Corresponding author: Shea M. Lambert ( firstname.lastname@example.org )
Academic editor: Johannes Penner
© 2017 Shea M. Lambert, Carl R. Hutter, Mark D. Scherz.
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.
Citation: Lambert SM, Hutter CR, Scherz MD (2017) Diamond in the rough: a new species of fossorial diamond frog (Rhombophryne) from Ranomafana National Park, southeastern Madagascar. Zoosystematics and Evolution 93(1): 143-155. https://doi.org/10.3897/zse.93.10188
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.
Amphibia, Anura, Microhylidae, Rhombophryne nilevina, taxonomy, osteology, micro-CT, endemicity, herpetology
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 estimates approaching 600 species; 99.9% of which are endemic to the island (reviewed in
Rhombophryne Boettger, 1880 is a particularly enigmatic cophyline genus consisting of 16 valid nominal species (
Phylogenetic relationships between Rhombophryne species estimated using maximum likelhood in RaxML using the mitochondrial 16S rRNA barcode fragment. Rhombophryne nilevina sp. n. is highlighted with blue bold text. Outgroups are removed from the tree figure for aesthetic purposes.
We herein describe Rhombophryne nilevina sp. n., discovered during herpetofaunal surveys of moist montane forest in the remote north-western corner of Ranomafana National Park, southeastern Madagascar, in January of 2015. We diagnose R. nilevina sp. n. from its congeners using morphological and osteological characters collected 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.
We collected specimens during the day through targeted searching, using the advertisement call to locate males. We euthanized specimens using 20% benzocaine, fixed them in ~10% formalin solution buffered 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 (
Immediately following euthanasia, we removed the tongue and placed it in 95% EtOH. We extracted genomic DNA using standard phenol-chloroform extraction protocol and amplified a fragment of the mitochondrial rRNA marker 16S using a previously published protocol (
GenBank Accession numbers for all sequences used in phylogenetic analysis. Asterisks indicate newly generated sequences.
|Platypelis tuberifera*||Vohidrazana||CRH 286||KY288470|
|Rhombophryne alluaudi||Andasibe||ZSM 3/2002||DQ019606|
|Rhombophryne alluaudi||Torotorofotsy||ZCMV 968||EU341105|
|Rhombophryne alluaudi||Tsararano||MRSN A 2620||AY594105|
|Rhombophryne cf. coronata*||Vohidrazana||
|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||
|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|
Raw genetic distances at the 16s rRNA gene fragment between analysed taxa and Rhombophryne nilevina sp. n. (KU 340893).
|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%|
We took morphological measurements using a digital caliper 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
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 filter. 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 files 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 production of figures. The osteological information presented is based on volume rendering. Only slightly calcified 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 Communications in Medicine (DICOM) stack of the scan files and rotational video produced in VG Studio Max 2.2 are available at the following MorphoSource http://morphosource.org/Detail/ProjectDetail/Show/project_id/263
We exported the volume as an “Analyze Volume” under standard settings in VG Studio Max 2.2, and imported the resulting .hdr file into Amira 6.1 (FEI Visualization Sciences Group, Burlington MA, USA), where a surface model was produced essentially following
We note that skeletal comparisons to other cophylines are based on largely unpublished micro-CT data produced 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, Stumpffia, Anilany, and Plethodontohyla are found in
We recorded calls attributed to the holotype on two occasions using an Olympus LS-10 Linear PCM Field Recorder and a Sennheiser K6-ME66 super-cardioid shotgun 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 available, 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 individual, however, only a single individual at a time was heard calling from this location, and the collected individual was found with distended vocal sac shortly after the second recordings. Additionally, the measured call parameters from the two occasions are nearly completely overlapping (Fig.
The advertisement call recorded for Rhombophryne nilevina in comparison with that of R. testudo. Calls were recorded 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 appropriate. The call recording of R. testudo is from
|Species||R. nilevina||R. testudo|
|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)
(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)|
The electronic version of this article in Portable Document Format (PDF) will represent a published work according to the International Commission on Zoological Nomenclature (ICZN), and hence the new names contained in the electronic version are effectively 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 Identifiers) can be resolved and the associated information viewed through any standard web browser by appending the LSID to the prefix 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.
We discovered a large-bodied cophyline microhylid frog near Andemaka within Ranomafana National Park in eastern Madagascar. Several obvious differences in morphology exist between the collected specimens and all known described and undescribed cophyline microhylids. 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
A frog assigned to the cophyline genus Rhombophryne on the basis of its divided vomer, the possession of clavicles and knob-shaped terminal phalanges (see
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, respectively). This species differs 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
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 distinguished from P. inguinalis by its smaller size (SVL 57.2 vs. 62.2–99.1 mm), the absence of enlarged fingertips, absence of dark inguinal spots (vs. occasional presence), and absence of a strong dorsolateral color border (vs. occasional presence); from P. notosticta, P. guentheri, P. fonetana, and P. mihanika by the absence of enlarged fingertips, 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 presence 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 being strongly amplitude modulated (Fig.
Morphology of the holotype. 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 subtle 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, directed 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.
Photos in life of Rhombophryne nilevina sp. n. (a) Dorsolateralview of the holotype (
Arms strongly built, relatively short; fingers without webbing, short, with distinct, rounded subarticular tubercles, relative lengths 1<2<4<3, the second finger marginally shorter than the fourth (and marginally longer than the first), without enlarged terminal discs; inner metacarpal tubercle strong, oblong, 28.1% of hand length; outer metacarpal tubercle indistinct, round. Legs relatively long and thick (HIL 152.5% of SVL; TIBL 42.2% of SVL), position of the tibiotarsal articulation when adpressed along the body not possible to assess without breaking the hindlimbs; toes long, unwebbed, with indistinct round subarticular tubercles, relative toe lengths 1<2<5<3<4, third toe distinctly longer than fifth; inner metatarsal tubercle present and distinct, 12.7% of foot length; outer metatarsal tubercle absent.
Coloration of the holotype. In preservative, the holotype is chocolate brown dorsally with a loosely reticulated 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 present before the terminus of each finger. 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 flecked with cream, and also possess a light annulus before the terminal phalanges. The ventral abdomen is brown with numerous small cream flecks. 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.
Osteology of the holotype (Fig.
Anterior braincase laterally closed by the sphenethmoid. Interior braincase containing calcified material. Nasal in medial contact with contralalteral and posterior contact with frontoparietal. Frontoparietal broadening anteriorly from narrow waist anterior to lateral flanges, 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 minute. Otic capsule dorsally poorly ossified.
Sternum not ossified. Clavicle robust, curved. Humerus proximally broad, distally rather narrow; possessing a well-developed crista ventralis along roughly 50% of its length; crista lateralis weak. Terminal phalanges of fingers and toes with small distal knobs. Phalangeal formula of fingers 2-2-3-3; of toes 2-2-3-4-3. Femur without cristae. Prepollex strong, blade-like, half length of first metacarpal. Prehallux strong, approximately half length of first 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
We analysed a total of seven calls from R. nilevina, and compared these to the call of R. testudo (Fig.
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 first harmonic frequency is 775.2–818.3 (796.8 ± 17.6) Hz (Fig.
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.
The specific epithet “nilevina” is a Malagasy word meaning “buried.” This name was chosen to recognize the fossorial habits of this species. It is to be treated as an invariable noun in apposition.
Due to morphological and size similarities, as well as geographic distribution, two existing names must be considered for this species: Phrynocara laeve Boettger, 1883, and Plethodontohyla laevis tsianovohensis Angel, 1936. Both of these names are currently considered to be junior synonyms of Rhombophryne alluaudi. We examined the morphology and osteology of the holotypes of both of these taxa (P. laeve: SMF 4286; P. laevis tsianovohensis: MNHN 1936.47), and our new species differs 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 discussed in a future article, and we here simply rule out the possibility that they are conspecific with R. nilevina sp. n. based on the presence vs. absence of a clavicle. The type specimen of P. laevis tsianovohensis was collected 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 flat, 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 flatter, 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 (
Rhombophryne nilevina has thus far been detected at a single site, near the former village of Andemaka, in the north-west of Ranomafana National Park (Fig.
Although the type locality of R. nilevina is within Ranomafana National Park, its occupancy within the park is potentially highly restricted, elevationally 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 overlooked. Given this large uncertainty in area of occupancy, we suggest an initial IUCN categorization of Data Deficient. If R. nilevina is for instance, restricted to the type locality, then habitat destruction, chytrid fungus (recently detected in Madagascar,
The discovery of Rhombophryne nilevina—never previously identified as a candidate species despite being found in one of the most well-surveyed National Parks of Madagascar—highlights the importance of continued field work for the advancement of systematics in Malagasy anurans. In particular, field surveys should help reveal diversity in clades containing species with small ranges and secretive life histories, including Rhombophryne and other cophyline frogs. Cophylines have already shown great promise as a model system for studying ecomorphological and reproductive mode evolution (e.g.
Rhombophryne nilevina is remarkable in several respects, including its morphology. Most obvious is its large size, the largest recorded for the genus, narrowly exceeding 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 sufficiently divergent from all other Rhombophryne species that it cannot be immediately assigned to a complex or species cluster.
In addition to morphological distinctiveness, Rhombophryne nilevina is currently the southernmost distributed species of Rhombophryne, excluding records of Rhombophryne alluaudi from the far south of Madagascar, which are due to confusion surrounding the identity of that species (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 affinities with Rhombophryne sp. Ca3 from Tsaratanana in northern Madagascar, but we consider this relationship tentative and ongoing multi-locus analyses suggest that R. nilevina represents a relatively early-diverging, phylogenetically distinct species of Rhombophryne (A. Crottini, pers. comm.). Given the limited information available at this time, the phylogenetic affinities of R. nilevina will need to be clarified in a future revision of the genus.
We thank the Malagasy authorities for issuing permits; field 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 fieldwork. Finally, SML would like to thank Ralaivao Jean Fulgence and Emile Rajeriarison for their exceptional work in the field during the Andemaka expedition. If not for their dedication and ability, R. nilevina would surely remain undiscovered.