Gene Flow and Differential Mortality in a Contact Zone Between Two

Biological Journal of the Linnean Society (2000), 71: 755–770. With 2 ﬁgures doi:10.1006/bijl.2000.0473, available online at http://www.idealibrary.com on

Gene ﬂow and diﬀerential mortality in a contact zone between two Albinaria species (Gastropoda; Clausiliidae)

SINOS GIOKAS1∗, MOYSIS MYLONAS2 AND KONSTANTINOS SOTIROPOULOS1 1Zoological Museum, Department of Biology, University of Athens, GR-15784, Athens, Greece and 2Natural History Museum of Crete, Department of Biology, University of Crete, GR-71110, Irakleion, Crete, Greece

Received 3 August 1999; accepted for publication 11 July 2000

This paper reports on the disruption of normally effective ecological and behavioural barriers, at a contact zone on Monemvasia peninsula (Peloponnese, Greece), between parapatric populations of the land snail species Albinaria discolor and Albinaria campylauchen. Detected outcomes were the increase of gene flow between these two species at the contact zone, the occurrence of rare alleles, as well as the occurrence of morphologically and ethologically intermediate A. campylauchen specimens. Furthermore, there was high genetic variability along with deficiency of heterozygotes, probably due to inbreeding. Additionally, the study of mortality of these populations, during the adverse summer aestivation period, indicated that A. campylauchen individuals with an inappropriate combination of morphological and behavioural characteristics suffered heavily. The maladaptation and disadvantage of these specimens revealed the possible action of a postzygotic isolation mechanism between these two morphologically, molecularly and electrophoretically distinct species, that can, however, mate under laboratory conditions. Finally, the above situation is discussed in terms of exogenous versus endogenous selection.

 2000 The Linnean Society of London

ADDITIONAL KEY WORDS:—hybridization – heterozygote deﬁciency – aestivation ethology – premating barriers – exogenous selection.

CONTENTS Introduction ...... 756 Material and methods ...... 757 The study area ...... 757 The studied populations ...... 758 Sampling ...... 759 Allozyme electrophoresis ...... 760 Allozyme data analysis ...... 760 Mortality analysis ...... 761 Results ...... 761

∗ Corresponding author. E-mail: [email protected] 755 0024–4066/00/120755+16 $35.00/0  2000 The Linnean Society of London 756 S. GIOKAS ET AL.

Allozymes ...... 761 Mortality ...... 761 Density ...... 764 Discussion ...... 764 Acknowledgements ...... 767 References ...... 768

INTRODUCTION

Contact zones, especially those in which hybridization occurs, are of special interest, providing data to test theories of speciation and the effects of gene flow and of selection on genetics dynamics (Woodruff, 1973; Endler, 1977; Barton & Hewitt, 1981, 1985; Hewitt, 1989; Harrison & Rand, 1989). In particular, studies of gene flow are integral to interpretation of microevolutionary patterns and geographic structure. In terrestrial molluscs the majority of studies on natural hybridization concern the well-known genera Cerion (Gould & Woodruff, 1978; Galler & Gould, 1979; Woodruff & Gould, 1987; Woodruff, 1989), Partula (Clarke, 1968; Johnson, Murrey & Clarke, 1993) and Cepaea (Cain & Currey, 1963; Johnson, 1976) and have been proved very informative in the resolution of former ambiguities on taxonomy and evolutionary processes. During the last decade evolutionary aspects of the pulmonate genus Albinaria have been studied by several scientists, (e.g. Ayoutanti et al., 1987, 1993; Mylonas et al., 1987; Gittenberger, 1988, 1991; Kemperman, 1992; Ayoutanti, 1994; Schilthuizen, 1994a; Douris et al., 1995; Schilthuizen, Gittenberger & Gultyaev, 1995; Giokas, 1996; Douris et al., 1998a,b). Albinaria is distributed in Greece, Asia Minor, Cyprus and The Lebanon and over 80 species and 200 subspecies have been described within this area (Nordsieck, 1977, 1999). Most populations of the genus are very localized, occurring allopatrically, or more seldom parapatrically, with other congeneric species, and cases of genuine sympatry are uncommon (Schilthuizen, 1994a; Giokas, 1996; Douris et al., 1998b). However, there is considerable dis- agreement on the taxonomic status of the taxa within the genus. Some data indicate that some Albinaria species are allozymically very close or occasionally indistinguishable (Ayoutanti et al., 1987; Kemperman & Degenaars, 1992; Schilt- huizen & Gittenberger, 1996), and under laboratory conditions prove to be inter- fertile (Mylonas et al., 1987; Giokas, 1996). Additionally, hybridization and introgression, based on morphological characters, have been reported (Fuchs & Kau¨fel, 1936; Gittenberger, 1979, 1991; Nordsieck, 1979, 1984; Ra¨hle, 1979). Ribosomal RNA (Schilthuizen et al., 1995) and mitochondrial DNA analyses (Douris et al., 1998a,b) reinforced these arguments and revealed interegeneric introgression, especially between the genera Albinaria and Isabellaria. Contact, (or more seldom, hybrid) zones within or between taxa belonging to those genera are known (Nordsieck, 1984; Kemperman, 1992; Giokas, 1996; Douris et al., 1998b). Several genetic studies on Albinaria have investigated: population structure and levels of gene flow of Albinaria corrugata (Schilthuizen & Lombaerts, 1994), reproductive isolation of the parapatric species A. hippolyti, A. spratti and A. ulrikae (Schilthuizen, 1994b), hybridization between adjoining populations of Albinaria taxa on Ionian islands (Kemperman & Degenaars, 1992), and structure of hybrid zones between subspecies of Albinaria hippolyti (Schilthuizen & Gittenberger, 1994; Schilthuizen, 1995a,b). The results of these studies indicated that the population structure of A. corrugata corresponds closely to the so-called stepping stone model (Schilthuizen & GENE FLOW AND DIFFERENTIAL MORTALITY IN A CONTACT ZONE 757

Figure 1. The sampling area on the Monemvasia peninsula and the distribution of Albinaria specimens. (Χ) A. discolor,(Β) A. campylauchen,(_) intermediates.

Lombaerts, 1994), and that complete reproductive isolation between certain nominal species is present (Schilthuizen, 1994b) or introgression occurs (Kemperman & Degenaars, 1992). Furthermore, it has been proposed that certain hybrid zones between subspecies may have resulted from secondary contact and are maintained by a balance between dispersal and selection against hybrids, supporting a peripatric mode of (sub)speciation in small isolated populations (Schilthuizen, 1994a). Ad- ditionally, there are several studies on the geographical structure of genetic variability of Albinaria populations indicating either the presence of heterogygote deﬁciency (Kemperman & Degenaars, 1992; Ayoutanti, 1994) or its absence (Schilthuizen & Gittenberger, 1996). Our paper aims to contribute to the discussion of microevolutionary processes in Albinaria. The partially contradictory results from previous studies and the rarity of contact or hybrid zones in Albinaria make their study, when they do exist, more important. We studied a contact zone in a small area adjacent to a road connecting Monemvasia peninsula and the Peloponnese mainland in Greece, between two parapatric populations of A. discolor and A. campylauchen (former Isabellaria campylauchen). These two species are distinct, whether measured by molecular or morphological markers (Giokas, 1996; Nordsieck, 1997; Douris et al., 1998b). This example was of particular interest because we had indications of hybridization and selection. There- fore, in order to investigate the disruption of isolating mechanisms, we examined alterations in the level of gene ﬂow level and departures from Hardy–Weinberg equilibrium. We also estimated the specimens’ viability during adverse hot and arid summer conditions.

MATERIAL AND METHODS

The study area

Field experiments and sampling were carried out on Monemvasia peninsula (Fig. 1). This peninsula, a former islet of 300 m altitude and 1.8 km long) is connected to the Peloponnese mainland by a narrow man-made bridge. The area is extremely arid and hot, especially during summer (Mavrommatis, 1978). The terrain is barren, 758 S. GIOKAS ET AL. without large-scale vegetation; there are only small shrubs (phrygana) and abandoned ﬁelds. Limestone boulders, with many crevices, are scattered on the peninsula. These are the preferred habitat of Albinaria (Giokas, 1996).

The studied populations

Albinaria campylauchen from Monemvasia had originally been recorded as Isabellaria campylauchen. However, recent studies of mtDNA and rRNA (Schilthuizen et al., 1995; Douris et al., 1998b) contradicted the established discrimination of the genera Albinaria and Isabellaria, that was based on the form of the clausilial apparatus (CA) (a complex of folds and lamellae in the aperture which characterizes a fully developed Albinaria shell). Moreover, Douris et al. (1998b) proved that A. grisea and I. campylauchen are indistinguishable on the basis of their mtDNA; morphologically, after the exclusion of certain homoplasious characters associated with the CA, they are also quite close. Consequently, we consider I. campylauchen to be A. camplylauchen. A. campylauchen has a very localized allopatric distribution as it is found in only a few localities at the southernmost part of Parnon Mts. A. discolor has a broader distribution as it occurs allopatrically in certain localities in the south-east Pelo- ponnese, in Attiki and on Kea, Aigina and Velopoula islands. A. discolor and A. campylauchen are well discriminated by a set of morphological characters and this has been supported by cladistic analysis (Giokas, 1996; Douris et al., 1998b). At Monem- vasia A. discolor has a prominent basal keel and a normal type clausilial apparatus (NCA), whereas A. campylauchen has a prominent dorsal keel and a well developed Graciliaria type clausilial apparatus (GCA). Like most land snails in southern Greece, Albinaria is active in winter, feeding on the microflora; it spends the period from late April to late October in aestivation. During aestivation, clusters of specimens are often formed, sometimes reaching several tens of individuals (Schilthuizen, 1994a; Giokas, 1996). There are two basic aestivation strategies (Gittenberger, 1991; Giokas, 1996). Species with a thick white shell attach, after formation of a thick epiphragm, to rocks or plants in full sunlight, the thick white shell providing protection from radiation and evaporation. This strategy is found in A. discolor. Species with thinner (often mottled) shells aestivate deep in crevices, under stones or in the soil. This strategy is adopted by A. campylauchen, which lacks an epiphragm. Populations of A. discolor and A. campylauchen on the Monemvasia peninsula exhibit a parapatric distribution. A. discolor has a wider distribution in the north of the peninsula, while A. campylauchen is more localized in the south-west (Fig. 1). A zone of abandoned former fields, with low population densities, demarcates these populations. However, there is a zone, within the A. campylauchen distribution, where specimens belonging to both species occur syntopically. This zone is very small (about 30 m long, 3 m wide) (arrowed, Fig. 1), occupying a small area beside the road connecting Monemvasia village with the castle. There we found several morphologically intermediate specimens (alive and empty shells) which could be distinguished in the field from the two parental types by the relative lack of prominence of either keel (Fig. 2). Additionally, we found specimens which although resembling A. campylauchen more closely in their shell characteristics (including shell thickness and GCA), had aestivation behaviour that resembled that of A. discolor. Some of these specimens were in less exposed sites, including rock crevices and GENE FLOW AND DIFFERENTIAL MORTALITY IN A CONTACT ZONE 759

Figure 2. From left to right (dorsal aspect): A. discolor, three intermediate specimens, A. campylauchen; scale bar=5 mm.

holes. Juveniles were harder to characterize as intermediate specimens from their shell characteristics, as they had not completed their shell development and many diagnostic characters (lamellae, keels and peristome) associated with the aperture had not formed. Consequently, besides viability analysis during aestivation, an estimation of gene ﬂow at the contact zone was considered necessary in order to obtain data on possible introgression.

Sampling

After a thorough investigation of the peninsula in May 1991 (first month of aestivation), we traced the area of syntopic occurrence of A. discolor and A. campylauchen (Fig. 1). We used non-toxic paint to mark the aestivating positions of 42 A. campylauchen and 51 A. discolor specimens in the contact zone. All aestivating specimens were classified individually as adults or juveniles, and their aestivation behaviour was recorded. Since these snails do not move once aestivation has started, positions rather than shells were marked to avoid disturbance. A further set of aestivation positions for 70 A. discolor specimens was also marked nearby (Site 1), where A. campylauchen specimens did not occur. It was impossible to mark A. campylauchen individuals that were aestivating deep in crevices, because these crevices were extremely deep and narrow. In the middle of September 1991, about a month before the end of the aestivation period, the sites were revisited, and the status of each marked specimen (alive, dead, missing, adult, juvenile) was recorded. After the end of our field observations, two samples of specimens from Monemvasia 760 S. GIOKAS ET AL.

T 1. Enzymes assayed, E.C. numbers, scored loci and number of alleles per locus detected in the electrophoretic analysis

Enzyme system E.C. Loci scored No. of Number alleles

Glucose-6-phosphate Dehydrogenase 1.1.1.49 G6pdh 2 Octanol Dehydrogenase 1.1.1.73 Odh 2 Esterase 3.1.1.– Est-1, Est-2 1, 4 Superoxide Dismutase 1.15.1.1 Sod-1, Sod-2 1, 1 Isocitrate Dehydrogenase 1.1.1.42 Idh-2 3 Peptidase (substrate: glycil-L-leucine) 3.4.–.– Pep-A 4 Malate Dehydrogenase 1.1.1.37 Mdh-1, Mdh-2 2, 4 Aspartate Aminotransferase 2.6.1.1 Aat-1 3 Phosphoglucomutase 5.4.2.2 Pgm-2 2

peninsula (A. campylauchen and A. discolor from the contact zone and A. discolor from Site 1) were collected. Additionally, a random sample from a very localized population (sampling area 25 m long, 5 m wide) of A. campylauchen from Sykea (8 km from Monemvasia) was collected. Forty sampling quadrats of 50 cm×50 cm were used. The shape and size of each quadrat was determined after preliminary sampling work to determine relative cost×relative variance (Krebs, 1989). A 20% conﬁdence limit was deliberately chosen in order to set the sample size, according to Elliot (1971). Specimens of were analysed electrophoretically.

Allozyme electrophoresis

Specimens were homogenized whole in an equal volume of grinding buﬀer (0.05 M Tris-HCl pH 7.1) and centrifuged under 5°Cat15000g for 5 min. The supernatant was stored at −80°C until subjected to electrophoresis. Vertical poly- acrylamide gel electrophoresis (PAGE) was performed and nine enzyme systems representing 12 presumptive loci were scored for each individual in all populations (Table 1). Zymograms were visualized under standard staining procedures (Murphy et al., 1996).

Allozyme data analysis

Allele frequencies were calculated and Nei’s (1978) unbiased expected heterozygosity was estimated in order to measure genetic variability of the examined populations. Departure from Hardy–Weinberg expectations was checked. Estimation of the Hardy–Weinberg exact probability was performed by the complete enumeration method (Louis & Dempster, 1987) for each locus in each population. A Fischer test was used to combine information over loci and over populations. In order to quantify the effect of migration on the genetic differentiation of these populations, gene flow was estimated between each pair of populations. Estimation of gene flow based on the degree of subdivision between populations (Fst) according = − ff to the formula: Nm (1/Fst 1)/4 (Wright, 1931, 1978), where N is the e ective population size and m the effective migration rate. All calculations were performed GENE FLOW AND DIFFERENTIAL MORTALITY IN A CONTACT ZONE 761 with the computer programs GENEPOP v. 3.1 c (Raymont & Rousset, 1995) and POPGENE v. 1.31 (Yeh & Boyle, 1997).

Mortality analysis

Contingency table analysis (including 2, G, Yates corrected 2 and Fisher exact (two tailed) tests, Zar (1984)) was used to test the statistical significance of differences in mortality. We examined differences in the survival between: (a) A. discolor and A. campylauchen marked specimens, (b) the age classes of the A. campylauchen marked specimens, (c) the aestivation categories of the A. campylauchen marked specimens. Additionally, we tested the difference in the proportions of age classes between A. discolor and A. campylauchen marked specimens at the contact zone.

RESULTS

Allozymes

Three out of 12 loci were monomorphic in all populations (Sod-1, Sod-2 and Est- 1). Allele frequencies of the nine polymorphic loci in all examined populations are presented in Table 2. Averaged observed heterozygosity (Ho) ranged from 0.092 (Adc) to 0.158 (Acc) while average expected heterozygosity (He) ranged from 0.140 (Ad) to 0.241 (Acc). Three cases of introgressed alleles were detected. In A. campylauchen, Idh-2B, Pep- AC and Pgm-2B alleles have been introgressed to the population of A. discolor (Adc) at the zone of contact (Table 2). Rare alleles (Woodruff, 1989) have been observed in the two contact zone populations in two loci: Mdh-2C and Aat-1C alleles are restricted in the contact populations in almost equal frequencies (Table 2). Significant departures from Hardy–Weinberg equilibrium against heterozygotes are clearly indicated (Table 3). Significant deficiency of heterozygotes occurs in the two contact populations (Adc, Acc) in G6pdh, Idh-2 and Pgm-2 loci which code for introgressed alleles (Table 2). Heterozygote deficiency has also been observed in the A. campylauchen ‘reference’ population in G6pdh and Est-2 loci (Table 3). High values of Nm were observed in several gene loci between contact zone populations (G6pdh, Nm=24.25; Odh, Nm=6.50; Idh-2, Nm=4.69; Aat-1, Nm= 35.41). Pairwise estimates of gene flow (Nm) indicate a relatively high interspecific gene exchange (Adc–Acc, Nm=0.933) compared to this between conspecific populations (Ad–Adc, Nm=1.866; Ac–Acc, Nm=1.512) (Table 4). The threshold value of Nm= 1 has been indicated by Wright (1931) as the limit to maintain genetic homogeneity among populations.

Mortality

The absolute numbers and the percentages of live and dead snails found at the end of the ﬁeld experiment compared to those recorded at the start of the observation are shown in Table 5. One A. campylauchen and 11 A. discolor marks were not rediscovered. 762 S. GIOKAS ET AL.

T 2. Table of polymorphic loci of four Albinaria populations showing allele frequencies, sample size (n), average observed and expected heterozygosity and standard errors, and proportion of polymorphic loci (P). Ad: A. discolor, Adc: A. discolor (contact zone), Acc: A. campylauchen (contact zone), Ac: A. campylauchen

Locus Allele Ad Adc Acc Ac

G6pdh A 0.900 0.444 0.545 0.825 B 0.100 0.556 0.455 0.175 n 1091120 Odh A 1.000 1.000 0.929 1.000 B 0.000 0.000 0.071 0.000 n 10 10 7 20 Est-2 A 0.900 0.900 0.250 0.316 B 0.100 0.000 0.750 0.000 C 0.000 0.100 0.000 0.605 D 0.000 0.000 0.000 0.079 n 10 10 10 19 Idh-2 A 1.000 0.667 0.400 0.289 B 0.000 0.333 0.400 0.132 C 0.000 0.000 0.200 0.579 n 109519 Pep-A A 0.400 0.500 0.182 0.125 B 0.600 0.250 0.000 0.000 C 0.000 0.250 0.682 0.825 D 0.000 0.000 0.136 0.050 n 10 10 11 20 Mdh-1 A 1.000 1.000 0.864 1.000 B 0.000 0.000 0.136 0.000 n 10 10 11 20 Mdh-2 A 0.700 0.900 0.773 0.950 B 0.300 0.050 0.000 0.025 C 0.000 0.050 0.045 0.000 D 0.000 0.000 0.182 0.025 n 10 10 11 20 Aat-1 A 0.700 0.850 0.909 0.975 B 0.300 0.050 0.000 0.025 C 0.000 0.100 0.091 0.000 n 10101120 Pgm-2 A 1.000 0.800 0.000 0.000 B 0.000 0.200 1.000 1.000 n 10 10 11 19 Av. He 0.140 0.209 0.241 0.152 SE 0.056 0.064 0.066 0.062 Av. Ho 0.133 0.092 0.158 0.116 SE 0.071 0.054 0.057 0.054 P 0.416 0.583 0.667 0.500

Whereas 97.5% of A. discolor still in place had survived, as had 95.7% of the 70 A. discolor specimens in Site 1, 41.5% of the A. campylauchen specimens had died, probably because of desiccation (their shells were not broken or drilled) (Table 5). The difference was statistically significant (in all tests P<0.001) implying that these intermediates suffered heavily during aestivation. Age appeared to have no effect on mortality of A. campylauchen specimens since juveniles and adults had quite similar mortality (Table 5) (in all tests P>0.9). GENE FLOW AND DIFFERENTIAL MORTALITY IN A CONTACT ZONE 763 13.2 24.9 38.9 25.3 102.3 0.0320 0.0004 0.0056 0.0000 = = = = N.S. = 10 10 14 14 Weinberg 28 2 2 2 2 = = = = 2 – Fisher test P P P P s method used to ’ ∗ − − − + d Weinberg expectation (exact Hardy – N.S. N.S. 0.185 0.265 0.320 0.096 0.049 0.420 0.420 ) from Hardy − − − + D 0.480 cant deviations ( fi A. campylauchen. 20.4 N.S. ∗ ∗ − d d 0.0024 = 6 2 = P (contact zone), Ac: 25.5 ∗ d 0.0013 = 8 0.180 0.444 0.625 0.528 0.564 0.301 0.180 combine loci, population and populations/loci test results 2 = P are quasi-monomorphic loci. A. campylauchen ) mean heterozygosities and signi Pgm-2 He , + − − − <0.001. P Mdh-1 ∗∗ , Odh (contact zone), Acc: <0.01, 44.4 ∗ ∗∗ ∗ P d d d ∗ 0.0000 = 0.000 0.000 0.000 0.600 0.200 0.300 0.000 0.000 0.143 0.300 0.000 0.545 0.273 0.455 0.182 0.494 0.289 0.180 0.496 0.133 0.375 0.640 0.483 0.236 0.368 0.165 0.000 0.000 0.600 0.600 0.400 0.050 0.263 0.579 0.350 0.100 0.050 ) and expected ( G6pdh Odh Est-2 Idh-2 Pep-A Mdh-1 Mdh-2 Aat-1 Pgm-2 10 2 = P Ho A. discolor ciency; fi D D D D He He He He Ho Ho Ho Ho , Adc: 3. Observed ( A. discolor Analysis not possible because Ad: d, heterozygote de + test  T Population Ad probability following the complete enumeration method of Louis & Dempster (1987) was performed for each locus in each population. Fisher Fisher Adc Acc Ac 764 S. GIOKAS ET AL.  = T 4. Pairwise esimates of Nm by Fst method (Nm 0.25 (1-Fst/Fst)

Population A. discolor A. campylauchen Site 1 Contact Contact Sykea

A. discolor — 1.8664 0.4657 0.5630 Site 1 — 0.9336 0.6605 Contact A. campylauchen — 1.5126 Contact − Sykea

T 5. Survival of the marked Albinaria specimens found at the end of the experiment in the contact zone. In each column the ﬁrst number indicates the live specimens, the second refers to the recaptured specimens in total. In parentheses the survival perentage

Total Adults Juveniles

A. discolor 39/40 (97.5%) 23/23 (100%) 16/17 (94.1%) A. campylauchen 24/41 (58.5%) 7/12 (58.3%) 17/29 (58.6%) Unexposed A. campylauchen 17/26 (65.4%) 5/9 (55.5%) 12/17 (70.6%) Exposed A. campylauchen 7/15 (46.7%) 2/3 (66.6%) 5/12 (41.6%)

A. campylauchen individuals (in both age classes and overall) aestivating in exposed sites on the rock surface suﬀered higher mortality than those found in crevices (Table 5). However, this was not formally signiﬁcant (in all tests P>0.2).

Density

Population mean densities were extremely low at the zone of contact (4.2 specimens/m2 and 5.3 specimens/m2 for A. campylauchen and A. discolor respectively), considering that densities of Albinaria populations are usually much higher (Schilt- huizen & Lombaerts, 1994; Giokas, 1996). Densities were quite high at Site 1 and Sykea. At Site 1 the density of A. discolor was 19.2 specimens/m2 and in Sykea the density of A. campylauchen was 9.7 specimens/m2. There was a signiﬁcant diﬀerence (P<0.01 in all tests) in the proportions of the size classes (adults, juveniles) that participated in the formation of the populations of A. discolor and A. campylauchen (Table 5) in the contact zone, with 57.5% of A. discolor, but only 29.3% of A. campylauchen, being adult.

DISCUSSION

The results of the field observations showed that A. campylauchen snails in exposed sites in the contact zone suffered heavier mortality during the summer than A. discolor specimens. This mortality was considerably higher in comparison with that detected in several other Albinaria populations. Giokas (1996) reports mortality percentages varying from 6% to 17%. Rock-dwelling snails, as a group, have a low GENE FLOW AND DIFFERENTIAL MORTALITY IN A CONTACT ZONE 765 physiological capacity to resist desiccation and they compensate for this by cryptic ethology (Arad, Goldenberg & Heller, 1995). Additionally, it is suggested that clausiliids have some morphological, physiological and behavioural mechanisms for regulating water balance (Warburg, 1972; Kemperman & Gittenberger, 1988; Arad et al., 1995; Giokas, 1996; Douris et al., 1998b). However, there is no general agreement on the significance of these mechanisms. Warburg (1972) and Nordsieck (1982) have stressed the role of the door-like clausilium. Kemperman & Gittenberger (1988), Arad et al. (1995), Gittenberger & Schilthuizen (1996) and Giokas (1996) have proposed that water loss is reduced by adherence to the rock or plant surface combined with epiphragm formation and in this way evaporation occurs mainly through the shell wall. Cryptic behaviour, especially in crevices, has also been suggested (Heller & Dolev, 1994; Arad et al., 1995; Giokas, 1996) as an effective response to arid conditions. The latter authors also pointed that aggregating can lessen mortality during aestivation. Therefore, we believe that a re-evaluation of the adaptive role of certain morphological characters is necessary. In particular, the adaptive role of the clausilial apparatus (CA) during aestivation appears less important. The GCA is now considered as a parallelism which originated several times within Clausiliidae (Gittenberger & Schilthuizen, 1996; Douris et al., 1998b; Gittenberger, 1998). Some populations of A. discolor and A. grisea (mtDNA analysis places A. campylauchen into A. grisea, Douris et al., 1998b) may have a GCA whereas others may lack it. Furthermore, the difference in age-structure between the populations of A. discolor and A. campylauchen suggested that probably few exposed A. campylauchen specimens survive to maturity. Given the tendency of A. campylauchen specimens aestivating unexposed to survive better than those which are exposed, it is reasonable to conclude that desiccation is prohibited mainly by the thick shell, the formation of the epiphragm and the contagious and cryptic ethology; three features that A. camplylauchen specimens at the contact zone are lacking. Additionally, it is probable that the widening of the road has resulted to the modification of the normal cryptic aestivation behaviour of A. campylauchen specimens which led to the higher mortality that was observed for these individuals. The deficiency of heterozygotes was the second outcome of our study. With the exception of the sample of A. discolor at Site 1, all samples showed significant departures from the Hardy–Weinberg equilibrium. Similarly, Kemperman & Degenaars (1992) report that 71% of 42 Albinaria populations, belonging to four nominal species from Ionian islands, exhibit deficiency of heterogygotes. The same result is revealed from the study of Ayoutanti (1994) on 31 Albinaria populations from southern Greece. However, Schilthuizen & Gittenberger (1996) did not observe significant departures from Hardy–Weinberg equilibrium. A deficit of heterogygotes has also been reported in some other lands snails such as Chondrina clienta (Baur & Klemm, 1989), Helix aspersa (Guiller, Coutellec-Vreto & Madec, 1996), and Zonitoides nitidus ( Jordaens et al., 1998). Heterozygote deficiency in land snails has been attributed to a breeding system with incomplete panmixia and inbreeding. Facultative self-fertilizing breeding systems are known for Rumina decollata (Selander & Kaufman, 1973), Partula gibba ( Johnson, Clarke & Murray, 1977), some Arion species (McCracken & Selander, 1980), Chondrina clienta (Baur & Klemm, 1989) and Zonitoides nitidus ( Jordaens et al., 1998). Giokas (1996) reports that specimens from several Albinaria species (such as A. discolor and A. grisea) produced, in the laboratory, offspring without copulation. However, Kemperman & Degenaars (1992) did not conclude whether the observed deficiency of heterozygotes in Ionian Albinaria taxa is caused by facultative apomixis 766 S. GIOKAS ET AL. or by inbreeding. Ayoutanti (1994) attributed deficiency of heterozygotes in Albinaria populations either to Wahlund effect (with low mobility of Albinaria specimens attributed to the formation of panmictic subdemes) or to mating with relatives. Schilthuizen & Lombaerts (1994) and Giokas (1996) reported mean lifetime dispersal of Albinaria specimens amounting to 2 m. Additionally, Giokas (1996) reported that indiviuals of Albinaria tend to cluster together during aestivation and that there is a temporal synchronization of awakening, caused by autumn rains, and a consequent synchronization of copulation. Hybridization between A. discolor and A. campylauchen was indicated by morphological (lack of prominent keels) and ethological features (resting behaviour during aestivation) of specimens found at the zone of sympatry, and the results of the genetic analysis reinforced this view. High values of Nm were observed in several gene loci between contact zone populations and three cases of introgressed alleles from A. campylauchen to A. discolor have been detected. Additionally, pairwise estimates of gene flow (Nm) indicated a relatively high interspecific gene exchange compared to this between conspecific populations (Table 4), quite close to the threshold value of Nm=1. The fact that the three introgressed alleles that have been detected at the contact zone pass exclusively from A. campylauchen to A. discolor (Table 2) can be explained by the hypothesis that as A. discolor specimens are the minority within the zone of sympatry (they occur within the distribution of A. campylauchen), and there is a higher probability to receive alleles that are common to A. campylauchen specimens. The presence of rare alleles (in two loci) only in the populations in sympatry makes the hypothesis of hybridization more plausible (Barton & Hewitt, 1985; Woodruff, 1989). Rare alleles (Slatkin, 1985), and their products (‘hybrizymes’) have been also detected in other cases in Albinaria (Kemperman & Degenaars, 1992; Schilthuizen & Gittenberger, 1994). The possible mechanisms by which hybrizymes could arise, such as intracistronic recombination, gene conversion and relaxed suppression of mutation, have yet to be confirmed. The high percentage of polymorphic loci and the high expected heterozygosity (He) (Table 2), especially at the contact zone, is indicative of high genetic variability. The percentages detected are higher than those reported for other Albinaria species and populations. Kemperman & Degenaars (1992) report that the polymorphic loci per Albinaria population from Ionian islands varies between 0.0% and 17.9%, with an average of 7.5%, whereas Schilthuizen & Gittenberger (1996) report percentages that are varying between 0.0% and 35.3%, with an average of 18.1%. However, their estimated polymorphic loci percentages for A. discolor and A. grisea populations are 29.4% and 35.3% respectively. The major questions concern the factors that generate and maintain this hybrid zone between these Albinaria species that are morphologically and molecularly distinct and have sufficient differences in morphology and behaviour to make such sympatry explicable in terms of conventional niche theory. We suggest the following hypothesis. Increased genetic variability, as is indicated by introgressed and rare alleles and the percentage of polymorphic loci at the contact zone, is the result of increased gene flow because of the higher probability for inter-specific copulation (due to the modification of aestivation ethology of A. campylauchen specimens). Additionally, the significant deficiency of heterozygotes is probably the result of inbreeding between A. discolor and A. campylauchen specimens that forms ‘mixed-demes’ constituted by individuals belonging to both species. In Albinaria, no mate recognition mechanisms are known, as shown by laboratory experiments (Mylonas et al., 1987; Schilthuizen, GENE FLOW AND DIFFERENTIAL MORTALITY IN A CONTACT ZONE 767 1995b; Giokas, 1996) and field observations (Giokas, 1996). The deficit of heterozygotes in the population of A. campylauchen at Sykea could also be the result of inbreeding. Another less possible cause of heterozygote deficiency could be high mortality during adverse periods, which in small populations such those in the contact zone and Sykea, could result in a short-term bottleneck. The observations made here, and the fact that the two species were copulating freely in the laboratory producing F1 offspring identical to the mother (Giokas, 1996), suggested that the primary premating isolating mechanisms in Albinaria are ecological and behavioural. The association of the restricted area of contact with a road suggests that human disturbance may upset normally effective ecological barriers (Hewitt, 1989). We suggest that ecological, habitat, and ethological premating isolation mechanisms, that normally prevent inter-population crosses, are modified in this zone and postzygotic isolation comes to the forefront in the form of a reduced viability of specimens exhibiting an abnormal aestivation behaviour. However, there is only indirect evidence for hybrid disadvantage at this zone in the form of the occurrence of rare alleles. This case is similar to the narrow, border-like hybrid zones which exist between subspecies of A. hippolyti (Schilthuizen & Lombaerts, 1994), where direct evidence of hybrid disadvantage is missing. We have no data to correlate directly the suspected morphological or ethological hybrids with actual genetic hybrids. Selection is acting against specimens having the morphology of A. campylauchen and the aestivation ethology of A. discolor. However, it is tempting to interpret the above situation in terms of exogenous versus endogenous selection (Moore & Price, 1993). The above case could be considered as evidence for exogenous selection with environmental selection acting directly on the two phenotypes producing a hybrid zone at the interface between the two habitats (rocks and bare former cultivated fields). The alternative hypothesis is that this zone may be a tension zone (Key, 1968; Barton & Hewitt, 1985), in which there is a balance between gene flow and selection against individuals of mixed ancestry maintains narrow hybrid zones. The association of the hypothesized hybrids with an ecotone favours the first hypothesis. Harrison (1993) suggests several outcomes for hybrid zones such as speciation, fusion, extinction of taxa, weakening or strengthening of barriers to gene exchange. Even though our study suffers from the fact that we do not have adequate estimates of survival of A. campylauchen found deep in crevices, or of the fertility of adult hybrids, and an accurate estimate of the panmictic unit, it does provide evidence for a possible scenario concerning species which are normally ecologically distinct and effectively reproductively isolated. Consequently, we can suggest that identity and cohesion within Albinaria may be maintained by the dynamic combination of demographic and genetic exchangeability, especially if we consider the role of the life history characteristics and the population dynamics of Albinaria, such as contagious distribution, limited mobility and aestivation ethology.

ACKNOWLEDGEMENTS

We are grateful to Dr R. M. Polymeni (University of Athens, Department of Biology, Section of Zoology & Marine Biology) for providing us the facilities of her laboratory, and to the editor and anonymous referees for their valuable comments. 768 S. GIOKAS ET AL.

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