Proc. Natl. Acad. Sc?. USA Vol. 77, No. 1, pp. 684-688, January 1980 Population Biology

Self-fertilization and monogenic strains in natural populations of terrestrial (breeding systems/hermaphroditic molluscs/colonization/general-purpose genotypes) GARY F. MCCRACKEN* AND ROBERT K. SELANDERt Department of Biology, University of Rochester, Rochester, New York 14627 Communicated by Ernst Mayr, October 22, 1979

ABSTRACT Electrophoretic studies of genetic variation in MATERIALS AND METHODS 14 of terrestrial slugs of the families Arionidae, Phil- omycidae, and Limacidae in the eastern United States indicate Collections. Specimens were collected in forests, woodlots, that self-fertilization, either facultative or obligatory, is the old fields, greenhouses, and roadside dumps. Most samples were normal breeding system in six of the species. Three of these six taken from areas smaller than 10 M2. In all, 2812 specimens species are single monogenic strains; one consists of three mo- from 80 populations were subjected to electrophoresis. nogenic strains; one includes a highly heterozygous form and Electrophoresis. Frozen were chopped into small two monogenic strains; and one has a moderate amount of pieces, and the pieces were soaked for 20 min in cold buffer polymorphism but little heterozygosity and strong linkage disequilibrium. Eight species are outcrossers, being highly (1:1, vol/vol). The mixture was centrifuged at 50,000 X g for polymorphic and panmitftic within local populations. Niche 30 min at 4°C to produce a clear supernatant, which was stored breadth, assessed in terms of extent of geographic distribution at -70°C until analyzed by electrophoresis. Starch gel elec- and variety of habitats oceuipied and measured on an experi- trophoresis and biochemical staining of enzymes were carried mental plot of woodland; is greater in some monogenic strains out by the methods of Selander et al. (40). From 9 to 20 enzyme than in highly heterozygous, outcrossing species. Colonizing loci were studied in each species. success apparently is independent of the amount of genetic Statistics. Observed heterozygosity per individual in a variation carried by a species. sample (Ho) was determined by direct count, and mean ob- It is common knowledge that self-fertilization (automixis) is the served heterozygosity for a strain or species (Ho) was obtained normal breeding system of many. hermaphroditic plants in a by averaging values for all samples. Expected heterozygosity large number of taxa, and there is convincing evidence that per individual in a sample (He) was calculated from allele self-fertilization in plants has evolved as a complex ecogenetic frequencies, with correction for small sample size (41), and adaptation in response to various physical and biotic environ- averaged over all loci assayed. Coefficients of deviation of ob- mental factors (1-15). In contrast, it is widely believed that served (ho,) from expected (he) numbers of heterozygotes at hermaphroditic animals either cannot self-fertilize or do so only each locus were calculated as D = (ho- he)/he, weighted by as an auxiliary mode of reproduction when opportunities for expected numbers, and summed over all loci examined in a outcrossing (amphimixis) are unavailable (16-19). This notion sample to obtain mean heterozygote deviation (D). Mean de- persists despite continuing reports of direct and circumstantial viation for a strain or species (D) was obtained by averaging the evidence of regularly occurring self-fertilization in various Ds for all samples. animals (refs. 20-29 and refs. cited in refs. 30 and 31). The application, in our laboratory, of electrophoretic techniques RESULTS to the study of polymorphism in genes encoding enzymes has Information on genetic polymorphism and heterozygosity in shown that many taxonomic species of terrestrial molluscs 14 taxonomic species of terrestrial slugs belonging to three consist of one or more invariant strains that are known or pre- families (Arionidae, , and Limacidae) is sum- sumed to be maintained by self-fertilization. The first example marized in Table 1. Our results demonstrate that some species to be worked out in detail was the pulmonate land snail Rumina of all three families are strongly polymorphic at their structural decollata (32-34). gene loci, with high levels of heterozygosity and Hardy- Here we report that many species of terrestrial slugs of several Weinberg proportions of genotypes in local populations. Sur- families, including some of the more ubiquitous and familiar veys of electrophoretically detectable variation in enzymes in forms, are monogenic or nearly so, apparently as a consequence various pulmonate land snails-notably Helix aspersa (42, 43), of frequent or obligatory self-fertilization. With regard to Cepaea nemoralis (44-46), Triodopsis albolabris (47), Partula breeding systems, genetic structure of populations, and patterns spp. (48), Cerion spp. (49), and Theba pisana (50)-have re- of speciation, these hermaphroditic animals are closely similar vealed similarly high levels of genetic variation, H being ap- to self-compatible plants. Our findings have implications for proximately 0.15. This and other information on the repro- current theoretical work on the evolution of breeding systems ductive biology of these species indicates that they are either (17, 35) and ecological polymorphism, which involves the predominantly (47, 48) or obligatorily (42-46) outcrossing. The fundamental problem of the relationships between environ- agreement between observed and expected proportions of mental heterogeneity and the quantity and organization of heterozygotes in our genetically variable populations of slugs genetic diversity in populations (36-39). indicates that they, too, are largely if not completely am- phimictic. These species are Arion hortensis, A. subfuscus The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "ad- * Present address: Department of Zoology, University of Tennessee, vertisement" in accordance with 18 U. S. C. § 1734 solely to indicate Knoxville, TN 37916. this fact. t To whom reprint requests should be addressed. 684 Downloaded by guest on October 2, 2021 Population Biology: McCracken and Selander Proc. Natl. Acad. Sci. USA 77 (1980) 685

Table 1. Genetic variation and breeding systems in terrestrial slugs Observed Number of Number of loci heterozygosity Heterozygote Popula- Individ- Exam- Poly- per individual deviation Species Region tions uals ined morphic (H0) (A) Breeding system Family Arionidae Arion fasciatus New York 10 814 18 0 0 Self-fertilizing A. circumscriptus New York 4 312 18 0 0 Self-fertilizing A. silvaticus New York 3 29 17 0 0 Self-fertilizing A. intermedius: Strain 1 New York 3 148 20 0 0 Self-fertilizing Strain 2 New York 2 45 20 0 0 Self-fertilizing Strain 3 New York 1 9 20 0 0 Self-fertilizing A. hortensis New York 4 135 12 8 0.159 -0.023 Outcrossing A. subfuscus Form 2 New York, 6 105 11 7 0.197 -0.053 Outcrossing Connecticut Form 4 Strain 1 New York 4 213 18 0 0 - Self-fertilizing Strain 2 New York 1 11 19 0 0 Self-fertilizing Family Philomycidae carolinianus New York 1 48 12 3 0.062 -0.015 Outcrossing P. species 1 Tennessee 2 15 12 9 0.198 +0.019 Outcrossing P. species 2 Georgia 4 40 12 8 0.109 -0.096 Outcrossing P. species 3 Georgia 3 13 11 1 0.005 0 Outcrossing (?) Family Limacidae Limax maximus New York, 3 52 11 5 0.166 +0.071 Outcrossing Tennessee, N. Carolina Lehmannia valentiana Georgia, 4 140 12 4 0.077 +0.014 Outcrossing Tennessee Deroceras reticulatum New York 13 367 10 8 0.188 -0.048 Outcrossing D. Iaeve New York, 12 316 9 7 0.005 -0.902 Mixed Virginia, N. Carolina, Tennessee, Georgia, Florida

(form 2; 51-53), Philomycus carolinianus and three uniden- that a distinctive color form of A. circumscriptus has been re- tified species of Philomycus, Limax maximus, Lehmannia ported (51, 52). valentiana, and Deroceras reticulatum. Of all the species, the most complex pattern of variation is In contrast to these highly variable forms, several other seen in the limacid Deroceras laeve. Many loci were poly- species listed in Table 1 showed no detectable heterozygosity, morphic in populations of this species, but observed levels of despite our extensive surveys of both individuals and loci. heterozygosity invariably were much lower than expected on Within two of these taxa, we detected two (A. subfuscus, form the basis of random mating, as indicated by the large overall 4) and three (A. intermedius) distinctive monogenic strains. heterozygote deficiency (6 = -0.902). Table 2 summarizes the This pattern of population structure, involving an absence or genetic data for all 12 populations of D. laeve sampled. Two low level of heterozygosity and the distribution of existing populations were monogenic, but all others had two or more variation largely among monogenic strains, has been described polymorphic loci and expected levels of heterozygosity ranging in the snail R. decollata (34), in other hermaphroditic animals from 0.04 to 0.12. However, heterozygotes were observed in (29, 54), and in many species of plants (5, 13). It is the expected only four populations, all of which had large heterozygote de- result of frequent self-fertilization and the consequent decay ficiencies. In several polymorphic populations there was also of heterozygosity. strong linkage disequilibrium among loci. A similar pattern was The strains within species are closely related to one another. observed in R. decollata in France, where strains occurring Expressed in terms of Nei's coefficient (55), the mean genetic sympatrically sometimes show low levels of outcrossing (34); identity (or similarity) (i) of pairs of the three strains of A. in- and linkage disequilibrium is also characteristic of self-fertil- termedius was 0.867 (range, 0.80-0.95), and that of the two izing plants. strains of A. subfuscus was 0.94. The monogenic species A. There is other evidence that D. laeve is capable of both out- fasciatus, A. circumscriptus, and A. silvaticus (members of the crossing and self-fertilization. Copulation has been observed "A. fasciatus complex"), which are morphologically similar and a spermatheca has been described by some authors (56, 57); and until recently were classified as one species (51, 52, 56, 57), others have maintained populations in the laboratory through are genetically more dissimilar (I = 0.65; range, 0.62-0.69) than 12 or more generations without outcrossing (26). Anatomically, are the strains of either A. intermedius or A. subfuscus. Al- adult individuals in both Europe and North America vary in though we have identified only one strain of each of these the extent of development of the male parts of the genitalia (23, species, we suspect that others exist; and, in this regard, we note 26, 56, 57); and uniformly aphallic populations have been re- Downloaded by guest on October 2, 2021 686 Population Biology: McCracken and Selander Proc. Natl. Acad. Sci. USA 77 (1980) Table 2. Genetic polymorphism and heterozygosity in populations of Deroceras laeve Number of loci Heterozygosity (H) Heterozygote Locality Indiviluals Examined Polymorphic Expected Observed deviation (D) Tompkins County, NY 39 10 4 0.119 0.011 -0.902 Monroe County, NY (1) 29 10 2 0.071 0 -1.000 Monroe County, NY (2) 43 9 3 0.061 0.008 -0.872 Pulaski County, VA 8 9 2 0.044 0 -1.000 Knox County, TN 43 10 4 0.056 0 -1.000 Chatham County, NC 26 9 2 0.058 0 -1.000 Burke County, NC 19 9 2 0.063 0.029 -0.534 Tift County, GA 37 9 2 0.055 0.009 -0.710 Alachua County, FL (1) 9 9 0 0 0 Alachua County, FL (2) 18 9 0 0 0 Alachua County, FL (3) 13 9 4 0.046 0 -1.000 Alachua County, FL (4) 32 9 4 0.044 0 -1.000

ported. Abeloos (23) concluded that the phallic and aphallic argued that the optimal genotype for colonizing individuals or conditions exist as a polymorphism in populations. We have those that encounter widely fluctuating environments should found that phallic and aphallic individuals are otherwise in- be broadly adaptive ("general purpose"), with great phenotypic distinguishable phenotypically and with respect to genotypes plasticity (4, 6, 37, 63), whereas adaptations to specific mi- at structural gene loci. croniches should develop in more stable situations (19, 64). There are indications that many species of terrestrial slugs There is some empirical evidence that self-fertilization in plants we have not yet studied also self-fertilize and that some we have helps to fashion and to preserve both types of genomes (5-7, examined have different breeding systems in other regions. A. 10, 14). A likely disadvantage of self-fertilization is that all in- ater in Europe may consist of both outcrossing and self-fertil- dividuals become homozygous, and populations consisting of izing strains, as does A. subfuscus in North America. A. ater one monogenic strain may be unsuccessful in complex, stable rufus is highly heterozygous and amphimictic (ref. 58; un- habitats where rapid adaptive response to a diversity of re- published observations), but breeding experiments with a ge- combining, coevolving competitors, predators, parasites, and netic marker have shown that A. ater ater preferentially self- pathogens is required (6, 8, 34, 65). However, this condition fertilizes when mates are available (28); and British populations may be alleviated to the extent that genetic variation is retained have little or no heterozygosity (58). All populations of the in different monogenic strains and periodically reshuffled by species of the A. fasciatus complex that we sampled were outcrossing. Most of the numerous and often conflicting hy- monogenic, but inasmuch as both copulation and a spermato- potheses that attempt to relate genetic heterozygosity to habitat phore have been described for at least one species of the com- diversity in animals do not consider selection on the breeding plex (57), we suspect that outcrossing can occur. The breeding system (38, 39), but we propose that theories developed from systems of species of the A. fasciatus complex have not been work on plants apply with equal force to hermaphroditic ani- studied experimentally, but A. intermedius is capable of re- mals. producing when reared in isolation in the laboratory (52). A. With the exceptions of D. laeve and Philomycus spp., all cineroniger is also able to reproduce uniparentally (59). Lanza species of slugs that we have studied have colonized North and Quattrini (60) demonstrated uniparental reproduction in America after introduction by man (51-53,56). Among these, laboratory strains of Vaginulus borellianus (family Veroni- an outcrossing species, D. reticulatum, is the most numerous cellidae) and noted that copulation rarely occurs in this species. and widespread (51-53), but extensive collection records by All philomycids that we examined were amphimictic, but Ikeda Chichester and Getz (51, 53) and our own field experience in (21) demonstrated, by use of a genetic marker, that P. bilineatus the eastern United States indicate that this species is common frequently reproduces by self-fertilization, even after copula- in only a limited range of disturbed habitats, including horti- tion. Individuals of Milax gagates isolated at hatching can re- cultural areas (old fields, gardens, greenhouses). Although much produce (27); and both D. agrestes and D. meridionale show less abundant, the outcrossers Limax maximus and Lehmannia genital polymorphism similar to that in D. laeve and have been valentiana are also restricted to disturbed habitats. The same maintained in the laboratory by uniparental reproduction over collection records and our experience indicate that A. fasciatus many generations (26). is the most widely distributed automictic species, and that it In the absence of extensive experimentation using marker occurs more frequently in relatively undisturbed habitat types loci, we cannot exclude the possibility that reproduction in some (forests, woodlots) than does D. reticulatum. Among the in- individuals or monogenic strains involves parthenogenesis troduced species, A. subfuscus (the self-fertilizing and out- rather than, or in addition to, self-fertilization. This is unlikely, crossing forms were not distinguished) and A. intermedius are however, because parthenogenesis is rare in molluscs in general found most frequently in relatively undisturbed habitats (51, and unknown in pulmonates (31, 61). Moreover, only a special 53). A. hortensis, which is amphimictic and unusually poly- homozygosity-enforcing type (18, 62) could account for the morphic, is the least abundant arionid we have studied and is monogenic character of populations. largely restricted to disturbed habitats. In sum, although no unequivocal pattern emerges from these observations, it appears DISCUSSION that the homozygous, self-fertilizing species actually occupy Self-fertilization in plants is generally regarded not only as an a wider range of habitats and have more successfully invaded adaptation ensuring reproduction when population densities natural areas than have the heterozygous, outcrossing species. of the plants themselves or their pollinators are very low but also Attempts to correlate colonizing success with breeding system as a mechanism for assembling adaptive genomes and pro- and level of genetic heterogeneity in slugs have been hampered tecting them from dissolution by recombination. It has been because (i) many of the strains we have discovered are not Downloaded by guest on October 2, 2021 Population Biology: McCracken and Selander Proc. Natl. Acad. Sci. USA 77 (1980) 687

distinguished in the literature on geographic and ecological 5. Allard, R. W., Jain, S. K. & Workman, P. L. (1968) Adv. Genet. distribution, (ii) other, as yet undetected, stains undoubtedly 41,55-131. occur within some taxa, and (iii) we have no information on 6. Baker, H. G. (1972) in , Phytogeography and Evolu- various strains and species were intro- tion, ed. Valentine, D. H. (Academic, New York), pp. 327- when and how often 347. duced. We have at least partially circumvented these problems 7. Grant, V. (1975) Genetics ofFlowering Plants (Columbia Univ. in a study of the ecological distributions of seven species of slugs Press, New York). on a plot of woodland near Ithaca, NY (unpublished observa- 8. Levin, D. A. (1975) Am. Nat; 109,437-452. tions). A total of 1695 individuals was collected, and the niche 9. Jain, S. K. (1976) Annu. Rev. Ecol. Syst. 7,469-495. breadth of each species was estimated by the method of Colwell 10. Antonovics, J. (1976) Ann. Mo. Bot. Gard. 63, 224-247. and Futuyma (66) from data on 88 physical, chemical, and 11. Allard, R. W., Kahler, A. L. & Clegg, M. T. (1977) in Measuring floristic environmental parameters that previously had been Selection in Natural Populations, eds. Christiansen, F. B. & species that are monogenic Fenchel, T. M. (Springer, Berlin), pp. 1-19. measured (67). Three (in rank order) 12. Clegg, M. T., Kahler, A. L. & Allard, R. W. (1978) in Ecological had relatively broad niches: A. fasciatus, A. circumecriptus, Genetics: The Interface, ed. Brussard, P. F. (Springer, Berlin), and A. subfuscus (form 4, strain 1). Of the four other species, pp. 173-188. D. reticulatum (He = 0.200; D = -0.083) ranked fourth in 13. Brown, A. D. H. (1979) Theor. Pop. Biol. 15, 1-42. niche breadth, D. laeve (He = 0.119; D = -0.902) ranked fifth, 14. Hamrick, J. L (1979) in Topics in Plant Population Biology, eds. and the monogenic species A. intermedius and A. sdlvatius had Solbrig, 0. T., Jain, S., Johnson, G. B. & Raven, P. H. (Columbia the narrowest niches. These results are concordant with those Univ. Press, New York), pp. 84-113. of an earlier study of the distribution of species of slugs in the 15. Solbrig, 0. T. (1976) Ann. Mo. Bot. Gard. 63,363-376. 16. Mather, K. (1973) Genetical Structure of Populations (Chapman Ithaca region by Beyer and Saari (68, 69) who found that A. and Hall, London). fasciatus and A. subfuscus (almost certainly the self-fertilizing 17. Williams, G. C. (1975) Sex and Evolution (Princeton Univ. Press, form) occupy wider ranges of habitats and are more common, Princeton, NJ). particularly in native vegetation types, than are D. reticulatum 18. White, M. J. D. (1978) Modes of Speciation (W. H. Freeman, and P. carolinianus. Relative to the highly heterozygous out- San Francisco). crossing species D. reticulatum, monogenic strains of A. fas- 19. Roughgarden, J. (1979) Theory of Population Genetics and ciatus, A. circumscriptus, and A. subfuscus apparently have Evolutionary Ecology: An Introduction (Macmillan, New broadly adaptive, general-purpose genotypes. There is no ap- York). between the amount of genetic variation 20. Richards, C. S. (1969) Malacologia 9, 339-348. parent relationship 21. Ikeda, K. (1937) J. Sci. Hiroshima Univ. Ser. B Div. 1 5, 67- in a population and the diversity of the habitats it occupies. 123. Much further work on both native and introduced species 22. Larambergue, M. (1939) Bull. Biol. Fr. Belg. 73, 1-231. of slugs will be required (i) to determine whether self-fertil- 23. Abeloos, M. (1945) Bull. Soc. Zool. Fr. 70, 135-139. ization is more often a characteristic of colonizing species than 24. Paraense, W. L. (1956) Evolution 10, 403-407. of those confined to relatively undisturbed, natural habitats, 25. Wu, S. (1972) Malacolog. Rev. 5, 95-164. and (ii) to understand spatial and temporal variation in 26. 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