Microhyla laterite sp. nov., A New Species of Microhyla Tschudi, 1838 (Amphibia: Anura: Microhylidae) from a Laterite Rock Formation in South West India

In recent times, several new species of amphibians have been described from India. Many of these discoveries are from biodiversity hotspots or from within protected areas. We undertook amphibian surveys in human dominated landscapes outside of protected areas in south western region of India between years 2013–2015. We encountered a new species of Microhyla which is described here as Microhyla laterite sp. nov. It was delimited using molecular, morphometric and bioacoustics comparisons. Microhyla laterite sp. nov. appears to be restricted to areas of the West coast of India dominated by laterite rock formations. The laterite rock formations date as far back as the Cretaceous-Tertiary boundary and are considered to be wastelands in-spite of their intriguing geological history. We identify knowledge gaps in our understanding of the genus Microhyla from the Indian subcontinent and suggest ways to bridge them.


Introduction
The Indian subcontinent supports a rich biological diversity despite high human population density [1][2][3]. This is attributed to the presence of a varied environment and habitats [1,4], unique biogeographical and geological history [5] and a strong tradition of conservation of nature [1,6]. The biodiversity across the country is not evenly distributed and is mainly concentrated in and around the Eastern Himalaya and the Western Ghats [4,7]. Both these regions are known for a high level of endemism across taxa [8,9].
The Indian subcontinent is one of few regions in the Old World to harbor a rich diversity of amphibians [10]. Currently, there are 391 species recorded from India, predominantly from the Western Ghats and Eastern Himalaya. A total of 220 amphibian species are recorded from the Western Ghats and several more are expected to be described in the near future. The species novelties being described are often from genera that are endemic to the Western Ghats (viz., Beddomixalus, Ghatixalus, Indirana, Mercurana, Micrixalus, Nyctibatrachus, Raorchestes) but such discoveries from widespread genera like Microhyla, Hoplobatrachus, Fejervarya and Euphlyctis are uncommon.
We encountered a species of Microhyla during surveys between years 2013-2015 as part of a citizen science initiative, 'My laterite: My habitat' led by one of the authors (Ramit Singal). This species of Microhyla did not match descriptions of the eight known species from the region. We undertook further studies to determine its identity and here, we report: 1. the description of Microhyla laterite sp. nov., ascertained using molecular (12S and 16S rRNA genes), morphology and bioacoustic comparisons and 2. Assess the threat status of this species using IUCN Red List criteria.

Ethics Statement
Fieldwork and sampling was carried out in Manipal, Udupi District of Karnataka. All specimens were collected with permission from the Principal Chief Conservator of Forests and Chief Wildlife Warden, Karnataka State Forest Department (Permission No. PCCF(WL)/E2/ CR-23/2015-16). Specimen collection and tissue sampling protocol followed guidelines for use of live amphibians and reptiles in field research by the American Society for Ichthyologists and Herpetologists and was approved by the Gubbi Labs Internal Committee on Animal Welfare and Ethics (Approval No. 2015-16/GLICAWE/01). All collections and tissue sampling adhered to the ethical standards put forth by the committee. Minimum samples were collected and used only for scientific work (M. laterite sp. nov: N = 4). Animals were located and gently picked up by hand and placed in moist cotton bags and transported to field station within 30 min. Individuals were then euthanized using 20% Benzocaine gel, a topical anesthetic. A small volume of the gel (< 0.5 cm 3 ) was squeezed out on a swab and applied on the animals' ventral region. After the animal ceased to show any signs of movement, a small portion of thigh muscle tissue (ranging between 0.1 to 0.25 cm 3 ) was extracted for molecular analysis. A sterilized stainless steel scissor and forceps were used to incise tissue. Tissue was stored in molecular grade ethanol. Specimens were later fixed in 4% formalin for 24h and then transferred to 70% alcohol. Our animal handling protocols strictly following the guidelines for euthanasia of amphibians and use of Benzocaine 20% is well known to effectively minimize any pain or distress to the animal.

Nomenclatural Acts
The electronic edition of this article conforms to the requirements of the amended International Code of Zoological Nomenclature, and hence the new names contained herein are available under that Code from the electronic edition of this article. This published work and the nomenclatural acts it contains have been registered in ZooBank, the online registration system for the ICZN. The ZooBank LSIDs (Life Science Identifiers) can be resolved and the associated information viewed through any standard web browser by appending the LSID to the prefix "http://zoobank.org/". The LSID for this publication is: urn:lsid:zoobank.org:pub: BBBDC86C-FE35-4929-A484-62C25C497ACB. The electronic edition of this work was published in a journal with an ISSN, and has been archived and is available from the following digital repositories: PubMed Central, LOCKSS.

Study Area
This study was conducted along the West coast of India (Fig 1). The new species of Microhyla was observed between years 2013-2015 in laterite habitats in and around the coastal town of Manipal, Udupi District, Karnataka State, India (13.2868°-13.3757°N and 74.7795°-74. 8731°E , 50 m amsl). The region receives an annual rainfall of about 4000 mm. These laterite formations are a part of the 'Deccan Traps' flood plain and are believed to have originated sometime during the mid-Tertiary [13]. The overall habitat comprises of grasses, herbs, shrubs and stunted trees interspersed with agricultural fields and houses.   16). Specimen collection and tissue sampling protocol followed guidelines for use of live amphibians and reptiles in field research by the American Society for Ichthyologists and Herpetologists and was approved by the Gubbi Labs Internal Committee on Animal Welfare and Ethics (Approval No. 2015-16/GLICAWE/01). All collections and tissue sampling adhered to the ethical standards put forth by the committee. Minimum samples were collected and used only for scientific work (M. laterite sp. nov: N = 4). Animals were located and gently picked up by hand and placed in moist cotton bags and transported to field station within 30 min. Individuals were then euthanized using 20% Benzocaine gel, a topical anesthetic. A small volume of the gel (< 0.5 cm 3 ) was squeezed out on a swab and applied on the animals' ventral region. After the animal ceased to show any signs of movement, a small portion of thigh muscle tissue (ranging between 0.1 to 0.25 cm 3 ) was extracted for molecular analysis. A sterilized stainless steel scissor and forceps were used to incise tissue. Tissue was stored in molecular grade ethanol. Specimens were later fixed in 4% formalin for 24h and then transferred to 70% alcohol. Our animal handling protocols strictly following the guidelines for euthanasia of amphibians and use of Benzocaine 20% is well known to effectively minimize any pain or distress to the animal.
We photographed the individuals before and after they were euthanized. Four individuals (three males and one female) of the M. laterite sp. nov. were collected on 26 th June 2015 between 19:00-21:00 h by KSS and RS from Manipal.

Molecular Analysis
We followed the protocol by Vences et al. [14] for DNA extraction from thigh muscle tissue. PCR amplification and sequencing of 16S and 12S rRNA genes was carried out following Gururaja et al. [15]. Amplified products were then sequenced at Chromous Biotech, Bangalore, India. The sequences were manually checked using Chromas lite 2.01 (http://www. technelysium.com.au/chromas_lite.html). Sequence alignment was carried out using the MAFFT algorithm [16] and manually corrected using MEGA1 V5.10 [17]. Sequences are deposited in GenBank (Accession numbers: KT600663, 664-KT600670, 671). The dataset was 1357 base pairs in length. Combined sequence data of 12S and 16S rRNA of 23 species of Microhyla are used for phylogenetic tree construction with Uperodon variegatus as an outgroup (S1 Table).
Maximum likelihood (ML) algorithm and Bayesian inference methods were used for phylogenetic analysis. The ML analysis was executed in RaxML v1.3 [18] with GTR+I+G model selected as the best-fit nucleotide substitution model in jModel test [19] for 1000 bootstrap replicates. The Bayesian analysis was performed in MrBayes 3.2.4 [20]. The combined data set of 16S and 12S gene fragments were used for the analysis with GTR+I+G selected as best fit models in jModel test. The Markov chain Monte Carlo analysis for the dataset was run for 50 million generations and trees were sampled every 500 cycles. The convergence of the runs was analyzed by assessing the split frequency standard deviations (<0.001) and potential scale reduction factor (PSRF~1.0). The first 10% of the sampled trees were discarded as burn-in and remaining samples were used to generate majority rule consensus tree. For estimating the genetic divergence, un-corrected pairwise genetic distance between the species was calculated in MEGA 5.10.

Morphological Measurements
Individuals were measured using a Mitutoyo1 digital caliper to the nearest 0.1 mm. Measurements and terminology follow Seshadri, Gururaja [21] and measured features are abbreviated and listed in S1 Appendix. Measurements of fingers and toes were made in ImageJ1 from photographs taken using a Nikon1 D90 Digital camera with a Nikkor1 105 mm micro lens. QGIS1 Pisa Ver. 2.10 was used for illustrating hand and foot.

Statistical Analysis
For bioacoustic comparison, a Mann-Whitney U test in R v.3.1.3 [22] was used to compare the means of call characteristics.

Call Recordings and Analysis
Calls were recorded using Sennheiser K61 unidirectional microphone coupled with a Marantz PMD 6601 solid state recorder. Vocalizations with relatively higher signal to noise ratio were chosen and analyzed using Audacity Ver.1.3 (Beta) and Raven Pro Ver.1.5. Eight calls from two individuals of M. laterite sp. nov., were used. Call terminology follows Kok and Kalamandeen [23]. Duration, dominant frequency and number of pulse of each call were analysed. Air temperature and relative humidity were recorded using a Kestrel 1 4500 pocket weather tracker.

Maps and Geographic Range Estimation
QGIS1 Pisa Ver. 2.10 was used to generate maps of range and distribution of M. laterite sp. nov. Data was sourced from www.gadm.org for administrative boundary and SRTM 90 m Database (http://srtm.csi.cgiar.org) for elevation. Area under minimum convex hull was computed on occurrence points of frogs to estimate extent of occurrence for IUCN assessment as per existing criteria [24].

Molecular Identification of Microhyla laterite sp. nov.
The intra-specific un-corrected pairwise genetic distance (UPGD) of M. laterite sp. nov. was 0.0% (n = 2) and the inter-specific UPGD of M. laterite sp. nov. was lowest with M. sholigari (range: 4.8-5.03%, n = 5) and was most with M. petrigena (14.14%, n = 2). The UPGD between the 23 species of Microhyla used in the analysis varied from 4.8% to 14.14% (S2 Table). Microhyla laterite sp. nov. is a sister species of M. sholigari, forming a distinct clade as inferred from the phylogenetic tree (Fig 2). However, the relationships with other species are not fully resolved as indicated by the low bootstrap support. Therefore, M. laterite sp. nov. was described as a new species and compared with M. sholigari. Paratypes: Two males (BNHS 5965, 5966) and one female (BNHS 5967) were collected in same locality, date and time as holotype by KSS and RS.

Species Description
Diagnosis. The new species is assigned to the genus Microhyla owing to the following set of characters sensu Parker (25), Dutta and Ray (26) and Matsui, Hamidy [27]: Small sized adults with circular pupil; dorsal skin smooth, with markings from back of eye to vent; supratympanic fold present; paratoid glands absent; fingers without webbing, free with or without dilations; tongue oval, entire and free at the base; snout less than twice the diameter of eye; tympanum hidden by skin; palmar tubercles distinct; distinct oval shaped inner metatarsal tubercle and rounded outer metatarsal tubercle; rudimentary webbing in foot.
Microhyla laterite sp. nov. can be distinguished from all other congeners in the Indian subcontinent by the following suite of characters: (i) A very small sized adult frog (Male: 15.3-16.6 mm, n = 3 and Female: 18.4 mm, n = 1); (ii) snout obtuse in dorsal and ventral view with indistinct canthus rostralis, snout protrudes beyond mouth in ventral view (iii) tongue obovate, margin irregular, without lingual papilla (iv) tympanum hidden; (v) head wider than long; (vi) skin smooth on dorsum and venter; (vii) short, dark horizontal band on dorsum on the same plane as forelimbs. (viii) Throat with dense purplish-black pigmentation, reducing in intensity towards belly; (ix) reduced webbing in feet; (x) discs with circum-marginal groves on fingers and toes.
Description of holotype.      Skin texture in preservative. Skin on snout, inter-orbital space and sides of head smooth; Dorsum smooth, interspersed with tubercles increasing in intensity towards vent; Dorsal surface of forelimb and hind limb smooth with tubercles on upper arm, thigh, shank and foot. Skin on ventral side smooth, throat shagreened.
Color in preservative. Dorsal coloration pale brown; tubercles pale red; a short dark horizontal band on dorsum along the same plane as forelimbs; a pair of black spots at the urostyle bone projections; forelimbs reddish brown with black cross bands. Tympanic region grayish black. Flanks with a black band starting above shoulder and terminating just before the groin. Anterior part of thigh with distinct black band starting from knee and terminating short of groin. Dorsal surface of hind limbs brownish with black cross bands. Vent with a black triangular marking. Anterior and posterior portions of pupil black. Ventral region pale cream colored; throat with dense purplish-black pigmentation, reducing in intensity towards belly; tarsus to tip of toes brownish with pale buff colored webbing.
Color in life. Overall pale brown with prominent black markings on dorsum, hands, feet and flanks. Distinct black horizontal band on dorsum along the same plane as forelimbs. The band has a red leading edge. Deep purplish black vocal sac when calling. Iris golden yellow with brown mottling. Pupil black. Ventral parts cream white except throat (Fig 4B).
Etymology. This species is named after the laterite rock formations in the type locality and other parts of its geographic range (Fig 4A). The specific name is an invariable noun in the nominative singular in apposition to generic name.

Ecology and Natural History Observations
Microhyla laterite sp. nov. was observed in and around the type locality during the south west monsoon seasons between April-August from 2013-2015. The species was found to be restricted to laterite habitats near rural and peri-urban areas. This species inhabits ephemeral ponds and other marshy areas in laterite habitats. They also occur in wet paddy fields where they were observed to vocalize from the embankment. Vocalization begins about 18:00 h and peaks between 19:15-21:30 h. The vocalization is very similar to that of ground crickets. Males can be located on leaf litter and other debris in dense grass clusters and often change their position while vocalizing. Upon disturbance, a few males were observed to stop calling and retreat backwards into small cavities in laterite rock formations where they lay low and hide for a few minutes.

Discussion
Members of the family Microhylidae comprise about 8% of all amphibians [31]. Having a pan tropical distribution, they have been of considerable interest in taxonomy and systematics. The classification and understanding of their evolutionary relationships has been in flux since Parker's comprehensive monograph in 1934. Several studies have been undertaken but have resulted in contradictory findings [32,33]. Further, identifying species in this genus based solely on morphology has proven to be difficult, owing to the small size, high morphological similarity and general diminutive habits of Microhyla. Hence, most species discoveries in South and Southeast Asia rely on integrative approach using morphology, molecular and bioacoustic data [34]. Recent studies on Microhyla from Indian subcontinent report several cryptic and un-described lineages [28,31]. Currently, there are three species of Microhyla found in south India viz., M. ornata, M. rubra and M. sholigari in addition to M. laterite which is described here.
Microhyla laterite is named after the 'lateritie' or 'ferricrete duricrust' habitats where they commonly occur. Laterite formations are a common feature along the western slopes of the Western Ghats and West coast where they are native habitats and not degraded lands [13,35,36]. Laterite rocks are anisotropic in nature giving a heterogeneous composition of ferruginous compounds that form a rigid skeletal framework which is often impregnated with relatively softer clay; Cavities and pores are a characteristic structural irregularity of these rocks [36]. Recent evidence suggests that these rock formations formed between late Cretaceous and early Tertiary and have a complex geological history and are of paleontological importance [37].
These laterite plateaus are broadly considered as 'rocky areas' as they are usually devoid of trees and other woody vegetation and are therefore classified as wastelands [38]. Though devoid of tertiary vegetation, these areas are rich in microhabitats as there is an abundance of ephemeral pools and associated vegetation. Vegetation assessments have revealed laterite habitats to harbor a high diversity of plants, dominated by Poaceae with over 45 endemics [39]. Utricularia spp. and Drosera spp. are also common in these habitats.
The localities where we observed the frog (Fig 1), are very few. They are dominated by shrubs, grasses and woody trees like Strychnos nux-vomica, Careya arborea, Macaranga indica, Mangifera indica and plantations of Acacia spp., Gliricidia spp. and Syzygium spp. The geographic range of M. laterite is small (146.13 km 2 ) and warrants immediate conservation attention. We list this species as endangered (EN) under the IUCN Red List criteria B1ab(iii)(iv) where the estimated extent of occurrence is less than 500 km 2 , the habitat is severely fragmented, there is a continued decline of area, extent and/or quality of habitat and the species is present in less than five locations. Surveys in other regions along the West coast have not detected this species. Laterite habitats receive very little protection from any legislation and are sought after for developmental works and are heavily mined for construction material in form of bricks [36]. They are also subject to destructive anthropogenic activities like dumping of municipal solid wastes; fuel wood collection; conversion to plantations; encroachments; use of ephemeral pools for domestic ablutions and washing of vehicles (Fig 6). All these severely impact the already fragmented habitat and cause irreparable damages.

Conservation Interventions
Laterite areas in India receive no protection and are considered as wastelands [38]. Given the threats these fragile habitats are facing, it is imperative to conserve them. The Wildlife Protection Act (1972) in conjunction with the National Wildlife Action Plan (2002-2016) provides opportunities for conserving such areas where they can be declared as 'Conservation Reserves' and/or 'Community Reserves'. These interventions will prevent further degradation by halting land conversion. With Community Reserves, the local community members are empowered to be key stakeholders and be greatly involved in conservation action [40]. Further, other legislations like the Biological Diversity Act (2002), enable areas of biological importance to be protected as 'Biological Heritage Areas' and several regions have already been listed in this framework [41]. These opportunities need to be explored in order to conserve the fragile laterite habitats which are vital for M. laterite.

Future Directions
The phylogenetic tree generated in this study provided low bootstrap support for almost all species except M. sholigari. The overall weak bootstrap support across species and clades is indicative of a spurious phylogenetic association because of missing species. Several attempts to understanding the phylogeny of species across taxa have encountered this issue. This arises either out of 'Linnean shortfall', where many un-described species exist or of 'Wallacean shortfall' where the geographic ranges are poorly understood due to lack of systematic sampling [42,43]. Another added challenge here is the lack of relevant phylogenetic information of taxonomically valid species, known as the 'Darwinian shortfall' [44]. Therefore, it is imperative that systematic surveys are undertaken across Indian subcontinent, including Sri Lanka for a robust phylogeny and systematics of the genus Microhyla. These efforts would help in overcoming some of the limitations we currently face in understanding amphibian ecology and evolution.
With molecular tools becoming increasingly reliable and affordable; studies could shed light into the population dynamics of these small frogs found in isolated and severely fragmented landscapes. In context of laterite habitats, studies have estimated the early diversification period of Microhylidae to be at the late Cretaceous period and that of Microhyla to be in the lower Tertiary period; signifying that several lineages survived through the KT boundary [45]. Since M. laterite appears to be restricted to laterite rock formations along the West coast, further research on determining divergence times of M. laterite and testing for an association with laterite formations would enable a better understanding of biogeography, systematics and paleo-ecology. This will enable us to explore interesting evolutionary ecology questions in Microhyla.
Supporting Information S1 Appendix. Details of abbreviations used for morphometric measurements.  Table. List of species and accession numbers used for phylogenetic analysis. List was based on Howlader, Nair (28). (DOCX) S2 Table. Un-corrected pairwise genetic distances of Microhyla species used for analysis. Uperodon variegatus is used as an out-group. Genetic distances are in percentage. (DOCX)