New genus of diminutive microhylid frogs from Papua New Guinea

A new genus of diminutive (10.1–11.3 mm) microhylid frogs is described from New Guinea that is unique in its combination of having only seven presacral vertebrae, a reduced phalangeal formula that leaves the fi rst fi ngers and fi rst toes as vestigial nubs, and reduction of the prepollex and prehallux to single elements. Relationships to other genera are unknown, but overall similarity suggests some relationship to Cophixalus, although that genus also diff ers in some muscle characters and likely remains paraphyletic. Th e new genus contains two species, which are among the smallest known frogs in the world. Th eir miniaturization may be related to their inhabiting leaf litter, exploitation of which may select for small size. Th e new genus is currently known only from one mountaintop in the southeasternmost portion of New Guinea and another on a nearby island. Th is region is part of the East Papuan Composite Terrane and, should this lineage prove endemic to that region, it may suggest that it originated prior to that geological unit’s docking with mainland New Guinea at 23–29 MY.


Introduction
Th e Asterophryinae is one of 11 subfamilies of microhylid frogs, but it contains more than half the species diversity within that family (Frost 2010).Th is subfamily is largely endemic to the Papuan region (comprising New Guinea and satellite islands, the Ad-miralty and Bismarck Archipelagos, and the Solomon Islands), contains 250+ species, is monophyletic (Savage 1973, van Bocxlaer et al. 2006, Frost et al. 2006, Roelants et al. 2007, van der Meijden et al. 2007), and is relatively young: molecular evidence suggests the extant members of this clade arose within the past ~30 MY (van Bocxlaer et al. 2006, Roelants et al. 2007), approximately consistent with geological evidence for the origin of New Guinea (Davies et al. 1996(Davies et al. , 1997)).Taxonomy within Asterophryinae has been fl uid, and the number of recognized genera has expanded from nine in the monograph of Parker (1934) to 20 at present (Günther 2009a, Frost 2010), mostly due to partitioning of recognized genera into more tractable, morphologically cohesive (e.g., Zweifel 2000) or monophyletic (Günther 2009a) groups.Nonetheless, additional partitioning is necessary to achieve a completely monophyletic taxonomy of Asterophryinae (Köhler and Günther 2008), and species diversity within this clade, though great, is severely underestimated (FK, unpubl. data).
Asterophryinae is morphologically and ecologically diverse, encompassing a variety of fossorial, terrestrial, semi-aquatic, scansorial, and arboreal forms (Burton 1986, Menzies 2006, FK, unpubl. data).Th is range of ecological diversity is greater than that for any other microhylid clade, and is rare within amphibian families generally.Furthermore, many of these adaptive ecotypes have clearly been derived independently, appearing in otherwise unrelated asterophryine lineages (Köhler and Günther 2008).However, the exact number of independent origins of each ecotype has yet to be determined, and our understanding of asterophryine morphological diversity continues to expand and evolve.
During the course of conducting biotic surveys in Papua New Guinea I discovered two new species of a diminutive, morphologically unique asterophryine frog that expand the ecomorphological range already known for the subfamily.Closer examination indicates that these species represent a lineage showing a combination of osteological and myological attributes that do not allow them to be accomodated within any existing genus.I take this opportunity to place these frogs in a new genus and describe the constituent species.

Materials and methods
All measurements were made with digital calipers or an optical micrometer to the nearest 0.1 mm, with the exception that disc widths were measured to the nearest 0.01 mm.Measurements, terminology, and abbreviations follow Zweifel (1985) and Kraus and Allison (2006): body length from snout−vent (SV); tibia length from heel to outer surface of fl exed knee (TL); horizontal diameter of eye (EY); distance from anterior corner of eye to center of naris (EN); internarial distance, between centers of external nares (IN); distance from anterior corner of eye to tip of snout (SN); head width at widest point, typically at the level of the tympana (HW); head length, from tip of snout to posterior margin of tympanum (HL); horizontal tympanum diameter (TY); width of the third fi nger disc (3 rd F); and width of the fourth toe disc (4 th T).I confi rmed skeletal and musculature features by dissection, X-ray, and double clearing and staining of select specimens; I follow the terminology of Fabrezi and Alberch (1996) for the carpals.Type specimens are deposited in the Bernice P. Bishop Museum, Honolulu (BPBM).All latitude and longitude coordinates use the Australian Geodetic Datum, 1966 (AGD 66).
Comparisons with other genera.Th e new genus diff ers from all known genera within the Asterophryinae in having the phalanges on the fi rst digits of each hand and foot reduced to tiny cartilaginous elements, imparting to these digits the external appearance of a rudimentary nub (Fig. 4D, E, 5D, E), and in having the prepollex and prehallux reduced to single elements.Its diminutive size also distinguishes it from the vast majority of other asterophryines: although a few small species of Albericus, Aphantophryne, Choerophryne, Cophixalus, and Oreophryne approach this small a size, only one species of Oreophryne attains it.Paedophryne further diff ers from all other asterophryine genera except Aphantophryne, some Albericus (Menzies 1997), and occasional aberrant individuals of Cophixalus (Zweifel & Parker 1989) in having seven (instead of eight) presacral vertebrae; and the length and narrowness of the straplike tongue is only approximated in some other very small species of Cophixalus and some Oreophryne.
Th e small species of Albericus, Choerophryne, and Oreophryne are most readily distinguished from Paedophryne by having expanded digital discs; as well, the last-named retains clavicles and a procoracoid cartilage.Th e new genus is superfi cially most similar to Aphantophryne (in vertebral number, short legs, and having the M. depressor mandibulae overlying the posterior margin of tympanum, Fig. 3G) and to smaller species of Cophixalus (in general appearance and tendency toward digital reduction in some species).In addition to the characters noted above, Paedophryne diff ers from Aphantophryne in its more gracile habitus (squat in Aphant ophryne); in having the tips of the digits fl attened (rounded in Aphantophryne); in having a long, straplike tongue (ovoid in Aphantophryne); in its more widely expanded sacral diapophyses; in lacking neural crests on the presacral vertebrae (present in Aphantophryne); and in having the M. adductor mandibularis anterior longus small and inserting only on lateral portions of frontoparietals (M.adductor mandibularis anterior longus hypertrophied and inserting on the medial portions of frontoparietals in Aphantophryne, Fig. 3B, Table 1).Paedophryne further diff ers from all Cophixalus species in lacking neural crests on the presacral vertebrae (present in Cophixalus), diff ers from all Cophixalus species except C. sphagnicola Zweifel & Allison in having the M. submentalis hypertrophied (M.submentalis a small strap in Cophixalus, Fig. 3C), and diff ers from all Cophixalus except C. cryptotympanum Zweifel and C. kaindiensis Zweifel in having the M. depressor mandibulae overlying the posterior margin of the tympanum (M.depressor mandibulae runs entirely behind posterior margin of tympanum in all other Cophixalus, Fig. 3D, Table 1).
Distribution.Known from one mountain in the southeastern tip of New Guinea and from one mountain on nearby Fergusson Island, D'Entrecasteaux Islands (Fig. 6).

Paedophryne kathismaphlox
Comparisons with other species.Th e new species diff ers from other asterophryines in the characters that typify the genus and in its unique burnt-orange rump patch.
In preservative, dorsum medium brown, irregularly and vaguely marked with darker brown, but pattern indistinct (Fig. 4A); darker markings somewhat more obvious on limbs.Face and behind eye brown spotted with light straw brown (Fig. 4C).Venter and under legs dark brown with scattered light straw-brown fl ecks; rear of thighs same (Fig. 4B).Dirty gray-white patch below anus and proximal portion of thighs (Fig. 4B).Iris black.Variation.Th ere is little mensural variation in the small type series (Table 2); the same is true for color pattern.Th e holotype is marginally lighter in ground color than the three paratypes; two paratypes have a vague, lighter brown interorbital bar; and the light ventral fl ecks in the type series vary from minute to small in size.Th e skin of the three paratypes is more rugose than seen in the holotype, with each animal appearing rather warty in preservative.Each of these three also clearly had warty or rugose skin in life (Fig. 7).
Seven presacral vertebrae relatively stout, with length approximately one-third to onehalf width; longer anteriorly, progressively narrowing posteriorly; all but the fi rst with transverse processes, these longer anteriorly, progressively decreasing posteriorly, most with chondrifi ed tips (Fig. 2B).Neural crests absent.Sacrum with widely expanded diapophyses.

A B
Hyoid plate cartilage, with slight mineralization anteromedially and posteriorly, and with recurved anterolateral processes and thinner posterolateral processes; hyales recurved, chondrifi ed; posteromedial processes ossifi ed, elongate, slightly curved, chondrifi ed at distal ends; ventromedial portion of hyoid proximal to the posteromedial processes with a pentagonal thickening (Fig. 1B).
Hand with six carpal elements: small distal carpal 2, distal carpals 3-5 fused into a single large element, prepollex reduced to a single element, and a large radiale lying between a small Element Y and moderate-sized ulnare (Fig. 1C).Metacarpals long and relatively wide; phalanx of F1 unossifi ed, reduced to a vestigial spot of cartilage; two phalanges on F2, three on F3, two on F4 (Fig. 1C).Terminal phalanx of F3 with expanded, T-shaped tip; those of F2 and F4 pointed, unexpanded (Fig. 1C).Foot with four tarsal elements, with prehallux reduced to a single element.Metatarsals long and relatively narrower than metacarpals; phalanx of T1 unossifi ed, reduced to a vestigial spot of cartilage; two phalanges on T2 and T5, three on T3, four on T4 (Fig. 1D).Terminal phalanges of T3 and T4 with expanded, T-shaped tips; that of T2 pointed, unexpanded; those of T1 and T5, vestigial and rounded (Fig. 1D).7).Each had a narrow reddish rim around the pupil.
Etymology.Th e name is a noun in apposition derived from the Greek roots "kathisma", meaning "rump", and "phlox", meaning "fl ame".It alludes to the distinctive burnt-orange patch beneath the anus of living animals.
Range.Known only from the type locality on the N side of Mt.Simpson, Milne Bay Province, Papua New Guinea (Fig. 6, fi lled circle).
Ecological notes.Animals were collected during the day from wet Dicranopteris linearis leaf litter at a former hunting camp cleared from D. linearis scrub at an ecotone between montane forest and D. linearis shrubland.Th e area appeared to be a former  5C), medium-sized eye (EY/SV = 0.13, EY/SN = 1.07,Fig. 5C), dorsal pattern of a pair of obscure dark-brown chevrons on a lighter-brown ground (Fig. 5A), and ventral pattern of scattered light-gray spots on a dark-brown ground (Fig. 5B).
Dorsum brown with obscure, darker brown chevron markings, one in the scapular region, the other in the lumbar region (Fig. 5a).Face and behind eye dark brown with few distinct light-gray spots (Fig. 5C).Venter and under legs dark brown with scattered, moderately large light-gray spots (Fig. 5B).Rear of thighs brown.Iris black.Color in life."Dorsum light gray brown with two obscure black chevrons on back -one above shoulders, one above groin -and a black mark between the eyes.Forelimbs reddish brown.Entire ventral side black with light-gray fl ecks."

Measurements (in mm)
Etymology.Th e species is named for its sole known location of occurrence on the highest mountain on Fergusson Island.
Range.Known only from Oya Tabu (Mt.Kilkerran), Milne Bay Province, Papua New Guinea (Fig. 6, star).Ecological notes.Th e sole specimen was retrieved by a native collector under a log during the day in mid-elevation (1400 m) montane forest.Th e area was suff ering drought due to an El Niño, so additional frogs or ecological information could not be obtained.

Discussion
Th e most immediately striking feature of Paedophryne is its diminutive size.Lehr and Colima (2008) provided a summary of body sizes seen in what they claimed to be the 40 smallest species of frogs then known.Of these, only two were of smaller average size than P. kathismaphlox.Similarly, only fi ve of their listed species may prove smaller than P. oyatabu, although the paucity of specimens for this latter species makes comparison less certain.However, Lehr and Colima's (2008) brief review overlooked fi ve species of equally small-sized asterophryines in the genera Aphantophryne, Choerophryne, Cophixalus, and Oreophryne (Zweifel and Parker 1989, Richards and Iskandar 2000, Richards and Burton 2003, Kraus and Allison 2006, Richards et al. 2007), and four additional diminutive species in Austrochaperina, Choerophryne and Cophixalus have been described since (Günther 2008, 2009b, Kraus and Allison 2009).Of these nine, only Oreophryne minuta Richards & Iskandar is as small as the two Paedophryne species, with males of the former ranging from 9.2-11.5 mm SV.All these species, as well as most others mentioned by Lehr and Colima (2008), are diminutive outliers in genera having larger average body sizes.Paedophryne may be an exception to this pattern.It clearly constitutes one of the most miniaturized frog clades in the world, yet close relatives of larger size are not apparent (see below).
Aside from overall body size, this new genus expands the range of morphological variation within asterophryine microhylids in two other dimensions.First, it is the only asterophryine to have such an extreme reduction in skeletal elements in the hands and feet.Members of Aphantophryne, Choerophryne, Cophixalus, and Copiula examined by me (Appendix I, II) typically have phalangeal formulae of 2-2-3-3 (but may have 1-2-4-2, 2-2-4-2, or 2-2-3-4) for hands and 2-2-3-4-3 or 2-2-3-4-2 for feet, whereas Paedophryne has 1-2-3-2 on hands and 1-2-3-4-2 on feet.Th is marks a net loss of 1-3 phalanges on each hand and 2-4 phalanges on each foot vs. the conditions seen in other small asterophryines.But phalangeal reduction is not limited solely to number: in Paedophryne the phalanges that remain on F1 and T1 are vestigial and chondrifi ed, and the second phalanx of T5 is also vestigial (Fig. 1) relative to those seen in other asterophryines.Lastly, all related asterophryines examined by me (Appendix I, II) have the prepollex and prehallux each consisting of two or three elements, whereas Paedophryne has only a single element for each, which is uncommon in frogs generally (Fabrezi 2001).Losses in these bony elements produce a unique pattern of reduced digital development that goes considerably beyond what is known in other small asterophryines.Th is presumably limits the fl exibility of the hands and feet and, hence, the degree to which Paedophryne species can grasp and climb.
Secondly, seven presacral vertebrae, instead of eight, appear rarely within Asterophryinae, being diagnostic for Aphantophryne (Zweifel and Parker 1989) but also present as individual variants in some Cophixalus and Albericus (Zweifel andParker 1989, Menzies 1997).Th e shared state between Paedophryne and Aphantophryne is likely to have been independently acquired inasmuch as the vertebrae of the two genera diff er in other features: Aphantophryne has well-developed neural crests while Paedophryne lacks them, and the sacral diapophyses are far more expanded in Paedophryne than they are in Aphantophryne.What, if any, functional consequences attend reduced vertebral number for these frogs remain to be ascertained, but the presence of variation in some other asterophryine species makes the subject liable to direct investigation.
Given the morphological uniqueness exhibited by Paedophryne it is not immediately certain wherein its relationships might lie.As noted earlier, it is superfi cially most similar to several species of small, semi-fossorial or terrestrial species of Cophixalus in general appearance and in having indications of digital reduction, such as reduced discs or reduced-sized digits (Kraus and Allison 2000, 2009, Günther 2006).However, this digital reduction in Cophixalus is not accompanied by similar loss of bony elements (see, e.g., Günther 2006), and the two genera diff er in several other features (Table 1), so it seems unlikely that Paedophryne represents merely an extreme form of Cophixalus.Further, Cophixalus has historically been a dumping ground for asterophryines having an eleutherognatine jaw but lacking clavicles, procoracoid cartilages, and omosternum (Parker 1934).In recent decades, the genera Albericus, Aphantophryne, and Copiula have been removed from this genus (Menzies and Tyler 1977, Zweifel and Parker 1989, Burton and Zweifel 1995), and the morphological diversity still remaining within Cophixalus makes its monophyly questionable.Until such monophyly is demonstrated, it is unclear what a "close relationship" between Paedophryne and a paraphyletic Cophixalus would signify.None of the other asterophryine genera of small size appears particularly similar to Paedophryne: Albericus is arboreal or scansorial and has expanded digital discs; the scansorial or terrestrial Choerophryne has expanded digital discs and a long snout involving horizontal orientation of elongated alary processes of the premaxillae; the terrestrial Copiula has expanded digital discs and a serous rostral gland; Aphantophryne is squat with rounded digits and exhibits several other diff erences from Paedophryne (Table 1); and Austrochaperina and Oxydactyla have clavicles, procoracoids, and an omosternum.None seems a likely candidate for sister-taxon to Paedophryne, and resolution of this question will require a comprehensive assessment of asterophryine relationships.
What accounts for the extremely small size of Paedophryne?As the name suggests, Paedophryne has several features that suggest morphological juvenilization, or paedomorphosis.Among these are the loss of bony elements in the hands and feet, chondrifi cation of many of the cranial elements, overall size miniaturization, and reduction in vertebral number.Each of these is suggestive of curtailed development relative to other asterophryines via early truncation of either osteosis or somitogenesis.Of course, to demonstrate whether this is true and which particular heterochronic mechanism might be involved requires interpretation from a resolved cladogram of asterophryine rela-tionships (Alberch et al. 1979).Th is is currently lacking, so at present paedomorphosis can only be presumed as likely.Should paedomorphic evolution in Paedophryne prove true, as it has for other miniaturized frogs (e.g., Trueb andAlberch 1985, Yeh 2002), similar loss of bony elements in diminutive species of Cophixalus and in Aphantophryne may refl ect independent acquisition in each lineage via paedomorphosis.Th is may be a consequence of shared lifestyles: each lineage consists of small frogs with relatively short legs and somewhat reduced digits that live in leaf litter and moss.Hence, it may be that exploitation of those constrictive habitats selects for small size and its attendant vertebral, limb, and phalangeal reduction.Based on present evidence, this lifestyle and some of the associated morphological changes were acquired independently within Asterophryinae at least twice (to wit, in Aphantophryne and Cophixalus, Köhler and Günther 2008), and possible more often.Clarifying this exact number will depend on determining the phylogenetic position of Paedophryne and additional miniaturized asterophryines in densely sampled asterophryine phylogenetic investigations.
Th e biogeographic origins of Paedophryne also remain uncertain.New Guinea has been formed over the past 60 million years by the sequential accretion of at least four prior island-arc systems or composite off shore terranes (Pigram and Davies 1987, Davies et al. 1996, 1997).As a result, New Guinea is composed of several dozen geological terranes many with emergent off shore origins and these can be grouped into fi ve major geological regions that refl ect diff erent periods of accretion history.Relevant to understanding the history of Paedophryne is that the genus is currently known only from the southeastern end of one of these major geological regions: the East Papuan Composite Terrane (EPCT), which comprises the present-day Papuan Peninsula and its immediately adjacent islands.Th is composite was assembled offshore from a variety of terranes and later sutured in toto onto the New Guinea mainland at about 23-29 MY (Davies et al. 1996(Davies et al. , 1997)).Should additional search prove Paedophryne to be restricted to the EPCT, it may suggest that this lineage arose during off shore construction of the EPCT, prior to its docking with the remainder of New Guinea.If true, this could indicate that the Asterophryinae is either older than the estimated date of ~30 MY provided by van Bocxlaer et al. (2006) and Roelants et al. (2007), that Paedophryne is one of the earlier lineages of Asterophryinae, or both.Long evolutionary isolation may help explain some of the morphological oddities of the genus as well as present uncertainty about its closest relatives.However, the small size and inconspicuous habits of these frogs have delayed their discovery until the present time, and it may be that the genus is more widespread in New Guinea than currently appreciated.If true, the biogeographic origins of Paedophryne would then presumably postdate the origin of the EPCT.specimen processing and documentation; and Shep Myers for preparing the fi gures.I thank Fred Malesa, Dolasi Salepuna, Jeff rey Tekwae, Brian Uruwa, Dage, Genta Sr., Genta Jr., Munda, Peter, Tanunu, and many other inhabitants of the Bunisi area for fi eld assistance; and Aussiya, Bendo Salepuna, Helen Kurage, Dyson Libai, David Mitchell, Bena Seta and the people of Basima, Bunisi, and Ulua for providing logistical assistance and/or permission to work in the Mt.

Figure 1 .Figure 2 .
Figure 1.A Pectoral girdle B hyoid C ventral view of bones of right hand, and D dorsal view of bones of right foot of Paedophryne kathismaphlox (BPBM 35353).

Figure 4 .
Figure 4.A Dorsum B ventrum C side of head D palmar view of left hand, and E plantar view of left foot of holotype of Paedophryne kathismaphlox (BPBM 17977).

Figure 5 .
Figure 5.A Dorsum B ventrum C side of head D palmar view of left hand, and E plantar view of right foot of holotype of Paedophryne oyatabu (BPBM 16433).
Simpson and Oyatabu areas.I thank the PNG National Museum and Art Gallery for providing in-country collaborative assistance and the Department of Environment and Conservation, National Research Institute, and Milne Bay Provincial Government for permission to conduct this research.Th is research was supported by National Science Foundation grants DEB-0103794 and DEB-0743890.Th is is contribution 2010-009 from the Pacifi c Biological Survey at the Bishop Museum.Appendix I Cleared-and-stained specimens examined for musculature and skeletal attributes Aphantophryne pansa Fry: Papua New Guinea: Morobe Province: Bulldog Road, 15.5 km.SSW of Wau, 7.477355°S, 146.6757246°E, 2800 m (BPBM 25278, 25286).Cophixalus balbus Günther: Papua New Guinea: West Sepik Province: 3.2 km SSE Mt.Sapau summit, Torricelli Mts., 550 m (BPBM 22701).Cophixalus cheesmanae Parker: Papua New Guinea: Morobe Province: 5.3-5.6 km NW summit Mt Shungol, 750-780 m (BPBM 18411).Cophixalus cryptotympanum Zweifel: Papua New Guinea: Morobe Province, Mt.Kaindi summit, 2360 m (BPBM 9532

Table 1 .
Features diagnosing Paedophryne gen.n. from its most similar-appearing relatives.Features given for Cophixalus exclude mention of uncommon variants discussed in the text.

Table 2 .
Mensural data for type series of Paedophryne kathismaphlox sp.n.