Biological Journal of the Linnean Society (1998), 63: 349–365. With 6 figures

Evidence of a reproductive barrier between two forms of the marine periwinkle fabalis ()

ANDREY TATARENKOV1,2,3 AND KERSTIN JOHANNESSON1

1Tja¨rno¨ Marine Biological Laboratory, S-452 96 Stro¨mstad, Sweden 2Institute of Marine Biology, Vladivostok 690041, Russia

Received 12 April 1997; accepted for publication 18 September 1997

Studies of allozyme variation may reveal unexpected patterns of genetic variation which challenge earlier conclusions of delimitations based on morphological data. However, allozyme variation alone may not be sufficient to resolve this kind of problem. For example, populations of the marine intertidal snail Littorina fabalis (=Littorina mariae) from wave exposed parts and from protected parts of the same shores are distinguished by different alleles of arginine kinase (Ark) while indifferent, or very nearly so, in another 29 loci. Intermediate populations have large deficiencies of exposed/sheltered heterozygote classes of Ark and we have earlier suggested habitat-related selection in this locus as the explanation. In this study we estimated growth rate of individual snails of different Ark-genotypes in three different habitats (exposed, sheltered and intermediate). In all habitats the snails homozygous for alleles of ‘exposed’ type grew faster and matured at a larger size than did snails homozygous for alleles of ‘sheltered’ types. This relationship was indirectly confirmed in three additional sites of intermediate exposure where exposed Ark-genotypes dominated among large (>8 mm) snails while the sheltered genotypes dominated among small (<5 mm) snails of truly sympatric samples. We furthermore found small differences in allele frequencies of two other loci (Pgi and Pgm-2) and in shell colour frequencies, comparing sympatric snails of exposed and sheltered Ark-homozygotes. Although we found no signs of habitat-related selection among snails of different Ark-genotype, or selection against heterozygotes, we cannot reject selection in Ark, as our experiments only covered one island, one season and grown-up snails. The coupling between allozyme and phenotypic characters in strictly sympatric samples of snails suggests the presence of two gene pools. Perhaps the large and small forms of L. fabalis represent very closely related cryptic taxa. However, introgression between them seems a possible explanation for the striking similarities in the vast majority of morphological and allozyme characters.

 1998 The Linnean Society of London

ADDITIONAL KEY WORDS:—allozymes – arginine kinase – colour variation – cryptic species – growth rate – reproductive isolation – wave exposure.

3 Present address: Department of Ecology and Evolutionary Biology, University of California, Irvine, CA 92697, U.S.A. Correspondence to Kerstin Johannesson. E-mail: K. [email protected] 349 0024–4066/98/030349+17 $25.00/0/bj970184  1998 The Linnean Society of London 350 A. TATARENKOV AND K. JOHANNESSON

CONTENTS

Introduction ...... 350 Swedish populations of L. fabalis ...... 351 Material and methods ...... 352 Transplant experiment ...... 352 Ark-differences between small and large snails ...... 354 Variation in Pgi, Pep and Pgm between sheltered and exposed Ark-genotypes 354 Statistics ...... 354 Results ...... 355 Transplant experiment ...... 355 Ark-differences between small and large snails ...... 359 Variation in Pgi, Pep and Pgm between sheltered and exposed Ark-genotypes 359 Discussion ...... 360 Acknowledgements ...... 363 References ...... 364

INTRODUCTION

A number of marine gastropods show limited gene flow among populations because they lack pelagic larval development. As a consequence, direct developing gastropods often reveal high levels of among-population variation in phenotypic and allozymic characters (e.g. Janson & Ward, 1984; Day, 1990; Boulding, Buckland- Nicks & Van Alstyne, 1993; Johnson & Black, 1996; Reid, 1996). Eventually, the differentiation may become large enough to provoke reproductive barriers between conspecific populations ( Johannesson, Rola´n-Alvarez & Ekendahl, 1995a; Hull, Grahame & Mill, 1996; Day, Leinaas & Anstensrud, 1994). By adopting the biological species concept, as defined by Mayr (1963), many of us use gene flow estimates to try to define species boundaries. Molecular techniques have rightly proven useful to disentangle the genetic relatedness of populations in taxa which are either morphologically very similar (see Knowlton, 1993) or have unusually high levels of intraspecific morphological variation (e.g. Janson & Ward, 1984; Johannesson & Johannesson, 1990a and b). However, Ehrlich & Raven (1969) argue that the pattern of selection, rather than the amount of neutral genes flowing among populations of a species, delimits the units of evolution. They give three categories of observations in support of this: (i) restricted gene flow among populations in many species; (ii) isolated populations of some species showing little differentiation; and (iii) populations of other species joined by high gene flow sometimes showing marked genetic differentiation. Templeton (1989) supports this view and adds another evolutionary force—genetic drift—being a possible way of driving new mutations to fixation, which is, according to him, ‘the genetic essence’ of an evolutionary lineage. Thus according to Templeton (1989), gene flow, selection and genetic drift are all important microevolutionary mechanisms which may be used to define species boundaries. In this study we report a case in which estimates of gene flow are not enough to answer the question of species delimitations. Although a coupling between a phenotypic and an allozyme character does indicate a reproductive barrier between two groups of the marine snail Littorina fabalis, we need to understand much more about the nature and role of the barrier before we will reach a taxonomic conclusion. Littorina fabalis is a European winkle confined to the macroalgal zone of intertidal fucoids. It was described as a separate species under the name Littorina mariae by A REPRODUCTIVE BARRIER BETWEEN TWO FORMS OF LITTORINA 351 Sacchi and Rastelli (1966), and has since mostly been considered a good taxonomic unit. There is, however, a long systematic history involved and this led Reid (1996) to conclude that the name L. fabalis has preference over the junior synonym L. mariae. There have also been some concerns about the taxonomic relationship between some morphologically divergent populations of L. fabalis. Thus, for example, Rola´n & Templado (1987) describe three forms of L. fabalis from the coast of Galicia (Spain). One, which they name ‘L. fabalis’, is found in along estuarine coastlines. Two deviating forms, one found in wave-exposed sites among the red- alga Gigartina stellata (‘A’), and the other present in protected lagoons among Zostera marina (‘B’). Despite marked differences in size, all three forms are considered conspecifics (Rola´n & Templado, 1987), and this is supported for two of them using information on allozyme variation (Rola´n-Alvarez, 1993). Reimchen (1981) describes what he names a ‘dwarf’ and a ‘large’ form of L. fabalis in Wales. These two forms differ in size of maturity (dwarf form 5–8 mm and large form 11–14 mm), frequencies of colour varieties, micro-ornamentation of the shell, and microhabitat distribution (the dwarf form being most common in sheltered microhabitats and the large form in exposed habitats). Reimchen (1981) suggests that these two forms should be treated as separate entities but hesitates to consider their taxonomic status as they intergrade in all characters in intermediate mi- crohabitats.

Swedish populations of L. fabalis

The distribution of L. fabalis in Sweden is somewhat different from that found in tidal areas (e.g. UK). Instead of being confined to low tidal levels, and living mainly on Fucus serratus, it is found throughout the eulittoral zone on the substratum or in the canopy of F. serratus, F. vesiculosus, nodosum and F. spiralis. It prefers moderately exposed shores and is replaced by L. obtusata in areas of decreasing wave-action. Still L. fabalis spans a certain range of wave exposure, from populations in moderately sheltered sites characterized by the presence of the macroalga Ascophyllum nodosum to populations in moderately exposed sites lacking Ascophyllum (this alga is an indicator of wave-protected sites; Lewis, 1972). In comparing allozyme frequencies between sheltered and exposed sites (defined by the presence/absence of Ascophyllum), we have earlier found that in 30 enzyme loci both groups of populations are monomorphic for identical alleles in 22 loci. Among the polymorphic loci, two show habitat-related variation. In one locus (Pgi), these differences are small in some areas and absent in others. In arginine kinase (Ark), however, huge systematic differences occur between sheltered and exposed Swedish sites, sometimes only a few tens of meters apart (Tatarenkov & Johannesson, 1994). The same differences are also found between exposed and sheltered sites in Wales and in France, but not in Spain (Tatarenkov & Johannesson, unpubl.) Six alleles are present in the Ark-locus, and three of these Ark130 (rare), Ark120 (common), Ark110 (rare) are typical of sheltered sites (pooled frequencies around 0.7–0.9 in nine sheltered Swedish sites, Tatarenkov & Johannesson, 1994), while the alleles Ark100 and Ark80 (both common) characterize exposed populations (pooled frequencies around 0.9 in 12 exposed sites, Tatarenkov & Johannesson, 1994). One very rare allele, Ark70 is found both in a few exposed and a few sheltered sites. In sites of intermediate wave exposure both sheltered and exposed alleles are 352 A. TATARENKOV AND K. JOHANNESSON present. Here we have found pronounced deficiencies of sheltered/exposed hetero- zygotes (e.g. Ark120/ Ark100 and Ark120/ Ark80; Tatarenkov & Johannesson, 1994). In spite of the heterozygote deficiency, the variation in Ark is smoothly clinal over gradual environmental transitions (Tatarenkov & Johannesson, unpubl.). Although we discussed the possibility of two reproductively isolated taxa which would easily explain the strong deficiencies of sheltered/exposed heterozygotes in Ark, we earlier suggested that the differentiation in Ark was due to strong habitat- related selection (Tatarenkov & Johannesson, 1994). The reason we believed this was the essential lack of differentiation in all the other loci examined. With the results of the present study we have evidence of a strong coupling between growth rate or size, and Ark-genotype, in snails of sympatric sites. Furthermore, we found slight differentiation between sheltered and exposed Ark-homozygotes in colour frequencies and in allozyme frequencies of two additional polymorphic loci. The new results strongly supports an alternative explanation to the observed deficiency of Ark-heterozygotes; the presence of two, although possibly communicating, gene pools.

MATERIAL AND METHODS

Transplant experiment

To assess growth rate and survival in snails of different Ark-genotype in different microhabitats we sampled and transplanted snails along a boulder shore of a small island (SE shore of Lo¨kholmen, Fig. 1). This boulder shore is about 150 m long and its north-eastern parts are less affected by wave action than are the south-western parts. The brown macroalgae present in the sheltered parts are Fucus spiralis, F. vesiculosus, F. serratus and Ascophyllum nodosum, while in the exposed parts only the Fucus species are present. Because we had already mapped the allozyme cline of this boulder shore at intervals of 20 m (Tatarenkov & Johannesson, unpubl.) we were able to choose a site with roughly equal proportions of sheltered (Ark110, Ark120 and Ark130) and exposed alleles (Ark80 and Ark100). This site was intermediately exposed to wave action as judged from microtopography and the distribution of Ascophyllum, and situated in the middle of the boulder shore (Fig. 1). From a small area (3×3 m) in this intermediate site we picked 771 snails ([2 mm) which we divided in four groups; one of 111 snails (a control group) and three of 220 snails each. We immediately froze the control group, while all snails of the three other groups were given a band of enamel paint along the outside of the aperture. All painted snails were placed in aquaria over 2 days to monitor survival and paint adherence and they all appeared vital after the laboratory treatment. Four days after sampling (on 17 May 1995) the three painted groups were released on the boulder shore, one in the most exposed part of the boulder shore, another in the most protected part, and the third in the sampling area, the intermediate site (Fig. 1). After 38–39 days we recaptured as many of the released snails as possible over several hours of intensive search in 2 days (24–25 June 1995). We measured the distance from the release spot that each snail had moved during the experiment. In the laboratory we measured shell size (we used the distance from the outer aperture A REPRODUCTIVE BARRIER BETWEEN TWO FORMS OF LITTORINA 353

Figure 1. Map of sampling sites in two small islands (Lo¨ckholmen and La˚ngholmen), SW Stro¨mstad (Swedish West Coast). Grey bands indicate the distribution of Littorina fabalis along the shores of the islands. Snails were picked at an intermediately wave-exposed site (open square) and transplanted to one sheltered ‘S’ one exposed ‘E’ and the intermediate site in Lo¨kholmen. Black squares show four intermediate sites from which sympatric samples of small (<5 mm) and large (>8 mm) snails were taken.

lip to the opposing edge of the last whorl as a measure of size) and shell increment of unpainted shell (both with an accuracy of 0.1 mm). We furthermore recorded the shell colour of each recaptured snail and the genotype of both control and recaptured snails at four polymorphic loci (arginine kinase, Ark; peptidase, Pep-1, phosphoglucose isomerase, Pgi; and phosphoglucomutase, Pgm-2) using the same methods of starch gel electrophoresis as described in Tatarenkov & Johannesson (1994). In accordance with earlier results (Tatarenkov & Johannesson, 1994) we dis- tinguished between alleles typically found in sheltered habitats and those characteristic of exposed habitats. Two compound alleles were designated, the sheltered (S-) allele including Ark130 (rare), Ark120 (common), Ark110 (rare), and the exposed (E-) allele including Ark80 (common), and Ark100 (common). One very rare allele (Ark70) could not be characterized as sheltered or exposed, and two snails heterozygous for this allele were excluded. Heterozygotes between the sheltered and exposed compound alleles were designated SE. 354 A. TATARENKOV AND K. JOHANNESSON We used the compound genotypes, SS, SE and EE, when looking for relationships between genotype and size, colour, growth rate, dispersal and survival. Snails of an additional sample picked in July 1995 at the intermediate site in Lo¨kholmen were sexed and their sexual status (mature or immature) and genotypes were recorded. This sample was not transplanted but used to assess the distribution of mature and immature individuals among different size classes of the three compound genotypes.

Ark-differences between small and large snails

Four samples from different shores of two islands were taken from intermediately exposed sites (Fig. 1). These samples included sympatric snails of SS and EE homozygotes and we used them to look for associations between size and Ark- genotype. We compared Ark-genotype distributions of two size classes of snails; small (<5 mm) and large (>8 mm). To assure a sympatric microdistribution, snails were taken from within 1–2 m2 in each site.

Variation in Pgi, Pep and Pgm between sheltered and exposed Ark-genotypes

Genetic associations between Ark-genotype and allele frequencies of Pgi, Pep-1, and Pgm-2 were assessed in five samples of L. fabalis picked at different dates (August 1993, September 1994, March 1995, May 1995 and July 1995) in the intermediate area of the south shore of Lo¨kholmen. Each sample was divided into one group of exposed Ark-homozygotes (EE) and one of sheltered Ark-homozygotes (SS), the heterozygotes (SE) were discarded. Allele frequencies of Pgi, Pep-1, and Pgm-2 were compared between the two groups of each sample, as well as between pooled groups of EE and SS homozygotes. All five samples were from the same 4×40 m area of shore.

Statistics

Genotype distributions of Ark within each sample were compared with Hardy- Weinberg distributions using Selander’s D (D=(Hobs−Hexp)/Hexp) (Selander, 1970); negative values of D indicating a deficiency of heterozygotes. In this comparison we used the pooled genotypes of Ark (SS, SE and EE). G-test (Sokal & Rohlf, 1995) statistics was applied to assess significant deviations from the expected distributions. Heterogeneity among samples was tested using a pseudoprobability test (Herna´ndez & Weir, 1989) with the program CHIHW (Zaykin & Pudovkin, 1993). This test avoids problems of small expected numbers. In cases of only two samples and two alleles we used Fischer’s exact test (Sokal & Rohlf, 1995). If the same hypotheses were tested repeatedly we used the sequential Bonferroni correction (Rice, 1989) to correct the P-values for multiple testing. A REPRODUCTIVE BARRIER BETWEEN TWO FORMS OF LITTORINA 355

0.9 0.8 0.7 0.6 0.5 0.4 * * 0.3 Allele frequency 0.2 0.1 0 Ark-S Pgi-100 Pep-100 Pgm-100

Figure 2. Allele frequencies of four polymorphic loci in three transplanted and one control groups of Littorina fabalis. All snails were originally from the intermediately exposed site in Lo¨kholmen (see Fig. 1). The transplanted groups were marked and released in exposed (Φ), sheltered (∆) and intermediate (Γ) sites, and recaptured after 38–39 days. The exposed and intermediate groups differed from the control group (Ε) in frequencies of the S-allele (see text) of Ark (P<0.05).

RESULTS

Transplant experiment

Allele and genotype distributions of recaptured snails After 38–39 days, we recaptured 112 snails (59%) in the intermediate site, 82 (37%) in the exposed and 89 (37%) in the sheltered site. We found no differences in allele frequencies in the three loci Pep-1, Pgi and Pgm-2 among any of the recaptured samples and the control sample. In Ark, the three samples recaptured in different habitats did not differ from each other (v2-test, P=0.95; Fig. 2). The two samples recaptured in the exposed and the intermediate sites had lower frequencies of the sheltered compound allele (S) than the control sample (v2-test, P=0.02 in both), while the sample recaptured in the sheltered site did not differ from the control (v2-test, P=0.20) (Fig. 2). The decrease was due to a lower number of Ark120/ Ark120 homozygotes in the recaptured samples (23%, all samples pooled) compared to the control sample (37%) (Fig. 3). Thus we found some evidence of differential survival of different genotypes but no suggestion of habitat-related selection or of heterozygotes being less fit than any of the homozygotes.

Size and growth of different Ark-genotypes As all the recaptured snails originally came from the same intermediate site, and as they did not differ significantly at any of the four loci, we pooled all the recaptured snails to look for associations between shell increment over 38–39 days and Ark- genotype, and between size and Ark-genotype. We used the sheltered (S) and exposed (E) compound alleles and the corresponding genotypes SS, SE and EE. Growth rates ranged from no growth at all to an increment of the last whorl of more than 10 mm over the experimental period. Most individuals belonged to one of two categories; a group of slow or non-growing snails (increment <2 mm), or a group of fast growing snails (increment >5 mm) (Fig. 4). Nearly all of the SS homozygotes 356 A. TATARENKOV AND K. JOHANNESSON

40 35 30 25 20 15 10

Genotype proportion (%) 5 0 120/120 120/110 120/100 120/80 100/100 100/80 80/80 Others SS-genotypes SE-genotypes EE-genotypes

Figure 3. Genotype frequencies in the three recaptured groups (Φ)ofLittorina fabalis pooled (see Fig. 2 and text) in comparison with frequencies of the control group (Ε).

were slow growing, while the EE homozygotes were either slow or fast growing. The heterozygotes also included both categories, although the majority were slow growing (Fig. 4). The difference between the two homozygote classes is particularly interesting when snails less than 10 mm in size are compared. Among these nearly all SS grew slowly while nearly all EE grew rapidly (Fig. 4). Another difference between the two groups of homozygotes was that no SS homozygotes were larger than about 12 mm, while the maximum size of the EE homozygotes was about 14 mm (Fig. 4). Size distribution of mature and immature snails sampled in July in the intermediate locality at Lo¨kholmen revealed that among the small sheltered homozygotes nearly all were sexually mature. Among the exposed homozygotes, on the other hand, there were two size classes, one immature class of similar sizes to the sheltered homozygotes, and one mature class of larger snails (Fig. 5). Comparing Figures 4 and 5 suggests that the slow growing snails of Figure 4 are sexually mature while the fast growing ones are immature.

Dispersal The recaptured snails had only moved rather short distances in the 38–39 days, on average 1–2 m. The average distance was slightly longer in the intermediate site compared to the exposed site, and still longer in the sheltered site (Table 1, differences between means are all significant, t-test, P <0.05). The distance moved by snails of SS, SE and EE genotypes of Ark did not differ in the exposed and intermediate sites, while in the sheltered site SS snails moved on average further than EE (P <0.05), and heterozygotes moved much less than both homozygotes (P <0.05 in both, Table 1).

Shell colour Shells of L. fabalis were either brown, yellow, banded in brown and yellow, or grey. The banded and grey forms were, however, rare (2%). We compared the frequencies of brown and yellow among snails of exposed (EE) and sheltered (SS) Ark-genotypes of all the recaptured snails. Yellow was the most frequent colour but A REPRODUCTIVE BARRIER BETWEEN TWO FORMS OF LITTORINA 357

12 SS

10

8

6

4 Shell increment (mm) 2

0 246810121614

12 EE

10

8

6

4 Shell increment (mm) 2

0 246810121614

12 SE

10

8

6

4 Shell increment (mm) 2

0 246810121614 Shell size (mm)

Figure 4. Shell increment of the last whorl, reflecting growth rate, of Littorina fabalis of different Ark- genotypes. ‘SS’ indicates individuals homozygote for alleles which dominate sheltered sites, ‘EE’ indicates exposed homozygotes, and ‘SE’ heterozygotes of one sheltered and one exposed allele. All the recaptured snails have been pooled in this figure (see Fig. 2). 358 A. TATARENKOV AND K. JOHANNESSON

25 Males

20

15

10

5

0 –7–8 –9 –10 –11 –12 –13 –14 –15 25 Females No. of individuals No. 20

15

10

5

0 –7–8 –9 –10 –11 –12 –13 –14 –15 Size class (mm)

Figure 5. Size distribution of mature and immature Littorina fabalis homozygote for the sheltered or the exposed Ark-alleles. All snails were from one sample picked at the intermediately exposed site on Lo¨khomen (see Fig. 1). Only upper range values of each size class are indicated. ‘SS’-mature (Φ); ‘SS’-immature (∆); ‘EE’-mature (Ε); ‘EE’-immature (C).

T 1. Mean migration distances over 38–39 days for sheltered (SS), heterozygote (SE) and exposed (EE) Ark-genotypes of L. fabalis released in three different microhabitats (exposed, intermediate and sheltered) in Lo¨kholmen (see Fig. 1). All snails were from the intermediate site in Lo¨kholmen Genotype: SS1 SE EE Mean N dist.±SE N dist.±SE N dist.±SE dist.±SE (m) (m) (m) (m)

Site: Exposed 15 1.0±0.2 14 1.4±0.3 47 1.1±0.1 1.1±0.1 Intermediate 21 1.3±0.1 21 1.6±0.2 61 1.4±0.1 1.4±0.1 Sheltered 23 2.1±0.1 5 0.7±0.1 47 1.6±0.1 1.7±0.1

1 Two additional snails (both of SS genotype) were found in the intermediate site, one after 55 days at a distance of 10.8 m, and the other after 59 days at a distance of 8.9 m. These are not included in the table. A REPRODUCTIVE BARRIER BETWEEN TWO FORMS OF LITTORINA 359

T 2. Allele frequencies in four polymorphic loci of sympatric samples of small and large L. fabalis from four localities (see Fig. 1). Each pair of samples as well as the combined samples were tested for heterogeneity with a pseudo-probability test (Zaykin & Pudovkin, 1993) or, in case of two alleles, with Fisher’s exact test. Significant P-values are shown in bold; one value non-significant when corrected for multiple testing is indicated.

South La˚ngholmen North La˚ngholmen South Lo¨kholmen North Lo¨kholmen Combined Locus small large small large small large small large P

Ark 47 38 36 36 41 41 40 40 N 130 0.000 0.000 0.000 0.000 0.037 0.000 0.050 0.000 120 0.809 0.250 0.889 0.278 0.744 0.256 0.825 0.188 110 0.043 0.013 0.028 0.014 0.061 0.037 0.050 0.000 100 0.085 0.592 0.083 0.583 0.122 0.561 0.075 0.688 80 0.032 0.145 0.000 0.097 0.037 0.146 0.000 0.125 70 0.032 0.000 0.000 0.028 0.000 0.000 0.000 0.000 P 0.000 0.000 0.000 0.000 0.000 Pgi 47 38 36 36 41 41 40 40 N 100 0.745 0.789 0.708 0.819 0.756 0.841 0.775 0.813 93 0.255 0.211 0.292 0.181 0.232 0.159 0.225 0.188 85 0.000 0.000 0.000 0.000 0.012 0.000 0.000 0.000 P 0.59 0.17 0.24 0.70 0.42 Pep-1 47 38 22 22 27 27 26 26 N 100 0.819 0.750 0.932 0.773 0.796 0.870 0.865 0.885 80 0.181 0.250 0.068 0.227 0.204 0.130 0.135 0.115 P 0.34 0.07 0.44 1.00 0.33 Pgm-2 46 38 35 36 41 41 40 40 N 100 0.565 0.737 0.800 0.778 0.598 0.659 0.600 0.512 93 0.424 0.250 0.200 0.222 0.402 0.341 0.400 0.488 87 0.011 0.013 0.000 0.000 0.000 0.000 0.000 0.000 P 0.021 0.84 0.52 0.32 0.16

1 Non-significant if corrected for multiple testing with the sequential Bonferroni correction (Rice, 1989).

it was less frequent among snails of sheltered genotype (62% yellow) compared to exposed genotype (79% yellow; v2=6.9, df=1, P <0.01).

Ark-differences between small and large snails

Frequencies of Ark-alleles differed substantially between small (<5 mm) and large (>8 mm) individuals of all the four samples picked in intermediately exposed sites. The sheltered allele (Ark120) was much more frequent among the small snails (mean 0.82 of the four samples) than among the large snails (mean 0.24) (Table 2). No differences were found among small and large snails in the other three loci (Table 2). The strong coupling between Ark-genotype and snail size caused heavy deficiencies of heterozygotes (SE) in the four samples, while less so if the samples were divided into subsamples of small and large snails (Table 3). Similar levels of deficiency in the groups of small and large snails suggested heterozygotes larger than 5 mm being as viable as homozygotes of the same size. 360 A. TATARENKOV AND K. JOHANNESSON

T 3. Heterozygote deficiency of Ark in samples of small and large individuals of L. fabalis from four sites on two islands (see Fig. 1). Deficiencies are indicated by negative values of Selander’s D (Selander, 1970) and deviations from Hardy-Weinberg expected distributions are assessed by a G-test (Sokal & Rohlf, 1981); ∗P <0.05; ∗∗P <0.01; ∗∗∗P <0.001

Lo¨kholmen La˚ngholmen South shore North shore South shore North shore

Small individuals; Selander’s D −0.360 −0.352 −0.689 −0.634 Result of G-test 4.15∗ 2.05 10.59∗∗ 5.61∗ Large individuals; Selander’s D −0.293 −0.640 −0.593 −0.139 Result of G-test 4.90∗ 12.19∗∗∗ 13.10∗∗∗ 1.42 Small and large; Selander’s D −0.528 −0.790 −0.775 −0.560 Result of G-test 24.47∗∗∗ 40.64∗∗∗ 46.7∗∗∗ 21.20∗∗∗

Variation in Pgi, Pep and Pgm between sheltered and exposed Ark-genotypes The five samples from the intermediate area on Lo¨kholmen sampled on different occasions were all in Hardy–Weinberg equilibrium in the three polymorphic loci Pgi, Pep-1, and Pgm-2. There was furthermore no significant heterogeneity among samples in these three loci, although we found significant differences among samples in Ark. We separated the homozygotes of sheltered (SS) and exposed (EE) Ark- genotype and compared the allele frequencies of the other three polymorphic loci between the homozygote groups. We found significant differences in Pgi both in two of the five individual samples and in the pooled sample. Part of the difference was caused by the allele Pgi85 being present in all five samples of SS snails while absent in all samples of EE snails (Table 4). Also the allele frequencies of Pgm-2 differed between exposed and sheltered homozygotes in one sample and in the pooled sample. In Pep-1, however, we found no signs of differences related to Ark- genotype (Table 4).

DISCUSSION

The exposed and sheltered forms of Littorina fabalis are morphologically very similar, and we have not found any character which is diagnostic between them. Apart from the marked differences in Ark-allele frequencies and growth rate, all other differences are more or less subtle. Only with careful comparisons between large truly sympatric samples were the differences in colour frequencies, Pgi and Pgm-2 allele frequencies, and migratory behaviour between the two homozygote classes of the intermediate site in Lo¨kholmen discovered. Despite the extremely small differences we argue that there is a gene-flow barrier between the exposed and sheltered forms in strictly sympatric sites. There is a strong correlation between growth and Ark-genotype; the fact that SS-homozygotes cease growing and become sexually mature at a much smaller size than do EE-homozygotes (Figs 4 & 5) resulting in high frequencies of S-alleles (Ark110,120,130) among small and of E-alleles (Ark80,100) among large snails in samples from intermediate sites (Table 2), could not easily be explained otherwise. Likewise, the small differences in colour A REPRODUCTIVE BARRIER BETWEEN TWO FORMS OF LITTORINA 361 0.001 from the intermediary wave-exposed -values are shown in bold and values P L. fabalis -genotype Ark 0.005 0.005 0.000 erent occasions. Each pair of samples as well as the combined samples were tested for heterogeneity ff -2 in groups of exposed (EE) and sheltered (SS) non-significant when corrected for multiple testing are indicated Pgm 0.84 0.66 0.07 0.16 -1 and Pep , Pgi 1 0.160.39 0.56 0.51 0.26 0.39 0.14 0.17 0.32 0.026 August 1993 Sept. 1994 March 1995 May 1995 July 1995 Groups pooled SS EE SS EE SS EE SS EE SS EE SS EE kholmen. The samples were picked at five di ¨ 4. Allele frequencies of -2  Non-significant if corrected for multiple testing with the sequential Bonferroni correction (Rice, 1989). T Pgi N1009385P 0.809 34 0.162 0.029 0.841 66 0.159 0.000 0.778 0.204 0.019 27 0.846 0.154 0.000 0.7341 26 0.252 0.014 0.824 0.176 111 0.000 0.798 0.177 34 0.025 0.822 0.177 0.000 0.715 99 0.279 0.006 0.833 200 0.167 0.000 0.767 0.215 86 0.018 0.833 0.167 0.000 123 357 449 Pep-1 N10080P Pgm 0.769 26N 0.231100 0.83693P 55 0.164 0.794 0.809 34 0.206 0.191 17 0.875 0.644 0.125 66 0.356 0.814 16 0.648 0.186 0.352 0.765 27 0.615 0.235 110 0.385 0.770 0.694 26 0.230 0.306 34 0.832 0.662 0.168 0.338 108 0.872 63 0.684 0.128 0.316 34 0.821 0.606 152 0.179 0.394 0.804 0.659 98 0.196 0.341 86 0.826 0.585 0.174 199 0.415 123 0.699 0.301 85 0.622 302 0.378 123 380 352 448 with a pseudo-probability test (Zaykin & Pudovkin, 1993) or, in case of two alleles, with Fisher’s exact test. Significant site in Lo 362 A. TATARENKOV AND K. JOHANNESSON frequencies, Pgi and Pgm allele-frequencies and migratory behaviour between the two homozygote classes maintained in microsympatry suggest distinct gene pools. The pronounced deficiencies of SE-heterozygote individuals in most sites, and in particularly in the intermediate sites, may obviously be a consequence of this reproductive barrier. In an earlier study, however, we suggested the deficiency being due to selection against heterozygotes (Tatarenkov & Johannesson, 1994). In this study we found no suggestion of heterozygotes being less viable than homozygotes among the adult snails (Table 3, Fig. 3). However, selection may take place at other seasons of the year, or at juvenile and embryonic stages. Possibly selection contributes, but the major part of the heterozygote deficiency seems more readily explained by the impeded gene-flow. A further topic of concern is the dramatic habitat-related differentiation in Ark- allele frequencies. Earlier we suggested this was a result of strong selection, referring to the fact that the variation in this locus is in sharp contrast to that of the other polymorphic loci (Tatarenkov & Johannesson, 1994). Despite the fact that we now have more data, it is not yet possible to reject selected or neutral variation. Among the transplanted snails we found no signs of habitat-related survival of snails of different Ark-genotype but selection may be seasonal or take place at early post- settlement stages. It seems possible that the differentiation in Ark is, at least in part, a result of habitat-related selection, otherwise the genetic structure of this locus would not have contrasted that clearly against the others. Indeed, the situation in Ark is superficially similar to what is found in a related species, L. saxatilis.In that species, strong habitat-related selection is present in one locus (aspartate aminotransferase, Aat) ( Johannesson, Johannesson & Lundgren, 1995b), generating steep genetic clines between two microhabitats only meters apart ( Johannesson & Johannesson, 1989). Further indications of varying degrees of microhabitat-related (presumably selected) variation are found in a few other loci in this species ( Johan- nesson & Tatarenkov, 1997). To test the possibility of selection in Ark is, unfortunately, difficult because we are hampered by the fact that we use the Ark alleles as markers of the two gene-pools and thus cannot assess the variation in Ark within each group. Until we find an independent marker, any difference found among the Ark-homozygote classes is likely to be due to differentiation in other characters. The loss of sheltered alleles during the transplant experiment could, for example, be explained as size selection (favouring large sizes) rather than selection against sheltered alleles. The loss of small snails may either reflect a bias in the recapture of different sizes or, size-specific viabilities during the experimental period. The presence of a reproductive barrier between an exposed and a sheltered form of L. fabalis is surprising although not unique for this genus. Recent findings have revealed gene-flow barriers also in L. saxatilis. Ecotypes of different forms of this species overlap in narrow transitional zones where reproductive barriers have been observed ( Johannesson, Johannesson & Rola´n-Alvarez, 1993; Hull et al., 1996). In Galician L. saxatilis, for example, there is a partial reproductive barrier between an upper and a lower rocky-shore ecotype due to strong assortative mating within the zone of overlap ( Johannesson et al., 1995a). There are, however, important differences between the Galician L. saxatilis and the L. fabalis groups of the present study. One notable difference is that the two ecotypes of L. saxatilis differ in a number of phenotypic characters many of which are evidently partly inherited (e.g. shell size, shell form, colour, physiological tolerance, behaviour, embryo characters and radula) A REPRODUCTIVE BARRIER BETWEEN TWO FORMS OF LITTORINA 363 ( Johannesson et al., 1993; Rola´n-Alvarez, Rola´n & Johannesson, 1996) while the two L. fabalis groups show a remarkable phenotypic similarity. In contrast, there is no allozyme differentiation between the Galician L. saxatilis forms if populations at a scale of kilometers are compared ( Johannesson et al., 1993), while there is a dramatic divergence between exposed and sheltered habitats in the Ark-loci of L. fabalis which is consistent over sites in Sweden (Tatarenkov & Johannesson, 1994), France and Wales (Tatarenkov & Johannesson, unpubl.). One important difference between the intraspecific structures of L. saxatilis and L. fabalis is that although both have reproductive barriers and a genetic divergence between their forms, the characters which are correlated in microsympatric samples of L. saxatilis are mor- phological characters while in L. fabalis, allozyme characters are involved. The reproductive barrier in Galician L. saxatilis is maintained by non-random mating ( Johannesson et al., 1995a). We have investigated this possibility in L. fabalis but found no signs of assortative mating between the homozygote classes of arginine kinase at two different localities ( Johannesson & Tatarenkov, unpubl.), although this does not exclude the possibility of such behaviours other times of the year. Unfortunately, with our present data we cannot assess the extent of the reproductive barrier in L fabalis. The admittedly small differences in Pgi and Pgm-2, as well as what seems to be random mating, suggests introgression between the groups. Past or present introgression is also supported by the fact that the variation in Pgi, Pep- 1 and Pgm-2 over populations from Sweden, Wales, France and Spain do not group exposed and sheltered populations but rather group populations according to their geographic relatedness (Tatarenkov & Johannesson, unpubl.). Our evidence of a reproductive barrier in L. fabalis is restricted to a small area in Sweden but results from earlier studies of Reimchen (1981 and 1982) suggest a similar phenomenon in Britain. He describes increasing average shell sizes from protected to exposed shores in L. fabalis from Ireland and Britain (Reimchen, 1982) as well as from micro-habitats of different exposures on Welsh shores (Reimchen, 1981). In the latter study he also describes differences between the small and large adults in other morphological traits, for example shell colour and micro-sculpturing of the periostracum. Despite intergrading characters in sites of intermediate exposure he suggests that these groups should be treated separately, possibly as distinct taxa. Although frustrating for the taxonomist and challenging for the evolutionary biologist, the existence of reproductive barriers in L. fabalis, as well as in L. saxatilis, is not all that surprising. Two species of the genus Littorina entered the Atlantic from the Pacific after the opening of the Bering Strait 3.5 to 4 Mya (Reid, 1990). The one that dispersed poorly due to direct development has now evolved into what is presently thought to be five distinct species, two of which are L. saxatilis and L. fabalis. The clade with planktonic larvae still remains a single species (L. littorea) (Reid, 1996). The combination of poor dispersal and a heterogeneous habitat (the intertidal) certainly promotes divergence. Indeed, the formation of reproductive barriers within L. fabalis and L. saxatilis is evidence for a continuing differentiation within this clade which sooner or later will lead to the formation of new species. Obviously, much additional work remains before we will be able to draw final conclusions about the evolutionary structure of L. fabalis. Estimating the variation in allozyme and phenotypic characters in widely spaced areas is required, as is the inclusion of neutral DNA-markers which will be used to assess gene-flow without inferring from selection. Furthermore, cross-breeding exposed and sheltered forms is probably required to estimate the strength of the reproductive barrier. 364 A. TATARENKOV AND K. JOHANNESSON

ACKNOWLEDGEMENTS

Both our families helped in the search for marked snails, D. Reid and one anonymous referee made a number of useful comments without which this paper would not have been publishable, and we wish to thank them all. Financial support was given from the Royal Swedish Academy of Sciences, the Swedish Institute and the Swedish Natural Science Research Council without which this work would not have been performed.

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