Bioacoustics of Gephyromantis mafy

Etymology. The species name is derived from the Malagasy word mafy meaning strong and difficult, and is used as a noun in apposition. It makes reference to the persistence of species like G. m a f y in degraded areas subject to intensive illegal wood exploitation, and also to the difficulty to find them and collect them in the field. Natural history. A chorus of three or four specimens of the new species was heard at a single site of the Mahasoa forest during our survey. The site was in a highly disturbed rainforest, next to a large fallen tree. Specimens were sitting in dense shrub between branches of the falling tree, on a relatively steep slope, about 50 cm perch height above the ground. They were calling during the day and at dusk. Advertisement call. The single collected specimen was collected after it was observed emitting its advertisement call. Because the observation was made from some distance, no clear observation of the shape of the inflated vocal sac (single, bilobed or paired) could be made. Due to logistic reasons we did not record the temperature, but we estimate that during the recording this was around 25°C. The advertisement call of Gephyromantis mafy recorded next to our campsite in Mahasoa forest, on 14 February 2008 at 16:45, consists of a series of 15–16 (n=3) pulsed notes, repeated at regular intervals ( Fig. 3–4 ). Numerical parameters are as follows (range followed by mean ± standard deviation): note duration, 233–321 ms (258 ± 24; n = 15); inter-note interval, 601–1359 ms (807 ± 258; n = 14); pulse rate in notes, ca. 385 ± 23.7 pulses/second (332–415, n=15), dominant frequency, 4306–4392 Hz (4332 ± 39; n = 14). Overall frequency is distributed in a narrow band from ca. 3270–5860 Hz, with some lowintensity band at around 3000 Hz probably representing the fundamental frequency. Notes exhibit some overall amplitude modulation, starting with high calling energy which continuously decreases towards the end of the note. Pulses are not very regular and not in all cases clearly discernible, but at an order of magnitude of about 100 pulses per note. Molecular differentiation. A comparison of DNA sequences of the mitochondrial 16S rRNA gene and Rag1 as available from the work of Kaffenberger et al. (2011) yielded distinct genetic divergences between G. m a f y and its close relatives. Kimura 2 parameter genetic distances between all species of the genus Gephyromantis yielded values between 2.4% and up to 25% divergence in 16S, and 0.1 and 4.7 in Rag1 ( Fig. 5 ). When comparisons are made within the nominal subgenus Gephyromantis , the pairwise comparisons give values between 2.6% and 13.9% in 16S, and 0.1 and 2.1 in Rag1. The distances between G. m a f y and it closest relatives ( G. eiselti and G thelenae ) range between 3.4–4.8% in 16S and 0.1–0.3% in Rag1, being similar or slightly higher than other species pairs comparisons ( Fig. 5 ). In contrast, the morphologically similar G. e n k i was very strongly differentiated (10-11.7% to the other three species), in agreement with its placement in a different subclade of the subgenus Gephyromantis (see Kaffenberger et al. 2011 ). The analysis of cytochrome b sequences (542 bp) showed that the mitochondrial divergence of the three closely related species, G. eiselti , G. mafy , and G. thelenae , is also found in additional specimens. All studied individuals of G. eiselti (three specimens from Ambavaniasy) had very similar or identical sequences different from those of the other species, and the same was true for all G. thelenae (ten specimens from two nearby sites, Andasibe and Camp Prolemur). Cytochrome b distances were 7.0–7.2% between G. eiselti and G. t h e l e n a e, 6.8–7.0% between G. eiselti and G. m a f y, and 4.6–4.8% between G. thelenae and G. mafy . Gephyromantis enki had 16.6–18.1% divergence to the other three species. Partial sequences of the nuclear Rag-1 gene (573 bp) were available from seven specimens of G. thelenae , two specimens of G. eiselti , and the holotype of G. m a f y . Not counting sites with heterozygous positions (which were usually variable also within species), all G. thelenae were separated by two substitutions from G. eiselti and G. m a f y, and G. eiselti by one position from G. m a f y and G. thelenae .

Diagnosis. A member of the subgenus Gephyromantis in the genus Gephyromantis on the basis of (1) presence of intercalary elements between ultimate and penultimate phalanges of fingers and toes (verified by external examination), (2) small size (SVL below 35 mm ), (3) distinctly enlarged terminal discs of fingers, (4) presence of outer metatarsal tubercle (even if barely recognizable), (5) absence of webbing on hands and presence of only rudimentary webbing on feet, (6) tight connection of tissue surrounding the two lateral metatarsalia, (7) presence of femoral glands in males, (8) presence of paired or bilobed blackish vocal sacs in males, (9) diurnal calling behaviour not concentrated at water bodies, (10) phylogenetic position, as supported by nuclear and mitochondrial genes, within the genus Gephyromantis . Within the subgenus, distinguished from all other species by combination of (1) small size (male SVL 20 mm ), (2) dorsum smooth, without ridges or tubercles, (3) upper lip uniformly whitish, (4) vomerine teeth absent, (5) lower lip ventrally with a yellowish tint in life; (6) a reddish tint on ventral side of thighs in life; (7) body slender, forelimbs and hindlimbs relatively short (tibiotarsal articulation reaching nostril when hindlimbs are adpressed along body), (8) calls consisting of a slow series of about 15 notes of distinctly pulsed structure with a low note repetition rate of about 1 per second (9) showing genetic differences in nuclear and mitochondrial genes that characterize it as genetically distinct from other species in the genus. The species is morphologically most similar to Gephyromantis eiselti , G. enki , and G. t h e l e n a e, and is closely related to two of these species ( G. eiselti and G. thelenae ) according to the multigene phylogenetic analysis of Kaffenberger et al. (2011) . It differs from G. e n k i by absence of dorsolateral ridges (vs. presence of weakly recognizable outer dorsolateral ridges), absence of vomerine teeth (vs. presence of weakly expressed vomerine teeth), distinctly longer note duration (233–321 vs. 31–34 ms), and distinctly lower note repetition rate (1 vs. 2.2–2.3 per second) in advertisement calls; from G. eiselti by a pulsed structure of advertisement call (vs. tonal); and from G. thelenae by a distinctly shorter note duration (233–321 vs. 560–610 ms) and higher note repetition rate (1 vs. ca. 0.5 per second) in advertisement calls. Other species in the subgenus Gephyromantis have different advertisement calls as well: G. b l a n c i, G. boulengeri , G. leucocephalus and G. runewsweeki emit series of short and largely melodious notes (note duration typically <100 ms) with short inter-note intervals (<100 ms). G. decaryi is the only other nominal species in the subgenus having calls of a somewhat pulsed structure, but also of short note duration (<100 ms) and inter-note interval duration (<200 ms) and thus very different from G. m a f y (note duration> 200 ms, inter-note interval duration> 500 ms). The new species is furthermore differentiated from all other species of Gephyromantis by a significant molecular differentiation (see Vieites et al. 2009 , reported as G. sp. 26 therein; and below).