Distribution of erythrocytic allozymes in two sibling species of greater galago [Galago crassicaudatus E. Geoffroy 1812 and G. garnettii (Ogilby 1838)]код для вставкиСкачать
American Journal of Primatology 14:235-245 (1988) Distribution of Erythrocytic Allozyrnes in Two Sibling Species of Greater Galago [Galago crassicaudatus E. Geoffroy 1812 and G. garnettii (Ogilby 1838)] AND DAVID S. DU"' 'Department of Zoology and 2MRCHuman Ecogenetics Unit, Department of Human Genetics, School of Pathology, South African Institute for Medical Research and University of the Witwatersrand, Johannesburg, South Africa JUDITH C. MASTERS' This study investigated the use of erythrocyte enzymes as indicators of the presence or absence of gene flow between the sibling species G. crassicaudatus and G. garnettii. Fifty-five animals deriving from 14 different source populations were included in the analyses. In addition to hemoglobin, eight enzyme systems were examined acid phosphatase, adenylate kinase, carbonic anhydrase II, esterase D, glucose-6-phosphatedehydrogenase, 6-phosphogluconate dehydrogenase, peptidase A, and peptidase B. Of these, adenylate kinase, glucose-6-phosphate dehydrogenase, hemoglobin, peptidase A, and peptidase B showed no interspecific or intraspecific variation. Esterase D was polymorphic in certain populations of G. crassicaudatus but not in others or in G. garnettii. Acid phosphatase and 6-phosphogluconate dehydrogenase were polymorphic in G. garnettii but monomorphic in all G. crassicaudatus populations. The taxa showed fixation for different alleles at the carbonic anhydrase I1 locus, indicating a lack of gene exchange betweea the taxa. We suggest that acid phosphatase, 6-phosphogluconate dehydrogenase, and carbonic anhydrase I1 may be used as genetic markers in the identification of these two taxa. Key words: gene flow, allozymes, Galago crassicaudatw, Galago garnettii INTRODUCTION The identification of genetic species requires information from a variety of sources in order to detect the presence or absence of gene flow between populations. Within the greater galagines (classified as a single species, Galago crassicaudatus, by Schwarz  and Hill ),two distinct populations have been identified on the grounds of morphology [Dixson & Van Horn, 1977; Olson, 1979; Masters, 1985, Received May 22, 1987; revision accepted October 7, 1987. Judith C. Masters is now at the Museum of Comparative Zoology, Harvard University, Cambridge, MA 02138. Address reprint requests there. 0 1988 Alan R. Liss, Inc. 236 I Masters and Dunn 1986; Masters & Lubinsky, 19881, karyology [De Boer, 1973; Pasztor & Van Horn, 1977; Masters, 1985, 19861, and reproductive behavior [Buettner-Janusch, 1964; Pasztor & Van Horn, 1976; Dixson & Van Horn, 1977; Eaglen & Simons, 19801. Olson 119791, following Thomas , advised the use of the specific name garnettii for the sister species of G. crassicaudatus, sensu stricto. Multiple enzyme forms are known generally as Ysozymes” [Markert & Mdler, 19591. Prakash et a1 [19691 introduced the term “allozymes” to refer particularly to those enzyme forms produced by different alleles at the same locus. Allozymes can be extremely informative in tracing possible gene flow between populations. Discrete gene pools could well show differences in proportional representation of various alleles. In the most favorable situations, different alleles may be fixed in different populations, enabling specific identification by means of genetic markers and making the presence of any hybrids relatively easy to detect. In this contribution we present the results of a series of electrophoretic analyses of erythrocytic allozymes, which were undertaken to investigate the possibility of gene flow between the designated taxa G. crassicaudatus and G. garnettii. MATERIALS AND METHODS Sampling Methods The reliability and significance of investigations of allozyme distributions depend chiefly on two aspects of the sampling method: sample size and the level of inbreeding within the sample. In galagos, these limitations are not easily overcome; wild populations are not readily accessible, and captive colonies tend to be highly inbred. Our sample consisted of 55 greater galagos: 40 G. crassicaudatus, 14 G. garnettii, and one interspecific hybrid. Twenty seven of the G. crassicaudatus were wildcaught in southern Africa (see Masters  for details), and one of the two G.c. monteiri was also taken from the wild, although details are not available of the locality. The remaining animals were all laboratory-reared (see Table I). Care was taken, however, t o ensure that they were derived from a number of source populations. Laboratory-bred galagos were classified by their curators on the basis of external morphological features (size, head and ear morphology, and pelage characteristics), according to the descriptions of Hill  and Olson . Specific identifications were verified by chromosomal analysis [Masters, 1986; Masters et al, 19871. TABLE I. Details of Animals Used in the Studv Species Crassicaudatus Garnettii GarnettiU crassicaudatus Totals Subspecies Argentatus Loennbergi Monteiri Umbrosus Z uluensis Garnettii Lasiotis 55 (28,27) No. source populations Locality reference 1 3 Buettner-Janusch  Masters  2 - 3 3 1 1 Masters  Masters  Buettner-Janusch  14 - Greater Galago ErythrocyticAllozymes I 237 A 5-ml aliquot of blood was drawn from the femoral vein of each galago using a heparinized disposable syringe and a 21-gauge sterile needle, and was stored prior to use in a glycerol-citrate buffer solution at -20°C. The procedure involved, first, packing the erythrocytes by light centrifugation and removing the supernatant plasma. This was followed by drop-by-drop addition of the glycerol-citrate solution until the volume of preservative equalled that of the cells. Addition of preservative was accompanied by gentle agitation of the mixture to ensure that all cells were well-coated. Preliminary tests comparing frozen and fresh samples demonstrated that this storage process did not substantially affect the electrophoretic performance of the proteins investigated here, even after a period of approximately 18 months. Electrophoretic Methods Hemolysates were prepared as follows: an aliquot of cells suspended in glycerolcitrate was removed and packed by light centrifugation. The supernatant was removed, and a volume of distilled water equal to that of the cells was added. On the basis of previous work [Buettner-Janusch & Buettner-Janusch, 1963, 1964; Buettner-Janusch & Wiggins, 1970; Barnicot & Hewett-Emmett, 19741 and preliminary tests, eight enzyme systems were chosen for investigation. These included acid phosphatase (AP), adenylate kinase (AK), carbonic anhydrase 11(CA esterase D (ESD), glucose-6-phosphate dehydrogenase (GGPD), 6-phosphogluconate dehydrogenase (GPGD), peptidase A (PEPA) and peptidase B (PEPB). In addition, hemoglobins were examined on the CA I1 and G6PD systems prior to staining. The methods used for particular enzyme systems were as follows. Acid phosphatase. We employed a modified version of that proposed by Hopkinson et a1  for human red cell AP. In the staining procedure, we used 0.5 M NaOH in place of NHBOH because of the lower volatility of the former compound. Adenylate kinase. The procedure followed was that devised by Fildes and Harris  for examining AK in human red blood cells. Carbonic anhydrase 11. The method of Hopkinson et a1  was employed, and the resultant fluorescent bands were visualized under ultraviolet illumination. Esterase D. For this analysis, we employed the staining procedure of Hopkinson et a1  and the gel that had been used in the AK analysis. After this gel had been sliced, the upper half was removed and the cut surface was exposed for staining-hence the reversed sample order in Figure 4. As for CA 11, gels were inspected under ultraviolet illumination. Glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. These two systems were run on the same vertical gel system (see below). The method used was that originally put forward by Fildes and Parr  and modified by Harris and Hopkinson . Peptidase A and peptidase B. The method used for characterizing these enzymes was a slightly modified version of that devised by Lewis and Harris . We used crude snake venom of Bitis arietuns rather than the purified enzyme L-amino acid oxidase. The samples were loaded on to the gels using double filter paper inserts of approximately 5 x 7 mm. For PEPA, PEPB, and AP, Whatman’s No. 17 was used. For AK, ESD, and CA 11, however, smaller samples were required, and thinner inserts (Whatman’s No. 3) were employed. In the case of G6PD and GPGD, wells were made in the starch, into which dilutions of the hemolysates were added (1:3 hemo1ysate:cathode buffer). The starch used was the starch hydrolysate for electrophoresis produced by Merck. All systems were run horizontally apart from GGPD and GPGD, which required vertical beds for better separation of isozymes. Gels ran for approximately 18 hours at 6 4 ° C . Results were photographed using a variable a, 238 I Masters and Dunn ASA film (Ilford XPl), because of the high ASA required to photograph enzymes visualized with a fluorescent stain under ultraviolet illumination. An ASA rating of 1250 was used, and the exposures were made at 1/15 to 1/30 second at f 1.8. RESULTS AND DISCUSSION For ease of interpretation, the results and discussion sections are presented together. Acid Phosphatase AP phenotypes were first examined in greater galagos by Buettner-Janusch and Wiggins , who investigated breeding colonies of “G.c. crassicaudatus” (ie, G. garnettii) and “G.c. argentatus” (ie, G. crassicaudatus, s.s.) maintained at the primate facility of Duke University. Electrophoresis of hemolysates from “G.c. crassicaudatus” indicated four bands of phosphatase activity, which they labeled 1-4 on the basis of their anodal mobilities. Band 1migrated the greatest distance from the origin, and band 4 migrated the least. The following phenotypes were defined for this group: 1. PA described individuals displaying bands 1 and 2 (AP A in more recent terminology). 2. PBreferred to those exhibiting bands 3 and 4 (ie, AP B). 3. PABanimals showed bands 1,2,3, and 4 (ie, AP AB). An examination of colony pedigrees suggested that bands 1 and 2 were inherited as a single unit, as were bands 3 and 4. This led the authors to the conclusion that the patterns were produced by simple codominant alleles. Hopkinson et a1 , studying human AP phenotypes, found similarly that a single allele appeared to give rise to more than one monomeric isozyme (see HSA, Fig. lb). Subsequent studies of these bands in human samples by Fisher and Harris [1971a,b] suggested that they represented conformational isozymes that were interconvertible. This explanation would similarly account for the patterns exhibited by the greater galago phosphatases. All of Buettner-Janusch and Wiggins’ G.c. argentatus individuals yielded an AP B phenotype (bands 3 and 4),although additional very faint bands of the same mobility as band 1, and infrequently, band 2, were also observed. The authors suggested these to be artifacts, as are sometimes observed in stored human samples. The results of our study (Fig. 1) accord very well with those of the previous authors. Two of the ten G.g. garnettii animals showed the phenotype AP A, four individuals were AP B, and four were heterozygous. In the case of the G.g. lasiotis sample, one animal had the phenotype AP A, one was AP B, and two were heterozygous. All G. crassicaudatus samples, without exception, showed the phenotype AP B. The faint bands observed by Buettner-Janusch and Wiggins in the band 1position were observed only in the Zimbabwe sample, and it seems likely that they are indeed artifactual, ie, not part of the same enzyme system. The hybrid individual (Fig. la) had an AP B phenotype but showed in addition a faint band with a mobility intermediate between bands 1and 2. This same band was seen on two independent runs. It is unclear whether this band represents a breakdown product caused by storage or a protein produced by the animal, and, if it is a protein, whether it is actually part of the AP enzyme system. On finding no polymorphisms in their G. crassicaudatus sample, BuettnerJanusch and Wiggins suggested that this could be due to a colony “founder effect”: wild populations would be expected to demonstrate polymorphism. However, the Greater Galago Erythrocytic AIIozymes 1 239 G G G G G C C C C H Y B G C G CHSAG C G C 1 2 3 4 5 30 31 32 33 6 34 7 35 8 36 9 37 G G G G C 2 3 4 5 30 31 32 33 C C C G CHSAG C G 6 34 7 35 a 36 9 CHYBG C 37 Fig. 1. a,b: Gels stained for acid phosphatase activity: C = G. crassiccmdutus; C = G. garnettic HSA = Homo sapiens; HYB = G.gwnettii x 0. crassicaudatus hybrid. Fig. 2. a.b: Gels stained for adenylate kinaso: species codes as in Figure 1. results of this study indicate that the allele AP*B may well be fxed in G. cmssicuu&us and that the polymorphism is restricted to G. garnettii. A review of acid phosphatase phenotypes among the prosimians by Barnicot and Hewett-Emmett  showed the greater galagos to be vastly different from C 31 C G 32 3 G 33 2 G HYBC G 1 4 30 C G 5 C C 4 G 30 31 G 5 C C H 3 G 2 G 32 33 1 G Y G C C 19 34 C 6 20 B C 18 17 C G C H 7 35 C 16 S 15 C 8 A 14 C 36 G C 13 C 12 C C 37 G 9 . Greater Galago Erythrocytic Allozymes / 241 their congeners, G. senegalensis and G. demidouii. However, the phenotype displayed by the G. alleni sample was similar to A P B. Adenylate Kinase Barnicot and Hewett-Emmett [19741 reported a complex banding pattern for this enzyme in prosimian species and demonstrated a degree of polymorphism in “G. crassica~datus’~ with regard to minor bands. Unfortunately, the starch gels used in this study did not yield the necessary resolution for firm conclusions regarding the position of minor bands, but the results did indicate a single common phenotype in all specimens (Fig. Zbprobably the second Gc phenotype shown in Barnicot & Hewett-Emmett’s Figure 3. Electrophoretic patterns were consistent with a simple monomeric protein, which is the subunit structure recorded previously for the enzyme in human samples by Harris and Hopkinson . Carbonic Anhydrase I1 Carbonic anhydrases are the slowest migrating of the red cell esterases and occur in human erythrocytes as two distinct molecular forms: CA I and CA 11 pashian, 19651. When the latter author undertook a comparison of esterases in primate species, including three lorisid taxa, he found both of these systems to be absent in G. crassicaudatus and t?potto. He noticed as well the presence of two CA I bands in the slow loris, Nycticebus coucang. However, in this study only CA I was not detected in the greater galagos, and CA I1 proved to be the most interesting enzyme system examined from the point of view of specific discrimination. CA II was visualized as a bright yellow fluorescent band migrating cathodally (Fig. 3). All G. crassicaudatus individuals examined showed the same band (S), migrating at a rate intermediate between that of the human sample (HSA) and those of the G. garnettii specimens. This latter group also showed bands of identical mobility (F), which moved at a somewhat faster rate than those observed in G. crassicaudatus. The hybrid individual showed both bands. It would appear, therefore, that the two CA I1 alleles are fixed in their respective populations; G. crassicaudatus individuals are homozygous for the “slower” allele; G. garrzettii animals are homozygous for the “faster” allele; and the hybrid is heterozygous. This result is strongly indicative of discrete gene pools in the two taxa. Esterase D Esterase D is an acetylesterase discovered fortuitously in human erythrocytes by Hopkinson et a1  while investigating the use of the fluorogenic substrate 4methyl-umbelliferyl acetate in the visualization of known esterases. Our results are very similar to those obtained by the above authors from human hemolysates (Fig. 4). The observed pattern is typical of a dimeric protein showing heterozygosity. Three phenotypes are immediately discernible: ESD 1 has a relatively slow major band (eg, C 31); ESD 2 has a relatively fast major isozyme (eg, G1G5); and ESD 2-1 has both of these bands plus one of intermediate mobility (eg, C 33). Phenotypes ESD 1 and ESD 2 presumably represent the homodimers and correspond with the two outer isozymes of the ESD 2-1 triplet. The intermediate band would then be heterodimeric, made up of polypeptide subunits determined by each of the two alleles [Hopkinson et al, 19731. -~ ~~ ~~~ ~ Fig. 3. a,b: Gels stained for carbonic anhydrase II: species codes as in Figure 1. Fig. 4. a,b: Gels stained for esterase D: species codes as in Figure 1. 242 I Masters and Dunn The heterozygote phenotype was observed only in G. crassicaudatus individuals and not in G. garnettii or the hybrid. However, this may be the result of small sample size in the latter species. Hopkinson and coworkers found good agreement between observed numbers of the three phenotypes in humans and those expected, assuming a Hardy-Weinberg equilibrium, although presumably one allele can become more common, or even fixed, in a semi-isolated population through genetic drift. This appeared to be the case with the G.c. umbrosus (Transvaal) and G.c. zuluensis (Zululand) populations sampled, all of which showed the ESD 1phenotype, although the sample sizes are too limited for any firm conclusions. Polymorphism was seen in the G.c. argentatus (East Africa) and G.c. loennbergi (Zimbabwe)samples only. Phenotype frequencies are recorded in Table 11. The human isozyme included as a control was indistinguishable from the ESD 1obtained from the greater galagos. Glucose-6-PhosphateDehydrogenase and 6-PhosphogluconateDehydrogenase G6PD is monomeric, and GPGD is a dimeric protein. No variation was detected in GGPD, all individuals showing a single major band migrating at a rate somewhat slower than that of our human control. In contrast, GPGD was polymorphic, but only in one population (Table ID. All G. crassicaudatus specimens investigated, as well as the hybrid, showed the same single homodimeric band (A) moving at an equivalent rate to the human control. In the G.g. garnettii sample, three individuals displayed homodimeric bands identical with the G. crassicaudatus and human phenotypes; three showed homodimers for an allele that migrated at a much slower rate (C); and four were heterozygous Wig. 5). The G.g. lasiotis animals were of uniform phenotype (C). Peptidase A and Peptidase B Neither interspecific nor intraspecific variation was detected in PEPA, a dimeric protein, or in PEPB, a monomer. The galagine PEPA isozyme was observed to migrate at a slightly slower rate than that of the human sample, while the mobility of galagine PEPB was considerably faster than that of its human counterpart. Hemoglobin In their review of primate hemoglobins, Buettner-Janusch and Buettner-Janusch  concluded that “All of the Lorisiformes . . . have two major haemoglobin components,” and indeed two distinct bands were observed in all individuals studied. However, an earlier contribution by the same authors [Buettner-Janusch & Buettner-Janusch, 19631 described the existence of a third minor component in approximately 50% of the specimens they examined. The component had a lower mobility than the two major bands. The fastest band was thought by the authors to be analogous to human A3 (‘‘senescent” hemoglobin). None of the animals studied here exhibited the slow minor component, even after the application of a benzidine stain, as recommended by Buettner-Janusch and Buettner-Janusch 119631. Barnicot and Hewett-Emmett , examining hemoglobins in 19 greater galagos, obtained the same result that we did. CONCLUSIONS 1.The results obtained in this study demonstrate the utility of electrophoretic investigations for prosimian systematics. 2. Several enzyme systems supported the hypothesis of separate gene pools for G. crassicaudatus and G. garnettii; this was particularly true for CA 11, but also applied to AP, GPGD, and possibly ESD. However, small sample sizes-particularly in the case of G. garnettii-indicate a need for further work in this area. 1 4 10 9 9 2 11 9 n 2(.20) 1(.25) 2(.50) 4(.40) *Brackets indicate phenotypic frequencies. G. c argentatus Loennbergi Monteiri Umbrosus Zuluensis G.g. garnettii Laswtis Hybrid Taxon 9 9 2 11 9 Acid phosphatase A AB B 4 10 1 10 11 9 9 2 9 Carbonic anhydrase 11 F FS S 1 4 11 9 7(.78) 4(.44) 2 1 1(.11) 5(.56) Esterase D 2-1 U.11) 2 TABLE 11. Summary of Phenotypic Data for Enzyme Systems Showing Inter- or Intraspecific Variation* 1 3(.30) 11 9 9 2 9 4(.40) 4 3(.30) 6-phosphogluconate dehydrogenase A AC C v. 0 9 2 f? 0 w B m ;;f 244 I Masters and Dunn G C G CHSAG C G C 8 36 9 37 6 34 7 35 C 38 G C HSA G C 10 39 12 4 0 G 13 G 14 Fig. 5. Gels stained for 6-phosphogluconatedehydrogenase:species codes as in Figure 1 3. CA II, AP,and 6PGD may be used as genetic markers for identifying these species in problematic circumstances. 4. Enzyme electrophoresis lacks many of the complications of comparative karyology (eg, facilities for tissue culture and the need for sterile conditions) and lends itself far more to field population studies. Samples may be frozen and stored with relative ease for analysis a t a later date. The greater use of this technique in prosimian systematics may aid considerably in the elucidation of several species problems currently facing workers in this field; eg, diverse diploid chromosome numbers have been observed within the taxa G.senegalensis and G.demidovii and may be indicative of cryptic species [De Boer, 19731. 5. The small volume of whole blood required for enzyme electrophoresis (approximately 2 ml) is a n additional fador that points to the usefulness of this technique for genetic studies in smaller-bodied animals. ACKNOWLEDGMENTS This project was supervised by Professor H.E. Paterson, and the electrophoresis was conducted under the auspices of Professor T. Jenkins. Capture of wild bushbabies was made possible by the Department of Environment Affairs in South Africa and by the Nature Conservation Council in Zimbabwe. N. Caithness and M. Longridge provided indispensible help on field trips. Facilities for animal maintenance were provided by the Primate Behaviour Research Group, University of the Witwatersrand, and the Department of Medical Microbiology, University of Zimbabwe. Special thanks are due to Dr. C. Bielert and N. Lyons for their assistance in this regard. Blood from captive animals was provided by the Duke University Center for Primate Biology and History (courtesy of M. Stuart) and Kassel University (courtesy of Dr. C. Welker). Special thanks are due to Professor B. Chiarelli and Drs. C. Welker and R. Stanyon for their assistance. Dr. R. Rayner and N. Caithness helped with the photography, and several people assisted in the transport of frozen samples, especially Professors R.M. Crewe and W. Maier, Dr. D. Romagno, and Mrs. E. Rayner. 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