Download of C. hortensis and C. nemoralis

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
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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
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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.