A golden jackal (Canis anthus) from Serengeti National Park, Tanzania. Based on genomic results, the researchers suggest this animal be referred to as the African golden wolf, which is distinct from the Eurasian golden jackal (Canis aureus). Credit: D. Gordon E. Robertson Despite their remarkably similar appearance, the "golden jackals" of East Africa and Eurasia are actually two entirely different species. The discovery, based on DNA evidence and reported in the Cell Press journal Current Biology on July 30, increases the overall biodiversity of the Canidae—the group including dogs, wolves, foxes, and jackals—from 35 living species to 36. 
 
 "This represents the first discovery of a 'new' canid species in Africa in over 150 years," says Klaus-Peter Koepfli of the Smithsonian Conservation Biology Institute in Washington, DC. 
 
 The new study, led by Koepfli and Robert Wayne of the University of California, Los Angeles, was inspired by recent reports suggesting that the African golden jackal was actually a cryptic subspecies of gray wolf. Those studies were based on an analysis restricted to mitochondrial DNA, which is passed along via the maternal lineage. 
 
 To expand the DNA evidence in the new study, Wayne retrieved DNA samples of golden jackals collected two decades ago in Kenya from his laboratory freezers. Koepfli and Wayne also established collaborations with colleagues, who provided them with samples from golden jackals in other parts of Africa and Eurasia. That genome-wide DNA evidence told a different story of the canids' evolutionary past. 
 
 "To our surprise, the small, golden-like jackal from eastern African was actually a small variety of a new species, distinct from the gray wolf, that has a distribution across North and East Africa," Wayne says. The researchers have named this previously unrecognized species the African golden wolf. 
 
 Koepfli and Wayne suspect that zoologists had mistaken African and Eurasian golden jackals for the same species because of a high degree of similarity in their skull and tooth morphology. However, the genetic data supports the idea that they are in fact two separate lineages that have been evolving independently for at least a million years. In fact, the new canid family tree suggests that these two lineages aren't even closely related. The African species is more closely related to the lineage leading to gray wolves and coyotes than jackals, which explains their new designation as African golden wolves. 
 
 A golden jackal (Canis aureus) from Israel. Based on genomic results, the researchers suggest this animal, the Eurasian golden jackal, is distinct from Canis anthus, which they propose be referred to as the African golden wolf. Credit: Eyal Cohen 
 
 The findings come as a reminder that "even among well-known and widespread species such as golden jackals, there is the potential to discover hidden biodiversity," with the help of genomic evidence, Koepfli says. The researchers say they will continue to study the relationships among golden jackal and wolf lineages in Africa, Eurasia, and the Middle East. 
 
 Explore further: The cryptic African wolf: Canis aureus lupaster is not a golden jackal 
 
 More information: Current Biology, Koepfli and Pollinger et al., "Genome-wide Evidence Reveals that African and Eurasian Golden Jackals Are Distinct Species" dx.doi.org/10.1016/j.cub.2015.06.060 
 
 ||||| Abstract The recent discovery of a lineage of gray wolf in North-East Africa suggests the presence of a cryptic canid on the continent, the African wolf Canis lupus lupaster. We analyzed the mtDNA diversity (cytochrome b and control region) of a series of African Canis including wolf-like animals from North and West Africa. Our objectives were to assess the actual range of C. l. lupaster, to further estimate the genetic characteristics and demographic history of its lineage, and to question its taxonomic delineation from the golden jackal C. aureus, with which it has been considered synonymous. We confirmed the existence of four distinct lineages within the gray wolf, including C. lupus/familiaris (Holarctic wolves and dogs), C. l. pallipes, C. l. chanco and C. l. lupaster. Taxonomic assignment procedures identified wolf-like individuals from Algeria, Mali and Senegal, as belonging to C. l. lupaster, expanding its known distribution c. 6,000 km to the west. We estimated that the African wolf lineage (i) had the highest level of genetic diversity within C. lupus, (ii) coalesced during the Late Pleistocene, contemporaneously with Holarctic wolves and dogs, and (iii) had an effective population size of c. 80,000 females. Our results suggest that the African wolf is a relatively ancient gray wolf lineage with a fairly large, past effective population size, as also suggested by the Pleistocene fossil record. Unique field observations in Senegal allowed us to provide a morphological and behavioral diagnosis of the African wolf that clearly distinguished it from the sympatric golden jackal. However, the detection of C. l. lupaster mtDNA haplotypes in C. aureus from Senegal brings the delineation between the African wolf and the golden jackal into question. In terms of conservation, it appears urgent to further characterize the status of the African wolf with regard to the African golden jackal. 
 
 Citation: Gaubert P, Bloch C, Benyacoub S, Abdelhamid A, Pagani P, Djagoun CAMS, et al. (2012) Reviving the African Wolf Canis lupus lupaster in North and West Africa: A Mitochondrial Lineage Ranging More than 6,000 km Wide. PLoS ONE 7(8): e42740. https://doi.org/10.1371/journal.pone.0042740 Editor: Sergios-Orestis Kolokotronis, Barnard College, Columbia University, United States of America Received: December 8, 2011; Accepted: July 12, 2012; Published: August 10, 2012 Copyright: © Gaubert et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: PG received funding for lab work and field surveys from Action Transversale “Biodiversité actuelle et fossile” and Société des Amis du Muséum National d’Histoire Naturelle et du Jardin des Plantes (MNHN, Paris, France). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. 
 
 Materials and Methods Observations of wolf-like canids (hereafter referred to as Canis sp.) living at the near periphery of packs of golden jackals (Canis aureus) were made in July 2011 by CB, twelve km east from Parc National du Djoudj, in northern Senegal. At this site, Canis sp. individuals were distinctively larger and darker than the golden jackals (Fig. 1). They also behaved differently, presenting solitary and shy demeanors (see Discussion). Hair samples were collected from a wolf-like individual at one of the observation sites. The samples were gathered at a specific resting site right after the animal left. We also collected hair samples from one golden jackal individual from the same observation site and a fourth generation-captive bred specimen originating from a wild-born Senegalese golden jackal pack (Zoo du Parc de Hann, Dakar). Six additional tissue samples of Canis sp. were obtained from the coastal region of northeastern Algeria and Adrar des Iforas, eastern Mali (Fig. 1). To improve the accuracy of phylogenetic assignment among African Canis, we also sampled tissues of side-striped jackals (Canis adustus) from western Africa (Table 1). DNA from hair and tissue samples was extracted using a standard CTAB extraction protocol [35] and an ABI PRISM© 6100 Nucleic Acid PrepStation (Applied Biosystems, Carlsbad, CA) following manufacturer’s recommendations, respectively. Two mtDNA fragments from the cytochrome b (CYTB; 402 bp) and control region (CR; 287–337 bp) were amplified by PCR, using the primer pairs GVL14724-H15149 [36] and CR1F-CR2R [37], respectively. PCR amplification protocols followed Gaubert et al. [36]. PCR products were visualized on a 1.5% agarose gel, purified and directly sequenced in both directions on 3730xl DNA Analyzer 96-capillary sequencers (Applied Biosystems, Foster City, CA) at Genoscope (Evry, France) and Eurofins MWG Operon (Ebersberg, Germany). All the sequences produced from this study were deposited in Genbank under the accession numbers JQ088650–JQ088665 and JQ088669–JQ088684 (Table 1). Nucleotide sequence alignments were preliminarily reconstructed using Clustal X 2.1 [38] and completed with BioEdit 7.0.9 [39]. The taxonomic assignment of the newly generated sequences of African Canis was done using a Bayesian framework, assessing sequence assignment through the Statistical Assignment Package 1.0.6. (SAP; [40]). The latter statistically assesses the level of confidence of sequence assignment by using a Bayesian phylogenetic reconstruction among a set of similarity-selected nucleotide sequences [40]. Consequently, the accuracy of the method is greater when (i) the genetic and geographic diversity of the taxa under study is well represented in Genbank, and (ii) the genetic diversity is structured into well-supported clades. We determined the minimum identity parameter to be 0.9, which corresponds to the lower bound (90%) of the level of similarity among retrieved sequences. We ran a phylogenetic analysis of the wolf-like clade using the Bayesian Markov Chain Monte Carlo (MCMC) procedure implemented in BEAST v. 1.6.2 [41]. CYTB and CR were arranged into a single ‘coherent’ segment, to which we applied the model HKY + I + Γ [42] as selected by jModelTest v. 0.1 with the AIC and BIC criteria [43]. The sample set was restricted to our generated sequences and a sub-sample of the wolf-like clade representatives for which this coherent segment (CYTB + CR) was covered in Genbank. We did not use outgroups since BEAST samples the root position (along with the rest of the tree nodes). We assumed a constant size coalescent model [41]. Operators were tuned manually after screening of effective sample size values from preliminary runs, to maximize their efficiency and obtain convergence in poorly estimated parameters [44]. Chain lengths were 100,000,000, sampled every 10,000 generations. Analyses were run twice independently. Log files were concatenated under LogCombiner 1.6.2 [44] with a final burn-in of 1,000. Convergence and stability of estimated parameters were checked using Tracer 1.5 [45]. In order to assess the jackal – wolf haplogroup relationships in a comprehensive framework, we built a median-joining network from the CYTB sequences representing Canis lupus and the three jackal species. We did not include the CR sequences in our network analysis because only one species of jackals was represented in GenBank (Canis aureus from Eurasia). All the CYTB sequences representing gray wolves and jackals that aligned with our CYTB fragment with a reasonable amount of nucleotide coverage (>75%) were downloaded from Genbank, resulting in datasets of 346 (CYTB) (alignment available upon request to the corresponding author). We used Network 4.6.0.0 (http://www.fluxus-engineering.com), with ε fixed to 0 in order to minimize alternative median networks. We used MEGA5 [46] to calculate the CYTB mean distances among the main phylogenetic lineages within the wolf-like clade, using the Kimura 2-parameter model (K2P) in order to provide estimates referable to the framework of the Genetic Species Concept [47], [48]. All positions with less than 95% site coverage were eliminated before analysis. Standard error was estimated by a bootstrap procedure (500 replicates). We used DnaSP 5.10 [49] to calculate the number of polymorphic sites (S), nucleotide diversity (π), number of haplotypes (h) and haplotype diversity (Hd) among the four main mtDNA lineages of Canis lupus found in our analyses (lupus/familiaris, pallipes, chanco and lupaster; see Results). Since DnaSP automatically considers missing data and indels as unique features, we removed nucleotide sequences and/or alignment blocks containing missing data and indels before analysis [36]. We calculated time to most recent common ancestor (TMRCA) in the four Canis lupus lineages independently, following the same analytical procedure in BEAST as described above. We used the rough evolutionary rate of 5.48% per million years estimated for the mitochondrial fragment encompassing CYTB to CR in canids [50], under a strict clock model. Given the straightforward calculation of this evolutionary rate, estimates derived from the latter should be regarded with some level of cautiousness. We used LAMARC 2.1.6 [51] to estimate the parameter Θ in the four Canis lupus lineages with a coalescent, maximum likelihood approach using a Markov Chain Monte Carlo (MCMC) genealogy sampler. CYTB and CR were again arranged into a single ‘coherent’ segment. We used the F84 model of evolution [52] and ran two simultaneous searches twice, including 1,000,000 steps for the heating scheme representing the 10 initial chains and 20,000,000 steps for the two final chains (burn-in = 10,000 steps). Percentage profile likelihoods were used to calculate the 95% confidence interval for Θ. From the relationship Θ = 2N ef mu (where N ef is the effective population size of females and mu is the mutation rate expressed as number of mutations per site per generation), we derived N ef from the evolutionary rate of Li et al. [50] (see above), fixing the generation time to three years [5]. 
 
 Acknowledgments We thank the following persons for contributing to the sampling effort: Séverin Tchibozo (Centre de Recherche pour la Gestion de la Biodiversité, Cotonou, Benin), Géraldine Veron and Raphaël Cornette (Muséum National d’Histoire Naturelle, Paris, France), and Gauthier Dobigny (Institut de Recherche pour le Développement, Montpellier, France). We are grateful to Baba Sarr (Direction des eaux et forêts et de la chasse) for giving permission to carry out fieldwork in Senegal, and to Lamine Gueye (Parc de Hann, Dakar) and René Bancal (Ranch de Bango, Saint-Louis) for providing logistical support. Laboratory work was supported by the ‘Consortium National de Recherche en Génomique’, and the ‘Service de Systématique Moléculaire’ of the Muséum National d’Histoire Naturelle (CNRS - UMS 2700). It is part of agreement 2005/67 between Genoscope and the Muséum National d’Histoire Naturelle on the project “Macrophylogeny of life” directed by Guillaume Lecointre. We thank Joseph Brennan, Mike Hoffmann and three anonymous reviewers for their useful comments on an earlier draft of the manuscript. 
 
 Author Contributions Conceived and designed the experiments: PG CB SD. Performed the experiments: PG CB SB AA PP CAMSD AC. Analyzed the data: PG CB. Contributed reagents/materials/analysis tools: PG CB AC SD. Wrote the paper: PG. ||||| View Images Africa's golden jackal (pictured, an animal in Tanzania's Ngorongoro Conservation Area) is actually a type of wolf. Photograph by Jason Edwards, National Geographic Creative 
 
 Jackals, the tricksters of traditional folklore , have fooled us yet again. 
 
 The golden jackal, which lives in East Africa and Eurasia, is actually two distantly related species—and one of them is a new species of wolf, a new study says. (Also see " Wolves Identified by Unique Howls, May Help Rare Species .") 
 
 Dubbed the African golden wolf, it's the first new species of canid—a group that includes wolves, coyotes , and jackals—discovered in 150 years. Africa is also home to two other wolf species, the gray wolf and Ethiopian wolf. ( Read "Africa's Last Wolves" in National Geographic magazine .) 
 
 Though golden jackals look mostly the same—the Eurasian animals are slightly smaller than the African ones, with a narrower skull and slightly weaker teeth—in-depth analysis of their DNA revealed two species that have evolved separately for millennia. 
 
 "I was very surprised," said study leader Klaus-Peter Koepfli , a biologist with the Smithsonian Conservation Biology Institute in Front Royal, Virginia. 
 
 Koepfli proposes renaming the African golden jackal the African golden wolf (Canis anthus), while retaining the original species name for the Eurasian golden jackal (C. aureus). 
 
 Sniffing Out Species 
 
 Scientists have wondered if golden jackals were more than one species for years. 
 
 Gaubert used snippets of jackal mitochondrial DNA, which is passed on by mothers, for his analysis. ( Watch a video of gray wolves hunting .) 
 
 Koepfli found these results interesting, and wanted to verify them using more samples from a broader geographic area and more data from across both the jackal and gray wolf genomes. 
 
 In doing so, he expected to replicate Gaubert's earlier work—but that's not what happened. 
 
 By examining 38 different genetic markers of 128 canid specimens—including golden jackals from Kenya, North Africa, and Eurasia; African gray wolves; and domestic dogs—Koepfli confirmed that African and Eurasian golden jackals are two separate species. Yet he also discovered that the African golden jackal is not a gray wolf subspecies. 
 
 Jackal vs. Ostrich Eggs vs. Vulture A 3-pound (1.4-kilogram) ostrich egg is a tough meal to crack—unless you're a tool-wielding vulture with just the right technique. 
 
 
 
 
 
 Instead, he discovered the African golden jackal is a new wolf species of its own, and that this species and the Eurasian golden jackal are distant cousins, having last shared an ancestor about a million years ago. The research appeared July 30 in the journal Current Biology . 
 
 "Airtight Case" 
 
 Gaubert stands by his original work, saying that although he finds the new study to be high-quality work, he isn't yet convinced that the African golden wolf is a new species. For instance, he says scientists have yet to tease apart some conflicting results in the DNA analyses. 
 
 "There's still a lot of work to be done," he said. 
 
 Greger Larson , a bioarchaeologist at the University of Oxford in the U.K., is convinced by the new research. 
 
 "They have phenomenal data and they do a nice series of analyses. It's a super airtight case," Larson said. (See National Geographic's gray wolf pictures.) 
 
 So why do the African golden wolf and the Eurasian jackal look the same if they're distantly related? 
 
 Study leader Koepfli says that the same evolutionary pressures likely influenced the animals' evolution. For instance, the canids' harsh desert habitats could have led to their small, lean bodies and light coats, which don't absorb as much sunlight. 
 
 "We're finding that the genetic information can tell us a very different story about animals," Koepfli said. 
 
 Follow Carrie Arnold on Twitter .
– Africa, home to the Ethiopian wolf and the gray wolf, can now lay claim to the African golden wolf—the first new species of canid (which also encompasses jackals, foxes, and coyotes) to be discovered in 150 years, reports National Geographic. The new species has long been misunderstood to be a golden jackal, which lives in both Eurasia and East Africa. But a new DNA analysis reveals that in spite of their convincingly similar appearances, they've evolved over millennia into two distinct species, and that the East African jackal is actually a new type of wolf. "I was very surprised," study leader Klaus-Peter Koepfli says. The finding, however, isn't entirely without precedent. In 2012, a French biologist reported in PLoS ONE that an analysis of mitochondrial (as opposed to nuclear) DNA suggested the African golden jackal may actually be a subspecies of gray wolf separate from its Eurasian doppelganger. But rather than arrive at the same conclusion per his expectations, Koepfli discovered that Africa's golden jackal is a new wolf species altogether, the so-called golden wolf. It appears to have parted genetic ways from the Eurasian jackal about a million years ago, he reports this week in the journal Current Biology, even though the two look so remarkably similar. That shared form was perhaps facilitated by "intense competition from a uniquely diverse carnivoran community including species larger and smaller than jackals, thus inhibiting size divergence." While his findings need to be confirmed, some call it an "airtight" case, and thus the number of living Canidae species could go from 35 to 36, reports PhysOrg. (Another species is down to its final four members.)