Appendix B
Fietz et al. (submitted)

Journal of Phycology (awaiting editor´s decision after revision)

First record of Nannochloropsis limnetica (Eustigmatophyceae) in the autotrophic picoplankton from Lake Baikal

S. Fietz, W. Bleiß, D. Hepperle, H. Koppitz, L. Krienitz, and A. Nicklisch

Abstract

Three new strains of eukaryotic picoplankton, isolated from Lake Baikal, were identified as Nannochloropsis limnetica Krienitz, Hepperle, Stich & Weiler. To date, N. limnetica had only been detected in small German and North American inland water bodies. On determination of the 18S rDNA sequence, the three new strains were found to be identical to each other as well as to the type strain KR 1998/3 (GenBank Acc. No. AF251496). RAPD-PCR revealed that the genotypes were different, although the Baikalian eustigmatophycean strains were more similar to each other compared to the type strain KR 1998/3 from Germany. Ecophysiological differences were also evident between the new strains from Lake Baikal and the type strain from growth rate determinations. The morphological characteristics were similar to that of a previous description of the species. However, while the cells of Eustigmatophyceae usually propagate by autosporulation, in these newly detected species germination of single daughter cells from thick-walled cells were observed for the first time. Based on pigment analysis, the occurrence of Eustigmatophyceae in Lake Baikal was estimated. Eustigmatophyceae were established to be a common member of the phytoplankton community of this large oligotrophic Siberian lake and occurred throughout the year, even under the ice cover during winter. Moreover, they peaked during early summer and in the South Basin. Hence, the widely accepted opinion that Chlorophyceae solely comprise the eukaryotic picoplankton should be changed and consider the Eustigmatophyceae.

INTRODUCTION

The widespread occurrence of eukaryotic, autotrophic picoplankton in Lake Baikal was previously established using light and epifluorescence microscopy (Boraas et al. 1991, Nagata et al. 1994, Belykh and Sorokovikova 2003, Fietz and Nicklisch 2004). However, to date, identification of Baikalian eukaryotic picoplanktonic species is still very rare (Belykh et al. 2000) and the Chlorophyceae were suggested to dominate the eukaryotic picoplankton of Lake Baikal (Nagata et al. 1994, Semenova and Kuznedelov 1998, Belykh et al. 2000). A chlorophycean strain from Lake Baikal was identified as Choricystis minor (Skuja) Fott by 18S rDNA sequencing. (Belykh et al. 2000). Moreover, due to a high violaxanthin content and a very low Chla/violaxanthin ratio in Lake Baikal, one would expect Eustigmatophyceae to be present in the phytoplankton (Fietz and Nicklisch 2004), although none have as yet been described (Kozhova 1987, Bondarenko 1995, Kozhova and Izmest’eva 1998).

Eustigmatophycean picoplankters exhibit a simple morphology, but they have unique ultrastructural features and photosynthetic pigments (Andersen et al. 1998, Krienitz et al. 2000). To accurately determine the composition of eukaryotic picoplankton communities, Hooks et al. (1988) suggested that microscopic observations should generally be complemented by HPLC-aided pigment analysis. Eustigmatophyceae are well known from marine systems (Volkman et al. 1993, Gladu et al. 1995, Andersen et al. 1998, Lubián and Montero 1998, and others), but only a few studies have been carried out in freshwater (Krienitz et al. 2000, Phillips and Fawley 2000, Fawley et al. 2004). However, Krienitz et al. (2000) described a new species, Nannochloropsis limnetica Krienitz, Hepperle, Stich and Weiler, from a hypertrophic freshwater pond, which was also found in a polytrophic lake in Germany.

Here, we characterize three new eustigmatophycean strains isolated from Lake Baikal in regard to their morphology, growth rates, pigment composition, fluorescence and absorption characteristics. Genetic similarities of these strains were established by RAPD-PCR and nuclear encoded 18S rDNA sequence analyses. Finally, we estimated the regional and seasonal occurrence of eustigmatophycean picoplankton in Lake Baikal from pigment data.

METHODS

Sampling. Water samples were collected in the main basins of Lake Baikal (South, Centre and North) and in the Selenga Delta during cruises within the framework of the CONTINENT project in July 2001, March 2002, July 2002 and July 2003 (EVK2-CT-2000-0057). Water samples were collected from 0.5 m, 5 m, 10-20 m, 20-30 m and/or 45-85 m water depths at all three sites and on all aforementioned dates. Samples were also collected biweekly by members of the Scientific Research Institute of Biology, State University Irkutsk from May 2002 to June 2003 at 5 m depth, 2.8 km offshore from Bolshy Koti near the western shore of the South Basin.

Isolation of algae. Diluted subsamples (containing 50 particles with chlorophyll fluorescence per 50 µL) from water samples taken in March and July 2002 were streaked on 0.8 % agar plates containing mineral nutrient solution complemented with vitamins. Agar Noble (Difco Lab., Detroit, Michigan, USA) was rinsed twice with supra pure water. Then, a 4 % agar solution was prepared in supra pure water, autoclaved and combined with either MIIIKS or M99F nutrient solution at a ratio of 1:5. Both solutions are modifications of M III (Nicklisch 1992) and contained 0.5 mmol·L^-1 CaSO₄, 0.5 mmol·L^-1 CaCl₂, 0.25 mmol·L^-1 MgSO₄, 0.1 mmol·L^-1 KCl, 0.75 mmol·L^-1 HCl, 2 mmol·L^-1 NaHCO₃ as well as a trace element solution (Nicklisch 1999) added at a dilution of 1:1000. Additionally, MIIIKS contained 0.4 mmol·L^-1 Na₂SiO₃, 0.5 mmol·L^-1 NaNO₃, 0.05 mmol·L^-1 KH₂PO₄, 0.01 mmol·L^-1 FeCl₃ and 0.02 mmol·L^-1 Na₂EDTA, whereas M99F contained 0.3 mmol·L^-1 Na₂SiO₃, 0.2 mmol·L^-1 NaNO₃, 0.01 mmol·L^-1 KH₂PO₄, 0.002 mmol·L^-1 FeCl₃ and 0.004 mmol·L^-1 Na₂EDTA. In equilibrium with air, the pH was 8.3 ± 0.2. Finally, the vitamins cobalamin (1 µg·L^-1), biotin (1 µg·L^-1) and thiamine (100 µg·L^-1) were added to both solutions.

Twenty-five phytoplankton strains were isolated and stored in the culture collection of the Leibniz Institute of Freshwater Ecology and Inland Fisheries, Berlin, Germany. Strains were grown under saturating nutrient (i.e. in the aforementioned solutions) and light conditions at 15° C in semi-continuous cultures (cf. Nicklisch 1992). From those strains, the three eustigmatophycean ones will be discussed here in detail: (1) strain baik03 was isolated in March 2002 from a sample collected in the South basin (104.41 °E, 51.76 °N); (2) strain baik43 from a sample collected in July 2002 in the North basin (109.00 °E, 54.08 °N); and (3) strain baik85 from a sample collected in July 2002 in the South Basin.

Light and electron microscopy. Living and formaldehyde-fixed (0.5 %) samples of cultures were investigated under a Jenalumar Contrast light microscope (Carl Zeiss AG, Oberkochen, Germany) by means of bright field illumination, epifluorescence and differential interference contrast. Staining was performed with DAPI (4’,6-diamidino-2-phenyl-indol, Sigma-Aldrich Co., St. Louis, MO, USA) and Fluorescent Brightener 28 (Sigma-Aldrich Co., St. Louis, MO, USA). The Fluorescent Brightener, also named Calcofluor White (M2R powder from Polysciences or Blankophor BA from Bayer), is used as whitening agent and selectively binds to cellulose and chitin.

For transmission electron microscopy (TEM), ca. 1 mL cell suspensions were concentrated by centrifugation (10 min, 600 x g, laboratory centrifuge 203, Sigma-Aldrich Co., St. Louis, MO, USA) in 1.5 mL microcentrifuge tubes. Pellets were resuspended and fixed in freshly prepared primary fixative containing 2.5 % glutaraldehyde, 2.0 % paraformaldehyde and 3 mM CaCl₂ in 0.1 M Na-cacodylate buffer, pH 7.4 for 2 h at room temperature. The samples were centrifuged and the pellets were mixed with a small volume of melted 2.5 % agarose (Carl Roth GmbH & Co., Karlsruhe, Germany) in 0.1 M Na-cacodylate buffer and immediately chilled on ice. The solidified agarose was cut into ca. 1 mm³ blocks. These were rinsed three times for 20 min each with 4° C cold 0.1 M Na-cacodylate buffer, and subsequently fixed in Karnovsky's mixture of 1 % osmium tetroxide and 1.5% potassium hexacyanoferrate (II) in double distilled water for 2 h (Karnovsky 1971). The samples were then rinsed in cold Na-cacodylate buffer solution, post-stained with 1 % uranyl acetate in 0.05 M sodium maleate buffer solution, pH 5.2 for 1 h at 4° C, dehydrated in a graded ethanol series, infiltrated, and finally embedded in Spurr's epoxy resin and polymerized for 24 h at 70° C (Spurr 1969). Ultrathin sections were cut with a diamond knife using an Ultracut S microtome (Leica, Vienna, Austria). The sections were sampled on uncoated 300 mesh grids and post-stained with uranyl acetate and Reynold's lead citrate. They were viewed in a Zeiss EM 900 electron microscope (Carl Zeiss AG, Oberkochen, Germany).

Growth rates. Cultures were grown in 500 mL Erlenmeyer flasks filled with 100 mL suspension and sealed with aluminium foil caps and mixed by circular shaking (60 ± 10 rpm) in a thermostat-controlled water bath (± 0.5° C). Illumination was supplied from the bottom by “cool white” fluorescent tubes on a 12/12 hours light/dark cycle and with a scalar irradiance of 100 ± 5 µmol quanta·m^-2·s^-1. M99F medium with or without vitamins was used as nutrient solution.

Determination of growth rates was performed in semi-continuous cultures according to the turbidostat-principle (Nicklisch 1992). First, the cultures were acclimated to the preselected conditions of temperature and light for three generation times. Biomass was measured and the culture was diluted to a constant starting concentration every two to three days. Specific growth rates were calculated from dilution rates during several weeks (Nicklisch 1999). The reported specific growth rate (µ) is the natural logarithm of biomass increase (start biomass / end biomass) normalized to time (day^-1).

Before the dilutions, the absorbance was determined with a photometer (type1101M, Eppendorf Inc., Hamburg, Germany) at 436 nm in a cuvette of 5 cm path-length as an optical measure of biomass concentration. Furthermore, the fluorescence signs F_o and F_m (Krause and Weis 1991) were determined with a pulse amplitude modulated fluorometer (Xenon-PAM, Heinz Walz GmbH, Effeltrich, Germany) as measures of the physiological state. By maintaining the maximum biomass below 100 µg·L^-1 Chla during cultivation, self-shading and nutrient limitation could be excluded.

Pigment composition. 30 – 50 mL of algal cultures were filtered through 25 mm Whatman GF/F-filters. The filters were frozen at – 80° C and then freeze-dried. Pigments were extracted with dimethylformamide by vibration shaking at a frequency of 2000 min^-1 for 1.5 h with a supplement of glass beads (0.75 – 1 mm). The extract was centrifuged for 20 min at 2500 g in a cooled centrifuge at 4° C (Biofuge Fresco, Heraeus Instruments, Hanau, Germany). The separation, identification and quantification of pigments were carried out according to Woitke et al. (1994) with a Waters HPLC system (Waters, Millford, MA, USA) as described previously by Fietz and Nicklisch (2004).

Fluorescence and absorption characteristics. Fluorescence of dark-adapted cells (F_o-value) was measured with a Phyto-PAM (Phytoplankton Analyzer, Heinz Walz GmbH, Effeltrich, Germany) using a diode array emitting light at four different wavelengths (470, 535, 620 and 650 nm). In vivo absorption spectra were measured with a spectrophotometer (UV-2101 PC, Shimadzu Corp., Kyoto, Japan) equipped with an integrating sphere from 400 to 750 nm.

Genetic analyses. RAPDs: About 1 mg freeze-dried (vacuum evaporator Christ, Osterode, Germany, at –20° C) algae were homogenized for 3 min in liquid nitrogen at a frequency of 1500 min^-1 (swing–mill MM 2000, Retsch, Haan, Germany). Total DNA was extracted using the CTAB method described by Rogers and Bendich (1985). After adding 600 µL of pre-warmed (60° C) 2 x CTAB buffer and vortexing, the solution was incubated at 60° C for 30 min, then 1 volume chloroform / isoamylalcohol (24:1) was added, and the sample centrifuged at 10000 rpm for 10 min at room temperature. The upper phase was removed and the DNA was precipitated with cold isopropanol. After 10 min centrifuging at 10000 rpm at 4° C and washing with 70 % ethanol the pellet was resuspended in distilled water.

RAPD-PCR was carried out as described by Koppitz et al. (1999) in 50 µL volumes containing approximately 25 ng genomic DNA, 1 x Taq DNA polymerase buffer (Boehringer Mannheim GmbH, Mannheim, Germany), 3 mM magnesium acetate, 0.2 mM each of dATP, dCTP, dGTP and dTTP (Boehringer Mannheim GmbH, Mannheim, Germany), 20 ng primer ([GACA]₄ or M13: 5’-GAGGGTGGCGGTTCT) and 2.5 units of Taq DNA polymerase (Boehringer Mannheim GmbH, Mannheim, Germany). PCR amplifications were performed in a MJ Research-Multicycler PTC 200 for 40 cycles of 20 s denaturation at 93° C, 60 s annealing at 50° C and 20 s extension at 72° C, and a final extension step at 72° C for 6 min. Amplification products were separated in 1.4 % agarose gels using 1 x TAE buffer and detected by staining with ethidium bromide.

For data evaluation, band positions on the gels were determined visually and the fingerprint pattern were transformed into a binary character matrix with 1 for presence or 0 for absence of a band at a particular position in a lane. Genetic similarity (GS) was estimated according to Nei and Li (1979) as GS = 2n_xy / (n_x+n_y) in which n_x and n_y are the total numbers of bands in the lanes of the sample x and y, respectively, and n_xy is the number of bands shared by the two samples.

Sequencing: Genomic DNA was extracted from small algal pellets derived from liquid cultures using DynaBeads (Deutsche Dynal GmbH, Hamburg, Germany) following the manufacturer's protocol for plant species. The genomic 18S rDNA - ITS 1 - 5.8S rDNA - ITS 2 region was amplified in 25 µL PCR reactions using primers 18S-PCR-5'F (5'-CCgAATTCgTCgACAACCTggTTgATCCTgCCAgT-3', Hepperle unpubl.) and ITS-PCR-3'R (5'-CCCggggggATCCATATgCTTAAgTTC AgCgggT -3', Coleman et al. 1994) and puReTaq™ Ready-to-Go™PCR Beads (Amersham Biosciences, Freiburg, Germany) in a standard PCR protocol. At least two PCR reactions were pooled and DNA purified on GFX columns (Amersham Biosciences, Freiburg, Germany). Before sequencing on an ABI3100 prism sequencer (Applied Biosystems, Darmstadt, Germany), about 40 ng purified PCR product, 2 µL 0.8 pmol·µL sequencing primer, and Big Dye Terminator™ Version 3.1 (Applied Biosystems, Darmstadt, Germany) were cycle-sequenced in final volumes of 10 µL using a standard cycle sequencing protocol.

Sequencing reactions were purified by ethanol precipitation using a final concentration of 66 % EtOH, rinsed in 70 % EtOH and air-dried. The pellet was resuspended in 20 µL HiDi-formamid™ (Applied Biosystems, Darmstadt, Germany) and run on a 50 cm capillary using standard settings. Partial sequences were assembled and subjected to proof-reading using SeqAssem^© Version 01/2004 (Hepperle 2004a).

The obtained 18S rRNA gene sequences were compared with the Nannochloropsis limnetica 18S rDNA sequence (GenBank Acc. no. AF251496, Krienitz et al. 2000) using Align Version 01/2004 (Hepperle 2004b).

Occurrence in Lake Baikal. The determination of total phytoplankton and autotrophic picoplankton biomass was described in Fietz and Nicklisch (2004). Total nano- and microphytoplankton were counted according to the settling technique (Utermöhl 1958). Samples for cyanobacterial and eukaryotic autotrophic picoplankton (50 mL) were fixed with formaldehyde (0.7 % final concentration) and filtered through black Nuclepore^© polycarbonate filters (0.2 µm pore size). The filters were placed on a microscope slide, briefly dried and covered with a drop of fluorescence-free immersion oil and a coverslip. Nano- and microphytoplankton were enumerated using a light microscope and autotrophic picoplankton were counted using an epifluorescence microscope. Eukaryotic autotrophic picoplankton fluoresced deep red (>665 nm) when excited with blue or green light. Phycobilin-containing procaryotic autotrophic picoplankton were identified by their light-red autofluorescence (<665 nm) when excited with green light. Cell counts were converted to biovolume according to their size and geometric form.

	Figure 1. Light microscopic pictures (interference contrast) of new strains of Nannochloropsis and a chlorophycean picoplankton strain (baik90) isolated from Lake Baikal. (a) baik03 with giant cells, (b) baik85, (c) baik90. Scale bar, 10 µm.

Based on the pigment data set (n=89) of water samples collected in Lake Baikal in July 2001, Chla/marker pigment ratios were calculated by multiple linear regressions as described by Fietz and Nicklisch (2004). The ratios were checked by the CHEMTAX matrix factorisation program. These Chla/marker pigment ratios allow us to calculate the contribution of the distinct chemotaxonomic groups to the total Chla, and thereby to estimate the phytoplankton composition of the standing crop. The final regression equation was: total Chla = 2.43·Fuco+2.52·Chlb+ 1.11·Zea^*+5.57·Viola^*, where Fuco (fucoxanthin) was the marker pigment of Bacillariophyceae and Chrysophyceae, Chlb was the marker pigment of the Chlorophyceae, Zea^* was the marker pigment of cyanobacterial picoplankton, and Viola^* was the marker pigment of Eustigmatophyceae. Zea^* included only the cyanobacterial part of zeaxanthin, which was at least 94 % of the total zeaxanthin. Chlorophyceae might also contain low amounts of zeaxanthin (5.3 % of lutein, Nicklisch and Woitke 1999). Therefore, the calculated proportion of zeaxanthin attributed to the Chlorophyceae was subtracted from the total zeaxanthin of each sample. Similarly, the eustigmatophycean part of violaxanthin (Viola^*) was calculated as the difference between the total violaxanthin and the chlorophycean part. The chlorophycean part of violaxanthin was estimated as being 15 % of Chlb (Nicklisch and Woitke 1999).

	Figure 2. Epifluorescent microscopic pictures of (a) autofluorescence of chloroplasts, (b) DAPI-staining and (c) staining with Fluorescent Brightner 28 (Calcofluor White) – two cells show a stained cell wall and the others the red autofluorescence of chloroplasts. Scale bar, 10 µm

	Figure 3. Ultrastructure of the Nannochloropsis strains isolated from Lake Baikal. (a) TEM images of strain baik03, (b) strain baik03 with the chloroplast endoplasmatic reticulum connected to the nuclear envelope (arrow), (c) strain baik43 with a lamellate vesicle, (d) a cell with lipid droplets. Abbreviations: C – chloroplast, G – Golgi body, LV – lamellate vesicle, L –lipid droplets, M – mitochondrium, N – nucleus, PG – plastoglobulus, PY – pyrenoid-like bodies , cm – cell membrane, cw – cell wall, cwp – cell wall papilla. Scale bar, 1 µm.

	Figure 4. Strain baik43 cells with badly fixed thick cell walls (a and b) and with a germinating (assumed resting) cell (c). Abbreviations: B – bacteria cell. Scale bar, 1 µm

RESULTS

Morphology and ultrastructure.The eustigmatophycean cells in exponential growing cultures were spherical to oval with diameters of 1.5 – 6 µm (Fig. 1, a and b). Using autofluorescence and DAPIstaining, the cells were determined to contain 1 to 8 chloroplasts and 1 to 8 nuclei with numbers of nuclei correlating to cell volume (Fig. 2, a and b). Larger cells with a diameter of up to 10 µm were found, although seldom, which contained many chloroplasts and nuclei (Fig. 1a). In contrast to the chlorophycean picoplankter (baik90, Fig. 1c) small refractive, rod-like bodies were frequently visible in the eustigmatophycean cells (Fig. 1, a and b). All typical morphological characteristics of Nannochloropsis were found in the TEM preparations of the eustigmatophycean strains isolated from Lake Baikal: chloroplasts with up to four thylakoid double layers (Fig 3a), plastoglobuli (Fig. 3b), a chloroplast endoplasmatic reticulum connected to the nucleus envelope (Fig. 3b), lamellate vesicles and Golgi bodies (Fig. 3c), pyrenoid-like structures (Fig. 3a), lipid droplets (Fig. 3d) and cell wall papilla (Fig. 3a). A cell membrane was also well visible along with a cell wall of variable thickness (Fig. 3a). Additionally, filament-like structures were found on the outside of cell wall (Fig. 3, a and e).

Some cells were not stainable with DAPI after formaldehyde fixation, indicating a less permeable cell wall. Less permeable cell walls were also observed in TEM preparations in which the fine structures were badly preserved in some cells, indicating insufficient fixing (Fig. 4, a and b). These small cells with thick, less permeable walls were considered as resting stages, because single cells – instead of several autospores – germinated from them (Fig. 4c). The special characteristics of the cell wall of these cells was also demonstrated with the fluorochrome Fluorescence Brightener 28, which selectively binds to cellulose. Although cellulose is unique to the cell walls of Chlorophyta and Dinophyta, some cells (assumed resting stages) of our cultures were also stained by this fluorochrome (Fig. 2c). Additionally, some cell walls of chlorophycean picoplankton cultures (baik90), which should contain cellulose, were not stained by this fluorochrome. Taken together, it was therefore impossible to distinguish cells of chlorophycean from eustigmatophycean picoplankton using this staining technique.

	Figure 5. Specific growth rates of the new strains of Nannochloropsis limnetica at 10 and 15° in comparison to the reference strain KR 1998/3 and a chloro-phycean picoplankter (baik90). C.I.: confi-dence interval

Growth rates. The specific growth rates of the new isolated strains from Lake Baikal were 0.3 – 0.7 d^-1 at 10° or 15° C under conditions of nutrient and light saturation (Fig. 5). However, the Baikal strains (including the chlorophycean one) had these high rates only when the nutrient solution was supplemented with vitamins, whereas the strain KR1998/3 exhibited no difference in growth whether vitamins were supplied or not. At 15° C the strain KR1998/3 grew with a significantly higher rate than all Baikal strains (Fig. 5). The differences among the Baikalian eustigmatophycean strains as well as between the Baikalian eustigmatophycean strains and the chlorophycean one were not significant at 15° C (Fig. 5). The coefficient of variation was relatively high for the strain baik43 (25 % in contrast to 8-20 % for all other strains and growing temperatures). On the one hand, this was caused by variable amounts of assumed resting stages, with Fluorescence Brightener 28; on the other hand, the growth rate determination was also influenced by a variable tendency to form small aggregates, which influenced the optical biomass measurement.

At 10° C the strain baik03 had the highest growth rate compared to the other Baikalian eustigmatopyhcean strains and the chlorophycean picoplankter (baik90) (Fig. 5). The difference between baik03 and KR 1998/3 was not significant (Fig. 5). The changing growth abilities at both different applied temperatures (10° and 15° C) were well reflected by the Q10 (Fig. 5), which was calculated based on a linear extrapolation of growth rates to a difference of 10° C. The Q10 was very high for KR 1998/3, the German strain, and very low for the Baikalian baik03, which was isolated from the water under the ice in March.

Pigment composition. The pigment composition clearly demonstrated that the eustigmatopyhcean strains differed greatly from the other phytoplankton groups (Table 1). Eustigmatophyceae lack chlorophylls other than Chla and were therefore clearly distinguishable from Bacillariophyceae and Chlorophyceae. Also, Eustigmatophyceae were characterized by a very high violaxanthin content (Table 1), a pigment that is often related only to the Chlorophyceae. The vaucheriaxanthin content, including vaucheriaxanthin-like (esterified) pigments, was much lower than that of violaxanthin in Eustigmatophyceae (Table 1). Vaucheriaxanthin is characteristic for Eustigmatophyceae but occur also in Xanthophyceae (Table 1). However, Eustigmatophyceae were also easily distinguishable from Xanthophyceae by the high Chla/violaxanthin ratio and absence of diadinoxanthin in Eustigmatophyceae (Table 1).

Table 1. Pigment content as % per Chla of selected strains isolated from Lake Baikal in July and March 2002. For the Eustigmatophyceae pigment contents in exponential growing cultures are given as well as in batch cultures with high biomasses in parenthesis. Abbrev.: Ant – antheraxanthin, ß-C – ß-carotene, Can – canthaxanthin, Ddx – diadinoxanthin, Dia – diatoxanthin, Ech – echinenon, Fuc – fucoxanthin, Lut – lutein, Neo – neoxanthin, Vau – vaucheriaxanthin, Vio – violaxanthin, Zea – zeaxanthin.

Fluorescence and absorption spectra: Fluorescence (Fig. 6, a and b) and absorption (Fig. 6, c and d) characteristics confirmed that the eustigmatophycean photosynthetic system differed from that of Chlorophyceae and Bacillariophyceae. However, both characteristics were similar for Eustigmatophyceae and Xanthophyceae. The fluorescence excitation of the Eustigmatophyceae is characterized by its maximum at 470 nm (Fig. 6a). The fluorescence at 535 nm was also high and decreased strongly towards 620 nm and 650 nm (Fig. 6a). The shape of the absorption cross-section was similar to that of the Chlorophyceae but differed in the region around 475 nm and 630 nm (Fig. 6c). Hence, Eustigmatophyceae can be distinguished from other phytoplankton groups, such as Chlorophyceae, Bacillariophyceae and also cyanobacteria (including phycobilin containing picoplankton), by use of fluorescence probes or spectral measurements.

	Figure 6. Fluorescence and in vivo absorption cross section of different strains isolated from Lake Baikal; (A) Fluorescence of Bacillariophyceae (strains baik03, baik43 and baik85) and Chlorophyceae, (B) Fluorescence of Eustigmatophyceae and Xanthophyceae. (C) Absorption of (strains baik03, baik43 and baik85) Bacillariophyceae and Chlorophyceae, (D) Absorption of Eustigmatophyceae and Xanthophyceae. Results are means for 5 to 20 samples taken at different times during culturing.

Genetic analyses. RAPD-PCR determined that the eustigmatophycean strains (baik03, baik43, baik85) from Lake Baikal differed among one another and from the reference strain Nannochloropsis limnetica (German strain KR 1998/3) as well as from a macrophyte sample (Phragmites australis (Cav.) Trin. ex Steud.) from NE Germany (Fig. 7). The genetic similarities among the three new eustigmatophycean strains were about 55 % (Table 2), but a lower similarity of about 35 % was found between these Baikal strains and the German KR 1998/3strain (Table 2).

The determined 18S rDNA - ITS1 - 5.8S rDNA - ITS2 sequences for the three eustigmatophycean strains from Lake Baikal were identical to each other as well as to the 18S rDNA of the type strain Nannochloropsis limnetica (GenBank Acc. no. AF251496, Krienitz et al. 2000). Phylogenetic analyses were therefore not performed as resulting phylogenetic tree reconstructions from 18S rDNA data would be identical to those published by Krienitz et al. (2000). No comparable analysis of the ITS1 and ITS2 sequences with other Eustigmatophyceae was possible as no ITS sequences are currently deposited in GenBank.

Occurrence. The contribution of eukaryotic picoplankton biomass, which included Eustigmatophyceae, to the total phytoplankton, was highest in the South compared to the Centre, North and Selenga Delta (Table 3). Eustigmatophyceae also had significantly higher contribution to total Chla in samples taken from the South (15 %) compared to the North and Selenga Delta (9-10 %; Table 3). The contribution of eukaryotic picoplankton to total biomass or of Eustigmatophyceae to total Chla did not show significant differences in connection with depth within the upper 30 m (Table 3). At several sites, deep Chla maxima (45-85 m) were detected with a fluorescence probe. At those depths, the contribution of Eustigmatophyceae to the total Chla was very low (<5 %, Table 3).

These regional and vertical differences relate to the summer communities only, since samples were collected in July 2001, July 2002 and July 2003. From May 2002 to April 2003, weekly samples were taken 2.8 km offshore from Bolshye Koti, located at the western shore of the South basin. Here, the contribution of the Eustigmatophyceae to the total Chla was highest in summer (14 % from mid-July to August) and much lower in all other seasons (Table 3). The percentage contri-bution of eukaryotic picoplankton, studied as spot checks, appeared also to be highest from mid-July to August (Table 3). However, even under the ice, Eustigmatophyceae were present and contributed about 3 % to the total biomass and 2.5 % to the total Chla (Table 3). Nevertheless, considering the regional and seasonal data, the occurrence of the Eustigmatophyceae (here, its contribution to total Chla) was weakly, but significantly correlated to the temperature (r = 0.276, P < 0.01, n = 214, Spearman-Rho correlation).

Table 2. Matrix of similarity coefficients of three Nannochloropsis strains (baik03, baik43, baik85) from Lake Baikal, Nannochloropsis limnetica KR1998/3 from Germany and a sample of Phragmites australis from Germany derived from PCR fingerprinting analysis with primer (GACA)4 and M13 (see Figure 7).

	Figure 7. PCR fingerprints of Nanochloropsis strains, primed with (GACA). Lane1 and 7: 1 kb DNA-ladder, lane 2-4 from the left to the right: strains baik03, baik43 and baik85, lane5: KR1998/3, lane 6: Phragmites australis.

DISCUSSION

The determined 18S rDNA sequences were identical for all three Baikalian eustigmatophycean strains as well as to the previously published Nannochloropsis limnetica sequence (Krienitz et al. 2002). Therefore, we conclude that the Baikalian eustigmatophycean strains belong to the species Nannochloropsis limnetica Krienitz et al. (2001). However, the PCR clearly showed that the genotypes of these strains were not identical either among the strains isolated from Lake Baikal or between these strains and the German strain KR 1998/3. Nevertheless, the genotypes of the Baikalian eustigmatophycean strains were more similar to each other than to the reference strain KR 1998/3.

Interestingly, Nannochloropsis limnetica was identified in a hypertrophic pond and a polytrophic lake in Germany (Krienitz et al. 2000), in different lakes of North America (Fawley and Fawley 2004, Fawley et al. 2004) and in the oligotrophic to mesotrophic regions of Lake Baikal. However, analyses of the genotypic and ecophysiological differences between the strains established that distinct ecotypes exist. ´Ecotypes´ describe genetically determined differences between populations within a species that reflect local matches between the organisms and their environments (Turesson (1922) in Begon, Harper and Townsend 1996).

Fawley and Fawley (2004) and Fawley et al. (2004) detected seven different genotypes of Nannochloropsis in lakes of Arrowwood National Wildlife Refuge, North Dakota and Itasca State Park, Minnesota; one of them possessed 18S rDNA and rbcL sequences identical to those of N. limnetica from Europe and the other belong to new taxa. The new types varied in both 18S rDNA and rbcL sequences and some morphological characters that distinguish them from N. limnetica. No type could be further differentiated by the rbcL sequence alone. These findings support the phenomenon that even though eukaryotic picoplankton exhibit a uniformed coccoid morphology, i.e. ‘green balls’, an extraordinarily high genetic diversity is hidden both in marine (Potter et al. 1997) and in freshwater (Krienitz et al. 1999) ecosystems. One explanation for this, is an adaptative advantage of the coccoid morphology in the ecosystem (Potter et al. 1997).

Table 3. Regional and vertical (July 2001, July 2002 and July 2003) as well as seasonal variation (2002/ 2003) of A) temperature, B) contribution of eukaryotic autotrophic picoplankton, which included Eustigmato-phyceae, to the total phyto-plankton biomass (in %) and C) contribution of Eustig-matophyceae to total Chla (in %). Mean values with 95 % C.I. are listed where normal distributed data sets were considered, and otherwise median values with minimum and maximum were given.

The ecophysiological difference between the genotypes of N. limnetica was demonstrated by different (1) growth rates, (2) Q10 values and (3) vitamin demand. The growth rate at 15° C, for example, was significantly highest for the German strain KR 1998/3 which also did not require vitamins. The Q10 was also highest for KR 1998/3, whereas it was lowest for the strain baik03. The unusual low Q10 of baik03, which was isolated from below the ice in March, could mean that its temperature optimum is between 10° and 15° C. This is nearing that of Synechocystis limnetica Popovskaya, a widespread cyanobacterial picoplankter in Lake Baikal, which was determined to be 8° C (Richardson et al. 2000).

The existence of distinct ecotypes may explain the occurrence of the same Eustigmatophyceae species (Nannochloropsis limnetica) in all seasons and regions including the cold waters in the North basin and under the ice (Table 3). Nevertheless, the most important development occurred in the warmer South basin and during the early summer. This is in contrast to observations in North America where Nannochloropsis are more common during cold water periods (Fawley and Fawley 2004, Fawley et al. 2004).

The morphological characteristics of the new strains of Nannochloropsis limnetica correspond to former descriptions concerning form, size of cells and inclusions of refractive bodies (Krienitz et al. 2000). Additionally, giant cells with diameters of up to 10 µm were found in exponential growing cultures (Fig. 1a) as well as cells with thick cell walls (Fig. 4, a and b), which were stainable with Calcofluor White (Fig. 2c), but badly stainable with DAPI and badly preserved by fixation for electron microscopy. They were considered as resting stages since the germination of single cells were observed and a higher number of such cells was found during depressed growth. The ultrastructural analyses identified lamellate vesicles in the cytoplasma (Fig. 3c) and that the chloroplast endoplasmatic reticulum was connected to the nuclear envelope (Fig. 3b); both are common characteristics in all eustigmatophycean species (Santos and Leedale 1995, Krienitz et al. 2000, Suda et al. 2002). The chloroplasts contained up to four stacked thylakoids and plastoglobuli (Fig. 3) as described by Krienitz et al. (2000), and pyrenoid-like structures and cell wall papilla (Fig. 3a) were also found as reported for Nannochloropsis oceanica by Suda et al. (2002). The interpretation of the pyrenoid-like structure in our material is difficult because this structure is located outside of the chloroplast and exhibits similarities to some of the ‘electron-dense bodies’ found in N. granulata Karlson & Potter (Karlson et al. 1996). We identified filament-like structures on the outside of the cell wall (Fig. 3, a and e) that, to our knowledge, have never been previously described. Their function is not yet defined. They could be involved in the formation of cell aggregates as observed in our cultures or could serve for moving of cells out of its old envelopes (Fig. 4c). Assumed resting stages and germination of single daughter cells were observed for the first time in Nannochloropsis as well as generally for the Eustigmatophyceae. These stages are comparable with the aplanospores of the closely related Xanthophyceae (Ettl 1978) and the germination stages of the green alga Marvania geminata Hindák (Hindák 1976, Sluiman and Reymond 1987).

Although distinction between chlorophycean and eustigmatophycean picoplankton using the epifluorescence microscope is not possible, more sophisticated measurements which profit from differences in absorption and fluorescence spectra can determine this (Fig. 5). Additionally, due to exceptional high violaxanthin/Chla ratio and the occurrence of vaucheriaxanthin and its esters, pigment analysis also allows eustigmatophycean and chlorophycean cells to be distinguished from each other.

However, besides the Eustigmatophyceae, also Chlorophyceae, Xanthophyceae (Table 2) and a few Chrysophyceae (Mackay et al. 1996) contain violaxanthin, and Xanthophyceae also contain vaucheriaxanthin. Software algorithms, as used in the CHEMTAX matrix factorization, can also help to discriminate between chemotaxonomic groups, which contain the same marker pigment in different amounts (Mackey et al. 1996), but detailed knowledge of the phytoplankton composition and its pigmentation is essential (Fietz and Nicklisch 2004). In this study, we did not identify violaxanthin-containing Chrysophyceae, and thus, these violaxanthin-containing Chrysophyceae species might be of lesser importance in Lake Baikal. Additionally, Xanthophyceae were not detected in earlier microscopic analyses (Fietz and Nicklisch 2004). However, they are known to occur in Lake Baikal, but only in sors (locally “lagoon”) and river mouths (Kozhova 1987, Kozhova and Izmest’eva 1998). Thus, the estimation of the Eustigmatophyceae occurrence, which is based on the violaxanthin/Chla ratio, seems reasonable.

The most common prokaryotic autotrophic picoplankters in Lake Baikal belong to Synechocystis limnetica (Popovskaya 2000) and Synechococcus spp. (Belykh and Sorokovikova 2003), and the most common eukaryotic picoplanktersbelong to Choricystis minor (Belykh et al. 2000). Although abundant in marine systems (Andersen et al. 1998), Nannochloropsis has only been rarely found in freshwater systems (Krienitz et al. 2000); however, this could be because identification might need a combination of microscopic, HPLC-aided and preferably molecular analyses (Fawley et al. 2004). The wide range of habituation of picoplankton was already demonstrated for the chlorophyte Choricystis minor, which is common in Lake Baikal and also found in different German inland waters along a nutrient gradient from oligotrophic to hypertrophic state (Hepperle and Krienitz 2001). However, now, it is evident that eustigmatophycean picoplankton may also be commonly found in freshwaters.

The marine Nannochloropsis is grown as a food source because of its high content of polyunsaturated fatty acids, particularly eicosapentaenoic acid (Sukenik et al. 1993). The German type strain (Krienitz et al. 2000) as well as the Baikalian strains (unpublished data) of the freshwater Nannochloropsis limnetica are also rich in polyunsaturated fatty acids (about 25 % of total fatty acids). Nevertheless, the content of eicosapentaenoic acid was much lower for Baikalian strain baik03 (1.6 %, unpublished data) than in the German type strain (24 %, Krienitz et al. 2000); however, it was similarly high as reported for marine strains (Sukenik et al. 1993). In contrast to the German type strain (Krienitz et al. 2000, Wacker et al. 2002), docosahexaenoic acid was found in the Baikalian strain baik03 in similar high amounts as eicosapentaenoic acid (1.3 %, unpublished data). Nannochloropsis limnetica is therefore very important for the food-web quality and should be included in the phytoplankton community surveys of Lake Baikal

ACKNOWLEDGMENTS

Thanks are due to members of the Scientific Research Institute of Biology, State University Irkutsk as well as of the Limnological Institute of Irkutsk, Siberian Branch Russian Academy of Sciences for their help on cruises and seasonal monitoring. We acknowledge H. Oberhänsli for the organisation and permanent support of the CONTINENT project. We also thank M. Wirth for the preliminary determination of fatty acid composition.

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