Research Article |
Corresponding author: Ananthanarayanan Sankar ( a.sankar@nus.edu.sg ) Academic editor: Raffael Ernst
© 2022 Ananthanarayanan Sankar, Ingg Thong Law, Ing Sind Law, Rasu Shivaram, Robin K. Abraham, Kin Onn Chan.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Sankar A, Law IT, Law IS, Shivaram R, Abraham RK, Chan KO (2022) Morphology, phylogeny, and species delimitation of Micryletta (Anura: Microhylidae) reveals a new species from Singapore. Vertebrate Zoology 72: 457-467. https://doi.org/10.3897/vz.72.e85020
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The genus Micryletta, also known as paddy frogs, ranges across much of south, east, and southeast Asia. Due to their relatively broad distribution and overall morphological similarities, many species have gone undetected until recently, largely owing to the use of molecular data. Consequently, the species diversity within this genus has quadrupled in just three years from three species prior to 2018, to 12 species in 2021, indicating that the systematics of this genus is still poorly understood. As such, we assembled the most comprehensive molecular phylogeny of Micryletta hitherto including novel sequences from a previously unsampled population from Singapore to assess the species diversity within this genus. In particular, we investigate the population from Singapore whose specific identity remains in question due to the lack of voucher specimens and genetic material. Our results show that the Singapore population represents a strongly supported and distinct lineage that is most closely related to M. inornata sensu stricto from Sumatra, Indonesia. Morphological and species delimitation analyses corroborate its distinction as a new species, which we describe herein as M. subaraji sp. nov. This and recent new taxon discoveries in Singapore demonstrate that the biodiversity of the highly urbanized island-state is still far from being fully realized and underscores the need for continued systematic surveys and protection of remaining habitats.
Amphibian, Kranji Marshes, Micryletta subaraji sp. nov., Systematics, Taxonomy
The amphibian genus Micryletta Dubois, 1987 (family: Microhylidae), commonly referred to as paddy frogs, is a relatively small genus comprising twelve species, namely M. aishani Das, Garg, Hamidy, Smith and Biju, 2019; M. dissimulans Suwannapoom, Nguyen, Pawangkhanant, Gorin, Chomdej, Che and Poyarkov, 2020; M. erythropoda (Tarkhnishvili, 1994); M. hekouensis Liu, Hou, Mo and Rao, 2021; M. immaculata Yang and Poyarkov, 2021; M. inornata (Boulenger, 1890); M. lineata (Taylor, 1962); M. melanops Poyarkov, Nguyen, Yang, and Gorin, 2021; M. menglienica (Yang and Su, 1980); M. nigromaculata Poyarkov, Nguyen, Duong, Gorin and Yang, 2018; M. steinegeri (Boulenger, 1909); and M. sumatrana Munir, Hamidy, Matsui, Kusrini and Nishikawa, 2020. These small-sized terrestrial frogs range from northern Sumatra through the Malay Peninsula, Indochina, southern China (Yunnan and Taiwan), and northeastern India (
Micryletta inornata was described from Sumatra, Indonesia, and is the type species of the genus. Due to morphological similarities with other species of Micryletta, M. inornata was historically considered a widespread species that occurred in Sumatra and throughout the Malay Peninsula and Indochina. However, a recent study by
In 2019, the occurrence of Micryletta was reported for the first time in Singapore (Central Catchment Nature Reserve, CCNR) and was putatively identified as M. cf. inornata based on cursory morphological examination in the field and live photographs of the specimen taken prior to its release (
A Distribution of confirmed localities of Micryletta inornata s.s. from Sumatra and Myanmar (blue diamonds) and M. cf. inornata from Kranji Marshes (green circle), and Central Catchment Nature Reserve (white circle) in Singapore. B Photo showing M. cf. inornata in life. C Degraded habitat at Kranji Marshes that M. cf. inornata specimens were detected in. Photos by Law Ingg Thong.
We collected 11 specimens during fieldwork in Kranji Marshes, Singapore (1°25.1150’N, 103°43.2641’E; Fig.
We took the following measurements of collected specimens using Mitutoyo digital calipers to the nearest 0.01 mm: SVL (snout-vent length; tip of snout to cloaca), HL (head length; tip of snout to posterior margin of mandible), HW (head width; measured across mandibular articulations), HD (head depth; measured at the occiput), UEW (upper eye-lid width; widest distance measured from upper margin of orbit to medial margin of upper eye lid), EL (eye length; measured across anterior and posterior corners of orbit), IND (internarial distance; distance across medial margins of nostrils), IOD (inter-orbital distance; smallest distance between medial margins of upper eye lid), SL (snout length; tip of snout to anterior-most point of orbit), N-EL (nostril-eye length; posterior margin of nostril to anterior-most pint of orbit), S-NL (snout-nostril length, anterior margin of nostril to tip of snout), FAL (forearm length; base of outer palmar tubercle to posterior margin of elbow inflection), HAL (hand length; base of outer palmar tubercle to tip of third finger), THL (thigh length; cloaca to outer margin of knee inflection), AGL (axilla-groin length; distance between arm and leg insertions), TFL (tibiofibula length; measured across the outer margins of knee and ankle inflections), and FL (foot length; outer margin of ankle inflection to tip of fourth toe).
A Levene’s test was performed on each dependant variable (character) to test for equal variance. However, the p-values of the Levene’s tests were >0.05 for each dependant variable. Therefore, t-tests were performed to determine whether the Singapore specimens were statistically different from Micryletta inornata s.s. collected from Sumatra following the framework outlined by
Advertisement call recordings were cleaned of background noise using the Noise Reduction tool in Audacity version 2.3.3 (Audacity, GNU General Public License). Noise reduction settings are as follow: Noise Reduction (dB) = 40; Sensitivity=20; Frequency Smoothing (bands) = 3. A total of 12 calls (n=12) from a single individual was analyzed. All calls were recorded at an ambient temperature range of 26–27 ºC. We generated oscillograms and spectrograms using the R package of SEEWAVE (
We amplified a ~900 bp fragment of the 16S rRNA mitochondrial gene for four specimens from Singapore (ZRC 1.13323, ZRC 1.13369, ZRC 1.13370, ZRC 1.13389) using the primers 16SD-L (5′-GTRGGCCTAAAAGCAGCCAC-3′), and 16SD- H (5′-CTCCGGTCTGAACTCAGATGACGTAG- 3′) (
Sequences were assembled and aligned (MUSCLE algorithm) using Geneious v5.6.7 (
We also performed two species delimitation analyses using the programs mPTP v.0.2.4 (
Raw measurements and summary statistics of all 11 specimens collected in this study are presented in Table
Measurements (in mm) of collected Micryletta cf. inornata from Singapore. Abbreviations are defined in Methods; the asterisk (*) denotes the holotype specimen. Mensural data of juvenile specimens, which are not in the type series, are also provided.
ZRC 1.13370* | ZRC 1.13369 | ZRC 1.13389 | ZRC 1.13469 | ZRC 1.13470 | Mean ± SD (n =5) | ZRC 1.13323 | ZRC 1.13466 | ZRC 1.13467 | ZRC 1.13468 | Mean ± SD (n =4) | ZRC 1.13390 | ZRC 1.13465 | Mean ± SD (n =2) | |
Sex | Male | Male | Male | Male | Male | Female | Female | Female | Female | Juvenile | Juvenile | |||
SVL | 18.90 | 17.86 | 17.73 | 17.45 | 18.09 | 18.01±0.49 | 23.04 | 20.69 | 16.94 | 18.06 | 19.68±2.37 | 15.05 | 15.40 | 15.23±0.18 |
HL | 6.23 | 5.82 | 5.88 | 5.97 | 6.31 | 6.04±0.19 | 6.46 | 6.56 | 4.99 | 5.90 | 5.98±0.62 | 4.76 | 5.17 | 4.97±0.21 |
HW | 6.55 | 5.92 | 6.48 | 6.26 | 6.17 | 6.28±0.23 | 5.91 | 5.50 | 4.81 | 5.25 | 5.37±0.4 | 4.86 | 4.94 | 4.9±0.04 |
HD | 3.85 | 3.6 | 3.35 | 4.04 | 3.82 | 3.73±0.24 | 2.02 | 3.32 | 3.19 | 2.46 | 2.75±0.53 | 2.71 | 2.50 | 2.61±0.11 |
UEW | 1.54 | 1.31 | 1.43 | 1.21 | 1.25 | 1.35±0.12 | 1.32 | 1.14 | 1.65 | 1.29 | 1.35±0.19 | 1.09 | 1.20 | 1.15±0.05 |
EL | 2.39 | 2.12 | 2.46 | 2.12 | 2.37 | 2.29±0.14 | 2.92 | 2.95 | 2.45 | 2.50 | 2.71±0.23 | 1.54 | 2.20 | 1.87±0.33 |
IND | 1.76 | 1.45 | 1.91 | 1.64 | 1.57 | 1.67±0.16 | 1.41 | 1.85 | 1.62 | 1.84 | 1.68±0.18 | 1.27 | 1.50 | 1.39±0.12 |
IOD | 2.26 | 2.31 | 2.61 | 2.50 | 2.00 | 2.34±0.21 | 2.96 | 3.04 | 2.29 | 2.70 | 2.75±0.29 | 2.67 | 2.38 | 2.53±0.15 |
SL | 2.41 | 2.12 | 2.26 | 2.31 | 2.59 | 2.34±0.16 | 2.95 | 3.30 | 2.73 | 2.82 | 2.95±0.22 | 2.26 | 2.88 | 2.57±0.31 |
N-EL | 1.26 | 1.26 | 1.36 | 1.33 | 1.40 | 1.32±0.06 | 2.38 | 2.48 | 2.22 | 2.34 | 2.36±0.09 | 1.96 | 1.93 | 1.95±0.02 |
S-NL | 1.15 | 0.86 | 0.9 | 0.98 | 1.19 | 1.02±0.13 | 1.50 | 1.22 | 1.14 | 1.00 | 1.22±0.18 | 1.06 | 1.26 | 1.16±0.1 |
FAL | 4.23 | 4.49 | 4.43 | 4.27 | 3.82 | 4.25±0.23 | 10.63 | 8.95 | 7.23 | 8.46 | 8.82±1.22 | 6.53 | 7.22 | 6.88±0.35 |
HAL | 5.13 | 4.86 | 4.57 | 4.37 | 5.03 | 4.79±0.28 | 5.72 | 5.68 | 4.16 | 4.58 | 5.04±0.68 | 3.65 | 3.94 | 3.8±0.15 |
THL | 8.24 | 8.24 | 7.41 | 7.99 | 8.04 | 7.98±0.3 | 9.12 | 8.29 | 7.16 | 6.89 | 7.87±0.89 | 6.81 | 6.55 | 6.68±0.13 |
AGL | 6.91 | 6.53 | 8.25 | 9.75 | 9.08 | 8.1±1.23 | 12.5 | 8.73 | 8.37 | 8.27 | 9.47±1.76 | 7.4 | 7.27 | 7.34±0.07 |
TFL | 8.59 | 8.15 | 7.98 | 7.92 | 8.01 | 8.13±0.24 | 9.47 | 9.49 | 7.82 | 8.11 | 8.72±0.76 | 6.78 | 7.1 | 6.94±0.16 |
FL | 9.4 | 8.55 | 7.37 | 9.30 | 10.75 | 9.07±1.11 | 7.76 | 9.96 | 7.77 | 8.72 | 8.55±0.9 | 7.21 | 7.38 | 7.3±0.09 |
Summary statistics of male specimens (mean ± standard deviation) and results of the t-test between Micryletta inornata s.s. from Sumatra and Micryletta cf. inornata from Singapore. The t-test was performed on the size-adjusted data (see materials and methods for more details). Size-adjustments were applied for all parameters except SVL. Data from Sumatra were obtained from
Mean ± SD | Mean ± SD | p-value | |
(Sumatra, n=3) | (Singapore, n=5) | ||
SVL | 18.1±1.7 | 18.01±0.49 | 0.9797 |
HL | 4.83±0.21 | 6.04±0.19 | 0.0000** |
HW | 5.4±0.36 | 6.28±0.23 | 0.0050** |
UEW | 0.97±0.12 | 1.35±0.12 | 0.0014** |
EL | 2.17±0.09 | 2.29±0.14 | 0.1962 |
IND | 1.4±0.08 | 1.67±0.16 | 0.0603 |
IOD | 2.4±0.24 | 2.34±0.21 | 0.6226 |
SL | 1.93±0.12 | 2.34±0.16 | 0.0042** |
N-EL | 1.2±0.22 | 1.32±0.06 | 0.0268* |
S-NL | 0.57±0.09 | 1.02±0.13 | 0.0001** |
FAL | 4.1±0.24 | 4.25±0.23 | 0.4344 |
HAL | 4.53±0.37 | 4.79±0.28 | 0.0864 |
TFL | 8.67±0.56 | 8.13±0.24 | 0.0313* |
FL | 8.43±0.52 | 9.07±1.11 | 0.3709 |
Comparisons of uncorrected p-distances revealed that M. cf. inornata from Singapore is 3.5–4.2% divergent from M. inornata s.s., which is comparable to divergences between M. steinegeri and M. immaculata (2.9–4.5%) and between M. hekouensis and M. immaculata (2.6–4.5%) but higher than divergences between M. erythropoda and M. lineata (2.3–2.9%) and between M. steinegeri and M. hekouensis (1.6–1.9%; Fig.
The mPTP species delimitation analysis inferred the Singapore population as a distinct species with strong support (average support value = 99; Fig.
The advertisement call of Micryletta cf. inornata from Singapore is a pulsatile sweeping metallic call that comprises of an average of 21 pulses (mean=21; range=16–28; n=12; Fig.
Maximum likelihood phylogeny based on 1,097 bps of the 16S rRNA mitochondrial gene. Nominal species and their regional distribution ranges are labeled to the right of the phylogeny. The clade containing M. cf. inornata from Singapore is highlighted in green. BS = ultrafast bootstrap support values.
Results of the ASAP and mPTP species delimitation analyses. Rankings for the ASAP analysis are based on ASAP-scores (for more information on ASAP-scores, see https://bioinfo.mnhn.fr/abi/public/asap/FAQ_asap.html). Numbers within bars represent the number of samples contained within each partition.
Results from all morphological, phylogenetic, and species delimitation analyses support the recognition of Micryletta cf. inornata from Singapore as a distinct species. Applying an integrative taxonomic framework based on multiple lines of support, we describe Micryletta cf. inornata from Singapore as a new species below:
Micryletta inornata Law, Thomas, and Law, 2019: 5
Subaraj’s Paddy Frog.
ZRC1.13370 (Fig.
ZRC 1.13369, adult male; ZRC 1.13389, adult male; ZRC 1.13469, adult male; ZRC 1.13470, adult male; ZRC 1.13323, adult female; ZRC 1.13466, adult female; ZRC 1.13467, adult female; ZRC 1.13468, adult female; all collected from the same location as the holotype between January and October 2021 by Law Ing Sind, Law Ingg Thong, and Sankar Ananthanarayanan (Fig.
Micryletta subaraji sp. nov. is a member of Micryletta based on its sister relationship to Micryletta inornata s.s. (Fig.
Adult male, SVL 18.9 mm; habitus slender; head slightly wider than long (HW/HL 1.05), acuminate in dorsal profile with abruptly rounded snout; snout length (SL 2.41 mm) roughly equal to eye diameter (EL 2.39 mm), and slightly longer than interorbital distance (IOD 2.26 mm); nostrils closer to tip of snout (S-NL 1.15 mm) than to anterior tip of eye (N-EL 1.26 mm); interorbital distance larger than internarial distance (IND 1.76 mm); eyes large, 38% of head length; pupil round and moderately dilated; tympanum not visible, and supratympanic fold not present. Skin on dorsal and ventral surfaces smooth; forelimbs and hindlimbs slender; F3>F4>F2>F1, T4>T3>T5>T2>T1; fingertips and toetips lacking in discs; no webbing between fingers and toes; circular basal subarticular tubercles on all fingers and toes; circular supernumerary metacarpal tubercles present at the base of F2, F3, and F4, palmar tubercle well-developed and circular, thenar tubercle and outer metacarpal tubercle circular; inner metatarsal tubercle circular, outer metatarsal tubercle absent (Fig.
Dorsum greyish brown; forelimbs and hindlimbs light brown with dark brown mottling; one dark brown broken vertebral stripe, with broken paravertebral stripes on each side; strong dark brown cephalic mottling in between eyelids A solid black lateral stripe from the tip of snout, past eye, remains unbroken until forearm, becomes a dark mottling between forelimb and hindlimb, where it terminates; a thin cream stripe runs parallel below the eyestripe, starts at nostril, terminates at forelimb. Venter cream with dark brown to grey mottling under sides of belly; inner legs are immaculate and uniformly light cream, tibiofibula is moderately mottled, bottom of tarsus is light brown. Stippling is more apparent under a microscope. Black gular sac (Fig.
Raw mensural data are presented in Table
Micryletta subaraji sp. nov. is so far only known from Singapore where it occurs in Kranji Marshes and putatively in the Central Catchment Nature Reserve. It may also occur in other parts of the island where suitable habitat is present.
The specific epithet honours the late Mr. Subaraj Rajathurai, who is a pioneer of conservation in Singapore.
In addition to the collected specimens, male advertisement calls were heard at an ephemeral pool in a depression created by an uprooted tree. The calling individuals were perched amidst dense undergrowth surrounding this puddle. These ephemeral depressions may be used by male frogs as a means to amplify their advertisement calls. Several other Microhylids (both native and introduced) were recorded in sympatry at the type locality including Microhyla heymonsi, Microhyla butleri, Microhyla cf. mukhlesuri, and Kaloula pulchra.
Although our molecular data is based on a single mitochondrial gene, all phylogenetic and species delimitation analyses strongly and unambiguously supported the Singapore population as a new species that is distinct from its sister lineage, M. inornata s.s. from Sumatra. This distinction is further corroborated by morphological data. Unfortunately, we were unable to compare advertisement calls because no recordings are available for M. inornata s.s. Nevertheless, we present and describe the advertisement call of the new species to facilitate future comparisons. We were also unable to obtain Micryletta specimens from the Central Catchment Nature Reserve (CCNR) where it was previously documented but not collected. Based on digital photographs, the CCNR specimen has less prominent dorsal markings and lacks a solid lateral stripe along the side of the head and flank (
Our phylogeny also indicates that M. immaculata is a complex that is not yet adequately understood. This species is currently considered to be endemic to Hainan Island, China (
Despite being a small and highly urbanized city-state, the occurrence of Micryletta in Singapore was only discovered in 2019 (
We thank the Singapore National Parks Board for facilitating our research under the permit (NP/RP20-116). We are also grateful to Johnathan Hruska for help with call analyses.