A new ancient lineage of frog (Anura: Nyctibatrachidae: Astrobatrachinae subfam. nov.) endemic to the Western Ghats of Peninsular India Author Vijayakumar, Seenapuram Palaniswamy Author Pyron, Robert Alexander Author Dinesh, K. P. Author Torsekar, Varun R. Author Srikanthan, Achyuthan N. Author Swamy, Priyanka Author Stanley, Edward L. Author Blackburn, David C. Author Shanker, Kartik text PeerJ 2019 2019-03-12 6457 1 28 journal article 10.7717/peerj.6457 f324a471-6180-4bc0-9dcd-b9c2a3b07ca6 PMC6419720 30881763 2608984 Type species. — Astrobatrachus kurichiyana sp. nov. urn:lsid:zoobank.org:act: A4A21F51-2A11-41C6-AA2A-B6F390070868, by present designation Holotype . — ZSI / WRC/A/2131 ( male , Fig. 3C– E ) by present designation, collected by S.P. Vijayakumar , K.P. Dinesh , and a team from Kurichiyarmala ( 11.602807 ° N 75.968025 ° E , alt. 1420 m ), Wayanad Plateau, WG during an expedition on 21st June 2010 . Paratypes . — ZSI / WRC/A/2132 ( CESF 1565 ) ( male ), and ZSI / WRC/A/2133 ( CESF 2897 ) ( female ); site same as holotype collection. Diagnosis. —This diagnosis applies to the subfamily, genus, and species. The following combination of characters can be used to diagnose this lineage from its close relatives Nyctibatrachus and Lankanectes : small to medium size (∼ 20–27 mm SVL); soft skin without ridged or wrinkled folds; fingers and toe tips with discs that are triangular in shape ( Figs. 3 and 4 ) without circummarginal groove; upper jaws having distinct teeth; distinct and angular canthus rostralis; distinct tympanum with a prominent supra-tympanic ridge ( Fig. 3 ); tongue lacking median papilla; short hind and fore-limbs; oblong subarticular tubercles on the fingers and toes that sometimes nearly coalesce (e.g., pedal digit III in Figs. 3 and 4 ); interdigital webbing on foot does not attain most proximal subarticular tubercle; absence of femoral glands; absence of nuptial pads in males; widely spaced nasal bones; a vomer separated into an anterior portion adjacent to the choana and a posterior dentigerous vomer fused to a neopalatine; omosternum not bifurcating posteriorly; a single narrow sternal element; lacking a large dorsal crest on the ilium; bluish-white spots ( Figs. 3 and 4 ), more prominent and scattered along the lateral sides of jaws, eyelids, belly, forearms and hind limbs, and on the throat; oval-shaped pupil; orange coloration of ventral sides of belly, forelimbs and hind limbs; elliptical pupil ( Fig. 3 ). The lineage is diagnosed easily in the field from species of Nyctibatrachus that occur sympatrically. Description of holotype ZSI/WRC/A/2131 (male) A small-sized frog (SVL ¼ 20.2 mm) with a squat, lean body (see Table 2 ); head length approximately equal to head width (HL ¼ 7.2 mm; HW ¼ 7.3 mm); snout acutely pointed in dorsal and obtusely pointed in ventral views; snout length (SL ¼ 3.0 mm) approximately equal to eye diameter (EL ¼ 2.9 mm); canthus rostralis angular, loreal region concave; inter orbital space flat and wide (IUE ¼ 2.8 mm), 2.3 times more than upper eye lid (UEW ¼ 1.2 mm) and subequal to inter-narial distance (IN ¼ 2.6 mm); distance between back of eyes two times more than front of eyes (IFE ¼ 3.8 mm; IBE ¼ 7.8 mm); nostrils oval, nearer to tip of snout and lateral in position; symphysial knob weak; tympanum distinct visible below supratympanic fold (TYD ¼ 1.2 mm); weak vomerine ridges present with three to four teeth; tongue bifid without papilla; lower jaw without teeth, upper jaw with distinct sharp teeth. Forearm slender (FLL ¼ 5.8 mm) longer than hand (HAL ¼ 4.0 mm); fingers short and thin without dermal fringes; relative finger length IV <I <II <III (FL1 ¼ 2.3 mm, FL2 ¼ 2.8 mm, FL3 ¼ 3.4 mm, FL4 ¼ 2.0 mm); finger tips triangular and blunt without circummarginal groove; webbing between fingers rudimentary; subarticular tubercles of fingers oblong with indistinct margins; pollex tubercle absent; supernumerary tubercles absent. Hindlimbs short, just touching when adpressed, and tibio-tarsal articulation reaches back of eyes; thigh length approximately equal to crus length (TL ¼ 9.3 mm; CL ¼ 9.0 mm); foot length is greater than tarsus length (FOL ¼ 9.3 mm, TAL ¼ 6.8 mm), relative toe length I <II <V <III <IV (TL4 ¼ 4.8 mm; ITL ¼ 2.0 mm); webbing rudimentary (not reaching the most proximal subarticular tubercle); inner metatarsal tubercle (IMTL ¼ 1.0) small and nearly continuous with subarticular tubercle of first Figure 3 Live images of Astrobatrachus kurichiyana . Profile (A), close-up of head (B), ventral (C), dorsal (D), side-profile (E). Photo Credit: S.P. Vijayakumar (A and B; reference collection CESF 1567), K.P. Dinesh (C, D and E; ZSI/WRC/A/2131) Full-size DOI: 10.7717/peerj.6457/fig-3 Figure 4 Paratype of Astrobatrachus kurichiyana - ZSI/WRC/A/2133 (female): Ventral head (A), dorsal head (B), ventral (C), ventral fingers (D), lateral head (E), ventral toes (F). Drawing Credit: Achyuthan N. Srikanthan Full-size DOI: 10.7717/peerj.6457/fig-4 toe; subarticular tubercles of toes oblong and indistinct, coalescing on some toes; outer metatarsal tubercle absent and supernumerary tubercles absent. Overall skin on dorsum soft, finely glandular, ventrally smooth glandular. Rictal gland and ocular gland absent. Table 2 Morphometric data for Astrobatrachus kurichiyana .

ZSI/WRC/A/2131 (Holotype)	ZSI/WRC/A/2132 (CESF 1565) (Paratype)	ZSI/WRC/A/2133 (CESF 2897) (Paratype)
Sex	M	M	F
SVL	20.2	27.3	27.2
HW	7.3	9.2	9.4
HL	7.2	9.3	8.9
IN	2.6	3.2	3.2
NE	1.6	2.0	1.8
MN	6.0	7.2	7.2
MFE	4.9	6.1	5.9
MBE	2.2	2.7	2.8
SL	3.0	4.2	4.0
EL	2.9	4.0	4.0
IUE	2.8	3.7	3.8
UEW	1.2	2.0	1.8
IFE	3.8	5.0	4.8
IBE	7.8	8.6	8.4
TYD	1.2	1.8	1.8
TE	1.0	1.4	1.4
FLL	5.8	6.8	6.6
HAL	4.0	5.5	5.1
FL1	2.3	2.8	2.8
FL2	2.8	3.1	3.2
FL3	3.4	3.8	3.8
FL4	2.0	2.1	2.1
TL	9.3	12.2	12.2
CL	9.0	12.0	12.0
TAL	6.8	8.2	8.2
FOL	9.3	11.0	10.8
TL4	4.8	6.4	6.2
ITL	2.0	2.6	2.6
IMT	1.0	1.4	1.2

Coloration of holotype In life, overall color on dorsum rufous brown, darker in anterior region of body and light in posterior part of body. Small pale bluish-white spots present all over body but larger and more concentrated on lateral, gular, anterior ventrum, and around tympanic region, jaws, and eyelid. In ventral region, arm, belly, thigh, and crus bright orange in color; gular region is dark gray with bluish white spots. Under surface of hand and foot dark gray. In preservative, color on dorsum dark brown with cream spots. Gular and anterior ventrum dark gray interspersed with cream spots, with melanocytes becoming widely spaced posteriorly; arm, posterior belly, thigh, and crus cream colored; hand and foot dark gray with pale gray skin under digit tips and subarticular tubercles. Figure 5 Female of Astrobatrachus kurichiyana (reference collection CESF 2898). Female of Astrobatrachus kurichiyana (CESF 2898) as visualized via microCT-scanning with skull in dorsal (A), right lateral (B), and ventral (C) views, entire skeleton in dorsal view (D), right hand in dorsal view (E), pectoral girdle in ventral view (F), and pelvis in right lateral view (G). Note that distal extent of the fourth and fifth toes on both feet were truncated during CT-scanning. Abbreviations of anatomical terms are as follows: av, anterior vomer; clav, clavicle; cor, coracoid; np, neopalatine; omo, omosternum; pro, prootic; prsph, parasphenoid; pv, posterior vomer; scap, scapula; sph, sphenethmoid; sq, squamosal; xiph, xiphisternum. Full-size DOI: 10.7717/peerj.6457/fig-5 Osteology Based on micro-CT scan of ZSI/WRC/2132 (paratype; male) and CESF 2898 and 2899 (reference collections, females) ( Figs. 5 and 6 ). The skull is lightly constructed, slightly longer than wide, lacking ornamented dermal bones, and having jaw joints that are anterior to the occiput. The prootics are incompletely ossified and not synostosed to the exoccipital or frontoparietals. The lateral walls of the neurocranium are not ossified such that the optic fenestrae and prootic foramina are not bounded in bone. The frontoparietals are broad, approximately 3/5 the width of the length, and do not articulate at the midline. The nasals are widely spaced. The premaxillae bear teeth, and each has a prominent pars dentalis and a robust alary process that is taller than wide and widely separated from the nasal. The maxillae bear teeth for approximately two-thirds of their length and are nearly straight. The quadratojugals are elongate, extending anterior to the body of the squamosal. The pterygoids are triradiate, each with a long anterior ramus that articulates with the maxilla near the posterior level of the maxillary teeth, a long posterior ramus extending to the jaw joint, and a short and thin medial ramus extending towards but not articulating with the prootic. The anterior portion of the vomer is small and distinct, forming a C-shape around the anteromedial surface of the choanae. This anterior portion is not connected to the dentigerous posterior portion that is synostosed to the neopalatine. The sphenethmoid is located anterior to the rostral extent of the parasphenoid, either abuts or is synostosed to the neopalatine, and has well-ossified tectum and septum nasi that separate prominent circular olfactory foramina. The parasphenoid is triradiate with the rostral extent of the cultriform process being V-shaped and Figure 6 Comparisons of male paratype (ZSI/WRC/2132) of Astrobatrachus kurichiyana to representatives of other WG endemics. Exemplars from Nyctibatrachidae , Micrixalidae , and Ranixalidae in different views ((A–F) entire skeleton in dorsal view; (G–L) dorsal view of skull; (M–R) ventral view of skull; (S–X) ventral view of pectoral girdle). Abbreviations of anatomical terms are as follows: av, anterior vomer; clav, clavicle; np, neopalatine; omo, omosternum; pv, posterior vomer. Full-size DOI: 10.7717/peerj.6457/fig-6 not attaining the sphenethmoids, and the lateral alae underlying the otic capsule. The squamosals are prominent with a zygomatic ramus that tapers rostrally, and a posterior ramus forming a small shelf just lateral to, but not synostosed to, the proootic. A thin distally tapering stapes (or columella) is present, terminating just below the posterior ramus of the squamosal; an ossified operculum is not present. The posteromedial processes of the hyoid are short and weakly expanded at their posterior ends. There are eight distinct, procoelous, non-imbricating presacral vertebrae that are not fused to one another and all of which have a large neural canal. The atlas lacks transverse processes and has widely separated cotyles. The transverse processes of Presacrals II are directly laterally, whereas those of Presacrals III–VII are directed posteriorly. Presacral VIII has weakly anteriorly directed transverse processes and a biconvex centrum, making the vertebral column displasiocoelous. The sacrum is procoelous with short transverse processes and has two condyles posteriorly. The urostyle bears two cotyles, which are not confluent, for articulation with the urostyle and a tall dorsal ridge that tapers posteriorly, ending approximately at the anteroposterior midpoint. The pectoral girdle is firmisternal and no elements are in direct articulation or synostosed. The scapula has a defined waist, expanding dorsally, and has an expanded and rounded acromial process. The clavicle is thin and nearly perfectly straight. The coracoid is widely expanded medially, with the medial extent approximately twice as wide as the lateral extent. A small omosternum is expanded posteriorly and tapers anteriorly. A stout and nearly rectangular bony xiphisternum projects distally from the coracoids. In CESF 2898, the three elements of the pelvic girdle—ilium, ischium, and pubis— are ossified and synostosed to one another. The shaft of the ilium is long and bears a weakly developed supraacetabular fossa, a well-defined dorsal protuberance, and a short dorsal crest for most of its length. The synostosis of the pelvic elements creates a broad sheet of bone posterior, ventral, and anterior to the acetabulum. In CESF 1565, the ventral acetabular expansion projects anteriorly, but not ventrally below the point at the margin of the acetabulum at which the ilium and pubis meet; the ischium and pubis are not fully ossified in this specimen. The humerus is thin and straight with a weakly developed ventral crest and entepicondyle. The forearm is approximately two-thirds the length of the humerus. The radiale and ulnare are large and elongate, and subequal in size. The distal carpals are fused into two groups: Element Y + distal carpal 2, and distal carpals 3+4+5. The phalangeal formula for the manus is 2–2–3–3 and there are two small ossified elements of the prepollex that are more ossified and discernable in the larger specimens (CESF 2898 and 2899). The terminal manual phalanges are blunt distally, and in some cases tapering. The tibiofibula and femur are similar in length. There is a tarsal sesamoid on the ventral surface at the articulation of the tibiofibula and the tibiale and fibulare. There are two distal tarsals, two small ossified elements in the prehallux, and a sesamoid on the plantar surface at the base of metatarsals IV and V. The phalangeal formula for the pes is 2–2–3–4–3. The terminal pedal phalanges taper and then expand into a small blunt tip. Comparisons Astrobatrachus is clearly distinguishable from other members of the Nyctibatrachidae as well as other families endemic to the WG ( Table 3 ). No information is available on the larval biology or reproductive modes of Astrobatrachus . Astrobatrachus differs from Lankanectes in lacking dorsal skin ridges, lacking a lateral line system as an adult, having a distinct tympanum and canthus rostralis, lacking odontoid fangs, having widely spaced nasals, having a long ridge of vomerine teeth, having separate anterior and posterior portions of the vomer with the latter fused to the neopalatine, lacking a forked omosternum, lacking a prominent dorsal crest on the ilium, and lacking both a tarsal fold and webbed pedal phalanges ( Dubois & Ohler, 2001 ; Senevirathne et al., 2018 ; Figs. 5 and 6 ). Similar to Lankanectes , Astrobatrachus lacks a median lingual papilla and femoral glands, lacks circummarginal grooves on the digits (present in some Nyctibatrachus ), has a second finger longer than the first finger, and indistinct subarticular tubercles. Table 3 Morphological character

Characters/ species	N. grandis
Size (mm)	62.2–76.9
Skin	Wrinkled with granular projections
Webbing between fingers	Absent
Webbing between toes	Webbed/extends beyond 3rd subarticular
Dorsoterminal groove on fourth toe disc	Present, cover distally
Subarticular tubercles	Prominent, oval,
Toe discs	Well developed
Coloration	Dorsum uniform gray with light patches
Tympanum	Indistinct
Femoral glands	Present
Subocular gland (proposed by Biju et al., 2011 )	Present
Tarsal fold	Tarsal fold present
Black or bluish- black liver— visible on the ventral side through skin in life	Yes
Ridge extending from the lip over the tip of the snout to between the nostrils	Present

Astrobatrachus differs from Nyctibatrachus in lacking rugose or wrinkled skin with longitudinal folds, having a distinct tympanum and canthus rostralis, lacking a subocular gland, having widely spaced nasals, having a long ridge of vomerine teeth, having separate anterior and posterior portions of the vomer with the latter fused to the neopalatine, lacking a forked omosternum, lacking femoral glands, lacking a prominent dorsal crest on the ilium, lacking both a tarsal fold and webbed pedal phalanges, lacking a rhomboidal pupil ( Biju et al., 2011 ; Chandramouli & Dutta, 2015 ). Similar to Nyctibatrachus , Astrobatrachus lacks a median lingual papilla (present in some Nyctibatrachus ), lacks a lateral line system as an adult, and has a second finger longer than the first finger and indistinct subarticular tubercles. The skeleton of Astrobatrachus is also distinguishable from genera of Micrixalidae ( Micrixalus ) and Ranixalidae ( Indirana , Walkerana ). Astrobatrachus differs from Micrixalus by having vomerine teeth and a posterior portion of the vomer, and lacking T- or Y-shaped terminal phalanges ( Dubois, 1986 ), but is similar by having widely spaced nasals, an unforked omosternum, and thin, straight clavicles. Astrobatrachus differs from Indirana by having widely spaced nasals, having a separate posterior vomer fused to the neopalatine, lacking a T- and Y-shaped terminal phalanges, but is similar by having an unforked omosternum, and thin, straight clavicles. Astrobatrachus differs from Walkerana by having widely spaced nasals, a separate posterior vomer fused to the neopalatine, a clavicle (absent in Walkerana ), a well-defined sternum, and lacking Y-shaped terminal phalanges, but is similar by having an unforked omosternum. Habits and habitat The new species is nocturnal and found below decayed leaf litter within montane forests in the vicinity of water. One individual was caught moving in a grassland adjoining the forest tract ( Fig. 7 ). On the forest floor, where most individuals were sampled, they hid under leaf litter when disturbed. Because individuals were secretive and difficult to spot, sampling involved an intensive search of the forest floor. Individuals were found to be shy of torch light and upon disturbance, made quick hopping movements to hide. No individuals were found exposed during the night during either rainy or non-rainy periods. As a general observation, most sympatric anurans in the region usually emerge in the dark and call during the rain or post rain seasons. Leaf-litter dwelling and habitat distinguishes A. kurichiyana from many species of Nyctibatrachus that are torrential frogs and prefer to live in water or next to perennial streams ( Biju et al., 2011 ). While its terrestrial habits are somewhat similar to some small-bodied Nyctibatrachus species (see Garg et al., 2017 ), the new lineage differs strongly from the two Lankanectes species which are aquatic ( Senevirathne et al., 2018 ). Distribution All known populations of this species occur in Kurichiyarmala on the Wayanad Plateau, in the WG Escarpment ( Fig. 1 ). The geographical range of Nyctibatrachinae, widespread across the WG, overlaps with Astrobatrachinae ( Fig. 1 ). However, both lineages have a disjunct distribution with respect to Lankanectinae, which is restricted to the Figure 7 Type locality of Astrobatrachus kurichiyana . Most individuals were sighted in the montane forests except for a single individual in the grassland. Locality: Kurichiyarmala, Wayanad Plateau. Photo taken: June 2010. Photo Credit: S.P. Vijayakumar. Full-size DOI: 10.7717/peerj.6457/fig-7 mountains of Sri Lanka ( Fig. 1 ). The new species occurs in syntopy and in broad sympatry with Nyctibatrachus grandis , N. minimus , N. vrijeuni, and N. kempholeyensis. Etymology From ‘Kurichiyana,’ a local tribal community residing near the type locality and currently known geographic range of the species. Species epithet is treated as a noun in apposition to the generic name. We suggest the common English name of the Starry Dwarf Frog. Conservation status Based on the lack of information on the abundance of the species, and inadequate data on the geographical range of the lineages, using the IUCN Red List criteria, we suggest A. kurichiyana be assigned to the Data Deficient category.