Appearance and establishment of diazotrophic cyanobacteriain Lake Kinneret (Sea of Galilee)

O. HADAS*, R. PINKAS*, N. MALINSKY-RUSHANSKY*, A. NISHRI* , A. KAPLAN †, A. RIMMER*

AND A. SUKENIK*

*Israel Oceanographic and Limnological Research, Yigal Allon Kinneret Limnological Laboratory, Migdal, Israel†Department of Plant and Environmental Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel1

SUMMARY

1. We propose that the appearance and establishment of Nostocales (cyanobacteria) species of the

genera Aphanizomenon and Cylindrospermopsis in the warm subtropical Lake Kinneret (Sea of

Galilee, Israel) from 1994 was linked to changes in climate conditions and summer nitrogen (N)

availability.

2. From 1994 to 2009, an increase in frequency of events of elevated water temperature (>29 �C) in

summer, and to some extent a greater frequency of lower summer wind speed events, affected

water turbulence and water column stratification, thus providing better physical conditions for the

establishment of these populations.

3. In recent years, N-depleted conditions in Lake Kinneret in early summer have promoted the

development and domination of Nostocales that could gain an ecological advantage owing to their

N2-fixing capability.

4. Nitrogen fixation rates coincided both with heterocyst abundance and with Nostocales biomass.

The N supplied to the lake via nitrogen fixation ranged from negligible quantities when

Nostocales represented only a minor component of the phytoplankton community to 123 tonnes

when Cylindrospermopsis bloomed in 2005. This high N2 fixation rate equals the average summer

dissolved inorganic nitrogen load to the lake via the Jordan River.

Keywords: cyanobacteria bloom, Lake Kinneret, nitrogen fixation, Nostocales

Introduction3

Cyanobacterial blooms of the order Nostocales (mainly

the genera Aphanizomenon and Cylindrospermopsis) have

intensified over the last decade in many freshwater lakes

and reservoirs worldwide (Ryan, Hamilton & Barnes,

2003; Wiedner et al., 2007; Mehnert et al., 2010; Paerl, Hall

& Calandrino, 2011; Kosten et al., 2011; Sukenik et al.,

2012).

For example, the presence of C. raciborskii has been

documented in central Asia, African lakes and Central

America, and it has proliferated in rivers, lakes, swamps

and water reservoirs in Australia (Padisak, 1997). This is

considered a tropical species, but over the last 20 years, it

has been found as far north as Constance Lake, Ottawa

(Canada), with maximal biomass in July–August and

contributing 63% of the total phytoplankton biomass

when water temperatures were high (Hamilton et al.,

2005). This geographical dispersion has been attributed to

a global warming trend. A northward expansion of

Nostocales species was also reported for Anabaena bergii

and Sphaerospermum sp. in German temperate lakes

(Stuken et al., 2006). The high temperature requirements

of C. raciborskii (>24 �C) and the build-up of a bloom only

in warm years can make this species an indicator of global

climate change in temperate zones (Padisak, 1997). The

spread and persistance of Nostocales species into both

higher northern and southern latitudes (Vidal & Kruk,

2008; Kosten et al., 2011) has led to several hypotheses as

to the underlying causes. These invasions may be linked

to global processes or to regional (including anthropo-

genic) changes. Here, we present Lake Kinneret as a case

F W B 2 7 9 2 B Dispatch: 26.3.12 Journal: FWB CE: Suganya

Journal Name Manuscript No. Author Received: No. of pages: 14 PE: Malini

Correspondence: O. Hadas, Israel Oceanographic and Limnological Research, Yigal Allon Kinneret Limnological Laboratory P.O. Box, 447,

Migdal 14950, Israel. E-mail: orah@ocean.org.il 2

Freshwater Biology (2012) doi:10.1111/j.1365-2427.2012.02792.x

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study that supports the hypothesis that an interaction

between global and regional processes led to the sudden

appearance of Nostocales in a subtropical lake.

Lake Kinneret, a warm freshwater monomictic lake,

used to be characterised by a stable annual phytoplankton

composition with a typical winter–spring bloom of the

dinoflagellate Peridinium gatunense and a summer–au-

tumn population consisting mostly of chlorophytes and a

minor population of cyanobacteria (Pollingher, 1981,

1986; Berman, Yacobi & Pollingher, 1992; Berman et al.,

1995). This pattern of phytoplankton assemblage changed

in 1994 when an exceptional bloom of a filamentous

nitrogen-fixing cyanobacterium, identified as Aphanizom-

enon ovalisporum Forti (Nostocales), occurred in the lake

(Pollingher et al., 1998; Hadas et al., 1999). Cylindrosperm-

opsis raciborskii Seenayya et Subba Raju was observed in

the lake for the first time in summer 1998 (Zohary, 2004;

Alster et al., 2010), formed a major summer bloom in 2005

and has since codominated the summer phytoplankton

with A. ovalisporum, in some years contributing between

60 and 80% of the monthly phytoplankton biomass. This

suggests that A. ovalisporum and C. raciborskii expand

their ranges similarly. The occurrence of A. ovalisporum

in 1994 was originally thought to be the first record of

Nostocales in Lake Kinneret, but a detailed search in the

Kinneret Limnological Laboratory Data Base revealed a

record of Nostocales from the early 1970s albeit with

lower biomass. In both cases, Nostocales appeared two

years after an exceptional rainy winter. It is not clear why

the appearance of Nostocales in 1994 led to persistence,

whereas that in the 1970s did not. Was it related to both

changes in climatic parameters and, as deduced from

long-term records, to limited availability of dissolved

inorganic N? The shift in the phytoplankton composition

since 1994 towards enhanced diazotrophic activity in

summer could be driven by gradual changes in many

environmental variables that, cumulatively, support the

persistence of nitrogen-fixing cyanobacteria (Hadas et al.,

2002).

Lake Kinneret serves as the main freshwater reservoir

of Israel, providing about 30% of the domestic water

demand and also serves for recreational activities. There-

fore, the appearance and persistence of the filamentous

toxic and non-toxic cyanobacteria, A. ovalisporum and

C. raciborskii, respectively, is of serious concern. Here,

we present a 15-year record of the appearance and

persistence of Nostocales in Lake Kinneret and their

contribution to the nitrogen budget via diazotrophic

activity, and we analyse changes in environmental and

climate conditions that could account for the recent

dominance of these species.

Methods

Study site

Lake Kinneret, Israel (32�42¢–32�55¢ N; 35�31¢–35�39¢ E), is

a warm monomictic freshwater lake, stratified for

8 months every year from April to December. During

stratification, an aerobic warm (24–30 �C) epilimnion and

an anoxic colder (14–16 �C) hypolimnion are formed.

Annual primary production in the epilimnetic euphotic

layer averages c. 1.8 g C m)2 day)1 (Berman et al., 1995).

Winter floods are the main sources of dissolved inorganic

nitrogen (DIN) to the lake ecosystem; they wash the peat

area in the northern part of the watershed before entering

the lake via the Jordan River. In an average hydrological

year, 75% of about 1200 tonnes of total N entering the lake

during winter is nitrate. Because of the lack of precipita-

tion in the summer (between May and September), water

inflows and nutrient loads are then drastically reduced

and the N load (ca 120 tonnes) is <15% of the winter load.

The main internal N load is the result of mineralisation of

organic material (in late spring) to ammonium with

subsequent oxidation to nitrate (during overturn in late

December–January). During drought years, much lower

amounts of nitrogen enter the lake. The Lake Kinneret

nitrogen balance reveals that 60% of the nitrogen entering

the lake is denitrified; thus, denitrification is the main

process regulating the DIN budget and preventing the

accumulation of nitrate (Serruya, 1978a).

In situ measurements of nitrogen fixation

Nitrogen fixation rates were measured by assessing

nitrogenase activity using the acetylene reduction assay

(Stewart, Fitzgerald & Burris, 1967). Depth profiles (0, 1, 2,

3, 5, 7, 10, 15 m) of epilimnetic water samples from station

A (32�49.305¢N; 35�35.5441¢ E), the deepest part in the lake

(42 m), were collected once every two weeks using a 5-L

polyvinyl chloride (PVC) sampler and then transferred to

the laboratory. We followed the method of Capone (1993)

with a few modifications. Water from each depth was

filtered using a 120-lm net in order to remove zooplank-

ton, followed by filtration of 1 L sample through a 47-mm-

diameter, pre-combusted GF ⁄C filter (which retained the

phytoplankton biomass). The filter was then transferred

into 28-mL serum bottles filled with 5 mL of 0.2 lm

(Nucleopore) filtered water sample from the correspond-

ing depth. Bottles were sealed with a grey rubber septum

and reinforced with an aluminium closure (Montoya et al.,

1996) and were flushed for 5 min with argon followed by

the addition of C2H2 (20% of head space volume). The

bottles were returned to the original site and depth of

2 O. Hadas et al.

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sampling in the lake and incubated in situ for between 12

to 48 h. At each depth, an additional bottle treated as

above but covered with aluminium foil was used as a

dark control. After incubation, the samples from each

depth were transferred to the laboratory in a dark box and

analysed immediately for C2H4 accumulated in the

sample by injection of 1 mL of the head space gas to a

GC-FID (Shimadzu) using Durapak phenyl isocyanate on

80 ⁄100 Porasil C in a 6¢ · 1 ⁄8¢¢ column, (Supelco), cali-

brated with ethylene (100 ppm standard, Supelco, Cat No:

22572; Sigma – Aldrich4 ).

Chlorophyll a

Chlorophyll a was measured by fluorometry after 90%

acetone extraction using the method of Holm-Hansen

et al. (1965).

Filament and heterocyst counts

Subsamples of water from the various depths were fixed

with Lugol solution, sedimented in aliquots of either 10 or

1 mL (depending on filament concentration) and counted

by the method of Utermohl (Utermohl, 1958) as described

by Berman & Pollingher (1974) using an Axioscope

inverted microscope5 . Biovolume was calculated from

geometric data of an individual species as described in

Hillebrand et al. (1999). The conversion of biovolume to

wet weight (WW) was performed by the expression of

total volume 106 l3 L)1, which equals to lg L)1 (or

mg m)3) based on the assumption that the specific gravity

of the algae is 1 (Berman & Pollingher, 1974).

DIN concentration

Lake water samples were filtered through a 0.45-lm filter

(Schleicher & Schuell6 ), and the concentrations of nitrogen

as NH4+, NO2

) and NO3) were measured using standard

methods (APHA, 2001) or by a Flow Injection autoana-

lyzer (QuikChem 8000, Automated Ion Analyser; Lachat

Instruments7 ) according to the manufacturer protocol. The

detection limit for N forms is 0.3 lMM .

Water temperature and stratification pattern

Temperature profiles were measured once a week down

the entire water column at Station A, located in the centre

of the lake, where lake depth is �42 m. Measurements

were taken from 1969 to 1986 using an underwater

thermometer (Montedoro-Whitney, CA, U.S.A.) and since

1987 using a STD-12 Plus (Applied Microsystems, Sidney,

B.C. Canada). Currently, measurements are taken every

1 cm with an error of ±0.005 �C and averaged for every

1 m. Long-term stratification patterns were calculated

using a simple empirical temperature–depth function

(Rimmer et al., 2011), which systematically defines the

thickness of the epilimnion, metalimnion and hypolim-

nion, the temperature gradient, the depth of the thermo-

cline, and the mean temperatures in the epilimnion and

hypolimnion. Moreover, the degree of stratification for

each profile was evaluated by calculating z (in cm), the

difference between lake volumetric centre Zv and lake

gravimetric centre Zg, (Imberger & Patterson, 1990). The

results were then averaged over each period (�14

profiles), providing an average seasonal stratification

pattern for 39 years (‘‘spring’’ containing data from April

to June; ‘‘summer’’ from July to September; and ‘‘au-

tumn’’ from October to December).

Wind measurements

Meteorological data were obtained from the Tabgha

Meteorological Station located above the lake surface, �

700 m offshore (32�52¢N and 35�33¢ E), at an elevation of

210 ASL. Full meteorological data (air temperature,

relative humidity, global short-wave radiation, down-

welling long-wave radiation, wind speed and direction),

at a time interval of 10 min, are available since 1996

(Rimmer, Samuels & Lechinsky, 2009), with wind speed

and direction measured using a wind monitor MA-05106

(R.M. Young, MI, U.S.A.) at �8 m above lake level.

Another set of wind speed measurements in the same

location is available from 1969 to 1986 obtained using a

mechanical Woelfle chart-recording anemometer. At Lake

Kinneret, a daily Mediterranean westerly sea breeze

commences soon after midday from April to October.

This sea breeze lasts for �5 h during April, �10 h during

July and �5 h during October. At 10 m above the water

surface, it may reach an hourly average speed of 4, 10 and

4 m s)1 during April, July and October, respectively.

Wind speed measurement data for � 40 years are avail-

able from different locations in the Lake Kinneret region.

Results

Appearance of Nostocales

The filamentous nitrogen-fixing cyanobacterium Aphani-

zomenon ovalisporum was first detected in Lake Kinneret in

summer 1994 (Fig. 1). Thereafter, it has appeared each

summer, albeit at a lower abundance than the maximal

(3000 filaments per mL)1, biomass of about

Appearance of Nostocales in Lake Kinneret 3

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63 g WW m)2) observed that year (Pollingher et al., 1998;

Hadas et al., 1999). Cylindrospermopsis raciborskii was first

detected in Lake Kinneret in the summer of 1998 and

reached a maximum of about 27 000 filaments mL)1, with

a biomass of 65 g WW m)2, in August 2005, contributing

82% to the phytoplankton biomass (Fig. 1). The C. raci-

borskii bloom of summer 2005 collapsed in early Septem-

ber followed by a peak in the A. ovalisporum population in

November. The long-term record (Fig. 1) suggests two

alternating periods since the 1990s where A. ovalisporum

dominated the summer–autumn population between 1994

and 2002 and again since 2007, whereas C. raciborskii was

prominent between 2003 and 2006. The repetitive appear-

ance of A. ovalisporum in Lake Kinneret and lakes in

Europe suggests that it expands its range similar to

C. raciborskii. A thorough scrutiny of the long-term phy-

toplankton record revealed a single event of Nostocales

presence in Lake Kinneret during the early 1970s,

although with a smaller biomass of 6 g m)2, which did

not persist in following years (insert in Fig. 1).

In Lake Kinneret, vegetative cells of A. ovalisporum and

C. raciborskii form straight filaments of 25–400 lm length

and 3–4 lm in diameter. A vegetative cell may differen-

tiate into two cell types: (i) heterocyst, capable of fixing

atmospheric dinitrogen into combined nitrogen; and (ii)

akinete, a dormant cell that can survive harsh conditions

and germinate when favoured conditions return. In both

species, heterocysts were abundant at the early stages of

the bloom, enabling the development of the population

under N-depleted conditions (Fig. 2). Interestingly, the

A. ovalisporum heterocyst biomass, both areal and per

filament, was several folds higher than that of C. racibor-

skii, and the heterocyst biomass per unit of Aphanizomenon

total biomass was in most cases 2- to 20-fold higher than

that of Cylindrospermopsis (Fig. 2). Aphanizomenon appears

later in summer, which may suggest that Aphanizomenon is

more dependent on N2 fixation than Cylindrospermopsis.

N2 fixation

Detailed studies of N2 fixation rates in the water column

have been carried out since 2001. For clarity, monthly

Aphaniz

om

enon

, C

ylin

dro

sp

erm

op

sis

bio

ma

ss g

m–

2

0

10

20

30

40

50

60

70

To

tal b

iom

ass g

m–

2

0

100

200

300

400

500

600

700A. ovalisporum

C. raciborskii

Total biomass

94 95 98 9996 97 00 01 02 03 04 05 06 07 08 0993929190

88

27

31

26 46

65

82

31

4039

Year

Year

Bio

mass

g m

0

2

4

6

8

10

Anabaena sp.

A. flos aquae

71 73 75 77 79 81 83 85 87 89 91

Fig. 1 Multiannual variations in the abundance of two Nostocales species and total phytoplankton biomass in Lake Kinneret. Numbers depicted

represent percentage of Nostocales of total phytoplankton biomass.

0

5

10

15

20

25

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

Akinetes

Heterocysts

Filaments

Aphanizomenon ovalisporumFilamentsbiomassg m –2Filamentsbiomassg m –2

H et erocyst s ,aki net es bi omassgm–2H et erocyst s ,aki net es bi omassgm–2

0

5

10

15

20

0.005

0.01

0.015

0.02

Akinetes

Heterocysts

Filaments

Cylindrospermopsis raciborskii(b)

0

0

5

10

15

20

25

J M S J M S J M S J M S

He

tero

cyst

bio

ma

ss

To

tal b

iom

ass

–1 ×

10

00

Year

Aphanizomenon

Cylindrospermopsis

2006 200920082007

(c)

(a)

Fig. 2 Annual variations in heterocysts, akinetes and filaments

(biomass units), areal abundance for (a) Aphanizomenon ovalisporum,

(b) Cylindrospermopsin raciborskii and (c) the ratio of heterocyst bio-

mass per unit filament biomass for both species in Lake Kinneret

during summer–autumn 2006–09.

4 O. Hadas et al.

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profiles for the years 2001 and 2005 are shown for both

Aphanizomenon and Cylindrospermopsis dominance (Fig. 3).

Hourly N2 fixation rates were expressed per unit of

chlorophyll and ⁄or per heterocyst. Nitrogen fixation

occurred within the upper 7 m, with maximal rates

ranging between the water surface and 3 m depth.

Maximal N2 fixation equivalent to 2.4 nmol

C2H4 lg)1 chl h)1 and 73 fmol C2H4 Het)1 h)1 was mea-

sured during the Aphanizomenon bloom in 2001, as

compared with 3.3 nmol C2H4 lg)1 chl h)1 and 26 fmol

C2H4 Het)1 h)1 during the Cylindrospermopsis bloom in

2005 (Fig. 3). Fixation usually started in June and reached

maximum rates in August or September (Fig. 4). The

maximal fixation rate of about 2.5 tonnes per lake day)1

were recorded in August 2005, whereas in 2001, 2004 and

2009, the highest rates observed in September were about

1.81, 1.66 and 1.05 tons per lake day)1, respectively. The

contribution of nitrogen fixation to the lake N budget

during the summer–autumn period was as little as

0.5 tonnes in 2003 and as much as 123 tonnes in 2005

(Table 1). During the summer–autumn months of 2001,

2004, 2005 and 2009, Lake Kinneret was provided with

‘‘new N’’, which during these years was between 63 and

150% of summer DIN inflows from the watershed via the

Jordan River (Table 1).

Dissolved inorganic nitrogen

The annual pattern for DIN concentrations (the sum of

nitrate-N, nitrite-N and ammonium-N) in the upper water

column of Lake Kinneret (Fig. 5) is characterised by high

winter–spring values, mainly due to external loads

Aug 2001

0.5 1.0 1.5 2.0 2.5

De

pth

(m

)

0

3

6

9

12

1520 40 60 80 100

Sep 2001

20 40 60 80 100

0.5 1.0 1.5 2.0 2.5

Oct 2001

20 40 60 80 100

0.5 1.0 1.5 2.0 2.5

nmol C2H4 mg–1chl h–1

fmol C2H4 Het–1 h–1

fmol C2H4 Het–1 h–1

1 2 3 4

De

pth

(m

)

0

3

6

9

12

1510 20 30 40

C2H4 chla–1

h–1

C2H4 Het–1

h–1

Jun 2005

nmol C2H4 µg–1 chl h–1

1 2 3 4

10 20 30 40

Jul 2005

1 2 3 4

10 20 30 40

Aug 2005

Aphanizomenon

Cylindrospermopsis

Fig. 3 Depth profiles for N2 fixation rate measured in Lake Kinneret during the 2001 and 2005 summer bloom events. Hourly N2 fixation rates

are presented per unit of chlorophyll and per heterocyst.

Appearance of Nostocales in Lake Kinneret 5

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(winter floods entering the lake mainly via the Jordan

River). The DIN levels decline rapidly in late spring to

early summer owing to biological assimilation and deni-

trification processes (indicated as a transition period in

Fig. 5). The DIN concentration gradually increases in

November–December as the thermocline deepens and

hypolimnetic ammonium reaches the epilimnion where it

is subsequently oxidised to nitrate (Fig. 5). During the two

representative years, 2001 and 2005, NH4+-N concentra-

tions ranged from traces (mid-July) to 0.35 mg N L)1 at

the end of December. Although the same trend was

observed in both years, differences in summer ammonium

concentrations were recorded. Small pulses of ammonium

were observed in August 2001, whereas ammonium was

hardly detected in the epilimnetic euphotic zone in

August 2005, probably due to the high affinity for

ammonium and its consumption by the C. raciborskii

bloom. Ammonium concentration sharply increased in

September 2005 (Fig. 5), in accordance with the collapse

and degradation of the Cylindrospermopsis population.

The multiannual DIN record (Fig. 6) was divided into

two periods: first, prior to the invasion of Nostocales to

Lake Kinneret (1969–93, empty boxes in Fig. 6) and,

second, after the invasion (1994–2009, filled boxes in

Fig. 6). The data are for three annual stages: (i) February–

April representing the winter–rainy season with high DIN

concentrations, (ii) July–September representing the sum-

mer with low DIN levels and (iii) June representing, in

most years, a transition phase in which DIN concentra-

tions gradually decreased (Figs 5 & 6). This transition

period slightly varied among years, but generally started

in late April and ended in July (Fig. 5). No significant

difference in the concentrations of the various DIN species

during the winter–spring season was observed for both

periods. In the second annual stage (July–September) and

in the transition stage (June), NO3) and NH4

+ concentra-

tions differed significantly between the two periods

(Fig. 6).

The decline in DIN concentration during the transition

period (April to June) can be described by an exponential

removal equation:

0.0

0.5

1.0

1.5

2.0

2.5

Jun Jul Aug Sep Oct Nov

To

nn

es N

2 f

ixe

d p

er

La

ke

d–

1

Month

2001

2002

2003

2004

2005

2006

2008

2009

Fig. 4 Estimated monthly summer atmospheric N2 fixation to Lake

Kinneret for the years 2001–09.

Table 1 The contributions of nitrogen fixation and external river

loading to the Lake Kinneret N budget during summer–autumn

(June–October) 2001–09

Year

N2 fixed per

lake (Tonnes)

External load (tonnes)

via the Jordan River

2001 97.5 65

2002 12.5 136

2003 0.5 335

2004 83.4 279

2005 123 195

2006 35.1 153

2007 1.6 152

2008 59 44

2009 81.1 94

DIN

, N

H4,

NO

2,

NO

3 (

mg

N L

–1)

0.0

0.1

0.2

0.3

0.4

DIN

NO3

NH4

NO2

2005

Winter Transition Summer – Autumn Winter

Month

0.0

0.1

0.2

0.3

0.42001

J F M A M J J A S O N D

Winter Transition Summer – Autumn Winter

Fig. 5 The annual pattern for dissolved inorganic nitrogen in the

upper water column of Lake Kinneret for two representative years,

2001 and 2005.

6 O. Hadas et al.

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DINt ¼ DIN0 � exp�kt

where DIN0 is the initial DIN concentration in the

epilimnion at the end of the winter period (usually April),

DINt is the DIN concentration at time t at the beginning of

summer and k is the removal rate. Lake Kinneret DIN data

during the annual transition period were used to estimate

the removal rate for the last 20 years (Fig. 7). The removal

rate varied among years as did the time by which a DIN

threshold of 30 lg N L)1 was measured and might have

triggered the growth of Nostocales (Vrede et al., 2009)

(Table 2). The annual DIN removal rate (k) was calculated

for the periods 1987–2009. The multiannual average

removal rates were significantly different for the periods

before and after Nostocales invasion (0.014 ± 0.004 and

0.023 ± 0.007 day)1, respectively, P £ 0.05). The period

until 1994 was characterised by more rainy years with

the lowest annual removal rate (0.008 day)1) in 1992. In the

Feb – Apr

NH

4 (

mg

N L

–1)

NO

3 (

mg

N L

–1)

DIN

(m

g N

L–

1)

0.00

0.05

0.10

0.15

0.20

0.00

0.15

0.30

0.45

0.60

0.00

0.15

0.30

0.45

0.60

NH

4 (

mg

N L

–1)

NO

3 (

mg

N L

–1)

DIN

(m

g N

L–

1)

0.00

0.05

0.10

0.15

0.20

0.00

0.15

0.30

0.45

0.60

0.00

0.15

0.30

0.45

0.60

NH

4 (

mg

N L

–1)

NO

3 (

mg

N L

–1)

DIN

(m

g N

L–

1)

0.00

0.05

0.10

0.15

0.20

0.00

0.15

0.30

0.45

0.60

0.00

0.15

0.30

0.45

0.60

69-93

94-09

June Jul – Sep

T value 3.34P value 0.0009DF 459

T value 3.43P value 0.0006DF 454

T value 3.82P value 0.00015DF 460

T value 2.4P value 0.017DF 461

T value 1.358P value 0.174DF 461

T value 2.06P value 0.039DF 463

T value 3.06P value 0.0025DF 164

T value 2.42P value 0.016DF 164

T value 2.65P value 0.0086DF 164

Fig. 6 Box plot presentation for the multiannual dissolved inorganic nitrogen (DIN) concentrations (NO3, NH3 and total dissolved inorganic N)

in the upper 10-m layer of Lake Kinneret. Data are divided into two periods: (i) 1969–93 prior to the invasion of Nostocales to Lake Kinneret and

(ii) 1994–2009 after the invasion. The data are presented for three annual stages: (i) February–April representing the winter–rain season with

high DIN concentrations; (ii) July–September representing the summer with low DIN concentrations and (iii) June representing the DIN

transition phase. This transition period slightly varied among years but generally started in late April and terminated in July. The lower

boundary of the box indicates the 25th percentile, a line within the boundary marks the average, a dashed horizontal line marks the median, and

the upper boundary of the box indicates the 75th percentile. Whiskers above and below the box indicate the 95th and 5th percentiles. The

parameters of a t-test (T, P and DF) are presented.

Appearance of Nostocales in Lake Kinneret 7

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period after the appearance of Nostocales, low DIN

concentrations in April were recorded owing to consecu-

tive drought years with minor or no winter flood events,

which resulted in low nitrogen loads entering the lake via

the Jordan River. A high removal rate for DIN

(0.037 day)1) was calculated for 1997 with a low DIN

concentration (13 lg N L)1) recorded in early June.

Temperature, wind and stratification data

The physical properties in Lake Kinneret have been

changed in recent decades as demonstrated by Rimmer

et al. (2011). They showed changes in the stratification

pattern of the lake that were the result of lake level

decrease, a slight increase in air temperature during the

Year19

87

1988

1989

1990

1991

1992

1993

1994

1995

1996

1997

1998

1999

2000

2001

2002

2003

2004

2005

2006

2007

2008

Exponential re

moval ra

te (

d–

1)

0.00

0.01

0.02

0.03

0.04

Average = 0.014 d–1

Average = 0.023 d–1

Rainy year

Dry year

Average year

Fig. 7 The exponential removal rate for dissolved inorganic nitrogen concentration calculated for each year (1987–2008) for the spring–summer

period. The bar pattern represents the overall winter precipitation as described in Table 4: Rainy year (white bar), Dry year (diagonal pattern)

and Average year (horizontal line pattern).

Table 2 Summary of the annual distribution of dissolved inorganic nitrogen in the upper water layer in Lake Kinneret for the years 1987–2008.

An exponential removal rate of dissolved inorganic nitrogen (DIN) was calculated for each year for the spring–summer period. The maximal

and minimal DIN values are given together with the timing (Julian days) they were measured. Rainy year–the monthly water inflow exceeded

80 MCM at least during one winter month. Average year – the monthly water inflow ranged between 40 and 80 MCM at least during one winter

month. Dry year – the monthly water inflow never exceeded 40 MCM during the winter months

Year

Exponential removal rate Max. DIN concentration Min. DIN concentration

Value (day)1) Corr. Coef. mg L)1 Timing (day) mg L)1 Timing (day)

1987 (Rainy) 0.0151 0.9 0.414 41 0.017 221

1988 (Rainy) 0.0192 0.819 0.366 115 0.010 262

1989 (Dry) 0.0155 0.882 0.317 50 0.018 211

1990 (Day) 0.0107 0.884 0.276 45 0.026 203

1991(Dry) 0.0148 0.93 0.271 45 0.029 230

1992 (Rainy) 0.0082 0.945 1.011 96 0.175 327

1993 (Rainy) 0.0110 0.903 0.505 52 0.030 283

1994 (Average) 0.0139 0.903 0.341 35 0.017 205

1995 (Average) 0.0159 0.79 0.453 64 0.021 227

1996 (Average) 0.0331 0.89 0.433 91 0.026 200

1997 (Average) 0.0366 0.857 0.317 76 0.013 166

1998 (Average) 0.0136 0.848 0.222 95 0.020 220

1999 (Dry) 0.0283 0.9025 0.340 52 0.020 160

2000 (Dry) 0.0282 0.903 0.382 65 0.014 180

2001 (Dry) 0.0257 0.889 0.296 45 0.017 150

2002 (Average) 0.0225 0.773 0.373 83 0.016 190

2003 (Rainy) 0.0146 0.86 0.558 103 0.048 257

2004 (Rainy) 0.0242 0.91 0.437 68 0.017 228

2005 (Rainy) 0.0236 0.91 0.445 72 0.020 170

2006 (Average) 0.0258 0.834 0.305 85 0.016 169

2007 (Average) 0.0179 0.677 0.311 42 0.01 175

2008 (Dry) 0.0186 0.768 0.312 76 0.030 174

8 O. Hadas et al.

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spring and summer, and the reduction in inflows to the

lake. The observed changes are elaborated here:

Temperature. Detailed analyses of the average temperature

in the epilimnion of Lake Kinneret from 1969 to 2009

revealed an increased temperature of �1 �C, with average

trends of 0.015, 0.034 and 0.022 �C yearr)1 during the

spring (April–June), summer (July–September) and au-

tumn (October–December), respectively. The 800 temper-

ature measurements from June to October in the database

for this period were compiled for the upper layer of the

water column (0–5 m). Table 3 shows that water temper-

atures above 28 �C were recorded 263 times out of the 800

measurements. Between 1994 and 2009, out of the 300

available records, 50%were equal to or exceeded 28 �C, as

compared to only 22% between 1969 and 1993 (Table 3).

Furthermore, water temperatures of 29 �C and above were

recorded 91 times in the upper layer between 1994 and 2009

but only few times during 1969–93 (Table 3). An extreme

example for this trendwas recordedduring a 21-dayperiod

in August 2010 with daily water temperatures above 31 �C

in the whole epilimnetic water column (data not shown).

Wind. Two periods of three consecutive years of hourly

wind speed data (1970–72 and 1997–99) were analysed.

Those were selected, as an example, owing to the few gaps

(96 and 90% of the data were available for the first and

second periods, respectively) and high measuring quality.

The hourly wind speed distribution was calculated for the

two periods and scaled to produce similar distributions of

low wind speed. The scaling was necessary because

measurement devices were different, as well as the

accurate height of the devices above lake level. The scaled

distribution for both periods were in good agreement for

the lower wind speed, but a clear difference in the upper

40% can be identified, indicating higher wind speed

events during the 1970s, reaching wind velocities of

12 m s)1 (Fig. 8). On average, the wind speed events

larger than 2 m s)1 were 6.4 ± 1.5% higher during the

1970s than during the 1990s. It was also found that these

high wind speed events were typical for June, July and

August. These results are in agreement with Saaroni et al.

(2010), who indicated a probable reduction in the fre-

quency of peak wind speed events from the 1970s to the

present.

Stratification. Analysis of a 39-year record from 1969 to

2008 (Table 4) showed that epilimnion thickness was

Table 3 High-temperature events (equal to or above 28 �C) in the

upper water layer (average values for 0–5 m) at Station A in Lake

Kinneret before (1969–93) and after the invasion of Nostocales (1994–

2009). Values indicate the number of events in which the average

temperature was equal to or higher than the specified temperature.

Values in parentheses are the percentages of these events out of the

entire measurement record

Temperature

(�C)

1969–93 (total of 500

measurements)

1994–2009 (total of 300

measurements)

‡28.0 112 (22%) 151 (50%)

‡28.5 58 (12%) 113 (38%)

‡29.0 24 (5%) 67 (22%)

‡29.5 7 (1.4%) 39 (13%)

‡30.0 2 (0.4%) 11 (4%)

1970–1972

1997–1999

00 . 20 . 40 . 60 . 81 . 00481 21 6

0 . 6 0 . 7 0 . 8 0 . 9 1D i f f e r e n c e

Quantiles (fraction)

Win

d s

pe

ed

(m

s–

1)

Win

d s

pe

ed

diffe

ren

ce

(m

s–

1)

Fig. 8 Scaled distribution of hourly wind speed during 1970–72

compared to 1997–99 and the difference between them (note the

different vertical axis).

Table 4 Average and standard deviation (SD) of epilimnion and metalimnion thickness, epilimnion temperature and stratification strength (z)

in Lake Kinneret during spring (Sp), summer (Su) and autumn (Au) from 1969 to 1993 and from 1994 to 2008

Epilimnion thickness (m) Epilimnion thickness (m)

Epilimnion temperature

(�C) z (cm)

Sp Su Au Sp Su Au Sp Su Au Sp Su Au

1969–93 AVG 9.21 14.82 21.50 11.36 7.63 4.61 22.69 27.86 22.16 0.58 1.05 0.43

SD 1.20 1.29 1.82 1.79 0.96 0.88 0.65 0.44 0.46 0.08 0.12 0.08

1994–2008 AVG 8.78 14.31 20.32 10.35 7.09 4.18 23.43 28.59 22.82 0.60 1.08 0.45

SD 0.87 0.87 1.54 1.34 0.66 0.82 0.68 0.61 0.55 0.06 0.05 0.06

Appearance of Nostocales in Lake Kinneret 9

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lower following 1994 (the difference varied between

0.48 m during the spring, 0.51 m during the summer

and 1.18 m during the autumn). A similar trend was

observed in the metalimnion thickness. The epilimnion

temperature was higher in all seasons during the second

period (Table 4), in accordance with the higher frequency

of extreme temperature events (Table 3). The average lake

stratification strength (z, cm) during the spring increased

from 0.58 cm in the early period to 0.60 cm in the later

period, and a similar trend was observed in summer and

autumn (Table 4), suggesting stronger stratification in the

second period. These differences between the averages in

the two periods are not statistically significant, but are

consistent trends demonstrated by Rimmer et al. (2011).

Discussion

The expansion of Nostocales cyanobacteria into meso-

trophic and oligotrophic lakes in temperate zones appears

to be a worldwide phenomenon (Padisak, 1997; Briand

et al., 2004; Wiedner et al., 2007; Mehnert et al., 2010;

Sukenik et al., 2012). This could be explained by the

tolerance of Nostocales to a wide range of environmental

conditions and cellular differentiation during their life

cycle (Padisak & Reynolds, 1998; Vidal & Kruk, 2008).

Their ability to form dormant cells (akinetes) that stay in

the sediment and germinate to produce vegetative cells

following improved environmental conditions is an

important advantage. The vegetative cells are then

brought to the water column by gas vesicles (Hense &

Beckman, 2006; Kaplan-Levy et al., 2010). Furthermore,

the gas vesicles within the vegetative filaments of Nosto-

cales enable migration between the nutrient-rich meta-

limnion and the illuminated upper epilimnion (Hadas

et al., 1999). Akinetes in Lake Kinneret develop as Nosto-

cales populations reach their maxima (Fig. 2) (Pollingher

et al., 1998; Hadas et al., 1999).

The previous appearance of Anabaena sp. and Aphani-

zomenon sp. in the early 1970s (Fig. 1) was not repeated in

subsequent years, which raises the question ‘‘Why were

Nostocales not a common component of the summer

phytoplankton population in Lake Kinneret before 1994¢¢?

The answer may be related to changes in the environmen-

tal conditions during the last 20 years, some of which are

presented in this study. The rising water temperature

(Table 3), lower water turbulence (driven by wind, Fig. 8),

reduced DIN level (Fig. 5), pulses of phosphorus during

summer (Hadas et al., 1999) and other biotic and abiotic

parameters may all contribute to the success of Nostocales.

Changes in the physical properties in Lake Kinneret in

recent decades were demonstrated by Rimmer et al.

(2011), who showed changes in the stratification pattern

of the lake that were the result of lake-level decrease, a

slight increase in air temperature during the spring and

summer and the reduction in inflows to the lake. Lower

wind speed might also contribute to a stronger stratifica-

tion and a shallower mixed depth. All the above long-

term changes (lake-level reduction, inflow reduction,

higher air temperature) affect the stratification pattern in

the same direction, by increasing the lake epilimnetic

temperature, reducing the epilimnion depth and increas-

ing the thermal gradient across the metalimnion.

One of the physiological advantages of Nostocales is

their ability to fix atmospheric dinitrogen and thus

proliferate in an environment depleted in combined

inorganic nitrogen, conditions that prevail in Lake Kin-

neret during the summer. Nitrogen depletion has been

evident in the lake since the 1990s, developing earlier in

the summer. The absence of Nostocales before 1994 is the

main reason why nitrogen fixation in Lake Kinneret was

rather low and was mostly attributed to photosynthetic

bacteria in the metalimnion (Cavari, 1978; Butow &

Bergstein-Ben Dan, 1992). High rates of nitrogen fixation

at 0–7 m depth were observed after the invasion of

Nostocales in 1994 (Fig. 3). Because of the strong summer–

autumn stratification, almost no N is supplied to the

epilimnion via the metalimnic layer, although some

intrusions may occur. The low d15N of A. ovalisporum

samples collected in the lake during its 1994 bloom

(0.32&) cannot be accounted for by vertical transport from

the hypolimnion of NH4+, which had higher d15N (Hadas,

Altabet & Agnihotri, 2009), and instead indicates that

nitrogen fixation was being used as an important source

of nitrogen (Minagawa & Wada, 1986; Montoya et al.,

2004). The increase in total particulate N (TPN) recorded

in Lake Kinneret by the end of 1994, following the

A. ovalisporum bloom, was attributed to high rates of N2

fixation (Gophen et al., 1999).

The tendency for alternation between A. ovalisporum

and C. raciborskii in Lake Kinneret during recent years

may suggest that C. raciborskii, which appears at the

beginning of summer, has higher affinity for ammonium

and may utilise it, whereas Aphanizomenon must fix N2 via

an energetically costly process (Figueredo, Giani & Bird,

2007). The threefold higher N2 fixation we measured by

Aphanizomenon heterocysts may be due to a more efficient

supply of carbon substrata by neighbouring vegetative

cells, from both sides, whereas the terminal Cylindro-

spermopsis heterocysts may be substratum-limited. C. rac-

iborskiiwas found to maintain high net growth rates under

fluctuating DIN conditions by using facultative diazotro-

phy (Moisander et al., 2012) and in the case of Lake

10 O. Hadas et al.

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Kinneret gained an advantage over A. ovalisporum in

which diazotrophy is inhibited by ammonium (Hadas,

unpublished data).

Is the measured nitrogen fixation rate sufficient to

support the summer Nostocales population in Lake

Kinneret? Based on the Redfield ratio (C: N 6.6 molar

ratio), about 20 mmol N m)2 day)1 are required to sup-

port Lake Kinneret summer primary production of about

133 mmol C m)2 day)1 (Berman et al., 1995). Considering

Nostocales biomass and the maximal nitrogen fixation

rates (1.6 mmol N m)2 day)1), nitrogen fixation can sup-

ply between 10 and 80% of the Nostocales demand for

nitrogen. This suggests that other sources such as NH4+

pulses or DON were used to support the maximal

biomass of the cyanobacterial population.

The invasion of Nostocales into Lake Kinneret coincided

with summers of higher temperatures than in earlier years.

Degradation of the massive Peridinium bloom (twice the

average) in June 1994 provided a pulse of phosphorus into

the lake and, combined with temperatures of 29 �C in the

water column, may have created the conditions for the first

bloom of A. ovalisporum in late summer–autumn 1994. The

appearance of C. raciborskii in 1998 was rather expected

owing to a further rise in water temperatures since 1998 to

above 30 �C. This species is known to appear at temper-

atures >24 �C (Padisak, 1997; Briand et al., 2004) and, like

Aphanizomenon (Bar-Yosef et al., 2010), may have an

advantage under phosphate limitation (Isvanovics et al.,

2000; Sprober et al., 2003; Posselt, Burford & Shaw, 2009).

These elevated ambient temperatures during the summer,

deeper in the epilimnetic water column, might have been

one of the variables supporting the establishment of the

Nostocales community in Lake Kinneret as well as in

various temperate lakes and may be indicative of global

warming (Briand et al., 2004).

Shifts in species composition of phytoplankton com-

munities may be induced by changes in the turbulence of

lake waters (Huisman et al., 2004; Spigel & Imberger, 1987;

Reynolds, 1998). Intensified mixing in lakes could cause

shifts in dominance from cyanobacteria towards chloro-

phytes and diatoms (Reynolds et al., 1983). In Lake

Kinneret, wind storms in February lasting for 2–4 days

(Serruya, 1978b; Shilo et al., 2007) might have helped the

diatom Aulacoseira granulata to outcompete the dinofla-

gellate Peridinium gatunense. In addition, a daily westerly

wind (maximum velocities of 12 m s)1) starting in late

May could explain why until 1994 Nostocales were not

part of the phytoplankton community in the lake. Com-

paring the wind strength at the two periods before and

after the invasion of Nostocales to Lake Kinneret (Fig. 8)

showed that calmer weather was observed after the

invasion, benefitting cyanobacteria and especially Nosto-

cales. Saaroni et al. (2010) suggested that the considerable

warming in the summer results from a rapid increase in

the occurrence of the weak Persian Trough (which is a

warm type) with a relatively weak maritime advection

occurring between May and October with a main peak in

August. Furthermore, Berman & Shteinman (1998) com-

puted the dissipation of turbulent kinetic energies (TKE)

and the intensity of turbulent mixing in the lake, showing

that the first appearance of A. ovalisporum in Lake

Kinneret in August–September 1994 coincided with a

period of lower rates of TKE dispersion and a shift from

vertical to horizontal dominance of the turbulent eddy

spins. This suggests that the changes in the turbulence

climate of Lake Kinneret were an important factor in

determining shifts in the phytoplankton assemblage.

The expansion and establishment of Nostocales in Lake

Kinneret provides a special example for the widely

accepted hypothesis about cyanobacterial dominance

under low N ⁄P ratio (Schindler, 1977; Smith, 1983). Lake

Kinneret was thought to be P-limited during summer–

autumn (Serruya et al. (1974). Nostocales appearance can

be predicted in response to the diminishing DIN, and the

intensity of the bloom is associated with the availability of

phosphate. We suggest that the recent appearance and

persistence of the Nostocales population in Lake Kinneret

(since 1994) was supported by a combination of their

diazotrophic activity with regional climatic and limnolog-

ical conditions such as increased temperatures, changes in

wind regime that affect physical mixing processes and

water turbulence, and variations in nutrient availability.

Acknowledgments

We thank Miki Schlichter for data from the Kinneret

Limnological Laboratory database and T. Zohary for

biomass data. This study was part of the Joint German-

Israeli-Project (FKZ 02WT0985, WR803) ‘‘Life-cycle of

Nostocales – An intrinsic dynamic component essential

to predict cyanobacterial blooms in lakes and reservoirs’’,

funded by the German Ministry of Research and Tech-

nology (BMBF) and Israel Ministry of Science and Tech-

nology (MOST). The continuous support of the Israel

Water Authority is acknowledged.

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Appearance of Nostocales in Lake Kinneret 11

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