Hoplobatrachus litoralis, Hasan, Mahmudul, Kuramoto, Mitsuru, Islam, Mohammed Mafizul, Alam, Mohammad Shafiqul, Khan, Mukhlesur Rahman & Sumida, Masayuki, 2012

Hasan, Mahmudul, Kuramoto, Mitsuru, Islam, Mohammed Mafizul, Alam, Mohammad Shafiqul, Khan, Mukhlesur Rahman & Sumida, Masayuki, 2012, A new species of genus Hoplobatrachus (Anura, Dicroglossidae) from the coastal belt of Bangladesh, Zootaxa 3312, pp. 45-48 : 47-56

Hoplobatrachus litoralis sp. nov.

Holotype. IABHU 3993, adult female (SVL: 100.6 mm) collected from Teknaf, Cox’s Bazar district (20° 52ʹ N, 92° 18ʹ E,> 5 m asl.), Bangladesh on 20 June 2011 by M. Hasan ( Figs. 1 A, 1B).

Paratypes. IABHU 3974, adult female (SVL: 121.3 mm), IABHU 3979, adult female (SVL: 109.1 mm), and IABHU 3980, adult female (SVL: 119.3 mm) collected from Ukhia, Cox’s Bazar district, Bangladesh on 19 June 2011 by M. Hasan. IABHU 3989, adult male (SVL: 89.5 mm), IABHU 3992, adult male (SVL: 83.7 mm), IABHU 3994, adult male (SVL: 84.7 mm) and IABHU 3997, adult female (SVL: 96.1 mm) collected from Teknaf, Cox’s Bazar district, Bangladesh on 20 June 2011 by M. Hasan.

Diagnosis. Large frog with SVL of 81.3–102.1 mm in males and 83.2–121.3 mm in females. A broad black band from anterior corner of eye through the nostrils to anterior edge of upper jaw, and another band along the lateral margin of upper jaw ( Fig. 1 C) are more distinct than in its close relatives H. tigerinus and H. rugulosus where the band widths are uneven and often discontinuous ( Fig. 1 D). There is a distinct black margin in the inner side of the upper arm in the new species, but such margin absent in H. tigernius ( Fig. 1 E-F). The inner metatarsal tubercle of the new species is black ( Fig. 1 G), whereas it is pigmentless in H. tigerinus ( Fig. 1 H). Inter-orbital distance is much narrower than eyelid width and inter-nostril distance in the new species (ELW/E-E = 1.875, N-N/E-E = 1.575, on average), whereas these values are nearly the same in H. tigerinus (ELW/E-E = 1.060, N-N/E-E = 1.002) and H. rugulosus (ELW/E-E = 1.021, N-N/E-E = 1.004).

Description of holotype (measurements in mm). Vomerine teeth, long oblique lines between choanae. Tongue tip bifurcated. Distinct symphysial knob on anterior edge of lower jaw.

Head longer than wide (HL: 43.9; HW: 40.2), obtusely pointed. Canthus rostralis blunt. Loreal region concave. Nostril nearer to tip of snout than to eye (S-N: 6.6; N-E: 10.7). Tympanum large, slightly smaller than eye (TD: 8.3; ED: 8.9). Inter-orbital space much narrower than eyelid width and inter-nostril space (E-E: 3.9; ELW: 7.2; N-N: 6.5).

Fingers free, finger tips blunt without disk. Finger length F3> F1> F2> F4 (F1: 12.9; F2: 10.3; F3: 13.2; F4: 7.7). Subarticular tubercles moderate. Thenar and palmar tubercles distinct.

Hindlimb about 1.6 times SVL (HLL: 165.3; SVL: 100.6). Femur length subequal to tibia length (FEL: 51.0; TIL: 51.8). Toe tips blunt, slightly rounded. Toe length T4> T5> T3> T2> T1 (T1: 11.8; T2: 17.8; T3: 26.2; T4: 34.6; T5: 27.9). Wide web, reaching the base of toe-tip disk ( Fig. 1 D). Subarticular tubercles were weak. Inner metatarsal tubercle moderate (IMT: 4.6). No outer metatarsal tubercle.

Many thin longitudinal ridges on the dorsum ( Fig. 1 A). Small round warts over dorsal and lateral side. Supratympanic fold from behind eye to posterior margin of tympanum. Weak tarsal ridge extending from proximal end of inner metatarsal tubercle to heel. Ventral side including thigh and tibia smooth.

Color in alcohol. Dorsum dark gray with many large black spots ( Fig. 1 A). Thin whitish mid-dorsal stripe from tip of snout to vent. Lateral side with many small black dots. Wide black band from anterior corner of eye through the nostrils to anterior margin of upper jaw ( Fig. 1 C). Another band on the lateral margin of upper jaw ( Fig. 1 C). Ventral side immaculate, except for large black blotches along the edge of lower jaw to the base of forelimb ( Fig. 1 B).

Large transverse black bands on the upper surface of thigh, tibia, and tarsus to the outer edge of foot. Rear side of thigh heavily mottled. Outer side of tarsal ridge dark, whereas inner side yellowish with several dark irregular blotches. Web dark gray, except for whitish upper side of inner web (between toes 1–4).

Color in life. Dorsal ground color varies from yellowish to dark brown with many dark brown to black spots. Large transverse black bands are present on the dorsal surface of the thigh, tibia, and tarsus region ( Fig. 2 A). Middorsal stripe is yellowish white. Bands running from the anterior part of eye to upper jaw margin and on the lateral margin of upper jaw are black. Many fused spots on the posterior surface of thigh are black with thin yellow reticulations between them. Tympanum is dark gray with pale central circle. A short discontinuous black line is present below the eye. Venter is creamy white with a few black blotches from the margin of lower jaw to base of forelimb ( Fig. 2 B).

Variation. Males have a pair of gray (in alcohol) subgular vocal sac on the underside of the jaw angle, and a well-developed nuptial pad on the base of 1st finger.

Of the 26 specimens examined, three (12%) lacked a mid-dorsal stripe. A broad mid-dorsal stripe, often found in Fejervarya species, was absent. Number and size of dark spots along the margin of the lower jaw varied, and only one specimen lacked these spots. Usually, the lateral upper jaw stripe is continuous, but in 6 specimens the stripe is fragmented into irregular markings.

Morphological comparisons. Among the three Hoplobatrachus species in Bangladesh ( H. litoralis , H. tigerinus , and H. crassus ), H. crassus is easily distinguishable from the other two by large shovel-like inner metatarsal tubercle and usual occurrence of large dark dots in the gular and pectoral region. In the following, we compare the morphological characters of H. litoralis with those of H. tigerinus and H. rugulosus . Hoplobatrachus rugulosus is so similar to H. tigerinus in that it was previously regarded as a subspecies of H. tigerinus . Because H. rugulosus occurs in Myanmar, just adjacent to the distribution range of H. litoralis , comparisons of the two are relevant.

All specimens (n = 15) of H. tigerinus from Mymensingh, Bangladesh had a thin mid-dorsal stripe. Five specimens (33%) had a thin yellowish stripe along the inner side of the tibia, usually extending over the upper side of the thigh to the base of the thigh. This line was not observed in H. litoralis . The underside of the mandible is finely dotted in most specimens. A marginal stripe along the jaw margin is usually fragmented into narrow irregular markings. The stripe from the eye through the nostrils to the anterior edge of the upper jaw is not as broad as in H. litoralis and is usually fragmented. In H. tigerinus , the broad light line from behind the eye to the groin is more distinct than in H. litoralis . Webs are mottled with an irregular dark pattern, in contrast to the uniform gray in H. litoralis .

Specimens of H. rugulosus from Thailand (n = 7) lacked a mid-dorsal stripe. The snout was rounded rather than pointed. The dorsal dermal ridges were wider and shorter than that of H. litoralis and H. tigerinus .

There are several old nomina which have been treated as junior synonyms of currently recognized Hoplobatrachus species. Rana brama Lesson, 1834 , a large frog described from Bengale, was reported as having very smooth surface, depressed dorsum, and short depressed head. Femur is much shorter than tibia. Obviously, H. litoralis differs from R. brama in these characteristics. Pyxicephalus frithi Theobald, 1868 described from Jessore, SW Bangladesh was reported to have quite smooth skin and uniform vinous coloration. These do not fit H. litoralis . Rana burkilli Annandale, 1910 was described from Tavoy, Myanmar. This species differs from H. litoralis in having ventral surface “marked with black, the markings sometimes taking on a reticulate character all over the belly”. The inner metatarsal tubercle was described as feebly developed, but H. litoralis has well-developed inner metatarsal tubercle. Rana gracilis (not of Gravenhorst 1829) Boulenger, 1920, a small frog (SVL 50 mm for male, 41 64 mm for female) described from Sri Lanka, was disclosed as having an oval inner metatarsal tubercle and smooth skin or feebly granulate above. Rana gracilis var. pulla (Stocliczka 1870) described from Penang hill, Malaysia was reported to be a very small frog (SVL is 7/ 8 inch [<23 mm] and HLL is 1 1/ 2 inch [nearly 38.1 mm]). Hoplobatrachus littoralis is distinguished from both R. gracilis and R. gracilis var. pulla considering its very large size (SVL = 89.96 mm and 101.42 mm; and HLL = 143.85 mm and 159.74 mm for male and female, respectively). The skin of H. litoralis is rough and its inner metatarsal tubercle is comparatively elongated rather than oval. Rana picta Gravenhorst, 1829 whose type locality is unknown ( Frost 2011) was reported to have three spots below one eye and there is no trace of tubercle on dorsal skin. These characteristics do not fit with the new species of H. litoralis .

Morphometric comparisons. Measurements of 29 body parts of H. litoralis , H. tigerinus , and H. rugulosus are summarized in Table 1. Mean SVL of males differs significantly (U test, P <0.05) between the three species, but that of females does not. Size of H. litoralis is slightly smaller than that of the other two species. SVL of females is larger than that of males (P <0.05) in H. litoralis and H. rugulosus .

The three species are clearly separated by canonical discriminant analysis ( Fig. 3 A). Eigenvalues are 11.606 for function 1, and 5.684 for function 2. Coefficients for function 1 are large especially in E-E, N-N, N-E, HAL, and ED for function 1 and in E-E, HAL, TD, and TIL for function 2. In principal component analysis ( Fig. 3 B), H. litoralis made completely separate cluster from H. rugulosus , but the scores of H. litoralis and H. tigerinus overlapped considerably.

Body ratios relative to SVL (e.g., HL/SVL and HW/SVL) and 10 other ratios (e.g., HL/HW and S-N/N-E) are shown in Table 2, and the results of the Mann-Whitney U test between ratios of H. litoralis vs. H. tigerinus and H. rugulosus are shown in Table 3. Mean values of ED/SVL, ELW/SVL, ED/E-E, N-N/E-E, and ELW/E-E are significantly large (P <0.01) in H. litoralis compared with those in H. tigerinus and H. rugulosus . These are apparently derived from relatively large ED, ELW, and N-N and relatively small SVL and E-E of H. litoralis .

Advertisement calls. Advertisement calls of H. litoralis are low-pitched groans emitted at about 4.4 s interval. Mean call duration is 0.28 s (n = 34). The call is composed of about 20 rapidly repeating pulses ( Fig. 4 A). Fundamental frequency is 0.30 kHz and the 3rd and 4th harmonic bands (at about 1.2 kHz) are dominant ( Fig. 4 B).

Roy and Elepfandt (1993) and Kanamadi et al. (1994) reported the acoustic features of H. tigerinus from northeast India (Assam and Meghalaya) and southwest India (Karnataka), respectively. Call durations are reported as 0.30 s (NE India) and 0.22 s (SW India); these values do not differ significantly from those of H. litoralis . Dominant frequency bands in H. tigerinus are 1.65 kHz and 0.52 kHz in NE India populations and 1.5–2.2 kHz and 0.2– 1.2 kHz in SW India populations. These dual-dominant bands are absent in H. litoralis . The number of pulses in a call is larger in H. litoralis than in H. tigerinus (16 in NE India and 12.6 in SE India).

Advertisement calls of H. crassus ( Kanamadi et al. 1992) are different from those of H. litoralis and H. tigerinus with regard to distinctly few number of pulse groups (2-4 per call) and show many intense harmonic bands.

Divergence in mitochondrial 16S rRNA and Cytb gene sequences. The average sequence divergence for 16S and Cytb was 3.2% and 14.2%, respectively, between H. litoralis from Cox’s Bazar and H. tigerinus from Mymensingh, while these values were only 2.1% and 9.1%, respectively, between the Mymensingh and Western Ghats populations of H. tigerinus ( Fig. 5 ). Hoplobatrachus litoralis is diverged from H. tigerinus of the Western Ghats by 3.3% for 16S and 12.0% for Cytb. Hoplobatrachus litoralis clearly differ from H. tigerinus from Mymensingh and the Western Ghats (BP: 92 for ML, 93 for MP, and 100 for BI). All H. litoralis specimens formed one clade supported by high bootstrap values (BP: 100 for ML, MP, and BI), while the H. tigerinus clade from Mymensingh and the Western Ghats was supported by medium bootstrap values (BP: 54 for ML, 81 for MP, and 67 for BI).

Distribution. Hoplobatrachus litoralis occurs in the southeastern coastal belt in Ukhia, Teknaf Upazilla (subdistrict) and Cox’s Bazar town of Cox’s Bazar district (21° 45ʹ N, 91° 97ʹ E,> 3 m asl.) in Bangladesh. The preferable habitat of this species is a vegetated, marshy ditch/ pond, beside the wetland created by hill stream (locally called “Jiri”) and/or sometimes the base of mountains having different soil texture from the mainland Bangladesh. This new species is sympatrically occurs with other anuran species such as H. crassus , as well as some Euphlyctis , Fejervarya , and Polypedates species. Hoplobatracus tigerinus is widely distributed in mainland Bangladesh ( Alam et al., 2008) as well as also in coastal region ranging from southwestern Shatkhira to southeastern Bandarban including Shatkhira, Barguna, Patuakhali, Bhola, Sandwip and Raojan of Chittagong and Bandarban districts (Islam et al. unpublished); but no H. tigerinus specimens have been obtained from Cox’s Bazar district area. Thus we have no confirmation about the overlapping region between this two species, but we speculate that this species is endemic in Cox’s Bazar district. The adjacent area of Myanmar (Teknaf) is only separated by the Naaf river, which probably does not constitute a strong barrier to H. litoralis migration. Thus it is possible that our new species may also occur in the adjacent coastal geographic region of Myanmar. But as we have no access for sampling in that region, we cannot confirm whether our new species is distributed in that region of Myanmar or not. Approximate distribution of all Asian Hoplobatrachus species is shown in Fig. 6 A, whereas the detail distribution of the three genetically different Hoplobatrachus species in Bangladesh is shown in Fig. 6 B.

The Teknaf-Ukhia peninsula is long, narrow and forested area rising up to 300 meters above the sea level and, encompassed by the Bay of Bengal to the west and the Naaf river to the east. This is a diverse habitat for many flora and fauna due to its special characteristics i.e. estuarine habitat and wetland at the base of the mountain. In April 1999, the Department of Environment (DOE) of Bangladesh declared that Teknaf peninisula is a Ecologically Critical Area (ECA) due to adverse change of its ecosystems by human activities. It is an important nesting site for at least four species ( Chelonia mydas , Eretmochelys imbricata , Lepidochelys olivacea and Dermochelys coraicea ) of marine turtle listed as globally threatened by IUCN. A few globally threatened shorebirds ( Eurynorhynchus pygmeus , Limnodromus semiplamatus , Tringa guttife r) also prefer this region for their habitat ( GoB/GEF/UNDP 1999).

Etymology. The specific name is derived from the Latin litoralis meaning coastal, in reference to the distribution range of this species; the Coastal belt of Bangladesh.

Figures

FIGURE 1. Holotype (IABHU 3993) of Hoplobatrachus litoralis sp. nov. after preservation in alcohol. (A) Dorsal aspect. (B) Ventral aspect. (C) Frontal aspect of head showing distinct black bands, compared with (D) that of H. tigerinu s (IABHU 3940). (E) Coloration of upper arm of H. litoralis (IABHU 3993), compared with (F) that of H. tigerinus (IABHU 3940). (G) Foot of paratype (IABHU 3980), compared with (H) that of H. tigerinus (IABHU 3940). Scale bar = 10 mm.

FIGURE 2. Holotype (IABHU 3993) of H. litoralis sp. nov. in life. (A) Dorsal view. (B) Ventral view. Scale bar = 25 mm.

FIGURE 3. (A) Scatterplot of individual discriminant scores on the first (CA 1) and second canonical axes (CA 2) for H. litoralis (circle), H. tigerinus (triangle), and H. rugulosus (rectangle). (B) Scatterplot of principal component 1 (PRN 1) versus principal component 2 (PRN 2) from the principal component analysis of H. litoralis (circle), H. tigerinus (triangle), and H. rugulosus (rectangle). In each species males were marked as solid and females were marked as open icons.

FIGURE 4. Sound spectrograms showing advertisement call structure of H. litoralis. The same call analyzed by FlatTop (A) and Hamming window (B). Abscissa: time in s. Ordinate: frequency in kHz. The continuous broad band at about 2.5 kHz is chirps of an insect. The call of several male individuals of H. litoralis was recorded from Ukhia, Cox’s Bazar district on 19 June, 2011 just after sunset and their voucher specimen numbers are involved in those of molecular analyses, but not specified.

FIGURE 5. Maximum likelihood (ML) tree based on the nucleotides sequence of 1078 bp of mitochondrial (16 S + Cytb) genes with Euphlyctis cyanophlyctis and E. hexadactylus as out groups. The most parsimonious and Bayesian analyses reconstructed the same tree topology. Numbers near branches represent bootstrap support for ML and MP inferences, and Bayesian posterior probability (ML – BPs / MP – BPs / BPP). The scale bar represents 0.1 nucleotide substitutions per site. The superscript letters indicate that the 16 S and Cytb data were taken from Alam et al. (2008) for constructing this tree. a) AB 272594, AB 274137; b) AB 290412, AB 274139; c) AB 290417, AB 290601; d) AB 290414, AB 290603; e) AB 272596, AB 274144; f) AB 290413, AB 290597; g) AB 272599, AB 274148; h) AB 272600, AB 274150; i) AB 272601, AB 274151 and j) AB 272605, AB 274163.

FIGURE 6. (A) Map showing the approximate distribution areas of all Asian Hoplobatrachus species based on Frost et al. (2011) and Alam et al. (2008). (B) Bangladesh map showing the collecting localities of H. litoralis is indicated by (closed triangle) and, the collecting localities of H. tigerinus and H. crassus are indicated by (closed circle) and (closed square), respectively. Each locality was used for molecular analysis, and the boxed locality was used for morphology.