Naturwissenschaften (2010) 97:525–543 DOI 10.1007/s00114-010-0667-x
ORIGINAL PAPER
Living within fallen palm leaves: the discovery of an unknown Blommersia (Mantellidae: Anura) reveals a new reproductive strategy in the amphibians of Madagascar
Franco Andreone & Gonçalo M. Rosa & Jean Noël & Angelica Crottini & Miguel Vences & Christopher J. Raxworthy
Received: 29 December 2009 /Revised: 23 March 2010 /Accepted: 25 March 2010 /Published online: 17 April 2010 # Springer-Verlag 2010
Abstract We describe a new mantelline frog of the genus occur. Thus, B. angolafa n.sp. represents a new evolution- Blommersia found in rainforest in North East Madagascar, ary lineage of Malagasy frogs in which phytotelmy is from the protected areas of Ambatovaky, Betampona, known. Up to now, reproduction in phytotelmata in Masoala, and Zahamena. Blommersia angolafa n.sp. is a Malagasy frogs has been reported for many cophyline small frog, with a body size of 17–21 mm, expanded finger microhylids, most species of Guibemantis, Mantella laevi- and toe tips, and colouration ranging from yellow to dark gata, and possibly in a still-undescribed species belonging brown, with pale-bluish spots on the flanks and light tips of to the genus Spinomantis. We consider the reproductive fingers and toes. A peculiar aspect characterising this new mode of B. angolafa as a derived character, having evolved species is its novel life history and reproductive mode. Both from the more typical reproduction in lentic water bodies. sexes live and breed in a phytotelmic habitat of water The general scarcity of lentic habitats in Malagasy rain- accumulated within fallen prophylls and fallen leaf sheaths forests may have provided the conditions that favoured the of at least three species of Dypsis palms. Within these evolution of this phytotelmic breeding strategy. The new phytotelmata, egg laying and complete larval development species, being specialised to a habitat represented by a few
Electronic supplementary material The online version of this article (doi:10.1007/s00114-010-0667-x) contains supplementary material, which is available to authorized users. F. Andreone (*) M. Vences Museo Regionale di Scienze Naturali, Division of Evolutionary Biology, Via G. Giolitti, 36, Technical University of Braunschweig, 10123 Torino, Italy Spielmannstrasse 8, e-mail: [email protected] 38106 Braunschweig, Germany e-mail: [email protected]
G. M. Rosa A. Crottini Departamento de Biologia Animal, Dipartimento di Biologia, Universitá degli Studi di Milano, Faculdade de Ciências da Universidade de Lisboa, Sezione di Zoologia e Citologia, Via Celoria 26, Lisbon 1749-016, Portugal 20133 Milano, Italy email: [email protected] e-mail: [email protected] e-mail: [email protected] C. J. Raxworthy J. Noël Department of Herpetology, Madagascar Fauna Group, American Museum of Natural History, BP 442, Morafeno, Central Park West at 79th St., Toamasina 501, Madagascar New York, NY 10024, USA e-mail: [email protected] e-mail: [email protected] 526 Naturwissenschaften (2010) 97:525–543 selected Dypsis species, potentially suffers the selective phytotelmata, such as tree holes, leaf axils, bamboo nodes, exploitation of these palms. more rarely on the ground or in leaf axils of Ravenala spp., Typhonodorum spp., Crinum spp., and Pandanus Keywords Amphibia . Anura . Mantellidae . New species . spp. (Glaw and Vences 1994). Cophyline frogs have non- Madagascar . Phytotelmic breeding . Dypsis palms feeding endotrophic tadpoles that develop within these habitats using yolk reserves. Although the deep phyloge- netic relationships of cophyline lineages are not yet fully understood, this specialisation to phytotelmic habitats, Introduction together with arboreality, seems had evolved several times independently within this group (Andreone et al. 2005; The currently known richness of over 6,660 species of Wollenberg et al. 2008). The situation within mantellids anuran amphibians (AmphibiaWeb 2010) also exhibit an appears to be quite different, and in general, phytotelmy impressive diversity of breeding strategies (Duellman and seems to be an exception, rather than the rule. The most Trueb 1986; Haddad and Prado 2005). According to well-known phytotelmic breeders are five described and Wells (2007), there are 39 different breeding strategies several undescribed species of the genus Guibemantis, known in anurans. In temperate regions, the diversity of whicharespecialisedtobreedinginPandanus leaf axils reproductive modes is comparatively low, yet tropical (Blommers-Schlösser 1979). Then, one poison frog, rainforests show a conspicuously high number of species Mantella laevigata, lays eggs in tree holes and bamboo that deviate from the “typical” and well-known reproduc- nodes, where the exotrophic tadpoles develop and are fed tive behaviour of egg deposition and tadpole development by the parents with fertilised and unfertilised eggs (Glaw in free water. Probably, the high atmospheric humidity and Vences 1992;Glawetal.2000b;Heying2001, 2004). and high rainfall in these tropical regions has permitted Recently, one undescribed Spinomantis species (S. sp. the progressive evolutionary tendencies of independence “aff. bicalcaratus”; see Glaw and Vences 2007) has also from breeding in free water (Summers and McKeon been found in Pandanus leaf axils and may use these 2004). phytotelmata for reproduction, although obligate breeding One anuran strategy of breeding in water, but in this habitat is yet to be demonstrated. independent from free water, is to use live or dead In contrast to the lineages mentioned above, all the parts of plants and the water accumulated within them species of the genus Blommersia follow a reproductive (usually known as “phytotelmata” or “phytotelms”)as mode that is rather typical of pond-breeding mantellines: selective breeding habitats (Lehtinen et al. 2004a, b). It eggs are laid on leaves or other structures overhanging is generally accepted that the adaptation to phytotelmata is ponds or puddles (rarely terrestrially in the leaf litter), and an important ecological innovation and a key evolutionary the tadpoles drop into the pond once they reached an path for many frog species. Following the review by adequate stage of embryonic development. It was there- Lehtinen et al. (2004b), there are at least 102 frog species fore surprising when we discovered a new species of (belonging to eight families/phylogenetic lineages) around mantellid frog belonging to the genus Blommersia that the world showing this habitat specialisation, with new reproduces in a still unknown phytotelmic habitat, repre- phytotelmic (or phytotelmatic) species still being de- sented by dead fallen leaves and prophylls of Dypsis scribed (e.g., Osteocephalus castaneicola,seeMoravec palms. To our knowledge, this is the first record of a frog et al. 2009). selectively using this kind of habitat for reproduction, and Madagascar is one of the most important biodiversity this is also the first Blommersia species showing larval hot spots for amphibian biodiversity, since it harbours a development in phytotelmata. In this paper, we formally striking number of endemic frog species, with more than describe this new species and discuss the trends of parallel 240 described species, and many undescribed (around adaptations to phytotelmic reproduction in Malagasy 130) but already identified candidate species (Vieites et al. frogs. 2009). This large island has also been flagged as an ideal area to study processes of species diversification (Vences et al. 2009). Specialisation to phytotelmata in Malagasy Materials and methods frogs has been achieved in both of the two largest adaptive radiations, namely in the cophyline microhylids and in the Study sites mantelline mantellids, which are also the most specious groups (Glaw and Vences 2007). Arboreal cophylines The new Blommersia species was found in four rainforest belong to the genera Anodonthyla, Cophyla, Platypelis, areas in Northern Central East and North East regions and Plethodontohyla and reproduce in a wide array of (Glaw and Vences, 2007) of Madagascar (Fig. 1): (1) Naturwissenschaften (2010) 97:525–543 527
Réserve Naturelle Intégrale (RNI) de Betampona, (2) Parc The RS (Special Reserve) d'Ambatovaky is located National (PN) de Zahamena, (3) Réserve Spéciale (RS) about 50 km west of the coastal town of Soanierana- d'Ambatovaky, and (4) PN de Masoala; all these sites Ivongo. Its area sums 60,050 ha and has an altitude ranging belonging to the protected area network of Madagascar from 400–1,185 m asl. Its vegetation is typical low- and (ANGAP 2003). mid-altitude rainforest, with sclerophyllous forest on ex- Created in 1927 and now completely surrounded by posed ridges. Many streams drain eastwards through the deforested areas, the RNI (Strict Nature Reserve) de site and create many small valleys and ridges between Betampona is a 2,228 ha remnant forest fragment located occasional steep-sloped, sharp-peaked mountains. Two between 17°15′–17°55′ S and 49°12′–49°15′ E. About 50% main rivers flow through the reserve, the Iampirano and of the reserve includes primary-mature forest, which the Sandrangato. The reserve hosts many regionally represents a rare surviving relict of the former extensive endemic amphibians and reptiles (Raxworthy et al. 1991). evergreen lowland rainforest of eastern Madagascar (Britt et The Masoala Peninsula is one of the major rainforest al. 2003; Glessner and Britt 2005). The canopy typically blocks of Madagascar. Located in the Northeast, it includes reaches 20–30 m. The altitude ranges from 275 to 650 m one of Madagascar's top conservation priorities for its above sea level (asl), and the landscape is characterised by extensive rainforest and high biodiversity of plant and hills and ridges. The reserve has a humid tropical climate, animal life. The 410,000 ha PN (National Park) de Masoala with rainy year precipitations ranging from 2,000 mm was created in the late 1990s. The altitude at Masoala (Jenkins 1987) to around 5,000 mm per year (Welch 2002). ranges 0–1,224 m. The site where the new Blommersia The PN (National Park) de Zahamena has an extension species was found bordered the Ankavanana River on the of 42,300 ha and is located around 30 km northwest from eastern side of the park. Betampona. This park is a hilly region, ranging 200– 1,500 m asl in altitude (Byg and Balslev 2001). According Collecting methods and used acronyms to Cornet (1974), this area is classified as humid to perhumid, with a rainfall between 1,500–2,000 mm per Frogs were searched both day and night during survey year. Precipitations occur throughout the year, with rainfall works carried out at the four areas during the rainy season. peaking from November to March. The vegetation is Night searches were carried out with the aid of headlamps characterised by evergreen humid forest and some second- and flashlights. After understanding the peculiar habitat ary forest, with some cultivations and secondary grasslands used by the new Blommersia species (given in detail later), (Thompson et al. 1987; Raxworthy 1988). we actively searched for it within fallen dead leaves, dead
Fig. 1 Distribution of B. ango- lafa n.sp. based upon specimens described in the text, overlaid on a landcover satellite map (from CABS 2004). The yellow polygons show the combined distribution of the three Dypsis palm species (D. hovomantsina, D. lastelliana, and D. tsaravoa- sira) where B. angolafa n.sp. has been found. The green polygons highlight the main protected areas 528 Naturwissenschaften (2010) 97:525–543 prophylls (the lowest tract of a palm inflorescence), and extracted using proteinase K (10 mg/ml) digestion followed dead leaf sheaths of palms belonging to the genus Dypsis. by a standard salt-extraction protocol (Bruford et al. 1992). Live individuals were photographed, and a few of them A fragment of ca. 550 bp of the 3′ terminus of the were euthanized (by immersion in a solution of MS222 or mitochondrial 16S rRNA gene, which has proven to be in chlorobutanol) and fixed in 10% buffered formalin or suitable in anuran species identification (Vences et al. 90% ethanol, and later transferred to 70–75% ethanol 2005), was sequenced for seven individuals (MRSN (metamorphosed individuals), or maintained in 4% formalin A6650.2, MRSN A6648, MRSN A6650.1, FAZC 13613, (larval individuals). Some egg clutches were also collected MRSN A6376, MRSN 6314, and UMMZ 213418) using and preserved in 4% formalin. standard cycling protocols and primers (Vences et al. 2003). All the voucher specimens are deposited at the Museo To assess genetic distinctness of the new species from all Regionale di Scienze Naturali, Torino, Italy (MRSN); other Malagasy frogs and ascertain its belonging to University of Michigan, Museum of Zoology, Ann Arbor, Blommersia, sequences were compared with a database USA (UMMZ); American Museum of Natural History, New containing homologous sequences of reliably identified York, USA (AMNH); and University of Antananarivo adult specimens of almost all Malagasy frog species, using Department of Animal Biology, Antananarivo, Madagascar the BLAST algorithm, and the alignment as published by (UADBA). Through the text, we also report (when available) Vieites et al. (2009; the species is included therein as the original field numbers, preceded by the collector's confirmed candidate species as Blommersia sp. 7). acronym, as follows: Franco Andreone Zoological Collec- To assess the phylogenetic position of the new species tion (FAZC), Madagascar Fauna Group (MFG), Ronald A. among described species of Blommersia, we compiled a Nussbaum (RAN), and Christopher J. Raxworthy (RAX). multigene dataset of all described Blommersia species using as outgroup representative phytotelmic and non- Morphometric measurements phytotelmic species of mantellids. We amplified mitochon- drial DNA fragments of the 5′ terminus of the 16S rRNA Measurements were taken following standard methods, gene, the cytochrome b, cytochrome oxidase subunit 1 using digital callipers and a dissecting microscope with (cox1), and 12S rRNA genes (mtDNA) and a nuclear DNA ocular micrometre (to the nearest 0.1 mm): Snout-vent fragment of the Rhodopsin gene. Besides three individuals length (SVL); head width (HW); head length (HL, from the (MRSN A6648, A6650.1, and A6650.2) of the new maxillary commissure to the snout tip); horizontal tympa- Blommersia species, we included sequences (some of them num diameter (TD); horizontal eye diameter (ED); eye- obtained from Genbank) of all described Blommersia nostril distance (END); nostril-snout tip distance (NSD); species (non-phytotelmic species), of M. laevigata (phyto- inter-narial distance (NND); hand length, from the carpal- telmic species), Guibemantis bicalcaratus (phytotelmic metacarpal articulations to the tip of the longest finger species), Guibemantis liber (non-phytotelmic species), (HAL); hind limb length, from the cloaca to the tip of the Spinomantis aglavei (non-phytotelmic species), and S. sp. longest toe (HLL); foot length including tarsus, from the aff. bicalcaratus possible (phytotelmic candidate species) tibiotarsal articulation to the tip of the longest toe (FOTL); for the phylogenetic analyses. Primer and PCR conditions foot length, from the tarsal-metatarsal articulations to the follow Vences et al. (2003). PCR products were resolved on tip of the longest toe (FOL); tibia length (TL); femoral automated sequencers (Applied Biosystems: ABI 3130XL). gland length (FGL), femoral gland width (FGW). Webbing Newly determined sequences were submitted to Genbank formula is given according to Blommers-Schlösser and (accession numbers: GU983096–GU983168). Sequences Blanc (1991) and Glaw and Vences (1994). were aligned in MEGA (Kumar et al. 2004) using the Tadpoles were measured with a stereo microscope using Clustal algorithm. Alignments of the 12S and 16S rRNA a micrometre to the nearest 0.1 mm. Developmental stages genes were adjusted manually and parts where alignment were given according to Gosner (1960). Terminology of required assumption of multiple indels were excluded from larval morphology follows McDiarmid and Altig (1999) phylogenetic analysis. and Altig and McDiarmid (1999). We performed maximum parsimony (MP), maximum likelihood (ML), and Bayesian inference searches based on Molecular analysis the full concatenated dataset. PAUP* 4.0b10 (Swofford 2002) was used to conduct MP analyses [100 random Tissue samples from adults and at least one specimen from addition sequence replicates, equal character weighting, tree each tadpole series of the new Blommersia species were bisection and reconnection (TBR) branch swapping, and collected and stored in 99% ethanol. Sampling was done gaps coded as missing data]. Nodal support was calculated using sterilised equipment to prevent potential contamination using the bootstrap analyses, with 1,000 replicates, ten and infection (Dejean et al. 2007). Total genomic DNA was random addition sequences replicates, and TBR branch Naturwissenschaften (2010) 97:525–543 529 swapping. ML analyses were carried out in Treefinder high bootstrap and Bayesian support values for almost all (Jobb et al. 2004), including the determination of the best- clades within the genus. Blommersia blommersae, B. fit model of evolution based on the Akaike information domerguei, and B. wittei form a monophyletic group, but criterion (AIC). Support for the resulting ML topology was relationships within this clade lack strong support. A obtained using the bootstrap analyses as implemented in second clade contains (1) a subclade with B. sarotra and Treefinder, with 1,000 replicates, ten random addition B. kely and (2) a clade with B. grandisonae and the new sequence replicates, and TBR branch swapping. On the species. The sister relationship of B. grandisonae and the basis of the test of maximum-likelihood models, the general new species is well supported, with 98% bootstrap and time reversible (GTR) model and a discrete (four rate 100% posterior probability Bayesian support. A previous categories) gamma distribution of among site rate hetero- molecular survey (Vieites et al. 2009), based on partial geneity were selected for ML analysis in Treefinder. sequences of the mitochondrial 16S rRNA gene, also Bayesian analyses were performed in MrBayes 3.1.2 placed sequences of B. angolafa (referred to as B. sp. 7 (Ronquist and Huelsenbeck 2003). The GTR+I+G model Betampona) in the genus Blommersia and largely agree was determined by AIC in MrModeltest (Nylander 2002)as with the phylogenetic tree presented here. the best-fitting model of substitution. We performed two runs of 10 million generations (started on random trees) and Intraspecific genetic differentiation The uncorrected pair- four incrementally heated Markov chains (using default wise genetic divergence at the 16S rRNA gene locus heating values), sampling the Markov chains at intervals of between individuals of B. angolafa from Betampona 1,000 generations. Stabilisation and convergence of likeli- (MRSN 6314, MRSN A6376 MRSN A6648, MRSN hood values occurred after 1 million generations. The first A6650.1-2, and FAZC 13613) and UMMZ 213418 (RAN two million generations were conservatively discarded, and 42751) from Masoala (Fig. 1) ranges from 2.7% to 3.4% eight million trees were retained post burn-in and summed (SM1), indicating differentiation between these two geo- to generate the majority rule consensus tree. graphically separated populations (separated by about 320 km). Interestingly, the genetic distinction observed Bioacoustics between these two populations fits the combined geograph- ic distribution of the three Dypsis palm species (Fig. 1). The recording was performed in September 2008, at the According to unpublished data provided by J. Dransfield beginning of the reproductive season. We recorded only a and M. Rakotoarinivo (Royal Botanical Gardens, Kew), few calls of Blommersia angolafa emitted by a single male, there is a gap of distribution between the northern and using a digital recorder (Boss Micro BR), accessorised with central–eastern distribution. It is currently unclear whether a semi-directional microphone, analysed with the acoustic this Dypsis gap is real or is a sampling artefact, but software Adobe Audition 3.0, and then compared to an potentially it could represent a barrier to gene flow for B. existing database of frog vocalisations (Vences et al. 2006). angolafa. By contrast, B. angolafa individuals from The male (non-collected) was recorded by one of us (J.N.) Betampona are highly homogeneous, with a genetic at its calling place, within a fallen prophyll already filled distance at the fragments of the 3′ terminus of the 16S with water, and it was unequivocally identified as B. rRNA gene ranging from 0% to 0.4% (SM1). Only three angolafa according to our former experience and to the individuals of B. angolafa from Betampona have been fact that no other frog was calling at the same site. Calls processed for the other loci, and similarly, the genetic were edited with a sampling rate of 44.100 Hz and 16 bits divergence within B. angolafa individuals is very low. In per sample in the mono-pattern. Frequency information was the fragment of the 5′ terminus of the 16S rRNA gene, the obtained through fast Fourier transformation (width 1,024 intraspecific genetic variation ranges from 0.3% to 0.4% points); the audiospectrogram was obtained at Hanning (SM2). In the 12S rRNA gene, these values range from window function with 256-band resolution. Temporal 0.1% to 0.4% (SM3), and also, in the cytochrome b (SM4) measurements in seconds (s) or milliseconds (ms) are and rhodopsin (SM5) genes, the intraspecific genetic provided as range, followed by mean, standard deviation, variation is very low, ranging from 0% to 0.2% and 0% and number of analysed units (N; notes, calls, or intervals). to 0.3%, respectively. Among the analysed genes, the cox1 (SM6) fragment is the most variable, and the intraspecific genetic variation of the analysed B. angolafa from Results Betampona at this locus ranges from 1.1% to 1.2% (SM6).
Molecular phylogeny The multigene phylogeny provided Interspecific genetic differentiation The genetic distance at here (Fig. 2) confirms that the new species belongs into the the fragments of the 3′ terminus of the 16S rRNA gene genus Blommersia. This tree is fully resolved, with mostly (SM1) between the sequence of the B. angolafa holotype 530 Naturwissenschaften (2010) 97:525–543
Fig. 2 Molecular phylogeny of representative phytotelmic and non- recorded by F. Glaw on 19.II.1994, at Ankeniheny, 21°C; B. phytotelmic species of mantellids based on 3,474 bp of mitochondrial domerguei: recorded by F. Glaw and M. Vences on 15.I.1994 at and nuclear DNA gene sequences. The non-phytotelmic S. aglavei Andringitra, 19°C; B. grandisonae, recorded by F. Glaw on 1.II.1995 was used as outgroup. Values at nodes are bootstrap values in percent at Andasibe, 23.8°C; B. kely: recorded by M. Vences and F. Glaw on from ML and MP (in italics), respectively; values below 80% not 11.II.2000 at Ambatolampy, 23.2°C; B. sarotra: recorded by F. Glaw shown. Asterisks denote Bayesian posterior probabilities values: on 1.I.1995 at Andasibe, 22°C; B. wittei: recorded by M. Vences on **= 99–100%. Blommersia live colouration and call oscillograms 20.III.2000 at Sambava, 24.7°C) are also shown (taken from Vences et al. 2006: B. blommersae:
(MRSN A6314) from Betampona and those of other similar to B. sarotra (8.5%) and to B. kely (12.5%), Blommersia species is high, ranging from 7.6% (B. respectively. Additional information on the level of diver- angolafa–B. grandisonae) to 11.5% (B. angolafa–B. kely). gence within and among mantellid genera for each locus is Similarly, the genetic distance between the sequence of B. available in the online supplementary material (SM1–SM6). angolafa from Masoala (UMMZ 213418) and the other Blommersia species is also high, ranging from 6.0% (B. angolafa–B. grandisonae) to 14.3% (B. angolafa–B. kely). Description of B. angolafa: new species Also at the fragments of the 5′ terminus of the 16S rRNA gene (SM2), at the cytochrome b (SM4) and rhodopsin Diagnosis A species belonging to the Mantellidae, subfam- (SM5) genes, the lower genetic distances between B. ily Mantellinae, based upon the following set of characters: angolafa and other congeneric species is found between (1) absence of nuptial pads, (2) presence of distinct femoral B. angolafa and B. grandisonae (i.e., 12.5%, 14.5%, and glands in males, (3) egg laying out of water, and (4) its 9.5%, respectively), while at the 12S rRNA gene (SM3) molecular genetic relationships. Assigned to the genus and at the cox1 (SM6) fragments, B. angolafa is more Blommersia as defined by Glaw and Vences (2002, 2006, Naturwissenschaften (2010) 97:525–543 531
2007), based upon: (1) small body size (SVL<21 mm); (2) less darkened on the head and the dorsum; brown tympanic patch-like femoral glands of type 2 (Glaw et al. 2000a)in region; this colour continues on to the flanks, and it is males (absent in females); (3) presence of weakly expressed limited below by a white band that extends to the upper webbing between toes; (4) presence of small-sized inner and lip...”]. The dark colouration on the head and tympanic outer metatarsal tubercles; (5) single and moderately disten- region is still visible in the holotype and type specimens, sible subgular vocal sac. The attribution to the genus despite their preservation in ethanol for about 40 years, and Blommersia is also supported by the molecular phylogenetic general colouration fading. It also represents the most analysis. Blommersia angolafa is further distinguished by all relevant chromatic trait when compared with the rather the other congeneric species for peculiar aspects regarding homogeneous colouration observed in B. angolafa (in morphology, colouration, calls, and life history. In particular, which the lateral whitish spots are visible as well). B. angolafa has enlarged tips on fingers and toes (less Although both B. angolafa and B. grandisonae have developed in the other species), and lacks any dark area in enlarged finger tips, they are much more developed in B. the tympanic and frenal region, present in the other angolafa. Blommersia. Moreover, B. angolafa has a rather uniform With the exception of B. angolafa, no other Blommersia dorso-lateral colouration, shading from yellowish–light was found in phytotelmata so far, although individuals brownish to dark brown, with light-bluish spots on the attributed to B. grandisonae (but possibly belonging to a flanks and light-bluish terminal parts of the fingers and still-undescribed candidate species) from Masoala were toes. The species also appears to be chromatically sexually found calling from a small water basin accumulated within dimorphic. In fact, males differ from females in having a an hollow tree trunk. light colouration, while females are more brownish. No other Blommersia species share any these characters Holotype MRSN A6314 (FAZC 13505) (Fig. 3a), male (Table 1). (ethanol-fixed and DNA sequenced), RNI de Betampona, Blommersia angolafa is also distinguished by B. Region Atsinanana, Commune Rurale de Sahambala, blommersae for the state of the lateral metatarsalia campsite locally known as Sahambendrana, 17°53′54,5″S– (separated vs. connected); by B. domerguei by the uniform 49°12′55,2″E, 458 m, leg. G.M. Rosa, J. Noël, and F. dorsum (vs. a contrasted pattern of three longitudinal dorsal Andreone, 6.II.2007. bands) and for the metatarsalia (separated vs. connected); by B. kely for the larger size (17–21 vs. 14–16 mm), the Paratypes MRSN A6249 (FAZC 13535), male (ethanol- uniform colouration vs. brownish often with light median fixed), RNI de Betampona, Commune Rurale de Saham- band and for the texture of the dorsum (smooth vs. warty) bala, campsite locally known as Sahambendrana, 17°53′ and lateral metatarsalia (separated vs. connected); by B. 54,5″S–49°12′55,2″E, 458 m, leg. G.M. Rosa, J. Noël, F. sarotra for the larger size (17–21 vs. 15–17 mm), the lateral Andreone 7.II.2007; MRSN A6376 (FAZC 13852), male metatarsalia (separated vs. connected), the vomerine teeth (ethanol-fixed), RNI de Betampona, administrative data as (absent vs. present), by B. wittei for the uniform colouration for the holotype, campsite locally known as Sahamben- (vs. bicoloured), the vomerine teeth (absent vs. present). drana, 17°53′54,5″S–49°12′55,2″E, 458 m, leg. G.M. Rosa, We provide additional information here concerning the J. Noël, and F. Andreone, 8.XI.2007; MRSN A6248 (FAZC comparison between B. angolafa and B. grandisonae 13585) male (ethanol-fixed), RNI de Betampona, adminis- because they share the most similar morphology, including trative data as for the holotype, campsite locally known as similar body size, the smooth texture of dorsal skin, the Sahambendrana, 17°53′54,5″S-49°12′55,2″E, 458 m, leg. separated metatarsalia, the presence of webbing on the foot, G.M. Rosa, J. Noël, and F. Andreone 10.II.2007; MRSN and the absence of vomerine teeth. Because the description A5848 (FAZC 13901), and MRSN A5865 (FAZC 13913), of B. grandisonae was written by Guibé (1974) based only one male (formalin-fixed) and one female (ethanol-fixed), on preserved specimens, there are no reliable observations RNI de Betampona, administrative data as for the holotype, of colouration in life. Thus, there was the possibility that campsite locally known as Maintimbato, 17°53′50,6″S–49° the B. grandisonae holotype referred to the same species as 13′39,9″E, 379 m, 1eg. G.M. Rosa, J. Noël, F. Andreone 8. individuals attributed to B. angolafa. However, we firmly XI.2007, and 18.XI.2007; AMNH A175391 (RAX 7602), reject this possibility, since we compared not only the male (formalin-fixed), RNI de Betampona, administrative original description, but also the type series. In the species data as for the holotype, campsite locally known as description, Guibé (1974) clearly stated that B. grandisonae Rendrirendry, 17°55′51,0″S—49°12′11,00″E, 300 m, leg. was “...plus ou moins rembruni sur la tête et le dos; région N. Rabibisoa, M.O. Randriambahiniarime, A.F. Ranjana- tympanique brune; cette teinte se prolonge en s’atténuant harisoa, C.J. Raxworthy 29.II.2004; AMNH A175392–93 sur les flancs, elle est limité en dessous par une bande (RAX 7978–79), males (formalin-fixed), RNI de Betam- blanche étendue sur la lèvre superieure...” [:“...more or pona, administrative data as for the holotype, campsite 532 Table 1 Main diagnostic characters of nominal species in Blommersia
Species Habitat SVL Dorsal colour Ventral Lateral Dorsal Finger Lateral Foot Vomerine Call features colour dark line texture and toe metatarsalia webbing teeth tips
B. angolafa n.sp. Phytotelms 17–21 Light–dark Uniformly Absent Smooth Enlarged Separated Present Absent Long pulsed note followed by 13–29 brownish brownish– short notes of variable length and with white yellowish number (note repetition rate ca. 7/s) spots B. blommersae Stagnant 18–21 Brownish Whitish Absent Smooth Narrow Connected Present Absent Series of usually 2–3 longer chirps water (8/s) B. domerguei Stagnant 16–20 Brownish Whitish Absent Smooth Narrow Connected Rudimentary Absent Long note followed by series of water with three 4–8 double-click notes (14/s) longitudinal lines B. grandisonae Stagnant 18–23 Brownish– Yellowish Black, Smooth Enlarged Separated Present Absent Long note followed by series of water greyish with bordered 5–6 short notes (11/s) paired with dark white markings B. kely Stagnant 14–16 Brownish, Whitish Absent Warty Narrow Connected Rudimentary Absent Single long note with slow pulse water often with repetition rate median light line B. sarotra Stagnant 15–17 Brownish Whitish Absent Smooth Narrow Connected Rudimentary Present Single long note with fast pulse water repetition rate B. wittei Stagnant 22–26 Brownish Whitish Absent Smooth Narrow Separated Present Present Fast regular series of up to 25 clicks water that in some populations have a melodious sound (10–15/s) auwseshfe 21)97:525 (2010) Naturwissenschaften – 543 Naturwissenschaften (2010) 97:525–543 533
Fig. 3 Blommersia angolafa n.sp. and its natural habitats. a Adult Rendrirendry; i tadpole in advanced development, with the hindlegs male (holotype) MRSN A6314 from Réserve Naturelle Intégrale de already visible (Gosner stage 39) from Rendrirendry; j tadpole, ventral Betampona (RNI), campsite locally known as Sahambendrana; b adult side, from Rendrirendry; k fallen Dypsis prophyll full of water, male MRSN A5848 (part of paratypes) from RNI de Betampona, selective habitat of B. angolafa, at Sahabefoza; l particular of a fallen campsite locally known as Maintimbato; c adult male (not collected), prophyll of Dypsis sp., from Sahambendrana; m a fallen prophyll of ventral side, from RNI de Betampona, campsite locally known as Dypsis sp. and surrounding habitat, at Masoala; n a living palm of the Sahabefoza; d couple in pre-mating habit from Sahabefoza; e two species D. tsaravoasira, whose dead phrophylls and leaf sheaths unsexed individuals and some tadpoles within a Dyspsis phophyll constitute the breeding habitats of B. angolafa, at Rendrirendry; o a filled with water from Masoala; f two egg clutches a few hours after living palm of the species D. lastelliana, from Sahambendrana. the deposition, from RNI de Betampona, campsite locally known as Photographs: F. Andreone (c), J. Noël (f, g, h, i, j, k, l, n and o), C.J. Rendrirendry; g developing egg clutch from Rendrirendry (about Raxworthy (e, m), G.M. Rosa (a, b and d) 5 days after the egg laying); h tadpole (Gosner stage 25), from 534 Naturwissenschaften (2010) 97:525–543 locally known as Sahambendrana, 17°55′53,0″S–49°12′ Vohitsivalana, 17°53′20,2″S–49°12′10,9″E, 508 m, leg. 56,0″E, 490 m, leg. N. Rabibisoa, M.O. Randriambahiniar- G.M. Rosa, J. Noël, and F. Andreone 28.II.2007; MRSN ime, A.F. Ranjanaharisoa, C.J. Raxworthy 16.III.2004; A6710 (MFG004), two eggs (ethanol-fixed), RNI de UADBA 26566 (RAX 7933), female (formalin-fixed), Betampona, administrative data as for the holotype, RNI de Betampona, administrative data as for the holotype, campsite locally known as Vohitsivalana, 17°53′20,2″S– campsite locally known as Rendrirendry, 17°55′51,0″S–49° 49°12′10,9″E, 508 m, leg. J. Noël 10.II.2009; MRSN 12′11,0″E, 450 m, leg. N. Rabibisoa, M.O. Randriambahi- A6711 (MFG005), two eggs (formol-fixed), RNI de niarime, A.F. Ranjanaharisoa, C.J. Raxworthy, 13.III.2004; Betampona, administrative data as for the holotype, UADBA 26594 (RAX 8040), female (formalin-fixed), RNI campsite locally known as Vohitsivalana, 17°53′20,2″S– de Betampona, campsite locally known as Sahambendrana, 49°12′10,9″E, 508 m, leg. J. Noël 10.II.2009. FAZC 13613, administrative data as for the holotype, 17°55′53,0″S–49° tadpole (ethanol-fixed), RNI de Betampona, administrative 12′56,0″E, 450 m, leg. N. Rabibisoa, M.O. Randriambahi- data as for the holotype, the campsite locally known as niarime, A.F. Ranjanaharisoa, C.J. Raxworthy, 17.III.2004; Sahambendrana, 17°53′53,4″S–49°12′58,0″ E, 400 m, leg. UADBA 26604 (RAX 7885), juvenile (formalin-fixed), and G.M. Rosa, J. Noël, and F. Andreone, 8.II.2007 [tissue RNI de Betampona, administrative data as for the holotype, sample, specimen not preserved]; UMMZ 228053 (RAN campsite locally known as Sahabefoza, 17°54′51,0″ S–49° 42754), 29 tadpoles (formalin-fixed) from PN de Masoala, 12′28,0″E, 490 m, leg. N. Rabibisoa, M.O. Randriambahi- Ankavanana River, 15°18′29,0″S–50°13′59″E, 100 m, leg. niarime, A.F. Ranjanaharisoa, C.J. Raxworthy, 11.III.2004; C.J. Raxworthy, A. and A. Razofimanantsoa, 12.I.1993. UMMZ 191625-27 (RAN 33745, 33754, 33811), males (formalin-fixed), RS d’Ambatovaky, 6 km NNW of Description of the holotype Male specimen in good state of Amberomanitra Village, 16°51′05,0″S–49°13′11,0″E, preservation. Fifth toe of the right foot lacking (taken for 450 m, leg. C.J. Raxworthy, 10-20.III.90; UMMZ genetic analysis). SVL = 19.5 mm. For other measure- 213418-22 (RAN 42751-53, 42756-57), two males, two ments, see Table 2. The body was slender, the head females and one juvenile (formalin-fixed), PN de Masoala, distinctly longer than wide, but not wider than body; snout Ankavanana River, 15°18′29,0″S–50°13′59,0″E, 90–100 m, pointed but slightly rounded in dorsal and lateral views, leg. C.J. Raxworthy, A. and A. Razofimanantsoa, 12-13. with nostrils directed laterally, not protuberant, nearer to tip I.1993; UMMZ 213423-25 (RAN 45928-30), two males of snout than to eye; canthus rostralis rather indistinct and and one juvenile, PN de Zahamena, Namarafana River, 17° straight; loreal region straight; tympanum distinct and 43′58,0″S–48°58′30,0″E, 450 m, leg. J.B. Ramanamanjato, rounded; supratympanic fold indistinct; tongue ovoid, A.P. Raselimanana, C.J. Raxworthy, A. and A. Razofima- slightly notched posteriorly but not bifid; vomerine teeth nantsoa, 17.III.1994. absent and maxillary teeth rudimentary; choanae rounded. Arms slender, with distinct, single subarticular tubercles, Additional material MRSN A6646 (FAZC 13558a), tad- the inner and outer metacarpal tubercles distinct, fingers pole (formalin-fixed), RNI de Betampona, campsite locally without webbing, relative length of fingers 1<2<4<3, with known as Sahambendrana, administrative data as for the the second finger shorter than the fourth one; finger discs holotype, 17°53′53,4″S–49°12′58,0″E, 400 m, leg. G.M. enlarged, and nuptial pads absent. Hindlimbs relatively Rosa, J. Noël, and F. Andreone 8.II.2007; MRSN A6647 robust; tibiotarsal articulation reaching the nostril when the (FAZC 13558b), metamorphosed (formalin-fixed), RNI de hindlimb is adpressed along the body; lateral metatarsalia Betampona, administrative data as for the holotype, separated; inner metatarsal tubercle small and outer meta- campsite locally known as Sahambendrana, 17°53′53,4″S– tarsal tubercle indistinct; toe discs enlarged; webbing 49°12′58,0″E, 400 m, leg. G.M. Rosa, J. Noël, and F. between toes weakly developed [1(0.5–1), 2i(0.5–1), 2e Andreone 8.II.2007; MRSN A6648 (FAZC 13559), two (1–1.5), 3i(2), 3e(1.5–2), 4i(2.5–3), 4e(2.5–3), 5(0.5–1)], tadpoles (ethanol-fixed), RNI de Betampona, administrative and the relative length of toes 1<2<5<3<4. Skin on the data as for the holotype, campsite locally known as dorsal surface smooth, without folds or ridges. Ventral skin Sahambendrana, 17°53′53,4″S–49°12′58,0″E, 400 m, leg. uniformly smooth. Femoral glands distinct in life, but not G.M. Rosa, J. Noël, and F. Andreone 8.II.2007; MRSN easily recognisable in ethanol in external view (well-visible A6649 (FAZC 13671), three tadpoles (formalin-fixed), RNI in internal view; Fig. 4a). de Betampona, administrative data as for the holotype, campsite locally known as Vohitsivalana, 17°53′20,2″S–49° Colouration of the holotype In life, the back was almost 12′10,9″E, 508 m, leg. G.M. Rosa, J. Noël, and F. brownish, with whitish–bluish, small spots on the flanks Andreone 28.II.2007. MRSN A6650 (FAZC 13670), two and tips of fingers and toes white on their superior surface. tadpoles (ethanol-fixed), RNI de Betampona, administrative The iris was dark brown with lighter brown speckling. The data as for the holotype, campsite locally known as ventral skin was also uniformly brownish, shading to auwseshfe 21)97:525 (2010) Naturwissenschaften Table 2 Morphometric measurements (all in millimetres) of the analysed specimens of B. angolafa n.sp.
CATALOGUE NUMBER Field Number Sex SVL HW HL TD ED END NSD NND HAL HLL FOTL FOL TL FGL FGW
MRSN A6314 FAZC 13505 HT M 19.5 6.6 6.6 1.8 2.7 2.4 1.8 2.1 5.1 33.6 14.8 8.9 11.4 2.7 1.2 MRSN A5848 FAZC 13901 PT M 18.7 6.6 7.5 1.6 2.7 1.8 1.9 2.1 5.8 31.1 14.1 9.2 11.4 3.6 1.6 MRSN A5865 FAZC 13913 PT F 19.2 6.6 7.5 1.8 2.3 1.8 1.8 2.3 4.9 31.5 14.5 9.5 10.4 - - MRSN A6248 FAZC 13585 PT M 17.3 5.7 6.7 1.6 2.2 1.9 1.6 2.1 4.1 27.8 11.9 6.9 9.2 3.3 1.4 MRSN A6249 FAZC 13535 PT M 21.1 6.8 7.3 1.4 2.4 2.3 1.9 2.8 5.5 34.1 15.4 9.1 11.6 3.9 1.7 MRSN A6376 FAZC 13852 PT M 18.5 5.8 6.9 1.4 2.8 2.0 1.6 2.1 5.1 29.7 12.1 8.9 10.7 3.6 1.7 –
UMMZ 191625 RAN 33745 PT M 19.6 6.5 6.5 1.2 2.4 1.9 1.3 2.8 5.5 37.8 15.4 9.6 11.4 2.5 1.1 543 UMMZ 191626 RAN 33754 PT M 19.6 6.5 6.4 1.0 2.6 2.1 1.4 2.6 5.5 35.6 14.8 9.1 10.9 3.0 1.4 UMMZ 191627 RAN 33811 PT M 19.8 6.5 6.2 1.1 2.6 1.9 1.2 2.5 6.0 39.0 16.8 10.1 11.5 3.0 1.2 UMMZ 191628 RAN 33812 PT M 18.0 6.3 6.0 0.9 2.4 1.9 1.3 2.5 5.2 33.8 13.7 8.6 10.3 3.2 1.1 UMMZ 213418 RAN 42751 PT F 20.3 6.5 6.2 0.9 2.3 1.9 1.2 2.4 5.4 35.6 14.4 9.5 11.2 - - UMMZ 213419 RAN 42752 PT M 20.1 6.6 6.5 1.1 2.5 1.8 1.4 2.7 5.6 36.5 15.0 9.6 10.9 3.0 1.5 UMMZ 213420 RAN 42753 PT F 19.3 6.7 6.2 1.0 2.5 1.8 1.3 2.4 5.2 34.0 14.2 9.0 10.2 - - UMMZ 213421 RAN 42756 PT M 18.1 6.6 6.0 1.1 2.3 1.8 1.3 2.6 5.2 33.7 13.3 8.8 10.2 3.0 1.5 UMMZ 213422 RAN 42757 PT J 9.4 3.6 2.9 0.4 1.3 0.8 0.7 1.4 2.6 16.2 6.0 3.6 5.0 - - UMMZ 213423 RAN 45928 PT M 19.4 6.4 6.5 1.0 2.6 1.8 1.3 2.6 5.6 34.3 14.2 8.4 10.5 2.5 1.1 UMMZ 213424 RAN 45929 PT J 16.5 5.7 5.6 0.8 2.3 1.9 1.2 2.0 4.5 31.9 13.6 7.9 9.6 - - UMMZ 213425 RAN 45930 PT M 19.6 6.2 6.5 0.9 2.4 1.8 1.4 2.6 5.2 33.1 13.6 8.5 10.1 2.0 0.7 AMNH A175391 RAX 7602 PT M 17.8 6.0 5.7 0.9 2.6 1.7 1.3 2.5 4.1 30.0 13.2 8.3 8.0 NM NM AMNH A175392 RAX 7978 PT M 18.4 6.4 6.0 1.0 2.5 1.6 1.5 2.4 4.9 29.9 13.7 8.4 9.7 3.5 1.3 AMNH A175393 RAX 7979 PT M 18.8 6.5 5.9 0.9 2.6 1.6 1.4 2.6 4.8 32.9 13.0 8.1 9.9 3.3 1.3 UADBA 26566 RAX 7933 PT F 19.7 6.1 6.6 1.2 2.6 2.7 1.6 2.5 5.1 32.4 15.0 10.2 9.8 NM NM UADBA 26594 RAX 8040 PT F 17.3 6.8 7.2 1.2 2.6 1.9 1.3 2.5 4.7 31.7 13.8 9.5 8.5 NM NM UADBA 26604 RAX 7885 PT J 11.3 4.3 4.7 0.5 1.5 1.4 1.2 1.6 2.7 13.8 6.5 4.4 4.9 NM NM
HT holotype, PT paratypes, M male, F female, J juvenile, NM not measured. Other abbreviations are given in the text 535 536 Naturwissenschaften (2010) 97:525–543
Variation The range of measurements of the paratypes is given in Table 2. They are similar to the holotype for general morphology and size. The variation observed mostly relates to differences in colour pattern. Metamor- phosed individuals in the wild showed a rather different colouration, with males almost yellow and females and newly metamorphosed froglets much darker (Fig. 3d). In males just after capture, the belly is mostly yellow and the breast whitish, while females are darker. We suspect that this difference in colouration between males and females is variable, and it might be subject to changes due to temperature, light conditions or behavioural interactions. In fact, the holotype (a male) showed a yellowish colouration (Fig. 3a), while other males when captured became darker (Fig. 3b, c). Individuals from Masoala and Ambatovaky (UMMZ 213418-22 and UMMZ 191625-27) had very pale blue spots (as opposed to white in the frogs from the other localities) on the finger and toe pads, and the flank spots were pale yellow rather than white. After several years in alcohol, the types have faded and are becoming uniformly whitish–yellowish. Three specimens (UMMZ 191625, 213419-20) now exhibit a pale brown throat in preservative.
Fig. 4 Detail of the internal side of the “type 2” femoral gland in Eggs and tadpoles The egg clutches of B. angolafa are MRSN A6249 (a) and the mouthparts in a tadpole of B. angolafa (b) rather small (15–30 mm in diameter), containing a limited number of eggs (2–10, counted on a series of nine greyish, except on the chest, which was light and almost clutches). The external jelly capsule is transparent brown- silverish. The throat was brownish. When found, it had a ish, and eggs are yellow (see Fig. 3). Eggs are laid outside rather dark tonality, but, after capture, it turned out to a the water, on inner walls of Dypsis prophylls, usually at lighter colouration (as visible in Fig. 3a). After 2 years in some millimetres from the water surface. The tadpoles are preservative, the back has faded to light–yellowish shading of the benthic type (McDiarmid and Altig 1999)andshow to whitish, with a rather indistinct pattern of white spots on typical ranoid morphology; according to the classification the flanks. The white-coloured tips of fingers and toes are of phytotelmic tadpoles drawn by Lannoo et al. (1987), no longer evident. thetadpoleofB. angolafa belongs to group IV (Table 3).
Table 3 Measurements (all in millimetres) of tadpoles of B. CATALOGUE NUMBER MRSN A6646 MRSN A6649.1 MRSN A6649.2 MRSN A6649.3 angolafa n.sp. Gosner stage 25 25 25 39 TL 28.75 19.13 23.13 33.00 HTL 11.00 7.88 8.75 11.00 TH 4.13 3.38 3.63 4.00 TW 6.88 5.38 6.00 7.00 Abbreviations are given in the HWD 3.50 3.00 3.00 3.38 text. TL total length, HTL head DW 2.25 1.75 2.13 2.63 and trunk length, TH trunk HT 4.50 4.13 3.75 4.63 height, TW trunk width, HWD head width at the oral disc, DW HTM 2.50 1.63 1.63 2.63 disc width, HT height of the tail, SND 1.63 1.25 1.38 1.63 HTM height of tail muscles, NED 1.38 1.25 1.25 1.38 – SND snout nostril distance, SSP 6.38 4.63 4.88 6.25 NED nostril–eye distance, SSP snout–spiracle distance, ID ID 1.13 1.00 0.88 1.13 inter-nostril distance, IIED inner IIED 1.75 1.13 1.38 1.63 inter-eye distance, OIED OIED 4.00 2.63 3.00 4.00 outer inter-eye distance Naturwissenschaften (2010) 97:525–543 537
The body is elliptical in lateral view and dorso-ventrally Natural history Blommersia angolafa appears as a rather flattened. In dorsal view, the body appears sub-ovoidal, secretive species specialised to live in the dead fallen leaves slightly squared, with almost parallel lateral profiles. The and/or prophylls of a particular genus of Malagasy palms. snout is dorsally rounded, and in lateral view, it slopes We found egg clutches, tadpoles, newly metamorphosed gently to the oral region and then turns strongly. The froglets and adult males and females exclusively within the external nares are located dorsolaterally, closer to snout tip phytotelmata of three palms belonging to the genus Dypsis, than to eyes. The eyes are small and directed latero- namely D. lastelliana (locally known as “menavozona”), dorsally. The tail fins are low. The origin of dorsal fin Dypsis tsaravoasira (“tsaravoasira”)andDypsis hovomant- starts at about 1/3 of the tail from its base, while ventrally, sina (“hovomantsina”). Of 57 records of the species in the fin is almost equally high. The maximum tail height is Betampona, 45 (79%) were located within prophylls of D. reached at the middle of the tail. The tail tip is rather tsaravoasira, nine (16%) of D. hovomantsina and three rounded, and the tail muscle reaches the tip of the fins. (5%) of D. lastelliana. Blommersia angolafa was found The spiracle is sinistral with a midlateral opening directed both in primary and secondary forest, due to the tolerance posteriorly. The inner wall of the spiracle is present and of some Dypsis palms to occur in secondary rainforest at free from the body. The vent tube is parallel to the ventral Betampona. The observed elevational distribution at margin of the fin, tubular in shape, and displaced dextrally Betampona ranged from 332–548 m asl. with a medial aperture. The oral disc is anteroventral, sub- The Dypsis species occupied by B. angolafa at Masoala, elliptical, with a uniserial row of marginal papillae in the Ambatovaky and Zahamena were not identified in the field, lower labium and on the lateral side of the upper labium. but we believe that it was the same species at all three sites, A few submarginal papillae are present in the lateral and photographs and field notes suggest this might be D. portions of the upper labium. The papillae are conical, lastelliana. These palms were mostly found in primary with rounded tips, unpigmented and translucent. Labial forests and had a height of 20–30 m, with a diameter at tooth row formula: 5(2−5)/3 (Fig. 4b). In life, the tadpoles breast height of 25–30 cm. The distribution of these three are uniformly brownish shading to reddish, especially on palms is summarised in Fig. 1, which shows an interesting the dorsal body. In early stages of development, the congruence with the distribution of B. angolafa. Their peripheral parts of the body are quite transparent. The elevational range at these three sites was 90–450 m. overall dorsal colouration recall the one of the substrate We never observed B. angolafa individuals outside dead upon which they live, notably the dead palm leaves and fallen Dypsis phytotelmata that were laying on the forest detritus accumulated on the bottom, resulting highly floor or that contained rainfall. It is also important to stress cryptic and very difficult to be localised. The belly is that most fallen leaves or prophylls of Dypsis do not almost transparent, and through the body walls the orientate on the forest floor in such a manner as to catch intestine and other organs are well visible. In tadpoles rainfall within the leaf bases, and thus they are dry in advanced stage of development and with the hind internally. In such cases, they were not occupied by frogs. limbs already developed, the tip of the fingers and toes At Masoala, we examined the growing crown of an are already whitish. After about 2 years of preservative occupied palm (with B. angolafa in fallen prophylls) and (4% formalin) the tadpoles have lost their original colour found that only the outermost attached prophylls trapped and became rather whitish on the back, with sparse rainwater. However, the prophyll space with water appeared darker melanophores. The belly has become translucent. to be too narrow to be occupied by adult frogs. No frogs Intestines are blackish and visible through the body were seen in the living crown of the palm. walls. We found breeding individuals of B. angolafa only during the peak of the rainy season. The call started at the Etymology One of us (C.J.R.), first used this name when he end of September, as confirmed by our recording in 2008, found, for the first time, the new Blommersia at Masoala in and continued mostly continuously until the end of 1993. The term “angolafa” or “angolafo” is the Malagasy February to early March. Calls were usually heard during vernacular name used at Masoala by the Betsimisaraka the dusk and early at night. We did not find any other frog people for the Dypsis palm species (mostly for Dypsis species within these prophylls. Egg clutches were found lastelliana), whose leaves and prophylls are used by the only from December onwards; they were glued to the inside new Blommersia species. We decided to use the name walls of palms' dead prophylls filled with water and fallen “angolafa” (pronounce “un-goo-LAF-ah”)tostressthe on the ground. Several different stages of tadpoles shared close relationship that existed between this frog and the the water accumulated within the same fallen prophyll. phytotelmic habitats formed by dead and fallen leaves of Since the water level within the dead leaves is highly Dypsis palms. The term is here used as specific epithet and variable and mostly dependent on the rainfall rate, the eggs is to be considered as a noun in apposition. were frequently found submerged. In other cases, especially 538 Naturwissenschaften (2010) 97:525–543 in periods of relative and prolonged dryness, the water pulses that are repeated with a pulse repetition rate of depth within the prophyll became very low, forming a thin 44–52 per second (n=3). One such pulsed note is followed water film, a few millimetres deep. In such case, tadpoles by an irregular series of up to 13–29 notes of type 2 which congregate in the remnant water and detritus and may even are shorter and of irregular structure. Notes of type 2 can be be only partly submerged. short clicks consisting of a single main pulse and with a Regarding the adults, we cannot confirm whether within duration of 10–17 ms (n=2) or can consist of up to eight each prophyll there is always only a couple (male and distinctly separated pulses each and then have durations of female) plus tadpoles and metamorphosed juveniles. In 63–86 ms (n=2), with all intermediate states occurring so some cases, we observed one or more females, but, within that a clear distinction of further different note types is not our collection, there is an evident bias of collected males possible and computing average values for note duration (ratio number of males/number of females=3.2), which and number of pulses makes little sense. Intervals between indicates that males are much more abundant at breeding notes of type 2 in a note series have a duration of 73– sites. This suggests that the males stay with eggs and 191 ms (91.8±35.3 ms; n=10). This kind of call structure, juveniles and may even guard eggs and offspring. To with one longer note, followed by a rather long series of follow-up a detailed observation of egg deposition and short clicks or “chirps” is known in other species of the larval development, we collected a clutch composed of genus Blommersia. This is evident for B. grandisonae and seven eggs. These were put in a small plastic terrarium, B. domerguei as shown in Fig. 2. Unfortunately, the rather where we recreated the same habitat situation, putting a poor quality of the original call recording cannot allow us layer of dead leaves on the bottom and using rainwater to to make detailed sound considerations on the sonogram fill it. The terrarium was kept in a shaded position at RNI structure. However, the frequency appears as a more or less de Betampona, under the same climatic conditions. Eggs regular band between about 2,500–5,500 Hz; although, the were checked at least once a day. After a period of 7– fundamental frequency may be higher than 2,500 Hz. 10 days all the eggs hatched. Development from Gosner's stage 20 until stage 25 (largest tadpole size without legs) Distribution Blommersia angolafa occurs at four forest lasted 20–23 days. The development from stage 25 to stage blocks in eastern Madagascar: Masoala, Ambatovaky, 39 (with visible hindlegs) lasted 10–13 days. Total duration Zahamena and Betampona and occupies rainforest with an of the larval development was 57–70 days. elevation range between 90 m (Ankavanana River, PN de Masoala) and 508 m (Vohitsivalana, RNI de Betampona). Advertisement call The advertisement call of B. angolafa This region corresponds to one of the low elevation regions was recorded on September 24th, 2008 at 17:45, at an air of endemism identified by Raxworthy and Nussbaum temperature of 20.5°C (Fig. 5). It consisted of two notes, (1996). We suspect that this species may also occur at rather similar to those described for B. grandisonae and B. other rainforest sites that fall within this elevational and domerguei. Note of type 1 is a long and clearly pulsed note latitudinal range for eastern Madagascar, such as Makira with a duration of 221–233 ms and consisting of 9–11 and Mananara-Nord.
Fig. 5 Sonagram (top) and oscillogram (bottom) of the advertisement call in B. ango- lafa. Recorded at RNI de Betampona, on 24 September, 2008 (17:45h, temperature 20.5°C) Naturwissenschaften (2010) 97:525–543 539
Conservation The new species is currently known only Zahamena protected areas. In the long-term, this would from four localities and drawing an extent of occurrence help connect the populations of B. angolafa occurring in (EOO) of around 15,000–18,000 Km2. Since this is less these two areas that are currently surrounded by unprotect- than 20,000 Km2 and since it is known from less than ten ed areas with a high intensity of forest destruction. localities and there is an inferred decline in the quality of habitats, we classify it as “vulnerable”. The biology of this species requires it to coexist with an appropriate Dypsis palm. So far, we have found B. angolafa only in Discussion phytotelmata of D. lastelliana, D. tsaravoasira or D. hovomantsina, and it is well-known that Dypsis palms are This newly discovered species of Blommersia is relevant suffering from selective logging and deforestation not only in terms of biodiversity knowledge increase, but (Dransfield and Beentje 2006). It is also worth noting that also within a framework of improving our comprehension many of the observations of B. angolafa occurred in the of amphibian habitat specialisation. According to Lehtinen phytotelmata of D. hovomantsina and D. tsaravoasira, et al. (2004b), phytotelmy is known for eight anuran which are also classified as “critically endangered” species families (Bufonidae, Dendrobatidae, Hylidae, Hyperoliidae, (Dransfield and Beentje 1995, 1998a, b). Dypsis lastelliana Mantellidae, Microhylidae, Petropedetidae, and Rhacophor- is not considered as threatened, and this was the only palm idae; see Table 4), with 102 phytotelmic species from a occupied at Masoala, Zahemena and Ambatovaky. Thus, total of 2,902 species (estimate at March 2010), the conservation situation for B. angolafa is potentially and corresponding to 3.51%. Within these families, Dendroba- negatively affected by the palms' threatened status. Most of tidae show the highest incidence of phytotelmic species the Dypsis palms (or at least D. lastelliana) are also (9.19%), while Mantellidae rank third (after Petropedetidae, threatened by selective logging habitat destruction, fire, with one of 16 being phytotelmic), with seven of 186 harvesting for palm heart, and plant and seed collection by species (3.76%). If we limit our analysis to the subfamily palm enthusiasts (Byg and Balslev 2001, 2003; Dransfield Mantellinae (sensu Glaw and Vences 2007; currently and Beentje 2006). represented by around 113 species), the phytotelmic All the sites where B. angolafa was found are important percentage rises to 6.19%. fragments of low-altitude rainforests, and their known In such a context, B. angolafa is particularly interesting amphibian communities are particularly species-rich. They in being the only species exclusively breeding in dead are also part of the protected area network managed by fallen prophylls and the fifth to breed regularly in fallen dry Madagascar National Parks, and for this reason, their vegetal material. It is unknown why the number of conservation appears more certain than other areas. We phytotelmic frogs breeding in such materials (such as nuts also reinforce the suggestion made by Britt (1998) to create and leaves) is so small (0.17%) when compared with the a protected forest corridor between the Betampona and overall number of phytotelmic species (Wells 2007;
Table 4 Number of total species per family (from Amphibia Web at 14th March 2010), number and percentage (between round parentheses) of phytotelmic species (data from Lehtinen 2004), and species living/breeding within dead/dry vegetal material fallen onto the ground
Family Species Number of Species breeding Distribution Habitat number phytotelmic in fallen species vegetal items
Bufonidae 543 7 (1.29%) R. castaneotica Brazil Nut capsules Dendrobatidae 272 25 (9.19%) A. castaneoticus Brazil Nut capsules A. quinquevittatusa Brazil Hylidae 887 32 (3.61%) O. castaneicola Brazil Nut capsules Hyperoliidae 213 4 (1.88%) – – – Mantellidae 186 7 (3.76%) B. angolafa Madagascar Palm prophylls and leaves Microhylidae 468 17 (3.63%) P. mihanikaa Madagascar Palm leaves Petropedetidae 12 1 (8.33%) P. guineensis Ivory Coast, Sierra Leone Fruit capsules and other cavities Rhacophoridae 321 9 (2.80%) – – – Total 2,902 102 (3.51%) 5 (0.17%) [4.90%] a The species marked was only occasionally found in phytotelmata made of dead/dry vegetal materials 540 Naturwissenschaften (2010) 97:525–543
Moravec et al. 2009). In particular, three of these species: sented by confirmed candidate species, two are uncon- Rhinella castaneotica, Adelphobates castaneoticus,and firmed candidate species and two are deep conspecific Osteocephalus castaneicola breed in fruit capsules of the lineages. Moreover, together with Wakea, the genus Brazil nut tree (Bertholletia excelsa), while a fourth one, Blommersia includes the smallest frogs of the family Phrynobatrachus guineensis, is apparently less selective, Mantellidae, with adult SVL of 15–26 mm (Glaw and and breeds in tree holes and also in empty nuts, snail shells Vences 2007; Vences et al. 2002, 2006). and other tiny water bodies (Rödel and Schiøtz 2004). A All Blommersia species known previous to the discovery further Adelphobates species, A. quinquevittatus, was also of B. angolafa, including the candidate species, show a reported to breed occasionally within fallen nut capsules rather generalist life style in terms of habits and habitat (Moravec et al. 2009). choice. They live close to marshes and other still and slow- Among Malagasy frogs, an egg string assigned to a flowing water bodies, i.e., ditches, swamps and ponds. One guarding male of Plethodontohyla mihanika was found in a species, B. grandisonae, was reported to breed in slow- water-filled dead leaf on the ground, suggesting a similar moving stretches and side pools of streams (Glaw and reproductive mode as in B. angolafa (Glaw and Vences Vences 2007). Males call mainly at sunset and early night 1994). However, this behaviour appears as occasional for P. and climb at low elevation from the ground (usually mihanika, as this cophyline microhylid usually breeds at a between a few centimetres to 1–2 m), usually on or under certain height from the ground in water-filled cavities or leaves (Glaw and Vences 2007; Vences et al. 2002). As for within leaf axils (Glaw and Vences 2007). We suspect that all species of mantellids in the subfamily Mantellinae, eggs this paucity of phytotelmic species breeding in fallen are laid outside the water, often attached to vertical surfaces vegetal material is related to the presence of suitable such as leaves overhanging the water. material to aid breeding. In Madagascar, we are not aware Present knowledge supports phytotelmic breeding of empty nut shells that could be used for such a purpose, evolving independently at least three times in mantellids: and we never observed frog species breeding within snail's in Guibemantis, Mantella and Blommersia. All of these shells. The prophylls and leaf sheaths of the three Dypsis instances concern one mantellid clade, namely the pond- species likely share morphological and structural character- breeding lineage in the subfamily Mantellinae. The best- istics that allow the capture of rainfall (if they are orientated known phytotelmic group of mantelline frogs are the correctly) and thus provide a long-term permanence of water. species belonging to the subgenus Pandanusicola in the The discovery of a Blommersia species of limited genus Guibemantis. These species are arboreal and live morphological differentiation from its closest pond- almost exclusively and breed within Pandanus leaf axils, breeding and scansorial species, but living within a novel where they also spend their larval life (Blommers- kind of phytotelmic mesocosm marks an important point in Schlösser 1979;Lehtinen2002, 2004, 2009; Lehtinen et our comprehension and definition of reproductive strategies al. 2004b, 2007). A remarkable exception is G. liber and of Malagasy anurans. Including B. angolafa, the genus at least another closely related (but still undescribed) Blommersia currently contains seven nominal species (B. species that apparently has reverted its life history to breed angolafa, B. blommersae, B. domerguei, B. grandisonae, B. in free waters, named G. sp. aff. liber “South” (Glaw and kely, B. sarotra and B. wittei). Yet, similar to many other Vences 2007). The second phytotelmic lineage in the groups of Malagasy frogs, there are many candidate species Mantellinae is M. laevigata, which is the only semi- attributed to Blommersia that are awaiting formal descrip- arboreal Mantella. This mantella climbs trees and bamboo tion. For example, it is evident that under the name B. reeds and lays the eggs within small water cavities. Then, grandisonae there are several still-undescribed species that B. angolafa marks the third instance of independent are in need of taxonomic recognition. There are at least evolution of phytotelmic frogs within the Mantellinae. In three morphologically and genetically distinct taxa that particular, this species is unique within the phytotelmic have been associated with this nomen: B. grandisonae by species in colonising fallen palm leaves and prophylls Glaw and Vences (1994, 2007), B. aff. grandisonae filled by water, and it is the only Malagasy lineage “Marolambo” and a further species named B. aff. grandi- showing a preference for phytotelmic habitats available on sonae from Manombo and depicted (but not described) by the ground. A fourth possible case of phytotelmic species Glaw and Vences (2007, p. 446). Because the types of refers to a still-undescribed Spinomantis. This mantelline grandisonae come from Ambana, which is a locality in SE genus includes stream-breeding species, but it still remains Madagascar, quite close to Manombo, it is likely that the to be demonstrated that the new species (Spinomantis sp. name “grandisonae” will be assigned to individuals from aff. bicalcaratus, as given in Glaw and Vences 2007) this locality. Following Vieites et al. (2009), within the actually uses the leaf axils of Pandanus also for reproduc- genus Blommersia, eight genealogical lineages are repre- tion and not just as a shelter. Naturwissenschaften (2010) 97:525–543 541
Phytotelmata are among the simplest aquatic ecosys- path followed by B. angolafa towards a strict phytotelmic tems used by vertebrates (Lehtinen et al. 2004b). Their adaptation. small size and the common instances of parental care in From the available data, B. angolafa appears to be such habitats, usually determine a lower density of limited to the northern central east and north east of competitors and probably also reduce the risk of predation Madagascar, which largely agrees with the distribution of (Summers and McKeon 2004). The tadpoles of some the Dypsis palms (Dransfield and Beentje 1998a, b) utilised Guibemantis species that breed in Pandanus leaf axils are for breeding by this frog. Further work should be directed also able to climb along the leaf surface and to move to investigating if this pattern is an example of: (1) co- among axils (e.g., G. bicalcaratus and G. punctatus; evolution, with the three Dypsis palm distributions defining Lehtinen 2004). This climbing behaviour was not observed the potential distribution range of B. angolafa or (2) in larvae of B. angolafa. coincidental distribution of B. angolafa and the three Several factors may be invoked to explain the multiple palms, resulting from shared historical biogeographic evolution of phytotelmic breeding in pond-breeding mantel- processes, with other breeding habitat for B. angolafa lines. All the mantellines deposit their eggs outside of the occurring elsewhere. We assume that B. angolafa is fully water, and many pond-breeders deposit them on leaves dependent on dead prophylls and leaves of Dypsis palms overhanging the water from where the developing tadpoles for breeding sites. However, the rarity of female and drop into the water. By choosing egg-deposition sites on juvenile metamorphosed specimens that we collected plants containing potential phytotelmata, such as bamboo, suggests that this species may also occupy other habitats, Pandanus pines or Dypsis palms, it is easily conceivable which were not sampled effectively. Possibly, females, that, in some cases, the tadpoles dropped accidentally into juveniles and non-breeding males become more arboreal (as the phytotelmata and completed their development there. suggested by the enlarged finger and toe tips) and thus Slight changes of egg deposition mode that led to an occupy canopy, or are living in the forest leaf litter. Either increased probability of such accidental phytotelmic breed- of these possibilities would also explain how this species is ing may have been evolutionary favoured, thus leading able to find and colonise new Dypsis palms. The current from free water-breeding to accidental and eventually distribution area of B. angolafa coincides also with the obligatory phytotelmic reproduction. A second factor wettest region of Madagascar, with precipitations reaching favouring phytotelmic breeding might be the pond- 4,000 mm per year (Jenkins 1987). A high precipitation breeding mode itself. We assume that pond-dwelling rate, especially during the amphibian breeding period, tadpoles are able to better tolerate poor oxygen saturation could provide a critical limiting factor for the distribution than many stream tadpoles. Furthermore, Madagascar's of this species. Being obligated to breed in fallen palm rainforests are characterised by rather steep slopes and, in leaves and prophylls, it will be necessary to supply enough general, a limited amount of lentic water bodies which are water to last from egg laying till the end of the often temporary. This may constitute a particularly strong metamorphosing period (beginning of October to at least evolutionary pressure for pond-breeding species to use February–March). phytotelmic habitats for reproduction. The hypothesis of Masoala is about 320 km northwards of RNI de egg deposition outside water and pond breeding being Betampona and represents the northernmost site known factors favouring the evolution of phytotelmic breeding in for B. angolafa. Therefore, the relatively high 16S rRNA Madagascar leads to the prediction of non-obligatory gene uncorrected pairwise divergences found between the phytotelmic breeders, i.e., species that usually reproduce individuals of these localities is not surprising, although in ponds but sometimes also use phytotelmata, representing some of these pairwise divergences exceed the 16S rRNA an intermediate evolutionary stage. Such species may be gene 3% threshold that has been recently proposed to found in Guibemantis where apparently multiple shifts recognise candidate new species or deep conspecific between breeding in open waters and in phytotelmata have lineages (Vieites et al. 2009). A potential explanation for occurred (Lehtinen et al. 2007). It is also worth stating that, these deep divergences might be related to the historical in the phylogenetic tree reconstructed here, the phytotelmic distributions of the three Dypsis palms (Fig. 1), with B. angolafa is sister to B. grandisonae. At Masoala, we disjunct Dypsis distributions and corresponding disjunct found individuals assigned to B. grandisonae (as estab- amphibian populations isolating genetic lineages of B. lished by Glaw and Vences 2007) breeding within a rather angolafa, if this species is completely dependent on these large water-filled tree cavity (voucher specimens MRSN palms for providing habitat for reproduction. The other A3767-3768, collected by F.A. on 16 December 1999). two known sites for B. angolafa are separated from Thus, this could be a Blommersia species with facultative Betampona by far smaller geographic distances: 30 km phytotelmic adaptations and could highlight the adaptive for Zahamena and 110 km for Ambatovaky, and thus, we 542 Naturwissenschaften (2010) 97:525–543 suspect that genetic distances between these localities will CABS (Center for Applied Biodiversity Science) (2004) Madagascar. be lower than the 3% threshold. In: Miller L, Steininger M (Eds.) Conservation International hotspots high resolution basemap series. CD distributed by Center for Applied Biodiversity Science (CABS) and Conserva- Acknowledgements The Betampona field survey was carried out in tion International, Washington collaboration with the Parc Botanique et Zoologique de Tsimbazaza Cornet A (1974) Essai cartographique bioclimatique à Madagascar, (Antananarivo) and the Département de Biologie Animale (Antananarivo carte à 1/2’000’000 et notice explicative N 55. ORSTOM, University). F. Andreone's fieldwork was financially supported by Saint Paris Louis Zoo, Wildcare Institute and Gondwana Conservation and Dejean T, Miaud C, Ouellet M (2007) Proposition d’un protocole Research. C.J. Raxworthy's fieldwork was financially supported by d’hygiène pour réduire les risques de dissémination d’agents – – National Science Foundation (BSR 90 24505, DEB 93 22600, DEB infectieux et parasitaires chez les amphibiens lors d’intervention – 99 84496), CARE, and Conservation International. M. Vences did his sur le terrain. Bulletin de la Societé Herpétologique de France work through a co-operation with the Département de Biologie Animale 122:40–48 (Antananarivo University) and was supported by the Volkswagen Dransfield J, Beentje HJ (1995) The palms of Madagascar. Royal Foundation. We are grateful to the local staff of ANGAP and to the Botanic Gardens, Kew and The International Palm Society Malagasy authorities for permits supporting our research and collecting Dransfield J, Beentje HJ (1998a) Dypsis hovomantsina. In: IUCN activity. A. Ohler kindly allowed the loan of some crucial typic material 2010. IUCN Red List of Threatened Species. Version 2010.1. housed at Muséum national d'Histoire naturelle de Paris. J. Dransfield
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