Research Article |
Corresponding author: Mark D. Scherz ( mark.scherz@gmail.com ) Academic editor: Peter Bartsch
© 2015 Mark D. Scherz, Andolalao Rakotoarison, Oliver Hawlitschek, Miguel Vences, Frank Glaw.
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:
Scherz MD, Rakotoarison A, Hawlitschek O, Vences M, Glaw F (2015) Leaping towards a saltatorial lifestyle? An unusually long-legged new species of Rhombophryne (Anura, Microhylidae) from the Sorata massif in northern Madagascar. Zoosystematics and Evolution 91(2): 105-114. https://doi.org/10.3897/zse.91.4979
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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 km2, which is not yet incorporated into any protected area.
Rhombophryne longicrus sp. n., Integrative taxonomy, Rhombophryne minuta , Osteology, Cophylinae , X-ray micro-computed tomography
The microhylid frog genus Rhombophryne (Anura, Microhylidae, Cophylinae) is receiving renewed taxonomic attention in the wake of recent genetic barcoding efforts on the frogs of Madagascar. These DNA barcode studies revealed that less than 60% of the island’s amphibian diversity might so far have been formally described (
Repeated swapping of ecological niches (i.e. transitions between gross ways of life, such as between terrestrial and arboreal lifestyles) has led to high ecological diversity in the Cophylinae (
Here, we describe a new Rhombophryne clearly distinct from all other known species, although phylogenetically nested. We describe its skeleton by means of micro-computed tomography (micro-CT), revealing additional differences to the other Rhombophryne species for which skeletal data are available (
Specimens were collected, euthanized, fixed in 90% ethanol and transferred to 70% ethanol for long-term storage. One specimen was deposited in the Université d’Antananarivo Département de Biologie Animale (UADBA), and the other in the Zoologische Staatssammlung München (ZSM).
In this work we refer to the species R. ornata and R. tany. Their descriptions are in press (
Morphological measurements were taken with a digital calliper to 0.01 mm and rounded to the nearest 0.1 mm for presentation here. Fig.
Micro-CT scanning was carried out on a nanotom m (GE Measurement & Control, Wunstorf, Germany). The holotype of the new species, ZSM 1630/2012, was mounted on a polystyrene board inside a sealed polyethylene vessel, and secured in place using small wooden struts and additional polystyrene. A small volume of 80% ethanol was added to achieve air saturation, preventing desiccation of the specimen. The vessel was mounted on a polyvinylchloride tube, and placed inside the micro-CT scanner. Scanning was conducted at a voltage of 140 kV and a current of 80 mA, with a timing of 500 ms for 20 minutes (2440 projections). Scan data were assembled in phoenix DATOS|X 2 RECONSTRUCTION CT software (GE Measurement & Control, Wunstorf, Germany) and visualised in VG STUDIO MAX 2.2 (Volume Graphics GmbH, Heidelberg, Germany) and subsequently processed into a 3D surface render in AMIRA 5.4.5 (FEI Visualization Sciences Group, Burlington MA, USA). Skeletal description is based on both surface and volume renderings of micro-CT data, due to artefacts produced in surface rendering. Readers are advised that micro-CT scanning does not render poorly calcified structures, especially cartilage. Cartilages are therefore omitted from the osteological description below and the respective figures; additional specimens will need to be cleared and stained in order to assess cartilaginous characters of these frogs. We provide a PDF-embedded interactive 3D model of the skeleton of the holotype as Suppl. material
We extracted total genomic DNA from ethanol-preserved tissue samples using proteinase K digestion (final concentration 1 mg/mL) and a standard salt extraction protocol (
We calculated a phylogenetic tree from the 16S sequences by Bayesian inference (BI) with MRBAYES 3.2 (
The new species described below is phylogenetically nested in the genus Rhombophryne (Fig.
ZSM 1630/2012 (FGZC 3653), an adult female with immature oocytes, collected in the montane forest of the Sorata Massif, north Madagascar (ca. 13.675°S, ca. 49.4392°E, ca. 1580 m; datum = WGS84) on 28 November 2012 by A. Rakotoarison, A. Razafimanantsoa, T. Rajoafiarison, F. M. Ratsoavina, O. Hawlitschek and F. Glaw.
UADBA-A 60271 (FGZC 3651), an adult male with the same collection data as the holotype.
A microhylid assigned to the genus Rhombophryne on the basis of overall morphology, including the possession of maxillary and vomerine teeth, absence of expanded toe pads, and absence of an enlarged prepollex. Confirmed as a member of the genus Rhombophryne on the basis of its phylogenetic relationships as assessed by mitochondrial DNA, as there are no known morphological characters by which Rhombophryne may be distinguished from Plethodontohyla.
Rhombophryne longicrus sp. n. is distinguished from all other Madagascan frog species by the following set of characters: SVL 23.8–27.9 mm, head wider than long, horizontal tympanum diameter 47% of eye diameter, absence of superciliary spines, weak supratympanic fold, dark supratympanic region and nostril, tibiotarsal articulation reaching the nostril, total hindlimb length 183–185% of SVL, second finger shorter than fourth, and fifth toe shorter than third. It is also separated by a pairwise genetic distance of at least 6.8% in the 16S mitochondrial gene from all other known species of the genus Rhombophryne.
Within the genus, R. longicrus sp. n. may be distinguished from all Rhombophryne species, except R. laevipes, R. minuta, and R. vaventy, by tibiotarsal articulation reaching the nostril (versus not exceeding the eye); from R. coronata, R. ornata, R. serratopalpebrosa, R. tany, and R. vaventy by the absence of superciliary spines (versus presence); from R. alluaudi, R. laevipes, R. matavy, R. testudo, and R. vaventy by its smaller size (SVL 23.8–27.9 mm versus 32–53 mm); from R. minuta by its larger size (SVL 23.8–27.9 mm vs. up to 17.1 mm); from R. testudo by the absence of barbels on the throat and tympanum smaller than eye; from R. alluaudi, R. coronata, R. serratopalpebrosa, R. tany, and R. vaventy by its weak, almost absent supratympanic fold; from R. coudreaui and R. vaventy by smooth dorsal skin (versus granular/tubercular); from R. mangabensis by lack of paired dark dorsal tubercles; from R. laevipes, R. mangabensis, R. ornata, R. testudo, and R. vaventy by absence of dark crossbands on hindlimbs; and from R. coronata, R. minuta, R. testudo, and R. vaventy by dark supratympanic region.
Osteologically, a micro-CT scanned specimen of R. longicrus sp. n. tentatively differs from R. ornata (3 specimens: ZSM 1815/2010, 1816/2010, and 2859/2010), R. serratopalpebrosa (1 specimen: MNHN 1975.24), R. tany (1 specimen: ZSM 1814/2010), and R. vaventy (1 specimen: ZSM 375/2005) (as described in
Rhombophryne species can be confused with Plethodontohyla species. Rhombophryne longicrus sp. n. differs from them in the following ways: absence of a sharp dorsolateral colour border and expanded finger and toe pads (versus presence in P. notosticta, P. guentheri, P. mihanika, and P. inguinalis), absence of inguinal spots (versus presence in P. mihanika, P. inguinalis, P. ocellata, and P. bipunctata), tibiotarsal articulation reaching the nostril (maximally reaching to the mid-eye in all Plethodontohyla except P. mihanika), absence of crossbands on legs (versus presence in P. fonetana, P. inguinalis, P. notosticta, P. guentheri, and P. mihanika), and smooth skin (versus granular to rough in P. tuberata).
Adult female in an excellent state of preservation. A ventral incision was made in order to check the sex and access the stomach contents. The incision runs laterally and posteroventrally anterior to the pubis and up the middle of the venter.
Body gracile; dorsal and ventral skin smooth. Head wider than long (HW 122.5% of HL), snout rounded in dorsal view, squarish in lateral view; nostrils weakly protuberant, directed laterally, equidistant between eye and snout; canthus rostralis concave; loreal region concave; tympanum indistinct, oval, horizontally 47% of eye diameter; pupil round; supratympanic fold weak, almost absent; tongue unlobed, posteriorly free; vomerine teeth present in a straight row with a small medial gap (<1 mm; see Osteology below); choanae small, oval.
Arms slender and long; fingers without webbing, long, without distinct subarticular tubercles, relative lengths 1<2<4<3, second finger much shorter than fourth, without enlarged terminal discs; inner metacarpal tubercle present; nuptial pads absent. Legs exceptionally long and slender (HIL 185% of SVL), tibiotarsal articulation reaching the nostril when hindlimb is adpressed along body; toes long, unwebbed, with indistinct subarticular tubercles, relative toe lengths 1<2<5<3<4, third toe much longer than fifth; inner metatarsal tubercle present and indistinct.
Colouration of the holotype: (Fig.
After three years in 70% ethanol, all browns have faded to shades of grey. Dorsal areas that were lightest in shade are whitish, particularly between the eyes anterior to the dark inter-ocular bar. Ventrally, all areas that lack pigmentation and were pink in life are cream in preservation. Chin the same colour as the snout.
Osteology of the holotype: (Fig.
Osteology of the holotype of Rhombophryne longicrus, ZSM 1630/2012. Skull in (a) lateral, (b) dorsal, and (c) ventral view. Skeleton in (d) dorsal and (e) ventral view. Note: figures display only calcified structures; cartilages are omitted due to limitations of micro-CT scanning. Abbreviations: angspl = angulosplenial, col = columella, exoc = exoccipital, fpar = frontoparietal, max = maxillary, mmk = mentomeckelian, pmax = premaxilla, povom = postchoanal vomer+neopalatine, proot = prootic, prsph = parasphenoid, prvom = prechoanal vomer, pter = pterygoid, qj = quadratojugal, smax = septomaxilla, spheth = sphenethmoid, sq = squamosal.
Mandible slim, edentate. Mentomeckelian small, in narrow medial contact with counterpart (possibly artefactual), and in dorsolateral contact with dentary. Dentary long and thin with a sculpted outer face and smooth inner face, overlapping angulosplenial for much of its length. Angulosplenial broadening posteriorly, with a posterior dorsomedial crista; possessing a lateral channel running from the posterior into the sculpted outer edge of the dentary.
Posterolateral processes of hyoid shovel-like, a medial crista running along posteromedial process, the base of which is broad and flat with a rounded anteromedial edge and sharp anterolateral and posteromedial corners; parahyoid absent.
Humerus long, slim and straight; crista lateralis weak, crista ventralis short (~30% of humerus length), crista medialis absent. Radioulna broadening distally. Finger phalangeal formula 2-2-3-3. Terminal phalanges of fingers 2, 3, and 4 with distal knobs. Prepollex 31% of first finger.
Pectoral girdle composed of paired coracoids, clavicles, scapulae, cleithra and suprascapulae. Sternal characters not visible in CT render. Coracoids in medial contact; medially dorsoventrally flattened, laterally rounded, posterior surface straight, anterior surface strongly concave. Clavicle thin and curved approximately parallel to the anterior edge of the coracoid, its lateral end broadened, posteriorly in contact with ventral edge of scapular pars acromialis. Scapula thick, hourglass shaped, its posterior edge less strongly curved than its anterior edge, medioventrally bifurcated; pars acromialis distally rounded, in contact with the lateral end of the clavicle, its anterior surface concave; pars glenoidalis curved ventrally, in contact with lateral face of coracoid, posterior face concave; dorsal edge of scapula approaching cleithrum. Cleithrum thin and long, not possessing any cristae, anteriorly thicker than posteriorly. Suprascapula with highest X-ray absorption ventrally and posteriorly suggesting possible ossification in these areas.
Toe phalangeal formula 2-2-3-4-3; terminal phalanges without distinct distal knobs. Leg bones long and thin. Femur without any crests. Tibiofibula slightly longer than femur. Tibiale and fibulare proximally and distally fused, articulating distally with metatarsals V and IV, tarsals 1–3, and the centrale. Prehallux present, short.
Ilium, ischium, and pubis forming ossified acetabulum, each composed of paired, medially fused elements. Iliac shafts oval in cross-section, dorsal-ventral diameter larger, possessing a weak dorsal tubercle posterior to shallow oblique groove. Iliosacral articulation type IIA sensu
Eight presacrals present; no vertebrae fused. Posterior articular processes round. Transverse processes of presacrals II–IV broader than those of V–VIII. Neural spines decreasing in size from presacral II to absent by V. Sacrum wide, with broad diapophyses articulating with the ilia; anterior edge of each diapophysis roughly perpendicular to body axis, posterior edge oblique. Urostyle long and thin, with a dorsal ridge along a third of its length, beginning at its anterior end; articulation with sacrum bicondylar.
Holotype (paratype in brackets), measurements in mm: SVL 28.0 (23.8), HW 9.9 (9.7), HL 8.0 (7.0), ED 3.3 (2.8), END 2.0 (2.0), NSD 1.9 (1.7), NND 3.0 (2.2), TDH 1.5 (1.3), TDV 1.8 (1.4), HAL 8.4 (7.0), UAL 5.7 (4.7), LAL 6.8 (5.7), FORL 20.9 (17.4), THIL 13.2 (11.7), THIW 3.9 (3.4), TIBL 14.6 (11.7), TIBW 2.97 (2.64) TARL 8.6 (7.3), FOL 14.8 (12.5), FOTL 23.4 (19.8), HIL 51.2 (43.2), IMCL 1.0 (0.9), IMTL 1.3 (1.0).
Only two specimens are known. The paratype is male, and smaller than the holotype (SVL 23.8 mm). It agrees in all aspects of its morphology with the holotype, but differs strongly in colouration (see Fig.
The species epithet is an invariable noun in apposition to the genus name, derived from the Latin words longus (meaning long), and crus (meaning leg), and refers to the unusually long legs of this species.
This species has only been found at high altitude in the montane forests of the Sorata massif in north Madagascar. Its distinctiveness leads us to hypothesize that it has never been found elsewhere and misidentified, so it may be microendemic to this small area. Additional surveys are required in areas in and around Sorata to identify its full distribution.
Both specimens described here were captured in the early evening on the ground along a path through primary montane forest. The stomachs of both specimens contained remains of several small insects (mostly Coleoptera) and a spider (possibly belonging to the family Salticidae), mixed with moss, suggesting an opportunistic diet of arthropods. Calls of this species are unknown. The female holotype had more than twenty immature oocytes with the largest having diameters ranging from 1.3 to 1.6 mm. As a member of the Cophylinae, it is likely that R. longicrus lays its eggs away from running water or large water bodies, and has endotrophic tadpoles.
The forests of Sorata are currently unprotected. All locally endemic species are threatened by uninhibited deforestation and forest degradation. The greatest pressure on forests is at their edges. High altitude species like R. longicrus may therefore be the least threatened by this. However, a sustained rate of deforestation will increase the threat level to species at ever-higher altitudes. It is conceivable that a restriction of this species to high altitudes may mean that it is susceptible to climate change (
While this species is, at present, known from just two specimens collected on one expedition, the fact that it has not been collected by previous expeditions suggests it may be scarce, seasonal, or have a scattered distribution. Even if it were distributed throughout the forests of the Sorata massif, its distribution would still only constitute an area of ~250 km2 (as calculated in Google Earth® Pro 6.1.0.500, Google Inc., Mountain View, CA). Thus, because of its potentially limited range inside an unprotected forest, the on-going and intensifying threat of deforestation, potential threat by climate change, and potential scarcity or seasonality, it qualifies as Endangered B1ab(iii) under the IUCN Red List Criteria (2012).
Hindlimb length is significantly associated with habitat and mode of life in frogs (
Robust and at least partly burrowing frogs typify the genus Rhombophryne. Some species are specialised burrowers (R. matavy and R. testudo), while others are probably facultative burrowers (R. serratopalpebrosa group, and probably R. alluaudi, R. laevipes, and R. mangabensis). Two species however seem to have at least partly abandoned burrowing: R. minuta and R. longicrus. Rhombophryne minuta lives at high altitude (close to and above the tree line) on the Marojejy massif and calls from low bushes that make up the complex matrix of ground in its unusual habitat (
In addition to leg length, several other characters are also associated with more saltatorial locomotion.
The iliosacral articulation of Rhombophryne longicrus is almost the same as members of the R. serratopalpebrosa group (
The discovery of such a distinctive new species highlights the incompleteness and patchiness of herpetological survey work in Madagascar. Whilst some forests, particularly accessible, protected ones, are receiving a lot of research attention (e.g. Betampona:
We would like to thank Theo Rajoafiarison, Fanomezana M. Ratsoavina and Angeluc Razafimanantsoa for their enormous help during the fieldwork, Hanta Razafindraibe for the loan of the paratype from the UADBA collection, and Jennifer Luedtke for her assistance with IUCN Red List status assessment. Field research was conducted under permit No. 265/12/MEF/SG/DGF/DCB.SAP/SCB (dated 18 Oct. 2012), exportation of specimens under permit No. 163N-EA12/MG12 (dated 17 Dec. 2012), (both issued by the Direction Générale des Forêts de Madagascar), and funded by the Mohamed bin Zayed Species Conservation Fund (project 11253064) and BIOPAT. We are grateful to the three anonymous reviewers who helped us to improve this manuscript.
PDF-embedded 3D Skeletal Model
Data type: Adobe PDF file
Explanation note: This file contains a PDF-embedded interactive 3D model of the skeleton of the holotype of Rhombophryne longicrus sp. n., ZSM 1630/2012, generated via X-ray micro-Computed Tomography. The model can be opened in Adobe® Acrobat Pro or Reader, versions IX and above