Cytologia 41: 261-282, 1976

Karyotypes of Three Species of Lepidoptera Including an Investigation of B-chromosomes in Pieris

T. R. L. Bigger M. R. C. RadiobiologyUnit, Harwell, Oxon. OXll ORD, England, Great Britain ReceivedSeptember 17, 1974

The application of new chromosome banding techniques has enabled detailed karyotypes of several species of Lepidoptera to be constructed. In a previous paper (Bigger 1975) information was given for two species, Pieris brassicae L. and Polymmatus icarus Rott. and evidence was presented for the existence of a de finite centromere-like constriction (primary centromere) at certain stages of the mitotic cycle. This paper extends these observations to a species in another family of Lepidoptera, the Satyridae, Pyronia tithonas L. and to two further Pierids, Pieris napi L. and P. rapae L. Primary centromeres have been found in all three species. A detailed comparison of the chromosome complements of the two closely related Pieris species shows that clear differences exist in chromosome morphology. Comparison is also made with the P. brassicae karyotype. In addition, the study has led to the discovery of supernumerary or B-chromo somes in both Pierid populations sampled. Hitherto supernumeraries have not been found in other races of these species in Europe (except a small number of P. rapae from Finland), nor in the rest of the world with the exception of the Japanese races.

Materials and methods

Testis and ovary tissues from three species of butterfly were used in this in vestigation. The butterflies referred to as Pyronia tithonas L., Pieris napi L. and

P. rapae L. were all collected at three localities within 10 Km of one another. The first is near the MRC Unit at Harwell (H), the second at Segsbury camp on the downs 5Km south-west of Wantage, Oxfordshire (S), and the third on the north eastern edge of the town of Wantage itself (W).

The cytological and G-banding techniques used on the testes and ovarioles were the same as those described before (Bigger 1975). Briefly, adult insects were in jected in the abdomen with a small volume of colcemid solution in order to accumulate metaphases. The required tissues were dissected out, macerated in a 0.1% trypsin solution, then fixed in methanol/acetic acid (4:1). The cell suspensions were then dispensed on to dry, grease-free slides and air-dried. Slides for ASG banding were treated in a saline solution (1.75g NaCI, 0.88g trisodium citrate dissolved in 100ml distilled water) at 60•Ž for one hour prior to Giemsa staining. Suitable mitotic spreads were photographed and karyotypes were con structed from enlargements with subsequent checking against the original cells. 262 T. R. L. Bigger Cytologia 41

In order to eliminate bias when karyotyping the two Pieris species a three month interval elapsed between the construction of the P. rapae karyotype and the commencement of the P. napi study. During that three month period other species were studied. Cells from all the specimens of the Pieris species were scored for the presence and characteristics of supernumerary chromosomes, and the butterflies themselves examined for phenotypic variation.

Results

All three species provided good ASG banded mitotic preparations. However mitotic chromosomes from both Pieris species did tend to be a little less distinct

Figs. 7-9. 7, Pyronia tithonas (•‰) mitosis showing several constrictions (arrows). 8, Pieris rapae (•‰) mitosis with diffuse B-chromosome. 9, part of a Pieris napi ((•‰)meiosis showing B1/B2 bivalent (plus a normal small autosome bivalent). (Bars on Figs. are 5ƒÊ).

than those of Polyommatus icarus (Bigger 1975, Fig. 2) but nevertheless they were

quite adequate for karyotyping. It has been noted before (Bigger 1960) that chromo some preparations from Lycaenids are usually much clearer than from other Le

pidoptera groups, even when using squash techniques. The P. tithonas chromo somes tended to be less intensely stained, though the G-banding patterns were

slightly more distinct than in the Pierids.

Chromosome karyotypes for all three species, both male and female, are shown

in Figs. 1-3 together with diagrammatic representations Figs. 4-6. These are ar

ranged in exactly the same way as in the previous paper (Bigger 1975) and include

the B-chromosomes in the case of the Pieris species. The diagrams show the Fig. 1. Karyotypes of G-banded chromosomes of Pyronia tithonas. Fig. 2. Karyotypes of G-banded chromosomes of Pieris rapae. Fig. 3. Karyotypes of G-banded chromosomes of Pieris napi. 266 T. R. L. Bigger Cytologia 41 1976 Karyotypes of Thre e Species of Lepidoptera 267 Figs. 4-6. Diagrammatic representations of the chromosome complements showing G-bands. 4, Pyronia tithonas. 5, Pieris rapae. 6, Pieris napi. 1976 Karyotypes of Three Species of Lepidoptera 269

Table 1. Chromosome measurement data. The mean lengths are from measurements of 16 chromosomes except in the cases of the X, Y and B where 13, 3 and 8 chromosomes were used respectively 270 T. R. L. Bigger Cytologia 41

Table 1. (cont'd)

* The mean length of each chromosome expressed as a percent age of the total haploid auto some length. Table 2. Comparison of the chromosome complements of Pieris rapae and P. napi

* Centromeric indices significantly different . ** Chromosomes identical. 272 T. R. L. Bigger Cytologia 41 maximum number of G-bands seen per chromosome drawn to approximate the size and staining intensity. Bands which tend to fuse, and wide and/or unstained interband regions are indicated. Unambiguous constrictions (primary centromeres) were again observed on the early mitotic chromosomes (including the supernu meraries). The constrictions remained visible in the Satyrid species to a later stage of metaphase than in the other species so far studied and Fig. 7 shows a more con

Table 3. Number of cells scoredin normal (n=25) specimensof Pieris rapae and P. napi, and number of specimensstudied+and - B-chromosomes

tracted cell with some chromosomes whose constrictions are still quite clear. Table 1 gives chromosome measurements and primary centromeric indices for each species. The chromosome complements for the different species may be summarised as follows. P. tithonas: 14 acrocentric, 8 submetacentric and 6 metacentric autosomes, Table 4. Number, type and appearance of B-chromosomes in Pieris rapae and P. napi

* Indicates the number of well contracted metaphases . •õ A more detailed report of the types of B-chromosomes found in these specimens will be published later with further data from other populations 274 T. R. L. Bigger Cytologia 41

and 2 sub-metacentric sex chromosomes. Satellites are present on the short arm of

chromosome 22. The chromosome number n=29 (Lorkovic 1941) was confirmed

in at least 10 mitotic and/or meiotic cells scored from each of 18 d •‰and 7•Š speci

mens. None were found to have a non-modal chromosome count.

P. rapae: 13 acrocentric, 8 sub-metacentric and three metacentric autosomes,

and 2 sub-metacentric sex chromosomes. Satellites are present on the short arms of chromosomes 4, 12 and 22, and the long arm of chromosome 19. The B-chromo

some when present is a single, large diffuse metacentric chromosome (Fig. 8). The

Figs. 10, 11. Pieris rapae (•‰) meiotic metaphase cells. 10, normal n=25 meiosis. 11 , meiosis with contracted B-chromosome. (Bar on Fig. =5ƒÊ). chromosome number n=25 (Federley 1938) was confirmed.

P. napi: is very similar to the previous species with 10 acrocentric, 11 sub metacentric and 3 metacentric autosomes, and 2 sub-metacentric sex chromosomes . Satellites are seen on the short arms of chromosomes 8, 22 and 23. The B-chromo somes in this species are very small, one being acrocentric and the other meta centric (Fig. 9). Again the chromosome number n=25 (Federley 1938) was con firmed.

The sex chromosome pair for all three species is heteromorphic . 1976 Karyotypes of Three Species of Lepidoptera 275

A detailed interspecific comparison of the Pieris complements is given in Table 2. When the chromosomes are set out in order of decreasing length they may be seen to match closely although the absolute lengths of an individual chromosome varied between the two species due to the selection of the cells that were measured,

Figs. 12-16. Pieris napi (•‰) contracted mitotic metaphases. 12, normal 2n=50 cell. 13, cell

plus B2 chromosome. 14, plus two B2 chromosomes. 15, plus B1 and B2 chromosomes. 16, plus two B1 and two B2 chromosomes. (Bar=5ƒÊ).

those from P. napi tending to be less contracted than the P. rapae cells. Table 2

also shows the number of bands per chromosome, a t-test value and probability

comparing the centromeric indices of each chromosome pair, and a column sum marising any differences. 276 T. R. L. Bigger Cytologia 41

The total number of Pieris specimens studied and the number of cells scored are given in Tables 3 and 4. Supernumerary chromosomes were found in a proportion of specimens of both P. rapae and P. napi. Karyotypes of cells of both species showed that the 25 chromosome pairs of the normal complement were identical in cells with and with

Figs. 17-20. Pieris napi (•‰) meiotic metaphase cells. 17, normal n=25 meiosis. 18, cell plus B1/B2 bivalent. 19, cell plus separate B1 and B2 univalent chromosomes. 20, cell plus one B biva lent and one univalent B2 chromosome. (Bar=5ƒÊ).

out these extra chromosome elements. Consequently these extra chromosomes are additional or B-chromosomes and not fragments of the normal complement (Figs.

2, 3).

P. rapae possessed one univalent B-chromosome and this was found in just 1976 Karyotypes of Three Species of Lepidoptera 277 over 50% of the individuals studied. It was a large metacentric chromosome, usually less contracted than the other chromosomes in the cells in which it appeared. It was often very diffuse and faintly stained at mitosis (Fig. 8) though this was not the case at meiosis, here the B-chromosome tended to be at the same stage of con traction as the normal bivalents and was more often than not the smallest chromo some element (Figs. 10, 11). In P. napi on the other hand 10 out of 25 specimens had B-chromosomes, the number of which varied from individual to individual, and often between different cells of a particular butterfly. The B-chromosomes were of two types. Both were very small in comparison with the P. rapae supernumerary and at mitosis were usually as contracted as the other chromosomes (Figs. 12-16). At meiosis the B-chromosomes frequently formed bivalents. The supernumerary chromosomes have been called B, for the near metacentric one, and B2 for the acrocentric. The bivalents at meiosis were of the three possible combinations of the two types, that is B1/B1, B2/B2 and B1/B2. The B-chromosomes were always the smallest elements at meiosis (Figs. 17-20). Table 4 sets out in detail the occurrence, number, appearance and size of the supernumeraries for each species.

Discussion

1. Primary centromeres In a previous paper (Bigger 1975) I reported that the early mitotic chromo somes of two Lepidoptera species possessed a definite constriction similar to the centromere of other organisms which have monokinetic chromosomes. The re sults from the three species in this study confirm the previous findings. Constric tions were again found on the mitotic chromosomes not only in the two Pieris species, as was to be expected since they were found in Pieris brassicae, but also in Pyronia tithonas, a species of a different family of butterflies, the Satyridae. According to White (1973) the question of the holokinetic organisation of Lepidoptera chromosomes is still an open one, even though it appears to be generally accepted that they are holocentric (Bauer 1967). A recent report by Murakami and Imai (1974) also shows that the controversy still exists although their results using Bombyx mori gonads demonstrate cytologically that at certain stages of mitotic metaphase the chromosomes are clearly holokinetic. However they also report the presence of constrictions (which they call secondary constrictions) on some chromosomes, but conclude that the silkworm lacks a localized centromere. The presence of the constrictions on the butterfly species' chromosomes is therefore difficult to interpret. These constrictions (here termed primary centro meres) are only observed at early mitotic metaphase and after several cells have been karyotyped each chromosome is seen to possess one. As metaphase pro gresses the chromosomes become more contracted and the constrictions cease to be visible. It was suggested (Bigger 1975) that the primary centromere performed only a limited kinetic function early on in the mitotic cycle and that this was superseded by the action of the holokinetic organisation of the chromosomes as they contract. 278 T. R. L. Bigger Cytologia 41

Alternatively the primary centromeres may not have any function at all other than holding the chromatids together at pro-metaphase but the disappearance of the constrictions early in metaphase may simply reflect the evolutionary change from monokinetic to holokinetic chromosomes. However the presence of the constric tions does make it possible to construct a detailed karyotype of the chromosome complements and helps distinguishing between morphologically similar chromo somes.

Table 5. Chromosomes of P. rapae and P. napi which closely resemble chromosomes of the P. brassicae karyotype

2. Comparison of Pieris karyotypes Pieris rapae and P. napi both have basic chromosome numbers of n=25 (Feder ley 1938, Lorkovic 1941). A comparison of the karyotypes and the diagrammatic representations (Figs. 2, 3, 5 and 6) shows that the complements are strikingly similar and that in many cases this similarity extends to the G-banding patterns. From the data of Table 2 the following conclusions may be drawn. a) Eight chromosomes are seen to be visually identical in both species (nos. 2, 5, 6, 10, 11, 17, 18 and the Y). b) A further 4 chromosomes differ only marginally in the posi tion of the centromeric constriction but have similar banding patterns (nos. 4, 7, 8 and 21). It must be noted that these four chromosomes are all acrocentric types (with or without satellites) and due to the limitations of the measuring techniques their minor differences are exaggerated when the centromeric indices are compared. These four chromosomes can therefore be regarded as visually identical in the two species. c) The remaining 14 chromosomes have obvious, though often minor , differences. These differences may be classified into several types . Firstly chromo somes where the banding patterns are very similar but the centromeric constriction position is different. There are 4 of these (nos. 1, 3, 14 and 15). Secondly, 6 chromosomes with similar centromeric indices but different G-banding (nos . 9, 13, 1976 Karyotypes of Three Species of Lepidoptera 279

16, 20, 23 and the X). Thirdly one chromosome (no. 22) where the centromeric index and the banding pattern both differ. Three further chromosomes do not fit into any of the above categories; chromosome no. 12 where the short arms differ (in P. rapae it is very short with satellites, in P. napi an unsatellited short arm with one G-band), no. 19 which has satellited long arms in P. rapae only and different G-band patterns, and lastly no. 24 which is a smaller acrocentric chromosome in P. rapae but a metacentric in P. napi, also with differing G-bands. It is also of interest to compare the P. rapae and P. napi chromosome com plements with that of P. brassicae (Bigger 1975). Table 5 lists those P. brassicae chromosomes that most clearly resemble visually P. rapae and P. napi chromo somes. It shows that the majority of P. brassicae chromosomes do have corre spondingly similar ones in either or both of the other species' complements and only 4 do not compare at all. Bearing in mind that the P. brassicae chromosome number (n=15) is lower than the other two species (n=25) and that the mean individual chromosome lengths are very similar, there is obviously considerably less chromo some material in the former species. This lends support to the conclusions of Kudrna (1974) that the genus Pieris should in fact be split, the P. brassicae group of species (n=15) being separated from the other rapae and napi-groups of species (n=25-27), the latter being placed in a new genus.

3. Supernumerary chromosomes Supernumerary or B-chromosomes are known in many animal and plant groups (White 1973, Battaglia 1964). They tend to accumulate by a variety of genetic mechanisms at different stages of the cell cycle (Nur 1969) but because the data are so far insufficient it is not possible to determine how they have arisen in the two Pieris species under discussion here. As mentioned earlier the karyotypes of the normal chromosomes were identical in cells with and without B-chromosomes, thus proving that the supernumeraries are true additional chromosomes and not small fragments of the normal chromosomes. Supernumerary chromosomes are known in several species in the genus Pieris (Lorkovic 1968) but the occurrence in both P. rapae and P. napi has so far only been reported in the Japanese races of these species and in a small number of speci mens of P. rapae from Finland. There are no records of B-chromosomes in any races from continental Europe, Asia or North America where the two species are to be found. Maeki and Remington (1960) stated that European and American P. r. rapae has no B-chromosome but Japanese P. r. crucivora does possess a minute extra element. A similar situation exists in P. napi with Japanese P. n. nesis. Lorkovic (1941) also found n=25 in both species from Eastern European material, though Federley (1938) reported n=26 from some P. rapae from Finland. Lorko vie (1968) states that P. napi is always strictly n=25, although P. bryoniae, con sidered by many authorities as only a sub-species of P. napi, does possess B-chromo somes. Maeki and Remington (1960) suggest that the Japanese P. napi may in fact be a distinct species. The types of B-chromosome found in the British material so far are set out in Table 4. It is clear that the two B-chromosomes of P. napi are quite distinct from 280 T. R. L. Bigger Cytologia 41

the single one found in P. rapae, not only in their morphology and G-banding patterns, but also in their behaviour at mitosis and meiosis (Figs. 8-20). In P. rapae only one B-chromosome per cell was ever found in 178 cells and in the majority of mitoses it tended to contract after the normal chromosomes (Fig. 8), but in meioses at the same time as the bivalents (Figs. 10, 11). It was always strictly univalent. At mitosis the supernumerary was often very faint and diffuse, sometimes up to three times as long as the largest autosome. However it was present in almost every cell examined in those individuals carrying the B chromosome and this would suggest that although it appears to divide late it does not tend to be lost at mitosis. The two B-chromosomes in P. napi the B, metacentric and the B2 acrocentric, behaved in the same way as the other chromosomes at mitosis and meiosis. Mito tic cells contained normally 1-4 univalent Bs (one cell was found with 5) and various combinations of the two types were seen (Figs. 12-16). At meiosis where two or more B-chromosomes were present, they were seen to form bivalents (Figs. 9, 18) though there was no evidence of any chiasmata. Quite often the B-pairs appeared to separate before the other bivalents. The B-chromosome bivalent at meiosis in cells where they could be identified with certainty was 2B1, 2B2 or B1/B2(this suggests that B, and B2 have some homologous region or regions in common). At present the supernumerary chromosome data are from individuals in a relatively small area of central southern England. Whether a similar situation exists in other parts of the British Isles is a matter of conjecture and further samples will have to be studied. This applies in particular to P. napi which tends to be a more static species than the more migratory P. rapae. One might expect the former to show more variability in numbers and types of B-chromosome than the latter. Particularly interesting would be a chromosome analysis of the univoltine P. napi race recently found in the north of England (Lees 1970). There is also specu lation as to the origins of the Irish and Scottish populations of P. napi and analysis of G-banded chromosomes and the presence or absence of B-chromosomes may be of value in deciding whether these populations are relicts from pre-Pleistocene glaciations distinct from the southern English populations which have received influxes of specimens from continental Europe. Evans (1960) studying B-chromosomes in the Roman Snail, Helix pomatia , and Hewitt (1972) investigating supernumeraries in the Mottled Grasshopper , Myrmeleotettix maculatus both showed that continental races of these animals do not possess B-chromosomes and suggest that those in the British races have arisen because of isolation and inbreeding. Variations in B frequency in the grasshopper are said to reflect environmental conditions, the more favourable habitats giving rise to more Bs. Similar reasons may be partly responsible for the butterflies' supernumeraries and may also hold for the Japanese races which are in an identical position of being island races existing a relatively short distance away from a con tinental land mass. Phenotypic examination of both Pieris species did not reveal any differences between butterflies possessing Bs and those that did not. Data are insufficient to determine any effects that the accumulation of B-chromosomes might have , though 1976 Karyotypes of Three Species of Lepidoptera 281 their prevalence in both species seems to indicate that they do not effect the fertility or vitality of either. In P. rapae there is obviously some mechanism preventing the accumulation of more than one B. At gametogenesis the univalent B will occur in half the resultant spermatids or ova. Possibly fertilized eggs containing two Bs are incapable of reaching maturity, the resultant larvae less vigorous or on the other hand one B-chromosome may be eliminated during early embryonic mitotic divisions. However in P. napi no such limitation appears to occur and possession of up to 5 Bs is apparently normal. 4. Conclusions From the foregoing observations it is clear that the G-banding technique can provide a useful means of differentiating the chromosomes of closely related spe cies, a differentiation that is aided in the examples investigated by the presence (at least in uncontracted chromosomes) of a primary centromere. It now remains to be determined whether differentiation of this type can be made in other closely related species of the genus Pieris, for example, between the karyotypes in the Pieris napi and ergane pseudorapae groups (Lorkovic 1968), and the Pieris bras sicae-cheiranthi-wollastoni group all of which species have a chromosome number of n=15 and karyotypes so far indistinguishable (Kudrna 1973). This latter group has been shown to differ in many aspects from other Pieris groups (Kudrna 1974) and it is likely that the genus Pieris will eventually be subdivided. Bowden (1966, 1970) has described many cross-breeding experiments involving P. napi and other Pieris species. A detailed karyotyping of any hybrid chromo some complements would be of immense interest when interpreting the results of any future experiments along similar lines. Another interesting group is the Lysandra coridon Poda. complex of species which has a variable range of chromosome numbers over Europe. L. coridon itself varies from n=87-92 (de Lesse 1969). These techniques now offer the op portunity of determining whether certain chromosomes are deleted, added to or fused.

Abstract

Chromosome banding techniques have been used to compare the karyotypes of Pieris rapae and P. napi. The basic complements (both n=25) are very similar but clear differences are present and these are discussed in detail. (A brief comparison is made with Pieris brassicae, n=15). Cytological techniques have confirmed the presence of a constriction (primary centromere) in the Pieris species and also in a member of the Satyridae (Pyronia tithonas, n=29). Supernumerary B-chromosomes were discovered in both P. rapae and P. napi. Hitherto these have been reported only in the Japanese races of these species and in a small number of Finnish P. rapae individuals and it has usually been as sumed that they are absent from populations in the rest of the world. The characteristics of the B-chromosomes differ both numerically and struc turally between the two species and the variations are discussed. 282 T. R. L. Bigger Cytologia 41

No phenotypic differences exist between individuals with and without B chromosomes.

Acknowledgement

I am once again indebted to Dr. J. R. K. Savage for his advice in the prepara tion of the manuscript.

References

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