A new species of Micryletta frog (Microhylidae) from Northeast India

We describe a new species of frog in the microhylid genus Micryletta Dubois, 1987 from Northeast India based on molecular and morphological evidence. The new species, formally described as Micryletta aishani sp. nov., is phenotypically distinct from other congeners by a suite of morphological characters such as brown to reddish-brown dorsum; dorsal skin shagreened with minute spinules; snout shape nearly truncate in dorsal and ventral view; a prominent dark streak extending from tip of the snout up to the lower abdomen; ash-grey mottling along the margins of upper and lower lip extending up to the flanks, limb margins and dorsal surfaces of hand and foot; tibiotarsal articulation reaching up to the level of armpits; absence of outer metatarsal tubercles; and absence of webbing between toes. Phylogenetic relationships within the genus are inferred based on mitochondrial data and the new taxon is found to differ from all the recognised Micryletta species by 3.5–5.9% divergence in the mitochondrial 16S rRNA. The new species was found in the states of Assam, Manipur, and Tripura, from low to moderate elevation (30–800 m asl) regions lying south of River Brahmaputra and encompassing the Indo-Burma Biodiversity Hotspot. The discovery validates the presence of genus Micryletta in Northeast India based on genetic evidence, consequently confirming the extension of its geographical range, westwards from Southeast Asia up to Northeast India. Further, for nomenclatural stability of two previously known species, Microhyla inornata (= Micryletta inornata) and Microhyla steinegeri (= Micryletta steinegeri), lectotypes are designated along with detailed descriptions.


INTRODUCTION
The Northeast region of India encompasses two globally recognised biodiversity hotspots-Himalayas in the north and Indo-Burma towards the South (Mittermeier et al., 2004), and is home to unique and diverse array of amphibians (e.g. Pillai & Yazdani, 1973;Kiyasetuo & Khare, 1986;Kamei et al., 2012;Biju et al., 2016). Yet, in comparison to the Western Ghats hotspot in Peninsular India that has witnessed a systematic documentation of amphibian diversity, particularly with over two-fold increase in the number of known species during the past two decades (Biju et al., 2014a;Garg et al., 2017), the Northeast regions of the country have remained relatively neglected (Kamei et al., 2012;Biju et al., 2016). The known amphibian fauna of Northeast India belongs to 11 families (Frost, 2018;AmphibiaWeb, 2018), of which Microhylidae is represented by three genera (Microhyla Tschudi, Kaloula Gray, and Uperodon Duméril and Bibron) confirmed to be present on the basis of detailed morphological and/or molecular studies (e.g. Das et al., 2005;Sengupta et al., 2009;Garg et al., 2018aGarg et al., , 2018bGarg et al., , 2018c. Another enigmatic group of microhylid frogs, genus Micryletta Dubois is believed to occur in the northeast state of Manipur based on a cursory report of Micryletta inornata Boulenger (Mathew & Sen, 2010). This species was previously also reported from the Andaman Islands of India based on a subadult specimen (Pillai, 1977) that requires confirmation (Harikrishnan & Vasudevan, 2018). Hence, there are uncertainties about the identity of Micryletta species in India, and consequently the geographical distribution of the genus and its members (chiefly M. inornata) outside of East (Mainland China and Taiwan) and Southeast Asia (Cambodia, Indonesia, Laos, Malaysia, Myanmar, Thailand, and Vietnam) .
During surveys over a period of 10 years in Northeast India, our rare encounters with a seemingly secretive species of microhylid frog led us to investigate its identity using molecular and morphological tools. Based on both phenotypic and genotypic evidence, we conclude that this frog belongs to genus Micryletta and represents a distinct new species to which no previously available name could be applied. Here, we formally describe the new species based on collections from the states of Assam, Manipur, and Tripura.

Field surveys and specimen collection
Sampling was carried out at three localities in Northeast India (Fig. 1). Animals were located during opportunistic night searches around water bodies (streams, marshes, stagnant pools, and temporary puddles) inside secondary forests or degraded areas surrounding human settlements. Sampled individuals were photographed, euthanised in MS-222 (Tricaine methane sulphonate), fixed in 4% formalin, and preserved in 70% ethanol. Tissue samples for molecular studies were obtained from the thigh muscle or liver and stored in absolute ethanol at -20 C. Type specimens are deposited at Zoological Survey of India, Kolkata (ZSIC) and referred specimens are available at Wildlife Institute of India, Dehradun (WII) or Systematics Lab, University of Delhi (SDBDU). Geographical coordinates of the sampling localities were recorded using a Garmin GPS (WGS 84) and distribution maps were prepared in QGIS version 2.6.1 (http://www.qgis.org).
Fieldwork and sampling were carried out with permissions from responsible authorities in the State Forest Departments of Assam, Tripura, and Manipur (

Morphological study
The new collections were morphologically compared with all previously known Micryletta species, based on the original descriptions (Boulenger, 1890(Boulenger, , 1909Taylor, 1962;Tarkhnishvili, 1994;Poyarkov et al., 2018), examination of types and other museum specimens (for M. inornata and M. steinegeri), and/or type photographs (M. erythropoda available from Moscow State University, National Depository Bank of Live Systems website https://depo.msu.ru/, accessed 20 May 2018; M. inornata lineata available from FMNH; M. nigromaculata available from Poyarkov et al., 2018). Only adult animals were used for morphometric studies. Sex and maturity of the specimens were determined either by the presence of secondary sexual characters (such as vocal sacs in males; eggs in gravid females) or through gonadal examination with the help of a small lateral or ventral incision. Measurements and photographs were taken for the right side of the specimen, except when a character was damaged, in which case the measurement was taken on the left side. The following measurements were taken to the nearest 0.1 mm using digital slide-calipers or binocular microscope with micrometre ocular: snout-vent length (SVL), head width (HW, at the angle of the jaws), head length (HL, from the rear of the mandible to the tip of the snout), snout length (SL, from the tip of the snout to the anterior orbital border), eye length (EL, horizontal distance between the bony orbital borders), TYD (maximum tympanum diameter), EN (distance from the front of the eye to the nostril), NS (distance from the nostril to the tip of the snout), internarial distance (IN), inter upper eyelid (IUE width, shortest distance between the upper eyelids), UEW (maximum upper eyelid width), forearm length (FAL, from the flexed elbow to the base of the outer palmar tubercle), hand length (HAL, from the base of the outer palmar tubercle to the tip of the third finger), shank length (SHL), thigh length (TL), foot length (FOL, from the base of the inner metatarsal tubercle to the tip of the fourth toe), TFOL (distance from the heel to the tip of the fourth toe), OMTL (length of outer metatarsal tubercle), IMTL (length of inner metatarsal tubercle), FL (finger length, from tip of the digit to its base where it joins the adjacent digit), FD (disc width of finger), FW (width of finger, measured at the base of the disc), TD (disc width of toe), TW (width of toe, measured at the base of the disc); digit number is represented by roman numerals I-V. Number of additional tubercles on toes are represented by roman numerical followed by S (small) or L (large). Morphometric terminologies follow Garg et al. (2018c); description of foot webbing and the webbing formulae follow Biju et al. (2014b). All measurements provided in the taxonomy section are in millimetres (mm).

Molecular study
Genomic DNA was extracted from a tissue sample of the new species by using Qiagen DNeasy blood and tissue kit (Qiagen, Valencia, CA, USA). A fragment of ∼540 bp of the mitochondrial 16S rRNA gene (16S) was PCR-amplified using previously published primers 16Sar and 16Sbr (Simon et al., 1994). The purified PCR product was sequenced on both strands using BigDye Terminator v3.1 Cycle Sequencing Kit on ABI 3730 automated DNA sequencer (Applied Biosystems, Foster City, CA, USA). Sequences were assembled and checked in ChromasPro v1.34 (Technelysium Pty Ltd., Tewantin, Australia). Additionally, the mitochondrial 16S sequences of all previously published Micryletta populations, along with outgroup taxa  from five generic representatives of the subfamily Microhylinae (Glyphoglossus molossus Günther, Kaloula pulchra Gray, Microhyla achatina Tschudi, Mysticellus franki Garg and Biju, and Uperodon systoma (Schneider)) and a member of the subfamily Dyscophinae (Dyscophus insularis Grandidier) were retrieved from the GenBank (Table 1). A dataset of total 44 taxa was assembled, aligned using ClustalW, and manually optimised in MEGA 6.0 (Tamura et al., 2013). The new sequence is deposited in the GenBank under accession number MK889218. Maximum likelihood (ML) and Bayesian analyses were performed to reconstruct phylogenetic relationships. The appropriate model of sequence evolution was determined by implementing Akaike Information Criteria in ModelTest 3.4 (Posada & Crandall, 1998). ML searches were executed in PAUP Ã (Swofford, 2002) using the best-fit model (GTR+I+G) with all parameters estimated. Bayesian analysis was implemented in MrBayes (Ronquist & Huelsenbeck, 2003) using uniform priors and four Metropolis-Coupled Markov Chain Monte Carlo chains for 25 million generations. Trees were sampled after every 1,000 generations. Convergence of the runs was determined by average standard deviation of the split frequencies of <0.01 and potential scale reduction factors of ∼1.0, and stationarity was observed through log likelihood trends. Bayesian posterior probabilities (BPP) for the clades were summarised after discarding the first 20% trees as burn-in (Huelsenbeck et al., 2001). Clade support was also assessed by 10,000 rapid bootstrap replicates executed using GTRGAMMA model in RAxML 7.3.0 (Stamatakis, Hoover & Rougemont, 2008) as implemented in raxmlGUI 1.1 (Silvestro & Michalak, 2012). Uncorrected pairwise distances were computed in PAUP Ã (Swofford, 2002) to understand intra and interspecific genetic divergences. A Median-Joining network was constructed using the software Network 4.6.1.0 (www.fluxus-engineering.com). A 178 bp fragment available for all the 16S Micryletta sequences was used to evaluate relationships and possible mutation steps among 21 haplotypes recovered from 38 samples.

Nomenclatural acts
The electronic version of this article in portable document format will represent a published work according to the International Commission on Zoological Nomenclature (ICZN), and hence the new names contained in the electronic version are effectively published under that Code from the electronic edition alone (see Articles 8.5-8.6 of the Code). This published work and the nomenclatural acts it contains have been registered in ZooBank, the online registration system for the ICZN. The ZooBank Life Science Identifiers (LSIDs) can be resolved and the associated information can be viewed through any standard web browser by appending the LSID to the prefix http://zoobank.org/. The LSID for this publication is as follows: urn:lsid:zoobank.org:pub:F0C54D63-0B29-470F-BDCD-72F7E2511C51. The online version of this work is archived and available from the following digital repositories: PeerJ, PubMed Central, and CLOCKSS.

Genetic relationships and sequence divergence
Phylogenetically, the new frog is nested in the genus Micryletta where it forms a clearly divergent lineage ( Fig. 2) with >3% divergence from all previously known species in the studied fragment of the mitochondrial 16S rRNA ( Table 2) cf. inornata lineata (BPP 100, BS 98), were poorly resolved. However, our overall topology was largely consistent with the previous studies that include most of the available Micryletta sequence data Alhadi et al., 2019). The Median-Joining network based on 21 unique haplotypes representing 38 Micryletta sequences also recovered M. aishani sp. nov. as a distinct species (Fig. 2). All the major lineages and species-level clades observed in our phylogenetic analysis were demarcated as distinct clusters with similar haplotype relationships, consequently providing additional evidence for our findings. The haplotype clusters for all the known and potential candidate species were separated by minimum of four up to 18 mutation steps (hereafter steps) from closely related members, except M. steinegeri and M. cf. steinegeri that were separated by three steps. M. aishani sp. nov. was most closely linked to the M. steinegeri species complex (specifically M. cf. steinegeri) with minimum 10 steps, followed by minimum 14 steps each with M. steinegeri, M. erythropoda, and M. cf. nigromaculata, and minimum 18 steps each with M. inornata and M. nigromaculata.
The uncorrected sequence divergence in the studied 16S fragment showed M. aishani sp. nov. to be divergent from the previously recognised species by interspecific genetic distances of 3.5% for M. steinegeri, 4.5-5.9% for M. inornata, 4.7% for M. erythropoda, and 4.5-5.1% from M. nigromaculata. It was also divergent from the populations suspected to represent another available nomen M. cf. inornata lineata by 3.3%. The new species also showed a minimum of 3.1% up to 4.8% divergence from other potential candidate species identified in the genus ( Table 2). The trends of interspecific divergences between other congeners ranged from minimum 2.5% between M. erythropoda and M. cf. inornata lineata, up to a maximum of 9.1% and 9.4% (between M. inornata-M. steinegeri species complex and M. inornata-M. cf. nigromaculata, respectively). The M. steinegeri species complex was, however, an exception as it showed shallow interspecific distances of 1.2-2.7% among four closely related clades (M. steinegeri, M. cf. steinegeri, Micryletta sp. A, and Micryletta sp. B), as well as high intraspecific distances of up to 3.0% within the Micryletta sp. A clade, suggesting that all of these either represent a single widely distributed species or a complex of multiple taxa with at least two potential candidate species. Another candidate species, M. cf. nigromaculata with 2.3-4.4% divergence from M. nigromaculata, was identified. Further phylogenetic studies complemented with proper identification as well as morphological comparison among all the Micryletta populations known so far will be necessary to ascertain the taxonomic status of the yet unidentified populations as well as to understand the patterns of genetic differentiation within the genus.

Note:
Values indicate mean genetic distances with minimum and maximum distances in parenthesis.  Etymology. The species epithet, aishani, is an invariable feminine noun derived from the Sanskrit word 'aishani' or aiśānī (meaning north-east), referring to the Northeast regions of India where this frog was discovered.
Diagnosis. The new species is assigned to the genus Micryletta due to the following combination of morphological traits: small body size (SVL 22-28 mm); absence of vomerine teeth; prominent subarticular tubercles on fingers and toes; finger and toe tips slightly expanded in to small discs; and absence of webbing between fingers and toes (Dubois, 1987;Fei et al., 2009). Micryletta aishani sp. nov. differs from the other recognised species of the genus by the following suite of morphological characters: relatively small adult size (SVL 22.1-23.5 mm, male, N = 7; SVL 25.6-27.3 mm, female, N = 4); slender body; snout nearly truncate in dorsal and ventral view, acute in lateral view; tibiotarsal articulation reaching up to the level of armpit when stretched forward along the body axis; dorsal skin shagreened with minute spinules; outer metatarsal tubercles absent; webbing between toes absent; dorsum brown to reddish-brown with a faint brown median band extending from margins of the upper eye lids and tapering up to the vent, and few scattered blackish-brown spots on posterior parts of the back and near the groin; lateral surfaces of head blackish-brown; prominent blackish-brown streak extending from tip of the snout up to lower abdomen; ash-grey mottling along the margins of the upper and lower lip, extending up to the flanks and the limb margins; anterior and posterior parts of thigh, tarsus, and dorsal surfaces of hand and foot brown with ash-grey mottling; iris bicoloured, upper half light brown and lower half dark brown; belly ash-grey with a purplish tinge and brown mottling towards the margins.
Description of holotype (measurements in mm). Adult male (SVL 22.8); head wider (HW 6.2) than long (HL 5.7); snout nearly truncate in dorsal and ventral view, acute in lateral view, its length (SL 2.7) longer than horizontal diameter of the eye (EL 2.2); loreal region vertical with rounded canthus rostralis; interorbital space flat, wider (IUE 2.7) than the upper eyelid (UEW 1.3) and internarial distance (IN 1.6); nostril closer to tip of the snout (NS 0.9) than the eye (EN 1.2); tympanum weakly-developed (TYD 0.4), less than one-fifth (18.2%) of the eye diameter (EL 2.2); supratympanic fold extending from posterior corner of eye to the shoulder weakly-developed; vomerine teeth absent; tongue small, oval, without papillae. Forearm (FAL 5.3) shorter than hand length (HAL 5.9); fingers without dermal fringes, finger length formula: I < II < IV < III, finger discs slightly wide compared to finger width (FD I 0.4, FW I 0.3; FD II 0.4, FW II 0.3; FD III 0.5, FW III 0.4; FD IV 0.5, FW IV 0.4), finger discs without grooves; subarticular tubercles prominent, oval, all present, subarticular tubercle formula: F I 1, F II 1, F III 2, F IV 2; single rounded supernumerary palmar tubercle at the base of fingers II, III, and IV; three metacarpal (palmar) tubercles; inner metacarpal tubercle distinct, rounded, small; outer metacarpal tubercle rounded, larger; medial metacarpal tubercle rounded, large; nuptial pads absent. Hind limbs slender, tibiotarsal articulation reaches up to the level of armpit (well below the eye) when stretched forward along the body axis; thigh length (TL 10.4) shorter than shank (SHL 10.7) and foot (FOL 11.4); distance from heel to tip of toe IV (TFOL 16.8); relative toe lengths: I < V < II < III < IV, toe discs slightly wide compared to toe width (TD I 0.5, TW I 0.4; TD II 0.6, TW II 0.4; TD III 0.7, TW III 0.5; TD IV 0.6, TW IV 0.4; TD V 0.6, TW V 0.4), toe discs without grooves; toes free of webbing; subarticular tubercles on toes prominent, oval, alternating with additional smaller tubercles, formula: T I 1(L), T II 1 (S) + 1(L), T III 1(S) + 1(L) + 1(S) + 1(L), T IV 1(S) + 1(L) + 1(L) + 1(S) + 1(L), T V (1S) + 1(L) + 1(S) + 1(L); single inner metatarsal tubercle (IMTL 0.6), oval-shaped, prominent, much shorter than half the length of toe I; outer metatarsal, and supernumerary metatarsal tubercles absent (Fig. 3). Skin of snout, upper eyelids, sides of head, anterior and posterior parts of back, and upper and lower parts of flank shagreened with minute spinules; dorsal surfaces of forelimbs smooth, dorsal surfaces of hind limbs sparsely granular; ventral surface of throat smooth with minute spinules; chest, belly, and ventral surface of limbs smooth (Fig. 3). Colour of holotype. In life. Dorsum reddish-brown with few scattered blackish-brown spots on posterior parts of the back and near the groin; a faint brown median band extending from margins of the upper eye lids and tapering up to the vent; lateral surfaces of head blackish-brown; a prominent blackish-brown streak extending from tip of the snout up to lower abdomen; ash-grey mottling along the margins of the upper and lower lip, extending up to the flanks, limb margins and dorsal surfaces of hand and foot; dorsal surface of forearms light reddish-brown; fingers brown with ash-grey mottling; dorsal surface of hind limbs reddish-brown with faint dark brown mottling, without dark transverse bands; toes I-IV brown with ash-grey mottling; groin, anterior and posterior parts of thigh, and lateral surfaces of shank and tarsus brown with ash-grey mottling; iris bicoloured, upper half light brown and lower half dark brown; ventral surface of throat greyish-brown; chest ash-grey with a purplish tinge and brown mottling; belly and fore-and hind limbs ash-grey with a purplish tinge and dark brown mottling towards the margins (Fig. 4). In preservation. Dorsum light greyish-brown with few scattered dark brown spots on posterior parts of the back, near the groin, and fore-and hind limbs; a faint greyish-brown median band extending from margins of the upper eye lids and tapering up to the vent; a dark brown streak extending from tip of the snout up to lower abdomen; lateral abdominal surfaces light grey with brown mottling; ventral surface of throat dark greyish-brown; chest light greyish-brown with brown mottling; belly, hand and foot greyish-brown with dark brown mottling towards the margins (Fig. 3).
Variations. Morphometric data from seven adult males and four adult females, including the holotype, is given in Table 3. Overall, the colour and meristic characters of the paratypes are similar to the holotype. Skin texture varies based on the sex: males with more prominent spinules on dorsal skin and throat. Ventral colouration varies from ash-grey to light purple with less or more prominent mottling (Figs. 4 and 5).
Distribution and natural history. Micryletta aishani sp. nov. is currently known from three Northeast Indian states of Assam, Tripura, and Manipur ( Fig. 1). At the type locality (Subhong), we came across a large aggregation of calling males in the month of May at around 8.30 pm. Individuals were calling from waterlogged fern banks located at the base of a small valley between hillocks (locally called Tillas). The area is characterised by degraded forest with areca nut plants and beetle vine cultivation located close to a human settlement (∼1 km). Two females were collected from the exposed slopes of the tillas close to the congregation. The species seems to have a narrow breeding season with very specific requirements as we failed to record any individuals either before waterlogging (during early April) or once the fern banks began to submerge in water (by June   Specifically, M. aishani sp. nov. differs from M. erythropoda by its relatively smaller adult snout-vent size, SVL 22.1-23.5 mm in males, N = 7, SVL 25.6-27.3 mm in females, N = 4 (vs. SVL up to 30 mm); outline of the snout nearly truncate in dorsal and ventral view (vs. obtuse); dorsum with few scattered blackish-brown spots on posterior part of the back and near the groin (vs. dark contrasting round or irregular shape spots irregularly scattered throughout the dorsum); dorsal skin shagreened with minute spinules (vs. skin smooth, slightly granulated on the lateral sides); tympanum much smaller than the eye diameter, TYD/EL 18.2-28.6% in males, TYD 32-41% in females (vs. relatively larger, equal to the half length of eye); tibiotarsal articulation reaching up to the level of armpit when stretched forward along the body axis (vs. tibiotarsal joint not reaching the hind part of tympanum); absence of outer metatarsal tubercles (vs. present); and webbing absent between toes (vs. rudimentary).
Micryletta aishani sp. nov. differs from M. inornata by its larger adult snout-vent size, SVL 22.1-23.5 mm in males, N = 7, SVL 25.6-27.3 mm in females, N = 4 (vs. smaller, SVL 16.8-20.5 mm in males, N = 3, SVL 19.5 mm in female, N = 1); outline of the snout nearly truncate in dorsal and ventral view (vs. nearly rounded); snout longer than the eye diameter, SL/EL 1.22-1.33 in males, N = 7, SL/EL 1.08-1.36 in females, N = 4 (vs. shorter, SL/EL 0.86-0.91 in males, N = 3, SL/EL 0.92 in female, N = 1); tibiotarsal articulation reaching up to the level of armpit when stretched forward along the body axis (vs. up to the level of eye); dorsal colouration reddish-brown with few scattered blackish-brown spots on posterior parts of the back and near the groin (vs. dark brown or brownish-grey with a silver tinge, and irregular blackish-brown blotches); dorsal skin shagreened with minute spinules (vs. smooth, covered with small tubercles or warts); anterior and posterior parts of thigh, lateral surfaces of shank and tarsus, and toes I-IV brown with ash-grey mottling (vs. light flesh-red without mottling); and ventral surface of belly, fore-and hind limbs ash-grey with a purplish tinge and brown mottling towards the margins (vs. light reddish-grey without mottling).
Micryletta aishani sp. nov. differs from M. nigromaculata by the outline of its snout nearly truncate in dorsal and ventral view (vs. rounded); eyes shorter than snout, EL/SL 0.75-0.82 in males, N = 7, EL/SL 0.73-0.93 in females, N = 4 (vs. relatively longer, EL/SL 0.86-1.07 in males, N = 18, EL/SL 0.91-1.03 in females, N = 3); males with a relatively smaller tympanum than the eye, TYD/EL 0.14-0.29 (vs. relatively larger, TYD/EL 0.36-0.46, N = 18); supratympanic fold weakly-developed (vs. thick and glandular); tibiotarsal articulation reaching up to the level of armpit when stretched forward along the body axis (vs. reaching up to the level of eye); dorsal skin shagreened with minute spinules (vs. slightly granular with small round flattened tubercles); dorsum reddish-brown with a faint median band (vs. dark brown irregular hourglass-shaped pattern having orange edges); a prominent dark blackish-brown streak from tip of the snout up to the lower abdomen on either side (vs. scattered dark spots or patches); ventral surface of belly ash-grey with a purplish tinge and brown mottling towards the margins (vs. whitish with indistinct light grey marbling).
Micryletta aishani sp. nov. differs from M. steinegeri by its relatively larger snout, SL/HL 0.43-0.52 in males, N = 7, SL/HL 0.39-0.47 in females, N = 4 (vs. shorter, SL/HL 0.34-0.38 in males, N = 3, SL/HL 0.37-0.38 in females, N = 2); eyes smaller than the snout, EL/SL 0.75-0.82 in males, N = 7, EL/SL 0.73-0.93 in females, N = 4 (vs. larger, EL/SL 1.04 in males, N = 3, EL/SL 1.07-1.14 in females, N = 2); tibiotarsal articulation reaching up to the level of armpit when stretched forward along the body axis (vs. reaching up to the level of tympanum); dorsum reddish-brown with few scattered blackish-brown spots on posterior parts of the back and near the groin (vs. dark grey to violet with irregular dark blotches or speckles); dorsal skin shagreened with minute spinules (vs. smooth to shagreened); anterior and posterior parts of thigh, lateral surfaces of shank and tarsus, and toes I-IV brown with ash-grey mottling (vs. light flesh-red without mottling); ventral surface of belly, fore-and hind limbs ash-grey with a purplish tinge and brown mottling towards the margins (vs. greyish-white); and webbing absent between toes (vs. rudimentary).
Furthermore, the new species M. aishani sp. nov. cannot be confused with another available nomen, M. inornata lineata, currently under the synonymy of M. inornata, due to its reddish-brown dorsum with a faint median band (vs. greyish-brown with three straight continuous or broken lines); snout longer than the horizontal diameter of eye, SL/EL 1.22-1.33 in males, N = 7, SL/EL 1.08-1.36 in females, N = 4 (vs. snout distinctly shorter than the eye length); dorsal skin shagreened with minute spinules (vs. smooth); and tibiotarsal articulation reaching up to the level of armpit when stretched forward along the body axis (vs. reaching up to the eye).
Diagnosis. Micryletta inornata differs from all other members of the genus by the following suite of morphological characters: smaller adult size, SVL 16.8-20.5 mm in males, N = 3, SVL 19.5 mm in female, N = 1; outline of the snout nearly rounded in dorsal and ventral view; tibiotarsal articulation reaching up to the level of eye when stretched forward along the body axis; dorsum brownish-grey with a silver tinge and irregular blackish-brown blotches; lateral surfaces of head blackish-brown with a silver white line along the upper lip; ventral surface of belly light reddish-grey without mottling; and its distribution restricted to the islands of Sumatra, Indonesia . For comparison with other congeners, see the 'Morphological comparison' section of M. aishani sp. nov.
Rationale for lectotypification. This species was originally described (Boulenger, 1890) based on three specimens (two males and one female) from "Deli, Sumatra". Subsequently, it was reported to occur widely across Mainland Southeast Asia (Thailand, Malaysia, Vietnam, Laos, Cambodia, and Myanmar) with few isolated records from South Asia (Manipur and Andaman Islands, India) and East Asia (southern China and Taiwan Island) (e.g. Berry, 1975;Pillai, 1977;Ohler, Swan & Daltry, 2002;Nguyen, Ho & Nguyen, 2005;Stuart & Emmett, 2006;Guo, Yang & Li, 2009;Mathew & Sen, 2010;Fei, Ye & Jiang, 2010;Frost, 2018;Poyarkov et al., 2018). However, the absence of any new collections of this species from its type locality or close vicinities since its original description had propagated confusions surrounding the identity of this taxon, as well as deterred meaningful taxonomic studies in the genus (e.g. Matsui et al., 2011;Poyarkov et al., 2018), until its recent rediscovery . Although, the distribution of this taxon has currently been restricted to Sumatra , the taxonomic status of a large number of populations identified as Micryletta inornata from other regions for over a century remains to be validated. Most reports of larger-sized (>20 mm) male as well as female specimens identified as M. inornata from other regions (Fig. 7) are all likely to be misidentifications requiring confirmation based on detailed morphological and molecular studies. Hence, in order to avoid any confusions surrounding the identity of Micryletta inornata (sensu stricto) and to define it objectively, in accordance with Article 74 of International Code of Zoological Nomenclature (International Commission on Zoological Nomenclature, 1999) we find it important to designate a lectotype from the syntypes (one of which remains unavailable) so that it can become the unique name bearer of this taxon. We examined the two currently available specimens (one male, NHM 1889.11.12.30 (ex. BMNH 1947.2.11.75) and one female, NHM 1889.11.12.4 (ex. BMNH 1947) at NHM, London. The female syntype NHM 1889.11.12.4 (ex. BMNH 1947 from "Deli, Sumatra" agrees with the original description, with respect to snout-vent size ("From snout to vent 20 million") and most other characters. Since this specimen was found to be in a relatively good condition, apart from bleached dorsal skin, we herein designate NHM 1889.11.12.4 (ex. BMNH 1947, an adult female, as the lectotype of Microhyla inornata Boulenger, 1890 (= Micryletta inornata), in order to stabilise the nomenclatural status of this species. The lectotype description provided below is largely consistent with the original description.
Description of lectotype (measurements in mm). Adult female (SVL 19.5); head wider (HW 5.9) than long (HL 5.3); snout nearly rounded in dorsal and ventral view, acute in lateral view, its length (SL 2.2) slightly shorter than horizontal diameter of the eye (EL 2.4); loreal region acute with rounded canthus rostralis; interorbital space flat, wider (IUE 2.5) than the upper eyelid (UEW 1.3) and internarial distance (IN 1.5); tympanum weakly-developed, supratympanic fold extending from posterior corner of eye to the shoulder weakly-developed; vomerine teeth absent; tongue small, oval, without papillae. Forearm (FAL 4.8) nearly equal to hand length (HAL 4.7); fingers without dermal fringes, finger length formula: I < II < IV < III, finger discs slightly wide compared to finger width, discs without grooves; fingers free of webbing; subarticular tubercles prominent, oval, all present, subarticular tubercle formula: F I 1, F II 1, F III 2, F IV 2; single rounded supernumerary palmar tubercles at the base of fingers II, III, and IV; three metacarpal (palmar) tubercles; inner metacarpal tubercle distinct, rounded, smaller; outer metacarpal tubercle rounded, larger; medial metacarpal tubercle rounded, large; nuptial pad absent. Hind limbs slender; tibiotarsal articulation reaches up to the level of eye when stretched forward along the body axis; thigh length (TL 8.1) shorter than shank (SHL 8.6) and foot (FOL 8.6); toe discs slightly wide compared to toe width, discs without grooves; toes free of webbing; subarticular tubercles on toes prominent, oval; single inner metatarsal tubercle (IMTL 0.4), prominent, rounded; outer metatarsal tubercle, and supernumerary tubercles absent.
Diagnosis. Micryletta steinegeri differs from all other members of the genus by the following suite of morphological characters: SVL 22.6-23.5 mm, N = 3 in males, 27.0-30.1 mm, N = 2 in females; outline of snout rounded to truncate in dorsal and ventral view; tibiotarsal articulation reaching up to the level of tympanum when stretched forward along the body axis; dorsum dark grey to violet with irregular dark blotches or speckles; dorsal skin smooth to shagreened; anterior and posterior parts of thigh light flesh-red without mottling; belly, fore-and hind limbs greyish-white; and rudimentary webbing between toes. For comparison with other congeners, see the 'Morphological comparison' section of M. aishani sp. nov.
Rationale for lectotypification. The original description was based on five specimens from "Kanshirei", "Formosa" (= Taiwan) (Boulenger, 1909 NHM 1909.10.29.92 (ex. BMNH 1947.76) from "Kanshirei", exactly agrees with the original description in terms to snout-vent size ("Total length 30 mm") and most other characters. Since this specimen is likely to have been the one used by Boulenger (1909) to describe the species, in accordance with Article 74 of International Code of Zoological Nomenclature (International Commission on Zoological Nomenclature, 1999) it is advisable for it to become the unique name bearer of the taxon and the standard for its application. This specimen was also found to be in a relatively good condition. Therefore, we herein designate NHM 1909.10.29.92 (ex. BMNH 1947, an adult female, as the lectotype of Microhyla steinegeri Boulenger, 1909 (= Micryletta steinegeri), in order to stabilise the nomenclatural status of this species. The lectotype description provided below is largely consistent with the original description.

DISCUSSION
The discovery of a new species of Micryletta from Northeast India highlights that although this region is considered as a 'gateway' between the Indian subcontinent and Eurasia, it harbours some unique microendemics, which are often overlooked due to presumed similarities with faunal elements in adjoining Southeast and East Asia (Mani, 1995;Kamei et al., 2012). It is therefore important to extensively document the biodiversity of Northeast Indian regions to gather a proper understanding of extant life forms and the evolutionary relationships between South and Southeast Asian faunal elements. Although rare to find due to its extremely short breeding season, the new Micryletta species is likely to be more widely distributed and common within the Northeast states. Apart from the confirmed records of M. aishani sp. nov. from regions south of River Brahmaputra in Assam, Manipur, and Tripura, one of us (AD) has also observed an unidentified Micryletta population (not collected) north of the river in Manas National Park, Assam. Further studies are required to ascertain the taxonomic status of this population as well as the role of physical barriers such as River Brahmaputra that divides Northeast India into two distinct biogeographical regions (Mani, 1995). After this study, it is now certain that the geographical distribution of genus Micryletta covers most of Indo-Burma biodiversity hotspot (including Northeast India) and extends towards Sundaland; the former appearing to be the major centre for species-level diversification. Further surveys in adjoining Himalayan regions will be necessary to understand the distribution limits of Micryletta. The biogeography of the genus could also shed light on various barriers of dispersal for amphibian species among the three adjoining, yet unique, biodiversity hotspots.
The present study also provides taxonomic insights on this morphologically complex genus of microhylid frogs with relatively conserved morphology at species-level despite As noted by previous researchers, members of the genus Micryletta have contrasting dorsal markings (e.g. Wang, Wu & Yu, 1989;Matsui et al., 2011;Poyarkov et al., 2018). However, Wang, Wu & Yu (1989) reported various colour morphs of M. 'inornata' categorised in to four types of dorsal markings, suggesting that dorsal colour and markings may have little taxonomic value in identification of Micryletta species. At the same time, they discussed size variations with Taiwanese populations shown to be larger than Thailand populations (Wang, Wu & Yu, 1989). Hence, the confusing morphology combined with overlapping distribution ranges of species makes delineation of the yet unidentified specimens and/or phylogenetically distinct lineages difficult. The rediscovery of M. inornata from Sumatra  and the proper identification of Northeast Indian populations in the present study, will aid future studies in providing a further resolution to the complicated taxonomy of Micryletta, consequently also enabling a better understanding of the patterns of diversification and geographical distribution in this group. A wider taxon sampling throughout the range of the genus in South, Southeast, and East Asia, especially from unexplored intervening regions, will also be necessary to gather a better understanding of intra-and interspecific variations, both morphologically and genetically (e.g. Matsui et al., 2011;Poyarkov et al., 2018). This could further result in the discovery of unidentified lineages and fill in the gaps to fully reconstruct the evolutionary history of this enigmatic group of microhylid frogs.

CONCLUSIONS
Our description of a new species of Micryletta from Northeast India contributes to a better understanding of the diversity in this genus. The discovery also genetically validates the presence of the genus Micryletta in India and the westward extension of its geographical range within South Asia. The study provides evidence for genotypic and phenotypic distinctness of the new species from all the previously recognised congeners, designates lectoptypes for nomenclatural stability of two previously known species, and confirms the presence of additional undescribed lineages within the genus. Altogether, our work will facilitate future taxonomic, phylogenetic, and biogeographical studies in this microhylid group.

Animal Ethics
The following information was supplied relating to ethical approvals (i.e. approving body and any reference numbers): Research received ethical approval from the Wildlife Institute of India, Dehradun (WII/ ESM/AD/AES-05) and Department of Environmental Studies, University of Delhi (DES/ 1020), India.

Field Study Permissions
The following information was supplied relating to field study approvals (i.e. approving body and any reference numbers): Fieldwork and sampling were conducted with permissions from responsible authorities in the State Forest Departments of Assam, Tripura, and Manipur (Study permits: No.

DNA Deposition
The following information was supplied regarding the deposition of DNA sequences: The DNA sequence is available at GenBank (MK889218).

Data Availability
The following information was supplied regarding data availability: Type specimens are deposited at Zoological Survey of India, Kolkata (ZSIC) under accession numbers ZSIC 14304-14312. The newly generated DNA sequence is available at GenBank: MK889218.