A-Glycerophosphate Dehydrogenase Within the Genus Drosophila (Dipteran Evolution/Unit Evolutionary Period) GLEN E
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Proc. Natl. Acad. Sci. USA Vol. 74, No. 2, pp. 684-688, February 1977 Genetics Microcomplement fixation studies on the evolution of a-glycerophosphate dehydrogenase within the genus Drosophila (dipteran evolution/unit evolutionary period) GLEN E. COLLIER AND Ross J. MACINTYRE Section of Genetics, Development and Physiology, Plant Science Building, Cornell University, Ithaca, New York 14853 Communicated by Adrian M. Srb, November 8,1976 ABSTRACT Antisera were prepared against purified a- least in D. melanogaster, for rapid production of the energy glycerophosphate dehydrogenase (EC 1.1.1.8) (aGPDH) from needed for flight (7-9). Drosophila melanogaster, D. virifis, and D. busckii. The im- munological distances between the enzymes from the 3 species The third criterion is that the protein should be evolving and those from 31 additional drosophilid species agree in gen- relatively slowly. Although cytogenetic analysis and interspe- eral with the accepted phylogeny of the genus. These data per- cific hybridization are adequate for est*blishing phylogenetic mit an estimate that the subgenus Sophophora diverged 52 relationships among closely related species, a protein that has million years ago from the line leading to the subgenus Droso- changed slowly is particularly useful for establishing the rela- phila. The antiserum against melanogaster aGPDH was ca- pable of distinguishing alielic variants of aGPDH. On the basis tionships among species groups, subgenera, genera, and even of presumed single amino acid substitutions, no-drosophilid families or orders. Brosemer et al. (10) and Fink et al. (11) have aGPDH tested differed from the melanogaster enzyme by more established with immunological tests that the structure of than eight or nine substitutions. The study was extended to in- aGPDH has been substantially conserved during the evolution clude representatives of six other dipteran families. The im- of the Hymenoptera. The results reported here show similar munological distances between zGPDH from Drosophila and conservatism within the genus Drosophila and among other aGPDH from these dipterans were reasonably consistent with a phylogeny of the order Diptera established by more conven- Diptera. tional means. The unit evolutionary period of this enzyme was This paper deals with the divergence of aGPDH within the estimated to be 18 million years. genus Drosophila and several related genera as measured by the immunological technique of microcomplement fixation Studies on the evolution of proteins have provided biologists (12). Differences between homologous proteins measured by with a powerful tool for constructing phylogenies, particularly this technique are directly related to percentage sequence di- of taza that are poorly represented in the fossil record. In gen- vergence (13, 14). The technique is sensitive enough to detect eral, the phylogenies derived from comparison of protein se- single amino acid differences (15, 16). quences have agreed well with phylogenies derived by more classical techniques (1-4). Since the insects have a fragmentary MATERIALS AND METHODS fossil record, our understanding of their evolution would greatly benefit from studies of their molecular evolution. Unfortu- Antiserum Preparation. aGPDH was purified to homoge- nately, the insects have been grossly underrepresented in studies neity from D. melanogaster, D. virilis, and D. buscki by the of molecular evolution (4). procedure of Collier et al. (17). Four New Zealand white male The genus Drosophila should be the initial focus of such rabbits were each injected with a total of 100 jig of the purified studies among the insects. It has the best understood phylogeny D. melanogaster enzyme. A total of 45 ;g of purified D. virils of any comparably large assemblage of species (5, 6) and its enzyme was injected into each of four additional rabbits. Three species are the easiest of the higher eukaryotes to manipulate rabbits each received a total of 15 ,g of purified enzyme from genetically. Agreement between a phylogeny derived from D. busckli. The antigen for each rabbit was divided into four molecular data and the generally accepted phylogeny provided 1-ml portions that were administered at weekly intervals. The by Throckmorton (6) would provide a basis of confidence for rabbits were bled from the ear 1 week after the last injection. molecular studies applied to related insect groups whose phy- The antisera exhibited a single precipitin line containing logenetic relationships are poorly understood. aGPDH activity in double diffusion and immunolectrophoretic The choice of a protein for such studies cannot be casual. analyses. Three criteria must be met. First, the protein should occur in Antigen Preparation. Each antigen used for microcomple- most insects. Second, because the study of structure-function ment fixation was partially purified as follows. Adult flies- (2 relationships in proteins can hardly be separated from studies g) were homogenized in 0.1 M sodium phosphate buffer, pH of their molecular evolution, the protein should be significant 7.1, containing 10-6 M NAD, 0.5 mM dithiothreitol, 1 mM functionally. The dimeric enzyme a-glycerophosphate dehy- EDTA, and 10 mM a-glycerophosphate. The extract was cen- drogenase (EC 1.1.1.8) (aGPDH) meets these criteria. Its role trifuged at 15,000 X g for 20 min and passed over carboxy- in intermediary metabolism ensures its presence in the tissues methyl cellulose and then DEAE-cellulose, each packed in small of most or all insects. Furthermore, this enzyme is essential, at sintered glass funnels and equilibrated with the homogenization buffer. Under these conditions, aGPDH is not retained on ei- Abbreviations: aGPDH, a-glycerophosphate dehydrogenase; ID, ther ion exchange resin, but the eye pigments and many other immunological distance. proteins are retained. About 20-fold purification for each an- 684 Downloaded by guest on October 2, 2021 Genetics: Collier and MacIntyre Proc. Natl. Acad. Sci. USA 74 (1977) 685 Table 1. Immunological distances between aGPDH from is the average of four determinations. Standard errors were D. melanogaster, D. busckii, and D. virilis and aGPDH from 41O% or less. various drosophilid species RESULTS AND DISCUSSION Antiserum* Antigent Immunological Distances Between Drosophilid aGPDH. mel. Table 1 shows IDs for Drosophila species arranged according A/A vir. busckii Species Species group to the generally recognized species groups. These data show that the three antisera differ in specificity. There is a lack of reci- 0 2 0 melanonaaster A/A procity of IDs among D. melanogaster, D. drilis, and D. 12 11 0 8 virilis busckii, perhaps due to errors associated with small IDs. 6 2 0 busckii However, because repeated determinations of these values 9 melanogaster B/B indicate an experimental error of only 10%, the lack of reci- 5 2 0 simulans procity could well be due to the different amounts of antigen 6 1 0 yakuba Ielanogaster used to produce the antisera. In other words, the small amounts 11 birchii A/A of enzymes from D. virilis and D. busckdi used to produce an- 17 birchii B/B tisera may not have been sufficient to elicit diverse families of 23 8 3 mimetica antibodies capable of recognizing all the antigenic determinants 13 affinis A/A on the enzyme surfaces. Thus, in addition to having low titers, 19 affinis B/B these antisera would be less discriminating than the antiserum 17 affinis C/C obscura against melanogaster enzyme. The remaining values in Table 15 12 7 algonquin 1 show that the antiserum against melanogaster enzyme is in 12 4 6 pseudoobscura fact the most discriminating of the three. In any case, because 16 miranda the data are not used to construct phylogenetic trees but only 29 10 6 willistoni willistoni 11 7 1 paulistorum to estimate degree of change in the enzyme during the evolu- 26 4 6 emarginata tion of the genus Drosophila, the lack of reciprocity among 19 2 17 sturtevanti saltans antisera is not critical. 17 6 14 Chymomyza procnemist Fig. 1, modified from Throckmorton (6), diagrams the re- 27 7 Chymomyza amoenat lationships of the species used in this study. These species rep- 25 5 9 funebris resent most of the major subdivisions of the genus. The IDs 16 0 8 gibberosa annulimana between the species' enzymes in Table 1 conform well with 21 0 8 montana virilis their phylogenetic relationships presented in Fig. 1. In general, 21 0 13 micromelanica with all three sera the smallest IDs are between enzymes from 20 paramelanica A/A melanica closely related species. This generalization has two major ex- 26. paramelanica B/B ceptions. First, D. mimetica is a member of the D. melano- 23 3 13 robusta A/A robusta gaster species group but immunologically, with each antiserum, 28 robusta B/B its enzyme is as different from the D. melanogaster enzyme 26 0 13 mercatorum repleta as are the enzymes from species of other subgenera. The second 21 3 8 hydci exception is D. busckii. Taxonomists have placed this species 26 5 11 immigrans A/A in the subgenus Dorsilopha. However, as seen in Table 1, the 30 immigrans B/B immigrans enzyme from busckii differs very little from that of melano- 16 nasuta A/A gaster, a member of the subgenus Sophophora. The basis of 12 nasuta B/B these anomalies may be differences in the rate of evolution of 23 11 13 Zaprionus vittigert 13 0 4 quinaria quinaria aGPDH in the lines of descent leading to D. mimetica and D. 19 1 8 putrida testaceae busCkui. 20 0 0 cardini cardini There are other instances of ID heterogeneity within species 17 0 11 pallidipennis pallidipennis groups. Until sequence data are available, it cannot be deter- 26 8 19 tripunctata tripunctata mined if this heterogeneity is due to rate differences or is a 19 6 5 duncani consequence of the approximate nature of the immunological measure. Therefore, the phylogenetic relationships revealed 41 28 28 Number of stocks tested by the data in Table 1 are more obvious when the average IDs * A dash indicates that an ID was not determined.