The Cameroon Volcanic Line, a mountain chain located between West and Central Africa, is a region of numerous endemic diversifications, including of puddle frogs (Phrynobatrachus). This study reviews the phylogeny and taxonomy of puddle frogs of the "Cameroon radiation," which is a clade containing mainly montane but also at least three lowland species. Molecular data revealed a novel evolutionary lineage from high altitudes in the northern part of the mountains. Puddle frogs from the new, minute-sized (SVL < 20 mm) lineage are identified using molecular, morphological and acoustic data and described as two new species, Phrynobatrachus arcanus sp. nov. (Gotel Mountains, Cameroon-Nigeria) and P. mbabo sp. nov. (Tchabal Mbabo, Cameroon). The tadpole of the first species is also described. Phylogenetic analyses placed the new lineage to the proximity of the recently described lowland small-sized taxa (P. horsti, P. ruthbeateae). Based on the inferred phylogeny, we propose five species groups within the Cameroon radiation: P. arcanus, P. chukuchuku, P. ruthbeateae, P. steindachneri, and P. werneri. The taxonomically enigmatic P. hylaios is proposed to be a member of the P. ruthbeateae species group. The basal radiation evolved during the late Miocene with subsequent diversifications occurring during the Pliocene, while closely related terminal taxa originated during the Pleistocene. We recommend that the newly described species are categorized as Critically Endangered due to their limited ranges and because recent surveys did not identify any individuals at the type localities. This further supports the need for conservation interventions in the mountains of Cameroon and Nigeria.

Department of Botany and Zoology Masaryk University Brno Czech Republic

Department of Zoology and Animal Physiology University of Buea Buea Cameroon

Department of Zoology National Museum Prague Czech Republic

Institute of Vertebrate Biology of the Czech Academy of Sciences Brno Czech Republic

Museum of Comparative Zoology Harvard University Cambridge MA USA

Natural History Museum Geneva Switzerland

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Altig R. A primer for the morphology of anuran tadpoles. Herpetological Conservation and Biology. 2007;2:71–74.

Altig R, McDiarmid RW. Body plan: development and morphology. In: McDiarmid RW, Altig R, editors. Tadpoles: The Biology of Anuran Larvae. Chicago: University of Chicago Press; 1999. pp. 24–51.

Amiet J-L. Ecologie et distribution des Amphibiens Anoures de la région de Nkongsamba (Cameroun) Annales de la Faculte des Sciences de Yaoundé. 1975;20:33–107.

Amiet J-L. Liste provisoire des amphibiens anoures du Cameroun. Yaoundé: Laboratoire de Zoologie, Faculté des Sciences de Yaoundé, Yaoundé University; 1978. pp. 1–20.

Amiet J-L. Phrynobatrachus manengoubensis. 2004. http://dx.doi.org/10.2305/IUCN.UK.2004.RLTS.T58125A11736272.en. [4 March 2018]. http://dx.doi.org/10.2305/IUCN.UK.2004.RLTS.T58125A11736272.en The IUCN Red List of Threatened Species 2004: e.T58125A11736272.

Amiet J-L, Goutte S. Chants d’Amphibiens du Cameroun. Saint-Just-la-Pendue: Imprimerie Chirat; 2017.

Angel F. Description de trois amphibiens nouveaux du Cameroun, matériaux de la mission P. Lepesme, R. Paulian et A. Villiers (2e note) Bulletin du Muséum national d’histoire naturelle, série. 1940;2(12):238–243.

Blackburn DC. A new puddle frog (Phrynobatrachidae: Phrynobatrachus) from the Mambilla Plateau in Eastern Nigeria. African Journal of Herpetology. 2010;59(1):33–52. doi: 10.1080/04416651003742160. DOI

Blackburn DC, Rödel M-O. A new puddle frog (Phrynobatrachidae: Phrynobatrachus) from the Obudu Plateau in eastern Nigeria. Herpetologica. 2011;67(3):271–287. doi: 10.1655/HERPETOLOGICA-D-10-00046.1. DOI

Bouckaert RR, Heled J. DensiTree 2: seeing trees through the forest. bioRxiv 012401. 2014. DOI

Boulenger GA. Fourth report on additions to the batrachian collection in the Natural-History Museum. Proceedings of the Zoological Society of London 1898; 1898. pp. 473–482.

Burgess N, Hales JDA, Underwood E, Dinerstein E, Olson D, Itoua I, Schipper J, Ricketts T, Newman K. Terrestrial ecoregions of Africa and Madagascar: a conservation assessment. Washington, D.C., Covelo, London: Island Press; 2004.

Burke K. Origin of the Cameroon Line of volcano-capped swells. Journal of Geology. 2001;109(3):349–362. doi: 10.1086/319977. DOI

Böhme W, Nikolaus G. Herpetological specimens from the Gotel Mountains and Mambilla Plateau. Nigeria Tauraco Research Report. 1989;1:28–30.

Castresana J. Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis. Molecular Biology and Evolution. 2000;17(4):540–552. doi: 10.1093/oxfordjournals.molbev.a026334. PubMed DOI

Center for Conservation Bioacoustics . Ithaca: The Cornell Lab of Ornithology; 2016. [11 June 2019].

Channing A, Rödel M-O. Field guide to the frogs & other amphibians of Africa. Cape Town: Struik Nature, Penguin Random House; 2019.

Channing A, Rödel M-O, Channing J. Tadpoles of Africa: the biology and identification of all known tadpoles in sub-Saharan Africa. Frankfurt am Main: Edition Chimaira; 2012.

Charles KL, Bell RC, Blackburn DC, Burger M, Fujita MK, Gvoždík V, Jongsma GFM, Kouete MT, Leaché AD, Portik DM. Sky, sea, and forest islands: diversification in the African leaf-folding frog Afrixalus paradorsalis (Anura: Hyperoliidae) of the Lower Guineo-Congolian rain forest. Journal of Biogeography. 2018;45(8):1781–1794. doi: 10.1111/jbi.13365. DOI

Cronin DT, Libalah MB, Bergl RA, Hearn GW. Biodiversity and conservation of tropical montane ecosystems in the Gulf of Guinea, West Africa. Arctic, Antarctic, and Alpine Research. 2014;46:891–904.

Darriba D, Taboada GL, Doallo R, Posada D. jModelTest 2: more models, new heuristics and parallel computing. Nature Methods. 2012;9(8):772. doi: 10.1038/nmeth.2109. PubMed DOI PMC

Deichmann JL, Mulcahy DG, Vanthomme H, Tobi E, Wynn AH, Zimkus BM, McDiarmid RW. How many species and under what names? Using DNA barcoding and GenBank data for west Central African amphibian conservation. PLOS ONE. 2017;12(11):e0187283. doi: 10.1371/journal.pone.0187283. PubMed DOI PMC

DeMenocal PB. Plio-Pleistocene African climate. Science. 1995;270(5233):53–59. doi: 10.1126/science.270.5233.53. PubMed DOI

DeMenocal PB. African climate change and faunal evolution during the Pliocene–Pleistocene. Earth and Planetary Science Letters. 2004;220(1–2):3–24. doi: 10.1016/S0012-821X(04)00003-2. DOI

Dewynter M, Frétey T. Liste taxonomique commentée et catalogue illustré des Amphibiens du Gabon. Les Cahiers de la Fondation Biotope. 2019;27:1–84.

Doherty-Bone TM, Gvoždík V. The Amphibians of Mount Oku, Cameroon: an updated species inventory and conservation review. ZooKeys. 2017;643:109–139. doi: 10.3897/zookeys.643.9422. PubMed DOI PMC

Drewes RC. A phylogenetic analysis of the Hyperoliidae (Anura): treefrogs of Africa, Madagascar, and the Seychelles Islands. Occasional Papers of the California Academy of Sciences. 1984;139:1–70.

Drummond AJ, Bouckaert RR. Bayesian evolutionary analysis with BEAST. Cambridge: Cambridge University Press; 2015.

Drummond AJ, Ho SYW, Phillips MJ, Rambaut A. Relaxed phylogenetics and dating with confidence. PLOS Biology. 2006;4(5):e88. doi: 10.1371/journal.pbio.0040088. PubMed DOI PMC

Drummond AJ, Suchard MA, Xie D, Rambaut A. Bayesian phylogenetics with BEAUti and the BEAST 1.7. Molecular Biology and Evolution. 2012;29(8):1969–1973. doi: 10.1093/molbev/mss075. PubMed DOI PMC

Duellman WE, Trueb L. Biology of Amphibians. Baltimore and London: The Johns Hopkins University Press; 1986.

Frost DR. Amphibian Species of the World: an Online Reference. Version 6.0http://research.amnh.org/herpetology/amphibia/index.html. [29 October 2019];2019

Gartshore ME. The status of the montane herpetofauna of the Cameroon Highlands. In: Stuart SN, editor. Conservation of Cameroon Montane Forests. Cambridge: Cambridge University Press; 1986. pp. 204–240.

Gingras B, Mohandesan E, Boko D, Fitch WT. Phylogenetic signal in the acoustic parameters of the advertisement calls of four clades of anurans. BMC Evolutionary Biology. 2013;13(1):134. doi: 10.1186/1471-2148-13-134. PubMed DOI PMC

Gosner KL. A simplified table for staging anuran embryos and larvae with notes on identification. Herpetologica. 1960;16:183–190.

Grandison AGC. Phrynobatrachus. In: Frost DR, editor. Amphibian Species of the World: A Taxonomic and Geographical Reference. Lawrence: Association of Systematics Collections and Allen Press; 1985. pp. 442–451.

Gridi-Papp M. SoundRuler: acoustic analysis for research and teaching. 2007. http://www.soundruler.sourceforge.net. [1 June 2019]. http://www.soundruler.sourceforge.net

Guindon S, Dufayard J-F, Lefort V, Anisimova M, Hordijk W, Gascuel O. New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. Systematic Biology. 2010;59(3):307–321. doi: 10.1093/sysbio/syq010. PubMed DOI

Heled J, Drummond AJ. Bayesian inference of species trees from multilocus data. Molecular Biology and Evolution. 2010;27(3):570–580. doi: 10.1093/molbev/msp274. PubMed DOI PMC

Herrmann H-W, Böhme W, Herrmann PA, Plath M, Schmitz A, Solbach M. African biodiversity hotspots: the amphibians of Mt. Nlonako, Cameroon. Salamandra. 2005;41:61–81.

Herrmann H-W, Schmitz A, Herrmann PA, Böhme W. Amphibians and reptiles of the Tchabal Mbabo Mountains, Adamaoua Plateau, Cameroon. Bonner Zoologische Beiträge. 2007;55:27–35.

Hirschfeld M, Blackburn DC, Doherty-Bone TM, Gonwouo LN, Ghose S, Rödel M-O. Dramatic declines of montane frogs in a Central African biodiversity hotspot. PLOS ONE. 2016;11(5):e0155129. doi: 10.1371/journal.pone.0155129. PubMed DOI PMC

Huelsenbeck JP, Rannala R. Frequentist properties of Bayesian posterior probabilities of phylogenetic trees under simple and complex substitution models. Systematic Biology. 2004;53(6):904–913. doi: 10.1080/10635150490522629. PubMed DOI

IUCN Standards and Petitions Subcommittee Guidelines for Using the IUCN Red List Categories and Criteria. Version 13http://www.iucnredlist.org/documents/RedListGuidelines.pdf. [16 June 2019];2017 Prepared by the Standards and Petitions Subcommittee.

Jackson K, Blackburn DC. The amphibians and reptiles of Nouabale-Ndoki National Park, Republic of Congo (Brazzaville) Salamandra. 2007;43:149–164.

Jackson K, Blackburn DC. A survey of amphibians and reptiles at degraded sites near Pointe-Noire, Kouilou Province, Republic of Congo. Herpetological Conservation and Biology. 2010;5:414–429.

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

Jongsma GFM, Tobi E, Dixon-MacCallum GP, Bamba-Kaya A, Yoga J-A, Mbega J-D, Mve Beh J-H, Emrich AM, Blackburn DC. Amphibians of Haut-Ogooué Province, southeastern Gabon. Amphibian & Reptile Conservation. 2017;11:1–23. (e144)

Katoh K, Standley DM. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Molecular Biology and Evolution. 2013;30(4):772–780. doi: 10.1093/molbev/mst010. PubMed DOI PMC

Kocher TD, Thomas WK, Meyer A, Edwards SV, Pääbo S, Villablanca FX, Wilson AC. Dynamics of mitochondrial DNA evolution in animals: amplification and sequencing with conserved primers. Proceedings of the National Academy of Sciences of the United States of America. 1989;86(16):6196–6200. doi: 10.1073/pnas.86.16.6196. PubMed DOI PMC

Kumar S, Stecher G, Li M, Knyaz C, Tamura K. MEGA X: molecular evolutionary genetics analysis across computing platforms. Molecular Biology and Evolution. 2018;35(6):1547–1549. doi: 10.1093/molbev/msy096. PubMed DOI PMC

Köhler J, Jansen M, Rodríguez A, Kok PJR, Toledo LF, Emmrich M, Glaw F, Haddad CFB, Rödel M-O, Vences M. The use of bioacoustics in anuran taxonomy: theory, terminology, methods and recommendations for best practice. Zootaxa. 2017;4251(1):1–124. doi: 10.11646/zootaxa.4251.1.1. PubMed DOI

Lanfear R, Frandsen PB, Wright AM, Senfeld T, Calcott B. PartitionFinder 2: new methods for selecting partitioned models of evolution for molecular and morphological phylogenetic analyses. Molecular Biology and Evolution. 2017;34:772–773. PubMed

Largen MJ, Dowsett-Lemaire F. Amphibians (Anura) from the Kouilou River basin, République du Congo. Tauraco Research Report. 1991;4:145–168.

Leaché AD, Portik DM, Rivera D, Rödel M-O, Penner J, Gvoždík V, Greenbaum E, Jongsma GFM, Ofori-Boateng C, Burger M, Eniang EA, Bell RC, Fujita MK. Exploring rain forest diversification using demographic model testing in the African foam-nest treefrog Chiromantis rufescens. Journal of Biogeography. 2019;46(12):2706–2721. doi: 10.1111/jbi.13716. DOI

Lefort V, Longueville JE, Gascuel O. SMS: smart model selection in PhyML. Molecular Biology and Evolution. 2017;34(9):2422–2424. doi: 10.1093/molbev/msx149. PubMed DOI PMC

Librado P, Rozas J. DnaSP v5: a software for comprehensive analysis of DNA polymorphism data. Bioinformatics. 2009;25(11):1451–1452. doi: 10.1093/bioinformatics/btp187. PubMed DOI

Marzoli A, Piccirillo EM, Renne P, Bellieni G, Iacumin M, Nyobe NB, Tongwa AT. The Cameroon Volcanic Line revisited: petrogenesis of continental basaltic magmas from lithospheric and asthenospheric mantle sources. Journal of Petrology. 2000;41(1):87–109. doi: 10.1093/petrology/41.1.87. DOI

Mbowou GIB, Lagmet C, Nomade S, Ngounouno I, Deruelle B, Ohnenstetter D. Petrology of the Late Cretaceous peralkaline rhyolites (pantellerite and comendite) from Lake Chad, Central Africa. Journal of GEOsciences. 2012;57:127–141. doi: 10.3190/jgeosci.118. DOI

Metscher BD. MicroCT for developmental biology: a versatile tool for high-contrast 3D imaging at histological resolutions. Developmental Dynamics. 2009;238(3):632–640. doi: 10.1002/dvdy.21857. PubMed DOI

Nieden F. Neue Reptilien und Amphibien aus Kamerun. Archiv für Naturgeschichte. 1910;76:234–246.

Palumbi S, Martin A, Romano S, McMillan WO, Stice L, Grabowski G. The simple fool’s guide to PCR. Vol. 2. Honolulu: University of Hawaii; 1991.

Perret J-L. Études herpétologiques africains. Bulletin de la Société Neuchâteloise des Sciences Naturelles. 1959;82:247–253.

Perret J-L. Les Amphibiens du Cameroun. Zoologische Jahrbücher. Abteilung für Systematik, Ökologie und Geographie. 1966;93:289–464.

Pfalzgraff T, Hirschfeld M, Barej MF, Dahmen M, Gonwouo LN, Doherty-Bone TM. The tadpoles of four Central Africa Phrynobatrachus species. Salamandra. 2015;51:91–102.

Portik DM, Bell RC, Blackburn DC, Bauer AM, Barratt CD, Branch WR, Burger M, Channing A, Colston TJ, Conradie W, Dehling JM, Drewes RC, Ernst R, Greenbaum E, Gvoždík V, Harvey J, Hillers A, Hirschfeld M, Jongsma GFM, Kielgast J, Kouete MT, Lawson LP, Leaché AD, Loader SP, Lötters S, Van Der Meijden A, Menegon M, Müller S, Nagy ZT, Ofori-Boateng C, Ohler A, Papenfuss TJ, Rößler D, Sinsch U, Rödel M-O, Veith M, Vindum J, Zassi-Boulou A-G, McGuire JA. Sexual dichromatism drives diversification within a major radiation of African amphibians. Systematic Biology. 2019;68(6):859–875. doi: 10.1093/sysbio/syz023. PubMed DOI PMC

Portik DM, Leaché AD, Rivera D, Barej MF, Burger M, Hirschfeld M, Rödel M-O, Blackburn DC, Fujita MK. Evaluating mechanisms of diversification in a Guineo-Congolian tropical forest frog using demographic model selection. Molecular Ecology. 2017;26(19):5245–5263. doi: 10.1111/mec.14266. PubMed DOI

Rambaut A, Drummond AJ, Xie D, Baele G, Suchard MA. Posterior summarization in Bayesian phylogenetics using Tracer 1.7. Systematic Biology. 2018;67(5):901–904. doi: 10.1093/sysbio/syy032. PubMed DOI PMC

Richards CM, Moore WS. A phylogeny for the African treefrog family Hyperoliidae based on mitochondrial rDNA. Molecular Phylogenetics and Evolution. 1996;5(3):522–532. doi: 10.1006/mpev.1996.0047. PubMed DOI

Ronquist F, Teslenko M, Van Der Mark P, Ayres DL, Darling A, Höhna S, Larget B, Liu L, Suchard MA, Huelsenbeck JP. MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Systematic Biology. 2012;61(3):539–542. doi: 10.1093/sysbio/sys029. PubMed DOI PMC

Ruddiman WF. Late Miocene to Pleistocene evolution of climate in Africa and the low-latitude Atlantic: overview of Leg 108 results. Proceedings of the Ocean Drilling Program: Scientific Results. 1989;108:463–484.

Rödel M-O, Burger M, Zassi-Boulou AG, Emmrich M, Penner J, Barej M. Two new Phrynobatrachus species (Amphibia: Anura: Phrynobatrachidae) from the Republic of the Congo. Zootaxa. 2015;4032(1):55–80. doi: 10.11646/zootaxa.4032.1.3. PubMed DOI

Rödel M-O, Doherty-Bone T, Kouete MT, Janzen P, Garrett K, Browne R, Gonwouo NL, Barej MF, Sandberger L. A new small Phrynobatrachus (Amphibia: Anura: Phrynobatrachidae) from southern Cameroon. Zootaxa. 2012;3431(1):54–68. doi: 10.11646/zootaxa.3431.1.4. DOI

Sabaj MH. 2019. https://asih.org/standard-symbolic-codes/about-symbolic-codes. [16 June 2019]. https://asih.org/standard-symbolic-codes/about-symbolic-codes

Stephens M, Scheet P. Accounting for decay of linkage disequilibrium in haplotype inference and missing-data imputation. American Journal of Human Genetics. 2005;76(3):449–462. doi: 10.1086/428594. PubMed DOI PMC

Stephens M, Smith JN, Donnelly P. A new statistical method for haplotype reconstruction from population data. American Journal of Human Genetics. 2001;68(4):978–989. doi: 10.1086/319501. PubMed DOI PMC

Stuart SN. Conservation of Cameroon Montane Forests. Cambridge: Cambridge University Press; 1986.

Tchassem FAM, Doherty-Bone TM, Kameni NMM, Tapondjou NWP, Tamesse JL, Gonwouo LN. What is driving declines of montane endemic amphibians? New insights from Mount Bamboutos, Cameroon. Oryx. 2019 doi: 10.1017/S0030605318001448. (in press) DOI

Vences M, Nagy ZT, Sonet G, Verheyen E. DNA barcoding amphibians and reptiles, DNA Barcodes: methods and protocols. In: Kress WJ, Erickson DL, editors. Methods in Molecular Biology. Vol. 858. Totowa: Humana Press; 2012. pp. 79–107. PubMed

White F. The vegetation of Africa: a descriptive memoir to accompany the UNESCO/AETFAT/UNSO vegetation map of Africa. Paris: United Nations; 1983.

Zimkus BM. 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. 2009;157:796–813.

Zimkus BM, Gvoždík V. Sky Islands of the Cameroon Volcanic Line: a diversification hot spot for puddle frogs (Phrynobatrachidae: Phrynobatrachus) Zoologica Scripta. 2013;42:591–611.

Zimkus BM, Larson JG. Phrynobatrachus ruthbeatae [sic] (Ruth-Beate’s Puddle Frog). Gabon: Haut Ogooé Province: Batéké Plateau National Park. Herpetological Review. 2013a;44:104.

Zimkus BM, Larson JG. Assessment of the amphibians of Batéké Plateau National Park, Gabon, including results of chytrid pathogen tests. Salamandra. 2013b;49:159–170.

Zimkus BM, Lawson LP, Barej MF, Barratt CD, Channing A, Dash KM, Dehling JM, Du Preez L, Gehring P-S, Greenbaum E, Gvoždík V, Harvey J, Kielgast J, Kusumba C, Nagy ZT, Pabijan M, Penner J, Rödel M-O, Vences M, Letters S. Leapfrogging into new territory: how Mascarene ridged frogs diversified across Africa and Madagascar to maintain their ecological niche. Molecular Phylogenetics and Evolution. 2017;106:254–269. doi: 10.1016/j.ympev.2016.09.018. PubMed DOI

Zimkus BM, Lawson L, Loader SP, Hanken J. Terrestrialization, miniaturization and rates of diversification in African puddle frogs (Anura: Phrynobatrachidae) PLOS ONE. 2012;7(4):e35118. doi: 10.1371/journal.pone.0035118. PubMed DOI PMC

Zimkus BM, Rödel M-O, Hillers A. Complex patterns of continental speciation: molecular phylogenetics and biogeography of sub-Saharan puddle frogs (Phrynobatrachus) Molecular Phylogenetics and Evolution. 2010;55(3):883–900. doi: 10.1016/j.ympev.2009.12.012. PubMed DOI

Congolius, a new genus of African reed frog endemic to the central Congo: A potential case of convergent evolution