16637525

1074

J. Phycol.38, 1074–1081 (2002)

EVOLUTION OF MICROALGAE IN HIGHLY STRESSING ENVIRONMENTS:

AN EXPERIMENTAL MODEL ANALYZING THE RAPID ADAPTATION OF

DICTYOSPHAERIUM CHLORELLOIDES

(CHLOROPHYCEAE) FROM

SENSITIVITY TO RESISTANCE AGAINST 2,4,6-TRINITROTOLUENE

BY RARE PRESELECTIVE MUTATIONS

Libertad García-Villada, Victoria López-Rodas

Genética, Facultad de Veterinaria, Universidad Complutense, E-28040, Madrid, Spain

Elena Bañares-España, Antonio Flores-Moya

Departamento de Biología Vegetal, Facultad de Ciencias, Universidad de Málaga, E-29071, Málaga, Spain

Mar Agrelo, Luis Martín-Otero

Departamento NBQ , F.N. La Marañosa, Ministerio de Defensa, P.O. Box 110, Madrid, Spain

and

Eduardo Costas

Genética, Facultad de Veterinaria, Universidad Complutense, E-28040-, Madrid, Spain

The increasing rates of global extinction due to

human activities necessitate studies of the ability of

organisms to adapt to the new environmental

condi-tions resulting from human disturbances. We

investi-gated the evolutionary adaptation of a microalga to

sudden environmental change resulting from

expo-sure to novel toxic chemical residues. A laboratory

strain of

Dictyosphaerium chlorelloides

(Naum.) Kom.

and Perm. (Chlorophyceae) was exposed to

increas-ing concentrations of the modern contaminant

2,4,6-trinitrotoluene (TNT). When algal cultures were

ex-posed to 30 mg

L

_{TNT, massive lysis of microalgal}

cells was observed. The key to understanding the

evolu-tion of microalgae in such a contaminated

environ-ment is to characterize the TNT-resistant variants that

appear after the massive lysis of the TNT-sensitive

cells. A fluctuation analysis demonstrated

unequivo-cally that TNT did not facilitate the appearance of

resistant cells; rather it was found that

TNT-resistant cells appeared spontaneously by rare

muta-tions under nonselective condimuta-tions, before

expo-sure to TNT. The estimated mutation rate was 1.4

10

_{mutants per cell division. Isolated resistant}

mu-tants exhibited a diminished fitness in the absence

of TNT. Moreover, the gross photosynthetic rate of

TNT-resistant mutants was significantly lower than

that of wild-type cells. Competition experiments

be-tween resistant mutants and wild-type cells showed

that in small populations, the resistant mutants were

driven to extinction. The balance between mutation

rate and the rate of selective elimination determines

the occurrence of about 36 TNT-resistant mutants

per million cells in each generation. These scarce

re-sistant mutants are the guarantee of potential for

ad-aptation.

Key index words:

adaptation; contamination;

fluctua-tion analysis; microalgae; mutafluctua-tion; photosynthesis;

resistance; 2,4,6-trinitrotoluene (TNT)

Abbreviations:

APS, apparent photosynthetic (rate);

DCMU, 3-(3,4-dichlorophenyl)-1,1-dimethylurea; DR,

dark respiration (rate); GPS, gross photosynthetic

rate; TNT, 2,4,6-trinitrotoluene

We live in a time in which global extinction rates

are 50–500 times background and are increasing

be-cause of human activities that are altering

biosphere-level processes. It is estimated that several million

populations and 300–30,000 species go extinct

annu-ally from a total of

10 million species (Woodruff

2001). Distinctive features of the future biosphere

could include a homogenization of biotas, a

prolifera-tion of opportunistic species, a pest-and-weed ecology,

and unpredictable emergent novelties (Myers and

Knoll 2001).

More investigations are needed to make sound

pre-dictions about the future and to determine actions to

mitigate the biodiversity crisis (Ehrlich 2001). The

biodi-versity crisis is reasonably well understood for

terres-trial vertebrates and a few other groups, but little is

known about organisms as abundant and important

as microbes. Studies of bacteria and protists are

neces-sary, because vitally important nutrient cycles may

be-come less predictable as essential microbes succumb

to anthropogenic toxins (Woodruff 2001). To this end,

we are investigating mechanisms of adaptation by

mi-croalgae after sudden and catastrophic

environmen-1_{Received 9 July 2001. Accepted 23 July 2002.}

1075

tal changes resulting from novel residual materials

polluting natural waters (Costas et al. 2001,

López-Rodas et al. 2001).

Among novel residual materials polluting water,

numerous chemical substances are of military use.

Contamination by chemicals from military sources is

not as frequently studied as that due to other

indus-trial pollutants. Yet today organisms are exposed to

such contaminants for the first time in their

evolu-tionary history, so studies on the adaptation of

organ-isms to these kinds of substances could provide a

gen-eral model for understanding the significance of this

type of pollution.

2,4,6-Trinitrotoluene (TNT) is the predominant

con-ventional explosive used by military forces, with an

an-nual production estimated at around one million

kilo-grams (Hartter 1985). Because TNT is only slightly

soluble in water (100

g

mL

_{), its disposal during}

manufacturing and testing uses large volumes of hot

water. As much as 500,000 gallons of

TNT-contami-nated water are generated per day by a single

ammu-nition plant (Walsh et al. 1973). Wastewaters are

di-rected to lagoons for settling of solid material before

being released to rivers and streams, and they reach

groundwater through leaching (Boopathy et al. 1994).

Other contamination sources include missile

produc-tion facilities, mining sites, military firing ranges, and

sites where outdated explosives are burned. Typical

contaminated sites may contain average

concentra-tions of 10,000 mg

kg

_{TNT in soil (Fernando et al.}

1990). As a consequence, TNT has caused extensive

damage to water and soil ecosystems because of its

pol-luting effect in the environment (Wyman et al. 1979).

It is known that TNT has toxic effects on a number

of organisms such as bacteria (Berthe-Corti et al. 1998,

Dodard et al. 1999, Sunahara et al. 1999), yeast and

fungi (Klausmeier et al. 1973, Spiker et al. 1992, Stahl

and Aust 1993), and plants (Palazzo and Leggett 1986).

It has been shown that TNT is cytotoxic to

mamma-lian cells (Honeycutt et al. 1996, Lachance et al. 1999)

and humans (Zlateva and Pavlova 1998). Furthermore,

there is evidence that TNT has had mutagenic effects

in a number of

in vitro

test systems (Kaplan and Kaplan

1982, Spanggord et al. 1982, Vaatanen et al. 1997,

Berthe-Corti et al. 1998). Finally, toxic effects of TNT

on unicellular freshwater algae have long been known

(Hudock and Gring 1970, Smock et al. 1976, Won et

al. 1976).

The main aims of this work were to determine 1)

the capacity for adaptation by microalgae to survive in

TNT-contaminated environments; 2) the nature of

the TNT-resistant cells that arise (i.e. resistant cells

arising by direct and specific acquired adaptation in

response to TNT vs. TNT-resistant cells arising by rare

spontaneous mutations arising randomly before the

TNT exposure); and 3) the main ecological–genetic

parameters of transformation from TNT sensitivity to

TNT resistance (i.e. mutation rate from TNT sensitivity

to TNT resistance, fitness of wild-type cells and

TNT-resistant mutants, competition between wild-type cells

and TNT-resistant mutants, the average number of

TNT-resistant mutants in the absence of TNT) and

the photosynthetic performance of both types of cells.

materials and methods

Experimental organism and growth conditions. Dictyosphaerium chlorel-loides (Naum.) Komárek and Perman (Chlorophyceae), wild-type strain DcG1wt, from the algal culture collection of the Fac-ultad de Veterinaria, Universidad Complutense de Madrid, was grown axenically in cell culture flasks (Greiner, Bio-One Inc., Longwood, N.J., USA) with 20 mL of BG-11 medium (Sigma, Aldrich Chemie, Taufkirchen, Germany) at 20 C with a photon irradiance of 60 mol photonsm2_s1 _{from fluorescent tubes}

under continuous light. Cells were maintained in balanced growth (Cooper 1991) by serial transfers of a cell inoculum to fresh medium once a month. Before the experiments, the cul-ture of DcG1wt cells was recloned (by isolating a single cell) to eliminate any spontaneous mutants that had already arisen in the cultures. Cultures were maintained as contaminant free as possible, and only cultures without detectable bacteria were used in the experiments.

Dose effect of TNT on fitness of wild-type cells. The effect of in-creasing doses of TNT on fitness under r-selection (MacArthur and Wilson 1967) was studied in laboratory cultures of DcG1wt wild-type cells as previously described (López-Rodas et al. 2001). Experimental cultures were inoculated each with 1.5 105 _cells

from mid-log exponentially growing cultures. A stock solution of TNT (kindly provided by Fábrica Nacional de Armas, La Marañosa, Ministerio de Defensa, Spain) was prepared in dis-tilled water, according to the standard security protocols, and introduced into the growth medium at concentrations of 0.6, 1.2, 2.0, 3.4, and 5.0 mgL1_{. Three replicates of each TNT}

con-centration and six unexposed controls were prepared. After 5 days, cell numbers in experiments and controls were counted using a hemocytometer (double Nuebauer, ruling, Fortuna W.G. Co., Wertheim, Germany). Cells were counted blind (i.e. the person counting the test did not know the identity of the tested sample) by at least two independent observers. The num-ber of counted samples of each experimental culture was deter-mined using the progressive mean procedure (Williams 1977) to ensure a counting error of 1%. Fitness (estimated as the Malthusian parameter m, defined as dN/dt mN) (Fisher 1958) was calculated as Nt N0emt (Crow and Kimura 1970),

where Nt and N0 are the cell number at time t and 0,

respec-tively, and t 5 days.

Isolation of TNT-resistant mutants. A Luria-Delbrück fluctua-tion analysis was used to investigate the transformafluctua-tion from TNT sensitivity to TNT resistance (i.e. to distinguish between TNT-resistant cells arising by rare spontaneous preselective mu-tations occurring randomly during cell division before expo-sure to TNT and TNT-resistant cells arising through physiologi-cal or specifiphysiologi-cally acquired postselective adaptation in response to the presence of TNT) and subsequently to estimate the rate of appearance of TNT-resistant cells. Since Luria and Delbrück (1943), in a seminal study, introduced the fluctuation test as a combined experimental and statistical procedure to analyze the occurrence of resistant variants in bacterial populations, subsequent theoretical and experimental work has modified it for application to a range of different organisms, from bacteria to human cells (Cole et al. 1976, Kendal and Frost 1988, Tlsty et al. 1989, Jones et al. 1994, Rosman et al. 1995, Asteris and Sarkar 1996, Crane et al. 1996). Recently, the theoretical basis and ex-perimental validation of a modified fluctuation analysis have also been described for application to liquid cultures of microalgae (Costas et al. 2001, López-Rodas et al. 2001).

In the first set of experiments, C 97 parallel culture tubes were inoculated each with N0 200 wild-type DcG1wt cells,

which were grown axenically under nonselective conditions. When each culture reached a convenient number of cells (Nt

1.35 105_{), they were supplemented with selective liquid}

me-dium to give a concentration of 30 mgL1 _{TNT. For the}

1076

DcG1wt cells from the same parental population were trans-ferred separately to tubes containing fresh liquid medium with 30 mgL1 _{TNT (45 tubes). The most suitable number of}

paral-lel cultures (C) and the final cell population (Nt) were

esti-mated before the experiment to obtain the maximum precision in estimation of mutation rates according to Li et al. (1983), within the limits of our laboratory facilities.

All set 1 cultures collapsed within about 5 days, but after al-most 4 weeks some cultures recovered due to the growth of TNT-resistant cells. Cultures were grown for 90 days and then analyzed by counting samples by inverted microscopy (Axiovert 35, Zeiss, Oberkóchen, Germany). The number of TNT-resistant cells in each tube was counted blindly by at least two indepen-dent persons. Cultures in which TNT-resistant variants were not detected were centrifuged (9000g) before observation to ensure that the progeny of even one resistant cell would be detected.

The proportion of cultures showing no mutant cells after TNT exposure in the first set of experiments was used to esti-mate the mutation rate (P0 estimator) as follows: P0 e(NtN0),

where P0 is the proportion of cultures showing no mutant cells,

is the mutation rate, N0 is the initial cell population size, and

Nt is the final cell population size (Luria and Delbrück 1943, Lea

and Coulson 1949, Tlsty et al. 1989).

Reliability, reproducibility, and precision of our procedures to estimate mutation rates were determined according to the British Standards Institute (1979) and Thrusfield (1995) rec-ommendations. Reliability was determined as the agreement between two iterations of the experiments, reproducibility was determined as the agreement among three sets of observations made on the same experiments by three different observers from different institutions (Genética and NBQ Departments), and precision was calculated as the minimum variation in muta-tion rates that can be detected using our procedure.

Characterization of the TNT-resistant mutants. TNT-resistant cells were isolated randomly from set 1 cultures and grown to mass populations. The TNT-resistant cultures so obtained were used in the following experiments: 1) after culture in the absence of TNT, fitness under conditions of r-selection was measured just as in a dose-effect study; 2) to check if mutants were able to re-tain the TNT-resistant phenotype throughout several genera-tions, they were exposed to 30 mgL1 _{TNT after culture in the}

absence of TNT for 60 days; and 3) the TNT-resistant cultures were treated with 10 M 3-(3,4- dichlorophenyl)-1,1-dimethyl-urea (DCMU; “diuron”) herbicide (Sigma), prepared in DMSO, which was introduced into the growth medium at a maximum concentration of 0.05% (v/v) as previously described (López-Rodas et al. 2001) to check for cross-resistance.

Competition between TNT-sensitive wild-type cells and TNT-resis-tant muTNT-resis-tants. A competition experiment between TNT-sensitive wild-type cells and TNT-resistant mutants was carried out as previously described (Costas et al. 1998). Four replicates of mixed cultures were established by mixing 75 105

TNT-resis-tant muTNT-resis-tants and 75 105 _{TNT-sensitive wild-type cells. The}

cultures were maintained by transferring an experimental cul-ture inoculum (1/8 v/v) to fresh BG-11 medium (7/8 v/v) without TNT once every 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-selec-tion). Samples from each replicate were grown in BG-11 me-dium containing 15 mgL1 _{TNT once every week to check for}

the presence of TNT-resistant mutants.

Photosynthetic characterization of TNT-sensitive wild-type cells and TNT-resistant mutants. Apparent photosynthetic (APS) and dark respiration (DR) rates were determined as oxygen exchange us-ing a Clark-type liquid-phase electrode, YSI 5331 (Yellow Sprus-ings Instruments Co., Yellowspring, OH, USA).

Culture samples of both cell types were incubated separately in an 8-mL temperature-controlled (20 0.1 C) magnetic gen-tle-stirring chamber. To measure APS versus photon irradiance, the chamber was illuminated with a slide projector, and the different values of irradiance (from 3.9 to 700 mol pho-tonsm2_s1_{) were obtained with PAR-transmitting}

neutral-density calibrated filters. Three replications of APS-irradiance

curves were carried out with both algal cell types. APS-irradi-ance curves were fitted to the Edwards and Walker (1983) model (using GraFit, Erithacus Software, Furrey, UK): APS APSmax (I

Ic) (I I0.5)1, where APSmax is the saturated APS rate, I is the

actual irradiance, Ic is the light compensation point, and I0.5 is

the light half-saturation constant. The initial slope of the APS-irradiance curves was obtained by the linear fit of the four initial values of the curves, and it was used as an estimator of photo-synthetic efficiency (). The DR rate was measured by covering the reaction chamber with two layers of aluminum foil. The gross photosynthetic rate (GPS) was calculated by the sum of APS and DR. Both APS and DR were obtained when the O2 concentration

changed linearly as a function of time (5–10 min).

The effect of increasing doses of TNT on GPS was analyzed on both TNT-sensitive wild-type cells and TNT-resistant mutants as explained above. The chamber was illuminated continuously with a saturating photon irradiance of 700 mol photonsm2_s1

dur-ing the experiments; this figure was derived from the measure-ments of APS irradiance that were run before the assays of dose effect. TNT from a stock solution was introduced into the cham-ber at concentrations of 0.3, 0.6, 1.0, 2.0, 3.5, 6.0, 11.0, and 15.0 mgL1_{. GPS decrease was used as an estimator of the toxic effect}

of TNT. The experiment was performed in triplicate.

The initial slope of the APS-irradiance curves as well as the slopes of the dose-effect experiments were compared by the test of equality of slopes (Sokal and Rohlf 1995).

results

Dose effect of TNT on fitness of wild-type cells.

Fitness of

wild-type DcG1wt cells under conditions of r-selection

in an uncrowded environment severely decreased with

increasing TNT concentration (Fig. 1). The

Malthu-sian parameter (

m

) was slightly affected by

concentra-tions of 1.2 mg

L

_{TNT but significantly reduced by}

concentrations as low as 2 mg

L

_{. Concentrations of}

3.4 mg

L

_{TNT caused destruction of cells. When}

mi-croalgal cultures were treated with 5 mg

L

_{, most}

cells were apparently destroyed, and we were unable

to detect algal growth.

Isolation of TNT-resistant mutants.

Our first aim was

to determine the nature of the TNT-resistant cells.

The data presented in Table 1 show that the low

varia-tion in set 2 cultures (variance/mean

0.85)

indi-Fig. 1. Relative fitness of Dictyosphaerium chlorelloides (wild-type DcG1wt cells) exposed to increasing TNT concentrations under conditions of r-selection. Relative fitness is represented as a fraction of untreated controls. Bars 1 SD.

1077

cated that the high variance in the number of

TNT-resistant cells per culture in set 1 must be due to

pro-cesses other than sampling error. In set 1 cultures,

variance significantly exceeded the mean (variance/

mean

32.0), as expected if TNT-resistant variants

arose by rare spontaneous mutation.

Our second aim was to estimate the rate of

muta-tion from TNT sensitivity to TNT resistance. The

spontaneous mutation rate (

), estimated with high

standards of reliability, reproducibility, and precision

via the P

method, was 1.4

10

(Table 1).

Characterization of the TNT-resistant mutants.

The

fit-ness of three randomly isolated mutants was estimated

under conditions of r-selection, both in the absence

and in the presence of TNT. In the absence of TNT,

TNT-resistant mutants showed a fitness value about

60% of that of the TNT-sensitive wild-type cells under

conditions of r-selection (Fig. 2). In contrast, when

the TNT-resistant mutants were grown in the

pres-ence of 3.4 mg

L

_{TNT, their fitness was about 14}

times higher than that of TNT-sensitive wild-type cells

(Fig. 2). Only the TNT-resistant mutants were able to

grow in medium containing TNT concentrations up

to 3.4 mg

L

_{(Fig. 2).}

Transmission of TNT resistance through successive

generations was examined by ascertaining the

mainte-nance of the TNT-resistant phenotype during

inter-vals of 30 generations of serial subculture in the

ab-sence of the selective agent.

No TNT-DCMU cross-resistance was observed. The

TNT-resistant mutants were unable to grow in a

me-dium containing 10

M DCMU herbicide. The

herbi-cide produced massive destruction of TNT-resistant

cells after 2 days of exposure.

Competition between TNT-sensitive wild-type cells and

TNT-resistant mutants.

The results of the competition

experiment between resistant mutants and

TNT-sensitive wild-type cells showed a rapid displacement

of the TNT-resistant mutants by the TNT-sensitive

wild-type cells in the absence of TNT (Table 2). After

only 4 weeks of competitive interaction in absence of

TNT, the wild-type cells apparently drove the

TNT-resistant genotype to extinction.

Photosynthetic characterization of TNT-sensitive wild-type

and TNT-resistant mutants.

The APS rate versus

pho-ton irradiance curves in the absence of TNT (Fig. 3)

showed that the net photosynthetic capacity of the

TNT-sensitive wild-type cells was 1.5 times that of the

Table 1. Fluctuation analysis of TNT-resistant variants in Dictyosphaerium chlorelloides wild-type strain DcG1wt.

Set 1 Set 2

No. of replicate cultures 97 45

N0 200

Nt 135,000 130,000

No. of cultures containing the following no. of TNT-resistant cellsmL1

0 (P0) 14

1–1000 56 1

1000 27 44 Variance/mean (of the no. of

TNT-resistant cells per replicate) 32 0.85

Mutation rate ()

(mutants per cell division) 1.4 105

Reliability of 93%

Reproducibility of P0 100%

Precision of (in mutants

per cell division) 0.051 105

Fig. 2. Relative fitness of resistant mutants and TNT-sensitive wild-type cells under conditions of r-selection in the absence and in the presence of TNT (3.4 mgL1_{). Bars 1 SD.}

Table 2. Presence of TNT-resistant mutants in mixed cultures (50% TNT-resistant mutants, 50% TNT-sensitive wild-type cells) evaluated at 1-week intervals under competition.

Weeks under competition

1 2 3 4

Replicate

I

II

III

IV

, ability to grow in liquid medium containing 15 mgL1

1078

TNT-resistant mutants. However, both cell types showed

the same saturating photon irradiance (700

mol

photons

m

_s

_).

The photosynthetic and respiratory data obtained

from APS versus photon irradiance curves in the

ab-sence of TNT are shown in Table 3. Saturated

photo-synthesis was 2.3 times greater in wild-type cells than

in TNT-resistant mutants. This was mostly due to the

wild-type DR value, which was 13.9 times that of the

TNT-resistant mutants. Because the photosynthetic

ef-ficiency (

) was similar in both strains (t

0.983, n

and n

12), compensation of the net photosynthesis

by respiration was achieved at almost six times lower

intensity in TNT-resistant mutants than in

TNT-sensi-tive wild-type cells (Table 3).

When photosynthetic characterization was

per-formed on TNT-exposed cultures, a severe gross

pho-tosynthetic rate decrease was observed with increasing

TNT concentrations (Fig. 4). No significant

differ-ences were observed in the dose-effect slopes achieved

for both cell types (t

0.340, n

and n

24). The

GPS value of both strains showed 50% inhibition

when cells were exposed to 2.0 mg

L

_{TNT and was}

completely inhibited in culture media containing 15.0

mg

L

_TNT.

discussion

We exposed a laboratory population of

D.

chlorel-loides

to increasing doses of a novel pollutant, TNT. As

has been reported in previous work carried out with

other microalgal species (Hudock and Gring 1970,

Smock et al. 1976),

D. chlorelloides

showed clear

sensi-tivity to TNT. In our experiment, the fitness of

D.

chlo-relloides

(wild-type strain) progressively decreased with

increasing concentrations of TNT, and even TNT

con-centrations as low as 3.4 mg

L

_{induced massive lysis}

of algal cells. Such levels of TNT can be found in

sev-eral aquatic ecosystems because ammunition plants,

mines, or old arsenals are sources of significant levels

of TNT pollution (Best et al. 1999a,b, Talmage et al.

1999). As a consequence, this contamination could be

an important challenge for microalgal populations.

When microalgal cultures were exposed to 30

mg

L

_{TNT, they became clear after some days due}

to the lysis of the sensitive cells by the toxic effect of

TNT. However, after further incubation, some

cul-tures became colored again due to the growth of

vari-ants that were resistant to the effect of TNT. A key to

understanding the adaptation of microalgae to

sur-vive in a TNT-contaminated environment is to

charac-terize the variants that appear after the lysis of the

TNT-sensitive cells. Therefore, once we had observed

the presence of such variants, our goal was to

distin-guish between TNT-resistant cells arising by rare

spontaneous mutations occurring randomly during

reproduction of the organisms under nonselective

conditions (i.e. before incorporating the TNT) and

TNT-resistant cells arising through specifically

ac-quired adaptations in response to environmental

se-lection.

Under a classic neo-Darwinist point of view, genetic

variability maintained in natural populations is

con-sidered as the pacemaker of adaptation in changing

environments. In contrast, evolutionary studies of

bac-terial populations suggest a new hypothesis, including

adaptive mutations (i.e. a process that, during

nonle-thal selection, produces mutations that relieve the

se-lective pressure whether or not other nonselected

mu-tations are also produced) resembling Lamarckism

(Cairns et al. 1988, Foster 2000). Fluctuation analysis

is the appropriate procedure to discriminate between

these alternatives (Luria and Delbrück 1943, Lea and

Coulson 1949, Cole et al. 1976, Cairns et al. 1988, Tlsty

et al. 1989, Dijkmans et al. 1994), and so it has been

previously applied to microalgae to investigate the

oc-currence of antibiotic-resistant cells (Sager 1962,

Gill-ham and Levine 1962, GillGill-ham 1965, Wurtz et al.

1979, Lee and Haughn 1980), cadmium-resistant cells

(Collard and Matagne 1990), and herbicide-resistant

Fig. 3. APS rate versus photon irradiance curve obtained for TNT-sensitive wild-type cells and TNT-resistant mutants of

Dictyosphaerium chlorelloides in absence of TNT. Data were fitted to the Edwards and Walker (1983) model. Bars 1 SD.

Table 3. Photosynthetic and respiratory parameters of the APS-irradiance curves of TNT-sensitive wild-type cells and TNT-resistant mutants of Dictyosphaerium chlorelloides in the absence of TNT.

Cell types APSmax DR GPSmax I0.5 IC R

Wild type-cells 68.6 5.2 38.8 15.6 107.4 30.1 8.2 17.1 2.9 1.3 0.5 0.954

TNT-resistant mutants 43.5 2.9 2.8 1.6 46.3 22.6 8.1 2.8 1.5 0.8 0.1 0.922 Units: APSmax, DR, and GPSmax (nmol O2h1106 cells1); I0.5 and IC (mol photonsm2s1); (nmol O2h1106 cells1[mol

1079

cells (López-Rodas et al. 2001). Our results suggest that

TNT resistance occurs due to rare spontaneous

muta-tions before exposure to TNT. Therefore, in these

mi-crobial populations, mutation seems to be the

pace-maker of evolution.

It is conceivable that adaptive mutation plays an

im-portant role in the evolution of microorganisms (Cairns

et al. 1988, Foster 2000). Adaptive mutations and other

related phenomena have been reported only in

micro-organisms, such as bacteria and yeast (Foster 1999).

However, adaptive mutation seems to have no influence

on adaptation of microalgae to catastrophic

environ-mental changes resulting from water contamination

(Costas et al. 2001, López-Rodas et al. 2001).

Appar-ently, if during a catastrophic environmental change

there is not a period of nonlethal selection, then only

rare spontaneous preadaptive mutation can ensure the

survival of the exposed microalgal population.

Our observations concerning the heritability of the

TNT-resistant phenotype also indicate that TNT

resis-tance is attributable to a mutant genotype, because

the maintenance of the resistant phenotype was

ob-served through 30 successive generations in the

ab-sence of TNT. Unfortunately, the molecular basis of

TNT-resistance remains unknown, and the results of

our experiments on cross-resistance of TNT resistance

with DCMU herbicide were insufficient to suggest any

hypothesis. The absence of cross-resistance only

im-plies that the mutation that confers TNT resistance is

different from that conferring DCMU resistance, but

this resistance could also be carried under the gene

coding for the D1 protein.

Because our experimental model with TNT

indi-cates that spontaneous TNT-resistant mutants are able

to survive in TNT-contaminated environments, we

es-tablish the mutation rates of TNT sensitivity to TNT

resistance. Although several uncertainties complicate

the calculation of mutation rate per gene (i.e. TNT

resistance could be encoded by nuclear or by

chloro-plasts genes), the value of 1.4

10

_{is an estimation}

of the number of TNT-resistant cells arising

spontane-ously per cell division, determined using high

stan-dards of reliability, reproducibility, and precision. The

rates of spontaneous mutation in microalgae usually

vary from 10

_{to 10}

_{mutants per cell division, and}

they vary among different species and from gene to

gene within the same species (López-Rodas et al.

2001). Consequently, the observed mutation rate for

TNT sensitivity to TNT resistance can be considered

as high. In this sense, it has been suggested that

or-ganisms possess the ability to regulate their mutation

rate in response to environmental conditions (Kepler

and Perelson 1995). Recent work indicates that the

genomic mutation rate seems to be adjusted to a level

that best promotes adaptation (Sniegowski et al. 2000).

If only TNT-resistant preselective mutants (which

appear spontaneously before TNT exposure) are able

to survive in a TNT-contaminated environment, then

the central question for understanding the rapid

ad-aptation of microalgae subsequent to catastrophic TNT

contamination is how these mutants are maintained

in natural populations in the absence of TNT.

TNT-resistant mutants exhibit a diminished fitness that

handicapped their surviving in natural populations in

the absence of TNT. In this sense, the competition

ex-periments between wild-type cells and TNT-resistant

mutants have shown that in small populations the

mu-tants are driven to extinction. Moreover, the

photo-synthetic rate of the TNT-resistant mutants was also

significantly lower than that of wild-type cells in

ab-sence of TNT, in agreement with the observed fitness.

In contrast, under selective conditions, the harmful

effects of TNT on photosynthesis were similar in

TNT-resistant mutants and TNT-sensitive wild-type cells.

The discrepancy between these results and the growth

rate measurements under TNT selection suggest that

TNT can also affect other metabolic pathways than

those of photosynthesis.

It is conceivable that there is a recurring mutation

from a normal wild-type allele to a TNT-resistant

al-lele that is detrimental in fitness under nonselective

conditions. In each generation, new resistance

mu-tants arise, but most of these mumu-tants disappear

sooner or later due to natural selection or chance

(Spiess 1989). At any one time, there will be a certain

number of mutant cells that are not yet eliminated.

The average number of such mutants will be

deter-mined as the balance between the mutation rate and

the rate of selective elimination:

(1

q)

q(1

s),

where

is the mutation rate, q is the allele frequency

of the mutant, and s is the selection coefficient of the

mutant (Crow and Kimura 1970, Spiess 1989). Our

data suggest that the average number of

TNT-resis-tant muTNT-resis-tants in the absence of TNT is about 36

mu-tants per million cells.

Fig. 4. Light-saturated GPS evolution versus TNT concen-tration obtained for sensitive wild-type cells and TNT-resistant mutants of Dictyosphaerium chlorelloides. Bars 1 SD.

1080

In conclusion (and using Occam’s razor), our

re-sults suggest that rare spontaneous preselective

mu-tants seem to be enough to ensure the survival of

microalgae after a catastrophic environmental change

resulting from modern water contamination, because

natural populations of microalgae consist of extremely

large numbers of cells. The large populations of

op-portunistic, generalist, r-selected microalgae will have

no problem proliferating in a polluted biosphere.

We thank Drs. J. Juste and C. Salgado for stimulating discussion of ideas. The manuscript was much improved by comments from Prof. M. J. Puertas. Supported by projects REN 2000-0771 (DGICYT) and Art 11 LRU F.N. Marañosa-U.C.M2000. We are grateful to Dr. Eric C. Henry (Herbarium, Department of Bot-any and Plant Pathology, Oregon State University, Corvallis, USA) for his help in improving the English style.

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