A new grass frog with rupicolous tadpoles from northern Zambia (Anura: Ptychadenidae) Author Channing, Alan Author Willems, Frank text Zootaxa 2018 2018-08-28 4462 3 349 366 journal article 29394 10.11646/zootaxa.4462.3.3 328adfa4-4626-461b-a14e-837fd43ed77f 1175-5326 1441683 55F9D6C8-6A28-4291-AF99-7250501508FB Ptychadena mutinondoensis sp. nov. Channing and Willems Mutinondo Grass Frog ( Figs. 3 , 4 , 6 ) Holotype . A male, ZMB 88379, ( Fig. 3 ) collected 7 January 2017 , from Mutinondo Wilderness , northern Zambia ( 12.456° S ; 31.293° E , 1430 m ) Paratypes . Two females, ZMB 88377 and ZMB 88380, and five males, ZMB 88381 – 88385, collected at the same locality. Other material . Two tadpoles ZMB 88374 and 88378, and two metamorphs ZMB 88375 – 76 all collected from the same locality. Sequences. 16S rRNA sequences were obtained from ZMB 88375–77 and ZMB 88379–80. Diagnosis. We assign the new species to the genus Ptychadena on the basis of the vomerine teeth reaching the anterior corners of the choanae, the outer metatarsals separated by webbing from the rest of the foot, and males with external lateral vocal sacs. Comparative data were drawn from Poynton and Broadley (1985) and Dehling and Sinsch (2013) . Ptychadena mutinondoensis was compared and distinguished from all other East, Central and Southern African Ptychadena species; the following diagnosis omits the West African species. The new species has no contrasting longitudinal bands on the posterior thigh, distinguishing it from P. subpunctata , P. taenioscelis , P. keilingi , P. upembae , P. guibei , P. nilotica , and P. anchietae . The foot is longer than half SVL, distinguishing it from P. schillukorum and P. mossambica . It has no light triangle on the snout, distinguishing it from P. subpunctata , P. obscura , P. anchietae and P. oxyrhynchus . It has three phalanges free of web on the fourth toe, distinguishing it from those with more webbing ( P. keilingi and P. bunoderma ). There is less than one phalanx of the fifth toe free of web, distinguishing from those with less webbing ( P. mossambica , P. keilingi , P. upembae , P. guibei , P. taenisocelis , P. bunoderma , P. ansorgii , P. porosissima and P. obscura ). The median skin folds are all similarly developed, distinguishing it from P. bunoderma , which has enlarged median folds. The snout is without skin folds, distinguishing it from P. uzungwensis and P. broadleyi . The similar Ptychadena broadleyi was characterised by a pair of short skin folds on the snout, and another between the eyes ( Stevens 1972 ). This feature is not present in all specimens. Stevens explains the smooth snout of some specimens as a note: "Snout ridges are very indistinct in some of (the) type specimens owing to the author's habit of injecting preservative into material during fixation, resulting in the skin being stretched and lifted from underlying muscles". Stevens was incorrect, as the skin over the top of the snout has not lifted in the type series. It would be better to state that the short skin folds on the snout are not always present in adults. There is no light longitudinal line on the tibia, distinguishing it from those with such a line ( P. subpunctata , P. keilingi and P. porosissima ). A light line is sometimes present in P. mossambica , P. guibei , P. taenioscelis and P. nilotica . There is no row of tubercles under the fourth metatarsal, although a row of flat white marks is present, distinguishing it from species with a row of tubercles under the fourth metatarsus ( P. mossambica , P. keilingi , P. upembae , P. guibei , P. bunoderma , P. ansorgii , P. uzungwensis , P. grandisonae , P. porosissima and P. obscura ). The snout is long, with the snout—nostril distance greater than the internarial distance, which distinguishes it from those without a long snout ( P. schillukorum , P. mossambica , P. keilingi , P. upembae , P. guibei , P. ansorgii , P. broadleyi , P. grandisonae , P. porosissima , P. nilotica , P. obscura and P. anchietae ). The foot is shorter than the tibia, distinguishing it from those with the foot equal to or longer than the tibia ( P. subpinctata , P. keilingi , P. guibei , P. taenioscelis , P. ansorgii and P. nilotica ). The gular pouch slits of males end below the arm insertion, similar to P. grandisonae . This character distinguishes the new species from those species where the slit ends in line with the lower arm insertion, to above the arm ( P. subpunctata , P. schillukorum , P. mossambica , P. keilingi , P. upembae , P. guibei , P. taeniscelis , P. bunoderma , P. ansorgii , P. uzungwensis , P. broadleyi , P. porosissima , P. nilotica , P. obscura , P. anchietae and P. oxyrhynchus ). The snout is longer and sharper in dorsal view compared to Ptychadena broadleyi ( Fig. 2 ). Description of holotype . A male ( Fig. 3 ), SVL 38.5, SUL 37.9; the body is gracile; head short (HL/SVL 0.34, HW/SVL 0.35), not wider than trunk, about as long as wide (HL/HW 0.97); snout long (SL/HL 0.58), sharply rounded in dorsal view, rounded in profile, projecting well beyond lower jaw, narrow (SL/IND 2.00); canthus rostralis rounded; loreal region slightly concave; nostrils situated on slight projections, closer to the eye than snout tip (END/SL 0.28); eyes directed anterolaterally, slightly protruding, small (ED/HL 0.29); eye diameter less than half snout length (ED/SL 0.50); interorbital distance is less than upper eyelid (IOS/EL 0.63), and nearly equal to internarial distance (IOS/IND 0.76); internarial distance just less than eye diameter (IOS/ED 0.76); vomerine teeth inconspicuous; tympanum visible, smaller than eye diameter (TYM/ED 0.63), the pale upper lip becoming a thickened gland below the tympanum, extending to above the arm insertion; upper jaw with small needle-like dentition; choanae small, rounded; tongue slightly notched posteriorly, 5.2 at widest part; median lingual process absent; vocal sac double, darkly pigmented through slit; vocal sac slit starts below arm insertion; skin folds present on back; median fold complete, runs from eye to thigh; post eyelid fold complete; dorsolateral fold interrupted anteriorly and posteriorly; lateral fold interrupted; skin rough laterally and between skin folds; ventral surface of limbs, gular and abdomen smooth. FIGURE 3 . A—Lateral view of holotype of Ptychadena mutinondoensis (ZMB 88379). B—Sharp snout of Ptychadena mutinondoensis (ZMB 88380) compared to C—snout of Ptychadena broadleyi (ZMB 88389). Scale bars = 10 mm. Arms slender; hand moderately large (HND/SVL 0.20); tips of fingers not enlarged into discs; relative length of fingers: II <IV <I <III; subarticular tubercles single and distinct, with one on fingers I, II and IV, and two on finger III; fingers without webbing; thenar tubercle distinct; metacarpals without supernumerary tubercles; pale nuptial pads present on upper surface of fingers I, II and III. Hind limbs gracile, proximal tarsal tubercle small, pale, distinct; tibia long (TIB/SVL 0.59); heels overlapping when knees are flexed and thighs are held at right angle to body; foot just shorter than tibia (TIB/FOT 1.15); relative length of toes: I<II<V<III<IV; toes without expanded discs; subarticular tubercles: one on toe I and II, two on toe III, three on toe IV and two on toe V; webbing formula I 2 –2.2 II 2– 3 III 2–3.2 IV 3– 1 V ; thin margin of webbing extending to tips; inner metatarsal tubercle prominent, oval smaller than eye diameter (IMT/ED 0.45); thin tarsal ridge present; outer metatarsal tubercle present as a small white bump. Measurements of the holotype and paratypes are presented in Appendix 2. Colour in life. The dorsal pattern of the holotype consists of small dark brown patches arranged along the skin folds on a beige background ( Fig. 3 ). Reddish-brown patches are present on the upper arm and laterally. There is a thin white vertebral line, enclosed in a broad beige vertebral band. A dark brown line runs from the tip of the snout, over the nostril, to the base of the eye. The iris is dark brown. The tympanum is transparent with a beige triangular central marking. The upper lip is lightly speckled with brown, and the lower lip is black posteriorly, with the pigment extending into the vocal pouch slit. The lateral skin fold is white, as is the gland extending from the upper lip to the arm insertion. The flanks have irregular brown and pale marbling. The upper surfaces of the arm are beige, with darker brown transverse bars on the lower arm. The leg has eight transverse irregular darker bars over the upper leg, thigh and foot. The back of the thigh is unmarked, apart from some faint speckling. The venter is white with a few black speckles on the yellow throat. Colour in preservative . The pattern is clearly visible as a darker brown on a paler brown background. Paratype variation. The paratypes are remarkably similar to the holotype in proportions, with the females larger, reaching a SVL of 43.5 mm . Between 3–3.5 phalanges of the fourth toe may be free of webbing on either side. Females had bright red infusions on the upper arm, above the eye, and in spots on the flanks. Advertisement call. The calls were recorded on 14 January 2016 between 19h00 and 20h00, air temperature 24°C. Males were heard calling during daytime, linked to rainy conditions, and after dark. The description is based on the call of males visually confirmed to be of this species, near the locality where the holotype was collected. The call consists of an irregular fast series of clicks, and brief trills of five to seven pulses ( Fig. 4 ). The pulse rate is approximately 35 s -1. The fundamental frequency is 1.1 kHz, with additional harmonics. The duration of the trills varies from 0.1– 0.3 s . Analysis of 93 calls from five males showed that the number of pulses varied from 1 to 8 (mean 3.7), call duration varied from 13–288 ms (mean 100), and pulse rate varied from 10.5– 58.1 s -1 (mean 29.2 s - 1, n=79). The emphasised frequency varied from 1033–3531 Hz, (mean 1738 Hz). In contrast, the advertisement call of P. broadleyi (n=3) ( Fig. 4 ) consists of longer loud trills of 9–24 (mean 17) pulses, with a call duration of 0.84– 2.25 s (mean 1.6 s ) and pulse rate 9.6– 10.2 s -1 (mean 10 s -1). The emphasised frequency varies from 1205–3531 Hz (mean 2670 Hz). The calls of other species of grass frogs consist of trills and chuckle calls. Breeding and tadpoles . Some notes on timing of calling and oviposition were taken during the 2016–2017 wet season. Rainfall occurred on multiple days in November and December. A few frogs were first noticed calling 22 December after dusk, the first warm evening after 48 hours of heavy rainfall and cold overcast weather. Freshly deposited eggs were found at two out of 10 known oviposition sites checked the following morning. No calling was noticed during consecutive days with mostly dry weather conditions, during which the seepage flow at oviposition sites dried up. Heavy rains set in again on 2 January leading to mass calling activity and oviposition at all known sites (checked from 4 January onwards). Calling activity continued throughout the wet season, with activity peaking during the first hours after dusk. Calling was also frequently noted at other times of day or night during warm and wet conditions. There was little if any calling after multiple days without precipitation. Eggs are deposited at the top of rock faces, where there is a film of water, under overhanging vegetation ( Figs. 6 , 7 ). The eggs are not covered by the film of water. Eggs were found until March, always during or shortly after a period of good rains. Four clutches contained 140, 180, 190 and 280 eggs ( 7 January 2016 ). Mean egg diameter was 9.3 mm , N=35. The following description is based on ZMB 88378, a stage 40 tadpole collected at Mutinondo Wilderness ( Fig. 5 ). Two others collected at the same time and place were found to have identical 16S rRNA sequences to the adults, confirming their identity. The tadpole has a total length of 32.9 mm . The head is bluntly rounded from above, and in lateral view. The eyes are large, positioned dorsolaterally. Extra-ocular proportion: width of head minus distance between lateral limits of eyes/distance between lateral limits of eyes = 0.9. The oral disc is anteroventral, with a single row of rounded marginal papillae below, with extra papillae laterally. The rostral gap in the papillae extends across the whole labium. The labial tooth row formula ( Altig 1970 ) is 2/1. The upper and lower jaw sheaths are deeply pigmented for more than half of their height. Both jaw sheaths are finely serrated. The external nares are closer to the eye than the tip of the snout. Rostronasal distance/ orbitonasal distance = 1.5. The narial aperture is circular without a distinct margin. No narial valve is apparent. Internarial ratio: width of nostril/internarial distance = 0.15. There is no orbitonasal line. No pineal spot is present. The spiracle opening is small, elliptical, slightly away from the body, and is visible dorsally. The opening is dorsolateral. FIGURE 4 . A—Part of the advertisement call of Ptychadena mutinondoensis , showing the clicks and brief trills. B—The advertisement call of Ptychadena broadleyi showing a typical trill of 10 pulses. Both recorded at 24°C. Tail height is less than head height. Height of tail muscles at base of tail/height of trunk = 0.50. Tails are low with little fin, dorsal and ventral fins equal; the dorsal fin originates at the junction of the body and the tail muscle. The fins are nearly parallel. Tail fins taper to the tip, with nearly straight margins. The tail tip is acute. The axis of the tail, extrapolated anteriorly, passes below the eye. Tail length/length of head and trunk = 2.7. The ratio exceeds 3.2 in other specimens. The vent tube is dextral, continuous with the ventral fin margin. The major measurements for the tadpole at Gosner stage 40, are: total length 32.9; tail length 24; maximum fin height 2.8; distance from highest point on fin to tail tip 7.0; distance from snout tip to spiracle 5.3; distance from snout tip to nostril 1.6; distance from snout tip to eye 2.8; distance from eye to nostril 1.1; oral disc width 1.7. In life the tadpoles are mottled grey and yellow-brown, with a lateral row of small white dots along the tail muscle, and additional small white dots along the base of the dorsal fin. The tadpoles can be darker, a mottled black and grey. The pale spots disappear once preserved. In preservative the specimen is pigmented dorsally ( Fig. 6 ). Dark areas are present around the nostrils, and between the eyes. A dark line runs from behind the eyes to the base of the tail. Fine black stipples are gathered in groups forming mottles along the caudal muscles and dorsal fin. The underside of the trunk, and the anterior half of the base of the tail, are unpigmented. The gut coils are clearly visible through the ventral skin. FIGURE 5 . Rainfall and timing of breeding of Ptychadena mutinondoensis at Lodge Rock, Mutinondo Wilderness, during the 2016/2017 season. Vertical arrows: initial two egg-laying events with % of known oviposition sites active; horizontal arrow: period eggs and/or tadpoles were present. Rainfall data collected at Lodge Rock, 200 m north of type locality. Tadpole development . Although no detailed data were systematically collected, development from hatching to metamorphosis takes 1–2 months. In both the 2015–2016 and 2016–2017 wet seasons, clear size cohorts could be distinguished within and between oviposition sites, suggesting multiple egg-laying periods within each season, presumably linked to periods of wet and warm conditions. Stage 33 tadpoles were found on 10 May 2017 , the latest in the season that tadpoles were found. Habitat. The frogs have only been found in association with granite slopes where there is a 1–2 mm film of slow-flowing water, seeping from vegetation higher up the slope. These conditions at Mutinondo are on or near large granite inselbergs, visible on the skyline in fig. 7. Both the eggs and tadpoles are found in the film of water, which does not cover them. Adults have been found in and under the surrounding vegetation. Calling males were typically observed at an oviposition site. A single adult female was found inside climax evergreen riverine forest 130 meters from a known oviposition site, during the hot dry season ( 8 September 2016 ). All other frog observations were in grassy vegetation on or very close to wet granite slopes during the wet November to April months. Plant species typical of oviposition sites and surrounding vegetation include Xerophyta equisetoides , Myrothamnus flabillifolia and Pentas nobilis at sub-shrub level, and Coleochloa setifera , Aeollanthus subacaulis , Polygala africana , Actiniopteris sp. , Oldenlandia herbacea , Scleria sp . and Pycreus sp . at herb level. Larger shrubs and emergent trees are typically absent. Amphibian community. Amphibian species recorded breeding at any of the 24 surveyed P. mutinondoensis breeding sites, include Tomopterna tuberculosa (1 site; calling males and tadpoles) and Mertensophryne taitana (2 sites; amplexus and tadpoles). P. mutinondoensis was the only breeding species at 22 out of 24 breeding sites surveyed. Other frogs recorded at the same general sites, but not observed breeding in association with the same seepage patches, are Arthroleptis xenodactyloides , A . xenochirus , A . stenodactylus , Sclerophrys gutturalis , S. pusilla , Kassina senegalensis , Leptopelis bocagii , Breviceps mossambicus , Amietia delalandii , Phrynobatrachus parvulus and Ptychadena oxyrhynchus . FIGURE 6 . Top—Eggs of Ptychadena mutinondoensis developing in a film of water under vegetation. Below — A stage 40 tadpole ZMB 88378 from Mutinondo Wilderness. Tadpole scale bar = 10 mm. Tadpole predation. An all-black, 10–15 mm long species of terrestrial carabid beetle was noted predating on P. mutinondoensis tadpoles on multiple occasions. The tadpoles also share the seepage waters with a range of carnivorous water insects such as small dragonfly (Anisoptera) larvae, which constitute likely predators. Population size. Lodge Rock and Campers Rock were systematically searched for tadpoles on 7 March 2015 . A total of 1,050 tadpoles was counted, spread over 20 seepage patches, 19 of which were located on Lodge Rock. Estimated numbers of calling males never exceeded 50 on a single evening during the 2015–2016 and 2016–2017 seasons. Repeated visits to all inselbergs in the reserve produced the following maximum numbers: Klipspringer Rock: remotely estimated at 20–50 calling males on the western flank alone (observers position Lodge Rock); Kankonde Rock: 50 tadpoles; Charlie’s Rock / Chipundu Stream: single juvenile; Mulinso Falls: 50 juveniles ; Mafonie: 100 tadpoles, single adult female; all other sites: none. The Mutinondo grass frog appears to be restricted to a small area, with a relatively small population. Etymology. The species is named for the Mutinondo Wilderness area.