APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 2005, p. 190–196
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Vol. 71, No. 1
Unveiling of Novel Radiations within Trichodesmium Cluster by hetR
Gene Sequence Analysis
Pernilla Lundgren,1Sven Janson,2* Sara Jonasson,1Alon Singer,1and Birgitta Bergman1
Department of Botany, Stockholm University, Stockholm,1and Department of Biology and Environmental Science,
University of Kalmar, Kalmar,2Sweden
Received 22 October 2003/Accepted 10 August 2004
The filamentous nonheterocystous cyanobacterial genus Katagnymene is a common diazotrophic component
of tropical and subtropical oceans. To assess the phylogenetic affiliation of this taxon, two partial 16S rRNA
gene sequences and 25 partial hetR gene sequences originating from the genera Katagnymene and Trichodes-
mium collected from open, surface waters of the Atlantic, Indian, and Pacific oceans were compared. Single
trichomes or colonies were identified morphologically by using light microscopy and then used directly as
templates in hetR PCR analyses. In addition, three cultured strains, identified as Katagnymene pelagica,
Katagnymene spiralis, and Trichodesmium sp., were examined. The data show that the genus Katagnymene is in
the Trichodesmium cluster and that K. pelagica Lemmermann and K. spiralis Lemmermann are most likely one
species, despite their different morphologies. Phylogenetic analyses also unveiled four distinct clusters in the
Trichodesmium cluster, including one novel cluster. Our findings emphasize the conclusion that known mor-
phological traits used to differentiate marine nonheterocystous cyanobacteria at the genus and species levels
correlate poorly with genetic data, and a revision is therefore suggested.
The world’s tropical and subtropical oceans are highly oli-
gotrophic and are therefore a suitable habitat for diazotrophic
cyanobacteria. In the open ocean, the nonheterocystous cya-
nobacterial genus Trichodesmium is very common. Trichodes-
mium was first discovered in 1830 (9), and in 1961 it was found
to be diazotrophic; its ecology and physiology have since been
characterized in numerous studies (6, 17). Recent estimates
suggest that Trichodesmium alone may account for 40 to 50%
of the global nitrogen sequestered through biological nitrogen
fixation (17). One key feature of Trichodesmium is its ability to
form colonies, but the cells may also exist as free trichomes.
The marine nonheterocystous genus Katagnymene occurs only
as free trichomes and was recently found to be diazotrophic
(22). Like Trichodesmium, its nitrogen fixation activity is re-
stricted to the photophase, and nitrogenase is confined to sub-
sets of cells termed diazocytes (10, 12), which in Katagnymene
account for about 7% of the total cells. Thus, Katagnymene and
Trichodesmium share a unique diazotrophic behavior (5, 15,
Katagnymene was first described by Lemmermann (19) and
was divided into two species, Katagnymene pelagica and Katag-
nymene spiralis, based on the degree of trichome coiling. Kat-
agnymene occurs in all major tropical and subtropical oceans
(18, 19, 22, 24, 30). The trichomes are characterized by cells
that are shorter than they are wide and by being surrounded by
a distinct mucilaginous sheath. K. spiralis is more or less spi-
rally coiled, and both Katagnymene species may form long
trichomes that sometimes are up to 15 mm long. In previous
taxonomic studies, Katagnymene was suggested to belong to the
oscillatoriacean family, together with Trichodesmium spp.; the
former genus was placed in the subfamily Hormoscilloidae, and
the latter genus was placed in the Phormidiaceae (1). Previ-
ously, Drouet (8) suggested that K. spiralis should be classified
as Microcoleus lyngbyaceus based on the thickened cell walls of
the trichome end cells and that K. pelagica var. major Wille
should be classified as Oscillatoria (Trichodesmium) erythraea.
Besides the two marine species, the genus Katagnymene also
includes five fresh or brackish water species (1, 2). It has
recently been suggested that K. spiralis is closely related to
Trichodesmium spp. based on certain gene sequence similari-
ties (22, 26).
Likewise, the taxonomy of Trichodesmium has often been
revised, and only one extensive genetic study has been per-
formed (14). This study showed that the genus harbors five
marine species in the following three main clusters: (i) a cluster
which includes Trichodesmium hildebrandtii and fusiform and
spherical colonies of Trichodesmium thiebautii; (ii) a cluster
which includes Trichodesmium erythraeum (including the lab-
oratory strain Trichodesmium sp. strain IMS 101); and (iii) a
cluster which includes Trichodesmium tenue and Trichodes-
In the present study, collected Katagnymene and Trichodes-
mium specimens first were identified by using light microscopy
and then were used as templates in PCR in order to generate
sequences that could be directly linked to a specific morphol-
ogy. A 448-bp fragment of the hetR gene, proposed to be
involved in diazocyte differentiation (10), was amplified and
analyzed for cyanobacteria identified as Katagnymene spp. or
Trichodesmium spp., which were collected in various geo-
graphic regions, and for a few cultured specimens. Our aim was
to determine the relationship between Katagnymene spp. and
Trichodesmium spp. using both 16S rRNA gene and hetR gene
sequences isolated from natural populations and cultures of
members of both genera.
* Corresponding author. Mailing address: Department of Biology
and Environmental Science, University of Kalmar, SE-391 82 Kalmar,
Sweden. Phone: 46 480447310. Fax: 46 480447305. E-mail: sven
MATERIALS AND METHODS
Sample collection. Cyanobacteria were collected during three cruises, in 1998
during a cruise of the R/V Roger Revelle in the southwestern Pacific Ocean from
New Zealand to Fiji, in 1999 during a cruise of the R/V Maurice Ewing off
northern Australia, and in 2001 during a cruise of the R/V Seward Johnson in the
midwestern Atlantic Ocean off Barbados. Samples were collected from the sur-
face down to a depth of 20 m by using plankton tows. The morphological
descriptions of Lemmermann (19), Wille (30), and Karsten (18) were used for
identification (Table 1). The samples obtained in 1999 and 2000 were identified
and photographed with a light microscope (Fig. 1 and Table 2). Then in each
case the same trichome was recovered and transferred in a micropipette in 4
droplets of filtered seawater and 1 droplet of Milli-Q water before it was placed
in a PCR tube containing 10 ?l of Milli-Q water. The samples were frozen,
thawed, and used directly as templates in PCR.
Cultures. Cultures PLA1 (preliminarily identified as K. pelagica) and PLA2
(no affiliation) were isolated (by P.L.) as single trichomes during the 2001 cruise
in the western Atlantic Ocean. The culture of K. spiralis (JWI1) was isolated by
J. Waterbury (Woods Hole Oceanographic Institution, Woods Hole, Mass.)
from the Zanzibar Channel in Tanzania in 1999. The cultures are maintained in
a nitrogen-free amended seawater (Sigma) medium containing trace metals,
EDTA, and vitamins as described previously (7) and supplemented with 15 ?M
phosphoric acid, and they are grown with a daily cycle consisting of 12 h of
darkness and 12 h of light (40 ?mol of photons ? m?2? s?1) at 27°C.
PCR amplification and sequencing. Partial 16S rRNA gene sequences were
obtained by using the cyanobacterium-specific primers CYA106F and CYA781R
(25). Each PCR was performed with an initial denaturation step consisting of
95°C for 4 min, followed by 30 cycles of 1 min at 93°C, 1 min at 50°C (57°C for
the 16S rRNA gene), and 1 min at 72°C and a final extension step consisting of
72°C for 4 min. For all of the samples except those obtained in 1998, the
trichomes were used directly as templates. Each PCR mixture (total volume, 25
FIG. 1. Representative morphologies of some of the species of Katagnymene and Trichodesmium included in the phylogenetic analyses. Further
details are given in Table 2. The morphotypes belong to cluster I (a to e), cluster II (f), cluster III (g to j), and cluster IV (k and l). (a) Cultured
JWI1 (K. spiralis). Bar, 100 ?m. (b) Specimen A27 (K. spiralis), with a pronounced mucilaginous sheath and with less pigmentation and
segmentation (arrow). Bar, 50 ?m. (c) Specimen B41-1 (K. spiralis). Bar, 100 ?m. (d) Cultured PLA1 (K. pelagica). Bar, 50 ?m. (e) Specimen A25,
puff-shaped colony of Trichodesmium sp. with curved trichomes. Bar, 100 ?m. (f) Specimen B49 (Trichodesmium sp.). Note the scattered gas
vacuoles appearing as light-reflecting objects. Bar, 50 ?m. (g) Cultured PLA2 (Trichodesmium sp.). Bar, 100 ?m. (h) Part of a trichome of specimen
B41-3 (Trichodesmium sp.) showing segmentation. Bar, 100 ?m. (i) Specimen A13 (Trichodesmium sp.). Bar, 100 ?m. (j) Part of a colony of T.
erythraeum. Bar, 100 ?m. (k) Part of a colony of T. tenue. Note the presence of the few light-reflecting large gas vesicles in the narrow cells. Bar,
50 ?m. (l) Specimen B46 (T. contortum). Note the presence of several clustered light-reflecting gas vesicles. Bar, 50 ?m.
TABLE 1. Morphological characteristics of K. spiralis and K.
pelagica given in the original descriptionsa
K. pelagica K. spiralis
Trichome SheathTrichome Sheath
aSee references 18, 19, 24, and 30. The value in parentheses is from excep-
b?, sheath present.
cND, not determined.
VOL. 71, 2005MOLECULAR PHYLOGENY OF TRICHODESMIUM CLUSTER191
?l) contained each deoxynucleoside triphosphate at a concentration of 200 nM,
0.5 mM MgCl2, 0.5 U of Dynazyme polymerase, Dynazyme reaction buffer, 0.5 ?M
primer PH1, and 0.5 ?M primer PH2. Primers PH1 (5?-TGY GCK ATT TAY ATG
ACC TA-3?) and PH2 (5?-ATG AAN GGT ATK CCC CAA GGA-3?) were con-
structed based on previously published Trichodesmium hetR sequences for amplifi-
cation of a 448-bp fragment. The PCR fragments were separated on a 1.5% agarose
gel. When a band was weak, the band was purified and used as a template in a
second PCR. The gel fragments were purified and subjected to one of the following
two treatments. The PCR products were either sequenced directly or cloned into the
pCR2.1 vector (Invitrogen, San Diego, Calif.) by using a Rapid DNA ligation kit
(Boehringer, Mannheim, Germany) and transformed by using One Shot competent
cells (Invitrogen). Plates were screened for white colonies. These colonies were
checked to make sure that they contained the correct insert by colony PCR, and
positive colonies were grown in Luria-Bertani medium overnight. Then DNA prep-
arations were checked for the correct insert by enzymatic restriction. For sequencing
of cloned inserts primers T7 and M13 reverse were used. When the purified PCR
products were sequenced directly, primers PH1 and PH2 were used. cDNA strands
were sequenced by using BigDye terminators (PE Applied Biosystems) with a Per-
kin-Elmer model ABI 377 automated sequencer.
Sequence analysis. When necessary, the alignments of sequences were edited
manually with Seaview (13). hetR trees were constructed by using PHYLIP
(version 3.6a; Department of Genetics, University of Washington, Seattle)
(http://evolution.genetics.washington.edu/phylip.html). The sequences of Apha-
nizomenon sp. strain KAC 15 and Leptolyngbya sp. strain PCC 73110 were used
as outgroups. When distance matrices were used to generate the trees, the
correction of Jukes and Cantor (16) was employed. For the nucleotide maxi-
mum-likelihood tree, the DNAML program was used (11), with the transition/
transversion ratio set to 1.69 and the base frequencies set to 0.291 (A), 0.189 (C),
0.214 (G), and 0.305 (T). The transition/transversion ratio and base frequencies
were determined by using PhyloWin. For maximum-likelihood analyses of the
translated amino acid sequences of hetR, TREE-PUZZLE 5.0 was used (27).
The analysis with TREE-PUZZLE was performed with the default settings,
FIG. 2. Phylogenetic comparison of F34-5 (Trichodesmium sp.),
JWI1 (K. spiralis), and the extant Trichodesmium cluster (14) based on
partial 16S rRNA gene sequences. The F34-5 (Trichodesmium sp.) and
JWI1 (K. spiralis) sequences both fall in the Trichodesmium cluster
with 100% bootstrap support. The sequences were analyzed by calcu-
lating the distances between pairs of sequences by using the distance
correction of Tajima and Nei (28), followed by construction of a
phylogenetic tree by the neighbor-joining method and bootstrap resa-
mpling with 500 replicates. The analysis was carried out with the
TREECON software package (29). Lyngbya majuscula was used as an
outgroup. Scale bar ? 0.05 substitution per sequence position.
TABLE 2. Morphological characteristics, collection sites, and accession numbers for the trichomes and colonies used for hetR and 16S rRNA
Current affiliation ClusterAccession no.
16S rRNA gene sequences
aThe origins of the samples are indicated as follows: F, 1998 southwest Pacific Ocean cruise; A, 1999 cruise off northern Australia (Indian Ocean or Pacific Ocean);
and B, 2001 cruise in the midwestern Atlantic Ocean.
bAbbreviations: AO, Atlantic Ocean; IO, Indian Ocean; PO, Pacific Ocean.
cND, not determined.
192LUNDGREN ET AL.APPL. ENVIRON. MICROBIOL.
except that the model of rate heterogeneity was chosen as a gamma distributed
The 16S rRNA gene sequences were analyzed by calculating distances between
pairs of sequences by using the distance correction of Tajima and Nei (28),
followed by construction of a phylogenetic tree by the neighbor-joining method
and bootstrap resampling with 500 replicates (Fig. 2). This analysis was carried
out by using the TREECON software package (29). Leptolyngbya sp. strain PCC
73110 was used as the outgroup in the 16S rRNA gene-based tree.
Nucleotide sequence accession numbers. The GenBank accession numbers for
the sequences determined in the present study are given in Table 2.
Microscopic observations. Our microscopic observations
and the origins of the collected specimens are summarized in
FIG. 3. Phylogenetic trees inferred from partial hetR sequences obtained in the present study from natural and cultured populations of
Katagnymene and Trichodesmium species and from the extant Trichodesmium cluster (14). Aphanizomenon sp. strain KAC15 and Leptolyngbya sp.
strain PCC 73110 were used as outgroups. The brackets with roman numbers indicate the four main clusters in the Trichodesmium cluster. The
brackets labeled K. pelagica/K. spiralis indicate the 13 sequences derived from trichomes originally identified as K. pelagica or K. spiralis.
(A) Maximum-likelihood analysis of partial hetR nucleotide sequences. (B) Maximum-likelihood analysis of partial hetR amino acid sequences. For
the analyses of the amino acid sequences encoded by hetR, TREE-PUZZLE 5.0 (27) was performed with the default settings, except that the model
of rate heterogeneity was chosen as a gamma distributed rate. Abbreviations: IMS, culture collection of the Institute of Marine Science, University
of North Carolina, Morehead City; KAC, Kalmar Algae Collection; PCC, Pasteur Culture Collection.
VOL. 71, 2005MOLECULAR PHYLOGENY OF TRICHODESMIUM CLUSTER 193
Table 2. For identification of Katagnymene we used the follow-
ing criteria described by Lemmermann (19) for the two marine
species: (i) trichomes in which the cell diameter varies from 12
to 35 ?m, with cells that are shorter than they are wide; (ii)
trichomes surrounded by a mucilaginous sheath; (iii) trichomes
coiled to straight; (iv) trichomes often segmented, yet retained
in the same sheath; and (v) trichomes solitary (trichomes do
not aggregate into colonies). Seven specimens, including the
cultured JWI1 specimen, were identified as K. spiralis. The
trichomes were coiled, and the cell diameters ranged from 12
to 30 ?m (Fig. 1a to d). The degree of coiling varied, and the
trichomes were surrounded by a thick mucilaginous sheath.
Regions with less pigmentation were often observed; this may
have been an initial step in the formation of necrotic cells,
leading to segmentation of trichomes (Fig. 1b and c). Six spec-
imens, including the cultured PLA1specimen, were identified
as K. pelagica. The trichomes were straight, had widths that
ranged from 15 to 35 ?m, and were surrounded by a mucilag-
inous sheath (Table 2 and Fig. 1e and f). Regions where there
was less pigmentation and segmentation of the trichome were
also observed in K. pelagica (Fig. 1e).
The remainder of the collected but unidentified single cya-
nobacterial trichomes and one colony were separated into
three different groups on the basis of morphological charac-
teristics (i.e., morphotypes). None of the morphotypes ob-
served clearly fit descriptions given previously. These included
A25, collected off northern Australia in 1999, which formed
loosely aggregated colonies composed of curled trichomes.
The trichomes ranged in diameter from 10 to 14 ?m (Fig. 1e).
Colonies with similar morphologies have also been observed in
the Zanzibar Channel in Tanzania (unpublished observations).
Another morphotype was observed in the midwestern Atlantic
Ocean in 2001. The trichomes were 20 to 25 ?m wide (B49 and
B51-3) and sheathless and had randomly scattered gas vacu-
oles. The trichomes were golden and did not form colonies
(Fig. 1f). Moreover, on all three cruises yet another morpho-
type was observed; in this morphotype the trichome diameters
varied from 18 to 60 ?m, and the cells were shorter than they
were wide (they were one-fifth to one-eighth times as long as
they were wide). The trichomes were often highly segmented,
and colonies were not observed. However, the cultured PLA2
organism is a representative of this morphotype, and it can
form loose colonies at least under laboratory conditions (Fig.
1g to i). This morphotype is identical to one described in
previous work (15) and does not correspond to the description
of either Katagnymene or Trichodesmium. Trichome morphol-
ogy identical to that of T. contortum (14) was detected in the
midwestern Atlantic Ocean in 2001. The trichomes were 30 to
35 ?m wide and pale brown (B46) (Fig. 1l). The trichome
morphologies of T. erythraeum and T. tenue are included for
comparative purposes in Fig. 1 (Fig. 1j and k), while no se-
quences are reported here for either of these species.
Genetic analyses. Part (584 bp) of the highly conserved gene
sequence of the small-subunit rRNA gene was sequenced for
some of the cyanobacteria collected. In the resulting phyloge-
netic tree the cultured JWI1 specimen (Table 2 and Fig. 1a),
assigned to the species K. spiralis, and the morphotype F34-5
(Table 2 and Fig. 1i to k) both fell in the Trichodesmium cluster
with 100% bootstrap support (Fig. 2). Furthermore, the phy-
logenetic analyses revealed that the 16S rRNA gene sequence
of JWI1 was closely related to those of T. thiebautii and T.
hildebrandtii, while F34-5 (Trichodesmium sp.) was closely re-
lated to T. erythraeum.
As Trichodesmium spp. display low genetic diversity, the
more variable gene hetR was used previously to analyze the
intrageneric phylogeny (14). In view of this, we used partial
hetR gene sequences (448 bp) to further resolve the relation-
ship between Katagnymene spp. and Trichodesmium spp. The
25 hetR sequences amplified in the present study were all from
organisms located in the Trichodesmium cluster with 100%
bootstrap support (Fig. 3a). Both the resulting hetR nucleotide
and amino acid trees, containing novel and previously de-
scribed Trichodesmium spp. (the latter sequences were in-
cluded for comparative purposes), contained four well-defined
clusters of related sequences (Fig. 3). Cluster I comprised T.
thiebautii, T. hildebrandtii, two of the novel sequences, and all
13 sequences from members of K. spiralis and K. pelagica.
Cluster II exclusively encompassed two identical sequences
from a novel morphotype. Cluster III comprised T. erythraeum
along with seven sequences from the large dark-pigmented
trichomes. Cluster IV contained T. tenue and T. contortum, as
well as one sequence from T. contortum from this study. As Fig.
3B shows, the 13 hetR sequences derived from trichomes mor-
phologically identified as K. spiralis or K. pelagica formed a
distinct radiation within Trichodesmium cluster I. These se-
quences included sequences from K. pelagica collected from six
different sampling sites on the three cruises, and seven se-
quences were generated from K. spiralis collected on the three
cruises and one cultured isolate from the Indian Ocean (Table
2 and Fig. 3). Morphologically, K. spiralis from the Pacific
Ocean had a larger cell diameter than the corresponding
trichomes from the Atlantic Ocean. Smaller morphological
differences were observed for K. pelagica from the different
geographic areas sampled (Table 2). The overall topologies of
the hetR nucleotide and deduced amino acid trees were similar
(Fig. 3). However, there was one exception in cluster I: in the
nucleotide-based tree (Fig. 3A) the sequence of the T. thie-
bautii tuft appeared to be ancestral to all other sequences in
cluster I with a weak supporting bootstrap value (72), but in
the amino acid tree (Fig. 3B) T. thiebautii was ancestral to the
A25 and T. thiebautii puff sequences only. This makes classifi-
cation of the different T. thiebautii phylotypes and morpho-
types difficult, and further studies are needed in order to re-
solve this issue. The different tree topologies observed for
cluster I could be explained by a high degree of homoplasy
within the group of sequences, to which the maximum-likeli-
hood method is less sensitive. The distance and parsimony
analyses of the hetR nucleotide sequences resulted in similar
topologies, but the sequence of the cultured PLA1 specimen
was grouped outside the cluster containing the other sequences
from K. pelagica and K. spiralis (data not shown).
Our data clearly demonstrate that the classification of the
marine cyanobacteria hitherto classified as belonging to the
genus Katagnymene is not supported by the 16S rRNA gene
and hetR phylogenies presented here. Rather, all the marine
nonheterocystous filamentous cyanobacteria collected and an-
alyzed fall in the same major cluster, the Trichodesmium clus-
194LUNDGREN ET AL.APPL. ENVIRON. MICROBIOL.
ter. Our data also for the first time demonstrate that Trichodes-
mium is divided into four main clusters, thereby extending
previous studies (14, 26) that identified three clusters by anal-
ysis of the same hetR sequence and identified two clusters with
a smaller number of strains by 16S-23S rRNA intergenic tran-
scribed sequence (ITS) analyses.
The large cluster I, besides known Trichodesmium species,
contained all the morphologically identified Katagnymene spp.
sequences. The partial hetR sequences from the 13 specimens
of K. pelagica and K. spiralis appeared to be randomly inter-
mixed in this cluster. Hence, our data suggest that these two
species should be merged into one species in spite of the
morphological diversity represented by trichomes whose
widths range from 12 to 35 ?m and the great differences in the
degree of coiling. Furthermore, it is clear that phenotypes such
as appearing in a noncolonial state, as solitary trichomes, and
as coiled trichomes are inconsistent with the genetic data pre-
sented here. The original descriptions of Katagnymene and
Trichodesmium (9, 19) relied on such phenotypic traits, and
revision is therefore needed. Consistent with this, analyses of
Baltic Sea Nodularia demonstrated that the distinction be-
tween coiled and noncoiled trichomes was not supported by
phycocyanin-intergenic sequence, gvpA-IGS, or rDNA-ITS ge-
notypic grouping (3). Furthermore, strains of the coiled cya-
nobacterium Arthrospira are not closely related to other coiled
cyanobacteria based on 16S rRNA gene sequences, and coiling
of trichomes can be lost during culturing (20, 23). The capacity
to form colonies and the colony shapes were not correlated to
the genetic data, since different colony shapes of the colony-
forming organisms (Trichodesmium) and the non-colony-form-
ing organisms (Katagnymene) were intermixed in cluster I.
Moreover, in cluster III the tuft-shaped T. thiebautii colonies
were also shown to be genetically identical to the puff-shaped
colonies of the same species when other genetic markers were
used (4, 26). However, the only partial hetR sequence from a
puff-shaped colony examined here clustered with the sequence
reported previously. The close relationship between Katag-
nymene spp. and Trichodesmium found here was substantiated
by previous data obtained by using partial nifH sequencing
(22), as well as sequencing of the 16S-23S rRNA ITS combined
with hetR denaturing gradient gel electrophoresis analyses and
HIP1 fingerprinting (26).
Cluster II, represented by gold-pigmented colonies (Table 2
and Fig. 3), is a novel cluster compared to the three clusters
identified by Janson et al. (14). The cluster II specimens most
likely represent a new species. However, due to a lack of
reference material other than the hetR sequences, a formal
description cannot be presented at this time.
Our data and those in previous analyses (14, 26) collectively
demonstrate that T. erythraeum is the most distinctly separate
cluster (cluster III) in the Trichodesmium radiation. Also in-
cluded in this cluster are sequences from large (?30-?m) dark-
pigmented trichomes with a high degree of segmentation, pre-
viously referred to as T. contortum due to the morphological
overlap (15). This was unexpected as T. erythraeum is charac-
terized by a cell width of 6 to 12 ?m (30) and forms raft-shaped
colonies (Fig. 1l), while the dark-pigmented morphotype ex-
amined here was typically more than 30 ?m wide and colonies
were not observed. Like cluster I, cluster III apparently en-
compasses sequences from a wide array of morphotypes,
which, however, showed more than 98% hetR nucleotide se-
Furthermore, a hetR sequence was retrieved from T. contor-
tum (sample B-46) (Fig. 1m and 3). The specimen was mor-
phologically and genotypically (100% sequence identity) ho-
mologous to the specimen described previously (14), and both
matched the original morphological description (30). Our data
therefore extended a recent study (26) by demonstrating a
morphological and genotypic difference between T. contortum
and K. spiralis.
In conclusion, the genus Trichodesmium was resolved into
four main clusters, which are composed of morphologically
diverse strains. One of these clusters (cluster I) also contains a
radiation comprising the two previously described marine Kat-
agnymene species, and cluster II is a novel Trichodesmium
lineage. One apparent link between the members of the genus
Katagnymene and the members of the genus Trichodesmium is
their nitrogen fixation behavior, which is unique among cya-
nobacteria (5, 15, 21, 22). We therefore propose that the genus
description of Trichodesmium must be extended to include
species and strains which predominantly live as individual
trichomes (organisms classified as Katagnymene) and that K.
pelagica and K. spiralis should be united into one species.
We are grateful to E. J. Carpenter (San Francisco State University,
San Francisco, Calif.) and D. Capone (The Wrigley Institute for En-
vironmental Studies, University of California at Los Angeles, Los
Angeles) for inviting us to participate in research cruises. J. Waterbury
(Woods Hole Oceanographic Institution, Woods Hole, Mass.) is ac-
knowledged for the cultured JWI1 specimen (K. spiralis).
We also acknowledge financial assistance from STINT (The Swedish
Foundation for International Cooperation in Research and Higher
Education) and Sida/SAREC (Swedish International Development
Cooperation Agency) to B.B. and the Swedish Research Council for
financial support provided to B.B. and S.J.
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