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Mitochondrial DNA Part ADNA Mapping, Sequencing, and Analysis
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Historical mitochondrial diversity in Africanleopards (Panthera pardus) revealed by archivalmuseum specimens
Corey Anco, Sergios-Orestis Kolokotronis, Philipp Henschel, Seth W.Cunningham, George Amato & Evon Hekkala
To cite this article: Corey Anco, Sergios-Orestis Kolokotronis, Philipp Henschel, Seth W.Cunningham, George Amato & Evon Hekkala (2017): Historical mitochondrial diversity in Africanleopards (Panthera pardus) revealed by archival museum specimens, Mitochondrial DNA Part A,DOI: 10.1080/24701394.2017.1307973
To link to this article: http://dx.doi.org/10.1080/24701394.2017.1307973
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RESEARCH ARTICLE
Historical mitochondrial diversity in African leopards (Panthera pardus) revealedby archival museum specimens
Corey Ancoa,b , Sergios-Orestis Kolokotronisb,c , Philipp Henscheld, Seth W. Cunninghama, George Amatob
and Evon Hekkalaa,b
aDepartment of Biological Sciences, Fordham University, Bronx, USA; bSackler Institute for Comparative Genomics, American Museum ofNatural History, New York, USA; cDepartment of Epidemiology and Biostatistics, School of Public Health, SUNY Downstate Medical Center,Brooklyn, USA; dPanthera, New York, USA
ABSTRACTOnce found throughout Africa and Eurasia, the leopard (Panthera pardus) was recently uplisted fromNear Threatened to Vulnerable by the International Union for the Conservation of Nature (IUCN).Historically, more than 50% of the leopard’s global range occurred in continental Africa, yet samplingfrom this part of the species’ distribution is only sparsely represented in prior studies examining pat-terns of genetic variation at the continental or global level. Broad sampling to determine baseline pat-terns of genetic variation throughout the leopard’s historical distribution is important, as thesemeasures are currently used by the IUCN to direct conservation priorities and management plans. Byincluding data from 182 historical museum specimens, faecal samples from ongoing field surveys, andpublished sequences representing sub-Saharan Africa, we identify previously unrecognized geneticdiversity in African leopards. Our mtDNA data indicates high levels of divergence among regional popu-lations and strongly differentiated lineages in West Africa on par with recent studies of other large ver-tebrates. We provide a reference benchmark of genetic diversity in African leopards against whichfuture monitoring can be compared. These findings emphasize the utility of historical museum collec-tions in understanding the processes that shape present biodiversity. Additionally, we suggest futureresearch to clarify African leopard taxonomy and to differentiate between delineated units requiringmonitoring or conservation action.
ARTICLE HISTORYReceived 30 December 2016Accepted 14 March 2017
KEYWORDSPanthera pardus; Africanleopard; museumcollections; geneticdiversity; phylogeography
Introduction
Africa’s complex and diverse biogeographic history has hadprofound impacts on its ecosystems. Savannas emerged forthe first time and glaciers began to form in Antarctica inresponse to gradually cooling temperatures in the Pliocene(Futuyma 2013). Global cooling accelerated in the Pleistocenetriggering the most recent ice age, or Last Glacial Maximum(LGM) (Steele 2007). Prolonged exposure to cool, dry condi-tions expanded more arid ecosystems (deserts and savannas)to the north and south and contracted forested regions,while shifts to warm, moist conditions gave rise to expansiverainforests across the equator, woodlands to the north andsouth, and the contraction of arid zones (Steele 2007).Repeated expansion and contraction of ecosystems frag-mented refugium distributions of habitat specialists (Futuyma2013), particularly in parts of Central and East Africa whereecosystems responded drastically to fluctuating climates(Lorenzen et al. 2012).
Recent genetic studies using the biogeographic history ofAfrican species have overturned previously held assumptionsabout population linkages in widespread vertebrates.Increasingly, research supports the theory that wide-ranging
species exhibiting discontinuity within their range may alsoexhibit variation at the molecular level; variation tends to cor-respond to geographic regions and major climatic events andis well documented in African taxa (Measey & Channing 2003;Moodley & Bruford 2007; Hekkala et al. 2011; Ishida et al.2011; Lorenzen et al. 2012; Smitz et al. 2013; Menegon et al.2014; Dowell & Hekkala 2016; Dowell et al. 2016; Fennessyet al. 2016) and across felids (Luo et al. 2004; McRae et al.2005; Haag et al. 2010; Charruau et al. 2011; Barnett et al.2014; Bertola et al. 2015). A review of two African felids, thelion (Panthera leo) and the cheetah (Acinonyx jubatus) demon-strate this pattern and the importance of revisiting conclu-sions drawn from previous research reliant on limitednumbers and spatial extent of samples.
Africa’s biogeographic history of expanding and contract-ing habitats has generated disjunct distributions amongbiota, including lions (Barnett et al. 2014). Due to severehuman persecution, habitat loss and loss of prey, lions dra-matically declined and many populations became geographi-cally isolated throughout the species’ range (Riggio et al.2013). Recent studies have found the African lion to harbourgreater genetic diversity than formerly recognized (Barnettet al. 2006; Bertola et al. 2011; Dubach et al. 2013;
CONTACT Corey Anco [email protected] Department of Biological Sciences, Fordham University, 441 East Fordham Road, Bronx, NY 10458, USASupplemental data for this article can be accessed here.
� 2017 Informa UK Limited, trading as Taylor & Francis Group
MITOCHONDRIAL DNA PART A, 2017http://dx.doi.org/10.1080/24701394.2017.1307973
Barnett et al. 2014), largely due to the limited spatial extentof earlier studies (O’Brien et al. 1987). These recent studies allcontributed to a gradual taxonomic revision of the lion, withthe most comprehensive study by Bertola et al. (2015) usingnuclear (nuDNA) and mitochondrial (mtDNA) markers. Theauthors confirmed deeply rooted phylogeographic breakswithin the African continent with West and Central Africanpopulations clustering with Indian lions (formerly recognizedas a separate subspecies; P. l. persica), and being geneticallydistinct from East and Southern African lions. The results oftheir analyses contested the recognized taxonomic status oflions by confirming the existence and clustering of independ-ent genetic lineages into several distinct geographic regions.Other recent phylogeographic studies are revealing of sub-regional genetic differentiation among wide-ranging Africantaxa (Moodley & Bruford 2007; Kadu et al. 2011; Demos et al.2014; Cunningham et al. 2016; Fennessy et al. 2016).
Early genetic analyses suggested the cheetah suffered agenetic bottleneck around ten thousand years ago (Ka) dur-ing the LGM rendering surviving populations depauperate atthe immunogenetic level (O’Brien et al. 1983, 1985; Menotti-Raymond & O’Brien 1993). However, Charruau et al. (2011)revealed distinct geographic clustering and divergence inextant populations predating the LGM using 94 samples frommost of the cheetah’s historical range. Additionally, Castro-Prieto et al. (2011) attributed the decreased diversityobserved by O’Brien et al. (1983) in the MHC class I alleles ofcheetahs to small sample sizes. Furthermore, Caro andLaurenson's (1994) critique largely dismissed linking geneticimpoverishment and juvenile mortality in cheetahs usingstrong supporting evidence from ecological studies to explainmortality rates (predation was responsible for �73% of cubdeaths). Together, these reports highlight the importance inrevisiting previous findings with increased sample sizes andgeographic breadth and in recognizing data limitations whileconsidering complementary fields of research. Recent atten-tion and revisions to genetic studies focusing on the Africanlion and cheetah bring into question the status of Africa’sremaining big cat, the leopard (Panthera pardus).
The leopard is a large, solitary carnivore once contiguousthroughout Africa and Eurasia, and possesses the greatest his-torical distribution (�34,850,000 km2) of any felid (Nowell &Jackson 1996; Sunquist & Sunquist 2002; Hunter et al. 2013;Jacobson et al. 2016). It is a habitat generalist (Nowell &Jackson 1996) and inhabits nearly every habitat type includ-ing savannas, woodlands, shrublands, temperate and tropicalforests, montane habitats, swamps, and semi-arid deserts(Sunquist & Sunquist 2002; Dutta et al. 2013; Hunter et al.2013) and ranges from sea level to �4500m above sea level(a.s.l.) (Aryal & Kreigenhofer 2009). Relative to other membersof the Panthera genus, the leopard is comparatively adapt-able, partly owing to its broader diet, one of the broadest ofany mammalian carnivore (Bailey 1993; Hayward et al. 2006)and given prey is abundant can develop a high tolerance tohuman disturbance in the absence of intense direct persecu-tion (Mondol et al. 2009; Athreya et al. 2013; Swanepoel et al.2013; Odden et al. 2014).
Leopards are vulnerable to numerous ongoing threatsincluding persecution for perceived and realized threats to
livestock and game animals, harvest for traditional and globaltrade in parts, habitat loss, the loss of prey populations, andunsustainable trophy hunting (Nowell & Jackson 1996;Henschel et al. 2011; Packer et al. 2011; Raza et al. 2012;Swanepoel et al. 2013). Compounded over time these threatshave resulted in a 63–75% contraction of their global distri-bution, while in Africa, 48–67% of leopard range has beenlost, with leopards in North and West Africa suffering themost catastrophic range reductions (�99% and 86-95%,respectively) (Jacobson et al. 2016). Remnant populations inthese regions may be geographically disconnected and arepotentially at risk of genetic isolation, as has been speculatedfor lions (Bj€orklund 2003; Riggio et al. 2013). Given the highrates of leopard range loss, it is critical for the species’ man-agement that we lack historical genetic data for leopards andare therefore unable to adequately assess the impacts rangefragmentation already had on extant populations.
Prior taxonomic hypotheses for the African leopardincluded descriptions of more than 20 subspecies (Table 1)(Stein & Hayssen 2013) spanning all major biomes except theextreme arid landscape of the Saharan Desert (Figure 1)(Olson et al. 2001). While these subspecific designations likelyoverestimated the actual number of genetically distinct leop-ard lineages, it is worth noting that descriptive characteristics(e.g. pelage morphology) are often associated with uniquegeographic features including the near fixation of melanismin Asiatic leopards south of the Isthmus of Kra (Kawanishiet al. 2010). With the advent and application of genetic analy-ses, we are better able to evaluate the taxonomic hypothesesproposed by previous researchers within the context of phy-logeography (Avise 2000).
The International Union for the Conservation of Nature(IUCN) recognizes nine subspecies based on molecular analy-ses (Miththapala et al. 1996; Uphyrkina et al. 2001) (Table 1).With the exception of the African (P. p. pardus) and Indianleopard (P. p. fusca), the IUCN lists or proposes all other sub-species as Endangered or Critically Endangered (Stein et al.2016). Previous genetic studies of the African leopard werelimited by small sample size and a geographic bias(Miththapala et al. 1996; Uphyrkina et al. 2001), and sufferfrom large gaps in geographic representation from the major-ity of Africa (Table S1), increasing the likelihood that previousassessments may have failed to capture the full spectrum ofgenetic diversity harboured in the African leopard.Furthermore, we observed conflicting information in samplesshared between the Miththapala et al. (1996) and Uphyrkinaet al. reports (2001) (Table S2). As a result, it is critical to reas-sess African leopards using larger sample sizes and widergeographic coverage.
In this study, we use DNA sequence data from 182 individ-uals including museum specimens (pre-1970, also referred toas archival or historical in this study) and contemporary(post-1990) faecal and tissue samples to provide a practicalreference benchmark of genetic diversity in African leopardsacross sub-Saharan Africa against which future monitoringcan be compared. We (a) determine if genetic diversity ofleopards found in museum collections is adequately repre-sented in previously published literature, (b) assess popula-tion structure and discuss phylogeographic patterns in the
2 C. ANCO ET AL.
Table 1. Description of classically described leopard subspecies adapted from Stein and Hayssen (2013).
Subspecies name Locality Region Source Notes
Panthera pardus – See below Felis pardus Linnaeus, 1758 IUCN status: Near Threatenedpardus North North Africa Felis pardus Linnaeus, 1758 IUCN status: Near Threatened (nomi-
nate form for continental Africa)adersi Zanzibar East Africa P[anthera] p[ardus]adersi Pocock, 1932a: 33.
Type locality ‘Zanzibar,’ restricted to ‘nearChuaka’ (Pocock 1932b:563).
adusta Abyssinia, Ethiopia East Africa Panthera pardus adusta Pocock, 1927: 214.Type locality ‘unknown.’
antinorii Somalia East Africa (Felis pardus) antinorii de Beaux, 1923: 276,278. Type locality ‘Keren, paese deiBogos,’ Somalia.
barbarus d’Alg�erie North Africa F(elis) pardus barbarus de Blainville, 1843:186. Type locality ‘d’Alg�erie’.
chui Northern Uganda East Africa Felis pardus chui Heller, 1913: 6. Type local-ity ‘Gondokoro, northern Uganda’.
fortis Kenya East Africa Felis pardus fortis Heller, 1913: 5. Type local-ity ‘Loita Plains, Southern Guaso Nyirodistrict, British East Africa’.
iturensis Belgian Congo Central Africa Panthera pardus iturensis J. A. Allen, 1924:259. Type locality ‘Niapu, Belgium Congo’.
leopardus (G€unther) Grahamstown Southern Africa Felis leopardus melanot[ica]. G€unther, 1885:plate xvi. Type locality ‘Grahamstown,’clarified to ‘about 20 miles fromGrahams-town’ by G€unther (1886:205).
leopardus (Scheber) Senegal West Africa Felis leopardus Schreber, 1775:plate CI;Schreber, 1777: 387. Type locality‘Senegal’.
melanosticta Unknown Southern Africa F(elis) pardus melanosticta Lydekker, 1908:430. Unjustified emendation of Felis mel-anotica G€unther, 1885.
melanotica Unknown Southern Africa F(elis) pardus melanotica: Pocock, 1907: 677.Name combination.
minor Sudan, South Sudan East Africa (Leopardus) pardus minor Matschie, 1895:199. Nomen nudum.
nanopardus Somalia East Africa Felis pardus nanopardus Thomas, 1904: 94.Type locality ‘40 miles west of Gorahai,’Somaliland.
palearia Algeria North Africa Felis palearia F. G. Cuvier, 1832: 3 for plateof panth�ere male. Type locality ‘Alger’.
panthera Algeria North Africa Felis panthera Schreber, 1775: plate XCIX;Schreber, 1777:384–385. Type locality‘Africa,’ restricted to ‘Algeria’ by Ellermanand Morrison-Scott (1951:316).
poecilura Gabon Central-West Felis poecilura Valenciennes, 1856: 1036.Type locality ‘Gabon’.
puella Namibia Southern Africa P[anthera] p[ardus]puella Pocock, 1932a: 33.Type locality ‘Kaokoveld’, Namibia.
reichenowi K�amerun Central-West Panthera pardus reichenowi Cabrera, 1918:481. Type locality ‘‘Yok�o (K�amerun)’.’
ruwenzorii Camerano Central-West F(elis) p(ardus) ruvenzorii de Beaux, 1923:275. Unjustified emendation of Felis par-dus ruwenzorii Camerano, 1906.
shortridgei Namibia Southern Africa P[anthera] p[ardus] shortridgei Pocock,1932a: 33. Type locality ‘Damaraland,’restricted to ‘Gangongo, 3560 ft. alt. onthe Okavango River some 120 milesabove the Okavango swamp in WesternCaprivi’ (Pocock 1932b: 584).
suahelicus Uganda East Africa Felis leopardus suahelicus Neumann, 1900:551. Type locality ‘Tanga, am Manjara-Seeund in den Loita-Bergen … In Nai(Nord-Ugogo), in Usandawe und inUganda’.
varia Unknown ? Felis leopardus varia Schreber, 1777: 387,plates CI and CIb. Vide Wagner 1841:479.Type locality unknown.
vulgaris Unknown ? Panthera vulgaris Oken, 1816: 1052.Unavailable name (InternationalCommission on Zoological Nomenclature1956: Opinion 417).
nimr Saudi Arabia Arabian Peninsula Felis nimr Hemrich and Ehrenberg,1833:plate xvii. Type locality ‘Arabia.’
IUCN status: Critically Endangered
(continued)
MITOCHONDRIAL DNA PART A 3
context of wide-ranging African taxa, and (c) explore a prob-able geographic origin and distribution of genetic diversity ofAfrican leopards across sub-Saharan Africa. We also discussthe role of DNA damage, and deamination in particular,regarding use and analysis of museum samples.
Materials and methods
Sample origin
Sample origin and locality data are detailed in Table 2. Forarchival specimens we sampled bone and tissue fragmentsfrom 94 leopard skulls (Department of Mammalogy, AmericanMuseum of Natural History [AMNH]), obtained 15 faecal sam-ples from field surveys, and retrieved an additional 126mtDNA sequences from NCBI GenBank (Ropiquet et al. 2015).Only archival samples where the entire ND-5 locus wassequenced were included in our analyses. Faecal samples forGabon, Nigeria, Senegal, and the Republic of Congo (n¼ 15)were provided by collaborators, including Panthera (NewYork, NY) and the Leibniz Institute for Zoo and WildlifeResearch (Berlin, Germany). Samples were collected between1905 and 2013. Samples from two earlier studies (Miththapalaet al. 1996; Uphyrkina et al. 2001) were excluded due to con-flicting sample origin and/or sequence discrepancies.
Laboratory work
DNA was isolated using the DNeasy Blood and Tissue Kit(Qiagen, Hilden, Germany) with the following modificationsfor archival samples. Sterilized samples were covered in200ll, of 1� Phosphate Buffer Saline (PBS) solution and incu-bated at room temperature for 48 h until tissues softenedprior to digestion according to the manufacturer’s protocol. Iftissues remained undigested, an additional 20 ll of proteinase
K were added and the incubation was repeated. All process-ing of archival samples was conducted in a PCR-free room,used specifically for degraded or low-quality DNA. Negativecontrols were used throughout the process.
We targeted a 611 bp region of the ND-5 mitochondrialgene, corresponding to positions 12,632–13,242 in the mito-chondrial genome sequence of the leopard (GenBank acces-sion EF551002.1) (Wei et al. 2011), known to harbourintraspecific variation specifically in leopards (Miththapalaet al. 1996; Uphyrkina et al. 2001; Farhadinia et al. 2015;Ropiquet et al. 2015). Due to the age and quality of the sam-ples used in this analysis, we generated amplicons <250bpusing eight primer pairs (Table S3). We used a 25 lL reactionvolume for PCR of archival specimens consisting of 11.5lL ofAmpliTaq Gold 360 (Thermo Fisher, Waltham, MA), 10 lM for-ward and reverse primer (0.7 lL each), 2lL of MgCl2, 8.6lL ofmolecular grade water, and 1.5 lL of template DNA.Contemporary samples followed the same recipe but used1lL of DNA template and 9.1 lL of molecular grade water.PCR was performed on Applied Biosystems 2720 (ThermoFisher, Waltham, MA) and Mastercycler ep gradient S(Eppendorf, Hamburg, Germany) thermal cyclers. Sangersequencing was carried out on an Applied Biosystems 3730xlDNA Analyzer (Thermo Fisher, Waltham, MA). Sequence chro-matograms were inspected and assembled in Sequencher 5.2.4 (Gene Codes Corp., Ann Arbor, MI). We then used BLAST onNCBI GenBank (Johnson et al. 2008; http://blast.ncbi.nlm.nih.gov/Blast.cgi) to confirm the genetic identity of the samples.
Data analysis
The final dataset was composed of 182 consensus DNAsequences: 41 archival, 15 contemporary faecal, and 126 from
Table 1. Continued
Subspecies name Locality Region Source Notes
Type specimen: ‘Arabian skin from themountains in the vicinity of Qunfida, Asir,Saudi Arabia’ (Spalton and Al Hikmani2006).
saxicolor Iran Southwest Asia P[anthera] p[ardus] saxicolor Pocock,1927:213. Type locality ‘Asterabad insouthern Iran’ (Spalton and Al Hikmani2006).
IUCN status: Endangered
fusca India Indian subcontinent Felis fusca Meyer, 1794. Type locality ‘Indiaorientali’.
IUCN status: Near Threatened
kotiya Ceylon Sri Lanka Panthera pardus kotiya Deraniyagala,1956:116. Type locality ‘Ceylon’.
IUCN status: Endangered
delacouri Annam Southeast Asia Panthera pardus delacouri Pocock,1930b:325. Type locality ‘Hu�e in Annam’.
IUCN status: Near Threateneda
melas Java Indonesia Felis melas G. Cuvier, 1809:152. Type locality‘Java’.
IUCN status: Critically Endangered
japonensis Japan North-Central China Leopardus japonensis Gray, 1862:262, plateXXXIII. Alleged type locality ‘Japan’.
IUCN status: Near Threateneda
orientalis Korea Northeast Asia Felis orientalis Schlegel, 1857:23, figure 13.Type locality ‘Korea’.
IUCN status: Critically Endangered
aRecent assessments by Laguardia et al. (2017) and Rostro-Garc�ıa et al. (2016) recommend uplisting of Indochinese and North Chinese leopards from ‘NearThreatened’ to ‘Critically Endangered’ and ‘Endangered’, respectively.
4 C. ANCO ET AL.
GenBank. PopART (Leigh & Bryant 2015; http://popart.otago.ac.nz) was used to construct a median-joining haplotype net-work with default parameters (e¼ 0) (Bandelt et al. 1999)edited and annotated with InkScape (free open-source SVGgraphics editor; Bah 2007). We retrieved additional ND-5sequences from GenBank for Arabian and Persian leopards(AY035277-79) used as outgroups. Haplotypes were assignedto one of the five populations using a combination of geo-graphic origin, haplotype clustering on network, and geneticsimilarity criteria (Figure 2). The five populations are: WestAfrica (WA), Coastal West-Central Africa (CWCA), Central-EastAfrica (CEA), Central-Southern Africa (CSA), and SouthernAfrica (SA). Assignment of haplotypes to WA and CWCA wasbased on haplotype clustering and genetic divergence fromneighbouring haplotype clusters. For CEA, the occurrence ofa dominant haplotype and multiple connections to otherhaplotypes representing leopard range countries predomi-nantly in equatorial Africa supports clustering these samples
together as one haplotype group. Haplotypes characterizingthe CSA population are represented by a cluster of leopardrange countries primarily located south of equatorial Africa.With the exception of two individuals, all samplescomprising the haplotype cluster designated as SA were fromSouth Africa.
We generated a geographical distribution map of the hap-lotypes using a binary matrix in ArcGIS 10.3 (ESRI, Redlands,CA) and displayed haplotypes over the historical leopard dis-tribution layer from Jacobson et al. (2016) (Figure 3). We usedexact localities and coordinate data where available. DNAsequences were aligned in MEGA 6.06 (Tamura et al. 2013)using ClustalW (Larkin et al. 2007). Geographical partitioningof haplotypes was quantified via analysis of molecular var-iance (AMOVA) (Excoffier et al. 1992). Populations wereplaced into one of the three continental regions associatedwith African phylogeography: (1) West Africa composed ofthe WA population representing the western extent of the
Figure 1. Distribution of 12 African leopard subspecies as described by Miththapala et al. (1996) and Uphyrkina et al. (2001). Hypothetical distributions displayedover biomes following Olson et al. (2001). Additionally described subspecies are listed in Table 1. For interpretation of terrestrial biomes the reader is referred to theonline version of this article.
MITOCHONDRIAL DNA PART A 5
Table 2. Origin and collection data of leopard samples.
Geographicregion Country No. of samples Sample identifier Specific locality Collection date Source
West Africa Nigeria 1 NI-17 Gashaka-Gumti NP 2009 Panthera, PhilippHenschel
Senegal 10 SEN-01, SEN-03, SEN-08,SEN-10, SEN-21, SEN-23, SEN-28, SEN-37,SEN-41, SEN-43
Niokolo-Koba NP 2011 Panthera, PhilippHenschel
Central Africa Cameroon 13 54334, 87236, 167352,170289, 170293,170294, 170295,170296, 170300,170301, 170302,170305, 170309
N/A 1923–1946 AMNH
Chad 2 164151 Fort Archambault District 1952 AMNH165802 N/A 1905 AMNH
DRC 9 52038 Akenge 1913 AMNH52006, 52021, 52023 Faradje 1911–1913 AMNH52048 Medje 191452044 Gamangui 1910 AMNH189390, 189391 Ubangi District, Karawa 1905 AMNH208770 Kivu District 1962 AMNH
Gabon 3 GAB-10, GAB-24, GAB-26 Lop�e NP 2011 Panthera, PhilippHenschel
Republic of Congo 1 T-Congo Domaine de Chasse deMboko HR
2013 Leibniz Institute for Zooand Wildlife Research,Torsten Bohm
East Africa Kenya 5 34745, 34746 Cherangangi Hills 1912 AMNH34747 Elgeyo Forest 1913 AMNH88628, 88629 N/A 1933 AMNH
Tanzania 6 81301, 81302, 81303 Rungwe 1929 AMNH85161 Serengeti Plains 1928 AMNH88393 Bamboo Forest 1933 AMNH42216 N/A 1913 AMNH
Southern Africa Angola 1 80610 Chitau 1925 AMNHBotswana 1 169460 Ngamiland, Bushman Pits 1950 AMNHMozambique 1 186944 N/A 1948 AMNH
32 leo01, leo03, leo04, leo05,leo06, leo08, leo09,leo10, leo11, leo12,leo13, leo14 leo15,leo16, leo17, leo75,leo77, leo79, leo80,leo81, leo82, leo84,leo85, leo88, leo89,leo90, leo91, leo94,leo95, leo97, leo98,leo99
Niassa Province 1998–2008 Ropiquet et al. (2015)
Namibia 1 165112 Kaokoveld 1953 AMNHSouth Africa 1 81845 Transvaal 1925–1930 AMNH
10 leo18, leo20, leo21, leo22,leo23, leo65, leo66,leo165, leo167, leo168
Eastern Cape 1998–2008 Ropiquet et al. (2015)
18 leo34, leo35, leo36, leo37,leo38, leo39, leo40,leo41, leo42, leo54,leo55, leo56, leo101,leo102, leo103, leo104,leo105, leo106
Kruger NP 1998–2008 Ropiquet et al. (2015)
43 leo44, leo110, leo121,leo122, leo123, leo124,leo125, leo126, leo127,leo128, leo129, leo130,leo131, leo132, leo133,leo134, leo135, leo136,leo137, leo138, leo139,leo140, leo141, leo142,leo143, leo144, leo145,leo146, leo147, leo148,leo149, leo150, leo151,leo152, leo153, leo154,leo155, leo156, leo157,leo158, leo159, leo160,leo161
Mkuze GR, Phinda GR 1998–2008 Ropiquet et al. (2015)
(continued)
6 C. ANCO ET AL.
leopard habitat; (2) Central-East-Southern Africa composed ofthe CWCA, CEA, and CSA populations representing equatorialleopard habitat; and (3) Southern Africa composed of the SApopulation representing the southern extent of leopard habi-tat. We assessed portions of genetic variance to divergenceeither among regions (West, Central-East/Central-Southern,Southern Africa), among populations within regions (CWCA,CEA, CSA) or within populations. Genetic diversity indices andpopulation statistics (pairwise FST analyses) were calculated inArlequin 3.5 (Excoffier & Lischer 2010) using the Kimura 2-parameter nucleotide substitution model (Kimura 1980) tocorrect for multiple hits accounting for transitions and trans-versions (Table 3). For population analyses, historical samplesrefer to archival or museum specimens collected pre-1970,and contemporary samples refer to faecal and tissue samplescollected after 1990.
Results
We identified 30 distinct haplotypes from 182 sequencedindividuals spanning sub-Saharan Africa. Haplotypes generallyfell into five distinguishable clusters as suggested by themedian-joining network with four haplotypes shared by72.5% of the samples (n¼ 132, Figure 2). Archival and mod-ern faecal samples accounted for 67% of observed haplotypes(n¼ 20), with another 10% (n¼ 3) shared between museum,modern faecal samples, and/or GenBank. GenBank sequencesaccounted for the remaining 23% (n¼ 7) of haplotypes.Private haplotypes were observed in each population, andH10, the dominant, i.e. most frequent, haplotype in CEA, con-tained the greatest number of network connections (n¼ 10),and had the fewest connections between each other clus-tered population (Figure 2). H10 was also the most
Table 2. Continued
Geographicregion Country No. of samples Sample identifier Specific locality Collection date Source
23 leo25, leo26, leo27, leo28,leo31, leo33, leo45,leo47, leo49, leo50,leo53, leo57, leo58,leo61, leo62, leo64,leo68, leo69, leo70,leo71, leo72, leo73,leo74
Western Cape 1998–2008 Ropiquet et al. (2015)
Zambia 1 89842 N/A 1939 AMNH
NP: National Park; N/A: Not Available; AMNH: American Museum of Natural History; DRC: Democratic Republic of Congo; GR: Game Reserve.
Figure 2. Median-joining network of 182 African leopards. Coverage spans 15 countries across sub-Saharan Africa. Three leopard sequences from GenBank are usedas outgroups: P. p. nimr (Arabian leopard: Saudi Arabia/Oman?) and P. p. saxicolor (Persian leopard: Afghanistan). Haplotypes are color-coded according to geogra-phy. Size of circle is proportional to the number of individuals sharing the same DNA sequence. Hash marks indicate mutations between haplotypes. Pie chart divi-sions indicate haplotype sharing between countries. Please refer to the online version of this article for interpretation of colored haplotypes.
MITOCHONDRIAL DNA PART A 7
geographically widespread of haplotypes spanning 53%(n¼ 8) of sampled countries, and likely the ancestral haplo-type among populations.
Haplotype diversity (Hd) exceeded 0.5 for all populationsexcept CSA (0.29) (Table 3). CWCA and CEA harboured thehighest nucleotide diversity per site (p) and per gene (k)(CWCA: p¼ 0.0066, k¼ 4; CEA: p¼ 0.0051, k¼ 3), and CWCAshowed the greatest genetic separation (six mutational stepsbetween H5 and H10) in the network (Figure 2). Similar diver-sity values were found in WA and SA, while CSA exhibitedthe lowest diversity values. Haplotype distributions exhibitedgeographic separation, although clinal variation was observedin every population except WA (Figures 2 and 3). In oneinstance between CEA and CSA (one leopard fromDemocratic Republic of Congo, DRC, grouped with CSA),eight instances between CEA and SA (eight leopards fromSouth Africa grouped with CEA), and four instances betweenCSA and SA (two leopards from Mozambique grouped withSA and two leopards from South Africa grouped with CSA).
Two leopards from Cameroon clustered with three leopardsfrom Gabon comprising the CWCA population.
There were 47 substitution sites, with a mean of 11.6 sub-stitutions per population (Table 3). The transition:transversionratio was 18.33:1, thus indicating a high transition bias forthis locus in Africa. The highest number of substitutionsoccurred within the CEA population (25 transitions and 1transversion), followed by SA (15 transitions and 1 transver-sion). We also examined incidences of singleton mutations inmuseum and faecal samples across populations (Table S4) toevaluate the potential of DNA sequence damage due tohydrolytic deamination (Hofreiter et al. 2001; Mitchell et al.2005; Zimmermann et al. 2008). Singleton mutations resultingin C!T transitions occurred at a total of five positionsbetween two populations, whereas G!A transitions occurredat a total of three positions between two populations.Variable sites are summarized in Table 4. The AMOVA analysisfound distinct population structuring at each scale of hier-archical partitioning. The greatest amount of variance was
Figure 3. Distribution of leopard haplotypes across sub-Saharan Africa. Haplotypes are displayed over historic and contemporary leopard distribution layers(Jacobson et al. 2016). Haplotypes are color-coded according to geographical origin and grouped into regional groups (see Figure 2). Pie chart divisions indicatepresence of more than one haplotype at a given locality. Please refer to the online version of this article for interpretation of colored haplotypes.
8 C. ANCO ET AL.
explained to be between groups, which accounted for 54%of observed variation (Table S5). Differences among popula-tions within groups represented 27% of the observed varia-tion, while 19% of the variation was explained withinpopulations.
Pairwise FST values were calculated between all popula-tions and compared to two Asiatic leopard subspecies (theArabian leopard (P. p. nimr) in the Middle East and thePersian leopard (P. p. saxicolor) in Southwest Asia) minedfrom GenBank (Table S6). Significant separation (p< .05) wasdetected between all populations (Figure 4), indicatingreduced gene flow and population structuring. Two popula-tions (WA and CWCA) showed almost complete differentia-tion from CSA (0.97 and 0.96, respectively). Populationaverage pairwise differences for African populations werealso calculated between and within populations (Figure 5).The corrected average pairwise differences (Nei’s D) werelargely congruent with results of the pairwise FST matrix(Figure 4). The average number of pairwise differencesbetween CEA and CSA was low (Nei’s D¼ 1.14, uncorrecteddistance¼ 2.97) and the average pairwise difference withinCSA was the lowest of all populations (0.3) (Figure 5).Pairwise FST values between African populations vary slightlywhen Asiatic sequences are omitted due to missing data(61 bp) in retrieved sequences from GenBank for Persianleopards. We tested for temporal changes by conductingpairwise FST comparisons between historical (museum) andcontemporary samples (faecal and tissue) from the same pop-ulation and found three of the four populations (CWCA, CSA,and SA) did not significantly differ between time periods(Table S7). WA only contained contemporary samples, andFST[CEAc-CEAh]¼ 0.27 (c¼ contemporary, h¼historical) wassignificant, however, contemporary samples for CEA wereonly represented by the haplotypes: H10 and H17 (Figure 2).
Discussion
Historical diversity of museum specimens
This work assembled 182 DNA sequences from 41 archivaland 15 contemporary faecal samples, as well as 126 GenBanksequences, and represents the most comprehensive mtDNAdataset for leopards. We reveal extensive, cryptic diversity inthe ND-5 locus among historical populations and retention ofindependent genetic lineages in extant populations. Distincthaplotypes are geographically clustered indicating a lack ofpanmixia. Overall haplotype diversity was high (0.84) withmoderate levels of nucleotide diversity (p¼ 0.0042) in leop-ards across sub-Saharan Africa (Table 3). Nucleotide diversityfell between values observed in mtDNA of other large felidsincluding jaguars (0.0077; Eizirik et al. 2001), lions (0.0066;Antunes et al. 2008), pumas (0.0032; Caragiulo et al. 2014),and tigers (0.0018; Luo et al. 2004).
While caution must be taken when drawing conclusionsfrom the analyses of individual mitochondrial loci, we haveidentified a greater degree of genetic diversity in the ND-5locus of the African leopard than previously recognized.Samples of known origin used in previous studies accountfor four countries (Botswana, Mozambique, Namibia, andTa
ble3.
Geneticvariatio
nin
theND-5
locusof
leop
ards
across
sub-SaharanAfrica.
Region
Popu
latio
ngrou
pN
No.
ofhaplotypes
Haplotype
diversity
(Hd)
Nucleotide
diversity
(p)
Diversity
pergene
(k)
Segregating
sites(S)
Parsimon
yinform
ative
sites
Transitio
nsTransversion
sSubstitutions
Indels
Private
substitutions
Sub-SaharanAfrica
AllS
amples:
sub-SaharanAfrica
182
300.841
0.0042
2.354
1224
11(M
ean)
0.6(M
ean)
11.6
(Mean)
1.2(M
ean)
7.2(M
ean)
West
West
104
0.533
0.0018
1.067
31
40
40
2Central-East-Southern
Central-East
4312
0.897
0.0051
37
1325
126
017
CoastalW
est-Central
55
10.0066
410
09
110
06
Central-Sou
thern
373
0.291
0.0005
0.303
02
20
21
1Southern
Southern
876
0.602
0.0014
0.796
24
151
165
10
MITOCHONDRIAL DNA PART A 9
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183
184
189
212
215
239
262
266
271
272
284
296
308
320
329
338
339
342
344
356
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(continued)
10 C. ANCO ET AL.
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MITOCHONDRIAL DNA PART A 11
Figure 4. Heatmap matrix of pairwise FST values between African, Arabian (P. p. nimr), and Persian leopards (P. p. saxicolor). WA: West Africa, CWCA: Coastal West-Central Africa, CEA: Central-East Africa, CSA: Central-Southern Africa, SA: Southern Africa. Dotted line separates populations of African leopards from Asiatic subspe-cies (P. p. nimr and P. p. saxicolor) along the y-axis.
Figure 5. Average number of pairwise differences between African leopard populations. WA: West Africa. CWCA: Coastal West-Central Africa; CEA: Central-EastAfrica; CSA: Central-Southern Africa; SA: Southern Africa. Values above the diagonal represent the average number of pairwise differences between populations(pXY). Diagonal values represent the average number of pairwise differences within populations (pX). Values below the diagonal represent the corrected averagenumber of pairwise differences between populations (pXY – (pXþpY)/2). Please refer to the online version of this article for interpretation.
12 C. ANCO ET AL.
South Africa) represented in this analysis (Kenya excludedfrom count because of ambiguous sample origin, see TableS1). This study includes samples from 11 additional countriesunrepresented in previous research (Table 2). Novel samplesaccount for 67% of all the observed haplotypes (n¼ 20),while uniquely sampled countries represent 63% of all theobserved haplotypes (n¼ 19) (Figure 2). Haplotypes recoveredonly from the museum specimens accounted for 43.33% ofall the haplotypes (n¼ 13) highlighting extensive geneticdiversity in historical leopard populations unrepresentedin contemporary populations sampled for this analysis(Figure 2).
Precautions were taken in sample preparation, extraction,and handling to minimize the risk of contamination fromexogenous DNA (P€a€abo 1989). However, degradation andfragmentation due to age, preservation method, storage con-ditions, and sample composition are known to impact thequality and amplification of DNA (Mitchell et al. 2005; Burrellet al. 2015). DNA repair mechanisms routinely identify andremove misincorporated lesions (Zimmermann et al. 2008),but repair ceases after organismal death resulting in struc-tural destabilization and the accumulation of damage (P€a€abo1989; Mitchell et al. 2005). Soft tissue is prone to oxidativedamage and degradation (P€a€abo 1989) whereas hard tissue(e.g. bone), which we primarily recovered during destructivesampling of museum specimens, limits the risk of exogenousDNA contamination and preserves the integrity of intactendogenous DNA (Burrell et al. 2015). Still, DNA damage,especially hydrolytic deamination of cytosine, could haveresulted in C!T and G!A transitions, which are predomi-nant substitutions associated with senescence (Hofreiter et al.2001; Mitchell et al. 2005; Burrell et al. 2015).
We evaluated the incidence of singleton substitutions inmuseum and faecal samples across populations to assess ifDNA damage due to cytosine deamination was present inour samples (Table S4) and found C!T transitions at fivepositions: one position in CEA and four positions in CWCA.Removal of C!T singletons in CEA did not affect networktopology or the number of haplotypes; however, removal ofC!T singletons in CWCA caused H6 and H9 to merge withH5. This decreased the number of haplotypes in CWCA fromfive to three, but network topology was unaffected withrespect to neighbouring population groups. G!A transitionsoccurred at three positions: two positions in CEA and one inWA. Removal of G!A singletons in CEA did not affect net-work topology or the number of haplotypes; however,removal of G!A singletons in WA resulted in merging H1and H2. This decreased the number of haplotypes in WAfrom four to three, but network topology was otherwiseunaffected.
Cytosine deamination is a common issue reported in therecovery of sequence data from ancient DNA (aDNA)(Binladen et al. 2006). Here, we have used the terms archivaland historical interchangeably to refer to DNA recoveredfrom museum specimens. In other studies, ‘historical’ DNArefers to aDNA. While there is no precisely defined chronolog-ical marker delimiting aDNA, aDNA generally refers to DNArecovered from specimens dating hundreds to thousands ofyears before present and has more recently included DNA
from Pleistocene-era subfossils (Hofreiter et al. 2001; Paaboet al. 2004; Mitchell et al. 2005; Binladen et al. 2006). Theaccumulation of mutations due to oxidative and hydrolyticdeamination occurs more slowly in museum specimens col-lected in the past couple hundred years than in archaeologi-cal specimens (P€a€abo 1989; Burrell et al. 2015), the latter ofwhich are described in the aforementioned studies. The old-est samples used in this analysis are <115 years old thus,DNA miscoding lesions associated with senescence areunlikely. However, DNA fragmentation commonly associatedwith museum specimens (Paabo et al. 2004; Binladen et al.2006; Burrell et al. 2015) was observed in our museumsamples.
Population structure and phylogeographic patterns
Leopards exhibited population structuring at large geo-graphic scales (West, Central-East/Central-Southern, andSouthern Africa), suggesting strong evidence against pan-mixia in this species. AMOVA and pairwise FST analyses sup-port differentiation in the ND-5 locus spanning five majorhaplogroups: West Africa, Coastal West-Central Africa, Central-East-Africa, Central-Southern Africa, and Southern Africa.Distinction between CEA and CSA as two independentregional populations is supported by pairwise FST analyses(Figure 4). Although still high, FST[CEA-CSA]¼ 0.40, was thelowest among all African leopard population comparisons.CSA showed higher levels of differentiation from WA andCWCA leopard populations, than the latter two did to CEA,indicating that CSA leopards are reproducing in isolationfrom neighbouring populations (Figure 4). Furthermore, CSAexhibited the highest levels of differentiation when comparedwith the two selected Asiatic subspecies: FST[CSA-nimr]¼ 0.98and FST[CSA-saxicolor]¼ 0.97 (Figure 4).
The haplotype network (Figure 2) and subsequent analysesrevealed deep divergence between WA and CWCA popula-tions and between all other defined population groups. Bothgroups yielded near absolute differentiation from CSA (0.97and 0.96, respectively) and very high differentiation fromCEA, the ancestral population (0.86 and 0.85, respectively)(Figure 4). Differentiation of West African leopards fromCentral and East African leopards is expected, as similar pat-terns exhibiting decreased gene flow across this region ofAfrica are well documented in other taxa (Arctander et al.1999; Alpers et al. 2004; Won & Hey 2005; Kadu et al. 2011;Dobigny et al. 2013; Dowell et al. 2016). Contemporary sam-ples from Nigeria and the Republic of Congo (2009 and 2013,respectively) retain the ancestral haplotype observed fromhistoric samples collected from Central and East Africabetween 1905 and 1962, whereas the contemporary samplesof leopards collected from Senegal in 2011 show significantgenetic differentiation (FST[WA-CEA]¼ 0.86) (Figure 4).
The CWCA population exhibited strong evidence forgenetic differentiation from CEA (FST [CWCA-CEA]¼ 0.85)despite the two populations residing in close geographicproximity (Figures 2 and 3). CWCA consists of three contem-porary samples from Gabon collected in 2011 and two histor-ical samples from Cameroon collected in 1934–1936. Thisindicates that highly divergent populations of leopards in
MITOCHONDRIAL DNA PART A 13
Cameroon (and possibly surrounding countries of Gabon andEquatorial Guinea) were present prior to earliest samplingefforts retrieved for this analysis and further suggests thatthis region may have acted as a refugial habitat for leopards.It is difficult to assess the historical and current geographicexpanse of the highly divergent locus observed in the CWCApopulation. We lack historical specimens from the surround-ing countries of Nigeria, the Central African Republic,Equatorial Guinea, Gabon, and the Republic of Congo.Additionally, historical mtDNA diversity of leopard popula-tions from West Africa is not represented in this dataset.Increased sampling efforts throughout the CWCA region andin West Africa may reveal similar patterns in clinal variation(as observed in CEA and CSA), particularly along the WestAfrican rainforest belt.
Haplotypes from Cameroon, DRC, Mozambique, and SouthAfrica were observed in more than one population group. Thisis not entirely unexpected, as these were also the four mostfrequently sampled countries. Samples from Cameroon areexclusively represented by museum specimens and constitutefive haplotypes. No locality data for the Cameroon sampleswere available in the museum records, which makes identifica-tion of a potential environmental barrier to gene flow delimit-ing CWCA from WA and CEA more difficult to assess. However,insights from phylogeographic analyses of primates indicate alikely genetic barrier of interest. Preliminary analyses usingmtDNA and microsatellite data identified two deeply divergentlineages of chimpanzees in western Africa and in central andeastern Africa formed a suture zone located in centralCameroon (Gonder & Disotell 2006; Gonder et al. 2006).Specifically, the Sanaga River or another historically proximalenvironmental discontinuity is hypothesized to be responsiblefor limiting distribution and restricting gene flow of severalspecies (Gonder & Disotell 2006; Anthony et al. 2007; Nicolaset al. 2011). These findings were supported by Bowden et al.(2012) using high-throughput sequencing techniques tounambiguously confirm the division of Cameroonian chimpan-zees into two genetically distinct populations separated mostprobably by the Sanaga River.
We recovered mtDNA from nine museum samples repre-senting six haplotypes in the DRC ranging from 1905 to 1962(�7.7 leopard generations) primarily collected from the north-western and northeastern parts of the country. Five samplesrecovered from eastern DRC exhibited variation from theancestral haplotype. These samples were recovered from theeastern edge of the Congo rainforest, and likely from a regioncharacterized by high environmental heterogeneity, includingthe formation and persistence of major waterways in recentgeologic history. While portions of densely vegetated regionsof central Africa are suspected to have maintained forestcover towards the end of the Pleistocene, these areas maynot have been as stable as previously hypothesized and weresurrounded by transitional and heterogeneous landscapesacross elevations (Nicolas et al. 2011). Mercader et al. (2000)use data from phytolith analyses to suggest core areas wereless characteristic of singular forested blocks and more closelyresembled a variety of vegetation compositions including for-ests (e.g. Ituri forest), forest-grassland mosaics, and ‘parklandenvironments’. As such, shifts in habitat stability may have
had additional impacts on resources (e.g. prey distributionsand availability) and thereby may explain observed patternsof diversity in leopard haplotypes recovered from the DRC.
Distribution of CEA haplotypes were primarily restricted toequatorial Africa with the exceptions South Africa andZambia (Figures 2 and 3). South of equatorial Africa, theemergence of another dominant haplotype (H20) of the CSApopulation group was observed in both historical and con-temporary samples. With the exception of H27 (museumspecimen), all Mozambique samples were obtained fromleopards from the Niassa province between 1998 and 2008(Ropiquet et al. 2015). Two samples from Mozambique, H26(leo95) and H27 (M-186944), clustered with SA. Inclusion ofH26 (leo95) with haplotypes from South Africa is consistentwith Ropiquet et al. (2015). Locality data beyond country oforigin was not recorded for H27. South Africa representedthe most frequently sampled country in this analysis (n¼ 95).Clustering of the two samples from South Africa (leo101 andleo104) with haplotypes from Mozambique is also consistentwith Ropiquet et al. (2015). Our analysis also clustered amuseum specimen (M-81845) and seven contemporary leop-ard samples from South Africa with CEA haplotypes. Inclusionof South Africa samples with haplotypes of equatorial Africais curious. In some instances, clustering or admixture of indi-viduals from varying geographic origins can be explained bytranslocation (Bertola et al. 2015), though this is not likely tobe the explanation in all observed cases. Another possibleexplanation could be that H10, the ancestral haplotype ismore widely distributed than currently represented in thenetwork. With the exception of Mozambique and SouthAfrica, all other leopard range countries characterizing CSAare only represented by museum specimens. The ancestralhaplotype co-occurs in the DRC and may also occur inAngola, Botswana, and Namibia in which case, the clusteringof samples from South Africa with the predominant and mostwidespread haplotype in spanning the majority of Centraland East Africa is not unexpected. The Arabian leopard(P. p. nimr) in the Middle East, is geographically, the closestrecognized genetically independent lineage to the Africanleopard, and is proximal to Persian leopard (P. p. saxicolor) inSouthwest Asia. When sequences from these two subspeciesof Asiatic leopards were compared to African sequences itbecame evident that populations of leopards within Africaexhibit genetic differentiation at levels comparable to, and insome cases exceeding levels observed between Asiatic andAfrican leopards at this locus (Figure 4). FST[saxicolor-nimr]¼ 0.91, whereas, FST[WA-CSA]¼ 0.97 and FST[CWCA-CSA]¼ 0.96. Additionally, FST[WA-CWCA], FST[WA-CEA], andFST[CWCA-CEA] were very similar when compared to P. p.nimr, and P. p. saxicolor. The results of the AMOVA and subse-quent pairwise FST analyses with Arabian and Persian leopardsstrongly suggest evidence for genetically differentiated popu-lations and population structuring in the African leopard.
Our use of natural history collections and contemporarysamples demonstrates that leopards are more geneticallydiverse across Africa than previously indicated. Here we havepresented the results from mixed populations (historical andcontemporary) for descriptive purposes. Pairwise analyses con-ducted temporally between samples from the same
14 C. ANCO ET AL.
population found three of the population groups (CWCA, CSA,and SA) did not significantly differ between time periods(Table S7). However, it is difficult to draw meaningful conclu-sions from the temporal analyses due to small sample sizesassociated with historical (n¼ 2) and contemporary samples(n¼ 3) of the CWCA population, historical samples of the CSApopulation (n¼ 4), and historical samples of the SA population(n¼ 1). There were no historical samples included in the WApopulation. By combining historical and contemporary sam-ples within the same population, we held temporal changesconstant in our FST analyses, highlighting the influence ofgeography on population dynamics. Temporal partitioningrevealed a significant difference in the CEA population, butadditional contemporary samples from CEA countries mightmore closely resemble the pattern and diversity of haplotypesfound in historic CEA samples. Additional analyses withincreased sample size and geographic breadth from both his-torical and contemporary leopard populations to corroboratefindings should be a point of focus in further studies.
Central-East Africa and origin of the ancestral haplotype
The distribution and evolutionary patterns of African carni-vores during the Pleistocene remains a subject of continueddebate due to the rarity of fossils and incomplete records(Turner 1990; Turner 1999; Werdelin & Lewis 2005; Werdelinet al. 2010). Fossils of two pantherines representing primitivelion and leopard lineages were recovered from Laetoli(Tanzania) dating 3.8–3.4Ma (Werdelin & Lewis 2005; Werdelinet al. 2010). The first confirmed fossils of P. pardus in Africawere recovered from the Olduvai, Bed I site in Tanzania anddate back �2Ma to the Pleistocene (Werdelin & Lewis 2005;Werdelin et al. 2010). Using 3.5Ma and 2Ma as fossil dates forcalibration, Uphyrkina et al. (2001) estimated an African originfor the modern leopard between 470 and 825 Ka, but did notspeculate a geographic region of origination.
Present-day habitat conditions in Central and East Africaare dictated primarily by rainfall. South of the Sahara desertto the west and extending to Uganda moist, lush rainforestsare surrounded by savannas, wetlands, and deciduous wood-lands, while grasslands, savanna, and mixed open canopywoodlands are more characteristic of East Africa (Olson et al.2001; Steele 2007; Lorenzen et al. 2012; Riggio et al. 2013).Vegetation zones have undergone major shifts in climate andecosystem structure over the past several epochs with someregions offering refugium for biota during the Plio-Pleistocene (Steele 2007; Futuyma 2013; Demos et al. 2014).Moist pluvials of the Pleistocene expanded forested habitatsalong the equatorial belt fragmenting savanna-adapted spe-cies, while dry interpluvials reconnected these semi-arid land-scapes (Dupont 2011; Lorenzen et al. 2012).
Central and East Africa are considered areas of highendemism and speciation due to the repeated expansion andcontraction of forest and savanna habitats and active historyof geologic activity (Anthony et al. 2007; Diamond &Hamilton 2009; Tolley et al. 2011; Lorenzen et al. 2012). Thecombined phylogeographic signatures from these taxa,including lions (Bertola et al. 2015; Bertola et al. 2016) high-light Central and East Africa as regions of high diversity.
While the recovery of fossils from lion and leopard predeces-sors in East Africa indicate overlap between these earlier spe-cies (Werdelin et al. 2014), lions predominately inhabitsavanna ecosystems whereas leopards are more habitat gen-eralists. Leopards may have initially evolved alongside lions,but their adaptability likely enabled the leopard to persist onforest-adapted species during moist pluvials, while the distri-bution of lions would have been more dependent on thoseof savanna-adapted herbivores, which shifted to accommo-date changes in vegetation availability (Lorenzen et al. 2012).In the context of leopard phylogeography and in assigning apossible geographic origin to leopard diversity a dominanthaplotype in CEA, H10, contains the greatest number of net-work connections, has the largest confirmed geographic dis-tribution of all haplotypes, and connects all other clusteredpopulations (Figure 2). Collectively, these attributes suggestthat H10, and consequently, the larger geographic region ofCentral-East Africa is the likely origin of diversity in the ances-tral haplotype for the ND-5 locus in leopards.
Studying a widespread species spanning numerous politi-cal borders can present challenges to sample coverage andretrieval. The remoteness and physical geography of someregions (e.g. Ahaggar Massif, Algeria) can make accessibilitydifficult (Busby et al. 2009). Secondly, expenses and bureau-cratic difficulties associated with organizing expeditions andcollection can impede timely recovery of samples (Burrellet al. 2015). Political instability can prohibit sampling inregions of species’ extant range, due to restricted access andsafety concerns (Dudley et al. 2002). In addition to risks posedto researchers, prolonged political turmoil intensifies theextraction of natural resources (e.g. Sudan) leading to habitatloss and decimated wildlife populations (UNEP 2007). Lastly,rapid urbanization and growth of developing countries inAfrica has led to a 48–67% contraction in leopard range,localized extinction events (Mauritania, Togo, and Zanzibar),and questionable status in four countries (Burundi, Gambia,Lesotho, and Mali) (Jacobson et al. 2016). Archival specimenslike those in the collections of the American Museum ofNatural History, thus become highly valuable and potentially,the only sources to study population demographics of a spe-cies from observed and possible extinction events.
Genetic diversity is recognized as a vital component toensuring the long-term preservation and biodiversity of wild-life populations (Diversity 2010). Combining both temporaland spatial components to genetic analyses is necessary toaid conservation efforts. In doing so, wildlife managers canmake informed management decisions based on patterns ofhow gene flow and genetic diversity have changed over timeand space (Mondol et al. 2013; Sharma et al. 2013). Archivalcollections when used in conjunction with samples of extantleopard populations help us to fill in genetic gaps caused byanthropogenic disturbance of habitats and populations, illu-minate patterns of variation, and better understand the rolehistorical processes have in shaping biodiversity.
Conclusion
Archival specimens, like those in the collections of theAmerican Museum of Natural History (AMNH) have frequently
MITOCHONDRIAL DNA PART A 15
been used to infer relationships among historical populations(P€a€abo 1989; Hekkala et al. 2011; Caragiulo et al. 2014;Dowell et al. 2016). Leopard collections from AMNH providedbroad spatial coverage of Central, East, and Southern Africa,and we supplemented coverage in West and Central Africawith faecal samples collected during field surveys andSouthern Africa with sequences ported from NCBI GenBank.This work represents the most comprehensive mtDNA datasetof leopards comprising data from 182 wild individuals repre-senting sub-Saharan Africa. We reveal extensive, cryptic diver-sity in the ND-5 locus among historical populations andretention of independent genetic lineages in extant popula-tions. Distinct African leopard haplotypes are geographicallyclustered indicating African leopards represent several geneti-cally differentiated populations. Our findings generally agreewith Miththapala et al. (1996) and Uphyrkina et al. (2001) thatleopards harbour high levels of genetic diversity, but illustrateadditional evidence of regional structure within Africa.
The African leopard harbours a greater degree of geneticdiversity than previously indicated and is partitioned in a pat-tern providing strong support for significant genetic subdivi-sion. Our pairwise FST analyses using mtDNA revealed leopardpopulations throughout sub-Saharan Africa retain highlydivergent copies of the ND-5 locus on levels approaching,and in some instances exceeding, FST values observedbetween Asiatic populations (Arabian and Persian leopards)presently recognized by the IUCN as separate subspecies(Figure 4). AMOVA revealed population structuring indicatinga lack of gene flow between larger geographic regions (WestAfrica, Central-East/Central-Southern Africa, and SouthernAfrica) and among all the populations within regions. Twopopulations, CEA and CSA showed decreased pairwise differ-ences relative to other populations, which could be an arti-fact of decreased sampling. Lastly, the star-like phylogeny,widespread distribution, and connectedness of the H10 hap-lotype points to a likely origin of diversity for the ancestralhaplotype of this locus in Central and East Africa. We cautionthis work may not fully express the degree of genetic diver-sity present in African leopards, especially given samplingdeficiencies in North Africa, parts of West Africa, and inNortheastern Africa.
This study has raised important questions regarding thetaxonomic status of leopards in Africa. First, these findingssupport a distinction between African populations andArabian and Persian leopard populations. We found additionalstrong support for an East-West split in African leopards, whichmay correspond to previously hypothesized taxonomic group-ings (Figure 1, Table 1) and is congruent with numerous recentphylogeographic analyses of widespread African taxa(Moodley & Bruford 2007; Lorenzen et al. 2012; Dobigny et al.2013; Smitz et al. 2013; Bertola et al. 2016; Fennessy et al.2016). More sampling is needed to accurately delineate geo-graphic features acting as potential barriers to gene flow (e.g.Sanaga River in Central Cameroon), while a suture zone hasbeen identified between CWCA and CEA populations (Figures2 and 3). In addition, we have identified previously unrecog-nized levels of genetic diversity in historical collections ofAfrican leopards not represented in contemporary leopardpopulations. While only based on mtDNA, the reconstruction
of a haplotype network using novel samples of African leop-ards has reopened a >15-year-old conversation regardingAfrican leopard diversity and taxonomy. We acknowledge thatour results are limited by the use of mtDNA, and consequentlysingle locus data. We therefore, strongly recommend multi-locus sampling to investigate whether African leopards exhibitevidence of discordance between mitochondrial and nuclearmarkers (Toews & Brelsford 2012). These findings will providethe foundation for our ongoing analysis of temporal changesin phylogeographic patterns using sequence capture from his-torical collections, which will contribute to management andplanning strategies to conserve remaining genetic diversity inthe African leopard.
Acknowledgements
The authors thank Panthera, the Global Felid Genetics Program, andTorsten Bohm for sample contributions from Gabon, Nigeria, Senegal,and Republic of Congo. All samples were collected with the authorizationand assistance of the respective statutory wildlife authorities in thosecountries, for which we are extremely grateful. Special thanks to EileenWestwig, the Department of Mammalogy, and the American Museum ofNatural History. We are extremely grateful for the assistance of theSackler Institute for Comparative Genomics including Angelica MenchacaRodriguez, Ashley Yang, Melina Giakoumis, Stephen Gaughran, RebeccaHersch, Mohammad Faiz, Dr. Anthony Caragiulo, Dr. Claudia Wultsch, andDr. Mark Siddall.
Disclosure statement
The authors report no declarations of interest.Support for this work was provided by Fordham University, the
American Museum of Natural History, and the Wildlife ConservationSociety.
Sequence data generated in support for this publication have beendeposited to GenBank with accessions KY292222-77.
ORCID
Corey Anco http://orcid.org/0000-0001-8132-2009Sergios-Orestis Kolokotronis http://orcid.org/0000-0003-3309-8465
References
Alpers DL, Van Vuuren BJ, Arctander P, Robinson TJ. 2004. Populationgenetics of the roan antelope (Hippotragus equinus) with suggestionsfor conservation. Mol Ecol. 13:1771–1784.
Anthony NM, Johnson-Bawe M, Jeffery K, Clifford SL, Abernethy KA, TutinCE, Lahm SA, White LJT, Utley JF, Wickings EJ, et al. 2007. The role ofPleistocene refugia and rivers in shaping gorilla genetic diversity incentral Africa. Proc Natl Acad Sci USA. 104:20432–20436.
Antunes A, Troyer JL, Roelke ME, Pecon-Slattery J, Packer C, WinterbachC, Winterbach H, Hemson G, Frank L, Stander P, et al. 2008. The evolu-tionary dynamics of the lion Panthera leo revealed by host and viralpopulation genomics. PLoS Genet. 4:3–4.
Arctander P, Johansen C, Coutellec-Vreto MA. 1999. Phylogeography ofthree closely related African bovids (tribe Alcelaphini). Mol Biol Evol.16:1724–1739.
Aryal A, Kreigenhofer B. 2009. Summer diet composition of the CommonLeopard Panthera pardus (Carnivora: Felidae) in Nepal. J Threat Taxa.1:562–566.
16 C. ANCO ET AL.
Athreya V, Odden M, Linnell JDC, Krishnaswamy J, Karanth U. 2013. Bigcats in our backyards: persistence of large carnivores in a humandominated landscape in India. PLoS One. 8:2–9.
Avise JC. 2000. Phylogeography: the history and formation of species.Cambridge, MA: Harvard University Press.
Bah T. 2007. Inkscape: guide to a vector drawing program. Upper SaddleRiver, NJ, USA: Prentice Hall Press.
Bailey T. 1993. The African leopard: ecology and behavior of a solitaryfelid. New York: Columbia University Press.
Bandelt HJ, Forster P, R€ohl A. 1999. Median-joining networks for inferringintraspecific phylogenies. Mol Biol Evol. 16:37–48.
Barnett R, Yamaguchi N, Barnes I, Cooper A. 2006. The origin, currentdiversity and future conservation of the modern lion (Panthera leo).Proc Biol Sci. 273:2119–2125.
Barnett R, Yamaguchi N, Shapiro B, Ho SYW, Barnes I, Sabin R, WerdelinL, Cuisin J, Larson G. 2014. Revealing the maternal demographic his-tory of Panthera leo using ancient DNA and a spatially explicit genea-logical analysis. BMC Evol Biol. 14:70.
Bertola LD, van Hooft WF, Vrieling K, Uit de Weerd DR, York DS, Bauer H,Prins HHT, Funston PJ, Udo de Haes HA, Leirs H, et al. 2011. Geneticdiversity, evolutionary history and implications for conservation of thelion (Panthera leo) in West and Central Africa. J Biogeogr. 38:1356–1367.
Bertola LD, Jongbloed H, van der Gaag KJ, de Knijff P, Yamaguchi N,Hooghiemstra H, Bauer H, Henschel P, White PA, Driscoll CA, et al.2016. Phylogeographic patterns in Africa and high resolution delinea-tion of genetic clades in the lion (Panthera leo). Sci Rep. 6:30807.
Bertola LD, Tensen L, Van Hooft P, White PA, Driscoll CA, Henschel P,Caragiulo A, Dias-Freedman I, Sogbohossou EA, Tumenta PN, et al.2015. Autosomal and mtDNA markers affirm the distinctiveness oflions in West and Central Africa. PLoS One. 10:1–15.
Binladen J, Wiuf C, Gilbert MTP, Bunce M, Barnett R, Larson G,Greenwood AD, Haile J, Ho SYW, Hansen AJ, et al. 2006. Assessing thefidelity of ancient DNA sequences amplified from nuclear genes.Genetics. 172:733–741.
Bj€orklund M. 2003. The risk of inbreeding due to habitat loss in the lion(Panthera leo). Conserv Genet. 4:515–523.
Bowden R, MacFie TS, Myers S, Hellenthal G, Nerrienet E, Bontrop RE,Freeman C, Donnelly P, Mundy NI. 2012. Genomic tools for evolutionand conservation in the chimpanzee: Pan troglodytes ellioti is a genet-ically distinct population. PLoS Genet. 8:1–10.
Burrell AS, Disotell TR, Bergey CM. 2015. The use of museum specimenswith high-throughput DNA sequencers. J Hum Evol. 79:35–44.
Busby GBJ, Gottelli D, Wacher T, Marker L, Belbachir F, De Smet K,Belbachir-Bazi A, Fellous A, Belghoul M, Durant SM. 2009. Geneticanalysis of scat reveals leopard Panthera pardus and cheetah Acinonyxjubatus in southern Algeria. Oryx. 43:412.
Caragiulo A, Dias-Freedman I, Clark JA, Rabinowitz S, Amato G. 2014.Mitochondrial DNA sequence variation and phylogeography ofNeotropic pumas (Puma concolor). J DNA Mapping, Seq Anal.25:304–312.
Caro TM, Laurenson MK. 1994. Ecological and genetic factors in conserva-tion: a cautionary tale. Science (80-). 263:485–486.
Castro-Prieto A, Wachter B, Sommer S. 2011. Cheetah paradigm revisited:MHC diversity in the world's largest free-ranging population. Mol BiolEvol. 28:1455–1468.
Charruau P, Fernandes C, Orozco-Terwengel P, Peters J, Hunter L, Ziaie H,Jourabchian A, Jowkar H, Schaller G, Ostrowski S, et al. 2011.Phylogeography, genetic structure and population divergence time ofcheetahs in Africa and Asia: evidence for long-term geographic iso-lates. Mol Ecol. 20:706–724.
Cunningham SW, Shirley MH, Hekkala ER. 2016. Fine scale patterns ofgenetic partitioning in the rediscovered African crocodile, Crocodylussuchus (Saint-Hilaire 1807). PeerJ. 4:e1901.
Demos TC, Kerbis Peterhans JC, Agwanda B, Hickerson MJ. 2014.Uncovering cryptic diversity and refugial persistence among smallmammal lineages across the Eastern Afromontane biodiversity hot-spot. Mol Phylogenet Evol. 71:41–54.
Diamond AW, Hamilton AC. 2009. The distribution of forest passerinebirds and Quaternary climatic change in tropical Africa. J Zool.191:379–402.
Diversity C. 2010. The Strategic Plan for Biodiversity 2011-2020 and theAichi Biodiversity Targets. Nagoya, Aichi Prefecture, Japan.
Dobigny G, Tatard C, Gauthier P, Ba K, Duplantier JM, Granjon L, KergoatGJ. 2013. Mitochondrial and nuclear genes-based phylogeography ofArvicanthis niloticus (Murinae) and sub-saharan open habitats pleisto-cene history. PLoS One. 8:e77815.
Dowell SA, Hekkala ER. 2016. Divergent lineages and conserved niches:using ecological niche modeling to examine the evolutionary patternsof the Nile monitor (Varanus niloticus). Evol Ecol. 53:1–15.
Dowell SA, Portik DM, de Buffr�enil V, Ineich I, Greenbaum E, KolokotronisS-O, Hekkala ER. 2016. Molecular data from contemporary and histori-cal collections reveal a complex story of cryptic diversification in theVaranus (Polydaedalus) niloticus Species Group. Mol Phylogenet Evol.94:591–604.
Dubach JM, Briggs MB, White PA, Ament BA, Patterson BD. 2013. Geneticperspectives on “Lion Conservation Units” in Eastern and SouthernAfrica. Conserv Genet. 14:741–755.
Dudley JP, Ginsberg JR, Plumptre AJ, Hart JA, Campos LC. 2002. Effects ofWar and Civil Strife on Wildlife and Wildlife Habitats. Conserv Biol.16:319–329.
Dupont L. 2011. Orbital scale vegetation change in Africa. Quat Sci Rev.30:3589–3602.
Dutta T, Sharma S, Maldonado JE, Wood TC, Panwar HS, Seidensticker J.2013. Fine-scale population genetic structure in a wide-ranging carni-vore, the leopard (Panthera pardus fusca) in central India. Austin J, edi-tor. Divers Distrib. 19:760–771.
Eizirik E, Kim JH, Menotti-Raymond M, Crawshaw PG, O’Brien SJ, JohnsonWE. 2001. Phylogeography, population history and conservationgenetics of jaguars (Panthera onca, Mammalia, Felidae). Mol Ecol.10:65–79.
Excoffier L, Lischer HEL. 2010. Arlequin suite ver 3.5: A new series of pro-grams to perform population genetics analyses under Linux andWindows. Mol Ecol Resour. 10:564–567.
Excoffier L, Smouse PE, Quattro JM. 1992. Analysis of molecular varianceinferred from metric distances among DNA haplotypes: application tohuman mitochondrial DNA restriction data. Genetics. 131:479–491.
Farhadinia MS, Farahmand H, Gavashelishvili A, Kaboli M, Karami M,Khalili B, Montazamy S. 2015. Molecular and craniological analysis ofleopard, Panthera pardus (Carnivora: Felidae) in Iran: support for amonophyletic clade in Western Asia. Biol J Linn Soc. 114:721–736.
Fennessy J, Bidon T, Reuss F, Vamberger M, Fritz U, JankeCorrespondence A, Kumar V, Elkan P, Nilsson MA, Janke A. 2016.Multi-locus Analyses Reveal Four Giraffe Species Instead of One. CurrBiol. 26:1–7.
Futuyma DJ. 2013. Evolution. 3rd ed. Sunderland (MA): SinauerAssociates, Inc.
Gonder KM, Disotell TR. 2006. Contrasting Phylogeographic Histories ofChimpanzees in Nigeria and Cameroon: A Multi-Locus GeneticAnalysis. In: Primate Biogeogr. [place unknown]: Springer US; p.135–168.
Gonder MK, Disotell TR, Oates JF. 2006. New genetic evidence on theevolution of chimpanzee populations and implications for taxonomy.Int J Primatol. 27:1103–1127.
Haag T, Santos AS, Sana DA, Morato RG, Cullen L, Crawshaw PG, DeAngelo C, Di Bitetti MS, Salzano FM, Eizirik E. 2010. The effect of habi-tat fragmentation on the genetic structure of a top predator: Loss ofdiversity and high differentiation among remnant populations ofAtlantic Forest jaguars (Panthera onca). Mol Ecol. 19:4906–4921.
Hayward MW, Henschel P, O’Brien J, Hofmeyr M, Balme G, Kerley GIH.2006. Prey preferences of the leopard (Panthera pardus). J Zool.270:298–313.
Hekkala E, Shirley MH, Amato G, Austin JD, Charter S, Thorbjarnarson J,Vliet KA, Houck ML, Desalle R, Blum MJ. 2011. An ancient icon revealsnew mysteries: mummy DNA resurrects a cryptic species within theNile crocodile. Mol Ecol. 20:4199–4215.
Henschel P, Hunter LTB, Coad L, Abernethy KA, M€uhlenberg M. 2011.Leopard prey choice in the Congo Basin rainforest suggests exploita-tive competition with human bushmeat hunters. J Zool. 285:11–20.
Hofreiter M, Jaenicke V, Serre D, von Haeseler A, P€a€abo S. 2001. DNAsequences from multiple amplifications reveal artifacts induced by
MITOCHONDRIAL DNA PART A 17
cytosine deamination in ancient DNA. Nucleic Acids Res.29:4793–4799.
Hunter L, Henschel P, Ray JC. 2013. Panthera pardus. In: Kingdon JS,Hoffmann M, editors. The Mamm Africa. Amsterdam, the Netherlands:Academic Press.
Ishida Y, Oleksyk TK, Georgiadis NJ, David VA, Zhao K, Stephens RM,Kolokotronis S-O, Roca AL. 2011. Reconciling apparent conflictsbetween mitochondrial and nuclear phylogenies in African elephants.PLoS One 6:e20642.
Jacobson AP, Gerngross P, Lemeris JRJ, Schoonover RF, Anco C,Breitenmoser-Wursten C, Durant SM, Farhadinia MS, Henschel P,Kamler JF, et al. 2016. Leopard (Panthera pardus) status, distribution,and the research efforts across its range. PeerJ. 36:1–28.
Johnson M, Zaretskaya I, Raytselis Y, Merezhuk Y, McGinnis S, Madden TL.2008. NCBI BLAST: a better web interface. Nucleic Acids Res.36:W5–W9.
Kadu CAC, Schueler S, Konrad H, Muluvi GMM, Eyog-Matig O, Muchugi A,Williams VL, Ramamonjisoa L, Kapinga C, Foahom B, et al. 2011.Phylogeography of the Afromontane Prunus africana reveals a formermigration corridor between East and West African highlands. Mol Ecol.20:165–178.
Kawanishi K, Sunquist ME, Eizirik E, Lynam AJ, Ngoprasert D, WanShahruddin WN, Rayan DM, Sharma DSK, Steinmetz R. 2010. Near fixa-tion of melanism in leopards of the Malay Peninsula. J Zool.282:201–206.
Kimura M. 1980. A simple method for estimating evolutionary rates ofbase substitutions through comparative studies of nucleotide sequen-ces. J Mol Evol. 16:111–120.
Laguardia A, Kamler JF, Li S, Zhang C, Zhou Z, Shi K. 2017. The currentdistribution and status of leopards Panthera pardus in China. Oryx.51:153–159.
Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA,McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, et al. 2007.Clustal W and Clustal X version 2.0. Bioinformatics. 23:2947–2948.
Leigh JW, Bryant D. 2015. Popart: full-feature software for haplotype net-work construction. Methods Ecol Evol. 6:1110–1116.
Lorenzen ED, Heller R, Siegismund HR. 2012. Comparative phylogeogra-phy of African savannah ungulates. Mol Ecol 21:3656–3670.
Luo SJS, Kim JHJ, Johnson WWE, Walt J, Van Der Walt J, Martenson J,Yuhki N, Miquelle DG, Uphyrkina O, Goodrich JM, et al. 2004.Phylogeography and genetic ancestry of tigers (Panthera tigris). PLoSBiol. 2:e442.
McRae BH, Beier P, Dewald LE, Huynh LY, Keim P. 2005. Habitat barrierslimit gene flow and illuminate historical events in a wide-ranging car-nivore, the American puma. Mol Ecol. 14:1965–1977.
Measey GJ, Channing A. 2003. Phylogeography of the genus Xenopus insouthern Africa. Amphibia-Reptilia. 24:321–330.
Menegon M, Loader SP, Marsden SJ, Branch WR, Davenport TRB,Ursenbacher S. 2014. The genus Atheris (Serpentes: Viperidae) in EastAfrica: phylogeny and the role of rifting and climate in shaping thecurrent pattern of species diversity. Mol Phylogenet Evol. 79:12–22.
Menotti-Raymond M, O’Brien SJ. 1993. Dating the genetic bottleneck ofthe African cheetah. Proc Natl Acad Sci USA. 90:3172–3176.
Mercader J, Runge F, Vrydaghs L, Doutrelepont H, Ewango C, Juan-Tresseras J. 2000. Phytoliths from Archaeological Sites in the TropicalForest of Ituri, Democratic Republic of Congo. Quat Res. 54:102–112.
Mitchell D, Willerslev E, Hansen A. 2005. Damage and repair of ancientDNA. Mutat Res Mol Mech Mutagen. 571:265–276.
Miththapala S, Seidensticker J, O’Brien SJ. 1996. Phylogeographic subspe-cies recognition in leopards (Panthera pardus): Molecular genetic varia-tion. Conserv Biol. 10:1115–1132.
Mondol S, Bruford MW, Ramakrishnan U. 2013. Demographic loss, geneticstructure and the conservation implications for Indian tigers. Proc BiolSci. 280:1–10. doi: 10.1098/rspb.2013.0496.
Mondol S, Navya R, Athreya V. 2009. A panel of microsatellites to individ-ually identify leopards and its application to leopard monitoring inhuman dominated landscapes. BMC Genet. 10:79.
Moodley Y, Bruford MW. 2007. Molecular biogeography: Towards an inte-grated framework for conserving Pan-African biodiversity. PLoS One.2:e454.
Nicolas V, Missoup AD, Denys C, Kerbis Peterhans J, Katuala P, Couloux A,Colyn M. 2011. The roles of rivers and Pleistocene refugia in shapinggenetic diversity in Praomys misonnei in tropical Africa. J Biogeogr.38:191–207.
Nowell K, Jackson P. 1996. Wild cats. Status Survey and ConservationAction Plan. Gland, Switzerland: IUCN.
O’Brien S, Roelke M, Marker L, Newman A, Winkler C, Meltzer D, Colly L,Evermann J, Bush M, Wildt D. 1985. Genetic basis for species vulner-ability in the cheetah. Science (80-). 227:1428–1434.
O’Brien SJ, Martenson JS, Packer C, Herbst L, Devos V, Joslin P, Ottjoslin J,Wildt DE, Bush M. 1987. Biochemical genetic-variation in geographicisolates of African and Asiatic Lions. Natl Geogr Res. 3:114–124.
O’Brien SJ, Wildt DE, Goldman D, Merril CR, Bush M. 1983. The cheetah isdepauperate in genetic variation. Science. 221:459–462.
Odden M, Athreya V, Rattan S, Linnell J. 2014. Adaptable neighbours:movement patterns of GPS-collared leopards in human dominatedlandscapes in India. PLoS One. 9:e112044.
Olson DM, Dinerstein E, Wikramanayake ED, Burgess ND, Powell GVN,Underwood EC, D’amico J. a, Itoua I, Strand HE, Morrison JC, et al.2001. Terrestrial ecoregions of the world: a new map of life on earth.Bioscience. 51:933.
P€a€abo S. 1989. Ancient DNA: extraction, characterization, molecular clon-ing, and enzymatic amplification. Proc Natl Acad Sci USA.86:1939–1943.
Paabo S, Poinar H, Serre D, Svante P, Jaenicke-despr V, Hebler J, RohlandN, Kuch M, Krause J, Vigilant L, et al. 2004. Genetic analyses fromancient DNA. Annu Rev Genet. 38:645–679.
Packer C, Brink H, Kissui BM, Maliti H, Kushnir H, Caro T. 2011. Effects oftrophy hunting on lion and leopard populations in Tanzania. ConservBiol. 25:142–153.
Raza RH, Chauhan DS, Pasha MKS, Sinha S. 2012. Illuminating the blindspot: A study on illegal trade in leopard parts in India (2001-2010).New Delhi, India:TRAFFIC India/WWF India.
Riggio J, Jacobson A, Dollar L, Bauer H, Becker M, Dickman A,Funston P, Groom R, Henschel P, de Iongh H, et al. 2013. Thesize of savannah Africa: A lion’s (Panthera leo) view. BiodiversConserv. 22:17–35.
Ropiquet A, Knight AT, Born C, Martins Q, Balme G, Kirkendall L, Hunter L,Senekal C, Matthee CA. 2015. Implications of spatial genetic patternsfor conserving African leopards. Comptes Rendus Biol. 338:728–737.
Rostro-Garc�ıa S, Kamler JF, Ash E, Clements GR, Gibson L, Lynam A,McEwing R, Naing H, Paglia S. 2016. Endangered leopards: range col-lapse of the Indochinese leopard (Panthera pardus delacouri) inSoutheast Asia. Biol Conserv. 201:293–300.
Sharma S, Dutta T, Maldonado E, Wood C, Panwar HS, Seidensticker J,Maldonado JE, Wood TC. 2013. Forest corridors maintain historicalgene flow in a tiger metapopulation in the highlands of central India.Proc Biol Sci. 280:20131506.
Smitz N, Berthouly C, Corn�elis D, Heller R, van Hooft P, Chardonnet P,Caron A, Prins H, van Vuuren BJ, de Iongh H, et al. 2013. Pan-Africangenetic structure in the African Buffalo (Syncerus caffer): investigatingintraspecific divergence. PLoS One. 8:e56235.
Spalton JA, Al Hikmani HM. 2006. The leopard in the Arabian Peninsula –distribution and subspecies status. Cat News Special Issue 1 - ArabianLeopard: 4–8.
Steele TE. 2007. Late Pleistocene of Africa. In: Elias SA, editor.Encyclopedia Quat Sci. Amsterdam: Elsevier; p. 3139–3150.
Stein AB, Athreya V, Gerngross P, Balme G, Henschel P, Karanth U,Miquelle D, Rostro S, Kamler JF, Laguardia A. 2016. Panthera pardus.IUCN Red List Threat Species.
Stein AB, Hayssen V. 2013. Panthera pardus (Carnivora: Felidae). MammSpecies. 900:30–48.
Sunquist M, Sunquist F. 2002. Wild Cats of the World. Chicago (IL):University of Chicago Press.
Swanepoel LH, Lindsey P, Somers MJ, van Hoven W, Dalerum F. 2013.Extent and fragmentation of suitable leopard habitat in South Africa.Anim Conserv. 16:41–50.
Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. 2013. MEGA6:Molecular evolutionary genetics analysis version 6.0. Mol Biol Evol.30:2725–2729.
18 C. ANCO ET AL.
Toews DPL, Brelsford A. 2012. The biogeography of mitochondrial andnuclear discordance in animals. Mol Ecol. 21:3907–3930.
Tolley KA, Tilbury CR, Measey GJ, Menegon M, Branch WR, Matthee CA.2011. Ancient forest fragmentation or recent radiation? Testing refu-gial speciation models in chameleons within an African biodiversityhotspot. J Biogeogr. 38:1748–1760.
Turner A. 1990. The evolution of the guild of larger terrestrial carnivoresduring the Plio-Pleistocene in Africa. Geobios 23:349–368.
Turner A. 1999. Evolution in African Plio-Pleistocene mammalian fauna:correlation and causation. In: African Biogeogr Clim Chang early HumEvol. Oxford: Oxford University Press; p. 76–87.
UNEP. 2007. Sudan Post-Conflict Environmental Assessment. Nairobi, Kenya.Uphyrkina O, Johnson WE, Quigley H, Miquelle D, Marker L, Bush M,
O’Brien SJ. 2001. Phylogenetics, genome diversity and origin of mod-ern leopard, Panthera pardus. Mol Ecol. 10:2617–2633.
Wei L, Wu XB, Zhu LX, Jiang ZG. 2011. Mitogenomic analysis of the genusPanthera. Sci China Life Sci. 54:917–930.
Werdelin L, Lewis ME. 2005. Plio-Pleistocene Carnivora of eastern Africa:Species richness and turnover patterns. Zool J Linn Soc. 144:121–144.
Werdelin L, Lewis ME, Haile-Selassie Y. 2014. Mid-Pliocene Carnivora fromthe Woranso-Mille Area, Afar Region, Ethiopia. J Mamm Evol.21:331–347.
Werdelin L, Yamaguchi N, Johnson E, Brien SJO. 2010. Phylogeny andevolution of cats (Felidae). In: Biol Conserv Wild Felids. Vol. 12. Oxford:Oxford University Press; p. 59–82.
Won YJ, Hey J. 2005. Divergence population genetics of chimpanzees.Mol Biol Evol. 22:297–307.
Zimmermann J, Hajibabaei M, Blackburn DC, Hanken J, Cantin E, Posfai J,Evans TC. 2008. DNA damage in preserved specimens and tissue sam-ples: a molecular assessment. Front Zool. 5:18.
MITOCHONDRIAL DNA PART A 19
