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 com- plement. The mammalian X and the avian Z are similar in size in mem- bers 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 microspectrophoto- metric 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-chromo- some, 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 magnifi- cation 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 mam- mals. 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 measure- ments 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).