Molecular and bioacoustic differentiation of Boophis occidentalis with description of a new treefrog from north-western Madagascar Author Vences, Miguel Author Andreone, Franco Author Glos, Julian Author Glaw, Frank text Zootaxa 2010 2544 54 68 journal article 10.5281/zenodo.196743 69e06eb0-113d-4a09-bfa8-f9bed8e01114 1175-5326 196743 Boophis tsilomaro sp. nov. (figures 2 and 4) Etymology. The specific name is used as a noun in apposition and is composed of the Malagasy words "tsilo", meaning "spine", and "maro", meaning "many". The name makes reference to the typical keratinized spicules on the dorsum and chest of breeding males. Remark. This species has been referred to as Boophis occidentalis by Andreone et al. (2002) and Vences et al. (2006), as Boophis sp. aff. occidentalis by Glaw & Vences (2007:166–167) and as Boophis sp. 4 in Vieites et al. (2009). 2002 ( FAZC 10667 , formalin fixed), adult male collected at Berara Forest (Sahamalaza Peninsula, north-western Madagascar ), 14°818.55'S and 47°854.92'E, 170 m a.s.l., Mahajanga Province, Analalava Fivondronana, Ambolobozo Firaisana and western part of the Befotaka Firaisana, on 20 February 2000 by F. Andreone, J. E. Randrianirina & M. Vences. Paratypes . MRSN A 1996 10667 , formalin fixed), adult male; MRSN A2004 ( FAZC 10666, formalin fixed), adult female; MRSN A1997 ( FAZC 10653), MRSN A1998 ( FAZC 10659), MRSN A 1999 ( FAZC 10661), MRSN A2003 ( FAZC 10695), four adult males. All the individuals have been collected at Berara Forest (Sahamalaza Peninsula, north-western Madagascar ), 14°818.55'S and 47°854.92'E, 170 m a.s.l., Mahajanga Province, Analalava Fivondronana, Ambolobozo Firaisana and western part of the Befotaka Firaisana, on 20–21 February 2000 by F. Andreone, J. E. Randrianirina & M. Vences. FIGURE 2. Preserved adult male specimens of (a) Boophis occidentalis (ZSM 2314/2007 from Isalo) and (b) Boophis tsilomaro (holotype MRSN A2002 from Berara forest). Both males were collected in breeding condition during emission of advertisement calls; note the slightly larger size of B. tsilomaro and the dense coverage of its dorsum with keratinized spicules. FIGURE 3. Sonagram and corresponding oscillogram of one note of a call of Boophis occidentalis (ZSM 2314/2007) from Isalo National Park. FIGURE 4. Photographs of Boophis tsilomaro in life, photographed at Berara forest in February 2000. (a) Two brownish-coloured and two green-coloured males as part of a breeding aggregation sitting in a shallow and slow-flowing part of a stream; (b) green-coloured male in the stream, visible on the stones are eggs of this species; (c) calling male; (d) brown-coloured male in dorsolateral view; (e) ventral view of same specimen as in (d). FIGURE 5. Photographs of Boophis occidentalis from Isalo National Park in life. (a–b) Male specimen ZSM 2314/2007 in dorsolateral and ventral view, and (c) same specimen at its calling position in a small cavity in a rock above a stream, photographed on 15 February 2007; (d) holotype (ZFMK 57383) collected on 29 January 1994. Additional material. Eggs and larval stages (all from Berara Forest): MRSN A2005 (tadpoles, preserved on 26 February 2000 , at 11 h); MRSN A2006 (tadpoles, preserved on 4 March 2000 , at 15 h); MRSN A2007 (tadpoles, preserved on 7 March 2000 , at around 14–16 h); MRSN A2008 (tadpoles, preserved on 9 March 2000 , at about 18 h); MRSN A2009 (tadpoles, preserved on 26 March 2000 , at about 20–22 h); MRSN A2010 (tadpole, preserved on 9 April 2000 ); ZSM 57/2001 (embryos, preserved on 22 February 2000 , at about 10 h); ZSM 54/2001 (embryos, preserved on 23 February 2000 , at about 9.15 h), ZSM 56/2001 (tadpoles, preserved on 24 February 2000 , at about 22 h); ZSM 192/2001 (eggs, laid on 20 February 2000 , fixed at about 24 h). Diagnosis. Assigned to the genus Boophis based on the presence of an intercalary element between ultimate and penultimate phalanges of fingers and toes (verified by external examination), enlarged terminal discs of fingers and toes, lateral metatarsalia separated by webbing, absence of outer metatarsal tubercle, molecular phylogenetic relationships (see Vieites et al. 2009 for a complete molecular analysis of Boophis ), and overall similarity to other Boophis species. Assigned to the Boophis albilabris group based on the following combination of characters: large size (male SVL 53–64 mm ); well developed webbing between fingers; presence of vomerine teeth; green colouration in life and colouration in preservative with a purple shade; presence of a white line along upper lip; molecular phylogenetic relationships (Vieites et al. 2009); and overall similarity to B. albilabris . Boophis tsilomaro differs from the other described species in the Boophis albilabris group by substantial genetic differentiation, with pairwise 16S divergences of 3.1–3.8% to B. occidentalis , and 4.5–6.0% to other species of the group. It further differs from B. praedictus and B. albilabris by the presence in most specimens of light dorsolateral lines which run from the eyes to the mid-flanks, by smaller size (adult SVL 53–64 mm vs. 62–89 mm and 76–93 mm , respectively) and by the colouration of the iris periphery (bluish vs. reddish, whitish or greenish). It mostly resembles its sister species B. occidentalis (see figure 5) from which it differs by a different iris colouration (outer iris ring orange-brown vs. metallic turquoise blue), a less distinct light dorsolateral stripe from the eye to mid-flanks (especially in preservative), larger size (SVL 53–64 mm vs. 46–52 mm SVL), a more distinct white line along the upper lip, and distinct differences in advertisement calls (see above). Description of the holotype . Adult male in breeding condition. Body moderately slender; head slightly longer than wide, slightly wider than body; snout rounded in dorsal view, obtuse in lateral view, nostrils directed laterally, nearer to tip of snout than to eye; canthus rostralis distinct, slightly concave in dorsal view, loreal region slightly concave; tympanum distinct, rounded, TD 67% of ED; supratympanic fold thin, distinct; vomerine odontophores distinct, well separated in two elongated patches, positioned posteromedial to choanae; choanae medium-sized, elongated. Tongue posteriorly bifid, free. Arms slightly thickened, with a white dermal edge from elbow to the lateral base of the finger. Subarticular tubercles single, round; metacarpal tubercles not recognizable; fingers broadly webbed; webbing formula 1(1), 2i (1.5), 2e(0), 3i (2), 3e(0), 4(0); relative length of fingers 1<2<4<3 (finger 2 distinctly shorter than finger 4); finger discs enlarged. A bony prepollex at the base of the first finger. Black keratinized nuptial pads on the base of the prepollex, and on the inner sides of fingers 1–3 as far as they are free of web, reaching the base of terminal finger discs. Hindlimbs slender; tibiotarsal articulation reaching centre of eye when hindlimb is pressed along body; lateral metatarsalia separated by webbing; inner metatarsal tubercle small, distinct, elongated; no outer metatarsal tubercle; toes almost fully webbed; webbing formula 1(0), 2i (0.25), 2e(0), 3i (0.5), 3e(0), 4i (0.5), 4e(0.5), 5(0); relative length of toes 1<2<5=3<4; toe discs enlarged. Skin granular on dorsal surfaces, especially where covered by black keratinized skin (almost whole head and back from snout to vent, external parts of lower arm, lower leg, tarsus and lateral sides of toes 4 and 5). Cloaca difficult to recognize, apparently concealed by a skin fold and oriented anteroventrally. Skin largely folded on the flanks, slightly granular on throat and chest, on belly and ventral surfaces of thighs except for a keratinized band along the lower lip and a second band of similar width ( 3–5 mm ). For measurements see table 1. After almost ten years in preservative, ground colour of dorsal upper surfaces is purple, which however is superimposed by large areas of skin covered by blackish keratinized spicules. A purple band below the canthus rostralis runs from snout tip to eye and is continued on ring surrounding the eye ventrally. The tympanum is purplish-brown. A narrow white band runs along the upper lip. Dorsal sides of fingers and toes pale yellowish. Upper flanks purple, lower flanks yellowish. A continuous pale yellowish narrow stripe on the lateral edges of lower arm and lateral finger and on the heel, and a discontinuous one along tarsus and lateral toe. All ventral surfaces pale yellowish except the areas along the lower lip, on the chest between the arms, and to a much lesser extent on the ventral side of the tarsus which are covered by blackish keratinized spicules. A light dorsolateral stripe from the eye to mid-flanks is not recognizable. Colouration in life unknown, but it might be expected that all purplish parts have been green. Variation. Measurements of the paratypes are given in table 1. Most of the paratypes generally resemble the holotype in morphology and colouration (especially all other males which have similar areas of black keratinized skin), but several differences are evident. MRSN A2001 has a brown colouration without any traces of purple. The dorsal skin is granular, but only fragmentary spicule cover is present on the sides of the head, along lower lip, on chest and elbows and on few other small patches of the dorsal surface. This may indicate that the level of sexual activity was already decreasing when the frog was collected, although black nuptial pads are still present on fingers 1–3. MRSN A2004 and MRSN A1996 are females with rather uniform purple dorsal colouration and distinct light dorsolateral stripes behind the eye. MRSN A1996 has well developed eggs with a light and a dark pole in the body cavity. The body cavity of most paratypes (all except MRSN A1997 and MRSN A2001) was opened for further studies. The life colouration of several specimens is shown in figure 4. According to these photographs dorsal colouration varies from green to olive grey, partly superimposed by blackish keratinized spicules in males. Inner iris ring brown, outer iris ring golden, posterior iris periphery blue. The webbing between the toes is red, the dorsal side of fingers and toes is yellow. Throat whitish, belly and ventral parts of limbs beige to orange-brown. Natural history. All available natural history data were published by Andreone et al. (2002): Individual age assessed by skeletochronology ranged from 4 to 11 years. Breeding behaviour was observed at a seasonal stream in subhumid forest after heavy rainfalls: Five choruses of eight to 90 males aggregated in shallow brook sections. Calling males were engaged in scramble battles. A description of the call is provided above and in Andreone et al. (2002). The call is documented in Vences et al. (2006) under the name Boophis occidentalis . Only two females were found. A pair laid a large number of eggs, attached as a single layer of isolated eggs on submerged stones. Tadpoles reared from these eggs had the typical morphology of streambreeding Boophis species with a 6(2–6)/3 keratodont formula and a relatively depressed body; see Andreone et al. (2002) for a detailed tadpole description. TABLE 2. Diagnostic characters and morphometric measurements (in mm) of adult males and females of Boophis occidentalis and B. tsilomaro sp. nov , summarized as mean (in bold) ± standard deviation, and minimum-maximum in parentheses. The population from the Tsingy de Bemaraha is not considered here (see text for a discussion of their characters). For abbreviations, see Materials and Methods and caption to Table 1. Summarized from original data in Table 1. States of the last three characters refer to both males and females of each species. B. tsilomaro B. occidentalis males females males females Distribution. Only known from the type locality at Berara (figure 6). The population from Tsingy de Bemaraha, which resembles both Boophis tsilomaro and B. occidentalis , needs further study but despite its similarity in external morphology is probably not referable to the new species described herein based on the combined evidence from its life colouration, relative small size of 44 mm (compare with table 1) and molecular relationships (see phylogenetic tree in figure 1).