Characterization of a photosynthetic Euglena strain isolated from an acidic hot mud pool

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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 : aurelio@cica.es (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.

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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

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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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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.

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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

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(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

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(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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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.

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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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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-

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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

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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

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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.

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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.

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(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

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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.

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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).

References [1] Brock, T.D. (1986) Introduction : An overview of the thermophiles.

In: Thermophiles, General, Molecular and Applied Microbiology (Brock T.D., Ed.), pp. 1^16. John Wiley and Sons, New York.

[2] Pace, N.R. (1997) A molecular view of microbial diversity and the biosphere. Science 276, 734^740.

[3] Weller, R., Walsh Weller, J. and Ward, D.M. (1991) 16S rRNA sequences of uncultivated hot spring cyanobacterial mat inhabitants retrieved as randomly primed cDNA. Appl. Environ. Microbiol. 57, 1146^1151.

[4] Wahlund, T.M., Woese, C.R., Castenholz, R.W. and Madigan, M.T. (1991) A thermophilic green sulfur bacterium from New Zealand hot springs, Chlorobium tepidum sp. nov.. Arch. Microbiol. 156, 81^90.

[5] Hiraishi, A., Umezawa, T., Yamamoto, H., Kato, K. and Maki, Y. (1999) Changes in quinone pro¢les of hot spring microbial mats with a thermal gradient. Appl. Environ. Microbiol. 65, 198^205.

[6] Miller, S.R. and Castenholz, R.W. (2000) Evolution of thermotolerance in hot spring Cyanobacteria of the genus Synechococcus. Appl.

Environ. Microbiol. 66, 4222^4229.

[7] Johnson, D.B. (1998) Biodiversity and ecology of acidophilic microorganisms. FEMS Microbiol. Ecol. 27, 307^317.

[8] Stetter, K.O. (1995) Microbial life in hyperthermal environments.

ASM News 61, 285^290.

[9] Roberts, D.M.L., (1999) Eukaryotic cells under extreme conditions.

In: Enigmatic Microorganisms and Life in Extreme Environments (Seckbach, J., Ed.), pp. 165^173. Kluver Academic Publishers, Dordrecht.

[10] Walsh, M.M. and Seckbach, J. (1999) The versatility of microorganisms. In: Enigmatic Microorganisms and Life in Extreme Environments (Seckbach, J., Ed.), pp. 153^162. Kluver Academic Publishers, Dordrecht.

[11] Moreira, D., Lo¤pez-Archilla, A.-I., Amils, R. and Mar|¤n, I. (1994) Characterization of two thermoacidophilic microalgae: Genome organization and comparison with Galdiera sulphuraria. FEMS Microbiol. Lett. 122, 109^114.

[12] Linton, E.W., Hittner, D., Lewandowski, C., Auld, T. and Triemer, R.E. (1999) A molecular study of euglenoid phylogeny using small subunit rDNA. J. Eukaryot. Microbiol. 46, 217^223.

[13] Kapfer, M. (1998) Assessment of the colonization and primary production of microphytobenthos in the littoral of acidic mining lakes in Lusatia (Germany). Water Air Soil Pollut. 108, 331^340.

[14] Lessmann, D., Deneke, R., Ender, R., Hemm, M., Kapfer, M., Krumbeck, H., Wollmann, K. and Nixdorf, B. (1999) Lake Plessa 107 (Lusatia, Germany) ^ an extremely acidic shallow mining lake.

Hydrobiologia 408/409, 293^299.

FEMSEC 1403 17-9-02

[15] Nakatsu, C. and Hutchinson, T.C. (1988) Extreme metal and acid tolerance of Euglena mutabilis and an associated yeast from Smoking Hills, Northwest Territories, and their apparent mutualism. Microb.

Ecol. 16, 355^363.

[16] Schlo«sser, U.G. (1994) SAG-Sammlung von Algenkulturen at the University of Go«ttingen. Catalogue of strains 1994. Bot. Acta 107, 113^186.

[17] Uzzo, A. and Lyman, H. (1969) Light dependence of temperatureinduced bleaching in Euglena gracilis. Biochim. Biophys. Acta 180, 573^575.

[18] Russell, G.K and Lyman, H. (1982) Production of chloroplast mutants in Euglena. In: Methods in Chloroplast Molecular Biology (Edelman, M., Hallick, R.B. and Chva, N.M., Eds.), pp. 39^50. Elsevier, Amsterdam.

[19] Montegut-Felkner, A.E. and Triemer, R.E. (1997) Phylogenetic relationships of selected euglenoid genera based on morphological and molecular data. J. Phycol. 33, 512^519.

[20] Alvarado, G. (2000) Los volcanes de Costa Rica: su geolog|¤a, historia y riqueza natural, 301 pp. Editorial UNED, San Jose¤, Costa Rica.

[21] Sueoka, N., Chiang, K.S. and Kates, J.R. (1967) Deoxiribonucleic acid and replication in meiosis of Chlamydomonas reinhardtii. Isotipic transfer experiments with a strain producing eight zoospores. J. Mol. Biol. 25, 47^66.

[22] Bishop, N.I. and Senger, H. (1971) Preparation and photosynthetic properties of Synchronous cultures of Scenedesmus. In: Methods in Enzymology, Vol. XXIII. Photosynthesis (San Pietro, A., Ed.), 53^66 pp. Academic Press, New York.

[23] McLellan, M.R., Cowling, A.J., Turner, M. and Day, J.G. (1991) Maintenance of algae and protozoa. In: Maintenance of Microorganisms (Kirshop, B.E. and Doyle, A., Eds.) 2nd edn., pp. 184^226.

Academic Press, London.

[24] Mackinney, G. (1941) Absorption of light by chlorophyll solutions.

J. Biol. Chem. 140, 315^322.

[25] Ortega, J.M., Roncel, M. and Losada, M. (1999) Light-induced degradation of cytochrome b559 during photoinhibition of the photosystem II reaction center. FEBS Lett. 458, 87^92.

[26] Porebski, S., Grant Bailey, L. and Baum, B.R. (1997) Modi¢cation of a CTAB DNA extraction protocol for plants containing high polysaccharide and polyphenol components. Plant Mol. Biol. Rep. 15, 8^ 15.

[27] Henze, K., Badr, A., Wettern, M., Cer¡, R. and Martin, W. (1955) A nuclear gene of eubacterial origin in Euglena gracilis re£ects cryptic endosymbioses during protist evolution. Proc. Natl. Acad. Sci. USA 92, 9122^9126.

[28] Valverde, F., Losada, M. and Serrano, A. (1997) Functional complementation of an Escherichia coli gap mutant supports and amphibolic role for NAD(P)-dependent glyceraldehyde-3-phosphate dehydrogenase of Synechocystis sp. strain PCC 6803. J. Bacteriol. 179, 4513^ 4522.

[29] Valverde, F., Losada, M. and Serrano, A. (1999) Engineering a central metabolic pathway: glycolysis with no net phosphorylation in an Escherichia coli gap mutant complemented with a plant Gap N gene.

FEBS Lett. 449, 153^158.

[30] Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F. and Higgins, D.G. (1997) The ClustalX windows interface: £exible strategies for multiple sequence alignment aided by quality analysis tools.

Nucleic Acids Res. 24, 4876^4882.

[31] Pe¤rez-Castin‹eira, J.R., Lo¤pez-Marque¤s, R.L., Losada, M. and Serrano, A. (2001) A thermostable K+-stimulated vacuolar-type pyrophosphatase from hyperthermophilic bacterium Thermotoga maritima.

FEBS Lett. 24819, 1^6.

[32] Jukes, T.H and Cantor, C.R. (1969) Evolution of protein molecules.

In: Mammalian Protein Metabolism, Vol. 3, (Nunro, H.N. Ed.), pp.

21^132. Academic Press, New York.

[33] Kimura, M. (1980) A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J. Mol. Evol. 16, 111^120.

Cyaan Magenta Geel Zwart

A. Sittenfeld et al. / FEMS Microbiology Ecology 42 (2002) 151^161 [34] Van de Peer, Y., Neefs, J.-M., De Rijk, P. and De Wachter, R. (1993) Reconstructing evolution from eukaryotic small ribosomal subunit RNA sequences: calibration of the molecular clock. J. Mol. Evol.

37, 221^232.

[35] Van de Peer, Y. and De Wachter, R. (1994) TREECON for Windows: a software package for the construction and drawing of evolutionary trees for the Microsoft Windows environment. Comput.

Appl. Biosci. 10, 569^570.

[36] Xia, X. (1997). DAMBE ver. 4.0.39 for Windows.

[37] Van den Hoek, C., Mann, D.G. and Jahns, H.M. (1993) Algae: An

FEMSEC 1403 17-9-02

161

Introduction to Phycology, 627 pp. Cambridge University Press, Cambridge.

[38] Inoue, Y. (1996) Photosynthetic thermoluminescence as a single probe of photosystem II electron transport. In: Biophysical Techniques in Photosynthesis (Amesz, J. and Of, A.J., Eds.), pp. 93^ 105. Kluwer Academic Publishers, Dordrecht.

[39] MuÂŤllner, A.N., Angeler, D.G., Samuel, R., Linton, E.W. and Triemer, R. (2001) Phylogenetic analysis of phagotrophic and osmotrophic euglenoids by using the nuclear 18S rDNA sequence. Int. J. Syst. Evol. Microbiol. 51, 783^791.

Cyaan Magenta Geel Zwart