Environmental Toxicology and Chemistry, Vol. 31, No. 5, pp.

977982, 2012
# 2012 SETAC
Printed in the USA
DOI: 10.1002/etc.1802

Critical Review

CONTAMINANT DRIVEN GENETIC EROSION: A CASE STUDY WITH DAPHNIA LONGISPINA

RUI RIBEIRO,*y DONALD J. BAIRD,z AMADEU M.V.M. SOARES, and ISABEL LOPES
yInstitute of Marine Research, Department of Life Sciences, University of Coimbra, Coimbra, Portugal
zEnvironment Canada, Canadian Rivers Institute, Department of Biology, University of New Brunswick, Fredericton, New Brunswick, Canada
Center for Environmental and Marine Studies, University of Aveiro, Aveiro, Portugal

(Submitted 13 May 2011; Returned for Revision 26 September 2011; Accepted 5 January 2012)

Abstract Natural populations exposed to pollutants are predicted to experience a loss of genetic diversity, especially through genetic
drift, gene ow (emigration), and/or selection (as sensitive genotypes may be lost). In the present study, the authors discuss the use of
selectable markers and neutral markers to evaluate a contaminant-driven loss of genetic diversity and possible implications of genetic
erosion on populations viability. Viability could be reduced by altering life history parameters, especially due to tness costs associated
with the acquisition of resistance and/or by compromising the resilience and adaptation to future environmental changes. This discussion
aims at an integrated and critical analysis of this topic; it is illustrated by several independent studies (each with its own specic
objectives) that were carried out at the same location with Daphnia longispina populations. To the best of the authors knowledge, this is
the most extensively documented case study on genetic erosion of a natural zooplankton population. Directional selection has been found
to be a main factor of microevolution; therefore, genetic erosion was detected by monitoring suitable phenotypic markers. Genetic drift
was found to be probably irrelevant or masked by other factors, especially gene ow. Although the acquisition of resistance apparently
did not entail genetically determined tness costs under uncontaminated conditions, the present case study suggests the possibility of a
further loss of genotypes due to some negative linkages between the sensitivity to potential ulterior toxicants. Environ. Toxicol. Chem.
2012;31:977982. # 2012 SETAC

KeywordsGenetic diversity Microevolution Directional selection Fitness costs Acid mine drainage

INTRODUCTION selection agent, thus conferring in a contaminated environment

Genetic diversity constitutes one of the three pillars of
biodiversity, which has been dened as the variety of living
organisms from all sources including inter alia, terrestrial,
marine, and other aquatic ecosystems and their habitats. It
exists as a continuum, including higher taxa (genera, families,
etc.), species, populations, subpopulations, individuals, and
genes of a population [1,2]. Accordingly, the following three
components of biological variability can be considered: (1) the
diversity of habitats, ecosystems, and landscapes; (2) species
diversity; and (3) gene variation within species.
Traditionally, ecological risk assessment has focused on the
effects that environmental stressors could pose to ecosystems
and species richness, and a number of factors (e.g., high
metal levels, acidication, climate changes [35]) have been
identied as being implicated in their decline or disappearance
[610]. More recently, the impact of contamination on genetic
diversity has begun to receive more attention. A loss of genetic
diversity may reduce populations viability by altering life
history parameters, especially due to tness costs associated
with resistance acquisition or by compromising the resilience
and adaptation to future environmental changes [1114]. Such
a loss has been detected in several contamination-exposed
populations [11,1519].
Two approaches are commonly used to detect changes in the
genetic diversity of populations inhabiting contaminated sites:
monitoring selectable markers versus neutral markers [2023].
Here, selectable markers are considered as genetically deter-
mined traits suspected to be affected by a specic directional
* To whom correspondence may be addressed
(rui.ribeiro@zoo.uc.pt).
Published online 9 March 2012 in Wiley Online Library
(wileyonlinelibrary.com).
977
advantages or disadvantages to an individuals tness (sensu
its contribution to the composition of subsequent generations
relative to the contribution of other genotypes or phenotypes)
[23]. Neutral markers are considered here as polymorphisms
that are not directly affected by the selective pressure [23].
Examples on the use of both types of markers at the biochem-
ical, physiological, or morphological level were reviewed by
van Straalen and Timmermans [23]. According to their work,
genetic erosion is the loss of genotypes determining a specic
trait or set of traits [23].
In the present study, we discuss the use of selectable markers
and neutral markers to evaluate the loss of genetic diversity and
possible implications of genetic erosion on populations via-
bility. This discussion aims at an integrated and critical analysis
of this topic; it is illustrated by several independent studies
(each with its own specic objectives) that were carried out
at the same location with Daphnia longispina O.F. Muller
(Cladocera, Crustacea) populations. To the best of our knowl-
edge, this is the most extensively documented case study on
genetic erosion of a natural zooplankton population.
STUDY SITE AND MODEL ORGANISM
The case study of the present work is located in southeastern
Portugal, near the abandoned cupric-pyrite mine of Sao Dom-
ingos [24], which continuously leaches acid mine drainage
(AMD) to an otherwise uncontaminated semilentic system,
the Chanca River Reservoir (Fig. 1). The AMD is produced
by the continuous oxidation of the abandoned mine tailings,
which is characterized by acidity (pH
2.1) and high dissolved
metals concentrations (Fe, Al, Zn, Cu, Mn, Co, Ni, Cd, Pb, Cr;
in decreasing amounts [2531]). The AMD impact in the
Chanca Reservoir is restricted to a small area (378370 N,
978 Environ. Toxicol. Chem. 31, 2012
Fig. 1. Schematic representation of the aquatic system of the abandoned Sao
Domingos mine region, including upstream reference ponds, the acid mine
drainage (AMD) efuent, and the Chanca River Reservoir (southeastern
Portugal) (adapted from Lopes et al. [38]).
078300 W), where the dilution promotes a rise in pH to circum-
neutral values and the precipitation of metals. Semilentic
reference sites free of metal contamination exist upstream
of the contaminated site (Fig. 1). Studies on evolutionary
ecotoxicology have been carried out with planktonic daphnid
populations inhabiting these impacted and reference habitats
D. longispina and, to a much lesser extent, Ceriodaphnia
pulchella Sars. Daphnids present relevant advantages as model
organisms, namely: (1) they are easy to culture under laboratory
conditions; (2) they have short life cycles producing many
offspring, which facilitates experimental studies; (3) they are
found in large numbers both at reference and impacted sites; and
(4) they reproduce by facultative ameiotic parthenogenesis,
which allows many individuals with the same genotype to be
obtained.
THE GENETIC EROSION HYPOTHESIS
Selectable markers
The Sao Domingos AMD is expected to eliminate the most
sensitive genotypes of aquatic organisms living in the Chanca
River Reservoir near the afuent entrance (hereafter designated
as impacted site), especially after rain events promoting runoff
of mine tailings. Indeed, a simulation of a 3% AMD pulse on
D. longispina laboratory populations at the carrying capacity,
comprising ve differentially AMD-resistant (multilocus allo-
zyme) genotypes with similar frequencies, cultured in the
laboratory for more than 500 generations, resulted in the
elimination of the two most sensitive genotypes, and the most
resistant one increased its frequency from 20 to approximately
50% [32].
R. Ribeiro et al.
Genetic erosion at the impacted site could also be enhanced
by the emigration of the most sensitive genotypes. The avoid-
ance response of 12 clonal lineages of D. longispina was
quantied in a constant gradient of copper (387 mg/L) [33].
Copper, instead of AMD, was chosen due to various reasons.
First, the pH of AMD dilutions is very unstable. Second, among
all measured metals, levels of copper showed the greatest
differences (also zinc) between reference and impacted sites,
which during the main rain season, were one to two orders of
magnitude greater at the impacted site (188 vs 2.8 mg/L [25]).
Third, the resistance to lethal levels of AMD and of copper was
found to be correlated (median clonal lethal concentration of
copper vs median clonal lethal time at 3% AMD: r 0.85,
p < 0.001, n 24 [34]). Avoidance from copper by sensitive
clonal lineages was found to occur much faster and at lower
concentrations than by resistant lineages [33]. The median
clonal avoidance time from the highest copper concentration
(87 mg/L; range: 24594 min) and the median clonal avoidance
copper concentrations after a 12-h exposure (range: 65110 mg/
L) versus the median clonal lethal copper concentrations after
a 48-h exposure (range: 60369 mg/L) were found to be corre-
lated (r 0.63, p < 0.05, n 12 and r 0.74, p < 0.01, n 12,
respectively) [33]. Taking into consideration the literature on
the spatial active avoidance from contaminants by zooplankton
[33,35,36], there are no reasons to doubt the possibility of
emigration by D. longispina individuals from the impacted site
to less contaminated areas of the Chanca Reservoir.
A sensitivity to lethal levels of AMD by D. longispina living
in the Chanca River Reservoir near the afuent entrance
(impacted site) lower than the sensitivity of those from nearby
reference sites was rst demonstrated by Lopes et al. [37]. The
authors showed that time-to-death of reference organisms
exposed to several AMD contaminated water samples was
signicantly lower than that of those collected at the impacted
site. Furthermore, it was demonstrated that resistance (i.e., the
genetically determined component of tolerance) to lethal levels
of AMD was responsible for the differences in sensitivity.
Therefore, AMD was proven to act as a directional selective
pressure resulting in microevolution, despite the counteraction
of two processes of genetic diversication. First, the continuous
gene ow (immigration from adjacent nonimpacted sites at the
Chanca River Reservoir) could have reestablished the original
genotypes frequencies. Second, this reestablishment could also
have been achieved through the hatching of new genotypes
from ephippial eggs, which are produced by sexual reproduc-
tion. Although Lopes et al. [37] remarked cautiously that it is
not possible to conclude if this (microevolution) took place
through the elimination of sensitive individuals (genetic erosion)
or through the spread of new genes (through mutation) or new
genotypes (through sexual recombination), their results appear
to indicate that the most sensitive D. longispina genotypes were
absent at the impacted site. This may be the case because when
exposed to AMD-contaminated samples, reference individuals
started to die well before any mortality was observed among
those collected at the impacted site.
The genetic erosion hypothesis was further supported in a
study explicitly addressing the possible elimination of sensitive
genotypes [38]. The sensitivity to lethal levels of copper for
265 clonal lineages, 136 from a reference site and 129 from the
impacted site, was characterized [38]. This characterization was
done after removing environmental inuences on tolerance
(i.e., previous acclimation to elevated levels of metals and/or
phenotypic plasticity) by keeping laboratory cultures for at least
15 generations under noncontaminated conditions. The most
Genetic erosion in a daphnid population
sensitive clonal lineages to lethal levels of copper were absent at
the impacted site, but extremely resistant lineages were present
at both the reference and impacted site [38]. Therefore, micro-
evolution at the impacted site took place through the elimination
of sensitive individuals (genetic erosion) and apparently not
through the spread of new genes or new genotypes conferring an
enhanced resistance. In a later study with the same species,
approximately 360 individuals from each of the two references
and the impacted site were collected, and their sensitivity (time-
to-death) to a lethal concentration of copper (250 mg/L) was
evaluated [39]. The 1,072 organisms were distributed into ve
categories of sensitivity, with the least tolerant organisms
almost absent at the impacted site (0.6%), whereas the highly
tolerant were represented at both reference sites (43 and 60%).
Again, the most tolerant organisms were not exclusively found
at the impacted site [39]. However, the near absence of very
sensitive organisms at the impacted site could also be accounted
for by processes other than genetic erosion, namely acclimation
and/or phenotypic plasticity, because individuals were main-
tained under laboratory conditions, without the contaminant
pressure, for just 6 h.
After removing environmental inuences on tolerance,
another study on microevolution, looking at the same species
and locations, investigated the time-to-death of at least 20
clonal lineages from each sitereference and impacted
exposed to lethal levels of copper (25 and 75 mg/L, respectively)
[25]. Only sensitive and resistant lineages to Cu were present at
the reference and contaminated site, respectively, thus support-
ing the genetic erosion hypothesis.
Another daphnid speciesCeriodaphnia pulchellawas
also targeted for evolutionary ecotoxicology studies at the same
location [27]. Originating from 15 egg-bearing females from
each of two references and from the impacted site, 20 neonates
from each site, after being kept in noncontaminated medium for
ve generations, were exposed to a locally collected AMD-
contaminated water sample and time-to-death was recorded.
The existence of genetically determined sensitivity differences
to AMD between references and the impacted site was attested.
Genetic erosion was considered the probable cause for those
differences because the frequency of the most sensitive organ-
isms at the impacted site (16%) was much lower than at the
references (73 and 100%) [27].
Beside copper, the comparison of D. longispina resistance to
lethal levels of zinc (median clonal lethal time values in a 96-h
exposure to 1.5 mg/L) between a reference and the impacted
site was also investigated [25]. No evidence of genetic erosion
was found because both populations had a similar distribution
pattern of sensitivities to zinc. Differences in resistance to lethal
levels of copper (addressed above) but not of zinc, between
the reference and the impacted site, were considered likely
to be related to differential selective pressures previously
experienced [25].
Given the above, evidence of genetic erosion in the present
case study has been gathered using resistance to lethal levels
of AMD and copper as selectable traits. However, the AMD is
expected not only to be lethal to the most sensitive organisms
but also to be a sublethal directional selective pressure. Several
of the aforementioned studies addressed genetic erosion looking
at sublethal markers in D. longispina, but decisive results could
not be obtained. Supporting evidence was found with the
reproductive output and the body growth in copper exposures,
after removing environmental inuences (at least ve gener-
ations under controlled conditions) by Lopes et al. [37].
Specically, after an exposure to a 60 mg/L copper solution
Environ. Toxicol. Chem. 31, 2012 979
in articial water and to the latter as control, until the release of
the fourth brood (12 replicates per concentration), organisms
originating from egg-bearing females collected at the impacted
site released as many neonates as the controls, while this copper
concentration signicantly inhibited the reproductive output of
organisms originating from the reference site [37]. In the same
experiment, both 15 and 30 mg/L copper solutions inhibited
body growth of reference organisms but not of impacted site
organisms [37]. On the contrary, no support was obtained either
for reproduction in an AMD exposure [37] or for feeding in
copper exposures [25,38]. The former study found that a
moderately toxic AMD-contaminated water sample (T6 in
Lopes et al. [37]) depressed reproduction both of organisms
originating from two references and from the most impacted site
with an equal intensity (4043% inhibition) [37]. The resistance
to lethal copper levels of 136 and 129 clonal lineages from a
reference and the impacted site, respectively, was investigated
in the latter study [38], as explained above. Feeding rates of
each clonal lineage exposed during 24 h to the respective
highest nonlethal copper concentration were quantied. In none
of the categories of resistance to lethal levels of copper,
organisms from the impacted site ate more than those from
the reference. Years later, feeding rates of clonal lineages from
the impacted site in 24-h exposures to 0.5 and 5 mg/L of copper
were found to be no higher than those from the reference [25].
Opposite results would be expected if genetic erosion at the
impacted site had eliminated those individuals most sensitive to
sublethal levels of contamination.
Metallothionein basal levels and their induction by copper as
potentially selectable traits were also quantied, but differences
between resistant and sensitive genotypes were not found [40].
Furthermore, the variation in 20 putatively polymorphic
enzyme systems was studied in 24 clonal lineages to evaluate
the possible relationship of allozyme proles with the differ-
ential resistance to lethal levels of copper and AMD [36]. The
lack of such a relationship and the fact that the clones were not
representative of each site (reference vs impacted) disallows
any speculation on possible genetic diversity differences
between sites [36].
Neutral markers
In the present case study, D. longispina individuals inhabit-
ing the impacted site are a very small fraction of the Chanca
River Reservoir population; therefore, microevolution through
random genetic drift would not be expected. Furthermore, even
large AMD amounts (produced after very strong rainstorms)
would not be enough to drastically reduce the population size
through a (genetic drift) bottleneck effect. These improbable
situations would be the ones for which the biomonitoring of
neutral markers has been shown to be an effective tool to detect
and quantify genetic differentiation [17,21,41]. Furthermore,
mutations due to genotoxicity of contaminants can mask a
possible decrease in genetic variation evaluated using neutral
markers [23].
To evaluate the impact of AMD on the genetic diversity and
structure of the D. longispina inhabiting the impacted site using
amplied fragment length polymorphisms (AFLP) was the
main objective of the study by Martins et al. [39]. From
1,072 organisms, 31 from each of two references and the
impacted site were selected, for AFLP analysis in a stratied
random sampling design. Neis genetic diversity observed
heterozygosity and Shannons information index revealed no
differences between each pair of sites (Kruskal-Wallis: p  0.7),
even though differences in time-to-death were evident (Neis
980 Environ. Toxicol. Chem. 31, 2012
index and median lethal time values of the two references vs the
impacted site: 0.38  0.08 and 0.33  0.16 vs 0.37  0.16, and
1 h 30 min and 6 h 17 min vs 22 h 58 min, respectively) [39].
More recently, genetic diversity at the reference and impacted
sites using 22 clonal lineages of D. longispina and a panel of
eight microsatellites was preliminarily evaluated [42]. The
results from this investigation did not even slightly point to a
reduction at the impacted site. Advantages and limitations of
using AFLP, microsatellites, and other molecular techniques
within the framework of assessing genetic erosion were
addressed by Van Straalen and Timmermans [23].
In summary, the present case study illustrates a situation
where directional selection is a main factor of evolution; there-
fore, genetic erosion is detected by monitoring suitable select-
able phenotypic markers, but where genetic drift is probably
irrelevant or masked by other microevolution factors, especially
gene ow. Therefore, neutral markers may fail to reveal the
loss of genotypes in the present AMD-impacted case study,
which may also be due to the possible genotoxicity of the AMD
leading to mutations. However, some caution should be taken
when choosing the selectable traits to be monitored. For exam-
ple, sublethal responses may be insensitive to detect loss of
genotypes.
POPULATIONS VIABILITY
The tness costs hypothesis
Due to its size, the D. longispina population of the Chanca
River Reservoir is not threatened, but gathered information will
be integrated to discuss genetic erosion implications in pop-
ulations viability. If resistance acquisition entails genetically
determined tness costs, including those due to negative plei-
otropy or genetic linkage, then in the absence of the former
stressor (i.e., after removing the contaminant source), the
growth rate of the remaining population could be dangerously
reduced. Several noted studies have investigated possible alter-
ations on life history parameters of resistant genotypes, under
no AMD or metal pressure, and have obtained contradictory
results. Evidence against the hypothesis of tness costs asso-
ciated with resistance acquisition was obtained in a work by
Lopes et al. [32], although in their work this topic is not debated.
A life history experiment with ve D. longispina genotypes,
with different multilocus allozyme proles, differing in their
sensitivity to lethal AMD levels (median genotype lethal time
values ranging from 50 to 1,529 min) was carried out [32].
Starting as neonates, 10 individuals from each genotype were
maintained individually under optimal conditions. They were
checked for the time each brood was released, the number of
neonates per brood, and longevity [32]. The intrinsic rate of
natural increase differed among genotypes, being higher in a
sensitive and in a tolerant clone, with no association whatsoever
with resistance [32]. A similar life history experiment was
performed by Saro et al. [43], in which 12 organisms from
each of 13 clonal lineages of D. longispina (median clonal lethal
time values in a 3% AMD dilution ranging from 49 to
1,530 min) were also maintained individually under optimal
conditions. They, too, were checked for the time each brood was
released, the number of neonates per brood, and longevity,
among other parameters. As previously discussed, tness costs
were not found.
Fitness costs evaluated under optimal conditions could be
unrealistic and misleading because negative effects might be
alleviated or aggravated by environmental stressors [44]. A
naturally occurring stressorintraspecic competitionwas
R. Ribeiro et al.
included in the controls of an experiment where a combination
of other stressors was investigated [32]. The controls were
laboratory populations comprising the ve aforementioned
genotypes in equal proportions (i.e., 20%). After 52 days, an
association between density and resistance was not found; both
the most sensitive and the most resistant genotypes presented
the highest population density proportions (28 and 26%, respec-
tively), and no differences were found among genotype fre-
quencies (p 0.20) [32]. If genetic costs were associated with
the acquisition of resistance, then in the absence of contami-
nants, and moreover, in the presence of competing sensitive
genotypes (with no tness costs), the density of resistant
genotypes would be at least reduced, which was not the case.
Besides Lopes et al. [32] and Saro et al. [43], evidence of
tness costs was also not found by Lopes et al. [37,38,45].
Nevertheless, the results obtained by Agra et al. [25] provided
two lines of evidence for tness cost: lower feeding rates of the
tolerant population compared to the reference population and
negative genetic correlations between mean clonal feeding rates
and median clonal survival time in control conditions (no added
Cu or Zn).
Even though the alteration of life history parameters due to
the AMD pressure is not fully evident in the present case study,
the loss of genotypes could still reduce population viability by
increasing susceptibility to future stressors [11,19].
Susceptibility to other stressors
Genetic erosion of selectable traits can have negative con-
sequences for population viability if the sensitivity to different
simultaneously or sequentially occurring directional selective
stressors is inversely related. The opposite scenariocoresist-
ance to lethal levels of toxicantswas reported for the present
D. longispina case study for the pair copper/zinc by both Agra
et al. [25] and Lopes et al. [45]. This result on coresistance can
be due either to specic resistance mechanisms associated with
previous exposure to both metals, which are present in the AMD
at elevated amounts, or to more generalist mechanisms that
counteract the effects of more than one contaminant [45]. No
correlations (positive or negative) were observed for the other
pairwise combinations of toxicants investigated by Lopes et al.
[45]AMD, copper, cadmium, zinc, hydrogen ions, and the
pyrethroid deltamethrin. However, crossing data on the same
clonal lineages from the studies of Lopes et al. [45] and Martins
et al. [28], it comes out that the most resistant to AMD (clonal
lineage A7) was one of the most sensitive to cadmium. Also, the
third most resistant to AMD (clonal lineage B11) was one of
the most sensitive to deltamethrin. Another clonal lineage very
resistant to AMD (clonal lineage B12) was very sensitive to
both cadmium and deltamethrin. These results suggest that the
organisms remaining at the Chanca River Reservoir impacted
site after a partially lethal AMD pulse could be at risk of further
genetic erosionif genetic drift and gene ow were absent
due to the probable elimination of the most sensitive genotypes
to other subsequently occurring toxicants (at least cadmium and
deltamethrin).
CONCLUSIONS
Results from the Sao Domingos mine case study provide
support for the genetic erosion hypothesis by the monitoring of
selectable markers. Nevertheless, it should be highlighted that
looking at sublethal markers did not yield consistent results.
Therefore, care should also be taken when choosing traits
potentially subjected to the pollutant directional pressure.
Genetic erosion in a daphnid population
The fact that the use of neutral markers revealed no signicant
loss of genetic diversity of D. longispina inhabiting the
impacted site is probably indicative that genetic drift is irrel-
evant or masked by other microevolution factors, especially
gene ow, and that AMD may lead to the appearance of new
alleles and genotypes through mutation. These contrasting
results from selectable markers and from neutral markers are
consistent with other studies that have failed to obtain signi-
cant agreement between molecular and quantitative measures of
genetic variation in daphnid populations [46,47]. Previous
studies have indicated that neutral markers are effective in
detecting loss of genetic variability in small or greatly reduced
(bottlenecked) isolated populations [21]. Therefore, we rec-
ommend that both approaches be considered simultaneously to
establish causality between exposure to contaminants in the
environment and population genetic diversity. Even though
acquisition of resistance apparently did not entail genetically
determined tness costs under uncontaminated conditions, the
present case study suggests the possibility of a further loss of
genotypes due to some negative linkages between the sensi-
tivity to potential ulterior toxicants. This latter topic has
received little attention and because of its potentially high
pertinence in real scenarios of contamination, it merits further
research.
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