Short-term adaptation of microalgae in highly stressful
environments : an experimental model analysing the resistance
of Scenedesmus intermedius (Chlorophyceae) to the heavy
metals mixture from the Aznalcollar mine spill
R . B AO S1 , L . G AR C I A -V ILLA D A2 , M . AG R EL O3 , V . L O P E Z - R O D A S2 , F . HI R A L D O1
A ND E . CO S TA S2
1 Estacion Biologica de Donana, Consejo Superior de Investigaciones Cientıficas, Avd. de Marıa Luisa s/n, Pabellon del Peru,
41013, Seville, Spain
2 Genetica, Facultad de Veterinaria, Universidad Complutense, 28040, Madrid, Spain
3 Departamento NBQ, F.N. La Maranosa, Ministerio de Defensa, PO Box 1105, Madrid, Spain
The toxic spill of acid wastes rich in heavy metals/metalloids (AWHM) from the Aznalcollar mine in April 1998, threatening
the Donana National Park, is considered to be the worst environmental disaster related to acute pollution in Spanish
history. The aim of this work was to study the adaptation of microalgae (which play an important role as primary
producers) from AWHM sensitivity to AWHM resistance by using the alga Scenedesmus intermedius as an experimental
model. The Malthusian parameter (m) and the carrying capacity (K) were reduced by mud and soil samples collected from
the affected area. A dose–effect analysis showed that fitness progressively diminished with increasing sample concentration. A
fluctuation analysis demonstrated that AWHM-resistant cells arose by rare spontaneous mutations that occurred randomly
prior to the incorporation of the AWHM. The rate of spontaneous mutation from AWHM sensitivity to AWHM resistance
was 2 12x10−O mutants per cell division. A competition experiment between wild-type AWHM-sensitive cells and AWHMresistant mutants showed that in small populations the AWHM-resistant mutants are driven to extinction in the absence of
selection for AWHM resistance. The resistant phenotypes are maintained in the absence of AWHM as the result of a balance
between spontaneous mutation and natural selection, so that about 43 AWHM-resistant mutants per million cells are present
in the absence of AWHM. Our experimental model suggests that mutation is essential for adaptation of microalgal
populations to environmental changes. Rare spontaneous pre-adaptive mutation is enough to ensure the survival of
microalgal populations in contaminated environments when the population size is large enough.
Key words : adaptation, Aznalcollar, Donana National Park, microalgae, mutation, resistance, toxic spill
Introduction
Human activities are altering biosphere-level processes and causing a biodiversity crisis (Woodruff,
2001), such as by water pollution resulting from
environmental catastrophes in rivers, marshes and
estuaries. As an example, the toxic spill of acid
wastes rich in heavy metals/metalloids (AWHM)
from the processing of pyrites at Aznalcollar mine is
considered to be the worst environmental disaster
related to acute pollution ever recorded in Spanish
history (Grimalt et al., 1999). The Aznalcollar mine
accident happened on April 1998 when the tailing
pond was breached, threatening the Donana
National Park, one of the most important wildlife
sites for the breeding and wintering of many birds
Correspondence to : E. Costas. Fax +34 9 3943769. e-mail
vlrodas@vet.ucm.es
ones. Approximately
including endangered
4x106 m3 of acid water and 2x106 m3 of toxic mud
containing large amounts of Fe (34–37 %), S (35–
40 %), Zn (0 8 %), Pb (0 8 %), As (0 5 %), Cu
(0 2 %), Sb (0 05 %), Co (0 0062 %), Tl (0 005 %), Bi
(0 005 %), Cd (0 0025 %), Ag (0 0025 %), Hg
(0 0015 %) and Se (0 001 %) were released into the
Agrio River and then entered the Guadiamar River,
which is a major tributary of the Guadalquivir
River (Grimalt et al., 1999). The toxic spill flooded
a zone of 400 m on both sides of these rivers. A mud
layer 1 7 m thick was left around the mine. Even
40 km downstream, the mud layer was still a few
centimetres thick, and affected approximately
4500 ha of adjacent land. The polluted water continued downstream for a further 20 km.
The Donana marshland is situated in the delta of
the Guadalquivir River (SW Spain). Because of its
R. Baos et al.
international importance, 132 000 ha of Donana
have been protected under national, EU, or international laws and conventions. Parts of the area
have been designated as a Ramsar Site (an internationally important wetland under the Ramsar
Convention) and Biosphere Reserve (UNESCO,
1981), and the area was inscribed as a World
Heritage Site in 1994 (Pain et al., 1998). In summary,
Donana National Park harbours 803 plant and 458
animal species, 361 of which are birds, representing
70 % of all European waterbird species.
The toxic spill of the Aznalcollar mine had a great
impact on the microalgal community of Agrio,
Guadiamar and Guadalquivir rivers. The spill
caused the complete disappearance of aquatic communities in the affected area, and 7 months later the
recovery of these communities was still poor (Prat et
al., 1999).
Numerous studies have shown that heavy metals
and metalloids are extremely toxic to microalgae in
both laboratory cultures and natural populations.
Microalgae are susceptible to arsenic (Blanck et al.,
1984, 1988), which has a significant effect on the
structure and physiology of the phytoplankton
community in lakes (Knauer et al., 1999). In
addition, organotin compounds have inhibitory
effects on the growth of Scenedesmus (Fargasova &
Kizlink, 1996). Uranium is toxic to Chlorella
(Franklin et al., 2000), mercury decreases photosynthetic efficiency of cyanobacteria even at micromolar concentrations (Lu et al., 2000), and copper
exposure has dramatic effects on algal communities
(Nor, 1987). Moreover, synergies among the components of the heavy metals could increase the
environmental hazards.
However, it has also been reported that microalgae from contaminated sites appear to have
adapted to high arsenic concentrations whereas
algae from unpolluted lakes remain sensitive
(Knauer et al., 1999). Rapid adaptation of microalgae to environmental changes resulting from
water pollution has been demonstrated recently
(Costas et al., 2001 ; Lopez-Rodas et al., 2001).
Unfortunately, the evolution of microalgae subsequent to a catastrophic environmental change is
insufficiently understood. Most of the theoretical
and experimental backgrounds to genetic mechanisms to adaptation have been carried out in
Mendelian populations of sexual diploid organisms
(Dobzhansky, 1955 ; Mayr, 1963 ; Lewontin, 1974 ;
Kimura, 1989 ; Spiess, 1989), whereas microalgae
are usually haploid with asexual reproduction and
form large populations of large clonal families
(Costas, 1990). Consequently, to determine whether
microalgae are able to adapt to acid wastes rich in
heavy metals is of considerable interest.
The main objectives of this work were : (i) to
explore the effects of this contamination episode on
594
microalgal populations and (ii) to estimate the
capability of microalgae to adapt to the toxic spill of
acidic heavy metals (AWHM) threatening the
Donana National Park. We studied unialgal laboratory populations of Scenedesmus intermedius
(Chlorophyceae) as an experimental model because
Scenedesmus spp. are among the most abundant
microalgal species in the affected area (Prat et al.,
1999). Furthermore, we have detected S. intermedius
in heavy-metal-contaminated tailing ponds of the
Aznalcollar mine. We analysed : (i) the impact of
mud and soil samples collected in the affected area
on the growth and survival of S. intermedius cells,
(ii) the occurrence of AWHM-resistant (AWHM r)
cells in cultures of AWHM-sensitive (AWHM s)
cells, including the rate of transformation from
AWHM sensitivity to AWHM resistance, and the
nature of the AWHMr cells (i.e. AWHMr cells
arising by direct and specific acquired adaptation in
response to the AWHM versus AWHMr cells
arising by rare spontaneous mutations occurring
randomly prior to AWHM exposure), and (iii) the
mechanisms of maintenance of AWHMr mutants in
microalgal populations (including the fitness of
wild-type cells and AWHMr mutants in the presence
or absence of AWHM and the competitive relations
between wild-type and AWHMr cells).
Materials and methods
Experimental organisms
Haploid vegetative cells of Scenedesmus intermedius
Chodat (Chlorophyceae), wild-type strain Si31Mwt from
the algal culture collection of Universidad Complutense
(Madrid), were grown axenically in cell culture flasks
(Greiner) with 20 ml of BG-11 medium (Sigma-Aldrich)
at 20 °C with a photon irradiance of 60 µmol m −2 s −1
from fluorescent tubes under continuous light. This strain
was isolated from a reservoir in Segovia (Spain). Cells
were maintained in mid-log exponential growth by serial
transfers of a cell inoculum to fresh medium once a
month. Prior to the experiments, the culture of Si31Mwt
was re-cloned (by isolating a single cell) to prevent
accumulation of previous spontaneous mutants. Cultures
were maintained as axenically as possible, and only
cultures without detectable bacteria were used in the
experiments.
AWHM sample collection
Four samples collected in the area affected by the spill of
AWHM were studied : near the mine (S1), Aznalcazar
(S2), Puente de Don Simon (S3) and Cangrejo Grande
(S4). One more sample, Palacio de Donana (S5), located
in an unaffected area, was used as a control site. The
sample characteristics (how the sample was affected by
the toxic spill and heavy metal/metalloid contents) are
summarized in Table 1. Following the procedure of
Simon et al. (1999), at all sampling points, two square
plots were laid out (25 mx25 m). At each corner and in
the centre of the plots, samples of mud (S1 and S3) or soil
Adaptation of Scenedesmus intermedius to the Aznalcollar mine spill
Table 1. Characterization of the samples from the toxic
spill at the Aznalcollar mine
Heavy metal
concentration
( µg g −1)
Co
Cu
Zn
As
Cd
Sb
Tl
Pb
S1
TM
S2
STM
S3
TM
S4
STM
S5
US
36
2095
12 925
2998
39
245
49
7661
10
113
353
89
1
5
2
252
35
850
4511
1815
20
39
25
5632
15
35
71
15
0
1
1
44
10
20
60
4
0
1
1
33
TM, toxic mud ; STM, soil affected by toxic mud ; STW, soil
affected by toxic water ; US, unaffected soil. The results are
expressed in dry weight.
at 0–10 cm depth (S2, S4 and S5) were taken. All samples
were dried and screened (2 mm screen size). Next, 250 g
of each sample category from the five sampling points per
plot were mixed and homogenized, stored in plastic
containers or polyethylene bags until analysis. S1 and S3
were taken in May/June 1998 (approximately 1 month
after the toxic spill), S2 and S4 in October/November
1998, after the first cleaning activities, and S5 was
collected in March 1999.
A two-step bulk sample digestion method, in Teflon
reactors, was used for heavy metal analysis of mud and
soils (S1–S5). This method was devised by Querol et al.
(1996). Solutions obtained from the digestion of samples
were analysed by inductively coupled plasma mass
spectrometry (ICP-MS). The accuracy of the analysis was
checked against certified reference materials (SO-4
Canadian Certified Reference Materials Projects) and
was expressed with a coefficient of variation of < 10 %.
Analytical precision, expressed as the relative standard
deviation, ranged between 3 % and 10 % for all the
elements studied.
Effect of AWHM
The effect of four different AWHM samples (S1, S2, S3,
S4) on algal cells was analysed as follows : A stock
solution was prepared by mixing 20 ml of each AWHM
sample and 200 ml of BG-11 medium (Sigma-Aldrich) for
18 h under continuous agitation ; after 6 h decanting, the
solution was filtered through 0 22 µm filters (Millipore) to
make it axenic. Each stock solution was inoculated with
10O±103 cells from mid-log exponentially growing
cultures. The control cultures contained S5, and S0
samples contained only BG-11 medium.
The effect of the different samples (S0–S5) was estimated by calculating the fitness under conditions of r and
K selection of triplicates of each AWHM sample and
controls as previously described (Lopez-Rodas et al.,
2001). In short, fitness under conditions of r selection in
an uncrowded environment was estimated as the
Malthusian parameter (m), in mid-log exponentially
growing acclimated cultures, as : Nt = N e mt, where Nt
°
595
environment was determined as the carrying capacity
(K), estimated as the maximal cell density reached by the
culture in saturated phase (about 22–24 days). Experiments and controls were counted blind (i.e. the person
sample), using a haemocytometer, by at least two independent persons. The number of samples in each case
was determined using the progressive mean procedure
(Williams, 1977), which assured a counting error of
±1 %.
In addition, a dose–effect study was performed similarly using 1/1000, 1/100 and 1/10 dilutions of stock
solution from S1.
Analysis of transformation from AWHM sensitivity to
AWHM resistance
A Luria–Delbruck fluctuation analysis was used to
investigate the nature of the transformation from
AWHM sensitivity to AWHM resistance (i.e. to distinguish between AWHMr cells arising by rare spontaneous pre-adaptive mutations occurring randomly during replication of organisms prior to the incorporation of
AWHM and AWHM r cells arising through physiological
or specifically acquired post-selective adaptation in response to AWHM) and, subsequently, to estimate the
rate of occurrence of AWHM r cells. Since Luria &
Delbruck (1943) introduced fluctuation analysis for
bacteria as a combined experimental and statistical
procedure based on the variation in the occurrence of
resistant variants, subsequent theoretical and experimental studies have modified the fluctuation test to be used
with organisms from bacteria to human cells (Cole et al.,
1976 ; Kendall & Frost, 1988 ; Tlsty et al., 1989 ; Jones et
al., 1994 ; Rossman et al., 1995 ; Asteris & Sarkar, 1996 ;
Crane et al., 1996). Recently, Lopez-Rodas et al. (2001)
have further modified the Luria–Delbruck fluctuation
analysis to be used with microalgae : plating is replaced by
the addition of liquid medium containing the selective
agent.
In the first set of experiments, C = 100 parallel culture
tubes were inoculated with N
102 Si31Mwt cells (a
°
number small enough to ensure that no pre-existing
mutants are present), and grown axenically as identically
as possible under non-selective conditions. At the end of
the growing period (when each culture reached a convenient number of cells, Nt 5 1x10 4), the cells were
transferred to selective liquid medium containing
S3 sample at 1/10 concentration. For the second set of
experiments, 50 aliquots of approximately 5 2x10 4 cells
from the same parental population were separately
transferred to tubes containing the same selective medium
as set 1.
Acid wastes rich in heavy metals/metalloids killed the
wild-type AWHMs cells but allowed the growth of
AWHM r cells. Liquid cultures were monitored using a
Zeiss Axiovert inverted microscope. After the cells were
detected in liquid cultures, they were counted blind using
settling chambers by at least three independent persons.
Cultures were monitored over at least 60 days (thereby
ensuring that a single mutant cell could establish a dense
culture).
The proportion of cultures showing no mutant colonies
after plating in the first set of experiments (P estimator)
°
R. Baos et al.
and N are the cell numbers at time t and 0 respectively.
°
Fitness under conditions of K selection in a crowded
596
was used to calculate the mutation rate as follows : P =
°
e−µ(Nt −N°) (Luria & Delbruck, 1943 ; Griffiths et al., 1996),
Adaptation of Scenedesmus intermedius to the Aznalcollar mine spill
597
where P is the proportion of cultures showing no mutant
°
colonies after plating, (Nt—N ) is the number of cell
°
divisions, and µ is the mutation rate.
fitness (r selection)
fitness (K selection)
Characterization of AWHM-resistant mutants
1
Fitness
Fitness of five randomly isolated (from different cultures)
AWHM r mutants and wild-type AWHMs cells was
characterized under conditions of r and K selection in
BG-11 medium without AWHM as well as in medium
containing 1/10 and 1/100 S3 sample/medium. The
experiments were carried out just as in the dose–effect
study.
Competition between wild-type and AWHM-resistant
mutants
A competition experiment between wild-type cells and
AWHM r mutants was carried out as previously described
(Costas et al., 1998). Five replicates of mixed cultures
were established by mixing 10O AWHM r mutants and 10O
AWHM s wild-type cells. The cultures were maintained
by adding 562 µl of the culture and 2438 µl of fresh BG11 medium (without AWHM) once a week. The objective
was to attain about 3 5 days of exponential growth
(competition under r selection) and about 3 5 days of
saturation (competition under K selection). Samples from
each replicate were grown in 1/10 AWHM/BG-11
medium once a week to check for the presence of AWHM r
mutants.
0
0·000
0·001
0·01
0·1
Dose
Fig. 2. Relative fitness under conditions of r and K
selection of Scenedesmus intermedius wild-type cells as
affected by different doses of AWHM (S1). Relative fitness
is represented as a fraction of untreated controls
(mean±SD).
Table 2. Fluctuation analysis of the occurrence of
AWHM-resistant variants in Scenedesmus intermedius
strain Si31Mwt
Results
Set 1
Fitness under conditions of r and K selection of
wild-type microalgae was significantly diminished
( p < 0 05, Kruskal–Wallis H-tests) by S2 and S4,
and dramatically reduced by S1, with respect to S0
and S5 controls (Fig. 1). When microalgal cultures
were treated with S3, most cells died in less than 5
No. of replicate cultures
No. of cultures containing the following
no. of AWHM resistant cells − 1:
ml
0
< 1000
1000–10 000
> 10 000
Variance/mean (of the no. of AWHMresistant cells per replicate)
Mutation rate (mutants per cell division)
100
34
35
22
9
> 100
Set 2
50
48
3
12
2 12x10 −O
fitness (r selection)
fitness (K selection)
Fitness
1
0
S0
S5
S2
S4
S1
S3
Samples
Fig. 1. Relative fitness under conditions of r and K
selection of Scenedesmus intermedius wild-type cells as
affected by the same dose of different AWHM samples
days ; there were no living cells after 7 days, and the
Malthusian parameter was m = 0.
A dose–effect analysis using the S1 sample shows
that the maximal cell density reached by the culture
in the saturated phase (carrying capacity, K) was
severely diminished even by concentrations as low
as 1/1000 S1 sample/medium (Fig. 2). As expected,
fitness under conditions of r selection of wild-type
cells was also progressively diminished with increasing S1 concentration.
A fluctuation analysis was carried out to study the
spontaneous occurrence of AWHM r variants in
cultures of AWHMs cells. Our first aim was to
(S1–S5). Relative fitness is represented as a fraction of
untreated controls (mean±SD).
R. Baos et al.
determine the nature of the resistance of
the
AWHMr cells. The data presented in Table 2
show that the low variation in set 2 experiments
indicates that any large fluctuations in set 1
must be due to processes other than sampling
error. In the set 1 experiment the variance
significantly exceeded the
598
Adaptation of Scenedesmus intermedius to the Aznalcollar mine spill
599
Table 3. Relative fitness under conditions of r and K selection of S. intermedius wild-type cells (strain Si31Mwt) and
AWHM-resistant mutants in medium with and without AWHM (sample S3)
AWHM concentration (v/v)
0
Fitness under
1/100
1/10
Wild-type
Resistant
mutant
Wild-type
Resistant
mutant
Wild-type
Resistant
mutant
1±0 07
1±0 17
0 51±0 07
0 56±0 15
0 96±0 19
0 53±0 15
0 53±0 11
0 53±0 09
0
0
0 31±0 16
0 38±0 19
r selection
K selection
Values are mean±SD.
Table 4. Presence of AWHM-resistant cells in mixed
cultures (50 % AWHM-resistant, 50 % AWHM-sensitive
wild-type) evaluated at 1 week intervals under competition
Weeks under competition
1
2
3
4
Replicate :
I
II
III
IV
V
+
+
+
+
+
+
—
—
+
+
+
—
—
—
—
—
—
—
—
—
Controls :
Wild-type
AWHMr mutants
—
+
—
+
—
+
—
+
important reduction in both the Malthusian parameter and the carrying capacity of AWHMr
mutants with respect to the wild-type cells was
observed in the absence of AWHM (S3). In contrast,
when both kinds of cells were grown in the presence
of 1/10 AWHM (S3)/medium, only the AWHMr
mutants were able to grow.
The results of the competition experiment between AWHMr mutants and wild-type cells show a
quick displacement of the AWHMr mutants by the
wild-type sensitive phenotype (Table 4). After only
4 weeks of competitive interaction in the absence of
AWHM, the AWHM r phenotype was driven to
extinction by the wild-type.
Discussion
+ indicates ability to grow in liquid medium containing AWHM.
Controls were pure cultures of wild-type and AWHM-resistant
cells respectively.
mean (variance/mean > 100 ; p < 0 05 Prescesenyi
test – Mayr, 1963) indicating that : (i) AWHMr
variants arose by rare spontaneous mutation, and
not through direct and specific acquired adaptation
in response to an environmental selection, and (ii)
AWHM is not facilitating the occurrence of
AWHMr cells. In addition, transmission of AWHM
resistance through successive generations has been
examined by ascertaining the maintenance of the
AWHMr phenotype (in five replicates of AWHMresistant mutants) for 45 generations of serial
subculture in the absence of the selecting AWHM.
Our second aim was to estimate the rate of
mutation from AWHM sensitivity to AWHM
resistance. Spontaneous mutation rate ( µ)
AWHMsensitivity —÷ AWHMresistance (using the P
°
estimator) was 2 12x10−O. This mutation rate was
estimated with high standards of reliability, reproducibility and precision (Table 2).
Fitness under conditions of r and K selection of
AWHMr mutants was estimated in the absence and
in the presence of two different AWHM concentrations (Table 3), in an attempt to characterize the
population behaviour of AWHMr mutants. An
We are working on an insufficiently studied aspect
of evolution : the adaptation of microalgae subsequent to a catastrophic environmental change. It
is known that low doses of a heavy metal (even in
the micromolar range) dramatically reduce the
growth and photosynthesis of microalgae from
cyanobacteria to Chlorophyceae (Knauer et al.,
1999 ; Franklin et al., 2000 ; Lu et al., 2000).
Complex interactions and synergies have been
revealed when several metals and other substances
act simultaneously. In addition, physical parameters modify the toxicity of heavy metals. As an
example, the toxicity of uranium and copper is
highly pH-dependent (Franklin et al., 2000). Consequently, we used the AWHM samples themselves in
an attempt to develop an experimental model as
close to reality as possible.
AWHM collected from different sites of the
Donana area were differentially toxic to
Scenedesmus intermedius in relation to their heavy
metal concentration. Mud samples, S1 and S3, were
maximally toxic to S. intermedius as well as having
the highest concentrations of heavy metals and As,
whereas the soil AWHM samples S4 and S2 showed
the lowest toxicity. S5, collected in a pristine
control site, did not exhibit any toxic effect on the
experimental organisms. As expected, the fitness
R. Baos et al.
of S. intermedius progressively decreased with
increasing concentrations of AWHM, but concentrations as low as 1/1000 AWHM/medium severely
diminished the cell density reached by the culture
in the saturated phase, and concentrations of
1/100 severely diminished cell growth, whereas
1/10 concentrations caused massive destruction of
algal cells.
In areas close to the mine, where the mud
thickness was greatest, the concentration of heavy
metals was probably similar to or even exceeded
those we assayed experimentally in this study
(Simon et al., 1999). Our soil AWHM samples were
collected 6 months after the spill, when sludge
removal was being carried out in some places, and,
in spite of that, we could see effects on cell growth.
All these factors suggest that there must have been
a massive destruction of algal populations and,
therefore, a heavy decrease in productivity throughout the whole river ecosystem. Indeed, in situ studies
carried out 7 months after the toxic spill showed
that the recovery of the aquatic communities was
still poor (Prat et al., 1999).
After establishing that AWHM samples were
toxic to algal cells, our main aim was to study the
adaptation of microalgae under comparable conditions to those occurring in situ. We therefore
treated S. intermedius laboratory cultures with
lethal concentrations of samples collected from the
affected area (AWHM samples). It is well established that algal populations can respond to the
chronic presence of a chemical by the development
of resistance. Communities established under arsenate stress in laboratory experiments are more
tolerant to arsenate than communities grown at
background levels of arsenate (Blanck et al., 1988).
In addition, increased tolerance to a pollutant could
be interpreted as evidence of a chemical impact at
the community level (Blanck, 1984).
When a microalgal culture was treated with the
S3 sample, the culture became clear after some days
due to destruction of the sensitive cells by the
sample. However, after further incubation for a few
days, the culture sometimes increased in density
again, due to growth of an algal variant that was
resistant to the action of the S3 sample. The key to
understanding the adaptation of microalgae to
AWHM-contaminated environments could be
analysis of this algal variant.
The main aim of the present study was to
distinguish between AWHMr cells arising by rare
spontaneous mutations occurring randomly during
replication of organisms in non-selective conditions
(i.e. prior to addition of the AWHM) and AWHMr
cells arising through specifically acquired adaptation in response to environmental selection (i.e.
through physiological adaptation after incorporating the AWHM). Although it has long been
600
assumed that
only pre-adaptive spontaneous
mutations occur, Cairns et al. (1988) and Hall
(1988) have proven the occurrence of adaptive
mutation. This is a process that, during selection,
produces mutations that relieve the selective pressure whether or not other, non-selected mutations
are also produced. Adaptive mutations and other
related phenomena have been reported in bacteria
and yeast, but not in other microorganisms, and
it is conceivable that adaptive mutation plays an
important role in evolution of microorganisms
(Foster, 2000).
Luria–Delbruck fluctuation analysis is an appropriate procedure to discriminate between preselective and post-selective mutations (Luria &
Delbruck, 1943 ; Lea & Coulson, 1949 ; Cole et al.,
1976 ; Cairns et al., 1988 ; Tlsty et al., 1989 ;
Dijkmans et al., 1994). It has been used widely in
Chlamydomonas to distinguish between spontaneous
and
induced
streptomycin-resistant
mutants (Gillham & Levine, 1962) ; to analyse Nmethyl-N ’-nitro-N-nitrosoguanidine- and 5-fluorodeoxyuridine-induced mutations (Gillham, 1965 ;
Wurtz et al., 1979) ; and to characterize the occurrence of cadmium-resistant mutants (Collard &
Matage, 1990). Recently, fluctuation analysis has
been used for studying adaptation from herbicide
sensitivity to herbicide resistance in microalgae
and for estimating mutation rates (Costas et al.,
2001 ; Lopez-Rodas et al., 2001). Here, fluctuation
analysis unequivocally demonstrated that the S3
AWHM sample was not inducing AWHMr cells,
but that AWHM resistance occurred by rare spontaneous mutation prior to addition of AWHM.
The observed heritability of the AWHMr phenotype over several generations of serial subculture in
the absence of the selecting agent also suggests that
AWHM resistance is attributable to mutant genotypes.
On the other hand, calculation of mutation rates
of AWHM sensitivity to AWHM resistance is not a
trivial question because our experimental model
suggests that, as AWHM was lethal to most wildtype strains, only spontaneously arising AWHMr
mutants would be able to survive in such environments. Consequently, mutation rates offer insights
into the evolutionary capabilities of microalgal
populations in AWHM-contaminated environments. The mutation rate was 2 12x10 −O AWHMr
cells per cell division. It has been theoretically and
experimentally demonstrated that P -based esti°
mation is very useful (Li & Chu, 1987 ; Mandelbrot,
1974). Although rates of spontaneous mutation to
resistance to water pollution have not been
measured in other microalgae, our rates are higher
than those for antibiotic resistance and for herbicide
resistance in microalgae (Sager, 1985 ; Lopez-Rodas
et al., 2001), and similar to that generating bleached
Adaptation of Scenedesmus intermedius to the Aznalcollar mine spill
mutants in Euglena (Nicolas et al., 1962). Such
mutation rates, coupled with rapid growth rates, are
presumably high enough to ensure the adaptation
of microalgae to water contamination.
In contrast to molecular evolution (which is a
continuous process that often occurs over long
periods of time at a nearly constant rate, even if
there are variations in rate among lineages), or to
phenotypic evolution (which is perceived as a highly
irregular process with long periods of stasis interrupted by short bursts of change) (Gould &
Eldredge, 1977), adaptation of algal populations to
modern pollution-derived environmental hazards
seems to be the result of a rare instantaneous event.
Adaptation of microalgae to environments with
modern contamination (such as herbicides or 2,4,6trinitrotoluene) by this type of rare instantaneous
event has been demonstrated recently (Costas et al.,
2001 ; Lopez-Rodas et al., 2001). Mutation seems to
be essential in understanding how algal populations
adapt to contaminated environments. Organisms
may possess the ability to regulate their mutation
rate in response to environmental conditions
(Kepler & Perelson, 1995), and differential mutation
rates across genes may be adaptations (Maley,
1997). The competitive ability of clonal cultures of
microalgae growing asexually is improved by selection acting on new genetic variation that has arisen
by mutation (Costas et al., 1998). What is more, in
asexual organisms, adaptation is fastest when the
rate of mutation equals the harmonic mean of
selection coefficients of mutants (Allen-Orrh, 2000).
Since algicide resistance presumably occurs by
random rare mutations before microalgae come
into contact with the algicide, the mechanism of
maintenance of the resistant mutants in natural
populations is a central problem for ecological
genetic studies of microalgae. The AWHMr mutants
exhibited a diminished fitness that limited their
survival in natural populations in the absence of
AWHM, so that in small populations, the AWHMr
mutants are driven to extinction by competition.
However, recurrent mutation occurs from a normal
wild-type allele to an AWHM-resistant allele that is
detrimental in fitness in the absence of the AWHM
contamination. New resistant mutants arise in each
generation, but most of these mutants are
eliminated sooner or later by natural selection, if
not by chance (Spiess, 1989). At any one time there
will be a certain number of AWHMr mutants as the
result of balance between new resistant cells arising
from spontaneous mutation and resistant cells
eliminated by natural selection. The average number of such mutants will be determined by the
balance of the mutation rate and the rate of selective
elimination as : µ(1—q) = q(1—s), where µ is the
mutation rate, q is the allele frequency of the mutant
and s is the selection coefficient of the mutant (Crow
601
& Kimura, 1970 ; Spiess, 1989). In our case, after
estimating the mutation rate as µ = 2 12x10−O and
the selection coefficient as s = 0 49, the average
number of AWHMr mutants in the absence of the
AWHM is about 43 mutants per million cells.
In conclusion, and employing ‘ Occam’s razor ’,
spontaneous pre-selective mutants (as ‘ hopeful
monsters ’) are enough to ensure the adaptation of
the enormous natural populations of microalga to
catastrophic environmental changes, as well as to
antibiotics, herbicides and 2,4,6-trinitrotoluene
(Costas et al., 2001 ; Lopez-Rodas et al., 2001).
However, contaminant-resistant phenotypes show
reduced growth and saturation density. Consequently, although microalgal populations are able
to survive events such as an AWHM spill, important
ecological parameters (such as primary production
and biomass) could be severely diminished.
Acknowledgements
This work was supported by Spanish DGES PB960576-C03-01 and DGI REN2000-0771 HID. We
thank Drs Javier Juste and Luis Ferrero, and Elena
Carrillo for help and advice. We are grateful to the
two anonymous referees for helpful comments on
the manuscript.
References
A
-O
, H. (2000). The rate of adaptation in asexuals.
Genetics, 155 : 961–968.
A
, G. & S
, S. (1996). Bayesian procedures for the
estimation of mutation rates from fluctuation experiments.
Genetics, 142 : 313–326.
B
, H., W
, G. & W
, S.A. (1984). Speciesdependent variation in algal sensitivity to chemical compounds.
Ecotoxicol. Environ. Safety, 8 : 339–351.
B
, H., W
, S.A. & M
, S. (1988). Pollution
induced community tolerance : a new ecotoxicological tool. In
Functional Testing on Aquatic Biota for Estimating Hazards of
Chemicals (Cairns, J. & Pratt, J.R., editors), 219–230. ASTM STP
988. American Society for Testing and Materials, Philadelphia.
C
, J., O
, J. & M
, S. (1988). The origin of
mutants. Nature, 335 : 142–145.
C , J., A
, C.F. & G
, M.H. (1976). The fluctuation test
as a more sensitive system for determining induced mutation in
L5178Y mouse lymphoma cells. Mutat. Res., 41 : 377–386.
C
, J.M. & M
, R.F. (1990). Isolation and genetic
analysis of Chlamydomonas reinhardtii strains resistant to cadmium. Appl. Environ. Microbiol., 56 : 2051–2055.
C
, E. (1990). Genetic variability in growth rates of marine
dinoflagellates. Genetica, 83 : 99–102.
C
, E., N
, B., L
-R
, V., S
, C. & T
, M.
(1998). Adaptation to competition by new mutation in clones of
Alexandrium minutum. Evolution, 52 : 610–613.
C
, E., C
, E., F
, L.M., A
, M., G
V
, L., J
, J. & L
-R
, V. (2001). Mutations of
algae from sensitivity to resistance against environmental selective
agents : the ecological genetics of Dictyosphaerium chlorelloides
under lethal doses of 3-(3,4-dichlorophenyl)-1,1-dimethylurea
(DCMU) herbicide. Phycologia, 40 : 391–398.
C
, G.J., T
, S.M. & J
, M.E. (1996). A modified
Luria–Delbruck fluctuation assay for estimating and comparing
mutation rates. Mutat. Res., 354 : 171–182.
R. Baos et al.
C
, J.F. & K
, M. (1970). An Introduction to Population
Genetics Theory. Harper & Row, New York.
D
, R., K
, S. & M
, M. (1994). Poisson-like
fluctuation patterns of revertants of leucine auxotrophy (leu-500)
in Salmonella typhimurium caused by delay in mutant cell division.
Genetics, 127 : 353–359.
D
, T. (1955). Genetics of the Evolutionary Process.
Columbia University Press, New York.
F
, A. & K
, J. (1996). Effect of organotin compounds on the growth of the freshwater alga Scenedesmus
quadricauda. Ecotoxicol. Environ. Safety, 34 : 156–159.
F
, P.L. (2000). Adaptive mutation : implications for evolution.
BioEssays, 22 : 1067–1074.
F
, N.M., S
, J.L., M
, S.J. & L , R.P. (2000).
pH-dependent toxicity of copper and uranium to a tropical
freshwater alga (Chlorella sp.). Aquat. Toxicol., 48 : 275–289.
G
, N.W. (1965). Introduction of chromosomal and nonchromosomal mutations in Chlamydomonas reinhardtii with Nmethyl-N’-nitro-N-nitrosoguanidine. Genetics, 52 : 529–537.
G
, N.W. & L
, R.P. (1962). Studies on the origin of
streptomycin resistant mutants in Chlamydomonas reinhardtii.
Genetics, 47 : 1463–1474.
G
, S.J. & E
, N. (1977). Punctuated equilibria : the
tempo and mode of evolution reconsidered. Paleobiology, 3 :
115–151.
G
, A.J.F., M
, J.H., L
, R.C. & G
,
W.M. (1996). An Introduction to Genetic Analysis. McGraw-Hill
Interamericana.
G
, J.O., F
, M. & M P
, E. (1999). The mine
tailing accident in Aznalcollar. Sci. Total Environ., 242 : 3–11.
H
, B. (1988). Adaptive evolution that requires multiple spontaneous mutations. I. Mutations involving an insertion sequence.
Genetics, 120 : 887–897.
J
, M.E., T
, S.M. & R
, A. (1994). Luria–Delbruck
fluctuation experiments : design and analysis. Genetics, 136 :
1209–1216.
K
, W.S. & F
, P. (1988). Pitfalls and practice of
Luria–Delbruck fluctuation analysis : a review. Cancer Res., 48 :
1060–1065.
K
, T.B. & P
, A.S. (1995). Modelling and optimisation
of populations subject to time-dependent mutation. Proc. Natl.
Acad. Sci. USA, 92 : 8219–8223.
K
, M. (1989). The neutral theory of molecular evolution and
the worldview of the neutralists. Genome, 31 : 24–31.
K
, K., B
, R. & H
, H. (1999). Toxicity of inorganic
and methylated arsenic to algal communities from lakes along an
arsenic contamination gradient. Aquat. Toxicol., 46 : 221–230.
L , D.E. & C
, C.A. (1949). The distribution of the numbers
of mutants in bacterial populations. J. Genet., 48 : 264–285.
L
, R.C. (1974). The Genetic Basis of Evolutionary Change.
Columbia University Press, New York.
L , I. & C , H.Y. (1987). Evaluation of methods for the estimation
of mutation rates in cultured mammalian cell populations. Mutat.
Res., 190 : 281–287.
L
-R
, V., A
, M., C
, E., F
, L.M.,
L
, A., M
-O
, L. & C
, E. (2001). Re-
602
sistance of microalgae to modern water contaminants as the result
of rare spontaneous mutation. Eur. J. Phycol., 36 : 179–190.
L , C.M., C
, C.W. & Z
, J.H. (2000). Acute toxicity of
excess mercury on the photosynthetic performance of cyanobacterium, S. platensis : assessment by chlorophyll fluorescence
analysis. Chemosphere, 41 : 191–196.
L
, S. & D
, M. (1943). Mutations of bacteria from virus
sensitivity to virus resistance. Genetics, 28 : 491–511.
M
, C. (1997). Mutation rates as adaptations. J. Theor. Biol.,
186 : 339–348.
M
, B. (1974). A population birth-and-mutation process.
I. Explicit distributions for the number of mutants in an old
culture of bacteria. J. Appl. Prob., 11 : 437–444.
M
, R. (1977). Ecologıa. Editorial Omega, Barcelona.
M
, E. (1963). Animal Species and Evolution. Harvard University
Press, Cambridge, MA.
N
, P., H
, P. & N
, V. (1962). Different phenotypes of non-photosynthetic (photo-)phenotypes in Euglena
gracilis : frequency of production by ultraviolet irradiation. C.R.
Seanc. Acad. Sci. III, 294 : 145–148.
N , Y.M. (1987). Ecotoxicity of cooper to aquatic biota : A review.
Environ. Res., 43 : 274–282.
P , D.J., S
, A. & M
, A.A. (1998). The Donana
ecological disaster : contamination of a world heritage estuarine
marsh ecosystem with acidified pyrite mine waste. Sci. Total
Environ., 222 : 45–54.
P
, N., T , J., S
, C., B
, M.D., P
, M. &
R
, M. (1999). Effect of dumping and cleaning activities on the aquatic ecosystems of the Guadiamar River
following a toxic flood. Sci. Total Environ., 242 : 231–248.
Q
, X., A
, A., L
-S
, A., M
, E. &
P
, F. (1996). Mineralogy of atmospheric particulates around
a large coal-fired power station. Atmos. Environ., 30 : 3557–3572.
R
, T.G., G
, E.I. & N
, A. (1995). Modelling and measurement of the spontaneous mutation rate in
mammalian cells. Mutat. Res., 328 : 21–30.
S
, R. (1962). Streptomycin as a mutagen for nonchromosomal
genes. Proc. Natl. Acad. Sci. USA, 48 : 2018–2026.
S
, R. (1985). Chloroplast genetics. BioEssays, 3 : 180–184.
S
, M., O
, I., G
, I., F
, E., F
, J.,
D
, C. & A
, J. (1999). Pollution of soils by the
toxic spill of a pyrite mine (Aznalcollar, Spain). Sci. Total
Environ., 242 : 105–115.
S
, E.B. (1989). Genes in Populations, 2nd edition. Wiley, New
York.
T
, T.D., M
, B.H. & L , K. (1989). Differences in the
rates of gene amplification in nontumorigenic cell lines as
measured by Luria–Delbruck fluctuation analysis. Proc. Natl.
Acad. Sci. USA, 86 : 9441–9445.
W
, M. (1977). Stereological techniques. In Practical Methods
in Electron Microscopy, vol. 6, part II (Glauvert, A.M, editor),
1–216. North-Holland America, Elsevier, Amsterdam.
W
, D.S. (2001). Declines of biomes and biotas and the
future of evolution. Proc. Natl. Acad. Sci. USA, 98 : 5471–5476.
W
, E.A., S
, B.B., R
, D.K., S
, H.S.,
G
, N.W. & B
, J.E. (1979). A specific increase in
chloroplast gene mutations following growth of Chlamydomonas
in 5-fluorodeoxyuridine. Mol. Gen. Genet., 170 : 235–242.