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Plankton Benthos Res 9(3): 168–175, 2014
Plankton & Benthos
Research
© The Plankton Society of Japan
Species composition of Skeletonema (Bacillariophyceae)
in planktonic and resting-stage cells in Osaka and
Tokyo Bays
MACHIKO YAMADA1,*, MAYUKO OTSUBO1, MASASHI KODAMA2, KEIGO YAMAMOTO3,
TETSUYA NISHIKAWA4, K AZUHIKO ICHIMI5, KUNINAO TADA6 & PAUL J. HARRISON7
1
Department of Environmental Science, International College of Arts and Sciences, Fukuoka Women s University,
Kasumigaoka, Higashi-ku, Fukuoka, 813–8529, Japan
2
Research Center for Fisheries Oceanography and Marine Ecosystem, National Research Institute of Fisheries Science,
Fukuura, Kanazawa-ku, Yokohama, Kanagawa, 236–8648, Japan
3
Marine Fisheries Research Center, Research Institute of Environment, Agriculture and Fisheries, Osaka Prefectural
Government, Tanigawa, Misaki, Sen-nan, Osaka, 599–0311, Japan
4
Hyogo Prefectural Technology Center for Agriculture, Forestry and Fisheries, Kasumi, Kami, Mikata, Hyogo 669–6541,
Japan
5
Aji Marine Station, Seto Inland Sea Regional Research Center, Kagawa University, Aji, Takamatsu, Kagawa 761–0130, Japan
6
Faculty of Agriculture, Kagawa University, Miki-cho, Kita-gun, Kagawa 761–0795, Japan
7
Department of Earth & Ocean Sciences, University of British Columbia, Vancouver BC V6T 1W3, Canada
Received 6 January 2014; Accepted 16 July 2014
Abstract: Species compositions of planktonic cells and benthic resting-stage cells of the marine diatom Skeletonema were investigated in Osaka and Tokyo Bays. Seven to 11 strains were isolated from each of the monthly water
samples for species identification by molecular analysis. The species frequency of the strains indicated that S. sp. cf.
marinoi-dohrnii complex was dominant throughout the year in Osaka Bay, whereas in Tokyo Bay the dominant species was replaced by S. japonicum during the cold season. The other species identified were S. costatum, S. japonicum,
S. pseudocostatum, and S. tropicum in Osaka Bay, and S. menzelii and S. tropicum in Tokyo Bay. For species identification of resting-stage cells, sediments in each bay were sampled at 2–3 stations and stored in the dark for ≥85 days.
Eight to 12 strains were isolated from each of the diluted sediments incubated at five temperatures (10–30°C).
S. costatum, S. japonicum and S. sp. cf. marinoi-dohrnii complex were identified in these stains; the resting stage of
S. japonicum was newly recognized. The dominant species of resting-stage cells in each bay largely agreed with
those of planktonic cells on a yearly basis. S. sp. cf. marinoi-dohrnii complex in the sediment could germinate at all
the temperatures but S. japonicum did not occur at 30°C. The results of seasonal occurrences and incubation experiments indicate that S. sp. cf. marinoi-dohrnii complex is eurythermal and S. japonicum prefers lower temperatures.
Possible factors for the difference in the community of Skeletonema between Osaka and Tokyo Bays are discussed.
Key words: Osaka Bay, resting-stage cell, Skeletonema, species composition, Tokyo Bay
Introduction
The marine diatom Skeletonema costatum (Greville)
Cleve was previously thought to be one of the most important phytoplankton species, because of its worldwide distribution in coastal waters except for Antarctic seas,
blooms sometimes causing red tides in eutrophic waters,
* Corresponding author: Machiko Yamada; E-mail, [email protected]
and its use in bioassays for physiological and ecological
experiments (Greville 1866, Zingone et al. 2005, Sarno et
al. 2005, Saggiomo et al. 2006, Sarno et al. 2007, Kooistra
et al. 2008). Recent taxonomic studies using scanning electron microscopic observations and molecular analyses
classified this species, which is henceforth referred to as S.
costatum sensu lato (s.l.), into eight species and thus the
genus currently consists of 11 species (Zingone et al. 2005,
Sarno et al. 2005, Sarno et al. 2007). Although S. costatum
Species of Skeletonema in Osaka & Tokyo Bays
s.l. had been well studied in both the laboratory and nature, our knowledge on species-specific characteristics of
the current 11 species is still very scarce. As for their biogeographic distributions, Kooistra et al. (2008) reported
that several species have global distribution. However,
studies on species composition of the genus following the
current taxonomy are still limited. In fact, species composition of the genus in the NW Pacific has been studied only
in the coastal waters of China (Chen et al. 2007, Cheng et
al. 2008), South Korea (Jung et al. 2009), and Japan
(Yamada et al. 2010, 2013).
Resting-stage cells, i.e. resting cells and resting spores,
are well-known in diatoms (Hargraves & French 1983,
Garrison 1984, McQuoid & Hobson 1996, Itakura et al.
1997, Ishii et al. 2012). Among the current 11 species of
Skeletonema, resting-stage cells have been reported for the
following six species: S. costatum (Grev.) Cleve emend.
Zingone & Sarno (sensu stricto) (Yamada et al. 2013), S.
dohrnii Sarno & Kooistra (Kooistra et al. 2008), S. marinoi
Sarno & Zingone (Godhe et al. 2006, Ellegaard et al. 2008,
Godhe & Härnström 2010, Härnström et al. 2011), S. menzelii Guillard et al. (Yamada et al. 2010), S. pseudocostatum Medlin emend. Zingone & Sarno (Kooistra et al.
2008, Yamada et al. 2010, Montresor et al. 2013), and
S. tropicum Cleve (Cleve) (Kooistra et al. 2008, Yamada et
al. 2009). The function of resting-stage cells of the tropical/subtropical species S. tropicum has been considered as
overwintering when they appear in the temperate coastal
zone (Yamada et al. 2010).
Considering the potential importance of resting-stage
cells for the ecology of microalgae, studies on both planktonic vegetative cells and resting-stage cells at the same
sites would give valuable information for better understanding heir ecology. However, such studies have been
limited to S. costatum s.l. (McQuoid 2002, McQuoid et al.
2002, McQuoid & Godhe 2004) and S. marinoi (Godhe &
Härnström 2010, Härnström et al. 2011, Smayda 2011). We
identified species for both planktonic vegetative cells and
resting-stage cells in Osaka and Tokyo Bays, where S.
costatum s.l. is the dominant phytoplankton species
throughout year and sometimes blooms (Nomura 1998,
Hoshika & Jou 2002, Yamaguchi 2011), in order to assess
species composition, dominant species, and seasonal succession in their two life stages in each bay. In this paper we
present the results and compare them between the two
bays.
Materials and Methods
Sampling of Skeletonema and water quality
The surface seawaters in Osaka Bay were sampled at
Stn 15 with a bucket once a month from April 2009 to
March 2010, and those in Tokyo Bay were sampled at Stn
T8 from August 2008 to July 2010 and Stn 2 from October
2008 to September 2010 (Fig. 1). Sampling was generally
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made in the first ten days of the month at Stns 15 and T8
and in the last ten days of the month at Stn 2, at sampling
intervals of ＞19 days at each station. In these surveys,
temperature of the surface water was measured using a
CTD (model ACL215, JFE Advantech, Japan) simultaneously with sampling. For measurement of vegetative cell
density of Skeletonema, 250 mL (Stns 15 and 2) or 2 L (Stn
8) of the water was fixed immediately after sampling with
glutaraldehyde at Stn 15, buffered formalin at Stn 2, and
Lugol s solution at Stn T8; samples for density measurements were not collected on two occasions (at Stn 15 in
Osaka Bay on April 2009 and at Stn 2 in Tokyo Bay in October 2009). For species identification, about 250 mL of the
water was kept in an ice box and brought to the laboratory
for culture. In addition to the monthly samples, a surface
water sample taken at Stn A in Osaka Bay on 16 June 2008
by the same method was used for species identification.
Bottom sediments for species identification of restingstage cells in Osaka Bay were sampled at Stn D on 29 July
2008, Stn 15 on 9 April 2009 and Stn 5 on 31 May 2010,
and those in Tokyo Bay were sampled at Stn T8 on 2 February 2010 and at Stn 2 on 28 May 2010. Sampling gears
used were a KK-type core sampler (Kimata et al. 1960) at
Stns D and 15, and a grab-type sediment sampler at the
other stations. The top (＜1.0 cm) of the sediment in the
sampler was taken by slicing for core samples or by scraping with a spoon for grab samples, and kept in a cool box
with refrigerant until processing in the laboratory.
Density, isolation and cultivation of Skeletonema cells
The fixed surface water samples were settled for
＞24 hours and examined under a light microscope to determine the cell density of Skeletonema. Enumeration was
done on a Sedgwick Rafter counting cell chamber for lowdensity samples and a hematocytometer for high-density
samples.
Seven to 11 clonal strains of Skeletonema were established from each of the seawater samples by isolating a
colony of 5–10 cells with a micropipette under an inverted
microscope. Since the colony densities of Skeletonema at
Stn 15 of Osaka Bay in December and Stn 2 of Tokyo Bay
in October, November, March, and April were too low to
be isolated directly from the water samples, cells in these
samples were grown in a batch culture under the same conditions as in the culture for clonal strains. Strains were
maintained in ESM medium (Okaichi et al. 1983) enriched
with silicate (88 μM Na2SiO3 · 9H2O) but without soil extract nor Tris buffer, and were incubated in test tubes on a
14 h light (100 μmol photons m−2 sec−1) : 10 h dark cycle at
15°C during the colder season from December to April and
23°C during the warmer season from May to November.
The sampled sediments were stored at 4°C in the dark
for ≥85 days until cells were isolated, by which we assume
that the viable cells isolated were from resting stages. To
stimulate resting-stage cells to germinate, the sediments
were suspended in water at five different dilutions and
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M. Yamada et al.
Fig. 1.
Maps showing the four sampling stations in Osaka Bay and the two stations in Tokyo Bay.
incubated under five different temperature conditions. Suspensions of the sediment were made using a stepwise dilution method as follows. One gram (approximately 1 mL) of
the sediment was suspended in 9 mL of ESM medium as
the initial (100) suspension, from which the suspension of
the five dilutions from 10 –1 to 10 –5 were made by adding
5 mL of the previous step suspension to 45 mL of ESM
medium. One milliliter of each dilution was pipetted to
each of five wells of five sterilized 48-well plates (i.e. in
total 25 wells for each dilution), and the five plates were incubated for at most 20 days at 10, 15, 20, 25, and 30°C, respectively, under the same light conditions as for the
strains isolated from seawater. One strain of Skeletonema
was isolated from each of the wells in which vegetative
cells occurred, and eight to 11 strains in Osaka Bay and
eight to 12 strains in Tokyo Bay were isolated from the 25
wells incubated at each temperature for each sediment
sample. Isolated colonies were maintained under the same
conditions as those for maintenance of the vegetative cells
as described above.
Species identification
Species of the strains were identified by molecular analysis of the large subunit (LSU) rDNA. Yamada et al. (2010)
found mysterious strains from Dokai Bay, which were
identified as S. dohrnii by LSU rDNA but as S. marinoi by
SSU rDNA. This species is considered to be a member of
the S. marinoi-dohrnii complex (Yamada et al. 2010). We
therefore applied the SSU rDNA analysis to 50 strains (27
from Osaka Bay and 23 from Tokyo Bay) of the 420 strains
identified as S. dohrnii by LSU rDNA. In previous papers
(Yamada et al. 2010, Kaeriyama et al. 2011) the name S.
marinoi-dohrnii complex was used for this species, but S.
sp. cf. marinoi-dohrnii complex is used in this paper because the term complex means a group of species. Protocols of DNA extraction, purification, PCR, and DNA sequence data analysis in the present study were the same as
those of Yamada et al. (2010), except for the four primers
(SSU471F: 5′-TAA CAA TGA CGG GCC TTT AC-3′;
SSU933F: 5′-TGC GAA AGC ATT TAC CAA GG-3′; SSU1400F: 5′-GAT GAA GGA AGA TGG CGG CA-3″; and
SSU-643R: 5′-CCT GCC AGA AAT CCA ACT ACG-3)
Species of Skeletonema in Osaka & Tokyo Bays
used for sequence analysis of SSU.
The species of the strains identified by molecular analysis were confirmed by morphological characters using a
scanning electron microscope (VE-7800, Keyence, Osaka,
Japan). Methodology for the observations was the same as
that in Yamada et al. (2010).
Results
The temperature of the surface water at Stn 15 in Osaka
Bay varied seasonally with a range of 8.8–26.9°C, and
at Stns T8 and 2 in Tokyo Bay between 10.9–26.1 and
10.1–27.8°C, respectively (Fig. 2). Seasonal ranges of
the cell densities of Skeletonema at Stns 15, T8 and 2
were 0–1.4×104, 5.3×10–1.1×104, and 4.0×10−2–1.4×
103 cells mL−1, respectively; the minimum density
(0 cells mL−1) at Stn 15 was recorded in December 2009
but vegetative cells increased in the cultured seawater from
the same month. The maximum cell densities occurred in
August in Osaka Bay and in July in Tokyo Bay. Blooms of
＞8.0×103 cells mL−1 were formed in March and August in
Osaka Bay and in July in the inner part (Stn T8) of Tokyo
Bay.
In total, 588 sequences were determined in this study, of
which 35 sequences were unique in each bay and have
171
been deposited in the DNA Data Bank of Japan (DDBJ). A
total of 115 strains of five species, Skeletonema costatum
(5 strains), S. japonicum (7 strains), S. pseudocostatum (3
strains), S. tropicum (3 strains), and S. sp. cf. marinoi-dohrnii
complex (97 strains), were isolated from the surface water
of Osaka Bay (Fig. 3). Skeletonema costatum occurred
only in three months from August to November, S. japonicum in February and March, and S. pseudocostatum and S.
tropicum each in a single month, June 2008 at Stn A and
November 2009 at Stn 15, respectively. Skeletonema sp. cf.
marinoi-dohrnii complex occurred throughout the year and
was most frequently isolated, comprising 87% of strains of
the four species at Stn 15.
A total of 109 strains of two species, S. japonicum (64
strains) and S. sp. cf. marinoi-dohrnii complex (45 strains),
were isolated from the surface water at Stn T8 in Tokyo
Bay (Fig. 3). Skeletonema japonicum appeared in nine
months from October to June and was more frequently isolated than S. sp. cf. marinoi-dohrnii complex during the
colder half of the year (from November to June) except for
December. Skeletonema sp. cf. marinoi-dohrnii complex
appeared in the seven months from June to January, and
was absent during the colder five months (November and
from February to May). At Stn 2 in Tokyo Bay, 116 strains
of four species, S. japonicum (30 strains), S. menzelii (1
Fig. 2. Seasonal variations in surface water temperature (squares, circles and triangles) and Skeletonema spp. cell density
(bars) at Stn 15 in Osaka Bay and Stns T8 and 2 in Tokyo Bay. Horizontal broken lines indicate the level of 8.0×
103 cells mL –1.
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M. Yamada et al.
Fig. 3. Seasonal variation in the strain numbers of Skeletonema spp. isolated from the surface waters of Osaka and Tokyo
Bays. Isolation was done on 7–11 strains for each sample.
strains), S. tropicum (6 strains), and S. sp. cf. marinoidohrnii complex (79 strains), were isolated. Skeletonema
japonicum was observed in the colder half of the year from
January to June, and was the most frequently isolated species in January, March and April. Skeletonema menzelii
and S. tropicum occurred only in June and September, respectively. Skeletonema sp. cf. marinoi-dohrnii complex
occurred throughout the year, except in January and
March, and was the dominant species except in the three
months when S. japonicum was dominant and in September when S. tropicum was dominant.
From the sediment collected in Osaka Bay, a total of 143
strains of three species, S. costatum (3 strains), S. japonicum
(2 strains), and S. sp. cf. marinoi-dohrnii complex (138
strains), were isolated (Fig. 4). Skeletonema sp. cf. marinoidohrnii complex was the only or the dominant species at
all incubation temperatures. Skeletonema costatum and
S. japonicum appeared only once at Stn D in the 25°C incubation and at Stn 5 in the 10°C incubation, respectively.
The strains from the sediment collected on 6 April 2009 at
Stn 15 were all S. sp. cf. marinoi-dohrnii complex.
From the sediment collected in Tokyo Bay, a total of 105
strains of two species, S. japonicum (55 strains) and S. sp.
cf. marinoi-dohrnii complex (50 strains), were isolated
(Fig. 4). The strain numbers collected at Stn T8 were equal
between the two species at 10°C. At higher temperatures,
S. japonicum strains decreased in number with temperature and did not occur at all at 30°C. The strain number of
S. sp. cf. marinoi-dohrnii complex increased with increasing incubation temperature, with the largest number (11
strains) at 25°C. At Stn 2, strains incubated at 10–25°C
were mostly S. japonicum, whereas those at 30°C were
contrastingly all S. sp. cf. marinoi-dohrnii complex.
Discussion
Species diversity of Skeletonema
Skeletonema costatum s.l. has been traditionally known
as the dominant phytoplankton species and a major cause
of red tides in Osaka and Tokyo Bays (Nomura 1998, Hoshika & Jou 2002, Yamaguchi 2011). The present study re-
Species of Skeletonema in Osaka & Tokyo Bays
173
Fig. 4. The strain numbers of Skeletonema spp. isolated from the overlying water in the sediment cultures taken from Osaka
and Tokyo Bays and incubated at five temperatures from 10–30°C. Isolation was done on 8–12 strains for each sample. Sampling date of each sediment sample in parentheses.
vealed that S. costatum s.l. consists of five species (S.
costatum, S. japonicum, S. pseudocostatum, S. tropicum,
and S. sp. cf. marinoi-dohrnii complex) in Osaka Bay and
four species (S. japonicum, S. menzelii, S. tropicum, and S.
sp. cf. marinoi-dohrnii complex) in Tokyo Bay. The species richness of Skeletonema identified by molecular analysis in other temperate bays are four in the Gulf of Naples
(Sarno et al. 2005, Saggiomo et al. 2006, Montresor et al.
2013), three in San Francisco Bay (Saggiomo et al. 2006),
two in Narragansett Bay (Saggiomo et al. 2006), and seven
in Dokai Bay (Yamada et al. 2010). The species richness in
Osaka and Tokyo Bays is lower than in Dokai Bay, but
comparable to or greater than in the other bays listed
above. However, the species richness of Skeletonema identified by molecular analyses in the previous and present
studies is probably underestimated, because the numbers
of cells or colonies used for identification were too few to
detect rare species. For example, the number of strains isolated for species identification in the present study was
seven to 11 in each month in Osaka and Tokyo Bays. In
this case, a rare species occupying 1% of the community,
which is not a low value for rare species in phytoplankton
communities, could not be detected with ≥90% probability.
The present study revealed that S. japonicum is one of
the dominant species in resting-stage cells in Tokyo Bay.
Since six other species of the genus are known to have a
resting stage, as mentioned above, S. japonicum is the seventh species having a resting stage. Among the species
identified in the present study, S. menzelii, S. pseudocostatum, and S. tropicum did not occur in the strains isolated
from the sediment, although their resting stages are known
(Kooistra et al. 2008, Yamada et al. 2010, Yamada 2013).
As suggested above, as with the underestimation of the
species richness in the plankton community, it is possible
that the absence of their resting-stage cells in the present
study might be an artifact due to rarity of their viable restingstage cells and to the small numbers of strains analyzed.
Comparison between Osaka and Tokyo Bays
The present study in Osaka Bay revealed that Skeletonema
sp. cf. marinoi-dohrnii complex outnumbered other congeners almost always throughout the year and that restingstage cells of the species could germinate at all temperatures from 10 to 30°C. Skeletonema japonicum occurred in
the plankton community only during the coldest two
months in Osaka Bay and was limited to the colder half or
three quarters of the year in Tokyo Bay. Resting-stage cells
of this species did not germinate at the highest incubation
temperature. These results indicate that S. sp. cf. marinoidohrnii complex is eurythermal while S. japonicum is less
eurythermal and prefers lower temperature than S. sp. cf.
marinoi-dohrnii complex.
The difference in the communities of Skeletonema between Osaka and Tokyo Bays is remarkable. The community in Osaka Bay is dominated only by S. sp. cf. marinoidohrnii complex, whereas that in Tokyo Bay is dominated
by two species, i.e. S. sp. cf. marinoi-dohrnii complex during the warm season and S. japonicum in the cold season.
The difference in the communities of Skeletonema between the two bays is probably attributable to differences
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M. Yamada et al.
in some environmental conditions such as temperature and
water quality.
The physiological preferences of the two dominant species described above suggests that temperature is the most
plausible factor explaining the different communities, although the present study showed no marked difference in
the surface water temperature between the two bays. According to published information, however, the bottom
temperature is considered somewhat higher in Osaka Bay
than in Tokyo Bay. Yamane et al. (1996) reported that the
average monthly bottom temperature for 20 years from
1973 to 1992 in the central part (about 50 m deep) of Osaka
Bay varied seasonally between 9 and 25°C (read from their
fig. 4). Although we could not find the corresponding average data for the same period in Tokyo Bay, the data for
bottom water temperature in Tokyo Bay in 1987 measured
by the Yokohama Environmental Science Research Institute (Ninomiya et al. 2009) are available for comparison.
According to their figure (Ninomiya et al. 2009, fig. 1), the
bottom water temperature at a station (30 m deep) located
in the central part of the inner bay ranged between 10 and
20°C. Considering that bottom water temperature is generally lower at deeper stations, the above information suggests that the bottom temperature is somewhat higher in
Osaka Bay than in Tokyo Bay. Accordingly, it is possible
that the higher bottom temperature in Osaka Bay is favorable for S. sp. cf. marinoi-dohrnii complex over to S.
japonicum. However, more detailed comparison of temperatures together with seasonal abundance data for each species for the two bays is necessary, because our data and the
above information on temperature is not enough to prove a
significant effect of temperature on the species.
Water quality may be another factor affecting the species composition of Skeletonema. According to recent reports of public organizations, however, water qualities are
similar between Osaka and Tokyo Bays. For example, annual mean CODs in recent years have ranged between 3
and 4 mg L –1 in the inner halves of both Osaka Bay (Osaka
Prefectural Government 2013) and Tokyo Bay (Ando et al.
2005).
Comparison between planktonic vegetative cells and
benthic resting cells
The present study revealed that the dominant species of
benthic resting-stage cells of Skeletonema largely agreed
with those of planktonic vegetative cells on a yearly basis
in each bay. In Tokyo Bay, however, the dominant species
in the resting-stage cells, which were collected in February
(Stn T8) and May (Stn 2), differed from those in the planktonic cells collected in the same months, i.e. the dominant
species in resting-stage cells and planktonic cells were S.
sp. cf. marinoi-dohrnii complex and S. japonicum, respectively, in February, and the reverse in May. The discrepancy in the species composition between the benthic resting-stage and planktonic cells is only natural because resting-stage cells in the sediment are considered to have ac-
cumulated over a long time.
Acknowledgements
We sincerely thank anomynous reviewers and the editor
for their critical comments, which improved the manuscript. We express our sincere appreciation to Dr. Hisako
Ogura of the Chiba Prefectural Environmental Research
Center for providing samples and water quality data. We
are very grateful to Mses. Tomoko Hidaka, Sana Tashiro,
and Yumi Fujino of Fukuoka Women s University, who
contributed to this study as their graduation theses. This
research was partially supported by the Osaka Bay Regional Offshore Environmental Improvement Center,
Grant-in-Aid for Osaka Bay Phoenix, 2009–2011,
20580210.
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