Field identification characters to diagnose Microhyla mukhlesuri from closely related M . mymensinghensis ( Amphibia : Microhylidae ) and range extension of M . mukhlesuri up to West Bengal State , India

Microhyla mymensinghensis and Microhyla mukhlesuri were recorded from West Bengal, also analyzed 12 characters to separate the species. We also hereby first time confirm the presence of Microhyla mukhlesuri from the main land in India


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Twenty-seven species have been described within the last 15 years (Frost 2021), yet Microhyla remains one of the most taxonomically exciting groups of Asian frogs. Microhylidae is one of the most species rich family of Anura, comprising 690 species in 12 subfamilies (Frost 2020  . Microhyla mymensinghensis was first described from Mymensingh, Bangladesh together with M. mukhlesuri (see Hasan et al. 2014). Microhyla mymensinghensis was recorded from Manipur, Meghalaya, Nagaland, Tripura, and West Bengal in India. But M. mukhlesuri has been listed only from Mizoram state in the northeastern India . No details were reported to confirm the finding. Our study confirms the presence of both species in West Bengal, by analyzing 16 morphometric characters to separate these species. We also hereby confirm the presence of Microhyla mukhlesuri from J TT the Gangetic Plain of West Bengal in India, as it was described from Bangladesh (Hasan et al. 2014).

Materials and Methods
Field work was conducted during late evening in the months of May-July 2020 in Kharagpur, Medinipur Sadar ( For species-level analysis we examined 16 morphological characters to elucidate species identification (1) Body size (SVL) (2) Head length and head width (HL/HW) (3) extent of Tibiotarsal articulation (4) Shape of Terminal phalanges on toes (5) Shape and size of Inner and outer metatarsal, metacarpal tubercle; (6) Webbing on toes (7) length of femur and tibia (8) Dorsal spotting. (9) extent of Lateral stripe (10) pattern on the anus. (11) FAL= Forearm length, LAL= Lower arm length, HAL= Hand length, FAW= Forearm width 12. Axilla-Groin distance (A-G) ( Table 1). We also have added HL, HW, FAW, TL and A-G ratios with SVL (Table no 2). We have grouped the species based on characters described by Hassan et al. (2014).
Principal Component Analysis (PCA) was performed on 16 morphometric measurements from specimens of both Microhyla mukhlesuri (n= 4) and Microhyla mymensinghensis (n= 5) using PAST 3.0 Software. Before doing the PCA, a normality test was done for all the variables. PCA factor scores for principal components (PC) with eigenvalues >1.0 were reported. Factor scores of the first two components were visualized on scatterplots to assess the degree of morphological differentiation between specimens of the two species.

Results
Two Microhyla species were collected during the present study Microhyla mukhlesuri (n= 4) and M. mymensinghensis (n= 5). The specimens were identified to belong to the genus Microhyla by the following morphological characters: absence of vomerine teeth, hidden tympanum, elliptical tongue, short snout, small eyes not protuberant and invisible from the ventral side, indistinct canthus rostralis and fingers free of webbing. Four specimens were identified as Microhyla mukhlesuri (Image 2a) based on size, extent of tibiotarsal articulation, mark on anus, forearm width, tibia length and shape of terminal phalanges Hasan et al. 2014). This is the first record of Microhyla mukhlesuri from entire Gangetic plains of India, West Bengal (Nayagram, 22.0361˚N, 87.1717˚E) (Image 1) which extends its range by 494 km from its type locality (Rhozan, Chittagong, Bangladesh) and by 568 km away from Mizoram, India (which was the only record from India Garg et al. 2019). Whereas M. mymensinghensis (Image 2b) is identified based on size, tibia length, mark on anus and forearm width Hasan et al. 2014). We found M. mukhlesuri differs from M. mymensinghensis by its relatively larger adult size ) and inverse U-shaped black mark above the anus for M. mukhlesuri whereas crescent-shaped black mark present above the anus in M. mymensinghensis as described by Hassan et al. (2014).
Identifying taxonomic characters to differentiate M. mukhlesuri and M. mymensinghensis is problematic. Both species exhibit similar extent of tibiotarsal articulation (when the hind leg is adpressed) up to the snout or between the eye and snout and a lateral stripe that also extends to near the snout. Garg (Table 1). Based on these characters we have identified and classified the specimens examined in this study. In addition to aforementioned described characters, we identified a few supplementary characters to differentiate two species more confidently: (a) shape of inner metacarpal tubercle elongated in M. mukhlesuri vs. rounded in M. mymensinghensis; (b) large inner metatarsal tubercle, i.e., about 0.76 (±.08) mm in M. mukhlesuri vs about 0.57 (±.06) mm in M. mymensinghensis; (c) most significantly, axilla-groin distance 47% of SVL in M. mukhlesuri vs 38% of SVL in M. mymensinghensis. Both these species were observed co-occurring in humanized / agricultural habitats in the sampled areas (Image 3a, b).
Microhyla mukhlesuri showed differences with M. mymensinghenis in terms of morphology. Principal Component Analysis (PCA) for the specimens of both the species (M. mukhlesuri, n= 4; M. mymensinghensis, n= 5) recovered two Principal Components (PC) with eigenvalues > 1.0 that accounted for 85.8% of the total variance. PC1 explained 74.09% variance with highest loadings for A-G, TL, SVL and FoL. PC2 explained 11.71% variance with highest loadings for A-G, and HW. The remaining factors explained 14.2% of the variations. For the combined data set for both male and female projections of the factor planes 1 and 2 showed distinct clusters for the two species (Figure 1). An inverse U shape mark on the anus for M. mukhlesuri (vs crescent shaped mark in M. mymensinghensis); larger SVL in M. mukhlesuri (19.92-20.86 mm) vs. shorter (17.22-18.86 mm) in M. mymensinghensis and terminal phalanges of toes knobbed but tip flattened in M. mukhlesuri (vs. knobbed but not flattened in M. mymensinghensis) are used to group the two species. In addition to this we have identified axilla-groin distance, size of metatarsal tubercle and shape of metacarpal tubercle to differentiate these two species more confidently. The PCA results reveal two different clusters on the scatterplot, representing two different species -Microhyla mukhlesuri (n= 4) and M. mymensinghensis (n= 5).
Microhyla mukhlesuri has been reported only from Mizoram state in the northeast India ). With two males and two female specimens we hereby for first time confirm the presence of Microhyla mukhlesuri from the Gangetic Plain in West Bengal, apart from Mizoram. We are optimistic about the wider distribution of Microhyla mukhlesuri in the Gangetic plains. Nevertheless we do encourage genetic studies of these Microhyla frogs, especially after finding geographic contact zones where two similar-looking, geneticallyallied congeners Microhyla mukhlesuri and M. mymensinghensis co-occur, throwing open necessity for further fine-scale diagnosis, preferably morphological, between them.