Metaphase Chromosome Configuration of the Immigrans Species Group of Drosophila

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 belonging 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 subgroups 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, immigrans 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 karyotype 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, quadrilineata 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 immigrans 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. D. curviceps showed 2R + 1V + 1D type and the Y chromosome was J-shaped as reported by Okada and Kurokawa (1957). D. formosana showed 3R + 1V, resulting from the rod-shape of Chromosome 4. This species exhibited a constriction in the middle part of Chromosome 4. The Y was rod-shaped and had clear secondary constrictions in both edges. The karyotype of D. ruberrima was 2R+ 1V + 1D type. The Y chromosome of this species was rod-shaped and clearly shorter than the X. In the D. nasuta subgroup, the karyotypes of 10 species and subspecies were investigated. Our observations generally agreed with the descriptions of Wilson et al. (1969), except for the following minor differences: 1) Chromo- some 4 of D, sulfurigaster bilimbata was a long dot. 2) Chromosome 4 of D. kepulauana was relatively longer than that of D. s, albostrigata. 3) In the Y chromosome of D. pulaua, a clear secondary constriction was seen. 4) The secondary constriction was also seen in Chromosome 4 of D. albomicans. Metaphase chromosomes of the immigrans species group 321

As the karyotype of one more species in this subgroup, D. s, neonasuta, Nirmala and Krishnamurthy (1973) reported 2n = 8, 2R + 1V + 1D type. Our observation well agreed with their description. The Y chromosomes of D. kohkoa and the D. sulfurigaster complex were polymorphic, showing some intraspecific variations previously pointed out by Wilson et al. (1969). In the D. quadrilineata subgroup, D. annul ipes presented the minimum chromosome number, 2n = 6, 2V + 1R. There was no dot chromosome, the two autosomal chromosomes were V-shaped, and the Y chromosome was a short rod. Lin et al. (1974) reported a different configuration for this species. According to them, the Y chromosome of this species was a metacentric and the shortest in the complement, the X was a metacentric and the longest in the complement, Pair II was submetacentric, and Pair III was telocentric with a secondary constriction. From these two quite different observations, it appears that there are two kinds of karyotypes of D. annulipes as in D. immigrans. In the D. lineosa subgroup, Bock (1966) and Bock and Baimai (1967) studied the karyotypes of D. argentostriata and D, silvistriata and reported that the former exhibited the most primitive chromosome type of the genus Drosophila, 5R + 1D, while the latter was 3R + 1V + 1D. Our observations of these two species agreed with theirs, but we found a clear secondary constriction in the Y chromosome of the former species. The karyotype of D, hypocausta belonging to the D, hypocausta subgroup was 2R+1V+1D, agreeing with the report of Pipkin (1956). The Y chromosome was a short rod. In the immigrans species group, generally speaking, species belonging to subgroups I, II and IV which we examined had a rod-shaped X chromosome and autosomes included a V-shaped pair, a pair of long rods and a pair of dots or short rods. However, the Y chromosome exhibited some variations, rod- shaped and a little shorter than the X in size, J-shaped, or small V-shaped. In the 2n = 6 karyotypes of D, albomicans and D. neohypocausta, the former resulted from fusion between the sex chromosomes and Chromosome 3 and the latter from the absence of the dot chromosomes. Most species of the immigrans species group presented karyotypes of 2n = 8, 2R + 1V + 1D (or short rod) which we hypothesize is the basic type of this species group. In the genus Drosophila, it has been accepted that the most primitive karyotype was five pairs of R's and a pair of dot chromosomes (Patterson and Stone 1952; Stone 1962). If this assumption is correct, in the immigrans species group, the V-shaped chromosome originated from fusion between two autosomal rods. The euchromatic regions of the other two autosomal rods either organized a double length rod by tandem fusion between them and/or the double length rod arose by a pericentric inversion of a two-element V or 322 K. I. WAKAHAMA et at. by a total translocation of one rod to the end of another (Wilson et at. 1969). If the basic karyotype of the immigrans species group was 2R + 1V + 1D (or a small rod), the karyotypes of D. annutipes belonging to the D. quadritineata subgroup and of D, argentostriata and D, sitvistriata of the D. tineosa subgroup were differed significantly. One characteristic feature of the species belonging to the immigrans species group, except for the species of D, hypocausta subgroup, was spinules on the inner side of the first femur. The above mentioned three species also exhibited this character, but from the view point of the difference in the karyotype, they seem remote from the species of the immigrans species group. Perhaps this characteristic of the fore-leg has a polyphyletic origin. In the salivary gland chromosomes of this species group, we have observed four long arms and one dot. Among the four long arms, one was the X chromosome and one of the other three arms was a double length. Therefore, the number of elements of the salivary chromosomes in this group was the same as those of D, viritis (5R + 1D) or of D. metanogaster (1R + 2V + 1D). Lin et at. (1973) analyzed the karyotypes of nine species of the immigrans species group collected in Taiwan and reported their phylogenetic relationships. In thier study, D, tongpua of the D, immigrans subgroup and D. xanthogaster of the D, hypocausta subgroup, neither of which we examined were included. The karyotype of the former was 3R + 1V containing dot chromosomes as long rods with additional heterochromatin and that of the latter was 2R + 1V + 1D. The X chromosome was a metacentric and Chromo- some 4 was a dot. A V-shaped X was also seen in D, quadritineata during our examination. D. xanthogaster was placed in the D. hypocausta subgroup (Duda 1924). Characteristic features of this subgroup are peculiar dimorphism in body color and absent or poorly developed spinules on the inner side of the first femur (Wilson et at. 1969). This species did not show such characteristic features (Lin et at. 1973), and Lin et at, stated that D. xanthogaster was not belong to the D. hypocausta subgroup. We agree with Lin's opinion. D. annutipes and D, neohypocausta, which lack the dot chromosome and distinct karyotypes, D. quadritineata, with the specific V-shaped X chromosome, and D. argentostriata and D. sitvistriata, with distinct karyotypes, might be remote from the other species of the immigrans group. When considering the D. nasuta subgroup, Ranganath and Hagele (1981) proposed the orthoselection theory. According to them, one metacentric chromosome was produced by one centric fusion between two primitive rods and a second centric fusion between other original elements yielded the second pair of metacentrics. These metacentric chromosomes were proposed to have subsequently undergone a pericentric inversion to form double length acrocentric chromosomes. A third centric fusion between these double length rods and the sex chromosomes produced the 2n = 6 karyotype of D. atbomicans. Metaphase chromosomes of the immigrans species group 323

Among 513 species and subspecies of the genus Drosophila examined so far, 2n = 6 is the minimum number which occur in about 50 species belonging to many species group. According to reports of karyotype evolution in the Primates, f usions repeatedly occurred in some branches of the genealogical tree, while in the other branches, fissions happened frequently and many kinds of patterns for evolution appeared branch by branch (Dutrillaux 1979). Although we are not yet in a position to discuss the exact process of speciation in the immigrans species group and we cannot determine whether f usions or fissions were more important during their evolution, it is possible to say that in the D. nasuta subgroup, chromosomal reconstitutions occurring by f usions were the main factors in their evolutionary process, as pointed by Ranganath and Hagele (1981). If this is true, D, albomicans is the most advanced species in this subgroup. The Y chromosome is one which shows intraspecific variations frequently, as pointed out by Baimai (1969) in D. birchii. Among related species, in general, intraspecific variations are often seen in chromosomes which contain a lot of heterochromatin. Wilson et al. (1969), reported three types of the Y chromosome, V-, J- and rod-shaped, in D, kohkoa and the D, sulfurigaster complex. According to them, D. s. albostrigata collected in Sarawak, Malaysia populations exhibited two types of J- and rod-shaped Y chromosomes and there was no V-shaped Y, while, in the Thailand populations, the V-shaped Y chromosome appeared in 23 lines, and the J-shaped in 16 lines; no rod-shaped Y was found. Further, in the Cambodian populations, J- and V-shaped Ys were found in almost equal frequencies. The Y chromosome in this species was polymorphic. Polymorphic features in some chromosome pairs have also been detected in black rat, Rattus rattus, by Yoshida and his collaborators (cf. Yoshida et al. 1971). They examined the karyotype of several species of black rats collected in different Asian and Oceanian localities with special emphasis on the relationships between the chromosome polymorphism and differentiation of the subspecies. Chromosomes of the number 1 pair of black rats were polymorphic, being either acrocentric (A) or subtelocentric (S). Frequencies of three types of the number 1 pair (A/A, A/S and S/S) differed locality by locality. The acrocentric number 1 chromosome was the original type of this chromosome pair. The subtelocentric one was formed by a pericentric inversion. Further, reduction of chromosome number (from 42 to 38) occurred by the Robert- sonian f usions of four acrocentric pairs (Nos. 4 and 7, and Nos. 11 and 12). Although we have observed polymorphism only in the Y chromosome, these phenomena seen in black rats are very similar to those in drosophilid species. The Robertsonian fusion in black rats compares to the fusion which might have occurred between the double length chromosomes and the sex chromo- 324 K. I. WAKAHAMA et al. somes and has produced D. al bomicans from species with the 2R + 1V + 1D karyotype. Although we cannot determine the species from which D, albomicans originated, it seems that D, nasuta is the most plausible one, because of the morphological similarities, the shape of the silvery marks on the male frons and darkness of the bands on the mesopleuron, good fertility in hybridization experiments, and geographical distribution of the two species (Kita- gawa et al. 1982). And further, Matsuzaki and Kitagawa (1982) reported strong commonality in the banding patterns in the salivary gland chromosomes of the two species. D. nasuta has dispersed further to India, Sri Lanka, Seychelles, Mauritius, Reunion, Madagascar and the southern countries of Africa without changing karyotype (Wakahama and Kitagawa 1972; Kitagawa et al. 1982) . Thailand and its adjacent areas seem to be the cradle of the black rat (cf. Southern 1964). Judging from the geographical distribution of the species of the immigrans group, especially those of the D. nasuta subgroup, it appears that the origin of the immigrans species group was in Borneo, southeast Asia, and that it has since dispersed to Hawaii and westward to Africa, with further speciation along the way (Wilson et al. 1969; Throckmorton 1975; Kitagawa et al. 1982). Among them, only D. immigrans has developed into a cosmo- politan species, distributed all over the world. The most peculiar phenomenon among the karyotype variations in the immigrans species group was the supernumerary chromosomes were found in D. albomicans (Hatsumi and Kitagawa 1980). The supernumerary chromosomes frequently observed in plants and called B chromosome, were reported independently for the first time in Drosophila by Clyde (1980) and Hatsumi and Kitagawa (1980). Previously, no supernumerary chromosomes had been reported (White 1978). According to Hatsumi (unpublished data), these B chromosomes were found in the Chiangmai population, Thailand, in collections of 1977 and 1981, and might be maintained consistently in this population. Recently, new cytological techniques, including C-, Q- and R-staining methods have been introduced for chromosome analyses in many kinds of organisms. It is possible to identify one chromosome from other morphological- ly similar chromosomes. In Drosophila species, these methods have been utilized and some experiments have been reported (Lee and Collins 1977; Tonomura and Tobari 1978; Ranganath and Hagele 1982). Henceforth, if these new techniques are applied for chromosomal analyses in Drosophila, chromosomal reconstitutions will be determined more correctly, particularly when combined with analyses of the salivary gland chromosomes. Analyses of chromosomes of members of the D. nasuta subgroup by differential staining methods are now in progress in our laboratories. Since our projects started several years ago, Professor Marshal R. Wheeler, University of Texas, has been most kind, giving valuable advice and criticism. D. pallidifrons and other Metaphase chromosomes of the immigrans species group 325 species collected in the southern Pacific were donated by him. We greatly appreciated his kind- ness. Professor Lynn H. Throckmorton, University of Chicago, was also a good adviser and we owed him much. We thank Emeritus Professor Toyohi Okada, Tokyo Metropolitan University, and Dr. Fei-Jan Lin, Academia Sinica, Taiwan, for their kind cooperation in the field collections. This work supported mainly by the funds of the Overseas Scientific Expedition in 1971 (No. 7114), 1979 (No. 40419), 1980 (No. 504344) and 1981 (No. 56041049) of the Ministry of Education, Science and Culture of Japan.

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