Reproductive patterns and their influence on the population genetics of sympatric species of the genus Crepidula (Gastropoda: Calyptraeidae).

ABSTRACT Crepidula dilatata (Lamark, 1822) and Crepidula fecunda (Gallardo, 1979) are sympatric species in southern Chile. Separating them by species is difficult because they are morphologically similar; an important character, which separates them is their comparative larval development. C. dilatata includes nutritive eggs within its egg capsules that are consumed by larvae in the capsule during their intracapsular development. Metamorphosis occurs in the capsule, followed by the hatching of crawling juveniles. The C. fecunda intracapsular development leads to the release of planktotrophic veliger larvae that persist in the water column for about 15 days prior to settlement and metamorphosis. These two contrasting reproductive strategies (direct and indirect development) may influence the capabilities of these species for dispersal, which could influence their comparative gene flow and population genetics. Newly developing molecular genetic techniques, such as RAPD used in the present study, were useful in identification of the two species studied and provided some initial data on their comparative population genetics. Greater gene flow and interpopulational gene diversity were found in C. fecunda (pelagic larva) compared with C. dilatata (direct development), with the latter showing populations to be the more genetically heterogeneous within the geographic range studied. It was thus evident that the pattern of larval development (direct or planktonic) influenced the comparative population genetic structure between these two species.

KEY WORDS: Crepidula dilatata, C. fecunda, Chiloe Island, reproductive patterns, RAPD, population genetic

INTRODUCTION

The basic patterns in reproductive strategies of marine benthic invertebrates include holobenthic cycles, with (a) direct larval development and no pelagic larval phase; (b) completely pelagic cycles and (c) pelagic-benthic cycles where larval development is mixed. The latter case includes the presence of a free-living larva in the water column prior to settlement and metamorphosis in the benthos (Thorson 1950, Mileikovsky 1971, Gallardo 1987, Gallardo 1989).

The presence of a pelagic larva may influence the characteristics of population structure, based on the impact of these stages on the capacity for dispersion of these species (gene flow) (Hunt 1993, Williams & Benzie 1993, Ayre et al. 1997, Hoskin 1997, Kyle & Boulding 2000, Collin 2001, Collin 2003, Nishikawa et al. 2003, Goffredo et al. 2004). A pelagic larva (lecithotrophic or planktotrophic), can remain in the water column for weeks, thus favoring its dispersion, in contrast with the direct mode of development where the individuals hatch from maternal incubatory structures as crawling juveniles (Collin 2001) thus remaining close to their sites of generation. Loss of the larval pelagic phase may favor evolutionary divergence in congeneric species with locally adapted populations; these could diversify in the expression of certain traits in their life histories, reflected in their gene frequencies (Strathmann 1980, Gallardo 1989, Pechenik 1999, Marshall & Keough 2003).

On the other hand, the reproductive strategy, which includes a pelagic larva would guarantee, in the absence of geographic barriers or restrictive oceanic currents, greater larval dispersal and thus greater gene flow. This might however diminish variability in the population structure (Strathmann 1986, Hunt 1993, Palumbi, 1994, Kyle & Boulding 2000, Collin 2001, Hellberg et al. 2002, Whalan et al. 2005), resulting in genetically homogeneous populations.

Species in the Family Calyptraeidae ("slipper limpets") are typically protandric hermaphrodites. They deposit egg capsules and exercise parental brooding as part of their reproductive strategies. Crepidula dilatata (Lamark 1822) and Crepidula fecunda (Gallardo 1979) coexist along a portion of the Chilean coast, having a distribution from 29[degrees]58'S to 41[degrees]52'S and 12[degrees]5'S to 41[degrees]31'S, respectively (Collin 2003); both species inhabit both intertidal and subtidal rocky substrates (Gallardo 1977). They can occur at high densities in estuaries and bays along the interior coast of the Island of Chiloe, as well as in protected bays and fjords in the southern channel region of Chile. The species closely resemble each other morphologically, and are best identified based on type of development undergone by their larvae, and secondarily by the maximum size of the adults (Gallardo 1976, 1977, 1979, Brown & Olivares 1996).

Taxonomic studies of the Chilean species of the genus Crepidula have been based historically on morphological criteria, where comparisons are made between morphometries of the shell and radula (Gallardo 1979, Brown & Olivares 1996). Also, these species can coexist sympatrically, which makes specific identification even more difficult, particularly when trying to determine the frequency of each species within a given habitat.

Capsules deposited by C. dilatata contain nutritive eggs that are consumed as extraembryonic nutrition by larvae developing in the capsules. This factor allows well developed crawling juveniles to hatch from the capsules, having shell lengths of between 0.9 and 1.2 mm (Gallardo 1976, 1977, 1979, Chaparro & Paschke 1990). With C. fecunda all the eggs deposited in the capsules develop into veliger larvae that hatch into the water column. They assume a planktotrophic life of about 15 days prior to settlement and metamorphosis, at which time they have a shell length of about 0.65 [+ or -] 0.028 mm (Chaparro et al. 2005).

Based on the reproductive mechanisms of these two species it was expected that C. dilatata would have a comparatively lower degree of dispersion in the absence of a pelagic larva, consequently reducing gene flow among its populations. On the other hand, C. fecunda has a wider dispersion because of its freely drifting larvae, the development of which continues in the water column for a period of about two weeks prior to settlement (Chaparro et al. 2005). In view of these considerations, the present research was carried out in an attempt to differentiate between these species at the genetic level using the technique of random amplified polymorphic DNA (RAPDs). A comparison was made on the level of dispersion and the degree of influence exercised by the different reproductive strategies of the two species, and their impact on the isolation of populations

MATERIALS AND METHODS

Population Sampling

About 50 specimens of the Crepidula species introduced earlier were collected from mixed populations inhabiting the intertidal and subtidal zones at sites along the interior shore of Chiloe Island in southern Chile. The sites sampled included: Quempillen (41[degrees]52'S; 73[degrees]46'W), Quemchi (42[degrees]08'S; 73[degrees]28'W), Dalcahue (42[degrees]22'S; 73[degrees]39'W), Queilen (42[degrees]52'S; 73[degrees]28'W), Chaiguao (43[degrees]08'S; 73[degrees]29'W) and Yaldad (43[degrees]08'S; 73[degrees]44'W) (Fig. 1).

[FIGURE 1 OMITTED]

As a means of corroborating later results obtained with molecular markers, limpets from each site were identified with species by examining the type of the larval development occurring in their associated egg capsules.

For the study of molecular markers, a tissue sample of about 1 g was taken from the foot of each specimen, fixed in 95% EtOH, and stored in Eppendorf tubes at -20[degrees]C until processed.

DNA Extraction 

Each specimen's DNA was obtained from a subsample of pedal tissue (100-150 mg), which was macerated and digested in lysis-buffer (50 mM Tris-HCL (pH 8.0); 1.0% SDS; 25 nM EDTA) with 200 [micro]g of protease K (Sigma). The samples were incubated at 37[degrees]C for 12 h in 1.5 ml Eppendorf tubes; each tube then received 500 [micro]L of a phenol/chloroform/isoamyl alcohol (24:24:1) solution. The mix in each tube was shaken with a vortex mixer for a few seconds and then centrifuged at x13,000 rpm for 15 min at 5[degrees]C. Following this, the supernatant containing the DNA was aspirated, taking care not to touch the intermediate layer. Each supernatant was transferred to a new 1.5 ml (autoclaved) Eppendorf tube. Each extracted DNA sample was resuspended in 200 [micro]L of 95% EtOH and placed in the cold at -18[degrees]C for 30 min to precipitate the sample. The EtOH was then removed, leaving the tubes containing the DNA pellet. Concentration and drying of samples was done using a SAVANT DNA model 110 concentrator. Each DNA pellet obtained was resuspended in 200 [micro]L of ultradistilled water (modified from Toro 1998), and the samples were cleaned with 200 [micro]L of 8M lithium chloride for 3 h at -20[degrees]C and then centrifuged at x10,400 rpm for 30 min at 4[degrees]C (Sambrook et al. 2001). The cleanup of the DNA was finalized by eliminating the supernatant and washing the pellet in 70% EtOH. Finally, each pellet was dried for 20 min using the above SAVANT concentrator, resuspended in 200 [micro]L of double-distilled water, and stored at -20[degrees]C until amplification.

DNA in the samples was quantified by reading absorbancies at 260 nm in a SUV 2120 UV/VIS spectrophotometer. This was carried out following dilution of sample aliquots to 1/20 in distilled water (Sambrook et al. 2001).

DNA Amplification 

Of a total of 49 primers, only three (5'-TAGCCCGCTT-3'; 5'-GTGCGTCCTC-3'; 5'-ACGACGTAGG-3' Biosource International) demonstrated polymorphism.

Amplification of the DNA samples was carried out using the polymerase chain reaction (PCR) employing a total mixture of 25 [micro]L in ultrathin (500 [micro]L) PCR tubes (Gordon Technologies). The reaction mixture was prepared using 1 [micro]L of genomic DNA (ca. 50 ng/[micro]L), 2.5 [micro]L of dNTPs (2 mM), 1.75 [micro]L of Mg[Cl.sub.2] (25 mM), 2.5 [micro]L PCR--buffer x10, 4.5 [micro]L of the primer (10 [micro]M/[micro]L), 1 unidad of Taq ADN polimerasa (5 U/[micro]L) (Invitrogen) and 12.55 [micro]L of double-distilled water (modified from Toro et al. 2004).

The mixture was covered by a drop of mineral oil to avoid evaporation and then each of the samples was placed in a thermo cycler (Amplitron II, Thermolyne Inc.). The program of the thermo-cycler began with generalized denaturation of the DNA at 95[degrees]C for 15 sec followed by 36 cycles, which included a partial denaturation at 94[degrees]C for 30 sec, an annealing process at 32[degrees]C for 30 sec, and finally a duplication period at 72[degrees]C for 1 min; after the 36 cycles the amplified product was maintained at 4[degrees]C (Toro et al. 2004).

Agarose Gel Electrophoresis 

All of the DNA fragments amplified with the primers used were separated in agarose gels (Invitrogen Ultra Pure 1000), at 1.2% in TBE 0.5 x buffer (44.5 mM tris base, 44.5 mM boric acid, 1M EDTA pH 8). Ethidium bromide (10 mg/mL) was added to the gel preparation at a concentration of 1%. Each gel was loaded with a pattern of 1000 bp standard bands (ladder DNA, Promega) for an approximate estimation of the molecular weight of the amplified bands. The gels with the samples incorporated were submerged in an electrophoretic chamber containing TBE 0.5 x buffer (44.5 mM tris base, 44.5 mM boric acid, 1 M EDTA pH 8). Each gel was run at 75 Volts for approximately 1.5 h. Visualization of the band patterns of each gel was done under UV light, and the image of the gel was obtained using a digital camera (modified from Toro 1998).

Data Analysis

The results of the RAPDs, were analyzed based on the band patterns recorded by photography of the agarose gels. Codification was made of the bands present (1) and those that were absent (0), which was then configured into a data matrix.

The identification of species was carried out based on the data obtained in the matrix, searching for bands or patterns of bands particular to each of the two species analyzed.

The statistical analysis was carried out using POPGENE 32 computational programs (Population Genetics Software) (Yeh & Boyle 1997) and the GenAlEx version 5.1 program (Peakall & Smouse 2001).

The gene flow (Nm) was calculated based on the coefficient of populational differentiation (Gst) by applying the formula (Nm = 0.5 (1--Gst)/Gst) (Mc. Dermott & Mc Donald 1993). Also determined was the genetic diversity (h), and genetic distance (D) based on the Nei (1978) formula, and also the number and percentages of polymorphic loci. Based on the pattern of genetic differentiation (genetic distance of Nei 1978), a dendrogram was generated, using the unweighted pair group method with arithmetic mean (UPGMA).

An analysis was carried out, by species, of the number of bands present in each of the populations examined, as well as the mean heterozygosity using the GenAlEx program (Peakall & Smouse 2001). An analysis of molecular variance was also carried out (AMOVA; Excoffier et al. 1992), to evaluate the inter and intrapopulational variation, and estimate the degree of panmixia of the populations (Holmes et al. 2004, Sands et al. 2003, Kruse et al. 2003). Use of a Mantel test (GenAlEx program, version 5.1) allowed for making paired comparisons between genetic distances and geographic distances, using a large number of random permutations. The geographic distances between the sampling sites were approximated by measurement from a nautical chart of the coastline.

RESULTS

The use of the RAPD technique identified 34 loci that were amplified starting with the three primers used.

The patterns of the bands used for each of the species allowed identification of discriminant bands in Crepidula dilatata and Crepidula fecunda (Fig. 2). The bands existing for C. fecunda had weights of about 700, 400 and 300 base pairs. In contrast, C. dilatata had bands of only 500 and 300 base pairs. Results from individuals that did not show the presence of discriminant bands were excluded from all analyses because these results may have been caused by technical complications.

[FIGURE 2 OMITTED]

Identifications of the samples from all the populations showed that 70% consisted of C. fecunda and 19% were C. dilatata; 11% of the samples were not identified to species by the molecular method and were eliminated from subsequent analyses (Fig. 3).

[FIGURE 3 OMITTED]

The highest percentage of C. dilatata occurred at Quempillen, whereas the highest percentages of C. fecunda occurred at Dalcahue and Yaldad. The remaining populations (Quemchi, Queilen and Chaiguao) contained both species, with a predominance of C. fecunda.

The number of bands amplified for each of the species and each of the populations studied gave an average value slightly lower for C. dilatata (22 [+ or -] 1.50) than for C. fecunda (25 [+ or -] 4.67).

At the population level, there was a smaller number of bands recorded for the Quempillen samples, with 18 bands found for C. fecunda. The remaining populations maintained equal numbers of bands per species. A few specific bands were identified for individuals of C. dilatata at Quempillen and Quemchi, which were encountered exclusively in limited populations, suggesting a degree of uniqueness of the limited population.

The gene flow estimated for C. dilatata was 0.91 and 1.86 for C. fecunda. A paired analysis of gene flow estimated between populations showed that for almost all the locations analyzed, the values were greater for C. fecunda; exception to this occurred at Quempillen-Queilen and Quempillen-Chaiguao (Fig. 4).

[FIGURE 4 OMITTED]

The intra and interpopulational genetic diversity was greater for C. fecunda than for C. dilatata at each of the locations sampled, with values of 0.20 and 0.14, respectively for the two species (Table 1). The population at Quempillen had lower values for genetic diversity, both for C. dilatata (0.09 [+ or -] 0.02) and for C. fecunda (0.11 [+ or -] 0.05).

In C. dilatata, the number of polymorphic loci was nearly constant, fluctuating between 19 and 22, whereas for C. fecunda, these fluctuated between 16 and 30; the percentage of polymorphic loci for each species was 85.2% and 100%, respectively. The populations that showed the lowest value for polymorphic loci were Chaiguao (19) for C. dilatata and Quempillen (16) for C. fecunda (Table 1).

For C. dilatata, the population at Yaldad was that presenting the greatest genetic distance among all the populations studied, whereas populations at Quempillen, Queilen, Quemchi and Chaiguao showed lower genetic distances. In the case of C. fecunda, the populations at Yaldad and Quempillen had the highest values for genetic distance in relation to the other populations studied. The populations at Quemchi, Chaiguao and Dalcahue had lower values for genetic distance, with Dalcahue the most outlayer from the group (Fig. 5, Table 2).

[FIGURE 5 OMITTED]

The magnitude of the genetic distance (Nei 1978) for C. dilatata was approximately double that for C. fecunda (Fig. 5).

The average heterozygosity of each of the populations studied was greater for C. fecunda than for C. dilatata. In the Quempillen population, C. dilatata and C. fecunda had lower values than all the other populations studied, suggesting that this location had unusual biological and oceanographic characteristics.

The analysis of molecular variance showed significant differences (P < 0.01) between C. dilatata and C. fecunda, explained by intrinsic variations in each population. In both species the percentage of interpopulational variation was greater than the intrapopulational variation, indicating that the total genetic variation was, for the most part explained by differences among populations (Table 3).

Correlation between geographic distance and genetic distance, using 5,000 permutations, showed significant correlations (P < 0.05) for both species. When the genetic distance was estimated for C. dilatata we obtained a Mantel value of -0.081, whereas the value obtained from the Mantel correlation for C. fecunda was 0.260.

DISCUSSION

The RAPD marker technique permitted identification of discriminant bands, which allowed species discrimination among samples of Crepidula spp. as reported for other taxa (Crossland et al. 1993, Andre et al. 1999, Rego et al. 2002, Costa et al. 2004, Liu & Cordes 2004)

The two species were found sympatrically in some of the populations studied. C fecunda was the only species identified from Dalcahue and Yaldad locations (marine conditions), whereas at Quempillen (estuarine conditions) more than 75% of the individuals were C. dilatata. This suggested a difference in distribution between the two species based on environmental differences in water mass. The percentage of unidentified individuals (11%), could be because of technical problems encountered during the DNA extraction, as well as in the amplification procedure.

The interpopulational gene flow for C. dilatata (Nm = 0.91) was less than that for C. fecunda (Nm = 1.86). Slatkin (1994) indicated that values for this parameter above unity indicated that populations were overcoming the effects of genetic drift, and thus impeding local differentiation.

Paired analyses for populations of the two species, suggested a higher gene flow for C. fecunda than for C. dilatata. The exceptions that favored the species with direct development (C. dilatata) occurred in comparisons between Quempillen and Chaiguao and Quempillen and Queilen. A similar situation has been described for the Chilean oyster Ostrea chilensis, where aquaculture activities have distorted the population genetic structure caused by transfer of juveniles from one culture location to another (Tort & Aguila 1996).

The greater degree of gene flow within the populations of C. dilatata from Quempillen, Queilen and Chaiguao, could be related to the presence of superficial marine currents (0-30 m), which run from the Gulf of Ancud to the Gulf of Corcovado having a net transport from the north toward the south of Chiloe Island (Silva et al. 1998).

This condition could facilitate the drift of juvenile individuals by rafting, as reported for Crepidula convexa (Collin 2001).

The greater genetic diversity found for C. fecunda in each of the populations studied may be explainable based on its having a pelagic larval stage lasting about two weeks. This amount of time in the waters along the coast of Chiloe would give the larvae of this species ample time for broad dispersion, promoting genetic interchange among populations. The populations of both species at Quempillen showed lower values for genetic diversity, suggesting reduced dispersion, or even larval retention (Bilton et al. 2002). This probability is supported by the hydrographic configuration of the Quempillen estuary, which has natural barriers to the entrance and exit of larvae.

The percentage of polymorphic loci fluctuated between 47.0% and 88.2% for C. fecunda and 55.8% to 64.7% for C. dilatata. These values were higher than average values for polymorphic loci reported for molluscs (41.2%, Fujio et al. 1983). The percentage of polymorphic loci in the oyster Ostrea edulis was between 18.2% and 40.9% (Saavedra et al. 1993), and in the prosobranchs Cominella lineolata and Bedeva hanleyi it was 24.1% and 41.7% respectively (Hoskin 1997). Based on the preceding, the values presented from the present study are comparatively high, which may be because of the use of DNA based molecular techniques.

The dendrograms for both species show that the genetic distance of C. fecunda is half that present in C. dilatata. This allows the inference that differences in their reproductive strategies (pelagic larva versus direct development) have an important effect on the genetic structure of the populations of each species. The genetic distances of species with direct development such as Crepidula cf. convexa (0.008-0.076) and Crepidula convexa (0.037--0.057) (Hoagland 1984) were lower than those presently reported for C. dilatata (0.0067-0.2004), which indicates that genetic interchange in the latter species is comparatively less. Greater or lesser genetic interchange may depend on factors such as the population density (Scheltema 1971, Kyle & Boulding 2000), or reproductive variables such as the effective population size (Hedgecock 1994) and presence and duration of the pelagic larval period (Hellberg 1996, Star et al. 2003). C. convexa produces from 8-20 capsules per female with each capsule containing 15-25 embryos (Hendler & Franz 1971), C. dilatata produces only 9-22 capsules per female with a range of 2-12 embryos per capsule (Penchaszadeh et al. 2002). These factors could strongly affect the dispersive capabilities of these species, because a greater number of descendents per generation would be more conducive to net gene flow between populations.

The genetic distance for C. fecunda (0.0131-0.1042) was mildly comparable with Crepidula fornicata (0.003-0.016) and Crepidula plana (0.052-0.097) (Hoagland 1984), all of which have a larval planktonic phase. The low values for genetic distance in C. fecunda could be because of local marine currents whose magnitude and direction of flow (Silva et al. 1998) impeded effective larval transport among some of the populations studied (Scheltema 1971).

The averages of heterozygosity for C. dilatata (0.09-0.16) and for C. fecunda (0.11-0.19), were within the ranges reported for different species of mollusks (0.05-0.21, Fujio et al. 1983). The average heterozygosity was low for C. dilatata, which indicated that the genetic interchange among its populations was very low, suggesting a future decline in the genetic diversity of the gene pool within these populations. This heterozygosity not only may be influenced by the mechanics of reproduction of the species (e.g., absence of pelagic larvae), but also by geographic and oceanographic factors, which may interrupt the flow of individuals among the populations (Palumbi 1994, Collin 2001, Wares et al. 2001, Hellberg et al. 2002, Zacherl et al. 2003, Whalan et al. 2005).

Species with direct development such as C. convexa and C. atrasolea, share similar characteristics as those of C. dilatata based on the results of the AMOVA. In these cases the interpopulational variation was greater than intrapopulation variation, indicating that the genetic variability was greater among the populations (Hellberg 1996). In contrast, the AMOVA data presently obtained for C. fecunda do not coincide with previously reported data for C. fornicata, as the latter species shows less interpopulational variation (Collin 2001). C. fecunda, releases intermediatesized larvae having shell lengths of 0.18-0.37 mm (Chaparro et al. 2002), whereas C. fornicata larvae are 0.44-0.48 mm in length (Collin 2001). Shell length at settlement in these species are 0.65 [+ or -] 0.028 mm (Chaparro et al. 2005) and 0.94-1.00 mm (Pechenik et al. 1996), respectively. As cited earlier C. fecunda has a pelagic life of only 15 days, whereas that of C. fornicata is 34 days (Thouzeau, 1991). Differences in the pelagic period of the life cycle imply different capabilities for in larval dispersal, which could have an impact on the genetic homogeneity of the populations (Ayre et al. 1997, Nishikawa et al. 2003).

The positive correlation between geographic and genetic distances, but low values for correlation between populations of C. fecunda and C. dilatata as shown by the Mantel test, may be because of the fact that the greatest distance between populations was only 230 km, which may influence the sensitivity of the analysis (Hellberg 1996, Exadactylos et al. 2003). Also, the physiographic configuration of the coastline may also have effected the low correlation given by the Mantel test (Wares et al. 2001, Hellberg et al. 2002, Zacherl et al. 2003). For example, whereas the populations at Yaldad and Chaiguao are closer geographically (39 km of coastline), their genetic distances are very high (C. dilatata = 0.2004; C. fecunda = 0.1042). Yaldad is a protected bay, and is not exposed to direct influence from subantarctic, and estuarine water produced eastward of Chiloe island (Silva et al. 1998). The Chaiguao region is more exposed to direct influences of the former water masses, and we suggest there is very low probability of larvae from Chaiguao entering Yaldad Bay.

To summarize, the present study focused molecular genetic techniques on the specific differentiation between C. dilatata and C. fecunda. These methods are a valuable complement to classical morphometric methods, particularly when there exists phenotypic plasticity influenced by environmental conditions at each locality (Seed 1992). The results of the present investigation support the hypothesis that the presence of a pelagic stage in the life cycle of a Crepidula species is conducive to comparatively greater interpopulational gene flow, which in turn produces greater heterozygosity in the populations. In contrast, direct development leads to an increase in intrapopulational homogeneity and isolation in populations because of constraints in dispersion encountered by the juvenile stages.

Continued research along the lines developed in the present study, supported by ecological and oceanographic studies, is of great interest because it can provide a more detailed vision of the factors affecting the population genetics of C. dilatata and C. fecunda. This study is of particular interest in the marine environment because it is not only an interesting, well defined model within a limited geographic area, but it can also serve as a general model of population genetic processes of systematic and evolutionary import by combining classical and newly molecular genetic methods.

ACKNOWLEDGMENTS

The authors thank Ms. J. Ojeda and Ms. M. Montoya for their help with the lab analysis. The work was supported by the Fondecyt-Chile research grant 1020171.

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ALFONSO J. SCHMIDT, JORGE E. TORO (1) * AND OSCAR R. CHAPARRO Instituto de Biologia Marina, Dr. J.E. Winter, Universidad Austral de Chile, Casilla 567, Valdivia, Chile

* Corresponding author. E-mail [e-mail]

TABLE 1.

Genetic population parameters for each sampled areas and species.

    Location                     Quempillen

Species                   C.d.                  C.f.
Heterozygosity
Mean [+ or -] SD    0.09 [+ or -] 0.02   0.11 [+ or -] 0.03
Polymorphic loci
  (No)                      20                   16
Polymorphic loci
  (%)                      58.8                 47.0
Genetic diversity
  (h) [+ or -] SD   0.09 [+ or -] 0.14   0.11 [+ or -] 0.15
Gene Flow (Nm)

    Location                       Quemchi

Species                   C.d.                  C.f.
Heterozygosity
Mean [+ or -] SD    0.15 [+ or -] 0.03   0.19 [+ or -] 0.02
Polymorphic loci
  (No)                      20                   30
Polymorphic loci
  (%)                      58.8                 88.2
Genetic diversity
  (h) [+ or -] SD   0.15 [+ or -] 0.17   0.18 [+ or -] 0.13
Gene Flow (Nm)

    Location                     Dalcahue

Species                    C.d.                 C.f.
Heterozygosity
Mean [+ or -] SD            --           0.16 [+ or -] 0.03
Polymorphic loci
  (No)                      --                   27
Polymorphic loci
  (%)                       --                  79.4
Genetic diversity
  (h) [+ or -] SD           --           0.16 [+ or -] 0.16
Gene Flow (Nm)

    Location                     Queilen

Species                    C.d.                 C.f.
Heterozygosity
Mean [+ or -] SD    0.16 [+ or -] 0.02   0.19 [+ or -] 0.03
Polymorphic loci
  (No)                      22                   29
Polymorphic loci
  (%)                      64.7                 85.2
Genetic diversity
  (h) [+ or -] SD   0.16 [+ or -] 0.14   0.18 [+ or -] 0.15
Gene Flow (Nm)

    Location                     Chaiguao

Species                   C.d.                  C.f.
Heterozygosity
Mean [+ or -] SD    0.14 [+ or -] 0.03   0.16 [+ or -] 0.03
Polymorphic loci
  (No)                      19                   29
Polymorphic loci
  (%)                      55.8                 85.2
Genetic diversity
  (h) [+ or -] SD   0.14 [+ or -] 0.16   0.17 [+ or -] 0.17
Gene Flow (Nm)

    Location                        Yaldad

Species                    C.d.                 C.f.
Heterozygosity
Mean [+ or -] SD            --           0.16 [+ or -] 0.03
Polymorphic loci
  (No)                      --                   21
Polymorphic loci
  (%)                       --                  61.7
Genetic diversity
  (h) [+ or -] SD           --           0.17 [+ or -] 0.19
Gene Flow (Nm)

    Location                        Total

Species                    C.d.                 C.f.
Heterozygosity
Mean [+ or -] SD            --                   --
Polymorphic loci
  (No)                      29                   34
Polymorphic loci
  (%)                      85.2                 100
Genetic diversity
  (h) [+ or -] SD   0.14 [+ or -] 0.14   0.20 [+ or -] 0.14
Gene Flow (Nm)             0.91                 1.86

C.d., Crepidula dilatata; C.f., Crepidula fecunda.

TABLE 2.

Genetic distances for Crepidula fecunda (above the diagonal) and
Crepidula dilatata (under the diagonal) according Nei (1978).

Location     Quempillen   Quemchi    Dalcahue

Quempillen      ****       0.0369     0.0805
Quemchi        0.0484       ****      0.0336
Dalcahue         --          --        ****
Queilen        0.0084      0.0216       --
Chaiguao       0.0178      0.0522       --
Yaldad         0.1700      0.1004       --

Location      Queilen     Chaiguao    Yaldad

Quempillen     0.0747      0.0880     0.0734
Quemchi        0.0131      0.0314     0.0670
Dalcahue       0.0367      0.0593     0.1018
Queilen         ****       0.0240     0.0976
Chaiguao       0.0067       ****      0.1042
Yaldad         0.1650      0.2004      ****

TABLE 3.

Molecular variance (AMOVA) for C. dilatata and C. fecunda
using 999 permutations.

Species             Level         D.f.   Mean        %
                                         Square   Variance

C. dilatata   Among population       3   14.268    75.5%
              Within population     45    3.365    24.5%
C. fecunda    Among population       5   41.543    73.9%
              Within population    171    3.734    26.1%

Species             Level           P        Mode of
                                           Development

C. dilatata   Among population    <0.01
              Within population           Direct
C. fecunda    Among population    <0.01   Planktotrophic
              Within population