Received: 27 March 2017 | Accepted: 6 August 2017 DOI: 10.1111/zsc.12257 ORIGINAL ARTICLE Deep divergence among mitochondrial lineages in African jackals Anagaw Atickem1,2 | Nils Chr. Stenseth2 | Marine Drouilly3 | Steffen Bock4 | Christian Roos5 | Dietmar Zinner6 1Cognitive Ethology Laboratory, Primate Genetics Laboratory, German Primate Recently, molecular analyses revealed that African and Eurasian golden jackals are Center (DPZ), Leibniz Institute for Primate distinct species. This finding suggests re- investigation of the phylogenetic relation- Research, Göttingen, Germany ships and taxonomy of other African members of the Canidae. Here, we provide a 2 Department of Biosciences, Centre for study on the phylogenetic relationship between populations of African jackals Ecological and Evolutionary Synthesis (CEES), University of Oslo, Oslo, Norway Lupulella mesomelas and L. adusta inferred from 962 bp of the mitochondrial cy- 3Department of Biological Sciences, tochrome b (cytb) gene. As expected from its disjunct distribution, with one popula- Institute for Communities and Wildlife in tion in eastern Africa and the other one in southern Africa, we found two mitochondrial Africa, University of Cape Town, lineages within L. mesomelas, which diverged about 2.5 million years ago (Ma). In Rondebosch, South Africa 4 contrast, in L. adusta with its more continuous distribution in sub-Saharan Africa, we Museum für Naturkunde Berlin, Berlin, Germany found only a shallower genetic diversification, with the exception of the West African 5Primate Genetics Laboratory, Gene Bank population, which diverged around 1.4 Ma from the Central and East African popu- of Primates, German Primate Center lations. Both divergence ages are older than, for example the 1.1–0.9 million years (DPZ), Leibniz Institute for Primate Canis lupus C. lupaster Research, Göttingen, Germany between the grey wolf and the African golden wolf . One 6Cognitive Ethology Laboratory, German taxonomic implication of our findings might be that the two L. mesomelas popula- Primate Center (DPZ), Leibniz Institute for tions warrant species status. However, genome-wide data with adequate geographi- Primate Research, Göttingen, Germany cal sampling are needed to substantiate our results. Correspondence Dietmar Zinner, Cognitive Ethology KEYWORDS Laboratory, German Primate Center (DPZ), Africa, Canidae, Canis, cytochrome b, Lupulella, mitochondrial DNA, phylogeny Leibniz Institute for Primate Research, Göttingen, Germany. Email: [email protected] Funding information Alexander von Humboldt-Foundation; The Rufford Foundation While endeavouring to throw some light into into the evolutionary history of many taxonomic groups in- the hopeless confusion the nomenclature of the cluding canids (e.g., Bardeleben, Moore, & Wayne, 2005; jackals of Africa is now in, I do not expect the vonHoldt et al., 2011, 2016; Lindblad-Toh et al., 2005; present communication to clear up all the dis- Wayne et al., 1997). In combination with the unified species putable points. concept of de Queiroz (2007), this approach has also led to De Winton (1899 p. 533). the discovery of cryptic species even within well-known gen- era such as Canis (Koepfli et al., 2015; Rueness et al., 2011). For instance, molecular analyses have shown that the African 1 | INTRODUCTION subspecies of the golden jackal C. aureus anthus is more closely related to the northern grey wolf (C. lupus) than to the The application of molecular methods in evolutionary biol- Eurasian golden jackal (C. aureus), and hence should be rec- ogy and population genetics has provided striking insights ognized as a distinct species within the genus Canis (Koepfli Zoologica Scripta. 2018;47:1–8. wileyonlinelibrary.com/journal/zsc © 2017 Royal Swedish Academy of Sciences | 1 2 | ATICKEM ET AL. et al., 2015; Viranta, Atickem, Werdelin, & Stenseth, 2017). It also became apparent that the genus Canis is a paraphyletic group, with the genera Lycaon (African wild dog) and Cuon (dhole) being more closely related to the wolf clade sensu lato (C. lupus including C. lupaster African golden wolf, C. la- trans Coyote, C. simensis Ethiopian wolf, C. aureus golden Ethiopia jackal and C. familiaris dog) than the African black- backed (C. mesomelas Schreber, 1775) and side- striped (C. adustus Sundevall, 1847) jackals (Lindblad- Toh et al., 2005; Wayne Guinea Benin & O’Brien, 1987). Because of this paraphyly, and as the two African jackal species fall significantly outside of the Canis Kenya Lupulella clade, Viranta et al. (2017) re-established the genus Angola Tanzania Hilzheimer, 1906, for these species. The question of the nomenclature and definition of Side-stripedjackal Black-backedjackal “lupaster” is far from being resolved as well as the ques- tion of whether Canis should encompass Lycaon, Cuon Namibia and Lupulella (Gippoliti & Groves, 2012; Gonzalez, 2012; Koepfli et al., 2015; Viranta et al., 2017). In this study, we 0 1,250 2,500 km S. Africa adopted the provisional taxonomy as proposed by Geraads (2011), Dinets (2015) and Viranta et al. (2017), that is Distribution map of African jackals (IUCN species Lupulella C. lupas- FIGURE 1 as a genus for the African jackals and distribution maps, Hoffmann, 2014a, 2014b). Arrows indicate ter for the African golden wolf (for a discussion of the taxo- countries from where samples or cytb sequences were available for the nomic implications, see Viranta et al., 2017). study The phylogenetic relationships among and within the two remaining African jackal species are not well resolved, partly because they have been used “only” as additional (Allen, 1939; Coetzee, 1977; Kingdon, 1997; Wozencraft, in- groups in phylogenetic analyses of canids, without a 2005). However, the validity of these taxa is questionable proper geographic sampling of the two species (Bardeleben (Loveridge & Macdonald, 2013; Sillero- Zubiri, 2009). et al., 2005; Koepfli et al., 2015; Lindblad- Toh et al., Lupulella mesomelas is found in southern Africa as well 2005; Wayne et al., 1997; Zrzavý & Řičánková, 2004) or, as in north- eastern Africa and the Horn of Africa (Figure 1) if the focus of a study, their sampling was geographically (Loveridge & Nel, 2013). Its two areas of occurrence are confined to Kenya and northern Tanzania (Wayne et al., separated by a distribution gap of 1,000 km from central 1990). Previous analyses based on nuclear markers sug- Tanzania to central Zimbabwe (Ansell, 1960; Loveridge & gested a common ancestry for L. adusta and L. mesome- Macdonald, 2002; Loveridge & Nel, 2013). Five subspecies las (Bardeleben et al., 2005; Lindblad- Toh et al., 2005), have been described (Coetzee, 1977), but given the disjunct whereas in analyses of mitochondrial markers, both jackal distribution of L. mesomelas, these were later reduced to only species did not form a monophyletic clade (Bardeleben two, the Cape jackal L. m. mesomelas in southern Africa et al., 2005; Koepfli et al., 2015; Viranta et al., 2017; and the East African jackal L. m. schmidti in eastern Africa Wayne et al., 1997; Zrzavý & Řičánková, 2004). When (Kingdon, 1997; Walton & Joly, 2003). comparing the genetic variation within the two species, So far no comprehensive study has been carried out to we expect contrasting phylogeographical signals due to the determine the genetic variation and phylogenetic relation- different distribution patterns of L. mesomelas (disjunct) ships between species and populations of African jackals. and L. adusta (continuous). But as de Winton nicely phrased it in 1899 (p. 533), we Lupulella adusta is found in large parts of sub- Saharan do not expect to clear up all the disputable points about Africa (Figure 1) from Senegal in the West to Eritrea in the the phylogeny and taxonomy of African Canini. We aim to East and South through East Africa to northern South Africa contribute the first data to close the gap in our knowledge and Angola (Hoffmann, 2014a; Loveridge & Macdonald, about the evolutionary history of canids by reconstructing 2013). While most authors consider a white stripe from the phylogenetic relationships among mitochondrial lin- elbow to hip and black side stripes as the most important eages of African jackals. We therefore generated sequence features in distinguishing the species as its name indicates data of a 962- bp- long fragment of the mitochondrial cyto- (Walton & Joly, 2003), this is not always conspicuous. Given chrome b (cytb) gene from modern and historic Lupulella the large distribution range of L. adusta, geographic varia- specimens and compared them with orthologous data from tion is expected and several subspecies have been proposed related species. ATICKEM ET AL. | 3 2 | MATERIALS AND METHODS predenaturation step at 95°C for 10 min, followed by 40–50 cycles, each with denaturation at 95°C for 30 s, annealing at 2.1 | Sample collection 50°C for 30 s and extension at 72°C for 30 s (museum sam- ples) or 60 s (modern tissue samples). At the end, a final We collected samples from 15 L. mesomelas individu- extension step at 72°C for 7 min was added. PCR perfor- als (three modern tissue samples from South Africa and 12 mance was checked on 1% agarose gels, and PCR products museum samples from Kenya, Tanzania and Namibia) and were excised from the gel and purified with the QIAquick 13 L. adusta samples (nine faecal samples from Ethiopia and Gel Extraction Kit (Qiagen). Sequencing was conducted four museum samples from Angola, Tanzania and Namibia) on an ABI 3130xL sequencer (Applied Biosystems) using (Table S1). The museum