Research Article |
Corresponding author: Sueny P. dos Santos ( palomavoava@hotmail.com ) Academic editor: Franco Andreone
© 2015 Sueny P. dos Santos, Roberto Ibáñez, Santiago R. Ron.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
dos Santos SP, Ibáñez R, Ron SR (2015) Systematics of the Rhinella margaritifera complex (Anura, Bufonidae) from western Ecuador and Panama with insights in the biogeography of Rhinella alata. ZooKeys 501: 109-145. https://doi.org/10.3897/zookeys.501.8604
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The Rhinella margaritifera species group consists of 17 species of toads distributed in tropical and subtropical South America and eastern Central America. The identity of some of its species is poorly understood and there are numerous undescribed cryptic species. Among them, the status of Rhinella margaritifera is one of the most problematic. Its range includes lowland rainforests separated by the Andes, the Chocoan rainforest to the west and the Amazonian rainforest to the east. This distribution is puzzling because the Andes are an old and formidable barrier to gene flow and therefore should generate vicariant speciation between disjunct lowland populations. Herein we clarify the taxonomy of populations of the R. margaritifera complex from Central America and the Chocó region of South America. The morphological and genetic variation of R. margaritifera was examined from 39 populations from Chocó, 24 from the upper Amazon region of Ecuador, and 37 from Panama, including the holotype of the Panamanian R. alata. Phylogenetic analyses were performed based on mitochondrial genes 12S rRNA, 16S rRNA, and cytochrome c oxidase I (COI) and the nuclear gene Tyrosinase (Tyr). The genetic and morphological data show that Panamanian and Chocoan populations are conspecific. In the phylogeny, populations from Chocó and Panama form a well-supported clade. The morphology of the holotype of R. alata falls within the variation range of Panamanian and Chocoan populations. Based on all this evidence, we assign the populations from western Ecuador and Panama to R. alata and demonstrate that the unusual distribution pattern of “R. margaritifera” on both sides of the Andes was an artifact of incorrectly defined species boundaries.
Andes, Biogeography, Chocó, Morphology, Panama, Phylogeny, Rhinella alata
Rhinella is a genus of bufonid frogs distributed from southern Texas, through southern Sonora (Mexico), south tropical Mexico, Central America, and South America. There are 87 recognized species of Rhinella (Frost, 2014) among which 17 belong to the R. margaritifera species group (
The R. margaritifera species group (formerly Bufo typhonius or Bufo margaritifer group) has one of the most complex histories in the systematics of Neotropical anurans (
Two species of the R. margaritifera group have been reported west of the Andes (Chocó region, humid forests west of the Andes in Colombia and Ecuador) and in eastern Panama: R. alata and R. margaritifera. R. alata was described by
Rhinella margaritifera was described by Laurenti in 1768. It occurs in eastern Panama (
The distribution of R. margaritifera in the humid lowlands west and east of the Andes is intriguing because, particularly for amphibians, the Andes represent a formidable barrier to gene flow (e.g.
Herein, genetic and morphological information were integrated to clarify the taxonomy of the populations of R. margaritifera from Panama and the Chocoan region. Populations from the western and eastern Andean slopes were compared to test the role of the Andes as a dispersal barrier in shaping the evolution of the R. margaritifera species complex.
Populations from Panama, the Ecuadorian Chocó, and the Amazon basin were sampled (Figs
Morphometric analyses were based on 120 adult specimens of R. margaritifera from Panama (14 specimens from 10 populations), Ecuadorian Chocó (74 specimens, 37 populations), and the Ecuadorian Amazon (32 specimens, 18 populations). Qualitative morphological characters were examined in the same specimens and 28 additional individuals from 27 Panamanian populations (Figs
Genetic analyses were based on newly generated sequences of R. margaritifera from 32 individuals and 19 populations: R. margaritifera from the Ecuadorian Chocó (12 individuals, 7 populations); R. margaritifera from Panama (3 individuals, 2 populations) and R. margaritifera from the Amazon basin (17 individuals, 10 populations), and six sequences for the outgroups (see Table
Examined specimens are deposited at the Museo de Zoología, Pontificia Universidad Católica del Ecuador (QCAZ, Quito, Ecuador), the American Museum of Natural History (AMNH, New York, USA), Círculo Herpetológico de Panama (CH, Panama, Panama), Centro de Ornitología y Biodiversidad (CORBIDI, Lima, Perú) and Museo de Vertebrados de la Universidad de Panama (MVUP). We also examined photographs of the holotypes of R. alata from Musée National d’Historie Naturelle (MNHN, Paris, France). Tissues were obtained from the QCAZ and CH collections. Tissues (liver or thigh muscle) were stored in 95% ethanol.
Morphological terminology and abbreviations follow
The goal of the morphological analyses was to compare three geographic regions: (1) Chocó (2) Panama, and (3) upper Amazon basin. Because the phylogeny showed that Panama and Chocó populations are conspecific, we also compared Chocó + Panama vs. upper Amazon. Morphometric analyses were based on adult and well-preserved specimens (
Principal Components Analysis (PCA) and Discriminant Function Analysis (DFA) were used to assess morphometric differentiation between Chocó, upper Amazon, and Panama. To remove the effect of body size (SVL), the MANOVA and PCA were applied to the residuals from the linear regressions between the measured variables and SVL, for males and females separately. For the PCA, only components with eigenvalues > 1 were retained. All measurements were first subjected to the Shapiro-Wilk normality to test for normal distribution (
Total DNA was extracted from muscle or liver tissue preserved in 95% ethanol or tissue storage buffer using standard guanidine thiocyanate protocol (M. Fujita, unpublished) with modifications. Polymerase Chain Reaction (PCR) was used to amplify the mitochondrial genes 12S rRNA, 16S rRNA, cytochrome c oxidase I (COI) and nuclear gene Tyrosinase (Tyr). PCR amplifications were carried out under standard protocols. Using standard primers developed by
Preliminary sequence alignment was done with Geneious Pro 5.4.6 (
The Bayesian search consisted of two parallel runs each with 130 × 106 generations with four Markov chains. The convergence of the runs was assessed with Tracer 1.5 (
For the ML analysis, we carried out 20 replicate searches and increased the setting “genthreshfortopoterm” until all searches resulted in similar likelihood values, indicating an efficient search (
Uncorrected pairwise (p) genetic distances were obtained for gene 16S using software Mesquite 2.75 (
Localities of the Rhinella margaritifera group from Chocó (triangles) and Amazon (squares). Gray for specimens analyzed morphologically, black for specimens analyzed both genetically and morphologically. Specimens (listed in Appendix
Panamanian populations of the Rhinella margaritifera group included in this study. White crosses for specimens analyzed morphologically, black crosses analyzed both morphologically and genetically. The type locality of R. alata is shown with a triangle. Specimens (listed in Appendix
GenBank accession numbers for DNA sequences used in the phylogenetic analysis.
Museum No. | Species | Country | Locality | GenBank Accession No. | Reference | |||
---|---|---|---|---|---|---|---|---|
TYR | 16S | 12S | COI | |||||
QCAZ10253 | R. alata | Ecuador | Reserva La Chiquita | KR012523 | KR012615 | KR012605 | KR012568 | This study |
QCAZ10254 | R. alata | Ecuador | Reserva La Chiquita | KR012524 | KR012616 | KR012601 | KR012567 | This study |
QCAZ10255 | R. alata | Ecuador | Reserva La Chiquita | KR012525 | KR012617 | KR012602 | KR012570 | This study |
QCAZ11598 | R. alata | Ecuador | Reserva La Chiquita | KR012526 | KR012618 | KR012603 | KR012550 | This study |
QCAZ13882 | R. alata | Ecuador | Manta Real | KR012527 | KR012619 | KR012597 | KR012571 | This study |
QCAZ13896 | R. alata | Ecuador | Manta Real | - | DQ158471 | DQ158471 | - | |
QCAZ14607 | R. alata | Ecuador | Borbón | KR012528 | KR012620 | KR012578 | KR012552 | This study |
QCAZ37244 | R. alata | Ecuador | Valle Hermoso | KR012539 | KR012632 | KR012592 | KR012576 | This study |
QCAZ37248 | R. alata | Ecuador | Valle Hermoso | KR012540 | KR012633 | KR012595 | KR012544 | This study |
QCAZ 23161 | R. alata | Ecuador | San Lorenzo | KR012534 | KR012626 | KR012577 | KR012562 | This study |
QCAZ25023 | R. alata | Ecuador | La Tortuga | KR012536 | KR012629 | KR012596 | KR012572 | This study |
QCAZ25025 | R. alata | Ecuador | La Tortuga | KR012537 | KR012630 | KR012582 | KR012573 | This study |
QCAZ25032 | R. alata | Ecuador | La Pedorrera | KR012538 | KR012631 | KR012604 | KR012569 | This study |
CH9104 | R. alata | Panama | Cana, Boca Cupé | KR012507 | KR012610 | KR012598 | KR012560 | This study |
MVUP2299 | R. alata | Panama | Río Chico Masambí, Parque Nacional Soberanía | KR012511 | KR012613 | KR012600 | KR012561 | This study |
CH9192 | R. alata | Panama | Parque Nacional Soberanía | KR012521 | KR012611 | KR012599 | KR012559 | This study |
QCAZ11597 | R. alata | Ecuador | Reserva La Chiquita | - | DQ15872 | DQ15872 | - | |
104mc | R. castaneotica | French Guyana | Tibourou | EF364355 | EF364289 | EF364263 | - | |
110pg | R. castaneotica | French Guyana | Moint Saint Marcel | EF364353 | EF364285 | EF364259 | - | |
QCAZ38477 | R. dapsilis | Ecuador | Villano B | KR012513 | KR012634 | KR012586 | KR012554 | This study |
QCAZ38512 | R. dapsilis | Ecuador | Villano BII | KR012514 | KR012635 | KR012587 | KR012558 | This study |
QCAZ38560 | R. dapsilis | Ecuador | Villano B | KR012515 | KR012636 | KR012588 | KR012555 | This study |
QCAZ38621 | R. dapsilis | Ecuador | Villano K4 | KR012516 | KR012637 | KR012606 | KR012556 | This study |
QCAZ38688 | R. dapsilis | Ecuador | Villano K4 | KR012517 | KR012638 | KR012607 | KR012575 | This study |
QCAZ38755 | R. dapsilis | Ecuador | Villano BII | KR012518 | KR012639 | KR012589 | KR012548 | This study |
QCAZ38892 | R. dapsilis | Ecuador | Comunidad Kutintza 2 | KR012519 | KR012640 | KR012608 | KR012566 | This study |
QCAZ38998 | R. dapsilis | Ecuador | Comunidad Kurintza 3 | KR012520 | KR012641 | KR012590 | KR012549 | This study |
MTR19199 | R. hoogmoedi | Brazil | Bahia, Camacan | - | JN867571 | JN867545 | - | |
112bm | R. lescurei | French Guyana | Litany | EF364343 | EF217473 | EF364279 | - | |
3027t | R. lescurei | French Guyana | Mitaraka | JN692065 | EF364305 | EF364279 | - | |
108mc | R. margaritifera | French Guyana | Kaw | EF364333 | EF364292 | EF364266 | - | |
136mc | R. margaritifera | French Guyana | Crique Margot | EF364335 | EF364292 | EF364266 | - | |
389MC | R. margaritifera | French Guyana | Camp Canopé | JN692029 | - | - | - | |
374MC | R. margaritifera | French Guyana | Régina | JN692038 | JN691389 | JN690782 | - | |
390MC | R. margaritifera | French Guyana | St Georges | JN692037 | JN691388 | JN690781 | - | |
2559T | R. margaritifera | French Guyana | Pic Matecho | JN690780 | JN691387 | JN690780 | - | |
4482T | R. margaritifera | French Guyana | Angoulème | JN692042 | JN691379 | JN690772 | - | |
163bm | R. margaritifera | French Guayana | Guatemala | EF364320 | EF364292 | EF364266 | - | |
164bm | R. margaritifera | French Guyana | Montagne des Singes | EF364321 | EF364292 | EF364266 | - | |
176bm | R. margaritifera | French Guyana | Crique Grand Leblond | EF364323 | EF364292 | EF364266 | - | |
195mc | R. margaritifera | French Guyana | Kaw | EF364325 | EF364292 | EF364266 | - | |
2034at | R. margaritifera | French Guyana | Nouragues | JN692033 | EF364292 | EF364266 | - | |
204mc | R. margaritifera | French Guyana | Saul | EF364328 | EF364295 | EF364269 | - | |
217mc | R. margaritifera | French Guyana | Grant Santi | EF364329 | EF364299 | EF364273 | - | |
225mc | R. margaritifera | French Guyana | Road St. Elie | EF364330 | EF364292 | EF364266 | - | |
284mc | R. margaritifera | French Guyana | St Elie | EF364336 | EF364292 | EF364266 | - | |
288ag | R. margaritifera | French Guyana | St Georges | JN692021 | JN691380 | JN690773 | - | |
294mc | R. margaritifera | French Guyana | Camp Canope | JN692029 | EF364292 | EF364266 | - | |
2bm | R. margaritifera | French Guyana | Cisame | EF364313 | EF364293 | EF364267 | - | |
307pg | R. margaritifera | French Guyana | Lac Toponowini | JN692022 | EF364292 | EF364266 | - | |
361mc | R. margaritifera | French Guyana | Lucifer | JN692031 | EF364292 | EF364266 | - | |
408pg | R. margaritifera | French Guyana | Mont Kotika | JN692023 | EF364292 | EF364266 | - | |
66mc | R. margaritifera | French Guyana | Monts Bakra | EF364334 | EF364298 | EF364272 | - | |
74af | R. margaritifera | French Guyana | St Georges | JN692020 | EF364266 | EF364292 | - | |
92bm | R. margaritifera | French Guyana | Cisame | EF364314 | EF364301 | EF364275 | - | |
KU215143 | R. margaritifera | Peru | Madre de Dios | - | AY819461 | AY819331 | - | |
13872MTR | R. margaritifera | Brazil | Amapá, Lourenço | JN692016 | JN691390 | JN690783 | - | |
13873MTR | R. margaritifera | Brazil | Amapá, Lourenço | JN692017 | JN691391 | JN690784 | - | |
13874MTR | R. margaritifera | Brazil | Amapá, Lourenço | JN692018 | JN691393 | JN690786 | - | |
13878MTR | R. margaritifera | Brazil | Amapá, Lourenço | JN692019 | JN691392 | JN690785 | - | |
MRT6313 | R. margaritifera | Brazil | Pará, Serra do Kukoinhokren | JN692075 | JN691394 | JN690787 | - | |
MRT6317 | R. margaritifera | Brazil | Pará, Serra do Kukoinhokren | JN692076 | JN691395 | JN690788 | - | |
KU215146 | R. margaritifera | Peru | Madre de Dios | - | - | HM563858 | JN867978 | |
CORBIDI5840 | R. margaritifera | Peru | Curupa | KR012522 | KR012612 | KR012594 | KR012564 | This study |
USNM268828 | R. margaritifera | Peru | Madre de Dios | - | DQ158490 | DQ158490 | - | |
KU215145 | R. cf. margaritifera | Peru | Madre de Dios | - | DQ158491 | DQ158491 | - | |
ZUEC-DCC3393 | R. cf. margaritifera | Brazil | Rio de Janeiro, Santo Aleixo | - | - | AY680262 | - | |
QCAZ17775 | R. margaritifera | Ecuador | 244 km of Indanza | KR012529 | KR012621 | KR012581 | KR012551 | This study |
QCAZ17989 | R. margaritifera | Ecuador | Estación Biológica JatunSacha | KR012530 | KR012622 | - | KR012565 | This study |
QCAZ17990 | R. margaritifera | Ecuador | Estación Biológica JatunSacha | KR012531 | KR012623 | KR012593 | KR012557 | This study |
QCAZ17991 | R. margaritifera | Ecuador | Estación Biológica JatunSacha | KR012532 | KR012614 | - | KR012543 | This study |
QCAZ23632 | R. margaritifera | Ecuador | 7Km North of Cosanga | KR012535 | KR012627 | KR012583 | KR012542 | This study |
QCAZ23917 | R. margaritifera | Ecuador | Gualaquiza-El Ideal | KR012512 | KR012628 | KR012591 | KR012547 | This study |
QCAZ10601 | R. margaritifera | Ecuador | Parque Nacional Yasuní | - | DQ15870 | DQ15870 | - | |
QCAZ18241 | R. margaritifera | Ecuador | Shaime | KR012533 | KR012625 | KR012585 | KR012553 | This study |
10226MSH | R. margaritifera | Brazil | Amazonas, Anavilhanas | JN692056 | JN691364 | JN690757 | - | |
10339MSH | R. margaritifera | Brazil | Amazonas, Anavilhanas | JN692057 | JN601365 | JN69058 | - | |
QCAZ42269 | R. margaritifera | Ecuador | Reserva Yachana | KR012541 | KR012642 | KR012584 | KR012563 | This study |
111af | R. martyi | French Guyana | Brownsberg | JN692045 | EF364303 | EF364277 | - | |
156mc | R. martyi | French Guyana | Trijonction | EF364337 | EF364303 | EF364277 | - | |
LAJ210 | R. ocellata | Brazil | Tocantins, Lajeado | - | JN867572 | JN867546 | - | |
MZUSP103261 | R. ocellata | Brazil | Tocantins, Peixe | - | DQ158479 | DQ158479 | - | |
SMF88237 | R. cf. paraguayensis | Bolivia | - | - | JF790186 | - | - | |
MNKA9691 | R. cf. paraguayensis | Bolivia | - | - | JF790185 | - | - | |
ESTR00173 | Rhinella sp. | Brazil | Amazonas, Carolina | - | JN867574 | JN867548 | - | |
AF7275337 | Rhinella sp. | Brazil | Mato Grosso, APM Manso | - | JN867575 | JN867549 | - | |
Outgroup | ||||||||
QCAZ50698 | Rhinella marina | Ecuador | Puerto Cayo | KR012508 | KR012643 | KR012579 | KR012545 | This study |
QCAZ50702 | Rhinella marina | Ecuador | San Andrés de Rocafuerte | KR012509 | KR012644 | KR012580 | KR012546 | This study |
QCAZ18203 | Rhinella festae | Ecuador | Estación Biológica Jatun Sacha | KR012510 | KR012624 | KR012609 | KR012574 | This study |
KU217501 | Rhinella festae | Ecuador | Pastaza | - | DQ158423 | DQ158423 | - | |
MTD43789 | Rhinella chavin | Peru | Palma Pampa | - | DQ158441 | DQ158441 | - | |
UTA53310 | Rhinella nesiotes | Bolivia | La Paz | - | DQ158478 | DQ158478 | - |
The complete matrix contained up to four genes and 3045 bp for 92 samples. For the complete data set, PartitionFinder chose seven partitions as the best strategy (best model in parenthesis): 12S (GTR + I + G), 16S (GTR + I + G), COI 1st position (TIMef + G), COI 2nd position (TVM + I + G), COI 3rd position (TrN + G), Tyr 1st and 2nd position (TrN + G), Tyr, 3rd position (TrN + I + G). For the mitochondrial analyses, the same five partitions were chosen, one for each ribosomal RNA gene and each codon position in COI. For the nuclear analysis, two partitions were chosen: Tyr, 1st and 2nd position and Tyr, 3rd position.
The tree topologies for the Maximum likelihood and Bayesian phylogenies were similar except for weakly supported nodes (posterior probability < 0.95 and bootstrap < 75). The Maximum Likelihood tree (Fig.
Maximum Likelihood phylogram depicting relationships within the Rhinella margaritifera species group. The phylogram was derived from the analysis of 3045 bp of mitochondrial (12S, 16S, COI) and nuclear (Tyr) genes. Numeric codes on terminals are individual collection numbers (associated data listed in Table
The sister clade to Chocó-Panama + Upper Amazon has weak support and includes other members of the R. margaritifera group (R. dapsilis, R. hoogmoedi, R. lescurei, R. martyi, R. ocellata, R. paraguayensis and “R. margaritifera”) from the Guiana region and Amazonian Brazil, Ecuador and Peru. Relationships among them are weakly supported on most branches.
The Maximum Likelihood tree based on mitochondrial genes (Fig.
Maximum Likelihood phylogram depicting relationships within the Rhinella margaritifera species group. The phylogram was derived from the analysis of 2495 bp of mitochondrial gene fragments (12S, 16S, COI). Numeric codes on terminals are individual collection numbers (associated data listed in Table
Morphometric comparisons. Morphometric data from adults are summarized in Table
Box and whisker plots showing snout-vent length variation in adult Rhinella margaritifera (upper Amazon) and R. alata (Chocó and Panama). The central bar indicates the median, the interquartile range is shown by the box length, and the range is shown by the short horizontal lines (whiskers). SVL = snout-vent length. The black cross is the holotype of R. alata.
Descriptive statistics for morphometric measurements of adults from Rhinella margaritifera from Amazonian Ecuador and R. alata from Chocó and Panama. Mean ± SD is given, with the range below. Abbreviations are: SVL = Snout-Vent Length; TL = Tibia Length; FL = Femur Length; HL= Head Length; HW = Head Width; STCH = Supratympanic Crest Height; SOCH = Supraorbital Crest Height; NSD = Nostril-Snout Distance; IND = Inter-Nostril Distance; TD = Tympanum Diameter; FT = Foot Length. All measurements are in mm.
R. margaritifera | R. alata | |||||||
---|---|---|---|---|---|---|---|---|
Amazon | Chocó | Panamá | combined | |||||
Morphometric measurements | Males (n = 16) |
Females (n = 16) |
Males (n = 43) |
Females (n = 31) |
Males (n = 6) |
Females (n = 8) |
Males (n = 49) |
Females (n = 39) |
SVL | 45.6 ± 4.11 (54.36–39.88) |
68.90 ± 8.26 (77.97–55.42) |
36.66 ± 2.42 (43.25 –31.84) |
44.82 ± 4.42 (56.19–38.55) |
38.03 ± 0.59 (39.20–37.54) |
42.38 ± 3.82 (49.69–37.78) |
36.83 ± 2.31 (43.25–31.84) | 44.27 ± 4.37 (56.19–37.78) |
TL | 18.73 ± 1.97 (23.13–15.14) |
29.36 ± 2.97 (34.26–24.01) |
15.98 ± 1.14 (18.72–13.69) |
18.26 ± 1.24 (20.73–16.22) |
15.86 ± 1.16 (18.12–15.09) |
17.79 ± 0.75 (18.99–16.41) |
15.97 ± 1.13 (18.72– 13.69) | 18.17 ± 1.16 (20.73–16.22) |
FL | 19.67 ± 1.97 (23.84–16.15) |
29.33 ± 3.67 (35.34–22.75) |
15.69 ± 1.34 (19.28–13.09) |
18.16 ± 1.72 (22.04–15.18) |
16.39 ± 0.37 (17.01 –16.03) |
17.46 ± 0.67 (18.72–16.72) |
15.77 ± 1.27 (19.28–13.09) | 18.02 ± 1.58 (22.04–15.18) |
HW | 16.9 ± 1.59 (19.93–14.77) |
25.88 ± 2.73 (30.69–21.01) |
12.57 ± 0.95 (15.14–10.31) |
15.10 ± 1.6 (18.94–12.49) |
12.98 ± 0.17 (13.3–12.8) |
14.90 ± 1.12 (17.23–13.79) |
12.63 ± 0.91 (15.14–10.31) | 15.06 ± 1.50 (18.94–12.49) |
HL | 14.6 ± 1.28 (17.44–13.27) |
22.27 ± 2.71 26.51–17.94) |
11.61 ± 0.8 (13.88–10.29) |
13.67 ± 1.19 (16.84–11.85) |
11.85 ± 0.21 (12.2–11.54) |
13.18 ± 1.12 (15.45–11.77) |
11.64 ± 0.76 (13.88–10.29) | 13.57 ± 1.17 (16.84–11.77) |
SOCH | 9.46 ± 0.86 (11.13–8.19) |
15.43 ± 2.02 (18.33–12.06) |
7.71 ± 0.59 (8.87–6.45) |
9.28 ± 0.86 (11.40–7.77) |
8.39 ± 0.21 (8.67–8.2) |
9.13 ± 0.49 (9.87–8.53) |
7.79 ± 0.59 (8.87–6.45) | 9.25 ± 0.79 (11.4–7.77) |
STCH | 8.78 ± 1.55 (12.27–6.78) |
17.73 ± 3.26 (22.7–12.35) |
6.27 ± 0.54 (7.97–5.36) |
7.96 ± 0.68 (9.71–6.63) |
6.59 ± 0.31 (6.99–6.31) |
7.38 ± 0.39 (8.01–6.99) |
6.31 ± 0.52 (7.97–5.36) | 7.84 ± 0.67 (9.71–6.63) |
NSD | 2.08 ± 0.44 (2.64–1.41) |
2.45 ± 0.42 (3.37–1.79) |
1.63 ± 0.29 (2.23–1.05) |
1.70 ± 0.21 (2.08–1.25) |
1.47 ± 0.17 (1.60–1.18) |
1.66 ± 0.16 (1.89–1.35) |
1.61 ± 0.28 (2.23–1.05) | 1.69 ± 0.20 (2.08–1.25) |
IND | 3.35 ± 0.35 (3.89–2.70) |
3.12 ± 0.37 (3.73–2.59) |
2.50 ± 0.33 (3.23–1.86) |
2.80 ± 0.43 (3.98–2.16) |
2.41 ± 0.11 (2.59–2.31) |
2.42 ± 0.23 (2.63–2.08) |
2.48 ± 0.32 (3.23–1.86) | 2.72 ± 0.43 (3.98–2.08) |
TD | 3.48 ± 0.24 (3.93–3.18) |
4.14 ± 0.21 (4.48–3.65) |
3.34 ± 0.47 (4.03–1.95) |
3.46 ± 0.59 (4.45–2.5) |
3.38 ± 0.20 (3.60–3.13) |
3.79 ± 0.25 (4.05–3.31) |
3.33 ± 0.45 (4.03–1.95) | 3.52 ± 0.55 (4.45–2.5) |
FT | 16.87 ± 2.145 (21.85–13.76) |
24.87 ± 3.64 (28.86 –19.13) |
13.46 ± 1.12 (15.88–11.43) |
15.33 ± 1.52 (19.40–13.15) |
13.72 ± 0.68 (14.70–12.82) |
14.96 ± 0.76 (16.54–14.39) |
13.48 ± 1.07 (15.88–11.43) | 15.25 ± 1.39 (19.4–13.15) |
Significant differences were observed in relative crest size between the Chocó-Panama and upper Amazon clades (Fig.
Box and whisker plots showing relative size of supratympanic crests for adult Rhinella margaritifera (upper Amazon) and R. alata (Chocó-Panama). The central bar indicates the median, the interquartile range is shown by the box length, and the range is shown by the short horizontal lines (whiskers). STCH = supratympanic crest height, HL = head length. The yellow cross is the holotype of R. alata.
Three components with eigenvalues > 1.0 were extracted from the PCA for females (Table
Character loadings and eigenvalues for Principal Components (PC) Analysis. The analysis was based on ten size-corrected morphometric variables measured in Amazonian, Chocoan and Panamanian populations of the R. margaritifera species group. Abbreviations are: TL = Tibia Length; FL = Femur Length; HL = Head Length; HW = Head Width; STCH = Supratympanic Crest Height; SOCH = Supraorbital Crest Height; NSD = Nostril-Snout Distance; IND = Inter-Nostril Distance; TD = Tympanum Diameter; FT = Foot Length. Bold figures indicate highest loadings.
Variable | PCA Females | PCA Males | ||||
---|---|---|---|---|---|---|
PC I | PC II | PC III | PC I | PC II | PC III | |
FL | 0.330 | 0.165 | 0.167 | 0.272 | 0.159 | 0.322 |
FT | 0.334 | 0.214 | 0.418 | 0.061 | -0.038 | 0.661 |
HL | 0.350 | -0.065 | 0.153 | 0.448 | -0.268 | -0.078 |
HW | 0.343 | 0.132 | -0.288 | 0.446 | -0.222 | -0.045 |
IND | -0.203 | 0.381 | 0.512 | 0.280 | 0.502 | -0.142 |
NSD | 0.217 | 0.155 | -0.580 | 0.262 | 0.386 | -0.186 |
SOCH | 0.368 | -0.067 | 0.190 | 0.423 | -0.071 | -0.082 |
STCH | 0.411 | -0.154 | -0.039 | 0.409 | -0.290 | -0.045 |
TD | 0.071 | 0.817 | -0.159 | 0.099 | 0.557 | -0.128 |
TL | 0.368 | -0.200 | 0.232 | 0.134 | 0.228 | 0.610 |
Eigenvalue | 4.411 | 1.192 | 1.128 | 2.800 | 1.947 | 1.585 |
Cumulative variance (%) | 44.11 | 56.03 | 67.31 | 28.00 | 47.47 | 63.32 |
In the DFA classification for females, 51 out of 55 females were assigned correctly to their geographic region. The four misclassified females from Ecuadorian Chocó were assigned to Panamanian populations. All specimens from the upper Amazon were correctly classified. In the DFA for males, 56 out of 65 males were correctly classified. The eight misclassified males from Ecuadorian Chocó were assigned to Panamanian populations and only one from upper Amazon to Panamanian populations. All males and females from Panama were correctly classified. The DFA analyses indicate that populations from the Ecuadorian Chocó are morphometrically very similar with those from Panama, both groups being markedly different from R. margaritifera from the upper Amazon.
Finally, evidence of sexual dimorphism was found in relative crest size: females have larger cephalic crests than males (Fig.
The upper Amazon clade differs from the Chocó-Panama clade in having protruding vertebral apophyses in the dorsum and bony knobs at angle of jaws (both absent in the Chocó-Panama clade; Figs
Dorsolateral and ventral views of Rhinella alata from the Chocó region. A and C QCAZ 50568 (SVL 40.37 mm), adult female, La Concordia, Santo Domingo Province, Ecuador B and D QCAZ 37248 (SVL 40.23 mm), adult male, Valle Hermoso, El Oro Province, Ecuador. Not shown at the same scale. Photos by S.R. Ron.
Dorsolateral views of Rhinella margaritifera from the Ecuadorian Amazon. Females: A QCAZ 55930 (SVL 80.15 mm) B QCAZ 55914 (SVL 72.49 mm), Lorocachi, Pastaza Province, Ecuador; males: C QCAZ 52343 (SVL 37.59 mm) D QCAZ 52344 (SVL 36.66 mm), Cascada San Rafael, Sucumbíos Province, Ecuador. Photos by S.R. Ron. Not shown at the same scale.
The holotype of R. alata (Thominot, 1884) (Fig.
Bufo alatus Thominot, 1884. Holotype: MNHN 84285, adult male from Obispo, Panama.
Rhinella alata is a small-sized (Table
Rhinella alata is most similar to R. acutirostris. Both species differ from other members of the R. margaritifera group by the absence of protruding vertebral apophyses, canthus rostralis not raised, snout projected, and low cranial crests. Rhinella acutirostris differs from R. alata in having a bony knob at the angle of jaws (bony knob absent in R. alata [
Rhinella alata is most closely related to populations of R. margaritifera from the upper Amazon basin in Ecuador and Peru. They can be easily distinguished by differences in body size (Fig.
The holotype is an adult male with SVL = 39.2 mm (Fig.
Variation in dorsal and ventral coloration of preserved specimens is shown in Figures
Rhinella alata from Ecuador showing variation in dorsal and ventral coloration of preserved specimens. Left to right, males: QCAZ 6733 (SVL 38.23 mm), QCAZ 10279 (SVL 35.08 mm); females, QCAZ 11598 (SVL 42.13 mm), QCAZ 14607 (SVL 50.95 mm), QCAZ 10439 (SVL 47.06 mm). See Appendix
Rhinella alata from Panama showing variation in dorsal and ventral coloration of preserved specimens. Left to right, male: AMNH 89459 (SVL 37.54 mm); females, AMNH 88694 (SVL 41.21 mm), AMNH 55476 (SVL 41.19 mm), AMNH 104454 (SVL 49.69 mm), AMNH 88689 (SVL 42.75 mm), AMNH 20896 (SVL 42.98 mm). See Appendix
Ventral surfaces of preserved specimens have a cream to yellowish-cream background color with irregular darker marks arranged in diverse patterns; marks can be light gray (QCAZ 6734, AMNH 88689), light brown (QCAZ 6732, AMNH 104454), dark gray (QCAZ 31606) or dark brown (QCAZ 6733, AMNH 89459), and vary from being restricted to the anterior half of the body (QCAZ 31604, AMNH 89459) to being present over the entire venter (QCAZ 4445, AMNH 88694). A longitudinal mid-ventral cream thin stripe can be present in the gular region (QCAZ 31602, 31606) or from the gular region to the mid-venter (QCAZ 6731, 11598).
Head shape in dorsal view varies from elongated (QCAZ 11598, AMNH 89459) to subtriangular (QCAZ 4447, AMNH 55475); in lateral view it varies from rounded (QCAZ 31605, AMNH 52749) to protruding (QCAZ 11393, AMNH 55475). Canthal region coloration varies from light gray or light brown to dark gray or dark brown. In some individuals the area below the eye and tympanum is yellowish cream (QCAZ 4447, AMNH 20896) or brown (QCAZ 31603, AMNH 88694) and differs from the color of the dorsum. Cloacal tubercles vary from yellowish cream (QCAZ 4441, AMNH 20896), to gray (QCAZ 31606) or brown (QCAZ 31602, AMNH 88695).
Based on digital photograph of an adult female QCAZ 50568 (Fig.
Based on a digital photography of an adult male QCAZ 37248 (Fig.
Rhinella alata has been recorded at 37 localities in the Ecuadorian Chocó (Cañar, Carchi, El Oro, Esmeraldas, Manabí, Pichincha, and Santo Domingo Provinces; Fig.
The examined specimens from Chocoan populations contain 21 gravid females (average SVL = 45.37 mm, SD = 4.05 mm): QCAZ 4262, QCAZ 4441, QCAZ 4442, QCAZ 4443, QCAZ 7065, QCAZ 10296, QCAZ 11597, QCAZ 11598 collected in January; QCAZ 50568 collected in February; QCAZ 11392, QCAZ 31601, QCAZ 31603, QCAZ 31605 collected in April; QCAZ 25023 collected in June; QCAZ 10439 collected in August; QCAZ 14607 collected in November; QCAZ 10301 collected in December. This suggests year round reproductive activity with a peak between January and April, a period that corresponds to the rainy season in the Ecuadorian Chocó.
In Panamanian populations gravid females were found in January (AMNH 104454), September (AMNH 55461), November (AMNH 88689), and December (AMNH 53699). In central Panama, R. alata breeds in ponds and pools along permanent streams or swamps. Reproduction is explosive and most takes place from the middle of the rainy season to early dry season (
Most of the Ecuadorian specimens are from Reserva Mayronga and Reserva Ecológica Cotacachi-Cayapas. They were found in the leaf litter of secondary forest and in agricultural lands. Some adults were observed at night within the forest in vegetation above the ground and some were found in amplexus (QCAZ 10271, QCAZ 10274, QCAZ 10275 in November 1996, and QCAZ 31604, QCAZ 31605 in February 1996). All the specimens collected in Reserva Ecológica Cotacachi-Cayapas were found in secondary forest. At some collecting sites, the forest has been cleared for cacao plantations (QCAZ specimen database).
According to the classification of
The main vegetation types for Panamanian localities are (following
Based on morphological characters,
In contrast,
The taxonomic status and phylogenetic position of populations traditionally ascribed to R. margaritifera (= Bufo typhonius; e.g.
The identity of the upper Amazon clade (Ecuador-Peru) remains unresolved. It was not possible to ascribe it unequivocally to any described species of the R. margaritifera species group and it is unlikely to be R. margaritifera sensu stricto (as defined by
These results raise some rather interesting questions. For instance, the complete distribution range of R. alata is yet to be determined. Extensive and explicit studies are necessary to reveal whether the species is continuously distributed from Ecuador to Panama or if it consists one, two (or more) disjoint population nuclei. This would be an indispensable step before planning further studies on the evolutionary history or conservation status of the species. Moreover, future studies including a larger number of samples, more representative of the geographic range of each species within the R. margaritifera group, from Colombia, Venezuela and Suriname, will help to clarify their evolutionary identity. It will also be necessary to re-evaluate, using molecular, morphological, ecological, behavioral, and phylogenetic analyses, the taxonomic status of species that have been previously described only morphologically such as R. acutirostris, R. magnussoni, R. proboscidea, R. roqueana, R. sclerocephala, R. scitula and R. stanlaii. Integrative approaches like the one we pursued in this study will help to disentangle the complex evolutionary history, systematics, and taxonomy of this species group.
Because all species in the R. margaritifera species group are distributed in South America, it is reasonable to assume that the presence of R. alata in Central America is the result of a single dispersal event from South America. The genetic distances between Chocoan and Panamanian populations are low (range 1.2–1.9%) and suggest that their divergence was recent and occurred after the closure of the Panamanian isthmus during the late Pliocene. Assuming a rate of evolution of the gene 16S of 0.00249–0.00277 substitutions per site per lineage per Myr (
Rhinella alata is sister to populations of R. margaritifera from the Ecuadorian and Peruvian Amazon and the eastern Andean slopes, up to 2000 m of elevation, forming altogether a robust clade. The two lineages are highly divergent from each other (uncorrected p distances 3.0–5.5%, mitochondrial gene 16S) and are morphologically distinctive. Therefore, both clades clearly represent separate species. Previously, R. margaritifera was considered to occur on lowland rainforests east and west of the Andes of Ecuador. This distribution was atypical because out of 174 amphibian species inhabiting the Amazonian rainforests of Ecuador below 600 m of elevation, only three also occur in the rainforests of the Chocó region west of the Andes: Hypsiboas boans, Rhinella marina and Trachycephalus typhonius (
This work was funded by Secretaría Nacional de Educación Superior, Ciencia, Tecnología e Innovación (Arca de Noé initiative) and Pontificia Universidad Católica del Ecuador. RI was supported by the Sistema Nacional de Investigación de Panama. We are particularly thankful to Annemarie Ohler, the Curator of Muséum National d’Histoire naturelle, who provided data and photos of the holotype of R. alata. David Buckley for valuable comments on the text. Ángel Sosa and Mario Yánez-Muñoz shared photographs of R. alata. Darrel Frost, Curator of AMNH, loaned specimens of R. alata from Panama. Pablo Venegas, curator of CORBIDI, provided a tissue sample from Peru. Nadia Páez for help in taking measurements of specimens. We are thankful to Andrea Manzano and María Ordoñez for carrying laboratory work. For specimen collection and locality data, we are indebted to Néstor Acosta, Silvia Aldás, Alejandro Arteaga, Fernando Ayala, Alvaro Barragán, Paola Buitrón, David Cannatella, Edwin Carrillo, Luis Coloma, Rafael de Sa, Alexandra Endara, Jorge García, Stella de la Torre, Mireya Dimas, Sehoya Harris, César Jaramillo, Fidel Jaramillo, Diego Lombeida, Alfredo Lopez, Luis E. Lopez, Fernando Nogales, Giovanni Onore, Aida Ortiz, Alexandra Quiguango, Fabián Sáenz, Frank Solís, Samuel Sucre, Italo Tapia, Queti Tapia, Eduardo Toral, J. M. Touzet, Ana M. Velasco, and Tania Villegas.
Examined material. Numbers in bold indicate specimens analyzed genetically and morphometrically.
Rhinella alata.— ECUADOR: PROVINCIA CAÑAR: Manta Real, Río Patul (2.5679°S, 79.3666°W), 350–400 m (QCAZ 3437, 3551, 4757–758); Manta Real (2.5537°S, 79.3642°W), 500 m (QCAZ 12778–779). PROVINCIA CARCHI: Vía Zumba–El Chota, 1500 m (QCAZ 12233). PROVINCIA EL ORO: Valle Hermoso, Parroquia Bella María (3.5019°S, 79.8172°W), 379 m (QCAZ 37244, 37248); El Progreso, vía Pasaje–Pan de Azúcar (3.2883°S, 79.7581°W), 180 m (QCAZ 10366). PROVINCIA ESMERALDAS: Lagarto, Mayronga Reserve (1.042°S, 79.28°W), 100 m (4262–4264, 4441–4451, 4709–4717, 6637–6642); Reserva Ecológica Bilsa (0.6202°S, 79.931°W), 534 m (QCAZ 6731–6743); Corriente Grande, Río Cayapas (0.6895°S, 78.9589°W), 70 m (QCAZ 10271, 10274–281, 10289, 10290, 10292, 10295–299, 10299, 10301); Reserva Ecológica Cotacachi Cayapas, Charco Vicente (0.6962°S, 78.9109°W), 60 m (QCAZ 3338–3339, 11391–396); Pichiyacu, Comunidad Chachi, Río Cayapas (0.9081°S, 78.998°W), 260 m (QCAZ 31602–609); Reserva Ecológica Cotacachi–Cayapas o Playa de Oro (0.8285°S, 78.722°W), 179 m (QCAZ 49381–382, 49387, 49391); Las Golondrinas near Río Canandé (QCAZ 12651–652); Durango, Río San José (1.054°S, 78.625°W), 33 m (QCAZ 24968–978); Río Onzole (0.712°S, 79.092°W), 110 m (QCAZ 10440–443); Comunidad Loma Linda, Río Onzole (0.8754°S, 79.0511°W), 95 m (QCAZ 10439); La Concordia (0.0022°S, 79.4105°W), 144 m (QCAZ 50573, 50568); San Lorenzo, Protectora La Chiquita (1.2333°S, 78.76°W), 60 m (QCAZ 10253, 10254–255, 11597, 11598); San Lorenzo, La pera del Guarapo (1.2684°S, 78.8067°W), 253 m (QCAZ 23161); La Pedorrera (0.4667°S, 79.9833°W), 53 m (QCAZ 25032); La Tortuga (0.591°S, 79.957°W), 86 m (QCAZ 25023); Borbón (1.0667°S, 79.05°W), 70 m (QCAZ 14607); Viche (0.6615°S, 79.5387°W), (QCAZ 4674); Durango (1.0427°S, 78.6245°W), (QCAZ 8549, 35250); 7 km western of Durango (1.0133°S, 78.6682°W) 220 m, (QCAZ 23164, 23623); Viruela, Rio Cayapas (1.1142°S, 78.9936°W), 45 m (QCAZ 10289); Al Tambo (0.9169°S, 79.5662°W) 253 m, (QCAZ 21138); El Milagro, La Mayronga (1.003°S, 79.326°W). PROVINCIA MANABÍ: El Carmen (0.274°S, 79.459°W). 300 m (QCAZ 7038–7039, 7065). PROVINCIA PICHINCHA: Reserva Forestal ENDESA (0.1667°S, 79,1667°W), 720 m (QCAZ 1659); Río Canoi (0.075°S, 79.051°W), 570 m (QCAZ 2745); 1 km E of Pedro Vicente Maldonado (0.0833°S, 79.039°W), 670 m, (QCAZ 2752); San Miguel de los Bancos (0.0166°S, 78.8833°W), (QCAZ 3813, 3815–818); San Miguel de los Bancos, Río Pitzará, 130 m (QCAZ 50846); km 9 San Miguel de los Bancos–Puerto Quito road (0.072°S, 78.9599°W), (QCAZ 5860); Puerto Quito, ENDESA (0.098°S, 79.117°W), (QCAZ 36827). PROVINCIA SANTO DOMINGO: Bosque Protector La Perla (0.057°S, 79.359°W), (QCAZ 3500–504); km 8 road to Santo Domingo (0.2005°S, 79.1924°W), 528 m (QCAZ 23621). PANAMA: COMARCA GUNA YALA: Dad Nakue Dubpir, Río Ogandí (9.2477°N, 78.1744°W), 150 m (CH 8842); Udirbi, Reserva Forestal (9.3167°N, 78.9833°W), 342 m (CH 1706); PROVINCIA COCLÉ: La Mina, Río Indio (8.9382°N, 80.1469°W), 48 m (CH 4922); near Río Tife cascade, Parque Nacional General de División Omar Torrijos Herrera (8.7065°N, 80.6352°W), 460 m (CH 0065); Obispo (9.1167°N, 79.6833°W) (MNHN 84285); Quebrada La Tiburcia, Cascajal (8.7158°N, 80.4605°W), 180 m (CH 5042); Quebrada La Varona, near Palmarazo (8.7342°N, 80.6565°W), 125 m (CH 5139). PROVINCIA COLÓN: Chitra, Santa Isabel (9.5186°N, 79.1534°W), 90 m (CH 7783); El Limón, Río Indio (8.9919°N, 80.1701°W), 19 m (CH 4967); Rinconcito, Punta Rincón (9.0135°N, 80.6884°W), 52 m (CH 1412); Río Caimito, Petaquilla (8.9706°N, 80.671°W), 54 m (CH 5476); Río Boquerón (9.3857°N, 79.4826°W), 150 m (AMNH 89459); Río Frijoles, Camino del Oleoducto, Parque Nacional Soberanía (9.1523°N, 79.7347°W), 67 m (CH 0307); road to Piña, after the represa Gatún (9.2603°N, 79.94°W), 34 m (CH 1679); Sta. Rosa and Guayabalito (9.1833°N, 79.65°W), 36 m (AMNH 55475); PROVINCIA DARIÉN: between Dos Bocas de Antaral and campsite on Serranía de Jingurudó (7.6564°N, 77.9986°W), <675 m (CH 4641); Cerro Tacarcuna, Río Pucuro (8.0011°N, 77.4852 °W), 640 m (AMNH 104454); Cana, trail to Boca de Cupé, Pinogana (7.7661°N, 77.6752°W), 518 m (CH 9104); Estación Pirre, Río Peresénico (8.0192°N, 77.7325°W), 90 m (CH 4057); Laguna Purriche (7.7222°N, 77.6555°W), 475 m (CH 6376); PROVINCIA PANAMA: Altos de Majé (AMNH 88689–8690, 88694); Barro Colorado (9.1636°N, 79.8378°W), 79 m (AMNH 20896, 5274, 55461–462); Parque Nacional Soberanía, Ancón (9.0764°N, 79.6594°W), 130 m (CH 9192); Chiva Chiva Road, Parque Nacional Camino de Cruces (9.0284°N, 79.5899°W), 41 m (CH 0491); Cruces trail (9.0453°N, 79.5892°W), 77 m (AMNH 55460); Finca Santa Bárbara, Nuevo Emperador, Arraiján (9.0011°N, 79.7235°W), 135 m (CH 1158); near Boquerón, Candelaria and Peluca (9.3671°N, 79.5546 °W) (AMNH 53699); near entrance to Chilibrillo Cave (9.1833°N, 79.6167°W) (AMNH 55476); Pacora (9.0833°N, 79.2833°W), 20 m (QCAZ 55481); Río Arraijancito (8.983°N, 79.6361°W), 110 m (CH 3980); Río Chico Masambí, Parque Nacional Soberanía, Ancón (9.0787°N, 79.6601°W), 135 m (MVUP 2299); Río Indio Arriba (8.6562°N, 80.1144°W), 645 m (CH 5005); San Juan de Pequení (9.3841°N, 79.5227°W), 100 m (CH 3702); stream near ACP Estación Río Chico (9.2636°N, 79.5097°W), 116 m (CH 6825); Tortí (8.9389°N, 78.4573°W), 95 m (MVUP 2256); Trinidad (8.7321°N, 79.9617°W), 420 m (CH 4313); Altos de Cerro Azul, Cerro Jefe (9.2284°N, 79.4046°W), 800 m (CH 3441).
Rhinella margaritifera.— ECUADOR: PROVINCIA ORELLANA: Parque Nacional Yasuní, Estación Científica Yasuní (0.6772°S, 76.4012°W), 230 m (QCAZ 8415, 17736, 17740, 41011); Parque Nacional Yasuní, Bloque 31 (0.942°S, 75.905°W), (QCAZ 11909); Parque Nacional Yasuní, Rio Yasuní (0.9248°S, 75.9152°W), 206 m (QCAZ 11940); Parque Nacional Yasuní, Via Pompeya-Iro (0.6536°S, 76.4536°W), 287 m (QCAZ 17216, 17329, 43011, 22401); Parque Nacional Yasuní, Apaika (0.8656°S, 75.9245°W), (QCAZ 33545); Estación Biológica Tiputini (0.0639°S, 76.1493°W), 250 m (QCAZ 10207); Nuevo Rocafuerte (0.8967°S, 75.437°W),186 m (QCAZ 39466); Añangu (0.5249°S, 76.3844°W), 255 m (QCAZ 43952–953); Chiroisla (0.58°S, 75.9177°W), 207 m (QCAZ 44318–319; Huiririma (0.7116°S, 75.6239°W), 194 m (QCAZ 44563–565). PROVINCIA PASTAZA: Río Bobonaza (1.8056°S, 77.3313°W), 250 m (QCAZ 10650); Kapawi Lodge (2.5387 °S, 76.8583°W), 239 m (QCAZ 25476, 25488–489); Pomona (1.625°S, 77.9072°W), 846 m (QCAZ 25631). PROVINCIA SUCUMBIOS: Reserva Limoncocha (0.4062°S, 76.6195°W), 261 m (QCAZ 43104, 43108); Pañacocha (0.4712°S, 76.0667°W), 255 m (QCAZ 44098–099). PROVINCIA NAPO: Reserva Yachana (0.8333°S, 77.1667 °W), 350 m (QCAZ 42269); Cascada de San Rafael (0.1036°S, 77.5808°W), 1300 m (QCAZ 31708). PROVINCIA MORONA SANTIAGO: Plan de Milagro (3.0011 °S, 78.5052°W), 1950 m (QCAZ 48242).
Bayesian consensus phylogram depicting relationships within the Rhinella margaritifera species group. The phylogram was derived from the analysis of 550 bp of nuclear gene Tyrosinase. Museum catalog numbers are shown in Table