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Systematics and Biodiversity
ISSN: 1477-2000 (Print) 1478-0933 (Online) Journal homepage: http://www.tandfonline.com/loi/tsab20
An integrative assessment of the taxonomic status
of putative hybrid leopard frogs (Anura: Ranidae)
from the Chortís Highlands of Central America,
with description of a new species
Ileana Luque-Montes, James D. Austin, Kayla D. Weinfurther, Larry David
Wilson, Erich P. Hofmann & Josiah H. Townsend
To cite this article: Ileana Luque-Montes, James D. Austin, Kayla D. Weinfurther, Larry David
Wilson, Erich P. Hofmann & Josiah H. Townsend (2018): An integrative assessment of the
taxonomic status of putative hybrid leopard frogs (Anura: Ranidae) from the Chortís Highlands of
Central America, with description of a new species, Systematics and Biodiversity
To link to this article: https://doi.org/10.1080/14772000.2017.1415232
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Published online: 04 Jan 2018.
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Research Article
An integrative assessment of the taxonomic status of putative hybrid
leopard frogs (Anura: Ranidae) from the Chort
ıs Highlands of Central
America, with description of a new species
ILEANA LUQUE-MONTES
1,2
, JAMES D. AUSTIN
1
, KAYLA D. WEINFURTHER
2,†
, LARRY DAVID WILSON
3
,
ERICH P. HOFMANN
2,z
& JOSIAH H. TOWNSEND
2
1
Department of Wildlife Ecology and Conservation, University of Florida, Gainesville, Florida 32611, USA
2
Department of Biology, Indiana University of Pennsylvania, Indiana, Pennsylvania 15705–1081, USA
3
Centro Zamorano de Biodiversidad, Escuela Agr
ıcola Panamericana Zamorano, Departamento Francisco Moraz
an, Honduras
(Received 19 September 2016; accepted 28 November 2017; published online 5 January 2018)
Integrative taxonomy seeks to approach the complex topic of species diagnosis using independent, complementary lines of
evidence. Despite their ubiquity throughout North and Central America, taxonomy of the American leopard frogs (Anura:
Ranidae: Rana: subgenus Pantherana) remains largely unresolved, and this is arguably nowhere truer than in the Central
American country of Honduras, where there are two nominal species, the taxonomy of which remains unresolved. Leopard
frogs from several mountainous areas along the continental divide in Honduras have previously been considered putative
hybrids between Rana brownorum and R. cf. forreri, as opposed to two alternate hypotheses: one that they represent a high-
altitude eco-morph of a single widespread species that included both lowland forms, or a second that there is an
undescribed highland species distinct from either of the recognized lowland forms. We examine this set of hypotheses
using three independent lines of evidence. First, we used species distribution modelling to examine potential geographic
isolation of the highland form and the two putative parental lowland species, and found strong ecological separation
between the highland and lowland forms. Second, mitochondrial and nuclear DNA supports the distinction of the highland
form from both putative parental species, with mtDNA data refuting the hypothesis that representatives of either species
may represent a matrilineal founder. Morphologically, the highland form is significantly smaller than, and otherwise readily
differentiated from, both R. brownorum and R. cf. forreri, as well as all other Rana found in Honduras and adjacent areas.
As a result, we formally describe the highland leopard frog as a new species.
http://zoobank.org/urn:lsid:zoobank.org:pub:BE53F587-3618-4433-9651-E495808E5474
Key words: Amphibia, integrative taxonomy, mitochondrial DNA, Pantherana, phylogeny, rhodopsin, species distribution
modelling
Introduction
Systematics and taxonomy constitutes the core of the life
sciences, providing the foundation upon which biological
research is structured (Mayr, 1942). Throughout most of
the Linnaean era of taxonomy traditional approaches to
species identification and delimitation have relied largely
on analysis of phenotypic variation, and the subjectivity
of the individual researcher (Dayrat, 2005). Increasingly,
taxonomists are taking an expressly integrative approach
to the problem of species delimitation that uses multiple
lines of evidence, including population and evolutionary
genetics, genomics, morphology, behaviour, geography,
and ecology (Padial, Miralles, De la Riva, & Vences,
2010; Schlick-Steiner et al., 2010).
Phylogenetic analysis of genetic data has become the
leading method for revealing previously unknown taxo-
nomic diversity, with analyses of morphology and other
phenotype-linked traits often being carried out within the
context of a phylogenetic hypothesis (Dayrat, 2005;
Fujita, Leach
e, Burbrink, McGuire, & Moritz, 2012). To
Correspondence to: Josiah Townsend. E-mail: josiah.
townsend@iup.edu
†
Current address: Department of Biology, East Carolina Univer-
sity, Greenville, North Carolina 27858, USA.
z
Current address: Department of Biological Sciences, Clemson
University, Clemson, South Carolina 29634, USA.
ISSN 1477-2000 print/ 1478-0933 online
ÓThe Trustees of the Natural History Museum, London 2018. All Rights Reserved.
https://doi.org/10.1080/14772000.2017.1415232
Systematics and Biodiversity (2018), 1–17
Downloaded by [71.61.69.106] at 16:20 04 January 2018
complement molecular phylogenetics, species distribution
modelling (SDM) is increasingly used to evaluate ecologi-
cal and physical characteristics that are associated with
the distributions of species, and for evaluating ecological
isolation amongst species (Leach
e et al., 2009; Pelletier,
Crisafulli, Wagner, Zellmer, & Carstens, 2015; Rissler &
Apodaca, 2007). SDM combines occurrence and environ-
mental data to predict the fundamental niche of a species.
As part of an integrative taxonomy, SDM provides a
quantitative platform for assessing geographic and eco-
logical isolation between and amongst nominal and candi-
date taxa, and can be used as a means of identifying
allopatry by inferring contemporary and historical barriers
to gene flow (Pelletier et al., 2015;R
€
odder, Weinsheimer,
& Loetters, 2010). Integrative taxonomy has the potential
to vastly improve our understanding in areas where taxo-
nomic research is most needed, particularly in the tropics
where levels of both biodiversity and habitat loss are high,
and amongst taxonomic groups with an elevated conserva-
tion priority, such as amphibians (Crawford, Lips, &
Bermingham, 2010).
New species of amphibians have been discovered at a
remarkable rate over the past two decades, with the overall
number of recognized species increasing more than 60%
from 1992 through 2016 (AmphibiaWeb, 2016;Duellman,
1993;K
€
ohler et al., 2005). While new amphibians are fre-
quently discovered in remote tropical regions, previously
unknown taxonomic diversity has also been uncovered in
densely populated urban areas (Biju et al., 2014;Newman,
Feinberg, Rissler, Burger, & Shaffer, 2012) highlighting
the need for continued taxonomic research across a wide
range of amphibian groups and habitats.
Leopard frogs (Anura: Ranidae: Rana:subgenus
Pantherana) are a widespread clade consisting of approxi-
mately 28 recognized species distributed from the Arctic
Circle in North America south to Panama (Yuan et al.,
2016). Despite their ubiquity throughout North and Central
America, the taxonomic status of many Pantherana popu-
lations remains understudied and unresolved, as evidenced
by the recent discovery and description of a new Rana
from the New York City metropolitan area (Feinberg
et al., 2014;Newmanetal.,2012). At least 15 unnamed
lineages of Rana have been identified from Mexico, Costa
Rica, and Panama, calling into question the taxonomic sta-
tus of some recognized species and underscoring the need
for a comprehensive and integrative taxonomic evaluation
of Neotropical ranids (Hillis & Wilcox, 2005;Zald
ıvar-
River
on, Le
on-Regagnon, & Nieto-Montes De Oca, 2004).
The Central American Republic of Honduras is home to
a diverse amphibian fauna, including five nominal species
of Rana:R. brownorum,R. forreri,R. maculata,R. vail-
lanti, and R. warszewitschii (Sol
ıs, Wilson, & Townsend,
2014). The taxonomy of at least two of these taxa remains
unresolved, with R. brownorum (previously referred to
as R. berlandieri; McCranie & Wilson, 2002) being
recognized as occurring in association with watersheds
draining into the Caribbean Sea, and the name R. forreri
being applied to populations associated with Pacific drain-
ages (Hillis, 1988; McCranie & Wilson, 2002; Zald
ıvar-
River
on et al., 2004). Based on the two previously pub-
lished phylogenetic studies that include samples from this
region (Hillis & Wilcox, 2005; Zald
ıvar-River
on et al.,
2004), application of those two names to populations in
Honduras has been shown to be a matter of convenience
rather than a reflection of evolutionary history. From this
point forward, we refer to the Pacific lowland populations
in Central America as R. cf. forreri, to reflect the existing
evidence that these populations are not conspecific with
R. forreri sensu stricto.
McCranie and Wilson (2002, pp. 478–479) reported
specimens of leopard frogs from several mountainous
areas along the continental divide in Honduras that they
considered putative hybrids between R. berlandieri (DR.
brownorum) and R. cf. forreri (Fig. 1). These intermediate
specimens are characterized by their small adult size rela-
tive to the former species, and possess a range of variation
in dorsolateral ridges that encompasses both the broken
and medially inset ridges of R. brownorum and the contin-
uous ridges of R. cf. forreri. The putative hybrids, herein
referred to as ‘intermediate’ populations, are reported
from elevations of 930 to 2,200 m in departments from
Ocotepeque in the west to El Para
ıso in the south-east
(McCranie & Wilson, 2002, p. 481, map 54). McCranie
and Wilson (2002) chose the working hypothesis that
these populations represent hybrids as opposed to two
other hypotheses, namely that the intermediate forms
were either (1) a high-altitude eco-morph of a single wide-
spread species that included both lowland forms (i.e.,
R. berlandieri [DR. brownorum] and R. cf. forreri) and
exhibited a wide range of morphological variation, or (2)
an undescribed highland species distinct from either of
the recognized lowland forms.
Fig. 1. Map showing localities for Rana brownorum,R. cf. for-
reri, and intermediate populations used for generating species
distribution models.
2 I. Luque-Montes et al.
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Other than interpreting what they viewed as intermedi-
ate phenotypes to be indicative of a hybrid zone, McCra-
nie and Wilson (2002) provided no evidence of
hybridization between the two lowland taxa. Phylogenetic
information available for Pantherana populations in Cen-
tral America is surprisingly limited, with representation
restricted to novel samples included in three studies (Frost
et al., 2006; Hillis & Wilcox, 2005;Zald
ıvar-River
on
et al., 2004). In a study of Pantherana from Mexico,
Zald
ıvar-River
on et al. (2004) resurrected the name
R. brownorum to refer to populations from southern Mex-
icopreviouslyreferredtoasR. berlandieri. The mitochon-
drial phylogeny provided by Hillis and Wilcox (2005)
recovered two samples that they referred to as R. macro-
glossa as the sister lineage to R. taylori from Nicaragua
and Costa Rica, together forming a clade that is, in turn,
sister to a clade containing three unnamed species. These
three unnamed lineages include one from intermediate ele-
vations on the Pacific slope of Panama (as R. sp. 4), one
from the highlands of central Costa Rica (as R. sp. 5), and
one from the Pacific lowlands represented by a single
sample from north-western Costa Rica (as R. sp. 6; Hillis
& Wilcox, 2005). Leopard frogs from north-western Costa
Rica and western Nicaragua were referred to as R. forreri
by Savage (2002)andK
€
ohler (1999,2001), respectively.
Given that the intermediate populations can be morpho-
logically differentiated from both lowland forms, and
appear to have geographically and ecologically isolated
distributions, the intermediate populations require an inte-
grative investigation of their taxonomic status. In an
attempt to resolve the status of the intermediate popula-
tions, we analysed the two nominal taxa, R. brownorum
and R. forreri, along with the intermediate populations
defined by McCranie and Wilson (2002), using three inde-
pendent lines of evidence: SDM, molecular phyloge-
netics, and comparative morphology.
We used SDM to assess the ecological distributions of
all three entities, following the assignment of localities
provided in McCranie and Wilson (2002), and evaluate
the potential for existence of natural contact zones that
could be inferred to represent a zone of hybridization. We
then conducted phylogenetic analyses using two widely
used mitochondrial loci, 12S and 16S rRNA, as well as
the nuclear protein-coding gene Rhodopsin, in order to
evaluate the relationships of Honduran populations of all
three entities. If these intermediate populations represent
a hybrid zone between R. brownorum and R. forreri, then
we would expect to recover haplotypes associated with
one or both nominal taxa amongst samples from interme-
diate populations, based on direct matrilineal inheritance
of the mitochondrial genome. Finally, we evaluated the
morphological variation of all three entities to determine
the extent of diagnosable morphological differentiation.
Finding strong evidence for taxonomic distinction based
on congruence amongst all assessed characters, we
consider the intermediate populations to represent a new
species and provide a formal taxonomic description.
Materials and methods
Taxon and gene sampling
We followed Yuan et al. (2016) in their use of a genus and
subgenus-level taxonomy that recognizes a single wide-
spread genus, Rana, divided into eight subgenera that
largely follow the taxonomy proposed by Hillis (2007),
placing the clade that contains leopard frogs and gopher
frogs into the subgenus Pantherana. We recognize that the
generic taxonomy of Ranidae has been a matter of active
debate amongst authorities (see Frost, 2016;Frost,
McDiarmid, Mendelson, & Green, 2012;Yuanetal.,2016
for summaries of the arguments). We herein refer to popu-
lations from the Pacific Lowlands of El Salvador, Hondu-
ras, Nicaragua, and Costa Rica as R. cf. forreri, noting that
(1) Zald
ıvar-River
on et al. (2004) showed that R. forreri is
a polytypic taxon with respect to populations in Mexico,
and (2) populations in Costa Rica presently referred to as
R. forreri were considered unnamed ‘species 6’ by Hillis
and Wilcox (2005) and Yuan et al. (2016). Localities and
voucher information for samples used in the phylogenetic
analyses are presented in Table S1 (see online
supplemental material, which is available from the article’s
Taylor & Francis Online page at (https://doi.org/10.1080/
14772000.2017.1415232). Institutional abbreviations fol-
low those standardized by the American Society of Ich-
thyologists and Herpetologists (Sabaj, 2016). Samples of
leopard frogs were collected across Honduras and northern
Nicaragua between 2005 and 2011. Prior to preservation, a
tissue sample was preserved in SED buffer (20% DMSO,
0.25 M EDTA, pH 7.5, NaCl saturated; Seutin, White, &
Boag, 1991; Williams, 2007). Specimens were preserved in
10% formalin and stored in 70% ethanol. We selected gene
loci for use in phylogenetic analyses in order to maximize
the comparability of our dataset with those previously pub-
lishedbyZald
ıvar-River
on et al. (2004) and Hillis and
Wilcox (2005), the only available comparative datasets that
include significant representation by Mesoamerican leopard
frogs. Both aforementioned datasets, however, use different
primer sets for the gene 12S that produced fragments that
only overlap by »397 bp. In order for our dataset to be
comparable to both the Zald
ıvar-River
on et al. (2004)and
Hillis and Wilcox (2005), datasets, we chose to sequence a
large fragment of 12S (809 bp; as was done by Zald
ıvar-
River
on et al., 2004 for Mexican leopard frogs) and a
widely used fragment of 16S (538 bp; included in the Hillis
& Wilcox (2005) dataset); we also sequenced a portion of
the nuclear protein-coding gene rhodopsin (»316 bp). For
the outgroups, we included the available sequences of
R. forreri complex from Pacific slope of western and south-
ern Mexico (Hillis & Wilcox, 2005;Zald
ıvar-River
on
Integrative taxonomy of putative hybrid leopard frogs from Honduras 3
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et al., 2004), as well as samples of R. capito,R. chirica-
huensis,andR. yavapaiensis to represent the three other
clades of Pantherana (Hillis & Wilcox, 2005; Yuan et al.,
2016).
Species distribution modelling
Species distribution models for Rana brownorum,R. cf.
forreri, and intermediate populations were constructed
using the maximum entropy method (implemented in
Maxent 3.3; Phillips, Anderson, & Schapire, 2006), using
presence-only data and predictor variables in the form of
grid files representing bioclimatic variables. The maxi-
mum entropy method has been demonstrated to perform
effectively with a relatively small number of presence-
only sample points (Cayuela et al., 2009; Pearson,
Raxworthy, Nakamura, & Peterson, 2007), providing the
best option for constructing SDM with our limited dataset.
Occurrence data were compiled from published records
(K€
ohler et al., 2005; McCranie, 2006; McCranie &
Castaneda, 2005; McCranie & Wilson, 2002) and unpub-
lished localities from our own work during 2008
(Table S1, S2, see supplemental material online).
Geographic coordinates (WGS 84) were organized in a
comma separated values file (.csv) using decimal fractions
(decimal degrees – DD) format (data included as Table
S2, see supplemental material online). Grid data layers at
a 30 second resolution were obtained from the WorldClim
database (http://www.worldclim.org). Monthly climate
data from weather stations around the world is interpo-
lated to produce layers of 1 km
2
resolution for monthly
total precipitation, and monthly mean, minimum and max-
imum temperature and 19 derived bioclimatic variables
(Hijmans, Cameron, Parra, Jones, & Jarvis, 2005). Pre-
and post-processing of species distribution models was
performed in SDMToolbox, a package of analytical tools
for ArcGIS (Brown, 2014). Mean, maximum, minimum,
and standard deviation were calculated along with pair-
wise correlation and covariance coefficients for each input
raster layer; a Pearson correlation coefficient of 0.75 was
used to identify and remove highly correlated variables
and thus reduce over-fitting of the model, reducing the
dataset to the following six variables: annual mean tem-
perature (Bio1), mean diurnal range (mean of monthly
(max temp–min temp); Bio2), isothermality (mean diurnal
range/temperature annual range; Bio3), annual precipita-
tion (Bio12), precipitation seasonality (coefficient of vari-
ation; Bio15), and precipitation of coldest quarter
(Bio19). We eliminated spatially correlated occurrence
records based in topographic heterogeneity, using the
‘Spatially Rarefy Occurrence Data’ function in SDMTool-
box. The resulting data were reduced from 127 samples to
106 spatially independent samples, maximizing spatial
independence of occurrence points. Predicted distribution
values were obtained using the mean and standard
deviation from 1000 bootstrap replicates, using 33% of
the data as testing data and a maximum of 5000 iterations
in MaxEnt 3.3 (Phillips et al., 2006). Output files in the
form of ASCII were imported into ArcGIS 10.2 for exam-
ination and post-processing. ASCII files were converted
to raster and then reclassified to obtain binary files depict-
ing suitable (1) and non-suitable (0) habitat using the
maximum training sensitivity plus specificity logistic
threshold as the cut off value (Phillips et al., 2006)(R.
brownorum D0.3381, R. cf. forreri D0.3859, intermedi-
ate populations D0.1544). Performance of the distribution
models was evaluated using the values of the area under
the curve of the receiver operating characteristic (ROC)
curve (AUC).
In order to identify contact zones between potential
distributions for R. brownorum and R. cf. forreri,we
reclassified the binary files to obtain individual occur-
rence codes (0 Dall absent, 1 DR. brownorum,3D
only R. cf. forreri,4DR. brownorum and R. cf. for-
reri,5Donly intermediates, 6 DR. brownorum and
intermediates, 8 DR. cf. forreri and intermediates, 9
Dall present) and then added the values together in a
single raster layer. We then converted the identified
contact zone into a polygon, generated 1000 random
points within the polygon and then extracted the prob-
ability values from all three distribution models for
comparison and determination of statistical significance
using an Analysis of Variance (ANOVA).
DNA extraction, PCR amplification, and
sequencing
Fragments of the 12S and 16S mitochondrial RNA and the
nuclear protein-coding gene Rhodopsin (rhod) were ampli-
fied using the primers 12SJ-L and 12SK-H for 12S (Goebel,
Donnelly, & Atz, 1999), 16Sar-L and 16Sbr-H for 16S (Pal-
umbi et al., 1991), and Rhod1A and Rhod1C for Rhodopsin
(Bossuyt & Milinkovitch, 2000). Template DNA was
extracted from muscle tissue using the GentraÒPureGeneÒ
DNA Isolation Kit (QIAGENÒ, Hilden, Germany) following
manufacturer’s instructions. Amplification was carried out in
25mL-volume reactions with the following cycling parame-
ters: 12S:2minat94
C, 45 s at 50C,and2minat72
C, fol-
lowedby39cyclesof:94
C for 30 s, 50C for 45 s, and
72C for 90 s, with a final extension of 10 min at 72C; 16S:
3minat94
C,followedby35cyclesof45sat94
C, 45 s at
50C, and 45 s at 72C, with a final extension of 5 min at
72C; rhod:5minat95
C, followed by 40 cycles of 30 s at
95C, 40 s at 60C, and 90 s at 72C, with a final extension
of 5 min at 72C. PCR products were verified using electro-
phoresis on a 1.5% agarose gel stained with ethidium bro-
mide. Unincorporated nucleotides were removed from PCR
products using 1 or 2 mLofExoSAP-IT
Ò(USB, Santa Clara,
CA, USA) per 10 mL of template. We cycle sequenced both
forward and reverse strands using the BigDyeÒTerminator
4 I. Luque-Montes et al.
Downloaded by [71.61.69.106] at 16:20 04 January 2018
3.1 Cycle Sequencing kit, followedbyspincolumnfiltration
through Sephadex before being electrophoresed on an ABI
3130xl (Applied BiosystemsTM Inc).
Extraction, amplification, and sequencing for some 16S
data were carried out at Smithsonian Institution Labora-
tory of Analytical Biology (Suitland, Maryland). Tem-
plate DNA was obtained using phenol-chloroform
extraction implemented by an AutoGen Geneprep 965
(AutoGen, Holiston, MA) automated DNA isolation
robot. PCR product was cycle sequenced using BigDyeÒ
Terminator v3.1 Cycle Sequencing kit (ABI), cleaned
using spin column filtration through Sephadex, and elec-
trophoresed on an ABI 3730xl DNA Analyser.
Sequence divergence and phylogenetic
analyses
Sequence divergence and haplotype diversity for the
target taxa were calculated using data from 16S for 61
samples, which included 19 samples from three locali-
ties representing intermediate populations, 32 samples
of Rana brownorum from seven localities, and 10 sam-
ples of R. cf. forreri from seven localities (Table S1,
see supplemental material online). We estimated pair-
wise sequence divergence for each gene and for all
samples using both the p-distance and Kimura 2-
parameter model in the program MEGA7 (Kumar,
Stecher, & Tamura, 2016). Phylogenetic analyses were
carried out on a concatenated 12S and 16S dataset and
a concatenated mtDNA (12S and 16S) and nuclear
(Rhodopsin) dataset. For ingroups, our 12SC16S data-
set included seven novel samples from three intermedi-
ate populations, eight novel samples of R. brownorum
from three localities, six novel samples of R. taylori
from four localities, and four novel samples of Rana
sp. from the central highlands of Costa Rica. Our sam-
ples were combined with 24 comparative samples from
Zald
ıvar-River
on et al. (2004), six from Hillis and
Wilcox (2005), and four from Frost et al. (2006); the
12SC16SCrhod dataset included six intermediate, and
six R. brownorum,fiveR. taylori, and four Rana sp.
samples, in addition to one R.cf.forreri from Frost
et al. (2006). We aligned sequences using ClustalW
(Thompson, Higgins, & Gibson, 1994) as implemented
in the program MEGA 7 (Kumar et al., 2016). Final
alignments for both datasets were deposited in Tree-
BASE (http://purl.org/phylo/treebase/phylows/study/
TB2:S21867). Prior to Bayesian phylogenetic analysis,
we used PartitionFinder v1.1.1 (Lanfear, Calcott, Ho,
& Guindon, 2012) to select optimal substitution mod-
els for each gene (12S, 16S) and each codon (rhod),
using the ‘greedy’ algorithm and ‘mrbayes’setof
models, and evaluating the best model using Akaike
Information Criterion scores. Phylogenetic analyses
were performed on the 12SC16S dataset, and the
12SC16SCrhod dataset. Maximum likelihood (ML)
was performed in RAxMLv8 (Stamatakis, 2014)with
raxmlGUIv1.5 (Silvestro & Michalak 2012), consisting
of 1,000 pseudoreplicates under the default
GTRCGAMMA model of nucleotide substitution, with
the dataset partitioned by gene (12S and 16S) and by
codon (rhod). Bayesian inference (BI) was performed
using MrBayes 3.1.2 (Huelsenbeck & Ronquist, 2001),
and consisted of two parallel runs of four Markov
chains (three heated, one cold) run for 20 £10
6
gener-
ations and sampled every 1,000 generations, with a
random starting tree and the first 2 £10
6
generations
discarded as burn-in. Convergence and stationarity
within chains were visualized with Tracer 1.6 (Ram-
baut, Suchard, Xie, & Drummond, 2014)and30%of
generations were discarded as burn-in. We estimated
the 50% majority-rule consensus topology and poste-
rior probabilities for each node with the remaining
trees.
Morphological systematics
Morphological terminology and descriptive format for
adults follow McCranie and Wilson (2002), and for tad-
poles follow Altig and McDiarmid (1999). Measurements
taken from 34 specimens (11 adult males, 11 adult
females, and 12 subadults) from intermediate populations
included snout–vent length (SVL), shank length (SHL;
from knee to ankle), foot length (FL; from posterior-most
portion of inner metatarsal tubercle to tip of longest toe),
head length (HL; tip of snout to posterior angle of jaw),
head width (HW; greatest width), width of upper eyelid
(EW; perpendicular to outer edge of eyelid), interorbital
distance (IOD; measured at midlength of upper eyelids),
tympanum length (TPL), and eye length (EL). Measure-
ments taken from four tadpoles included body length (BL;
from tip of snout to junction of posterior body wall with
axis of tail), tail length (TAL; from body terminus to tail
tip), total length (TL), eye diameter (ED), interorbital dis-
tance (IOD; distance between eyes, measured between
dorsal margins of eyes), internasal distance (IND; distance
between nostrils, measured dorsal margins of nostril open-
ings) oral disc width (ODW; greatest width), snout width
(SW; measured at level of posterior end of oral disc),
maximum tail height (MTH; measured in profile at tallest
point), tail muscle height (TMH; measured at tallest point
near anterior terminus of tail), and tail muscle width
(TMW; measured at widest point near anterior terminus
of tail). All measurements were taken with digital cal-
lipers and rounded to the nearest 0.1 mm. Raw measure-
ment data are provided in Table S3 (see supplemental
material online). Comparative data for Honduras speci-
mens other than those in the type series are from
McCranie and Wilson (2002). Differences between the
observed mean values for measurements from each taxon
Integrative taxonomy of putative hybrid leopard frogs from Honduras 5
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and sex were calculated with a comparison of means test,
which calculates a significance value (P) representing the
probability obtaining the observed means if the difference
between them is zero.
Results
Species distribution modelling
The relative contribution of the variables to the models is
shown in Table 1 and Fig. S1 (see supplemental material
online). The variables that contributed most (83.5%) to
model the distributions were the same for R. brownorum
and R. forreri, Precipitation Seasonality (37.2 and
34.2%), Mean Diurnal Range (34.2 and 31.1%), and to a
lesser degree, Annual Precipitation (12.1 and 7.6). While
the most important variables for predicting the distribu-
tion of the intermediate populations (94.1) were
Table 1. Analysis of variable contributions. Estimates of
relative contributions of the environmental variables to the
MaxEnt model.
Species Bio1 Bio12 Bio15 bio19 Bio2 Bio3
R. brownorum 5.7 12.1 37.2 5.1 34.2 5.7
R. forreri 0.3 7.6 52.9 7.1 31.2 1
Intermediate 76.9 0.7 4.9 4.7 0.1 12.5
Fig. 2. Species distribution models for Rana cf. forreri (top), intermediate populations (middle), and R. brownorum (bottom), showing
the predicted fundamental niche for each and associated plots showing the receiver operating curve (ROC) for both training and test data.
6 I. Luque-Montes et al.
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Isothermality (12.5), Annual Mean Temperature (76.9),
and Precipitation of Coldest Quarter (4.7). Each distribu-
tion model performed well based on high AUC (Fig. 2,
Table 2).
Binary maps showing presence/absence of suitable hab-
itat revealed only one area of overlap containing optimal
habitat and probability of occurrence for both R.
brownorum and R. cf. forreri and is extralimital to the
SDM for the intermediate populations; several other areas
of potential co-distribution were identified but completely
contained within the optimal habitat of one species or
the other (Figs 3, S2, see supplemental material online).
Several areas were identified as optimal habitat and proba-
bility of occurrence for both R. brownorum and intermedi-
ates (Figs 3, S2, see supplemental material online). The
values extracted from each contact zone for each species
SDM are shown in Table S4 (see supplemental material
online). The mean values of the distribution probability
extracted from the distribution model in the R. browno-
rum–R. cf. forreri contact zone, were 0.56 for R. browno-
rum and 0.49 for R. cf. forreri, with a significant P-value
of <0.0001. The average values for the R. brownorum–
intermediate contact zone were 0.48 for R. brownorum
and 0.31 for intermediate populations with a significant
P-value of <0.0001 (Table 3, Fig. S3, see supplemental
material online).
Sequence divergence and phylogenetic
analyses
Samples representing the three target taxa for this study,
R. brownorum,R. cf. forreri, and ‘intermediate’ popula-
tions heretofore considered hybrids of the former taxa,
Table 2. Performance of distribution models for R. brownorum,R. forreri, and intermediate populations. Mean values are averages of
1000 bootstrap replicates, with 33% of data as test localities. The mean value for equal training sensitivity and specificity was used as a
threshold to create a range map of predicted presences/absences.
Localities
Train (Test)
Mean
Test AUC (S.D.)
Mean
Training AUC
Maximum training
sensitivity plus specificity
(logistic threshold)
R. brownorum 54 (16) 0.8547 (0.0258) 0.8947 0.3381
R. forreri 7 (2) 0.9036 (0.0401) 0.928 0.3859
intermediate populations 21 (6) 0.9791 (0.0106) 0.9847 0.1544
Fig. 3. Presence/absence maps showing maximum extent of fundamental environmental niche for Rana cf. forreri (top), intermediate
populations (middle), and R. brownorum (bottom), and combined map showing potential contact zones.
Integrative taxonomy of putative hybrid leopard frogs from Honduras 7
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were well-differentiated by mitochondrial sequence data.
For our in-group taxa, we calculated p-distance and
sequence divergence under the K2P model for 61 samples
using a 538 bp segment of 16S, recovering seven unique
haplotypes (intermediates D1, R. brownorum D3, R. cf.
forreri D3). Intraspecific sequence divergence was low
in all three groups: intermediate populations (0.0–0.2%;
nD19), R. brownorum (0.0–0.5%; n D32), and R. cf. for-
reri (0.0–0.7%; n D10). Interspecific divergence between
the intermediate samples and Honduran R. brownorum
was 2.8–3.3% and R. cf. forreri was 2.3–3.3%. Based on
16S data, assignments of all 61 samples from 17 localities
correspond both to the assignments made by our SDM
and to the morphological assignments of McCranie and
Wilson (2002).
For the combined 12SC16S dataset, the best-fit models
(based on AIC scores) of nucleotide divergence for each
partition separately were: 12S – GTRCICG; 16S –
GTRCICG. For the 12SC16SCrhod dataset, the best-fit
models of nucleotide divergence were: 12S – GTRCICG;
16S – GTRCICG; rhod (1st codon position) – GTRCI,
rhod (2nd codon position) – K80CI; rhod (3rd codon posi-
tion) – GTRCICG.
Bayesian and ML phylogenetic analyses of both the
12SC16SCrhod (Fig. 4) and mtDNA-only datasets
(Fig. 5) produced congruent results. Both sets of analyses
recovered the intermediate populations as a monophyletic
group that is the sister lineage to Rana sp., an undescribed
species from the highlands of Costa Rica that includes a
single sample referred to as R. sp. 5 by Hillis and Wilcox
(2005). Together, these lineages are sister to the Pacific
lowland samples from Honduras and Costa Rica referred
to as R. cf. forreri (R. sp. 6 from Hillis & Wilcox, 2005).
Rana brownorum populations from Honduras form a
monophyletic group, and in the 12SC16S analyses were
recovered as conspecific with the majority of R. browno-
rum samples from Mexico (Zald
ıvar-River
on et al., 2004)
and two samples considered by Hillis and Wilcox (2005)
Table 3. Occurrence probability means and variance for three input data points, and 1000 random points within the identified contact
zone. Analysis of Variance was used to determine non-significant differences.
Contact Zone Sample Points Model Mean Min Max Variance P-value
R. brownorum–R. forreri 638 R. brownorum 0.56 0.338 0.817 0.016 <0.0001
R. forreri 0.49 0.070 0.740 0.040
R. brownorum–Intermediates 3146 R. brownorum 0.48 0.338 0.758 0.009 <0.0001
Intermediate 0.31 0.154 0.722 0.015
Fig. 4. Bayesian phylogram inferred from concatenated dataset of mtDNA (12S and 16S) and nuclear (rhodopsin) sequence data, with
corresponding species distribution models shown; maximum likelihood bootstrap values (0–100) and Bayesian posterior probabilities
(0–1.0) are shown for each clade.
8 I. Luque-Montes et al.
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to represent R. macroglossa; all referred R. brownorum
samples formed a sister clade to samples of R. taylori
from Nicaragua. Two samples from the western end of the
Central Depression of Chiapas (MZFC 12196, from
Tuxtla Guti
errez, Chiapas; and MZFC 12895, from San
Antonio Chimalapa, Oaxaca) formed a divergent sister
clade to the remaining R. brownorum samples, consistent
with results previously reported by Zald
ıvar-River
on et al.
(2004).
Comparative morphology
Standard morphological measurements and morphometric
ratios differentiate the intermediate populations from those
representing R. brownorum and R. cf. forreri,aswellasall
other Rana found in Honduras and adjacent areas (Table
S4, see supplemental material online). Most notably, the
intermediate populations are significantly smaller than
either R. brownorum (males t-stat 8.484, P<0.0001, d.f.
D20; females t-stat 4.284, P<0.001, d.f. D16) or R. cf.
forreri (males t-stat 5.484, P<0.0001, d.f. D17; females
t-stat 3.769, P<0.003, d.f. D12), with adult males mea-
suring 46.6–64.3 mm (57.9 §5.4; mean §standard devia-
tion) and adult females measuring 43.7–76.3 mm
(60.3 §12.3), versus 73.0–88.4 mm (76.9 §4.5) and 76.0–
91.1 mm (81.7 §5.7) for R. brownorum and 65.4–
81.9 mm (73.8 §7.2) and 78.9–94.1 mm (89.3 §9.0) for
R. cf.forreri. Comparisons of the morphological character-
istics of the intermediate populations with the other
regional Rana are detailed in the taxonomic account below
and summarized in Table S4 (see supplemental material
online).
Based on phylogenetic analysis of 12S, 16S, and rho-
dopsin genes that recover all intermediate samples as a
single monophyletic group, morphological differences
between intermediate samples and both nominal taxa, and
ecological isolation revealed by SDM, we consider the
intermediate populations previously referred to as hybrids
between R. brownorum and R. cf. forreri to represent a
previously unrecognized species, which we formally
describe in the taxonomic account below.
Taxonomy
Rana lenca sp. nov.
(Figs 6,8,9,10,12)
Common English name. Lenca Leopard Frog
Fig. 5. Bayesian phylogram inferred from combined 12S and 16S dataset; maximum likelihood bootstrap values (0–100) and Bayesian
posterior probabilities (0–1.0) are shown for each clade.
Integrative taxonomy of putative hybrid leopard frogs from Honduras 9
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Common Spanish name. La Rana Lenca
Rana pipiens: Dunn & Emlen, 1932:25 (in part); Lynch &
Fugler, 1965:12 (in part); Meyer, 1969:176 (in part);
Meyer & Wilson, 1971:32 (in part). Rana ‘pipiens’:
McCranie et al., 1986:560; Wilson & McCranie, 1993;
Wilson, Porras, & McCranie, 1986:3:2 (in part).
Rana berlandieri: McCranie, Wilson, & Williams,
1993:257; Heyer, de S
a, McCranie, and Wilson
‘1996’ (1997):169; Wilson & McCranie, 1998:14 (in
part). Rana berlandieri £Rana forreri hybrid:
McCranie & Wilson, 2002: 475; McCranie & Wilson,
2004: 40. Lithobates brownorum £Lithobates forreri:
McCranie, 2006: 115; McCranie & Casta~
neda, 2007:
251.
Holotype. An adult male (Fig. 6; UF 166670) from Cerro
San Pedro La Loma (Fig. 7; 14.3118N, 88.0993W),
2,010 m elevation, Departamento de Intibuc
a, Honduras,
collected 5 February 2008 by I.R. Luque-Montes, J.H.
Townsend, and L.D. Wilson. Original field number JHT
2327.
Paratypes. 34; 11 males, including seven paratopotypes
(UF 166663–65, 166670–74), two (UF 166639, 166641)
from Thomas Cabot Biological Station (14.0349N,
87.0761W), 1,640 m elevation, Reserva Biol
ogica Cerro
Uyuca, Departamento de Francisco Moraz
an, collected 17–
18 March 2007 by J.M. Butler, L.P. Ketzler, J.H. Townsend,
S.R. Travers, and L.D. Wilson, and two (UF 166658,
166687) from Thomas Cabot Biological Station (same
locality), collected 17 July 2007 by L.P. Ketzler, J.H. Town-
send, and L.D. Wilson; 11 females, including one paratopo-
type (UF 166685), and 10 from Thomas Cabot Biological
Station (UF 166638, 166642 [Fig. 8.1], 166644–45, 166647,
collected 17–18 March 2007; UF 166657, 166659–62, col-
lected 17 July 2007); 12 juveniles, including five paratopo-
types (UF 166667–69, 166675, 166680), six from Thomas
Cabot Biological Station (UF 166640, 166643 [Fig. 8.2],
166646, collected 17–18 March 2007; UF 166650, 166654–
55, collected 17 July 2007), and one (UF 166686) from
Zacate Blanco (14.3395N, 88.2469W), 2,080 m elevation,
Departamento de Intibuc
a, collected 30 July 2008 by I.R.
Luque-Montes, J.H. Townsend, L.D. Wilson, and L.P. Ket-
zler; 4 tadpoles (1 lot; UF 166637), from Thomas Cabot
Biological Station, collected 17–18 March 2007 by J.M.
Butler, L.P. Ketzler, J.H. Townsend, S.R. Travers, and L.D.
Wilson.
Fig. 8. Paratypes of Rana lenca from Reserva Biol
ogica Cerro
Uyuca, 1,640 m elevation, Departamento de Francisco Moraz
an,
Honduras: (1) adult female paratype (UF 166642; 64.3 mm SL);
(2) Subadult female paratype (UF 166643) and tadpole (UF
166637). Photos by Jason M. Butler.
Fig. 6. Adult male holotype (UF 166670) of Rana lenca.
Fig. 7. Type locality of Rana lenca, San Pedro La Loma,
2,010 m elevation, Departamento de Intibuc
a, Honduras, shown
in January 2008 (top) and during drought conditions in May
2015 (bottom). Photos by JHT.
10 I. Luque-Montes et al.
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Etymology. The name lenca is given in honour of the
indigenous Lenca people, the traditional inhabitants of the
mountainous region of south-western Honduras.
Diagnosis and comparisons. Rana lenca can be distin-
guished from congeners by the following combination of
characters: well-defined dorsal spots, relatively small adult
size in males and females, head relatively long and broad,
tympanum same relative size in males and females, genetic
differences, and geographic distribution. There are two spe-
cies of the Rana berlandieri group presently considered to
occur in northern Central America, R. brownorum and R. cf.
forreri,fromwhichR. lenca may be distinguished by having
smaller adult size (46.6–64.3 mm [57.9 §5.4] in males,
43.7–76.3 mm [60.3 §12.3] in females, versus 73.0–
88.4 mm [76.9 §4.5] and 76.0–91.1 mm [81.7 §5.7] for R.
brownorum and 65.4–81.9 mm [73.8 §7.2] and 78.9–
94.1 mm [89.3 §9.0] for R. cf.forreri), and a relatively
Fig. 9. Unvouchered examples of Rana lenca; (1) adult male from the type locality; (2) adult female from the type locality; (3) adult
male from the Reserva Biol
ogica Cerro Uyuca; (4) adult female from the Reserva Biol
ogica Cerro Uyuca; (5) adult male at edge of
pond at type locality; (6) adult female floating amongst Pinus oocarpa needles in a spring-fed pool at Reserva Biol
ogica Cerro Uyuca.
Photos by JHT.
Integrative taxonomy of putative hybrid leopard frogs from Honduras 11
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longer and broader head (HL/SVL 0.413–0.459 [0.430 §
0.01] in males and 0.390–0.484 [0.431 §0.03] in females,
versus 0.355–0.423 [0.385 §0.02] and 0.365–0.410 [0.388
§0.02] in R. brownorum and 0.364–0.399 [0.384 §0.01]
and 0.342–0.368 [0.354 §0.01] in R. cf. forreri;HW/SVL
0.346–0.389 [0.364 §0.01] in males and 0.334–0.379
[0.358 §0.02] in females, versus 0.299–0.355 [0.331 §
0.02] and 0.321–0.347 [0.332 §0.01] in R. brownorum and
0.340–0.352 [0.346 §0.01] and 0.311–0.336 [0.326 §0.01]
in R. cf. forreri). One species, R. maculata, is known to occur
in sympatry with R. lenca,andR. lenca can be distinguished
from R. maculata by having a relatively longer head (HL/
SVL 0.413–0.459 [0.430 §0.01] in males and 0.390–0.484
[0.431 §0.03] in females, versus 0.342–0.396 [0.367 §
0.02] and 0.356–0.399 [0.373 §0.01] in R. maculata), larger
tympanum (TPL/EL 0.566–0.828 [0.733 §0.08] in males,
0.707–0.932 [0.775 §0.09] in females, versus 0.537–0.646
[0.597 §0.03] and 0.475–0.630 [0.628 §0.03] in R. macu-
lata), and distinct dark dorsal spots (dorsal spots absent or
indistinct in R. maculata). Two other species, R. vaillanti
and R. warszewitschii, are found in lowland habitats in Hon-
duras; R. lenca differs from R. vaillanti in having a smaller
adult size (46.6–64.3 mm [57.9 §5.4] in males, 43.7–
76.3 mm [60.3 §12.3] in females, versus 65.9–84.1 [78.6 §
6.5] and 85.4–116.8 [101.4 §11.1] in R. vaillanti) and dis-
tinct dark dorsal spots (dorsal spots absent or indistinct in R.
vaillanti); and from R. warszewitschii by having larger adult
males (46.6–64.3 mm [57.9 §5.4] in males, versus 33.7–
37.4 mm [36.5 §1.7] in R. warszewitschii), relatively longer
shanks (SH/SVL 0.575–0.638 [0.606 §0.02] in males,
0.525–0.658 [0.597 §0.04] in females, versus 0.488–0.520
[0.503 §0.01] and 0.508 in R. warszewitschii), longer feet
(FL/SVL 0.539–0.618 [0.574 §0.02] in males, 0.513–0.620
[0.565 §0.03] in females, versus 0.470–0.501 [0.487 §
0.01] and 0.465 in R. warszewitschii), a broader head (HW/
SVL 0.346–0.389 [0.364 §0.01] in males and 0.334–0.379
[0.358 §0.02] in females, versus 0.295–0.319 [0.311 §
0.02] and 0.302 in R. warszewitschii), by lacking an outer
metatarsal tubercle (present in R. warszewitschii), by lacking
lateral grooves along margins of toe tips (lateral grooves
present on toe discs of R. warszewitschii),andbylargeyel-
low spots or vertical bars on the inner thighs (large yellow
spots or vertical bars present on inner thighs of R.
warszewitschii).
Description of holotype in preservative. An adult male
(Fig. 6; UF 166670) with a snout that is subovoid in dorsal
aspect and rounded in lateral profile. The canthus is distinct
and the loreal region concave. The nostrils are directed post-
erolaterally, being situated at a point »40% the distance
between the tip of the snout and anterior border of the orbit.
No supratympanic fold is apparent. The tympanum is promi-
nent, being separated from the eye by a distance of less than
one-third the length of the tympanum. The upper arm is
shorter and more slender than the relatively robust forearm.
Transverse dermal fold present on upper surface of wrist,
and vertical dermal fold absent from elbow. Tubercles or
dermal ridge absent along posterior ventrolateral edge of
forearm. Finger tips are not expanded and lack pads and dig-
ital grooves. Subarticular tubercles on fingers round and
globular in shape. Fingers lacking supernumerary tubercles.
The palmar tubercle is not well-defined and smaller than the
thenar tubercle, and accessory palmar tubercles are absent.
The thenar tubercle is elongate and elevated, the pollex is
not enlarged, and a well-developed grey pad is present on
the thumb and visible in dorsal view. Relative length of fin-
gers II<I<IV<III. The fingers are unwebbed and lack lat-
eral keels. The heels overlap when hind limbs held at right
angles to body. Vertical dermal fold present on outer lateral
edge of heel, and the heel is smooth; a row of poorly defined
Fig. 12. Malformed metamorphosed juvenile (Gosner stage 45)
of Rana lenca (UF 166681) from the type locality, exhibiting
polyphalangy and complete unreduced tail typical of Gosner
stage 42.
Fig. 11. Waveform visualization (top) and audiospectrogram
(bottom) of male vocalization of Rana lenca from Reserva
Biol
ogica Cerro Uyuca.
Fig. 10. Diagramatical illustration of a tadpole of Rana lenca
(JHT 3820, Gosner Stage 37) from the type locality, Cerro San
Pedro La Loma.
12 I. Luque-Montes et al.
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tubercles present along posterior ventrolateral edge of tar-
sus; ventral surface of tarsus smooth. Inner tarsal fold
absent. Subarticular tubercles on toes ovoid and globular in
shape. Supernumerary tubercles and plantar tubercles absent
from toes. Inner metatarsal tubercle elongated and elevated;
outer metatarsal tubercle absent. Relative length of toes
I<II<V<III<IV. The toe tips are not expanded and lack
pads. Digital groove absent around tips of toes. Fleshy
fringes present laterally on unwebbed portions of toes; web-
bing formula of feet I 1/4–2 II 1–2 1/2 III 1–3 IV 3–1/4 V.
The cloacal opening is directed posteriorly, with the skin
below cloaca granular. The skin of dorsal and ventral surfa-
ces is smooth. Short dorsolateral ridges present, terminating
anterior to the level of the thighs. The pupil is horizontally
elliptical. The edge of the palpebral membrane is dark
brown, with the membrane itself being translucent. The
bicornuate tongue is ovoid with a shallow notch between
two distinct fleshy lobes at the posterior tip, and is free pos-
teriorly for about three-quarters of its length. Vomerine
tooth patches on paired, elevated, medial ridges; tooth
patches separated by distance approximately one-third the
width of either patch. Choanae ovoid, positioned to the out-
side of vomerine tooth patches. Maxillary teeth present.
Poorly developed paired vocal slits present. Lateral paired
vocal sacs present, located posterior to the angle of the jaw.
Measurements of holotype (in mm). SVL 58.5; SHL
35.8; FL 32.5; HL 25.0; HW 21.3; EW 5.0; EL 6.9; IOD
3.4; TPL 5.6.
Colouration. Colour in alcohol of holotype after four
years of preservation: dorsal surfaces of head and body
dark brown with irregular, somewhat elongate dorsal spots
that are paler above the dorsolateral ridges than below,
with spots contacting or below the dorsolateral ridges
being very dark brown; dorsolateral ridges lacking contin-
uous dark borders; dorsal surface of forelimbs brown,
paler than dorsum, bearing two dark brown crossbars on
each forearm and a brown spot covering each elbow; dor-
sal surface of hind limbs brown with regularly spaced
darker brown crossbars on thighs, shanks, and feet; lateral
surfaces of body grey-brown, paler than dorsum, with
some darker brown mottling; posterior surface of thighs
brown with paler brown to cream mottling; ventral surface
of body cream with some pale grey-brown mottling cen-
trally; chin with scattered grey-brown mottling; palms
medium grey. Variation in dorsal colouration and pattern
are illustrated for live paratypes of an adult female, sub-
adult female, and tadpole (Fig. 8), preserved paratypes of
five adult females and six adult males (Fig. S4, see sup-
plemental material online), and three adult males and
three adult females photographed in situ (Fig. 9).
Description of tadpole. A typical tadpole in Gosner
Stage 29 (UF 166637) from Thomas Cabot Biological Sta-
tion, 1,640 m elevation, Reserva Biol
ogica Cerro Uyuca,
Departamento de Francisco Moraz
an, has the following
measurements: BL 25.9; TAL 44.1; TL 69.6; ED 3.1;
IOD 9.6; IND 4.1; ODW 6.8; SW 7.8; MTH 14.5; TMH
7.2; TMW 6.1. Body depressed, wider than high; snout
semicircular in dorsal view, rounded in lateral profile;
eyes moderately large (ED/BL 0.12), directed dorsolater-
ally; nostrils situated at point slightly closer to tip of snout
than eyes, directed anterolaterally; spiracle sinistral,
directed posterodorsally, situated slightly below midline
at point about half the distance from tip of snout to poste-
rior end of body; vent tube dextral; tail musculature slen-
der, extending nearly to tip of acutely rounded tail; height
of tail musculature slightly less than height of either dor-
sal or ventral fins at midlength of tail; dorsal fin terminat-
ing on tail before reaching the posterior end of body. Oral
disc emarginate, relatively small (ODW/SW 0.87) and
positioned anteroventrally; oral disc with broad dorsal gap
in marginal papillae extending most of the width of the
oral disc, rest of oral disc bordered by one row of large
marginal papillae (4–5 per mm); two to three irregular
rows of submarginal papillae present lateral to lower jaw
sheath and between marginal papillae and P-3 tooth row;
keratinized jaw sheaths wide, not serrated; upper jaw
sheath arched, with large, round lateral processes; lower
jaw sheath widely V-shaped; A-2 tooth row gap broad,
with length of lateral segments less than that of medial
gap; A-1 tooth row long, extending just beyond the termi-
nus of the marginal papilla on each side, A-2 row extend-
ing laterally about the same distance as the A-1 row; P-1
tooth row narrowly interrupted medially; P-1 and P-2
tooth rows subequal, shorter than A-1 row, P-3 row
shorter than P-1 and P-2 rows. Colouration after four
years preserved in formalin: body brown dorsally and lat-
erally, dark grey ventrally; tail musculature brown,
heavily spotted or mottled with darker brown throughout;
tail fins translucent brown throughout, becoming darker
posteriorly. A tadpole at Gosner Stage 37 (JHT 3820)
from Cerro San Pedro La Loma (Fig. 7), 2,010 m eleva-
tion, Departamento de Intibuc
a, Honduras, is illustrated
(Fig. 10).
Male vocalization. A male advertisement vocalization
representing Rana lenca from Reserva Biol
ogica Cerro
Uyuca, 1,560 m elevation, was recorded with an Olympus
WS-823 digital recorder and a Sennheiser MKE 400
microphone at an air temperature of 23.6C and barometer
pressure of 24.78 Hg. Recordings were sampled at a 16-bit
resolution and rate of 44.1 kHz and analysed using Raven
Pro version 1.4 (Cornell Laboratory of Ornithology,
Ithaca, NY, USA). The call possessed the following char-
acteristics: call length 240 ms, pulse number 4, and domi-
nant frequency 516.8 Hz (Fig. 11).
Distribution and natural history. Rana lenca is known
from 1,560 to 2,080 m elevation on the Pacific versant of
south-central and south-western Honduras, from localities
Integrative taxonomy of putative hybrid leopard frogs from Honduras 13
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within the Lower Montane Moist Forest formation (Hol-
dridge, 1967). The type locality is an impoundment
(Fig. 7) near the top of Cerro San Pedro La Loma to the
east of La Esperanza, Depto. Intibuc
a. Small patches of
remnant cloud forest are found in the vicinity of the pond;
however most of the area is converted to pasture and the
pond is used as a water source for livestock. The majority
of the remaining paratypes were collected around a small
pond in the transitional zone between pine-oak and cloud
forest just to the south of the Thomas Cabot Biological
Station, which is managed by the nearby Escuela Agr
ıcola
Panamericana Zamorano in Reserva Biol
ogica Cerro
Uyuca, Depto. Francisco Moraz
an. One subadult paratype
(UF 166686) was collected in a marshy area near a reser-
voir in the highland community of Zacate Blanco to the
west of La Esperanza, an area that also possesses patches
of remnant cloud forest but has been largely deforested.
Adults, metamorphs, and tadpoles of other anurans col-
lected in sympatry at these two localities include the con-
gener R. maculatus and representatives of the families
Bufonidae (Incilius ibarrai,I. porteri,Rhinella horribilis)
and Hylidae (Exerodonta catracha,Ptychohyla salvador-
ensis,Tlalocohyla loquax). In addition to these taxa,
McCranie and Wilson (2002) reported tadpoles of this
species in association with tadpoles of Hypopachus bar-
beri (Microhylidae), Leptodactylus silvanimbus (Lepto-
dactylidae), Ptychohyla hypomykter, and Scinax staufferi
(both Hylidae).
Remarks. Specimens considered by McCranie and Wil-
son (2002) to represent ‘R. brownorum XR. forreri’
hybrids are reported from a broader elevational (930–
2,200 m), ecological (Premontane Moist Forest, Premon-
tane Dry Forest, and Lower Montane Moist Forest forma-
tions), and geographic (departments of El Paraiso,
Francisco Moraz
an, Intibuc
a, La Paz, and Ocotepeque)
distribution than is represented in the type series. We con-
sider populations in the departments of Francisco
Moraz
an, Intibuc
a, and La Paz to be conspecific with R.
lenca, and tentatively refer populations from El Paraiso
and Ocotepeque to the new taxon, with the understanding
that further sampling may reveal additional undocu-
mented evolutionary diversity.
A partially metamorphosed specimen from San Pedro
La Loma (UF 166681; 33.3 mm SVL, 41.1 mm tail
length; Fig. 12) exhibits some developmental irregulari-
ties, most notably polyphalangy on the left hind limb. The
mouth appears almost completely metamorphosed,
extending posteriorly beyond the level of the eye (DGos-
ner Stage 45); however, the tail remains essentially com-
plete and shows no sign of atrophy or reduction (D
Gosner Stage 42). Empty pesticide bottles were observed
floating in the pond where this specimen was collected
(the type locality), which is also used by and openly
accessible to livestock.
Discussion
Contact zones and potential for hybridization
The contact zones identified in the SDM analyses repre-
sent the potential of occurrence of the species in their fun-
damental niche, rather than their realized niche. Our SDM
and contact zone analyses identified a narrow band of
potential sympatry between Rana brownorum and
R. lenca, one large and several peripheral areas of poten-
tial sympatry between R. brownorum and R. cf. forreri,
and no predicted contact zones between R. lenca and R.
cf. forreri (Figs 3, Fig. S2, see supplemental material
online). In reference to the McCranie and Wilson (2002)
hypothesis that populations of R. lenca represented
hybrids between R. brownorum and R. cf. forreri,our
results suggest that zones of potential contact between
those two taxa are essentially limited to the Honduran
Depression, a wide valley that interrupts the continental
divide and provides a corridor of subhumid habitat
between the Caribbean and Pacific versants (Figs 3, Fig.
S2, see supplemental material online). While R.
brownorum has a significantly higher probability of occur-
ring in the Honduran Depression than R. cf. forreri
(Table 3), this contact zone is ecologically unsuitable for
R. lenca. Furthermore, across the study area the modelled
distribution of R. cf. forreri does not contact the distribu-
tion of R. lenca, providing support that they are ecolog-
ically isolated and allopatric.
Areas of potential contact between R. brownorum
and R. lenca aresomewhatmorewidespread(Figs 3,
Fig. S2, see supplemental material online). Within the
potential contact zones, the intermediate populations
are significantly less likely to occur than R. browno-
rum (Table 3). In the cases of both contact zones (R.
brownorum and R. cf. forreri,andR. brownorum and
R. lenca),morefocusedsamplingisneededtodeter-
mine if either of these two pairs occur in sympatry,
and, if so, to investigate the potential for hybridization.
Based on our SDM and phylogenetic results, we find
no support for the hybrid hypothesis of McCranie and
Wilson (2002) with respect to populations assigned to
R. lenca, and suggest that any potential hybridization
would be the result of secondary contact between
R. brownorum and R. lenca.
Patterns of diversification in Mesoamerican
Ranids
Our phylogenetic analyses (Figs 4,5) provide clarity in
understanding the diversification for leopard frogs in
Central America. There are at least six species-level
lineages of Pantherana that occur south of Mexico and
belong to one of two geographically and ecologically
discrete clades: a ‘Caribbean’ clade and a ‘Pacific/
Highland’ clade. This topology suggests a model of
14 I. Luque-Montes et al.
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initial invasion of Central America by a single leopard
frog ancestor, followed by vicariance across the conti-
nental divide and subsequent diversification within
each of the two clades.
The Caribbean clade contains at least two species: Rana
brownorum, which we presently consider to inhabit low to
mid elevations in south-eastern Mexico, Guatemala, and
Honduras, and R. taylori, which we consider to occur at
low and mid elevations in Nicaragua and Costa Rica.
Two samples included by Zald
ıvar-River
on et al. (2004), as
R. brownorum 4 and 7 from the Central Depression of Chia-
pas, are sufficiently divergent from other R. brownorum
samples from Mexico and Honduras to warrant further
investigation. To date, the distributional limits and differen-
tiation between R. brownorum and R. taylori remain
uncharacterized, but would inferentially include a hypothet-
ical contact zone in eastern Honduras and/or northern
Nicaragua.
The Pacific/Highland clade is made up of at least
three species: Rana lenca; its sister lineage, Rana sp.
from the central highlands of Costa Rica (species 5
from Hillis & Wilcox, 2005); and the undescribed
Pacific lowland species referred to as R. cf. forreri
(species 6 from Hillis & Wilcox, 2005;Lithobates for-
reri from Frost et al., 2006), with a distribution that
apparently extends from at least El Salvador through
southern Honduras and western Nicaragua to north-
western Costa Rica. This topology suggests initial iso-
lation between the lowland species and the ancestor of
the two highland lineages, followed by allopatric spe-
ciation between the northern (R. lenca)andsouthern
(Costa Rica) lineages. The phylogenetic relationships
of the remaining Central American lineage are some-
what ambiguous; the undescribed species from western
Panama (species 4 from Hillis & Wilcox, 2005)
appears to represent the sister lineage to the clade con-
taining both the Caribbean and Pacific/Highland clades.
It is indicative of the lack of taxonomic resolution
in Mesoamerican ranids that R. lenca is part of a
clade that contains two other unnamed species (R. cf.
forreri and Rana sp.fromCostaRica)and,before
this study, contained no accurately named taxa. Con-
trary to the intention of integrative taxonomy, around
half of all published studies that delimit new species
using integrative methods do not formally diagnose
and name the new species that they delimit, thus fail-
ing to realize the fundamentally critical role that tax-
onomy plays within the biological sciences (Pante,
Schoelinck, & Puillandre, 2014). This paper marks the
first in a series of studies aimed at exploring the evo-
lutionary diversification of Neotropical Ranidae, and
providing taxonomic resolution in support of establish-
ing a secure framework for developing strategies to
conserve and manage overlooked populations in the
Chort
ıs Highlands. While this study has dealt
specifically with resolving the status of the putative
hybrid populations in Honduras, we see this as a first
step towards addressing the remaining unresolved line-
ages in Mesoamerica, with the goal that this and sub-
sequent papers will provide a much-needed measure
of stability to the species-level taxonomy of Neotropi-
cal true frogs.
Acknowledgements
This study was carried out as partial fulfilment for a Mas-
ter of Science degree by IRL; fieldwork in 2007 and 2008
was supported by a grant from the Critical Ecosystem
Partnership Fund (CEPF) to JHT; laboratory work at Indi-
ana University of Pennsylvania (IUP) was supported by
grants from the IUP School of Graduate Studies and
Research and an IUP Department of Biology Undergradu-
ate Research Experience fellowship to KDW; logistical
support for our work in Honduras was provided by the
Centro Zamorano de Biodiversidad. Carla C
arcamo de
Mart
ınez and Iris Acosta O. (Departamento de Areas Pro-
tegidas y Vida Silvestre) assisted in the acquisition of per-
mits for research and exports, and fieldwork in 2007 and
2008 was completed under permits AFE-COHDEFOR
Resolucion GG MP- 055-2006 and Dictamen DAPVS
0091-2006. Field assistance was provided by Jason M.
Butler, T.J. Firneno, Alexander Hess, Michael Itgen, Lor-
raine Ketzler, Catherine Krygeris, Fatima Pereira, and
Scott Travers. Samantha Wisely and Steve Johnson pro-
vided helpful comments on a draft of this manuscript. We
thank Jimmy McGuire, Carol Spencer, and David Wake
(MVZ) and Rafe Brown, Richard Glor, and Luke Welton
(KU) for grants of tissues from their respective collec-
tions. We are grateful to Karli M. Rogers for providing
the tadpole illustration.
Disclosure statement
No potential conflict of interest was reported by the
authors.
Funding
This work was supported by the Critical Ecosystem Part-
nership Fund, Indiana University of Pennsylvania (IUP)
School of Graduate Studies and Research, IUP College
of Natural Science and Mathematics, IUP Department of
Biology and Pennsylvania State System of Higher Educa-
tion (PASSHE) Faculty Professional Development Fund.
Supplemental data
Supplemental data for this article can be accessed here https://
doi.org/10.1080/14772000.2017.1415232.
Integrative taxonomy of putative hybrid leopard frogs from Honduras 15
Downloaded by [71.61.69.106] at 16:20 04 January 2018
ORCID
Erich P. Hofmann http://orcid.org/0000-0001-6209-
6800
Josiah H. Townsend http://orcid.org/0000-0002-6232-
4065
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Associate Editor: Mark Wilkinson
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