Species composition of Skeletonema (Bacillariophyceae) in planktonic ...

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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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)


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

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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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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-


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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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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. References Ando H, Kashiwagi N, Ninomiya K, Ogura H, Kawai T (2005) Changes in the state of water pollution in Tokyo Bay since 1980̶Trend analysis of water quality using monitoring data obtained by local governments̶. Ann Rep Tokyo Metropolitan Res Inst Environ Protection 2005: 141–150. (in Japanese) Chen GF, Wang GC, Zhang BY, Fan XL (2007) Morphological and phylogenetic analysis of Skeletonema costatum-like diatoms (Bacillariophyta) from the China Sea. Eur J Phycol 42: 163–175. Cheng J, Yang L, Junrong L, Yahui G, Peng W, Ho K-C, Xin L (2008) Morphological variability and genetic diversity in five species of Skeletonema (Bacillariophyta). Prog Natural Sci 18: 1345–1355. Ellegaard M, Godhe A, Härnström K, McQuoid M (2008) The species concept in a marine diatom: LSU rDNA-based phylogenetic differentiation in Skeletonema marinoi/dohrnii (Bacillariophyceae) is not reflected in morphology. Phycologia 47: 156–167. Garrison DL (1984) Chapter 1 Planktonic Diatoms. In: Marine Plankton Life Cycle Strategies (eds Steidinger KA, Walker LM). CRC Press, Florida, pp. 1–17. Godhe A, McQouid MR, Karunasagar I, Karunasagar I, RehnstamHol A-S (2006) Comparison of three common molecular tools for distinguishing among geographically separated clones of the diatom Skeletonema marinoi Sarno et Zingone (Bacillariophyceae). J Phycol 42: 280–291. Godhe A, Härnström K (2010) Linking the planktonic and benthic habitat: Genetic structure of the marine diatom Skeletonema marinoi. Mol Ecol 19: 4478–4490. Greville RK (1866) Description of new and rare diatoms. Series 20. Transactions of The Microscopical Society & Journal 14: 77–86. Hargraves PE, French FW (1983) Diatom resting spore: significance and strategies. In: Survival strategies of the algae (ed Fryxell GA). Cambridge University Press, New York, pp. 49– 68. Härnström K, Ellegaard M, Andersen TJ, Godhe A (2011) Hundred years of genetic structure in a sediment revived diatom


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