Bioacoustics of Phrynobatrachus horsti

Distribution. Phrynobatrachus horsti sp. nov. is known from several localities across south-western Republic of the Congo (Kouilou and Niari Departments) and adjacent Gabon ( Fig. 4 ). The latter locality has been published as a record of the morphologically similar P. ruthbeateae . However, the photos and genetics of the Gabon individuals ( Zimkus & Larson 2013a , b ), confirm that these frogs are P. horsti sp. nov. (Appendix 1, Table 2 ). In contrast, it is not clear if the small puddle frogs termed Phrynobatrachus sp. 2 by Burger et al . (2004) , originating from mid-altitude forests on the Monts Doudou, Gabon , are P. ruthbeateae , P. horsti sp. nov. or a third species. So far it seems that the new species may have a distribution in Central African lowland to mid-altitude rainforests inbetween the Ogooué and the Congo Rivers. Habitat and natural history. Our P. horsti sp. nov. records stem from leaf litter in disturbed and undisturbed lowland to mid-altitude rainforests, including swamp forests ( Fig. 5 ). The altitudinal range of P. horsti covers altitudes from 554–728 m a.s.l. in the Mayoko area, but drops below 100 m in the Kouilou Department. The species seems to breed in small and shallow stagnant pools, including holes excavated by artisanal gold miners. We heard many calling males at puddles in a logged forest patch on 20 and 21 October 2012 . Three males and a female were found active on the forest floor near the edge of a stream on 26 July 2012 . This is comparable to the habitat choice and calling sites of the closely related P. r u t h be a t e a e ( Rödel et al. 2012a ). It is not entirely clear in which type of habitat P. horsti was recorded on the near-by Batéké Plateau in Gabon ( 400–840 m a.s.l.) by Zimkus & Larson (2013a , b ). From the site descriptions it seemed to be a mix of savannah and gallery forest. In their table these authors mark the species for savannah, farmbush, gallery forest and forest ( Zimkus & Larson 2013b ). A range of habitats from savannah to forest is unusual for the genus. Most other congeners show clear species specific habitat preferences (e.g. Rödel & Ernst 2002b ; Rödel et al. 2004 ; Channing & Howell 2006 ; Kouamé et al. 2008 ). Comparable to us, Zimkus & Larson (2013b) state that they collected the frogs from leaf litter.

Description of holotype [measures in mm]. Typical, small adult male Phrynobatrachus with short oval, compact body shape; snout-vent length: 14; short snout, rounded to slightly truncate in dorsal and rounded to slightly protruding in lateral view; canthus rostralis rounded; loreal region straight to very slightly convex; headwidth directly behind the eyes: 5; medium sized eyes positioned laterally; eye-diameter: 2.1; distance anterior corner of eye to nostril: 1.5; nares small and roundish to slightly vertically elliptical, positioned laterally; straight distance nostril-snout tip: 1.1; nostril closer to snout than to eye; inter-narial distance: 2.4, larger than inter-orbital distance: 1.8; pupil round; supratympanic ridge curving down from posterior corner of eye to forearm base; tympanum diameter: 1.1, with distinct tympanic annulus, annulus round with central round papilla; tympanum much smaller than diameter of eye; upper and lower maxillae and praemaxillae with minute teeth, hidden by lips; vomerine teeth absent; elongated, almost rectangular tongue, tip with distinct v-shaped notch, median conical papilla near anterior attachment of tongue; tongue densely beset with small round papillae; small round choanae positioned at lateral edges of mouth roof. Forelimbs slender; upper arm: 3.4; lower arm: 3.6; hand to tip of finger III: 4.6; hand with two large tubercles, small and oval thenar tubercle, large and round palmar tubercle; carpal gland absent; fingers with round, nondivided, protruding subarticular tubercles, no further tubercles on hand and fingers; relative finger length: IV<I=II<III; manual webbing absent; finger tips broadened, forming small roundish to slightly pointed discs, being almost double as wide as subarticular tubercles; no distinct nuptial pads; hind limbs short and slender; femur: 7.1, slightly shorter than tibia: 8.5; tibia length 60.7% of SVL; foot including longest toe: 10.3; femoral glands absent; tarsal tubercle present, small and round; large inner metatarsal tubercle: 0.8, long and narrow, approximately 1/3 of length of toe I: 2.8; small round outer metatarsal tubercle; long and slender toes; relative toe length: I<II<V<III<IV; digits with basal webbing; webbing formula I(1), II(1), III(2), IV(3), V(2); lateral skin fringes extending further towards toe tips; toe tips enlarged to roundish discs, being approximately double as wide as subarticular tubercles. Dorsal skin fine granular (in life) to smooth (in preservation); a pair of long comma-shaped scapular ridges, ranging from about posterior corner of upper eyelid to slightly beyond level of forearm bases, converging towards vent; dorsolateral ridges faintly indicated; posterior edge of eyelid with few flat round warts, spiny eyelid tubercle absent; no white spines or warts on flanks; skin on outer parts of thighs and dorsal part of lower legs granular; skin on lower parts of limbs, belly and pectoral region smooth; throat without skin folds running parallel to mandibles; anterior third of lower mandible and anterior third of throat densely beset with rounded spines. Colouration in ethanol after 1.5 years. Back and dorsal part of flanks more or less uniform brown; snout slightly clearer, dorsal with dark median spot, snout tip including anterior part of upper lip and loreal region light brown; clear spot between anterior part of eyelids; posterior part of inter-orbital space with dark bar; eyelids dark grey; lateral part of upper lip, sub-orbital region, supratympanic ridge and temporal area including tympanum dark brown to black, extending into a similarly coloured blotch on the anterior part of the upper arm; flanks posterior to forearm bases with large dark axillary blotch; brown flanks fainting ventrally into whitish belly; arms coloured as back, lower arm towards hand with two dark transversal bars; hands dorsally lighter coloured with dark crossbands; lower legs brown as back; thighs with 3-4 faintly indicated dark cross-bars; anterior part of thigh with dark longitudinal stripe, being the border to whitish ventral parts; lower leg dorsally with three (right) and four (left) dark cross-bars; feet and toes with dark cross-bands; dark blotch around vent, dorsally bordered by narrow yellowish line; lower parts of feet and hands greyish brown; remaining ventral parts of limbs and posterior part of belly uniform whitish; lateroventral parts of venter, anterior part of belly and pectoral region whitish but densely beset with minute black spots; throat and lower mandibles dark grey to black; lower mandibles with few small clear spots. Colouration in life ( Fig. 1 ). Basic colour of dorsal surface of head, upper part of flank, back and dorsal surfaces of extremities light brown; snout, loreal region and anterior part of eyelids orange brown; snout with median longitudinal black spot; black inter-orbital bar; back with irregular darker patterning; lower arms, thighs, lower legs and feet with dark crossbars; anterior part of upper mandible brown with a dark bar; a V-shaped darker figure in scapular region, pointing anteriorly; black lateral face mask extending from below the eye and supratympanic ridge to forearm bases, covering tympanum; continuing below arms as a lateroventral black patch to axillar region; eye posterodorsally bordered by fine blue line; black pupil; iris copper red; central belly dirty white; belly laterally and pectoral area dark grey, no distinct black ventrolateral line or band; ventral part of forelimbs and hands dark grey; groin area and lower surfaces of thighs, lower leg and tarsus yellowish to orange; lower part of lower legs bordered by blackish spots; ventral surfaces of toes blackish. Variation ( Figs. 1–2 ). Snout-vent length in males with spiny black throats (presumably indicating maturity) 13.5–15.8 mm and 13.1–19.7 mm in females (smallest one presumably not adult); body dimensions generally as in holotype , for small deviations see Table 1 . The scapular ridges may differ in lengths and distinctness, but are always discernible and comma-shaped. Short dorsolateral ridges, composed of a very dense row of small warts, are visible in most specimens. Scapular and dorsolateral ridges are usually coloured dark. The webbing formulae showed some slight variation ( Table 1 ). The black face mask may cover either the entire lateral parts of the head (sometimes with minute white spots; Figs. 2 c, d), extending from snout-tip to dorsal of the forearm insertion, or the parts anterior to the eye are coloured in clearer brown ( Figs. 1 , 2 ), sometimes with darker spots. Individuals with clearer coloured loreal regions also have dark brown upper mandible with black bars instead of entirely black mandibles. The back might be uniform dark or reddish brown, show some darker patterning, or carry a broad central dark band ( Fig. 2 a). A light inter-orbital stripe is usually present. The iris colour varies from golden to copper red. The flank might be either uniform brown, grey or mottled with darker dots and lines ( Fig. 2 c). The black axillary spot may be distinct and extending as far caudally as to about mid-body. Usually, the edges of this spot are irregularly shaped. In other individuals it is nothing but a rather indistinct mottling of blackish spots with clearer dots. We neither observed in males, nor in females distinct black bordering of belly and lower shanks as in P. ruthbeateae . The inguinal region is yellowish. The belly seems to be dirty white in males and females (in preserved specimens it appears pure white); the border of the belly being dark grey in males. In some males, all with distinct gular spines, only the anterior half of the throat is coloured black. Females with dirty white (life) or white (in preservation) throats. Their lower mandibles largely coloured black. This black colour often interrupted along mandibles and always interrupted at tip of mouth; the margins of black colour to throat skin are irregular, almost wavelike. Only indistinctly indicated lateral skin folds running parallel to mandible in males. Female throat skin smooth. Genetics. The mean genetic difference in the sampled region of the 16S rRNA gene between Phrynobatrachus horsti sp. nov. and 40 other West African and Central African congeners ranged from 3.66–18.10% ( Table 2 ). The species with highest genetic similarity to P. horsti sp. nov. all occur in Central Africa. With the exception of P. ruthbeateae and P. b at e s i i , all belong to a clade of species being endemic to restricted mountainous regions along the Cameroon Volcanic Line (Zimkus et al. 2010; Zimkus & Gvoždík 2013 ). The uncorrected pairwise sequence divergence to the most similar species were P. ruthbeateae (mean: 3.66%), P. chukuchuku (3.78%), P. schioetzi (3.86%), P. steindachneri (3.89%), P. w er n e r i (4.09%), P. n j i om oc k (4.22%), P. jimzimkusi (4.28%), P. danko (4.43%), P. manengoubensis (4.48%), and P. b at e s i i (4.70%), respectively. A comparison of our sequences with the published sequences for Gabonese “ P. ruthbeateae ” records ( Zimkus & Larson 2013a , b ; see Table 2 ), revealed that these frogs are conspecific with P. horsti sp. nov. The intraspecific variation of all available P. horsti sp. nov. sequences ranged from 0–1.56% (mean ± sd: 0.51 ± 0.43%; N= 105). Mean intraspecific variation in the morphologically similar P. ruthbeateae was 0.66% (0–1.20%, sd: 0.47, N= 6). ZMB# sex SVL HW ED IO IN DEN DNS TD FL TL FLT IMT Webbing formulae 81698 m 14.0 5.0 2.1 1.8 2.4 1.5 1.1 1.1 7.1 8.5 10.3 0.8 I(1), II(1), III(2), IV(3), V(2) 81699 m 14.2 5.7 2.4 1.9 1.9 1.6 1.1 0.8 8.0 8.7 11.5 0.7 I(1), II(1), III(2), IV(3), V(2) 81700 m 14.7 5.5 2.4 2.2 2.6 1.8 1.1 1.0 8.3 8.7 13.0 1.2 I(1), II(1), III(2), IV(3), V(2) 81701 f 18.4 6.8 2.3 2.2 2.3 1.8 1.4 1.1 10 10.1 14.0 1.0 I(1), II(1), III(2-1.75), IV(3), V(1.5) 81702 m 14.2 5.5 2.0 2.4 2.3 1.4 1 0.9 8.5 9.5 12.0 1.0 I(1), II(1), III(2), IV(3), V(2) 81703 f 19.1 5.5 2.5 2.2 2.2 1.9 1.1 1.4 9.5 10.3 13.4 1.0 I(1), II(1), III(2), IV(3), V(2) 81704 f 18.1 5.7 2.3 2.0 2.2 1.7 1.2 1.0 8.8 8.4 12.1 1.1 I(1), II(1), III(2), IV(3), V(2) 81705 m 13.9 4.1 1.8 1.5 1.9 1.5 1.0 1.3 7.5 8.0 10.4 0.8 I(1), II(1), III(2), IV(3), V(2) 81706 m 14.9 5.3 2.2 2.0 2.4 1.4 1.0 1.0 7.0 7.1 10.1 0.8 I(1), II(1), III(2), IV(3), V(2) 81707 f 18.3 6.0 2.2 2.5 2.2 2.0 1.0 1.0 8.4 8.0 12.1 1.0 I(1), II(1), III(2), IV(3), V(2) 81708 m 15.2 5.4 2.0 1.9 2.2 1.5 1.4 1.1 7.8 7.5 1.0 I(1), II(1), III(2), IV(3), V(2) 81709 f 16.0 5.5 2.3 1.9 2.0 1.5 1.1 1.2 8.1 8.4 10.1 0.8 I(1), II(1), III(2), IV(2.75), V(1) 81710 m 15.2 4.1 1.9 2.1 2.1 1.5 1.4 1.1 7.1 7.4 10.8 0.8 I(1), II(1), III(2.5-2), IV(3), V(2) 81711 f 17.4 6.2 2.4 1.5 2.0 1.4 1.2 1.4 8.1 9.1 13.4 0.9 I(1), II(1), III(2), IV(3), V(2) 81712 m 13.7 4.0 1.9 2.1 2.0 1.4 1.1 1.1 7.0 7.0 10.9 0.7 I(1), II(1), III(2), IV(3), V(2) 81713 m 15.2 4.3 1.8 1.8 1.8 1.6 1.2 1.0 8.1 7.6 12.0 1.0 81714 f 18.1 6.1 2.5 2.3 2.3 2.0 1.4 1.0 8.6 9.4 13.4 1.1 I(1), II(1), III(2), IV(3), V(2) 81715 m 14.1 4.0 1.9 2.0 1.9 1.5 1.1 1.0 7.4 7.9 11.4 0.8 81716 f 17.5 5.7 2.2 1.9 1.8 1.7 1.2 1.3 9.9 9.5 14.0 0.8 I(1), II(1.5-1), III(2), IV(3), V(2) 81717 f 13.9 5.1 2.2 2.1 2.2 1.4 1.1 1.2 7.3 8.4 11.4 0.7 I(1), II(1), III(2), IV(3), V(2) 81718 m 15.5 4.1 1.9 2.1 1.8 1.1 1.0 1.1 7.2 8.2 11.8 0.8 I(1), II(1), III(2), IV(3), V(2) 81719 m 15.8 5.5 2.5 2.2 2.2 1.4 1.2 1.0 7.4 7.9 10.2 0.9 I(1), II(1), III(2), IV(3), V(2) 81720 m 13.9 4.2 2.1 2.2 1.9 1.4 1.0 1.2 7.8 0.8 I(1), II(1), III(2), IV(3), V(2) 81721 m 13.9 4.3 1.9 2.1 2.1 1.5 1.0 1.0 8.2 8.3 0.9 81722 f 19.1 5.3 2.2 2.6 1.8 1.9 1.5 1.2 9.0 9.8 9.4 0.9 I(1), II(1), III(2), IV(3), V(2) 81723 f 17.5 6.3 2.3 2.1 2.3 1.8 1.0 1.5 8.9 9.1 12.5 0.9 I(1), II(1), III(2), IV(3), V(2) 81724 m 15.1 5.4 2.1 2.5 2.1 1.4 1.1 1.3 8.1 8.1 10.2 1.0 I(1), II(1), III(2), IV(3), V(2) 81725 m 14.4 5.1 2.1 2.1 1.8 1.5 1.1 1.0 7 7.4 7.6 0.7 I(1), II(1), III(2), IV(3), V(2) 81726 f 16.3 6.0 2.2 1.8 2.0 1.6 1.0 1.0 8.1 8.8 12.2 0.7 I(1), II(1), III(2), IV(3), V(2) 81727 m 15.5 5.4 2.0 1.9 2.0 1.4 1.1 1.0 8.1 8.1 11.5 0.8 I(1), II(1), III(2), IV(3), V(2) 81728 m 13.5 5.5 1.9 2.3 2.3 1.4 1.3 1.2 7.4 8.1 10.1 81729 m 14.8 4.1 2.1 2.2 2.0 1.8 1.0 1.1 7.5 8.5 10.1 81730 m 15.5 4.7 2.1 1.8 2.1 1.4 1.2 1.0 8.2 7.8 81731 m 14.3 5.1 2.0 2.1 2.1 1.5 1.1 1.2 7.3 8.8 10.9 1.0 I(1), II(1), III(2), IV(2.75), V(1) min max mean sd N acridoides 10.27 11.78 10.70 0.33 15 africanus 8.66 9.85 9.03 0.28 15 alleni 13.05 14.08 13.23 0.27 15 annulatus 11.93 12.53 12.33 0.15 15 auritus 11.07 12.46 11.34 0.33 15 batesii 4.31 4.97 4.70 0.18 15 calcaratus 11.93 13.16 12.37 0.26 15 chukuchuku 3.22 4.23 3.78 0.23 15 cornutus 11.73 12.35 12.00 0.16 15 cricogaster 4.84 5.45 5.09 0.16 15 danko 4.04 4.85 4.43 0.20 15 dispar 11.67 12.34 12.08 0.19 15 francisci 10.06 11.40 10.36 0.32 15 fraterculus 11.55 11.96 11.75 0.12 15 ghanensis 12.80 14.41 13.08 0.40 15 guineensis 12.01 13.36 12.29 0.33 15 gutturosus 12.32 13.86 12.92 0.32 15 hylaios 15.30 17.83 15.75 0.59 15 intermedius 8.27 9.17 8.70 0.22 15 jimzimkusi 3.82 4.92 4.28 0.25 15 latifrons 13.76 15.56 14.03 0.46 15 leveleve 10.75 11.55 11.16 0.19 15 liberiensis 6.93 8.40 7.43 0.32 15 maculiventris 13.30 14.26 13.84 0.23 15 manengoubensis 4.22 4.88 4.48 0.17 15 mayokoensis sp. nov. 11.70 12.72 12.11 0.21 30 natalensis 10.89 12.11 11.40 0.26 15 njiomock 3.62 4.63 4.22 0.23 15 cf. ogoensis * 12.22 13.31 12.65 0.23 15 phyllophilus 11.43 12.04 11.74 0.16 15 pintoi 13.72 15.01 14.19 0.27 15 plicatus 10.70 12.52 11.10 0.42 15 rainerguentheri 11.97 12.99 12.12 0.27 15 ruthbeateae 2.62 4.74 3.66 0.44 60 sandersoni 17.57 18.50 18.10 0.23 15 schioetzi 3.42 4.67 3.86 0.30 15 steindachneri 3.43 4.44 3.89 0.22 15 tokba 10.12 11.62 10.75 0.30 15 villiersi 12.33 13.60 12.95 0.26 15 werneri 3.83 4.44 4.09 0.16 15 Call# DoFr minFr maxFr FrBW Pu# PuRa PuDu PuInS PuInE CD 1 3852.7 2282.0 4510.0 2227.0 37 11.6 9.2 235.9 82.5 3.2 2 3161.1 2093.0 4699.0 2606.0 20 7.9 10.3 240.6 103.9 2.5 3 3823.2 1912.0 5435.0 3523.0 35 9.2 7.1 415.1 51.4 3.8 4 3486.6 2098.0 5398.0 3300.0 28 9.6 8.2 237.5 52.8 2.9 5 3725.7 1986.0 5583.0 3597.0 36 7.9 8.0 465.5 30.4 4.5 6 3644.5 1875.0 5212.0 3337.0 32 7.6 8.2 529.9 69.1 4.2 7 3513.3 1986.0 5324.0 3337.0 33 8.4 7.9 363.5 16.6 3.9 8 3658.7 2172.0 4397.0 2225.0 26 12.6 10.0 15.1 68.3 2.1 9 3692.7 2283.0 4359.0 2077.0 24 8.8 8.1 266.5 25.0 2.7 10 3959.6 2839.0 4359.0 1520.0 17 8.0 8.6 22.3 188.7 2.1 11 3707.2 2320.0 4285.0 1965.0 19 8.7 8.4 451.7 82.1 2.2 12 3641.7 2283.0 4248.0 1965.0 30 12.8 10.8 17.8 34.8 2.3 Min 3161.1 1875.0 4248.0 1520.0 17.0 7.6 7.1 15.1 16.6 2.1 Max 3959.6 2839.0 5583.0 3597.0 37.0 12.8 10.8 529.9 188.7 4.5 Mean 3655.6 2177.4 4817.4 2639.9 28.1 9.4 8.7 271.8 67.1 3.0 SD 204.9 259.4 524.9 734.2 6.9 1.9 1.1 181.3 46.5 0.9 Acoustics ( Fig. 4 ) . The advertisement call of Phrynobatrachus horsti sp. nov. consists of numerous short single pulses, ranging from 7–10 ms (mean ± sd, 8.7 ± 1.1 ms, N= 337). The entire call has a duration of 2.1–4.5 sec (3.0 ± 0.9 sec, N= 12), with a pulse rate of 7.6–12.8 pulses/sec (9.4 ± 1.9 pulses/sec, N= 12). The calls start with a longer interval between the first and second pulse (15.1–529.9 ms; mean ± sd: 271.8 ± 181.3 ms, N= 12). These intervals then become shorter (16.6–188.7 ms; 67.1 ± 46.5 ms, N= 12). The dominant frequency of the calls range between 3161–3960 Hz, with the maximum call energy at 3656 Hz (N=12). The frequency range covers 2640 Hz (N=12), with a minimum frequency at 2177 Hz and a maxium frequeny at 4817 Hz. We observed neither harmonics nor any frequency modulation. Call parameters are summarized in Table 3 .