Skip to main content Skip to main navigation menu Skip to site footer
Responsive image
Issue: Vol. 4455 No. 1: 1 Aug. 2018
Type: Article
Published: 2018-08-01
DOI: 10.11646/zootaxa.4455.1.3
Page range: 68–98
Abstract views: 868
PDF downloaded: 416

Integration of nuclear and mitochondrial gene sequences and morphology reveals unexpected diversity in the forest cobra (Naja melanoleuca) species complex in Central and West Africa (Serpentes: Elapidae)

Molecular Ecology and Fisheries Genetics Laboratory, School of Biological Sciences, Bangor University, Bangor LL57 2UW, United Kingdom.
14 Rue des Roses, 06130 Grasse, France.
Laboratoire de Paludologie et Zoologie médicale, Institut de Recherche pour le Développement (IRD), MIVEGEC, B.P. 1386, Dakar, Sénégal.
Institut Systématique Evolution Biodiversité (ISYEB), Muséum national d'Histoire naturelle, CNRS, Sorbonne Universités, EPHE, 57 rue Cuvier, CP 30, 75005 Paris, France.
Department of Biology, Whitman College, 345 Boyer Avenue, Walla Walla, WA 99362, USA.
Department of Biological Sciences, University of Texas at El Paso, 500 W University Avenue, El Paso, TX 79968, USA.
Department of Biological Sciences, University of Texas at El Paso, 500 W University Avenue, El Paso, TX 79968, USA.
Laboratoire d’Herpétologie, Département de Biologie, Centre de Recherche en Sciences Naturelles, Lwiro, DR Congo.
Hielscherstr. 25, D-13158 Berlin, Germany.
Molecular Ecology and Fisheries Genetics Laboratory, School of Biological Sciences, Bangor University, Bangor LL57 2UW, United Kingdom.
Molecular Ecology and Fisheries Genetics Laboratory, School of Biological Sciences, Bangor University, Bangor LL57 2UW, United Kingdom.
Molecular Ecology and Fisheries Genetics Laboratory, School of Biological Sciences, Bangor University, Bangor LL57 2UW, United Kingdom.
Molecular Ecology and Fisheries Genetics Laboratory, School of Biological Sciences, Bangor University, Bangor LL57 2UW, United Kingdom. Evolutionary and Adaptive Genomics Group, Institute for Biochemistry and Biology, University of Potsdam, Karl-Liebknecht-Str. 24-25, Haus 29, 14476 Potsdam (Golm), Germany.
Natural History Museum of Zimbabwe, PO Box 240, Bulawayo, Zimbabwe.
Integrative taxonomy Africa Naja melanoleuca Naja guineensis sp. nov. Naja savannula sp. nov. Elapidae systematics Reptilia

Abstract

Cobras are among the most widely known venomous snakes, and yet their taxonomy remains incompletely understood, particularly in Africa. Here, we use a combination of mitochondrial and nuclear gene sequences and morphological data to diagnose species limits within the African forest cobra, Naja (Boulengerina) melanoleuca. Mitochondrial DNA sequences reveal deep divergences within this taxon. Congruent patterns of variation in mtDNA, nuclear genes and morphology support the recognition of five separate species, confirming the species status of N. subfulva and N. peroescobari, and revealing two previously unnamed West African species, which are described as new: Naja (Boulengerina) guineensis sp. nov. Broadley, Trape, Chirio, Ineich & Wüster, from the Upper Guinea forest of West Africa, and Naja (Boulengerina) savannula sp. nov. Broadley, Trape, Chirio & Wüster, a banded form from the savanna-forest mosaic of the Guinea and Sudanian savannas of West Africa. The discovery of cryptic diversity in this iconic group highlights our limited understanding of tropical African biodiversity, hindering our ability to conserve it effectively.

References

  1. Arévalo, E., Davis, S.K. & Sites, J.W. (1994) Mitochondrial DNA sequence divergence and phylogenetic relationships among eight chromosome races of the Sceloporus grammicus complex (Phrynosomatidae) in Central Mexico. Systematic Biology, 43, 387–418.
    https://doi.org/10.1093/sysbio/43.3.387

    Avise, J.C. (2000) Phylogeography. Harvard University Press, Cambridge, Massachusetts, 447 pp.

    Babik, W., Branick, W., Crnobrnja-Isailović, J., Colgălniceanu, D., Sas, I., Olgun, K., Poyarkov, N.A. Garcia-París, M. & Arntzen, J.W. (2005) Phylogeography of two European newt species—discordance between mtDNA and morphology. Molecular Ecology, 14, 2475–2491.
    https://doi.org/10.1111/j.1365-294X.2005.02605.x

    Barlow, A., Pook, C.E., Harrison, R.A. & Wüster, W. (2009) Co-evolution of diet and prey-specific venom activity supports the role of selection in snake venom evolution. Proceedings of the Royal Society B, 276, 2443–2449.
    https://doi.org/10.1098/rspb.2009.0048

    Bickford, D., Lohman, D.J., Sodhi, N.S., Ng, P.K.L., Meier, R., Winkler, K., Ingram, K.K. & Das, I. (2007) Cryptic species as a window on diversity and conservation. Trends in Ecology and Evolution, 22, 148–155.
    https://doi.org/10.1016/j.tree.2006.11.004

    Boulenger, G.A. (1896) Catalogue of the Snakes in the British Museum (Natural History) III. British Museum (Natural History), London, 727 pp.
    https://doi.org/10.5962/bhl.title.54273

    Broadley, D.G. (1958) Mount Silinda Expedition, December 1957. Journal of the Herpetological Association of Rhodesia, 2, 2.
    https://doi.org/10.1080/0440730X.1958.9650560

    Broadley, D.G. (1968) A review of the African cobras of the genus Naja (Serpentes: Elapinae). Arnoldia, 3, 1–14.

    Broadley, D.G. (1983) FitzSimons’ Snakes of Southern Africa. Delta Books, Johannesburg, 376 pp.

    Broadley, D.G. & Blaylock, R. (2013) The Snakes of Zimbabwe and Botswana. Edition Chimaira, Frankfurt am Main, 387 pp.

    Broadley, D.G. & Wüster, W. (2004) A review of the southern African ‘non-spitting’ cobras (Serpentes: Elapidae: Naja). African Journal of Herpetology, 53, 101–122.
    https://doi.org/10.1080/21564574.2004.9635504

    Burbrink, F.T., Lawson, R. & Slowinski, J.B. (2000) Mitochondrial DNA phylogeography of the polytypic North American rat snake (Elaphe obsoleta): a critique of the subspecies concept. Evolution, 54, 2107–2118.
    https://doi.org/10.1554/0014-3820(2000)054[2107:MDPOTP]2.0.CO;2

    Butler, J.A. (1982) Capture au Nigeria d’un Naja noir et blanc de grande taille et notes sur les autres Najas de l’Ouest Africain. Notes Africaines, 176, 110–111.

    Casewell, N.R., Harrison, R.A. & Wüster, W. (2011) Gene tree parsimony of multi-locus snake venom protein families reveals species tree conflict as a result of multiple parallel gene loss. Molecular Biology and Evolution, 28, 1157–1172.
    https://doi.org/10.1093/molbev/msq302

    Casewell, N.R., Wagstaff, S.C., Wüster, W., Cook, D.A.N., Bolton, F.M.S., King, S.I., Pla, D., Sanz, L., Calvete, J.J. & Harrison, R.A. (2014) Medically important differences in snake venom composition are dictated by distinct postgenomic mechanisms Proceedings of the National Academy of Sciences of the USA, 111, 9205–9210.
    https://doi.org/10.1073/pnas.1405484111

    Casewell, N.R., Wüster, W., Vonk, F.J., Harrison, R.A. & Fry, B.G. (2013) Complex cocktails: the evolutionary novelty of venoms. Trends in Ecology and Evolution, 28, 219–229.
    https://doi.org/10.1016/j.tree.2012.10.020

    Ceríaco, L.M.P., Marques, M.P., Schmitz, A. & Bauer, A.M. (2017) The “Cobra-preta” of São Tomé Island, Gulf of Guinea, is a new species of Naja Laurenti, 1768 (Squamata: Elapidae). Zootaxa, 4324 (1), 121–141.
    https://doi.org/10.11646/zootaxa.4324.1

    Ceríaco, L.M.P., de Sá, S.d.A.C., Banderira, S., Valério, H., Stanely, E.L., Kohn, A.L., Marques, M.P., Vindum, J.V., Blackburn, D.C. & Bauer, A.M. (2016) Herpetological survey of Iona National Park and Namibe Regional Natural Park, with a synoptic list of the amphibians and reptiles of Namibe Province, southwestern Angola. Proceedings of the California Academy of Sciences, 63, 15–61.

    Chabanaud, P. (1920) Contribution à l’étude de la faune herpétologique de l’Afrique Occidentale. Bulletin du Comité d’Etudes Historiques et Scientifiques de l’Afrique Occidentale Française, 1921, 445–472.

    Chirio, L. (2003) Inventaire des reptiles de la région de la Réserve de Biosphère Transfrontalière du W (Niger/Bénin/Burkina Faso: Afrique de l’Ouest). Bulletin de la Société Herpétologique de France, 132, 13–41.

    Chirio, L. (2013) Inventaire des reptiles de la région de Sangarédi (Guinée maritime). Bulletin de la Société Herpétologique de France, 144, 67–100.

    Chirio, L. & Ineich, I. (2006) Biogeography of the reptiles of the Central African Republic. African Journal of Herpetology, 55, 23–59.
    https://doi.org/10.1080/21564574.2006.9635538

    Chirio, L. & LeBreton, M. (2007) Atlas des reptiles du Cameroun. Muséum National d’Histoire Naturelle, Paris, 686 pp.

    Clause, A.G., Pavón-Vázquez, C.J., Scott, P.A., Murphy, P.M., Schaad, E.W. & Gray, L.N. (2016). Identification uncertainty and proposed best-practices for documenting herpetofaunal geographic distributions, with applied examples from southern Mexico. Mesoamerican Herpetology, 3, 977–1000.

    Conradie, W., Bills, R. & Branch, W.R. (2016) The herpetofauna of the Cubango, Cuito, and lower Cuando river catchments of south-eastern Angola. Amphibian & Reptile Conservation, 10, 6–36.

    Department of the Navy, Bureau of Medicine and Surgery. (1965) Poisonous Snakes of the World. A Manual for Use by US Amphibious Forces. Government Printing Office, Washington, D.C., 212 pp.

    De Queiroz, K. (1998) The general lineage concept of species, species criteria, and the process of speciation. In: Howard, D.J. & Berlocher, S.H. (Eds.), Endless Forms. Species and Speciation. Oxford University Press, New York, pp. 57–75.

    De Queiroz, K. (2007) Species concepts and species delimitation. Systematic Biology, 56, 879–886.

    https://doi.org/10.1080/10635150701701083

    Eniang, E.A., Egwali, E.C., Luiselli, L.M., Ayodele, I.A., Akani, G.C. & Pacini, N. (2006) Snake bushmeat from the forest markets of south-eastern Nigeria. Natura (Milano), 95, 33–46.

    Ewing, B., Hillier, L., Wendl, M.C. & Green, P. (1998) Base-calling of automated sequencer traces using phred. I. Accuracy assessment. Genome Research, 8, 175–185.
    https://doi.org/10.1101/gr.8.3.175

    Fischer, J.G. (1885) Ichthyologische und herpetologische Bemerkungen. V. Herpetologische Bemerkungen. Jahrbuch der Hamburgischen Wissenschaftlichen Anstalten, 2, 82–121.

    Flot, J.-F. (2010) SEQPHASE: a web tool for interconverting PHASE input/output files and FASTA sequence alignments. Molecular Ecology Resources, 10, 162–166.
    https://doi.org/10.1111/j.1755-0998.2009.02732.x

    Fry, B.G., Winkel, K.D., Wickramaratna, J.C., Hodgson, W.C. & Wüster, W. (2003) Effectiveness of snake antivenom: species and regional venom variation and its clinical impact. Journal of Toxicology -Toxin Reviews, 22, 23–34.
    https://doi.org/10.1081/TXR-120019018

    Greenbaum, E. (2017) Emerald Labyrinth: A Scientist’s Adventures in the Jungles of the Congo. ForeEdge, Lebanon, New Hampshire, xv + 329 pp.

    Hallowell, E. (1857) Notes of a collection of reptiles from the Gaboon country, West Africa, recently presented to the Academy of Natural Sciences of Philadelphia, by Dr. Henry A. Ford. Proceedings of Academy of Natural Sciences, Philadelphia, 9, 48–72.

    Hare, M.P. (2001) Prospects for nuclear gene phylogeography. Trends in Ecology and Evolution, 16, 700–706.
    https://doi.org/10.1016/S0169-5347(01)02326-6

    Harvey, M.B., Barker, D.G. Ammerman, L.K. & Chippindale, L.K. (2000) Systematics of pythons of the Morelia amethistina complex (Serpentes: Boidae) with the description of three new species. Herpetological Monographs, 14, 139–185.
    https://doi.org/10.2307/1467047

    Hebert, P.D., Cywinska, A., Ball, S.L. & deWaard, J.R. (2003) Biological identifications through DNA barcodes. Proceedings of the Royal Society B, Biological Sciences, 270, 313–321.
    https://doi.org/10.1098/rspb.2002.2218

    Hekkala, E., Shirley, M.H., Amato, G., Austin, J.D., Charter, S., Thorbjarnarson, J., Vliet, K.A., Houck, M.L., Desalle, R. & Blum, M.J. (2011) An ancient icon reveals new mysteries: mummy DNA resurrects a cryptic species within the Nile crocodile. Molecular Ecology, 20, 4199–4215.
    https://doi.org/10.1111/j.1365-294X.2011.05245.x

    Hughes, B. (2013) Snakes of Bénin, West Africa. Bulletin de la Société Herpétologique de France, 144, 101–159.

    Jackson, K., Zassi-Boulou, A.-G., Mavoungou, L.-B. & Pangou, S. (2007) Amphibians and reptiles of the Lac Télé Community Reserve, Likouala Region, Republic of Congo (Brazzaville). Herpetological Conservation and Biology, 2, 75–86.

    Joly, S. & Bruneau, A. (2006) Incorporating allelic variation for reconstructing the evolutionary history of organisms from multiple genes: an example from Rosa in North America. Systematic Biology, 55, 623–636.
    https://doi.org/10.1080/10635150600863109

    Jones, P.J. (1994) Biodiversity in the Gulf of Guinea: an overview. Biodiversity and Conservation, 3, 772–785.
    https://doi.org/10.1007/BF00129657

    Kaiser, H. (2014) Best practices in herpetological taxonomy: errata and addenda. Herpetological Review, 45, 257–268.

    Kaiser, H., Crother, B.I., Kelly, C.M.R., Luiselli, L., O’Shea, M., Ota, H., Passos, P., Schleip, W.D. & Wüster, W. (2013) Best practices: in the 21st Century, taxonomic decisions in herpetology are acceptable only when supported by a body of evidence and published via peer-review. Herpetological Review, 44, 8–23.

    Kimura, M. (1980) A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. Journal of Molecular Evolution, 16, 111–120.
    https://doi.org/10.1007/BF01731581

    Laurent, R.F. (1955) Diagnoses préliminaires de quelques serpents venimeux. Revue de Zoologie et Botanique Africaine, 51, 127–139.

    Laurent, R.F. (1956) Contribution à l’herpétologie de la région des Grands Lacs de l’Afrique centrale. I. Généralités. II. Chéloniens. III. Ophidiens. Annales du Musée royal du Congo Belge, 48, 1–390.

    Laurent, R.F. (1973) Le concept de sous-espèce et la variation géographique chez Naja melanoleuca et Naja nigricollis. Revue de Zoologie et Botanique Africaine, 87, 198–204.

    Lauridsen, L.P., Laustsen, A.H., Lomonte, B. & Gutiérrez, J.-M. (2017) Exploring the venom of the forest cobra snake: toxicovenomics and antivenom profiling of Naja melanoleuca. Journal of Proteomics, 150, 98–108.
    https://doi.org/10.1016/j.jprot.2016.08.024

    Leaché, A.D. & Fujita, M.K. (2010) Bayesian species delimitation in West African forest geckos (Hemidactylus fasciatus). Proceedings of the Royal Society B., 1697, 3071–3077. https://doi.org/10.1098/rspb.2010.0662

    Leviton, A.E., Gibbs, Jr., R.H., Heal, E. & Dawson, C.E. (1985) Standards in Herpetology and Ichthyology: Part I. Standard symbolic codes for institutional resource collections in herpetology and ichthyology. Copeia, 1985, 802–832.

    Luiselli, L. & Angelici, F.M. (2000) Ecological relationships of two Afrotropical cobra species (Naja melanoleuca and Naja nigricollis). Canadian Journal of Zoology, 78, 191–198.
    https://doi.org/10.1139/z99-200

    Malhotra, A., Dawson, K., Guo, P. & Thorpe, R.S. (2011a) Phylogenetic structure and species boundaries in the mountain pitviper Ovophis monticola (Serpentes: Viperidae: Crotalinae) in Asia. Molecular Phylogenetics and Evolution, 59, 444–457.
    https://doi.org/10.1016/j.ympev.2011.02.010

    Malhotra, A., Thorpe, R.S., Mrinalini & Stuart, B.L. (2011b) Two new species of pitviper of the genus Cryptelytrops Cope 1860 (Squamata: Viperidae: Crotalinae) from Southeast Asia. Zootaxa, 2757, 1–23.

    Marais, J. & Jubber, W. (2010) Geographic Distributions. Naja melanoleuca Hallowell, 1857. Forest Cobra. African Herp News, 52, 24–25.

    Matschie, P. (1893) Die Reptilien und Amphibien des Togogebietes. Mittheilungen von Forschungsreisenden und Gelehrten aus den Deutschen Schutzgebieten, 6, 207–215.

    May, R.M. (1990) Taxonomy as destiny. Nature, 347, 129–130.
    https://doi.org/10.1038/347129a0

    Measey, J.G. (2013) Taxonomic publishing, vandalism and best practice: African Journal of Herpetology makes changes that will safeguard authors. African Herp News, 60, 2–4.

    Melzer, S., Bell, T. & Patterson, G.B. (2017) Hidden conservation vulnerability within a cryptic species complex: taxonomic revision of the spotted skink (Oligosoma lineoocellatum; Reptilia: Scincidae) from New Zealand. Zootaxa, 4300 (3), 355–379.
    https://doi.org/10.11646/zootaxa.4300.3.2

    Menzies, J.I. (1966) The snakes of Sierra Leone. Copeia, 1966, 169–179.
    https://doi.org/10.2307/1441123

    Monasterio, C., Alvarez, P., Trape, J.-F. & Rödel, M.-O. (2016) The herpetofauna of the Dindefelo Natural Community Reserve, Senegal. Herpetology Notes, 9, 1–6.

    Mrinalini, Thorpe, R.S., Creer, S., Lallias, D., Dawnay, L., Stuart, B.L. & Malhotra, A. (2015) Convergence of multiple markers and analysis methods defines the genetic distinctiveness of cryptic pitvipers. Molecular Phylogenetics and Evolution, 92, 266–279.
    https://doi.org/10.1016/j.ympev.2015.06.001

    Myers, N., Mittermeier, R.A., Mittermeier, C.G., da Fonseca, G.A.B. & Kent, J. (2000) Biodiversity hotspots for conservation priorities. Nature, 403, 853–858. https://doi.org/10.1038/35002501

    Ogden, R. & Thorpe, R.S. (2002) Molecular evidence for ecological speciation in tropical habitats. Proceedings of the National Academy of Sciences of the USA, 99, 13612–13615.
    https://doi.org/10.1073/pnas.212248499

    Padial, J.M., Miralles, A., De la Riva, I. & Vences, M. (2010) The integrative future of taxonomy. Frontiers in Zoology, 7, 16.
    https://doi.org/10.1186/1742-9994-7-16

    Palumbi, S.R. (1996) Nucleic acids II: the polymerase chain reaction. In: Hillis, D.M., Moritz, C. & Mable, B.K. (Eds.), Molecular Systematics, Second Edition. Sinauer, Sunderland, Massachusetts, pp. 205–247.

    Puorto, G., Salomão, M.G., Theakston, R.D.G., Thorpe, R.S., Warrell D.A. & Wüster, W. (2001) Combining mitochondrial DNA sequences and morphological data to infer species boundaries: phylogeography of lanceheaded pitvipers in the Brazilian Atlantic forest, and the status of Bothrops pradoi (Squamata: Serpentes: Viperidae). Journal of Evolutionary Biology, 14, 527–538.
    https://doi.org/10.1046/j.1420-9101.2001.00313.x

    Rannala, B. & Yang, Z. (2003) Bayes estimation of species divergence times and ancestral population sizes using DNA sequences from multiple loci. Genetics, 164, 1645–1656.

    Sauvage, H.E. (1884) Notice sur une collection de reptiles et de poissons recueillie à Majumba, Congo. Bulletin de la Société Zoologique de France, 9, 199–204.

    Schmidt, K.P. (1923) Contributions to the herpetology of the Belgian Congo based on the collection of the American Museum Congo Expedition, 1909–1915. Part II. Snakes. Bulletin of the American Museum of Natural History, 49, 1–146.

    Shaney, KJ., Wostl, E., Hamidy, A., Kurniawan, N., Harvey, M.B. & Smith, E.N. (2017) Conservation challenges regarding species status assessments in biogeographically complex regions: examples from overexploited reptiles of Indonesia. Oryx, 51, 627–638.
    https://doi.org/10.1017/S0030605316000351

    Slowinski, J.B. & Wüster, W. (2000) A new cobra (Elapidae: Naja) from Myanmar (Burma). Herpetologica, 56, 257–270. [http://www.jstor.org/stable/3893276]

    Spawls, S. & Branch, B. (1995) The Dangerous Snakes of Africa. Blandford, London, 192 pp.

    Spawls, S., Howell, K., Drewes, R. & Ashe, J. (2002) A Field Guide to the Reptiles of East Africa. Academic Press, London, 543 pp.

    Spawls, S., Howell, K., Hinkel, H. & Megegon, M. (2018) A Field Guide to East African Reptiles. 2nd Edition. Bloomsbury, London, 634 pp.

    Stephens, M., Smith, N. & Donnelly, P. (2001) A new statistical method for haplotype reconstruction from population data. American Journal of Human Genetics, 68, 978–989.
    https://doi.org/10.1086/319501

    Stephens, M. & Scheet, P. (2005) Accounting for decay of linkage disequilibrium in haplotype inference and missing data imputation. American Journal of Human Genetics, 76, 449–462.
    https://doi.org/10.1086/428594

    Stucki-Stirn, M.C. (1979) Snake Report 721. Herpeto-Verlag, Teuffenthal, 650 pp.

    Swofford, D.L. (2002) PAUP*—Phylogenetic Analysis Using Parsimony (*and Other Methods). Beta Version 4.0b10. Sinauer, Sunderland.

    Tamura, K., Stecher, G., Peterson, D., Filipski, A. & Kumar, S. (2013) MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Molecular Biology and Evolution, 30, 2725–2729.
    https://doi.org/10.1093/molbev/mst197

    Thorpe, R.S. & Richard, M. (2001) Evidence that ultraviolet markings are associated with patterns of molecular gene flow. Proceedings of the National Academy of Sciences of the USA, 98, 3929–3934.
    https://doi.org/10.1073/pnas.071576798

    Thorpe, R.S., Surget-Groba, Y. & Johansson, H. (2010) Genetic tests for ecological and allopatric speciation in anoles on an island archipelago. PLoS Genetics, 6 (4), e1000929.
    https://doi.org/10.1371/journal.pgen.1000929

    Tolley, K.A., Alexander, G.J., Branch, W.R., Bowles, P. & Maritz, B. (2016) Conservation status and threats for African reptiles. Biological Conservation, 204, 63–71.
    https://doi.org/10.1016/j.biocon.2016.04.006

    Townsend, T.M., Alegre, R.E, Kelley, S.T., Wiens, J.J. & Reeder, T.W. (2008) Rapid development of multiple nuclear loci for phylogenetic analysis using genomic resources: an example from squamate reptiles. Molecular Phylogenetics and Evolution, 47, 129–142.
    https://doi.org/10.1016/j.ympev.2008.01.008

    Trape, J.-F & Baldé, C. (2014) A checklist of the snake fauna of Guinea, with taxonomic changes in the genera Philothamnus and Dipsadoboa (Colubridae) and a comparison with the snake fauna of some other West African countries. Zootaxa, 3900 (3), 301–338.
    https://doi.org/10.11646/zootaxa.3900.3.1

    Trape, J.-F., Chirio, L., Broadley, D.G. & Wüster, W. (2009) Phylogeography and systematic revision of the Egyptian cobra (Serpentes: Elapidae: Naja haje) species complex, with the description of a new species from West Africa. Zootaxa, 2236, 1–25.

    Trape, J.F. & Mané, Y. (2006) Guide des Serpents d’Afrique Occidentale. Savane et Désert. IRD Editions, Paris, 226 pp.

    Wallach, V., Williams, K.L. & Boundy, J. (2014) Snakes of the World. A Catalogue of Living and Extinct Species. CRC Press, Boca Raton, 1227 pp.
    https://doi.org/10.1201/b16901

    Wallach, V., Wüster, W. & Broadley, D.G. (2009) In praise of subgenera: taxonomic status of cobras of the genus Naja Laurenti (Serpentes: Elapidae). Zootaxa, 2236, 26–36.

    Williams, D.J., Gutiérrez, J.-M., Calvete, J.J., Wüster, W. Ratanabanangkoon, K., Paiva, O., Brown, N.I., Casewell, N.R., Harrison, R.A., Rowley, P.D., O’Shea, M., Jensen, S.D., Winkel, K.D. & Warrell, D.A. (2011) Ending the drought: new strategies for improving the flow of affordable, effective antivenoms in Asia and Africa. Journal of Proteomics, 74, 1735–1767.
    https://doi.org/10.1016/j.jprot.2011.05.027

    Wüster, W. (1996) Taxonomic changes and toxinology: systematic revisions of the Asiatic cobras (Naja naja species complex). Toxicon, 34, 399–406.
    https://doi.org/10.1016/0041-0101(95)00139-5

    Wüster, W. & Broadley, D.G. (2003) A new species of spitting cobra from northeastern Africa (Serpentes: Elapidae: Naja). Journal of Zoology, London, 259, 345–359.
    https://doi.org/10.1017/S0952836902003333

    Wüster, W. & Broadley, D.G. (2007) Get an eyeful of this: a new species of giant spitting cobra from eastern and north-eastern Africa (Squamata: Serpentes: Elapidae: Naja). Zootaxa, 1532, 51–68.

    Wüster, W., Crookes, S., Ineich, I., Mané, Y., Pook, C.E., Trape, J.-F. & Broadley, D.G. (2007) The phylogeny of cobras inferred from mitochondrial DNA sequences: evolution of venom spitting and the phylogeography of the African spitting cobras (Serpentes: Elapidae: Naja nigricollis complex). Molecular Phylogenetics and Evolution, 45, 437–453.
    https://doi.org/10.1016/j.ympev.2007.07.021

    Wüster, W. & McCarthy, C.J. (1996) Venomous snake systematics: implications for snakebite treatment and toxinology. In: Bon, C. & Goyffon, M. (Eds.), Envenomings and their Treatments. Fondation Mérieux, Lyon, pp. 13–23.

    Wüster, W., Peppin, L., Pook, C.E. & Walker, D.E. (2008) A nesting of vipers: phylogeny, historical biogeography and patterns of diversification of the Viperidae (Squamata: Serpentes). Molecular Phylogenetics and Evolution, 49, 445–459.
    https://doi.org/10.1016/j.ympev.2008.08.019

    Wüster, W. & Thorpe, R.S. (1991) Asiatic cobras: systematics and snakebite. Experientia, 47, 205–209.
    https://doi.org/10.1007/BF01945429

    WWF & IUCN. (1994) Centres of Plant Diversity. A Guide and Strategy for Their Conservation. Vol. 1. Europe, Africa, South West Asia and the Middle East. IUCN Publications Unit, Cambridge, 354 pp.

    Yang, Z. & Rannala, B. (2010) Bayesian species delimitation using multilocus sequence data. Proceedings of the National Academy of Sciences of the USA, 107, 9264–9269.
    https://doi.org/10.1073/pnas.0913022107

    Yang, Z. & Rannala, B. (2014) Unguided species delimitation using DNA sequence data from multiple loci. Molecular Biology and Evolution, 31, 3125–3135.
    https://doi.org/10.1093/molbev/msu279