Abstract
Bufo preussi Matschie, 1893 from Mt. Cameroon was the first African Torrent toad to be described, soon followed by Atelopus africanus Werner, 1898 from Bismarkburg in Togo. Shortly after, both species were considered conspecific and, currently, both fall under the name Werneria preussi following taxonomic revisions. The type locality of A. africanus was consequently believed to be an error, especially due to the absence of reports of Werneria individuals in Togo in subsequent years. However, it remained uncertain if this synonymy is justified or if A. africanus may indeed represent a valid but potentially extinct species. To answer this question, we generated the first mitochondrial data (12S, 16S and COI) for name-bearing types of W. preussi and A. africanus using a museomic approach. This was further complemented by mitochondrial and nuclear data of contemporary relatives. Our multi-gene phylogeny inferred from mitochondrial data showed that W. preussi and A. africanus constitute a well-supported monophyletic group, which further includes W. submontana. Together with distance values falling below the commonly used species threshold of 3% for 16S and 6% for COI, this result provides strong evidence for A. africanus being a junior synonym of W. preussi, and the absence of this genus in Togo. It further indicates that W. submontana does not represent a species distinct from W. preussi, but rather a geographic variety differing morphologically from topotypic W. preussi. Due to this morphological differentiation, we propose to consider the W. submontana populations as a distinct subspecies and herein introduce the new name Werneria preussi submontana comb. nov. We further designate a lectotype for W. preussi, in order to increase nomenclatural stability and facilitate future research focusing on African Torrent Toads.
References
- Amiet, J.-L. (1971) Les batraciens orophiles du Cameroun. Annales de la Faculté des Sciences du Cameroun, 5, 83–102.
- Amiet, J.-L. (1972) Description de trois bufonidés orophiles du Cameroun appartenant au groupe de Bufo preussi Matschie (Amphibiens Anoures). Annales de la Faculté des Sciences du Cameroun, 11, 121–140.
- Amiet, J.-L. (1975) Écologie et distribution des amphibiens anoures de la région de Nkongsamba (Cameroun). Annales de la Faculté des Sciences de Yaoundé, 20, 33–107.
- Amiet, J.-L. (1976a) Voix d’amphibiens camerounais. V.– Bufonidae: genres Bufo, Werneria et Nectophryne. Annales de la Faculté des Sciences du Cameroun, 21–22, 139–157.
- Amiet, J.-L. (1976b) Observations anatomique et biologique sur le genre Werneria Poche, 1903. Revue Zoologique Africaine, 90, 33–45.
- Amiet, J.-L. & Goutte, S. (2017) Chants d’Amphibiens du Cameroun. J.-L. Amiet & Editions Petit Génie, Nyons & Saint Nazaire, 280 pp., 4 audio CDs.
- Blackburn, D.C., Kosuch, J., Schmitz, A., Burger, M., Wagner, P., Gonwouo, L.N., Hillers, A. & Rödel, M.-O. (2008) A new species of Cardioglossa (Anura: Arthroleptidae) from the Upper Guinean forests of West Africa. Copeia, 2008, 603–612. https://doi.org/10.1643/ch-06-233
- Boulenger, G.A. (1906) Report on the batrachians collected by the late L. Fea in West Africa. Annales du Museo Civico di Storia Naturali Genova, 42, 157–172, 2 pls.
- Burrell, A.S., Disotell, T.R. & Bergey, C.M. (2015) The use of museum specimens with high-throughput DNA sequencers. Journal of Human Evolution, 79, 35–44. https://doi.org/10.1016/j.jhevol.2014.10.015
- Cairns, N., Cicchino, A., Stewart, K., Austin, J. & Lougheed, S. (2021) Cytonuclear discordance, reticulation and cryptic diversity in one of North America’s most common frogs. Molecular Phylogenetics and Evolution, 156, 107042. https://doi.org/10.1016/j.ympev.2020.107042
- Channing, A. & Rödel, M.-O. (2019) Field Guide to the Frogs & other Amphibians of Africa. Penguin Random House, Cape Town, 408 pp.
- Clement, M., Posada, D.C. & Crandall, K.A. (2000) TCS: a computer program to estimate gene genealogies. Molecular Ecology, 9, 1657–1659. https://doi.org/10.1046/j.1365-294x.2000.01020.x
- Dayrat, B. (2005) Towards integrative taxonomy. Biological Journal of the Linnean Society, 85, 407–415. https://doi.org/10.1111/j.1095-8312.2005.00503.x
- De la Riva, I. (1994) Anfibios anuros del Parque Nacional de Monte Alén, Río Muni, Guinea Ecuatorial. Revista Española de Herpetología, 8, 123–139.
- Dewynter, M., Chirio, L., Melki, F., Cordier, J. & Frétey, T. (2017) Premières données herpétologiques (Amphibiens et Reptiles) sur le massif du Koumouna-Bouali (Gabon). Les cahiers de la fondation Biotope, 11, 1–42.
- Dufresnes, C., Pribille, M., Alard, B., Gonçalves, H., Amat, F., Crochet, P.-A., Dubey, S., Perrin, N., Fumagalli, L., Vences, M. & Martínez-Solano, I. (2020) Integrating hybrid zone analyses in species delimitation: lessons from two anuran radiations of the western Mediterranean. Heredity, 124, 423–438. https://doi.org/10.1038/s41437-020-0294-z
- Fong, J.J., Blom, M.P., Aowphol, A., McGuire, J.A., Sutcharit, C. & Soltis, P.S. (2023) Recent advances in museomics: revolutionizing biodiversity research. Frontiers in Ecology and Evolution, 11, 1188172. https://doi.org/10.3389/978-2-8325-2433-6
- Frétey, T., Dewynter, M. & Blanc, C. (2012) Amphibiens d’Afrique Centrale et d’Angola – Clé de détermination illustrée des amphibiens du Gabon et du Mbini. Illustrated identification key of the amphibians from Gabon and Mbini. Biotope, Mèze (Collection Parthénope), Muséum national d’Histoire naturelle, Paris, 232 pp.
- Frost, D.R. (2024) Amphibian Species of the World: an online reference. Version 6.2. Electronic database. American Museum of Natural History, New York, New York. Available from: https://amphibiansoftheworld.amnh.org/index.php (accessed 15 April 2024) https://doi.org/10.5531/db.vz.0001
- Fulton, T.L. & Shapiro, B. (2019) Setting up an ancient DNA laboratory. Ancient DNA: methods and protocols, 1963, 1–13. https://doi.org/10.1007/978-1-4939-9176-1_1
- Gansauge, M.T., Gerber, T., Glocke, I., Korlevic, P., Lippik, L., Nagel, S., Riehl, L.M., Schmidt, A. & Meyer, M. (2017) Single-stranded DNA library preparation from highly degraded DNA using T4 DNA ligase. Nucleic Acids Research, 45, e79. https://doi.org/10.1093/nar/gkx033
- Griesbaum, F., Jongsma, G.F.M., Penner, J., Kouamé, N.G., Doumbia, J., Gonwouo, N.L., Hillers, A., Glos, J., Blackburn, D.C. & Rödel, M.-O. (2023) The smallest of its kind: Description of a new cryptic Amnirana species (Amphibia, Anura, Ranidae) from West African rainforests. Zootaxa, 5234 (3), 301–339. https://doi.org/10.11646/zootaxa.5254.3.1
- Hillers, A., Boateng, C.O., Segniagbeto, G.H., Agyei, A.C. & Rödel, M.-O. (2009) Assessment of the amphibians in the forests of southern Ghana and western Togo. Zoosystematics and Evolution, 85, 127–141. https://doi.org/10.1002/zoos.200800019
- Hirschfeld, M., Barej, M.F., Loader, S.P. & Rödel, M.-O. (2012) Description of two Werneria tadpoles from Cameroon (Amphibia: Anura: Bufonidae). Zootaxa, 3172 (1), 65–68. https://doi.org/10.11646/zootaxa.3172.1.5
- Hirschfeld, M., Blackburn, D.C., Doherty-Bone, T.M., Gonwouo, L.N., Ghose, S. & Rödel, M.-O. (2016) Dramatic declines of montane frogs in a Central African biodiversity hotspot. PLoS ONE, 11, e0155129. https://doi.org/10.1371/journal.pone.0155129
- Hoang, D.T., Chernomor, O., von Haeseler, A., Minh, B.Q. & Vinh, L.S. (2018) UFBoot2: Improving the ultrafast bootstrap approximation. Molecular Biology and Evolution, 35, 518–522. https://doi.org/10.1093/molbev/msx281
- IUCN SSC Amphibian Specialist Group (2018) Werneria preussi. The IUCN Red List of Threatened Species, 2018, e.T54895A96227185. https://doi.org/10.2305/IUCN.UK.2018-1.RLTS.T54895A96227185.en
- Lanfear, R., Frandsen, P.B., Wright, A.M., Senfeld, T. & Calcott, B. (2017) PartitionFinder 2: new methods for selecting partitioned models of evolution for molecular and morphological phylogenetic analyses. Molecular Biology and Evolution, 34, 772–773.
- https://doi.org/10.1093/molbev/msw260
- Larsson, A. (2014) AliView: a fast and lightweight alignment viewer and editor for large data sets. Bioinformatics, 30, 3276–3278. https://doi.org/10.1093/bioinformatics/btu531
- Lasso, C.A., Rial, A.I., Castroviejo, J. & de la Riva, I. (2002) Herpetofauna del Parque Nacional de Monte Alén (Río Muni, Guinea Ecuatorial). Graellsia, 58, 21–34. https://doi.org/10.3989/graellsia.2002.v58.i2.276
- Li, H. & Durbin, R. (2009) Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics, 25, 1754–1760. https://doi.org/10.1093/bioinformatics/btp324
- Li, H., Handsaker, B., Wysoker, A., Fennell, T., Ruan, J., Homer, N., Marth, G., Abecasis, G., Durbin, R. & 1000 Genome Project Data Processing Subgroup (2009) The sequence alignment/map format and SAMtools. Bioinformatics, 25, 2078–2079. https://doi.org/10.1093/bioinformatics/btp352
- Liedtke, H.C., Müller, H., Hafner, J., Penner, J., Gower, D.J., Mazuch, T., Rödel, M.-O. & Loader, S.P. (2017) Terrestrial reproduction as an adaptation to steep terrain in African toads. Proceedings of the Royal Society B: Biological Sciences, 284, 20162598. https://doi.org/10.1098/rspb.2016.2598
- Liedtke, H.C., Müller, H., Rödel, M.-O., Menegon, M., Gonwouo, L.N., Barej, M.F., Gvozdik, V., Schmitz, A., Channing, A., Nagel, P. & Loader, S. (2016) No ecological opportunity signal on a continental scale? Diversification and life‐history evolution of African true toads (Anura: Bufonidae). Evolution, 70, 1717–1733. https://doi.org/10.1111/evo.12985
- Martin, M. (2011) Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet Journal, 1, 10–12. https://doi.org/10.14806/ej.17.1.200
- Matschie, P. (1893) Einige anscheinend neue Reptilien und Amphibien aus West-Afrika. Sitzungsberichte der Gesellschaft Naturforschender Freunde zu Berlin, 1893, 170–175. https://doi.org/10.5962/bhl.part.9925
- Mertens, R. (1938) Herpetologische Ergebnisse einer Reise nach Kamerun. Abhandlungen der senckenbergischen naturforschenden Gesellschaft, 442, 1–52.
- Miller, C.A., Tasse Taboue, G.C., Ekane, M.M., Robak, M., Sesink Clee, P.R., Richards-Zawacki, C., Fokam, E.B., Fuashi, N.A. & Anthony, N.M. (2018) Distribution modeling and lineage diversity of the chytrid fungus Batrachochytrium dendrobatidis (Bd) in a central African amphibian hotspot. PloS ONE, 13 (6), e0199288. https://doi.org/10.1371/journal.pone.0199288
- Nguyen, L.-T., Schmidt, H.A., von Haeseler, A. & Minh, B.Q. (2015) IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Moecular Biology and Evolution, 32, 268–274. https://doi.org/10.1093/molbev/msu300
- 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
- Paijmans, J.L.A., Baleka, S., Henneberger, K., Taron, U., Trinks, A., Westbury, M. & Barlow, A. (2017) Sequencing single-stranded libraries on the Illumina NextSeq 500 platform. ArXiv, 2017, arXiv:1711.11004. https://doi.org/10.48550/arXiv.1711.11004
- Pauwels, O.S.G. & Rödel, M.-O. (2007) Amphibians and national parks in Gabon, western Central Africa. Herpetozoa, 19(3/4), 135–148. https://doi.org/10.1163/15685381-00002942
- Perl, R. G. B., Nagy, Z. T., Sonet, G., Glaw, F., Wollenberg, K.C. & Vences, M. (2014) DNA barcoding Madagascar’s amphibian fauna. Amphibia-Reptilia, 35, 197–206. https://doi.org/10.1163/15685381-00002942
- Penner, J., Wegmann, M., Hillers, A., Schmidt, M. & Rödel, M.-O. (2011) A hotspot revisited – a biogeographical analysis of West African amphibians. Diversity and Distributions, 17, 1077–1088. https://doi.org/10.1111/j.1472-4642.2011.00801.x
- Perret, J.-L. (1966) Les amphibiens du Cameroun. Zoologische Jahrbücher, Systematik, 8, 289–464.
- Perret, J.-L. & Amiet, J.-L. (1961) Remarques sur les Bufo (Amphibiens Anoures) du Cameroun. Annales de la Faculté des Sciences du Cameroun, 5, 47–55.
- Plath, M., Herrmann, H.-W. & Böhme, W. (2006) New frog species of the genus Phrynobatrachus (Anura: Phrynobatrachidae) from Mt. Nlonako, Cameroon. Journal of Herpetology, 40, 486–495. https://doi.org/10.1670/0022-1511(2006)40[486:nfsotg]2.0.co;2
- Rambaut, A., Drummond, A.J., Xie, D., Baele, G. & Suchard, M.A. (2018) Posterior summarization in Bayesian phylogenetics using Tracer 1.7. Systematic Biology, 67, 901–904. https://doi.org/10.1093/sysbio/syy032
- Raxworthy, C.J. & Smith, B.T. (2021) Mining museums for historical DNA: advances and challenges in museomics. Trends in Ecology & Evolution, 36, 1049–1060. https://doi.org/10.1016/j.tree.2021.07.009
- Rödel, M.-O. & Ernst, R. (2000) Bufo taiensis n. sp., eine neue Kröte aus dem Taï-Nationalpark, Elfenbeinküste. herpetofauna, 22 (125), 9–16.
- Rödel, M.-O., Kosuch, J., Veith, M. & Ernst, R. (2003) First record of the genus Acanthixalus Laurent, 1944 from the Upper Guinean rain forest, West Africa, with the description of a new species. Journal of Herpetology, 37, 43–52. https://doi.org/10.1670/0022-1511(2003)037[0043:frotga]2.0.co;2
- Rödel, M.-O., Schmitz, A., Pauwels, O.S.G. & Böhme, W. (2004) Revision of the genus Werneria Poche, 1903, including the descriptions of two new species from Cameroon and Gabon (Amphibia: Anura: Bufonidae). Zootaxa, 720 (1), 1–28. https://doi.org/10.11646/zootaxa.720.1.1
- Rödel, M.-O., Adum, G. B., Aruna, E., Assemian, N.E., Barej, M. F., Bell, R.C., Doherty-Bone, T., Burger, M., Demare, G., Doumbia, J., Ernst, R., Gonwouo, N.L., Hillers, A., Hirschfeld, M., Jongsma, G.F.M., Kouamé, N.G., Kpan, T.F., Mohneke, M., Nago, G.A., Ofori-Boateng, C., Onadeko, A., Pauwels, O.S.G., Sandberger-Loua, L., Segniagbeto, G.H., Fokoua, A.M.T., Tobi, E., Tohé, B., Zimkus, B.M. & Penner, J. (2021) Diversity, threats and conservation of western and central African amphibians (Senegal, The Gambia, Guinea Bissau, Mali, Guinea, Sierra Leone, Liberia, Ivory Coast, Burkina Faso, Ghana, Togo, Benin, Nigeria, Niger, Cameroon, Gabon, São Tome & Principe, Equatorial Guinea, Central African Republic, Chad, Republic of the Congo, Democratic Republic of the Congo, northern Angola). In: Heatwole, H., Rödel, M.-O. (Eds.), Status and Threats of Afrotropical Amphibians – Sub-Saharan Africa, Madagascar, Western Indian Ocean Islands. Amphibian Biology. Vol. 11. Part 7 Status of Conservation and Decline of Amphibians: Eastern Hemisphere. Frankfurt Contributions to Natural History. Vol. 78. Edition Chimaira, Frankfurt/M, pp. 11–101.
- Rohland, N., Siedel, H. & Hofreiter, M. (2004) Nondestructive DNA extraction method for mitochondrial DNA analyses of museum specimens. Biotechniques, 36, 814–821. https://doi.org/10.2144/04365st05
- Ronquist, F., Teslenko, M., Van Der Mark, P., Ayres, D.L., Darling, A., Höhna, S., Larget, B., Liu, L., Suchard, M.A. & Huelsenbeck, J.P. (2012) MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Systematic Biology, 61, 539–542. https://doi.org/10.1093/sysbio/sys029
- Sánchez-Vialas, A., Calvo-Revuelta, M., Castroviejo-Fisher, S. & De la Riva, I. (2020) Synopsis of the amphibians of Equatorial Guinea based upon the authors’ field work and Spanish natural history collections. Proceedings of the California Academy of Sciences, 66, 137–230.
- Scherz, M.D., Rakotoarison, A., Hawlitschek, O., Vences, M. & Glaw, F. (2015) Leaping towards a saltatorial lifestyle? An unusually long-legged new species of Rhombophryne (Anura, Microhylidae) from the Sorata massif in northern Madagascar. Zoosystematics and Evolution, 91, 105–114. https://doi.org/10.3897/zse.91.4979
- Scherz, M.D., Crottini, A., Hutter, C.R., Hildebrand, A., Andreone, F., Fulgence, T.R., Köhler, G., Ndriantsoa, S.H., Ohler, A., Preick, M., Rakotoarison, A., Rancilhac, L., Raselimanana, A.P., Riemann, J.C., Rödel, M.-O., Rosa, G.M., Streicher, J.W., Vieites, D.R., Köhler, J., Hofreiter, M., Glaw, F. & Vences, M. (2022) An inordinate fondness for inconspicuous brown frogs: Integration of phylogenomics, archival DNA analysis, morphology, and bioacoustics yields 24 new taxa in the subgenus Brygoomantis (genus Mantidactylus) from Madagascar. Megataxa, 7, 113–311. https://doi.org/10.11646/megataxa.7.2.1
- Segniagbeto, G.H., Bowessidjaou, J.E., Dubois, A. & Ohler, A. (2007) Les Amphibiens du Togo: état actuel des connaissances. Alytes, 24, 72–90.
- Segniagbeto, G.H., Ohler, A., Rödel, M.-O., Luiselli, L. & Dubois, A. (2024) Amphibians of Togo: taxonomy, distribution and conservation status. Zoosystema, 46, 631–670. https://doi.org/10.5252/zoosystema2024v46a25
- Stephens, M., Smith, N.J. & 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
- Straube, N., Lyra, M.L., Paijmans, J.L. A., Preick, M., Basler, N., Penner, J., Rödel, M.-O., Westbury, M.V., Haddad, C.F.B., Barlow, A. & Hofreiter, M. (2021) Successful application of ancient DNA extraction and library construction protocols to museum wet collection specimens. Molecular Ecology Resources, 21, 2299–2315. https://doi.org/10.1111/1755-0998.13433
- Streicher, J.W., Sadler, R. & Loader, S.P. (2020) Amphibian taxonomy: early 21st century case studies. Journal of Natural History, 54, 1–13. https://doi.org/10.1080/00222933.2020.1777339
- Vences, M., Miralles, A., Brouillet, S., Ducasse, J., Fedosov, A., Kharchev, V., Kostadinov, I., Kumari, S., Patmanidis, S., Scherz, M.D., Puillandre, N. & Renner, S.S. (2021) iTaxoTools 0.1: Kickstarting a specimen-based software toolkit for taxonomists. Megataxa, 6, 77–92. https://doi.org/10.11646/megataxa.6.2.1
- Vences, M., Patmanidis, S., Kharchev, V. & Renner, S.S. (2022) Concatenator, a user-friendly program to concatenate DNA sequences, implementing graphical user interfaces for MAFFT and FastTree. Bioinformatics Advances, 2, vbac050. https://doi.org/10.1093/bioadv/vbac050
- Vences, M., Patmanidis, S., Schmidt, J.-C., Matschiner, M., Miralles, A. & Renner, S.S. (2024) Hapsolutely: a user-friendly tool integrating haplotype phasing, network construction and haploweb calculation. Bioinformatics Advances, vbae083. [published online] https://doi.org/10.1093/bioadv/vbae083
- Vieites, D.R., Wollenberg, K.C., Andreone, F., Kohler, J., Glaw, F. & Vences, M. (2009) Vast underestimation of Madagascar’s biodiversity evidenced by an integrative amphibian inventory. Proceedings of the National Academy of Sciences of the United States of America, 106, 8267–8272. https://doi.org/10.1073/pnas.0810821106
- Wandeler, P., Hoeck, P.E. & Keller, L.F. (2007) Back to the future: museum specimens in population genetics. Trends in Ecology & Evolution, 22, 634–642. https://doi.org/10.1016/j.tree.2007.08.017
- Werner, F. (1898) Über Reptilien und Batrachier aus Togoland, Kamerun und Tunis aus dem kgl. Museum für Naturkunde in Berlin II. Verhandlungen der kaiserlich-königlichen zoologisch-botanischen Gesellschaft in Wien, 48, 191–213. https://doi.org/10.5962/bhl.part.11464
- Zimkus, B.M. (2009) Biogeographical analysis of Cameroonian puddle frogs and description of a new species of Phrynobatrachus (Anura: Phrynobatrachidae) endemic to Mount Oku, Cameroon. Zoological Journal of the Linnean Society, 157, 795–813. https://doi.org/10.1111/j.1096-3642.2009.00579.x
- Zimkus, B.M., Baláž, V., Belasen, A.M., Bell, R.C., Channing, A., Doumbia, J., Gonwouo, L. N., Fokam, E.B., Zassi-Boulou, A.-G., Greenbaum, E., Gvozdik, V., James, T., Hirschfield, M., Jackson, K., Kusamba, C., Larson, J.G., Mavoungou, L.-B., Rödel, M.-O. & Penner, J. (2020) Chytrid pathogen (Batrachochytrium dendrobatidis) in African amphibians: a continental analysis of occurrences and modeling of its potential distribution. Herpetologica, 76, 201–215. https://doi.org/10.1655/0018-0831-76.2.201