Hydrobiologia 435: 83–90, 2000.© 2000Kluwer Academic Publishers. Printed in the Netherlands.

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A eukaryotic alga from picoplankton of Lake Baikal: morphology,ultrastructure and rDNA sequence data

Olga I. Belykh, Ekaterina A. Semenova, Konstantin D. Kuznedelov, Elena I. Zaika &Nina E. GuselnikovaLimnological Institute of Siberian Branch of Russian Academy of Sciences, P.O. Box 4199, 664033 Irkutsk, RussiaE-mail: belykh@lin.irk.ru

Received 10 May 1999; in revised form 14 October 1999; accepted 28 May 2000

Key words:Lake Baikal, picoplankton,Choricystis minor, morphology, ultrastructure, rDNA sequence data

Abstract

An eukaryotic alga (strain BAC 9708) from the picoplankton of Lake Baikal (Eastern Siberia) has been cultivatedin order to investigate its morphology, ultrastructure, growth requirements, pigment composition and nuclear-encoded 18S and chloroplast-encoded 16S rDNA sequences. Cells of strain BAC 9708 contain a nucleus, a singlechloroplast without a pyrenoid, one mitochondrion, a poorly visible dictyosome, a large vacuole and a cell wall withan outer sporopollenin-containing layer. Ellipsoidal cells, 1.5–2.0× 1.0–1.5µm in size, propagate usually by two,or infrequently by four autospores. Sequence analysis of nuclear-encoded rDNA showed that all 581 nucleotides ofthe 5′-end of the 18S rDNA were identical to that ofChoricystis minor(Skuja) Fott (Chlorococcales, Chlorophyta)obtained earlier (Krienitz et al., 1996). The chloroplast-encoded rDNA sequence of this strain showed identitywith one of three chloroplast sequences, selected by PCR-amplification with 16S rDNA specific primers from totalpicoplanktonic DNA, obtained previously.

Introduction

During the long-term study of phytoplankton in LakeBaikal, extremely high values of primary produc-tion to biomass ratio were found, leading severalauthors to hypothesize the existence of planktonic or-ganisms, which play an important role in primaryproduction but were undetected by the standard set-tling method (Votintsev et al., 1963, 1975; Antipova,1963; Glazunov & Kozhova, 1966).

A more detailed analysis of Baikalian phytoplank-ton allowed Popovskaya (1968) to discover picoplank-ton containing a great diversity of algal species andto describe a new cyanobacteria,Synechocystis lim-neticaPopovsk. measuring of 1.5–1.6µm in diameteras dominant picoplanktonic species. To date, inform-ation regarding the species composition of Baikalianpicoplankton are limited to the species described byPopovskaya (Popovskaya, 1968).

Recently, epifluorescence microscopy has been ap-plied to study the autotrophic picoplankton in South-

ern Baikal. Unicellular cyanobacteria generally dom-inate the picoplankton community, although uniden-tified picoplankton that fluoresce red under blue ex-citation (eukaryotic green or prochlorophytic algae)contributed up to 40% of the total autotrophic pico-plankton at certain times (Nagata et al., 1994).

More concrete data on the species variety ofBaikalian phytopicoplankton were obtained by com-parative analysis of nucleotide sequences of rDNAfragments isolated from total picoplanktonic DNA.The sequences identified from those in the EMBLdatabase belonged to four cyanobacteria and three taxaof green algae (Semenova & Kuznedelov, 1998).

Obviously, the latter approach provides us withthe possibility to infer the closest sequence alreadytaxonomically determined. But we have no real (non-inferential) information on the morphological charac-teristics of the organisms. The present paper describesa green picoplanktonic alga isolated from Lake Baikalin terms of morphology, ultrastructure, growth re-quirements and pigment composition, as well as the

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determination of the taxonomic position of the algaby partial nucleotide sequence data from nuclear- andchloroplast-encoded small subunit ribosomal DNA.

Materials and methods

Field samples were collected from the offshore watersin the southern sector of Lake Baikal. Water sampleswere passed through 3-µm Nuclepore filters. Indi-vidual picoplankton cells were isolated by the agarstreak method using the solid medium Z8 (Rippka,1988). Agar plate colonies were microscopically ex-amined to select cells with sizes less than 2µm forstreaking onto agar again.

Experimental cultures were grown in liquid me-dium Z8 or Gromova 6 (Gromov, 1984). Vitamin B12(1 mg/l) and thiamin (5 mg/l), and soil extract stock(mix 1 part air-dried, sieved soil with 2 parts distilledwater, 10 ml/l) were added to study the vitamin re-quirements and heterotrophic capabilities. The cellswere incubated without shaking at 24◦C and illu-minated with cool-white fluorescent light of differentintensity (500–2000 lux) at the surface of the culture,on a 16:8 h light:dark cycle. Cells were examinedwith an Olympus BH2 microscope equipped with anepifluorescence illumination system. For autofluores-cence, a 470 nm (blue) excitation filter was used. Thepresence of sporopollenin in the cell wall was testedby acetolysis in a mixture of sulphuric and acetic acids(Atkinson et al., 1972).

For scanning electron microscopy (SEM), cellswere fixed with 1% glutaraldehyde in 0.1 M caco-dylate buffer (pH 7.2) at 4◦C. They were placed onto a0.2-µm Nuclepore filter and dehydrated in an ethanolseries. After critical point drying, the cells were coatedwith gold and observed with Philips 525 M SEM.

For transmission electron microscopy (TEM), thecultured algae were fixed with 1% glutaraldehyde and0.25% OsO4 in 0.1 M cacodylate buffer for 30 minat 4 ◦C, washed twice and postfixed in 1% OsO4 for1 h at 4◦C. After washing in the buffer, the sampleswere dehydrated in an ethanol series and embedded inEpon. Thin sections were stained with uranyl acetateand lead citrate and examined using a Jeol 100B TEM.

Chlorophylls were analyzed at the end of the ex-ponential growth phase when the cell density reachedapproximately 105 cells ml−1. The presence of chloro-phylls was tested by the fluorescence emission spec-trum of the acetonic crude extract with a Hitachi650-10S fluorometer. The excitation wavelength was

adjusted at 4200–470 nm and compared to the knownstandard spectra of different chlorophylls. Chloro-phylls a and b were measured using hot ethanol asan extraction solvent, as described by Jespersen &Christoffersen (1987) with a Lomo SF-46 spectropho-tometer.

Total DNA extraction was performed accordingto Schmidt et al. (1991). The polymerase chain re-action (PCR) was performed in 20-µl reactions con-taining 10 mmol/l TRIS–HCl (pH 8.9); 2.5 mmol/lMgCl2; 40 mmol/l KCl; 0.1 mg/ml BSA; 0.2 mmol/lof each dGTP, dATP, dTTP, dCTP; 1µmol/l of eachprimer; 1–20 ng total DNA; 1 U Taq polymerase.Amplification was carried out with 35 cycles of de-naturation at 94◦C for 60 s, annealing at 55◦Cfor 70 s, and extension at 72◦C for 2 min. Twoprimers, 5′-TACCTGGTTGATCCTGCCAGTA-3′ &5′-ATTACCGCGGCTGCTGGCACC-3′ (Kuznedelovet al., 1996) homologous to nucleotide positions 1–22 and 630–610, respectively, of the human 18SrDNA, were used for amplification of the target rDNAfragment. Two other primers specific to nucleotide po-sitions 50–68 (5′-AACACATGCAAGTCGAACG-3′ -forward primer) and 536–519 (5′-GWATTACCGCG-GCKGCTG-3′ - reverse primer) ofE. coli 16S rDNA,and used earlier by Schmidt et al. (1991), were usedfor amplification of the chloroplast-encoded rDNAfragment.

The PCR-amplified sequences were purified by gelelectrophoresis and recovered as described by Gyllen-sten (1989). The resulting DNA fragments were sub-jected to direct sequencing by the dideoxynucleotide-terminated Sanger’s method according to Murray(1989). In addition to PCR primers, the internal primer5′-GTTTCTCAGGCTCCCTCTC-3′ (Kuznedelov &Timoshkin, 1993) was used in the sequencing reac-tions.

For analysis of the sequence data obtained, weused NCBI sequence searching services such as theBLASTN (Altschul et al., 1990) program that allowscomparisons of DNA sequences against the nucleotidesequence databases from GenBank, EMBL and DDBJ.

Results

Light microscopy

The strain BAC 9708 has a bright grass-green col-our. The vegetative cells are ellipsoidal with roundedends, 1.5–2.0× 1.0–1.5µm in size. The algae propag-ate mostly by two, or infrequently four autospores of

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equal size, developing into ellipsoidal autosporangia.Under blue excitation, red autofluorescence typical ofgreen algae was noted in the peripheral area of thecytoplasm due to the chloroplast of the cell. The pres-ence of sporopollenin in the cell wall was detected bythe resistance to acetolysis.

Electron microscopy

SEM preparations of strain BAC 9708 showed cellsto have a smooth surface (Figure 1). Mainly, al-gae were represented by vegetative cells. Sometimescells at different stages of division were observed.Mostly, the cells were divided in two (Figure 2).Under TEM the ultrastructure of vegetative cells, auto-spores and autosporangia was studied. Details of theultrastructure of cells are shown in Figures 3 and4. Cell organization is very simple. Each cell con-tains a single parietal chloroplast without a pyrenoid.The cup-shaped chloroplast generally contains one ormore starch granules (Figure 5), which sometimes arelarge. The lamellae are composed of bundles of 3–5 thylakoid sacs. The large nucleus (0.8–1.0µm indiameter) is located in the lateral part of the cell nearthe plasma membrane. Often a nucleolus is observedin the center of the nucleus (Figure 4). The mitochon-drion, one per cell, is approximately 0.5–0.7µm wide.It is usually located in the center of the cell, betweenthe chloroplast and the nucleus, so that the membraneof the nucleus is close to the membrane of the mi-tochondrion. Dictyosomes are poorly visible in thecells. Vacuoles are present in all cells (Figures 3–5)and contain homogeneous electron-transparent mater-ial. There are electron-dense round bodies in some ofthe vacuoles (Figure 6). Chloroplast, nucleus and va-cuole occupy a major portion of the cell; the remainderis filled by many ribosomes. The cell wall is strictlyregular. The outer part was represented by a so-calledtrilaminar layer (TL-layer) composed of two dark lay-ers with an electron-transparent layer between them(Figure 7). The inner region is an electron-dense mi-crofibrillar cellulose layer, 10–15 nm in width. Ageingresulted in an irregular thickening of the envelope, upto 100 nm in some cases. Many cells were observedat different stages of division (Figures 8–10). Thecells multiply by autospore formation during whichtwo daughter cells are formed within the mother cellwall. The mother cell wall does not form part of thewall of the autospores. Before the protoplast division,the chloroplast elongates, and then divides into two

chloroplasts. Autospores are of equal size, ellipsoidalwith parietal chloroplasts, mitochondrion and nucleus.

Chlorophylls and growth characteristics

The algae contain chlorophyllsa andb. The ratio ofchlorophyll b/a is 0.49. To test for the presence ofa chlorophyllc-like pigment, the crude extract wasanalyzed by fluorescence excitation at 440 nm. Thechlorophyllc-like pigment can be detected by its fluor-escence emission at 630 nm, which is not quenched byother pigments. In our case, the spectrum showed onlychlorophyllsa andb emission without any chlorophyllc-like emission. The algae do not require vitamin B12,thiamin or organic supplementary nutrients for theirgrowth. The Baikalian strain BAC 9708 grows well atlow to middle light intensity.

Molecular data

To determine the taxonomic position of the algae stud-ied, we applied nucleotide sequence comparison inthe rDNA locus encoding the 5′-end domain of thesmall subunit (SSU) ribosomal RNA, covering thehighly variable regions V1, V2 and V3 (Neefs et al.,1993). One sequence, obtained with primers specificto eukaryotic SSU rDNA and consisted of 581 read-able nucleotide positions, has been deposited in theEMBL/GenBank/DDBJ nucleotide sequence databaseunder the accession number Y15431. The sequencewas identical to the target region of the 18S rDNAsequence ofChoricystis minor(Krienitz et al., 1996;the EMBL database accession number: X89012). An-other sequence was amplified with primers specificto eubacterial SSU rDNA, contained 316 readablenucleotide positions, and was identified as one ofthree unidentified green algal chloroplast sequences(the EMBL database accession number: AJ011163)earlier obtained from Baikal picoplankton bulk DNA(Semenova & Kuznedelov, 1998).

Discussion

The picoplanktonic alga described in the present workshould be classified as a coccoid chlorophyte withautosporulation. The pigment composition, exclud-ing prasinophycean attribution by the lack ofc-likechlorophyll, showed a chlorophycean pattern withchlorophyll b/a ratio close to that ofNanochlorumeucaryotumand Chlorella fusca(Wilhelm & Wild ,1982), and slightly less than that of marine green

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Figures 1–2.Scanning electron microscopy of the cells of strain BAC 9708. (1) Ellipsoidal cells with smooth surfaces on a 0.2-µm Nucleporefilter. (2) Cells dividing into two autospores.Figures 3–4. Transmission electron microscopy. General organization of cells of strain BAC 9708.(3) Thin-section showing the nucleus (N), nucleolus (Nc), vacuoles (V), chloroplast (Ch); starch grain (S), mitochondrion (M). Note remnantsof the mother cell wall (arrows). (4) Vegetative cell with thickened microfibrillar cellulose layer. Plasmalemma have an irregular line (asterisks).

picoplanktonic algae (Hooks et al., 1988). TEM-observations of our strain demonstrated a minimal setof the organelles with their location typical for uni-cellular picoplanktonic chlorococcalean algae such asChlorella nana(Andreoli et al., 1978; Thinh & Grif-fiths, 1985),Nanochlorum eucaryotum(Wilhelm etal., 1982) andNannochloris-like algae (Sarokin &Carpenter, 1982; Brown & Elfman, 1983; Menzel &Wild, 1989).

On the basis of the ultrastructural examination ofNannochloris-like algae isolated from marine habitats,Sarokin & Carpenter (1982) discussed the invalid-ity of the generic nameNannochlorisfor them. ThegenusNannochlorishas historically been character-ized by Naumann (1921) as a genus with a simplebinary division into two equal halves and classifiedas a member of the Ulotrichales. However, the nameNannochloriswas used both for definition and de-scription of green coccoid algae in which autospor-

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Figures 5–6.Transmission electron microscopy (5) Vegetative cell with a large vacuole and cup-shaped chloroplast containing six starchgranules. (6) Cells with electron-dense bodies in the vacuoles.

ulation in pure cultures was found (Brown & Elf-man, 1983; Turner & Gowen, 1984), and for othersmall eukaryotes in a natural population of pico-plankton (Johnson & Sieburth, 1982). Ultrastructuralinvestigations ofNannochloris bacillarisNaumann,an iconotype species of this genus, clearly demon-strated the propagation of this species by binary di-vision (Shimada et al., 1993; Ogawa et al., 1995).Identification of coccoid picoplanktonic algae is veryproblematic with traditional methods. Krienitz et al.(1996), in order to determine the taxonomic positionof Nannochloris-like algae, performed a study of thecultured algae and field material using LM, TEM,and molecular methods. Cells of field samples fromLake ‘Große Fuchskuhle’, two strains isolated fromthis lake andChoricystis minor(Skuja) Fott (=Nanno-chloris coccoidesNaumann, strain SAG 251-1) storedin the Algensammlung Göttingen, Germany were mor-phologically similar toNannochloris-like algae. Theresults of 18S rRNA gene sequences of three strainsshowed that they belong to one species,Choricystisminor (Skuja).

The Baikalian chlorophyte is similar in the mor-phology, ultrastructure, mode of reproduction toChoricystis minor, strain SAG 251-1 described byMenzel & Wild (1989) asNannochloris coccoides.Strain SAG 251-1 grows well, as does our strain, ininorganic medium without vitamins at low to middlelight intensity. The cell shape, the absence of a pyren-oid in the chloroplast and, in particular, the construc-tion of the cell wall are features which characterizeC.

minor and our Baikalian isolate. The cell wall struc-ture of C. minor and of our alga differs from thatobserved in marineNannochloris-isolates (Sarokin &Carpenter, 1982; Menzel & Wild, 1989). The cell wallof freshwater species has, in addition to an outer tri-laminar layer, an inner microfibrillar layer consistingof cellulose and closely associated with the TL-layer.The width of the inner layer is 30 nm inC. minor,strain SAG 251-1, in BAC 9708 it is 100 nm. In con-trast, marineNannochloris-algae have only a separatetrilaminar ‘dark-light-dark’ layer. However, there aresome differences in the ultrastructural details betweenthe Baikalian isolate andC. minor, strain SAG 251-1.Cells of our alga always have well-expressed vacuoles,considerably larger in size than those ofC. minorandother picoplanktonic chlorococcalean algae. In addi-tion, cell vacuoles of strain BAC 9708 are often filledwith electron-dense material and in the vacuoles ofsome cells there was a dark body. Similar bodies havebeen described by Sarokin & Carpenter (1982) in one(C1) of five strains of marineNannochloris-like al-gae. Strain C1 displayed a large electron-dense bodylocated near the chloroplast or associated with the tri-laminar cell wall that may be involved in the synthesisof new sporopollenin-containing cell wall.

The partial 18S rRNA gene sequence of strainBAC 9708 from Lake Baikal was identical to thatof Choricystis minor(Krienitz et al., 1996) withinall 581 base positions used in the comparison ana-lysis. Closely related species usually have at leasta few mutations within this part of the 18S rRNA

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Figures 7–10.Transmission electron microscopy (7) Autospore showing the cell wall and associated trilaminar (sporopollenin) layer of themother cell (arrow). Note the microfibrillar layer (asterisks) located between the plasmalemma and TL-layer of the cell wall. (8–10) Divisionstages of strain BAC 9708. (8) Cell in an early division stage. Autospores within the mother cell wall. (9) Two daughter cells still surroundedby the parental trilaminar wall. (10) Two daughter cells still in contact after division.

gene (coding the highly variable regions V1, V2 andV3). For instance, there are six differences in the 18SrRNA gene sequence ofChlorella sorokinianain thisarea compared with closely relatedChlorella vulgaris(Huss et al., 1989). Krienitz et al. (1996) concludedthat the threeChoricystisstrains investigated shouldbe regarded as conspecific on the basis that there wasnot a single mutation in the highly variable regionsV2, V4, V7 (and V9) of the 18S rDNA. There isevidence that the partial sequence data are usually suf-ficient for species identification based on phylogenetic

analysis. However, for more reliable species identi-fication, the divergence extent among closely relatedspecies should be estimated. By now, there are notenough sequence data to tell about the divergence ex-tent by this locus among different species of the genusChoricystis. Hence, our strain can be considered asbelonging to the genusChoricystisand might be con-specific withC. minor. However, it is necessary toobtain some additional data to ensure the conspecifityof our alga andChoricystis minor, by comparing, forexample, more variable genes.

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The genusChoricystis(Skuja) Fott includes threepicoplanktonic species:C. minor (Skuja) Fott, C.hindakii (Tell) and C. coccoides(Rodhe et Skuja)Fott (Hindak, 1984). The most characteristic featuresof the genusChoricystis sensuFott are the prevalentformation of two autospores, the absence of a pyren-oid, and the absence of any mucilage; all species areplanktonic, free-living species (Fott, 1976). The cellshape of strain BAC 9708 from Baikal is ellipsoidallike cells ofC. hindakiiTell, described by Tell (1979)from the Danube River and distinct fromC. minorwith asymmetrical cells, having one side concave, theother side convex (Fott, 1976). As described above,the cell shape of the picoplanktonic alga studied hereis similar toC. minor(Krienitz et al., 1996) from theculture collection and is characterized by lack of thekidney-like shape in the culture such as seen forC.hindakii in field samples. The cell size of our algais close to that ofC. coccoides(Tell, 1979) and lessthan otherChoricystis-species. In field samples fromLake ‘Große Fuchskuhle’, a high abundance ofC.minorwas observed: its cells are ellipsoidal or slightlykidney-shaped with rounded ends, 1.5–3.5× 1.0–2.5µm in size. In contrast, high abundance of cells mor-phologically similar to the ones studied here is notseen in Lake Baikal, making considerable difficultiesfor their description in field samples.

The partial 16S rRNA gene sequence from thechloroplast was identical to one of three types ofchloroplast sequences obtained from Baikalian pico-plankton bulk DNA with eubacteria specific primers(Semenova & Kuznedelov, 1998). So, using com-parative chloroplast sequence analysis, the taxonomicstatus of one unidentified green alga was determinedto the genus level.

In conclusion, the alga isolated from Lake Baikalis characterized here by morphology, ultrastructure,pigment composition and rRNA gene sequences andhas been determined to be a species belonging tothe genusChoricystis. This alga has characterist-ics typical of the genusChoricystis, described bySkuja (1948) and Fott (1976). Sequence analysis ofthe Baikalian algae showed partial 18S rRNA genesequence identical toC. minor. Morphology and ul-trastructure of the Baikalian isolate are similar toC.minor (Skuya) (Krienitz et al., 1996). However, thereare some morphological, ultrastructural and ecologicaldifferences between the Baikalian isolate and this spe-cies, that does not allowed to identify our strain asC.minor.Representatives of the genusChoricystisare themost common species of green picoplanktonic algae in

many freshwater ecosystems, but undescribed in LakeBaikal until now. In the latest species list of Baikalianphytoplankton there is no genusChoricystis(Bondar-enko, 1995). The genusChoricystisis new for LakeBaikal.

Acknowledgements

The present study has been supported in part by a grantfor young scientists from the Siberian Branch of Rus-sian Academy of Sciences and by grant 97-04-96178-Baikal from the Russian Foundation for FundamentalResearch.

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