Zoological Journal of the Linnean Society, 2020, XX, 1–32. With 13 figures.
MARCEL A. CAMINER1,2, and SANTIAGO R. RON1*,
Museo de Zoología, Facultad de Ciencias Exactas y Naturales, Pontificia Universidad Católica del
Ecuador, Av. 12 de Octubre y Roca, Aptdo. 17-01-2184, Quito, Ecuador
2
Institute of Organismic and Molecular Evolution, Johannes Gutenberg University Mainz, Hanns-DieterHüsch-Weg 15, 55128 Mainz, Germany
1
Received 17 April 2019; revised 13 December 2019; accepted for publication 8 January 2020
The combination of genetic and phenotypic characters for species delimitation has allowed the discovery of many
undescribed species of Neotropical amphibians. In this study, we used DNA sequences (genes 12S, 16S, ND1 and
COI) and morphologic, bioacoustic and environmental characters of the Boana semilineata group to evaluate their
phylogenetic relationships and assess their species limits. In addition, we included DNA sequences of several species
of Boana to explore cryptic diversity in other groups. We found three Confirmed Candidate Species (CCS) within the
B. semilineata group. Holotype examination of Hyla appendiculata shows that it is a valid species that corresponds to
one of the CCS, which is here transferred to Boana. We describe the two remaining CCS. Our phylogeny highlights a
number of secondary but meaningful observations that deserve further investigation: (1) populations of B. pellucens
from northern Ecuador are more closely related to B. rufitela from Panama than to other Ecuadorian populations of
B. pellucens; (2) we report, for the first time, the phylogenetic relationships of B. rubracyla showing that it is closely
related to B. rufitela and B. pellucens; and (3) B. cinerascens and B. punctata form two species complexes consisting of
several unnamed highly divergent lineages. Each of these lineages likely represents an undescribed species.
ADDITIONAL KEYWORDS: Amazon – amphibian – candidate species – cryptic diversity – phylogeny – taxonomy.
INTRODUCTION
The genus Boana Gray, 1825 is distributed in the
Neotropical region from Nicaragua to Argentina
and Trinidad and Tobago. Boana is the second most
species-rich genus of the family Hylidae, containing
93 recognized species (Frost, 2019) divided into
seven species groups (Faivovich et al., 2005): B.
alopunctata, B. benitezi, B. faber, B. pellucens,
B. pulchella, B. punctata and B. semilineata. A putative
synapomorphy for Boana is the presence of a prepollical
spine (Faivovich et al., 2005; but see: Peloso et al.,
2018). Recent integrative systematic reviews of small
clades of Amazonian Boana (e.g. Caminer & Ron, 2014;
*Corresponding author. E-mail: santiago.r.ron@gmail.com
[ Ve r s i o n o f r e c o r d , p u b l i s h e d o n l i n e 2 1 M a r c h
2020; http://zoobank.org/ urn:lsid:zoobank.
org:pub:AD80E63E-EF0E-42FB-8AE3-47B705CA4454]
Fouquet et al., 2016; Peloso et al., 2018) have shown
that undescribed cryptic species can even outnumber
described species. These results highlight the need for
systematic reviews of Boana based on genetic data.
One clade that has been the subject of recent
systematic reviews based on molecular phylogenies
is the B. semilineata species group (Fouquet et al.,
2016; Peloso et al., 2018). It occurs in the Atlantic
Forests, the Amazon, the Chocó region of Colombia and
Ecuador, and in eastern Panama (Fouquet et al., 2016).
The group contains nine nominal species: B. boans
(Linnaeus, 1758), B. diabolica (Fouquet et al., 2016),
B. geographica (Spix, 1824), B. hutchinsi (Pyburn &
Hall, 1984), B. icamiaba Peloso et al., 2018, B. pombali
(Caramaschi et al., 2004), B. secedens (Lutz, 1963),
B. semilineata (Spix, 1824) and B. wavrini (Parker,
1936). One putative morphological synapomorphy for
the group is the presence of a reticulated lower lid
(Faivovich et al., 2005). A species with a challenging
© 2020 The Linnean Society of London, Zoological Journal of the Linnean Society, 2020, XX, 1–32
1
Downloaded from https://academic.oup.com/zoolinnean/advance-article-abstract/doi/10.1093/zoolinnean/zlaa002/5810752 by University of Rochester user on 22 March 2020
Systematics of the Boana semilineata species group
(Anura: Hylidae), with a description of two new species
from Amazonian Ecuador
2
M. A. CAMINER and S. R. RON
2016). The only species left with a trans-Andean
distribution in lowland rainforests is B. boans. Genetic
and morphological analyses of populations on both
sides of the Andes are needed to determine whether
they represent two or one species.
Here, we present a new phylogeny of Boana,
including all Ecuadorian species of the genus, some of
which had unknown phylogenetic relationships. The
main aim of our study is to delimit species boundaries
among populations of the B. semilineata species group
by combining genetic, morphologic, environmental
and bioacoustic characters. In addition, we explore
the existence of candidate species in other species
groups of Boana by adding genetic characters from
additional populations. Finally, we determine whether
populations from opposite sides of the Andes of
B. boans belong to a single or more species. We predict
that populations on opposite sides of the Andes have
high genetic divergence as a result of the isolating
effect of the Andes.
MATERIAL AND METHODS
DNA extraction, sequencing and analyses
We selected available mitochondrial sequences in
GenBank (until October 2018) attributed to 63 of the
93 species formally described for the genus Boana
(Frost, 2019). We also included sequences from 118
individuals of Boana from Ecuador (Supporting
Information, Table S1). Total genomic DNA was
extracted from muscle or liver tissue preserved in
95% ethanol or tissue storage buffer using standard
phenol-chloroform extraction protocols (Sambrook
et al., 1989). We amplified the mitochondrial genes
12S rRNA, 16S rRNA, NADH dehydrogenase 1 (ND1)
and cytochrome c oxidase I (COI). PCR amplification
was carried out using standard protocols and primers
are presented in Supporting Information, Table S2.
PCR products were cleaned using Exo I/SAP digest
and sequenced in both directions by Macrogen Co. Ltd
(South Korea). Sequences were edited and aligned
with GENEIOUS v.5.4.4 software (GeneMatters Corp.)
using the GENEIOUS alignment algorithm. Leading
and trailing ends were trimmed manually to remove
low-quality sequences. The matrix was imported into
MESQUITE (v.3.04; Maddison & Maddison, 2015) and
the alignment was checked and manually revised. The
final combined DNA matrix contained sequence data
for: 159 samples for 12S (923 bp, 5% of missing data),
290 for 16S (1767 bp, 56.9%), 136 for ND1 (1072 bp,
4.8%), 138 for COI (670 bp, 1.4%) and 44 for cytochrome
b (385 bp, 2.6%).
Because it is likely that variation in each of our
sampled genes (or codon positions within proteincoding genes) were shaped by different evolutionary
© 2020 The Linnean Society of London, Zoological Journal of the Linnean Society, 2020, XX, 1–32
Downloaded from https://academic.oup.com/zoolinnean/advance-article-abstract/doi/10.1093/zoolinnean/zlaa002/5810752 by University of Rochester user on 22 March 2020
taxonomy in this group is Boana geographica. Its
distribution range has ~10.4 million km 2, the 13 th
largest among the ~8000 described amphibian
species (Pimm et al., 2014). Duellman (1973) reported
significant geographic variation in life coloration
and advertisement call, suggesting the existence
of cryptic species. Fouquet et al. (2007) and Jansen
et al. (2011) reported deeply divergent lineages
within B. geographica, providing additional evidence
of the existence of cryptic species. More recently,
Fouquet et al. (2016) and Peloso et al. (2018) carried
out systematic reviews based on morphological,
genetic and bioacoustic evidence that resulted in the
identification of seven unnamed candidate species
and the description of B. diabolica and B. icamiaba.
The unnamed candidate species belong to a clade that
includes B. diabolica, B. geographica, B. hutchinsi
and B. semilineata. A putative synapomorphy for this
clade is the absence of an enlarged prepollex forming a
projecting spine (Peloso et al., 2018).
The reviews of Fouquet et al. (2016) and Peloso et al.
(2018) are significant advances to solve the taxonomy
of B. geographica. However, those studies only
include one genetic sample of ‘B. geographica’ from
Ecuador. We have found morphological differences
between sympatric individuals of B. geographica in
the Ecuadorian Amazon that suggest the existence
of at least two cryptic species there (Ron & Read,
2018). Therefore, a thorough review of Ecuadorian
populations of the B. semilineata group is needed.
An enigmatic member of the Boana semilineata
species group is B. boans. This large hylid is unique
among neotropical amphibians, because of its
geographic distribution. Unlike other amphibians
inhabiting lowland Amazonian rainforests, B. boans
also occurs in lowland Chocoan rainforests, on the
other side of the Andes (La Marca et al., 2010; Ron
et al., 2019). This distribution is unexpected, because
the Andes appear to be an impassable barrier for
lowland amphibians. Only three other amphibians
were thought to be shared between lowland
rainforests on opposite sides of the Andes: Rhinella
marina (Linnaeus, 1758), R. margaritifera (Laurenti,
1768) and Trachycephalus typhonius (Laurenti, 1768).
Although a few anuran species are shared across the
Merida Cordillera in Venezuela [e.g. Boana pugnax
(Schmidt, 1857), B. xerophylla (Duméril & Bibron,
1841); Escalona et al., 2019], their populations
likely connect through adjacent dry lowlands along
the Andes. Systematic reviews of Rhinella marina,
R. margaritifera and Trachycephalus typhonius have
shown that their shared distribution across the Andes
was an artefact of incorrect delimitation of species
boundaries because Chocoan populations turned out
to be distinct species from those in the Amazon (dos
Santos et al., 2015; Acevedo et al., 2016; Ron et al.,
SYSTEMATICS OF BOANA SEMILINEATA GROUP
Morphological analyses
Our morphological analyses focus on Ecuadorian
populations of the Boana semilineata group. The
specimens examined for the description of the new
species (listed in the type-series and Appendix), were
fixed in 10% formalin, preserved in 70% ethanol and
deposited in the collections of the Museo de Zoología
at Pontificia Universidad Católica del Ecuador (QCAZ)
and the Herpetology Division at the Ecuadorian
Instituto Nacional de Biodiversidad, INABIO
(DHMECN, Quito, Ecuador). We also examined the
type material of Hyla appendiculata Boulenger,
1882 (lectotype BMNH 1947.2.13.1) deposited at the
Natural History Museum (NHM, London, UK) and
specimens identified as Boana from the herpetological
collection of Universidad de los Andes (ANDES,
Bogotá, Colombia).
To evaluate morphometric variation, we measured
133 specimens of ‘Boana geographica’ from Ecuador
with digital callipers (± 0.01 mm) according to the
methodology described in Duellman (1970, 1973).
Webbing formulae of hands and feet follow Savage &
Heyer (1967) as modified by Myers & Duellman (1982).
Acronyms for morphological measurements are: SVL
(snout–vent length), HL (head length), HW (head
width), ED (eye diameter), TD (tympanum diameter),
TL (tibia length), FEL (femur length), FL (foot length)
and CL (calcar length). Sex was determined by gonadal
examination or by the presence of vocal sac folds and
nuptial excrescences in males.
We also examined colour and patterning differences
between the new species and their closest relatives.
In the Diagnosis sections, coloration always refers
to preserved specimens unless otherwise stated.
Eleven qualitative morphological characters were
evaluated: (1) dorsal skin texture (i, smooth; ii,
shagreen; iii, finely granular); (2) dorsal coloration
(X-shaped mark on scapular region: i, present; ii,
absent); (3) marks on flanks and hidden surfaces of
thighs (i, dark vertical bars: ii, dark vertical stripes;
iii, uniform black; iv, speckled pattern); (4) ventral skin
texture (i, coarsely areolate; ii, areolate; iii, smooth);
(5) ventral coloration; (6) in life, webbing coloration;
(7) palpebrum reticulated (i, present; ii, absent); (8)
nuptial excrescences in males (i, present; ii, absent);
(9) projecting prepollices (i, present; ii, absent); (10)
tympanum (i, present; ii, absent); and (11) calcar
size and shape (i, small and triangular; ii, small and
tubercular; iii, absent). Coloration in life was obtained
from digital photographs.
Bioacoustic analyses
We analysed advertisement calls from five individuals
of ‘Boana geographica’ recorded at Yasuní (Provincia
Orellana), Sardinayacu (Provincia Morona Santiago)
and Miazi-Nuevo Paraíso (Provincia Zamora
Chinchipe) in Ecuador. Recordings were made with
a Sennheiser K6-ME67 directional microphone and
Olympus LS-10 and WS-802 digital recorders. Calls
© 2020 The Linnean Society of London, Zoological Journal of the Linnean Society, 2020, XX, 1–32
Downloaded from https://academic.oup.com/zoolinnean/advance-article-abstract/doi/10.1093/zoolinnean/zlaa002/5810752 by University of Rochester user on 22 March 2020
processes, we partitioned the data according to gene
and codon position to analyse each partition under
separate models of evolution. We used software
PartitionFinder v.1.1.1 (Lanfear et al., 2012) to
simultaneously estimate both the best-fit model for
each partition and the best-partition strategy for
our data. The phylogenetic relationships among the
species were inferred from the concatenated matrix of
the mitochondrial genes 12S rRNA, 16S rRNA, ND1,
COI and cytochrome b (Cytb). For the outgroup, we
added samples of Dendropsophus bifurcus (Andersson,
1945) and D. sarayacuensis (Shreve, 1935).
Phylogenetic analyses were conducted using
maximum likelihood (ML) and Bayesian inference
(BM). Bayesian phylogenetic analyses were carried
out in MrBayes v.3.2 (Ronquist et al., 2012). Each
Bayesian analysis consisted of four parallel runs of
the Metropolis coupled Markov chain Monte Carlo for
60 × 106 generations. Each run had four chains with
a temperature of 0.1. For each analysis, the chain
was sampled every 1000 generations. The first 25%
of sampled trees were discarded as burn-in and the
remaining trees were used to estimate the Bayesian
tree, posterior probabilities and other model parameters.
Convergence of the Markov chain Monte Carlo
simulations was assessed by checking if the average
standard deviation of split frequencies were < 0.05
between runs. Search convergence and stationarity was
visualized and effective using TRACER v.1.5 (Rambaut
& Drummond, 2007). TRACER was also used to obtain
estimate sample-size values (ESS) for parameter
estimates. We assumed that ESS values > 200 were
indicative of an adequate search.
Maximum likelihood analyses were conducted using
GARLI v.2.0 (Genetic Algorithm for Rapid Likelihood
Inference; Zwickl, 2006) under default settings.
Analysis was terminated after 500 000 generations
without topology improvement. We ran a total of 20
independent searches and used random starting trees
(streefname = random) to reduce the probability of
inferring a suboptimal likelihood solution. Likelihood
values of the 20 searches differed by less than 1.0
likelihood units between each other, indicating that
all searches converged on similar optimal trees. Nodal
support was obtained using 100 bootstrap replicates.
MESQUITE (Maddison & Maddison, 2015) was used
to generate a majority-rule consensus tree from the
bootstrap replicates. Nodes corresponding to partitions
present in less than 50% of the bootstrap replicates
were collapsed.
3
4
M. A. CAMINER and S. R. RON
Environmental analyses
We considered environmental differentiation as
additional evidence to define species boundaries (e.g.
Rissler & Apodaca, 2007; Aguilar-Puntriano et al.,
2016). We obtained bioclimatic values at localities of the
B. semilineata species group using software QGIS v.3.4
(QGIS Development Team, 2018). Rasters of bioclimatic
variables, with 1 km resolution, were downloaded
from WorldClim v.2.0 (http://www.worldclim.org; Fick
& Hijmans, 2017). The variables were: (1) Annual
Mean Temperature, (2) Mean Diurnal Range, (3)
Isothermality, (4) Temperature Seasonality (standard
deviation *100), (5) Max. Temperature of Warmest
Month, (6) Min. Temperature of Coldest Month, (7)
Temperature Annual Range, (8) Mean Temperature
Table 1. Character loadings, eigenvalues, and percentage
of explained variance for Principal Components (PC) I–II.
The analysis was based on eight acoustic variables from
the advertisement calls of Boana appendiculata, B. nigra
and B. ventrimaculata. Bold figures indicate highest
loadings
Variable
PC I
PC II
Call duration
Number of notes per call
Rise time of the call
Pulses per note
Pulse rate
Note duration
Dominant frequency call
Frequency bandwidth
Eigenvalue
%
0.86
0.94
0.65
–0.2
0.24
–0.40
0.77
0.84
3.64
45.58
0.40
–0.17
0.71
0.90
–0.84
0.89
–0.26
0.36
3.23
40.46
of Wettest Quarter, (9) Mean Temperature of Driest
Quarter, (10) Mean Temperature of Warmest Quarter,
(11) Mean Temperature of Coldest Quarter, (12) Annual
Precipitation, (13) Precipitation of Wettest Month,
(14) Precipitation of Driest Month, (15) Precipitation
Seasonality (Coefficient of Variation), (16) Precipitation
of Wettest Quarter, (17) Precipitation of Driest
Quarter, (18) Precipitation of Warmest Quarter and
(19) Precipitation of Coldest Quarter. To avoid spatial
autocorrelation, we reduced the number of localities
using a buffer with a 5-km radius around each point.
If two or more localities of the same species were at a
distance < 10 km from each other, we randomly chose
one of them. We obtained a total of 88 localities from
individuals sequenced or morphologically identified
as belonging to the B. semilineata species group.
Since species identification in the literature could be
incorrect, we only included geographic records based
on specimens examined by us. We used Principal
Component Analysis (PCA) to examine environmental
differentiation between candidate species using
software JMP v.9.0.1 (SAS Institute, 2010).
Species limits
Species limits were evaluated under the framework
of integrative taxonomy (Dayrat, 2005; Vieites et al.,
2009). We identified undescribed species by using
the categories defined by Vieites et al. (2009). During
the first stage, we partitioned genetic lineages into
candidate species using two criteria. First, we used
a Poisson Tree Processes (PTP) model (Zhang et al.,
2013) to infer the most likely number of species within
the Boana geographica–B. semilineata complex in our
dataset, as implemented in the bPTP server (http://
species.h-its.org/ptp/). The input tree was obtained
© 2020 The Linnean Society of London, Zoological Journal of the Linnean Society, 2020, XX, 1–32
Downloaded from https://academic.oup.com/zoolinnean/advance-article-abstract/doi/10.1093/zoolinnean/zlaa002/5810752 by University of Rochester user on 22 March 2020
of B. semilineata recorded by Axel Kwet from Brusque
(Santa Catarina, Brazil) and B. geographica described
by Duellman & Pyles (1983) from Santa Cecilia
(Sucumbíos, Ecuador) were also analysed (downloaded
from Fonoteca Zoológica of MNCN, http://www.fonozoo.
com and the Macaulay Library at the Cornell Lab of
Ornithology, http://macaulaylibrary.org). Spectrograms
were generated with RAVEN v.1.5 software (2010)
using a Fast Fourier Transformation (FFT) from a
sample of 1024 points and a frequency resolution of
44.1 Hz. Recordings are deposited at the QCAZ audio
archive (available at the Anfibios del Ecuador website:
https://bioweb.bio/faunaweb/amphibiaweb/).
Ten acoustic parameters (modified from: Cocroft &
Ryan, 1995) were measured to describe the structure
of each call: (1) call duration = time from beginning
to end of the call, measured in the oscillogram; (2)
notes per call = number of notes per call; (3) note
duration = average time from beginning to end of a
note; (4) pulses per call = number of pulses per call;
(5) pulses per note = number of pulses per note; (6)
pulse rate = number of pulses repeated in a defined
period of time within a note; (7) interval between
notes = average time from ending of a note and
beginning of next note; (8) call rise time = time from
beginning of call to point of maximum amplitude; (9)
dominant frequency = frequency in call containing
the greatest amount of energy, measured along the
entire call; and (10) frequency bandwidth = difference
between the upper and lower frequencies measured
along the entire call. Several calls of each individual
were measured to calculate an average. The species
comparisons were based on a single value (average)
per male.
Eight call variables (listed in Table 1) were used to
run a Principal Components Analysis (PCA) to assess
acoustic differentiation between calls from candidate
species using software JMP v.9.0.1 (SAS Institute,
2010).
SYSTEMATICS OF BOANA SEMILINEATA GROUP
without character data to compare, were assigned to
the Unconfirmed Candidate Species category.
As a general reference we also obtained uncorrected
mean genetic distances using MEGA v.6.06 (Tamura
et al., 2011) for gene 16S, given that this is a reference
standard to identify candidate species (e.g. Fouquet
et al., 2007; Jansen et al., 2011; Caminer et al., 2017).
We only used sequences that overlapped by more than
330 bp to calculate the distances.
RESULTS
Phylogenetic relationships
The complete DNA sequence data matrix has 4925 bp
for 304 samples. PartitionFinder chose four partitions
as the best strategy (best model in parentheses):
12S, 16S, COI 3 rd , ND1 3 rd and Cytb 3 rd position
(GTR + I + G), COI 1st, ND1 1st and Cytb 1st position
(SYM + I + G), COI 2nd position (F81), ND1 2 nd and
Cytb 2 nd position (HKY + I + G). The tree topologies for
the maximum likelihood and Bayesian phylogenies are
similar except for weakly supported nodes (posterior
probability, pp < 0.5 and bootstrap < 52).
The phylogeny shows strong support for the
B. semilineata species group (Fig. 1A, B).Within it, a wellsupported clade includes all species except B. pombali
and B. icamiaba. The next most-inclusive clade is
composed of all species except B. boans. We also found
strong support for the B. geographica–B. semilineata
clade (sensu Peloso et al., 2018). Hereafter we refer to it
as the B. geographica–B. semilineata species complex.
This clade also includes ‘B. hutchinsi’, B. diabolica and
several undescribed species. Their status is assessed
in the ‘Identification of candidate species’ section.
Surprisingly, populations of B. boans on both sides
of the Andes are separated by short branches and low
genetic distances. Uncorrected-p distances for gene
16S range from 0.7 to 0.9% (Chocó vs. Amazonian
Ecuador). Chocoan populations form a well-supported
clade. Samples from Amazonian Ecuador also form
a clade, sister to Chocoan samples. Samples from
Amazonian Brazil, Colombia, Peru and the Guianas
also show short branches and weak phylogenetic
structure.
Regarding other species groups of Boana, our
phylogeny (Fig. 1C) is consistent with the strongly
supported species groups found by Duellman et al.
(2016). A notable difference occurs in the Boana
calcarata species complex, which shows a clade
containing B. almendarizae (Caminer & Ron, 2014),
B. calcarata (Troschel, 1848), B. fasciata (Günther,
1858) and B. maculateralis (Caminer & Ron, 2014).
However, the Duellman et al. (2016) tree shows
B. fasciata and B. maculateralis more closely related to
© 2020 The Linnean Society of London, Zoological Journal of the Linnean Society, 2020, XX, 1–32
Downloaded from https://academic.oup.com/zoolinnean/advance-article-abstract/doi/10.1093/zoolinnean/zlaa002/5810752 by University of Rochester user on 22 March 2020
from the 16S gene matrix alignment using maximum
likelihood. We ran 100 000 MCMC generations, with a
thinning value of 100 and a burn-in of 10%.
Second, we partitioned genetic lineages into
candidate species using the Automatic Barcode Gap
Discovery (ABGD) method (available at https://bioinfo.
mnhn.fr/abi/public/abgd; Puillandre et al., 2012).
This distance-based approach identifies a barcode
gap in the frequency distribution of pairwise genetic
distances among samples. Based on that gap, it
proposes candidate species among samples. ABGD has
shown good performance to identify candidate species
compared to other DNA barcode methods (Paz &
Crawford, 2012). The analyses were based on the 16S
gene matrix, Kimura two-parameter distances, a prior
for the maximum value of intraspecific divergence
from 0.001 to 0.05, ten recursive steps and a gap width
of 0.5. A consensus between the candidate species
found by bPTP and ABGD was obtained by applying
a conservative criterion to minimize type I errors
(e.g. one species incorrectly split into two or more). In
case of disagreements between bPTP and ABGD, we
chose the alternative, implying the lower number of
candidate species.
On a second stage, we assigned the candidate species
to two mutually exclusive categories: (1) Unconfirmed
Candidate Species (UCS) and (2) Confirmed Candidate
Species (CCS; Vieites et al. 2009). Confirmed Candidate
Species were those showing species-level differences
in adult morphological, bioacoustic or environmental
characters when compared to its most closely related
species. We assumed that species-level differences in
morphology were: (1) fixed differences in qualitative
morphological characters between sister species or (2)
quantitative morphological differences between sister
species. For both categories we evaluated morphological
characters of known diagnostic value in Boana. For
comparisons of bioacoustic characters we applied
Kohler et al. (2017) criteria to define species limits.
Specifically, we assumed that qualitative call differences
(Kohler et al., 2017: 46) or quantitative differences
in note duration, dominant frequency or pulse rate,
which are useful to assess species limits (Kohler et al.,
2017: 51), were indicative of species-level differences.
We also compared environmental envelopes between
sister species (see ‘Environmental analyses’ section) as
additional evidence of species distinctiveness. Finally,
the existence of sympatric regions between candidate
species was also considered evidence of lineage
independence. This is because deep genetic divergence
in sympatry is indicative of lack of gene flow and the
existence of non-geographic reproductive barriers (and,
therefore, lineage independence).
Candidate species not showing those differences
(morphological, bioacoustical or environmental), or
5
6
M. A. CAMINER and S. R. RON
B. tetete and B. alfaroi. Our topology for those species
is the same as that reported by Caminer & Ron (2014).
The Boana pellucens species group has strong
support and includes samples of B. rubracyla
(Cochran & Goin, 1970), which are sister to the clade
B. rufitela + B. pellucens (pp = 1 and bootstrap = 100).
Populations from Durango and Lita, Ecuador, of
‘B. pellucens’ are more closely related to B. rufitela
© 2020 The Linnean Society of London, Zoological Journal of the Linnean Society, 2020, XX, 1–32
Downloaded from https://academic.oup.com/zoolinnean/advance-article-abstract/doi/10.1093/zoolinnean/zlaa002/5810752 by University of Rochester user on 22 March 2020
Figure 1. A, Maximum likelihood phylogram depicting relationships within Boana. The phylogram was derived from
analysis of 4916 bp of mitochondrial DNA (gene fragments 12S, 16S, ND1, COI and Cytb). Museum catalogue numbers
and localities are shown for each sample. Bootstrap values are above the branches and Bayesian posterior probabilities are
below; asterisks indicate values > 99 (bootstrap) or > 0.99 (posterior probability); missing values indicate values < 50 or 0.5.
Outgroup species are not shown. Abbreviations are: AR = Argentina, BO = Bolivia, BR = Brazil, CO = Colombia, COS = Costa
Rica, EC = Ecuador, FG = French Guiana, GU = Guyana, PA = Panama, PE = Peru, SU = Suriname, VE = Venezuela. Vertical
bars in the B. semilineata group indicate species partitions inferred using genetic (bPTP and ABGD), morphologic, and
bioacoustic criteria. Horizontal grey boxes in 1c indicate uncorrected genetic distances > 2% for the mitochondrial gene 16S.
See text for details.
SYSTEMATICS OF BOANA SEMILINEATA GROUP
7
than to other populations of B. pellucens (pp = 1 and
bootstrap = 99; Fig. 1C).
Identification of candidate species
The bPTP analysis recovered 17 species (Figs 1A, B;
Supporting Information, Fig. S1). The ABGD method
recovered 14 species across a wide range of priors for
maximal intraspecific thresholds (0.024–0.088; Figs 1A,
1B and Supporting Information, Fig. S2). The results
of both methods are highly similar except for ABGD
lumping into a single species, four species detected in
bPTP (part of the B. semilineata species complex in
Fig. 1B). Based on our conservative criterion, we chose
the 14 species partition for subsequent analyses. Of
the 14 species, six correspond to B. boans, B. diabolica,
B. geographica, B. icamiaba, B. semilineata and
B. wavrini; its assignment has been documented before
(Fouquet et al., 2016; Peloso et al., 2018). Boana pombali
is of uncertain assignment, because our bPTP and
© 2020 The Linnean Society of London, Zoological Journal of the Linnean Society, 2020, XX, 1–32
Downloaded from https://academic.oup.com/zoolinnean/advance-article-abstract/doi/10.1093/zoolinnean/zlaa002/5810752 by University of Rochester user on 22 March 2020
Figure 1. B, Continued.
8
M. A. CAMINER and S. R. RON
ABGD analyses identified two candidate species within
populations identified as ‘B. pombali’. Resolution of the
status of those populations falls outside the scope of
our review and we leave it unresolved.
Therefore, six candidate species could be undescribed
(clades A, B, C, D, E and F in Fig. 1A, B). Morphological,
acoustic and environmental data are available for
clades A, B and D, which allows us to assign them to the
© 2020 The Linnean Society of London, Zoological Journal of the Linnean Society, 2020, XX, 1–32
Downloaded from https://academic.oup.com/zoolinnean/advance-article-abstract/doi/10.1093/zoolinnean/zlaa002/5810752 by University of Rochester user on 22 March 2020
Figure 1. C, Continued.
SYSTEMATICS OF BOANA SEMILINEATA GROUP
Morphometric comparisons
The following comparisons pertain to male SVL. There
are significant differences between the three species.
Boana ventrimaculata sp. nov. (clade D) is larger than
B. appendiculata (clade A) and B. nigra sp. nov. (clade
B) (all P values for t tests < 0.001), while B. nigra
sp. nov. is smaller than B. appendiculata (t = 2.92,
d.f. = 56.4, P = 0.005).
Relative tympanum size of males and females
of B. ventrimaculata sp. nov. is larger than that of
B. appendiculata and B. nigra sp. nov. (all P values
for t tests < 0.001), while B. nigra sp. nov. presents a
smaller tympanum compared to B. appendiculata (all
P values for t tests < 0.03; comparisons were made on
the residuals of the regression between the tympanum
diameter and SVL).
In the DFA classification, most specimens are assigned
to the correct species. In B. appendiculata, 29 out of 35
males are correctly classified (four are misclassified as
B. nigra sp. nov. and two as B. ventrimaculata sp. nov.);
in B. nigra sp. nov., three out of 26 males are incorrectly
classified (all as B. appendiculata); in B. ventrimaculata sp.
nov., two out of 20 males are incorrectly classified (as
B. appendiculata) Among females, in B. appendiculata,
27 out of 30 specimens are correctly classified (three are
misclassified as B. nigra sp. nov.); in B. nigra sp. nov., all
five females are correctly classified; in B. ventrimaculata,
only one out of 16 females is incorrectly classified (as
B. nigra sp. nov.). Overall, the DFA show morphometric
differentiation among species, specially between
B. ventrimaculata sp. nov. and B. nigra sp. nov.
Bioacoustic comparisons
Our PCA of advertisement calls from six males result
in two PCs with eigenvalues > 1.0. These two PCs
Figure 2. Phylogenetic relationships within the Boana semilineata group showing the Confirmed Candidate Species (CCS)
and Unconfirmed Candidate Species (UCS).
© 2020 The Linnean Society of London, Zoological Journal of the Linnean Society, 2020, XX, 1–32
Downloaded from https://academic.oup.com/zoolinnean/advance-article-abstract/doi/10.1093/zoolinnean/zlaa002/5810752 by University of Rochester user on 22 March 2020
Confirmed Candidate Species category (Fig. 2). Clade
A is sister to clade B; they differ from each other in
environmental envelope and qualitative morphological
characters (see species descriptions for details). Clade
A corresponds to Hyla appendiculata Boulenger, 1882,
a binomen mistakenly considered a junior synonym of
B. geographica (see ‘Taxonomic review’ section). Clade
B is a new species that we describe below as Boana
nigra sp. nov.
Clade D is sister to a clade that includes B. semilineata
and Clade C. Calls of clade D and B. semilineata show
quantitative differences in note duration, dominant
frequency and pulse rate (ranges do not overlap
between species; Table 2). Although our sample size
was low (one male in B. ventrimaculata sp. nov. and
two males in B. semilineata), note duration, dominant
frequency and pulse rate are static variables and have
low intraspecific variation (Kohler et al., 2017).
Clades C, E and F are left as Unconfirmed Candidate
Species due to lack of data. The following sections show
the comparisons between B. appendiculata, B. nigra
sp. nov. and B. ventrimaculata sp. nov.
9
© 2020 The Linnean Society of London, Zoological Journal of the Linnean Society, 2020, XX, 1–32
2.07 ± 1.6 (1.22–3.92)
5.16 ± 1.04 (4–6)
0.17 ± 0.02 (0.15–0.17)
94 ± 26.8 (75–113)
20.5 ± 1.6 (18–22)
120.4 ± 18.2 (107.6–133.3)
0.16 ± 0.1 (0.1–0.27)
0.44 ± 0.13 (0.35–0.53)
1387.6 ± 65.7 (1341.2–1434)
1841.9 ± 46.8 (1808.8–1875)
0.45 ± 0.04 (0.43–0.51)
1
0.45 ± 0.04 (0.43–0.51)
33.9 ± 2.6 (31–37)
33.9 ± 2.6 (31–37)
75.8 ± 9.2 (62.5–78.7)
NA
0.34 ± 0.03 (0.32–0.39)
974.2 ± 183.3 (795.2–1205.6)
1316.2 ± 258.1 (1076.6–1645.2)
Type II
890.1 ± 65.8 (818.3–947.5)
0.25 ± 0.11 (0.15–0.22)
1044.2 ± 41.4 (996.6–1063.8)
87.9 ± 17.4 (76.3–108)
NA
28.3 ± 8.1 (21–37)
0.33 ± 0.11 (0.25–0.46)
1
0.33 ± 0.11 (0.25–0.46)
28.3 ± 8.1 (21–37)
B. nigra
(N = 1)
680.5 ± 93.4 (559.9–818.3)
0.08 ± 0.04 (0.02–0.11)
561.4 ± 79.1 (475.2–685.4)
57.5 ± 3.3 (53.5–61.9)
NA
13.8 ± 1.9 (11–16)
0.24 ± 0.03 (0.18–0.28)
1
0.24 ± 0.03 (0.18–0.28)
13.8 ± 1.9 (11–16)
B. ventrimaculata
(N = 1)
1068.1 ± 146.2 (964.6–1171.4)
0.14 ± 0.01 (0.13–0.15)
1240.3 ± 194.8 (1102.5–1378.1)
258.8 ± 30.1 (237.6–280.1)
0.04 ± 0.002 (0.04–0.05)
11.7 ± 0.6 (9–15)
0.14 ± 0.01 (0.13–0.15)
2.1 ± 0.14 (1–3)
0.04 ± 0.002 (0.04–0.05)
23.6 ± 0.28 (14–32)
B. semilineata
(N = 2)
Downloaded from https://academic.oup.com/zoolinnean/advance-article-abstract/doi/10.1093/zoolinnean/zlaa002/5810752 by University of Rochester user on 22 March 2020
Call duration (s)
Notes per call
Note duration (s)
Pulses number per
call
Pulses number per
note
Pulse rate
Interval between
notes (s)
Call rise time (s)
Dominant
frequency (Hz)
Frequency
bandwidth (Hz)
Type I
B. appendiculata
(N = 5)
Table 2. Descriptive statistics for calls parameters of members of the Boana semilineata group. Values for B. nigra and B. ventrimaculata were obtained from
five calls from a single male. Call vouchers: B. appendiculata (KU 143144–45, QCAZ 67113–34), B. nigra (DHMECN 12130) and B. ventrimaculata (QCAZ 49233).
The N values indicate the number of males analysed. If available, several calls of each individual were measured to calculate an average. When N > 1, descriptive
statistics are based on those averages only (one value per male); when N = 1, we report descriptive statistics for several calls of the same male. Mean ± SD is
given with range in parentheses. Calls of B. nigra, B. ventrimaculata and B. semilineata are of Type I only
10
M. A. CAMINER and S. R. RON
SYSTEMATICS OF BOANA SEMILINEATA GROUP
Environmental comparisons
Two components with eigenvalues > 1.0 are extracted
from the PCA for 88 localities. The two components
account for 81.2% of the total variation. The first
component (PC I) explains 41.9% of the total variation
and the second component (PC II) explains 39.3%. There
is partial segregation between B. nigra sp. nov. and the
other species along PC I but specially PC II, a component
Figure 3. Axes I and II from Principal Components Analysis. A and B: based of six size–corrected morphological variables for
81 males (A) and 52 females (B) of Boana. C: based on eight advertisement calls variables of B. appendiculata (four males), B.
nigra (1), B. ventrimaculata (1), and B. semilineata (2). The only calls type II of B. appendiculata are shown in hollow green
triangles. D: based on 19 bioclimatic variables for 88 localities. See Tables 1, 3, and 4 for character loadings on each component.
© 2020 The Linnean Society of London, Zoological Journal of the Linnean Society, 2020, XX, 1–32
Downloaded from https://academic.oup.com/zoolinnean/advance-article-abstract/doi/10.1093/zoolinnean/zlaa002/5810752 by University of Rochester user on 22 March 2020
account for 86.05% of the total variation. PC I (45.6%
of the variance) has high loadings on number of notes
and call duration; PC II (40.5% of the variance) has
high loadings on number of pulses per note and note
duration (Table 1). The acoustic space (as represented
by PC I and PC II) shows segregation among the
advertisement calls of the three species (Fig. 3C). We
also included calls of B. semilineata, which separate
from the other species along PC II.
11
12
M. A. CAMINER and S. R. RON
Variable
Foot length
Head length
Head width
Tympanum diameter
Tibia length
Femur length
Eigenvalue
%
PCA Males
PCA Females
PC I
PC II
PC I
PC II
0.31
–0.10
0.47
0.16
0.85
0.90
1.91
31.94
–0.74
0.43
0.37
0.82
0.02
0.01
1.56
26.06
0.39
0.31
0.68
0.13
0.89
0.83
2.14
37.41
0.41
–0.57
0.35
0.69
–0.05
–0.32
1.21
20.26
Table 4. Character loadings, eigenvalues and percentage
of explained variance for Principal Components (PC) I–
II of environmental variables. The analysis was based
on 88 localities of Boana appendiculata, B. nigra and
B. ventrimaculata. Bold figures indicate highest loadings
Variable
PC I
PC II
BIO1 = Annual Mean Temperature
BIO2 = Mean Diurnal Range
BIO3 = Isothermality (BIO2/BIO7) (* 100)
BIO4 = Temperature Seasonality (standard
deviation *100)
BIO5 = Max Temperature of Warmest Month
BIO6 = Min Temperature of Coldest Month
BIO7 = Temperature Annual Range
(BIO5–BIO6)
BIO8 = Mean Temperature of Wettest
Quarter
BIO9 = Mean Temperature of Driest Quarter
BIO10 = Mean Temperature of Warmest
Quarter
BIO11 = Mean Temperature of Coldest
Quarter
BIO12 = Annual Precipitation
BIO13 = Precipitation of Wettest Month
BIO14 = Precipitation of Driest Month
BIO15 = Precipitation Seasonality
(Coefficient of Variation)
BIO16 = Precipitation of Wettest Quarter
BIO17 = Precipitation of Driest Quarter
BIO18 = Precipitation of Warmest Quarter
BIO19 = Precipitation of Coldest Quarter
Eigenvalue
%
0.34
–0.11
–0.2
0.24
0.04
–0.24
–0.12
0.09
0.34
0.33
–0.01
0.07
0.08
–0.2
0.34
0.03
0.34
0.34
0.06
0.04
0.34
0.04
–0.05
–0.07
–0.08
0.04
0.35
0.33
0.34
–0.19
–0.05
–0.09
–0.09
–0.08
7.96
41.94
0.34
0.33
0.33
0.33
7.46
39.26
with high loadings in precipitation-related variables
(all P values for t tests < 0.03; Fig. 3D; Table 4).
Taxonomic review of the Boana geographica–
Boana semilineata species complex
Fouquet et al. (2016) assigned the binomen ‘Hypsiboas
geographicus’ to populations from Tefé, Purus, Serra do
Divisor (Brazil) and Concepción (Bolivia), which form
a single clade. We agree with the assignment of Boana
geographica, given its type locality ‘flumen Teffé’.
Fouquet et al. (2016) assigned Boana semilineata to
two specimens from Rio de Janeiro (e.g. specimen
CFBH 5424 in Fig. 1B). We follow that assignment and
highlight that our bPTP and ABGD analyses indicate
that the sister clade of B. ventrimaculata is a species
complex. The assignment of other currently recognized
binomials is shown in Figure 1 (A, B).
Available junior synonyms in the Boana
geographica–Boana semilineata species complex
Junior synonyms are: Hyla appendiculata
Boulenger, 1882; Hylella punctatissima Reinhardt
& Lutken, 1862; Centrotelma cryptomelan Cope,
1867; and Cophomantis punctillata Peters, 1870.
Except for Hyla appendiculata, all type localities
are in the Atlantic Forest of Brazil and are at a
distance of > 2000 km from the distribution range of
our Confirmed Candidate Species. Therefore, those
binomials are unlikely to be applicable to them. The
type locality of Hyla appendiculata, erroneously
synonymized under B. geographica, overlaps with
the distribution range of two Confirmed Candidate
Species: clades A and D (Fig. 6). Examination of
the lectotype and published descriptions from
the literature allows us to assign the name Hyla
appendiculata to clade A (Fig. 1). We document this
assignment in the following section.
Taxonomic status of Hyla appendiculata
The lectotype of Hyla appendiculata is an adult
female with SVL = 63.70 mm (BMNH 1947.2.13.1
formerly 80.12.5.168; Fig. 4) from Canelos, Provincia
Pa s t a z a , E c u a d o r. H y l a a p p e n d i c u l a t a w a s
synonymized under Hyla geographica geographica by
Parker (1935; for a detailed discussion see: Duellman,
1973). The lectotype closely resembles individuals
of B. geographica s.s. and clade A, which also have
brown-paired vertical stripes on the flanks and hidden
surfaces of the thighs (Fig. 5). The type locality lies
within the distribution range of clade A (Fig. 6) and
at a distance of 780 km from the geographic range of
B. geographica s.s. Based on its resemblance to the
holotype and its geographic distribution, we conclude
that clade A is conspecific with Hyla appendiculata,
which we remove from synonymy of B. geographica.
This taxonomic decision results in the new binomial
Boana appendiculata.
© 2020 The Linnean Society of London, Zoological Journal of the Linnean Society, 2020, XX, 1–32
Downloaded from https://academic.oup.com/zoolinnean/advance-article-abstract/doi/10.1093/zoolinnean/zlaa002/5810752 by University of Rochester user on 22 March 2020
Table 3. Character loadings, eigenvalues, and percentage
of explained variance for Principal Components (PC)
I–II. The analysis was based on six morphometric
variables of adult Boana appendiculata, B. nigra and
B. ventrimaculata. Bold figures indicate highest loadings
SYSTEMATICS OF BOANA SEMILINEATA GROUP
13
Figure 5. Adult preserved specimens of Boana appendiculata showing variation in dorsal and ventral coloration. From left
to right, first and second rows: QCAZ 64085, 25955, 28227, 30921 (males), 31035 (subadult female), 36932, 36934, 39416,
43850 (males); third and fourth rows: QCAZ 44160, 44168, 44174, 52610, 52734 (males), 37873, 5076, 25797 (females). See
Appendix for locality data. All specimens are shown at the same scale.
Identity of Boana hutchinsi
According to Peloso et al. (2018), specimens of clade D
(clade S5 in that publication) represent B. hutchinsi. This
assignment was based on comparisons between specimens
from Vaupés (Colombia), Amazonas (Brazil) and the type
material of B. hutchinsi (holotype UTA-A 24819 and
paratypes 24830 and 24835). Nevertheless, a thorough
review of the description of B. hutchinsi indicates that it
© 2020 The Linnean Society of London, Zoological Journal of the Linnean Society, 2020, XX, 1–32
Downloaded from https://academic.oup.com/zoolinnean/advance-article-abstract/doi/10.1093/zoolinnean/zlaa002/5810752 by University of Rochester user on 22 March 2020
Figure 4. Dorsal and ventral views of the lectotype of Hyla appendiculata (BMNH 1947.2.13.1; adult female).
14
M. A. CAMINER and S. R. RON
is not conspecific with clade D. We base this conclusion
on the original description of type material (Pyburn &
Hall, 1984), which differs from that of clade D: webbing
between second, third and fourth fingers black in adults
(yellowish white, brown or cream in clade D), dorsum
lavender-grey in recently metamorphosed individuals
(brown with black flecks in clade D) and advertisement
calls having four to six notes with a mean duration of
0.68 s (one note with mean duration of 0.24 s in clade D).
The available evidence indicates that B. hutchinsi have
not been included in any molecular phylogeny.
Systematic accounts
Boana appendiculata (Boulenger, 1882) comb.
nov.
(Figs 4, 5, 7B; Table 5)
Hyla appendiculata Boulenger, 1882.
Lectotype (Fig. 4): BMNH 1947.2.13.1 (designated by
Parker, 1933), an adult female from ‘Canelos’, Ecuador.
Common names: Proposed standard English name:
Canelos treefrog. Proposed standard Spanish name:
rana arbórea de Canelos
Diagnosis (Figs 5, 7B): Boana appendiculata is
characterized by: (1) mean SVL 45 mm in males (range
38.5–53.1; N = 96), 63.8 mm in females (range 50.2–72.6;
N = 40; Table 5); (2) thighs long (TL/SVL 0.45–0.56); (3)
dorsal skin finely granular; (4) dorsum coloration dull
tan, brown, dark brown or cream usually with irregular
dark brown markings frequently including an X-shaped
mark in scapular region; (5) flanks and hidden surfaces
of thighs creamy or pale grey with black vertical
stripes (either single or paired); (6) areolate skin on the
posterior surface of throat, belly and ventral surfaces of
thighs; (7) venter vary from creamy white to yellowish
white or orange, with or without dark brown spots; (8)
in life, webbing yellow, orange or brown; (9) palpebrum
reticulated; (10) nuptial excrescences present in
breeding males; (11) projecting prepollices absent in
both sexes; (12) tympanum large with distinct tympanic
ring; (13) small, triangular calcar on heel, often coloured
© 2020 The Linnean Society of London, Zoological Journal of the Linnean Society, 2020, XX, 1–32
Downloaded from https://academic.oup.com/zoolinnean/advance-article-abstract/doi/10.1093/zoolinnean/zlaa002/5810752 by University of Rochester user on 22 March 2020
Figure 6. Distribution of Boana geographica, B. appendiculata, B. nigra, B. ventrimaculata, and B. sp. (clade F in Fig. 1B).
Localities are based on adult and sequenced specimens deposited at Museo de Zoología of Pontificia Universidad Católica
del Ecuador and from Fouquet et al. (2016).
F
M
F
M
F
Mean ± SD is given with range in parentheses. Abbreviations are: SVL = Snout–vent length; HL = Head length; HW = Head width; ED = Eye diameter; TD = Tympanum diameter; TL = Tibia length;
FEL = Femur length; FL = Foot length; CL = Calcar length; F = females; M = males. All measurements are in mm.
1.5 ± 0.4
(0.7–2.27)
2.1 ± 0.4
(1.1–2.7)
1.1 ± 0.5
(0.9–1.6)
2.3 ± 0.4
(1.9–2.8)
1.7 ± 0.4
(1–2.4)
2.5 ± 0.5
(1.6–3.7)
16.3 ± 1.8
(13–19.8)
24 ± 2.1
(18.4–28)
16.4 ± 0.9
(14.9–18.6)
25.2 ± 2.8
(21.2–28.8)
19.2 ± 1.9
(15.8–23.6)
31 ± 2.3
(27.8–34.4)
B. appendiculata
(N = 35)
B. appendiculata
(N = 30)
B. nigra
(N = 26)
B. nigra
(N = 5)
B. ventrimaculata
(N = 20)
B. ventrimaculata
(N = 17)
M
44.3 ± 3.5
(38.5–51.9)
64.4 ± 4.8
(53.5–72.6)
42.2 ± 2.0
(38.5–46.3)
64.8 ± 7.9
(56.4–76.7)
52.7 ± 4.6
(44.5–60.7)
81.6 ± 5.4
(72.9–90.5)
11.5 ± 1.5
(8–14.3)
15.6 ± 1.9
(12.1–18.6)
10.8 ± 1.3
(8.8–13.9)
15.2 ± 3.1
(11.2–19.5)
12.9 ± 1.9
(9.8–17.3)
18.9 ± 2.6
(14.6–22.9)
16.6 ± 1.5
(11.9–18.4)
22.7 ± 1.5
(19.1–25)
14.5 ± 1.2
(10.2–16.4)
23.5 ± 2.8
(21–27.9)
18.8 ± 1.6
(16.2–22.2)
29.9 ± 2.3
(26.6–34.9)
4.6 ± 0.5
(3.7–5.6)
5 ± 0.5
(4–5.9)
4.1 ± 0.5
(3.4–5.5)
5.3 ± 0.5
(4.6–5.8)
5.3 ± 0.6
(3.9–6.3)
6.7 ± 0.8
(5.3–8.4)
3.9 ± 0.4
(3–4.8)
5.1 ± 0.5
(4–5.8)
3.3 ± 0.3
(2.8–3.9)
4.6 ± 0.3
(4.3–5.1)
4.8 ± 0.5
(3.7–5.6)
6.4 ± 0.8
(5.1–7.7)
21.8 ± 2.1
(18.1–25.8)
32.9 ± 2.7
(26.9–37.8)
20.7 ± 1.2
(18.5–23.5)
33 ± 4.1
(29.3–38.2)
26.8 ± 2
(22.8–29.8)
41.9 ± 2.9
(36.3–47.8)
22.8 ± 2
(19.1–26.2)
33.9 ± 2.2
(26.9–37.7)
21.9 ± 0.8
(20.4–23.6)
34.5 ± 4.4
(31.1–40.2)
27.3 ± 1.9
(23.8–30.9)
42.4 ± 2.8
(36.9–47.9)
CL
FL
FEL
TL
TD
ED
HW
HL
SVL
Species
15
with a cream spot; (14) advertisement call consists of
two types of calls: Type I with one note and Type II
with four to six notes; (15) pulses per note varying from
31 to 37 (Type I) and 18 to 22 (Type II); and (16) call
dominant frequency varying from 1076–1645 Hz (Type
I) and 818–947 Hz (Type II).
Comparisons with other species: Boana appendiculata
is most similar to B. semilineata, B. diabolica,
B. geographica and B. hutchinsi. It differs from the
last three species by having yellow to orange or brown
webbing in life (webbing black in B. hutchinsi, red
in B. diabolica and B. geographica; Fouquet et al.,
2016). Although webbing between fingers III–IV and
toes IV–V is grey dorsally in B. geographica, colour
between the other fingers differs as indicated above.
Bright webbing colours fade to cream in preservative
and become indistinguishable among species. Boana
appendiculata can be further distinguished by the
presence of black vertical stripes on the flanks
(speckled pattern in B. diabolica, B. hutchinsi
and B. semilineata; Fouquet et al., 2016). Boana
appendiculata differs from B. ventrimaculata sp. nov. in
having black vertical stripes on the flanks (a speckled
pattern or dark blotches in B. ventrimaculata sp. nov.)
and by its smaller size (differences are significant in
males: Student’s t = 7.16, d.f. = 31.9, P < 0.001). Boana
appendiculata is similar to B. nigra sp. nov. but it differs
in having a combination of yellow or orange ventral
coloration in life (cream to pale orange in B. nigra sp.
nov.) and black vertical stripes on the flanks and
hidden surfaces of thighs (uniform black in males of
B. nigra sp. nov.; Fig. 7A, 10). However, small adult
specimens of B. appendiculata (< 42 mm) may present
a similar colour pattern to B. nigra sp. nov. (cream
ventral coloration with uniform black flanks). Both
species also differ in environmental envelope (Fig. 3D;
scores along PC I and PC II are significant different:
PC I, t = 8.51, P < 0.001; PC II, t = 2.55, P = 0.034).
Boana appendiculata occurs in warmer localities than
B. nigra sp. nov. as shown by annual mean temperature
(bio01): B. appendiculata mean = 24.28 ˚C (SD = 1.53,
N = 59) vs. B. nigra mean = 19.48 ˚C (SD = 1.78, N = 8).
Despite high genetic distances (4.2–5.2% for gene
16S; Table 6), both species are sympatric in at least
one locality in Pastaza Province. This suggests the
existence of non-geographic reproductive barriers and,
therefore, lineage independence.
Boana appendiculata is easily distinguished from
B. boans, B. icamiaba, B. pombali and B. wavrini by
its prepollex not modified into a projecting spine and
by the presence of pigmented nuptial excrescences.
Boana appendiculata further differs from B. boans
in having well-defined vertical stripes on the flanks
and hidden surfaces of thighs (diffuse vertical bars
and barely distinguishable in B. boans), and webbing
© 2020 The Linnean Society of London, Zoological Journal of the Linnean Society, 2020, XX, 1–32
Downloaded from https://academic.oup.com/zoolinnean/advance-article-abstract/doi/10.1093/zoolinnean/zlaa002/5810752 by University of Rochester user on 22 March 2020
Table 5. Descriptive statistics for morphometric of Boana appendiculata, B. nigra and B. ventrimaculata used for Principal Component Analysis
SYSTEMATICS OF BOANA SEMILINEATA GROUP
16
M. A. CAMINER and S. R. RON
extends to about the middle of the outer finger (fully
webbed hands in B. boans).
Variation in preservative: Morphometric data for adult
specimens are summarized in Table 5, while variation
in dorsal and ventral coloration of preserved specimens
is shown in Figure 5. The most noticeable variation
occurs in the dorsal coloration and in the shape of the
calcars. Calcar size varies from inconspicuous (e.g.
QCAZ 44174) to prominent (e.g. QCAZ 43850). Sexual
dimorphism was observed in SVL, with 38.5–51.9 mm
(44.3 ± 3.5 mm, N = 35) in males and 53.5–72.6 mm
(64.4 ± 4.8 mm, N = 30) in females. Females are
significantly larger than males (t = –20.13, d.f. = 52,
P < 0.001).
Head shape varies between subacuminate and
rounded in dorsal view; in lateral view it varies
between truncate and short, rounded. The head is
slightly wider than the body in most of the specimens.
Breeding males have keratinized nuptial excrescences
on the inner surfaces of the thumbs. Webbing on the
hand varies slightly among specimens: fingers I basal
II (1+–1½)—(2–3–) III (1½–2½)—(1–2) IV. Variation of
webbing between toes is I (1–2–)—(1–2–) II (1–1½)—
(1–2+) III (1–1+)—(1–2+) IV (1–2+)—(1––1+) V. Vomerine
teeth (N = 15): 10–14 (right = 11.5) and 10–15
(left = 12.1).
Background dorsal coloration varies from dull
tan (e.g. QCAZ 36932), brown (e.g. QCAZ 25955),
dark brown (e.g. QCAZ 64085) to cream (e.g. QCAZ
44167) with irregular faint to well-defined dark
brown marks in diverse patterns. Most frequently
there is an X-shaped mark on the scapular region
(e.g. QCAZ 25797, 44168, 52734). A dark brown
transversal band (e.g. QCAZ 36934, 52610) or
sometimes two narrow transversal stripes
interconnected (e.g. QCAZ 44168) are usually
present on the sacral region. In some individuals
© 2020 The Linnean Society of London, Zoological Journal of the Linnean Society, 2020, XX, 1–32
Downloaded from https://academic.oup.com/zoolinnean/advance-article-abstract/doi/10.1093/zoolinnean/zlaa002/5810752 by University of Rochester user on 22 March 2020
Figure 7. Dorsal and ventral views of adults of: A, Boana nigra, QCAZ 58786 (holotype; SVL = 42.53 mm), 58783
(SVL = 45.95 mm), 59114 (SVL = 45 mm), adult males, 58798 (SVL = 68.03 mm), adult female; B, B. appendiculata, QCAZ
64085 (SVL = 45.67 mm), adult male, 52845 (SVL = 66.82 mm), adult female; C, B. ventrimaculata, QCAZ 51241 (holotype;
SVL = 57.57 mm), adult male, 39137 (SVL = 79.98 mm), adult female.
N=2
0
0.086
0.077
0.068 (0.061–0.074)
0.062 (0.057–0.069)
0.082 (0.079–0.088) 0.061 (0.059–0.067)
0.078 (0.074–0.086) 0.059 (0.057–0.064)
0.044 (0.032–0.059) 0.049 (0.049–0.052) 0
0.045 (0.035–0.059) 0.049 (0.044–0.052) 0.035
N=9
N=9
0.09 (0.088–0.093)
0.079 (0.064–0.089)
0.086 (0.079–0.091) 0.07 (0.064–0.077)
0.046 (0.029–0.066) 0.008 (0–0.017)
N = 41
N = 41
N = 48
0.080 (0.069–0.093)
0.068 (0.054–0.082)
0.078 (0.064–0.094) 0.060 (0.052–0.076)
0.022 (0–0.054)
N=4
N=4
N = 11
0
0.046 (0.042–0.052)
0.062 (0.059–0.069) 0.059 (0.057–0.061)
N = 43
N = 17
N = 12
N = 25
N = 17
N = 12
N = 25
N = 24
N = 19
N = 32
N = 19
N = 14
N = 27
N = 27
0.004 (0–0.012)
0.047 (0.039–0.057)
B. diabolica
B. geographica
B. appendiculata
Clade A
B. nigra
Clade B
B. semilineata
species complex
B. ventrimaculata
Clade D
Clade E
Clade F
0.001 (0–0.005)
0.045 (0.04–0.052)
0.06 (0.052–0.071)
N = 30
N = 35
0.017 (0–0.03)
N = 56
N = 51
N = 64
Clade F
Clade E
B. ventrimaculata
Clade D
B. semilineata
species complex
B. nigra
Clade B
B. appendiculata
Clade A
B. geographica
B. diabolica
17
there are scattered minute black flecks on the
dorsal surfaces (e.g. QCAZ 25797, 36932, 39416).
A dark mid-dorsal line may extend from the tip of
the snout to the mid-sacrum, but in some specimens,
the line is restricted to the head. Individuals may
display asymmetrically distributed small to large
cream or dark blotches on the dorsum (e.g. QCAZ
5076, 28227, 44160, 64085). The coloration of flanks
and hidden surfaces of thighs varies from creamy
to pale grey, with vertical dark stripes to black bars
(e.g. QCAZ 30921, 36934, 39416); bars on the flanks,
and less frequently on the thighs, are sometimes
arranged in pairs. Flanks are uniform black in
juveniles, some subadults and a few small adults
(< 42 mm). In some individuals, the bars can also be
present on the hidden surfaces of the shanks (e.g.
QCAZ 25797). Dark brown transversal bands 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). The heels are usually
cream, contrasting with the darker brown coloration
of the dorsum (e.g. QCAZ 5076, 64085), but in some
individuals, heels have the same colour as the rest
of the body (e.g. QCAZ 36932, 44160).
Ventral surfaces of preserved specimens vary from
creamy white (e.g. QCAZ 30921) to yellowish white
(e.g. QCAZ 39416). In some individuals there are dark
brown spots on the belly (e.g. QCAZ 5076). Coloration
of webbing and discs varies from yellowish white to
cream, brown or grey. Coloration of bones is white or
green (e.g. QCAZ 64085).
Coloration in life (Fig. 7B): Dorsal surfaces vary from
brown (e.g. QCAZ 51182) to dark brown (e.g. QCAZ
23855), reddish brown (e.g. QCAZ 26267) or yellowish
tan (e.g. QCAZ 52845); transversal bands on dorsal
surfaces on limbs and marks on dorsum vary from
light brown to dark brown, yellow or white (e.g. QCAZ
52733, 61767, 64101); in some individuals there are
scattered minute black flecks on dorsal surfaces (e.g.
QCAZ 36934); flanks and hidden surfaces of thighs are
cream or yellowish with dark brown vertical stripes or
bars (e.g. QCAZ 51179, 51182, 64085), uniform black
in some small adult individuals; belly and ventral
surfaces of limbs vary from cream to yellowish or
orange (e.g. QCAZ 37879, 41009, 64099); throat cream
white or yellowish; webbing varies from yellow to
orange or brown; fingers and toes are dorsally grey or
with same coloration of webbing; iris brown or bronze
(e.g. QCAZ 52845, 55356); palpebrum finely reticulated
with golden yellow.
In recently metamorphosed individuals, flanks,
hidden surfaces of thighs and webbing are black;
venter is grey; dorsal surfaces of the limbs and sides
of the head are dark brown; dorsum is brown with
scattered black flecks (Fig. 8A).
© 2020 The Linnean Society of London, Zoological Journal of the Linnean Society, 2020, XX, 1–32
Downloaded from https://academic.oup.com/zoolinnean/advance-article-abstract/doi/10.1093/zoolinnean/zlaa002/5810752 by University of Rochester user on 22 March 2020
Table 6. Pairwise genetic distances (uncorrected p) of 16S DNA sequences (409 pb) among members of the Boana semilineata group. Mean is given with range in
parentheses (below diagonal). Number of individuals compared is shown above diagonal. Diagonal shows intra-clade genetic distances
SYSTEMATICS OF BOANA SEMILINEATA GROUP
18
M. A. CAMINER and S. R. RON
Vocalization: Based on one male, not collected, from
Yasuní on the road to Pompeya at km 73 (recorded
by Morley Read); two males from Santa Cecilia (KU
143144–45; recorded by W. E. Duellman on 10 October
1971; 20:50–21:00 h; 23 °C) and two males from MiaziNuevo Paraíso (QCAZ 67113–14; collected by D. Paucar
on 4 March 2017; 21:00 h). The call that lacks voucher
specimen was assigned to Boana appendiculata based
on the similarity of its structure compared to vouchered
recordings of B. appendiculata. The call varies from a
long groan (Type I, mean duration of 0.45 s and 31–37
pulses/note) to a series of short chuckle-like notes
(Type II, mean 2.07 s and four to six notes/call) with
fewer pulses per note (18–22 pulses/note). The long
note (Fig. 9A, B) has a mean dominant frequency of
973.2 Hz, rise time of 0.34 s and frequency bandwidth
1316.2 Hz. The serial of short notes (Fig. 9C, D) has a
mean dominant frequency of 1387.6 Hz, rise time of
0.44 s, frequency bandwidth of 1841.9 Hz and interval
between notes of 0.16 s. Other call parameters are
listed in Table 2.
Distribution and ecology: Boana appendiculata occurs
in the Amazon basin of Ecuador (Morona Santiago,
Napo, Orellana, Pastaza, Sucumbíos and Zamora
Chinchipe provinces), Brazil (Amazonas, Pará and
Rondônia states) and Colombia (Fig. 6). Localities with
known elevation range from 14 to 1050 m above sea
level. The elevation at Shell (1050 m) is the highest,
while Caxiuanã (14 m) is the lowest. Specimens of
B. appendiculata occur in terra firme forest, swamps,
semi-flooded and flooded forests and artificial open
areas. They are generally found at night in primary
and secondary forest, perching on vegetation 20 to
600 cm above the ground, next to lakes, flooded areas
and temporary ponds in clearings. Few individuals
were found in flooded areas with pastures or forest
away from water bodies. Their occurrence in secondary
forests and artificial open areas suggest at least some
tolerance of anthropogenic habitat disturbance.
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) Amazonian lowland evergreen forest,
characterized by high plant alphadiversity and a canopy
height of 30 m with emergent trees that reach 40 m; (3)
floodplain lowland forest of white-waters, characterized
by periodical flooding with white-waters from large
rivers, with the vegetation reaching 35 m in height
and several horizontal strata of vegetation; and (4)
lowland forest of palms and black-waters characterized
by a canopy height of 30 m with dense understory and
dominance of the palm Mauritia flexuosa L.f. (Sierra
et al., 1999). Vegetation types at localities in Brazil
include Purus-Madeira moist forests, Purus Varzea and
Xingu-Tocantins-Araguaia moist forests (according to
the World Wildlife Fund, 2017 classification scheme;
available at http://www.eoearth.org/view/article/151948).
Remarks: A 16S sequence of ‘Boana cf. geographica’
from Valle del Guamuez, Colombia, published by
Meza-Joya et al. (2019; GenBank accession number
MF583739) is identical to the homologous sequence
of specimen QCAZ 61767 of B. appendiculata from
Limoncocha, Ecuador (539 bp). This similarity indicates
the presence of B. appendiculata in Colombia. The
Meza-Joya et al. (2019) publication became available
while our manuscript was under review. Therefore, we
did not include its sequence into our phylogeny.
Boana nigra sp. nov.
(Figs 7A, 10; Table 5)
Lsid: urn:lsid:zoobank.org:act:FE5D2127-D2BD4F4D-A4E5-4CE742A32F21
Holotype (Figs 7A, 10): QCAZ 58786 (field no. SC
48236), adult male from Ecuador, Provincia Morona
Santiago, Parque Nacional Sangay, Sardinayacu
(2.1028° S, 78.1521° W), 1348 m a.s.l., collected by
© 2020 The Linnean Society of London, Zoological Journal of the Linnean Society, 2020, XX, 1–32
Downloaded from https://academic.oup.com/zoolinnean/advance-article-abstract/doi/10.1093/zoolinnean/zlaa002/5810752 by University of Rochester user on 22 March 2020
Figure 8. Dorsolateral views of juveniles of: A, B. appendiculata, QCAZ 39339 (SVL = 28.04 mm); B, Boana nigra, QCAZ
61883 (SVL = 25.04 mm); C, B. ventrimaculata, QCAZ 38663 (SVL = 27.06 mm).
SYSTEMATICS OF BOANA SEMILINEATA GROUP
19
Daniel Rivadeneira, David Velalcázar, Javier Pinto,
Francy Mora, Darwin Núñez, Juan Carlos Sánchez
and Andrea Correa on 29 January 2015.
Paratopotypes: QCAZ 58783–85, 58787–89, 58791–
97, adult males, 58790, 58798, adult females, same
collectors as the holotype, 15–29 January 2015;
© 2020 The Linnean Society of London, Zoological Journal of the Linnean Society, 2020, XX, 1–32
Downloaded from https://academic.oup.com/zoolinnean/advance-article-abstract/doi/10.1093/zoolinnean/zlaa002/5810752 by University of Rochester user on 22 March 2020
Figure 9. Calls of: A–D, Boana appendiculata from Yasuní, Province Orellana; E, F, B. nigra from Sardinayacu, Province
Morona Santiago; G, H, B. ventrimaculata (QCAZ 49233) from Yasuní, Province Napo. A, C, E, G are oscillograms and B, D,
F, H spectrograms.
20
M. A. CAMINER and S. R. RON
Paratypes: ECUADOR: PROVINCIA MORONA
SANTIAGO: Bosque Protector Abanico (2.2583° S,
78.1983° W), 1560 m, QCAZ 48984, adult female,
collected by D. Morocho on 1 June 2007; bridge N of
Macas (2.3017° S, 78.1137° W), 944 m, QCAZ 47044,
collected by S. Poe, F. Ayala, L. Gray, J. Davis and
I. Latella, on 15 December 2009; Parque Nacional
Sangay (1.786° S, 77.979° W), 982 m, QCAZ 54605–6,
51776, 51780, tissues, collected by L. MacLean and
A. Almendáriz, on 4 December 1991 and 29 August
2011; PROVINCIA PASTAZA: Centro Ecológico Zanja
Arajuno, km 6 on the road San Ramón-El Triunfo
(1.373° S, 77.860° W), 910 m, QCAZ 36929, 36931,
40959, 45282, adult males, 45283, female, 37903,
37988, tadpoles, 40265, 40958, subadult females,
collected by S. R. Ron, I. Tapia, L. Coloma, A. Carvajal,
A. Merino, E. Arbeláez, A. Tapia, D. Salazar, D. Acosta
and C. Korfel, on 29 April 2004 and 2 February 2009.
Common names: Proposed standard English name:
black-flanked treefrog. Proposed standard Spanish
name: rana arbórea de flancos negros.
Diagnosis (Figs 7A, 10): Boana nigra is characterized
by: (1) mean SVL 42.2 mm in males (range 38.5–46.3;
N = 26), 64.8 mm in females (range 56.4–76.7; N = 5;
Table 5); (2) thighs long (TL/SVL 0.44–0.53); (3) dorsal
skin finely granular; (4) dorsum coloration brown,
dark brown, cream or grey usually with an X-shaped
mark on scapular region or rounded marks over the
entire dorsum; (5) flanks and hidden surfaces of
thighs vary from uniform black to dark vertical bars;
(6) areolate skin on the posterior surface of throat,
belly and ventral surfaces of thighs; (7) venter creamy
white to pale orange, plain or spotted with dark brown;
(8) in life, webbing pale orange, orange or black; (9)
palpebrum reticulated; (10) nuptial excrescences
present in breeding males; (11) projecting prepollices
absent in both sexes; (12) tympanum large with distinct
tympanic ring; (13) small triangular calcar on heel,
often coloured with a cream spot; (14) advertisement
call with one note; (15) pulses per note varying from 21
to 37; and (16) call dominant frequency varying from
996 to 1063 Hz.
Comparisons with other species: Boana nigra can
be distinguished from B. ventrimaculata sp. nov.,
B. hutchinsi, B. diabolica and B. semilineata by having
uniform black or dark vertical stripes (in females) on
flanks (speckled pattern or blotches in all the four
species; Fouquet et al., 2016). In addition, the hidden
surfaces of thighs and webbing in life are red in
B. diabolica (uniform black in B. nigra, sometimes with
dark vertical bars on the hidden surfaces of thighs).
The individuals of B. nigra with dark vertical bars
pattern on flanks and hidden surfaces of the thighs can
be confused with B. geographica and B. appendiculata.
However, B. nigra differs in having orange or black
webbing in life (red webbing except between fingers
III–IV and toes IV–V in B. geographica) and a cream to
pale orange ventral coloration in life (yellow or orange
in B. appendiculata).
Some small adult B. appendiculata (< 42 mm) can
also have a cream venter with uniform black flanks.
Ventral and webbing bright coloration fade to cream in
preservative and become indistinguishable among the
three species. Boana nigra and B. appendiculata also
differ in environmental envelope (Fig. 3D; for details
see ‘Comparisons with other species’ in the description
of B. appendiculata). Despite being separated by high
genetic distances (Table 6), they occur in sympatry,
which indicates the existence of non-geographic
reproductive barriers.
Boana nigra is easily distinguished from B. boans,
B. icamiaba, B. pombali and B. wavrini by its prepollex
not modified into a projecting spine and by the
presence of pigmented nuptial excrescences. Boana
boans further differs from B. nigra in having diffuse
vertical bars on the flanks and barely distinguishable
on the hidden surfaces of the thighs (well-defined
vertical bars or uniform black in B. nigra) and by its
fully webbed hands (webbing extends to about the
middle of the outer finger in B. nigra).
Description of the holotype: Adult male, 42.5 mm
SVL, foot length 16.5 mm, head length 9.9 mm, head
width 14.5 mm, eye diameter 4.5 mm, tympanum
diameter 3.7 mm, tibia length 22.4 mm, femur length
22.2 mm, calcar length 0.9 mm, arm length 7.7 mm,
eye–nostril distance 4.3 mm, head wider than long
and wider than body; snout sloping in lateral view,
subacuminate in dorsal view; distance from nostril
to eye shorter than diameter of eye; canthus rostralis
rounded; loreal region concave; internarial region
subtly depressed; nostrils not protuberant, directed
laterally; interorbital area slightly convex; eye
large, protuberant; lower eyelid finely reticulated;
diameter of eye 1.2 times diameter of tympanic
annulus; supratympanic fold inconspicuous; tympanic
membrane roundish, slightly longer than high;
tympanic annulus evident, rounded, separated
from eye by c. 0.5 times its diameter. Arm slender,
axillary membrane absent; relative length of fingers
I < II < IV < III; finger discs broadly expanded, round,
© 2020 The Linnean Society of London, Zoological Journal of the Linnean Society, 2020, XX, 1–32
Downloaded from https://academic.oup.com/zoolinnean/advance-article-abstract/doi/10.1093/zoolinnean/zlaa002/5810752 by University of Rochester user on 22 March 2020
QCAZ 59110–12, 59114, 59116–17, adult males,
59113, metamorph, 59115, 59118, tadpoles, 61881–87,
juveniles, collected by S. R. Ron, D. Paucar, P. Venegas,
P. Baldeón, M. Caminer and E. Nusirquia, 27 February
2015; QCAZ 58337–8, tissues, collected by J. Brito on 29
October 2014; DHMECN 12130, adult male, collected
by D. R. Batallas and P. Bejarano on 16 June 2014.
SYSTEMATICS OF BOANA SEMILINEATA GROUP
Colour of holotype in preservative (Fig. 10): Dorsum
light grey with extensive dark brown rounded marks,
sometimes interconnected; faint brown transversal
bands on the dorsal surfaces of shanks; flanks, hidden
surfaces of limbs and dorsal surfaces of thighs, upper
arms, hands and feet black; venter cream with dark,
diffuse, minute dots on the belly; ventral surfaces of
thighs, forearms and hands cream; ventral surfaces of
shanks and upper arms black; discs and webbing grey.
Colour of holotype in life (Fig. 7A): Dorsum brown with
dark brown rounded marks over the entire dorsum;
dorsal and hidden surfaces of limbs and flanks black;
faint brown transversal bands on the dorsal surface of
hindlimbs; venter and ventral surfaces of thighs and
hands cream; belly cream with brown diffused dots;
ventral surfaces of upper limbs, shanks and feet black;
discs and webbing black; iris bronze.
Etymology: The specific epithet nigra is a Latin
adjective in the nominative case meaning black, and
refers to the black coloration on the flanks of these
frogs.
Variation in preservative: Morphometric data for
adult specimens are summarized in Table 5, and
variation in dorsal and ventral coloration of preserved
specimens is shown in Figure 10. Sexual dimorphism
is observed in SVL, with 38.5–46.3 mm (42.2 ± 2 mm,
N = 26) in males and 56.4–76.7 mm (64.8 ± 7.9 mm,
N = 5) in females. Females are significantly larger
than males (t = –6.40, d.f. = 4.1, P = 0.002).
Head shape varies between subacuminate and
rounded in dorsal view; in lateral view it varies between
truncate and short, rounded. The head is wider than
long and slightly wider than the body in most of the
specimens. Breeding males have keratinized nuptial
excrescences on the inner surfaces of the thumbs.
Calcar size ranges from inconspicuous (e.g. QCAZ
58783) to prominent (e.g. QCAZ 45283). Webbing on
the hand varies slightly among specimens: fingers
I basal II (1 +–2 –)—(2½–3 –) III (2 ––2½)—(2–2 +) IV.
Variation of webbing between toes is I (1–2–)—(1–2–)
II (1–1+)—(1–2+) III (1–1½)—(1+–2+) IV (2––2+)—(1–1+)
V. Vomerine teeth (N = 15): 8–12 (right = 9.14) and
7–12 (left = 9.71).
Most variation involves dorsal coloration and pattern.
Background dorsal coloration varies from brown (e.g.
QCAZ 45282), dark brown (e.g. QCAZ 48984), cream
(e.g. QCAZ 45283) to grey (e.g. QCAZ 47044) with a
dark brown, X-shaped mark on the scapular region
or dark brown rounded marks over the entire dorsum
(e.g. QCAZ 59114); sometimes rounded marks are
interconnected (e.g. QCAZ 59117). Scattered, minute,
black or white flecks are present on the dorsal surfaces
(e.g. QCAZ 40958). A dark mid-dorsal line extends
from the tip of the snout to the mid-sacrum (e.g. QCAZ
45283). Dark brown or grey transversal bands 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). The coloration of flanks
and hidden surfaces of thighs and shanks are uniform
black in males (e.g. QCAZ 58783) or grey with dark
vertical bars in females (e.g. QCAZ 48984). However, in
some cases males can present dark vertical bars on the
flanks (e.g. QCAZ 6478). The heels have usually the
same colour as the rest of the body (e.g. QCAZ 45282),
but in some individuals heels are cream, contrasting
with the background dorsal coloration (e.g. QCAZ
40958).
Ventral surfaces of preserved specimens vary from
creamy white (e.g. QCAZ 40958) to yellowish white
(e.g. QCAZ 48984). In some individuals, there are dark
brown spots on the belly (e.g. QCAZ 58783). Coloration
of webbing and discs vary from yellowish white to
cream, black or grey.
Coloration in life (Fig. 7A): Dorsal surfaces vary from
brown (e.g. QCAZ 58783) to dark brown (e.g. QCAZ
59110) or reddish brown (e.g. QCAZ 59111) with
brown marks on the dorsum; brown transversal bands
on dorsal surfaces on limbs; brown rounded spots and
blotches over the entire dorsum (e.g. QCAZ 59110,
59111). In some individuals, there are scattered
minute black or white flecks on dorsal surfaces (e.g.
© 2020 The Linnean Society of London, Zoological Journal of the Linnean Society, 2020, XX, 1–32
Downloaded from https://academic.oup.com/zoolinnean/advance-article-abstract/doi/10.1093/zoolinnean/zlaa002/5810752 by University of Rochester user on 22 March 2020
as wide as long; subarticular tubercles prominent,
round to ovoid, single; supernumerary tubercles
absent; palmar tubercle inconspicuous; large and
rugose dark P-shaped nuptial pad on dorsal surface of
finger I; prepollex not modified into a projecting spine;
webbing formula of fingers I basal II1½—3–III2½—
2IV. Toes bearing discs broadly expanded, rounded
and slightly smaller than those of fingers; relative
length of toes I < II < V < III < IV; inner metatarsal
tubercle large, elliptical; outer metatarsal tubercle
absent; subarticular tubercles single, round, large and
protuberant; supernumerary tubercles small, round;
webbing formula of toes I1+—2–II1—2–III1+—2+IV2+—
1+V. Vocal sac single and subgular. Skin on dorsum,
head and dorsal surfaces of limbs finely granular;
throat smooth anteriorly without mental gland;
posterior surface of throat, belly and ventral surfaces
of thighs areolate, those of shanks smooth. Cloacal
opening directed posteriorly at upper level of thighs,
round tubercles below and on sides of vent. Tongue
slightly cordiform, widely attached to mouth floor;
vomerine odontophores situated parallel to choanae,
in two oblique series narrowly separated, each bearing
9–11 vomerine teeth; choanae trapezoidal, oblique.
21
22
M. A. CAMINER and S. R. RON
© 2020 The Linnean Society of London, Zoological Journal of the Linnean Society, 2020, XX, 1–32
Downloaded from https://academic.oup.com/zoolinnean/advance-article-abstract/doi/10.1093/zoolinnean/zlaa002/5810752 by University of Rochester user on 22 March 2020
Figure 10. Adult preserved specimens of Boana nigra showing variation in dorsal and ventral coloration. From left to
right, first and second rows: QCAZ 58786 (holotype), 58783, 45282 (males), 40958, 2657 (subadult females); third and fourth
rows: QCAZ 6478, 58793, 59114 (males), 48984 (female); fifth and sixth rows: QCAZ 45283, 47044, 58790, 58798 (females).
See the type-series list for locality data. All specimens are shown at the same scale.
SYSTEMATICS OF BOANA SEMILINEATA GROUP
Vocalization: Based on one male (DHMECN
12130) recorded at Sardinayacu (Provincia Morona
Santiago) by D. R. Batallas on 16 June 2014 at 22:30 h,
air temperature 14.8 °C. The call (Fig. 9E, F; Table 2)
consists of one groan-like note with a mean duration
of 0.33 s and dominant frequency of 1044.2 Hz. Other
parameters are available in Table 2. The individual
was perched on vegetation 40 cm above the ground
near a lake.
Distribution and ecology: Boana nigra occurs on
the eastern Andean slopes of Ecuador (provinces of
Morona Santiago, Napo, Pastaza, Sucumbíos and
Zamora Chinchipe) (Fig. 6). Localities with known
elevation range from 910 to 1847 m a.s.l. The elevation
at Sardinayacu (1847 m) is the highest known for
B. nigra, while Centro Ecológico Zanja Arajuno
(910 m) is the lowest. It is generally found next to
streams, swamps, ponds and lakes. Individuals have
been recorded at night perching on vegetation 30 to
400 cm above ground or water. The specimen from 9
de Octubre was found next to a highway on the leaf
litter.
Vegetation types at known localities include: (1)
Amazonian evergreen foothill forest, characterized by
a mixture of Amazonian and Andean vegetation with a
canopy of 30 m; (2) montane cloud forest of the eastern
Andes, characterized by trees covered by mosses and
abundant epiphytes; (3) evergreen lower montane
forest of the east of the northern and central Andes,
characterized by a canopy height of 25 to 30 m, with
abundant epiphytes, and by the absence of species
of trees characteristic of the lowlands (e.g. species
from the family Bombacaceae and Myristicaceae);
and (4) lowland floodplain forest of white-waters,
characterized by periodical flooding with white-waters
from large rivers, with the vegetation reaching 35 m
in height, and several horizontal strata of vegetation
(Sierra et al., 1999).
Boana ventrimaculata sp. nov.
(Figs 7C, 11; Table 5)
Lsid: urn:lsid:zoobank.org:act:34C45CDE-5F974AE8-A655-E2C1DA6E9787
Holotype (Figs 7C, 11): QCAZ 51241 (field no. SC37210),
adult male from Ecuador, Provincia Orellana, Estación
Científica Yasuní PUCE (0.673° S, 77.407° W), 245 m
a.s.l., collected by Italo Tapia, Teresa Camacho, Francy
Mora and Santiago R. Ron, on 1 June 2011.
Paratopotypes: QCAZ 51236, adult male, collected
with the holotype; QCAZ 17517, juvenile, collected by
O. Pérez and M. Bustamante on 17 February 2000.
Paratypes: ECUADOR: PROVINCIA MORONA
SANTIAGO: Pankints (2.9019° S, 77.894° W), 332 m,
QCAZ 46429, adult female, collected by J. Brito on 26
December 2010; PROVINCIA NAPO: Huino (0.6449° S,
77.149° W), 273 m, QCAZ 30928, adult male, collected
by D. Alvarado and G. Buitrón on 3 February 2003;
Misahuallí (1.0343° S, 77.6685° W), 401 m, QCAZ 20000,
adult female, collected by F. Ayala and M. Díaz on 26
March 2002; PROVINCIA SUCUMBÍOS: Río Aguarico,
San Pablo (0.255° S, 76.426° W), 220 m, QCAZ 6314–15,
adult females, collected on 2 August 1994; PROVINCIA
PASTAZA: Campamento K10, Campo Villano (1.476°
S, 77.5347° W), 475 m, QCAZ 38772, adult male,
39247, 56429, 56307, adult females, 38663, juvenile,
collected by Y. Mera, D. Paucar, F. Ayala, E. Carrillo,
G. Díaz, J. Brito and D. Velalcázar on 14 February
2008 and 18 November 2013; Comunidad Tarangaro,
Campo Villano (1.3951° S, 77.3838° W), 338–1035
m, QCAZ 39130, 54233, 54238, adult males, 39137,
adult female, collected by Y. Sagredo, A. Barahona,
D. Paucar, G. Díaz, Y. Mera and F. Ayala on 23 August
2008 and 20 June 2012; Juyuintza, 4.5 km SE (2.1132°
S, 76.1646° W), 164 m, QCAZ 53533, adult male,
collected by M. Ortega and F. Timias on 26 June 2012;
Lorocachi (1.6386° S, 75.9711° W), 207 m, QCAZ 56114,
adult male, collected by S. R. Ron, D. Paucar, F. Ayala,
D. Velalcázar, M. J. Navarrete, G. Galarza, J. Pinto,
V. Chasiluisa, S. Arroyo and D. Zurita on 23 June 2013;
PROVINCIA ORELLANA: Nuevo Rocafuerte, Río
Napo (0.9165° S, 75.423° W), 194 m, QCAZ 44845–46,
adult males, collected by S. R. Ron, E. Toral and I. Tapia
on 12 July 2009; Parque Sumaco Balsayacu (0.3724°
S, 77.3411° W), 600 m, QCAZ 23059, adult male,
collected by P. Salvador on 16 January 2002; Estación
de Biodiversidad Tiputini USFQ (0.633° S, 76.1473°
W), 231 m, QCAZ 20398, adult male, collected by M. D.
Proaño on 2 July 2002; Estación Científica Yasuní
PUCE, km 8 on the road Tivacuno (0.6815° S, 76.3897°
W), 251 m, QCAZ 18280, adult female, collected by
© 2020 The Linnean Society of London, Zoological Journal of the Linnean Society, 2020, XX, 1–32
Downloaded from https://academic.oup.com/zoolinnean/advance-article-abstract/doi/10.1093/zoolinnean/zlaa002/5810752 by University of Rochester user on 22 March 2020
QCAZ 36929, 58783). Flanks and hidden surfaces of
thighs are uniform black (in males) or grey with dark
vertical bars (in females); belly and ventral surfaces
of limbs vary from cream to pale orange (e.g. QCAZ
59111, 59114); throat cream white or pale orange;
webbing vary from pale orange to orange or black;
fingers and toes dorsally vary from black to grey or
have the same coloration of webbings; iris brown (e.g.
QCAZ 58797) or bronze (e.g. QCAZ 36929); palpebrum
finely reticulated with golden yellow.
In recently metamorphosed individuals, flanks,
hidden surfaces of thighs, dorsal surfaces of the limbs,
sides of the head and webbing are black; venter is grey
sometimes with scattered dark brown spots; dorsum is
cream with black spots and irregular blotches (Fig. 8B).
23
24
M. A. CAMINER and S. R. RON
Common names: Proposed standard English name:
Yasuní treefrog. Proposed standard Spanish name:
rana arbórea del Yasuní.
Diagnosis (Figs 7C, 11): Boana ventrimaculata is
characterized by: (1) mean SVL 52.7 mm in males
(range 44.5–60.7; n = 20), 81.6 mm in females (range
72.9–90.5; n = 17; Table 5); (2) thighs long (TL/SVL
0.45–0.56); (3) dorsal skin finely granular; (4) dorsum
brown, dark brown, cream or dull tan usually with
an X-shaped mark on scapular region; (5) flanks grey
with white speckles, and hidden surfaces of thighs
grey with black vertical bars; (6) areolate skin on the
posterior surface of throat, belly and ventral surfaces
of thighs; (7) venter creamy white to yellowish white,
plain or with brown blotches; (8) in life, webbing
brown or orange; (9) palpebrum reticulated; (10)
nuptial excrescences present in breeding males;
(11) projecting prepollices absent in both sexes; (12)
tympanum large with distinct tympanic ring; (13)
small, triangular calcar on heel, often with a cream
spot; (14) advertisement call with one note; (15) 11 to
16 pulses per note; and (16) call dominant frequency
varying from 475 to 685 Hz.
Comparisons with other species: Boana ventrimaculata
differs from B. geographica, B. appendiculata and
B. nigra by having a speckled pattern or blotches on
the flanks (black flanks or dark vertical bars or stripes
in the three species) and brown blotches on the ventral
surfaces (immaculate or spotted with dark brown in the
three species). Boana ventrimaculata is larger (male
mean SVL = 52.72 mm; female mean SVL = 81.62 mm)
than B. diabolica (male mean SVL = 43.50 mm,
female mean SVL = 56.30 mm; Fouquet et al.,
2016), B. geographica (male mean SVL = 43.00 mm,
female mean SVL = 48.60 mm; Fouquet et al., 2016),
B. appendiculata (t = 7.16, d.f. = 31.9, P < 0.001) and
B. nigra (t = 9.61, d.f. = 24.9, P < 0.001) (Table 5).
Among species of the B. geographica–B. semilineata
species complex, B. ventrimaculata is morphologically
more similar to B. diabolica and B. hutchinsi.
However, it can be distinguished by having a speckled
pattern on the hidden surfaces of thighs and brown
to orange webbing in life (hidden surfaces of thighs
and webbing are immaculate red in B. diabolica;
black in B. hutchinsi; Pyburn & Hall, 1984; Fouquet
et al., 2016). Boana ventrimaculata can be further
distinguished from B. hutchinsi by having a brown
dorsum with black flecks in recently metamorphosed
individuals (lavender-grey in B. hutchinsi), and a
shorter duration and lower number of notes in the
advertisement call (one groan-like note with a mean
call duration of 0.24 s in B. ventrimaculata; four to six
chuckle-like notes with a mean call duration of 0.68 s
in B. hutchinsi; Pyburn & Hall, 1984). Boana hutchinsi
has not been included in molecular phylogenies and,
therefore, current diagnosis are based exclusively on
morphological and bioacoustic characters.
Specimens of B. ventrimaculata can be easily confused
with B. semilineata due to their similar colour pattern
(brown webbing, black flanks with white speckles and
brown spots on the abdomen; Fouquet et al., 2016; Peloso
et al., 2018), but these species can be clearly distinguished
by having markedly differentiated advertisement calls
(one note with a mean duration of 0.24 s, and mean
dominant frequency of 561 Hz in B. ventrimaculata
vs. one to three notes with a mean duration per note
of 0.04 s, and mean dominant frequency of 1240 Hz in
B. semilineata). The type localities of both species are
separated by ~4500 km making it extremely unlikely
that they represent the same species.
Boana ventrimaculata is easily distinguished from
B. boans, B. icamiaba, B. pombali and B. wavrini by
its prepollex not modified into a projecting spine and
by the presence of pigmented nuptial excrescences.
Boana boans further differs from B. ventrimaculata
in having diffuse vertical bars on the flanks (speckled
pattern in B. ventrimaculata) and by its fully webbed
hands (webbing extends to about the middle of the
outer finger in B. ventrimaculata).
Description of the holotype: Adult male, 57.6 mm SVL,
foot length 21.5 mm, head length 12.4 mm, head width
© 2020 The Linnean Society of London, Zoological Journal of the Linnean Society, 2020, XX, 1–32
Downloaded from https://academic.oup.com/zoolinnean/advance-article-abstract/doi/10.1093/zoolinnean/zlaa002/5810752 by University of Rochester user on 22 March 2020
D. Paucar on 20 December 2001; Yasuní (0.6771° S,
76.4011° W), 230 m, QCAZ 13962, adult male, 30271,
adult female, collected by D. Prado on 18 November
1999; Yasuní, Apaika (0.8111° S, 76.704° W), 200 m,
QCAZ 27788–89, adult males, collected by F. Nogales
on 11 October 2000; Parque Nacional Yasuní, km 38 on
the road Pompeya Sur-Iro (0.6535° S, 76.4535° W), 238
m, QCAZ 17251, adult male, collected by S. de la Torre
on 25 February 1994; Parque Nacional Yasuní, km 45
on the road Pompeya Sur-Iro (0.6940° S, 76.4562° W),
247 m, QCAZ 8181, adult female, collected by M. Read
on 29 January 1995; km 74 on the road Pompeya SurIro (0.8358° S, 76.3533° W), 257 m, QCAZ 5105, adult
female, collected by S. de la Torre on 20 May 1994;
Parque Nacional Yasuní, Ginta-Bloque 16 (0.9981° S,
76.1963° W), 243 m, QCAZ 20465, adult male, collected
by M. Díaz on 27 January 2001; Parque Nacional
Yasuní, on the road Pompeya Sur-Iro km 21 (0.6504°
S, 76.5201° W), 306 m, QCAZ 13301, adult female,
collected by F. Sornoza on 21 August 1996; km 38 on
the road Pompeya Sur-Iro (0.6539° S, 76.4518° W), 230
m, QCAZ 49129, adult female, collected by M. Read on
11 January 1996; km 107 on the road Pompeya SurIro (0.9811° S, 76.2476° W), 249 m, QCAZ 6821, 49233,
adult males, 8325, 30972, adult females, collected by
S. Harris and M. Read on 12 November 1994 and 4
November 1996.
SYSTEMATICS OF BOANA SEMILINEATA GROUP
Colour of holotype in preservative (Fig. 11): Dorsum
brown with a dark brown X-shaped mark on the
scapular region and a dark brown transversal band
on the sacrum; dark brown triangle with its base in
the interorbital area and its apex between the nostrils;
dark brown transversal bands on the dorsal surfaces
of the limbs; flanks grey with dark brown blotches;
hidden surfaces of the thighs grey with brown vertical
bars; venter cream with brown rounded blotches on
the belly; discs and webbing grey.
Colour of holotype in life (Fig. 7C): Dorsum background
brown with dark brown marks; dark brown transversal
bands on the dorsal surface of limbs; flanks grey with
brown blotches; hidden surfaces of thighs and shanks
grey with brown vertical bars; venter and ventral
surfaces of thighs yellowish with brown rounded
blotches on the belly; throat cream; webbing orange;
iris brown; bones green.
Etymology: The specific name is derived from the
Latin words ventriculus = belly, and macula = spot or
stain, in reference to the brown blotches on the chest
and belly of these frogs.
Variation in preservative: Morphometric data for
adult specimens are summarized in Table 5. Variation
in dorsal and ventral coloration of preserved specimens
is shown in Fig. 11. Sexual dimorphism is observed in
SVL, with 44.5–60.7 mm (52.7 ± 4.6 mm, N = 20) in
males and 72.9–90.5 mm (81.6 ± 5.4 mm, N = 17) in
females. Females are significantly larger than males
(t = –17.39, d.f. = 31.5, P < 0.001).
Head shape varies between subacuminate and
rounded in dorsal view; in lateral view it varies between
truncate and short, rounded. The head is slightly wider
than the body, but in some cases equal (e.g. QCAZ 39137).
Breeding males have keratinized nuptial excrescences
on the inner surfaces of the thumbs. Calcar size ranges
from inconspicuous (e.g. QCAZ 30928) to prominent
(e.g. QCAZ 51241). Webbing on the hand varies slightly
among specimens: fingers I basal II (1––1+)—(2––2+) III
(1+–2–)—(1–1½) IV. Variation of webbing between toes
is I (1––1+)—(1–1+) II (1–1–)—(1–2) III (1––1+)—(1–2–)
IV (1–2–)—(1––1+) V. Vomerine teeth (N = 10): 14–21
(right = 16.9) and 12–22 (left = 18).
Variation in dorsal coloration of preserved specimens
is extensive. Dorsal coloration varies from brown (e.g.
QCAZ 30928) to dark brown (e.g. QCAZ 56307), cream
(e.g. QCAZ 49233) or dull tan (e.g. QCAZ 39137) with
irregular faintly or well-defined dark brown marks
in diverse patterns, but most frequently consist of
an X-shaped mark on the scapular region (e.g. QCAZ
13962). Individuals usually have a dark brown
transversal band (e.g. QCAZ 39247) or two narrower
transversal bars usually interconnected on the sacral
region (e.g. QCAZ 13962). In some specimens there
are scattered minute black flecks and white rounded
spots on the dorsal surfaces (e.g. QCAZ 38772, 56114).
Individuals may display asymmetrically distributed
large cream blotches on the dorsum (e.g. QCAZ 44845).
The coloration of flanks is grey with white speckles
or sometimes with brown blotches. Vertical bars are
present on the hidden surfaces of the thighs and
sometimes on the shanks (e.g. QCAZ 39137). Dark
transversal bands 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). The
© 2020 The Linnean Society of London, Zoological Journal of the Linnean Society, 2020, XX, 1–32
Downloaded from https://academic.oup.com/zoolinnean/advance-article-abstract/doi/10.1093/zoolinnean/zlaa002/5810752 by University of Rochester user on 22 March 2020
19.3 mm, eye diameter 6.2 mm, tympanum diameter
4.8 mm, tibia length 27.8 mm, femur length 26.4 mm,
calcar length 2.3 mm, arm length 9.9 mm, eye–nostril
distance 7.1 mm, head wider than long and wider than
body; snout truncate in lateral view, subacuminate in
dorsal view; distance from nostril to eye larger than
diameter of eye; canthus rostralis rounded; loreal region
concave; internarial region subtly depressed; nostrils
not protuberant, directed laterally; interorbital area
slightly convex; eye large, protuberant; lower eyelid
finely reticulated; diameter of eye 1.3 times diameter of
tympanic annulus; supratympanic fold inconspicuous;
tympanic membrane roundish, slightly longer than
high; tympanic annulus evident, rounded, separated
from eye by c. 0.4 times its diameter. Arm slender,
axillary membrane absent; relative length of fingers
I < II < IV < III; finger discs broadly expanded, round,
as wide as long; subarticular tubercles prominent,
round to ovoid, single; supernumerary tubercles
absent; palmar tubercle inconspicuous; large and
rugose dark P-shaped nuptial pad on dorsal surface
of finger I; prepollex not modified into a projecting
spine; webbing formula of fingers I basal II1—2–III2–
—1+IV. Toes bearing discs broadly expanded, rounded
and slightly smaller than those of fingers; relative
length of toes I < II < V < III < IV; inner metatarsal
tubercle large, elliptical; outer metatarsal tubercle
absent; subarticular tubercles single, round, large and
protuberant; supernumerary tubercles small, round;
webbing formula of toes I1—1II1–—1III1—1IV1— 1–V.
Vocal sac single and subgular. Skin on dorsum, head
and dorsal surfaces of limbs finely granular; throat
smooth anteriorly without mental gland; posterior
surface of throat, belly and ventral surfaces of thighs
areolate, those of shanks smooth. Cloacal opening
directed posteriorly at upper level of thighs, round
tubercles below and on the sides of vent. Tongue
slightly cordiform, widely attached to mouth floor;
odontophores situated parallel to choanae, in two
oblique series closely set, each bearing 19–21 vomerine
teeth; choanae trapezoidal, oblique.
25
26
M. A. CAMINER and S. R. RON
heels have usually the same colour as the rest of the
body (e.g. QCAZ 8181, 13962), but in some individuals,
heels are cream contrasting with the background
dorsal coloration (e.g. QCAZ 49233).
Ventral surfaces of preserved specimens vary from
creamy white (e.g. QCAZ 30928) to yellowish white
(e.g. QCAZ 8181) with a belly usually yellowish. Lips,
throat, chest, belly and ventral surfaces of the limbs
usually with brown blotches (e.g. QCAZ 39247, 49233,
56114). Coloration of webbing and discs vary from
yellowish white to brown or cream. Coloration of bones
is white or green.
Coloration in life (Fig. 7C): Dorsal surfaces vary from
brown (e.g. QCAZ 56114) to dark brown (e.g. QCAZ
56307) or reddish brown (e.g. QCAZ 38772); brown
transversal bands on dorsal surfaces of limbs; brown
marks and white or yellow blotches on dorsum (e.g.
QCAZ 56114, 56429); in some individuals, there are
scattered minute black dots on dorsal surfaces (e.g.
QCAZ 39130); flanks are grey or bluish with white
speckles; hidden surfaces of thighs and shanks are
bluish with dark vertical bars; venter and ventral
surfaces of thighs vary from yellowish to cream white
usually with brown blotches on the belly (e.g. QCAZ
39130, 56429); throat cream white or yellowish with or
without brown blotches; webbings vary from brown to
orange; fingers and toes dorsally vary from brown to
grey; iris copper (e.g. QCAZ 51236); palpebrum finely
reticulated with golden yellow.
© 2020 The Linnean Society of London, Zoological Journal of the Linnean Society, 2020, XX, 1–32
Downloaded from https://academic.oup.com/zoolinnean/advance-article-abstract/doi/10.1093/zoolinnean/zlaa002/5810752 by University of Rochester user on 22 March 2020
Figure 11. Adult preserved specimens of Boana ventrimaculata showing variation in dorsal and ventral coloration. From
left to right, first and second rows: QCAZ 51241 (holotype), 13962, 30928, 38772, 56114, 49233 (males); third and fourth
rows: QCAZ 44845 (male), 56307, 39137, 39247, 8181 (females). See listed in the type-series for locality data. All specimens
are shown at the same scale.
SYSTEMATICS OF BOANA SEMILINEATA GROUP
Vocalization: One male (QCAZ 49233) was recorded
at Yasuní, on the road Pompeya Sur-Iro, km 108
(Provincia Orellana) by Morley Read on 4 November
1996 at 20:00 h. The call (Fig. 9G, H; Table 2) consists
of one groan-like note with a mean duration of 0.24 s
and dominant frequency of 561.4 Hz. Other call
parameters are shown in Table 2. The individual was
perched on vegetation 1 m above a stream.
Distribution and ecology: Boana ventrimaculata
inhabits the Amazon basin of Ecuador (provinces
of Morona Santiago, Napo, Orellana, Pastaza and
Sucumbíos) and Brazil (Estado do Amazonas) (Fig. 6).
Localities with known elevation range vary between
64 and 1035 m of elevation. Among localities with
known elevation, Comunidad Tarangaro (1035 m)
is the highest and Rio Içá (64 m) is the lowest. Most
specimens of B. ventrimaculata were found in terra
firme forest and semi-flooded forests. Frogs have
been found at night in primary and secondary forest,
perching on vegetation 20 to 500 cm above the ground,
near streams or along trails away from water bodies.
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) Amazonian lowland evergreen
forest, characterized by high plant alpha-diversity and
a canopy height of 30 m with emergent trees that reach
40 m; (3) lowland floodplain forest of white-waters,
characterized by periodical flooding with white-waters
from large rivers, with the vegetation reaching 35 m in
height and several horizontal strata of vegetation; and
(4) lowland forest of Mauritia flexuosa palms and blackwaters characterized by a canopy height of 30 m with
dense understory (Sierra et al., 1999). Localities in Brazil
fall within the Purus Varzea vegetation type (according
to the World Wildlife Fund, 2017 classification scheme;
available at http://www.eoearth.org/view/article/151948).
DISCUSSION
Similar to recent reviews of Neotropical hylids based
on genetic characters (e.g. Ron et al., 2012, 2018;
Caminer & Ron 2014; Ferrão et al., 2016; Caminer
et al., 2017; Rivadeneira et al., 2018), our results and
those of Fouquet et al. (2016) and Peloso et al. (2018)
show that Boana geographica, which until recently
was considered a single widespread species, is a
species complex. Four new species have been described
and one resurrected since 2016 [herein and in Fouquet
et al. (2016) and Peloso et al. (2018)]. Nevertheless,
there are at least six seemingly undescribed species
that would require further systematic work (in
addition of B. hutchinsi that remains to be included
in a molecular phylogeny), especially in Amazonian
Brazil. The B. semilineata group exemplifies the vast
work on systematics needed to solve the Linnean
shortfall in Neotropical amphibians.
The phylogeny and geographic distribution of species
of the Boana semilineata group suggest an origin in
the Amazon basin. This scenario is suggested by the
Amazonian distribution of most species of the group,
including those diverging more basally: B. boans,
B. icamibia and B. wavrini. The only exception is
B. pombali, which occurs in the Atlantic forest.
An Amazonian origin would suggest that species
occurring in the Atlantic forest and the Guianan
region dispersed from the Amazon region. Boana
nigra is the only species of the group occurring almost
exclusively above 1000 m of elevation (range from 910
to 1847 m in B. nigra; < 1100 m in all other species).
This implies a recent dispersal from lowland tropical
rainforests to Andean montane forests. Boana nigra
has a parapatric distribution with its sister species,
B. appendiculata. Other example of recent dispersion
from the Amazon lowlands to the Andean slopes have
been shown in the Boana calcarata–fasciata species
complex (Caminer & Ron, 2014), where the sister
species B. almendarizae and B. fasciata are mainly
distributed in Andean montane forests (range from
500 to 1950 m), while the rest of the group occurs in
adjacent Amazonian lowlands (< 650 m). These types
of distribution pattern could result from parapatric
speciation along the elevation gradient.
We combine molecular, morphological, bioacoustic
and environmental data to delimit species within the
B. semilineata group. Although we find differences
in advertisement calls and morphology (large males
and females are easy to differentiate), there is
remarkable similarity between small individuals of
B. appendiculata and B. nigra. Subadults, and small
adults of both species, show uniform black coloration
on flanks and hidden surfaces of the thighs that make
their identification challenging. Without genetic data
it is difficult to recognize some specimens (e.g. QCAZ
52610, 52734) as distinct.
The scant ecological data suggest differences in
habitat preference between the sympatric species
B. appendiculata and B. ventrimaculata. Boana
ventrimaculata has been recorded in forests near lotic
waters. Boana appendiculata, in contrast, usually
occurs near lentic waters in open areas. Additional
data on habitat preferences are needed to test these
presumed differences.
Our results allow us to determine the status of
trans-Andean Chocoan populations of B. boans.
© 2020 The Linnean Society of London, Zoological Journal of the Linnean Society, 2020, XX, 1–32
Downloaded from https://academic.oup.com/zoolinnean/advance-article-abstract/doi/10.1093/zoolinnean/zlaa002/5810752 by University of Rochester user on 22 March 2020
In recently metamorphosed individuals, flanks,
hidden surfaces of thighs and webbing are black;
venter is grey; dorsal surfaces of the limbs are dark
brown; dorsum is brown with black flecks over the
entire surface. (Fig. 8C).
27
28
M. A. CAMINER and S. R. RON
ACKNOWLEDGEMENTS
Field and laboratory work were funded by grants
from the Secretaría Nacional de Educación Superior,
Ciencia, Tecnología e Innovación del Ecuador
SENESCYT (Arca de Noé initiative; SRR and Omar
Torres principal investigators) and PUCE-DGA (SRR
principal investigator). Charlie Barnes, D. Flores,
G. Nicholls, A. Manzano and S. Espinoza carried out
extractions and amplification of DNA. Diego PáezMoscoso helped organizing sequences from GenBank.
William E. Duellman, Diego R. Batallas and The
Cornell Lab of Ornithology provided call recordings.
Andrea Varela provided photographs of the lectotype
of Hyla appendiculata. Andrew Crawford provided
access to the amphibian collection of Universidad
de los Andes, Colombia. Mario Yánez-Muñoz
from INABIO loaned a tissue of Boana rubracyla.
Ministerio de Ambiente del Ecuador provided collect
and research permits (001-11 IC-FAU-DNB/MA;
005-12-IC-FAU-DNB/MA; 003-15-IC-FAU-DNB/
MA; 002-16-IC-FAU-DNB/MA; 003-17-IC-FAU-DNB/
MA; 005-2009-INVESTIGACIÓN-B-DPMS/MAE;
006-2012-FAU-MAE-DPO-PNY; 010-2013-FAU-MAEDPAO-PNY; 008-IC-FAU-DPPZ/MA; 016-2015-FAUMAE-DPAO; MAE-DNB-ARRGG-CM-2014-0002).
For collection of specimens of the new species we are
indebted to D. Rivadeneira, D. Velalcázar, J. Pinto,
F. Mora, D. Núñez, J. C. Sánchez, A. Correa, D. Paucar,
P. Venegas, P. Baldeón, E. Nusirquia, J. Brito, D. R.
Batallas, P. Bejarano, D. Morocho, S. Poe, F. Ayala,
L. Gray, J. Davis, I. Latella, L. MacLean, A. Almendáriz,
I. Tapia, L. Coloma, A. Carvajal, A. Merino, E. Arbeláez,
A. Tapia, D. Salazar, D. Acosta, C. Korfel, T. Camacho,
O. Pérez, M. Bustamante, D. Alvarado, G. Buitrón,
M. Díaz, V. Ruales, Y. Mera, E. Carrillo, G. Díaz,
Y. Sagredo, A. Barahona, M. Ortega, F. Timias, M. J.
Navarrete, G. Galarza, V. Chasiluisa, S. Arroyo,
D. Zurita, E. Toral, P. Salvador, M. D. Proaño, M. Read,
D. Prado, S. de la Torre, F. Nogales and S. Harris.
REFERENCES
Acevedo AA, Lampo M, Cipriani R. 2016. The cane or
marine toad Rhinella marina (Anura: Bufonidae): two
genetically and morphologically distinct species. Zootaxa
4103: 574–586.
Aguilar C, Wood PL Jr, Belk MC, Duff MH, Sites JW Jr.
2016. Different roads lead to Rome: integrative taxonomic
approaches lead to the discovery of two new lizard
lineages in the Liolaemus montanus group (Squamata:
Liolaemidae). Biological Journal of the Linnean Society
120: 448–467.
Caminer MA, Ron SR. 2014. Systematics of treefrogs of
the Hypsiboas calcaratus and Hypsiboas fasciatus species
© 2020 The Linnean Society of London, Zoological Journal of the Linnean Society, 2020, XX, 1–32
Downloaded from https://academic.oup.com/zoolinnean/advance-article-abstract/doi/10.1093/zoolinnean/zlaa002/5810752 by University of Rochester user on 22 March 2020
Unexpectedly, we find low genetic distances between
samples from the Chocó region and the Amazonian
region of Ecuador, suggesting that they represent a
single species. This is a surprising finding, because the
presence of the Andean barrier between them should
have prevented gene flow for an extended period. All
other amphibian species from lowland rainforests
occur either in the Chocó or in Amazonia, but not in
both. The few species that were believed to have a
trans-Andean distribution turned out to represent
separate species, one on each side of the mountain
range. Rhinella marina, for example, was split in
R. horribilis for populations west of the Andes and
R. marina for population on the east (Acevedo et al.,
2016). Boana boans and B. xerophylla (Escalona et al.,
2019) appear to be the only two species in which a
trans-Andean distribution has been confirmed with
genetic evidence. However, B. boans is the only species
occurring in Chocoan rainforests.
At a broader scale, our phylogenetic analysis
indicates the existence of a high number of potential
new species in the genus Boana in Ecuador and
Colombia. Ecuadorian populations of ‘B. pellucens’
from Durango and Lita (Provincia Esmeraldas) are
most closely related to B. rufitela from Panama than
to other Ecuadorian populations of B. pellucens. High
genetic distances between both clades of Ecuadorian
populations (5%) also hint that Ecuadorian populations
encompass two species. Our results suggest that
populations from Esmeraldas are B. rufitela or an
undescribed species. Until this issue is solved, we
consider B. rufitela as occurring in Ecuador.
The non-monophyly of the B. punctata group is
congruent with results by Pyron & Wiens (2011)
and Duellman et al. (2016). Paraphyly is due to the
position of B. picturata, which has weak support.
Although other species have been assigned to
this group (i.e. B. alemani, B. atlantica, B. hobbsi,
B. jimenezi and B. liliae) on the basis of morphological
similarity (Faivovich et al., 2005; Kok, 2006; Señariz
& Ayarzagüena, 2006), their position based on genetic
characters needs to be determined.
Our phylogenetic analyses indicate that Boana
cinerascens and B. punctata form two species
complexes consisting of four to six divergent lineages
each (uncorrected p-distances among lineages range
from 4.1 to 11% in B. cinerascens and 2.9 to 6.6% in
B. punctata). Some of these lineages were reported
by Faivovich et al. (2004, 2005), Fouquet et al. (2007),
Jansen et al. (2011) and Guarnizo et al. (2015).
Elucidation of the status of candidate species outside
the B. semilineata species group is beyond the scope
of this publication. Therefore, we leave them as
mtDNA highly divergent lineages that could represent
undescribed species (Fig. 1).
SYSTEMATICS OF BOANA SEMILINEATA GROUP
Fouquet A, Martinez Q, Zeidler L, Courtois EA,
Gaucher P, Blanc M, Dias Lima J, Marques Souza S,
Rodrigues M, Kok PJR. 2016. Cryptic diversity in the
Hypsiboas semilineatus species group (Amphibia, Anura)
with the description of a new species from the eastern
Guiana Shield. Zootaxa 4084: 79–104.
Frost DR. 2019. Amphibian species of the world: an online
reference, version 6.0. New York: American Museum of
Natural History. Electronic Database accessible. Available
at: http://research.amnh.org/herpetology/amphibia/index.
html (accessed 14 May 2019).
Guarnizo CE, Paz A, Muñoz-Ortiz A, Flechas SV, MéndezNarváez J, Crawford AJ. 2015. DNA barcoding survey
of anurans across the Eastern Cordillera of Colombia and
the impact of the Andes on cryptic diversity. PLoS One 10:
e0127312.
Jansen M, Bloch R, Schulze A, Pfenninger M. 2011.
Integrative inventory of Bolivia’s lowland anurans reveals
hidden diversity. Zoologica Scripta 40: 567–583.
Kohler J, Jansen M, Rodriguez A, Kok PJR, Toledo LF,
Emmrich M, Glaw F, Haddad CFB, Rodel MO, Vences M.
2017. The use of bioacoustics in anuran taxonomy: theory,
terminology, methods and recommendations for best practice.
Zootaxa 4251: 1–124.
Kok P. 2006. A new species of Hypsiboas (Amphibia: Anura:
Hylidae) from Kaiateur National Park, eastern edge of the
Pakaraima mountains, Guyana. Bulletin de l’Institut Royal
des Sciences Naturelles de Belgique 76: 191–200.
La Marca E, Azevedo-Ramos C, Coloma LA, Solis F,
Ibáñez R, Jaramillo C, Fuenmayor Q, Ron SR,
Hardy J. 2010. Hypsiboas boans. The IUCN Red List of
Threatened Species 2010: e.T55415A11304871. Available
at: https://dx.doi.org/10.2305/IUCN.UK.2010-2.RLTS.
T55415A11304871.en (accessed 15 October 2018).
Lanfear R, Calcott B, Ho SYW, Guindon S. 2012.
PartitionFinder: combined selection of partitioning schemes
and substitution models for phylogenetic analyses. Molecular
Biology and Evolution 29: 1695–1701.
Linnaeus C. 1758. Systema naturae per regna tria naturae,
secundum classes, ordines, genera, species, cum characteribus,
differentiis, synonymis, locis, 10th edn, Vol. 1. Stockholm: L.
Salvius.
Lutz B. 1963. New species of Hyla from southeastern Brazil.
Copeia 1963: 561–562.
Maddison WP, Maddison DR. 2015. Mesquite: a modular
system for evolutionary analysis, version 3.04. Available at:
http://mesquiteproject.org/ (accessed 20 August 2015).
Meza-Joya FL, Ramos-Pallares E, Hernández-Jaimes C.
2019. Hidden diversity in frogs within Boana calcarata–
fasciata and Boana geographica species complexes from
Colombia. Herpetology Notes 12: 391–400.
Myers CW, Duellman WE. 1982. A new species of Hyla from
Cerro Colorado, and other tree frog records and geographical
notes from Western Panama. American Museum Novitates
2752: 1–32.
Parker HW. 1933. A list of the frogs and toads of Trinidad.
Tropical Agriculture 10: 8–12.
© 2020 The Linnean Society of London, Zoological Journal of the Linnean Society, 2020, XX, 1–32
Downloaded from https://academic.oup.com/zoolinnean/advance-article-abstract/doi/10.1093/zoolinnean/zlaa002/5810752 by University of Rochester user on 22 March 2020
complex (Anura, Hylidae) with the description of four new
species. ZooKeys 370: 1–68.
Caminer MA, Milá B, Jansen M, Fouquet A, Venegas PJ,
Chávez G, Lougheed SC, Ron SR. 2017. Systematics of
the Dendropsophus leucophyllatus species complex (Anura:
Hylidae): cryptic diversity and the description of two new
species. PLoS One 12: e0171785.
Caramaschi U, Pimenta BVS, Feio RN. 2004. Nova
especie do grupo de Hyla geographica Spix, 1824 da floresta
Atlântica, Brasil (Amphibia, Anura, Hylidae). Boletim do
Museu Nacional. Nova Serie, Zoologia. Rio de Janeiro 518:
1–14.
Cocroft RB, Ryan MJ. 1995. Patterns of advertisement call
evolution in toads and chorus frogs. Animal Behaviour 49:
283–303.
Dayrat B. 2005. Towards integrative taxonomy. Biological
Journal of the Linnean Society 85: 407–415.
Dos Santos SP, Ibáñez R, Ron SR. 2015. Systematics of
the Rhinella margaritifera complex (Anura, Bufonidae)
from western Ecuador and Panama with insights in the
biogeography of Rhinella alata. ZooKeys 501: 109–145.
Duellman WE. 1970. Hylid frogs of Middle America.
Monograph of the Museum of Natural History University of
Kansas 1: 1–753.
Duellman WE. 1973. Frogs of the Hyla geographica group.
Copeia 1973: 515–533.
Duellman WE, Pyles RA. 1983. Acoustic resource partitioning
in anuran communities. Copeia 1983: 639–649.
Duellman WE, Marion AB, Hedges B. 2016. Phylogenetics,
classification, and biogeography of the treefrogs (Amphibia:
Anura: Arboranae). Zootaxa 4104: 1–109.
Escalona MD, Acuña Juncá F, Giaretta AA, Crawford AJ,
La Marca E. 2019. Contrasting genetic, acoustic, and
morphological differentiation in two closely related gladiator
frogs (Hylidae: Boana) across a common Neotropical
landscape. Zootaxa 4609: 519–547.
Faivovich J, García PCA, Ananias F, Lanari L, Basso NG,
Wheeler WC. 2004. A molecular perspective on the
phylogeny of the Hyla pulchella species group (Anura,
Hylidae). Molecular Phylogenetics and Evolution 32:
938–950.
Faivovich J, Haddad CFB, García PC, Frost DR,
Campbell JA, Wheeler WC. 2005. Systematic review of
the frog family Hylidae, with special reference to Hylinae:
phylogenetic analysis and taxonomic revision. Bulletin of the
American Museum of Natural History 294: 1–240.
Ferrão M, Colatreli O, de Fraga R, Kaefer IL, Moravec J,
Lima AP. 2016. High species richness of Scinax treefrogs
(Hylidae) in a threatened Amazonian landscape revealed by
an integrative approach. PLoS One 11: e0165679.
Fick SE, Hijmans RJ. 2017. Worldclim 2: new 1-km
spatial resolution climate surfaces for global land areas.
International Journal of Climatology 37: 4302–4315.
Fouquet A, Gilles A, Vences M, Marty C, Blanc M,
Gemmell NJ. 2007. Underestimation of species richness in
neotropical frogs revealed by mtDNA analyses. PLoS One 2:
e1109.
29
30
M. A. CAMINER and S. R. RON
Ron SR, Venegas PJ, Ortega-Andrade HM, GagliardiUrrutia G, Salerno P. 2016. Systematics of Ecnomiohyla
tuberculosa with the description of a new species and
comments on the taxonomy of Trachycephalus typhonius
(Anura, Hylidae). ZooKeys 630: 115–154.
Ron SR, Duellman WE, Caminer MA, Pazmiño D. 2018.
Advertisement calls and DNA sequences reveal a new
species of Scinax (Anura: Hylidae) on the Pacific lowlands of
Ecuador. PLoS One 13: e0203169.
Ron SR, Merino-Viteri A, Ortiz DA. 2019. Anfibios del
Ecuador, version 2019.0. Available at: https://bioweb.bio/
faunaweb/amphibiaweb/ (accessed 2 February 2019).
Ronquist F, Teslenko M, van der Mark P, Ayres DL,
Darling A, Höhna S, Larget B, Liu L, Suchard MA,
Huelsenbeck JP. 2012. MrBayes 3.2: efficient bayesian
phylogenetic inference and model choice across a large model
space. Systematic Biology 61: 539–542.
Sambrook J, Fritsch EF, Maniatis T. 1989. Molecular
cloning: a laboratory manual. New York: Cold Spring Harbor
Laboratory Press.
SAS Institute. 2010. User guide, version 9.01. Cary: SAS
Institute. Available at: http://www.jmp.com/ (accessed 12
December 2010).
Savage JM, Heyer WR. 1967. Variation and distribution in
the tree-frog genus Phyllomedusa in Costa Rica, Central
America. Studies on Neotropical Fauna and Environment 5:
111–131.
Señariz JC, Ayarzagüena J. 2006. A new species of
Hypsiboas (Amphibia; Anura; Hylidae) from the Venezuelan
Guyana, with notes on Hypsiboas sibleszi (Rivero, 1972).
Herpetologica 62: 308–318.
Sierra R, Cerón C, Palacios W, Valencia R. 1999. Mapa de
vegetación del Ecuador Continental 1:1’000.000. Quito: Proyecto
INEFAN/GEF-BIRF, Wildlife Conservation Society y Ecociencia.
Spix JB. 1824. Animalia nova sive species novae Testudinum et
Ranarum quas in itinere per Brasiliam annis MDCCCXVII–
MDCCCXX jussu et auspiciis Maximiliani Josephi
I. Bavariae Regis. München: F. S. Hübschmann.
Tamura K, Peterson D, Peterson N, Stecher G, Nei M,
Kumar S. 2011. MEGA5: molecular evolutionary genetics
analysis using maximum likelihood, evolutionary distance,
and maximum parsimony methods. Molecular Biology and
Evolution 28: 2731–2739.
Vieites DR, Wollenberg KC, Andreone F, Köhler J,
Glaw F, Vences M. 2009. Vast underestimation of
Madagascar’s biodiversity evidenced by an integrative
amphibian inventory. Proceedings of the National
Academy of Sciences of the United States of America 106:
8267–8272.
Zhang J, Kapli P, Pavlidis P, Stamatakis A. 2013. A
general species delimitation method with applications to
phylogenetic placements. Bioinformatics 29: 2869–2876.
Zwickl DJ. 2006. Genetic algorithm approaches for the
phylogenetic analysis of large biological sequence datasets
under the maximum likelihood criterion. Unpublished Ph.D.
Thesis, University of Texas, Austin, 125.
© 2020 The Linnean Society of London, Zoological Journal of the Linnean Society, 2020, XX, 1–32
Downloaded from https://academic.oup.com/zoolinnean/advance-article-abstract/doi/10.1093/zoolinnean/zlaa002/5810752 by University of Rochester user on 22 March 2020
Parker HW. 1935. The frogs, lizards and snakes of British
Guiana. Proceedings of the Zoological Society of London
1935: 505–530.
Parker HW. 1936. A collection of reptiles and amphibians
from the Upper Orinoco. Bulletin du Musée Royal d’Histoire
Naturelle de Belgique 12: 1–4.
Paz A, Crawford AJ. 2012. Molecular-based rapid inventories
of sympatric diversity: a comparison of DNA barcode clustering
methods applied to geography-based vs clade-based sampling
of amphibians. Journal of Biosciences 37: 887–896.
Peloso PLV, De Oliveira RM, Sturaro MJ, Rodrigues MT,
Lima-Filho GR, Bitar YOC, Wheeler WC, Aleixo A. 2018.
Phylogeny of map tree frogs, Boana semilineata species
group, with a new Amazonian species (Anura: Hylidae).
South American Journal of Herpetology 13: 150–169.
Pimm SL, Jenkins CN, Abell R, Brooks TM, Gittleman JL,
Joppa LN, Raven PH, Roberts CM, Sexton JO. 2014.
The biodiversity of species and their rates of extinction,
distribution, and protection. Science 344: 1246752.
Puillandre N, Lambert A, Brouillet S, Achaz G. 2012.
ABGD, automatic barcode gap discovery for primary species
delimitation. Molecular Ecology 21: 1864–1877.
Pyburn WF, Hall DH. 1984. A new stream inhabiting treefrog
(Anura: Hylidae) from southeastern Colombia. Herpetologica
40: 366–372.
Pyron RA, Wiens JJ. 2011. A large-scale phylogeny of
Amphibia including over 2800 species, and a revised
classification of extant frogs, salamanders, and caecilians.
Molecular Phylogenetics and Evolution 61: 543–583.
QGIS Development Team. 2018. QGIS geographic
information system. Open Source Geospatial Foundation
Project. Available at: http://qgis.osgeo.org (accessed 11 April
2019).
Rambaut A, Drummond A. 2007. TRACER. MCMC trace
analysis tool, version 1.4. University of Oxford. Available
at: http://tree.bio.ed.ac.uk/software/tracer (accessed 10
September 2010).
Rissler LJ, Apodaca JJ. 2007. Adding more ecology
into species delimitation: ecological niche models and
phylogeography help define cryptic species in the black
salamander (Aneides flavipunctatus). Systematic Biology 56:
924–942.
Rivadeneira CD, Venegas PJ, Ron SR. 2018. Species limits
within the widespread Amazonian treefrog Dendropsophus
parviceps with descriptions of two new species (Anura,
Hylidae). ZooKeys 726: 25–77.
Ron SR, Read M. 2018. Boana geographica. In: Ron SR,
Merino-Viteri A, Ortiz DA, eds. Anfibios del Ecuador, version
2018.0. Museo de Zoología, Pontificia Universidad Católica
del Ecuador. Database accessible. Available at: https://
bioweb.bio/faunaweb/amphibiaweb/FichaEspecie/Boana%20
geographica (accessed 11 October 2018).
Ron SR, Venegas PJ, Toral E, Read M, Ortiz DA,
Manzano AL. 2012. Systematics of the Osteocephalus
buckleyi species complex (Anura, Hylidae) from Ecuador and
Peru. ZooKeys 229: 1–52.
SYSTEMATICS OF BOANA SEMILINEATA GROUP
31
SUPPORTING INFORMATION
Table S1. Genbank accession numbers for DNA sequences used in the phylogenetic analysis.
Table S2. Primers used for DNA amplification.
Figure S1. Species delimitation tree based on the Poisson Tree Process (PTP) model, using a fragment of the 16S
mitochondrial gene. The blue lines indicate branching processes among species, while red lines indicate taxa that
should be considered as part of the same lineage.
Figure S2. ABGD species delimitation.
APPENDIX
EXAMINED SPECIMENS
Boana appendiculata. ECUADOR: PROVINCIA
MORONA SANTIAGO: on the road Puerto Morona-San
José de Morona (2.8802° S, 77.6788° W), 200 m (QCAZ
52610); PROVINCIA ZAMORA CHINCHIPE: Centro
Shuar Yawi (4.4495° S, 78.6489° W), 945 m (QCAZ
31035–36); PROVINCIA NAPO: Ahuano (1.0545° S,
77.5483° W), 385 m (QCAZ 36723); Cotundo, Huasquila
Lodge (0.8237° S, 77.8015° W), 804 m (QCAZ 57191);
Huino (0.6449° S, 77.149° W), 273 m (QCAZ 30910,
30921); Reserva Ecológica Yachana (0.8457° S, 77.2287°
W), 317 m (QCAZ 48846); Serena (1.0961° S, 77.9243°
W), 551 m (QCAZ 25849–50); on the road Miazi-Nuevo
Paraíso (4.3091° S, 78.645° W) 1006 m (QCAZ 67108–
24); PROVINCIA SUCUMBÍOS: Reserva Faunística
Cuyabeno (0.0885° S, 76.142° W), 222 m (QCAZ 2157,
2061, 2123–24, 2208, 2213–14, 2216, 2220, 2222, 2798,
3357, 4930–31, 6210–16, 25955); Zábalo (0.3181° S,
75.7662° W), 220 m (QCAZ 27729, 27937); Comunidad
Chonta Yacu (0.1115° S, 77.3743° W), 610 m (QCAZ
25797); Selva Lodge (0.4981° S, 76.3738° W), 250 m
(QCAZ 12025); Rey de los Andes (0.2082° S, 76.2368°
W), 270 m (QCAZ 28477); Puerto Libre (0.1912° N,
77.4882° W), 700 m (QCAZ 21406); Parque Pacayacu
(0.0378° S, 76.5857° W), 262 m (QCAZ 23055); Puerto
Bolívar (0.0886° S, 76.142° W), 240 m (QCAZ 28227);
bridge of the Río Cuyabeno, on the road Tarapoa-Palma
Roja (0.032° S, 76.3205° W), 240 m (QCAZ 6060–61);
Limoncocha (0.4062° S, 76.6194° W), 261 m (QCAZ
3357–62, 4679, 8016, 8019, 43082, 43095, 43112,
43136, 43139, 43141, 61766–68, 63542); PROVINCIA
PASTAZA: Shell (1.5064° S, 78.0607° W), 1050 m
(QCAZ 36088–90, 36095, 36100, 36201–3, 36209–14,
36217, 36220); Sarayacu (1.7245 °S, 77.4755 °W), 383
m (QCAZ 52727, 52733–34); Río Tiguino, Bataburo
Lodge (1.25° S, 76.6667° W), 238 m (QCAZ 20015,
20029–30, 20080–81, 39416); Pomona, Fundación
Hola Vida (1.625° S, 77.9072° W), 846 m (QCAZ
25701, 25714, 25626, 25636, 37161–62, 37197); 5 km
N of the Puyo, on the road to the Tena Hostería Safari
(1.443° S, 77.9966° W), 954 m, (QCAZ 26255, 26267);
Bobonaza (1.4945° S, 77.8696° W), 660 m, (QCAZ
40749); Canelos (1.5793° S, 77.7321° W), 650 m (QCAZ
48704–7, 52845, 56610, 56586–87); Lorocachi (1.6377°
S, 75.9703° W), 210 m (QCAZ 55943, 55973); Centro
Etnoturístico Indichuris (1.6509° S, 77.9213° W), 799
m (QCAZ 57791); Fátima (1.4113° S, 78° W), 1023 m
(QCAZ 6244–45, 6248, 49320–21, 49324, 49333, 49341,
49353, 30323); Chunitayo (1.9625° S, 77.98° W), 1000
m (QCAZ 11063); Estación Científica Oglán (1.3235°
S, 77.9912° W), 568 m (QCAZ 58102); Centro Ecológico
Zanja Arajuno (1.3567° S, 77.8594° W), 903 m (QCAZ
36932–34, 62064, 62081); PROVINCIA ORELLANA:
Estación Científica Yasuní PUCE (0.6771° S, 76.4011°
W), 250 m (QCAZ 2385, 8419–20, 14819, 16619, 16801–
2, 16804–8, 16809, 16812, 18293, 19197–200, 19678,
20833, 22461–63, 22500, 22572–75, 22673–74, 22676,
22769, 23073, 36027, 41004, 41007–10); Añango, Parque
Nacional Yasuní (0.53333° S, 76.45° W), 243 m (QCAZ
5528–30, 5086–87, 17253); on the road Pompeya-Iro,
Parque Nacional Yasuní (0.6535° S, 76.4535° W), 238
m (QCAZ 5076–77, 5083–84, 5099, 5102, 5145, 5150,
5154, 5162, 5175, 5228–30, 5239, 5242–44, 5247, 5376–
78, 5383, 5418–20, 8204–9, 8211–12, 9678–79, 9700–1,
12382, 13311–12, 17252, 32719, 36028, 46499, 51170,
51179–82, 53399); Río Yasuní, Parque Nacional Yasuní
(0.9047° S, 76.144° W), 228 m (QCAZ 64083–107);
Edén, Río Napo (0.4983° S, 76.0711° W), 216 m (QCAZ
44159–75, 44226); Tambococha (0.9783° S, 75.4256°
W), 177 m (QCAZ 55227, 55234, 55242, 55253, 55261,
55315, 55356, 55360–61, 55393); Huiririma, Río Napo
(0.7116° S, 75.6239° W), 194 m (QCAZ 44578–83);
Pompeya, Sacha Lodge (0.4705° S, 76.4597° W), 247
m (QCAZ 37873, 37879, 39339); Reserva Biológica Río
Bigal (0.5525° S, 77.4199° W), 907 m (QCAZ 48491);
Río Huataraco, 70 km E of Hollín (0.747° S, 77.354°
W), 380 m (QCAZ 7117); Chiroisla, Río Napo (0.5756°
S, 75.8998° W), 203 m (QCAZ 44434); Comunidad
Sinchichicta, Río Napo (0.6793° S, 75.8454° W), 220
m (QCAZ 57927); La Primaver, Río Napo (0.4442° S,
76.868° W), 244 m (QCAZ 43850); Río Pusuno (0.7263°
S, 76.7266° W), 266 m (QCAZ 14078); bridge over
the Río Tiputini, near campamento ELF (0.7417° S,
76.8611° W), 295 m (QCAZ 7898, 7913); Pindo (0.7489°
S, 76.7693° W), 304 m (QCAZ 49738); Coca (0.4691°
© 2020 The Linnean Society of London, Zoological Journal of the Linnean Society, 2020, XX, 1–32
Downloaded from https://academic.oup.com/zoolinnean/advance-article-abstract/doi/10.1093/zoolinnean/zlaa002/5810752 by University of Rochester user on 22 March 2020
Additional Supporting Information may be found in the online version of this article at the publisher's web-site.
32
M. A. CAMINER and S. R. RON
Nacional Yasuní (0.4716° S, 76.5832° W), 243 m (QCAZ
43039); PROVINCIA SUCUMBÍOS: Puerto Bolívar
(0.0886° S, 76.142° W), 240 m (QCAZ 28184).
Boana pellucens. ECUADOR: PROVINCIA EL
ORO: route from Huaquillas to La Cuca Arenillas
(3.5357° S, 80.0671° W), 53 m (QCAZ 23680); 8 km
on the road La Avanzada-Playó (3.588° S, 79.919° W),
230 m (QCAZ 30594); near Valle Hermoso (3.5116° S,
79.8202° W), 282 m (QCAZ 37254); Carabota (3.3283°
S, 75.581° W), 395 m (QCAZ 45317); PROVINCIA
ESMERALDAS: Durango (1.0724° N, 78.6534° W), 120
m (QCAZ 13192, 40270); PROVINCIA PICHINCHA:
La Unión del Toachi (0.3318° S, 78.9379° W), 901 m
(QCAZ 24359); PROVINCIA SANTO DOMINGO DE
LOS TSÁCHILAS: Alluriquín-La Florida (0.2777° S,
79.0181° W), 971 m (QCAZ 13875).
B o a n a p i c t u r a t a . E C UA D O R : P R OV I N C I A
ESMERALDAS: 3 km of Durango (1.0643° N, 78.6443°
W), 158 m (QCAZ 15549); PROVINCIA PICHINCHA:
Amanecer Campesino, Río Santiago (0.0485° S, 78.9641°
W), 838 m (QCAZ 32452); PROVINCIA SANTO
DOMINGO DE LOS TSÁCHILAS: Alluriquín-La
Florida (0.2777° S, 79.0181° W), 971 m (QCAZ 13894).
B o a n a p u n c t a t a . E C UA D O R : P R OV I N C I A
MORONA SANTIAGO: Pankints (2.9019° S, 77.894°
W), 332 m (QCAZ 46423); PROVINCIA NAPO:
Estación Biológica Jatun Sacha (1.0645° S, 77.6128°
W), 405 m (QCAZ 43790); PROVINCIA ORELLANA:
La Primavera, Río Napo (0.4442° S, 76.7868° W), 244
m (QCAZ 43790); PROVINCIA SUCUMBÍOS: Reserva
Biológica Limoncocha (0.4062° S, 76.6194° W), 261 m
(QCAZ 43105).
Boana rosenbergi. ECUADOR: PROVINCIA
COTOPAXI: La Maná (0.9433° S, 79.2647° W), 163
m (QCAZ 26288); PROVINCIA ESMERALDAS: 5 km
W of Durango (1.0724° N, 78.6534° W), 120 m (QCAZ
13194); Alto Tambo (0.9169° N, 78.5661° W), 253 m
(QCAZ 17081); PROVINCIA LOS RÍOS: Patricia
Pilar (0.5726° S, 79.369° W), 200 m (QCAZ 19770);
PROVINCIA MANABÍ: Estero Ancho, 52 km W of
El Carmen (0.0677° S, 79.7961° W), 329 m (QCAZ
23186); PROVINCIA PICHINCHA: Pedro Vicente
Maldonado (0.0789° N, 79.6194° W), 678 m (QCAZ
35454, 45244).
B o a n a r u b r a c y l a . E C UA D O R : P R OV I N C I A
CARCHI: Tobar Donoso (1.1643° N, 78.5071° W), 257
m (QCAZ 26206–7).
© 2020 The Linnean Society of London, Zoological Journal of the Linnean Society, 2020, XX, 1–32
Downloaded from https://academic.oup.com/zoolinnean/advance-article-abstract/doi/10.1093/zoolinnean/zlaa002/5810752 by University of Rochester user on 22 March 2020
S, 78.9863° W), 256 m (QCAZ 416); km 78 on the
road Maxus (0.8346° S, 76.3198° W), 257 m (QCAZ
6822), Loreto-Avila Viejo (0.6833° S, 77.3166° W), 434
m (QCAZ 10740); COLOMBIA: DEPARTAMENTO
AMAZONAS: km 9–10 on the road Leticia, Río Takana
(4.136° S, 69.9412° W), 83 m (ANDES-A 881–82, 906–
07, 909–10, 1631–33).
Boana boans. ECUADOR: PROVINCIA BOLÍVAR:
La Pretoria (1.7621° S, 79.2889° W), 122 m (QCAZ
51309, 51319); Santa Rosa de Agua Clara (2.1856° S,
79.113° W), 424 m (QCAZ 32593–94); PROVINCIA
ESMERALDAS: Durango (1.0427° N, 78.6245° W),
211 m (QCAZ 32049, 32062); PROVINCIA MORONA
SANTIAGO: Limón Indanza (2.9266° S, 78.407° W),
1100 m (QCAZ 41810); Pankints (2.9019° S, 77.894°
W), 332 m (QCAZ 46489); PROVINCIA NAPO:
Ahuano (1.0545° S, 77.5483° W), 385 m (QCAZ 27029);
Cotapino, near Río Pucuno (0.8011° S, 77.4318° W),
600 m (QCAZ 13682); Tena (0.9888° S, 77.8124° W),
508 m (QCAZ 16033); PROVINCIA ORELLANA:
Estación Científica Yasuní PUCE (0.6771° S, 76.4011°
W), 250 m (QCAZ 19248); PROVINCIA PASTAZA:
Campo Villano (1.5048° S, 77.5106° W), 345 m (QCAZ
39038, 38455, 38783); PROVINCIA PICHINCHA: 10
km W Pedro Vicente Maldonado (0.0999° N, 79.1329°
W), 422 m (QCAZ 45246).
Boana cinerascens. ECUADOR: PROVINCIA
MORONA SANTIAGO: Payapas Santiago (3.0465°
S, 78.0129° W), 300 m (QCAZ 52536); PROVINCIA
PASTAZA: Lorocachi (1.6407° S, 76° W), 185 m (QCAZ
56088); Canelos (1.592° S, 77.6932° W), 420 m (QCAZ
56580).
Boana hutchinsi. COLOMBIA: DEPARTAMENTO
VAUPÉS: Cano Kuduyari, Comunidad Pirasemo,
(1.317° N, 70.3889° W), 199 m (ANDES-A 2508).
Boana nigra. ECUADOR: PROVINCIA MORONA
SANTIAGO: on the road Nueve de Octubre (2.2492°
S, 78.2025° W), 1532 m (QCAZ 52557); Parque
Nacional Sangay, Río Volcán (2.1035° S, 78.1571°
W), 1350 m (QCAZ 2656–59); PROVINCIA NAPO:
San Francisco (0.4206° S, 77.8458° W), 1730 m
(QCAZ 7019, 7029–34); PROVINCIA SUCUMBÍOS:
Reventador town (0.0462° S, 77.5291° W), 1400 m
(QCAZ 6478–79).
Boana nympha. ECUADOR: PROVINCIA MORONA
SANTIAGO: Pankints (2.9019° S, 77.894° W), 332 m
(QCAZ 46426); PROVINCIA ORELLANA: Parque