Phylogeny and phylogeography of critically endangered Gyps species based on nuclear and mitochondrial markers

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O R I G I N A L A R T I C L E

Phylogeny and phylogeography of critically endangered

Gyps

species based on nuclear and mitochondrial markers

Muhammad ArshadÆJavier GonzalezÆ

Abdel Aziz El-SayedÆ Tim OsborneÆ

Michael Wink

Received: 1 March 2008 / Revised: 22 October 2008 / Accepted: 23 October 2008 / Published online: 3 December 2008 ÓDt. Ornithologen-Gesellschaft e.V. 2008

Abstract Populations of Oriental White-backed Vulture (Gyps bengalensis) and Long-billed Vulture (Gyps indicus) dramatically declined by 95–100% on the Indian subcon-tinent in mid-1990s. The present study was conducted to discover the phylogeny and phylogeography ofGyps spe-cies based on nuclear (recombination activating gene, RAG-1) and mitochondrial (cytochrome b, cytb) markers. Gypsspecies showed monophyly and no geographic par-tition was observed within the three groups ofGypsspecies (G. bengalensis, G. indicusandG. fulvus) despite the large sample size available (n=149). Our study supports the treatment of G. indicus and G. tenuirostris as separate species. In all analyses, the earliest divergence separated G. bengalensis from all other Gyps taxa while a sis-ter relationship was supported between G. fulvus and G. rueppellii, and these two taxa together were sister group to a clade consisting of G. indicus, G. tenuirostris and G. coprotheres.Molecular clock estimates of both nuclear and mitochondrial (RAG-1, cytb) genes indicated a rapid and recent diversification within theGypsspecies.

Keywords PhylogenyPhylogeographyGypsspecies

Cytochromeb RAG-1

Introduction

Two main evolutionary lineages are found within the group of Old World vultures (subfamilies Aegypiinae and Gypaetinae). The subfamily Gypaetinae includes Gypo-hierax angolensis, Neophron percnopterus and Gypaetus barbatus, while the Eutriorchis astur has recently been found to be a member of this clade (Lerner and Mindell 2005). The second subfamily includes the genera Necro-syrtes and Gyps which form a monophyletic clade with Aegypius, Torgos, Trignoceps and Sarcogyps as a sister group (Wink 1995; Wink and Sauer-Gu¨rth 2004). According to a new classification proposed by Griffiths et al. (2007), the subfamilies Aegypiinae and Gypaetinae were placed in the subtribe Gypina and Gypaetina, respectively.

There are eight species in the genusGyps:G. africanus, G. coprotheresandG. rueppelliiin Africa;G. bengalensis, G. indicus, G. tenuirostrisandG. himalayensisin Asia; and G. fulvus in Europe, Africa and Asia (Ferguson-Lees and Christie 2001; Pain et al. 2003). AllGyps species forage over wide areas, and the ranges of many of these taxa overlap. Juveniles may disperse more widely or are more nomadic than adults (Houston1974,1983).

Johnson et al. (2006) expanded the molecular data set by using the previously published mitochondrial cytochromeb sequences (Seibold and Helbig 1995; Lerner and Mindell 2005) and investigated the phylogenetic relationships among 60 individuals from the genusGyps. Mitochondrial sequence data indicate a recent and rapid diversification within this genus. Phylogenetic analyses and conservative Communicated by J. Fjeldsa˚.

Electronic supplementary material The online version of this article (doi:10.1007/s10336-008-0359-x) contains supplementary material, which is available to authorized users.

M. Arshad (&)J. GonzalezA. A. El-SayedM. Wink Department of Biology, Institute of Pharmacy and Molecular Biotechnology (IPMB), University of Heidelberg,

Im Neuenheimer Feld 364, 69120 Heidelberg, Germany e-mail: arshadbzu@yahoo.com

T. Osborne

Tandala Ridge, PO Box 22, Okaukuejo, Outjo, Namibia DOI 10.1007/s10336-008-0359-x

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estimates suggest that the diversification ofGypstaxa took place within the past 6 million years.

Vultures are important not only for environmental hygiene but also for their considerable cultural and reli-gious significance in India and elsewhere (Pain et al.2003). In the 1980s, vultures were so abundant around human settlements that they were considered a serious hazard to aircraft (Grubh et al.1990). According to Houston (1985), G. bengalensiswas regarded as possibly the most abundant large bird of prey in the world. However, the populations of threeGypsvulture species (G. bengalensis, G. indicusand G. tenuirostris) in the Indian subcontinent have dramati-cally declined within the past 10 years and have recently been listed as critically endangered by the World Conser-vation Union (IUCN2006). These threeGypsspecies share similar feeding behavior, and they are extremely effective and efficient scavengers of the soft tissues of large mam-mals (Houston1983; Mundy et al.1992). In Pakistan, the proximate cause of the decline of G. bengalensis was identified as being due to high rates of mortality from renal failure (Gilbert et al.2002) caused by the toxic effect of diclofenac, a non-steroidal anti-inflammatory drug com-monly used to treat domestic livestock (Oaks et al.2004). Among Gyps species, the extent of diclofenac suscepti-bility is not well known. Direct evidence indicates that diclofenac causes mortality in at least four of the Gyps species (G. africanus, G. bengalensis, G. indicus and G. fulvus); however, the toxicity of this drug to non-domesticated animals is not yet known (Swan et al. 2006; Shultz et al. 2004).

Captive breeding programs and other conservation efforts depend on the identification of the genetical lin-eages (Karl and Bowen 1999; Purvis et al. 2005). Since phylogenies can assist to set conservation priorities among critically endangered species of Gyps with similar life histories, in the present study we have included more samples (n =260) from different localities especially from the current declining populations of South Asia along with previously published work (Wink 1995; Seibold and Hel-big 1995; Lerner and Mindell2005; Johnson et al. 2006). The focus of the present investigation was an analysis of the phylogeographical structure among Gyps populations and the corroboration of phylogenetic relationships among vulture species by using nucleotide sequences of both cytochromeband nuclear recombination activating gene 1 (RAG-1).

Methods

To resolve phylogenetic relationships amongGypstaxa, a total of 260 samples was analyzed that correspond to eight species of the genus Gyps throughout a large pro-portion of their geographical range (Fig.1). The sequences generated in this study were based at least on two indi-viduals per species with some taxa having as many as 77 representative individuals. They were deposited at the GenBank under the accession numbers listed in Table 1 (Electronically Supplementary Material, ESM). We retrieved other sequences from GenBank (Seibold and

Fig. 1 Approximate location of sample sites (filled circle) across the range ofGypsspecies (after Mundy et al.1992; del Hoyo et al.1994; Ferguson-Lees and Christie2001; Rasmussen and Anderton2005); theshaded areais the distributional range of the species.1Cambodia,

2Vietnam,3Thailand,4China,

5Nepal,6India,7Pakistan,

8Kazakhstan,9Iran,10Asia Minor,11Israel,12Palestine,

13Cyprus,14Spain,15

Gambia,16Namibia,17South Africa

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Helbig 1995; Lerner and Mindell 2005; Johnson et al. 2006; see Table 2 in ESM).

DNA isolation

The DNA was obtained from blood, feather or muscle. The samples were stored at -20°C and preserved in EDTA buffer or 70% ethanol. The isolation of total genomic DNA followed standard protocols (Sambrook and Russell2001) as follows: tissues were digested for 48 h at 37°C using 20% of SDS and 1 mg of proteinase K. Cell fragments and proteins were precipitated by centrifugation with a satu-rated solution of NaCl. The DNA was extracted from the supernatant using a denaturing buffer containing guanidine thiocyanate and mercaptoethanol (1%) at 50°C overnight. The extraction of the DNA was performed with phenol/ chloroform and chloroform/isoamyl alcohol. From the supernatant, the DNA was precipitated by adding 0.8 vol-umes of ice-cold isopropanol. The precipitated DNA was washed with 70% ethanol, dried, and dissolved in TE buffer.

Amplification ofcytb andRAG-1

The mitochondrial cytb gene [1,026 base pairs (bp)] and the nuclear RAG-1 (recombination activating gene 1;

1,847 bp) were amplified using nested PCR. The amplifi-cations were performed in 50-ll reaction volumes containing 1.5 mM MgCl2, 10 mM Tris (pH 8.5), 50 mM KCl, 100lM dNTPs, 0.6 units Taq DNA polymerase (Bioron, Ludwigshafen), 200 ng of DNA, and 5 pmol of primers. The primers used for amplification and sequencing are listed in Table1.

Optimal annealing temperatures were found by gradient PCR in a Tgradient thermocycler (Biometra). The cycle protocol consisted of: (1) an initial denaturation at 94°C for 8 min, (2) 32 cycles including denaturation at 94°C for 45 s, annealing at 48–58°C for 1 min and extension at 72°C for 1 min, followed by (3) a final extension period at 72°C for 10 min. PCR products were precipitated with 4 M NH4Ac and ethanol (1:1:12) and centrifugation (13,000 rpm) for 15 min. The PCR products were visual-ized on 1.4% agarose gels using k-Pst I. PCR re-amplifications were performed under similar conditions as above with 1ll of the initial PCR reaction (previously diluted 10 times) as template and only 20 cycles.

Sequencing was performed using the DYEnamic ET Terminator Cycle Sequencing Kit (GE Healthcare) and a cycle sequencing protocol as follows: 31 cycles of 20 s at 96°C, 15 s at 50°C, and 1 min at 60°C. SephadexTM G-50 columns (Amersham Biosciences) and MultiScreen filter plates (Millipore) were used for the purification of

Table 1 Primers used in this study for amplification (amp) and sequencing (seq) of mitochondrial (cytb) and nuclear (RAG-1) genes

Primer Sequence (50–30) Use Reference

cytb

mta1 CCC CCT ACC AAC ATC TCA GCA TGA TGA AAC TTC G amp/seq (L) Dietzen et al. (2003)

mtc TGA GGA CAA ATA TCA TTC TGA GG amp/seq (L) Dietzen et al. (2003)

L14764 TGR TAC AAA AAA ATA GGM CCM GAA GG amp/seq (L) Sorenson et al. (1999)

mtb TTG TGA TTA CTG TAG CAC CTC AAA ATG ATA TTT GTC amp/seq (H) Modified from Kocher et al. (1989)

mte GCA AAT AGG AAG TAT CAT TCT GG amp/seq (H) Fritz et al. (2006)

mtfr CAT AGA AGG GTG GAG TCT TCA GTT TTT GGT TTA CAA amp/seq (H) Modified from Wink et al. (2002)

mtfsh TAG TTG GCC AAT GAT GAT GAA TGG GTG TTC TAC TGG amp/seq (H) Dietzen et al. (2003)

RAG-1

R13B CTC CCT GAA GAG ATT CAG CAT CC amp (L) Groth and Barrowclough (1999)

R13 TCT GAA TGG AAA TTC AAG CTG TT amp (L) Groth and Barrowclough (1999)

R17 CCC TCC TGC TGG TAT CCT TGC TT amp/seq (L) Groth and Barrowclough (1999)

R50 CTG ATC TGG TAA CCC CAG TGA AAT CC amp/seq (L) Irestedt et al. (2001)

R21 GGA TCT TTG AGG AAG TAA AGC CCA A amp/seq (L) Groth and Barrowclough (1999)

R1920 CTC TGA CGG CAA TCC TGA GYC C amp/seq (L) El-Sayed (2007)

R2815 GGC ATT CAT TTT TCG GAA CCT CC amp/seq (H) El-Sayed (2007)

R22 GAA TGT TCT CAG GAT GCC TCC CAT amp/seq (H) Groth and Barrowclough (1999)

R2B GAG GTA TAT AGC CAG TGA TGC TT amp (H) Groth and Barrowclough (1999)

R2 TTC CAC TGA ATC ATT GCT TTC CA amp (H) Groth and Barrowclough (1999)

R51 GAC CCT CTT TCT GCT ATG AGG GGG C amp/seq (H) Irestedt et al. (2001)

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sequence products. Sequences were analyzed by capillary electrophoresis using a MegaBACETM 1000 sequencer (Molecular Dynamics, Amersham Pharmacia).

Sequence analysis, networks and phylogenetic reconstruction

Sequences were aligned manually with BioEdit version 7.0.9.0 (Hall2004). Next, the sequences were checked for unexpected stop codons using the program MEGA version 4.0 (Tamura et al. 2007). Basic statistics and average uncorrectedp-distances were calculated with MEGA ver-sion 4.0 (Tamura et al.2007). The analyses were performed on individual genes and both genes concatenated (cytb?RAG-1). Phylogenetic trees were reconstructed using maximum likelihood (ML) and maximum parsimony (MP) in PAUP* version 4.0b10a (Swofford 2002), and Bayesian inference (BI) in MPIMrBayes version 3.1.2. (Ronquist and Huelsenbeck2003; Altekar et al.2004). MP and ML heuristic searches were performed with 10 random stepwise additions, tree-bisection-reconnection branch-swapping, and ‘multrees’ option. We explored the model of sequence evolution that fits the data best with Modeltest version 3.7 (Posada and Crandall 1998). The best model was then used with the ML analyses. Robustness of nodes was assessed by 1,000 bootstrap replicates. For BI analy-ses, two independent runs of 6,000,000 generations each were performed along with four Markov chains. The evo-lutionary model selected for BI analysis was the GTR?C?I. Trees were sampled every 500 generations and the first 3,000 samples were discarded as ‘burn in’.

Considering the cytb dataset, as well as the combined dataset (cytb ?RAG-1) analyses, Hooded Vulture (Nec-rosyrtes monachus), Red-headed Vulture (Sarcogyps calvus), Black Vulture (Aegypius monachus), Lappet-faced Vulture (Torgos tracheliotos) and White-headed Vulture (Trigonoceps occipitalis) from Aegypiinae and Bearded Vulture (Gypaetus barbatus), Egyptian Vulture (Neophron percnopterus) and Palm-nut Vulture (Gypohierax angol-ensis) from Gypaetinae were used as the sister group, while Secretary Bird Sagittarius serpentarius was used as an outgroup to root the trees.

Median-joining networks were reconstructed for mito-chondrial haplotypes with Network version 4.2.0.1 (Bandelt et al. 1999). To obtain age estimates for the divergence events within the Gyps species, we used two calibrations: (1) forcytbsequences a rate of 0.6%9Myr-1 (Pereira and Baker 2006) was calculated assuming the diversification ofGypstaxa have occurred within the past 6 million years (My) (Johnson et al. 2006), and (2) from RAG-1 sequences a rate of 0.056%9My-1 based on RAG-1divergences reported by Groth and Barrowclough (1999). The split between Haliaeetus-Buteo 17 My ago

yields a very similar RAG-1divergence rate of 0.058% 9 My-1(Helbig et al. 2005).

Results

Sequence variation

Including the haplotypes published by Seibold and Helbig (1995), Lerner and Mindell (2005) and Johnson et al. (2006), a dataset of 260 representative individuals was generated with the cytbgene. Among the genus Gyps, 58 haplotypes (Table 2; Fig.4) were distinguished based on 108 variable sites (94 transitions and 14 transversions). The combined data set (cytb?RAG-1) consisted of 2,873 bp and exhibited 71 variable sites among ingroup taxa (Fig.3). Uncorrected p-distances between taxa were different across loci (cytb and RAG-1) with cytb sequences showing higher divergence estimates as expec-ted (Table3). Nucleotide composition of the cytb sequences contained low levels of guanine (13.6%) and high levels of cytidine (34%) and maximum average p-distances ranged from 0.6 to 2.8% including only ingroup taxa, while the distance was 9.8% between the ingroup (Gypsspecies) and the sister group taxa (Aegypiinae). Phylogenetic analysis

For the cytb sequences, the model of sequence evolution that fits the data best consisted of the TrN?G=0.1848 (Tamura and Nei 1993). MP, ML and BI analyses gener-ated the same topology forcytband both mitochondrial and nuclear genes concatenated (Figs.2,3). In all the analyses, the Old World vultures form two main evolutionary lin-eages, Aegypiinae and Gypaetinae. Phylogenetic analysis based on 1,026 bp of cytb revealed eight clades of Gyps species which are closely related and can be distinguished with bootstrap values: 71 and 70% for MP and ML, respectively, and 0.98 for BI posterior probability. All recognized Gyps species showed monophyly and almost each clade was supported with high bootstrap proportions and posterior probability values (Fig.2). No geographic partition was observed within the three groups of Gyps species (G. bengalensis, G. indicus, andG. fulvus) despite the large sample size available (n =149). The similar phylogeographical structure is reflected in a minimum spanning network based on cytb haplotypes (Fig.4). Our study supports the treatment of G. indicus and G. tenui-rostris as separate species with a sequence divergence of 1%. In addition, the historically proposed grouping of bengalensis and africanus together in the genus Pseudo-gyps cannot be corroborated by mitochondrial cytb and nuclearRAG-1data with 2.4% sequence divergence.

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In all analyses, the earliest divergence separatedG. ben-galensisfrom all otherGypstaxa and the next divergence is forG. himalayensisfollowed byG. africanus. A sister rela-tionship was supported betweenG. fulvusandG. rueppellii, and these two taxa together were sister group to a clade consisting ofG. indicus,G. tenuirostris, andG. coprotheres.

Discussion

Phylogenetic relationships amongGypsspecies

In the present study, we performed a phylogenetic analysis amongGyps species in order to update current conserva-tion efforts. Along with the haplotypes published by Seibold and Helbig (1995), Lerner and Mindell (2005), and Johnson et al. (2006) more extensive samplings were obtained from the field (Fig.1).

Our results strongly support the change to the tradi-tional taxonomy of Gyps species mentioned by Johnson et al. (2006). The phylogenetic distinctiveness of the Long-billed and Slender-billed Vultures as separate spe-cies recommended previously (Rasmussen et al. 2001; Rasmussen and Anderton 2005; Johnson et al. 2006) was supported in our study based on cytb sequence data. Additionally, average sequence divergence between G. indicus and G. tenuirostris (1%) is similar to their respective divergence estimates from G. coprotheres (1.1%, 1.7%) and to those reported between various other broadly recognized species within the family Accipitridae (Lerner and Mindell 2005). These results highlight the utility of molecular phylogenetic methods in identifying independent evolutionary lineages within a group that has a long history of taxonomic uncertainty (Hume 1869; Jerdon 1871; Hume 1873; Amadon 1977; Mundy et al. 1992; Helbig et al. 2005).

Table 2 Variable site matrix among eight species of the genusGypshaplotypes

1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 2 3 4 5 5 6 6 8 8 9 9 9 0 1 2 3 3 4 6 7 8 8 0 1 1 2 2 2 3 4 4 5 6 6 7 9 1 1 1 2 2 4 5 9 9 1 4 5 6 6 6 7 7 7 8 3 7 9 9 2 7 7 8 2 6 0 2 4 2 4 9 5 8 4 8 7 3 9 4 3 6 2 5 8 4 2 6 2 1 5 9 1 1 5 8 1 7 8 7 6 9 4 4 1 3 4 9 2 4 7 0 Haplotype N GA1 32 G G A C C A A C C C T C T T A G A C T C T C G C T C C A T G A A C G A T T A A C C G A C A A C G T G T G G C T T GA2 19 . . . T . . . . GA3 10 . . . G T . . . . GA4 3 . . . T . . . . GA5 2 . . . T . . . . GA6 2 . . . G . . . C . . . . GA7 2 . . . T . . . . GA8 1 . . . G . T . . . . GA9 1 . . . T . . . GA10 1 . . . T . . . G . T . . . . GA11 1 . . . C . . T . . . . GA12 1 . . . T . . . . GA13 1 . . . T . . . C GA14 1 . . . . GB15 18 A . . . . G . . T . . . C . . . C . T . . . T . . . G . . . . GB16 7 A . . . . G . . T . . . C . . . C . T . . . T . . . C . . . G . . . . GB17 3 A A . T . G . . T . . . C . . . C . T . . . T . . . C . . . . GB18 2 A . . . . G . . T . . . G . C . . . C . T . . . T . . . G . . . . GB19 1 A . . . . G . . T . . . C . . . C T T . . . T . . . G . . . . GB20 1 A . . . . G . . T . . . C . . . C . T . . . T . . . G . . G . . . . GB21 1 A . . . . G . . T . . . C . . . C . T . . . T . . . C . . . G . . G . . . . GB22 1 A . . . . G . . T . . . C . . . C . T . . . T . G . C . . . . GB23 1 A . . . . G . . T . . . C . . . C . T . . . T . . . C . . . . GB24 1 A . . . . G . . T . . . C . . . C . T . . . T . . C . . . . GB25 1 A . . . . G . . T . . . T C . . . C . T . . . T . . . C . . . . GB26 1 A . . . . G . . T . . . C . . . C . T . . . T . . . ? . . . G . . . . GF27 46 . . . T . . . A . . C . . . A . C . . . T . . . G . . . G T . . . T . . GF28 3 . . . T . . . A . . C . . . A . C . . . T . . . . C G . . . G T . . . T . . GF29 2 . . . T . . . A . . C . . . A . C . . . T . . . G . . . G T . . . T . . GF30 1 . . . T . . . A . . C . . . A . C . . . C . . . T . . . G . . . G T . . . T . . GF31 1 . . . T . . . A . . C . . . A . C . . . T . . . G . . . G T . . . T . . GF32 1 . . . T . . . A . . C . . . A . C . . . T . . . G . . . G T . . . T . . GF33 1 . . . T . . . A . . C . . . A . C . . . T . . . G . . . G T . . . T . . GF34 1 . . . T . . . A . . C . . . A . C . . . T . . . G . . . G T . . . T . . GF35 1 . . . T . . . A . . C . . . A . C . . . T . . . G . . . G T . . . T . . GF36 1 . . . T T . . . A . . C . . . A . C . . . T . . . G T . . . G T . . . T . . GF37 1 . . . T . . . A . . C . . . A . C . . . T . . . G . . . G G T . . . T . . GF38 1 . . . T . . . A . . C . . . A . C . . . T . . . G . . . G T . . . C . . T . . GI39 40 A . . . T . . . C . . . A . C . . . T . . . T . . . T . . GI40 6 . . . T . . . C . . . A . C . . . T . . . T . . . T . . GI41 3 A . . . T . . . C . . . A . C . . . T . . . T . . . T . . GI42 1 . . . T . . . C . . . A . C . . . T . . . T . . . . A . T . . GI43 1 A . . . T . . . C . . . A . C . . . T . . . T . . . T . . GTE44 12 . . . T . C . . . C . . . A . C . . G . . . . T A . . . T . . . T . . GTE45 1 . . . T T . C . . . C . . . A . C . . G . . . . T A . . . T . . . T . . GCO46 4 . . G . . . T . . . . C . . . . C . . . A T C . . . T . . . T A . . . T . . GCO47 1 . . G . . . T . . . . C . . . . C . . . A T C . . . T . . . T . . . T A . . . T . . GCO48 1 . . G . . . T . . . . C . . . . C . . . A T C . . . T . . . T A . . . T . . GCO49 1 . . G . . . T . . . . C . . . . C . . . A T C . . . T . . . ? . . . T . . . T . . GR50 2 ? . . . T . . . C . . . A . C . . . T . . . A . . . G T . . . T C . GR51 1 ? . . . T . C . T . . . C . . . C . . . A . C . . . T . . . A . . . G T . . . T C . GR52 1 ? . . . T . C . T . . . C . G . A . C . . . T . . . A . . . G T . . . T C . GR53 1 ? . . . T . . . C . . . A . C . . . T . . . A . . . G T . . . T C . GR54 1 ? . . . T . . . C . . . A . C . . . T . . . A . . . G T . . . T C . GH55 5 A . . . T . . . G . . . C T . . A . C . T . . . T . . C . . . T . G . A . A . . . T . . GH56 1 A . . . T . . . G . . . C T . . A . C . T . . . T . . C . . . T . G . A . A . . . T . . GH57 1 A . . . T . . . G . . . C T . . A . C . T . . . T . . C . . . T . G . A . A . . . T . . GH58 1 A . . . T . . . G . . . C T . . A . C . T . . . T . . C . . . T . G . A . A . . . T . . Alignment positiona

cytochrome b Sample site

16 16 16 16 16 16 16 16 16 16 16 16 16 17 1,3,7 5,7 1 1 7 7 7 1 7 7 2 6 7,8,9,11,12,13,14,15 14 14,15 14 14 14 11 9 * 10 6 11 7 6,7 7 7 7 3,5,6,15 6 17 17 17 17 * 17 * 15 15 4,5,6 6 4 *

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Old World vultures have been proposed to be mono-phyletic (Brown and Amadon 1968; Thiollay 1994) or polyphyletic (Wink and Sauer-Gu¨rth 2004; Lerner and Mindell2005; Griffiths et al.2007). Sister groups had not been identified for the Old World vultures, although Gypohierax angolensis was proposed to represent the transition from vultures to sea eagles (Brown and Amadon 1968). In our study, we find strong support for two separate subfamilies. The Gypaetinae is the earlier divergent group includingGypohierax, Gypaetus, andNeophron while the remaining vultures form a separate group, the Aegypiinae. Molecular clock and evolutionary scenery

Although we observed the monophyly for the majority of Gypsspecies and the estimates of sequence difference were relatively small (0.6–2.8%; Table3), multiple relationships were unresolved due to low nodal support. This shows that

there was probably a rapid and recent diversification within theGypsspecies. According to a generally supported avian mitochondrial DNA divergence rate of 2%9My-1(Shields and Wilson 1987; Lovette 2004), our mitochondrial cytb sequence divergence estimates indicate that the radiation of Gypsspecies occurred 1.1 My ago. These estimates could be violated in some avian taxa and genes (Mindell and Thacker 1996; Pereira and Baker2006). By assuming that the above divergence estimate rates were too high (Pereira and Baker 2006), a lower rate (e.g., 0.6%9My-1) still yields diver-gence times predating the Pleistocene (3.67 My). ForRAG-1 sequences the rate of 0.056%9My-1reported by Groth and Barrowclough (1999) also supports a recent radiation ofGypsspecies (3.57 My ago). For the case ofButeo, Helbig et al. (2005) also reported a similar molecular clock for RAG-1sequences.

Similarly to Johnson et al. (2006), the divergence esti-mates of the present study do not correspond with

Table 2 continued 1 4 4 5 5 5 5 5 5 5 5 6 6 6 6 6 6 6 6 6 6 7 7 7 7 7 7 7 7 8 8 8 8 8 8 8 8 8 8 8 9 9 9 9 9 9 9 9 9 9 9 9 0 9 9 3 3 4 5 6 9 9 9 0 0 2 3 4 5 5 5 7 8 0 1 2 2 3 3 8 8 2 2 3 3 3 4 5 6 8 8 9 0 0 1 2 3 3 4 5 5 6 7 7 0 2 6 4 9 6 8 4 4 7 8 7 9 1 0 9 1 4 7 8 4 5 4 0 3 2 8 1 6 2 8 0 4 7 7 8 8 3 5 7 1 3 5 4 6 9 8 4 7 8 0 1 0 Haplotype N GA1 32 T T T C T A C G C G C G A C T A C C A G C A T T G T T T A A C A G G T C T A T A C C A G C C C A T A C C GA2 19 . . . . GA3 10 . . . . GA4 3 . C . . . . GA5 2 . . . T . . . A . . . . GA6 2 . . . . GA7 2 . . . . GA8 1 . . . A . GA9 1 . . . . GA10 1 . . . . GA11 1 . . . C . . . . GA12 1 . . . C . . . . GA13 1 . . . T . . . C . . . . GA14 1 . . . ? . . . C . . . . GB15 18 . . . T A . . . A . . . G A . G C . . . . C . . . A C . C . C . . . G C . T T . . . . . GB16 7 . . . T A . . . A . . . G A . G C . . . . C . . . A C . C . C . . . G C . T T . . . . . GB17 3 . . . T A . . . A . . . G A . G C . . . . C . . . A C . C . C . . . G C . T T . . . . . GB18 2 . . . T A . . . A . . . G A . G C . . . . C . . . A C . C . C . . . G C . T T . . . . . GB19 1 . . . T A . . . A . . . G A . G C . . . . C . . . A C . C . C . . . G C . T T . . . . . GB20 1 . . . T A . . . A . . . G A . G C . . . . C . . . A C . C . C . . . G C . T T . . . . . GB21 1 . . . T A . . . A . . . G A . G C . . . . C . . . A C . C . C . . . G C . T T . . . . . GB22 1 . . . T A . . . A . . . G A . G C . . . . C . . . A C . C . C . . . G C . T T . . . . . GB23 1 . . . T A . . . A . . . G A . G C . . . . C . . . A C . C . C . . . G C . T T . . . . . GB24 1 C . . . T A . . . A . . . G A . G C . . . . C . . . A C . C . C . . . G C . T T . . . . . GB25 1 C . . . T A . . . A . . . G A . G C . . . . C . . . A C . C . C . . . G C . T T . . . . . GB26 1 . . . T A . . . A . . . A . G C . . . . C . . . A C . C . C . . . G C . T T . . . . . GF27 46 . . . . C . . . A . . . A . . . A A . . C . C . . . G C . . . . GF28 3 . . . . C . . . A . . . A . . . A A . . C . C . . . G C . . . . GF29 2 . . . . C . . . A . . . A . . . A A . . C . C . . T G C . . . . GF30 1 . . . . C . . . A . . . A . . . A A . . C . C . . T G C . . . . GF31 1 . . . . C . . . A . . . A . . . A A . . C . C . . T G C . . . A . GF32 1 . . . . C . . . A . . . A . . . A A . . C . C . . . G C . . T . . . . . GF33 1 . . . . C . . . A . . . A . . . A A . . C . C G . . G C . . T . . . . . GF34 1 . . . . C . . . A . . . A . . . A . . C . C . . . G C T . . . . GF35 1 . . . . C . . . A . . . A . . . A A . A C . C . . . G C . . . . GF36 1 . . . . C T . . . A . . . A . . . A A . . C . C . . . G C . . . T GF37 1 . . . . C . . . A . . . A . . . A A . . C . C . . . G C . . . . GF38 1 . . . . C . . . A . . . A . . . A A . . C . C . . . G C . . . . GI39 40 . . . . C . . . T A . . . A . . . A . . C . C . . . G C . . . . GI40 6 . . . . C . . . T A . . . A . . . A . . C . C . . . G C . . . . GI41 3 . . . . C . . . T A . . . A . . . A . . C . C . . . G C . . . . C . . . GI42 1 . . . . C . . . T A . . . A . . . A . . C . C . . . G C . . . . GI43 1 . . . . C . . . T . T A . . . A . . . A . . C . C . . . G C . . . . GTE44 12 . . C . C . . . A T . . . A . . . C . . . A . . C . C . . . G C T . . . . GTE45 1 . . C . C . . . A T . . . A . . . C . . . A . . C . C . . . G C T . . . . GCO46 4 . . . . C . . . A . . C G . . . A T . . . A . . . G . A . . C . C . . . G C . . . G . . GCO47 1 . . . . C . . . A . . C G . . . A T . . . A . . . G . A . . C . C . . . G C . . . G . . GCO48 1 . . . T C . . . A . . C G . . . A T . . . A . . . G . A . . C . C . . . G C . . . G . . GCO49 1 . . . . C . . . A . T . . A . . A T . . . A . . . A . . C . C . . . G C . . . G . . GR50 2 . . . . C . . . A . . . A . . . C . . . . A . . C . C . . T G C . . . . GR51 1 . . . . C . . . A . . . A . . . C . . . . A . . C . C . . . G C . . . . GR52 1 . . . . C . . . A . . . A . . . C . . . . A . . C . C . . . G C . . . . GR53 1 . . . . C . . . A . . . A . . . C . . . . A . . C . C . . . G C . . . . GR54 1 . . . . C . . . A . . . A G . . . C . . . . A . . C . C . T . G C . . . . GH55 5 . . . A . . . T . A . G . . . C C . . G . . . A . . C T C . . . . C . . . G . . . . GH56 1 . . . A . A . . . T . A . G . . . C C . . G . . . A . . C T C . . . . C . . . G . . . . GH57 1 . . . A . . . . T T . A . G . . . C C . . G . . . A . . C T C . . . . C . . . G . . . . GH58 1 . . . A . . . T . A . G . . . C C . . G T . . A . . C T C . . . . C . . G G . . . . Alignment positiona

cytochromeb Sample site

16 16 16 16 16 16 16 16 16 16 16 16 16 17 1,3,7 5,7 1 1 7 7 7 1 7 7 2 6 7,8,9,11,12,13,14,15 14 14,15 14 14 14 11 9 * 10 6 11 7 6,7 7 7 7 3,5,6,15 6 17 17 17 17 * 17 * 15 15 4,5,6 6 4 *

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geographic proximity or the current distributions of the species. For example, between G. indicus and both G. coprotheresandG. rueppelliidivergence estimates are relatively low (1.1–0.6% incytb), yet the compared species occupy different continents (Asia and Africa). In contrast, species with geographically proximate distributions, G. coprotheres and G. africanus in Africa and G. tenui-rostris and G. himalayensis in South Asia, present divergence estimates which are relatively high (2.4 and 2.6%, respectively). Marine records of African climate variability document that important evolutionary changes during the Pliocene–Pleistocene interval were mediated by changes in African climate or shifts in climate variability (de Menocal 1995; de Menocal 2004). According to the analysis of best dated and most complete African mammal fossil databases, speciation changes occurred during the Pliocene–Pleistocene suggesting more varied and open habitats (humid taxa were replaced by arid adapted taxa). East African vegetation shifted from closed canopy to open savannah vegetation starting in the mid-Pliocene. Accord-ing to the variability selection hypothesis, climatic variability plays a significant role on faunal adaptation, selection, and evolution. InGypsspecies, divergence time that predates the Pleistocene suggesting that some Plio-cene–Pleistocene events may have been climatically mediated. The historic radiation of the genusGypslikely evolved under environmental conditions that no longer exist to the same extent throughout their current distributions.

Among Old World vultures, Gyps species are unique due to exclusive scavenger habits, while other vultures (Gypohierax angolensis) mainly feed on fruits, although they occasionally eat meat (Houston 1983; Mundy et al.

1992). This specialized feeding behavior among Gyps vultures seems to have evolved due to their close associ-ation with ungulate populassoci-ations, particularly migratory populations in Africa and Asia. In fact, the observed tem-poral and geographic diversification of Gyps vultures coincides with the diversification of Old World ungulates, especially in the family Bovidae (Vrba 1985; Arctander et al.1999; Matthee and Davis2001) and the expansion of grass-dominated ecosystems in Africa and Asia (Jacobs et al. 1999). These close associations likely played a sig-nificant role in adaptation and rapid diversification ofGyps vultures. According to Houston (1983), the large body size ofGypsand their ability to cover larger distances in search for food are related to the associated migrant distributions and seasonal fluctuations in mortallity of ungulates, and as a consequence they become incapable of hunting their own prey.

Conservation

The Oriental White-backed Vulture (Gyps bengalensis), once one of the most common raptors on the Indian sub-continent, has declined by over 95% within the past decade. This decline is mainly due to incidental con-sumptions of diclofenac, commonly used to treat domestic livestock. In South Asia, the conservation status of other Gypsvultures is also of immediate concern, given the lack of knowledge regarding status of their population and the continuing existence of taxonomic uncertainties. There-fore, an accurate assessment of the phylogenetic relationships among Gyps species should aid in their conservation by clarifying taxonomic uncertainties, and

Table 3 Average uncorrectedp-distances among Aegypiinae based oncytb(below diagonal) andRAG-1(above diagonal) sequences

Speciesa 1 2 3 4 5b 6 7 8 9 10 11 12 13 1. G. africanus(77) 0.002 0.000 0.000 – 0.000 0.000 0.002 0.005 0.004 0.004 0.004 0.005 2. G. bengalensis(38) 0.024 0.002 0.002 – 0.002 0.002 0.002 0.005 0.003 0.004 0.004 0.005 3. G. fulvus(60) 0.019 0.021 0.000 – 0.000 0.000 0.002 0.005 0.004 0.004 0.004 0.005 4. G. indicus(51) 0.016 0.017 0.006 – 0.000 0.000 0.002 0.005 0.004 0.004 0.004 0.005 5b G. tenuirostris(13) 0.021 0.024 0.011 0.010 – – – – – – – 6. G. coprotheres(7) 0.024 0.027 0.014 0.011 0.017 0.000 0.002 0.005 0.004 0.004 0.004 0.005 7. G. himalayensis(8) 0.028 0.026 0.021 0.019 0.026 0.027 0.002 0.005 0.004 0.004 0.004 0.005 8. G. rueppellii(6) 0.020 0.021 0.007 0.006 0.012 0.015 0.021 0.007 0.005 0.006 0.006 0.007 9. N. monachus(4) 0.081 0.078 0.075 0.076 0.078 0.079 0.083 0.074 0.003 0.003 0.003 0.004 10. S. calvus(2) 0.091 0.090 0.094 0.091 0.095 0.092 0.098 0.096 0.098 0.001 0.001 0.002 11. T. tracheliotos(2) 0.096 0.086 0.090 0.093 0.096 0.096 0.092 0.089 0.090 0.096 0.000 0.001 12 A. monachus (2) 0.084 0.076 0.082 0.083 0.087 0.086 0.080 0.080 0.088 0.084 0.041 0.001 13 T. occipitalis(1) 0.086 0.085 0.081 0.081 0.090 0.089 0.086 0.083 0.093 0.093 0.064 0.061

a Sample size in parentheses forcytbsequences b Missing data forRAG-1

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Fig. 2 Phylogenetic relationship among haplotypes of eight Gyps

species based oncytb gene sequences. The topology shown is the Bayesian tree and these are also congruent with MP and ML analysis.

MP and ML bootstrap nodal support values ([50 and[70) are above the branches and Bayesian posterior probabilities are below

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Fig. 3 Phylogeny amongGypsspecies based on mitochondrialcytb

and nuclearRAG-1 sequence. The topology shown is the Bayesian inference and these are congruent with MP and ML analysis as well.

MP and ML bootstrap nodal support values ([50 and[70) are above the branches and Bayesian posterior probabilities are below

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enabling inference of their respective relatedness to the susceptibleG. bengalensis.In the present study, phyloge-netic relationships for all recognized species were assessed within the genusGyps. Dry forest landscapes in Cambodia still support vulture populations, because the effect of diclofenac was not so severe due to lack of free ranging domestic cattle (Handschuh 2007). In Cambodia, Gyps bengalensis populations share many haplotypes with the declining populations in Pakistan and India. Due to the absence of a phylogeographical structure within the studied taxa, reintroduction programs could be achieved by restocking individuals from the remaining populations. The continuing veterinary use of diclofenac is an unknown but potential risk to related species with similar feeding habits asGyps bengalensis.

Zusammenfassung

Stammesgeschichte und Phylogeographie kritisch bedrohter Gyps-Arten basierend auf Kern- und mitochondrialen Markern

Die Populationen vonGyps bengalensisundGyps indicus haben auf dem indischen Subkontinent zwischen der Mitte der 80er Jahre und dem Ende der 90er Jahre um 95% bis 100% dramatisch abgenommen. Das Ziel der vorliegenden Studie war die Untersuchung der Stammesgeschichte und Phylogeografie von Geiern der Gattung Gyps anhand von Kern- und mitochondrialen Markern (RAG-1 bzw. cyt b). Die Gattung Gyps ist monophyletisch und trotz des großen Stichprobenumfangs konnte keine geographische

Fig. 4 Minimum spanning network representing the relationships between haplotypes of eight species of the genusGypsmentioned in Table2. The size of eachcircleis approximately proportional to the number of individuals sharing those haplotypes

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Aufgliederung innerhalb den 3 Arten (G. bengalensis, G. indicus und G. fulvus) nachgewiesen werden. Unsere Untersuchung unterstu¨tzt die Trennung vonG. indicusund G. tenuirostrisin zwei verschiedene Arten. In allen Ana-lysen wird zuna¨chstG. bengalensisvon allen anderenGyps Taxa abgetrennt, wa¨hrend G. fulvus und G. rueppellii Geschwistertaxa repra¨sentieren, die wiederum eine Schwestergruppe zu G. indicus, G. tenuirostris und G. coprotheresbilden. Die molekulare Uhr, basierend auf Kern- und mitochondrialen Genen weist auf eine rasche und rezente Auftrennung innerhalb derGypsArten hin.

Acknowledgments This Project was supported by a fellowship of the Deutscher Akademischer Austausch Dienst to M.A. We are very grateful to many people who helped us with the field work, especially the Ministry of Environment and Tourism Namibia for permission to collect and export the samples. We thank Hedi Sauer-Gu¨rth for her help in the laboratory as well as Theodor C. H. Cole for improving this manuscript. We thank Prof. Dr. H. Bock (Managing Director of IWR) and S. Friedel for access to parallel computing facilities at the interdisciplinary center for Scientific Computing (IWR, Heidelberg University). Finally, we greatly appreciate the constructive comments of anonymous referee to improve the final manuscript.

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