Bioacoustics of Boulenophrys rubrimera

Distribution and conservation status: This species is known from between 1400 m asl and 1722 m asl at two localities, 2 km apart in Sa Pa District, Lao Cai Province , Vietnam and 50 km north-west at Maandi, Jinping County, Ailao Mountain Range, Yunnan Province , China ( Fig. 1 ; Chen et al . 2017 ). The species' area of occupancy (AOO) and extent of occurrence (EOO) are currently predicted to be 385 km 2 and 2298 km 2, respectively. The Vietnamese and Chinese portions of this species’ range comprise two threat-defined locations; land use likely differs between the two countries, and habitat loss probably affects the species independently in each. Ongoing disturbance to the species’ habitat due to forest clearance for agriculture has been observed in Sa Pa District. We recommend that Megophrys rubrimera sp. nov. is listed as Endangered in accordance with the IUCN Red List of Threatened Species categories and criteria B1ab(iii). Comparisons: Megophrys ( Panophrys ) rubrimera sp. nov. can be distinguished from all other congeners found in mainland southeast Asia, north of the Isthmus of Kra and neighbouring provinces of China on the basis of morphology, and from all congeners for which comparable data is available on the basis of molecular and acoustic data. Comparisons with each subgenus are discussed separately below.

Description of holotype : Sexually mature male ( Figs. 2 & 3 ). Head small, wider than long; the snout rounded in dorsal view and obtusely protruding in lateral view, rostral appendage absent ( Figs. 3A & 4G ); loreal region vertical and concave; canthus rostralis angular; dorsal region of the snout is slightly concave; eye diameter nearly twice the maximum tympanum diameter and subequal to snout length; eye-tympanum distance is smaller than the maximum tympanum diameter; tympanum round and orientated vertically. Pupil in life oval, vertically orientated when dilated; nostril orientated laterally, closer to eye than snout; internarial distance exceeds eyelid width, and subequal to narrowest point between upper eyelids; weakly defined vomerine ridges present, oblique and barely separated anteriorly with small vomerine teeth; maxillary teeth present. Tongue moderately large and not clearly notched posteriorly. Forelimbs short and stocky, forearm length shorter than hand length; fingers short and wide without lateral fringes ( Fig. 3B ), finger length formula I <II <IV <III; interdigital webbing absent, subarticular tubercles absent, palmar tubercles absent; supernumerary tubercles absent, but skin raised on articulations of Fingers III, IV; thenar tubercle absent; finger tips slightly expanded and flattened to oval pads; terminal grooves absent. Hind limbs relatively short and stocky; thigh longer than shank, and longer than foot; toes relatively short and wide, lateral fringes present ( Fig. 3C ); toe tips very slightly dilated, terminal groves absent; toes not webbed; outer metatarsal tubercle, subarticular and supernumerary tubercles absent; inner metatarsal tubercle prominent. Skin of dorsal surfaces of body, limbs, and dorsal and lateral surfaces of head weakly granular; tympanum granular with its borders slightly raised; very small pointed tubercle present on outer edge of upper eyelid; supratympanic fold narrows as it passes above the tympanum, terminating above axilla, supratympanic fold with many dark asperities along its crest ( Fig. 4A ); flanks with small scattered tubercles, some of which terminate in darkened asperities; thin dorsolateral fold extending from behind supratympanic fold to approximately threequarters distance to groin; a weak, “V”-shaped parietoscapular ridge present, its two sides extending posteriorly from above tympanum and meeting medially beyond level of axilla; a second inverted “U”-shaped ridge present on mid-dorsum which joins laterally with dorsolateral folds ( Figs. 2A & 4A ); ridges with some dark asperities; small tubercles tipped with dark asperities on dorsal surface of shanks, and arranged into distinct transverse rows on the thighs and forearms. Large distinct tubercles present in the inguinal region; gular region, chest and ventral surfaces of limbs smooth; pectoral glands obvious, small, slightly raised, positioned on level with axilla ( Figs. 2B & Fig. 4D ); femoral glands small, slightly raised, one positioned equidistant from knee and cloaca on the posterior surface of each thigh. Colour of holotype in life ( Figs. 4A–B, D & F ): Dorsally light tan; darker “Y”-shaped marking on dorsum between the eyes, border of marking beige; vertical dark brown bar below eyes; lateral surface of the eyelid dark with a single medial light bar; tympanum dark brown; hind and forelimbs possess dark tan bars, darker on the forelimbs than the hind limbs; tubercles on flanks encircled by darker tan; groin red-orange; gular and pectoral region grey-brown; abdomen light grey with dark grey blotches and white speckling; ventral surface of thighs pinkish grey with darker blotches; inner thighs and outer surface of shanks red-orange; underside of Fingers III, IV orange; thenar tubercles red-orange; inner metatarsal tubercle on feet red-orange; iris metallic brown. SVL VNMN 2017.002* 29.8 VNMN 2017.003** 27.9 AMS R177676** 28.7 AMS R177675** 30.5 HNLP 2016062200001^ 30.2 AMS R177677** 26.7 AMS R177678** 27.4 AMS R177679** 30.4 Hw HL IFE 11.2 9.4 3.5 11.1 10.1 3.8 11.7 11.6 4.2 11.6 11.2 4.0 11.6 11.2 4.1 11.1 10.6 3.9 10.4 9.8 3.7 12.2 11.1 4.1 IBE EL TYD 3.8 3.3 2.0 3.6 3.6 2.1 4.0 3.4 2.0 4.4 3.5 2.3 3.7 3.5 2.2 3.5 3.2 2.1 3.7 3.3 2.5 4.0 3.2 2.2 TYE SL EN 1.9 3.9 1.6 2.4 3.8 1.9 2.0 4.4 1.5 2.1 4.1 1.9 1.8 4.0 1.9 2.2 3.6 1.9 1.5 2.8 1.9 1.9 3.9 1.9 SN IUE IN 2.0 4.0 4.0 2.2 3.7 3.3 2.7 4.0 4.3 1.9 3.3 3.3 1.9 3.4 3.5 1.8 3.2 3.0 1.5 3.3 2.6 1.7 3.2 3.1 UEw FAL HAL 3.1 6.5 7.5 3.1 6.4 7.8 3.9 7.5 9.2 3.7 6.6 8.5 3.5 7.1 8.8 3.1 6.3 8.4 3.1 6.3 7.8 3.3 7.3 8.1 FIL FIIL FIIIL 2.4 2.8 4.1 3.3 3.5 4.8 3.6 4.4 5.6 3.5 3.5 4.9 3.8 4.2 5.1 3.0 2.9 5.1 3.1 3.4 4.7 3.8 3.5 5.7 FIVL SHL TL 2.9 14.4 14.6 3.1 14.8 14.9 3.3 16.1 15.6 3.6 15.6 15.9 3.4 15.5 15.8 3.3 14.9 14.8 3.4 13.6 14.7 3.6 15.9 15.9 FOL IMT Mass in life (g) 13.5 2.7 2.4 12.4 2.4 2.0 15.4 2.1 2.3 13.9 2.6 2.5 13.3 2.5 2.5 12.9 2.4 2.3 14.1 2.4 2.3 14.7 2.41 2.6 Colour of holotype in preservative: ( Figs. 2 & 3 ): Majority of dorsal and lateral surfaces of the head, body, forelimbs and hind limbs brown; darker brown “Y”-shaped marking between eyes; darker brown “X”-shaped marking over opposing “V”-shaped and “U”-shaped ridges; dorsolateral folds and flank tubercles bordered by darker margin; front of snout and lateral canthus rostralis dark brown; narrow vertical dark brown bar below eyes and dark brown blotch covering tympanum; upper jaw with darker blotches; two dark brown blotches on the dorsal surface of forearms; dorsal surface of Fingers II, III and IV with dark brown blotches. Gular region, chest and anterior part of abdomen primarily creamy-grey, with grey brown speckling on gular region; abdomen blotched with dark brown; ventral surfaces of thighs with grey brown mottling, and shanks with grey brown speckling; ventral surfaces of feet grey brown; area surrounding vent with dark brown blotches; forelimbs ventrally mottled and blotched with light and dark brown; extending to the ventral surface of hands; inner metatarsal tubercles unmarked and beige, tips of fingers unmarked and beige; tips of toes only lightly speckled with grey brown, if at all; lateral fringe on toes, beige, unmarked; pectoral and femoral glands beige. Measurements (in mm): Holotype . SVL 29.8, HW 11.2, HL 9.4, SL 3.9, SN 2.0, EN 1.6, EL 3.3, IUE 4.0, UEW 3.1, IFE 3.5, IBE 3.8, TYD 2.0, TYE 1.9, FAL 6.5, HAL 7.5, FIL 2.4, FIIL 2.8, FIIIL 4.1, FIVL 2.9, TL 14.6, SHL 14.4, FOL 13.5, IMT 2.7, weight in life 2.4 g . Variation: Measurements of the type series are shown in Table 3 . Paratypes and referred specimens generally agree with the holotype morphologically, but with the following exceptions: in VNMN 2017.003, AMS R177676 the middorsal “U”-shaped ridge (on the holotype ) is replaced by an inverted “V”-shaped ridge. AMS R177676 with distinct dark grey blotches on ventral surface of thighs, this is lacking in VNMN 2017.003 and the holotype specimen. In life, AMS R177676 has a vivid red-orange flash on the axillary region ( Fig. 4G ). Gular region of VNMN 2017.002 and AMS R177678 lack dark blotches, present in all other specimens. Ventral surface of thighs (on AMS R177679 and AMS R177676) are covered in dark blotches, which are not present on any of the other specimens. Tongue weakly notched in one specimen (HNLP2016062200001). Secondary sexual characters: Females of Megophrys rubrimera sp. nov. are currently unknown. Two male specimens (AMS R177678 and AMS R177679) possessed raised oval nuptial pads covered in microspinules on the base of Finger II; protruding fleshy projection (secondary sexual characteristic of some male Megophrys e.g. M . caudoprocta , M . koui and M . pachyproctus ) above the cloaca absent from all specimens. After fixation it was very difficult to open the mouths of the specimens without damaging them due to the small head size. It was therefore not possible to determine the position, presence or absence of internal vocal slits which are a secondary sexual characteristic used as a diagnostic character to the species level (e.g. Mahony et al . 2013 ). Tadpole: ( Fig. 5 ): The following tadpole description is based on a single specimen (AMS R177680) at Stage 37, the tadpole was confirmed as that of M . rubrimera sp. nov. by molecular analysis. Body elongated and slender; nares oval and are closer to the eye than to the snout; internarial distance is subequal to the interorbital distance; eyes positioned dorsolaterally, the pupils are round; the spiracle is sinistral and the spiracular tube protrudes from midway along the body wall just posterior to maximal trunk width and opens laterally; the tail makes up 68% of the total body length; the dorsal tail fin is low, particularly at the proximal half of the tail length; the basal tail width is 60% of the maximal trunk width; the oral disk is subterminal and antero-dorsal; the width of the umbelliform oral disc makes up ~80% of the maximal width of the trunk; the upper and lower lips each have deep medial emarginations; submarginal papillae are present on both sides between the oral orifice and the emarginations of the upper and lower lip, concentrated in a row around the margins; keratodonts are absent. Colour in life: Dorsally, the head and body are brown with darker speckles; obvious pale neuromasts; the oral disk is translucent yellowish brown with dark brown submarginal papillae; the dorsal and ventral fins opaque, pale yellowish brown with speckles; the venter is speckled white and brown; iris orange, speckled with black dots. Colour in preservative: Body brown with darker speckles; oral disc translucent grey brown with dark brown submarginal papillae; the dorsal and ventral fins opaque, pale grey brown with darker speckles; the venter is speckled grey and brown. Tadpole body measurements (in mm): BL 10.5, BH 4.4, BS 4.3, ES 1.7, IND 1.7, IOD 4.1, BW 5.4, ED 1.2, LFH 1.3, MTH 5.9, NE 1.9, ODW 4.5, SN 0.6, SS 5.7, TAL 22.8, TTL 33.3, TMH 3.1, TMW 5.4, UFH 1.3). Advertisement call: Call descriptions are based on the calls of one paratype (AMS R177677) and two unvouchered individuals. Advertisement calls were recorded at 21.0–22.9 °C ambient temperature. Calls were an average of 73.3 ms (62–85 ms) in duration ( Table 4 ; Fig. 6 ). Calls of unvouchered individual (b) contained an average of 23.05 pulses (19–25), whereas the calls of AMS R177677 and another unvouchered individual (a) were not distinctly pulsed. The average dominant frequency of calls was 3.3 kHz (3.2–3.4 kHz). Calls were repeated at a rate of approximately 3.25 (3.05–3.37) calls per second, and had an average intercall interval of 221.3 ms (190–261 ms). The number of calls within each call group ranged from 16–51, with an average of 38.7. Most call groups began at a relatively low amplitude, increasing with each call up to approximately a quarter to a third of the duration, after which amplitude remained relatively constant ( Fig. 6A i –ii & B i–ii). Individual calls either began with a medium relative amplitude and peaked near the middle before declining towards the end ( Fig. 6A iii), or peaked at the beginning, steadily declining throughout ( Fig. 6B iii). AMS R177677 Unvouchered individual (a) Unvouchered individual (b) Number of call groups measured 3 3 5 Total number of calls analysed 20 20 20 Call duration (ms) 74.2 (56–83) 79.6 (72–85) 66.0 (62–70) Intercall interval (ms) 207.0 (190–235) 232.8 (217–261) 225.6 (208–324) Call repetition rate (calls/s) 3.3 3.1 3.4 Calls/call group 39.3 (34–50) 45.6 (42–51) 31.2 (16–47) Pulses/call Not distinctly pulsed Not distinctly pulsed 23.05 (19–25) Dominant frequency (kHz) 3.2 (3.2–3.4) 3.4 (3.4–3.4) 3.2 (3.2–3.4) Temperature (°C) 22.9 21.0 22.1 Natural history: All specimens of Megophrys rubrimera sp. nov. were found in disturbed evergreen secondary forest. Males were calling during June 2012 and 2016, and tadpoles (Stage 37) were collected in June 2016 , suggesting a prolonged breeding season or that the species has a long larval period. Males were observed calling on stream-side vegetation (Fig. 7A–B) and a steep north-eastern facing roadside bank with seepages and a small stream (Fig. 7C–E). Females were not observed.

Diagnosis: Assigned to the genus Megophrys on the basis of tadpole morphology; tadpoles have dorsally orientated umbelliform oral discs (Dubois & Ohler 1998; Li et al . 2011), which is diagnostic for the genus within Megophryidae ; and to the subgenus Panophrys on the basis of molecular data. Megophrys rubrimera sp. nov. differs from its congeners by a combination of the following morphological characters (based on eight adult males): (1) small size (SVL 26.7–30.5 mm ); (2) very small palpebral horn on upper eyelid; (3) toes lacking interdigital webbing but possessing narrow lateral fringes; (4) tympanum diameter: eye diameter 58.0–76.0 mm; (5) shank length: snout vent length 48.0–56.0%; (6) groin, inner surface of thighs and outer surface of shanks red-orange; (7) absence of subarticular tubercles on fingers and toes; (8) red-orange inner metatarsal tubercle; (9) head width greater than head length; (10) head width: snout vent length 38.0–42.0%; (11) weakly defined vomerine ridges with teeth; and (12) an advertisement call with a dominant frequency of 3.23 kHz.