Chlamydomonas pitschmannii Ettl, a Little Known Species ... · ORIGINAL PAPER Chlamydomonas...

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Protist, Vol. 156, 287—302, August 2005

1

Correspondinfax 39 081 450e-mail anpollio

& 2005 Elsevdoi:10.1016/j

elsevier.de/protis
http://www.Published online date 03 August 2005
ORIGINAL PAPER

Chlamydomonas pitschmannii Ettl, a Little KnownSpecies from Thermoacidic Environments

Antonino Pollioa,1, Paola Cennamob, Claudia Cinigliaa, Mario De Stefanoc, Gabriele Pintoa,and Volker A.R. Hussd

aDipartimento di Biologia Vegetale dell’Universita di Napoli "Federico II", Via Foria 223, 80139 Napoli, ItalybFacolta di Lettere dell’Universita degli Studi ‘‘Suor Orsola Benincasa’’, Via S. Caterina da Siena, 80135Napoli, Italy

cDipartimento di Scienze Ambientali, Seconda Universita di Napoli, Via Vivaldi, 81100 Caserta, ItalydLehrstuhl fur Molekulare Pflanzenphysiologie der Universitat, Staudtstrasse 5, 91058 Erlangen, Germany

Submitted November 8, 2004; Accepted April 24, 2005Monitoring Editor: Robert A. Andersen

Three Chlamydomonas strains were isolated from the soils of a hot spring located in the Campi FlegreiCaldera (Naples, Italy). Ecophysiological, morpho-cytological and molecular features were used tocharacterize these isolates and to compare them with chlamydomonax acidophila strains from algalculture collections. The strains were collected from three points of the volcanic site, differing in theirphysico-chemical conditions. Among the examined Chlamydomonas strains, only the isolates from CampiFlegrei could grow optimally at pH values r3.0. These isolates also showed a high tolerance to desiccationand high temperatures, not evidenced by the other Chlamydomonas strains included in the study. 18SrDNA phylogeny indicates that the isolates from Campi Flegrei are closely related to Chlamydomonaspitschmannii and two strains isolated in Canada and Europe, that have been designated asChlamydomonas acidophila. A Chlamydomonas acidophila strain isolated from the type locality in Japanis less closely related according to its molecular phylogeny, and can also be discerned by light and electronmicroscopy. Moreover, vegetative cells and sporangia of Chlamydomonas acidophila from Japan showed amedian trilaminar structure not observed in the other strains. Our results show that Chlamydomonaspitschmannii could represent a hitherto unknown extremophilic Chlamydomonas species.& 2005 Elsevier GmbH. All rights reserved.

Key words: Chlamydomonas pitschmannii; Chlamydomonas acidophila; thermoacidic environments; 18SrRNA.

Introduction

Autotrophic flagellates belonging to chrysophytes,euglenophytes, and chlorophytes dominate thephytoplankton communities of acidic waters at

g author;[email protected] (A. Pollio)

ier GmbH. All rights reserved..protis.2005.04.004

pHo3.0 (Lessmann et al. 2000). The occurrenceof Chlamydomonas in these extreme habitats haslong been known (Lackey 1939), and two speciesof Chlamydomonas inhabiting very low pH envir-onments have so far been erected.

Pascher (1930) described the species Chlamy-domonas sphagnophila which occurs in very

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ARTICLE IN PRESS288 A. Pollio et al.

acidic humic bog waters of the Erzgebirge(Germany). However, no authentic culture of thisspecies is available; only one strain labeled as C.sphagnophila is maintained in culture (CCAP 11/31, isolated by Neish in Canada). This strain hasnever been studied in detail, and its attribution toC. sphagnophila needs further investigation. An-other strain (SAG 56.72 ¼ UTEX 293 ¼ CCAP 11/57) previously classified as C. sphagnophila hasbeen assigned by Ettl & Schlosser to Chlorococ-cum elkhartiense Archibald & Bold (Starr andZeikus 1993). The CCAP collection also maintainsfour strains labeled as Chlamydomonas sphagno-phila var. dysosmos which were obtained by R.Lewin (Neilson et al. 1972) from mutations of strainUTEX 2399 ( ¼ SAG 11/36a). This strain, formerlyidentified as C. dysosmos Moewus, was subse-quently reclassified as C. applanata Pringsheim(Starr and Zeikus 1993). The attribution of thesefour strains to C. sphagnophila has not beenrecently re-examined but little confidence can beplaced on their specific identification. Therefore,presently C. sphagnophila seems to be a poorlydistinguished species and it is not clear whether itshould be regarded as a synonym of Chlorococ-cum elkhartiense.

The other acid tolerant Chlamydomonas is C.acidophila Negoro (1944), isolated from thevolcanic lake Katanuma, Japan, at pH 1.7 and at211C. Again, an authentic strain is not available,but the presence of this species in acidic sites ismore firmly established. Fott and McCarthy (1964)isolated C. acidophila from artificial water bodiesoriginating from peat removal, where the pHreached values as low as 1.0. This strain ispresently maintained in two algal collections(UTCC strain 354 ¼ CCAP 11/136). Later, Har-greaves et al. (1975) in a survey of the vegetationof highly acidic streams of England, reported thecommon occurrence of C. acidophila (sub C.applanata var. acidophila), but no strain from thesesites is presently held in culture. Twiss (1990)isolated three Chlamydomonas strains from acidicsoils of Sudbury, Canada, which he assigned to C.acidophila (UTCC 121, 122, and 123). Morerecently, Nishikawa and Tominaga (2001) studiedthe ultrastructure and metal tolerance of a C.acidophila strain (OU 030/a; Okanomizu Univer-sity, Tokyo) isolated from the type locality of thisspecies.

During the exploration of the Pisciarelli hotsprings, an extremely acidic site located in thehydrothermal system of Campi Flegrei Caldera,Italy, we isolated three Chlamydomonas strainsfrom three different microhabitats, the most

extreme one with a temperature of 501C and apH of 0.8. These isolates are most remarkablebecause among microalgae only Cyanidialesand diatoms are reported to live under suchextreme conditions, and no green algae haveever been recorded to tolerate such a combinationof high temperature and low pH (Ciniglia et al.2005).

Recently, Proschold et al. (2001) proposed amulti-method approach to study the taxonomyand phylogeny of Chlamydomonas. Adopting thismethod, light microscopy, life history, and 18SrDNA sequence analyses were used to identify theChlamydomonas isolates from Pisciarelli at thespecies level. A first microscopical examinationled us to exclude their assignment to C. sphagno-phila. Pascher (1930) described for this speciesvegetative cells up to 9mm wide�18 mm long,whereas the cells of our isolates were consider-ably smaller. To ascertain whether the Pisciarelliisolates belong to C. acidophila, the strains OU030/a, UTCC 121, and UTCC 354 of C. acidophila,originating from acidic sites of Japan, Canada,and the Czech Republic respectively, and the typestrain SAG 14.73 of C. pitschmannii were includedin this study (Table 1).

Results

Field Observations

The hydrothermal system of Pisciarelli is a smallarea (about 30� 10 m) formed by spring watersrising from a deep boiling aquifer. Hot vapors,mainly composed of H2S and CO2, feed thegeothermal waters (Valentino and Stanzione2003). The hot pools are concentrated in a smallarea of the site (Fig. 1A). Two rivulets from thepools cross the entire site and converge into asingle stream, running for 10 m (Fig. 1C) andending in a small pond (Fig. 1E), which is dryduring the summer season. For this study, the siteof Pisciarelli has been divided into three micrositescharacterized by different environmental condi-tions: (1) superficial layers of the rocks surround-ing the hot pools with a temperature of 25—701Cand a pH of 0.5—1.5 (microsite HR ¼ hot rocks;Fig. 1A); (2) soils found within about 10 m from thehot pools and 1 m from the pond with a tempera-ture of 18—651C and a pH of 0.5—3.5. These aresandy soils, irregularly covered by opal and/oralunite layers generated by the hydrothermalactivity (microsite SS ¼ sandy soils; Fig. 1C); and(3) soils along the border of the cold acidic pond

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Chlamydomonas pitschmannii from Extreme Environments 289

with a temperature of 20—221C and a pH of 1.5(microsite BP ¼ border of the pond; Fig. 1E). Thealgal distribution over the entire site was irregular;sulfur and silica depositions were intermingledwith dense local aggregations of algal cells, whichcould be macroscopically recognized by theirgreen-brown color. In samples from micrositeHR, Galdieria sulphuraria was the dominantspecies; whereas in all the samples from micro-sites SS and BP, Cyanidiales, diatoms, and greenalgae were found in the upper layer of thesubstratum (0—3 mm).

The superficial samples collected over the entiresite of Pisciarelli revealed an ubiquitous presenceof Chlamydomonas, although in the vicinity of thehot springs, only a few Chlamydomonas cells weredetected. Three Chlamydomonas strains wereisolated, one from each microsite, at very low pHvalues (1.0—1.5), and at temperatures rangingfrom 221C (borders of cold pond) to 481C (rocksaround the hot pool). X-ray microanalyses of thesamples collected from different micrositesshowed the same chemical elements for all themicrosites: oxygen, silica, sulfur, and aluminumwere the major chemical elements detected inmicrosites HR and SS; potassium and iron werealso present but in smaller percentages (Fig.1B,D). Higher amounts of iron were detected inmicrosite BP, even though there was less alumi-nium (Fig. 1F). Opale and, in minor amounts,alunite were the main minerals found across theentire site.

Chlamydomonas cells were always observed inassociation with other algae: at temperaturesabove 351C the dominant algal species belongedto Cyanidiales, and Chlamydomonas was a minorcomponent (o5% of the algal population). Algalsamples from microsite HR (rocks located up toabout 40 cm from the hot spring; 501C, pH 1.0)contained a peculiar algal population. Chlamydo-monas cells were rarely observed, while Cyani-diales accounted for not more than 15% of thealgal cells. About 80% of the population wascomposed of anomalous rounded cells, typically5—10mm in diameter, solitary, aflagellated, palegreen or colorless, and surrounded by a thick cellwall. Within the cells, only an irregular greenishbody was observed occupying about 1

8213 of the

cellular volume, which exhibited a weak fluores-cence, probably due to the presence of a reducedamount of chlorophyll. The rest of the cells had ahyaline appearance without any recognizablefeatures. It was not possible from the lightmicroscopic observations to attribute these cellsto Cyanidiales or to Chlamydomonas. Moreover,

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Figure 1. Details of the three sampling sites. A. The rocks around hot boiling pools (site HR). B. EDX analysisof microelements found in the sample shown in Fig. 1A. C. Sandy soil surrounding hot pools (site SS). D. EDXanalysis of microelements found in the sample shown in Fig. 1C. E. Border of the acidic pond (site BP). F. EDXanalysis of microelements found in the sample shown in Fig. 1E.

290 A. Pollio et al.

about half of these anomalous cells were notstained by neutral red, suggesting that they werevital. However, staining results were often ambig-uous. In an attempt to identify these anomalouscells, samples from the hot spring rocks weresuspended either in Allen (1959) medium at pH 1.5(the standard medium for Cyanidiales) or in

modified Bold Basal Medium (BBM; Nichols1973) at pH 3.0, and the development of cultureswas followed daily by light microscopy. Whengrown in Allen medium at pH 1.5 and at 351C,50% of the anomalous cells remained unchangedduring the course of the experiment (30 days),whereas the other half resumed a green appearance

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in about 15 days. All of these cells could beattributed to Cyanidiales, mainly to the genusGaldieria. On the other hand, anomalous cellsgrown in modified BBM at pH 3.0 remained in theiraltered state throughout the duration of the tests.These results seem to indicate that the anomalouscells should mainly belong to Galdieria, andthat Chlamydomonas cells play little part in thealgal population living on the rocks around the hotpool.

At lower temperatures Chlamydomonas grew inassociation with Pinnularia sp., which was themost widespread alga of microsites SS and BP,and formed easily recognizable macroscopicaggregations. Occasionally, some chlorococca-lean species such as ‘‘Chlorella’’ saccharophila(Kruger) Migula, Viridiella fridericiana Albertano etal. and Pseudococcomyxa simplex (Mainx) Fottwere also found in both microsites. In all thesamples collected, Chlamydomonas cells werenon-motile and had a rounded shape. India inkstaining did not indicate the presence of amucilaginous sheath. Sporangia containing 4—8autospores were frequently observed, whereas inall the samples, except those from microsite HR,phases of sexual reproduction were never ob-served. When samples were suspended in a liquidmedium, Chlamydomonas cells acquired flagellawithin 48 h.

Molecular Phylogenetic Analyses

The 18S rRNA gene sequences of all threePisciarelli isolates were identical and, togetherwith the morphological and ecophysiologicalfeatures listed in Tables 2 and 3, suggest that allisolates could belong to the same species.Phylogenetic analyses place the isolates with highbootstrap support next to the type strain SAG14.73 of Chlamydomonas pitschmannii, togetherwith strains UTCC 354 and 121, formerly classifiedas ‘‘C. acidophila’’ (Fig. 2). In contrast, strain OU030/a, which represents the "classic" C. acido-phila, is clearly distinct taking a more basalposition in the tree ancestral to the sister cladescontaining C. pitschmannii and Chlorococcumelkhartiense/Tetracystis aeria, although this posi-tion is supported only by low bootstrap values of53—55%. The branching order within the C.pitschmannii cluster is not consistent concerningthe relative position of UTCC strains 354 and 121.While NJ analyses cluster strain UTCC 121 with60% bootstrap support together with the typestrain of C. pitschmannii and the Pisciarelli isolatesto the exclusion of strain UTCC 354 (data not

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shown), MP and ML analyses suggest an oppositearrangement with bootstrap values of 81% and94% respectively (Fig. 2). The latter view is moreconsistent with the morphological features andthe optimum pH for growth (Tables 2 and 3). Therelatively large number of nucleotide substitutionsfound between the strains of the C. pitschmanniicluster might be caused by a ‘‘fast clock’’, as mostsubstitutions are unequally distributed among the18S rRNA gene and concentrated in two smallareas, the E21 region (V4) and helix 23 accordingto Neefs and De Wachter (1990). All acid-tolerantstrains investigated in this study are part of the‘‘Moewusii’’-Clade proposed by Proschold et al.(2001).

Morphological Characters

Based on the results of the molecular analyses,the three Chlamydomonas isolates from Pisciarelliwere compared with the authentic strain (SAG14.73) of C. pitschmannii and with strains UTCC121 and 354 of ‘‘C. acidophila’’, isolated fromacidic soils near Sudbury, Canada, and fromFrantiskovy Lazne, Czech Republic, as well aswith strain OU 030/a, isolated from KatanumaLake, Japan, the type locality of C. acidophila. Themost important morphological characters ob-served for all strains are summarized in Table 2.Relevant similarities were an ovoid cell shape withbroadly rounded ends (Fig. 3A,B) and having alarge, parietal chloroplast, occasionally incised atmargins (Fig. 3a). Only the Japanese C. acidophilastrain OU 030/a was different with a lanceolateshape and a considerably smaller size of cells andgametangia (Table 2; Fig. 4A,B). All strains had twoanterior contractile vacuoles, including strain OU030/a, which previously was described to haveonly one contractile vacuole (Nishikawa andTominaga 2001). Further common characteristicsincluded one lateral and median pyrenoid, and alaterally located stigma in the anterior part of thecell.

Asexual reproduction in all strains examinedoccurred by formation of four zoospores. Thesporangia were aflagellated and the cellulardivision was preceded by chloroplast division(Fig. 3C). The first protoplast division was long-itudinal, leading to a two-celled sporangium(Figs 3D, 4C,D). The second division occurredasynchronously in the two spores and was perpen-dicular to the first one (Figs 3E—G, 4E). Zoosporeswere liberated through an apical opening of theparental cell wall (Fig. 3H) in the strains fromPisciarelli, in the type strain of C. pitschmannii

ARTICLE IN PRESS

Figure 2. Phylogenetic position of acid-tolerant Chlamydomonas species based on nuclear-encoded 18SrRNA (SSU) gene sequences. Representatives of the ‘‘Moewusii’’-Clade, as well as of all other CW(clockwise-displaced basal bodies) clades within the Chlorophyceae (Proschold et al. 2001) were included asreference organisms, and rooted with DO (direct opposite basal bodies) representatives as outgroup. Strainsfor which SSU sequences were determined in this study are printed in bold. The tree topology is from a MLanalysis with settings corresponding to the TrN+I+G model chosen as best-fit model by Modeltest (Posadaand Crandall 1998). Numbers above branches are bootstrap values for MP and NJ analyses, respectively,computed with 1000 replicates. ML bootstrap values for 100 replicates were determined only for the‘‘Moewusii’’-Clade for reasons of computation time, and are indicated below branches. Only bootstrapvalues450% are shown.


(SAG 14-73), and in strains UTCC 121 and 354 of‘‘C. acidophila’’. The cell walls of these strains lacksporopollenin as shown by TEM analysis (Fig. 3I).A peculiar situation was observed in the sporangiaof C. acidophila OU 030/a. During the course ofsporangial maturation, autospores apparentlymove towards opposite ends within the sporan-gium, which is clearly stretched (Fig. 4E—H)

before completely bursting. This strain possessesa trilaminar cell wall containing sporopollenin(Fig. 4I)

To ascertain the presence of a sexual cycle,selected isolates were grown under N-deficiencyin a 16:8 h light-dark cycle; all isolates revealedsexual reproduction. Gametangia (Fig. 5A—C)formed 8—16 gametes, which were released

ARTICLE IN PRESS

Figure 3. Optical, SEM, and TEM micrographs of Chlamydomonas pitschmannii strain DBV 238. A.Vegetative cell. Scale bar: 5 mm. B. SEM micrograph of a vegetative cell. Scale bar: 5 mm. C. Early stage ofchloroplast division. Scale bar: 5mm. D. Two-celled sporangium. Scale bar: 5mm. E—G. Four-celledsporangia. In G, the cell wall is already partially lacerated and the autospores are tightly held together. Scalebars: E, 5 mm; F, G, 2 mm. H. Sporangium releasing autospores through an apical pore. Scale bar: 10 mm. I.TEM micrograph of cell wall. Scale bar: 0.1 mm.


through an apical opening of the cell wall (Fig. 5D)in all strains except OU 030/a, where the parentalcell wall was completely lysed (Fig. 5E). Sexualreproduction occurred by isogamy (Fig. 5F,G) andthe zygote was protected by a thick cell wall (Fig.5H,I).

Ecophysiological Features

Results concerning ecophysiological features ofChlamydomonas isolates and reference strainsare presented in Table 3. The tolerance to low pHwas different between strains. Strains UTCC 121and 354, as well as strain SAG 14.73 of C.

pitschmannii were unable to grow at pH lowerthan 2.0. The isolates 238, 239, and 292 fromPisciarelli were the only ones with an optimal pHfor growth lower than 3.0 and the highestresistance to acidic conditions. All strains wereable to grow heterotrophically. Optimal growthtemperature was 371C for the Pisciarelli isolatesand 261C for all other strains. Moderate thermo-tolerance up to 341C was observed for allreference strains, whereas the isolates 238, 239,and 292 from Pisciarelli showed an upper tem-perature limit of growth at 421C. Moreover, theywere unable to grow on nitrate as a nitrogensource.

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Figure 4. Optical, SEM, and TEM micrographs of Chlamydomonas acidophila strain OU 030/a. A. Vegetativecell. Scale bar: 5mm. B. SEM micrograph of a vegetative cell. Scale bar: 5mm. C,D. Two-celled sporangium.Scale bar: 5mm. E—H. Four-celled sporangia. Autospores progressively move towards opposite directionswithin the sporangium which is clearly stretched. Scale bars: E—G, 5 mm; H, 2mm. I. TEM micrograph of cellwall. The arrow indicates the sporopollenin layer. Scale bar: 0.1 mm.


The dehydration tests revealed a differenttolerance to desiccation between strains. About90% of the cells of the reference strains were notviable after 48 h of dehydration, while the isolates238, 239, and 292 were able to grow even after 5days of dehydration; thus, confirming their abilityto survive at extreme conditions.

Discussion

Environmental Factors

Pisciarelli is a heterogeneous environment inwhich spatial and temporal variations occur.Temperature is probably the most important factordetermining the distribution of microalgae in thissite, as was recently reported (Huss et al. 2002).However, nutrient availability can also play acrucial role: the emissions of the geothermal

system represent the main source of both CO2

and H2S (Chiodini et al. 2001), and are concen-trated mainly in subsite HR. Due to a considerableincrease in volcanic activity, the fluid emissions in2002 were much higher than in previous years(unpubl. results) and the pH of the soil of the entiresite stayed between 1.0 and 2.0. Values betweenpH 3.5 and 4.0 were recorded for the years 2000and 2001 in microsites SS and BP, thus allowingthe distribution of acid-tolerant chlorococcaleanspecies such as ‘‘Chlorella’’ saccharophila andViridiella fridericiana (Huss et al. 2002). Theoccurrence of a Chlamydomonas species in thesoils of the Pisciarelli hydrothermal system wasreported in the mid-1970s (Pinto and Taddei1976). However, subsequent investigations carriedout on this site over the following decades onlyoccasionally indicated the presence of Chlamy-domonas. The bloom observed during 2002seems to involve not only pH, but is the result of

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Figure 5. Sexual reproduction of Chlamydomonas pitschmannii strain DBV 238. Only in E, a gametangium ofChlamydomonas acidophila strain OU 030/a is shown. A—C. Maturation of gametangia. Scale bars: A,B,5 mm; C, 10 mm. D. Liberation of gametes from an apical pore of cell wall. Scale bar: 5 mm. E. Liberation ofgametes through laceration of the cell wall in Chlamydomonas acidophila OU 030/a. Scale bar: 10 mm. F,G.Fusing gametes. Scale bars: F, 5 mm; G, 10 mm. H. Maturing zygote. Scale bar: 5 mm. I. Mature zygote withthickened secondary cell wall. Scale bar: 5 mm.


a combination of several factors. The scarcity ofrainfall in this year seems to be another environ-mental constraint well tolerated by our Chlamy-domonas isolates, which has shown a significantresistance to dehydration stress in laboratoryexperiments. Moreover, the ability of Chlamydo-monas cells to adhere to soil particles which canbe transported by wind (McKenna-Neumann et al.1996) might account for the very fast colonizationof the entire site by this alga. The absence ofChlamydomonas in acid water streams of Pisciar-elli might be due to the reduced CO2 availabilitycompared to terrestrial or endolithic habitats,where CO2 can diffuse very quickly to the cells(Gross 2000).

Chlamydomonas in Pisciarelli seems to behaveas a highly specialized organism, which thriveswhen the environmental conditions become parti-cularly severe. Experiments have confirmed thatthe isolates of Pisciarelli are highly resistant toboth high temperature and low pH values, whiletheir pH optimum for growth of 2.0—2.5 iscomparable to that of a C. acidophila strainisolated by Hargreaves and Whitton (1976) from

an English acid stream. Other strains attributed tothis species tolerated pH values of 2.0, butshowed significant growth only at pH43.0 andgrew optimally at pH values around 6.0 (Cassin1974; Visviki and Palladino 2001). Other acido-philic chlamydomonads such as C. sphagnophila(Cassin 1974) and C. applanata (Visviki andSantikul 2000), as well as four strains of acid-ophilic snow algae belonging to Chloromonas(Hoham and Mohn 1985), did not grow atpHo3.0 and showed pH optima higher than 4.0.

This specialization has an adaptation cost:when the environmental conditions are less severein Pisciarelli, Chlamydomonas occupies only re-sidual, very narrow niches, mainly located insubsite BP. These findings confirm the predictionthat ‘‘prolonged selection for specialization leadsto increasingly narrow niche widths’’ (Kassen2002). Another distinctive feature of our isolatestaken from Pisciarelli is that they are unable togrow on nitrate. The same phenomenon has beenobserved in a Galdieria sulphuraria strain isolatedfrom the same site (Pinto et al. 2003) and could berelated to the presence of ammonium as the sole

ARTICLE IN PRESSChlamydomonas pitschmannii from Extreme Environments 297

source of nitrogen in the area (Chiodini et al.2001).

Morphology and Reproductive Features

The three Chlamydomonas strains isolated fromPisciarelli, as well as the strains UTCC 121 and354, are morphologically identical to the typestrain of C. pitschmannii when cultured in thesame growth medium. Their characters fit thedescription of C. pitschmannii by Ettl (1976a),whereas the C. acidophila strain OU 030/a fromthe type locality differs from the others by itsshape and smaller size. However, some uncer-tainty on the specific attribution of our isolates stillremains due to a few differences observed withdiagnosis and type illustrations of C. pitschmanniiEttl, 1976a,b). Noteworthy is the absence of thepapilla both in field and in cultured Pisciarellistrains, whereas Ettl (1976a) described the pre-sence of a large, squared papilla. However, thepapilla was not observed also in C. pitschmanniitype strain under our laboratory conditions.Further data such as the sequences of the InternalTranscribed Spacers (ITS) and cross experimentsbetween Pisciarelli isolates and the type strain ofC. pitschmannii could definitively clarify theirtaxonomical position.

In their natural environment, the Pisciarellistrains have shown different morphological char-acters, the cells being larger than those inlaboratory cultures, and aflagellate. Ettl (1976a)did not mention the presence of non-motile,ovoidal cells in field material. The absence offlagella in other species of Chlamydomonas isrelated to pH: e.g. C. applanata becomes non-motile at pH lower than 3.4 (Visviki and Santikul2000), but all available strains of C. acidophilawere motile even at pH 1.9 in liquid culture. On theother hand, non-motile cells were observed inageing laboratory cultures of both C. pitschmanniiand ‘‘C. acidophila’’ strains. The absence offlagella from field samples of Pisciarelli could bedue to the reduced water content of soils,although Twiss (1990) isolated C. acidophilasamples from acidic soils of Sudbury, Ontario,Canada, at pH lower than 3.9, but did not reportthe presence of non-motile cells in field material.In situ observations at Pisciarelli have only rarelyshown the presence of Chlamydomonas sporan-gia. Day-night cycles play a key role in sporangiaproduction: all our field observations were carriedout in late morning (10—11 am); whereas celldivision, formation of sporangia, and liberation ofautospores in Chlamydomonas might mainly

occur during the night, as suggested by McAteeret al. (1985).

According to Ettl (1988), three types of vegeta-tive division can be observed in the genusChlamydomonas: longitudinal, semi-transverse,and transverse. In the first case, a 901 rotation ofthe protoplast may occur before the first division(false transverse division). All strains included inthis study showed this feature, which is adistinctive character of the genus Chlamydomo-nas sensu Proschold et al. (2001), and also thechloroplast division followed the pattern de-scribed for other Chlamydomonas species (Ettl1976b).

Schlosser (1976) showed that different modesof zoospore release occur in the genus Chlamy-domonas. The sporangia of all strains, exceptstrain OU 030/a, liberate the zoospores throughthe opening of an apical hole in the cell wall. Thismode of zoospore release corresponds to Schlos-ser type B-C (1976). The sporangia of C. acid-ophila OU 030/a showed a thicker and roughcovering and the presence of a trilamellar layer inthe cell wall, not observed in the other Chlamy-domonas strains examined. The occurrence of atrilamellar layer in the cell walls of unicellular greenalgae is associated with the presence of spor-opollenin, which is thought to ensure stability andstrength in the cell wall (Atkinson et al. 1972;Brunner and Honegger 1985). It is well known thatduring zoosporogenesis, the sporangial cell wall ofChlamydomonas is hydrolyzed by vegetative lyticenzymes (Schlosser 1984). The presence of atrilamellar layer in the sporangial cell wall of C.acidophila OU 030/a might cause the differentpattern of zoospore liberation by complete dis-solution of the parental cell wall (Fig. 5E).

Negoro (1944) and Ettl (1976a) did not describethe sexual reproduction of C. acidophila and C.pitschmannii. The first evidence of sexual repro-duction in C. acidophila was reported by Rhodes(1981), who observed gametes, isogamy, andformation of a spiny zygote in laboratory testscarried out on isolates from acidic mines of Ohio,USA. Although the strains 238, 239, and 292 fromPisciarelli, like the authentic strain of C. pitsch-mannii (SAG 14.73), showed the same featureswhen grown under N deficiency, no phase ofsexual reproduction was observed in the field. Theoccurrence of zygospores among the few Chla-mydomonas cells found in the subsite HR isprobably connected to the ability of these restingstages to survive environmental stresses (VanWinkle-Swift and Rickoll 1997). This might indicatethat specific environmental factors govern sexual


reproduction in the field such as humidity, avail-able nutrients, photoperiod, and temperature, andthat only very extensive samplings carried outduring 24 h and in different seasons could shedlight on the life cycle of Chlamydomonas isolatesin their habitat.

Molecular Features

The morphological similarities of the Pisciarelliisolates with the type strain SAG 14.73 of C.pitschmannii and with two strains of C. ‘‘acid-ophila’’ (UTCC 354 and 121) are reflected also bythe phylogenetic analyses of their 18S rRNA genesequences, implying that they all could belong toC. pitschmannii (Fig. 2). In contrast, the "classic"C. acidophila strain OU 030/a has some differentmorphological features and is more distantlyrelated based on its 18S rRNA gene sequence.Chlamydomanas pitschmannii forms a sister cladeto that containing, among others, C. moewusii andChlorococcum elkhartiense UTEX 293, anotherstrain able to grow at low pH. Thus, all acid-tolerant Chlamydomonas/Chlorococcum strainsincluded in this study belong to the ‘‘Moewusii’’-Clade, one of several subclades proposed byProschold et al. (2001) for the Chlorophyceae. Anuncultured eukaryote (strain RT1n1), isolated fromthe Spanish river Rio Tinto with a pH of 2 (Amaral-Zettler et al. 2002), also belongs to the ‘‘Moewu-sii’’-Clade (Fig. 2). However, other species such asChlamydomonas applanata (Visviki and Santikul2000), which belongs to the ‘‘Polytoma’’ clade(Fig. 2), may also be tolerant to low pH suggestingthat the acquisition of acid-tolerance occurredmultiple times independently within green algae(e.g. Cassin 1974; Huss et al. 2002).

Conclusions

Our study has shown that the strains isolated fromPisciarelli can be tentatively assigned to Chlamy-domonas pitschmannii and that this species isable to thrive at low pH. The presence of C.pitschmannii in acidic environments was neverrecorded, and all Chlamydomonas strains so farisolated from acidic sites have been assigned toC. acidophila. The species C. pitschmannii waserected by Ettl (1976a) on a culture isolated fromBrezova, Czech Republic. According to the author,the alga grew on a Tribonema bed, near a waterspring. Ettl (1976a) did not mention whether it wasa thermal spring, although Brezova is very close toCarlovy Vary, the most important thermal locality

of Moravia. However, the authentic strain of C.pitschmannii (SAG 14.73) showed a remarkableacid tolerance (growth at pH 2.5), whereas it wassensitive to moderately high temperatures(4301C). The morphological characters of Chla-mydomonas strains from Pisciarelli are very similarnot only to the authentic strain of C. pitschmannii(SAG 14.73), but also to Canadian and Europeanisolates (strains UTCC 121 and 354) so farassigned to C. acidophila, and their attribution tothis species needs to be re-examined.

On the other hand, C. acidophila strain OU 030/a is clearly different from the other strains includedin this study, as shown by morphological andmolecular analyses. The authentic strain of C.acidophila Negoro is not available, but the strain030/a has been isolated from Katanuma Lake, thetype locality of C. acidophila. According toNishikawa and Tominaga (2001), temperatureand pH of the lake were the same as reportedby Negoro (1944); the main morphological char-acters of this isolate correspond to those de-scribed by Negoro (1944), although his descriptiondid not include all the characters used in moderntaxonomic treatments of Chlamydomonas spe-cies. On the other hand, a comparison with theiconotypus would be meaningless on account ofthe lack of details in the original drawing; thus, anew typification of this species with a moredetailed description and drawings is necessary.Moreover, it should be recalled that the ‘‘Moewu-sii’’ Chlamydomonas lineage, to whom both C.acidophila and C. pitschmannii belong, is notmonophyletic with C. reinhardtii, the proposedneotype of Chlamydomonas (Proschold et al.2001); for this reason, all members of the‘‘Moewusii’’ clade should be assigned to othergenera.

Strain OU 030/a should be examined in furtherdetail before being considered as a possibleformal authentic strain of C. acidophila and moreinformation is needed also on its geographicaldistribution, since its occurrence in acidic placesother than Katanuma lake is not clearly ascer-tained. However, according to Finlay (2002), thepresence of endemic microbial species is ques-tionable, and a more careful exploration of acidicsites might shed light on this point. At themoment, there is a problem of undersampling,and particularly for the Southern hemisphere, thegeographical records of acidophilic Chlamydomo-nas are scanty.

The results from this study suggest that C.acidophila and C. pitschmannii could be twodistinct species coexisting in the same habitats.


It is noteworthy that Hargreaves and Whitton(1976) observed in some acidic streams ofEngland the occurrence of two Chlamydomonaspopulations differing in their cell sizes; however,extensive samplings in different acidic environ-ments are needed to confirm this observation.

Methods

Sampling procedures: The sampling area includ-ing the three subsites defined in ‘‘Results’’ wasdivided by nylon strings into 140 numbered squaremeters. A random number table was used toselect 9 squares, three for each subsite. For eachselected square, three sampling points (5 cmdiameter) were chosen, where algae superficiallycovered more than 50% of the sampling point.Temperature was measured for each sample witha digital thermometer and the pH was measuredwith a portable pH-meter or with pH indicatorstrips. The soils were collected with a coresampler, which was driven to a depth of 2.5 cm.Each core was transferred into a vial and stored at41C. In the laboratory, the depth of each algal corewas measured, and two sections, respectively atthe top and bottom of the algal layers, were cutout, suspended in liquid medium, and examinedunder a light microscope.

Isolation of strains and morphological ob-servations: The medium used for the isolation ofthe strains and for the maintenance of stockcultures was designated as ABM. It is based on amodification of Bold Basal Medium (BBM) (Ni-chols 1973) obtained by replacing nitrate with(NH4)2SO4 as nitrogen source to give a finalconcentration of 250 mg/l. All natural samplescontaining Chlamydomonas cells were suspendedin ABM medium adjusted to pH 2.0 with a 7%H2SO4 solution, and were grown at 251C undercontinuous fluorescent light. At this pH, all greenalgae occurring in the samples, except Chlamy-domonas, did not grow. On the other hand, thetemperature chosen represented the limiting con-ditions for the growth of cyanidiales, which arethermophilic. After seven days, Chlamydomonasrepresented over 80% of the total algal populationof these cultures, and was separated from theother components by serial dilution. UnialgalChlamydomonas cultures were obtained within 1month. Then, axenic clones were obtained bystreaking agarized ABM with a drop of a liquidculture and subsequent aseptic isolation of theresulting colonies. The Chlamydomonas isolatesand reference strains used in this study are listed

in Table 1. The strains isolated from Pisciarelli aremaintained in the Algal Collection of the Depart-ment of Biological Sciences of the University‘‘Federico II’’ of Naples (DBV), and are availableon request. Moreover, two of these strains, DBV238 and 239, were also deposited in the Universityof Toronto Culture Collection as UTCC 611 and612 respectively.

Chlamydomonas cells were observed with aNikon Eclipse E800 microscope, equipped withNomarski interference optics, and morphometricmeasurements were made on 100 cells fromeach isolate by means of an image analysissoftware (LUCIA). The observations were madeat different stages of the life cycle. Sexualreproduction was induced as indicated byProschold et al. (2001): 5 ml of a dense cellsuspension was centrifuged, the supernatantdiscarded and the pellet washed two times in anitrogen-depleted culture medium beforebeing resuspended in either ABM or BBM med-ium. The suspension was incubated in ashallow watch glass, which was placed on a glasstriangle in a Petri dish and filled with 20 ml ofdistilled water to reduce evaporation. Sampleswere observed each day. Chloroplast morphologyof the different Chlamydomonas strains wasdetected by fixation according to Proschold etal. (2001): 500ml of algal suspension were fixedfor 30 min on ice with 50ml 2% OsO4, 20ml 25%glutaraldehyde, and 430ml BBM, and washedonce in culture medium. Cells were then observedwith Nomarsky interference optics. For SEMobservations, the specimens were fixed with 1%formalin, washed three times with distilledwater by centrifugation, dehydrated in a gradedalcohol series, and critical-point dried. Thesamples were then mounted on Aluminum stubs,sputter-coated with gold, and examined at anaccelerating voltage of 20 kV. For qualitative andquantitative microanalyses, aliquots of soil sam-ples from each core were fixed with the sameprocedure and collected on carbon-coated gra-phite stubs. The main chemical elements weredetected by using a quantitative microanalysissystem (ZAFPB). For TEM analyses, the algal cellswere first collected by centrifugation (2 000 rpm for5 min) and then suspended in 4% glutaraldehydebuffered with 0.2 M Na-cacodylate (pH 7.5, ratio1:1). The cells were fixed for 1 h at roomtemperature, washed three times with 0.1 M Na-cacodylate (pH 6.3), concentrated by centrifuga-tion, and embedded in 1.5% agar. The agar blockswere then cut into small pieces and immersedin a mixture of 0.2 M OsO4 and 2% potassium


ferrocyanide (1:1). The postfixation was performedovernight at 41C. Subsequently, the material waspelleted and washed three times with 0.1 M Na-cacodylate and placed in 4% uranyl acetateovernight at 41C. The blocks were then rinsedwith distilled water, dehydrated, and embedded inepoxy resin before examination.

Ecophysiological characterization: The algaewere grown in 100 ml Erlenmeyer flasks at23711C under continuous light with a totalirradiance of 100mE m�2 s�1 provided by daylightfluorescent Philips lamps (TLD 30w/55). The flaskswere placed on a plexiglas shaking apparatus. Allecophysiological tests were carried out in12� 120 mm test tubes containing 6 ml of ABMat pH 3.0, except for the tests on pH tolerance.For the heterotrophic growth tests, glucose wasadded to the medium to give a final concentrationof 2.5 mM. In the experiments on pH limits ofgrowth, H2SO4 was added in different amounts toABM medium to obtain final pH values of 0.5, 0.7,1.5, 3.0, 3.5, 5.0, 6.0, and 7.0. Each strain wasgrown in triplicate in 100 ml Pyrex Erlenmeyerflasks containing 50 ml of culture medium. The pHvalue was controlled daily in each flask using aMettler Seveneasy pH meter, and corrected iffound to have changed for more than 0.1 pH unitby adding a few drops of dilute solutions of H2SO4

or NaOH. The pH probe was sterilized with ethanoland washed several times with sterile distilledwater prior to taking pH readings. For growth testson nitrate as a sole source of nitrogen, algalcultures which had previously been centrifuged(5 000 rpm�10 min) and washed two times with anitrogen-free medium were inoculated in BBMmedium containing 3 mM NaNO3. In addition, acontrol for each Chlamydomonas strain containingonly ABM medium at pH 3.0 was performed. Aftersterilization by autoclaving, the test tubes wereinoculated with several drops of enrichmentcultures of each strain to be tested, to give 0.05units of absorbance at 550 nm corresponding toabout 100 000 cells ml�1. The growth of algae wasfollowed either as absorbance increase at 550 nmwith a Baush and Lomb spectronic 20 colorimeter,or by counting the cell number with a Burkerbloodcounting chamber. Growth experimentswere carried out in triplicate and the results wereevaluated on the basis of three tests. Specificgrowth rates were calculated for each individualtest tube by linear regression of logarithmic celldensity data obtained during the experiments. Theresults were evaluated on the average of all threetests and the relative standard error neverexceeded 5%.

Dehydration stress was induced by mild heatingaccording to Hsu and Hsu (1988). One milliliter ofeach Chlamydomonas culture in the exponentialphase of growth (500 000 cells ml�1) was collectedby centrifugation and dehydrated in a convectionoven at 401C for 1—7 days. A sample of each strainwas stained daily with the vital stain neutral red toestimate the number of dead cells. At the end of thetests, the dehydrated cells were resuspended at pH3.5 and the growth of cultures was followed asdescribed above. The percentages of viability forliving cells from an exponential growing culture andfrom cells killed in boiling water for 15 min were99.97% and 0.03% respectively.

DNA isolation, PCR and sequencing: Allstrains were harvested by centrifugation(6 000 rpm for 5 min) and total DNA extractedusing the DNeasy Plant Mini Kit (Qiagen). 18SrRNA genes were amplified by PCR. PCR condi-tions and sequencing primers were the same aspreviously described by Huss et al. (1999). PCRproducts were purified with the QIAquick PCRPurification Kit (Qiagen); both strands weredirectly sequenced using the BigDyeTM TerminatorCycle Sequencing Kit (PE-Applied Biosystems)and an ABI Prism 310 Genetic Analyzer (AppliedBiosystems). The 18S rRNA gene sequences ofChlorococcum elkhartiense UTEX 293, Chlamy-domonas acidophila OU 030/a, UTCC 354 andUTCC 121, and of Chlamydomonas pitschmanniiDBV 238, 239, and 292 were deposited in theEMBL database under the accession numbersAJ628976-82 respectively.

Phylogenetic analyses: 18S rRNA gene se-quences were aligned manually under considera-tion of their secondary structure (Huss and Sogin1990; Neefs and De Wachter 1990). Parts of the50- and 30- ends for which sequence informationwas not available for all strains, as well aspositions which could not be unambiguouslyaligned, were excluded from the data set, resultingin a total of 1700 positions that were used for theanalyses. Phylogenetic trees were inferred by theneighbor-joining (NJ), the maximum parsimony(MP), and the maximum likelihood (ML) methods.For all methods, heuristic bootstrap analyses(Felsenstein 1985) with 1 000 (NJ, MP) or 100(ML) replicates were conducted with the PAUPprogram package 4.0b10 (Swofford 2002). Theprogram Modeltest 3.06 (Posada and Crandall1998) was used to select the evolutionary modelthat fitted our data set best. The best-fit model TrNof Tamura and Nei (1993) was chosen byModeltest with a proportion of invariable sites (I)of 0.5893 and a gamma shape parameter (G) of


0.6326. This model (TrN+I+G) was used forinferring trees using the likelihood optimalitycriterion with ML. For NJ analyses, pairwisesequence similarities were converted with thesame model into evolutionary distances, startingtrees were obtained via neighbor-joining, and theTBR branch-swapping algorithm was selected. Inthe MP analyses, starting trees were obtained viarandom stepwise addition of taxa repeated 10times, gaps were treated as ‘‘fifth base’’, and TBRwas selected.

Acknowledgements

We would like to thank Dr. Kahoko Nishikawa forher generous gift of the strain OU 030/a ofChlamydomonas acidophila.

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Chlamydomonas pitschmannii Ettl, a Little Known Species ... · ORIGINAL PAPER Chlamydomonas...

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