JOURNAL OF PLANKTON RESEARCH j VOLUME 27 j NUMBER 4 j PAGES 323–330 j 2005

Distribution of the cyanobacterium Richelia intracellularis as an epiphyte of the compressus in the western Pacific Ocean

FERNANDO GO´ MEZ1*, KEN FURUYA2 AND SHIGENOBU TAKEDA2 1 STATION MARINE DE WIMEREUX, UNIVERSITE´ DES SCIENCES ET TECHNOLOGIES DE LILLE, CNRS-UMR 8013 ELICO,28 AVENUE FOCH, BP 80, F-62930 2 WIMEREUX, FRANCE, DEPARTMENT OF AQUATIC BIOSCIENCES, THE UNIVERSITY OF TOKYO,1-1-1 YAYOI, BUNKYO, TOKYO 113–8657, JAPAN.

*CORRESPONDING AUTHOR: fernando.gomez@fitoplancton.com

Received November 30, 2004; accepted in principle February 10, 2005; accepted for publication February 21, 2005; published online March 3, 2005

The few available records on the association of the heterocystous cyanobacterium Richelia intracellularis epiphytically with Chaetoceros compressus are restricted to the Indian and western Pacific Oceans, whereas the association of R. intracellularis as an in other is ubiquitous in warm oceans. From ten cruises in the western Pacific Ocean, the Richelia-Chaetoceros consortia were exclusively observed in the periphery of the geographic proliferations of C. compressus, coinciding with the overlapping area of the populations of asymbiotic C. compressus and R. intracellularis as an endosymbiont in Rhizosolenia clevei.

INTRODUCTION verified owing to lack of illustrations (Marumo and The fixation of atmospheric in the sea is an Asaoka, 1974; Hallegraeff and Jeffrey, 1984). important source of new nitrogen to ocean surface Some confusion exists on the first record of R. intracellularis. waters, stimulating phytoplankton productivity and fuel- Lemmermann (Lemmermann, 1899) described the hetero- ling the biological pump. In tropical waters the non- cystous cyanobacterium Calothrix rhizosoleniae as an epiphyte heterocystous cyanobacterium Ehrenberg growing on Rhizosolenia, and no further records are available ex Gomont is largely responsible for nitrogen fixation in the literature. Schmidt (Schmidt, 1901) found a similar (Karl et al., 2002), but unicellular diazotrophic cyano- cyanobacterium as an endosymbiont in Rhizosolenia.Being may also play a role (Zehr et al., 2001). High unable to decide if this was effectively conspecific with temperatures exclude heterocystous from C. rhizosoleniae, he described it as R. intracellularis. Later, warm oceans (Staal et al., 2003), with the exception of Lemmermann (Lemmermann, 1905) reported C. rhizosole- the diazotroph heterocystous cyanobacterium Richelia niae to occur as an endosymbiont in Rhizosolenia and Hemi- intracellularis Schmidt. This cyanobacterium has been aulus and as an epiphyte on Chaetoceros.Carpenter shown to provide quantitatively substantial inputs of (Carpenter, 2002) used the name Calothrix for a cyanobac- nitrogen on regional scales (Carpenter et al., 1999). terium found growing epiphytically on Chaetoceros,as Richelia intracellularis is an extracellular endosymbiont opposed to the name Richelia which was used when the which locates itself in the periplasmic space between the cyanobacterium occurred as an endosymbiont. This con- plasmalemma and silica wall in the diatoms Rhizosolenia trasts with previous usage by the same author (Janson et al., clevei Ostenfeld, Hemiaulus spp. and Guinardia cylindrus 1999). According to Janson et al.(Jansonet al., 1999) the (Cleve) Hasle (Sundstro¨m, 1984; Villareal, 1992). Records filaments of R. intracellularis seen on Chaetoceros matched those of it growing on cells of Chaetoceros Ehrenberg are rarer described by Lemmermann. Several reviews also reported (Okamura, 1907; Karsten, 1907; Norris, 1961; Sournia, R. intracellularis growing as an epiphyte on Bacteriastrum Shad- 1968; Janson et al., 1999), and not all reports can be bolt (Villareal, 1992; Rai et al., 2000; Carpenter, 2002),

doi:10.1093/plankt/fbi007, available online at www.plankt.oupjournals.org Journal of Plankton Research Vol. 27 No. 4 Ó Oxford University Press 2005; all rights reserved JOURNAL OF PLANKTON RESEARCH j VOLUME 27 j NUMBER 4 j PAGES 323–330 j 2005

although this does not appear to have been recorded photo- 160 W. Samples were collected from 9 stations at 14 depths graphically. between 5 and 200 m depth; (iv) Six cruises were carried out Janson et al.(Jansonet al.,1999) analysed the hetR at Station H on board R/V Oshoro Maru and Station A7 on sequence of R. intracellularis growing intracellularly in several board R/V Wakataka Maru in the Oyashio area during the hosts and also epiphytically on Chaetoceros. These authors spring and summer of 2003. pointed out that R. intracellularis is the only marine species of All samples were collected using Niskin bottles, pre- its genus (Richelia sinica Shen et Li is a freshwater species), and served with acidified Lugol’s solution (Hasle and Syvertsen, it is able to form associations with diatoms belonging to 1997)andstoredat5C. Subsamples (400 mL) were several different genera besides showing a considerable var- allowed to settle in glass sedimentation cylinders. The top iation in size and morphology (Janson et al., 1999). 350 mL of each sample was siphoned off progressively over In warm oceans, the occurrence of R. intracellularis 5 days using a length of small-bore tubing. The remaining growing as an epiphyte on Chaetoceros is rare. Based on 50 mL was settled in composite sedimentation chambers an analysis of several locations in the western Pacific and observed using a Nikon inverted microscope equipped Ocean, we found some spatio-temporal trends of the with a Nikon digital camera. In all cruises except for the occurrence of the Chaetoceros-Richelia consortia. The May 2002 one, cells of Rhizosolenia with and without results of this analysis are reported here. R. intracellularis were counted separately. From each sampled depth, 5 mL of water was frozen at METHOD 20 C for dissolved inorganic nutrient analysis. After thaw- ing, nutrient concentration was determined according to Samples were collected during ten cruises in the western Parsons et al. (Parsons et al., 1984), using a TRAACS 800 Pacific Ocean (Fig. 1): (i) Two cruises were carried out on for the Soyo Maru cruises (http://ss.nrifs.affrc.go.jp/eiyo/ board R/V Soyo Maru (13–20 May and 3–10 July 2002) cruise/index.html) or TRAACS 2000 (Nishida, 2004). along the 138 E meridian in the vicinity of the Kuroshio The section plots of the distribution of the variables were Current. Nine stations were sampled from 30300 to 34150 produced by interpolating between casts using the kriging N in May and 10 stations from 30 to 34200N during the as the gridding method in the Surfer software (Golden July cruise. At each station, 15 depths from 5 to 200 m were Software Inc.). sampled; (ii) R/V Hakuho Maru visited the Celebes, Sulu and SouthChinaSeasfrom7Novemberto18December2002. RESULTS Samples were collected from 10 stations at six depths from 0 to 150 m depth. The Sulu Sea is a poorly studied region of the world’s oceans, and this study sampled three stations in that area; (iii) A cruise was carried out on board R/V Mirai Based on the distribution of the physical variables along (15–28 January 2003) along the Equator from 160 Eto the 138 E meridian in the south of Japan, three regions

Fig. 1. Map of the station locations in the western Pacific Ocean. The insets show the Kuroshio and Oyashio Currents surrounding Japan and the Celebes, Sulu and South China Seas.

324 GO´ MEZ ET AL. j RICHELIA INTRACELLULARIS AS AN EPIPHYTE OF C. COMPRESSUS

were identified in the study area: the slope waters, the detection limit (0.1 mM) (Fig. 3E). Subsurface fluorescence Kuroshio Current and the offshore subtropical waters. maxima were shallower in the Sulu Sea compared to the The hydrographical conditions differed between the two South China and Philippine Seas. In the Celebes Sea at cruises. In May, the cyclonic gyre along the slope waters station 3, fluorescence was highest at 30 m depth and (Station 3) made the Kuroshio Current more narrow C. compressus reached an abundance of up to 1150 cell L–1. around Station 6 compared to the July cruise, when Microplankton vertical distribution showed a deep (75– the Kuroshio area extended from station B2 (Fig. 2A 100 m) maximum dominated by Chaetoceros atlanticus var. and B). In May, the Kuroshio Current salinity (34.7) neapolitanus (Schro¨der) Hustedt, Thalassionema nitzschioides did not differ from the surrounding offshore subtropical (Grunow) Mereschkowsky, Pseudo-nitzschia spp. and Bacter- waters (Fig. 2C). In July, the Kuroshio Current was iastrum spp. In the surface waters only dinoflagellates, the wider, warmer and salinity was lower (<34.2) (Fig. 2D). nitrogen-fixer Trichodesmium and R. clevei were found, the In both cruises, nitrate and phosphate concentrations latter with a low abundance (<5cellL–1). In the southern were highest in the onshore waters. The Kuroshio Cur- Sulu Sea one colony of the Chaetoceros-Richelia consortium rent was associated with a strong decline in nutrients, was found in the surface waters both at stations 4 and 6 especially nitrate at and around the surface (Fig. 2E–H). (Table I, Fig. 4J and K). In station 4, C. compressus reached Nitrate was exhausted in offshore waters especially in maximum abundances of 55 and 235 cells L–1 at 30 and 50 July (Fig. 2E and F). A slight surface maximum was m depth, respectively; at station 6 it ranged from 15 to 75 observed at Station 10 in May (Fig. 2E). cells L–1 between 20 and 75 m depth. In May, chlorophyll fluorescence was highest near the surface in the slope waters whereas in July a deep max- Other areas imum was present at 70–80 m depth in the offshore In the western equatorial Pacific and Oyashio cruises, no subtropical waters (Fig. 2I). In July, fluorescence was occurrence of the Richelia-Chaetoceros consortium was lower in the slope waters whereas in May a deeper and found and C. compressus did not proliferate either. thinner maximum was present at 90–100 m depth in the offshore waters (Fig. 2J). During both cruises, microphy- Morphological observations toplankton abundance was highest in the most inshore Colonies of C. compressus usually contained 10–16 diatom stations. In May the diatom community was dominated cells and 1–9 epiphytic filaments of R. intracellularis, with by Chaetoceros denticulatus and Guinardia delicatula and it the shorter diatom colony containing more numerous reached a maximum abundance of 60300 cell L–1. Richelia filaments per cell (Fig. 4M and N; Table I). Chaetoceros compressus reached 8080 cell L–1 (Fig. 2K). In The epiphytic filaments of R. intracellularis were strongly July, maximum diatom abundance was 5260 cell L–1 attached to the diatom cells in the spaces between and C. compressus reached a maximum of 670 cell L–1 adjacent cells of the colony, and were oriented transver- in subsurface waters (Fig. 2L) (Table I). sely with respect to the axis of the colony (Fig. 4G–N). In May, two colonies of the Richelia-Chaetoceros consor- Typically, the single terminal Richelia was tium were observed at 30 m depth (33 N, 138 E) (Table I, similar in size to the vegetative cells or slightly smaller Fig. 2). The maximum abundance (10 colonies L–1) (Fig. 4I), but larger ones were also seen (Fig. 4K). occurred in July at 33450 N, 138 E at 20 m depth. One Chaetoceros compressus colonies containing epiphytic R. consortium was observed at 5, 10 and 30 m depth intracellularis filaments lacked the usual numerous small (Table I). In May, the population enumerated as R. clevei plate-like chloroplasts and therefore appeared to be in the inshore waters probably corresponded to Rhizosolenia senescent. The Richelia-Chaetoceros consortia appeared to styliformis (Fig. 2M). In July, Rhizosolenia specimens contain- be associated with unidentified epiphytic naviculoid dia- ing R. intracellularis were counted independently of non- toms on Chaetoceros cells (Fig. 4G–N). In the Sulu Sea, R. intracellularis-containing cells. The maximum abundance one of the colonies examined also contained small green of the Richelia-Chaetoceros consortia coincided with the coccoid cells (Fig. 4J and K). Unattached filaments of maximum abundance of the R. intracellularis-containing R. intracellularis (Fig. 4C and D) occurred in locations R. clevei population (30 cell L–1) and also the maximum where filaments were also found on the surface of sym- abundance of unattached R. intracellularis filaments biotic diatom plasmalemma lacking the (Fig. 4E (Fig. 2N). and F) and as endosymbiont (Fig. 4A and B). In addition, in other stations in the Sulu Sea, two Sulu/Celebes Seas filaments of a heterocystous free-living cyanobacterium Alowsalinity(<33.1) and warm (>29C) water layer was joined at the level of the were found at 100 m present at the surface (Fig. 3A and C). In the surface waters depth (Station 10; 8500 N, 121480 E) (Fig. 4O–Q). of stations 4 and 6, nitrate concentrations were below These filaments, which appeared to be in a viable

325 JOURNAL OF PLANKTON RESEARCH j VOLUME 27 j NUMBER 4 j PAGES 323–330 j 2005

Fig. 2. Section plots of (A and B) temperature (C), (C and D) salinity, (E and F) nitrate (mM), (G and H) phosphate (mM), (I and J) fluorescence (relative units), (K and L) abundance of Chaetoceros compressus (cell L–1), (M) Rhizosolenia clevei with and without Richelia intracellularis (that corresponded to R. styliformis in coastal waters) and (N) R. clevei with R. intracellularis (cell L–1) along the meridian 138E in the south of Japan in May and July, respectively. The black circles point to the occurrence of Richelia-Chaetoceros consortia.

326 GO´ MEZ ET AL. j RICHELIA INTRACELLULARIS AS AN EPIPHYTE OF C. COMPRESSUS

Table I: Date, geographical coordinates (latitude, longitude), depth in meters, size of the colony of Chaetoceros compressus and number of filaments of Richelia intracellularis per colony of Chaetoceros

Date Latitude (north) Longitude (east) Depth (m) Chaetoceros compressus Richelia intracellularis (cell per colony) (filaments per colony)

11 May 2002 33 138 –30 11 4 11 May 2002 33 138 –30 10 5 8July2002 33450 138 –5 10 3 8July2002 33450 138 –10 18 5 8July2002 33450 138 –20 11 4 8July2002 33450 138 –20 14 9 8July2002 33450 138 –20 16 6 8July2002 33450 138 –20 12 1 8July2002 33450 138 –30 3 6 21 November 2002 7250 121120 025 7 22 November 2002 6540 119110 020 5

Fig. 3. Section plots of (A) temperature (C), (B) fluorescence (relative units), (C) salinity, (D) abundance of Chaetoceros compressus (cell L–1), (E) nitrate (mM) and (F) Rhizosolenia clevei (cell L–1) in the Celebes, Sulu and South China Seas. Please note that the distance among the stations is not proportional. The black circles point to the occurrence of Richelia-Chaetoceros consortia. condition, occurred at 100 m depth, with no other heterocyst (Fig. 4O–Q). Their colour, size and morphol- filamentous cyanobacteria present in the surrounding ogy was reminiscent of R. intracellularis but this has never waters. The smaller (50 mm) filament had one terminal been documented to possess intercellular heterocysts. heterocyst, analogous to R. intracellularis, whereas the This is consistent with our observation that filaments of larger (170 mm) one had a 9-mm intercellular spherical R. intracellularis rarely appeared below 50 m depth.

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Fig. 4. Photomicrographs of filamentous heterocyst-forming cyanobacteria. (A and B) Richelia intracellularis as an endosymbiont in Rhizosolenia clevei var. clevei.(C and D) Unattached filaments of R. intracellularis.(E and F) R. intracellularis as an epiphyte on the plasmalemma of Rhizosolenia lacking the frustule. (G–N) R. intracellularis as an epiphyte on Chaetoceros compressus.(I) R. intracellularis with heterocyst similar in size and (K) larger than the vegetative cells. The arrows point to epiphytic green coccoid cells. (M, N) Note the thin hyaline sheath in some specimens (see arrow). (O–Q)An unknown free-living cyanobacterium with intercellular heterocyst. Scale bars represent 10 mm.

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DISCUSSION as well as apparently free-living was often found in the regions where the consortia were observed (Fig. 4E and F). Occurrence of the Richelia-Chaetoceros We hypothesize that unattached filaments of R. intracellu- consortia laris (Fig. 4C–D) originating from R. clevei are able to colonise senescent colonies of Chaetoceros lacking epiphytic Richelia-Chaetoceros The consortia appeared in a restricted R. intracellularis. Observations on R. intracellularis growing as range of environmental conditions. During both cruises an endosymbiont in R. clevei showed that gas-vesicles were their occurrence was restricted to the transition zone absent and therefore the cyanobacterium cells might be between the slope waters and the Kuroshio Current in unable to regulate their position in the water column a transect of more than 500 km. (Janson et al., 1995). Consequently, unattached filaments Obviously, the occurrence of the Richelia-Chaetoceros should not be viable and therefore a would be consortia requires the preliminary presence of both the required. After the contact between the cyanobacterium Chaetoceros diatom host cells and the cyanobacterium. and the diatom, one filament of R. intracellularis colonises a compressus is a common member of the neritic diatom cell of the C. compressus colony (Fig. 4G) and then spreads assemblage in the Japanese coastal waters, whereas it is out to other diatom cells (Fig. 4H and J). less common in open waters (Okamura, 1907; Furuya The presence of Richelia in Chaetoceros is often associated and Marumo, 1983). Its presence in the Kuroshio Cur- with epiphytic pennate diatoms, tentatively identified as a rent can be related to mixing with coastal waters during species of the Pseudo-nitzschia americana complex (Lundholm the eastward transit of the Kuroshio Current along et al., 2002). It is possible that these epiphytic diatoms southern Japan (Furuya and Marumo, 1983). In the consume part of the N2-origin nitrogen excreted by Richelia Sulu Sea, a semi-enclosed deep basin isolated from the (Fig. 4M and N). This exchange, which does not exist in surrounding ocean and connected to the Celebes Sea via the case of Richelia growing as an endosymbiont inside many passages through the Sulu Archipelago, it is pos- diatom cells, could decrease the efficiency of the transfer sible that the colonies of C. compressus found in the sur- of nitrogen to C. compressus. Chaetoceros colonies cannot face nitrate-limited waters were associated with an survive in oligotrophic conditions and likely decompose plume. During the boreal summer, surface as a result. In these conditions Richelia continues to grow. water drifts southwards through the Sulu Archipelago Higher number of Richelia filaments per diatom cell was into the Celebes Sea, whereas in winter this drift is found in shorter colonies (Table I). reversed (Metzger and Hurlburt, 1996). Prevailing local This could be the first stage of a future successful sym- winds can alter the flow in the surface waters, resulting biotic association when the efficiency in the exchanges of C. compressus in moderate upwelling and export of to the nutrients between the nitrogen fixer and the host increase. southern Sulu Sea from the Sulu Archipelago. This association is now temporal and it may not facilitate that the host survive in oligotrophic waters expanding to other geographical areas. The association is restricted to Hypothesis on the origin of epiphytic Richelia the transition area where the distribution of R. intracellularis growing on Chaetoceros as an endosymbiont in diatoms and that of C. compressus From our observations, it appears that the occurrence of overlaps. To the best of our knowledge, records of Richelia- Richelia-Chaetoceros consortia was associated with the con- Chaetoceros consortia are restricted to the Indian and wes- temporary presence of C. compressus and R. intracellularis as tern Pacific Oceans. Chaetoceros compressus is widely distrib- an endosymbiont in other diatoms. Chaetoceros compressus is uted in these waters, and R. intracellularis growing as an a tropical species originally described from Hong Kong, endosymbiont in R. clevei is ubiquitous in warm oceans. which is not to be confused with C. contortum,atemperate Although the distribution of the Richelia-Chaetoceros consor- to northern species (Rines, 1999). Janson et al.(Janson tia in the world oceans is less understood compared to et al.,1999) reported that hetR gene sequences of R. intra- endosymbiotic cyanobacteria, further studies of the nature cellularis growing endosymbiotically in R. clevei are closely of the Chaetoceros-Richelia consortia will contribute to a related to those of R. intracellularis growing epiphytically on better knowledge of and nitrogen fixation in Chaetoceros. Non-attached filaments of R. intracellularis, the sea. which appeared to be free-living, were found in the same samples where R. intracellularis appeared as an endosym- ACKNOWLEDGEMENTS biont (Fig. 4C and D). The origin was primarily considered as recently released filaments from the surface of symbiotic This study was supported by a Grant-in-aid for Creative diatom plasmalemma lacking the frustule. Diatom- Basic Research (12NP0201, DOBIS) from the MEXT, plasmalemma with Richelia filaments lacking the frustule Japan. Dr A. Shiomoto provided samples and data from

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the Kuroshio area within the SOYO programme, Lemmermann, E. (1899) Ergebnisse einer Reise nach dem Pacific National Research Institute of Fisheries Science, Japan. (H. Schauinsland 1896/97). Planktonalgen, Abhandl Aus Naturw. Ver. We are grateful to the scientists and crew of R/V Hakuho Bremen, 16, 313–398. Maru (Ocean Research Institute, the University of Lemmermann, E. (1905) Die Algenflora der Sandwich-Inseln. Ergebnisse einer Reise nach dem Pacific, H. Schauinsland 1896/97. Engler’s Bot. Tokyo), R/V Mirai (JAMSTEC), R/V Oshoro Maru Jahrbu¨cher Syst, Pflanzengeschichte Pflanzengeographie, 34, 607–663. (Hokkaido University) and R/V Wakataka Maru (Tohoku Lundholm, N., Hasle, G. R., Fryxell, G. A. et al. (2002) Morphology, National Fisheries Research Institute) for their kind help phylogeny and of species within the Pseudo-nitzschia americana with sampling during the Sulu Sea (KH02-4); western complex (Bacillariophyceae) with descriptions of two new species, equatorial Pacific Ocean (MR02-K06) and Oyashio area Pseudo-nitzschia brasiliana and Pseudo-nitzschia linea. Phycologia, 41, 480–497. cruises. We thank Dr G. Novarino (Natural History Marumo, R. and Asaoka, O. (1974) Distribution of pelagic blue-green Museum, London) for improving on the manuscript. algae in the North Pacific Ocean. J. Oceanogr. Soc. Jpn., 30, 77–85. F.G. acknowledges the financial support of the European Metzger, E. J. and Hurlburt, H. E. (1996) Coupled dynamics of the Commission (ICB2-CT-2001–80002). This is a contri- South China Sea, the Sulu Sea and Pacific Ocean. J. Geohys. Res., bution to the French IFB ‘Biodiversite´ et Changement 101, 12331–12352. Global’ program. Nishida, S. (ed.) (2004) Preliminary Data Report of the Hakuho Maru cruise KH-02–4. Ocean Research Institute, The University of Tokyo, Tokyo. Norris, R. E. (1961) Observations on phytoplankton organisms col- REFERENCES lected on the N.Z.O.I. Pacific cruise, September, 1958. New Zealand. J. Sci., 4, 162–188. Carpenter, E. J. (2002) Marine cyanobacterial symbioses. Biol. Environ., 102B, 15–18. Okamura, K. (1907) Some Chaetoceras and Peragallia of Japan. Bot. Mag., Tokyo, 21, 89–106. Carpenter, E. J., Montoya, J. P., Burns, J. et al. (1999) Extensive bloom Parsons, T. R., Maita, Y. and Lalli, C. M. (1984) A Manual of Chemical of a N2-fixing diatom/cyanobacterial association in the tropical . Mar. Ecol. Prog. Ser., 185, 273–283. and Biological Methods for Seawater Analysis. Pergamon Press, Oxford. Furuya, K. and Marumo, R. (1983) The structure of the phytoplankton Rai, A. N., So¨derba¨ck, E. and Bergman, B. (2000) Cyanobacterium- community in the subsurface chlorophyll maxima in the western plant symbioses. New Phytol., 147, 449–481. North Pacific Ocean. J. Plankton Res., 5, 393–406. Rines, J. E. B. (1999) Morphology and taxonomy of Chaetoceros contortus Hallegraeff, G. M. and Jeffrey, S. W. (1984) Tropical phytoplankton Schu¨tt 1895, with preliminary observations on Chaetoceros compressus species and pigments of waters of North and North- Lauder 1864. Bot. Mar., 42, 539–551. West Australia. Mar. Ecol. Prog. Ser., 20, 59–74. Schmidt, J. (1901) Ueber Richelia intracellularis, eine neue in Plankton- Hasle, G. R. and Syvertsen, E. E. (1997) Marine Diatoms. In Tomas, Diatomeen lebende Alge. Hedwigia, 40, 112–115. C. R. (ed.), Identifying Marine Phytoplankton. Academic Press, San Sournia, A. (1968) Diatome´es planctoniques du canal de Mozambique Diego, pp. 5–385. et de I’ıˆle Maurice. Me´m. O.R.S.T.O.M., 31, 1–120. Janson, S., Rai, A. N. and Bergman, B. (1995) The intracellular Staal, M., Meysman, F. J. R. and Stal, L. J. (2003) Temperature Richelia intracellularis: ultra–structure and immuno–locali- excluded N2-fixing heterocystous cyanobacteria in the tropical sation of phycoerythrin, , Rubisco and glutamine synthe- oceans. Nature, 425, 504–507. tase. Marine Biology, 124, 1–8. Sundstro¨m, B. G. (1984) Observations on Rhizosolenia clevei Ostenfeld (Bacil- Janson, S., Wouters, J., Bergman, B. et al. (1999) Host specificity in the lariophyceae) and Richelia intracellularis Schmidt. Bot. Mar., 27, 345–355. Richelia-diatom symbiosis revealed by hetR gene sequence analysis. Villareal, T. A. (1992) Marine nitrogen-fixing diatom-cyanobacterial Environ. Microbiol., 1, 431–438. symbioses. In Carpenter, E. J., Capone, D. G. and Reuter, J. (eds), Karl, D., Michaels, A., Bergman, B. et al. (2002) Dinitrogen fixation in Marine Pelagic Cyanobacteria. Trichodesmium and other . Kluwer the world’s oceans. Biogeochemistry, 57/58, 47–98. Academic Press, Dordrecht, The Netherlands, pp. 163–175. Karsten, G. (1907) Das Indische Phytoplankton nach dem Material der Zehr, J. P., Waterbury, J. B., Turner, P. J. et al. (2001) Unicellular Deutschen Tiefsee-Expedition 1898–1899. Dtsch. Tiefsee-Exped. cyanobacteria fix N2 in the subtropical North Pacific Ocean. Nature, 1898–1899, 2, 423–548. 412, 635–638.

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