Localization of nucleolar organizing regions in pinniped karyotypes

Transcription

1 Hereditas!M: 2%34 (1981) Localization of nucleolar organizing regions in pinniped karyotypes ULFUR ARNASON Institute of Genetics, University of Lund, Sweden ARNASON, U Localization of nucleolar organizing regions in pinniped karyotypes. -Hereditus 94: Lund, Sweden. ISSN 001&0661. Received April 9, 1980 A silver-staining technique was used for determining the localization of nucleolar organizing regions (NORs) in the four principal pinniped karyotypcs. The otariid and odobenid karyotypes were rep resented by Arctocephalus pusillus (2n=36) and Odobenus rosmarus (2n=32), respectively. The 2n=34 phocid karyotypc was represented by Cystophora crisrata and the 2n=32 karyotype by Phoca largha and P. fasciuta. In each of the karyotypes, only one pair carries a NOR site. This is consistent with the presence of a secondary constriction in one pair only. The limited differences that separate the otariid and odobenid karyotypcs involve the NOR pair. The otariid NOR pair is a rather small metacentric with the NOR site in the shorter arm. The odobenid NOR pair is a rather large submetacentric with the NOR site in the middle of the long arm. The translocation separating the 2n=34 and 2n=32 phocid karyotypes also involves the NOR pair. Thus, the chromosomal localion of NOR is not rigid. Together with recent advances in pinniped paleontology, the present results were interpreted as consistent with diphyletic origin of the pinnipeds from two ancestral lineages, one otariid-odobenid and one phocid, which entered the marine environment separately. 6'!fur Amason. Institute of Genetics, University of Lund, S Lund, Sweden Four principal karyotypes are found in the Pinnipedia. These karyotypes comprise the 2n=36 otariid, the 2n=32 odobenid and the 2n=34 and 2n = 32 phocid karyotypes. The karyological similarities of the Pinnipedia were described by FAY et al. (1967) and established further by ARNASON (1974a, 1977) and ANBINDER (1974). In ARNASON (1977) the G-band patterns of the principal karyotypes were compared in some detail. The G-band comparisons revealed a very close relationship between the otariid and odobenid karyotypes. The otariid and phocid karyotypes showed considerable similarities. Of the 2n=34 and 2n=32 phocid karyotypes the 2n=34 form is the most ancient one. The karyotypes differ by a single translocation. The present paper describes the localization of nucleolar organizing regions in the pinniped karyotypes. The otariid and odobenid karyotypes are represented by Arctocephalus pusillus and Odobenus rosmarus, respectively. The 2n=34 phocid karyotypes are represented by Cystophora cristata and the 2n=32 karyotype by Phoca largha and Phoca fasciata. The karyotype of P. largha has not been presented previously. The P. fasciata karyotype differs from other 2n=32 phocid karyotypes with respect to the sex chromosomes. The pinniped phylogeny is discussed in the light of the karyological findings. Material and methods The A. pusillus material was collected at Elephant Rock, South Africa, by Dr. Peter B. Best. The 0. rosmarus, P. largha and C. fasciata materials were collected in the eastern Bering Sea by Dr. F. H. Fay and established in culture by Mr. Don Ritter, Alaska Dept. of.health and Social Services, Fairbanks. The coat pattern of the mature female P. largha was anomalous, somewhat resembling P. hispida (FAY, in prep.). In other respects it resembled a typical P. largha and its pup was typical of that species. The C. cristata material originated from Kulusuk, Greenland, where it was collected by a local hunter, Mr. Torvald Mikaelsen. All chromosome analyses were performed on cells grown in tissue culture. The Ag-I technique of BLOOM and GOODPASTURE (1976) was used for staining of the nucleolar organizing regions (NORs). The incubation time was h at 37 C. The karyotype arrangements and designations follow the system proposed by ARNASON (1977) based on G-band comparisons. Results The South African fur seal, Arctocephalus pusillus

2 30 ~.ARNASON Hereditas 94 (1981) t X Y sml Fig. 1. Ag-I stained karyotype of the South African fur seal,drcrocephalus pusillus, 2n=36. Distinct deposit of silver in satp. Bar: 10prn. is a representative of the 2n=36 otariid karyotypes. With minor modifications due to different accumulation of C-heterochromatin, this karyotype is found in all sea lions and fur seals studied so far. In the Arctocephalus karyotype, only one pair is Ag-positive, Fig. 1. This pair, designated sat, is characterized by a prominent secondary constriction in the short arm, p. The two Sat homologues usually showed considerable differences in the amount of Ag-deposition but whether one specific homologue always showed the largest deposits could not be decided. ml x x wl 2 sml Fig. 2. Ag-I stained karyotype of the walrus, Odobenus rosrnarus, 211~32. The NOR site is located in w2q. Bar: 10 Pm.

3 Hereditas 94 (I9811 NUCLEOLAR ORGANIZING REGIONS IN PINNIPEDS li 3 in w2 was Ag-positive, Fig. 2. One of the homologues frequently showed a discontinuous deposit of silver. In the 2n=34 phocid karyotypes, one small metacentric pair, sat, is characterized by a secondary constriction in the short arm. This is the only site of the karyotype that is Ag-positive. Fig. 3 shows an Ag-I banded karyotype of the hooded seal, Cystophora cristata. The silver deposit was very limited in all preparations. 9 xx The 2n=32 phocid karyotypes are characterized by one large metacentric pair, s, having a secondary constriction in the p arm. The homogenously stained karyotype of the spotted seal, Phoca largha, is indistinguishable from other 2n= Fig. 3. Ag-I stained karyotype of the hooded seal, Cystophora cristaiu, 2n=34. The silver deposit in satp is indistinct. Bar: 10pm. The karyotype of the walrus, Odobenus rosrnarus, is closely related to the otariid karyotype, a great majority of the pairs showing identical G-band patterns (ARNASON 1977). The most distinctive chromosome of the walrus karyotype is w2. This chromosome has a wide secondary constriction in the middle of the long arm. In the walrus karyotype, only the secondary constriction phocid karyotypes except that of P. fasciata. A positive silver-staining reaction was limited to the secondary constriction in sp, Fig. 4. The autosomal complement of the ribbon seal, Phoca fasciata, is identical to that of other 2n=32 phocids. The X is particularly large, comprising almost 9% of the complement. Also the Y is larger than in other phocids. The silver staining of the ribbon seal chromosomes gave the same results as in P. largha, only the secondary constriction in sp being Ag-positive, Fig. 5. In the A. pusillus metaphases, associations between the sat homologues were occasionally observed. In 0. rosmarus, associations between the w2 homologues virtually never occurred. In the phocid karyotypes associations between Ag-I positive homologues did not seem to exist. x 6 i smt m l x x s ml X Y & sml Fig. 4. Ag-I stained karyotype of the spotted seal, Phoca lurgha, 2n=32. The NOR site is located in sp. Bar: IOpm. Fig. 5. &-I stained karyotype of the ribbon seal, Phoca fasciata, 2n=32. Bar: 1Opm.

4 32 ~.ARNASON Hereditas 94 (1981) Discussion The pinniped karyology is known in considerable detail. In this group, four principal karyotypes occur, viz. the 2n=36 otariid, the 2n=32 odobenid and the 2n=34 and 2n=32 phocid karyotypes. The G-band patterns of the principal karyotypes have been studied and compared (ARNASON 1974a, 1977); in these two papers, C-band studies of the principal karyotypes were also presented. The picture of the pinniped karyology has now been completed with NOR studies and the accumulated information permits a karyological survey of the group. Since the original contribution of FAY et al. ( 1967). more detailed karyological information has become available, in particular after the introduction of the G-band technique. G-banded karyotypes of several pinnipeds were presented by ARNASON (1974a) and the comprehensive studies of WURSTER-HILL and GRAY (1975) on the banding patterns in procyonids, viverrids and felids allowed them to make comparisons with the banded otariid and phocid karyotypes presented by ARNASON (1974a). The close relationship between the otariid and odobenid karyotypes was demonstrated by ARNA- SON (1977) who concluded that the otariid karyotype was the more ancestral of the two forms. This conclusion was primarily based on the fact that the otariid pair, sat, having a secondary constriction in the short arm, was apparently identical to a pair occumng in various carnivore karyotypes whereas the large odobenid pair, w2, having a secondary constriction in the long arm, is unique. The G-band pattern of w2 suggested that this chromosome had originated from a, fusion of two otariid chromosomes, sat and m6 (ARNASON 1977). The present demonstration of a single NOR site in both the otariid and odobenid karyotypes confirms this view. The Enaliarctidae (MITCHELL and TEDFORD 1973) are considered as representing a lineage from which first the Odobenidae and later the Otariidae evolved (REPENNING 1976). The separation of the Enaliarctidae into the lineages leading to the recent Otariidae and Odobenidae occurred in the North Pacific in early Miocene about 21 million years ago. The odobenids flourished in the North Pacific during the late Miocene and early Pliocene but became extinct in that area 3-4 million years ago. The recent Odobenus rosmarus is a successor to Prorosmarus, a branch that invaded the Atlantic Ocean through the Central American Seaway possibly as much as 8 million years ago (REPENNING and TEDFORD 1977). The extant Odobenus thus evolved in the North Atlantic but returned to the Pacific through the Arctic Ocean about 0.5 million years ago. The karyotype conservatism in the marine mammals has been demonstrated and discussed earlier in considerable detail (ARNASON 1972, 1974a-c). The presumed low degree of inbreeding among these animals was considered to be the basis for their karyotype uniformity. A conservation of repetitive DNA sequences in cetaceans also has been demonstrated (ARNASON et al. 1978; ARNASON et al., in preparation). When the otariid-odobenid situation is considered, a remarkable karyotype uniformity becomes evident. All otariids studied so far (Z. californianus, E. jubatus, A. pusillus, C. ursinus) show very similar G-band patterns, and the odobenid karyotype is strikingly similar to them. Great similarities exist between the otariid-odobenid karyotypes and those of the carnivores, particularly the pro- cyonids (ARNASON 1974a, 1977; WURSTER-HILL and GRAY 1975). It seems natural to conclude that the otariid karyotypes have been more or less unchanged since the initiation of the radiation of the existing genera about 5 million years ago (REPEN- NING and TEDFORD 1977). It is of course impossible to answer the question when the differences between the otariid and odobenid karyotypes arose. Since new karyotype forms are rarely established in marine mammals, it is likely that the differences arose early in the evolution of the odobenid lineage. However, colonization of the Atlantic Ocean by a small isolated group could also have provided conditions for the establishment of a new karyotype form. Like the odobenid and otariid karyotypes, the phocid karyotypes have a secondary constriction in one pair. In the 2n=34 phocids the morphology of this pair is similar to the otariid sat. In the 2n=32 phocids the secondary constriction is located in the short arm in the largest chromosome of the complement. As shown earlier, this chromosome, s, characteristic for the 2n=32 phocid karyotypes, originated by a fusion between the sat and t chromosomes in the 2n=34 karyotypes. The present silver staining procedure confirmed this view since it was shown that only a single NOR site is present in both karyotype forms. No other NOR regions could thus be involved in the origination of the s chromosome. Apart from the sat + t translocation, the 2n=34 and 2n=32 karyotypes are identical except for minor deviations caused by different accumulation of C-heterochromatin. In the autosomes of all

5 Hereditas 94 (1981) NUCLEOLAR ORGANlZlNG REGIONS IN PINNIPEDS 33 2n=32 phocids, very little C-heterochromatin is present, and the hooded seal (2n=34) shows the same picture. In the bearded seal, 2n=34, the centromeric heterochromatin is markedly more conspicuous; in addition, terminal heterochromatin is present in a few pairs. The accumulation of heterochromatin in the ribbon seal X has been mentioned earlier (ARNASON 1977). The karyotype conservatism in the phocids is as marked as in the otariid-odobenid lineage. The Hawaiian monk seal, Monuchus schauinslundi belongs to the 2n=34 phocids. The karyotype of this species has not been subjected to banding analysis but chromosome measurements have shown that the karyotype of this species is hardly distinguishable from the other 2n=34 karyotypes, the only difference being a higher arm ratio in one pair, m5, in the monk seal than in the other species studied (ARNASON 1977). According to REPENNING and by, (1977) the Hawaiian monk seal presumably became separated from an ancestral population more than 15 million years ago. The origin of the 2n=32 phocid karyotype is only tentative but Phoca hispida, which has a typical 2n=32 karyotype, seems to have been isolated for as much as 10 million years (REPENNING, pers. comm.). Both these figures strongly underline the karyotype stability of the marine mammals. The similarity between the otariid and phocid G-band patterns was demonstrated by ARNASON (1977). Based on these comparisons, it was concluded that the karyological agreement was compatible with a monophyletic origin of the pinnipeds. The possibility was, however, not excluded that the recent pinnipeds evolved from different carnivore ancestors that shared closely similar karyotypes. The banding studies showed a close karyological relationship of both groups with the procyonid karyotypes presented by WURS- TER-HILL and GRAY (1975). Recent advances in pinniped paleontology ( by 1976; REPENNING 1976) have indicated separated origins for the Otariidae and Phocidae. Taking these into consideration, it seems less likely that the karyological differences between the otariid and phocid lineages could have originated after these animals entered the sea. This suggests that the ancestors of the recent lineages had developed karyological differences before they acquired their marine habits. Thus the present survey of karyological data does not seem to support the earlier view of a common lineage entering the sea from which the odobenids, otariids and the phocids later diverged. The karyological similarities between the ota- riid-odobenid lineage and the procyonids are easily demonstrated. The similarity between the phocid and procyonid karyotypes is not as extensive but, nevertheless, the similarity between these two groups seems greater than the similarities between the phocids and any other terrestrial carnivore group. It is probable that the terrestrial ancestors of the phocids had a faster karyotype evolution than the procyonids. The karyological ancestry of the phocids among the terrestrial carnivores is, therefore, difficult to establish. From a karyological point of view it is worth noting that the chromosomes carrying the NORs seem particularly prone to participate in karyotype rearrangement. This has occurred both in the otariid-odobenid and in the phocid lineages. In both instances, the NORs have established new chromosomal localizations. Evidently, the localization of the NORs is, thus, not particularly rigid. Analogous observations have been made in another group, the Cetacea, which also exhibit pro- nounced karyotype uniformity (ARNASON in preparation). Acknowledgments. - I am greatly indebted to Drs. F. H. Fay, Institute of Marine Science, University of Alaska, Fairbanks and C. A. Repenning, U.S. Geological Survey, Menlo Park, California, for valuable comments and suggestions in the preparation of the manuscript. I am very grateful to F. H. Fay, P. B. Best and T. Mikaclsen for the materials collected and to D. Ritter for growing the Alaskan samples. The work was supported by grants Nos. B and B from the Swedish Natural Science Research Council. Literature cited ANBINDER, E. M Comparative karyology of the pinnipeds. - Thesis, Acad. Sci., YIadivosrok ARNASON, U The role of chromosomal rearrangement in mammalian speciation with special reference to Cetacea and Pinnidedia. - Hereditas 70: 1 I34 18 ~NASON, U. 1974a. Comparative chromosome studies in Pinnipcdia. - Hereditas 76: 17%226 ~NASON, b. Comparative chromosome studies in Ce- tacea. - Hereditas 77: 1-36 ARNASON, c. Phylogeny and speciation in Pinnipedia and Cetacea - A cytogenetic study. - Thesis, Institute of Genetics, University of Lund, Sweden ~NASON, The relationship between the four principal pinnipcd karyotypes. - Hereditas 87: hnason, 0.. PURDOM, I. F. and JONES, K. W Conservation and chromosomal localization of DNA satellites in balenopterid whales. - Chromosoma 66: BLOOM, S. A. and GOODPASTURE. C An improved technique for selective silver staining of nucleolar organizer regions in human chromosomes. -Hum. Genet. 34: FAY, F. H., RAUSCH, V. R. and FELTZ, E. T Cytogenetic comparison of some pinnipeds (Mammalia: Eutheria). -Can. J. Zool. 4s:

6 34 ~.ARNASON Hereditas 94 (1981) MITCHELL, E. D. and TEDFORD, R. H The Enaliarctinae, a new group of extinct aquatic Carnivora and a consideration of the origin of the Otariidae. -Bull. Am. Museum Nar. Hist. 151: RAY, C. E Geography of phocid evolution. - Syst. Zool. 25: REPENNING, C. A Adaptive evolution of sea lions and walruses. - Sysr : REPENNING, C. A. and RAY, C. E The origin of the Hawaiian monk seal. - Proc. Biol. SOC. Wash. 89 (58): REPENNING, C. A. and TEDFORD, R. H Otarioid seals of the Neogene. - U.S. Geol. Survey Professional Paper 992 WURSTER-HILL, D. H. and GRAY, C. W The interrelationships of chromosome banding patterns in procyonids. vivemds and felids. - Cytogenet. Cell Genet. 15: 36331