Detection of Cryptic taxa in the genus Leptophryne (Fitzinger, 1843) (Amphibia; Bufonidae) and the description of a new species from Java, Indonesia Author Hamidy, Amir Author Munir, Misbahul Author Rahmania, Mila Author Kholik, Azis Abdul text Zootaxa 2018 2018-07-26 4450 4 427 444 journal article 29307 10.11646/zootaxa.4450.4.2 f8b727ad-d5df-41f8-ba9a-9910d42abed1 1175-5326 1444900 D62364EC-2DEA-462B-BC9B-1AD557BA785A Leptophryne javanica new species ( Figs. 3 A , 4 , 5 , 6 A , 7 A ) Leptophryne cruentata ( Tschudi, 1838 ) : Mumpuni 2014 Holotype : an adult male MZB Amph 15946 ( Figs. 3A & 4 ) (SVL 23.2 mm ), collected by Mumpuni and Mulyadi on October 2009 2000 h from the north-western slope of Mt. Slamet, Curug [water fall] Awu, Desa [village] Guci, Kecamatan [sub district] Bumi Jawa , Kabupaten [regency] Tegal, Provinsi [Province] Jawa Tengah ( S 7°11'57.51" , E 109°10'4.94" , elevation 1400 m a.s.l.) ( Fig. 1 ). Paratypes : Three adult males, MZB Amph 15941 (SVL 23.3 mm ), MZB Amph 15942 (SVL 22.5 mm ), and MZB Amph 15943 (SVL 23.9 mm , Fig. 6.I & 7.A ) collected from same locality, time and same collectors as the holotype ; two adult males MZB Amph 28565 (SVL 24.0 mm), MZB Amph 28566 (SVL 23.7 mm ) and an adult female MZB Amph 28564 (SVL 29.6 mm , Figs. 3B & 5 ) collected by Farist Alhadi , Didi Saiful Mahdi , Aldio Dwi Putra and Ahmad Nabil Faturahman on 10 March 2017 at 1900 h from Curug Cisurian , Mt. Ciremai , Desa Sukamukti , Kecamatan Jalaksana , Kabupaten Kuningan , Provinsi Jawa Barat ( S 06°56’14.6” , E 108°25’47.0” , elevation 1158 to 1201 m a.s.l. ) ( Fig. 1 ). FIGURE 2. Maximum Likelihood tree of ~473 bp fragment of 16S rRNA gene for Leptophryne javanica sp. nov. , along with representatives all Leptophryne species. Values above and below branch indicate Bayesian Posterior Probabilities (BPP), Maximum Likelihood Bootstrap Proportion (MLBP), and Neighbour Joining Bootstrap Proportion (NJBP). Referred specimens : Four adult males, MZB Amph 15940, 15944, 15945, 15947 collected from the same locality, time and same collector as the holotype . Etymology. The specific epithet is in reference to Java , the island where the type series was collected. Diagnosis. The new species is placed in the genus Leptophryne on the basis of it possessing the following characteristics: small size (< 50 mm SVL); body and limbs slender; firmisternal pectoral girdle present; tympanum visible; distinct paratoid gland present; tip of the fingers and toes rounded (not dilated); hands with basal webbing between fingers; fourth toe not broadly webbed; and large, elongate tubercle near base of each toe ( Davis 1935 ; Inger 1996; Malkmus et al . 2002). Moreover, the new species is assigned to the genus Leptophryne on the basis of a well-supported phylogenetic hypothesis based upon 473 bp of the mitochondrial 16S rRNA gene ( Fig. 2 ). The new species differs from its congeners in having a small body size (adult males 22.2–24.0 mm and single adult female 29.6 mm in SVL; Table 3 ), moderately slender body, dark grey dorsum mottled with distinct yellow, tip of the fingers and toes blunt, hindlimbs short, forth toe short; basal webbing on the hands, well developed webbing on toes; snout very protruding, pupils horizontal, parotoid gland present, tympanum indistinct and vomerine teeth absent; adult males with a median subgular vocal sac, enlarged arm, and nuptial pad. Description of holotype (measurements in mm). Small size (SVL 23.2); habitus moderately slender; head slightly longer (HL 7.6) than broad (HW 7.1), without bony crest; snout obtusely pointed, protruding in profile, projecting beyond lower jaw, longer (SL 2.9) than upper eyelid (UEW 2.0); canthus rostralis angular, loreal region vertical, concave; nostril nearer the tip of snout (SNL 0.9) than to the eye (NEL 2.0), latter much shorter than eye diameter (ED 3.0); interorbital space (IOD 2.9) much broader than the upper eyelid (UEW 2.0); tympanum indistinct, small (TD 0.8); no vomerine teeth; a median subgular vocal sac present; tongue mushroom-shaped, without papillae, notched posteriorly. Brachium length (BL 5.1) shorter than hand length (HAL 6.9), and much shorter than lower arm length (LAL 12.6); lower arm enlarged, much wider than brachium. Hands with basal webbing, fingers moderately slender, first finger (fin1L 1.5) shorter than second, which is shorter than fourth, the third finger is much longer (fin3L 3.9) (fin1L<fin2L<fin4L<fin3L), fingertip blunt, slightly swollen; inner palmar tubercle absent; outer palmar tubercle large (OPTL 0.9); supernumerary tubercle prominent, located near base of each finger; subarticular tubercle distinct; a distinct nuptial pad at the base of the first finger. Hindlimb slender (HLL 39.5); heels meeting when legs held at right angles body; tibia (TBL 11.9) longer than foot length (FL 10.4); tarsus (TSL 5.9) longer than fourth toe length (Toe4L 5.7); first toe (Toe1L 1.1)<second toe<third toe<fifth toe (Toe5L 3.4)<fourth toe (Toe4L 5.7), tibio-tarsal articulation of adpressed limb reaching to the nostril, toe tips blunt and slightly swollen, toe webs rather well developed, webbing not fleshy, formula I 0˗ 1 II 0˗1½ III 0˗ 2 IV 2˗½ V. Inner metatarsal tubercle distinct (IMTL 1.2) more than twice size of outer metatarsal tubercle (OMTL 0.5); large supernumerary tubercle located near the base of each toe; subarticular tubercle distinct; tarsal fold present. FIGURE 3. Photos in life of (A) male holotype (MZB Amph 15946) and (B) Female paratype (MZB 28564) of Leptophryne javanica sp. nov. , (C) male (MZB Amph 22257) (photographs by Farist Alhadi), and (D) female (uncatalogued) (photographs by Arief Tajali), of Leptophryne cruentata from Cibereum waterfall, Mt. Gede. FIGURE 4. Dorsal and ventral views of preserved male holotype Leptophryne javanica sp. nov. (MZB Amph 15946) (Above). Lateral view of head, ventral surfaces of left foot and right hand, and dorsal surface of right hand of the male holotype (MZB Amph 15946) (Below). Scale bars = 5 mm. Skin above with small granular tubercles, scattered on all parts, including the dorsal surface of the limbs; mandibular spines present; paratoid glands distinct, consisting of a pair of oval glands in a line, continuous with an oblique row of conspicuously enlarged warts along the dorsolateral surface ( Fig. 7 ); supratympanic fold absent; ventrum weakly granulated, particularly on chest; nuptial pads present; pectoral glands, femoral glands, dermal ridges on the limbs, and pineal spot are absent. Colour. In life, dorsally and laterally dark grey with distinct mottled yellow on head, back and limbs; groin and ventral surface of limbs, tibia, foot, hand, and webbing reddish; belly with a suffusion of yellow, chest and throat reddish; limbs with distinct yellow bars dorsally; posterior surface of thigh with yellow bars, barring continuing to the posterior surfaces of the flank; upper lip with yellow bars, loreal region dark grey with small yellow spots; pupil horizontal; iris golden with reticulated black pattern. In preservative, some yellow patterning fades to white, while the dark grey coloration on the dorsum darkens. Variations. Morphometric variation is shown in Table 3 . Male body size ranges from 22.2 mm to 24.0 mm. The male paratype MZB Amph 15943 is the largest and male paratype MZB Amph 15940 is the smallest in the type series. Four type specimens (MZB Amph 15942, 15943, 15946, 15947) have distinct black nuptial pads on the basal of the first finger while other type specimens (MZB Amph 15940, 15941, 15945, 15944) have brown nuptial pads. The tibio-tarsal articulation of adpressed hindlimbs reaches to the nostril in all but two type specimens (MZB Amph 15944, 15945), where it reaches the snout. The female paratype (MZB Amph 28564) has a much larger body size (SVL 29.6 mm ) compared to males. The irregular black spots and hour glass marking are present on the dorsum of the female paratype but absent in all male specimens. TABLE 3. Morphological measurements (mm) of Javanese Leptophryne species. Abbreviations defined in text.

Characters	Leptophryne javanica sp. nov.			Leptophryne cruentata		Leptophryne barbonica (Java)
Holotype	Males (n = 10)		Female (n=1)	Males (n = 12)		Females (n = 12)		Males (n = 12)		Females (n = 13)
MZB Amph 15946	Mean±SD	Range	MZB Amph 28564	Mean±SD	Range	Mean±SD	Range	Mean±SD	Range	Mean±SD	Range
SVL	23.2	23.0±0.68	22.2–24.0	29.6	25.1±1.03	23.3–26.7	34.4±2.06	31.1–37.4	23.8±1.18	21.7–25.8	30.3±2.13	26.1–33.3
HL	7.6	7.5±0.18	7.3–7.7	8.7	8.1±0.34	7.7–8.9	10.2±0.62	9.1–11.1	7.6±0.25	7.1–8.0	8.4±0.53	7.5–9.1
HW	7.1	7.0±0.27	6.7–7.6	8.3	7.3±0.39	6.8–8.2	9.2±0.68	8.0–10.0	6.8±0.34	6.4–7.5	7.5±0.47	6.7–8.3
SL	2.9	3.0±0.21	2.6–3.3	3.2	3.2±0.13	3.0–3.5	3.9±0.37	3.3–4.5	2.9±0.17	2.7–3.4	3.3±0.36	2.8–4.0
SNL	0.9	0.9±0.23	0.6–1.4	1.5	1.1±0.20	0.8–1.6	1.5±0.86	1.1–4.2	0.8±0.15	0.5–1.1	0.8±0.11	0.7–1.0
NEL	2.0	1.8±0.21	1.5–2.1	1.8	2.0±0.17	1.8–2.3	2.4±0.61	1.1–3.2	1.9±0.26	1.5–2.5	2.1±0.33	1.3–2.5
IND	2.1	3.4±0.26	3.1–3.8	2.6	1.9±0.19	1.6–2.3	2.3±0.27	1.9–2.7	1.7±0.17	1.5–2.1	1.8±0.18	1.5–2.2
IOD	2.9	2.9±0.30	2.3–3.2	4.2	3.0±0.26	2.5–3.5	3.7±0.76	3.1–5.9	2.5±0.27	2.1–2.9	3.1±0.34	2.3–3.8
UEW	2.0	2.1±0.16	1.8–2.3	2.8	2.2±0.18	1.9–2.6	2.8±0.20	2.5–3.2	2.5±0.23	2.2–3.0	3.0±0.18	2.6–3.2
ED	3.0	2.9±0.19	2.5–3.2	3.4	3.0±0.23	2.7–3.3	3.7±0.28	3.3–4.1	3.3±0.22	3.0–3.8	3.8±0.34	3.2–4.1
TD	0.8	0.7±0.17	0.4–0.9	0.9	0.7±0.19	0.4–1.0	0.9±0.13	0.7–1.1	1.2±0.15	0.8–1.3	1.4±0.18	1.2–1.8
BL	5.1	5.7±0.47	4.7–6.2	7.4	5.3±0.42	4.5–6.0	6.6±0.34	6.2–7.2	5.7±0.40	4.8–6.3	7.8±0.78	6.5–8.9
LAL	12.6	12.5±0.28	12.1–13.0	16.3	12.9±0.58	12.2–14.2	17.5±1.18	15.5–19.5	13.6±0.38	12.9–14.1	17.3±0.88	15.7–18.8
HAL	6.9	6.8±0.19	6.5–7.2	9.2	7.1±0.32	6.7–7.6	9.5±0.73	8.1–10.5	7.6±0.35	6.9–8.1	9.1±0.51	8.2–9.7
OPTL	0.9	1.1±0.15	0.8–1.3	1.3	1.2±0.18	0.9–1.5	1.5±0.33	1.1–2.3	1.2±0.28	0.4–1.4	1.4±0.26	1.0–2.0
fin1L	1.5	1.3±0.13	1.1–1.6	2.1	1.4±0.20	1.1–1.7	2.2±0.43	1.0–2.7	2.0±0.24	1.7–2.5	2.7±0.21	2.2–3.0
fin3L	3.9	3.8±0.31	3.4–4.3	5.5	3.9±0.28	3.5–4.4	5.4±0.62	4.5–6.7	4.3±0.33	3.8–5.1	5.4±0.35	4.8–6.3
HLL	39.5	39.3±1.22	37.0–40.9	50.1	40.9±2.13	37.9–45.6	52.9±2.9	47.2–56.8	45±1.37	43.0–47.0	57.3±2.23	54.3–62.2
TBL	11.9	12.0±0.23	11.7–12.5	15.6	12.4±0.52	11.7–13.8	16±0.62	15.2–17.0	14.1±0.46	13.4–14.9	18.3±0.73	17.5–19.6
TSL	5.9	6.0±0.30	5.6–6.6	7.6	6.4±0.31	5.9–6.8	8.3±0.53	7.7–9.6	6.9±0.38	6.3–7.5	8.4±0.46	7.6–9.3
FL	10.4	10.3±0.54	9.1–11.0	13.9	10.9±0.63	10.1–12.0	14.7±1.29	12.2–17.3	12.0±1.90	10.5–17.7	14.5±0.72	13.4–16.0
OMTL	0.5	0.7±0.11	0.5–0.8	0.9	0.8±0.15	0.6–1.0	1.0±0.12	0.8–1.2	0.9±0.13	0.7–1.2	1.0±0.18	0.7–1.2
IMTL	1.2	1.3±0.20	0.9–1.5	1.6	1.3±0.25	0.8–1.6	1.7±0.27	1.2–2.2	1.2±0.14	1.0–1.5	1.2±0.20	0.9–1.5
Toe1L	1.1	1.2±0.22	0.9–1.6	1.6	1.3±0.13	1.1–1.5	1.9±0.34	1.4–2.6	1.5±0.17	1.2–1.8	2.0±0.42	1.3–2.5
Toe4L	5.7	5.5±0.54	4.3–6.0	7.2	5.7±0.38	5.2–6.5	7.8±0.92	5.5–9.3	6.0±0.30	5.6–6.6	7.7±0.46	7.0–8.3
Toe5L	3.4	3.3±0.27	2.7–3.6	4.9	3.5±0.33	2.7–4.0	5.1±0.53	4.4–6.0	3.9±0.28	3.6–4.6	5.0±0.42	4.5–6.1

Comparisons. Morphologically and genetically, L. javanica sp. nov. is most similar to L. cruentata . It can be distinguished from L. cruentata by the following characteristics: smaller body size, SVL 22.2–24.0/n=11 (versus 23.3–26.7/n=12) in males, SVL 29.6 n=1 (versus 31.1–37.4/n=12) in females ( Table 3 ); shorter hindlimbs, HLL 37.0–40.9/n=11 (versus 37.9–45.6/n=12) in males, HLL 50.1/n=1 (versus 47.2–56.8/n=12) in females ( Table 3 ); shorter fourth toe length, Toe4L 4.3–6.0/n=11 (versus 5.2–6.5/n=12) in males, but longer fourth toe length, Toe4L 7.2/n=1 (versus 5.2–6.5/n=12) in females ( Table 3 ); more extensive toe webbing, I 0˗ 1 II 0˗1½ III 0˗ 2 IV 2˗½ V (versus I ½ ˗ 1 II ½˗1½ III ½˗ 2 IV 3˗ 1 V ) ( Fig. 6C ); hand with basal webbing (versus no webbing) ( Fig. 6D ); dorsum mottled with distinct yellow ( Figs. 3A, 3B ) [versus dorsum mottled with red ( Fig. 3C ) or combination of red and yellow ( Fig. 3D )]; and very protruding snout (versus less protruding snout) ( Figs. 7A, 7B ). The new species can be distinguished from L. borbonica by possessing the following combination of characteristics: dark grey or black dorsum mottled with distinct yellow ( Figs. 3A, 3B ) (versus brown with black spots); more extensive toe webbing, I 0˗ 1 II 0˗1½ III 0˗ 2 IV 2˗½ V (versus I ½ ˗ 1 II 1˗ 2 III 1½˗ 3 IV 3˗ 1 V ) ( Fig. 6C ); hand with basal webbing (versus no webbing) ( Fig. 6D ); shorter hindlimbs, HLL 37.0–40.9/n=11 (versus 43.0–47.0/n=12) ( Table 3 ); shorter fourth toe length, Toe4L 4.3–6.0/n=11 (versus 5.6–6.6/n=12) ( Table 3 ); tympanum indistinct (versus distinct) ( Figs. 7A, 7C ). FIGURE 5. Dorsal body, ventral hand, ventral foot and ventral body of a female paratype Leptophryne javanica sp. nov. (MZB Amph 28564). Scale bars = 5 mm. Photos by Farist Alhadi. Characteristics of male advertisement call. Call duration from 1.84–3.41 s (mean±SD=2.81±0.59, n=4), call interval of 48–82.94 s (mean±SD=62.83±14.71, n=4), call period of 51.11–84.78 s (mean±SD=65.43±14.20, n=4). The call contains a pulse and a continuous pure tone, which are divided into three types: single pulse repeated (type I), double pulses repeated (type II), and continuous pure tone (type III) ( Fig. 8 ). The calls are ascending, mostly beginning with a long type I, followed by type II and type III, ended by short type I ( Fig. 8 ). Type I contains15–18 pulses, pulse durations of 4.0–45.0 ms (mean±SD=26.08±9.47, n=4/1 male), pulse interval of 31.0– 1.38.0 ms (mean±SD=84.27±22.25, n=4/1 male), pulse periods of 51.0–169.0 ms (mean±SD=109.25±27.14, n=4/1 male), pulse rate of 5.9–19.6 (mean±SD=9.86±3.03, n=4/1 male), dominant frequency of 3.56–4.13 kHz (mean±SD=3.88±0.14, n=4/1 male). Type II contains 2–6 repeated double pulses, pulse durations of 8.0–28.0 ms (mean±SD=17.89±4.68, n=3/1 male), pulse rate of 6.9–7.9 (mean±SD=7.32±0.30, n=3/1 male), dominant frequency of 3.75–3.94 kHz (mean±SD=3.78±0.07, n=3/1 male). Type III contains 35–80 pulses (mean±SD=52.0±21.6, n=4/1 male), pulse durations of 144.0–417.0 ms (mean±SD=335.00±129.42, n=4/1 male), dominant frequency of 3.75–4.50 kHz (mean±SD=4.13±0.0.34, n=4/1 male). Range. Leptophryne javanica sp. nov. is currently known only from the northwestern slope of Mt. Slamet and Mt. Ciremai ( Fig. 1 ). Since L. cruentata is endemic to mountains in the west of Java , Leptophryne javanica sp. nov. is likely endemic to mountains in the central part of the island with an elevation of 1,200–1,500 m a.s.l. Natural history. Eggs and larvae are unknown. The type series was collected from a swift, rocky stream with waterfalls in secondary montane forest. In Mt. Ciremai, the streams are 1.5–2.0 m in width and 0.4–0.6 m in depth. Specimens were found on rocks within the stream and on the stream banks, 0.3–0.5 m from the water ( Fig. 9 ). Other species found in the same habitat as L. javanica sp. nov. are Limnonectes kuhlii (Tschudi) , Limnonectes macrodon (Duméril and Bibron) , Odorrana hosii (Boulenger) , Huia masonii (Boulenger) , Chalcorana chalconota (Schlegel) , Phrynoides asper (Gravenhorst) , Megophrys montana (Kuhl and Van Hasselt) , Rhacophorus margaritifer (Schlegel) , and Rhacophorus reinwardtii (Schlegel) .