Hydrobiologia 378: 11–19, 1998. R. M. O’Riordan, G. M. Burnell, M. S. Davies & N. F. Ramsay (eds), Aspects of Littorinid Biology. 11 © 1998 Kluwer Academic Publishers. Printed in Belgium. Electrophoretic heterogeneity within and between flat periwinkles (Mollusca: Gastropoda) along an intertidal transect at Ria Ferrol, northwest Spain C. Olabarria1, J.-M. Timmermans2 & T. Backeljau2,∗ 1 Dept. Biology, Faculty of Biology, University of Santiago de Compostela, E-15706 Santiago de Compostela, Spain 2 Royal Belgian Institute of Natural Sciences, Vautierstraat 29, B-1000 Brussels, Belgium (∗ author for correspondence) Key words: esterases, Littorinidae, myoglobin, population genetics, Spain, systematics Abstract Using isoelectric focusing of esterases (EST), general proteins (GP) and myoglobin (Mb), we surveyed intra- and interspecific differentiation in flat periwinkles along a vertical intertidal transect in the Ensenada do Baño at Ria Ferrol, N.W. Spain. In this region, L. obtusata occurs in four algal belts, although it is rare in the lowest zone defined by Fucus serratus. L. fabalis is common in the F. serratus and F. vesiculosus belt, but is absent higher up on Ascophyllum nodosum and F. spiralis. Our data show that (1) EST and GP consistently differentiate between L. obtusata and L. fabalis, without however providing useful diagnostic markers, (2) L. fabalis is the less variable (heterozygous), but more heterogeneous species, (3) Mb patterns show significant heterogeneity in L. obtusata between the F. serratus zone and the other algal belts, but not in L. fabalis, and (4) the data on littorinid Mb appear inconsistent with a dimeric protein controlled by a single locus. Yet, assuming two loci coding for a monomeric (or dissociated dimeric) protein produces for the flat periwinkles a data set in which no significant deviations from Hardy-Weinberg expectations were detected. Nevertheless, this speculative interpretation fails to explain all littorinid Mb data. Hence the genetics and structure of littorinid Mb need further study. Introduction L. obtusata is a perennial species that lives upon the macrophytes themselves (e.g. Reid, 1996). The closely related and highly similar flat periwin- Besides these ecological differences, L. obtusata kles Littorina obtusata (Linnaeus, 1758) and L. fabalis and L. fabalis show a number of other diagnostic (Turton, 1825) [formerly L. mariae Sacchi & Rastelli, features involving morphology, life history (both re- 1966] are common and widely distributed in the eu- viewed by Reid, 1996), and allozymes (reviewed by littoral of the European Atlantic coasts, where they Tatarenkov, 1995a). Some of these features are subject live sympatrically on fucoids (e.g. Reid, 1996). This to considerable geographic variation and interspecific obligatory macrophyte-association is unique within overlap so that their value as species markers can vary the genus Littorina, but at the same time differen- between populations (Reid, 1996). This may be partly tiates both flat periwinkles since L. obtusata usually due to the fact that both species have direct devel- lives in the middle to upper eulittoral on Ascophyllum opment, without a planktonic dispersal stage. Under nodosum and Fucus vesiculosus, whereas L. fabalis such conditions, gene flow between populations is ex- prefers F. serratus in the lower to middle eulittoral. In pected to be insufficient to counteract differentiation addition, L. fabalis is an annual species that feeds on provoked by genetic drift at neutral loci (e.g. Ward, the micro-epiphytes growing on the macroalgae, while 1990; Tatarenkov & Johannesson, 1994). In contrast, Article: hy-r2 Pips nr. 180355 (hydrkap:bio2fam) v.1.1 hy-r2.tex; 21/11/1998; 22:29; p.1 12 natural selection can impose substantial differentiation Materials and methods in any species, even those with high dispersal abil- ity (e.g. Koehn et al., 1983; Johnson & Black, 1982, A total of 88 specimens of L. obtusata and 54 spec- 1984; Hilbish, 1985, 1996). imens of L. fabalis was collected along a vertical Several allozyme studies have investigated the ef- intertidal transect in the Ensenada do Baño at Ria Fer- fects of drift and selection in Littorina spp., including rol, N.W. Spain. The transect comprised four algal flat periwinkles, by describing population differentia- levels, with the uppermost zone defined by F. spiralis, tion and structuring at ‘horizontal’ micro- and macro- and the three lower levels by, in descending order, As- geographic scales (e.g. Vuilleumier & Matteo, 1972; cophyllum nodosum, F. vesiculosus and F. serratus.In Berger, 1977; Newkirk & Doyle, 1979; Janson & each level an area of 0.5 m2 was sampled. Specimens Ward, 1984; Janson, 1987a; Mill & Grahame, 1988; were transported alive to the laboratory in Brussels, Dytham et al., 1992; Johannesson, 1992; Mill & where they were stored at –80 ◦C until prepared for Grahame, 1992; Tatarenkov & Johannesson, 1994; electrophoresis. Specimens were identified by the pe- Tatarenkov, 1995b). Yet, relatively few papers deal nis morphology for the males (e.g. Sacchi & Rastelli, with genetic differentiation between vertical intertidal 1966; Warmoes et al., 1988; Reid, 1996) or the struc- zones (e.g. Johannesson et al., 1993, 1995a), al- ture of the pallial oviduct for the females (Reid, 1990, though at least in rough periwinkles selection seems 1996). to maintain sharp allozyme frequency differences at Individual tissue homogenates were prepared by an aspartate aminotransferase (AAT-1) locus between crushing the shells and dissecting the radular muscles intertidal levels (Johannesson & Johannesson, 1989; and digestive gland. Visibly parasitised animals were Johannesson et al., 1995b). discarded. Tissues were kept separately and were ho- Against this background we present here prelimi- mogenised in a 20% (w/v) aqueous sucrose solution nary data on the population structure and differentia- (a fixed volume of 5 µl sucrose solution for radular tion of L. obtusata and L. fabalis along a vertical in- muscles and a proportional volume of 5 µlsucrose tertidal transect in N.W. Spain. We particularly aimed solution per mg digestive gland tissue). Crude ho- at (1) assessing whether the zonation pattern of both mogenates were centrifuged for 30 min at 19 000×g species could be correlated with electrophoretic vari- and at 4 ◦C. Digestive gland supernates were directly ation in three protein markers for which a functional stored at –80 ◦C, radular muscle supernates were first relationship with habitat, feeding and diet might be as- further diluted by adding 4 µl sucrose solution per µl sumed, viz. esterases (EST), radular myoglobins (Mb) supernate. and general proteins (GP) (e.g. Berger et al., 1975; Horizontal IEF in 3–9 pH gradients for digestive Oxford, 1975, 1978; Alyakrinskaya, 1989, 1994; gland EST and 4–6.5 pH gradients for radular muscle Medeiros et al., this volume), (2) testing whether these proteins was performed as outlined by Medeiros et al. markers support the allozyme-based observation that (1998). EST were stained with the recipe of Backeljau L. obtusata is the genetically more variable of the et al. (1994). Mb/GP were revealed by Coomassie and two flat periwinkles (e.g. Janson, 1987b; Zaslavskaya silver staining as described by Medeiros et al. (1998) et al., 1992; Backeljau & Warmoes, 1992; Rolán- and Backeljau et al. (1994), respectively. Alvarez et al., 1995; Tatarenkov, 1995a), and (3) EST and Mb/GP patterns were analysed by means evaluating to what extent these markers can be used of band counting and calculating Jaccard’s match- to diagnose both species. ing coefficients (SJ) between adjacent protein profiles Because EST, Mb and GP are complex systems, (Backeljau et al., 1994). SJ values were used to con- requiring a high-resolution separation technique, we struct UPGMA trees with the program NTSYS (Rohlf, applied isoelectric focusing (IEF) as described by 1993). Mean numbers of protein bands per individ- Backeljau et al. (1994) and Medeiros et al. (1998). ual (NB) were calculated and tested for intra- and The latter authors were also the first to report on the interspecific differences with Kruskal-Wallis tests. In- presence of Mb in L. fabalis. traspecific band heterogeneity was assessed with the Shannon-Wiener index (H 0) as described by Mill & Grahame (1988, 1992). Mb profiles were also used for a population ge- netic treatment involving allele frequency estimation, Hardy-Weinberg (HW) equilibrium testing with a hy-r2.tex; 21/11/1998; 22:29; p.2 13 Table 1. Distribution of the Mb patterns (A–G) defined in Figure 3 OforL. obtusata (Figure 1B). However, some rarer over the algal belts in L. obtusata and L. fabalis. N = total number of specimens bands were also species-specific. The mean number of Mb/GP bands per individual was significantly larger Population N ABCDEFG in L. obtusata (NB = 22) than in L. fabalis (NB = 18) L. obtusata 68 2 54 7 1 1 2 1 (p<0.05), but within species there were no signifi- cant differences between algal levels or sexes. Because F. spiralis 20 1 18 1 – – – – 0 A. nodosum 21 1 19 – – 1 – – the Mb/GP profiles were too complex, no H values F. vesiculosus 19 – 12 6 – – 1 – were calculated. Yet, the mean SJ value between in- F. serratus 8– 5 –1–11 dividuals of L. fabalis (SJ = 0.61) was lower than the corresponding value for L. obtusata (SJ = 0.71). Once L. fabalis 40 5 30 5 – – – – again clustering of the interpopulation SJ values sep- F. vesiculosus 20 3 14 3 – – – – arated both flat periwinkles (Figure 2B). Hence the F. serratus 20 2 16 2 – – – – Mb/GP and EST results were highly consistent, even though they yielded different topologies for the L. Total 1087 84121121 obtusata populations (Figure 2A–B). The Mb profiles revealed five bands (a–e) and seven patterns (A–G) (Figure 3). Pattern D (one in- dividual of L. obtusata from F. serratus) consisted of a Fisher exact test corrected for multiple testing by the single band. All other profiles comprised two, three or sequential Bonferroni procedure (Rice, 1989), con- four bands (Figure 3).