A new species of the genus Euphlyctis (Anura: Dicroglossidae) from West Bengal, India

A new species of green frog of the genus Euphlyctis Fitzinger, 1843 is described from West Bengal, a state of eastern India. A robust frog, SVL of male 86mm and that of female 132mm. The species is diagnosed by the presence of following characters: green dorsum and female with a greenish white mid-dorsal line, tibiotarsal articulation reaches eye, male with two vocal sac openings at the junction of jaw, female is larger than male though body parameters is proportionately longer in male, nostril snout length 3.45% of SVL, nostril much closer to snout tip than eye, units of hind limb i. e. thigh, shank and foot are almost equal in length, relative length of finger: II < IV < I < III. The new species is compared with existing eight species of the genus Euphlyctis.


Materials and methods
Two live specimens of Euphlyctis (one male and one female) were collected at night from Bhadirban (six km. west of Suri), Birbhum district, West Bengal, India and kept in a large plastic jar with a few pores at the top. Next day, the specimens were killed by chloroform. Morphological data were taken with the help of a digital slide caliper, a divider and a millimeter scale. Photographs were taken immediately after killing. The specimens were preserved in 4% formaldehyde solution for further investigation.
The abbreviations used for morphometric analysis and comparison with other species are as follows: SVL= Snout vent length, HL= Head length (from the rear of mandible to the tip of snout), HW= Head width (length at the angle of jaws), HD= Head depth (vertical length posterior to orbit), UAL= Upper arm length (from the base of upper arm to elbow), FAL= Fore arm length (from the fixed elbow to the base of outer palmer tubercle), HAL= Hand length (from the of outer palmer tubercle to the tip of the third finger), THL= Thigh length (from the vent to the knee), SHL= Shank length (from knee to heel), FOL= Foot length (from the base of inner metatarsal tubercle to the tip of the fourth toe), TFOL= Distance from the heel to the tip of the fourth toe, TFLL= Total fore limb length, THLL= Total hind limb length, EN= Distance from the front of the eye to the nostril, EL= Eye length (horizontal distance between the orbital borders), SL= Snout length (from the tip of the snout to the anterior border), NS= Distance from the nostril to the tip of the snout, IN= Internarial distance (shortest distance in between two nostrils), IUE= Inter upper lid width (shortest distance between the upper eye lids), UEW= Maximum upper eye lid width, IFE= Interval front of the eyes (shortest distance between the anterior orbital borders), IBE= Interval back of the eyes (shortest distance between the post orbital borders), MN= Distance from the back of mandible to nostril, MFE= Distance from the back of mandible to the front of eye, MBE= Distance from the back of mandible to the back of eye, IOL= Inter orbital length, OTL= Posterior of orbit to anterior of tympanum length, TD= Tympanum diameter, FLI-IV= Finger length of I to IV, NP= Nuptial pad, TLI-V= Toe length of I to V, IMTL= Length of inner metatarsal tubercle, MTFF= Distance from distal edge of metatarsal to maximum incurvature of web between fourth and fifth toe, MTTF= Distance from distal edge of metatarsal to maximum incurvature of web between third and fourth toe, TFTF= Distance from maximum incurvature of web between third and fourth toe to tip of fourth toe, FFTF= Distance from maximum incurvature between fourth and fifth toe to tip of fourth to tip of fifth toe.
E. bengalensis was compared with other eight species of Euphlyctis on the basis of different morphometric characters by paired t-test. Spearman's correlation was performed to separate different morphometric characters of male and female frogs. Different body parameters (HL, HW, etc.) of nine species of Euphlyctis were compared by using ANOVA to find out if the mean values of different body parameters are significantly different or not.
Principal component analysis (PCA) and construction of heatmap was performed on the multivariate data of nine species of Euphlyctis (see Table S1 in Supplementary Informations). PCA and heatmap analyses were performed using ClustVis: https://biit.cs.ut.ee/clustvis/ (Metsalu & Vilo, 2015). PCA does a linear transformation on the multivariate data to extract variables (principal components) that are uncorrelated. The first few components (otherwise known as principal components and denoted as PC1, PC2) capture the data with most variability. Plotting PC1 vs PC2 approximate the distance between points and if the points on the 2D PC plots are not overlapping then these points form separate clusters. Before PCA analyses, the original values are transformed using ln(x+1) function. Unit variance scaling was applied on the row values of the dataset and singular value decomposition (SVD) with imputation was used to calculate PCs.
Heatmap uses color coding to visualize multivariate data matrix. Looking at the phenogram integrated with heatmap one can get a helicopter view of how corresponding variables of different species are separated better than others. Euphlyctis bengalensis sp. nov. (Fig. 1).
Etymology. Species name is an adjective to the 'bengal', the state (West Bengal) from where holotype was collected.

Diagnosis.
Large and robust Euphlyctis species. The species is characterized by a combination of the following characters: SVL of holotype male 86 mm and that of paratype female 132 mm, dorsal dark green with a greenish white middorsal line in female only, nostril snout length 3.45% of SVL (n= 2), nostril much closer to snout tip than eye (more than double in length), nostril snout length 42.77% of distance from eye to nostril, fore arm length 18.89% of SVL, foot length 48.81% of SVL. Relative length of fingers: II< IV< I< III. Thigh, shank and foot are almost equal in length in both sexes. Relative length of toes: I< II< III< V< IV.
Description. (Table 1).Adult male (Holotype): Large sized frog (SVL 86 mm). Head large, conical, broader than long (HW: HL= 1.25), a little depressed, canthus rostralis indistinct, loreal region oblique and concave, snout obtusely pointed both dorsally and ventrally, projecting beyond mouth. In lateral view, snout bluntly pointed. Snout length equal to eye length (EL= SL= 12 mm). Nostril much closer to snout than eyes (NS= 3.0, EN= 7.4), round and covered with a small flap (Fig. 3). Internarial distance slightly greater than interorbital length (IN= 4.3 mm, IOL= 4.0 mm) which is half of eye length (EL= 8 mm). Eye is moderately large (EL 28.57% of HL and 9.30% of SVL), protruding, pupil horizontal, iris blackish. Upper eye lid width is 1.5 times greater than interorbital length (UEW 21.43% of HL, IOL 14.29% of HL). Tympanum is round, large and close to eye (TD 30% of HL, OTL 10.99% of HL) and slightly larger than eye (EL 95% of TD). Supratympanic fold distinct. A distinct fold of skin runs from posterior of orbit to the base of shoulder covering the tympanum (Fig. 3). A pair of vocal sac openings on both sides of lower jaw near jaw angle in the form of 8 mm long slit (Fig. 4). The slit is as long as the length of eye. Vomerine teeth are oblique in position, in the anterior margin of choanae. Spatuate bifid tongue is with lingual papilla (Fig. 4). Lower jaw with three tooth like bony projections that fit in fossa on the upper jaw (Fig. 2).
Forelimb: Strong and stout, 69.19% of SVL. Fore arm length 1.3 times the hand length (FAL= 17.5 mm, HAL= 23 mm), fingers free, gradually tapering to pointed tips though base if first finger is very wide (Fig. 4). First finger is longer than the second. Red blister like oval nuptial pad (4.2 mm long and 3.1 mm width) is at the base of first finger (according to Flores, 1985, nuptial pad is Type I). Second and third fingers slightly fringed laterally. Subarticular tubercles are small and indistinct. Relative lengths of fingers: II< IV< I< III (FLI= 17.5 mm, FLII= 15.5 mm, FLIII= 20.5 mm, FLIV= 16.5 mm).
Hindlimb: Long and stout, 1.74 times of SVL. Heels overlap when folded at right angles to body, tibiotarsal articulation reaching eyes. Thigh, shank and foot length almost equal in length and more than half of SVL (52% of SVL) with cap like skin fold in knee and ankle (Fig. 3). Heel to tip of fourth toe 1.7 times longer than fourth toe length (TFOL= 69.5 mm, TLIV= 40.2 mm). Inner metatarsal tubercle is small and digitiform, about one-fifth of first toe length (IMTL= 3.8 mm, TLI= 19.5 mm), outer metatarsal tubercle absent. Toe tip small and slightly pointed. Subarticular tubercles are small and indistinct. Basal half of each toe is broad with skin folds while distal half is narrow (Fig. 3). First and fifth toe with well develop fringe (wider in the first toe). Relative toes lengths: I< II< III< V< IV (TLI= 19.5 mm, TLII= 25.5 mm, TLIII= 31.5 mm, TLIV= 40.2 mm, TLV= 34.2 mm). Fully developed web reaching toe tip and incised sharply, MTTF greater than MTFF (1.12 times) and FFTF is also greater than TFTF (1.33 times).
Adult female (Paratype): Most of the body parameters in female are proportionately lower compared to those of male. Both the fore and hind limbs in female is shorter than those of male (26% and 10% respectively). Moreover, finger and toe length in percentage of SVL is longer in male compared to those of female. Webs of hind limbs are also well developed in male. Some characters exhibited significant differences between sexes though proportionate values are within a reasonable range. 18 characters (HL, HW, UAL, FAL, HAL, THL, SHL, FOL, TFOL, EL, SL, IUE, IFE, MN, OTL, TD, FLI and TLI) of male and female frog are significantly different (Spearman's correlation, r s = 0.35).
Skin: Dorsum with small warts mostly on the posterior one-third and lateral side of the body. Fore limb smooth while hind limb with small warts except from heel to toes. A 'U'-shaped line of warts from upper portion of thigh to vent is present in both sexes (Fig. 4). Ventral is with porous warts, more distinct and large on the thorax and posterior of thigh.
Colour (in live): Dorsal leafy green, female with a greenish white mid-dorsal line from vent to snout tip ( Fig. 1 and 4), a few white spot on the lateral side of the body and upper portion of hand. Tympanum is also dark green and difficult to separate from body colour (Fig. 3). A few white spots are present at the base of hand and leg. Sides of body from hand to leg and lower part of the dorsum (in between two thighs) are with distinct green warts. Ventral portion of 1st and 2 nd finger are greenish while 3 rd and 4 th finger yellowish red. 1 st 2 nd and 3 rd toes reddish white while 4 th and 5 th toes are green. A white spotted line is present from ankle to base of first toe (Fig. 4). Ventral is yellowish in female and yellowish brown in male. In female, brownish patches are present particularly in thigh, shank and lower half of abdomen. Yellowish white large round warts are distinct on throat and breast (Fig. 3). Brownish 'V'shaped patch with white spot in thigh and shank is present in male (pale in female) (Fig. 1). Abdomen also have pale brownish patch. Posterior of thigh is brownish in colour with white irregular roundish spots (Fig.  2). Greenish narrow fold of skin is present around the vent (Fig. 3). Tongue reddish. Web bears dark marblings (Fig. 3).
Natural history. E. bengalensis sp. nov. a semi aquatic frog usually live half submerged in water throughout the day but at night often come to land for roaming and collection of food. They are very common in ponds and wetlands with full of water hyacinth and other species of floating plants.
The present species is clearly distinguished from its closest congener E. hexadactylus in having a smaller ratio of HL/HW (0.  15 vs. 1.11). NS in the present species is less than half of that of E. hexadactylus (3.45% SVL vs. 8.10% SVL). Snout in E. hexadactylus is longer compared to E. bengalensis (11.35% SVL vs. 20% SVL). In E. hexadactylus IOD is 23% less and TD is more than 10% greater compared to E. bengalensis. Moreover, all the fingers and toes are proportionately longer in E. bengalensis. In E. hexadactylus a prominent black central stripe from base of fore limb is present vs. absent in the present species. E. hexadactylus was reported to have a white or pale yellow venter (Dutta & Manamendra-Arachchi, 1996;Chanda, 2002;Daniel, 2002;Daniels, 2005) and the specimens of Mangalore have a finely mottled pattern on the venter (Joshy et al., 2009). But in the present species, venter is dark green with a few white spots around it.  (7.50 vs. 3.45, 6.10 vs. 4.40 and 11.30 vs. 8.68 respectively). Except first finger all other fingers and toes are slightly longer in the present species.

E. bengalensis
E. ghoshi not only restricted to Manipur but also very distinct from other species of Euphlyctis by combination of characters including rounded head, snout length 15% of SVL, tympanum diameter 6.67% of SVL and with minimum TD/ED ratio (only 0.50). Except the recently discovered E. karaavali, snout length in percentage of SVL is minimum in all other species (< 15%). Pair-wise comparisons of E. bengalensis with other eight species of Euphlyctis are presented in Table 2. The result shows that except two foreign species, E. kalasgramensis (from Bangladesh) and E. ehrenbergi (from Saudi Arabia) all six Indian species are significantly different from the present species.  (Table 3). Heat map including phenogram is presented in Fig. 5. Phenogram shows that for some morphological features E. bengalensis is morphologically more closure to E. aloysii however further genetic studies are necessary to compare these species beyond morphological characteristics.

Discussion
The type locality of all the Indian species of Euphlyctis are south and south-western part of India (Boulenger, 1920;Daniels, 1992;Biju, 2001;Joshy, 2009;Priti et al. 2016) except E. ghosi, the type locality of which is Manipur only (Chanda, 1990). Among these, only two species, E. cyanophlyctis and E. hexadactylus are also reported from eastern part of India including West Bengal (Srinivasulu et al., 2006). So far eastern part of India especially West Bengal have not been explored fully for amphibian species. In this context, this is the first report of new discovery of an amphibian species of the genus Euphlyctis from West Bengal.
The discovery of the present species from pond of agricultural land indicates that there is high diversity of amphibian species outside of forest. In this region, random use of chemical fertilizer and pesticides in agriculture is the major cause of dwindling of different animal species including amphibians. To explore the animal species in any locality, one of the best ways is to engage common man of that locality. On this principle, I have already started to explore different animal species by citizen engagement in my locality (Birbhum district, West Bengal) and reported four new species of freshwater fish and a new species of land planarian, Bipalium bengalensis. Moreover, I have collected a few species of frogs (of the genus Euphlyctis, Polypedates, Microhyla and Fejervarya), millipedes (Polydesmida and Harpagophorida), dipteran fly and aquatic insects. The present report is an outcome of this drive.