Hidden hotspots of amphibian biodiversity in China.

Identifying and protecting hotspots of endemism and species richness is crucial for mitigating the global biodiversity crisis. However, our understanding of spatial diversity patterns is far from complete, which severely limits our ability to conserve biodiversity hotspots. Here, we report a comprehensive analysis of amphibian species diversity in China, one of the most species-rich countries on Earth. Our study combines 20 y of field surveys with new molecular analyses of 521 described species and also identifies 100 potential cryptic species. We identify 10 hotspots of amphibian diversity in China, each with exceptional species richness and endemism and with exceptional phylogenetic diversity and phylogenetic endemism (based on a new time-calibrated, species-level phylogeny for Chinese amphibians). These 10 hotspots encompass 59.6% of China's described amphibian species, 49.0% of cryptic species, and 55.6% of species endemic to China. Only four of these 10 hotspots correspond to previously recognized biodiversity hotspots. The six new hotspots include the Nanling Mountains and other mountain ranges in South China. Among the 186 species in the six new hotspots, only 9.7% are well covered by protected areas and most (88.2%) are exposed to high human impacts. Five of the six new hotspots are under very high human pressure and are in urgent need of protection. We also find that patterns of richness in cryptic species are significantly related to those in described species but are not identical.

Chemogenetic Inhibition Reveals That Processing Relative But Not Absolute Threat Requires Basal Amygdala.

While our understanding of appetitive motivation has benefited immensely from the use of selective outcome devaluation tools, the same cannot be said about aversive motivation. Findings from appetitive conditioning studies have shown that basal amygdala is required for behaviors that are sensitive to updates in outcome value, but similar results in aversive motivation are difficult to interpret due to a lack of outcome specificity. The studies reported here sought to develop procedures to isolate sensory-specific processes in aversive learning and behavior and to assess the possible contribution of the basal amygdala. Post-training changes to outcome value produced commensurate changes to subsequently tested conditioned responding in male rodents. Specifically, increases in shock intensity (i.e., inflation) augmented, while repeated exposure to (i.e., habituation of) an aversive sound (klaxon-horn) reduced freezing to conditioned stimuli previously paired with these outcomes. This was extended to a discriminative procedure, in which following revaluation of one event, but not the other, responding was found to be dependent on outcome value signaled by each cue. Chemogenetic inactivation of basal amygdala impaired this discrimination between stimuli signaling differently valued outcomes, but did not affect the revaluation process itself. These findings demonstrate a contribution of the basal amygdala to aversive outcome-dependent motivational processes.SIGNIFICANCE STATEMENT The specific content of pavlovian associative learning has been well studied in appetitive motivation, where the value of different foods can be easily manipulated. This has facilitated our understanding of the neural circuits that generate different forms of motivation (i.e., sensory specific vs general). Studies of aversive learning have not produced the same degree of understanding with regard to sensory specificity due to a lack of tools for evaluating sensory-specific processes. Here we use a variant of outcome devaluation procedures with aversive stimuli to study the role of basal amygdala in discriminating between aversive stimuli conveying different degrees of threat. These findings have implications for how we study generalized threat to identify dysregulation that can contribute to generalized anxiety.

A novel multilocus phylogenetic estimation reveals unrecognized diversity in Asian horned toads, genus Megophrys sensu lato (Anura: Megophryidae).

The horned toad assemblage, genus Megophrys sensu lato, currently includes three groups previously recognized as the genera Atympanophrys, Xenophrys and Megophrys sensu stricto. The taxonomic status and species composition of the three groups remain controversial due to conflicting phenotypic analyses and insufficient phylogenetic reconstruction; likewise, the position of the monotypic Borneophrys remains uncertain with respect to the horned toads. Further, the diversity of the horned toads remains poorly understood, especially for widespread species. Herein, we evaluate species-level diversity based on 45 of the 57 described species from throughout southern China, Southeast Asia and the Himalayas using Bayesian inference trees and the Generalized Mixed Yule Coalescent (GMYC) approach. We estimate the phylogeny using both mitochondrial and nuclear DNA data. Analyses reveal statistically significant mito-nuclear discordance. All analyses resolve paraphyly for horned toads involving multiple strongly supported clades. These clades correspond with geography. We resurrect the genera Atympanophrys and Xenophrys from the synonymy of Megophrys to eliminate paraphyly of Megophrys s.l. and to account for the morphological, molecular and biogeographic differences among these groups, but we also provide an alternative option. Our study suggests that Borneophrys is junior synonym of Megophrys sensu stricto. We provide an estimation of timeframe for the horned toads. The mitochondrial and nuclear trees indicate the presence of many putative undescribed species. Widespread species, such as Xenophrys major and X. minor, likely have dramatically underestimated diversity. The integration of morphological and molecular evidence can validate this discovery. Montane forest dynamics appear to play a significant role in driving diversification of horned toads.

Hebbian and neuromodulatory mechanisms interact to trigger associative memory formation.

A long-standing hypothesis termed "Hebbian plasticity" suggests that memories are formed through strengthening of synaptic connections between neurons with correlated activity. In contrast, other theories propose that coactivation of Hebbian and neuromodulatory processes produce the synaptic strengthening that underlies memory formation. Using optogenetics we directly tested whether Hebbian plasticity alone is both necessary and sufficient to produce physiological changes mediating actual memory formation in behaving animals. Our previous work with this method suggested that Hebbian mechanisms are sufficient to produce aversive associative learning under artificial conditions involving strong, iterative training. Here we systematically tested whether Hebbian mechanisms are necessary and sufficient to produce associative learning under more moderate training conditions that are similar to those that occur in daily life. We measured neural plasticity in the lateral amygdala, a brain region important for associative memory storage about danger. Our findings provide evidence that Hebbian mechanisms are necessary to produce neural plasticity in the lateral amygdala and behavioral memory formation. However, under these conditions Hebbian mechanisms alone were not sufficient to produce these physiological and behavioral effects unless neuromodulatory systems were coactivated. These results provide insight into how aversive experiences trigger memories and suggest that combined Hebbian and neuromodulatory processes interact to engage associative aversive learning.

A New Species of the Genus Pseudocalotes (Squamata: Agamidae) from Southwest Yunnan, China.

In this study, a new species of the genus Pseudocalotes is described from Yingjiang County, Dehong Dai and Jingpo Autonomous Prefecture, Yunnan Province, China, based on four female specimens. It can be distinguished from its congeners by the following combination of characters: (1) interoculabials 3 or 4; (2) canthals 5-7; (3) cicrcumorbitals 8-11; (4) 1 scale between rostral and nasal; (5) interparietal 1; (6) superciliaries 4-6; (7) supralabials 6-7, the 1st in contact with the nasal; (8) infralabials 6-8; (9) transverse gular fold and antehumeral fold present; (10) 2-3 enlarged scales between eye and ear; (11) nuchal crest single, consists of 3-5 erected spines; (12) dorsal crest row single, discontinuous and low, located between two keeled, parallel and enlarged scale rows; (13) enlarged postrictals absent; (14) scales around midbody 53-62, dorsal body scales heterogenous in size and shape; (15) midventrals smaller than dorsals; (16) subdigital scales on the 4th finger 20-26, and on the 4th toe 24-29; (17) dorsal background coloration light taupe with four irregular brown patches along the middle of dorsal; (18) inner lips wathet, tongue aurantiacus, throat bluish black. The population from Yingjiang County was nested within a highly supported lineage, formed a sister taxon with P. kakhienensis (SH 97/UFB 100) and according to the p-distance, the new species differed from its congeners by 14.5% to 35.2% for NADH dehydrogenase subunit 2 (ND2) and 15.5% to 25.0% for NADH dehydrogenase subunit 4 (ND4).

A New Species of Diploderma (Squamata, Agamidae) from the Valley of Dadu River in Sichuan Province, with a Redescription of Topotypes of D. splendidum from Hubei Province, China.

This study describes a novel species of Diploderma (Squamata, Agamidae) from the lower valley of the Dadu River of the Sichuan Province of Western China based on its distinct morphological features and molecular evidence. D. daduense sp. nov. can be distinguished from its congeners by its tympanum concealed; head mainly green-yellow, supplemented by black; skin folds under the nuchal and dorsal crest obviously present in adult males only, its vertebral crest discontinuous between nuchal and dorsal sections with a distinct gap; transverse gular fold present but not obvious in some individuals; gular spot absent in both sexes; dorsolateral stripes green-yellow anteriorly, cyan in the center and blurry off-white posteriorly in adult males, the upper edge of dorsolateral stripes strongly jagged in adult males; no radial stripes around the eyes; inner-lip coloration smoky-white, and the coloration of the tongue and oral cavity as a light-flesh color in life; bright green-yellow transverse stripes on dorsal body in males; black patches are evenly distributed along the vertebral line between the dorsolateral stripes from the neck to the base of the tail in males; beech-brown or gray-brown line along the vertebral line with heart-shaped or diamond-shaped black patches on the dorsal body in females; and supratemporals fewer than four on at least one side. The phylogenetic tree based on mitochondrial ND2 sequences indicates that D. daduense sp. nov. forms an independent clade with strong support 1/100 in ML bootstrap/Bayesian posterior probability and is the sister group to D. splendidum. At the inter-species level, the p-distance is at least 6.95%, further confirming that an independent species had been identified. Our work raises the number of species within the genus Diploderma to 47.

Aberrant TSC-Rheb axis in Oxytocin receptor+ cells mediate stress-induced anxiety.

Stress is a major risk for the onset of several maladaptive processes including pathological anxiety, a diffuse state of heightened apprehension over anticipated threats1. Pathological anxiety is prevalent in up to 59% of patients with Tuberous Sclerosis complex (TSC)2, a neurodevelopmental disorder (NDD) caused by loss-of-function mutations in genes for Tuberin (Tsc2) and/or Hamartin (Tsc1) that together comprise the eponymous protein complex. Here, we generated cell type-specific heterozygous knockout of Tsc2 in cells expressing oxytocin receptor (OTRCs) to model pathological anxiety-like behaviors observed in TSC patient population. The stress of prolonged social isolation induces a sustained negative affective state that precipitates behavioral avoidance, often by aberrant oxytocin signaling in the limbic forebrain3,4. In response to social isolation, there were striking sex differences in stress susceptibility in conditional heterozygote mice when encountering situations of approach-avoidance conflict. Socially isolated male mutants exhibited behavioral avoidance in anxiogenic environments and sought more social interaction for buffering of stress. In contrast, female mutants developed resilience during social isolation and approached anxiogenic environments, while devaluing social interaction. Systemic and medial prefrontal cortex (mPFC)-specific inhibition of downstream effector of TSC, the integrated stress response (ISR), rescued behavioral approach toward anxiogenic environments and conspecifics in male and female mutant mice respectively. Further, we found that Tsc2 deletion in OTRCs leads to OTR-signaling elicited network suppression, i.e., hypofrontality, in male mPFC, which is relieved by inhibiting the ISR. Our findings present evidence in support of a sexually dimorphic role of prefrontal OTRCs in regulating emotional responses in anxiogenic environments, which goes awry in TSC. Our work has broader implications for developing effective treatments for subtypes of anxiety disorders that are characterized by cell-autonomous ISR and prefrontal network suppression.

Four new species of the genus Diploderma Hallowell, 1861 (Squamata, Agamidae) from China.

Four new species of Diploderma are described from Sichuan and Yunnan provinces, southwestern China, based on an integrative taxonomic approach, combining morphological and genetic data. The first new species from Danba County, Sichuan Province, is morphologically most similar and phylogenetically closely related to D.flaviceps, but it can be diagnosed from the latter by having a relatively much shorter tail and by a genetic distance of 4.4% in the ND2 gene; the second new species from Muli County, Sichuan Province, is phylogenetically closely related to D.daochengense, D.yongshengense, and D.yulongense, but it can be diagnosed from the latter three species by having a pale yellow gular spot and by genetic distances of 5.6-6.7% in the ND2 gene; the third new species from Jiulong County, Sichuan Province, is morphologically most similar and phylogenetically closely related to D.angustelinea, but it can be diagnosed from the latter by having a relatively much longer tail and by a genetic distance of 2.8% in the ND2 gene; and the last new species from Weixi County, Yunnan Province, is phylogenetically closely related to D.aorun, but it can be diagnosed from the latter by having a pale yellow gular spot and by a genetic distance of 2.9% in the ND2 gene. Our work brings the number of species within the genus Diploderma to 46.

Taxonomic review of the Calotesversicolor complex (Agamidae, Sauria, Squamata) in China, with description of a new species and subspecies.

Caloteswangisp. nov., a new species of the agamid genus Calotes Cuvier, 1817, from southern China and northern Vietnam, is described. This species can be distinguished from all known congeners by a combination of morphological characteristics and genetic divergence in the mitochondrial tRNA, ND2, and CO1 genes. Molecular phylogenetic analysis revealed that the new species was formed as a monophyletic group and that considerable genetic divergence existed between its congeners (minimum p-distance, 4.6%). Caloteswangisp. nov. is distinguished by a combination of the following characteristics: average SVL < 90 mm for adult males; 10-14 dorsal eyelid scales; scales on side of neck and adjacent shoulder area pointing obliquely upward; keels on neck scales weakly to strongly developed; fold in front of the shoulder absent; pair of dark triangular patches extending from the front of the shoulder to the jaw angles; and orange coloration of the tongue. Caloteswangisp. nov. is similar to C.irawadi but differs in having scales between the nasal shield and the orbit and a fourth toe with a claw that can reach between the eyes and tympanum (even to the snout when hind the limbs are adpressed forward). Phylogenetic analyses revealed two well-supported subspecies, Lineages A and B in C.wangisp. nov., with mean uncorrected p-distances between them of 2%. We propose that Lineage A, which is mainly from the central and southern Wuzhi Mountains on Hainan Island, is a subspecies, C.w.hainanensisssp. nov. Lineage B mainly comprises individuals from other sites on the island plus the adjacent mainland, and is described as subspecies, C.w.wangissp. nov. A diagnostic key to all Calotes species of China is also provided.

Identity of the holotype and type locality of Rhabdophis leonardi (Wall, 1923) (Colubridae: Natricinae), with notes on the morphology and natural history of the species in southwestern China.

The original description of Natrix leonardi (currently Rhabdophis leonardi) by Frank Wall in 1923, based on a specimen from the "Upper Burma Hills," lacked important morphological details that have complicated the assignment of recently collected material. Furthermore, although the holotype was never lost, its location has been misreported in one important taxonomic reference, leading to further confusion. We report the correct repository of the holotype (Natural History Museum, London), together with its current catalog number. We also describe key features of that specimen that were omitted from the original description, and provide new details on the morphology of the species, including sexual dichromatism unusual for the genus, based upon specimens from southern Sichuan, China. Rhabdophis leonardi is distinguished from its congeners by the following characters: 15 or 17 DSR at midbody and 6 supralabials; distinct annulus around the neck, broad and red in males, and narrow and orange with a black border in females; dorsal ground color light green or olive; some lateral and dorsal scales possessing black edges, the frequency of black edges gradually increasing from anterior to posterior, forming irregular and ill-defined transverse black bands; eye with prominent green iris; black ventral spots with a red edge, most numerous at midbody but extending halfway down the length of the tail. In southwestern China, this species is frequently found at 1730-2230 m elevation. It has been documented to prey upon anuran amphibians, including toads. A recently published phylogenetic analysis showed this species to be deeply nested with the genus Rhabdophis, as a member of the R. nuchalis Group. That analysis also revealed the existence of two closely related but geographically distinct subclades in the molecular analysis, one of which may represent an unnamed taxon.

Out of Asia: natricine snakes support the Cenozoic Beringian Dispersal Hypothesis.

Based on a combination of six mitochondrial gene fragments (12S RNA, cyt b, ND1, ND2, ND4 and CO1) and one nuclear gene (c-mos) from 22 genera we infer phylogenetic relationships among natricine snakes and examine the date and area of origin of these snakes. Our phylogenetic results indicate: (1) the subfamily Natricinae is strongly supported as monophyletic including a majority of extant genera, and a poorly known and previously unassigned species Trachischium monticola, (2) two main clades are inferred within Natricinae, one containing solely taxa from the Old World (OW) and the other comprising taxa from a monophyletic New World (NW) group with a small number of OW relatives. Within the first clade, the genera Xenochrophis and Amphiesma are apparently not monophyletic. Divergence dating and ancestral area estimation indicate that the natricines originated in tropical Asia during the later Eocene or the Oligocene. We recover two major dispersals events out of Asia, the first to Africa in the Oligocene (28 Ma) and the second to the Western Palearctic and the New World at 27 Ma. This date is consistent with the dispersal of numerous other OW groups into the NW.

Three new species of Diploderma Hallowell, 1861 (Squamata, Agamidae) from the Hengduan Mountain Region, south-western China.

Three new species of Diploderma are described from the Hengduan Mountain Region in south-western China, based on morphological and genetic data. The first new species from Yulong County, Yunnan Province is morphologically most similar and phylogenetically closely related to D.brevicauda, but it can be diagnosed from the latter by having a relatively longer tail; the second new species from Xiangcheng County, Sichuan Province is phylogenetically closely related to D.bowoense, but it can be diagnosed from the latter by the absence of a distinct gular spot; and the third new species from Yongsheng County, Yunnan Province is phylogenetically closely related to D.yulongense, but it can be diagnosed from the latter by having different colourations of the ventral and ventrolateral surfaces of the body. Taxonomy and diversity survey are the basis of species conservation, our discoveries contributing to better conservation of the species of this genus.

Confirmation of Tylototritonziegleri Nishikawa, Matsui & Nguyen, 2013 in China, with discussion on the relationship between T.verrucosus Anderson, 1871 and T.panwaensis Grismer, Wood, Quah, Thura, Espinoza & Murdoch, 2019 (Caudata, Salamandridae).

Background: The distribution of the Ziegler's Crocodile Newt Tylototritonziegleri Nishikawa, Matsui & Nguyen, 2013 in China has been controversial. This species was originally recorded uncertainly from Guangxi Autonomous Region, China. Subsequently, this species was recorded from Yunnan and Guangdong provinces, China. Thereafter, the record from Guangdong was denied and the record from Yunnan was questioned. New information: Two specimens of Tylototriton Anderson, 1871 were collected from Wenshan Prefecture, Yunnan Province, China, in 2020. Phylogenetically, the sequences of these two specimens clustered with the sequences of T.ziegleri (including the holotype) from its type locality with strong support, and morphologically agree well with the original description of T.ziegleri. We confirm the record of T.ziegleri in China and present detailed collection site and morphological description of the specimens from China. In addition, we found that T.panwaensis Grismer, Wood, Quah, Thura, Espinoza & Murdoch, 2019 may be the synonym of T.verrucosus Anderson, 1871. We discussed the relationship between T.verrucosus and T.panwaensis.

A cryptic species of the Amolopsricketti species group (Anura, Ranidae) from China-Vietnam border regions.

It was supposed that the current records of Amolopsricketti might be a species complex composed of multiple species. In this study, on the basis of wide sampling, we found that the records of A.ricketti from Yunnan, China, and northern Vietnam actually represent a cryptic species based on morphological and molecular evidence. Amolopsshihaitaoi sp. nov. can be distinguished from other members of the A.ricketti species group by its moderate body size (SVL 35.5‒37.3 mm in males and 39.2‒45.7 mm in females); white spines on the temporal region, loreal region, snout, and lips in breeding males but absent in females; overlapping heels; tibiotarsal articulation reaching tip of snout; indistinct longitudinal glandular folds on the skin of the shoulders; presence of supernumerary tubercles below the base of fingers II‒IV, distinct pineal body; presence of vomerine teeth; and absence of vocal sacs. Phylogenetic analysis supports that the new species is sister to Amolopsyatseni and the populations from Jingxi, Guangxi and Lào Cai, Vietnam previously reported as A.yatesni also belong to it. Additionally, our results indicate that more cryptic species may exist within the A.ricketti species group, implying that more studies are needed to achieve a complete understanding of the species diversity of this group.

General and Specific Aversive Modulation of Active Avoidance Require Central Amygdala.

Three studies provide evidence that the central nucleus of the amygdala, a structure with a well-established role in conditioned freezing, is also required for conditioned facilitation of instrumental avoidance in rats. First, the immediate early gene c-Fos was measured following the presentation of a previously shock-paired tone in subjects trained either on an unsignaled avoidance task or not (in addition to tone only presentations in naïve controls). Significantly elevated expression of c-Fos was found in both the avoidance trained and Pavlovian trained conditions relative to naïve controls (but with no difference between the two trained conditions). In a subsequent study, intracranial infusions of muscimol into the central amygdala significantly attenuated the facilitation of shock-avoidance by a shock-paired Pavlovian cue relative to pre-operative responding. The final study used a virogenetic approach to inhibit the central amygdala prior to testing. This treatment eliminated the transfer of motivational control over shock-avoidance by both a shock-paired Pavlovian stimulus, as well as a cue paired with a perceptually distinct aversive event (i.e., klaxon). These findings provide compelling support for a role of central amygdala in producing aversive Pavlovian-instrumental transfer.

Antagonism of lateral amygdala alpha1-adrenergic receptors facilitates fear conditioning and long-term potentiation.

Norepinephrine receptors have been studied in emotion, memory, and attention. However, the role of alpha1-adrenergic receptors in fear conditioning, a major model of emotional learning, is poorly understood. We examined the effect of terazosin, an alpha1-adrenergic receptor antagonist, on cued fear conditioning. Systemic or intra-lateral amygdala terazosin delivered before conditioning enhanced short- and long-term memory. Terazosin delivered after conditioning did not affect consolidation. In vitro, terazosin impaired lateral amygdala inhibitory postsynaptic currents leading to facilitation of excitatory postsynaptic currents and long-term potentiation. Since alpha1 blockers are prescribed for hypertension and post-traumatic stress disorder, these results may have important clinical implications.

Neotype designation and redescription of Tropidophorus guangxiensis Wen, 1992 (Squamata: Sauria: Scincidae), with description of a new subspecies from central South China.

The genus Tropidophorus consists of small semi-aquatic lizards that dwell in lowland forest steams (Barbour, 1921; Bauer & Jackman, 2008). Here, we designate the neotype and re-describe T. guangxiensis Wen, 1992 based on newly collected topotypic specimens. We also describe a new subspecies from Xuefeng Mountain, Hongjiang County, Hunan Province, central South China. Based on two mitochondrial genes (12S rRNA and 16S rRNA), the phylogenetic position of T. guangxiensis is allocated for the first time. Additionally, our data strongly support that the new subspecies is phylogenetically closely related to T. g. guangxiensis. We also present a morphological identification key for known species and subspecies of Tropidophorus in China.

A new snake species of the genus Gonyosoma Wagler, 1828 (Serpentes: Colubridae) from Hainan Island, China.

A new species of the genus Gonyosoma Wagler, 1828 is described herein based on six specimens from the Diaoluoshan Mountains, Hainan Island, Hainan Province, China. The new species, Gonyosoma hainanensesp. nov., is most similar to its continental sister species, Gonyosoma boulengeri (Mocquard, 1897). Both taxa have a scaled protrusion on the anterior portion of the rostrum, distinct from other congeners. However, Gonyosoma hainanensesp. nov. can be distinguished from G. boulengeri by two significant morphological characters: (1) black orbital stripe absent in adults (vs. present in G. boulengeri); and (2) two loreals (vs. one loreal in G. boulengeri). The new species is also genetically divergent and forms a unique clade from its sister species and all other congeners based on sequences of the mitochondrial gene cytochrome b (cyt b).

A new species of the genus Acanthosaura (Squamata, Agamidae) from Yunnan, China, with comments on its conservation status.

A new species of Acanthosaura from Yunnan, China, is described based on morphological and genetic data. The new species can be separated from all other species of the genus by having a different shape of the black eye patch, a different coloration of the postorbital and occipital spines and nuchal crest, and a different color of the gular pouch. Genetically, uncorrected sequence divergences of COI between the new species and investigated congeners ranged from 16.12% to 24.11%. The conservation status of the new species is also discussed.