The reed frog genus Hyperolius (Afrobatrachia, Hyperoliidae) is a speciose genus containing over 140 species of mostly small to medium-sized frogs distributed in sub-Saharan Africa. Its high level of colour polymorphism, together with in anurans relatively rare sexual dichromatism, make systematic studies more difficult. As a result, the knowledge of the diversity and taxonomy of this genus is still limited. Hyperolius robustus known only from a handful of localities in rain forests of the central Congo Basin is one of the least known species. Here, we have used molecular methods for the first time to study the phylogenetic position of this taxon, accompanied by an analysis of phenotype based on external (morphometric) and internal (osteological) morphological characters. Our phylogenetic results undoubtedly placed H. robustus out of Hyperolius into a common clade with sympatric Cryptothylax and West African Morerella. To prevent the uncovered paraphyly, we place H. robustus into a new genus, Congolius. The review of all available data suggests that the new genus is endemic to the central Congolian lowland rain forests. The analysis of phenotype underlined morphological similarity of the new genus to some Hyperolius species. This uniformity of body shape (including cranial shape) indicates that the two genera have either retained ancestral morphology or evolved through convergent evolution under similar ecological pressures in the African rain forests.

Biodiversity Monitoring Centre Department of Ecology and Biodiversity of Aquatic Resources University of Kisangani Avenue Munyororo 550 Kisangani Democratic Republic of the Congo

Czech Academy of Sciences Institute of Theoretical and Applied Mechanics Prosecká 76 190 00 Prague Czech Republic

Czech Academy of Sciences Institute of Vertebrate Biology Květná 8 603 65 Brno Czech Republic

Department of Botany and Zoology Faculty of Science Masaryk University Kotlářská 2 611 37 Brno Czech Republic

Department of Zoology National Museum Cirkusová 1740 193 00 Prague Czech Republic

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Portik DM, et al. Sexual dichromatism drives diversification within a major radiation of African amphibians. Syst. Biol. 2019;68:859–875. doi: 10.1093/sysbio/syz023. PubMed DOI PMC

Frost, D. R. Amphibian Species of the World: an Online Reference. Version 6.1https://amphibiansoftheworld.amnh.org/index.php; American Museum of Natural History, New York, USA. 10.5531/db.vz.0001 (2020).

Portik DM, Blackburn DC. The evolution of reproductive diversity in Afrobatrachia: A phylogenetic comparative analysis of an extensive radiation of African frogs. Evolution. 2016;70:2017–2032. doi: 10.1111/evo.12997. PubMed DOI PMC

Laurent RF. Diagnoses préliminaires de treize batraciens nouveaux d’Afrique centrale. Rev. Zool. Bot. Afr. 1950;44:1–18.

Laurent RF. Deux reptiles et onze batraciens nouveaux d’Afrique centrale. Rev. Zool. Bot. Afr. 1951;44:360–381.

Laurent RF. Description d’un rhacophoride nouveau du Congo Belge (Batracien) Rev. Zool. Bot. Afr. 1940;33:313–316.

Conradie W, et al. New insights into the taxonomic status, distribution and natural history of De Witte’s Clicking Frog (Kassinula wittei Laurent, 1940) Afr. Zool. 2020;55:311–322. doi: 10.1080/15627020.2020.1821771. DOI

Drewes RC. A phylogenetic analysis of the Hyperoliidae (Anura): treefrogs of Africa, Madagascar, and Seychelles Islands. Occas. Pap. California Acad. Sci. 1984;139:1–70.

Channing A. A re-evaluation of the phylogeny of old World treefrogs. Afr. Zool. 1989;24:116–131.

Laurent RF, Combaz J. Sur l’attribution générique de certains batraciens appartenant à la sous-famille de Hyperoliinae. Rev. Zool. Bot. Afr. 1950;43:269–280.

Rödel M-O, et al. A new tree-frog genus and species from Ivory Coast, West Africa (Amphibia: Anura: Hyperoliidae) Zootaxa. 2009;2044:23–45. doi: 10.11646/zootaxa.2044.1.2. DOI

Channing, A. & Rödel, M.-O. Field Guide to the Frogs & Other Amphibians of Africa. (Struik Nature, 2019).

Schiøtz, A. Treefrogs of Africa (Edition Chimaira, 1999).

Laurent RF. Description de deux Hyperolius nouveaux du Sankuru (Zaire) Rev. Zool. Bot. Afr. 1979;93:779–791.

Schiøtz A. Notes on the genus Hyperolius (Anura, Hyperoliidae) in central République Démocratique du Congo. Alytes. 2006;24:40–60.

Laurent RF. Phennogrammes d’anoures bases sur la morphométrie. Monit. Zool. Ital. 1981;15:1–22.

Bell RC, et al. Idiosyncratic responses to climate-driven forest fragmentation and marine incursions in reed frogs from Central Africa and the Gulf of Guinea Islands. Mol. Ecol. 2017;26:5223–5244. doi: 10.1111/mec.14260. PubMed DOI

Soro N, Kouamé AM, Kouamé NG, Adepo-Gourène AB, Rödel M-O. Morerella cyanophthalma (Anura: Hyperoliidae) in south-eastern Ivory Coast: Additional data and implications for species’ conservation. Herpetol. Notes. 2019;12:1215–1223.

Rage J-C. The amphibians and reptiles at the Eocene-Oligocene transition in Western Europe: An outline of the faunal alterations. Dev. Palaeontol. Stratigr. 1986;9:309–310. doi: 10.1016/S0920-5446(08)70135-3. DOI

Prothero DR. The Late Eocene-Oligocene extinctions. Annu. Rev. Earth Planet. Sci. 1994;22:145–165. doi: 10.1146/annurev.ea.22.050194.001045. DOI

Pan AD, Jacobs BF, Dransfield J, Baker WJ. The fossil history of palms (Arecaceae) in Africa and new records from the Late Oligocene (28–27 Mya) of north-western Ethiopia. Bot. J. Linn. Soc. 2006;151:69–81. doi: 10.1111/j.1095-8339.2006.00523.x. DOI

Zachos JC, Quinn TM, Salamy KA. High-resolution (104 years) deep-sea foraminiferal stable isotope records of the Eocene-Oligocene climate transition. Paleoceanography. 1996;11:251–266. doi: 10.1029/96pa00571. DOI

Zachos JC, Pagani M, Sloan L, Thomas E, Billups K. Trends, rhythms, and aberrations in global climate 65 Ma to present. Science. 2001;292:686–693. doi: 10.1126/science.1059412. PubMed DOI

DeConto RM, Pollard D. Rapid Cenozoic glaciation of Antarctica induced by declining atmospheric CO2. Nature. 2003;421:245–249. doi: 10.1038/nature01290. PubMed DOI

Coxall HK, Wilson PA, Pälike H, Lear CH, Backman J. Rapid stepwise onset of Antarctic glaciation and deeper calcite compensation in the Pacific Ocean. Nature. 2005;433(7021):53–57. doi: 10.1038/nature03135. PubMed DOI

Liu Z, et al. Global cooling during the Eocene-Oligocene climate transition. Science. 2009;323(5918):1187–1190. doi: 10.1126/science.1166368. PubMed DOI

Plana V. Mechanisms and tempo of evolution in the African Guineo-Congolian rainforest. Philos. Trans. R. Soc. Lond. B. 2004;359:1585–1594. doi: 10.1098/rstb.2004.1535. PubMed DOI PMC

Zachos JC, Shackleton NJ, Revenaugh JS, Pälike H, Flower BP. Climate response to orbital forcing across the Oligocene-Miocene boundary. Science. 2001;292(5515):274–278. doi: 10.1126/science.1058288. PubMed DOI

Greenbaum E, Kusamba C. Conservation implications following the rediscovery of four frog species from the Itombwe Natural Reserve, Eastern Democratic Republic of the Congo. Herpetol. Rev. 2012;43:253–259.

Ernst R, Lautenschläger T, Branquima MF, Hölting M. At the edge of extinction: A first herpetological assessment of the proposed Serra do Pingano Rainforest National Park in Uíge Province, northern Angola. Zoosyst. Evol. 2020;96:237–262. doi: 10.3897/zse.96.51997. DOI

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

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

Badjedjea G, et al. A preliminary survey of the amphibian fauna of Kisangani ecoregion, Democratic Republic of the Congo. J. Adv. Bot. Zool. 2015;3:V3I407. doi: 10.5281/zenodo.1000085. DOI

Badjedjea G, et al. Contribution to the knowledge of amphibians of Kponyo village (DR Congo) J. Adv. Bot. Zool. 2016;4:V4I104. doi: 10.5281/zenodo.898031. DOI

Masudi FM, et al. Preliminary data on amphibian diversity of the Okapi Wildlife Reserve (RFO) in Democratic Republic of the Congo. Am. J. Zool. 2019;2:38–43. doi: 10.11648/j.ajz.20190203.11. DOI

Fišer C, Robinson CT, Malard F. Cryptic species as a window into the paradigm shift of the species concept. Mol. Ecol. 2018;27:613–635. doi: 10.1111/mec.14486. PubMed DOI

Bickford D, et al. Cryptic species as a window on diversity and conservation. Trends Ecol. Evol. 2007;22:148–155. doi: 10.1016/j.tree.2006.11.004. PubMed DOI

Losos JB. Convergence, adaptation, and constraint. Evolution. 2011;65:1827–1840. doi: 10.1111/j.1558-5646.2011.01289.x. PubMed DOI

Bravo GA, Remsen JV, Jr, Brumfield RT. Adaptive processes drive ecomorphological convergent evolution in antwrens (Thamnophilidae) Evolution. 2014;68:2757–2774. doi: 10.1111/evo.12506. PubMed DOI

Scheffer M, van Nes EH. Self-organized similarity, the evolutionary emergence of groups of similar species. Proc. Natl. Acad. Sci. U.S.A. 2006;103:6230–6235. doi: 10.1073/pnas.0508024103. PubMed DOI PMC

Sabaj MH. Codes for natural history collections in ichthyology and herpetology. Copeia. 2020;108:593–669. doi: 10.1643/ASIHCODONS2020. DOI

Palumbi, S. et al. The Simple Fool’s Guide to PCR (University of Hawaii, 1991).

Bossuyt F, Milinkovitch MC. Convergent adaptive radiations in Madagascan and Asian ranid frogs reveal covariation between larval and adult traits. Proc. Natl. Acad. Sci. U.S.A. 2000;97:6585–6590. doi: 10.1073/pnas.97.12.6585. PubMed DOI PMC

Smith SA, Stephens PR, Wiens JJ. Replicate patterns of species richness, historical biogeography, and phylogeny in Holarctic treefrogs. Evolution. 2005;59:2433–2450. doi: 10.1111/j.0014-3820.2005.tb00953.x. PubMed DOI

Wiens JJ, Fetzner JW, Jr, Parkinson CL, Reeder TW. Hylid frog phylogeny and sampling strategies for speciose clades. Syst. Biol. 2005;54:778–807. doi: 10.1080/10635150500234625. PubMed DOI

Shen XX, Liang D, Feng YJ, Chen MY, Zhang P. A versatile and highly efficient toolkit including 102 nuclear markers for vertebrate phylogenomics, tested by resolving the higher level relationships of the Caudata. Mol. Biol. Evol. 2013;30:2235–2248. doi: 10.1093/molbev/mst122. PubMed DOI

Gvoždík V, et al. Evolutionary history of the Cameroon radiation of puddle frogs (Phrynobatrachidae: Phrynobatrachus), with descriptions of two critically endangered new species from the northern Cameroon Volcanic Line. PeerJ. 2020;8:e8393. doi: 10.7717/peerj.8393. PubMed DOI PMC

Kearse M, et al. Geneious Basic: An integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics. 2012;28:1647–1649. doi: 10.1093/bioinformatics/bts199. PubMed DOI PMC

Katoh K, Misawa K, Kuma K, Miyata T. MAFFT: A novel method for rapid multiple sequence alignment based on fast Fourier transform. Nucleic Acids Res. 2002;30:3059–3066. doi: 10.1093/nar/gkf436. PubMed DOI PMC

Katoh K, Standley DM. MAFFT multiple sequence alignment software version 7: Improvements in performance and usability. Mol. Biol. Evol. 2013;30:772–780. doi: 10.1093/molbev/mst010. PubMed DOI PMC

Kozlov AM, Darriba D, Flouri T, Morel B, Stamatakis A. RAxML-NG: A fast, scalable and user-friendly tool for maximum likelihood phylogenetic inference. Bioinformatics. 2019;35:4453–4455. doi: 10.1093/bioinformatics/btz305. PubMed DOI PMC

Ronquist F, et al. MRBAYES 3.2: efficient Bayesian phylogenetic inference and model selection across a large model space. Syst. Biol. 2012;61:539–542. doi: 10.1093/sysbio/sys029. PubMed DOI PMC

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. Mol. Biol. Evol. 2017;34:772–773. doi: 10.1093/molbev/msw260. PubMed DOI

Huelsenbeck JP, Rannala B. Frequentist properties of Bayesian posterior probabilities of phylogenetic trees under simple and complex substitution models. Syst. Biol. 2004;53:904–913. doi: 10.1080/10635150490522629. PubMed DOI

Ogilvie HA, Bouckaert R, Drummond AJ. StarBEAST2 brings faster species tree inference and accurate estimates of substitution rates. Mol. Biol. Evol. 2017;34:2101–2114. doi: 10.1093/molbev/msx126. PubMed DOI PMC

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

Feng Y-J, et al. Phylogenomics reveals rapid, simultaneous diversification of three major clades of Gondwanan frogs at the Cretaceous-Paleogene boundary. Proc. Natl. Acad. Sci. U.S.A. 2017;114:E5864–E5870. doi: 10.1073/pnas.1704632114. PubMed DOI PMC

Yuan Z-Y, et al. Natatanuran frogs used the Indian Plate to step-stone disperse and radiate across the Indian Ocean. Nat. Sci. Rev. 2018;6:10–14. doi: 10.1093/nsr/nwy092. PubMed DOI PMC

Hime PM, et al. Phylogenomics reveals ancient gene tree discordance in the amphibian tree of life. Syst. Biol. 2020 doi: 10.1093/sysbio/syaa034. PubMed DOI PMC

Rambaut A, Drummond AJ, Xie D, Baele G, Suchard MA. Posterior summarisation in Bayesian phylogenetics using Tracer 1.7. Syst. Biol. 2018;67:901–904. doi: 10.1093/sysbio/sys032. PubMed DOI PMC

Bouckaert R, et al. BEAST 2.5: an advanced software platform for Bayesian evolutionary analysis. PLOS Comput. Biol. 2019;15:e1006650. doi: 10.1371/journal.pcbi.1006650. PubMed DOI PMC

Dehling JM, Sinsch U. Partitioning of morphospace in larval and adult reed frogs (Anura: Hyperoliidae: Hyperolius) of the Central African Albertine Rift. Zool. Anz. 2019;280:65–77. doi: 10.1016/j.jcz.2019.04.003. DOI

Channing A, et al. Taxonomy of the super-cryptic Hyperolius nasutus group of long reed frogs of Africa (Anura: Hyperoliidae), with descriptions of six new species. Zootaxa. 2013;3620:301–350. doi: 10.11646/zootaxa.3620.3.1. PubMed DOI

Conradie W, et al. A new Reed Frog (Hyperoliidae: Hyperolius) from coastal northeastern Mozambique. Zootaxa. 2018;4379:177–198. doi: 10.11646/zootaxa.4379.2.2. PubMed DOI

Watters JL, Cummings ST, Flanagan RL, Siler CD. Review of morphometric measurements used in anuran species descriptions and recommendations for a standardized approach. Zootaxa. 2016;4072:477–495. doi: 10.11646/zootaxa.4072.4.6. PubMed DOI

Mosimann JE. Size allometry: Size and shape variables with characterizations of lognormal and generalized Gamma distributions. J. Am. Stat. Assoc. 1970;65:930–945. doi: 10.1080/01621459.1970.10481136. DOI

Gvoždík V, Moravec J, Kratochvíl L. Geographic morphological variation in parapatric Western Palearctic tree frogs, Hyla arborea and Hyla savignyi: Are related species similarly affected by climatic conditions? Biol. J. Linn. Soc. 2008;95:539–556. doi: 10.1111/j.1095-8312.2008.01056.x. DOI

Dolinay M, et al. Gene flow in phylogenomics: sequence capture resolves species limits and biogeography of Afromontane forest endemic frogs from the Cameroon Highlands. bioRxiv. 2020 doi: 10.1101/2020.10.09.332767. PubMed DOI

R Core Team. R: A Language and Environment for Statistical Computing (R Foundation for Statistical Computing, https://R-project.org, 2020).

Oksanen, J. et al. vegan: Community Ecology Package. R Package Version 2.5–6. https://CRAN.R-project.org/package=vegan (2019).

Paluh DJ, Stanley EL, Blackburn DC. Evolution of hyperossification expands skull diversity in frogs. Proc. Natl. Acad. Sci. U.S.A. 2020;117:8554–8562. doi: 10.1073/pnas.2000872117. PubMed DOI PMC

Adams, D. C., Collyer, M. L. & Kaliontzopoulou, A. Software for Geometric Morphometric Analyses. R Package Version 3.2.1.https://cran.r-project.org/package=geomorph (2020).

Arino, O. et al. Global Land Cover Map for 2009 (European Space Agency & Université catholique de Louvain; 10.1594/PANGAEA.787668 (2012).

Cignoni et al. MeshLab: an open-source mesh processing tool. Sixth Eurographics Italian Chapter Conference 129–136 (2008).