Structural rearrangements of chromosomes have played a decisive role in the karyotypic evolution of species. It is also known that inversions, translocations, fusions, fissions, heterochromatin variations and other chromosomal changes occur as transient events in natural populations. Herein we report the occurrence of a rare event of centric fission of a metacentric chromosome in a laboratory population of Drosophila, called Cytorace 1. This centric fission has been fixed in a sub-population of Cytorace 1, resulting in a new chromosomal lineage called Fissioncytorace-1.

Research on hybrid zones is a relatively recent development in evolutionary biology. Hybrid zones occur in nature when genetically distinct groups of individuals meet and mate, resulting in at least some offspring of mixed ancestry (Harrison 1990; Barton and Hewitt 1989). The evolutionary significance of hybrid zones, particularly in animals, has been studied in different groups of organisms such as grasshoppers (Hewitt 1993), butterflies (Mallet 1993), crickets (Rand and Harrison 1989), toads (Szymura 1993), birds (Moore 1977) and mammals (Searle 1988, 1991; Patton 1993). This has lead to the realization of its importance which is reflected in treating hybrid zones as "windows on the evolutionary process" (Harrison 1990) and as "natural laboratories" (Hewitt 1988; Barton and Hewitt 1989).

Drosophila nasuta nasuta with 2n = 8 (figure 1a, b) has a pair of metacentrics, representing chromosome 2, a pair of acrocentric chromosomes 3, an acrocentric X, a submetacentric Y and a pair of dot chromosomes. D. n. albomicans with 2n = 6 (figure 1c, d) has two pairs of metacentrics and a pair of long dot chromosomes. Of the two pairs of metacentrics, the smaller one represents chromosome 2. The larger metacentric, believed to be a product of centric fusion between autosome 3 and the sex chromosomes of a nasuta-like parent, is referred to as X3 and Y3 chromosomes (Ranganath and Hagele 1981). D. n. nasuta and D. n. albomicans are morphologically indistinguishable members of the nasuta subgroup of the immigrans species group of Drosophila. They are allopatric in distribution (Ranganath and Ramachandra 1987). These karyotypically different sibling forms can be crossed in the laboratory and hybrid progeny produced that can be maintained for many generations. The F₁’s of the cross between D. n. nasuta and D. n. albomicans have 2n = 7, while the F₂’s and subsequent hybrid individuals show various karyotypic combinations, referred to as ‘karyotypic mosaicism’ (Rajasekarasetty et al 1979; Ramachandra and Ranganath 1986). In many of the crosses, the karyotypic mosaicism disappears due to fixation of karyotypes, the composition of which differs between crosses. Such recombinant populations, each with a new and a different karyotypic combination are called Cytoraces. Sixteen such Cytoraces have evolved between 1985–1996, differing by the contribution of D. n. nasuta and of D. n. albomicans chromosomes (Ramachandra and Ranganath 1996).

The assemblage containing D. n. nasuta, D. n. albomicans and the 16 Cytoraces is recognized as a new evolving cluster and referred to as the ‘nasuta–albomicans complex’ (Ramachandra and Ranganath 1996). Tanuja et al (1998) treated this complex as an artificial hybrid zone of Drosophila with allo-sympatric populations.

We herein report the emergence of a new karyotypic lineage within this lineage through a very rare event of centric fission.

2. Materials and methods

2.1 Drosophila stocks

(i) D. n. nasuta (2n = 8) (Coorg, south India), (ii) D. n. albomicans (2n = 6) (Okinawa, University of Texas collections, 3045.11) and (iii) Cytorace 1 (males, 2n = 7; females, 2n = 6) (Ramachandra and Ranganath 1986), produced by interracial hybridization between the two races. The metaphase chromosomes from neural ganglion were prepared following the method of Ramachandra and Ranganath (1986). The C-banding technique of Sumner (1972) and the Q-banding procedure adopted by Ranganath et al (1982) were followed.

3. Results

Cytorace 1 is the product of hybridization between the males of D. n. nasuta and the females of D. n. albomicans (Ramachandra and Ranganath 1986). The females of Cytorace 1 have 2n = 6 (2ⁿ 2^a, X3^a X3^a, 4ⁿ 4ⁿ) while the males have 2n = 7 (2ⁿ 2^a, Yⁿ X3^a 3ⁿ, 4ⁿ 4ⁿ) (the superscripts represent the parent from which the chromosome has been inherited: n = nasuta; a = albomicans, figure 2).

The uniqueness of Cytorace 1 is that although different diploid numbers are present in males and females, it breeds true for these karyotypes. Females of Cytorace 1 produce only one type of egg with 2, X3^a, 4ⁿ chromosomes, while the males can produce 6 types of sperm. Of these, only two carry haploid sets of chromosomes, namely 2, X3^a, 4ⁿ and 2, Yⁿ, 3ⁿ, 4ⁿ, while the others are aneuploid for either chromosome 3 or the sex chromosomes. The eggs with 2, X3^a, 4ⁿ chromosomes, if fertilized by a sperm with the 2, X3^a, 4ⁿ complement, result in a female with 2n = 6 (2 2, X3^a X3^a, 4ⁿ 4ⁿ); on the other hand, if this egg is fertilized by a sperm with 2, Yⁿ, 3ⁿ, 4ⁿ, it results in a male with 2n = 7 (2 2, Yⁿ X3^a 3ⁿ, 4ⁿ 4ⁿ). Since 1985, the karyotype of Cytorace 1 has been regularly screened, and except for the very rare occurrence of one or two aneuploids, all males and females showed the 2n = 7 and 2n = 6 karyotypes respectively as described above.

During 1996–1998, in one of the replicate cultures of Cytorace 1, in addition to the expected 2n = 6 female and 2n = 7 male karyotypes, five new karyotypic combinations were recorded (figure 3), the frequency of which is provided in table 1. Among these new karyotypes, two are aneuploids (figure 3), which occurred in very low frequency and only during 1996. The other three types are euploid, more frequent and have been consistently recorded since 1996. Of these, one was a male with 2n = 8 (figure 3) with a pair of metacentric chromosomes (chromosome 2), a pair of acrocentric chromosomes 3, a submetacentric X and a Y chromosome, along with a pair of dot chromosomes. The other two new karyotypes were of females. One had 2n = 8 (figure 3) with a pair of metacentric chromosomes 2, a pair of acrocentric chromosomes 3, a pair of submetacentric X chromosomes and a pair of dots. The other female had 2n = 7 (figure 3), with a pair of chromosomes 2, one chromosome 3, one submetacentric X chromosome, one metacentric X3 chromosome and a pair of dots. The submetacentric nature of the new X chromosome is resolved by looking at its anaphase configuration (figure 6E, F). Thus, this culture of Cytorace 1 is polymorphic with two expected and three new karyotypes. The interesting observation is that the frequency of the expected female karyotype with 2n = 6, gradually declined to 4% while each of the four other types was represented with a frequency above 20% (table 1).

4. Discussion

The segregation pattern of chromosomes found in Cytorace 1 males with 2n = 7 and females with 2n = 6 cannot produce these new karyotypes, as the latter have
a submetacentric X chromosome which is absent in Cytorace 1.

What is the source of this ‘submetacentric X’ chromosome? One possibility is that this culture was contaminated with individuals of D. n. nasuta which have unfused X chromosomes. To resolve this, a cross was made between D. n. nasuta males and Cytorace 1 females with 2n = 8, to provide F₁’s in which a direct comparison between the X chromosomes could be made (figure 4). A marked difference is seen between these chromosomes, wherein the acrocentric X chromosome of nasuta is much shorter with significantly less heterochromatin than that of the "new submetacentric X" chromosome. Therefore, the appearance of the latter in Cytorace 1 is not due to contamination with D. n. nasuta flies.

The other possibility involves a centric fission of the metacentric X3 chromosome present in Cytorace 1 resulting in a submetacentric X chromosome and an acrocentric chromosome 3 (figure 5). Centric fission would have been followed by addition of heterochromatin in the form of a short arm to produce a submetacentric X chromosome. The original metacentric ‘X3’ chromosome was inherited from D. n. albomicans. The analysis of the karyotypic phylogeny of the nasuta subgroup has shown that the X3 and the Y3 metacentric chromosomes of

D. n. albomicans have evolved through centric fusion between the acrocentric chromosomes 3 and the sex chromosomes (X and Y) of a D. n. nasuta like karyotype (Ranganath and Hagele 1981). The new X chromosome derived through fission has a nearly 59% content of heterochromatin whereas that of D. n. nasuta contains about 47% (figure 4). Since these new karyotypes with submetacentric X chromosomes appeared during the early part of 1996, the fission in one of the X3 metacentric chromosomes of either a male or a female of Cytorace 1 might have occurred during 1995. The products of this fission were retained in the population of Cytorace 1, sometimes at a frequency higher than that of others (table 1).

During 1998, about 200 isofemale lines from this polymorphic culture of Cytorace 1 were established. One of the isofemale lines thus established had males and females with 2n = 8 only (figure 6), indicating that the fixation of the new submetacentric X chromosome derived from the fission of the X3 chromosome had occurred. This new chromosomal strain is named "Fissioncytorace-1" and its karyotypic phylogeny is depicted in figure 7. With the submetacentric X chromosome, the karyotype of Fissioncytorace-1 (2n = 8) differs from that of D. n. nasuta (2n = 8) which has acrocentric X chromosomes. However, Lakhotia and Roy (1981) have reported the occurrence of a submetacentric X chromosome in the Varanasi strain of D. n. nasuta. But for this rare report, studies made by our group since 1975 as well as those of Professor O Kitagawa’s group in Japan (Wakahama and Kitagawa 1980; Wakahama et al 1983) and of Wilson et al (1969), have shown only acrocentric X chromosomes in D. n. nasuta.

The present finding is unique in that a fission not only has occurred but also that the products of this fission have been fixed in a new chromosomal lineage of Drosophila. To our knowledge, this is a first report for the genus Drosophila. Further, the X3 chromosome of D. n. albomicans, an earlier product of centric fusion, and the X chromosomes, the present products of centric fission, are suitable candidates to probe the status of centromeres. Similarly, Imai et al (1994) have shown the importance of chromosomal fission during the karyotypic evolution of the Myrmecia pilosula species complex of ants. The cytogenetic relationship between Fissioncytorace-1 and its parent namely the Cytorace 1 as well as with its grandparents namely D. n. nasuta and D. n. albomicans is being investigated.

Acknowledgements

We thank the Department of Biotechnology and the Department of Science and Technology, New Delhi, for financial assistance. We thank the reviewers profusely for their constructive comments and criticisms.

MS received 1 March 1999; accepted 23 August 1999