Research

Adaptation of Spirogyra insignis (Chlorophyta) to an

Blackwell Publishing, Ltd.

extreme natural environment (sulphureous waters)

through preselective mutations
Antonio Flores-Moya1, Eduardo Costas2, Elena Bañares-España1,2, Libertad García-Villada2, María Altamirano1
and Victoria López-Rodas2
1
Departamento de Biología Vegetal (Botánica), Facultad de Ciencias, Universidad de Málaga, E-29071 Málaga, Spain; 2Departamento de Producción Animal,
Genética, Facultad de Veterinaria, Universidad Complutense, E-28040 Madrid, Spain

Summary

Author for correspondence: • Adaptation of Spirogyra insignis (Chlorophyceae) to growth and survival in an
A. Flores-Moya extreme natural environment (sulphureous waters from La Hedionda Spa, S. Spain)
Tel: +34 952 13 33 41 was analysed by using an experimental model.
Fax: +34 952 13 19 44
Email: floresa@uma.es
• Photosynthetis and growth of the alga were inhibited when it was cultured in La
Hedionda Spa waters (LHW), but after further incubation for several weeks, the
Received: 10 September 2004
culture survived due to the growth of a variant that was resistant to LHW.
Accepted: 23 November 2004
• A Luria-Delbrück fluctuation analysis was carried out to distinguish between
resistant filaments arising from rare spontaneous mutations and resistant filaments
arising from other mechanisms of adaptation. It was demonstrated that the resistant
filaments arose randomly by rare spontaneous mutations before the addition of
LHW (preselective mutations). The rate of spontaneous mutation from sensitivity to
resistance was 2.7 × 10−7 mutants per cell division.
• Since LHWresistant mutants have a diminished growth rate, they are maintained in
nonsulphureous natural waters as the result of a balance between new resistants
arising from spontaneous mutation and resistants eliminated by natural selection.
Thus, recurrence of rare spontaneous preselective mutations ensures the survival of
the alga in sulphureous waters.

Abbreviations

Fm′ , maximum fluorescence of light-adapted cells; Ft, steady state fluorescence of

light-adapted cells; LHW, La Hedionda Spa water; N0, initial number of cells in a cul-
ture; Nt, final number of cells in a culture after a time period, P0, proportion of cul-
tures showing no mutant cells in a fluctuation analysis; ΦPSII, effective quantum yield
from PSII; µ, mutation rate.

Key words: adaptation, fluctuation analysis, mutation, quantum yield, Spirogyra,

sulphureous water.
New Phytologist (2005) 166: 655–661

© New Phytologist (2005) doi: 10.1111/j.1469-8137.2005.01325.x

values of temperature, salinity, pH, or mineral deficiency or

Introduction excess) is an interesting topic from both biochemical and
Survival and growth of phytoplankton living in extreme physiological points of view (Fogg, 2001). Because algae and
natural environments (i.e. habitats characterised by extreme cyanobacteria are the principal primary producers of aquatic

www.newphytologist.org 655
656 Research
ecosystems (Kirk, 1994; Falkowski & Raven, 1997), the
tolerance of these organisms to stressed environments is also
very relevant from an ecological point of view. However, little
is known about the mechanisms allowing algal adaptation to
such extreme conditions. Within limits, algal species should
survive in stressed environments as a result of two different
processes: physiological adaptation, usually resulting from
modifications of gene expression (Bradshaw & Hardwick,
1989; Belfiore & Anderson, 2001); and, if appropriate genetic
variability is available, classic evolutionary changes should
occur in populations subject to consistent stress, due to
genetic mutations that confer resistance (Belfiore & Anderson,
2001). It is accepted that short-term or fluctuating stress is
best met by phenotypic response systems, while continuous or
predictable stress can be met by genetically determined response
systems (Bradshaw & Hardwick, 1989; Davison & Pearson,
1996). Because physiological adaptation is bounded by the
types of conditions commonly encountered by organisms, it
remains for genetic adaptation to overcome extreme environ-
mental conditions (Hoffmann & Parsons, 1991).
La Hedionda Spa (Casares, Málaga; S Spain) is an example of
a natural extreme environment, characterised by sulphureous
waters. Its history goes back to the Roman period, when
Julius Caesar recovered his health after bathing there. Since
then, La Hedionda waters have been used for curative purposes,
mainly as a remedy for skin conditions. The sulphide ion
in these waters acts as a powerful fungicide and bactericide.
Therefore it might be expected that proliferation of aquatic
microorganisms should be restricted in La Hedionda
Spa; nevertheless the waters host a planktonic community,
mainly composed of cyanobacteria and microalgae. The pres-
ence of cyanobacteria in La Hedionda Spa is not very surpris-
ing because this group of prokaryotes is known for its ability
to occupy extreme habitats (Schopf, 1994). In fact, the major-
ity of organisms that are usually found in extreme environ-
ments are bacteria (Fogg, 2001). However, the presence of
eukaryotic algae in La Hedionda Spa suggests that they have
undergone some adaptive process conferring tolerance to such
adverse conditions.
The aim of this work was to assess the mechanisms (i.e.
physiological vs genetic adaptation) allowing tolerance of
algae to an extreme natural environment such as La Hedionda
Spa waters. For this purpose, a modified Luria-Delbrück
fluctuation analysis was carried out, using as experimental
organism the green alga Spirogyra insignis (Hassall) Kützing,
isolated from nonsulphureous waters. Luria & Delbrück (1943)
proposed fluctuation analysis as a combined experimental
and statistical method to first distinguish among variant
cells arising through direct and specific adaptation in response
to environmental selection, and second measure the rate
of spontaneous mutation. Although Luria-Delbrück fluc-
tuation analysis has been proved to be the most sensitive
approach to distinguish between rare spontaneous mutation
and direct adaptation to environmental selection (Cole et al.,
New Phytologist (2005) 166: 655–661
1976), the study of mutation rates in groups other than
bacteria has been hampered by the cumbersome aspects of
the analysis (Rossman et al., 1995).
This study is a complement to other work carried out in a
framework focused on understanding algal adaptation to
novel, anthropogenic chemical water pollutants, such as
antibiotics, herbicides and substances of military use (Costas
et al., 2001; López-Rodas et al., 2001; Baos et al., 2002;
García-Villada et al., 2002, 2004), but in this study we
applied similar procedures to investigate algal adaptation
to extreme natural conditions. In this way, we show that the
survival of algae in extreme natural habitats could be achieved
by rapid adaptation via rare, preselective mutations.
Materials and Methods
Phytoplankton community from La Hedionda Spa
In order to qualitatively characterise the phytoplankton
inhabiting La Hedionda Spa water ( LHW ), samples from this
locality were collected in July 2003, and phytoplankton was
identified using an inverted microscope (Axiovert 35, Zeiss,
Oberkóchen, Germany).
Determination of sulphide levels in La Hedionda
Spa waters
Four drops of Zn(CH3CO2)2 2 N were added to a 100 ml
LHW sample; immediately, the sampling bottle was closed
avoiding bubbles and any remaining air volume. The sample
was stored at 4°C and in dark conditions until sulphide
determination was carried out. Sulphide level was determined
by titration with Na2S2O3 0.1 N, in accordance with APHA-
AWWA-WPCF (1992).
Experimental organism: isolation and culture
Experiments were performed with Spirogyra insignis (Hassall)
Kützing haploid vegetative cells, from the algal culture
collection of the Facultad de Veterinaria, Universidad Com-
plutense de Madrid. The alga was isolated from a water
sample collected at a nonsulphureous lagoon in Doñana
National Park (SW Spain). This strain was founded from
a single filament, which assured that there was no genetic
variability within the strain. Before the experiments, S. insignis
was grown axenically in cell-culture flasks (Greiner, Bio-One
INC., Longwood, NJ, USA) with 20 ml of BG-11 medium
(Sigma, Aldrich Chemie, Taufkirchen, Germany) at 20°C and
a photosynthetic photon fluence rate of 60 µmol m−2 s−1 over
the waveband 400 –700 nm, provided by cool white fluorescent
tubes, under continuous light. Cultures were maintained in
balanced growth (acclimated, mid-log exponential growth)
(Cooper, 1991) by periodically transferring an inoculum to
fresh medium.
www.newphytologist.org © New Phytologist (2005)

Fig. 1 Schematic diagram of the experiment
modified from the classic Luria-Delbrück
fluctuation analysis. Set 1 examined the
variance of mutational events in different
cultures (each started from a small inoculum)
propagated under nonselective conditions
and finally supplemented with liquid medium
containing the selective agent. In this
example, a mutational event occurred late in
the propagation of culture 1 and early in the
propagation of culture 3; no mutational
events occurred in culture 2. Set 2 sampled
the variance of parental populations (the
distribution should be Poisson).
Toxicity test: effect of La Hedionda Spa water on
quantum yield
In order to demonstrate the toxic effect of the sulphureous
waters on S. insignis isolated from nonsulphureous waters, we
measured the change in photosynthetic performance of the
alga when it was cultured in LHW by assessing the changes
in the effective quantum yield of photosystem II (ΦPSII).
Water samples (5 l) from La Hedionda Spa were collected and
homogeneously mixed. The resulting integrated water sample
was filtered (0.22 µm, Stericup, Millipore Corporation, Billerica,
MA, USA) and kept in a plastic can in cold and dark
conditions until the experiments. Culture samples of S. insignis
wild-type were placed in experimental tubes containing 1.5 ml
of the integrated water sample from La Hedionda Spa.
Controls in BG-11 medium were also prepared.
Chlorophyll a fluorescence of photosystem II was meas-
ured on three replicates of both exposed and control cultures,
using a PAM-2000 fluorimeter ( Walz, Effeltrich, Germany) at
five different time periods (0, 0.5, 1, 24 and 48 h). Effective
quantum yield was calculated as follows:
ΦPSII = (F m′ − Ft )/F m′ ,
where F m′ and Ft are the maximum and the steady state
fluorescence of light-adapted cells, respectively (Schreiber
et al., 1986; Maxwell & Johnson, 2000).
Fluctuation analysis
In order to distinguish between genetic (i.e. spontaneous
mutations occurring before exposure to the selective agent) and
physiological adaptation to LHW, a modified Luria-Delbrück
analysis was performed, as described in López-Rodas et al.
(2001) ( Fig. 1). The modification of the analysis involves the
use of liquid medium containing the selective agent rather
© New Phytologist (2005) www.newphytologist.org
Research 657
than plating on a solid medium, as was done by Luria &
Delbrück (1943) with bacterial cultures. In short, two different
sets of experimental cultures were prepared. The first one
(set 1) consisted of 71 parallel cell-culture flasks, each inoculated
with 102 S. insignis wild-type filaments (i.e. a number small
enough to make it likely that no pre-existing mutants were
present). These cultures were grown (as previously detailed)
until they reached c. 105 filaments each, when filaments from
all cultures were isolated and transferred to selective liquid
medium containing LHW. The second set of cultures (set 2)
consisted of 36 parallel cell-culture flasks containing the same
selective medium as set 1. Set 2 cultures were each inoculated
with c. 105 S. insignis wild-type filaments from the same
parental population used to found set 1 cultures. Both sets
were inoculated simultaneously. Cultures were grown for
30 d and then visually assessed by inverted microscope. The
number of LHW resistant filaments in each cell-culture flask
was counted blindly by at least two different persons.
Overall mean cell number per filament was 9.6 ± 2.2
(n = 50). Thus, a mean value of 10 cells per filament was used
for the calculations. That is to say, the number of cells in the
experiments was one order of magnitude higher than that of
filaments (103 cells at the start of the experiment in the set 1,
and 106 cells at the end of the experiment in the set 1, and in
the initial inoculum in the set 2).
According to Luria & Delbrück (1943), two distinctive
results can be found when conducting a fluctuation analysis,
each of them being interpreted as the independent conse-
quence of two different phenomena of adaptation. In the
first case, cells per culture variance in set 1 could be found to be
low (i.e. variance/mean c. 1) if resistant cells arose by physio-
logical adaptation or specific postselective mutations, since
every cell is likely to have had the same chance of developing
resistance. Thus, interculture (flask-to-flask) variation would
be consistent with the Poisson model. By contrast, if high
variation in the interculture number of resistant cells is found
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(i.e. variance/mean > 1), it means that resistant cells appeared
by random preselective mutations occurring before selection,
and the interculture variation would not be consistent with
the Poisson model. That is to say, they occurred during the
time in which the cultures reached Nt from N0 cells, before the
exposure to LHW. In set 2 cultures, if resistants arose by prese-
lective mutations, variance is expected to be low, because
set 2 samples the variance of the parental population. Thus,
despite the way resistants appear, interculture variance of
resistants in set 2 should be similar to the average of resistants
in set 2 cultures. Because this set is the experimental control
of the analysis of fluctuation, if a similar variance/mean ratio
between set 1 and set 2 is found, it confirms that resistant
cells appeared by physiological adaptation or postselective
mutations, rather than by preselective mutations.
In addition, the fluctuation analysis allows estimation of
the rate of appearance of resistant cells. There are different
approaches for accomplishing this estimation (Rosche &
Foster, 2000). Due to the methodological limitations imposed
by a fluctuation analysis using liquid cultures, the proportion
of set 1 cultures showing no mutant cells after LHW exposure
(P0 estimator) was the parameter used to calculate the muta-
tion rate (µ). The P0 estimator (Luria & Delbrück, 1943) is
defined as follows:
P0 = e −µ(N t −N 0 )
where P0 is the proportion of cultures showing no mutant
cells, and N0 and Nt are the initial and the final cell population
sizes (103 and 106 cells, respectively). Therefore, µ was calculated
as:
µ = −Loge P0/(Nt − N0).
Reliability, reproducibility and precision of the procedure for
estimating mutation rate was determined a posteriori (British
Standards Institute 1979; Thrusfield, 1995, see details in
López-Rodas et al., 2001). Reliability was determined based
on the agreement among three iterations of the experiments;
reproducibility was determined as the agreement among three
sets of observations made on the same experiments by three
different observers; finally, precision was calculated as the
minimum variation in mutation rate that can be detected
using the procedure.
Results
The phytoplankton community supported by La Hedionda
Spa has a very low microalgal species richness, Spirogyra being
the most abundant genus. The cyanophycean genus Oscillatoria,
as well as the diatoms Navicula, Nitzschia and Achnanthes,
were also represented, although in lower numbers.
Sulphide levels in LHW in July 2003 was 7 ± 0.3 mg l−1
(n = 5); this S2– concentration figure exerted a highly toxic
New Phytologist (2005) 166: 655–661
Fig. 2 Changes in the effective quantum yield from PSII (ΦPSII) of
Spirogyra insignis cultured in BG-11 medium (open bars) or La
Hedionda Spa water (LHW, hatched bars), after different time period
of incubations. Vertical lines on bars shown SD ( n = 5).
effect on ΦPSII from wild-type S. insignis: the parameter dra-
matically decreased immediately after being exposed to LHW
and, 1 h later, no significant ΦPSII values were detected (Fig. 2).
When conducting the fluctuation analysis to differentiate
between physiological and genetic adaptation to LHW, every
experimental culture of both sets 1 and 2 apparently collapsed
following LHW exposure. In the case of set 1, some cultures
recovered after 20–30 d of LHW exposure, apparently due to
the growth of LHWresistant cells ( Table 1). By contrast, every
set 2 culture recovered, and LHWresistant cells were observed in
all cultures ( Table 1). In addition, low LHWresistant cell variation
was observed in set 2 (variance/mean c. 1, consistent with
Poisson variability; P < 0.05, using χ2 as a test of goodness
of fit), which indicated that the high variance found in set 1
cultures should be due to processes other than sampling
error ( Table 1). As in set 1 cultures, the variance significantly
exceeded the mean (variance/mean = 73.3; P < 0.001, using χ2
as a test of goodness of fit), it could be inferred that LHWresistant
cells arose by rare, preselective spontaneous mutations
rather than by specific physiological adaptation or postselective
mutations (i.e. adaptive mutations specifically appearing in
response to LHW) in response to the environmental change.
The second aim was to estimate the rate of mutation
(µ) from sensitivity to resistance for LHW. The estimated µ
of LHWsensitive → LHWresistant using the P0 estimator was
2.7 × 10−7 mutants per cell division (Table 1). This value of µ
was estimated with high standards of reliability, reproducibility
and precision (Table 2).
A randomly selected resistant mutant culture from set 1
was isolated and maintained in the algal culture collection.
It was confirmed that the culture was able to retain a resistant
phenotype throughout successive generations of serial sub-
cultures in the absence of LHW. However, the growth rate
of the resistant culture (0.49 ± 0.04 divisions per day, n = 20)
was significantly lower (, P < 0.05) than that of the
wild-type, sensitive filaments (0.64 ± 0.03 divisions per day,
n = 20).
www.newphytologist.org © New Phytologist (2005)

Table 1 Fluctuation analysis of LHWresistant variants in Spirogyra
insignis wild-type strain
Set 1 Set 2
No. of replicate cultures 71 36
No. of cultures containing the following no. of LHWresistant filaments:
0 54 0 The rate of mutation of LHWsensitive → LHWresistant
1–100 10 36
> 100 7 0
Variance/mean (of the no. of 73.3 0.8
resistant filaments per replicate)
µ (mutants per cell division) 2.7 × 10−7
Table 2 Reliability, reproducibility and precision of the procedure to
estimate mutation rate (µ) of LHWsensitive → LHWresistant in Spirogyra
insignis
Reliability of µ (%) 89
Reproducibility of P0 observed value (%) 100
Precision of µ (mutants per cell division) 0.1 × 10−7
Discussion
The predominance of Spirogyra in the sulphureous waters
from La Hedionda Spa was somewhat surprising, because it
is widely accepted that eukaryotes rarely inhabit extreme
environments (Zettler et al., 2002). Our results suggest
that the Spirogyra population from La Hedionda Spa might
well have undergone adaptation to these adverse conditions.
For example, the toxic effect of LHW on ΦPSII of S. insignis
isolated from nonsulphureous waters clearly showed that the
LHW toxicity might account for the low phytoplankton
species richness and density observed in La Hedionda Spa,
and that the survival of phytoplankton could only be achieved
by physiological or genetic adaptation. Moreover, growth of S.
insignis was inhibited when it was cultured in LHW, but after
further incubation for several weeks, the cultures survived due
to the growth of variants that were resistant to LHW. The key
to understanding evolution of the alga in sulphureous waters
is to analyse the rare algal variants that occur after the massive
destruction of the sensitive cells by LHW.
Fluctuation analysis is an appropriate procedure to
discriminate between spontaneous preselective mutations
and physiological adaptation to an environmental selection,
postselective mutations, or other causes (Luria & Delbrück,
1943; Cairns et al., 1988; Tlsty et al., 1989). The variation in
LHWresistant filament number (set 1 vs set 2) unequivocally
demonstrates the spontaneous nature of the LHW resistance.
Moreover, LHW did not stimulate the occurrence of resistant
filaments; on the contrary, LHW resistance develops by rare
and random spontaneous mutations. It is noteworthy that
using the modified fluctuation analysis we developed, it
would be difficult to observe postselective mutations if the
rate of these kinds of mutations for LHWresistant were smaller
© New Phytologist (2005) www.newphytologist.org
Research 659
than 10−7. Despite this, it seems very unlikely that adaptive
mutations occurred in our cultures, because these kinds of
mutations are usually observed in nonproliferating microbial
populations after being incubated on nonlethal selective
medium plates (Foster, 2000). The rapid lethal effect of LHW
seems unlikely to allow the appearance of adaptive mutations.
(2.7 × 10−7 mutants per cell division) was one to two orders
of magnitude lower (from 2.12 × 10−5–1.76 × 10−6 mutants
per cell division) than those we have described for other
microalgal species (Costas et al., 2001; López-Rodas et al.,
2001; Baos et al., 2002; García-Villada et al., 2002, 2004).
This is somewhat surprising, since the microalgal species used
in these studies (the cyanobacteria Microcystis aeruginosa
(Kützing) Kützing and Pseudanabaena sp., and the chloro-
phyceans Dictyosphaerium chlorelloides (Naumann) Komárek
and Perman, Dunaliella tertiolecta Butcher, Scenedesmus sp.
and Scenedesmus intermedius Chodat) reproduce asexually,
whereas Spirogyra reproduces both asexually and sexually.
In a wide-ranging study of mutation rates in DNA-based
microbes (from bacteriophages to filamentous fungus), Drake
(1991) asserted that the spontaneous mutation rate (per
genome per replication) of these groups of organisms is a
nearly invariant value, despite the ample variability in genome
size and mode of reproduction. Different explanations have
been proposed for such observations. The most likely is that
spontaneous mutation rates have evolved equally in all DNA-
based microbes to a limit determined by both the mutational
load they cause and the physiological cost of reducing these
rates. Despite this, theoretical studies have suggested that, as
long as asexuals cannot evade mutational load by means of
sexual recombination, lower rates of spontaneous mutations
at equilibrium are expected in such organisms because they
are thought to be subjected to stronger indirect selection
(Sniegowski et al., 2000). Thus, it has also been suggested
that the rate of recombination in asexual organisms (such as
bacteria) could be higher than is commonly supposed, and
so their mutation rates have evolved similar values to that of
sexual ones (Drake et al., 1998). In the present case, the opposite
also applies because Spirogyra is known to have sexual repro-
duction, but the frequency or the conditions necessary for this
to occur are not known. In this sense, it has been suggested
that most of the microorganisms that show both asexual and
sexual reproduction have clonal rather than sexual population
structures, sexual reproduction being an unusual phenome-
non ( Tibayrenc et al., 1991). In any event, sexual reproduc-
tion, an event easily detectable because Spirogyra turns
noticeably brownish in colour from the previous bright green,
reflecting the loss of chlorophyll pigments from zygotes and
development of brown zygote walls (Graham & Wilcox,
2000) did not take place during the experiment.
The present study is just a simple model of algal adaptation
to natural stressful environments. The results reported here
suggest that preselective mutations are sufficiently frequent in
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660 Research
algal populations to allow them to adapt to extreme natural
environments. Nevertheless, genetic mutations usually imply
an energetic cost that may affect the survival of adapting
populations, so it could also be interesting to analyse the genetic
variability of algal species. In a series of preliminary studies
analysing the genetic variability in populations of the cyano-
bacterium Microcystis aeruginosa, significant variability was
found for morphological and physiological (fitness and
photosynthetic performance) traits (Rico et al., 2004; E. Costas
et al., unpublished; V. López-Rodas et al., unpublished). This
kind of approach can also cast some light on the ability of
algae to adapt to environmental changes.
Stressful environments sometimes sustain populations
of species living at the extreme limits of their physiological
tolerance. Although the genetic variability of marginal popu-
lations has been studied and compared with that of central
populations (Hoffmann & Parsons, 1991), little is known
about how species adapted to more favourable habitats can
extend to extreme environments. Despite this, it has been
pointed out that it is important for evolutionary biology to
study the ability of species to evolve in response to shifts in
environmental conditions, especially in recent years, when
drastic or rapid environmental changes resulting from human
activities have occurred (Hoffman & Parsons, 1991). Our
results suggest that rare preselective mutants can be sufficient
to ensure the adaptation of eukaryotic algae to extreme natural
habitats.
Acknowledgements
This work was financially supported by REN2000 –0771HID,
REN2001–1211HID and Parques Nacionales 093/2003
grants. We thank Dr Eric C. Henry (Herbarium, Department
of Botany and Plant Pathology, Oregon State University,
USA) for his kind revision of the English style and usage. Dr
Miguel Hernández and Ms. Magalí Roberto (Departamento
de Química Analítica, Universidad de Málaga) kindly helped
us to measure sulphide concentration in water.
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