Abstract
Mitochondrial introgression has been reported between red fox (Vulpes vulpes) and Rüppell’s fox (Vulpes rueppellii). While an evolutionary scenario of old divergence followed by recent mitochondrial introgression has been proposed, the directionality, prevalence, and timing of this event remain unclear. To further investigate this scenario, we analysed mitogenomes (n=85), including four newly generated red fox mitogenomes from Türkiye and a new Rüppell’s fox mitogenome from the United Arab Emirates, and partial mitochondrial DNA sequences (n=320) from both species across their ranges, including a newly comprehensive sampling in the Anatolian Peninsula (n=80). Our results are consistent with unidirectional mitochondrial introgression from the desert-adapted Rüppell’s fox into the generalist red fox, likely driven by climatic shifts promoting secondary contact and asymmetrical reproductive behaviour. Phylogenetic analysis unveiled two deeply divergent mitochondrial lineages of Rüppell’s fox sampled among red fox individuals: one distributed across Türkiye, Iran and Tunisia, and another restricted to Iran, suggesting that likely now-extinct (ghost) mitochondrial lineages may have introgressed into red foxes at least twice. Estimates of the time to the most recent common ancestor indicate that introgressed and contemporary Rüppell’s fox mitochondrial lineages diverged approximately 230 kya, predating the current intraspecific mitochondrial diversification of Rüppell’s fox lineages (~72 kya). This study highlights how comprehensive mitogenomic data and exhaustive regional surveys are critical for elucidating the complexity of introgression patterns in closely related canids.
Data availability
The datasets generated during the this study are available in the GenBank repository, under the accession numbers: PV802399–PV802404 and PV800565–PV800662.
References
Mallet, J. Hybridization as an invasion of the genome. Trends. Ecol. Evol. 20, 229–237 (2005).
Taylor, S. A. & Larson, E. L. Insights from genomes into the evolutionary importance and prevalence of hybridization in nature. Nat. Ecol. Evol. 3, 170–177 (2019).
Toews, D. P. L. & Brelsford, A. The biogeography of mitochondrial and nuclear discordance in animals. Mol. Ecol. 21, 3907–3930 (2012).
Currat, M., Ruedi, M., Petit, R. J. & Excoffier, L. The hidden side of invasions: Massive introgression by local genes. Evolution (N Y) 62, 1908–1920 (2008).
Petit, R. J. & Excoffier, L. Gene flow and species delimitation. Trends. Ecol. Evol. 24, 386–393 (2009).
Lehman, N. et al. Introgression of coyote mitochondrial DNA into sympatric North American gray wolf populations. Evolution 45, 104–119 (1991).
Mercure, A., Ralls, R. K., Koepfli, K. P. & Wayne, R. K. DNA differentiation of swift, kit and Arctic foxes. Evolution (N Y) 47, 1313–1328 (1993).
Koepfli, K. et al. Genome-wide evidence reveals that african and eurasian golden jackals are distinct species report genome-wide evidence reveals that african and eurasian golden jackals are distinct species. Curr. Biol. 25, 2158–2165 (2015).
von Holdt, B. M. et al. Whole-genome sequence analysis shows that two endemic species of North American wolf are admixtures of the coyote and gray wolf. Sci. Adv. 2, e1501714 (2016).
Gopalakrishnan, S. et al. Interspecific gene flow shaped the evolution of the genus Canis. Curr. Biol. 28, 3441-3449.e5 (2018).
Basuony, A. E., Saleh, M. & Hailer, F. Mitogenomic analysis of Rüppell’s fox (Vulpes rueppellii) confirms phylogenetic placement within the Palaearctic clade shared with its sister species, the red fox (Vulpes vulpes). Mitochondrial DNA. A. DNA Mapp. Seq. Anal. 0, 1–7 (2024).
Basuony, A. E. et al. Paraphyly of the widespread generalist red fox (Vulpes vulpes): introgression rather than recent divergence of the arid-adapted Rüppell ’ s fox (Vulpes rueppellii)?. Biol. J. Linnean Soc. 138, 453–469 (2023).
Geffen, E., Mercure, A., Girman, D. J., Macdonald, D. W. & Wayne, R. K. Phylogenetic relationships of the fox-like canids: Mitochondrial DNA restriction fragment, site and cytochrome b sequence analyses. J. Zool. 228, 27–39 (1992).
Leite, J. V., Álvares, F., Velo-Antón, G., Brito, J. C. & Godinho, R. Differentiation of North African foxes and population genetic dynamics in the desert—Insights into the evolutionary history of two sister taxa, Vulpes rueppellii and Vulpes vulpes. Org. Divers. Evol. 15, 731–745 (2015).
Hoffmann, M. & Sillero-Zubiri, C. Vulpes vulpes (amended version of 2016 assessment). The IUCN Red List of Threatened Species 2021: e.T23062A193903628. (2021).
Mallon, D., Murdoch, J. D. & Wacher, T. Vulpes rueppelli. The IUCN Red List of Threatened Species 2015: e.T23053A46197483 (2015).
Statham, M. J. et al. Range-wide multilocus phylogeography of the red fox reveals ancient continental divergence, minimal genomic exchange and distinct demographic histories. Mol. Ecol. 23, 4813–4830 (2014).
Geraads, D. A revision of the fossil Canidae (Mammalia) of North-western Africa. Palaeontology 54, 429–446 (2011).
Larivière, S. & Seddon, P. J. Vulpes rueppelli. Mamm. Species 678, 1–5 (2001).
Larivière, S. & Pasitschniak-Arts, M. Vulpes vulpes. Mamm. Species 537, 1–11 (1996).
Williams, J. B., Muñoz-Garcia, A., Ostrowski, S. & Tieleman, B. I. A phylogenetic analysis of basal metabolism, total evaporative water loss, and life-history among foxes from desert and mesic regions. J. Comp. Physiol. B 174, 29–39 (2004).
Rocha, J. L. et al. North-African fox genomes show signatures of repeated introgression and adaptation to life in deserts. Nat. Ecol. Evol. https://doi.org/10.1038/s41559-023-02094-w (2023).
Wang, M. S. et al. Ancient hybridization with an unknown population facilitated high-altitude adaptation of canids. Mol. Biol. Evol. 37, 2616–2629 (2020).
Lipshutz, S. E. Interspecific competition, hybridization, and reproductive isolation in secondary contact: missing perspectives on males and females. Curr. Zool. 64, 75–88 (2018).
Chan, K. M. A. & Levin, S. A. Leaky prezygotic isolation and porous genomes: Rapid introgression of maternally inherited DNA. Evolution (N Y) 59, 720–729 (2005).
Schuster, M. et al. The age of the sahara desert. Science (1979) 311, 821 (2006).
Masojć, M. et al. Saharan green corridors and Middle Pleistocene hominin dispersals across the Eastern Desert. Sudan. J Hum Evol 130, 141–150 (2019).
Ghane-Ameleh, S. et al. Mid-Pleistocene transition as a trigger for diversification in the Irano-Anatolian region: evidence revealed by phylogeography and distribution pattern of the eastern three-lined lizard. Glob. Ecol. Conserv. 31, e01839 (2021).
Sacks, B. N. et al. Pleistocene origins, western ghost lineages, and the emerging phylogeographic history of the red wolf and coyote. Mol. Ecol. 30, 4292–4304 (2021).
Edwards, C. J. et al. Ancient hybridization and an Irish origin for the modern polar bear matriline. Curr. Biol. 21, 1251–1258 (2011).
Rocha, J. L. et al. African climate and geomorphology drive evolution and ghost introgression in sable antelope. Mol. Ecol. 31, 2968–2984 (2022).
Andrews, S. FastQC: A quality control tool for high throughput sequence data. http://www.bioinformatics.babraham.ac.uk/projects/fastqc/ (2010).
Schubert, M., Lindgreen, S. & Orlando, L. AdapterRemoval v2: Rapid adapter trimming, identification, and read merging. BMC. Res. Notes. 9, 88 (2016).
Lopez Colom, R. & O’Brien, M. The genome sequence of the red fox, Vulpes vulpes (Linnaeus, 1758). Wellcome Open Res. 10, 13 (2025).
Li, H. Aligning sequence reads, clone sequences and assembly contigs with BWA-MEM. Preprint at arXiv http://arxiv.org/abs/1303.3997 (2013).
Li, H. et al. The sequence Alignment/Map format and SAMtools. Bioinformatics 25, 2078–2079 (2009).
Korneliussen, T. S., Albrechtsen, A. & Nielsen, R. ANGSD: Analysis of next generation sequencing data. BMC. Bioinformatics 15, 356 (2014).
Lucas, T., Vincent, B. & Eric, P. Translocation of mitochondrial DNA into the nuclear genome blurs phylogeographic and conservation genetic studies in seabirds. R Soc Open Sci 9 (2022).
Ronquist, F., van der Mark, P. & Huelsenbeck, J. P. Bayesian phylogenetic analysis using MrBayes. In The Phylogenetic Handbook: A Practical Approach to Phylogenetic Analysis and Hypothesis Testing (eds Lemey, P. et al.) 111–141 (Cambridge University Press, 2009).
Darriba, D., Taboada, G. L., Doallo, R. & Posada, D. jModelTest 2: More models, new heuristics and parallel computing. Nat. Methods. 9, 772 (2012).
Rambaut, A. FigTree v1.4.4. http://tree.bio.ed.ac.uk/software/figtree/ (2018).
Rozas, J. et al. DnaSP 6: DNA sequence polymorphism analysis of large data sets. Mol. Biol. Evol. 34, 3299–3302 (2017).
Leigh, J. W. & Bryant, D. PopART: full-feature software for haplotype network construction. Methods Ecol. Evol. 6, 1110–1116 (2015).
Kimura, M. A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J. Mol. Evol. 16, 111–120 (1980).
Kumar, S. et al. MEGA12: Molecular evolutionary genetic analysis version 12 for adaptive and green computing. Mol. Biol. Evol. 41, msae263 (2024).
Suchard, M. A. et al. Bayesian phylogenetic and phylodynamic data integration using BEAST 1.10. Virus Evol. 4, vey016 (2018).
Gernhard, T. The conditioned reconstructed process. J. Theor. Biol. 253, 769–778 (2008).
Drummond, A. J., Ho, S. Y. W., Phillips, M. J. & Rambaut, A. Relaxed phylogenetics and dating with confidence. PLoS Biol. 4, e88 (2006).
Perini, F. A., Russo, C. A. M. & Schrago, C. G. The evolution of South American endemic canids: a history of rapid diversification and morphological parallelism. J. Evol. Biol. 23, 311–322 (2010).
Bartolini Lucenti, S. & Madurell-Malapeira, J. Unraveling the fossil record of foxes: an updated review on the Plio-Pleistocene Vulpes spp. from Europe. Quat. Sci. Rev. 236, 106296 (2020).
De Bonis, L. et al. The oldest African fox (Vulpes riffautae n. sp., Canidae, Carnivora) recovered in late Miocene deposits of the Djurab desert, Chad. Naturwissenschaften 94, 575–580 (2007).
Zhao, C., Zhang, H., Liu, G., Yang, X. & Zhang, J. The complete mitochondrial genome of the Tibetan fox (Vulpes ferrilata) and implications for the phylogeny of Canidae. C. R. Biol. 339, 68–77 (2016).
Rambaut, A., Drummond, A. J., Xie, D., Baele, G. & Suchard, M. A. Posterior summarization in Bayesian phylogenetics using Tracer 1.7. Syst. Biol. 67, 901–904 (2018).
Acknowledgments
We are grateful to the Senior Editor, Dr. Francesco Nardi, and the two reviewers for valuable comments on the previous version of this manuscript.
Funding
RGR and RG were supported by the research contracts (https://doi.org/10.54499/2022.00505.CEECIND/CP1730/CT0002 and https://doi.org/10.54499/2022.07926.CEECIND/CP1730/CT0008, respectively) from the Portuguese Foundation for Science and Technology (FCT). İG and AAH were supported by the Scientific Research Coordination Unit of Ondokuz Mayıs University (No. PYO.FEN.1904.19.005). This work was funded by FCT through the GENCLIM project (https://doi.org/10.54499/2022.05474.PTDC).
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RGR, IG and RG conceived and designed the study; AH, SD, and IG were responsible for sampling in Turkey; AH and MMR conducted laboratory output; JLR contributed with the partial mitogenomic dataset; RGR and RG analyzed the data and wrote the first draft of the manuscript. All authors actively contributed and approved the final version of the manuscript, and agree to be held accountable for the content therein.
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Rocha, R.G., Hassan, A.A., Demirtaş, S. et al. Multiple introgression events from ghost Rüppell’s fox mitochondrial lineages into red fox.
Sci Rep (2026). https://doi.org/10.1038/s41598-026-45528-8
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DOI: https://doi.org/10.1038/s41598-026-45528-8
Keywords
- Anatolian Peninsula
- Canidae
- Ghost introgression
- Mitogenomes
- Phylogeny
Source: Ecology - nature.com
