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 in- trogression, 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, espe- cially 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