Bioacoustics of Kalophrynus limbooliati

Description of holotype (measurements in mm). Medium sized ( SVL 26.2); habitus moderately stocky ( Figs. 1 , 3 ); head triangular, slightly shorter (8.6) than wide (9.0); snout pointed, directed downwards in profile ( Fig. 3 ), projecting beyond lower jaw; eye moderate, as long (3.5) as snout (3.5); canthus rostralis not sharply defined, straight; loreal region vertical, very slightly concave; nostril below canthus rostralis, directed laterally, much closer to tip of snout (1.1) than to eye (2.0); interorbital distance (3.7) much wider than internarial distance (2.1), the latter subequal to upper eyelid (2.2); pineal spot absent; tympanum distinct, diameter (2.3) more than three-fifths that of eye, and very close to eye (0.3); upper jaw edentate; tongue entire, without papillae; a crenulated ridge of skin on palate posterior to eye, preceded by a shorter, similarly notched one between posterior halves of eye; a median, subgular vocal sac; vocal openings posterior to rictus. Forelimb long (18.0, 72% of SVL) and slender; fingers thick, basally slightly webbed; tips rounded, not dilated; relative length of fingers: IV<I<II<III; portion of fourth finger projecting from palm (0.9) shorter than length of terminal phalanx of third finger (1.4); outer palmar tubercle large and fleshy, inner indistinct; subarticular tubercles between finger tip and palmer tubercle rounded, two on fingers I, II and IV, three on III ( Fig. 2 A); humeral spine absent. Hindlimb moderately long (35.5, 135% of SVL) and slender; tibia moderately long (10.9, 42% of SVL), heels not overlapping when folded limbs held at right angles to body; tibiotarsal articulation of adpressed limb reaching to the center of tympanum when held alongside body; foot (8.7) much shorter than tibia (FL, 80% of TL); toe tips rounded; relative length of toes: I<V<II<III<IV; webbing poorly developed ( Fig. 2 B), formula: I 1–2 II 2–3 III 2– 3 IV 3–1 V ; subarticular tubercles numbering one on first and second toes, two on third toe, and three on fourth toe; distal tubercle on third toe and distal and middle ones on fourth toe prominent and rounded, others small and less distinct; no subarticular tubercles on fifth toe; inner metatarsal tubercle oval, length (1.0) more than half of first toe length (1.5); an indistinct outer metatarsal tubercle ( Fig. 2 B). Skin coarsely granular dorsally, with small tubercles scattered posteriorly from behind upper eyelid to vent; tips of tubercles forming white asperities; an indistinct gland on side of head behind tympanum, not delimited by a sinuous groove; upper lateral surfaces of body with scattered minute tubercles; lower lateral surfaces of body, posterior half of chin, abdomen, and posterior side of thighs with large, flattened glandules; skin of gular region not modified for vocal sac; inner and outer margins of fourth finger without skin fringes; nuptial pads absent. Color. In life, ground color of dorsum highly variable from light orange brown to dark chocolate brown, with several small dark brown spots and obscure hour-glass shaped marking from upper eyelid to shoulder; a light dorsolateral line extending from snout tip through margin of upper eyelid to groin, forming a boundary between lighter dorsum and darker sides of head and shoulder; tips of dorsal granules whitish; limbs dorsally brown with an obscure darker bar ( Figs. 1 A, 3); ventrum pinkish gray, with scattered white flecks ( Fig. 1 B); inguinal spot entirely black without lighter borders or pattern; iris golden with black pigmentation. In preservative, color and pattern have generally faded but not obviously changed. Variation. Individuals of the type series are generally similar in morphology ( Table 1 ). In both adult male paratypes (KUHE 53314, 53315), internarial distance is larger than upper eyelid width. They have darker throat and more developed asperities on dorsum than the holotype . The inguinal black spot varies in size, and is lacking on the left side in one individual (KUHE 53315). The single juvenile (KUHE 52061) has smooth dorsum, with several black spots scattered from shoulder, some continuing to the inguinal marking. Chin and anterior part of ventrum are dotted with dark spots in this juvenile paratype . SVL RHL RHW RIND RIOD RUEW UKMHC 705 ( holotype ) 26.2 32.6 34.3 8 13.9 8.2 KUHE 53314 ( paratype ) 27 32.6 32.2 9.6 12.2 7.8 KUHE 53315 ( paratype ) 28.7 31.9 32.6 9.1 11.7 7.3 mean+SD/median 27.3+1.28 32.6 32.6 9.1 12.2 7.8 UKMHC 705 (holotype) RSL REL 13.4 13.4 RN-EL 7.6 RTD 8.8 RT-EL 1 RLAL 47.3 KUHE 53314 (paratype) KUHE 53315 (paratype) median 12.8 17.4 12 13.4 12.8 13.4 6.4 6.8 6.8 8.1 9.4 8.8 0.4 0.7 0.7 46.7 46 46.7 UKMHC 705 (holotype) KUHE 53314 (paratype) KUHE 53315 (paratype) median RFLL RTL 68 41.4 63 41.3 63.4 41.5 63.4 41.4 RFL 33.2 35.6 35.2 35.2 RHLL 135.5 134.1 128.6 134.1 RIMTL 3.8 3.3 3 3.3 R1TL 5.7 5.6 5.7 5.7 Range. Southern part of Western (Peninsular) Malaysia : Johor (Panti, Kota Tinggi; Gunung Pulai, Kulai [ 350– 605 m a.s.l.]; Gunung Lambak, Kluang [ 02°00' E , 103°22' E , 75–185 m a.s.l.]; Trail Lagenda, Gunung Ledang, Tangkak [ 02°22' N , 102°37' E , 750–895 m a.s.l.: recorded by calls]) and Negeri Sembilan (Sungai Kenaboi, Kenaboi: recorded by calls). Singapore : Bukit Timah Nature Reserve ( Lim & Lim 1992: see below, discussion ). Possibly Janda Baik, Pahang, Western (Peninsular) Malaysia ( Dring 1979: see below, discussion ). Natural history. On Gunung Pulai, Gunung Ledang, and Gunung Lambak, males were found calling in widely scattered choruses at dry nights (from 1930 h) in early September in the dense secondary broad-leaf forest. Calling males hid themselves among dead leaves and were very difficult to locate, but responded to playback of recorded calls and/or whistles imitating their calls. There were no large bodies of water at the calling sites. The associated anuran species observed at the nearest waters were Hylarana laterimaculata and Microhyla heymonsi Vo g t. At Sungai Kenaboi, calls were heard in secondary and bamboo forests on dry night. Microhyla heymonsi , Fejervarya limnocharis (Gravenhorst) , Humerana miopus (Boulenger) , Hylarana labialis (Boulenger) , and Polypedates leucomystax (Gravenhorst) were observed at the same locality around pools along a forest road. The single juvenile paratype from Panti (KUHE 52061) was found walking at a dry night at the edge of secondary forest in late July. In early September, no further specimens of this species or calls were detected at this locality. Associated anuran species observed were Phrynoidis aspera (Gravenhorst) , Kaloula pulchra Gray , Micryletta inornata (Boulenger) , Limnonectes blythii (Boulenger) , H. miopus , and H. labialis . Call characteristics. We recorded the calls of K. limbooliati at Gunung Pulai, Kulai on 6 September 2009 (air temperature 23.9 C), Gunung Lambak, Kluang on 7 September 2009 (air temperature 27.2 C), and at Kenaboi, Negeri Sembilan on 10 and 11 September 2009 (air temperature 26.4 C). The advertisement call ( Fig. 4 ) consists of a very long series of 24–61 (mean±SD = 41.1±11.2, n = 62/seven males) unpulsed notes and lasts about 6.3–15.2 (mean±SD = 10.9±2.8) s. The note repetition rate is 3.31–4.20 (mean±SD =3.72±0.30) notes per s. Each note lasts about 61–76 (mean±SD = 70.0±6.0) ms, and time interval between two notes varies from 238–310 (mean±SD = 271.7±25.4) ms. The dominant frequency lies at 1632–2008 (mean±SD = 1909±156) hz, and harmonics are at about 3000–4000 and 7000–8000 hz ( Fig. 4 A). The call has marked frequency and intensity modulations, and frequencies abruptly increase to the middle and then decrease towards the end of a note. Comparisons. Males of K. limbooliati sp. nov. with a SVL of 26.2–28.7 mm SVL (mean = 27.3 mm ) are of similar size to K . heterochirus Boulenger ( 24.1–27.2 mm , mean = 26.3 mm ), K. eok Das & Haas ( 26.3 mm ), and K. punctatus Peters ( 28.3 mm ); it can be readily differentiated from these species as follows: white spots in a black inguinal marking absent (present in K. heterochirus ); second finger with two subarticular tubercles (with single subarticular tubercle in K. eok ); fifth toe is not projecting as far as or farther than the third toe unlike K. punctatus (data from Matsui 2009 ).

Diagnosis. The new species is assigned to a member of Kalophrynus through mitochondrial DNA genealogy ( Matsui et al. 2011 ) together with the following morphological characteristics: No spine-like projection of skin at heel and elbow; belly without a network pattern; tips of fingers not expanded; underside of fingers without greatly enlarged tubercles; snout less than twice diameter of eye; inner metatarsal tubercle low, not shovel-like; tympanum visible. A medium-sized species that can be distinguished from all congeneric species by the following combination of characters; adult males 26.2–28.7 mm SVL (mean = 27.3, n = 3); snout pointed, directed downwards; third toe longer than fifth; projection of fourth finger from palm shorter than length of terminal phalanx of third finger; two subarticular tubercles under fourth finger; no subarticular tubercles under fifth toe; indistinct gland dorsal to arm insertion; no nuptial pads or asperities; forelimb not very stout, without a humeral spine in males; outer metatarsal tubercle absent; light lateral stripe; usually with distinct inguinal dark spot without white rim or spotting; calls similar to Hylarana laterimaculata with a long series of high-pitched whistle like notes.

Remarks. Previously allocated to K. pleurostigma , K. limbooliati was redescribed as a new species based on acoustic, morphological, and molecular data ( Matsui et al. 2011 ; Matsui et al. 2012), after the authors noticed that populations from southern Peninsular Malaysia and Singapore emit different calls from northern populations. The calls of K. limbooliati highly resemble Pulchrana laterimaculata (Matsui et al. 2012) with which it occurs sympatrically over a large part of its range. Flower (1896) collected the first specimen for Singapore , and Peninsular Malaysia , at BTNR. In describing vertical forest distribution of vertebrates, Murphy (1973) described K. limbooliati as a common leaf-litter inhabitant, but did not provide any data or details. Leaving this remark aside, and unpublished museum records, another specimen of K. limbooliati was not reported until 95 years later when one was seen in a drain near the entrance of BTNR on 11 January 1991 ( Lim 1991a ). Certainly, this observation was downplayed and not regarded as a rediscovery or novel since the species is perceived as common. Yet, the first occurrence of it outside of BTNR was not until 28 September 1993 when a single individual was found at MNF (Subaraj 1994). Occurrence. Restricted to CNR and surrounding Nature Parks and forests. Common.