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THE CHROMOSOMES OF THE ALPINE MARMOT MARMOTA MARMOTA L. (RODENTIA: SCIURIDAE)' Ry K. FERNANDEZ-DONOSO, M. FRACCARO, J. LINIISTEN, F. POKT.4, SUSI SCAPPATICCI end L. TIEPOLO EURATOM UNIT FOR HUMAN RADIATION AND CYTOGENETICS, UNIVERSITY OF I'AVIA, ITALY, DEPARTMENT OF ENDOCRINO1A)GY AND hlETAROLIShI, KAROLINSKA SJUKHUSET, STOCKHOLM, SWEDEN AND CENTRAL LARORATORY, THE HOSPITAL, SONDRIO, ITALY (Received February llth, 1969) HI: Alpine marmot, iVlttrmotn mtrrmotcr, now confined to the Alps T and the Carpathians, and the steppe marmot, Mtrrniotn bobrtk, that occurs from Southern European Russia to Asia, are two representatives of the genus Mctrmotrt in the Palearctic Zone (BURTON, 1965) for which no chroniosonie studies have been reported. Studies on several north American members of the genus, have shown that the chromosome number of this group varies from 2n=36 of Illfrrmotti broiiwri to 2n=42 of Mtrrmoftr crrli!yrttn (RAUSCH and RAUSCH, 1965). The fundamental number (FN), here defined as the number of nutosome arms, is reported to be 62 for all of them (HOFF- MANN and NADLER, 1968). Material and methods Two :inimals, :I male and a female, were trapped in i'altellina, Italy, at the beginning of the hibernation. The female weighed 2.6 kg arid the male 1.4 kg. Chroniosome preparations were obtained from bone marrow with a direct method, from blood cultures processed after 48 and 72 hours and from cell cultures established from explants of kidney. The testes for meiotic preparations were processed by the method described by HUL- TEN et rtl. (1966). Sex chroniatin was investigated in nuclei from the kidney cultures stained with Feulgen. For autoradiography, 0.5 ,ire of 3H-TdR (hmersham, s.a. 6.2 c/mM) was added per in1 of medium for ' This publication i\ contribution N. 457 of the Euratom Biology Division.

THE CHROMOSOMES OF THE ALPINE MARMOT MARMOTA MARMOTA L. (RODENTIA: SCIURIDAE)

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THE C H R O M O S O M E S OF THE A L P I N E MARMOT MARMOTA MARMOTA L.

(RODENTIA: SCIURIDAE)' Ry K . FERNANDEZ-DONOSO, M . FRACCARO, J . LINIISTEN, F. POKT.4,

SUSI SCAPPATICCI end L. TIEPOLO

EURATOM UNIT FOR HUMAN RADIATION AND CYTOGENETICS, UNIVERSITY OF I'AVIA,

ITALY, DEPARTMENT OF ENDOCRINO1A)GY AND hlETAROLIShI, KAROLINSKA SJUKHUSET,

STOCKHOLM, SWEDEN A N D CENTRAL LARORATORY, THE HOSPITAL, SONDRIO, ITALY

(Received February l l t h , 1969)

HI: Alpine marmot, iVlttrmotn mtrrmotcr, now confined to the Alps T and the Carpathians, and the steppe marmot, Mtrrniotn bobrtk, that occurs from Southern European Russia to Asia, are two representatives of the genus Mctrmotrt in the Palearctic Zone (BURTON, 1965) for which no chroniosonie studies have been reported.

Studies on several north American members of the genus, have shown that the chromosome number of this group varies from 2n=36 of Illfrrmotti broiiwri to 2n=42 of Mtrrmoftr crrli!yrttn (RAUSCH and RAUSCH, 1965). The fundamental number ( F N ) , here defined as the number of nutosome arms, is reported to be 62 for all of them (HOFF- MANN and NADLER, 1968).

Material and methods

Two :inimals, :I male and a female, were trapped in i'altellina, Italy, at the beginning of the hibernation. The female weighed 2.6 kg arid the male 1.4 kg.

Chroniosome preparations were obtained from bone marrow with a direct method, from blood cultures processed after 48 and 72 hours and from cell cultures established from explants of kidney. The testes for meiotic preparations were processed by the method described by HUL- TEN et r t l . (1966). Sex chroniatin was investigated in nuclei from the kidney cultures stained with Feulgen. For autoradiography, 0.5 ,ire of 3H-TdR (hmersham, s.a. 6.2 c/mM) was added per in1 of medium for

' This publication i \ contribution N. 457 of the Euratom Biology Division.

CHROMOSOMES OF MARMOTA 171

A

B

C

1 2 3 4 5 6

7 8 9 10 11 12

1 3 14 15 16 1 7 1 8

x x

Fig. 1. Female karyotype from a cultured kidney cell. The chromosomes are arranged in three groups A, B, C (see text).

the last four and a half hours to the blood and kidney cultures, with colcemide during the last two hours of culture. The slides were coated with Kodak AR-10 stripping film, and exposed for the appropriate time. Details of procedures were as in TIEPOLO d r t l . (1967).

RESULTS

1. Mitotic chromosomes

The 68 metaphases analysed from ,the three tissues studied showed consistently a chromosome number of 2n= 38. The karyotype was subjectively arranged by size and position of the centroniere into three groups of chromosomes, as shown in Figures 1 and 2. None of the chro- mosomes had obvious sattelites or secondary constrictions.

1 i 2 DONOSO, FRACCARO, LINDSTEN, PORTA, SCAPPATICCI, TIEPOLO

1 2 3 4 5 6

7 . . 8 9 10 1 1 12

13 14 16 17 18 15 ?i

Y X

Fig. 2. hlale keryotype from a bone marrow cell.

Group A (N. 1-6-X) is composed of seven pairs, four of them nieta- centrics (numbers l , 4, 5 and 6) and two quasinietacentrics (numbers 2 and 3). To this group belongs the X chromosome, a metacentric, which has a size approximately equal to chromosome N. 5. The X could not be morphologically identified except at autoradiography. It was then found to be 5 % in absolute length of the entire Complement, both in the female and the male, without including the Y chromosome in thc measurements. Group B (N. 7-12) is composed of six pairs of sub- metacentrics. Group C (N. 13-18) is composed of six pairs of acro- centrics. We had some difficulty in classifying N. 18 because of some variation in the relative length of the short arm. In some cell the N. 18 could be defined as a submetacentric. The Y chromosome is the small- est of the complement and appears as a metacentric, although the position of the centromere is sometimes difficult to establish (Fig. 2) .

According to this classification the F.N., as defined in the Introduc- tion, is 60.

CHROMOSOMES OF MARMOTA 173

Fig. 3. - Phases of the first meiotic division in the mal,e. Arrows point to the XY bivalent.

2. Meiotic chromosomes

Various phases of meiotic divisions are shown in Figures 3 and 4. The sex bivalent becomes clearly visible in the early pachytene, as a fuzzy structure peripherally located in the nucleus (Fig. 3 a) and later

1 74 DONOSO. FRACCARO. LINDSTEN. PORTA. SCAPPATICCI. TIEPOLO

1 2 3 4 5 6

7 8 9 10 1 1 12

13 14 1 5 16 1 7 18

II- X ’

Fig. 4. Haploid karyotype from an X-carrying metaphase of the second meiotic division.

as zl thread with a slight positive heteropycnosis which contrasts with the autosome (Fig. 3 b). In the late pachytene, the sex chromosomes appear as a sex vesicle (Fig. 3 c). At dialrinesis-first metaphase the XY bivalent had in all cells a characteristic end to end association (Fig. 3, d , e , f ) . Bivalents at first metaph:ise and chroniosonies at second metaphzlse (Fig. 4 ) were consistently 19 in the 37 cells analysed. The number of chiasniata between autosomes (Fig. 5) was estimated to 37, 37, 38, 39, 40, 41, 41 and 45 in the eight diakinesis-first metaphases studied in this respect.

3. Radioautography

In the female cells, both from kidney and blood cultures, a single metacentric chromosome was found to be clearly late-replicating. This chromosome was interpreted as an X (Fig. 6) . In several cells among those with a high grain count there were conspicuous clusters of grains :it the centromeric regions. In these cells the long acrocentric chromo-

CHROMOSOMES OF MARMOTA 155

4

4

2

3

2

2

3

2

1

2 2

2 2

b 1 1

Fig. 5. Cut-out bivalents from a first spermatocyte. The lowest number of chinsniata considered to explain the shape of each bivalent is stated.

somes (Nos. 13 and 14) had characteristic clusters of grains both at the centromeric and at the telomeric region of their long arms (Fig. 5, a) . Another chromosome beside the X was late replicating. This was an acrocentric, N. 16 or 17 which in several cells appeared at the very end of S, late replicating in synchrony with the X. This pair showed, in a number of cells, a remarkable asynchrony between the two homologues (Figs. 5 c, d ) . The centromeric regions of several autosomes were also labelled towards the end of S. In the male, we obtained only 3 labelled cells. There was no indication of a late replicating pattern of the Y, but the low number of labelled cells did not allow a definite conclusion.

176 DONOSO, FRACCARO, LINDSTEN, PORTA, SCAPPATICCI, TIEPOLO

b

d Fig. 6. Radioautographs of female metaphases f rom kidney cell cultures. a : The late replicating X chromosome indicated by arrow and the same chroniosome with- out grains in the upper frame. The bent arrow indicates an example of long acro- centric chroniosome with characteristic pattern. The same chromosome without grains in the lower frame. b: Late replicating X chromosome (arrow) and the same without grains (frame). c: Cell with a late replicating X chromosome (straight arrow and lower frame) and one short acrocentric chromosome (16-17) heavily labelled (bent arrow and upper f rame). d: Cell labelled a t the very end of S . Note labelled X

chromosome (straight arrow) and short acrocentric chromosonies (bent arrows) .

CHROMOSOMES OF MARMOTA 1 7 7

Fig. 7 . Kidney cultured female nuclei. Arrows point to a single, peripheral hetero- pycnotic body. Feulgen stain.

4. Sex chromatin

A large proportion of female epithelial-like cells from kidney cul- tures displayed a single heteropycnotic mass which appeared as a V- shaped structure near the nuclear membrane (Fig. 7 ) . No similar struc- ture was observed in the male.

DISCUSSION

The karyotype of the Alpine marmot differs from those of four of the five North-American representatives of this genus so far described, both in chromosome number and in the size and morphology of the different groups of chromosomes. Thus, M . caligata and M . flaviventris both have 2n=42 with 22 metacentric and submetacentric chromo- somes and 18 acrocentrics (excluding the sex pair), while M . broweri has 2n=36 with 28 metacentrics and 6 acrocentrics (RAUSCH and RAUSCH, 1965; HOFFMAN and NADLER, 1968). Conversely the karyotype of M. mctrmota is very similar to that of the Northeastern American woodchuck, Mnrmotn monax. This species also has 38 chromosomes and the distribution of the different groups of chromosomes, namely 26 metacentrics and submetacentrics and 10 acrocentrics is similar to that of M. mnrmotcl (COUSER et al., 1963; HENIRSCHKE and HSU, 1967). Thus, our results do formally support one of the two hypotheses of HOFF-

MANN and NADLER (1968) who, assuming that the primitive marmot of the Pliocene had 38 chromosomes, postulated that M . marmotn should also have 38 chromosomes. However, a comparison between the karyo- 12 - II?redilas 63

178 DONOSO, FRACCARO, LINDSTEN, PORTA, SCAPPATICCI, TIEPOLO

type of ,If. mcirmottr as we defined it and the published ones of Mnr- motri monnx is difficult mainly because of the definition of chronio- some N . 11 in M . monux which can be classified either as a long acro- centric or as a subtelocentric and the shorter N. 18 in M. mctrmotcc, which we classified as acrocentric but with some doub8ts. In fact, the acrocentric group of M. momix appears to be composed of five pairs of chromosomes as it could be in M. mcirmotci, provided that in the latter species the pair N. 18 is not classified as an acrocentric but as a sub- metacentric. This probleni of classification is not trivial because if chromosome N . 11 of M. m o m x and N. 18 of M. mcirmotci are classified as acrocentrics then the F.N. in both species would be 60. If on the contrary both chromosomes are classified as submetacentrics, then the F.N. becomes 62. Ihrthermore, the smallest autosome N. 18 of M. mrir- motri looks identical to the pair of M. monrix which has been placed as N . 18 by COUSER et crl. (1963) and placed in the same position in the Atlas of Hsu and RENIRSCHKE (1967). Thus, if these two pairs should both be classified as submetacentrics then .M. mcrrmotn would have a F . N . of 62 and M . moncix of 64. The hypothesis of HOFFMANN and NADLER (1968) on the evolution of the genus Mrirmotn is based on several zoological, ecological and karyotypical characteristics, but one of the cornerstones of the hypothesis is the common F . N . of 62 for the group. It follows that it could be iniportarit that further studies :ire per- formed on both iM. monux and M. mcirmotrr in order to define the F.N. and/or to discover whether there are chroniosonie polymorphisnis with- in the animal population. To our mind these uncertainties indicate that while the concept of the F . N . is certainly useful in the classification of large groups of species it becomes less efficient when there are difficul- ties in the classification of individual chromosomes in closely related species.

The sex chroniosomes appear identical in M. mcirmotrr and M. monnx. It should be noted that the X chromosome is about the 5 % in length of the total karyotype. The homogeneity of the morphology of the X found in the species of this group, with the probable exception of M. flciuicentris, seems to us to indicate that the X chromosome has not undergone relevant changes in the evolution of the karyotype of this genus.

Finally, the pattern of replication of one acrocentric that we found in M. mnrmotn, which is late replicating and often asynchronous within the two honiologues could be a useful marker for cytotaxononiic studies in the genus Mrirmotu.

CHROMOSOMES OF MARMOTA 179

Acknoioledgernenf. - R. FERNANDEZ-DONOSO is at the Euratom IJnit in Pavia on leave from the Department of Genetics, University of Chile, Santiago, Chile, with a Fellowship of the International Atomic Energy Agency, Vienna.

SUMMARY

The Alpine niarmot (Mmmotcc murmotu) has 38 chromosomes. The X chromosome is nietacentric, five per cent of the complement in length and late replicating. The Y chromosome is a tiny metacentric. The number of autosonie arms is 60, but this number is uncertain because chroniosome N. 18 which was considered acrocentric could be alterna- tively a sub-metacentric. The number of chiasmata varied between 37- 45 in eight first spermatocytes analysed. These findings are discussed and compared with what is already known about other species of the genus Murmota.

Literature cited BURTON, M. 1965. Systematic dictionary of mammals of the world. - Museum Press,

London. COUSER, W., SARGENT, P., BROWHILL, L. E. and BENIRSCHKE, K. 1963. The somatic

chromosomes of the Northeastern American woodchuck, Marrnota Inonax. - Cytologia 28: 108-111.

HOFFMANN, R. S. and NADLER, C. F. 1968. Chromosomes and systematics of some North American species of the genus Mormota (Rodentia Sciuridae). - Ex- perientia 24: 740-742.

Hsu, T. C. and BENIRSCHKE, K. 1967. An atlas of mammalian chromosomes. - Vol. 1 : Folio 9, Springer, New York.

HULTEN, M., LINDSTEN, J., PEN-MING L. MINC and FRACCARO, M. 1966. The XY bivalent in human male meiosis. - Ann. Hum. Genet. 30: 119-123.

RAUSCH, R. I. and RAUSCH, V. R. 1965. Cytogenetic evidence for the specific distinc- tion of an Alaskan marmot, Marrnota broweri HALL and GILMORE (Mammalia: Sciuridae). - Chromosoma 16: 618-623.

TIEPOLO, L., FRACCARO, M., HULTEN, M., LIKDSTEN, J., MANNINI, A. and PEN-MINC L. M I N G . 1967. Timing of sex chromosome replication in somatic and germ-line

cells of the mouse and the rat. - Cytogenetics 6: 51-66.