Evidence for Introgressive Hybridization of Captive Markhor (Capra falconeri) with Domestic Goat: Cautions for Reintroduction

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1 Biochem Genet (2008) 46: DOI /s y Evidence for Introgressive Hybridization of Captive Markhor (Capra falconeri) with Domestic Goat: Cautions for Reintroduction Sabine E. Hammer Æ Harald M. Schwammer Æ Franz Suchentrunk Received: 2 May 2007 / Accepted: 2 November 2007 / Published online: 29 January 2008 Ó Springer Science+Business Media, LLC 2008 Abstract Markhors (Capra falconeri) are among the most endangered mammal species, and several conservation measures, including ex situ breeding, are implemented to prevent their extinction. We studied sequence diversity and differentiation of the first hypervariable segment of the mitochondrial DNA control region among C. f. heptneri and C. f. megaceros kept in four zoos in relationship to lineages of other wild and domestic goats, to assess for the first time the level of molecular distinctness and variability among those subspecies, and to check for possible introgression by related Capra taxa, such as domestic goats. Levels of differentiation between some Capra falconeri lineages and modern domestic goats were similar to levels between other wild goat species (i.e., Capra aegagrus, Capra ibex) and domestic goats. Among pure markhor lineages, paraphyly was observed for C. f. heptneri, suggesting occurrence of shared ancestral polymorphism among markhor subspecies and/or ancient or recent gene exchange between subspecies. Interestingly, 35.7% of all studied markhors from three zoos are introgressed by the domestic goat. Furthermore, despite relatively small breeding group sizes, markhors have maintained a relatively high proportion of mtdna variation within zoo groups. In any case, the existence of markhors introgressed with domestic goat DNA in zoos should be considered when selecting markhors for ex situ breeding programs with the aim of building up a stock for later reintroduction into the wild. S. E. Hammer (&) Clinical Immunology, University of Veterinary Medicine Vienna, Veterinärplatz 1, A-1210 Vienna, Austria sabine.hammer@phylo-dat.net H. M. Schwammer Zoo Vienna, Maxingstrasse 13 B, A-1130 Vienna, Austria F. Suchentrunk Research Institute of Wildlife Ecology, University of Veterinary Medicine Vienna, Savoyenstrasse 1, A-1160 Vienna, Austria

2 Biochem Genet (2008) 46: Keywords Capra falconeri Markhor Mitochondrial control region Introgression Conservation genetics Introduction Markhors, Capra falconeri (Wagner 1839), are today struggling for survival in semiarid, cliffside mountain areas (at an elevation of 600 3,600 m) of Tajikistan, Afghanistan, Pakistan, in the southern border region of Uzbekistan and Turkmenistan, as well as in northern India (Fig. 1). The majority of the total world population is found in Pakistan and is estimated to encompass about 3,200 3,700 animals, with numbers generally decreasing (Shackleton 1997; Weinberg et al. 1997). However, certain conservation measures, such as community-based conservation or ex situ breeding, have been implemented in recent years and appear to Fig. 1 Distribution of the markhor, C. falconeri, modified after Brent Huffman, ungulate.com (compiled from Shackleton 1997 and Weinberg et al. 1997). The gray shading indicates areas in which markhor occurrence has been recorded. The Bukharan markhor, C. f. heptneri, occurs in Uzbekistan, Turkmenistan, Tajikistan, and Afghanistan, whereas the straight-horned markhor, C. f. megaceros, can be found mainly in Afghanistan and Pakistan. The arrows indicate markhor populations that might have gone extinct already (Weinberg et al. 1997)

3 218 Biochem Genet (2008) 46: have a positive effect on at least some markhor populations (Virk 2000; see also Rosser et al for a critical review of regulated trophy hunting in markhor). The subspecies C. f. falconeri (the Astor or flare-horned markhor) and C. f. megaceros (the Kabul or straight-horned markhor) are endangered, and C. f. heptneri (the Tadjik or Bukharan markhor) is critically endangered (Shackleton 1997). The subspecies C. f. cashmiriensis (the Cashmere markhor) and C. f. jerdoni (the Suleiman markhor) are not listed in the 2004 IUCN Red List, but C. f. jerdoni and C. f. megaceros are registered as endangered in the CITES Appendix I, U.S. ESA (cf. Grubb 2005). Markhor occurs mainly in highly fragmented populations of relatively small local size (\100 individuals) that are threatened by habitat loss, uncontrolled poaching, illegal trophy hunting, and disturbance, together with forage competition from domestic livestock (Shackleton 1997; Weinberg et al. 1997). A captive breeding program for the Tadjik markhor (C. f. heptneri) was running in Tajikistan in the late 1980s, but the civil war in Afghanistan caused extensive damage to the ecology of the reserve. Currently, a captive breeding program (ARES markhor breeding program) is aimed at developing a group of genetically diverse C. f. heptneri in semiopen habitat in the Lesser Carpathians, Slovakia (Pokoradi 2005). Most of the markhor currently held in western zoos or wildlife parks are considered to belong to this latter subspecies (Shackleton 1997). The objective of this study is to investigate diversity and differentiation patterns within and among C. f. heptneri and C. f. megaceros (C. f. falconeri samples could not be obtained) kept in zoos in relationship to lineages of other wild and domestic goats. The extent of molecular lineage differentiation within and between subspecies or groups held in zoos should be relevant for decisions associated with possible ex situ breeding programs. In particular, we ask how divergent mitochondrial lineages are in individuals kept in zoos, and whether zoo individuals represent pure lineages or show introgression by domestic goat; the latter cannot be excluded a priori for all zoo groups. Materials and Methods Studied Animals Total genomic DNA was isolated from whole blood samples of 28 captive markhor from four zoos in Europe, representing two different subspecies: the Bukharan markhor C. f. heptneri and the straight-horned markhor C. f. megaceros (Table 1). Whole blood samples of captive markhor were collected from 1996 to 1998, and the study presented here was carried out in the years 2005 and DNA Extraction and Sequencing Total DNA was extracted using the GenElute Mammalian Genomic DNA Miniprep Kit (Sigma, MO), following the manufacturer s instructions. Approximately 1 lg of extracted DNA was subjected to a first round of PCR to amplify the entire mtdna

4 Biochem Genet (2008) 46: Table 1 Wild specimens included in the analysis of the phylogenetic relationship among Capra species, with special reference to the markhor (C. falconeri) Species Common name Geographic origin Accession number Reference b mtdna HVI haplotype c C. [ibex] caucasica West Caucasian tur Georgia AJ C. [ibex] sibirica Siberian ibex Pakistan AJ C. [ibex] nubiana Nubian ibex Israel AJ C. cylindricornis Daghestan tur Dagestan (Russia) AJ Dagestan (Russia) AJ C. aegagrus blythi Sindh ibex Kirthar Mountains (Pakistan) AB C. aegagrus Bezoar ibex Japan a AB Turkmenistan or AJ France a Turkmenistan or AJ France a Turkmenistan or France a AJ C. falconeri heptneri Bukharan markhor Tajikistan or Turkmenistan Tajikistan or Turkmenistan Zoo Berlin-1 Zoo Berlin-2 Zoo Berlin-3 Zoo Berlin-4 Zoo Berlin-5 Zoo Berlin-6 Zoo Berlin-7 Zoo Berlin-8 Zoo Berlin-9 Zoo Blackpool-10 (UK) Zoo Blackpool-11 (UK) Zoo Blackpool-12 (UK) Zoo Helsinki-14 AJ AJ HVI_CF7 AM HVI_CF1 AM HVI_CF5 AM HVI_CF2 AM HVI_CF5 AM HVI_CF3

5 220 Biochem Genet (2008) 46: Table 1 continued Species Common name Geographic origin Accession number Reference b mtdna HVI haplotype c C. falconeri megaceros Straight-horned markhor Zoo Helsinki-15 Zoo Helsinki-16 Zoo Helsinki-17 Zoo Helsinki-18 Zoo Helsinki-19 Zoo Helsinki-20 Zoo Helsinki-21 Zoo Helsinki-22 Zoo Helsinki-23 Zoo Helsinki-24 Zoo Vienna-25 (Austria) Zoo Vienna-26 (Austria) Zoo Vienna-27 (Austria) Zoo Vienna-28 (Austria) Zoo Vienna-29 (Austria) Ovis aries Domestic sheep AB AM HVI_CF5 AM HVI_CF5 AM HVI_CF5 AM HVI_CF4 a Details for the geographic origin of the C. aegagrus specimens: Japan, Gumma Safari Park, Tomioka (Japan); Turkmenistan or France, Turkmenistan or captive (Paris, France) b References: 1, Luikart et al. (2001); 2, Takada et al. (1997); 3, Sultana et al. (2003); 4, this study c Whole blood samples of captive markhor were collected from 1996 to C. falconeri private haplotypes are indicated in bold. Ovis aries served as outgroup in the phylogenetic reconstruction control region by using universal primers H00651 and L15926 (Kocher et al. 1989). To target 372 bp at the 5 0 end of the mtdna HVI region, a nested PCR was performed with the primers CAPFI and CAPFI (Luikart et al. 2001), tagged with M13-specific tails for automated sequencing. PCR reactions were performed on an Eppendorf Mastercycler Gradient; the thermal cycling parameters and protocols are given in Kocher et al. (1989) and Luikart et al. (2001), respectively. PCR reactions were analyzed on 1.5% agarose/etbr gels in 19 TAE, purified and subjected to automated sequencing on the LiCor Long Readir 4200 (LiCor, Inc., NE).

6 Biochem Genet (2008) 46: Capra hircus Subset for Phylogenetic Reconstruction We constructed a DNA sequence data matrix (372 bp), comprising the seven newly found C. falconeri haplotypes of the mtdna HVI region, 11 Capra spp. originating either from wild populations or from zoos (Table 1), and 428 C. hircus haplotypes, by using the alignment program Clustal X (Thompson et al. 1997). The computed sequence alignment was checked by eye and manually edited if necessary. Although we used a shorter mtdna HVI alignment of 372 bp instead of 469 bp (Luikart et al. 2001), neither a loss of potentially informative haplotypes nor a significant reduction of the phylogenetic signal (i.e., parsimony informative sites) or important alterations in the topologies of the resulting trees (data not shown) were detected, when comparing the downloaded sequences of the two alignment sizes (372 vs. 469 bp). Calculating the percent sequence identity of the two C. f. heptneri (Luikart et al. 2001) (accession nos. AJ and AJ317873) based on either 469 bp or on our presently used 372 bp segment did not show a significant difference ( and ). Thus, we concluded that our reduced alignment length would not have reduced the overall information in the study significantly. The C. hircus subset included 314 haplotypes from the Middle and Near East, southern and northern Europe, Asia, and sub-saharan Africa (Luikart et al. 2001) (accession nos. AJ AJ317875); 36 haplotypes from Europe, Africa, India, and Asia (Amills et al. 2004) (AY AY424949); 49 haplotypes from Pakistan (Sultana et al. 2003; Sultana and Mannen 2004) (AB AB110591, AB AB162217); 14 haplotypes from China (Chen et al. 2005) (AY AY853301); 9 haplotypes from Laos (Mannen et al. 2001) (AB AB044304); and 6 haplotypes from zoos or national livestock breeding centers in Japan (Takada et al. 1997) (AB AB004082). Phylogenetic Reconstruction Analyses Neighbor-joining (NJ) analysis was conducted with Mega version 3.1 (Kumar et al. 2004) under the TrN93+I+G model (Tamura and Nei 1993) optimized for this data set by Modeltest 3.06 (Posada and Crandall 1998). Additionally, maximumlikelihood and parsimony analyses were conducted using PAUP version 4.02b (Swofford 2001), implementing the settings of the TrN93+I+G model in the respective analysis. The robustness of the phylogenies was assessed by the bootstrap percentage (Felsenstein 1985) and by the reliability percentages, i.e., the number of times the group appears after 10,000 puzzling steps. Pairwise distances and net distances between groups were computed using Mega version 3.1 (Kumar et al. 2004) based on the previously selected model (Tamura and Nei 1993). Results and Discussion From the 28 studied markhor specimens, we obtained seven mtdna HVI haplotypes, occurring solely among C. falconeri and including four private ones:

7 222 Biochem Genet (2008) 46: HVI_CF1, 2, 3, and 4 (Table 1; Fig. 2). All mtdna HVI haplotypes were found among C. f. heptneri, except HVI_CF4, which seems to be restricted to C. f. megaceros. The mtdna HVI haplotypes HVI_CF5 and 7 are the only ones shared by C. f. heptneri and C. f. megaceros. In the NJ bootstrap consensus tree, those particular haplotypes occur only among the markhor forming the distinct cluster clearly separated from the domestic goats (Fig. 2). The NJ analysis further revealed that the C. falconeri haplotypes HVI_CF2, 3, and 6 arise among the C. hircus-a cluster, displaying HVI_CF3 as a basal offshoot in this cluster. For the domestic goat sequences downloaded, we found four mtdna HVI haplogroups (C. hircus-a, -B, -C, and D). The large star-shaped cluster (C. hircus-a) comprises 398 C. hircus mtdna HVI haplotypes found in specimens from all geographic sampling locations, together with one C. aegagrus haplotype (Takada et al. 1997) (accession no. AB004076). The three smaller clusters (C. hircus-b, -C, and -D) contain only 3, 18, and 9 C. hircus mtdna HVI haplotypes, respectively, found mainly in Pakistan, Cashmere, Laos, Mongolia, and China. Maximum-likelihood and parsimony-based Fig. 2 NJ bootstrap consensus tree of 447 haplotypes of Capra spp. The tree was constructed under the TrN93+I+G model (base frequencies: A = , C = , G = , T = ; proportion of invariable sites: ; shape parameter of the gamma distribution: ). The branch lengths are proportional to the amount of evolutionary change that has occurred along them. Numbers at the major clades denote the bootstrap percentages of 1,000 replicates. The large star-shaped cluster (C. hircus-a) contains 398 mtdna HVI haplotypes, whereas the three smaller lineages (C. hircus-b, -C, and -D) contain only 18, 3, and 9 mtdna HVI haplotypes, respectively. The inset (a) shows the haplotype composition of the first C. falconeri cluster in more detail. The inset (b) displays the position of C. falconeri haplotypes in the large star-shaped cluster (C. hircus-a) in more detail. Private C. falconeri haplotypes are indicated in bold

8 Biochem Genet (2008) 46: trees were nearly identical in shape, with similarly high bootstrap supports (data not shown). Three C. f. heptneri haplotypes (HVI_CF2, 3, and 6) occurred together with one C. aegagrus (Takada et al. 1997) (accession no. AB004076) within the large starshaped cluster of C. hircus-a, pointing toward a retained ancestral variation, or introgression of domestic goat genes into markhors. Among pure markhor lineages, paraphyly was observed for C. f. heptneri, which might indicate occurrence of shared ancestral polymorphism within markhor and/or ancient or recent gene exchange between these subspecies in the wild. The wild-living C. f. heptneri (Luikart et al. 2001) (accession no. AJ317872) showed nearly the same phylogenetic distance from the neighboring C. f. heptneri haplotypes as was observed for the C. hircus-d clade and C. ibex caucasica (Luikart et al. 2001) (accession no. AJ317875) (Fig. 2; Table 2). Among the two C. cylindricornis sequences (Luikart et al. 2001) (accession nos. AJ AJ317869), a slightly lower divergence was found than among nonintrogressed markhor (Table 2). That latter comparison might suggest a similar divergence in the mtdna of wild-living markhors as compared to other wild-living goats (e.g., C. aegagrus; Fig. 2). Genetic distances between wild goats and wild-living and captive markhors were 11.1 and 12.4%, respectively, whereas pairwise comparison between captive and wild-living markhors exhibits 8.1% (Table 3). To determine the proportion of sequence variation of markhor due to the investigated zoo groups and the two studied subspecies (C. f. heptneri and C. f. megaceros), we used the Arlequin version 2.0 (Schneider et al. 2000) software package to run two separate AMOVAs (Excoffier et al. 1992), one based on the two subspecies and one based on the five zoo groups. Nevertheless, our two AMOVA models showed that sequence variation was predominantly due to variation within subspecies rather than to partitioning into the two studied subspecies (82.48 vs %, P = , first AMOVA model) and due to variation within single zoo groups rather than between zoo groups (71.63 vs %, P \ , second AMOVA model). The present results indicate levels of differentiation between some C. falconeri lineages and modern domestic goats (C. hircus) similar to levels between other wild goat species (i.e., C. aegagrus, C. ibex) and domestic goats (Table 2; cf. Bruford et al. 2003). The basic body pattern of all wild and domestic goats is similar, and moreover, they can freely interbreed in captivity (Manceau et al. 1999). The close gene pool relationships among diverse taxa within the genus Capra and the possibility of (repeated) introgressive hybridization of ancestral or modern populations render the whole group difficult taxonomically (see also Grubb 2005). Our current results suggest that 35.7% of all studied individuals from three zoos are introgressed by domestic goat mtdna. Hybridization of markhor or other species (e.g., C. aegagrus, C. ibex) held in zoos with domestic goats (or wild goats) is plausible; however, it can be speculated that in some cases introgressed wildliving ancestral C. falconeri herds may have been the source populations of captive markhors. The possibility of introgressed animals or whole groups in zoos should be considered when selecting markhors for ex situ breeding programs, with the aim of building up a stock for later reintroduction into the wild. We expect introgression of

9 224 Biochem Genet (2008) 46: Table 2 Tamura Nei distances a of wild Capra spp. and captive markhor (C. falconeri) [1] [0.028] [0.028] [0.021] [0.025] [0.027] [0.010] [0.009] [0.028] [0.028] [0.009] [0.030] [0.029] [0.047] [0.041] [0.025] [0.031] [0.035] [2] [0.000] [0.010] [0.024] [0.030] [0.028] [0.025] [0.027] [0.029] [0.027] [0.027] [0.027] [0.030] [0.035] [0.027] [0.031] [0.034] [3] [0.010] [0.024] [0.030] [0.028] [0.025] [0.027] [0.029] [0.027] [0.027] [0.027] [0.030] [0.035] [0.027] [0.031] [0.034] [4] [0.022] [0.031] [0.021] [0.019] [0.028] [0.029] [0.020] [0.029] [0.026] [0.030] [0.032] [0.022] [0.025] [0.030] [5] [0.024] [0.024] [0.025] [0.025] [0.023] [0.023] [0.022] [0.024] [0.032] [0.028] [0.023] [0.021] [0.028] [6] [0.031] [0.029] [0.009] [0.003] [0.030] [0.008] [0.019] [0.031] [0.033] [0.031] [0.032] [0.030] [7] [0.008] [0.030] [0.030] [0.005] [0.028] [0.027] [0.039] [0.038] [0.025] [0.028] [0.033] [8] [0.029] [0.028] [0.006] [0.029] [0.026] [0.039] [0.038] [0.023] [0.030] [0.034] [9] [0.008] [0.028] [0.005] [0.018] [0.026] [0.031] [0.029] [0.030] [0.025] [10] [0.029] [0.008] [0.018] [0.030] [0.032] [0.029] [0.031] [0.029] [11] [0.027] [0.025] [0.039] [0.036] [0.024] [0.027] [0.031] [12] [0.017] [0.025] [0.030] [0.029] [0.029] [0.024] [13] [0.028] [0.029] [0.019] [0.024] [0.028] [14] [0.034] [0.033] [0.035] [0.036] [15] [0.029] [0.026] [0.036] [16] [0.023] [0.028] [17] [0.027] [18] Distance values (Tamura and Nei 1993) are given below the diagonal, and standard errors (computed by 1,000-bootstrap replicates) are given in brackets above the diagonal. Specimens: 1 4, C. aegagrus (Bezoar ibex, accession nos. AB004076, AJ317867, AJ317866, AJ317864); 5, C. aegagrus blythi (Sindh ibex, AB110591); 6 12, C. falconeri HVI_CF1-CF7 (captive markhor); 13, C. f. heptneri (Bukharan markhor, AJ317872); 14, C. [ibex] sibirica (Sibirian ibex, AJ317874); 15, C. [ibex] nubiana (Nubian ibex, AJ317871); 16, C. [ibex] caucasica (West Caucasian tur, AJ317875); 17, C. cylindricornis (Daghestan tur, AJ317868); 18, C. cylindricornis (Daghestan tur, AJ317869) a

10 Biochem Genet (2008) 46: Table 3 Tamura Nei distances a between wild and captive Capra groups Wild Capra spp. b Captive markhor c Wild markhor d Wild Capra spp / /0.008 Captive markhor 0.124/ / /0.003 Wild markhor 0.111/ / /0.002 a Distance values (Tamura and Nei 1993) between groups/net between groups are given below the diagonal, and standard errors (computed by 1,000 bootstrap replicates) are given above the diagonal. Distance values for nonintrogressed captive versus wild-living markhors are given in italics b Wild Capra spp.: C. aegagrus (Bezoar ibex, accession nos. AB004076, AJ317867, AJ317866, AJ317864); C. aegagrus blythi (Sindh ibex, AB110591); C. falconeri heptneri (Bukharan markhor, AJ317872); C. [ibex] sibirica (Sibirian ibex, AJ317874); C. [ibex] nubiana (Nubian ibex, AJ317871); C. [ibex] caucasica (West Caucasian tur, AJ317875); C. cylindricornis (Daghestan tur, AJ317868); C. cylindricornis (Daghestan tur, AJ317869) c Captive markhor: C. falconeri HVI_CF1-8 d Wild markhor: C. f. heptneri (Bukharan markhor, AJ and AJ317873) nuclear DNA as well in markhors, but identification of such cases would necessitate information on characteristic nuclear alleles (e.g., at microsatellite loci) or at least frequency estimates of such alleles for pure markhor. Apart from the presence of introgressed markhors among assumed pure animals, one of our AMOVA models revealed that despite relatively small group sizes in zoos, markhors obviously have maintained a relatively high proportion of mtdna variation within breeding groups, as well as no strong drift effect due to breeding in separate zoo groups. This might be the result of frequent transfers (in earlier and/or recent days) of markhor among zoos or breeding groups, and might have also affected the differentiation pattern of lineages in the two subspecies studied. To investigate whether markhor individuals kept in zoos, wildlife parks, or breeding stations are indeed representative genetically for wild-living populations to be useful for liberation programs, noninvasive genetic diversity surveys of wildliving markhor populations based on nuclear markers (e.g., microsatellites) and mtdna should be considered. Acknowledgments For collaboration and help provided in collecting samples we thank Wolfgang Zenker. We also thank Anita Haiden for her extensive laboratory assistance. This work was part of a project funded by the Verein der Freunde des Tiergartens Schönbrunn, granted to Franz Suchentrunk. References Amills M, Capote J, Tomas A, Kelly L, Obexer-Ruff G, Angiolillo A, Sanchez A (2004) Strong phylogeographic relationships among three goat breeds from the Canary Islands. J Dairy Res 71: Bruford MW, Bradley DG, Luikart G (2003) DNA markers reveal the complexity of livestock domestication. Nat Rev Genet 4: Chen SY, Su YH, Wu SF, Sha T, Zhang YP (2005) Mitochondrial diversity and phylogeographic structure of Chinese domestic goats. Mol Phylogenet Evol 37:

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