Science of the Total Environment 325 (2004) 59–69

The population genetic structure of Littorina littorea (Mollusca: Gastropoda) along a pollution gradient in the estuary (The ) using RAPD analysis

Hans De Wolfa, *, Ronny Blustaa,b , Thierry Backeljau

aDepartments of Biology, Ecofysiology, Biochemistry and Toxicology, University of – RUCA, Groenenborgerlaan 171, B-2020 Antwerp, Belgium bRoyal Belgian Institute of Natural Sciences, B-1000 Brussels, Belgium

Received 24 June 2003; received in revised form 23 October 2003; accepted 16 November 2003

Abstract

The population genetic structure of the periwinkle Littorina littorea was analysed using random amplified polymorphic DNA (RAPD).Three primers, coding for six putative polymorphic loci were surveyed to infer the genetic structure of seven populations located along the heavily polluted Western (i.e. in order of decreasing pollution load W1, W2, W3 and R1) and the relatively clean (E1, E2 and E3) estuary (The Netherlands).A genetic distance based UPGMA (Unweighted pair group method with arithmetic mean) dendrogram revealed an estuary-related structuring, as Eastern and sites formed two separate clusters.The Western Scheldt cluster was, however, much more heterogeneous, with three RAPD loci revealing a significant genetic heterogeneity compared to none when the Eastern Scheldt sites were compared.Overall mean heterozygosity levels were high, but did not reveal a difference between the estuaries.The current data (1) confirm the patterns of variation previously observed with electrophoretic analyses of esterases and (2) strongly support that these patterns of variation have a genetic basis, in the presence of intense gene flow.In addition, it is suggested that selection, rather than bottleneck effects, induced by the less favourable living conditions at W1, W2 and W3 are responsible for the genetic patterning. ᮊ 2003 Elsevier B.V. All rights reserved.

Keywords: Estuary; Heavy metals; Littorina littorea; Random amplified polymorphic DNA (RAPD); Salinity

1. Introduction DNA level (direct mutagenic effect) or via toxi- cant-mediated mortality andyor curtailment of Environmental toxicants are known to affect the reproduction (population genetic effects)(e.g. genetic structure of natural populations (e.g. Mor- ) Becerril et al., 2001; Belfiore and Anderson, 2001; ton, 1993; Belfiore and Anderson, 2001 , either Theodorakis et al., 2001).The latter may indirectly through the direct action of the toxicant at the result from (1) mutagenic effects (e.g. Hebert and ) *Corresponding author.Tel.: q32-3-218-04-78; fax: q32- Luiker, 1996; Bickham et al., 2000 , but may also 3-218-04-97. be achieved when (2) the toxicant interferes with E-mail address: [email protected] (H.De Wolf ). the physiology of an organism (physiological

0048-9697/04/$ - see front matter ᮊ 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.scitoten.2003.11.004 60 H. De Wolf et al. / Science of the Total Environment 325 (2004) 59–69 effects)(e.g. Farag et al., 1994; Depledge and bottlenecks have the potential to lower the genetic Billinghurst, 1999; Troncoso et al., 2000), or when variability (heterozygosity and polymorphism) in (3) it alters the environment in which the organism populations, thus reducing their future adaptive has to live (ecological effects)(e.g. Lande, 1998; potential (e.g. Hebert and Luiker, 1996; Bickham Beasley and Kneale, 2002; Edwards, 2002). et al., 2000; Staton et al., 2001). A mutagenic toxicant may affect both germ and Hence, toxicant-related mutagenic, physiological somatic cells.When mutagenic effects occur in andyor ecological effects may affect the genetic somatic cells, they may lead to cell death or may population structure indirectly via population level transform the cell into malignancy (Becerril et al., mediated processes, while mutagenic substances 1999; Bickham et al., 2000).These effects are may affect the genetic structure in a direct manner often delayed until later in life and result in a as well (e.g. Theodorakis et al., 1998, 2001; reduced viability or health of the affected individ- Bickham et al., 2000; Belfiore and Anderson, ual (Hebert and Luiker, 1996).In contrast, muta- 2001). tions in germ cells can be passed on to the Obviously, effects of environmental toxicants offspring, introducing new genetic variation into are difficult to discern in the field from other the population, thus directly affecting its genetic factors that might equally well affect the genetic structure (Staton et al., 2001).Germ cell mutations variability.Indeed, the genetic make-up of popu- with large effects, however, will often lower the lations has always been affected by natural factors, viability of gametes, embryos and neonates, lead- leading to extinctions in the past and resulting in ing to a fitness reduction (e.g. Hebert and Luiker, the evolution of new species (Bickham et al., 1996; Bickham et al., 2000).Likewise, toxicants 2000).However, due to the accelerated speed in that interfere with the animals’ physiology, affect- which man currently changes the environment, ing the integrity of membranes, homeostasis and including the production of environmental toxi- other physiological processes will result in a fitness cants, it becomes increasingly more difficult for decrease (Nony and Schnellmann, 2001), while populations to adapt (Guttman, 1994).As a con- toxicants that affect ecological resources such as sequence, the present extinction rate is estimated food availability, presence of other species, etc., to be 10–100 times the historical background may indirectly affect survival andyor reproduction (Pimm et al., 1995), thus seriously threatening the of animals in contaminated areas as well (e.g. biodiversity (Depledge, 1996; Lande, 1998). Beasley and Kneale, 2002; Edwards, 2002). Against this background a population genetic If toxicant-mediated mutagenic, physiological or survey, using isoelectric focussing of esterase ecological effects do result in large-scale mortality (EST) loci was performed, using populations of or if they reduce the population recruitment, a the periwinkle Littorina littorea, collected along genetic bottleneck may occur, decreasing the the Scheldt estuary (The Netherlands)(De Wolf genetic variation as low frequency, and rare alleles et al., 2001a).This estuary consists of two adja- may be lost during this process (e.g. Gillespie and cent, but separated arms (i.e. Eastern and Western Guttman, 1998; Belfiore and Anderson, 2001; Scheldt) of which the western one is highly pol- Theodorakis et al., 2001).However, if certain luted, while the eastern one is relatively clean genotypes are more susceptible to mutagenicity (Gerringa et al., 1996).A few years ago, the andyor toxicant-related physiological or ecological western arm was even placed among the most effects than others, the toxicant may act as a heavily metal polluted estuaries of the world (Bay- selective agent upon loci that are crucial for the ens, 1998), as it is a major drain for industrial and survival of the animal (e.g. Gillespie and Guttman, domestic wastes.Pollution decreases from the 1998; Belfiore and Anderson, 2001).Allele fre- mainland towards the North Sea (i.e. downstream), quencies at these loci may be shifted as well in creating complex pollution gradients, which are relatively short periods of time, depending on the also reflected in the soft tissues of L. littorea (De strength of the selective force.Besides causing Wolf et al., 2000).In addition, and in contrast to shifts in allele frequencies, both selection and the eastern part, there is a downstream increasing H. De Wolf et al. / Science of the Total Environment 325 (2004) 59–69 61 salinity gradient, which sets the distribution limits Western (W) and Eastern (E) Scheldt.In the for L. littorea along the Western Scheldt (De Wolf Western Scheldt these sites were, in order of et al., 2001b).Despite the periwinkle’s gene flow decreasing pollution and increasing salinity: Han- potential (via planktonic development) and thus sweert (W1), Ellewoutsdijk (W2) and Borssele ability to continuously introduce lost andyor new (W3).Westkapelle (R1), located at the very end genetic variation into populations, a non-isolation of the Western Scheldt, facing the North Sea, was by distance-related genetic structuring was appar- chosen as a marine reference site, while Krabben- ent (De Wolf et al., 2001a).Indeed, a multi- dijke (E1), Yerseke (E2) and Kattendijke (E3) dimensional scaling procedure, performed on an were sampled in the Eastern Scheldt (Fig.1 ). electrophoretic EST profile based Dice similarity After collection, the shells were removed and the distance matrix, revealed two clusters, separating animals were sexed based on the presenceyabsence the Eastern and Western Scheldt sampling sites of the vesicula seminalis.Because intersex or the (De Wolf et al., 2001a).In addition, genetic maculinisation condition in which the paliale ovi- variability, expressed as mean number of isoelec- duct of females is gradually transformed into a tric focussed EST bands per site, differed signifi- prostate gland and sperm groove and penis may cantly, as the least polluted and most marine-like develop, has been known to occur along the sites revealed the highest variable EST profiles Scheldt estuary (De Wolf et al., 2001c), females (De Wolf et al., 2001a).Apparently, pollution were further grouped into ‘normal’ and ‘intersex’ andyor natural factors like low salinity (1) affected individuals.Genomic DNA was extracted from the genetic population structure and (2) seemed to individual columellar muscles following the CTAB decrease genetic variability, thus decreasing the (Cetyltrimethylammonium bromide) protocol of adaptive potential of L. littorea along the estuary. Winnepenninckx et al. (1996), modified so that The mechanisms responsible (i.e. selection andyor tissues were not ground under liquid nitrogen, but bottleneck effects) for this structuring remained, instead, directly incubated in CTAB buffer. however, to be determined.In addition to selection andyor bottleneck effects, the expression of protein 2.2. RAPD amplification markers such as EST loci may be affected as well through gene regulation, secondary modifications PCR reactions were performed in a final volume of the EST enzyme, etc., which could equally well of 25 ml, containing 25 pmol RAPD primer, 50– explain the observed molecular patterning.There- 100 ng template DNA and a standard quantity fore, additional genetic markers and information ‘Ready To Go’ RAPD analysis mixture (Amer- are needed to confirm the EST structuring.Hence, sham Pharmacia Biotech Inc.).PCR conditions we used RAPD analysis to describe the population were: 5 min at 95 8C followed by 45 cycles, of genetic structure of L. littorea at different sites 30sat958C, 60 s at 40 8C, and 120 s at 72 8C. ( q ) along the Scheldt estuary east west , comparing After the last cycle, a final step of 5 min at 72 8C the current results with the previously obtained was added to allow complete extension of all EST results, enabling us to discriminate between amplified fragments.The amplified products (10 ecophysiological and genetic effects.In addition, ml) were separated for 90 min at 100 V on 0.8% the current analysis will help us to formulate ( ) agarose, 0.025% ethidium bromide TAE gels. Gels hypotheses regarding the underlying mechanism s were photographed under UV light. that are responsible for the population genetic Forty random decamer primers were screened structure of L. littorea along the polluted Western (Kits H and G of Operon Technologies), three of and relatively clean Eastern Scheldt estuary. which OPG08 (59-TCACGTCCAC-39), OPG20 2. Materials and methods (59-TCTCCCTCAG-39) and OPH10 (59- CCTACGTCAG-39) were retained for further anal- 2.1. Sample collection and DNA extraction ysis.RAPD bands were scored as present yabsent On 8th August, 1998, L. littorea was collected and only well-resolved bands were considered. at seven sites (20 individuals per site) along the The reproducibility of the RAPD profiles was 62 H. De Wolf et al. / Science of the Total Environment 325 (2004) 59–69

Fig.1.Study area and sampling sites (i.e. Hansweert (W1), Ellewoutsdijk (W2), Borssele (W3), Westkapelle (R1), Krabbendijke (E1), Yerseke (E2) and Kattendijke (E3)). tested for each primer, by reanalysing five individ- unbiased estimates of the recessive allele frequen- uals from each site. cies were obtained using the method of Lynch and Milligan (1994), as implemented by the software 2.3. Data analysis package RAPDFST v.4.0.1 (Black, 1997).This package was used to estimate genetic population 2 Under the assumption that each RAPD band differentiation, by means of FST and x contingen- ( 2s represents a single diallelic locus in Hardy–Wein- cy table analysis x 2NFST; degrees of freedom berg equilibrium, a condition which seems war- (d.f.)snumber of subpopulationsy1; Nsnumber ) ranted for L. littorea, given previous allozyme data of individuals . FST values were calculated within (e.g. Fevolden and Garner, 1987; Foltz et al., both Scheldt arms and were used to estimate gene 1993), the presentyabsent (1y0) data set can be flow levels (Nm) according to Wright (1965). converted into allele frequencies (e.g. Apostol et Neutrality was tested at each locus, using the al., 1996; De Wolf et al., 1998).However, because Popgene v1.32 package (Yeh and Boyle, 1996), RAPD bands behave as dominant markers (i.e. implementing the Ewens–Watterson test for neu- 1sAA or Aa and 0saa), allele frequency esti- trality.Upper and lower boundaries of the 95% mates are less accurate than with codominant confidence region were calculated after 1000 per- markers (Lynch and Milligan, 1994).Therefore, mutations.Pairwise linkage disequilibrium among H. De Wolf et al. / Science of the Total Environment 325 (2004) 59–69 63

Table 1 Allele frequencies (0srecessive; 1sdominant allele; Nsnumber of individuals analysed) at all analysed loci from all sampling sites; (for abbreviations of sampling sites see Section 2)

W1 W2 W3 R1 E1 E2 E3 OPG08-1 N 20 20 20 20 20 20 20 0 0.8367 0.9220 0.6882 0.8944 0.7071 0.8367 0.8944 1 0.1633 0.0780 0.3118 0.1056 0.2929 0.1633 0.1056 OPH10-1 N 20 20 20 20 20 20 20 0 0.8660 0.9747 0.8885 0.7746 0.8367 0.9220 0.8944 1 0.1340 0.0253 0.1115 0.2254 0.1633 0.0780 0.1056 OPH10-2 N 20 20 20 20 20 20 20 0 0.9747 1.0000 1.0000 0.8944 0.8944 0.8367 0.8944 1 0.0253 0.0000 0.0000 0.1056 0.1056 0.1633 0.1056 OPG20-1 N 20 20 20 20 20 20 20 0 0.8660 0.8660 0.9733 0.7746 0.8062 0.7454 0.7071 1 0.1340 0.1340 0.0267 0.2254 0.1938 0.2546 0.2929 OPG20-2 N 20 20 20 20 20 20 20 0 0.6325 0.7071 0.6489 0.8367 0.9220 0.9129 0.9220 1 0.3675 0.2929 0.3511 0.1633 0.0780 0.0871 0.0780 OPG20-3 N 20 20 20 20 20 20 20 0 0.9487 0.9487 0.9733 0.7746 0.9487 0.8819 0.9487 1 0.0513 0.0513 0.0267 0.2254 0.0513 0.1181 0.0513 alleles at all RAPD loci were calculated with the OPG08; two with OPH10 and three with OPG20 RAPDLD v2.0 software (Black, 1996), using a (Table 1).None of these RAPD bands was diag- x2 goodness-of-fit test. nostic for sexual status, estuary or specific sam- Heterozygosity levels were computed at each pling sites, although OPH10-2 was only expressed locus for each population, while mean heterozy- in one individual from the upstream-located West- gosity levels and standard errors were calculated ern Scheldt sites, whereas it frequently occurred for each sampling site as well, using the Popgene in the downstream Western Scheldt part (i.e. West- v1.32 package (Yeh and Boyle, 1996).Possible kapelle) and in the Eastern Scheldt (Table 1). differences in heterozygosity levels, between both The Ewens–Watterson test revealed acceptable estuaries were evaluated for each locus by means fits for neutral expectations, except for OPH10-2 of the non-parametric Mann–Whitney U-test, in Hansweert (W1), OPH10-1 in Ellewoutsijk ( using the software package Statistica Statsoft, (W2), OPG20-1 and OPG20-3 in Borssele (W3) ) 1998 . (Table 2).However, given the large number of Prevosti genetic distances were calculated tests performed (42), these four departures from among all sampling sites and were used to con- neutrality were not considered significant.Linkage struct a UPGMA topology, with the BIOSYS was observed in 16 out of 100 pairwise loci package (Swofford and Selander, 1989).A signif- comparisons (Table 3).After Bonferroni correction icance level of 5% was used throughout, while a Bonferroni correction was applied to correct for for multiple testing, only the combination OPG20- false assignments of significance by chance alone 2 OPG20-3 remained significant and this at all ( ) (multiple test problem). three Eastern Scheldt sampling sites Table 3 . FST values indicated a high degree of population 3. Results differentiation in the Western Scheldt, with three out of six RAPD loci revealing a significant The three primers retained for further analysis genetic heterogeneity (Table 4).In contrast, no yielded six polymorphic and reproducible RAPD significant population differentiation could be bands, representing six putative loci: one with detected among the sampling sites within the 64 H. De Wolf et al. / Science of the Total Environment 325 (2004) 59–69

Table 2 ( s s Results of the Ewens–Watterson test for neutrality Fobs sum of squared observed allele frequencies, L95% lower boundary of the 95% confidence region, U95%supper boundary of the 95% confidence region. (for abbreviations of sampling sites see Section 2)

Locus W1 W2 W3 R1

Fobs L95% U95% Fobs L95% U95% Fobs L95% U95% Fobs L95% U95% OPG08-1 0.7267 0.5000 0.9050 0.8561 0.5000 0.9050 0.5709 0.5014 0.9003 0.8111 0.5050 0.9050 OPH10-1 0.7679 0.5000 0.9050 0.9506 0.5000 0.9050 0.8019 0.5014 0.9003 0.6508 0.5050 0.9050 OPH10-2 0.9506 0.5000 0.9050 1.0000 – – 1.0000 – – 0.8111 0.5050 0.9050 OPG20-1 0.7679 0.5000 0.9050 0.7679 0.5000 0.9050 0.9481 0.5014 0.9003 0.6508 0.5050 0.9050 OPG20-2 0.5351 0.5000 0.9050 0.5858 0.5000 0.9050 0.5443 0.5014 0.9003 0.7267 0.5050 0.9050 OPG20-3 0.9026 0.5000 0.9050 0.9026 0.5000 0.9050 0.9481 0.5014 0.9003 0.6508 0.5050 0.9050 Locus E1 E2 E3

Fobs L95% U95% Fobs L95% U95% Fobs L95% U95% OPG08-1 0.5858 0.5050 0.9050 0.7267 0.5000 0.9050 0.8111 0.5050 0.9050 OPH10-1 0.7267 0.5050 0.9050 0.8561 0.5000 0.9050 0.8111 0.5050 0.9050 OPH10-2 0.8111 0.5050 0.9050 0.7267 0.5000 0.9050 0.8111 0.5050 0.9050 OPG20-1 0.6875 0.5050 0.9050 0.6204 0.5000 0.9050 0.5858 0.5050 0.9050 OPG20-2 0.8561 0.5050 0.9050 0.8409 0.5000 0.9050 0.8561 0.5050 0.9050 OPG20-3 0.9026 0.5050 0.9050 0.7917 0.5000 0.9050 0.9026 0.5050 0.9050

Eastern Scheldt (Table 4).Average gene flow Scheldt (Ps0.026), whereas, heterozygosity at estimates (Nm) are nonetheless high, ranging from locus OPG20-2 is significantly lower in the Eastern 4.5 within the Western Scheldt to 17.8 within the Scheldt (Ps0.032).Although not significant (Ps Eastern Scheldt (Table 4). 0.074), heterozygosity at locus OPG20-1 is more Locus-specific heterozygosity levels, mean het- elevated in the Eastern Scheldt.The heterozygosity erozygosity levels and standard errors are graphi- levels at the remaining loci are comparable cally represented in Fig.2.Although mean between both Scheldt arms. heterozygosity levels are comparable between the The Prevosti genetic distance based UPGMA different sites, locus-specific heterozygosity levels topology is shown in Fig.3.The topology reflects differ significantly.Indeed, heterozygosity at locus the geographical positioning of the different sam- OPH10-2 is significantly lower in the Western pling sites, yielding two clusters, one joining the

Table 3 Results of the linkage disequilibrium tests performed at each sampling site for all possible pairwise loci combinations (only signif- icant results are tabulated; for abbreviations of sampling sites see Section 2)

Locus W1 W2 W3 R1 E1 E2 E3 OPG08-1 OPG20-2 0.017 OPH10-1 OPH10-2 0.0062 OPH10-1 OPG20-1 0.0311 OPH10-1 OPG20-2 0.0103 OPH10-1 OPG20-3 0.0227 0.0388 OPH10-2 OPG20-1 0.0186 OPH10-2 OPG20-3 0.0426 OPG20-1 OPG20-2 0.0100 0.0105 0.0393 OPG20-1 OPG20-3 0.0421 0.0144 OPG20-2 OPG20-3 0.0004* 0.0007* 0.0004* *Significant after Bonferroni correction. H. De Wolf et al. / Science of the Total Environment 325 (2004) 59–69 65

Table 4 ( ) Summary of gene flow estimates Nm , FST-statistics and results of a x2 test for possible genetic heterogeneity within the Eastern and Western Scheldt

Western Scheldt Eastern Scheldt

Locus FST PNmFST PNm OPG08-1 0.059 0.0261 4.0 0.040 0.0891 6.0 OPH10-1 0.047 0.0588 5.0 0.012 0.4766 20.6 OPH10-2 0.059 0.0256 4.0 0.007 0.6655 35.5 OPG20-1 0.043 0.0801 5.6 0.009 0.5845 27.5 OPG20-2 0.031 0.1763 7.8 0.000 0.9863 OPG20-3 0.078 0.0063 3.0 0.014 0.4370 17.6 Mean 0.053 0.0001 4.5 0.014 0.6255 17.6 Fig.3.Prevosti’s genetic distance UPGMA topology, based on all six RAPD loci. 4. Discussion Western Scheldt sites, except for Westkapelle, and one joining the Eastern Scheldt sites and L. littorea has been the subject of many popu- Westkapelle. lation genetic surveys, using allozyme data.Most

Fig.2.Mean (A) and locus-specific heterozygosity levels (i.e. A: OPG08-1; B: OPH10-1, 2; C: OPG20-1, 2, 3) and (D) genotypic distribution at locus OPH10-2 observed in the Eastern and Western Scheldt sampling. 66 H. De Wolf et al. / Science of the Total Environment 325 (2004) 59–69 of these studies failed, however, to detect genetic differs between both estuaries and which displays structuring both at micro- and macro-geographical a downstream increasing gradient along the West- scales (e.g. Berger, 1973; Wilkins and O’Reagan ern Scheldt, is known to affect the physiology 1980; Janson, 1987; Johannesson, 1992).This has andyor ecological resources of estuarine organ- been explained by the planktonic development and isms, while it may interact with the heavy metal thus presumed gene flow potential of the species uptake as well (e.g. Blust et al., 1992; Bjerregaard (Johannesson, 1992).Indeed, L. littorea is a plank- and Depledge, 1994; Jackson et al., 2000).At high tonic developer, producing long-lived (4–7 weeks) salinities, Cd will, for instance, be complexed with free-floating larvae which are presumed to disperse chlorides making it less available for uptake (Blust over long distances.Consequently, new recruits et al., 1992; Jackson et al., 2000).In addition, the may arise from a variety of parental genotypes, uptake of the remaining uncomplexed bioavailable thus counteracting possible local genetic differen- Cd2q fraction will be hampered at higher salinities, tiation by genetic drift.This seems to hold for the due to a more intense competition with Ca2q for populations of L. littorea in the Eastern Scheldt, uptake via the Ca2q channels (Bjerregaard and but does not seem to apply to the populations from Depledge, 1994).Given the higher ambient heavy the Western Scheldt, where three loci revealed metal levels and (for many trace metals) increased significant intra estuary differences.These results uptake in the lower saline Western Scheldt sam- are in line with previous EST data from the same pling points, we expect more metal mutagenicity sampling sites (De Wolf et al., 2001a), revealing and adverse physiological andyor ecological (1) an Eastern–Western Scheldt related genetic effects in the Western Scheldt, especially at differentiation; (2) a genetic structuring within the upstream located sites. Western Scheldt which seems to correlate with a Increased mutagenic activity could explain the salinityypollution gradient; and (3) an Eastern near absence of OPH10-2 amplification in the Scheldt positioning of the Western Scheldt ‘refer- upstream parts of the Western Scheldt, if this ence’ sampling site Westkapelle.Hence, the EST activity is directed towards the OPH10-2 priming patterns of L. littorea along the Scheldt estuary sites andyor interpriming site distance.However, seem to have a genetic basis.In order to ensure chromosomal andyor DNA damage research is that the current patterns of genetic variation are needed to investigate this hypothesis in the future. temporarily stable, the RAPD analysis will have Alternatively, selection and or bottlenecks, either to be performed again in the future, on individuals related to the mutagenic, physiological andyor collected at the same sampling sites, using the ecological effects might be implicated. same RAPD loci.Nevertheless, the fact that strik- A bottleneck is expected to affect most loci, ingly similar patterns of genetic variation emerge reducing their heterozygosity and polymorphism at different points in time using EST and RAPD levels (e.g. Gillespie and Guttman, 1998; Belfiore analysis, suggests temporal stability. and Anderson, 2001; Theodorakis et al., 2001), The question that arises now is how to explain while selection is expected to affect particular loci these patterns of variation among and within the only (e.g. Gillespie and Guttman, 1998; Belfiore Eastern and Western Scheldt.Heavy metals such and Anderson, 2001), not necessarily reducing as Cd, Cr, Cu, Fe and Ni, of which the ambient heterozygosity (i.e. heterosis).Examples of both and soft tissue concentrations differ between both selection and bottleneck-related effects can be estuaries and display upstream increasing soft tis- found in RAPD-related toxicological literature sue concentration gradients along the Western (e.g. Theodorakis and Shugart, 1997; Ma et al., Scheldt (De Wolf et al., 2000), are known to be 2000).Based on our results it is, however, difficult mutagenic (Forni, 1994; Reid et al., 1994).They to differentiate between both mechanisms.When may also affect the physiology (e.g. Farag et al., we compare the overall mean heterozygosity levels 1994; Nicholson, 2001) andyor the resources of from the Western and Eastern Scheldt (respectively the animals (e.g. Clements et al., 2000; Beasley 0.21 and 0.23) with previously published allozyme and Kneale, 2002).Likewise, salinity which also based heterozygosity levels for L. littorea (0.07) H. De Wolf et al. / Science of the Total Environment 325 (2004) 59–69 67

(Foltz et al., 1993), (0.04)(Fevolden and Garner, (2) heterozygosity levels are elevated and compa- 1987; Janson, 1987), it is clear that they are rable between polluted and pristine sites; and (3) elevated and of the same magnitude at both estu- alleles and genotypes at OPH10-2 are not random- aries.Given these high heterozygosity values and ly distributed along the estuary, suggest that selec- considering the gene flow levels (Nm)1),itis tion, rather than a bottleneck, is responsible for rather unlikely that the genetic patterning resulted the genetic patterning. from a genetic bottleneck.Nevertheless, the current In order to test this hypothesis, laboratory exper- high heterozygosity levels cannot completely iments will have to be set up, determining geno- refute the bottleneck hypothesis, as heterozygosity types, DNA damage, chromosomal damage, levels obtained via allozyme analysis have, in physiological and fitness parameters in periwinkles some cases, been reported to underestimate the that are challenged to a wide salinity andyor heavy genetic variability, when compared to DNA - metal range.In addition, all sampling sites will ers (Apostol et al., 1996). have to be ecologically characterised in order to Evidence for selection-related effects might, on look for possible environmentally affected ecolog- the other hand, be found when considering the ical differences among the sites, which could affect allelicygenotypic distribution at locus OPH10-2 the life history of the periwinkles’ as well. (Fig.2 ).Indeed, OPH10-2 was expressed in 28% of the individuals from the Eastern Scheldt, where- Acknowledgments as it could only be detected in one individual from the Western Scheldt when Westkapelle was omitted This research was supported by the Fifth Frame- from the analysis.Consequently, all but one indi- work Programme of the European Commission vidual from the three upstream-located Western under contract number EVK3-CT-2001-00048, by Scheldt sampling sites shared the same homozy- an F.W.O. ‘Krediet aan navorsers’ and RAFO1 gotic recessive genotype at OPH10-2.The fact DEWOH KP02 grant.HDW is a Postdoctoral that the recessive allele in OPH10-2 is almost Fellow of the Fund for Scientific Research-Flan- completely fixed in the upstream-located Western ders (Belgium)(F.W.O . ). Scheldt sampling sites, while both the dominant as well as the recessive allele are frequently found References in the downstream part of the Western and in the Eastern Scheldt, suggests a selection-related effect Apostol BL, Black WC, Reiter P, Miller BR.Population through hitchhiking (Yan et al., 1998; Belfiore and genetics with RAPD-PCR markers: the breeding structure Anderson, 2001). of Aedes aegypti in Puerto Rico.Heredity 1996;76:325 – 334. 5. Conclusion Bayens W.Evolution of trace metal concentrations in the Scheldt estuary (1978–1995).A comparison with estuarine and ocean levels.Hydrobiologia 1998;366:157 –167. In conclusion, one can say that the genetic Beasley G, Kneale P.Reviewing the impact of metals and patterning obtained with the protein EST marker PAH’s on macro invertebrates in urban watercourses.Prog is confirmed in the current study, using DNA Phys Geogr 2002;26(2):236 –270. markers.Hence, the EST patterns did not result Becerril C, Ferrero M, Sanz F, Castano˜ A.Detection of mitomycin C-induced genetic damage in fish cells by use from an ecophysiological-induced phenotypic of RAPD.Mutagenesis 1999;14:449 –456. expression, but truly reflected the periwinkles’ Becerril C, Acevedo H, Ferrero M, Sanz F, Castano˜ A.DNA genome.These genetic patterns may result from fingerprint comparison of rainbow trout and RTG-2 cell line the direct mutagenic activity at metal polluted using random amplified polymorphic DNA.Ecotoxicology sites, but may also result from population-mediated 2001;10:115 –124. processes, induced by salinityyheavy metal pollu- Belfiore NM, Anderson SL.Effects of contaminants on genetic patterns in aquatic organisms: a review.Mutat Res tion-related mutagenic, physiological andyor eco- 2001;489:97 –122. logical adverse effects.The fact that (1) genetic Berger EM.Gene–enzyme variation in three sympatric species patterns persist in time, despite intense gene flow; of Littorina.II The Roscoff population, with a note on the 68 H. De Wolf et al. / Science of the Total Environment 325 (2004) 59–69

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