Zootaxa 4066 (3): 331–342 ISSN 1175-5326 (print edition)

http://www.mapress.com/j/zt/
Copyright © 2016 Magnolia Press
Article ZOOTAXA
ISSN 1175-5334 (online edition)
http://doi.org/10.11646/zootaxa.4066.3.9
http://zoobank.org/urn:lsid:zoobank.org:pub:61398241-F4AA-4E46-A85A-0434419E7914

A new species of Microhyla (Anura: Microhylidae) from Sri Lanka:

an integrative taxonomic approach

NAYANA WIJAYATHILAKA1, SONALI GARG2, GAYANI SENEVIRATHNE1, NUWAN KARUNARATHNA1,

S.D. BIJU2 & MADHAVA MEEGASKUMBURA1,3
1
Department of Molecular Biology and Biotechnology, Faculty of Science & Postgraduate Institute of Science, University of
Peradeniya, Sri Lanka
2
Systematics Lab, Department of Environmental Studies, University of Delhi, Delhi 110 007, India
3
Corresponding author. E-mail: madhava88m@gmail.com

Abstract

Species boundaries of Microhyla rubra of India and Sri Lanka were assessed using the following criteria: genetic barcod-
ing, morphology, and vocalization. We use a ca. 500 bp fragment of the 16S rRNA mitochondrial gene and show that there
is an uncorrected pairwise distance of 2.7−3.2% between the Indian and Sri Lankan populations of M. rubra. We show
that they are different in several call characteristics such as, dominant frequency, call duration, call rise time and pulse
rate. Morphologically, the Sri Lankan population can be distinguished from the typical M. rubra described from southern
India, by a combination of characters: body size, skin texture, and feet dimensions. We recognize the population from Sri
Lanka as a new species, Microhyla mihintalei sp. nov., a widely distributed lowland species with an elevational distribu-
tion of up to 500 m a.s.l.

Key words: Amphibia, taxonomy, barcoding, bioacoustics, multiple criteria, Sri Lanka

Introduction

Sri Lanka is an amphibian hotspot containing a significant amount of newfound diversity (Meegaskumbura et al.
2002). A lion’s share of the species described so far from the island are contained in the genus Pseudophilautus, the
Old World shrub frogs, which constitutes about 40 new species described since 2002 (Manamendra-Arachchi &
Pethiyagoda 2005; Meegaskumbura & Manamendra-Arachchi 2005, 2011; Meegaskumbura et al. 2007, 2009,
2012; Wickramasinghe et al. 2013). However, only a few of the other Sri Lankan anuran taxa have been subjected
to systematic analyses. These include descriptions for instance of, Ramanella nagaoi Manamendra-Arachchi &
Pethiyagoda, 2001, the newly erected Sri Lankan endemic tree frog genus Taruga Meegaskumbura et al., 2010,
revision of the genus Hylarana (Biju et al. 2014b) with description of H. serendipi Biju et al., 2014 from Sri Lanka
(and six new species from India) and revision of the genus Adenomus (Meegaskumbura et al. 2015). These recent
descriptions are based on using multiple criteria in the context of integrative taxonomy and the general lineage
concept of species (De Queiroz 1999). Apart from these, other frog taxa in Sri Lanka have only received meager
taxonomic attention over the last decade.
Given that much of the species described from Western Ghats-Sri Lanka biodiversity hotspot have been from
putatively previously well-known groups, the cryptic nature of the diversity has been highlighted many times (e. g.,
Manamendra-Arachchi & Pethiyagoda 2005; Biju et al. 2011, 2014a, 2014b). In this backdrop of species crypsis,
the utility of integrative approaches is pronounced (Meegaskumbura & Manamendra-Arachchi 2005; Vences et al.
2010; Biju et al. 2014b; Meegaskumbura et al. 2015).
Family Microhylidae currently comprises 13 subfamilies with a wide distribution across the world (Frost 2015;
Peloso et al. 2015). Sri Lankan microhylids belong to two genera Uperodon Duméril & Bibron, 1841 and
Microhyla Tschudi, 1838 (categorized under the subfamily Microhylinae; Peloso et al. 2015). Microhyla, “the rice
frogs” (Frank & Ramus 1995) contains four species in Sri Lanka, out of which, M. karunaratnei Fernando &

Accepted by M. Vences: 9 Dec. 2015; published: 15 Jan. 2016 331

Siriwardhane, 1996 and M. zeylanica Parker & Osman-Hill, 1949 are montane isolates endemic to the island while
M. ornata (Duméril & Bibron 1841) and M. rubra (Jerdon 1854) are non-endemic lowland species.
Microhyla rubra is considered to have an Indian-Sri Lankan distribution throughout the history of its identity.
Jerdon (1854) attributed the type locality of the species as the sandy riverbanks in Karnataka and mentioned that it
is also found in Ceylon (“in the Carnatic near rivers, in sandy banks . . . also Ceylon”). Subsequently several
authors also reported this species from Sri Lanka (e.g., Kirtisinghe 1957; Dutta & Manamendra-Arachchi 1996;
Dutta 1997; Frost 2015; Howlader et al. 2015).
Here we delineate the species boundaries of Indian-Sri Lankan populations of Microhyla rubra using genetic
barcoding, morphology and vocalization in the context of integrative taxonomy.

Material and methods

Field survey and specimen collection. Adult specimens were collected at night, mostly by locating calling males,
and sometimes by opportunistic surveys during both day and night. GPS coordinates of collection localities were
determined using a Garmin eTrex Summit and/or Garmin GPSmap62 hand-held GPS devices. Live frogs were
photographed in the wild, with a few exceptions in captivity. Animals were euthanized in tricaine methanesulfonate
(MS-222), fixed in 4% formalin and preserved in 70% ethanol. Tissue samples (thigh muscles or liver) were taken
immediately after euthanization and stored in absolute ethanol for subsequent molecular analyses at -20 °C in the
Department of Zoology, University of Peradeniya (DZ) or Systematics Lab, University of Delhi (SDBDU). Type
specimens are deposited in the Department of Zoology, University of Peradeniya (DZ).
External morphology. Measurements and associated terminology follow Biju et al. 2014b and
Meegaskumbura et al. 2015. The term shank is used here to refer to the part of the leg containing the tibia, and
thigh is used for the part containing the femur. Measurements of all specimens were taken by SDB, using a digital
slide-caliper, or a binocular microscope with a micrometre ocular, to the nearest 0.1 mm. All measurements
provided in the taxonomy section are in millimeters. Abbreviations: SVL (snout-vent length), HW (head width, at
the angle of the jaws), HL (head length, from the rear of the mandible to the tip of the snout), MN (distance from
the rear of the mandible to the nostril), SL (snout length, from the tip of the snout to the anterior orbital border), EL
(eye length, horizontal distance between the bony orbital borders), IUE (inter upper eyelid width, shortest distance
between the upper eyelids), UEW (maximum upper eyelid width), IN (internarial distance, least distance between
the inner margins of nares), NS (distance between middle of naris and snout tip, EN (distance between anterior
most point of orbit and middle of nostril), FAL (forearm length, from the flexed elbow to the base of the outer
palmar tubercle), HAL (hand length, from the base of the outer palmar tubercle to the tip of the third finger), SHL
(shank length), TL (thigh length), FOL (foot length, from the base of the inner metatarsal tubercle to the tip of the
fourth toe), digit number is represented by roman numerals I-V. For museums and frequently used terms,
abbreviations are as follows: DZ (Department of Zoology, University of Peradeniya), SDBDU (Systematics Lab,
University of Delhi), MM (Madhava Meegaskumbura), SDB (SD Biju) and NW (Nayana Wijayathilaka). In the
‘Material studied’ section and Table 1, the following abbreviations after specimen numbers refer to: (HT) holotype,
and (PT) paratype. Mann-Whitney U test was done to evaluate the separation of the morphometric variables
between the Indian and Sri Lankan populations (P ≤ 0.05).
Genetic comparison. Tissues were collected from four populations of “Microhyla rubra” in Sri Lanka
(Anuradhapura, Mihintale, Maakandura, Dambulla) and one population from India (Shivanahalli, Karnataka)
(Table 1). DNA extractions were done using Qiagen DNeasy blood and tissue kits. A fragment of ca. 500 bp of the
16S rRNA gene was amplified by PCR and sequenced using dye termination cycle sequencing. Palumbi (1996)
primers were used with the PCR conditions as follows: denaturation at 95 °C for 40 s, annealing at 46 °C for 40 s,
and extension at 72 °C for 50 s, 35 cycles, with a final extension of 72 °C for 7 min. PCR products were purified
using Qiagen Qiaquick PCR purification kit and sequenced on an ABI 3100 automated sequencer.
Chromatograms were edited using 4peaks (v. 1.7.1). Dataset was put together in MEGA v. 5.0 (Tamura et al.
2011) and aligned using ClustalW algorithm as implemented in MEGA. Uncorrected pairwise distances between
the individuals representing populations from India and Sri Lanka were calculated using PAUP* 4.0b10 (Swofford
2002) following default distance settings. There were no ambiguously aligned regions, and hence no portions were
removed prior to the pairwise distance comparison.

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 TABLE 1. Voucher numbers, measurements (in mm), collection localities and GenBank accession numbers of the specimens used in this study. All abbreviations as in Materials and Methods. Note:
GenBank accession numbers of 16S gene sequences are given only for the specimens used for interspecific pairwise comparisons.

Altitude Coordinates Accession

Voucher HW
Sex Locality SVL HL SL IUE UEW MN EN NS EL FAL HAL TL SHL FOL Latitude Longitude Number
Number (meters)
(oN) (oE) (16S)

Microhyla mihintalei sp. nov.

M Anuradhapura DZ 1553 24.8 7.4 6.2 2.7 2.6 1.5 5 1.4 1 2.3 5.1 6 11.3 11.5 11.6 90 8.3541 80.3967 -

A NEW MICROHYLA FROM SRI LANKA

M Anuradhapura DZ 1554 25.8 7.6 6.4 2.9 3 1.6 5.2 1.5 1 2.4 5 6.1 11.3 11.5 11.5 90 8.3541 80.3967 -
M Anuradhapura DZ 1555 26.7 8.1 6.7 3 2.9 1.9 6.1 1.7 1.3 2.5 4.9 6.4 12.3 12.4 11.9 90 8.3541 80.3967 -
M Anuradhapura DZ 1556 26.4 7.9 6.5 2.9 3 1.9 6.2 1.6 1.4 2.6 4.5 6.5 12.2 12.4 12.1 90 8.3541 80.3967 -
M Anuradhapura DZ 1557 27.3 8.1 6.7 3 3.3 2.1 6.1 1.7 1.6 2.8 5 6.9 12.5 12.7 12.2 90 8.3541 80.3967 -
M Anuradhapura DZ 1467 26.1 8 6.6 2.8 2.8 2 5.6 1.6 1.2 2.5 4.9 6.3 12.1 12.3 11.8 90 8.3541 80.3967 -
M Anuradhapura DZ 1468 24.3 7.2 5.6 2.6 2.7 1.6 4.9 1.5 1 2.4 4.8 5.9 11.1 11.2 11.3 90 8.3541 80.3967 KU214861
M Anuradhapura DZ 1473 26.7 8 6.1 2.7 2.6 1.9 5.7 1.5 1.2 2.5 5 6.5 12.1 12.2 12.1 90 8.3541 80.3967 -
M Maakandura DZ 1410 21.7 6.9 5 2.5 2.2 1.3 4.4 1.4 1 2.1 4.6 5.6 10.8 10.9 10.8 30 7.3245 79.9887 KU214857
M Ampara DZ 1457 25.4 7.7 6.4 2.7 2.8 2 5.7 1.8 1.5 2.5 4.9 6.3 11.3 11.4 11.8 30 7.2932 81.6704 -
M Mihintale DZ 1445 22.2 7.1 5.7 2.5 2.6 1.6 4.7 1.4 1.2 2.3 4.6 5.9 11.1 11.1 11.1 120 8.3548 80.5054 KU214858
M Mihintale DZ 1446 22.6 7.1 5.8 2.6 2.5 1.6 4.6 1.3 1.2 2.4 4.5 6.1 11.3 11.3 11.3 120 8.3548 80.5054 KU214860
M Dambulla DZ 1127 23.1 7.3 6.2 2.7 2.5 1.7 4.6 1.3 1.3 2.4 4.1 6.1 11.3 11.3 11.8 190 7.8584 80.6773 KU214856
Average 24.9 7.6 6.1 2.7 2.7 1.7 5.3 1.5 1.2 2.4 4.8 6.2 11.6 11.7 11.6
Standard deviation 1.9 0.4 0.5 0.2 0.3 0.2 0.6 0.2 0.2 0.2 0.3 0.3 0.6 0.6 0.4
F Maakandura DZ 1418 24.4 7.6 6.3 2.8 2.7 1.5 4.5 1.6 1.2 2.1 4.3 6.2 12.3 12.4 12.2 30 7.3245 79.9887 KU214859
Microhyla rubra
M Shivanahalli SDB 2561 28.6 8.3 7.1 3.4 3 2.1 5 1.7 1.2 2.2 5.3 6.9 14.3 12.1 12.8 908 12.7284 77.5790 -
M Shivanahalli SDB 2559 27.9 8.1 6.7 3.6 2.7 2.1 5.4 1.6 1.2 2.6 5.2 6.9 14.1 12 12.1 908 12.7284 77.5790 -
M Shivanahalli SDB 2560 27.9 8.2 8. 2 3.2 3.1 2.4 6 1.8 1.4 2.4 5.7 7 13 12.1 13.1 908 12.7284 77.5790 -
M BR Hills SDB 2548 29.6 9.2 7.1 2.9 3 2.5 6.6 1.7 1.2 3 5.3 7.1 14.3 12.2 12.6 1195 12.0028 77.1423 -
M Shivanahalli SDB 2558 29 9 8 3.3 2.9 2.5 6.5 1.7 1.2 2.6 5.7 6.4 13.2 12.2 13.1 908 12.7284 77.5790 KU214855
M Shimoga SDB 40134 28.4 8.7 7.1 3.1 2.8 1.9 5.8 1.5 1 2.6 4.7 6.6 13.6 12 11.5 640 13.984 75.1100 -
M Shimoga SDB 40132 27.2 8.2 6.8 3 2.9 2 5.7 1.4 1.1 2.7 5 6.6 12 12.4 12.3 640 13.984 75.1100 -
M Shimoga SDB 40133 25.8 7.4 6.9 2.7 2.5 1.8 4.9 1.6 1 2.2 4.6 6.2 12 11.9 11.6 640 13.984 75.1100 -
Karnataka* - - - - - - - - - - - - - - - - - - - AB201192
Average 28 8.4 7.1 3.1 2.9 2.2 5.7 1.6 1.2 2.5 5.2 6.7 13.3 12.1 12.4
Standard deviation 1.17 0.6 0.4 0.3 0.2 0.3 0.6 0.1 0.1 0.3 0.4 0.3 0.9 0.1 0.6

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 Vocalization. We recorded vocalizing males from Coimbatore, Tamil Nadu State, India (11.0826°N,
77.0617°E, 395 m a.s.l, 8 October 2014) and Mihintale, Sri Lanka (8.3548°N, 80.5054°E, 120 m a.s.l, 27
September 2014). A Marantz PMD 620 MKII solid-state recorder (sampling rate 44.1 kHz, 16-bit resolution) and a
directional SennheiserME66 microphone equipped with a foam windscreen were used for call recording.
Microphone was hand-held and its tip was placed approximately 1 m from the calling frog. To avoid clipping of the
call, gain-setting of the recorder was adjusted prior to start of each new recording. For a given recording, a fixed
gain-setting was used. A total of 60 calls were measured, ten calls each from three Sri Lankan animals and 30 calls
from one Indian frog, respectively. Following each recording, the calling animal was captured and its SVL was
measured using digital calipers. We measured the wet-bulb air temperature at the focal male’s calling site to the
nearest 0.1 °C using a Zeal thermometer (76 mm immersion, 305 mm length). Raven Pro 1.4 software (Cornell
Laboratory of Ornithology, Ithaca, NY, USA) was used for call analysis. Principal component analysis (PCA) for
the call variables was done using Systat version 11.0 (Systat Software, Inc. 2009. SYSTAT 11).

Results

Taxonomy

Microhyla mihintalei sp. nov.

(Figures 1–4; Table 1)

Suggested common name. Mihintale Red Narrow-mouthed Frog.

Etymology. The species is named after Mihintale, the point of unison for two ancient cultures when Mahinda
Thero (Son of Indian Emperor, Asoka) met Dewanampiya Tissa (the king of Anuradhapura, Sri Lanka) in 246 BC.
Mihintale is also considered to be one of the world’s earliest sanctuaries. The species epithet mihintalei is a noun in
apposition to the generic name.
Holotype. Adult male (DZ 1553), Anuradhapura (8.3541°N, 80.3967°E, 90 m a.s.l) Anuradhapura Sri Lanka,
collected by NW and team, 22 December 2014.
Paratypes. Four adult males (DZ 1554–1557), collected along with the holotype; three adult males (DZ 1467,
DZ 1468, DZ 1473) collected from the same locality, collected by NW and team, 12 October 2012; one adult male
(DZ 1410) and one female (DZ 1418), from Maakandura (7.3245°N, 79.9887°E, 30 m a.s.l.), Kurunegala district,
Sri Lanka, collected by MM and team, 28 August 2014; one adult male (DZ 1457), from Ampara (7.2932°N,
81.6704°E, 30 m a.s.l.), Ampara district, Sri Lanka, collected by NW and team, 10 October 2014; two adult males
(DZ 1445–1446), from Mihintale (8.3548°N, 80.5054°E, 120 m a.s.l.), Anuradhapura district, Sri Lanka, collected
by MM and team, 2 October 2014; one adult male (DZ 1127), from Dambulla (7.8584°N, 80.6773°E, 190 m a.s.l.),
Matale district, Sri Lanka, collected by MM and team, 4 January 2013.
Diagnosis. We allocate these specimens to Microhyla since they show the following diagnostic characters for
this genus (Parker 1934): narrow head; eyes reduced; maxillary and vomerine teeth absent; fingers without
webbing; toes with basal webbing; prominent inner metatarsal tubercle on foot (Fig. 1). Microhyla mihintalei sp.
nov. is diagnosable from its congeners by the combination of following characters: small-sized, slender body, adult
male SVL 21.7–27.3 mm (N = 13), adult female SVL 24.4 mm (N = 1); tympanum indistinct or obscured by a layer
of skin without clearly defined borders; vomerine ridge absent; tongue elliptical; finger and toe tips rounded
without marginal grooves; webbing between toes rudimentary; inner and outer metatarsal tubercles present.
Morphological comparisons. Microhyla mihintalei sp. nov. is closely related to Microhyla rubra based on
molecular evidence and overall external morphology, however differs from the latter by its relatively smaller adult
male size, SVL 21.7–27.3 mm, N = 13 (vs. larger SVL 25.8–29.6 mm, N = 8); dorsal skin shagreened to sparsely
granular (vs. granular); thigh equal to shank and foot length, male, TL 11.6 ± 0.6 mm, SHL 11.7 ± 0.6 mm, FOL
11.6 ± 0.6 mm, N = 14 (vs. longer, male, TL 13.8 ± 0.5 mm, SHL 11.9 ± 0.2 mm, FOL 12.4 ± 0.4 mm, N = 8).
Mann-Whitney U test results suggest that the two congeners are significantly different in SVL, HW, HL, SL, UEW,
TL, FAL, HAL and FOL (Appendix 1). The new species is not conspecific with any of the other known species
from the Western Ghats of India and Sri Lanka. However, for the purpose of more clarity and to avoid possible
confusions, a comparison with the other four valid species known from this region is being provided. Microhyla

334 · Zootaxa 4066 (3) © 2016 Magnolia Press WIJAYATHILAKA ET AL.

mihintalei sp. nov. differs from M. karunaratnei and M. zeylanica from Sri Lanka, and M. ornata and M. sholigari
Dutta and Ray, 2000 from the Western Ghats, by its relatively large snout-vent size of the adult male, SVL 21.7–
27.3 mm, N = 13 (vs. SVL 15.8–19.1 mm, N = 11; SVL 11.8–20.0 mm, N = 5; SVL 14.5–20.3 mm, N = 5; SVL
17.5–24.8 mm, N = 7, respectively). Specifically, the new species differs from M. karunaratnei and M. sholigari by
its non-dilated finger and toe tips without circum-marginal grooves (vs. dilated with circum-marginal grooves),
differs from M. karunaratnei by its finger and toe disc rounded without dorso-terminal groove (vs. dilated with
dorso-terminal groove), differs from M. ornata by its shovel-shaped metatarsal tubercle (vs. rounded nonshovel-
shaped).

FIGURE 1. Holotype of Microhyla mihintalei sp. nov. (DZ 1553, an adult male, SVL 24.8 mm). A. Dorsal view; B. ventral
view; C. lateral view of head; D. ventral view of foot; E. ventral view of hand.

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FIGURE 2. A–D. Oscillogram of advertisement calls (four consecutive calls and a single call) of males of Microhyla
mihintalei sp. nov. and Microhyla rubra. A. four consecutive calls (time shown: 3 seconds); B. a single call (time shown: 0.3
seconds) of M. mihintalei sp. nov.; C. four consecutive calls (time shown: 3 seconds); D. a single call (time shown: 0.3
seconds) of M. rubra; E. PC1 vs. PC2 factor scores of the Principal Components Analysis of call characters for the two species.

Genetic comparison. Comparison of the uncorrected pairwise divergences of generated sequences

representing four Sri Lankan populations, Dambulla (DZ 1127), Maakandura (DZ 1410, DZ 1418), Mihintale (DZ
1445, DZ 1446) and Anuradhapura (DZ 1468) of Microhyla mihintalei sp. nov., and one population of Microhyla
rubra from Karnataka (SDBDU 2558), along with one available GenBank sequence (AB201192) show that the
genetic divergences range between 2.7−3.2% for the two species (Appendix 2).
Acoustic comparison. PCA with unrotated axes on the correlation matrix of the call characters (involving
eight variables: Call Duration, Number of Pulses, Pulse Rate, Dominant Frequency, Call Rise Time, Call Fall time,
50% Call Rise Time, 50% Call Fall Time) for the two species shows that Microhyla rubra from India separates out

336 · Zootaxa 4066 (3) © 2016 Magnolia Press WIJAYATHILAKA ET AL.

well from M. mihintalei in the PC space (Fig. 2). The separation however is only attributable to the factors that
contribute to the PC1 axis, which is explained mostly by Call Duration, Dominant Frequency, Call Rise Time and
Pulse Rate (Pulse Rate loads negatively while the other three load positively). The two species overlap completely
on PC2 axis (Fig. 2), which is explained mostly by Call Fall time, Number of Pulses and Call Rise Time (Call Rise
Time loads negatively and the other two load positively).

FIGURE 3. A–G. Microhyla mihintalei sp. nov. in life. A–E. Holotype (DZ 1553, an adult male). A. dorsolateral view with a
light-grey band extending from behind the eye to the groin; B. dorsal view showing the light reddish-brown dorsum; C. ventral
view highlighting the dark blackish-brown calling patch; D. lateral view of thigh and groin; E. posterior view of thighs with
irregular black markings; F. Paratype (DZ 1127, an adult male) in dorsal view; G. Paratype (DZ 1457, an adult male); H.
Microhyla rubra in life (SDBDU 2559, an adult male), from Shivanahalli, Karnataka.

Holotype description (measurements in mm). Small-sized, adult male (SVL 24.8). Head small (HW 7.4, HL
6.2), shorter than wide, snout sub-ovoid in dorsal and ventral view, rounded in lateral view, its length (SL 2.7)
longer than horizontal diameter of eye (EL 2.3); loreal region rounded with vertical canthus rostralis; interorbital
space wider (IUE 2.6) than upper eyelid (UEW 1.5) and internarial distance (IN 1.8); nostril closer to tip of snout
(NS 1.0) than eye (EN 1.4); tympanum indistinct or obscured by a layer of skin without clearly defined borders,
supratympanic fold distinct, extending from posterior corner of upper eyelid to near the insertion of forelimb at

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axilla; eye diameter (EL 2.3); vomerine ridge absent; tongue elliptical. Forelimbs moderately short and thin;
forelimb (FAL 5.1) shorter than hand length (HAL 6.0); fingers short, finger length formula I<IV<II<III; tips of all
fingers without discs, rounded; subarticular tubercles prominent, rounded, all present (Fig. 1); palmar tubercles
three, well developed, oval. Hindlimbs relatively long and thin, thigh length (TL 11.3) subequal to shank (SHL
11.5), and foot (FOL 11.6); relative digit lengths I<II<V<III<IV; tips of all toes without discs, rounded; webbing
present: basal; weakly developed dermal ridge on all toes present; subarticular tubercles prominent, oval; two
metatarsal tubercles, oval, shovel-shaped, outer one slightly larger than inner.
Skin of dorsum, snout, between eyes, sides of head, posterior part of back, upper and lower part of flanks and
ventral surface smooth, dorsal parts of forelimb, thigh, tibia and tarsus smooth to shagreened.
Colour in preservation. Dorsum light brownish-grey with a broad light grey band extending from behind the
eye to the groin; dorsal surface of forelimbs lighter in colour compared to the dorsum and with incomplete black
cross-bands; lateral side of head and tympanic area dark grey; groin light grey with black patches; two distinct and
narrow black streaks extending from the snout along the side of head to near the groin; posterior parts of thigh and
tibia with irregular black markings; a narrow black patch extends from anal opening to near the knee; ventral
surface creamy-white, throat with dark blackish-brown calling patch.
Colour in life (Fig. 3). Dorsum light reddish-brown with a faint light grey band extending from behind the eye
to the groin (Fig. 3D); lateral side of head and tympanic area dark grey; groin light grey with black patches; two
distinct and narrow black streaks extending from the snout along the side of head to near the groin; posterior parts
of thigh and tibia light brown with irregular black markings; a narrow black patch extends from anal opening to
near the knee; ventral surface grey, throat with dark blackish-brown calling patch (Fig. 3).
Variation. See Table 1 for morphometric data from 13 adult males and one female. DZ 1127 (in life): dorsum
reddish-brown with brown band extending from behind the eye up to the groin, faint cross-bands on both limbs,
lateral side of head and tympanic area light grey (Fig. 3F); DZ 1457 (in life: dorsum dark reddish-brown, lateral
side of head and tympanic area dark grey; ventral surface light grey with dark grey reticulations especially on the
throat; in preservation, light grey dorsum (Fig. 3G); DZ 1467 (in life): dark greyish-brown with dark brown dorsal
band.
Secondary sexual characters. Males: Single vocal sac externally visible on lower jaw. Female (DZ 1418): ova,
pigmented on poles (diameter 0.7–1.1 mm, N =15).
Distribution and Natural History. Microhyla mihintalei sp. nov. is found in the lowland dry zone (Fig. 4),
mostly in shaded areas, often close to stream and river banks or regions that become fairly stable ephemeral pools
during the rainy season. They were observed emerging from borrows in ground to call by around 7.30 PM, slightly
after the initiation of calling of M. ornata, a smaller sympatric species. Compared to M. ornata, the abundance of
M. mihintalei sp. nov. is much lower, and so are the corresponding numbers of tadpoles in pools. In captivity, M.
mihintalei was observed digging rapidly into soil using their hind feet. This indicates that they are active
borrowers, and not only ground-burrow utilizers. They lay eggs as loosely arranged sheets on the water surface,
mainly in ephemeral pools and their tadpoles also form loose shoals. The tadpole of M. mihintalei was described by
Bowatte and Meegaskumbura (2011), however, as M. rubra from Sri Lanka.

Discussion

Microhyla mihintalei sp. nov. can be distinguished from M. rubra with respect to the criteria that were evaluated in
the present study. They differ from each other by 2.7−3.2% uncorrected genetic distances for 16S rRNA, which
conforms to species-level genetic distances in amphibian sister taxa in general (Vences et al. 2005). However, this
is surprising, given that for M. ornata, a closely related species, shows very low genetic distances between Indian
and Sri Lankan populations.
Bossuyt et al. (2004, 2005) highlighted the clade-level endemism and the non-vagility of per-humid forest
adapted and freshwater restricted taxa despite the existence of many land bridges between India and Sri Lanka
during the last 500,000 years. Microhyla mihintalei and M. rubra also seem to be species that did not have genetic
exchange across most of these land-bridge connections despite being predominantly lowland dry-adapted species.
A deeper analysis, involving greater taxon sampling of microhylids between the two regions will help ascertain the
patterns of dispersal and distribution between the two regions.

338 · Zootaxa 4066 (3) © 2016 Magnolia Press WIJAYATHILAKA ET AL.

FIGURE 4. Distribution of Microhyla mihintalei sp. nov. in Sri Lanka indicating its wide distribution, right from sea-level up
to 500 m a.s.l. Populations for which genetic data (16S rRNA) were generated are denoted in red circles. Arrow indicates the
call-recorded population from Mihintale.

A NEW MICROHYLA FROM SRI LANKA Zootaxa 4066 (3) © 2016 Magnolia Press · 339
 The tadpole of Microhyla mihintalei has already been described earlier (Kirtisinghe 1957, 1958; Bowatte &
Meegaskumbura 2011), but as the Sri Lankan population of M. rubra. The tadpole of Microhyla rubra so far has
only brief and early descriptions (Rao 1918; Parker 1928, 1934), which precludes finer scale comparison with the
Sri Lankan sister species. Larvae of M. mihintalei has a whip-like tail-end flagellum, a dorsoterminal mouth,
absence of flaps, presence of a median notch on upper lip and presence of papillae on lower lip (Bowatte &
Meegaskumbura 2011). Comparative study of the two tadpoles may provide further characters to distinguish the
two species.

Acknowledgements

We thank the National Research Council Sri Lanka (Grant # 11-124) for funding, and graduate studentships;
Department of Wildlife Conservation and Forest Department of Sri Lanka for research permits. Champika Bandara
for field work and other EES lab members who contributed; the State Forest Department, Government of
Karnataka, India for study permission to SDB.

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APPENDIXϭ͘Mann–Whitney U test results of all morphological measurements of two species, Microhyla rubra and
Microhyla mihintalei sp. nov. Significance was accepted at P ≤ 0.05 and U < 24.
Measurement P value U value Significant
SVL 0.00112 6.5 Yes
TL 0.00262 10 Yes
SHL 0.26272 36 No
FOL 0.0151 18 Yes
HW 0.00188 8.5 Yes
HL 0.00652 14 Yes
SL 0.00338 11 Yes
IUE 0.2187 34.5 No
UEW 0.00596 13.5 Yes
MN 0.15854 32 No
EN 0.12852 30.5 No
NS 0.5157 42.5 No
EL 0.3125 30.5 No
FAL 0.02034 19.5 Yes
HAL 0.0048 12.5 Yes

APPENDIX 2. Uncorrected pairwise distances (%) for 16S mtDNA gene fragment between two species.
Species 1 2 3 4 5 6 7
1. Microhyla rubra (SDB2558)
2. M. rubra (AB201192) 0.57
3. M. mihintalei sp. nov. (DZ 1127) 3.00 2.81
4. M. mihintalei sp. nov. (DZ 1410) 2.87 2.68 0
5. M. mihintalei sp. nov. (DZ 1418) 2.88 2.69 0 0
6. M. mihintalei sp. nov. (DZ 1445) 2.89 2.70 0 0 0
7. M. mihintalei sp. nov. (DZ 1446) 2.87 2.68 0 0 0 0
8. M. mihintalei sp. nov. (DZ 1468) 3.23 3.02 0.21 0.20 0.20 0.21 0.20

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