A new species of Micryletta Dubois, 1987 (Anura, Microhylidae) from Yunnan Province, China

A new species of the genus Micryletta Dubois, 1987 is described from Yunnan Province, China, based on morphological and molecular analyses. The most obvious differences between the new species and other species of this genus are small body size, unique coloration, and relatively longer hind limbs. In 16S rRNA gene sequences, the new species is diverged from all other congeners by 3.1%–8.0%.


Materials and methods
Field survey in Hekou County, Honghe Prefecture, Yunnan Province, China (Figure 1), was carried out under the permission of Daweishan National Natural Reserve Management and Protection Bureau. Specimens were fixed and preserved in 75% ethanol and deposited at Kunming Natural History Museum of Zoology, Kunming Institute of Zoology, Chinese Academy of Sciences (KIZ). Measurements were taken to the nearest 0.1 mm with digital calipers. Paired measurements were made on the left side. The descriptions of morphological characteristics followed Poyarkov et al. (2018) with minor modifications. SVL: snout-vent length, measured from the tip of the snout to cloaca; HL: head length, measured from the tip of snout to hind border of jaw angle; SL: snout length, measured from the anterior corner of eye to the tip of snout; EL: eye length, measured as the distance between anterior and posterior corners of the eye; NEL: nostrileye length, measured as the distance between the anterior corner of the eye and the nostril center; HW: head width, measured as the maximum width of head on the level of mouth angles in ventral view; IND: internarial distance, measured as the distance between the 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 maximum distance between the medial edge of eyeball and the lateral edge of upper eyelid; TMP: Tympanum length, measured as the horizontal tympanum diameter; FLL: forelimb length, measured as the length of straightened forelimb to the tip of third finger; LAL: lower arm and hand length, measured as the distance between elbow and the tip of third finger; HAL: hand length, measured as the distance between the proximal end of outer palmar (metacarpal) tubercle and the tip of third finger; 1FL: first finger length, measured as the distance between the tip and the distal end of inner palmar tubercle; IPTL: inner palmar tubercle length, measured as the maximum distance between proximal and distal ends of inner palmar tubercle; OPTL: outer palmar tubercle length, measured as the maximum diameter of outer palmar tubercle; 3FDD: third finger disk diameter; HLL: hindlimb length, measured as the length of straightened hindlimb from groin to the tip of fourth toe; TL: tibia length, measured as the distance between the knee and tibiotarsal articulation; FL: foot length, measured as the distance between the base of the inner metatarsal tubercle to the tip of the fourth toe; IMTL: inner metatarsal tubercle length, measured as the maximum length of inner metatarsal tubercle; 1TOEL: first toe length, measured as the distance between the distal end of inner metatarsal tubercle and the tip of first toe; 4TDD: fourth toe disk diameter. We compared morphological characters of the new species with other members of the genus relying on original species descriptions (Boulenger 1890(Boulenger , 1909Taylor 1962;Tarkhnishvili 1994;Poyarkov et al. 2018;Das et al. 2019;Munir et al. 2020;Suwannapoom et al. 2020;Yang and Poyarkov 2021) and the additional data from Yang and Poyarkov (2021).
Total genomic DNA was extracted from liver tissues using the standard phenol-chloroform method (Hillis et al. 1996;Sambrook and Russell 2001). A fragment encoding mitochondrial 16S rRNA gene was amplified and sequenced. The primers L2188 (Matsui et al. 2006): 5'-AAAGTGGGCCTAAAAGCAGCCA-3' and 16H1 (Hedges 1994): 5'-CTCCGGTCTGAACTCAGATCAC-GTAGG-3' were used in amplification and cycle sequencing. Amplified DNA was produced in 20 μl reactions after an initial denaturation step of 5 min at 94 °C and 43 cycles of denaturation for 1 min at 94 °C, annealing for 1 min with the TouchDown program from 65 °C to 55 °C reducing 1 °C every cycle, extension for 1 min at 72 °C, and final extension for 5 min at 72 °C. PCR products were isolated through electrophoresis using 1.5% agarose gel, and further purified using Millipore Microcon Kits. Purified PCR products were sequenced by Davis Sequencing using BigDye terminator 3.1 and sequences were edited and manually managed using SeqMan in Lasergene 7.1 (DNASTAR Inc., Madison, WI, USA) and MEGA X (Kumar et al. 2018 (Poyarkov et al. 2018;Alhadi et al. 2019;Das et al. 2019;Munir et al. 2020;Suwannapoom et al. 2020;Miller et al. 2021;Yang and Poyarkov 2021) were downloaded from Genbank (Table 1). Sequences of Mysticellus fran-  ki, Kaloula pulchra and Uperodon systoma were used as outgroups according to Suwannapoom et al. (2020) and Yang and Poyarkov (2021). Sequences were aligned using ClustalW (Thompson et al. 1994) integrated in MEGA X (Kumar et al. 2018) with default parameters. Genetic divergences (uncorrected p-distance) were calculated in MEGA X with the parameters Transitions + Transversions, Uniform rates, and Pairwise deletion (Kumar et al. 2018). The best substitution model GTR+F+G4 was selected using the Akaike Information Criterion (AIC) in ModelFinder (Kalyaanamoorthy et al. 2017). Maximum likelihood phylogenetic analysis was performed in IQ-TREE 1.6.12 (Nguyen et al. 2015), and nodal support was estimated by 1,000 ultrafast bootstrap (UFB) replicates. Nodes with UFB values of 95 and above were considered significantly supported (Minh et al. 2013). Bayesian Inference was performed in MrBayes 3.2.7 (Ronquist et al. 2012) based on the selected substitution model. Two runs were performed simultaneously with four Markov chains starting from a random tree. The chains were run for 1,000,000 generations and sampled every 100 generations. The first 25% of the sampled trees were discarded as burn-in after the standard deviation of split frequencies of the two runs was less than a value of 0.01, and then the remaining trees were used to create a 50% majority-rule consensus tree and to estimate Bayesian posterior probabilities (BPP). Nodes were considered well-supported if they had BPP of 0.95 or higher (Huelsenbeck et al. 2001;Wilcox et al. 2002).

Results
Bayesian inference and Maximum likelihood analyses recovered consistent topology ( Figure 2) and agreed essentially with earlier phylogenies of Micryletta (Poyarkov et al. 2018;Das et al. 2019;Suwannapoom et al. 2020;Yang and Poyarkov 2021). The two specimens from Hekou County were nested in the genus Micryletta and formed a distinct clade sister to a clade consisting of M. immaculata and M. steinegeri with strong support.
The genetic divergences between the two specimens from Hekou County and all other congeners ranged from 3.1% (with M. steinegeri) to 8.0% (with M. nigromaculata) ( Table 2).

Micryletta hekouensis sp. nov.
http://zoobank.org/1CEEDB31-AFF2-427B-A219-860D0C0C991E   following characters: small-sized within genus (SVL 20.5-20.8 mm); areas above canthus rostralis, upper eyelids, areas posterior to eyelids, and dorsum of upper arms golden, other parts of dorsum almost solid black or yellowish grey with brownish black stripes; lateral sides of head and body black or yellowish grey, a white stripe from lower front of eye along upper lip back to anterior forelimb insertion; ventral side of body and limbs pink brown, chin region in adult males brownish black, small and irregular white marbling patterns on chest and lateral belly; supratympanic fold indistinct; outer metatarsal tubercle absent; webbing between toes absent; tibiotarsal articulation adpressed limb reaching level of front of eye. Description of holotype. Adult male. SVL 20.5 mm; habitus relatively slender; head small and triangular, slightly wider (HW 6.9 mm) than long (HL 6.5 mm); snout (SL 2.9 mm) abruptly rounded in dorsal view and slightly acuminate in profile, projecting beyond margin of lower jaw; eyes relatively small, slightly protuberant, pupil oval, transverse, eye diameter (EL 2.5 mm) approximately equal to interorbital distance (IOD 2.4 mm).
Top of head flat, canthus rostralis rounded and distinct; loreal region weakly concave; nostril round, closer to tip Forelimbs slender (FLL 14.9 mm), lower arm and hand length (LAL 10.9 mm) more than a half of snoutvent length (LAL/SVL 0.53). Fingers slender with no webbing, rounded in cross-section, no lateral fringes; first finger well-developed, second finger slightly shorter than fourth, relative finger lengths: I<II<IV<III; tips of fingers round and not dilated; subarticular tubercles on fingers distinct, rounded and prominent, formula 1, 1, 2, 2; supernumerary tubercles on palm present and developed; three metacarpal tubercles, inner one rounded and smallest (IPTL 0.4 mm), median one (MPTL 0.7 mm) rounded and almost directly in front of elongated outer one (OPTL 0.8 mm); two rounded and one elongated prominent supernumerary palmar tubercles on the base of fingers II-IV, respectively, slightly larger than inner metacarpal tubercle; nuptial pad absent.
Hindlimbs slender and long (HLL 33.3 mm), more than two times longer than forelimbs (HLL/FLL 2.23); tibia (TL 10.9 mm) slightly shorter than one-third of hindlimb length; tibiotarsal articulation of adpressed limb reaching level of front of eye; foot (FL 11.4 mm) slightly longer than tibia. Relative toe lengths: I<II<V<III<IV; tarsus smooth, tarsal fold absent; tips of toes round and not dilated, slightly wider than those of fingers; webbing between toes absent; subarticular tubercles on toes oval and prominent, formula: 1, 1, 2, 3, 2; dermal ridges present under 2 nd to 4 th toes but indistinct; inner metatarsal tu-bercle oval, prominent, and small (IMTL 0.7 mm); outer metatarsal tubercle absent.
Dorsal skin smooth above, scattered with tiny and flat tubercles on dorsum of body, flanks, and hindlimbs; subtle longitudinal median ridge present on dorsum; dorsolateral fold absent; lateral sides of head smooth; ventral skin of body and limbs smooth.
Coloration of holotype in life. Areas above canthus rostralis, upper eyelids, areas just posterior to eyelids, dorsum of upper arms, and areas above tibiotarsal articulation golden; other parts of dorsum of body black with two indistinct parallel longitudinal grey stripes on back; other parts of dorsum of limbs black mottled with gray and yellow. Lateral sides of head and body black, from lower front of eye along upper lip back to anterior forelimb insertion white; one indistinct longitudinal grey stripe on each side of body. Ventral side of body and limbs pinkish brown, chin region brownish black; small and irregular white marbling patterns on chest and lateral belly; some small white spots on lower lip. Iris bicolored, with upper third bronze and lower two-thirds brownish black.
Coloration of holotype in preservative. Colors faded; areas above canthus rostralis, upper eyelids, and areas just posterior to eyelids turned to dark grey; dorsum of upper arms turned to pink; ventral side turned to yellowish white with light gray marbling on chest and lateral sides of belly; colors of other parts of body almost the same as in life.
Variation. The female paratype is quite similar in appearance to the holotype (Table 3), but show some variations in coloration. The female paratype has a relatively lighter body color, areas above canthus rostralis, upper eyelids, areas posterior to eyelids, and dorsum of upper arms are golden, the same as holotype; however, midline of the back is brownish black, one discontinuous black  stripe dorsolateral on each side, lower parts of flanks greyish brown; other parts of dorsal and lateral body are yellowish grey; dorsa of lower arms and hindlimbs brownish grey with larger and more obvious irregular yellow spots. The color of the ventral side is similar to that of the holotype, except that the chin region is not brownish black but light yellow. Sexual dimorphism. Male has opening of vocal sac and single hypopharyngeal vocal sac, female has no vocal sac and opening of vocal sac. Besides this, there is no significant morphological character difference between males and females.
Etymology. The specific epithet hekouensis refers to Hekou County, the type locality of the new species. We propose "Hekou Paddy Frog" for the common English name and "河口小姬蛙" (Hé Kǒu Xiǎo Jī Wā) for the common Chinese name of the new species.
Natural history. Specimens of the new species were found in the grass on the ground at night. Once startled, they jumped away quickly. The collection site is surrounded by primary broad-leaved forest and bamboo. There are karst rocks nearby, no water body within a few hundred meters, and no courtship calls were heard. The collection site is in the nature reserve and the environment is not destroyed; this species is not threatened at present (Figure 6).
Distribution. This species is currently known only from the type locality, Nanxi Town, Hekou County, Honghe Prefecture, Yunnan Province, China. It is expected to be found in neighboring Northern Vietnam.
Comparisons. Micryletta hekouensis sp. nov. differs from M. aishani by relatively smaller body size ); snout abruptly rounded in dorsal view and slightly acuminate in lateral view (vs. snout shape nearly truncate in dorsal view and acute in lateral view); dorsum almost solid black or yellowish grey with brownish black stripes (vs. dorsum brown to reddish brown with several blackish brown spots present on posterior parts of back and near groin); tibiotarsal articulation adpressed limb reaching level of front of eye (vs. reaching to armpit).
Micryletta hekouensis sp. nov. differs from M. dissimulans by dorsum almost solid black or yellowish grey with brownish black stripes (vs. dorsum reddish brown with merging irregular shaped brown blotches edged in beige); flanks black or greyish brown (vs. large black spots on flanks and axillary and inguinal areas present); white stripes on upper lips present (vs. absent); tibiotarsal articulation adpressed limb reaching level of front of eye (vs. reaching to tympanum).
Micryletta hekouensis sp. nov. differs from M. erythropoda by relatively smaller body ; dorsum almost solid black or yellowish grey with brownish black stripes (vs. dorsum gray or beige to saturated ochre or brick red, dark contrasting round or irregular shape spots irregularly scattered throughout the dorsum); venter without dark patterns (vs. with relatively distinct dark and light marbled speckling); outer metatarsal tubercle absent (vs. present); tibiotarsal articulation adpressed limb reaching level of front of eye (vs. reaching to posterior edge of tympanum).
Micryletta hekouensis sp. nov. can be distinguished from M. immaculata by relatively smaller body 1 mm); dorsum almost solid black or yellowish grey with brownish black stripes (vs. dorsum bronze brown to reddish brown without dark patterns); supratympanic fold indistinct (vs. distinct); supratympanic fold indistinct (vs. distinct); webbing between toes absent (vs. basal and poorly developed); tibiotarsal articulation adpressed limb reaching level of front of eye (vs. reaching to tympanum).
Micryletta hekouensis sp. nov. can be distinguished from M. inornata sensu stricto from Sumatra, Indonesia, and from Tanintharyi, Myanmar, by dorsum almost solid black or yellowish grey with brownish black stripes (vs. dorsum brownish grey with irregular blackish brown blotches and blackish brown streak); ventral side of body and limbs pinkish brown or pinkish grey with small and irregular white marbling patterns on chest and lateral belly (vs. ventral side of body and limbs light reddish grey without mottling, nearly immaculate, or chin, chest, and lateral belly with a few dark marbling patterns); tibiotarsal articulation adpressed limb reaching level of front of eye (vs. reaching to eye).
Micryletta hekouensis sp. nov. can be diagnosed from M. lineata by supratympanic fold indistinct (vs. distinct); venter pink brown with small and irregular white marbling patterns on chest and lateral belly (vs. venter beige with light brown mottling along throat); tibiotarsal articulation adpressed limb reaching level of front of eye (vs. reaching to eye).
Micryletta hekouensis sp. nov. differs from M. nigromaculata by supratympanic fold indistinct (vs. distinct); dorsum almost solid black or yellowish grey with brownish black stripes (vs. dorsum brown to reddish brown with dark brown irregular hourglass shaped pattern and two large dark inguinal spots); flanks black or greyish brown (vs. flanks greyish white with dark patches or spots); white stripes on upper lips present (vs. absent); chin region in males brownish black (vs. whitish with light-gray marbling); tibiotarsal articulation adpressed limb reaching level of front of eye (vs. reaching to eye).
Micryletta hekouensis sp. nov. differs from M. sumatrana by supratympanic fold indistinct (vs. distinct); dorsum almost solid black or yellowish grey with brownish black stripes (vs. dorsum golden brown scattered with black spots); dark cross bands on tibia and tarsus absent (vs. present); venter without dark patterns (vs. with dark brown and cream mottling).
Micryletta hekouensis sp. nov. differs from M. steinegeri by relatively smaller body (SVL 20.5-20.8 mm vs. up to 30 mm); dorsum almost solid black or yellowish grey with brownish black stripes (vs. dorsum dark gray to violet with irregular dark blotches or speckles); venter without dark patterns (vs. with grayish white and brown spots); webbing between toes absent (vs. rudimentary webbing); tibiotarsal articulation adpressed limb reaching level of front of eye (vs. reaching to tympanum). Yang and Rao (2008), Fei et al. (2009), Fei et al. (2012, and AmphibiaChina (2021) all recorded Micryletta inornata distributed in Menglun Town, Mengla County, Xishuangbanna Prefecture, Yunnan Province, China. Micryletta inornata sensu lato was widely reported from mainland Southeast Asia; however, recent phylogenetic studies have indicated that M. inornata sensu stricto is restricted to Indonesia and southern Myanmar, and the populations of M. inornata sensu lato contain several undescribed paraphyletic lineages with respect to other named taxa (Das et al. 2019;Munir et al. 2020;Miller et al. 2021). Therefore, the population distribution in Xishuangbanna remains to be studied.