(Gyps Bengalensis, G. Africanus, G. Indicus and G. Fulvus) Based on Microsatellite Analysis

J. Raptor Res. 43(3):227–236 E 2009 The Raptor Research Foundation, Inc.

GENETIC VARIATION OF FOUR GYPS SPECIES (GYPS BENGALENSIS, G. AFRICANUS, G. INDICUS AND G. FULVUS) BASED ON MICROSATELLITE ANALYSIS

MUHAMMAD ARSHAD,1 INKEN PEDALL,JAVIER GONZALEZ, AND MICHAEL WINK Heidelberg University, Institute of Pharmacy and Molecular Biotechnology, INF 364, 69120 Heidelberg, Germany

OHAD HATZOFE Science and Conservation, Israel Nature and Parks Authority, 3 Am Veolamo Street, Jerusalem 95463, Israel

ALEEM AHMED KHAN Institute of Pure and Applied Biology (Zoology Division) University Multan, 60800 Pakistan

TIM OSBORNE Tandala Ridge, P.O. Box 22 Okaukuejo, via Outjo, Namibia

ABSTRACT.—Although several phylogenetic studies of Gyps species have been conducted, few studies have addressed the genetic diversity of these species on a finer scale such as microsatellite analyses. We collected samples of migratory adults and nestlings from four species of vultures in six different localities. We analyzed the samples using microsatellites in order to determine the genetic distance as well as the amount of variation within and among Gyps species populations. Low genetic diversity in Long-billed Vultures (G. indicus) was probably indicative of a single population with no immigration and low gene flow. As this species is critically endangered, future conservation programs should consider genetically suitable stock for a breeding and reintroduction program. High genetic diversity in African White-backed Vultures (G. africanus) was likely indicative of a number of populations, with immigration and gene flow. We confirmed previous findings of low genetic differentiation among Griffon Vulture (G. fulvus) populations, which indicated high mobility and gene flow among these populations.

KEY WORDS: Gyps; genetic variation; microsatellite analysis; vultures.

VARIACIO´ N GENE´ TICA CON BASE EN ANA´ LISIS DE MICROSATE´ LITES EN CUATRO ESPECIES DEL GE´ NERO GYPS (G. BENGALENSIS, G. AFRICANUS, G. INDICUS Y G. FULVUS)

RESUMEN.—Aunque se han realizado varios estudios filogene´ticos en especies del geńero Gyps,so´lo unos pocos han abordado la estructura gene´tica de esas especies a una escala ma´s fina con un ana´lisis de microsate´lites. En el presente estudio fueron colectadas muestras de adultos migratorios y de polluelos de cuatro especies de buitres en seis localidades diferentes. Utilizamos un ana´lisis de microsate´lites para determinar la divergencia y variacioń gene´tica dentro y entre poblaciones del geńero Gyps. La baja diversidad gene´tica en G. indicus se debe probablemente a la existencia de una sola poblacioń sin inmigracioń y bajo flujo geńico. Dado que esta especie se encuentra ‘‘En Peligro Crı´tico’’, los programas de conservacioń futuros deberıán considerar un stock gene´tico apropiado para programas de reintroduccioń y crıá. La alta diversidad gene´tica detectada en G. africanus se debe probablemente a una alta inmigracioń y flujo geńico entre las numerosas poblaciones cercanas. Confirmamos los hallazgos previos con relacioń a la baja va- riabilidad gene´tica en G. fulvus, lo que indico´ una alta movilidad y flujo geńico entre sus poblaciones. [Traduccioń del equipo editorial]

The vulture genus Gyps is widely distributed across Africa, Asia, and Europe and includes eight species: African White-backed Vulture (Gyps africa- 1 Email address: [email protected] nus), Oriental White-backed Vulture (G. bengalen-

227 228 ARSHAD ET AL.VOL. 43, NO.3 sis), Cape Vulture (G. coprotheres), Griffon Vulture (G. processes should be considered among the criteria fulvus), Himalayan Vulture (G. himalayensis), Long- for protecting habitats and species (Moritz 1999, billed Vulture (G. indicus), Rueppell’s Vulture (G. 2002). rueppellii), and Slender-billed Vulture (G. tenuirostris). Microsatellites are short tandem repeats (STR) Gyps species are efficient scavengers of the soft tissues that are probably the markers most frequently used of large mammals, usually ungulates (Houston 1983, to study population genetics. A number of recent Mundy et al. 1992). They tend to be colonial nesters studies used microsatellite markers in raptor species and communal feeders alongside conspecifics and and they have proved to be useful tools in the con- other vulture species. Gyps vultures reproduce slowly, servation and management of wild and captive pop- reaching maturity after 4–6 yr, and produce one egg ulations (Rudnick et al. 2005, Wink 2007, Banhos et during a breeding season (Mendelssohn and Leshem al. 2008). The evolution of simple repeats is far 1983, del Hoyo et al. 1994). In the phylogeographic from simple. One important implication is that context, almost no geographic partition was observed the mutational rate for microsatellite DNA is not within three Gyps species, G. bengalensis, G. indicus, uniform; the rate differs among loci and perhaps and G. fulvus, with respect to the analysed mtDNA also among species (Ellegren 2004). Consequently, sequences (Arshad et al. 2009a). the use of microsatellite data to address interspecific In the 1980s, G. bengalensis was regarded as ‘‘pos- relationships should be done only with caution. sibly the most abundant large bird of prey in the Although a number of phylogenetic studies of Gyps world’’ (Houston 1985). On the Indian subconti- species have been conducted based on nucleotide nent, populations of three Gyps species (G. bengalen- sequences of marker genes (Seibold and Helbig sis, G. indicus and G. tenuirostris) have dramatically 1995, Wink 1995, Lerner and Mindell 2005, Johnson declined within the past 10 yr and have recently et al. 2006), only a few studies have addressed Gyps’ been listed as critically endangered by the World genetic structure on a finer scale, by using methods Conservation Union (IUCN 2007). Because G. ben- of high resolution such as microsatellite analyses. In galensis and G. indicus are currently very rare or ex- the present study, we analysed samples using micro- tinct in many parts of their historic range (IUCN satellite DNA analysis, to determine the genetic dis- 2007), their biology is of considerable conservation tance as well as diversity within and among Gyps spe- interest. Griffon Vultures were widespread around cies populations from different locations. the Mediterranean Sea, but underwent a severe population decline at the end of the nineteenth century METHODS and beginning of the twentieth century, mainly due Sample Collection. To determine the genetic to direct and indirect human persecution (Gouar et structure among four species of the genus Gyps a al. 2007). In France, they went extinct in the Alps at total of 186 samples were studied from six native the end of nineteenth century and in the Massif colonies in Pakistan (Toawala and Nagar Parkar), Central in 1945. However, successful reintroduc- Namibia (Ethosa National Park), Spain, Israel and tions have occurred since the 1980s. In contrast to Cyprus. These colonies are scattered throughout other Old World vulture species, G. africanus appar- the geographical ranges of the species (Fig. 1). ently receives little attention from researchers, and For three species (G. bengalensis, G. indicus and G. there seems to be limited conservation concern re- africanus), we collected 3–5 drops of blood from garding this species (van Wyk et al. 2001). nestlings ca. 6 wk old using brachial puncture (Ta- Conservation should not only be directed toward ble 1). In three populations of G. fulvus, we collect- endangered species but should also encompass the ed blood and muscle samples from marked adults management of existing biodiversity. To ensure the and nestlings (Table 1). long-term persistence of a species, a conservation DNA Extraction. Blood was stored in EDTA buff- strategy should therefore include the assessment er (Arctander 1988) or in 70% ethanol. Total DNA and the preservation of the amount and pattern of was isolated from blood or muscle using standard genetic variation within current populations (May phenol-chloroform protocols (Sambrook and Rus- and Henry 1995, Helbig 1996); ecological data sell 2001). may also be relevant for defining population distinc- Microsatellite and Genotyping System. Several tiveness as evolutionarily significant units (Crandall known microsatellite primers were tested for their et al. 2000). Moreover, the maintenance and resto- degree of polymorphism. The following eight mi- ration of both ecological and microevolutionary crosatellite loci were selected to analyse the genetic SEPTEMBER 2009 GENETIC VARIATION OF GYPS SPECIES 229

Figure 1. Approximate location of sample sites (filled circles) across the range of Gyps species The shaded area is the distribution range of the species (after del Hoyo et al. 1994, Ferguson-Lees and Christie 2001 and Rasmussen and Anderton 2005). distance and diversity of a total of 186 individuals dCTP, dTTP, and dATP), 2.5 ml103 buffer with from six populations of the genus Gyps: Gf3F3, 15 mM MgCl2, 0.15 ml Taq-Polymerase (0.6 Units; Gf3H3, Gf11A4 (Mira et al. 2002), BV5, BV12, Bioron, Ludwigshafen, Germany), 0.1 ml 33P a–dATP BV13, BV14, and BV20 (Gautschi et al. 2000). (1 mCi). Thermal cycling was performed under the The PCR was performed with 60 ng of total DNA following conditions: 2 min at 94uC, followed by 35 in 25 ml of reaction volume containing 10 pmol of cycles of 30 sec at 94uC, 1 min at 50–58uC, 1 min at each primer, 1 ml nucleotide-mix (100 mM of dGTP, 72uC, with a final extension at 72uC for 10 min and then lowered to 4uC for further storage. PCR products Table 1. Species, origin, and number of individuals were separated electrophoretically on a denaturing sampled. Polyacrylamide gel electrophoresis (PAGE) at 65 W for 1.5 h (length 40 cm). After drying, the gel was exposed to an X-ray film (Hyperfilm-MP, Amersham) SPECIES ORIGIN N for 1–2 d and developed (X-ray developer and fixer; Gyps bengalensis Pakistan (Toawala) 32 Kodak, Rochester, New York, U.S.A.) Gyps africanus Namibia (Ethosa National 31 Data Analysis. To assess whether the selected loci Park) were sufficiently polymorphic to evaluate differenc- Gyps indicus Pakistan (Nagar Parker) 55 Gyps fulvus Spain 35 es between group structures, we calculated the prob- Gyps fulvus Israel 13 ability of identity PID, which is the probability that Gyps fulvus Cyprus 20 two individuals drawn at random from a population have the same genotype at multiple loci (Taberlet TOTAL 186 and Luikart 1999). We computed the multilocus 230 ARSHAD ET AL.VOL. 43, NO.3

unbiased PID among the 186 analyzed individuals 2000, Falush et al. 2003). The test considered the using the Waits et al. (2001) formulae implemented individuals of G. bengalensis, G. africanus, G. indicus, in the FAMOZ software (Gerber et al. 2003). and G. fulvus from Pakistan, Namibia, Israel, Cy- The evidence for the occurrence of null alleles prus, and Spain without any origin group informa- was calculated for each locus within each population. In order to identify correct number of groups tion. The frequency of null alleles was estimated (K) beside the maximum probability [LnP(D)], we following Brookfield (1996). The departure from used the DK according to Evanno et al. (2005). A Hardy-Weinberg equilibrium (HWE) and linkage series of ten independent runs with K (number of disequilibrium (exact tests) were compared among independent genetic groups) 5 1–7 were used with loci for each population using GENEPOP v. 3.4. A an admixture model with correlated allele frequen- sequential Bonferroni correction (Rice 1989) was cies. Throughout the analysis, the burn-in period used for these tests. Allele frequencies, Nei’s estima- was fixed at 100 000 and the number of further tion of heterozygosity and the allelic richness (RS) MCMC runs was set at 50 000. In addition, a sepa- per locus and per population were calculated with rate Bayesian clustering analysis was also performed FSTAT v. 2.9.3.2 (Goudet 2002). Standard diversity using only G. fulvus samples. indices (Nei 1987), like mean number of alleles RESULTS (Na), observed (HO) and expected (HE) heterozygosity were calculated using ARLEQUIN v. 3.1 (Ex- Microsatellite Analysis. The probability of identity coffier et al. 2005). (PID) for the eight-locus combinations was less than The fixation indices FIS and FST (Weir and Cock- 0.001, i.e., the eight loci in the present study were erham 1984, Nei 1987) were calculated with GENE- polymorphic enough to unambiguously character- POP v. 3.4 (Raymond and Rousset 1995). FIS and ize each individual by a unique multilocus geno- FST can explain deviation from HWE. Negative FIS type. values suggest outbreeding behavior due to higher The mean number of alleles among populations observed heterozygosity compared to Hardy-Wein- ranged from 4.0–6.5. No significant genotypic link- berg proportions and vice versa. FST shows the ge- age disequilibrium was detected after Bonferroni netic differentiation between two subpopulations corrections. For all populations in each of the four and also is used as a genetic distance measure. groups, HWE at each locus was not rejected except PAUP (Swofford 2002) was employed to calculate for Gf11A4 (G. fulvus; Cyprus), BV5 (G. fulvus; a neighbour-joining (NJ) tree based on pairwise Spain), and BV12 (G. indicus; Table 2). The one- FST-values. Other genetic distances according to Ca- tailed test (H1 5 heterozygote deficiency) for all valli-Sforza and Edwards (1967), Nei (1972) and loci deviating from HWE showed a significant het- Reynolds et al. (1983) were calculated with the pro- erozygote deficiency. This deviation suggests in- gram package PHYLIP v. 3.2 (Felsenstein 1989). breeding or assortative mating. This heterozygote The genetic affinities among the four groups (G. deficiency may be also due to null alleles. However, bengalensis, G. africanus, G. indicus, and G. fulvus) there was no evidence of null alleles for each locus were evaluated by a NJ tree. Bootstrap resampling within each species except for BV5 (G. fulvus; (1000 replicates) was performed to test the robust- Spain). ness of the tree topology. In order to determine the The mean number of alleles per polymorphic lo- level of genetic variability among populations, a cus, the allelic richness, and rates of heterozygosity paired t-test was performed; furthermore, the signif- (both observed and expected) did not differ signif- icance of the pairwise FST values were assessed with icantly different among G. fulvus populations (Ta- the program Arlequin (version 3.0) with 100 permu- ble 2). Pairwise FST values showed very low genetic tations. In order to examine the clustering pattern differences among the three populations of G. ful- of all individuals, a principal coordinate analysis vus (FST 5 0.033–0.043) and these differences were (PCoA) was performed using MVSP v. 3.13 (Kovach not statistically significant (P . 0.05). However, sig- 2005) with standardized Euclidean distance. Other nificant differences were observed among popula- genetic distances (average taxonomic, Braykurt and tions of different species (P , 0.05). Jaccard; Kovach 2005) were used as well. The greatest genetic variability (mean number of Bayesian Clustering Analysis. To infer the popu- alleles per polymorphic locus, the allelic richness, lation structure, a Bayesian clustering method was and rate of heterozygosity) was found in G. africa- selected using STRUCTURE v. 2.2 (Pritchard et al. nus, followed by G. bengalensis, G. fulvus, and G. in- SEPTEMBER 2009 GENETIC VARIATION OF GYPS SPECIES 231

Table 2. Genetic diversity in Gyps populations using multilocus microsatellite DNA genotypes.

MICROSATELLITE DIVERSITYa

POPULATIONS N Na HO HE RS FIS HWD G. bengalensis (Pakistan) 32 6.0 0.583 0.589 4.4 0.01 0 G. africanus (Namibia) 31 6.5 0.669 0.685 5.0 0.02 0 G. indicus (Pakistan) 55 4.0 0.467 0.475 2.6 0.07 1 (BV12) G. fulvus (Spain) 35 5.0 0.507 0.513 3.4 0.01 1 (BV5) G. fulvus (Israel) 13 5.0 0.502 0.544 4.3 0.08 0 G. fulvus (Cyprus) 20 5.1 0.570 0.551 3.7 0.07 1 (Gf11A4) a Na, mean number of alleles; HO,HE, observed and expected heterozygosity; RS, allelic richness for each population; FIS, the inbreeding coefficient and HWD, number of loci deviating from HWE. dicus. However, significantly low genetic variability The inferred proportions of ancestry for each pop- (allelic richness) among groups was found only in ulation into the different clusters were high, which G. indicus. According to Nei’s genetic distance as emphasizes the genetic distinctiveness of the four well as pairwise FST, the greatest distance to another groups (i.e., species). group was measured between G. bengalensis and G. According to the Bayesian clustering statistics, fulvus, whereas the distance between G. africanus most of G. bengalensis and G. indicus populations and G. fulvus was minimal (Table 3). from Pakistan do not share their pool of genes with The NJ method indicated the same tree topology other populations of G. africanus and G. fulvus. Only based on different genetic distances (Fig. 2). Gyps one individual from the G. fulvus population from bengalensis, G. africanus, and G. indicus cluster to- Spain was referred to the G. africanus cluster from gether and are separated from G. fulvus, with high Namibia, with a proportion of about 70%, whereas bootstrap values. Using principal coordinate analy- the gene pool of G. bengalensis and G. indicus were sis, we found a clustering pattern similar to the NJ also present in the G. fulvus population from Israel, dendrogram (Fig. 3). although minimally (Fig. 4). Bayesian Clustering Analysis. The estimation of the number of genetically distinct clusters achieved DISCUSSION a maximal probability with K 5 4 groups, corre- Genetic Variation within Populations of G. fulvus. sponding to the four species (G. bengalensis, G. afri- The measured genetic diversity was similar in all canus, G. indicus, and G. fulvus). We also performed native populations (Spain, Israel and Cyprus) of a separate Bayesian clustering analysis only on G. Griffon Vultures. Moreover, the levels of genetic fulvus populations and we did not find any structure diversity in the Griffon Vulture populations were among those three populations, which further sup- similar to the levels found in a previous study by ported our estimation that K 5 4. The groups were Gouar et al. (2007), but higher than those estimated associated with their cluster, with a proportion of for other species of vultures such as Gypaetus barba- membership of 0.976 (G. indicus), 0.943 (G. africa- tus and Neophron percnopterus in Europe (Gautschi et nus), 0.966 (G. fulvus), and (0.977) G. bengalensis. al. 2003, Kretzmann et al. 2003). Low FST among all

Table 3. Nei’s genetic distances among the six populations (above diagonal) and pairwise FST values below diagonal.

POPULATIONS 123456 1. G. bengalensis (Pakistan) — 0.393 0.740 0.793 0.753 0.761 2. G. africanus (Namibia) 0.180* — 0.580 0.350 0.354 0.411 3. G. indicus (Pakistan) 0.315* 0.260* — 0.615 0.559 0.736 4. G. fulvus (Spain) 0.340* 0.166* 0.326* — 0.057 0.073 5. G. fulvus (Israel) 0.317* 0.161* 0.313* 0.033 — 0.071 6. G. fulvus (Cyprus) 0.320* 0.165* 0.344* 0.043 0.038 —

* 5 significant (P , 0.05). 232 ARSHAD ET AL.VOL. 43, NO.3

Figure 2. Unrooted NJ dendrogram for six populations of Gyps vultures, based on Nei’s genetic distance. Numbers represent the bootstrap values. populations of G. fulvus confirmed the high dispers- ic results using mitochondrial cytb, nd2, and control al rates in these populations. Dispersal abilities of region sequence data indicate a recent and rapid Griffon Vultures are substantial; for example, in diversification within the genus Gyps (Seibold and Croatia, marked birds dispersed 400–600 km from Helbig 1995, Wink 1995, Lerner and Mindell 2005, their natal areas (Susic 2000). Johnson et al. 2006, Arshad et al. 2009a). When pop- In the present study, we found that G. fulvus populations differ significantly, FST and genetic distances ulations from Spain and Israel shared their gene have been overestimated in the presence of null al- pool with populations of G. africanus, G. bengalensis, leles (Chapuis and Arnaud 2007). In the present and G. indicus. This may indicate some degree of study the frequency of null alleles was estimated, hybridization. Hybridization has been reported be- and we found no evidence of null alleles except for tween Gyps species in captivity and under natural one locus for BV5 (G. fulvus; Spain). circumstances (McCarthy 2006). However, the ge- In the present study, pairwise FST values and differ- netic pattern demonstrated in our study may also ent distance matrixes were used to estimate the genet- result from homoplasy or retention of shared ances- ic distance among Gyps populations of the studied tral states. taxa. According to Nei’s genetic distance as well as Genetic Distance among Four Gyps Species Pop- pairwise FST, the smallest distance was between G. ben- ulations. Gyps species are closely related. Phylogenet- galensis and G. africanus, and the greatest distance be- SEPTEMBER 2009 GENETIC VARIATION OF GYPS SPECIES 233

Figure 3. Principal coordinate analysis (PCoA) plot for six populations of Gyps vultures, based on standardized Euclidean distance. tween G. bengalensis and G. fulvus. Our results agreed found in G. africanus, followed by G. bengalensis, with previous studies on Gyps species that employed G. fulvus,andG. indicus. However, low genetic var- mitochondrial sequences (Arshad et al. 2009a). In iability (allelic richness) was found only in G. indi- addition, the historically proposed grouping of G. ben- cus. Gyps africanus samples were collected from galensis and G. africanus together in the genus Pseudo- Ethosa National Park (Namibia), which represents gyps was not corroborated by our microsatellite data, only a small subset of the large population in although the two are very closely related. southern Africa. High genetic diversity in G. africa- Genetic Variation within Populations of the Four nus is probably due to frequent immigration and Gyps Species. The greatest genetic variability was gene flow.

Figure 4. Assignment test for six populations of Gyps vultures. Each individual is represented by a vertical column, subdivided into K fractions. K 5 4, which distinguishes between G. bengalensis (light gray), G. africanus (black), G. indicus (medium gray), and G. fulvus (dark gray), is most informative. 234 ARSHAD ET AL.VOL. 43, NO.3

Toawala represents the only remaining popula- anonymous referees, who reviewed earlier versions of tion of G. bengalensis in Punjab province (Pakistan), this manuscript. where a 95% decline has been recorded since 2000 LITERATURE CITED (Gilbert et al. 2006). Considerable levels of genetic ARCTANDER, P. 1988. Comparative studies on avian DNA variability were found for this population, despite its restriction fragment length polymorphism analysis: small size. These results agreed with the theoretical convenient procedures based on blood samples from expectations that long-lived species can retain rela- live birds. J. Ornithol. 129:205–216. tively high levels of genetic diversity in small popu- ARSHAD, M., M.J.I. CHAUDHARY, AND M. WINK. 2009b. High lations over short time periods (Hailer et al. 2006). mortality and sex ratio imbalance in a critically declin- These levels of genetic variability did not differ sig- ing Oriental White-backed Vulture population (Gyps nificantly from those in other Gyps species (except bengalensis) in Pakistan. J. Ornithol. 150:495–503. G. indicus). ———, J. GONZALEZ, A.A. EL-SAYED,T.OSBORNE, AND M. Gouar et al. (2007) used microsatellites to study WINK. 2009a. Phylogeny and phylogeography of critical- three native colonies of G. fulvus (Israel, Croatia, ly endangered Gyps species based on nuclear and mitochondrial markers. J. Ornithol. 150:419–430. and French Pyrenees, Ossau), one established rein- BANHOS, A., T. HRBEK,W.GRAVENA,T.SANAIOTTI, AND I.P. troduced colony in France and four captive founding FARIAS. 2008. Genomic resources for the conservation groups of G. fulvus. 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Cladistics 5:164–166. mibia Ministry of Environment and Tourism for the per- FERGUSON–LEES,J.AND D.A. CHRISTIE. 2001. Raptors of the mit to collect blood. Our thanks go to M.J.I. Chaudhary, world. Christopher Helm, London, U.K. D. Annand, H. Mumtaz, B. Bed’hom, and G. Blanco for ¨ help in collecting samples. We thank Hedi Sauer-Gu¨rth GAUTSCHI, B., J.P. MULLER,B.SCHMID, AND J.A. SHYKOFF. for her help in the laboratory. Guillermo Delgado Cas- 2003. Effective number of breeders and maintenance tro kindly revised our abstract in Spanish. Finally, we of genetic diversity in the captive bearded vulture pop- greatly appreciate the constructive comments of three ulation. Heredity 91:9–16. SEPTEMBER 2009 GENETIC VARIATION OF GYPS SPECIES 235

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