J. Plankton Res. 2005 Gómez 323 30

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Distribution of the cyanobacteriumRichelia intracellularis as an epiphyte

of the diatom Chaetoceros compressus in the western Pacific Ocean

FERNANDO GOMEZ1*, 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

WIMEREUX, FRANCE, 2DEPARTMENT OF AQUATIC BIOSCIENCES, THE UNIVERSITY OF TOKYO, 1-1-1 YAYOI, BUNKYO, TOKYO 113 –8657, JAPAN.

*CORRESPONDING AUTHOR: [email protected]

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.

I N T R O D U C T I O N

The fixation of atmospheric nitrogen in the sea is animportant source of new nitrogen to ocean surface

waters, stimulating phytoplankton productivity and fuel-

ling the biological pump. In tropical waters the non-

heterocystous cyanobacterium Trichodesmium Ehrenberg

ex Gomont is largely responsible for nitrogen fixation

(Karl et al ., 2002), but unicellular diazotrophic cyano-

bacteria 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 (Sundstrom, 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 existson thefirst recordof R. intracellularis .

Lemmermann (Lemmermann, 1899) described the hetero-

cystous 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. rhizosole-

niae to occur as an endosymbiont in Rhizosolenia and Hemi-

aulus and as an epiphyte on Chaetoceros . Carpenter

(Carpenter, 2002) used the name Calothrix for a cyanobac-

terium found growing epiphytically on Chaetoceros , asopposed to the name Richelia which was used when the

cyanobacterium occurred as an endosymbiont. This con-

trasts 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 Shad-

bolt (Villareal, 1992; Rai et al ., 2000; Carpenter, 2002),

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

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Journal of Plankton Research Vol. 27 No. 4 Oxford University Press 2005; all rights reserved

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although this does not appear to have been recorded photo-

graphically.

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

itsgenus ( Richelia sinica Shen et Li is a freshwater species),andit is able to form associations with diatoms belonging to

several different genera besides showing a considerable var-

iation 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.

M E T H O D

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 30300 to 34150

N in May and 10 stations from 30 to 34200N 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 poorlystudied 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

between5 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, pre-

served with acidified Lugol’s solution (Hasle and Syvertsen,1997) and stored at 5C. 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

20C for dissolved inorganic nutrient analysis. After thaw-

ing, 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.).

R E S U L T S

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 andthe Celebes, Sulu and South China Seas.


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http://ss.nrifs.affrc.go.jp/eiyo/cruise/index.html






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. 2Aand 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 Cur-

rent 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 max-

imum 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, microphy-

toplankton 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 consor-

tium 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 33450 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 contain-

ing R. intracellularis were counted independently of non-

R. 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).

Sulu/Celebes Seas

A low salinity ( <33.1) and warm ( >29C) water layer was

present at the surface (Fig. 3A and C). In the surface waters

of stations 4 and 6, nitrate concentrations were below

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 (Schroder) Hustedt, Thalassionema nitzschioides

(Grunow) Mereschkowsky, Pseudo-nitzschia spp. and Bacter-

iastrum 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 transver-sely 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 dia-

toms 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 locationswhere filaments were also found on the surface of sym-

biotic 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; 8500 N, 121480 E) (Fig. 4O–Q).

These filaments, which appeared to be in a viable

GOMEZ ETAL. j RICHELIA INTRACELLULARIS AS AN EPIPHYTE OF C. COMPRESSUS

325



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 (thatcorresponded 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 inMay and July, respectively. The black circles point to the occurrence of Richelia-Chaetoceros consortia.


326



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 morphol-

ogy 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.

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)

Richelia intracellularis

(filaments per colony)

11 May 2002 33

138

–30 11 4

11 May 2002 33 138 –30 10 5

8 July 2002 33450 138 –5 10 3

8 July 2002 33450 138 –10 18 5

8 July 2002 33450 138 –20 11 4

8 July 2002 33450 138 –20 14 9

8 July 2002 33450 138 –20 16 6

8 July 2002 33450 138 –20 12 1

8 July 2002 33450 138 –30 3 6

21 Nov ember 2002 7250 121120 0 25 7

22 Nov ember 2002 6540 119110 0 20 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 notproportional. The black circles point to the occurrence of Richelia-Chaetoceros consortia.


327



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 ) Anunknown free-living cyanobacterium with intercellular heterocyst. Scale bars represent 10 mm.


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D I S C U S S I O N

Occurrence of the Richelia-Chaetoceros

consortia

The Richelia-Chaetoceros consortia appeared in a restricted

range of environmental conditions. During both cruisestheir 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 Cur-

rent can be related to mixing with coastal waters during

the eastward transit of the Kuroshio Current along

southern Japan (Furuya and Marumo, 1983). In theSulu 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 pos-

sible that the colonies of C. compressus found in the sur-

face 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 con-

temporary 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. intra-

cellularis growing endosymbiotically in R. clevei are closelyrelated 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 endosym-

biont (Fig. 4C and D). The origin was primarily considered

as recently released filaments from the surface of symbiotic

diatom plasmalemma lacking the frustule. Diatom-

plasmalemma 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. intracellu-

laris (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 wereabsent 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 sym-

biotic 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 facilitatethat 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 Richelia-

Chaetoceros consortia are restricted to the Indian and wes-

tern Pacific Oceans. Chaetoceros compressus is widely distrib-

uted 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 consor-

tia in the world oceans is less understood compared to

endosymbiotic cyanobacteria, further studies of the nature

of the Chaetoceros-Richelia consortia will contribute to abetter knowledge of symbiosis and nitrogen fixation in

the sea.

A C K N O W L E D G E M E N T S

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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J. Plankton Res. 2005 Gómez 323 30

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