Campana et al. BMC Genomics (2016) 17:1013 DOI 10.1186/s12864-016-3368-9 RESEARCH ARTICLE Open Access Genome sequence, population history, and pelage genetics of the endangered African wild dog (Lycaon pictus) Michael G. Campana1,2*, Lillian D. Parker1,2,3, Melissa T. R. Hawkins1,2,3, Hillary S. Young4, Kristofer M. Helgen2,3, Micaela Szykman Gunther5, Rosie Woodroffe6, Jesús E. Maldonado1,2 and Robert C. Fleischer1 Abstract Background: The African wild dog (Lycaon pictus) is an endangered African canid threatened by severe habitat fragmentation, human-wildlife conflict, and infectious disease. A highly specialized carnivore, it is distinguished by its social structure, dental morphology, absence of dewclaws, and colorful pelage. Results: We sequenced the genomes of two individuals from populations representing two distinct ecological histories (Laikipia County, Kenya and KwaZulu-Natal Province, South Africa). We reconstructed population demographic histories for the two individuals and scanned the genomes for evidence of selection. Conclusions: We show that the African wild dog has undergone at least two effective population size reductions in the last 1,000,000 years. We found evidence of Lycaon individual-specific regions of low diversity, suggestive of inbreeding or population-specific selection. Further research is needed to clarify whether these population reductions and low diversity regions are characteristic of the species as a whole. We documented positive selection on the Lycaon mitochondrial genome. Finally, we identified several candidate genes (ASIP, MITF, MLPH, PMEL) that may play a role in the characteristic Lycaon pelage. Keywords: Lycaon pictus, Genome, Population history, Selection, Pelage Background Primarily a hunter of antelopes, the African wild dog is a The African wild dog (Lycaon pictus) is an endangered highly distinct canine. Wild dogs are differentiated from canid species (International Union for Conservation of other canine species by their anatomical adaptations re- Nature Red List Classification: C2a (i)) [1]. While the lated to hypercarnivory and cursorial hunting, including species formerly ranged over most of sub-Saharan high-crowned, sectorial teeth and the lack of dewclaws Africa, wild dogs suffer from a suite of threats including [4]. They have a highly specialized social structure in severe habitat fragmentation, human persecution, and which both males and females disperse to form new disease epidemics. They are now restricted to less than packs and only a single dominant pair in each pack re- seven percent of their former range [2], with only small, produces [5]. Wild dogs are also noted for their colorful and frequently declining, remnant populations in frag- pelage, from which they derive their species name pictus mented pockets of eastern and southern Africa (Fig. 1). (‘painted’), and the absence of an undercoat. They maintain enormous home ranges (varying between Eastern and southern populations of wild dogs are 200 and 2000 km2) and naturally live at very low genetically and morphologically distinct [6], although densities, even compared to other carnivores [3]. there is a large admixture zone covering Botswana, south-eastern Tanzania, and Zimbabwe [7]. Gene flow occurs across the species’ entire range [8], which is * Correspondence: [email protected] 1Center for Conservation Genomics, Smithsonian Conservation Biology unsurprising given the excellent dispersal capabilities Institute, 3001 Connecticut Avenue NW, Washington, DC 20008, USA documented in Lycaon pictus [1]. Nevertheless, Marsden 2Department of Environmental Science and Policy, George Mason University, et al. [2] documented both genetic structuring between 4400 University Drive, Fairfax, VA 22030, USA Lycaon Full list of author information is available at the end of the article extant populations (likely the result of habitat © The Author(s). 2016 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Campana et al. BMC Genomics (2016) 17:1013 Page 2 of 10 packs [9]. We sequenced a female (sampled July 2003) from this recolonized population. In contrast, wild dogs were reintroduced to Hluhluwe-Imfolozi Park, KwaZulu-Natal Province in 1980 and remained as a single pack for many years [10]. Eventually, Lycaon breeding effectively ceased until new animals were intro- duced in 1997 and afterwards (2001, 2003, etc.) [11]. The KwaZulu-Natal wild dogs are now managed as part of the South African Lycaon “metapopulation” [12, 13]. We sequenced a male (sampled October 2007), born in KwaZulu-Natal to parents that were translocated from Limpopo province in 2003. Therefore, the South African individual’s ancestry represents genes from the northeastern part of the country. The genomes from these two populations represent some of the first published wild canid genomes and are particularly valuable given the susceptibility of wild dogs to diseases and habitat fragmentation [9, 14]. We used our novel genome sequences to reconstruct the last 1,000,000 years of Lycaon genome demography and population history. We identified over a million poly- morphic sequence variants for further population-level study. These variants produced ~35 million predicted genic effects. We identified over 15,000 candidate genes that may have undergone adaptation since the Lycaon/ Canis divergence. We found evidence of positive selec- tion on the Lycaon mitochondrial genome. Finally, we examined genes involved in canid coat phenotype to Fig. 1 South African wild dog pack (top)andmapofextantandformer wild dog range (bottom). The sampling locations of the two individuals identify candidate genes underlying the characteristic are noted on the map. Ranges are modified from Woodroffe Lycaon pelage. and Sillero-Zubiri [1] and Marsden et al. [2]. Extant range data used with permission from the International Union for the Conservation Results and discussion of Nature [Woodroffe R, Sillero-Zubiri C 2012. Lycaon pictus.In:IUCN Genome sequencing of the African wild dog 2016. IUCN Red List of Threatened Species. Version 2016–2. http:// www.iucnredlist.org. Downloaded 12 July 2016]. Photograph by Based on alignment with the domestic dog genome, we Micaela Szykman Gunther have sequenced ~90% (5.8× mean read depth) of the Kenyan individual’s genome and ~93% (5.7×) of the South African Lycaon individual’s genome. We identified 16,967,383 autosomal sequence variants (including fragmentation) and a recent reduction in effective 14,360,480 single nucleotide polymorphisms [SNPs] and population size (Ne) since the 1980s. Furthermore, 2,606,903 indels) separating our Lycaon genomes from African wild dogs exhibit very little major histocom- the domestic dog (Canis familiaris) genome [GenBank: patibility complex variation, which may reflect popu- CanFam3.1] (Additional files 1 and 2) [15]. Of these, lation decline [8]. 1,092,450 (781,329 SNPs and 311,121 indels) were To better understand African wild dog genomic evolu- polymorphic in the African wild dog. The remaining tion, genetic variation and population history, we 15,874,933 autosomal sequence variants (13,579,151 shotgun sequenced whole genomes from two individuals SNPs and 2,295,782 indels) were monomorphic in the from widely separated populations with very different two Lycaon individuals. We identified 717,870 X- modern ecological histories: Laikipia County, Kenya and chromosomal variants (619,606 SNPs and 98,264 indels), Hluhluwe-Imfolozi Park, KwaZulu-Natal Province, of which 32,801 (23,001 SNPs and 9,800 indels) were South Africa. Wild dogs disappeared from Laikipia, polymorphic and 685,069 (596,605 SNPs and 88,464 Kenya in the 1980s and reappeared in 2000 – likely indels) were monomorphic in the African wild dog. recolonizing from a small population in neighboring Additionally, we sequenced the Kenyan and South Samburu district (~50 km distant). The population in African wild dog mitochondrial genomes to depths of Laikipia alone now numbers more than 150 dogs and 15 943× and 1021×, respectively. We annotated the Lycaon Campana et al. BMC Genomics (2016) 17:1013 Page 3 of 10 autosomal and X-chromosomal sequences using the do- Candidate selected processes and genomic regions mestic dog genome annotations [GenBank:CanFam3.1.81] Based on the domestic dog genome annotations, SnpEff and the mitochondrial genome [GenBank:NC_002008.4] 4.1 L predicted that the identified Lycaon autosomal and and MSY sequences [GenBank:KP081776.1] using their X-chromosomal sequence variants would cause 34,001,288 reference sequence annotations [15–17]. and 36,362,161 genic effects in the Kenyan and South African individuals, respectively (Additional files 1 and 2) [22]. The majority of sequence variant effects fell within Lycaon demographic history introns (Kenyan: 62.604%, South African: 63.383%) and We analyzed the two wild dogs’ autosomal population intergenic regions (Kenyan: 23.081%, South African: histories using PSMC (Fig. 2) [18]. Both genomes exhib- 23.162%). To discover candidate genes that have diverged ited a strong