Chromosome Replication in Four Species of Snakes*

Transcription

1 Chromosoma (Berl.) 26, (1969) Chromosome Replication in Four Species of Snakes* N. O. BTA~C~I, W. BngAX, MAlCTHA S. A. DE BIANCHI, MA~IA L. BEQAK and MAlCIA N. RABELLO Comisi6n de Investigaei6n Cientifica de la Provincia de Buenos Aires, La Plata, Argentina, and Secg~o de Gen6tica, Instituto Butantan, S~o Paulo, Brasil Received October 28, 1967 Abstract. Chromosome measurements were performed in four species of snakes related at the level of suborder (Boa constrictor amarali, Xenodon merremii, Philodryas patagoniensis, Bothrops ]araraca). The data obtained point out that pairs 1--3 were common to the four snakes and probably inherited from the ancestor of the suborder Serpentes. Pairs 5--8-W were characteristic of each snake; hence, it is possible to assume that they followed evolving after the appearing of the suborder Serpentes. Z-chromosomes were metacentric in B. constrictor amarali, X. merremii and B. jararaca and slightly submetacentric in P. patagoniensis. Area of these chromosomes varied from % of the haploid set in the four species studied.--the study of chromosome replication at the end of the S period points out that "shared chromosomes" have similar patterns of labeling. Therefore, it is proposed that the distribution of late replicating regions and heterochromatin in the genome is phylogenetically transmitted and probably genetically determined.--the analysis of the ending-sequence of chromosome replication shows that sex chromosomes finish earlier than macroautosomes. It is concluded that snakes probably have no mechanism of sex chromosome heterochromatinization in either sex. The absence of late replicating Z-chromosome in the males, favours the hypothesis that no mechanism of sex dosage compensation is acting in the suborder Serpentes. Introduction Several facts indicate a close karyological kinship between snakes and birds. Both have about the same DNA content, presence of microchromosomes, and similar Z-chromosomes representing about 10% of the haploid set. Moreover, the evolution from the primitive ZW pair to the remarkable heteromorphic ZW-chromosomes of birds can be step-by-step followed from the ancient down to the advanced families of Serpentes (BE~AK, BEqAK, NAZA~T~ and OHNO, 1964). The study of chromosome replication in birds shows that W-chromosomes finish replication late in the S period, together with late replicating * This work was partially supported by Public Health Service Research grant No. GM from the National Institute of General )~edical Sciences and by the Fundo de Pesquisas do Instituto Butantan.

2 Chromosome Replication in Snakes 189 autosomal regions. Furthermore, it has been reported that homologue asynchrony of replication is less striking in birds than in mammals (BIANCI~I and MOLI~A, ]967a). Until now, as far as we know, no studies on chromosome replication in reptiles have been communicated. Therefore, the present work aims at determining the similarities and differences in the pattern and time-sequence of chromosome replication between different species of snakes and between snakes and birds. Material and Methods The four species of Serpentes selected for this investigation were : Boa constrictor amarali (STULL) (2 ~ and 2 ~) of the family Boidae; Xenodon merremii (WAGLER) (2 3 and 2 ~) and Philodryas patagoniensis (GIRARD) (1 ~ and 1 ~) of the family Colubridae; Bothrops jararaca (WIED) (2 ~ and 2 ~) of the family Crotalidae. With the exception of Xenodon and Philodryas which are related at the level of family all the species are related at the level of suborder. Short term cultures of blood leucocytes and whole blood microteehniques were set up and harvested as described elsewhere (BEQAK, BE(~AK and NAZARETH, 1963; BEQAK, BEQAK, NAZARETE and PEOClNINI, 1964). 3H-TdR (Sp. ac. 6.9 C/m Mole) at a final concentration of 1 ~xe per ml of culture medium was added 6, 8 and 10 hours before harvesting. Autoradiographic methods have been previously reported (B~oHI, LI2VIA-DE-FAI~IA and JAWOlCSKA, 1964). Exposure time was 15 days for all the experiments. The total number of metaphases and autoradiograms analysed were 773 and 196 respectively. Chromosome areas were determined by the cut out method and expressed as percent of the haploid set. The accuracy of this method has been tested elsewhere (O~o, BEqA]~ and BEgAK, 1964). A total of 10 metaphases were measured in each species. Nomenclature for centromeric posit~ion of chromosomes was used according to :BE~AK (1965). Chromosomes with an arm index below 1.5 were considered metacentrics. When the arm index ranged between 1.5 and 2, chromosomes were classified as submetacentris. Finally those elements with an arm index above 2 were considered subterminal or aerocentrics. Results Comparison o/ Karyotypes The chromosomes of the snakes under analysis in this report have been described with considerable detail in previous publications (BE~A~:, BEqAK and NAZARETh, 1962; BE~AK, B~K, NAZARETH and 0I~O, 1964; Bn~AK, 1965, 1966). However, it is worth mentioning here some data indicating that the four species have several pairs of chromosomes in common (" shared chromosomes"). With the exception of X. merremii which has 30 chromosomes the remaining species--like most snakes--have complements of 36 chromosomes. When karyotypes were constructed in decreasing order of size

3 190 N. O. BIANCm et al. : Table. Arm ratios and chromosome lengths Species b Chromosome No Z L AR L AR L AR L AR B.c ~ L =~ X.m ~ P.p. 22 ~-I B.j :~ ~ W- and Z-chromosomes of Boa cannot be distinguished; consequently, no figures are given for the former. it was observed that the four species had eight pairs of macrochromosomes. Moreover, the measurements performed indicated that pairs 1, 2 and 3 were alike in the four snakes ("shared chromosomes") (Table). Pairs 1 and 3 being metacentric and pair 2 submetaeentrie. Z-chromosomes occupied the fourth place in the karyotype of the four snakes analysed. Although in B. constrictor amarali the sex chromosomes could not be identified due to absence of heteromorphism the fourth pair will be considered as the sex pair by homology to other species (BE~AK, BE~AK, NAZARETH and 0~o, 1964). In B. constrictor amarali, B. ]araraca and X. merremii Z-chromosomes were metacentric measur- /rig 8.6, 8.8 and 10.6% respectively of the haploid set (Table). In P. patagoniensis the Z-chromosome was slightly submetacentric comprising about 10.5% of the haploid complement (Table). Measurements of sex chromosomes in this paper differ by less than 10% with previously published data. This small variability can be explained taking into account that different investigators performed the two sets of measurements. Presumed W- and Z-chromosomes were alike in B. constietor amarali. In the other three species W-elements were distinctly smaller allowing an easy identification of the heteromorphic sex. The size, kinetochore location, and some other morphological features indicated that the four last pairs of macrochromosomes were characteristic of each species ("non-shared chromosomes") (Table and Fig. 10). In B. constrictor amarali pairs 5 and 6 were subterminal and pairs 7 and 8 terminal. In P. patagoniensis pair 5 was metacentric with a remarkable secondary constriction in the distal third of the long arm; pair 6 was terminal and pairs 7 and 8 subterminah X. merremii showed metacentric homologues in pair 5 and 8, submctacentric chromosomes in pair 7 and subterminal ones in pair 6. Finally, B. ]araraca had subterminal elements in pairs 6 and 8 and submetacentric chromosomes in pairs 5 and 7.

4 Chromosome Replication in Snakes 191 expressed as percentage o/the haploid set ~ W L AR L AR L AR L AR L AR 7.46 ~ ~: s ~: L s :j b B.C. Boa constrictor amarali, X.m. Xenodon merremi, P.p. Philodryas patagoniensis. B.j. Bothrops ]araraca. Patterns o/ Chromosome Replication The percentage of labeled metaphases in each of the 3H-TdR-treatments performed was about the same in the four species. In 6-hour labelings only 7--10% of metaphases exhibited radioactivity. When ah-tdr was added in the last 8 hours of culture the percentage of labeled mitosis increased to %. Finally, in the 10-hour treatments lebeling could be detected in % of metaphases. On account of these data it can be concluded that G 2 period in blood cultures of snakes lasts about 9 hours. Such a long G 2 phase can be a biological trait of Serpente8 or the result of the low temperature of culture (30 ~ C). The former assumption seems to be the most probable for it is supported by the duration of the G 2 phase in Leptodactylns ocellatus (Amphibia, Anura); in spite of the incubation temperatures (25 ~ C) the G 2 period in cultured blood cells of these frogs is 3.5 hours (BIA~CtII and MOLINA, 1967 b). Since continous ~H-TdR labeling was employed it is possible to assume that unlabeled chromosomes had finished DNA synthesis when the isotope was added to the cultures. Thus, metaphases with more than half of the complement labeled were considered to belong to late stages of the S phase (Sa stage) (Figs. 1 and 2). Moreover, when less than half of the complement was radioactive the cell was considered to stem from final stages of the S period (S 4 stage) (Figs. 3--6). Late labeling patterns in pairs 1, 2 and 3 were similar in the four species. Therefore the description of silver grain distribution on each of these pairs is valid for the four snakes. In the S a stage chromosomes No. 1 exhibited radioactivity throughout their lengths. However, a certain tendency to have a higher concentration of labeling in the paracentromeric and distal regions of both arms could be found. In the S a stage the density of silver grains progressively decreased until only a few grains could be detected on these chromosomes (Fig. 7).

5 192 N. 0. BIANCttI et al.: Figs. 1--6

6 Chromosome Replication in Snakes 193 Fig. 7 Figs Labeling of the largest macroautosomes ("shared chromosomes") in the S a and S 4 stages. Bc: Boa constrictor amarali; Xm: Xenodon merremii; Pp.: Philodryas patagoniensis; Bj.: Bothrops ]araraca. Notice the similar patterns of labeling in the four species. 1,200. Fig. 7. pair 1. Fig. 8. pair 2. Fig. 9. pair 3 Figs ~.r pre- and postautoradiography from three species of snakes. 1,200. Figs. 1 and 2. P. patagoniensis (female). Initial Sa stage. Notice the absence of labeling in the W-chromosome. Figs. 3 and 4. B. jararaca (male). Initial S 4 stage. Notice the late replicating regions on the complement. Figs. 5 and 6. B. constrictor amarali (female). Final S 4 stage. Notice the late replicating regions in on No. 3-chromosome and in pair 5

7 Fig. 8 (Legend see p. 193) Fig. 9 (Legend see p. 193)

8 N. O. BIANC~II et al. : Chromosome Replication in Snakes 195 Fig. 10. Labeling of pairs 5--8 ("non-shared chromosomes"), Be: Boa cor~strictor amarali (S t stage); Xm: Xenodon merremii (S a stage); Pp.: Philodryas patagoniensis (S t stage); B].: Bothrops ]araraea (S t stage). 1,200 Such a lack of well delimited replicating regions seemed at first sight to suggest an absence of delayed DNA synthesis on pair 1. However, since No. 1-homologues finished replication at the very end of the S period we were prone to accept that late replication was scattered instead of being condensed in small areas. In pair 2 the perieentromeric area was the first to become free from labeling. Afterwards, silver grains disappeared from the proximal half of the long arm and only the short arm and the distal region of the long arm kept replicating in the S 4 stage (Fig. 8). No. 3-chromosomes during the 83 stage were labeled along their full extent; in the S~ phase the distal half of both arms became unlabeled and only the pericentromeric areas showed delayed DNA synthesis (Fig. 9). "Non-shared maeroautosomes" (pairs 5--8) had radioactivity evenly distributed on their whole lengths during the Sa stage. In the $4 phase

9 196 I~. O. BIANC:ttI et al.. Fig. 11. Late replicating patterns of ZZ- and ZW-pairs. Be: Boa constrictor amarali; Xm: Xenodon merremii; Pp.: Philodryas patagoniensis; B].: Bothrops jararaca. 1,200 a progressive decrease of radioactivity with lack of intrachromosome asynchrony of synthesis was observed in most of these chromosomes; the only exception to this rule being the following late replicating regions : a) the centromeric region of chromosomes No. 5 in B. constrictor amarali; b) the distal half of pair 6 in Philodryas; c) the short arm of pair 7 in B. jararaca (Fig. 10). In the four snake species the short arm of Z-chromosomes was the last region of sex chromosomes to end DIqA synthesis. Furthermore, in X. merremii and P. patagoniensis, the telomeric region of the long arm was found to finish replication slightly later than the corresponding region in the other two species. In B. constrictor amarali the morphology and pattern of labeling of W-chromosomes were indistinguishable from that of Z-chromosomes. In Xenodon and Bothrops the short arms of W-chromosomes were the last regions to finish replication (Fig. 11). This same pattern was also observed in about half of the cells from Philodryas; the other half showing radioactivity in the long arm and the short arm unlabeled (Fig. ll). Chronology o/chromosome Replication The time-sequence of chromosome replication was obtained by determining the percentage of labeling for each pair of macrochromosomes in metaphases from S 3 and S 4 stages. The results from

10 9 Chromosome Replication in Sna~kes 197 Xm-- Be- i Pp- Xm Bc- Bj 3 Bj PP-[ ZZ Bj Bc- Pp- 1. Bc- L Xm-- 5 Bjpp, 6 Bj PP-! Illllllllllllllll f~l B Mic Pp B j-l, Fig. 12. Chronology of replication in the four species of snakes. Notice the ea~rly ending of replication of Z- and W-chromosomes and the similar ending-sequence of "shared chromosomes" 100% each species were compared with those from other snakes and the final data are indicated in the histogram of fig. 12. In this figure chromosomes from each snake are represented as a line plotted against the percentage of labeling. Full lines indicate labeling in both members of the pair. Dotted lines point out the existence of homologue asynchrony.s ince mierochromosomes could not be identified with accuracy they were grouped and represented as a black bar. The thinner the bar, the less is the number of microehromosomes labeled. "Shared macroautosomes" (pairs 1--3) finished DNA synthesis at about the same time in the four species. A similar behavior was also observed for some "non-shared macroautosomes" (pair 5 in

11 198 N.O. BIAI~CKI et al.: B. c. amarali, X. merremii and P. patagoniensis ; pair 6 in P. patagoniensis and B. jararaca ; pair 7 in X. merremii, P. patagoniensis and B. ]araraca ; pair 8 in B. c. amarali, P. patagoniensis and B. jararaca). Some other "non-shared pairs" and microautosomes ended replication at variable time depending on the species under consideration. Asynchrony of replication between homologues was variable depending on the pair and the species. However pairs 7 and 8 exhibited a striking homologue asynchrony in the four snakes (Fig. 12). The ending sequence of synthesis in Z- and W-chromosomes was variable according to the species. However, in all the cases these chromosomes finished replication before macroautosomes. In Philodryas this early ending was so extreme that W-chromosomes of this species could be observed unlabeled at the very beginning of the S 3 phase (Figs. 1 and 2). Discussion It has been reported that related species have in some cases one or more chromosome pairs in common (MA~aTIN and HAYMAN, 1967). In the present work the comparative study of complements indicates that the four species of snakes analysed have three autosomal pairs in common (pairs 1--3). Furthermore, the study of chromosome replication shows that "shared chromosomes" are not only morphologically alike hut also have similar late replicating regions. It is actually known that delayed DNA synthesis is one of the properties of the heterochromatin (LI~A-DE-FAI~IA and JAWOlCSXA, 1968). Therefore, by extrapolation it can be assumed that chromosomes common to several species have identical heterochromatin distribution. Since the snakes studied are related at the level of suborder it can be assumed that "shared chromosomes" were inherited from the ancestor of the suborder Serpentes. Hence it can be concluded that late replicating regions are phylogenetically transmitted and probably genetically determined. It also seems evident that chromosomal stability--at least in snakes--varies from pair to pair. In fact, pairs 1, 2 and 3 seem to have finished their evolution when the suborder Serpentes appeared (upper Cretacious). Conversely the last four pairs and the W-chromosomes complement have continued evolving until more recent times. This is well illustrated by P. patagoniensis and X. merremii which although being related at the level of family have a variable morphology of pairs Z-chromosomes show a defined tendency to maintain a similar morphology in the suborder Serpentes. However, the finding of a submetacentric Z-chromosome in P. patagoniensis probably indicate that at least in some species the Z-chromosome was able to undergo some

12 Chromosome Replication in Snakes 199 internal rearrangements. Such a caracteristic suggests that the Z-chromosome can be placed in an intermediate position between "shared" and "non-shared" chromosomes. "Shared chromosomes" and several "non-shared autosomal pairs" located in an equivalent position in the karyotype of the four snakes finish replication at the same moment. This similar ending-sequence of replication is not surprising for "shared pairs". However, the behavior of "non-shared autosomes" deserves some comment. It is actually known that chromosomal rearrangements modify is some cases and do not modify in others the degree of heteroehromatinization of the interchanged regions (WHITE, 1954; EVANS, FORD, LYON and GRAY, 1965; CATTANAC~, 1961; CATTANAC~ and ISAAOSON, 1965; SEARLE~ 1962; FORD and EvANs, 1964; GVSTAVSSON, FRACCARO, TIEPOLO and LINDSTEN, ]968). Since heterochromatin and ]ate replication are associated events, the time-sequence of chromosome replication may undergo a shift in the former instance and no change in the latter. The complement of the snakes analysed in this report may be considered as the final result of chromosomal rearrangements involving pairs 5--7-ZW and microehromosomes. Hence, "non-shared pairs" with similar ending-sequence of replication would not have undergone changes in their degree of heterochromatinization. The opposite being true for those corresponding pairs exhibiting different chronology of synthesis. Genetic evidence and the lack of a late replicating Z-chromosome in males point out the absence of dosage compensation in birds (BIANCm and MOLINA, 1967a; ScH~w, 1962). In snakes, since no sex-linked genes have been reported, there is no genetic evidence supporting or denying the existence of dosage compensation. However, the absence of a late replicating sex chromosome in either sex seems to strengthen the second possibility. If this is so, we have one more argument stressing the close karyologieal kinship between Ayes and Serpentes. References BEgAK, W.: Constitui~o cromoss6mica e mecanismo de determina~o do sexo em ofldios sul-americanos. I. Aspectos cariotlpicos. Mere. Inst. Butantan 32, (1965). -- Constitui~o~ sex cromoss6mica e mecanismo de determinag~o doo em ofidios sul-americanos. II. Cromossomos sexuais e evoln~o do cariotlpo. Mere. Inst. Butantan, Simp. Intern. 33, (1966). M. L. BEqAJ(, and H. g. S. NAZARE~: Karyotype studies of two species of South American snakes (Boa constrictor amarali and Bothrops jararaca). Cytogenetics 1, (1962) Chromosomes of snakes in short term cultures of blood leucocytes. Amer. Naturalist 97, (1963). 14 Chromosoma (Ber].) Bd. 26

13 200 N. O. BIA~Cm et al.: Chromosome Replication in Snakes BE~JAK, W., M. L. BE(~AK, H. l~. S. :NAZARETH, and S. 0I~o: Close karyological kinship between the reptilian suborder Serpentes and the class Ayes. Chromosoma (Berl.) 15, (1964)., and O. PECClm~r Chromosomes of cold-blood animals from whole blood short-term cultures. Microtechnique Mature. Chrom. Newsl. 14, (1964). BIANCm, N. 0., and O. J. MOLI~A: Chronology and pattern of replication in the bone marrow chromosomes of Gallus domestieus. Chromosoma (Berl.) 21, (1967a) DNA replication patterns in somatic chromosomes of Leptodaetylus ocellatus (Amphibia, Anura). Chromosoma (Berl.) 22, (1967b). -- A. LINA-DE~FAR~, and H. JAWO~SK~: A technique for removing silver grains and gelatin from tritium autoradiograms of human chromosomes. Hereditas (Lund) 31, (1964). CATTANAOI~, B. M. : A chemically induced variegated-type position effect in the mouse. Z. Vererbungsl. 92, (1961)., and J.H. ISA~CSON: Genetic control over the inactivation of autosomal attached to the X-chromosome. Z. Vererbungsl. 96, (1965). EVANS, H. J., C. E. Fo~D, M. F. LYon, and J. G~Au DNA replication and genetic expression in female mice with morphologically distinguishable X-chromosomes. Nature (Loud.) 206, (I965). FORD, C. :F., and E. P. EVANS: A reciprocal transloeation in the mouse between the X-chromosome and a short autosome. Cytogenetics 3, (1964). GUSTAVSSON, I., 1~r FR&CCARO, L. TIEPOLO, and J. LINDSTE~: Presumptive X-autosome translocation in a cow: preferential inactivation of the normal X chromosome. Nature (Loud.) (1968). LI~-Dn-FAR~, A., and H. JAWORSK~: Late DNA synthesis in heterochromatin. Nature (Lond.) 217, (1968). M~RTI~, P. G., and D. L. HAYMA~: Quantitative comparison between the karyotypes of australian marsupials from three different superfamilies. Chromosoma (Berl.) 20, (I967). O~No, S., W. BE~AK, and M.L. BE~AK: X-autosome ratio and the behavior pattern of individual X-chromosomes in placental mammals. Chromosoma (Berh) 15, 14~30 (1964). So~]:MI]), W.: DNA replication patterns of the heterochromosomes in Gallus domestieus. Cytogenetics 1, (I962). SEA~Ln, A. G.: Is sex linked tabby really recesive in the mouse? Heredity 17, 297 (1962). W~ITn, M. S. D.: Animal cytology and evolution, 2nd ed., 454 p. Cambridge: Cambridge University Press Dr. N. 0. BIANe~I Comisi6n de Investigaci6n Cientffica de la Provineia de Buenos Aires Calle 526 entre 10 y 11 La Plata Provineia de Bs. Aires, Rep. Argentina