Research
Rapid adaptation of phytoplankters to geothermal
waters is achieved by single mutations: were extreme
environments ‘Noah’s Arks’ for photosynthesizers
during the Neoproterozoic ‘snowball Earth’?
Blackwell
Oxford,
New
NPH
©
1469-8137
0028-646X
September
10.1111/j.1469-8137.2008.02620.x
2620
9
0
Original
932???
XX
22???
ThePhytologist
Authors
UK
Article
Publishing
2008(2008).Ltd
Journal compilation © New Phytologist (2008)
XX
Eduardo Costas1*, Antonio Flores-Moya2* and Victoria López-Rodas1*
1Genética
(Producción Animal), Facultad de Veterinaria, Universidad Complutense, E–28040 Madrid, Spain; 2Biología Vegetal (Botánica), Facultad de
Ciencias, Universidad de Málaga, Campus de Teatinos s/n, E–29071 Málaga, Spain
Summary
Author for correspondence:
A. Flores-Moya
Tel: +34 952131951
Fax: +34 952131944
Email: [email protected]
Received: 02 June 2008
Accepted: 21 July 2008
• Geothermal waters often support remarkable communities of microalgae and
cyanobacteria apparently living at the extreme limits of their tolerance. Little is
known about the mechanisms allowing adaptation of mesophilic phytoplankters to
such extreme conditions, but recent studies are challenging many preconceived
notions about this. The aim of this study was to analyse mechanisms allowing adaptation of mesophilic microalgae and cyanobacteria to stressful geothermal waters.
• To distinguish between the pre-selective or post-selective origin of adaptation
processes allowing the proliferation of mesophilic phytoplankters in geothermal
waters, several Luria–Delbrück fluctuation analysis were performed with the microalga
Dictyosphaerium chlorelloides and the cyanobacterium Microcystis aeruginosa,
both isolated from nonextreme waters. Geothermal waters from seven places in Italy
and five icebound places at Los Andes in Argentina were used as selective agents.
• Physiological adaptation was achieved in the least toxic waters. In contrast, rapid
genetic adaptation was observed in waters ostensibly lethal for the experimental
organisms. This adaptation was achieved as consequence of single mutations at
one locus.
• It was hypothesized that a similar mechanism of rapid genetic adaptation could
explain the survival of photosynthetic life during the Neoproterozoic ‘snowball
Earth,’ where geothermal refuges such as those studied could have been ‘Noah’s
Arks’ for microalgae and cyanobacteria.
Key words: adaptation, Dictyosphaerium, geothermal waters, Microcystis,
Neoproterozoic ‘snowball Earth’, survival photosynthesizers.
New Phytologist (2008) 180: 922–932
© The Authors (2008). Journal compilation © New Phytologist (2008)
doi: 10.1111/j.1469-8137.2008.02620.x
Introduction
Extreme aquatic environments often support remarkable
communities of phytoplankters living at the extreme limits of
their tolerance (Seckbach & Oren, 2007). Survival and growth
of such microorganisms in habitats characterized by extreme
*The three authors contributed equally to this work.
922
values of pH, toxic mineral concentrations, temperature,
salinity and other stress factors, is an interesting topic from
both biochemical and physiological points of view (Fogg, 2001).
Since algae and cyanobacteria are the principal primary
producers of aquatic ecosystems (Kirk, 1994; Falkowski &
Raven, 1997), the capability of phytoplankters to proliferate
in these extreme natural environments is very relevant for
understanding these intriguing communities.
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Research
Little is known about the mechanisms allowing adaptation
of phytoplankton to such extreme conditions. Phytoplankton
organisms can survive in adverse environments as a result of
physiological adaptation (i.e. acclimatization) supported
by modifications of gene expression (Bradshaw & Hardwick,
1989; Fogg, 2001). However, when the values of some environmental factors exceed the physiological limits, survival
depends exclusively on adaptive evolution, by the occurrence
of mutations that confer resistance (Sniegowski & Lenski,
1995; Belfiore & Anderson, 2001). It is accepted that shortterm or fluctuating stress is best met by physiological adaptation, while continuous or predictable stress can be met by
genetically determined response systems (Bradshaw &
Hardwick, 1989; Davison & Pearson, 1996). Moreover, it is
usually assumed that genetic adaptation to extreme environments is achieved very slowly. However, recent studies are
changing many preconceived notions about the adaptation of
microalgae to extreme environments. As an example, eukaryotic
microalgae (rather than extremophile bacteria) contributed
the highest fraction of the biomass (at least 60%) in the
Rio Tinto (Amaral Zettler et al., 2002), an extremely acidic
(pH 1.7–2.5) environment with a very high heavy metal
content (Gónzalez-Toril et al., 2003). Molecular studies show
that these microalgae are closely related to neutrophilic species
rather than acidophilic lineages. For this reason, it was proposed
that adaptation from neutral to extreme environments must
occur rapidly (Amaral Zettler et al., 2002). Moreover, it was
suggested that microalgae resistant to Rio Tinto waters arose
randomly by rare spontaneous mutations and, as a result, algal
populations were able to rapidly adapt to Rio Tinto water by
means of selection of resistant mutants growing in nonextreme
conditions (Costas et al., 2007). It also has been proposed that
rare pre-selective mutants can be sufficient to ensure the adaptation of mesophilic algae to other extreme natural habitats.
Flores-Moya et al. (2005) demonstrated that algae inhabiting
the extreme acidic, sulphurous water from La Hedionda Spa
(southern Spain) could originate by selection of pre-selective
mutants of mesophilic algal lineages inhabiting nonextreme
environments. Similarly, microalgal adaptation to the stressful
acidic, metal-rich mine waters from Mynydd Parys (North
Wales, UK) and Aguas Agrias stream (southwest Spain) is also
caused by selection of preselective mutants of mesophilic
algal lineages that arose previous to toxic water exposure
(López-Rodas et al., 2008a,b).
The aim of this study was to determine whether mesophilic
cyanobacteria and microalgae are capable of rapid adaptation
to extreme environments by single mutations. For this purpose,
cosmopolitan, mesophilic phytoplankton species isolated from
nonextreme waters were selected; specifically, Dictyosphaerium
chlorelloides was isolated from a pristine, slightly alkaline
(pH 8.0) mountain lake from Sierra Nevada (southern Spain)
while the cyanobacterium Microcystis aeruginosa was isolated
from a pristine pond with nonacidic waters (pH 8.1) in
Doñana National Park (south-west Spain). In both species, we
analysed adaptation to seven different geothermal systems
from Italy (including warm ponds, hot springs, seltzer springs
and extremely acid hot springs) and five icebound geothermal
waters at Los Andes in Argentina (including warm ponds, hot
springs, seltzer hot springs and geysers). We studied whether
the mesophilic chlorophyceans and cyanobacteria could adapt
to survive and grow in these geothermal waters, by using an
experimental design known as fluctuation analysis (Luria &
Delbrück, 1943). This experimental approach allowed us to
differentiate between physiological and genetic adaptation. It
was found that in the majority of the extreme geothermal
waters, adaptation was achieved by the photosynthetic
mesophiles through very rapid genetic adaptation caused by
single mutations. In addition, we hypothesize that a similar
mechanism of rapid genetic adaptation could explain the
survival of photosynthetic life during the ‘snowball Earth,’
where geothermal refuges, such as the ones studied in Italy
and Los Andes, could be ‘Noah’s Arks’ for cyanobacteria
and microalgae.
Based on geologic and paleomagnetic evidence, the ‘snowball
Earth’ hypothesis proposes that a series of global glaciations
occurred during the Neoproterozoic era, between c. 740 million
years ago (Mya) and 580 Mya, with the ice line reaching
the Equator and with a sea-ice cover > 100 m in thickness in
tropical latitudes (Kirschvink, 1992; Hoffman et al., 1998).
Although Hyde et al. (2000) suggested a climatic scenario in
which a partly frozen Earth had ice-free oceans at the Equator
(‘slushball Earth’), it seems that only the ‘snowball Earth’
hypothesis can explain all the geological and paleomagnetic
data of Neoproterozoic glacial deposits (Schrag & Hoffman,
2001). The ‘snowball Earth’ model implies a drastic survival
pressure on the photosynthetic biota because liquid water and
sunlight were not available simultaneously, for millions of
years, in any place on the Earth. In fact, it is supposed that
primary production collapsed (Schrag & Hoffman, 2001), as
revealed by negative carbon isotope anomalies in carbonate
rocks (Kaufman et al., 1997; Hoffman et al., 1998; Rothman
et al., 2003). However, photosynthetic prokaryotic (cyanobacteria) and most eukaryotic phyla (including green, red and
chromophytic algae) evolved before the late Neoproterozoic
glaciations (Knoll & Bauld, 1989; Knoll, 1992) and they
must have survived these extreme environmental conditions.
Recent evidence suggests that global Neoproterozoic
glaciations contributed only modestly to the major extinction
of autotrophic and heterotrophic eukaryotes (Corsetti et al.,
2006). Schrag & Hoffman (2001) proposed that the survival
of photosynthesizers during such extended glaciations was
achieved in ice-free refuges associated with volcanic activity,
such as hot springs and thermal ponds (geothermal areas). A
highly adapted community of photosynthetic microbes can
develop in geothermal waters – cyanobacteria if the pH is
> 4.8, as well as a few genera of eukaryotic ‘cyanidia’ (Brock,
1973; Ward et al., 1998; Castenholz, 2000; Donachie et al.,
2002; Sand, 2003; Ciniglia et al., 2005; Walker et al., 2005;
© The Authors (2008). Journal compilation © New Phytologist (2008) www.newphytologist.org
New Phytologist (2008) 180: 922–932
923
924 Research
Gaylarde et al., 2006; Jing et al., 2006; Lehr et al., 2007;
Pinto et al., 2007). However, these stressful environments are
lethal for mesophilic cyanobacteria and algae because of
extreme values of pH and high temperatures, as well as high
concentrations of dissolved heavy metals (Brock, 1978;
Dando et al., 1998; Webster & Nordstrom, 2003; Tyrovola
et al., 2006). Therefore, if the proposal of Schrag & Hoffman
(2001) about the survival of photosynthesizers in ice-free
volcanic refuges during the ‘snowball Earth’ is true, it could be
hypothesized that mesophilic cyanobacteria and algae have
the potential to develop adaptations allowing their survival and
growth in these refuges.
Materials and Methods
Sampling sites, in situ analysis of geothermal waters,
and algal identification
Seven different geothermal waters from Italy, and five different
icebound geothermal waters at Los Andes, Neuquén, Argentina,
were studied (descriptions of sampling points are given in
Table 1). The values of conductivity, temperature and pH at
the sampling sites were determined using a YSI 6820-C-M
probe (Yellow Springs, OH, USA). Cyanobacteria and
microalgae were identified in fresh samples (directly after
collection in each site) using a McArthur portable microscope
(Kirk Technology, Cambridge, UK). In addition, 3 l of water
was collected from each location, filtered (0.22 µm, Stericup;
Millipore Co., Billerica, MA, USA), kept in a closed bottle
excluding any air, and stored at 4°C in darkness until the
laboratory experiments (toxicity tests and fluctuation analysis;
see below) were performed.
Experimental organisms and culture conditions
A wild-type strain of the chlorophycean D. chlorelloides
(Naumann) Komárek and Perman and of the cyanobacterium
M. aeruginosa (Kützing) Lemmermann (both strains from
the Algal Culture Collection of the Veterinary Faculty,
Complutense University, Madrid, Spain) were grown in
100-ml cell culture flasks (Greiner, Bio-One Inc., Longwood,
NJ, USA) with 20 ml BG-11 medium (Sigma-Aldrich Chemie,
Taufkirchen, Germany), at 22°C under continuous light of
60 µmol m−2 s−1 over the waveband 400–700 nm. Although
D. chlorelloides usually forms two- or four-celled (rarely, 16celled) colonies, and is capable of sexual reproduction in nature,
this strain was exclusively propagated by asexual reproduction,
and it was represented by single-celled individuals. Cultures
were axenically maintained in mid-log exponential growth
(Cooper, 1991) by serial transfers of subcultures to fresh
medium, and only cultures without detectable bacteria were
used in the experiments. The absence of bacteria in the cultures
was confirmed periodically (once every week) by epifluorescence
microscopy after staining with acridine orange. Before the
New Phytologist (2008) 180: 922–932
experiments, the cultures were cloned by isolating a single
cell, to avoid including any previous spontaneous mutants
accumulated in the culture.
Toxicity test: effect of geothermal waters on Malthusian
fitness and effective quantum yield
The changes in effective quantum yield from photosystem II
(ΦPSII), and Malthusian fitness (m), were measured when the
wild-type strains of D. chlorelloides and M. aeruginosa were
cultured in the geothermal water samples. Samples (5 × 105
cells) from mid-log exponentially growing cultures of both
species were placed in culture tubes (Sarsted Co., Nümbrecht,
Germany) containing 5 ml of the geothermal water. Controls
were cultured in BG-11 medium.
The effective quantum yield (ΦPSII) of the strains was
measured in triplicates of each geothermal water sample and
controls using a ToxY-PAM fluorimeter (Walz, Effeltrich,
Germany) after 48 h exposure. Effective quantum yield was
calculated as follows:
Φ PSII = ( Fm′ − Ft )/ Fm′
Eqn 1
( Fm′ and Ft are the maximum and the steady-state fluorescence
of light-adapted cells, respectively; Schreiber et al., 1986).
Malthusian fitness values were also calculated in three
replicates in each geothermal water sample as well as in three
controls, using the equation of Crow & Kimura (1970):
m = Loge (Nt/N0)/t,
Eqn 2
(t = 5 d; N0 and Nt are the cell numbers at the start and at
the end of the experiment, respectively). Cell number in
experiments and controls was counted using a Beckman
(Brea, CA, USA) Z2 particle counter.
Adaptation to geothermal waters: fluctuation analysis
from sensitivity to resistance
A modified fluctuation analysis (Costas et al., 2001; LópezRodas et al., 2001) was carried out to study the adaptation of
the experimental strains to geothermal waters. The modification
involves the use of liquid culture medium rather than plating
on solid medium (Luria & Delbrück, 1943).
Briefly, for each geothermal water analysed, two different
sets of experimental cultures were prepared (Fig. 1). In the set
1 experiments, from 70 to 96 (depending on the strain and
geothermal water; see Table 2) culture tubes were inoculated
with approx. 102 cells of D. chlorelloides or M. aeruginosa (N0;
a number small enough to reasonably ensure the absence of
pre-existing mutants in the strain). Cultures were grown in
5 ml BG-11 medium at 22°C until c. 105 cells (Nt). Then,
cultures were centrifuged to form a pellet of cells in the tube,
the medium was decanted and 5 ml of one of the geothermal
waters at 30°C was added to each tube. For the set 2 control,
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© The Authors (2008). Journal compilation © New Phytologist (2008) www.newphytologist.org
Table 1 Physico-chemical characteristics and description of the geothermal waters sampled to study algal adaptation, algal community inhabiting in the geothermal waters, and inhibitory effect
of these waters on Malthusian fitness (m) and effective quantum yield from photosystem II (ΦPSII) of Dictyosphaerium chlorelloides (Dc; Chlorophyceae) and Microcystis aeruginosa (Ma;
Cyanobacteria), respectively
Sampling location
Description
Conductivity
(mS)
Temperature (°C)
pH
Algal community
Geothermal waters from Italy
Bagno Vignoni
Warm pond with fumaroles
3.7
38.6
6.0
Amiana Marni
Hot spring with sulphide
7.7
43.8
6.7
Pienzza
Infierno Sujo
Sufione Pisciarelly
Fangary
Warm pond with fumaroles
Seltzer spring with benzene
Acid hot spring and fumaroles
Warm pond with fumaroles
7.4
6.0
2.4
8.3
27.7
21.0
30.1 (in the adjacent pond)
32.1
2.5
2.9
2.0
5.7
Dense biofilms with high diversity
of cyanobacteria and microalgae
Biofilms with low diversity of
cyanobacteria and microalgae
Low diversity of chlorophyceans
Low diversity of chlorophyceans
Not detected
Low diversity of cyanobacteria
and chlorophyceans
Low diversity of chlorophyceans
6.7
30.1 (in the adjacent pond)
3.2
6.5
59.9
6.4
Aguas Calientes
Las Papas
Los Tachos
La Maquinita
3.4
8.2
6.3
1.2
50.4
36.6
46.1
37.1
5.6
6.5
7.0
4.0
Warm pond with fumaroles
Warm pond with sulphur
Geyser forming a pond
Hot spring forming a pond
High diversity of cyanobacteria
and microalgae
Mats of benthic microalgae
Floating mats of microalgae
Microalgae and cyanobacteria
Not detected
Inhibition of
ΦPSII (% control)
Dc
Dc
Ma
Ma
17
19
14
15
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
55
99
64
70
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
Research
New Phytologist (2008) 180: 922–932
Puzzoly
Hot spring with fumaroles
Geothermal waters from Argentina
Doña Sara
Seltzer hot spring forming a pond
Inhibition of
m (% control)
925
926 Research
Fig. 1 Schematic diagram of possible results obtained in the
experiment (modified from the classic Luria and Delbrück fluctuation
analysis). Set 1, different cultures of Dictyosphaerium chlorelloides
and Microcystis aeruginosa (each started from a small inoculum,
N0 = 102 cells) were propagated under nonselective conditions
(i.e. BG-11 medium) until a very high cell density was reached
(Nt ≈ 105 cells), and then transferred to the selective agent
(i.e. the different geothermal waters). If resistant cells arose during
the exposure to geothermal waters (physiological adaptation), the
number of resistant cells in all the cultures must be similar (Set 1A).
If resistant cells arose by rare mutations occurring in the period of the
propagation of cultures (i.e. before exposure to geothermal waters)
the number of resistant cells in all the cultures must be different
(Set 1B). In the figure, one mutational event occurred late in the
propagation of culture 1 (therefore, the density of geothermal
water-resistant cells found is low) and early in the propagation of
culture 3 (thus, density of geothermal-resistant cells found is higher
than in culture 1); no mutational events occurred in culture 2. Set 2,
Different replicates from the same parental culture sampling the
variance of the parental population are used as an experimental
control. In this case, the number of resistant cells in all the cultures
must be similar.
New Phytologist (2008) 180: 922–932
from 25 to 30 aliquots (depending on the species and geothermal
water; see Table 2) of c. 105 cells of D. chlorelloides or
M. aeruginosa from the same parental populations growing
in BG-11 medium at 22°C, were separately transferred to
culture tubes containing 5 ml of geothermal water at 30°C.
Cultures were observed for 75 d (thus ensuring that one
mutant cell could generate enough progeny to be detected, yet
not reach the stationary phase) and the resistant cells in each
culture were counted.
Two different results can be found in the set 1 experiment
when conducting a fluctuation analysis, each result being
interpreted as the independent consequence of two different
phenomena of adaptation. In the first case, if resistant cells
arose during the exposure to the selective agent (i.e. by
physiological adaptation), the variance in the number of cells
per culture would be low because every cell is likely to have
the same chance of developing resistance (Fig. 1, set 1A).
Consequently, inter-culture (tube-to-tube) variation would
be consistent with the Poisson model (i.e. variance : mean
ratio approx. 1). By contrast, if resistant cells arose before the
exposure to the selective agent (i.e. genetic adaptation by rare
spontaneous mutation occurring during the time in which the
cultures grew to Nt from N0 cells before the exposure to
geothermal water), a high variation in the interculture number
of resistant cells per culture would be found (Fig. 1, set 1B).
Consequently, the tube-to-tube variation would not be
consistent with the Poisson model (i.e. variance : mean ratio
> 1). Obviously, another result – 0 resistant cells in each culture
– could also be found, indicating that neither selection on
spontaneous mutations that occur before geothermal water
exposure, nor specific adaptation during the exposure to the
geothermal water, took place.
The set 2 cultures are the experimental controls of the
fluctuation analysis (Fig. 1). Variance is expected to be low,
because set 2 samples the variance of the parental population.
If the variance : mean ratio of set 1 is significantly greater than
the variance : mean ratio of set 2 (fluctuation), this confirms
that resistant cells arose by rare mutations that occurred before
exposure to the geothermal water. If a similar variance : mean
ratio between set 1 and set 2 is found, it confirms that resistant
cells arose during the exposure to the geothermal water.
The fluctuation analysis also allows estimation of the rate
of appearance of resistant cells. The proportion of cultures
of set 1 showing nonresistant cells after geothermal water
exposure (i.e. the first term of the Poisson distribution, named
the P0 estimator; Luria & Delbrück, 1943) was the parameter
used to calculate the mutation rate (µ) as:
µ = −LogeP0/(Nt − N0)
Eqn 3
Mutation-selection equilibrium
If the mutation from a normal wild-type geothermal watersensitive allele to a geothermal water-resistant allele is recurrent,
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Research
Table 2 Fluctuation analysis to study adaptation of Dictyosphaerium chlorelloides (Chlorophyceae) and Microcystis aeruginosa (Cyanobacteria)
to different geothermal waters from Italy and Argentina
Bagno Vignoni Amiana Marmi Pienza
Infierno Sujo Sufione Pisciarelly Fangary
Geothermal waters from Italy
Set 1
Set 2
Set 1
Set 2
Set 1 Set 2 Set 1 Set 2 Set 1
Set 2
Dictyosphaerium chlorelloides
Number of replicate cultures
74
28
70
30
70
Number of cultures containing the following no. of resistant cells:
0
0
0
33
0
< 104
0
0
5
0
104–2.5 × 104
0
0
21
0
> 2.5 × 104
74
28
11
30
Variance : mean ratio (of the number 0.9
1.2
60*
0.8
of resistant cells per replicate)
Adaptation process
Physiological Genetic
Microcystis aeruginosa
Number of replicate cultures
75
30
70
30
Number of cultures containing the following no. of resistant cells:
0
0
0
54
0
< 104
0
0
9
0
104–2.5 × 104
0
0
7
30
> 2.5 × 104
75
30
0
0
Variance/mean ratio (of the number 1.2
1.1
91*
1.1
of resistant cells per replicate)
Adaptation process
Physiological Genetic
28
70
59
0
62
2 28
7
1
0
1
8
0
0
31* 1.0 60*
70
30
70
30
70
0
30
0
0
1.1
70
0
0
0
−
30
0
0
0
−
19
17
15
19
100*
0
0
0
30
0.9
27
0
36 30
7
0
0
0
72* 0.9
Genetic
None
70
28
70
30
70
30
70
30
70
30
70
0
0
0
−
28
0
0
0
−
70
0
0
0
−
30
0
0
0
−
70
0
0
0
−
30
0
0
0
−
63
4
3
0
9*
0
30
0
0
1.2
70
0
0
0
−
30
0
0
0
−
None
None
Genetic
None
Aguas Calientes
Las Papas
Set 1
Set 2
Set 1
Set 2
Set 1
Set 2
Dictyosphaerium chlorelloides
Number of replicate cultures
70
25
25
70
25
0
0
25
0
0.8
38
23
4
5
37*
0
0
25
0
1.3
Number of cultures containing the following number of resistant cells:
0
36
0
96
< 104
41
0
0
104–2.5 × 104
11
25
0
> 2.5 × 104
8
0
0
Variance : mean (of the number
−
−
−
of resistant cells per replicate)
Adaptation process
Genetic
None
>
30
Genetic
Doña Sara
Number of cultures containing the following number of resistant cells:
0
70
25
23
< 104
0
0
27
104–2.5 × 104
0
0
14
> 2.5 × 104
0
0
6
Variance : mean ratio (of the number
1.2
1.1
> 100*
of resistant cells per replicate)
Adaptation process
Physiological
Genetic
Microcystis aeruginosa
Number of replicate cultures
96
25
96
Set 2 Set 1 Set 2
30
Icebound geothermal
waters from Argentina
70
Set 1
Puzzoli
Genetic
Los Tachos
Genetic
Genetic
Set 1
70
48
7
7
7
> 100*
None
La Maquinita
Set 2
Set 1
Set2
25
70
25
0
25
0
0
0.9
70
0
0
0
−
25
0
0
0
−
Genetic
None
25
96
25
96
25
96
25
25
0
0
0
−
96
0
0
0
−
25
0
0
0
−
47
39
6
4
29*
0
0
25
0
1.1
96
0
0
0
−
25
0
0
0
−
None
Genetic
None
The characteristics of the different geothermal waters are shown in Table 1.
*Variance/mean > 1; P < 0.001, using χ2 as a test of goodness of fit.
and the resistant allele is detrimental to fitness in the absence
of geothermal water, then new mutants arise in each generation,
but most of these mutants are eliminated sooner or later by
natural selection, if not by chance. Thus, at any given time
there will be a certain number of resistant cells that are not yet
eliminated. According to Kimura & Maruyama (1966), the
average number of such mutants will be determined by the
balance between µ and the rate of selective elimination (s):
© The Authors (2008). Journal compilation © New Phytologist (2008) www.newphytologist.org
New Phytologist (2008) 180: 922–932
927
928 Research
Table 3 Mutation rate (µ, mutants per cell per generation), coefficient of selection against resistant mutant (s) and frequency of the geothermal
water-resistance allele (q) in Dictyosphaerium chlorelloides (Chlorophyceae) and Microcystis aeruginosa (Cyanobacteria), during genetic
adaptation to different geothermal waters from Italy and Argentina (see Table 2 for the fluctuation analysis)
Dictyosphaerium chlorelloides
Italy
Amiana Marmi
Pienzza
Infierno Sujo
Fangary
Puzzoly
Argentina
Aguas Calientes
Las Papas
Los Tachos
Doña Sara
Microcystis aeruginosa
µ
s
q
µ
s
q
8.5 × 10–6
2.0 × 10–6
1.4 × 10–6
1.5 × 10–5
1.1 × 10–5
0.73
0.85
0.91
0.73
0.91
1.2 × 10–5
2.4 × 10–6
1.5 × 10–6
2.0 × 10–5
1.2 × 10–5
2.7 × 10–6
−
−
1.1 × 10–6
−
0.92
−
−
0.78
−
2.9 × 10–6
−
−
1.4 × 10–6
−
1.3 × 10–5
6.9 × 10–6
4.2 × 10–6
−
0.89
0.91
0.85
−
1.5 × 10–5
7.6 × 10–6
4.9 × 10–6
−
−
−
7.9 × 10–6
1.1 × 10–5
−
−
0.86
0.93
−
−
9.2 × 10–6
1.2 × 10–5
q = µ/(µ + s),
Eqn 4
where q is the frequency of the geothermal water-resistant
allele and s is the coefficient of selection calculated as:
r
s
s = 1 − (mGW
/ mGW
),
Eqn 5
r
s
where mGW
and mGW
are the Malthusian fitness of geothermal water-resistant and geothermal water-sensitive cells
measured in nonselective conditions (i.e. BG-11 medium),
respectively.
Results
The seven geothermal waters from Italy were collected from
different sites (Table 1) in an attempt to analyse the effects of
different geothermal waters on phytoplankton. Consequently
they showed substantial differences in pH (from 2.0 to 6.7),
conductivity (from 2.4 to 8.3 mS) and temperature (from
21.0 to 43.8°C) (Table 1). Usually the phytoplankton flora in
such geothermal waters is very poor, and indeed few species of
eukaryotic microalgae, at low densities (< 500 total cells ml−1),
were detected in most of the geothermal waters analysed.
Geothermal waters from Pienzza and Infierno Sujo contained
eukaryotic microalgae (mainly chlorophyceans), but cyanobacteria were not detected (Table 1). Neither microalgae nor
cyanobacteria were found in Sufione Pisciarelli (Table 1). By
contrast, dense and diverse communities of microalgae and
cyanobacteria inhabited the warm pond of Bagno Vignoni
(Table 1). Most of geothermal waters analysed were lethal for
the wild-type strain of D. chlorelloides and M. aeruginosa
in laboratory experiments. The extremely toxic effect of these
geothermal waters produced total inhibition of growth and
photosynthetic performance (ΦPSII) in cultures of both
species (Table 1). Only water from Bagno Vignoni caused
New Phytologist (2008) 180: 922–932
scant inhibition of growth and photosynthesis of laboratory
cultures (Table 1).
The five different geothermal ponds from Los Andes
remain icebound except for a few days in the summer, but do
not freeze during the winter, maintaining temperatures
≥ 30°C. Eukaryotic and prokaryotic photosynthesizers are
often represented in these ponds (Table 1). Doña Sara maintains
a high diversity of cyanobacteria and microalgae, and dense
algal populations also inhabit Aguas Calientes, Las Papas and
Los Tachos. Only in La Maquinita were we unable to detect
photosynthesizers (Table 1). Despite this, water from Aguas
Calientes, Las Papas, Los Tachos and La Maquinita were
lethal for our two strains of mesophilic photosynthesizers:
m and ΦPSII of the wild-type strains of D. chlorelloides and
M. aeruginosa were totally inhibited by these four geothermal
waters (Table 1). Water from Doña Sara was also toxic, but
not lethal (Table 1).
When conducting the fluctuation analysis, different results
were obtained (Table 2). In most of the geothermal waters
from Italy (Amiana Marmi, Pienza, Infierno Sujo, Fangary
and Puzzoly for D. chlorelloides, and Amiana Marmi and
Fangary for M. aeruginosa) and Los Andes (Aguas Calientes,
Las Papas and Los Tachos for D. chlorelloides, and Doña Sara
and Los Tachos for M. aeruginosa) the cell density was drastically
reduced in each experimental culture of sets 1 and 2 in both
species owing to destruction of sensitive cells. However, after
further incubation for several weeks, some cultures increased
in density again, apparently owing to growth of geothermal
water-resistant variants. In the case of set 1, some cultures
recovered after 75 d of geothermal water exposure (Table 1).
By contrast, every set 2 culture recovered, and geothermal
water-resistant cells were detected in all cultures (Table 2). In
addition, low fluctuation was observed in set 2 (variance :
mean ratio = 1, consistent with Poisson variability; P < 0.05,
using χ2 as a test of goodness of fit), which indicated that
www.newphytologist.org © The Authors (2008). Journal compilation © New Phytologist (2008)
Research
the high fluctuation found in set 1 cultures should be caused
by processes other than sampling error (Table 2). As in set 1
cultures, the variance significantly exceeded the mean
(variance : mean ratio > 1; P < 0.001 using χ2 as a test of
goodness of fit), so it could be inferred that geothermal
water-resistant cells arose by rare, pre-selective spontaneous
mutations rather than by specific physiological adaptation
appearing in response to geothermal waters. By contrast, both
strains used in the study proliferated in Bagno Vignoni as
result of physiological adaptation, and D. chlorelloides also
acclimatized to Doña Sara (variance : mean ratio = 1, consistent
with Poisson variability; P < 0.05, using χ2 as a test of
goodness of fit, in both set 1 and set 2 waters; Table 2).
When the species diversities observed in situ in the different
geothermal waters (Table 1) were compared with the results of
laboratory fluctuation analysis (Table 2), a correlation could
be observed. Bagno Vignoni is the location with the highest
diversity and abundance of cyanobacteria and microalgae;
D. chlorelloides and M. aeruginosa easily proliferated in Bagno
Vignoni by means of physiological adaptation. High microalgal
diversity was also observed in Doña Sara and D. chlorelloides
also proliferated without difficulty in Doña Sara by physiological adaptation. By contrast, algae and cyanobacteria were
detected in neither Sufione Pisciarelli nor in La Maquinita,
and our experiment found that neither D. chlorelloides nor
M. aeruginosa were able to adapt to the water from these sites.
A few species of microalgae (but not cyanobacteria), at low
densities, were detected in Pienzza, Infierno Sujo, Puzzoly,
Aguas Calientes and Las Papas; laboratory fluctuation analysis
showed that rare spontaneous pre-selective mutations allowed
adaptation of D. chlorelloides to these water samples, but
M. aeruginosa was unable to proliferate. Cyanobacteria were
detected in Amiana Marmi, Fangary, Doña Sara and
Los Tachos; fluctuation analysis showed that spontaneous
resistance-mutants of M. aeruginosa proliferated in these
water samples.
The estimated mutation rates (µ) from sensitivity to resistance
to the different geothermal waters ranged from 1.4 × 10–6 to
1.5 × 10–5 mutants per cell per division in D. chlorelloides,
and from 1.1 × 10–6 to 1.1 × 10–5 in M. aeruginosa
(Table 3). Isolated D. chlorelloides and M. aeruginosa geothermal
water-resistant mutants growing in the absence of the selective
agent (i.e. in BG-11 medium) showed fitness values lower
than those found in the wild-type strains (data not shown).
The relative values of fitness of resistant mutants and sensitive
wild types were used to compute the coefficient of selection (s)
of geothermal-resistant mutants and by using the values of µ
and s, the frequency (q) of resistant alleles as the consequence
of the balance between mutation and selection was calculated
(Table 3). A frequency of 1.5–20 resistant mutants per 106
cells in D. chlorelloides, and 1.4–12 resistant mutants per 106
cells in M. aeruginosa, could be maintained in the absence of
the selective agent as the consequence of the balance between
recurrent mutation and selection (Table 3).
Discussion
There is growing scientific interest in how inhabitants of
geothermal waters adapt to living in some of the most extreme
conditions on Earth (Sand, 2003). Geothermal areas are
often hazardous for mesophilic photosynthesizers because
of the presence of dissolved mineral components such as
arsenic and mercury, hydrogen sulphide, and highly acidic
or very alkaline conditions (Webster & Nordstrom, 2003;
Tyrovola et al., 2006; Lehr et al., 2007). Indeed, we unable
to detect cyanobacteria or microalgae in the geothermal
waters of Sufione Pisciarelly (Italy) and La Maquinita (Los
Andes), and most of geothermal waters analysed were
lethal for nearly all cells of our strains of D. chlorelloides and
M. aeruginosa. For example, six geothermal waters (from a total
of seven) analysed from Italy and four icebound geothermal
waters (from a total of five) analysed from Los Andes, totally
inhibited growth and photosynthetic quantum yield of
D. chlorelloides and M. aeruginosa. Consequently, the survival
of mesophilic cyanobacteria and microalgae in most geothermal
waters could only be achieved by some kind of genetic
adaptation.
Apparently, adaptation to geothermal waters is not easy.
The eukaryotic microalga D. chlorelloides was unable to adapt
to waters from Sufione Pisciarelly and La Maquinita, whereas
the cyanobacterium M. aeruginosa did not adapt to water
samples from Pienza, Infierno Sujo, Sufione Pisciarelly, Puzzoli,
Aguas Calientes, Las Papas and La Maquinita (7 geothermal
waters from a total of 12). Since adaptation to extreme
environments (such as geothermal waters) seems to be difficult,
the classic point of view assumes that genetic adaptation
at such extreme conditions is a gradual process. By contrast,
here we propose an alternative explanation for adaptation of
cyanobacteria and microalgae to geothermal waters. When
D. chlorelloides and M. aeruginosa were cultured in different
geothermal waters, usually cultures show that all the sensitive
cells are destroyed by the toxic effect of such waters. However,
after further incubation for 75 d, some cultures recovered,
owing to the growth of cells that were resistant to the toxic
effect of geothermal waters. The key to understanding
adaptation of mesophilic cyanobacteria and microalgae to the
extremely adverse conditions of the geothermal waters is to
analyse the rare variants that proliferate after the massive
destruction of the sensitive cells by this selective agent. Fluctuation analysis is an appropriate procedure to discriminate
between geothermal water-resistant cells arising by rare spontaneous mutations occurring randomly during replication of
organisms before exposure to this selective agent and geothermal
water-resistance arising through specifically acquired adaptation
induced by geothermal waters (Sniegowski & Lenski, 1995;
revised by Sniegowski, 2005).
Genetic adaptation by rare spontaneous mutation seems to
be the usual mechanism allowing adaptation of microalgae
and cyanobacteria to geothermal waters. The large fluctuation
© The Authors (2008). Journal compilation © New Phytologist (2008) www.newphytologist.org
New Phytologist (2008) 180: 922–932
929
930 Research
in number of resistant cells detected in the set 1 experiments
compared with the insignificant fluctuation in set 2 controls
(observed in 8 geothermal water samples from a total of 12
with D. chlorelloides as well as in 4 geothermal water samples
from a total of 12 in M. aeruginosa), unequivocally demonstrates
that these resistant cells arose by rare spontaneous single
mutations (which occur before geothermal waters exposure)
and not through direct and specific adaptation in response to
geothermal waters. Consequently, mesophiles can adapt to
such geothermal waters much more rapidly by single mutations
than if the ability to survive required multiple mutations.
Recent evidence suggests that mutation at one locus can enable
adaptation of mesophile cyanobacteria and microalgae to
other hostile natural environments (Flores-Moya et al., 2005;
Costas et al., 2007; López-Rodas et al., 2008a,b) as well as to
sudden anthropogenic chemical contamination (Costas et al.,
2001; López-Rodas et al., 2001; García-Villada et al., 2002,
2004; López-Rodas et al., 2007). This evidence suggests that
the different species of mesophilic algae and cyanobacteria
follow the same pattern. Consequently, it could be hypothesized
that mesophilic populations would be likely to survive
extreme environmental changes.
The mutation rates from geothermal water-sensitivity
to geothermal water-resistance in D. chlorelloides ranged from
1.4 × 10–6 to 1.5 × 10–5 mutants per cell per division. These
values were similar to those found in this species for the
resistance to the potent biocide 2,4,6-trinitrotoluene (GarcíaVillada et al., 2002), but higher than those we have described
for resistance to the stressful (pH < 2 and high levels of metals)
natural environments from Spain’s Rio Tinto (Costas et al.,
2007) or, in the case of the chlorophycean Spirogyra, to the
sulphurous water from La Hedionda Spa (southern Spain)
(Flores-Moya et al., 2005). In this sense, mutation rates from
geothermal water-sensitivity to geothermal water-resistance
in the cyanobacterium M. aeruginosa (from 1.1 × 10 –6 to
1.1 × 10–5) were higher than those for the algicide copper
sulphate (García-Villada et al., 2004) or the herbicide glyphosate (López-Rodas et al., 2007). It may be that the mutation
for geothermal water resistance is more common than other
kinds of mutation.
In contrast, physiological acclimatization only occurred in
two geothermal water samples from a total of twelve in
D. chlorelloides and only one geothermal water sample from a
total of twelve in M. aeruginosa (as demonstrated by the insignificant fluctuation in number geothermal water-resistant
cells observed in the set 1 experiment, and similar to the insignificant fluctuation in set 2 controls). In addition, adaptation
was not achieved by either D. chlorelloides in Suffione Pisciarelly
and La Maquinita, or by M. aeruginosa in seven geothermal
water samples from a total of twelve samples analysed.
In a few cases, geothermal waters are lethal for photosynthetic
microbes. Conversely, cyanobacteria and microalgae seem to
be able to adapt to stressful geothermal waters as a result of
rare spontaneous mutations and, if the toxicity is not too high,
New Phytologist (2008) 180: 922–932
by physiological acclimatization. Taking into account both
the relatively high number of resistant mutants and the countless cells comprising phytoplankton populations, apparently
enough geothermal water-resistant mutant cells are present
in natural populations of these organisms (before geothermal
water exposure) as consequence of the balance between recurrent
spontaneous mutations and selection.
This capability of mesophilic algae to adapt to geothermal
waters by means of mutation at one or a small number of loci
could have significant implications for the survival of algae
during the Neoproterozoic ‘snowball Earth.’ Since green, red
and chromophyte eukaryotic algae evolved before the late
Neoproterozoic glaciations (Knoll & Bauld, 1989; Knoll,
1992) they must have survived these extreme environmental
conditions. Although primary production collapsed during
the ‘snowball Earth’ period (Kaufman et al., 1997; Hoffman
et al., 1998; Schrag & Hoffman, 2001; Rothman et al., 2003),
just afterwards massive blooms of eukaryotic algae took place
(Elie et al., 2007). In addition, microfossils suggest that global
Neoproterozoic glaciations did not make a large contribution
to the extinction of autotrophic eukaryotes (Corsetti et al.,
2006). The survival of photosynthetic life during the Neoproterozoic ‘snowball Earth’ may have been achieved in ice-free
refuges of geothermal waters (Schrag & Hoffman, 2001).
Usually, icebound geothermal waters show a considerable
gradient of temperature (very hot within the spring, and
progressively colder, ultimately freezing). Consequently,
temperature is not a selective factor in these areas and only
selection for resistance to extreme values of pH, toxic and
other chemical stressors will occur. In addition there is no
evidence that the ‘snowball Earth’ ancestors of modern
mesophilic algae were thermophiles. A recent paper supports
the concept of continuous, numerous establishment events by
mesophilic microorganisms in volcanic areas, in contrast to
very rare arrivals of thermophilic strains (Portillo & González,
2008). It could be hypothesized that among these continuously
arriving mesophilic microalgae, resistant mutants easily
colonized numerous geothermal waters, which could be ‘Noah’s
Arks’ during the global glaciations of Neoproterozoic Earth.
Arriving mesophilic algae were able to colonize extremely
acid and toxic environments of Rio Tinto and La Hedionda
(Flores-Moya et al., 2005, Costas et al., 2007). Another
alternative could be the survival of microbial communities
living within and upon ice (Corsetti et al., 2006), if only
a thin (< 2 m) ice cover occurred in tropical latitudes (Pollard
& Kasting, 2005). Perhaps eukaryotic algae could proliferate
in both scenarios, with geothermal waters as the main refuge
of freshwater algae and the sea under a thin ice cover as refuge
for marine algae. However, after the Neoproterozoic ‘snowball
Earth’ period ended (with very high atmospheric CO2) the
water would be acid, warm and very rich in nutrients (Nisbet
et al., 2007). Consequently, the algae and cyanobacteria that
survived in acid geothermal waters would have been pre-adapted
to colonize these new environments.
www.newphytologist.org © The Authors (2008). Journal compilation © New Phytologist (2008)
Research
Acknowledgements
This work was financially supported by CGL 2005-01938
BOS, S-0505/AMB/0374 CAM and P05-RNM-00935 grants.
Dr Eric C. Henry (Herbarium, Department of Botany and
Plant Pathology, Oregon State University) kindly revised the
English style and usage. Dr Fernando Hiraldo (Estación
Biológica de Doñana, Consejo Superior de Investigaciones
Científicas) and Dr Antonio Delgado (Estación Experimental
de Zaidín, Consejo Superior de Investigaciones Científicas)
suggested the initial ideas. Manuel de la Riva, Sebastián
Dimartino and Obdulio Monsalvo helped us during sampling
in the field. Eva Salgado, Fernando Marvá and Mónica Rouco
contributed to the laboratory work.
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New Phytologist (2008) 180: 922–932
www.newphytologist.org © The Authors (2008). Journal compilation © New Phytologist (2008)