Evolution in the Genus Rhinella: A Total Evidence Phylogenetic Analysis of Neotropical True Toads (Anura: Bufonidae)

True toads of the genus Rhinella are among the most common and diverse group of Neotropical anurans. These toads are widely distributed throughout South America, inhabiting a great diversity of environments and ecoregions. Currently, however, the genus is defined solely on the basis of molecular characters, and it lacks a proper diagnosis. Although some phenetic species groups have traditionally been recognized within Rhinella, the monophyly of some of them have been rejected in previous phylogenetic analyses, and many species remain unassigned to these poorly defined groups. Additionally, the identity and taxonomy of several species are problematic and hinder the specific recognition and description of undescribed taxa. In this work, we first perform phylogenetic analyses of separate mitochondrial and nuclear datasets to test the possible occurrence of hybridiza-tion and/or genetic introgression in the genus. The comparative analysis of both datasets revealed unidirectional mitochondrial introgressions of an unknown parental species into R . horribilis (“ghost introgression”) and of R . dorbignyi into R . bernardoi; therefore, the mitochondrial and nuclear data-sets of these species were considered separately in subsequent analyses. We performed total-evidence phylogenetic analyses that included revised molecular (four mitochondrial and five nuclear genes) and phenotypic (90 characters) datasets for 83 nominal species of Rhinella, plus several undescribed and problematic species and multiple outgroups. Results demonstrate that Rhinella was nonmono-phyletic due to the position of R . ceratophrys, which was recovered as the sister taxon of Rhaebo nasicus with strong support. Among our outgroups, the strongly supported Anaxyrus + Incilius is the sister clade of all other species of Rhinella. Once R . ceratophrys is excluded, the genus Rhinellais monophyletic, well supported, and composed of two major clades. One of these is moderately supported and includes species of the former R . spinulosa Group (including R . gallardoi); the mono-phyletic R . granulosa, R . crucifer, and R . marina Groups; and a clade composed of the mitochondrial sequences of R . horribilis. The other major clade is strongly supported and composed of all the spe-cies from the non-monophyletic R . veraguensis and R . margaritifera Groups, the former R . acrolophaGroup, and R . sternosignata. Consistent with these results, we define eight species groups of Rhinella that are mostly diagnosed by phenotypic synapomorphies in addition to a combination of morpho-logical character states. Rhinella sternosignata is the only species that remains unassigned to any group. We also synonymize nine species, treat three former subspecies as full species, and suggest that 15 lineages represent putative undescribed species. Lastly, we discuss the apparently frequent occurrence of hybridization, deep mitochondrial divergence, and “ghost introgression”; the incomplete phenotypic evidence (including putative character systems that could be used for future phy-logenetic analyses); and the validity of the known fossil record of Rhinella as a source of calibration points for divergence dating analyses.Peer reviewe

Análise filogenética das rãs de desenvolvimento direto (Anura, Terrarana, Craugastoridae)

A major recent trend in systematics is the re-integration of morphological data into total evidence analysis. To date, few studies have explored the effects of incorporating morphological and molecular data into total evidence analyses; however, those that have done so have found that even a comparatively small phenomic dataset can have disproportionately large impacts on results. Given the demonstrated importance of morphological characters in testing the phylogenetic relationships, herein I provide a case in point for the exploration of the effects of incorporating morphological evidence as an independent source of phylogenetic evidence, and an important framework to test hypotheses supported by molecular evidence, in the superfamily Brachycephaloidea. In this study, 338 amphibian species are included, of which 318 species correspond to the superfamily Brachycephaloidea. All known genera within the superfamily were sampled. The complete dataset included 13,686 molecular characters (mitochondrial and nuclear markers) and 185 morphological characters, which were analyzed together. In addition, I performed additional analyses modifying the complete datasets to evaluate the effects of character and taxon sampling. As results, I found that the superfamily Brachycephaloidea does not represent a monophyletic as previously thought. Likewise, the phylogeny resulting from this analysis showed several taxa to be nonmonophyletic: Brachycephalidae and Craugastoridae as well as genera Craugastor, Psychrophynella, and Pristimantis. From the detailed examination of the impact of both character and taxon sampling on the phylogenetic relationships of the superfamily Brachycephaloidea, I found that the inclusion of a comparatively small phenomic dataset (185 character) as well as the inclusion of two key taxa (Atopophrynus syntomopus and Dischidodactylus duidensis) had disproportionately large impacts on the tree topology. Finally, I provide a new taxonomy for this group. In this, I recognize five families, of which three are morphologically diagnosable (Eleutherodactylidae, Hypodactylidae, and Strabomantidae) whereas the other two are diagnosable solely with molecular evidence (Ceuthomantidae and Craugastoridae). Likewise, 12 out of 26 genera included are morphologically diagnosable.Uma grande tendência em estudos de sistemática filogenética é a reintegração de dados morfológicos em análises de evidência total. Até o momento, poucos estudos exploraram os efeitos da incorporação de dados morfológicos conjuntamente com dados moleculares em análises de evidência total. No entanto, aqueles que o fizeram concluíram que mesmo uma pequena base de dados fenômicos pode ter grandes e desproporcionais impactos nos resultados. Dada a demonstrada importância de caracteres morfológicos em testes de relacionamentos filogenéticos, aqui apresento um caso de exploração dos efeitos da incorporação de evidência morfológica como uma fonte independente de evidência filogenética e como importante \"framework\" para testar hipóteses suportadas por evidência molecular na superfamília Brachycephaloidea. Nesse estudo, 338 espécies de anfíbios foram incluídas, das quais 318 correspondem a espécies da superfamília Brachycephaloidea. Todos os gêneros conhecidos da superfamília foram amostrados. A base de dados completa inclui 13.686 caracteres moleculares (marcadores mitocondriais e nucleares) e 185 caracteres morfológicos, que foram analisados conjuntamente. Ademais, realizei análises adicionais modificando as bases de dados para avaliar os efeitos de amostragem de caracteres e táxons. Como resultado, encontrei que a superfamília Brachycephaloidea não é monofilética, como anteriormente considerada. Da mesma forma, a filogenia resultante da análise de evidência total mostrou que vários outros táxons também não são monofiléticos: as famílias Brachycephalidae e Craugastoridae, assim como os gêneros Craugastor, Psychrophrynella e Pristimantis. Através do exame detalhado do impacto de diferentes amostragens de caracteres e táxons sobre as relações filogenética na superfamília Brachycephaloidea, encontrei que a inclusão de dois táxons chaves (Atopophrynus syntomopus e Dischidodactylus duidensis) tiveram grandes e desproporcionais impactos na topologia das árvores. Finalmente, eu apresento uma nova taxonomia para as rãs de desenvolvimento direto, reconhecendo cinco famílias, das quais três são morfologicamente diagnosticáveis (Eleutherodactylidae, Hypodactylidae e Strabomantidae), enquanto as outras duas são diagnosticáveis apenas com evidência molecular (Ceuthomantidae e Craugastoridae). Da mesma forma, 12 dos 26 gêneros inclusos são morfologicamente diagnosticáveis

Revisión morfológica de las ranas del género Strabomantis (Anura: Brachycephalidae Sensu frost et al. 2006)

Las Ranas de desarrollo directo o carentes de etapas larvales en su desarrollo (Eleutherodactylus sensu lato) han presentado significantes cambios en su clasificación, inicialmente basada en morfología y más recientemente en herramientas moleculares. En este contexto el siguiente trabajo empleó caracteres osteológicos y miológicos en los grupos de especies que conforman el género Strabomantis, con el objetivo de buscar homologías que puedan apoyar la monofilia de estos grupos, así como también la del género. Se encontró evidencia en donde se demuesta que los grupos de especies deben ser reorganizados, en cuanto al género Strabomantis se encontró que es parafilético con respecto al género Hypodactylus. Se hace necesario la reevaluación detallada del estatus genérico de Strabomantis zygodactylus, pues esta especie según la evidencia mostrada, está más relacionada con las especies del género Craugastor y Oreobates.Abstract. The Direct-developing frogs or frogs without larval stage development (Eleutherodactylus sensu lato) have undergone significant changes in their systematics, initially based on morphology and more recently on molecular data. In this context, this work used osteological and miological characters in the species groups that comprise the Strabomantis genus, aiming to search for homologies that could support the monophyly of these groups, as well as that of the genus. The evidence supports the reorganization of groups and demonstrates Strabomantis genus is paraphyletic with respect to the Hypodactylus genus. It is necessary a detailed evaluation of generic status of Strabomantis zygodactylus because this species, according to the evidence shown, is more closely related to the species of Craugastor and Oreobates genus.Maestrí

Pristimantis calima Ospina-Sarria, 2019, sp. nov.

Pristimantis calima sp. nov. (Figs. 1A, 2A,E, 3A; Table 1) Holotype. — KU 168849, adult male from the Río Calima, 1.5 km (by road) west of Lago Calima, 1230-m elevation (3°53 ′ N, 76°34 ′ W; datum ¼ WGS84), Departamento del Valle del Cauca, Colombia, one of a series collected by William E. Duellman and Linda Trueb on 31 May and 1 June 1975. Paratypes. — KU 168846–48, 168851, adult females collected with the holotype. Diagnosis. — Pristimantis calima is diagnosed by the following combination of characters: (1) skin on dorsum smooth, becoming finely shagreen laterally and coarse on the flanks; ventral skin areolate; discoidal fold present, well anterior to groin; dorsolateral folds and ridges absent in scapula region; (2) tympanic membrane differentiated, round; its length 36.3% of eye length in one male and 26.4–38.6% in four females; tympanic annulus prominent, not covered by supratympanic fold, which extends from posterior corner of orbit along upper edge of temporal region toward insertion of arm; (3) snout moderately long, subacuminate in dorsal view, rounded in profile, with papilla at tip; canthus rostralis straight; (4) upper eyelid bearing 3–6 tubercles small, narrower than IOD (53.5–73.3% IOD); small interocular tubercle present; cranial crests absent; (5) choanae small, ovoid; not concealed by palatal shelf of maxillary arch; dentigerous processes of vomers prominent, triangular in outline, separated medially by a distance equal to the width of the visible dentigerous process, located well behind posterior edges of choanae, each odontophore bearing 4–5 teeth; (6) male with vocal slits and subgular vocal sac evident externally; nuptial pads present on thumbs; (7) finger I shorter than finger II; discs and circumferential grooves present on all fingers; discs truncate, except on finger I, which is round; disc of finger I smaller than that of finger II and this in turn smaller than discs on fingers III and IV; (8) fingers with lateral fringes; palmar tubercle not divided; thenar tubercle oval, slightly smaller than palmar tubercle; supernumerary tubercles low, distributed on all fleshy parts of palm; subarticular tubercles round, two on thumb and second finger and three on third and fourth fingers, and larger than supernumerary tubercles; (9) two or three ulnar tubercles subconical, not coalesced (Fig. 2A); (10) heel and outer edge of tarsus lacking tubercles; inner tarsal tubercle indistinct; (11) inner metatarsal tubercle elongate, its length twice its width; low, conical outer metatarsal tubercle one-fourth size of inner metatarsal tubercle; subarticular tubercles round, two on toes I and II, three on toes III and V, and four on toe IV; supernumerary plantar tubercles low, rounded, on proximal segments of toes; (12) toes bearing prominent lateral fringes; basal webbing not encompassing basal subarticular tubercles, except on outer border of toe IV (Fig. 2E); toe III shorter than toe V; toe III extending to distal edge of the antepenultimate subarticular tubercle of toe IV; toe V reaching distal edge of penultimate subarticular tubercle of toe IV; discs and circumferential grooves present on all toes; discs of toes III – V equal to discs on fingers III – IV and larger than discs of toes I–II; (13) color in life: dorsum dull green, reddish brown, or olive brown; ventral surfaces of hind limbs pinkish lavender; groin and anterior surfaces of thighs cream brown; throat and belly surfaces yellow; iris pale creamy bronze with median red streak; (14) SVL in the only known adult male 24.0 mm; in adult females 37.7–40.0 mm (mean ± 1 SD ¼ 38.8 ± 0.90 mm; n ¼ 4). 3 CORRESPONDENCE: e-mail, [email protected] Comparisons. — Pristimantis calima differs from the other species in the genus by having basal webbing on outer edge of toe IV, subarticular tubercles beneath the joint between distal phalanges, papilla at the tip of snout, and uniform cream-brown coloration on the anterior surfaces of thighs and in the groin. It is most similar to three Ecuadorian species— P. eugeniae, P. n&eng;ctoph&eng;lax, and P. subsigillatus (Fig. 1) by having subarticular tubercles beneath the joint between distal phalanges, dorsal skin smooth, ventral skin areolate, discoidal fold well anterior to groin, snout subacuminate in dorsal view and rounded in profile, papilla at tip of snout, vocal slits and nuptial pads present, dentigerous processes of vomers prominent, tympanic membrane differentiated, tympanic annulus not covered by supratympanic fold, and by lacking of tubercles on medial surface of tarsus. However, P. calima has more webbing on outer edge of toe IV that encompasses the basal subarticular tubercle (not encompassing basal subarticular tubercle in those species; Fig. 2E–H). Furthermore, females of P. calima are larger (SVL 37.7–40.0 mm) than those of P. eugeniae (SVL 30.5–37.6 mm; Lynch and Duellman 1997) and P. subsigillatus (SVL 30.0– 33.4 mm; Lynch and Duellman 1997). Also, P. calima differs from P. eugeniae by having a row of three or four subconical tubercles on the postaxial surface of the forearm (only the antebrachial tubercle is present in P. eugeniae; Fig. 2A,B). Furthermore, P. calima differs from P. n&eng;ctoph&eng;lax and P. subsigillatus by having uniform cream-brown coloration on anterior surfaces of thighs and groin area (black bars or gray reticulations of the anterior surfaces of the thighs and groin in P. n&eng;ctoph&eng;lax and P. subsigillatus; Fig. 3). Finally, P. calima could be considered similar in appearance to P. mindo Arteaga et al. (2013); however, P. calima has basal webbing on the outer edge of toe IV, papilla at the tip of snout, nuptial pads present on thumbs, and females having uniform creambrown coloration on the anterior surfaces of thighs and in the groin (instead of flash marks on the thighs and groin in P. mindo). : Among species of Pristimantis in western Colombia, few have webbing between toes; these are P. albericoi (Lynch and Ruiz-Carranza 1996), P. bernali (Lynch 1986), P. diaphonus (Lynch 1986), P. diogenes (Lynch and RuizCarranza 1996), P. h&eng;botragus (Lynch 1992), P. jaimei (Lynch 1992), and P. loustes (Lynch 1979). In comparison with P. calima, P. albericoi is smaller, with a SVL of 20.6– 20.7 mm in adult males and 25.0– 29.3 mm in adult females (Lynch and Ruiz-Carranza 1996) and has low postocular folds (SVL 24.0 mm in adult males and 37.7–40.0 mm of SVL in adult females, and postocular folds absent in P. calima). Likewise, P. bernali lacks vocal slits, nuptial pads on thumb, and papilla on the tip of the snout (characters present in P. calima), whereas P. diaphonus and P. loustes differ by having toes half webbed and no tympanic membrane (basal webbing not encompassing basal subarticular tubercles except on outer border of toe IV and tympanic membrane evident in P. calima). Webbing encloses the basal subarticular tubercles on the toes and an inner tarsal fold characterizes P. diogenes and P. jaimei (basal webbing not encompassing basal subarticular tubercles except on outer border of toe IV and tarsal fold absent in P. calima). Finally, P. h&eng;botragus is distinguished by having a flaplike inner tarsal fold (inner tarsal fold absent in P. calima). Description of the holotype. —An adult male with head wider than body; head width 42.5% of SVL; head length 37.5% of SVL; snout moderately long, subacuminate in dorsal view, rounded in profile; snout having one papilla at its tip; eye–nostril distance 69.6% of diameter of eye; nostrils not protuberant, directly lateral at level of lower jaw. Canthus rostralis straight, not elevated; loreal region concave lacking tubercles; lips rounded; internarial region slightly depressed; top of head flat with small conical tubercle between upper eyelids; upper eyelid with three small tubercles; its width 66.6% of IOD; supratympanic fold slightly curved downward just posterior to orbit; tympanic membrane and tympanic annulus present, not covered by the supratympanic fold; two enlarged postrictal tubercles present. Choanae small, nearly round, not obscured by palatal shelf; dentigerous processes of vomers prominent, each process bearing four teeth; tongue as long as wide, its posterior border not notched, posterior third not adherent to floor of mouth; paired vocal slits present, longitudinal, lateral to base of tongue; median subgular vocal sac present. Skin on dorsum smooth, becoming finely shagreen on the flanks; skin on belly, throat, and ventral surfaces of the thighs areolate; discoidal fold present well anterior to groin; dorsolateral folds absent; cloacal sheath short; no tubercles in cloacal region. Three ulnar tubercles forming a row along the ventrolateral edge of the forearm; palmar tubercle not divided; thenar tubercle oval, slightly smaller than palmar tubercle; subarticular tubercles round, prominent, two on thumb and second finger and three on third and fourth fingers, and larger than supernumerary tubercles; supernumerary tubercles low, on all fleshy parts of palm; fingers having lateral fringes; relative lengths of fingers I <II <IV <III, all fingers having terminal ventral pads well defined by circumferential grooves; disc on thumb narrow, that on finger II slightly smaller than tympanic annulus; discs on fingers III and IV as wide as tympanic annulus; white, nonspinous nuptial pads are present on the dorsomedial surface of the base of the thumb. Hind limbs moderately robust; when hind limbs flexed perpendicular to axis of body, heels overlap; tibia length 50.4% of SVL; foot length 46.2% of SVL; heel and outer edge of tarsus lacking of tubercles; inner tarsal tubercle indistinct; inner metatarsal tubercle elongate, its length twice its width; oval outer metatarsal tubercle one-fourth size of inner; toes bearing lateral fringes and discs (about as wide as long) on expanded pads; basal webbing not encompassing basal subarticular tubercles, except on outer border of toe IV; relative lengths of toes I <II <III <V <IV; toe III much shorter than toe V; toe III extending to distal edge of the antepenultimate subarticular tubercle of toe IV; toe V reaching distal edge of penultimate subarticular tubercle of toe IV; all toes having terminal ventral pads well defined by circumferential grooves; discs of toes III–V equal to discs on fingers III–IV and larger than discs of toes I–II; subarticular tubercles prominent, rounded, two on toes I and II, three on toes III and V, and four on toe IV; supernumerary tubercles low, rounded, numerous on proximal segments of digits. In life, dorsum dull green, reddish brown, or olive brown (Fig. 1A). Ventral surfaces of hind limbs pinkish lavender, throat and belly surfaces yellow. Iris pale creamy bronze with medial red streak (WED, field catalog, 31 May 1974). Measurements of holotype (mm). —SVL 24.0, tibia length 12.1, foot length 11.1, HL 9.0, head width 10.2, IOD 3.0, internarial distance 2.0, width of upper eyelid 2.0, diameter of eye 3.3, eye–nostril distance 2.3, diameter of tympanum 1.2. Distribution and ecology. — Pristimantis calima is known only from the type locality in lower montane rain forest in a deep valley with small, cascading streams (Fig. 4). The frogs were found on leaves, on the ground, and on tree trunks 2–3 m above the ground at night. During the 2 d and nights spent at the type locality on 31 May and 1 June 1975, temperature varied from 14 to 25°C. The observed daily rainfall was 19 mm and 4 mm, respectively. Etymology. —The specific name is a noun in apposition. It is the name of the type locality, the Río Calima. This locality is recognized as one of the most important archaeological reserves in Colombia because it contains the prints of an ancient Colombian civilization that was identified as ‘‘Calima.’’ Remarks. —The humid Pacific slopes of the Cordillera Occidental and adjacent Chocoan lowlands harbor a great diversity of amphibians, especially centrolenid, dendrobatid, and strabomantid frogs. WED (with L. Trueb and J.E. Simmons) spent 3 d at the Río Calima site in September 1974 and 2 d there in May and June 1975. During these 5 d and nights they found five new species: N&eng;mphargus prasinus (Duellman 1981), Gastrotheca dendronastes Duellman 1983, H&eng;loscirtus simmonsi (Duellman 1989), Pristimantis calima, and P. diaphonus (Lynch 1986). Other sympatric species found were: P. achatinus (Boulenger 1898), P. er&eng;thropleura (Boulenger 1896), P. orpacobates (Lynch et al. 1994), N. grandisonae (Cochran and Goin 1970), and N. griffithsi (Goin 1961).Published as part of Ospina-Sarria, Jhon Jairo, 2019, Two New Species of Pristimantis (Amphibia: Anura: Strabomantidae) from Southwestern Colombia, pp. 85-95 in Herpetologica 75 (1) on pages 85-89, DOI: 10.1655/d-18-00019, http://zenodo.org/record/771726

A Taxonomic Review of Tan-Brown Glassfrogs (Anura: Centrolenidae), with the Description of a New Species from Southwestern Colombia

Thirty species of the glassfrogs genus Nymphargus, including a new species, are known to inhabit the cloud forest of the Andes of Colombia and Ecuador. Four of these species (Nymphargus anomalus, N. ignotus, N. rosada, and the new species) are unusual by having a tanbrown dorsal coloration instead of the widespread green coloration commonly found in glassfrogs. Herein, we review tan-brown glassfrogs species, providing information on their external morphology, color ontogeny, ecology, and distribution. The new taxon inhabits the Andes of southwestern Colombia, Departamento del Cauca, and it is distinguished from other centrolenids by having a tan-brown dorsum with dark spots lacking ocelli, reduced (basal) webbing between outermost fingers, and lacking iridophores on the digestive tract and hepatic peritonea. We also find that Nymphargus ignotus is a polymorphic species distributed along the western flank of the Cordillera Occidental in Colombia. The egg masses of N. ignotus are similar to those known in others Nymphargus species, except for the presence of supplementary empty capsules. The distribution, abundance and function of empty capsules and the nature of the tan-brown color pattern in glassfrogs are discussed. © 2017 Brazilian Society of Herpetology

The Complex Evolutionary History of the Tympanic Middle Ear in Frogs and Toads (Anura)

Most anurans possess a tympanic middle ear (TME) that transmits sound waves to the inner ear; however, numerous species lack some or all TME components. To understand the evolution of these structures, we undertook a comprehensive assessment of their occurrence across anurans and performed ancestral character state reconstructions. Our analysis indicates that the TME was completely lost at least 38 independent times in Anura. The inferred evolutionary history of the TME is exceptionally complex in true toads (Bufonidae), where it was lost in the most recent common ancestor, preceding a radiation of \u3e150 earless species. Following that initial loss, independent regains of some or all TME structures were inferred within two minor clades and in a radiation of \u3e400 species. The reappearance of the TME in the latter clade was followed by at least 10 losses of the entire TME. The many losses and gains of the TME in anurans is unparalleled among tetrapods. Our results show that anurans and especially bufonid toads, are an excellent model to study the behavioural correlates of earlessness, extratympanic sound pathways and the genetic and developmental mechanisms that underlie the morphogenesis of TME structures

Evolution in Rhinella (Anura: Bufonidae)

155 pages : illustrations (some color) ; 26 cm.True toads of the genus Rhinella are among the most common and diverse group of Neotropical anurans. These toads are widely distributed throughout South America, inhabiting a great diversity of environments and ecoregions. Currently, however, the genus is defined solely on the basis of molecular characters, and it lacks a proper diagnosis. Although some phenetic species groups have traditionally been recognized within Rhinella, the monophyly of some of them have been rejected in previous phylogenetic analyses, and many species remain unassigned to these poorly defined groups. Additionally, the identity and taxonomy of several species are problematic and hinder the specific recognition and description of undescribed taxa. In this work, we first perform phylogenetic analyses of separate mitochondrial and nuclear datasets to test the possible occurrence of hybridization and/or genetic introgression in the genus. The comparative analysis of both datasets revealed unidirectional mitochondrial introgressions of an unknown parental species into R. horribilis ("ghost introgression") and of R. dorbignyi into R. bernardoi; therefore, the mitochondrial and nuclear datasets of these species were considered separately in subsequent analyses. We performed total-evidence phylogenetic analyses that included revised molecular (four mitochondrial and five nuclear genes) and phenotypic (90 characters) datasets for 83 nominal species of Rhinella, plus several undescribed and problematic species and multiple outgroups. Results demonstrate that Rhinella was nonmonophyletic due to the position of R. ceratophrys, which was recovered as the sister taxon of Rhaebo nasicus with strong support. Among our outgroups, the strongly supported Anaxyrus + Incilius is the sister clade of all other species of Rhinella. Once R. ceratophrys is excluded, the genus Rhinella is monophyletic, well supported, and composed of two major clades. One of these is moderately supported and includes species of the former R. spinulosa Group (including R. gallardoi); the monophyletic R. granulosa, R. crucifer, and R. marina Groups; and a clade composed of the mitochondrial sequences of R. horribilis. The other major clade is strongly supported and composed of all the species from the non-monophyletic R. veraguensis and R. margaritifera Groups, the former R. acrolopha Group, and R. sternosignata. Consistent with these results, we define eight species groups of Rhinella that are mostly diagnosed by phenotypic synapomorphies in addition to a combination of morphological character states. Rhinella sternosignata is the only species that remains unassigned to any group. We also synonymize nine species, treat three former subspecies as full species, and suggest that 15 lineages represent putative undescribed species. Lastly, we discuss the apparently frequent occurrence of hybridization, deep mitochondrial divergence, and "ghost introgression"; the incomplete phenotypic evidence (including putative character systems that could be used for future phylogenetic analyses); and the validity of the known fossil record of Rhinella as a source of calibration points for divergence dating analyses