Microbiology (2000), 146, 2309–2315
Printed in Great Britain
A deeply branched novel phylotype found in
Japanese paddy soils
Hongik Kim,1,3 Daiske Honda,2 Satoshi Hanada,3 Norihiro Kanamori,1
Satoshi Shibata,1 Taro Miyaki,1 Kazunori Nakamura3 and Hiroshi Oyaizu1
Author for correspondence : Hongik Kim (National Institute of Bioscience and Human-technology).
Tel : j81 298 61 6590. Fax : j81 298 61 6587. e-mail : hongik!nibh.go.jp
1,2
Department of Global
Agricultural Sciences,
Graduate School of
Agricultural and Life
Sciences1, and Institute of
Molecular and Cellular
Biosciences2, The
University of Tokyo,
Yayoi 1-1-1, Bunkyo-ku,
Tokyo 113, Japan
3
Microbial Resources and
Chemotaxonomy
Research Group, National
Institute of Bioscience
and Human-technology,
Agency of Industrial
Science and Technology,
1-1 Higashi, Tsukuba,
Ibaraki 305-8566, Japan
Novel 16S rDNA clones which possibly constitute a sister clade from the two
known archaeal lineages, Crenarchaeota and Euryarchaeota, were found in
paddy soil environments. Overall signature sequences showed that the clone
sequences shared a majority of signature sequence features with the Archaea
and Eukarya. However, there were at least nine nucleotides which
distinguished the novel clones from the domains Archaea and Eukarya.
Phylogenetic trees, drawn by maximum-parsimony, neighbour-joining and
maximum-likelihood methods, also supported the unique phylogenetic
position of the clones. Both signature sequence and phylogenetic analyses
strongly suggest that the novel organisms constitute a new group and their
phylogenetic positions are distant from the Crenarchaeota and Euryarchaeota.
A specific primer set was designed to detect the presence of the novel group
of organisms in terrestrial environments. Specific DNA fragments were
amplified from all paddy soil DNAs, suggesting that the novel organisms are
widely distributed in rice paddy fields in Japan.
Keywords : novel soil clones, unculturable micro-organisms, paddy soil, novel
phylotype
INTRODUCTION
The three-domain system (Woese et al., 1990 ; Wheelis et
al., 1992) has been widely accepted, in contrast to the
eocyte tree (Lake, 1991), for describing the relationships
of the major lineages. Within this system, the three
phylogenetic lineages are categorized as the domains
Eukarya, Bacteria and Archaea (Woese et al., 1990).
Each domain is further subdivided into kingdoms. The
domain Archaea contains two kingdoms, Euryarchaeota
and Crenarchaeota. The kingdom Euryarchaeota
(Woese et al., 1990 ; Wheelis et al., 1992) includes
methane-producing archaea, extremely halophilic
archaea and some other genera, such as Archaeoglobus,
Thermococcus and Thermoplasma. The kingdom Crenarchaeota (Woese et al., 1990 ; Wheelis et al., 1992)
consists of extremely thermophilic and sulfur-dependent
archaea, such as Sulfolobus, Thermoproteus, Pyrodictium and Desulfurococcus.
.................................................................................................................................................
Abbreviations : ML, maximum-likelihood ; MP, maximum-parsimony ; NJ,
neighbour-joining.
The GenBank/EMBL/DDBJ accession numbers for the sequences of the
novel soil clones and their aligned data set are D88480–D88489 and
ds36901, respectively.
0002-4022 # 2000 SGM
Organisms in many disparate environments still remain
uncultured because of the difficulty of cultivation based
on conventional techniques. In recent years, the phylogenetic positions of uncultured bacteria have been
revealed by 16S rDNA sequence analyses (Ward et al.,
1990, 1992 ; Weller & Ward, 1991 ; Fuhrman et al.,
1992). Uncultured archaeal organisms have been detected in different biotopes, including marine environments (DeLong, 1992 ; Fuhrman et al., 1992), hot springs
(Barns et al., 1994, 1996), abyssal environments
(McInerney et al., 1995), freshwater (Schleper et al.,
1997) and soils (Bintrim et al., 1997 ; Großkopf et al.,
1998). Crenarchaeotal lineages, which are branched
very deeply from cultivated Crenarchaeota, were revealed to be predominant in marine and abyssal
environments (Fuhrman et al., 1992 ; McInerney et al.,
1995). In addition to this bunch of deeply branched
crenarchaeotal lineages, a number of phylotypes within
the Crenarchaeota have also been detected in nonextreme environments, such as marine, freshwater and
terrestrial soils (DeLong, 1998). These findings indicate
that members of this kingdom of the Archaea are
globally distributed and they are playing an ecologically
important role in their habitats. Euryarchaeotal clones
have also been detected in marine environments
2309
H. K I M a n d O T H E R S
(DeLong, 1992), hot springs (Barns et al., 1994) and
paddy fields (Großkopf et al., 1998). Discovering unknown organisms in environmental microbial communities is perhaps not as difficult as was once thought.
Recently, Barns et al. (1996) found an ancient lineage of
the Archaea in a hot spring and proposed a new
kingdom, ‘ Korarchaeota ’.
In the course of diversity analysis of paddy soil archaea,
we have found a number of unusual 16S rDNA clones
(Kudo et al., 1996, 1997). Small-subunit rRNA genes
were partially amplified by PCR from DNA extracted
directly from paddy soil, by using primer sets consisting
of reverse primers designed to amplify the 16S rRNA
encoding genes of a novel lineage selectively and
archaeal forward primer. In this report, we describe a
novel phylotype with no known close relatives deduced
from molecular phylogenetic analysis.
METHODS
Sample collection and DNA manipulations. Soil DNA was
extracted from several paddy soil samples collected at
Kagoshima-city (31m 34h N 130m 33h E), Yamaguchi-city (33m
57h N 130m 56h E), Kumagaya-city (36m 9h N 139m 23h E),
Tsukuba-city (36m 05h N 140m 08h E), Kumamoto-city (32m
49h N 130m 43h E), Tokyo-city (35m 42h N 139m 46h E) and
Yamagata-city (38m 15h N 140m 21h E). The DNA was purified
by agarose gel electrophoresis and submitted to PCR. A
volume of 10–50 ng DNA was used for each amplification.
Sampling procedures and PCR conditions are described in
detail by Kudo et al. (1996). Primer sets were synthesized
specifically to amplify the unusual 16S rDNA. The forward
primers synthesized were AS11F (5h-TTC GTC GAC GGT
TGA TCC YGS CRG AGG C-3h), which corresponds to
positions (in Escherichia coli) 11–23 of archaeal 16S rRNA ;
AS343F (5h-TTC GTC GAC TAC GGG GCG CAS CAG GC3h), which corresponds to positions 343–359 ; and AS564F (5hAAC CGT CGA CTG GGC CTA AAG CGY CCG TAG C3h), which corresponds to positions 564–584. Reverse primers
(complementary to 16S rRNA) were synthesized to be specific
to the unusual 16S rDNA based on the determined partial 16S
rDNA sequences (Kudo et al., 1996). The reverse primers,
which had sequences complementary to positions 1247–1229,
were SC13 (5h-CGG CGA ATT CTC CAC CAT TGT TGC
GCG-3h) and SW42 (5h-CGG CGA ATT CCC TAT CAT
TGT TGC GCG-3h). These primers had EcoRI or SalI
restriction sites on the 5h end. The DNA amplified by primer
sets of AS564F-SC13 and AS564F-SW42 was cloned into
pBluescript vector, and 10 clones were sequenced with a DNA
sequencer (Shimadzu DSQ-1000).
Phylogenetic analysis. Soil clone sequences were compared to
sequences in the available databases using the  algorithm (Altschul et al., 1997) to search for close evolutionary
relatives (the last survey was performed in March 21, 2000). As
no sequences in the DNA database showed similarities higher
than 80 %, representative organisms were selected from the
Archaea, Eukarya and Bacteria. Partial 16S rDNA sequences
of these organisms were then compiled for phylogenetic
analyses with those of soil clones. The data set was aligned
manually using 16S rRNA secondary structure to remove
some unalignable regions on the domain level and sequences
of identified homologous regions were realigned using the
program   version 1.7 (Thompson et al., 1994). The
compared sequences were positions 632–820, 880–996,
1041–1132 and 1142–1229. A maximum-parsimony (MP) tree
2310
reconstruction was performed using the  software version
3.1.1 (Swofford, 1991). The applied ratio of transition and
transversion was 2 : 1. A heuristic search was used with a
random stepwise addition sequence of 100 replicates, treebisection-reconnection branch swapping and the 
option. A further analysis was run with 100 bootstrap
replicates, each consisting of 10 additional random replicates.
A maximum-likelihood (ML) analysis was carried out using
a program package,  version 2.3b3 (Adachi &
Hasegawa, 1996). A ML distance matrix was calculated using
NucML, and a neighbour-joining (NJ) topology as the starting
tree for the ML method was reconstructed by NJdist in
. A ML tree was obtained using NucML with R (local
rearrangement search) option based on the HKY model
(Hasegawa et al., 1985). Local bootstrap probabilities were
estimated by the RELL (resampling of estimated log-likelihood) method (Kishino et al., 1990 ; Hasegawa & Kishino,
1994). NJ analysis was performed using the  software
version 3.572 (Felsenstein, 1995).  from this program
package was used to create a distance matrix based on the
two-parameter method of Kimura (1980). This distance matrix
was used to construct a NJ tree using the NJ algorithm from
 ( version 3.572), and the bootstrap analyses
utilized 1000 replicate data sets.
To assess the phylogenetic placement of the novel soil clones,
nucleotide signature analysis was done by using the 
program package (O. Strunk & W. Ludwig ; http :\\
www.mikro.biologie.tu-muenchen.de\pub\ARB\ ;
Technische Universita$ t Mu$ nchen, Germany) with data from the
Ribosomal Database Project (RDP) (Maidak et al., 1999).
Chimera check and prediction of rRNA secondary structure.
Each sequence was submitted to the - program
of the RDP (Maidak et al., 1999) to detect the presence of
possible chimeric artefacts, and the putative partial secondary
structure of 16S rRNA was predicted from the primary
structure using the program RNA structure version 2.52
(Mathews & Burkard, 1997). The Zuker algorithm (1989) was
used for RNA structure prediction based on the free energy
minimization, and the putative secondary structure was
constructed through comparison with that of published smallsubunit rRNA (Neefs et al., 1993).
Distribution of novel soil organisms in the environment. To
examine the distribution of the novel organisms in the
environment, we designed an oligonucleotide primer, 970F
(5h-AAT YYA ACT CAA CGC RGA G-3h), with a sequence
identical to positions 959–976 of 16S rRNA of the novel
clones. The presence of the novel soil organisms was tested
with a primer set of 970F and SC13 for paddy soils, upland
soils and forest soils collected in Japan. For each PCR reaction,
approximately 10 ng purified soil DNA was used, and
amplification was performed using AmpliTaq Gold (Perkin
Elmer) in a thermal cycler (Hybaid) programmed for 9 min at
94 mC, followed by 55 cycles of 30 s at 94 mC, 20 s at 57 mC, and
25 s at 70 mC, and an additional incubation at 70 mC for
10 min. After amplification, 7 µl product was electrophoresed
in a 1n5 % agarose gel and visualized by ethidium bromide
staining.
RESULTS AND DISCUSSION
Sequences of cloned 16S rDNA
Großkopf et al. (1998) analysed a number of archaeal
16S rDNA clones, covering novel euryarchaeotal groups,
which were detected on rice roots and in anoxic rice
paddy soil. Crenarchaeotal clones, which are divergent
A novel phylotype found in paddy soils
Euk
Euk
Euk
Euk
Euk
Euk
.................................................................................................................................................................................................................................................................................................................
Fig. 1. Putative secondary structure of the partial 16S rRNA of SC4. Bold letters were used to indicate the novel soil clone
unique nucleotide sequences. The corresponding nucleotides to these positions are highly conservative on the domain
level and they are indicated in boxes. Bac, Euk and Arc in boxes indicate Bacteria, Eukarya and Archaea, respectively, and
italic numerals are the numbers of the E. coli numbering system. The nucleotide 1 corresponds to the 585th nucleotide of
the 16S rDNA in the E. coli numbering system.
from this phylogenetic line of descent, were also detected
in soil environments (Bintrim et al., 1997). The discovery
of new lineages in the soil ecosystem is not surprising
considering the great genetic diversity of archaeal rDNA
clones retrieved from various natural environments. We
previously found novel 16S rDNA clones based on
sequence identity (Kudo et al., 1996, 1997). Although the
length of compared 16S rDNAs was approximately
270 bp, the sequences were extremely novel. Therefore,
extended phylogenetic analysis was necessary to clarify
the phylogenetic position of the novel soil clones.
To amplify nearly complete 16S rDNA fragments of the
novel soil organisms, PCR amplification was performed
with two primer sets, AS11F-SC13 and AS11F-SW42.
However, these primer sets did not give any amplification products. AS343F as a forward primer was
subjected to the PCR amplification instead of AS11F,
but these primer sets also failed to amplify the target
molecules. Partial 16S rDNA was obtained with primer
sets AS564F-SC13 and AS564F-SW42, and these products were cloned. Sequences of 10 cloned DNA samples
were determined for further analyses, and the sequences
of each clone were 82–98 % similar to each other. The
length of sequenced DNA ranged from 644 to 647
nucleotides. Each sequence was folded into a putative
secondary structure of 16S rRNA from positions 585 to
1228 (E. coli numbering system ; Fig. 1), and they were
found to be well-conserved in agreement with previously
published models (Neefs et al., 1993). The correct
secondary structure seemed to show some evidence that
the individual sequences are a natural occurrence,
although this evidence does not give complete proof that
the sequences are not chimeric. - also did
not detect chimeras among the novel soil sequences.
Phylogenetic analysis and signature analysis of the
novel soil clones
Phylogenetic analyses by MP, NJ and ML all showed
similar topologies (Fig. 2). Novel soil clones formed a
monophyletic clade which was supported by 99 %
bootstrap proportions, and these phylotypes formed a
sister group with the Euryarchaeota and Crenarchaeota
in all the trees. However, the domain Archaea was
ambiguously divergent by MP analysis. This unusual
2311
(b)
H. K I M a n d O T H E R S
2312
(a)
(c)
Crenarchaeota
Euryarchaeota
Novel soil clones
Eukarya
Bacteria
........................................................................................................................................................................................................................................................................................................................................................................
Fig. 2. Phylogenetic relationships of the novel soil clones inferred from the three analytical methods. (a) Strict consensus tree of the six most parsimonious
trees ; (b) NJ tree ; (c) ML tree (α/β l 2n942). Numbers in the MP and NJ trees indicate bootstrap percentage and numbers in the ML tree indicate local
bootstrap probabilities. Scale bars below the NJ and ML trees mean substitutions per site. The GenBank/EMBL/DDBJ accession numbers of the determined
sequences are D88480–D88489. The accession numbers of compared sequences are as follows : Archaeoglobus fulgidus, X05567 ; Aquifex pyrophilus, M8358 ;
Chloroflexus aurantiacus, D38365 ; Dictyostelium discoideum, X00134 ; Deinococcus radiodurans, M21413 ; E. coli, J01695 ; Giardia muris, X65036 ; Haloferax
volcanii, K00421 ; Homo sapiens, X03205 ; Methanobacterium thermoautotrophicum, Z37156 ; Methanobacterium bryantii, M59124 ; Methanococcus jannaschii,
M59126 ; Methanococcus thermolithotrophicus, M59128 ; Methanococcus vannielii, M36507 ; Methanococcus voltae, M59290 ; Methanogenium cariaci,
M59130 ; Methanospirillum hungatei, M60880 ; Petrotoga miotherma, L10657 ; Saccharomyces cerevisiae, M27607 ; Sulfolobus solfataricus, X03235 ;
Thermococcus celer, M21529 ; Thermofilum pendens, X14835 ; Thermoproteus tenax, M35966 ; Thermotoga maritima, M21774 ; Tritrichomonas foetus, U17509 ;
Vairimorpha necatrix, M24612 ; pjp27, L25852 ; pjp33, L25300 ; pjp41, L25301 ; pjp78, L25303 ; pjp89, L25305 ; JM2, L24196 ; and JM8, L24201. Clones pjp27,
pjp33, pjp41, pjp78 and pjp89 were isolated from Yellowstone National Park hot springs (Barns et al., 1994) ; JM2 and JM8 were isolated from an abyssal
deposit feeder (McInerney et al., 1995).
A novel phylotype found in paddy soils
Table 1. 16S rRNA sequence signature distinguishing Bacteria, Eukarya, Archaea and
novel soil clones
.....................................................................................................................................................................................................................................
Signatures for the Archaea, Eukarya and Bacteria were determined by  with a RDP database.
Mostly A, C, G and U are signatures with percentages of emergence lower than 97 %.
Position
688
693
699
716
756
912
931
946
952
962
966
973
975
1050
1086
1087
1109
1110
1114
1186
1208
1235
Novel soil
clones
Archaea*
Eukarya
Bacteria
C
A
G
C
G
U
G
G
C
Y
U
R
Mostly A
A
C
C
A
G
U
A
Mostly U
A
G
R
Y
C
G
Mostly U
G
A
C
G
U
C
G
G
C
C
A
G
C
G
C
U
G
G
C
Y
Mostly A
U
Mostly G
A
Mostly C
U
U
G
G
G
Mostly C
Mostly U
A
G
Y
Mostly A
Mostly C
U
G
Mostly G
C
A
Mostly C
Mostly C
C
A
Mostly U
Mostly C
G
G
Mostly A
G
Mostly U
Mostly G
C
A
C
G
C
U
* Including planktonic and abyssal clones and ‘ Korarchaeota ’.
branching was probably due to the limited number of
informative sites. The monophyletic relationship of the
Euryarchaeota and Crenarchaeota is supported by
comparatively high local bootstrap probabilities (90 %)
in the ML tree ; however, the bootstrap value for the NJ
topology was lower than that of the ML method. The
ML method is suitable for phylogenetic analysis among
organisms which have multiple substitutions. The NJ
method underestimates multiple substitutions, so it is
possible that its internal branch lengths are shorter than
those obtained by the ML method (Adachi & Hasegawa,
1995). In this case, the ML tree possibly estimates the
right internal branch length and true evolutionary tree.
The topology of phylogenetic trees is not reliable when
the bootstrap shows a moderate to low level. Deep
branchings, such as those between bacterial phyla,
usually show relatively low bootstrap values, and the
different branching topology has been presented in
various published papers, even though the same molecules were used for these analyses (Woese, 1987 ; Neefs
et al., 1993 ; Olsen et al., 1994). However, they are
clearly distinguished by signature sequences (Woese,
1987). We tried to find signature sequences (Woese,
1987) to distinguish the novel soil clones from archaea,
including deeply branched archaeal phylotypes. The
signature sequences are shown in Table 1. Among the
deeply branched archaeal phylotypes, marine planktonic
clones have been discovered from Pacific Ocean
bacterioplankton samples (Fuhrman et al., 1992). Later,
McInerney et al. (1995) also found clones belonging to
the marine planktonic archaeal lineage from the midgut
contents of a deep-sea marine holothurian. Fuhrman et
al. (1992) reported that the planktonic clones belonged
to the crenarchaeotal lineage, but McInerney et al.
(1995) reported that the planktonic and abyssal clones
formed a discrete phylogenetic cluster that was separate
from the Crenarchaeota and Euryarchaeota. Signature
sequence analysis (Table 1) revealed that the signature
nucleotides of planktonic and abyssal clones and the
‘ Korarchaeota ’ were identical to those of the Archaea.
In contrast, the novel soil clones showed signature
sequences that appeared to be different from those of the
Archaea (including planktonic and abyssal clones and
‘ Korarchaeota ’), Bacteria and Eukarya. The differences
that distinguish these novel soil clones from the Archaea,
Bacteria and Eukarya were found in positions 688, 693,
699, 946, 1050, 1114, 1186, 1208 and 1235 (Table 1).
Overall signature sequences showed that the novel clone
sequences shared more distinct signature nucleotides
with the Archaea and Eukarya than with the Bacteria.
Signature sequences that shared a majority of signature
2313
H. K I M a n d O T H E R S
Table 2. GjC contents of 16S rDNA from the novel soil
clones and the Archaea from position 586 to 1228
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
bp
Mean GjC content
(range) (mol%)
Novel soil clones
(10 clones)
55n9
(54n0–57n9)
Archaea
Euryarchaeota
(11 species)
Crenarchaeota
(three species and three clones)
‘ Korarchaeota ’
(two clones)
Planktonic and abyssal clones
(two clones)
55n8
(51n1–63n9)
65n2
(62n8–68n8)
64n5
(64n0–65n0)
53n4
(49n8–55n4)
sequence features with Archaea and Eukarya were
positions 716, 912, 931, 952, 966, 1086, 1109 and 1110.
The mean sequence similarity of the novel soil clones to
the Crenarchaeota and Euryarchaeota was 78n0 %, a
value much lower than the mean value (83n7 %) between
Crenarchaeota and Euryarchaeota.
The signature sequences shown in Table 1 are very
conservative, and some of the nucleotides, at positions
688, 699, 946, 1050, 1208 and 1235, have been recognized
as universally conserved among the domains Eukarya,
Archaea and Bacteria (Woese, 1987). Our finding that
the 10 novel soil clones exhibited non-universal
sequences supports their connection to an ancient
lineage of life ; thus the acquisition of their full 16S
rDNA sequences may help to resolve phylogenetic
relationships of major lineages in the tree of life.
Inference of novel soil micro-organisms
We know nothing about these organisms other than
their 16S rDNA sequences, so it might be difficult to
predict their phenotypic characteristics. However, it is
expected that sequences derived from the paddy fields
are from mesophilic organisms. Table 2 compares the
GjC contents of the sequenced regions of the 16S
rDNA of Archaea and the novel soil clones. The high
GjC content of the Crenarchaeota is correlated with
their thermophilic characteristic ; thermophiles usually
have rDNA GjC contents of 60 mol % whereas
mesophiles generally have rDNA GjC contents of
55 mol % or less (Dalgaard & Garrett, 1993 ; Woese et
al., 1991). Judging from the sampling environments and
the GjC contents of the novel soil clones, the sequences
probably come from mesophilic organisms.
The presence of the novel soil organisms was determined
by using a primer set of 970F and SC13 for paddy soils,
upland soils and forest soils collected in Japan. PCR
amplification (Fig. 3) showed that the novel organisms
were present in all paddy soil samples, but not in upland
and forest soil samples. The archaeal community
2314
489
267
80
.................................................................................................................................................
Fig. 3. PCR amplification of novel soil clones with a very specific
primer set 970F-SC13. Lanes : 1 and 13, pHY size maker (Takara
shuzo) ; 2, Kumagaya paddy soil no. 2 ; 3, Kumagaya paddy soil
no. 3 ; 4, Yamaguchi paddy soil ; 5, Kagoshima paddy soil ; 6,
Yamagata paddy soil ; 7, Tokyo upland soil ; 8, Yamagata
upland soil ; 9, Yamagata pasture land soil ; 10, Tokyo forest
soil ; 11, Yamagata forest soil no. 1 ; 12, Yamagata forest soil
no. 2 ; 14, DNA of Methanobacterium wolfei DSM 2970T ; 15,
DNA of Methanobrevibacter smithii DSM 861T ; 16, DNA of
Methanosaeta thermoacetophila DSM 4774T ; 17, DNA of
Methanosarcina mazei TMA.
structure changes according to soil conditions (Chin et
al., 1999). Likewise, the population of the novel microorganisms might change depending on the season and
weather conditions. In the PCR detection, novel soil
micro-organisms were detected in anoxic soils but not in
oxic soils. From these facts, we speculate that the
sequences would come from anaerobes or facultative
anaerobes.
Conclusions
In the present study, we discovered a novel phylogenetic
cluster of life in soil DNAs. This phylogenetic cluster
showed a unique signature sequence in the 16S rDNA.
The cluster was also diverse phylogenetically. We have
not yet recognized the organisms from which the novel
clones were derived. However, the organisms are
presumed to inhabit Japanese paddy soils and this
would indicate an important role in the soil anaerobic
ecosystem. If these organisms are cultured, they will
provide useful information, not only on microbial
ecology, but also on the phylogenetic relationships of
major lineages in the tree of life.
ACKNOWLEDGEMENTS
The authors gratefully acknowledge the support of the Yakult
Bioscience Foundation. We thank Dr Yoichi Kamagata for his
helpful discussion and for editing the English usage in the
manuscript. We are also grateful to Professor Wen-Tso Liu for
his valuable advice and comments on the . This study was
supported in part by a domestic research fellowship provided
by the Japan Science and Technology corporation (H. K.).
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Received 2 February 2000 ; revised 13 April 2000 ; accepted 4 May 2000.
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