A New Species of Microhyla (Anurα: Microhylidαe) from Nilphαmαri, Bαnglαdesh Author Howlader, Mohammad Sajid Ali Ecological Genetics Research Unit, Department of Biosciences, University of Helsinki, Helsinki, Finland, Author Nair, Abhilash Ecological Genetics Research Unit, Department of Biosciences, University of Helsinki, Helsinki, Finland, Author Gopalan, Sujith V. Molecular Ecology Laboratory, Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram, Kerala, India Author Merilä, Juha Ecological Genetics Research Unit, Department of Biosciences, University of Helsinki, Helsinki, Finland, text PLoS ONE 2015 e 0119825 2015-03-25 10 3 1 18 http://dx.doi.org/10.1371/journal.pone.0119825 journal article 10.1371/journal.pone.0119825 1932-6203 PMC4373918 25806804 12630525 Microhyla nilphamariensis sp. nov. urn:lsid:zoobank.org:act: 11E1D35F-7FC1–43C1–8318– 747F9FC0C882 Etymology. The species name is derived from the name of the type locality Nilphamari, where the type specimens were collected. Holotype . Adult male, MZH-2362, collected from grass-field (25° 48 0 06.12"N , 88° 53 0 59.21"E ), Koya Golahut, Saidpur, Nilphamari, Bangladesh ; collected by M. S. A. Howlader , June 9, 2012 . Paratopotypes . MZH-2360 (adult female), MZH-2361 (adult female), MZH-2363 (adult female), MZH-2364 (adult female), MZH-2365 (adult female), and MZH-2366 (adult female) collected from the same locality as the holotype ; collected by M. S. A. Howlader and Abdur Razzaque, June 9, 2012 . Diagnosis. Microhyla nilphamariensis sp. nov. is characterized by a combination of the following characters: HL 77% of HW , EN roughly six times greater than NS , IN roughly five times greater than NS , IOD two times greater than IN , MBE 15% of HL, small ovoid-shaped inner metacarpal tubercle, very small rounded outer metacarpal tubercle, toes with rudimentary webbing, absence of digital discs, inner metatarsal tubercle small and round, outer metatarsal tubercle ovoid-shaped, minute, and indistinct. Description of taxa Holotype (adult male). Small sized frog ( SVL 17.36 mm ). Head large, triangular, wider than long, HL 77% of HW , HW 27 % of SVL , HL 21 % of SVL , MFE 67 % of HL, MBE 15 % of HL. Snout nearly rounded in lateral view, SL 46% of HL; canthus rostralis indistinct, loreal region concave. Nostrils much closer to snout tip than to eyes, NS 16 % of EN ; NS 1 % of SVL , EN 7 % of SVL ; nostrils rounded and very small, NS 20 % of IN , MN 92 % HL. Eye large, EL 52 % of HL, EL 11 % of SVL ; interorbital distance greater than maximum width of upper eyelid greater, UEW 41 % of IOD , UEW 44 % EL , UEW 4 % SVL . Interorbital space convex, IN 49 % of IOD . Tympanum is hidden. Arms moderately long, FAL 74% HAL , FAL 16% of SVL , HAL 22% SVL . Fingers small, free of webbing, tips are flattened. Relative length of fingers, shortest to longest: 1 <2 <4 <3; fingers lacking dermal ridge. Palm with ovoid-shaped inner metacarpal tubercle, small rounded outer metacarpal tubercle. Subarticular tubercle prominent, rounded, single tubercle per digit. Hind limbs relatively long, TL 42% of SVL , THIGHL 86% of TL; FOL 49% SVL and TL 86% FOL, FOL 71% of TFOL . Toes long, thin, tips rounded; webbing between toes weakly developed [1(1), 2i (1.75), 2e (1), 3i (2.5), 3e (2), 4i (3), 4e (3.25), 5(1.75)]. Relative lengths of toes, shortest to longest: 1 <2 <5 <3 <4. Inner metatarsal tubercle small and round, present at base of first toe; outer metatarsal tubercle is ovoid-shaped, minute, indistinct; subarticular tubercles well-developed, nearly ovoid-shaped. Dorsal surface smooth with some tiny tubercles on the back and on the sides the body; tiny granules on upper eyelids, loreal, and cloacal region. Dorsal surface of forelimbs, thigh and tarsi glandular. Throat, chest, abdomen and ventral part of thigh and tibia smooth. Basic dorsal coloration light brown with distinct dark brown diamond-shaped marking over the back, beginning between the eyes and extending to both the eyelids, narrowing behind the head and widening above the shoulder, then narrowing again and finally broadening out, sending a stripe to the groin and thigh ( Fig. 2 ). A dark streak extends along the sides from back of the eye to shoulder. Limbs with dark cross bars. The belly is dull white; the throat and chest are brown. Measurements (in mm). Male ( holotype ): SVL 17.36; HL 3.74; HW 4.82; MN 3.47; SL 1.75; MFE 2.52; MBE 0.57; IN 1.03; IOD 2.09; EN 1.25; NS 0.21; EL 1.95; UEW 0.86; HAL 3.93; FAL 2.91; LAL 1.72; THIGHL 6.41; TL 7.45; TFOL 11.96; FOL 8.61. Female ( paratopotype ): SVL 17.84; HL 3.85; HW 5.02; MN 3.57; SL 1.79; MFE 2.61; MBE 0.6; IN 1.06; IOD 2.16; EN 1.27; NS 0.22; EL 1.98; UEW 0.89; HAL 4; FAL 2.97; LAL 1.81; THIGHL 6.44; TL 7.51; TFOL 12.07; FOL 8.71. Variation. Morphometric variability is described in Table 1 . Distribution. Microhyla nilphamariensis sp. nov. is known only from the type locality ( Fig. 1 ). However, Matsui et al . [ 18 ] and Hasan et al . [ 6 ] found individuals from Dinajpur district carrying haplotypes similar to those found from the type locality, suggesting that the distribution area might extend beyond the type locality. Natural history. The new species was observed only at night during the rain. At the type locality, specimens were found in a grass-field near temporary pools. Molecular phylogeny and genetic divergence of new species. The sequence divergences between Microhyla nilphamariensis sp. nov. and other congeneric species were significant, ranging from 5.7% to 13.2% for 16S rRNA ( Table 2 ). Intraspecific genetic divergence within the new species was estimated at 0.5%. Microhyla nilphamariensis sp. nov. formed a distinct clade in the phylogenetic analyses with high bootstrap ( ML method) and posterior probability support (Bayesian method; Fig. 3 ). Microhyla nilphamariensis sp. nov. was identified as a sister taxa to M . ornata ( Fig. 3 ). Molecular phylogeny suggests that M . nilphamariensis sp. nov. belongs to the Indian clade of Microhyla species group (including M . ornata and M . rubra ), rather than having closer affinity to Southeast Asian species ( Fig. 3 ). Fig 2. Photographs of Microhyla nilphamariensis sp. nov. (A) Dorso-lateral view of male (holotype, live), (B) Dorsal view of male (holotype). (C) Ventral view of foot and (D) palm. doi:10.1371/journal.pone.0119825.g002 Morphological comparison. M . nilphamariensis sp. nov. is morphologically distinct from the closely related species M . ornata and M . rubra in the following qualitative characters ( Fig. 4 ): inner metacarpal tubercle small and ovoid-shaped ( vs . large and goblet-shaped in M . ornata ; elongated in M . rubra ), outer metacarpal tubercle very small and rounded ( vs . very large, prominent and heart-shaped in M . ornata and M . rubra ), inner metatarsal tubercle small and round ( vs . elongated, large and very prominent in M . ornata ; shovel-shaped in M . rubra ), outer metatarsal tubercle ovoid-shaped, minute, and indistinct ( vs . compressed and large in size in M . ornata ; shovel-shaped and large in M . rubra ). Quantitative diagnostic characters include (see also: Table 1 , S 2 Fig.): head length 77% of head width ( vs . roughly equal to head width in M . ornata and M . rubra ), distance from front of eyes to the nostril roughly six times greater than nostril–snout length ( vs . over one and a half times greater than nostril–snout length in M . ornata ; over single time greater than nostril–snout length in M . rubra ), internarial distance five times greater than nostril–snout length ( vs . nearly two times greater than nostril– snout length in M . ornata ; less than two times greater than nostril–snout length in M . rubra ), interorbital distance two times greater than internarial distance ( vs . more than three times greater than internarial distance in M . ornata and M . rubra ), distance from back of mandible to back of the eye 15% of head length ( vs . more than 36% of head length in M . ornata and M . rubra ). These two closely related Microhyla species are morphologically clearly distinct from the new species also according to MANOVA (F 34,18 = 334.93, P <0.001), and according to a discriminant analysis which correctly classifies all individuals to their respective species along two significant (Eigenvalues 194.51, F 36.47, P <0.001) canonical axes ( Fig. 5A ). In a principal component (PC) analysis, the three species are significantly different from each other (Tukey’ s HSD ; P> 0.05 in all pairwise comparisons) along the first PC-axis (Eigenvalue = 14.07; 82.8% variance explained), which correspond to variation in overall size (all traits loading positively and roughly equally on this axis), M . nilphamariensis sp. nov. being the smallest species ( Fig. 5B ). The second PC-axis (Eigenvalue = 1.48; 8.7% variance explained) captures shape differences, but in this axis M . nilphamariensis sp. nov differs significantly only from M . ornata (Tukey’ s HSD , P <0.05). Nevertheless, that the M . nilphamariensis sp. nov. is clearly differentiated from M . ornata and M . rubra can also be depicted from bivariate scatterplots (S2 Fig.) showing that it’ s diagnostic ratios (see above) do not overlap with those of M . ornata and M . rubra . Table 1. Summary of quantitative and qualitative diagnostic characters in Microhyla nilphamariensis sp. nov. and its closest morphological and phylogenetic congeners.

Microhyla nilphamariensis sp. nov.		Microhyla ornate		Microhyla rubra
Male (n = 1)	Female (n = 6)	Male (n = 6)	Female (n = 4)	Male (n = 1)	Female (n = 3)
HL:HW	0.77	0.76±0.01	0.95±0.03	0.97±0.02	0.91	0.96±0.01
(0.74–0.77)	(0.89–0.97)	(0.95–1.00)	(0.95–0.97)
HL:SVL	0.21	0.22±0.01	0.28±0.01	0.28±0.02	0.3	0.34±0.01
(0.21–0.23)	(0.27–0.29)	(0.26–0.30)	(0.33–0.35)
MBE:HL	0.15	0.15±0.01	0.45±0.13	0.45±0.15	0.4	0.36±0.01
(0.14–0.16)	(0.31–0.64)	(0.31–0.59)	(0.35–0.38)
EL:HL	0.52	0.52±0.01	0.49±0.03	0.47±0.03	0.52	0.43±0.05
(0.51–0.54)	(0.45–0.54)	(0.43–0.50)	(0.37–0.46)
UEW:EL	0.44	0.42±0.02	0.39±0.03	0.39±0.02	0.4	0.53±0.04
(0.39–0.45)	(0.34–0.41)	(0.36–0.42)	(0.51–0.57)
EN:NS	5.95	5.92±0.08	1.51±0.23	1.60±0.22	1.08	0.97±0.13
(5.77–5.98)	(1.24–1.92)	(1.43–1.92)	(0.81–1.04)
IN:NS	4.9	4.84±0.04	1.68±0.36	1.77±0.41	0.91	1.03±0.12
(4.91–4.81)	(1.30–1.86)	(1.37–2.32)	(0.89–1.10)
IOD:IN	2.03	2.02±0.03	3.09±0.44	2.87±0.23	3.91	3.30±0.09
(1.98–2.05)	(2.79–3.65)	(2.6–3.12)	(3.19–3.38)
SL: HL	0.47	0.47±0.01	0.41±0.02	0.39±0.01	0.4	0.37±0.04
(0.45–0.49)	(0.38–0.43)	(0.38–0.41)	(0.32–0.39)
Metacarpal tubercle	Ovoid-shaped inner metacarpal tubercle; very small rounded outer metacarpal tubercle		Large goblet-shaped inner metacarpal tubercle; very large and prominent, heart shaped outer metatarsal tubercle which appears as two tubercles fusing to form a single one.		Elongated inner metacarpal tubercle; very large and prominent, heart-shaped outer metatarsal tubercle.
Metatarsal tubercle	Inner metatarsal tubercle small round shaped; outer metatarsal tubercle is ovoid, minute, and indistinct.		Inner metatarsal tubercle elongated, large and very prominent; outer metatarsal tubercle is compressed and large in size.		Inner metatarsal tubercle shovelshaped, bearing longitudinal groove, large and prominent; outer metatarsal tubercle is also shovelshaped and large.
Morphological ratios are given as mean ± standard deviation.

doi:10.1371/journal.pone.0119825.t001 Table 2. Pairwise genetic divergence (number of base substitutions per site) among Microhyla species based on 446 bp mtDNA (16S gene) sequences.

(a)	(b)	(c)	(d)	(e)	(f)	(g)	(h)	(i)	(j)	(k)	(l)	(m)	(n)	(o)	(p)	(q)	(r)	(s)	(t)	(u)	(v)	(w)
(a)	M . nilphamariensis sp. nov. (Holotype)
(b)	M . nilphamariensis sp. nov.	0.005
(c)	M . ornate	0.057	0.060
(d)	M . butleri	0.123	0.123	0.141
(e)	M . okinavensis	0.096	0.096	0.121	0.133
(f)	M . fissipes	0.105	0.108	0.110	0.134	0.057
(g)	M . heymonsi	0.123	0.119	0.137	0.151	0.090	0.087
(h)	M . perparva	0.150	0.150	0.132	0.124	0.140	0.139	0.147
(i)	M . rubra	0.075	0.082	0.101	0.129	0.128	0.115	0.141	0.152
(j)	M . berdmorei	0.095	0.102	0.107	0.135	0.107	0.104	0.112	0.139	0.113
(k)	M . mixture	0.102	0.102	0.120	0.116	0.019	0.057	0.087	0.120	0.122	0.110
(l)	M . malang	0.110	0.113	0.127	0.133	0.092	0.078	0.065	0.153	0.112	0.116	0.089
(m)	M . achatina	0.108	0.108	0.120	0.147	0.096	0.090	0.083	0.152	0.124	0.089	0.081	0.077
(n)	M . mantheyi	0.112	0.112	0.135	0.144	0.088	0.088	0.071	0.146	0.120	0.110	0.079	0.066	0.063
(o)	M . pulchra	0.114	0.107	0.119	0.134	0.112	0.115	0.121	0.159	0.121	0.086	0.115	0.138	0.115	0.132
(p)	M . superciliaris	0.114	0.114	0.112	0.099	0.121	0.110	0.129	0.119	0.115	0.125	0.115	0.120	0.133	0.125	0.147
(q)	M . fowleri	0.095	0.102	0.121	0.138	0.111	0.113	0.116	0.139	0.110	0.021	0.114	0.110	0.089	0.103	0.095	0.129
(r)	M . annectens	0.132	0.128	0.132	0.130	0.154	0.134	0.145	0.096	0.142	0.100	0.140	0.148	0.142	0.139	0.150	0.123	0.112
(s)	M . palmipes	0.129	0.129	0.121	0.133	0.144	0.113	0.156	0.110	0.143	0.143	0.129	0.153	0.157	0.155	0.147	0.112	0.142	0.135
(t)	M . marmorata	0.120	0.120	0.115	0.124	0.128	0.121	0.127	0.081	0.119	0.118	0.115	0.122	0.126	0.127	0.145	0.117	0.121	0.054	0.130
(u)	M . petrigena	0.130	0.130	0.121	0.137	0.151	0.132	0.132	0.065	0.129	0.120	0.129	0.145	0.137	0.142	0.142	0.115	0.125	0.082	0.110	0.085
(v)	M . mukhlesuri	0.099	0.103	0.098	0.127	0.055	0.014	0.079	0.130	0.105	0.108	0.055	0.070	0.081	0.089	0.113	0.098	0.118	0.141	0.121	0.115	0.122
(w)	M . mymensinghensis	0.098	0.098	0.103	0.138	0.059	0.039	0.081	0.129	0.124	0.112	0.053	0.085	0.085	0.089	0.121	0.112	0.116	0.139	0.127	0.114	0.127	0.034

doi:10.1371/journal.pone.0119825.t002 Fig 3. Phylogenetic relationships among all known species in the genus Microhyla . Analysis is based on 446 bp of mtDNA (16S gene) sequence, showing the position of Microhyla nilphamariensis sp. nov. Chaperina fusca was used as an outgroup. The Microyla ornata sequence is from Karnataka (Western Ghats, India). Numbers on branches represent bootstrap support values for Maximum-likelihood, and Bayesian posterior probabilities, respectively. doi:10.1371/journal.pone.0119825.g003 Genetic divergence of M . nilphamariensis sp. nov. from all other species in the Southeast Asian clade is 10% to 13% ( Fig. 3 ). Likewise, the new species is morphologically different from all known Southeast Asian species in comparison to the original descriptions [ 39 – 55 ]. Morphological characters of the other Southeast Asian species ( M . berdmorei , M . borneensis , M . achatina , M . butleri , M . palmipes , M . annamensis , M . annectens , M . heymonsi , M . mantheyi , M . superciliaris , M . malang , M . chakrapanii , M . marmorata , M . nanapollexa , M . pulverata , M . arboricola , M . darevskii , M . minuta , M . pineticola , M . pulchella , M . perparva , M . mixtura , M . fowleri , M . maculifera , M . petrigena , M . orientalis ) such as webbed toes with distinct digital discs, and leaf vain-type dorsal surface markings separate them from M . nilphamariensis sp. nov. , which has reduced webbing, absent discs and irregular dorsal surface markings. M . sholigari also differs from M . nilphamariensis sp. nov. as it has discs on fingers ( Fig. 4 ). Shovel-shaped inner metatarsal tubercle and a more rounded snout separate M . picta from M . nilphamariensis sp. nov. [ 56 ]. M . nilphamariensis sp. nov. lacks the minutely shagreened dorsum, mid-dorsal ridge, and deeply furrowed outer metacarpal tubercle of M . fusca [ 57 ]. M . nilphamariensis sp. nov. differs from M . pulchra [ 58 ] and M . erythropoda [ 59 ] in having a rounder snout, smaller body size, and rudimentary foot webbing ( M . pulchra and M . erythropoda have obtuse or obtusely pointed snouts, larger body size and half-webbed toes). M . karunaratnei [ 60 ] and M . zeylanica [ 61 ] are two species endemic to Sri Lanka , and differ from M . nilphamariensis sp. nov. by extensive digital webbing, presence of digital discs, IOD 1.6 times greater than UEW ( vs . rudimentary digital webbing, absence of digital discs, IOD 2.43 times greater than UEW ). Absence of digital discs differentiates M . nilphamariensis sp. nov. from M . mukhlesuri , M . fissipes and M . mymensinghensis , in which digital discs are present.