Ophryophryne elfina, Jr., Nikolay A. Poyarkov, Duong, Tang Van, Orlov, Nikolai L., Gogoleva, Svetlana S., Vassilieva, Anna B., Nguyen, Luan Thanh, Nguyen, Vu Dang Hoang, Nguyen, Sang Ngoc, Che, Jing & Mahony, Stephen, 2017

Jr., Nikolay A. Poyarkov, Duong, Tang Van, Orlov, Nikolai L., Gogoleva, Svetlana S., Vassilieva, Anna B., Nguyen, Luan Thanh, Nguyen, Vu Dang Hoang, Nguyen, Sang Ngoc, Che, Jing & Mahony, Stephen, 2017, Molecular, morphological and acoustic assessment of the genus Ophryophryne (Anura, Megophryidae) from Langbian Plateau, southern Vietnam, with description of a new species, ZooKeys 672, pp. 49-120 : 61-76

Ophryophryne elfina sp. n. Figs 6G, H, 8, 9, 10, 11, 12, 13C, 14A (right), 14B, C

Chresonymy:

" Ophryophryne sp. 1" - Orlov et al. 2008:82 (Chu Yang Sin N.P., Dak Lak Prov., Vietnam; ZISP 12880-12884).

[?] Ophryophryne gerti - Stuart et al. 2010:40 (eight uncataloged topotype females from Bidoup–Nui Ba N.P., Langbian Plateau, Vietnam).

" Ophryophryne sp." - Poyarkov [Paiarkov] and Vassilieva 2011:174, 202; fig. 5.6 (Bidoup Mt., Bidoup–Nui Ba N.P., Lam Dong Prov., Vietnam; ZMMU A-4716, ZMMU A-4788, ZMMU A-5674, ZMMU A-5675).

Ophryophryne gerti - Nguyen et al. 2014:148-149; fig. 2 ( partim–– Hon Ba Mt., Hon Ba Nature Reserve [hereafter N.R.], Khanh Hoa Prov., Vietnam; VNMN 983, ZFMK 94220).

Holotype.

ZMMU A-5669 (field number NAP-02658), adult male from the northern slope of Chu Pan Fan Mountain, Chu Yang Sin National Park, Bong Krang Commune, Lak District, Dak Lak Province, Vietnam (coordinates 12°22'31.90"N; 108°21'14.10"E, elevation 1725 m a.s.l.), collected along a mountain stream in mixed evergreen montane tropical forest by N.A. Poyarkov on 07 April 2012 (Figs 8 and 9).

Paratypes.

ZMMU A-5691 (field numbers ABV-00580; ABV-00581), two juveniles from the north-western slope of Chu Yang Sin Mountain, Chu Yang Sin N.P., Hoa Le Commune, Krong Bong Dist., Dak Lak Prov., Vietnam (12°24'47.70"N; 108°24'25.40"E, 1975 m a.s.l.), collected in leaf litter in mixed evergreen montane tropical forest by N.A. Poyarkov on 25 May 2014; ZMMU A-5692 (field number NAP-00582), adult male from the northern slope of Bidoup Mountain, Bidoup–Nui Ba N.P., Da Chais Commune, Lac Duong Dist., Lam Dong Prov., Vietnam (12°06'5.60"N; 108°39'34.20"E, 2035 m a.s.l.), collected along mountain stream in evergreen montane elfin forest by N.A. Poyarkov on 09 May 2009; ZMMU A-5675 (field numbers NAP-01456; NAP-01459), two adult males from the northern slope of Bidoup Mountain, Bidoup–Nui Ba N.P., Da Chais Commune, Lac Duong Dist., Lam Dong Prov., Vietnam (12°06'5.60"N; 108°39'34.20"E, 2035 m a.s.l.), collected on stones and vegetation along a mountain stream in mixed evergreen montane elfin forest by N.A. Poyarkov and A.B. Vassilieva on 25 June 2010; ZMMU A-4788 (field numbers NAP-01455; NAP-01449; NAP-01450; NAP-01460), four adult males from the southern slope of Hon Giao Mountain Ridge, Bidoup–Nui Ba N.P., Da Chais Commune, Lac Duong Dist., border of Lam Dong Prov. and Khanh Hoa Prov., Vietnam (12°11'33.10"N; 108°42'41.80"E, 1890 m a.s.l.), collected along a mountain stream sitting on stones and tree branches near the water edge, in mixed evergreen montane elfin forest by N.A. Poyarkov and A.B. Vassilieva on 30 June 2010; ZMMU A-5674 (field numbers NAP-01451; NAP-01452), two adult males from the southern slope of Hon Giao Mountain Ridge, Bidoup–Nui Ba N.P., Da Chais Commune, Lac Duong Dist., border of Lam Dong Prov. and Khanh Hoa Prov., Vietnam (12°11'33.10"N; 108°42'41.80"E, 1890 m a.s.l.), collected along a mountain stream on stones and vegetation in evergreen montane elfin forest by N.A. Poyarkov and A.B. Vassilieva on 29 June 2010; ZMMU A-5170 three adult males (field numbers ABV-00454; ABV-00472; ABV-00471), and one adult female (ABV-00455) from the northern slope of Bidoup Mountain, Bidoup–Nui Ba N.P., Da Chais Commune, Lac Duong Dist., Lam Dong Prov., Vietnam (12°06'5.60"N; 108°39'34.20"E, 2035 m a.s.l.), collected along a mountain stream in mixed evergreen montane tropical forest by A.B. Vassilieva on 16 April 2014.

Referred specimens. ITBCZ 2786, ITBCZ 2788, ITBCZ 2792, ITBCZ 2828, three adult females and one adult male collected along a mountain stream in evergreen mountain forest on the summit of Hon Ba Mountain, Hon Ba N.R., Dien Khanh Dist., Khanh Hoa Prov., Vietnam (12°07'10.60"N; 108°56'51.60"E, 1510 m a.s.l.), by Sang Ngoc Nguyen, Luan Thanh Nguyen and Vu Dang Hoang Nguyen on 22-24 December 2015; ITBCZ 2908-2909, ITBCZ 2918-2919, ITBCZ 3502, five adult males collected along a mountain stream in evergreen montane forest on the summit of Hon Ba Mountain, Hon Ba N.R., Dien Khanh Dist., Khanh Hoa Prov., Vietnam (12°07'28.80"N; 108°58'14.20"E, 950 m a.s.l.), by Sang Ngoc Nguyen, Luan Thanh Nguyen, and Vu Dang Hoang Nguyen on 22-28 March 2016; ZMMU A-5679 (field number NAP-01169), 7 larvae collected in a cascade mountain stream on the northern slope of Bidoup Mountain, Bidoup–Nui Ba N.P., Da Chais Commune, Lac Duong Dist., Lam Dong Prov., Vietnam (12°06'5.60"N; 108°39'34.20"E, 2035 m a.s.l.), by N.A. Poyarkov on 03 May 2009; ZMMU A-5684 (field number NAP-02673), 4 larvae collected in a cascade mountain stream on the northern slope of Chu Pan Fan Mt., Chu Yang Sin N.P., Bong Krang Commune, Lak Dist., Dak Lak Prov., Vietnam (coordinates 12°22'31.90"N; 108°21'14.10"E, elevation 1725 m a.s.l.), by N.A. Poyarkov on 07 April 2012.

Etymology.

The specific epithet is an adjective (in agreement with the genus name in feminine gender), derived from “elf”, the English spelling of “alfus” in Latin, referring to usually forest-dwelling supernatural mythological creatures in Germanic mythology and folklore; the name is given in reference both to the funny appearance and small size of the new species, as well as to the their endangered habitat, restricted to wet evergreen montane forests at high elevations of the Langbian Plateau; such forests are often called "elfin forests".

Recommended vernacular name.

The recommended common name in English is "Elfin Mountain Toad". The recommended common name in Vietnamese is “Cóc Núi Tiểu Yêu Tinh".

Diagnosis.

The species is allocated to Ophryophryne based on its obvious similarities with its sister taxa, its molecular phylogenetic affinities, and the absence of maxillary teeth considered diagnostic for the genus (previous authors, e.g. Ohler 2003, Delorme et al. 2006 and Fei et al. 2009 also indicated a horizontal pupil and the absence of vomerine teeth as diagnostic for Ophryophryne , this is reconsidered by Mahony et al. 2017). Ophryophryne elfina sp. n. is distinguished from its congeners by a combination of the following morphological attributes: (1) small adult body size, male SVL 26.9-33.9 mm (N = 29), female SVL 35.1-36.5 mm (N = 6); (2) snout sharply protruding in profile; (3) tympanum diameter approximately half of eye diameter; tympanum to eye distance approximately 70-90% of tympanum diameter; (4) finger length formula: F1 <F4 ≤ F2 <F3, or F1 ≤ F2 <F4 <F3; toe webbing rudimentary, toe length formula: T1 <T5 <T2 <T3 <T4; (5) short dorsolateral glandular ridge present above shoulder; (6) palpebral projection present as a small single tubercle to moderately developed single projection; (7) dermal cloacal protuberance and dermal flaps above cloacal opening absent; (8) skin of dorsal and lateral surfaces of head, body and limbs shagreened with numerous small tubercles, large warts on the flanks; (9) skin on dorsal and lateral surfaces of body, head and limbs with numerous bright orange-red (in life) asperities; (10) males with a red-orange (in life) nuptial pad on F1; (11) dorsal coloration light yellow-brown with dark hourglass-shaped marking on dorsum usually edged with white or beige (in life); (12) posterior suborbital light bar well-defined, usually clearly separated from dark-brown temporal triangular spot, uniformly covering temporal area and tympanum.

The new species is also markedly distinct from all congeners for which comparable sequences are available (16S rRNA mitochondrial gene; uncorrected genetic distance> 8.2%). The advertisement call of the new species consists of whistling notes uttered in series: average 12.84 ± 0.41 calls per series, with an average dominant frequency of 4645.94 ± 4.39 Hz, repetition rate per recording/series 1.18 ± 0.2 calls/s and 3.87 ± 0.07 calls/s, respectively, with average call duration 73 ± 0.23 ms and inter-call interval 207 ± 2.06 ms, also distinguishes the new species from Ophryophryne species for which calls are known, including the two species found in sympatry.

Description of holotype.

Mature male (SVL 27.2 mm); habitus slender (Figs 8A, B, and 9). Specimen in good state of preservation; median abdomen dissected, dissection length ca. 9.0 mm; liver and testes observable through incision, testes white, enlarged (testes length 3.9 mm; Fig. 8B); ventral right femur dissected for molecular sampling, dissection length 9.8 mm.

Head moderately large (HL/SVL 29.2%; HW/SVL 29.5%), almost as wide as long (HW/HL 101.0%), triangular in dorsal view; top of head flat; snout comparatively short (ES/HL 30.9%), narrow (IFE/HW 39.4%), somewhat truncated in dorsal view (Fig. 8A), sharply protruding in profile, without rostral appendage (Figs 8C, 9); snout projecting significantly beyond margin of lower jaw (Fig. 8C); loreal region slightly concave; canthus rostralis distinct, sharp; dorsal region of snout flat; eyes large (ED/HL 44.8%), slightly protuberant in dorsal view and in profile, eye less than twice as long as maximum tympanum diameter (TYD/ED 53.7%) and half times longer than snout (ED/SL 145.3%); eye-tympanum distance less than maximum tympanum diameter (TYE/TD 83.3%); tympanum distinct, almost circular shaped with vertical diameter slightly exceeding horizontal diameter, tympanum large (TYD/HL 24.1%); pupil in preservation diamond-shaped (Fig. 8C), horizontally orientated; nostril oval-shaped, vertical, laterally orientated, located as far from eye as from snout (EN/NS 100.8%); internarial distance greater than eyelid width (IN/UEW 119.5%), and subequal to narrowest point between upper eyelids (IN/IUE 103.7%); pineal ocellus not visible externally (Fig. 8A); vomerine ridges not absent; maxillary and vomerine teeth absent; vocal sac gular, its openings not discernable; tongue moderately large, with free posterior end, not notched posteriorly, lacking medial lingual process.

Forelimbs slender, forearm moderately long (FAL/SVL 25.7%), slightly enlarged relative to upper forelimb, and shorter than hand (FAL/HAL 96.1%); fingers long and narrow, dorsoventrally flattened, weak lateral fringes present on third and fourth fingers (Fig. 8D), finger length formula F1 <F4 <F2 <F3; fingers completely free of webbing; subarticular tubercles absent, replaced by low callous dermal ridges; supernumerary tubercles absent; outer palmar (metacarpal) tubercle small, round, elevated but with indistinct borders (Fig. 8D); thenar tubercle weak; finger tips in life rounded, weakly expanded relative to digit widths (wider than the distal-most finger articulation), with circular pads (Fig. 8D); terminal grooves absent.

Hindlimbs slender, relatively long, shanks overlap when thighs are held at right angle to body; shank length less than half of snout–vent length (SHL/SVL 48.7%); thighs shorter than shanks (SHL/TL 109.2%), and feet (FOL/TL 110.9%); toes long and slightly dorsoventrally flattened (Fig. 8E), relative toe lengths T1 <T5 <T2 <T3 <T4; toe tips slightly expanded relative to digit widths (wider than the distal-most toe articulation), with circular pads; terminal grooves absent; lateral fringes on toes, outer metatarsal tubercle, subarticular and supernumerary tubercles absent; inner metatarsal tubercle well developed with distinct borders, ca. two times longer than wide, oval-shaped (IMT/FOL 9.3%); well-developed dermal ridge of callous tissue present on ventral surface of all toes and continuing to metatarsus; rudimentary webbing present between all five toes, basal web distinct between toes T2-T3 and T3-T4, but is completely reduced between toes T1-T2 and T4-T5; tarsal fold absent.

Skin texture and skin glands.

Skin of dorsal and lateral surfaces of head, body and limbs shagreened, with numerous small skin asperities present on anterior two thirds of dorsum, sparse posteriorly, increasing in density along dermal ridges, densely covering dorsal and lateral surfaces of head, upper eyelids, and dorsal surfaces of thighs, shanks, upper forelimbs, forearms, hands, feet and digits, and absent from all remaining surfaces. Small tubercles finely and relatively evenly scattered on dorsal surfaces of trunk, head and limbs, including maxilla, mandible, eyelids and dorsal surfaces of head, forelimbs and hindlimbs (Figs 8A and 9); small tubercles present on temporal region, tympanum smooth, tympanic rim distinct but not elevated relative to skin of temporal region; on dorsal surfaces tubercles arranged in distinct longitudinal ridges on upper forelimbs, forearms, shanks and thighs, becoming less distinct on dorsal surfaces of hands, feet and digits (Fig. 9); six large tubercles on left flank and seven large tubercles on right flank irregularly scattered from axilla to groin, intermixed with smaller tubercles; central portion of outer edge of upper eyelid slightly thickened, with a distinct small single tubercle on a thickened ridge (Figs 8C, 9); distinct thick glandular supratympanic fold, narrow anteriorly, considerably widening posteriorly, extending from posterior corner of eye gently sloping down towards dorsal margin of tympanum (but not concealing it), where it broadly curves down, terminating above axilla (Figs 8C, 9); short dorsolateral glandular ridge present above shoulders, on anterior part of dorsum, its length comparable with eye diameter (Fig. 9); a weak ">-<"-shaped glandular dermal parietoscapular-sacral ridge present on dorsum (Figs 8A, 9); transverse fold at head basis absent; dermal projection above cloaca absent; gular region, chest, abdomen and ventral surfaces of limbs smooth to weakly shagreened (especially on the posterior surface of abdomen); nuptial pad present, single, covered with microgranules, covering entire dorsal metacarpal of first finger extending distally to ca. 3/4 basal phalange length; pectoral glands round, flat, of medium size, positioned level with axilla; femoral gland flat, indistinct, on posterior surface of thighs.

Color of holotype in life.

Entire dorsum light olive-brown to yellow-brown with large irregular brownish grey spots; dorsal surfaces of head yellowish brown from tip of snout to eyes; small oval-shaped spot with irregular borders on dorsal surface of snout between anterior canthi; a small dark dot on dorsal surface of snout tip; similar single dark dots on anterior parts of upper eyelids; brown “V” -shaped marking on crown between supraorbital horns with apex pointing posteriorly, outlined with thin light-beige edging; round brownish spot at head basis; ">-<"-shaped marking surrounded with dark olive-brown, outlined with thin light-beige edging forming a hourglass-shaped dorsal marking (Fig. 8A); two small roundish brown spots at sacrum (Fig. 8A); supratympanic fold dorsally light yellowish brown, ventrally dark-brown; front and lateral surfaces of snout and lateral canthus rostralis dark reddish brown; lateral surfaces of maxilla dark brown with four distinct orange-brown to yellowish beige bars extending from orbits towards edge of maxilla: smallest anteriormost light band borders nostril ventrally, with two posterior light bands extend from posterior corner of eye towards angle of mouth (Fig. 9); axilla purplish brown; tympanum uniform purplish brown; temporal region uniform dark purplish brown, clearly defined from light beige area on posterior part of maxilla; pupil black, outlined in copper-gold; iris golden dorsally and ventrally, copper-orange at medial part, with tiny dark reticulations spreading from pupil; sclera lemon-yellow; upper surface of limbs yellowish brown with irregular dark-brown spots on forearms and transverse spots forming dark-brown and greyish bands across shanks, thighs and tibio-tarsus (three complete transverse bands on left leg, two complete [both on shank and thigh] and one incomplete [on shank only] transverse bands on right leg: Figs 8 and 9), knee joint dark brown; sides beige-yellow with indistinct greyish white flecking and large black spots with irregular borders: marking location of large warts on each side of body, four smaller spots located dorsally, and two large brown-black spots located ventrally, top of larger flank tubercles brownish cream (Fig. 9); throat brownish to purplish grey with greyish white flecking and irregular dark-brown spots; chest and anterior half of abdomen purplish grey with whitish flecking and grey-brown blotches; posterior half of abdomen lighter greyish pink with irregular dark blotches; lower surface of limbs purplish grey with white and beige flecking; area surrounding vent and posterior surface of thighs dark black-brown with whitish spots, posterior surface of thighs near tibio-tarsal articulation black-brown with sparse whitish dots; dorsal surface of feet and shanks yellowish beige with brown flecking; ventral surface of feet and shanks brown-black. Pectoral and femoral glands creamy white. Nuptial pad, outer metacarpal (palmar) and metatarsal tubercles pink- red to orange-red. Asperities covering dorsal surfaces of body, head, limbs and digits, lateral sides of head and anterior part of chest in life bright orange-red, forming reddish rows and ridges on dorsal surfaces of limbs as well as on edge of upper eyelid, palpebral projection also with orange-red asperities.

Color of holotype in preservative.

In preservative coloration faded to light grey-brown on dorsum and flanks, with slightly paler limbs and greyish beige to whitish on venter; reddish and orange tints, as well as iris coloration, faded completely; dark markings on dorsum, sides and venter and other features remain without significant change (Fig. 8). Banding on limbs is less pronounced. Chest, abdomen, throat, interior portions of forelimbs and thighs are pale greyish brown; formerly brightly colored dorsal asperities and nuptial pads, palmar and metatarsal tubercles turned transparent or creamy white (Fig. 8C, E).

Measurements of the holotype

(all in mm, taken by NAP).SVL 27.2; HW 8.0; HL 7.9; IFE 3.2; IBE 6.8; ED 3.6; TYD 1.9; TYE 1.6; SL 2.5; EN 1.3; NS 1.3; IUE 2.3; IN 2.3; UEW 2.0; FAL 7.0; HAL 7.3; FIL 2.4; FIIL 2.8; FIIIL 4.6; FIVL 2.8; SHL 13.2; TL 12.1; FOL 11.9; TFOL 18.9; IMT 1.8; TIL 1.7; TIIL 3.8; TIIIL 5.5; TIVL 6.7; TVL 3.5.

Variation.

Morphometric variation within the type series and other referred specimens of the new species is shown in Table 5. Individuals of the type series are similar in morphology and body proportions (Figs 9, 10). There is a clear and significant difference in body size between males and females (Fig. 3): females (SVL 35.1-36.5 mm, N = 6) are significantly bigger than males (SVL 26.9-33.9 mm, N = 29) (Duncan test, p <0.05); sexual differences were not significant for other mensural characters possibly due to the small sample size of females. Certain variation is observed in finger lengths: most of the examined specimens have the finger length formula F1 <F4 <F2 <F3 (N = 14), in some specimens, including the holotype, the second and the fourth fingers are of equal length (F1 <F4 = F2 <F3; N = 5), or the fourth finger is longer than the second (F1 <F2 <F4 <F3; N = 5); rarely the second finger is as long as the first finger (F1 = F2 <F4 <F3; N = 2). Specimens vary in the number and size of black spots and blotches on flanks (Fig. 10A, B). In life, both sexes of the new species have lighter dorsum and belly coloration when nocturnally active. Other in-life variation was observed for throat coloration: throat can be dark brownish with clear dark-grey blotches (Fig. 10C) to almost uniform brown-violet to purple with dark blotches not discernable (Fig. 10D). There is significant variation in dorsal pattern: in some specimens the dorsum looks almost uniform yellowish brown with an indistinct hourglass-shaped figure (Figs 6G, 10A, B, 14 [right]) whereas in other specimens the hourglass-shaped figure is distinct, dark brown and edged with light beige (Figs 6H, 9). There is some variation in the length of palpebral projections, from a small almost indistinct tubercle (Figs 6H, 13C) to a moderately well-developed projection (Figs 6G, 14). Coloration of the lateral surfaces of the head vary, but on all specimens two light suborbital bars are distinct, clearly separated from the uniform dark-brown coloration of the tympanal area. Iris coloration shows insignificant variation: Nui Chua Mt. population appear to have copper-red coloration of the entire iris "(Fig. 14B), somewhat different from the coloration of the holotype (Fig. 9). Recently metamorphosed and juvenile specimens have numerous bright red-orange tubercles (Fig. 10E, F) which are more conspicuous than in adults. Excluding the presence of nuptial pads on males, the new species shows no significant variation in dermal characters among sexes (Fig. 10); in preservative smaller tubercles become flattened and less distinct.

Tadpole description.

Tadpoles were allocated to Ophryophryne elfina sp. n. based on the following evidence: (1) morphological features characteristic for megophryine larvae in general; Ophryophryne or Megophrys s. lato in particular (elliptical shaped body with long muscular tail, oral disk forms a dorsally oriented funnel); (2) collected in the stream where calling males of the new species were recorded; (3) species identification confirmed by mtDNA sequences of short 16S rRNA gene fragment (up to 500 bp) (GenBank Acession numbers: KY515232-KY515233, see Table 1).

Standard tadpoles measurements (mean ± SD, N = 5, Stage 25; ZMMU A-5679, field number NAP-01169, collected from Bidoup Mt., 1900 m a.s.l., Bidoup–Nui Ba N.P., Lam Dong Prov.) (all in mm, taken by NAP): TOL = 28.4 ± 1.3 (27.4-30.2); BL = 8.6 ± 0.1 (8.4-8.7); TAL = 19.8 ± 1.2 (18.9-21.5); BW = 4.4 ± 0.4 (3.8-4.6); BH = 3.6 ± 0.2 (3.4-3.7); TH = 4.5 ± 0.4 (4.0-4.8); SVL = 9.2 ± 0.3 (9.0-9.5); SSp = 4.8 ± 0.2 (4.5-4.9); UF = 1.4 ± 0.1 (1.3-1.5); LF = 1.1 ± 0.0 (1.1-1.1); IN = 2.6 ± 0.1 (2.5-2.6); IP = 2.8 ± 0.2 (2.7-3.2); RN = 1.7 ± 0.1 (1.7-1.8); NP = 0.8 ± 0.1 (0.7-0.8); ED = 0.8 ± 0.0 (0.8-0.9).

The following description is based on five tadpoles at stage 25 (ZMMU A-5679, field number NAP-01169). In lateral view (Fig. 11A), body slightly compressed dorsoventrally (BH/BW 83.5 ± 4.09%), especially anteriorly, convex both dorsally and ventrally. Body elliptical in dorsal view (Fig. 11B), with maximum width at middle of body (BW/BL 51.0 ± 4.2%); snout short, rounded, blunt. Eyes of moderate size (ED/BL 9.8 ± 0.3%), not bulging, separated by a distance which equals approximately 1.1 times internarial distance (IP/IN 110.2 ± 6.8%), directed and positioned dorsolaterally, not visible in ventral view; pupils oriented dorsolaterally. Nares tubular, positioned dorsally (near anterior edge of eye), much closer to pupils than to tip of snout; directed laterally. Spiracle sinistral, conical, very short, opening at half of distance from snout tip to vent (SSp/SVL 52.2 ± 3.4%); spiracle attached to body wall for most of its length, extremity is free, positioned at the level of longitudinal axis, oriented dorsoposteriorly, opening varies from rounded to oval. Tail long, more than two times longer than body (TAL/BL 231.3 ± 11.0%), lanceolate; almost equal in height along its length (point of maximum height of tail located just anterior to midlength of tail); tail tip bluntly rounded, without terminal filament; tail musculature strong, gradually tapering, almost reaching tail tip. Tail fins shallow, moderately well developed, not extending onto body: dorsal fin originating almost at body-tail junction, much shorter than lower fin proximally and nearly equal in height to it on middle of tail; dorsal fin slightly higher than ventral fin on distal half of tail (LF/UF 77.1 ± 4.9%); free margin of dorsal fin horizontal and shallow on anterior half of tail; free margin of ventral fin parallel to tail musculature. Vent opening medial, tubular, directed posteriorly, not linked to ventral tail fin. Neither skin glands nor neuromasts visible in preservative, but neuromasts of the lateral line system are distinct in life (Fig. 12A) forming two curved lines running from snout towards orbits and along orbital margins ventrally. Subterminal oral disk with lips expanded vertically forming a dorsally oriented funnel (Fig. 11B); lateral corners of funnel distinct; upper lip notably smaller than lower; lips lack keratodonts, but bear short, low ridges, more densely arranged on upper than on lower labium, arranged in 18-24 (mean = 22) longitudinal rows and from 2-4 (mean = 3) transverse rows on upper labium to 4-6 (mean = 5) transverse rows on lower labium. Marginal papillae absent. Width of expanded funnel comprises over 75% of body length (Fig. 12A) and just 30% when folded in preservation (Fig. 12B).

In life tadpoles have dorsal side of body and upper flanks uniform brownish red or brownish orange (Fig. 12A, B). Lower flanks weakly mottled with dark brown, few round blackish spots on tail and dorsum; with orange neuromasts visible on dorsal surface (Fig. 12A). Abdomen light brownish orange, intestine not visible through body wall. Caudal muscles pale; tail fins translucent with a few darker spots (more on upper than on lower fin); dorsally tail with indistinct middorsal orange line (Fig. 12A). Eyes golden with black reticulations. Oral funnel pinkish orange with brownish red papillae (Fig. 12B). In preservative tadpole coloration gets much duller, but the general coloration pattern is still visible after 7 years in ethanol.

Advertisement call characteristics.

Refer to the Acoustic differentiation section, Table 4 and Fig. 5, for bioacoustic comparison of the new species with O. synoria and O. gerti . Refer to Appendix 3 for call variation data in Ophryophryne elfina sp. n.

Position in mtDNA phylogeny and sequence divergence.

The new species is reconstructed as a member of the Ophryophryne Group II (Fig. 2), forming a sister group with respect to the clade joining O. gerti and O. synoria (see Fig. 2). Uncorrected genetic p-distances between Ophryophryne elfina sp. n. partial sequences for the 16S rRNA gene and all homologous sequences available on GenBank included in the analysis (see Table 1) varied from 8.2% (with O. gerti s. stricto, clade A) to 10.0% (with O. hansi and O. synoria , clade C) (see Table 2). This degree of pairwise divergence in the 16S rRNA gene is greater than that usually representing differentiation at the species level in anura ( Vences et al. 2005a, 2005b, Vieites et al. 2009, Poyarkov et al. 2015a, 2015b). Intraspecific variation in this gene fragment for Ophryophryne elfina sp. n. is significant - maximum sequence divergence between Nui Chua Mt. population (Fig. 2, clade D) and populations from the rest of the species range (Fig. 2, clade E) is p = 3.1%. Intraspecific variation in 16S rRNA gene fragment within one geographic population was lower, and ranged from 0.0% to 0.8% of substitutions.

Distribution.

Ophryophryne elfina sp. n. is found to be endemic to five provinces in (Lam Dong, Dak Lak, Khanh Hoa, Ninh Thuan and Phu Yen) in the northern and eastern part of the Langbian Plateau and its foothills in southern Vietnam (localities 6-12, Fig. 1). The new species is restricted to wet evergreen montane tropical and elfin forests, receiving high precipitation from the sea. Such wet forests are found only on high elevations in the central parts of the Langbian Plateau (e.g. 1900-2100 m a.s.l. on Bidoup Mt., Lam Dong Prov., Fig. 1, Loc. 6) or peripheral mountains remote from the sea (e.g. 1900-2300 m a.s.l. on Chu Pan Fan and Chu Yang Sin Mts., Dak Lak Prov., Fig. 1, Locs 10 and 11), but on the eastern foothills of the plateau which receive more precipitation, the new species is found at lower elevation (from 950 to 1510 m a.s.l. on Hon Ba Mt., Khanh Hoa Prov., Fig. 1, Loc. 8; 780 m a.s.l. on Nui Chua Mt., Ninh Thuan Prov., Fig. 1, Loc. 9; and 700 m in Phu Yen Prov., Fig. 1, Loc. 12).

Ecology.

All specimens were collected at night after heavy rains along montane cascade rocky streams, along small waterfalls, or intermittent rocky brooks; or found during the day time under tree-logs and within leaf litter in the limited fragments of primary montane wet polydominant evergreen tropical forests, with a high abundance of large rocks and fallen trees covered with a thick layer of mosses. This including high montane forests that are composed of the specific floral community known as “elfin” forests, with miniature trees (up to 10 m tall). These areas always have high precipitation and have much milder climate than other tropical forests in southern Vietnam: active breeding of the new species was recorded in February with temperatures of ca. 11-12°C.

On Bidoup Mt. summit (Lam Dong Prov.), Ophryophryne elfina sp. n. was recorded from 1890 to 2035 m a.s.l. in montane polydominant high canopy (trees up to 35 m tall) and elfin (trees up to 10 m tall) (sub)tropical forests with the predominance of trees of the family Fagaceae ( Lithocarpus sp., Castanopsis sp.), Elaeocarpaceae ( Elaeocarpus sp.), Lauraceae ( Machilus sp.), Magnoliaceae ( Magnolia sp., Michelia sp.), and occasional large trees of Fokienia hodginsii ( Cupressaceae ). These forests have thick leaf litter, numerous fallen logs and rocks covered with mosses, and an undergrowth that is predominated by ferns (mostly Asplenium sp., Aspleniaceae ) (see Kuznetsov and Kuznetsova 2011) (Fig. 15A). On Hon Giao mountain ridge, the new species was found along mountain streams from 1800 to 1900 m a.s.l. in montane polydominant elfin forests with the predominance of trees of the family Fagaceae ( Castanopsis sp., Lithocarpus sp.), Lauraceae ( Cinnamomum sp., Neolitsea sp.), Ericaceae ( Rhododendron sp.), Magnoliaceae ( Mangletia sp., Michelia sp.), Elaeocarpaceae ( Elaeocarpus sp.) and Podocarpaceae ( Podocarpus neriifolius ). This forest had a dense undergrowth of mosses, orchids ( Coelogyne sp., Dendrobium sp.; Orchidaceae ) and occasional ferns ( Cyathea sp., Cyatheaceae ) (see Kuznetsov and Kuznetsova 2011) (Fig. 15B). In Chu Yang Sin N.P. (Dak Lak Prov.), the new species was found from 1800 to 2100 m a.s.l. in montane forests with the predominance of trees of the families Pinaceae ( Pinus krempfii ; Pinus kesiya ) and Fagaceae ( Lithocarpus sp., Castanopsis sp.), with dense undergrowth of ferns, numerous rocks and fallen trees covered with mosses (Fig. 15C). In Hon Ba N.R. (Khanh Hoa Prov.), the new species was found from 950 to 1510 m a.s.l. along mountain streams in forests having polydominant composition including Fagaceae ( Lithocarpus sp., Quercus sp.), Elaeocarpaceae ( Elaeocarpus sp.), Theaceae (Thea sp., Camellia sp.), Lauraceae ( Cinnamomum sp., Neolitsea sp.), Araliaceae ( Schefflera sp.) and Rutaceae ( Euodia sp.).

On Bidoup Mt. summit (1890-2035 m a.s.l.; Lam Dong Prov.) Ophryophryne elfina sp. n. occurs in syntopy with Leptobrachium pullum (Smith, 1921), Leptobrachium leucops Stuart, Rowley, Tran, Le & Hoang, 2011, Leptolalax bidoupensis Stuart, Rowley, Tran, Le & Hoang, 2011, Leptolalax pallidus Rowley, Tran, Le, Dau, Peloso, Nguyen, Hoang, Nguyen & Ziegler, 2016, Ingerophrynus galeatus ( Günther, 1864), Hylarana montivaga (Smith, 1921), Rhacophorus vampyrus Rowley, Le, Thi, Stuart & Hoang, 2010, Theloderma palliatum Rowley, Le, Hoang, Dau & Cao, 2011 and Raorchestes gryllus (Smith, 1924). On Hon Giao Mt. (1900-2000 m a.s.l.; borders of Lam Dong and Khanh Hoa provinces), Ophryophryne elfina sp. n. occurs in syntopy with Leptobrachium leucops , Leptolalax bidoupensis , Duttaphrynus melanostictus (Schneider, 1799), Hylarana montivaga , Rhacophorus vampyrus and Raorchestes gryllus . On Chu Pan Fan and Chu Yang Sin Mts. (1900 m a.s.l., Dak Lak Prov.), the new species is found in syntopy with Xenophrys cf. maosonensis (Bourret, 1937), Leptobrachium sp., Leptolalax sp., Hylarana montivaga , Rhacophorus vampyrus and Raorchestes gryllus . On Chu Yang Sin Mt. (1700-1800 m a.s.l., Dak Lak Prov.), the new species is also found in syntopy with Ophryophryne gerti (Fig. 14A). On Hon Ba Mt. (1500 m a.s.l., Khanh Hoa Prov.), the new species was recorded in syntopy with Leptobrachium leucops , Leptolalax sp., Microhyla arboricola Poyarkov, Vassilieva, Orlov, Galoyan, Tran, Le, Kretova & Geissler, 2014, Theloderma truongsonense (Orlov & Ho, 2005) and Raorchestes gryllus .

Reproductively active males were found while calling along streams, usually sitting on leaves of ferns or on the stone banks, rarely on rocks or large stones (see Fig. 14B, C). Some specimens were collected hiding amongst fern stems and were difficult to locate. Females were found hiding under tree logs or in the forest litter.

The ovaries of females contained well-developed unpigmented eggs with a diameter of approximately 2.2-2.8 mm (N = 15; measured from ZMMU ABV-00455, gravid female). On Hon Ba Mt., calling males were observed between 22 to 24 December, 22 to 28 March and 15 to 18 October. On Bidoup, Hon Giao and Chu Yang Sin Mts., reproductive activity and calling males were recorded from 10 February until mid-July. Tadpoles were found from April until July in the same streams where calling males were recorded; during the day time tadpoles hide under flat stones or dead leaves on the stream bed, but come out and can be visible in the shallow sandy parts of the stream at night. Metamorphosed individuals were observed in Chu Yang Sin N.P. in May.

Conservation status.

The full extent of the distribution of Ophryophryne elfina sp. n. is unknown, and the discovery of new localities on mountain ridges at elevations above 1500 m are highly anticipated. To date the species’ range includes the following nature conservation areas: Bidoup–Nui Ba N.P. (Lam Dong Prov.), Chu Yang Sin N.P. (Lam Dong Prov.), Hon Ba N.R. (Khanh Hoa Prov.) and Nui Chua N.P. (Ninh Thuan Prov.). However, population size and population dynamics of the new species are unknown. Given the available information, we suggest Ophryophryne elfina sp. n. to be considered as a Data Deficient species following IUCN’s Red List categories ( IUCN Standards and Petitions Subcommittee 2016).

Comparisons.

Ophryophryne elfina sp. n. is one of the smallest species of its genus, with adult male size (SVL 26.9-33.9 mm) similar to that of O. pachyproctus (adult male SVL 28.0-30.0 mm).

Ophryophryne elfina sp. n. differs from allopatric O. hansi (central Vietnam and neighboring southeastern Laos and northeastern Cambodia; Ohler 2003, Stuart 2005, Stuart et al. 2006) by its much smaller adult size: Ophryophryne elfina sp. n. male SVL 26.9-33.9 mm, N = 29, female SVL 35.1-36.5 mm, N = 6 (vs. O. hansi male SVL 33.4-43.1 mm, N = 12, female SVL 45.1-53.9 mm, N = 5; our data), by skin of dorsal and lateral surfaces of head, body and limbs shagreened with numerous small tubercles (vs. notably tubercular and warty skin on dorsal surfaces), and short dorsolateral glandular ridge above each shoulder (vs. dorsolateral glandular ridges absent).

Ophryophryne elfina sp. n. differs from allopatric O. pachyproctus (Yunnan Prov. in southern China, central Vietnam and possibly adjacent regions of Laos; Bain et al. 2007, Fei et al. 2009, 2010, 2012) by lacking dermal protuberance with dermal flaps above cloacal opening (vs. present on O. pachyproctus ), short dorsolateral glandular ridge above each shoulder not connected to posterior tips of ">-<"-shaped glandular parietoscapular-sacral ridge (vs. dorsolateral glandular ridge connected to posterior tips of “H” - or “Y” -shaped glandular parietoscapular-sacral ridge), supratympanic fold light brown dorsally on males (vs. white or light beige dorsally on males), and males with nuptial pad on first finger only (vs. one nuptial pad each on first and second fingers).

Ophryophryne elfina sp. n. differs from allopatric O. microstoma (Guangxi and Yunnan Provs., southern China to northern Vietnam, and northern Thailand; Khonsue and Thirakhupt 2001, Chan-ard 2003, Fei et al. 2009, 2010, 2012, Nguyen et al. 2009), by much smaller adult size: Ophryophryne elfina sp. n. male SVL 26.9-33.9 mm, N = 29, female SVL 35.1-36.5 mm, N = 6 (vs. O. microstoma male SVL 34.3-44.4 mm, N = 10, female SVL 39.4-57.0 mm, N = 7; Ohler 2003, Fei et al. 2009, Stuart et al. 2010; our data), short anterior dorsolateral glandular ridge above each shoulder (vs. dorsolateral ridges long, ca. 65-90% trunk length), and large tubercles posteriorly on dorsal surface of body (vs. large tubercles absent).

Ophryophryne elfina sp. n. differs from allopatric O. poilani (found in the mountains of the Tay Nguyen Plateau of central Vietnam and in adjacent areas of northeastern Cambodia, and, possibly, Laos; Bourret 1937, Stuart et al. 2010) by having smaller adult body size: Ophryophryne elfina sp. n. male SVL 26.9-33.9 mm, N = 29, female SVL 35.1-36.5 mm, N = 6 (vs. O. poilani male SVL 32.6-38.1 mm, N = 14, female SVL 47.4-50.8 mm, N = 2; Stuart et al. 2010), short dorsolateral glandular ridge above the shoulder not connected to posterior tips of ">-<"-shaped parietoscapular-sacral glandular ridge (vs. dorsolateral glandular ridge connected to posterior tips of ">-<"-shaped parietoscapular-sacral glandular ridge), and lacking characteristic dark “mask-like” coloration of temporal region, and supratympanic fold being dark brown ventrally and light brown dorsally on males (vs. temporal region and lateral surfaces of head uniformly dark-colored forming a dark “mask”, extending posteriorly towards axilla; supratympanic fold edged with white on males).

Ophryophryne elfina sp. n. differs from sympatric O. synoria (found at lower elevation from 200 to 1500 m a.s.l. in the foothills of the Langbian Plateau in southern Vietnam and adjacent easternmost hilly Cambodia) by much smaller adults body size: Ophryophryne elfina sp. n. male SVL 26.9-33.9 mm, N = 29, female SVL 35.1-36.5 mm, N = 6 (vs. O. synoria male SVL 38.2-53.7 mm, N = 14, female SVL 51.4-70.7 mm, N = 3; our data; Fig. 3), red-orange nuptial pad (in life) on first finger only (vs. two nuptial pads, covered in brown microgranules, large on first finger, covering entire dorsal metacarpal extending to 3/4 basal phalange length, on second finger medium sized on metacarpal extending to mid basal phalange on inner dorsal side), numerous bright red-orange asperities (in life) on dorsal and lateral surfaces of body, head and dorsal surfaces of limbs (vs. black and white asperities, small sized, spinular, moderately dense in narrow band along lower jaw, and on posterior upper jaw, few on tympanic region [exlcuding tympanum], along supratemporal folds and on posterior upper eyelids; some on anterior dorsum, becoming moderately dense posteriorly, above and surrounding cloaca, few on dorsal shanks, and absent on remaining surfaces on holotype of O. synoria ), and smaller tympanum/eye diameter ratio, TYD/ED 48.9%-62.6%, N=29 (vs. TYD/ED 64.8%-85.2%, N=14).

Ophryophryne elfina sp. n. differs from sympatric O. gerti (found at mid-elevations from 700 to 2000 m a.s.l. in the central and northern parts of the Langbian Plateau in southern Vietnam) by typically smaller adults body size: Ophryophryne elfina sp. n. male SVL 26.9-33.9 mm, N = 29, female SVL 35.1-36.5 mm, N = 6 (vs. O. gerti male SVL 31.7-42.2 mm, N = 15, female SVL 43.1-47.4 mm, N = 3; our data; Fig. 3), bright red-orange nuptial pads on males in life (vs. grey or black-brown nuptial pads on males in life), short dorsolateral glandular ridge above each shoulder, not connected to posterior tips of ">-<"-shaped parietoscapular-sacral glandular ridge, see Figs 6 G–H, and 8 (vs. strong dorsolateral glandular ridge from above each shoulder to approximately 4/5 distance between axilla and groin, connecting with posterior tips of ">-<"-shaped parietoscapular-sacral glandular ridge; see Figs 6 A–C, 7), skin on dor sal and lateral surfaces of body shagreened with numerous small tubercles (vs. skin on dorsum and sides of body granular, with numerous small and medium-sized tubercles and larger warts, see Fig. 6 A–C), dark hourglass-shaped markings on dorsum normally edged with white, see Figs 6H and 8 (vs. dark hourglass-shape on dorsum indistinct or, if present, unclear and not edged with white, Fig. 6 A–C), and throat, chest and abdomen having generally lighter coloration than in O. gerti .

Though available information on tadpole morphology of Oprhyophryne is very limited ( Liu and Hu 1962, Huang et al. 1991, Grosjean 2003, Fei et al. 2009), the tadpoles assigned to the new species based on the analysis of short 16S rRNA gene sequences (Table 1) have certain morphological characteristics that could be useful for distinguishing the larval stage of Ophryophryne elfina sp. n. from other Ophryophryne species. From tadpoles of O. microstoma , described in detail by Grosjean (2003), tadpoles of Ophryophryne elfina sp. n. differ mainly by possessing a longer tail: TOL/BL ratio 231.3 ± 11%, N = 5 (vs. TOL/BL <210%, N = 52 on O. microstoma ), and tail tip rounded (vs. tail tip bluntly pointed), mean = 22 longitudinal rows of papillae and from 2-4 (mean = 3) transverse rows of papillae on the upper labium and 4-6 (mean = 5) transverse rows of papillae on the lower labium, N = 5 (vs. mean = 20 longitudinal rows of papillae, and two upper labium and four lower labium transverse rows of papillae [without clear limits], N = 52); however, some of these differences may relate to the fact that Grosjeans’ description was based on later developmental stages (Gosner stage 37) than our sampling ( Gosners’ stage 25).

DNA-barcoding using short sequences for 16S rRNA (Table 1) also enabled us to identify tadpoles of sympatric Ophryophryne species from the Langbian Plateau, and though our sampling is not big enough to provide detailed morphological descriptions of larval morphology for O. gerti and O. synoria , we found some differences in coloration of tadpoles which may be useful for preliminary diagnostics of the three sympatric Ophryophryne species in the wild. Despite overall morphological similarity, both O. gerti and O. synoria show the presence of light golden to copper blotches on dorsal surfaces of the body and tail, whereas Ophryophryne elfina sp. n. tadpoles always have distinctive uniform brownish coloration with small coppery dots (Fig. 12 A–B).

Despite overall similarity, advertisement calls of each Ophryophryne species inhabiting the Langbian Plateau are easily diagnosable based on acoustic parameters. Some parameter values clearly differ between all of the studied species (Fig. 5). For example, the highest values of the repetition rate per recording and per series are found for O. synoria (3.07 ± 0.13 calls/s, N = 3, and 5.34 ± 0.15 calls/s, N = 15, respectively) while the lowest are reported for O. gerti (0.35 ± 0.14 calls/s, N = 3, and 2.33 ± 0.03 calls/s, N = 108, respectively). Ophryophryne elfina sp. n. occupies an intermediate position between these two species (1.18 ± 0.2 calls/s, N = 5, and 3.87 ± 0.07 calls/s, N = 140, respectively; differences significant, F2.8 = 46.7, p <0.001 and F2.260 = 220.7, p <0.001, respectively, one-way ANOVA).

The call temporal parameters for Ophryophryne elfina sp. n. compared to sympatric O. synoria and O. gerti , also differ for series duration, which is the highest in calls of Ophryophryne elfina sp. n., comprising 3.42 ± 0.11 s, N = 140 (see Table 4) (differences with O. gerti significant, F2.267 = 40.4, p <0.001, one-way ANOVA; differences with O. synoria not significant, see Table 4 for details). The advertisement call of the new species is further significantly different from calls of O. gerti for values of a number of acoustic parameters (see Table 4), such as the number of calls per series (12.84 ± 0.41, N = 140, versus 4.64 ± 0.16, N = 115, for O. gerti ; differences significant, F2.267 = 151.4, p <0.001, one-way ANOVA), the call duration (73 ± 0.23 ms, N = 1797, versus 104 ± 0.5 ms, N = 533, for O. gerti ; differences significant, H2.2530 = 1345.1, p <0.001, Kruskal-Wallis ANOVA) and inter-calls interval (207 ± 2.06 ms, N = 1657, versus 421.54 ± 4.17 ms, N = 418, for O. gerti ; differences significant, H2.2260 = 1008.5, p <0.001, Kruskal-Wallis ANOVA). The advertisement call of Ophryophryne elfina sp. n. is further different from calls of O. synoria in the frequency of maximum amplitude (4645.94 ± 4.39 Hz, N = 1797, versus 3798.9 ± 4.87 Hz, N = 200; differences significant, H2.2530 = 1030.2, p <0.001 (U = 0), Kruskal-Wallis ANOVA).

Finally, the new species is markedly distinct from all other congeners for which comparable sequences are available, including it closest relatives O. gerti and O. synoria , by relatively large genetic distances in 16S rRNA mtDNA gene fragment (p ≥ 8.2%).