Ó Indian Academy of Sciences

Hybridization, transgressive segregation and evolution of new genetic systems in Drosophila

H. A. RANGANATH* and S. ARUNA Drosophila Stock Centre, Department of Studies in Zoology, University of Mysore, Manasagangotri, Mysore 570 006, India

Abstract

Introgressive hybridization facilitates incorporation of genes from one species into the gene pool of another. Studies on long-term effects of introgressive hybridization in animal systems are sparse. Drosophila nasuta (2n = 8) and D. albomicans (2n = 6)—a pair of allopatric, morphologically almost identical, cross-fertile members of the nasuta subgroup of the immigrans species group—constitute an excellent system to analyse the impact of hybridization fol- lowed by transgressive segregation of parental characters in the hybrid progeny. Hybrid populations of D. nasuta and D. albomicans maintained for over 500 generations in the laboratory constitute new recombinant hybrid genomes, here termed cytoraces. The impact of hybridization, followed by introgression and transgressive segregation, on chromosomal constitution and karyotypes, some fitness parameters, isozymes, components of mating behaviour and mating preference reveals a complex pattern of interracial divergence among parental species and cytoraces. This assemblage of characters in different combinations in a laboratory hybrid zone allows us to study the emergence of new genetic systems. Here, we summarize results from our ongoing studies comparing these hybrid cytoraces with the parental species, and discuss the implications of these findings for our understanding of the evolution of new genetic systems.

[Ranganath H. A. and Aruna S. 2003 Hybridization, transgressive segregation and evolution of new genetic systems in Drosphila. J. Genet. 82, 163–177]

Introduction mals is small because, even when fertile hybrids are pro- duced, there is severe selection against the genetically Hybridization is the crossing of individuals belonging to imbalanced gametes resulting through introgression (Mayr two unlike natural populations that have secondarily come 1963). Of late, however, this concept of hybridization as in contact (Mayr 1963). There are contradictory views an evolutionary dead-end in animals is being challenged regarding the potential role of hybridization in evolution. by reports of frequent hybridization between closely rela- In plants, hybridization is considered to be a widespread ted species (Barton and Hewitt 1989; Avise 1994; Arnold and potentially creative evolutionary force that is thought 1997; Goodman et al. 1999; Bruke and Arnold 2001), to have contributed to past diversification during environ- although hybridization between species that have diver- mental changes (Anderson 1949; Anderson and Stebbins ged considerably could result in embryonic or adult letha- 1954; Cruzan and Arnold 1993; Rieseberg et al. 1996; lity (Bock 1984), or male sterility even if the hybrids are Fritz 1999). On the other hand, it has been thought that viable (Haldane 1922). In situations where hybrids can the evolutionary role of interspecific hybridization in ani- survive and reproduce, giving rise to at least some off- spring of mixed ancestry, the region is recognized as a hybrid zone, and such regions are of considerable evo- *For correspondence. E-mail: [email protected]. This paper is dedicated to the memory of our teacher, Prof. N. B. lutionary interest (Barton and Hewitt 1989; Harrison Krishnamurthy. 1990).

Keywords. Drosophila nasuta; Drosophila albomicans; hybridization; cytoraces; new genetic systems.

Journal of Genetics, Vol. 82, No. 3, December 2003 163 H. A. Ranganath and S. Aruna

Many evolutionary biologists have viewed the hybrid ential role of hybridization in the formation of new gene- zone as an active site for evolutionary change, wherein tic systems and in speciation, using an artificial hybrid selection against hybridization shapes the causes and con- zone created in the laboratory and earlier described by sequences of genetic and ecological interaction between Tanuja et al. (1998). differentiated populations. The potential ways in which hybridization can promote the origin of new characteri- Drosophila nasuta and D. albomicans: a goldmine stics are transgressive segregation, establishment of new for evolutionary cytogenetics genetic background, and increased incidence of new muta- tions (Stebbins 1973; Rieseberg et al. 1999). To be suc- Over 90 per cent (perhaps 98 per cent) of all speciating cessful, these hybrid progeny must become stabilized events are accompanied by karyotypic changes and, in through the establishment of true-breeding intermediate a majority of these cases, structural chromosomal re- gene combinations, or introgressed genomes. Such intro- arrangements have played the primary role in initiating gressed systems can lead to production of recombinant divergence (White 1978). However, Carson (1982) is of genotypes that have properties different from those of the view that in most cases the fixation of particular karyo- either parent and, thus, this process can be an important types is likely to be merely an incidental accompaniment source of new variation leading to the establishment of of small-population effects and forced selection for re- new genetic systems or genomes (Anderson 1949). Natu- organization as the species is formed. ral selection would tend to favour hybrids that have The basic karyotype of the genus Drosophila is beli- formed coadapted gene complexes (Dobzhansky (1949, eved to be 2n = 12, with five pairs of acrocentric rods, 1970a,b) by overcoming genetic incompatibility and that and a pair of small dot chromosomes. During the evolu- are, consequently, more viable and fertile (Templeton tion of different groups of Drosophila from this so-called 1981; Barton 2001). Thus, in contrast to mutation, which primitive karyotype, centric fusions and inversions, as well has a constant rate across a range of species, hybridiza- as additions or deletions of heterochromatin, have played tion can provide a much faster mechanism for generation a major role in shaping the karyotypic phylogeny. Studies of genetic variations wherein favourably interacting gene tracing the steps involved in karyotypic evolution in the complexes are determined by hybrid founder events fol- immigrans species group of Drosophila, with particular lowed by natural selection, leading to the establishment emphasis on the nasuta subgroup, have shown involve- of a new evolutionary lineage with a novel genome. ment of centric fusions in transforming the acrocentric- A potential hybrid zone can provide insights into the dominated ancestral karyotype into a metacentric form mechanisms of speciation for it reveals the genetic dif- (Ranganath and Hägele 1981; Rao and Ranganath 1990), ferences that have accumulated during the early steps of an example of karyotypic orthoselection (sensu White 1973). speciation. Hybrid-zone studies can yield information D. nasuta Lamb with 2n = 8 diploid chromosomes appears about the possible state and degree of divergence bet- to have evolved from the putative ancestral karyotype via ween populations that may be ‘on the way’ to differentiat- two centric fusions among the ancestral rods leading to ing into races / species (Hewitt 1988). Many natural hybrid two metacentric chromosomes, followed by a pericentric zones are available for study of the evolutionarily signifi- inversion in one of them, forming a double-length acro- cant mechanisms of the creation of new genetic systems centric chromosome. Thus the karyotypic composition of via introgressive hybridization. However, one major draw- D. nasuta is a pair of metacentrics (chromosome 2), one back of these natural hybrid zones is the lack of informa- pair of double-length acrocentrics (chromosome 3), one tion about their time of origin. Moreover, it may take a pair of acrocentric sex chromosomes, and a pair of small considerable time for populations to differentiate into neo- dot chromosomes. A further centric fusion of the double- forms, even with fairly high levels of gene flow. Thus, length acrocentric with the sex chromosomes, leading to many such studies have been confined to estimating the a reduction in chromosome number, appears to have been age of the hybrid zone from the extent of differentiation involved in the formation of the D. albomicans Duda reported. There seems to be a need for a hybrid zone whose karyotype with 2n = 6. Thus in addition to a pair of meta- age is precisely known and in which the speciating mecha- centrics (chromosome 2), and a pair of long dot chromo- nism is traced and reported right from its origin. Such a somes, D. albomicans has another pair of metacentrics— system would help validate the accuracy of estimates pro- the product of centric fusion (X•3, Y•3 chromosomes), vided for natural hybrid zones. However, most experimen- also termed neo-sex chromosomes, where one arm is tal hybridization studies in the past, especially on animals, homologous to the double-length acrocentric (chromo- have been confined to a few generations. Thus, there is a some 3) and the other arm to the sex chromosome of need for long-term hybridization experiments wherein a D. nasuta (figure 1) (Ranganath and Ramachandra 1994). hybrid zone could be created in the laboratory as a paral- There are also differences in heterochromatin and satel- lel system simulating natural hybrid zones. In this paper, lite DNA contents of these two species. D. nasuta has 22% we describe results from experimental studies of the pot- heterochromatic region in the genome, whereas D. albo-

164 Journal of Genetics, Vol. 82, No. 3, December 2003 Evolution of new genetic systems in Drosophila micans has 12%. Further, nearly 80% of the long dot process of speciation. For example, even though D. moja- chromosome of D. albomicans is heterochromatic, com- vensis and D. arizonensis differ from one another by at pared to 50% heterochromatin in the small dot chromo- least seven inversions, they can produce fertile hybrid some of D. nasuta. The quantum of heterochromatin in offspring (Wasserman 1982). Similarly, D. virilis and X•3 chromosome of D. albomicans is less than the total D. americana texana, in spite of having different karyo- heterochromatin content of chromosomes X and 3 to- types owing to fusion between an autosome and the sex gether in D. nasuta. However, in terms of polytene band- chromosomes, produce fertile hybrids (Throckmorton 1982). ing, the euchromatic components of the two species are Likewise, significant karyotypic divergence involving effectively cytologically identical (Ranganath and Hägele centric fusions, major structural reorganization in dot 1982; Hägele and Ranganath 1983). D. nasuta has one major chromosomes, changes in the pattern and distribution of AT-rich satellite region with a density of 1.663 g/cm3, heterochromatin, and amplification of satellite DNA sequ- which constitutes 7–8% of the genome. D. albomicans, on ences has occurred during the evolution of D. albomi- the other hand, has three major AT-rich satellite regions cans. Yet D. nasuta and D. albomicans are cross-fertile with densities of 1.674, 1.665 and 1.661 g/cm3, which to- and hybrid progeny can be maintained for many genera- gether make up 28–30% of the total DNA. The in situ tions, and hence it has been suggested that they be treated hybridization of satellite DNA regions of the two taxa has as chromosomal races (Nirmala and Krishnamurthy 1972; revealed that they have common satellite sequences. Thus, Ramachandra and Ranganath 1987; cf. Ranganath 2002). there appears to have been an amplification of satellite On the other hand, Wilson et al. (1969) have felt that since sequences in D. albomicans in both the dot and Y chromo- D. albomicans has a different karyotype from D. nasuta somes, and a concomitant reduction in the satellite con- it should be treated as a distinct species in the nasuta tent of chromosomes 2 and X•3 (Ranganath et al. 1982). subgroup. Kitagawa et al. (1982) prefer to treat D. nasuta Structural changes in chromosomes and, hence, the kar- and D. albomicans as semi-species of the nasuta complex, yotype of species in a lineage illuminate the history and on the basis of sexual isolation and insemination test. Like- presumed phylogeny of the concerned group. Yet, it is wise, Chang and Ayala (1989) have opined that D. nasuta difficult to generalize the impact of such changes on the and D. albomicans fit the category of ‘super species’ of Mayr (1963). However, the biological species concept (Mayr 1963) requires reproductive isolation between spe- cies. Despite karyotypic divergence, since D. nasuta and D. albomicans enjoy mutual open genetic systems, they should, therefore, be treated as chromosomal races (Ranga- nath et al. 1974). On the other hand, according to the phy- logenetic species concept, species are recognized strictly in terms of their status as diagnosable evolutionary taxa (Cracraft 1983). Following this concept, two sister taxa could broadly hybridize and still be considered as species if each is diagnosable as a discrete taxon. By the criterion of the phylogenetic species concept, therefore, D. nasuta and D. albomicans, which are karyotypically different and occupy two distinct positions in the karyotypic phy- logenetic tree of the nasuta subgroup, have to be treated as species. The most important aspect of D. nasuta and D. albomicans to consider at this juncture is that hybridi- zation between D. nasuta and D. albomicans is observed only in the laboratory, because these two species are allo- patric in nature and, consequently, have no opportunity to hybridize, resulting in the protection of the distinct iden- tities of their respective gene pools.

Figure 1. Karyotypes of D. nasuta (A) and D. albomicans (B). The centric fusion between autosomes 3 and sex chromosomes has resulted in the evolution of the karyotype of D. albomicans, Origin of the hybrid zone: the nasuta– with reduction in diploid number (Ranganath and Hägele 1981). Therefore Mahesh et al. (2000, 2001) have redescribed the kar- albomicans complex yotype of D. albomicans and have treated the autosome – sex chromosome fusion products, the X3 and Y3 chromosomes, as Interracial hybridization between D. nasuta (2n = 8) and neo-sex chromosomes and the long dot chromosomes as chro- D. albomicans (2n = 6) leads to a hybrid gene pool. The mosome 3. chromosomes of D. nasuta and D. albomicans can be

Journal of Genetics, Vol. 82, No. 3, December 2003 165 H. A. Ranganath and S. Aruna identified on the metaphase plate with certainty on the Cytorace 2: (males 2n = 6, with 2n2a 4a4a Y•3a X•3a; basis of the heterochromatin content of each chromosome females 2n = 6, with 2n 2a 4a 4a X•3a X•3a) n a n n a a n n in the two parents. The F1 hybrids have 2n = 7, with four Cytorace 3: (males 2n = 8, with 2 2 3 3 4 4 Y X ; chromosomes of D. nasuta and three of D. albomicans. females 2n = 8, with 2n2a 3n3n 4a4a XnXn) n a a a n a n In F2 and subsequent generations, karyotypic mosaicism Cytorace 4: (males 2n = 7, with 2 2 4 4 3 Y•3 X ; is noticed, with a variety of individual karyotypes, such females 2n = 8 with 2n2a 3n3n 4a4a XnXn). as nasuta type, albomicans type, F type, and also new 1 These cytoraces are maintained in separate cages and combinations (Ranganath 1978; Rajasekarasetty et al. 1979; are, consequently, independent genetic and evolutionary Yu et al. 1997). To study the dynamics of chromosomal entities. In nature, following the formation of such hybrid segregation during gametogenesis in the F hybrids, and 1 races, intercrossing with parents and other hybrid races its impact on the fertility of the next generation, a sys- could result in clines for genetic variation across the tematic assessment was made by Ranganath and Krishna- hybrid zone, as has been reported for D. americana and murthy (1981). F males produced six different types of 1 D. texana (Patterson and Stone 1952; Throckmorton 1982; sperm, of which 39% had normal haploid chromosome McAllister 2002). Such transient clines are of consider- complement while the remaining 61% were aneuploids able interest for studying chromosomal rearrangements for the chromosomes X and 3. On the other hand, the F 1 following hybridization and possible introgression. With females produced only two types of eggs with the normal the intention of creating a laboratory system analogous to haploid complement of chromosomes, and aneuploid eggs such transient hybrid-zone clines, we carried out a second were not observed. Fertility test on F and backcross pro- 2 round of interracial hybridization in our laboratory system geny revealed males to be sterile more often than fema- by crossing the parental species D. nasuta and D. albo- les. Out of 400 males and 400 females, 177 males and only micans with the four newly evolved cytoraces in various 14 females were found to be sterile. Thus F luxuriance 1 combinations. In this manner, 28 new hybrid populations followed by F2 breakdown was noticed for a few para- meters of fitness, namely fecundity, rate of development and viability (Ranganath 1978). The F2 breakdown was attributed to abnormal chromosomal segregation during the hybrid meiosis (Ranganath and Krishnamurthy 1981). Even then, the few fertile individuals that could survive and reproduce contributed to the next generation and, con- sequently, these hybrid populations could be maintained in the laboratory for a number of generations. With this background, we initiated a series of long- term hybridization experiments to systematically assess the fate of chromosomes of D. nasuta and D. albomicans in hybrid populations over generations in a laboratory hybrid zone (figure 2). The F2 and later generations showed polymorphism with respect to chromosomal constitution. Each of these hybrid populations was independently cul- tured in a separate cage, often with population-size bot- tlenecks, especially during the early generations after hybridization. In some of the hybrid lineages, there was a gradual decline in the degree of karyotypic mosaicism, and karyotypically stabilized forms became established. These stabilized karyotypic neo-races were monomorphic for different novel combinations of parental chromosomes, and were termed cytoraces (Ramachandra and Ranganath 1985, 1988, 1990, 1996; Ranganath and Ramachandra 1987). Four such karyotypically stable hybrid cytoraces were obtained after the first round of hybridization, and their karyotypic compositions were as follows (The super- script on each chromosome indicates the parent from which it was inherited):

n a n n n n a Cytorace 1: (males 2n = 7, with 2 2 4 4 3 Y X•3 ; Figure 2. A schematic representation of the experimental set- females 2n = 6, with 2n2a 4n4n X•3a X•3a) up of hybridization between D. nasuta and D. albomicans.

166 Journal of Genetics, Vol. 82, No. 3, December 2003 Evolution of new genetic systems in Drosophila were established. To speed up the differentiation of these longed inbreeding of such hybrid lineages has resulted polymorphic populations, we prevented gene flow among in establishment of homozygous state for either dot chro- them, and their parents, by maintaining each population mosomes of nasuta or those of albomicans, but never a in a separate cage. These hybrid populations, consequ- heterozygous state. There is reason to suspect that dot chro- ently, experienced the genetic isolation characteristic of mosome heterozygotes have lower fitness than the homo- allopatry, while being reared under a common set of envi- zygotes owing to meiotic incompatibility between the dot ronmental conditions, a situation more similar to that chromosomes of D. nasuta and D. albomicans, which experienced by sympatric populations, and have thus are very different. The metaphase dot chromosomes of been referred to as being ‘allo-sympatric’ (Tanuja et al. D. albomicans are nearly five times larger than those of 1998). Over a period of a few years, some of these popu- D. nasuta and contain a huge amount of heterochromatin. lations resulting from the second round of hybridization Polytene banding patterns of these chromosomes have also lost their karyotypic mosaicism, resulting in the labo- also revealed that the dot chromosomes of D. albomicans ratory evolution of a new set of 12 karyotypically mono- are broader and shorter than those of D. nasuta. The large morphic cytoraces (Ramachandra and Ranganath 1996; quantum of heterochromatin in the metaphase dot chro- Tanuja et al. 1998; Tanuja 2000; Ranganath 2002). mosomes of D. albomicans is found in the chromocentre, The complete set of two parental species, the four initi- while in the euchromatic arm a few bands of the basal ally established cytoraces, and the 12 cytoraces esta- region are invertedly duplicated at the tip, resulting in a blished after the second round of hybridization now bending of the tip of the chromosome towards the base, constitute a laboratory hybrid zone. Since the entire gene making it broader as well as shorter. Moreover, in the pool of this hybrid zone is contributed by D. nasuta and polytene chromosomes of the F1 hybrids, the correspond- D. albomicans, we have termed this hybrid zone with 18 ing homologous chromosomes of nasuta and albomicans races the nasuta–albomicans complex of the nasuta sub- parents synapse completely whereas the arms of the nasuta group (Ramachandra and Ranganath 1996), whose mem- and albomicans dot chromosomes exist as two indepen- bers have been classified into eight categories on the basis dent entities without pairing (Hägele and Ranganath 1982). of their diploid chromosomal complement (figure 3). The most likely explanation for the observed homozy- gosity of dot chromosomes derived from D. nasuta or D. albomicans in hybrid cytoraces, therefore, is that of Patterns of divergence in the nasuta– heterozygote disadvantage, with the relative fitness advan- albomicans complex tage of the two homozygotes varying among cytoraces based on differences in the genomic background. In cyto- The members of the nasuta–albomicans complex have races where the albomicans dot chromosome homozy- been studied in detail to uncover cytogenetic and other dif- gotes are fitter than nasuta homozygotes, the albomicans ferences among them, and anagenetic changes leading to homozygotes get fixed, and vice versa. Of course, a role the establishment of different genetic systems have been for drift in the fixation of different dot chromosomes in documented. In this section, we summarize the more different cytoraces also cannot be ruled out. important results from this ongoing set of studies. On the other hand, the metacentric chromosome 2 of D. nasuta and D. albomicans exists polymorphically in Trends in the karyotypic evolution of cytoraces each of the cytoraces, with three types of individuals, D. nasuta, D. albomicans and their hybrids have become namely homozygous for nasuta (2n 2n), homozygous for a key system for understanding the implications and the albomicans (2a 2a), and heterozygous (2n 2a), suggesting impact of the hybridization in establishing new hybrid either heterozygote advantage (Tanuja et al. 2003) or, races through karyotypic repatterning. The chromosomes perhaps, selective neutrality of 2n and 2a. In either case, it of the parental races, namely D. nasuta and D. albomi- suggests compatibility of these second chromosomes com- cans, are differentially represented in the cytoraces. ing from the two different parents. The ‘dot’ chromosomes, in particular, present an inte- With regard to the sex chromosomes and autosome 3 resting scenario. Each of the cytoraces is homozygous for components of the karyotype, the females of cytoraces either the nasuta dot (two cytoraces) or the albomicans can, in principle, have Xn Xn 3n 3n (nasuta) or X•3a X•3a n n a dot (14 cytoraces) chromosomes. During the evolution of (albomicans) or X 3 X•3 (F1 type) chromosome com- each cytorace, the initial F1 was heterozygous for nasuta plements. Similarly, the males of cytoraces may have either and albomicans dot chromosomes. During the subsequent parental combinations such as Xn Yn 3n 3n (nasuta) or a a n n a n hybrid generations, a transient phase of karyotypic insta- X•3 Y•3 (albomicans) or the F1 type (X Y X•3 or 3 bility was noticed, with three types of individuals, namely Xn Y•3a). In none of the cytoraces did females exhibit n n a homozygous for nasuta dots, homozygous for albomi- the F1 karyotype X 3 X•3 . Females in seven cytoraces cans dots, and heterozygous for these dots, in varying showed the nasuta type of arrangement of sex chromo- frequencies (Ramachandra and Ranganath 1985). The pro- somes and chromosome 3, whereas nine cytoraces were

Journal of Genetics, Vol. 82, No. 3, December 2003 167 H. A. Ranganath and S. Aruna

Figure 3. Interracial hybridization between D. nasuta and D. albomicans followed by main- tenance of hybrid progeny for many generations has resulted in the emergence of populations with the introgressed stable karyotypes. Such populations with differential chromosomal repre- sentation of D. nasuta and D. albomicans are called cytoraces. This figure illustrates the karyotypic composition of the newly evolved assemblage called the ‘nasuta–albomicans com- plex’, which includes D. nasuta, D. albomicans and cytoraces. Note the different patterns of representation of the parental chromosomes in different cytoraces.

168 Journal of Genetics, Vol. 82, No. 3, December 2003 Evolution of new genetic systems in Drosophila fixed for the albomicans type of arrangement. On the diploid chromosome number. White (1973, 1978) and other hand, among the males of the cytoraces all the four King (1993) have discussed this at length to demonstrate expected combinations are seen, albeit in different cyto- the importance of chromosomal changes in the karyotype races. For these chromosomes, therefore, it appears that phylogeny of different animal and plant lineages. The evo- coassociation of parental elements was entertained in lution of the D. nasuta and D. albomicans karyotypes is males only while stringent selection has been observed in known to have involved centric fusion (Ranganath and females for homozygous association of nasuta or albomi- Hägele 1982). It has been postulated that the fusion of cans chromosomes. Thus there is a conflicting picture chromosomes and evolution of the new cytogenetic race between male and female karyotypes of these cytoraces D. albomicans occurred between 350,000 and 500,000 for the sex chromosomes and chromosome 3. years ago (Chang and Ayala 1989; Chang et al. 1989). We A critical analysis of the trends in the karyotypic evo- now discuss whether such major changes in karyotypes lution of hybrid cytoraces suggests that interchange of can take place even in laboratory populations. the chromosomal material between corresponding homo- In one of the subpopulations of the hybrid lineage logous chromosomes of D. nasuta and D. albomicans is cytorace 1, occurrence of a centric fission was recorded limited. Any genetic exchange between D. nasuta and (Tanuja et al. 1999b). The metacentric chromosome X•3a, D. albomicans chromosomes has to occur in heterozygo- which is considered to be derived from the fusion of X tes. The F1 heterozygote condition for the dot chromo- chromosome and chromosome 3 of a nasuta-like ances- somes is gradually replaced by homozygosity for one of tor, had undergone fission to result in independent units, the parental chromosomes. Even when these chromoso- namely an X chromosome and chromosome 3. This fission mes coassociate in the hybrid, complete synapsis may not event was associated with the addition of heterochro- occur because of cytogenetic divergence between them. matin in the form of a short arm to the X chromosome, With regard to the sex chromosomes and autosome 3, making it submetacentric. The euchromatic arm of this females of cytoraces have either the D. nasuta comple- submetacentric chromosome was homologous to the acro- ment (3n 3n Xn Xn) or the D. albomicans complement centric X chromosome of D. nasuta. The other product of (X•3a X•3a). Thus, in the final stabilized karyotype, females fission of X•3a, the acrocentric chromosome 3, was homo- are not heterozygous. Even when heterozygosity is pre- logous to chromosome 3 of D. nasuta. Thus, this cytorace sent for these chromosomes (Xn 3n X•3a), for example in 1 population was dimorphic, with individuals with two a the F1 and subsequent hybrid generations prior to karyo- types of chromosomal complements—the X•3 chromo- typic stabilization, the metacentric X•3a pairs with homo- some, or chromosomes 3 and X. From this population, logous acrocentric 3n and Xn chromosomes. Even during males and females possessing only the products of cen- this transient period, recombination between these elements tric fission were isolated and used to establish a new popu- might not have occurred freely, as also seen in the case of lation, fission cytorace 1 (figure 4), with 2n = 8 in both the genus Mus, where a crossover suppressor may ope- males and females (Tanuja et al. 1999b). This is yet an- rate in the vicinity of metacentric centromere (cf. Searle other addition to the nasuta–albomicans complex of Dro- 1998). Thus it seems likely that only the second chromo- sophila. The uniqueness of fission cytorace 1 is not only somes of the parental species are together regularly in that the products of fission are fixed in it, but also that it the hybrid genomes of the cytoraces, therefore limiting is the only member in this complex with a submetacentric meiotic recombination to only this component of the X chromosome. The establishment of cytorace 1 took hybrid genome. about 20 generations from the initial interracial hybri- An interesting facet reflected from the karyotypic stu- dization between D. nasuta and D. albomicans. Subsequ- dies on the hybrids of D. nasuta and D. albomicans is the ently, within a span of about 300 generations, we observed lack of complete parental type of karyotype in any of the a major evolutionary change in karyotype giving rise to a stabilized lines or cytoraces. Though there was preferen- new chromosomal lineage via centric fission. The obser- tial elimination or retention of specific chromosomes from vation of such karyotypic changes in regularly monitored either of the parents, D. albomicans chromosomes were laboratory systems is of great interest as it permits an more preferred than those of the D. nasuta parent. In so empirical study of these cytogenetic processes important far stabilized 16 cytoraces, overall 221 chromosomes are to raciation and speciation as they occur. This is an present of which 98 chromosomes are of D. nasuta and advance over the typical situation in which the past 123 are of D. albomicans. Is it a reflection of cytoraces occurrence of such processes must be inferred from the reverting to albomicans parental type? karyotypes of extant species.

Stepwise evolution of a new race through centric fission The trap of an autosome in males of a cytorace

Chromosomal fusion and fission are two basic mecha- One of the interesting aspects of study of the evolution nisms, apart from the ploidy, that result in alteration of the of sex chromosomes is to account for heteromorphism

Journal of Genetics, Vol. 82, No. 3, December 2003 169 H. A. Ranganath and S. Aruna

Figure 4. Karyotypic phylogeny of fission cytorace 1. The scheme is an extension of the karyotypic phylogeny of the nasuta subgroup given earlier by Ranganath and Hägele (1981). The primitive karyotypic constitution of Drosophila has 2n = 12 (A to F). From this primitive setup Ranganath and Hägele (1981) have drawn the successive stages in the karyotypic evolution of the members of the nasuta subgroup. In this lineage, the karyo- type of D. nasuta is a product of two centric fusions and a pericentric inversion. A third centric fusion has resulted in the evolution of the karyotype of D. n. albomicans. Cytorace I is the product of hybridization between males of D. nasuta and females of D. albomi- cans. Males and females of cytorace 1 have 2n = 7 and 2n = 6, respectively. The X3 chro- mosome has undergone centric fission to give rise to acrocentric chromosome 3 and a submetacentric X chromosome. Addition of heterochromatin after fission has occurred in this submetacentric X chromosome. These fission products are fixed in a subpopulation of cytorace 1 and this new lineage is referred to as fission cytorace 1.

170 Journal of Genetics, Vol. 82, No. 3, December 2003 Evolution of new genetic systems in Drosophila

(X and Y chromosomes) and associated dosage compen- is euchromatic, is just about 600 generations, and it pro- sation. The density of active loci on the Y chromosome is vides extremely good opportunities for future investiga- usually very low, and the process leading to such inacti- tions on evolution of dimorphism in sex chromosomes. vation of loci is called degeneration of Y chromosome. The dimorphic sex chromosomes (X, Y) are considered Isozymes to have evolved from a pair of autosomes which slowly differentiated over millions of years (Muller 1918; Luc- Isozymes are among the molecular markers employed in chesi 1978; Charlesworth 1996; Rice 1996a,b; Steine- evolutionary studies to assess the genetic variability within mann and Steinemann 1997; Marin et al. 2000). and genetic distance between races / species. Here we sum- Hybridization between D. nasuta and D. albomicans marize results from a survey of isozyme variation in has given rise to introgressed cytoraces, where chromo- D. nasuta, D. albomicans, and four of the cytoraces, car- somes of parents are represented in different combi- ried out to analyse the pattern of introgression in the nations. One of the most notable events is that one of the hybrid genomes of the cytoraces. The introgressed popu- autosomes of D. nasuta is restricted to only the male lations are expected to exhibit alleles of both parents as genome in cytorace 1. For instance, the acrocentric well as new single-locus and multilocus genotypes. There chromosome 3, which is seen in both males and females have also been reports of novel alleles or ‘hybrizymes’ in of D. nasuta, is restricted to males of cytorace 1. There- hybrid zones of mammals, birds, reptiles, amphibians and fore, in this cytorace, two chromosomes are limited to the insects (Woodruff 1989). In some studies, a moderate male genome—the regular Y chromosome, and the acro- increase in allelic polymorphism in introgressed popula- centric autosome 3 inherited from the D. nasuta parent tions, compared to the parental taxa, has also been obser- (figure 5). As this autosome 3 is cosegregating with the Y ved (Soltis 1985; De Pamphilis and Wyatt 1990). On the chromosome and is limited to males, it is called a recent other hand, however, stabilized introgressants that are re- neo-Y chromosome (Tanuja et. al. 1999a). The polytene productively isolated from their parental taxa are often banding pattern of this neo-Y chromosome is completely expected to have faced population bottlenecks, thus lead- homologous to the counterpart arm in X•3a chromosome ing to decreased genetic variability relative to the pro- of D. albomicans and to the chromosome 3n of D. nasuta, genitor populations (cf. Rieseberg and Wendel 1993). both of which are seen in females as well as males. The The degree of introgression in the nasuta–albomicans situation in cytorace 1 with reference to the 3n chromo- complex was assessed among D. nasuta, D. albomicans some or the euchromatic arm 3a of the X•3a chromosome, and cytoraces 1–4, taking into consideration 11 iso- and its homologue now found only in males, represents a zymes: 1-esterase, 2-esterase, alkaline phosphatase, acid classical case of sex chromosomes, wherein one of them phosphatase, glucose-6-phosphate dehydrogenase, 1-glycero- is shuttling between males and females while the other phosphate dehydrogenase, malate dehydrogenase, xanthine remains in males only. As discussed by Muller (1918), dehydrogenase, superoxide dismutase, octanol dehydro- Lucchesi (1978), Charlesworth (1996), Rice (1996a,b), genase and alcohol dehydrogenase (Aruna and Ranganath Steinemann and Steinemann (1997) and Marin et al. (2000), 2004). Overall, 122 alleles were identified, of which 52 the emergence of heteromorphic sex chromosomes from alleles were common to all the six races analysed, nine homomorphic predecessors can be due to many pheno- were unique to one race, and 70 were common to at least mena, such as the accumulation of deleterious mutations, two races. D. nasuta had 96 alleles, D. albomicans had absence of recombination in males, and accumulation of 93, and cytorace 1 and cytorace 2 had 92 alleles, while cy- transposable elements. In nature, these phenomena are torace 3 and cytorace 4 had 83 and 88 alleles, respec- believed to occur over vast periods of time and it is diffi- tively. A comparison between the isozyme profiles of the cult to study natural populations in the process of the parents, D. nasuta and D. albomicans, revealed that 16 emergence of heteromorphic sex chromosomes, leaving alleles from D. nasuta are not found in D. albomicans only the possibility of comparative studies of sex chromo- and 13 alleles from D. albomicans are not found in somes of different ages. For example, studies have com- D. nasuta. Thus these 29 alleles could, in principle, be pared the structure of the Y chromosome of D. americana used as diagnostic markers to trace the pattern of intro- americana (a few hundred years old) and D. miranda (two gression in the stabilized hybrids. But, of the 16 alleles found million years old) in an attempt to illustrate the inter- in D. nasuta and not in D. albomicans, those for 2Est1.61 mediate stages with different levels of degeneration in and G6PD1.20 were unique to D. nasuta, while of 13 alle- the evolution of dimorphic chromosomes (Charlesworth les found in D. albomicans and not in D. nasuta, four were et al. 1997; Steinemann and Steinemann 1997). In this unique to D. n. albomicans, namely those for 2Est1.3, context, the case of the neo-sex chromosome of cytorace Acph1.05, XDH1.06 and a-GPD0.91. Thus, only 23 alleles were 1 is unique in that one of the autosomes of the parental finally employed to examine the introgression pattern race is inherited in a manner similar to that of a classical of parental alleles among the cytoraces. Of the 16 alleles Y chromosome. The age of this neo-Y chromosome, which noticed in D. nasuta, 14 alleles were found to have

Journal of Genetics, Vol. 82, No. 3, December 2003 171 H. A. Ranganath and S. Aruna

Figure 5. Chromosomal phylogeny of the neo-Y chromosome of cytorace 1. Hybridization between males of D. nasuta and females of D. albomicans has led to the evolution of the karyotype of cytorace 1 which has 2n = 7 in males and 2n = 6 in females. Of the seven chromosomes of males, the acrocentric chromosome 3, an autosome of the nasuta par- ent, is now restricted only to the male genome of cytorace 1 (chromosome with a *). The female produces only one type of gamete while males can produce six types of sperms, two with normal haploid quota of chromosomes and four that are aneuploids. But anueploid adults are not recorded. Therefore generation after generation males with 2n = 7 and females with 2n = 6 are produced and the acrocentric chromosome is found only in males. This chromosome is labelled as neo-Y chromosome.

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introgressed into the genomes of cytoraces with different D. nasuta and D. albomicans, though separated about frequencies. However, not all 14 alleles were common to 500,000 years ago (Chang and Ayala 1989; Chang et al. all cytoraces; cytorace 1 had eight of the 14 alleles while 1989), still cluster together, while the cytoraces, which cytorace 2, cytorace 3 and cytorace 4 had 10, eight and are only 350–500 generations old, form another cluster, nine alleles, respectively. Of the nine alleles of D. albo- underscoring the major role of hybridization in generat- micans, six were represented in cytorace 1, and eight, five ing novel genetic variation. and six alleles were found to have introgressed into the genomes of cytorace 2, cytorace 3 and cytorace 4, respec- tively. If this comparison gave a measure of the extent of Incipient premating isolation introgression of parental genomes, then the presence of novel alleles in the introgressed cytoraces revealed an- In Drosophila sexual behaviour plays an important role other aspect of hybridization. Thirteen alleles were found in establishment of reproductive isolation between popu- to be novel to the introgressed systems, the ‘hybrizymes’. lations (Spieth 1968). Sexual behaviour includes a sequ- Of these, three alleles, namely those for 1Est0, 2Est1.54 and ence of events of male courtship attempts and female XDH0.98, are unique to cytorace 4 (frequency 4.8%), cyto- responsive reaction, and even a slight deviation from the race 2 (3.7%) and cytorace 1 (12.7%), respectively. Ten specific sexual behaviour can affect reproductive chances novel alleles were found in four cytoraces. The allele for and fitness. However, in an introgressed system variations Aph0.97 was noticed in all four cytoraces analysed, with in reproductive behaviour are forced into the hybrids. an overall frequency of 26.6% for cytoraces. These hybri- Thus the occurrence of hybridization between races / spe- zymes may be recombined products of either the genes cies constitutes a challenge to which they have to respond inherited from both the parents, or the products of genes either by developing or strengthening isolating mecha- obtained through recombination, or the reflection of gene nisms (Dobzhansky 1970), or by weakening isolation bar- expression in a coadapted hybrid genetic background. riers, thereby making the interacting races / species more Genetic distance estimates (Nei 1972) obtained from similar (Mayr 1963). the frequency of the 122 alleles among the six races ran- A detailed scrutiny of the mating behaviour among six ged from 0.091 to 0.219, with the greatest distance bet- races of the nasuta–albomicans complex showed the pre- ween cytorace 1 and cytorace 3, and the least distance sence of 24 different courtship elements (M. C. Shilpa between cytorace 1 and D. albomicans. Finally, the den- and H. A. Ranganath, unpublished data). Of these compo- drogram based on genetic distance obtained by combined nents of courtship, 15 were male specific, six were female results of 11 isozymes using UPGMA (figure 6) indicates specific, and three were due to both the sexes. The male- two clusters. In one clade, cytorace 1 and D. albomicans specific elements are anterior approach, posterior appro- cluster with D. nasuta, whereas in the other clade cyto- ach, transverse approach, tapping, anterior circle, posterior race 2 and cytorace 3 cluster with cytorace 4. The parents, circle, left circle, right circle, full circle, wing extension, wing rippling, wing flicking, wing scissoring, wing wav- ing, and attempt to copulate. The female-specific courtship elements are decamping, ignoring, kicking, wing flut- tering, wing flicking and wing spreading. Courtship lat- ency, courtship duration and copulation duration are due to both sexes involved. A study of these courtship elements revealed considerable divergence among the races stud- ied (parental species and four cytoraces). Members of cytorace 1 and cytorace 4 possess all the 24 courtship elements, whereas D. nasuta, cytorace 2 and cytorace 3 have 23 elements, and D. albomicans has only 21 ele- ments. Of the 24 courtship elements noticed, 20 were common to all the six races. Male anterior approach was not seen in males of cytorace 2 and cytorace 3, male wing waving was not seen in males of D. nasuta and D. al- bomicans, and female wing fluttering and female wing flicking were absent in females of D. albomicans. Thus, these cytogenetically closely related races of the nasuta– albomicans hybrid zone have shown symptoms of quanti-

tative divergence for a few components of mating behav- Figure 6. Dendrogram based on 11 isozymes for a few mem- iour. The dendrogram based on these values is shown in bers of the nasuta–albomicans complex. figure 7.

Journal of Genetics, Vol. 82, No. 3, December 2003 173 H. A. Ranganath and S. Aruna

Incipient prezygotic isolation from these experiments, described in detail by Tanuja et al. The widely accepted biological species concept empha- (2001a), is that these closely related races of the nasuta– sizes the importance of reproductive isolation to the pro- albomicans complex show initiation of the earliest stages cess of speciation. Therefore any analysis of speciation of prezygotic isolation, manifested as a tendency for mat- or raciation analysis should include a systematic study of ings to be initiated early and to last for a longer duration traits involved in prezygotic and postzygotic isolation. among homogamic rather than heterogamic matings. This The extent of sexual isolation decides the status of rela- is further substantiated in male-choice, female-choice and tionship between populations. Populations that appear to multiple-choice experiments. In these situations, the mat- be evolving either prezygotic or postzygotic reproductive ing was far from random. Males of D. albomicans, cy- isolation provide rare opportunities to follow the events torace 1 and cytorace 4 in male-choice experiments, females in acquisition or emergence, or both, of characters that pro- of cytorace 1 and cytorace 2 in female-choice experi- mote divergence between populations, facilitating repro- ments, and males and females of D. nasuta, D. albomicans, ductive isolation. Rather than looking at finished products cytorace 1 and cytorace 4 against males and females of of speciation to trace the evolutionary process involved in cytorace 2 in multiple-choice experiments had signifi- speciation, if one tries to unravel the events and proces- cantly more homogamic matings than expected (Tanuja ses of genetics of speciation in recently derived forms, et al. 2001b). Thus, by taking cognizance of divergence then one gets an opportunity to understand and provide in the components of mating behaviour and the findings direct evidence for the mechanism and stages in the deve- of mating-choice experiments, one can see the initiation lopment of reproductive isolation. In this respect, the of the earliest stages in the acquisition of reproductive nasuta–albomicans hybrid zone, where variations in court- isolation among the members of the nasuta–albomicans ship elements have been recorded, provides an excellent complex of Drosophila. We hope to continue to study the model system for investigating the reproductive-isolation process of the development of reproductive isolation in status of the members in the complex. this model system as it evolves further. The performance of six races of the nasuta–albomi- cans complex during homogamic matings is summed up Concluding remarks as follows: for mating latency cytorace 2 > D. nasuta > cytorace 3 > cytorace 4 > cytorace 1 > D. albomicans, which Stebbins (1973) has opined that mutation can never pro- gives a clear indication of interracial divergence. Simi- vide enough variability to allow major evolutionary advan- larly, in heterogamic matings under no-choice situation ces to take place. Genetic recombination can be a major males of D. nasuta had the least mating latency and the source of such variability, especially when accomplished longest copulation duration, while males of cytorace 2 by mass hybridization between populations with different showed exactly the opposite trend. On the other hand, adaptive norms. Templeton (1981) has argued that hybri- females of D. albomicans showed minimum mating lat- dization followed by production of unstable hybrids, in- ency with prolonged period of copulation. The message breeding and hybrid breakdown may result in a form of natural selection favouring F2 and those later generations that have better viability and fertility. This may result in the formation of a new rare recombinant class of geno- type. Experimental animal studies on hybridization do not usually extend beyond a few generations (Shaw and Wilkinson 1980; Scribner 1993; Price and Boake 1995). On the other hand, long-term effects should be consi- dered for a better understanding of the evolutionary consequences of hybridization (Rieseberg and Carney 1998). Interracial hybridization between D. nasuta and D. albo- micans, and maintenance of hybrid populations for over 600 generations, have resulted in an excellent model system providing evidence to substantiate many hypothe- ses laid out by Stebbins (1973), Templeton (1981) and Rieseberg and Carney (1998). Introgression of genomes of D. nasuta and D. albomicans and transgressive segre-

gation of parental features in different patterns in diffe- Figure 7. Dendrogram based on mating behaviour compo- rent lineages have given rise to different genetic systems nents for a few members of the nasuta–albomicans complex. called cytoraces. These cytoraces are passing through the

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