COPPER TOLERANCE TESTING ON Mimulus Cupreus DOMBR., SCROPHULARIACEAE, IN POPULATIONS EXPOSED AND NON EXPOSED TO COPPER MINE POLLUTION
Ginocchio R*, Toro I. (Departamento de Ecología, Pontificia Universidad Católica de
Chile, Alameda 340, Santiago, Chile), Schnepf D. (Institute for Soil Science, Agricultural University, Vienna, Austria)
ABSTRACT
Several
plant species have evolved metal tolerance as a response of increasing metal
concentration in soils either by natural causes or man-polluting activities. A well described example is the evolution of a tolerant
ecotype of the monkey flower (Mimulus
gutattus) on Copperopolis, an abandoned copper mine in California.
Although
copper mine activities have been intense in some areas of the Andes Range in
central Chile, presence of metal tolerant ecotypes has not been investigated
for any plant species. Therefore, two populations of Mimulus cupreus from a low perturbed basin (Río Cipreses) and a
highly copper mine perturbed one (e.g. El Teniente belonging to CODELCO-El
Teniente copper mine) were tested to determine their copper tolerance. Copper
tolerance was tested on cuttings under different concentrations of CuSO4
(0, 0.5, and 2.0 mg L-1) in Ca(NO3)2 0.5g L-1,
using the methodology described by Macnair (1993); copper tolerance of tolerant
individuals of Mimulus gutattus from
Copperopolis were also tested under the same methodology and experimental
conditions. Total copper concentration of water and sediment were also checked
to determine differences on copper levels in the inmediate growing environment
of M.
cupreus.
Results
indicated that copper content in water of streams of both basins were very low
(under detection limit, <0.05 mg L-1) but total copper levels in
sediment were higher in El Teniente than in Río Cipreses basin (1,025 and 135
mg Kg-1, respectively). Although root production and growth from
cuttings was strongly inhibited in Mimulus
cupreus from Río Cipreses, tolerant individuals were found at El Teniente
population, indicating evolution of copper tolerance due to the higher copper
content in soils of El Teniente basin. The degree of copper tolerance of M. cupreus from El Teniente was,
however, lower than individuals of Mimulus
gutattus from Copperopolis.
INTRODUCTION
Old
metal mine operations in Calavera County, California, have led to several
abandoned mine sites with high total metal concentrations in soils (Allen
&Sheppard, 1971; Macnair et al., 1993). Although these metal mines are
currently abandoned, several studies indicate the ocurrence of microevolution
in some species of local plants, such as the monkey flower Mimulus guttatus Fisher ex DC (Scrophulariacea). It has been shown
that the degree of metal tolerance in the monkey flower is correlated with the
level of metal contamination in the microenvironment in which plants are
located (Macnair, 1987; Macnair et al.,
1993). Specifically, it has been shown under laboratory conditions that root
growth from cuttings of non-tolerant individuals of M. guttatus is completely
inhibited at copper concentrations of 0.5 mg L-1 in calcium nitrate
while tolerant individuals are not affected (Allen &Sheppard, 1971; Macnair
et al., 1993). This example and others have indicated evolution of metal
tolerance in higher plants illustrates the rapidity of plant adoptions to an
ever changing environment and has become classic examples of microevolution (Tilstone et al,
1997).
The high andine region of the Cachapoal-river in central Chile represents a system of basins from highly perturbed because of strong mine-activities, to not perturbed ones. Therefore it presents an interesting system to evaluate the ocurrence of microevolutive processes in Chilean native species where selection of copper tolerant individuals may have occurred due to excess copper in soil, such as it has been described for M. guttatus in Copperopolis, California, U.S.A. Therefore, a population of Mimulus cupreus from a low perturbed basin (Río Cipreses) and from a highly copper mine perturbed one (El Teniente) were tested to determine their copper tolerance. Highly copper tolerant individuals of Mimulus guttatus, from Copperopolis, California (obtained as seeds from the laboratory of Dr. Mark Macnair, University of Exeter, England) were also tested to compare M. cupreus responses.
METHODS
Seeds of M.
cupreus collected in the field from Río Cipreses and El Teniente basins and
seeds of M. guttatus from
Copperopolis were germinated and grown until maturity in aereated hydroponic
solutions (1/5 strengh Hoagland-solution). Cuttings from 86-90 individual of
each population were harvested so that each individual could be tested at
copper concentrations of 0, 0.5 and 2.0 mg L-1 as CuSO4
in calcium nitrate (0.5 g L-1). Cuttings were grown in 1000 ml
plastic pots under 12:12 light/dark photoperiod, constant temperature (24+2
°C) and humidity (55+5 %) during seven days. Solutions
were changed after 3 days to maintain the desire copper concentration
throughout the experiment. The number of adventitious roots and the lengh of
the longest root were measured per cutting after one week.
Additionaly,
water and sediment samples (0-20 cm) were collected at Río Cipreses and El
Teniente basins in the summer of year 2000, in the same habitats were Mimulus cupreus grow and from where seed
were collected. Total copper content was determined by the USEPA Method 7210
(Atomic Absorption, Direct Aspiration) or by USEPA Method 7211 (Atomic
Absorption, Furnace Technique).
RESULTS AND DISCUSSION
Analysis
of water samples indicates that copper concentration in streams of Río Cipreses
and El Teniente were M. cupreus
plants grow are very low and below the detection limit (<0.05 mg L-1).
However, the analysis of the sediments indicated that the total copper content
in El Teniente basin is about ten times higher (1,025 mg Kg-1) than
in Río Cipreses one (15 mg Kg-1). The constant deposition of metal
rich particles from a copper smelter located nearby this M. cupreus population during the last decades should be responsible
for the increment of total copper level in soils.
The results of copper tolerance testing on M. cupreus from Río Cipreses indicated that both root production and root length were strongly inhibited when copper concentration was increased to 0.5 mg L-1 while individuals of M. gutattus does not show any strong inhibiton at all copper concentrations used (Figures 1 and 2). Although root production and length of M. cupreus from El Teniente were also inhibited at increasing copper concentration in comparison to M. gutattus, there were some tolerant individuals that produced adventitious roots at copper concentrations of 0.5 and 2.0 mg L-1 (Figures 1 and 2). The degree of copper tolerance of M. cupreus from El Teniente was, however, lower than individuals of Mimulus gutattus from Copperopolis. Therefore, selection of copper tolerant individuals at El Teniente population has been occuring due to the increasing bioavailable copper concentration in sediments from the settlement of metal rich particles released from the smelter.
Figure 1: Histogram of the root number produced from cuttings cultivated under 0 mg L-1 (white), 0.5 mg L-1 (grey), and 2.0 mg L-1 (black) for Mimulus cupreus from Río Cipreses (A) and El Teniente (B), and for Mimulus gutattus from Copperopolis (C).
Figure 2: Dose-response curves of root growth from cuttings of Mimulus cupreus from Río Cipreses and El Teniente and Mimulus gutattus from Copperopolis grown under increasing copper concentrations. Data was fitted according the curve y=a/(1+abx).
REFERENCES
Copper tolerance in some Californian populations of
the monkey flower, Mimulus guttatus:
Allen W.R.; Sheppard P.M. (1971), Proc. Roy. Soc. Lond. B. 177: 177-196
The effect of zinc contamination from electric
pylons-contrasting patterns of evolution in five grass species:Al-Hiyaly S.A.K.; Mc
Neilly T.; Bradshaw A.D. (1990), New Phytol. 114:183-190
Heritability and distribution of variation in degree
of copper tolerance in Mimulus guttatus at Copperopolis, California:Macnair M.R., Smith S.E., Cumbes Q.J. (1993),Heredity 71: 445-455
El genero Mimulus L. (Scrophulariacea) en Chile: Von Bohlen Ch. V. (1995), Gayana Bot. 52(1): 7-28
The consequence of selection for copper tolerance on
the uptake and accumulation of copper in Mimulus guttatus: Tilstone G.H., Macnair M.R. (1997), Annals of Botany 80: 747-751
Heavy metal tolerance in plants: a model evolutionary
system: Macnair M.R. (1987) Trends in Ecology and
Evolution 2: 354-359