Biologia 63/6: 843—851, 2008
Section Botany
DOI: 10.2478/s11756-008-0111-2
Freezing and desiccation injury resistance in the filamentous green
alga Klebsormidium from the Antarctic, Arctic and Slovakia*
Josef Elster1*, Peter Degma2, Ľubomír Kováčik3, Lucia Valentová3,
Katarína Šramková3 & Antonio Batista Pereira4
1
Institute of Botany, Academy of Sciences of the Czech Republic, CZ-379 82 Třeboň & Faculty of Science, University of
South Bohemia, Branišovská 31, CZ-37005 České Budějovice, Czech Republic; [email protected]
2
Department of Zoology, Faculty of Natural Sciences, Comenius University in Bratislava, Mlynská dolina B-1, SK-84215
Bratislava, Slovakia
3
Department of Botany, Faculty of Natural Sciences, Comenius University in Bratislava, Révová 39, SK-81102 Bratislava,
Slovakia
4
Universidade Federal do Pampa – UNIPAMPA, Campus São Gabriel, Av. Antônio Trilha, 1847, CEP: 97300-000/RS,
Brasil
Abstract: The freezing and desiccation tolerance of 12 Klebsormidium strains, isolated from various habitats (aeroterrestrial, terrestrial, and hydro-terrestrial) from distinct geographical regions (Antarctic – South Shetlands, King George
Island, Arctic – Ellesmere Island, Svalbard, Central Europe – Slovakia) were studied. Each strain was exposed to several
freezing (−4 ◦C, −40 ◦C, −196 ◦C) and desiccation (+4 ◦C and +20 ◦C) regimes, simulating both natural and semi-natural
freeze-thaw and desiccation cycles. The level of resistance (or the survival capacity) was evaluated by chlorophyll a content,
viability, and chlorophyll fluorescence evaluations. No statistical differences (Kruskal-Wallis tests) between strains originating from different regions were observed. All strains tested were highly resistant to both freezing and desiccation injuries.
Freezing down to −196 ◦C was the most harmful regime for all studied strains. Freezing at −4 ◦C did not influence the survival of studied strains. Further, freezing down to −40 ◦C (at a speed of 4 ◦C/min) was not fatal for most of the strains. RDA
analysis showed that certain Antarctic and Arctic strains did not survive desiccation at +4 ◦C; however, freezing at −40 ◦C,
as well as desiccation at +20 ◦C was not fatal to them. On the other hand, other strains from the Antarctic, the Arctic,
and Central Europe (Slovakia) survived desiccation at temperatures of +4 ◦C, and freezing down to −40 ◦C. It appears that
species of Klebsormidium which occupy an environment where both seasonal and diurnal variations of water availability
prevail, are well adapted to freezing and desiccation injuries. Freezing and desiccation tolerance is not species-specific nor
is the resilience only found in polar strains as it is also a feature of temperate strains.
Key words: Klebsormidium; green algae; algal adaptation; freezing and desiccation; Antarctic; Arctic; Slovakia
Introduction
Klebsormidium P.C. Silva, Mattox & Blackwell (Klebsormidiophyceae, Streptophyta) is one of the most
widespread green algal genera found around the world
in aero-terrestrial, terrestrial, and hydro-terrestrial
habitats (Lokhorst 1996; Poulíčková et al. 2001; Elster
2002; Škaloud 2006; Rindi 2007, Sluiman et al. 2008).
The genus Klebsormidium has posed a taxonomic challenge for generations of phycologists, because of its morphological and biological simplicity (simple construction of the thallus, unbranched uniseriate filament, with
undifferentiated cylindrical cells, that possess a single
parietal plate- or disc-shaped chloroplast, and the lack
of information about their sexual reproduction). The
many unresolved taxonomic questions, together with its
high ecological importance in various marginal and/or
extreme environments, brought Klebsormidium to the
attention of the currrent researchers.
Klebsormidium frequently occupies environments
where both a seasonal and diurnal variation of water
availability exists. These habitats range from aquatic
and semi-aquatic, to dry conditions, and differ in periodicity, amplitude, synchronicity, and regularity. Further, they initiate a number of ecological and physiological acclimations, as well as adaptation responses. For
several years we have studied the algal ecology from various aero-terrestrial, terrestrial, and hydro-terrestrial
habitats in the Arctic (Ellesmere Island: Elster & Svoboda 1996; Elster et al. 1997, Svalbard: Kubečková et
* Presented at the International Symposium Biology and Taxonomy of Green Algae V, Smolenice, June 26–29, 2007,
Slovakia.
This paper is dedicated to the memory of the late Dr. Bohuslav Fott (1908–1976), Professor of Botany at the Charles
University in Prague, to mark the centenary of his birth.
c 2008 Institute of Botany, Slovak Academy of Sciences
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Material and methods
Fig. 1. Map of the world with location of sampling sites.
al. 2001; Kaštovská et al. 2005, 2007), the Antarctic
(King George Island) and Central Europe (Slovakia:
Uher et al. 2004, 2005, 2006). In this study, unialgal
Klebsormidium strains were subjected (in their natural
conditions) to extremes of water availability gradients,
and to rapid fluctuations across these extremes. The
speed at which water states can change between liquid, ice, snow, and complete dryness, mainly at low
or high temperatures, is one of the most important
ecological and physiological factors. On a physiological level, changes in the temperature and water status provoke a series of adaptive responses such as
developing resistance and tolerance to cold, freezing,
drought, desiccation and salinity stress (Elster & Benson 2004). Most of these ecological and physiological
acclimation/adaptation strategies can also be found in
selected habitats at temperate latitudes (e.g. various
aero-terrestrial and mountainous habitats). However,
Elster & Benson (2004) have suggested that the physical and chemical conditions that are imposed, uniquely,
by polar environments, have selected for particularly
specific and resilient sets of adaptive biological characteristics.
The aim of this study was to assess the freezing and desiccation tolerances of 12 Klebsormidium
strains, isolated from various habitats: aero-terrestrial
(tombstone, gravelstone, limestone, green sand, whale
bone remains), terrestrial (edaphic and/or lithophytic)
and hydro-terrestrial (glacial stream periphyton, cryoconite, and subglacial river sediment) in distinct geographical regions (the Antarctic – South Shetlands,
King George Island, the Arctic – Ellesmere Island, Svalbard, and Central Europe – Slovakia). It is expected
that improved knowledge of resistance to freezing – desiccation injuries in the genus Klebsormidium will be
helpful in providing answers to the following questions:
(i) Why does this group of algae frequently occupy various marginal or extreme localities and habitats worldwide? (ii) Can we use such information to better understand the biology and taxonomy of this group of algae?
Sample collection, culturing and isolate maintenance
Algal mats from several types of habitats (aero-terrestrial,
terrestrial, and hydro-terrestrial) of the Antarctic (King
George Island), the Arctic (Svalbard and Ellesmere Island),
and Slovakia were collected (Fig. 1, Table 1). Algal samples collected in the field were transported to the laboratory in a frozen state. Isolation was performed on plates
with agarized Z medium (Staub 1961). Plates were streaked
with a small amount of field material, and cultivated at a
temperature ca. 18 ◦C, in dim light (ca. 50 µmol photons
m−2 s−1 ). After a few days of cultivation, algal colonies had
developed which were transferred separately to sterile agar
plates. Unialgal Klebsormidium strains were cultured at a
temperature of 8 ◦C and a photon fluence rate of 80 µmol
photons m−2 s−1 . The strains’ morphologies were studied
using a Leica DM 2500 light microscope (Table 1). Microphotographs were taken with an ARTCAM 300MI 3Mpxl
CMOS USB 2.0 Camera, equipped with Quick PHOTO MICRO 2.1 software.
Freezing and desiccation experiments
A dense suspension of each Klebsormidium strain (in 2 mL
cryovials) was placed in a programmable freezer (Planer
Kryo 10), and exposed to different cooling regimes (Table 2)
that simulated both natural and seminatural freeze-thaw cycles (Stibal & Elster 2005; Šabacká & Elster 2006; Machová
et al. 2008). In addition, two aliquots were desiccated for
three days in a desiccator at 4 ◦C and 20 ◦C, respectively.
After the exposure to the described regimes, the cryovials
with the algae were either quickly melted in a water bath
at 40 ◦C for ca. 2–5 min (if frozen), or re-suspended in ca.
0.5 mL of liquid medium (if desiccated).
After the freezing and desiccation experiments, the resuspended algal inoculum (1 mL) was uniformly spread by
a glass rod on a 2% agar plate, with nutrient solution Z.
The agar plates were maintained at a temperature of 8 ◦C
and a photon fluence rate of 80 µmol photons m−2 s−1 for 3
days.
The chlorophyll-a (Chl-a) content, both before and
after the freezing/desiccation experiments, was estimated
spectrophotometrically on Whatman GF/C filters. Filters
with algal suspension were covered with a mixture of 90%
acetone and methanol (5:1 by volume) (Pechar 1987) and
ground in a frictional mortar. The extract was transferred
into a vial, and kept in a dark refrigerator overnight. The
acetone – methanol extract was then adjusted to the set
volume, pooled, and centrifuged for 15 min at 6000 rpm to
remove fine sediments. Absorbance was measured by a spectrophotometer at 665 and 750 nm, before and after acidification with 10−3 M HCl for 1 h. Chl-a and phaeophytin were
then calculated, using the standard equations of Marker et
al. (1980).
Viability evaluations were done by controlled cultivation of algal colonies on agar plates (Lukavský 1975). Agar
plates were maintained under the same conditions as described above, and viabilities were determined under the
light microscope using the formula V = (NL × 100)/(NL +
ND ) where V is the viability in %, NL the number of living
cells in filaments, and ND the number of dead filament cells.
Chlorophyll fluorescence of the algal biomass was measured by a FluorCam 700 MF, version 3.013 (PSI, Brno,
Czech Republic). The slow (Kautsky) induction kinetics of
fluorescence was used (Kautsky & Hirsch 1934). Petri dishes
with algal biomass were initially kept in darkness for 10
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Table 1. Localities and habitat characteristics.
Strain (used acronym,
isolators, year/collection n.)
Morphometrical and morphological characteristics of the strains
Locality, habitat characteristics
Width (µm)
Length (µm)
Characteristics in culture
LUC 1 = UHER 2003/06
Slovakia, Bratislava, cemetery
Kozia brana, tombstone of Adolf
Orkonyi (1877), neogene calcarenite,
shadow and humid; 48◦ 08 87.9 N,
17◦ 05 97.1 E
5.1–8.1
(3)8.1–10.2(16.3) easy fragmentation, short filaments (up to 100 cells); in
liquid medium 1st type of
growth habit
LUC 2 = KOVÁČIK CHS
1998/13
Slovakia, Bratislava, subterranean jewish cemetery (crypt)
Chatam Sofer, gravestone of rabbi
Hirsch Spira (1727); 48◦ 08 52.3 N,
17◦ 05 46.4 E
6.1–8.1
(4)9.2–14.3(22.4) formation of H-pieces, filaments up to 500 cells; in
liquid medium 2nd type of
growth habit
LUC 3 = ŠRÁMKOVÁ GO
5/4
Slovakia, cave Gombasecka, in front
of the Herényi Hall – right near steps,
closely the lamp, limestone substrata;
48◦ 30 41.3 N, 20◦ 27 50.8 E
4.5–6.1
(4)7.5–11.2(16.3) formation of H-pieces, filaments up to 100 cells (sometimes up to 500 cells); in
liquid medium 1st type of
growth habit
LUC 4 = JANČUŠOVÁ ANT Antarctica, King George Island, Ad2004/1
miralty Bay, Crepin Point, green sand
near the bird nests; 62◦ 05 30.3 S,
58◦ 28 32.3 W
5.1–6.1
(5.1)8.1–13.2(20.4) filaments up to 1000 cells;
in liquid medium 1st type of
growth habit
LUC 5 = JANČUŠOVÁ ANT Antarctica, King George Island, Ad2004/2
miralty Bay, Crepin Point, green
sand near bird nests; 62◦ 05 30.3 S,
58◦ 28 32.3 W
5/4/2001 (7.1)10.2–17.3(22.4) filaments up to 1000 cells,
cultivated at 12 ◦C; in liquid
medium 1st type of growth
habit
LUC 6 = KOVÁČIK ANT
2003/87a
Antarctica, King George Island,
Keller Peninsula; green growth
on remainder of whale bone
near the seashore; 62◦ 05 02.9 S,
58◦ 25 19.0 W
6.1–8.1
(3)6.1–12.2(21.4) H-pieces, filaments made up
of about 500–1000 cells growing in very compact tutfs;
in liquid medium 1 type of
growth habit
LUC 7 = KOVÁČIK ANT
2003/87b
Antarctica, King George Island,
Keller Peninsula; green growth
on remainder of whale bone
near the seashore; 62◦ 05 02.9 S,
58◦ 25 19.0 W
7.1–8.1
(5.1)9.2–12.2(20.4) filaments up to 500 cells, formation of H-pieces; in liquid medium 1 type of growth
habit
LUC 8 = KOVÁČIK ANT
2003/31
Antarctica, King George Island,
Keller Peninsula, green growth
on the rocks near the seashore;
62◦ 03 48.5 S, 58◦ 24 56.4 W
6.1–8.1
(6.1)10.2–20.4(32.6) in submerged tufts filaments
made up to 500 cells, in surface layer up to 1000 cells; in
liquid medium 2nd type of
growth habit
LUC 9 = ELSTER 1991/3
Arctic, Canada, Ellesmere Island,
produce dark-green mats in streaming water, these mats were most
abundant in glacial stream close to
glacial front, central part of Sverdrup
Pass, moraine area, Teardrop Glacier;
(79◦ 08 N, 80◦ 30 W)
7.1–8.1
(5.1)8.1–13.2(18.3) filaments up to 1000 cells;
in liquid medium 1st type of
growth habit
5LUC 11 = KAŠTOVSKÁ
2002/45
Arctic, Svalbard, Ny-Alesund, Austre
Broeggerbreen, cryoconite sediment;
(78◦ 53 N, 11◦ 80 E)
7.1–9.2
(5.1)7.1–12.2(17.3) filaments up to 1000 cells;
in liquid medium 1st type of
growth habit
LUC 12 = KAŠTOVSKÁ
2002/43
Arctic, Svalbard, Ny-Alesund, Vestre
Broeggerbreen, subglacial, river sediment; (78◦ 53 N, 11◦ 80 E)
8.1–11.2
LUC 14 = KAŠTOVSKÁ
2002/5
Arctic, Svalbard, Ny-Alesund, Vestre
Broeggerbreen, cryoconite sediment;
(78◦ 53 N, 11◦ 80 E)
7.1–9.2
(4)6.1–10.2(20.4) filaments up to 1000 cells, formation of H-pieces; in liquid
medium 1st type of growth
habit
(7.1)10.2–15.3(25.5) filaments up to 1000 cells;
in liquid medium 1st type of
growth habit, Growth habits:
1st type = submerged tufts;
2nd type = submerged tufts
and surface layer
Growth habits: 1st type = submerged tufts; 2nd type = submerged tufts and surface layer
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Fig. 2. Growth habit of twelve examined strains of Klebsormidium; scale 10 µm.
90
12000
85
10000
80
8000
0,50
75
6000
0,45
70
4000
0,40
65
2000
0,35
60
0
0,30
55
Slovakia
Arctic
Antarctica
Continent
Mean
Lower Limit
Upper Limit
Fig. 3. 95% confidence intervals for mean Chl-a content (dashed/dotted line; in µg), fluorescence (dashed line; in relative units) and
viability (unbroken line; in %) of Klebsormidium strains from different geographical regions.
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Table 2. Experimental regimes used for freezing and desiccation. The freezing-melting regimes (−4 ◦C, −40 ◦C, and −196 ◦C) were
repeated three times. In addition, the regime down to −196 ◦C was done without a programmable freeze. The cryovials with the algae
suspension were simply submerged into liquid nitrogen, and kept there for 3–5 minutes, then again melted. Desiccation experiments
at +4 ◦C and +20 ◦C were performed only once and lasted three days.
Regime
Process
Starting temperature
Final temperature
Three repetitions
rate
time
frozen −4 ◦C
frozen −40 ◦C
direct in liquid N2
desiccated +4 ◦C
desiccated +20 ◦C
freezing
20 ◦C
−4 ◦C
4 ◦C/min
6 min
freezing
20 ◦C
−40 ◦C
4 ◦C/min
15 min
freezing
20 ◦C
−196 ◦C
desiccation
20 ◦C
4 ◦C
desiccation
20 ◦C
20 ◦C
3days
3days
min after which samples were transferred to the measuring chamber of the apparatus. A weak, modulated measuring radiation was applied to the sample, and the minimum
chlorophyll fluorescence yield (F0 ) in the dark-adapted state
(DAS) was recorded. After this irradiation, a short pulse of
a saturating radiation (saturation pulse) was applied, and a
maximum chlorophyll fluorescence yield, in DAS (FM ), was
recorded. Then, the actinic radiation was applied, and the
changes of fluorescence were recorded. From the parameters F0 and FM , the maximum variable chlorophyll fluorescence yield, in DAS, was estimated (FV = FM − F0 ) and the
maximum quantum yield of photosystem II (PS II) photochemistry was calculated (FV /FM ). This value, referred to
in the results as fluorescence, was used to quantify the algal
biomass.
Statistical analysis
The freezing-desiccation experiments for viability, fluorescence, and Chl-a content of the strains from different geographical regions were compared using the Kruskal-Wallis
test. Two-way ANOVA was used for the comparison of
strains and the various freezing-desiccation regimes according to the Chl-a content and three-way ANOVA for the same
comparisons whereby the third factor was represented by
duplicate experiments, according to fluorescence and viability. Redundancy analyses (RDA; Ter Braak & Šmilauer
2002) were used to analyse responses of the strains to freezing and desiccation regimes according to viability and fluorescence (non-standardized data). Three duplicates of each
freezing/desiccation regime were entered into these analyses.
Results
Kruskal-Wallis tests did not reveal statistical differences between strains originating from different geographical regions (the Antarctic, the Arctic and Slovakia) when evaluated by viability, Chl-a content, and
fluorescence (Fig. 3). Only the LUC 9 strain which originated from a shallow wetland (hydro-terrestrial) habitat at Ellesmere Island (Canadian Arctic) differed significantly from the others in its higher Chl-a content
when different freezing-desiccation regimes were tested
by two-factor ANOVA (F = 6.810, P < 0.001) (Fig. 4).
This wetland strain is freezing-desiccation stress resistant and fast growing. Arctic strain LUC 11 from a
soil habitat in Svalbard differed from the others by its
very low fluorescence (F = 15.387, P < 0.001) and viability (F = 11.758, P < 0.001) when compared by
three-factor ANOVA (Fig. 4).
All three parameters (Chl-a content, fluorescence,
and viability tests) showed that freezing down to
−196 ◦C was the most detrimental regime for all of the
studied strains (Fig. 5). This regime differed significantly from all others in viability and in fluorescence
when data of the different strains were used for the comparison of these regimes by three-factor ANOVA (F =
84.605, P < 0.001 for viability and F = 189.361, P <
0.001 for fluorescence). However, it significantly differed
in Chl-a content only from the control and −4 ◦C treatments, compared by two-factor ANOVA (F = 8.274,
P < 0.001). The control differed in all three parameters from all freezing and desiccation treatments, except
the −4 ◦C treatment.
When measurements of fluorescence were evaluated using RDA (Fig. 6) only freezing at −4 ◦C and the
control were not statistically significant, from the six
categories of experimental treatment. Two components
(canonical axes) captured 71.7% of the total variance
in strains, and 83.9% in the strain-freezing/desiccation
relationship. The first canonical axis was highly significant (Monte Carlo permutation test: F = 20.037, P
= 0.001). All strains studied showed zero fluorescence
at −196 ◦C which a lethal temperature for all strains.
Strains LUC 6, 7, and 8, isolated from aero-terrestrial
whale bones and rock on King George Island, Antarctica, and LUC 11 from cryoconite sediment from Svalbard in the Arctic showed similar features. In the RDA
diagram these strains appeared in the lower left corner. They did not survive desiccation at +4 ◦C. However, freezing at −40 ◦C and desiccation at +20 ◦C was
not fatal for these strains. The rest of strains (LUC
1, 2, 3, 4, 5, 9, 12 and 14), were located in the upper
left corner of the diagram. Strains LUC 1, 2, 3, 4 and
5 were aero-terrestrial strains from stones in Slovakia
(LUC 1, 2, 3), and from green sands near bird nests
on King George Island, Antarctica (LUC 4, 5). On the
contrary, strains 9, 12 and 14 originated from hydroterrestrial environments in the Arctic. Strains LUC 9,
from a glacial stream in Ellesmere Island, and UC 12
and 14, were from glacial river and cryoconite sediments
from Svalbard, respectively, and survived desiccation
better at lower temperatures (+4 ◦C) and freezing down
to −40 ◦C.
When measurements of viability were evaluated using RDA (Fig. 7), only freezing at −196 ◦C and desiccation at +20 ◦C were statistically significant from the
six categories of experimental treatments. Two comUnauthenticated
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848
Arctic
30000
18000
16000
25000
Slovakia
Chlorophyll [µg]
Chlorophyll [µg]
14000
20000
Antarctica
15000
10000
12000
10000
8000
6000
5000
4000
2000
LUC14
LUC12
LUC11
LUC9
LUC8
LUC7
LUC6
LUC5
LUC4
LUC3
LUC2
LUC1
0
0
CONT
Strain
MIN4
MIN40
MIN196
PLUS4
PLUS20
PLUS4
PLUS20
PLUS4
PLUS20
Freezing/desiccation regime
Mean
Lower Limit
Upper Limit
Mean
Antarctica
Lower Limit
Upper Limit
Arctic
0.55
0.65
0.50
0.60
0.45
Fluorescence [rel.units]
Fluorescence [rel.units]
0.60
Slovakia
0.40
0.35
0.30
0.25
0.20
0.55
0.50
0.45
0.40
0.35
0.15
0.30
LUC14
LUC12
LUC11
LUC9
LUC8
LUC7
LUC6
LUC5
LUC4
LUC3
LUC2
LUC1
0.10
0.25
CONT
MIN4
Strain
Average
100
Slovakia
Lower Limit
MIN40
MIN196
Freezing/desiccation regime
Upper Limit
Antarctica
Average
Arctic
Lower Limit
Upper Limit
110
100
90
80
Viability [%]
Viability [%]
90
80
70
60
70
60
50
40
50
30
20
LUC14
LUC12
LUC11
LUC9
LUC8
LUC7
LUC6
LUC5
LUC4
LUC3
LUC2
LUC1
40
Strain
Average
Lower Limit
10
CONT
MIN4
MIN40
MIN196
Freezing/desiccation regime
Upper Limit
Average
Lower Limit
Upper Limit
Fig. 4. 95% confidence intervals for mean Chl-a content, fluorescence and viability of Klebsormidium strains (see Table 1).
Fig. 5. 95% confidence intervals for mean Chl-a content, fluorescence and viability of Klebsormidium strains under different
freezing-desiccation regimes.
ponents (canonical axes) captured 54.5% of the total variance in the strains and 100.0% in the strainsfreezing/desiccation relationship. The first canonical
axis was highly significant (Monte Carlo permutation
test: F = 14.315, P = 0.001). Similarly, as in fluorescence, freezing at −196 ◦C was the most significant treatment. It was lethal for strains LUC 9, LUC
11, LUC 12 and LUC 14. These strains appeared farthest from the appropriate centroid (Fig. 7). Similarly,
strains LUC 2, 11, 12, and 14 suffered the most desiccation at +20 ◦C, and grouped together (see ellipse in
Fig. 7).
acterised by a certain genetically fixed level of resistance to low temperatures and desiccation. The level
of resistance (or survival capacity) varies among individual strains and/or species, and depends on the organism’s origin and phylogeny. Both cold stress (chilling and freezing) and desiccation stress strongly influence metabolic processes in the cells (Nishida & Murata
1996; Hudák & Salaj 1999; Potts 1999; Elster & Benson
2004; Alpert 2005, 2006).
In de-glaciated parts of both the Arctic and
Antarctic, the temperature during summer days may
reach 8 ◦C or more but rarely falls to less than 5 ◦C below
zero. In autumn, temperatures are more stable (around
0 ◦C) and numerous day-to-day freeze-thaw cycles occur (Davey et al. 1992; Elster & Komárek 2003). This
overnight freezing is restricted to the vegetation surface, and the temperature only drops to a few degrees
Discussion
As for vascular plants, eukaryotic microalgal species (in
this case, species of the genus Klebsormidium) are char-
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Freezing and desiccation injury resistance in Klebsormidium
Fluorescence
1.5
PLUS4
1
14
9
1
12
3
-1.5
0.5
2
4
5
MIN40
-1
7
0
-0.5
0
PLUS20
0.5
1
11
6 8
1.5
2
MIN196
-0.5
-1
-1.5
strains
centroids
Fig. 6. RDA biplot of the fluorescence response to freezing (−196,
and −40 ◦C) and desiccation (+4 and +20 ◦C) by Klebsormidium
strains (see Table 1).
Viability
1.2
PLUS20
0.8
9
5 1
4
3
8
0.4
7
6
0
-1.5
-1
-0.5
14
12
0
0.5
1
MIN196
1.5
2
-0.4
2
-0.8
11
-1.2
strains
centroids
Fig. 7. RDA biplot of the viability response to freezing (−196 ◦C)
and desiccation (+20 ◦C) of Klebsormidium strains (see Table 1).
below zero. This period of sub-lethal temperatures may
be of importance in promoting the cold-hardiness of the
organisms before the decline into the lower winter temperatures. During winter conditions temperatures fall
far below 0 ◦C (down to −30 to −35 ◦C, Davey et al.
1992); the water level also drops, and the terrestrial algal communities are completely desiccated and frozen
until liquid water is available again when spring returns.
In temperate regions, most perennial vascular
plants and other organisms undergo during their seasonal cycle periods of both drought and chill-freezemelts, and have the ability to develop a tolerance to
these hardening conditions. During the winter period
the temperatures fluctuate widely, and numerous dayto-day freeze-thaw cycles can occur (Elster et al. 2002;
Machová et al. 2008). The length of each season in polar and temperate regions depends on the geographical
position (latitude and altitude), as well as on several
other geographical and ecological factors.
849
It is clear that winter freezing is the major event,
and may be the only one, that can be expected to cause
significant mortality in the communities (Hawes 1990;
Davey et al. 1992). Studies based on field or laboratory
experiments have shown that some algae are able to
tolerate prolonged periods of desiccation (Davey 1989;
Hawes et al. 1992; Jacob et al. 1992) and/or freezing
(Davey 1989; Hawes 1990). It is also obvious that there
are strain/species specific differences in the overwintering strategies, and also between strains/species inhabiting different habitats (Davey 1989; Hawes et al. 1992;
Jacob et al. 1992).
In our experiments, freezing down to −4 ◦C did not
influence the survival of the 12 strains studied (evaluated as mean viability, fluorescence, and Chl-a content).
Two-factor and three-factor ANOVA tests (Fig. 5)
showed that there were no differences between the control and the −4 ◦C experimental treatment. Freezing
down to −4 ◦C commonly occurs in nature during the
polar summer periods (Elster & Komárek 2003; Elster
& Benson 2004) and also in temperate regions during autumn and spring (Elster et al 2002; Machová et
al. 2008). Down to that temperature, the liquid contents of cells is not frozen and this chilling in resistant
species results in survival, without injuries. Similar results (Hawes 1990) have been recorded for an Antarctic
Zygnema sp. during repeated overnight exposures to
temperatures of down to −4 ◦C, where photosynthetic
capacity was maintained without any cryoinjury effect.
Additionally, Hawes (1990) also demonstrated that during the Antarctic summer, diurnal changes were slow.
Slow temperature changes (0.5 ◦C/min) prevent intracellular ice nucleation.
At temperatures less than or equal to −40 ◦C homogenous ice nucleation occurs and all cell liquid water freezes (Convey 2000; Elster & Benson 2004). Two
types of freezing can occur in cells. Crystallisation is
the arrangement of liquid water molecules into orderly
ice structures (Luyet 1966). The formation of ice usually destroys membranes, particularly if the crystals are
formed intracellularly (Fuller 2004). Crystallisation is a
slow process, which is very common in nature. Vitrification is the process of solidification of the cellular
contents into a non-crystalline (amorphous) state. It
is the result of rapid freezing (greater than 3 ◦C/min)
which does not occur in nature. In our experiments, a
cooling speed of 4 ◦C/min was chosen. This is on the
boundary between vitrification and crystallization. In
our experiments most of the strains survived the freezing at −40 ◦C (at a speed of 4 ◦C/min) which was not
fatal for most of strains (Fig. 6). On the contrary, direct immersion into liquid nitrogen was fatal for the
most strains (Figs 5–7).
Water deficiency includes both osmotic and mechanical stresses, and the most common adaptation
against desiccation is the production of compatible
solutes (Mackay et al. 1984). This effect has been
examined in the mat-forming cyanobacterial species
in the cold environment of the McMurdo Ice Shelf,
continental Antarctica (de los Ríos et al. 2004).
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J. Elster et al.
850
Desiccation-adapted species form copious quantities of
mucopolysaccharides (exopolymeric substances, EPS).
This material likely slows down the flow of liquid water during desiccation. In addition, the present study
also showed that the photosynthetic recovery (PS II
effective quantum yield) process of dry colonies of Nostoc, during rehydration, was inhibited by higher solar irradiation, at the initial stage of desiccation. This
decline was invoked by high levels of photosynthetically active radiation (PAR). High levels of PAR led
to enhanced thermal dissipation of excessive excitation energy and damage to PS II (Franklin et al. 2003;
Gao & Ye 2007). In another study (Šabacká & Elster
2006), desiccation was more injurious than freezing, especially for green algae which showed high mortality.
Even more injurious was desiccation at +20 ◦C which
was fatal for some strains. In our experiments, certain of the Antarctic and Arctic strains did not survive (as evaluated by fluorescence response) desiccation at +4 ◦C (Fig. 6). However, desiccation at +20 ◦C
was not fatal for them (Fig. 6). Other strains, however, (Central European strains from Slovakia, the
Antarctic, and the Arctic) reacted differently to desiccation. They survived desiccation at a lower temperature +4 ◦C. It is difficult to explain these differences.
No statistical differences between freeze and desiccation stressed strains from the Antarctic, the Arctic,
and Slovakia were observed (Fig. 3). There were only
two strains which expressed their freezing-desiccation
resistances on the upper and lower survival amplitudes.
The strain from a shallow wetland (hydro-terrestrial)
habitat at Ellesmere Island (Canadian Arctic, LUC 9)
was highly freeze-desiccation stress-resistant, according to the Chl-a content (Fig. 4). On the contrary,
Arctic strain LUC 11 from soil habitats in Svalbard
stood out by its very low freeze-desiccation resistance
as indicated by fluorescence and viability (Fig. 4, 7).
From these results it can be concluded that Klebsormidium strains originating from various different
geographical zones (the Antarctic, the Arctic, Slovakia) and from various habitat types (shallow wetlands, edaphitic, lithophytic) have a very similar response to both freezing and desiccation stresses and
that they are particularly resistant to freezing and
desiccation injuries. These results are quite surprising since Šabacká & Elster (2006) demonstrated that
there are differences in the freezing tolerances among
cyanobacteria isolated from continental and maritime
Antarctica, and also among strains originating from
different habitat types. However, these authors also
showed that such a significant trend, which was observed in cyanobacteria, was not found for eukaryotic
algae. Šabacká & Elster (2006) argued that the group
of tested green algal strains (Chlorella sp., C. minutissima, Chlorosarcina sp., Pseudococcomyxa simplex and
Klebsormidium sp.) was taxonomically and morphologically diverse, comprising different genera and different life forms (coccoid, filamentous) which would
influence the freezing-desiccation resistance. Thus, it
is likely that the studied Klebsormidium strains (the
most widespread green algal genus) are well adapted for
life in aero-terrestrial, terrestrial, and hydro-terrestrial
habitats around the world. Most of the Klebsormidium
species occurring in polar and temperate regions are
well adapted against both freezing and desiccation injuries.
Acknowledgements
This work was supported by the Grant Agency of the Czech
Republic (Project No. 206/05/0253), the Grant Agency of
the Ministry of Education of the Czech Republic (Kontakt
– ME 934 and MEB 080822), the Slovak Grant Agencies
for Science VEGA (Project No. 1/0190/08), and APVV
(Project SK-CZ-0023-07), and by the Brazilian Antarctic
Programme CIRM/PROANTAR and CNPq/MMA/CIRM.
We are very grateful to Mrs. Jana Šnokhousová for her technical assistance. Peter Raphael Lemkin and Hans Sluiman
kindly improved the English. Finally, we acknowledge the
insightful comments of the reviewers which improved our
interpretations of the data.
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Received September 1, 2007
Accepted March 17, 2008
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