linear differentiation of the c-band pattern of the w...

Chromosoma (Bed.) 83, 275~87 (1981) CHROMOSOMA �9 Springer-Verlag 1981

Linear Differentiation of the C-Band Pattern of the W Chromosome in Snakes and Birds*

Gregory A. Mengden Department of Population Biology, Research School of Biological Sciences, Australian Nationai University P.O. Box 475, Canberra City, A.C.T. 2601

Abstract. Evidence is presented from C-banding studies that the W chromosome of eleven species of snakes is not homogeneous in nature but is differentiated linearly into alternating lighter and darker C positive regions. The same is true of the W chromosome of at least some birds. There is evidence from the literature indicating a similar differentiation of the Y chromosome of some mammals and here the intermediate C positive regions are deficient in highly repetitive DNA. The significance of heterochromatinization as a means of generating differentiated sex chromosomes is discussed in the light of these findings.

Introduction

Simple sex chromosome systems fall into two basic categories involving respec- tively male (XYff, XXg) or female (ZZff, ZWg) heterogamety. In vertebrates the former condition is found in most mammals, reptiles, some amphibians and fish, while the latter obtains in all birds and some reptiles, amphibians and fish. The development of such systems has always been assumed to be a secondary phenomenon. That is, cytologically differentiated sex chromosomes have arisen during the course of evolution from a progenitor pair of homologues indistinguishable in mitotic morphology and with complete pairing at meiosis. The lower vertebrates (fishes, amphibians and especially reptiles) offer good affirmatory evidence for such an assumption since related species are known in all three groups both with and without differentiated sex chromosomes.

Until recently it has also been assumed that the differentiation of sex chromosomes depended on the creation of a genetically differential segment between the two progenitor homologues which permits the differential accumulation of sex factors. This, it was argued, was a progressive process involving the gradual extension of genetically differential segments, which do not cross over

* Dedicated to Professor M.J.D. White on the occasion of his 70th Birthday

0009-5915/81/0083/0275/:$02.60

276 G.A. Mengden

(Darlington, 1958), at the expense of the pairing segments, which do cross over. Accompanying this there has often been a reduction in the size of the Y or the W element. As such there are two obvious mechanisms which might lead to the development of differentiated sex chromosomes: (1) chiasma localization and (2) pericentric inversion. Both of these processes lead directly to the production of both pairing and differential segments. No good case of the former is known although a possible example exists in the three-toed Congo Eel Amphiuma tridactylum where the long arms of one of the longest bivalents are invariably unpaired at male meiosis and hance do not cross over (Donnelly and Sparrow, 1965). The second mechanism is known in at least three species, namely Phyllodactylus marmoratus (King and Rofe, 1976). Cnemidophoris tigris (Bull, 1978) and Acrantophis dumereli (Mengden and Stock, 1980). In all three cases the sex pair is undifferentiated in overall length but the centromeres of the two chromosomes are located at different sites. Added to this, in all three cases, G banding indicates clearly that there has indeed been a pericentric inversion.

Recently Singh et al. (1976) have suggested that in snakes, and by extension other cases too (Schmid, 1979, 1980), heterochromatinization may be the critical step leading to the initial differentiation of the sex chromosome pair and that where other morphological differences occur between sex chromosomes these are secondary consequences of the differentiation process. This argument is based on three facts: (1) In Ptyas mucosus (Colubridae) the Z and W are indistinguishable in length and centromere position but they are differentiated in C-band characteristics. Whereas the Z has only a small centric C-band the whole of the W is C-band positive. (2) Using the technique of in situ hybridization it can be shown that a specific satellite DNA, sat III, extracted from female tissues of Elaphe radiata (Colubridae) is concentrated exclusively on the C-band positive W of Ptyas mucosus. The same is true of Natrix piscator (natricine Colubridae) as well as Bungarus caeruleus and B. fasciatus (Elapidae). (3) In Python reticulatus (Boidae) and Xenopeltis unicolor, without differentiated sex chromosomes, sex chromosome specific satellite III D N A does not hybridize to any of the chromosomes.

The case of Cnemidophoris tigris (Bull, 1978) makes it clear that not all systems conform to the Singh model. Neither can the argument of Singh et al. explain all snake systems. In Acrantophis dumereli (Boidae) there is a ZW system in which the two sex chromosomes are clearly differentiated by a pericentric inversion, as confirmed by G-banding, but only a small amount of centromeric C-banding obtains in both Z and W. Interestingly this is the only known boid with differentiated sex chromosomes.

Thus what is apparent is that there is no uniform solution applicable to all sex chromosome systems. Nevertheless there are clearly good grounds for regarding heterochromatinization as one mechanism by which differentiated sex chromosomes may have evolved and it is interesting therefore to consider what may have been involved in this process. To this end the present paper examines in some detail the properties of sex chromosomes in a number of amphibians, snakes and birds.

C-Band Pattern of the W Chromosome 277

Materials and Methods

Localities and sources for the specimens examined are provided in Table 1. Mitotic chromosomes were obtained from snakes by direct preparation of peripheral blood, following the technique of Baker et al. (1971), or from tissue culture (Stock and Mengden, 1975). Bird chromosomes were obtained directly by feather pulp preparation (Mengden and Stock, 1976) or from tissue culture (Stock and Mangden, 1975). The frog chromosome preparations are bone marrow flushes following exposure to PHA. All preparations were pretreated with colchicine and hypotonic 0.075 M KCL and then fixed in 1:3 acetic acid:methanol prior to air drying.

C-banding was carried out on air dried preps following exposure to saturated BaOH (see King, 1980). The G-banding was obtained after trypsin exposure (Sites et al., 1979) as well as with the combined trypsin-urea technique (Stock et al., 1974). Q-banding followed the procedure used by Stocker et al. (1978).

The silver staining technique employed was that of Goodpasture and Bloom (1975) using AgNO3.

Results

A. Snakes

Since the discovery o f he teromorphic sex chromosomes in snakes (Kobel, 1962) they have been found to be the most variable elements in the serpent genome (Beqak and Beqak, 1969; Baker et al., 1972 and Singh, 1972). Moreover , the extent o f the sex ch romosome differentiation found in any group tends to parallel the taxonomic posit ion o f that group. Thus, morphological ly "pr imi- t ive" families (most boids) do not have differentiated sex ch romosomes while the mos t extreme forms of sex ch romosome differentiation appear in the morpho- logically " a d v a n c e d " snakes (Viperidae and Elapidae). Colubrids occupy an intermediate posit ion with some species in a given genus having h o m o m o r p h i c sex chromosomes while other species in the same genus have he teromorphic sex chromosomes . In the present paper at tention has been directed to cases of both " in t e rmed ia t e " (Natracine Colubridae) and " a d v a n c e d " (Elapidae) families. In many snakes thus far studied the Z ch romosome has a small block of centromeric he terochromat in and many of them show " g r e y " blocks on both telomeres when examined by the C-banding method. Prel iminary G-band studies indicated that the Z is certainly not as conserved as is the mammal i an X (Mengden and Stock, 1980) and this observat ion is confirmed in the present study.

1. Natricine Colubrid Snakes. F r o m gross karyotypic preparat ions the sex chromosomes of the two New World natricine snakes examined here (Tharnnophis marcianus and T. elegans) have earlier been reported as the four th largest pair o f ch romosomes (Baker et al., 1972) and the data presented here confirms this. The W ch romosome is acrocentric in T. marcianus and metacentric in T. elegans, though intraspecific variat ion in centromere position occurs in some populat ions o f T. marcianus (Baker et al., 1972). The Old World natricine snake Amphiesma rnauri has a submetacentric W chromosome.

The W of all three species shows linear differential staining when C-banded, displaying species-specific patterns of lighter and darker C positive bands along its entire length. In T. marcianus the acrocentric W appears predominant ly

278 G.A. Mengden

Table 1. Specimens examined

Snakes Natricine Colubrid Snakes

Amphiesma mauri

Thamnophis elegans

T. marcianus

IF 1F 1M

1M 1F

3F

Rainbow Bay, Qld. Australia Tin Can Bay, Qld. Australia Bessey Springs, N.T. Australia

Salt Lake Co., Utah, U.S.A. Salt Lake Co., Utah, U.S.A.

Jeff Davis Co., Texas, U.S.A.

Elapid Snakes - All Australian Notechis ater ater

N. a. serventyi

N. seutatus

Pseudechis guttata

P. porphyriacus

Pseudonaja textilis

Tropideehis carinatus

Elapid Snakes - All African Aspidelaps lubricus lubricus Naja melanoleuca N. nigricollis

Anurans Rana clamitans

Birds Amazona ventralis

A. vittatus

A. guildingi

Buteo jamaicensis

1M 2F

1M 3M 3F

1F 4M 2F 2F 1M 1F 1F

2F

2M 1F IF

2M 3F 1M 1F 1M 1F

1F 1F 1M 1M 2F

IF 1F 1M 1F

3M 3F

2F 2M

3M 2F

2M IF

1F

Wilmington, S.A. Wilmington, S.A.

Flinder's Is., Tas. Chappell Is., Tas. Chappell Is., Tas.

Bungendore, N.S.W. Collector, N.S.W. Collector, N.S.W. Collector, N.S.W. Captains Flat, N.S.W. Captains Flat, N.S.W. Skeleton Cr., Vic.

Oakey, Qld.

Casuarina Sands, A.C.T. Casuarina Sands, A.C.T. Pine Is., A.C.T.

Canberra, A.C.T. Canberra, A.C.T. Nowra, N.S.W. Macquarie Marshes, N.S.W. Monto, Qld., Billiat, S.A.

Mt. Glorias, Qld. Springbrook, Qld. Springbrook, Qld. Mullumbimby, N.S.W. Mullumbimby, N.S.W.

San Antonio Zoological Gardens Houston Zoological Gardens San Diego Zoological Gardens San Diego Zoological Gardens

Nashville, Tenn., U.S.A. Nashville, Tenn., U.S.A.

Houston Zoological Gardens Houston Zoological Gardens

U.S.D.I. Field Station, Puerto Rico U.S.D.I. Field Station, Puerto Rico

Houston Zoologicai Gardens Houston Zoological Gardens

Houston Zoological Gardens


Fig. 1. a A C-banded preparation of the New World Natricine snake Thamnophis marcianus. The W sex chromosome exhibits a large "g r ey" band in the telomeric region distal to the centromere and other light gaps in an otherwise dark C-band positive heterochromatic region. Many of the autosomes also exhibit an intermediate "g r ey" reaction in the telomeric regions, b A C-banded preparation of the W chromosome of an Old World Natricine snake Amphiesma mauri demonstrat ing a light C-band reaction along the distal half of the long arm (bracket) as well as an interstitial light band (arrowed)

dark staining after C-banding, but exhibits a large " g r e y " band in the telomeric region distal to the centromere (Fig. 1 a). There is also evidence of lighter gaps in the otherwise dark staining, C-positive, heterochromatin, particularly near the centromere. Many of the autosomes exhibit a " g r e y " reaction in the telomeric regions when C-banded and these areas are easily distinguished from the darker centromeric heterochromatin (see also Mengden and Stock, 1980, Fig. 3 b). The metacentric W chromosome of T. elegans is largely dark staining when C-banded, but shows distinct lighter grey bands on both telomeres.

The submetacentric W of the Old World natricine snake Amphiesma mauri is dark 'C-band positive over the entire short arm. The long arm shows lighter C-positive heterochromatin on the distal half of the arm as well as a light interstitial band (Fig. l b).

2. Elapid Snakes. Data are presented here on six Australian and three African species. This represents but a small proportion of the chromosome banding data that will be discussed in a forthcoming cytotaxonomic treatment of the family and its relationships (Mengden unpublished). The gross karyotype for the Australian Tiger Snake Notechis scutatus has been previously published (Shine and Bull, 1977) and the sex chromosomes are again the fourth largest pair. The W chromosome of all specimens reported in the present paper also

280 G.A. Mengden

Fig. 2. a A comparison of C and G-banding patterns of the W chromosomes in two species of Austral ian elapid snakes, Notechis scutatus (N.s.) and Pseudechis porphyriacus (P.p.). Note that neither species exhibits a simple inverse relationship between dark C positive regions and G-band regions. C-band preparations of P.porphyriacus clearly demonstrate close sister chromatid apposition in the dark C-band regions, b C-banded W chromosomes from a series of Austral ian and African elapid snakes. All exhibit a specific banding pattern of lighter and darker C positive regions except Naja melanoleuca (N.m.) which appears darkly C-band positive throughout its length. Austra- lian Species: N.a.a=Noteehisaterater , N . A ? = N . aterserventyi, N.s. =N . scutatus, P.g. =Pseudechis guttata, P.p. = P. porphyriacus, P.t. = Pseudonaja textilis, T.c. = Tropidechis carinatus. African Species: A.1. = Aspidelaps lubricus, N.n. = Naja nigricollis, N.m. = Naja melanoleuca

exhibit a species-specific linear pattern of alternate lighter and darker C-band positive areas (Fig. 2). The lighter C-band positive regions, however, are darker than typical euchromatic areas. No intraspecific variation in the C-banding pattern of the W Chromosome has been detected except between subspecies of Notechis ater where the banding pattern is consistent within a subspecies

Fig. 3. a Silver staining in the Tiger Snake, Notechis scutatus in the largest pair of autosomes (/) and the W sex chromosome. Note the lack of silver staining on the W chromosome, b Silver staining in the Red Bellied Black Snake, Pseudechis porphj~riecus, in the largest pair of autosomes and the W sex chromosome. Note, again, there is no silver positive reaction on the W chromosome. The W chromosome is as large as the largest pair of autosomes, e Silver staining for the N O R in Notechis scutatus in situ. Note, the only silver positive region in the genome is on the largest pair of autosomes, d C-banded W sex chromosome of the Hispaniolan Parrot Amazona ventalis exhibiting a differential C-positive reaction with the darker C-positive material in a paracentric position, e C-banded preparation of the Red Tailed Hawk Buteo jamaicensis. Note the light C-band positive region at the distal end of the long arm of the W sex chromosome as well as lighter segments interstitially

282 G.A. Mengden

though varies between subspecies (Fig. 2). Whether the absence of variation in other cases constitutes a true taxonomic difference remains to be clarified.

Close sister chromatid apposition is sometimes observed in the regions of dark C-positive heterochromatic bands, particularly in the Red-bellied Black Snake Pseudechis porphyriacus (Fig. 2), but such close apposition has never been seen in the lighter C-band positive regions. When Q-banded, the dark C positive bands appear Q negative, or dull, as compared to the rest of the W chromosome and the autosomes.

All the elapids which have been studied in this laboratory show G-banding of the W chromosome. The G-band patterns also appear to be species-specific (Fig. 2) and there is no simple inverse relationship between the dark C-band regions of the W chromosome and the G negative or interbands, as seen in the entirely heterochromatic arms of some autosomes in the boid Sansinia mada- gascarensis (compare Figs. 3a and 10 in Mengden and Stock, 1980).

Silver staining demonstrates that the only Ag- positive area in the genome is found interstitially on the long arm of the largest pair of autosomes. This coincides with a large acromatic gap which I assume to be the NOR (Fig. 3 aa-c). No W chromosome of any elapid tested has shown any silver positive reaction. Clearly therefore, none of the lighter C or G-band areas of the W correspond to secondary constrictions.

B. Birds

While some ratites are said to lack differentiated sex chromosomes (Takagi et al., 1972), a majority of birds show ZW sex chromosome heteromorphy. There are relatively few published studies on C-banding in birds. The four species referred to here all show obvious heteromorphism and the W is once more clearly linearly differentiated in C-band characteristics. The pattern of banding along the W chromosome is very similar in the three species of Amazon Parrots (Hispaniolan Parrot Amazona ventralis, Puerto Rican Parrot A. vittatus and the St. Vincent's Parrot A. guildingi) and is shown for A. ventralis which has a grey telomeric region on the short arm and a large grey area on the distal two thirds of the long arm (Fig. 3 d). A re-examination of C-band characteristics of the Red Tailed Hawk, Buteojamaicensis (Mengden and Stock, 1976) shows that it too is linearly differentiated (Fig. 3e). In all four bird species studied in this paper some of the grey areas of the W are not easily distinguished in staining intensity from the euchromatic areas of the autosomes. In the case of the hawk, however, in addition to the grey telomeric region on one arm the other arm is segmented with a particularly dark area near the centromere and the telomere.

Discussion

Differences in staining intensity in sex chromosomes after C-banding, similar to the cases reported above, have been seen in the Y chromosome of Drosophila melanogaster (Hsu, 1971), Man (Jalal et al., 1974a), the Caribou (Pathak and Stock, 1974) and some hamster species, namely Cricetus cricetus, Cricetulus


griseus and Mesocricetus auratus (Vistorin et al., 1977). Jalal et al. have classified the three former cases as examples of a distinct category of C-band heterochromatin.

Both Arrighi et al. (1974) and Jalal et al. (1974b) have suggested that the phenomenon of differential C-banding may be related to the differential rate of fading of C-band preparations. However, my own observations indicate that the condition is consistently reproducible and moreover is evident in freshly prepared material from a variety of tissues.

A similar differentiation of heterochromatin into alternating lighter and darker C positive regions is apparent in C-band preparations of the W chromosomes of birds of the genus Larus published by Ryttman et al. (1979) and photographs published by Stock et al. (1974) of the W chromosomes of the ring-necked Dove Streptopelia risoria and the domestic pidgeon (Columba livia). Ryttman et al. (1979) made no reference to the phenomenon, while Stock et al. (1974) commented that while the W chromosome of the species they studied were darkly staining throughout it had a distinctly darker region near the centromere. ' ~.~

Singh et al. (1976 and 1980) on the other hand, did not report any sign of differential intensity in the character of C positive regions along the W chromosome of the snakes they studied, though they point out that the homomorphic W of the colubrid Ptyas mucosus gave a uniform " g r e y " C-band positive reaction. The W chromosomes of all snake species they studied with heteromorphic sex chromosomes were reported as dark C positive throughout their entire length. The consistent absence of any non-centric C blocks in the autosomes and the lack of even centric C-bands in some of their preparations suggests a possible over-exposure to the alkali treatment; this would be expected to blurr any differential staining that might be present (Schmid, 1979).

The results obtained from the in situ hybridization of cRNA from sex chromosome specific sat DNA of snakes appear somewhat ambiguous. Thus sat III RNA obtained from Elaphe radiata appears to label the W chromosome uniformly throughout its length in all species which show C-band differentiated sex chromosomes, with the exception of E. radiata itself. In this species it is claimed that the label localizes at the telomeres (Singh et al., 1980) though the figure offered in support of this claim suggests that label is distributed over most of both arms excluding only the immediate centromeric regions. On the other hand the BK minor cRNA derived from D N A of Bungarusfascia- tus, and which appears to be homologous with the minor sat IV of E. radiata, shows a more localized and distinct distribution in both E. radiata and Naja naja oxiana. On this evidence it is clear that there are areas in the W chromosomes of some snakes where neither sat III or BK minor are present and other areas where they may co-exist.

Some of the Australian members of the '" advanced" family Elapidae possess exceptionally large W chromosomes. Thus in Pseudechis porphyriacus (Fig. 3 b) the W is as large as the largest autosome and in most species the W is at least as large as the Z. Such W chromosomes, with their obvious diversity of C positive banding patterns between species, would be especially interesting material in which to examine the composition of the different C-band regions

284 G.A. Mengden

in terms of their capacity to hybridize with sex chromosome specific satellites, provided of course that in situ hybridization is sufficiently sensitive to distinguish between short adjacent regions with and without satellite DNA.

Comparing their own data on banding patterns in hamster species to the DNA data of Arrighi et al. (1974), Vistorian et al. (1977) have suggested that some areas of the differentially staining C positive regions of the Y chromosome may be deficient in highly repeated DNA sequences. Further evidence of the unique structure of the intermediate " g r e y " C-band material is found in the Y chromosome of the mouse Mus musculus. This chromosome shows no dark centromeric heterochromatin but is intermediate between centric heterochromatin and euchromatin with a grey appearance similar to the W of Ptyas rnucosus and the intermediate blocks on the elapid W chromosome reported here (Chen and Ruddle, 1971; Pardue, 1970; Arrighi and Hsu, 1971; Hsu et al., 1971). Pardue and Gall (1970) have shown the Y chromosome of the mouse is the only element in the genome which is totally deficient in highly repetitive satellite DNA. Whether this lighter staining C band material represents an intermediate stage in the process of heterochromatinisation is not clear.

Whatever the details of the heterochromatinization process of the W chromosome in snakes and birds there is a need to distinguish two categories of heterochromatinization in relation to sex chromosome differentiation:

(1) Primary heterochromatinization where the first step in the differentiation of the sex pair actually depends on the heterochromatinization event, though this does not pre'clude structural changes following on subsequently.

(2) Secondary heterochromatinization where heterochromatin changes follow from, rather than initially determine, the differentiation of the sex pair which would depend either on chisasma localization or else on primary structural change.

As far as primary heterochromatinization is concerned it is difficult to imag- ine how such a differential molecular transformation could have taken place between what must initially have been two homologous structures. Neither is it obvious how the presumed silencing of a whole chromosome has been compensated for in a developmental sense. Singh et al. (1976) have suggested that amplification and distribution of sex specific satellites has involved saltation coupled with multiple internal rearrangements which they assume to be inversions. If this is genuinely the case the inversions in question must have been paracentric because there is no change in the gross morphology between sex chromosome homologues in Ptyas rnucosus. Since the W chromosome of snakes has been shown to G-band satisfactorily, one test for the inversion distribution model of Singh et al. would be to compare the G-band pattern of the Z and W chromosomes in species where they are homomorphic yet differentiated in respect to C-bands and satellite.

King (1980)has drawn attention to the possibility of transforming euchromatin into heterochromatin without any accompanying structural changes. His argument is based on comparisons of the C-banding characteristics between the autosomes of closely related species of frogs. At least one case is on record within a species, however, where a known autosomal polymorphism is involved in which one homologue is dramatically different in C-band character from its partner (Bianchi and Ayers, 1971). A similar situation has been observed


Fig. 4. A C-banded preparation of the mitotic chromosomes of a female frog of the species Rana clamitans. The sixth largest pair of chromosomes exhibit a dark C-band block over the distal half of one arm of one homologue only. Included are two further examples of the same situation from distinct females collected at the same locality. See text for further discussion

for pair six in females o f a popula t ion o f the frog Rana clamitans. Here three female specimens f rom a single popula t ion o f R. clamitans examined by me exhibited a large dark staining C-band positive block in one a rm of one homologue of ch romosome pair six wi thout altering the length o f that arm. This homologous pair of ch romosomes is easily distinguished by size f rom all other autosomes (Fig. 4). Three male specimens studied f rom the same popula t ion did not possess this C-positive block. Addi t ional specimens need to be examined f rom this and other populat ions to determine if this sex limited po lymorph i sm in fact represents a genuine sex determining system. Interestingly, the only other anuran species with differentiated Z W chromosomes are also ranids. These are Pyxicephalus adspersus and P. delalandii both o f which show an increased heterochromat ic content in the W chromosome. In the first o f these species the increase is coupled with a radical reduct ion in the size of the W whereas in the other it is accompanied by a pericentric inversion but no size alteration. Schmid (1980), who described these cases, follows Singh et al. (1980) in assuming that the structural changes concerned are secondary to the heterochromat iniza- t ion process though there is no compell ing evidence to refute or conf i rm such an assumption. This case clearly highlights the difficulty o f distinguishing between pr imary and secondary heterochromatinizat ion.

As far as secondary heterochromat inizat ion is concerned there is only one suggestive example so far known. Saez (1963), has argued that progressive

286 G.A. Mengden

h e t e r o c h r o m a t i n i z a t i o n o f the Y c h r o m o s o m e has o c c u r r e d in s o m e o r t h o p t e r a n

n e o X Y sys tems, a sugges t i on t h a t has been s u p p o r t e d by W h i t e (1973). T h e i r

a r g u m e n t s h o w e v e r a re ba sed on a c o m p a r i s o n b e t w e e n la rge ly u n r e l a t e d species.

T h e r e is ce r t a in ly g o o d e v i d e n c e f r o m Cnernidophoris tigris t h a t C - b a n d m a t e r i a l can be los t f o l l o w i n g a p e r i c e n t r i c i n v e r s i o n l e ad ing to sex c h r o m o s o m e d i f fe ren-

t i a t i o n (Bull , 1978). T h e s a m e a p p e a r s to h a v e been the case in the e v o l u t i o n

o f t he n e o X Y sys tem o f the o r t h o p t e r a n Stenacatantops angustifrons ( K i n g

a n d J o h n , 1980). T h u s s e c o n d a r y d e h e t e r o c h r o m a t i n i z a t i o n m a y also o c c u r in

the e v o l u t i o n o f s o m e sex c h r o m o s o m e systems.

Acknowledgements. I am indebted to Prof. B. John for criticaily reviewing the manuscript. I wish to thank Drs J.J. Bull, B. John and M. King for many discussions on the subject of sex chromosome differentiation, in particular in the Australian elapid snakes. I am indebted to Dr J.J. Bull for permission to publish C-band data of one natricine snake species which represents joint work and to Dr A. Dean Stock who graciously provided the previously unpublished photograph of the hawk C-banded preparation.

M. Fitzgerald, R. Jenkins and J. Wombey assisted with the collection of Australian elapid snakes. The Parks and Wildlife Services of Queensland and New South Wales have provided collecting permits for some of the specimens examined in this study and their cooperation in obtaining material used in this and other studies currently underway is gratefully acknowledged.

I wish to thank Dr T.C. Hsu of M.D. Anderson Hospital and Tumor Research Institute, Houston; Dr K. Benirschke, Dr J. Beacon, and T. Schultz of the San Diego Zoo, Hugh Quinn and R. Berry of the Houston Zoo; J. Murphy of the Dallas Zoo and Dr N. Schnider of the U.S.D.I. who all provided space, equipment and/or assisted in obtaining samples from specimens in their charge.

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Received January 19, 1981 / Accepted by B. John Ready for press February 12, 1981

linear differentiation of the c-band pattern of the w...

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