Material and methods
Tadpoles were collected in the field, euthanised by immersion in chlorobutanol solution, and immediately sorted into homogeneous series based on morphological characters. From each series one specimen was selected and a tissue sample from its tail musculature or fin taken and preserved in 99% ethanol. This specimen is here named 'DNA voucher'. After tissue collection, all specimens were preserved in 4% formalin. Specimens were deposited in the Zoologische Staatssammlung München, Germany (ZSM); comparative specimens were examined from the herpetological collection of the Zoological Museum Amsterdam, Netherlands (ZMA).
Tadpoles were identified using a DNA barcoding approach based on a fragment of the mitochondrial 16S rRNA gene, which is known to be sufficiently variable among species of Malagasy frogs (Thomas et al., 2005). The 550 bp fragment was amplified using primers 16Sa-L and 16Sb-H from Palumbi et al. (1991) applying standard protocols, resolved on automated sequencers, and compared to a near-complete database of sequences of adult Malagasy frog species. Identification was considered to be unequivocal when the tadpole sequence was 99-100% identical to an adult specimen from the same geographical region, and not more similar to any sequence from another species. DNA sequences were deposited in Genbank (accession numbers DQ060232-DQ060237; accession numbers of comparative adult specimens are included in the sequence set AY847959-AY848683).
Drawings and descriptions of the tadpoles in this paper are based only on the DNA vouchers. All specimens of each series were examined to assess morphological variability and to complete the structures missing because of tissue sampling in the DNA vouchers. Developmental stages are based on Gosner (1960). The labial tooth row formula (LTRF) and the morphological descriptions in general follow Altig and McDiarmid (1999). Labial tooth row fomula is in addition also given according to the notation of Dubois (1995). The measurements taken using a stereomicroscope with measuring device and converted later into millimetres are: BL = body length (from the tip of the snout to the junction of the posterior body wall with the axis of the tail myotomes); BW = body width (the widest point of the ‘head’ right behind the eyes); ED = eye diameter; IOD = interorbital distance (from the centre of each pupil); TH = tail height (at the beginning of the tail); TMW = tail muscle width (at the beginning of the tail); MTH = tail height at mid-length of tail (including caudal fin); TMH = tail muscle height (at mid-length of tail); TAL = tail length; ODW = maximum oral disc width; DGMP = dorsal gap of the marginal papillae. The mouth parts include: MP = number of marginal and SMP = submarginal papillae; LMP = length of the marginal papillae; UTR = upper tooth row; LTR = lower tooth row; NT = number of teeth; LLTR = length of the tooth row.