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BiologicalJournal ofthe Linnean Society, 14: 293-301. With 3 figures N oveniber/December 1980 The karyotype of Cepaea sylvatica (Pulmonata: Helicidae)and its relationship to those of C. hortensis and C . nemoralis A J. J. B. GILL Department of Genetics, University ofliverpool, P . O . Box 147, Liverpool L693BX AND A. J. CAIN Department ofZoology, University of Liverpool, P.O. Box 147, Liverpool L693BX Acceptedfor publication April 1980 The karyotypes of Cepaea nemoralis (L.) and C. hortensis (Muller), with 2n=44 and a conspicuously large pair of chromosomes, are described and compared with that of C. syluatica (Draparnaud)which has 2n= 50. The karyotype of C. syluatica also has a conspicuously large pair ofchromosomes but the comparison suggests that these have an independent origin from those in the 2n=44 species. There is no evidence that the large chromosomes in C. nemoralis and C. hortensis have originated from simple fusion of chromosomes from a 2n=50 karyotype with chromosomes all sub-equal such as is reported for C. uindobonensis. It may be that such a karyotype with little size dih-entiation amongst the chromosomes is not a primitive feature in the Helicinae. The relationship of shell colour and handing polymorphism to the chromosome architecture is discussed. KEY WORDS :-Cepaeasyluatica- Karyotype-chromosome fusions. CONTENTS Introduction . Methods . . Results . . . Discussion . . Acknowledgements References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 293 295 295 299 30 1 30 1 I NTRODUCTI0N The presence of marked polymorphisms for both shell colour and banding pattern in PO ulations of both Cepaea hortensis and C . nemoralis has prompted a great deal o genetic and ecogenetic investigation in the species. These investigations, notably by Lamotte, Cain & Sheppard and their associates, and P 293 0024-4066/80/070293+09$02.00/0 0 1980 The Linnean Society of London 294 J. J. B. GILL AND A. J. CAIN Cook, have shown that the variation is largely genetic in origin and, at least in some cases, is subject to selection. It is known that in both species there are at least seven and possibly nine loci which are involved in the control of shell colour and banding pattern. In C . nemoralis at least six and in C . hortensis at least five of the loci are closely linked. The first accurate cytogenetic studies in Cepaea were carried out by Baltzer who, as early as 1913, determined the chromosome numbers of C . hortensis and C. uindobonensis as 2n=44 and 2n=50 respectively and noted that the meiotic plates of C. hortensis showed a single element which was very much larger than the other bivalents while C . uindobonensis did not. He suggested that this large bivalent in the 2n=44 species might have originated from fusion of some of the smaller chromosomes in the 2n=50 genome. Perrot 8c Perrot (1937) carried out an investigation of the chromosomes of C . hortensis, C . nemoralis, C . sylvatica and C . uindobonensis but, like Baltzer’s, all their preparations were of meiotic material. These authors confirmed that C . hortensis had a chromosome number of 2n=44 and showed that it shared this number with C . nemoralis. They also reported that C. nemoralis showed a very large bivalent similar to that of C. hortensis. They demonstrated that C. sylvatica, like C . vindobonensis, had a chromosome number of 2n=50 but, unlike the latter species, had a chromosome complement in which one of the bivalents was very much larger than the others. The size differential between this bivalent and the others was not, however, as great as that between the large bivalent in the 2n=44 s ecies and the other chromosomes in their complements. Perrot 8c Perrot con rmed Baltzer’s observation that there was no great size differential amongst the bivalents of C . vindobonensis. Although the variation in basic colour and pattern has been much studied in the 2n=44 species, there are very few data available for any such variations in C. syluatica and C . uindobonensis. Schilder 8c Schilder’saccount ( 1953, 195 7 ) suggests, however, that some of the gene loci governing banding and perhaps colour present in the 2n=44 species are also to be found in those with 2n=50. We know of no publication, however, which suggests that the variation in the 2n=50 species is organised into the distinct and extensive polymorphisms so characteristic of C. hortensis and C . nemoralis. It therefore appears unlikely that the loci governing colour and pattern in the 2n=50 pair are organized into a supergene. The arguments put forward by Cain & She pard ( 1954), Cain, King 8c Sheppard (1960) and Cook (1967)relating to the estab ishment of the linkage in the 2n=44 species all agree that the supergene was established in response to selection. Cook ( 1967) has calculated, for C. nemoralis, that the probability of finding six out of nine loci linked is either 4 x lo-’ or 2 x lo-’ depending on whether the supergene is on the large chromosome or one of the others. All the arguments obliquely suggest that the supergene may be on the large chromosome and indeed that the very reason for the existence of such gross chromosome inequality may be the establishment of the supergene. Although there is no hard evidence for such a view, the suggestion is not without its attractions, offering as it does some historicalhelectional reason for the massive architectural differentiation between the two chromosome levels. There is therefore some support for Baltzer’s suggestion that the large chromosome in the lower chromosome number species represents a number, presumably eight, of the chromosomes of the 2n=50 genome. K F KARYOTYPES OF CEPAEA SPECIES 295 We are unaware of any attempt to integrate the genetic and cytogenetic data and decided, therefore, to carry out as detailed an analysis as possible of the chromosomes of the various Cepaea species to see if any homologies between their genomes could be demonstrated and, with luck, any ancient translocations shown. METHODS For detailed karyotypic studies only mitotic chromosomes are of use and, as pointed out by Bantock (19721, these are not readily available. However, Page (1978) has carried out a detailed analysis of the mitotic chromosomes of C. nemorulis using embryos as a source of mitoses. We have also used embryos as a source of material but have used squash rather than air-drying techniques as we found that after our treatments, air-dried preparations were frequently excessively fuzzy. The embryos were dissected from five-day-old eggs and washed free of adhering yolk in 0.05%colchicine in saline (Lockwood, 1963), in which they were incubated for two hours at room temperature. The embryos were then transferred to 0.05% colchicine in quarter strength saline for 12 min before being fixed in 3 : 1 absolute alcoho1:glacial acetic acid. The h e d embryos were hydrolyzed in N.HC1 at 6OoC for 6 min and stained in Schiff s reagent. Usually about three slides could be made from any one embryo. Meiotic preparations were made from ovotestis by methods similar to those of Bantock (1972) and were stained in lactic-aceticorcein. All attempts to band the chromosomes by enzyme or alkali digestion or by using u.v. fluorescent dyes failed. RESULTS Our analysis of the chromosomes of C. nemorulis is in very close agreement with those of Bantock (1972) and Page (1978). The most obvious feature of any mitotic preparation (Fig. 1A) is the pair of very large, almost metacentric chromosomes. 0 n karyotyping the chromosomes, however, three pairs are readily discernible from the rest (Fig. 1C). These are the very large pair already mentioned, the next pair down which is also almost metacentric, and a pair of sub-metacentric or acrocentric chromosomes which are amongst the largest of the remainder but show an obvious secondary constriction about half-way along the long arm. The largest pair has been recognized by Bantock (1972) as his group ‘A’, the next as group ‘B’ and that with the secondary constriction has been reported by Page (1978). This latter author has also described some of the larger of the other 19 chromosome pairs as being individually recognizable but, in our experience, this is only possible in the very best of preparations. Even then, because of the continuum in size formed by these 38 chromosomes, the absolute recognition of any one may be difficult. We therefore believe that the most useful way to treat the chromosomes, other than the three pairs previously mentioned, is as a single group. Within this group many of the chromosomes are obviously nearly metacentric and others obviously not metacentric but because of their smallness and the existence of many which are intermediate in centromere position, any firm classification based on this criterion must be suspect. The chromosome architecture may be further complicated by the presence of pericentric inversions 296 J. J. B. GILL A N D A. J. CAIN Figure 1. A. Mitosis in C. nemoralis. B. Meiosis in C. nemoralis. C. Karyotype of C.ncmoralis. D . Mitosis in C. horlenszs. E. Meiosis in C.horfensis. KARYOTYPES OF CEPAEA SPECIES 29 7 in some populations as reported by Page (1978). Interspecific comparisons based on exact measurements of the chromosomes in the agglomerate groups are therefore difficult. After our methods of preparation, the longest pair of chromosomes varies in length from c. 6-10 pm with a mean of about 7 ym and, based on a sample of 38 cells, forms about 15% of the total karyotype length. The arm ratio for this chromosome is 0.872f 0.076, so that it is not quite metacentric. This pair is about two and a half times as long as the next pair, which is also nearly metacentric with an arm ratio of 0.798+0.097. The pair with the obvious secondary constriction cutting off a large satellite is amongst the largest of the acrocentric pairs and is Figure 2. Karyotypes of C. hortensis with secondary constrictions arrowed. 298 J . J. B. GILL AND A. J. CAIN Figure%Cepaeasyluatica. A, B,mitosis; C, karyotype;D,E, meiosis. KARYOTYPES OF CEPAEA SPECIES 299 about 2 pm long. The only chromosomes which can be easily and consistently recognized in meiotic preparations are those forming the largest pair (Fig. 1B). Both mitotic preparations and karyotypes of C. hortensis are extremely similar to those of C. nemorulis, with the most obvious feature again being an extremely large, nearly metacentric, pair of chromosomes (Fig. 1D). These, like those of C. nemorulis, form about 15% of the total length of the karyotype (Fig. 2A, B). Based on a sample of 34 cells they have an arm ratio of 0.836 f 0.078 and are about two and a half times as long as the next pair which are very similar in shape with an arm ratio of 0.865+0.131. In some preparations this second pair of chromosomes shows a secondary constriction about half-way along one of its arms. A great many satellites and secondary constrictions are apparent in the chromosomes of C. hortensis (Fig. 2A, B) but as these are not a consistent feature of the karyotype and their demonstration is much dependent on treatment, they are of use in karyotypic studies only when very large samples can be used. In some cells a pair of chromosomes very similar to the satellited pair in C.nemorulis is apparent (arrowed in Fig. 2A). In meiotic plates only the very large chromosome pair can be consistently recognized (Fig. 1D). Material of C. syluatica was not so plentifully available as that of the other species but mitotic preparations were obtained from 22 embryos. All showed a chromosome number of 2n=50. Superficially, the chromosomes of this species are very similar to those of C. nemoralis and C. hortensis with the most obvious feature of mitotic plates being the presence of a pair of chromosomes very much longer than any of the others (Fig. 3A). These chromosomes are, however, considerably shorter than the longest in the 2n=44 species, varying from c. 5 to 8 pm in length after our methods of preparation. They represent about 10%of the total length of the karyotype and are nearly metacentric with an arm ratio of 0.7 7 1 f 0.096. They are about one and a half times as long as the next pair which are almost exactly metacentric with an arm ratio of 0.93 k 0.07 1. The other 46 chromosomes in the karyotype (Fig. 3C)form a continuum in size similar to that of Bantock’s group C (Bantock, 1972). As in C. nemoralis it may be possible to recognize the biggest metacentric or acrocentric pairs in this group but, for reasons similar to those for C. nemoralis, this cannot be done consistently and we therefore do not separate them. None of the chromosomes of C. sylvatica appear to show secondary constrictions. In meiotic preparations of C. syluatica (Fig. 3B, D, El the largest chromosome pair is readily recognizable and frequently one of the smaller pairs shows satellite-type structures (Fig. 3D, El. These may occasionally be so far separated from the main body of the chromosomes that they can give the appearance of an extra, small chromosome pair (Fig. 3E). I t is not known which of the somatic chromosomes are represented by these nor is the significance of their shape understood. Unfortunately no material of C.uindobonenszs was available to us. DISCUSSION The more or less continuous gradation in chromosome size and the general lack of any constant morphological distinguishing features amongst most of the chromosomes of all three species available to us makes detailed karyotypic comparison between them difficult. In all cases, however, it is possible to recognize consistently the two largest chromosome pairs and it may be valid to 300 J. J. B. GILL AND A. J. CAIN use differences amongst these as a measure of the amount of karyotypic differentiation which has occurred. There are no significant differences either in absolute chromosome length or in arm ratios of the two largest pairs of chromosomes between C. hortensis and C. nemoralis. As far as can be judged all the other chromosomes are present in both species. The presence of so many secondary constrictions in C. hortensis may represent some important differences in genetic organization between this species and C. nemoralis but as these structures are notoriously variable in their expression, any conclusions on this must await a much more extensive study. The similarities of the karyotypes of C. hortensis and C nemoralis suggest that the species are of relatively recent origin from a common ancestor in which the characteristic chromosomal architecture was produced, or that the inequalities of chromosome size in the karyotype are a very old organization in Cepaea with a high, but at present not understood, selectivevalue. Superficially, the karyotype of C . syluatica suggests that the latter view may be correct but such detailed analyses as can be carried out, show that the chromosomal arrangement in this species is markedly distinct from those of the other two. The differences are particularly apparent in the largest chromosome pair. It occupies considerably less of the total karyotype length than the longest in the 2n=44 species and, as judged from arm ratios, its shape is considerably different. It may be tempting to suggest that the long chromosome in C. syluatica represents an early stage in the build up of the large chromosome in the 2n=44 species, presumably representing the physical evidence for the build up of an important linkage group. This may be a particularly attractive hypothesis if it is assumed that Baltzer’s ( 19 13) observation, that no large chromosome exists in C. uzndobonensis, is correct and that this species represents the ancestral karyotype in Cepaea. The evolutionary path would then be: small large {c.hortensis linkage linkage C . vindobonensis group * C. sylvatica group * C. nemoralis (or ancestor) The conversion of the large C. syluatica chromosome to that of the 2n=44 species and the reduction in chromosome number from 2n=50 to 2n=44 would require the translocation of three chromosomes on to the large one. If these were of the smallest types in the C. syluatica karyotype it is possible that a chromosome of the length of that in the 2n=44 species could be produced but it is difficult to envisage how one of the correct shape could arise, particularly if the translocations were involved in the production of the colour/banding supergene. To produce tight linkage these translocations would all have to be on to the same arm of the large chromosome at least and this would produce a chromosome very much less metacentric than that present in C. hortensis and C . nemoralis. Any firm conclusions on this must, however, await either a detailed analysis of the linkage relationships in C. syluatica or perhaps the production of some F, hybrids between the 2n=44 and 2n=50 species. I t may indeed prove that the absence of any major size differential in the chromosomes, as in C. vindobonensis, is a secondary characteristic and that some chromosomal arrangement similar to that in C. syluatica and the 2n=44 species represents the ancestral karyotype in Cepaea. Certainly some other species of KARYOTYPES OF CEPAEA SPECIES 30 I Helicinae such as Helix aspersa and Eobania vermiculata exhibit a pair of chromosomes considerably larger than the others in the karyotype. ACKNOWLEDGEMENTS We are grateful to Helena Kempski for assistance with chromosome preparations, and to Robert H. Cowie for his care in maintaining the cultures, to Dr J. S . Jones and D r W. Macdonald for collecting material, and to Drs C. Bantock and L. M. Cook for criticism of the paper. REFERENCES RALTZER, F., 1913. Uber die Chromosomen der Tachea (Helix) hortensis, T . ausfriaca und der sogenannten einseitigen Bastarde T . hortensis x T . ausfn’aca.Archiv.fur ZelCforschung, 11: 151-169. RANTOCK, C. R., 1972. Localization ofchiasmata in Cepaea nemoralis L. Heredity, 29: 213-221. CAIN, A. J., KING, J. M. B. & SHEPPARD, P. M., 1960. New data o n the genetics of polymorphism in the snail Cepaea nemoralis L. Genetics, Princefon, 4 5 : 393-41 1. CAIN, A. J . & SHEPPARD, P. M., 1954. Natural selection in Cepaea. Genetics, Princeton, 39: 89-1 16. COOK, L. M., 1967. The genetics of Cepaea nemaralir. Heredity, 22: 397-410. LOCKWOOD, A. P. M., 1963. Animal Body Fluids and their Regulation. London: Heineman. PAGE, C., 1978. The karyotype of the land snail Cepaea nemoralis (L.).Heredity, 4 1 : 321-327. PERROT, J . L. & PERROT, M., 1937. Monographie des Helix du groupe Cepaea; contribution a la notion de I’esp6ce. Bulletin Biologique de la France el de la Belgique, 73: 232-262. SCHILDER, F. A. & SCHILDER, M., 1953. Die Banderschnecken, eine Studie zur Euolution der Tiere. Jena: Fischer Verlag. SCHILDER, F. A. & SCHILDER, M., 1957. Die Banderschnecken, eine Studie zur Evolution der Tiere. Schluss: Die Banderschnecken Europas. Jena: Fischer Verlag.