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. BAO S1 , L . GARCI A-VILLAD A2 , M . AGREL O3 , V . L O PEZ-RODA S2 , F
. HIRALD O1
AND E. COSTA S2
1
Estacion Biologica de Donana, Consejo Superior de Investigaciones
Cientıficas, Avd. de Marıa Luisa s/n, Pabellon del Peru,
42 1013, Seville, Spain
3 Genetica, Facultad de Veterinaria, Universidad Complutense, 28040,
Madrid, Spain
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 AWHM-resistant
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
includ6ing3
endangered ones.
6 A3pproximately
Human activities are altering biosphere-level pro-
4x10
m of acid water and 2x10
m of toxic mud
cesses 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
[email protected]
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 con- tinued downstream for a
further 20 km.
The Donana marshland is situated in the delta of the Guadalquivir River
(SW Spain). Because of its
international importance, 132 000 ha of Donana have been protected
under national, EU, or in- ternational laws and conventions. Parts of the
area have been designated as a Ramsar Site (an in- ternationally
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 com- munities 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 photo- synthetic
efficiency of cyanobacteria even at micro- molar concentrations (Lu et
al., 2000), and copper exposure has dramatic effects on algal communities
(Nor, 1987). Moreover, synergies among the com- ponents of the heavy
metals could increase the environmental hazards.
However, it has also been reported that micro- algae 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 micro- algae 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 sub- sequent to a catastrophic environmental change is
insufficiently understood. Most of the theoretical and experimental
backgrounds to genetic mech- anisms 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
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 lab- oratory 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 r) cells in cultures of AWHM-sensitive
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)
2 1
at 20 °C with a photon irradiance of 60 µmol m s
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
Table 1. Characterization of the samples from the toxic spill at the
Aznalcollar mine
Heavy metal
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). Experi- ments and controls were counted blind (i.e. the
person
sample), using a haemocytometer, by at least two in-
concentration
( µg g )
S1 S2 S3 S4 S5
TM STM TM STM US
dependent persons. The number of samples in each case was determined
using the progressive mean procedure
Co 36 10 35 15 10
Cu 2095 113 850 35 20
Zn 12 925 353 4511 71 60
As 2998 89 1815 15 4
Cd 39 1 20 0 0
Sb 245 5 39 1 1
Tl 49 2 25 1 1
Pb 7661 252 5632 44 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.
(Williams, 1977), which assured a counting error of
±1 %.
In addition, a dose–effect study was performed simi- larly 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 dis-
r
tinguish between AWHM
cells arising by rare spon-
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
taneous pre-adaptive mutations occurring randomly dur- ing replication of
organisms prior to the incorporation of AWHM and r cells
arising through physiological or specifically acquired post-selective
adaptation in re- sponse to AWHM) and, subsequently, to estimate the
r
containers or polyethylene bags until analysis. S1 and S3
rate of occurrence of AWHM
cells. Since Luria &
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
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 experimen-
tal 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 = 2100 parallel culture
deviation, ranged between 3 % and 10 % for all the
tubes were inoculated with N° 10
Si31Mwt cells (a
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
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 cultur4e reached a con- venient number of
cells, Nt 5 1x10 ), the cells were
transferred to selective liquid medium containing
S3 sample at 1/10 concentration. For the second 4set of
sample and 200 ml of BG-11 medium (Sigma-Aldrich) for
experiments, 50 aliquots of approximately 5 2x10
cells
18 h under continuous agitation ; after 6 h decanting, the solution was
filtered through 0 22 µm filters (Millipore) to
maOke it 3axenic. Each stock solution was inoculated with
from the same parental population were separately transferred to tubes
containing the same selective medium as set 1.
10 ±10
cells from mid-log exponentially growing
Acid wastes rich in heavy metals/metalloids killed the
s
cultures. The control cultures contained S5, and S0
wild-type AWHM
r
cells but allowed the growth of
samples contained only BG-11 medium.
AWHM
cells. Liquid cultures were monitored using a
The effect of the different samples (S0–S5) was esti- mated 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
mt
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
growing acclimated cultures, as : Nt = N° e
, where Nt
after plating in the first set of experiments (P° estimator)
and N° are the cell numbers at time t and 0 respectively. Fitness under
conditions of K selection in a crowded
was used to calculate the mutation rate as follows : P° =
µ(N N )
e t ° (Luria & Delbruck, 1943 ; Griffiths et al., 1996),
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.
Characterization of AWHM-resistant mutants
Fitness of five randomly isolated (from different cultures)
fitness (r selection)
fitness (K selection)
r
AWHM
mutants and wild-type s
1
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
r
AWHM mutants was carried out as previously described
(Costas et al., 1998). Five reOplicates of mixed culturesO
0
0·000 0·001 0·01 0·1
Dose
were established by mixing 10
s
AWHM mutants and 10
AWHM
wild-type cells. The cultures were maintained
Fig. 2. Relative fitness under conditions of r and K
by adding 562 µl of the culture and 2438 µl of fresh BG-
11 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 r mutants.
Results
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
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
Set 1 Set 2
No. of replicate cultures 100 50
No. of cultures containing the following no. of AWHM resistant cells
:
ml
0 34
< 1000 35
1000–10 000 22 48
> 10 000 9 3
Variance/mean (of the no. of AWHM-
resistant cells per replicate)
> 100 1 2
O
fitness (r selection)
fitness (K selection)
1
Mutation rate (mutants per cell division) 2 12x10
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 in-
creasing S1 concentration.
A fluctuation analysis was carried out to study the
r
spontaneous occurrence of AWHM
0 s
variants in
S0 S5 S2 S4 S1 S3
cultures of AWHM
cells. Our first aim was to
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 (S1–S5). Relative fitness is represented as a
fraction of untreated controls (mean±SD).
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
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 1/100 1/10
Fitness under
Wild-type
Resistant
mutant Wild-type
Resistant
mutant Wild-type
Resistant mutant
"r selection "1±0 07 "0 51±0 "0 96±0 "0 53±0 "0 "0 31±0 16 "
" " "07 "19 "11 " " "
"K selection "1±0 17 "0 56±0 "0 53±0 "0 53±0 "0 "0 38±0 19 "
" " "15 "15 "09 " " "
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
+ + — —
important reduction in both the Malthusian par- ameter 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 be- tween 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
r
Controls :
AWHM, the AWHM
phenotype was driven to
Wild-type — — — —
AWHMr mutants + + + +
+ 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 AWHM- resistant 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°
O This mutation rate was
extinction by the wild-type.
Discussion
We are working on an insufficiently studied aspect of evolution : the
adaptation of microalgae sub- sequent 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 par- ameters modify
the toxicity of heavy metals. As an example, the toxicity of uranium
and copper is highly pH-dependent (Franklin et al., 2000). Conse-
quently, 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
estimator) was 2 12x10 .
estimated with high standards of reliability, repro- ducibility 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
concen-
trations (Table 3), in an attempt to characterize the population
behaviour of AWHMr mutants. An
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
of S. intermedius progressively decreased with increasing concentrations
of AWHM, but concen- trations 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 through- out
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
con- ditions 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 estab-
lished that algal populations can respond to the chronic presence of a
chemical by the development of resistance. Communities established under
ar- senate 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 adap- tation in response to environmental selection (i.e.
through physiological adaptation after incor- porating the AWHM).
Although it has long been
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 press- ure 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 ap- propriate procedure to
discriminate between pre- selective 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 spon- taneous and induced streptomycin-resistant mutants
(Gillham & Levine, 1962) ; to analyse N- methyl-N '-nitro-N-
nitrosoguanidine- and 5-fluoro- deoxyuridine-induced mutations (Gillham,
1965 ; Wurtz et al., 1979) ; and to characterize the occur- rence 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 spon- taneous mutation prior to addition of AWHM.
The observed heritability of the AWHMr pheno- type over several
generations of serial subculture in the absence of the selecting agent
also suggests that AWHM resistance is attributable to mutant geno- types.
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 wild- type strains, only
spontaneously arising AWHMr mutants would be able to survive in such
environ- ments. Consequently, mutation rates offer insights into the
evolutionary capabilities of microalgal populations in AWHM-contaminated
environ- ments. The mutation rate was 2 12x 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
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 in- terrupted 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 2,4,6- trinitrotoluene) 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 selec- tion 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 num- ber
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
& Kimura, 1970 ; Spiess, 1989). In our case, after estimating the mutation
rate as µ = 2 12x10 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. Conse- quently, 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 PB96-
0576-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.
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(AWHM
(AWHM
1
AWHM
Fitness
AWHM
r
AWHM
1
Fitness
10
O
O
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Adaptation of Scenedesmus intermedius to the Aznalcollar mine spill
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