates the
fraction of the total Chl apool belonging to each
pigment-determined group (Mackey et al. 1996,
DiTullio et al. 2003, Latasa 2007). Typically, a fixed
mass ratio between peridinin and Chl aof 1.06 is
used in these analyses for quantification of the
dinoflagellate portion of phytoplankton community
(Mackey et al. 1996, Latasa 2007, Wright et al. 2010,
Yanpei et al. 2014). However, Zapata et al. (2012)
examined the pigment composition of 64 dinoflag-
ellate species (112 strains) and found that the molar
peridinin: Chl aratio was variable and ranged from
0.54 to 2.06 among the different species. They ana-
lyzed five different Scrippsiella species (but not
S. lachrymosa) and found their molar ratios of peri-
dinin: Chl ato be 0.89–1.18, which converts to
0.62–0.83 as mass ratio and fits well with the ratios
given in Table 2. They described six different major
chloroplast types in dinoflagellates and cautioned
against using only peridinin for tracking photosyn-
thetic dinoflagellates in field samples. Using addi-
tional marker pigments offers oceanographers
greater power for resolving dinoflagellates in mixed
communities (Zapata et al. 2012). The significant
differences in the pigment ratios between life-stage
samples, for example with a higher peridinin to Chl
aratio for zygotes and cysts (Table 2), shows that
extra care should be taken to verify pigment data
with cell counts and microscopic identification
when sampling during dinoflagellate blooms. The
variability in the peridinin:Chl aratio of the differ-
ent life stages can affect the CHEMTAX allocation
of Chl ainto the dinoflagellate group.
It is thought that resting cysts do not contain
chloroplasts, and they often appear colorless under
light microscopy except for the accumulation body
DINOFLAGELLATE LIFE STAGE PIGMENTS 71
that is bright red in S. lachrymosa. As the zygote
encysts, a thick and impermeable cell wall is pro-
duced to prohibit harmful substances from entering
the cell during dormancy (for example during
anoxic conditions). The cell wall, however, may also
prevent the release of harmful breakdown products
(e.g., reactive oxygen species) within the cell pro-
duced during the cyst formation process and possi-
bly also by persistent (albeit slow) metabolism
during dormancy. As a result, there may well be a
need for cellular antioxidants as well as a closed
vesicle (i.e., “trash-compartment”) that can store
harmful products and release them when the cell
later germinates. This waste-product compartment
may be one function of the accumulation body,
which is not seen in actively growing vegetative cells,
but rather only in cells that are closed from free
exchange with the environment such as resting
cysts, pellicle cysts, and forming cysts. A similar situ-
ation occurs in symbiotic Symbiodinium cells within
coral tissues, which also have accumulation bodies
(Franklin et al. 2004). TEM examinations of Alexan-
drium tamarense have demonstrated that resting cysts
of this species do not contain any chloroplasts. The
accumulation body, however, was shown to contain
membranous and granular regions, suggesting that
it is formed from the fusion of chloroplasts (Fritz
et al. 1989). Spector (1984) suggested that the func-
tion of the accumulation body is the breakdown of
superfluous organelles and membranes. Zhou and
Fritz (1994) examined accumulation bodies in two
Prorocentrum species and concluded they were
dinoflagellate lysosomes; spherical vesicles contain-
ing acid hydrolases, capable of breaking down a
wide array of biomolecules, essentially a dinoflagel-
late “waste-disposal system.” The accumulation body
is present in the germinating planomeiozyte, inher-
ited by one cell after division and later discarded
(personal observations of germinating cysts of
S. lachrymosa, following this experiment; Fritz et al.
1989 on A. tamarense –formerly Gonyaulax tamaren-
sis). The presence of pigments in otherwise clear
S. lachrymosa cysts indicates that the pigments were
most likely found within the accumulation body.
The high proportion of photoprotective pigments
in the cysts (Fig. 4C) indicates that these could be
used to mitigate oxidative stress in the cyst while it
is being formed and sealed off from the external
environment. The details of the role of the accumu-
lation body in cyst formation and especially related
to protection functions within the cell need to be
studied further.
When dinoflagellate gametes are formed, they are
attracted to each other, and their swimming behavior
causes bio-convection patterns (Persson and Smith
2013). Gamete swimming patterns and the resulting
dense cell accumulations are concentrated further
and transported by water circulation patterns (e.g.,
fronts, currents; Persson and Smith 2013 and refer-
ences therein, Wyatt and Zingone 2014) which might
bring gametes and zygotes closer to the surface than
they would be found as vegetative cells. These differ-
ences in movements and transportation might elicit a
need for more photoprotection in zygotes, especially
for a species that thrives at lower light depths as vege-
tative cells. In S. lacrymosa, both the photoprotective
and photosynthetic cellular pool sizes were lower in
the zygote and cyst stages relative to the vegetative
and gamete stages (Figs. 3 and 4). The higher de-
epoxidation ratio and photoprotective pigment ratio
observed in the zygote and cyst stages, however, sug-
gests that combating oxidative stress within the cell
takes priority over synthesizing light-harvesting pho-
tosynthetic pigments. Recently, the xanthophyll cycle
has been suggested as a viable cellular process to
combat oxidative stress induced by various environ-
mental factors (other than light) leading to growth
limitation (McLenon and DiTullio 2012). The high
de-epoxidation ratio in zygotes and cysts suggests that
xanthophyll cycling is an important antioxidant strat-
egy for the cell to mitigate oxidative stress resulting
from metabolic changes accompanying sexual repro-
duction and transformation into a resting cyst.
CONCLUSIONS
Various life cycle stages of S. lachrymosa had
different pigment concentrations and compositions;
vegetative cells had the highest pigment content,
gametes lower, and zygotes the lowest. Resting cysts
contained some pigments, probably located in the
brightly colored accumulation body. Zygotes
appeared darker than other stages when viewed
under the microscope, but had significantly lower
pigment content. The larger size and denser cell
content of zygotes, with more organelles and
vesicles, may make them more optically dense and
therefore appear darker when viewed under a
microscope. Scrippsiella lachrymosa zygotes contained
significantly higher amounts of b-carotene than all
other life stages, and both zygotes and resting cysts
had pigment compositions dominated by carote-
noids, whereas in vegetative cells and gametes, chl
pigments dominated. Production of photoprotective
pigments and xanthophyll cycling may be important
mechanisms for protecting the zygote from irrepara-
ble oxidative damage during the major metabolic
changes that occur during sexual reproduction. The
differences in pigmentation between life stages may
have implications for the interpretation of pigment
data from field samples and demonstrates that pig-
ment data should be verified with cell counts and
microscopic identification when sampling during
dinoflagellate blooms.
The Signe och Olof Wallenius Foundation provided financial
support. We are grateful to Jennifer Alix for assistance with
culturing as well as two anonymous reviewers and Dr. Gary H.
Wikfors for manuscript advice. Mention of trade names does
not imply endorsement.
72 AGNETA PERSSON ET AL.
Anderson, D. M. 1980. Effects of temperature conditioning on
development and germination of Gonyaulax tamarensis (Dino-
phyceae) hypnozygotes. J. Phycol. 16:166–72.
Anderson, D. M., Cembella, A. D. & Hallegraeff, G. M. 2012. Pro-
gress in understanding harmful algal blooms: paradigm shifts
and new technologies for research, monitoring, and manage-
ment. Annu. Rev. Mar. Sci. 4:143–76.
Anderson, D. M., Chrisholm, S. W. & Watras, C. J. 1983. Impor-
tance of life cycle events in the population dynamics of
Gonyaulax tamarensis.Mar. Biol. 76:179–89.
Anderson, D. M., Kulis, D. M. & Binder, B. J. 1984. Sexuality and
cyst formation in the dinoflagellate Gonyaulax tamarensis: cyst
yield in batch cultures. J. Phycol. 20:418–25.
Anderson, D. M. & Wall, D. 1978. Potential importance of ben-
thic cysts of Gonyavlax tamarensis and G. excavata in initiating
toxic dinoflagellate blooms. J. Phycol. 14:224–34.
Cusick, K. D. & Sayler, G. S. 2013. An overview on the marine
neurotoxin, saxitoxin: genetics, molecular targets, methods
of detection and ecological functions. Mar. Drugs 11:991–
1018.
Dale, B. 1979. Collection, preparation, and identification of
dinoflagellate resting cysts. In Taylor, F. J. R. & Seliger, H.
H. [Eds.] Toxic Dinoflagellate Blooms. Elsevier, North Holland,
pp. 443–52.
Dale, B. 1983. Dinoflagellate resting cysts: ‘benthic plankton’. In
Fryxell, G. A. [Ed.] Survival Strategies of the Algae. Cambridge
University Press, Cambridge, pp. 69–136.
DiTullio, G. R. & Geesey, M. E. 2002. Photosynthetic pigments in
marine algae and bacteria. In Bitton, G. [Ed.] The Encyclope-
dia of Environmental Microbiology. John Wiley & Sons Inc., New
York, pp. 2453–70.
DiTullio, G. R., Geesey, M., Jones, D. R., Daly, K., Campbell, L. &
Smith, W. O. Jr 2003. Phytoplankton assemblage structure
and primary productivity along 170°W in the South Pacific
Ocean. Mar. Ecol. Prog. Ser. 255:55–80.
Figueroa, R. I., Garce0s, E. & Bravo, I. 2007. Comparative study of
the life cycles of Alexandrium tamutum and Alexandrium minu-
tum (Gonyaulacales, Dinophyceae) in culture. J. Phycol.
43:1039–53.
Franklin, D. J., Hoegh-Guldberg, O., Jones, R. J. & Berges, J. A.
2004. Cell death and degeneration in the symbiotic dinoflag-
ellates of the coral Stylophora pistillata during bleaching. Mar.
Ecol. Prog. Ser. 272:117–30.
Fritz, L., Anderson, D. M. & Triemer, R. E. 1989. Ultrastructural
aspects of sexual reproduction in the red tide dinoflagellate
Gonyaulax tamarensis.J. Phycol. 25:95–107.
Guillard, R. R. L. & Ryther, J. H. 1962. Studies of marine plank-
tonic diatoms. I. Cyclotella nana Hustedt and Detonula confer-
vacea (Cleve) Gran. Can. J. Microbiol. 8:229–39.
Head, M. J. 1996. Chapter 30. Modern dinoflagellate cysts and
their biological affinities. In Jansonius, J. & McGregor, D. C.
[Eds.]. Palynology: Principles and Applications, Vol. 3. American
Association of Stratigraphic Palynologists Foundation, Dallas
Texas, pp. 1197–248.
Henriksen, P., Riemann, B., Kaas, H., Munk Sørensen, H. & Lang
Sørensen, H. 2002. Effects of nutrient-limitation and irradi-
ance on marine phytoplankton pigments. J. Plankton Res.
24:835–58.
Ishikawa, A. & Taniguchi, A. 1996. Contribution of benthic cysts
to the population dynamics of Scrippsiella spp. (Dinophyceae)
in Onagawa Bay, northeast Japan. Mar. Ecol. Prog. Ser.
140:169–78.
Kremp, A., Shull, D. H. & Anderson, D. M. 2003. Effects of
deposit-feeder gut passage and fecal pellet encapsulation on
germination of dinoflagellate resting cysts. Mar. Ecol. Prog.
Ser. 263:65–73.
Latasa, M. 2007. Improving estimations of phytoplankton class
abundances using CHEMTAX. Mar. Ecol. Prog. Ser. 329:13–
21.
Latasa, M. & Berdalet, E. 1994. Effect of nitrogen and phospho-
rus starvation on pigment composition of cultured Hetero-
capsa sp. J. Plankton Res. 16:83–94.
Lewis, J. 1991. Cyst-theca relationships in Scrippsiella (Dino-
phyceae) and related orthoperidinioid genera. Bot. Mar.
34:91–106.
Mackey, M. D., Mackey, D. J., Higgins, H. W. & Wright, S. W.
1996. CHEMTAX - a program for estimating class abun-
dances from chemical markers: application to HPLC
measurements of phytoplankton. Mar. Ecol. Prog. Ser.
144:265–83.
McLenon, A.L. and DiTullio, G.R. 2012. Effects of increased tem-
perature on dimethylsulfoniopropionate (DMSP) concentra-
tion and methionine synthase activity in Symbiodinium.
Biogeochemistry 110:17–29.
Montresor, M., Sgrosso, S., Procaccini, G. & Kooistra, W. H. C.
F. 2003. Intraspecific diversity in Scrippsiella trochoidea
(Dinophyceae): evidence for cryptic species. Phycologia
42:56–70.
Nehring, S. 1994. Spatial distribution of dinoflagellate resting
cysts in recent sediments of Kiel Bight, Germany (Baltic
Sea). Ophelia 39:137–58.
Nehring, S. 1997. Dinoflagellate resting cysts from recent German
coastal sediment. Bot. Mar. 40:307–24.
Olli, K. 2001. High encystment of a dinoflagellate in batch cul-
ture. In Garce’s, E., Zingone, A., Montresor, M., Reguera, B.
& Dale, B. [Eds.] LIFEHAB Life Histories of Microalgal Species
Causing Harmful Blooms. European Commission Directorate
General, Science, Research and Development, Calvia’,
Majorca, Spain, pp. 53–6.
Olli, K. & Anderson, D. M. 2002. High encystment success of the
dinoflagellate Scrippsiella cf. lachrymosa in culture experi-
ments. J. Phycol. 38:145–56.
Persson, A., Godhe, A. & Karlson, B. 2000. Dinoflagellate cysts in
recent sediments from the west coast of Sweden. Bot. Mar.
43:69–79.
Persson, A. & Smith, B. C. 2009. Consumption of Scrippsiella
lachrymosa resting cysts by the eastern oyster (Crassostrea vir-
ginica). J. Shellfish Res. 28:221–5.
Persson, A. & Smith, B. C. 2013. Cell density-dependent swim-
ming patterns of Alexandrium fundyense early stationary phase
cells. Aquat. Microb. Ecol. 68:251–8.
Persson, A., Smith, B. C., Alix, J. H., Senft-Batoh, C. & Wikfors,
G. H. 2012. Toxin content differs between life stages of
Alexandrium fundyense (Dinophyceae). Harmful Algae 19:101–
7.
Persson, A., Smith, B. C., Dixon, M. & Wikfors, G. H. 2008b. The
eastern mud snail, Ilyanassa obsoleta, actively forages for, con-
sumes, and digests cysts of the dinoflagellate, Scrippsiella
lachrymosa.Malacalogia 50:341–5.
Persson, A., Smith, B. C., Wikfors, G. H. & Alix, J. 2008a.
Dinoflagellate gamete formation and environmental cues:
observations, theory, and synthesis. Harmful Algae 7:798–801.
Persson, A., Smith, B. C., Wikfors, G. H. & Alix, J. 2013. Differ-
ences in swimming pattern between life cycle stages of the
toxic dinoflagellate Alexandrium fundyense.Harmful Algae 21–
22:36–43.
Riseman, S. F. & DiTullio, G. R. 2004. Influence of iron on partic-
ulate DMSP and DMSO in the Peru upwelling regime. Can.
J. Fish Aquat. Sci. 61:721–35.
Rivkin, R. B., Seliger, H. H., Swift, E. & Biggley, W. H. 1982.
Light-shade adaptation by the oceanic dinoflagellates Pyrocys-
tis noctiluca and P. fusiformis.Mar. Biol. 68:181–91.
Schl€uter, L., Møhlenberg, F., Havskum, H. & Larsen, S. 2000.
The use of phytoplankton pigments for identifying and
quantifying phytoplankton groups in coastal areas: testing
the influence of light and nutrients on pigment/chlorophyll
aratios. Mar. Ecol. Prog. Ser. 192:49–63.
Smith, B. C. & Persson, A. 2004. Dinoflagellate cyst production in
one-liter containers. J. Appl. Phycol. 16:401–5.
Smith, B. C. & Persson, A. 2005. Synchronization of encystment
of Scrippsiella lachrymosa (Dinophyta). J. Appl. Phycol.
17:317–21.
Spear, A. H., Daly, K., Huffman, D. & Garcia-Rubio, L. 2009.
Progress in developing a new detection method for the
DINOFLAGELLATE LIFE STAGE PIGMENTS 73
harmful algal bloom species, Karenia brevis, through multi-
wavelength spectroscopy. Harmful Algae 8:189–95.
Spector, D. L. 1984. Unusual inclusions. In Spector, D. L. [Ed.]
Dinoflagellates. Academic Press, Orlando, Florida, pp. 365–
90.
Steidinger, K. A. & Tangen, K. 1997. Dinoflagellates. In Tomas,
C. R. [Ed.] Identifying Marine Phytoplankton. Academic Press,
San Diego, California, pp. 387–584.
Swift, I. E., Milborrow, B. V. & Jeffrey, S. W. 1982. Formation of
neoxanthin, diadinoxanthin and peridinin from [
14
C]zeax-
anthin by a cell-free system from Amphidinium carterae.Phyto-
chemistry 21:2859–64.
Takaichi, S. 2011. Carotenoids in algae: distributions, biosynthe-
ses and functions. Mar. Drugs 9:1101–18.
Underwood, A. J. 1997. Experiments in Ecology: Their Logical Design
and Interpretation Using Analysis of Variance. Cambridge
University Press, Cambridge, UK.
Wright, S. W., van den Enden, R. L., Pearce, I., Davidson, A. T.,
Scott, F. J. & Westwood, K. J. 2010. Phytoplankton commu-
nity structure and stocks in the Southern Ocean (30–801E)
determined by CHEMTAX analysis of HPLC pigment signa-
tures. Deep Sea Res. II 57:758–78.
Wyatt, T. & Zingone, A. 2014. Population dynamics of red tide
dinoflagellates. Deep Sea Res. II 101:231–6.
Yamamoto, H. Y., Nakayama, T. O. M. & Chichester, O. 1963.
Studies on the light and dark interconversions of leaf xan-
thophylls. Arch. Biochem. Biophys. 97:168–73.
Yanpei, Z., Haiyan, J., Hongliang, L., Jianfang, C., Bin, W., Fajin,
C., Youcheng, B., Yong, L. & Shichao, T. 2014. Phytoplank-
ton composition and its ecological effect in subsurface cold
pool of the northern Bering Sea in summer as revealed by
HPLC derived pigment signatures. Acta Oceanol. Sin.
33:103–11.
Zapata, M., Fraga, S., Rodr
ıguez, F. & Garrido, J. L. 2012. Pig-
ment-based chloroplast types in dinoflagellates. Mar. Ecol.
Prog. Ser. 465:33–52.
Zapata, M., Rodrıguez, F. & Garrido, J. L. 2000. Separation of
chlorophylls and carotenoids from marine phytoplankton: a
new HPLC method using a reversed phase C8 column and
pyridine-containing mobile phases. Mar. Ecol. Prog. Ser.
195:29–45.
Zhou, J. & Fritz, L. 1994. The PAS/accumulation bodies in Proro-
centrum lima and Prorocentrum maculosum (Dinophyceae) are
dinoflagellate lysosomes. J. Phycol. 30:39–44.
Zinssmeister, C., Keupp, H., Tischendorf, G., Kaulbars, F. &
Gottschling, M. 2011. Ultrastructure of calcareous dinophytes
(Thoracosphaeraceae,Peridiniales) with a focus on vacuolar crys-
tal-like particles. PLoS ONE 8:e54038.
74 AGNETA PERSSON ET AL.
