Zootaxa 1334: 27–43 (2006) ISSN 1175-5326 (print edition) www.mapress.com/zootaxa/ ZOOTAXA 1334 Copyright © 2006 Magnolia Press ISSN 1175-5334 (online edition) Discovery of a new basal relict lineage of Madagascan frogs and its implications for mantellid evolution FRANK GLAW1, SIMONE HOEGG2 & MIGUEL VENCES3 1Zoologische Staatssammlung, Münchhausenstr. 21, 81247 München, Germany 2Lehrstuhl für Evolutionsbiologie, Department of Biology, University of Konstanz, 78457 Konstanz, Germany 3Division of Evolutionary Biology, Zoological Institute, Technical University of Braunschweig, Spielmannstr. 8, 38106 Braunschweig, Germany Corresponding author. E-mail: Frank Glaw, [email protected] Abstract Frogs of the subfamily Mantellinae (Amphibia: Anura: Mantellidae) are a species-rich and diverse lineage endemic to the Madagascan region. The major synapomorphy of this clade is a derived reproductive mode including an unusual mating behaviour (loss of strong mating amplexus, egg deposition outside of water) and associated morphological adaptations (evolution of femoral glands, loss of nuptial pads). However, the evolutionary steps towards this unique character complex remain obscure. We here describe a recently discovered new frog, Tsingymantis antitra gen. nov., sp. nov. from the moderately dry karstic massif Tsingy de Ankarana in northern Madagascar. The new species is not referable to any existing genus or species groups. A phylogenetic analysis, based on DNA sequences of four mitochondrial genes (12S and 16S rRNA, tRNAVal, cytochrome b) and one nuclear gene (rhodopsin) placed Tsingymantis without significant support as sister taxon of the Mantellinae which was found to be a well-defined monophyletic group (100% Bayesian and 99% bootstrap support). The position of Tsingymantis as the most basal clade of the Mantellinae is in agreement with several morphological and osteological characters, suggesting that this subfamily including Tsingymantis may be a monophyletic group whereas the Boophinae could represent the most basal clade of the Mantellidae. We therefore include Tsingymantis in the Mantellinae in a preliminary way, pending further study. In contrast to the large majority of recent mantellid species which are adapted to humid rainforests, the most basal clades of the three subfamilies show adaptations to relatively dry conditions, indicating that the climate during the early radiation of mantellids (probably in the Eocene) may have been drier than in recent times. Key words: Anura, Mantellidae, Tsingymantis antitra gen. nov., sp. nov., molecular phylogeny, Madagascar Accepted by S. Carranza: 17 Aug. 2006; published: 16 Oct. 2006 27 ZOOTAXA Introduction 1334 The amphibian fauna of Madagascar includes more described species (231) than any other African country (e. g. http://amphibiaweb.org) and has by far the highest rate of endemism as well (> 99% at species level). Most of these species belong to the Mantellidae which is endemic to Madagascar and Mayotte Island. This family is divided into the three subfamilies Mantellinae, Boophinae, and Laliostominae which can be diagnosed independently by both, non-molecular and molecular characters (Vences & Glaw 2001; Glaw & Vences 2006). With 106 described species and an enormous diversity in morphology, behaviour and habits, the subfamily Mantellinae represents the most successful lineage of Malagasy frogs. This clade is mainly characterized by a derived reproductive mode including behavioural and morphological apomorphies, but the evolutionary steps towards this unique reproductive biology remain obscure. In the past 35 years a large number of new frog species has been discovered in Madagascar, most of them representatives of genera or species groups that were known since long. The most recent discoveries of new genera were Madecassophryne Guibé, 1974, based on a single species (Madecassophryne truebae), and Paradoxophyla Blommers-Schlösser & Blanc, 1991, based on a single species as well (Microhyla palmata). Both these species were discovered by Charles P. Blanc in 1971 in south-eastern Madagascar (Guibé 1974) and both are rather small (20–25 mm snout-vent length [SVL]). Very recently, the genus Wakea Glaw & Vences, 2006 was established. The single species of this genus was only discovered in 2001 and represents (with a SVL of 11–16 mm) the smallest mantellid known so far (Vences et al. 2002a). W. madinika is morphologically very similar to the species in the genus Blommersia, but turned out to be the sister group of the genus Mantella by genetic studies (Vences et al. 2003, see also Fig. 3). All other new genus group names that have been established for Madagascan frogs in the past decades (Dubois 1992; Glaw & Vences 1994, 2006; Glaw et al. 1998) were based on previously known species or species groups, indicating that all major lineages of Madagascan frogs might have been discovered. A similar situation is found on a global scale. Despite the enormous amount of amphibian species which have been described in recent years, the discovery of deeper new lineages, such as new genera, has been a rare event in the past decades and sometimes received remarkable attention (Biju & Bossuyt 2003; Min et al. 2005). Of the 35 extant (non-fossil) and valid amphibian genera named since 1990 (extracted from Dubois 2005) 22 include species that were discovered before 1990 (extracted from Frost 2004) and therefore do not represent discoveries of new lineages. They are the result of splitting of known genera, usually based on increased phylogenetic resolution (e.g., new genera erected by Frost et al. 2006). Only 13 genera were based on (and include only) species that were described since 1990 (Minervarya, Nasikabatrachus, Protohynobius, Karsenia, Churamiti, Metaphryniscus, Truebella, Aromobates, Rupirana, Bryobatrachus, Spicospina, Altigius, and Ericabatrachus). During surveys in the Ankarana reserve in northern Madagascar in the years 2003 and 28 © 2006 Magnolia Press GLAW ET AL. 2004 we discovered four female specimens of a large new frog species (SVL 66–67 mm) ZOOTAXA that was not referable to any genus or subgenus of Madagascan frogs. 1334 In the present paper we describe this remarkable new mantellid genus and species and investigate its phylogenetic relationships. We then discuss its potential to understand the evolution of the derived reproductive modes in mantellines and its relevance to resolve the relationships between the three mantellid subfamilies. Materials and methods Specimens were collected at night with the help of torches by opportunistic searching. Vouchers were fixed in 90% ethanol and subsequently stored in 70% ethanol. Museum acronyms used are UADBA (Université d’Antananarivo, Département de Biologie Animale), ZFMK (Zoologisches Forschungsmuseum A. Koenig, Bonn), and ZSM (Zoologische Staatssammlung, München). The following morphological measurements were taken with dial calipers to the nearest 0.1 millimeter: SVL (snout-vent length), HW (head width), HL (head length), ED (horizontal eye diameter), END (eye-nostril distance), NSD (nostril-snout tip distance), NND (nostril-nostril distance), TD (tympanum diameter), HAL (hand length), FORL (forelimb length), HIL (hindlimb length), FOL (foot length), FOTL (foot length including tarsus), TIBL (tibia length). DNA was extracted from muscle tissue samples preserved in ethanol. We complemented the data set of Glaw & Vences (2006) which consists of partial fragments of four mitochondrial genes (12S rRNA, 16S rRNA, tRNAVal, cytochrome b) and one nuclear gene (rhodopsin) by amplifying and sequencing these fragments for the new species from Ankarana, from one crucial species missing in the previous dataset (Gephyromantis pseudoasper, a representative of the subgenus Phylacomantis), and by filling gaps of sequences that were previously missing. Detailed information on DNA extraction, primers, PCR and sequencing are given in Glaw & Vences (2006) and Vences et al. (2003). Newly obtained sequences were submitted to Genbank (accession numbers DQ901378- DQ901396; see appendix for a complete list of accession numbers). Sequences were aligned using ClustalW (Thompson et al. 1994) as implemented in Bioedit (Hall 1999) and alignments were refined manually. Positions with gaps or positions that could not be aligned reliably were excluded from further analyses as were the third codon positions of cytochrome b. The complete dataset encompassed 58 sequences and 2303 positions. Bayesian inference was performed using MrBayes 3.1 (Huelsenbeck & Ronquist 2001; Ronquist & Huelsenbeck 2003) on a partitioned dataset with partitions for each single gene and 1st and 2nd positions from cytochrome b. Models of sequence evolution and the analysis parameters (proportion of invariable sites and gamma-shape parameter alpha) were estimated using Modelgenerator (Keane et al. 2004). We ran 100,000 MANTELLINAE © 2006 Magnolia Press 29 ZOOTAXA generations while sampling every 10th tree and a burn in value of 5000. The Maximum 1334 Likelihood analyses were performed with PhyML (Guindon & Gascuel 2003) with parameters estimated for the complete dataset. We ran 500 bootstrap replicates. Results Tsingymantis gen. nov. Type species and only included species. Tsingymantis antitra sp. nov. Etymology. Derived from "tsingy", the Malagasy word for eroded karstic limestone formations and the Greek word mantis = treefrog (see Vences et al. 1999 for the derivation of mantis). The genus name refers to the habitat, the tsingy formations of Ankarana. The gender of this genus is masculine.