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


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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
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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
Corresponding author: Shea M. Lambert (
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
Rhombophryne nilevina
Received 15 August 2016
Accepted 22 December 2016
Published 24 February 2017
Academic editor:
Johannes Penner
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
permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Lambert, S.M. et al.: A new Rhombophryne from southeastern Madagascar
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.
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.
Zoosyst. Evol. 93 (1) 2017, 143–155
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).
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
Lambert, S.M. et al.: A new Rhombophryne from southeastern Madagascar
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).
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-
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).
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
Zoosyst. Evol. 93 (1) 2017, 143–155
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
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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 The LSID for this
publication is:
B1B8-4B12-8FDF-1260C94B0AF8. The online version
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following digital repositories: CLOCKSS and Zenodo.
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.
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
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).
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)
(8.3 ± 2.1)
Call duration (ms) 505–544
(536 ± 1.7)
(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)
(263.8 ± 10.8)
Dominant frequency
throughout call (Hz)
(537.9 ± 9.2)
(542.8 ± 8.8)
Dominant frequency
at peak amplitude (Hz)
(537.9 ± 9.2)
(586.8 ± 10.8)
First Harmonic (Hz) 775.2–818.3
(796.7 ± 17.6)
(791.3 ± 10.8)
Lambert, S.M. et al.: A new Rhombophryne from southeastern Madagascar
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),
Zoosyst. Evol. 93 (1) 2017, 143–155
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
Lambert, S.M. et al.: A new Rhombophryne from southeastern Madagascar
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.
Zoosyst. Evol. 93 (1) 2017, 143–155
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.
Lambert, S.M. et al.: A new Rhombophryne from southeastern Madagascar
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;, 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;, pterygoid anterior ramus; pter.vr, pterygoid
ventral ramus;, 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.
Zoosyst. Evol. 93 (1) 2017, 143–155
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.
Lambert, S.M. et al.: A new Rhombophryne from southeastern Madagascar
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.
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.
Andreone F, Vences M, Vieites DR, Glaw F, Meyer A (2005) Recur-
rent ecological adaptations revealed through a molecular analysis
of the secretive cophyline frogs of Magascar. Molecular Phyloge-
netics and Evolution 34(2): 315–322.
Bletz MC, Rosa GM, Andreone F, Courtois EA, Schmeller DS, Rabibisoa
NHC, Rabemananjara FCE, Raharivololoniaina L, Vences M, Wel-
don C, Edmonds D, Raxworth CJ, Harris RN, Fisher MC, Crottini A
(2015) Widespread presence of the pathogenic fungus Batrachochy-
trium dendrobatidis in wild amphibian communities in Madagascar.
Scientic Reports 5: 8633.
D’Cruze N, Köhler J, Vences M, Glaw F (2010) A new fat fossorial frog
(Microhylidae: Cophylinae: Rhombophryne) from the rainforest of
the Forêt d’Ambre Special Reserve, northern Madagascar. Herpeto-
logica 66(2): 182–19.
Duellman WE, Trueb L (1994) Biology of Amphibians. Johns Hopkins
University Press, London, U.K., 696 pp.
Emerson SB (1979) The ilio-sacral articulation in frogs: form and
function. Biological Journal of the Linnaean Society 11: 153–168.
Glaw F, Vences M (2007) A Field Guide to the Amphibians and Reptiles of
Madagascar. Third Edition. Köln, Vences & Glaw Verlags GbR, 496 pp.
Glaw F, Köhler J, Vences M (2010) A new fossorial frog, genus
Rhombophryne, from Nosy Mangabe Special Reserve, Madagas-
car. Zoosystematics and Evolution, 86(2): 235–243. http://dx.doi.
Hutter CR, Guayasamin JM (2015) Cryptic diversity concealed in the
Andean cloud forests: two new species of rainfrogs (Pristimantis)
uncovered by molecular and bioacoustic data. Neotropical Biodi-
versity 1: 36–59.
Hutter CR, Lambert SM, Cobb KA, Andriampenomanana ZF, Vences
M (2015) A new species of bright-eyed treefrog (Mantellidae) from
Madagascar, with comments on call evolution and patterns of syn-
topy in the Boophis ankaratra complex. Zootaxa 4034(2): 531–555.
Katoh K, Standley DM (2013) MAFFT multiple sequence alignment
software version 7: improvements in performance and usability.
Molecular Biology and Evolution 30(4): 772–780. http://dx.doi.
Kearse M, Moir R, Wilson A, Stones-Havas S, Cheung M, Sturrock S,
Buxton S, Cooper A, Markowitz S, Duran C, Thierer T, Ashton B,
Mentjies P, Drummond A (2012) Geneious Basic: an integrated and
extendable desktop software platform for the organization and anal-
ysis of sequence data. Bioinformatics 28(12): 1647–1649. http://
Paradis E, Claude J, Strimmer K (2004) APE: analyses of phylogenetics
and evolution in R language. Bioinformatics 20(2): 289–290. http://
Perl RB, Nagy ZT, Sonet G, Glaw F, Wollenberg KC, Vences M (2014)
DNA barcoding Madagascar’s amphibian fauna. Amphibia-Reptilia,
35(2): 197–206.
Pyron RA, Wiens JJ (2011) A large-scale phylogeny of Amphibia includ-
ing over 2800 species, and a revised classication of extant frogs,
salamanders, and caecilians. Molecular Phylogenetics and Evolution
61 (2): 543–583.
R Development Core Team (2016) R: A language and environment for
statistical computing. R Foundation for Statistical Computing, Vienna,
Rakotoarison A, Crottini A, Müller J, Rödel M-O, Glaw F, Vences M
(2015) Revision and phylogeny of narrow-mouthed treefrogs (Co-
phyla) from nothern Madagscar: integration of molecular, oste-
ological, and bioacoustic data reveals three new species. Zootaxa
3937(1): 061–089.
Rosa GM, Crottini A, Noël J, Rabibisoa N, Raxworthy CJ, Andreone F
(2014) A new phytolemic species of Platypelis (Microhylidae: Co-
phylinae) from the Betampona Reserve, eastern Madagascar. Sala-
mandra 50(4): 201–214.
Ruthensteiner B, Heß M (2008) Embedding 3D models of biological
specimens in PDF publications. Microscopy Research and Tech-
nique 71: 778–786.
Scherz MD, Ruthensteiner B, Vences M, Glaw F (2014) A new microhylid
frog, genus Rhombophryne, from northeastern Madagascar, and a re-de-
scription of R. serratopalpebrosa using micro-computed tomography.
Zootaxa, 3860 (6): 547–560.
Scherz MD, Ruthensteiner B, Vieites DR, Vences M, Glaw F (2015a)
Two new microhylid frogs of the genus Rhombophryne with super-
ciliary spines from the Tsaratanana Massif in northern Madagascar.
Herpetologica, 71(4): 310–321.
Scherz MD, Rakotoarison A, Hawlitschek O, Vences M, Glaw F
(2015b): Leaping towards a saltatorial lifestyle? An unusually long-
legged new species of Rhombophryne (Anura, Microhylidae) from
the Sorata massif in northern Madagascar. Zoosystematics and Evo-
lution, 91(2): 105–114.
Scherz MD, Vences M, Rakotoarison A, Andreone F, Köhler J, Glaw F,
Crottini A (2016a): Reconciling molecular phylogeny, morpholog-
ical divergence and classifcation of Madagascan narrow-mouthed
frogs (Amphibia: Microhylidae). Molecular Phylogenetics and Evo-
lution 100: 372–381.
Scherz MD, Glaw F, Vences M, Andreone F, Crottini A (2016b) Two new
species of terrestrial microhylid frogs (Microhylidae: Cophylinae:
Rhombophryne) from northeastern Madagascar. Salamandra 52(2):
Zoosyst. Evol. 93 (1) 2017, 143–155
Scherz MD, Hawlitschek O, Andreone F, Rakotoarison A, Vences M,
Glaw F (in review) A review of the taxonomy and osteology of
the Rhombophryne serratopalpebrosa species group (Anura: Micro-
hylidae) from Madagascar, with comments on the value of volume
rendering of micro-CT data to t taxonomists. Zootaxa.
Stamatakis A (2014) RaxML version 8: a tool for phylogenetic analy-
sis and post-analysis of large phylogenies. Bioinformatics 30 (9):
Vences M, Glaw F, Marquez R (2006) The Calls of the Frogs of Madagascar.
3 Audio CD’s and booklet. Foneteca Zoológica, Madrid, Spain, 44 pp.
Vieites DR, Wollenberg KC, Andreone F, Köhler J, Glaw F, Vences
M (2009) Vast underestimation of Madagascar’s biodiversity evi-
denced by an integrative amphibian inventory. Proceedings of the
National Academy of Sciences, 106(20): 8267–8272. http://dx.doi.
Wollenberg KC, Vieites DR, Van Der Meijden A, Glaw F, Canatella DC,
Vences M (2008) Patterns of endemism and species richness in Mal-
agasy cophyline frogs support a key role of mountainous areas for
speciation. Evolution 62(8): 1890–1907.
Supplementary material 1
File S1
Authors: Shea M. Lambert, Carl R. Hutter, Mark D.
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.
... In a large-scale assessment of Malagasy amphibians using molecular taxonomic identification, Vieites et al. (2009) revealed the presence of 10 unnamed lineages within the genus Rhombophryne, later increased to 13 candidate species by Perl et al. (2014). Since 2009, 13 new species of Rhombophryne have been described (D'Cruze et al., 2010;Glaw et al., 2010;Lambert et al., 2017;Scherz, 2020;Scherz et al., 2016aScherz et al., , 2017Scherz et al., , 2019Scherz et al., , 2015aScherz et al., , 2014Scherz et al., , 2015b for a total number of 20 currently recognized species (AmphibiaWeb, 2021). This boost in taxonomic and systematic research is explained by the increased availability of specimens hosted in institutional collections, and by the application of an integrative taxonomic approach (Padial et al., 2010). ...
... Ca03 was established as a candidate species by Vieites et al. (2009). This lineage has a 16S3 0 genetic distance of 1.8% to R. nilevina Lambert et al., 2017, and of 2.0% to R. cf. nilevina (Table 1). ...
... (3) In the description of R. nilevina, Lambert et al. (2017) report both institutional collection codes KU 340893 and KU 340897 as the holotype, and associated the only sequence generated for this species (KY288475; 16S3 0 ) to KU 340893. After consulting with the authors, we confirm that the code KU 340897 corresponds to the specimen number of the holotype of R. nilevina and that the accession KY288475 was generated from this individual, whereas the code KU 340893 is not associated with any specimens of this species. ...
The study of diamond frogs (genus Rhombophryne, endemic to Madagascar) has been historically hampered by the paucity of available specimens, because of their low detectability in the field. Over the last 10 years, 13 new taxa have been described, and 20 named species are currently recognized. Nevertheless, undescribed diversity within the genus is probably large, calling for a revision of the taxonomic identification of published records and an update of the known distribution of each lineage. Here we generate DNA sequences of the mitochondrial 16S rRNA gene of all specimens available to us, revise the genetic data from public databases, and report all deeply divergent mitochondrial lineages of Rhombophryne identifiable from these data. We also generate a multi-locus dataset (including five mitochondrial and eight nuclear markers; 9844 bp) to infer a species-level phylogenetic hypothesis for the diversification of this genus and revise the distribution of each lineage. We recognize a total of 10 candidate species, two of which are identified here for the first time. The genus Rhombophryne is here proposed to be divided into six main species groups, and phylogenetic relationships among some of them are not fully resolved. These frogs are primarily distributed in northern Madagascar, and most species are known from only few localities. A previous record of this genus from the Tsingy de Bemaraha (western Madagascar) is interpreted as probably due to a mislabelling and should not be considered further unless confirmed by new data. By generating this phylogenetic hypothesis and providing an updated distribution of each lineage, our findings will facilitate future species descriptions, pave the way for evolutionary studies, and provide valuable information for the urgent conservation of diamond frogs.
... The BI and ML reconstructions of Rhombophryne largely agreed with one another (Fig. 1) and with most previous work on this genus (Scherz et al. 2015a(Scherz et al. , 2016a(Scherz et al. , 2017aLambert et al. 2017), although support was generally low, and it is evident that the phylogeny of the genus cannot be resolved based on these two short mitochondrial markers alone (R. coudreaui for example is very unstable in its placement, but on morphological grounds is thought to belong to the R. testudo+R. matavy clade). ...
... Fortunately, the detection probability of (semi-) fossorial frogs seems to increase in inclement weather. In Madagascar, cyclones appear to act as a particular stimulus for the activity of such frogs: three species that my colleagues and I have recently described, R. nilevina, R. ellae, and Anodonthyla eximia, were all collected during cyclonic weather (Lambert et al. 2017;Scherz et al. 2019). This emphasises the importance of rainy-season studies in even well-surveyed areas like Montagne d'Ambre in order to fully capture the diversity of these areas. ...
... Morphologically, Rhombophryne ellae sp. nov. is similar to R. laevipes, R. nilevinaLambert, Hutter &Scherz, 2017, and R. botabota Scherz, Glaw, Vences, Andreone & Crottini, 2016 ...
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Although taxonomic progress on the frogs of Madagascar is currently proceeding at an unprecedented pace, the goal of completing the amphibian inventory of this hyper-diverse island is still far off. In part this is because more new species continue to be discovered at a high rate, in some cases within well-studied areas. Here, I describe Rhombophryne ellaesp. nov. , a new species of diamond frog discovered in Montagne d’Ambre National Park in northern Madagascar in 2017. This new species is highly distinctive in having orange flash-markings on its hindlimbs (not known from any described species of Rhombophryne ), and large, black inguinal spots (larger than in all other described Rhombophryne species). It is separated from all named species of Rhombophryne by a substantial uncorrected pairwise distance in the 16S rRNA mitochondrial barcode marker (> 7%) and is most closely related to an undescribed candidate species from Tsaratanana in northern Madagascar. Rhombophryne ellaesp. nov. adds another taxon to the growing list of cophyline microhylids that have red to orange flash-markings, the function of which remains unknown and which has clearly evolved repeatedly in this radiation. The discovery of such a distinctive species within a comparatively well-studied park points toward the low detectability of semi-fossorial frogs and the role of inclement weather in increasing that detectability.
... n. is not the first species to have almost exactly this distribution. Rhombophryne vaventy Scherz, Ruthensteiner, Vences & Glaw was recently recovered from Sorata (Peloso et al. 2016, Scherz et al. 2016, Lambert et al. 2017) after initially having been described from the same type locality as G. lomorina sp. n. (Scherz et al. 2014). ...
... The environmental conditions of these two regions are similar (Brown et al. 2016), and various species originally described from one of the two areas have subsequently been discovered in the other, e.g. Rhombophryne vaventy (Peloso et al. 2016, Scherz et al. 2016, 2017a, Lambert et al. 2017, Gephyromantis (Asperomantis) tahotra (Glaw et al. 2011, and G. (D.) schilfi Vences 2000, Scherz et al. 2017b). These similarities are generally limited to species found above 1200 m, probably because forest below 1200 m in Sorata has been mostly eradicated. ...
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We describe a new species of the genus Gephyromantis, subgenus Vatomantis (Mantelli-dae, Mantellinae), from moderately high elevation (1164-1394 m a.s.l.) on the Marojejy, Sorata, and Andravory Massifs in northern Madagascar. The new species, Gephyromantis (Vatomantis) lomorina sp. n. is highly distinct from all other species, and was immediately recognisable as an undescribed taxon upon its discovery. It is characterised by a granular, mottled black and green skin, reddish eyes, paired subgular vocal sacs of partly white colour, bulbous femoral glands present only in males and consisting of three large granules, white ventral spotting, and a unique, amplitude-modulated advertisement call consisting of a series of 24-29 rapid, quiet notes at a dominant frequency of 5124-5512 Hz. Genetically the species is also strongly distinct from its congeners, with uncorrected pairwise distances ≥10 % in a fragment of the mitochondrial 16S rRNA gene to all other nominal Gephyromantis species. A molecular phylogeny based on 16S sequences places it in a clade with species of the subgenera Laurentomantis and Vatomantis, and we assign it to the latter subgenus based on its morphological resemblance to members of Vatoman-tis. We discuss the biogeography of reptiles and amphibians across the massifs of northern Madagascar, the evidence for a strong link between Marojejy and Sorata, and the role of elevation in determining community sharing across this landscape.
... In spite of the recent efforts in revising the systematics of this subfamily (e.g. D' Cruze et al. 2010, Rakotoarison et al. 2012, 2015, Rosa et al. 2014, Scherz et al. 2016b, Lambert et al. 2017), the genus Plethodontohyla has been relatively neglected. Plethodontohyla in its most updated definition ( Wollenberg et al. 2008, Scherz et al. 2016a) comprises moderately small to large terrestrial or scansorial forest frogs (snout-vent length [SVL] 25-100 mm, Glaw and Vences 2007b). ...
... The call of Plethodontohyla laevis is overall quite similar to the other species of this genus, and also members of the genus Rhombophryne (e.g. Lambert et al. 2017), but seems to have a lower repetition rate. Although we recorded only one of these long inter-call intervals, the recording was made on a rainy night, with more males calling simultaneously, all with long inter-call intervals. ...
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The systematics of the cophyline microhylid frog genera Plethodontohyla and Rhombophryne have long been intertwined, and their relationships have only recently started to become clear. While Rhombophryne has received a lot of recent taxonomic attention, Plethodontohyla has been largely neglected. Our study is a showcase of just how complex the taxonomic situation between these two genera is, and the care that must be taken to resolve taxonomic conundrums where old material, multiple genus transitions, and misattribution of new material obfuscate the picture. We assessed the identity of the historic names Dyscophus alluaudi (currently in the genus Rhombophryne), Phrynocara laeve and Plethodontohyla laevis tsianovohensis (both synonyms of Rhombophryne alluaudi) based on an integrative taxonomic approach harnessing genetics, external morphology, osteological data obtained via micro-Computed Tomography (micro-CT) and bioacoustics. We show that (1) the holotype of Dyscophus alluaudi is a member of the genus Plethodontohyla; (2) the Rhombophryne specimens from central Madagascar currently assigned to Rhombophryne alluaudi have no affinity with that species, and are instead an undescribed species; and (3) Phrynocara laeve and Dyscophus alluaudi are not synonymous, but represent closely related species, whereas Plethodontohyla laevis tsianovohensis is tentatively confirmed as synonym of D. alluaudi. We resurrect and re-describe Plethodontohyla laevis, and re-allocate and re-describe Plethodontohyla alluaudi on the basis of new and historic material.
... Anurans are also the most diverse in larval and adult cranial structure of the extant amphibian orders (Trueb 1993). Many cranial bones are lost as discernible elements repeatedly across this clade, including through fusion between elements or failure to ossify, as well as novel bones evolving in specific subclades (e.g., see Trueb, 1970Trueb, , 1973Hall and Larsen 1998;Campos et al. 2010;Schoch 2014;Pereyra et al. 2016;Lambert et al. 2017). Traditional morphometric approaches would fail to capture the morphology of these variably present bones. ...
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Evolutionary integration (covariation) of traits has long fascinated biologists because of its potential to elucidate factors that have shaped morphological evolution. Studies of tetrapod crania have identified patterns of evolutionary integration that reflect functional or developmental interactions among traits, but no studies to date have sampled widely across the species‐rich lissamphibian order, Anura (frogs). Frogs exhibit a vast range of cranial morphologies, life history strategies and ecologies. Here, using high‐density morphometrics we capture cranial morphology for 172 anuran species, sampling every extant family. We quantify the pattern of evolutionary modularity in the frog skull and compare patterns in taxa with different life history modes. Evolutionary changes across the anuran cranium are highly modular, with a well‐integrated ‘suspensorium’ involved in feeding. This pattern is strikingly similar to that identified for caecilian and salamander crania, suggesting replication of patterns of evolutionary integration across Lissamphibia. Surprisingly, possession of a feeding larval stage has no notable influence on cranial integration across frogs. However, late‐ossifying bones exhibit higher integration than early‐ossifying bones. Finally, anuran cranial modules show diverse morphological disparities, supporting the hypothesis that modular variation allows mosaic evolution of the cranium, but we find no consistent relationship between degree of within‐module integration and disparity. This article is protected by copyright. All rights reserved
... It is home to thousands of endemic species, 739 of which are endemic vertebrates, with many still to be and currently being described (Lambert et al. 2017;Perl et al. 2014;Scherz et al. 2017;Vieites et al. 2009) -see Table 1 for breakdown (Goodman and Benstead 2005). Many of these species are vulnerable to climate change (Bellard et al. 2014) and are under severe pressure from habitat loss and fragmentation , especially on the east coast (Geldmann et al. 2014). ...
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Invasive and introduced species can pose major ecological challenges to vulnerable native wildlife. Biodiversity hotspots, in particular, require protection from this significant cause of species loss. One hotspot, Madagascar, is experiencing the accidental introduction of a potentially ecologically damaging species – the toxin carrying bufonid toad, Duttaphyrnus melanostictus. The presence of these toxic invaders drives fears that if such a species gains a foothold widespread poisoning of Malagasy predators could occur, mirroring the invasion of Australia by Rhinella marina. This includes numerous endemic and endangered species. The mechanism by which the toxin acts upon organisms has been previously identified via the study of toxin resistant versus toxin non-resistant taxa. Specific amino acid substitutions are required on the organism’s Na+/K+–ATPase for them to be resistant to bufonid toxin. This solution to combat the toxin is widely consistent across taxa providing a method to discover and predict toxin resistance or vulnerability. Here I investigate the Na+/K+–ATPase gene to detect vulnerability of a selection of Malagasy fauna to the toxics of Duttaphrynus melanostictus. It is discovered that no tested species on Madagascar have the capacity to survive ingestion of the novel toxin. The vulnerability is found in all examined species, including snakes, frogs, lizards, lemurs and tenrecs. The results suggest that the invasive Duttaphrynus melanostictus is liable to have significant impact on Malagasy fauna.
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We describe a fantastic new species of forest frog (Mantellidae: Gephyromantis : subgenus: Laurentomantis ) from moderately high elevations in the vicinity of Andasibe, Madagascar. This region has been surveyed extensively and has a remarkably high anuran diversity with many undocumented species still being discovered. Surprisingly, by exploring areas around Andasibe that lacked biodiversity surveys, we discovered a spectacular and clearly morphologically distinct species, previously unknown to science, Gephyromantis marokoroko sp. nov. , documented for the first time in 2015. The new species is well characterised by a very rugose and granular dorsum, dark brown skin with bright red mottling, sparse light orange to white spots on the ventre, vibrant red eyes and femoral glands present only in males that consist of eight medium-sized granules. Bioacoustically, the new species has a quiet advertisement call that differs from related species by having a moderate call duration, 2–4 strongly pulsed notes and a slow note repetition rate. Furthermore, it has substantial differentiation in mitochondrial DNA, with pairwise distances of 7–9% to all other related species in sequences of the mitochondrial 16S rRNA marker. Additional evidence is given through a combined four mitochondrial markers and four nuclear exons concatenated species tree, strongly supporting G. striatus as the sister species of the new species in both analyses. The discovery of this new species highlights the need for continued inventory work in high elevation rainforests of Madagascar, even in relatively well-studied regions.
Aim The diversity of brood size across animal species exceeds the diversity of most other life‐history traits. In some environments, reproductive success increases with brood size, whereas in others it increases with smaller broods. The dominant hypothesis explaining such diversity predicts that selection on brood size varies along climatic gradients, creating latitudinal fecundity patterns. Another hypothesis predicts that diversity in fecundity arises among species adapted to different microhabitats within assemblages. A more recent hypothesis concerned with the consequences of these evolutionary processes in the era of anthropogenic environmental change predicts that low‐fecundity species might fail to recover from demographic collapses caused by rapid environmental alterations, making them more susceptible to extinctions. These hypotheses have been addressed predominantly in endotherms and only rarely in other taxa. Here, we address all three hypotheses in amphibians globally. Location Global. Time period Present. Major taxa studied Class Amphibia. Methods Using a dataset spanning 2,045 species from all three amphibian orders, we adopt multiple phylogenetic approaches to investigate the association between brood size and climatic, ecological and phenotypic predictors, and according to species conservation status. Results Brood size increases with latitude. This tendency is much stronger in frogs, where temperature seasonality is the dominant driver, whereas salamander fecundity increases towards regions with more constant rainfall. These relationships vary across continents but confirm seasonality as the key driver of fecundity. Ecologically, nesting sites predict brood size in frogs, but not in salamanders. Finally, we show that extinction risk increases consistently with decreasing fecundity across amphibians, whereas body size is a “by‐product” correlate of extinction, given its relationship with fecundity. Main conclusions Climatic seasonality and microhabitats are primary drivers of fecundity evolution. Our finding that low fecundity increases extinction risk reinforces the need to refocus extinction hypotheses based on a suggested role for body size.
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On a recent expedition to eastern Madagascar, we discovered a distinct new species of the genus Calumma that we describe here using an integrative approach combining morphology, coloration, osteology and molecular genetics. Calummaroalokosp. n. has a dermal rostral appendage and occipital lobes, and belongs to the C.boettgeri complex, within the Madagascar-endemic phenetic C.nasutum species group. It is readily distinguished from other species of the C.boettgeri complex by a characteristic two-toned body coloration and small body size with a snout-vent length of 45.6 mm in an adult male. The osteology of the skull, with a prominent maxilla and broad parietal, is similar to the closest related species, C.uetzi . Analysis of uncorrected genetic distances within the C.nasutum group using the mitochondrial gene ND2 shows a minimum pairwise distance of 11.98% to C.uetzi from the Sorata massif and Marojejy National Park >500 km north of the type locality of C.roalokosp. n. . Given an apparently small range (potentially <300 km ² ), located entirely outside of any nationally-protected areas, we recommend this new species be classified as Endangered under criterion B1ab(iii) of the IUCN Red List. The discovery of clearly distinct species like C.roalokosp. n. in an area of Madagascar that is comparatively thoroughly surveyed highlights the critical role of continued field surveys for understanding the true extent of Madagascar’s spectacular biodiversity.
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Background: The Portable Document Format (PDF) is the standard file format for the communication of biomedical information via the internet and for electronic scholarly publishing. Although PDF allows for the embedding of three-dimensional (3D) objects and although this technology has great potential for the communication of such data, it is not broadly used by the scientific community or by clinicians. Objective: The objective of this review was to provide an overview of existing publications that apply 3D PDF technology and the protocols and tools for the creation of model files and 3D PDFs for scholarly purposes to demonstrate the possibilities and the ways to use this technology. Methods: A systematic literature review was performed using PubMed and Google Scholar. Articles searched for were in English, peer-reviewed with biomedical reference, published since 2005 in a journal or presented at a conference or scientific meeting. Ineligible articles were removed after screening. The found literature was categorized into articles that (1) applied 3D PDF for visualization, (2) showed ways to use 3D PDF, and (3) provided tools or protocols for the creation of 3D PDFs or necessary models. Finally, the latter category was analyzed in detail to provide an overview of the state of the art. Results: The search retrieved a total of 902 items. Screening identified 200 in-scope publications, 13 covering the use of 3D PDF for medical purposes. Only one article described a clinical routine use case; all others were pure research articles. The disciplines that were covered beside medicine were many. In most cases, either animal or human anatomies were visualized. A method, protocol, software, library, or other tool for the creation of 3D PDFs or model files was described in 19 articles. Most of these tools required advanced programming skills and/or the installation of further software packages. Only one software application presented an all-in-one solution with a graphical user interface. Conclusions: The use of 3D PDF for visualization purposes in clinical communication and in biomedical publications is still not in common use, although both the necessary technique and suitable tools are available, and there are many arguments in favor of this technique. The potential of 3D PDF usage should be disseminated in the clinical and biomedical community. Furthermore, easy-to-use, standalone, and free-of-charge software tools for the creation of 3D PDFs should be developed.
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Over the last three years, three new species of saw-browed diamond frogs (Rhombophryne serratopalpebrosa species group)—a clade of cophyline microhylid frogs native to northern and eastern Madagascar—have been described. We here review the taxonomy of these frogs based on a new multi-gene phylogeny of the group, which confirms its monophyly but is insufficiently resolved to clarify most intra-group relationships. We confirm Rhombophryne guentherpetersi (Guibé, 1974) to be a member of this group, and we re-describe it based on its type series and newly collected material; the species is characterised by small superciliary spines (overlooked in its original description), as well as large tibial glands and an unusually laterally compressed pectoral girdle. We go on to describe two new species of this group from northern Madagascar: both R. diadema sp. nov. from the Sorata Massif and R. regalis sp. nov. from several sites in the northeast of the island possess three superciliary spines, but they are characterised by several subtle morphological and osteological differences. The new species are separated from all known congeners by an uncorrected pairwise distance of at least 5.1% in a ca. 550 bp fragment of the 16S rRNA gene. In order to highlight the significance of the skeleton in the taxonomy of this group, we provide a detailed description of its generalized osteology based on volume-rendered micro-CT scans of all described members, revisiting al- ready-described skeletons of some species, and describing the skeletons of R. guentherpetersi, R. coronata, and the new taxa for the first time. Use of volume rendering, instead of surface rendering of micro-CT data, resulted in some discrepancies due to the properties of each method. We discuss these inconsistencies and their bearing on the relative value of surface and volume rendering in the taxonomist’s toolkit. We argue that, while surface models are more practical for the reader, volumes are generally a more objective representation of the data. Thus, taxonomic description work should be based on volume rendering when possible, with surface models presented as an aid to the reader.
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We describe two new microhylid frog species of the genus Rhombophryne from the humid forests of northeastern Madagascar: Rhombophryne botabota sp. n. and R. savaka sp. n. The former is a medium-sized species, characterised by darkened lateral sides of the head (present in only one other congener, R. laevipes) and a unique combination of morphological , osteological, and molecular characters. The latter is a rather small species, characterised by medially undivided vomerine teeth with two large lateral diastemata, and presence of inguinal spots. Rhombophryne savaka sp. n. is the first species of the genus known from Makira Natural Park, and is reported also from Marojejy National Park and Amboloko-patrika (Betaolana Forest). Although its distribution range is relatively large compared to those of congeners, its known extent of occurrence is less than 2,000 km². As deforestation and habitat degradation persist as threats despite formal legal protection, we suggest an IUCN Red List status of Vulnerable for this species. Rhombophryne savaka sp. n. is possibly en-demic to the Marojejy National Park, like several other Rhombophryne species, but is unusual in being found at a relatively low altitude. As such, it is likely to be at high risk of habitat loss and decreasing range, and we propose a status of Endangered for it. We discuss the affinities of these new species and the variability of calls in this genus.
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The Rhombophryne serratopalpebrosa (Guibe ́ 1975) species complex consists of numerous cryptic narrow-mouthed frogs (Microhylidae) mostly restricted to northern Madagascar. We here provide an updated phylogeny of the genus Rhombophryne, and formally define the R. serratopalpebrosa group, characterized by the possession of small, fleshy superciliary spines. We describe two new species belonging to this group from the Tsaratanana Massif in northern Madagascar: Rhombophryne ornata sp. nov. and R. tany sp. nov. Both of the new species have fewer superciliary spines above each eye than any other member of the group (2 vs. 3–4). Rhombophryne ornata sp. nov. is unusual in having fairly elaborate dorsal markings, and is unique in having reddish thighs. In contrast, R. tany sp. nov. is a truly cryptic species characterized by a combination of characters unique within the R. serratopalpebrosa group. Micro-CT scans revealed differences between the skeletons of these species and other members of the group: the pubis is unossified in both species, and R. ornata sp. nov. lacks ossification in the epiphyses of its limb bones and in many of its small bones. We give a full osteological description of these frog species to facilitate ongoing research concerning this group. Genetic data suggest that these two syntopic species might be sister to each other, but they show a substantial genetic divergence of 3.9% and 8.4% uncorrected pairwise distance in the mitochondrial 16S rRNA and cytochrome oxidase subunit I genes, respectively. As these species have a limited extent of occurrence, and are known only from a single location in a forest that is declining in quality, we propose they be listed as Vulnerable B1ab(iii) on the International Union for Conservation of Nature Red List.
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Two new species of frogs previously confused with Pristimantis calcarulatus are identified using molecular phylogenet-ics and described using an integrative taxonomic approach. The species are distributed in montane cloud forests of the northwestern Ecuadorian Andes. Pristimantis pahuma n. sp. and Pristimantis cedros n. sp. differ from their closest relatives by strongly supported reciprocal monophyly in mitochondrial genetic data (16S). Additionally, the genetic distance among P. cedros n. sp., P. pahuma n. sp. and P. calcarulatus (sensu stricto) is 7.1–9.5%. The advertisement call of P. pahuma n. sp. also differs from that of P. calcarulatus; the former emits single-note calls irregularly, whereas P. calcaru-latus always calls in a series of 8–24 notes (calls for P. cedros n. sp. not recorded). Morphologically, the three species are almost undistinguishable; however, P. cedros n. sp. differs in life by having an iris with more numerous and smaller black reticulations, whereas the other two species have larger and fewer reticulations. Biogeographically, P. cedros n. sp. is separated from P. pahuma n. sp. and P. calcarulatus by the climatically dry and low elevation Río Guayllabamba Valley, which acts as a strong barrier to dispersal in these frogs. The results of this and other studies suggest that the true species richness of Pristimantis in the Andes is vastly underestimated. This underestimation may lead to declining protection for such cryptic species, many of which have smaller ranges than previously assumed. Species distributed across potential geographic barriers should be studied to detect the existence of cryptic species.
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A new species of bright-eyed treefrog (Mantellidae) from Madagascar, with comments on call evolution and patterns of syntopy in the Boophis ankaratra complex Abstract We describe a new species of Boophis treefrog from Ranomafana National Park in the southern central east of Madagascar. This region has remarkably high anuran diversity, and along with neighbouring sites, hosts more than 35 Boophis species. Boophis boppa sp. nov. is part of the B. ankaratra sub-clade (herein named the B. ankaratra complex), previously identified within the monophyletic B. albipunctatus species group. It occurs sympatrically with two other species of the complex (B. ankaratra and B. schuboeae). Morphological differentiation of species within the B. ankaratra clade remains elusive, but species are well characterized by distinct advertisement calls, with B. boppa having the longest note duration and inter-note intervals when compared to closely related species. Furthermore, it has moderate differentiation in mitochondrial DNA, with pairwise distances of 1.9–3.7% to all other species in sequences of the mitochondrial 16S rRNA marker. Additional evidence is given by the lack of haplotype sharing with related species for the nuclear exon DNAH-3. All examples of syntopic occurrence in this complex involve species with strongly different advertisement calls, while allopatric species have more similar calls. Such a pattern might result from adaptive call co-evolution but could also be the result of non-adaptive processes. Thorough clarification of the systematics of the B. ankaratra sub-clade is required, and we outline future directions for both bioacoustic and genetic research.
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The Madagascar-endemic microhylid genus Rhombophryne consists of a range of partly or completely fossorial frog species. They lead a poorly known, secretive lifestyle, and may be more diverse than previously thought. We describe a new species from the high altitude forests of the Sorata massif in north Madagascar with unusual characteristics for this genus; R. longicrus sp. n. has long, slender legs, unlike most of its fossorial or semi-fossorial congeners. The new species is closely related to R. minuta, a much smaller frog from the Marojejy massif to the southeast of Sorata with similarly long legs. We discuss the morphology of these species relative to the rest of the genus, and argue that it suggests adaptation away from burrowing and toward a more saltatorial locomotion and an accordingly more terrestrial lifestyle. If this is the case, then these frogs represent yet more ecological diversity within the already diverse Cophylinae. We recommend an IUCN Red List status of Endangered B1ab(iii) for R. longicrus sp. n., because it is known only from a single site in a forested area of roughly 250 km 2 , which is not yet incorporated into any protected area.
A recent study clarified several aspects of microhylid phylogeny by combining DNA sequences from Sanger sequencing and anchored phylogenomics, although numerous aspects of tree topology proved highly susceptible to data partition and chosen model. Although the phylogenetic results of the study were in conflict with previous studies, the authors made several changes to the taxonomy of Madagascar's cophyline microhylids. We re-analysed part of their data together with our own molecular and morphological data. Based on a supermatrix of 11 loci, we propose a new phylogeny of the Cophylinae, and discuss it in the context of a newly generated osteological dataset. We found several sample misidentifications, partially explaining their deviant results, and propose to resurrect the genera Platypelis and Stumpffia from the synonymy of Cophyla and Rhombophryne, respectively. We provide support for the previous genus-level taxonomy of this subfamily, and erect a new genus, Anilany gen. nov., in order to eliminate paraphyly of Stumpffia and to account for the osteological differences observed among these groups. Deep nodes in our phylogeny remain poorly supported, and future works will certainly refine our classification, but we are confident that these will not produce large-scale rearrangements.