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Systematics of treefrogs of the Hypsiboas calcaratus and Hypsiboas fasciatus

species complex (Anura, Hylidae) with the description of four new species
Article in ZooKeys · January 2014

DOI: 10.3897/zookeys.370.6291 · Source: PubMed
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A peer-reviewed open-access journal
ZooKeys 370: 1–68 (2014)
Systematics of treefrogs of the Hypsiboas calcaratus and Hypsiboas fasciatus species complex... 1
doi: 10.3897/zookeys.370.6291 RESEARCH ARTICLE
www.zookeys.org Launched to accelerate biodiversity research
Systematics of treefrogs of the Hypsiboas calcaratus and

Hypsiboas fasciatus species complex (Anura, Hylidae)
with the description of four new species
Marcel A. Caminer1,2,†, Santiago R. Ron1,‡
1 Museo de Zoología, Escuela de Biología, Pontificia Universidad Católica del Ecuador, Av. 12 de Octubre y
Roca, Aptdo. 17-01-2184, Quito, Ecuador 2 Current address: Museo Nacional de Ciencias Naturales, Depar-
tamento de Biologia Evolutiva y Biodiversidad, José Gutierrez Abascal 2, Madrid 28006, Spain
† http://www.zoobank.org/6AABCD30-765B-4428-A918-854EDB7CACB7
‡ http://www.zoobank.org/ACF9C463-F771-459C-B22B-AF6B9902DF57
Corresponding author: Santiago R. Ron ([email protected])
Academic editor: F. Andreone | Received 23 September 2013 | Accepted 25 December 2013 | Published 15 January 2014
http://www.zoobank.org//1CAC7714-D905-48F1-873A-997D4109A590
Citation: Caminer MA, Ron SR (2014) Systematics of treefrogs of the Hypsiboas calcaratus and Hypsiboas fasciatus species
complex (Anura, Hylidae) with the description of four new species. ZooKeys 370: 1–68. doi: 10.3897/zookeys.370.6291
Abstract
We review the systematics of the Hypsiboas calcaratus species complex, a group of widely distributed
Amazonian hylid frogs. A comprehensive analysis of genetic, morphological, and bioacoustic datasets un-
covered the existence of eleven candidate species, six of which are confirmed. Two of them correspond to
Hypsiboas fasciatus and Hypsiboas calcaratus and the remaining four are new species that we describe here.
Hypsiboas fasciatus sensu stricto has a geographic range restricted to the eastern Andean foothills of southern
Ecuador while Hypsiboas calcaratus sensu stricto has a wide distribution in the Amazon basin. Hypsiboas
almendarizae sp. n. occurs at elevations between 500 and 1950 m in central and northern Ecuador; the
other new species (H. maculateralis sp. n., H. alfaroi sp. n., and H. tetete sp. n.) occur at elevations below
500 m in Amazonian Ecuador and Peru. The new species differ from H. calcaratus and H. fasciatus in
morphology, advertisement calls, and mitochondrial and nuclear DNA sequences. Five candidate species
from the Guianan region, Peru, and Bolivia are left as unconfirmed. Examination of the type material of
Hyla steinbachi, from Bolivia, shows that it is not conspecific with H. fasciatus and thus is removed from
its synonymy.
Copyright M.A. Caminer, S.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.
2 Marcel A. Caminer & Santiago R. Ron / ZooKeys 370: 1–68 (2014)
Resumen
Revisamos la sistemática del complejo de especies Hypsiboas calcaratus, un grupo de ranas hílidas de am-
plia distribución en la Amazonía. Un análisis exhaustivo de datos genéticos, morfológicos y bioacústicos
revelaron la existencia de once especies candidatas, de las cuales seis son confirmadas. Dos de ellas corre-
sponden a Hypsiboas fasciatus e Hypsiboas calcaratus y las cuatro restantes son especies nuevas que describi-
mos aquí. Hypsiboas fasciatus sensu stricto tiene un rango geográfico restringido a las estribaciones orientales
de los Andes del sur del Ecuador, mientras que Hypsiboas calcaratus sensu stricto tiene una distribución
amplia en la cuenca amazónica. Hypsiboas almendarizae sp. n. se encuentra a alturas entre los 500 y 1950
m en el centro y el norte del Ecuador, mientras que las otras nuevas especies (H. maculateralis sp. n., H.
alfaroi sp. n. y H. tetete sp. n.) se encuentran a elevaciones bajo 500 m en la Amazonía de Ecuador y Perú.
Las nuevas especies difieren de H. calcaratus e H. fasciatus en morfología, cantos de anuncio y secuencias
de ADN mitocondrial y nuclear. Cinco especies candidatas de la región de las Guayanas, Perú y Bolivia
quedan como no confirmadas. La examinación del material tipo de Hyla steinbachi indica que no es cone-
specífica con H. fasciatus y por lo tanto se remueve de su sinonimia.
Keywords
Advertisement call, cryptic diversity, conservation status, morphology, new species
Palabras clave
Anura, canto de anuncio, diversidad críptica, estado de conservación, morfología, nuevas especies
Introduction
Management and conservation planning of biodiversity requires proper species iden-

tifications and comprehensive inventories. Recent DNA-based studies of Amazonian
amphibians have shown the existence of a large proportion of undescribed species that
have escaped detection in morphology-based assessments (Elmer et al. 2013; Elmer
and Cannatella 2008; Fouquet et al. 2012; Fouquet et al. 2007; Funk et al. 2012; Jun-
gfer et al. 2013; Ron et al. 2012). The discovery of these taxonomic voids highlights
the need of renewed and intensive efforts to discover and catalogue amphibians in the
Amazon region. This is particularly urgent because recent evaluations of the potential
impact of climate change have shown that Amazonian amphibians are highly vulner-
able (Foden et al. 2013). Conservation measures could be ineffective without reliable
baseline data on species identity and distribution.
Among the species complexes that have been proven to contain a large proportion
of hidden species richness is the Hypsiboas calcaratus-fasciatus species complex (Funk et
al. 2012). Both species are widely distributed in the Amazon basin (Azevedo-Ramos et
al. 2010; Icochea et al. 2004) and are characterized by having brown dorsal coloration,
basal hand webbing, and presence of dark marks or bars on the flanks and concealed
surfaces of the thighs (Duellman 1973). According to most current accounts Hypsiboas
calcaratus is a species with large calcars and vertical bars on the flanks and thighs while
H. fasciatus has small calcars and irregular black spots on the flanks and thighs (e.g.,
De la Riva et al. 2000; Duellman 2005; Rodríguez and Duellman 1994). Presumably,
these accounts are based on the taxonomic review by Duellman (1973), who included
them in the Hyla geographica group. In their phylogeny of Hylidae, Faivovich et al.
(2005) demonstrated that the “Hyla geographica” group was paraphyletic. Hypsiboas
calcaratus and H. fasciatus were assigned to the Hypsiboas albopunctatus group, which
was diagnosed by genetic characters. The H. albopunctatus group, according to the defi-
nition of Faivovich et al. (2005), contains nine formally described species: H. albopunc-
tatus (Spix, 1824), H. calcaratus (Troschel, 1848), H. dentei (Bokermann, 1967), H.
fasciatus (Günther, 1858), H. heilprini (Noble, 1923), H. lanciformis (Cope, 1871),
H. leucocheilus (Caramaschi & Niemeyer, 2003), H. multifasciatus (Günther, 1859)
and H. raniceps (Cope, 1862). Wiens et al. (2010) found strong support for the H.
albopunctatus group with H. calcaratus and H. fasciatus being sister to each other. Pyron
and Wiens (2011), however, show H. fasciatus as sister of H. dentei and H. calcaratus
as sister of a clade formed by H. lanciformis, H. multifasciatus and H. albopunctatus.
Recently published genetic, morphological and bioacoustic evidence suggests that
H. fasciatus and H. calcaratus are a species complex. A phylogenetic analysis based
on mitochondrial DNA (mtDNA) sequences revealed the presence of unconfirmed
candidate species (Fouquet et al. 2007a). More recently, Funk et al. (2012) analyzed
variation in mitochondrial and nuclear DNA, acoustic and morphological characters
of populations of both species and found the existence of nine candidate species. Here
in we incorporate additional genetic and morphological data and identify 11 candidate
species of which six are confirmed candidate species. We describe four of them includ-
ing their advertisement calls and variation in external morphology.
Methods
Morphological terminology follows Duellman (1970 and 1973). Examined specimens
(listed in Appendix) are housed in Museo de Zoología de la Pontificia Universidad
Católica del Ecuador (QCAZ), Museo de Historia Natural Gustavo Orcés at Escuela
Politécnica Nacional (EPN), National Museum of Natural History (USNM), and Nat-
ural History Museum (BMNH). We also examined the type material of Hyla leptoscelis
(holotype BMNH 1947.2.23.10), Hypsiboas fasciatus (holotype BMNH 58.4.25.22)
and Hyla steinbachi (syntypes BMNH 1947.2.13.61–63) deposited at the British Mu-
seum of Natural History.
Adult specimens were measured for the following variables: (1) snout-vent length;
(2) head length; (3) head width; (4) femur length; (5) tibia length; (6) foot length;
(7) tympanum diameter; and (8) calcar length. Measurements were made according
to the methodology described in Duellman (1970) with digital calipers (nearest 0.01
mm) from specimens fixed in 10% formalin and preserved in 70% ethanol. Sex was
determined by gonadal inspection or by the presence of prepollical spines and/or vocal
sac folds in males.
Multivariate analyses of variance (MANOVA) and Principal Components Analysis
(PCA) were used to assess the degree of morphometric differentiation among adult
individuals of all species considered. To remove the effect of covariation in size, the
MANOVA and PCA were applied to the residuals from the linear regressions between
six morphometric variables and SVL; this procedure was performed separately for
males (n = 136) and females (n = 34). For the PCA, only components with eigenvalues
> 1 were retained. Finally, variables were compared between species with Student’s t-
tests after ensuring their fit to a normal distribution.
Multivariate morphometric analyses were based in 170 adult specimens (popula-
tions of origin in parentheses): 25 H. fasciatus (including the holotype; Centro Shuar
Yawi, La Pradera, Las Orquídias, Limón, Romerillos Alto, Miazi Alto, Tink and Zamo-
ra), 27 H. almendarizae sp. n. (Baños, El Rosario, Guamote, Limón Indanza and Río
Hollín), 39 H. calcaratus (Añangu, Canelos, Chiroisla, Comunidad Santa Rosa, Edén,
El Coca, Kapawi, Estación Científica Yasuní PUCE, La Primavera, Puerto Bolivar,
San Vicente, Selva Lodge and Tena), 28 H. maculateralis sp. n. (Bataburo Lodge, Chi-
roisla, Comunidad Santa Rosa, Cuyabeno, Edén, El Coca, Huiririma, La Primavera,
Selva Lodge, San Vicente, Santa Teresita and Zábalo), 44 H. alfaroi sp. n. (Añangu,
Chiroisla, Cuyabeno, Edén, El Coca, Estación Científica Yasuní PUCE, Huiririma,
La Primavera, Lago Agrio, Nuevo Rocafuerte, Pañacocha, Puerto Bolivar, San Vicente
and Selva Lodge), and 7 H. tetete sp. n. (Comunidad Santa Rosa and Jatun Sacha). See
Tables 1 and 2 and Appendix for detailed locality information. Statistical analyses were
carried out in SPSS ® (2009, version 17.0 for Windows).
In the Diagnosis sections, coloration refers to preserved specimens unless other-
wise noted. Seven qualitative morphological characters were evaluated: (1) dorsal col-
oration, (2) ventral coloration, (3) iris coloration, (4) middorsal stripe [i. present, ii.
absent], (5) black flecks on the neck and chest [i. present, ii. absent], (6) marks on flank
and hidden surfaces of the thighs [i. dark transversal bars, ii. thin dark transversal bars,
iii. dark blotches, iv. dark dots] and (7) size and shape of calcar [i. large and triangular,
ii. large and conical, iii. small and conical, iv. small and tubercular, v. absent]. Color in
life was obtained from color photographs.
Sound recordings were made with a Sennheiser K6–ME67TM directional micro-
phone with digital recorders Olympus LS-10TM and Marantz PMD660TM. Calls were
analyzed using Raven 1.3 software (Cornell Lab of Ornithology 2003-2008) at a sam-
pling rate of 44.1 kHz and a frequency resolution of 10.8 Hz. Measured call variables
are defined in Table 3. If available, several calls or notes were analyzed per individual
to calculate an individual average. Temporal variables were measured on the oscillo-
gram, spectral variables on the power spectrum. Five call variables were used to run a
Principal Components Analysis (PCA) to assess the degree of acoustic differentiation
between calls from five males of Hypsiboas fasciatus (from La Pradera and Zamora), five
males of H. almendarizae sp. n. (from Limón Indanza), seven males of H. calcaratus
(from Estación Científica Yasuní PUCE and Tena), one male of H. maculateralis sp. n.
(from Comunidad Santa Rosa), four males of H. alfaroi sp. n. (from Estación Cientí-
fica Yasuní PUCE), and four males of H. tetete sp. n. (from Comunidad Santa Rosa).
Original recordings are deposited in the audio archive of the QCAZ and are available at
the AmphibiaWebEcuador website (http://zoologia.puce.edu.ec/vertebrados/anfibios/).
Table 1. Descriptive statistics for morphometric measurements of male Hypsiboas alfaroi, H. almendarizae, H. calcaratus, H. fasciatus, H. maculateralis, and H. tetete
used for Principal Component Analysis. Mean ± SD is given with range below. Bold figures represent combined values for males of all populations. Abbreviations
are: SVL = Snout-vent length; FOOT = Foot length; HL = Head length; HW = Head width; ED = Eye diameter; TD = Tympanum diameter; TL = Tibia length; FL
= Femur length; CL = Calcar length. All measurements are in mm.
Species SVL FOOT HL HW ED TD TL FL CL

H. alfaroi 32.80 ± 1.97 12.54 ± 0.65 8.95 ± 0.98 10.07 ± 0.61 3.25 ± 0.36 2.28 ± 0.28 18.50 ± 0.95 16.25 ± 0.96 Calcar
(n = 32) 27.91–36.27 11.33–13.80 7.13–11.84 8.72–10.95 2.52–4.03 1.70–3.01 16.54–20.50 14.71–17.91 absent
Yasuní PUCE 32.20 ± 1.98 12.73 ± 0.61 8.35 ± 0.60 10.10 ± 0.53 3.29 ± 0.36 2.24 ± 0.11 18.66 ± 1.10 16.25 ± 0.98
Calcar absent
(n = 7) 27.91–35.19 11.33–13.46 7.40–8.99 8.79–10.80 2.90–4.03 2.08–2.50 16.54–20.50 14.81–17.89
Huiririma
33.61–34.22 12.95–13.80 9.69–9.75 10.37–10.77 3.25–3.32 2.22–2.51 18.10–19.32 15.50–17.17 Calcar absent
(n = 2)
Nuevo Rocafuerte 33.99 ± 2.13 12.51 ± 0.81 9.91 ± 1.16 10.23 ± 0.63 3.11 ± 0.41 2.05 ± 0.23 18.82 ± 1.23 16.96 ± 1.22
Calcar absent
(n = 5) 30.34–35.88 11.41–13.71 8.98–11.84 9.26–10.95 2.52–3.61 1.70–2.28 16.95–20.05 14.92–17.91
Pañacocha 33.04 ± 0.52 12.10 ± 0.19 9.58 ± 0.10 9.65 ± 0.68 3.34 ± 0.33 2.29 ± 0.05 17.97 ± 0.29 16.27 ± 0.20
Calcar absent
(n = 3) 32.64–33.63 11.95–12.31 9.46–9.64 8.87–10.14 2.97–3.62 2.25–2.34 17.66–18.22 16.04–16.41
Selva Lodge
32.11–33.85 12.72–13.14 8.58–9.06 9.18–10.53 2.73–3.61 2.11–2.91 18.47–18.90 14.71–16.21 Calcar absent
(n = 2)
H. almendarizae 37.64 ± 2.01 15.04 ± 0.82 9.74 ± 0.86 11.8 1 ± 0.60 4.02 ± 0.34 2.27 ± 0.24 21.27 ± 0.94 19.24 ± 1.06 1.51 ± 0.24
(n = 23) 34.31–44.56 12.99–16.00 8.33–11.35 10.80–13.07 3.36–4.56 1.63–2.74 19.53–23.04 15.84–21.52 1.09–1.88
Baños
37.39–37.98 13.43–15.16 9.51–9.69 11.88–12.00 4.22–4.32 2.31–2.30 19.53–21.21 18.48–19.38 1.32–1-39
(n = 2)
El Rosario 38.90 ± 1.79 15.29 ± 0.40 11.06 ± 0.46 12.40 ± 0.27 4.42 ± 0.12 2.30 ± 0.20 22.45 ± 0.66 19.37 ± 0.23 1.64 ± 0.07
(n = 3) 37.56–40.92 14.89–15.70 10.52–11.34 12.15–12.68 4.35–4.56 2.13–2.53 21.74–22.58 19.10–19.50 1.56–1.68
Limón Indanza 36.72 ± 1.23 14.97 ± 0.88 9.19 ± 0.39 11.73 ± 0.56 3.94 ± 0.34 2.25 ± 0.23 21.01 ± 0.73 19.13 ± 1.19 1.51 ± 0.28
(n = 12) 34.31–38.60 12.99–16.00 8.33–9.74 10.80–12.68 3.36–4.54 2.02–2.74 19.64–22.24 15.84–20.23 1.09–1.88
Río Hollín
38.46–44.56 15.73–15.75 10.20–11.35 11.72–13.07 3.60–4.38 1.63–2.25 21.80–22.62 18.83–21.52 1.33–1.57
(n = 2)
H. calcaratus 36.82 ± 2.59 15.00 ± 1.31 10.14 ± 1.06 11.93 ± 1.01 3.63 ± 0.37 2.48 ± 0.21 22.10 ± 1.57 19.03 ± 1.48 2.03 ± 0.33
(n = 35) 27.61–42.50 10.68–17.44 7.71–12.63 9.16–13.72 2.47–4.52 1.92–2.94 16.76–24.61 13.82–21.44 1.32–2.65
Canelos 36.59 ± 1.49 14.64 ± 0.59 10.48 ± 0.55 11.68 ± 0.59 3.87 ± 0.30 2.43 ± 0.24 21.78 ± 0.34 18.28 ± 0.53 2.06 ± 0.25
(n = 3) 34.89–37.69 14.08–15.26 10.05–11.10 11.01–12.13 3.69–4.22 2.18–2.66 21.39–22.03 17.67–18.66 1.89–2.34
Tena 35.23 ± 2.80 13.87 ± 1.10 9.12 ± 1.15 11.95 ± 1.24 3.71 ± 0.31 2.57 ± 0.23 20.69 ± 1.29 18.28 ± 1.13 2.05 ± 0.39
(n = 5) 32.10–39.15 12.65–15.20 8.18–10.69 10.69–13.72 3.33–4.04 2.39–2.94 18.77–21.98 16.51–19.43 1.56–2.54
Systematics of treefrogs of the Hypsiboas calcaratus and Hypsiboas fasciatus species complex...
Edén
36.88–37.09 14.39–14.57 9.75–10.72 11.30–12.24 3.29–3.55 2.41–2.71 20.70–21.85 17.42–17.63 1.84–2.13
5
(n = 2)
6

El Coca
34.83–34.96 14.21–14.42 9.05–9.16 11.19–11.31 2.47–2.94 2.10–2.37 20.04–22.52 16.46–19.22 1.68–1.98
(n = 2)
Estación Científica Yasuní
37.78 ± 1.53 15.73 ± 0.89 10.43 ± 0.89 12.20 ± 0.90 3.71 ± 0.30 2.48 ± 0.17 22.99 ± 0.98 19.91 ± 0.85 2.06 ± 0.36
PUCE
35.21–40.10 14.54–17.44 8.99–12.63 9.59–13.33 3.36–4.52 2.23–2.87 21.12–24.61 18.40–21.44 1.32–2.47
(n = 18)
Parque Nacional Yasuní,
Pompeya 36.11–36.31 13.50–15.52 10.42–11.43 11.02–13.13 3.60–4.12 2.43–2.63 21.14–23.47 19.38–19.55 1.90–2.04
(n = 2)
H. fasciatus 35.40 ± 1.65 14.00 ± 0.59 9.651 ± 0.66 11.39 ± 0.59 3.71 ± 0.34 2.42 ± 0.25 20.07 ± 0.89 18.18 ± 0.87 1.35 ± 0.15
(n = 19) 32.65–37.74 12.85–15.24 8.42–11.53 10.41–12.33 3.15–4.42 2.03–2.78 18.63–21.71 16.12–19.94 1.08–1.63
Centro Shuar Yawi 35.79 ± 2.14 13.81 ± 1.02 9.69 ± 0.57 11.60 ± 0.71 3.91 ± 0.38 2.40 ± 0.25 20.55 ± 1.34 18.14 ± 1.42 1.35 ± 0.07
(n = 4) 36.22–37.72 12.85–15.24 9.03–10.43 10.66–12.33 3.50–4.42 2.20–2.73 18.63–21.71 16.12–19.42 1.25–1.41
La Pradera 34.69 ± 0.82 14.18 ± 0.14 9.61 ± 0.31 10.98 ±0.43 3.66 ± 0.16 2.31 ± 0.33 19.27 ± 0.43 18.00 ± 0.53 1.50 ± 0.10
(n = 4) 33.74–35.74 14.00–14.22 9.36–10.06 10.41–11.44 3.56–3.82 2.03–2.78 19.06–19.83 17.30–18.44 1.39–1.63
Tiink 34.79 ± 1.89 13.96 ± 0.52 9.72 ± 1.06 11.17 ± 0.63 3.55 ± 0.42 2.50 ± 0.19 20.30 ± 0.80 18.44 ± 1.00 1.23 ± 0.16
(n = 6) 32.65–37.50 13.46–14.77 8.42–11.53 10.60–12.10 3.15–4.09 2.26–2.72 19.25–21.12 17.29–19.94 1.08–1.47
Zamora
34.55–36.95 13.52–13.58 8.94–9.55 11.54–11.85 3.85–3.95 2.51–2.78 19.21–19.61 17.71–18.04 1.25–1.40
(n = 2)
H. maculateralis 36.00 ± 1.92 13.55 ± 1.03 9.59 ± 1.18 11.08 ± 0.70 3.55 ± 0.49 2.17 ± 0.28 20.67 ± 1.48 18.18 ± 1.40 1.67 ± 0.32
(n = 22) 31.86–39.17 11.10–16.22 6.62–11.19 9.21–12.29 2.63–4.39 1.26–2.63 16.94–23.23 15.31–20.57 1.15–2.52
Bataburo Lodge
34.20–38.18 13.02–13.90 9.58–11.10 10.27–11.81 3.36–4.24 2.09–2.14 18.60–22.21 17.77–19.19 1.24–1.47
(n = 2)
Edén 34.74 ± 2.68 12.65 ± 1.37 7.93 ± 1.34 10.38 ± 1.02 3.29 ± 0.63 2.12 ± 0.16 18.83 ± 1.64 16.85± 1.35 1.81 ± 0.22
(n = 3) 31.86–37.17 11.10–13.68 6.62–9.30 9.21–11.09 2.63–3.88 1.94–2.24 16.94–19.82 15.31–17.84 1.56–1.96
Reserva de Producción
Faunística Cuyabeno 36.40 ± 1.53 13.59 ± 0.60 10.23 ± 0.75 11.32 ± 0.45 3.97 ± 0.26 2.29 ± 0.15 21.65 ± 0.69 19.56 ± 0.83 1.89 ± 0.35
PUCE 33.71–38.69 12.57–14.19 9.15–11.19 10.69–12.14 3.67–4.39 2.10–2.57 20.63–22.43 18.36–20.57 1.55–2.52
(n = 7)
Marcel A. Caminer & Santiago R. Ron / ZooKeys 370: 1–68 (2014)
La Primavera
35.26–38.49 13.41–14.58 9.36–11.01 10.90–11.14 3.02–3.67 1.26–2.28 20.84–20.91 17.20–17.69 1.27–1.86
(n = 2)
Selva Lodge
37.48–39.17 12.98–16.22 8.77–9.94 11.72–12.29 3.24–3.33 2.43–2.63 20.86–23.23 16.12–19.90 1.61–1.90
(n = 2)
H. tetete
31.72 ± 0.42 12.22 ± 0.15 8.13 ± 0.53 10.32 ± 0.24 3.60 ± 0.25 2.62 ± 0.35 17.62 ± 0.24 16.50 ± 0.36 Calcar
Comunidad Santa Rosa
31.15–32.24 12.01–12.40 7.48–8.75 9.97–10.64 3.38–4.02 2.25–3.01 17.30–17.93 16.09–17.00 absent
(n = 5)
Table 2. Descriptive statistics for morphometric measurements of female Hypsiboas alfaroi, H. almendarizae, H. calcaratus, H. fasciatus, H. maculateralis, and H.
tetete used for Principal Component Analysis. Mean ± SD is given with range below. Bold figures represent combined values for females from all populations. Ab-
breviations are: SVL = Snout-vent length; FOOT = Foot length; HL = Head length; HW = Head width; ED = Eye diameter; TD= Tympanum diameter; TL = Tibia
length; FL = Femur length; CL = Calcar length. All measurements are in mm.

H. alfaroi 44.51 ± 3.09 16.72 ± 1.96 11.59 ± 1.17 14.21 ± 1.33 4.12 ± 0.56 2.96 ± 0.32 25.47 ± 1.93 22.77 ± 1.64
Calcar absent
(n = 12) 39.68–49.21 11.39–18.94 8.90–13.09 1.55–15.76 3.23–5.00 2.22–3.44 22.88–9.44 19.88–25.66
Estación Científica
42.81 ± 2.08 15.82 ± 2.99 11.71 ± 0.66 13.93 ± 1.05 4.12 ± 0.36 2.87 ± 0.23 24.83 ± 1.52 22.91 ± 1.32
Yasuní PUCE Calcar absent
40.27–45.37 11.39–17.71 11.03–12.28 12.41–14.70 3.59–4.34 2.60–3.14 23.18–26.80 21.17–24.35
(n = 4)
Playas de Cuyabeno
44.51–48.01 16.60–17.41 12.15–13.09 14.99–15.71 4.91–5.00 3.18–3.44 24.61–25.11 21.16–23.38 Calcar absent
(n = 2)
H. almendarizae 48.11 ± 6.88 19.36 ± 2.92 11.59 ± 1.96 14.77 ± 2.45 4.08 ± 0.66 2.52 ± 0.41 25.81 ± 6.88 24.46 ± 3.41 1.89 ± 0.55
(n = 4) 37.80–51.94 15.11–21.64 8.70–12.99 11.15–16.59 3.45–4.68 2.07–3.00 15.51–29.74 19.40–26.62 1.20–2.53
Nueve de Octubre 51.54 ± 0.35 20.78 ± 0.88 12.55 ± 0.43 15.98 ± 0.54 4.28 ± 0.62 2.68 ± 0.34 29.25 ± 0.44 26.15 ± 0.60 2.12 ± 0.36
(n = 3) 51.26–51.94 19.87–21.64 12.14–12.99 15.61–16.59 3.57–4.68 2.32–3.00 28.87–29.74 25.47–26.62 1.87–2.53
H. calcaratus 50.92 ± 4.80 20.56 ± 2.05 13.08 ± 2.03 16.46 ± 1.85 4.14 ± 0.86 3.12 ± 0.50 31.00 ± 3.13 26.05 ± 2.15 2.62 ± 0.16
(n = 4) 45.94–56.29 18.44–23.17 11.04–15.83 14.59–18.42 3.57–5.42 2.67–3.77 28.10–35.29 24.47–29.09 2.42–2.78
H. fasciatus 51.89 ± 3.18 20.44 ± 1.23 13.91 ± 0.79 16.57 ± 0.72 4.83 ± 0.43 3.25 ± 0.38 29.58 ± 1.46 27.02 ± 1.34 1.95 ± 0.15
(n = 5) 47.16–54.84 18.79–21.98 12.59–14.53 15.80–17.38 4.28–5.32 2.70–3.77 28.55–32.09 25.74–29.20 1.73–2.09
H. maculateralis 45.18 ± 9.39 16.27 ± 3.29 11.76 ± 2.40 14.21 ± 2.95 3.78 ± 0.26 2.43 ± 0.52 25.23 ± 4.42 21.90 ± 4.22 1.61 ± 0.40
(n = 6) 32.04–55.31 11.25–19.77 8.28–14.10 10.01–17.37 3.34–4.06 1.70–3.00 18.04–29.66 15.48–26.54 1.09–2.22
Zábalo
32.04–38.26 11.25–14.58 8.28–9.28 10.01–12.38 3.34–3.61 1.70–2.03 18.04–22.82 15.48–19.15 1.09–1.39
(n = 2)
H. tetete
Jatun Sacha 45.33–45.85 16.81–18.17 11.15–12.96 13.66–14.11 4.09–4.96 3.56–3.85 25.45–25.78 21.18–21.81 Calcar absent
(n = 2)
7
Table 3. Call traits of Hypsiboas spp. analyzed in this study. See text for details.
Character Description
Time from the beginning of the first note to the end of the last note
Call duration
of the call
Number of notes Number of notes in the call
Time from the beginning of the call to the point of its maximum
Rise time
amplitude
Call dominant frequency The frequency with the greatest amount of sound energy along all the call
The frequency with the greatest amount of sound energy in the first
Call fundamental frequency
harmonic, measured along all the call
Dominant frequency at the The frequency with the greatest amount of sound energy measured at
beginning to the third note the beginning of the third note
Fundamental frequency at the The frequency of the first harmonic measured at the beginning of the
beginning to the third note third note
Dominant frequency at the The frequency with the greatest amount of sound energy measured at
end to the third note the end of the third note
Fundamental frequency at the The frequency of the first harmonic measured at the end of the third
end to the third note note
Number of pulses Number of pulses per note
Pulse repetition rate Number of pulses per second
Interval between calls Time from end of call to the beginning of next call
The conservation status of each species was evaluated according to the IUCN Red
List categories and criteria (IUCN 2001). The estimates of the distribution area were
based on the minimum convex polygon (the smallest polygon in which no internal an-
gle exceeds 180 degrees and which contains all known localities). Vegetation types are
based on WWF Ecoregions (available at http://www.eoearth.org/view/article/151948)
except for Ecuadorian localities, which are based in the more detailed classification of
Sierra et al. (1999).
Phylogenetic analyses
Phylogenetic analyses are an expansion of those presented by Funk et al. (2012) to in-
clude additional sequences of H. calcaratus, H. dentei, H. fasciatus and H. raniceps pub-
lished by Darst and Cannatella (2004), Faivovich et al. (2004, 2005), Salducci et al.
(2002, 2005), Jansen et al. (2011), Wiens et al. (2005, 2006) and 19 sequences of the
nuclear gene RAG-1 (recombination activating gene 1). GenBank accession numbers
for the sequences not included in Funk et al. (2012) are shown in Table 4. The same
matrix and alignment of mitochondrial DNA sequence data (genes 12S rRNA, 16S
rRNA and COI mtDNA) was employed. For the nuclear DNA, preliminary align-
ment of RAG-1 and POMC sequences were done with CLUSTAL X 2.0 (Larkin et al.
2007). Manual adjustments to the alignment were made using Mesquite version 2.75
(Maddison and Maddison 2009).
Table 4. Genbank accession numbers for DNA sequences used in the phylogenetic analysis. Accession numbers for mitochondrial sequences not included here are
listed in Funk et al. (2012).
Genbank Accession No.

Museum No. Species Reference
12S 16S COI POMC RAG1
QCAZ 44351 Hypsiboas alfaroi JN970413 JN970549 JN970682 JN970804 KF955320 Funk et al. 2012; This study
QCAZ 44425 H. alfaroi JN970415 JN970551 JN970684 JN970806 KF955321 Funk et al. 2012; This study
QCAZ 44858 H. alfaroi JN970469 JN970605 JN970737 JN970860 KF955322 Funk et al. 2012; This study
QCAZ 50785 H. alfaroi KF955303 KF955305 KF955306 KF955307 -- This study
QCAZ 31452 H. almendarizae JN970482 JN970618 -- JN970873 KF955311 Funk et al. 2012; This study
QCAZ 32645 H. almendarizae JN970386 JN970522 JN970658 JN970777 KF955312 Funk et al. 2012; This study
QCAZ 39650 H. almendarizae JN970394 JN970530 JN970665 JN970785 KF955313 Funk et al. 2012; This study
QCAZ 51809 H. almendarizae KF955304 -- -- -- -- This study
QCAZ 43256 H. calcaratus JN970444 JN970580 JN970713 JN970835 KF955314 Funk et al. 2012; This study
KU 202911 H. calcaratus AY326056 AY326056 -- -- -- Darst and Cannatella 2004
KU 221856 H. calcaratus DQ380352 -- -- -- -- Wiens et al. 2006
13MC H. dentei EF376018 AF467270 -- -- -- Salducci et al. 2002; Salducci et al. 2005
QCAZ 17030 H. fasciatus JN970399 JN970535 JN970669 JN970790 KF955310 Funk et al. 2012; This study
QCAZ 48583 H. fasciatus JN970490 JN970626 -- JN970881 KF955308 Funk et al. 2012; This study
QCAZ 48584 H. fasciatus JN970388 JN970524 -- JN970779 KF955309 Funk et al. 2012; This study
QCAZ 20641 H. lanciformis JN970512 JN970648 JN970767 JN970898 KF955325 Funk et al. 2012; This study
QCAZ 30936 H. lanciformis JN970510 JN970646 JN970765 JN970896 KF955326 Funk et al. 2012; This study
QCAZ 40082 H. maculateralis JN970405 JN970541 JN970675 JN970796 KF955317 Funk et al. 2012; This study
115MC H. raniceps EF376021 AF467269 -- -- -- Salducci et al. 2002; Salducci et al. 2005
MACN 37795 H. raniceps AY843657 AY843657 -- -- -- Faivovich et al. 2005
USNM 174173 H. raniceps AY819375 -- -- -- -- Wiens et al. 2005

QCAZ 40080 H. tetete JN970403 JN970539 JN970673 JN970794 KF955323 Funk et al. 2012; This study
9
10
Genbank Accession No.

Museum No. Species Reference
12S 16S COI POMC RAG1
QCAZ 40081 H. tetete JN970404 JN970540 JN970674 JN970795 KF955324 Funk et al. 2012; This study
MNKA 9467 Hypsiboas sp. -- JF790135 -- -- -- Jansen et al. 2011
AMNH-A
Hypsiboas sp. -- AY549335 -- -- -- Faivovich et al. 2004
164081
NMP6V 71250 Hypsiboas sp. -- AY843613 -- -- -- Faivovich et al. 2005
Because it is likely that each of our sampled genes (or codon positions in protein
coding genes) evolved under different processes, we partitioned the matrices according
to gene and codon position to analyze each partition under separate models of evolu-
tion. We used PartitionFinder v. 1.1.1 (Lanfear et al. 2012) to find the best-fit model
for each partition and also the best partition strategy. In the mitochondrial matrix, we
defined five a priori partitions (12S, 16S and one partition for each codon position of
COI). In the nuclear matrix we defined six a priori partitions (corresponding to each
codon position of our two genes).
Phylogenetic relationships were inferred separately for mitochondrial and nuclear
genes using maximum-likelihood and Bayesian inference. Maximum-likelihood and
Bayesian analyses were conducted in GARLI v. 2.0 (Zwickl 2006) and MRBAYES v.
3.2.1 (Ronquist et al. 2012) respectively using the same methodology described by
Funk et al. (2012).
Results
Phylogenetic relationships
The phylogenetic relationships recovered from the analysis of the mitochondrial

DNA sequences (Fig. 1) were consistent with those reported by Funk et al. (2012).
The following sections describe the relationships of the samples not included in
Funk et al. (2012).
We found strong support for a clade that includes Hypsiboas dentei and the H. calca
ratus-H. fasciatus species complex. This clade is sister to H. lanciformis + H. multifasciatus.
There are eleven candidate species, two more than those reported by Funk et al. (2012).
The population from Bolivia (clade J) is sister to clades I and H from French Guiana,
Guyana, and Suriname. The branch lengths in the phylogeny and the genetic distances
between J and I-H (uncorrected p-distance range 3.1–5.8% in gen 16S; Table 5) suggest
that J is not conspecific to any of the candidate species reported by Funk et al. (2012).
An additional unconfirmed candidate species (clade K) is represented by a single sample
(NMP6V 71250; reported as Hypsiboas calcaratus by Faivovich et al. 2005) from 50 km
W of Iquitos (Peru). The genetic distance relative to its closest relative (clade D) ranges
from 4.8 to 5.9% (16S), strongly suggesting that it is a separate species. The branch
lengths that separate clade K from clade D are longer than the lengths that separate pairs
of some confirmed candidate species (e.g., Hypsiboas fasciatus vs. Hypsiboas almendarizae
sp. n.) further implying that K is a valid undescribed species.
The phylogeny based on the nuclear genes (RAG-1 and POMC) is generally con-
sistent with the mitochondrial phylogeny but is less resolved (Fig. 2). Similarly to the
mitochondrial phylogeny, it shows strong support for two basal clades (Clades A–D
and Clades E–F). At the species level, it provides strong support for clades A and D.
The only strongly supported disagreement between the nuclear and the mitochon-
drial phylogeny is the placement of individual QCAZ 44177 because in the nuclear
Figure 1. Maximum likelihood phylogram depicting relationships within Hypsiboas. The phylogram
was derived from analysis of 2400 bp of mitochondrial DNA (gene fragments 12S, 16S, COI). Museum
catalog number and locality are shown for each sample. Bootstrap values are shown above the branches
and Bayesian posterior probabilities are shown below; missing values indicate values below 50 (bootstrap)
or 0.5 (posterior probability). Outgroup species (Hypsiboas lanciformis, H. pellucens and H. rubracylus)
are not shown. Abbreviations are: ARG Argentina, BO Bolivia, EC Ecuador, FG French Guiana, GUY
Guyana, PE Peru, SU Suriname.
tree it appears within a clade formed by individuals from the mitochondrial clades A
and B, while in the mitochondrial tree it appears as a member of clade C. The mor-
phological data shows that QCAZ 44177 is a member of clade C because it has its
distinctive characters (e.g., large and triangular calcar).
Figure 1. Continue
Taxonomic review
The available names for the populations sampled in our phylogenies are Hypsiboas cal-
caratus (Troschel, 1848), Hypsiboas fasciatus (Günther, 1858), Hyla leptoscelis Bouleng-
er, 1918, and Hyla steinbachi Boulenger, 1905. Examination of three of the holotypes
and published descriptions from the literature allowed us to assign the available names
to clades A and C (Fig. 1). We document those assignments in the following section.
Taxonomic status of H. fasciatus and H. calcaratus

The holotype of H. fasciatus is an adult female (SVL = 58.59 mm; BM 1858.7.25.22,
reported as “BM 58.4.25.22” by Duellman 1973; Fig. 3) collected by Louis Fraser. It
14
Table 5. Pairwise genetic distances (uncorrected p) of 16S DNA sequences among members of the Hypsiboas calcaratus species complex. Mean ± SD is given with
range in parentheses (below diagonal). Number of individuals compared is shown above diagonal. Diagonal shows intra-clade genetic distances.
Clade A Clade B Clade C Clade D Clade E Clade F

Clade G Clade H Clade I Clade J Clade K
H. fasciatus H. almendarizae H. calcaratus H. maculateralis H. alfaroi H. tetete
Clade A 0.004 ± 0.004
n = 31 n = 49 n = 36 n = 45 n = 18 n = 17 n = 19 n = 17 n = 19 n = 16
H. fasciatus (0.007–0.017)
Clade B 0.036 ± 0.009 0.008 ± 0.0006
n = 50 n = 37 n = 46 n = 19 n = 18 n = 20 n = 18 n = 20 n = 17
H. almendarizae (0.035–0.049) (0.001–0.012)
Clade C 0.057 ± 0.011 0.041 ± 0.003 0.009 ± 0.008
n = 55 n = 64 n = 37 n = 36 n = 38 n = 36 n = 38 n = 35
H. calcaratus (0.038–0.078) (0.034–0.050) (0.001–0.031)
Clade D 0.077 ± 0.003 0.074 ± 0.003 0.086± 0.006 0.002 ± 0.0008
n = 51 n = 24 n = 23 n = 25 n = 23 n = 25 n = 22
H. maculateralis (0.072–0.100) (0.069–0.086) (0.064–0.102) (0.001–0.004)
Clade E 0.082 ± 0.004 0.080 ± 0.003 0.089 ± 0.004 0.107 ± 0.004 0.002 ± 0.002
n = 33 n = 32 n = 34 n = 32 n = 34 n = 31
H. alfaroi (0.074–0.100) (0.072–0.090) (0.073–0.105) (0.097–0.120) (0.003–0.017)
Clade F 0.085 ± 0.002 0.078 ± 0.001 0.084 ± 0.003 0.100 ± 0.002 0.028 ± 0.002 0.013 ± 0.009
n=5 n=7 n=5 n=7 n=4
H. tetete (0.083–0.095) (0.073–0.082) (0.074–0.096) (0.093–0.107) (0.023–0.037) (0.001–0.019)
0.082 ± 0.002 0.077 ± 0.002 0.085 ± 0.003 0.097 ± 0.003 0.039 ± 0.002 0.041 ± 0.001
Clade G 0 n=6 n=4 n=6 n=3
(0.081–0.092) (0.073–0.079) (0.075–0.091) (0.091–0.107) (0.033–0.045) (0.039–0.043)
0.088 ± 0.003 0.082 ± 0.002 0.095 ± 0.003 0.098 ± 0.003 0.055 ± 0.003 0.053 ± 0.002 0.052 ± 0.002 0.004 ± 0.002
Clade H n=6 n=8 n=5
(0.083–0.101) (0.077–0.087) (0.088–0.108) (0.094–0.107) (0.050–0.068) (0.049–0.056) (0.050–0.055) (0.001–0.007)
0.088 ± 0.004 0.083 ± 0.003 0.098 ± 0.004 0.097 ± 0.005 0.059 ± 0.002 0.056 ± 0.001 0.058 ± 0.001 0.031 ± 0.002
Clade I 0.003 n=6 n=3
(0.081–0.097) (0.077–0.089) (0.093–0.115) (0.091–0.112) (0.056–0.067) (0.055–0.058) (0.057–0.059) (0.028–0.034)
0.081 ± 0.002 0.082 ± 0.003 0.084 ± 0.004 0.094 ± 0.004 0.053 ± 0.004 0.052 ± 0.002 0.035 ± 0.0008 0.038 ± 0.005 0.056 ± 0.002
Clade J 0 n=5
(0.077–0.087) (0.079–0.090) (0.079–0.097) (0.091–0.108) (0.049–0.075) (0.048–0.055) (0.034–0.035) (0.031–0.045) (0.054–0.058)
0.073 ± 0.002 0.068 ± 0.001 0.072 ± 0.008 0.051 ± 0.004 0.101 ± 0.004 0.099 ± 0.001 0.102 ± 0.002 0.086 ± 0.0002
Clade K 0.097 0.103 0
(0.071–0.081) (0.065–0.071) (0.047–0.085) (0.048–0.059) (0.094–0.112) (0.098–0.100) (0.100–0.105) (0.085–0.086)
Figure 2. Maximum likelihood phylogram depicting relationships within Hypsiboas. The phylogram was
derived from analysis of 1272 bp of nuclear DNA (gene fragments RAG-1 and POMC). Museum catalog
number and locality are shown for each sample. Bootstrap values are shown above the branches and Bayes-
ian posterior probabilities are shown below; values below 50 (bootstrap) or 0.5 (posterior probability) are
not shown. Colors refer to clades identified using mtDNA (see Figure 1).
closely resembles individuals from clade A and B (Fig. 4A–B), which also have a small
calcar and black bars on the flanks and thighs (Figs 5 and 6). We assign the binomial H.
fasciatus to clade A based on morphometric data showing a closer affinity to the holotype
(Fig. 7A). Clade B (H. almendarizae sp. n.) further differs from the holotype in having
narrower transversal dark bars on the flanks and thighs. The assignment of H. fasciatus to
clade A is also supported by geographic distribution. Although in the species description
by Günther (1858) the type locality is vaguely stated as “Anden von Ecuador”, it is almost
certain that the holotype was collected either in Gualaquiza or Zamora, Morona Santiago
and Zamora Chinchipe provinces, Ecuador. We infer this from letters and records of the
collections of the travels of Louis Fraser (Gardner 1983 and references therein). During
his trip, which lasted from 1857 to 1859, the only collection sites within the distribution
range of the H. fasciatus-H. calcaratus species complex are Gualaquiza (elevation 1100
m) and Zamora (1000 m). Both localities overlap with the distribution range of clade A
exclusively. Clade A has an elevation range from 700 to 1600 m.
Duellman (1973) characterized H. fasciatus as having, in the majority of the speci-
mens, brown flecks on the throat and chest and black spots on the flanks and thighs.
The holotype of H. fasciatus differs from this diagnosis because it has dark bars instead
of dots on the flanks and thighs and lacks flecks on the throat and chest. Most of the
specimens reported by Duellman (1973) as “H. fasciatus” are from localities below 700
m and resemble individuals from our clades D, E, and F (Figs 4D–E and 8D–F). Al-
though Duellman (1973) noted the aforementioned differences between the holotype
and the other individuals, he considered them conspecific.
Figure 3. Dorsolateral (top), ventral (left), and dorsal (right) views of the holotype of Hypsiboas fasciatus
(BMNH 58.4.25.22).
The holotype of H. calcaratus could not be examined because it is lost (Duellman

1973; Frost 2013). Nevertheless, the description of the holotype of H. calcaratus by
Troschel (1848) and the location of the type locality (British Guiana) suggest that the
binomial H. calcaratus is conspecific to clade C (Fig. 1). The holotype was described
as having a long calcar, perpendicular short black stripes on the flanks, and transversal
bars on the legs, which are characteristic of clade C. This designation is also supported
by the type locality “British Guiana” because it overlaps with the distribution of clade
C. The presence of clade C in the Republic of Guyana (formerly known as British
Guiana) is documented by photographs and the morphological description of “Hypsi-
boas calcaratus” by Kok and Kalamandeen (2008). Clade C is the only species with
developed calcar and dark bars on the legs and flanks that occurs below 500 m (Fig. 9).
Based on the available evidence, we assign the binomial Hypsiboas calcaratus to clade C.
Taxonomic status of Hyla leptoscelis and Hyla steinbachi

Other available names for the Hypsiboas fasciatus-Hypsiboas calcaratus species group are
Hyla leptoscelis Boulenger, 1918 and Hyla steinbachi Boulenger, 1905. Hyla leptoscelis
was synonymized under H. calcaratus by Duellman (1973). The holotype is a juvenile
female with SVL = 27 mm (BM 1947.2.23.10; Fig. 10). As noted by Duellman (1973)
and Lutz (1973), the specimen is in poor condition, discolored, and predominantly gray.
However, it has large calcars and lacks webbing on the hands, indicating that it belongs to
H. calcaratus and, thus, the synonymy proposed by Duellman (1973) seems to be correct.
Hyla steinbachi Boulenger, 1905 (Fig. 11) was synonymized under H. fasciata by
De la Riva (1990). There are three syntypes, two are adults of unknown sex (BM
1947.2.13.61and BM 1947.2.13.62; SVL 36.20 and 32.85 mm, respectively) and one
is a juvenile (BM 1947.2.13.63, SVL 22.04 mm). The adult specimens have ill-defined
calcars and prominent and abundant supernumerary tubercles on the hands (Fig. 11).
These characters distinguish it from Hypsiboas fasciatus, H. calcaratus, and the new
species described herein. Thus, we remove it from its synonymy with H. fasciatus and
assign it to the genus Hypsiboas: Hypsiboas steinbachi new. comb.
Systematic accounts
Hypsiboas calcaratus (Troschel, 1848)
http://species-id.net/wiki/Hypsiboas_calcaratus
Hyla calcarata Troschel, 1848:660. Type material not designated and likely lost. Type
locality “Britisch-Guiana” (= Guyana; Frost 2013).
Diagnosis. Hypsiboas calcaratus (Figs 4C, 8C, and 9) is characterized by: (1) mean
SVL 36.82 mm in males (range 27.61–42.50; n = 36), 50.92 mm in females (range
45.94–56.29; n = 4); (2) basal webbing on the fingers; (3) calcar large and triangular;
(4) dorsal background color ranging from reddish brown to cream, pinkish white or
grayish brown, in most cases dark marks are present (e.g., broad transversal marks,
Figure 4. Dorsolateral views of adult females of A Hypsiboas fasciatus, QCAZ 48611, SVL = 51.79 mm
B H. almendarizae, QCAZ 32638, SVL = 51.26 mm C H. calcaratus, QCAZ 24282, SVL = 51.26 mm
D H. maculateralis, QCAZ 43825, SVL = 55.31 mm E H. alfaroi, QCAZ 43252, SVL = 45.37 mm.
large black stains); (5) often middorsal dark brown line present; (6) flanks pale cream
or gray (in life, blue in large females and light blue or white in males) with dark brown
vertical bars; (7) hidden surfaces of thighs pale cream or gray (in life, blue in large
females and light blue or white in males) with dark brown transversal bars; (8) ventral
surfaces of thighs creamy white, yellowish white or brown; (9) venter creamy white or
yellowish white; (10) webbing on feet; (11) in life, iris creamy silver or bronze with
upper yellow to orange band; (12) prepollical spine present in males.
Hypsiboas calcaratus is most similar to H. fasciatus and H. almendarizae sp. n. It
differs from both species by the shape of the calcar (large and triangular in H. calcara-
tus, small and conical in H. fasciatus, and large and conical in H. almendarizae sp. n.;
Fig. 14C–D) and by the number of notes in the advisement call (Fig. 12). Hypsiboas
calcaratus can be further distinguished from H. fasciatus by the color of the upper band
in the iris: red to reddish brown in H. fasciatus, yellow to orange in H. calcaratus. Hypsi-
boas almendarizae sp. n. differs from H. calcaratus in having narrower transversal dark
bars on the flanks and thighs (mean width of bars on thighs = 5.05% of femur length,
Figure 5. Adult Hypsiboas fasciatus showing variation in dorsal and ventral coloration of preserved speci-
mens. From left to right, first and second rows: QCAZ 24866, 26497, 41575 (females); third and fourth
rows: QCAZ 18271, 41488, 41576, 48584 (males). See Appendix for locality data. All specimens are
shown at the same scale.
SD = 0.61, in H. almendarizae sp. n. vs. 7.89%, SD = 1.2, in H. calcaratus; differences

are significant: t = -6.72, df = 18, P < 0.001) and smaller calcars.
Hypsiboas calcaratus differs from H. maculateralis sp. n. in advertisement call (lower
dominant frequency, higher fundamental frequency; Figs 12E–F and 13A–B) and by
Figure 6. Adult Hypsiboas almendarizae showing variation in dorsal and ventral coloration of preserved
specimens. From left to right, first and second rows: QCAZ 32638, 32645 (females), 24392 (male); third
and fourth rows: QCAZ 39647, 39650, 24394 (males). See Appendix for locality data. All specimens are
the presence of transversal bars on the flanks and hidden surfaces of the thighs (dark
blotches instead of bars in H. maculateralis sp. n.) Hypsiboas calcaratus can be distin-
guished from H. alfaroi sp. n. and H. tetete sp. n. by the presence of a calcar (instead of
Figure 7. Principal components from analysis of six size-corrected morphological variables for A 34
females and B 136 males of Hypsiboas. See Table 8 for character loadings on each component.
a small tubercle on the heel) and by the absence of dark flecks on the gular region and
chest (present in H. alfaroi sp. n. and H. tetete. sp. n.) Morphological characters useful
to differentiate H. calcaratus from other species are shown in Table 6.
Variation. Variation in dorsal and ventral coloration of preserved specimens is shown
in Figure 9. Background dorsal coloration varies from cream (e.g., QCAZ 40085) to
pinkish white (e.g., QCAZ 44530), reddish brown (e.g., QCAZ 14957, 43256, 44422),
pale reddish brown (e.g., QCAZ 43259) or pale grayish brown (e.g., QCAZ 48718).
Irregular dorsal marks may be present in diverse patterns. A dark middorsal line extends
from the tip of the snout to the mid-sacrum (e.g., QCAZ 43256), but in some specimens
it only extends along the head (e.g., QCAZ 25514) or on the anterior half of the body
(e.g., QCAZ 43131). There is variation in the number, size, and shape of dorsal marks.
Some individuals (e.g., QCAZ 43256) have five to seven brown diffuse transversal bands
(sometimes interconnected). Brown transversal bars are present on the dorsal surfaces
of the limbs (one or two on the upper arm and forearm and three to five on the thigh,
shank, and foot). In some individuals, the dorsum and dorsal surfaces of the forearms
and shanks have large black stains (e.g., QCAZ 14957) or scattered brown or white dots
(e.g., QCAZ 40085, 44178, 14971). The coloration of flanks and hidden surfaces of
thighs vary from pale cream to creamy white or light gray, with dark brown transversal
bars. The number of bars on the flank varies from 4 to 13; the number of bars on the
thigh varies from 4 to 9. The extent of the area with bars varies from the groin to the mid
flank (e.g., QCAZ 43259) to from the groin to the axilla (e.g., QCAZ 43256). In some
individuals, the bars can also be present on the hidden surfaces of the shanks, ventral
surfaces of the forelimbs, and dorsal surfaces of the feet (e.g., QCAZ 43256).
Figure 8. Dorsolateral views of adult males of A Hypsiboas fasciatus, QCAZ 48584, SVL = 33.77 mm
B H. almendarizae, QCAZ 39649, SVL = 36.54 mm C H. calcaratus, QCAZ 43256, SVL = 40.07 mm
D H. maculateralis, QCAZ 40082, SVL = 36.16 mm E H. alfaroi, QCAZ 43260, SVL = 30.35 mm F H.
tetete, QCAZ 40081, SVL = 31.15 mm.
Ventral surfaces of preserved specimens vary from creamy white (e.g., QCAZ
44530) to yellowish white (e.g., QCAZ 43256). In some individuals, scattered minute
pale brown blotches are present on the lips (e.g., QCAZ 31446, 44178). Coloration
of webbing and discs vary from yellowish white to brown or gray. Coloration of bones
is white or green.
Coloration in life. (based on photographs; Figs 4C and 8C). Dorsal surfaces
vary from light brown (e.g., QCAZ 40056) to reddish brown (e.g., QCAZ 36869) or
brown (e.g., QCAZ 24282) with a middorsal dark brown line (e.g., QCAZ 40985);
some individuals have brown diffuse transversal bands (e.g., QCAZ 43256); the dorsal
surfaces of the limbs have pale brown transversal bars (e.g., QCAZ 43256); scattered
Table 6. Diagnostic characters of male specimens of the Hypsiboas calcaratus species complex. Coloration corresponds to preserved specimens unless otherwise noted.
Characters H. alfaroi H. almendarizae H. calcaratus H. fasciatus H. maculateralis H. tetete

Creamy white or Pale cream or creamy Pale cream, creamy white Pale cream, creamy white, Pale cream, creamy white Creamy white or gray
Flank Coloration gray with dark brown white with thin dark or light gray with dark brown or gray with dark or gray with dark brown with dark brown irregular
irregular spots brown vertical bars brown vertical bars brown vertical bars blotches spots
Creamy white, gray or Pale cream or creamy Pale cream, creamy white Pale cream, creamy white, Pale cream, creamy white Creamy white or brown
Coloration of the hidden
brown with dark brown white with thin dark or light gray with dark brown or gray with dark or gray with dark brown with dark brown irregular
surfaces of thighs
irregular spots brown transversal bars brown transversal bars brown transversal bars blotches spots
Size and shape of the Calcar absent. Only small Calcar absent. Only small
Large and conical Large and triangular Small and conical Large and triangular
calcar tubercle present tubercle present
Brown flecks on the neck
Present Absent Absent Absent Sometimes present Present
and chest
Well-defined, yellow to Well-defined, yellow to Well-defined, red to
Iris upper band, in life Ill-defined, yellow Ill-defined, yellow Ill-defined, yellow
orange orange reddish brown
Figure 9. Adult Hypsiboas calcaratus showing variation in dorsal and ventral coloration of preserved
specimens. From left to right, first and second rows: QCAZ 44422, 44530 (females), 14957 (male); third
and fourth rows: QCAZ 43259, 40085, 43256 (males). See Appendix for locality data. All specimens are
minute white and black dots can be present on the dorsum (e.g., QCAZ 40056); in
some individuals there are large dark brown blotches on the dorsum, dorsal surfaces of
the forearms and shanks (e.g., QCAZ 43245); flanks are white, light blue or blue with
dark brown vertical bars (e.g., QCAZ 40083); hidden surfaces of thighs and shanks
are white, light blue or blue with dark brown transversal bars (e.g., QCAZ 43034);
in some specimens there are dark brown transversal bars on the hidden surfaces of
the shanks, ventral surfaces of the upper arms, and dorsal surfaces of the feet (e.g.,
QCAZ 43034); a faint creamy white stripe usually is evident on the outer edge of the
feet, tarsus, forearms, and hands (e.g., QCAZ 26062); venter creamy white with belly
yellowish white; ventral surfaces of hindlimbs and forelimbs translucent white (e.g.,
QCAZ 43824) or yellowish (e.g., QCAZ 40085); in some individuals, ventral surfaces
of the thighs are creamy white (e.g., QCAZ 43047); discs and webbing yellowish (e.g.,
QCAZ 40085) or brown (e.g., QCAZ 40985); iris creamy silver (e.g., QCAZ 40056)
or bronze (e.g., QCAZ 40085) with an upper yellow to orange band (e.g., QCAZ
43047); bones are white (e.g., QCAZ 40083) or green (e.g., QCAZ 43824).
In the examined adult series, the largest male has a SVL of 42.50 mm, and the larg-
est female 56.29 mm; mean male SVL = 37.08 mm (n = 35; SD = 2.09), mean female
SVL = 50.92 mm (n = 4; SD = 4.80). Females are significantly larger than males (t =
-5.71, df = 3, P = 0.009). Inter-population variation in size and other morphometric
variables is shown in Tables 1 and 2.
Advertisement call. Two males were recorded at Tena (Provincia Napo) on 1
March 2009 and five males at Estación Científica Yasuní PUCE (Provincia Orellana)
on 20 June 2009, in vegetation next to streams or ponds. Acoustic parameters of the
advertisement call are shown in Table 7. The call (Fig. 12E–F) consists of a single quack
note with a mean duration of 0.05 s (SD = 0.00) and mean rise time of 0.04 s (SD
= 0.01). The mean dominant frequency is 1780.50 Hz (SD = 112.73) and the mean
fundamental frequency is 557.13 Hz (SD = 46.21).
Distribution and ecology. Hypsiboas calcaratus has confirmed records (based on DNA
sequences and specimens listed in Appendix) from French Guiana, Guyana and the Ama-
zon basin of Brazil, Ecuador, and Peru (Fig. 17). A photograph published by De la Riva
et al. (2000) confirms its presence in Bolivia. Records from Colombia and Venezuela need
confirmation. Known localities range in elevation from sea level (Kaw) to 650 m (Canelos).
Hypsiboas calcaratus occurs in Terra Firme forest, flooded forests (Várzea and Ig-
apó), and swamps. It is generally found next to streams, ponds, and lakes. Individuals
have been recorded at night perching on vegetation 15 to 200 cm above the ground.
Their occurrence in secondary forests and artificial open areas suggest at least some
tolerance of anthropogenic habitat disturbance.
Vegetation types at known localities include Southwest Amazon Moist Forest and
Napo Moist Forest for the Peruvian and Ecuadorian localities, Guianan Moist Forest
for the Guyana and French Guiana localities, and Madeira-Tapajós Moist Forest for
the Brazilian locality (according to the World Wildlife Fund, 2012).
Conservation status. Its distribution polygon has 3’586,597 km2 and overlaps
with protected areas and large regions of pristine forest. Hypsiboas calcaratus is rela-
tively frequent in scientific collections suggesting that, at least in part of its range, it is
not a rare species. For these reasons we propose assigning H. calcaratus to the Red List
category of Least Concern.
26
Table 7. Descriptive statistics for call parameters of Hypsiboas alfaroi (QCAZ 43260–63), H. almendarizae (QCAZ 39645, 39647–50), H. calcaratus (QCAZ
40084–85, 43247, 43256–59), H. fasciatus (QCAZ 48583–86, 48633), H. maculateralis (QCAZ 40082), and H. tetete (QCAZ 40060, 40080–81, 48095). The n
values indicate the number of males analyzed. Mean ± SD is given with range in parentheses. Values for H. maculateralis were obtained from three calls from a single
male. See Table 3 for a description of each parameter.
H. tetete
H. alfaroi H. almendarizi H. calcaratus H. fasciatus H. maculateralis
(n = 4)
(n = 4) (n = 5) (n = 7) (n = 5) (n = 1)
Type 1 Type 2
0.20 ± 0.05 0.48 ± 0.07 0.05 ± 0.00 0.52 ± 0.24 0.35 ± 0.04 0.10 ± 0.02 0.11 ± 0.02
Call Duration (s)
(0.16–0.19) (0.39–0.55) (0.05–0.06) (0.27–0.90) (0.317–0.398) (0.08–0.12) (0.09–0.13)
Number of notes 4–5 3–4 1 3–5 3–4 1 1
0.07 ± 0.03 0.30 ± 0.10 0.04 ± 0.01 0.24 ± 0.06 0.19 ± 0.10 0.03 ± 0.02 0.05 ± 0.02
Rise time (s)
(0.05–0.08) (0.20–0.45) (0.03–0.05) (0.17–0.30) (0.084–0.289) (0.02–0.06) (0.03–0.07)
2079.53 ± 83.43 1954.43 ± 128.43 1780.50 ± 112.73 1855.81 ± 148.08 2217.93 ± 56.94 1938.47 ± 26.24 1829.12 ± 12.61
Call dominant frequency (Hz)
(1924.50–2032.74) (1828.18–2115.65) (1619.32–1927.25) (1712.00–2088.70) (2174.92–2282.51) (1830.31–1959.23) (1815.97–1841.10)
2036.31 ± 130.30 951.76 ± 61.38 557.13 ± 46.21 884.89 ± 105.52 488.10 ± 12.47 1940.14 ± 28.29 1821.96 ± 16.96
Call fundamental frequency (Hz)
(1951.05–2146.50) (882.86–1029.95) (512.60–651.38) (722.60–1001.30) (473.71–495.33) (1830.31–1964.23) (1808.80–1841.10)
14.85 ± 3.03 14.12 ± 0.74 11.80 ± 1.69 10.22 ± 1.68
Number of pulses – – –
(11.90–19.17) (12.60–14.75) (9.60–13.56) (8.67–12.00)
200.41 ± 31.89 268.46 ± 11.08 179.53 ± 18.08 92.97 ± 2.26
Pulse repetition rate (pulses/s) – – –
(168.95–249.22) (250.71–280.11) (150.31–195.95) (90.72–95.24)
Dominant frequency at the 1869.09 ± 52.61 1827.51 ± 175.70
– – – – –
beginning to the third note (Hz) (1808.80–1905.70) (1722.70–2140.10)
Fundamental frequency at the
826.16 ± 58.21 841.64 ± 91.67
beginning to the third note – – – – –
(766.58–882.90) (728.30–969.00)
(Hz)
Dominant frequency at the end 2058.93 ± 33.98 1962.32 ± 190.41

– – – – –
to the third note (Hz) (2026.28–2094.10) (1798.80–2282.50)
Fundamental frequency at the 937.43 ± 83.53 950.18 ± 144.22
– – – – –
end to the third note (Hz) (882.90–1033.60) (709.50–1055.10
7.75 ± 0.95 16.2 ± 6.37 12 ± 2.64 51 ± 24.72 152.5 ± 3.53 9 ± 4.24
Interval between calls (s) –
(7–9) (10–15) (6–23) (13–78) (150–155) (6–15)
Recording temperatures (oC) 25.9 21.5 23.9–25.6 20.1–23.6 21.5 22.2–25 22.2–25
Figure 10. Holotype of Hyla leptoscelis (BM 1947.2.23.10). Above: dorsolateral view; below: ventral
(left) and dorsal (right) views.
Figure 11. Syntype of Hyla steinbachi (BM 1947.2.13.61). Above: dorsal (left) and ventral (right) views;
below: lateral view of head and ventral view of left hand.
Hypsiboas fasciatus (Günther, 1858)

http://species-id.net/wiki/Hypsiboas_fasciatus
Hyla fasciata Gunther, 1858:327. Holotype: BMNH 58.4.25.22, a female from “An-
den von Ecuador” (Andes of Ecuador).
Diagnosis. A member of the genus Hypsiboas characterized by: (1) mean SVL 35.40
mm in males (range 32.65–37.74; n = 19), 51.89 mm in females (range 47.16–54.84;
n = 5); (2) basal webbing on fingers; (3) calcar small and conical; (4) dorsal colora-
tion varying from cream to grayish brown, pinkish white or brown, with dark marks
in some individuals (e.g., broad transversal bands); (5) middorsal brown stripe often
present; (6) flanks pale cream or gray (in life, blue in large females and light blue or
white in males) with dark brown vertical bars; (7) hidden surfaces of thighs pale cream
or gray (in life, blue in large females and light blue or white in males) with dark brown
transversal bars; (8) ventral surfaces of thighs creamy white, yellowish white or brown;
(9) venter creamy white or yellowish white; (10) webbing on feet; (11) in life, iris
creamy silver or bronze with upper red to reddish brown band; (12) prepollical spine
present in males.
Hypsiboas fasciatus is most similar to H. almendarizae sp. n. and H. calcaratus.
Hypsiboas calcaratus can be distinguished from H. fasciatus by the shape of the calcar
(large and triangular in H. calcaratus vs. small and conical in H. fasciatus), structure
of the advertisement calls (Fig. 12A–B and 12E–F), and by the yellow to orange color
of the upper band in the iris (red to reddish brown in H. fasciatus). Hypsiboas almen-
darizae sp. n. differs from H. fasciatus in having narrower transversal dark bars on the
flanks and thighs (mean width of bars on thighs = 5.05% of femur length, SD = 0.61,
in H. almendarizae sp. n. vs. 8.58%, SD = 1.56, in H. fasciatus). The color of the upper
band in the iris also distinguishes both species (red to reddish brown in H. fasciatus vs.
yellow to orange in H. almendarizae sp. n.; Fig. 14A–B).
Hypsiboas fasciatus differs from H. maculateralis sp. n. in advertisement call (Figs
12A–B and 13A–B) and in the presence of transversal bars on the flanks and hidden
surfaces of the thighs (dark blotches instead of bars in H. maculateralis sp. n.) Hypsi-
boas fasciatus can easily be distinguished from H. alfaroi sp. n. and H. tetete sp. n. by
the presence of a calcar (instead of a small tubercle on the heel) and by the absence of
dark flecks on the gular region and chest (present in H. alfaroi sp. n. and H. tetete sp.
n.) Morphological characters useful to differentiate H. fasciatus from other species are
shown in Table 6.
Variation. Variation in dorsal and ventral coloration of preserved specimens is
shown in Figure 5. Background dorsal coloration varies from cream (e.g., QCAZ
41488, 48584) to pale grayish brown (e.g., QCAZ 18271), grayish brown (e.g.,
QCAZ 27258), pinkish white (e.g., QCAZ 41575), pale brown (e.g., QCAZ 23148)
or brown (e.g., QCAZ 26497). Irregular dorsal marks can be present in diverse pat-
terns. A dark middorsal stripe extends from the tip of the snout to the mid-sacrum
(e.g., QCAZ 26497) or to the vent (QCAZ 23144). In a few individuals, the mid-
dorsal line is absent (QCAZ 17123, 31040). In some individuals (e.g., QCAZ 41575)
broad transversal bands are present on the dorsum; narrower brown transversal bars are
present on the dorsal surfaces of the limbs (one or two on the upper arm and forearm
and three to five on the thigh, shank, and foot). In some individuals, scattered minute
brown dots may be present on dorsum (e.g., QCAZ 48584). The coloration of flanks
and hidden surfaces of thighs vary from pale cream to creamy white, brown or gray,
with dark brown vertical bars (4 to 12 on each flank and 6 to 12 on each thigh). The
area with vertical bars extends from the groin to the axilla (e.g., QCAZ 41575) or to
the mid flank (e.g., QCAZ 41576, 18271). In some individuals, similar transversal
Figure 12. Calls of the Hypsiboas calcaratus species complex. A–B H. fasciatus (QCAZ 48585) from
La Pradera, Provincia Morona Santiago C–D H. almendarizae (QCAZ 39650) from Limón, Provincia
Morona Santiago E–F H. calcaratus (QCAZ 40085) from Tena, Provincia Napo. A, C, E are oscilograms
and B, D, F spectrograms.
bars can be present on the hidden surfaces of the shanks, ventral surfaces of the fore-
limbs, and dorsal surfaces of the feet (e.g., QCAZ 41575). The shape of the calcar is
small and conical and shows little variation among individuals.
Ventral areas vary from creamy white (e.g., QCAZ 48584) to yellowish white (e.g.,
QCAZ 41576, 41488). A narrow to wide brown stripe can be present on the outer
edge of the hands, forearms, thighs, feet, and tarsal folds (e.g., QCAZ 48584, 41576,
41575, 26497). In some specimens, scattered minute pale brown blotches can be pre-
sent on the lips (e.g., QCAZ 26497, 24866). Coloration of webbing and discs vary
from yellowish white to brown or gray. Bones are white.
In the examined series of adults, the largest male has a SVL of 37.74 mm, and the
largest female 54.84 mm; mean male SVL = 35.40 mm (n = 19; SD = 1.65), mean
female SVL = 51.89 mm (n = 5; SD = 3.18). Females are significantly larger than males
(t = -16.24, df = 22, P < 0.001). Inter-population variation in size and other morpho-
metric variables is shown in Tables 1 and 2.
Coloration in life. (based on digital photographs; Figs 4A and 8A). Dorsal sur-
faces vary from pale yellowish tan (e.g., QCAZ 47070) to brown (e.g., QCAZ 48611)
with a middorsal dark brown line (e.g., QCAZ 48585) and scattered minute black
dots (e.g., QCAZ 47051); some individuals have diffuse broad pale brown transversal
bands on the dorsum (e.g., QCAZ 48583); pale brown transversal bars are present
on the dorsal surfaces of the limbs; flanks and hidden surfaces of thighs are white,
light blue or blue with dark brown vertical bars (e.g., QCAZ 47070, 47051); in some
specimens, dark brown vertical bars are present on the hidden surfaces of the shanks,
ventral surfaces of the upper arms and dorsal surfaces of the feet (e.g., QCAZ 48671);
venter creamy white, sometimes with yellowish white on its posterior half (e.g., QCAZ
48670); ventral surfaces of hindlimbs and forelimbs translucent pinkish white (e.g.,
QCAZ 47051) or brown (e.g., QCAZ 48611); in some specimens, ventral surfaces of
the thighs creamy white (e.g., QCAZ 47070); discs and webbing pale yellowish tan
(e.g., QCAZ 48584) or brown (e.g., QCAZ 48611); a narrow to wide brown stripe
is present on the outer edge of the hands, forearms, thighs, feet, and tarsal folds (e.g.,
QCAZ 48611); iris creamy silver (e.g., QCAZ 48584) or bronze (e.g., QCAZ 48611)
with upper red to reddish brown band (e.g., QCAZ 48628); bones vary from green
(e.g., QCAZ 48671) to white (e.g., QCAZ 48628).
Advertisement call. We recorded calls of one male at Bombuscaro (Provincia
Zamora Chinchipe) on 9 June 2010, three males at La Pradera (Provincia Morona
Santiago) on 10 June 2010, and one male at Comunidad San Luis (Provincia Morona
Santiago) on 13 June 2010. The advertisement call consists of three to five quack notes
(Fig. 12A–B). Mean call duration is 0.52 s (SD = 0.24) and mean rise time is 0.24 s
(SD = 0.06). Sound frequency increases from the beginning to the end of the note;
mean dominant frequency is 1855.81 Hz (SD = 148.08) and mean fundamental fre-
quency is 884.89 Hz (SD = 105.52). Other call parameters are listed in Table 7.
Distribution and ecology. Hypsiboas fasciatus has been recorded in the Ecuado-
rian and Peruvian Amazon basin (Morona Santiago and Zamora-Chinchipe provinces
in Ecuador and Región Amazonas in Peru) (Fig. 15). Localities with known elevation
Figure 13. Calls of the Hypsiboas calcaratus species complex. A–B H. maculateralis (QCAZ 40082) from
Comunidad Santa Rosa, Provincia Napo C–D H. alfaroi (QCAZ 43260) from Estación Científica Yasuní
PUCE, Provincia Orellana E–H H. tetete (QCAZ 40081) from Comunidad Santa Rosa, Provincia Napo.
A, C, E, G are oscilograms and B, D, F, H spectrograms.
range from 730 to 1593 m above sea level. The elevation at Romerillos Alto (1593 m)
is the highest known locality for H. fasciatus, while Tink (730 m) is the lowest.
Specimens from Comunidad San Luis, La Pradera, La Pituca, Limón, Miazi Alto,
Nueva Principal, and Tiink were found in primary and secondary forest, perching
on vegetation 30 to 200 cm above the ground, in flooded areas, ponds, swamps, and
near streams. Individuals in Zamora were found in grassy swamps roosting in shrubs
between 30 and 110 cm above ground.
Vegetation types for Ecuadorian localities are: (1) Amazonian Evergreen Foothill
Forest, characterized by a mixture of Amazonian and Andean vegetation with a canopy
of 30 m, (2) Evergreen Lower Montane Forest of the Amazonian Range, character-
ized by trees reaching 20–30 m of height and abundant epiphytes and hemiepiphytes,
with Dictyocaryum lamarckianum (Arecaceae) as dominant species, and (3) Evergreen
Lower Montane Forest of the East of the Southern Andes, characterized by abundant
epiphytes, trees reaching 30 m of height with Podocarpus as dominant species.
Conservation Status. Its distribution polygon has 8,572 km2 of which 2,198 km2
(25.6%) have been degraded by human activities, especially agriculture and cattle rais-
ing (estimated from Ministerio de Ambiente del Ecuador 2013). Because habitat deg-
radation is increasing, we assign H. fasciatus to the Red List category Near Threatened.
Its distribution polygon overlaps with three protected areas: Parque Nacional Podocar-
pus, Reserva Biológica el Quimi and Refugio de Vida Silvestre el Zarza. Its distribution
polygon does not overlap with the polygon reported in the Red List assessment for this
species by Icochea et al. (2004). The new range represents < 1% of the range reported
in the IUCN Red List.
Hypsiboas almendarizae sp. n.

http://zoobank.org/5D68CEA5-D2D1-44A9-AA7C-D30081D92C7D
http://species-id.net/wiki/Hypsiboas_almendarizae
Common name. English: Almendariz’s treefrog; Spanish: Rana arbórea de Almendáriz

Holotype. (Fig. 14E) QCAZ 39650 (field no. SC-PUCE 23213), adult male from
Ecuador, Provincia Morona Santiago, General Leonidas Plaza, “Limón”, on the road
to Gualaceo (2.9796°S, 78.4415°W), 1237 m above sea level, collected by Marcel A.
Caminer on 17 January 2009.
Paratopotypes. (Fig. 8B) QCAZ 39638–40, 39645–49, adult males, collected
with the holotype.
Paratypes. (Figs 4B, 6) ECUADOR: PROVINCIA NAPO: Pacto Sumaco
(0.6339°S, 77.5922°W), 1560 m, QCAZ 10910, adult male, collected by L. E. López
on 1 January 1996; Río Hollín (0.6998°S, 77.6665°W), 1068–1950 m, QCAZ 22365–
66, adult males, collected by M. R. Bustamante on 27 January 2002; Río Hollín, on
the road Jondachi-Loreto (0.7707°S, 77.7820°W), 1100 m, QCAZ 6905, 6910, adult
males, 6889, juvenile, collected by P. Ordoñez, P. Guarderas, J. F. Freile, M. C. Terán
and O. Torres-Carvajal on 3 December 1994; Río Hollín, on the road Hollín-Loreto
Figure 14. Variation in color of the iris of A Hypsiboas fasciatus (QCAZ 47051), and B H. almendari-
zae (QCAZ 42055). Calcar in C Hypsiboas almendarizae (QCAZ 39639) and D H. calcaratus (QCAZ
40055). Note that the calcar in H. almendarizae is large and conical while in H. calcaratus is large and
triangular. Dorsolateral views of the holotypes of E H. almendarizae, adult male (QCAZ 39650), SVL =
36.72 mm F H. maculateralis, adult male (QCAZ 40082), SVL = 36.16 mm; and G H. tetete, adult male
(QCAZ 40081), SVL = 31.15 mm.
Figure 15. Distribution of species of the Hypsiboas calcaratus complex. Localities are based on museum
specimens from Museo de Historia Natural Gustavo Orcés of Escuela Politécnica Nacional, Museo de
Zoología of Pontificia Universidad Católica del Ecuador, Natural History Museum University of Kansas,
and Smithsonian Institution National Museum of Natural History.
(0.9666°S, 77.7632°W), 600 m, QCAZ 283, 782, 4177–78, adult males, collected by
A. Flachier, F. Campos-Yánez, L. A. Coloma and G. Onore on 5 December 1987 and
1 February 1988; Río Hollín (0.6998°S, 77.6665°W), 1068–1950 m, QCAZ 21942,
22364, adult males, 17944, collected by M. R. Bustamante, I. G. Tapia, G. Onore,
F. Ayala-Varela, S. Valdivieso, D. Tirira and J. J. Wiens on 20 March 1990 and 9 De-
cember 2001; Archidona, Estacíon Sarayacu (0.6910°S, 77.8208°W), 1320 m, QCAZ
51809, juvenile female, collected by D. Rivadeneira and X. Salazar on 23 August 2011;
PROVINCIA TUNGURAHUA: Baños, Río Lagarto (1.4025°S, 78.2980°W), 1472
m, QCAZ 24392, 24394, adult males, collected by I. G. Tapia and D. Hill on 5 May
2001; Río Verde (1.4026°S, 78.2979°W), 1514 m, QCAZ 47047–48, adult males,
collected by S. Poe, F. Ayala-Varela, L. Gray, J. Davis and I. Latella on 14 December
2009; Río Negro (1.4135°S, 78.2110°W), 1220 m, QCAZ 4029–30, 4907, 21273–
74, adult males, 24357, adult female, 4034, metamorph, collected by F. Campos-
Yánez, L. A. Coloma, C. Proaño and J. J. Camacho on 1 May 1993 and 24 September
2002; mouth of the Pastaza river (1.4128°S, 78.2688°W), 1440 m, QCAZ 31449,
adult male, 31450, adult female, collected by D. Paucar on 9 March 2006; Baños, Río
Lagarto (1.4025°S, 78.2980°W), 1472 m, QCAZ 24386–91, 24393, 24395, adult
males, collected by I. G. Tapia and D. Hill on 5 May 2001; mouth of the Pastaza river
(1.4128°S, 78.2687°W), 1440 m, QCAZ 31452, juvenile, collected by D. Paucar on
8 March 2006; PROVINCIA PASTAZA: Pomona, Reserva de Bosque Tropical Hola
Vida (1.6250°S, 77.9072°W), 831 m, QCAZ 37163, adult male, collected by I. G. Ta-
pia, L. A. Coloma, C. Proaño and M. Páez on 9 July 2007; PROVINCIA MORONA
SANTIAGO: 2.2 km N San Juan Bosco (2.0070°S, 77.9348°W), 1013 m, QCAZ
26438, adult male, collected by L. A. Coloma on 12 August 2003; Parque Nacional
Sangay, Sardinayacu river (2.0597°S, 78.1564°W), 1600 m, EPN 7740, adult male,
collected by A. Almendáriz; Central Hidroeléctrica Abanico (2.2457°S, 78.1958°W),
1600 m, EPN 11435–36, 11438, adult males, 11437, adult female, collected by Y. Sa-
gredo and J. Brito; 7.6 km W from Nueve de Octubre, along the road Guamote-Macas
(2.2163°S, 78.2898°W), 1571 m, QCAZ 32638–39, 32645, adult females, collected
by A. Pounds, L. A. Coloma, M. R. Bustamante and I. G. Tapia on 30 November
2006; 4.8 km N from Rosario (2.8858°S, 78.3880°W), 841 m, QCAZ 26474–77,
adult males, 26480, metamorph, collected by L. A. Coloma, A. Merino and E. R. Wild
on 13 August 2003; N from Mendez, 4 km NW from Patuca (airline distance) on the
road to Logroño (2.7291°S, 78.2849°W), 600 m, QCAZ 23810, adult male, collected
by S. R. Ron and G. E. Romero on 6 April 2003; El Rosario (2.9282°S, 78.4082°W),
1100 m, QCAZ 30590–92, adult males, collected by I. G. Tapia, M. R. Bustamante
and A. Merino on 23 March 2004; N from General Leonidas Plaza, “Limón”, on the
road Limón-Méndez (2.9046°S, 78.3869°W), 879 m, QCAZ 26300, adult male, col-
lected by S. R. Ron, M. Guerra and I. G. Tapia on 13 January 2004; Limón Indanza,
on the road to Ayanza (2.9899°S, 78.4260°W), 994 m, QCAZ 39642, adult male, col-
lected by M. A. Caminer on 17 January 2009; near General Leonidas Plaza, “Limón”,
km 74 on the road to San Antonio (2.9266°S, 78.4070°W), 1070 m, QCAZ 8573,
adult male, collected by N. Acosta-Buenaño and J. Bosch on 16 July 1995.
Referred specimens. ECUADOR: PROVINCIA MORONA SANTIAGO: 2.2

km N San Juan Bosco (2.0070°S, 77.9348°W), 1013 m (QCAZ 26429–37, 26439);
Nueve de Octubre, on the road Guamote-Macas (2.2445°S, 78.2074°W), 1527 m
(QCAZ 32268, 37639); Méndez (2.7145°S, 78.3153°W), 500 m (QCAZ 1006–07);
Km 65 on the road Macas-Méndez (2.68083°S, 78.23341°W), 540 m (QCAZ 15904–
06, 15999); 13 km N from Limón (2.8895°S, 78.3952°W), 800 m (QCAZ 27317–23,
27325); Limón Indanza (2.9796°S, 78.4414°W), 800–1560 m (QCAZ 8572, 25958,
26407, 26555, 26562, 31866, 32831, 32834, 32836, 32842, 39642, 40027, 41932–
33, 41992, 42001, 42017–18, 42022, 42026, 42037, 42039–40, 42055, 42236);
Plan de Milagro-Limón, 4.3 km WSW Limón (3.0118°S, 78.4784°W), 1373–1560 m
(QCAZ 26376–85, 26400–01, 41894, 41897, 41898); 3 km Limón-Indanza, on the
road to Gualaquiza (3.0489°S, 78.5007°W), 950 m (QCAZ 48176–78); on the road
Indanza-Gualaquiza (3.1435°S, 78.5359°W), 1277 m (QCAZ 26421–22); Bosque
Protector Abanico, 1646–1720 m (QCAZ 49029, 49034).
Diagnosis. A member of the genus Hypsiboas characterized by: (1) mean SVL
37.64 mm in males (range 34.31–44.56; n = 23), 48.11 mm in females (range 37.80–
51.94; n = 4); (2) basal webbing on fingers; (3) calcar large, conical; (4) dorsal colora-
tion varying from cream to reddish brown, grayish brown or pale brown, sometimes
with dark marks (e.g., broad transversal bands or narrow longitudinal lines); (5) dark
brown middorsal line often present; (6) flanks pale cream or creamy white (in life, blue
in large females and light blue or white in males) with thin dark brown vertical bars;
(7) hidden surfaces of thighs pale cream or creamy white (in life, blue in large females
and light blue or white in males) with thin dark brown transversal bars; (8) ventral
surfaces of thighs creamy white or yellowish white; (9) venter creamy white or yellow-
ish white; (10) webbing on feet; (11) in life, iris cream or creamy silver with an upper
yellow to orange band; (12) prepollical spine present in males.
Hypsiboas almendarizae is most similar to H. fasciatus and H. calcaratus. It can be
distinguished from H. calcaratus by the shape of the calcar (large and conical in H.
almendarizae vs. large and triangular in H. calcaratus; Fig. 14 C–D), width of the trans-
versal lines on the flanks (thin in H. almendarizae vs. wide in H. calcaratus), and adver-
tisement call (Fig. 12 C–F). Hypsiboas almendarizae differs from H. fasciatus in having
narrower transversal dark bars on the flanks and thighs (mean width of bars on thighs
= 5.05% of femur length in H. almendarizae vs. 8.58% in H. fasciatus; differences are
significant: t = 7.05, df = 12, P < 0.001) and in color of the upper band of the iris (red
to reddish brown in H. fasciatus vs. yellow to orange in H. almendarizae; Fig. 14 A–B).
Hypsiboas almendarizae differs from H. maculateralis sp. n. in advertisement call
(Figs 12 C–D and 13 A–B) and in the presence of vertical lines on the flanks and
hidden surfaces of the thighs (dark blotches instead of lines in H. maculateralis sp. n.)
Hypsiboas almendarizae can be distinguished from H. alfaroi sp. n. and H. tetete sp. n.
by the presence of a calcar (instead of a small tubercle on the heel) and by the absence
of dark flecks on the gular region and chest (present in H. alfaroi sp. n. and H. tetete sp.
n.) Morphological characters useful to differentiate H. almendarizae from other species
are shown in Table 6.
Description of the holotype. Adult male, 36.72 mm SVL, foot length 15.93 mm,
head length 9.18 mm, head width 11.95 mm, eye diameter 4.26 mm, tympanum di-
ameter 2.41 mm, tibia length 21.78 mm, femur length 20.03 mm, calcar length 1.10
mm, arm length 6.17 mm, eye-nostril distance 2.7 mm, head wider than long and
wider than body; snout round in lateral view, truncate in dorsal view; distance from
nostril to eye shorter than diameter of eye; canthus rostralis indistinct, rounded; loreal
region concave; internarial area convex; nostrils not protuberant, directed laterally;
interorbital area slightly convex; eye large, strongly protuberant; diameter of eye 1.8
times diameter of tympanic annulus; tympanum concealed beneath skin; tympanic an-
nulus evident, ovoid, longer dorsoventrally, concealed dorsally by supratympanic fold,
separated from eye by ca. 1.2 times its diameter; posterior end of supratympanic fold
reaches anterior border of arm insertion. Arm slender, axillary membrane absent; indis-
tinct low tubercles present along ventrolateral edge of forearm; relative length of fingers
I<II<IV<III; fingers bearing large, oval discs, that of third finger about three fourths of
tympanum diameter; subarticular tubercles prominent, round to ovoid, single; super-
numerary tubercles present; palmar tubercle small, elongated; prepollical tubercle large,
flat, elliptical; prepollex enlarged, claw shaped; nuptial excrescences absent; webbing
absent between fingers. Large conical calcar on tibiotarsal articulation; ill defined, scat-
tered tubercles on tarsus and along ventrolateral edge of foot; toes bearing discs slightly
wider than long, smaller than those of fingers; relative length of toes I<II<V<III<IV;
outer metatarsal tubercle ill defined, small, round; inner metatarsal tubercle large, el-
liptical; subarticular tubercles single, round, flat; supernumerary tubercles restricted
to the soles; webbing formula of toes I2—2¾II1¾—3-III2-—3+IV3+—2-V. Skin on
dorsum, head, and dorsal surfaces of limbs smooth; skin on flanks smooth with weak
longitudinal wrinkles posterior to the arm; skin on venter coarsely granular; skin on
ventral surfaces of head and thighs granular, those of shanks smooth. Cloacal opening
directed posteriorly at upper level of thighs; cloacal sheath short simple, covering cloa-
cal opening; round tubercles below and on the sides of vent, larger proximally. Tongue
cordiform, widely attached to mouth floor; vomerine odontophores triangular, nar-
rowly separated, posteromedial to choanae, bearing six vomerine teeth on each side;
choanae trapezoidal, oblique.
Color of holotype in preservative. Dorsum pale grayish brown with five to seven broad
brown transversal bands and scattered minute black dots; few small cream dots on the
posterior one third of the dorsum; dark brown middorsal line from tip of the snout to
the mid-dorsum; brown transversal bars on dorsal surfaces of the limbs (two on the up-
per arm, five on the thigh, and three on the shank and foot); flanks pale cream with ten
thin dark brown transversal bars; hidden surfaces of thighs pale cream with seven to nine
thin dark brown transversal bars; narrow cream stripe present above the anus; venter yel-
lowish white becoming creamy white on its posterior half; ventral surfaces of hindlimbs,
forelimbs, and webbing yellowish white; faint narrow to wide pale brown stripe on the
outer edge of the hands and forearms; minute brown blotches on lips; bones white.
Color of holotype in life. (Fig. 14E). Dorsum pale yellow tan with pale broad brown
transversal marks and scattered minute black and white dots; dark brown middorsal
line is present; pale brown transversal bars on the dorsal surfaces of the limbs; flanks
white with faint thin dark brown vertical bars; hidden surfaces of thighs white with
thin dark brown transversal bars; venter creamy white; ventral surfaces of hindlimbs
and forelimbs translucent pinkish; discs and webbing yellowish; iris cream with upper
yellow band; bones white.
Etymology. The specific name is a noun in the genitive case and is a patronym
for Ana Almendáriz, Ecuadorian herpetologist who for more than three decades has
contributed to the study of Ecuadorian amphibians and reptiles. Ana Almendáriz is
curator of Herpetology in the Museo de Historia Natural Gustavo Orcés at Escuela
Politécnica Nacional del Ecuador.
shown in Figure 6. Background dorsal coloration varies from creamy white (e.g.,
QCAZ 32638, 32645) to reddish brown, grayish brown (e.g., QCAZ 24394, 39647),
pale grayish brown (e.g., QCAZ 24392, 39650) or pale brown (e.g., QCAZ 24386).
A dark middorsal line extends from the tip of the snout to the middle of the sacrum
(e.g., QCAZ 39647) or to the vent (e.g., QCAZ 32645), but in some specimens is
restricted to the head (e.g., QCAZ 26429) or to the anterior half of the body (e.g.,
QCAZ 39650). In few specimens, the middorsal line is faint or absent (e.g., QCAZ
24392). Irregular dorsal marks may be present in varying number, size, and shape. Five
to seven broad transversal bands (sometimes interconnected) can be present on the
dorsum. Some individuals (e.g., QCAZ 24394, 39650, 39647) have narrow brown
transversal bars on the dorsal surfaces of the limbs (one or two on the upper arm and
forearm, and three to five on the thigh, shank, and foot). Faint to well-defined narrow
longitudinal lines may be present on the dorsum (e.g., QCAZ 32638, 32645). In some
individuals (e.g., QCAZ 32645), there are few scattered brown or white dots on the
dorsum, and dorsal surfaces of forearms, shanks and tarsi. The coloration of the flanks
and hidden surfaces of the thighs vary from pale cream to creamy white, with thin dark
brown transversal bars. The number of bars on the flanks and thighs varies from six to
ten on each flank and six to thirteen on each thigh; the area with vertical bars extends
from the groin to the mid-flank. In few individuals, the dark transversal bars of the
thighs and flanks are faint (e.g., QCAZ 32645).
39650) to yellowish white (e.g., QCAZ 39647). A narrow to wide faint pale brown
stripe is present in some individuals on the outer edge of the hands, forearms, feet,
thighs, and tarsal folds (e.g., QCAZ 39647). In some individuals, scattered minute
pale brown blotches may be present on the lips (e.g., QCAZ 32638, 39647). Colora-
tion of webbing and discs vary from yellowish white to brown or gray. Vomerine odon-
tophores are triangular (with arched base in some specimens). Bones white.
In the adult type series, the largest male has a SVL 44.56 mm, and the largest fe-
male 51.94 mm; mean male SVL = 37.64 mm (n = 23; SD = 2.01), mean female SVL
= 48.11 mm (n = 4; SD = 6.88). Females were not significantly larger than males (t =
-3.02, df = 3, P = 0.055). Inter-population variation in size and other morphometric
variables is shown in Tables 1 and 2.
Coloration in life. (based on photographs; Figs 4B and 8B). Dorsal surfaces vary
from pale yellowish tan (e.g., QCAZ 39640) to reddish brown (e.g., QCAZ 32638)
with a middorsal dark brown line (e.g., QCAZ 42055); sometimes broad pale brown
transversal bands are present on the dorsum; the dorsal surfaces of the limbs often
have pale brown transversal bars (e.g., QCAZ 39640); in some specimens, the dorsum
has faint to well-defined narrow brown longitudinal lines (e.g., QCAZ 39649); scat-
tered minute black, yellow or white dots can be present on the dorsum (e.g., QCAZ
39648); flanks and hidden surfaces of thighs white, light blue or blue with thin dark
brown vertical bars (e.g., QCAZ 39646, 42055); in some individuals there are thin
dark brown transversal bars on the hidden surfaces of the shanks (e.g., QCAZ 42055);
venter creamy white with yellowish white belly (e.g., QCAZ 48177); ventral surfaces
of hindlimbs and forelimbs translucent pinkish (e.g., QCAZ 39650) or yellowish (e.g.,
QCAZ 39638); in some specimens, ventral surfaces of the thighs creamy white (e.g.,
QCAZ 49029); discs and webbing yellowish (e.g., QCAZ 48177); iris cream (e.g.,
QCAZ 39648) or creamy silver (e.g., QCAZ 39639) with upper yellow to orange band
(e.g., QCAZ 39646); bones white (e.g., QCAZ 48177).
Advertisement call. We recorded calls from five males at Limón Indanza (Provin-
cia Morona Santiago) on 17 January 2009 (Table 7). The advertisement call consists of
three to four quack notes (Fig. 12C–D). Mean call duration is 0.48 s (SD = 0.007) and
mean rise time is 0.30 s (SD = 0.10). Sound frequency increases from the beginning
to the end of the note; mean dominant frequency is 1954.43 Hz (SD = 128.43) and
mean fundamental frequency is 951.76 Hz (SD = 61.38). The advertisement calls of H.
almendarizae and H. fasciatus are similar to each other (Fig. 12 A–D). The distribution
ranges of these species are parapatric with a small area of sympatry in the surroundings
of Leonidas Plaza (Provincia Morona Santiago). The analyzed calls from H. fasciatus
were from a region of allopatry. Future studies should aim to compare calls from the
region of sympatry to explore the possibility of reproductive character displacement.
Distribution and ecology. Hypsiboas almendarizae occurs on the eastern Andean
slopes of central and southern Ecuador (Morona Santiago, Napo, and Tungurahua
provinces) (Fig. 15). Localities with known elevation range from 500 to 1950 m above
sea level. The elevation at Río Hollín (1950 m) is the highest known locality for H.
almendarizae, while Méndez (500 m) is the lowest.
Most specimens of H. almendarizae were collected at Río Napinaza, a river sur-
rounded by secondary forest, pastures, and agricultural lands. Frogs were perching on
vegetation 25 to 80 cm above the ground. Individuals from Limón Indanza, Río Hol-
lín, Río Lagarto, Méndez, Río Pastaza, and Nueve de Octubre were found in flooded
areas with pastures, on vegetation 30 to 70 cm above the ground. Few individuals were
found near river Hollín, in small ponds in primary and secondary forest, roosting on
branches and leaves 20 to 150 cm above the ground. All the specimens from Rosario
and Plan de Milagro were found next to a highway, in swamps and streams with grass.
Vegetation types are: (1) Amazonian Evergreen Foothill Forest, characterized by
a mixture of Amazonian and Andean vegetation with a canopy of 30 m, (2) Ever-
green Lower Montane Forest of the Amazonian Range, characterized by trees reaching
20–30 m of height and abundant epiphytes and hemiepiphytes, with dense vegeta-
tion, and Dictyocaryum lamarckianum (Arecaceae) as dominant species, (3) Evergreen
Lower Montane Forest of the East of the Southern Andes, characterized by abundant
epiphytes, trees reaching 30 m of height with Podocarpus trees as dominant species, (4)
Evergreen Lower Montane Forest of the East of the Northern and Central Andes, char-
acterized by a canopy of 25 to 30 m, with abundant epiphytes and by the absence of
species of trees characteristic of the lowlands like the family Bombacaceae and Myris-
ticaceae, and (5) Amazonian Lowland Evergreen Forest, characterized by high plant
alpha-diversity and a canopy of 30 m with emergent trees that reach 40 m.
Conservation status. Its distribution polygon has 14,983 km2. Within this area,
4,864 km2 (32.4%) of its habitat has been degraded by human activities, especially
agriculture and cattle raising (estimated from Ministerio de Ambiente del Ecuador
2013). Current habitat degradation within the range of H. almendarizae is extensive
and its increase may threaten its survival in the future. Therefore, we propose that H.
almendarizae is assigned to the Near Threatened category. Its distribution polygon
overlaps with Parque Nacional Sangay, Parque Nacional Llanganates, and Parque Na-
cional Sumaco Napo-Galeras.
Hypsiboas maculateralis sp. n.

http://zoobank.org/BB94092B-695D-4747-ADFD-313747D7FAE1
http://species-id.net/wiki/Hypsiboas_maculateralis
Common name. English: Stained treefrog; Spanish: Rana arbórea manchada

Holotype. (Fig. 14F) QCAZ 40082 (field no. SC-PUCE 23221), adult male from Ec-
uador, Provincia Napo, Comunidad Santa Rosa (1.02337°S, 77.48359°W), 354 m above
sea level, collected by Marcel A. Caminer and Edwin Carrillo Ponce on 28 February 2009.
Paratypes. ECUADOR: PROVINCIA ORELLANA: Río Napo, Santa Teresita,
4 km NW from Nuevo Rocafuerte (0.9008°S, 75.4135°W), 186 m, QCAZ 44651,
adult female, 44673, adult male, collected by S. R. Ron, E. Toral, and I. G. Tapia on
9 July 2009; Río Napo, Huiririma (0.7116°S, 75.6239°W), 194 m, QCAZ 44636,
adult male, collected by S. R. Ron, E. Toral and I. G. Tapia on 8 July 2009; Río Napo,
San Vicente (0.6790°S, 75.6511°W), 203 m, QCAZ 44531, adult female, 44532,
adult male, collected by S. R. Ron, E. Toral and I. G. Tapia on 7 July 2009; Río
Napo, Chiroisla (0.5756°S, 75.8998°W), 203 m, QCAZ 44452, adult male, collected
by S. R. Ron, E. Toral and I. G. Tapia on 5 July 2009; Río Napo, Edén (0.4983°S,
76.0711°W), 216 m, QCAZ 44182–83, 44186, 44188–90, adult males, collected
by S. R. Ron, E. Toral and I. G. Tapia on 4 July 2009; Río Napo, sector La Prima-
vera (0.4442°S, 76.7868°W), 244 m, QCAZ 43825, adult female, 43827, adult male,
QCAZ 43800, juvenile, collected by S. R. Ron, E. Toral and I. G. Tapia on 29 June
2009; Río Napo, El Descanso (0.4310°S, 76.7864°W), 244 m, QCAZ 43897, adult
male, collected by S. R. Ron, E. Toral and I. G. Tapia on 30 June 2009; El Coca
(0.4778°S, 76.9898°W), 267 m, QCAZ 43712, adult male, 43709–10, 43715, ju-
veniles, collected by S. R. Ron, E. Toral and I. G. Tapia on 27 June 2009; Guiyero

community (0.6248°S, 76.4944°W), 227 m, EPN 10319, adult male, collected by
A. Almendáriz and J. Hernández; Guiyero community, Nambai km 32 (0.6248°S,
76.4944°W), 240 m, EPN 10887, adult male, collected by A. Almendáriz, J. Awa
and P. Ima; Parque Nacional Yasuní, km 74 on the road Pompeya-Iro (0.8331°S,
76.3416°W), 257 m, QCAZ 5238, adult female, collected by S. de la Torre and S. R.
Ron on 20 February 1994; Nuevo Rocafuerte, Tambococha (0.9783°S, 75.4256°W),
177 m, QCAZ 55359, adult male, 55395, 55374, adult females, collected by F. Ayala-
Varela, E. Carillo, J. Brito, A. Varela and D. Quirola on 13 March 2013; PROVIN-
CIA SUCUMBÍOS: Zábalo, Familia Criollo (0.3181°S, 75.7662°W), 220 m, QCAZ
28004, 28024, adult females, collected by M. R. Bustamante, N. Acosta-Buenaño, M.
Guerra and C. Proaño on 30 September 2003 and 2 October 2003; Playas de Cuy-
abeno (0.2654°S, 75.8917°W), 230 m, QCAZ 28401, adult female, collected by M.
R. Bustamante, N. Acosta-Buenaño, M. Guerra and C. Proaño on 8 October 2003;
Playas de Cuyabeno, Agurico Pañacoha (0.3106°S, 76.0335°W), 200 m, EPN 13452,
adult male, collected by P. Meza Ramos; Putumayo, Güepi camp 20 km N of Zábalo
(0.1779°S, 75.6767°W), 220 m, EPN 7189, adult female, collected by A. Almendáriz;
Reserva Faunística Cuyabeno (0.0849°N, 76.1344°W), 273 m, QCAZ 2148, 29454,
adult males, 2156, adult female, collected by S. de la Torre and F. Campos on 13
May 1990 and 9 May 2004; Río Napo, La Selva Lodge (0.5086°S, 76.3649°W), 229
m, QCAZ 44020–21, adult males, collected by S. R. Ron, E. Toral and I. G. Ta-
pia on 2 July 2009; Indillana, (0.43370°S, 76.5419°W), 265 m, QCAZ 24672, sub-
adult, collected by F. Ayala-Valera on 11 January 2002; Campo Vinita, on the road
Palma Roja-Pto El Carmen de Putumayo (0.1220°N, 75.8611°W), 217 m, QCAZ
29280–82, adult males, collected by S. Aldás-Alarcón on 7 May 2004; PROVINCIA
PASTAZA: Bataburo Lodge, S of the road from Cononaco (1.2083°S, 76.7166°W),
250 m, QCAZ 39430, 39452, adult males, collected by S. D. Padilla and C. Meyer on
10 October 2008 and 13 October 2008.
Referred specimens. ECUADOR: PROVINCIA SUCUMBÍOS: Reserva de Pro-
ducción Faunística Cuyabeno, Estación Científica Universidad Católica del Ecuador,
Laguna Grande (0.0195°S, 76.1712°W), 220 m (QCAZ 6141–43, 6146–51); PERU:
REGION MADRE DE DIOS: Manu National Park, Cocha Cashu Biological Station,
Río Manu (11.9166°S, 71.3°W), 340 m (USNM 299774).
Diagnosis. A member of the genus Hypsiboas characterized by: (1) mean SVL
55.31; n = 6); (2) basal webbing on fingers; (3) calcar large, triangular; (4) dorsal col-
oration varying from creamy white to reddish brown, pinkish white or brown, some-
times with dark markings (e.g., narrow longitudinal lines, broad transversal marks, or
large black stains); (5) dark brown middorsal line restricted to the head or the anterior
half of the body often present; (6) flanks pale cream or creamy white (in life, blue in
large females and light blue or white in males) with dark brown blotches; (7) hidden
surfaces of thighs pale cream or gray (in life, blue in large females and light blue or
white in males) with dark brown blotches; (8) ventral surfaces of thighs yellowish white
or brown; (9) ventral coloration varying from creamy white to yellowish white with
or without brown flecks on the neck and chest; (10) webbing on feet; (11) in life, iris
cream silver, yellowish or cream with faint yellow to orange color on its upper quarter;
(12) prepollical spine present in males.
Hypsiboas maculateralis (Figs 4D, 8D and 16) differs from H. fasciatus, H. almen-
darizae, and H. calcaratus in advertisement call (Figs 12 and 13A–B) and by having
dark blotches on the flanks and thighs (dark lines in H. almendarizae, H. calcaratus,
and H. fasciatus). Hypsiboas maculateralis can be distinguished from H. alfaroi sp. n.
and H. tetete sp. n. by the presence of a calcar (instead of a small tubercle on the heel).
Morphological characters useful to differentiate H. maculateralis from other species are
shown in Table 6.
Description of holotype. Adult male, 36.16 mm SVL, foot length 14.33 mm,
head length 8.98 mm, head width 11.71 mm, eye diameter 4.07 mm, tympanum diam-
eter 2.44 mm, tibia length 20.29 mm, femur length 18.44 mm, calcar length 1.44 mm,
arm length 6.36 mm, eye-nostril distance 2.86 mm, head wider than long and wider
than body; snout rounded in lateral view, truncate in dorsal view; distance from nostril
to eye shorter than diameter of eye; canthus rostralis indistinct, rounded; loreal region
concave; internarial area convex; nostrils not protuberant, directed laterally; interorbital
area slightly convex; eye large, strongly protuberant; diameter of eye 1.7 times diameter
of tympanic annulus; tympanum concealed beneath skin; tympanic annulus evident,
ovoid, longer dorsoventrally and concealed dorsally by supratympanic fold, separated
from eye by ca. 1.1 times its diameter; posterior end of supratympanic fold reaches
anterior border of arm insertion. Arm slender, axillary membrane absent; indistinct
low tubercles along ventrolateral edge of forearm; relative length of fingers I<II<IV<III;
fingers bearing large, oval discs, that of third finger about three fourths of tympanum
diameter; subarticular tubercles prominent, round to ovoid, single; supernumerary
tubercles present; palmar tubercle small, elongated; prepollical tubercle large, flat, el-
liptical; prepollex enlarged, claw shaped; nuptial excrescences absent; webbing absent
between fingers. Large triangular calcar on tibiotarsal articulation; ill defined, scattered
tubercles on tarsus and along ventrolateral edge of foot; toes bearing discs slightly wider
than long, smaller than those of fingers; relative length of toes I<II<V<III<IV; outer
metatarsal tubercle ill defined, small, round; inner metatarsal tubercle large, elliptical;
subarticular tubercles single, round, flat; supernumerary tubercles restricted to the soles;
webbing formula of toes I2—2+II1¾—3-III2-—3IV3—1½V. Skin on dorsum, head,
and dorsal surfaces of limbs smooth; skin on flanks smooth with weak longitudinal
wrinkles posterior to the arm; skin on venter coarsely granular; skin on ventral surfaces
of head and thighs granular, those of shanks smooth. Cloacal opening directed posteri-
orly at upper level of thighs; cloacal sheath short and simple, covering cloacal opening;
round tubercles below and on sides of vent, larger proximally. Tongue ovoid, widely
attached to mouth floor; vomerine odontophores triangular with arched base, narrowly
separated, posteromedial to choanae, each bearing 8–12 teeth; choanae ovoid.
Color of holotype in preservative. Dorsum pale reddish brown with two faint brown
narrow longitudinal lines interconnected in the scapular region (Fig. 16); scattered min-
Figure 16. Adult Hypsiboas maculateralis showing variation in dorsal and ventral coloration of preserved
specimens. From left to right, first and second rows: QCAZ 43825, 44531, 44651, 28004, 28401 (fe-
males); third and fourth rows: QCAZ 40082 (holotype), 43897, 44020–21, 44184 (males). See Appendix
for locality data. All specimens are shown at the same scale.
ute black dots distributed on dorsum; dark brown middorsal line along snout; dorsal
surfaces of limbs pale reddish brown with five pale brown narrow transversal bars on
each thigh; flanks and hidden surfaces of thighs pale cream with dark brown blotches;
narrow white stripe above vent; venter creamy white; ventral surfaces of limbs and web-
bing yellowish white; narrow to wide pale brown stripe on the outer edge of the hands,
forearms, thighs, tarsal folds, and feet; minute brown blotches on lips; bones white.
Color of holotype in life. (Fig. 14F). Dorsum creamy white with six narrow pale
brown longitudinal lines; a dark brown middorsal line; five pale brown faint transversal
bars on dorsal surface of each thigh and two narrow pale brown longitudinal lines on
dorsal surface of each shank; scattered minute black dots on dorsum; flanks and hidden
surfaces of the thighs light-bluish white with dark brown blotches; discs and webbing
pale yellowish tan; iris cream with faint yellow coloration on its upper margin.
Etymology. The specific name is derived from the Latin words macula = stain, and
lateralis = lateral, in reference to the brown dark blotches on the flanks of these frogs.

QCAZ 44020) to reddish brown (e.g., QCAZ 28401), pale reddish brown (e.g.,
QCAZ 28004), pale pinkish white (e.g., QCAZ 44021, 43897), pinkish white (e.g.,
QCAZ 44531, 44651, 44184) or brown (e.g., QCAZ 43825). A dark middorsal line
extends from the tip of the snout to the middle of dorsum (e.g., QCAZ 2156), but in
some specimens is restricted to the snout (e.g., QCAZ 43825, 44651, 28004) or is ab-
sent (e.g., QCAZ 44531). There is variation in the number, size, and shape of the dor-
sal marks. Five to seven broad transversal bands on the dorsum (sometimes intercon-
nected) and narrower brown transversal bars on the dorsal surfaces of the limbs (one or
two each on upper arm and forearm and three to five each on thigh, shank and foot)
may be present (e.g., QCAZ 28401). In some individuals, faint, narrow longitudinal
dark lines may be present (e.g., QCAZ 43897). In a few specimens, the dorsum, dorsal
surfaces of forearms, and shanks may present large black stains (e.g., QCAZ 44021)
and/or few scattered brown dots (e.g., QCAZ 43897, 44020). The coloration of the
flanks and hidden surfaces of thighs vary from pale cream to creamy white or gray, with
dark brown blotches. There is variation in the distribution of blotches on flanks; the
extent covered by the blotches ranges from groin to the mid-flank (e.g., QCAZ 28004)
or to the axilla (e.g., QCAZ 55374). In some individuals, similar blotches can also be
present on the hidden surfaces of the shanks, ventral surfaces of the forelimbs, and
dorsal surfaces of the feet (e.g., QCAZ 43825). In few individuals, the dark blotches of
the thighs and flanks are faint (e.g., QCAZ 43897).
44020, 44184; Fig. 16) to yellowish white (e.g., QCAZ 28004). Some individuals
have brown flecks on the gular region, chest, and lips (e.g., QCAZ 43825). A narrow
to wide brown stripe is present in some individuals on the outer edge of the hands,
forearms, thighs, tarsal folds and feet (e.g., QCAZ 28004, 28401, 44021). Colora-
tion of webbing and disc varies from yellowish white to brown or gray. Vomerine
odontophores are triangular (with arched base in some specimens). Coloration of
bones is white.
In the adult type series, the largest male has a SVL of 39.17 mm, and the largest
female 55.31 mm; mean male SVL = 36 mm (n = 22; SD = 1.92), mean female SVL =
45.18 mm (n = 6; SD = 9.39). Females are significantly larger than males (t = -3.43, df
= 4, P = 0.025). Inter-population variation in size and other morphometric variables
are shown in Tables 1 and 2.
Coloration in life. (based on photographs; Figs 4D and 8D). Dorsal surfaces vary
from pale yellowish tan (e.g., QCAZ 43827) to yellowish cream (e.g., QCAZ 44636)
with a middorsal dark brown line (e.g., QCAZ 43827); sometimes, ill-defined broad
pale brown transversal bands (e.g., QCAZ 43827) or narrow pale brown longitudinal
lines are present on the dorsum (e.g., QCAZ 44636); in some individuals, there are
pale brown transversal bars on the dorsal surfaces of the limbs (e.g., QCAZ 44636);
scattered minute black dots may be present on the dorsum (e.g., QCAZ 44636);
flanks white, light blue or blue with dark brown blotches (e.g., QCAZ 43825, 43827,
Figure 17. Distribution of Hypsiboas calcaratus and H. maculateralis. Localities are based on museum
specimens from Museo de Historia Natural Gustavo Orcés of Escuela Politécnica Nacional, Museo de
Zoología of Pontificia Universidad Católica del Ecuador, Natural History Museum University of Kansas,
and Smithsonian Institution National Museum of Natural History.
44636); hidden surfaces of thighs white, gray, orange, light blue or blue with dark
brown blotches (e.g., QCAZ 43825, 43825, 44636); in some specimens, hidden sur-
faces of the shanks, ventral surfaces of the upper arms and dorsal surfaces of the feet are
blue with dark brown blotches (e.g., QCAZ 43825); venter creamy, sometimes with
yellowish on its anterior half and with brown flecks on the neck, chest, and lips (e.g.,
QCAZ 43827); ventral surfaces of hindlimbs and forelimbs translucent white (e.g.,
QCAZ 43827) or translucent orange (e.g., QCAZ 44636); in some specimens, ventral
surfaces of the thighs creamy white (e.g., QCAZ 43825); discs and webbing brown
(e.g., QCAZ 43825), orange (e.g., QCAZ 44636) or pale yellowish tan (e.g., QCAZ
43827); a narrow to wide brown stripe may be present on the outer edge of the hands,
forearms, thighs, feet, and tarsal folds (e.g., QCAZ 43825, 43827); iris yellowish (e.g.,
QCAZ 43715) or cream with faint yellow to orange color on its upper quarter (e.g.,
QCAZ 43825); bones vary from white to green (e.g., QCAZ 43827, 44636). Juveniles
have similar coloration to adults, except that the blotches on the flanks and hidden
surfaces of thighs are absent (e.g., QCAZ 43709–10, 43715, 43800).
Advertisement call. A call from a male (QCAZ 40082) at Comunidad Santa Rosa
(Napo Provincia) recorded on 28 February 2009 (Table 7) consists of three to four
notes that sound like a high-pitched chuckle. The mean call duration is 0.35 s (SD =
0.04) and the mean rise time is 0.19s (SD = 0.1). The dominant frequency is higher
(mean = 2217.93 Hz, SD = 56.94) and the fundamental frequency lower (mean =
488.10 Hz, SD = 12.47) compared to H. fasciatus, H. almendarizae, H. calcaratus, H.
alfaroi sp. n. and H. tetete sp. n.
Distribution and ecology. Hypsiboas maculateralis inhabits the Amazon basin of
Ecuador (Napo, Orellana, Pastaza, and Sucumbíos provinces) and Peru (Región de
Madre de Dios) (Fig. 17). Localities with known elevation range vary between 186 and
354 m of elevation. The elevation of Comunidad Santa Rosa (354 m) is the highest
known and Santa Teresita (186 m) is the lowest.
All the specimens in Zábalo, Reserva Cuyabeno, Santa Teresita, Bataburo Lodge,
and km 74 Pompeya-Iro road were found at night, in primary or secondary forests,
perching on vegetation between 40 and 200 cm above the ground, in areas of pasture
and flooded forest.
Vegetation types for Ecuadorian localities are: (1) Amazonian Lowland Evergreen
Forest, characterized by high plant alpha-diversity and a canopy of 30 m with emer-
gent trees that reach 40 m, (2) Floodplain Lowland Forest of White-Waters, character-
ized by periodical flooding with white-waters from large rivers, the vegetation reaches
35 m of height, and there are several horizontal strata of vegetation, and (3) Lowland
Forest of Palms and black-waters, swamps characterized by a canopy of 30 m with dens
e understory and a dominance of the palm Mauritia flexuosa.
Vegetation type of the Peruvian locality is Southwest Amazon Moist Forest.
Conservation status. The distribution polygon of H. maculateralis has an area of
209,304 km2. Because its distribution range is large and includes extensive areas of undis-
turbed forest, we propose that H. maculateralis be assigned to the category Least Concern.
Hypsiboas alfaroi sp. n.

http://zoobank.org/6D92050D-8BC3-44FD-A749-5B50DD8D2F73
http://species-id.net/wiki/Hypsiboas_alfaroi
Common name. English: Alfaro’s treefrog; Spanish: Rana arbórea de Alfaro

Holotype. QCAZ 43262 (field no. SC-PUCE 23245), adult male from Ecuador,
Provincia Orellana, Parque Nacional Yasuní, 1 km from Northern Production Facili-
ties (NPF) oil camp (0.6893°S, 76.4290°W), 238 m above sea level, collected by Mar-
cel A. Caminer and Edwin Carrillo Ponce on 20 June 2009.
Paratopotypes. QCAZ 43260–61, 43263, adult males, collected with the holotype.
Paratypes. ECUADOR: PROVINCIA ORELLANA: Río Napo, Nuevo Roca-
fuerte, (0.9192°S, 75.4010°W), 187 m, QCAZ 44788–89, 44790–91, adult males,
collected by S. R. Ron, E. Toral and I. G. Tapia on 10 July 2009; Nuevo Rocafuerte,
Alta Florencia (0.8966°S, 75.4370°W), 187 m, QCAZ 39510, adult male, collected by
S. D. Padilla and P. Vargas Mina on 29 October 2008; Río Napo, Huiririma (0.7116°S,
75.6239°W), 194 m, QCAZ 44634–35, adult males, collected by S. R. Ron, E. Toral
and I. G. Tapia on 8 July 2009; Río Napo, San Vicente (0.6790°S, 75.6511°W), 203
m, QCAZ 44527, adult male, 44528, adult female, collected by S. R. Ron, E. Toral
and I. G. Tapia on 7 July 2009; Río Napo, Chiroisla (0.5756°S, 75.8998°W), 203 m,
QCAZ 44351, adult female, 44425, adult male, collected by S. R. Ron, E. Toral and
I. G. Tapia on 5 July 2009; Río Napo, Edén (0.4983°S, 76.0711°W), 216 m, QCAZ
39631, 44180, adult females, 44191, adult male, collected by S. Aldas, S. R. Ron,
E. Toral and I. G. Tapia on 21 January 2009 and 4 July 2009; Río Napo, Añangu
(0.5249°S, 76.3844°W), 255 m, QCAZ 43977–78, adult males, collected by S. R.
Ron, E. Toral and I. G. Tapia on 1 July 2009; Estación Científica Yasuní, Universi-
dad Católica del Ecuador (0.6748°S, 76.3844°W), 231 m, QCAZ 43249–50, 43253,
43255 adult males, 43251–52, 43254, adult females, same collectors as the holotype
between 18–19 June 2009; Estación Científica Yasuní, Universidad Católica del Ec-
uador (0.6748°S, 76.3844°W), 219 m, QCAZ 19328, adult female, collected by I. G.
Tapia and G. Carotti on 20 February 2002; Río Napo, sector La Primavera (0.4442°S,
76.7868°W), 244 m, QCAZ 43826, adult male, collected by S. R. Ron, E. Toral and
I. G. Tapia on 29 June 2009; Río Napo, sector La Primavera, El Descanso (0.4310°S,
76.7864°W), 244 m, QCAZ 43894, adult female, collected by S. R. Ron, E. Toral and
I. G. Tapia on 30 June 2009; El Coca (0.4778°S, 76.9898°W), 267 m, QCAZ 559,
adult male, collected by Giovanni Onore on 1 April 1984, QCAZ 43682, adult male,
43683–84, adult males, collected by S. R. Ron, E. Toral and I. G. Tapia and 27 June
2009; PROVINCIA SUCUMBÍOS: Playas del Cuyabeno (0.2654°S, 75.8917°W),
230 m, QCAZ 28272, 28398, adult females, 28278, adult male, collected by M. R.
Bustamante, N. Acosta-Buenaño, M. Guerra and C. Proaño between 7–8 October
2003; Puerto Bolívar (0.0886°S, 76.1420°W), 240 m, QCAZ 28240, adult male, col-
lected by P. Menéndez and M. R. Bustamante on 5 August 2003; Río Napo, La Selva
Lodge (0.5086°S, 76.3649°W), 229 m, QCAZ 44025, 44027, adult males, collected
by S. R. Ron, E. Toral and I. G. Tapia on 2 July 2009; Río Napo, 2.5 km S from Paña-
cocha (0.4712°S, 76.6667°W), 225 m, QCAZ 44851, 44853, 44858, adult males,
44856, adult female, collected by S. R. Ron, E. Toral and I. G. Tapia on 3 July 2009;
2.5 km N from Nueva Loja (0.0917°N, 76.8901°W), 350 m, QCAZ 33983, adult
female, 33984, adult male, collected by S. Valdivieso, D. Tirira, J. Wiens and L. A.
Coloma on 17 March 1990; Pañacocha, Moretal Sur (0.2758°S, 75.9352°W), 212 m,
QCAZ 50785, juvenile, collected by R. Betancourt on 11 February 2011.
Referred specimens. ECUADOR: PROVINCIA ORELLANA: Río Napo,
Nuevo Rocafuerte, (0.9192°S, 75.4010°W), 187 m (QCAZ 44789); Río Napo, Hu-
iririma (0.7116°S, 75.6239°W), 194 m (QCAZ 44632–33); Río Napo, San Vicente
(0.6790°S, 75.6511°W), 203 m (QCAZ 44525–26); Río Napo, Chiroisla (0.5756°S,
75.8998°W), 203 m, (QCAZ 44424, 44426–27, 44429); Río Napo, Edén (0.4983°S,
76.0711°W), 216 m, (QCAZ 44181, 44249); Estación Científica Yasuní, Universidad
Católica del Ecuador (0.6702°S, 76.4376°W), 231 m (QCAZ 8466, 8469, 11883,
19203, 49153); Río Napo, sector La Primavera, El Descanso (0.4310°S, 76.7864°W),
244 m (QCAZ 43895–96); Parque Nacional Yasuní, Pozo SPF, 250 m (QCAZ
31257–59); Nuevo Rocafuerte, Tambococha (0.9783°S, 75.4256°W), 177 m (QCAZ
55226, 55232, 55296, 55363, 55432–33); PROVINCIA SUCUMBÍOS: Playas de
Cuyabeno (0.2654°S, 75.8917°W), 230 m (QCAZ 28383–84, 28391, 28397); Puer-
to Bolívar (0.0886°S, 76.1420°W), 240 m (QCAZ 27815, 28190, 28211, 28230,

28232, 28310, 28315); Río Napo, entrance to La Selva Lodge (0.5086°S, 76.3649°W),
229 m (QCAZ 44022–24, 44026, 44028); La Selva Lodge, Limoncocha (0.4981°S,
76.3738°W), 245 m (QCAZ 25401, 25408, 25410, 25417); Río Napo, 2.5 km S from
Pañacocha (0.4712°S, 76.6667°W), 225 m (QCAZ 44848–50, 44852, 44854–55,
44857); 2.5 km N from Nueva Loja (0.0917°N, 76.8901°W), 350 m (QCAZ 2797).
Diagnosis. Hypsiboas alfaroi (Fig. 4E, 8E and 18) is characterized by: (1) mean
SVL 32.80 mm in males (range 27.91–36.27; n = 32), 44.51 mm in females (range
39.68–49.21; n = 12); (2) basal webbing on fingers; (3) tubercle on heel; (4) dorsal col-
oration varying from creamy white to grayish brown or brown, sometimes with dark
markings (e.g., broad transversal bands, narrow longitudinal lines); (5) faint brown
middorsal line often present; (6) flanks creamy white or gray (in life, blue in large fe-
males and light blue or white in males) with dark brown dots or blotches; (7) hidden
surfaces of thighs creamy white, gray or brown (in life, blue in large females and light
blue or white in males) with dark brown dots or blotches; (8) ventral surfaces of thighs
creamy white, yellowish white or brown; (9) ventral areas creamy white or yellowish
white with brown flecks on the head, neck, and chest; (10) webbing on feet; (11) in
life, iris yellowish, bronze or cream with faint yellow coloration on its upper margin;
(12) prepollical spine present in males.
Hypsiboas alfaroi is most similar to H. tetete sp. n. but it can be distinguished by
having a markedly different advertisement call (Fig. 13C–H). Although with overlap-
ping values, H. alfaroi has a smaller tympanum (relative to SVL, mean male TD/SVL =
0.06, SD = 0.01, n = 32; H. tetete sp. n. mean male TD/SVL = 0.08, SD = 0.009, n = 5;
differences are significant: t = –3.21, df = 35, P = 0.003). Hypsiboas alfaroi differs from
H. fasciatus, H. almendarizae, H. calcaratus, and H. maculateralis in advertisement call
(Figs 12A–F and 13A–D) and by having a small tubercle on the heel instead of a large
calcar. Hypsiboas alfaroi further differs from H. fasciatus, H. almendarizae, and H. cal-
caratus by having dark brown dots on the flanks (vertical dark lines in H. fasciatus, H.
almendarizae, and H. calcaratus). Morphological characters useful to differentiate H.
alfaroi from other species are shown in Table 6.
Description of holotype. Adult male, 32.06 mm SVL, foot length 13.22 mm,
head length 8.77 mm, head width 10.02 mm, eye diameter 3.69 mm, tympanum
diameter 2.19 mm, tibia length 18.74 mm, femur length 16.24 mm, arm length 5.71
mm, eye-nostril distance 2.59 mm, head wider than long and wider than body; snout
rounded in lateral view, truncate in dorsal view; distance from nostril to eye shorter
than diameter of eye; canthus rostralis indistinct, rounded; loreal region concave; inter-
narial area convex; nostrils not protuberant, directed laterally; interorbital area slightly
convex; eye large, strongly protuberant; diameter of eye 1.7 times diameter of tym-
panic annulus; tympanum concealed beneath skin; tympanic annulus evident, ovoid,
longer dorsoventrally and concealed dorsally by supratympanic fold, separated from
eye by ca. 1.01 times its diameter; posterior end of supratympanic fold reaches anterior
border of arm insertion. Arm slender, axillary membrane absent; indistinct low tuber-
cles present along ventrolateral edge of forearm; relative length of fingers I<II<IV<III;
Figure 18. Adult Hypsiboas alfaroi showing variation in dorsal and ventral coloration of preserved speci-
mens. From left to right, first and second rows: QCAZ 28272, 28398, 44180 (females); third and fourth
rows: QCAZ 43262 (holotype), 43263, 43826 (males). See Appendix for locality data. All specimens are
fingers bearing large, oval discs, that of third finger about three fourths of tympanum
diameter; subarticular tubercles prominent, round to ovoid, single; supernumerary
tubercles present; palmar tubercle small, elongated; prepollical tubercle large, flat, el-
liptical; prepollex enlarged, claw shaped; nuptial excrescences absent; webbing absent
between fingers. Small tubercle on tibiotarsal articulation; ill defined, scattered tuber-
cles on tarsus and along ventrolateral edge of foot; toes bearing discs slightly wider than
long, smaller than those of fingers; relative length of toes I<II<V<III<IV; outer meta-
tarsal tubercle ill defined, small, round; inner metatarsal tubercle large, elongated, el-
liptical; subarticular tubercles single, round, flat; supernumerary tubercles restricted to
the soles; webbing formula of toes I2—2II2—3III2—3IV3—1¾V. Skin on dorsum,
head, and dorsal surfaces of limbs smooth; skin on flanks smooth with weak longitu-
dinal wrinkles posterior to the arm; skin on venter coarsely granular; skin on ventral
surfaces of head and thighs granular, those of shanks smooth. Cloacal opening directed
posteriorly at upper level of thighs; short simple cloacal sheath covering cloacal open-
ing; round tubercles below and on the sides of vent. Tongue ovoid, widely attached to
mouth floor; vomerine odontophores triangular, narrowly separated, posteromedial to
choanae, bearing 10 vomerine teeth on each side; choanae trapezoidal, oblique.
Color of holotype in preservative. Dorsum grayish brown with five to six broad dif-
fuse brown transversal bands; scattered minute black dots on dorsal surfaces; few small
cream dots restricted to the posterior dorsum; faint brown narrow middorsal line re-
stricted to the head; flanks creamy white with dark brown dots; dorsal surfaces of
hindlimbs and forelimbs grayish brown with narrow transversal brown bars (one or
two on each upper arm and forearm, and three or four on each thigh, shank, and
tarsus); hidden surfaces of thighs grayish brown with dark brown dots; venter creamy
white with brown flecks on head, neck, and chest; ventral surfaces of hindlimbs and
forelimbs yellowish white with a narrow to wide brown stripe on the outer edge of the
hands, forearms, tarsal folds, and feet; bones white.
Etymology. The specific name is a noun in the genitive case and is a patronym for
Eloy Alfaro Delgado, former Ecuadorian president (1897–1901 and 1906–1911) and
leader of the liberal revolution in Ecuador. His government promoted the separation
between church and state and the modernization of Ecuador by supporting education
and large-scale systems of transportation and communication.
QCAZ 44429) to pale grayish brown (e.g., QCAZ 19328), grayish brown (e.g.,
QCAZ 44180), pale brown (e.g., QCAZ 28398) or brown (e.g., QCAZ 28272). Ir-
regular dorsal marks may be present in diverse patterns. A faint dark middorsal line
extends from the tip of the snout to the mid-dorsum (e.g., QCAZ 43895) or the sacral
region (QCAZ 44025), but in some specimens it is restricted to the head (e.g., QCAZ
55926) or is altogether absent (e.g., QCAZ 28272). There is variation in the number,
size, and shape of the dorsal marks. Five to seven diffuse broad transversal bands on
the dorsum (sometimes interconnected), and narrower brown transversal bars on the
dorsal surfaces of the limbs (one or two each on the arm and forearm, and three to
five each on the thigh, shank, and foot) may be present (e.g., QCAZ 28272). Faint,
narrow longitudinal lines may be present on the dorsum (e.g., QCAZ 44858, 43263).
The dorsum and dorsal surfaces of limbs can have scattered black or white dots (e.g.,
QCAZ 43263, 28398). The coloration of flanks varies from creamy white to gray with
irregular dark brown spots distributed from the groin to the mid-flank. In some indi-
viduals, similar spots can also be present in the hidden surfaces of the thighs, shanks,
ventral surfaces of the forelimbs, and dorsal surfaces of the feet (e.g., QCAZ 28272).
The hidden surfaces of thighs sometimes have dark brown blotches (QCAZ 28272).
Ventral areas of preserved specimens vary from creamy white (e.g., QCAZ 28398,
44180) to yellowish white (e.g., QCAZ 43263) with dark flecks on the head, neck, and
chest (Fig. 18). In some individuals, the flecks are also present on hindlimbs, forelimbs,
and belly (e.g., QCAZ 28272, 28398). A narrow to wide brown stripe is present in
some individuals on the outer edge of the hands, forearms, thighs, tarsal folds, and
feet (e.g., QCAZ 43263). Coloration of webbing and disc vary from brown to gray.
Vomerine odontophores are triangular (with arched base in some specimens). Bones
are white.
In the adult type series, the largest male has a SVL of 36.27 mm, and the largest
female 49.21 mm; mean male SVL = 32.80 mm (n = 32; SD = 1.97), mean female
SVL = 44.51 mm (n = 12; SD = 3.09). Females were significantly larger than males (t =
-14.94, df = 42, P < 0.001). Inter-population variation in size and other morphometric
variables are shown in Tables 1 and 2.
Coloration in life. (based on photographs; Figs 4E and 8E). Dorsal surfaces vary
from pale creamy white (e.g., QCAZ 39631) to yellowish tan (e.g., QCAZ 43261),
reddish brown (e.g., QCAZ 43254) or brown (e.g., QCAZ 43978) with a faint brown
middorsal line (e.g., QCAZ 43978); sometimes, broad pale brown transversal bands
are present on the dorsum (e.g., QCAZ 43978); narrow pale brown transversal bars
(e.g., QCAZ 43683) or narrow pale brown longitudinal lines (e.g., QCAZ 44635)
may ornament the dorsal surfaces of the limbs; scattered minute black dots on the
dorsum may be present (e.g., QCAZ 44635); flanks are white, light blue or blue with
irregular dark brown spots (e.g., QCAZ 43683) or blotches (e.g., QCAZ 43252); hid-
den surfaces of thighs are gray, white, light blue or blue with dark brown irregular spots
(e.g., QCAZ 43683) or blotches (e.g., QCAZ 39631); in some specimens, the hidden
surfaces of the shanks and dorsal surfaces of the feet have similar brown spots (e.g.,
QCAZ 43254); venter creamy white (e.g., QCAZ 43254) or yellowish with creamy
white belly (e.g., QCAZ 43260); conspicuous brown flecks are present on the ventral
areas of the head, neck, and chest (e.g., QCAZ 44635); ventral surfaces of hindlimbs
and forelimbs are translucent white (e.g., QCAZ 43683) or yellowish (e.g., QCAZ
43260); in some specimens, the ventral surfaces of the thighs are creamy white (e.g.,
QCAZ 43254); discs and webbing are brown (e.g., QCAZ 43978), yellowish (e.g.,
QCAZ 43261) or pale cream (e.g., QCAZ 39631); a narrow to wide brown stripe may
be present on the outer edge of the hands, forearms, thighs, feet, and tarsal folds (e.g.,
QCAZ 43978); iris yellowish (e.g., QCAZ 43683), bronze (e.g., QCAZ 43254) or
cream with a subtle yellow tone on its upper quarter (e.g., QCAZ 44635); coloration
of bones varies from white (e.g., QCAZ 43252) to green (e.g., QCAZ 43683).
Advertisement call. Three males were recorded at Estación Científica Yasuní
PUCE (Provincia Orellana) on 20 June 2009. The call (Fig. 13C–D) consists of four
to five trill-like notes with a mean duration of 0.20 s (SD = 0.05) and mean rise time
of 0.07 s (SD = 0.03). Other call parameters are listed in Table 7.
Distribution and ecology. Hypsiboas alfaroi occurs in the Ecuadorian north-

ern Amazon region (Napo, Orellana, and Sucumbíos provinces; Fig. 15). Localities
with known elevation range from 176 m (Nuevo Rocafuerte) to 350 m (Nueva Loja).
Nuevo Rocafuerte is on the border between Ecuador and Peru; the occurrence of H.
alfaroi in Peru is highly likely.
Specimens from Nuevo Rocafuerte, Playas de Cuyabeno, Puerto Bolívar, Estación
Científica Yasuní of Universidad Católica del Ecuador, and Nueva Loja were found at
night in primary and secondary forest, perching on vegetation 50 to 180 cm above the
ground, in flooded areas, swamps, near streams and in forest away from water bodies.
Individuals at San Vicente were found in pastures, secondary forests, flooded grassland,
and ponds.
Vegetation types for Ecuadorian localities are: (1) Amazonian Lowland Evergreen
Forest, characterized by high plant alpha-diversity and a canopy of 30 m with emer-
gent trees that reach 40 m, (2) Floodplain Lowland Forest of White Waters, charac-
terized by periodical flooding with white waters from large rivers and vegetation that
reaches 35 m of height with several vegetation strata, and (3) Lowland Forest of Palms
and black-waters, swamps characterized by a canopy of 30 m with dense understory
and a dominance of the palm Mauritia flexuosa.
Conservation status. The distribution polygon of H. alfaroi has 47,524 km2.
Within it, 4,287 km2 (9.0%) have been degraded by human activities, especially agri-
culture and cattle raising (estimated from Ministerio de Ambiente del Ecuador 2013).
Because its distribution range is relatively large and has a low proportion of degraded
habitat, we propose that H. alfaroi be assigned to the Least Concern category.
Hypsiboas tetete sp. n.

http://zoobank.org/D9448B7C-4778-4912-A21A-3E32E4266EF1
http://species-id.net/wiki/Hypsiboas_tetete
Common name. English: Tetete’s treefrog; Spanish: Rana arbórea de los Tetetes
Holotype. (Fig. 14G) QCAZ 40081 (field no. SC-PUCE 23220), adult male
from Ecuador, Provincia Napo, Comunidad Santa Rosa, road to Tena (1.0214°S,
77.4782°W), 344 m above sea level, collected by Marcel A. Caminer and Edwin Car-
rillo Ponce on 28 February 2009.
Paratopotypes. QCAZ 40060–61, 40079–80, adult males, collected with the
holotype.
Paratypes. ECUADOR: PROVINCIA NAPO: Jatun Sacha (1.0649°S,
77.6142°W), 420 m, QCAZ 48094, adult male, 48095–96, adult females, collected
by S. R. Ron and Morley Read on 31 March 2010.
Referred specimens. PERU: REGIÓN LORETO: San Jacinto (2.3125°S,
75.8628°W), 180 m (KU 221864).
Diagnosis. Hypsiboas tetete (Figs 8F and 19) is characterized by: (1) mean SVL
Figure 19. Adult Hypsiboas tetete showing variation in dorsal and ventral coloration of preserved speci-
mens. From left to right, first and second rows: QCAZ 48095–96 (females), 40060 (male); third and
fourth rows: QCAZ 40079, 40081 (holotype), 48094 (males). See Appendix for locality data. All speci-
mens are shown at the same scale.
45.85; n = 2); (2) basal webbing on the fingers; (3) tubercle on the heel; (4) dorsal
background coloration varying from grayish brown to pale brown, sometimes with dark
markings (e.g., diffuse broad transversal bands); (5) faint brown middorsal line often
present; (6) flanks creamy white or gray (in life, light blue in large females and white in
males) with dark brown irregular spots; (7) hidden surfaces of thighs creamy white or
brown with dark brown irregular spots or blotches; (8) ventral surfaces of thighs creamy
white or yellowish white; (9) ventral areas creamy white or yellowish white with brown
flecks on the head, neck, and chest; (10) webbing on the feet; (11) in life, iris yellow or
cream with yellow on its anterior half; (12) prepollical spine present in males.
Hypsiboas tetete differs from H. fasciatus, H. almendarizae, H. calcaratus, and H.
maculateralis in advertisement call (Figs 12A–F and 13A–H) and by having a tubercle
on the heel instead of a calcar. Hypsiboas tetete is most similar to H. alfaroi from which
differs in advertisement call (Fig. 13C–H). Although with overlapping values, Hypsi-
boas tetete has a statistically significant larger tympanum than H. alfaroi (see H. alfaroi
diagnosis). Morphological characters useful to differentiate H. tetete from other species
are shown in Table 6.
Description of holotype. Adult male, SVL 31.15 mm, foot length 12.01 mm,
head length 7.48 mm, head width 10.31 mm, eye diameter 3.51 mm, tympanum
diameter 2.25 mm, tibia length 17.76 mm, femur length 16.09 mm, arm length 5.50
mm, eye-nostril distance 1.97 mm, head wider than long and wider than body; snout
rounded in lateral view, truncate in dorsal view; distance from nostril to eye shorter
than diameter of eye; canthus rostralis indistinct, rounded; loreal region concave; inter-
narial area convex; nostrils not protuberant, directed laterally; interorbital area slightly
convex; eye large, strongly protuberant; diameter of eye 1.6 times diameter of tympanic
annulus; tympanum concealed beneath skin; tympanic annulus evident, ovoid, longer
dorsoventrally and concealed dorsally by supratympanic fold, separated from eye by ca.
1.03 times its diameter; posterior end of supratympanic fold reaches anterior border
of arm insertion. Arm slender, axillary membrane absent; indistinct low tubercles pre-
sent along ventrolateral edge of forearm; relative length of fingers I<II<IV<III; fingers
bearing large, oval discs, that of third finger about three fourths of tympanum diam-
eter; subarticular tubercles prominent, round to ovoid, single; supernumerary tuber-
cles present; palmar tubercle small, elongated; prepollical tubercle large, flat, elliptical;
prepollex enlarged, claw shaped; nuptial excrescences absent; webbing absent between
fingers. Small tubercle on tibiotarsal articulation; ill defined, scattered tubercles on
tarsus and along ventrolateral edge of foot; toes bearing discs slightly wider than long,
smaller than those of fingers; relative length of toes I<II<V<III<IV; outer metatarsal
tubercle ill defined, small, round; inner metatarsal tubercle large, elongated and ellipti-
cal; subarticular tubercles single, round, flat; supernumerary tubercles restricted to the
soles; webbing formula of toes I2-—2½II2—3+III2—3+IV3+—2-V. Skin on dorsum,
head, and dorsal surfaces of limbs smooth; skin on flanks smooth with weak longitu-
dinal wrinkles posterior to the arm; skin on venter coarsely granular; skin on ventral
surfaces of head and thighs granular, those of shanks smooth. Cloacal opening directed
posteriorly at upper level of thighs; short simple cloacal sheath covering cloacal open-
ing; round tubercles below and on the sides of vent. Tongue ovoid, widely attached
to mouth floor; vomerine odontophores triangular with arched base, barely separated,
posteromedial to choanae, bearing eight vomerine teeth on each side; choanae ovoid.
Color of holotype in preservative. Dorsum grayish brown with scattered minute

black dots; faint brown narrow middorsal line extends from the tip of the snout to the
mid-sacrum; dorsal surfaces of hindlimbs and forelimbs grayish brown with narrow
transversal brown bars (one or two on each forearm and three or four on each thigh,
shank, and tarsus); flanks creamy white with dark brown irregular spots; hidden surfac-
es of thighs grayish brown with dark brown irregular spots; venter creamy white with
brown flecks on the neck, chest, and lips; ventral surfaces of hindlimbs and forelimbs
yellowish white with a narrow to wide brown stripe on the outer edge of the hands,
forearms, thighs, tarsal folds, and feet; bones white.
Color of holotype in life. (Fig. 14G). Dorsum pale yellowish tan with four nar-
row pale brown longitudinal lines; a dark brown middorsal line extends from the tip
of snout to mid-sacrum; dorsal surfaces of hindlimbs yellowish tan with pale brown
transversal bars; scattered minute dark brown dots on the dorsal surfaces of limbs and
dorsum; flanks white with well-defined dark brown irregular spots; hidden surfaces of
thighs pale yellowish tan with dark brown spots; venter creamy white with yellowish
white belly; ventrally, scattered brown flecks on the chest, gular region, and jaw mar-
gin; ventral surfaces of hindlimbs and forelimbs creamy white; discs and webbing pale
yellow tan; iris cream with faint yellow coloration on its upper half; bones white.
Etymology. The specific name is a noun and refers to the Tetete, a Western Tu-
canoan indigenous group that inhabited the Colombian and Ecuadorian Amazon. It
was decimated by the rubber exploitation and became extinct during the 1970s (Was-
serstrom et al. 2011). Its recent disappearance parallels the destruction of increasingly
large areas of forest in the Ecuadorian Amazon with the ensuing decline of biodiversity.
shown in Figure 19. Background dorsal coloration varies from grayish brown (e.g.,
QCAZ 48094) to pale grayish brown (e.g., QCAZ 48096) or pale brown (e.g., QCAZ
48094). Irregular dorsal marks may be present in diverse patterns. A faint middorsal
line extends from the tip of snout to mid-dorsum (e.g., QCAZ 40079), mid-sacrum
(e.g., QCAZ 40060) or to the cloaca (e.g., QCAZ 48095–96). There is variation in
the number, size and shape of the dorsum marks. Five to seven broad transversal bands
(sometimes interconnected) may be present on the dorsum; the dorsal surfaces of the
limbs have brown transversal bars (one or two each on upper arm and forearm and
three to five each on thigh, shank, and foot) (e.g., QCAZ 40080). Some individuals
have scattered black or white dots on the dorsum (e.g., QCAZ 40060). The coloration
of flanks varies from creamy white to gray with conspicuous dark brown irregular spots
distributed from the groin to mid-flank. In some individuals, similar spots can also
be present on the hidden surfaces of shanks and dorsal surfaces of feet (e.g., QCAZ
48096). The coloration of the hidden surfaces of thighs varies from creamy white to
brown, with dark brown spots (e.g., QCAZ 40060) or blotches (QCAZ 48096).
Ventral areas of preserved specimens vary from creamy white (e.g., QCAZ 48094,
48096) to yellowish white (e.g., QCAZ 40079) with scattered flecks on the head and
chest. In some individuals, the flecks are also present on hindlimbs, forelimbs, and
belly (e.g., QCAZ 48095). Some individuals (e.g., QCAZ 48096) have a narrow to
wide brown stripe on the outer edge of the hands, forearms, thighs and tarsal folds.
Coloration of webbing and discs vary from brown to gray. Vomerine odontophores are
triangular (with arched base in some specimens). Bones white.
In the adult type series, the largest male has a SVL of 32.24 mm, and the largest fe-
male 45.85 mm; mean male SVL = 31.72 mm (n = 5; SD = 0.42), female SVL range is
45.33 to 45.85 mm (n = 2). Inter-population variation in size and other morphometric
variables are shown in Tables 1 and 2.
Coloration in life. (based on photographs; Fig. 8F). Dorsal surfaces vary from pale
yellowish tan (e.g., QCAZ 40060) to reddish brown (e.g., QCAZ 40079), with a brown
middorsal line (e.g., QCAZ 48096); narrow pale brown longitudinal lines (e.g., QCAZ
40060, 48094) may be present. Some individuals have pale brown transversal bars on
the dorsal surfaces of hindlimbs (e.g., QCAZ 40080); scattered minute black dots can
be present on the dorsal surfaces of limbs and dorsum (e.g., QCAZ 40060); flanks are
white (light blue in large females) with dark brown irregular marks with rounded (e.g.,
QCAZ 40060) or elongated shapes (e.g., QCAZ 48096); hidden surfaces of thighs
are white or gray with dark brown spots (e.g., QCAZ 40060); in some specimens, the
hidden surfaces of the shanks and dorsal surfaces of feet also have dark brown irregular
spots (e.g., QCAZ 48096); venter creamy (e.g., QCAZ 48096) or yellowish white (e.g.,
QCAZ 40080) with scattered brown flecks on the ventral surfaces of the head and chest
(e.g., QCAZ 48094); ventral surfaces of hindlimbs and forelimbs creamy white (e.g.,
QCAZ 40060) or yellowish white (e.g., QCAZ 40061); discs and webbing pale yellow
tan; iris bronze (e.g., QCAZ 48096) or cream with faint yellow coloration on its upper
half (e.g., QCAZ 40060); bones are white (e.g., QCAZ 40080).
Calls. We recorded the calls of four males at Comunidad Santa Rosa (Provincia
Napo) on 28 February 2009, in flooded areas of secondary forest. Call parameters are
shown in Table 7. Two call types were recorded. Type one (Fig. 13 E–F) was the most
common and consisted of a beep-like note with a mean duration of 0.10 s (SD = 0.02),
average rise time 0.03 s (SD = 0.02) and average dominant frequency 1938.47 Hz (SD
= 26.24). Type two (Fig 13 G–H) consisted of a single pulsed note with a mean dura-
tion of 0.11 s (SD = 0.02), mean rise time 0.05 s (SD = 0.02), and mean dominant
frequency 1829.12 Hz (SD = 12.61). Call type two was alternated between calls of
type one. Only males QCAZ 40060, 40080–81 produced this type of call.
Distribution and ecology. Hypsiboas tetete is distributed in the Ecuadorian (Pro-
vincia Napo) and Peruvian Amazon basin (Región Loreto) (Fig. 15). Known localities
range in elevation from 180 m (San Jacinto) to 420 m (Jatun Sacha). It is likely to have
a larger distribution. Unfortunately, the lack of distinctive morphological characters
relative to H. alfaroi, preclude the unequivocal identification of museum specimens
not associated with advertisement calls or genetic data. All specimens from Comu-
nidad Santa Rosa and Jatun Sacha were found in flooded areas, in secondary forest,
roosting on vegetation, 50 to 80 cm above ground.
Vegetation type for the Ecuadorian localities is Amazonian Lowland Evergreen
Forest characterized by high plant alpha-diversity and a canopy of 30 m with emergent
trees that reach 40 m.
Vegetation type at the Peruvian locality is Napo Moist Forest.

Conservation status. The distribution polygon has 2,950 km2 of which 106
(3.5%) have been degraded by human activities (estimated from Ministerio de Ambi-
ente del Ecuador 2013). Because its known distribution range is small with less than
five localities and habitat degradation is increasing, H. tetete is assigned to the Endan-
gered category under criteria B1ab(iii).
Morphometric comparisons between species

The following comparisons pertain to male SVL. There are significant differences be-
tween most species pairs. Hypsiboas alfaroi and H. tetete are smaller than H. almendarizae,
H. fasciatus, H. calcaratus, and H. maculateralis (all P values for t tests < 0.001); H. alfaroi
is larger than H. tetete (t = 2.74, df = 31, P = 0.01); H. fasciatus is smaller than H. almen-
darizae (t = -3.87, df = 40, P < 0.001) and H. calcaratus (t = -3.02, df = 52, P = 0.004);
while H. almendarizae is larger than H. maculateralis (t = 2.79, df = 43, P = 0.008).
Two components with eigenvalues > 1.0 were extracted from the PCA for 136
males. The two components accounted for 61.30% of the total variation. The highest
loadings for PC I were tibia length, femur length, and foot length; for PC II it was tym-
panum diameter (Table 8). The morphometric space shows high overlap among species
(Fig. 7B). The first PC shows a partial segregation between H. maculateralis (shorts
limbs) and H. calcaratus (long limbs). Comparisons of PC I between males of the six
species show significant differences between H. fasciatus and H. almendarizae relative to
H. calcaratus and H. alfaroi (all P values for t tests < 0.02); H. calcaratus is significantly
different from H. maculateralis (t = 4.56, df = 55, P < 0.001), H. alfaroi (t = 6.29, df =
65, P < 0.001), and H. tetete (t = 3.43, df = 20, P = 0.003); H. fasciatus is different from
H. maculateralis (t = 2.14, df = 39, P = 0.038); sister species H. alfaroi and H. tetete are
also significantly different along PC I (t = -3.35, df = 15, P = 0.004). Comparisons of
PC II (mainly tympanum diameter) shows that H. tetete differs from all the others spe-
cies (all P values for t tests < 0.05); while H. fasciatus and H. almendarizae are different
relative to H. calcaratus, H. maculateralis, and H. alfaroi (all P values for t tests < 0.05).
Two components with eigenvalues > 1.0 were extracted from the PCA for 34 fe-
males. The two PCs accounted for 55.89% of the total variation. Principal Component I
(35.06% of the variance) is positively correlated with head width, tibia length, and femur
length, while PC II (20.83% of the variance) is correlated with head length (Table 8).
Overall, there is wide overlap in morphometric space among species. However, there is
segregation between H. almendarizae and H. calcaratus along PC I and H. almendarizae
and H. maculateralis along PC II (Fig. 7A). In the PCA for females, PC I (mainly head
shape and tibia length) showed significant differences between H. maculateralis and H.
calcaratus (t = 3.24, df = 8, P = 0.012) and H. alfaroi (t = -2.39, df = 16, P = 0.029);
PC II (foot length) shows significant differences between H. almendarizae relative to
H. fasciatus (t = 2.66, df = 7, P = 0.032), H. calcaratus (t = -2.62, df = 6, P = 0.039), H.
maculateralis (t = -4.39, df = 8, P = 0.002), and H. alfaroi (t = -2.84, df = 14, P = 0.013).
Table 8. Character loadings, eigenvalues, and percentage of explained variance for Principal Components
(PC) I–II. The analysis was based on six morphometric variables of adult Hypsiboas alfaroi, H. almen-
darizae, H. calcaratus, H. fasciatus, H. maculateralis, and H. tetete. Bold figures indicate highest loadings.
PCA Males PCA Females

Variable
PC I PC II PC I PC II
Foot length 0.75 –0.35 0.61 –0.58
Head length 0.22 0.01 0.17 0.87
Head width 0.66 0.32 0.70 0.33
Tympanum diameter 0.30 0.89 0.39 0.01
Tibia length 0.81 –0.37 0.68 0.12
Femur length 0.81 0.10 0.75 –0.15
Eigenvalue 2.49 1.18 2.10 1.25
% 41.55 19.74 35.06 20.83
Table 9. Character loadings, eigenvalues, and percentage of explained variance for Principal Components
(PC) I–II. The analysis was based on five acoustic variables from the advertisement calls of Hypsiboas
alfaroi, H. almendarizae, H. calcaratus, H. fasciatus, H. maculateralis, and H. tetete. Bold figures indicate
highest loadings.
Character Loading
Variable
PC I PC II
Rise time 0.64 0.28
Call dominant frequency 0.35 0.75
Call duration 0.95 0.06
Number of notes –0.42 0.78
Call fundamental frequency 0.82 –0.23
Eigenvalue 2.31 1.32
% 46.27 26.47
Call comparisons between species
Two components with eigenvalues > 1.0 were extracted from the PCA of calls from 25
males. Both PCs accounted for 72.74% of the total variation. Principal Component I
(46.27% of the variance) has high loadings on call duration and fundamental frequency;
PC II (26.47% of the variance) has high loadings on number of notes and dominant fre-
quency (Table 9). The acoustic space (as represented by PC I and PC II) shows segregation
among the advertisement calls of all species except H. fasciatus and H. almendarizae, which
are overlapping (Fig. 20). There are also qualitative differences among the advertisement
calls. The closely related H. fasciatus, H. almendarizae, and H. calcaratus share calls consist-
ing of varying numbers of quack-like notes. These calls are markedly different from those
of H. maculateralis, H. alfaroi, and H. tetete. Although the calls of H. alfaroi and H. tetete
appear close in acoustic space, they have a markedly different structure (Fig. 13C–H).
Variation in recording temperature between species was low (< 6 C degrees; Table 7) and
cannot explain the marked interspecific structural differences in advertisement calls.
Figure 20. Axes I and II from Principal Components Analysis based on five acoustic variables from the ad-
vertisement calls of Hypsiboas fasciatus (5 males), H. almendarizae (5), H. calcaratus (7), H. maculateralis (1),
H. alfaroi (4), and H. tetete (4). See Table 9 for character loadings on each component.
Discussion
The use of genetic characters in the study of the systematics of Amazonian amphib-
ians has resulted in the discovery of an unexpectedly large proportion of undescribed
species. Most of those new species were previously considered populations of a single
species with a large distribution range. Examples of these complexes include Bolito-
glossa peruviana, Engystomops petersi, Osteocephalus buckleyi, Osteocephalus taurinus,
Pristimantis ockendeni, Rhinella margaritifera, and Scinax ruber (Elmer et al. 2013;
Elmer and Cannatella 2008; Fouquet et al. 2007; Funk et al. 2012; Jungfer et al. 2013;
Ron et al. 2012). Hypsiboas calcaratus and Hypsiboas fasciatus show a similar pattern
because both were considered to have a widespread distribution comprising most of
the Amazon Basin (IUCN 2011). Our results and those from Funk et al. (2012) in-
dicate that they form a complex of eleven candidate species. Available morphological
and advertisement call data allowed us to confirm the species status of six of them.
Two correspond to H. calcaratus and H. fasciatus and four are new species described
herein. The five remaining candidate species remain unconfirmed although geographic
distribution suggests that clade J from Buenavista, Bolivia (Fig. 1) could correspond to
H. steinbachi (type locality Sara province, Departamento Santa Cruz, Bolivia). Ichilo
province (where Buenavista is found, the source of genetic samples for this study) is
adjacent to Sara province.
Implications of the discovery of hidden species richness in our current under-

standing of the biology of Amazonian amphibians
The discovery of hidden species richness could require a review of our current under-
standing of the biogeography, evolution and conservation status of Amazonian am-
phibians (Icochea and Angulo 2010, Peloso 2010). This need arises from the antici-
pated increase in the total number of described species and the availability of more ac-
curate assessments of the geographic distribution of the species. Although the number
of genetic-based studies on the systematics of Amazonian amphibians is still limited,
our results and those from other species complexes (see above) suggest that the changes
could be substantial. In the H. calcaratus-H. fasciatus complex the number of species
increased three to five times and there was a sizeable decrease in the distribution range
of Hypsiboas fasciatus. According to the distribution polygon of the IUCN Red List
(Icochea et al. 2004), H. fasciatus has a distribution of 5’867,000 km2. Our results
show that the distribution polygon of H. fasciatus, based on the localities shown in Fig.
15, does not overlap with the Red List polygon and has less than 10,000 km2, about
0.014% of Red List estimate. If these changes are typical among Amazonian amphib-
ians, the reliability of large global databases, like those of the IUCN Red List, Global
Amphibian Assessment (http://www.iucnredlist.org/initiatives/amphibians) could be
compromised. The same problem would affect large scale analyses of biogeography,
evolution, and conservation of amphibians that have relied those databases (e.g., Buck-
ley and Jetz 2007; Pyron and Wiens 2013).
The conservation status of Amazonian amphibians will also require a reassessment
under the new taxonomy. The aggregated results for the H. fasciatus species complex
show a decrease in the proportion of Least Concern species from 100% to 50%. If this
amount of change is representative of Amazonian amphibians, we would expect an
increase in the proportion of species under threatened categories.
The use of molecular tools is revolutionizing and reinvigorating the fields of tax-
onomy and systematics. By combining genetic data with other sets of independent
characters, a practice that has been recently referred as “integrative taxonomy” (Dayrat
2005; Padial and De la Riva 2009), species boundaries can be defined objectively.
These new tools have allowed documenting large numbers of undescribed Amazonian
amphibians, which highlight the need for dense scientific collections (including ge-
nome samples) and comprehensive molecular-based taxonomic reviews. The comple-
tion of the inventory of species should be considered a priority because other scientific
endeavors cannot succeed without reliable taxonomic data.
Acknowledgments
This study was supported by grants from the Secretaría Nacional de Educación Superior,
Ciencia, Tecnología e Innovación del Ecuador SENESCYT (PI-C08-0000470), and the
Pontificia Universidad Católica del Ecuador. William E. Duellman (Natural History Mu-
seum, University of Kansas), Antoine Fouquet, and Ross MacCulloch (Royal Ontario Mu-
seum) provided tissues or DNA sequences from Peru and Guiana. The Ecuadorian Minis-
terio de Ambiente provided research and collection permits numbers 004-IC- FAU-DPF,
006-IC-FAU-DBAP/MA, 001-10 IC-FAU-DNB/ MA, and 008-09 IC-FAU-DNB/MA.
Ana Almendáriz (EPN), Barry Clarke (BMNH), and Roy McDiarmid (USNM) provided
access to their amphibian collections. Edwin Carrillo, Juan Fernando Dueñas, and Elicio
Tapia assisted fieldwork. For specimen collection we thank Néstor Acosta-Buenaño, A.
Almendáriz, F. P. Ayala, M. R. Bustamante, J. Bosch, J. Brito, F. Campos, E. Carrillo, L. A.
Coloma, S. de la Torre, M. A. Guerra, D. Kizirian, M. Mejía, A. Merino-Viteri, T. Panchi,
A. Pounds, G. Onore, J. Parrales, M. Read, F. Robledo, G. E. Romero, Y. Sagredo, E.
Tapia, I. G. Tapia, E. Toral, O. Torres-Carvajal, and J. J. Wiens. Teresa Camacho-Badani
made a review of the literature that helped to determine the type locality of H. fasciatus.
We thank Diego Páez-Moscoso, Diego Morales Briones, Mario Yánez-Muñoz and three
anonymous reviewers for providing helpful comments on the manuscript.
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Appendix
Examined specimens
Hyla leptoscelis. BRAZIL: AMAZONAS: Lago do Tachy, above São Paulo Olivença,
Rio Solimöes (BMNH 1947.2.23.10, holotype).
Hyla steinbachi. BOLIVIA: PROVINCIA SARA: Departamento Santa Cruz de la Si-
erra (BMNH 1947.2.13.61–63, syntypes).
Hypsiboas calcaratus. ECUADOR: PROVINCIA ORELLANA: Río Napo, San Vi-
cente (0.6790°S, 75.6511°W), 203 m (QCAZ 44529–30); Río Napo, Chiroisla
(0.5756°S, 75.8998°W), 203 m (QCAZ 44422); Río Napo, Añangu (0.5249°S,
76.3844°W), 255 m (QCAZ 43933–36, 43979); Río Napo, Edén (0.4983°S,
76.0711°W), 216 m (QCAZ 44176–79, 44225, 44246–47, 44286); Estación
Científica Tiputini, Universidad San Francisco de Quito (0.6387°S, 76.1492°W),
230 m (QCAZ 12343–44); Río Rumiyacu (0.89566°S, 75.94783°W), 250 m
(QCAZ 20547–51); Parque Nacional Yasuní, between km 80 and km 75, on the
road Pompeya-Iro (0.8401°S, 76.3024°W), 243 m (QCAZ 43057, 43060, 43062–
63); Pompeya-Iro road, 38.8 km from Pompeya (0.6535°S, 76.4535°W), 237 m
(QCAZ 8201); Parque Nacional Yasuní, km 9 Pompeya (0.4598°S, 76.5931°W),
253–271 m (QCAZ 43034–35, 43038, 43046–47); Estación Científica Yasuní,
Universidad Católica del Ecuador (0.6713°S, 76.4005°W), 230–250 m (QCAZ
8210, 8812, 11912–13, 12386, 14815–17, 16792–97, 16798–99, 17825,
18264, 19202, 19204–05, 20290, 20305, 20837, 22488–89, 22561–66, 22866,
22988, 23038–39, 23065–69, 23848–54, 24214, 24282, 31445–46, 36869–
78, 40985–86, 41005–06, 41015, 43242–48, 43256–59, 49205); Río Napo,
sector La Primavera (0.4442°S, 76.7868°W), 244 m (QCAZ 43824); El Coca
(0.4778°S, 76.9898°W), 257 m (QCAZ 43713, 43789); La Belleza, Comuni-
dad Bocana del Suno (0.6922°S, 77.1353°W), 309 m (QCAZ 33522, 33524);
Aguarico, confluence of the Yasuní and Jatuncocha rivers (0.9836°S, 75.4183°W),
200 m (EPN 5085–86); Aguarico, Ishpingo II oil well (1.0947°S, 75.6494°W),
178 m (EPN 3865); Loreto, San José Daguano (0.8255°S, 77.4347°W), 450 m
(EPN 5674); San Luis de Armenia (0.4822°S, 77.0683°W), 300 m (EPN 11739);
PROVINCIA SUCUMBÍOS: Cuyabeno, Caña de Canangüeno (0.3990°S,
75.8753°W), 222 m (QCAZ 11924); Reserva Faunística Cuyabeno (0.08498°N,
76.13444°W), 273 m (QCAZ 2034, 2046–49, 2242, 4612); Laguna de Mate-
ococha (0.01846°N, 76.22155°W), 220 m (QCAZ 26062); Puerto Bolívar
(0.0886°S, 76.1420°W), 240 m (QCAZ 28181, 28185, 28197); La Selva Lodge
(0.4981°S, 76.3738°W), 245 m (QCAZ 4333, 25419, 25434); La Selva Lodge,
Mandi Cocha (0.41666°S, 76.1333°W), 250 m (QCAZ 11540–41, 11545,
12005–07, 12009); Reserva Limoncocha (0.4062°S, 76.6194°W), 261 m (QCAZ
43100, 43131,43268); Shushufindi (0.0331°S, 76.6535°W), 270 m (QCAZ
15188); Zancudococha (QCAZ 4535); Tarapoa (QCAZ 23095); Cofán Duvuno
community (0.0333°S, 77.1166°W), 340 m (EPN 4917); PROVINCIA NAPO:
Reserva Yachana (0.8333°S, 77.1666°W), 300 m (QCAZ 48833); Comunidad

Santa Rosa, on the road to Tena (0.9895°S, 77.4412°W), 341–439 m (QCAZ
40055–56, 40083); Tena, on the road to Jatun Sacha (1.0449°S, 77.76951°W),
428 m (QCAZ 18173, 40084–85); on the road Hollín-Loreto (QCAZ 649);
La Selva (QCAZ 7436–40, 7443); PROVINCIA PASTAZA: Canelos Sacha
(1.5822°S, 77.7155°W), 500–650 m (QCAZ 14956–75, 48712, 48718–20);
Bataburo Lodge (1.2083°S, 76.7166°W), 220 m (QCAZ 39442); Kapawi Lodge
(2.5386°S, 76.8583°W), 239 m (QCAZ 9018, 25513–14); Misión (2.2143°S,
76.5142°W), 240 m (EPN 885); Montalvo (2.0666°S, 76.9666°W), 305 m (EPN
884); Shionayacu (2.1001°S, 76.6334°W), 360 m (EPN 886); FRENCH GUI-
ANA: Kaw (4.7166°N, 52.1333°W), 3 m (105PG); Cayenne, Crique Arataye
(4°N, 52.66°W), 70 m (USNM 247780); Lac Toponow (3.0527°N, 52.7102°W),
152 m (123PG); Trinite (4.5833°N, 53.35°W), 290 m (192BM); Crique Margot
(5.4666°N, 53.95°W), 44 m (131MC); GUYANA: Rupununi, Iwokrama For-
est Reserve, 5 hrs S (downstream) of Kurupukari (4.285°N, 58.50944°W), 99
m (USNM 531370, 531372); BRAZIL: RONDONIA: Nova Brasilia (11.15°S,
61.566 °W), 431 m (USNM 304048); PERU: REGIÓN MADRE DE DIOS:
Cuzco Amazónoico, 15 km E Puerto Maldonado (12.583°S, 69.083°W), 200 m
(MUSM 14456, 14547, KU 215205); Río Tambopata, W bank of Zona Reser-
vada Tambopata-Candamo (13.1416°S, 69.6066°W), 211 m (USNM 332415);
Pakiza, Manu National Park (11.866°S, 71.3°W), 360 m (USNM 345216–17);
REGIÓN LORETO: Río Lagarto Cocha, Aguas Negras (0.5230°S, 75.2569°W),
183 m (USNM 520924).
Hypsiboas fasciatus. ECUADOR: Andes of Ecuador (BMNH 58.4.25.22; holotype);
PROVINCIA MORONA SANTIAGO: 7.9 km N Limón, on the road Limón-
Macas (2.8858°S, 78.3970°W), 1571 m (QCAZ 26496–97); Comunidad Nueva
Principal, 3 km near to the town (3.1686°S, 78.3870°W), 1059 m (QCAZ 48611,
48628); Tiink, Río Zamora (3.3330°S, 78.4518°W), 730 m (QCAZ 17016,
17123, 17128, 17030–31, 17040–41, 17148); Comunidad San Luis (3.3420°S,
78.4677°W), 792 m (QCAZ 48633, 48636, 48639, 48670–71); La Pradera, on
the road to Gualaquiza (3.3365°S, 78.6504°W), 1015–1036 m (QCAZ 18270–71,
48584–93); road to Río Abanico Jimbitano, 1000 m (QCAZ 21188–89); road be-
tween Gualaquiza and Limón, 3 km from Limón (3.3990°S, 78.5786°W), 1140 m
(QCAZ 27610); San Juan Bosco, Cóndor Mirador (3.5192°S, 78.4314°W), 1100
m (EPN 14060–65); 3 km Gualaquiza-Limón (3.3700°S, 78.5680°W), 1140 m
(QCAZ 21707, 21957, 33976); PROVINCIA ZAMORA-CHINCHIPE: Gravel
road East to Sarsa (3.8078°S, 78.6059°W), 1500 (QCAZ 47051); Piuntza, Finca
Don Mesías (3.8564°S, 78.8646°W), 1154–1192 m (QCAZ 40797, 40802); near
to Zamora (4.0491°S, 78.9256°W), 927 m (QCAZ 27258); Zamora (4.0669°S,
78.9508°W), 1060 m (QCAZ 23144, 23147–49, 48583); Bombuscaro (4.0893°S,
78.9609°W), 1091 m (QCAZ 12444, 12446, 12448, 12452, 12494–97); La
Pituca (4.1461°S, 78.9446°W), 1343 m (QCAZ 24650); Romerillos Alto, 26 km
of Zamora (4.1850°S, 78.9352°W), 1300–1593 m (QCAZ 24866, 47070); Las
Orquidias, around the town (4.2290°S, 78.6577°W), 874 m (QCAZ 41488); Mi-
azi Alto, “Tepuy” camp (4.2562°S, 78.6222°W), 1250–1300 m (QCAZ 41575–
76, 41659, 41518); Destacamento Militar Miasi (4.2833°S, 78.6333°W), 800 m
(EPN 4112–13, 4115–16, 4118); Paquisha, Blanco river (3.8945°S, 78.5161°W),
1630 m (EPN 14218); Yantzaza, Colibrí Goldmarca (3.7665°S, 78.5055°W),
1377 m (EPN 12672, 12674, 13288, 13290–92); Yantzaza, Concesión Colibrí
(3.7588°S, 78.5009°W), 1424 m (EPN 11387–91, 12316); Yantzaza, Sachavaca
sector (3.7042°S, 78.4827°W), 1280 m (EPN 14216–17); Yantzaza, road to Pin-
dal (3.7522°S, 78.5528°W), 1224 m (EPN 13677); Centro Shuar Yawi (4.4301°S,
78.6489°W), 945 m (QCAZ 31037–40); near to Tundaime (QCAZ 16466);
PERU: REGIÓN AMAZONAS: Cordillera del Cóndor, Upper Río Comainas,
Puesto Vigilancia (4.1°S, 78.3833°W), 1100 m (USNM 525495, 525499).
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Systematics of treefrogs of the Hypsiboas calcaratus and Hypsiboas fasciatus

Article in ZooKeys · January 2014

Marcel Caminer Santiago Ron

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Systematics of treefrogs of the Hypsiboas calcaratus and

Marcel A. Caminer1,2,†, Santiago R. Ron1,‡

Corresponding author: Santiago R. Ron ([email protected])

Academic editor: F. Andreone | Received 23 September 2013 | Accepted 25 December 2013 | Published 15 January 2014

Management and conservation planning of biodiversity requires proper species iden-

Species SVL FOOT HL HW ED TD TL FL CL

Species SVL FOOT HL HW ED TD TL FL CL

Species SVL FOOT HL HW ED TD TL FL CL

Genbank Accession No.

USNM 174173 H. raniceps AY819375 -- -- -- -- Wiens et al. 2005

Genbank Accession No.

The phylogenetic relationships recovered from the analysis of the mitochondrial

Taxonomic status of H. fasciatus and H. calcaratus

Clade A Clade B Clade C Clade D Clade E Clade F

The holotype of H. calcaratus could not be examined because it is lost (Duellman

Taxonomic status of Hyla leptoscelis and Hyla steinbachi

SD = 0.61, in H. almendarizae sp. n. vs. 7.89%, SD = 1.2, in H. calcaratus; differences

Characters H. alfaroi H. almendarizae H. calcaratus H. fasciatus H. maculateralis H. tetete

Dominant frequency at the end 2058.93 ± 33.98 1962.32 ± 190.41

Hypsiboas fasciatus (Günther, 1858)

Hypsiboas almendarizae sp. n.

Common name. English: Almendariz’s treefrog; Spanish: Rana arbórea de Almendáriz

Referred specimens. ECUADOR: PROVINCIA MORONA SANTIAGO: 2.2

Hypsiboas maculateralis sp. n.

Common name. English: Stained treefrog; Spanish: Rana arbórea manchada

veniles, collected by S. R. Ron, E. Toral and I. G. Tapia on 27 June 2009; Guiyero

Variation. Variation in dorsal and ventral coloration of preserved specimens is

Hypsiboas alfaroi sp. n.

Common name. English: Alfaro’s treefrog; Spanish: Rana arbórea de Alfaro

to Bolívar (0.0886°S, 76.1420°W), 240 m (QCAZ 27815, 28190, 28211, 28230,

Distribution and ecology. Hypsiboas alfaroi occurs in the Ecuadorian north-

Hypsiboas tetete sp. n.

Color of holotype in preservative. Dorsum grayish brown with scattered minute

Vegetation type at the Peruvian locality is Napo Moist Forest.

Morphometric comparisons between species

PCA Males PCA Females

Call comparisons between species

Implications of the discovery of hidden species richness in our current under-

Reserva Yachana (0.8333°S, 77.1666°W), 300 m (QCAZ 48833); Comunidad

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