A biochemical genetic study of allozyme polymorphism in two natural populations of the cape griffon vulture (Gyps coprotheres) and individuals held in captivity

, pp. 481493, 1992

ICAL G E N E T I C S T U D Y M IN TWO N A T U R A L l I F F O N V U L T U R E (GYP~ ~IDIVIDUALS H E L D IN ( WYz,* F. H. VAN DER BANK* and G. *Sanlam Research Unit for Environmental Conservation, [Dcpartment Chemistry and Biochemistry, Rand Afrikaans University, P.O. P. Box 524 South Africa. Fax: (011) 489-2411

DZYME I'IONS OF

~THERES) FY ad tDepartment of ; 2000, Republic of

(Received 24 February 1992; accep)ted 25 Mar A~traet--1. The detection of genetic variation by starch aand polyacr3 specie applied to the problem of preserving genetic variation in avian a 2. Allele frequency data, assessed at 34 structural gene loci, were q population genetic structure of Gyps coprotheres. 3. Measures of variability included percentage polymorp)hic loci (1 l, (0.021). 4. Two population simulation programmes were utilised utilis to pred variability present in this species. 5. The importance of the preservation of genetic variation variati in the C

INTRODUCTION he Cape vulture (Gyps coprotheres), is one of eight Th, ulture species found in southern Africa (Brown vulture ett al., 1982; Brown and Amadon, 1989). This species is the heaviest endemic accipitrid in southern Africa ?,obertson, 1986), belonging to the group of "Old (Robertson dorld", griffon vultures (Mundy and Steyn, 1977). World". 'he Cape vulture nests and roosts colonially on The cliff faces between latitudess 20 ° 07'S and 34 ° 22'S eds almost exclusively on (Benson et al., 1990) and feeds tgulates (Robertson and carcasses of migratory ungulates Boshoff, 1986). entury the Cape vulture During the nineteenth eentur was the most common vulture found in many mdy, 1982; Steyn, 1985). parts of South Africa (Mundy, ed as "vulnerable" in the Today G. coprotheres is listed ok--Birds and Birds (Tarboton i South African Red Data Book--Bir( Allan, 1982; Brooke, 1984~) and as "rare" in the ta Book (Benson et al., I.C.B.P./I.U.C.N. Red Data aumber of Cape vultures 1990). The decline in the number (Brown, 1985; Komen, 1988;I; Donnay, 1989)has been ascribed to various factors.s, of which several have chart, 1990). been induced by man (Butchart, The majority of recorded negative affecting factors )us human encroachment are caused by the continuous he birds. The elimination on the natural habitat of the rom the foraging areas of of indigenous game herds from arbanisation, industrialisthe Cape vulture due to urbanisat lopment, has led to ensuation and agricultural develo ich the birds have to cope ing food shortages with which trophy or metabolic bone (Butchart, 1989). Osteodystrophy disease has resulted in the Cape vulture since the number of bone-crushing spotted hyenas (Crocuta crocuta) and other large carmvores These carnivores provide adult Ca bone fragments, which are fed to tI

•trophoresis can be 5er to describe the :age heterozygosity ity of the current discussed.

lundy aand Ledger, 1976). additional calcium source (Mundy addit f¢ healthy skeletal The additional calcium is essential for rowth during the development of the 1 vulture chick. grow Due to the fact that hyenas and c~~rnivores are not and have ac~ comI)atible with most farming activities been eliminated from these areas, the supplemental habits have calcium fe calcil sources provided by their feeding ;nee of bone fragtherefore diminished. In the absen there ments, ment a calcium shortage develop)s and causes defortuities in the primary bones of the chick. This proves fatal during attempts to fly, if not during the maiden flight (Butchart, 1990). The lack of a natural ;vident in the crop source of bone fragments is evid contents of nestlings. Cape vulture nestlings regurgimechanism tate their crop contents as a defensive defe hers at 1their nesting sites. when approached by researchers as well as Fragments of bone china and clear c coloured glass are often found tol~gether with bone fragments in the crop contents. Such Suci foreign material may also cause internal injuries and an eventually lead to premature death. The frequency of such material nestlings was profoundly more in the Scheerpoort Sche xdoon et al., 1992). than in the Manoutsa nestlings (Ver~ critical consideration Poisoning of vultures is another crit of deliberate as the birds often become the victims vict Jaarsveld, 1987) or are poisoning by poachers (Van Jaars~ subjected to the indiscriminate use us of highly toxic substances by landowners (Vernon, 1987; Brown and Piper, 1988). Whilst perching on high-voltage electrical pylons short-circuits the wings of a vulture can cause cal 1984). Inexresulting in immediate death (Ledger, (Led perienced juveniles are also known 1to collide with the power lines (Steyn, 1985) adding electrocution to tors. Due to agricultural irs have become regular for vultures. In the event

ERIKA VAN W Y K et al.

reservoir, the bird's ad the sheer walls of scape. The birds are d ultimately drown turbance caused by helicopters, as well collectors and persedetrimentally affect These factors could ~ess within a colony. s one of the bestorld (Ledger, 1988; utchart, 1989), most researchers cannot reach a conusion concerning the severity of one, or all, of the miting factors the bird has to cope with (Tarboton nd Allan, 1982; Friedman and Mundy, 1983; ~omen, 1986; Benson et al., 1990). The fact remains mt the accelerating decline in parts of the popu~tion indicates that failure in curbing the current rate f mortality due to human interference could lead to le extinction of G. coprotheres within the next 60 ,~ars (Brown, 1985). In light of this statistic, urgent 9nservation attention is warranted for the Cape alture (Steyn, 1985). The first step in any management programme atails observation and experimentation to secure ackground information regarding the biology and abitat of the animal in question (Maguire, 1986). .esearchers have realised that in order to initialise a conservation 3nservation strate strategy for the Cape vulture, the influence ace of all the limiting factors should be accurately assa, ~sayed (Mundy, 1982). An aspect that has been n e~glected, however, as is the case with many other st~ecies (Cohn, 1990), is the analysis of the genetic structure ructure of the bird (Eitniear, 1989). All biologically important characteristics of populations .tions, including their size and productive efficiency, a r ~' e e determined by the historically established gene pools (Altukhov, 1981). The."adaptability of a species is dependent on the amount ant of genetic variation present in populations of the species, with which the animal can respond to environmental or biotic changes (Meffe, 1990). It has as been noted that a loss 'en rise to depressions in of genetic variation has given fitness traits of individualss (such as growth rate, survival and fecundity), thuss affecting the short-term berg, 1990). Therefore, in viability of populations (Leberg, order to ensure the long-term m survival of a species, a conservation strategy shouldd include the assessment and preservation of the aamount m o u n t and pattern of genetic variation within the species (May et al., 1975; Meffe, 1990). The conservation of genetic aetic variation within a natural population is a com~ nplex problem. The first step is to find an indication as to the amount, pattern r e n t genetic diversity; in and distribution of the current other words, to establish thee genetic structure of the involves implementation population. The second step involves ve these genetic resources of actions, in order to preserve for the future. No action cann be successful without a thorough initial understandin ing of the present genetic ndorf and Phelps, 1981). structure of a species (Allendorf Fluctuations in the size of breeding populations can be accompanied by a reduction in the ~enetic diversity (Shields, 1990) and the an variation can be severely reduced wit r

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of time (Ryma ). Keeping this in mind, it was decided the genetic structure of the Cape vultm assess whether or not the a m o u n t of gen, within the species could be considered a Lctor. Due to the fact that captive manage rimes are planned for the Cape vulture ( ), knowledge concerning the genetics ot is essential to maintain optimum varia he progeny. Gel-electrop] ~chnique which has been successfully ap past to accurately assay protein variati organisms (Avise and Aqu~ aadro, 1982 electrophoresis has been used less freql amine genetic variation withi within and an of birds than in other verte vertebrates (Gt 1983). Although studies of geenetic vari~ are steadily increasing, they are still i permit many kinds of com[ ~arative al son and Marten, 1991). The majority 1 o ,'tic studies of birds have, furth furthermore, b< able in many ways due to the Iuse of dis dques, non-standardised nom~ nomenclature t methods of data interpr ~retation (1~ The aim of this paper is to describe d~ th riation found in blood samp9les of th ture as determined by meas measurement c riation patterns. The resuits obtained from an electrophoret ~horetic analysis will be used to make predictions regard garding the survival cour,, of G. coprotheres. Furtherm course ermore, reference to appn appropriate avian literature was made throughout this study in order to ensure tha that the new data, obtai obtained for the Cape vulture, contribute c to the grow growing general fund of avian gen enetic information. r

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MATERIALS AND METH ODS

A total of 42 birds were sampled during duri 1990-1991. The samples were obtained from wild birds bir located in two natural populations and captive birds from four different institutions (Table 1). Referring to the t~ terminology used by Piper et al. (1989), the age classes of the vultures in captivity varied from immature birds (categorise orised into young- and nestlings, which were old-immature) to adult birds. The nes sampled on the nests, were more than 40 4 days old and fell into the N3-N4 nestling group. Disturbances at the nesting cliff are highly ill-advised during this period as adults may take flight, flig leaving the nests unprotected (Piper et al., 1989). Samp Sampling was, however, performed in such a way, that parent I~ birds appeared very calm and were often observed sitting in close proximity of their nest whilst their offspring were handled 1 (Verdoorn et al., 1992). vps coprotheres sampled Table 1. Distribution o f 42 specimens o f Gyp this st study at 6 localities during : this

Coordinates

Age groups

Localities

Latitude

De Wildt

Captive individuals 25° 41'S, 27° 56'E Immature--Adult Immature-Adult 26 ° 10'S, 27 ° 47'E 33° 46'S, 18° 48'E I m m a t u r e - A d u l t

Johannesburg Tygerberg World o f Birds

34 ° 0YS,

Longitude

18 ° 20'E

Immature-Adult

N 9 3 7 2

Natural populations Manoutsa

24 ° 26"S,

30 ° 41"E 5'E

Nestlings Nestlings Adult

7 13 I

Genetic variation in Cape Griffon vultt

ay is situated on the 0) in the Magaliesberg Hartebeespoort Dam approximately I0 km heetah Research and stitution is home to a volved in an extensive , 1988). Wild vultures, vided with food at the Station. Pretoria lies on cliffs formed by e Drakensberg escarpment in the eastern Transvaal ~undy, 1982). The colony is situated on the farm anoutsa which is located approximately 30 km south of oedspruit (Benson et al., 1990). The Kruger National Park s east of the colony and forms part of the foraging area the birds (Van Jaarsveld, 1988). The Johannesburg Zoo is located within the boundaries the Hermann Eckstein Park which is an approximate ,e-minute drive from the central city area of Johannesburg. Cape Province. The Tygerberg Zoopark is Cape Town's o and reptile park. The zoo is situated along the N1 route, tween Kraaifontein and Paarl. Whereas most captive tltures are housed in enclosures which contain other raptor ecies, the vultures in this zoo are managed in an exclusive ca. Attention is focused on establishing successful breedg pairs (J. Spence, personal communication), The World o f Birds is one of the largest bird parks in the )rid, with uniquely landscaped aviaries. The park is lOCateu :ated in the Atlantic coastal area of Hout Bay, which is situated uated approximately 20 km south of Cape Town. The two natural locations that were chosen as study areas were ~'re selected on account of the environmental differences presented esented to the respective populations by the surrounding are;eas. The Magaliesberg area is under direct influence of man in as it is in, in close proximity of major cities and is regularly fre¢ ;quented for recreational purposes. The natural foraging rannge o f the vulture is constantly declining due to extensive animal imal husbandry and the birds are largely dependent on vultur~ lture restaurants in the area as food sources, In contrast, the Manoutsa colon, ~lony is situated in a fairly natural area, detached from larg(e cities and severe industrial activities. The vast expanse of thee K r Kruger National Park and numerous game farms, found in the eastern Transvaal, ensure that the vultures are provided rovided with natural food sources. It has been noted that fewer in incidents of osteodystrophy and adult deaths are reported orted for these birds than is the case with the Scheerpoort colon olony (Mundy et al., 1980). We decided to determine whether ether a genetic limitation, in conjunction with other ne gative factors (Van Wyk, Van der Bank and Verdoorn, un republished data) could be contributing to this phenomenon. The birds in captivity were analysed to determine the amount o f genetic variation that: is irr immediately available to researchers, for establishing calc~tive breeding programmes, The ring numbers on captive birds irds were noted next to the sample numbers (list available on m request) to identify birds possessing high levels of diversit~ ity. Whilst sampling of the nestlings was underway, the opportunil ~ortunity was taken to ring the birds. Coloured tarsal rings were used in combination with a numbered metal ring. The numbers were also recorded for later reference. . . . . . . . .

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Sampling Due to the conservation status; of G" Gyps coprotheres, it was not feasible to sacrifice any birds Is for research purposes. It was therefore imperative that the,~sampling process involved a non-critical tissue type and that the ( caused minimal stress to the birds. Blood a favourable tissue source for electrop

researchers in th n, 1984). Birds have been od loss than mammals and reported to toler (estimated to be six percent 50-70% of total a from birds without longof body weight) Le use of feather pulp has term effects (Ev; rsden and May, 1984) but yielded satisfactc tdy, as sampling could not was impractical es of the birds. Due to the be coincided witl ~ed in the literature (Cooper variety of anticoa Corbin, 1978), three differet al., 1969b; Bar duate the respective preserent substances w, vation efficiencies samples were obtained by r et al., 1962) of the underbranchial venipul and 59) after disinfection o f the wing (Sibley l leg veins in large species is area. Although s recommended (E, ,' veins of the underwing are recom locate. A 5 ml sample was more visible and taken from each i directly separated into glass taken test-tubes and a test-tube. The glass tubes test-t~ respectively contl leparin (Barrowclough and respec Corbin, 1978), 0. 9% NaCI) (Manwell et al., Corbi: 1963) and eth etracetate (EDTA 10%) Corbin et al., I~ 1 ml blood was added. No (Curb anticoagulants w~ ae polypropylene test-tubes antico into 2 ml b] ,'d. The glass test-tubes were into which w immediately plac ice and the polypropylene imme( ansportation to the laboratubes into liquid anticoagulants were centory. The sampl (Evans, 1987) to separate trifug(ed for 10n dasma and red c la was drawn from the red plasm test-! The plasma, cells aand transferred to alternative test-tubes. red cells ce and whole blood samples were frozen and stored Vohs and Carr, 1969). at -- 22 0 ° C to await electrophoresis (Voh ,lU~illll

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Biochemical techniques Tw( Two methods of one-dimensional zone zo electrophoresis, namel:ty starch- and polyacrylamide gel-electrophoresis, gel-elq were applie, flied. Several similar genetic studies (Baker et al., 1966; Vohs and Carr, 1969) were also based oon these techniques. It is cc common knowledge that the compos msition and pH of the buffer system (Smithies, 1959), the size, size shape and ionic charg~ "ge o f the protein molecules (Johns lohnson, 1979) and the percentage, pH and composition of the gel (Ferreira et al., 1984) are variables affecting the separat ~aration and resolution qualities of the electrophoresis techniq ue. In this study, it was found that the anticoagulant used also played a role in the results obtained, especially while using acrylamide gel-electrophoresis. Therefore, a series of test gels were run to experiment with the different variables. variab A total of 29 enzyme systems were stained on both starch sta and polyacrylamide gels. Starch gels are more reacdily adaptable for enzyme localisation than polyacrylamide gels (Baker et al., 1966) and proved to afford better results resul than polyacrylamide gels in this study. In addition, samples showing variation were always run in several different di buffers, to ascertain whether the observed variatio ation was true or an artifact, as well as to find optimum conditions con for further experiments. (a) Horizontal starch gel-electrophore ohoresis. The standard protocol of flat bed starch gel-electrophc )horesis, as described by Evans (1987) and Grant (1989) was uutilized. The starch percentage was varied from 10% (Cooper )er et aL, 1969), 11% (Zink and Avise, 1990), 12% (Fergusl uson and Bamford, 1973), 13% (Vuorinen, 1984), 14% (Matson, (M," 1989b) and 15% (Quinteros and Miller, 1968). The 10% I and 11% gels proved to be most successful. After preliminary studies, using various vark buffer combinations to determine optimal electrophor¢ ~horetic conditions, five systems were isolated and applied (Table (Tat 2). The buffer systems were adapted from those used kby various authors molarity of the substances )ted from Shaw and Prasad 31). System 2 was adapted

ER1KA VAN WYK el aL Buffer systems used to resolve enzyme systems ir r

Electrode components

pH

Continuous buffer systems Tris 0.15 M 6.9 18.171 g/l Citric acid 0.05 M 9.456 g/I orate

3.

4a.

4b

Phosphate

Tris 0.18 M 21.052 g/I EDTA 0.004 M 1.48 g/l Boric acid 0.10 M 6.183 g/I

8.6

Na:HPO 4 0.10M 26.825 g/l NaH2PO 4 0.10 M 13.900 g/l

6•5

Discontinuous buffer systems LiOH 0.06 M 8.0 2•518 g/1 Boric acid 0.30 M 18.549 g/l

Lithium-hydroxide

Lithium-hydroxide

LiOH 0.03 M 1.259 g/l Boric acid 0.19 M 11.748 g/I

8.1

s

11 bt

50 ml

75 bL

50 ml

32 bu

52 ml

Tr 32 Ci 9.~ ele Di sol

pH

8.7

al

Tr 6.( Ci 1.-~ ele Dilute 35 ml of total solution to 350 ml

8.4

Note: All dilutions are performed with distilled water•

f r'ore o

Evans (1987) and Markert and Faulhaber (1965). S3ystem 3 was adapted from Harris and Hopkinson (1976). $5ystem 4a is the most widely used discontinuous buffer sy/stem for dehydrogenases (Grant, 1989) and is used by Manwell lanwell and Baker (1975). System 4b was adjusted from tat applied by Evans (1987) and Vuorinen (1984). tha (b) Polyacrylamide gel-electrophoresis (PAGE). Pro•d n r ~ d p ~ r r i h p d ffor n r vvertical ortlc,al ~ l a r t r c ~ t ~ h n r o ~ i ~ im n polyacrylnolv~c~rvl. electrophoresis cedures described electro amide gels by Ferreira et al. (1984) were followed• The "pore" size of the gel, which affects the separation of the protein molecule, is effected by the crosslinking agent, bisacrylamide (Johnson, 1979)•~. Therefore the a m o u n t of al acrylamide percentage was bisacrylamide varied, as the total acr altered in order to vary the gel concentrations. The gel percentages that were tested to, obtain optimum resolution were 5% (Johnson, 1979), 6% (Avtalion, 1982), 7% (Samways and Lelyveld, 1982), 8% (Ferreira et al., 1984)• Although satisfactory 1984) and 9% (Perrier et aL, 1984 results were obtained on 5% gels, the 6% gels proved to be optimal for this study.. The pH of the gels was adjusted by a Tris-HCl solution m to a pH of 8.9 (Samways and Lelyveld, 1982, Ferreira et al., 1984)• The running buffer solution was composed of Tris (6.0 g/l) and glycine rreira et aL, 1984). Different (28.80 g/l) with a pH of 8.3 (Ferreira sample volumes were tested. The best resolution was ts of 0.01 ml, diluted with obtained from sample aliquots 0.03 ml sucrose solution (40%).• The gels were run at 180 V for 30 rain and then set at 2500 V for the duration of the run (Ferreira et at., 1984), which ch lasted for approximately 7 hr.

Histochemical techniques Following electrophoresis, thee starch gels were sliced into sayed for a different protein. four slabs and each slab was assa, ;tains frequently gave weak Standard recipes for specific stains activity for these birds. Consequently, eoenzyme concentrations were varied conditions (Barrowclough and Corbin, 1 .

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Ha and Hopkinson were adapted from those detailed by Harris (1976), Ferreira et al. (1984) and Evans 1~ (1987). Two meth, methods were used to apply the stain to t the gels. The first method soh containing the meth~ entailed soaking the gel in a solution stammg components, followed by destaining dest the gel in a staini secon solution which was devoid of the staining agent second Grant, 1989). The second method was performed by using (Gral ~ % agar overlay in which the compaonents were mixed. a 92% This method is used for detection of the majority of enzyme polyacrylamide systems in starch gels (Evans, 1987). The "] gels were all stained by stain solutions• Staining was done for 21 different proteins and afforded g ood electrophoretic in bexokinase resolution (Table 3). Activity was also found f Table 3. Enzyme Commission Numbers (EC No.) of the proteins analyses in assayed and the abbreviations used for eleetrophoretic elect Gyps coprotheres Abbreviations EC No. Protein name Aspartate aminotransferase AAT 2.6.1.1 Adenylate kinase AK 2.7.4.3 ALB Albumin CK 2.7.3.2 Creatine kinase E 3.1.1.8 Cholinesterase EST 3.1.1.-Esterase GPI 5.3.1.9 Glucose-6-phosphate isomerase HB Haemoglobin 1.1.1.42 Isocitrate dehydrogenase IDH 1.1.1.27 L-Lactate dehydrogenase LDH 1.1.1.37 Malate dehydrogenase sMDH ME 1.1.1.40 Malic enzyme PEP(LA) 3.4.--.-Dipeptidas¢ PEP(LT) 3.4.--.-Dipeptidase PEPB 3.4.--.-Tripeptide aminopeptidase PEPD 3.4.13.9 Proline dipeptidas¢ enase PGDH 1.1.1.44 Phosphogluconate dehydrogena PGM 5.4.2.2 Phosphoglucomutase Aq Otl.da~_~tlnlmaeirl~ ~ h n esphorylase ~hnrvlae PNP PROT SOD

~

1

Genetic variation in Cape Griffon vul ies, band resolution was OVlanwell and Baker, electropherograms were graphed. The gels were Rion for 3 hr and dried servation.

o.e-

~ 0.4 ~ o.s

m ssion numbers, names allocated according to aational Union of Bio(I.U.B.N.C.) and the as proposed by Evans 1987) and Shaklee et aL (1989). We assume that our :lectrophoretic detectable variants at a locus differ genetially from one another and refer to them as alleles (Gutirrez et al., 1983). Alleles at each locus were designated Llphabetically by the electrophoretic mobilities of the gene ]roducts they encode, relative to the mobility of the most ommon allelic product at the locus. Allele products, exdbiting cathodal mobility, were indicated by a minus sign Avise et aL, 1980). In the case of multiple isozymes for a jven protein, the most anodal locus was designated "1", ¢ith more cathodal loci indicated by progressively higher mmbers (Yang and Patton, 1981). :tatistical analysis

The proportion of polymorphic loci (p), was estimated as ratio of the counted number of polymorphic loci to the 3tal number of loci examined and expressed as a percentage ~orbin, 1983). Although some authors label a protein etas monomorphic when the most common allele has a fre, •equency of 0.95 or greater (Manwell and Baker, 1975), a cut-off at-off point of 0.99 was used in this study (Evans, 1987). The enumeration of genotypes provided genotypic frequency uency distributions, which were used to calculate the observed bserved frequencies of alleles and the expected genotype fre( 'equencies were distributed according to the HardyWeinbe! eeinberg equilibrium for binomial expansion (Grant, 1989 989). Significant departures from Hardy-Weinberg proportions ortions were detected via the maximum likelihood analysis (G) 7)-test. This statistic is distributed as the Chi-square test and critical values can be foundd in in aa Chi-square table with the same degrees of freedom (Ferreira et al., 1984), where the rejection criterion for one deegree of freedom has a value of 3,841 (Stoker, 1977). The G-test was calculated with ,ith the formula: G = 2[~Oi. In(O/)~-~'Oi.ln(E/)] - ~'Oi. h )i. lnq where Oi and Ei are the observ~'ed and expected numbers of the individuals with a particular flar phenotype, respectively (Ferreira et aL, 1984).

0.8" i 0.8........................

0.4" i 0.1100

___

0,2

~ 0.1 0

Im

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80 SS ¢ERATION

40

60

the B FFrooueooy ~ 600; 8ot= O, Q2

Fig. 2. The pr( enetic variatio~ gene

of natural selection on the otheres over 50 generations.

TIT observed The lated as the rati( the individual i 1 that individual. was determined heter heterozygotes, l equit uifibrium disU for each c locus (] beterozygosity (l beter of of hl hl over all loc formula form~ of Nei

of individuals (hi) was calcur of beterozygous loci within umber of loci assayed for bserved heterozygosity (Ho) Lhe expected frequencies of calculated Hardy-Weinberg Limates of the beterozygosity )83). The average calculated ned by averaging the values ce, hi was determined by the hl = 1 - Z x i 2

where xi is the frequency of the ith allele at locus x. when Heterozygosity was depicted according to suggestions offered by Corbin (1983). T~ population genetic simulation programmes were Two used to illustrate the effects of stoch~ stochastic (random) and deten deterministic processes on the genetics of q populations. For this purpose l the selection coefficients (s) were calculated from the formula: s=l-w

where w represents Darwinian fitness or o relative reproductive efficiency (Ayala, 1982). The geno genotype producing the highest reproductive efficiency was assigned a fitness of one and was employed to determine w value values for the remaining genotypes, according to methods followed follc by Price and Boag (1987). The first programme (DRFTMUSL).) utilises the population size, mutation rate and select selection coefficients as parameters, in order to simulate the ccombined effects of random genetic drift, mutation and natural selection (Fig. 1). These microcomputer simulatition programmes of Prof. M. Gilpin (Dept. of Biology, University Univ of California at San Diego, La Jolla, CA 92093) were used to predict the probability of survival in Gyps coproth~ vrotheres due to genetic variability. The selection coefficients as well as a the initial allele frequency of the B allele are specified in the second programme (SELECTIN). The effect o f , natural selection, a deterministic process, on a two allele sy.,stem is modeled by this programme (Fig. 2). Each run was repeated r( 10 times to obtain a reliable average value. RESULTS

6

10

lS

2O 2S SO

ss

(o

(s

6o

C~NERM~

n-I 600; u-O000000~, s'O, 0.237, 0 3 3 3

ve effect (nf randnm oan,..tie Fig. 1. The predicted cumulative drift and mutation on the genetic vari~ protheres over 50 generati(

Electrophoretic conditions

(a) S t a r c h gel-electrophoresis. O OIf the 21 protein systems that were analysed, 17 were w resolved o n hvdrnlwe.d ~t~reh T h , , ~ ~.,,~,,,,,,,~ ;, ymes include AAT, AK, d E , NP, the peptidases )NP. A l b u m i n s (ALB)

ERIKA VAN WYK

et

al.

was stained for E d SOD banding also ar overlay technique the exception of E olutions. ate isomerase (GPI), ctorily resolved by oteins were stained )ed by Grant (1989) ~, as anticoagulant erns. Haemoglobins run but could also e detected after gels were stained for PROT. The ,'paration of EST during P A G E was good but starch el-electrophoresis gave similar results and was used )r the remainder of the study. (c) Histochemical buffers. Three different stain uffers were required for the respective staining mixires. The pH and molarity of the buffers were varied zcordingly. Tris was used as main- or co-component t most situations but a phosphate buffer also proved seful (Evans, 1987). Overlays were prepared by lixing 15 ml Agar with volumes of 10-15 ml buffer. he combinations of variables yielding optimum ectrophoretic conditions for enzyme resolution for 3 of the protein systems are listed in Table 4. rotein variation

(a) Variation at loci. Structural enzymes yielded clear ear banding patterns for a number of 34 genetic loci ~ci. Proteins encoded by multiple loci and polymorphic hic loci are given in Table 5, along with the relative allelic frequencies for the loci. The remaining loci [ A A T * , A L B * , E*, H B * , PEP(LA)*, PEPD*, IDH*, sMDH*, ME*, PG GDH*, D H * P G M * and PNP*] are encoded by a single

loct ~cus and no variation was observed. The percentage polymorphic alymorphic loci (P) was calculated at 11.76% for isted in Table 5. the four polymorphic loci listed (b) Heterozygosity. Deviation from expected as for a natural panmictic Hardy-Weinberg proportions hree of the polymorphic population was found at three :us that confirmed to the loci (Table 6). The only locus parity was E S T * , where thes value of the G-test was calculated at 0.454 with a deLegree of freedom of one. erozygosity (Ho) was deThe average observed heteroz' he mean population bettermined to be 0.039 and the ated as 0.021 (+0.0113). erozygosity (He) was calculated

Table 4. Electrophoresisand rprotein staining conditions Eleetrophoresis Protein systems Buffer pH Anticoagulant AAT 4b Phosphate (0.1 M) 7.5 Wholeblood AK 3 Tris-HCI (0.3 M) 8.0 Wholeblood CK 2 Tris-HC1 (0.3 M) 7.1 Wholeblood E 4a Phosphate (0.2 M) 7.1 Heparin EST 2 Phosphate (0.2 M) 7.1 Wholeblood GPI * Tris-HC1 (0.3 M) 8.0 Saline IDH 1 Tris (0.2 M) 8.0 Wholeblood LDH 4a Tris (0.2 M) 9.0 Wholeblood EDTA MDH * Tris (0.2 M) 9.8 ME l Tris (0.2 M) 9.8 Wholeblood PEP 4b Phosphate (0.2 M) 7.5 Wholeblood PGD 2 Tris-HCl (0.3 M) 7.1 Wholeblood PGM 2 Tris-HC1 (0.3 M) 8.0 Wholeblood Eleetrophoretic systems are described in Tabl • PAGE as described in Material and Method

Table 5 poly

=ies of multiple and

Locus Ag-l*

."

Gyps coprotheres

Frequency 1.000 1.000

AK-2* AK-3* CK*

0.564 0.436 0.951 0.049 0.613 0.387

EST* GPI-I*

1.000 1.000 1.000 1.000 I .oo0 1.000

GP1_2o LDH-1 LDH-2 LDH-3 LDH-4 PEPBPEPBPEP(L

1.000 1.000

0.763 0.237

SOD-I SOD-2 SOD -3 SOD-4 PROT. PROTPROT. PROT.

1.000 1.000 1.000

1.000 1.000

1.000 1.000 1.000

outer simu stmulattons Com~Tuter sel intensities (a) Selection coefficients. The selection

Lained for relative were determined by values obtai fitness fitnes (Table 7), using the genotyr tic data obtained t by Price from the electrophoretic study as outlined and Boag (1987). Computer simulation programm ramme 1. This proimulates the combined gramme ( D R F T M U S L ) simulate effects mut~ and natural effect of random genetic drift, mutation selection. Population size is entered as a whole numselecl ber, mutation rate as a decimal and the selection coefficients as decimals separated by b) commas. Values for s from Table 7 are entered into the programme, along with an estimated value for electrophoresis of (u). By default, the 0.0000001 for the mutation tempo (~ allelic frequency of each run starts at 0.5. The estistudied mated population size (n) of the individuals in in the colonies is 1600. Accordin Lg to the results (Fig. 1), the genetic variation within the Cape vulture population will reach zero-levels within the next 35-40 generations of the birds. Computer simulation programm ramme 2. This programme ( S E L E C T I N ) models natural nat selection, a deterministic process, on a two-allele two-al system. The selection coefficients (s) are specific ~ecified, as well as the initial allele frequency of the B allele. allel This value was calculated to be 0.0326 from the ddata obtained via the effect electrophoresis. The programme simulated sin natural selection will have on the B allele over 100 Table 6. Loci of Gyps coprotheres exhibiting significant deviations (P ~<0.05) from Hardy-Weinberg expectations ¢ Locus AK-3* CK* PEP(LT}*

Heterozygotes observed 34 0 0

Expected G-Test 19.179 29.840 3.805 15.983 13.737 41.603 instances and the critical value

Genetic variation in Cape Griffon vultt for the genotypes present ic variation in adults and

AB 3

nB 1-0.667 0.333

locus, s. indicate that the B than 35 gencrations, DISCUSSION

rlood as sample source The four most common tissue types analysed uring allozyme electrophoresis of avian species are sually obtained from the muscle, heart, kidney and ver of sacrificed individuals (Johnson and Marten, 991). Egg white proteins have given favourable .'sults (Baker and Manwell, 1967) and research has ven included the use of beak proteins and feather ulp (Matson, 1984). A combination of two or rare tissues is employed in most cases to enable tamination of a maximum amount of proteins, lood (haemoglobin, plasma and serum) has been the 9urce of many studies. A literature survey has r e;vealed v e a l c o [that n a t tthe h e majority of researchers have concen•ated on a specific fraction of blood (Mueller et aL, trated 962; Manwell et aL, 1963; Ferguson and Bamford, 1962 1973 973). There seems to be a paucity in publications ealing with data acquired for haemoglobin, plasma dealin nd serum in combination. With this in mind, in and ddition to the fact that blood was the only tissue addition available vailable for this study, the selection of proteins ssayed was random. An attempt was made to score assa' all !1 detectable loci present in the samples. Evans (1987) tabulated a revnew of'past avian studies, where the number of loci examined :d ranged between 14 and 46. Included in the summary¢ ((Evans, 1987) is a survey conducted on the heart, liver :er, muscle and serum of zh revealed 31 loci in total four species of parulids which (Barrowclough and Corbina, 1978). It can thus be inferred that blood, which gave 34 protein coding loci during the present studdy, is an excellent tissue source when analysed under~r optimal electrophoretic conditions. I ~ f l ~ x

z _ l _

_ l _ z _

a

--

_.]_

_ P

. . . .

z

_

_] . . . .

.__

] r

.

.

.

.

I . . . .

Banding patterns No electrophoretic datat from previous studies of G. coprotheres could be.~ obtained. The banding patterns found in this stud,ly and the interpretation thereof, is therefore compared red with results obtained for other bird species and other vertebrates, The order Passeriformes consists :onsists cof 75 families and is the largest of the 27 aviann orders. Birds belonging to this group are generally referred fferred to as "passerines". The remaining 26 orders, ofbf which the Cape vulture is classified under Falconiformes ~rmes, are represented by 99 families of "non-passerines" Les" (Howard and Moore, 1984). The majority of aviann ~ genetic studies has been conducted on passerines 0(Guti6rrez et al., 1983). nn~ h a v e b e e n i n c l u d e d in Representatives of both groups have comparisons between other species Cape vulture. The enzyme subunil

derived from t] ledged by Evans (1987). From the comt follow, the Cape vulture r a number of protein exhibits monol coding loci wh ly found to be polymorphic in avian s Aspartate an :e. This enzyme appears to be encoded 1 ~mal loci, sAAT*, which F*, which migrates in the migrates anoda al., 1980). One intense cathodal direct ,~r a 24-hr staining period zone of bandinl in the anodal r aably consistent with the sAAT* locus th passerines and non80) reported two AAT* passexines. Avi bited three banded hetloci, of which rphic for two species of eroz3:ygotes and passe passerines (Mu ld Mimidae). This strutbrines, where variability ture is also fol [y at the mAAT* locus was detected i Lese results confirm that (Gut: ~uti6rrez et AAT L AA'[ is of dim zones of activity were Adenylate k A~ buffer system (Table 2, resolved on th resol )f the results was verified No. 3) i and the the Tris-ci (Table 2, No. 1). It is on tl ;sumed that ~ding areas are the prodprest el. Adenylate kinase-l* ucts of three ~phic for all individuals. and AK-2* we Aden Adenylate kinase-3* was polymorpl~hic and displayed double-banded heterozygotes, consistent con with the doul: ported monomeric structure of~the enzyme. Not repol man:y avian studies which were nreviewed included AK. Cole and Parkin (1981) stated that other species to exhibit several encoding em A K loci. are known i Theyy identified three areas of stair staining in the house ~arrow (Passer domesticus) but failed fail to confirm the sparz existence of three loci in this pas passerine. Adenylate exist~ tse-2* and AK-3* were examined examir kina~ in the brown trout (Salmo trutta; Pisces), (Ryma ,man et al., 1979). In his review, Evans (1987) listed A AK K as being monomorphic for 22 avian taxa in an analysis an of 23 taxa. Albumin. One single band of homozygotes 1~ was for E. In detected anodally on the gel stained st~ this study, no variation was obs ~bserved in the 42 individuals examined. Albumin* was w also noted as the most anodal locus on gels stained st~ for PROT. Egg-white ALB are fractioned into inte various staining areas by Baker and Manwell (196 (1967), which proved to be polymorphic for the migra gratory or common quail (Coturnix coturnix) and the ring-necked pheasant (Phasianus colchicus L.).) according ac to Baker et al. (1966). Both of these speci :ties belong to the Galliformes and are representatives of non-passerine birds. One monomorphic, anodal locus is reported for two passerine species; the Chin~golo, or Rufous, collared sparrow (Zonotrichia cap, ~ensis) in a study by Nottebohm and Selander (1972) (197: and species of Galapagos finches (Yang and Pa Patton, 1981) after analysis of heart, liver, kidney and muscle tissue. Creatine kinase. At least two encoding e CK loci have been reported in birds, of which wt one is found predominantly in brain and heart hear tissue and the other in skeletal muscle. Freezing and a thawing could lead to two zones of activity at a single zone of h e a r t C I ( ( A r i s e at a l t0Rfl)). T h ~ e authors resolved These Mimidae and Vireonidae ttion between species of L ] _

--

E R I K A VAN W Y K et

). Both the heartd monomorphic in ~idonax hammondii) en (1991). One area towards the anode was found to be e. 3rotein is not menrveyed during this trate specificity that EST (Baker et al., mainly in plasma r serum and is probably a tetramer (Evans, 1987). wo autosomal loci were reported by Harris and [opkinson (1976). In this study one monomorphic ~cus, namely E*, was found migrating slowly, aodally. This result coincides with results obtained ~, Lawrence et al. (1960). Esterase. The review of avian studies presented by vans (1987), verifies the existence of four genetic acoding E S T * loci. Of 77 taxa studied, 44 proved to polymorphic for EST-I*. Thus it can be derived mt EST is a highly variable enzyme, which is also ue for fish taxa (Mulder, 1989). Products of the liver ~ecific Est-l* migrate rapidly towards the anode; roducts of the heart-predominant Est-3* migrate tthodaily and a third zone, EST-2* can be found roving anodally at a slower pace than E S T - 1" (Avise e l l al., 1980). Two zones of activity were found in the Caape vulture, but only one was accurately and consistentl' stently scorable. This EST migrated rapidly towards the te anode and is probably homologous to the EST-1" r e :gion described in many studies. This locus dislayed polymorphism, the other zone, supposedly played E5ST-2* showed variants which could not be scored )nsistently and was thus excluded during the hetconsistentl erozygosity calculations. None main regions of serum-EST was detected n ~sant• and• considerable for the ring-necked pheasant quantitative and qualitative individual variation was reported (Baker et al., 1966).. Variation at the E S T - l * and EST-2* loci was found during the analysis of 10 species of Galliformes (Gutirrrez irrrez et al., 1983). In the lines ;eriformes), only E S T - I * New Guinea starlings (Passeriforme could accurately be recordedi in plasma, heart, muscle and liver. This locus was pollymorphic in both species ~nted by three and six examined and was represented orbin et al., 1974). phenotypes, respectively (Corbin erase. One monomorphic Glucose-6-phosphate isomerase. locus of GPI was scored in this study. This enzyme vo structural loci in most appears to be encoded by two fish species (Mulder, 1989) but most avian studies reveal only one controllinlg locus. Variation was forms at GPI* (Gutirrrez found in two species of galliforms sm was also found in et al., 1983). Polymorphism Zonotrichia capensis (Nottebohm and Selander, amorphic for five species 1972). This locus was monomor !., 1982) and for Passer of Vireonidae (Avise et al., domesticus (Cole and Parkinn, 1981). Haemoglobin. The haemo~globin of Phasianus colher galliform and anserichicus resembles that of other form birds. These haemc ~globins consist of two Lponent, which migrates components; a major comp ~tinn A slightly in the cathodal direction arn d ~ r n i n n r t w a i n ponent which migrates slowly, anc same electrophoretic conditions. Tt

al.

and blood appc )nservative characteristic in the pheasan ,' major HB* locus only one of 129 bir howed deviation (Baker et al., 1966). t bands were shown on zymograms sco American thrushes and their allies, on] tal band could be interpreted accurate table mobility differences occurring only cies (Arise et al., 1980). Isocitrate deJ This enzyme is encoded by two loci in 3dal migrating sIDH-I*, is the soluble 1 9rotein, whilst the mitochondrial form grates cathodally (Evans, 1987). 198T One ze ty was detected in the cathc cathodal regio~ duals of the Cape vulture and is i: presumal us to the mIDH-2* locus. Lactate La dehy is a well-established fact that birds 1 and ssess five major forms of LDH. ~r, which results from the LDH Each for rand~ random combi Jo types of polypeptide subunits, know * (muscle) and L D H - H * subu] (heart) r/-2*. An extensive poly(hear or LDI~ the L D H - H * locus of morl:~hism was AploJ olonis metallid ). Among 357 specimens were observed and acsamp9led, five hat three allelic forms of coun counted for by synthesis of the heart the L D H - H * Lonomorphic for all but subu].nits. This two of coJ (Corbin c 108 individuals of Aplonic eontoroides et al., al. 1974). This proves the usefuh :fulness of LDH as a biocl~ biochemical marker to distinguish between species Mulder, 1989). Matson (1989) identified id a testes(Mul mbiformes and points )ecific locus L D H - X * in Columbif speci: in most vertebrate out that t] LDH is routinely assayed in population genetic and systematic stematic studies. Serum popu LDH is reported to migrate towards towa the cathodal on in Galliformes, but the resolution rest was poor regio pands were difficult to and individual i LDH isozyme band,. detec:t (Baker et al., 1966). A total of five loci was _~ brown trout (Pisces; examined in populations of bro~ Salmo trutta) and the absence of heterozygotes at L D H - I * was ascribed to the lack lac of gene flow between two demes of the species (Ryman et al., 1979). The present study revealed four areas of banding, one migrated of which three appeared anodally and a cathodally. At each zone staining occurred at the could be same intensity and no mobility differences difl was therefore predetected between individuals. It w ~resented by a single, sumed that each zone was represe homozygous locus. locus, Malate dehydrogenase. One monomorphic mor migrating anodally was detected in i~ G. coprotheres. 3us to the anodally This locus is presumably homologot migrating, soluble form ( s M D H - I * ), of the enzyme. migration The second form, which exhibits cathodal ca (mitochondrial mMDH-2*), was not encountered in this study. The fact that the s M D H - I * locus for this was monomorphic is not a rare occurrence oc enzyme, as it has proved to be ver y conservative in et al. (1974) birds. This has been reported by Corbin C¢ and Avise et al. (1980). Where Wher~ variation was recorded, it was usually detected at a the mMDH-2* locus (Avise et al., 1982; Yang and an Patton, 1981). ~ q n e ~ i e ~ n f t h e t ~ e n u ~ . 4 m m o odramus, dramu,, however, were 14DH-I* locus (Zink and

Genetic variation in Cape Griffon vultut not well-reported isted the two loci tributed in 13 taxa h exhibiting partial n North American he- 1" was found to birds (Avise et al., between species of k/E-l* locus is the mitochondrial form solated from liver, (Evans, 1987). One ~nomorphic locus was found in this study. Peptidase. There are a minimum of six presumptive ne loci encoding for peptidase in birds. Products most loci have multiple, overlapping substrate ~nities (Matson, 1989a). The product obtained }m utilising the dipeptidase leueyl-alanine, PE'~LA)*, appears as a monomorphic locus which igrates rapidly towards the anode. The dipeptidase bstrate leucyl-tyrosine PEP(LT)* results in a zone activity that is polymorphic in the Cape vulture, ae heterozygote displayed two bands, which is nsistent with the monomeric structure reported for e enzyme (Mulder, 1989). Peptidase-B* is the oduct of the tripeptidase substrate leucylglycylycine (LGG). This peptidase appears to be rela~ely substrate-specific in avian species (Matson, )89a). Two zones of activity were recorded in this stud' ady and the presumption was made that these areas w e~re r e rrepresented e by two encoding loci, PEPB- 1* and PEPB-2*. EPB-2*. These electromorphs migrated slowly in thee anodal direction. The products of PEPD* exhibit a high degree of substrate specificity. This peptidase acts :ts only on carboxytermal prolines (in this case phen aenylalanyl-proline). Due to the specificity of the pel,'ptidase, a separate EC number of 3.4.13.9 was all( lotted to PEPD* (Matson, 1989a). This locus was monomorphic for the present study and was found to have a slightly faster er anodal migration than PEPB*. In the house sparrow (Passer domesticus), five zones of activity were detected ed when LA and LT were used as substrates. Two of thtese zones are represented by two polymorphic peptidases, Lses, whilst two cannot be accurately resolved. Peptidase-B* re-B* appears to be polymorphic and reveals double-ban .le-banded heterozygotes (Cole and Parkin, 1981). Ina the Chingolo sparrow, two systems appear when LA is used as substrate, Peptidase(LA)-l*, which mi migrates further anodal than PEP(LA)-2* is found too be weakly polymorphic (Nottebohm and Selander, 1972 1972). Two loci are found to control the expression of )f the peptidase product when LT is used as substrate te in six genera of Cichlidae (Pisces) according to Van B~ m derr Bank Bank, et al. (1989). Other than the mention of'this substrate in results obtained for the house sparrow, row, no specific reference to the products of this substrate :rate is found in the avian literature surveyed. Barrowclou flough and Corbin (1978) used L G G as substrate to det etect PEPB* products in the Parulidae. Matson (1989a 1989a) observed tissuespecific mobility in the PEPD* 'D* locus but scored only one encoding locus. This author thor men mentioned the possibility that two PEPD* locii mav .y exist in birds, Phosphogluconate dehydrogenase. consistently one of the most poly PIlD

I I'l'~ll/~M

n birds. Although the systems routine by one gene locus only, protein appears~ ~nomorphic in 49 avian this locus was f¢ ther taxa (Evans, 1987). taxa and polym( s, represented by threeA total of nine round in Muscicapidae banded hetero~ e (Avisc et al., 1980). and one specie Genetic variatio )H* locus was detected and Vireonidae (Avise between species et al., 1982), P, rowclough and Corbin, 1978), individl tsser domesticus and Zonotriehia cat dae), (Nottebohm and Selander, Selam 1972; kin, 1981), Columbidae te (Fleischer et al., 1991), (Coo[ )er et al., 1 Phasi; Phasianidae ant (Guti~rrez et al., 1983). Johnson Johns and M: vported monomorphism for PGDH* P, in als of Empidonax hammond~ mondi (Tyranni )ne of activity was detected namely. tected, G. coprotheres and was also interpreted fi zomorphic in this study. Phosphogluco P& ee loci were isolated from muscle ar cts of parulids. A total of 15 species v [ and PGM-I* showed variat variation betw( ~ies, PGM-2* between nine species aJ between three species (Barr( 'Barrowclough 1978). Three loci were also identified] i m and Selander (1972), but the tl majority of examinations re..ported two loci. This iis found to be consistent with the summary of Evan~ (1987). In this study a singl( single, monomorphic locus was resolved. Pu~ Purine-nucleoside phosphorylase. High levels of polyn polymorphism were exhibited in sp¢ ~¢cies of thrushes, where the heterozygote was repr( where ~resented by four band, typical of a trimeric structure bands, struct~ (Avise et al., 1980)). This result coincides with the summary of P N P distribution among avian taxa, PNP* ta~ where 11 out of 24 taxa were polymorphic for this thk, enzyme (Evans, 1987). At least two phenotypes were wer detected in the house sparrow and were termed ked fast or slow, but were interpreted as a single homogeno genous genetic locus (Cole and Parkin, 1981). Variation was I found at five populations (out of nine) for !the Hammond's flycatcher (Johnson and Marten, 1991). 19 In the Cape vulture PNP was resolved as a singgle monomorphic zone of weak activity, migrating ra[ rapidly towards the anode. General proteins. Ferguson and Bamford (1973) resolved the plasma PROT found in Columba species into 23 fractions. Baker et aL (1966) (1~ reported 21 fractions in the plasma of ring-neck( ~-necked pheasants and compared the fractions as similar to t those found in the chicken. Three zones of P R( PROT activity were accounted for by Avise et al. (1982), where PRO T- 1" was the only locus showing variat variation. Intraspecific variation was found in species of ol the Galapagos finches at PROT-I* (Yang and Patton, 1981). Although several zones of activity were resolved on gels stained for PROT, only fou four non-enzymatic PROT* loci appeared consistent an could be accustent and rately scored. No variation was vas dete detected at any of the loci assayed in the Cape vulture. Superoxide dismutase. Two zone,, zones of activity were detected in the house snarrow, botl~ appearing as an ,w, both anodally migrating subreports for other species

E R I K A

V A N

No loci (mitochononly the latter is Passer domesticus :i were reported in ~, where SOD-I* and SOD-2* was 990). From the list and SOD-2* are enzyme appears to ccurrence of polyand no account of ybrid goldfish have en reported to have four loci encoding for SOD anzmann and Down, 1982). Three anodal zones d one cathodal zone of activity was detected in the :sent study. Once again, the four zones exhibited nilar intensities. No sub-bands or mobility differces were detected. Four encoding loci were thus :sumed. . . . . . . . . . .

o .

.

.

.

.

.

.

.

.

.

.

.

.

.

J-

-

w

,viations from expected Hardy-Weinberg equirium As can be seen in Table 6, three polymorphic :i deviated significantly from expected H a r d y einberg proportions. The deviations at the CK* d PEP-(LT)* loci were represented by a deficit the heterozygotes observed whereas the deviation the AK-3* locus was caused by an excess. Various :tors can contribute to deviations from expected zactors Hardy-Weinberg proportions. The most likely cause for deficiencies in the amount of heterozygotes, is the absence of panmixia in a ecies ((Barrowclough and Corbin, 1978). The breedspecies ingg biology of the Cape vulture suggests that the birds aly represent a non-random mating system. When trub male remains with a single female for at least the lenagth of the breeding season, the male is termed nonogamous". This male is then excluded from "mono the pool of available mates~, thereby affecting subsequent mating frequencies (Findlay, 1987). Cape assified as monogamous vultures can definitely be class due to the fact that matinag is generally for life. A species is considered too be philopatric when they remain faithful to at breeding territory or he chances of inbreeding colony, which will increase the 2ape vultures are known (Greenwood, 1987). Adult Ca tes of the previous year. to reoccupy their nesting sites rents until independency Progeny remain with the parents is reached (approximately six months) after which they leave the colony (Mundy, 1982). In the hey reach sexual matufour to six years before the' rity, the young birds may wander far from their birth site but are reported to return and breed at their natal colonies (Pickford ett al., 1989). Inbreeding explanation for the curcould thus be a plausible ex :epancies as this usually rent Hardy-Weinberg discrel ~mozygotes (Greenwood, results in an increase of homoz, 1987). tmpling error may have The possibility that a sam aould not occurred during this study should no be ignored. The factor responsible for this error is most likely the 983). This occurs when Wahlund effect (Corbin, 1983 individuals having differenttt allelic frequencies are included in a single sample (Grant. (Grant, 1989). The oresence of both immature and adult bir for the Wahlund effect as well as t a

W Y K

et

al.

nd female individuals in uneven distribut the population,, , 1987) and selection Genetic mut~ existence of null alleles against heterozy 978) are also conceived (Barrowciough~ -Weinberg deviations. as foundations nutation rate in birds, Little is known excluded as a causal thus this phen 1984). Selection against factor (Barrowc Lo stable polymorphism heterozygotes d~ 1978). The presence of (Barrowclough conforms to expected the EST-I* 1o s is an indication that Hard) r-Weinber ~t have occurred. It is, couk this event e ment as only one stable however, not a howe~ Nas found in the Cape polymorphism polym Corbin (1978) reported flture. Barro~ vultur in avian studies, thus of the absence al arrence of such alleles as tentatively exem tentat deviations found in this plausi )lausible explan study. Dm variation found at the Due to the f the expectations of the ~T-I* locus c ESTon in the present study, Hard,~¢-Weinber EST alleles were, and ~ossibility e the p~ t. This was suggested by still are, al selectiv LDH-H* and the EST* Corbin et al. (1! Corbi found in starlings of the genus Aplonis. loci f( . . . . . . . . . . . .

x . . . .

J

-

Polymorphism and heterozygosity :tween the percent correlation exists bet~ A positive l emorphism and the overall level of genetic varipolyn abilit~y within a taxon (Corbin, 1987)). A high percenta high value retie age of o polymorphism generally reflects Values for these thes entities in G. in heterozygosity. he ~rotheres tend to be lower than the majority of copro percentage values av valuer listed by Evans (1987). The average ,morphic loci calculated for 174 bird species was polyn found to be 24.0% (Evans, 1987), mc~re than twice the amount (11.76%) calculated for the tl: Cape vulture. trrent study can be The estimate for P in the currer considered accurate based on the fact that ~olymorphism depends the accuracy of percentage polymc and on the number on the number of loci examined an~ mmum of 14 loci is of individuals sampled. A minimu commonly taken, although at least 20 is advisable, and a minimum of 30 individuals iis recommended, but preferably at least a 100 should shouk be sampled for lculated heterozygosity two or more regions. The calculate for 174 bird species averaged 0.044 0.04 (Evans, 1987); once again, this amount represents more than twice the value (0.021) calculated for G. coprotheres. Cape vulture The low genetic variability in the t Ltions for this species. could have the following implication As mentioned in the introduction, the t loss of genetic resslons in fitness traits variation can give rise to depressior may explain of individuals (Leberg, 1990). This Tl the presence of low reproductive success and the )orted by Komen associated mortality factors repo (1986). Long-term adaptability of populaitions and species is dependent on a base of genetic varriation with which to respond to environmental or biotic novelties (Merle, 1990). An example worth nmentioning in this ker et al. (1966). Results ;vealed that resistance to

Genetic variation in Cape Griffon vultl a high degree of ace it was suggested ads to the cheetah's s against the feline ). The occurrence of sidered examples of ailure to adapt may ttinction, Jlation programmes apearance of genetic ariation in a relatively short time-span. The first rogramme ( D R F T M U S L ) is based on the assumpon that the alleles are distributed equally within opulations. The result plotted in the second graph , however, a more realistic representation of the 'ue situation due to the fact that equal distribution f alleles in natural populations is not a c o m m o n henomenon (Grant, 1989). The results in Fig. 2 tcorporate the calculated allelic frequencies of the resent electrophoretic analysis. The variable alleles Lay be lost in less than 35 generations, although atural selection may be favourable for the remaining eterozygotes. In the absence of genetic variation the ~ecies will be left vulnerable in the face of extreme avironmental changes. Observed monomorphism for G. coprotheres at loci sually polymorphic in birds, the comparatively low usuall :vel of heterozygosity (less than half of the value level veraged for 174 bird species) and the predictions aver~ resultin ,'suiting from the computer simulations all point to a frailty in the genetic structure insofar as adaptabilit~ ility is concerned. It appears that birds located ini captivity (in particular at Tygerberg Zoo), possess le highest level of genetic variability. Cooperation the etween areas practising captive management probetween g.,rammes of this species is essential. This is of great importance as a time ma y come when the Cape vulture will be extinct in the,' wild and survival of this species will depend on successful bre ssful breedin breeding procedures in zoological parks and wildlife tdlife preserves. In anticipation of this event, the hitighest possible variation of levels found in currentt populations should be maintained in birds held inI captivity. 3rs would like to thank the Acknowledgements--The authors ironmental Conservation for Sanlam Research Unit for Environment~ financial assistance during this study. We would also like to ~rd Burroughs, Ms Anne van thank Dr Lynn Colly, Dr Richard )anie Terblanche, Mr Albert Dyk, Mr Walter Neser, Mr Danie dr Richard Froneman, Ms Ingrid Becker, Mr Richar ~hard Anker-Simmons, Anker-Simmons [angold and Mr Koos Meyer Mr John Spence, Mr Walter Man ance duri during durin the collection of for their cooperation and assistance blood samples from Cape vultures. REFERENCES

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