CLOSE KARYOLOGICAL KINSHIP BETWEEN the REPTILIAN SUBORDER SERPENTES and the CLASS AVES* by WILLY BEQAK, MARIA LUIZA B]~Qak, H

Home , Anomalepis, Spilotes, Spilotes pullatus, Xenodon merremii

Chromosoma (Berl.) 15, 606--617 (1964)

From the Section of Genetics, Instituto Butantan, S~o Paulo (Brasil) and the Department of Biology, City of Hope Medical Center, Duarte, California (USA) CLOSE KARYOLOGICAL KINSHIP BETWEEN THE REPTILIAN SUBORDER SERPENTES AND THE CLASS AVES* By WILLY BEQAK, MARIA LUIZA B]~qAK, H. R. S. NAZARETH, and SustTMu OE•o With 16 Figures in the Text (Received August 21, 1964)

Introduction An elaboration of the karyologieal relationships among the classes Reptilia, Ayes, and Mammalia assumed great interest after we found that mammals and birds constitute two independent groups with regard both to absolute size of the chromosomes governing development of the homogametic sex as well as the total genetic content of the diploid complement. The mammalian X and the avian Z are similar in size in members of their respective classes, and mammals have twice the amount of genetic material possessed by birds (0~o et al. 1964a, 1964b). Three postulates were based on these findings: 1. Fossil records indicate that modern forms of birds already existed when protoinsectivores, ancestral to all placental mammals, first appeared at the dawn of the Cenozoic era. This long separation of the two classes is reflected in the finding that birds and mammals have different amounts of genetic material and different sex-determining mechanisms. 2. Speciation within each class of warm-blooded vertebrates depended mainly on chromosome rearrangements and mutations of individual genes, not on drastic changes in total genetic content. 3. In each class, the direct ancestor already contained in its diploid chromosome complement a particular chromosomal pair serving as sex elements. Despite subsequent refinement of the chromosomal sex- determining mechanism, the element accumulating factors governing development of the homogametic sex retained the original size and genetic * In S~o Paulo, this work was supported by Fundae~o de Amparo a Pesquisa do Estado de S~o Paulo e Fundo de Pesquisas do Instituto Butantan. In Duarte, this work was supported in part by grant CA-05138-05, National Cancer Institute, U.S. Public Health Service. Contribution No. 36--64, Department of Biology, City of Hope Medical Center. KaryologicM kinship between snakes and birds 607 constitution of the ancestral chromosome. Original genes extraneous to sex determination persisted as Xdinked genes in mammals, Z-linked genes in birds. These postulates led us to study members of the class Reptilia by measuring total chromosome area and by making microspectrophotometric measurements of DNA content (to be reported separately by ATKIN et al.). We found that the class ReptiIia is not uniform; instead, members fall into two distinct categories: one comprised of the orders Crocodylia and Chelonia, the other constituted of the order Squamata. Within the latter, morphologically distinct sex chromosomes have been found and femMe heterogamety established among snakes, the suborder Serpentes (KoBEL 1962; ]~EgAK et al. 1962, 1963b), but not lizards, the suborder Sauria (MATT~Eu and VAN BnINK 1956; VAN BRI~: 1959). Kerein are described cyto]ogicM studies of eight species of snakes. With regard to total genetic content as well as absolute size of the Z-chromosome, snakes and birds may be regarded as similar.

Materials and Methods The eight snakes chosen for this investigation are listed in Table 1. Chromosome preparations of seven of the eight were made in S~o Paulo (Brasil) from cultured peripheral blood leukocytes fixed in a 3 : 1 mixture of methanol and acetic acid, then Mr-dried (B~gAK et al. 1963a). Chromosome preparations from the

Table 1. Species o/snalces investigated ]or the present study An asterisk (*) indicates the one from which chromosome preparations were made in Duarte (USA). Order Family Species (diploid number) Reference

Serpentes Boidae Boa constrictor amarali (2n = 36) BECAK et al. 1962 Epicrates cenchria crassus (2n = 36) Present study Colubridae *Dr ymarehon corais couperi (2n ~ 36) Present study Spilotes pullatus anomalepis (2 n = 36) Present study Clelia occipitolutea (2n = 50) Present study Xenodon merremii (2n = 30) Present study Crotalidae Bothropsjararaca (2n -- 36) BEC•K et aL 1962 Bothrops atrox (2n = 36) Present study gopher snake, Drymarchon corais couperi, were made in Duarte (USA) from fresh spleen and gonads fixed in 50 per cent acetic acid and squashed, the same procedure used in the previous studies of mammals and birds (0~o et al. 1964a, 1964b). The method for measuring total chromosome area as well as absolute size of the Z has been described in considerable detail (0~o etal. 1964a). Summarized briefly, photomicrographic negatives of colchieinized metaphase figures made at the same magnification ( • 2200) are selected, each negative placed in the photo- graphic enlarger, the image projected onto a sheet of white paper at a final magnification of • 6300, the outline of each chromosome traced with a sharp hard pencil, 608 BEgAx, BzgAx, NAZA~T~ and Omvo: and the images cut out and weighed on a precision balance. The conversion factor was found to be 0.3332 g ~ 100 #e. As five metaphase figures from the homogametie male sex of each species were measured, the value for total chromosome area given in Table 2 is the mean of five measurements, that for the Z-chromosome the mean of 10 measurements.

Table 2 Weight in grams o/the paper cutouts o/chromosomes, both minimum and maximum The mean of the measurements has been converted to #~. [An asterisk (*) indicates the one species studied in Duarte; others were done in S~o Pau]o.]

Family, species Total area Z-chromosome Z:AZ (diploid number) Weight (g) Weight (g) ~ (%)

Boidae: Boa constrictor amarali 0.2737 82.14 0.0130 3.90 9.61 (2n=36) (0.2400) (0.0113) (0.3092) (0.0140) Epicrates cenchria crassus 0.2969 85.86 0.0125 3.61 8.49 (2n=36) (0.2496) (0.0105) (0.3363) (0.0143) Colubridae : *Drymarchon corai8 couperi 0.2087 62.64 0.0117 3.51 11.02 (2n = 36) (0.1740) (0.0079) (0.2269) (0.0116) Spilote8 pullatus anoma- 0.2758 79.76 0.0109 3.15 7.84 lepis (2n = 36) (0.2440) (0.0090) (0.3077) (0.0138) Clelia occ@itolutea 0.2654 79.65 0.0131 3.93 10.37 (2n = 50) (0.2476) (0.0125) (0.2892) (o.o136) Xenodon merremii 0.2626 78.81 o.o156 4.68 11.46 (2n : 30) (0.2537) (0.0137) (0.2813) (0.0182) Crotalidae : Bothrops jararaca 0.2689 80.70 0.0128 3.84 9.57 (2n~36) (0.2542) (0.0110) (0.2941) (o.o155) Bothrops atrox (2n = 36) 0.2778 80.34 o.o117 3.80 8.30 (0.2574) (0.0096) (0.3111) (o.o15o)

Observations and Discussion 1. Karyological relationships among mammals, birds, and three orders o/reptiles Figs. 1--6 contains somatic metaphase figures printed at the same magnification of the rat (Rattus norvegieus, 2n=42), class Mammalia, and the pigeon (Columba livia domestica, 2n: 80 • class Ayes, together with four representative species of the class Reptilia: the South American Karyological kinship between snakes and birds 609

Figs. 1--6. Colchicinized somatic metaphase figures obtained directly from spleen of a mammal, a bird, and four species of reptiles. All are printed at the same magnification. The sex chromosomes are so marked. All photomicrographs were taken by Leitz Panphot (lenses used: 100 x 10). -- Fig. 1. Female Rattus norvegieus (2n =42), family Muridae, order Rodentia, class Mammalia -- Fig. 2. Male Columba livla domestica (2n=80 • family Columbidae, order Columbi]ormes, class Ayes. -- Fig. 3. Female Caiman sclerops (2n =42), family Alligatoridae, order Crocodylia, class Reptilia. -- Fig. 4. Female ~lmyda ]erox (2 n -- 66), family Trionyehidae, order Chelonia, class Reptilia. -- Fig. 5. Female Anolis carolinensis (2n --36), family Tguanidae, suborder Sauria, class Reptilia. -- Fig. 6. Female Drymarchon corais couperi (2 n = 36), family Colubridae, suborder Serpentes, class Reptilia alligator (Caiman sclerops, 2n~42), order Crocodylia; the fresh-water soft-shelled turtle (Amyda ]erox, 2n= 66), order Chelonia;the chameleon lizard (Anolis carolinensis, 2n~36)~ suborder Sauria, order Squamata; and the gopher snake (Drymarchon corais couperi, 2n~36), suborder 610 BEVAK,BEgAK, NAZARETH and OHIO:

Serpentes, order Squamata. Comparison of the six metaphase figures shows that the class Reptilia consists of two different karyological groups. Members of the orders Crocodylia and Chelonia (Figs. 3 and 4) present karyologicM characteristics similar to those of placental mammals. The chromosomal constitution of Caiman sclerops (Fig. 3) closely resembles that of Rattus norvegicus (Fig. 1), while that of Amyda/erox (Fig. 4) is similar to certain mammals with a high diploid chromosome number, such as the guinea pig (Cavia cobaya, 2n= 62). Our measurements of total chromosome area, as well as microspectrophotometric determinations of DNA content, have both shown that the total genetic content of the orders Crocodylia and Chelonia is about 80 per cent of that found in mammals. The mean total chromosome area for Rattus norvegicus was 156 #2 while Caiman sclerops measured ]31 #2 and Amyda lerox, 118# 2. Since morphologically distinct sex chromosomes could not be discerned in members of these two orders (MATTHEY, and vA~ B~INK 1956; VAS Bt~INK 1959) and the heterogametic sex remains unknown, the evolutional relationship of these orders to placental mammals cannot be established. On the other hand, the close karyologicM kinship among birds (Fig. 2), lizards (Fig. 5), and snakes (Fig. 6), is quite evident. Not only are their Chromosome complements characterized by the presence of microehromosomes, they are also similar in total genetic content. The mean total chromosome area for Columba livia domestica was 63/~2 (O~No st al. 1964b), for Anolis carolinensis, 82 #2, and for Drymarchon corals couperi, 63 #2 (Table 2).

2. Uni/ormity of total chromosome area in the suborder Serpentes A basic karyotype consisting of eight pairs of macrochromosomes and 10 pairs of microchromosomes appeared to be possessed by a great majority of snakes. Deviations from this were encountered only in the family CoIubridae, such as Clelia occipitolutea (2n~-50) shown in Fig. 11, and Xenodon merremii (2n= 30), in Fig. 12. KoB~L (1962) found the same basic karyotype in Vipera berus in the family Viperidae. Uniformity of the suborder Serpentes with regard to the total genetic content was expected in those species possessing the basic karyotype. Actual measurements not only substantiated this expectation, but also revealed that two deviations in chromosome number were unrelated to total genetic content (Table 2). Total chromosome areas of eight snake species in the present study varied from 62.64/~ for Drymarchon corals couperi, to 85.56 #2 for Epicrates cenehria crassus (Table 2). As total chromosome areas of various avian species in the previous study (OHio et al. 1964b) varied Karyological kinship between snakes and birds 611

from 62.42 #2 to 67.95 #2, the total genetic content of the reptilian suborder Serpentes appears only slightly larger than that for the class Aves. 3. Uni]ormity o[ the Z-chromosome Recognizable heteromorphism between the Z and the W in the female permitted the unequivocal recognition of the sex chromosomes in six of the eight snakes studied. Of those, Drymarchon corais eouperi and Spilotes pullatus anomalepis of the family Colubridae, and Bothrops ]araraca and Bothrops atrox of the family Crotalidae possessed the basic karyotype. In each of these four species, the Z-chromosome is the fourth largest element having a median eentromere (Figs. 6, 9, 10, 13, and 14). Furthermore, when the ZZ-pair of the homogametic male of one of these four species is compared with the fourth largest pair of either sex of Boa constrictor amarali (Fig. 7) and Epicrates cenchria crassus (Fig. 8) of the family Boidae, they are seen to be morphologically identical. From this may be inferred that in the suborder Serpentes, the fourth largest pair of the basic karyotype is always the sex pair, regardless of family. In Boidae, however, the Z and W are still homomorphie to each other, this primitive state of differentiation perhaps reflecting the antiquity of the family. Fossils dating from the Eocene epoch nearly 50 million years ago have been found. In the family Boidae, then, if one member of the fourth largest pair is assumed to be the Z-chromosome, measurements reveal that the Z-chromosomes of various snake species are almost alike in absolute size, comprising nearly 10 per cent of the homogametic haploid (AZ) set (Table 2). This also applies to the Z of the two remaining members of the family Colubridae, Clelia occipitolutea, with 50 chromosomes in the diploid set, and Xenodon merremii, with 30. In the former (Fig. ll), the Z ranked third or fourth in absolute size and was the only metacentrie in the complement, while in the latter (Fig. 12), the Z was still the fourth largest element. The snake family Colubridae can be compared to rodents among mammals, being geographically widespread and containing numerous species with many diverse characteristics. Among its members, initial steps toward the development of heteromorphism between the male- determining Z-chromosome and the femMe-determining W-chromosome could be seen. In Drymarchon corais couperi (Figs. 6 and 9) and Spilotes pullatus anomalepis (Fig. 10), both members of the fourth largest pair in the female were still the same in absolute size. However, a perieentric inversion appeared to have occurred in the W-chromosome, which was a subterminal element. Another approach toward heteromorphism was taken in Clelia occipitolutea (Fig. 11) in which the W was twice as large as the Z. b~

Figs. 7--14. Serial alignments of the diploid ehromoso]I~e sets of the heterogametic female of eight species of snakes. Karyotypes already published (BEQA!K et al. 1962) are presented here as schematic drawings in which the ailtosomes are outlizled, the Z is solid black, and the W is shaded. The rebirth l~rges~ pair of each species is the Z~V pait,. -- l~ig, 7, Boa constrictor amarali (2n=36), family Boidae.--Fig. 8. Epicrates cenchria crassus (2n=36), family Boidae. -- Fig, 9. Drymarehon corals couperi (2n=36), family Colubridae, -- Fig. 10. Spilotes pullatus anomalepis (211~36), family Col~bridae o

g g

Fig. 1i, Clelia occipiloZuteet (2n=50), f~rnily Colubridae. - Fig. 12. Xenodon merremii (2n~30, family Colubridae. -- Fig. 13. Bothro'ps jararaca (2 n = 36 ), f~mily Crotalidae. -- Fig. 1 ~:. Bo~hrops atrox (2 n = 36), fa~mily CrotaZidae 614 B]~;~tK, BEgA~:, NAz~_t~:t~ 0mqo:

Differential accumulation of factors governing the development of opposite sexes by two members of an originally homologous pair is possible only ff free crossing-over between the two is prevented during meiosis of the heterogametic sex. Pericentric inversion and duplication are equally effective in accomplishing partial isolation of the W from the Z. A perieentrie inversion isolates one arm of the W from the Z, leaving it free to accumulate female-determining factors; specialization follows. Duplication of the W leaves half of the duplicated W in an unpairable state. In this half, accumulation of the factors governing development of the heterogametic sex is now possible. In amphibians, in fact, duplication of the W-chromosome appears to have occurred sporadically, having been reported in Xenopus laevis of the family Pipidae (WEILE~, and 0~o 1962), as well as in Bu]o paracnemis of the family BuJonidae (unpublished data). In the evolution of the sex-determining mechanism, however, duplication appears to be a step down a dead-end street. In Xenodon merremii of the family Colubridae (Fig. 12), and all species of the family Crotalidae, the highly advanced poisonous snakes of the New World, the W-chromosome has become a distinctly smaller element (Fig. 13 and 14) comparable in degree of specialization to the minute W of birds. Thus, within the reptilian suborder Serpentes, the step-by-step differentiation of sex chromosomes from the originally homomorphic elements was witnessed. Heteromorphism was gradually established by changes occurring in the W-chromosome which accumulated factors governing development of the heterogametic sex. Throughout this process of differentiation, the Z-chromosome retained its original size (Table 2). Further, the Z-chromosome of snakes appears to be only slightly larger in absolute size than the Z of birds when judged from the Z:AZ ratio. Although only New World snakes were covered in the present study, KOBEL'S study (1962) on the family Viperidae clearly shows that what applies to species of the New World applies with equal relevance to species of the Old World.

Conclusions The fact that two classes of warm-blooded vertebrates constitute independently uniform groups with regard to total genetic content (MANDELetal. 1950; AL~gr,Ey etal. 1955; OK~r 1964a, 1964b) obviously reflects the long separation of the lineages which eventually gave rise to mammals and birds. As both are derived from reptiles, which have been in existence since the dawn of the Mesozoic era, it is no surprise that surviving members sort into two distinct categories. Karyological kinship between snakes and birds 615

What is striking is the close karyological kinship between snakes and birds. Not only is the total genetic content of both groups very similar but the mieroehromosomes characteristic of avian species are also present in snakes, the fundamental snake karyotype containing 10 pairs, that of birds about 30 pairs. Furthermore, female heterogamety of the ZZ-ZW type operates in both. Our observations of lower vertebrates have shown that lack of interstitial chiasmata is characteristic of male meiosis. In fishes, amphibians, and most reptiles, two homologous chromosomes at diplotene and diakinesis were usually held together only by two terminal chiasmata (W]~IL~R and 0HN0 1962). Within the order

Figs. 15 and 16. Diakiuesis figures from direct squash preparations of the testes of a lizard and a snake. -- Fig. 15. Anolis carolinensis, suborder Sauria, order Squamata. -- Fig. 16. Drymarchon corals couperi, suborder Serpentes, order Squamata

Squamata, this was certainly true of the suborder Sauria (Fig. 15); only in snakes occurred great frequency of interstitial chiasmata (Fig. 16). This is one more factor apparently setting snakes apart from other reptiles, and revealing their close kinship to avian species, which have an extremely high frequency of interstitial chiasmata (OHio 1961). In view of these findings, the close similarity in absolute size of the snake Z and the avian Z appears to be more than accidental. We prefer to interpret this fact as an indication that when a specific homologous pair of antosomes in an ancient reptile ancestral to both snakes and birds elected to become the sex pair, they were then of the size still maintained by the Z-chromosome. WI~scgI (1959) believes that the sex chromosomes of vertebrates have been in existence since the Jurassic period of the Mesozoic era, possibly even earlier. Despite later branching of this lineage into snakes and into birds, that member of the sex pair which had accumulated factors governing development of the homogametic male sex to become the Z-chromosome did not change substantially, retaining to this day the size of the originM autosome. If Chromosoma (Berl.), Bd. 15 41 616 BEgAK, BEgAK, NAZARETH,~nd On~o:

indeed this is the case, the difference between the snake Z and the avian Z should be due mostly to different kinds of mutations which occurred to individual genes on this chromosome.

Summary In contrast to the situation found in two classes of warm-blooded vertebrates, mammals and birds, the class Reptilia is not uniform with regard to total genetic content ; rather, it contains two distinct categories. The close cytological kinship between snakes and birds was revealed. Both are almost identical in total genetic content, which is about 50per cent that of placental mammals. Both have microchromosomes, as well as Z-chromosomes very similar in absolute size, comprising nearly 10 per cent of the homogametie haploid (AZ) set. This leads to the implication that snakes and birds originated from the same lineage, and that their Z-chromosomes have not changed substantially since the Jurassic period of the Mesozoic era, about 180 million years ago. Within the reptilian suborder Serpentes, the step-by-step differentiation from the primitive ZW pair to the grossly heteromorphie ZW pair could be observed. In the ancient family Boidae, the sex chromosomes were still homomorphic to each other. In the family Colubridae, the beginning of heteromorphism was manifested in two ways. In some species, a pericentric inversion on the W caused it to differ from the Z; in others, duplication of the W occurred. In the family Crotalidae, the W had apparently achieved its very specialized status ; it was a distinctly smaller element.

References ALLFREY, V. G., A. E. M~RSKu and H. STERN: The chemistry of the cell nucleus. Advane. Enzymol. 16, 411--500 (1955). BE~AK, W., M.L. BE~AK, and lt. R. S. NAZARET~I: Karyotypic studies of two species of South American snakes (Boa con.strictor amarali and Bothrops ]araraca). Cytogenetics 1,305--3]3 (1962) ; -- Chromosomes of snakes in short term cultures of blood leucocytes. Amer. Naturalist 97, 253--256 (1963a) ; -- Karyo- typic studies of South American snakes. Presented before the XI Internat. Cong. Genet., The Hague 1963b. BRINx, J. M. VAN : L'expression morphologique de la digametie chez les sauropsid~s et les monotr6mes. Chron~losoma (Berl.) 1O, 1--72 (1959). KOBEL, H. 1%. : Heterochromosomen bei Vipera berus L. (Viperidae, Serpentes). Experientia (Basel) 18, 173--174 (1962). I~/~ANDEL,P., P. MkTAIS et S. Cv~Y: Les quantit~s d'acide d4soxypentose-nucl4ique par leucocyte chez diverse esp~ces de Mammif~res. C. R. Aead. Sci. (Paris) 231, 1172--1174 (1950). MATTHEu ]~., et J. M. VAN BEINK: La question des h6t4rochromosomes chez les Sauropsid~s. I. Reptiles. Experientia (Basel) 12, 53 (1956). Karyological kinship between snakes and birds 617

OHIO, S. : Sex chromosomes and microchromosomes of Gallus domesticus. Chromo- soma (Berl.) 11, 48r (1961). -- W. B~gA~, and M. L. BEgAK: X-autosome ratio and the behavior pattern of individual X-chromosomes in placental mammMs. Chromosoma (Berl.) 15, 14--30 (1964a). -- C. STn~Ivs, L.C. CHRISTIAn% W. BE~AK, and M.L. BE~AK: Chromosomal uniformity in the avian subclass Carinatae. Chromosoma (Berl.) 15, 280-288 (1964b). WEIr,~R, C., and S. OHio : Cytological confirmation of female heterogamety in the African water frog (Xenopus laevis). Cytogenetics 1, 217-223 (1962). WiTscm, E. : Age of sex-determining mechanisms in vertebrates. Science 130, 372--375 (1959). Dr. Svsu~v 0~o Dept. of Biology, City of Hope Medical Center Duarte, Calif. (USA)

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