FEMS Microbiology Ecology 42 (2002) 151^161
www.fems-microbiology.org
Characterization of a photosynthetic Euglena strain isolated from
an acidic hot mud pool of a volcanic area of Costa Rica1
Ana Sittenfeld a , Marielos Mora a , Jose¤ Mar|¤a Ortega b , Federico Albertazzi a ,
Andre¤s Cordero a , Mercedes Roncel b , Ethel Sa¤nchez c , Maribel Vargas c ,
Mario Ferna¤ndez d , Ju«rgen Weckesser e , Aurelio Serrano b;
a
Centro de Investigacio¤n en Biolog|¤a Celular y Molecular (CIBCM), Universidad de Costa Rica, Ciudad Universitaria Rodrigo Facio, San Jose¤, Costa Rica
b
Instituto de Bioqu|¤mica Vegetal y Fotos|¤ntesis (IBVF), Universidad de Sevilla y CSIC, Americo Vespucio s/n, 41092 Sevilla, Spain
c
Unidad de Microscop|¤a Electro¤nica, Universidad de Costa Rica, Ciudad Universitaria Rodrigo Facio, San Jose¤, Costa Rica
d
Escuela Centroamericana de Geolog|¤a, Universidad de Costa Rica, Ciudad Universitaria Rodrigo Facio, San Jose¤, Costa Rica
e
Universita«t Freiburg, Institut fu«r Biologie II, Mikrobiologie, Scha«nzlestraMe 1, D-79104 Freiburg i.Br., Germany
Received 4 April 2002; received in revised form 18 July 2002; accepted 19 July 2002
First published online 26 August 2002
Abstract
Conspicuous green patches on the surface of an acidic hot mud pool located near the Rinco¤n de la Vieja volcano (northwestern Costa
Rica) consisted of apparently unialgal populations of a chloroplast-bearing euglenoid. Morphological and physiological studies showed
that it is a non-flagellated photosynthetic Euglena strain able to grow in defined mineral media at temperatures up to 40‡C and exhibiting
higher thermotolerance than Euglena gracilis SAG 5/15 in photosynthetic activity analyses. Molecular phylogeny studies using 18S rDNA
and GapC genes indicated that this strain is closely related to Euglena mutabilis, another acid-tolerant photosynthetic euglenoid, forming a
clade deeply rooted in the Euglenales lineage. To our knowledge this is the most thermotolerant euglenoid described so far and the first
Euglenozoan strain reported to inhabit acidic hot aquatic habitats. = 2002 Federation of European Microbiological Societies. Published
by Elsevier Science B.V. All rights reserved.
Keywords : Photosynthetic euglenoid ; Acidic hot mud pool; Volcanic spring ; 18S rDNA ; GapC; Euglena
1. Introduction
Active volcanic geothermal sites in the Earth’s surface
and mid-ocean ridges provide niches for organisms that
grow in extreme conditions in terms of temperature, pH,
chemical and other environmental characteristics [1]. Thermophilic bacteria and archaea are widely distributed in
these areas [2]. Microbial mats with communities of prokaryotes such as chemotrophic sulfur bacteria, cyanobac-
* Corresponding author. Tel. : +34 (95) 4489524;
Fax : +34 (95) 4460065.
E-mail address : [email protected] (A. Serrano).
1
This paper and the new species described herein are dedicated to His
Royal Highness the Prince of Asturias, Don Felipe de Borbo¤n y Grecia,
for his constant interest in environmental a¡airs and outstanding
contribution to the scienti¢c research cooperation between Spain and
Latinoamerican nations.
teria, and phototrophic bacteria have been studied extensively [3^8]. Considering that the vast majority of eukaryotes cannot survive prolonged exposure to temperatures
above 40^45‡C ^ the upper temperature limit for eukaryotes is in the region of 60‡C [1,9] ^ eukaryotic microorganisms are less diverse than prokaryotes in the thermal
acid habitats of volcanic areas [10]. The photosynthetic
production in these hot acid environments is carried out
almost exclusively by micro-algae of the family Cyanidiaceae, which grow at extremely low pH ( 6 3) and at temperatures up to 57‡C [11].
Among eukaryotes, euglenoids are included within the
most ancient free-living protists, which inhabit a variety of
environments including marine and freshwater, soil and
parasitic environments [12]. Euglena mutabilis Schmitz
(Euglenophyceae) has been described as one of the dominant phytobenthic species in acid mining lakes [13,14].
E. mutabilis is well known for its high metal and acid
tolerance, and is able to grow at a pH of 1.3 [15]. In
relation to temperature, moderate thermotolerance up to
0168-6496 / 02 / $22.00 = 2002 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 2 ) 0 0 3 2 7 - 6
FEMSEC 1403 17-9-02
Cyaan Magenta Geel Zwart
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A. Sittenfeld et al. / FEMS Microbiology Ecology 42 (2002) 151^161
32‡C has been described for Euglena gracilis var. bacillaris
[16]. Growth in liquid medium at 34‡C is known to be
e¡ective in bleaching (irreversible chloroplast loss) Euglena: after 10 cell divisions in the light, e¡ectively all of the
cells yield bleached (white) colonies on plating [17,18].
Photosynthetic euglenoids constitute a group united by
common characteristics including a surface pellicle composed of proteinaceous strips underlain by microtubules,
an intranuclear mitotic spindle, chloroplasts and eyespots
[12]. Reliable methods for determining microbial phylogenies based on gene sequences have been developed [2], and
recently utilized for the study of euglenoids [12,19].
In this report, we describe a photosynthetic non-£agellated Euglena strain (abbreviated name, CRRdV) isolated
from a boiling mud pool with temperatures ranging from
38 to 98‡C and pH between 2 and 4. The hot mud pool is
located in an area characterized by di¡erent forms of geothermal activity at Las Pailas, Rinco¤n de la Vieja volcano,
northwestern Costa Rica [20]. The morphological characterization of the euglenoid performed by light and electron
microscopy (EM), as well as photosynthetic activity studies and molecular phylogenetic analyses using two di¡erent gene markers are presented. Although, as was mentioned above, there are several reports on euglenoids
living at low pH, to our knowledge this study represents
the ¢rst report of a Euglena strain inhabiting a hot spring
volcanic area, showing a capacity to colonize low-pH environments and thermotolerance levels (of up to a maximum of 50‡C under natural conditions at the study site)
not found for any other euglenoid studied so far.
2. Materials and methods
2.1. Description of the study site
The Rinco¤n de la Vieja Volcano (Rincon), a Costa Rican National Park located within the Area de Conservacion de Guanacaste, is a complex and elongated ridge,
comprising at least nine eruptive vents, which lies within
the Cordillera de Guanacaste, 24 km NNE of the city of
Liberia in northern Costa Rica. The maximum height of
this volcano is 1895 m a.s.l. and its area is 400 km2 , which
means that Rincon is the biggest volcano of Costa Rica.
The eruptive vents are pyroclastic cones with incidental
lava £ows in the volcanic sequence [20]. Most of the volcanoes at Rincon have been destroyed by erosion, are
covered with volcanic ash and some of them have exuberant vegetation. The historic activity seems to have been
limited to episodic eruptions of steam, ashes and pyroclasts. The current activity in the active crater consists of
degassing through fumaroles and boiling in the hot lake.
At the base of the southern £ank of the volcano there is an
area known as Las Pailas, which covers 50 ha, where different forms of geothermal activities are present: hot
springs form small streams with very hot water, sulfurous
FEMSEC 1403 17-9-02
lakes, fumaroles, small hollows with constantly bubbling
muddy water and vapour holes, and mud cones of di¡erent shapes and size which become specially active during
the rainy season. One of the features is a small circular
area of 200 m2 known as Pailas de Agua Caliente, which
contains three acidic boiling mud pools or little mud volcanoes composed of dense mud and small amounts of
water. In the central mud pool several green patches
were observed on the surface during various visits to the
site and were sampled for the present work.
2.2. Sample collection and physico-chemical
characterization of the study site
Samples were collected in the area of Pailas de Agua
Caliente from di¡erent sites of the central boiling mud
pool (denoted ES) that showed green-grass-colored
patches on the surface (Fig. 1). Samples from adjacent
locations in the pool showing no green color and from
other mud pools with no green patchiness were also obtained for microscopy and physical and chemical characterization. Several samples of mud from green patches and
from 2^5 cm depth from the surface were obtained from
ES during the rainy season on June 25, 1998, September 3,
1998 and July 13, 1999 and in the dry season on March
18, 2000. Temperature was measured at di¡erent points of
the pool with a digital thermometer with the aid of an
extended probe (Type K Thermo-couple) ; pH was measured on site with pH indicator strips (Merck, Germany)
and con¢rmed with a portable pH meter (Orion, CA,
USA). Mud samples were collected and observed on site
with a light microscope (Reichert, Austria) and a vital dye
solution (Brilliant cresyl blue, Merck) to estimate viable
cells, and transported to the laboratory at room temperature. Measurements of light intensity were made using a
digital handheld light meter (Extech, Taiwan).
Data on mud physico-chemical composition was limited
to samples taken from ES during March, 2000. These
samples were analyzed by gamma ray spectrometry, by
X-ray di¡ractometry and by £uorescence induction.
Chemical analyses of mud samples were performed at
the analytical facilities of the University of Freiburg i.Br.
(Germany).
2.3. Isolation and culture of the photosynthetic
euglenoid
At the laboratory, mud samples were observed by light
microscopy for the presence of microorganisms. Samples
containing green euglenoid-like protists were separated in
Percoll gradients (Pharmacia, Uppsala, Sweden) : 1 ml of
mud was added to 4 ml Percoll and centrifuged at 26 000
rpm using an SW.50.1 rotor in an ultracentrifuge (Beckman Instruments Inc., CA, USA) at 4‡C for 1 h. Fractions
from the gradients were observed by light microscopy, and
those containing protist cells were washed several times
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A. Sittenfeld et al. / FEMS Microbiology Ecology 42 (2002) 151^161
with saline solution (NaCl 0.85%, w/v) until Percoll was
eliminated. Clean fractions were initially inoculated into
biphasic media consisting of Sueoka media (SO) [21] supplemented with 5 Wg of vitamin B12 and 1 mg of vitamin
B6 l31 and 50 Wg ml31 of ampicillin (Sigma, St. Louis,
MO, USA). The pH of the medium was adjusted to 6.6.
The solid phase for biphasic and solid media was prepared
by the addition of 2% (w/v) agar (Difco). Cultures were
incubated at room temperature under white £uorescent or
natural light as described [18,22]. Cells from biphasic cultures were transferred to 125-ml glass £asks containing 50
ml of liquid SO or to solid media in plates and sub-cultured until pure cultures were obtained. Maintenance of
the isolates was performed as described [23] or by keeping
the samples in glass £asks containing mud from the study
site.
Determination of thermal tolerance was performed by
examining samples maintained at di¡erent temperatures,
either in hot mud at the study site (35^60‡C) or in de¢ned
mineral media (liquid and solid SO media) at the laboratory (25^45‡C), for various time periods (1 h to 3 weeks)
and using the vital dye referred to above for cell viability
detection. E. gracilis var. bacillaris (strain SAG 1224-5/
15 = ATCC 10616) that grows rapidly at temperatures up
to 32‡C [16] was used as a control organism.
2.4. Optical light-microscopy (OM) and EM
OM was used to observe aliquots of mud samples directly on site, and of mud, liquid and solid cultures at the
laboratory, using both bright-¢eld and re£ected-light £uorescence techniques (450^480 nm excitation ¢lter, s 550
nm emission ¢lter ; for chlorophyll a/b red £uorescence;
Olympus BX 50, Japan). For EM, aliquots of mud samples and cultures were ¢xed in 2.5% glutaraldehyde, 2.0%
p-formaldehyde (Karnovsky solution) in 0.1 M phosphate
bu¡er, pH 7.4 (PB). Samples for transmission electron microscopy (TEM) were included in 2.0% agarose (Sigma) in
PB and cut in 1.0-mm3 sections. The samples were post¢xed with 1% osmium tetroxide. The preparations were
dehydrated in ethanol, then propylene oxide was used as
intermediate solvent, before ¢nally embedding in Spurr
resin. Ultra-thin sections (60^70 nm) were obtained with
a Reichert Ultracut’s ultramicrotome (Leica Wien, Austria), stained with 4% uranyl acetate and 2% Sato’s triple
lead and observed with Hitachi H-7.100 or H-7.000 transmission electron microscopes working at 100 kV. The samples for scanning electron microscopy (SEM) were ¢xed in
Karnovsky solution. Formvar0 membrane-covered grids
were laid over 25-Wl drops of the samples for 3 min. Grids
were then negatively stained in 0.5% phosphotungstic acid,
pH 7.0, for 10 s, excess liquid drained o¡ and the grids
dried. Grids were put over double-face tape and ion
coated with 20 nm Au-Pd and observed with Hitachi
S-570 and Hitachi S-2360N scanning microscopes working
at 15 kV.
FEMSEC 1403 17-9-02
153
2.5. UV-visible (VIS) absorption spectroscopy and
photosynthetic activity studies
UV-VIS absorption spectra were performed at 20‡C in
an SLM Aminco DW-2000 UV-VIS spectrophotometer in
a 3-ml cuvette containing either cell suspensions in SO
medium or solutions of methanol-extracted photosynthetic
pigments (mostly chlorophylls a and b) [24]. Photosynthetic activity studies were performed with both the green
euglenoid CRRdV and E. gracilis SAG 1224-5/15
( = ATCC 10616) that was used as a strain control.
Light-dependent oxygen evolution activity of intact cells
in SO medium was determined polarographically at various temperatures using a Clark-type oxygen electrode and
saturating white light. Thermoluminescence glow curves of
intact cells were measured essentially as described [25].
Chlorophyll concentration was determined by the method
of MacKinney [24].
2.6. DNA isolation, ampli¢cation and sequencing of
18S rDNA and GapC genes
Total genomic DNA was isolated from the euglenoid
CRRdV essentially according to Porebski et al. [26].
Brie£y, cells were grown until late exponential phase (1^
2U105 cells ml31 ) in liquid SO medium and harvested by
gentle centrifugation (3000Ug for 10 min). The cell pellet
was resuspended in TE (10 mM Tris^HCl and 1 mM
EDTA, pH 7.4) and washed twice under the same conditions. The cell pellet was then resuspended in 400 Wl TE
and 500 Wl of bu¡er CTAB 2U (2% v/v hexadecyltrimethyl^ammonium bromide (Sigma), 1.4 M NaCl, 100 mM
Tris^HCl, 20 mM EDTA, pH 8). After Proteinase K (Sigma) addition (10 mg ml31 ), the preparation was incubated
at 37‡C for 30 min. Subsequently, L-mercaptoethanol and
sodium dodecyl sulfate were added to a ¢nal concentration
of 1% (v/v) and 2% (w/v), respectively, followed by 1.5 h
incubation at 60‡C. The supernatant was extracted twice
with phenol^chloroform^isoamyl alcohol (25:24:1 v/v)
and the aqueous phase precipitated with 0.1 volume of
3 M sodium acetate and 2 volumes of 95% (v/v) ethanol.
The precipitate was washed twice with ice-cold 70% (v/v)
ethanol. The RNA was digested by adding 5 Wl of an
RNase solution (10 mg ml31 RNase, 100 mM Tris^HCl,
10 mM EDTA, pH 8), with incubation for 30 min at 37‡C,
and the DNA extracted as above.
Puri¢ed DNA was ampli¢ed by PCR using di¡erent
combinations of the following oligonucleotide primers:
528F, 449F/Eug, 516R, 120R and 1637F/Eug as described
[19], and primers 18S F (5P-AA(C/T)TGGTTGATCCTGCCAG(C/T)-3P) and 18S R (5P-TGATCCT(G/C)TGCAGGTTCACC-3P), which correspond to regions in the published 18S rDNA sequence of E. gracilis (bases 1^21 for
the forward primer (18S F) and 2281^2303 for the reverse
primer (18S R), GenBank accession M12677). PCR ampli¢cations were performed using a GeneAmp XL PCR kit
Cyaan Magenta Geel Zwart
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A. Sittenfeld et al. / FEMS Microbiology Ecology 42 (2002) 151^161
(Perkin Elmer, CA, USA) according to manufacturer speci¢cations. 50-Wl ampli¢cation reaction mixtures containing
2 pmol of each primer, approx. 10 ng of genomic DNA,
200 WM each deoxynucleotide triphosphate, 0.5 U Taq
polymerase (Life-Technologies, MD, USA) or Taq XLarge
kit (Perkin-Elmer, CA, USA) were performed according to
the respective primers requirements. All reactions were incubated in a thermal cycler (Perkin-Elmer Cetus, CA,
USA) for 4 min at 94‡C, followed by 25 ampli¢cation
cycles of 30 s at 94‡C, 30 s at 50‡C and 6 min at 72‡C.
The very last step was extended by an additional 15 min at
72‡C. Ampli¢cation products were puri¢ed with a QIAquick-spin or QIAEX II puri¢cation kit (Qiagen, Germany) and their expected size veri¢ed in agarose gels.
Cleaned ampli¢cation products were sequenced directly
by cycle sequencing kit Big Dye Terminators1 (Applied
Biosystems, CA, USA) and an ABI PRISM 377 DNA
sequencer (Applied Biosystems).
An internal region of approx. 0.94 kb comprising about
95% of the coding sequence of a GapC gene encoding
cytosolic NAD-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDHC, EC 1.2.1.12, a key enzyme of
glycolysis) was PCR-ampli¢ed using a cDNA preparation
(‘Time Saver’ cDNA synthesis kit, Pharmacia Biotech)
from CRRdV as the template and two degenerate oligonucleotides (gap4, forward, 5P-AAT(C)GGA(CGT)TTC(T)GGA(CGT)A(C)GA(G)ATA(CT)GGA(CGT)A(C)G3P; and gap2, reverse, 5P-ACCATG(A)CTG(A)TTG(A)CTC(T)ACCCC-3P) constructed from two highly conserved amino acid regions of GAPDH proteins located
near the N- and C-termini (NGFGRIG and WYDNEWG, respectively) [27^29]. The ampli¢cation products
were processed and analyzed as described above. Automatic sequencing of three independent clones performed
as described above yielded identical sequences.
2.7. Sequence alignment and phylogenetic analysis
For multiple-alignment of 18S rDNA sequences of various Euglenophycean taxa, CLUSTAL X 1.81 [30] was
used. The secondary structure of E. gracilis SSU rRNA
(database at the University of Antwerp, http://rrnawww.uia.ac.be) was used to improve the alignments. The
estimation of the identity matrix was done with the
aligned sequences. Sequences used were: Petalomonas cantuscygni (GenBank accession U84731), Peranema trichophorum (U84733), Khawkinea quartana (U84732), Phacus
pyrum (AF112874), Ph. splendens (AF190814), Ph. similis
(AF119118), Ph. oscillans (AF181968), Ph. pusillus
(AF190815), Ph. orbicularis (AF283315), Ph. alatus
(AY014999), Ph. megalopsis (AF090870), Ph. pleuronectes
(AF081591), Ph. acuminata (AF283311), Ph. aenigmaticus
(AF283313), Ph. pseudonordstedtii (AF283316), Ph. brachykentron (AF286209), Eutriptiella sp. (AF112875), Lepocinclis ovum (AF110419), L. ovata (AF061338), L. buetschlii (AF096993), Astasia longa (AF283306), A. curvata
FEMSEC 1403 17-9-02
(AY004245), Strobomonas sp. (AF096994), Trachelomonas
(AF090377), T. hispida (AF090377), T. volvocina
(AF096995), Euglena agilis (AF115279), E. gracilis
(M12677), E. acus (AF152104), E. stellata (AF150936),
E. spirogyra (AF150935), E. mutabilis (AF096992), E. viridis (AF112872), E. oxyuris (AF090869), E. tripteris
(AF286210), E. chlamydophora (AY029407), E. anabaena
(AF081593), E. intermedia (AY029408), Euglena UTEX
sp. (AF112873), Distigma curvata (AF099081), Gyropaigne
lefevrei (AF110418), Crithidia fasciculata (Y00055). The
sequence of Bodo caudatus (X53910), a representative of
kinetoplastids (a sister group of euglenoids), was used as
an outgroup.
For multiple-alignment of amino acid sequences of
GAPDH proteins deduced from Gap genes, the CLUSTAL X 1.81 program was used as described [31]. Only
protein sequences between the conserved regions used to
design the degenerate oligonucleotides described above
were considered for the alignment and subsequent construction of the phylogenetic tree. The following amino
acid sequences (named from its corresponding gene but
in standard style) were used: Anabaena variabilis Gap1
(L07497) and Gap2 (L07498) ; Synechocystis sp. PCC
6803 Gap1 (X86375) and Gap2 (P49433); Arabidopsis
thaliana GapA (P25856), GapB (P25857) and GapC
(P25858) ; Nicotiana tabacum GapA (P09043), GapB
(P09044) and GapC (P09094); Pinus sylvestris GapCp
(CAA04942) and GapC (P34924); Selaginella lepidophylla
GapC (AAB59010); Chlamydomonas reinhardtii GapA
(P50362) and GapC (P49644) ; Chorella fusca GapA
(AJ252208) and GapC (AJ252209) (F. Valverde and A.
Serrano, unpublished results); E. gracilis GapA (P21904)
and GapC (P21903); Chondrus crispus GapA (P34919) and
GapC (P34920) ; Gracillaria verrucosa GapA (P30724) and
GapC (P54270) ; Guillardia tetha GapCp (U40032) and
GapC (U39873); Pyrenomonas salina GapCp (U40033)
and GapC (U39897); Ochromonas danica GapCp and
GapC (F. Valverde and A. Serrano, unpublished results) ;
Cyanophora paradoxa GapC (AJ313316) (F. Valverde and
A. Serrano, unpublished results) ; Cyanidium caldarium
GapC (AJ313315) (F. Valverde and A. Serrano, unpublished results); Saccharomyces cerevisiae GapC1 (P00360);
Monocercomonas sp. GapC (AAC63603); Haemophilus in£uenzae Gap1 (U32898); Homo sapiens GapC (NP002037);
Trypanosoma brucei GapC (P10097) and GapCg (P22512);
Leishmania mexicana GapC (X65220) and GapCg
(X65226); Trypanosoma cruzi GapCg (P22513); and Trypanoplasma borelli GapC1 (CAA52631) and GapC2
(CAA52632).
Polymorphic sites (681 nucleotides) were selected to
construct the phylogenetic trees for the 18S rDNA. The
calculation of the distance matrix for the 18S rDNA sequences was done with the aligned sequences according to
Jukes and Cantor [32], Kimura’s 2-parameter distance [33]
and Van de Peer et al. [34] (TREECON ver 1.3b program
[35]) and maximum-parsimony (DAMBE ver.4.0.39 pro-
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A. Sittenfeld et al. / FEMS Microbiology Ecology 42 (2002) 151^161
Fig. 1. View of the central acidic hot mud pool (ES) at the Pailas de
Agua Caliente area near the Rinco¤n de la Vieja volcano (northwestern
Costa Rica). Note the green patches on the mud surface (arrowed) that
revealed the algal growth (bar = approx. 30 cm).
gram [36]). The calculation of the distance matrix for the
GAPDH was done with the aligned full protein sequences
according to Kimura’s 2-parameter distance [33]. The evolutionary trees were constructed with the neighbor-joining
method [32,33,35]. The bootstrap con¢dence levels for the
interior branches of the trees were performed with 1000
resamplings for neighbor-joining and for maximum-parsimony analysis [36].
3. Results and discussion
3.1. Presence of a photosynthetic euglenoid in an acidic hot
mud pool and physico-chemical characteristics of the
study site
Las Pailas de Agua Caliente basically comprises three
dense mud pools or little mud volcanoes with small
amounts of water on the surface, characterized by constant bubbling and boiling spots. Only the central mud
pool (ES) contained the green patchiness. Only a greenpigmented, presumably photosynthetic, euglenoid-like microorganism (abbreviated strain name, CRRdV) was observed by OM (on site and at the laboratory, directly and
using a vital dye) or by EM in samples obtained from
green patches on the surface of ES (Fig. 1). The occurrence of the green patchiness varied considerably over
short distances in the mud pool and over short time intervals. In general, a higher density of the green algal mats
was observed at the periphery of the pool during the wet
season, in contrast to small patches observed towards the
center during the dry season. Since no other eukaryotic
microorganism (either micro-alga or protozoan) was observed by microscopic examinations, these green patches
can be considered as unialgal microbial communities of
CRRdV. However, further studies were required to con¢rm this observation, in view of the limitations of OM for
distinguishing closely related species.
FEMSEC 1403 17-9-02
155
The temperature and pH of ES ranged from 35‡C to
98‡C and between 2 and 4, respectively, and varied due to
changes in steam sources over short periods of time and in
di¡erent sectors of the pool. Seasonal variations in temperature, but not in pH, were detected. Collections made
during the rainy season revealed lower temperatures (between 35‡C and 48‡C) on the borders of the mud pool and
higher temperatures (up to 98‡C) at the center. Temperature measurements obtained during the dry season indicated lower temperatures at the center, with the higher
temperatures at the borders. As stated above, the grassgreen-colored areas on the surface of the pool were indicative of the presence of CRRdV: only CRRdV cells were
observed in the samples by OM on site. The temperature
of the green patches containing the euglenoid varied from
34‡C to 45‡C (during both the dry and rainy seasons).
More detailed measurements obtained in March, 2000 at
25 cm and 1 m from the grass-green areas were 48‡C and
98‡C respectively. In consequence, the euglenoid population was observed at the lower end of the temperature
range (35^40‡C) and the location in the mud pool varied
according to the temperature changes. Euglenoid cells
were only found within 2 cm of the surface. Although
the upper temperature boundary for the presence of the
euglenoid in the mud pool was around 42^45‡C, due to
the constant appearance of boiling spots on the surface of
the mud pool, a thermotolerance of up to 50‡C for 1^2 h
was estimated when a series of samples obtained from a
particular mud spot were observed on site by OM using a
vital dye. The same result was obtained on site, when
green-colored mud samples were kept in thermal containers at 50‡C for up to 2 h.
It is essential to obtain pure cultures of dominant organisms from extreme environments, but also to study the
activity of organisms directly in nature, because they often
behave in a di¡erent way in laboratory cultures [1]. The
euglenoid CRRdV was maintained in SO mineral medium
under £uorescent white light. The di¡erences observed in
the temperature boundaries detected for CRRdV in the
mud and with cultures in de¢ned mineral media in the
laboratory suggest di⁄culties in replicating the conditions
and growth characteristics observed in nature. Although it
was not possible to maintain cultures for more than 7^
10 days at 45‡C in SO mineral medium, it can be suggested that CRRdV is a thermotolerant protist capable
of tolerating up to 50‡C for 1^2 h in the natural conditions
observed at the study site.
Temperature and pH conditions were similar at the
three mud pools of Las Pailas de Agua Caliente area.
Data on inorganic matter composition from mud is limited to samples taken from the central boiling mud pool of
Las Pailas during March, 2000. By gamma ray spectrometry the mud contains signi¢cant amounts of K, Bi, Pb,
Rn, Ra, Ac, Th, U, Nb and Ac (ranging from 2.369114 for
Ac-228 to 7.773401 Bq kg31 for K-40). Fluorescence induced by X-ray irradiation of the same sample determined
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A. Sittenfeld et al. / FEMS Microbiology Ecology 42 (2002) 151^161
Fig. 2. Morphological characterization of the euglenoid CRRdV.
A: Bright-¢eld light microscopy of euglenoid cells growing photoautotrophically in SO medium (bar = approx. 20 Wm). The insert shows
bright-¢eld (left) and £uorescence micrographs of a cell observed in a
mud sample; note the red £uorescence due to the photosynthetically active chlorophylls a/b contained in the green chloroplasts. B: SEM of a
CRRdV cell. Note the clear composition of the helically distributed
strips of the pellicle and the apparent absence of £agella (bar = approx.
6 Wm). C: TEM showing a longitudinal cross-section of a CRRdV cell.
Note the strips of the pellicle around the cell, the chloroplasts (with
three membrane lamellae) located at the cell periphery, and the paramylon granules (white areas) that together with the nucleus are located in
the central region of the cell. Ch, chloroplasts; N, nucleus ; P, paramylon granule. Again, no evidence for £agella was found (bar = approx.
7 Wm).
that Ti, Fe and Sr were also present, the most abundant
element of these three being Sr whose intensity was more
than 7000 (counts per 2000 s). The analysis of mud by
X-ray di¡ractometry distinguished three important minerals: kaolinite (Si4 O10 )All4 (OH)8 , quartz (SiO2 ) and cristobalite (a SiO2 polymorph). Chemical analysis of mud indicated that Al2 O3 and SiO2 were by far the major
components (22.2% and 31.6% of dry weight, respectively),
with minor amounts of SO3 , P2O5 , Fe2 O3 , MgO, K2 O,
Na2 O, Ti, Ba and Sr, and traces of Zr, Rb, Cu, Ni, Cr,
Rh and Pd. In solution, Ca2þ and Mg2þ were the most
abundant cations (274 and 113 mg l31 , respectively) and
Cl3 and NO3
3 the most abundant anions (22 and 23 mg
l31 , respectively). No signi¢cant amounts of organic compounds were found in mud samples, a fact that speaks in
favor of the photosynthetic capability of the CRRdV euglenoid in its natural habitat. In this context it is interesting to note that surface radiation measurements on site
revealed that light on the surface of ES (in the range of
1300 lux) should be enough for photosynthesis, despite the
dense vegetation that surrounded the acidic hot mud pool
during the rainy season.
3.2. Morphological and structural description of the
euglenoid
OM observation of samples from green ¢lms on mud
samples and laboratory cultures showed only one type of
solitary, apparently a£agellated (palmelloid), green eukaryotic cell devoid of cell wall (surrounded by a thin
pellicle), typically about 45 Wm long (range from 30 to
60 Wm) and 6^10 Wm wide, containing several green chlo-
FEMSEC 1403 17-9-02
roplasts of varying size that exhibited strong red £uorescence due to chlorophylls (Fig. 2A and inserts). Since these
characteristics are very similar to those described for the
Euglenophyceae [19,37], the CRRdV protist was classi¢ed
from the very early steps of this work as a euglenoid.
More detailed observation indicated that morphological
characteristics were in general shared with the greencolored genus Euglena, particularly with the previously
described species E. mutabilis (www.lifesci.Rutgers.edu/
~triemer). However, some di¡erences observed with this
Euglena species included size, movement and the apparent
absence of £agella in CRRdV (E. mutabilis has a single
short £agellum), a morphological feature that was con¢rmed by EM (see below). Active CRRdV cells exhibit
movements characterized by wriggling and constant bending (euglenoid movement) when observed directly in mud
samples on site, as well as in liquid and solid laboratory
cultures. This movement is typical of euglenoids that lack
£agella or are not using them to swim [37], a fact that is in
agreement with our morphological data (see below). Thus,
CRRdV cells display dramatic size variations and distortion of body shape, from elongated nearly cylindrical
shape to round cells and encapsulated stages (see Fig.
2A). One orange^red eyespot free in the cytoplasm (independent of chloroplasts) was observed in cells from mud
samples (Fig. 2A, insert), whereas several independent
dark inclusions appeared in cells cultured in SO (Fig. 2A).
By SEM, CRRdV fusiform cells showed a size of 30U8
Wm; neither £agella nor basal bodies were observed. The
protist is surrounded by a pellicle (periplast) composed of
well-developed strips called myonems that covers it helicoidally (Fig. 2B). These pellicular strips have a thickness
of approx. 0.2 Wm and are present over the entire cell
surface. The periplast is £exible and allows the microorganism to elongate and contract. TEM revealed two to
four large chloroplasts of cylindrical or elongated shape
that are located at the cell periphery in close contact with
the plasma membrane (Fig. 2C). The chloroplasts have an
envelope of three membranes, as previously described for
other euglenoids [37], and lamellae formed by threestacked thylakoid membranes. The nucleus has a spherical
shape and can be central or located at the periphery of the
cell. The nucleolus has a central nuclear position and was
surrounded by electron-dense bodies, which should correspond to condensed chromosomes (Fig. 2C). Although
several preparations of cells from independent cultures
and di¡erent types of ultra-thin sections were analyzed,
a canal/reservoir complex (ampulla) with £agellar apparatus was not observed. It was common to ¢nd in the cytoplasm vacuoles of di¡erent size, as well as numerous granules of the reserve polysaccharide paramylon (Fig. 2C).
Spherical osmophilic circular structures are seen in the
cytoplasm and probably correspond to the eyespots. Membrane systems of endoplasmic reticulum are located close
by, but not connected to, the nucleus. The Golgi apparatus is composed of multiple membranes forming cisternae
Cyaan Magenta Geel Zwart
A. Sittenfeld et al. / FEMS Microbiology Ecology 42 (2002) 151^161
Fig. 3. Growth on plates of SO medium of photoautotrophic cultures of
E. gracilis SAG 5/15 and the euglenoid CRRdV. Note the clearly di¡erent growth patterns on solid medium: CRRdV (an evidently a£agellated
strain) forms well-de¢ned colonies while E. gracilis (a very motile, £agellated strain) covers the whole plate.
and vesicles. Elongated mitochondria are often seen in
large numbers and have a structure typical for eukaryotic
organisms. When several isolates collected at di¡erent
times were studied, no di¡erences in morphology were
observed by OM or EM in the original ¢eld samples and
the corresponding derived laboratory cultures.
3.3. Culture and photosynthetic capacity
Slow growth (doubling time 24^48 h) in both liquid and
solid SO media was observed at 25, 35 and 40‡C. Faster
growth (doubling time about 20 h) was obtained only
when liquid cultures were shaken and ¢ltered air bubbled
through the media. Cultures maintained at 45‡C produced
bleaching and death of CRRdV after 7^10 days. The
growth pattern on SO solid medium showed clear di¡erences to that of E. gracilis (Fig. 3): whereas CRRdV
formed well-de¢ned circular colonies, E. gracilis ^ a very
motile, £agellated strain ^ covered the whole plate in a
spread pattern fashion. This di¡erence was to be expected
if £agella were actually absent from CRRdV. The viability
of the cells was maintained for over 2 years at room temperature in samples preserved in mud from the study site.
The room temperature absorption spectra of methanolextracted cell pigments and intact cell suspensions of strain
CRRdV are shown in Fig. 4A. The major visible absorption maxima occur at positions characteristic of higher
plants, green algae and photosynthetic euglenoids, and
are due mostly to chlorophylls a and b and some carotenoid species absorbing between 450 and 500 nm. Cells suspensions showed peaks at 683, 620 and 446 nm. Methanolextracted cell pigments exhibited peaks at 666, 620, 477
and 446 nm. These absorption spectra are virtually identical to those obtained with E. gracilis (data not shown).
Fig. 4B shows the temperature dependence of oxygen evolution activity of both CRRdV and E. gracilis cell suspensions. In both cases, the activity at 25‡C (270 and 210
Wmol O2 mg Chl31 h31 , respectively) exhibited a gradual
FEMSEC 1403 17-9-02
157
increase with temperature to reach the maximum rate at
approx. 40‡C for CRRdV (450 Wmol O2 mg Chl31 h31 )
and at 35‡C for E. gracilis (350 Wmol O2 mg Chl31 h31 ).
Above these temperatures, activity abruptly decreased
with increasing temperature, being completely inhibited
at 45‡C in the case of E. gracilis, but still remaining at
about 40% of the maximal activity in CRRdV cells. Thermoluminescence serves as a convenient probe of both the
donor and acceptor chemistry in the photosystem II (PSII)
reaction center [25,38]. Fig. 4C shows the thermoluminescence glow curves of both E. gracilis and CRRdV cell
suspensions grown at 25‡C. Upon excitation with a single
saturating £ash, PSII exhibited a thermoluminescence
band peaking at an emission temperature of around
25‡C and 33‡C for E. gracilis and CRRdV, respectively.
This band was assigned to the B-band originating from
both the processes of charge recombination between the
secondary quinones acceptor QB and S2 (B2 -band) and S3
(B1 -band) states of the manganese cluster of the water
oxidation complex [38]. In consequence, B-band is emitted
in CRRdV cells at about 8‡C higher than for E. gracilis.
This implies a deeper stabilization of charge-separated
states in the PSII reaction center of the CRRdV strain.
Overall, these photosynthetic activity analyses showed that
the optimal capability of the PSII reaction center in
CRRdV occurs at a higher temperature than for the
most thermotolerant strain of Euglena, E. gracilis 5-15,
that was used as a control organism. Therefore, concerning photosynthesis, CRRdV can be considered as the most
thermotolerant euglenoid described so far.
Fig. 4. Photosynthetic characterization of the euglenoid CRRdV.
A: Room temperature UV-visible absorption spectra of whole cells
(^ ^ ^) and photosynthetic pigments (mostly chlorophyll a/b) extracted with methanol (9). B: Temperature dependence of oxygen evolution by cells of CRRdV (b) and E. gracilis SAG 5/15 (a). Activity
was measured in 50 mM Mes^NaOH (pH 6.5), 0.1 M sucrose, 5 mM
CaCl2 and 5 mM MgCl2 , with 0.4 mM 2,5-dimethyl-1,4-benzoquinone
and 1 mM potassium ferricyanide as electron acceptors. The values corresponding to 100% were 450 and 350 Wmol O2 mg Chl31 h31 for
CRRdV and E. gracilis cells, respectively. C: Thermoluminescence glow
curves of CRRdV and E. gracilis cells. The glow curves were recorded
after one saturating £ash at 0‡C.
Cyaan Magenta Geel Zwart
158
A. Sittenfeld et al. / FEMS Microbiology Ecology 42 (2002) 151^161
Fig. 5. Phylogenetic distance trees of 18S rDNA, constructed by the neighbor-joining method with (A) Jukes and Cantor [32], (B) Kimura [33] two parameter formulas for distance estimations, showing the phylogenetic position of the euglenoid CRRdV in the context of the Euglenoids group. Bold
type indicates the Euglenales taxa. The kinetoplastid euglenozoan B. caudatus was used as outgroup. Numbers on the tree indicate the bootstrap percentage of each node (based on 1000 resamplings) for each consensus tree. Note that CRRdV form a well supported clade deeply rooted in the Euglenales lineage. In general, grouping of other species is the same as reported in recent phylogenetic studies on euglenoids [39].
3.4. PCR ampli¢cation of 18S rDNA and GapC genes
3.5. Phylogenetic analysis of sequenced data
3.4.1. Ampli¢cation and sequencing of 18S rDNA fragment
A DNA fragment was successfully ampli¢ed by PCR
from genomic DNA isolated from CRRdV. The nucleotide sequence of the ampli¢ed fragment was determined
for both strands in their entirety. The length (ignoring
the amplifying primers) was 2407 bp. The sequence was
deposited in the GenBank nucleotide sequence database
with the accession number AY029278.
The 18S rDNA sequence of CRRdV was preliminarily
matched with previously published 18S rDNA sequences
using WWW BLAST at the NCBI home page http://
www.ncbi.nlm.nih.gov). The phylogenetic trees were constructed using a subset of 46 euglenoid sequences and
B. caudatus (Bodonidae, non-parasitic kinetoplastids) as
outgroup, from maximum-parsimony [36] and distance
[35] methods.
The similarity matrix identity value using the aligned
18S rDNA sequences of CRRdV and E. mutabilis was
51.1%, whereas the maximal similarity was between
E. gracilis and E. intermedia at 72.7% (percent similarity
table not shown). These results suggest that CRRdV could
be a di¡erent species from E. mutabilis and possibly a new
Euglena species altogether. The three distance estimation
methods [32,33] produced identical topologies (Fig. 5) for
the E. mutabilis and CRRdV clade. The acidophilic euglenoids E. mutabilis and CRRdV clade posed in a basalbranching in the Euglenales lineage that is well supported
with a bootstrap value of 100% (Fig. 5). The same relationship for E. mutabilis and CRRdV was con¢rmed using
the maximum-parsimony analysis with the same matrix
data (Fig. 6). In general, the topology of the trees was
concordant with recent data on euglenoid phylogeny,
namely, the polyphyletic character of the Euglena taxa,
3.4.2. Ampli¢cation and sequencing of GapC fragment
PCR-ampli¢cation using oligonucleotides from conserved regions of GAPDH proteins successfully generated
a cDNA fragment of the expected size (approx. 0.94 kb),
corresponding to about 95% of the complete coding region
of typical Gap genes. So far, Gap genes of only one photosynthetic euglenoid (E. gracilis) have been reported,
namely, a plant-like GapA gene encoding the chloroplastic
anabolic GAPDHA and a GapC encoding the cytosolic
glycolytic GAPDHC [27]. Several Gap clones of CRRdV
were sequenced and all of them were found to correspond
to a single GapC-type gene similar to the homologue from
E. gracilis (approx. 70% identity at the DNA level and
85% identity at protein level). The sequence was deposited
in the EMBL/GenBank nucleotide sequence database with
the accession number AJ312943.
FEMSEC 1403 17-9-02
Cyaan Magenta Geel Zwart
A. Sittenfeld et al. / FEMS Microbiology Ecology 42 (2002) 151^161
159
(Fig. 7), a result in agreement with the current accepted
view that Euglenozoa are an early-branching primitive
group of protists within the phylogeny of eukaryotes.
The molecular phylogeny studies performed with the
two gene markers ^ 18S rDNA and GapC, which encodes
an enzyme of central metabolism ^ are therefore consistent
with the morphological and physiological data reported in
this work and suggest that CRRdV is a Euglena strain
di¡erent to those previously described.
Summarizing, we have shown that a photosynthetic Euglena strain exclusively colonized an extreme habitat of
acidic hot mud pools in a volcanic area surrounded by
tropical forest, in which euglenozoan protists have never
been found before. This euglenoid was isolated and cultured photoautrophically in de¢ned mineral media, showing thermotolerance. The morphological, physiological
and molecular phylogeny studies strongly suggest that it
must be considered a new Euglena species, for which the
name of Euglena pailasensis (Eukaryota ; Euglenozoa ; Euglenida; Euglenales) is proposed.
Fig. 6. Phylogenetic tree of 18S rDNA, constructed by the maximumparsimony method, showing the phylogenetic position of the euglenoid
CRRdV in the context of the Euglenoids group. Bold type indicates the
Euglenales taxa. B. caudatus was used as outgroup. Numbers on the
tree indicate the bootstrap percentage of each node (based on 1000 resamplings) for the consensus tree.
the phagotrophic strains anchoring the base of the euglenoids lineage and the multiple origins of osmotrophic euglenales [19,39]. However, in previous euglenoid trees,
E. mutabilis was not included.
A phylogenetic analysis was also performed using the
deduced amino acid sequence of GAPDHC, a key enzyme
of the glycolytic/gluconeogenic pathways, which is present
in all organisms studied so far (Fig. 7). As stated above,
the PCR-ampli¢ed CRRdV sequences correspond to a single GapC gene encoding a GAPDHC protein very similar
to the cytosolic E. gracilis GAPDHC, being the second
euglenoid GapC gene characterized so far. It should be
the relevant Gap marker for molecular phylogeny studies
of the host eukaryotic cell (the common ancestor of all
Euglenozoa, both euglenoids and kinetoplastids) that established the secondary endosymbiosis with a green alga
that gave rise to present-day photosynthetic euglenoids. In
agreement with this, the two Euglena GapC-deduced protein sequences clearly cluster in the phylogenetic tree with
the GAPDHs of Trypanoplasma, a member of the Bodonidae group (free-living kinetoplastids) and the glycosomal
GAPDHs (GAPDHCg) of parasitic trypanosomatids,
characteristic enzymes of these parasites that probably
evolved from the cytosolic GAPDHC of the Euglenozoa
common ancestor [27]. This clade appears therefore as a
deep (primitive) branch in the glycolytic GapC lineage
closely related to the cyanobacterial Gap1 GAPDHs
FEMSEC 1403 17-9-02
Fig. 7. Phylogenetic distance tree, constructed by the neighbor-joining
method with the Kimura formula, of GAPDH proteins including the
amino acid sequence deduced from the CRRdV GapC gene. Bootstrap
values (over 1000 replicates) in the nodes indicate the statistical support
of the corresponding group. Note the robust clustering (1000, in bold)
of the CRRdV sequence which corresponds to the glycolytic/cytosolic
GAPDHC, with its homologue from E. gracilis and the glycosomal
GAPDH sequences (GapCg) of kinetoplastids (Trypanosomatidae and
Bodonidae) in a clade (shadowed box) branching deeply in the lineage
of glycolytic GAPDH proteins (cytosolic, plastidic (Cp) and glycosomal
(Cg) eukaryotic GapC, and bacterial Gap1). The lineage formed by the
anabolic GAPDHs ^ GapA/B (chloroplastic) and (ciano)bacterial Gap2
^ is clearly di¡erent to the GapC family and was used as an outgroup.
The parabasalid protozoa, that have a bacterial-like cytosolic
GAPDHC, are represented here by the Monocerconomas sp. GapC.
Cyaan Magenta Geel Zwart
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A. Sittenfeld et al. / FEMS Microbiology Ecology 42 (2002) 151^161
Acknowledgements
The authors thank Professors Manuel Losada (University of Sevilla, Spain) and Rodrigo Ga¤mez (National Institute of Biodiversity, INBio, Costa Rica) for generous
encouragement and help. Part of this work was supported
by a collaborative grant Costa Rica-Spain (‘Plan de Cooperacio¤n Cient|¤¢ca con Iberoame¤rica’, 1998-2000, MAE,
Spain). The Spanish team also acknowledges grants
PB97-1135 (MCYT, Spain) and PAI CVI-261 (Junta de
Andalucia, Spain). The Costa Rica team was supported
in part by Grant VI 801-98-507 from Vicerrector|¤a de Investigacio¤n, Universidad de Costa Rica (San Jose¤ Costa
Rica).
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