chloroparva pannonica gen. et sp. nov. (trebouxiophyceae, chlorophyta) – a new picoplanktonic...

Chloroparva pannonica gen. et sp. nov. (Trebouxiophyceae, Chlorophyta) – a new

picoplanktonic green alga from a turbid, shallow soda pan

BOGLARKA SOMOGYI1*, TAMAS FELFOLDI

2, KATALIN SOLYMOSI3, JUDIT MAKK

2, ZALAN GABOR HOMONNAY2,

GYORGYI HORVATH4, ERIKA TURCSI

5, BELA BODDI3, KAROLY MARIALIGETI

2AND LAJOS VOROS

1

1Balaton Limnological Research Institute of the Hungarian Academy of Sciences, H – 8237, Tihany, Klebelsberg Kuno 3, Hungary2Department of Microbiology, Eotvos Lorand University, H – 1117, Budapest, Pazmany Peter 1/c, Hungary

3Department of Plant Anatomy, Eotvos Lorand University, H – 1117, Budapest, Pazmany Peter 1/c, Hungary4Department of Pharmacognosy, University of Pecs, Medical School, H – 7624, Pecs, Rokus 2, Hungary

5Department of Biochemistry and Medical Chemistry, University of Pecs, Medical School, H – 7624, Pecs, Szigeti 12, Hungary

SOMOGYI B., FELFOLDI T., SOLYMOSI K., MAKK J., HOMONNAY Z.G., HORVATH G., TURCSI E., BODDI B., MARIALIGETI

K. AND VOROS L. 2011. Chloroparva pannonica gen. et sp. nov. (Trebouxiophyceae, Chlorophyta) – a newpicoplanktonic green alga from a turbid, shallow soda pan. Phycologia 50: 1–10. DOI: 10.2216/10-08.1

We describe Chloroparva pannonica Somogyi, Felfoldi & Voros gen. et sp. nov., a new trebouxiophycean picoplanktonicalga isolated from a turbid, shallow soda pan in Hungary. The cells are spherical to oval, less than 2 mm in diameter,with simple ultrastructure typical to small green algae. Cells divide by autosporulation, forming two daughter cells perautosporangium. Cell wall structure consists of an outer trilaminar layer, an inner microfibrillar layer and an electron-transparent layer covering the plasma membrane. The trilaminar layer of the mother cell wall often persists around theautospores. Typical chlorophyte pigments have been found, including chlorophyll a and b and lutein as the dominantcarotenoid. The main fatty acid was oleic acid. The phylogenetic position of the new chlorophyte confirms the proposalof a new genus within the Trebouxiophyceae. Based on its 18S rRNA gene sequence, this isolate is distantly related toNannochloris eucaryotum UTEX 2502, Chlorella minutissima C-1.1.9 and C. minutissima SAG 1.80 (# 97.6% 18S rRNAgene pairwise similarities).

KEY WORDS: Chloroparva pannonica, New chlorophyte species, 18S rRNA gene, Picoplankton, Turbid soda pan

INTRODUCTION

Photoautotrophic picoplankton comprises small (, 2 mm)

prokaryotic picocyanobacteria and eukaryotic photo-

trophs. These small cells dominate photosynthetic biomass

and primary production in many marine ecosystems and

can also play an important role in freshwaters (Callieri

2008; Vaulot et al. 2008). Among freshwater ecosystems –

in some low transparent or turbid lakes/pans – extremely

high picoplankton abundances (106–108 cells ml21) and

contributions (90–100%) have been detected (Carrick &

Schelske 1997; Hepperle & Krienitz 2001; Voros et al. 2008;

Felfoldi et al. 2009; Somogyi et al. 2009). During the last

decades, the eukaryotic component of picophytoplankton

in freshwaters has received much less attention than

picocyanobacteria, presumably due to their characteristic

autumn–winter appearance in the temperate zone (Callieri

2008; Voros et al. 2009).

Picoeukaryotic algal species had been described formerly

based on morphological and biochemical characters. As

these tiny cells have limited morphological features, some

studies simply referred them to as ‘little green balls’,

‘Chlorophyta isolates’, ‘Chlorella-like cells’ or ‘Nanno-

chloris-like algae’ (Stockner 1991; Huss et al. 1999; Henley

et al. 2004; Krienitz et al. 2004). The ‘classical’ taxonomy

was based on the following criteria and methods: cell

morphology and ultrastructure, cell division pattern, cell

wall structure and composition and biochemical and

physiological characters (Huss et al. 1999; Henley et al.

2004; Krienitz et al. 2004). The introduction of phylogenetic

methods into their taxonomy based mainly on 18S rRNA

gene analysis, led to the upset of the original morphology-

based classification and clarifies that these small cells

presumably evolved by convergent evolution (Lewin et al.

2000; Callieri 2008). Nevertheless, the combination of

molecular and morphological approaches offers the most

favourable approach to understanding picophytoplankton

diversity (Callieri 2008).

The introduction of molecular methods into phycology

caused algologists to change their way of thinking as

particular algal strains instead of species would be the basic

taxonomical items in case of some groups. During the last

years, more and more algal taxa have been revised, and

many examples demonstrated that different algal strains

identified morphologically as the same species proved to

belong to different taxa based on phylogenetic analysis

(Huss et al. 1999; Krienitz et al. 1999; Fawley et al. 2004;

Henley et al. 2004; Krienitz et al. 2004). In the absence of

sufficient information, taxonomical status of some algal

strains remained uncertain as they could not be referred to

any existing taxa. One of these examples is the taxonomic

revision of ‘Nannochloris-like’ algae executed by Henley

et al. (2004) proposing the new genus Picochlorum Henley,

Hironaka, Guillou, M. Buchheim, J. Buchheim, M. Fawley

& K. Fawley for 13 marine or saline autosporing taxa and* Corresponding author ([email protected]).

Phycologia (2011) Volume 50 (1), 1–10 Published 7 January 2011

1

providing a new species diagnosis of Picochlorum oklaho-

mensis Hironaka. The ‘original, morphology-based’ Nanno-

chloris genus was restricted to Nannochloris bacillaris

Naumann and Chlorella sp. Yanaqocha RA1., while

Nannochloris eucaryotum (Wilhelm, Eisenbeis, Wild &

Zahn) Menzel & Wild UTEX 2502 and Chlorella minu-

tissima Fott & Novakova C-1.1.9 formed a problematic

sister group (branching deeper than the other isolates). At

this time, neither can be definitively assigned to any existing

genus (Henley et al. 2004).

Besides the study of Henley et al. (2004), several

taxonomic revisions could be cited, constructing monophy-

letic taxa within the phylum Chlorophyta; however, further

development needs more information by means of isolation

of new algal strains or application of culture independent

molecular phylogenetic techniques (Huss et al. 1999;

Krienitz et al. 1999; Fawley et al. 2004; Henley et al.

2004; Krienitz et al. 2004; Luo et al. 2010). Here we present

the formal description of a new picoeukaryotic algal

species, Chloroparva pannonica, isolated from Boddi-szek

pan (Hungary), whereby this study contributes to the

continuous advance of the molecular phylogenetics and

taxonomy of Trebouxiophyceae (Chlorophyta).

MATERIAL AND METHODS

Site description

Boddi-szek pan is a turbid soda pan located in the Danube-

Tisza Interfluve in Hungary (46u469N, 19u089E; for a

geographical map see Felfoldi et al. 2009), with a total

surface area of about 117 ha. The pan is an intermittent

shallow water body that frequently dries out entirely by the

end of the summer. The maximum water depth is less than

30 cm, and due to the high concentration of inorganic

suspended particles (up to 2900 mg l21) and dissolved

coloured (humic) substances, the Secchi-disk transparency

ranges only between 3 and 7 cm (Voros et al. 2006, 2008).

The pan has alkaline water, characterised by the dominance

of Na+ and HCO32 ions with pH values between 8.9 and 9.8

(Voros et al. 2006). The salinity varies between hypo-

and mesosaline ranges corresponding to the season and

water level with conductivity values between 2200 and

16,500 ms cm21 (Voros et al. 2006).

Isolation and culture methods

The picoeukaryotic strain ACT 0608 was isolated from the

water of Boddi-szek pan in December 2005. The isolation

was carried out on a modified brackish water medium as

previously described (Somogyi et al. 2009). Unialgal

cultures were established by serial streakings on 1.5% agar

plates and single colony isolations. For maintenance, the

cultures were transferred to modified BG11 medium, in

which only one tenth of the recommended micronutrient

solution was used (Rippka et al. 1979). Maintenance

cultures in liquid media were kept at 21uC and

100 mmol m22 s21 of cool-white fluorescent light (Tungs-

ram F33) on a 14:10 h light:dark cycle.

Microscopical methods

Living cells from both young and old cultures were

examined under Olympus BX51 microscope equipped with

differential interference or phase-contrast optics and

Olympus DP71 digital camera. For cell size calculations,

approximately 400 cells were manually measured using

Olympus CellD software. For scanning electron microscopy

(SEM), samples from a growing culture were fixed in

glutaraldehyde (5% in 0.1 M phosphate buffer) and filtered

onto 0.2 mm polycarbonate filter (Millipore). Filters were

dehydrated in acetone dilution series (30, 50, 70, 80, 90 and

100% twice), then critical point dried with liquid CO2 after

being infiltrated with amyl-acetate and finally coated with

gold. Cells were examined using HITACHI S-2600N

scanning electron microscope at an accelerating voltage of

20 kV. For transmission electron microscopy (TEM),

samples were fixed in 2.5% glutaraldehyde overnight at

4uC. For this, 3 ml of algal culture were mixed with a

fixative solution containing 250 ml glutaraldehyde (50%)

and 1.75 ml Na-K phosphate buffer (70 mM, pH 7.2). The

samples were harvested by centrifugation (4000 3 g, 5 min,

20uC), then the pellet was resuspended and mixed into

drops of solidifying agar solution (2%) according to

Solymosi et al. (2006). The solidified agar plates were cut

into small pieces and postfixed in 1% OsO4 for 2 h. Fixing

solutions were diluted in 70 mM Na-K phosphate buffer

(pH 7.2), and the same buffer was used for rinsing after

fixation steps. After dehydration in alcohol series, the

samples were embedded in Durcupan ACM epoxy resin

(Fluka Chemie AG). Ultrathin (70 and 50 nm) sections

were cut with a Reichert Jung ULTRACUT E microtome

(Reichert-Jung AG). The sections were stained with 5%

uranyl acetate dissolved in methanol for 5 min and treated

with Reynold’s lead citrate solution for 5 min. Cells were

investigated using Hitachi 7100 TEM at 75-kV accelerating

voltage.

Pigment analysis

For pigment analysis, the culture was grown at 25uC in a

14:10 h light:dark cycle under cold fluorescent illumination

(Tungsram F33) of about 60 mmol m22 s21. The isolation,

identification and quantification of chlorophyll a and b

were performed according to Mantoura & Llewellyn (1983).

Cells were harvested by centrifugation (15,000 3 g, 15 min),

and pigments were extracted in acetone helped with

ultrasonic homogenizator. After centrifugation (15,000 3

g, 10 min), chlorophyll a and b were analysed by high-

pressure liquid chromatography (HPLC), equipped with

fluorescence detector (Waters) on a 10-mm, 3.9 3 300-mm

inner-diameter (i.d.) mBondapack C18 reverse-phase column

[Waters system using solvent as 10% ion-pairing reagent

(Mantoura & Llewellyn 1983), 10% water and 80%

methanol (MeOH); flow rate: 1 ml min21]. The excitation

wavelength of the detector was 420 nm, and the emission

wavelength was 650 nm. The wet weight (biomass) of the

algal suspension was calculated on the basis of cell density

[determined by epifluorescence microscopy according to

MacIsaac & Stockner (1993)] and cell volumes assuming a

specific gravity of 1.0.

2 Phycologia, Vol. 50 (1), 2011

The isolation, identification and quantification of carot-

enoids were performed according to Haugan et al. (1995). A

lyophilised alga suspension was extracted three times with

MeOH and once with diethyl ether, then the methanolic

extracts were combined and transferred into the mixture of

toluene and hexane in a separatory funnel. After evapora-

tion of this solution by rotary evaporator, the residue was

dissolved in diethyl ether. The ethereal solutions were

combined, and the total extract was saponified with 30%

KOH/MeOH in heterogeneous phase (Molnar & Szabolcs

1979). The carotenoid composition was analysed by HPLC

on the basis of their UV/VIS spectroscopic properties

(gradient pump: Dionex P680; detector: Dionex PDA-100;

t 5 45 min; l 5 450 nm) on an end-capped C18 column (250

3 4.6-mm i.d.; Merck LiChrospher 100 RP-18; 5 mm) using

as solvents 12% H2O/MeOH (A), MeOH (B), 50% acetone/

MeOH (C) [gradient program: 100% A (0–2 min); 80% A

and 20% B (2–10 min); 50% A and 50% B (10–18 min); 100%

B (18–27 min); 100% C (27–34 min; 100% B (34–43 min);

100% A (43–56 min), flow rate: 1.250 ml min21]. To specify

the carotenoid composition, the total extract was distribut-

ed between MeOH:H2O (9:1) and hexane. The distribution

resulted in a hypophasic and epiphasic fraction (hypophasic

and epiphasic carotenoids; Molnar & Szabolcs 1979), which

were reanalysed separately by HPLC providing more strict

quantitative analysis. For the identification of pigments,

authentic reference samples were used.

Fatty acid analysis

For fatty acid analysis, the culture was grown at 8 and 21uCin a 14:10-h light:dark cycle under cold fluorescent

illumination (Tungsram F33) of about 60 mmol m22 s21.

Cells were harvested by centrifugation (15,000 3 g, 15 min)

in the stationary phase (after 3 weeks). Cellular fatty acids

were extracted, and methylation was performed according

to Stead et al. (1992). Nonadecanoic acid (19:0; Sigma-

Aldrich) was added as an internal standard. Fatty acid

methyl esters (FAME) were analysed by gas-liquid chro-

matography (HP 5890 GC). The separation of FAMEs was

performed using HP-1 dimethylpolysiloxane column, and

37-Component FAME Mix (Supelco) was used as quality

standard. Fatty acids were identified through comparison

with the retention times of FAME standards.

Phylogenetic analysis

Genomic DNA was extracted according to the procedure

described previously by Somogyi et al. (2009). Polymerase

chain reaction (PCR) amplification of the 18S rRNA gene

was performed with a final volume of 50 ml using

approximately 2 ml of genomic DNA, 0.2 mM of each

deoxynucleotide, 2 mM MgCl2, 1 U LC Taq DNA

polymerase (Fermentas), 13 PCR buffer (Fermentas),

0.325 mM of Euk328f and Euk329r primers (Table 1) and

400 ng of BSA (Fermentas). PCR amplicon was purified

with the PCR-MTM Clean Up System (Viogene). Sequenc-

ing was carried out with the BigDyeH Terminator v3.1

Cycle Sequencing Kit (Applied Biosystems) using the

primers listed in Table 1. Chromatograms were corrected

manually with Chromas 1.45 software (Technelysium Pty

Ltd). The generated sequences were compared to the

GenBank nucleotide database using the Blast program

(Altschul et al. 1997). The obtained 18S rRNA gene

sequence was submitted to GenBank under the accession

number FJ013257. Alignment of various trebouxiophycean

algal sequences was generated with ClustalW (Thompson et

al. 1994) and corrected manually using the MEGA4

software (Tamura et al. 2007). Phylogenetic analysis was

performed with the software PAUP* version 4.0b10

(Swofford 2002) and MrBayes version 3.1 (Huelsenbeck

& Ronquist 2001) using 1671 nucleotide positions. Likeli-

hood settings were calculated with Modeltest version 3.7

(Posada & Crandall 1998).

RESULTS

Chloroparva pannonica Somogyi, Felfoldi & Voros gen. et

sp. nov.

Cellulae virides, forma ab rotunda ad ovalis, 1–2.3 3 1.2–

2.6 mm diametro, in lacibus alcalius, semisalinis viventes.

Nucleus unicus, mitochondrius unicus, chloroplastus unicus

lateraliter positus sine pyrenoide, granulis amyli interdum

praesentibus. Inter pigmenta chloroplasti chlorophyllae a,b,

luteinum, violaxanthinum, noexanthinum et b-carotenum

adsunt. Flagella nulla. Paries cellulae tristratus cum strato

microfibrillaris internus. Reproductio asexualis autosporis in

Table 1. List of oligonucleotide primers used in this study.

Primer name Sequence Position1 Reference

Euk328f2 59-ACC TGG TTG ATC CTG CCA G-39 2–20 Moon-van der Staay et al. (2000)Euk329r2 59-TGA TCC TTC YGC AGG TTC AC-39 1797–1778 Moon-van der Staay et al. (2000)18S rRNA 59-PCR 1F3 59-ACC TGG TTG ATC CTG CCA GT-39 2–21 Yamamoto et al. (2003)18S rRNA 59-PCR 1R3 59-CGT AGG CYT GCT TTG AAC AC-39 786–767 Yamamoto et al. (2003)18S rRNA 59-PCR 2F3 59-CMA TTG GAG GGC AAG TCT GG-39 544–563 Yamamoto et al. (2003)18S rRNA 59-PCR 2R3 59-TAA GAA CGG CCA TGC ACC AC-39 1289–1270 Yamamoto et al. (2003)18S rRNA 59-PCR 3F3 59-AAG TTR GGG GMT CGA AGA CG-39 980–999 Yamamoto et al. (2003)18S rRNA 59-PCR 3R3 59-CCT TCY GCA GGT TCA CCT AC-39 1793–1774 Yamamoto et al. (2003)18S-1270F3 59-GTG GTG CAT GGC CGT TCT TA-39 1270–1289 this study18S-544R3 59-CCA GAC TTG CCC TCC AAT TG-39 563–544 this study

1 Annealing sites refer to nucleotide positions of the Chlorella vulgaris SAG 211-11b 18S rRNA gene sequence (X13688; Huss & Sogin1989).

2 For PCR amplification, thermal profile as described in Moon-van der Staay et al. (2000).3 For sequencing.

Somogyi et al.: Chloroparva pannonica gen. et sp. nov. 3

partes duas; reproductio sexualis ignota. 18S rRNA sequen-

tia genetica (FJ013257) demonstrat differentias a speciebus

ceteris Trebouxiophycearum.

Cells green, spherical to oval, with a diameter of 1–2.3 3

1.2–2.6 mm, growing in alkaline, soda waters. One nucleus,

one mitochondrion, one lateral chloroplast without pyre-

noid, starch grains sometimes present. Chloroplast pig-

ments comprise chlorophylls a and b, lutein, violaxanthin,

neoxanthin and b-carotene. Flagella absent. Cell wall

trilaminate, with an inner microfibrillar layer. Reproduc-

tion by autosporulation into two daughter cells. Sexual

reproduction unknown. Analysis of 18S rRNA gene

sequence (FJ013257) shows differences from sequences of

other trebouxiophycean species.

HOLOTYPE: A lyophilised specimen has been deposited in

the Algological Collection of the Hungarian Natural

History Museum (under BP 7964).

ISOTYPE: Live culture has been submitted to the SAG

Culture Collection of Algae (University of Gottingen)

under SAG 2358.

ICONOTYPE: Fig. 9.

TYPE LOCALITY: An alkaline soda pan in the Danube-Tisza

Interfluve, Hungary.

Morphology, ultrastructure and reproduction

The cell size ranged from 1 to 2.3 mm in width and from 1.2

to 2.6 mm in length, with an average size of 1.4 3 1.7 mm.

SEM observation showed that the cell surface was either

smooth or wrinkled, but the surface of the dividing cells was

always wrinkled (Figs 1, 2). TEM observation revealed that

the cell organization was very simple. There were no

flagella, no basal bodies detectable by electron microscopy.

Each cell had a single nucleus and a single, parietal, usually

cup-shaped and bilobed chloroplast, containing stacked

thylakoid lamellae composed typically of four to six

thylakoid sacs (Figs 3, 8). Often one or two electron-dense

plastoglobuli could be observed within the plastids (Fig. 3),

but starch grains were only rarely found under the

experimental conditions (i.e. in the relatively young algal

culture). A single elongated mitochondrion, with cristae,

lied in the central cytoplasm, closely associated with the

concavity of the chloroplast and also with the nucleus

(Figs 3, 5). Vacuoles were also observed that contained

either electron-transparent material or sometimes electron-

dense bodies (Figs 3, 9). Peroxisome was also found

(Fig. 5). Chloroplast, nucleus and vacuole occupy a major

portion of the cell; the remainder is filled by the cytoplasm

containing several ribosomes (Figs 3, 5).

The cell wall was strictly regular, consisted of electron-

dense and electron-transparent layers and had an average

thickness of 28–63 nm. An electron-transparent layer

covered the plasma membrane, and this layer was followed

by a denser, fibrillar and relatively thick layer (inner

microfibrillar sheet) above which a trilaminar sheath was

observed consisting of an electron-dense, an electron-

transparent and another, outermost electron-dense layer

(Figs 4, 6). No mucilaginous envelope was observed on the

outer surface of the cells (Figs 4, 6).

Cells divided by autosporulation, generally giving rise to

two daughter cells (Figs 7–10). The remnants of the mother

cell wall (usually the trilaminar layer) were often observed

loosely surrounding the daughter cells after autosporulation

(Fig. 10). The remnants of the grandmother cell wall were

occasionally found around the autospores outside the

mother cell wall (Fig. 9). After the release of the daughter

cells, a ghost-like, curling, empty wall was sometimes

observed. No evidence for sexual reproduction was observed.

Figs 1–2. Scanning electron micrographs of Chloroparva pannonica(stub ACT 0608).

Fig. 1. Vegetative cell with a smooth surface. Scale bar 5 1 mm.Fig. 2. Vegetative cell with a wrinkled surface. Scale bar 5 1 mm.

Figs 3–6. Transmission electron micrographs of Chloroparvapannonica. C, chloroplast; CW, cell wall; M, mitochondrion; N,nucleus; P, peroxisome; PG, plastoglobule; PM, plasma membrane;V, vacuole.

Fig. 3. Vegetative cell. Scale bar 5 0.5 mm.Fig. 4. A close-up from Fig. 3 showing the cell wall structure:trilaminar outer layer of the vegetative cell (arrowhead), granulo-fibrillar inner layer and electron-transparent layer covering theplasma membrane (arrow). Scale bar 5 0.2 mm.Fig. 5. Vegetative cell. Scale bar 5 0.5 mm.Fig. 6. A close-up from Fig. 5 showing the cell wall structure.Notation as in Fig. 4. Scale bar 5 0.2 mm.


Pigment composition

Chloroparva pannonica contained typical chlorophyte pig-

ments, including chlorophylls a and b and lutein as the

dominant carotenoid. The chlorophyll a (tr: 6.5 min) and b

(tr: 9.1 min) content of C. pannonica was 4.66 and

2.27 mg g21 wet weight (biomass), respectively. The chloro-

phyll b:a ratio was 0.48. During the carotenoid analysis, the

proportion of the hypophasic and epiphasic fraction was

2.5. The main carotenoids were (all-E)- lutein, (all-E)-

violaxanthin, (99Z)-neoxanthin and b-carotene (b,b-

carotene) (Table 2).

Fatty acid content

The total fatty acid content of C. pannonica was 45 mg g21

dry cell mass at 8uC and 35 mg g21 dry cell mass at 21uC(Table 3). At 8uC, saturated fatty acids (SFA) constituted

7.2%, monounsaturated fatty acids (MUFA) 88.9% and

polyunsaturated fatty acids (PUFA) 3.8% of the total fatty

acid content. At 21uC, SFA composed 5.7%, MUFA 93.7%

and PUFA 0.5% of the total fatty acid content. The main

fatty acid of C. pannonica was oleic acid (18:1n-9)

constituting approximately 90% of the total extracted fatty

acid content.

18S rRNA gene analysis

Sequence analysis of C. pannonica (ACT 0608) resulted in

2329 nucleotides. Based on the database search, the closest

relatives were N. eucaryotum UTEX 2502, C. minutissima

C-1.1.9 and C. minutissima SAG 1.80 with 97.5–97.6%

pairwise similarities. Molecular analysis of the 18S rRNA

gene sequence placed the new picoalgal isolate on a dis-

tinct branch within the Trebouxiophyceae, Chlorophyta

(Fig. 11). The new isolate contains an intron in its 18S

rRNA gene with the same insertion position but different

length as in Koliella spiculiformis (Vischer) Hindak,

Actinastrum hantzschii Lagerheim SAG 2015 and Micracti-

nium sp. Fresinius CCAP 211/92 (Fig. 12).

DISCUSSION

Molecular biological methods appear to be useful tools to

assign taxa with a limited number of morphological

features. The introduction of DNA-based phylogenetic

analysis in the taxonomy of freshwater and saline

planktonic green algae resulted in the construction of new

genera (Henley et al. 2004; Krienitz et al. 2004; Fawley et al.

2005); although the nomenclature and systematics of

several small coccoid chlorophyte isolates are still waiting

for revision (Henley et al. 2004). Due to these uncertainties,

currently there is no general pairwise similarity value of 18S

rRNA gene that defines chlorophyte genera. Krienitz et al.

(2004) found that the taxa of Chlorella and Parachlorella

clade (Trebouxiophyceae, Chlorophyta), including several

genera (Dicloster, Didymogenes, Closteriopsis, Parachlor-

ella, Micractinium etc.), showed a range of 18S rRNA

sequence similarities from 97.4 to 99.5%. The phylogenetic

analysis of the ‘Nannochloris-like’ algae (Trebouxiophyceae,

Chlorophyta) revealed that these isolates (the Nannochloris-

like clade, containing the genera Picochlorum, Nannochloris,

Marvania, etc.) possess only 3.75% 18S rRNA gene

sequence heterogenity (Henley et al. 2004). Sequence

analysis of C. pannonica revealed that this isolate is

distantly related to other isolates of the Trebouxiophyceae

(# 97.6% 18S rRNA gene pairwise similarities); therefore,

the phylogenetic position of the new chlorophyte confirms

the proposal of a new genus (Fig. 11).

The closest relatives of C. pannonica are the members of

the ‘problematic group’ according to the work of Henley et

al. (2004): N. eucaryotum UTEX 2502, C. minutissima C-

1.1.9 and C. minutissima SAG 1.80. In contrast to the

members of this group, C. pannonica possesses one intron

(presumably a group I intron) in its 18S rRNA gene,

located in an insertion position previously described in

other related trebouxiophycean isolates (Fig. 12). Group I

intron insertions are relatively frequent in the ribosomal

RNA of green algae and have sporadic, highly biased

distribution (Haugen et al. 2005; Hoshina & Imamura

2008). The majority of these mobile genetic elements is

predicted to spread with reverse splicing (Woodson & Cech

1989; Bhattacharya et al. 2005) and has insertion positions

Figs 7–10. Transmission electron micrographs of Chloroparvapannonica during autosporulation. C, chloroplast; GMCW, grand-mother cell wall; MCW, mother cell wall; N, nucleus; PM, plasmamembrane; V, vacuole.

Fig. 7. Mother cell with two autospores. Scale bar 5 0.5 mm.Fig. 8. A close-up from Fig. 7 showing the structure of themother cell wall and the evolving daughter cell wall. Thedaughter cell wall has a fine trilaminar layer (arrowhead), a firmgranulo-fibrillar inner layer and a thin electron-transparentlayer. Scale bar 5 0.2 mm.Fig. 9. Mother cell with two autospores. Asterisk indicates anelectron-dense body. Scale bar 5 0.5 mm.Fig. 10. Two daughter cells at a late phase of autosporulation.Scale bar 5 0.5 mm.


restricted to only a few sites within the rRNA genes

(Hoshina & Imamura 2008). In addition to the two

distantly related chlorophyte genera, Choricystis (Treboux-

iophyceae) and Mychonastes (Chlorophyceae), the new

Chloroparva is the third freshwater picoeukaryotic algal

genus that contains introns in the 18S rRNA gene (Krienitz

et al. 1996; Hepperle & Schlegel 2002; Luo et al. 2010). Due

to the uneven occurrence (Fig. 12), introns in the 18S

rRNA gene should not be regarded as useful phylogenetic

markers among chlorophytes or among picoeukaryotic

algae.

The taxonomy of the members of the ‘problematic group’

is somewhat confusing. The type strain – Mainz 1 – of N.

eucaryotum was renamed based on its 18S rRNA gene

sequence to Picochlorum eukaryotum (Wilhelm, Eisenbeis,

Wild & Zahn) Henley, Hironaka, Guillou, M. Buchheim, J.

Buchheim, M. Fawley & K. Fawley by Henley et al. (2004).

According to Wilhelm et al. (1982), Mainz 1 strain had

small cells (1.5 mm on average) surrounded by a smooth and

thin cell wall, but dividing cells showed a rougher surface.

Menzel & Wild (1989) described that the cells divided by

autosporulation and that the two daughter cells remained

enveloped by the mother cell wall. After the release of the

daughter cells, the empty parental envelope displayed a

characteristic curling. Nannochloris eucaryotum UTEX

2502 is supposed to be the relative of N. eucaryotum SAG

55.87 according to the catalogue of SAG and also UTEX

Culture Collections. As described by Yamamoto et al.

(2001), N. eucaryotum UTEX 2502 had spherical cells of

2.5 mm in diameter, divided by autosporulation, which

resulted in two, three and four round daughter cells and a

persistent mother cell wall. In a latter study, Yamamoto et

al. (2003) noted that N. eucaryotum UTEX 2502 was

Table 3. Selected fatty acid content (in mg g21 dry weight) ofChloroparva pannonica in the stationary phase of growth at 8uCand 21uC in comparison to Choricystis minor and Pseudodictyos-phaerium jurisii in the stationary phase of growth at 20uC.

Fatty acids (FA)

C. pannonicaC. minor1 P. jurisii1

8uC 21uC 20uC 20uC

Saturated fatty acids (SFA)

14:0 0.90 0.55 1.09 0.2016:0 0.92 0.44 6.59 3.1317:0 0.00 0.08 — —18:0 0.99 0.91 0.95 0.4020:0 0.23 0.00 — —21:0 0.20 0.00 — —

Monounsaturated fatty acids (MUFA)

14:1 n-5 0.34 0.30 — —16:1 n-7 0.00 0.00 0.64 0.1017:1 n-7 0.37 0.28 — —18:1 n-9 39.26 31.94 0.68 0.45

Polyunsaturated fatty acids (PUFA)

18:2 n-6 0.49 0.12 0.90 0.8518:3 n-6 0.36 0.00 0.04 0.0818:3 n-3 0.00 0.00 1.80 0.7720:3 n-3 0.16 0.06 — —20:3 n-6 0.30 0.00 — —20:4 n-6 0.00 0.00 0.14 0.1420:5 n-3 0.00 0.00 0.46 0.1722:2 n-6 0.42 0.00 — —22:5 n-3 0.00 0.00 0.04 0.1522:6 n-3 0.00 0.00 0.22 0.15

S FA 44.95 34.69 18.99 10.37S SFA 3.25 1.99 8.63 3.73S MUFA 39.98 32.52 1.32 0.55S PUFA 1.73 0.18 3.60 2.31

1 According to Krienitz & Wirth (2006).

Table 2. The carotenoid composition of the total extract, the hypophasic and the epiphasic fractions isolated from Chloroparva pannonicalyophilised cell mass.

Peak Carotenoid UV/VIS (nm)

Total extract Hypophasic fraction Epiphasic fraction

tr (min) % tr (min) % tr (min) %

1 (all-E)-neoxanthin 468 441/4421 416 12.00 t2 10.73 1.1 — —2 (99Z)-neoxanthin 463/4641 435 412 14.26 7.4 12.92 5.8 — —3 (all-E)-violaxanthin 467 438 415 15.33 12.6 14.00 12.3 13.95 t2

4 epimers of luteoxanthin 448 420 398 16.75 17.15 t2 15.58 16.00 t2 — —5 (all-E)-lutein-5,6-epoxide 467 439/4411 416 19.85 t2 18.71 1.0 — —6 (Z)-isomers of lutein-5,6-epoxide 465 438 413 21.65 22.04 t2 20.58 21.00 t2 — —7 (all-E)-lutein 470 443 (420) 23.58 66.4 22.79 73.5 22.78 43.18 (9Z) + (99Z)-lutein 465 438 (417) 26.54 2.8 26.10 3.3 26.09 1.29 (13Z) + (139Z)-lutein 463 436 (415) 3303 27.08 2.4 26.71 3.0 26.69 2.0

10 (15Z)-lutein 466 439 (417) 3303 27.50 t2 27.16 t2 27.15 t2

11 (all-E)-b-cryptoxanthin 474 447 424 36.17 1.6 — — 36.15 36.234 9.04

12 (9Z) + (99Z)-b-cryptoxanthin 470 443 420 36.29 t2 — —13 c (b,y)-carotene + d (e,y)-carotene5 487 458/4576433 37.85 t2 — — 37.93 5.314 a-carotene (b,e-carotene) 473 445 (420) 4206 38.95 t2 — — 39.12 2.215 b-carotene (b,b-carotene) 477 452 39.55 6.8 — — 39.58 37.216 (9Z)-b-carotene 472 446 40.00 t2 — — 39.95 t2

1 In case of the hypophasic fraction.2 In traces.3 Peak 330 (cis-peak) with high intensity is characteristic to the (13Z)-, (139Z)- and (15Z)-isomers of carotenoids.4 (all-E) + (9Z) + (99Z)-b-cryptoxanthin, UV/VIS: 475 447 (422) nm.5 Tentatively identified.6 In case of the epiphasic fraction.


heterogeneous, and they purified it by single colony

isolation resulting in KSW0203. Interestingly, N. eucaryo-

tum KSW0203 was shown to be an autosporic taxon with

larger cells compared to their earlier results (autospores 3–

4 mm, mother cells 5–6 mm in diameter). Notwithstanding,

Henley et al. (2004) obtained the identical 18S rRNA gene

sequence for N. eucaryotum UTEX 2502 as Yamamoto et

al. (2003) for N. eucaryotum KSW0203. According to

Tschermak-Woess (1998), N. eucaryotum SAG 55.87 had

ellipsoidal cells somewhat less than 2 3 2 mm, and the size

Fig. 11. Maximum likelihood (ML) tree of 18S rRNA gene sequences retrieved from Chloroparva pannonica isolate ACT 0608 and theclosest relatives (Trebouxiophyceae, Chlorophyta). ML analysis was conducted heuristically using the tree-bisection-reconnection optionwith likelihood settings of the best-fit model [TrN+I+G model; Lset Base 5 (0.2533 0.2111 0.2705), Nst 5 6, Rmat 5 (1.0000 2.0131 1.00001.0000 5.0758), Rates 5 gamma, Shape 5 0.6136; Pinvar 5 0.6952; the same values were used for maximum parsimony (MP)]. Bootstrapvalues greater than 70 (based on 100 and 1000 replicates for ML and MP, respectively) and Bayesian (B) posterior probabilities (3 100)from 500,000 generations are shown (order: ML/MP/B). Asterisk marks sequences that are also present in Fig. 12.


of mother cells reached 3.5 mm. The long persistence of the

mother cell wall surrounding the autospores was also

described (Tschermak-Woess 1998). Cell division was

found by autosporulation, with two, three or rarely four

autospores per mother cell resulting from two temporally

separated processes of autosporulation (Tschermak-Woess

1998). As Henley et al. (2004) suggested, sequencing of

strain SAG 55.87 would be necessary to reveal the relation

between these two strains.

The type strain – Lefevre no. 87 – of C. minutissima was

renamed based on its 18S rRNA gene sequence to

Mychonastes homosphaera Kalina & Puncocharova by Huss

et al. (1999). For strain C-1.1.9 (and there are other algal

strains misinterpreted as C. minutissima, e.g. SAG 1.80),

which is unrelated to M. homosphaera, Huss et al. (1999)

proposed keeping the name C. minutissima provisionally

until more information becomes available. According to our

knowledge, detailed description is available only for strain

Lefevre no. 87. Nevertheless, C. minutissima C-1.1.9 is

absent in any current culture collection as implied by Henley

et al. (2004). The detailed study of C. minutissima SAG 1.80

would be necessary – parallel with sequencing N. eucar-

yotum SAG 55.87 – to clarify the taxonomical status of the

closest relatives of the proposed novel genus, C. pannonica.

Based on the above descriptions, C. pannonica have

smaller cells (usually less than 2 mm) than N. eucaryotum

UTEX 2502 and SAG 55.87 (Figs 1, 2). Unfortunately,

detailed electron microscopy–based description is not

available in the case of these strains; however, the

autosporulation and the persistent mother cell wall

surrounding the autospores suggest similarity to C.

pannonica (Figs 9, 10). According to Yamamoto et al.

(2003), autosporulation is an ancestral character, but until

now the taxonomical value of the mode of cell division is

not clear. In C. pannonica, formation of only two

autospores per mother cell was observed, in contrast to

the three or four autospores in the other strains; however,

the number of autospores may depend on culture condi-

tions (Krienitz et al. 1996). The cell wall structure of C.

pannonica (Figs 4, 6) is very similar to that of Choricystis

minor (Skuja) Fott as described by Menzel & Wild (1989)

and Krienitz et al. (1996). According to Krienitz et al.

(1996), the cell wall of C. minor consists of a thin (10–20 mm)

trilaminar layer, which is closely associated with an inner

fibrillar layer, and a relatively diffuse layer of low electron

optical density is often located between this inner layer and

plasmalemma. According to Menzel & Wild (1989), in the

course of autosporulation, the inner layer (presumably

cellulose) of the still closed mother cell wall disappears,

leaving only the trilaminar sheath. We assume that in case

of C. pannonica, the inner microfibrillar layer of the mother

cell wall disappears similarly to C. minor, and only the

trilaminar layer (of the mother cell wall or occasionally the

grandmother cell wall) persists for a long time around the

autospores (Figs 9, 10). Based on SEM micrographs, the

surface of the cells was either smooth or wrinkled (Figs 1,

2). TEM micrographs clearly indicated that the wrinkled

cell surface was not the result of a network of ribs on the

cell wall as in M. homosphaera (Hanagata et al. 1999). We

hypothesize that the long persistence of the mother cell wall

around the daughter cells – which is clearly observable on

TEM micrographs (Figs 9, 10) – results in this wrinkled

appearance (Fig. 2). In contrast with this, cells after the

detachment of the mother cell wall remnants have a smooth

surface (Fig. 1).

Menzel & Wild (1989) and Krienitz et al. (1996)

described the typical curling of the mother cell wall after

autosporulation, as it is observable in the case of many

trebouxiophycean species (e.g. P. eukaryotum) and also in

the case of C. pannonica. This curling suggests that the

trilaminar cell wall layer contains sporopollenin, which

provides a defence mechanism against drying out and

enzymatic disintegration (Menzel & Wild 1989). In the case

of C. pannonica, the toleration of desiccation is presumably

a very important feature, as this alga has to survive the

summer period, when the pans usually dry out.

The fatty acid content of C. pannonica was higher at 8uCthan at 21uC. The relative contribution of polyunsaturated

fatty acids increased significantly (7.4 times) due to the

temperature decrease (Table 3). Picoeukaryotes show char-

acteristic seasonal appearance in the temperate zone: they

dominate the pico-fraction from autumn to spring (Callieri

Fig. 12. Position and length of introns within the 18S rRNA gene presented in Chloroparva pannonica isolate ACT 0608 and in its closestrelatives. Introns are indicated with black boxes; nucleotide positions are shown according to the Chlorella vulgaris SAG 211-11b 18S rRNAgene sequence (X13688; Huss & Sogin 1989); nt, nucleotides.


2008; Voros et al. 2009). This characteristic seasonal

dynamic was also observable in the case of Hungarian

soda pans, and C. pannonica was isolated from a winter

phytoplankton sample. The winter predominance of

picoeukaryotes was correlated with their lower light and

temperature requirement (Somogyi et al. 2009). Besides

this, the increased PUFA content at lower temperature

might be an important feature in the temperature acclima-

tization. Krienitz & Wirth (2006) analysed the fatty acid

content of the most abundant freshwater picoplanktonic

green algal species, C. minor and Pseudodictyosphaerium

jurisii (Hindak) Hindak. According to their results, C.

minor contained 64% palmitic acid (16:0) and 13% alpha-

linolenic acid (18:3 n-3) similarly to P. jurisii, which

contained 57% palmitic acid and 12% alpha-linolenic acid

(Table 3). Several studies suggest that the fatty acid content

and composition of nanoplanktonic green algae could be

notably different; however, saturated fatty acids [palmitic

acid or stearic acid (18:0)] were dominant (29–70%) in the

case of the studied species (Chlorella sp., Monoraphidium

sp., Scenedesmus sp. and Stichococcus sp.) at 20uC (Makulla

2000; Teoh et al. 2004; Krienitz & Wirth 2006). Based on

these results, the extremely high monounsaturated oleic

acid ratio of C. pannonica seems to be a distinctive feature.

In conclusion, the new chlorophyte isolate originating

from a turbid, shallow soda pan proved to be a new species

– belonging to a new genus within the Trebouxiophyceae –

based on its 18S rRNA gene sequence. Chloroparva

pannonica has a minimal set of cell organelles similarly to

other pico-sized chlorophytes. The pigment composition

showed a typical chlorophycean pattern. The long persis-

tence of the mother cell wall surrounding the autospores

proved to be somewhat unique.

ACKNOWLEDGEMENTS

The study was sponsored by the Hungarian Research Fund

(OTKA K 73369 and partially by K 76176, K 60121).

Gyorgyi Horvath was supported by a scholarship from the

University of Pecs, Medical School (PTE AOK KA). Tamas

Felfoldi was supported by a scholarship from the Ministry of

Education and Culture, Hungary (DFO 0051/2009). The

authors thank Emil Boros for his help with fieldwork, Attila

Kovacs for helpful discussions and Balazs Nemeth and

Erzsebet Lakatos for their technical assistance. The authors

are grateful to Csilla Jonas for skillful assistance in electron

microscopic sample preparation and ultrathin sectioning and

to Laszlo Hiripi for chlorophyll a and b analysis. We also

thank Peter Takacs for his help with the Latin diagnosis.

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Received 3 February 2010; accepted 11 May 2010

Associate editor: Lothar Krienitz


chloroparva pannonica gen. et sp. nov. (trebouxiophyceae, chlorophyta) – a new picoplanktonic...

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