Contaminant driven genetic erosion: A case study with Daphnia longispina

Authors


Abstract

Natural populations exposed to pollutants are predicted to experience a loss of genetic diversity, especially through genetic drift, gene flow (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 fitness 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 specific 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 flow. Although the acquisition of resistance apparently did not entail genetically determined fitness 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: 977–982. © 2012 SETAC

INTRODUCTION

Genetic diversity constitutes one of the three pillars of biodiversity, which has been defined 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, acidification, climate changes 3–5) have been identified as being implicated in their decline or disappearance 6–10. 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 fitness costs associated with resistance acquisition or by compromising the resilience and adaptation to future environmental changes 11–14. Such a loss has been detected in several contamination-exposed populations 11, 15–19.

Two approaches are commonly used to detect changes in the genetic diversity of populations inhabiting contaminated sites: monitoring selectable markers versus neutral markers 20–23. Here, selectable markers are considered as genetically determined traits suspected to be affected by a specific directional selection agent, thus conferring in a contaminated environment advantages or disadvantages to an individual's fitness (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 biochemical, 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 specific 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' viability. This discussion aims at an integrated and critical analysis of this topic; it is illustrated by several independent studies (each with its own specific objectives) that were carried out at the same location with Daphnia longispina O.F. Müller (Cladocera, Crustacea) populations. To the best of our knowledge, 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 São Domingos 24, which continuously leaches acid mine drainage (AMD) to an otherwise uncontaminated semilentic system, the Chança 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 25–31). The AMD impact in the Chança Reservoir is restricted to a small area (37°37′ N, 07°30′ W), where the dilution promotes a rise in pH to circumneutral 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.

Figure 1.

Schematic representation of the aquatic system of the abandoned São Domingos mine region, including upstream reference ponds, the acid mine drainage (AMD) effluent, and the Chança River Reservoir (southeastern Portugal) (adapted from Lopes et al. 38).

THE GENETIC EROSION HYPOTHESIS

Selectable markers

The São Domingos AMD is expected to eliminate the most sensitive genotypes of aquatic organisms living in the Chança River Reservoir near the affluent 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 five differentially AMD-resistant (multilocus allozyme) 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.

Genetic erosion at the impacted site could also be enhanced by the emigration of the most sensitive genotypes. The avoidance response of 12 clonal lineages of D. longispina was quantified in a constant gradient of copper (3–87 µg/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 µg/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 µg/L; range: 24–594 min) and the median clonal avoidance copper concentrations after a 12-h exposure (range: 65–110 µg/L) versus the median clonal lethal copper concentrations after a 48-h exposure (range: 60–369 µg/L) were found to be correlated (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 Chança Reservoir.

A sensitivity to lethal levels of AMD by D. longispina living in the Chança River Reservoir near the affluent entrance (impacted site) lower than the sensitivity of those from nearby reference sites was first demonstrated by Lopes et al. 37. The authors showed that time-to-death of reference organisms exposed to several AMD contaminated water samples was significantly 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 diversification. First, the continuous gene flow (immigration from adjacent nonimpacted sites at the Chança 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 reproduction. 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 influences 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 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, microevolution 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 µg/L) was evaluated 39. The 1,072 organisms were distributed into five 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 maintained under laboratory conditions, without the contaminant pressure, for just 6 h.

After removing environmental influences 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 site—reference and impacted—exposed to lethal levels of copper (25 and 75 µg/L, respectively) 25. Only sensitive and resistant lineages to Cu were present at the reference and contaminated site, respectively, thus supporting the genetic erosion hypothesis.

Another daphnid species—Ceriodaphnia pulchella—was 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 five 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 organisms 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 influences (at least five generations under controlled conditions) by Lopes et al. 37. Specifically, after an exposure to a 60 µg/L copper solution in artificial 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 significantly inhibited the reproductive output of organisms originating from the reference site 37. In the same experiment, both 15 and 30 µg/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 (40–43% 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 quantified. 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 µg/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 quantified, 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 profiles with the differential 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 inhabiting the impacted site are a very small fraction of the Chança 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 amplified 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 stratified random sampling design. Nei's genetic diversity observed heterozygosity and Shannon's information index revealed no differences between each pair of sites (Kruskal-Wallis: p ≥ 0.7), even though differences in time-to-death were evident (Nei´s 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; therefore, genetic erosion is detected by monitoring suitable selectable phenotypic markers, but where genetic drift is probably irrelevant or masked by other microevolution factors, especially gene flow. 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 example, sublethal responses may be insensitive to detect loss of genotypes.

POPULATIONS' VIABILITY

The fitness costs hypothesis

Due to its size, the D. longispina population of the Chança River Reservoir is not threatened, but gathered information will be integrated to discuss genetic erosion implications in populations' viability. If resistance acquisition entails genetically determined fitness costs, including those due to negative pleiotropy 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 alterations on life history parameters of resistant genotypes, under no AMD or metal pressure, and have obtained contradictory results. Evidence against the hypothesis of fitness costs associated 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 five D. longispina genotypes, with different multilocus allozyme profiles, 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, fitness 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 stressor—intraspecific competition—was included in the controls of an experiment where a combination of other stressors was investigated 32. The controls were laboratory populations comprising the five 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%, respectively), and no differences were found among genotype frequencies (p = 0.20) 32. If genetic costs were associated with the acquisition of resistance, then in the absence of contaminants, and moreover, in the presence of competing sensitive genotypes (with no fitness 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 fitness 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 fitness 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 consequences for population viability if the sensitivity to different simultaneously or sequentially occurring directional selective stressors is inversely related. The opposite scenario—coresistance to lethal levels of toxicants—was 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 specific 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 Chança River Reservoir impacted site after a partially lethal AMD pulse could be at risk of further genetic erosion—if genetic drift and gene flow 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 São 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. The fact that the use of neutral markers revealed no significant loss of genetic diversity of D. longispina inhabiting the impacted site is probably indicative that genetic drift is irrelevant or masked by other microevolution factors, especially gene flow, 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 significant 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 recommend 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 fitness 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. 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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