Two new cryptic species of Microhyla Tschudi, 1838 (Amphibia, Anura, Microhylidae) related to the M. heymonsi group from central Vietnam

Abstract 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 daklakensissp. nov. and Microhyla ninhthuanensissp. 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.

Heymon's narrow-mouthed frog, Microhyla heymonsi Vogt, 1911, was originally described from Taiwan based on eight male specimens . 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; Frost 2021). Recently, Garg et al. (2019) demonstrated that M. heymonsi represents a complex of species, and that the current number of recognized Microhyla species may be underestimated. Garg et al. (2019) revealed six and Gorin et al. (2020) revealed seven or eight genetic lineages within M. 'heymonsi'.
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 . Two further populations remained that could not be assigned to any known species of Microhyla. Herein, we describe these two populations of Microhyla from central Vietnam as two new species.

Sampling
Field surveys were conducted in Kon Tum, Dak Lak, Lam Dong, and Ninh Thuan provinces, Vietnam (  Hoang et al.). Geographic coordinates and elevations were obtained using a Garmin GPSMAP 78S (WGS 84 data). After photographing specimens in life, they were euthanized in a closed vessel with a piece of cotton wool containing ethyl acetate (Simmons 2002). Specimens were fixed in 70% ethanol for five hours, and then later transferred to 70% ethanol for permanent storage. Tissue samples were preserved separately in 70% ethanol prior to specimen fixation. Specimens referred to in this paper are deposited in the collections of the Vietnam National Museum of Nature (VNMN), Hanoi, Vietnam; Institute of Ecology and Biological Resources (IEBR), Hanoi, Vietnam; Chengdu Institute of Biology (CIB), Chengdu, Sichuan, China; and the Zoological Institute of the Russian Academy of Sciences (ZISP), St. Petersburg, Russia. Sex was determined by the direct observation of calling males in life or by gonadal dissection. Further information on the specimens is provided in Suppl. material 1: Table S1.
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 (Van Bocxlaer et al. 2006). Locality information and accession numbers for all sequences included in the analysis can be found in Suppl. material 1: Table S2.
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 (Thompson et al. 1997) option in MEGA 7.0 (Kumar et al. 2016) with default parameters and subsequently optimized manually in BioEdit 7.0.5.2 (Hall 1999). We then checked the initial alignments by eye and made slight adjustments. Prior to Bayesian tests, phylogenetic analyses were performed in MrBayes 3.2 (Ronquist et al. 2012). We chose the optimum substitution models for entire sequences using Kakusan 4 (Tanabe 2011) based on the Akaike information criterion (AIC). The best model selected for ML was the general time reversible model (GTR: Tavaré 1986) with a gamma shape parameter (G: 0.202 in ML and 0.226 in BI). The BI summarized two independent runs of four Markov Chains for 10 000 000 generations. A tree was sampled every 100 generations and a consensus topology was calculated for 70 000 trees after discarding the first 30 001 trees (burn-in = 3 000 000) (Nguyen et al. 2017). We checked parameter estimations and convergence using Tracer version 1.5 (Rambaut and Drummond 2009). The strength of nodal support in the ML tree was analyzed using non-paramet-ric bootstrapping (MLBS) with 1000 replicates. We regarded tree nodes in the ML tree with bootstrap values of 75% or greater as sufficiently resolved (Hillis and Bull 1993;Huelsenbeck and Hillis 1993), and nodes with a BPP of 95% or greater as significant in the BI analysis (Leaché and Reeder 2002).

Morphological analysis
All measurements were taken from 63 preserved specimens (Suppl. material 1: Table S4) with a digital caliper to the nearest 0.01 mm under a dissecting microscope. The following morphological characteristics were taken following Matsui (2011); Matsui et al. (2013), andPoyarkov et al. (2014), with some modifications:

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 Matsui 2011); 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 Glaw and Vences (1997) and webbing formula followed Savage (1975).

Principal component analysis (PCA)
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 (Hammer et al. 2001).

Sequence variations
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).

Interspecific uncorrected p-distances
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 1: Table  S3). Our results support those of Garg et al. (2019) and Gorin et al. (2020), clearly indicating that the M. 'heymonsi' clade represents a complex of several species, either representing previously available names or so far unrecognized diversity.

Phylogenetic relationships
The BI and ML analyses produced topologies with -ln L = 15327.579 and 12672.916, respectively. BI and ML analyses obtained similar topologies ( Fig. 2) that differed only at several poorly supported basal nodes. Our matrilineal genealogy was consistent with previous studies based on mt DNA (e.g., Matsui et al. 2005Matsui et al. , 2011Hasan et al. 2012;Matsui et al. 2013;Howlader et al. 2015;Wijayathilaka et al. 2016;Seshadri et al. 2016;Yuan et al. 2016;Khatiwada et al. 2017;Nguyen et al. 2019;Garg et al. 2019;and Hoang et al. 2020). As shown in previous molecular analyses Peloso et al. 2016), Asian Microhyla species were divided into two geographical subgroups: the Southeast Asian subgroup was recently named as Nanohyla by Gorin et al. (2021), with eight known species: N. annamensis, N. annectens, N. hongiaoensis, N. marmorata, N. nanapollexa, N. perparva, N. petrigena, and N. pulchella; and Pan-Asian, including all other South, Southeast, and East Asian members (Fig. 2). Among the known taxa, molecular data are presented for the first time for four poorly known species: N. annamensis, N. hongiaoensis, N. pulchella, and M. pineticola. Molecular results further revealed M. heymonsi to represent a complex consisting of multiple species.

Morphological analysis
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 1: Table S5).

Taxonomic conclusions
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. Diagnosis. 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.

Microhyla ninhthuanensis
Description of holotype. 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. 4G); dorsal surface of head flat, loreal region acute; indistinct canthus rostralis; nostril oval, lateral, closer to tip of snout (N-EL 1.33) than to eye (EL 1.95); interorbital distance wide, greater than internarial distance (IOD/IND 1.28); and internarial distance wide, greater than upper eyelid width (IND/UEW 1.18); tympanum hidden, supratympanic fold weak, from posterior corner of eye to arm insertion; vomerine teeth absent, tongue without papillae, oval and free at the rear half of its length; slit-like openings to a small vocal sac.
Dorsal surface of head and body smooth, flank shagreened, dorsal surface smooth, including fingers and toes, fore and hind limbs; ventral surfaces smooth.
Coloration of holotype in life. 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.
Coloration of holotype in preservative. After preservation in ethanol, dorsal coloration changed from light brown to greyish pink (Fig. 4G), ventral surface of chest, belly, and limbs changed from creamy white to whitish beige (Fig. 4H); dorsal pattern: dark spots on dorsum and stripes on dorsal surfaces of limbs unchanged, dark brown pattern changed to dark grey; iris completely black, pupil round, white.
Variation. 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 1: Table S4).
Comparisons. Microhyla ninhthuanensis sp. nov. is morphologically most similar to M. heymonsi sensu stricto (Fig. 2), but differs by having: 1) a snout round in profile (vs. snout obtusely pointed in M. heymonsi sensu stricto), 2) pinkish brown dorsal surface with a dark brown marking in X-shape between eyes to insertion of the arms (vs. dorsal surface red to grayish red with a dark brown marking in X-shape and a vague V-shape dark brown marking in M. heymonsi sensu stricto (Vogt, 1913). Detailed comparisons between M. ninhthuanensis sp. nov. and other members of the M. heymonsi group are shown in Table 1 and Suppl. material 1: Table S5. Etymology. 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.
Natural history. 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. 6B). Larval stages and eggs of the new species are unknown.
Distribution. Microhyla ninhthuanensis sp. nov. is currently only known from the type locality in Phuoc Binh National Park, Ninh Thuan Province, Vietnam (Fig. 1). The species was recorded at an elevation of ca. 300 m a.s.l. Conservation status. 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).
Description of holotype. 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. 5G); dorsal surface of head flat, canthus rostralis round; loreal region steep, weakly concave; nostril round, lateral, above canthus rostralis, closer to tip of snout (N-EL 1.20) than to eye (EL 1.79), interorbital distance wide (IOD 1.90) much greater than the internarial distance (IND 1.62) and the upper eyelid width (UEW 1.15); pineal spot absent, tympanum hidden, supratympanic fold weak, running from posterior corner of eye to arm insertion; vomerine teeth absent, tongue without papillae, roundly spatulate and free at the rear half of its length; slit-like openings to a median vocal sac (Fig. 6G).
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. 3K, L).
Coloration of holotype in life. 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 3K, 4L).

A B
Coloration of holotype in preservative. After preservation in ethanol, the dorsal coloration changed from brown to whitish grey (Fig. 4E), and the ventral surface of chest, belly, and limbs changed from creamy white to whitish beige (Fig. 4F). The dorsal pattern, dark spots on the dorsum and stripes on the dorsal surfaces of the limbs are unchanged, dark brown pattern on the dorsum changed to dark grey; iris completely black, pupil round, white.
Variation. (Suppl. material 1: Table S4, Fig. 3). Paratypes vary in body size, dorsal color pattern, and shape of black scapular spots. Adult males are smaller than adult females and have a distinct vocal sac (Suppl. material 1: Table S4).
Comparisons. Microhyla daklakensis sp. nov is morphologically similar to M. ninhthuanensis from Ninh Thuan Province and M. heymonsi sensu stricto (Fig. 2), but differs by having: 1) a snout round in profile (vs. snout obtusely pointed in M. heymonsi sensu stricto), 2) finger I longer than one-half the length of the finger II (vs. finger I shorter than one-half the length of the finger II in the new form from Ninh Thuan and M. heymonsi sensu stricto), 3) dorsal surface yellowish brown with a dark brown marking in V-shape between the eyes and the insertion of the arms (vs. pinkish brown dorsal surface with a dark brown marking in X-shape between eyes and insertion of the arms in the new form from Ninh Thuan, and dorsal surface red to grayish red with a dark brown X-shaped marking and a vague V-shaped dark brown marking in M. heymonsi sensu stricto (Vogt, 1913). Detailed comparisons between Microhyla daklakensis sp. nov. and other members of the M. heymonsi group are shown in Table 1 and Suppl. material 1: Table S5.
Etymology. 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.
Natural history. 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. 6A). The new species was found in sympatry with four congeners including M. berdmorei, M. butleri, M. mukhlesuri, and M. pulchra, all of which were reproducing simultaneously with the new species in the same breeding site. Other anurans such as Fejervarya limnocharis, Occidozyga cf. lima, and Occidozyga martensii also occurred in sympatry. Larval stages and eggs of the new species are unknown.
Distribution. 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. 1). The species was recorded at an elevation of ca. 500 m a.s.l.
Conservation status. 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 (IUCN Standards and Petitions Subcommittee 2017).

Discussion
Our matrilineal genealogy is consistent with those of Matsui et al. (2011, Nguyen et al. (2019), Garg et al. (2019), and Gorin et al. (2020Gorin et al. ( , 2021. The BI and ML genealogy showed that the monophyly of Microhylinae was not supported and the relationships among microhylid subfamilies remained unresolved (Nguyen et al. 2019) (Fig. 2). In our phylogenetic analyses using a 12S rRNA-16S rRNA gene fragment, M. ninhthuanensis was recovered as sister to M. heymonsi sensu stricto with high nodal support values (1.00/99.2) and in turn, this clade was sister to M. daklakensis with nodal support values (1.00/54.1). Furthermore, the results of morphometric analyses (PCA) indicated M. ninhthuanensis to be distinct from M. daklakensis and M. 'heymonsi'.
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 (Rowley et al. 2010a(Rowley et al. , 2010b(Rowley et al. , 2016. The need for further biological exploration in this region in concert with improved conservation measures is urgent due to intensified logging and road construction, along with increasing agricultural pressure and other human activities (De Koninck 1999;Kuznetsov and Kuznetsova 2011;Laurance 2007;Meijer 1973;Meyfroidt and Lambin 2008).