Materials and methods Directory UMM :Data Elmu:jurnal:J-a:Journal of Experimental Marine Biology and Ecology:Vol246.Issue1.MAr2000:

70 H . De Wolf et al. J. Exp. Mar. Biol. Ecol. 246 2000 69 –83 ability e.g. Crisp, 1978; Palumbi, 1994, 1996. In marine gastropods, for example, species with planktonic dispersing larvae display higher levels of gene flow and less population genetic differentiation e.g., Mitton et al., 1989; Benzie and Williams, 1992; Brown and Murray, 1995, than nonplanktonic developing species with poor dispersal capacity e.g. Johannesson et al., 1993; Johnson and Black, 1995; Rolan-Alvarez et al., 1995; Trussell, 1996. Nevertheless, even in species with high dispersal abilities there are several factors that may limit actual dispersal and or gene flow, thus creating opportunities for genetic differentiation as well Palumbi, 1994, 1996. These limitations include: invisible gene flow barriers, isolation by distance, behavioral limits to dispersal, selection, and the recent history of a species Palumbi, 1994, 1996. For instance, in the Macaronesian i.e. Azores [AZ], Madeira [MA], Canary Islands [CA] and Cape Verde Islands [CV] planktonic developing periwinkle, Littorina striata, King and Broderip, 1832, no genetic population differentiation is detected at mi- crogeographical scales i.e. 5–100 m De Wolf et al. 1998a, whereas preliminary allozyme data tentatively suggest a tendency towards an isolation by distance IBD relationship among more distant populations i.e. up to 500 km within an archipelago AZ Backeljau et al., 1995. In addition, esterase and random amplified polymorphic DNA RAPD patterns reveal a higher degree of genetic variability in the CV than in the other archipelagos De Wolf et al. 1998b,c. In this paper we explore the population genetics of L . striata at macrogeographic scales by surveying allozyme data from populations covering the entire known geographic distribution of the species i.e. up to 2000 km. We particularly aim at assessing whether the allozyme variation reveals a macrogeographical patterning and if so, whether this patterning is indeed related to IBD.

2. Materials and methods

2.1. Sampling sites Forty-two populations of L . striata n 5 1640 were collected at comparable wave- exposed sites in 13 Macaronesian Islands, including the archipelagos of the AZ, MA, ˜ CA and CV. Sampled islands included: in AZ, Sao Miguel AZ1–12, Pico: AZ13–14, ˜ Santa Maria AZ15, Faial AZ16–18, and Sao Jorge AZ19–20; in MA, Madeira MA1–2, Porto Santo MA3 and Deserta Grande MA4; in CA, Tenerife CA1–4 ˜ and Gran Canaria CA5–6; and finally in CV, Sao Nicolau CV1–2, Sal CV3–7, and ˜ Sao Vicente CV8–12 Table 1. 2.2. Allozyme electrophoresis After field collecting, periwinkles were starved for 4 days and subsequently frozen and stored at 2 808C. Sample preparation and protocols for vertical polyacrylamide gel electrophoresis were as described by Backeljau and Warmoes 1992. Five polymorphic enzyme loci were analyzed. Glucose phosphate isomerase GPI, E.C. 5.3.1.9, 6- phosphogluconate dehydrogenase PGD, E.C. 1.1.1.44, and malate dehydrogenase H . De Wolf et al. J. Exp. Mar. Biol. Ecol. 246 2000 69 –83 71 Table 1 Populations that were analysed with their corresponding abbreviations Archipelago Island Sampling site Abbreviation ˜ Azores AZ Sao Miguel SM Mosteiros AZ1–AZ2 Capellas AZ3 Santana AZ4 Villa de Nordeste AZ5 Faial da Terra AZ6 ˜ Povoc¸ao AZ7 Caloura AZ8 Lagoa AZ9 Santa Clara AZ10 Feteiras AZ11 Ferereia AZ12 Santa Maria SAM Anjos AZ13 ˜ Sao Jorge SJ F. da St. Cristo AZ14 Urzelina AZ15 Pico PI Lajes AZ16 Calhau AZ17 Faial FA P. da Almoxariffe AZ18 Capelinhos AZ19 P. da Espalama AZ20 Madeira MA Madeira MA Canic¸io de Baixo MA1 P. de St. Caterina MA2 Porto Santo POS Porto Santo MA3 Deserta Grande DEG Doca MA4 Canary Is. CA Gran Canaria GRC Agoustin CA1–CA4 Tenerife TEN Playa da America CA5–CA6 ˜ Cape Verde Is. CV Sao Nicolau SAN Harbour CV1–CV2 Sal SAL Santa Maria CV3–CV4 Jose Fonseca CV5–CV6 Pedro de Lumen CV7 ˜ Sao Vicente SAV Baia das Gatas CV8–CV9 Mindelo CV10–CV11 Calhau CV12 MDH, E.C. 1.1.1.37 were resolved in a continuous gel and tray buffer are identical Tris–citric acid buffer at pH 8.0, while hydroxybutyric acid dehydrogenase HBDH, E.C. 1.1.1.30 was resolved in a continuous Tris–boric acid EDTA buffer at pH 5 8.9. Mannose phosphate isomerase MPI, E.C. 5.3.1.8 was analyzed using a discontinuous buffer system with a Tris–glycine tray buffer and a Tris–HCl gel buffer, both at pH 9.0. Enzyme stainings were adapted from Harris and Hopkinson 1976. 2.3. Statistical analysis Allele frequencies, mean numbers of alleles per locus MNA and heterozygosity levels H 5 observed, H 5 expected were estimated with the BIOSYS program obs exp Swofford and Selander, 1989. Genotype frequency departures from Hardy–Weinberg 72 H . De Wolf et al. J. Exp. Mar. Biol. Ecol. 246 2000 69 –83 HW equilibrium conditions were tested with exact probability tests, implemented by the GENEPOP software v. 1.1 Raymond and Rousset, 1995, which applies the Markov chain method proposed by Guo and Thompson 1992. Interarchipelago differences in MNA or H were investigated by means of two obs analyses of variance, using the software package STATISTICA v. 5.0 Statsoft, 1995. Genetic population differentiation can be expressed by means of hierarchical F- statistics. When a hierarchical arrangement of populations is assumed, in this case populations P being placed within islands I, archipelagos A and total distribution area T, the variance of the observed genetic differentiation among the populations var can be split up into its variance components. A series of F-statistics can be ST obtained e.g., F , F , F and F , of which the terms in the following equation PI IA PT AT 1 2 F 5 1 2 F ? 1 2 F ? 1 2 F PT PI IA AT represent respectively: total differentiation, differentiation among populations within islands, differentiation among islands within archipelagos, and differentiation among archipelagos. These F-values are not additive. Hence, F reflects only the additional IA variance among islands beyond that which exists among populations, and F reflects AT only additional variance among archipelagos beyond that which exists among islands Wolf and Campbell, 1995. F-values and corresponding variance components were calculated with the WRIGHT78 option in BIOSYS Swofford and Selander, 1989. Allele frequency heterogeneities among the four archipelagos, were evaluated with Fisher exact tests applied to R 3C contingency tables as implemented by the GENEPOP v. 1.1 software Raymond and Rousset, 1995. The differentiation among islands was further analyzed by means of correspondence analysis, executed with the NTSYS v. 1.80 software Rohlf, 1993. Gene flow Nm among archipelagos was estimated using private allele frequencies Slatkin, 1985; Slatkin and Barton, 1989. Pairwise Nm values were plotted against pairwise geographical distances, enabling us to calculate the correlation coefficient and corresponding regression equation. A significance level of 5 was used throughout. The sequential Bonferroni technique was employed to correct for false assignments of significance by chance alone multiple test problems Rice, 1989.

3. Results