Drosophila willistoni polytene chromosomes. I. Pericentric inversion

CARYOLOGIA Vol. 58, no. 3: 249-254, 2005 Drosophila willistoni polytene chromosomes. I. Pericentric inversion on X chromosome Rohde Cláudia, Tiago H. Degrandi, Daniela C. De Toni and Vera L. S. Valente* Departamento de Genética, Instituto de Biociências, Universidade Federal do Rio Grande do Sul, P.O. Box 15053, CEP 91501-970, Porto Alegre, RS, BRAZIL. Fax: + 55.51.3316.7311. Abstract A first case of polymorphism of a pericentric inversion on X-chromosome was detected in six wild populations of Drosophila willistoni from islands and the mainland of Santa Catarina State, southern Brazil. The high representativeness of this inversion, with frequencies between 10 to 42 % in samples of wild populations from Santa Catarina State, suggests that it could be adaptive and that the establishment of such polymorphism was probably due to crossing over restriction in the chromosomal region involved in the rearrangement. Key words: Drosophila willistoni; pericentric inversion; X polytene chromosome. INTRODUCTION * Corresponding author. e-mail: vera.gaiesky@ufrgs.br. Pericentric inversions are rare in wild populations of Drosophila (reviews in Miller 1939 in D. algonquin; Carson and Stalker 1949; Etges 1998 in D. robusta; Freire-Maia 1960; Hinton and Downs 1975 in D. ananasse; Roberts 1967; Coyne et al. 1993 in D. melanogaster) while paracentric inversion polymorphisms are common (Sperlich and Pfriem 1986; Krimbas and Powell 1992). This is because crossing over in the inverted region can produce duplications and deletions, which can cause the loss of up to 50% of the offspring of heterozygote carriers. Protective mechanisms that prevent such losses in paracentric inversions were cytologically demonstrated in Sciara impatiens (Carson 1946) and in D. melanogaster (Hinton and Lucchesi 1960). In these organisms, which successfully exploit this type of polymorphism, the dicentric chromatid resulting from crossing over within the inverted segment is incorporated into one of the polar bodies while the innermost chromatid (which is not involved in crossing over) remains to be included in the functional egg nucleus. The acentric fragment disintegrates in the egg cytoplasm. Hinton and Downs (1975), while studying pericentric inversions in D. ananassae suggested that the crossing over in heterozygous females is restricted to region of exchange outside the inverted loop. This is a way of preventing damage to the offspring of heterozygous females, while in males recombination is usually absent (Stevens 1908; Morgan 1912; 1914). Drosophila willistoni is a Neotropical species that shows high chromosomal polymorphism for paracentric inversions (Da Cunha et al. 1950; 1959; Da Cunha and Dobzhansk 1954; Dobzhansky 1957; Valente and Araújo 1985; 1986; Valente et al. 1993; 2001; 2003; Rohde 2000). The species also present high allozyme variability (Ayala et al. 1970; 1972a; 1972b; Ayala and Tracey 1974). This species is extremely useful for evolutionary studies, since it has a wide geographical distribution (Spassky et al. 1971; Dobzhansky and Powell 1975), several levels of speciation (Cordeiro and Winge 1995), great ecological versatility (Carson 1965; Dobzhansky 1965), and high genetic variability - measured as concealed genic variability (Pavan et al. 1951; Cordeiro and Dobzhansky 1954). In the course of the study of paracentric inversion polymorphism in 25 wild populations of D. willistoni from different American regions (Rohde 2000), we observed the occurrence of a pericentric inversion in individuals collected in some islands and in mainland from Santa Catarina State, southern Brazil. Here we report for the first time this unusual phenomenon in D. willistoni and describe one pericentric inversion and its frequencies in wild populations where the geographical

250 rohde, degrandi, de toni and valente distribution is large enough to suggest lack of under-dominance in carriers of this inversion. MATERIALS AND METHODS Populations of Drosophila willistoni were collected at six different sites in the southern Brazilian State of Santa Catarina as indicated in the map of Fig. 1. Two sites were on the Santa Catarina island: Canto da Lagoa and Sertão do Peri. Three were on other little islands, near the Santa Catarina island: Ratones Grande Island, Ratones Pequeno Island and Arvoredo Island. The sixth collection was at Park Serra do Tabuleiro on the mainland. Fig. 1 Map of South America, showing Brazil and state of Santa Catarina with the collection sites of Drosophila willistoni. A) Canto da Lagoa, Santa Catarina Island; B) Ratones Grande Island; C) Ratones Pequeno Island; D) Sertão do Peri, Santa Catarina Island; E) Arvoredo Island; F) Park Serra do Tabuleiro, on the mainland. Each D. willistoni isofemale line collected at each site was grown in uncrowded cultured bottles at 17 C on standard medium (Marques et al. 1966). To prepare slides of polytene chromosomes, each salivary gland of third instar female larvae were dissected out in Ephrussi and Beadle (1936) saline solution, transferred to 45% acetic acid solution for a few seconds, transferred to a drop of solution of water: acetic acid: lactic acid (3:2:1) for three minutes and squashed under a coverslip in a drop of acetic orcein (2% of orcein in 51% acetic acid and 15% lactic acid). Photomicrographs were made using a Zeiss photomicroscope, 1000 x magnification. All photos were compared with the photomap of Regner et al. (1996) for south Brazilian population of D. willistoni and with photomap of Rohde et al. (in preparation) for D. willistoni species. RESULTS AND DISCUSSION The new pericentric inversion here first described, named Inv. XLXR-p, includes 16.86 % of the proximal section of the left arm XL and 12.87 % of right arm XR (or about 14.8 % of X- chromosome) according to detailed cytological analysis make by Rohde (2000). According to Table 1, this pericentric inversion was well represented in frequencies between 10 to 42 % in all D. willistoni population samples from Santa Catarina and was found in both homozygous (Fig. 2a) and heterozygous (Fig. 2b) states in individuals of all populations recorded. The inversion breakpoints were recognized at 3D section on the left arm and at 19A section on the right arm of the chromosome X according the photomaps of Regner et al. 1996 and Rohde 2000. More recently, Aulard et al. (2004) detected one pericentric inversion on the second chromosome of transgenic lines of D. melanogaster generated by the injection of hobo elements into the strain which previously lacked them. The authors suggest that hobo element can induce inversions leading to new homokaryotypic population. In accordance with these results and those by other authors (Cáceres et al. 2001; Casals et al. 2003; Puig et al. 2004), several observations suggest a possible impact of transposable elements in the generation of chromosomal inversions in natural populations. By in situ hybridization technique, however, we tested eight different isofemales lines (collected in Ratones Grande Island) for the presence of canonical P element in their polytene chromosomes (data not shown). Not one insertion site of P was detected inside or near the breakpoints of XLXR-p inversion. Besides the fact that many other sites of canonical P element were detected along the euchromatic arms (data not shown) we suggest that this transposable element has no asso-

drosophila willistoni pericentric inversion 251 Table 1 Description of the collection sites and frequencies of the pericentric inversion XLXR-p in populations of Drosophila willistoni from the island and mainland of the Santa Catarina State, southern Brazil. Samples on Santa Catarina State, Brazil (Coordinates) Canto da Lagoa - Santa Catarina Island (27 o 35í32ííS-48 o 28í32ííW) Ratones Grande Island (27 o 29í30ííS-48 o 36í42íW) Ratones Pequeno Island (27 o 29í31ííS-48 o 34í04íW) Sertão do Peri - Santa Catarina Island (27 o 45í11ííS-48 o 32í34ííW) Arvoredo Island (27 o 17í26ííS-48 o 28í32ííW) Park Serra do Tabuleiro (27 o 42í09ííS-48 o 34í09ííW) Number of X-chromosomes analyzed Number of XLXR-p inversion Estimated frequency (%) of XLXR-p inversion 40 7 17 36 15 42 26 5 19 10 1 10 26 7 27 16 4 25 Fig. 2 X polytene chromosomes of Drosophila willistoni. (a) chromosomal arm XR from female larvae of D. willistoni homozygous for the pericentric inversion XLXR-p. Sections 1, 2 and 3 of the XL arm are shown after the basal sections 21 and 22 on the XR chromosomal arm. (b) chromosomal arms XL and XR obtained from one female larvae of D. willistoni, heterozygous for one pericentric inversion (XLXR-p) and for two paracentric inversions (Inv. XL-e and Inv. XR-e). Breaking lines indicate the extension and breakpoints of pericentric inversion XLXR-p.

252 rohde, degrandi, de toni and valente ciation with the X pericentric inversion in D. willistoni. The ubiquity of the XLXR-p inversion in a great number of populations from Santa Catarina State suggests that under-dominance is not present in carriers of this inversion. It appears that the populations of the small islands which we studied (in which genetic drift operates) have overcome the deleterious effects of this type of inversion by natural selection, producing an innocuous or adaptive variant, a mechanism first suggested by Coyne (1989). It also seems that carriers of this variant were able to migrate to neighboring islands and to the continent (or in the opposite direction), finally establishing themselves throughout this territory. The fact that the inversion breakpoints are close to the centromere suggests that all the recombination which can occur in this chromosome takes place outside the inverted segment, since the whole inversion is around 15 % of the total length of X-chromosome. A restriction of crossing over in the pericentromeric regions of chromosomes appears to be here detected as also occurring in other organisms in which pericentric inversion polymorphism has been successful; e.g. grasshoppers (White 1958; Nur 1968; Haines et al. 1978), and rodents (Ohno et al. 1966). The increasing body of data on pericentric inversions in wild populations of Drosophila has been reviewed by Lemeunier and Aulard (1992) for D. melanogaster, by Levitan (1992) for D. robusta and by Matsuda et al. (1983) for D. ananassae, and it thus seems that pericentric inversions are not very rare. The challenge that remains is to discover their maintenance mechanisms. Coyne et al. (1993) developed the hypothesis that the degree of semi-sterility of pericentric inversion heterokaryotypes depends far more on the position of the breakpoints than on the physical inversion length. Navarro and Ruiz (1997) tested this hypothesis and suggested that both the genetic and physical length of the inversion are the main factors influencing the degree of under-dominance of pericentric inversions. They also described the effect of the mechanical inhibition of crossingover exerted by the tested inversions, and found that shorter inversions were particularly effective in this type of inhibition. Perhaps the short length and pericentromeric feature of the XLXR-p inversion described in the current paper could explain the successful establishment of this polymorphism in the Santa Catarina populations of Drosophila willistoni. Acknowledgements The authors are grateful to Dr. Paulo R. P. Hofmann, from Federal University of Santa Catarina (UFSC) for the facilities provided during sampling trips to the islands; to Miss Nena Basílio Morales for technical assistance; to CNPq and CAPES for fellowships; and to FAPERGS, FINEP, CNPq and PROPESQ-UFRGS for research grants. REFERENCES Aulard S., Vaudin P., Ladevèze V., Chaminade N., Périquet G. and Lemeunier F., 2004 Maintenance of a large pericentric inversion generated by the hobo transposable element in a transgenic line of Drosophila melanogaster. Heredity, 92: 151-155. Ayala F.J. and Tracey M.L., 1974 Genetic differentiation within and between species of the Drosophila willistoni group. PNAS USA, 71: 999-1003. Ayala F.J., Mourao C.A., Pérez-Salas S., Richmond R. and Dobzhansky T., 1970 Enzyme variability in the Drosophila willistoni group. I. Genetic differentiation among sibling species. PNAS USA, 67: 225-232. Ayala F.J., Powell J.R. and Tracey M.L. 1972a Enzyme variability in the Drosophila willistoni group. V. Genic variation in natural populations of Drosophila equinoxialis. Genet. Res., 20: 19-42. Ayala F.J., Powell J.R. and Tracey M.L., 1972b Enzyme variability in the Drosophila willistoni group. IV. Genic variation in natural populations of Drosophila willistoni. Genetics, 70: 113-139. Cáceres M., Ranz J.M., Barbadilla A., Long M., and Ruiz A., 1999 Generation of a widespread Drosophila inversion by a transposable element. Science, 285(5426): 415-418. Cáceres M., Puig M., and Ruiz A., 2001 Molecular characterization of two natural hotspots in the Drosophila buzzatii genome induced by transposon insertions. Genome Research, 11(8): 1353-1364. Carson H.L. and Stalker H.D., 1949 Seasonal variation in gene arrangement frequencies over a three-year period in Drosophila robusta. Evolution, 3: 322-329. Carson H.L., 1946 The selective elimination of inversion dicentric chromatids during meiosis in the eggs of Sciara impatiens. Genetics, 31: 95-113. Carson H.L., 1965 Chromosomal morphism in geographically widespread species of Drosophila. In: Baker H.G. and Stebbins G.L. (Eds), The Genetics of Colonizing Species, pp. 508-531. Academic Press, New York. Casals F., Cáceres M., and Ruiz A., 2003 The foldback-like transposon Galileo is involved in the generation of two different natural chromosomal inversions of Drosophila buzzatii. Molecular Biology Evolution, 20(5): 674-685. Cordeiro A.R. and Dobzhansky T., 1954 Combining ability of certain chromosomes in Drosophila

drosophila willistoni pericentric inversion 253 willistoni and invalidation of the wild type concept. American Naturalist, 88: 75-86. Cordeiro A.R. and Winge H., 1995 Levels of evolutionary divergence of Drosophila willistoni sibling species. In: Levine L. (Ed), Genetics of Natural Populations: the Continuing Importance of Theodosius Dobzhansky, vol. 19, pp. 262-280. Columbia University Press. New York. Coyne J.A., Meyers W., Crittenden A. and Sniegowski P., 1993 The fertility effects of pericentric inversions in Drosophila melanogaster. Genetics, 134: 487-496. Coyne J.A., 1989 A test of the role of meiotic drive in fixing a pericentric inversion. Genetics, 123: 241-243. Da Cunha A.B. and Dobzhansky T., 1954 A further study of chromosomal polymorphism in Drosophila willistoni in its relation to environment. Evolution, 8: 119-134. Da Cunha A.B., Burla H. and Dobzhansky T., 1950 Adaptive chromosomal polymorphism in Drosophila willistoni. Evolution, 4: 212-235. Da Cunha A.B., Dobzhansky T., Pavlovsky O. and Spassky B., 1959 Genetics of natural populations. XXXVIII. Supplementary data on the chromosomal polymorphism of Drosophila willistoni in its relation to the environment. Evolution, 13: 389-404. Dobzhansky T. and Powell J.R., 1975 The willistoni group of sibling species of Drosophila, In: King R.C. (Ed.), Handbook of Genetics, pp. 589-622. Plenum Press, New York. Dobzhansky T., 1957 Genetics of natural populations. XXVI. Chromosomal variability in island and continental populations of Drosophila willistoni from Central America and the West Indies. Evolution, 11: 280-293. Dobzhansky T., 1965 Wild and domestic species of Drosophila. In: Baker H.G. and Stebbins L.G. (Eds), The Genetics of Colonizing Species, pp. 533-545. Academic Press, New York. Ephrussi B. and Beadle G.W., 1936 A technique of transplantation for Drosophila melanogaster. American Naturalist, 70: 6-8. Etges W.J., 1998 A new pericentric inversion in a natural population of Drosophila robusta. Drosophila Information Service, 81: 142-143. Freire-Maia N., 1960 Peculiar gene arrangements in Brazilian natural populations of Drosophila ananassae. Evolution, 15: 486-495. Haines R.L., Roberts P.A. and Lattin J.D., 1978 Pericentric inversion polymorphism in the grasshopper Boonacris alticola. Chromosoma, 65: 185-197. Hinton C.W. and Downs J.E., 1975 The mitotic, polytene and meiotic chromosomes of Drosophila ananassae. J. Heredity, 66: 353-361. Hinton C.W. and Lucchesi J.V., 1960 A cytogenetic study of crossing over in inversion heterozygotes of Drosophila melanogaster. Genetics, 45: 87-94. Krimbas C.D. and Powell J.R., 1992 Drosophila Inversion Polymorphism. CRC Press, Boca Raton. Lemeunier F. and Aulard S., 1992 Inversion polymorphism in Drosophila melanogaster. In: Krimbas C.D. and Powell J.R. (Eds), Drosophila Inversion Polymorphism, pp: 339-405. CRC Press, Boca Raton. Levitan M., 1992 Chromosomal variation in Drosophila robusta Sturtevant. In: Krimbas C.D. and Powell J.R. (Eds), Drosophila Inversion Polymorphism, pp. 221-338. CRC Press, Boca Raton. Marques E.K., Napp M., Winge H. and Cordeiro A.R., 1966 A cornmeal, soybean flour, wheat germ medium for Drosophila. Drosophila Information Service, 41: 187. Matsuda M., Imai H.T. and Tobari Y.N., 1983 Cytogenetic analysis of recombination in males of Drosophila ananassae. Chromosoma, 8: 286-292. Miller D., 1939 Structure and variation of the chromosomes in Drosophila algonquin. Genetics, 28: 699-708. Morgan T.H., 1912 Complete linkage in the second chromosome of the male of Drosophila. Science, 36: 719-720. Morgan T.H., 1914 No crossing over in male of Drosophila of genes in the second and third pairs of chromosomes. Biol. Bull., 26: 195-204. Navarro A. and Ruiz A., 1997 On the fertility effects of pericentric inversions. Genetics, 147: 931-933. Nur U., 1968 Synapsis and crossing over within a pericentric inversion in the grasshopper Camnula pellucida. Chromosoma, 25: 198-214. Ohno S., Weiler C., Poole J., Christian L. and Stenius C., 1966 Autosomal polymorphism due to pericentric inversions in the deer mouse (Peromyscus maniculatus) and some evidences of somatic segregation. Chromosoma, 18: 177-187. Pavan C., Cordeiro A.R., Dobzhansky T., Malagolowkin C., Spassky B. and Wedel M., 1951 Concealed genic variability in Brazilian populations of Drosophila willistoni. Genetics, 36: 13-30. Puig M., Cáceres M., and Ruiz A., 2004 Silencing of a gene adjacent to the breakpoint of a widespread Drosophila inversion by a transposon-induced antisense RNA. PNAS USA, 101(24): 9013-9018. Regner L., Pereira M.S.O., Alonso C.E.V., Abdelhay E., and Valente V.L.S., 1996 Genomic distribution of P elements in Drosophila willistoni and a search for their relationship with chromosomal inversions. J. Heredity, 87: 191-198. Roberts P.A., 1967 A positive correlation between crossing over within heterozygous pericentric inversions and reduced egg hatch of Drosophila females. Genetics, 56: 179-187. Rohde C., 2000 Polimorfismo cromossômico e elementos transponíveis em Drosophila willistoni. PhD Thesis, pp. 238. Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil. Spassky B., Richmond R.C., Pérez-Salas S., Pavlovsky O., Mourao C.A., Hunter C.A., Hoenigsberg H., Dobzhansky T. and Ayala F.J.,

254 rohde, degrandi, de toni and valente 1971 Geography of the sibling species related to the Drosophila willistoni and the semi-species of the D. paulistorum complex. Evolution, 25: 129-143. Sperlich D. and Pfriem P., 1986 Chromosomal polymorphism in natural and experimental populations. In: Ashburner M., Carson H.L. and Thompson Jr J.N. (Eds.), The Genetics and Biology of Drosophila, vol. 3c, pp. 257-309. Academic Press, New York. Stevens N.M., 1908 A study of the germ cells of certain Diptera, with reference to the heterochromosomes and the phenomenon of synapsis. J. Exp. Zool., 5: 359-374. Valente V.L.S. and Araújo A.M., 1985 Observations on the chromosomal polymorphism of natural populations of Drosophila willistoni and its association with the choice of feeding and breeding sites. Rev. Brasil. Genet., 8: 167-173. Valente V.L.S. and Araújo A.M., 1986 Chromosomal polymorphism, climatic factors, and variation in population size of Drosophila willistoni in Southern Brazil. Heredity, 57 (2): 149-160. Valente V.L.S., Goni B., Valiati V.H., Rohde C. and Morales N.B., 2003 Chromosomal polymorphism in Drosophila willistoni populations from Uruguay. Genet. and Molec. Biol. 26 (2): 163-173. Valente V.L.S., Ruszczyk A. and Santos R.A., 1993 Chromosomal polymorphism in urban Drosophila willistoni. Rev. Brasil. Genet., 16 (2): 307-319. Valente, V.L.S., Rohde C., Valiati V.H., Morales N.B. and Goni B., 2001 Chromosome inversion occurring in Uruguayan populations of Drosophila willistoni. Drosophila Information Service, 84: 55-59. White, M.J.D., 1958 Restrictions on recombination in grasshopper populations and species. Cold Spring Harbor Symp. Quant. Biol., 23: 307-317. Received 15.2.2005; accepted 28.6.2005