Description of two new species of the microhylid frog genus Oreophryne ( Amphibia : Anura : Microhylidae ) from southern Papua New Guinea

Two new species of the microhylid genus Oreophryne are described on the basis of material collected from the Kikori River Basin in southern Papua New Guinea. Both species belong to the group of Oreophryne that have a ligamentous connection between the procoracoid and scapula. One of the new species is small (snout-urostyle length in males 19.7 – 23.3 mm and in females 23.8 – 27.4 mm), and males can be distinguished from congeners in this group by having one or a few conspicuous yellow spots in the inguinal region and an advertisement call consisting of a loud rattle lasting 1.4 – 2.3 seconds. The second species is larger (males 25.9 – 31.4 mm and females 30.1 – 34.7 mm) and in life has a uniform brownish-grey dorsum with numerous tiny white dots. Its advertisement call is a series of 9 – 15 ‘whistling’ or ‘peeping’ notes. Both species are predominantly arboreal, normally calling from perches more than 2 m above the ground.


Introduction
The Kikori River Basin in southern Papua New Guinea (PNG) encompasses a broad range of habitats, from low altitude coastal mangroves and rainforest to lush hill forest, montane moss forest and high-montane grassland.The frog fauna in this area is extremely diverse and, despite recent taxonomic studies of microhylid (RichaRds & OliveR 2010;GüntheR et al. 2012) and hylid (Rich-aRds & OliveR 2006; RichaRds 2007) species from the catchment nearly half of the frogs known from there remain undescribed (e.g.RichaRds 2002).
Frogs of the genus Oreophryne reach their greatest diversity on the island of New Guinea where numerous new species have been described in recent years (e.g.GüntheR & RichaRds 2011;GüntheR et al. 2009GüntheR et al. , 2012GüntheR et al. , 2014;;KRaus 2011KRaus , 2013;;KRaus & allisOn 2009), and they currently represent one of the most speciose group of microhylid frogs known from the region.During the course of three surveys in the Kikori Basin between 2001 and 2003 the junior author obtained material of two Oreophryne species that do not match the descriptions of any known taxon.They were illustrated as Oreophryne sp.nov. 2 and Oreophryne sp.nov. 3 by RichaRds (2002) and we here provide formal descriptions of these two new species.

Material and methods
Male frogs were collected at night after they were located by their advertisement calls; females were detected when they were exposed on foliage.Representative specimens were photographed in life the next day, and all specimens were then anaesthetised in an aqueous chlorobutanol solution and subsequently fixed in 5 % formalin.Liver samples were taken from some specimens before fixation, and stored in 96 % ethanol to enable later DNA sequencing.All specimens were transferred to 75 % ethanol within two days of fixation.One specimen of the first new species was cleared and stained as an osteological preparation according to a method modified from dinG-eRKus & uhleR (1977) and the pectoral region was dissected from a specimen of the second new species.The following measurements were taken with a digital calliper (> 10 mm) or with a binocular dissecting microscope fitted with an ocular micrometer (< 10 mm) to the nearest 0.1 mm from preserved specimens only: SUL snout-urostyle length from tip of snout to distal tip of urostyle bone; SUL is generally slightly shorter than snout-vent length (SVL).As the measurement error is higher in the latter, we prefer to use the former.Both measurements are sufficiently similar (unpublished data) that, where relevant, we compare our SUL measurements with SVL's presented for members of the genus in some papers; TL tibia length: external distance between knee and ankle; TaL length of tarsus: external distance between tarsal and ankle joints held at right angles; T4L length of 4 th toe: from tip of toe to proximal end of inner metatarsal tubercle; T4D transversal diameter of disc of 4 th toe; T1D transversal diameter of disc of first toe; F3L length of 3 rd finger; F3D transversal diameter of disc of 3rd finger; F1D transversal diameter of disc of first finger; HL head length, from tip of snout to posterior margin of tympanum; HW head width, taken in the region of the tympana; SL snout length, from an imaginary line connecting the centres of the eyes to tip of the snout; END distance from anterior corner of orbital opening to centre of naris; IND internarial distance between centres of nares; ED eye diameter, from anterior to posterior corner of orbital opening; TyD horizontal diameter of tympanum.
Advertisement calls were recorded under natural conditions with a Sony Pro-Walkman or a Sony TCM 5000EV tape recorder and a Sennheiser ME66 Microphone with K6 power module, and analysed with Avisoft-SAS Lab Pro software.
Material studied for comparative purposes is listed by GüntheR (2015).tympanum small (about one-third of eye diameter) and hardly visible; no prominent supratympanic fold.Legs moderately long (TL/SUL 0.48).Fingers unwebbed and with broad and grooved terminal discs, their relative lengths 3 > 4 ~ 2 > 1 (Fig. 4); disc of third finger twice width of penultimate phalanx, no prominent metacarpal or subarticular tubercles.All toes with wide and grooved terminal discs, discs of third toe same width as disc of fourth toe; no webs be- tween toes and no prominent metatarsal or subarticular tubercles; relative lengths of toes 4 > 5 > 3 > 2 > 1 (Fig. 5).

Abbreviations of collections
A conspicuous tubercle between angle of jaw and insertion of upper arm; no other distinct tubercles on dorsal or ventral surfaces in preservative but in life some inconspicuous tubercles were evident on flanks and dorsal surfaces of limbs.

Colour in preservative:
Ground colour of all dorsal surfaces off-white with diffuse brown stipples and flecks on fore and hind legs; head also irregularly pigmented with brown laterally; a conspicuous brown postocular fleck merges dorsolaterally with a large but less intensely pigmented blotch that reaches nearly as far as the inguinal region and that has a posterioventrally directed branch.An off-white mid-dorsal line divides a pair of small brown spots between scapular and occipital region and a brown area between the eyes.Ventral surfaces off-white with diffuse brown mottles mainly on chest, lower arms and thighs.Yellow inguinal spot(s) barely visible.
Colour in life: Ground colour of body yellowish laterally, yellowish-red dorsally, and of dorsal limbs reddish.Dorsally limbs covered by a dense pattern of diffuse brown pigmentation except on upper arm.A dark brown and posteriorly bifid (compare Fig. 2) blotch on upper flank; this blotch is connected by a broad band of less intense brown with a dark brown postocular fleck.Two pairs of dark brown spots between eyes and one pair between scapular region and occiput.Largest (lower) portion of tympanum yellowish; tubercle between corner of mouth and insertion of upper arm appears as a short but conspicuous whitish streak.Pupil horizontally oval with reddish inner margin, iris silvery with brown venation (Fig. 6).
Morphological variation in the type series.Measurement and body ratios of the type specimens are presented in Table 1.Six of the ten paratypes are coloured similarly to the holotype, with a broad pale mid-dorsal band and dark dorsolateral patches.A typical example of this colour pattern is exhibited by SAMA R69682, which is illustrated in Fig. 7.The remaining four specimens have pale dorsal and lateral surfaces while the largest female, ZMB 83556, exhibits a rather uniform brownish dorsum in preservative.All males have one or several more or less conspicuous yellow spots in the inguinal region (Fig. 8).
Female ZMB 83556 does not show such spots and the      character was not noted before female 83557 was cleared and stained, so it is possible that females do not exhibit this feature.Ventral surfaces vary from uniform off-white to finely speckled and roughly mottled with brown.
Distribution and ecological notes.The new species is known only from two locations at 900 m and 1,300 m asl in very wet, lower-montane rainforest in the Kikori River Basin of Southern Highlands Province, PNG (Fig. 1).Males normally called from leaves ~ 3 -5 m high in the forest understorey at night during and after heavy rain.This species was abundant at the type locality in mossy forest at the summit of Iagifu Ridge near Moro, but was difficult to capture there because of its tendency to call from elevated perches in steep, broken limestone karst terrain.
Vocalisation.Four calls from two males, recorded at an air temperature of 18 °C, were analysed.The advertise-  ment call is a loud rattle of 1.40 -2.30 s duration, mean 1.85 ± 0.39 s.The calls contain 34 -49 strongly pulsed notes (Fig. 9), mean 41.8 ± 6.2 notes/call.Notes consist of about 5 irregular pulses that are too close together to measure accurately (Fig. 9).

Diagnosis.
A medium-to large-sized species of the genus Oreophryne with a snout-urostyle length in adult males (n = 5) from 25.9 -31.4 mm and in adult females (n = 2) from 30.1 -34.7 mm.Connection between procoracoid and scapula ligamentous.No webs between fingers, distinct basal webs between all toes; fifth toe same length as third; finger discs generally wider than toe discs (ratio T4D/F3D 0.83 -1.00); shanks fairly short (TL/SUL 0.39 -0.43).Eyes of medium size (ED/SUL 0.110 -0.131), eye-naris distance greater than internarial distance (END/IND 1.00 -1.25).Dorsal and lateral body surfaces in life uniform brownish-grey with numerous tiny white dots (tubercles).In preservative, lateral and dorsal body surfaces uniform light grey to middle-brown with less contrasting whitish dots.Advertisement call a series of 9 -15 whistling or 'peeping' notes of 2.8 -4.8 s duration, with notes unpulsed and lasting on average 80 ms; note repetition rate is 2.9 -3.4 notes/s and dominant frequency is at 2.6 kHz.
Description of the holotype .Adult male with a SUL of 27.2 mm.Additional measurements and ratios are listed in Table 2. Head broader than long (HL/ HW 0.83); tip of snout truncate with a small median protuberance at the tip in dorsal view (Fig. 11) and truncate in lateral view; nostrils very near tip of snout, directed laterally and not visible from above, distance between nares clearly less than distance between eye and naris (END/ IND 1.25); canthus rostralis in dorsal view straight; loreal region sloped, weakly concave and its upper margin (canthus rostralis) rounded; tongue wide, long, with a very small posterior indentation; prepharyngeal ridge with only hints of denticles; vocal slit visible on left side of mouth floor, that on right side not clearly recognizable; tympanum small (about one-third of eye diameter), its upper margin covered by skin; a weak supratympanic fold.Legs moderately short (TL/SUL 0.42); fingers unwebbed and with broad and grooved terminal discs (disc of third finger twice width of penultimate phalange), their relative lengths 3 > 4 > 2 > 1; metacarpal and subarticular tubercles weakly expressed.All toes with wide and grooved terminal discs, discs of third toe same width as disc of fourth toe (T4D/F3D 1.0), distinct basal webs between toes, weakly developed metatarsal and subarticu-lar tubercles, relative lengths of toes 4 > 5 ~ 3 > 2 > 1.A few low, inconspicuous tubercles on all dorsal surfaces, ventral surfaces smooth.

Colour in preservative.
All dorsal surfaces uniform brown with some small white tubercles, mainly on lateral surfaces of body.Tympana somewhat lighter than surrounding surfaces.Some small light spots on margin of upper eye lid.Ventral surfaces uniform off-white.
Colour in life: Dorsally head and body uniform greybrown, limbs lighter grey-brown; all dorsal and lateral surfaces with small whitish tubercles.Iris golden with dark brown venation that is mainly longitudinally oriented.
Morphological variation in the type series.Measurements and body ratios of the type specimens are presented in Table 2. Paratypes differ in colour only slightly from the holotype.All specimens have a uniform dorsal colouration, six are brownish-grey and two are greyishbrown in preservative; in life parts of the dorsal surfaces may be more brownish and parts may be more greyish as in SAMA R69687 (Fig. 15).In most specimens the    whitish tubercles disappeared in preservative to a large extent.In about half of the specimens the head is lighter dorsally than the remaining dorsal surfaces.In only one specimen (ZMB 83562) the middle dorsum is covered by a reticulum of whitish flecks.Ventral surfaces in all specimens are uniformly off-white.
Distribution and ecological notes.Oreophryne pseud unicolor is abundant in the lowland and hill forests of the Kikori Basin, where males normally call from elevated perches on leaves up to 30 m above the ground.Males also call from within hollow vines and small holes in branches and tree trunks, and calling males have occasionally been found guarding clutches of eggs within these well-protected calling and nesting sites.The loud whistling or 'peeping' sound produced by this species is a conspicuous component of the acoustic environment at night throughout the Kikori Basin at altitudes between ~ 200 and at least 900 m a.s.l.The taxonomic status of populations of whistling Oreophryne from east and west of the Kikori Basin (e.g.RichaRds et al. 2015) requires confirmation; based on currently available information O. pseudunicolor is known with certainty only from the lowlands and foothills of Gulf and Southern Highlands Provinces of south-central Papua New Guinea (Fig. 1).
Vocalisation.Fifteen calls from two males recorded at an air temperature of 22 °C were analysed.The advertisement call is a series of loud and melodious whistling or 'peeping' notes.Mean call length is 3.85 ± 0.61 s, range   Etymology.Pseudunicolor is a compounded Latin adjective meaning "seemingly unicolor" and refers to the similarity of the new species to Oreophryne unicolor.
Comparisons with other species.About 30 Oreophryne species have a cartilaginous connection (vs. a ligamentous connection as in the new species) between procoracoid and scapula and are not considered further in this paragraph.About 20 species have a ligamentous connection between procoracoid and scapula.The probable maximum size range of O. pseudunicolor sp.nov. is from 24 -38 mm SUL, and the species has a whistling advertisement call.Therefore, species smaller or larger than this range and/or with rattling, squeaking, or trilling calls can be separated from the new species on the basis of these characters.There remain seven species in   In its possession of a ligamentous connection between the procoracoid and scapula, in having webbing between the toes, and in sharing broadly overlapping values of most measurements and ratios, O. ampelos is difficult to distinguish from O. pseudunicolor.Unfortunately the advertisement call of O. ampelos is not known, precluding comparisons of this important diagnostic character.However several consistent differences in body shape and colouration indicate that the two species are distinct from each other.O. ampelos can be readily distinguished from the new species by its distinctly wider head (ratio HW/ SVL 0.37 -0.42 vs 0.33 -0.37 in O. pseudunicolor) and there are also smaller but statistically significant differences in limb length (TL/SUL p = 0.017) and size of toe discs (T4D/SUL; p = 0.023).There are also consistent differences in dorsal colouration between the species.O. am pelos was described as having a "dorsal pattern of brown punctations on a pale strawyellow ground color which gives an overall appearance of a uniformly light tan, uni formly dark tan, or mottled light and dark tan animal" (KRaus 2011).Two of the paratypes examined by KRaus (2011) also had a wide, light tan mid-vertebral stripe bor-dered by narrower dark brown stripes.O. pseudunicolor has never a straw-yellow colour (in life or preservative), always exhibiting a uniform grey-brown dorsum with tiny pale tubercles and flecks (Fig. 15) with, at most (in very rare cases), scattered blotches of tan on the mid-dorsum.
Another species very closely related to the new species is O. unicolor.There are no clear differences in measurements and body colouration except colour of the iris which is reddish in O. unicolor and golden in O. pseudu nicolor.However, differences in advertisement calls and in sequences of the 16S rDNA gene are evidence for the specific status of both species.The call notes of O. uni color last 149 -194 ms (mean 167 ± 10.6 ms, n = 66) those of O. pseudunicolor last 61 -90 ms (mean 78 ± 3.6 ms, n = 184).Note repetition rate in calls from O. unicolor varied between 1.5 -1.8 notes/s, mean 1.68 notes/s and in calls from O. pseudunicolor between 2.9 -3.4 notes/s, mean 3.2 ± 0.13 notes/s.A pairwise distance calculation of 417 sites of the 16S RNA gene showed a p-distance of 8.6 % in each case between O. unicolor from the type locality (ZMB 70188 from the Wondiwoi Mts, Wandammen Peninsula, West Papua Province) compared with three specimens of O. pseudunicolor (SAMA R 69684, ZMB 83561, and ZMB 83562) whereas p-distances between these three specimens of P. pseudunicolor were 0.2 -0.7 % (stelbRinK & vOn Rintelen, pers.comm.).
We found other frogs of the genus Oreophryne in the Kikori region that are smaller than both O. pseudunicolor and O. unicolor, but are otherwise as similar to O. uni color in their body ratios as O. pseudunicolor is to that species.Furthermore, the smaller Kikori frogs have whistling advertisement calls more similar to the geographically distant O. unicolor than to sympatric O. pseudunicolor.Their p-distances of the 16S RNA gene to O. unicolor varied between 4.8 and 5.5 % and to O. pseudunicolor between 7.7 and 8.2 %.Thus, according to calls and 16S rDNA these frogs are more closely related to O. unicolor than O. pseudunicolor is.We have also examined a morphologically similar series of Oreophryne from the southern slopes of the central cordillera to the north-east of the Kikori Basin that has a more yellowish ground colour (as described for O. ampelos) and whistling calls.
It appears that a number of morphologically and acoustically similar Oreophryne species with whistling calls occur in the forests of southern New Guinea.On the basis of present knowledge the taxonomic status of these frogs is not clear and we refrain from naming additional populations until additional acoustic and genetic studies have been undertaken.

Fig. 1 .
Fig. 1.Map of known locations for O. flavomaculata (white circles) and O. pseudunicolor (black circles).The half-black circle is the only known site where the two species occur in sympatry.
) and there are some weak upper harmonics up to 8 kHz.
support for this project was received from the Mark Mitchell Research Foundation and the South Australian Museum Board.We are extremely grateful to R. sinGadan and P. Kei (UPNG) who provided numerous courtesies in Port Moresby and to B. ROy and B. wilmOtt (Department of Environment and Conservation) who assisted with export permits.J. RObins (PNG National Research Institute) kindly assisted with SJR's research visas.M. hutchin-sOn and C. KOvach (SAMA) provided access to specimens in their care and L. capOn kindly produced Figure1.We are grateful to F. tillacK (ZMB) for taking Figures 2 -5 and 11 -14 and for various technical help.

Table 1 .
Body measurements and body ratios of the type series of Oreophryne flavomaculata sp.nov.SAMA R69679 is the holotype; all types are adult males except ZMB 83556 and ZMB 83557 which are adult females.
The specific epithet flavomaculata is a feminine compounded Latin adjective; flavus, -a, -um means yellow and maculatus, -a, -um means spotted.It refers to the conspicuous yellow spots in the inguinal region exhibited by most specimens.
Comparisons with other species.About 30 Oreo phry ne species have a cartilaginous connection (vs. a li gamen tous connection as in the new species) between pro coracoid and scapula and are not considered further in this paragraph.About 20 species have a ligamentous con nection between procoracoid and scapula.The probable maximum size range of O. flavomaculata sp.nov.males is from 19 -25 mm SUL, and the species has a rattling advertisement call.Therefore, males smaller or larger than this range and/or with peeping, squeaking, or trilling calls can be separated from the new species on the basis of these characters.There remain 14 species in the size range of O. fla vo maculata and with rattling or unknown advertisement calls which will be compared more closely: O. albopunctata has noticeable basal webs on toes (no webs in fla vo maculata) call duration in O. flavomaculata 1.4 -2.3 s and repetition rate 21.3 -24.3 notes/s; O. ezra has webbed toes, a blue iris (silvery in O. flavomaculata) and advertisement calls with a long introductory note (missing in O. fla vomaculata); O. furu has basal webs between toes, no yellow spots in the inguinal region and most advertisement call notes consist of three pulses (five pulses in Oreophryne pseudunicolor sp.nov.Holotype.SAMA R69686 (Field number: FN SJR 3199), adult male, Darai Plateau, Gulf Province, Papua New Guinea (07°07.771'S,143°36.806'E;400 m a.s.l.), collected by S. RichaRds on 28 July 2003.

Table 2 .
Body measurements and body ratios of the type series of Oreophryne pseudunicolor sp.nov.SAMA R69686 is the holotype; all types are adult males except PNGNM unregistered and SAMA R69687 which are adult females and ZMB 83562 which is subadult.-90 ms; mean of means of internote length 255.4 ± 16.4 ms, range of means 234 -280 ms, total range of 157 internote intervals 223 -365 ms.Mean note repetition rate 3.2 ± 0.13, range 2.97 -3.36 notes/s.Most frequencies scatter between 2.4 and 2.8 kHz.The dominant frequency is at 2.6 kHz (Fig. 17 2.8 -4.8 s.The calls contain 9 -15 notes, mean 12.3 ± 1.62 notes/call.Call notes are unpulsed and without frequency modulation (Fig.16).Mean of means of note length 77.9 ± 3.6 ms, range of means 72 -83 ms, total range of 184 notes 61 the size range of O. pseudunicolor that have whistling calls, or for which the advertisement calls are unknown, and these will be compared more closely: O. ezra has a blue iris (golden in O. pseudunicolor) and, among other characters, a smaller body size (males 22.3 -26.Oreophryne most similar to O. pseudunicolor are O. ampelos and O. unicolor.O. am pelos was described by KRaus (2011) from the Hindenburg Range and Star Mountains of far western Papua New Guinea at altitudes between 840 and 1,280 m a.s.l.