Diamond in the rough: A new species of fossorial diamond frog (Rhombophryne) from Ranomafana National Park, southeastern Madagascar

Abstract

We describe a new species from the cophyline microhylid genus Rhombophryne, a group of fossorial and terrestrial frogs endemic to Madagascar. Found during herpetofaunal surveys of moist montane forest in the remote north of Ranomafana National Park, Rhombophryne nilevina sp. n. exemplifies two difficulties that hinder taxonomic progress in Malagasy cophyline frogs: micro-endemicity and highly secretive habits. Known from only two adult male specimens, this new species is nonetheless easily distinguishable from all other known Rhombophryne using morphological data, and osteological data collected here via X-ray Micro-Computed Tomography, or " micro-CT ". This species is now the largest known Rhombophryne, and the only one known from Ranomafana National Park, which will make it the southern-most member of the genus pending a forthcoming taxonomic revision involving Plethodontohyla and Rhombophryne. Pairwise distances of the mitochondrial 16s rRNA marker show a minimum genetic distance of 4.9% from other nominal Rhombophryne. We also describe recordings of an advertisement call, emitted from a burrow by the holotype. Rhombophryne nilevina sp. n. is not known to be found syntopically with other Rhombophryne, nor to be present elsewhere in Ranomafana National Park, but it probably does co-occur with a few ecologically similar Plethodontohyla species. Although the type locality is within a protected area, we suggest an IUCN listing of Data Deficient for R. nilevina sp. n., as its area of occupancy is largely undetermined within the park. Key Words Amphibia Anura Microhylidae Rhombophryne nilevina taxonomy osteology micro-CT endemicity herpetology

Full text

museum für naturkunde
Diamond in the rough: a new species of fossorial diamond frog
(Rhombophryne) from Ranomafana National Park,
southeastern Madagascar
Shea M. Lambert1, Carl R. Hutter2, Mark D. Scherz3
1 Department of Ecology and Evolutionary Biology, University of Arizona, Tucson, AZ 85721, USA
2 Biodiversity Institute and Department of Ecology and Evolutionary Biology, University of Kansas, Lawrence, KS 66045–7561, USA
3 Zoologische Staatssammlung München (ZSM-SNSB), Münchhausenstr. 21, 81247 Munich, Germany
http://zoobank.org/ACD2A947-B1B8-4B12-8FDF-1260C94B0AF8
Corresponding author: Shea M. Lambert (slambert1@email.arizona.edu)
Abstract
We describe a new species from the cophyline microhylid genus Rhombophryne, a group
of fossorial and terrestrial frogs endemic to Madagascar. Found during herpetofaunal sur-
veys of moist montane forest in the remote north of Ranomafana National Park, Rhom-
bophryne nilevina sp. n. exemplies two diculties that hinder taxonomic progress in
Malagasy cophyline frogs: micro-endemicity and highly secretive habits. Known from
only two adult male specimens, this new species is nonetheless easily distinguishable
from all other known Rhombophryne using morphological data, and osteological data
collected here via X-ray Micro-Computed Tomography, or “micro-CT”. This species is
now the largest known Rhombophryne, and the only one known from Ranomafana Na-
tional Park, which will make it the southern-most member of the genus pending a forth-
coming taxonomic revision involving Plethodontohyla and Rhombophryne. Pairwise
distances of the mitochondrial 16s rRNA marker show a minimum genetic distance of
4.9% from other nominal Rhombophryne. We also describe recordings of an advertise-
ment call, emitted from a burrow by the holotype. Rhombophryne nilevina sp. n. is not
known to be found syntopically with other Rhombophryne, nor to be present elsewhere in
Ranomafana National Park, but it probably does co-occur with a few ecologically similar
Plethodontohyla species. Although the type locality is within a protected area, we suggest
an IUCN listing of Data Decient for R. nilevina sp. n., as its area of occupancy is largely
undetermined within the park.
Key Words
Amphibia
Anura
Microhylidae
Rhombophryne nilevina
taxonomy
osteology
micro-CT
endemicity
herpetology
Received 15 August 2016
Accepted 22 December 2016
Published 24 February 2017
Academic editor:
Johannes Penner
Introduction
Over the past several decades, integrative approaches to
taxonomy have shown that Madagascar’s anuran fauna
is one of the most spectacular on earth, with current es-
timates approaching 600 species; 99.9% of which are en-
demic to the island (reviewed in Vieites et al. 2009, Perl et
al. 2014). This estimate continues to rise as more candidate
species are newly discovered, with ~465 species estimated
in Vietes et al. (2009), and ~530 in Perl et al. (2014). Many
recent candidate species have been found from very few
localities, and are presumably restricted to small ranges
(e.g., Rosa et al. 2014, Hutter et al. 2015). Among clades
of Malagasy frogs, the subfamily Cophylinae Cope, 1889
(family Microhylidae Günther, 1858) faces one of the
steepest taxonomic gaps, with more candidate species ex-
isting than described species (Vieites et al. 2009, Perl et
al. 2014, Scherz et al. 2016a). This phenomenon is likely
explicable by the many challenges they present to system-
atists, including secretive habits, small range sizes, and
numerous morphologically cryptic species.
Rhombophryne Boettger, 1880 is a particularly enig-
matic cophyline genus consisting of 16 valid nominal spe-
cies (Scherz et al. 2016a,b), found primarily in rainforest
habitats of northern and eastern Madagascar. In addition
to fossorial or otherwise secretive habits, the apparently
Zoosyst. Evol. 93 (1) 2017, 143–155 | DOI 10.3897/zse.93.10188
Copyright Shea M. Lambert et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which
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Lambert, S.M. et al.: A new Rhombophryne from southeastern Madagascar
144
small ranges of many species have historically impeded
data collection, and thus taxonomic progress, in the genus
(Glaw et al. 2010). However, fueled by the application
of integrative taxonomic approaches, the number of de-
scribed Rhombophryne has swelled in recent years, with 8
species described since 2010 (D’Cruze et al. 2010, Glaw
et al. 2010, Scherz et al. 2014, 2015a,b, 2016b), and only
a few already-published candidate species left to be de-
scribed (Scherz et al. 2016b), although several more have
been identied and will be described soon (Fig. 1 and
Scherz, Crottini, et al. unpubl. data).
We herein describe Rhombophryne nilevina sp. n., dis-
covered during herpetofaunal surveys of moist montane
forest in the remote north-western corner of Ranomafa-
na National Park, southeastern Madagascar, in January of
2015. We diagnose R. nilevina sp. n. from its congeners
using morphological and osteological characters collect-
ed with the help of X-ray Micro-Computed Tomography
(micro-CT). We also describe the male advertisement call
of the new species, which is distinct from all other known
vocalizations in the genus.
Methods
Specimen collection
We collected specimens during the day through targeted
searching, using the advertisement call to locate males. We
euthanized specimens using 20% benzocaine, xed them
in ~10% formalin solution buered with sodium phosphate
to pH 7.0, and transferred them to 70% ethanol for long-
term storage after approximately two weeks. We deposited
the holotype in the Biodiversity Institute of the University
of Kansas (KU) and a paratype in the Mention Zoologie
et Biodiversité Animal, Faculté des Sciences, Université
d’Antananarivo (formerly Département de Biologie Ani-
male of the Universiteé d’Antananarivo; UADBA).
DNA barcoding and phylogenetic analysis
Immediately following euthanasia, we removed the
tongue and placed it in 95% EtOH. We extracted genomic
DNA using standard phenol-chloroform extraction proto-
col and amplied a fragment of the mitochondrial rRNA
marker 16S using a previously published protocol (Hutter
et al. 2015). We include a total of seven newly generated
16S sequences in this study; one of R. nilevina sp. n., one
tentatively assigned to R. coronata, and ve outgroup se-
quences (Table 1). We acquired sequences of the same 16S
fragment for other Rhombophryne from Genbank. Prior
to alignment, we removed identical sequences using the
“Find Duplicates” option in Geneious version 6 (Kearse
et al. 2012). All retained sequences and their accession
numbers are listed in Table 1. We aligned sequences with
the MAFFT (Katoh and Standley 2013) plugin version
1.3 for Geneious, using the the “E-INS-i” algorithm and
otherwise default settings. We inferred phylogenetic re-
Figure 1. Phylogenetic relationships between Rhombophryne species estimated using maximum likelhood in RaxML using the mi-
tochondrial 16S rRNA barcode fragment. Rhombophryne nilevina sp. n. is highlighted with blue bold text. Outgroups are removed
from the tree gure for aesthetic purposes.

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Table 1. GenBank Accession numbers for all sequences used in phylogenetic analysis. Asterisks indicate newly generated sequences.
Species Locality Voucher number Accession
Platypelis barbouri* Ambatomandondona KU 340681 KY288471
Platypelis pollicaris* Torotorofotsy KU 340614 KY288472
Platypelis tuberifera* Vohidrazana CRH 286 KY288470
Plethodontohyla inguinalis* Vohidrazana KU 340642 KY288474
Rhombophryne alluaudi Andasibe ZSM 3/2002 DQ019606
Rhombophryne alluaudi Torotorofotsy ZCMV 968 EU341105
Rhombophryne alluaudi Tsararano MRSN A 2620 AY594105
Rhombophryne cf. coronata* Vohidrazana KU 340732 KY288476
Rhombophryne coronata Mandraka ZSM 694/2001 EU341103
Rhombophryne coudreaui Betampona FAZC 13887 FJ559299
Rhombophryne coudreaui Betampona MRSN A 6271 HM364771
Rhombophryne coudreaui Betampona MRSN A 6347 HM364772
Rhombophryne guentherpetersi Tsaratanana ZCMV 12401 KU937796
Rhombophryne laevipes Montagne d’Ambre ZSM 218/2004 EU341104
Rhombophryne laevipes Montagne d’Ambre FGZC 1052 KM509189
Rhombophryne longicrus Sorata forest FGZC 3651 KR025897
Rhombophryne mangabensis Nosy Mangabe ZCMV 886 KU724181
Rhombophryne matavy Foret’d Ambre FGZC 1888 FJ559298
Rhombophryne matavy Foret’d Ambre FGZC 1890 GU195641
Rhombophryne cf. mangabensis Antsiranana, Andapa AMNH 181903 KM509192
Rhombophryne minuta Marojejy FGZC 2897 EU341100
Rhombophryne minuta Marojejy FGZC 2899 EU341106
Rhombophryne ornata Tsaratanana Camp Matsaborimaika DRV 6456 KP895582
Rhombophryne ornata Tsaratanana Camp Matsaborimaika ZCMV 12382 KP895583
Rhombophryne ornata Tsaratanana Camp Matsaborimaika ZCMV 12384 KP895584
Rhombophryne savaka Marojejy ZCMV 2065 KU724176
Rhombophryne serratopalpebrosa Ambolokopatrika FAZC 7292 EU341111
Rhombophryne sp. Ca01 Ilampy FAZC 10314 FJ559295
Rhombophryne sp. Ca03 Tsaratanana MRSN A 2631 AY594107
Rhombophryne sp. Ca03 Tsaratanana ZSM 667/2001 FJ559296
Rhombophryne botabota Ambolokopatrika MRSN A 2640 AY594104
Rhombophryne botabota Marojejy FGZC 2866 EU341102
Rhombophryne botabota Marojejy ZCMV 2065 FJ559297
Rhombophryne sp. Ca07 Tsaratanana 2001 G46 EU341108
Rhombophryne sp. Ca09 Masoala MRSN A 2115 AY594110
Rhombophryne sp. Ca10 Ilampy MRSN A 2610 AY594111
Rhombophryne nilevina sp. n. * Andemaka KU 340893 KY288475
Rhombophryne botabota Makira ZCMV 11473 KU724173
Rhombophryne tany Tsaratanana Camp Matsaborimaika ZCMV 12359 KP895585
Rhombophryne testudo Nosy Be ZSM 474/2000 KC180070
Rhombophryne testudo Nosy Be ZSM 475/2000 EU341110
Rhombophryne vaventy Antsiranana AMNH A167315 DQ283409
Rhombophryne vaventy Marojejy FGZC 2842 EU341107
Scaphiophryne marmorata* Torotorofotsy KU 340620 KY288473
lationships with RaxML 8.2.6 (Fig. 1; Stamatakis 2014),
using the -f a option to search for a maximum likelihood
tree and conduct 1000 rapid bootstrap replicates, under the
GTR model of sequence evolution and with gamma dis-
tributed rate variation. Finally, we calculated raw pairwise
genetic distances from the alignment using the dist.dna
function of the ape package in R (Table 2, Paradis et al.
2004, R Development Core Team 2016).
Morphology
We took morphological measurements using a digital cali-
per to 0.01 mm, rounded to 0.1 mm. We note that only the
holotype was measured, as the paratype was unavailable
for study. Measurements follow the standard for this genus
and are repeated here verbatim from Scherz et al. (2015b):
“SVL (snout–vent length), HW (maximum head width),
HL (head length, from the maxillary commissure to the
anterior-most point of the mouth), ED (horizontal eye di-
ameter), END (eye–nostril distance), NSD (nostril–snout
tip distance), NND (internarial distance), TDH (horizontal
tympanum diameter), TDV (vertical tympanum diame-
ter), HAL (hand length, from the metacarpal–radioulnar
articulation to the tip of the longest nger), LAL (lower
arm length, from the carpal–radioulnar articulation to the

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Lambert, S.M. et al.: A new Rhombophryne from southeastern Madagascar
146
Table 2. Raw genetic distances at the 16s rRNA gene frag-
ment between analysed taxa and Rhombophryne nilevina sp. n.
(KU 340893).
Taxon Distance
Rhombophryne sp. Ca03 (Tsaratanana) 3.80%
Rhombophryne alluaudi
(Andasibe, Torotorofotsy, Tsararano) 4.89–5.98%
Rhombophryne botabota
(Ambolokopatrika, Marojejy, Makira) 5.98%
Rhombophryne sp. Ca01 (Ilampy) 7.61%
Rhombophryne minuta (Marojejy) 9.78–10.32%
Rhombophryne sp. Ca10 (Ilampy) 10.87%
Rhombophryne tany
(Tsaratanana Camp 2 Matsaborimaika) 11.41%
Rhombophryne laevipes (Montagne d’Ambre) 11.41%
Rhombophryne guentherpetersi (Tsaratanana) 12.50%
Rhombophryne vaventy (Antsiranana, Marojejy) 11.96–12.50%
Rhombophryne testudo (Nosy Be) 11.96%
Rhombophryne coronata (Mandraka) 11.96%
Rhombophryne sp. Ca07 (Tsaratanana) 12.50%
Rhombophryne mangabensis (Nosy Mangabe) 13.04%
Rhombophryne sp. “Ambolokopatrika”
(Ambolokopatrika) 13.04%
Rhombophryne longicrus (Sorata) 11.96%
Rhombophryne cf. mangabensis (Andapa) 12.50%
Plethodontohyla inguinalis (Vohidrazana) 13.59%
Rhombophryne ornata
(Tsaratanana Camp 2 Matsaborimaika) 13.59%
Rhombophryne coudreaui (Betampona) 14.13%
Rhombophryne sp. Ca09 (Masoala) 13.59%
Platypelis pollicaris (Torotorofotsy) 15.76%
Rhombophryne cf. coronata (Vohidrazana) 15.22%
Platypelis barbouri (Ambatomandondona) 16.30%
Rhombophryne matavy (Forêt d’Ambre) 19.02%
Platypelis tuberifera (Vohidrazana) 18.48%
Scaphiophryne marmorata (Torotorofotsy) 23.37%
center of the radioulna–humeral articulation), UAL (upper
arm length, from the center of the radioulna–humeral ar-
ticulation to the trunk, measured along the posterior aspect
of the arm), FORL (forelimb length, given by the sum of
HAL, LAL, and UAL), FOL (foot length, from the tarsal–
metatarsal articulation to the tip of the longest toe), TARL
(tarsal length, from the tarsal–metatarsal articulation to
the tarsal–tibiobular articulation), FOTL (foot length in-
cluding tarsus, from the tibiotarsal articulation to the tip
of the longest toe, given by the sum of FOL and TARL),
TIBL (tibiobula length), TIBW (tibiobula width at
thickest point, measured in dorsal aspect), THIL (thigh
length, from the vent to the femoral–tibiobular articula-
tion), THIW (thigh width at thickest point, measured in
supine position), HIL (hindlimb length, given by the sum
FOL, TARL, TIBL, and THIL), IMCL (maximum length
of inner metacarpal tubercle), IMTL (maximum length
of the inner metatarsal tubercle).” A gure depicting the
measurement scheme is presented in Scherz et al. (2015b).
Osteology
We performed micro-CT scanning on a phoenix|x
nanotom m cone-beam scanner (GE Measurement &
Control, Wunstorf, Germany), using a tungsten target and
a 0.1 mm Cu lter. We employed settings of 140 kV and
80 µA, with a timing of 750 ms, for 2440 projections and a
total scan time of 30 minutes. We assembled the scan les
in datos|x 2 reconstruct CT software (GE Measurement &
Control, Wunstorf, Germany), and imported them as an
unsigned 8-bit volume into VG Studio Max 2.2 (Volume
Graphics GMbH, Heidelberg, Germany). We used the
phong renderer with a custom color palate and rendering
curve to register and visualize the scan. Using the built-in
function, we took high-resolution screenshots for the pro-
duction of gures. The osteological information present-
ed is based on volume rendering. Only slightly calcied
cartilage can be visualized using micro-CT, so we omit
descriptions of the cartilaginous structures of the pectoral
girdle (sternal features and most of the suprascapula) and
those associated with the skull (the hyoid plate and nasal
cartilages in particular). A Digital Imaging and Commu-
nications in Medicine (DICOM) stack of the scan les
and rotational video produced in VG Studio Max 2.2 are
available at the following MorphoSource http://morpho-
source.org/Detail/ProjectDetail/Show/project_id/263
We exported the volume as an “Analyze Volume” un-
der standard settings in VG Studio Max 2.2, and imported
the resulting .hdr le into Amira 6.1 (FEI Visualization
Sciences Group, Burlington MA, USA), where a surface
model was produced essentially following Ruthenstein-
er and Heß (2008). This model is embedded in a Sup-
pl. material 1. The model is provided solely for reader
comprehension; surface models carry inherent bias due
to the manual thresholding are therefore less reliable for
osteological description than volume renderings (Scherz
et al. in review).
We note that skeletal comparisons to other cophylines
are based on largely unpublished micro-CT data pro-
duced by MDS, which will be involved in revisions of
the genera of this subfamily over the next few years.
However, micro-CT-based osteological accounts for
Rhombophryne, Stumpa, Anilany, and Plethodontohyla
are found in Scherz et al. (2016a) and for Cophyla and
Platypelis in Rakotoarison et al. (2015).
Bioacoustics
We recorded calls attributed to the holotype on two oc-
casions using an Olympus LS-10 Linear PCM Field Re-
corder and a Sennheiser K6-ME66 super-cardioid shot-
gun microphone. The calls were recorded at a sampling
rate of 44.1 kHz and 16 bits resolution in WAV format.
Recordings were made at mid-day in overcast weather
conditions. No precise temperature recordings are avail-
able, but we estimate that the ambient temperature was
approximately 20° C at the time of recording. We note
that the individual was not visible during the recordings,
as it was calling from a burrow. We therefor e cannot be
completely certain that the recordings are of the same in-
dividual, however, only a single individual at a time was
heard calling from this location, and the collected indi-
vidual was found with distended vocal sac shortly after

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147
the second recordings. Additionally, the measured call
parameters from the two occasions are nearly completely
overlapping (Fig. 4; Table 3).
We follow Rakotoarison et al. (2015) and dene a call
as individual temporally distinct segments separated by a
return to the background noise between each of these seg-
ments. This denition is equivalent to single notes used in
other call denitions (Duellman and Trueb 1994; mantel-
lids: Hutter et al. 2015). We dene calls as “amplitude mod-
ulated” when there are two or more clear amplitude peaks.
Following Rakotoarison et al. (2015) and Hutter and
Guayasamin (2015), we report the following call vari-
ables: call duration (ms); inter-call interval (s); number
of amplitude peaks; note envelope shape (time at peak
amplitude / call duration); dominant frequency (Hz),
measured throughout call and at peak amplitude; fun-
damental frequency (Hz); and rst harmonic frequency
(Hz). Call rate was not calculated because of insucient
sample size. We used Raven Pro 1.4 to measure tempo-
ral and spectral call characteristics. Digital recordings are
deposited at the University of Kansas Biodiversity Insti-
tute digital archive and are available upon request.
Registration of nomenclature
The electronic version of this article in Portable Document
Format (PDF) will represent a published work according
to the International Commission on Zoological Nomen-
clature (ICZN), and hence the new names contained in
the electronic version are eectively published under that
Code from the electronic edition alone. This published
work and the nomenclatural acts it contains have been
registered in ZooBank, the online registration system for
the ICZN. The ZooBank LSIDs (Life Science Identiers)
can be resolved and the associated information viewed
through any standard web browser by appending the
LSID to the prex http://zoobank.org/. The LSID for this
publication is: urn:lsid:zoobank.org:pub:ACD2A947-
B1B8-4B12-8FDF-1260C94B0AF8. The online version
of this work will be archived and made available from the
following digital repositories: CLOCKSS and Zenodo.
Results
We discovered a large-bodied cophyline microhylid
frog near Andemaka within Ranomafana National Park
in eastern Madagascar. Several obvious dierences in
morphology exist between the collected specimens and
all known described and undescribed cophyline mi-
crohylids. Analysis of a fragment of its mitochondrial
16S rRNA gene recovered it with a close relationship
to an undescribed population of Rhombophryne from
northern Madagascar (sp. Ca03 from Vieites et al. 2009;
Fig. 1). However, this population is quite distinct from
the newly collected frogs morphologically (Scherz et al.,
unpubl. data). We also note that the 16S tree is largely
unresolved, likely due to a limited number of characters
it includes. Ongoing multi-locus analyses suggest that
R. nilevina is quite phylogenetically distinct from all
known Rhombophryne, including sp. Ca03 (A. Crottini,
pers. comm.). Our 16S analysis also shows a minimum
genetic distance of 4.9% between our new taxon and
all valid, nominal Rhombophryne species (Table 2). We
therefore describe it as a new species:
Rhombophryne nilevina sp. n.
http://zoobank.org/DAD2876A-D5C4-4D7B-B712-B22013161FC4
Suggested common English name: The buried diamond frog
Suggested common Malagasy name: Sahona diamondra nilevina
Suggested common French name: La grenouille de diamant enterré
Holotype. KU 340897 (CRH 798), an adult male collect-
ed at mid-day on February 8th 2015 by Shea Maddock
Lambert, Emile Rajeriarison, and Ralaivao Jean Ful-
gence in montane rainforest near the former village of
Andemaka in Ranomafana National Park (ca. 21.1287°S,
47.5054°E, elevation ca. 1240m a.s.l.; Fig. 2).
Paratype. UADBA-A Uncatalogued (CRH 799), an
adult male collected the morning of February 7th 2015
by Shea Maddock Lambert and Ralaivao Jean Fulgence,
otherwise with the same collection information as the
holotype.
Diagnosis. A frog assigned to the cophyline genus
Rhombophryne on the basis of its divided vomer, the
Table 3. The advertisement call recorded for Rhombophryne ni-
levina in comparison with that of R. testudo. Calls were record-
ed from males calling during the day that were subsequently
collected as vouchers. Note envelope is the ratio of the time of
peak amplitude to note duration. Data are the range and then the
mean ± two standard deviations in parentheses, when appropri-
ate. The call recording of R. testudo is from Vences et al. (2006).
Parameters
Species R. nilevina R. testudo
Specimen number KU 340897 NA
Locality Ranomafana Nosy Be
N – calls 7 4
Inter-call interval duration (s) 42.5–99.5
(68.77 ± 24.0)
5.98–10.1
(8.3 ± 2.1)
Call duration (ms) 505–544
(536 ± 1.7)
828–896
(853 ± 2.9)
Call envelope 0.601–0.787
(0.663 ± 0.073) -
Number of amplitude peaks 3–5
(3.4 ± 0.5) 1
Fundamental frequency (Hz) 236.9–279.9
(261.5 ± 22.9)
258.4–279.9
(263.8 ± 10.8)
Dominant frequency
throughout call (Hz)
528.3–538.8
(537.9 ± 9.2)
538.3–555.9
(542.8 ± 8.8)
Dominant frequency
at peak amplitude (Hz)
528.3–538.8
(537.9 ± 9.2)
581.4–602.9
(586.8 ± 10.8)
First Harmonic (Hz) 775.2–818.3
(796.7 ± 17.6)
775.2–796.7
(791.3 ± 10.8)

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Lambert, S.M. et al.: A new Rhombophryne from southeastern Madagascar
148
Figure 2. Map of Ranomafana National Park and the type locality of Rhombophryne nilevina sp. n.. Map is a composite of Landsat
8 satellite imagery and a hillshade layer created from SRTM 1 Arc-Second Global digital elevation data. Data available from the
U.S. Geological Survey.
possession of clavicles and knob-shaped terminal phalanges
(see Scherz et al. 2016a). This species is characterized by
the following suite of characters: large size (SVL at least
up to 57.2 mm), wide, short head (HW 180.7% of HL),
tympanum 58.6% of eye, forelimb 51.1% of SVL, tibia
42.2% of SVL, hindlimb 152.5% of SVL, large inner
metacarpal and metatarsal tubercles, supratympanic fold
distinct and raised, running from the posterior corner of
the eye straight over the tympanum, then sharply down
behind it, extending to join the front of the arm, distinct
vomerine teeth forming curved rows posteromedial to
the oblong choanae, separated medially by a small cleft,
second nger shorter than fourth nger, fth toe distinctly
shorter than third, without nger or toe reduction, nger
and toe tips not enlarged. Additionally, R. nilevina is
separated from all nominal species of Rhombophryne by
an uncorrected pairwise distance of at least 4.9% in the
fragment of the 16S rRNA gene, and by at least 3.8% from
all known candidate species in this genus.
Rhombophryne nilevina is the largest species in the
genus Rhombophryne, and can be distinguished based on
this character alone from all other described species (SVL
57.2 mm vs. maximums of 56.3 mm and 52.9 mm for the
next two largest species, R. laevipes and R. vaventy, re-
spectively). This species diers from all of its congeners
as follows: from all members of the R. serratopalpebrosa
group (R. serratopalpebrosa, R. coronata, R. vaventy, R.
ornata, R. tany, and R. guentherpetersi, plus two species
under description by Scherz et al. in review) by the ab-
sence of superciliary spines (vs. presence); from R. testu-
do, R. coudreaui, and R. matavy by less wide head (HW
180.7% vs. 187.6–242.4% of HL in R. testudo and R.
matavy), longer forelimb (FORL 51.1% vs. 35.4–49.8%
of SVL), longer hindlimb (HIL 152.5% vs. 117.4–140.8%
of SVL), and the possession of a clavicle (vs. lack there-
of); from R. longicrus and R. minima by its wider head
(HW 180.7% vs. 122.5–142.8% of HL), shorter forelimb
(FORL 51.1% vs. 70.4–74.7% of SVL), and shorter hind-
limb (HIL 152.5% vs. 178.5–183.8% of SVL); from R.
savaka and R. mangabensis by its longer forelimb (FORL
51.1% vs. 40.9–47.9% of SVL), well ossied clavicles
(vs. poorly ossied), and absence of black inguinal spots
and a mid-vomerine diastema (vs. presence in R: savaka);
and from R. alluaudi, R. laevipes, and R. botabota by its
wider head (HW 180.7% vs. 144.2–173.8% of SVL), ab-
sence of light dorsolateral stripes (vs. presence in R. allu-
audi), absence of a stark color border between the dorsal
and lateral parts of the head (vs. presence in R. botabota),

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absence of inguinal ocellations (vs. presence in R. laevi-
pes and R. alluaudi).
Rhombophryne nilevina is morphologically similar
to terrestrial members of the genus Plethodontohyla, but
aside from being distinguishable from this genus by the
combination of the possession of clavicles with knob-
shaped terminal phalanges, this species can be distin-
guished from P. inguinalis by its smaller size (SVL 57.2
vs. 62.2–99.1 mm), the absence of enlarged ngertips,
absence of dark inguinal spots (vs. occasional presence),
and absence of a strong dorsolateral color border (vs. occa-
sional presence); from P. notosticta, P. guentheri, P. fonet-
ana, and P. mihanika by the absence of enlarged ngertips,
absence a strong dorsolateral color border (vs. presence in
all but P. fonetana), and shorter forelimb (FORL 51.1% vs.
57.5–71.9% of SVL); and from P. bipunctata, P. tuberata,
P. brevipes, and P. ocellata by the absence of inguinal spots
(vs. presence in all but P. tuberata) and larger size (SVL
57.2 vs. 24.6–44.7 mm) and from P. tuberata by the pres-
ence of smooth skin (vs. granular skin).
Although the bioacoustic repertoires of cophylines is
far from completely known, bioacoustically, this species’
call is strongly distinct from the other known calls by be-
ing strongly amplitude modulated (Fig. 4). To the human
ear, this call most closely resembles the genetically dis-
tant R. testudo (Table 2), but the call of R. testudo diers
by having a much longer duration and lacking signicant
amplitude modulation (Fig 4). No other known calls can
be confused with those of this species.
Description of the holotype. Morphology of the holo-
type. An adult male specimen in an excellent state of
preservation. The vocal sac is still somewhat loose and
malleable. The tongue was removed as a tissue sample.
Body rotund; dorsal and ventral skin smooth, with sub-
tle bumps on the dorsal skin (more rugose in life). Head
considerably wider than long (HW 180.7% of HL), snout
rounded in dorsal and lateral view; nostrils protuberant, di-
rected laterally, closer to the snout than the eye; canthus
rostralis distinct and concave; loreal region concave and
oblique; tympanum indistinct, oval, horizontally 58.6% of
eye diameter; pupil dilated in preservative but more or less
round in life (Fig. 3a, 3d); supratympanic fold distinct and
raised, running from the posterior corner of the eye straight
over the tympanum, then sharply down behind it, extend-
ing to join the front of the arm; tongue removed as a tissue
sample, was attached anteriorly and posteriorly free; vom-
erine teeth distinct, forming curved rows posteromedial to
the choanae; choanae relatively large, oblong.
Arms strongly built, relatively short; ngers without
webbing, short, with distinct, rounded subarticular tuber-
cles, relative lengths 1<2<4<3, the second nger margin-
ally shorter than the fourth (and marginally longer than the
rst), without enlarged terminal discs; inner metacarpal
tubercle strong, oblong, 28.1% of hand length; outer meta-
carpal tubercle indistinct, round. Legs relatively long and
thick (HIL 152.5% of SVL; TIBL 42.2% of SVL), posi-
tion of the tibiotarsal articulation when adpressed along the
body not possible to assess without breaking the hindlimbs;
toes long, unwebbed, with indistinct round subarticular tu-
bercles, relative toe lengths 1<2<5<3<4, third toe distinctly
longer than fth; inner metatarsal tubercle present and dis-
tinct, 12.7% of foot length; outer metatarsal tubercle absent.
Coloration of the holotype. In preservative, the holo-
type is chocolate brown dorsally with a loosely reticulat-
ed pattern of ebony to burnt umber markings, including
an indistinct interocular bar. There are no inguinal spots.
The loreal region has a grey marking in it. The forelimb
is as the dorsum, with dark patches on the elbow and a
crossband on the forearm. A distinct light annulus is pres-
ent before the terminus of each nger. The hindlimb is
dorsally as the back, with three dark crossbands on the
thigh and shank. The posterodorsal thigh has weak cream
spots, as does the anterior thigh. The dorsal foot is brown
speckled with cream. The toes are even more ecked with
cream, and also possess a light annulus before the termi-
nal phalanges. The ventral abdomen is brown with nu-
merous small cream ecks. The chin is darker and mostly
solid dark brown. The ventral arms are as the trunk. The
subarticular and metacarpal tubercles are lighter in color
than the rest of the hand. The ventral hindlimbs are as the
abdomen. The color in life was as in preservative (Fig. 3).
Osteology of the holotype (Fig. 5, Suppl. material 1).
The skeleton of the holotype is typical of Rhombophryne.
It is well ossied and robust. The right femur shows signs
of an old break toward its distal end that has healed.
Anterior braincase laterally closed by the spheneth-
moid. Interior braincase containing calcied material.
Nasal in medial contact with contralalteral and posteri-
or contact with frontoparietal. Frontoparietal broadening
anteriorly from narrow waist anterior to lateral anges,
possessing a strong, posteriorly elongated dorsal process.
Prechoanal vomer simple, triradiate. Neopalatine and
postchoanal vomer distinguishable. Vomerine teeth not
medially fused, without diastemata, oriented oblique to
antero-posterior body axis, curved. Maxillary teeth min-
ute. Otic capsule dorsally poorly ossied.
Sternum not ossied. Clavicle robust, curved. Humer-
us proximally broad, distally rather narrow; possessing
a well-developed crista ventralis along roughly 50% of
its length; crista lateralis weak. Terminal phalanges of
ngers and toes with small distal knobs. Phalangeal for-
mula of ngers 2-2-3-3; of toes 2-2-3-4-3. Femur without
cristae. Prepollex strong, blade-like, half length of rst
metacarpal. Prehallux strong, approximately half length
of rst metatarsal.
Neural spines decrease in size posteriorly, the sixth
and seventh lacking spines altogether. Neural arches of
atlas fused. Dorsal crest of urostyle running roughly 80%
along its shaft. Iliosacral articulation type IIA sensu Em-
erson 1979. Iliac shafts with well developed dorsal tuber-
cles and deep oblique grooves; dorsal crests running most
of their length. Pubis partially ossied.
Variation. The paratype UADBA-A Uncatalogued (CRH
799) strongly resembles the holotype, but has a slightly

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Lambert, S.M. et al.: A new Rhombophryne from southeastern Madagascar
150
Figure 3. Photos in life of Rhombophryne nilevina sp. n. (a) Dorsolateralview of the holotype (KU 340893). (b) Dorsal view of the
holotype. (c) Ventral view of the holotype. (d) Dorsolateral view of the paratype (CRH 799, UADBA-A Uncatalouged). (e) Dorsal
view of the paratype. (f) Ventral view of the paratype.

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more distinct color border between the lateral and dorsal
head (see Fig. 3 for comparison).
Bioacoustics. We analysed a total of seven calls from
R. nilevina, and compared these to the call of R. testudo
(Fig. 4; Table 3). We presume that the calls we recorded
come from one individual, the holotype (see Materials
and methods). We further assume that the recorded call
is an advertisement call, as no other call types (except dis-
tress calls) are known from cophylines. This call sounds
like a slow groan to the human ear.
Each call is rapidly pulsed, with 3–5 (3.5 ± 0.534)
amplitude modulated peaks occurring throughout the
call, and peak amplitude occuring in the last 50% of
the call. The call duration is 505–544 (536 ± 1.7) ms
with an inter-call interval duration of 42.5–99.5 (68.8
± 24.0) s. The fundamental frequency is 236.9–279.9
(261.5 ± 22.9) Hz. The mean dominant frequency
throughout the call was 528.3–555.9 (537.9 ± 9.2) Hz
and the rst harmonic frequency is 775.2–818.3 (796.8
± 17.6) Hz (Fig. 4).
Etymology. The specic epithet “nilevina” is a Malagasy
word meaning “buried.” This name was chosen to recog-
nize the fossorial habits of this species. It is to be treated
as an invariable noun in apposition.
Available names. Due to morphological and size simi-
larities, as well as geographic distribution, two existing
names must be considered for this species: Phryno-
cara laeve Boettger, 1883, and Plethodontohyla laevis
tsianovohensis Angel, 1936. Both of these names are
currently considered to be junior synonyms of Rhombo-
phryne alluaudi. We examined the morphology and os-
teology of the holotypes of both of these taxa (P. laeve:
SMF 4286; P. laevis tsianovohensis: MNHN 1936.47),
and our new species diers critically from both in the
possession of a well-developed clavicle (vs. absence/
strong reduction; Scherz unpubl. data). Their taxonomy,
as well as that of Rhombophryne alluaudi, will be dis-
cussed in a future article, and we here simply rule out
the possibility that they are conspecic with R. nilevina
sp. n. based on the presence vs. absence of a clavicle.
The type specimen of P. laevis tsianovohensis was col-
lected from Tsianovoha, which is around 60 km south of
Ranomafana, suggesting the possibility of sympatry or
parapatry with R. nilevina.
Natural history. Both known specimens of R. nilevina
were obtained from a relatively at, poorly drained
section of moist montane forest adjacent to a stream,
with the holotype found along the bank of this stream.
Nearby habitats include a swamp with many large
Pandanus and steep forested slopes with relatively
smaller trees. However, the calls of R. nilevina seemed
to emanate mostly from the atter, forested area. Males
were heard calling during the day, particularly during
overcast conditions and after rainfall. Advertisement
calls were not heard at night, however, the night-time
chorus of other frogs, including Boophis, Spinomantis,
Gephyromantis, and Anodonthyla, may have interfered
with detection. When heard from a distance, the call is
reminiscent of that of an owl. When heard from close
proximity, the call sounds like a groan, and is far less
melodic. Both specimens were both located by auditory
tracking, and found calling from underground: one
from a cavity under the roots of a large tree, and the
other from a burrow in soft, moist soil alongside the
stream. In order to collect the holotype from its burrow,
excavation was required. Based on these observations
and suggestive morphology, we presume that R. nilevina
spend much of their lives underground, possibly
coming to the surface for short periods during rainfall,
similar to other fossorial Rhombophryne species (Glaw
and Vences 2007, D’Cruze et al. 2010). We also note
that R. nilevina was discovered in the middle of the wet
season, and after a week-long period of particularly
heavy, sustained rain.
Distribution. Rhombophryne nilevina has thus far been
detected at a single site, near the former village of An-
demaka, in the north-west of Ranomafana National Park
(Fig. 2). This locality is relatively high-elevation for
Ranomafana National Park (ca. 1240 m). To our knowl-
edge, R. nilevina has not been detected by any previous
survey, including several conducted by CRH and SML
at similarly high-elevation sites in the northern (Miara-
nony), central (Vohiparara), and southern (Maharira) re-
gions of Ranomafana. Nevertheless, we do not rule out
here the possibility that R. nilevina occurs elsewhere in
the park. This is in large part due to the secretive hab-
its and potentially ephemeral activity periods of this
species (see Natural history). In addition, much of the
high-elevation forest of Ranomafana is dicult to ac-
cess and thus remains sparsely or completely unsurveyed
for herpetofauna. Although it is possible that R. nilevina
has been overlooked in other eastern rainforest patches,
current information suggests that this species is endemic
to Ranomafana National Park, and potentially to a much
smaller area within the park.
Conservation status. Although the type locality of R.
nilevina is within Ranomafana National Park, its occu-
pancy within the park is potentially highly restricted, ele-
vationally and geographically, as it has not been detected
in any other herpetological surveys of the park. However,
its secretive lifestyle means that it icould be easily over-
looked. Given this large uncertainty in area of occupancy,
we suggest an initial IUCN categorization of Data De-
cient. If R. nilevina is for instance, restricted to the type
locality, then habitat destruction, chytrid fungus (recently
detected in Madagascar, Bletz et al. 2015), and/or climate
change could easily place the only population of R. nile-
vina sp. n. at risk of extinction.

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Lambert, S.M. et al.: A new Rhombophryne from southeastern Madagascar
152
Figure 4. The osteology of Rhombophryne nilevina sp. n. Skull in (a) lateral, (b) dorsal, and (c) ventral view; and full skeleton in
(d) dorsal and (e) ventral view. Abbreviations: angspl, angulosplenial; angspl.cp, angulosplenial coronoid process; col, columella;
exoc, exoccipital; fpar, frontoparietal; fpar.dop, frontoparietal dorsal process; max, maxilla; max.pf, maxillary pars fascialis; mmk,
mentomeckelian bone; npl, neopalatine; pmx, premaxilla; povom, postschoanal vomer; proot, prootic; prvom, prechoanal vomer;
prsph.ap, parasphenoid alary process; prsph.cp, parasphenoid cultriform process; pter.ar, pterygoid anterior ramus; pter.vr, pterygoid
ventral ramus; pter.mr, pterygoid medial ramus; qj, quadratojugal; qj.pvp, quadratojugal posteroventral process; smx, septomaxilla;
spheth, sphenethmoid; sq, squamosal; sq.or, squamosal otic ramus; sq.zr, squamosal zygotic ramus.

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Discussion
The discovery of Rhombophryne nilevina—never pre-
viously identied as a candidate species despite being
found in one of the most well-surveyed National Parks
of Madagascar—highlights the importance of continued
eld work for the advancement of systematics in Mal-
agasy anurans. In particular, eld surveys should help
reveal diversity in clades containing species with small
ranges and secretive life histories, including Rhombo-
phryne and other cophyline frogs. Cophylines have al-
ready shown great promise as a model system for study-
ing ecomorphological and reproductive mode evolution
(e.g. Andreone et al. 2005, Wollenberg et al. 2008), and
the continued discovery and description of novel species
will only further this potential.
Rhombophryne nilevina is remarkable in several re-
spects, including its morphology. Most obvious is its
large size, the largest recorded for the genus, narrowly ex-
ceeding R. laevipes (Glaw & Vences, 2007; Scherz et al.
unpubl. data). In addition, the relatively long legs, wide
head, and rotund body shape contribute to the distinctive
appearance of this species. In total, the morphology of R.
nilevina is suciently divergent from all other Rhombo-
phryne species that it cannot be immediately assigned to
a complex or species cluster.
In addition to morphological distinctiveness, Rhombo-
phryne nilevina is currently the southernmost distributed
species of Rhombophryne, excluding records of Rhombo-
phryne alluaudi from the far south of Madagascar, which
are due to confusion surrounding the identity of that spe-
cies (Scherz, Bellati, Crottini et al. unpubl data). It also
has a strongly amplitude-modulated call unlike that of
any congeners (although few call recordings are available
for this genus).
Our limited genetic data suggests that R. nilevina
may have anities with Rhombophryne sp. Ca3 from
Tsaratanana in northern Madagascar, but we consider
this relationship tentative and ongoing multi-locus anal-
yses suggest that R. nilevina represents a relatively ear-
Figure 5. Comparative spectrograms (top), oscillograms (center) and power spectra (bottom) between the calls of (A) Rhombo-
phryne nilevina sp. n. and (B) R. testudo (from Vences et al. 2006). Spectrogram was created using a Hanning window size of 1024.

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Lambert, S.M. et al.: A new Rhombophryne from southeastern Madagascar
154
ly-diverging, phylogenetically distinct species of Rhom-
bophryne (A. Crottini, pers. comm.). Given the limited
information available at this time, the phylogenetic an-
ities of R. nilevina will need to be claried in a future
revision of the genus.
Acknowledgements
We thank the Malagasy authorities for issuing permits;
eld research was conducted under permit number 303/14/
MEF/SG/DGF/DCB.SAP/SCB; specimens were exported
under 017N-EV01/MG14. We also thank MICET and
Centre ValBio for facilitating eldwork. Finally, SML
would like to thank Ralaivao Jean Fulgence and Emile
Rajeriarison for their exceptional work in the eld during
the Andemaka expedition. If not for their dedication and
ability, R. nilevina would surely remain undiscovered.
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Supplementary material 1
File S1
Authors: Shea M. Lambert, Carl R. Hutter, Mark D.
Scherz
Data type: Adobe PDF le
Explanation note: This le contains a PDF-embedded in-
teractive 3D model of the skeleton of the holotype of
Rhombophryne nilevina sp. n., KU 340897, generated
via X-ray micro-Computed Tomography. The model
can be opened in Adobe® Acrobat Pro or Reader, ver-
sions IX and above. To activate it, click the image.
Copyright notice: This dataset is made available under
the Open Database License (http://opendatacommons.
org/licenses/odbl/1.0/). The Open Database License
(ODbL) is a license agreement intended to allow us-
ers to freely share, modify, and use this Dataset while
maintaining this same freedom for others, provided
that the original source and author(s) are credited.