The Population Genetic Structure of Littorina Littorea (Mollusca: Gastropoda) Along a Pollution Gradient in the Scheldt Estuary (The Netherlands) Using RAPD Analysis
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Science of the Total Environment 325 (2004) 59–69 The population genetic structure of Littorina littorea (Mollusca: Gastropoda) along a pollution gradient in the Scheldt estuary (The Netherlands) using RAPD analysis Hans De Wolfa, *, Ronny Blustaa,b , Thierry Backeljau aDepartments of Biology, Ecofysiology, Biochemistry and Toxicology, University of Antwerp – 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 Eastern Scheldt (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 Western Scheldt 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