Distribution of the cyanobacterium

Richelia intracellularis as an epiphyte

of the diatom Chaetoceros compressus
in the western Pacic Ocean
FERNANDO GO

MEZ
1
*, KEN FURUYA
2
AND SHIGENOBU TAKEDA
2
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,
2
DEPARTMENT OF AQUATIC BIOSCIENCES, THE UNIVERSITY OF TOKYO, 1-1-1 YAYOI, BUNKYO, TOKYO 1138657, JAPAN.
*CORRESPONDING AUTHOR: fernando.gomez@toplancton.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 Pacic
Oceans, whereas the association of R. intracellularis as an endosymbiont in other diatoms is ubiquitous
in warm oceans. From ten cruises in the western Pacic 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 NTRODUCTI ON
The xation of atmospheric nitrogen in the sea is an
important 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 xation
(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
veried owing to lack of illustrations (Marumo and
Asaoka, 1974; Hallegraeff and Jeffrey, 1984).
Some confusionexists onthe rst 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 conspecic 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, as
opposed 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
laments of R. intracellularis seenon Chaetoceros matchedthose
described by Lemmermann. Several reviews also reported
R. intracellularis growing as an epiphyte on BacteriastrumShad-
bolt (Villareal, 1992; Rai et al., 2000; Carpenter, 2002),
JOURNAL OF PLANKTON RESEARCH
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doi:10.1093/plankt/fbi007, available online at www.plankt.oupjournals.org
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
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 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 Pacic
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
Pacic Ocean (Fig. 1): (i) Two cruises were carried out on
board R/V Soyo Maru (1320 May and 310 July 2002)
along the 138

E meridian in the vicinity of the Kuroshio

Current. Nine stations were sampled from 30

30
0
to 34

15
0
N in May and 10 stations from 30

to 34

20
0
N during the
July cruise. At each station, 15 depths from5 to 200 mwere
sampled; (ii) R/VHakuho Maru visited the Celebes, Sulu and
South China Seas from7 November to 18 December 2002.
Samples were collected from 10 stations at six depths from
0 to 150 mdepth. The Sulu Sea is a poorly studied region of
the worlds oceans, and this study sampled three stations in
that area; (iii) A cruise was carried out on board R/V Mirai
(1528 January 2003) along the Equator from 160

E to
160

W. Samples were collected from9 stations at 14 depths

between 5 and 200 mdepth; (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 acidied Lugols 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 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.).
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 identied 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 Cur-
rent was associated with a strong decline in nutrients,
especially nitrate at and around the surface (Fig. 2EH).
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 uorescence was highest near the
surface in the slope waters whereas in July a deep max-
imum was present at 7080 m depth in the offshore
subtropical waters (Fig. 2I). In July, uorescence was
lower in the slope waters whereas in May a deeper and
thinner maximum was present at 90100 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-
tiumwere observed at 30 mdepth (33

N, 138

E) (Table I,
Fig. 2). The maximum abundance (10 colonies L
1
)
occurred in July at 33

45
0
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 maximumabundance
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 laments
(Fig. 2N).
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
detection limit (0.1 mM) (Fig. 3E). Subsurface uorescence
maxima were shallower in the Sulu Sea compared to the
South China and Philippine Seas. In the Celebes Sea at
station 3, uorescence 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 dinoagellates, the
nitrogen-xer 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 Pacic 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 1016 diatom
cells and 19 epiphytic laments of R. intracellularis, with
the shorter diatom colony containing more numerous
Richelia laments per cell (Fig. 4M and N; Table I).
The epiphytic laments 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. 4GN).
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 laments lacked the usual numerous small
plate-like chloroplasts and therefore appeared to be
senescent. The Richelia-Chaetoceros consortia appeared to
be associated with unidentied epiphytic naviculoid dia-
toms on Chaetoceros cells (Fig. 4GN). In the Sulu Sea,
one of the colonies examined also contained small green
coccoid cells (Fig. 4J and K). Unattached laments of
R. intracellularis (Fig. 4C and D) occurred in locations
where laments 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
laments of a heterocystous free-living cyanobacterium
joined at the level of the heterocysts were found at 100 m
depth (Station 10; 8

50
0
N, 121

48
0
E) (Fig. 4OQ).
These laments, which appeared to be in a viable
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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) uorescence
(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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condition, occurred at 100 m depth, with no other
lamentous cyanobacteria present in the surrounding
waters. The smaller (50 mm) lament had one terminal
heterocyst, analogous to R. intracellularis, whereas the
larger (170 mm) one had a 9-mm intercellular spherical
heterocyst (Fig. 4OQ). 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 laments 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 laments of Richelia intracellularis per colony of Chaetoceros
Date Latitude (north) Longitude (east) Depth (m) Chaetoceros compressus
(cell per colony)
Richelia intracellularis
(laments per colony)
11 May 2002 33

138

30 11 4
11 May 2002 33

138

30 10 5
8 July 2002 33

45
0
138

5 10 3
8 July 2002 33

45
0
138

10 18 5
8 July 2002 33

45
0
138

20 11 4
8 July 2002 33

45
0
138

20 14 9
8 July 2002 33

45
0
138

20 16 6
8 July 2002 33

45
0
138

20 12 1
8 July 2002 33

45
0
138

30 3 6
21 November 2002 7

25
0
121

12
0
0 25 7
22 November 2002 6

54
0
119

11
0
0 20 5
Fig. 3. Section plots of (A) temperature (

C), (B) uorescence (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.
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Fig. 4. Photomicrographs of lamentous heterocyst-forming cyanobacteria. (A and B) Richelia intracellularis as an endosymbiont in Rhizosolenia clevei
var. clevei. (C and D) Unattached laments of R. intracellularis. (E and F) R. intracellularis as an epiphyte on the plasmalemma of Rhizosolenia lacking
the frustule. (GN) 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). (OQ) An
unknown free-living cyanobacterium with intercellular heterocyst. Scale bars represent 10 mm.
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DI SCUSSI ON
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 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 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 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 ow 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 closely
related to those of R. intracellularis growing epiphytically on
Chaetoceros. Non-attached laments of R. intracellularis,
which appeared to be free-living, were found in the same
samples where R. intracellularis appeared as an endosym-
biont (Fig. 4Cand D). The origin was primarily considered
as recently released laments from the surface of symbiotic
diatom plasmalemma lacking the frustule. Diatom-
plasmalemma with Richelia laments 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 laments of R. intracellu-
laris (Fig. 4CD) 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 laments
should not be viable and therefore a host would be
required. After the contact between the cyanobacterium
and the diatom, one lament 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 identied as a
species of the Pseudo-nitzschia americana complex (Lundholm
et al., 2002). It is possible that these epiphytic diatoms
consume part of the N
2
-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 efciency 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 laments per diatom cell was
found in shorter colonies (Table I).
This could be the rst stage of a future successful sym-
biotic association when the efciency in the exchanges of
nutrients between the nitrogen xer 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 Richelia-
Chaetoceros consortia are restricted to the Indian and wes-
tern Pacic 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 a
better knowledge of symbiosis and nitrogen xation 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 Pacic 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 nancial support of the European
Commission (ICB2-CT-200180002). This is a contri-
bution to the French IFB Biodiversite et Changement
Global program.
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JOURNAL OF PLANKTON RESEARCH
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NUMBER 4
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PAGES 323330
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