FEMS Microbiology Ecology 34 (2000) 45^56
www.fems-microbiology.org
Identi¢cation of novel Archaea in bacterioplankton of a boreal
forest lake by phylogenetic analysis and £uorescent in situ
hybridization1
German Jurgens a; *, Frank-Oliver Glo«ckner b , Rudolf Amann b , Aimo Saano a ,
Leone Montonen a , Markit Likolammi c , Uwe Mu«nster c
a
Department of Applied Chemistry and Microbiology, Division of Microbiology, University of Helsinki, P.O. Box 56, Biocenter 1A, Viikinkaari 9,
FIN-00014 Helsinki, Finland
b
Max-Planck-Institute for Marine Microbiology, Celsiusstrasse 1, D-28359 Bremen, Germany
c
Lammi Biological Station, University of Helsinki, FIN-16900 Lammi, Finland
Received 24 March 2000; received in revised form 7 July 2000; accepted 11 August 2000
Abstract
We report here on novel groups of Archaea in the bacterioplankton of a small boreal forest lake studied by the culture-independent
analysis of the 16S rRNA genes amplified directly from lake water in combination with fluorescent in situ hybridization (FISH). Polymerase
chain reaction products were cloned and 28 of the 160 Archaea clones with around 900-bp-long 16S rRNA gene inserts, were sequenced.
Phylogenetic analysis, including 642 Archaea sequences, confirmed that none of the freshwater clones were closely affiliated with known
cultured Archaea. Twelve Archaea sequences from lake Valkea Kotinen (VAL) belonged to Group I of uncultivated Crenarchaeota and
affiliated with environmental sequences from freshwater sediments, rice roots and soil as well as with sequences from an anaerobic digestor.
Eight of the Crenarchaeota VAL clones formed a tight cluster. Sixteen sequences belonged to Euryarchaeota. Four of these formed a cluster
together with environmental sequences from freshwater sediments and peat bogs within the order Methanomicrobiales. Five were affiliated
with sequences from marine sediments situated close to marine Group II and three formed a novel cluster VAL III distantly related to the
order Thermoplasmales. The remaining four clones formed a distinct clade within a phylogenetic radiation characterized by members of the
orders Methanosarcinales and Methanomicrobiales on the same branch as rice cluster I, detected recently on rice roots and in anoxic bulk
soil of flooded rice microcosms. FISH with specifically designed rRNA-targeted oligonucleotide probes revealed the presence of
Methanomicrobiales in the studied lake. These observations indicate a new ecological niche for many novel `non-extreme' environmental
Archaea in the pelagic water of a boreal forest lake. ß 2000 Federation of European Microbiological Societies. Published by Elsevier
Science B.V. All rights reserved.
Keywords : Archaea; Bacterioplankton; Boreal forest lake; 16S rRNA; Phylogeny; Fluorescent in situ hybridization
1. Introduction
Freshwaters and wetlands in boreal environments have
been identi¢ed as important sources for radiatively relevant trace gases [1,2] and can contribute by up to 34%
from total global wetlands CH4 £uxes [3]. Prokaryotes in
* Corresponding author. Tel. : +358 (9) 19159277;
Fax: +358 (9) 19159322; E-mail : german.jurgens@helsinki.¢
1
The sequences of environmental Archaea from freshwater SSU rDNA
VAL clones obtained in this study have been submitted to the DDBJ/
EMBL/GenBank database under accession numbers AJ131263^
AJ131278 and AJ131311^AJ131322.
soil, sediments and water play an essential role in this
scenario by their contribution to CO2 and CH4 cycling
and £uxes [4]. In addition to their global role in biogeochemical cycles [5], prokaryotes play a key role in the
transformation of natural organic matter, which in many
boreal freshwaters is of primarily allochthonous origin and
recalcitrant [6,7]. However, it is still little known about the
community structure and function of indigenous microorganisms in boreal freshwaters [7]. Except for the abundance and the role of methanogens in some boreal lakes
[8,9] microorganisms in boreal freshwater are lumped together and still treated as a black box. For a better understanding of indigenous bacterioplankton in boreal freshwaters, we are interested in the development of new
0168-6496 / 00 / $20.00 ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
PII: S 0 1 6 8 - 6 4 9 6 ( 0 0 ) 0 0 0 7 3 - 8
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G. Jurgens et al. / FEMS Microbiology Ecology 34 (2000) 45^56
molecular tools and approaches to follow their role in
nutrient cycling, biodegradation and microbial food web
interactions. Recently, we reported the phylogenetic positions of novel groups of Archaea obtained from boreal
forests in southern Finland [10,11]. There may be phylogenetic and functional links between the boreal terrestrial
and aquatic environments and we are now searching for
similar archaeal groups in boreal lake water by developing
phylogenetic probes in order to trace them under in situ
conditions in their speci¢c habitats.
Most phylogenetic studies on freshwater Archaea have
so far been made from sediment samples. It has been
shown that some of the 16S rDNA sequences of Archaea
belong to di¡erent sub-clusters of Group I uncultured
Crenarchaeota [12,13]. Others were a¤liated with the
orders Methanosarcinales [14] and Methanomicrobiales
[15,16]. Only a few non-cultured indigenous planktonic
Archaea have been described by Òvrea®s et al. [9] in the
meromictic lake Saelenvannet (Norway) with a salinity
gradient in the chemocline.
In addition to polymerase chain reaction (PCR)-based
techniques, £uorescent in situ hybridization (FISH) with
rRNA-targeted £uorescently labeled oligonucleotide
probes has been widely used to study microbial communities in various environments [17,18]. However, the number of detected Archaea cells often remained below the
detection limit (i.e. 1% of the cells stained by DAPI
(4P,6-diamidino-2-phenylindole)) when e.g. the microbial
community composition of the Wadden Sea sediments
from the German North Sea coast were investigated [19].
In a study on seasonal variation in the community
structure and cell morphology of pelagic prokaryotes in
a high mountain lake in Austria, it has been shown that
Archaea represented from less than 1% to a maximum of
5% of all DAPI-stained cells and reached their highest
densities in September and November. A distinct maximum of a ¢lamentous fraction of Archaea was observed
during September and October, which was probably an
e¡ect of grazing pressure by phagotrophs [18]. All these
investigations were made by using probe ARCH915 [20],
which is speci¢c for all Archaea. Only a few attempts have
yet been made to develop phylogenetic trees of indigenous
planktonic Archaea in boreal freshwater and to use them
as a basis for probe design in FISH.
In our studies on the phylogenetic relatedness and distribution of Archaea in the bacterioplankton of the boreal
forest lake Valkea Kotinen we used two basic molecular
approaches : phylogenetic analysis of the ampli¢ed 16S
rRNA gene sequences and FISH. In this paper we report
the phylogenetic placement of new freshwater clones
among numerous recently discovered environmental 16S
rDNA Archaea sequences. New sequences could be used
to design probes, suitable for the study of these indigenous
Archaea under in situ conditions. We show here that it
is possible to detect the abundance, cell size and morphotype of novel Archaea in the plankton by FISH, using
FEMSEC 1169 18-10-00
some of these new probes. In the future we intend to
follow their fate and role in the microbial food web of
this lake and to place them in a phylogenetic and functional context.
2. Materials and methods
2.1. Basic physico-chemical and biological aspects in
Valkea Kotinen
Valkea Kotinen is a typical small boreal lake located in
the Evo forest area in southern Finland (61³24PN,
24³07PE). It has a water surface area of 3.6 ha, a catchment/surface ratio of 8.3, a maximum depth of 7 m and an
average depth of 3 m.
This mesohumic forest lake has typical seasonal thermal
and chemical strati¢cation patterns with, on average, a 1^
2-m-deep epilimnion, a 0.5^1-m-deep metalimnion and a
deeper (3^4 m deep) anoxic hypolimnion, from early May
until late September. The thermal stability of the water
column can vary from May until September between 5
and 25 J m32 (Birgean wind work), and 2 and 18 J m32
(Schmidt thermal stability), respectively [21], with very few
mixing events during the summer stagnation [22]. The
water was slightly acid with a pH = 4.9^6.2, a low bu¡er
capacity (alkalinity 6 0.2 meq m33 ) and a watercolor of
100^250 g platinum units m33 . The total organic carbon
varied between 7.8 and 17.4 g C m33 and the DOC between 7.5 and 16.5 g C m33 indicating a high level of
DOC sources, which were mainly of allochthonous origin.
Inorganic nutrients were low and the phosphorus and nitrogen resources were available primarily as organic resources. Phytoplankton biomass (Chl-a) varied between
2.2 and 75.4 mg m33 with a major contribution from
Raphidiophyceae [21]. Gonyostomum semen is the dominant
phytoplankton species in this lake from mid-July until late
September, which covers on average 40^90% of all the
Chl-a [23]. The ratio of net primary production to community respiration (P:R) was on average 1.1:1 and in late
summer and autumn decreased even below 1, which is a
major e¡ect of bacterioplankton activity on the whole lake
carbon metabolism [21].
2.2. Sampling and nucleic acid extraction
Lake water was taken with a Limnos sampler from the
surface (0.1 m) down to the deepest (7 m) water layers in
0.5-m intervals from the pelagial zone.
For microbial DNA extraction, isolation and puri¢cation, integrated water samples were taken from the pelagial zone in July and stored in pre-cleaned, sterile Nalgene
bottles in the dark on crushed ice prior to ¢ltration. The
cells from 250 ml of water were then collected on a 0.2Wm-pore-size polycarbonate (hydrophilic) ¢lter (diameter,
47 mm; Cyclopore1 Track Etched Membrane, Whatman,
Cyaan Magenta Geel Zwart
G. Jurgens et al. / FEMS Microbiology Ecology 34 (2000) 45^56
UK) and immediately frozen at 325³C until nucleic acid
extraction.
After 3 days storage at 325³C, extraction began with
the addition of 5 ml of lysis bu¡er (1 mg ml31 lysozyme,
40 mM EDTA, 50 mM Tris^HCl, 0.75 M sucrose,
pH = 8.0) and incubation at 37³C for 30 min [24]. Then
proteinase K (0.5 mg ml31 ) and sodium dodecyl sulfate
(1% SDS w/v) were added and the ¢lter was incubated at
55³C for 2 h. The lysate was recovered from the ¢lter to a
fresh tube. The ¢lter was rinsed with an additional 2 ml
lysis bu¡er, incubated for 10 min at 55³C and the lysates
were pooled. To the pooled lysate, 5 M NaCl (¢nal concentration 0.7 M) and hexadecyltrimethyl ammonium bromide (¢nal concentration 1% w/v in the presence of 0.7 M
NaCl) were added. The mixture was incubated at 65³C for
20 min and then extracted with chloroform^isoamyl alcohol (24:1). The upper water-DNA phase was collected into
a fresh tube and DNA was precipitated by adding 0.6
volume of isopropanol. The pellet was washed with 70%
(v/v) ethanol, dried and dissolved in 100 Wl of water. This
crude DNA sample was puri¢ed using a Wizard DNA
clean up kit (Promega, USA) [25] and the resulting
DNA was used as a template for the PCR.
2.3. Sampling for in situ hybridization
Samples for FISH were collected during the period from
March to September from three depths: the epilimnion (0^
47
1-m depths), the metalimnion (1^3-m depths) and the hypolimnion (from 3 to 7 m, near the bottom sediment).
Water samples were taken in 0.5-m intervals with a 1-l
Limnos sampler in the pelagial of the lake at the deepest
point. Lake water samples from the epi-, meta- and hypolimnetic sub-samples were pooled and stored in sterile 5-l
Nalgene bottles immersed in crushed ice during sampling
and transportation to the laboratory. Within 2^3 h after
sampling the microbial cells were concentrated from 15^20
ml of water on a 0.2-Wm-pore-size polycarbonate ¢lter
(diameter, 47 mm; Cyclopore1 Track Etched Membrane,
Whatman, UK) by vacuum ¢ltration at 6 100 P m32 . The
¢lters were prepared and ¢xed for FISH as described previously [26,27] and stored at 325³C until further processing.
2.4. PCR ampli¢cation and cloning
A region of the 16S rRNA gene was ampli¢ed using a
nested PCR approach with primers described in Table 1.
As a ¢rst step, PCR was performed using two sets of
primers: (i) Ar4F^Un1492R and (ii) Ar4F^Ar958R, amplifying respective regions of the 16S rRNA gene of
around 1500 and 1000 bp in length. As a second step,
both PCR products were used separately in new PCR reactions using an Ar3F^Ar9R `internal nested' primer set.
This pair of Archaea-speci¢c primers was located inside
the products from the ¢rst round and ampli¢ed a 16S
Table 1
PCR primers and FISH probes used in this study
Fd
Name
Sequence (5P^3P)
Comments
Refs.
PCR primers
Ar4Fa (8^25)b
Ar958Ra (958^967)
Ar9R (906^927)
TCY GGT TGA TCC TGC CRG
YCC GGC GTT GAV TCC AAT T
CCC GCC AAT TCC TTT AAG TTT C
[12]
[53]
[10]
Ar3F (7^26)
TTC CGG TTG ATC CTG CCG GA
Un1492R (1492^1510)
GGT TAC CTT GTT ACG ACT T
FISH probesc
ARCH915 (915^934)
EURY498 (498^511)
CREN499 (499^515)
CREN512 (512^527)
forward primer, speci¢c to Archaea 16S rRNA gene
reverse primer, speci¢c to Archaea 16S rRNA gene
reverse `internal nested' primer, speci¢c to Archaea
16S rRNA gene
forward `internal nested' primer, speci¢c to Archaea
16S rRNA gene
universal reverse primer, speci¢c to all types of
16S rRNA gene
GTG CTC CCC CGC CAA TTC CT
CTT GCC CRG CCC TT
CCA GRC TTG CCC CCC GCT
C GGC GGC TGA CAC CAG
[20]
[33]
[33]
this work
20
0
0
0
CREN569 (569^585)
GCT ACG GAT GCT TTA GG
this work
0
EURY496 (496^512)
GTC TTG CCC GGC CCT TTC
this work
20
EURY499 (499^514)
CGG TCT TGC CCG GCC CT
this work
20
EURY514 (514^528)
G CGG CGG CTG GCA CC
speci¢c to most Archaea sequences
speci¢c to part of Euryarchaeota sequences
speci¢c to part of Crenarchaeota sequences
speci¢c to most Crenarchaeota sequences in the
updated ARB database
speci¢c to most environmental Crenarchaeota
sequences in the updated ARB database
speci¢c only to a Methanomicrobiales group and
new VAL 47, VAL 1, VAL9, VAL78 clones
speci¢c to Methanosarcina, Methanosaeta,
Methanomicrobiales groups and new VAL 47,
VAL 1, VAL9, VAL78 clones
speci¢c to most Euryarchaeota sequences in the
updated ARB database
this work
20
a
Modi¢cations in primers Ar4F and Ar958R compared to reference primers were made by G. Jurgens.
Target site (position numbers refer to the E. coli 16S rRNA).
c
FISH probe overlaps shown in bold.
d
Formamide concentration in % (v/v) for the in situ hybridization bu¡er.
b
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[10]
[53]
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G. Jurgens et al. / FEMS Microbiology Ecology 34 (2000) 45^56
rRNA gene region around 900 bp long. The reampli¢ed
PCR products were puri¢ed from agarose gel using PrepA-Gene DNA Puri¢cation Kits (Bio-Rad Laboratories,
USA) and were separately cloned in the pGEM-T-Easy
vector (Promega, USA).
In order to ensure the archaeal origin of the 160 clones
obtained, Southern hybridization with an Archaea-speci¢c
probe was performed [11]. Inserts were digested with AvaII
and MspI restriction enzymes to determine restriction
fragment length polymorphism [10] patterns and as a result 28 of them were chosen for sequencing. We have not
found any speci¢c di¡erences between the inserts reampli¢ed from the two initial PCR products and therefore consider all clones independent and equivalent.
2.5. Phylogenetic analysis
The phylogenetic analysis was performed using the program package ARB [28]. In order to ensure the correct
phylogenetic placement of the obtained sequences and to
give a comprehensive up-to-date evaluation of the Archaea phylogeny, new sequences from freshwater (VAL)
were added to the ARB database consisting of 642 complete or partial Archaea 16S rRNA sequences from public
databases. Only the sequences with the su¤cient length for
phylogeny (minimum of 400 nucleotides) were included in
the analysis. The ARB_EDIT tool was used for automatic
sequence alignment and then the sequences were corrected
manually. Regions of ambiguous alignment were excluded
from the analysis. A 50% invariance criterion for the inclusion of individual nucleotide sequence position in the
analysis was used to avoid possible treeing artifacts. The
program CHIMERA_CHECK version 2.7 from Ribosomal Database Project [29] was used to detect possible chimera among studied sequences.
Many clones in the database were sequenced only partially and some of them do not overlap with the others. To
avoid reconstruction artifacts arising from partial sequences we constructed two `backbone' maximum-likelihood
trees : one consisting of 84 Crenarchaeota and the other
of 79 Euryarchaeota clones for which sequence data between Escherichia coli 16S rDNA positions 7 and 927 were
available. All the VAL sequences were already included at
this stage of the analysis, therefore their placement was
determined by the more robust and sophisticated maximum-likelihood method. The partial sequences were then
added using the parsimony option of the program package
ARB to those trees without changing the overall tree
topology.
Several trees di¡ering in the set of alignment positions
as well as reference sequences were used during tree con-
struction. Phylogenetic trees were inferred by performing
maximum likelihood (fastDNAml) [30], maximum parsimony [31] and neighbor-joining with Jukes^Cantor distance correction method [32] included in the ARB package. Similarities between 16S rDNA sequences were
determined by using the multiple sequence comparison
tool of the ARB program package.
2.6. In situ hybridization and probe description
FISH followed the protocol of Amann [26] and Glo«ckner et al. [17]. Oligonucleotides labeled with carbocyanine
dye CY3 were purchased from Interactiva (Germany). A
list of tested probes, their speci¢city, nucleotide sequences
and respective formamide concentrations in the in situ hybridization bu¡ers are summarized in Table 1.
To detect Archaea in the samples the previously described 16S rRNA gene probes EURY498, CREN499
[33] and ARCH915 [20] were used as well as ¢ve probes,
newly designed using the ARB `Probe_design' program
[28].
The probe EURY498 was previously thought to be speci¢c to most Euryarchaeaota [33]. However, since it was
designed, many new indigenous Archaea sequences were
found. After inclusion into the ARB database recently
published archaeal 16S rRNA sequences, it appeared
that EURY498 was not able to detect more than 50% of
the Euryarchaeota sequences. This probe does not cover
sequences obtained in this study from VAL-clones: VAL
47, VAL 1, VAL9 and VAL78, related to Methanomicrobiales, as well as many other sequences of uncultured Archaea. Therefore, in addition to the EURY498 probe,
three new probes for Euryarchaeota (EURY514,
EURY496 and EURY499) were designed taking into account new Archaea sequences, including VAL clones. Two
new probes, CREN512 and EURY514, targeted sequences
at the kingdom level and were speci¢c to kingdoms Crenand Euryarchaeota, respectively. The new probe
CREN569 was speci¢c to most environmental sequences
of Crenarchaeota. New probes CREN512, EURY499 and
EURY514 were tested for the optimal formamide concentration on pure cultures of Crenarchaeota (Pyrobaculum
neutrophilum, Sulfobococcus zilligii, Thermoproteus tenax)
and Euryarchaeota (Methanosarcina barkery, Methanobacterium formicium). Members of the Bacteria (E. coli and
Sinorhizobium) were used as negative controls for all Archaea probes. Probe-speci¢c cell counts were calculated
taking into consideration counts obtained with the negative control NON338 [34].
For hybridization, a section of the ¢lter was placed on a
glass slide, overlaid with 20 Wl of hybridization solution
C
Fig. 1. Phylogenetic analysis of the VAL freshwater Crenarchaeota clones from lake Valkea Kotinen (shown as bold). Scale bar: 0.1, estimated number
of substitutions per nucleotide position.
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G. Jurgens et al. / FEMS Microbiology Ecology 34 (2000) 45^56
[0.9 M NaCl, 20 mM Tris/HCl (pH 7.4), 0.01% SDS w/v,
0^35% (v/v) formamide (for the reference see Table 1) and
50 ng of probe] and incubated at 46³C for 2 h in an equilibrated humid chamber [27]. Optimal formamide concentrations for the new probes were determined empirically
by formamide series from 0 to 50% (v/v) with a 10% (v/v)step.
The ¢lters were transferred to a pre-warmed (48³C) vial
containing 50 ml of washing solution [70 mM NaCl, 20
mM Tris/HCl (pH 7.4), 5 mM EDTA and 0.01% SDS w/v]
and incubated freely £oating at 48³C for 15 min. Filters
were dried on Whatman paper and overlaid with 50 Wl of
DAPI solution (1 mg ml31 in water) for 5^10 min at room
temperature in the dark. Subsequently, they were shortly
washed in 70% (v/v) ethanol and then in distilled water,
dried on Whatman paper and mounted on slides with
Citi£uor AF1 (Citi£uor Ltd., Canterbury, UK). Epi£uorescence microscopy was carried out as described before
[27] using a Zeiss Axioplan equipped with speci¢c ¢lter
sets (DAPI : Zeiss01 and CY3 : Chroma HQ41007, Chroma Tech. Corp., Brattleboro, VT, USA).
Table 2
Environmental sequences of Group I Crenarchaeota included in the
phylogenetic analysis and shown on the tree as numbers in boxes
3. Results and discussion
Group I.1c
Name of the box Sequence name Accession No.
Group I.1a
Group I.1b
TS
FIN
ANT12
WHARQ
SBAR
pB1-100
JBT, JTA
Mariana
JM
PM, C
p712
Fosmid 4B7
C. symbiosum
NH49-9
PVA_OTU
LMA
SB95
P17, M17
KBS
SCA
pLemA8
pGrfA
FFS
AF052943, AF052946, AF052948,
AF052949
AF052954
U11043
M88079
M88075, M88076
U86488
AB015274^AB015278
D87348^D87350
L24195^L24201
U71109, U71110, U71112^U71118
U81531
U39635
U51469
Z11573
U46678^U46680
U87517^U87520
U78197^U78200
U68650, U68604
AF058719^AF058730
U62811^U62820
U59987
U59968^U59982, U59999
X96688^X96692, X96694^X96696,
AJ006919^AJ006922
3.1. Microbial community aspects in Valkea Kotinen
Due to its thermal stability the Valkea Kotinen hypolimnetic water is anaerobic during the whole growing season. About 43^94 WM methane has been measured in
epilimnetic water and 140^13000 WM methane in hypolimnetic water from spring to winter, respectively [35].
Signi¢cant PCR products were obtained from hypolimnetic water layers with mcr-primers [36] coding for the
methyl-coenzyme reductase (MCR) in methanogens [37].
This could imply that methanogens make up a considerable part of the archaeal population, and play a crucial
role in the C1-carbon £ux that via grazing phagotrophs
¢nally ends up in zooplankton as suggested by Jones et al.
[38].
Bacterial cell numbers in the lake varied between 1 and
5.7U106 cells ml31 with a bio-volume between 0.009 and
0.04 Wm3 cell31 . The generally low variation of bacterial
numbers and the observed small size of bacteria was probably a major e¡ect of grazing pressure by phagotrophs
[39]. Bacterial production measured as [14 C]leucine incorporation varied on average between 15.4 and 19.7 Wg C l31
d31 all over the water column [21]. A mean value of 15.4
Wg C l31 d31 was measured in epilimnetic water, 18.5 Wg C
l31 d31 in metalimnetic water and 19.7 Wg C l31 d31 in
hypolimnetic water. These data are within a similar range
as those found for marine waters, lakes and other studied
Finnish forest lakes [7,40].
3.2. Phylogenetic placement of archaeal clones recovered
from lake Valkea Kotinen among other environmental
sequences
A total of 28 clones (named VAL) recovered from lake
Valkea Kotinen were sequenced. We found (i) that VAL
sequences contained representatives of both Cren- and
Euryarchaeota, (ii) no cultivated Archaea were closely related to the VAL sequences and (iii) the archaeal sequences most closely related to the VAL clones (with similarity
around 96%) inhabited a very wide range of environments : marine and lake sediments, di¡erent types of soils,
rice roots and an anaerobic digestor. Two separate trees,
one for Crenarchaea and one for Euryarchaea, were generated (Figs. 1 and 2). Sequences listed in Tables 2 and 3
represent uncultured Archaea indicated on tree ¢gures as
numbers in the boxes. Tables 2 and 3 were made in order
to provide detailed information on all environmental sequences used in tree reconstruction. The names of the
clones, references and environments are given in Table 4.
We found that 12 freshwater VAL-sequences fall within
C
Fig. 2. Phylogenetic analysis of the VAL freshwater Euryarchaeota clones from lake Valkea Kotinen (shown as bold). Scale bar: 0.1, estimated number
of substitutions per nucleotide position. Numbers in the boxes indicate how many of the sequences are from uncultured Archaea.
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G. Jurgens et al. / FEMS Microbiology Ecology 34 (2000) 45^56
a subcluster (named `freshwater cluster' [41] or Group I.3
[13] of Group I uncultured non-thermophilic Crenarchaeota (Fig. 1). Eight of the sequences form a tight VAL I
lineage inside of the cluster which also includes sequences
previously detected in lake sediments [12,15], rice roots
and soil [42], and in an anaerobic digestor [43].
The analysis of 16 VAL sequences identi¢ed four distinct lineages related to di¡erent orders of Euryarchaeota
(Fig. 2). Four clones were a¤liated with environmental
sequences previously detected in anoxic sediments from
Rotsee (Switzerland) [44], post-glacial profundal freshTable 3
Environmental sequences included in the analysis and shown on the
Euryarchaeota tree as numbers in boxes
Name of
the box
Sequence
name
Accession No.
Methanosarcina
ARR
AJ227936, AJ227944, AJ227945, AJ227922,
AJ227940
Y15394, Y15387
U81777, U81776
U87515, U87516
AF029207^AF029211
AF015986, AF015987, AF015974,
AF015975, AF015972, AF015966,
AF015991, AF015977, AF015986
AB015279
AF134393, AF134384
ABS
vadin
LMA
ARC
2MT, 2C,
1MT
Methanosaeta
JTA
Eel-BA,
Eel-TA
ABS,
A5.1-A
2MT, 2C
Y15391^Y15393, Y15386, Y15389, Y15398,
Y15396
AF015990, AF015980, AF015993,
AF015973
Rot
Y18082, Y18083, Y18087, Y18090,
Y18091, Y18094
Rice cluster I ARR
AJ227924, AJ227950, AJ227923, AJ227930,
AJ227928, AJ227937, AJ227942, AJ227943,
AJ227933, AJ227926, AJ227921, AJ227927,
AJ227920, AJ227935
ANME-1
Eel-BA,
AF134380-AF134382, AF134383,
Eel-TA
AF134385^AF134387, AF134391,
AF134392
Extreme
2C,1MT,
AF015984, AF015967, AF015976,
halophiles
2MT
AF015988, AF045182, AF045164
AF045189
Group II
TS
AF052944, AF052945 AF052947
FF
AF052953, AF052951
FIN
AF052952
GIN492
AF052950
pB1
U86502, U86508
pN1, p712
U86455, U81547
PVA_OTU_1 U46677
ANT5
U11044
WARN
M88078
SB95, SBAR U78206, M88074, M88077
OARB
U11042
MethanoABS, A5.1-B Y15388, Y15385, Y15390, Y15399
bacteriales
ARR
AJ227934, AJ227938, AJ227947, AJ227949,
AJ227939
vadinDC06 U81775
ARC
AF029172^AF029205
Cd30
AB008900
FEMSEC 1169 18-10-00
Table 4
Archaea sequences by their environments
Clone name
Environment description
Hot environments
pSL
hot spring, Yellowstone National Park, USA
pJP
hot spring, Yellowstone National Park, USA
PVA_OTU
hydrothermal vent microbial mat, Paci¢c Ocean,
Hawaii
Marine and salty environments
ANT, OARB
marine picoplankton, Arthur Harbor, Antarctica
WHARQ,
marine picoplankton, Atlantic ocean, USA
WARN
SBAR, SB95
marine picoplankton, St. Barbara Channel, USA
pB1
marine picoplankton, Atlantic ocean
p712, pN1
marine plankton, Paci¢c ocean
PM, C
marine plankton, Atlantic ocean
Fosmid 4B7
marine picoplankton, Paci¢c Ocean
NH
marine picoplankton, Paci¢c Ocean, USA
TS
suspended particulate matter, North Sea, Netherlands
FIN
£ounder digestive tract
FF
£ounder feces
GIN492
grey mullet digestive tract
JM
sea cucumber midgut, Atlantic ocean
C. symbiosum
marine sponge tissue, Paci¢c Ocean
JBT, JTA, JTB marine sediment cold-seep area, Japan Trench
Mariana
marine sediment, Mariana Trench
BBA
marine sediment, Cape Cod, USA
1MT, 2MT, 2C salt marsh sediments, UK
Eel-BA, Eel-TA marine sediment, Eel river, CA, USA
Freshwater environments
pLaw
freshwater lake sediment, Lawrence Lake, USA
LMA
freshwater lake sediment, Lake Michigan, USA
pLem
freshwater lake sediment, Lake Lemon, USA
pGrf
freshwater lake sediment, Lake Gri¡y, USA
Winarc
freshwater lake sediment, Lake Windermere, UK
Rot
freshwater lake sediment, Lake Rotsee, Switzerland
VAL
freshwater lake water, Valkea Kotinen Lake, Finland
Soils
P17, M17
forest soil, Brazil
FFSB
forest soil, Finland
FFSA, FFSC
forest soil, Finland
KBS
agricultural soil, Michigan, USA
SCA
agricultural soil, Wisconsin, USA
ABS, A5.1
anoxic rice paddy soil
Arc.
deep subsurface paleosol, Washington State, USA
Other environments
R
Peat bogs
ARR
rice roots
Vadin
bio¢lm of a £uidized-bed anaerobic digestor
ARC
bovine rumen
Cd30
intestinal micro£ora of the termite gut
water sediment [16] and peat bogs [36] within the order
Methanomicrobiales. Another four clones formed a group
among sequences inhabiting marine sediments [45,46], rice
roots and soil [42] and the deep-sea [47] (see Group III on
Fig. 2). Clone VAL147 clustered together with clone EelTA1c9 found by Hinrichs et al. [48] in marine sediments
related to a decomposing methane hydrate, which is very
convincingly, albeit based on circumstantial evidence, proposed to represent methanotrophic anaerobes.
The remaining seven clones formed two distinct lines of
Cyaan Magenta Geel Zwart
G. Jurgens et al. / FEMS Microbiology Ecology 34 (2000) 45^56
53
Fig. 3. Identi¢cation of Archaea in a water sample taken from lake Valkea Kotinen by FISH. Combination of hybridization with CY3-labeled, rRNAtargeted oligonucleotide probes EURY496 (A) and EURY499(B) and DAPI staining. Identical microscopic ¢elds have been visualized with an epi£uorescence microscope using ¢lter set speci¢c for CY3 (right, up and down) and DAPI (left, up and down). Bar, 10 Wm.
descent which all three methods (maximum likelihood,
maximum parsimony and neighbor-joining) of phylogenetic analysis placed in the kingdom Euryarchaeota,
although the exact placement within this kingdom could
vary because of the large phylogenetic distances between
members of these clades and the rest of the analyzed sequences. Clade VALII, consisting of four clones, was only
distantly related to the Methanosaeta group. Sequence
VAL33-1 allowed placement of this clade on the same
branch with the rice root and soil cluster I [42] using all
methods on the basis of 85.9% similarity between sequence
and cluster. Levels of rDNA similarity between the other
three VAL II clade sequences and sequences from the
closest rice cluster I were extremely low (65.8 to 69.2%).
A similar situation was observed for three clones from
cluster VAL III, which were related to the order Thermoplasmales, including two marine environmental Groups II
and III [13] and rice cluster V. We found that the level of
similarity between members of this clade, compared to
members of Thermoplasmales and Methanobacteriales,
FEMSEC 1169 18-10-00
ranged from 71.3% (Thermoplasma acidophilum, Thermoplasmales) to 76.3% (Methanothermus fervidus, Methanobacteriales). The rice cluster V, which was most close to
the three VAL III clade sequences, is believed to comprise
non-methanogenic anaerobic Archaea [42].
We found that the new VAL sequences shared major
parts of the archaeal domain signatures at homologous
and nonhomologous positions [49,50]. However, clones
with unstable placement (VAL90, VAL35-1, VAL31-1,
VAL84, VAL125, VAL112) showed non-archaeal signatures for the several strong signature nucleotides at the
positions 338, 367, 393 and 867 (E. coli numbering). Intradomain signature analysis con¢rmed a¤liation of 12 VAL
sequences with the Crenarchaeota and 16 VAL sequences
with the Euryarchaeota kingdom of Archaea.
3.3. FISH in Valkea Kotinen
In this study, we developed new archaeal probes to be
used with the FISH technique. This enabled us to quanti-
Cyaan Magenta Geel Zwart
54
G. Jurgens et al. / FEMS Microbiology Ecology 34 (2000) 45^56
tatively study the indigenous Archaea distribution in the
water column and its potential contribution to the microbial food web. Initially, we used the probes ARCH915 [20],
CREN499 and EURY498 [33] in the FISH experiments.
With the general Archaea-speci¢c probe ARCH915 we
were able to detect 7 þ 2% of the DAPI-stained cells (data
for hypolimnium). Interestingly, no signi¢cant di¡erences
were detected between the samples taken at di¡erent times
during the sampling period from spring to autumn.
Bright positive hybridization signals and cell morphologies (Fig. 3) received with four Euryarchaeotal probes
(EURY498, EURY514, EURY496 and EURY499) were
identical to the signals obtained with the general Archaea
probe ARCH915. The most speci¢c of all tested Euryarchaeota probes was EURY496, which covers the order
Methanomicrobiales and related environmental sequences.
Therefore, we suspect that the Euryarchaeota clones detected in the FISH experiments belong to the order Methanomicrobiales. The size and the morphotypes of detected
cells (Fig. 3) show similarities to some methanogens (for
example Methanospirillum hungatei). Such morphotypes
could have ecological aspects in the microbial food web
as was discussed by Pernthaler et al. [18] who showed that
Archaea represented 1^5% of all DAPI-stained cells in
lake Gossenko«llesee, Austria. Despite a high background
£uorescence, maximum intensity of the signals and maximum cell counts for the EURY496 probe (7 þ 2% of the
DAPI-stained cells) were observed in samples taken from
the hypolimnion, which is in agreement with our methane
measurement data and the results for the mcr-gene. Cell
counts for epilimnion were below the detection limit (1%).
In comparison to the success with Euryarchaeota
probes, none of the Crenarchaeota probes including
CREN499, as well as newly designed CREN512 and
CREN569 probes, gave positive hybridization signals.
The negative FISH result is in contrast to our obtained
PCR products but could be due to low numbers of Crenarchaeota cells in the samples and/or low ribosome contents in the bacterial cells [51,52]. Other reasons could
arise from physical^chemical aspects, i.e. low penetration
of probe material through the cell wall and the secondary
structure of the rRNA molecule.
In summary, the results of the phylogenetic analyses
and FISH suggest that a large number of previously `unknown' Archaea were found in non-extreme habitats, i.e.
in the pelagic water of a boreal forest lake, which has
temperature amplitudes between 4 and 20³C and partial
oxic/anoxic conditions over the water column. Sequences
detected in lake Valkea Kotinen by the rRNA approach
belong to members of the Crenarchaeota and Euryarchaeota branch of the domain Archaea. The existence of
Archaea in the freshwater environment was expected, if
we take into consideration the proven presence of methanogens in freshwater ecosystems. However, such large
diversity and low 16S rRNA sequence similarity of the
obtained new freshwater Archaea to the existing environ-
FEMSEC 1169 18-10-00
mental sequences in pelagic interfaces as shown in this
study was surprising.
It has been shown earlier that representatives of Crenarchaeota inhabit boreal forest soils [10,11]. In this paper,
we have shown that they also occupy pelagic habitats in a
boreal forest lake. We found that in both biotopes, Crenarchaeota were diverse, forming clusters that do not fall
into those known from other environments. Crenarchaeota found in the boreal forest lake water di¡ered signi¢cantly from Crenarchaeota found in the boreal forest
soil samples. This may be due to the di¡erent geographical
location of the two sites in Finland or due to speci¢c
habitat properties such as the di¡erent nutrient availability
and/or selection- and grazing pressure in aquatic environments compared to forest soil. However, a possible phylogenetical^ecological link between Crenarchaeota in boreal forest soil and boreal forest lake water remains to be
studied through sampling at various sites from lake Valkea Kotinen and from soils within its water catchment
area. Euryarchaeota detected in our experiments occupy
a very speci¢c place in the environment. As many of them
seem to be very distantly related to any of the main metabolic subgroups of Archaea, namely, the methanogens or
the halophiles, their way of living and metabolic properties
remain unclear.
The outcome of this study, together with other recent
work on Archaea in marine and terrestrial environments,
supports our idea and working hypothesis that these organisms inhabit a wide variety of niches and are ecologically much more successful and important then previously
thought. We suggest that they play a speci¢c role in boreal
aquatic food webs and in the biochemical/geochemical cycling of carbon in their habitats. We expect that a variety
of new physiological phenotypes will be discovered, as
soon as some of these organisms can be successfully cultivated and their metabolic and genetic potentials will be
available for further characterization.
Acknowledgements
We thank Bernhard Fuchs, Wilhelm Scho«nhuber, Katrin Ravenschlag and Jakob Pernthaler (Max Plank Institute, Bremen, Germany) for their great support and helpful advice, and Robin Sen (Biocenter, University of
Helsinki, Finland) for revising the English. This work
was supported by a three-month grant from Max Planck
Institute, Bremen, Germany, a grant from the Mai and
Tor Nessling Foundation, Finland, a grant from the University of Helsinki, Finland and by the Finnish Academy
within the Finnish Biodiversity Programme (FIBRE).
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