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Distribution of the cyanobacterium Richelia intracellularis as an epiphyte of the diatom Chaetoceros compressus in the western Pacific Ocean
´ MEZ1*, KEN FURUYA2 AND SHIGENOBU TAKEDA2 FERNANDO GO
1

´ DES SCIENCES ET TECHNOLOGIES DE LILLE, CNRS-UMR 8013 ELICO, STATION MARINE DE WIMEREUX, UNIVERSITE
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AVENUE FOCH, BP 80, F-62930

WIMEREUX, FRANCE, DEPARTMENT OF AQUATIC BIOSCIENCES, THE UNIVERSITY OF TOKYO, 1-1-1 YAYOI, BUNKYO, TOKYO *CORRESPONDING AUTHOR:

113–8657, JAPAN.

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 diazotroph 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 endosymbiont in other diatoms 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
The fixation of atmospheric nitrogen in the sea is an important source of new nitrogen to ocean surface waters, stimulating phytoplankton productivity and fuelling the biological pump. In tropical waters the nonheterocystous cyanobacterium Trichodesmium Ehrenberg ex Gomont is largely responsible for nitrogen fixation (Karl et al., 2002), but unicellular diazotrophic cyanobacteria may also play a role (Zehr et al., 2001). High temperatures exclude heterocystous cyanobacteria from warm oceans (Staal et al., 2003), with the exception of the diazotroph heterocystous cyanobacterium Richelia intracellularis Schmidt. This cyanobacterium has been shown to provide quantitatively substantial inputs of nitrogen on regional scales (Carpenter et al., 1999). Richelia intracellularis is an extracellular endosymbiont which locates itself in the periplasmic space between the plasmalemma and silica cell wall in the diatoms Rhizosolenia clevei Ostenfeld, Hemiaulus spp. and Guinardia cylindrus (Cleve) Hasle (Sundstro ¨ m, 1984; Villareal, 1992). Records of it growing on cells of Chaetoceros Ehrenberg are rarer (Okamura, 1907; Karsten, 1907; Norris, 1961; Sournia, 1968; Janson et al., 1999), and not all reports can be

verified owing to lack of illustrations (Marumo and Asaoka, 1974; Hallegraeff and Jeffrey, 1984). Some confusion exists on the first record of R. intracellularis. Lemmermann (Lemmermann, 1899) described the heterocystous cyanobacterium Calothrix rhizosoleniae as an epiphyte growing on Rhizosolenia, and no further records are available in the literature. Schmidt (Schmidt, 1901) found a similar cyanobacterium as an endosymbiont in Rhizosolenia. Being unable to decide if this was effectively conspecific with C. rhizosoleniae, he described it as R. intracellularis. Later, Lemmermann (Lemmermann, 1905) reported C. rhizosoleniae to occur as an endosymbiont in Rhizosolenia and Hemiaulus and as an epiphyte on Chaetoceros. Carpenter (Carpenter, 2002) used the name Calothrix for a cyanobacterium found growing epiphytically on Chaetoceros, as opposed to the name Richelia which was used when the cyanobacterium occurred as an endosymbiont. This contrasts with previous usage by the same author (Janson et al., 1999). According to Janson et al. (Janson et al., 1999) the filaments of R. intracellularis seen on Chaetoceros matched those described by Lemmermann. Several reviews also reported R. intracellularis growing as an epiphyte on Bacteriastrum Shadbolt (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

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although this does not appear to have been recorded photographically. Janson et al. (Janson et al.,1999) analysed the hetR gene sequence of R. intracellularis growing intracellularly in several hosts and also epiphytically on Chaetoceros. These authors pointed out that R. intracellularis is the only marine species of its genus (Richelia sinica Shen et Li is a freshwater species), and it is able to form associations with diatoms belonging to several different genera besides showing a considerable variation in size and morphology (Janson et al., 1999). In warm oceans, the occurrence of R. intracellularis growing as an epiphyte on Chaetoceros is rare. Based on an analysis of several locations in the western Pacific Ocean, we found some spatio-temporal trends of the occurrence of the Chaetoceros-Richelia consortia. The results of this analysis are reported here.

METHOD
Samples were collected during ten cruises in the western Pacific Ocean (Fig. 1): (i) Two cruises were carried out on board R/V Soyo Maru (13–20 May and 3–10 July 2002) along the 138 E meridian in the vicinity of the Kuroshio Current. Nine stations were sampled from 30 300 to 34 150 N in May and 10 stations from 30 to 34 200 N during the July cruise. At each station, 15 depths from 5 to 200 m were sampled; (ii) R/V Hakuho Maru visited the Celebes, Sulu and South China Seas from 7 November to 18 December 2002. 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 (15–28 January 2003) along the Equator from 160 E to

160 W. Samples were collected from 9 stations at 14 depths between 5 and 200 m depth; (iv) Six cruises were carried out at Station H on board R/V Oshoro Maru and Station A7 on board R/V Wakataka Maru in the Oyashio area during the spring and summer of 2003. All samples were collected using Niskin bottles, preserved with acidified Lugol’s solution (Hasle and Syvertsen, 1997) and stored at 5 C. Subsamples (400 mL) were allowed to settle in glass sedimentation cylinders. The top 350 mL of each sample was siphoned off progressively over 5 days using a length of small-bore tubing. The remaining 50 mL was settled in composite sedimentation chambers and observed using a Nikon inverted microscope equipped with a Nikon digital camera. In all cruises except for the May 2002 one, cells of Rhizosolenia with and without R. intracellularis were counted separately. From each sampled depth, 5 mL of water was frozen at 20 C for dissolved inorganic nutrient analysis. After thawing, nutrient concentration was determined according to Parsons et al. (Parsons et al., 1984), using a TRAACS 800 for the Soyo Maru cruises (http://ss.nrifs.affrc.go.jp/eiyo/ cruise/index.html) or TRAACS 2000 (Nishida, 2004). The section plots of the distribution of the variables were produced by interpolating between casts using the kriging as the gridding method in the Surfer software (Golden Software Inc.).

RESULTS
Kuroshio Current
Based on the distribution of the physical variables along 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.

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were identified in the study area: the slope waters, the Kuroshio Current and the offshore subtropical waters. The hydrographical conditions differed between the two cruises. In May, the cyclonic gyre along the slope waters (Station 3) made the Kuroshio Current more narrow around Station 6 compared to the July cruise, when the Kuroshio area extended from station B2 (Fig. 2A and B). In May, the Kuroshio Current salinity (34.7) did not differ from the surrounding offshore subtropical waters (Fig. 2C). In July, the Kuroshio Current was wider, warmer and salinity was lower (<34.2) (Fig. 2D). In both cruises, nitrate and phosphate concentrations were highest in the onshore waters. The Kuroshio Current was associated with a strong decline in nutrients, especially nitrate at and around the surface (Fig. 2E–H). Nitrate was exhausted in offshore waters especially in July (Fig. 2E and F). A slight surface maximum was observed at Station 10 in May (Fig. 2E). In May, chlorophyll fluorescence was highest near the surface in the slope waters whereas in July a deep maximum was present at 70–80 m depth in the offshore subtropical waters (Fig. 2I). In July, fluorescence was lower in the slope waters whereas in May a deeper and thinner maximum was present at 90–100 m depth in the offshore waters (Fig. 2J). During both cruises, microphytoplankton abundance was highest in the most inshore stations. In May the diatom community was dominated by Chaetoceros denticulatus and Guinardia delicatula and it reached a maximum abundance of 60300 cell L–1. Chaetoceros compressus reached 8080 cell L–1 (Fig. 2K). In July, maximum diatom abundance was 5260 cell L–1 and C. compressus reached a maximum of 670 cell L–1 in subsurface waters (Fig. 2L) (Table I). In May, two colonies of the Richelia-Chaetoceros consortium were observed at 30 m depth (33 N, 138 E) (Table I, Fig. 2). The maximum abundance (10 colonies L–1) occurred in July at 33 450 N, 138 E at 20 m depth. One consortium was observed at 5, 10 and 30 m depth (Table I). In May, the population enumerated as R. clevei in the inshore waters probably corresponded to Rhizosolenia styliformis (Fig. 2M). In July, Rhizosolenia specimens containing R. intracellularis were counted independently of nonR. intracellularis-containing cells. The maximum abundance of the Richelia-Chaetoceros consortia coincided with the maximum abundance of the R. intracellularis-containing R. clevei population (30 cell L–1) and also the maximum abundance of unattached R. intracellularis filaments (Fig. 2N).

detection limit (0.1 mM) (Fig. 3E). Subsurface fluorescence maxima were shallower in the Sulu Sea compared to the South China and Philippine Seas. In the Celebes Sea at station 3, fluorescence was highest at 30 m depth and C. compressus reached an abundance of up to 1150 cell L–1. Microplankton vertical distribution showed a deep (75– 100 m) maximum dominated by Chaetoceros atlanticus var. neapolitanus (Schro ¨ der) Hustedt, Thalassionema nitzschioides (Grunow) Mereschkowsky, Pseudo-nitzschia spp. and Bacteriastrum spp. In the surface waters only dinoflagellates, the nitrogen-fixer Trichodesmium and R. clevei were found, the latter with a low abundance (<5 cell L–1). In the southern Sulu Sea one colony of the Chaetoceros-Richelia consortium was found in the surface waters both at stations 4 and 6 (Table I, Fig. 4J and K). In station 4, C. compressus reached maximum abundances of 55 and 235 cells L–1 at 30 and 50 m depth, respectively; at station 6 it ranged from 15 to 75 cells L–1 between 20 and 75 m depth.

Other areas
In the western equatorial Pacific and Oyashio cruises, no occurrence of the Richelia-Chaetoceros consortium was found and C. compressus did not proliferate either.

Morphological observations
Colonies of C. compressus usually contained 10–16 diatom cells and 1–9 epiphytic filaments of R. intracellularis, with the shorter diatom colony containing more numerous Richelia filaments per cell (Fig. 4M and N; Table I). The epiphytic filaments of R. intracellularis were strongly attached to the diatom cells in the spaces between adjacent cells of the colony, and were oriented transversely with respect to the axis of the colony (Fig. 4G–N). Typically, the single terminal Richelia heterocyst was similar in size to the vegetative cells or slightly smaller (Fig. 4I), but larger ones were also seen (Fig. 4K). Chaetoceros compressus colonies containing epiphytic R. intracellularis filaments lacked the usual numerous small plate-like chloroplasts and therefore appeared to be senescent. The Richelia-Chaetoceros consortia appeared to be associated with unidentified epiphytic naviculoid diatoms on Chaetoceros cells (Fig. 4G–N). In the Sulu Sea, one of the colonies examined also contained small green coccoid cells (Fig. 4J and K). Unattached filaments of R. intracellularis (Fig. 4C and D) occurred in locations where filaments were also found on the surface of symbiotic diatom plasmalemma lacking the frustule (Fig. 4E and F) and as endosymbiont (Fig. 4A and B). In addition, in other stations in the Sulu Sea, two filaments of a heterocystous free-living cyanobacterium joined at the level of the heterocysts were found at 100 m depth (Station 10; 8 500 N, 121 480 E) (Fig. 4O–Q). These filaments, which appeared to be in a viable

Sulu/Celebes Seas
A low salinity (<33.1) and warm (>29 C) water layer was present at the surface (Fig. 3A and C). In the surface waters of stations 4 and 6, nitrate concentrations were below

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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 138 E in the south of Japan in May and July, respectively. The black circles point to the occurrence of Richelia-Chaetoceros consortia.

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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 (cell per colony) 11 May 2002 11 May 2002 8 July 2002 8 July 2002 8 July 2002 8 July 2002 8 July 2002 8 July 2002 8 July 2002 21 November 2002 22 November 2002 33 33 33 450 33 450 33 450 33 450 33 450 33 450 33 450 7 250 6 540 138 138 138 138 138 138 138 138 138 121 120 119 110 –30 –30 –5 –10 –20 –20 –20 –20 –30 0 0 11 10 10 18 11 14 16 12 3 25 20 Richelia intracellularis (filaments per colony) 4 5 3 5 4 9 6 1 6 7 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 filamentous cyanobacteria present in the surrounding waters. The smaller (50 mm) filament had one terminal heterocyst, analogous to R. intracellularis, whereas the larger (170 mm) one had a 9-mm intercellular spherical

heterocyst (Fig. 4O–Q). Their colour, size and morphology was reminiscent of R. intracellularis but this has never been documented to possess intercellular heterocysts. This is consistent with our observation that filaments of 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
Occurrence of the Richelia-Chaetoceros consortia
The Richelia-Chaetoceros consortia appeared in a restricted range of environmental conditions. During both cruises their occurrence was restricted to the transition zone between the slope waters and the Kuroshio Current in a transect of more than 500 km. Obviously, the occurrence of the Richelia-Chaetoceros consortia requires the preliminary presence of both the diatom host cells and the cyanobacterium. Chaetoceros compressus is a common member of the neritic diatom assemblage in the Japanese coastal waters, whereas it is less common in open waters (Okamura, 1907; Furuya and Marumo, 1983). Its presence in the Kuroshio Current can be related to mixing with coastal waters during the eastward transit of the Kuroshio Current along southern Japan (Furuya and Marumo, 1983). In the Sulu Sea, a semi-enclosed deep basin isolated from the surrounding ocean and connected to the Celebes Sea via many passages through the Sulu Archipelago, it is possible that the colonies of C. compressus found in the surface nitrate-limited waters were associated with an upwelling plume. During the boreal summer, surface water drifts southwards through the Sulu Archipelago into the Celebes Sea, whereas in winter this drift is reversed (Metzger and Hurlburt, 1996). Prevailing local winds can alter the flow in the surface waters, resulting in moderate upwelling and export of C. compressus to the southern Sulu Sea from the Sulu Archipelago.

Hypothesis on the origin of epiphytic Richelia growing on Chaetoceros
From our observations, it appears that the occurrence of Richelia-Chaetoceros consortia was associated with the contemporary presence of C. compressus and R. intracellularis as an endosymbiont in other diatoms. Chaetoceros compressus is a tropical species originally described from Hong Kong, which is not to be confused with C. contortum, a temperate to northern species (Rines, 1999). Janson et al. (Janson et al.,1999) reported that hetR gene sequences of R. intracellularis growing endosymbiotically in R. clevei are closely related to those of R. intracellularis growing epiphytically on Chaetoceros. Non-attached filaments of R. intracellularis, which appeared to be free-living, were found in the same samples where R. intracellularis appeared as an endosymbiont (Fig. 4C and D). The origin was primarily considered as recently released filaments from the surface of symbiotic diatom plasmalemma lacking the frustule. Diatomplasmalemma with Richelia filaments lacking the frustule

as well as apparently free-living was often found in the regions where the consortia were observed (Fig. 4E and F). We hypothesize that unattached filaments of R. intracellularis (Fig. 4C–D) originating from R. clevei are able to colonise senescent colonies of Chaetoceros lacking epiphytic R. intracellularis. Observations on R. intracellularis growing as an endosymbiont in R. clevei showed that gas-vesicles were absent and therefore the cyanobacterium cells might be unable to regulate their position in the water column ( Janson et al., 1995). Consequently, unattached filaments should not be viable and therefore a host would be required. After the contact between the cyanobacterium and the diatom, one filament of R. intracellularis colonises a cell of the C. compressus colony (Fig. 4G) and then spreads out to other diatom cells (Fig. 4H and J). The presence of Richelia in Chaetoceros is often associated with epiphytic pennate diatoms, tentatively identified as a species of the Pseudo-nitzschia americana complex (Lundholm et al., 2002). It is possible that these epiphytic diatoms consume part of the N2-origin nitrogen excreted by Richelia (Fig. 4M and N). This exchange, which does not exist in the case of Richelia growing as an endosymbiont inside diatom cells, could decrease the efficiency of the transfer of nitrogen to C. compressus. Chaetoceros colonies cannot survive in oligotrophic conditions and likely decompose as a result. In these conditions Richelia continues to grow. Higher number of Richelia filaments per diatom cell was found in shorter colonies (Table I). This could be the first stage of a future successful symbiotic association when the efficiency in the exchanges of nutrients between the nitrogen fixer and the host increase. 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 the transition area where the distribution of R. intracellularis as an endosymbiont in diatoms and that of C. compressus overlaps. To the best of our knowledge, records of RicheliaChaetoceros consortia are restricted to the Indian and western Pacific Oceans. Chaetoceros compressus is widely distributed in these waters, and R. intracellularis growing as an endosymbiont in R. clevei is ubiquitous in warm oceans. Although the distribution of the Richelia-Chaetoceros consortia in the world oceans is less understood compared to endosymbiotic cyanobacteria, further studies of the nature of the Chaetoceros-Richelia consortia will contribute to a better knowledge of symbiosis and nitrogen fixation in the sea.

ACKNOWLEDGEMENTS
This study was supported by a Grant-in-aid for Creative Basic Research (12NP0201, DOBIS) from the MEXT, Japan. Dr A. Shiomoto provided samples and data from

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the Kuroshio area within the SOYO programme, National Research Institute of Fisheries Science, Japan. We are grateful to the scientists and crew of R/V Hakuho Maru (Ocean Research Institute, the University of Tokyo), R/V Mirai (JAMSTEC), R/V Oshoro Maru (Hokkaido University) and R/V Wakataka Maru (Tohoku National Fisheries Research Institute) for their kind help with sampling during the Sulu Sea (KH02-4); western equatorial Pacific Ocean (MR02-K06) and Oyashio area cruises. We thank Dr G. Novarino (Natural History Museum, London) for improving on the manuscript. F.G. acknowledges the financial support of the European Commission (ICB2-CT-2001–80002). This is a contribution to the French IFB ‘Biodiversite ´ et Changement Global’ program.

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