Zoosyst. Evol. 96 (2) 2020, 313-323 | DOI 10.3897/zse.96.51372 eee BERLIN Diamond frogs forever: a new species of Rhombophryne Boettger, 1880 (Microhylidae, Cophylinae) from Montagne d’Ambre National Park, northern Madagascar Mark D. Scherz!:2 1 Zoologische Staatssammlung Miinchen (ZSM-SNSB), Miinchhausenstr. 21, 81247, Miinchen, Germany 2 Zoologisches Institut, Technische Universitat Braunschweig, Mendelssohnstr. 4, 38106, Braunschweig, Germany 3 Department of Biology, Universitat Konstanz, Universitdtstr. 10, 78464, Konstanz, Germany http://zoobank. org/5 EE0689E-27DD-41B1-B0AA-AF300COBS5BA4 Corresponding author: Mark D. Scherz (mark.scherz@gmail.com) Academic editor: Johannes Penner # Received 21 February 2020 # Accepted 4 May 2020 Published 15 June 2020 Abstract 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 ellae sp. 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 ellae sp. 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. Key Words Amphibia, Anura, micro-CT, molecular genetics, osteology Rhombophryne ellae sp. nov., systematics, taxonomy Introduction The diamond frogs, genus Rhombophryne Boettger, 1880, are a group of fossorial and terrestrial microhylid frogs in the subfamily Cophylinae. These frogs show sub- stantial ecological variation, with most species being ter- restrial or semi-fossorial, but several species being fully fossorial (R. testudo Boettger, 1880, R. matavy D’ Cruze, Kohler, Vences & Glaw, 2010, and R. coudreaui (Angel, 1938)), and one being highly miniaturised (RX. proportion- alis Scherz, Hutter, Rakotoarison, Riemann, Rodel, Ndri- antsoa, Glos, Roberts, Crottini, Vences & Glaw, 2019). In this respect they capture a significant portion of the variation within the Cophylinae (Andreone et al. 2005; Wollenberg et al. 2008). DNA barcoding studies revealed considerable undescribed diversity within Rhombophryne (Vieites et al. 2009; Perl et al. 2014), triggering increased taxonomic attention. The known species diversity of the genus has more than doubled in the last 10 years, as nu- merous new species have been described from the rain- forests of northern and northeastern Madagascar (Vences and Glaw 2003; D’Cruze et al. 2010; Scherz et al. 2014, 2015a, 2015b, 2016a, 2016b, 2017a, 2019). Despite the increased rate of taxonomic description of Rhombo- phryne species, several undescribed candidate species of Rhombophryne are still awaiting description (Vieites et Copyright 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. 314 al. 2009; Scherz et al. 2016b), and recent fieldwork efforts in well-studied and poorly studied areas (both in terms of herpetofaunal survey work and biological survey work in general) have continued to yield new discoveries. Montagne d’Ambre National Park in northern Mada- gascar 1s the island’s oldest protected area (Goodman et al. 2019). In general it is considered well studied, but only two published herpetofaunal studies have been conducted in the park, one focussing on the rainforest at elevations above 700 m a.s.l. (Raxworthy and Nussbaum 1994), and the other on drier, more deciduous forest at elevations be- low 700 maz.s.l. in the former Forét d’ Ambre Special Re- serve (now subsumed within the national park) (D’ Cruze et al. 2008). Until now, two Rhombophryne species, R. laevipes (Mocquard, 1895) and R. matavy, have been known from the park, although reference to a Plethodon- tohyla from the park in the past (Raxworthy and Nuss- baum 1994) may have referred to other Rhombophryne species, given that these two genera were taxonomically re-arranged only afterwards (Andreone et al. 2005; Wol- lenberg et al. 2008) and there are no known Plethodonto- hyla species present in the park (Glaw and Vences 2007; my own unpublished data). During fieldwork in 2017, a relatively small Rhombophryne was collected that was immediately recognisable as an undescribed species. Not only was it considerably smaller than an adult R. /aevipes, but it also differed considerably in colouration and mor- phology from that species. I show here that this animal is genetically highly distinct from all other Rhombophryne species and describe it as a new species. Methods The new specimen was collected during fieldwork in Mon- tagne d’Ambre in December 2017. It was photographed in life before being anaesthetised and subsequently eu- thanised with an aqueous solution of MS-222. A tissue sample was taken from the right thigh and deposited in 99% ethanol. The specimen was fixed with 90% ethanol and transferred to 70% ethanol for long-term storage. The specimen has been deposited in the Zoologische Staats- sammlung Miinchen (ZSM) in Munich, Germany (ZSM 76/2018). Other institutional and field numbers are used in Table 1, and these are: AMNH-A are the amphibian se- ries numbers of the American Museum of Natural Histo- ry, New York NY, USA; FAZC and FN are field numbers of Franco Andreone; FG/MV, FGZC, ZCMV, and MV are field numbers of Frank Glaw and Miguel Vences; KU is the zoological collection of Kansas University, Lawrence KS, USA; MSZC is my own field number series; and RAX are the field numbers of Christopher J. Raxworthy. Morphological examination of the specimen followed that used in my previous work on this genus; an illus- trated measurement scheme is presented in Scherz et al. (2015a). For practicality, I reiterate this measurement scheme here: SVL, snout-vent length; HW, head width at the widest point; HL, head length, measured diagonally from the jaw commissure to the anterior-most point of the zse.pensoft.net Mark D. Scherz: A new diamond frog from Montagne d’Ambre, Madagascar jaw; ED, eye diameter; END, eye-nostril distance; NSD, nostril-snout-tip distance; NND, nostril-nostril distance; TDH, horizontal tympanum diameter; TDV, vertical tym- panum diameter; HAL, hand length, from the base of the hand to the tip of the longest (third) finger; UAL, upper arm length, from the insertion of the arm to the elbow; LAL, lower arm length, from the elbow to the base of the hand; FORL, forelimb length, given by the sum of HAL, UAL, and LAL; FARL, forearm length, given by the sum of HAL and LAL; THIL, thigh length, from the cloaca to the knee; THIW, width of the thigh at its widest point; TIBL, tibia length, from the knee to the tibiotarsal articu- lation; TIBW, width of the tibia at its widest point; TARL, tarsus length, from the tibiotarsal articulation to the base of the foot (end of the inner metatarsal tubercle); FOL, foot length, from the base of the foot to the end of the longest (fourth) toe; FOTL, given by the sum of FOL and TARL; HIL, given by the sum of THIL, TIBL, TARL, and FOL; IMCL, inner metacarpal tubercle length; OMCL, outer metacarpal tubercle length; IMTL, inner metatarsal tubercle length; OMTL, outer metatarsal tubercle length. A micro-computed tomography (micro-CT) scan of the specimen was produced using a phoenix|x nanotom m cone beam scanner (GE Measurement & Control, Wun- storf, Germany). The scan was made at a voltage of 140 kV and current of 80 uA, using a tungsten target with a 0.1 mm copper filter. 2440 projections each were captured at 750 ms for a total scan duration of 30 minutes. The scan was reconstructed in datos|x reconstruct (GE Meas- urement & Control) and examined in VG Studio Max 2.2 (Volume Graphics GmbH, Heidelberg, Germany). Screenshots used here are from volumetric renderings of the skeleton, following recommendations laid out in Scherz et al. (2017a). A DICOM stack of the scan has been deposited on MorphoSource.org at http://www.morpho- source.org/Detail/ProjectDetail/Show/project_id/1005. Total genomic DNA was extracted from the tissue sam- ple using a standard salt extraction (Bruford et al. 1992). A segment of the 3' end of the 16S rRNA mitochondrial bar- coding marker was amplified using the primers 16Sar-L and 16Sbr-H (Palumbi et al. 1991) following protocols used by Vences et al. (2003), and a further segment from the cytochrome oxidase I (COI) marker with the primers dgL.CO1490 and dgHCO2198 (Meyer et al. 2005) follow- ing protocols used by Perl et al. (2014). Sequences were resolved on an ABI 3130x!l automated DNA sequencer (Applied Biosystems, Foster City, CA, USA). The newly determined sequences are deposited in GenBank under ac- cession numbers MT371794 (16S) and MT372330 (COI). A phylogeny of the genus Rhombophryne was con- structed using the newly determined sequences and a ma- trix of all other species and several candidate species for the same genes from GenBank (Table 1). This resulted in a matrix of 27 ingroup terminals plus Anodonthyla mon- tana Angel, 1925 used as an outgroup. 16S sequences were available from all included terminals, and COI sequences from 18 terminals. Sequences of the two loci were aligned in AliView 1.21 (Larsson 2014) using MUSCLE (Edgar 2004), and the alignment visually proofed. 16S rRNA and Zoosyst. Evol. 96 (2) 2020, 313-323 315 Table 1. Uncorrected pairwise distance (p-distance) in the 3' fragment of the 16S rRNA mitochondrial marker, and GenBank ac- cession numbers of sequences used to reconstruct phylogenetic relationships within Rhombophryne in Figure 1. For the full p-dis- tance table (all comparisons) see Appendix II, which is based on the more comprehensive sampling of specimens for 16S given in Appendix I. Genus Species Specimen number Anodonthyla montana FG/MV 2001-530 Rhombophryne botabota FGZC 2896 Rhombophryne coronata MV2001-199 Rhombophryne coudreaul FAZC 13887 Rhombophryne diadema FGZC 3631 Rhombophryne ellae sp. nov. MSZC 0534 Rhombophryne guentherpetersi ZCMV 12401 Rhombophryne laevipes FGZC 1052 Rhombophryne longicrus FGZC 3654 Rhombophryne mangabensis ZCMV 886 Rhombophryne matavy FGZC 1888 Rhombophryne minuta FGZC 2897 Rhombophryne nilevina KU 340893 Rhombophryne ornata ZCMV 12384 Rhombophryne proportionalis ZCMV 12404 Rhombophryne regalis FN 7292 Rhombophryne savaka ZCMV 2065 Rhombophryne tany ZCMV 12359 Rhombophryne testudo FG/MV 2000-277 Rhombophryne vaventy FGZC 2842 Rhombophryne sp. aff. coronata KU 340732 Rhombophryne sp. aff. vaventy AMNH-A167315 Rhombophryne sp. CaQl FAZC 10314 Rhombophryne sp. CaQ3 MV2001-131 Rhombophryne sp. CaO7 MV2001-G46 Rhombophryne sp. Cal0 FAZC 10312 Rhombophryne sp. ex-alluaudi ZCMV 968 Rhombophryne sp. RAX 10368 RAX 10368 16S p-distance 3'16S col - AY594090 GU177056 7.1-7.9 EU341102 KF611585 11.5-11.6 EU341103 KM509856 11.8 FJ559299 - 10.4 KU724171 - - MT371794 MT372330 9.0-9.4 KU937796 - 8.5-9.6 KM509189 KM509857 925 KRO25898 - 12.7-13.0 EU341109 KF611588 11:0 FJ559298 KF611589 10.9-11.3 EU341100 KF611590 Tad. KY288475 - 7.4-9.5 KP895584 KF611583 8.8 KU937808 KF611640 10.3 EUSAd ile) - 11.4 KU724176 KF611594 9.1 KP895585 KF611582 9.1-9.9 AY594125 EF396066 9.8 EU341107 KF611595 10.6 KY288476 - 8.8 DQ283409 KM509853 8.7 FI559295 - 7.5-8.0 FJ559296 KF611592 6.0 EU341108 - 74 AY594111 - 7.6-8.1 EU341105 KF611584 9.6 KM509192 KM509860 COI alignments were concatenated to increase phylogenet- ic signal, yielding an alignment of 1134 positions, of which 327 were parsimony informative. GTR + I + G was deter- mined to be the best model using model testing in MEGA X (Kumar et al. 2018). Phylogenetic analysis was performed under Maximum Likelihood (ML) in MEGA X and Bayes- ian inference (BI) in MrBayes 3.2 (Ronquist et al. 2012). ML analysis was conducted with 500 nonparametric boot- strap replicates, using SPR level 5 branch swapping. For BI analysis, two parallel runs were carried out, each with four heated chains, for a total of 10 million generations. Trees were sampled every 10,000 generations and 10% were dis- carded as burn-in after checking for convergence in Tracer 1.5 (Rambaut and Drummond 2007). A second matrix of 51 ingroup terminals plus A. montana was constructed for the 3' 16S marker alone, including a larger number of spec- imens for each species (full list in Appendix I). This too was aligned in AliView 1.21 using MUSCLE, yielding 530 positions, of which 159 were parsimony informative, and from it uncorrected pairwise distances (p-distances) were calculated in MEGA X (Appendix IT). The electronic version of this article in Portable Docu- ment Format (PDF) represents a published work according to the International Commission on Zoological Nomen- clature (ICZN), and hence the new name contained in the electronic version 1s effectively published under that Code from the electronic edition alone. This published work and the nomenclatural act it contains have been registered in ZooBank, the online registration system for the ICZN. The ZooBank LSIDs (Life Science Identifiers) can be re- solved 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:1- sid:zoobank.org: pub: SEE0689E-27DD-41B1-BOAA-AF- 300COB5BA4. The online version of this work will be archived and made available from the following digital repositories: CLOCKSS and Zenodo. Results 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, 2016a, 2017a; Lambert 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). The BI tree was better resolved and better matched previous hypothe- ses, SO it is shown in Figure 1. It is intended here to serve as a guide only to illustrate the degree of divergence of the new taxon from other Rhombophryne species, and not as an evolutionary hypothesis. The new specimen collected in Montagne d’Ambre (ZSM 76/2018) is recovered in a clade together with R. sp. Ca07 from Tsaratanana with high support (bootstrap support (BS) = 94%, posterior probability (PP) = 1). The position of this clade within the genus 1s variable; in BI zse.pensoft.net 316 Mark D . Scherz: A new diamond frog from Montagne d’Ambre, Madagascar Rhombophryne tany ZCMV 12359 54/0.41 50/0.86 85/0.33 78/0.93 Rhomb 100/1 54/1 10.51 -/0.70 4 -/0.36 99/1 Rhombophryne -/0.37 0.03 94/1 -/0.46 -/0.42 98/1 27/0.91 Rhombophryne sp. Ca07 MV2001-G46 Rhombophryne sp. Rhombophryne diadema FGZC 3631 Rhombophryne guentherpetersi ZCMV 12401 Rhombophryne ornata ZCMV 12384 ophryne vaventy FGZC 2842 Rhombophryne sp. aff. vaventy AMNH-A167315 Rhombophryne regalis FN 7292 Rhombophryne sp. aff. coronata KU340732 Rhombophryne coronata MV2001-199 Rhombophryne longicrus FGZC 3653 Rhombophryne minuta FGZC 2897 proportionalis ZCMV 12404 00/1 Rhombophryne ellae sp. nov. MSZC 0534 Ca10 FAZC 10312 Rhombophryne matavy FGZC 1888 Rhombophryne testudo FG/MV2000-277 Rhombophryne laevipes FGZC 1052 -/10.59 -/0.62 97/0.98 24/0.51 -10.68 Rhombophryne sp 81/0.65 Rhombophryne botabota FGZC 2896 Rhombophryne sp. Ca03 MV2001-131 Rhombophryne nilevina KU340893 . Ca01 FAZC 10314 Rhombophryne sp. (formerly ‘alluaudi’) ZCMV 968 83/0.99 78/1 Rhombophryne coudreaui FAZC 13887 Rhombophryne mangabensis ZCMV 886 Rhombophryne savaka ZCMV 2065 Rhombophryne sp. RAX 10368 Figure 1. Bayesian inference phylogeny of Rhombophryne based on concatenated sequences of the mitochondrial markers 16S tRNA and COI (1134 bp). Values at nodes indicate percent boot clades not recovered in the ML tree. Anodonthyla montana was strap support (BS)/Bayesian posterior probability (PP); ‘-’ indicates used as outgroup (not shown for graphical reasons). Rhombophryne serratopalpebrosa is the only described species missing from this phylogeny, as no sequences of that species are available. analysis it was found to be sister to R. sp. Cal0, whereas in ML analysis it was found to be sister to the R. /aevipes spe- cies group. The placement of Rhombophryne sp. Ca07 has been problematic before (see Scherz et al. 2016b, 2017b), so its affinities will need further work to clarify. However, it is unambiguously clear that the specimen from Mon- tagne d’ Ambre 1s highly distinct from all described species in the genus, and indeed from all known candidate species. The new frog from Montagne d’Ambre is separated from R. sp. Ca07 by an uncorrected pairwise distance (p-distance) of 6% in the 3' fragment of the mitochon- drial 16S rRNA gene analysed here, and from all other species of Rhombophryne by at least 7.1% (Table 1; Ap- pendix I). This distance is much higher than the 3% 16S p-distance threshold usually used for candidate species recognition in Madagascar’s frogs (Vieites et al. 2009), and within the genus Rhombophryne is comparable with several species pairs (e.g. R. minuta (Guibé, 1975) and R. longicrus Scherz, Rakotoarison, Hawlitschek, Venc- es, & Glaw, 2017 at 6.2-6.3%) and much greater than some others (e.g. R. ornata Scherz, Ruthensteiner, Vie- ites, Vences & Glaw, 2015 and R. guentherpetersi (Guibé, 1974) at 2.2-3.8%; see Scherz et al. 2017a). The specimen of the new frog is an adult or subadult fe- male, based on the presence of small eggs in its ovaries and thickened, white oviducts. Morphologically, the individual from Montagne d’Ambre most closely resembles R. sava- ka Scherz, Glaw, Vences, Andreone & Crottini, 2016 and R. mangabensis Glaw, Kohler & Vences, 2010 among de- scribed species but differs from them in a number of aspects that I detail below. Most characteristically, its thighs have zse.pensoft.net bright flash-markings, which are not present in any described Rhombophryne so far (but see the Discussion for more on this topic), and it has distinct black inguinal spots, which are rare among Rhombophryne species. Both flash-mark- ings and inguinal spots are notably present in R. sp. Ca07, further supporting the close affinities of these two species (R. sp. Ca07 will be described elsewhere in the context of a larger revision). Rhombophryne species show little sexual dimorphism, and as bioacoustic recordings of males are rare, species are described based on holotypes of either sex. Based on the congruence of strong genetic divergence and morphological differences from all nominal species, the new specimen from Montagne d’Ambre unambigu- ously represents an undescribed species, and I here pro- vide its diagnosis and description. Rhombophryne ellae sp. nov. http://zoobank. org/A656F828-D640-4C7D-B3D3-BCCE6D0E11B8 Figures 1-3 Holotype. ZSM 76/2018 (MSZC 0534), adult or subadult female, collected on 28 December 2017 in Montagne d’Ambre National Park (12.5066°S, 49.1746°E, 892 m a.s.1.), Antsiranana Region, northern Madagascar by M. D. Scherz, J. H. Razafindraibe, A. Razafimanantsoa, O. Randriamalala, S. M. Rasolonjavato, R. Tiavina, E. Z. Lattenkamp, and A. Rakotoarison. Diagnosis. Rhombophryne ellae sp. nov. is assigned to the genus Rhombophryne based on its plump body shape, Zoosyst. Evol. 96 (2) 2020, 313-323 presence of vomerine and maxillary teeth, curved clav- icle, knob-shaped terminal phalanges, and phylogenetic relationships. It is distinguished by the following unique combination of characters: (1) adult or subadult female SVL 24.9 mm, (2) distinctly enlarged inner metatarsal tubercle, (3) absence of superciliary spines, (4) orange flash-markings on its posterior thighs in life, and (5) pres- ence of large and distinct black inguinal spots. The new species can be distinguished from all de- scribed members of the genus Rhombophryne by the or- ange colouration on its posterior thighs. Additionally, it may be distinguished from all members of the Rhombo- phryne serratopalpebrosa species group (R. serratopal- pebrosa (Guibé, 1975), R. guentherpetersi, R. coronata (Vences & Glaw, 2003), R. vaventy Scherz, Ruthensteiner, Vences & Glaw, 2014, R. ornata, R. tany Scherz, Ruthen- steiner, Vieites, Vences & Glaw, 2015, R. diadema Scherz, Hawlitschek, Andreone, Rakotoarison, Vences & Glaw, 2017, and R. regalis Scherz, Hawlitschek, Andreone, Ra- kotoarison, Vences & Glaw, 2017) with ease by absence of superciliary spines; from R. testudo, R. matavy, and R. coudreaui by smoother dorsal skin, a longer and less broad head (HW/HL 1.48 vs 1.88—2.42), relatively longer legs (HIL/SVL 1.77 vs 1.17—1.41), and less-developed inner metatarsal tubercle; from R. savaka and R. manga- bensis by its longer forelimb (FORL/SVL 0.55 vs 0.41- 0.48), longer hindlimb (HIL/SVL 1.77 vs 1.49—1.60), and less broad head (HW/HL 1.48 vs 1.54—1.86); and from R. minuta and R. longicrus by its shorter hindlimb (HIL/SVL 1.77 vs 1.79-1.84), shorter forelimb (FORL/SVL 0.55 vs 0.70-0.75), and wider head (HW/HL 1.48 vs 1.22—1.43). Morphologically, Rhombophryne ellae sp. nov. is simi- lar to R. laevipes, R. nilevina Lambert, Hutter & Scherz, 2017, and R. botabota Scherz, Glaw, Vences, Andreone & Crottini, 2016, especially young individuals, but in ad- dition to large genetic distances, it differs by absence of ocelli on the hidden portions of the legs (vs presence), presence of large black inguinal spots (vs absence), and absence of distinct colouration of the lateral surface of the head (vs presence in R. botabota and some R. /aevipes), and also in its smaller body size from R. /aevipes and R. nilevina (presumed adult female SVL 24.9 mm vs at least 28.6 mm and generally > 30 mm). Rhombophryne ellae sp. nov. is also distinguished from all described Rhombophryne species by an uncor- rected p-distance of > 7.1% in a fragment of the 16S rRNA gene (Table 1; Appendix II). The new species can also be distinguished from all known members of the morphologically similar but not closely related genus Plethodontohyla on the basis of the orange colouration on its posterior thighs. Holotype description. Morphology. An adult or sub- adult female specimen in a good state of preservation, its hindlimbs and toe tips slightly dehydrated. Tissue sam- ples taken from left thigh for sequencing. A small incision is present on the right side and in the wall of the gut. De- veloping eggs are visible in the ovaries, and the oviduct is thick and white. S17 Body robust. Head wider than long (HW/HL = 1.48). Pupils more or less round. Snout rounded in dorsal and lateral view. Canthus rostralis distinct, concave. Loreal region concave, without dermal folds. Nostrils nearer to snout tip than to eye (END/NSD = 0.87), directed laterally, slightly protuberant. Tympanum distinct, TDH/ED = 0.6. Supratympanic fold distinct, rounded over the tympanum from the posterior corner of the eye, ending anterior to the insertion of the forelimb. Superciliary spines absent. Vomerine teeth distinct, in a straight row on either side of the palate, varying in height, approaching each other me- dially but separated by a small gap. Choanae diminutive, unusually close to the neopalatine. Arms rather slender. Fingers without webbing, rela- tive lengths 1<4<2<3; fourth finger slightly shorter than second; finger tips not expanded; fingers not reduced (Fig. 2c); nuptial pads absent; inner metacarpal tubercle present, outer metacarpal tubercle absent; subarticular tu- bercles round and flat, undivided. Hindlimbs fairly slen- der (also in life, not an artefact of preservation); tibiotarsal articulation reaches the eye when the hindlimb is ad- pressed forward along the body; TIBL/SVL = 0.46. Inner metatarsal tubercle present, round, slightly enlarged, outer metatarsal tubercle present, rather weak, distinct, round. Toes unwebbed; relative lengths 1<2<5<3<4, fifth toe dis- tinctly shorter than third. Toe tips not expanded, second, third, and fourth toe tips slightly pointed. Dorsal and ven- tral skin smooth in preservative, but with a few dispersed pustules in life (Fig. 2). Dorsolateral folds absent. Colouration. After just over a year in preservative, specimen dorsally brown, with two darker spots above the suprascapulae. A faint dark brown chevron is pres- ent on the posterior portion of the dorsum. Distinct black inguinal spots present. A russet brown spot is present on the posterodorsal portion of the tympanum, which 1s oth- erwise dirty cream. On the left side of the snout there is a further lighter area that 1s not present on the right side. A light interocular bar is present. The dorsal hindlimbs are as the dorsum in colour, with faint, dark-grey crossbands on the thigh and shank. The feet are highly mottled with cream and grey-brown, with a whitish annulus before each toe tip. The hidden surfaces of the thigh are cream, and there is a distinct black trapezoid in the cloacal region. The forelimbs are similar to the dorsum in dorsal colour- ations, with a dark-grey crossband on the antebrachium, followed by a white spot distally. The hands are mottled like the feet, also with light annuli before each fingertip. Ventrally, the abdomen is translucent cream, the scapu- lar region and chin are mottled cream and olive-brown, and the hindlimbs and forelimbs are brown, flecked with cream, forming larger blotches more distally. The soles of the hands and feet are dark-grey, mottled with cream. The subarticular and carpal and tarsal tubercles are likewise cream. Colouration in life is shown in Figure 2. Osteology. Skeleton resembling other Rhombophryne species (Scherz et al. 2017a); what follows is thus a brief summary of remarkable features of the skeleton (Fig. 3). Frontoparietal robust, bearing dorsal processes nearly forming a ridge. Otic capsule not dorsally ossified. Nasal zse.pensoft.net 318 Mark D. Scherz: A new diamond frog from Montagne d’Ambre, Madagascar Figure 2. The holotype of Rhombophryne ellae sp. nov. in life. a. In dorsolateral view; b. In dorsal view; ¢. In ventral view; d. In posterior view showing the orange flash-markings on the thighs and the distinctive inguinal spots; e. On leaf litter. broad with a long and cuneate maxillary process, widely separated from the contralateral. Post-choanal vomer bear- ing teeth with a distinct diastema either side of the cultri- form process of the parasphenoid. Parasphenoid alae near- ly the breadth of its cultriform process. Neopalatine broad. Sphenethmoid strongly ossified, bounding around half the length of the braincase. Exoccipitals widely separated dor- zse.pensoft.net sally. Premaxilla and maxilla bearing teeth. Maxilla with a broad connection to the quadratojugal. Quadratojugal anteriorly broad, its bulbous posteroventral process thick, dorsally with broad contact to the squamosal. Squamosal with a distinctly flared ventral ramus, a nearly vertical otic ramus, and a shorter, anteromedially curving zygomatic ramus. Pterygoid anterior ramus distinctly bowed, ventral Zoosyst. Evol. 96 (2) 2020, 313-323 319 +f — Oh g2-7 Figure 3. Volumetric renderings of micro-CT scans of the skeleton of the holotype of Rhombophryne ellae sp. nov. (ZSM 76/2018). a—c. Full skeleton in (a) dorsal, (b) lateral, and (c) ventral view; d—g. Skull in (d) dorsal, (e) ventral, (f) anterior, and (g) lateral view; h. Foot in ventral view; i. Hand in ventral view. Abbreviations: angspl — angulosplenial; angspLcp — coronoid process of the angulo- splenial; col.pip — pars interna plectri of columella; col.pmp — pars media plectra of columella; exoe — exoccipital; exoc.oc — occipital condyle of exoccipital; fpar — frontoparietal; fpar.dp — dorsal process of frontoparietal; fpar.If— lateral flange of frontoparietal; max — maxilla; max.pf-— pars facialis of maxilla, mmk — mentomeckelian; nasal.mp — maxillary process of nasal; neopal — neopalatine; pmx — premaxilla; pmx.ap — ascending process of premaxilla; pmx.Ip — lingual process of premaxilla; pmx.palproc — palatine pro- cess of premaxilla; povom — postchoanal portion of vomer; proot — prootic; prsph.al — parasphenoid alae; prsph.ep — cultriform pro- cess of parasphenoid; prsph.pp — posterior process of parasphenoid; prvom — prechoanal portion of vomer; pter.ar — anterior ramus of pterygoid; pter.mr — medial ramus of pterygoid; pter.pr — posterior ramus of pterygoid; qj — quadratojugal; smx — septomaxilla; spheth — sphenethmoid; sq.or — otic ramus of squamosal; sq.vr — ventral ramus of squamosal; sq.zr — zygomatic ramus of squamosal. ramus very deep. Mandible robust, the receiving surface of the angulosplenial (coronoid process) somewhat lat- erally flared to receive the quadratojugal. Clavicle robust and curved. Coracoid slender at its midpoint. Humerus with a strong crista ventralis and a broad epicondylus ul- naris. Urostyle with a dorsal crest running circa two-thirds of its length. Ilium with a low dorsal crest and a rather shallow oblique groove. Pubis semi-ossified. Centrale of the foot large, but the prehallux is not enlarged. Terminal phalanges of fingers and toes knobbed. Prepollex short and triangular. Hand bone configuration as in the R. serra- topalpebrosa species group (Scherz et al. 2017a). Measurements (all in mm). SVL 24.9, HW 9.6, HL 6.5, ED 2.5, END 1.3, NSD 1.5, NND 3.1, TDH 1.5, TDV 1.6, HAL 5.3, VAL3.6, LAL 4.7, FORL 13.7, FARL 10.0, THIL 12.5, THIW 3.5, TIBL 11.5, TIBW 3.3, TARL 6.96, FOL 11.6, FOTL 18.6, HIL 42.5, IMCL 1.0, OMCL 1.1, IMTL 1.0, OMTL 0.6. Natural history, distribution, and conservation status. The holotype was collected at 892 m a.s.l. in rainforest on Montagne d’Ambre during the day actively jumping away from trampling feet during moderate to heavy rain brought about by Cyclone Ava. Its gut contents included three whole ants and one ant head, seemingly belonging to two different species (one of the whole ants 1s diminutive), the head of a jumping spider (Salticidae), and the elytra and other body parts of a beetle. Nothing more is known of the ecology of this species, though it is probable that its reproductive mode and ecology is similar to other lit- zse.pensoft.net 320 ter-dwelling Rhombophryne species (Scherz et al. 2016a). As the species is known from a single individual, its Red List status cannot be confidently estimated. However, the syntopically occurring Stumpffia species have been sug- gested to be Near Threatened due to their small range and presumed micro-endemicity within a well-protected for- est, and this likely applies to R. ellae sp. nov. as well. Etymology. It is with great pleasure that I dedicate this charming little frog to my partner, Dr Ella Z. Lattenkamp, in appreciation of her love, support, and infinite patience, and in celebration of the completion of her PhD. Available names. No names are currently available for the family Cophylinae that could refer to this species. Discussion The bright orange flash-markings on the thighs of R. e//ae are a particularly notable character. It is the first described Rhombophryne species to possess this trait, though it should be noted that it is also present in R. sp. Ca07 (depicted by Glaw and Vences 2007: 119, photograph 5, as R. guenth- erpetersi, though not showing the flash markings), as well as another undescribed species from Marojeyy that seems more closely allied with R. mangabensis and R. savaka (unpublished data). Rhombophryne ornata also has dark reddish colouration on its legs, although that colouration is less clearly demarcated and may serve some other purpose. These three lineages are not closely related, and the species between them do not have flash-markings, indicating re- peated evolution of this trait within Rhombophryne. Beyond Rhombophryne, red to orange thighs are also present in various other Malagasy microhylids (e.g. Stump- ffia be Kohler, Vences, D’Cruze & Glaw, 2010 and several Platypelis species; Glaw et al. 2020), not to mention man- tellids, and indeed numerous other groups of frogs world- wide. The fact that this trait is so widespread, and has clear- ly been evolved numerous times, implies some functional significance. Yet, at present I am not aware of any tests of its function, which must be imagined to be either intraspe- cific communication (sexual selection) or, more likely, in predator deterrence. The pairing of the bright flash-mark- ings with inguinal ‘eye’ spots, as is the case for R. ellae, would tend to support the latter hypothesis. Yet, in most other Malagasy frogs with orange thighs, such inguinal markings are missing. Behavioural observations and de- tailed studies will be needed on individual groups of frogs to establish the function of the flash-markings and whether that function is common to all taxa that have developed it, even when the shade of red can vary substantially (e.g. the recently described Platypelis ranjomena Glaw, Scherz, Rakotoarison, Crottini, Raselimanana, Andreone, Kohler & Vences, 2020 has deep blood-red flash-markings, as op- posed to the bright orange of R. e//ae; see Glaw et al. 2020). Rhombophryne ellae is another highly distinct member of the genus Rhombophryne, discovered in 2017. It is of comparable divergence to R. Jongicrus, which was discov- zse.pensoft.net Mark D. Scherz: A new diamond frog from Montagne d’Ambre, Madagascar ered in 2012 and described in 2015 (Scherz et al. 2015a). Unlike the latter, which was found in an area that has sel- dom been surveyed before, the new species described here was found in Montagne d’Ambre National Park, an area that is generally considered well surveyed. Fossorial and semi-fossorial frogs are particularly prone to being missed by short-term and localised survey efforts, and are still more so when they are limited to extremely small rang- es, as 1s often the case among Madagascar’s microhylids, including several Rhombophryne species; I presume that this will also be the case of R. e/lae. This often means that specimen numbers are few, and in the present case, only a single specimen of the new species has so far been col- lected (examples of other recently described frog species based on singletons include R. tany, Anodonthyla eximia Scherz, Hutter, Rakotoarison, Riemann, Rédel, Ndriant- soa, Glos, Roberts, Crottini, Vences & Glaw, 2019, Oreo- bates yanucu Kohler & Padial, 2016, and all three species of Vietnamophryne Poyarkov, Suwannapoom, Pawang- khanant, Aksornneam, Duong, Korost & Che, 2018). Although the usage of singletons for species descrip- tions is not ideal, it can be necessary, given that rarity is natural and common (Lim et al. 2012). The combined substantial improvement in our knowledge of the genus Rhombophryne over the last decade (D’ Cruze et al. 2010; Scherz et al. 2014, 2015a, 2015b, 2016a, 2016b, 2017a, 2019) and the establishment of DNA barcoding in these frogs (Vieites et al. 2009), allows us to be highly confident in the recognition of species-level lineages in this genus. Micro-CT even allows us to access data on the internal anatomy of such singletons without harming the speci- mens, yielding a wealth of data that would formerly have required at least two specimens, with one intact and one dissected or cleared and stained. Kohler and Padial (2016) discussed this point in some detail, highlighting the com- parative robustness of species described using multiple lines of evidence compared to those based solely on mor- phology, even when specimens are plentiful (although, as they say, ‘large series of specimens are always desirable’ ). In the case of R. e/lae, the diagnosis and description was facilitated by the highly unique appearance of the frog and its very large genetic distances from congeners. Of course, much remains unknown about singleton species, such as variability, sexual dimorphism, ecology, and adult body size distributions (for further discussion see Lim et al. 2012 and Kohler and Padial 2016); future work must continue to build on the taxonomic foundation, though it may be difficult when they are so rarely encoun- tered. Fortunately, the detection probability of (semi-) fossorial frogs seems to increase in inclement weather. In Madagascar, cyclones appear to act as a particular stim- ulus 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. Even so, many taxa are liable to be overlooked; continuous monitoring is Zoosyst. Evol. 96 (2) 2020, 313-323 probably the most effective way to ensure near-total sam- pling of such areas, but even with substantial manpower and resources, some rare and ephemeral species may go unnoticed. Conservation strategies must account for this detectability problem among potentially undescribed spe- cies by protecting habitat at the landscape level. Acknowledgements I thank F. Glaw and M. Vences for their support and su- pervision over the last six years and for the many ways in which they have aided in my work on the herpetofauna of Madagascar. I thank J. Glos, J. Kohler, and one anonymous reviewer for their helpful feedback on this manuscript. For their assistance during fieldwork in Montagne d’ Ambre, I thank J. H. Razafindraibe, A. Razafimanantsoa, O. Randri- amalala, S. M. Rasolonjavato, E. Z. Lattenkamp, R. Tiavi- na, and A. Rakotoarison. Extraction and sequencing were performed at the Technische Universitat Braunschweig by M. Kondermann, for which I am very grateful. I thank the Malagasy authorities for providing permits to conduct this research, which was carried out under permit no. 191 17- MEEF/SG/DGF/SDAP/SCB.Re. The fieldwork was fund- ed by a grant of the Deutsche Forschungsgemeinschaft (grant VE 247/13-1 to MDS and M. Vences). The speci- men was exported under permit no. 032-EA02.MG18. References Andreone F, Vences M, Vieites DR, Glaw F, Meyer A (2005) Recurrent eco- logical adaptations revealed through a molecular analysis of the secre- tive cophyline frogs of Madagascar. Molecular Phylogenetics and Evo- lution 34(2): 315-322. https://doi.org/10.1016/j-ympev.2004.10.013 Angel F (1925) Liste des reptiles et batraciens rapportés de Madagascar par M. G. 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Evolution 62(8): 1890-1907. https://do1.org/10.1111/ j.1558-5646.2008.00420.x Appendix I GenBank numbers for all 16S sequences included in Appendix II. Species Genbank Number Anodonthyla montana AY594090 Rhombophryne botabota AY594104 EU341102 KU724172 KU724173 KU724174 KU724175 Rhombophryne sp. CaO1 AY594106 FI559295 Rhombophryne sp. Ca03 AY594107 FJ559296 Rhombophryne sp. CaQ7 EU341108 Rhombophryne sp. CalO AY594111 Rhombophryne coronata EU341103 KM509188 Rhombophryne coudreaui FIB59299 HM364771 Rhombophryne diadema KU724171 Rhombophryne ellae MT371794 Rhombophryne guentherpetersi KU724178 KU937796 Rhombophryne laevipes EU341104 KM509189 Rhombophryne longicrus KRO25897 KRO25898 Rhombophryne mangabensis EU341109 KU724179 KU724180 KU724182 Rhombophryne matavy FJ559298 GU195641 Rhombophryne minuta EU341100 EU341106 Rhombophryne nilevina KY288475 Rhombophryne ornata KP895582 KP895583 KP895584 Rhombophryne proportionalis KU937808 Rhombophryne regalis EU341111 Rhombophryne savaka KU724176 KU724177 Rhombophryne sp. aff. coronata KU340732 Rhombophryne sp. aff. vaventy DQ283409 Rhombophryne sp. ex-alluaudi AY594105 AY594112 EU341105 KU724170 Rhombophryne sp. RAX 10368 KM509192 Rhombophryne tany KP895585 Rhombophryne testudo AY594125 KC180070 Rhombophryne vaventy EU341107 323 Zoosyst. 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