Research Article |
Corresponding author: Tao Thien Nguyen ( nguyenthientao@gmail.com ) Corresponding author: Jianping Jiang ( jiangjp@cib.ac.cn ) Academic editor: Johannes Penner
© 2021 Chung Van Hoang, Tao Thien Nguyen, Hoa Thi Ninh, Anh Mai Luong, Cuong The Pham, Truong Quang Nguyen, Nikolai L. Orlov, Youhua Chen, Bin Wang, Thomas Ziegler, Jianping Jiang.
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:
Hoang CV, Nguyen TT, Ninh HT, Luong AM, Pham CT, Nguyen TQ, Orlov NL, Chen YH, Wang B, Ziegler T, Jiang JP (2021) Two new cryptic species of Microhyla Tschudi, 1838 (Amphibia, Anura, Microhylidae) related to the M. heymonsi group from central Vietnam. ZooKeys 1036: 47-74. https://doi.org/10.3897/zookeys.1036.56919
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The Microhyla heymonsi species complex from central Vietnam was examined, and based upon morphological and molecular evidence, two new species are described. The discovery of Microhyla daklakensis sp. nov. and Microhyla ninhthuanensis sp. nov. brings the total number of known species in the genus to 46 and the species number of Microhyla in Vietnam to 13. The Truong Son Range harbors the highest diversity of the genus Microhyla with 11 recorded species so far. However, this apparent micro-endemic diversity is at risk because of habitat loss by deforestation, which highlights the necessity of further research leading to improved conservation measures.
Microhyla, new species, central Vietnam, morphology, molecular phylogeny
The genus Microhyla Tschudi, 1838 currently contains 44 species, which are distributed from India and Sri Lanka eastwards to the Ryukyu Archipelago of Japan and southwards to Indonesia (
In Vietnam, nine species of Microhyla have been recorded to date (
Heymon’s narrow-mouthed frog, Microhyla heymonsi Vogt, 1911, was originally described from Taiwan based on eight male specimens (ZMB 54906–54913). Apart from Taiwan, this species has a wide distribution (Jang-Liaw and Chou 2015), and is reported from across East and Southeast Asia, from mainland China through Sumatra (Amphibia Web 2020;
During our recent field surveys in Kon Tum, Dak Lak, Lam Dong, and Ninh Thuan provinces in central Vietnam between 2016 and 2019, a number of microhylid frog specimens were collected that morphologically resembled M. ‘heymonsi’. However, morphological and molecular analyses showed that these populations represent independent evolutionary lineages. The population from Lam Dong Province was recently described as M. neglecta (
Field surveys were conducted in Kon Tum, Dak Lak, Lam Dong, and Ninh Thuan provinces, Vietnam (Fig.
Extraction of genomic DNA from 41 tissue samples (Suppl. material
In addition to the 41 sequences of the collected samples in this work, we used 76 available sequences of 12S rRNA–16S rRNA from GenBank (Garg et al. 2019) for phylogenetic analyses. Sequences of Kaloula pulchra Gray, 1831 were included in the analysis as outgroup (
Phylogenetic trees were constructed by using Maximum Likelihood (ML) and Bayesian Inference (BI) analyses. Chromas Pro software (Technelysium Pty Ltd., Tewantin, Australia) was used to edit the sequences, and then aligned using the ClustalW (
All measurements were taken from 63 preserved specimens (Suppl. material
SVL snout-vent length (measured from the tip of snout to cloaca);
HL head length (measured from tip of snout to hind border of jaw angle, but not measured parallel with the median line as done by
SL snout length (measured from the anterior corner of eye to the tip of snout);
EL eye length (measured as the distance between the anterior and posterior corners of the eye);
N-EL nostril-eye length (measured as the distance between the anterior corner of the eye and the nostril);
HW head width (measured as the maximum width of the head on the level of mouth angles in ventral view);
IND internarial distance (measured as the distance between central points of nostrils);
IOD interorbital distance (measured as the shortest distance between the medial edges of eyeballs in dorsal view);
UEW upper eyelid width (measured as the widest distance from the medial edge of eyeball to the lateral edge of the upper eyelid);
FLL forelimb length (measured as length of straightened forelimb to tip of third finger);
LAL lower arm and hand length (measured as distance from elbow to tip of third finger);
HAL hand length (measured from proximal end of outer palmar [metacarpal] tubercle to tip of third finger);
IPTL inner palmar tubercle length (measured as maximal distance from proximal to distal ends of inner palmar tubercle);
OPTL outer palmar tubercle length (measured as maximal diameter of outer palmar tubercle);
HLL hindlimb length (measured as length of straightened hindlimb from groin to tip of fourth toe);
TL tibia length (taken as the distance between the knee and tibiotarsal articulation);
FL foot length (measured from distal end of tibia to tip of toe IV);
IMTL inner metatarsal tubercle length (taken as maximal length of inner metatarsal tubercle);
1TOEL first toe length (from distal end of inner metatarsal tubercle to tip of first toe);
OMTL outer metatarsal tubercle length;
1FW first finger width (measured at the distal phalanx);
1–3FLO finger lengths, outer side (O) of the first-third;
2–4FLI finger lengths, inner side (I) of the second-fourth;
2–4FDW finger disk diameters;
1–5TDW toe disk diameters.
Terminology for describing eye coloration in life followed
A total of 45 species (Suppl. material
Measurements were used to compare the morphometric difference between the four males and six females of the population from Dak Lak Province and the nine males and two females of the population from Ninh Thuan Province vs. the eight males and two females of M. ‘heymonsi’ from Kon Tum Province. All statistical analyses were performed using PAST 2.17b software (
The final alignment of 12S rRNA–16S rRNA contained 1979 numbers of characters. Of these, 1222 sites were conserved and 731 sites exhibited variation, with 587 characters being parsimony-informative. The transition-transversion bias (R) was estimated as 1.279. Nucleotide frequencies were A = 31.64%, T = 23.96%, C = 23.09%, and G = 21.31% (data for ingroup only).
The genetic divergence of the population from Ninh Thuan Province and its congeners ranged from 3.6% (M. heymonsi sensu stricto from Taiwan) to 13.2–13.4% (M. laterite). The genetic divergence of the population from Dak Lak Province and its congeners ranged from 2.4–2.9% (the M. ‘heymonsi’ population from Kon Tum Province) to 12.4–13.4% (M. nanapollexa). These values were higher than the genetic distances between other recognized species of Microhyla (i.e., 2.4% between M. fanjingshanensis and M. beilunensis, M. borneensis and M. malang; 2.4% between M. okinavensis and M. beilunensis; 2.2% between M. okinavensis and M. mixtura) (Suppl. material
The BI and ML analyses produced topologies with –ln L = 15327.579 and 12672.916, respectively. BI and ML analyses obtained similar topologies (Fig.
Bayesian Inference of the matrilineal genealogy of Microhyla derived from the analysis of 1979 bp of 12S rRNA–16S rRNA mtDNA sequences. Numbers above and below branches are Bayesian posterior probabilities and ML bootstrap values. The scale bar represents 0.05 nucleotide substitutions per site.
The two new forms could be assigned to the genus Microhyla based both on the molecular phylogenetic data and the following morphological characters: size relatively small; maxillary and vomerine teeth absent; vomer divided into two parts, disappearing at the posterior edge of the choana; tongue round posteriorly; skin smooth or with tubercles; tympanum hidden; palate with one or two rows of horizontal skin ridges; fingers without webbing; toes slightly webbed or free of webbing; metacarpal tubercles two or three; and the absence of skin ridge or skin projection between the subarticular tubercles of toes III and IV.
The two new forms differ from other known species of Microhyla by having a medium body size, stocky habit, round snout, smooth skin on dorsum, disks on distal end of toes present, dorsal median longitudinal grooves on finger disks present, superciliary tubercles absent, light dorsomedial vertebral line present (morphological characters and distribution data for each species are summarized in Suppl. material
The two new forms of Microhyla from Dak Lak and Ninh Thuan provinces can also be separated from M. ‘heymonsi’ based on morphometric data. We extracted three principal component axes in the PCA result with eigenvalues greater than 0.01, the first two component axes accounted for 93.49% (in males) and 81.80% (in females) of the variation (Suppl. material
Based upon the phylogenetic analyses of 12S rRNA–16S rRNA sequences, the two populations clearly differ from all other species of Microhyla for which comparable genetic data are available, and the observed differences in mtDNA sequences were congruent with other morphological data. Accordingly, we describe the two new species as follows.
(n = 10) All collected by C.V. Hoang et al. at the same location as the holotype: IEBR.A 4841–4843 (HAO74, HAO76, HAO77),
Selected diagnostic characters for the comparisons between the species of the Microhyla heymonsi group.
M. ‘heymonsi’ | Microhyla daklakensis sp. nov. | Microhyla ninhthuanensis sp. nov. | |
---|---|---|---|
SVL M | 16.5–22.0 | 17.7–20.1 | 17.3–18.8 |
SVL F | 18.0–26.5 | 22.9–26.8 | 21.6–23.6 |
Habit | Stocky | Stocky | Stocky |
Snout profile | rounded, obtusely pointed | rounded | rounded |
Dorsal skin | smooth | smooth | smooth |
F1 vs. F2 | F1 ≤ ½ F2 | F1 > ½ F2 | F1 ≤ ½ F2 |
Disks on distal end of fingers | present | present | present |
Dorsal median longitudinal line grooves on finger disks | usually present | present | present |
Disks on distal end of toes | present | present | present |
Dorsal peripheral grooves on toe disks | usually present | present | present |
Presence or absence of superciliary tubercles | absent | absent | absent |
Presence or absence of light dorsomedial (vertebral) line | present | present | present |
Tibiotarsal articulation | shorter than snout | shorter than snout | shorter than snout |
Foot webbing | I2 – 2½II2 – 3III3 – 4IV4⅓ – 3V | I2 – 2½II2 – 3III3 – 4IV4⅓ – 3V | I2 – 2½II2 – 3III3 – 4IV4⅓ – 3V |
Distribution | S China, NE India, SE Asia to Sumatra | Dak Lak | Ninh Thuan |
Microhyla ninhthuanensis sp. nov. is distinguished from its congeners by a combination of the following morphological characters: 1) body stocky, size medium (SVL 17.3–18.8 mm, n = 9 males; 21.6–23.6 mm, n = 2 females). 2) dorsum smooth; 3) head triangular, snout round in profile; 4) finger I shorter than one-half the length of finger II; 5) tips of all outer fingers dilated, forming disks, with a median longitudinal groove visible dorsally; 6) tips of all toes distinctly dilated into disks, with a weak median longitudinal groove visible dorsally, producing the appearance of two scutes; 7) inner metacarpal tubercle oval and prominent, paired outer metacarpal tubercle divided by a waist into two equal-sized parts: outer part quite round, inner part quite crescent; 8) tibiotarsal articulation of straightened limb not reaching snout; 9) webbing basal: I2 – 2½II2 – 3III3 – 4IV4⅓ – 3V; 10) inner metatarsal tubercles oval, prominent and outer metatarsal tubercles round; 11) upper eyelid without supraciliary spines; 12) narrow faint brown stripe extending from rear corner of eye to axilla; 13) light thin vertebral stripe present, canthus rostralis with dark lines; 14) small dark round spot at mid-dorsum, divided by a light vertebral stripe; 15) dorsum pinkish brown with dark brown marking in X-shape between eyes and arm, along vertebral and dorsolateral region stripes form wavy dust strip towards the groin, a small dark marking ‘ ()’-shaped in the center of the dorsum and mid-dorsal line; 16) an even black lateral stripe from above arm, almost reaching groin; 17) chin dark grey; throat white with scattered dark grey dusting; chest and belly creamy white.
Dorsolateral and ventral views of the specimens in life: comparative specimen of Microhyla neglecta (
Habitus stocky, size medium, SVL 18.20 mm; head wider than long (HL/HW 0.83); snout long, abruptly round in dorsal view, projecting beyond margin of lower jaw, longer than diameter of eye (SL/EL 1.24); eyes small, slightly protuberant, pupil round (Fig.
Dorsolateral and ventral views of the specimens in preservative: comparative specimen of Microhyla neglecta (
Forelimbs short, about three times shorter than hindlimbs (FLL/HLL 0.31); hand two times shorter than forelimb length (HAL/FLL 0.43); fingers slender, free of webbing, round in cross-section, skin fringes of fingers weak; first finger shorter than one-half the length of the second finger (1FLO/2FLO 0.44), second finger slightly shorter than fourth (2FLI/4FLI 0.98), latter much longer than first, and much shorter than third (2FLI/3FLI 0.74); relative finger lengths: I < II < IV < III (Fig.
Lateral view of the head, right hand and right food of Microhyla neglecta (
Hindlimbs slender and slightly short (HLL 31.62), tibia length longer than half of snout-vent length (TL/SVL 0.58); tibiotarsal articulation at straightened limb not reaching snout; foot longer than tibia (FL/TL 1.32); relative toe lengths: I<II<V<III<IV; tarsus smooth, inner tarsal fold absent; tips of all toes distinctly dilated into disks, slightly wider than those of fingers (3TDW 0.53, 3FDW/3TDW 0.93), dorsally all toes with median longitudinal grooves at disks; relative toe disk widths: I<V<II<III=IV; webbing between toes basal and poorly developed, webbing formula: I2 – 2½II2 – 3III3 – 4IV4⅓ – 3V; subarticular tubercles prominent, all present, circular, formula 1, 1, 2, 3, 2; inner metatarsal tubercle elongated, oval, large and prominent, length (IMTL 0.78); outer metatarsal tubercle round, elevated and very well distinct, smaller (OMTL 0.38) than length of inner metatarsal tubercle (Fig.
Dorsal surface of head and body smooth, flank shagreened, dorsal surface smooth, including fingers and toes, fore and hind limbs; ventral surfaces smooth.
Dorsal surface of head and trunk pinkish brown with a dark brown marking in X-shape between eyes to arm, dorsolateral stripes form wavy dust strip towards the groin; a small dark marking in ‘ ()’-shape in the center of the dorsum and a mid-dorsal line extending from the tip of snout to vent. Flanks and lateral surface of head dark, a dark lateral stripe running from snout tip to nostril, fading towards upper jaw. Chin dark grey; throat white with scattered dark grey dusting; chest and belly creamy white. Limbs dorsally with narrow indistinct dark brown cross-bars; fingers and toes dorsally brown with dark brown cross-bars; forelimbs ventrally creamy white, hindlimbs ventrally with creamy white thigh becoming dark grey toward shank, foot. Iris bicolored, golden in upper one-third, dark copper in its lower two-thirds; pupil oval, horizontal, black.
After preservation in ethanol, dorsal coloration changed from light brown to greyish pink (Fig.
Specimens vary in body size, dorsal markings, and black scapular spots. Adult males smaller than adult females, adult males with small vocal sac (Suppl. material
Microhyla ninhthuanensis sp. nov. is morphologically most similar to M. heymonsi sensu stricto (Fig.
Specific epithet is in reference to the type locality, Ninh Thuan Province. We recommend “Ninh Thuan narrow-mouth frog” as the common English name and “Nhái bầu ninh thuận” as the Vietnamese name.
All specimens were collected at night from 19:00 to 23:00 h on the ground near the banks of a small stream in the forest and on the sides of a recently constructed road next to the devastated forests (Fig.
Microhyla ninhthuanensis sp. nov. is currently only known from the type locality in Phuoc Binh National Park, Ninh Thuan Province, Vietnam (Fig.
Currently, the evergreen forest in Phuoc Binh National Park is connected with other forests in Tay Nguyen Plateau. Based on its habitat and altitudinal range, the new species is likely to be endemic to Tay Nguyen Plateau. However, the extent of its actual distribution range requires further study. Given the available information, we suggest Microhyla ninhthuanensis sp. nov. be considered as Data Deficient following IUCN’s Red List categories (IUCN Standards and Petitions Subcommittee 2001).
(n = 9) All collected by C.V. Hoang et al. at the same location as the holotype: IEBR.A 4845-4746 (
(1) Microhyla daklakensis sp. nov. is distinguished from its congeners by a combination of the following morphological characters: 1) body stocky, size medium (SVL 17.7–20.1 mm n = 4 males; 21.1–23.8 mm, n = 6 females), 2) dorsum smooth; 3) snout round in profile; 4) finger I longer than one-half the length of the finger II; 5) tips of all outer fingers dilated, forming disks, with a median longitudinal groove visible dorsally; 6) tips of all toes distinctly dilated into disks, with a weak median longitudinal groove visible dorsally, producing the appearance of two scutes; 7) inner metacarpal tubercle oval and prominent, paired outer metacarpal tubercle divided by a waistline into two equal-sized parts: outer part quite round, inner part crescent-shaped; 8) tibiotarsal articulation of straightened limb not reaching snout; 9) webbing basal: I2 – 2½II2 – 3III3 – 4IV4⅓ – 3V; 10) inner metatarsal tubercles oval, prominent and outer metatarsal tubercles round; 11) upper eyelid without supraciliary spines; 12) narrow faint brown stripe extending from rear corner of eye to axilla; 13) thin, pale vertebral stripe present, canthus rostralis with dark lines; 14) small dark round spot at mid-dorsum, divided by a light vertebral stripe; 15) dorsal surface yellowish brown, a dark brown marking in V-shape between eyes to insertion of arms; 16) vertebral and dorsolateral stripes form wavy dust strip towards the groin; 17) a small dark marking in ‘ ()’-shape on the center of the dorsum and mid-dorsal line; 18) an evenly colored black lateral stripe from above the insertion of the arms, almost reaching groin; 19) chin dark grey; throat white with scattered dark grey dusting; chest and belly creamy white.
Habitus stocky, size medium SVL 19.07 mm; head wider than long (HL/HW 0.82); snout long, abruptly round in dorsal view, projecting beyond margin of lower jaw, longer than diameter of eye (SL/EL 1.34); eyes comparatively small, slightly protuberant, pupil round (Fig.
Forelimbs comparatively short, about three times shorter than hindlimbs (FLL/HLL 0.33); hand two times shorter than forelimb length (HAL/FLL 0.44); fingers slender, free of webbing, round in cross-section, no skin fringes on fingers present, dorsoventrally flattened, skin fringes of fingers weak; first finger well-developed, longer than one-half the length of the second finger (1FLO/2FLO 0.57), second finger slightly shorter than fourth (2FLI/4FLI 0.89), latter much longer than first, and much shorter than third (2FLI/3FLI 0.60), relative finger lengths: I < II < IV < III (Fig.
Hindlimbs slender and slightly short (HLL 30.99), tibia length longer than half of snout-vent length (TL/SVL 0.53); tibiotarsal articulation at straightened limb not reaching snout; foot longer than tibia (FL/TL 1.36); relative toe lengths: I<II<V<III<IV; tarsus smooth, inner tarsal fold absent; tips of all toes distinctly dilated into disks, slightly wider than those of fingers (3TDW 0.63, 3FDW/3TDW 0.83), dorsally all toes with median longitudinal grooves at disks; relative toe disk widths: I<V<II<III<IV; webbing between toes basal and poorly developed (Fig.
Skin: Dorsal surface of head and body smooth, flanks smoothly shagreened, dorsal surface of fore and hind limbs, including fingers and toes, smooth; ventral surfaces smooth (Fig.
Dorsal surface of head and trunk yellowish brown to light brown with a dark brown marking in a V-shape between eyes to insertion of arms. Vertebral and dorsolateral stripes forming a wavy dust stripe towards the groin. A small dark brown marking in ‘ ()’-shape in the center of the dorsum and mid-dorsal line. Flanks and lateral surface of head dark, a darker lateral stripe running from snout tip to nostril, fading towards the upper jaw and the belly, fading into belly as dusting. Chin dark grey; throat white with scattered dark grey dusting; chest and belly creamy white. Limbs dorsally with narrow indistinct dark brown cross-bars; fingers and toes dorsally brown with dark brown cross-bars; forelimbs ventrally creamy white, hindlimbs ventrally with creamy white thigh changing to dark grey toward shank and foot. Iris bicolored, golden in upper one-third, dark copper in lower two-thirds; pupil oval, horizontal, black (Figs
After preservation in ethanol, the dorsal coloration changed from brown to whitish grey (Fig.
(Suppl. material
Microhyla daklakensis sp. nov is morphologically similar to M. ninhthuanensis from Ninh Thuan Province and M. heymonsi sensu stricto (Fig.
Specific epithet is in reference to the type locality, Dak Lak Province. We recommend “Dak Lak narrow-mouth frog” as the common English name and “Nhái bầu dak lak” as the Vietnamese name.
All specimens were collected at night from 19:00 to 23:00 h on the ground near the banks of small temporary ponds formed after heavy rain, along the edges of the forest and on the sides of a recently constructed road next to the devastated forests (Fig.
Microhyla daklakensis sp. nov. is currently known only from the type locality in Nam Ka Nature Reserve, Krong No District, Dak Lak Province, Vietnam (Fig.
Currently, the evergreen forest in Nam Ka Nature Reserve, Dak Lak Province, is connected with other forests in the Tay Nguyen Plateau. The extent of its actual distribution range requires further study. Given the available information, we suggest Microhyla daklakensis sp. nov. be considered as Data Deficient following IUCN’s Red List categories (
Our matrilineal genealogy is consistent with those of
The discovery of M. ninhthuanensis and M. daklakensis brings the total number of known species in the genus Microhyla to 46 and the species number in Vietnam to 12. The Truong Son Range harbors the highest diversity of the genus Microhyla with ten recorded species so far. It shows that there is still an underestimation of species diversity in the Microhyla genus (especially M. heymonsi group). We strongly recommend focused research to elucidate the taxonomic issues of the M. heymonsi group.
In terms of conservation concern, habitat loss is one of the greatest threats to amphibians in Southeast Asia, and the amphibians of the region appear to be particularly vulnerable to habitat alterations (
We would like to thank the directorates of the Bi Doup-Nui Ba National Park, Phuoc Binh National Park, Nam Ka Nature Reserve and Forest Protection Department of Kon Tum Province for supporting our work and issuing relevant permits. Many thanks to L. Iogansen, A. Ostroshabov (St. Petersburg), and T. Y. Nguyen (Hanoi) for their assistance. Thanks to E. J. Sterling (New York) and K. Koy (Berkeley) for providing the map. Research of TTN is funded by the Vietnam National Foundation for Science and Technology Development (NAFOSTED, Grant Number 106.05-2019.334). This research is supported by the Strategic Priority Research Program of the Chinese Academy of Sciences (XDA23080101), NSFC (31471964), The World Academy of Sciences (TWAS), CAS- TWAS president fellowship program, RFBR 19-54-54003 Vietnam, the State programm AAAA-A19-119082990107-3 and Ideal Wild.
Tables S1–S6
Data type: species data
Explanation note: Table S1. Specimens of Microhyla and Nanohyla used in the morphological and molecular analyses. Table S2. Specimens and sequences of Microhyla and outgroup Microhylidae representatives used in molecular analyses. Table S3. Uncorrected (“p”) distance matrix showing percentage pair-wise genetic divergence 12S rRNA16S rRNA between members of the Microhyla species group. Table S4. Measurements (in mm) and proportions of the type series of M. neglecta, M. pineticola, Microhyla ninhthuanensis sp. nov., Microhyla daklakensis sp. nov., and M. 'heymonsi'. Table S5. Selected diagnostic characters for the species in the genus Microhyla (modified from Poyakov et al. 2014;