Genetica78: 63-72, 1989 © KluwerAcademicPublishers, Dordrecht - Printedin the Netherlands 63 Taxonomic identification of Drosophila nasuta subgroup strains by glue protein analysis S. R. Ramesh 1 & W.-E. Kalisch 2 Institut fiir Genetik, Medizinische Fakultiit, Ruhr-Universitiit Bochum, Postfach 102148, D-4630 Bochum 1, Federal Republic of Germany 1Permanent address: Dept. of P.G. Studies & Research in Zoology, University of Mysore, Manasagangotri, Mysore-570 006, India 2To whom all correspondence may be addressed Received15.1.1988 Acceptedin revisedform 14.9.1988 Abstract Protein fractions of salivary glands were analyzed from 30 wildtype strains of eight species belonging to the Drosophila nasuta subgroup by SDS-polyacrylamide gel electrophoresis. The electrophoretic patterns indicated several prominent bands which could be shown to represent the major glue protein fractions. The glue protein fractions are species-specific as well as wildtype strain-specific. Wildtype strain specificities are characterized by variations of the species-specific patterns. The patterns of the different wildtypes, species, and hybrids were used for taxonomic identification within the nasuta subgroup, in which the females are morphologically indistinguishable and the males differ only by the markings of their frons. The hybrids provide evidence for gonosomal as well as autosomal linkage of individual genes coding for the major glue protein fractions. Introduction The nasuta subgroup of the immigrans species group of Drosophila has been studied considerably from the genetic and evolutionary points of view. In several cases the dose relationship of the members allows for the obtainment of fertile hybrid progeny (cf: Ramesh & Rajasekarasetty, 1980; Ramachandra & Ranganath, 1986). The Drosophila nasuta subgroup is, however, an assemblage of morphologically almost identical species. Females of this subgroup are morphologically indistinguishable, while the males have silvery markings on the frons, the extent of which varies (Nirmala & Krishnamurthy, 1972). Taxonomic identification of species obtained in the wild as well as those from population genetic studies have so far been based on cytological investigation (metaphase plates, polytene chromosomes), or hybridization studies (Ramachandra & Ranganath, 1986). In this paper we describe a simple procedure to identify different wildtype strains of the same species, different species, and interspecific hybrids through their major glue protein fractions. The technique is based on SDS-polyacrylamide gel electrophoresis of the salivary gland proteins. We discuss probable reasons for the wildtype strain-specific and species-specific patterns together with evolutionary aspects from data in the literature. Material and methods Stocks The Drosophila melanogaster wildtype (Berlin) strain has been used in our lab for about twenty years, the D. nasuta nasuta strain (Mysore I in Table 64 1) was caught in Coorg, Karnataka, India and has been used and maintained in the lab for the past ten years. The other Drosophila strains of the nasuta subgroup were obtained from the National Drosophila Species Resource Center, Bowling Green, Ohio, U.S.A. Further details regarding the different wildtype strains of the species used in the present investigation as well as kD-values of their major glue protein fractions are given in Table 1. Standard cornmeal food was used together with extra baker's yeast for the feeding of the larvae. Cultures were maintained at 22+1 °C. Table 1. List of Drosophila stocks used in the experiments. Nr a 15112-1781 15112-1781.1 15112-1781.2 15112-1781.6 15112-1751 15112-1751.1 15112-1751.2 15112-1751.4 15112-1751.5 15112-1761.1 15112-1771 15112-1771.1 15112-1771.2 15112-1771.3 15112-1771.4 15112-1801 15112-1831 15112-1831.1 15112-1831.2 15112-1831.3 15112-1831.4 15112-1821 15112-1821.1 15112-1821.5 15112-1821.6 15112-1821.7 15112-1811 15112-1811.1 15112-1811.3 15112-1811.5 15112-1811.7 Species D. melanogaster D. n. nasuta D . n . nasuta D. n. nasuta D. n. nasuta D. n. nasuta D. n. albomicans D. n. albomicans D. D. D. D. K. D. D. n. albomicans n. albomicans n. albomicans n. kepulauana kohkoa kohkoa kohkoa D. kohkoa D. kohkoa D. pulaua D. s. sulfurigaster D. s. sulfurigaster D . s . sulfurigaster D. s. sulfurigaster D. s. sulfurigaster D. D. D. D. D. s. s. s. s. s. bilimbata bilimbata bilimbata bilimbata bilimbata D. D. D. D. D. s. s. s. s. s. albostrigata albostrigata albostrigata albostrigata albostrigata Original collection locality of wildtype strains b Berlin, Germany Coorg, India (Mysore I) Mysore, India (Mysore II) Seychelles Is., Africa Seychelles Is., Africa Mombasa, Kenya, Africa Okinawa, Japan Alishan, Taiwan Makung, Peng Hu Is., Pescadores Bon Chakkrarat, Thailand Komi, Ishigaki Is., Japan Semongok For. Res., Sarawak, Malaysia Bon Chakkrarat, Thailand Sarawak, Malaysia Mount Makiling, Luzon, Philippines Samut Songkhram, Thailand Rizal, Philippines Semongok For. Res., Sarawak, Malaysia Queensland, Australia Kavieng, New Ireland Wau, New Guinea Papua, New Guinea Kavieng, New Ireland Tantalus, Oahu, Hawaii Pago Pago, Tutuila, American Samoa Tongatapu, Tonga Islands Nadarivatu, Viti Levu, Fiji Savusavuitangga, Vahua Levu, Fiji Kuala Lumpur, Malaysia Ari Ksatr, Cambodia Semongok For. Res., Sarawak, Malaysia Brunei, Borneo Singapore, Indonesia kD-valuescof protein fractions in domains: II III 43.0 43.0 40.0 40.0 40.0 35.0 37.0 35.0 40.0 37.0 30.0 35.0 33.0 36.0 35.0 27.0 d 28.0 31.5 34.0 32.5 31.5 34.0 34.0 34.0 34.0 34.0 34.0 28.5; 27.0 d 26.0d 34.0 28.5; 28.0 d 34.0 30.0; 31.0; 26.0; 26.5; 27.5; 25.5; 24.0; 25,5; 18.5; 27.0; 23.0; 20.0; 25.0; 28.0; 21.0; 25.0; 25.5; 22.0; 25.5; 24.5; 24.0; 25.5; 21.5; 25.5; 25.5; 25.5; 25.5; 26.5; 25.0 25.0; 25.0; 25.0; IV 28.0 30.0 25.0 25.5 25.5 23.0 23.0 23.0 18.0 25.5 22.0 19.0 24.0 27.0 19.5 24.0 23.0 21.0 24.5 23.0 22.0 24.5 20.0 24.0 24.0 24.0 24.0 24.5 23.0 23.0 23.0 14.0 14.5; 14.0 14.0 14.5; 14.0 14,0 14.0 14.0 14.0 14.0 14.0; 14.0; 14.0; 14.0; 14.0; 14.0; 14.0; 13.5 14.0; 14.0; 13.5 14.0; 14.0; 14.0; 14.0; 14.0; 14.0 14.0 14.0 14.5 14.0 14.0 14.0 13.0 13.0 13.0 13.0 13.0 13.5 13.5 13.5 13.5 13.0 13.0 13.5 13.0 13.0 a Code numbers from the Drosophila Species Stock List (1984) of the National Drosophila Species Resource Center, Bowling Green State University, Bowling Green, Ohio, U.S.A. b For more details see Wilson et aL (1969). c Approximate kD-values of the dot-labeled bands in Figs. 2 - 8 , according to the present results from several experiments with different protein extracts. d Major glue protein fractions which could belong to the adjacent domain on the basis of their kD-values. 65 Preparation of samples Mid third-instar larvae were used in all investigations in order to rule out the possibility of any age-specific variation in the pattern of glue protein (Ramesh & Kalisch, 1988). Larvae were washed and dissected in a medium (Ashburner, 1970) containing 0.03 M Na2PO 4 buffer (pH 6.8), 0.04 M KCI, 0.011 M NaC1, 0.003 M CaCI2, and 0.021 M MgCI2 at 20+_1 °C. Glands were transferred for 20 min to a 1.5 ml Eppendorf tube filled with cold 10% TCA (= Trichloracetic acid). The TCA was pipetted from the Eppendorf tube and the glands were washed (each time for 20 min) with 95°7o ethanol, as well as with a mixture of methanol and chloroform (l:l) by pipetting it several times using a Pasteur pipette. The tissue was finally allowed to dry at 37 °C. 30 ~l (for single pair extracts) or 50/xl (for multiple pair extracts) of a sample buffer [0.0625 M Tris-HCl (pH 6.8), 1% SDS, 1% /3-mercaptoethanol, 10% Glycerol, and 0.001% Bromophenol blue] was added to the dried tissue. The Eppendorf tubes were tightly closed and the contents were heated in a boiling water bath for 10 min. Tubes were then cooled to room temperature and centrifuged for 5 min at 1000 rpm. These extracts could be stored at - 2 0 ° C for a few days. Electrophoresis 20 #I samples were loaded in each slot on a 13.7°70 SDS-PAAG (with 0.8o7o bisacrylamide) of 1 mm thickness. Using a 0.05 M Tris, 0.384 M glycine buffer (pH 8.3) containing 0.1070 SDS as electrode (tray) buffer, the electrophoresis was performed at 165 volts for 3½ h at 20+_1°C, until the Bromophenol blue migrated to 8 cm in the separation gel. The gel was treated overnight in a prestaining solution [50o7o TCA and isopropanol (hl)], stained in 0.15 % Coomassie brilliant blue R-250 for 2 h, and destained (25% methanol and 7.5% acetic acid). For separating closely migrating double bands, as for example the 30 kD and the 28 kD bands in D. n. nasuta (compare Fig. I and 2) we used diluted running buffer (1"1 with distilled water) and 15/~1 of the sample per slot. Polyacrylamide gels were selected instead of starch gels as supporting material for electrophoresis, because of the higher power of resolution, the possibility to change the pore size by changing the monomer concentrations and other advantages (Gordon, 1969). Marker proteins For calculation of kD-values we used the following combination of marker proteins (electrophoretic pattern in Fig. 9): Phosphorylase a (97 kD), Conalbumin (86 kD), Bovine serum Albumin (68 kD), Ovalbumin (45 kD), Carbonic Anhydrase (29 kD), Myoglobin from whale (14 kD), and Cytochrome C (12.4 kD). Results Salivary gland protein fractions in Drosophila nasuta and D. melanogaster Patterns of the protein fractions from the salivary glands of mid third-instar larvae were compared in Drosophila nasuta nasuta and in D. melanogaster using 13.7070 SDS-polyacrylamide gel electrophoresis (Fig. 1). Both patterns differ remarkably due to the presence of at least five prominent bands in D. n. nasuta (labeled: > 100 kD, 43 kD, 30 kD, 28 kD and 14 kD; for separation of the 30 kD/28 kD double band see Fig. 2). In further studies (Ramesh & Kalisch, 1987; 1988), we were able to show that these prominent protein fractions belong to the major fractions of glue proteins. We were also able to demonstrate that besides the 30 kD/28 kD double band, the prominent bands of > 100 kD and 43 kD are composed of tightly neighbored multiple bands. The total amount of glue proteins produced in D. n. nasuta is roughly twice that in D. melanogaster when estimated in relation to the total amount of proteins including the glue proteins (Ramesh & Kalisch, 1988). Comparison of the glue protein patterns in eight members of the nasuta subgroup Eight members belonging to the nasuta subgroup 66 Fig. 2. Salivarygland protein fractions in different members of the nasuta subgroup: (A) Drosophila nasuta nasuta (Mysore I); - (B) 1). n. albomicans (-1751; for code numbers see Table 1); - (C) D. n. kepulauana (-1761.1); - (D) D. kohkoa (-1771); - (E) D. pulaua (-1801); - (F) D. sulfurigaster sulfurigaster (-1831.2); - (G)D.s. bilimbata(-1821.6); - (H)D. s. albostrigata [ - 1811;same strain as in Fig. 11Eand F]. Three pairs of salivary Fig. 1. Patterns of salivary gland protein fractions in (A) Drosophila melanogaster (wildtype strain Berlin) and (B) Drosophila nasuta nasuta (wildtype strain Mysore I) in a 13.7°/0 SDS-polyacrylamidegel. Eight pairs of salivary glands of mid third-instar larvae wereused for each protein extract. Coomassieblue staining, kD-valuesare givenfor the major glue protein fractions of D. n. nasuta. were studied for their patterns of glue protein fractions. Figure 2 shows the patterns of total salivary glands (together with the glue proteins in them) to indicate the extent of species-specific differences of glue as well as the differences of the cellular proteins if any. Evidence from electrophoretic data of isolated saliva plugs showing that in all species the prominent bands represent glue protein fractions are given elsewhere (Ramesh & Kalisch, 1988 and in preparation). It is obvious from the patterns in Figure 2 that the prominent glue protein fractions in different species show homologies and can be grouped at least into four domains: Domain-I band(s) [homologous with the < 100 kD band(s) inD. n. nasuta] represent fractions o f more than 100 kD in all the different species analyzed. Since on one hand, the glue protein fractions o f this domain represent very tight multiple glands of mid third-instar larvae were used for each protein extract (in Figs. 2 - 8). I - IV indicatedomains of'homologous'protein fractions in the different species(seetext). Dot-labeledbands (in Figs. 2-8) indicate glue protein fractions in domains II-IV as far as analyzed and for which kD-valuesare given in Table 1. Cf. Material and methods. bands (two bands in D. n. nasuta; Ramesh & Kalisch, 1988) and show little variability on the 13.7% SDSpolyacrylamide gels of this study on the other, we have not included the domain-I bands in the discussion of the present investigation. It is even obvious from Figure 2, however, that there are differences between the domain-I bands in the individual species of the n a s u t a group. Domain-II bands [homologous with the 43 kD band(s) ofD. n. nasuta] vary between one band in D. k o h k o a (Fig. 2D) and m a n y bands in D. n. a l b o m i c a n s (Fig. 2B; for comparison see also Figs. 4 and 5). D o m a i n - I l l band(s) [homologous with the 30 kD and 28 kD bands ofD. n. nasuta] exist mostly as two fractions, of which the one with the higher mobility (with the lower kD-value) is the more prominent one. Domain-IV band(s) [homologous with the 14 kD band of D. n. nasuta] are represented by one or two fractions. Bands of domain IV show little variation with regard to the kDvalues in different species and wildtypes (Figs. 3 - 8 ; Table 1). 67 The species-specific characteristics of the patterns of glue protein fractions are mainly represented by bands of the domains II and III. Whether or not bands of homologous domains in different species represent protein fractions with 'homologous' functions in the glue molecules is so far uncertain. Furthermore, it is still uncertain what the differences are between homologous bands on the molecular genetic level. The glue protein patterns from different larvae belonging to the same wildtype and species are reproducible. To minimize differences based on gland sizes, species-specific developments, and preparation of protein samples, however, we used more than one pair of salivary glands in most of experiments of the present study. Fig. 3. Salivary gland protein fractions o f D. n. nasuta v, ildtx pc Glue protein patterns in different wildtype stocks From the comparative studies in D. melanogaster one knows that there is a great variability in the major glue protein patterns among different wildtype stocks (Beckendorf & Kafatos, 1976; Korge, 1977; Velissariou & Ashburner, 1980). Therefore, in addition to the strains in Fig. 2, we analyzed several wildtype strains of six species of the nasuta subgroup (Table 1). Figure 3 shows five different D. n. nasuta wildtype strains. The characteristic nasuta pattern was found in all the strains but they also showed minor differences. The 'homologous' bands of domain III are slightly different in all of the wildtype patterns depicted. In domain IV, only the Mysore II and the Kenya strains show double bands. Furthermore, the geographical relationship of the original collection sites is obvious from the comparison of the domain II bands. The two strains from southern India have a greater similarity with each other than with the three strains from Africa and vice versa. In Figure 4 we have shown the patterns in five different wildtype strains of D. n. albomicans. Although a characteristic D. albomicans pattern is presented by all of the wildtype strains, there is also a lot of wildtype strain-specific variation. This could be simply correlated, at least partially, to the fact that D. albomicans has the highest number of bands strains from different collections: (A) Mysore I, India [same strain as in Fig. 1B and 2A]; - (B) Mysore II, India; - (C) Seychelles Is. I, Africa; - (D) Seychelles Is. II, Africa; - (E) Mombasa, Africa. Cf. legend of Figure 2. in domains II and III of all species checked so far. Figure 5 represents five different D. kohkoa wildtype strains. The two strains from Thailand (A and D in Fig. 5) are only slightly different, whereas the Fig. 4. D. n. albomicans wildtypes: (A) Okinawa, Japan [same strain as in Fig. 2B]; s (B) Alishan, Taiwan; - (C) Makung, Peng Hu Is., Pescadores; - (D) Bon Chakkrarat, Thailand; - (E) Komi, Japan. Cf. legend of Figure 2. Unlabeled prominent bands in domains II and III so far have not been analyzed. 68 Fig. 5. 1). k o h k o a wddtypc~; (A) BOLl Chakkarat, lhailand [same strain as in Fig. 2D]; - (B) Sarawak, Malaysia; - (C) Mount Makiling, Phillippines; - (D) Samut Songkhram, Thailand; - (E) Rizal, Philippines. Cf. legend in Figure 2. Fig. 7. D. s. bilimbata wildtypes: (A) Nadarivatu Viti Levu, Fiji [same strain as in Fig. 2G]; - (B) Tantalos, Hawaii; - (C) Pago Pago, Am. Samoa; - (D) Tongatapu, Tonga Islands; - (E) Savusavuitangga, Fiji. Cf. Figure 2. remaining three strains seem to have little in common as far as domains II and III are concerned. Figure 6 indicates characteristic differences in the mobility of similar patterns of the protein fractions in domains II and III between D. s. sulfurigaster strains. The D. s. bilimbata wildtype strains in Figure 7 show the highest degree of pattern similarity, even though they are from different islands from all over the Pacific. All of them, among minor differences show one characteristic additional band between the prominent bands of domains II and III. The four wildtype strains of D. s. albostrigata in Figure 8 can be divided into two groups: A 'typical' albostrigata pattern with the characteristic narrow set of major glue protein bands of domains II and III (A and C in Fig. 8), and the remaining two strains Fig. 6. D. s. sulfurigaster wildtypes: (A) Queensland, Australia; - (B) Kavieng, New Ireland; - (C) Papua, New Guinea; - (D) Kavieng, New Ireland, Cf. Figure 2. Fig. 8. D. s. albostrigata wildtypes: (A) Ari Ksatr, Cambodia; - (B) Semongok Forest Reserve, Sarawak; Malaysia; - ((2) Brunei, Borneo; - (D) Singapore, Indonesia. Cf. Figure 2. 69 (B and D in Fig. 8) which are almost identical with each other and look like the D. s. sulfurigaster (Fig. 6) and D. s. bilimbata (Fig. 7) patterns with slightly different mobilities, but are identical with none of these. The differences found concern bands in the overlapping regions of domains II and III as well as in domain IV. Identification o f hybrids and location o f genes coding f o r major glue protein fractions In interspecific hybrid larvae, one can determine the genetic background of both parents by the characteristic pattern shown in Figures 2 - 8 . This technique could be used for taxonomic identification of individual larvae. Experiments of this type could also be of use in population genetic studies and should even provide an easy means to examine hybridization in nature. In the following, we give three examples. In Figure 9C, D. n. nasuta x D. n. albomicans hybrid females show the full set of protein bands from both parents (A and E). In connection with the hybrid females shown in Figures 10D and liD, each hybrid can be identified and characterized by the set of its major glue protein fractions. The technique, however, is limited to female hybrids because the major glue protein fractions of domains II and III are X-chromosomally linked in all the members of the nasuta subgroup tested so far. Evidence for the X-chromosomal linkage of genes coding for the major glue protein fractions is given in Figure 9 for D. n. nasuta and D. n. albomicans: The patterns of protein fractions of one pair of salivary glands are shown for one D. n. nasuta male (A) and one D. n. albomicans male (E) as well as for reciprocal Fl-hybrid males (B and D) and a hybridfemale (C). In reciprocal crosses (Fig. 9) the Flhybrid males show, in comparison with Fl-hybrid females, only the glue protein fractions of their mothers. This involves the 43 kD, 30 kD, and 28 kD bands in D. n. nasuta (arrows in Fig. 9B; compare also Fig. 1B) and at least the 35.0 kD, 25.5 kD, 23 kD bands in D. n. albomicans (arrows in Fig. 9D). Since the Fl-hybrid males receive their Xchromosome from their mothers, the X-chromo- Fig. 9. Salivary gland protein fractions of D. n. n a s u t a (Mysore I) x D. n. a l b o m i c a n s ( - 1 7 5 1 ) hybrids as well as evidence for Xchromosomal linkage of major glue protein fractions in D. n. n a s u t a and D. n. a l b o m i c a n s : (A) male of D. n. nasuta; - (B) male- and (C) female-hybrid from a D. n. n a s u t a mother and a D. n. a l b o m i c a n s father; - (D)male-hybrid from a D. n. a l b o m i c a n s mother and a D. n. n a s u t a father; - (E) male of D. n. alb o m i c a n s . Each protein extract from one pair of salivary glands from a mid third-instar larva. Arrow-labeled bands indicate major glue protein fractions of both species which are coded by X-chromosomal genes. (M) Marker proteins [listed in Material and methods]. Fig. 10. Salivary gland protein fractions of D. s. s u l f u r i g a s t e r ( - 1831.2) x D. s. b i l i m b a t a ( - 1821.6) hybrids as well as evidence for X-chromosomal linkage of the major glue protein fractions: (A) male and (B) female of D. s. sulfurigaster, - (C) maleand (D) female-hybrid from a D. s. s u l f u r i g a s t e r mother and a D. s. b i l i m b a t a father; - (E) male and (F) female ofD. s. b i l i m b a ta. Cf. Figure 9. 70 Salivarygland protein fractions in D. s. sulfurigaster (-1831.2) x D. s. albostrigata (-1811) hybridsand evidencefor X-chromosomal linkage of genes: (A) male and (B) female of D. s. sulfurigaster; - (C) male-and (D) female-hybridfrom a D. s. sulfurigaster motherand a D. s. albostrigata father; - (E) male and (F) femaleof D. s. albostrigata. Cf. Figure9. Fig. 11. somal linkage of genes can be confirmed. In Figure 10, comparable results are given for O. s. s u l f u r i g a s t e r and D. s. b i l i m b a t a , i.e., the major glue protein fractions of the domains II and III (labeled in Fig. 10) are coded by X-chromosomal linked genes. In this case, the pattern of a reciprocal F lhybrid is not shown. Figure 11 shows evidence for the X-chromosomal linkage of genes coding for bands of domains II and III in D. s. s u l f u r i g a s t e r and D. s. a l b o s t r i g a t a . Discussion The present pattern analysis of glue protein fractions in the n a s u t a subgroup indicates that the 30 strains as well as the hybrids tested so far, can be identified individually by the technique used. It can not be excluded, however, that additional wildtype strains may exist which are even more similar than the D. s. b i l i r n b a t a strains (Fig. 7). In this case, our technique would have to be extended to the minor glue and tissue protein fractions to be sensitive enough for an individual identification. Also in those hybrids in which protein fractions overlap in their mobility with each other, it would be more difficult to tell the genotypes of the parents apart. Nevertheless, our technique is, so far, easier to handle for taxonomic identification than any technique based on cytological and/or morphological differences. Furthermore, our technique can even be used for the identification of wildtype strains and hybrids. The n a s u t a subgroup-specific pattern of the glue protein fractions (domains I - I V ) with its speciesspecific differences on the one hand and the wildtype strain-specific variations on the other is of interest from the evolutionary and the molecular genetic point of view. Species-specific differences include (besides quantitative differences) variations in the number of fractions within the four domains (Fig. 2) as well as variations of the electrophoretic mobility of the fractions within each of the domains (Fig. 3 - 8). The different number of 'homologous' protein fractions in wildtype strains and in species could be based upon chromosome mutations like gene duplications, whereas the different mobilities could be based among other possibilities on point mutations. One knows that even the substitution of a single amino acid can already significantly change the mobility of a protein in a gel (Noel e t al., 1979). However, so far little is known about the phylogenetic relationships between different members of the n a s u t a subgroup on the basis of isoenzyme variations (Ramesh & Rajasekarasetty, 1980) and nothing is known about the gene structure of the n a s u t a subgroup on the DNA level. In another study (Ramesh & Kalisch, 1988) we were able to show that in D. n. n a s u t a the prominent glue proteins of domains I - III are PAS positive. So, the differences in the mobility of these protein fractions could also be based on differences in the glycosylation of the protein fractions. Even though the 30 strains tested are different from each other, it is surprising how similar the general pattern of prominent glue protein fractions (domains I - I V ) is within the n a s u t a subgroup (Fig. 2). There are several other proteins in nature which have been shown to be very similar in different species, as for example the hemoglobin, insulin and histone molecules. The need of conserving the essential function of these proteins has been obviously a selecting factor during evolution, leading only to 71 minor changes between different species. In the case of glue proteins, however, it is hard to believe that the survival of the flies is dependent on a specific composition of glue, because completely different patterns have been reported in wildtype strains of D. melanogaster (Beckendorf & Kafatos, 1976; Korge, 1977; Velissariou & Ashburner, 1980), in D. hydei (Ramesh & Kalisch, unpublished), and in others (Manousis, 1985). In this connection, one also has to consider the possibility that the genome posesses the ability to regulate differentially the rate of evolution of DNA sequences in different chromosomal locations (Martin & Meyerowitz, 1986). Finally, we know from Figures 9-11 as well as from a cytologic and formal genetic study (Ramesh & Kalisch, 1988) that the X-chromosomal genes coding for fractions of domains II and III are tightly linked and probably form a cluster of tandemly arranged genes within the huge puff of subdivision 10 (in the reference map of D. n. nasuta, published by Ranganath & Krishnamurthy, 1974). This tandem arrangement of the glue protein genes could probably indicate a situation similar to the location of the glue polypeptides sgs-3, sgs-7, and sgs-8 of the 68C puff in D. melanogaster (Garfinkel et al., 1983), i.e., a factor which conserved the basic pattern. Results of the present study can be compared with evolutionary genetic data from the literature. Wilson et al. (1969) have shown that the three D. sulfurigaster species are more related with each other than with the remaining species of the nasuta complex. Based on our data in Figure 2 this agrees with the results in D. s. sulfurigaster and D. s. bilimbata, but not with the results in D. s. albostrigata. However, the wildtype strains from Malaysia and Indonesia (B and D in Fig. 8) in comparison with Figures 6 and 7 indicate that there is in fact a stronger relationship between the three sulfurigaster species than to the Cambodia strain (H in Fig. 2 and A in Fig. 8) or the Borneo strain (C in Fig. 8). By this, the two albostrigata wildtype strains, which show the bilimbata and sulfurigaster-like pattern (B and D in Fig. 8), have to be seen as the more original ones, whereas the two characteristic narrow-banded patterns (A and C in Fig. 8) could be based on more recent evolutionary events. Acknowledgements S.R.R. is grateful to the Univ. Grants Commission, New Delhi, India, for sponsoring and to the Deutscher Akademischer Austauschdienst, FR Germany, for the award of a scholarship. 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