Leptolalax pyrrhops, Poyarkov, Nikolay A., Rowley, Jodi J. L., Gogoleva, Svetlana I., Vassilieva, Anna B., Galoyan, Eduard A. & Orlov, Nikolai L., 2015

Poyarkov, Nikolay A., Rowley, Jodi J. L., Gogoleva, Svetlana I., Vassilieva, Anna B., Galoyan, Eduard A. & Orlov, Nikolai L., 2015, A new species of Leptolalax (Anura: Megophryidae) from the western Langbian Plateau, southern Vietnam, Zootaxa 3931 (2), pp. 221-252 : 232-246

Leptolalax pyrrhops sp. nov.

Holotype. ZMMU A- 5208 (field number ABV-00148), adult female from Loc Bac forest (operated by Loc Bac Forest Enterprise), Loc Bao Commune, Bao Lam District, Lam Dong Province, Vietnam (coordinates 11°44' 17" N, 107° 42' 25" E, elevation 830 m. a.s.l.), collected by E.A. Galoyan and A.B. Vassilieva on 10 of April 2013.

Paratypes. ZMMU A- 4873, two adult males (individual field numbers ABV-00213 and ABV-00215) collected by E.A. Galoyan and A.B. Vassilieva on 15 of April 2013 in mountain forest approximately 1700 m from the area of holotype collection, within the Loc Bac forest, Lam Dong Province, Vietnam (11°44'07" N, 107° 43' 17" E, elevation 1100 m. a.s.l.), and four adult females collected in the same area as male paratypes on 13 of April 2013 (individual field numbers ABV-00157–00158) and on 15 of April 2013 (individual field numbers ABV-00176 and ABV-00214). ZISP 12041 (field number ABV-00212), adult female from Loc Bac forest, Loc Bao Commune, Bao Lam District, Lam Dong Province, Vietnam (coordinates 11°44' 17" N, 107° 42' 25" E, elevation 830 m. a.s.l.), collected by E.A. Galoyan and A.B. Vassilieva on 15 of April 2013. VNMN A2015.02 (field number ABV-00177), adult female from Loc Bac forest, Loc Bao Commune, Bao Lam District, Lam Dong Province, Vietnam (coordinates 11°44' 17" N, 107° 42' 25" E, elevation 830 m. a.s.l.), collected by E.A. Galoyan and A.B. Vassilieva on 15 of April 2013.

Etymology: The specific epithet is a noun in the nominative case, derived from Greek " pyrrhos " for "firecolored" and Greek " ops " for "eye", in reference to the iris color of the new species.

Recommended vernacular name. The recommended common name in English is “ Orange-eyed litter frog ”, referring to the beautiful iris coloration of the new species. The recommended common name in Vietnamese is “ Cóc Mày Mắt Cam ”.

Diagnosis. The species is assigned to the genus Leptolalax based on the following characters considered to be diagnostic for the genus: (1) comparatively small size; (2) rounded finger tips, the presence of an elevated inner metacarpal tubercle not continuous on to the thumb; (3) presence of macroglands on body (including supraaxillary, pectoral, femoral and ventrolateral glands, chest glands are present but they do not form teats); (4) vomerine teeth absent; (5) tubercles on eyelids and (6) anterior tip of snout with whitish vertical bar (Inger 1966; Dubois 1980, 1983; Matsui 1997, 2006; Lathrop et al. 1998; Delorme et al. 2006). Leptolalax pyrrhops sp. nov. is distinguished from its congeners by a combination of the following morphological attributes: (1) presence of distinct dark brown/black distinct dorsolateral markings including blackish spots on the flank and dark canthal and/ or temporal streaks; (2) rudimentary webbing on toes; (3) externally distinct tympanum, (4) dorsal skin finely shagreened with numerous small tubercles and pustules; (5) medium size for the genus (30.3–33.9 mm in 2 adult males, 30.8–34.3 mm in 7 females); (6) grey-pinkish to dark brownish-violet chest and belly with numerous whitish speckles also covering the lateral sides of body; (7) ventrolateral glands small, indistinct, do not form a distinct line; (8) comparatively small pectoral glands, comprising 1–3% of adult SVL; (10) a bicolored iris, typically bright orange-red in upper two-thirds, fading to silvery green in lower third. The new species is also markedly distinct from all congeners for which comparable sequences are available (16S rRNA mitochondrial gene; uncorrected genetic distance>10.3%). The advertisement call of the new species, consisting of a single long 'introductory' note, comprising 5–12 pulses, followed by of 4–5 predominantly single-pulsed notes, and with an average dominant frequency of 1.91–2.23 kHz, also distinguishes the new species from Leptolalax species for which calls are known.

Description of holotype. Medium-sized specimen in good state of preservation for head and body and partially dehydrated limbs due to ethanol preservation; habitus slender ( Figs. 3 , 4 A, 4B). The holotype is dissected on left side of trunk, dissection length 9.3 mm; left ovary with small ovocytes (diameter 0.85–0.95 mm) clearly seen in the dissection. The ventral side of the right femur is also dissected, dissection length 11.9 mm. Head. Head slightly longer than wide (HDW/HDL 0.90), flattened, triangular in dorsal view; top of head flat; snout comparatively short (SNT/HDL 0.37), obtusely rounded in dorsal view ( Fig. 3 A) and slightly truncate but gently rounded in profile ( Fig. 3 C), snout slightly projecting beyond margin of the lower jaw; nostril oval-shaped, vertical, located much closer to tip of snout than to eye ( Fig. 3 C); loreal region slightly concave; canthus rostralis distinct, bluntly rounded; eyes large (EYE/HDL 0.36), eye diameter almost equal to snout length (EYE/SNT 0.98), notably protuberant in dorsal view and in profile, pupil vertical, diamond-shaped; tympanum distinct, round with vertical diameter slightly exceeding the horizontal diameter (vertical diameter to horizontal diameter (TMP) ratio 1.14), tympanum comparatively small with diameter over two times smaller than that of the eye (TMP/EYE 0.46); tympanic rim notably elevated relative to skin of temporal region; vomerine teeth absent; pineal ocellus absent; vocal sac and vocal sac openings absent; tongue long, wide, with free posterior end, heart-shaped with a shallow medial notch at posterior tip; supratympanic fold forming a distinct wide ridge, running from the posterior corner of eye posteriorly towards dorsal edge of tympanum, sharply curving down towards axillary gland posteriorly to corner of mouth, supratympanic ridge comparatively smooth with few flat tubercles ( Fig. 3 C). Forelimbs. Forelimbs thin, slender; tips of fingers in life rounded and somewhat swollen, but appear to be truncate, notably flattened in preservative, slightly widened distally (wider than distal finger articulation) ( Fig. 3 D); relative finger lengths IV <I = II <III; nuptial pad absent; subarticular tubercles absent, replaced by low dermal ridges, distinct on toes II–IV; a large, flat oval-shaped inner metacarpal tubercle not separated from small, laterally compressed drop-shaped outer metacarpal tubercle (inner metacarpal tubercle is about three times wider than the outer metacarpal tubercle, width rate 0.39), they touch each other on almost full length of the outer metacarpal tubercle, but the borders of both tubercles are clearly marked by a thin medial groove; fingers completely free of webbing, in life with no distinct fringing, no signs of fringes on fingers I, II and IV; in preservative all fingers look somewhat flattened ( Fig. 3 D). Hindlimbs. Hindlimbs slender, tibia half of snout-vent length (TIB/SVL ration 0.5); tibiotarsal articulation of adpressed limb reaching snout, but not beyond snout tip, posterior edge of tibiotarsal articulation reaches well beyond anterior margin of eye. Tips of toes rounded and slightly swollen in life, but look truncate, notably flattened in preservative possibly to dehydration in ethanol, slightly widened distally, similar to that of fingers; relative toe length I <II <V <III <IV; subarticular tubercles absent, replaced by clear, well-developed dermal ridges, distinct on all toes and continuing to metatarsus; large, oval-shaped inner metatarsal tubercle well pronounced, 2.3 times longer than wide, outer metatarsal tubercle absent; basal webbing present between all five toes, web especially clear between toes II–III and III–IV, greatly reduced between toes I–II and IV–V; in life no clear lateral fringes seen on any toe ( Fig. 3 E). Skin texture and skin glands. Skin shagreened with numerous small tubercles and pustules finely and relatively evenly scattered on dorsal surfaces of trunk, head and limbs, tubercles absent on sides ( Fig. 3 A, Fig. 4 A), upper eyelid with numerous small tubercles ( Fig. 3 C); skin on ventral surfaces of trunk, head and limbs smooth ( Fig. 3 B, Fig. 4 B); pectoral gland located at forelimb basis on ventral surface of axillary region, rounded, 0.78 mm in diameter, distinct in preservative and in life ( Fig. 3 B, Fig. 4 B); femoral gland oval, elongated, on posteroventral surface of thigh, approximately three times closer to knee than to vent, approximately 1.62 mm in diameter, more distinct in life than in preservative ( Fig. 3 B, Fig. 4 B); supraaxillary gland located in axillary region dorsally from insertion of forelimb, rounded, raised, 1.54 mm in diameter; ventrolateral glands small (diameter less than 0.7 mm), indistinct, rounded and flat, irregularly scattered on flanks, not forming a distinct line.

Colour of holotype in life. Dorsal surfaces of head and trunk dark reddish-brown with no distinct darker markings, but several dark-ochre blotches of irregular shape and indistinct borders located at base of head and on the posterior part of the dorsum. Dorsal surfaces of forelimbs (lower arms) and hindlimbs somewhat lighter brownish, forearms much lighter with orange to light reddish-brown background color. Faint transverse brownish grey bars are darker than the background color and are located on the dorsal surface of the thighs, tibia, tarsus, lower arms, fingers and toes ( Fig. 4 A). Tiny whitish to light bluish flecks scattered on dorsolateral sides of body, dorsal surfaces of lower arms, thighs, tibia, tarsus, and limb insertion areas; flecks are especially numerous on lateral sides of body, being densely scattered ( Fig. 4 A). On lateral sides of body several distinct blackish spots (1–2 mm in diameter) located in two lines running from axilla to groin (5 large spots on each side of body; the most posterior black spot located in the groin area is about 1.5 times larger than the other spots) and ventrally of this line (one larger and one smaller spot on each side of the body). Two smaller blackish elongated spots located on the sacrum. The dorsolateral surfaces of body, fingers, toes and elbow to upper arm bluish-grey to dusty pinkish. A distinct creamy-white spot on the edge of the lower jaw under the eye and a smaller indistinct whitish blotch located anteriorly to the latter; tiny white stripe on the snout creamy-white, with irregular borders. Tympannic area dark blackish-brown from the supratympanal ridge to the ventral edge of tympanum; the dark spot has indistinct borders and extends from the posterior corner of eye towards the supra-axillary gland. Ventral surfaces of forelimbs, hindlimbs and belly opaque pinkish to light bluish-purple; throat transparent pinkish, somewhat warmer in color compared to the belly ( Fig. 4 B). Ventral surface of chest bluish-violet (due to transparent skin and underlying liver). Ventral surfaces of head, limbs, chest and belly covered with irregular bluish-white speckles and dusting; speckles larger and more densely scattered on belly and chest, on head speckles are larger along the anterior margin of throat. Ventral surface of arms pinkish-purple with whitish dusting along lateral margins; ventral surface of thighs, tibia, and tarsus brownish-purple with faint bluish-white flecking. Metatarsal tubercle bluish with darker distal edge ( Fig. 4 B). Supra-axillary gland pale copper-red; pectoral glands bluish-white; femoral glands whitish edged with purplish-brown color; ventrolateral glands indistinct, bluish white, indistinct from whitish lateral flecking. Iris bicolored: bright orange-gold in upper half, fading to greenish-silver in lower third; orange color brighter in upper half, lower half showing silvery greenish speckles; fine black reticulations throughout the iris, but denser on its periphery. Anterior part of iris bearing a notably darker (brownish-orange) stripe, running from middle of pupil to anterior corner of eye. Iris periphery lined with black ( Fig. 4 B). Sclera light whitish-cream.

Colour of holotype in preservative. In preservative coloration fades to dark grey-brown on dorsum and flanks, with slightly paler limbs and yellowish-grey to whitish on the venter; reddish and pinkish tints, as well as iris coloration, fades completely; other features remain without significant change ( Fig. 3 A). Banding on limbs are less pronounced, white flecks on body flanks and ventral sides are more pronounced ( Fig. 3 C). Ventral surface of chest, belly, throat, interior portions of arms and thighs are pale greyish brown; macrogrands turn creamy white ( Fig. 3 B).

Measurements of the holotype (all in mm). SVL 33.8; HDL 14.1; HDW 12.6; SNT 5.2; EYE 5.1; IOD 4.0; TMP 2.4; TEY 1.6; TIB 17.0; ML 8.9; PL 16.2.

Variation. All individuals in the type series are generally similar in morphology and body proportions (see Fig. 5 ); variation of the type series in morphometric characters is shown in Table 3. There is no clear difference in body size between the sexes based upon the series examined. Specimens vary in the number and size of black ventrolateral blotches. In life, both sexes of the new species show much lighter coloration of belly and throat nocturnally ( Fig. 4 B). Diurnally the belly is much darker, coloured brown-violet to purple with white speckles. Whitish gonads can be easily seen through the semitransparent skin on the belly of gravid females ( Fig. 4 D). Compared to the nocturnal coloration, the diurnal coloration of the dorsum has more brownish, ochre and orange tints, and the dark brownish and blackish blotches on the dorsum are more obvious ( Fig. 4 C; Fig. 6 A). At night the dorsum appears almost uniformly dark grayish or blackish with contrasting bluish-white speckling on flanks ( Fig. 4 A; Fig. 6 B). Iris coloration varies slightly in the balance of coppery-orange versus pale greenish-silver; in one individual (ZMMU A-4873, field number ABV-00213), however, the lower silvery-green third of iris looks almost the same uniformly golden-orange as the upper two thirds ( Fig. 6 B). The new species shows no significant variation in skin structure among sexes (see Fig. 6 ); in preservative large and elongated tubercles become flatter than in life, but remain distinct; smaller pustules become less distinct in preservative.

successive calls the calling rate was 0.13 calls per second. Note duration varied from 11 to 67 ms. The first ‘introductory’ note always was longer than successive notes (45 ± 4 [32–67] ms versus 15 ± 0.1 [11–20] ms). The first note of each call consisted of 5–12 pulses repeated at a rate of approximately 123–165 pulses per note ( Table 4, Fig. 8 ). In most cases, first note had lower amplitude than other notes, the difference between amplitude values of the first ‘introductory’ note and successive notes was 80.2 ± 0.8 (45.7–97.9) mV ( Table 4). The frequency modulation was absent and the harmonics were not clearly visible in the recording ( Fig. 8 ).

Recording Parameter Registration No. ZMMU A-4873,

ABV-00213

Temperature (ºC) 25

Number of calls 7

Call duration (ms) 254 ± 11 (208–297) Intercall interval (s) 4.42 ± 0.43 (3.26–5.63) Notes/call 5.71± 0.18 (5–6) Internote interval (ms) 29 ± 1 (16–36)

Call repetition rate (calls/s) 0.13

Introductory note Note duration (ms) 45 ± 4 (32–67)

Pulses/note 7.43 ± 0.90 (5–12) Dominant frequency (Hz) 1990 ± 20 (1910–2060) Amplitude (mV) 356.6 ± 0.8 (298.6–440) Pulse repetition rate (pulses/s) 141.45 ± 6 (123.5–164.9)

Clicks/Notes 2–X Note duration (ms) 15 ±0.1 (11–20)

Pulses/note 1

Dominant frequency (Hz) 2070 ± 20 (1930–2230) Amplitude (mV) 436.8 ± 0.8 (375.1–485.8) Note repetition rate (notes/s) 18.61 ±0.37(16.84–19.38) Position in mtDNA phylogeny and sequence divergence. Uncorrected genetic p -distances between Leptolalax pyrrhops sp. nov. 16S rRNA sequences and all homologous sequences available on GenBank included in the analysis (see Table 1) varied from 13.5% (with L. applebyi ) to 20.8% (with L. bourreti ), with the exception of the sister species L. bidoupensis , which showed an uncorrected sequences divergence of 10.3% (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). Intraspecific variation in this gene fragment for Leptolalax pyrrhops sp. nov. sampled from the type locality was <0.31%.

Distribution. The new species has been so far recorded from only two sites (<2 km from each other) in the mountain forests of the Loc Bac forest, Bao Lam District, Lam Dong Province, Vietnam; on the western edges of the Langbian Plateau at altitudes from 800 to 1100 m a.s.l. The distribution of the new species may be quite narrow, possibly restricted to a small mountain ridge on the borders of Loc Bao and Loc Lam communes, Bao Lam District; less than 10 km in length. The known localities of the new species are located ca. 40 km southwards from the southernmost locality of L. bidoupensis (Bidoup Mt., Bidoup—Nui Ba N.P., Lam Dong Province), thus marking the southernmost record of the genus Leptolalax from Vietnam and Indochina known to date.

Ecology. All specimens were collected at night after heavy rains along the small intermittent, rocky streams on the limited parcels of primary montane high polydominant evergreen tropical forest with a high abundance of large rocks and the predominance of trees of the families Magnoliaceae , Sapindaceae , Podocarpaceae , Euphorbiaceae , Fagaceae , Theaceae , Sapotaceae , Caesalpiniaceae , Anacardiaceae , Altingiaceae , Rhodoleiaceae , Elaeocarpaceae , Lauraceae , Sterculiaceae and Dipterocarpaceae ( Fig. 7 ). Leptolalax pyrrhops sp. nov. occurs in syntopy with Leptobrachium pullum ( Smith, 1921) , Ophryophryne sp., Limnonectes limborgi ( Sclater, 1892) , Limnonectes sp., Kurixalus sp., Polypedates megacephalus Hallowell, 1861 , Hylarana milleti ( Smith, 1921) . Calling males were found along the stream, sitting on large mossy rocks, some specimens were hiding under the rocks and stones and were difficult to locate ( Fig. 6 B). Females were found hiding under rocks on the edge of water, or in the dry riverbed of a mountain stream filled with forest litter and leaves. The ovaries of all females contained welldeveloped, unpigmented eggs of a diameter of approximately 1.6–2.1 mm. Reproductive activity and calling males were recorded in April (10–15 of April). Amplexus occurred while keeping males and females in a plastic container after capture ( Fig. 6 A). Despite our intensive searches we could not find the tadpoles of the new species; larvae of Limnonectes sp. were found in the same streams where the new species was observed.

Conservation status. Leptolalax pyrrhops sp. nov. is presently known from two closely located sites in Loc Bac forest (currently operated by Loc Bac Forest Enterprise) in Lam Dong Province. The actual distribution of the new species is unknown but probably is quite narrow. Given the available information, we suggest Leptolalax pyrrhops sp. nov. to be be considered as a Data Deficient species following IUCN’s Red List categories ( IUCN 2001).

Comparisons. Leptolalax pyrrhops sp. nov. is both most morphologically and molecularly similar to smallbodied Leptolalax species from southern and central Vietnam and northeastern Cambodia: L. applebyi , L. bidoupensis and L. melicus (Clade B), and L. botsfordi from northern Vietnam. In having a dark brownish red ventral surface with white speckling, Leptolalax pyrrhops sp. nov. can be differentiated from all other Leptolalax in mainland Southeast Asia, except for L. applebyi , L. bidoupensis , L. melicus and L. botsfordi ( L. aereus , L. alpinis , L. arayai , L. bourreti , L. dringi , L. firthi , L. fuliginosus , L. fritinniensis , L. gracilis , L. hamidi , L. khasiorum , L. lateralis , L. laui , L. liui , L. marmoratus , L. minimus , L. nahangensis , L. nokrekensis , L. oshanensis , L. pelodytoides , L. pictus , L. playcephalus , L. sabahmontanus , L. solus , L. sungi , L. tamdil , L. tuberosus and L. zhangyapingi have mostly white or pale greyish or brownish venters, with or without dark spots or mottling; L. croceus has a bright orange venter; L. pluvialis has a dirty white or grey belly with dark brown or grey marbling, and uniform pale dirty white or grey throat with pale speckling only around the margins; L. melanoleucus and L.

ventripunctatus show large patches of distinct brown or black and white marbling, L. heteropus displays a grey venter, speckled with black; L. maurus has a black or dark grey brown venter, with indistinct small light patches, and L. kecil displays a uniformly dark venter with large, dark orange pectoral glands).

In having an externally distinct tympanum the new species can be easily distinguished from two Vietnamese species L. sungi and L. tuberosus (versus tympanum externally indistinct in L. sungi and L. tuberosus ).

In having toes with rudimentary webbing and poor lateral fringing, Leptolalax pyrrhops sp. nov. can also be readily differentiated from L. alpinus , L. firthi , L. laui and L. liui , which have wide lateral fringing on toes, and from L. pelodytoides , which has more extensive webbing and wide lateral fringes between toes.

In typically having a bicoloured iris, with the upper half bright orange-red and the lower half fading to greenish-silver, Leptolalax pyrrhops sp. nov. can be further distinguished from L. aereus , L. applebyi , L. botsfordi , L. croceus , L. kajangensis , L. kecil , L. laui , L. liui , L. maurus , L. melicus , L. nahangensis , L. sungi and L. tuberosus , all of which typically have a uniform iris coloration with black reticulations.

In having the dorsal skin finely shagreened with numerous small tubercles and pustules finely and relatively evenly scattered on dorsum, the new species can be differentiated from L. arayai , L. croceus , L. khasiorum , L. lateralis , L. maurus , L. minimus , L. solus , L. tamdil , L. tuberosus and L. ventripunctatus (roughly granular or tuberculate skin texture with skin ridges and large tubercles) and from L. alpinus , L. applebyi , L. bidoupensis , L. bourreti , L. eos , L. fuliginosus , L. melanoleucus and L. pluvialis (smooth or otherwise less tuberculate dorsal skin in these species).

……continued on the next page Mean 32.10 13.09 11.14 4.99 4.62 3.89 1.95 1.58 15.78 8.41 15.03

ΖMMU A-5208; ABv-00148 ♀ 33.8 14.1 12.6 5.2 5.1 4.0 2.4 1.6 17.0 8.9 16.2 Hοlοtype ΖMMU A-4873; ABv-00157 ♀ 32.3 13.3 11.7 5.2 5.1 4.0 2.2 1.7 16.2 8.7 15.6 Paratype ΖMMU A-4873; ABv-00158 ♀ 30.8 13.7 12.1 4.4 5.1 4.5 2.3 1.3 15.9 8.6 15.6 Paratype ΖMMU A-4873; ABv-00176 ♀ 33.6 13.6 12.0 5.0 5.0 3.9 2.2 1.8 17.0 8.7 16.1 Paratype vNMN A2015.02; ABv-00177 ♀ 34.3 13.2 11.2 4.5 5.4 4.1 2.2 2.0 16.3 7.8 15.0 Paratype ΖISP 12041; ABv-00212 ♀ 33.9 15.0 12.0 4.7 4.8 4.0 2.2 1.9 16.2 8.1 15.6 Paratype ΖMMU A-4873; ABv-00214 ♀ 33.5 15.9 12.1 5.7 5.3 4.3 2.1 1.7 17.2 8.3 16.3 Paratype

Mean 33.16 14.10 11.96 4.97 5.12 4.10 2.20 1.71 16.55 8.44 15.76

St.dev. 1.23 1.01 0.44 0.46 0.17 0.20 0.10 0.23 0.50 0.37 0.44

Max 34.3 15.9 12.6 5.7 5.4 4.5 2.4 2.0 17.2 8.9 16.3

Min 30.8 13.2 11.2 4.4 4.8 3.9 2.1 1.3 15.9 7.8 15.0

The presence of black markings (spots or blotches) on the flanks further distinguishes the new species from L. aereus , L. arayai , L. croceus , L. eos , L. firthi , L. laui , and L. tuberosus (no distinct black lateral markings on the flanks), L. zhangyapingi (indistinct brown lateral spots and flecks).

In having an indistinct ventrolateral glandular line, Leptolalax pyrrhops sp. nov. is differentiated from L. alpinus , L. botsfordi , L. fuliginosus , L. khasiorum , L. liui , L. oshanensis , L. pelodytoides , L. pluvialis and L. tamdil , which all have distinct, more complete ventrolateral glandular lines.

The new species can be further easily distinguished from L. botsfordi ( Lao Cai Province, northern Vietnam), also with dark brownish red ventral surface with white speckling, by the following: (1) comparatively larger head size (HDL:SVL 0.41–0.47 versus 0.34–0.36 in L. botsfordi ), (2) dense white speckling on the belly (versus only faint white spotting on the belly in L. botsfordi ) and (3) relatively small femoral glands (1.0–3.0 mm, 2–4% SVL versus 2.4–4.3 mm, 7–14% SVL in L. botsfordi ).

Leptolalax pyrrhops sp. nov. can be further differentiated from the three most morphologically similar species from Kon Tum and Langbian Plateaus with dark reddish brown ventral surfaces (Clade B), L. applebyi , L. bidoupensis and L. melicus , in the following ways. From L. applebyi (central Vietnam, Kon Tum Plateau, see Fig. 9 A, B) the new species is markedly different by having (1) shagreened skin on dorsum with numerous small tubercles (versus more smooth skin on dorsum lacking large tubercles, only scarcely scattered small tubercles in L. applebyi , see Fig. 9 A, B), (2) bicolored orange-silvery iris (versus uniformly coppery red iris in L. applebyi ), (3) notably larger body size in adults (30.3–33.9 mm in males; 30.8–34.3 mm in females) (versus 19.6–22.3 mm for males; 21.7–26.4 for females in L. applebyi ), and (4) by comparatively longer tibia in the new species (male TIB:SVL 0.48–0.52 versus 0.42–0.48 in L. applebyi ).

From L. melicus (Kon Tum Plateau in Ratanakiri, north-eastern Cambodia,see Fig. 9 C, D) the new species can be diagnosed by the following: (1) in having bicolored orange-silvery iris (versus uniformly dark golden iris in L. melicus ), (2) by a larger body size in adults (30.3–33.9 mm in males; 30.8–34.3 mm in females) (versus 19.5–22.7 mm for males in L. melicus ), (3) by a slightly longer tibia in the new species (male TIB:SVL 0.48–0.52 versus 0.46–0.48 in L. melicus ), and (4) smaller relative pectoral gland size (PEC/SVL 1–3% versus 3–6% in L. melicus ).

From its sister species, L. bidoupensis (southern Vietnam, eastern and northern edges of Langbian Plateau, see Fig. 9 E, F), Leptolalax pyrrhops sp. nov. can be distinguished by having (1) shagreened skin on dorsum with numerous small tubercles (versus mostly smooth skin texture with no skin ridges or obvious tubercles in L. bidoupensis ), (2) comparatively larger head (HDL:SVL 0.41–0.47 versus 0.36–0.42 in L. bidpoupensis ), (3) notably larger body size in adults (30.3–33.9 mm in males; 30.8–34.3 mm in females) (versus 18.5–25.4 mm for males; 28.3–29.4 mm for females in L. bidoupensis ), (4) longer tibia in the new species (male TIB:SVL 0.48–0.52 versus 0.42–0.50 in L. bidoupensis ), and (5) smaller relative pectoral gland size (PEC/SVL 1–3% versus 3–7% in L. bidoupensis ). The two sister species can also be distinguished by mtDNA (see above) and acoustic properties (see below).

The advertisement call of Leptolalax pyrrhops sp. nov. differs structurally from all 24 Leptolalax species with described calls: L. aereas , L. alpinis , L. applebyi , L. arayai , L. bidoupensis , L. botsfordi , L. croceus , L. dringi , L. firthi , L. fuliginosus , L. fritinniens , L. gracilis , L. hamidi , L. heteropus , L. kecil , L. liui , L. marmoratus , L. melanoleucus , L. melicus , L. oshanensis , L. pictus , L. solus , L. sabahmontanus and L. tuberosus . Although our comparisons are based upon only a single individual, Leptolalax pyrrhops sp. nov. is the only species of Leptolalax with a call containing a single long introductory note containing 5–12 pulses approximately 82% the amplitude of successive notes, followed by 4–5 single-pulsed notes (‘clicks’). Compared to species in the L. applebyi group, the advertisement call of Leptolalax pyrrhops sp. nov. most closely resembles that of L. melicus , in having a distinct introductory note, but differs by having 4–6 notes/call (versus 4–11 in L. melicus ), and 5–12 (mean 7.4) relatively high amplitude pulses in a introductory note (versus 8–50 [mean 22.7 and 26.5)] relatively low-amplitide pulses in L. melicus ). Leptolalax pyrrhops sp. nov. also appears to differ from L. melicus in terms of dominant frequency (1.9–2.2 kHz versus 2.6–4.0 kHz in L. melicus ), but this may be at least partially related to body size (eg. Rowley et. al. 2010a). More detailed comparisons between Leptolalax pyrrhops sp. nov. and L. melicus are not possible given our sample size (n= 1 in L. pyrrhops sp. nov. and n= 2 in L. melicus ). Differences in the advertisement call between the new species and both L. applebyi and L. bidoupensis are much clearer. The advertisement call of Leptolalax pyrrhops sp. nov. differs from L. applebyi in having a long introductory note and relatively high number of pulses (versus no introductory note in L. applebyi ), 5–6 notes call (versus 4–5 notes/call in L. applebyi ), a single pulse in all non-introductory notes (versus 4–5 pulses/note in L. applebyi ), and a dominant frequency of 1.9–2.2 kHz (versus 4.0–4.3 kHz for L. applebyi ; although this may at least partially reflect body size differences). The advertisement call of Leptolalax pyrrhops sp. nov. differs from the call of its sister species, L. bidoupensis , by having a long introductory note and relatively high number of pulses (versus no introductory note in L. bidoupensis ), and 4–5 notes/call (versus 5–9 notes/call in L. bidoupensis ).

Finally, the new species is markedly distinct from all other congeners for which comparable sequences are available, including it closest relative—the sister species L. bidoupensis —by relatively large genetic distances in 16S rRNA mtDNA gene fragment (p = 10.3%). The only known locality of the new species is 100 km from the closest known locality of L. bidoupensis ; the ranges of the two species appear to be confined to different mountain massifs within the Langbian Plateau. In addition, L. pyrrhops sp. nov. and L. bidoupensis may inhabit different altitudinal ranges (the new species is known from 800 to 1100 m a.s.l.; L. bidoupensis has been reported from 1550 to 2100 m a.s.l., our data). Based on this data we assume that the two species have allopatric distributions.

Figures

FIGURE 3. Holotype Leptolalax pyrrhops sp. nov. (ZMMU A- 5208, female; filed number ABV- 00148) in preservative: (A) dorsal view, (B) ventral view (C) head, lateral view, (D) volar view of the left hand, (E) plantar view of the left foot. Scale bar 3 mm. Photos by N. A. Poyarkov.

FIGURE 4. Coloration of the Leptolalax pyrrhops sp. nov. in life: female holotype (ZMMU A- 5208; filed number ABV- 00148): (A) dorsolateral view, (B) ventral view; female paratype (ZMMU A- 4873, filed number ABV- 00158) in life: (C) dorsolateral view, (D) ventral view. Photos by E. A. Galoyan.

FIGURE 5. Paratypes of Leptolalax pyrrhops sp. nov. in preservative. Scale bar 10 mm. Photo by N. A. Poyarkov.

FIGURE 6. Leptolalax pyrrhops sp. nov. in situ: (A) a pair in amplexus (male ZMMU A- 4873, field number ABV- 00215; female ZISP 12041, field number ABV- 00212); (B) calling male (ZMMU A- 4873, field number ABV- 00213) showing night coloration. Photos by E. A. Galoyan.

FIGURE 7. Natural habitat of Leptolalax pyrrhops sp. nov. in Loc Bac woodland, Lam Dong Province, typical breeding site at small forest stream. Photo by E. A. Galoyan.

FIGURE 8. Advertisement call of the male paratype advertisement calls of Leptolalax pyrrhops sp. nov. (ZMMU A- 4873, field number ABV- 00213) recordered at air temperature of 25 ºC. (A) waveform of high-pass filtered call, (B) wave form and (C) corresponding spectrogram of the same call, unfiltered.

FIGURE 9. Members of Leptolalax applebyi species group. L. applebyi (Ngoc Linh N. P., Kon Tum Province, Vietnam): (A) adult male AMS R 173635; (B) AMS R 173777; L. melicus (Virachey N. P., Ratanikiri Province, Cambodia): (C) adult male holotype, MVZ 258198; (D) adult male paratype MVZ 258199; L. bidoupensis: (E) ZMMU A- 5215, Chu Yang Sin N. P., Dak Lak Province, Vietnam; (F) adult female paratype NCSM 77320, Bidoup—Nui Ba N. P., Lam Dong Province, Vietnam (photos A – D and F by J. J. L. Rowley; E—photo by N. A. Poyarkov).

Tables

TABLE 2. Uncorrected p - distance (percentage) between 16 S rRNA sequences of Leptolalax species included in phylogenetic analyses (below the diagonal) and standard error estimates (above the diagonal). The ingroup mean uncorrected p - distances are shown on the diagonal and shaded with grey (continues next page).

	Species	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17
1	L. minimus	2.6	1.2	1.2	1.1	1.3	1.3	1.7	1.6	1.6	1.7	1.4	1.6	1.4	1.6	1.7	1.6	1.9
2	L. pluvialis	5.4	0.0	1.2	1.3	1.4	1.3	1.7	1.7	1.7	1.6	1.5	1.7	1.6	1.7	1.9	1.6	1.9
3	L. aereus	7.0	5.7	1.1	1.1	1.4	1.3	1.8	1.7	1.8	1.7	1.3	1.6	1.2	1.5	1.7	1.5	1.9
4	L. nyx	6.0	6.0	5.8	–	1.3	1.2	1.7	1.6	1.8	1.8	1.4	1.7	1.4	1.6	1.8	1.6	2.0
5	L. ventripunctatus	7.8	7.2	7.7	6.2	0.5	0.4	1.6	1.6	1.9	1.5	1.6	1.9	1.5	1.8	1.9	1.8	2.2
6	L. cf. ventripunctatus	7.4	6.5	7.3	5.9	0.8	0.0	1.6	1.5	1.8	1.5	1.5	1.8	1.5	1.8	1.9	1.7	2.1
7	L. firthi	13.9	13.8	13.5	11.8	12.1	11.8	0.4	1.8	2.2	1.8	1.9	2.1	1.9	2.0	2.3	2.1	2.1
8	L. liui	12.2	11.6	12.1	10.7	13.8	12.5	15.0	1.2	1.5	1.8	1.8	1.9	1.7	1.9	2.3	1.8	2.1
9	L. laui	11.5	11.3	13.4	11.8	14.1	12.8	16.7	6.9	0.2	1.7	1.7	1.8	1.8	1.7	2.3	1.7	2.2
10	L. zhangyapingi	12.7	10.7	12.1	11.1	12.1	11.7	14.3	12.9	12.1	–	1.7	1.8	1.8	1.8	2.1	1.7	2.0
11	L. oshanensis	11.7	11.8	11.4	10.5	11.9	11.6	15.8	12.1	12.5	13.7	1.1	0.7	1.0	1.4	1.0	1.3	2.0
12	L. cf. oshanensis 1	11.3	11.2	11.0	9.9	12.3	11.7	15.2	11.3	11.5	13.0	3.6	–	1.2	1.4	1.1	1.4	2.1
13	L. cf. oshanensis 2	11.0	10.7	9.5	10.3	11.8	11.2	14.3	9.5	11.9	12.4	5.3	4.4	–	1.3	1.0	1.3	2.0
14	Leptolalax sp. 1	11.6	11.6	12.7	11.8	12.9	12.5	16.0	12.9	13.5	14.0	9.0	7.8	5.9	–	1.8	1.5	2.0
15	L. bourreti	12.1	11.6	11.4	11.2	13.6	12.7	16.6	12.7	14.4	15.4	5.1	3.4	3.0	8.3	0.0	1.5	2.4
16	L. eos	12.1	11.7	12.1	11.4	13.4	12.7	15.5	10.8	12.1	12.0	7.3	6.2	4.7	8.7	5.6	0.0	2.1
17	L. applebyi	15.4	14.2	14.9	14.9	16.4	16.1	18.4	17.4	17.4	15.2	16.3	15.8	13.7	13.4	18.1	14.9	0.0
18	L. melicus	13.4	12.7	11.4	11.2	13.7	13.4	17.2	18.1	18.4	15.5	15.2	15.2	12.5	13.5	16.6	14.6	7.1
19	L. bidoupensis	15.5	15.4	15.8	14.8	17.5	17.3	17.9	16.3	17.7	17.3	17.8	18.2	15.2	15.9	20.6	15.7	9.3
20	L. pyrrhops sp. nov.	16.8	15.9	15.5	15.7	17.2	16.7	18.2	16.5	16.6	17.8	18.7	17.8	17.2	16.1	20.8	16.8	13.5
21	Leptolalax sp. 2	14.9	14.5	15.5	14.5	17.3	17.0	18.9	18.2	17.8	17.3	16.1	15.2	14.9	16.3	18.1	15.8	14.0
22 23	L. arayai L. gracilis	15.2 20.6	16.6 20.6	15.6 18.8	16.0 21.4	15.9 22.3	16.0 21.7	19.7 22.4	18.4 23.8	18.3 22.0	18.7 21.7	17.8 21.1	17.8 19.9	14.8 19.4	15.6 19.1	19.0 23.4	16.3 20.5	14.5 18.1
24	L. maurus	16.9	17.6	15.7	17.0	17.1	17.3	19.2	19.7	20.3	17.3	17.3	17.0	14.6	15.6	20.2	17.0	15.2
25	L. pictus	17.8	17.7	16.3	16.2	17.6	17.1	16.6	18.6	20.4	19.0	19.0	18.1	16.0	17.6	19.9	16.5	17.5
26	L. frittiniens	16.4	16.1	15.4	15.8	16.3	15.8	18.4	18.5	19.8	19.3	18.0	17.7	16.6	16.6	20.0	15.5	18.1
27	L. dringi	16.3	16.6	15.7	16.1	16.4	16.1	18.0	18.3	17.8	18.5	17.8	17.6	16.8	16.2	19.9	16.7	16.4
28	L. sabahmontanus	15.6	16.5	16.0	16.3	16.0	15.5	17.8	18.3	18.6	18.1	17.6	16.8	15.3	15.8	19.2	15.5	14.6
29	L. heteropus	17.3	17.2	17.7	17.2	18.7	18.4	22.2	20.9	21.9	20.9	21.5	21.5	18.8	20.6	23.5	20.6	16.0
30	L. marmoratus	16.1	15.7	15.8	16.4	15.6	15.1	18	18.2	19.3	17	18.1	17.6	14.6	15.3	18.7	15.1	13.6
31	L. hamidi	16.6	17	16.1	16.7	16.6	16.1	18.4	19.1	17.8	17	17.4	16.7	15.2	15.9	19.5	14.5	15.2
32	Brachytarsophrys	22.4	21.9	20.4	20.1	19.5	19.8	23.3	21.7	22.1	21.7	21.7	21.1	21.2	23.1	23.3	21.4	22.4