Two new endemic genera and a new species of toad (Anura: Bufonidae) from the Western Ghats of India

Background Bufonidae are a large family of toads with a subcosmopolitan distribution. Recent molecular phylogenetic analyses have revealed a radiation of toads (Adenominae) with distinct adult and larval ecomorphs on the Southern parts of the Indian subcontinent. The Indian torrential species "Ansonia" ornata has a basal position in this clade and does not group with South-East Asian Ansonia. Additionally, the nested position of "Bufo" koynayensis and an undescribed sister species, and their distinct ecologies including a non-typical egg-laying strategy within bufonids, support the recognition of a second distinct genus. In this paper we describe two new genera and one new species from the Adenominae clade. Findings Ansonia ornata Günther, 1876 "1875" is transferred to Ghatophryne gen. nov., a genus of torrentially adapted toads that are endemic to the Western Ghats of India. On the basis of close morphological resemblance and distribution, Ansonia rubigina Pillai and Pattabiraman, 1981 is provisionally transferred to this new genus. The Western Ghats endemic toad Bufo koynayensis Soman, 1963 is transferred to a new genus Xanthophryne gen. nov. Based on molecular and morphological evidence, we additionally describe a new species, Xanthophryne tigerinus sp. nov., from Amboli in the Western Ghats. Conclusion The descriptions and subsequent taxonomic changes we propose result in three genera of bufonids recognised as being endemic to the Western Ghats (Ghatophryne gen. nov., Xanthophryne gen. nov. and Pedostibes), and one to Sri Lanka (Adenomus). The spatial distribution, and arrangement of these lineages at the base of Adenominae diversification, reflects their Early Neogene isolation in the Western Ghats-Sri Lanka hotspot.


Introduction
Bufonidae are a family of toads with over 500 extant species distributed on most continents. Current opinions on bufonid taxonomy are very divergent, and range from the recognition of multiple genera [1], to favouring a subcosmopolitan genus Bufo with plenty of subgenera [2]. Although the phylogenetic relationships of bufonids have been studied intensively [1,3,4], the evolutionary position of species on the Indian subcontinent had remained unclear. However, recent molecular phylogenetic analyses revealed that these toads belong to a radiation containing distinct ecomorphs in adult and/or larval forms [5]. The early diversification of this clade, for which the name Adenominae is available [6], has been reconstructed as occurring on the Southern parts of the Indian subcontinent. Early diversification of Adenominae, timed at the early Miocene, led to several endemic lineages ("Ansonia" ornata, Pedostibes tuberculosus, the "Bufo" koynayensis group and Adenomus) [5] and to the origin of the more widespread Duttaphrynus clade. The unexpected evolutionary position of "Ansonia" ornata and "Bufo" koynayensis (see abstract) requires recognition of two new genera to accommodate these Western Ghats endemic species and their relatives.

Methods
Collection of amphibian specimens was made during fieldtrips in the Western Ghats between 1997 and 2002. Specimens were preserved in 5% formaldehyde for 2 days, and subsequently transferred to 70% ethanol. Measurements were taken to the nearest 0.1 mm, using a digital slide-caliper or a binocular microscope with a micrometer ocular. The description (all measurements in mm) of the types follows terminology used elsewhere [7]. To comply with regulations of the International Code of Zoological Nomenclature we have deposited copies of this article at the following publicly accessible libraries: Bombay Natural History Society, Mumbai, India (BNHS); Koninklijk Belgisch Instituut voor Natuurwetenschappen, Brussels, Belgium (KBIN); Natural History Museum, London, UK (BMNH); American Museum of Natural History, New York, USA (AMNH); Museum National d'Histoire Naturelle, Paris, France (MNHN); Russian Academy of Sciences, Moscow, Russia (RAS).

Ghatophryne gen. nov. (Figure 1) Etymology
The generic epithet is derived from the words 'Ghats' and 'phryne'. The former is the Sanskrit word for 'steps' and refers to the Western Ghats mountain range, where this genus is endemic, while 'phryne' means toad in Greek.

Diagnosis and comparison
Ghatophryne can be distinguished from other bufonid genera by the combination of the following characters: small-sized adults (male SVL 26.9-30.1, N = 5; female 35.0, N = 1); dorsum reddish brown, ventrum dark brownish black with prominent yellowish-orange spots; head without cranial ridge, lack of parotoid glands; fingers free, toes medium webbed; skin on dorsum with sparsely granular projections especially on anterior half of the body; eggs non-pigmented, tadpoles with a suctorial disk, adapted to mountain streams throughout the life cycle (SD Biju, personal observations). Ghatophryne can be characterized in a phylogenetic framework as the most inclusive clade that contains Ansonia ornata Günther, "1876" 1875, but not Bufo melanostictus Schneider, 1799 and Ansonia hanitschi Inger, 1960 [5].
Ghatophryne is morphologically very similar to species of Ansonia, but is endemic to the southern part of the Western Ghats, while Ansonia is known only from South-East Asia (Sumatra, Borneo and Philippines).

Distribution
Ghatophryne has only been reported from Kerala and Karnataka in the Western Ghats of India.
Colour of lectotype: in preservation dorsally uniform light greyish brown with a dorsal light yellowish brown patch; ventral side light brown with white irregular spots.

Diagnosis and comparison
Xanthophryne can be distinguished from other bufonid genera by the combination of the following characters: small-sized adults (male SVL 26.5-32.9, N = 12; female SVL 33.3-35.3, N = 3) having light brown dorsum with a suffusion of dull chrome-yellow; head with discontinuous and weak canthal and preorbital ridges on the anterior part, flanks and sides of the abdomen have chromeyellow patches, or sometimes 2-4 continuous bands; tympanum indistinct, rather weak parotoid glands; toes and fingers without webbing, tips rounded; eggs in clutches. Xanthophryne can be characterized in a phylogenetic framework as the most inclusive clade that contains Bufo koynayensis Soman, 1963 but not Bufo melanostictus Schneider, 1799 and Bufo kelaartii Günther, 1858.

Distribution
Xanthophryne has only been reported from the northern part of the Western Ghats of India.

Description of the holotype
Color: in preservation: dorsum light-brown, loreal and tympanic region light brown, lateral region dark grey with light colour stripes; fingers I and II, and toes I and II creamy white; ventral side light brownish grey with dark grey irregular spots; in life: dorsum golden yellow, lateral region brown with light yellow stripes extending from dorsum; fingers I and II, and toes I and II whitish; ventral side light grey with dark greyish brown irregular spots, lower jaw margins white.

Variation
Measurements of eight type specimens are given in Table  1. During the breeding season, both male and female toads have a bright yellow dorsum with light yellow and light blue lateral stripes. Outside the breeding season, they have a light brownish grey dorsum with light yellow lateral stripes.

Distribution and natural history
Xanthophryne tigerinus sp. nov. is known only from the type locality Amboli. The type series was collected from the ground near disturbed evergreen forest patches during a rainy evening after 19:00 h. Amplexus is axillary. Egg clutches contain 30-35 eggs per clutch (N = 4; VG, per-sonal observation). The eggs are laid in temporary puddles on laterite rocks (Figure 4).

Notes
Bufo koynayensis Soman, 1963 was described twice based on the same type series but bearing different numbers. Soman first described Bufo koynayensis, without designating any type other than mentioning, "was collected in good numbers" [9]. Almost 40 years later, specimen ZSI A.1784 was recognized as the holotype of Bufo koynayensis Soman, 1963 [10]. This specimen was examined and is badly damaged (SDB, personal observation). The samples collected by Soman were utilized to describe Bufo sulphureus (holotype, BNHS 377 and 26 paratypes including 5 males, 11 females and 10 juveniles) [11], which as a consequence is a junior synonym of Bufo koynayensis. As described in the original description, the type series of Bufo sulphureus were deposited in the British Museum of Natural History, the Indian Museum and the Bombay Natural History Society. In this study, we examined the holotype and 18 paratypes (14 adult males -SVL 24.0-31.8 mm and 4 subadults -SVL 20.9-25.0 mm) deposited in BNHS and five paratypes in BMNH under the nomen 'Bufo sulphureus'. In addition, recent collections of 'Bufo' koynayensis from the type locality were compared. All available material of this species is consistent with the original description.

Conclusion
Ghatophryne gen. nov. and Xanthophryne gen. nov. are both endemic to the Western Ghats of India. Together with the arboreal toad genus Pedostibes (Western Ghats endemic; species from South-East Asia belong to another clade) and the genus Adenomus (Sri Lanka endemic), they form the oldest lineages in the Adenominae clade. The recognition of each of these lineages as distinct genera highlights their early diversification on the southern parts of the Indian subcontinent [5].