Highly Endangered African Wild Dogs (Lycaon Pictus) Lack Variation at the Major Histocompatibility Complex

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Journal of Heredity 2009:100(Supplement 1):S54–S65 Ó The American Genetic Association. 2009. All rights reserved. doi:10.1093/jhered/esp031 For permissions, please email: [email protected]. Advance Access publication June 16, 2009 Highly Endangered African Wild Dogs (Lycaon pictus) Lack Variation at the Major Histocompatibility Complex

CLARE D. MARSDEN,BARBARA K. MABLE,ROSIE WOODROFFE,GREGORY S. A. RASMUSSEN, SARAH CLEAVELAND,J.WELDON MCNUTT,MASENGA EMMANUEL,ROBERT THOMAS, AND LORNA Downloaded from https://academic.oup.com/jhered/article/100/suppl_1/S54/892226 by guest on 01 October 2021 J. KENNEDY

Division of Ecology and Evolutionary Biology, Graham Kerr Building, University of Glasgow, Glasgow, G12 8QQ, UK (Marsden, Mable, and Cleaveland); Centre for Integrated Genomic Medical Research, University of Manchester, Manchester, UK (Kennedy); Institute of Zoology, Regent’s Park, London, UK (Woodroffe); Wildlife Conservation Research Unit, Department of Zoology, University of Oxford, Tubney, Oxon, UK (Rasmussen); Painted Dog Research, Natural History Museum, Bulawayo, Zimbabwe (Rasmussen); Botswana Predator Conservation Trust, Maun, Botswana (McNutt); Tanzanian Wildlife Research Institute, Arusha, Tanzania (Emmanuel); and Royal Zoological Society of Scotland, Edinburgh zoo, Corstorphine Road, Edinburgh, EH12 6TS, UK (Marsden and Thomas).

Address correspondence to Clare Marsden at the address above, or e-mail: [email protected].

Abstract The major histocompatibility complex (MHC) is a set of highly polymorphic genes involved in the immune response. Extensive research on the canid MHC has found moderate-to-high levels of diversity at the DLA-DRB1, DLA-DRA, DLA- DQA1, and DLA-DQB1 class II loci with frequent transspeciﬁc polymorphism among Canis species. In this study, we assessed MHC variation in the more distantly related and highly endangered African wild dog (Lycaon pictus). We screened 168 African wild dogs from Eastern and Southern Africa as well as 200 samples from the European captive population for variation at MHC class II loci. As for all other canids screened to date, we found a single allele at DLA-DRA, which was the same as that found in Canis species. In contrast, we found 17 DLA-DRB1 alleles, one DLA-DQA1 allele, and two DLA- DQB1 alleles, all of which were unique to African wild dogs. At DLA-DRB1, African wild dogs were found to have comparable numbers of alleles but less overall amino acid variation than other canids. However, the low numbers of alleles at DLA-DQA1 and DLA-DQB1 are surprising, given that in other canids, these loci are also highly variable. Overall, our data suggest that African wild dogs are genetically depauperate at the MHC relative to other canids. These data are indicative of a loss of genetic variation, possibly as a result of population bottlenecks and declines experienced by this species. Key words: adaptive variation, DLA, Lycaon pictus, MHC, population bottleneck

The major histocompatibility complex (MHC) is a highly adaptive genetic variation for evolutionary change and rising diverse set of vertebrate genes that code for molecules concerns about infectious diseases in the conservation of involved in the recognition of intra- and extracellular endangered species (Daszak et al. 2000), assessments of antigens and, therefore, form a fundamental component MHC variation are increasingly incorporated into endan- of immune responses (Eggert et al. 1998; Hedrick 2003; gered species research (e.g., giant panda Ailuropoda melano- Piertney and Oliver 2006). MHC genes are renowned for leuca (Wan et al. 2006), crested ibis Nipponia nippon (Zhang their high allelic diversity and heterozygosity, which is et al. 2006), and Mexican wolf Canis lupus baileyi (Hedrick thought to be the result of pathogen-driven balancing et al. 2000)). selection (Van Den Bussche et al. 1999). Diversity at the Considerable research has been conducted on the canid MHC is adaptively signiﬁcant in disease resistance; high MHC (known as the dog leukocyte antigen, DLA) in the diversity has been shown to allow response to a wider range domestic dog Canis familiaris and more recently, wild Canis of parasites and pathogens than low diversity (Hedrick et al. species: Gray wolf (Canis lupus) (Seddon and Ellegren 2002; 2001, 2003; Sommer et al. 2002). Given the importance of Kennedy et al. 2007), Coyote (Canis latrans) (Seddon and

S54 Marsden et al. African Wild Dogs (Lycaon pictus) Lack Variation at the MHC

Ellegren 2002), Ethiopian wolf (Canis simensis) (Kennedy LJ, packs; study population size ;300); Serengeti, Northern unpublished data), and Mexican wolf (Hedrick et al. 2000). Tanzania (n 5 14 from 4 packs; study population size Research has focused on variation at 3 MHC class II loci: ;160); Okavango, Northern Botswana (n 5 53 from 8 DLA-DRB1, DQA1, and DQB1, which are physically packs; study population size ;200); and Hwange, Western tightly linked and inherited as a haplotype (Kennedy et al. Zimbabwe (n 5 15 from 7 packs; study population size 2007). MHC class II loci are involved in the recognition of ;250). The sampled Serengeti population, hereafter re- antigens of extracellular pathogens and parasites. However, ferred to as New Serengeti, represents a population that is strong linkage disequilbrium has been found between MHC thought to have naturally re-established in the early 2000s, class I and II loci in humans (Sanchez-Mazas et al. 2000), rather than the Serengeti population assessed in previous domestic dogs, and many other species studied, suggesting genetic studies (Girman et al. 1997, 2001), which was that variation at MHC class II loci can also reﬂect variation extirpated with the last pack disappearing in 1991 (Woodroffe

at MHC class I loci, which are involved in the recognition of et al. 1997). South African samples were derived from a set Downloaded from https://academic.oup.com/jhered/article/100/suppl_1/S54/892226 by guest on 01 October 2021 intracellular pathogens such as viruses (Piertney and Oliver of animals artificially reintroduced and translocated between 2006). To date, 134 DLA-DRB1 alleles, 26 DLA-DQA1 game reserves in South Africa and included some captive alleles, and 68 DLA-DQB1 alleles have been assigned animals of South African origin (A. Bastos, n 5 43). This official names by the DLA Nomenclature Committee South African sample set is considered as a managed (Kennedy LJ, unpublished data). These genes have been group of animals rather than a free-ranging population. shown to be polymorphic across the Canis genus, with A further 6 samples from this managed group were collected particularly high levels of polymorphism in both the from a set of 16 wild dogs that were translocated from domestic dog and Gray wolf (Seddon and Ellegren 2002; Pilanesberg Game Reserve, South Africa, to Hwange Kennedy 2007; Kennedy et al. 2007). Transspecific poly- National Park, Zimbabwe, in 2006. These animals were morphism (allele sharing) has been found to be a recurring analyzed as part of the South African sample set rather feature among Canis species at all 3 loci. A fourth locus, thantheHwangesampleset.The15Hwangesamplesdo DLA-DRA, appears to be monomorphic for allele DLA- not include any animals recently translocated from South DRA*0101 in all canids screened to date (Kennedy LJ, Africa or their offspring. Ear or muscle samples were also unpublished data). Given the focus of research on the genus provided from carcasses collected in Kajiado district in Canis, it is not currently known if these patterns of MHC Southern Kenya (R. Woodroffe, n 5 1), Ghanzi district in polymorphism are specific to these species or a characteristic Western Botswana (M. Swarner, n 5 1), Northern Sofala of canids in general. province in Central Mozambique (J.-M. Andre´, n 5 3from African wild dogs are the sole member of the Lycaon one pack), and Mangetti district in North Western Namibia genus and a distantly related member of the wolf-like canid (F. Stander, n 5 1). We sampled 200 captive African wild clade, to which the genus Canis belongs (Girman et al. dogs (75% of the total population) from the European 1993). This highly endangered social species has suffered Endangered Species Program, which are derived from extensive declines in the wild to ,6000 individuals founders from Southern Africa. This sample set was distributed across a few remaining small and fragmented analyzed together and is hereafter referred to as EU zoos. populations (Figure 1) (Woodroffe and Ginsberg 1997; Details of the 31 contributing institutions are given in Sillero-Zubiri et al. 2004). Disease is argued to represent Supplementary table 1. a significant threat to African wild dogs, which share DNA was extracted from samples using DNeasy susceptibility to diseases of common sympatric canids such extraction kits (Qiagen, Crawley, UK) according to the as jackals and domestic dogs (Alexander et al., forthcoming), manufacturer’s instructions, with the following modifica- outbreaks of which have resulted in both pack and tions: Tissue samples were lysed for 18 h rather than 3; population extinctions in the past (reviewed in Woodroffe blood spots and hair samples were lysed for 3 h rather than et al. 2004). Consequently, knowledge of the MHC is one. A negative control was conducted with all extractions particularly pertinent to African wild dog conservation. to detect contamination. In this study, we have characterized MHC class II DLA- Sequence-based typing was conducted on exon 2 of the DRB1, DRA, DQA1, and DQB1 variation in African wild DLA-DRB1, DLA-DQA1, and DLA-DQB1 loci using dogs to extend knowledge of the canid MHC to more locus-specific intronic domestic dog primers that gave distantly related canid species (Lindblad-Toh et al. 2005). products of 303 bp (DLA-DRB1), 345 bp (DLA-DQA1), Specifically, we assessed levels of polymorphism at MHC and 300 bp (DLA-DQB1). Primers were as follows (M13 and class II loci and looked for evidence of allele sharing T7 tails are underlined): DRBln1: ccg tcc cca cag cac att tc between African wild dogs and species in the genus Canis. (Wagner et al. 1996); DRBln2M13r: cag gaa aca gct atg acc tgt gtc aca cac ctc agc acc a (Wagner et al. 1996); DQAln1: taa ggt tct ttt ctc cct ct (Wagner et al. 1996); DQAIn2: gga cag att Methods cag tga aga ga (Wagner et al. 1996). DQB1BT7 taa tac gac tca cta tag gg ctc act ggc ccg gct gtc tc (Wagner et al. 1996); Blood, tissue, hair, and serum samples were provided from DQBR2: cac ctc gcc gct gca acg tg (Kennedy, Barnes, Happ, free-ranging study populations in Eastern and Southern Quinnell, Bennett, et al. 2002). A fourth MHC class II locus Africa (Figure 1): Laikipia, Central Kenya (n 5 56 from 13 DLA-DRA, which has been shown to be monomorphic in all

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Figure 1. Historic (light gray) and present (dark gray) range of African wild dogs according to McNutt et al. (2008). Sampling locations are shown with circles. Carcass samples are depicted with smaller circles and italics. Country codes: Kenya, KNY; Tanzania, TNZ; Zimbabwe, ZIM; Botswana, BOT; Mozambique, MOZ; South Africa, SAF; and Namibia, NAM.

other canids tested to date (Kennedy LJ, unpublished data), increased from 20 to 30 cycles for DNA derived from hair, was examined using locus-specific exonic primers: DRAF: blotting paper, and serum samples, which typically yielded gag cac gta atc atc cag gc; DRAR: ggt gtg gtt gga gcg cgc ttt lower quantities of DNA. a (Wagner JL, personal communication) and gave products of PCR products were cleaned using ExoSAP-IT (USB) approximately 261 bp. according to the manufacturer’s instructions and sequenced Polymerase chain reactions (PCR) were performed in on an ABI 3730 sequencer. Sequencing was conducted in 25 ll reactions containing 1 Â Q solution (Qiagen), 1 Â PCR both directions for DLA-DRB1, using primers DRBln1 and buffer containing 15 mM MgCl2 (Qiagen), 1 mM MgCl M13r (cag gaa aca gct atg acc). To reduce costs, (Qiagen), 0.4 mM of each DNTP (Invitrogen, San Diego, unidirectional sequencing was used for DLA-DQA1 and CA), 0.04 uM of each primer, 0.1 lg/ll BSA (Promega), 1 unit DLA-DQB1, using primers DQAln1 and DQB1BT7, of Hot Startaq (Qiagen), and approximately 25 ng of template respectively. Sequence data were analyzed using Match DNA. To detect contamination, each PCR was run with both Tools and Match Tools Navigator (Applied Biosystems), as the DNA extraction negative and a PCR-negative control described in Kennedy, Barnes, Happ, Quinell, Courtenay, containing no template DNA. Reactions were run on et al. (2002). This method relies on an allele library built PTC-200 DNA engine machines (MJ Research Inc.). PCR from homozygotes. We had 6 heterozygous individuals amplifications were conducted with a touchdown protocol: 15 (South Africa 5 3, EU zoos 5 3) that did not match any min at 95 °C; 14 touchdown cycles of 95 °C for 30 s; followed pair of known alleles. Therefore, we cloned these 6 by 1 min annealing, starting at 62 °C (DLA-DRB1), 62 °C individuals using the TOPO TA cloning system (Invitrogen) (DLA-DRA), 52 °C (DLA-DQA1), 68 °C (DLA-DQB1), and and identified a single new allele DRB1*90301. This allele reducing at 0.5 °C per cycle; and 72 °C for 1 min. This was was subsequently found in a further 12 heterozygotes. There followed by 20 cycles of 95 °C for 30 s, 60 °C (DLA-DRB1), were 3 pairs of alleles, which could not be distinguished 55 °C (DLA-DRA), 50 °C (DLA-DQA1), 65 °C (DLA- using the Match tools analytical method because some allele DQB1) for 1 min, and 72 °C for 1 min. The protocol ended pairs gave the same heterozygous sequence (DRB1*90601/ with a final extension of 72 °C for 10 min. The number of 90202 and DRB1*90602/90201; DRB1*90101/90201 amplifications in the second stage of the PCR protocol was and DRB1*90102/90202; DRB1*90101/90601 and

S56 Marsden et al. African Wild Dogs (Lycaon pictus) Lack Variation at the MHC

DRB1*90102/90602). These ambiguous combinations were Sample sizes varied from 14–56 for nonmanaged resolved using a combination of reference strand-mediated populations. Therefore, we used rarefaction to compensate conformation analysis (RSCA) and pedigree information from for sampling disparity between study populations by the zoo populations. RSCA is a genotyping method that standardizing to a population size of 10 using the program separates allelic variants based on conformation-dependent HP-Rare v.4.1 (Kalinowski 2005). We calculated nucleotide mobility through a gel (Kennedy et al. 2005) and was used to diversity in populations as the average number of distinguish between ambiguous DLA-DRB1 heterozygous segregating sites h and pairwise diversity p, in DnaSP 4.20 sequences by running ambiguous samples alongside a set (Rozas and Rozas 1995), using a Jukes–Cantor model of of candidate alleles in homozygous form. For the EU substitutions and standard errors calculated with 5000 zoo samples, individuals with ambiguous allele combinations bootstrap replications. We tested for an excess of could be resolved using pedigree information to examine the heterozygosity relative to Hardy–Weinberg proportions,

alleles of siblings, parents, and offspring. For example, which is indicative of selection on the current generation, Downloaded from https://academic.oup.com/jhered/article/100/suppl_1/S54/892226 by guest on 01 October 2021 individual #P20791 was found to be heterozygous for using the U test in Genepop 4.0 (Raymond and Rousset either 1) DRB1*90601/DRB1*90202 or 2) DRB1*90602/ 1995). Synonymous and nonsynonymous genetic distances DRB1*90201. Five of its siblings were found to have the were calculated separately for putative peptide-binding following 4 alleles DRB1*90101, DRB1*90201, DRB1*90301, region (PBR) sites and non-PBR sites using the Nei– and DRB1*90602, which means that #P20791 must be Gojobori method with a Jukes–Cantor model of substitu- heterozygous for DRB1*90602/DRB1*90201. Pedigree data tions in Mega 4.0 (Tamura et al. 2007). Putative PBR sites were also used to examine segregation of DLA-DRB1 alleles were based on the human HLA-DRB1 (Brown et al. 1993). and lineages within families. Chi-square goodness-of-fit tests Due to the recombining nature of MHC genes, phylogenetic were used to compare observed segregation patterns to trees are not strictly appropriate for analysis of the MHC expected genotype combinations under random segregation at and there is too much variation to allow a network a single locus. Pedigree information for the EU zoo samples approach. However, MHC allele trees are a useful tool for were provided by H. Verberkmoes. Pedigrees were drawn displaying relationships among alleles. Phylogenetic trees using SmartDraw 2009. were constructed using African wild dog sequences Preliminary sequencing of 30 individuals for DLA- alongside 105 alleles from Canis species made available by DQA1, DLA-DQB1, and DLA-DRA revealed just 1, 2, and LJ Kennedy, who collates these data on behalf of the DLA 1 alleles, respectively. Consequently, we used RSCA nomenclature committee (Kennedy et al. 2001). We also (together with sequenced samples as controls) to screen tested alternative phylogenetic models but these did not for further variation at these loci. For DLA-DQA1, DLA- affect the resolved relationships within the tree. Therefore, DQB1, and DLA-DRA, RSCA analysis was conducted on we have only shown neighbor joining trees with Kimura’s 2- samples from EU zoos (n 5 92), Laikipia (n 5 56), New parameter model as implemented in Mega 4.0 to demon- Serengeti (n 5 9), Okavango (n 5 53), Hwange (n 5 13), strate relationships. Following Seddon and Ellegren (2002), South Africa (n 5 6), and the 6 carcass samples. DNA from a human HLA sequence with ;80% similarity to dog DLA- 5 New Serengeti, 2 Hwange, and 43 South Africa samples DRB1 alleles was used as an out-group (HLA-DRB1*03011, were not available in time for RSCA analysis; however, accession number AF352294). Bootstrapping was con- sequence-based typing detected no new DLA-DRB1 alleles ducted with 5000 replicates. in these samples. DLA-DQB1 typing was conducted on an additional 25 Okavango samples that were not successfully typed at the DLA-DRB1 due to low-quality DNA and Results RSCA failures. Because RSCA was used to screen for new variants and the EU zoos included large family groups, we African wild dogs were found to have 17 DLA-DRB1 alleles did not type offspring if we typed both parents and screened (n 5 368), 1 DLA-DQA1 allele (n 5 234), 2 DLA-DQB1 a maximum of 3 animals per litter. In total, we typed 92 alleles (n 5 234), and 1 DLA-DRA allele (n 5 234). Fewer individuals representative of 36 sibling groups from the 200 samples were analyzed at DLA-DQB1, DLA-DQA1, and captive samples. DLA-DRA because of the lack of variation found. The new alleles identified in this study were submitted to However, we did type representative individuals for all the DLA nomenclature committee. Those that met the DLA-DRB1 alleles. This is important because in domestic appropriate criteria were recognized and assigned official dogs and other Canis species, there is strong linkage between names by the committee. Prior to this study, preliminary MHC class II loci. Therefore, new DLA-DQB1, DLA- data (Kennedy LJ, Bacon H, Radford A, unpublished data) DQA1, and DLA-DRA variants would be most likely found based on 4 African wild dog museum samples provided by in individuals with new DLA-DRB1 alleles. There was no the National Museums of Scotland (A. Kitchener) had evidence of pseudogenes (stop codons or frameshift identified 3 DLA-DRB1 alleles (DLA-DRB1*90101, 90102, mutations), indicating that functional genes were being and 90201), 1 DLA-DQA1 allele (DLA-DQA1*01901), and amplified. All DLA-DRB1, DQA1, and DQB1 alleles 2 DLA-DQB1 alleles (DLA-DQB1*90101 and 90201). One detected in African wild dogs were new and have not been allele did not fulfill the naming criteria and is referred to by identified in any other canid species to date; accession its local name ‘‘fmut.’’ numbers DQA1 (AM182470), DQB1 (FJ648575,

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FJ648576), and DRB1 (FJ648559-FJ648574). As with all were shared among lineages, and 2 of which were specific to other surveyed wolf-like canids (Kennedy LJ, unpublished Lineage B. Overall, the average numbers of nucleotide data), African wild dogs were monomorphic at DLA-DRA differences within alleles of the same lineage were 6.0 for allele DRA*00101, which was originally identified in (lineage A) and 6.8 (lineage B), compared with an average of domestic dogs (Wagner et al. 1995). 22.9 nucleotide differences between alleles from different African wild dog DLA-DRB1 alleles varied at 31 lineages. Because RSCA analysis, DNA cloning, and se- polymorphic sites across 95 codons, with 14 substitutions quencing did not detect more than 2 alleles in any individual at the first codon position, 10 at the second codon position, and less than half of the individuals sampled (46%) had and 7 at the third codon position. These changes alleles from both lineages, we are confident that these 2 corresponded to 17 amino acid differences among alleles allelic lineages are derived from a single locus. Furthermore, (Figure 2). This included unique amino acid residues at 2 pedigree data clearly show cosegregation of the 2 allelic

codons not seen in other canids and 1 new polymorphic site lineages within families (Figure 3). Downloaded from https://academic.oup.com/jhered/article/100/suppl_1/S54/892226 by guest on 01 October 2021 at a putative PBR residue which is monomorphic in all Phylogenetic analyzes on African wild dog DLA-DRB1 other canids. All DLA-DRB1*907011 alleles differed from alleles were conducted alongside alleles from Canis species. each other at the amino acid level, except for DLA- The highly polymorphic nature of these genes resulted in DRB1*907011 and DRB1*907012, which indicates a high insufficient resolution to determine specific relationships level of nonsynonymous substitutions. The majority of between groups of alleles; however, they were used to nucleotide (22/31) and amino acid (14/17) differences indicate the positioning of African wild dog alleles relative between DLA-DRB1 alleles were found to occur within the to the alleles of other canids (Supplementary Figure 1). 3 hypervariable regions (HVR) (Figure 2) (Kennedy et al. African wild dog DLA-DRB1 alleles were clearly shown to 2007). Nine of the 22 functionally important putative PBR cluster into 2 distinct and separate monophyletic branches sites of DLA-DRB1 based on human HLA-DRB1 were rather than being scattered across branches, as found with variable in African wild dogs. The ratio of nonsynonymous Gray wolf and Ethiopian wolf alleles. Furthermore, African to synonymous substitutions at the putative PBR sites was wild dog alleles were clearly positioned within, rather than greater than 1.0 and larger than in non-PBR, but it was not peripheral to, the canid DLA-DRB1 allele tree, indicating found to be significant (PBR: dN 5 0.2, dS 5 0.117, dN/dS 5 similarity to other canid alleles. In particular, comparison of 1.709, P 5 0.073; non-PBR: dN 5 0.031, dS 5 0.022, dN/dS amino acid sequences highlight that certain African wild dog 5 1.409, P 5 0.307). DLA-DRB1 lineage B alleles and certain Ethiopian wolf DLA-DRB1 alleles consisted of two highly divergent alleles differ by just one amino acid at HVR1 and are allelic lineages, which we have called A (7 alleles) and B (10 identical at HVR2 (data not shown). alleles). Alleles within lineages were relatively similar, DLA-DRB1 alleles from both A and B lineages were whereas alleles from different lineages were highly divergent found in all populations with more than 3 samples. Four of (Figure 2). Lineage A alleles have identical HVR1 and HVR2 7 lineage A and 5 of 9 lineage B DLA-DRB1 alleles were sequences. Lineage B alleles have the same HVR1 sequence detected in two or more sampling areas, which were often (which is different from that in lineage A) and 1 of 2 very separated by large geographic distances (see Table 1). For similar HVR2 sequences which differed by just one amino example, DLA-DRB1*90202 was found in countries acid. At HVR3, there were 5 different sequences, 3 of which across Eastern (Laikipia, Kenya; New Serengeti, Tanzania)

Figure 2. African wild dog DLA-DRB1 alleles aligned to domestic dog DLA-DRB1*00101 sequence. Matching amino acids are indicated with a dash, varying amino acids are indicated by single letter amino acid codes. Alleles are grouped into 2 phylogenetically divergent allelic lineages, A (above the line) and B (below the line). The 3 canid HVR, which code for the PBRs, are shown in gray.

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Figure 3. Segregation analysis of DLA-DRB1 alleles according to sequence-based typing data of captive African wild dog samples from European zoos. African wild dog DLA-DRB1 alleles comprise 2 highly divergent allelic lineages, A and B. Lineage B alleles are underlined to demonstrate segregation of these allelic lineages. Family 1 represents an example where the mother has 2 lineage B alleles and the father 2 lineage A alleles. Each offspring is seen to inherit one lineage A allele from their mother and one lineage B allele from their father. The 2 expected genotype classes (90401/90101 and 90401/90102) occur at a frequency of 8 and 7, respectively, which is not signiﬁcantly different than expected for a single locus (P . 0.95). Family 2 is an example of segregation where both parents have one lineage A and one lineage B allele. Although the expected frequency of each genotype class (1.75 for each of the four possible combinations of parental alleles) is too low to reliably apply a chi-square goodness-of-ﬁt test, each expected genotype occurs at least once. Three of 7 offspring are shown to inherit a lineage A allele from both parents, 2 offspring inherit a lineage B allele from both parents, and 2 offspring inherit a lineage A allele from one parent and a lineage B allele from the other parent. and Southern Africa (Hwange, Zimbabwe; Okavango, were generally high, there was not an excess of heterozy- Botswana; NW Namibia; and South Africa). gosity relative to Hardy–Weinberg expectations in any African wild dog populations were found to differ from nonmanaged population. The South African sample set each other in DLA-DRB1 allelic composition, allelic consisted almost entirely of heterozygotes (46/49). How- diversity, and heterozygosity. For nonmanaged populations, ever, this is a managed group of animals derived from the number of alleles per population varied between 3 and 9 multiple sources rather than a natural population. Together, and average observed heterozygosity varied from 53.6% to the EU zoos were found to have 12 of the 14 DLA-DRB1 92.9% (see Table 1). Despite being the most thoroughly alleles detected in Southern African populations and levels sampled population, Laikipia had the smallest number of of heterozygosity (82%) comparable to nonmanaged wild alleles (3 alleles, n 5 56) and, correspondingly, also had the populations (53.6–92.9%). One allele (DRB1*90101) was lowest observed heterozygosity (53.6%). However, nucleo- found at high frequency among the zoo samples (33.5%). tide diversity was actually highest in this population (p 5 The 2 DLA-DQB1 alleles differed at 8 sites within 0.0758, h 5 0.0716), suggesting that the 3 alleles are highly HVR2, resulting in 5 amino acid differences. This included 1 divergent; there were 29 variable sites among these 3 alleles. new polymorphic amino acid site that is monomorphic in In contrast, nucleotide diversity was lower in the 3 other other canids tested to date and 4 unique amino acid residues. populations, which had between 7 and 9 alleles (p 5 0.0509, DLA-DQB1*90101 was considerably more frequent 0.0595, 0.0613; h 5 0.0435, 0.0435, 0.0484). Rarefaction was (87.5%) than DLA-DQB1*90201 (12.5%) resulting in used to standardize population sample sizes to n 5 10 and a predominance of DLA-DQB1*90101 homozygotes showed Hwange to be most diverse in terms of numbers of (81%). In fact, we found just 6 DLA-DQB1*90201 alleles expected with that sample size (7.8 alleles), although homozygotes in 234 samples. Both DLA-DQB1 alleles New Serengeti and Okavango had only slightly lower levels were found across Eastern and Southern Africa (Table 2); of diversity (6.1 and 5.8 alleles, respectively). All 3 of these however, DLA-DQB1*90201 was noticeably absent from populations had at least 50% more diversity than Laikipia Hwange, Zimbabwe. Previous research has shown strong (2.9 alleles). Although levels of observed heterozygosity linkage disequilibrium between the canid DLA-DRB1,

S59 S60 Table 1. Frequency of DLA-DRB1 alleles and lineages across sampling localities, subdivided into free-ranging nonmanaged populations, and samples from a managed population, and Heredity of Journal carcass and captive samples

Nonmanaged populations Managed Carcass samples Captive Downloaded fromhttps://academic.oup.com/jhered/article/100/suppl_1/S54/892226bygueston01October2021 S. Africa, Kajiado, Ghanzi, Sofala, Mangetti, EU Locus Laikipia, KNY N. Serengeti, Hwange, Okavango, SAF KNY BOT MOZ NAM zoos DRB1* (n 5 56) TNZ (n 5 14) ZIM (n 5 15) BOT (n 5 28) (n 5 49) (n 5 1) (n 5 1) (n 5 3) (n 5 1) (n 5 200) 1) 2009:100(Supplement Lineage A 90101 6.7 8.9 5.1 33.5 90102 3.6 32.1 12.3 90201 3.3 32.1 24.5 100.0 9.5 90202 57.1 14.3 10.0 7.1 25.5 100.0 5.8 90203 42.9 50.0 100.0 90204 14.3 90301 4.1 3.5 Lineage B 90401 10.0 13.0 90402 33.3 1.8 7.14 1.5 90501 15.2 3.3 90602 3.6 6.5 90601 27.7 12.5 4.1 50.0 907011 16.7 21.4 4.0 907012 13.3 0.3 90702 3.3 9.8 90801 17.9 5.4 8.2 0.5 fmut 3.6 Total number of alleles 3 7 9 7 8 2 1 1 1 12 % Lineage A 57.1 75.0 20.0 80.4 59.2 50.0 100.0 100.0 100.0 57.1 % Lineage B 42.9 25.0 80.0 19.6 40.8 50.0 0.0 0.0 0.0 42.9 Ho % 53.6 92.9 73.3 82.9 93.9* NA NA NA NA 82.0 He % 57.9 76.7 84.4 77.5 82.0 NA NA NA NA 82.9 Standardized number 2.9 6.1 7.8 5.8 of alleles, n 5 10 p 0.0758 (0.024) 0.0613 (0.009) 0.0509 (0.0001) 0.0595 (0.011) p (syn) 0.0482 0.0450 0.0294 0.0391 p (nonsyn) 0.0810 0.0632 0.0552 0.0628 h 0.0716 (0.0019) 0.0435 (0.0001) 0.0484 (0.0001) 0.0435 (0.0004)

n 5 number of individuals typed. Ho 5 observed heterozygosity (%). Asterisk denotes signiﬁcant excess to Hardy–Weinberg proportions (HWE), P , 0.001. He 5 expected heterozygosity under HWE (%). Nucleotide diversity was calculated as pairwise diversity p and segregating sites h in DnaSP. p (syn) 5 nucleotide diversity at synonymous sites. p (nonsyn) 5 nucleotide diversity at nonsynonymous sites. Population diversity metrics were not calculated where less than four individuals were sampled in a population, or for the managed South African sample set and captive samples, which do not represent true populations. NA, not applicable. Country codes—Kenya, KNY; Tanzania, TNZ; Zimbabwe, ZIM; Botswana, BOT; Mozambique, MOZ; South Africa, SAF; and Namibia, NAM. Marsden et al. African Wild Dogs (Lycaon pictus) Lack Variation at the MHC

DQA1, and DQB1 loci (Kennedy et al. 2007). There was

234) insufﬁcient variation at the DLA-DQA1 and DQB1 loci for 5 haplotype designation in African wild dogs. However, we n did detect an association between DLA-DQB1*90201 and DLA-DRB1 lineage A alleles. Six of 7 individuals 92) ( homozygous for DLA-DQB1*90201 had only lineage A 5 2.2 12.5

n DLA-DRB1 alleles (DRB1*90101, *90201, *90202, or tion, and carcass and EU zoos ( *90204). Furthermore, all DLA-DQB1*90201 heterozygotes

1) had at least one DLA-DRB1 lineage A allele, most

5 commonly DRB1*90101, DRB1*90201, or DRB1* 90202. n a, NAM. Downloaded from https://academic.oup.com/jhered/article/100/suppl_1/S54/892226 by guest on 01 October 2021 Mangetti, NAM ( Discussion Research on MHC class II loci in Canis species has shown

3) moderate-to-high levels of diversity at the DLA-DRB1,

5 DLA-DQA1, and DLA-DQB1 class II loci with frequent n Sofala, MOZ ( transspeciﬁc polymorphism (allele sharing) among Canis species. In this study, we conducted a geographically widespread survey of MHC class II variation in the highly

1) endangered African wild dog to extend knowledge of the

5 canid MHC to more distantly related canid species. African n Ghanzi, BOT ( wild dogs belong to a monotypic genus that is phylogenetically and morphologically divergent from Canis species (Wayne et al. 1997; Bardeleben et al. 2005). In total, we found 17 alleles at the DLA-DRB1 locus, 1 allele at the 1) DLA-DQA1 locus and 2 alleles at the DLA-DQB1 locus, all 5

n of which are currently unique to African wild dogs. At Kajiado, KNY ( DLA-DRA, African wild dogs were monomorphic for the

6) same allele found in other canids.

5 Balancing selection is a key mechanism in the mainte- n nance of variation at MHC loci (reviewed in Garrigan and

S. Africa, SAF ( Hedrick 2003) and is indicated by an increased ratio of nonsynonymous (dN) to synonymous (dS) substitutions at the amino acid residues of the functionally important PBR 53)

5 (Seddon and Ellegren 2002). Although dN/dS was elevated at

n putative PBR sites of DLA-DRB1 alleles in African wild dogs, there was not a signiﬁcant excess of nonsynonymous

Okavango, BOT ( substitutions (P 5 0.073). This is not typical of canid DLA- DRB1 alleles; there was a signiﬁcant excess of dN/dS at PBR sites in Gray wolves, Coyotes, and domestic dogs (Seddon and Ellegren 2002). Whereas d /d ratios provide in- 13) N S

5 formation on historical selection, excess heterozygosity can

Hwange, ZIM (n provide an indication of current selection at a locus (Garrigan and Hedrick 2003; Aguilar et al. 2004). Despite

9) the high heterozygous frequencies found in nonmanaged 5 free-ranging populations, the observed heterozygosity did n not exceed Hardy–Weinberg expectations. This is not atypical for MHC studies (Garrigan and Hedrick 2003). N. Serengeti, TNZ ( The distribution of alleles from polymorphic loci under balancing selection are predicted to show very different distributions from that of neutral loci. In particular, they are

56) expected to show lower levels of differentiation in allele

5 composition between populations (Schierup et al. 2000). Frequency of DLA-DQB1 alleles across sampling localities, subdivided into free-ranging nonmanaged populations, and samples from a managed popula n Nonmanaged populationsLaikipia, KNY ( Neutral Managed Carcassgenetic samples markers show strong structuringand Captive Total differentiation between African wild dogs populations, in number of individuals typed. Country codes—Kenya, KNY; Tanzania, TNZ; Zimbabwe, ZIM; Botswana, BOT; Mozambique, MOZ; South Africa, SAF; and Namibi particular between Eastern and Southern Africa (Girman 5 9010190201 89.3 10.7 72.2 27.8 100.0 70.8 29.2 100.0 100.0 100.0 100.0 100.0 97.8 87.5 captive samples Locus DQB1* Table 2. n et al. 2001). At the MHC, we found 17 DLA-DRB1 alleles,

S61 Journal of Heredity 2009:100(Supplement 1) which clustered into 2 highly distinct lineages. These 2 issue. Nonetheless, it is valuable to evaluate the impact of lineages showed no evidence of geographic structuring; all management actions, such as translocations and captive areas where more than 3 animals were sampled had alleles breeding, on adaptive genes. In 2006, 16 African wild from both lineages. Similarly, individual DLA-DRB1 alleles dogs were translocated from South Africa to Hwange, were not geographically restricted, with many alleles Zimbabwe. Sampling of 6 of these South African trans- detected in populations spanning Eastern and Southern located animals detected 1 allele (DLA-DRB1*90301) not Africa. The discordance between patterns of MHC and present in the 15 resident Hwange samples. This may neutral variation could indicate that selective forces are indicate that the translocation has introduced new MHC shaping patterns of MHC diversity across African wild dog diversity into the Hwange population. Our results show that populations; for example, selection for alleles which confer 12 of the 14 DLA-DRB1 alleles found in Southern African resistance to diseases common to most populations. populations and both DLA-DQB1 alleles are represented in

Two DLA-DQB1 alleles were detected in African wild the European zoo African wild dog population. Nonethe- Downloaded from https://academic.oup.com/jhered/article/100/suppl_1/S54/892226 by guest on 01 October 2021 dogs. However, allele DLA-DQB1*90201 was considerably less, allele DLA-DRB1*90101 clearly dominates this rarer (12.5%). This rare allele was found across Eastern and population (33.8%). High frequency of this allele does not Southern Africa but was absent from Hwange. This may be appear typical to Southern Africa where the EU zoo the result of the low frequency of DLA-DRB1 lineage A founders originated; it has less than 10% representation in alleles in Hwange (20%), which appear to be associated with Hwange, Okavango, and South Africa. Mapping DLA- DLA-DQB1*90201. The stark differences in frequency of DRB1 alleles onto the EU zoo pedigree (data not shown) the 2 DLA-DQB1 alleles may be indicative of selection on shows that overrepresentation of this allele is the result of adaptive differences between these alleles or haplotypes. an extreme bias in founder contributions and is a major High MHC allelic diversity in a population and high cause of homozygosity in the EU zoos (21/35 homozygotes heterozygosity in individuals is thought to be important were homozygous for DRB1*90101). Management of because it theoretically expands the range of pathogens to this population is now focusing on equalizing founder which a population or individual can respond (Doherty and representation. Zinkernagel 1975; Sommer et al. 2002). We found that the The patterns of MHC variation detected in African wild number of DLA-DRB1 alleles and levels of heterozygosity dogs are best interpreted through comparison with other varied between populations (Table 1), even after population canids. Extensive research on the MHC in Canis species sample sizes were standardized using rarefaction. This may shows frequent transspecific polymorphism at DLA-DRB1, reflect differences in demographic history and connectivity. DQA1, and DQB1 loci (Kennedy et al. 2001; Seddon and The highest allelic diversity in nonmanaged populations was Ellegren 2002; Kennedy et al. 2007). By contrast, all alleles found in Hwange (9 alleles, n 5 15), which is a long- characterized at these 3 loci in African wild dogs were standing stable population located within an admixture zone unique to this species and not yet found in any species of (Girman et al. 2001). In contrast, the lowest number of Canis. Furthermore, phylogenetic analyses of African wild alleles was found in Laikipia (3 alleles, n 5 56), a recently dog DLA-DRB1 alleles showed clustering into 2 distinct recolonized population, which is also relatively isolated branches (species-specific allelic clustering) rather than (Woodroffe et al. 2007). Clearly, however, recolonization a scattered distribution throughout the DLA-DRB1 tree does not always result in low numbers of alleles because the indicative of transspecific polymorphism (as seen in Gray recently recolonized New Serengeti population was consid- wolves and Ethiopian wolves). Such a distribution may erably more diverse than Laikipia. However, the New suggest that the canid DLA-DRB1, DLA-DQA1, and DLA- Serengeti is linked to a number of other African wild dog DQB1 allele lineages diverged prior to speciation within the populations and therefore may have been recolonized by genus Canis 1–2 Ma (Seddon and Ellegren 2002) but after a mixture of founders from multiple source populations. the divergence of the Lycaon and Canis genera approximately Despite lower allele numbers, nucleotide diversity among 4–5 Ma (Wayne et al. 1997). However, given that allele alleles was higher in the 2 recently recolonized populations sharing is most common among species of Canis at DLA- (Laikipia and New Serengeti) than in two long-standing DQA1 and DLA-DQB1 loci (Seddon and Ellegren 2002; populations (Hwange and Okavango) (Table 1). The lower Kennedy et al. 2007), whereas there are 1 and 2 alleles, nucleotide diversity measures in Hwange and Okavango respectively, in African wild dogs, it is also possible that likely reflect the presence of closely related or similar alleles shared alleles have been lost. and may indicate that in these populations, new diversity has Allelic diversity at DLA-DQA1 and DLA-DQB1 was been accumulating, whereas in the recently recolonized much lower in African wild dogs than expected based on the populations, there has been insufficient time for the pattern found in other canids (Table 3, Hedrick et al. 2000; evolution of new variants. More research is required to Seddon and Ellegren 2002; Kennedy, Barnes, Happ, explore whether differences in MHC diversity between Quinnell, Courtenay, et al. 2002; Kennedy 2007; Kennedy populations reflect differences in disease characteristics of et al. 2007). This cannot be explained by the endangered populations or neutral processes such as size of historical status of African wild dogs or differences in sampling bottlenecks. because they had lower levels of DLA-DQA1 and DLA- The use of fitness-related genes, such as the MHC, in DQB1 variation than Ethiopian and Mexican wolves; 2 endangered species management remains a contentious other endangered canids sampled from single populations

S62 Marsden et al. African Wild Dogs (Lycaon pictus) Lack Variation at the MHC

Table 3. Comparison of DLA alleles found in different canid populations

Study species/population DRB1 DQA1 DQB1 Reference Species sampled in multiple populations African wild dog: n 5 368 17 1 2 Gray wolfa: Canada, Alaska: n 5 194 17 12 15 Kennedy et al. (2007) Gray wolf: Northern Europe: n 5 163 17 9 10 Seddon and Ellegren (2002) Gray wolf: Total n 5 407 26 18 21 Kennedy et al. (2001, 2007); Seddon and Ellegren (2002) Species sampled from single populations African wild dogb: Single population, n 5 14–56 3–10 1 2 Mexican wolfc: Single population, n , 7 4 5 3 Hedrick et al. (2000); Kennedy et al. (2001, 2007) Ethiopian wolfd: Bale Mountains population, n 5 99 4 2 5 Kennedy LJ, unpublished data Downloaded from https://academic.oup.com/jhered/article/100/suppl_1/S54/892226 by guest on 01 October 2021 a Gray wolves are not an endangered species (Mech & Boitani 2008). b Numbers of alleles detected in individual African wild dog populations where 14–56 animals were sampled per population. c Mexican wolves, Canis lupus baileyi, are a critically endangered subspecies of the Gray wolf Canis lupus. They are thought to have gone extinct in the wild in the c.1970. All individuals extant today have been bred in captivity and are derived from 7 founders (Hedrick et al. 2000; Kennedy et al. 2001, 2007). d Ethiopian wolves are highly endangered, with just 500 individuals. MHC surveys were conducted on 99 samples from the largest (n 5 250) of the 7 extant Ethiopian wolf populations (Kennedy LJ, unpublished data).

(Table 3). It is particularly striking that 5 DLA-DQA1 alleles DRB1 locus and low numbers of alleles at the DLA-DQA1 were found in fewer than 7 Mexican wolves sampled from and DQB1 loci, for a canid, even for an endangered one. a single population, whereas in this study, we found just African wild dogs may have lost allelic diversity across all a single allele in 234 African wild dogs sampled across MHC class II genes due to historical bottlenecks, with strong Eastern and Southern Africa. The lack of variation at these disease pressures subsequently maintaining or generating loci does not appear to be the result of nonmatching MHC variation at the least conserved region, in this case the primers as all samples ampliﬁed successfully; if a mutation DLA-DRB1 locus. The presence of just 2 highly divergent had occurred in the primer site, homozygous individuals monophyletic allelic lineages for both DLA-DRB1 and DLA- for these alleles should fail to amplify. African wild DQB1 is consistent with the hypothesis that this species dogs showed the most variation at the DLA-DRB1 loci, suffered severe bottlenecks, resulting in the loss of alleles and where they had the same number of DLA-DRB1 alleles subsequent evolution of new diversity (Van Den Bussche et al. to Gray wolves sampled across a similar geographic range 1999). However, both DLA-DQB1 alleles and both DLA- in both European and North American regions (Table 3) DRB1 lineages were represented across African wild dog and slightly higher numbers of DLA-DRB1 alleles in populations. A range-wide bottleneck would be unlikely to single populations than other endangered canids. However, produce such a consistent pattern of diversity loss across because African wild dog DLA-DRB1 alleles are derived populations because this would result in the random loss of from just 2 allelic lineages, amino acid diversity among variation. It is more likely that African wild dogs suffered local alleles was considerably lower than for other canids: 17 population extinctions across most of the African wild dog variable amino acid sites across 17 DLA-DRB1 alleles in range with remnant populations retaining both allelic lineages African wild dogs compared with 26 variable amino acid and subsequently expanding to recolonize their former range. sites across 17 alleles in the North American Gray wolf It is clear from our study that African wild dogs are (Kennedy et al. 2007) and 22 variable amino acids sites atypical in their patterns of MHC diversity among the canids, amongst just 4 alleles in a single Ethiopian wolf population which have been studied to date. However, all canids (Kennedy LJ, unpublished data). Furthermore, there was previously studied at the MHC (domestic dogs, Gray wolves, less variation at the putative PBR site residues, which are Coyotes, Ethiopian wolves, Red wolves, and Mexican thought to be primarily responsible for functional differ- wolves) have been closely related species of Canis, which ences between alleles (Sommer 2005), in African wild dogs have a long history of hybridization (Lehman et al. 1991; compared with Ethiopian wolves (Kennedy LJ, unpublished Gottelli et al. 1994; Garcia-Moreno et al. 1996; Roy et al. data) and North American Gray wolves (Kennedy et al. 1996; Vila et al. 1997; Verginelli et al. 2005). Consequently, we 2007): total number of variable PBR sites, 9, 11, and 15, cannot distinguish whether African wild dogs show different respectively; average number of residues/PBR site, 1.5, 1.7, patterns of MHC polymorphism to Canis species because of and 2.2, respectively. Consequently, one might speculate that factors related to African wild dog demographic history, although African wild dogs have a large number of DLA- rather than their distant phylogenetic relationship to the Canis DRB1 alleles, they may have little functional diversity. Overall, genus, or the fact that they lack extensive hybridization in our data suggest that African wild dogs are genetically their recent evolutionary history. Future work is planned on depauperate at the MHC relative to other canids. They have other nonhybridizing species of the wolf-like clade to uncharacteristically low amino acid diversity at the DLA- investigate these alternative hypotheses.

S63 Journal of Heredity 2009:100(Supplement 1)

Adaptive genetic variation is of primary interest in Brown JH, Jardetzky TS, Gorga JC, Stern LJ, Urban RG, Strominger JL, conservation genetics; therefore, MHC data have particular Wiley DC. 1993. Three-dimensional structure of the human class II application to endangered species programs (Aguilar et al. histocompatibility antigen HLA-DR1. Nature. 364:33–39. 2004). In this study, we have shown that the highly Daszak P, Cunningham AA, Hyatt AD. 2000. Emerging infectious diseases endangered African wild dog has a reduced level of MHC of wildlife; threats to biodiversity and human health. Science. 287:443–449. variation compared with other canids, perhaps as a result of Doherty PC, Zinkernagel RM. 1975. A biological role for the major historical bottlenecks. Among African wild dog populations, histocompatibility antigens. Lancet. 305:1406–1409. levels of MHC diversity were found to vary, but more Eggert F, Muller-rucholtz W, Fertsi R. 1998. Olfactory cues associated with research is required to investigate the signiﬁcance of this in the major histocompatibility complex. Genetica. 104:191–197. relation to differences in disease incidence and exposure. Garcia-Moreno J, Matocq MD, Roy MS, Geffen E, Wayne RK. 1996. Our data have shown that the distribution of MHC variation Relationships and genetic purity of the endangered Mexican wolf based on analysis of microsatellite loci. Conserv Biol. 10:376–389.

does not match the pattern of neutral genetic variation Downloaded from https://academic.oup.com/jhered/article/100/suppl_1/S54/892226 by guest on 01 October 2021 highlighted in previous studies (Girman et al. 2001), Garrigan D, Hedrick PW. 2003. Perspective: detecting adaptive molecular indicating that conservation plans based on neutral genetic polymorphism: lessons from the MHC. Evolution. 57:1707–1722. data alone may not adequately conserve adaptive genetic Girman DJ, Kat PW, Mills MGL, Ginsberg JR, Borner M, Wilson V, variation. It is promising, however, that such a high pro- Fanshawe JH, Fitzgibbon C, Lau LM, Wayne RK. 1993. Molecular genetic portion of MHC diversity from free-ranging populations has and morphological analyses of the African wild dog (Lycaon pictus). J Hered. 84:450–459. been successfully conserved within the European captive breeding programme. This high diversity is likely the result Girman DJ, Mills MGL, Geffen E, Wayne RK. 1997. A molecular genetic analysis of social structure, dispersal, and interpack relationships of the of the diverse origin of individuals that founded the African wild dog (Lycaon pictus). Behav Ecol Sociobiol. 40:187–198. European captive population. Girman DJ, Vila C, Geffen E, Creel S, Mills MGL, McNutt JW, Ginsberg J, Kat PW, Mamiya KH, Wayne RK. 2001. Patterns of population Supplementary Material subdivision, gene ﬂow and genetic variability in the African wild dog (Lycaon pictus). Mol Ecol. 10:1703–1723. Supplementary material can be found at http://www.jhered. Gottelli D, Sillero-Zubiri C, Applebaum GD, Roy MS, Girman DJ, Garcia- oxfordjournals.org/. Moreno J, Ostrander EA, Wayne RK. 1994. Molecular genetics of the most endangered canid: the Ethiopian wolf Canis simesis. Mol Ecol. 3:301–312. Hedrick PW. 2003. The major histocompatibility complex (MHC) in declining Funding populations: an example of adaptive variation. In: Holt WV, Pickard AR, Rodger JC, Wildt DE, editors. Reproduction science and integrated Joint grant between Natural Environment Research Council conservation. Cambridge (UK): Cambridge University Press. p. 97–113. and the Royal Zoological Society of the Scotland-Edinburgh Hedrick PW, Kim TJ, Parker KM. 2001. Parasite resistance and genetic Zoo (NER/S/A/2006/14139); The Royal Zoological variation in the endangered Gila topminnow. Anim Conserv. 4:103–109. Society of Scotland. Hedrick PW, Lee RN, Buchanan C. 2003. Canine parvovirus enteritis, canine distemper, and major histocompatibility complex genetic variation in Acknowledgments Mexican wolves. J Wildl Dis. 39:909–913. Hedrick PW, Lee RN, Parker KM. 2000. Major histocompatibility complex We would like to thank the following African wild dog ﬁeld projects for (MHC) variation in the endangered Mexican wolf and related canids. contributing samples: African Wild Dog Conservation Project Mozambique, Heredity. 85:617–624. Botswana Wild Dog Project, Painted Dog Research Project Zimbabwe, Kalinowski ST. 2005. HP-Rare 1.0: a computer program for performing Samburu-Laikipia Wild Dog Project Kenya, Serengeti Wild Dog Conser- rarefaction on measures of allelic richness. Mol Ecol Notes. 5:187–189. vation Project Tanzania. In addition, we thank Matt Swarner, Jean-Marc Andre, Tshepo Matjila, Amanda Bastos, Hanru Strydom, Laurence Frank, Kennedy LJ. 2007. 14th International HLA and Immunogenetics Flip Stander, and Andrew Kitchener for assistance with sample provision. Workshop: report on joint study on canine DLA diversity. Tissue Antigens. We are very grateful to all of the European zoos that contributed samples to 69:269–271. this research and would like to thank Hanny and Wim Verberkmoes for Kennedy LJ, Angles JM, Barnes A, Carmichael LE, Radford AD, Ollier coordinating captive sampling and providing assistance with analysis of stud WER, Happ GM. 2007. DLA-DRB1, DQA1, and DQB1 alleles and book data. haplotypes in North American Gray Wolves. J Hered. 98:491–499. Kennedy LJ, Angles JM, Barnes A, Carter SD, Francino O, Gerlach JA, Happ GM, Ollier WER, Thomson W, Wagner JL. 2001. Nomenclature for References factors of the dog major histocompatibility system (DLA), 2000: second Aguilar A, Roemer G, Debenham S, Binns M, Garcelon D, Wayne RK. 2004. report of the ISAG DLA Nomenclature Committee. Anim Genet. High MHC diversity maintained by balancing selection in an otherwise 32:193–199. genetically monomorphic mammal. Proc Natl Acad Sci USA. 101:3490–3494. Kennedy LJ, Barnes A, Happ GM, Quinnell RJ, Bennett D, Angles JM, Day Alexander KA, McNutt JW, Briggs MB, Standers PE, Funston P, Hemson MJ, Carmichael N, Innes JF, Isherwood D, et al. 2002. Extensive G, Keet D, M van Vuuren. 2008. Forthcoming. 2008. Multi-host pathogens interbreed, but minimal intrabreed, variation of DLA class II alleles and and carnivore management in Southern Africa. Comp Immunol Microbiol haplotypes in dogs. Tissue Antigens. 59:194–204. Infect Dis. doi:10.1016/j.cimid.2008.10.005. Kennedy LJ, Barnes A, Happ GM, Quinnell RJ, Courtenay O, Carter SD, Bardeleben C, Moore RL, Wayne RK. 2005. A molecular phylogeny of the Ollier WER, Thomson W. 2002. Evidence for extensive DLA poly- Canidae based on six nuclear loci. Mol Phylogenet Evol. 37:815–831. morphism in different dog populations. Tissue Antigens. 60:43–52.

S64 Marsden et al. African Wild Dogs (Lycaon pictus) Lack Variation at the MHC

Kennedy LJ, Quarmby S, Fretwell N, Martin AJ, Jones PG, Jones CA, Van Den Bussche RA, Hoofer SR, Lochmiller RL. 1999. Characterization Ollier WER. 2005. High-resolution characterization of the canine DLA- of Mhc-DRB allelic diversity in white-tailed deer (Odocoileus virginianus) DRB1 locus using reference strand-mediated conformational analysis. J provides insight into Mhc-DRB allelic evolution within Cervidae. Hered. 96:836–842. Immunogenetics. 49:429–437. Lehman N, Eisenhawer A, Hansen K, Mech LD, Peterson RO, Gogan PJP, Verginelli F, Capelli C, Coia V, Musiani M, Falchetti M, Ottini L, Palmirotta Wayne RK. 1991. Introgression of coyote mitochondrial-DNA into R, Tagliacozzo A, De Grossi Mazzorin I, Mariani-Costantini R. 2005. sympatric North-American gray wolf populations. Evolution. 45:104–119. Mitochondrial DNA from prehistoric canids highlights relationships Lindblad-Toh K, Wade CM, Mikkelsen TS, Karlsson EK, Jaffe DB, Kamal between dogs and South-East European wolves. Mol Biol Evol. M, Clamp M, Chang JL, Kulbokas EJ, Zody MC, et al. 2005. Genome 22:2541–2551. sequence, comparative analysis and haplotype structure of the domestic Vila C, Savolainen P, Maldonado JE. 1997. Multiple and ancient origins of dog. Nature. 438:803–819. the domestic dog. Science. 276:1687–1689. McNutt JW, Mills MGL, McCreery K, Rasmussen G, Robbins R, Woodroffe Wagner JL, Burnett RC, DeRose SA, Storb R. 1996. Molecular analysis and

R. 2008. Lycaon pictus. In: IUCN 2008. 2008 IUCN Red List of Threatened polymorphism of the DLA-DQA gene. Tissue Antigens. 48:199–204. Downloaded from https://academic.oup.com/jhered/article/100/suppl_1/S54/892226 by guest on 01 October 2021 Species. Available from: http://www.lucnredlist.org. Wagner JL, Burnett RC, Works JD, Storb R. 1996. Molecular analysis of Mech LD, Boitani L. 2008. Canis lupus. In: IUCN 2008. 2008 IUCN Red List DLA-DRBB1 polymorphism. Tissue Antigens. 48:554–561. of Threatened Species. Available from: http://www.iucnredlist.org. Wagner JL, Derose SA, Burnett RC, Storb R. 1995. Nucleotide-sequence Piertney SB, Oliver MK. 2006. The evolutionary ecology of the major and polymorphism analysis of canine DRA cDNA clones. Tissue Antigens. histocompatibility complex. Heredity. 96:7–21. 45:284–287. Raymond M, Rousset F. 1995. GENEPOP (Version 1.2): population Wan Q-H, Zhu L, Wu HUA, Fang S-G. 2006. Major histocompatibility genetics software for exact tests and ecumenicism. J Hered. 86:248–249. complex class II variation in the giant panda (Ailuropoda melanoleuca). Mol Roy MS, Geffen E, Smith D, Wayne RK. 1996. Molecular genetics of pre- Ecol. 15:2441–2450. 1940 red wolves. Conserv Biol. 10:1413–1424. Wayne RK, Geffen E, Girman DJ, Koepﬂi KP, Lau LM, Marshall CR. Rozas J, Rozas R. 1995. DnaSP, DNA sequence polymorphism: an 1997. Molecular systematics of the Canidae. Syst Biol. 46:622–653. interactive program for estimating population genetics parameters from Woodroffe R, Cleaveland S, Courtenay O, Laurenson MK, Artois M. 2004. DNA sequence data. Comput Appl Biosci. 11:621–625. Infections disease in the management and conservation of wild canids. In: Sanchez-Mazas A, Djoulah S, Busson M, de Gouville IL, Poirier JC, Dehay Macdonald DW, Sillero-Zubiri C, editors. Biology and conservation of wild C, Charron D, Excofﬁer L, Schneider S, Langaney A, et al. 2000. A linkage canids. Oxford (UK): Oxford University Press. p. 123–142. disequilibrium map of the MHC region based on the analysis of 14 loci Woodroffe R, Ginsberg J. 1997. Past and future causes of wild dogs’ haplotypes in 50 French families. Eur J Hum Genet. 8:33–41. population decline. In: Woodroffe R, Ginsberg J, Macdonald DW, editors. Schierup MH, Vekemans X, Charlesworth D. 2000. The effect of The African wild dog: status survey and action plan. Gland (Switzerland): subdivision on variation at multi-allelic loci under balancing selection. IUCN. p. 58–73. Genet Res. 76:51–62. Woodroffe R, Ginsberg J, Macdonald DW, editor. 1997. The African wild Seddon JM, Ellegren H. 2002. MHC class II genes in European wolves: dog: status survey and conservation action plan. Gland (Switzerland): World a comparison with dogs. Immunogenetics. 54:490–500. Conservation Union. Sillero-Zubiri C, Hoffman M, Macdonald DW, editor. 2004. Canids: Woodroffe R, Lindsey PA, Romanach SS, Ranah S. 2007. African wild dogs foxes, wolves, jackals and dogs. Status survey and action plan. Gland (Lycaon pictus) can subsist on small prey: implications for conservation. J (Switzerland): IUCN. Mammalogy. 88:181–193. Sommer S. 2005. The importance of immune gene variability (MHC) in Zhang B, Fang SG, Xi YM. 2006. Major histocompatibility complex evolutionary ecology and conservation. Front Zool. 2:16. variation in the endangered crested ibis Nipponia nippon and implications for Sommer S, Schwab D, Ganzhorn JU. 2002. MHC diversity of endemic reintroduction. Biochem Genet. 44:113–123. Malagasy rodents in relation to geographic range and social system. Behav Ecol Sociobiol. 51:214–221. Received November 22, 2008; Revised January 26, 2009; Tamura K, Dudley J, Nei M, Kumar S. 2007. MEGA4: Molecular Accepted April 22, 2009 Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol. 24:1596–1599. Corresponding Editor: Francis Galibert

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