Incipient reproductive isolation between two sympatric morphs of the intertidal snail Littorina saxatilis.
Received December 14, 1993. Accepted March 3, 1994.
Speciation is considered by many authors a key to understanding the evolutionary process (Templeton 1981; Sluys 1991; Coyne 1992). However, the study of speciation presents impressive constraints to experimental work, because of the complexity of the material as well as the time scale involved (Endler 1977; Barton and Charlesworth 1984). This explains in part, why there still is no consensus about the species concept or the evolutionary process that causes speciation (e.g., Otte and Endler 1989). The biological species concept (BSC) states that a species represents a reproductively isolated unit (e.g., Barton and Hewitt 1985) and is the theory accepted by most evolutionists (Coyne 1992; for alternatives, see Otte and Endler 1989 and Sluys 1991). The BSC is sometimes interpreted in slightly different ways, contributing to the confusion surrounding this topic (e.g., Barton and Hewitt 1985, 1989). In this study and an earlier one (Johannesson et al. 1993), we followed the BSC, assuming that evidence of gene flow or potential gene flow between populations shows that they are conspecifics.
Mechanisms of speciation are likewise much debated, probably because they are closely related to species concepts (Barton and Charlesworth 1984; Templeton 1989; Coyne 1992). Following the BSC most evolutionists define speciation as the evolution of reproductive isolation (Coyne 1992). A universally accepted mechanism of speciation is allopatric speciation (White 1978; Littlejohn and Watson 1985) or adaptive divergence (sensu Templeton 1981). In allopatric speciation, reproductive isolation evolves mainly as a by-product of genetic isolation, caused by any extrinsic barrier between two taxa. Nevertheless, the classical interpretation assumes that natural selection can assist speciation directly, increasing prezygotic isolation by reinforcement (sensu Loftus-Hills and Littlejohn 1992) if, during secondary contact, both morphs present some postzygotic isolation and hybrids between them are less fit than parental taxa (Barton and Hewitt 1985; Butlin 1987; Loftus-Hills and Littlejohn 1992). However, as Butlin (1987) noted, reinforcement can support speciation only if postzygotic isolation is not already complete between incipient species. The reinforcement hypothesis is also involved in other speciation mechanisms, for example, sympatric speciation (Barton and Hewitt 1985; Butlin 1987) or habitat divergence (sensu Templeton 1981). However, true reinforcement is difficult to distinguish from enhanced reproductive isolation due to pleiotropic effects of selection on other characters (Templeton 1981; Butlin 1987 and 1989).
To test reinforcement and related hypotheses, it is of interest to find examples in nature in which conspecific morphs that still exchange genes show some degree of prezygotic reproductive isolation. These situations, however, are expected to be rare (Butlin 1987 and 1989). Only a few examples have been proposed as possible candidates of speciation events mediated by reinforcement (Barton and Hewitt 1985; Littlejohn and Watson 1985; Butlin 1987). We present below a natural example in which two sympatric morphs, which still maintain an important gene flux between them, have developed an incipient reproductive isolation.
Mating is usually one of the main behaviors capable of producing prezygotic reproductive isolation between incipient species (Spieth and Ringo 1983; Coyne 1992). Mating behavior mechanisms that may contribute to reproductive isolation can be partitioned in three independent components: relative mating success for males (male sexual selection), relative mating success for females (female sexual selection), and assortative mating (sexual isolation). In this study, we consider sexual selection as one of several selection components acting on any organism during its lifetime. Sexual selection is then the component that can be observed during mating within a species (O'Donald 1980; Hedrick and Murray 1983; Endler 1986; Hartl and Clark 1989). This approach is consistent with Darwin's definition of sexual selection (O'Donald 1980), and it does not have the problems associated with other definitions (see Zahavi 1991). However, the most efficient and most frequently observed component of mating behavior contributing to reproductive isolation is sexual isolation (Spieth and Ringo 1983; Gilbert and Starmer 1985; Marin 1991).
The three mating components are frequently used to study mate choice and sexual selection within species but may also be applied to situations of incipient species (Spieth and Ringo 1983). However, these mating components are difficult to observe in nature (Endler 1986), especially between incipient species (Littlejohn and Watson 1985; Butlin 1987, 1989). Therefore, most studies have either used indirect indexes of the components or have not estimated the three components simultaneously (see, e.g., Barton and Hewitt 1985; Littlejohn and Watson 1985; Endler 1986; Butlin 1987). We present below an example where all three components were measured directly between sympatric morphs of a natural population of snail. The results suggest female sexual selection and support the theory that sexual isolation contributes significantly to the partial reproductive barrier found between the two parental morphs.
The Organism and the Problem
Littorina saxatilis (Olivi) is a marine shore-dwelling prosobranch gastropod that lacks pelagic larvae and reproduces through direct development of the embryos. In this species, gene flow is maintained by slow diffusion of genes between neighboring demes along shores and through occasional transports (see, e.g., Janson 1987a; Johannesson and Warmoes 1990) between island populations. Adults migrate in the order of 1-4 m per 3 mo (Janson 1983), as in a related species (Hamilton 1978).
This snail have separate sexes and internal fertilization, and mating pairs are found during most of the year. Males climb the partners (which are not always females) in a counterclockwise manner before the penis is inserted into the mantle cavity of the partner (Saur 1990). Although an early study reported that males mated indiscriminately both with other males and with females of other Littorina species (Raffaelli 1977), Saur (1990) has since shown that male-female pairs remain in a copulating position longer than male-male pairs. This suggests some ability to discriminate between true and false mates. Similar patterns of mating behavior have also been observed in other related species, for example, L. mariae and L. obtusata (Rolan-Alvarez 1993).
Littorina saxatilis is highly polymorphic in shell characters, probably because of a low genetic exchange among populations (see, e.g., Janson and Ward 1984; Janson 1987b), and because the littoral zone is an environment of strong physical and biotic gradients. Different morphs may be found both in adjacent shores (e.g. Raffaelli 1982; Janson and Ward 1984 and 1985), and at different levels of the same shores (Johannesson and Johannesson 1990a; Johannesson et al. 1993). Between-shore variation is characterized by more or less continuous gradations from one morph into the other in areas of environmental clines (e.g. Raffaelli 1979; Janson and Sundberg 1983). This is also true for within-shore variation (between different shore levels) in many but not all places (Johannesson and Johannesson 1990a), as shown below.
In the exposed rocky shores of Galicia, Spain, a smooth unbended form of L. saxatilis is mainly confined to a low-shore zone of blue mussels, whereas 10-20 m higher up the shore, a ridged and banded form is present in a zone of barnacles (Johannesson et al. 1993). The distributions of the ridged and banded and the smooth and unbended morphs overlap in a narrow zone of the midshore on the border of the mussel and barnacle zones. The two pure morphs of L. saxatilis are also most numerous in the midshore, but individuals with mixed characters are present at frequencies of about 8%-29% (Johannesson et al. 1993; this study). Hybrids seems vital, and females of hybrid morphs carry embryos in their brood pouches, indicating that they are not sterile. We do not know, however, the actual survival rates of the hybrids compared to the parental morphs.
This microdistribution of the two parental morphs and one hybrid are in areas of heavy wave exposure on the Atlantic coast of Galicia, areas that sometimes extend for tens of kilometers.
Allozyme characters indicate that the two pure morphs share a common gene pool and are thus conspecifics (Johannesson et al. 1993). That is, over a distance of tens of kilometers, morphological and genetical distances are uncorrelated. However, on a microscale (tens of meters), gene flow between morphs is reduced (mean of Nei's genetic distances based on five polymorphic loci is 0.046; data from Johannesson et al. 1993) compared to gene flow within morphs (mean genetic distance = 0.007). The morphological and genetic patterns of Galician Littorina saxatilis are best explained by strong disruptive selection assisted by partial interruption of gene flow between the subpopulations of pure morphs living in adjacent microhabitats (Johannesson et al. 1993).
The impeded gene flow between pure morphs may be a consequence of a partial reproductive isolation between the morphs. Such a reproductive barrier may also explain the low frequency of mixed types in the midshore where the pure morphs are both present. Possibly, or alternatively, phenotypic selection against mixed-morph individuals may contribute to this. To test the hypothesis of a partial reproductive barrier, we have analyzed three components of mating behavior in two midshore areas, where the two pure morphs were present in nearly equal numbers, As a comparison, we have also analyzed mating behaviors of upper- and lower-shore areas almost completely dominated by the ridged and banded and the smooth and unbended morphs, respectively. With the sampling design we chose, we were also able to analyze the microgeographic distribution of the different morphs in relation to the distribution of different combinations of mating pairs in the midshore areas.
We found that snails of different morphs do not mate at random in the midshore zone. This is partly due to an assortative reproductive behavior and partly to microhabitat selection. Female sexual selection may also contribute to this pattern. This result may explain the deficiency of mixed-type individuals in the midshore, and the impeded gene flow between subpopulations of different morphs. As this partial mating barrier is between intraspecific morphs living more or less sympatrically in the same shores, it first suggests an example of incipient sympatric speciation, given that the reproductive barrier will evolve into complete sexual isolation. Second, it seems possible that the reproductive barrier is strengthened by reinforcement. Alternatively, assortative mating is a pleiotropic effect caused by selection on other characters.
MATERIALS AND METHOD
This study was performed at Silleiro Cape (42[degrees]6'N, 8[degrees]53'W), on the Atlantic coast of Galicia, Spain. We collected pairs of copulating snails and surrounding noncopulating individuals from two different areas (I and II) 100 m apart. At each area, one lower-shore strip (within the blue mussel belt), one midshore strip (on the border between the zones of blue mussels and barnacles), and one upper-shore strip (within the barnacle belt) were sampled. The strips were a few meters wide and 10 to 50 m in length, depending on the density of snails and the occurrence of mating pairs. We sampled October 7-21, 1991. In the midshore, the same two strips were sampled on three and four consecutive days, and the data for each strip were pooled to one sample. In area I, the upper-and lower-shore strips were sampled both on October 7 and October 21, and these data were not pooled (samples Ia and Ib) as the time elapsed between the two sampling dates was so long. The lower- and upper-shore strips of area II were sampled just once (for details, see fig. 1).
The only other littorinid species found was Melarhaphe neritoides, which was present in high numbers in the upper-shore barnacle zone. In no case, however, did we find any mating activity between M. neritoides and L. saxatilis.
We considered snails to be copulating when an active male was attached to the right-hand side of the partner's shell and his penis was inserted into the mantle cavity of the partner. When sampled, the active male and the partner were separated and stored in individual bags. All noncopulating individuals within the area of one or a few petri dishes surrounding the mating pair were also sampled as reference individuals. The number of reference individuals collected around each mating pair ranged from 0 to 15 (average 4.2) in the upper shore, 2 to 46 (average 12.5) in the midshore, and 2 to 46 (average 11.2) in the lower shore.
We sexed the snails as mature males (if they had a fully developed penis), mature females (if they had developing eggs or embryos in the brood pouch), and juveniles (all others).
We used the same two qualitative shell characters as in Johannesson et al. (1993) to separate the two pure morphs and the mixed morph from each other. An individual was defined as ridged and banded if it had continuous ribs and dark bands parallel to the growth spiral of the last whorl. If these characters were both absent, the individual was considered a smooth and unbended morph. Individuals with only one of the characters, were defined as mixed morphs. We mainly considered the morphology of the last whorl because sometimes older parts of the shell were eroded. Furthermore, as reported by Johannesson et al. (1993), juveniles reared in the laboratory were all smooth and banded until a size of about 3 mm. At this size, ridges begin to develop in the offspring of ridged and banded parents, whereas, in the offspring of smooth and unbended parents, the bands of successive whorls break up into a pattern of tessellation. Although these morphs are in general distinct, we used the same reference of classification in all samples by letting all individuals be classified by one of us. Shell length of each adult snail was measured with an accuracy of 0.02-0.04 mm.
In the midshore zone, where both pure and the mixed morph were present, we analyzed the distribution of the different morphs among the mating pairs to look for assortative mating. Furthermore, we compared the frequencies of copulating and noncopulating individuals of each sex and morph to reveal male and female (or partner) sexual selection (O'Donald 1980; Arnold and Wade 1984). We mean "potential for sexual selection" when we use the term "sexual selection," as we cannot prove that every male-female copulation resulted in sperm transfer (Arnold and Wade 1984). Furthermore, male-male and male-juvenile matings are obviously meaningless in an evolutionary sense, but analyzing "sexual selection" of these pairs might suggest behavioral patterns.
The observed distribution of pairs were compared with the distributions expected from complete random mating of the three morphs with contingency-table [chi square] tests (see, e.g., Anderson and McGuire 1978; Santos et al. 1986). As there were few individuals of the mixed morph present, we had several expected values below 5. This problem disappeared if we pooled the mixed category with any of the others but, because we might have lost information in this way, we present both unpooled and pooled results.
We also used a sexual-isolation index, Yule's V(e.g., Pielou 1977; Gilbert and Starmer 1985) to quantify the degree of assortative mating between the ridged and banded and the smooth and unbended morphs. This index is estimated according to the formula: V = (RR*SS - RS*SR)/ [(FR*FS*MR*MS).sup.1/2], where RR is the number of homotypic (within morph) ridged and banded pairs SS is the homotypic pairs of smooth and unbended snails, and RS and SR are the two types of heterotypic (between morph) pairs. Furthermore, FR is the total number of ridged and banded females; FS, total smooth and unbended females; MR, total ridged and banded males; and MS, total smooth and unbended males.
The possibility of sexual selection was tested by comparing the frequencies of copulating and noncopulating individuals of each sex, with a [x.sup.2] contingency test (Anderson and McGuire 1978; Santos et al. 1986). We also used the cross-product estimate (Cook 1971; Knoppien 1985) as a direct measurement of relative fitness for one morph compared to a reference morph (with fitness 1) in each sex separately. The fitness of morph A relative to the reference morph (R) is according to the cross product estimate given by:
[f.sub.A] = (CopA * Non-copR)/(Non-copA * CopR),
where Cop is the number of copulating individuals of one sex, and Non-cop the number of noncopulating individuals of the same sex within the same area. Confidence intervals for each fitness value against its reference value were obtained with the percentile method using bootstrap simulations (Efron 1982). This nonparametric approach often has more statistical power at small sample sizes than other traditional alternatives, and it is not constrained by assumptions of normality, etc. (Efron 1982). Every fitness value was resampled 1000 times using a compiled BASIC program (QuicBasic, ver 2.0) running in a PC computer (Rolan-Alvarez 1993).
The three components of mating behavior (male and female sexual selection and sexual isolation) were also analyzed in relation to snail sizes. Correlation coefficients were estimated in order to search for size-assortative mating at any of the three shore levels (e.g., Moore 1987). Analyses of variances were used to test size differences between copulating and noncopulating individuals of the upper and lower-shore samples. Before these analyses, we tested for heterogeneity of variances using Cochran's test (see, e.g., Underwood 1981). In no case did we find the variances to deviate significantly from homogeneity (P > 0.05).
We also analyzed the microgeographic distribution of the two pure morphs in the two midshore areas where they were both present. We defined a morph index as the number of ridged and banded snails divided by the total number of pure morph snails. Values of this morph index were obtained for each reference group of noncopulating snails surrounding a mating pair. We generated 1000 random sets of morph indexes, using a bootstrapping procedure, from the total frequency of each of the two morphs in each area, taking into account the different sizes of the reference groups. The variance of the morph indexes of each set was estimated, and the 95% confidence interval of all 1000 variances was found. The variance of the observed morph indexes were then compared to this confidence interval. This procedure was repeated for both study areas.
Distribution of Morphs and Pair Types
The upper-shore samples were completely dominated by ridged and banded individuals, whereas in the lower-shore samples, the majority of snails (92%-94%) were smooth and unbended (table 1). In the two midshore samples, the two pure morphs (ridged and banded and smooth and unbended) appeared in nearly equal numbers, whereas 8%-14% were of mixed type (table 1).
[TABULAR DATA 1 OMITTED]
All pairs found were intraspecific L. saxatilis pairs. Male-female pairs were most frequent, but a varying number of pairs were either male-male or male-juvenile pairs (table 1). The distribution of maladaptive pairs differed between the upper-, mid-, and lower-shore samples (contingency-table analysis, all samples from the same shore level pooled; [chi square] = 34.8, df = 4, P < 0.001). Male-male pairs were most frequent in the midshore and least frequent in the lower shore, whereas male-juvenile pairs were least frequent in the upper shore (table 1).
The microgeographic distribution of the two pure morphs were significantly different from random in both midshore areas. That is, the variance of the observed morph indexes were outside the 95% confidence interval of the bootstrap variances in both areas (table 2). These results show that, although both pure morphs were common in the midshore, their microdistribution was nonrandom. This is not surprising to us, as during sampling in the midshore zones, we observed that the two microhabitats (the mussel belt and the barnacle belt) formed a mosaic with different-sized patches in this area.
[TABULAR DATA 2 OMITTED]
Mating Behavior among Morphs
Ridged and banded and smooth and unbended morphs occurred with nearly equal frequency in the sympatric strips that we sampled in the midshore (see table 1), suggesting that heterotypic pairs would be fairly common if the two morphs mated at random and if mates of both morphs were about equally attractive. Heterotypic pairs of males and females were, however, infrequent. In the midshore strip of area I, only I of 60 pairs was a ridged and banded snail and a smooth and unbended one. In area II, 10 of 48 pairs were heterotypic (ridged and banded with smooth and unbended) (see Appendix 1). A contingency [chi square] test confirmed that mate choice deviated significantly from random among these pair types, and that this pattern was most pronounced in area I (table 3). The same pattern was obvious among the male-juvenile pairs observed, with only 8 of 34 being heterotypic pairs, although the assortative mating was significant in pooled data only (table 3). Among the male-passive male pairs, a majority (14 of 25) were a smooth and unbended male with a ridged and banded passive male, but no pairs were found in which the active male was ridged and banded and the passive male smooth and unbended (see Appendix 1). The male-male mating pattern did not deviate significantly from random mating.
[TABULAR DATA 3 OMITTED]
With contingency tests, we did not find significant sexual selection, except among the passive male partners, where we found a highly significant effect (table 3). Obviously, this "sexual selection" has no evolutionary sense but may suggest a certain mating pattern because males mating other males prefer ridged and banded males over smooth and unbanded.
The contingency test can detect selection only if pronounced fitness differences exist among all included morphs. Somewhat different results were obtained with a direct fitness estimator, which evaluates the relative fitness value for the males and females of the three morphs. The cross-product fitness estimates suggested that ridged and banded females were more successful than mixed females during matings (table 4). We found no trends of male sexual selection against the mixed morphs, although the ridged and banded males in site I were significantly more fit than the smooth and unbanded males (table 4, see also the component of male sexual selection in table 3).
[TABULAR DATA 4 OMITTED]
Assortative mating among male-female pairs in the midshore zone may be caused by active mate choice. Alternatively, the two morphs may mate assortatively because of their nonrandom microdistribution. To test this, we reanalyzed the male-female mating pairs and divided the pairs (both sites pooled) into pairs found in "smooth and unbended patches," in which the noncopulating individuals surrounding those pairs were mainly of the smooth and unbended morph (morph indexes of less than 0.333), "ridged and banded patches" (morph indexes of 0.667 or more), and "sympatric patches" (morph indexes 0.333-0.667).
In both allopatric and sympatric patches, the observed numbers of homotypic pairs were higher than expected by chance (see Appendix 2). The results deviated significantly from random mating analyzed either by applying a t-test to the different estimates of Yule's V testing its deviation from an expected values of V = 0 (no sexual isolation) (see Gilbert and Starmer 1985), or by using a pseudo-probability approach to estimate the significance of the [chi square] values (Zaykin and Pudovkin 1993) (table 5). However, Yule's V's estimated for pairs of one pure morph and one mixed morph did not differ significantly from zero, which suggests random mating between pure and mixed morphs. Thus an important part of the genetic exchange between pure morphs in the midshore occurs through mixed-morph individuals.
[TABULAR DATA 5 OMITTED]
We also estimated the degree of reproductive isolation between the pure morphs in the low-shore areas, where in fact both were present (see table 1). Interestingly, we found no indications of nonrandom mating in this zone (table 5).
Mating Behavior and Size Variation
Size of mature snails (both mating and nonmating) differed significantly between the two pure morphs and between sexes in all shore levels and samples (except in one of the upper-shore samples, table 6). The ridged and banded morphs were, on average, smaller in the midshore than in the upper shore (P < 0.05, for all cases), but there was no significant size difference between mid- and lower-shore smooth and unbanded snails.
[TABULAR DATA 6 OMITTED]
Sizes of mates were positively correlated in male-female pairs of the three upper-shore samples, one midshore sample, and at least one of the lower-shore samples (table 7). This might suggest a size-dependent mate-choice mechanism. However, average sizes of noncopulating males and females within each reference sample surrounding a mating pair were also correlated in most cases (table 7). This indicated that snails of similar sizes were aggregated on the shore, in particularly in the upper shore, and thus the positive size correlation of mates may have only been a consequence of this. Nevertheless, this size aggregation results in size-assortative mating.
[TABULAR DATA 7 OMITTED]
We also tested the possibility of sexual selection on size among the upper- and lower-shore snails. The analysis included two orthogonal factors, mode (copulating versus noncopulating; a fixed factor) and sample (Ia, Ib, II; a random factor). The ANOVA suggested that copulating lower-shore males are larger than noncopulating males (P = 0.07). No such trend was evident when sizes of copulating and noncopulating snails of the other groups were compared in the same way: P = 0.76 for lower-shore females, P = 0.94 for upper-shore males, and P = 0.93 for upper-shore females.
On exposed rocky shores of Galicia, natural populations of L. saxatilis split because of disruptive selection in two conspecific morphs, one upper-shore and one lower-shore, that still maintain an important gene flux between them (Johannesson et al. 1993). Spectacular adaptive responses to different environmental pressures have been described for L. saxatilis on sheltered and exposed shores (Raffaelli 1982; Janson 1982 and 1983; Grahame et al. 1990) and on upper-and lower-shore microenvironments (Johannesson and Johannesson 1990a), as well as for other related gastropods (Boulding 1990; Palmer 1990; Reid 1993).
Ecotypes of L. saxatilis, sometimes only a few meters apart, can maintain important phenotypic (sometimes known to be genetic) differences in spite of gene flow between them (Janson and Ward 1984 and 1985; Johannesson and Johannesson 1990b). Endler (1977) has shown that, with strong opposing selection pressures over environmental shifts, this is indeed possible, in spite of a substantial gene flow. Despite pronounced polymorphisms in both shell characters and allozymes, any reproductive barrier between different morphs has never been reported in this species before. Furthermore, there are few good examples of incipient reproductive isolation between conspecific morphs of other taxa (Barton and Hewitt 1985; Butlin 1987 and 1989). In this study, however, we have found a partial reproductive barrier between the upper- and lower-shore Galician ecotypes of L. saxatilis.
Three different mechanisms contribute to maintain this barrier. The first one is the use of different microhabitats that prevent the majority of snails from meeting and mating a snail of the contrasting morph. Even in the midshore areas, the pure morphs were nonrandomly distributed, suggesting some extension of habitat selection related to the patchy environment of mussels and barnacles. Similar adaptive behaviors are used to explain reproductive isolation among species or incipient species (see, e.g., Templeton 1981; Butlin 1987). The enhancement of reproductive isolation by this mechanism is probably a pleiotropic effect caused by natural selection favoring different morphs in different habitats (Templeton 1981; Butlin 1987). Alternatively, habitat choice may be a favored behavior because of selection against hybrids (i.e., reinforcement).
The second mechanism contributing to incipient reproductive isolation is assortative mating. This is supposed to be the most important behavioral mechanism producing reproductive isolation among incipient species (Spieth and Ringo 1983; Gilbert and Starmer 1985; Marin 1991). We have found conspicuous but not complete reproductive isolation between the two pure morphs of L. saxatilis in the midshore zone (see, e.g., table 5) which cannot solely be explained by the nonrandom microdistribution found in this zone. This as the patterns of assortative mating were similar in sympatric and allopatric patches, and thus a behavioral component of mate choice seems most likely. Moreover, the male-male pairs did not show assortative mating (see table 3), as would have been expected if the microdistribution of the pure morphs would have been the most important factor explaining the assortative mating behavior.
Sexual isolation caused by intrinsic behavioral mechanisms is observed between species (Spieth and Ringo 1983; Barton and Hewitt 1985; Loftus-Hills and Littlejohn 1992) and, less frequently, between conspecific morphs (see, e.g., Barton and Hewitt 1985; Butlin 1987; Ritchie et al. 1989). These intrinsic behavioral mechanisms may have evolved as a consequence of the reduced fitness of hybrid production--that is, through reinforcement--or they may be pleiotropic effects of disruptive selection on other characters. Either of these possibilities, or a combination of both, explains the role of reproductive isolation resulting from assortative mating between the pure morphs of Galician L. saxatilis. However, at present, we are not able to distinguish between the two morphs (see below).
The origin of the pure morphs is essentially unknown to us, although their genetic cohesiveness (see Johannesson et al. 1993) strongly suggests that the morphological cline is primary rather than secondary (Wake et al. 1989). Furthermore, as mentioned above, different ecotypes of L. saxatilis are described from many areas of this species distribution (Heller 1976; Raffaelli 1982; Janson and Sundberg 1983; Janson and Ward 1985; Grahame et al. 1990; Johannesson and Johannesson 1990a). This suggests that local selection pressures are capable of substantial morphological divergence between populations (or rather subpopulations) only tens of meters apart (e.g., Janson and Sundberg 1983) or even less (e.g., Johannesson and Johannesson 1990a). There is no reason to believe that in any of these cases, including the Galician populations, the different morphs or ecotypes have evolved in allopatry and thereafter have come into secondary contact.
In contrast to what we found in Galicia, most populations of L. saxatilis in which the variation in morphology over different microhabitats has been studied show a gradual change from one pure morph into the other with a dominance of phenotypically intermediate individuals in the transition zones (Raffaelli 1979; Janson and Sundberg 1983; Johannesson and Johannesson 1990a). A small daily action radius of the snails (Janson 1983) results in nonoverlapping distributions of the pure morphs in these cases. From viability estimates of one such gradual cline, we know that exposed, sheltered, and intermediate morphs differ substantially in survival rates over the three types of habitats, each morph being the fittest one within its own habitat (Janson 1983; Johannesson and Sundberg 1992).
Indeed, it also seems probable that the two pure Galician morphs present survival advantages to each other in their own microenvironments. Although we have no estimates of relative survival rates, the complete, or nearly complete, dominance of the pure morphs in the upper and lower shores, respectively, supports strong differential survival. The low rate of the mixed morph in the midshore, however, may perhaps suggest this morph to be at a disadvantage also in this area. If so, this may support the reinforcement model (see Butlin 1987, 1989). Moreover, a patchy distribution of pure morphs, that is, habitat selection, has been suggested as favorable for reinforcement (Diehl and Bush 1989; Harrison and Rand 1989). The lack of assortative mating within the low shore (table 5) may be a support for reinforcement, too, but more probably this is the result of a behavioral switch when the density of homotypic mates falls under a certain threshold value.
A third mechanism may contribute to the partial reproductive isolation between the pure morphs of L. saxatilis in Galicia as hybrid females tended to have lower sexual fitness than at least one of the pure morphs. Some theoretical models have shown the importance of sexual selection in relation to the evolution of reproductive isolation (Butlin 1987).
In light of the presence of assortative mating mechanisms, it is perhaps surprising to find such a high rate of maladaptive mating attempts (i.e., male-male and at least some of the male-juvenile pairs being formed). The answer may be that snails of Littorina saxatilis have no capacity for mate recognition at a distance, although L. littorea males are able to distinguish between male and female mucus tracks (Erlandsson and Kostylev 1995). It seems instead that mate choice in L. saxatilis takes place when the attempt to copulate has already started, that is, when the active male tries to insert the penis into the mantle cavity of the partner. Interrupted matings may explain why the frequency of maladaptive matings at any moment is lower than the frequency of adaptive matings, although the encounter of potential mates may be at random. In L. littorea, the duration of a copulation is of major importance and directly correlated to the adaptiveness of a mating (Saur 1990; Erlandsson and Johannesson 1994).
Although few smooth and unbended males mated ridged and banded females in the midshore zones, the most common male-male pairs were those between a smooth and unbended active male and a ridged and banded male partner. Perhaps this suggests that any mate choice in relation to morph may be due to the female rather than to the male in a pair. Some female behaviors that suggest active mate choice have been documented in the laboratory with related species of Littorina (Saur 1990; Rolan-Alvarez 1993). Moreover, the phenomenon seems to be common in different groups of organisms (O'Donald 1980; Borgia 1981; Berglund et al. 1986). On the other hand, in L. saxatilis, male choice has also been suggested among parasitized females (Saur 1990).
Although male-male matings are obviously not evolutionarily reasonable, it may be interesting to note that while smooth and unbended males mated ridged and banded males as partners, no ridged and banded males mated smooth and unbended males as partners (and this gave a significant effect in the sexual-selection component of the male partners of table 3, and see Appendix 1). This suggests that although males do not choose morph, they do in fact choose the size of their partner, large mates being favored over small. This is also found in L. Iittorea, where large females are favored over small as mates (Erlandsson and Johannesson 1994).
Size has been suggested to be of fundamental importance in local adaptation of L. saxatilis populations (Sundberg 1988), and the differences found in Galicia seem likely to be an effect of adaptation to different microenvironments. As, however, size is also a factor that influences mate choice, one possibility is that the assortative mating between the pure morphs in Galicia is due to pleiotropic effects rather than to reinforcement.
In upper-shore samples, in particular, we found a positive correlation between sizes of mates. This has been observed in some populations of L. saxatilis (Saur 1990) and also in related Littorina species (Rolan-Alvarez 1993; Erlandsson and Johannesson 1994), but is missing in other populations of L. saxatilis (Raffaelli 1977; Saur 1990) or related Littorina species (Raffaelli 1977; Erlandsson and Johannesson 1994). In our study, we found that the distribution of noncopulating snails also were related to size, that is, smaller snails and larger snails tended to group separately in many samples (see table 7). This may suggest that pair correlations are a consequence of nonrandom distribution of snails of different sizes. We do not know the reason for this grouping, possibly it is a consequence of natural selection for different sizes in different patches, or it is because similarly sized snails aggregate to find mating partners. Nevertheless the result will be size-assortative mating.
Among females of upper and lower zones, we found no trends of sexual selection in any morph related to size, although this has been found in populations of L. Iittorea (Erlandsson and Johannesson 1 994). Among males of the lower zone, however, rate of copulation tended to be higher among larger males (P = 0.07). This may suggest occasional patterns of size-dependent male-male competition, female rejection of small males, or both.
In summary, the pure morphs of Galician L. saxatilis do, in many cases, mate size assortatively in both sympatric and allopatric zones. Whether this pastern of size-assortative mating within morphs can predict the partial reproductive isolation between morphs in the midshore zone is hard to say, as size and morph are so much confounded in our data. That is, we cannot today distinguish between what might be a pleiotropic effect of natural selection on size in combination with size-assortative mating and reinforcement favored by a lowered fitness of hybrid matings. The result, however, is reproductive isolation in both cases.
A species like L. saxatilis in which an effective dispersal mechanism is absent, because of the lack of a pelagic stage, may obviously be sensitive to factors that further limit the exchange of genes among demes. Incipient reproductive isolation between different morphs mainly confined to different microenvironments seems to be a potential for further evolutionary changes. Although Johannesson et al. (1993) have shown that, at present, gene flow between the two pure morphs is large enough to warrant them being conspecifics, improvements of the assortative mating mechanisms may eventually lead to a complete reproductive barrier and thus to sympatric speciation.
We thank R. Butlin and B. Johannesson for useful comments on earlier versions of the manuscript. We had financial support from the Swedish Natural Science Research Council, the Xunta de Galicia, and the foundation of Collianders.
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Observed numbers of matings between three different morph (ridged and banded, smooth and unbended, and mixed) of Littorin, saxatilis in the midshore of exposed Galician shores. Two areas (and II) 100 m apart were sampled. Numbers of noncopulating individuals surrounding the mating pairs are indicated in bold and numbers of matings expected from random mating are indicated in brackets.
[TABULAR DATA OMITTED]
Observed matings among three morphs of Littorina saxatilis as in Appendix 1. Here, however, the material has been comparted into three groups. "Smooth and unbended patches" were those patches ([nearly equal to]10 [cm.sup.2]) dominated by the smooth and unbended parental morph (morph index < 0.33, see text), "sympatric patches" had about equal proportions of the two parental morphs (0.33 < morph index < 0.67), and "ridged and banded patches" were dominated by the ridged and banded parental morph (morph index > 0.67). Data from two midshore areas 100 m apart (I and II) have been pooled. Numbers of pairs expected from random mating are indicated in brackets.
[TABULAR DATA OMITTED]
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|Author:||Johannesson, Kerstin; Rolan-Alvarez, Emilio; Ekendahl, Anette|
|Date:||Dec 1, 1995|
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