Jpn. J. Genet. (1983) 57, pp. 315-326 Metaphase chromosome configuration of the immigrans species group of Drosophila BY Ken-Ichi WAKAHAMA*,Tamiko SHINOHARA**,Machiko HATsUMI***, Satoko UCHIDA**** and Osamu KITAGAWA*** *Department of Biology , Shimane University, **Department of Human Cytogenetics, Japan Red Cross Medical Center, ***Department of Biology, Tokyo Metropolitan University and ****Ono Pharmaceutical Co. Ltd. (Received February 11, 1983) ABSTRACT Metaphase chromosome configurations of 23 species and subspecies belong- ing to five subgroups of the immigrans species group of Drosophila were examined by the Giemsa staining method, from the point of view of the phylogenetic relationships in this species group. New to science are the descriptions of the karyotypes of five species, Taxon-C, Taxon-F, D, nivei f rons,, D. quadrilineata and D, neohypocausta which are 2n=8, 2R+1V+1D; 2n=8, 2R+1V+1D; 2n=8, 2R+1V+1D; 2n =12, 4R+ 1V+ 1D; and 2n = 6, 2R+ 1V, respectively. The karyotypes of the other species were also examined and compared with earlier descriptions. Some of our observations agreed with earlier ones but the remainder showed some major or minor differences from the previous reports. The basic karyotype of the species belonging to the immigrans species group was 2n=8, 2R+1V+1D (or short rod). The karyotypes of D. annul ipes and D. quadrilineata of the D. quadrilineata subgroup, of D, argentostriata and D, silvistriata of the D, lineosa subgroup and of D, neohypocausta of the D. hypocausta subgroup differed fundamentally from the basic type of the immigrans species group. These five species might be somewhat remote from the other species of the immigrans group. Some species exhibited intraspecific variations among the forms of the Y chromosome and Chromosome 4, with additional heterochromatin. In the D. nasuta subgroup, it seemed that fusions were important factors in their evolution. D, albomicans is the most advanced species, and from considerations of morphological similarities, results of hybridization tests, karyotypes, and geographical distribution, D, nasuta is the most plausible species from which D, albomicans originated. D, albomicans collected from the Chiangmai population in Thailand had supernumerary chromosomes, the most peculiar phenomenon among the karyotype variations of the immigrans species group. These chromosomes may be maintained consistently in this population. 1. INTRODUCTION Since Patterson and Stone (1952) has been reviewed the configurations of metaphase chromosomes of 215 different species in the genus Drosophila, 316 K. I. WAKAHAMA et al. many investigators have reported the karyotype surveys, and metaphase karyotypes of more than 500 species and subspecies have been analyzed up to the present (Clayton and Wheeler 1975). Generally, fusion, pericentric and paracentric inversions and translocation are well known as the factors to cause the structural changes of chromosomes and all of these rearrangements lead to changes of the configurations of the chromosome set, the reconstitution of karyotype. Although structural variations within a species, such as inversion, are commonly observed in the salivary gland chromosomes of most species of Drosophila, it was thought that the Drosophila karyotype was comparatively stable from species to species. However, intraspecific variations of karyotype have been often reported for some chromosomes such as the Y chromosome, the shortest autosomal elements (usually dots) and the one arm of the V- shaped (metacentric) X chromosome which are largely heterochromatic (Ward 1949; Baimai 1969; Wilson et al. 1969; Tonomura and Tobari 1978). It is very important to examine the karyotypes of all species belonging to one species group to study the genetic mechanisms of speciation, since, in all organisms, related species show common karyotypes. It is well understood chromosomal reconstitutions are very effective and significant in establishing the fitness of each species. In this study, we present the karyotypes of the immigrans species group of Drosophila and discuss the phyletic relationships within this group. 2. MATERIALSAND METHODS Almost all the specimens used in this experiment were collected by the Overseas Scientific Research of the Ministry of Education, Science and Culture of Japan in 1971, 1979 and 1981. In total, 38 strains belonging to 23 species and subspecies of 5 subgroups of the immigrans species group were used (Table 1). These specimens have been maintained in our laboratories on the usual cornmeal-molasses-agar medium under uncrowded conditions. The karyotypes were observed in larval ganglia cells. Preparations were made by the following method: 1) Third instar larvae were dissected in Ringer's solution and the ganglia were removed. The sex of individuals was distinguished by the size of the gonad. 2) A drop (ca. 0.02 ml) of colcemide solution (colcemide 1 mg J1 ml distilled water) was dropped into 2 ml of Ringer's solution and the ganglia were placed in it. 3) Ringer's solution was replaced by 2-3 ml of hypotonic 0.075 M KCI. The ganglia were transferred into this solution and left for 8 minutes. 4) The ganglia were then transferred into the 3: 1 Carnoy's solution and Metaphase chromosomes of the immigrans species group 317 Table 1, Species and strains belonging to the immigrans species group used in this experiment 318 K. I. WAKAHAMA et al. fixed for 30 minutes. 5) They were frayed on a glass slide which had been cooled and soaked in 50% ethanol in the refrigerator. 6) The slide was then dried over a gas-burner. 7) The dried slide was stained with fresh 10% Giemsa in 1/15 M phosphate buffer (pH 6.4) for 30-40 minutes, rinsed with tap water, and dried. 8) The slide was observed without a cover slip under Nikon or Olympus Optophoto Microscope, using 40 X or 100 X (oil immersion method was applied at this magnification) object lenses and 10 X occular lens. 9) Photographs of well spread chromosomes were taken. 3. RESULTS AND DISCUSSION According to Wilson et al. (1969), the immigrans species group contains about 70 "nominal" species, which they assign to five subgroups, I, the D. immigrans subgroup; II, the D, nasuta subgroup; III, the D. quadrilineta subgroup; IV, the D. lineosa subgroup and V, the D. hypocausta subgroup, plus a sixth group containing species of uncertain relationships. In the present study, we used 23 species and subspecies belonging to sub- groups I, II, III, IV and V. Results are shown in Fig. 1. Wilson et al. (1969) reviewed the karyotypes of 6 species and subspecies of the D, nasuta subgroup and reported that their basic type was 2n=8, two pairs of R's (2R, one is the sex chromosome and the other is the double length autosomal chromosome), one pair of V's (1V) and one pair of dot chromosomes (1D). This karyotype is also common in species belonging to the D. immigrans subgroup, but, in some cases, heterochromatin is added to the dot chromosome, making it a heavy heterochromatic rod, and there appeared to be three pairs of R's and one pair of V's. However, if we regard the long rod as the dot chromosome, the basic metaphase configuration of the species of the D, immi- grans subgroup becomes 2R + 1V + 1D. Results of the Observations: 1) In the D, immigrans subgroup, the karyotype of Taxon-C (Lin 1974, personal communication) is newly reported. This species showed the basic karyotype of 2n = 8, 2R + 1V + 1D. However, a secondary constriction was detected in Chromosome 4 and the Y chromosome was J-shaped (acrocentric) in this species. 2) In the D. nasuta subgroup, two species, D. niveifrons and Taxon-F, were newly examined karyologically. The male frons of D. niveifrons was entirely covered with a silvery white mark and could not be distinguished from D. nasuta, D, albomicans or D. kepulauana in this point. However, the karyo- type of this species clearly differed from those of the above three species. Metaphase chromosomes of the immigrans species group 319 Fig. 1. Diagrammatic representations of metaphase configurations o f the immigrans species group. Males are shown. 320 K. I. WAKAHAMA et al. Chromosome 4 was a long rod with added heterochromatin almost as large as the longer type of D, albomicans collected in Taiwan and Okinawa. The sex chromosomes separated from Chromosome 3 and the Y chromosome showed two secondary constrictions on both its edges like those of the Y chromosome of D. formosana. In the D. nasuta subgroup, all species showed the basic configuration of 2R + 1V + 1D (or 1 short rod), although D, albomicans uniquely showed 2n = 6, resulting from fusion between the sex chromosomes and Chromosome 3. 3) Previously, only D. hexastriata had been examined cytologically in the D. quadrilineata subgroup and its karyotype was reported as 2n = 8, 2R + 1V+1D (Tan et al. 1949). In this experiment, the karyotype of D, quadri- lineata was studied anew. It showed a quite different type, 2n=12, 4R+ 1V+1D. Among the immigrans species group, only the X chromosome of this species was V-shaped. The Y and the other autosomal chromosomes, except for the dot, were rod-shaped. 4) In the D, hypocausta subgroup, D, neohypocausta was newly investigated, showing 2n = 6, 2R + 1V. The X chromosome and Chromosomes 2 and 3 of this species were similar to those of D. hypocausta but no dot chromosome was observed. Six is the minimum chromosome number in the genus Drosophila, also found in D, albomicans and some others. In addition to these newly described species, 18 other species of the immi- grans group were also examined cytologically. Two karyotypes have been reported for D. immigrans (Patterson and Stone 1952). Our observations in this experiment agreed with their Type-I, 3R+ 1V, but the following points differed from their description: The Y chromosome was not small V-shaped, but J shaped and there was a secondary constriction in the long arm of this chromosome as that in the report of Le Calvez (1953) from the European collection.