Detection of cryptic taxa in Leptobrachium nigrops (Amphibia, Anura, Megophryidae), with description of two new species Author Hamidy, Amir Author Matsui, Masafumi Author Nishikawa, Kanto Author Belabut, Daicus M. text Zootaxa 2012 3398 22 39 journal article 10.5281/zenodo.281817 9c482ced-fa54-4bb6-a31c-2e8f0b205f40 1175-5326 281817 Leptobrachium ingeri sp. nov. Synonymy: L. nigrops : Inger 1966 , p. 37 (part). Holotype . KUHE 53848, an adult female collected from Matang Wildlife Centre ( 01°36’34’’N , 110°09’36’’E , 60 m a.s.l.), Kuching Division, Sarawak , Borneo, Malaysia , collected by K. Eto and K. Nishikawa at 20:00 h on 30 August 2010 . Paratypes . SRC 0 0 0 1 (former KUHE 53833) and KUHE 53834, two adult males, and KUHE 53832, an adult female, all collected from Santubong ( 01°44’37’’N , 110°19’11’’E , 60 m a.s.l.), Kuching Division, Sarawak , Borneo, Malaysia on 29 August 2010 , collectors same as for holotype ; MZB Amp 11790, an adult female collected from marshy area of Tanjung Padang, Belitung, Indonesia , collected by D. Gower, A. Hamidy, and K. Sanders on December 2005 . Referred specimens. KUHE 12087 and KUHE 12099, two adult males, collected from Niah National Park ( 03°49’N , 113°45’E , ca. 50 m a.s.l.), Miri Division, Sarawak , Borneo, Malaysia , collected by M. Matsui on 26 December 1990 . Etymology. The specific name is dedicated to Dr. Robert F. Inger of Field Museum, Chicago, who was the first to notice morphological differences between populations of L. nigrops from Borneo and Malay Peninsula. Diagnosis. A small-sized Leptobrachium of the L. nigrops group, with SVL of 39.3–47.1 mm in females and 28.6–36.8 mm in males; finger tip sharply pointed; toe web relatively well developed; inner metatarsal tubercle large; dorsum brown with distinct dark brown markings; tympanum usually at least upper one-third covered by dark brown marking; body laterally with black spots scattered from axilla to groin; belly and limbs heavily blotched ventrally; limbs, including fingers and toes, distinctly barred dorsally; large dark blotches at groin continuing to ventral and posterior sides of thigh; femoral gland in a large white blotch. Description of holotype (measurements in mm). Small sized (SVL 44.6); habitus moderately stocky, body tapering to groin, head broad and depressed, slightly longer (HL 19.6: 43.9% SVL) than wide (HW 18.0: 40.4% SVL); snout obtusely pointed, rounded triangle from above, truncate in profile, projecting beyond lower jaw; eye large, obviously projecting from sides of head, slightly smaller (EL 6.8: 15.3% SVL) than snout (SL 7.3: 16.4% SVL); canthus rostralis sharp, loreal region oblique, moderately concave; nostril lateral, below canthus, closer to snout (S-NL 3.9: 8.7% SVL) than to eye (N-EL 4.1: 9.2% SVL); internarial distance ( IND 3.4: 7.6% SVL) much narrower than interorbital distance, (IOD 5.7: 12.8% SVL), latter slightly narrower than upper eyelid (UEW 6.0: 13.5% SVL); pineal spot absent; tympanum visible, diameter (TD 3.2: 7.2% SVL) almost half that of eye and separated from eye by about two-third of its diameter (T-EL 2.2: 4.9% SVL); no vomerine teeth; tongue heart-shaped, without papillae, notched posteriorly. Forelimb slender and long (FLL 31.3: 63.7% SVL), about three-fifth of hindlimb; fingers moderately slender, unwebbed; first finger (1FL 5.9: 10.7% SVL) slightly longer than fourth and second, third much longer (3FL 8.9: 16.2% SVL); finger tip sharply pointed, slightly hooked, not swollen; inner palmar tubercle large (IPTL 2.5: 4.5% SVL), not extending onto first metacarpal, and outer palmar tubercle smaller (OPTL 2.0: 3.6% SVL); subarticular tubercles indistinct, replaced by low callous tissue. FIGURE 3. Three species of the Leptobrachium nigrops group in life. (A) A female of L. nigrops from Malay Peninsula (KUHE 42243); (B) A female paratype of L. ingeri sp. nov. from Santubong (KUHE 53832); (C) A female holotype of L. kanowitense sp. nov. from Kanowit (KUHE 42575). FIGURE 4. Ventral views in males (top row) and females (bottom row) of (A) L. nigrops : KUHE 15658 (male) and KUHE 42243 (female); (B) L. ingeri sp. nov. : a paratype (KUHE 53834: male) and the holotype (KUHE 53848: female); (C) L. kanowitense sp. nov. : a paratype (KUHE42574: male) and the holotype (KUHE 42575: female). Scale indicates 5 mm. Hindlimb slender and relatively short (HLL 65.6: 119.2% SVL); heels not meeting when legs held at right angles to body; tibia distinctly longer (TL 20.0: 36.3% SVL) than foot (FL 17.0: 30.9% SVL); tibiotarsal articulation of adpressed limb reaching to middle of tympanum; third toe longer than fifth; toe tips similar to those of fingers; toe webs rather well developed, webbing formula I 1–1 1/ 2 II 1/4–2 3/ 4 III 1 1/4–3 2/ 3 IV 3 2/3–1 1/ 2 V ; inner metatarsal tubercle oval, low but large (IMTL 2.2: 4.9% SVL) more than half of first toe (1TOEL 3.2:7.2%); outer metatarsal tubercle absent; subarticular tubercles obscure, elongate, replaced by low callous tissue. Skin above nearly smooth, scattered with minute granules on top of head, especially around interorbital region, granules denser on upper eyelid; ventrum weakly granular, particularly on belly; low supratympanic ridge from posterior corner of eye to axilla; low dermal ridges in longitudinal rows on upper arm; flat pectoral gland at medial border of axilla behind arm insertion; femoral gland small, concentrated in one cluster on right side, but diffused on left side. Colour. In life, dorsally brown with dark brown blotches in interorbital region, medial one followed by continuous longitudinal band and lateral ones by more or less interrupted bands; dorsal brown laterally with distinct dark spots fading to lighter towards belly; loreal region, anterior to eye, covered by dark brown; upper onethird of tympanum and supratympanic ridge covered by dark band; groin marked with irregular dark blotches; ventrum cream heavily blotched all-over; limbs with distinct black bars dorsally and irregular dark blotches ventrally; posterior thigh banded by black bars, banded pattern continuing to posterior flank; iris totally black, surrounded by light blue sclera visible in maximally opened eye. In preservative, aspects of the colour pattern remain, but dorsal brown colour has been darkened. FIGURE 5. Ventral sides of thigh (top row), hand (middle row), and foot (bottom row) of (A) L. nigrops (KUHE 42243), (B) the holotype of L. ingeri sp. nov. (KUHE 53848), and (C) the holotype of L. kanowitense sp. nov. (KUHE 42575). FIGURE 6. Spectrograms (above) and wave form (below) of advertisement call of (A) L. nigrops (KUHE 15430) from Kuala Lumpur, Malay Peninsula and (B) L. ingeri sp. nov. (KUHE 53834) from Santubong, Sarawak. Variation. Morphological variations are shown in Table 3. The male paratype (KUHE 53834) is smaller in body size, and has an internal vocal sac and a pair of vocal sac openings. The female paratype (KUHE 53832) is larger in body size with much darker throat than the holotype . It has distinct white dots around vent and posterior thigh. The female paratype from Belitung (MZB Amp 11790) is smaller than holotype . Although the color slightly faded, blotches on groin and posterior thigh can be still recognized. Specimens from Belitung have larger femoral gland and more rugose dorsal skin than specimens from Borneo. Like the holotype , tibio-tarsal articulation in one female reaches only to middle of tympanum, but it reaches to points beyond anterior border of tympanum in the remaining one female and all males. Calls. The calls recorded at Santubong on 29 August 2010 (air temperature = 26.2°C; Fig. 6 B) had the duration from 305–763 (mean+SD = 485.9+243.8, n = 3/ 1 male ) ms, and emitted intermittently. The call consisted of 5–9 (6.3+2.3, n = 3) short notes, each with the duration of 16–33 (20.7+5.1, n = 18) ms, and the note gap of 88–102 (93.7+3.8, n = 15) ms, with the note repetition rate of 11.8–16.4 (13.7+2.4, n = 3) per s. Each note is composed of 4–11 (6.1+2.0, n = 18) fine pulses. Frequency bands spread over 1.5 to 2.2 kHz range, with the dominant from 1,650 to 1,950 (1,792+75, n = 18) Hz. Each note showed weak frequency modulation, with the initial and middle frequencies (1,650 to 1,950 [1,792+75, n = 18] Hz) falling towards the end of the note (1,600–1,900 [1,760+99, n = 18] Hz). The calls recorded on 26 December 1990 at Niah (air temperature = 25.9°C) were basically similar in temporal structure (call duration 539–697 [618.2+111.7, n = 2/ 1 male ] ms; number of notes 8–10 [9.0, n = 2]; note duration 21–39 [25.8+5.1, n = 18] ms; note gap 72–105 [76.2+7.9, n = 16] ms; note repetition rate 14.3–18.4 [14.6, n = 2] per s; number of pulse per note 5–10 [7.0+1.5, n = 18]). However, frequencies were lower than in Santubong calls, with the dominant from 1,300 to 1,500 (1,392+62, n = 18) Hz. Lowering of the initial frequency (1,350 to 1,600 [1,450+132, n = 3] Hz) towards the end of the note (1,250–1,350 [1,300+41, n = 4] Hz) was similar to the Santubong calls. Three harmonic frequencies were present at approximately 1,875–1,930, 2,250–2,300, and 3,900–4,000 Hz. Comparisons. Leptobrachium ingeri is placed in the L. nigrops group by having small body size. This character distinguished it from all other species of Leptobrachium , except for the other two species of the group ( L. nigrops and L. kanowitense ). Leptobrachium ingeri has relatively larger eye than L. nigrops (15.0–18.5% SVL vs.12.0–17.3% SVL in L. nigrops ), and relatively larger values than L. kanowitense in snout to nostril length (7.9–10.4% SVL vs. 7.0–8.5% SVL in L. kanowitense ), internarial distance (7.2–8.7% SVL vs. 6.2–7.0% SVL), intercanthal distance (17.6–20.7% SVL vs. 16.9–18.5% SVL), and inner metatarsal tubercle length (4.8–6.3% SVL vs. 4.3–4.4% SVL). In addition to morphometric differences, L. ingeri differs from L. kanowitense in more developed toe webs: phalanges free of web L . ingeri samples were 1–1 3/4 on the outer side of first (vs. 1 3/4–2 on L. kanowitense ), 1/4–2/3 on the outer side of second (vs. 1 1/4–1 3/4), 2 2/3-3 on the inner side of third (vs. 3–3 1/4), 1 1/4–1 3/4 on the outer side of third (vs. 2), 3 2/3 on the inner side of fourth (vs. 3 3/4–4), 3 2/3–3 3/4 on the outer side of fourth (vs. 3 3/4–4), and 1 1/2–1 2/3 on the inner side of fifth (vs. 2) toes. Leptobrachium ingeri further differs from the other two species in much more sharp finger tips, and in more distinct markings on dorsum and ventrum than the other two species. In calls, L. ingeri closely resembles L nigrops from Malay Peninsula: Calls of L. nigrops , recorded on 30 December 1992 (air temperature = 26.4°C) and on 21 January 1993 (air temperature = 25.9°C) at the campus of University of Malaya , Selangor, West Malaysia were short trills (Fig. 11). The call varied in length from 618–851 (617.9+131.4, n = 3) ms and consisted of 8–12 (mean+SD = 9.7+2.1, n = 26/ 2 males ) short notes, each with 4–26 (8.4+5.2, n = 29) pulses. The note duration varied from 14–85 (29.8+17.8, n = 29) ms, and the note gap from 72–111 (78.6+10.4, n = 26) ms. The note repetition rate varied from 12.9–14.3 (13.8+0.7, n = 3). Frequency band spread over 1,700 to 1,950 Hz range, with the dominant from 1,700 to 1,950 (1,852+75, n = 29) Hz. Within a note, the initial frequency (1,800–1,900 [1,840+39, n = 10] HZ) slightly lowered at the end (1,700–1,950 [1,777+82, n = 11] Hz). These characteristics overlap those of L. ingeri , and the two species cannot be differentiated acoustically. Range. The coastal area of Sarawak in Malaysian Borneo (Santubong, Matang [Kuching Division], and Niah [Miri Division]); Belitung Island, Indonesia (Tanjung Padang) ( Figure 1 ). Natural history. Larval, and other ecological data are lacking, but breeding season seems to include late August and late December, as evidenced by calling males. Matang, Santubong, and Tanjung Padang, where the type series of L. ingeri were collected, are lowland (< 60 m a.s.l.). The holotype was collected under a dead tree in an old trail near a small river in secondary forest. Other species found on this trail were: Limnonectes kuhlii (Tschudi) , Limnonectes leporinus Andersson , Limnonectes kenepaiensis (Inger) , Fejervarya limnocharis (Gravenhorst) , Ansonia leptopus (Günther) , Meristogenys jerboa (Günther) , Hylarana signata (Günther) , Hylarana raniceps (Peters) , Polypedates leucomystax (Gravenhorst) , Rhacophorus fasciatus Boulenger , Kalophrynus heterochirus Boulenger , and Microhyla borneensis Parker.