Zoosyst. Evol. 94(1) 2018, 109-135 | DOI 10.3897/zse.94.14698 ® PENSOFT. museum fur naturkunde Resurrection and re-description of Plethodontohyla laevis (Boettger, 1913) and transfer of Rhombophryne alluaudi (Mocquard, 1901) to the genus Plethodontohyla (Amphibia, Microhylidae, Cophylinae) Adriana Bellati?’", Mark D. Scherz*", Steven Megson?, Sam Hyde Roberts’, Franco Andreone®, Goncalo M. Rosa®”.®, Jean Noél?, Jasmin E. Randrianirina’®, Mauro Fasola', Frank Glaw’, Angelica Crottini?? Dipartimento di Scienze della Terra e dell'Ambiente, Universita di Pavia, Via Ferrata 1, I-27100 Pavia, Italy Zoologische Staatssammlung Miinchen (ZSM-SNSB), Miinchhausenstr. 21, 81247 Miinchen, Germany School of Science and the Environment, Manchester Metropolitan University, Manchester, MI 5GD, UK SEED Madagascar, Studio 7, 1A Beethoven Street, London, W10 4LG, UK Museo Regionale di Scienze Naturali, Sezione di Zoologia, Via G. Giolitti, 36, I-10123, Torino, Italy Department of Biology, University of Nevada, Reno, Reno NV 89557, USA Institute of Zoology, Zoological Society of London, Regent's Park, London, NW1 4RY, UK Centre for Ecology, Evolution and Environmental Changes (CE3C), Faculdade de Ciéncias da Universidade de Lisboa, Bloco C2, Campo Grande, 1749-016 Lisboa, Portugal 9 Madagascar Fauna and Flora Group, BP 442, Morafeno, Toamasina 501, Madagascar ON DO RW DY 10 Pare Botanique et Zoologique de Tsimbazaza, BP 4096, Antananarivo 101, Madagascar 11 CIBIO, Research Centre in Biodiversity and Genetic Resources, InBIO, Universidade do Porto, Campus Agrario de Vairdo, Rua Padre Armando Quintas, N° 7, 4485-661 Vairdo, Portugal http://zoobank.org/AFA6C1FE-1627-408B-9684-F6240716C62B Corresponding author: Angelica Crottini (tiliquait@yahoo. it) Abstract Received 26 June 2017 The systematics of the cophyline microhylid frog genera Plethodontohyla and Rhom- Accepted 19 January 2018 bophryne have long been intertwined, and their relationships have only recently started Published 2 February 2018 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 Academic editor: the taxonomic situation between these two genera is, and the care that must be taken to Johannes Penner resolve taxonomic conundrums where old material, multiple genus transitions, and mis- attribution of new material obfuscate the picture. We assessed the identity of the historic Key Words names Dyscophus alluaudi (currently in the genus Rhombophryne), Phrynocara laeve and Plethodontohyla laevis tsianovohensis (both synonyms of Rhombophryne alluaudi) based Amphibia on an integrative taxonomic approach harnessing genetics, external morphology, osteologi- Anura cal data obtained via micro-Computed Tomography (micro-CT) and bioacoustics. We show Phrynocara laeve that (1) the holotype of Dyscophus alluaudi is a member of the genus Plethodontohyla, Plethodontohyla alluaudi (2) the Rhombophryne specimens from central Madagascar currently assigned to Rhombo- Madagascar Phryne alluaudi have no affinity with that species, and are instead an undescribed species; Integrative taxonomy and (3) Phrynocara laeve and Dyscophus alluaudi are not synonymous, but represent close- ly related species, whereas Plethodontohyla laevis tsianovohensis is tentatively confirmed as synonym of D. alluaudi. We resurrect and re-describe Plethodontohyla laevis, and re-al- locate and re-describe Plethodontohyla alluaudi on the basis of new and historic material. * Both authors contributed equally Copyright Adriana Bellati 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. 11) Introduction The microhylid subfamily Cophylinae Cope, 1889, en- demic to Madagascar, is today recognised as possessing eight genera according to Scherz et al. (2016a): Pletho- dontohyla Boulenger, 1882, Rhombophryne Boettger, 1880, Stumpffia Boettger, 1881, Madecassophryne Guibé, 1974, Platypelis Boulenger, 1882, Anodonthyla Miller, 1892, Cophyla Boettger, 1880 and Anilany Scherz, Venc- es, Rakotoarison, Andreone, Kohler, Glaw & Crottini, 2016. Recent studies (Vieites et al. 2009, Perl et al. 2014) have provided DNA barcoding reference sequences for almost all described and undescribed frog species of Madagascar and have highlighted the presence of a large taxonomic gap in the Cophylinae, which currently com- prises over 100 described species (Scherz et al. 2016a, AmphibiaWeb 2018), but which has an estimated further > 40 species yet to be formally described. In spite of the recent efforts in revising the systematics of this subfamily (e.g. D’Cruze et al. 2010, Glaw et al. 2010, Rakotoarison et al. 2012, 2015, 2017, 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 (Wol- lenberg et al. 2008, Scherz et al. 2016a) comprises mod- erately small to large terrestrial or scansorial forest frogs (snout-vent length [SVL] 25—100 mm, Glaw and Vences 2007b). Despite sharing a large number of morphological features (e.g. the presence of connected lateral metatar- salia, vomerine teeth, inner [and sometimes outer] meta- tarsal tubercles, short hindlimbs and males with a single subgular vocal sac; Scherz et al. 2016a), an osteological circumscription to the genus still remains difficult. The ecomorphologies correlate with different skeletal adapta- tions; for instance, all semi-arboreal taxa (P. notosticta, P. mihanika) and three predominantly terrestrial species (P. guentheri, P. inguinalis and P. fonetana) possess ex- panded terminal phalanges (T- or Y-shaped), while all other terrestrial and fossorial taxa possess knob-shaped terminal phalanges. Whereas the clavicle is always ab- sent or highly reduced in terrestrial or fossorial species, it can be present or absent in arboreal species, suggesting repeated loss of these bones (Scherz et al. 2016a, unpub- lished data), but also the monophyly of these phenetic groups (arboreal and terrestrial/fossorial) has yet to be es- tablished. In addition to being a morphologically diverse genus, Plethodontohyla has had an exceptionally convo- luted taxonomic history. Plethodontohyla was originally erected by Boulenger (1882) to contain Callula notosticta Gunther, 1877 (the type species of the genus), P. inguinalis Boulenger, 1882 and P. brevipes Boulenger, 1882. This circumscription was before its time in recognizing the phenotypic diver- sity of Plethodontohyla, as Boulenger (1882) established the genus containing terrestrial/fossorial (P brevipes) and arboreal and semi-terrestrial (P. notosticta, P. ingui- nalis) species. Peters (1883) later erected two genera, Phrynocara Peters, 1883 (type species Ph. tuberatum zse.pensoft.net Bellati, A. et al.: Taxonomy and re-descriptions of Plethodontohyla laevis and P. alluaudi Peters, 1883) and Mantipus Peters, 1883 (type species M. hildebrandti Peters, 1883), each monotypic. Some years later Mocquard (1895) erected another monotypic genus, Mantiphrys Mocquard, 1895 (type species Man- tiphrys laevipes Mocquard, 1895). Over the 20th century, six more species of the genus Plethodontohyla were de- scribed, plus two in the genus Phrynocara and seven in Mantipus. Noble and Parker (1926) synonymised Man- tiphrys with Mantipus, and Phrynocara with Plethodon- tohyla, and transferred P. inguinalis to Mantipus. Guibé contributed repeatedly to the species-level taxonomy of these genera (e.g. Guibé 1947, 1974, 1975), but made only one small change at genus-level, which he later reversed (Guibé 1947, 1952, 1978; see below). Blom- mers-Schlosser and Blanc (1991) later synonymised Mantipus with Plethodontohyla in their comprehensive monograph on the amphibians of Madagascar (Mantipus hildebrandti becoming a junior synonym of Plethodon- tohyla inguinalis). At this point, the genus Plethodonto- hyla contained twelve nominal species, encompassing a wide range of ecomorphologies (terrestrial, fossorial and semi-arboreal) and sizes, from the 22 mm P. minuta to the 100 mm P. inguinalis. Andreone et al. (2005) produced the first compre- hensive molecular dataset for the subfamily Cophyli- nae, where it became clear that several members of the genus Plethodontohyla were more closely related to Rhombophryne testudo Boettger, 1880—which had, until that point, been alone in the monotypic genus Rhombo- phryne—but refrained from making any taxonomic ar- rangements until more data were available. Frost et al. (2006) also recovered the paraphyly of Plethodontohyla first identified by Andreone et al. (2005), and transferred three species to Rhombophryne (R. alluaudi, R. coudreaui and R. /aevipes). Based on the more comprehensive mo- lecular analysis of the subfamily (Wollenberg et al. 2008) another four species (R. minuta, R. coronata, R. guenther- petersi, R. serratopalpebrosa) were transferred to Rhom- bophryne (Glaw and Vences 2007b). Since then, no more species have moved between these two genera except R. matavy which was erroneously transferred to Plethodon- tohyla by Peloso et al. (2016), but returned to Rhombo- phryne by Scherz et al. (2016a). The taxon Dyscophus alluaudi Mocquard, 1901 was originally described with the type locality ‘Fort Dau- phin’ (or Tolagnaro; Fig. 1), but was later transferred to Plethodontohyla by Noble and Parker (1926). It was then moved to Mantipus by Guibé (1947), but was later returned by him to Plethodontohyla without comment (Guibé 1978), presumably based on the similarity of its pectoral girdle to that of Mantipus angeli Guibé, 1947, which he synonymised with Plethodontohyla tuberata (Guibé 1952). Another taxon, Phrynocara laeve Boett- ger, 1913, was described from Sakana, East Madagascar, a locality reported to be a magnificently preserved piece of jungle (Boettger 1913). This taxon was transferred to Plethodontohyla by Noble and Parker (1926), initially with an incorrect emendation (Plethodontohyla laeve), Zoosyst. Evol. 94 (1) 2018, 109-136 111 Species Altitude Plethodontohyla alluaudi 0 Plethodontohyla laevis Plethodontohyla cf. laevis Plethodontohyla sp. Ca01 Plethodontohyla sp. ? Type Locality 0 100 A mbodiriana Analalava ivorano Est pasinambo ,; Ranomafana EA 2 rie BK, A. 7 P_|Tsitongambarika (Anosyenne Chain) -—Ste Luce Figure 1. Locality records of Plethodontohyla laevis, P. alluaudi, and P. sp. Ca01, including the uncertain records of two members of this complex (P. cf. /aevis and P. sp.) from Blommers-Schlosser (1975). zse.pensoft.net iZ Bellati, A. et al.: Taxonomy and re-descriptions of Plethodontohyla laevis and P. alluaudi later corrected to P. /aevis by Parker (1934). A supposed subspecies of Plethodontohyla laevis, P. 1. tsianovohen- sis Angel, 1936, was later described from Tsianovoha (approximately 22°07’60.00”S, 047°19°60.00"E; 112 m above sea level [a.s.l.], collected by R. Heim be- tween 1934 and 1935, Fig. 1), a lowland forest in the Vatovavy-Fitovinavy region in eastern Madagascar, but was synonymised with P. /aevis by Guibé (1978). Pletho- dontohyla laevis was then synonymised with P. alluaudi by Blommers-Schlosser and Blanc (1991). Following Blommers-Schlosser (1975), who assigned a specimen collected in Mandraka to Mantipus alluaudi, Glaw and Vences (1992), in their first edition of the field guide to the amphibians and reptiles of Madagascar, attributed a specimen of a cophyline frog from Andasibe in central eastern Madagascar also to P. alluaudi. Subsequently collected specimens from this region became the genetic reference material for the taxon (Andreone et al. 2005). On the basis of genetic data from these specimens, P. alluaudi was transferred to Rhombophryne by Frost et al. (2006), where it has since remained. Recently, individuals of a species of terrestrial co- phyline microhylid were found in Betampona Special Reserve (Fig. 1), a small but relatively well-maintained lowland rainforest fragment in eastern Madagascar (Rosa et al. 2012). Genetic analysis revealed these specimens to belong to the genus Plethodontohyla, and they were referred to as P. sp. Ca3 by Vieites et al. (2009) and Scherz et al. (2016a) (also called P. sp. aff. brevipes [Ca FJ559294] by Rosa et al. 2012). In this study, we examined these specimens from Bet- ampona, and found them to have strong affinities with the holotype of Phrynocara laeve. This suggested that at least Phrynocara laeve was incorrectly attributed to the genus Rhombophryne, and prompted questions regard- ing the genus-level assignment of Dyscophus alluaudi. To resolve these questions, we investigated the morphol- ogy and osteology of the type material of Dyscophus alluaudi, Phrynocara laeve, and Plethodontohyla laevis tsianovohensis (Fig. 2). We then studied the morpholo- gy, osteology and genetics of the Rhombophryne species from Andasibe currently assigned to R. alluaudi, recent- ly collected material of P. sp. Ca3 from Betampona, and of specimens of ‘R. alluaudi’ from near Tolagnaro. We base our study on the integration of data from external and internal (osteological) morphology, natural history, congruence between mitochondrial and nuclear DNA differentiation and bioacoustic analyses. Materials and methods In anticipation of the main outcomes of our research, we hereafter use Plethodontohyla laevis and P. alluaudi in reference to these two names, except when discuss- ing the type material, where we refer to the species by their original names (Phrynocara laeve and Dyscophus alluaudi, respectively). zse.pensoft.net Voucher specimen collection New specimens were collected either during the day by searching the leaf litter, or at night using torches and headlamps, sometimes guided by the male advertisement call. Representative voucher specimens were euthanized, and then fixed in 90% ethanol or 10% buffered formalin, rinsed in water and preserved in 70% ethanol. Live col- ouration was photographed at the time of capture. Locality information were recorded using a GPS, da- tum WGS84. Field numbers FAZC, ACZC and ACZCYV, FGZC, and PBZT-RJS refer to F. Andreone, A. Crottin1, F. Glaw, and J.E. Randrianirina, respectively. Tissue sam- ples (taken before specimen fixation) were obtained from hindlimb muscle or tongue, and preserved separately in 99% ethanol. Institutional abbreviations used herein are as follows: ZSM = Zoologische Staatssammlung Munchen, Germa- ny; SMF = Naturmuseum Senckenberg in Frankfurt am Main, Germany; UADBA-A = Amphibian collections of the Université d’Antananarivo Département de Biolo- gie Animale, Madagascar (currently Mention Zoologie et Biodiversité Animale, Faculté des Sciences, Univer- sité d’Antananarivo, Antananarivo); MRSN = Museo Regionale di Scienze Naturali, Torino, Italy; MNHN = Muséum National d’ Histoire Naturelle de Paris, France; ZFMK = Zoologisches Forschungsmuseum Alexander Koenig, Bonn, Germany; ZMA = Zooélogisch Museum Amsterdam (transferred to Naturalis Biodiversity Center in Leiden), Netherlands; BMNH = Natural History Mu- seum, London, United Kingdom. Morphological measurements Measurements of preserved specimens were taken by MDS with a calliper to the nearest 0.01 mm, rounded to the nearest 0.1 mm (ratios calculated before rounding to avoid compound rounding errors): SVL (snout-vent length), HW (maximum head width), HL (head length, from the rictus to the snout tip), ED (horizontal eye di- ameter), END (eye-nostril distance), NSD (nostril-snout tip distance), NND (internarial distance), TD (horizontal tympanum diameter), HAL (hand length, from the radi- oulnar-carpal articulation to the tip of the longest finger), FORL (forelimb length, given by the sum of HAL, low- er arm length [LAL] and upper arm length [UAL]), FOL (foot length, from the tarsal-metatarsal articulations to the tip of the longest toe), TARL (tarsus length), FOTL (foot length including tarsus, given by the sum of FOL and TARL), TIBL (tibia length), HIL (hind-limb length, given by the sum of FOL, TARL, TIBL and thigh length [THIL]), IMCL (maximum length of inner metacarpal tu- bercle). Examined specimens are listed in Table 1. Note that measurements of Plethodontohyla brevipes are from specimens that match the original description of that spe- cies in having a uniform brown dorsum and slightly gran- ular dorsal skin. Terminology and description scheme fol- low Vences et al. (2003), Glaw and Vences (2007a) and Glaw et al. (2007) to allow for better comparison to other Plethodontohyla species. Zoosyst. Evol. 94 (1) 2018, 109-136 113 Figure 2. Photographs of the holotypes of (a—b) Phrynocara laeve (SMF 4286), (c—d) Dyscophus alluaudi (MNHN 1901.235) and (e-f) Plethodontohyla laevis tsianovohensis (MNHN 1936.47) in dorsal (a, ¢, e) and ventral (b, d, f) view. Osteological analyses The holotypes of Phrynocara laeve (SMF 4286), Dyscophus — alluaudi (MNHN ~— 1901.235) — and Plethodontohyla laevis tsianovohensis (MNHN 1936.47), and one specimen assigned below to Plethodontohyla laevis (MRSN A6340) from Betampona, one specimen assigned below to P. alluaudi (ZSM 89/2004) from Andohahela and one specimen of the Rhombophryne species formerly assigned to P. alluaudi by Glaw and Vences (1992) from Andasibe (ZSM 3/2002) were scanned using X-ray micro-Computed Tomography (micro-CT), in a phoenix|x nanotom m _ cone-beam micro-CT scanner, following protocols used previously zse.pensoft.net Taxonomy and re-descriptions of Plethodontohyla laevis and P. alluaudi Bellati, A. et al.: 114 cz |ovt|veo|coz|zzt| 6 | ovo | eqisepuy | ‘ds euA1ydoquioyy | G9ZzS MINAZ QT | 2c. |vss|zre| 2st] se] Ivo aqisepuy | ‘ds auAsydoqwiouy | Z00Z/E€ WSZ 02 Fest [org OLZ ect] 26 | 170 aqisepuy | ‘ds auAsydoquioyy ee ee z vel |e ea oe ; j sadiaalq 9002/2580 vt | vst }osr|zoz}ret| 89 | zo ve EYE EMOUEH || Te Me onouior Nee 2 he Uail te, eed, ¥ , : eyojeu| sadiaalq 9002/17S80 Sie || oe ee ea ele AL|| eer (estos lv ser SaMiatoty | -Glucnhtaaleac Wee ¥ ir neta al euswouey sadinalgq 900Z/€S80 ie €or|LZZ1}v It] €9 | Tro ve EES IOHERT METROID Sta ee = = = ie sadiaalq 9002/TS80 a Pe Ajayeyele | sadinalgq £00Z/S092 s ‘euelewiouey | e/Ayoluopoyie/d WSZ a wed JeuoeN sadinalg €002/61790 ¥ ‘euejeuouey | e/AyouopoYjely WSZ q eyo|eul| Pe 9002/958 vt ‘euejewouey TOeD “ds ‘d WSZ zs eyo|eu| me 9002/SS8 [1 €9 ‘euejeuuouey 1080 “ds “¢ WSZ =| SINBE| €10Z/086 d 0'z vv'0|9 CUOCIMESE | e/AyojUOpoy}eld SZ "a me ; ; SINae| ST | TE1 91zZ| ZI Zv| 88 |rr'0 SJ OURIONUY | piKuoruopoypajg | 28L9V NSU nal. a SINae| d piles = SINae| , fa : ro ' 7 SIAQE] oz | OV'T e€z| 6ST €OL oye Ace pdonopouary: |ELoow Ned Uz | v2 euodwiejag aes ISTOW NSU ; ejAYOJUOPOY}a/q Py SINBE| 910Z/681 yt | sgt PAPIPIECW | eyAyoquopoypald WSZ 6'T 9S'T | S'OT | S'9T eueyeS anak] e4e20UAIYd | 987 JINS . nodes vale : Ipnenjje 7002/68 GT O'zT| 19 | ZEO 6L'I BISUBYOPUY | a ¢ygruopoyteld Wee Saul 2 sala Be an | 2 Ipnenj|e v66LZ elsayYeUOpU GT ZZ1| 19 | 6E'0 LOT| 62 |Z et IPUEUOPUY \\|- ernconuomotnsia ace coaplee p SISU@YOAOURIS} 1POEEt e¢ | Zy'T | v's8|9'ze|o0'Sz|9°'7T| 6E'0 |9'zz| ES°0 0/z ‘olg'z| ¢ ‘O|Z'v| TZ'T | Z'ST | 8'9z ee8i04 SIAR] Shit e/Ayoyuopoyja}d ela eee : Ar rs Ipnenyje GEZ'TO6T Lz | set |z6s|tsz|z9T| 68 | veo |O'9T| ZrO L¢| Sst |Z0T|8'6T| vZy | 4 | LH | ulydneg x04 anydooska NGAI : AS TA | sn} Jaquinn TOW! | ll |1i03) 103 Tavs oi | Tat ,| 1404 ) avi fava) wa , | GL|GNN | GSN | ONa | ee a3] aH | ma TAS | X8S] 21g] Aa112907 salsads Giasties ¢ < 4 ‘gqruoant “f Sopeuu “Py Sayeuloy “y SodAyoyoy “LH :7X9} OU} Ul popnyoUl JOU SUOTIVIADIQGY ‘SpouJoW pue sTelIoyep] Oy) JO UOTDIS SJUSLDINSVOU SY} 99S “SUOTILIADIQG” JO (SANA Sore Jd9d9X9 SOU] [IU UI SJUSUOINSvOUU [[e) Apnys sty} 10} posAjeue ‘ds auduydoqwoyy pue sadiaaig g ‘|QeD ‘dS J ‘Ipnwnypyo J ‘siaav] d 0} pousisse suswiideds pajoa][09 ATJUIIOI OY} JO pUe S7sUaYOAOUDIS] SlAaD] DjAYOJUOpOYjald “Ipnonyyo snydoasaq ‘adav] vAévIoUcAY J JO SAdAjOJOY dy} JO sJUdWIOINSeOW [eOISOTOYdIo/ *[ I[qQuVy ¢ zse.pensoft.net Zoosyst. Evol. 94 (1) 2018, 109-136 oes Figure 3. Individuals of Plethodontohyla laevis in life, illustrating the diversity of colour patterns from different sites of the species known distribution range: (a) MRSN A6188 from Betampona in dorsolateral and (inset) ventral view; (b) MRSN A6181 from Betampona in dorsolateral view; (c) FAZC 13898 from Betampona in dorsolateral view (Photos by Gongalo M. Rosa); (d) MRSN A6787 from Aniv- orano Est in dorsolateral and (inset) ventral view (Photo by Jasmin E. Randrianirina); (e) ZSM 189/2016 from Analalava-Foulpointe in dorsolateral view (Photo by Frank Glaw); (f) individual (not collected) from Ambodiriana in dorsolateral view (Photo by Lauric Reynes). for Madagascan microhylids (e.g. Scherz et al. 2014, 2015a, b, 2016a, b , 2017). The specimens were scanned individually at 140 kV and 80 uA, with a timing of 750 ms, for a total of 20 or 30 minutes (1440 or 2440 images respectively). Reconstruction methods were the same as those used previously (see the aforementioned literature, and especially Scherz et al. 2017). Examination of the internal anatomy of the specimens was conducted in VG STUDIO MAX 2.2 (Volume Graphics GmbH, Heidelberg, Germany). DICOM stacks of the scans and rotational videos are deposited in MorphoSource at the following URL: http://morphosource.org/Detail/ ProjectDetail/Show/project_id/396. Portable document file (PDF)-embedded 3D models of select specimens were produced using AMIRA 6.1 (FEI Visualization Sciences Group, Burlington, MA), and are provided as digital Suppl. materials 1-6. Osteological terminology follows Trueb (1968, 1973). Molecular analyses Ten samples of P. /aevis from four different localities (Fig. 3) (seven from Betampona Natural Reserve, one from Marovato, one from Anivorano Est and one from near Analalava-Foulpointe); three samples of P. a//uaudi (in its new definition but until now referred to as ‘P. bipuncta- ta’; Fig. 4a) from Andohahela (EU341068, Wollenberg et al. 2008), Tsitongambarika (Anosyenne Chain) and Sainte Luce; one sample of P. sp. Cal Ranomafana (Ambatolahy, EU341067, Wollenberg et al. 2008; Fig. 4b); three samples of Rhombophryne sp. (formerly assigned to R. alluaudi) zse.pensoft.net 116 Bellati, A. et al.: Taxonomy and re-descriptions of Plethodontohyla laevis and P. alluaudi to as P. bipunctata; from Andohahela); (b) P. sp. CaO1 in dorsolateral view (ZCMV 555; from Ambatolahy); (c) P. brevipes in dor- solateral view (ZSM 649/2003; from Ranomafana); and (d) Rhombophryne sp. (formerly identified as R. alluaudi) in lateral view (ZFMK 52765 from Andasibe) (Photos by Frank Glaw and Miguel Vences). from Torotorofotsy (ZCMV 968; EU341105, Wollenberg et al. 2008), Andasibe (ZSM 3/2002; AY594112, Andre- one et al. 2005) and Tsararano (MRSN A2620; AY594105, Andreone et al. 2005); one sample of all nominal species of the genus Plethodontohyla (with the exception of P. no- tosticta for which we included sequences from individuals from two localities, one in the North and one in the South) were used in the molecular analyses (see Table 2 for more details). A homologous sequence of R. testudo, the type species of the genus Rhombophryne, was also included. For the newly obtained samples, total genomic DNA was extracted using Proteinase K (10 mg/ml) digestion followed by a standard salt-extraction protocol (Bruford et al. 1992). We amplified one mitochondrial gene frag- ment (rrnL large ribosomal RNA or 16S rRNA gene) for all newly obtained tissues samples, and one nuclear gene fragment, the pro-opiomelanocortin (POMC) gene, for a subset of samples (see details in Table 2). Standard pol- ymerase chain reactions were performed in a final vol- ume of 25 ul and using 0.75 ul each of 10 pmol primer, 0.4 ul of total dNTP 10 mM (Promega), 0.1 wl of 5 U/ml GoTagq (Promega), 5 ul 5X Green GoTaq Reaction Buffer (Promega) and 4 wl of MgCl, 25mM (Promega). To se- quence a fragment of ca. 550 bp of the 3’ terminus of the mitochondrial large ribosomal RNA gene (16S), proven to be suitable for amphibian identification (Vences et al. 2005), we used the primers AC_16S_AR 5’°CGCCTGT- TTATCAAAAACATS’ and AC_16S_BR 5’°CCGGTYT- GAACTCAGATCAYGT3’ modified from Kocher et al. zse.pensoft.net (1989) and Palumbi et al. (1991), using standard protocols. To sequence the POMC fragment, we used the primers POMC DRVF1 5’ATATGTCATGASCCAYTTYCGCT- GGAA3’ and POMC DRVRI 5’GGCRTTYTTGAAWA- GAGTCATTAGWGG3’ (Vieites et al. 2007) as in Venc- es et al. (2010). Successfully amplified fragments were purified and sequenced at Macrogen Inc., where labelled fragments were analysed on an ABI 3730XL automated DNA sequencer (Applied Biosystems). Sequences were compared with GenBank sequenc- es, and chromatographs were visually checked and edit- ed, when necessary, using BIOEDIT 7.0.5.3 (Hall 1999). Gaps were included in the hypervariable regions of the 16S to account for indels in the final alignment. All new- ly determined sequences have been deposited in GenBank (MG273701—MG273723; Table 2). Uncorrected pairwise distances (p-distance transformed into percentage using the complete deletion option) amongst individuals of the same species and between ingroup analysed species were computed using MEGA 7.0.21 (Kumar et al. 2016). Bayesian analyses were conducted in MRBAYES 3.2.2 (Ronquist et al. 2012). The GTR+I+G model was deter- mined by AIC in jModelTest2 (Darriba et al. 2012) as the best-fitting model of substitution. We performed two runs of 10 million generations (started on random trees) and four incrementally heated Markov chains (using default heating values), sampling the Markov chains at intervals of 1,000 generations. Stabilization and convergence of likelihood values were checked by visualizing the log Zoosyst. Evol. 94 (1) 2018, 109-136 117 Table 2. List of samples included for the molecular analyses with their respective localities, voucher and/or field number informa- tion, institutional catalogue number (where available) and GenBank accession numbers. Sample ID Field Institutional catalogue | Accession | Accession number number nos. 16S | nos. POMC . HM364769/ ACP1109 Plethodontohyla laevis Betampona - MRSN A6340 F J559204 MG273712 ACP1901 Plethodontohyla laevis Betampona ACZCV 0066 ZSM 980/2013 MG273701 |MG273713 ACP1108 Plethodontohyla laevis Betampona - MRSN A6189 MG273702 | MG273714 ACP 1107 Plethodontohyla laevis Betampona - MRSN A6181 MG273703 - ACP2196 Betampona _| ACZC 6262 - MG273704 | MG273715 ACP2214 Betampona _| ACZCV 0268 z MG273705 | MG273716 ACP2066 Betampona _| ACZC 5923 - MG273706 | MG273717 ACP1362 ieee MRSN A6674 MG273707 | MG273718 ACPS1 Fl | Plethodontohyla laevis | laevis Analalava MG273708 - ACP1368 | Plethodontohyla laevis | Anivoranoest | PECUIS MRSN A6787 MG273709 | MG273719 FGZC161 Plethodontohyla alluaudi Andohahela FGZG 164. ZSM 89/2004 EU341068 | MG273720 ACP1056 | Plethodontohylaalluaudi |, Sitongambarika | en79 15493 MG273710 | MG273721 (Anosyenne Chain) SE47 Plethodontohyla alluaudi Sainte Luce - MG273711 ~ ZCMV555 _ | Plethodontohyla sp. CaOl Ambatolahy ZCMV 555 EU341067 | MG273722 ZCMV968 Rhombophryne sp. Torotorofotsy EU341105 | MG273723 AYSO4105 | na avso4i12 | na Plethodontohyla ocellata Ambohitsara ZCMV 88 UADBA uncatalogued EU341062 - na Plethodontohyla brevipes Maharira ZCMV 270 - EU341063 - na Plethodontohyla fonetana Bemaraha FGZC S17 ZSM 123/2006 EU341058 - na Plethodontohyla guentheri Marojejy FGZC 2814 ZSM 61/2005 EU341059 - na Plethodontohyla inguinalis Vohiparara - ZMA 20223 EU341057 - na Ranomafana £u341056 | __— na Nosy Mangabe cu34i061 | na £U341060 | __— na Plethodontohyla bipunctata Tolagnaro RAX 10726 - KM509181 - na Plethodontohyla tuberata Manjakatompo ZSM 375/2000 EU341064 - na Rhombophryne testudo Nosy Be, Lokobe ZSM 474/2000 KC180070 - na Scaphiophryne marmorata Andasibe ZSM 4/2002 AY834191 = likelihoods associated with the posterior distribution of trees in the software TRACER 1.5 (Rambaut and Drum- mond 2007), and occurred after about 3—3.5 million gen- erations. The first four million generations were conserv- atively discarded, and six million trees were retained post burn-in and summed to generate the majority rule con- sensus tree (Fig. 5a). The purpose of the presented phy- logenetic analyses 1s: 1) to show that the four analysed populations of P. /aevis form a monophyletic group; 2) to show the closest phylogenetic relationship of this species to P. alluaudi in its new definition and P. sp. CaO1 (which might prove to be conspecific with P. alluaudi), and 3) to show that the specimens formely assigned to “Rhom- bophryne alluaudi’ (from Torotorofotsy, Andasibe, and Tsararano) do not belong to the genus Plethodontohyla, rather than provide a phylogenetic hypothesis of the phy- logenetic relationships of Plethodontohyla species and support for the genus monophyly. Alternative alleles of the analysed POMC gene frag- ment were inferred using the PHASE algorithm (Ste- phens et al. 2001) implemented in the software DNASP 5.10.3 (Librado and Rozas 2009). Haplotype network re- construction of POMC phased sequences (Fig. 5b) was performed using the software TCS 1.21 (Clement et al. 2000). This software employs the method of Templeton et al. (1992) and calculates the number of mutational steps by which pairwise haplotypes differ, computing the probability of parsimony for pairwise differences until the probability exceeds 0.95 (no manual adjustment of threshold was necessary). The minimum number of mu- tational steps required to connect the two networks ob- tained using the parsimony method of Templeton et al. (1992) was identified using the 'fix connection limit’ op- tion as implemented in TCS. Bioacoustic analyses Vocalizations of P. /aevis were recorded in the field with a Marantz PMD 660 digital recorder, accessorized with a semi-directional microphone. Calls were successively analysed with the acoustic software ADOBE AUDITION 3.0. Definition of variables and terminology in call de- scriptions follow Rosa and Andreone (2010), Rosa et zse.pensoft.net 118 Bellati, A. et al.: Taxonomy and re-descriptions of Plethodontohyla laevis and P. alluaudi R. testudo Nosy Be KC180070 a me (*)-R. sp. Torotorofotsy EU341105 kK R. sp. Andasibe AY594112 R. sp. Tsararano AY594105 Kk kk P. alluaudi Andohahela EU341068 aap P. alluaudi Tsitongambarika MG273710 P. alluaudi Sainte Luce MG273711 P. sp. Ca01 Ambatolahy EU341067 P. laevis Betampona HM364769 P. laevis Betampona MG273703 P. laevis Betampona MG273702 P. laevis Betampona MG273705 P. laevis Analalava MG273708 P. laevis Betampona MG273704 P. laevis Marovato MG273707 #k P. laevis Anivorano Est MG273709 (*) P. laevis Betampona MG273701 P. laevis Betampona MG273706 Kk Kk P. tuberata Manjakatompo EU341064 P. mihanika Ranomafana EU341056 P. ocellata Ambohitsara EU341062 =* |_-——— P. brevipes Maharira EU341063 P. bipunctata Tolagnaro KM509181 xk P. inguinalis Vohiparara EU341057 ak P. fonetana Bemaraha EU341058 («) P. guentheri Marojejy EU341059 Ped P. notosticta Nosy Mangabe EU341061 P. notosticta Manombo EU341060 0.02 i P laevis Betampona Hi P laevis Marovato WP Iaevis Anivorano Est WS P. alluaudi Tsitongambarika GP alluaudi Andohahela WP sp. Ca01 Ambatolahy HR. sp. Torotorofotsy Figure 5. a) Bayesian inference tree based on 529 bp of the mitochondrial 16S. Asterisks denote Bayesian posterior probabilities val- ues: one asterisk enclosed in parentheses, > 90%; one asterisk, > 98%; two asterisks, > 99—100%. b) Haplotype network reconstruction for the phased alleles of the nuclear POMC gene fragment in P. /aevis from Betampona, Marovato and Anivorano Est, P. alluaudi from Tsitongambarika and Andohahela, P. sp. CaO1 from Ambatolahy and Rhombophryne sp. from Torotorofotsy. al. (2010, 2011) and Kohler et al. (2017), and calls are compared to described Plethodontohyla vocalizations available in the literature (see Table 3). Recordings were re-sampled at 44,100 Hz and 16 bit resolution in mono and with the ‘Waveform’ extension. Frequency informa- zse.pensoft.net tion was obtained through Fast Fourier Transformation (FFT, width 1024 points); the audiospectrogram was ob- tained with a Hanning window function resolution of 256 bands. Zoosyst. Evol. 94 (1) 2018, 109-136 119 Table 3. Comparative measurements from advertisement calls of Plethodontohyla species. Species Temp. | Series Note duration Duration of inter- Note Dominant Visible Reference (°C) of (ms) note intervals (s) | repetition | frequency | harmonics notes rate (n/s) (Hz) ; 391-422 P. laevis (407412.7, n=d) 47 (n=1) 0.04 1820-2530 this study . 320-560 ; * _.). —$$ | = P. alluaudi (4784109, n=4) unknown unknown | 1400-2100 this study ix Pin tae 133-191 0.85-1.15 P, inguinalis (148418, n=10) | (1£0.085, n=10) 800-1300 Vallan et al. (2005) P. mihanika 24.5 ISO D2 abe Vences et al. (2003) (159+6, n=12) (5.341, n=11) 280-340 2 unknown P. notosticta 19 1 365-412 (391413.3, n=15) 3.9-5.4 (3.740.74, n=14) Glaw and Vences (1992) Rosa et al. (2011); this study 1900-2200 4 1000 unknown 0.26 930-1330 9 * due to the low quality of the available recordings not all the parameters were possible to obtain. Temporal measurements are provided as range, fol- lowed by mean, standard deviation and number of ana- lysed units (n). We measured air temperature (to the near- est 1 °C) with digital devices at close distance to calling frogs (i.e. temperature information refers to air temper- ature at the time of recording, not body temperature of the calling specimen). The number of recordings did not allow for temperature corrections. Results and discussion We here present evidence that (1) Dyscophus alluaudi and Phrynocara laeve are both members of the genus Plethodontohyla, (2) Plethodontohyla laevis tsianovo- hensis is more similar to D. alluaudi than Ph. laevis, (3) the osteology and morphology of the holotypes of Dy- scophus alluaudi and Phrynocara laeve indicate that they are not conspecific; (4) the species of Rhombophryne cur- rently called R. ‘alluaudi’ from the Andasibe region has no affinity with that species; (5) populations of P. sp. Ca3 from Betampona are conspecific with Phrynocara laeve; and (6) populations of a species of Plethodontohyla from southern Madagascar, until now referred to as ‘P. bipunc- tata’ (ZSM 89/2004) are conspecific with Dyscophus al- luaudi. Based on these findings, we resurrect and re-de- scribe Plethodontohyla laevis, we transfer Dyscophus alluaudi to the genus Plethodontohyla and re-describe it. Identity of the holotypes of Dyscophus alluaudi, Phryno- cara laeve and Plethodontohyla laevis tsianovohensis We examined the type material of Dyscophus alluaudi, which is currently assigned to the genus Rhombophryne, and its junior synonyms Phrynocara laeve and Plethodon- tohyla laevis tsianovohensis (depicted in Fig. 2). As we have intimated previously (Scherz et al. 2016a, b), an in- creasing body of evidence suggests that the name D. allu- audi is misapplied. Our investigation resulted in strong ev- idence for taxonomic placement of the respective names: (1) The holotype of Dyscophus alluaudi, MNHN 1901.235 (Fig. 2c, d), is an adult ovigerous female specimen measuring 47.4 mm in SVL (for all other measurements see Table 1). It has knob-like terminal phalanges, an unossified pubis, tri-radiate prechoanal vomer with the lateral ramus situated at the midpoint, a strongly descending lateral flange of the frontoparietal, a short maxillary facial process, fron- toparietals not extending beyond the level of the neopalatine and a well-developed transverse dorsal ridge on the fronto- parietal (Fig. 6, see Suppl. material 1). The pectoral girdle has highly reduced clavicles (remaining just as short thin bony elements near the glenoid socket; indicated by arrows in Fig. 6) and a facet near the middle of the anterior edge of the coracoid. The pectoral girdle has been exposed on the specimen, and a thin cartilaginous extension of the pro- coracoid runs from the anterior glenoid socket to the facet on the leading edge of the coracoid, and then broadens and runs along it to the omosternum (intact only on the left side); the bony remnants of the clavicles are barely discernable through the dissecting microscope, as they are transparent and very thin. A similar condition to that seen in Dyscophus alluaudi was described for the type specimen of Mantipus angeli by Guibé (1974; confirmed by M.D. Scherz, personal observation), which is a synonym of Plethodontohyla tu- berata (Peters, 1883). This state was unknown to Scherz et al. (2016a), suggesting the diagnostic value of the ‘absence of clavicles’ paired with absence of nasal-frontoparietal contact for Plethodontohyla recognition must be refined to include also these reduced lateral elements. The configura- tion is nevertheless clearly different from Rhombophryne, including the reduced clavicles of species like R. mangab- ensis (M.D. Scherz et al. unpublished data). (2) The holotype of Phrynocara laeve, SMF 4286 (Fig. 2a, b), is probably also an adult female (with develop- ing eggs), measuring 38.1 mm in SVL (for all other meas- urements see Table 1). It lacks clavicles, has knob-like terminal phalanges, an unossified pubis, a tri-radiate pre- choanal vomer with the lateral ramus displaced anteriorly, lateral flange of frontoparietal not descending strongly, a short maxillary facial process, frontoparietals extending beyond the level of the neopalatine and a well-developed if discontinuous transverse dorsal ridge on the frontopa- rietal (Fig. 6, Suppl. material 2). It differs strongly from MNHN 1901.235 in its smaller size (SVL 38.1 vs. 47.4 mm), much narrower head (HW/HL 1.56 vs. 1.85), longer zse.pensoft.net 120 Bellati, A. et al.: Taxonomy and re-descriptions of Plethodontohyla laevis and P. alluaudi MNHN 1936.47 Plethodontohyla alluaudi MNHN 1901.235* = © j=) SS oO [oe] = i¢p) N je) wz 9] ide) ~} 6 & 2 % ®o = ay g =. < Q S 8 = b ne?) <3 WL = ep) jes9}e7] jesioq BIPIID) [e10}08q Figure 6. Skull and pectoral girdle morphology of Plethodontohyla alluaudi and P. laevis. Asterisks indicate the holotypes of each species. Arrows on the pectoral girdle of P. alluaudi MNHN 1901.235 indicate the clavicles. Scale bars indicate 1 mm. zse.pensoft.net Zoosyst. Evol. 94 (1) 2018, 109-136 relative forelimb length (FORL/SVL 0.56 vs. 0.47), longer relative hindlimb length (HIL/SVL 1.43 vs. 1.25), the ab- sence of large dark inguinal spots (vs. presence), lateral ra- mus of the prechoanal vomer displaced anteriorly (vs. cen- tral), frontoparietals exceeding the level of the neopalatine (vs. not exceeding the neopalatine) and the weakly de- scending lateral flange of the frontoparietal (vs. strongly). The pectoral girdle is similar to MNHN 1901.235, but has also been damaged, obscuring the state—it nevertheless lacks the ossified clavicles of that specimen. We therefore conclude that it is not a synonym of D. alluaudi, but in- stead a valid species in need of resurrection. Its osteology also suggests that it is a member of the genus Plethodon- tohyla, on the basis of the absence of clavicles and lack of nasal-frontoparietal contact. (3) The type specimen of Plethodontohyla laevis tsiano- vohensis, MNHN 1936.47 (Fig. 2e, f), 1s also an adult, ovi- gerous female measuring 58.0 mm in SVL (for all other measurements see Table 1). It lacks clavicles, but has the same cartilaginous pectoral arrangement as the holotype of D. alluaudi, but on the left side it has been damaged so that a second pectoral fenestra is formed medial to the anterior facet of the coracoid, where it ought not to be. In addition, it has knob-like terminal phalanges, an ossified pubis, an al- most crescentic prechoanal vomer with a weak lateral ramus, a strongly descending lateral flange of the frontoparietal, a long maxillary facial process, frontoparietals not extending beyond the level of the neopalatine and a well-developed transverse dorsal ridge on the frontoparietal (Fig. 6, Suppl. material 3). Its affiliations are not quite clear; it is larger in size than either D. alluaudi or Ph. laeve (SVL 58.0 vs. 47.4 and 38.1 mm, respectively), its arms are longer than those of D. alluaudi but shorter than those of Ph. laeve (FORL/ SVL 0.53 vs. 0.47 and 0.56, respectively), it has longer legs than both (HIL/SVL 1.47 vs. 1.25 and 1.43, respectively), its tympanum is broader than both (TD/ED 0.58 vs. 0.53 and 0.40, respectively), and its osteology shares some elements with either species and differs from both in others (e.g. the length of the facial process). Overall, the skeleton and ex- ternal morphology more closely resembles that of MNHN 1901.235, and we therefore tentatively conclude that P /. tsianovohensis should be left in the synonymy of D. allu- audi. However, we emphasise that the D. alluaudi and P. 1. tsianovohensis type specimens do not agree in all aspects of their morphology, and their type localities are separated by at least 300 km (Fig. 1), so this taxon may eventually be recognized as a valid, species-level name (possibly it may represent P. sp. Ca01, whose osteology has not been studied here, but see below). It must therefore be re-visited in future treatments of the taxonomy of the P. alluaudi complex. In any case, it is the most junior of the available names, and its identity can remain unresolved for the time being. Identity of recently collected specimens As a next step, we analysed the osteology and mor- phology of three more recently collected specimens: (1) ZSM 3/2002, a specimen from Andasibe of the species currently referred to as ‘Rhombophryne alluaudi’ following 121 Blommers-Schlosser (1975) and Glaw and Vences (1992); (2) MRSN A6340, a specimen of a species of Plethodon- tohyla collected from near the potential type locality of Phrynocara laeve that agrees strongly with the original de- scription of that species; and (3) ZSM 89/2004, a specimen of a species of Plethodontohyla collected in Andohahela, near to the type locality of Dyscophus alluaudi. (1) ZSM 3/2002 is genetically a member of the genus Rhombophryne (Figs 4d, 5a). Osteologically, it resembles other published and unpublished Rhombophryne skeletons (Scherz et al. 2014, 2015a, b, 2016a, b, 2017, unpublished data) and it differs unambiguously from the holotype of Dyscophus alluaudi. it has fully developed clavicles (vs. rudimentary clavicles present in the holotype of D. a/luau- di), two independent dorsal processes on the frontoparietal (rather than a more or less continuous ridge) and a fused presacral VIII and sacrum (Suppl. material 4). Addition- ally, it lacks inguinal spots and any trace of the pattern originally described for Dyscophus alluaudi. Thus, it 1s clear that the taxon Dyscophus alluaudi is currently mis- applied. Based on its molecular phylogenetic identity (Ta- ble 2; Fig. 5a), as well as the presence of curved clavicles and knobbed terminal phalanges, this species 1s a member of the genus Rhombophryne. It does not match any other described species of Rhombophryne, and will therefore be described in a forthcoming revision of that genus. (2) MRSN A6340 is a specimen of Plethodontohyla collected at Betampona and genetically similar to oth- er specimens collected at Marovato, Anivorano Est and Analalava-Foulpointe (Table 2; Fig. 5). It is an adult male (collected when calling), measuring 33.0 mm in SVL (for all other measurements see Table 1). It lacks clavicles, has knob-like terminal phalanges, a mostly unossified pubis, a tri-radiate vomer with a lateral ramus displaced anteriorly, lateral flange of frontoparietal not descending strongly, a moderately short maxillary facial process, frontoparietals extending beyond the level of the neopala- tine and a well-developed transverse dorsal ridge on the frontoparietal (Fig. 6, Suppl. material 5). In all of these re- spects, it strongly resembles the osteology of the holotype of Phrynocara laeve. Its external morphology also resem- bles that species, though it differs somewhat in ratios (but note the variability of measurements shown in Table 1). It differs clearly from Dyscophus alluaudi and PI. laevis tsianovohensis on the same grounds given above from Ph. laeve, 1.e. the absence of large dark inguinal spots (vs. presence in D. alluaudi), lateral ramus of prechoanal vomer displaced anteriorly (vs. central), frontoparietal exceeding the level of the neopalatine (vs. not exceeding the neopalatine) and the weakly descending lateral flange of the frontoparietal (vs. strongly). We therefore conclude that this species is assignable to Plethodontohyla laevis, distinct from Plethodontohyla alluaudi, and we resurrect and re-describe it below based on data from the holotype and our new material. (3) ZSM 89/2004 is a specimen collected in Andoha- hela and genetically belonging to the genus Plethodonto- hyla (Table 2; Figs 4a, 5). This specimen is molecularly zse.pensoft.net 22 similar to other specimens collected at Tsitongambarika and Sainte Luce and moderately similar to the sequence of a specimen collected at Ambatolahy (Fig. 5). ZSM 89/2004 has close genetic affinities to the specimens that we here confer to P. alluaudi, representing a closely re- lated clade (see Fig. 5a). Osteologically, it differs from the holotype of Phrynocara laeve (and other specimens conferred to that taxon) in the following respects: lat- eral ramus of prechoanal vomer central (vs. displaced anteriorly), and lateral flange of frontoparietal descend- ing strongly (vs. not descending strongly). Its coracoids show distinct facets for the attachment of cartilage, more strongly developed than in P. /aevis. By comparison, it differs from Dyscophus alluaudi in the narrower skull, frontoparietals exceeding the level of the neopalatine, ab- sence of clavicle remnants and the proportions of some skull elements (compare the skulls in Fig. 6, Suppl. ma- terial 6). However, we hypothesise that these differences between this specimen and the holotype of Dyscophus alluaudi are due to the considerable difference in body size (SVL 29.1 vs. 47.4 mm) and that the proportions of the skull and its ossification are correlates of age and size. The differences to Phrynocara laeve appear more sub- stantial, despite the similarity in size. Both ZSM 89/2004 and UADBA-A 27994 (FGZC 160) possess inguinal spots and agree in external morphology with Dyscophus alluaudi. We therefore attribute these populations from southern Madagascar to Plethodontohyla alluaudi, and we re-describe this species below. Remarks on the identity of P sp. Ca01 and P. brevipes Fig. 4b, c We note that the specimen representing P. sp. Ca0l (ZCMV 555) from Ambatolahy in eastern Madagascar (21°14’37.92”S, 047°25’34.38”E) is genetically very similar to the samples attributed to P. alluaudi and phy- logenetically represents the sister taxon of the specimens here attributed to P. alluaudi. A picture of a specimen of P. sp. Ca01, ZCMV 555 (or 556, a second not yet sequenced individual) was depicted as Plethodontohyla brevipes on page 125 of Glaw and Vences (2007b) and in Fig. 1 of Scherz et al. (2016a). Two additional specimens belong- ing to this taxon are currently present in the ZSM collec- tion: ZSM 855/2006 and ZSM 856/2006. Elements of the overall appearance of specimens ZCMV 555 (based on the photograph) and ZSM 855/2006 and ZSM 856/2006 disagree with the description of that species, most nota- bly by the presence of a distinct marking over the back of the head (originally described as ‘uniform dark brown above’). In contrast, they bear a remarkable resemblance to P. alluaudi and P. laevis. These specimens may there- fore be closely related to the holotype of P. /aevis tsiano- vohensis, which is from an area comparatively near to Ambatolahy. Nevertheless, the taxonomic status of P. sp. Ca01, and also the relationships of Plethodontohyla bre- vipes based on its holotype (BMNH 1947.2.10.42), clear- ly need to be revised. This is however beyond the scope of zse.pensoft.net Bellati, A. et al.: Taxonomy and re-descriptions of Plethodontohyla laevis and P. alluaudi the current study, and must be conducted in the context of a larger revision of the genus. In the diagnoses against P. brevipes presented for the following re-descriptions, we included only measurements from specimens that resem- ble the original description in having uniformly brown dorsal colouration and slightly granular dorsal skin. Molecular analyses Among representatives of the genus Plethodontohyla, the mean uncorrected p-distance (for the 16S fragment) of P. laevis varies between 5.5% (comparison with P. sp. Ca0l which may be conspecific with P. alluaudi) and 11.5% (comparison with P. guentheri and with P. brevi- pes). Our data also reveal some genetic differentiation between the four known populations of P. /aevis, with an intraspecific mean uncorrected p-distance of 1.1% (Table 4). For other intraspecific comparisons and comparisons with other Plethodontohyla species see Table 4. The two Bayesian analyses resulted in largely identi- cal trees, with only minor changes in posterior probability values, and showed that P. /aevis from the four analysed localities forms a robust monophyletic group (posterior probability [PP] 0.99). Our analyses recovered a moder- ately supported sister relationship (PP 0.94) for P. laevis and the clade composed of P. alluaudi in its new definition and P. sp. CaOl from Ambatolahy (Fig. 5a). The mean uncorrected p-distance of P. sp. CaOl and P. alluaudi is 2.9% and these taxa might indeed represent two popula- tions of the same species. Similarly, the three specimens from Torotorofotsy, Andasibe and Tsararano belonging to the genus Rhombophryne apparently are the same taxon (mean uncorrected p-distance 0.7%; PP 1.0), although a more extensive phylogenetic analysis of Rhombophryne will be required to further confirm this result. The haplotype network reconstruction of the nuclear POMC gene (Fig. 5b) shows no haplotype sharing be- tween Plethodontohyla laevis (from Betampona, Maro- vato and Anivorano Est) and P. alluaudi. Wide haplotype sharing is observed between the three analysed popula- tions of P. /aevis used in this analysis, with at least two haplotypes (haplotype H1 and H2; Fig. 5b) found in all three populations; and haplotype sharing is observed also between P. alluaudi from Andohahela and P. sp. Ca01 from Ambatolahy (haplotype H7; Fig. 5b). The analysis of haplotype network reconstruction fails to recover a single statistically significant haplotype net- work for the analysed dataset that comprises representa- tive samples of P. /aevis from three localities, P. alluaudi from two localities, P. sp. CaOl and the Rhombophryne species from Torotorofotsy, and a minimum of 18 substi- tutions are required to join these two haplotype networks (see Fig. 5b for details). Plethodontohyla laevis (Boettger, 1913), bona species Figs 2a, b, 3, 6-8, Suppl. materials 2, 5 Remarks. This species has been referred to as Plethodon- tohyla sp. 3 ‘Betampona’ by Vieites et al. (2009), Pletho- Zoosyst. Evol. 94 (1) 2018, 109-136 123 Table 4. Estimates of evolutionary divergence over sequence pairs within- (bold) and between-species for the analysed 16S rRNA mitochondrial gene fragment. The number of base differences per site averaged over all sequence pairs within and between groups are shown. The analysis involved 28 nucleotide sequences. All positions containing gaps and missing data were eliminated. There were a total of 300 positions in the final dataset. Evolutionary analyses were conducted in MEGA7 (Kumar et al. 2016). The pres- ence of n/c (not computed) in the results denotes cases in which it was not possible to estimate evolutionary distances. “R. sp.’ refers to the undescribed Rhombophryne species formerly assigned to R. alluaudi. BES Ho alluaudi | CaQ1 ocellata | brevipes | fonetana | guentherl | inguinalis | mihanika | notosticta | bipunctata tuberata testudo P.alluaudi | 0.7% r ore Wr wi P. sp. CaOl P. laevis R. sp. P. ocellata P. brevipes P. fonetana . P. guentheri >| 10. P. tuberata 56% | 5.7% Ti08% 3.7% 10.0% | 9.7% 10.0% 8.7% 8.3% 10.7% 9.0% n/c R. testudo 12.8% | 14.0% 11.70% | 13.0% 9.3% 12.3% 11.3% n/c dontohyla sp. aff. brevipes [Ca FJ559294] by Rosa et al. (2011, 2012) and Plethodontohyla sp. Ca03 Betampona by Scherz et al. (2016a). Blommers-Schloésser (1975) re- ferred to a male specimen from ‘near Tampoketsa d’ An- kazobe’ (approx. 18°19’05.5”S, 047°06°42.8”E , Fig. 1) as P. laevis. This locality from the central highlands is indeed closer to the distribution range of P. /aevis than to P. alluaudi (as refined here), but we consider this re- cord uncertain until the specimen (ZMA 6688) has been examined. Resurrection of Plethodontohyla laevis. As we have shown above, several osteological and morphological characters exist to distinguish the holotypes of Dyscophus alluaudi and Phrynocara laeve. Osteological characters suggest that both taxa are members of the genus Pletho- dontohyla. Specimens recently collected in Betampona Special Reserve closely match the morphology, osteolo- gy and appearance of the holotype of Phrynocara laeve. A village named Sakana (in the Vavatenina commune, Toamasina Province) was located by using Google Earth roughly 70 km North of Betampona (17°18’00.00"S, 049°01°59.99”E), and we suspect that this may have been the forest to which Boettger was referring in the origi- nal description (Boettger 1913). The newly collected material in Betampona, Marovato, Anivorano Est and Analalava-Foulpointe is genetically and morphologically relatively uniform and it is distinct both from Plethodon- tohyla alluaudi (in its new definition), and from all other nominal Plethodontohyla species. We therefore resurrect Plethodontohyla laevis (Boettger, 1913) from the synon- ymy of P. alluaudi and we provide a re-description of it based on the re-examination of its holotype (including its osteology via micro-CT scanning), and examination of additional, newly collected material. Holotype. SMF 4286, an adult female collected by A. Voeltzkow in 1905 in Sakana, East Madagascar. Referred material. Adult male (ethanol-fixed, DNA sequenced and included in Rosa et al. 2012: Accession number HM364769) MRSN A6340 (FAZC_ 13902), collected by G.M. Rosa and J. Noél on 18 November 2007 at Betampona Nature Reserve, campsite Main- timbato (17°53’35.5”S, 049°13°41.3"E, 283 m a.s.l.), Toamasina Province, eastern Madagascar. MRSN A6189 (FAZC 13643), adult female (ethanol-fixed and DNA sequenced), collected by G.M. Rosa and J. Noél on 21 February 2007 at Betampona Reserve campsite Main- timbato (17°53’36.9"S, 049°13737.2”E, 295 m a.s.l.); MRSN A6181 (FAZC 13494), adult female (etha- nol-fixed and DNA sequenced), collected by G.M. Rosa and J. Noél on 4 February 2007 at Betampona Reserve, Piste Principal (17°55’40.5”S, 049°12’07.4"E, 355 m a.s.l.); ZSM 980/2013 (ACZCV 0066), adult male (eth- anol-fixed and DNA sequenced), collected by A. Crot- tint, D. Salvi, E. Scanarini and J.H. Velo on the morn- ing of 9 November 2013 at Betampona Nature Reserve, campsite Sahaindrana (17°53’55.50”’S, 049°12’02.4”E, 327 m as.l.); UADBA uncatalogued (ACZCV 0268), adult unsexed (ethanol-fixed and DNA sequenced), col- lected by A. Crottin1, D. Salvi, E. Scanarini, F. Andre- one, S. Faravelli, J. Noél and Georges on the evening of 20 November 2013 at Betampona Nature Reserve, campsite Sahabefoza (17°54’54.82”S, 049°12’32.31”E, 349 m a.s.l.); MRSN A6674 (PBZT-RJS 2020), adult female (ethanol-fixed and DNA sequenced), collected by J.E. Randrianirina on 17 October 2008 at Marovato (18°41°09.60”S, 048°36719.80”E, 692 m a.s.1.); MRSN A6787 (PBZT-RJS 1830), adult male (ethanol-fixed and DNA sequenced), collected by J.E. Randrianirina on 12 zse.pensoft.net 124 Bellati, A. et al.: Taxonomy and re-descriptions of Plethodontohyla laevis and P. alluaudi maxilla angspl Figure 7. The skeleton of Plethodontohyla laevis (SMF 4286) rendered via micro-CT scanning. (a—b) Full skeleton in (a) dorsal and (b) ventral view. (c—e) Skull in (c) dorsal, (d) ventral and (e) lateral view. Abbreviations: angspl, angulosplenial; col, columel- la; exoc, exoccipital; exoc.oc, occipital condyle of exoccipital; fpar, frontoparietal; fpardop, frontoparietal dorsal process; max, maxilla; max.pf, pars facialis of maxilla; max.parspal, pars palatina of maxilla; mmk, mentomeckelian; npl, neopalatine; pmax, premaxilla; povom, postchoanal vomer; proot, prootic; prsph, parasphenoid; prsph.al, alary process of parasphenoid; prvom, pre- choanal vomer; pter, pterygoid; pter.mr, medial ramus of pterygoid; pter.vr, ventral ramus of pterygoid; qj, quadratojugal; qj.pvp, posteroventral process of quadratojugal; smax, septomaxilla; sq.or, otic ramus of squamosal; sq.vr, ventral ramus of squamosal; sq.zr, zygotic ramus of squamosal; spheth, sphenethmoid. For a 3D rotational model, see Suppl. material 2. October 2008 at Anivorano Est, village d’ Ambalatenina, Forét d’?Andrarihitra (18°45’56.94”S, 048°57°07.20”E, 270 ma.s.l.); ZSM 189/2016 (FGZC 5239), juvenile (eth- anol-fixed and DNA sequenced), collected by F. Glaw, D. Protzel and L. Randriamanana, on Ist January 2016 near Analalava-Foulpointe (17°42’25.39”S, 049°27°35.59”E, ca. 30 ma.s.l.). Diagnosis (see also Tables 1, 5 and Figs 2—3, 6-8). A microhylid belonging to the subfamily Cophylinae, with connected lateral metatarsalia, short hindlimbs, tibiotar- sal articulation not exceeding the nostril, inner metatarsal tubercle present, vomerine teeth present, clavicle absent, knob-shaped terminal phalanges, and males with a sin- gle subgular vocal sac; therefore attributed to the genus Plethodontohyla (see Appendix A of Scherz et al. 2016a). The attribution to the genus Plethodontohyla 1s also sup- ported by molecular phylogenetic evidence from newly collected material (see Fig. 5). The species is character- ised by the following suite of characters: (1) moderately large size (male SVL 33.0-40.3 mm; female SVL 36.8— zse.pensoft.net 42.2 mm); (2) HW/HL 1.41—1.79; (3) FORL/SVL 0.50— 0.57; (4) HIL/SVL 1.24-1.58; (5) TIBL/SVL 0.36-0.40; (6) rounded snout tip; (7) toe tips not enlarged; (8) finger tips not enlarged; (9) knob-shaped terminal phalanges of the fingers and toes; (10) smooth dorsal skin; (11) absence of a distinct dorsolateral colour border; (12) presence of a supratympanic dermal fold; (13) presence of a typical- ly bold and generally white-bordered brown ‘X’ marking on head; (14) tibiotarsal articulation reaching at least the tympanum and (15) TD/ED 0.33-0.52. Furthermore, the species 1s separated from all nominal taxa in this genus by an uncorrected pairwise distance of at least 5.5% in the sequenced 16S fragment (comparison with P. alluaudi in its new definition and P. sp. Ca01). Plethodontohyla laevis may be distinguished from other members of the genus Plethodontohyla as follows: from P. inguinalis, P. notosticta, P. guentheri, P. mihani- ka and P. fonetana by non-expanded terminal digits (vs. moderately to strongly expanded) and by its knob-shaped terminal phalanges of the fingers and toes (vs. T- or Y-shaped) and from all these species except P. fonetana Zoosyst. Evol. 94 (1) 2018, 109-136 by the absence of a dorsolateral colour border (present in all of these species but only some specimens of P. in- guinalis). It also differs from P. notosticta, P. guentheri and P. mihanika by having a rounded snout tip (vs. gen- erally pointed); from P. ocellata, P. bipunctata, P. bre- vipes, P. inguinalis and P. tuberata by smooth skin (vs. granular or tubercular); from P. inguinalis, P. notosticta, P. guentheri and P. mihanika by the presence of a supra- tympanic fold running from the posterior border of the eye backward until the forelimb (vs. absence); from all species of Plethodontohyla except P. alluaudi and P. sp. Ca01 by the presence of a bold, mostly white-bordered *X’ marking (see Fig. 3 for its variation) on the head (vs. absence); from P. tuberata, P. bipunctata, P. brevipes and P. mihanika by a tibiotarsal articulation reaching at least the tympanum (vs. reaching the insertion of the arms or going beyond the tip of snout in P. mihanika), and from P. ocellata, P. bipunctata, P. fonetana and most individuals of P. brevipes by lacking two symmetrical and concave thin dorsal folds (vs. presence). Plethodontohyla alluaudi (as newly circumscribed) and P. sp. Ca01 are morphologically the most similar spe- cies to P. laevis (see also Figs 3-4). For distinction from P. alluaudi, see the re-description of that species, below. Plethodontohyla laevis differs from P. sp. Ca01 by larger body size (SVL 33.0—42.2 vs. 27.7—31.9 mm) and smaller tympanum (TD/ED 0.33-0.52 vs. 0.55—0.63). Plethodontohyla laevis also resembles Rhombophryne botabota, R. laevipes, R. mangabensis and R. savaka in external morphology and in some aspects of its colour- ation. It may be distinguished from all species by the presence of a bold, mostly white-bordered *X’ marking on the head (vs. absence), but additionally it may be dis- tinguished from all four of these species by the absence of clavicles; from R. /aevipes by its smaller size (SVL 36.8-42.2 mm vs. 44.5-56.3 mm), much shorter leg length (HIL/SVL 1.24—1.58 vs. 1.75—1.86) and absence of white ocelli in the inguinal region (vs. presence); from R. laevipes and R. mangabensis by its smaller tympanum (TD/ED 0.33-0.52 vs. 0.57—0.73); from R. botabota, R. mangabensis and R. savaka by its larger size (SVL 36.8— 42.2 mm vs. 20.4—32.2 mm); from R. savaka by its slight- ly narrower head (HW/HL 1.41-1.79 vs. 1.80), longer relative forelimb length (FORL/SVL 0.50-0.57 vs. 0.43), raised supratympanic fold (vs. not raised) and absence of a diastema in the vomerine teeth (vs. presence); and from R. botabota and R. mangabensis by generally shorter rel- ative tibia length (TIBL/SVL 0.36—40 vs. 0.38—0.45). Re-description of the holotype (SMF 4286). Speci- men in relatively good state of preservation (Fig. 2a, b). Right forelimb fractured (Fig. 7a, b). Ventrally slit down the midline of the whole body. SVL 38.1 mm (for other measurements, see Table 1). Body moderately enlarged and flattened dorsoventrally; head much wider than long and almost as wide as body; snout rounded in dorsal and lateral view; nostrils directed laterally, slightly protuber- ant, nearer to tip of snout than to eye; canthus rostralis ZS: distinct, concave; loreal region concave; tympanum in- distinct, rounded, roughly 40% of eye diameter; supra- tympanic fold from eye to shoulder distinct and straight; tongue ovoid, very broad, posteriorly free and slightly notched; maxillary teeth present; vomerine teeth distinct, forming oblique transverse rows posterior to choanae, laterally approaching the maxillae and medially almost in contralateral contact; choanae ovoid. Arms robust, fingers bearing marked single subarticular tubercles and hands bearing indistinct paired outer metacarpal tuber- cles; large, slightly protruding inner metacarpal tuber- cle; fingers without webbing; relative length of fingers 1<2=4<3, fourth finger roughly equal in length to second; finger disks not enlarged; nuptial pads absent. Hindlimbs robust; tibiotarsal articulation reaching the tympanum when hindlimb adpressed along body; tibia length 38.8% of SVL; lateral metatarsalia connected; distinct inner and less distinct outer metatarsal tubercles present; only traces of webbing between toes; relative length of toes 1<2<5<3<4; third toe distinctly longer than fifth. Skin on dorsum and venter smooth; supratympanic fold whitish. Colour of iris indistinguishable. Colouration. After more than a century in preservative (holotype collected in September 1904) colouration is fad- ed. Dorsum light brown with darker brown-black spots, markings and presence of a bold X-shaped marking bor- dered with a white line on the head behind the eye. Col- ouration of the proximal dorsal portion of the hindlimbs mottled with dark brown markings on a cream background colour; the same colouration extends into the inguinal re- gion. Colouration of the distal dorsal portion of the hind- limbs light brown with faint brown crossbands. Sides of head and tympanic region brownish, with darkener flecks. Ventral skin markedly pigmented: throat and chest mot- tled brown and cream, abdomen and ventral legs cream, becoming increasingly mottled with faint brown distally. The colouration in life of this specimen 1s not known. Osteology. In the following, we describe notable and 1m- portant diagnostic characters of Plethodontohyla laevis based on SMF 4286 (Figs 2, 6-7) and the newly collect- ed specimen MRSN A6340 (Fig. 8). PDF-embedded 3D models of these skeletons are provided as Suppl. materi- als 2 and 5. Right humerus fractured in SMF 4286. Skeleton of SMF 4286 relatively poorly ossified, such that the carpals, knee and heel joints and pubis are not visible in the mi- cro-CT scans. The skeleton of MRSN A6340 is compar- atively well ossified. Vomerine teeth anteriorly convex, long, occupying the whole postchoanal vomer, separated medially by a small gap. Palatine processes of premaxilla subequal in width and length. Prechoanal vomer flat and triradiate, the lateral ramus closer to the anterior end. Pre- maxilla and maxilla bearing teeth. Nasals large, broad, not in contact with other bones. Sphenethmoid only lat- erally ossified. Extensive calcification present inside the braincase of MRSN A6340. Posterior ramus of pterygoid zse.pensoft.net 126 Bellati, A. et al.: Taxonomy and re-descriptions of Plethodontohyla laevis and P. alluaudi Frequency (kHz) 0 200 400 600 800 1000 Relative amplitude oO 200 400 600 800 Time (ms) 1000 Figure 8. Audiospectrogram and corresponding oscillogram of the advertisement call of Plethodontohyla laevis recorded at Betampona Strict Nature Reserve on 18 November 2007 (22:30 h, 21 °C). Calling male MRSN A6340 (top). extremely long and broad. Transverse ridge across poste- rior of frontoparietals. Weakly descending lateral flange of frontoparietal. Clavicle absent. Coracoids with weak notches for the attachment of the procoracoid cartilage. Cleithrum broad. Terminal phalanges of fingers knobbed. Finger phalangeal formula 2-2-3-3. Neural spines present on presacrals 2 and 3. Sacrum thin. Iliosacral articulation type ITA/B sensu Emerson (1979). Urostyle bearing a strong, straight dorsal ridge for almost its entire length; articulation bicondylar. Iliac shafts bearing weak dorsal crests; possessing a shallow oblique groove and lacking a dorsal tubercle. Leg bones lacking crests. Toe phalangeal formula 2-2-3-4-3. Colouration in life based on recently collected material. The colouration tn life is remarkably variable. Dorsal col- ouration is a range of brown tones, while ventral coloura- tion contains elements of grey, black and beige. The most consistent element is a more or less bold, X-shaped mark- ing on the back of the head, with the anterior arms of the X reaching onto the posterior of each eye, and the posterior arms reaching posteriorly to either join the dorsal pattern (typically fading) or remaining clearly distinct in the su- prascapular region. This distinctive marking is present in the dorsal colouration also in juveniles (B. Ferrier, per- sonal communication) and is clear in most adults, though it can sometimes be faded and not clearly distinct from the rest of the dorsal colouration. Posterior to this ‘Xx’, the zse.pensoft.net dorsum is typically dark brown mottled with black, with generally clearly defined, subsymmetrical light brown markings distributed over the dorsum. In some specimens there are also black spots on the flanks. The lateral head is typically light brown (the colour of the dorsal markings), the supratympanic fold somewhat lighter and the tympa- num containing a dark spot. In the inguinal region, black spots and light brown spots merge to form lines, but do not form a clearly defined, single black spot as in e.g. P. allu- audi. The dorsal thigh is mottled black with light brown, semi-regular spots, or with moderately defined dark and light crossbands, whereas the dorsal shank is a murky brown with irregular light brown spots or light brown with dark crossbands. The dorsal forelimb is as the shank. The ventral body is beige in base colour, with irregular dark brown mottling over the chin, which can be totally black in males, becoming less regular posteriorly over the abdomen and being free of maculations over the hip. The ventral shank is mostly beige, sometimes with dark spots. A light brown annulus is present before the tip of each finger. Variation. Morphometric variation is given in Table 1. No remarkable variation in general morphology exists between the holotype and the newly collected material, except that the second finger is shorter than the fourth in newly collected specimens. Colouration variation has been discussed in the above section, and is merely more vivid and distinct in the new material than the holotype. Females lack the single subgular vocal sac that can be highly extensible (Fig. 8). MRSN A6189 and MRSN A6181 are slightly less pigmented than MRSN A6340 and the colouration of the proximal dorsal portion of the hindlimbs (mottled with black markings on a cream back- ground colour) extends forward to lateral midbody and backwards to the tibiotarsal articulation on the ventral side of the hindlimb. Natural history. Little information is available on this species. The eight new specimens were found active on the ground during the day or during the night, and this species was also found moderately active in dry condi- tions (after several days of no rainfall). In November 2013 one individual (not collected) was encountered along a drift fence during a rainy night. ZSM 980/2013 was encountered hidden in the leaf litter at the base of a Ravenala madagascariensis and ZSM 189/2016 was ac- tive in the leaf litter during the day. No information on the reproductive biology of this species is currently known. In 2007, a group of males were heard calling during rain- fall. Individuals were calling from within the leaf litter or at the opening of a burrow, into which they can easily dis- appear upon detection. The holotype has a nearly intact millipede in its stomach (see Fig. 7). This is the only diet record currently available for this species. Distribution and conservation status. Plethodontohyla laevis is known from (1) the type locality Sakana (whence no recent records for the species are known), (2) Betam- Zoosyst. Evol. 94 (1) 2018, 109-136 pona Natural Reserve, (3) Marovato, (4) Anivorano Est, (5) Analalava-Foulpointe and (6) the Réserve Privée (RP) d’Ambodiriana (only photographic records are available from this locality) (Figs 1-3). All these sites are distrib- uted at low altitudes in the central- to North-East of Mad- agascar. Altitudinal distributional range reaches from sea level to ca. 700 m a.s.l.. Due to the severe habitat degra- dation of the lowland rainforest in the northeast of Mad- agascar it is likely that the species 1s locally extinct at its type locality. Surveys in Zahamena Natural Reserve so far failed to report this species, but a more thorough in- vestigation of the area is required to confirm the presence or absence of this species in that area. As noted above, the record of the species from the central highland (Anka- zobe) 1s dubious and must be confirmed by further field- work in this area and/or examination of the material cited by Blommers-Schlosser (1975). The species occurs at least in two protected areas, where it seems to be a relatively abundant although it is a species with secretive habits. Nevertheless, its distribu- tion is highly fragmented, its extent of occurrence is quite limited (minimum convex polygon = 9770.92 km?) and it is threatened by on-going habitat destruction. These fac- tors (range under 20,000 km/, severely fragmented distri- bution and on-going habitat destruction) qualify P. /aevis as Vulnerable under criterion Blab(iii) of the IUCN Red List (IUCN 2012). Acoustic repertoire. Advertisement calls were recorded from a single male (MRSN A6340) at Betampona (Main- timbato: 17°53’35.50”S, 049°13’41.30”E, 283 m a.s.l.) on 18 November 2007, at 22:30 h at an air temperature of 21 °C (Rosa et al. 2011, track #50) (Fig. 8, Table 3). Each call consisted of a single loud note repeated after long, regular intervals, starting as unharmonious sound, but be- ing tonal for most of its duration. They were slightly fre- quency-modulated in their tonal part and lasted 391-422 ms (407 + 12.7, n=4). We recorded one inter-call interval of 47 s. The fundamental frequency of the tonal part was 0.89-1.40 kHz with a dominant frequency band (frequen- cy containing the greatest sound energy) between 1.82— 2.53 kHz. Up to 10 harmonics were visible on the spec- trogram and no attenuation of any of the harmonics was observed. 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. Plethodontohyla alluaudi (Mocquard, 1901) Figs 2c, d, 4a, 6, 9, Suppl. materials 1, 3, 6 Remarks. Sequences of this species have been referred to as Plethodontohyla bipunctata Andohahela by Wol- lenberg et al. (2008), Vieites et al. (2009), Perl et al. Ia (2014) and Scherz et al. (2016a). Blommers-Schloss- er (1975) referred to a specimen from Ampasinambo (20°31°25.0”S, 048°01713.7”E) as P. brevipes, but later corrected this to P. alluaudi (Blommers-Schlosser and Blanc 1991). This locality is between the distributions of P. alluaudi (as refined here) and P. /aevis, and we there- fore consider this record uncertain until the specimen (ZMA 6689) has been re-examined. Identity and redefinition. The original description of Dyscophus alluaudi is based on a single specimen of 47.4 mm SVL from the generic locality ‘Fort Dauphin’. After the examination and comparison of the type material with recently collected material in south-eastern Madagascar close to the type locality of Dyscophus alluaudi, we here reassign this species to the genus Plethodontohyla. We therefore re-describe and redefine Plethodontohyla allu- audi based on the holotype (including its osteology via micro-CT scanning), on the holotype of P. /aevis tsiano- vohensis and the recently collected material from Ando- hahela, Tsitongambarika and Sainte Luce. Holotype. MNHN 1901.235, an adult female collected by M. Alluaud in ‘Fort Dauphin’. Referred material. MNHN 1936.0047, holotype of P. laevis tsianovohensis, an adult female collected by R. Heim between 1934 and 1935 in Tsianovoha, East Mad- agascar. ZSM 89/2004 (FGZC 161), an unsexed adult individual (DNA sequenced and included in Wollenberg et al. 2008: Accession number EU341068), collected by F. Glaw, M. Puente, M. Thomas and R. Randriani- aina on 31 January 2004 at Andohahela, (between Isa- ka-Ivondro and Eminiminy; 24°45’00”S, 046°51’00”E, ca. 230 m a.s.l.), Toliara Province, south-eastern Mad- agascar; UADBA-A 27994 (FGZC 160), an unsexed adult individual, collected by F. Glaw, M. Puente, M. Thomas and R. Randrianiaina on 31 January 2004 at Andohahela (between Isaka-Ivondro and Eminiminy; 24°45°00”S, 046°51’°00”E, ca. 230 m as.l.), Toliara Province, south-eastern Madagascar, MRSN_ uncata- logued (FAZC 15423), unsexed adult individual (etha- nol-fixed and DNA sequenced), collected by F. Andreone and G.M. Rosa on 29 February 2012 at Tsitongambari- ka Forest Reserve (Anosyenne Chain; 24°33’32.10’S, 047°11°24.90”E, 32 m a.s.l.); UADBA-A 62219, an unsexed adult individual, collected by S. Megson on 22 July 2013 at Sainte Luce (24°47'12”S, 047°09’°45”E, ca. 19 m as.l.), Toliara Province, south-eastern Madagas- car, UADBA-A 62224, an unsexed juvenile individual, collected by S. Megson on 17 July 2013 at Sainte Luce (24°46°87°S, 047°10°24”E, ca. 7 ma.s.l.), Toliara Prov- ince, south-eastern Madagascar. Diagnosis (see also Tables 1, 5 and Figs 2, 4, 6, 9). A large microhylid belonging to the subfamily Co- phylinae, with connected lateral metatarsalia, short forelimbs (FORL/SVL 0.47—0.58), short hindlimbs, zse.pensoft.net 128 Bellati, A. et al.: Taxonomy and re-descriptions of Plethodontohyla laevis and P. alluaudi max.parspal rvom fj : SN i maxilla Figure 9. The skeleton of Plethodontohyla alluaudi (MNHN 1901.235) rendered via micro-CT scanning. (a—b) Full skeleton in (a) dorsal and (b) ventral view. (c—e) Skull in (c) dorsal, (d) ventral and (e) lateral view. Abbreviations are as in Fig. 7. For a 3D rotational model, see Suppl. material 1. tibiotarsal articulation reaching the insertion of the arms, inner metatarsal tubercle present, maxillary and vomerine teeth present, clavicle absent or highly re- duced, knob-shaped terminal phalanges and males with a single subgular vocal sac; therefore attributed to the genus Plethodontohyla (see Appendix A of Scherz et al. 2016a). The attribution to the genus Plethodontohyla is also supported by phylogenetic molecular evidence from newly collected material (see Fig. 5). The species is characterised by the following suite of characters: (1) moderately large size (SVL 28.8-58.0 mm); (2) HW/ HL 1.67-1.85; (3) FORL/SVL 0.47-0.58; (4) HIL/ SVL 1.25-1.47; (5) TIBL/SVL 0.34—0.39; (6) round- ed snout tip; (7) toe tips not enlarged; (8) finger tips not enlarged; (9) knob-shaped terminal phalanges of the fingers and toes; (10) smooth dorsal skin; (11) ab- sence of a distinct dorsolateral colour border; (12) pres- ence of a supratympanic fold; (13) presence of a bold zse.pensoft.net white-bordered ‘X’ marking on head; (14) tibiotarsal articulation reaching the insertion of the arm; (15) TD/ ED 0.33—0.66. Furthermore, the species is separated from all nominal taxa in this genus by an uncorrected pairwise distance of at least 5.6% (comparison with P. tuberata, genetic distance of 5.8% with P. /aevis). The genetic distance with P. sp. Ca01 is 2.9%. Plethodontohyla alluaudi may be distinguished from other members of the genus Plethodontohyla as fol- lows: from P. inguinalis, P. notosticta, P. guentheri, P. mihanika and P. fonetana by non-expanded terminal digits (vs. moderately to strongly expanded) and by its knob-shaped terminal phalanges of the fingers and toes (vs. T- or Y-shaped) and from all these species except P. fonetana by the absence of a dorsolateral colour bor- der (present in all of these species but only some speci- mens of P. inguinalis). It also differs from P. notosticta, P. guentheri and P. mihanika by having a rounded snout Zoosyst. Evol. 94 (1) 2018, 109-136 tip (vs. generally pointed); from P. ocellata, P. bipuncta- ta, P. brevipes, P. inguinalis and P. tuberata by smooth skin (vs. granular or tubercular); from P. inguinalis, P. notosticta, P. guentheri and P. mihanika by the presence of a supratympanic fold running from the posterior bor- der of the eye backward until the forelimb (vs. absence); from all species of Plethodontohyla except P. laevis and P. sp. CaO1 by the presence of a bold white-bordered *X’ marking on the head (vs. absence); from P. /aevis, P. sp. Ca0l, P. ocellata, P. inguinalis, P. notosticta, P. guen- theri, P. fonetana and P. mihanika by a tibiotarsal artic- ulation reaching the insertion of the arms (vs. see Table 5); and from P. brevipes (n = 6) by a generally wider head (HW/HL 1.67—1.85 vs. 1.53—1.71, Mann-Whitney U-test, P = 0.032), a generally smaller tympanum (TD/ ED 0.33-0.66 vs. 0.60-0.79, Mann-Whitney U-test, P = 0.025), tendency toward larger relative hand size (HAL/ SVL 0.23-0.28 vs. 0.21-0.24, Mann-Whitney U-test, P = 0.051), larger inner metatarsal tubercle (IMTL/FOL 0.13-0.17 vs. 0.09-0.13) and presence of a bold ‘X’ marking on the head (vs. absence). Plethodontohyla laevis and P. sp. Ca01 are morpho- logically the most similar species to P. alluaudi. Pletho- dontohyla alluaudi can be distinguished from P. laevis by frequent presence of inguinal spots (vs. general absence), generally larger tympanum size (TD/ED 0.33—0.66 [0.53—0.66 for three of the four examined specimens] vs. 0.33—0.52) and tendency toward larger relative hand size (HAL/SVL 0.23—0.28 vs. 0.21—0.25, Mann-Whitney U-test, P = 0.085). Re-description (based on MNHN 1901.235). Specimen in relatively good state of preservation (Figs 2, 6, 9). A cross-shaped incision made over the pectoral girdle, a lat- eral incision on the left side and a number of incisions on the lower back. A strong transverse fold is present at the posterior head, certainly a fixation artefact. SVL 47.4 mm (for other measurements, see Table 1). Body large and robust; head much wider than long (HW/HL 1.85) and almost as wide as body; snout rounded in dorsal and lateral view; nostrils directed laterally, slightly protu- berant, nearer to tip of snout than to eye; canthus ros- tralis distinct, concave; loreal region concave, oblique; tympanum slightly distinct, rounded, TD/ED 0.53; supratympanic fold from eye to shoulder distinct and curved; tongue ovoid, very broad, posteriorly free and not notched; mandible damaged at the midline so that the two halves are distinguishable externally; maxillary teeth present; vomerine teeth distinct, forming oblique curved rows posterior to choanae, laterally approaching the max- illae and medially almost in contralateral contact; cho- anae ovoid. Arms robust, fingers bearing marked single subarticular tubercles and hands bearing indistinct outer metacarpal tubercles; large, protruding inner metacarpal tubercle; fingers without webbing; relative length of fin- gers 1<2<4<3, fourth finger slightly longer than second; finger disks not enlarged; nuptial pads absent. Hindlimbs robust; tibiotarsal articulation reaching the insertion of 1S the arm when hindlimb adpressed along body; TIBL/ SVL 0.34; lateral metatarsalia connected; distinct inner and less distinct outer metatarsal tubercles present; only traces of webbing between toes; relative length of toes 1<2<5<3<4; third toe distinctly longer than fifth. Skin on dorsum smooth; supratympanic fold whitish. Colour of iris indistinguishable. Colouration. After over a century in preservative, the colouration is strongly faded, and several details of the pattern originally illustrated by Mocquard (1901) are only barely distinguishable. The base colouration of the whole specimen is cream-brown. Large dark brown markings in the inguinal region remain, as do oblique dark brown bars on the anterior thigh and faint brown spots on the posterior thigh. A faint trace of the large X-shaped mark- ing on the posterior head is present only as the outlines of this shape. The ventral skin is translucent cream, and muscles are visible through it. No other traces of colour- ation remain. The colouration in life of this specimen 1s not known. Osteology. In the following, we describe notable and im- portant diagnostic characters of Plethodontohyla alluaudi based on MNHN 1901.235, MNHN 1936.47 and ZSM 89/2004 (Fig. 6). PDF-embedded 3D models of these skeletons are provided as Suppl. materials 1, 3 and 6. Ossification 1s variable, but lowest in ZSM 89/2004 where the knees and carpals are not visible. Vomerine teeth anteriorly convex, with a distinct angle in MNHN 1936.47 not present in the other two specimens, cover- ing the whole postchoanal vomer, separated medially by a small gap. Palatine processes of premaxilla sube- qual in length, the medial process thinner than the lat- eral process. Prechoanal vomer flat and triradiate, the lateral ramus around its midpoint, but weak or missing in MNHN 1936.47. Nasals large and broad, not 1n con- tact with other bones. Sphenethmoid laterally closed, brain case of ZSM 89/2004 and MNHN1901.235 with some internal mineralisation. Posterior ramus of pterygoid extremely long and broad. Strong transverse ridge across posterior of frontoparietals most raised at its lateral extremities, strongly descending lateral flange of frontoparietal. The right coracoid of MNHN 1901.235 is fractured mid-way along its length. Clav- icles are present only in MNHN 1901.235, where they are reduced to thin slivers. The coracoid possesses a strong notch for the attachment of the procoracoid car- tilage. Cleithrum broad. Terminal phalanges of fingers knobbed. Finger phalangeal formula 2-2-3-3. Neural spines present on presacrals 2 and 3. Sacrum relative- ly thin, broadening laterally. Iliosacral articulation type ITA/B sensu Emerson (1979). Urostyle bearing a strong, straight dorsal ridge for almost its entire length; articulation bicondylar. Iliac shafts bearing weak dor- sal crests; possessing a shallow oblique groove and lacking a dorsal tubercle. Leg bones lacking crests. Toe phalangeal formula 2-2-3-4-3. zse.pensoft.net 130 Bellati, A. et al.: Taxonomy and re-descriptions of Plethodontohyla laevis and P. alluaudi Table 5. Morphological variation in the analysed specimens of Plethodontohyla spp. Abbreviations not identified in the text: TT, Toe Tips (1, not enlarged, 2, enlarged); FT, Finger Tips (1, not enlarged, 2, enlarged); TP, Terminal Phalanges (K, knob-shaped, T, T-shaped, Y, Y-shaped); DDL, Dorsolateral Line, a narrow white dorsolateral line delimiting a sharp difference between the dorsal colouration and the uniformly dark flanks, extending from the tip of the snout backward until the inguinal region (+, presence, - absence, (+), not always present); Sk, Skin (1, smooth, 2, granular, 3, with tubercles); RID, Ridge, supratympanic dermal fold (+, presence, - absence); X, bold ‘X’ marking on the head between the eye bordered by a thin while line (+, presence, - absence); DDF, Dorsal Dermal Folds, two symmetrical and concave thin dorsal folds (+, presence, - absence); TTA, Tibiotarsal Articulation (1, reaching the tympanum, 2, reaching the insertion of the arms, 3, reaching the eye, 4, extending beyond the eye, * at least); ST, Snout tip (1, rounded, 2, pointed). ID code Genus | __ Species | ___—Locality_—_‘[Sex/ Status /TT| FT | TP | DLL Sk | RID] X | DDF | TTA|ST SMF4286 _[Phiynocara__iaeve «| _—sSakana—| F | HT |i lil K|-|1|+|+| - [nal i MRSN A6189__|Plethodontohyla ra MRSN A6181 Plethodontohyla_ |laevis Betampona F fy ES | VK 1 | + il MRSN A6340 Plethodontohyla |laevis Betampona M Md Ae els 1 | + 1 ZSM 980/2013_ |Plethodontohyla_ |laevis Betampona M iad Me 8 1 | + ah MRSN A6674 Plethodontohyla |laevis Marovato F Ly ek kK 1 | + 1 MRSN A6787 Plethodontohyla |laevis Anivorano Est M oe Neel | Le 1 | o+ lk MNHN 1901.235 | Dyscophus alluaudi Fort Dauphin F LA al 1 + 1 MNHN 1936.47 | Plethodontohyla a Fane Tsianovoha F 1]/1/k 1 1 75M 69/2004 __[Plethodontohyla lalluauci_ | __Andohahela_|-| - |ili/k|-|1i]|+| i FAZC 15423 Plethodontohyla_ |alluaudi Ape c ar = | Sa |jeiie (peat (Pa (Pee 1 FEEGG™ [rst ow | aanae [== [a [a ea [|= [onl ZCMV 555 Plethodontohyla_|sp. Ca01 Ambatolahy | - | - [1] 1 [n/al - [n/a] + 1 ZSM 855/2006_ =| Plethodontohyla_|sp. CaO1 Imaloka ile] | Am) he 1 | + ik ZSM 856/2006_ =‘|Plethodontohyla |sp. CaO1 Imaloka el ek 1 | + i MRSN A3221 Plethodontohyla_ |tuberata Manjakatompo 4 | hls. | Mi 3 / + 1 MRSN A2859 Plethodontohyla_ |ocellata Ranomafana ileal Pele nee 2 | + 1 ACZCV 0101 Plethodontohyla_ |ocellata Betampona SS SS eT eae eae ees Te | al ZSM 5204/2005 [Plethodontohyla [bipunctata | Andohahela__|-| - |i|1/k|-|2]|+| l ZSM 854/2006__|Plethodontohyla |brevipes «| ——s Imaloka «=| - | - [itil k | - | 244 1 MRSN A2476 Plethodontohyla_ |inguinalis Kalambatritra - - Z|) 42h Wiel: 2 1 MRSN A5653 Plethodontohyla_ |notosticta Sahavontsira - 2.| 2° \ 1 1 2 ZSM 61/2005 Plethodontohyla_ | guentheri Marojejy F FAN |e | 225 |) CT 1 Zs ZSM 123/2006_ |Plethodontohyla |fonetana Tsingy de Bemaraha] F a ee | ee 1 il ZSM 1087/2001 [Plethodontohyla 7 Colouration in life based on recently collected material. Dorsally a greenish brown colouration, with a distinct dark brown ‘*X’ marking on the posterior head, with the anterior arms of the X over the eyes, and the posterior arms reaching the suprascapular region, bordered with a fine, light brown line. The dorsal colouration is flecked with white, with es- pecially large white spots over the ends of the iliac shafts. A large blackish inguinal spot bordered with a white line is present. The flank colouration is marbled brown with white spots. The supratympanic fold is whitish, and forms a weak colour border between the dorsal colouration and the rich- er brown lateral head. The arm is anteriorly darker brown, almost blackish. The dorsal thigh is also darker brown, but not blackish. The ventral colouration is translucent and thus peach over the chin and pinkish over the abdomen, invaded on the sides behind the pectoral girdle by brown flecks, but not meeting medially. The ventral thighs are also pinkish, anteriorly and posteriorly with brown flecks. A yel- low-cream annulus is present before the tip of each finger. zse.pensoft.net Variation. Morphometric variation is given in Table 1. The holotype and MNHN 1936.47 are considerably larger than the newly collected material. MNHN 1936.47 apparently lacks inguinal spots, but its colouration is faded to the point where these might have disappeared. UADBA-A 27994 has a considerably smaller tympanum diameter than the other specimens (TD/ED 0.34 vs. 0.53—0.66). The holotype has the shortest forelimbs (FORL/SVL 0.47 vs. 0.53-0.58) and hindlimbs (HIL/SVL 1.25 vs. 1.41—1.47). Natural history. At Andohahela specimens were found in the leaf litter of rainforest during the day. In Anosyenne Chain (Tsitongambarika) the specimen was found at night under leaf litter not far from the edge of the forest patch. Males from the Sainte Luce population have been heard calling in large choruses from hidden positions after heavy rainfall during the day and night, both inside the forest and in more open areas. During such periods choruses consist of many dozens of individuals. Individuals are extremely hard to detect and cease calling if they notice any distur- Zoosyst. Evol. 94 (1) 2018, 109-136 bance, retreating into their burrows and hiding places in the forest floor. Individuals may be seen travelling above ground on rainy nights, particularly in areas in close prox- imity to small water bodies such as shallow forest streams, the margins of swamps and even in ephemeral mud pud- dies. At Sainte Luce, one adult specimen was found during the day, in light rainfall, underneath a log in littoral forest; and a juvenile specimen was found during the day in dry weather in severely degraded habitat, inside the shell of a deceased large land snail. The body of this individual became bloated during initial handling. In mature forest, adult individuals have also been observed beneath dead Pandanus \eaves, under dead fallen trees and dead logs. No feeding or reproductive behaviour has been observed. Distribution and conservation status. Plethodontohyla alluaudi is known from (1) the type locality “Fort Dau- phin’ (or Tolagnaro, whence there are no recent records for the species, although this collection site was probably a very generic one), (2) Andohahela National Park, (3) Tsitongambarika Forest Reserve, (4) Sainte Luce and (5) Tsianovoha. Observations in Sainte Luce have been made in two of the largest forest fragments (fragments S7 and S9). All these sites are distributed at low altitudes in the East or south-eastern of Madagascar. Altitudinal distribu- tional range extends from sea level to ca. 230 mas.L. It is not clear where the type locality of this species is, but if it was a forest in the vicinity of Tolagnaro, then it is quite possible that it has been extirpated due to forest destruc- tion there. Surveys in nearby Nahampoana and Mandena forests have so far failed to report this species, but a more thorough investigation of the area is required to confirm the presence or absence of this species in that area. The species occurs at least in three protected areas, where it seems to be a relatively abundant although it has very secretive habits. Nevertheless, its distribution is highly fragmented, its extent of occurrence is quite limited (minimum convex polygon = 5372.81 km’) and it is threatened by on-going habitat destruction. As for P. laevis, it therefore qualifies as Vulnerable under IUCN Red List criterion Blab(iti) (UCN 2012). Acoustic repertoire. Advertisement calls were recorded from a chorus of males at Sainte Luce (24°46’51.72”S, 047°10°13.14”"E; 10 m as.l.) on 30 June 2015, around 15:00 h at an air temperature of 24 °C (Table 3). This is a preliminary acoustic description due to the low quality of the available recordings (background noise and overlap- ping of several calls), which has compromised the obtain- ment of some parameters (Table 3). The following param- eters could be assessed: the call consisted of a single note (soft whistle) repeated after apparently regular intervals. Calls lasted 320-560 ms (478 + 109, n=4). The dominant frequency seems to range from 1.4 to 2.1 kHz, however these values should be interpreted with caution since the distance from the calling individuals might complicate the distinction of harmonics. A more accurate bioacoustic analysis will be needed when new data are available. ibe a Taxonomic challenges in cophyline taxonomy The resurrection of Plethodontohyla laevis and transfer of Dyscophus alluaudi from Rhombophryne to the genus Plethodontohyla brings this genus to 11 nominal species (not including the dubious P. angulifera Werner, 1903), and Rhombophryne down to 18. At present, only two other candidate species are known from Plethodontohyla (one from Tsaratanana and P. sp. Ca01 from Ambatolahy and Imaloka, which might be conspecific with P. alluau- di), but preliminary results suggest that the undescribed diversity in this genus 1s still widely unexplored and it will probably wind up being as great as it was for Rhom- bophryne (Vieites et al. 2009, Perl et al. 2014), with at least four additional undescribed species still awaiting formal description (A. Crottini et al. unpublished data). With this much-needed clarification of these historical names, we are now finally able to make larger progress on the taxonomy of this genus. The cophyline microhylids are a case study of the need for an integrative taxonomic approach (Dayrat 2005). Taxonomic action like the synonymisation of Pletho- dontohyla laevis with P. alluaudi was made on the ba- sis of external morphological differences and the state of the pectoral girdle, but could not take into account oth- er aspects of skeletal morphology, nor could it account for genetics, as it was done before micro-CT and genet- ic methods were widely available, and based on single individuals (Blommers-Schlosser and Blanc 1991). Our approach, combining external morphology and osteology without damaging the type specimens of old and recently collected material, and the availability of genetic samples from several populations in Madagascar largely resem- bling the holotypes of P. /aevis and P. alluaudi, provides a more robust hypothesis on the identities of these species than has been possible in the past. Cophyline microhylids are still the least understood amphibians of Madagascar and the recent major advanc- es in cophyline taxonomy would not have been possible without the collection of new material. However, more extensive and widespread collection of specimens from across Madagascar is still needed to fully characterize species distribution ranges and clarify their systematics. At least two new genera are still in need of description (Scherz et al. 2016a), basal relationships among the dif- ferent genera are still poorly resolved, and even at the intra-generic level there are still several unresolved re- lationships (Scherz et al. 2016a). The intra-genus rela- tionships in Plethodontohyla are no exception to this. Although the monophyly of the genus is now relatively well established (Andreone et al. 2005, Wollenberg et al. 2008, Scherz et al. 2016a), one study (Pyron and Wiens 2011) has found them to be polyphyletic, with one poorly supported group (containing P. inguinalis, P. tuberata, P. bipunctata, P. brevipes and P. ocellata) found to be the sister clade of all cophylines but Anodonthyla, and the other group (although with no support) composed of P. mihanika, P. fonetana, P. guentheri and P. notosticta, falling sister to the genus Cophyla. The former group zse.pensoft.net PZ Bellati, A. et al.: Taxonomy and re-descriptions of Plethodontohyla laevis and P. alluaudi would have the name Mantipus Peters, 1883 available for it, while the latter would retain the name Plethodonto- hyla Boulenger, 1882. On the other hand, if the species with T- or Y-shaped terminal phalanges would result in a monophyletic group (i.e. if P. inguinalis were to move to the group containing P. notosticta, P. fonetana, P. guentheri and P. mihanika), the oldest available name for the terrestrial species with knobbed phalanges would be Phrynocara. The morphology of the genus combined with the latest available multi-gene phylogeny (Scherz et al. 2016a) suggests however that this group is an eclectic but monophyletic radiation, consisting of several species groups. However, due to the variable external morpholo- gy, ecological plasticity, conflicting phylogenetic studies and the availability of many old names and synonyms, an in depth phylogenetic analysis that will assess the species phylogenetic relationships and provide a taxonomic revi- sion of the genus is needed. Acknowledgments We are grateful to the Malagasy authorities, in particular the Ministere de Il’ Environnement et des Foréts, for issuing research and export permits. We extend our thanks to Ka- ren Freeman and Ingrid Porton of Madagascar Fauna and Flora Group. Fieldwork was financially supported by the Saint Louis Zoo’s Field Research for Conservation pro- gram (FRC# 12-12) of the Wildcare Institute and Gond- wana Conservation and Research. We thank Chantal Mis- andeau and Lauric Reynes for sharing their photographs of the amphibians of Ambodiriana. The Institute for the Conservation of Tropical Environments (ICTE-MICET) provided crucial logistic support. We are grateful to the teams of the Muséum National d’Histoire Naturelle de Paris (France), the Naturmuseum Senckenberg in Frank- furt am Main (Germany) and the Mention Zoologie et Biodiversité Animale, Université d’ Antananarivo (Mad- agascar) for the loan of material pertaining to this paper. We thank the reviewers for their useful comments that significantly improved the manuscript. We are indebted to Miguel Vences for having provided three unpublished POMC sequences used in this work. This work was fun- ded by Portuguese National Funds through FCT - Founda- tion for Science and Technology under the IF/00209/2014/ CP1256/CT0011 Exploratory Research Project. Work of the authors over the past 25 years has been made possible by collaboration accords with the Université d’ Antanana- rivo (Mention Zoologie et Biodiversité Animale) and with the Parc Botanique et Zoologique de Tsimbazaza. 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Proceedings of the National Academy of Sciences of the United States of America 106(20): 8267-8272. https://doi.org/10.1073/pnas.0810821106 Wollenberg KC, Vieites DR, van der Meijden A, Glaw F, Cannatella DC, Vences M (2008) Patterns of endemism and species richness in Malagasy cophyline frogs support a key role of mountainous areas for speciation. Evolution 62(8): 1890-1907. https://doi.org/10.1111/ j.1558-5646.2008.00420.x Supplementary material | PDF-embedded 3D model of the skeleton of Plethodontohyla alluaudi holotype (MNHN 1901.235) Authors: Adriana Bellati, Mark D. Scherz, Steven Meg- son, Sam Hyde Roberts, Franco Andreone, Goncalo M. Rosa, Jean Noél, Jasmin E. Randrianirina, Mauro Fasola, Frank Glaw, Angelica Crottini Data type: Adobe PDF file 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. Link: https://doi.org/10.3897/zse.94.14698 suppl] Supplementary material 2 PDF-embedded 3D model of the skeleton of Plethodontohyla laevis holotype (SMF 4286) Authors: Adriana Bellati, Mark D. Scherz, Steven Meg- son, Sam Hyde Roberts, Franco Andreone, Goncalo Zoosyst. Evol. 94 (1) 2018, 109-136 M. Rosa, Jean Noél, Jasmin E. Randrianirina, Mauro Fasola, Frank Glaw, Angelica Crottini Data type: Adobe PDF file 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. Link: https://do1.org/10.3897/zse.94.14698 suppl2 Supplementary material 3 PDF-embedded 3D model of the skeleton of Plethodontohyla laevis tsianovohensis holo- type (MNHN 1936.47) Authors: Adriana Bellati, Mark D. Scherz, Steven Meg- son, Sam Hyde Roberts, Franco Andreone, Gongalo M. Rosa, Jean Noél, Jasmin E. Randrianirina, Mauro Fasola, Frank Glaw, Angelica Crottini Data type: Adobe PDF file 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. Link: https://do1.org/10.3897/zse.94.14698 suppl3 Supplementary material 4 PDF-embedded 3D model of the skeleton of ZSM 3/2002, a specimen of an undescribed Rhombophryne species formerly called Rhombophryne ‘alluaud?’ Authors: Adriana Bellati, Mark D. Scherz, Steven Meg- son, Sam Hyde Roberts, Franco Andreone, Gongalo M. Rosa, Jean Noél, Jasmin E. Randrianirina, Mauro Fasola, Frank Glaw, Angelica Crottini Data type: Adobe PDF file Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons. 135 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. Link: https://do1.org/10.3897/zse.94.14698 suppl4 Supplementary material 5 PDF-embedded 3D model of the skeleton of MRSN A6340, a specimen assigned to Plethodontohyla laevis Authors: Adriana Bellati, Mark D. Scherz, Steven Meg- son, Sam Hyde Roberts, Franco Andreone, Gongalo M. Rosa, Jean Noél, Jasmin E. Randrianirina, Mauro Fasola, Frank Glaw, Angelica Crottini Data type: Adobe PDF file 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. Link: https://do1.org/10.3897/zse.94.14698 suppl5 Supplementary material 6 PDF-embedded 3D model of the skeleton of ZSM 89/2004, a specimen assigned to Plethodontohyla alluaudi Authors: Adriana Bellati, Mark D. Scherz, Steven Meg- son, Sam Hyde Roberts, Franco Andreone, Gon¢alo M. Rosa, Jean Noél, Jasmin E. Randrianirina, Mauro Fasola, Frank Glaw, Angelica Crottini Data type: Adobe PDF file 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. Link: https://do1.org/10.3897/zse.94.14698 suppl6 zse.pensoft.net