Abstract
We describe a new species of dendrobatid frog, Leucostethus bilsa sp. nov., using molecular, morphological, and acoustic evidence. We also comment on the taxonomic status of four similar Hyloxalus and Colostethus. We provide an updated phylogeny of Leucostethus that corroborates previous hypotheses of relationships of nine species. Phylogenetic analysis using mitochondrial (i.e., 7095 bp of combined data from NADH1, NAHD2, cytochrome c oxidase I, cytochrome b and 12S-16S rRNA) and seven nuclear genes (i.e., 4739 bp) indicate a close relationship of L. bilsa to an undescribed species from Gorgona Island, Colombia, both of which apparently diverged in the Pliocene about 3 million years ago with about 6.25% (i.e., 146/2335 bp) differences for the section of 12S-16S mitochondrial fragment. Leucostethus bilsa is diurnal and riparian, characterized by distinctive bright mustard-yellow flash marks in the axillar and groin regions, posterior belly, and in the hindlimbs, the presence of dark gray lower labial stripe or marks, sexual dimorphism in ventral pattern, and by having male uniparental care. We describe its osteology and the male advertisement call, which is a series of peep notes. Osteological microCT images of representatives of each of the Colostethinae genera reveal a number of intriguing characters that may prove to be useful in phylogenetic studies. In terms of its distribution, Leucostethus bilsa is currently known only from a very small area within the Reserva Biológica Bilsa, located within the Cordillera Mache-Chindul in the Chocoan region of northwestern Ecuador, which was a Pliocene-Pleistocene refugium. This region is highly threatened with habitat degradation and remains as the last surviving refuge for a forest community known for a high proportion of endemic species of both flora and fauna.
References
Almendáriz, A. & Carr, J. (1992) Herpetofauna of Bilsa Area. In: Parker III, T. & Carr, J. (Eds.), Status of forest remanents in the cordillera de la costa and adjacent areas of southwestern Ecuador. Conservation International, Washington, pp. 1–28.
Anderson, R.P. & Jarrín, P. (2002) A new species of spiny pocket mouse (Heteromyidae: Heteromys) endemic to western Ecuador. American Museum Novitates, 3382, 1–26.
https://doi.org/10.1206/0003-0082(2002)382<0001:ANSOSP>2.0.CO;2
Andrews, S.M. (2010) FastQC: a quality control tool for high throughput sequence data. Babraham Institute. Available from: http://www.bioinformatics.babraham.ac.uk/projects/fastqc (accessed 6 August 2020)
Bankevich, A., Nurk, S., Antipov, D., Gurevich, A.A., Dvorkin, M. & Kulikov, A.S. (2012) SPAdes: A new genome assembly algorithm and its applications to single-cell sequencing. Journal of Computational Biology, 19, 455–477.
https://doi.org/10.1089/cmb.2012.0021
Barbour, T. (1905) The vertebrata of Gorgona Island, Colombia: Reptilia; Amphibia. Bulletin of the Museum of Comparative Zoology. Cambridge, Massachusetts, 46, 98–102.
Bolger, A.M., Loshe, M. & Usadel, B. (2014) Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics, 30, 2114–2120.
https://doi.org/10.1093/bioinformatics/btu170
Boulenger, G.A. (1898) An account of the reptiles and batrachians collected by Mr. W. F. H. Rosenberg in western Ecuador. Proceedings of the Zoological Society of London, 1898, 107–126, 9 pls.
Camacho, C., Coulouris, G., Avagyan, V., Ma, N., Papadopoulos, J., Bealer, K. & Madden, T.L. (2009) BLAST plus: architecture and applications. BMC Bioinformatics, 10, 9.
https://doi.org/10.1186/1471-2105-10-421
Cantalamessa, G., Di Celma, C. & Ragaini, L. (2005) Sequence stratigraphy of the Punta Ballena Member of the Jama Formation (Early Pleistocene, Ecuador): insights from integrated sedimentologic, taphonomic and paleoecologic analysis of molluscan shell concentrations. Palaeogeography Palaeoclimatology Palaeoecology, 216, 1–25.
https://doi.org/10.1016/j.palaeo.2004.09.012
Di Celma, C., Cantalamessa, G., Landini, W. & Ragaini, L. (2010) Stratigraphic evolution from shoreface to shelf-indenting channel depositional systems during transgression: Insights from the lower Pliocene Sua Member of the basal Upper Onzole Formation, Borbon Basin, northwest Ecuador. Sedimentary Geology, 223, 162–179.
https://doi.org/10.1016/j.sedgeo.2009.11.009
Cerón, C., Palacios, W., Valencia, R. & Sierra, R. (1999) Las formaciones naturales de la Costa del Ecuador. In: Sierra, R. (Ed.), Propuesta preliminar de un sistema de clasificación de vegetación para el Ecuador continental. Proyecto INEFAN/GERF-BIRF y Ecociencia, Quito, pp. 55–74.
Cisneros-Heredia, D., Delia, J., Yánez, M.H. & Ortega, H.M. (2010) Endemic Ecuadorian glassfrog Cochranella mache is Critically Endangered because of habitat loss. Oryx, 44, 114.
https://doi.org/10.1017/S0030605309990640
Clark, J., Neill, D. & Asanza, M. (2006) Florisitic checklist of the Mache-Chindul mountains of northwestern Ecuador. Department of Botany, National Museum of Natural History, Smithsonian Institution, contribution of the United States National Herbarium, Washington D.C., 180 pp.
Clay, R., Jack, S. & Vincent, J. (1995) A stronghold for long-wattled umbrellabird discovered in Ecuador. Cotinga, 1995, 6–7.
Coloma, L.A. (1995) Ecuadorian frogs of the genus Colostethus (Anura: Dendobatidae). University of Kansas, Natural History Museum. Miscellaneous Publications, 87, 1–75.
https://doi.org/10.5962/bhl.title.16171
Cuesta-Camacho, F., Peralvo, M., Ganzenmüller, A., Sáenz, M., Novoa, J., Rifrío, G. & Beltrán, K. (2007) Identificación de vacíos y prioridades para la conservación de la biodiversidad terrestre en el Ecuador continental. In: Beltrán, K., Campos F., Clark, K., Cuesta-Camacho, F., Denkinger, J., Ganzenmüller, A., Jiménez, P. Luna, S., Novoa, J., Peralvo, M., Riofrío, G., Ruiz, D., Sáenz, M., Suárez, C. & Terán, M.C. (Eds.), Análisis de vacíos y áreas prioritarias para la conservación de la biodiversidad en el Ecuador continental. Instituto Nazca de Investigaciones Marinas, EcoCiencia, Ministerio del Ambien-te, The Nature Conservancy, Conservación Internacional, Quito, pp. 1–83.
Dingerkus, G. & Uhler, L.D. (1977) Enzyme clearing of alcian blue stained whole small vertebrates for demostration of cartilage. Stain Technology, 52, 229–232.
https://doi.org/10.3109/10520297709116780
Dodson, C.H. & Gentry, A.H. (1991) Biological extinction in western Ecuador. Annals of the Missouri Botanical Garden, 78, 273–295.
https://doi.org/10.2307/2399563
Duellman, W.E. & Trueb, L. (1986) Biology of Amphibians. McGraw-Hill Co., New York, 670 pp.
https://doi.org/10.2307/1445022
Dunn, E.R. (1933) Amphibians and reptiles from El Valle de Anton, Panama. Occasional Papers of the Boston Society of Natural History, 8, 65–79.
Fabrezi, M. & Alberch, P. (1996) The carpal elements of anurans. Herpetologica, 52, 188–204.
Frost, D.R. (2020) Amphibian species of the world: an online reference. Version 6.0. American Museum of Natural History, New York. Available from: http://research.amnh.org/herpetology/amphibia/index.html (accessed 6 August 2020)
Funkhouser, J.W. (1956) New frogs from Ecuador and southwestern Colombia. Zoologica. New York, 41, 73–80.
Giraldo, A. (2012) Isla Gorgona: paraíso de biodiversidad y ciencia. Universidad del Valle, Cali, 224 pp.
Grant, T., Frost, D.R., Caldwell, J.P., Gagliardo, R., Haddad, C.F.B., Kok, P.J.R., Means, D.B., Noonan, B.P., Schargel, W.E. & Wheeler, W.C. (2006) Phylogenetic systematics of dart-poison frogs and their relatives (Amphibia: Athesphatanura: Dendrobatidae). Bulletin of the American Museum of Natural History, 299, 1–262.
https://doi.org/10.1206/0003-0090(2006)299[1:PSODFA]2.0.CO;2
Grant, T., Rada, M., Anganoy-Criollo, M., Batista, A., Dias, P.H., Jeckel, A.M., Machado, D.J. & Rueda-Almonacid, J.V. (2017) Phylogenetic systematics of dart-poison frogs and their relatives revisited (Anura: Dendrobatoidea). South American Journal of Herpetology, 12, S1–S90.
https://doi.org/10.2994/SAJH-D-17-00017.1
Guayasamin, J.M. & Bonaccorso, E. (2004) A new species of glass frog (Centrolenidae: Cochranella) from the lowlands of northwestern Ecuador, with comments on the Cochranella granulosa group. Herpetologica, 60, 485–494.
IUCN (2019) Guidelines for using the IUCN Red List Categories and Criteria. Version 14. IUCN, Gland and Cambridge.
IUCN Species Survival Commission (2012) Guidelines for application of IUCN Red List Criteria at regional and national levels. Version 4.0. IUCN, Gland and Cambridge.
Kerr, A.C. (2005) La Isla de Gorgona, Colombia: A petrological enigma? Lithos, 84, 77–101.
https://doi.org/10.1016/j.lithos.2005.02.006
Kerr, A.C. & Tarney, J. (2005) Tectonic evolution of the Caribbean and northwestern South America: The case for accretion of two Late Cretaceous oceanic plateaus. Geology, 33, 269–272.
https://doi.org/10.1130/G21109.1
Li, D.H., Liu, C.M., Luo, R.B., Sadakane, K. & Lam, T.W. (2015) MEGAHIT: an ultra-fast single-node solution for large and complex metagenomics assembly via succinct de Bruijn graph. Bioinformatics, 31, 1674–1676.
https://doi.org/10.1093/bioinformatics/btv033
Liu, K., Raghavan, S., Nelesen, S., Linder, C.R. & Warnow, T. (2009) Rapid and accurate large-scale coestimation of sequence alignments and phylogenetic trees. Science, 324, 1561–1564.
https://doi.org/10.1126/science.1171243
Lynch, J.D. (1971) Evolutionary relationships, osteology, and zoogeography of leptodactyloid frogs. University of Kansas Museum of Natural History Miscellaneous Publications, University of Kansas, Museum of Natural History Lawrence, Kansas, 238 pp.
Marin, C.M., Molina-Zuluaga, C., Restrepo, A., Cano, E. & Daza, J.M. (2018) A new species of Leucostethus (Anura: Dendrobatidae) from the eastern versant of the Central Cordillera of Colombia and the phylogenetic status of Colostethus fraterdanieli. Zootaxa, 4461 (3), 359–380.
https://doi.org/10.11646/zootaxa.4464.3.3
Ministerio de Ambiente del Ecuador (2012) Sistema de Clasificación de los Ecosistemas del Ecuador Continental. s.n., Quito, 143 pp.
Morales, V.R. & Schulte, R. (1993) Dos especies nuevas de Colostethus (Anura, Dendrobatidae) en las vertientes de la Cordillera Oriental del Perú y del Ecuador. Alytes, 11, 97–106.
Neill, D.A. (1997) Coastal moist and wet forests, Ecuador. In: Davis, S.D., Haywood, V., Herrera-MacBryde, O., Villa-Lobos, J. & Hamilton, A.C. (Eds.), Centres of Plant Diversity: A Guide and Strategy for their Conservation. Vol. 3. The Americas. International Union for the Conservation of Nature, London, pp. 508–512.
Noble, G.K. (1921) Five new species of Salientia from South America. American Museum Novitates, 29, 1.
Ortega-Andrade, H.M., Meza-Ramos, P., Cisneros-Heredia, D.F. & Yánez-Muñoz, M. (2010a) Los anfibios y reptiles del Chocó Esmeraldeño. In: Altamirano-Benavides, M., Cisneros-Heredia, D.F. & Meza-Ramos, P. (Eds.), Serie Herpetofauna del Ecuador: El Chocó Esmeraldeño. Museo Ecuatoriano de Ciencias Naturales, Quito, pp. 95–252.
Ortega-Andrade, H.M., Rojas-Soto, O. & Paucar, C. (2013) Novel data on the ecology of Cochranella mache (Anura: Centrolenidae) and the importance of protected areas for this critically endangered glassfrog in the Neotropics. PLoS ONE, 8, 1–13.
https://doi.org/10.1371/journal.pone.0081837
Ortega-Andrade, M.H., Bermingham, J., Aulestia, C. & Paucar, C. (2010b) Herpetofauna of the Bilsa Biological Station, province of Esmeraldas, Ecuador. Check List, 6, 119–154.
https://doi.org/10.15560/6.1.119
Posada, D. (2008) jModelTest: Phylogenetic Model Averaging. Molecular Biology and Evolution, 25, 1253–1256.
https://doi.org/10.1093/molbev/msn083
Pyron, A.R. & Wiens, J.J. (2011) A large-scale phylogeny of Amphibia including over 2800 species, and a revised classification of extant frogs, salamanders, and caecilians. Molecular Phylogenetics and Evolution, 61, 543–583.
https://doi.org/10.1016/j.ympev.2011.06.012
Pyron, R.A. (2014) Biogeographic analysis reveals ancient continental vicariance and recent oceanic dispersal in amphibians. Systematic Biology, 63, 779–797.
https://doi.org/10.1093/sysbio/syu042
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, Oxford, pp. 57–75.
de Queiroz, K. (1999) The general lineage concept of species and the defining properties of the species category. In: Wilson, R.A. (Ed.), Species, New Interdisciplinary Essays. Bradford/MIT Press, Cambridge, Massachusetts, pp. 48–89.
Rivero, J.A. & Granados-Díaz, H. (1990) “1989”. Nuevos Colostethus (Amphibia, Dendrobatidae) del Departamento de Cauca, Colombia. Caribbean Journal of Science, 25, 148–152.
Rivero, J.A. & Serna, M.A. (1986) Dos nuevas especies de Colostethus (Amphibia, Dendrobatidae). Caldasia, 15, 525–531.
Rivero, J.A. & Serna, M.A. (1991) Tres nuevas especies de Colostethus (Anfibia [sic], Dendrobatidae) de Colombia. Trianea, 4, 481–495.
Rivero, J.A. & Serna, M.A. (2000) “1995”. Nuevos Colostethus (Amphibia, Dendrobatidae) del Departamento de Antioquia, Colombia, con la descripción del renacuajo de Colostethus fraterdanieli. Revista de Ecología Latino-Americana, 2, 45–58.
Sabaj Pérez, M.H. (Ed.) (2014) Standard symbolic codes for institutional resource collections in herpetology and ichthyology: an Online Reference. Version 5.0 (22 September 2014). American Society of Ichthyologists and Herpetologists, Washington, D.C. [program]
Sanderson, M.J. (2002) Estimating absolute rates of molecular evolution and divergence times: A penalized likelihood approach. Molecular Biology and Evolution, 19, 101–109.
https://doi.org/10.1093/oxfordjournals.molbev.a003974
Santos, J.C., Baquero, M., Barrio Amorós, C.L., Coloma, L.A., Erdtmann, L.K., Lima, A.P. & Cannatella, D.C. (2014) Aposematism increases acoustic diversification and speciation in poison frogs. Proceedings of the Royal Society B: Biological Sciences, 281, 20141761.
https://doi.org/10.1098/rspb.2014.1761
Santos, J.C. & Cannatella, D.C. (2011) Phenotypic integration emerges from aposematism and scale in poison frogs. Proceedings of the National Academy of Sciences, 108, 6175–6180.
https://doi.org/10.1073/pnas.1010952108
Santos, J.C., Coloma, L.A. & Cannatella, D.C. (2003) Multiple, recurring origins of aposematism and diet specialization in poison frogs. Proceedings of the National Academy of Sciences, 100, 12792–12797.
https://doi.org/10.1073/pnas.2133521100
Santos, J.C., Coloma, L.A., Summers, K., Caldwell, J.P., Ree, R. & Cannatella, D.C. (2009) Amazonian amphibian diversity is primarily derived from late Miocene Andean lineages. PLoS Biology, 7, 0448–0461.
https://doi.org/10.1371/journal.pbio.1000056
Santos, J.C., Tarvin, R.D., O’Connell, L.A., Blackburn, D.C. & Coloma, L.A. (2018) Diversity within diversity: Parasite species richness in poison frogs assessed by transcriptomics. Molecular Phylogenetics and Evolution, 125, 40–50.
https://doi.org/10.1016/j.ympev.2018.03.015
Sierra, R. (1996) La deforestación del noroccidente del Ecuador, 1983-1993. EcoCiencia, Quito, 20 pp.
Silverstone, P.A. (1971) Status of certain frogs of the genus Colostethus, with descriptions of new species. Los Angeles County Museum Contributions in Science, 1971, 1–8.
Spix, J.B. von (1824) Animalia nova sive species novae Testudinum et Ranarum, quas in itinere per Brasiliam annis MDCCCXVII - MDCCCXX jussu et auspiciis Maximiliani Josephi I. Bavariae Regis. Seraph. Hübschmanni, Monachii. [unkown pagination]
https://doi.org/10.5962/bhl.title.63182
Trueb, L. (1993) Patterns of cranial diversity in Lissamphibia. Vol. 2. Patterns of structural diversity. In: Hanken, J. & Hall, B.K. (Eds.), The skull. The University of Chicago Press, Chicago, Illinois, pp. 255–343.
Vasquez-Velez, A.I. (2014) Structure and diversity of the vegetation of the Gorgona Island National Natural Park, Colombia. Revista de Biología Tropical, 62, 13–26.
https://doi.org/10.15517/rbt.v62i0.15976
Vences, M., Kosuch, J., Boistel, R., Haddad, C.F.B., La Marca, E. & Lötters, S. (2003) Convergent evolution of aposematic coloration in Neotropical poison frogs: a molecular phylogenetic perspective. Organisms Diversity & Evolution, 3, 215–226.
https://doi.org/10.1078/1439-6092-00076
Zwickl, D.J. (2006) Genetic algorithm approaches for the phylogenetic analysis of large biological sequence datasets under the maximum likelihood criterion. Ph. D. thesis, University of Texas, Austin, 115 pp.