Corresponding author: Sueny P. dos Santos (
Academic editor: F. Andreone
The
dos Santos SP, Ibáñez R, Ron SR (2015) Systematics of the
The
Two species of the
The distribution of
Herein, genetic and morphological information were integrated to clarify the taxonomy of the populations of
Populations from Panama, the Ecuadorian Chocó, and the Amazon basin were sampled (Figs
Morphometric analyses were based on 120 adult specimens of
Genetic analyses were based on newly generated sequences of
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
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tissue preserved in 95% ethanol or tissue storage buffer using standard guanidine thiocyanate protocol (M. Fujita, unpublished) with modifications.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 (
Localities of the
Panamanian populations of the
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 |
|
Ecuador | Reserva La Chiquita |
|
|
|
|
This study |
QCAZ10254 |
|
Ecuador | Reserva La Chiquita |
|
|
|
|
This study |
QCAZ10255 |
|
Ecuador | Reserva La Chiquita |
|
|
|
|
This study |
QCAZ11598 |
|
Ecuador | Reserva La Chiquita |
|
|
|
|
This study |
QCAZ13882 |
|
Ecuador | Manta Real |
|
|
|
|
This study |
QCAZ13896 |
|
Ecuador | Manta Real | - |
|
|
- |
|
QCAZ14607 |
|
Ecuador | Borbón |
|
|
|
|
This study |
QCAZ37244 |
|
Ecuador | Valle Hermoso |
|
|
|
|
This study |
QCAZ37248 |
|
Ecuador | Valle Hermoso |
|
|
|
|
This study |
QCAZ 23161 |
|
Ecuador | San Lorenzo |
|
|
|
|
This study |
QCAZ25023 |
|
Ecuador | La Tortuga |
|
|
|
|
This study |
QCAZ25025 |
|
Ecuador | La Tortuga |
|
|
|
|
This study |
QCAZ25032 |
|
Ecuador | La Pedorrera |
|
|
|
|
This study |
CH9104 |
|
Panama | Cana, Boca Cupé |
|
|
|
|
This study |
MVUP2299 |
|
Panama | Río Chico Masambí, Parque Nacional Soberanía |
|
|
|
|
This study |
CH9192 |
|
Panama | Parque Nacional Soberanía |
|
|
|
|
This study |
QCAZ11597 |
|
Ecuador | Reserva La Chiquita | - |
|
|
- |
|
104mc |
|
French Guyana | Tibourou |
|
|
|
- |
|
110pg |
|
French Guyana | Moint Saint Marcel |
|
|
|
- |
|
QCAZ38477 |
|
Ecuador | Villano B |
|
|
|
|
This study |
QCAZ38512 |
|
Ecuador | Villano BII |
|
|
|
|
This study |
QCAZ38560 |
|
Ecuador | Villano B |
|
|
|
|
This study |
QCAZ38621 |
|
Ecuador | Villano K4 |
|
|
|
|
This study |
QCAZ38688 |
|
Ecuador | Villano K4 |
|
|
|
|
This study |
QCAZ38755 |
|
Ecuador | Villano BII |
|
|
|
|
This study |
QCAZ38892 |
|
Ecuador | Comunidad Kutintza 2 |
|
|
|
|
This study |
QCAZ38998 |
|
Ecuador | Comunidad Kurintza 3 |
|
|
|
|
This study |
MTR19199 |
|
Brazil | Bahia, Camacan | - |
|
|
- |
|
112bm |
|
French Guyana | Litany |
|
|
|
- |
|
3027t |
|
French Guyana | Mitaraka |
|
|
|
- |
|
108mc |
|
French Guyana | Kaw |
|
|
|
- |
|
136mc |
|
French Guyana | Crique Margot |
|
|
|
- |
|
389MC |
|
French Guyana | Camp Canopé |
|
- | - | - |
|
374MC |
|
French Guyana | Régina |
|
|
|
- |
|
390MC |
|
French Guyana | St Georges |
|
|
|
- |
|
2559T |
|
French Guyana | Pic Matecho |
|
|
|
- |
|
4482T |
|
French Guyana | Angoulème |
|
|
|
- |
|
163bm |
|
French Guayana | Guatemala |
|
|
|
- |
|
164bm |
|
French Guyana | Montagne des Singes |
|
|
|
- |
|
176bm |
|
French Guyana | Crique Grand Leblond |
|
|
|
- |
|
195mc |
|
French Guyana | Kaw |
|
|
|
- |
|
2034at |
|
French Guyana | Nouragues |
|
|
|
- |
|
204mc |
|
French Guyana | Saul |
|
|
|
- |
|
217mc |
|
French Guyana | Grant Santi |
|
|
|
- |
|
225mc |
|
French Guyana | Road St. Elie |
|
|
|
- |
|
284mc |
|
French Guyana | St Elie |
|
|
|
- |
|
288ag |
|
French Guyana | St Georges |
|
|
|
- |
|
294mc |
|
French Guyana | Camp Canope |
|
|
|
- |
|
2bm |
|
French Guyana | Cisame |
|
|
|
- |
|
307pg |
|
French Guyana | Lac Toponowini |
|
|
|
- |
|
361mc |
|
French Guyana | Lucifer |
|
|
|
- |
|
408pg |
|
French Guyana | Mont Kotika |
|
|
|
- |
|
66mc |
|
French Guyana | Monts Bakra |
|
|
|
- |
|
74af |
|
French Guyana | St Georges |
|
|
|
- |
|
92bm |
|
French Guyana | Cisame |
|
|
|
- |
|
KU215143 |
|
Peru | Madre de Dios | - |
|
|
- |
|
13872MTR |
|
Brazil | Amapá, Lourenço |
|
|
|
- |
|
13873MTR |
|
Brazil | Amapá, Lourenço |
|
|
|
- |
|
13874MTR |
|
Brazil | Amapá, Lourenço |
|
|
|
- |
|
13878MTR |
|
Brazil | Amapá, Lourenço |
|
|
|
- |
|
MRT6313 |
|
Brazil | Pará, Serra do Kukoinhokren |
|
|
|
- |
|
MRT6317 |
|
Brazil | Pará, Serra do Kukoinhokren |
|
|
|
- |
|
KU215146 |
|
Peru | Madre de Dios | - | - |
|
|
|
CORBIDI5840 |
|
Peru | Curupa |
|
|
|
|
This study |
USNM268828 |
|
Peru | Madre de Dios | - |
|
|
- |
|
KU215145 |
|
Peru | Madre de Dios | - |
|
|
- |
|
ZUEC-DCC3393 |
|
Brazil | Rio de Janeiro, Santo Aleixo | - | - |
|
- |
|
QCAZ17775 |
|
Ecuador | 244 km of Indanza |
|
|
|
|
This study |
QCAZ17989 |
|
Ecuador | Estación Biológica JatunSacha |
|
|
- |
|
This study |
QCAZ17990 |
|
Ecuador | Estación Biológica JatunSacha |
|
|
|
|
This study |
QCAZ17991 |
|
Ecuador | Estación Biológica JatunSacha |
|
|
- |
|
This study |
QCAZ23632 |
|
Ecuador | 7Km North of Cosanga |
|
|
|
|
This study |
QCAZ23917 |
|
Ecuador | Gualaquiza-El Ideal |
|
|
|
|
This study |
QCAZ10601 |
|
Ecuador | Parque Nacional Yasuní | - |
|
|
- |
|
QCAZ18241 |
|
Ecuador | Shaime |
|
|
|
|
This study |
10226MSH |
|
Brazil | Amazonas, Anavilhanas |
|
|
|
- |
|
10339MSH |
|
Brazil | Amazonas, Anavilhanas |
|
|
|
- |
|
QCAZ42269 |
|
Ecuador | Reserva Yachana |
|
|
|
|
This study |
111af |
|
French Guyana | Brownsberg |
|
|
|
- |
|
156mc |
|
French Guyana | Trijonction |
|
|
|
- |
|
LAJ210 |
|
Brazil | Tocantins, Lajeado | - |
|
|
- |
|
MZUSP103261 |
|
Brazil | Tocantins, Peixe | - |
|
|
- |
|
SMF88237 |
|
Bolivia | - | - |
|
- | - |
|
MNKA9691 |
|
Bolivia | - | - |
|
- | - |
|
ESTR00173 | Brazil | Amazonas, Carolina | - |
|
|
- |
|
|
AF7275337 | Brazil | Mato Grosso, APM Manso | - |
|
|
- |
|
|
|
||||||||
QCAZ50698 |
|
Ecuador | Puerto Cayo |
|
|
|
|
This study |
QCAZ50702 |
|
Ecuador | San Andrés de Rocafuerte |
|
|
|
|
This study |
QCAZ18203 |
|
Ecuador | Estación Biológica Jatun Sacha |
|
|
|
|
This study |
KU217501 |
|
Ecuador | Pastaza | - |
|
|
- |
|
MTD43789 |
|
Peru | Palma Pampa | - |
|
|
- |
|
UTA53310 |
|
Bolivia | La Paz | - |
|
|
- |
|
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
The sister clade to Chocó-Panama + Upper Amazon has weak support and includes other members of the
The Maximum Likelihood tree based on mitochondrial genes (Fig.
Maximum Likelihood phylogram depicting relationships within the
Box and whisker plots showing snout-vent length variation in adult
Descriptive statistics for morphometric measurements of adults from
|
|
|||||||
---|---|---|---|---|---|---|---|---|
Amazon | Chocó | Panamá | combined | |||||
Morphometric measurements | Males |
Females |
Males |
Females |
Males |
Females |
Males |
Females |
|
45.6 ± 4.11 |
68.90 ± 8.26 |
36.66 ± 2.42 |
44.82 ± 4.42 |
38.03 ± 0.59 |
42.38 ± 3.82 |
36.83 ± 2.31 (43.25–31.84) | 44.27 ± 4.37 (56.19–37.78) |
|
18.73 ± 1.97 |
29.36 ± 2.97 |
15.98 ± 1.14 |
18.26 ± 1.24 |
15.86 ± 1.16 |
17.79 ± 0.75 |
15.97 ± 1.13 (18.72– 13.69) | 18.17 ± 1.16 (20.73–16.22) |
|
19.67 ± 1.97 |
29.33 ± 3.67 |
15.69 ± 1.34 |
18.16 ± 1.72 |
16.39 ± 0.37 |
17.46 ± 0.67 |
15.77 ± 1.27 (19.28–13.09) | 18.02 ± 1.58 (22.04–15.18) |
|
16.9 ± 1.59 |
25.88 ± 2.73 |
12.57 ± 0.95 |
15.10 ± 1.6 |
12.98 ± 0.17 |
14.90 ± 1.12 |
12.63 ± 0.91 (15.14–10.31) | 15.06 ± 1.50 (18.94–12.49) |
|
14.6 ± 1.28 |
22.27 ± 2.71 |
11.61 ± 0.8 |
13.67 ± 1.19 |
11.85 ± 0.21 |
13.18 ± 1.12 |
11.64 ± 0.76 (13.88–10.29) | 13.57 ± 1.17 (16.84–11.77) |
|
9.46 ± 0.86 |
15.43 ± 2.02 |
7.71 ± 0.59 |
9.28 ± 0.86 |
8.39 ± 0.21 |
9.13 ± 0.49 |
7.79 ± 0.59 (8.87–6.45) | 9.25 ± 0.79 (11.4–7.77) |
|
8.78 ± 1.55 |
17.73 ± 3.26 |
6.27 ± 0.54 |
7.96 ± 0.68 |
6.59 ± 0.31 |
7.38 ± 0.39 |
6.31 ± 0.52 (7.97–5.36) | 7.84 ± 0.67 (9.71–6.63) |
|
2.08 ± 0.44 |
2.45 ± 0.42 |
1.63 ± 0.29 |
1.70 ± 0.21 |
1.47 ± 0.17 |
1.66 ± 0.16 |
1.61 ± 0.28 (2.23–1.05) | 1.69 ± 0.20 (2.08–1.25) |
|
3.35 ± 0.35 |
3.12 ± 0.37 |
2.50 ± 0.33 |
2.80 ± 0.43 |
2.41 ± 0.11 |
2.42 ± 0.23 |
2.48 ± 0.32 (3.23–1.86) | 2.72 ± 0.43 (3.98–2.08) |
|
3.48 ± 0.24 |
4.14 ± 0.21 |
3.34 ± 0.47 |
3.46 ± 0.59 |
3.38 ± 0.20 |
3.79 ± 0.25 |
3.33 ± 0.45 (4.03–1.95) | 3.52 ± 0.55 (4.45–2.5) |
|
16.87 ± 2.145 |
24.87 ± 3.64 |
13.46 ± 1.12 |
15.33 ± 1.52 |
13.72 ± 0.68 |
14.96 ± 0.76 |
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
Three components with eigenvalues > 1.0 were extracted from the PCA for females (Table
Principal components extracted from the analysis of ten size-corrected morphological variables of adult
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
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 |
|
HL | 0.350 | -0.065 | 0.153 |
|
-0.268 | -0.078 |
HW | 0.343 | 0.132 | -0.288 |
|
-0.222 | -0.045 |
IND | -0.203 |
|
|
0.280 |
|
-0.142 |
NSD | 0.217 | 0.155 | - |
0.262 | 0.386 | -0.186 |
SOCH |
|
-0.067 | 0.190 | 0.423 | -0.071 | -0.082 |
STCH |
|
-0.154 | -0.039 | 0.409 | -0.290 | -0.045 |
TD | 0.071 |
|
-0.159 | 0.099 |
|
-0.128 |
TL |
|
-0.200 | 0.232 | 0.134 | 0.228 |
|
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
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
Dorsolateral views of
Dorsolateral views of
The holotype of
Dorsal (
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
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.
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,
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
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
These results raise some rather interesting questions. For instance, the complete distribution range of
Because all species in the
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
Examined material. Numbers in bold indicate specimens analyzed genetically and morphometrically.
Bayesian consensus phylogram depicting relationships within the