PRIMARY RESEARCH PAPER

Harmful and parasitic unicellular eukaryotes persistin a shallow lake under reconstruction (L. Karla, Greece)

Eleni Nikouli • Konstantinos Ar. Kormas •

Panagiotis Berillis • Hera Karayanni •

Maria Moustaka-Gouni

Received: 11 February 2013 / Revised: 8 June 2013 / Accepted: 15 June 2013 / Published online: 29 June 2013

� Springer Science+Business Media Dordrecht 2013

Abstract The reconstructed Lake Karla, Greece, has

been undergoing its water-filling period since Novem-

ber 2009. In this paper, we aimed at investigating

whether the unicellular eukaryotes, including the

toxic/parasitic ones, that have been found during mass

fish kills in the lake (March–April 2010), persist

during the first warm period of the lake (May, August,

November 2010). Given that microscopic character-

ization of some of these eukaryotes is not adequate for

their identification, we analysed the 18S rRNA gene

diversity of plankton samples. All the found phylo-

types belonged to the phyla of Mesomycetazoa,

Chlorophyta, Fungi, Alveolata, Cercozoa, Crypto-

phyta and Stramenopiles. Some members of these

groups seem to persist in Lake Karla as they have been

found in early spring as well. These microscopic

eukaryotes are either ichthyotoxic/parasitic (e.g.

Pfiesteria sp./Pseudopfiesteria shumwayae, some

Fungi, Mesomycetazoa, Lagenidium sp., Cercozoa)

or indicative of hyper-eutrophic conditions (e.g.

Oocystis sp., Scenedesmus spp.) and were rather

abundant during the first spring–autumn period of

the lake’s refilling process. These complex micro-

scopic communities are expected to shape highly

dynamic and variable food webs with the risk of

repeated fish kills.

Keywords 18S rDNA � Plankton � Unicellular

eukaryotes � Shallow � Lake � Karla � Greece

Introduction

Practically all known biota of shallow lakes has been

the focus of numerous studies, starting from the old-

known strong relationship between the nutrient regime

and the lakes’ primary producers to conceptual and

mathematical models describing seasonal succession

of phytoplankton (Lampert & Sommer, 2007). The

latter, not just has a pivotal role in the shallow lake

ecosystem structure and function, but its community

composition, abundance and biomass are now recog-

nized as universal tools for assessing the ecological

quality of these systems (WFD, 2000). However,

ongoing research on these systems is still vivid and

Handling editor: Stefano Amalfitano

Electronic supplementary material The online version ofthis article (doi:10.1007/s10750-013-1604-8) containssupplementary material, which is available to authorized users.

E. Nikouli � K. Ar. Kormas (&) � P. Berillis

Department of Ichthyology and Aquatic Environment,

School of Agricultural Sciences, University of Thessaly,

384 46 Volos, Greece

e-mail: kkormas@uth.gr

H. Karayanni

Department of Biological Applications and Technology,

University of Ioannina, 451 10 Ioannina, Greece

M. Moustaka-Gouni

School of Biology, Department of Botany, Aristotle

University of Thessaloniki, 541 24 Thessaloniki, Greece

123

Hydrobiologia (2013) 718:73–83

DOI 10.1007/s10750-013-1604-8

rewarding, mostly due to its often unpredictable

temporal and spatial changes. Due to their small water

volume and their frequently unstable hydrological

budget, they are influenced by variable environmental

parameters more pronounced than other lake ecosys-

tems (Padisak & Reynolds, 2003). These changes are

usually more pronounced in eutrophic shallow lakes

(e.g. Fernandez et al., 2012). Such great variance is

very difficult to incorporate in realistic experiments

which in most cases do not last long enough to

enlighten us with their result on the lakes’ microalgal

community succession (Waters et al., 2012). Shallow

lakes are of great interest to humans, as it is shown by

the large number of constructed ones. Their usage

includes mostly reservoirs for drinking water or

irrigation purposes and recreation sites (Padisak &

Reynolds, 2003). Worldwide, a majority of lakes are

categorized as shallow ones, whilst 70% of the lakes in

Greece are shallow (Coops et al., 2003).

In the study of shallow lakes, phytoplankton is

traditionally studied as it is highly responsive to

environmental changes and several phytoplankton

species serve as indicators for, e.g. eutrophication,

ecological water quality (Padisak et al., 2006). More-

over, we now know that some unicellular eukaryotes,

like nanoflagellates, dinoflagellates and ciliates, are

key players in the transfer of energy in aquatic food

webs (Sherr & Sherr, 2002). However, their precise

identification and enumeration can be a dubious task at

least in some cases (Caron et al., 2009; Christaki et al.,

2011). Nowadays, it is well accepted that the identi-

fication of unicellular eukaryotic species should

integrate both morphological traits and molecular

markers. This approach circumvents identification

problems of ecological importance, such as the cryptic

species of several morphospecies that exist in eukary-

otic lineages (Weisse, 2008) or the small (B5 lm)

freshwater eukaryotes which remain highly under-

investigated and are best studied only with molecular

tools (Sime-Ngando et al., 2011). In addition, in cases

of microorganisms with complex but unknown or

inefficiently described life cycles, the phylotype

approach assists in clarification of unknown cell

morphologies, thus rendering the trophic relationships

and ecophysiological traits more explicable.

Undesired blooms of harmful cyanobacteria and

algae, which are common in shallow eutrophic lakes,

pose the question of safety for these habitats, not only

for human, animal and ecosystem health but also for

economic growth and development of the areas where

they are found (Bakker, 2012). In a previous study,

provoked by fish kills in the recently reconstructed

L. Karla, Greece, associated with the occurrence of

toxic/parasitic unicellular eukaryotes and/or cyano-

bacteria, we had identified a high diversity of unicel-

lular eukaryotes and some of them could be related to

the fish kills (Oikonomou et al., 2012). In this study, we

aimed at (a) investigating whether these eukaryotes

persist during cyanobacterial blooms and (b) extending

our knowledge in planktic single-celled eukaryotes

biodiversity. For this, we investigated the unicellular

eukaryotes’ diversity in May (following the reported

blooms of Prymesium parvum and Pfiesteria piscicida

and the start of cyanobacteria blooms), August (peak of

cyanobacterial blooms) and November (end of cyano-

bacterial blooms) 2010, the lake’s first year of water

filling. Identifying the common, if any, 18S rDNA

phylotypes between different periods will permit us to

detect whether the lake’s planktic life kicks off with

opportunistic or typical shallow lake species.

Materials and methods

The water filling of Lake Karla, central Greece

(Fig. 1), which started in 2009, is the last major phase

of its reconstruction. With a 288-km perimeter. It

occupies ca. 1/4 of the old Lake Karla which covered

180 km2 and which was dried up in 1962 for

reclaiming agricultural land. The hydrological basin

of Lake Karla covers 1,171 km2 of which more than

600 km2 make up a southern flat plain, whilst the east

part is surrounded by mountains and hills with a

perimeter of 228 km. Elevation ranges from 50 m to

more than 2,000 m, and the mean elevation of the

region is about 230 m. It is a shallow lake with a

current maximum water depth of 2.5 m.

Sampling took place at the end of spring, summer

and autumn, spanning the lake’s pre-warm, warm and

late-warm phase (Table 1). Water samples of 1–2 l

were collected in 4-L collapsible plastic bottles

(Nalgene, USA) from ca. 0.5 m depth at the water-

level pier at the southeast end of the lake (Fig. 1).

Water temperature, dissolved oxygen, salinity and

pH were measured in situ using a WTW sensor

(Weilheim, Germany).

Water samples for DNA extraction were transferred

to the laboratory and processed\2 h after collection.

74 Hydrobiologia (2013) 718:73–83

123

After a 180-lm mesh net pre-screening to exclude

larger organisms and particles, a volume of

250–400 ml of water was filtered through a 0.2-lm

pore size Polycarbonate Isopore filter (Sartorius,

Germany). The filtration was conducted under reduced

pressure (B100 mmHg) to prevent cell damage and

before filters were clogged. Filters were stored imme-

diately at -80 �C until further analysis.

Bulk DNA was extracted using the UltraClean Soil

DNA isolation kit (MoBio Laboratories, USA) accord-

ing to the manufacturer’s protocol after aseptically

slicing the filters. Concentration of bulk DNA, as

estimated in a NanoDrop ND-1000 spectrophotometer

(NanoDrop Technologies, USA), was 10.2, 5.7 and

2.6 ng ll-1 for the May, August and November 2010

samples, respectively. For the May and August sam-

ples, the 18S rRNA gene was amplified using the

eukaryote-specific primers EukA (50-AACCTGGTTG

ATCCTGCCAGT-30; Medlin et al., 1988) and

Euk1633r (50-GGGCGGTGTGTACAARGRG-30;Dawson & Pace, 2002). Each PCR mix of the 50-ll

final volume contained ca. 10 ng of environmental

DNA, 10 ll of 59 Green GoTaq Flexi buffer

(Promega, USA), 5 ll of 2 mM dNTPs, 3 ll of

25 mM MgCl2, 0.25 ll of 100 pmol/ll of each primer

and 0.25 ll of 5 U of GoTaq DNA polymerase

Fig. 1 Map of Lake Karla, Greece, and sampling point (black dot). Black squares show points of inflowing water for reconstruction

purposes

Table 1 Basic

environmental parameters

in Lake Karla, Greece

Sampling date pH Temperature (�C) Salinity (PSU) Dissolved oxygen

(mg ml-1)

28–05–2010 8.0 19.7 9.1 5.6

28–08–2010 8.5 31.7 13.2 4.8

25–11–2010 8.8 12.2 2.9 6.9

Hydrobiologia (2013) 718:73–83 75

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(Promega, USA). PCR reactions for the May and

August samples included an initial denaturation step at

95�C for 2 min followed by 40 cycles of denaturing at

95�C for 40 s, annealing at 50�C for 40 s, and

elongation at 72�C for 2 min and 15 s, with an

additional step of final elongation at 72�C for 7 min.

For the November samples, nested PCR was used, with

the first primers being EukA and EukB (50-GATCCT

TCTGCAGGTTCACCTAC-30; Medlin et al., 1988),

whilst in the second PCR, EukB was replaced by

Euk1633r. The latter included an initial denaturation

step at 95�C for 15 min, which was followed by 40

cycles consisting of denaturation at 95�C for 45 s,

annealing at 55�C for 1 min, and elongation at 72�C for

2 min and 30 s; a final 7-min elongation step at 72�C

was included.

PCR for each sample was repeated with different

cycle numbers (between 18 and 39). The lowest

number of cycles with a positive signal, i.e. 28, 31 and

20 cycles for the May, August and November samples,

respectively, was used for cloning in order to eliminate

some of the major PCR innate limitations (Wintzin-

gerode et al., 1997; Spiegelman et al., 2005) and to

avoid differential representation of low and high 18S

rRNA gene copy numbers.

PCR products were visualized on 1% agarose gel

under UV light, purified by the Montage purification kit

(Millipore Inc., Molsheim, France). The purified PCR

products were ligated into the PCR XL TOPO Vector

(Invitrogen-Life Technologies, USA) and transformed

in electrocompetent Escherichia coli cells according to

the manufacturer’s specifications. For each clone

library, a maximum of 72 clones were sequenced, each

containing an insert of ca. 1,600 bp. These clones were

grown in liquid Luria–Bertani medium with kanamycin

and their plasmids were purified using the Nucleospin

Plasmid Quick-Pure kit (Macherey-Nagel GmbH and

Co. KG, Duren, Germany) for DNA sequencing.

Capillary electrophoresis sequencing (Macrogen Inc.,

Seoul, Korea) was performed using the BigDye Termi-

nator kit (Applied Biosystems-Life Technologies, USA)

with the M13F (50-GTAAAACGACGGCCAG-30) and

M13R (50-CAGGAAACAGCTATGAC-30) primers.

Each sequence read was ca. 950–1,000 bp. For each

individual clone, forward and reverse reads were

assembled, and then the assembled sequences were

checked for chimeras. The Pintail program (http://

www.bioinformatics-toolkit.org/Web-Pintail/) was

used for the detection of putative chimeric sequences.

Chimeras were discarded from the dataset. Using the

multiple alignment program CLUSTALW2 (http://www.

ebi.ac.uk/Tools/clustalw2/index.html/) and based on

98% gene similarity as a phylotype cutoff (Caron et al.,

2009; Nebel et al., 2011), clones were grouped together

and considered members of the same phylotype. All

sequences were compared with the BLAST function

(http://www.ncbi.nlm.nih.gov/BLAST/) for the detec-

tion of closest relatives. Sequence data were compiled

using the MEGA5 software (Tamura et al., 2011) and

aligned with sequences obtained from the GenBank

(http://www.ncbi.nlm.nih.gov/) database, using the

ClustalX aligning utility. Phylogenetic analyses were

performed using the MEGA 5 software (Tamura et al.,

2011) and the topology of the tree was based on

neighbour joining according to Jukes-Cantor. Boot-

strapping under parsimony criteria was performed with

1,000 replicates. Sequences of unique phylotypes found

in this study have GenBank accession numbers

KC315804–KC315844.

Library clone coverage was calculated by the

formula of the Good’s C estimator [1 - (ni/N)] [Good,

1954], where ni is the number of phylotypes repre-

sented by only one clone and N is the total number of

clones examined in each library. The number of

predicted phylotypes for each clone library was

estimated after the abundance-based richness formula

SChao1 (Chao, 1984, 1987).

Results

pH (Table 1) showed little variation and always

remained above 8.0. Temperature (Table 1) showed

the expected seasonal pattern for Mediterranean shal-

low lakes, with its highest value in August (31.7�C) and

a lower value in November (12.2�C). Conductivity

(Table 1) showed high values in May and especially in

August (23.01 mS cm-1), but it decreased down to

5.51 mS cm-1 in November. Salinity values (in PSU)

were proportional to conductivity (Table 1). Dissolved

oxygen (Table 1), although minimum in August

(4.8 mg ml-1), never reached hypoxic values.

A total of 72, six and 54 sequences were analysed

for the May, August and November samples, respec-

tively, corresponding to 24, five and 12 phylotypes,

based on a C98% similarity cutoff limit. All phylo-

types but one, i.e. Copepoda, represented unicellular

eukaryotes of the following taxa: Mesomycetazoa,

76 Hydrobiologia (2013) 718:73–83

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Chlorophyta, Fungi, Alveolata, Cercozoa, Crypto-

phyta and Stramenopiles (Fig. 2). The ratio of

observed to predicted phylotypes according to the

SChao1 index ranged between 35.4% (November) and

77.4% (May), whilst clone coverage based on the

Good’s C estimator was considered satisfactory for

May and November clone libraries (Fig. S1).

In May, the most abundant taxa were Fungi (Fig.

S1a, b) and Chlorophyta (Fig. S5). The August clone

library consisted only of five phylotypes belonging to

the Cryptophyta (Fig. S4), Fungi (Fig. S1a, b),

Mesomycetazoa (Fig. S2) and Stramenopiles (Fig.

S7), none of which dominated. In November, the

dominant phylum was the Stramenopiles (Fig. S7). The

vast majority of the remaining phylotypes belonged to

the Alveolata (Fig. S8) and Cercozoa (Fig. S6).

No phylotype was found to occur in all three

samplings, but there were some shared between

different samplings (Fig. 3). Two unknown Fungi-

related phylotypes (Fig. S1a, b; KRL03E17,

KRL03E21) and one from Mesomycetazoa (Fig. S2;

KRL03E02) related to Ichthyophonida sp., which were

detected in May were also found in August. Between

August and November, there were no common phyl-

otypes. The highest number of common phylotypes

occurred between May and November. These included

three Alveolata (Fig. S8; the unknown KRL03E03,

KRL03E57 and the Pseudopfiesteria shumwayae-like

KRL03E38), three Stramenopiles (Fig. S7, S9; the

Cyclotella sp.-like KRL03E08, the Goniochloris sp.-

like KRL03E85 and the Lagenidium sp.-like

KRL03E75) and the Scenedesmus subspicatus-related

KRL03E40 from the Chorophyta (Fig. S5).

Discussion

We investigated the existing 18S rRNA gene diversity

during the first warm period, after the water-filling

process started in the shallow, reconstructed Mediter-

ranean Lake Karla, in May, August and November

2010. These periods coincided with the start (May),

peak (August) and end (November) of the lake’s

cyanobacterial blooms (Papadimitriou et al., 2013).

Apart from adding to the very limited knowledge of

the unicellular eukaryotes’ diversity in reconstructed

lakes, and especially the small (B5 lm) eukaryotes in

freshwater, we also aimed at investigating whether the

microorganisms following the fish kills which

occurred in March/April 2010 (Oikonomou et al.,

2012) were the same with the ones recorded during

these fish kills.

Chlorophyta (Fig. S5, S9) were found in May and

November. Phylotype KRL03E07 dominated in May

and is practically identical to Scenedesmus pectinatus.

Another three phylotypes (KRL09E46, KRL03E19

and KRL03E40) were related to Scenedesmus spp.,

rendering this genus an important component of the

autotrophic community. Members of the Scenedesm-

aceae prefer lotic (Bellinger & Sigee, 2010) and

hypertrophic (Tavera & Dıez 2009) waters. The

family Oocystaceae was represented by phylotypes

Fig. 2 Collapsed

phylogenetic tree of the 18S

rDNA relationships (ca.

1600 bp) of the

representative unique for

each sample (grouped on

C98% similarity) phylotype

found in the Lake Karla

water column in May,

August and November 2010.

The tree is based on the

neighbour-joining method

as determined by distance

Jukes–Cantor analysis. One

thousand bootstrap analyses

(distance) were conducted.

Scale bar represents 2%

estimated distance. For

detailed sub-trees per

phylum, see Supplementary

Material

Hydrobiologia (2013) 718:73–83 77

123

KRL03E42 and KRL03E76. Oocystis sp. are fre-

quently found in freshwaters (Hepperle et al., 2000),

whilst dominant in heavily polluted water bodies like

the shallow brackish lake Koronia (Michaloudi et al.,

2012).

A total of 10 Stramenopiles phylotypes (Fig. S7)

were found. In August and November, they were most

abundant and closely related to the marine diatom

Chaetoceros gracilis. This could be due to the

increased salinity in summer. Similarly, KRL09E17

was related to marine phytoplankton centric diatoms,

whereas phylotypes KRL09E05 and KRL03E08 were

related to the frequently occurring freshwater genus

Cyclotella (Fig. S9; Hasle & Syvertsen, 1997; Stoer-

mer & Julius, 2003). Phylotype KRL03E60 repre-

sented Pseudotetraedriella kamillae which has been

found in the oligotrophic Lake Stechlin, Germany, and

is considered a low abundance species during spring

and autumn (Hegewald et al., 2007). The two Oomy-

cetes phylotypes (KRL03E75 and KRL09E14) repre-

sent Lagenidium caudatum, which has been isolated

from a nematode host originating from rabbit and cow

faecal material (Beakes et al., 2006).

Six phylotypes were found in the Alveolata (Fig.

S8), all in May and November. Four of them

(KRL03E03, -57, KRL09E06, -21) were not close to

any cultivated members of the phylum. Their closest

(93.7–94.1%) known relative is the predatory flagellate

Colpodela edax (Leander et al., 2003), which can be

found both in freshwaters and marine habitats

(Myl’nikov, 1991). It consumes smaller protists (Arndt

et al., 2000) by sucking the attacked cells (Brugerolle,

2002). Phylotypes KRL03E38 and KRL09E10 are

closely related to the heterotrophic dinoflagellates

Pseudopfiesteria shumwayae and Pfiesteria piscicida.

Pfiesteria piscicida has been associated with mass fish

kills (Burkholder et al., 1992; Noga et al., 1996;

Oikonomou et al., 2012), caused by the release of

potent ichthyotoxins (Smith et al., 1988; Burkholder &

Glasgow, 1995). It is also considered responsible for

cases of human diseases through the consumption of P.

piscicida-toxicated fish (Burkholder & Glasgow,

1997; Steidinger et al., 2006). Pseudopfiesteria shu-

mwayae is similar to P. piscicida, although its patho-

genicity, growth characteristics and food preference

are not completely known. In general, this and related

species become abundant after increased nutrient

loadings due to the increase of their prey, i.e. phyto-

plankton (Burkholder & Glasgow, 1997).

The Cryptophyta include both auto- and heterotro-

phic species (Mignot, 1965; McFadden et al., 1994).

Phylotype KRL06E73 is distantly (94.2%) related to

the heterotrophic Chilomonas paramecium (Cavalier-

Smith et al., 1994) and KRL09E02 was highly similar

to the photosynthetic Cryptomonas curvata (Marin

et al., 1998). The ecological importance of the

Cryptophyta lies in their potential as prey for fresh-

water plankton (Chen & Folt, 1993) and some inver-

tebrates like Dreissena polymorphya (Vanderploeg

et al., 1996). In a study that included 11 mountain lakes,

Fig. 3 Venn diagram

showing the common

unicellular eukaryotes

occurring during the first

year of Lake Karla’s,

Greece, water refilling

78 Hydrobiologia (2013) 718:73–83

123

Cryptophyta phylotypes were most abundant in the

most eutrophic lake (Triado-Margarit & Casamayor,

2012), indicating the preference of this group for

eutrophic systems, such as L. Karla.

In total, nine Fungi phylotypes (Fig. S1a, b) were

found, and all were related to yet uncultivated

members of the phylum with their closest relatives

originating from marine and freshwater sediments.

Two identical phylotypes from May (KRL03E21) and

August (KREL06E206) were related to Fungi found in

the water column of the severely degraded, hypertro-

phic L. Koronia, Greece (Genitsaris et al., 2009). Four

phylotypes (KRL03E17, -32, -80, KRL06E139),

along with similar phylotypes from the deep, oligo-

to mesotrophic L. Pavin, France (Lefevre et al., 2007)

and Ketelmeer, The Netherlands (Van Hannen et al.,

1999) with heavy metals polluted sediments (Hulscher

et al., 2002), form a discrete novel clade. The Fungi-

like phylotypes found in May belong most likely to a

novel clade. Whether these new Fungi are actual

parasites or not is not clarified from this study,

although their ecological role might be important

since the Fungi are considered to alter the prevailing

food web structure and function through the consump-

tion of zoospores by mesozooplankton and micro-

plankton (Niquil et al., 2011), especially in eutrophic

ecosystems (Miki et al., 2011).

All members of the Mesomycetazoa are considered

parasites of fish, crustaceans and amphibians (Van

Hannen et al., 1999). In L. Karla, such phylotypes

were found in May and August (Fig. S2), belonging to

the Ichthyophonida order and are related to Sphaero-

forma arctica, Ichthyophonida fragrantissima, iso-

lated from the digestive tract of marine invertebrates

(Marshall et al., 2008) and Anurofeca richardsi, a

known parasite of Anura (Baker et al., 1999).

Cercozoa (Fig. S6) where found only in May and

probably their occurrence is limited to spring, as they

were also found in March and April (Oikonomou et al.,

2012). Phylotype KRL03E11 is a yet uncultivated

taxon, but it belongs to a clade that includes Platyreta

germanica and Arachnula impatiens which are para-

sites of fungi, algae nematodes and possible bacteria

(Bass et al., 2009). Phylotype KRL03E39 was close to

the Cryothecomonas longipes and Protaspis grandis,

isolated from the marine environment, implying the

association of this phylotype with increased salinities

as is the case with other Cercozoa found in L. Karla

previously (Oikonomou et al., 2012). C. longipes is a

selective parasitic flagellate, infecting members of the

Stramenopiles (Kuhn et al., 2000) and can cause mass

death of phytoplankton (Drebes et al., 1996; Tillmann

et al., 1999).

The Copepoda phylotype found was closely related

to the predatory genus Cyclops. The genetic material

might have come from eggs, nauplii or body fragments

which occurred in the May sample. Members of the

genus Cyclops are widespread in freshwaters (Box-

shall & Defage, 2008). In L. Karla, in particular,

Cyclops sp. is the one and only—but highly abun-

dant—copepod species that has been observed to date

(Kagalou, unpublished data). Such metazoan

sequences have also been found before in similar

studies targeting unicellular eukaryotes (Luo et al.,

2011).

The major taxa found in our study were also found

in a parallel study which focused on the microscopic-

based analysis of phytoplankton between April and

October 2010 (Papadimitriou et al., 2013). However,

our study revealed the existence of some unicellular

eukaryotes that were not identified by microscopy, like

the phylotypes associated with the Mesomycetazoa

(Fig. S2), the Fungi (Fig. S1b) and the novel clades

within the Cercozoa (Fig. S6) and the Alveolata (Fig.

S8). The structure of unicellular eukaryotic commu-

nities in L. Karla, as revealed by their 18S rDNA

analysis, was found to vary from spring to autumn. At

the end of spring, Fungi (25/72 sequences, 6/24

phylotypes) and Chlorophyta (24/72 sequences, 6/24

unique phylotypes) dominated. On the contrary, at the

end of summer, only five low abundance eukaryotic

phylotypes were retrieved, indicating that unicellular

eukaryotes were not abundant, as was also reported by

microscopic analysis of phytoplankton during the

warm period in L. Karla in 2010 (Papadimitriou et al.,

2013). In addition, the relationship of the initial

extracted bulk DNA concentration with the DNA

concentration after the 18S rDNA-specific PCR indi-

cated that only a small fraction of the bulk DNA was of

eukaryotic origin, with its major part being prokary-

otic (data not shown). During the warm period in 2010,

i.e. April–October, Cyanobacteria dominated on aver-

age 75% in the phytoplankton community, and in

August this dominance was [90% (Papadimitriou

et al., 2013), as is the case in several other eutrophic

shallow lakes (Oliver & Ganf, 2000; Steven et al.,

2012). This could explain the low amount of eukary-

otic DNA and low number of eukaryotic phylotypes

Hydrobiologia (2013) 718:73–83 79

123

retrieved, especially the autotrophic ones through light

and nutrient limitation. Such a seasonal decline in

unicellular eukaryotic species richness has been

reported for the oligotrophic L. Stechlin as well (Luo

et al., 2011), implying that the increased trophic state

of a lake brings reduction in the prevailing phylotype

number. Moreover, another likely reason for the low

number of both autotrophic and hetero-/mixotrophic

microscopic eukaryotes is allelopathy. Allelopathy

contributes to promoting, maintaining and succeeding

cyanobacterial and microalgal blooms in freshwater

and marine environments (Gross, 2003; Legrand et al.,

2003). For example, Microcystis (Singh et al., 2001;

Sukenik et al., 2002), Anabaena species (Graneli et al.,

2008), Nodularia spumigena (Zak et al., 2012),

Planktothrix and Aphanizomenon species (Keating,

1987) are known to inhibit growth of microalgae and

other cyanobacteria as well.

A substantial part of the found phylotypes is related

to parasitic/saprophytic and not to bacterivorous

species, as is often revealed by similar studies

(Sime-Ngando et al., 2011 and references therein).

This is also indicated by the exceptionally high

prokaryotic cell counts measured during the first year

of the L. Karla’s refilling (Fig. S10) with its second

highest value (79.6 9 106 cells ml-1) in August,

when the lowest number of unicellular eukaryotic

phylotypes occurred.

Based on microscopy in freshwater lake ecosys-

tems, the Heterokonta, the kathablepharids and the

Choanozoa contribute in excess of 90% of the

flagellate protozoa diversity observed (Arndt et al.,

2000). However, microscopy with molecular studies

do not always corroborate (e.g. Luo et al., 2011), but

are rather complementary, especially for the smaller

protists and some specific groups like the cryptomo-

nads (e.g. Richards et al., 2005; del Campo &

Massana, 2011). In our study, we used a 0.2-lm filter

in order to include picoplanktic forms, whilst detailed

microscopic investigations of L. Karla’s first year are

reported elsewhere (Papadimitriou et al., 2013).

Previously, in L. Karla in March and April 2010

(Oikonomou et al., 2012; Papadimitriou et al., 2013),

we identified some ecologically important microor-

ganisms not found in the present study. For example,

Prymnesium parvum, a potentially toxic haptophyte,

reached 99 9 106 individuals ml-1 and represented

44.6% of all phytoplankton species abundance. In the

present study, P. parvum was not detected, probably

due to its low growth rate when nutrient concentra-

tions are high (Guo et al., 1996). In addition, in most

cases, its blooms occur at the beginning of spring,

suggesting its preference for lower temperature

regimes (Baker et al., 2007). Still, L. Karla seems to

be plagued by microscopic eukaryotes that are either

ichthyotoxic/parasitic (e.g. Pfiesteria sp./Pseudopfies-

teria shumwayae, some Fungi, Mesomycetazoa, La-

genidium sp., Cercozoa) or indicative of hyper-

eutrophic conditions (e.g. Oocystis sp., Scenedesmus

spp.), at least during its first spring–autumn period.

These complex communities are likely to be interact-

ing mostly by allelopathic or harshly competitive

trophic relationships. The question that remains is

whether these microorganisms are ‘vagabonds’ (sensu

Newton et al. (2011) and will persist and plague the

lake’s future or whether their occurrence is simply

opportunistic, ‘tourists’ (sensu Newton et al., 2011),

and the ongoing succession will result in typical lake

microorganisms, ‘natives’ (sensu Newton et al., 2011).

Ongoing, targeted and multiphasic monitoring of the

lake’s plankton as in every continuously changing

system is required, especially since several of these

microorganisms cannot be identified/quantified by a

single approach.

Acknowledgments This part of this work was supported by

the John S. Latsis Public Benefit Foundation, Research

Programs 2011. Part of the laboratory work was financed by

the MSc programme ‘‘Sustainable Management of Aquatic

Environment’’ by the Department of Ichthyology and Aquatic

Environment, University of Thessaly. Peter McGee http://

anglais.webs.com/englishotherversions.htm provided editorial

and linguistic assistance. We thank the two anonymous reviewers

for their comments on the originally submitted manuscript.

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