Not all little brown frogs are the same: a new species of secretive and cryptic Gephyromantis (Anura: Mantellidae) from Madagascar Author Vieites, David R. Author Wollenberg, Katharina C. Author Vences, Miguel text Zootaxa 2012 3344 34 46 journal article 10.5281/zenodo.281463 dfa09c44-1ef4-4d36-addc-b2decc0e1611 1175-5326 281463 Gephyromantis mafy sp. nov. ( Fig. 1 ) Remark. This species has been included as confirmed candidate species Gephyromantis sp. 26 in the molecular survey of Malagasy frog diversity published by Vieites et al. (2009) , and under the same name by Wollenberg et al. (2011) and Kaffenberger et al. (2011) . Holotype . ZSM 1889/2008 (fieldnumber ZCMV 8100), adult male collected at Mahasoa campsite, near Ambodisakoa village, northeast of Vohimena at the northeastern edge of Lake Alaotra, Faritany (province) of Toamasina, northern central east of Madagascar , 17.29769ºS , 48.70199ºE , 1032 m asl, on 14 February 2008 , by David R. Vieites, J. Patton, C. Patton, P. Bora and M. Vences. 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). Description of the holotype . Adult male, in good state of preservation; muscles from right thigh removed as tissue sample. SVL 20.4 mm (see table 1 for complete list of measurements). Body slender; head slightly longer than wide; snout acuminated in dorsal view, truncate in lateral view; nostrils directed laterally, slightly protuberant, much nearer to tip of snout than to eye; canthus rostralis distinct, concave; loreal region concave; tympanum distinct, elliptical (slightly higher than wide), less than half of eye diameter; supratympanic fold very distinct, straight; tongue ovoid, distinctly bifid posteriorly; vomerine teeth absent. A dark dermal fold (the inflatable parts of the vocal sacs) is running along each lower jaw from the commissure of the mouth to one third of the lower jaw. Arms slender; a very poorly developed inner and outer metacarpal tubercles, subarticular tubercles single; fingers without webbing; relative length of fingers 1<2<4<3, second finger distinctly shorter than fourth finger; finger disks distinctly enlarged; nuptial pads absent. Hindlimbs slender; tibiotarsal articulation reaching nostril when hindlimb is adpressed along body; lateral metatarsalia connected; inner metatarsal tubercle very distinct, outer metatarsal tubercle almost not recognizable; webbing formula between toes 1(1), 2i (1.5), 2e(1), 3i (2.5), 3e(2), 4i /e(3), 5(2); relative toe length 1<2<3≤5<4. Toe disks enlarged. Skin on the upper surface smooth; ventral skin smooth on throat and limbs, granular on belly. Femoral glands well delimited, with 12 conspicuous granules per gland of yellow color in life (less distinct in preservative), situated in the third proximal part of the thigh. FIGURE 1. Gephyromantis mafy sp. nov. , (A) dorsolateral and (B) ventral view of male holotype. FIGURE 2. Distribution map of Gephyromantis mafy , G. eiselti and G. t h e l e n a e . TABLE 1. Morphometric measurements (all in mm) of examined voucher specimens of Gephyromantis mafy (holotype specimen), G. thelenae , G. eiselti , and G. enki . For abbreviations of morphometric measurements and collection acronyms, see Materials and Methods. For measurements of the types of G. eiselti , G. thelenae , and G. e n k i, see Glaw & Vences 2002b). RHL (relative hindlimb length) is coded as follows: when hindlimb is adpressed along body, tibiotarsal articulation reaches (1) between eye and nostril, (2) nostril, (3) snout tip, (4) beyond snout tip. Locality Field No Collection No SVL HW HL TD ED END Gephyromantis mafy sp. nov. Mahasoa forest ZCMV 8100 ZSM 1889/2008 20.4 6.4 7.7 1.4 2.9 2.0 The overall coloration in life is light brown with pinkish legs and a distinct facial dark mask contrasting with pale upper lips. Dorsal body color light brown, not uniform but consisting of three different tones (between brown and green) arranged in small inconspicuous patches, becoming darker towards the head (see Fig. 1 ). Dorsal color of forelimbs has the same pattern as the dorsum but with lighter tones of brown, which is more evident after two years in preservative. Dorsal color of hindlimbs as body but with a pinkish tone towards the sides and the ventral side, which is lost in preservative. The dorsal part of the head shows a contrasting color pattern, with an increasingly darker proximal region towards the eyes, where the color changes abruptly to light brown ca. 1 mm before the anterior edge of the eye towards the nostril. A broad lateral dark brown line goes from the posterior part of the head towards the eye, including the tympanum, becoming narrower and less conspicuous between the eye and the nostril where it ends. The area between this dark brown and the upper jaw is very distinct with a contrasting bright white coloration from the posterior section of the head to the anterior part of the eye, where it turns into a duskier and less conspicuous white towards the nostril. The lower lips are creamy white alternating with flecks of light brown. Ventral side of chest is light pink with a poorly defined and faint white stripe, belly light pink with white granular spots not present on limbs. Ventral side of arms and legs uniform light pink, but with vermiculations of red, especially on the thighs, giving the overall impression of reddish legs. Iris golden with dark discontinuities in inner and posterior iris areas.

Gephyromantis thelenae	Mean±SD 20.5±1.3	6.7±0.4	8.4±0.5	1.4±0.2	2.9±0.3	2.2±0.3
Andasibe ZCMV 8137	UADBA uncatalogued 20.6	6.7	8.1	1.3	2.6	2.3
Andasibe ZCMV 8135	ZSM 1883/2008 19.6	6.2	8.0	1.5	2.7	2.1
Andasibe ZCMV 8114	UADBA uncatalogued 19.0	6.6	9.7	1.5	2.7	2.5
Andasibe ZCMV 8131	ZSM 1881/2008 20.0	6.3	7.8	1.3	2.6	2.0
Andasibe ZCMV 8136	ZSM 1884/2008 20.0	6.2	7.9	1.4	2.6	2.4
Andasibe ZCMV 8134	ZSM 1882/2008 19.5	6.9	8.3	1.4	3.2	2.4
Andasibe ZCMV 8144	ZSM 1885/2008 21.6	7.4	8.9	1.5	3.0	2.5
Camp Prolemur ZCMV 8765	UADBA uncatalogued 20.9	6.5	8.1	1.2	2.7	2.2
Camp Prolemur ZCMV 8793	ZSM 1887/2008 21.9	6.4	8.1	1.4	3.1	2.3
Camp Prolemur ZCMV 8147	ZSM 1888/2008 20.8	7.5	8.4	1.4	2.8	2.6
Camp Prolemur ZCMV 8148	ZSM 1886/2008 18.7	6.4	8.7	1.3	2.9	2.0
Camp Prolemur ZCMV 8764	UADBA uncatalogued 23.8	7.1	8.5	1.3	3.5	1.7
Camp Prolemur ZCMV 8149	UADBA uncatalogued 20.2	7.0	8.8	2.0	3.0	1.9
Camp Prolemur ZCMV 8150 Gephyromantis eiselti	UADBA uncatalogued 20.8 Mean±SD 20.5±1.0	6.8 6.2±0.4	8.8 7.7±0.3	1.3 1.1±0.1	3.0 2.8±0.2	2.0 1.9±0.3
Ambavaniasy ZCMV 8145	ZSM 1856/2008 19.8	6.3	7.8	1.0	3.1	1.6
Ambavaniasy ZCMV 8146	ZSM 1857/2008 20.0	6.6	7.9	1.2	2.8	1.9
Ambavaniasy ZCMV 8113 Gephyromantis enki	ZSM 1855/2008 21.6 Mean±SD 20.0±0.7	5.8 6.8±0.5	7.4 8.7±0.8	1.1 1.4±0.2	2.7 2.8±0.3	2.3 2.3±0.2
Sakaroa ZCMV 8160	UADBA uncatalogued 20.1	6.6	8.4	1.6	3.3	2.1
Talatakely 2 ZCMV 8199	UADBA uncatalogued 19.8	6.0	8.2	1.5	3.0	2.3
Ranomafanakely ZCMV 5323	UADBA uncatalogued 18.7	6.7	7.7	1.4	2.4	1.9
Ranomafanakely ZCMV 5318	UADBA uncatalogued 20.8	7.6	9.5	1.4	3.0	2.5
Ranomafanakely ZCMV 5317	UADBA uncatalogued 20.0	6.9	8.1	1.0	2.4	2.2
Valbio Campsite ZCMV 5327	UADBA uncatalogued 20.7	6.9	9.4	1.3	2.8	2.3
Kidonavo ZCMV 5345	UADBA uncatalogued 19.8	7.0	9.8	1.3	2.6	2.4

continued.
Locality END	NSD	NND	FORL	HAL	HiL	FOTL	FOL	TL	RHL
Gephyromantis mafy sp. nov.
Mahasoa forest 2.0	1.3	2.1	12.7	6.2	35.1	15.7	9.4	11.5	2
Gephyromantis thelenae 2.2±0.3	1.5±0.3	2.1±0.2	13.3±0.7	6.3±0.4	35.3±1.6	16.0±0.8	9.6±0.7	11.9±0.6
Andasibe 2.3	1.6	2.0	12.9	6.0	34.9	15.8	9.8	11.7	3
Andasibe 2.1	1.2	2.1	13.0	5.7	34.2	15.3	9.2	11.1	3
Andasibe 2.5	1.9	2.1	12.7	6.0	33.6	15.4	9.3	11.1	3
Andasibe 2.0	1.4	2.0	13.1	6.3	36.5	16.0	9.5	12.7	4
Andasibe 2.4	1.3	2.1	13.2	6.4	35.8	16.5	9.7	12.0	4

Andasibe	2.4	1.7	2.5	12.8	6.4	35.7	15.7	9.6	11.9	4
Andasibe	2.5	2.0	2.5	14.3	7.1	36.7	16.4	10.4	12.1	3
Camp Prolemur	2.2	1.4	2.0	12.0	6.2	32.9	15.3	10.3	11.4	2
Camp Prolemur	2.3	1.6	2.1	14.6	6.3	34.3	15.7	8.1	12.6	2
Camp Prolemur	2.6	1.3	2.2	13.6	6.2	35.4	15.8	9.6	11.3	3
Camp Prolemur	2.0	1.2	1.9	13.2	6.3	32.5	16.1	9.3	12.1	3
Camp Prolemur	1.7	1.3	2.0	13.3	6.1	37.0	16.3	9.7	12.1	2
Camp Prolemur	1.9	1.5	2.0	14.4	7.0	37.6	18.5	10.9	12.8	4
Camp Prolemur	2.0	1.1	2.0	13.0	6.2	36.4	15.5	9.7	11.6	4
Gephyromantis eiselti	1.9±0.3	1.4±0.1	1.9±0.1	12.0±0.5	6.3±0.4	35.9±0.3	16.6±0.6	9.9±0.8	11.5±0.6
Ambavaniasy	1.6	1.5	2.0	11.7	6.8	35.8	16.7	9.8	11.5	3
Ambavaniasy	1.9	1.2	1.9	12.7	6.2	36.3	17.2	10.7	12.1	3
Ambavaniasy	2.3	1.5	1.9	11.7	6.0	35.7	16.0	9.2	11.0	2
Gephyromantis enki	2.3±0.2	1.2±0.2	2.0±0.1	11.0±2.4	7.0±2.8	34.6±2.0	16.0±0.9	9.7±0.4	12.0±0.5
Sakaroa	2.1	1.4	2.0	5.6	13.2	36.4	16.1	9.7	11.7	4
Talatakely 2	2.3	1.5	2.0	11.3	5.7	34.6	15.1	9.3	11.6	3
Ranomafanakely	1.9	1.2	2.0	11.7	5.1	33.3	14.7	9.0	11.6	2
Ranomafanakely	2.5	1.2	2.0	11.4	6.1	36.4	16.8	10.2	12.4	2
Ranomafanakely	2.2	1.0	2.0	11.4	6.0	37.0	16.8	10.0	12.4	3
Valbio Campsite	2.3	1.0	2.0	13.0	6.8	32.4	17.0	10.0	12.8	3
Kidonavo	2.4	1.4	1.8	12.4	5.7	32.3	15.8	10.0	11.4	1

In preservative the overall coloration is similar to that in life but colors are considerably faded; in particular, the the reddish color on the ventral side of arms and thighs, and the faint white stripe present on the ventral side of the chest, are lost. 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. FIGURE 3. Spectrogram and corresponding oscillogram of the advertisement call of Gephyromatis mafy sp. nov. from Mahasoa, recorded on 14 February 2008. The lower oscillogram is a close up of a single note, showing its clearly pulsed structure. 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. FIGURE 4. Comparison of oscillograms between G. m a f y sp. nov. , G. eiselti , G. thelenae and G. enki , and corresponding oscillogram of the advertisement call of Gephyromatis mafy sp. nov. from Mahasoa, recorded on 14 February 2008. Note the clear differences between species and how the call of G. m a f y is intermediate between the calls of G eiselti and G. thelenae . Calls of G. eiselti , G. thelenae and G. enki are from Vences et al. (2006). 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 ). FIGURE 5. Kimura 2 parameter pairwise genetic comparisons, based on the loci 16S and RAG1 between all species of the genus Gephyromantis , all species of the subgenus Gephyromantis , and between G. m a f y sp. nov. and the rest of species of the subgenus Gephyromantis . Data for these comparison is taken from 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 .