Composition and temporal dynamics of planktonic archaeal assemblages from anaerobic sulfurous environments studied by 16S rDNA denaturing gradient gel electrophoresis and sequencing

Abstract

The planktonic archaeal assemblages of several anaerobic, sulfide-rich, aquatic environments were analyzed in space and time by PCR-denaturing gradient gel electrophoresis and sequencing of 16S rRNA gene fragments. The systems were sampled in different years between 1992 and 1998. PCR products were obtained directly from the original DNA without previous nested amplification and yielded successful fingerprints with mostly sharp bands in the gel. Nineteen samples from the anaerobic hypolimnia of 8 lakes and 1 coastal lagoon in NE Spain, Mallorca and Switzerland were compared and a temporal survey was carried out in one of the lakes (Lake Vilar). Between 4 and 14 well-defined bands appeared. All the sequenced bands belonged to Archaea. Although most of the water bodies shared the same climatic conditions and presence of sulfide, the limnological parameters were different among them and different finger-prints were observed in different lakes. Euryarchaeota, i.e., methanogen- and thermoplasma-related sequences, appeared in all the samples but crenarchaeota were recovered only from Lake Vilar. A temporal shift in the predominant members of the archaeal assemblage from crenarchaeota to members of the cluster of thermoplasmales and relatives took place in Lake Vilar between February and June. Sequences related to thermoplasmales and crenarchaeota were distantly related to cultured strains (81 % similarity in 16S rDNA) and clustered with branches represented only by environmental clones, whereas sequences related to methanogens grouped with a sequence from an endosymbiont of 1 anaerobic ciliate. A new branch of freshwater euryarchaeota appeared within the cluster of thermoplasmales and relatives. Our study indicates the presence of dynamic archaeal populations in the water column of nonthermophilic, sulfide-rich environments, further extending the diversity and distribution of Archaea in nature. The temporal shift in community composition in Lake Vilar suggests that Archaea grow under in situ conditions. If this is the case, Archaea would be active players in the anaerobic biogeochemical cycles of these environments.

Full text

AQUATIC MICROBIAL ECOLOGY
Aquat Microb Ecol
Vol. 25: 237–246, 2001 Published September 28
INTRODUCTION
Archaea illustrate better than any other group of
organisms how dramatically our perception of the liv-
ing world has changed since the biosphere has been
examined from a molecular point of view (Pace 1997).
First, analysis of ribosomal RNA sequences surpris-
ingly revealed that Archaea constitute a third domain
of life (Woese et al. 1990, Woese 2000). Second, when
molecular studies were carried out with natural sam-
ples, Archaea were shown to be ubiquitous (DeLong
1992, Fuhrman et al. 1992, Hershberger et al. 1996,
Stein & Simon 1996). The phenotypic range of culti-
vated Archaea had restricted these organisms to habi-
© Inter-Research 2001
**Present addresses:
**Observatoire Ocèanologique de Banyuls-CNRS, BP 44, 66651
Banyuls-sur-Mer, France. E-mail: emilio@obs-banyuls.fr
**Kluyver Institute of Biotechnology, Delft University of Tech-
nology, 2628 BC Delft, The Netherlands
Composition and temporal dynamics of planktonic
archaeal assemblages from anaerobic sulfurous
environments studied by 16S rDNA denaturing
gradient gel electrophoresis and sequencing
Emilio O. Casamayor1,*, Gerard Muyzer2,** , Carlos Pedrós-Alió1
1Departament de Biologia Marina i Oceanografia, Institut de Ciències del Mar, CMIMA-CSIC,
Pg. Maritim de la Barceloneta, 08003 Barcelona, Spain
2Max Planck Institute for Marine Microbiology, Celsiusstr. 1, 28359 Bremen, Germany
ABSTRACT: The planktonic archaeal assemblages of several anaerobic, sulfide-rich, aquatic envi-
ronments were analyzed in space and time by PCR-denaturing gradient gel electrophoresis and
sequencing of 16S rRNA gene fragments. The systems were sampled in different years between 1992
and 1998. PCR products were obtained directly from the original DNA without previous nested
amplification and yielded successful fingerprints with mostly sharp bands in the gel. Nineteen sam-
ples from the anaerobic hypolimnia of 8 lakes and 1 coastal lagoon in NE Spain, Mallorca and
Switzerland were compared and a temporal survey was carried out in one of the lakes (Lake Vilar).
Between 4 and 14 well-defined bands appeared. All the sequenced bands belonged to Archaea.
Although most of the water bodies shared the same climatic conditions and presence of sulfide, the
limnological parameters were different among them and different fingerprints were observed in dif-
ferent lakes. Euryarchaeota, i.e., methanogen- and thermoplasma-related sequences, appeared in all
the samples but crenarchaeota were recovered only from Lake Vilar. A temporal shift in the predom-
inant members of the archaeal assemblage from crenarchaeota to members of the cluster of thermo-
plasmales and relatives took place in Lake Vilar between February and June. Sequences related to
thermoplasmales and crenarchaeota were distantly related to cultured strains (81% similarity in 16S
rDNA) and clustered with branches represented only by environmental clones, whereas sequences
related to methanogens grouped with a sequence from an endosymbiont of 1 anaerobic ciliate. A new
branch of freshwater euryarchaeota appeared within the cluster of thermoplasmales and relatives.
Our study indicates the presence of dynamic archaeal populations in the water column of nonther-
mophilic, sulfide-rich environments, further extending the diversity and distribution of Archaea in
nature. The temporal shift in community composition in Lake Vilar suggests that Archaea grow under
in situ conditions. If this is the case, Archaea would be active players in the anaerobic biogeochemi-
cal cycles of these environments.
KEY WORDS: Archaea · DGGE · Fingerprinting · 16S rDNA · Sulfurous lakes · Diversity
Resale or republication not permitted without written consent of the publisher

Aquat Microb Ecol 25: 237–246, 2001
tats with high temperature, extreme values of pH, high
salinity or anaerobic environments allowing methano-
genesis. Thus, the genetic and metabolic diversity of
Archaea and their ecological distribution seemed more
limited than those of Bacteria. Uncultured Archaea,
however, extend from marine planktonic environ-
ments to soils and sediments (see a review in DeLong
1998), showing up also in close association with ani-
mals (Preston et al. 1996, Van der Maarel et al. 1998)
and bacteria (Boetius et al. 2000). Therefore, the
expected metabolic capabilities of Archaea have in-
creased greatly. Recently, a combination of in situ
hybridization and microautoradiography has shown
that marine Archaea are active and able to take up
amino acids (Ouverney & Fuhrman 2000).
During the last few years, more and more sequences
have been deposited in databases and new groups of
uncultured Archaea have been described (DeLong
1992, Fuhrman et al. 1992, Barns et al. 1996, Jurgens et
al. 1997, 2000, Buckley et al. 1998). These groups are
the marine and freshwater sediments cluster (Crump &
Baross 2000), the terrestrial cluster (Buckley et al.
1998) and some cosmopolitan marine clusters (Mas-
sana et al. 2000). However, ecological studies covering
temporal and spatial variability in the archaeal assem-
blages are still scarce (DeLong et al. 1994, Massana
et al. 1998, 2000, Murray et al. 1998, 1999, Pernthaler
et al. 1998, Crump & Baross 2000, Cytryn et al. 2000,
Karner et al. 2001). Such studies provide insight into
changes in community structure and population
dynamics and yield information useful to investigate
their functional roles. In Archaea most of these studies
have concentrated on marine and terrestrial samples,
in a hypersaline lake or in sediments. Only a handful of
studies have been carried out in the water column of
freshwater habitats: 2 meromictic lakes (Øvreås et al.
1997, Casamayor et al. 2000b), a high mountain lake
(Pernthaler et al. 1998) and a boreal forest lake (Jur-
gens et al. 2000). Whether Archaea are a common com-
ponent of freshwater plankton is still uncertain.
In the present work, we have carried out a compara-
tive survey on the composition of the archaeoplankton
from several anaerobic sulfurous water bodies and a
temporal study centered on one of them. We used
denaturing gradient gel electrophoresis (DGGE) fin-
gerprinting of 16S rRNA genes, PCR-amplified directly
from the original DNA (Muyzer et al. 1998). This tech-
nique allows extensive comparison on a spatio-tempo-
ral scale. In addition, bands were excised from the gel
and sequenced, and the identity of the populations was
investigated. DGGE has been successfully applied to
Bacteria but, to our knowledge, direct DGGE analysis
of Archaea has been shown in only 1 recent study
(Cytryn et al. 2000) besides our own work (Casamayor
et al. 2000b). Sequence analysis of the 600 bp 16S
rDNA fragments indicated the presence of a new lin-
eage within euryarchaeota. For the first time, we
describe temporal changes in the composition of fresh-
water archaeal assemblages involving a shift between
distantly related phylogenetic groups.
MATERIALS AND METHODS
Environments, sampling and analysis. The systems
sampled are located in karstic regions of northeastern
Spain (Banyoles and Cuenca), in the coastal area of
Mallorca (El Cibollar, Balearic Islands), and in the
Swiss Alps (Lake Cadagno) and were sampled in dif-
ferent years between 1992 and 1998. Basins III, IV and
VI from Lake Banyoles, Lake Vilar and Lake Cisó are
in the Banyoles karstic area (42° 8’ N, 2° 45’ E) and the
microbial communities inhabiting these systems have
been extensively studied by both conventional (for
reviews see Guerrero et al. 1987, Pedrós-Alió & Guer-
rero 1993) and molecular methods (Casamayor et al.
2000a,b). Lake Banyoles, covering a surface area of
1.1 km2, is a polje comprising 6 main basins. The 3
basins studied are located in the northern area of the
lake. Basins III and IV are meromictic with 25 and 18 m
maximal depth, respectively, and incoming water
seeps through bottom springs (Casamitjana & Roget
1993). The chemocline oscillates between 16 and 21 m
in Basin III and between 12 and 17 m in Basin IV
depending on the season. Basin VI is 17 m deep and
holomictic, with a thermocline situated around 12 to
13 m, and does not have bottom seepage. Blooms of the
photosynthetic bacteria Chorobium phaeobacteroides
and Thiocystis minor (formerly Chromatium minus)
have been described in the 3 basins (Garcia-Gil et al.
1996). Lake Vilar is a meromictic lake formed by 2
basins with a maximum depth of 9 m and a surface
area of 11 000 m2, and is connected to Lake Banyoles
through a small and shallow channel. High sulfide con-
centrations are found throughout the year, although
sulfide is restricted to the deeper, high-conductivity
waters. A stable chemocline exists at 4.5 m, where
dense populations of photosynthetic sulfur bacteria
develop. Lake Cisó is a small holomictic lake (650 m2),
1 km away from the former, with a maximum depth of
6.5 m. The thermocline is at 1.5 m, where dense popu-
lations of photosynthetic sulfur bacteria develop. The
lake becomes anoxic during winter holomixis (com-
plete mixing) and high sulfide concentrations (up to
500 µM) are present in the entire water column
(Pedrós-Alió & Guerrero 1993). Lake El Tobar and
Lake La Cruz belong to the Cuenca karstic system
(39°59’ N, 1° 52’ W). Biological and physico-chemical
properties of these lakes have been well described
(Miracle et al. 1992 and references therein, Casamayor
238

Casamayor et al.: Archaea in sulfurous lakes
et al. 2000a). Lake El Tobar is meromictic, 19.5 m in
depth, and contains permanently anoxic saline waters
(consisting mostly of a sodium chloride brine) starting
at about 12 m depth, where blooms of photosynthetic
bacteria develop (Garcia-Gil et al. 1999). Lake La Cruz
is a meromictic freshwater lake, 24 m deep, with bot-
tom waters enriched in calcium, magnesium and iron
bicarbonates, and low amounts of sulfide; a thermo-
cline exists at 8 to 12 m and gas-vacuolate species of
photosynthetic bacteria Pelodictyon and Amoebobac-
ter develop there.
El Cibollar is a small coastal lagoon on the northeast
of the S’Albufera of Mallorca, 750 m from Alcudia Bay
(39° 50’N, 3° 10’E), for which some limnological studies
have been carried out (Moyà et al. 1987, Borrego et al.
1997). The lagoon has a surface area of almost 4 ha, and
average depth is 3.3 m and maximal depth 8.7 m. The
oxygen-sulfide interface is located at 3 to 4 m with max-
imum H2S concentrations up to 2000 µM. Finally, Lake
Cadagno (46° 33’N, 8° 43’ E) is a permanently stratified,
sulfide-rich meromictic lake (H2S concentrations
around 1000 µM), located at 1923 m above sea level in
the Piora valley (Southern Switzerland). It is about
850 m long and 420 m wide, with a maximum depth of
21 m and a permanent chemocline found between 9
and 14 m. The lower layer is rich in dissolved salts
brought by sulfurous sublacustrine springs. Several bio-
geochemical and molecular studies have been carried
out in this lake (Wagener et al. 1990, Putschew et al.
1996, Schanz et al. 1998, Bosshard et al. 2000, Fritz &
Bachofen 2000, Tonolla et al. 2000), reporting blooms of
the purple sulfur bacteria Chromatium okenii and
Amoebobacter purpureus at the chemocline.
For most of the samples, depth profiles of water tem-
perature, conductivity and oxygen concentration were
measured in situ using a portable multisensor probe
(Hydrolab DS-3; Hydrolab Instruments, TX, USA).
Sampling depths were selected according to physico-
chemical parameters. Water samples were taken using
a PVC cone connected through tubing to a battery-dri-
ven pump as described elsewhere (Miracle et al. 1992).
Sulfide was measured with the methylene blue colori-
metric method in 10 ml subsamples fixed quickly by
addition of 100 µl of 10 N NaOH and Zn-acetate to a
final concentration of 0.1 M (Golterman et al. 1978).
Cells were counted in samples fixed with 4%
formaldehyde (v/v) and stained with DAPI (Porter &
Feig 1980) using epifluorescence microscopy. For DNA
analysis, plastic containers were filled with 1 to 5 l of
water and were kept in the dark on ice until processed
in the laboratory a few hours later. Sampling depths for
molecular studies are indicated in Table 1. Samples
were concentrated using a refrigerated centrifuge at
8000 ×g(Sorvall Instruments, DuPont, DE, USA) and
cell pellets were kept frozen at –70°C until further use.
Microscopic observation of the supernatants showed
that only between 1 and 3% of the cells were not
recovered by this method.
239
Table 1. Temperature, conductivity, sulfide concentration and DAPI counts of prokaryotes (values ×106cells ml–1) for the depths
sampled for molecular analysis. The depth of the oxic-anoxic interface in each lake is also indicated. ND: not determined.
*Sample from the oxic-anoxic interface (presence of 1.4 mg l–1 oxygen)
Sample Date Interface Depth Temperature Conductivity pH H2S Total
(d.mo.yr) (m) (m) (°C) (µS cm–2) (µM) cells
El Cibollar lagoon 26.05.98 4.0 6.00 25 48000 7–8 present ND
Lake Cadagno 02.07.98 11.5 13.00 8 320 7–8 1050 ND
Lake La Cruz 29.09.94 11.8 12.25 7 470 7–8 20 18.2
Lake El Tobar 30.09.94 11.5 14.00 13 16120 7–8 15 ND
Banyoles B-III 14.09.95 16.5 17.00 13 1366 7–8 6 ND
Banyoles B-IV 29.09.95 11.5 13.25 18 1870 7–8 30 21.3
Banyoles B-VI 29.09.95 12.5 14.00 16 1400 7–8 10 4.3
Lake Cisó 25.09.92 1.5 1.75 16 2200 7.1 400 17.3
Lake Cisó 19.02.96 0.0 0.10 9 2000 7.6 150 19.1
Lake Cisó 09.04.96 1.5 1.75 11 2280 7.1 230 41.2
Lake Vilar 30.09.93 4.0 4.40 16 2000 7.6 819 22.6
Lake Vilar 19.02.96 4.25 4.75 13 2380 7.5 190 8.0
Lake Vilar 19.02.96 4.25 7.00 13 2370 7.5 986 10.5
Lake Vilar 09.04.96 3.75 4.25 13 2430 7.5 720 17.5
Lake Vilar 09.04.96 3.75 7.00 13 2770 7.5 707 15.5
Lake Vilar* 23.04.96 3.75 3.75 15 1180 7.3 4 8.3
Lake Vilar 23.04.96 3.75 4.25 15 1660 7.6 480 42.1
Lake Vilar 23.04.96 3.75 7.00 14 2040 7.5 837 16.3
Lake Vilar 08.05.96 3.8 4.40 16 1600 7.5 550 28.0
Lake Vilar 21.05.96 4.0 4.40 17 1910 7.5 844 31.0
Lake Vilar 20.06.96 4.0 6.00 16 2420 7.6 800 17.2

Aquat Microb Ecol 25: 237–246, 2001
DNA extraction and analysis. DNA was extracted
with hot SDS-phenol and purified with phenol-chloro-
form-isoamylalcohol (25:24:1 v/v) followed by ethanol
precipitation as reported elsewhere (Casamayor et al.
2000b). Fragments of the 16S rDNA suitable for subse-
quent DGGE analysis were obtained with the primer
combination ARC344F (ACG GGG YGC AGC AGG
CGC GA) (Stahl & Amann 1991) and ARC915R (GTG
CTC CCC CGC CAA TTC CT) (Raskin et al. 1994). A
40 nucleotide GC-rich sequence was attached to the
5’ end of the forward primer according to Muyzer et al.
(1998). Annealing temperature was 61°C. PCR condi-
tions and DGGE were as described before (Casamayor
et al. 2000b), running for 3.5 h at a constant voltage of
200 V and at 60°C in a 20 to 80% vertical denaturant
gradient (100% denaturant agent is 7 M urea and 40%
deionized formamide). After electrophoresis, the gels
were stained with ethidium bromide and photo-
graphed with UV transillumination (314 nm) with a Po-
laroid camera. The photographs were scanned, and
the digitized images were processed with the NIH Im-
age software (National Institutes of Health, Bethesda,
MD, USA) to measure relative band intensities. A
DGGE band was defined as an ethidium bromide sig-
nal higher than 0.2% of the total intensity of all bands
in each lane. The variability in intensities measured
among replicate analyses was less than 4%. Prominent
bands were excised from the gels and sequenced as re-
ported elsewhere (Casamayor et al. 2000b).
Sequences were submitted to basic local alignment
search (Altschul et al. 1990) to get a first indication of
what sequences were retrieved. Then, sequences were
aligned by using the ARB program package (Technical
University of Munich, Munich, Germany; http://www.
arb-home.de). Partial sequences were inserted into the
optimized and validated tree available in ARB (derived
from complete sequence data), by using the maximum-
parsimony criterion and a special ARB parsimony tool
that did not affect the initial tree topology. Nucleotide
sequence accession numbers at EMBL are as follows:
AJ239988 to AJ239990 (CIARC-1 to CIARC-3),
AJ240004 to AJ240006 (VIARC-1 to VIARC-3),
AJ306414 (VIARC-0), AJ306415 (VIARC-4), AJ306416
(VIARC-5), AJ306409 (CIBARC-1), AJ306410 (TOARC-
2), AJ306411 (CRARC-3), AJ306412 (CRARC-4) and
AJ306413 (BANARC-5 to BANARC-7).
RESULTS
All systems studied had 2 water layers separated
according to gradients of temperature or conductivity.
The top water layer (epilimnion) was oxygenated and
with light. The bottom water layer (hypolimnion) was
anoxic, sulfide was present, and it was in the dark.
Very little archaeal PCR product (or no product at all)
was obtained from the upper aerobic layers. Even at
the oxic-anoxic interface most of the DGGE bands
found in the hypolimnia were absent. Thus, we
focused all the analyses in the anoxic hypolimnia. Dif-
ferent sulfide concentrations were measured for the
hypolimnion of each system, ranging from 6 µM in
Basin III of Lake Banyoles to 1050 µM in Lake Cadagno
(Table 1). Temperature in the study sites ranged
between 7 and 25°C. Conductivity ranged between
1600 and 48 000 µS cm–2 and pH was between 7 and 8.
Most systems were sampled in summer (July to Sep-
tember), whereas Lake Cisó and Lake Vilar were sam-
pled several times and in different seasons. The con-
centration of prokaryotes in the bottom waters was
around 107cells ml–1 (Table 1). This count includes all
DAPI-stained particles with a prokaryote-like mor-
phology. Morphologically conspicuous cells such as
green and purple photosynthetic sulfur bacteria
(Chlorobium sp. and Thiocystis minor, respectively)
were counted separately (see results in Casamayor et
al. 2000a).
Lake Vilar was sampled twice each month, from
February to June 1996. No marked differences in con-
ductivity, temperature or pH were found during the
survey in the hypolimnion of the lake (Table 1). Sulfide
concentrations, however, increased from winter to
summer in the hypolimnion (from 800 to 1200 µM at
around 4.5 m). Lake Cisó was sampled at the end of
summer 1992 and in winter and early spring 1996.
Small differences were found in the physicochemical
parameters measured in this lake, and sulfide ranged
between 150 and 400 µM (Table 1). Lake Cisó becomes
240
Fig. 1. Negative image of the ethidium bromide-stained dena-
turing gradient gel electrophoresis (DGGE) gel containing the
archaeal 16S rDNA fragments from the different environ-
ments. Bands that were cut off from the gel are labeled but
only those that yielded a clean sequence have a number.
92 and 93 samples were taken in 1992 and 1993, respectively
B-IV
B-VI
B-III
La Cruz
Vilar93
El Tobar
Cadagno
El Cibollar
Cisó92
DGGE run
1
23
4
0567

Casamayor et al.: Archaea in sulfurous lakes
anoxic during winter holomixis because of its
special limnological conditions, and sulfide
concentrations up to 400 µM were measured
from the bottom of the lake to the surface.
Anaerobic archaeal assemblages inhabiting
these systems were compared by DGGE fin-
gerprinting. The number of well-defined sharp
bands ranged between 4 and 14 among lakes
(Fig. 1), and in most cases just a few bands
(1 to 3) appeared as the most intense. The fin-
gerprints showed differences in the mobility
of the DGGE bands among hypolimnia of
lakes (Fig. 1). Very similar fingerprints were
observed in winter and in spring for Lake
Vilar (Fig. 2) and Lake Cisó (see Fig. 3 in
Casamayor et al. 2000b). In Lake Vilar, how-
ever, a shift was apparent in summer from the
rest of the year (lane for 20 June in Fig. 2).
Very little archaeal PCR product was obtained
from the upper aerobic layers of Lake Vilar.
As was the case with other lakes, most of the
archaeal DGGE bands appearing in the
hypolimnetic samples were absent from the
oxic-anoxic interface. As an example of the
DGGE band pattern from such an interface,
the 3.75 m sample is presented in Table 1 and
in the last lane in Fig. 2.
The most prominent bands in the profiles
(in terms of intensity and frequency of ap-
pearance) were excised, re-amplified, puri-
fied and sequenced (27 bands). From these, only 17
bands yielded good-quality sequences. Some of them
were obtained from the same position in different
lanes and gave the same sequence (e.g., VIARC-2 in
Fig. 2). Thirteen sequences were included in a phylo-
genetic tree (Fig. 3) together with bands previously
obtained from Lake Cisó in 1996 (CIARC-1, -2 and -3;
Casamayor et al. 2000b). The sequences were named,
in addition to the number shown in Figs 1 & 2, with a
code for the lake and a code for the primer used
(ARC). The components of the archaeal assemblages
were distributed among 3 groups: methanogens, ther-
moplasmales and crenarchaeota (Fig. 3). Sequences
from thermoplasmales and relatives cluster and cre-
narchaeota were very distantly related to cultured
organisms (e.g., 81% to Desulfurococcus mobilis or
Thermoplasma acidophilum) whereas methanogens
where more similar to cultured strains (e.g., 90 % to
Methanospirillum hungatei). The sequences belong-
241
Fig. 3. Phylogenetic affiliation within the domain Archaea of excised
bands obtained from the gels in Figs 1 & 2. The tree was constructed by
adding the partial sequence data to a previously validated and opti-
mized tree by parsimony analysis. Scale bar = 0.10 mutations per
nucleotide position
23.04-3.75m
20.06-6.00m
21.05-4.40m
08.05-4.40m
23.04-7.00m
09.04-7.00m
23.04-4.25m
09.04-4.25m
19.02-7.00m
19.02-4.75m
DGGE run
VIARC-2VIARC-2
VIARC-1
VIARC-3
VIARC-3
VIARC-4
VIARC-5
VIARC-5
VIARC-1
Winter----------->summer
Fig. 2. Negative image of the ethidium bromide-stained
DGGE gel containing the archaeal 16S rDNA fragments from
the temporal survey in Lake Vilar. The same sequence was
recovered when bands with identical position in the gel and
from different dates were analyzed

Aquat Microb Ecol 25: 237–246, 2001
ing to methanogens formed a closely related group
and were all related to endosymbionts of anaerobic
ciliates (97% similarity). Thermoplasmales-related se-
quences obtained from different lakes grouped in a
single branch that was not related to previously
described sequences. Lake Vilar was the only water
mass where a planktonic crenarchaeota-like popula-
tion was detected.
The relative band intensities were used as an esti-
mate of abundance to compare the composition of the
archaeal assemblages among samples that were am-
plified in the same PCR run. For this comparison, the
sequences were pooled into 3 phylogenetic groups:
methanogens, thermoplasmales-related and crenar-
chaeota. An average of 22% of the total band intensity
in each lane could not be sequenced and assigned to
known organisms and, therefore, these bands were
assigned to an ‘unidentified’ class (black bars in Figs 4
& 5). In general, this fraction consisted of minor com-
ponents of the community (i.e., weak bands). It must
be kept in mind that these bands may correspond to
any of the 3 identified groups or to other groups of
Archaea. On the basis of these results, we found envi-
ronments where thermoplasmales-related organisms
reached >50% of the total intensity (Lake Vilar in sum-
mer and the 3 basins of Lake Banyoles), environments
where methanogens reached > 50% of the total inten-
sity (Lake Cisó) and environments dominated by 1 cre-
narchaeota (Lake Vilar in winter). In Lake La Cruz
40% was assigned to thermoplasmales-related organ-
isms and 35% to methanogens. In El Cibollar coastal
lagoon, methanogens were 40% but the composition of
the remaining 60% was not possible to allocate. Lake
El Tobar had a very high unidentified fraction too and
thermoplasmales-related organisms were found at
20% of the total intensity.
When a temporal survey was carried out in Lake
Vilar a shift from crenarchaeota to thermoplasmales-
related sequences was observed from winter to sum-
mer (Fig. 5). Crenarchaeota accounted for up to 80% of
the total band intensity in February and progressively
decreased to 10% in June. In parallel, thermoplas-
males-related sequences increased from <10% in Feb-
ruary up to 60% in June. The methanogen contribu-
tion was largest in spring (around 45% of the total
band intensity) but similar percentages were mea-
sured in winter and summer (between 5 and 10%).
Only 1 sequence of crenarchaeota was recovered from
Lake Vilar (VIARC-2). On the other hand, 2 closely
related sequences within the thermoplasmales cluster
(VIARC-1 and VIARC-5, 99% similarity in 16S rDNA
between them) and 2 of methanogens (VIARC-3 and
VIARC-4, 98% similarity) were recovered from Lake
Vilar. These sequences showed temporal changes in
their respective band intensities (Fig. 6).
242
B-IV B-VI
% recovered from DGGE
100
80
60
40
20
0Tb
Ci
96
wi
Vi
96
wi
Vi
96
su
B-III Ci
96
sp
Cr Cb Vi
93
not determined
methanogens
thermoplasmales
crenarchaeota
Fig. 4. Relative abundance of archaeal groups in the different
lakes. Quantitative data were obtained from band intensities
in the gels in Figs 1 & 2, and from a previous study in Lake
Cisó (Casamayor et al. 2000b). Samples: basins from Lake
Banyoles (B-III, B-IV and B-VI); Lake Vilar in summer (Vi96su)
and in winter (Vi96wi) 1996 and September 1993 (Vi93); Lake
Cisó in spring (Ci96sp) and winter (Ci96wi); Lake La Cruz
(Cr); Lake El Tobar (Tb); and El Cibollar coastal lagoon (Cb)
not determined
methanogens
thermoplasmales
crenarchaeota
JunMayAprMarFeb
100
80
60
40
20
0
100
80
60
40
20
0
month
% recovered from DGGE
WINTER SUMMER
Fig. 5. Survey of the relative abundance of different archaeal
groups along the temporal study in Lake Vilar. Quantitative
data were obtained from the gel in Fig. 2

Casamayor et al.: Archaea in sulfurous lakes
DISCUSSION
Up to now, freshwater planktonic culturable Ar-
chaea were restricted to methanogens and sulfur-
metabolizing thermophiles. Archaea inhabiting sul-
furous environments had been restricted mostly to
crenarchaeotes with thermophilic and acidophilic
metabolisms (Brock 1997, Huber et al. 1998, Vásquez
et al. 1999). Methanogens have also been detected
coexisting with sulfate reducers in sulfurous environ-
ments although at very low rates of activity (Senior et
al. 1982) and were expected not to be very abundant in
such systems because sulfate-reducing bacteria are
better competitors for available acetate and H2(Abram
& Nedwell 1978). Recently, a planktonic crenar-
chaeota-related 16S rDNA sequence was obtained
from a Norwegian meromictic lake (Lake Sælenvan-
net; 150 µM sulfide, salinity 2%) and methanogens
were also present (Øvreås et al. 1997). Methanogens
and thermoplasmales-related sequences have also
been detected in the sulfide-rich water layers of the
saline Solar Lake (1 mM sulfide; salinity 19%) (Cytryn
et al. 2000). Obviously, members of these groups are
able to thrive in a wide range of environments and are
not limited to the extremophiles known from cultures.
In the present work we have compared archaeal as-
semblages from several anaerobic, sulfide-rich hypo-
limnia. In situ conditions in these habitats match nei-
ther with the physiological demands of cultured ar-
chaeal strains nor with the environments from where
clones deposited in databases were obtained. There-
fore, these systems are potential habitats for unknown
groups of Archaea. For this comparison we have used a
fingerprinting technique (DGGE) and sequence analy-
sis of the resulting bands. The small number of pub-
lished studies using DGGE separation of 16S rDNA
PCR products of Archaea reflects that this separation
has been difficult to perform. Most of these publications
show DGGE profiles characterized by highly complex
and mostly diffuse bands. In several cases, a previous
nested PCR step was introduced (e.g., Øvreås et al.
1997) that might increase the potential biases intro-
duced by PCR. We have described a primer set and
PCR conditions that yielded successful fingerprints with
mostly sharp bands in the gel (Casamayor et al. 2000b).
The PCR products were obtained directly from the orig-
inal DNA and allowed us to carry out a semi-quantita-
tive approximation. In addition, the length of the PCR
product (600 bp) allowed more reliable identification of
the sequences than using shorter 16S rDNA fragments
for comparison in databases. A recent work targeting
the same 16S rDNA region (Cytryn et al. 2000) reported
nonspecific amplification using a similar primer set
when annealing temperature decreased to 52°C. On
the contrary, our results showed that all the sequences
we recovered from the fingerprints belonged to Ar-
chaea. Some of the bands excised from our gel, how-
ever, did not yield useable sequence data. This diffi-
culty, intrinsic to DGGE, may be related to the presence
of different sequences in the same DGGE band or to the
fact that weak bands may not have enough template
DNA for their reamplification. A more detailed discus-
sion on potentials and limitations of DGGE has been
previously published (Casamayor et al. 2000b).
Archaeal DGGE fingerprints are considerably het-
erogeneous among systems and differences within
each lake were smaller than those between lakes. A
temporal survey indicated, however, strong changes in
time for the archaeal assemblage in Lake Vilar. There-
fore, these assemblages appear to be different among
lakes, but also summer and winter populations can be
different within a lake. This and the identity of the
main populations were confirmed after sequencing of
the DGGE bands. As expected, the archaeal sequences
retrieved did not share close similarity with any cul-
tured organisms. The sequences recovered belonged
to 3 different groups. First, the methanogens that we
recovered were closely related to a methanogenic
endosymbiont of the anaerobic ciliate Plagiopyla. This
is a well-known symbiosis (Wagener et al. 1990, Fenchel
& Finlay 1991) and in our study sites anaerobic ciliates
such as Plagiopyla and Metopus were also seen by
epifluorescence microscopy carrying endosymbiontic
243
Fig. 6. Changes in the relative abundance of closely related
sequences (≥98% similarity in 16S rDNA sequence) detected
in the temporal study of Lake Vilar. Quantitative data were
obtained from the gel in Fig. 2

Aquat Microb Ecol 25: 237–246, 2001
methanogens (Massana & Pedrós-Alió 1994). Second,
we found that the previously described crenarchaeotal
sequence VIARC-2 (Casamayor et al. 2000b) was pre-
sent in Lake Vilar throughout the temporal study. And,
third, the phylogenetic affiliation showed a new lin-
eage of organisms within the thermoplasmales and rel-
atives group that contained only our sequences from
karstic lakes (Lake Vilar, Lake Banyoles, Lake El Tobar
and Lake La Cruz). This lineage is included in a bigger
cluster containing sequences from freshwater environ-
ments (clones from sediments and plankton).
The same fingerprinting and identical sequences
(BANARC-5, -6 and -7) were obtained from the 3 basins
of Lake Banyoles (B-III, B-IV and B-VI). This is not sur-
prising because all 3 basins are interconnected within
this multibasin lake (Casamitjana & Roget 1993). Inter-
estingly, sequences from Lake Banyoles and Lake
Vilar, which were only 10 m apart, were not the same.
All these sequences were distantly related to se-
quences CIARC-3 and CIARC-2 obtained from Lake
Cisó (1 km away from the former). These results are in
line with previous results, suggesting that anaerobic
hypolimnia of stratified lakes may act as islands in an
aerobic world for anaerobic microorganisms (Casa-
mayor et al. 2000b). We cannot exclude, however, that
different limnological conditions for each lake may
select different microbial assemblages.
Recent reports show that the group of thermoplas-
males and relatives is more widely spread than previ-
ously thought and can be found in both marine and
freshwater environments (DeLong 1998, Van der Maarel
et al. 1998, Bowman et al. 2000, Jurgens et al. 2000, Mas-
sana et al. 2000). We found sequences related to the ther-
moplasmales and relatives in 7 out of 9 study sites, but
we cannot discount that they were also present in the
high percentage of undetermined sequences from Lake
Cadagno and the El Cibollar coastal lagoon. Therefore,
they appear to be a widespread lineage. The sequences
we retrieved were not related to clones from marine en-
vironments and in some cases were close to sequences
from lake sediments. Unfortunately, these organisms
have not been recovered in pure culture and phenotypic
properties cannot be inferred either, due to their distant
genotypic relation with cultured archaea. Therefore,
their ecological significance, biochemistry, physiology
and impact on biogeochemical cycles remain unknown.
Some of the archaeal sequences are closely related to
endosymbionts and we cannot rule out that some others
also correspond to endosymbiontic microorganisms. It is
also possible that new anaerobic metabolisms or micro-
bial interactions are involved, as was shown recently for
the anaerobic oxidation of ammonium and methane (Jet-
ten et al. 1998, Strous et al. 1999, Boetius et al. 2000) and
the association with metazoan species (Preston et al.
1996, Van der Maarel et al. 1998).
Using the relative band intensity as an estimate of
abundance, we found a striking dominance of thermo-
plasmales-related sequences within the archaeal
assemblage of sulfurous lakes, especially in the Bany-
oles karstic area, and a temporal change in the abun-
dance of populations. These changes where higher
than the methodological error of 4% intensity (e.g.,
70% range of variability for crenarchaeota and 50%
for thermoplasmales; see Fig. 5). We were cautious in
the number of PCR cycles run to avoid the ‘plateau’
phase and in using the same amount of template in
each reaction. The samples that we compared were
run in the same PCR run and analyzed in the same
DGGE gel. If there was any PCR bias, it should have
been the same in all lanes. Therefore, comparison
among these samples is valid. In addition, throughout
the seasonal study, gradual changes were seen and
were not a random distribution of the intensity of the
band for each population, adding further credibility to
the changes observed. Moreover, we and others have
published acceptable correlation between quantitative
information obtained by microscopy and by DGGE
(Casamayor et al. 2000b and references therein). Thus,
although not free of limitations, signal intensity of
DGGE bands obtained after ethidium-bromide stain-
ing is a useful tool to calculate relative percentages of
the different groups. Obviously, absolute quantitative
data on archaeal abundance require the use of other
techniques.
Our study found qualitative and quantitative tempo-
ral changes in the archaeal assemblage of a lake, and
we were able to identify the populations involved by
DGGE and sequencing. Temporal changes in archaeal
assemblages were recently described affecting mor-
phology, abundance and identity of archaeal assem-
blages in several systems. Thus, successive peaks of 2
archaeal morphotypes were observed during autumn
in a high mountain lake by fluorescence in situ
hybridization (FISH) (Pernthaler et al. 1998). A maxi-
mum of 5% of all DAPI-stained cells (equivalent to
around 1.7 ×105archaeal cells ml–1) hybridized with
the general archaeal probe ARCH915 in this lake but
archaeal populations were hardly present thereafter.
In Antarctic coastal waters, a strong seasonality of
archaeal rRNA concentrations has been reported, with
the highest values occurring during the austral winter
and early spring (Massana et al. 1998, Murray et al.
1998). Marine group I (crenarchaeota-affiliated) was
shown to be more abundant in Antarctic coastal waters
in late winter and early spring, whereas the marine
group II (thermoplasmales-affiliated) was a minor
component but peaked in abundance in summer and
early autumn (Murray et al. 1998). Intriguingly, this
temporal partitioning of the system between
crenarchaeota and thermoplasmales-related micro-
244

Casamayor et al.: Archaea in sulfurous lakes
organisms matches what we have found in a karstic
lake. The environmental factors responsible for these
changes in time, however, remain unknown.
Our results indicate that the genetic diversity of Ar-
chaea is relatively large in non-thermophilic, sulfurous
environments. We were able to identify shifts in
sequences at the microdiversity scale (more than 97%
similarity in 16S rDNA) after sequencing DGGE bands.
Several investigators have indicated that such small
differences in 16S rRNA genes may belong to closely
related but ecologically different populations (Fuhr-
man & Campbell 1998, Moore et al. 1998). The abun-
dance of these Archaea remain to be determined but
these data suggest that they are novel types of organ-
isms that may be widely distributed and contribute to
the anaerobic biogeochemical cycles of karstic lakes
and coastal lagoons. The vertical distribution (accumu-
lating in sulfide-rich layers) and the temporal dynam-
ics indicate that these archaeal assemblages are au-
tochthonous. The challenge now is to determine the
physiological capabilities and environmental roles of
these uncultured but ecologically relevant Archaea.
Acknowledgements. This work was financed by the Max
Planck Society in Germany, by DGICyT grant number PB95-
0222 from the Spanish Ministerio de Educación y Cultura and
by project MIDAS (Microbial Diversity in Aquatic Systems,
MAS3-CT97-0154) from the European Union. We thank R.
Amann for his generous and continuous support and L.
Bañeras, C. M. Borrego, R. Rosselló-Mora and R. Bachofen for
sampling facilities and sharing field data. H. Schäfer is
thanked for optimization of DGGE conditions and R. Massana
for helpful comments on the manuscript. We also thank 3
anonymous reviewers for comments and suggestions. E.O.C.
benefited from the exchange program between CSIC and the
Max Planck Society.
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246
Editorial responsibility: Dittmar Hahn,
Newark, New Jersey, USA
Submitted: March 21, 2001; Accepted: June 13, 2001
Proofs received from author(s): September 7, 2001