Multiple Data Sources Reveal a New Asian Leaf Litter Toad of Leptobrachella Smith, 1925 (Anura, Megophryidae) from Southwestern China

A new species of Leptobrachella is described from Sichuan Province and Chongqing Municipality,China.Molecular phylogenetic analyses based on mitochondrial and nuclear gene sequences indicated that the new species is genetically divergent from its congeners.It could be identified from its congeners by a combination of followings characters:body size of male 29.1-34.1 mm(n=14),female 34.1-34.9 mm(n=4);dorsal skin rough with large tubercles in size of humeral glands,without conical spines;fringes on toes narrow;ventral body basically floral white with deep grey pigments all over;dorsal body deep greyish brown with smoky black markings;iris gold above,gradually silver bellow;tibia-tarsal articulation reaches the level of the middle of the eye when leg being stretched forward;the main call type with dominant frequency4.08 ± 0.16 kHz(14.1-14.9℃),call duration 170.35± 15.19ms,the number of pulses for the first note in a call 3.50±0.89,and the number of pulses for the second note in a call 5.08±0.77.

A New Species of the Genus Hebius Thompson,1913(Serpentes:Natricidae)from Yunnan,China

A new keelback snake of the genus Hebius,H.jingdongensis sp.nov.,is reported from Jingdong in Yunnan,China based on a combination of molecular and morphological evidence.Although closest to H.khasiensis and H.boulengeri phylogenetically,this new species is genetically distinct amongst congeners with the mitochondrial Cyt b uncorrected p-distance ranging from 9.5%(vs.H.octolineatus)to 14.8%(vs.H.andreae).In addition,this new species can be distinguished from its congeners by a combination of the following morphological characters:(1)body cylindrical,tail relatively short,TaL/TL 0.235–0.243;(2)dorsal scale rows 19-19-17;(3)ventrals 163–166,cloacal plate divided,subcaudals 71–74 pairs;(4)supralabials 8,the first two contact with the nasals;(5)infralabials 9 or 10,preocular 1,postoculars 3;(6)body dorsum sandy beige with the sides yellowish-brown decorated with two brick-red stripes on the dorsolateral scale rows in life;(7)sutures between the labials brick-red in life and taupe in preservative;(8)ventral light yellow with the outer part of each scale brick-red in life;(9)ventrolateral stripes present.

A New Species of the Genus Nanorana Günther,1896(Anura:Dicroglossidae)from Hengduan Mountains of China

An integrative study based on morphological data and mitochondrial molecular data of all known Nanorana(sensu stricto)species have revealed a new species from the Hengduan Mountains,N.huangi sp.nov.The new species can be distinguished from its congers by its:(1)medium body size,SVL 31.3–40.6 mm in adult males(n=12),39.2–48.1 mm in adult females(n=8);(2)absence of subarticular tubercles on fingers,with supernumerary tubercles below the base of the fingers small but distinct;(3)presence of dark dorsal patches without pale margins,generally creamy yellow ventral body basically,bright yellow ventral limbs;(4)small but prominent tympanum;(5)tibio-tarsal joint that reaches to the tympanum or the shoulder when hindlimbs adpressed forward towards body;(6)head length more than 30%of SVL.In addition,the new species can be separated from all other congeners by uncorrected genetic distances:ranging from 2.8%–7.5%of the mitochondrial 12S rRNA,1.1%–2.1%of the 16S rRNA,and 7.5%–11.5%of the COI.

A New Leaf Litter Toad of Leptobrachella Smith, 1925(Anura, Megophryidae) from Sichuan Province, China with Supplementary Description of L. oshanensis

A new species of the Asian leaf litter toad genus Leptobrachella is described from Sichuan Province, China. Molecular phylogenetic analyses based on mitochondrial and nuclear gene sequences clustered the new species as an independent clade nested into L. oshanensis species group. The new species could be distinguished from its congeners by a combination of following characters: body size moderate(25.8–32.6 mm in male, 33.7–34.1 mm in female);distinct black spots present on flanks;toes rudimentary webbed, with narrow lateral fringes, dermal ridges under toes interrupted at articulations;ventral belly cream white with variable brown specking;skin on dorsum relatively smooth with fine tiny granules or short ridges;iris copper above, silver bellow;greyish black patches on posterior thigh absent or small;spines on surface of chest absent in male during breeding season;nasals entirely or partially separated from sphenethmoid in male;dorsal surface of tadpoles semitransparent light brown, spots on tail absent, keratodont row formula I: 3+3(2+2)/2+2: I;calls simple, call series basically consist of repeated long calls, at dominant frequency(4831.9 ± 155.8) Hz and call duration(544.5 ± 146.8) ms. In addition, we made supplementary description on L. oshanensis including holotype, variations, tadpoles, skull and bioacoustics. Besides, this paper reports cases of femoral adipose glands in the genus Leptobrachella as first known sexual dimorphism skin glands for males of Megophryidae.

A New Species of the Gekko japonicus Group(Squamata:Gekkonidae)from Southwest China

A new species of the Gekko(Squa ma ta:Gekkonidae)is described from the border of Sichuan and Yunnan Province,southwest China,based on distinct morphological and molecular features.Gekko jinjiangensis sp.nov.is distinguished from congeners by a combination of the following characters:size small(SVL 50.2–61.6 mm,n=13);nares in contact with rostral;interorbital scales between anterior corners of the eyes 20–24;ventral scales between mental and cloacal slit 146–169;midbody scale rows 111–149;ventral scale rows 31–47;subdigital lamellae on first toe 8–11,on fourth toe 11–15;no webbing in the fingers and toes;with tubercles on uapper surface of fore and hind limbs;precloacal pores 4–5 in males;postcloacal unilateral tubercles 1–2;dorsal surface of body with 8–9 large greyish brown markings between nape and sacrum.In molecular analyses,the new species is sister to G.scabridus,but separated from them by approximately 9.9%–12.2%in genetic divergence as shown by a fragment of the partial mitochondrial ND2 gene.The new species is the highest Gekko with elevation range from 2000 to 2476 m.Further surveys are recommended to better understand the occurrence and population status of the new species.

A New Species of Trimeresurus Lacépède, 1804(Squamata: Viperidae) from Southwestern China, Vietnam, Thailand and Myanmar

The pit vipers of the genus Trimeresurus Lacépède, 1804 is one of the largest groups of Asian snakes, distributed from India to China and Indonesia. Recent surveys in Jiangcheng and Simao, Yunnan Province, China resulted in a new species previously allocated to T. albolabris. Combining morphological and molecular data, we describe it as Trimeresurus guoi sp. nov. The new species morphologically differs from T. albolabris in the yellow green ventral color;an indistinct ventrolateral line;the a bsence of a postocular stripe;the firebrickred iris;a dark red stripe on dorsal tail;hemipenes with relatively weak sparse papillae, reaching 23 rd subcaudal when unextruded. Molecularly, the new species forms a clearly divergent lineage(BPP 1.00/UFB 100). Uncorrected pairwise distances of mitochondrial gene Cyt b between the new species and other known species of the subgenus Trimeresurus range from 0.052(T. albolabris) to 0.071(T. insularis).

Endocast Morphological Variation and Its Driving Forces in Scutiger boulengeri

Cranialendocasts canbeusedasareliable proxy for brain size,reflecting the evolutionary and environmental selection pressure of species.Although studies on endocasts in amphibians have increased in recent years,those performed on endocasts of Anura are comparatively rare,especially at the intraspecific level.Here,using a high-altitude endemic toad-Scutiger boulengeri-as a model,through the application of integrative methods(morphology,anatomy,phylogeny,and ecology),we studied intraspecific variations in endocast morphology and explored its driving forces.Three-dimensional reconstruction and the brain-to-endocranial cavity(BEC)index suggested that the endocast of S.boulengeri can reflect brain morphology to a large extent.Elliptic Fourier analysis andprincipal componentanalysis revealedgreat variability in the cranial endocast morphology among individuals,as well as the variation concentrated in the regions of telencephalon and optic tectum.In the species,individuals with large bodies are accompanied by a larger endocast size;the relative endocast sizes have significant cladedifferences but no sexual dimorphism.Additionally,the relative endocast sizes of S.boulengeri were not associated with phylogenetic history and aquatic preference but were positively correlated with altitude and negatively correlated with oxygen content,temperature,and precipitation factors(annual mean temperature,temperature seasonality,annual precipitation,and precipitation seasonality).Thesefindings suggested that high-altitude and extreme environmental conditions acted as important selectiveforces in morphological variation of the cranial endocastof S.boulengeri.

A New Species of the Genus Rhabdophis Fitzinger,1843(Squamata:Colubridae)in Southwestern Sichuan,China

The genus Rhabdophis is a group of widely distributed snakes with more than 20 species.Recent field surveys uncovered a species in southwestern China,which has long been considered as R.pentasupralabialis.Combined molecular and morphological analyses revealed it as a new species Rhabdophis chiwen sp.nov.Based on 12 specimens,this new species is distinguished by the following characters:1)dorsal body saddlebrown,dorsal scales typically with black margins forming spots and stripes,the margin of the outer row forming two faint dorsolateral black cross-bars alongside body;2)ventral scales 151–159,the outer margin of ventral scales and several lateral rows of dorsal scales forming ventrolateral longitudinal brownish-red coloration,with faint black spots in the middle of ventral scales;3)a black oblique stripe present below eyes,often with a black spot between the 2 nd and 3 rd supralabial and a black stripe on the 5 th supralabial;4)eyes dark khaki,pupils black;5)infralabials usually 7,the first four in contact with anterior chin-shields;6)temporal scales 1+1;7)dorsal scales in 15 rows,feebly keeled except the outer 1–2 rows;8)anal scale divided;subcaudals 45–59;9)preocular 1 and postoculars 3(occasionally 2);10)body medium-sized(snout-vent length:adult males 404–431 mm,adult females 409–476 mm);11)tail moderate(tail length/total length in adult males 0.205–0.238,in adult females0.172–0.193).With the discovery of this new species,the total number of species in genus Rhabdophis is 28 with 12 th species known to occur in China.

Detection of weak non-covalent cation-π interactions in NGAL by single-molecule force spectroscopy

Cation-π interaction is an electrostatic interaction between a cation and an electron-rich arene.It plays an essential role in many biological systems as a vital driving force for protein folding,stability,and receptor-ligand interaction/recognition.To date,the discovery of most cation-π interactions in proteins relies on the statistical analyses of available three-dimensional(3D)protein structures and corresponding computational calculations.However,their experimental verification and quantification remain sparse at the molecular level,mainly due to the limited methods to dynamically measure such a weak non-covalent interaction in proteins.Here,we use atomic force microscopy-based single-molecule force spectroscopy(AFM-SMFS)to measure the stability of protein neutrophil gelatinase-associated lipocalin(also known as NGAL,siderocalin,lipocalin 2)that can bind iron through the cation-π interactions between its three cationic residues and the iron-binding tri-catechols.Based on a site-specific cysteine engineering and anchoring method,we first characterized the stability and unfolding pathways of apo-NGAL.Then,the same NGAL but bound with the iron-catechol complexes through the cation-π interactions as a holo-form was characterized.AFM measurements demonstrated stronger stabilities and kinetics of the holo-NGAL from two pulling sites,F122 and F133.Here,NGAL is stretched from the designed cysteine close to the cationic residues for a maximum unfolding effect.Thus,our work demonstrates high-precision detection of the weak cation-π interaction in NGAL.

Combination of Click Chemistry and Enzymatic Ligation for Stable and Efficient Protein Immobilization for Single-Molecule Force Spectroscopy

Protein immobilization is an essential method for both basic and applied research for protein,and covalent,site-specific attachment is the most desirable strategy.Classic methods typically rely on a heterobifunctional cross-linker,such as N-hydroxysuccinimide(NHS)-linker-maleimide,or a similar two-step process.It utilizes the amino-reactive NHS and the thiolreactive maleimide to conjugate protein to the solid support.However,NHS as a chemical is susceptible to hydrolysis during storage and handling,and maleimide reacts nonspecifically with all cysteines available in the protein,leading to an inconsistent result.To solve these problems,we have developed a method by combining a strain-promoted azide–alkyne cycloaddition(SPAAC)click reaction and an OaAEP1(C247A)-based enzymatic ligation.The method was demonstrated by the successful immobilization of enhanced green fluorescent protein(eGFP),which was visualized by fluorescent imaging.Moreover,the correct folding and stability of the immobilized protein were verified by atomic force microscopy-based single-molecule force spectroscopy(AFM-SMFS)measurement with a high success rate(89%).Finally,the strength of the 1,2,3-triazole linkage from the azide-dibenzocyclooctyne(DBCO)-based SPAAC reaction was quantified with an ultrahigh rupture force>1.7 nN.Thus,this stable,efficient,and site-specific immobilization method can be used for many challenging systems,especially SMFS studies.