Phylogeographic patterns and conservation implications of the endangered Chinese giant salamander

Understanding genetic diversity patterns of endangered species is an important premise for biodiversity conservation. The critically endangered salamander Andrias davidianus, endemic to central and southern mainland in China, has suffered from sharp range and population size declines over the past three decades. However, the levels and patterns of genetic diversity of A. davidianus populations in wild remain poorly understood. Herein, we explore the levels and phylogeographic patterns of genetic diversity of wild‐caught A. davidianus using larvae and adult collection with the aid of sequence variation in (a) the mitochondrial DNA (mtDNA) fragments (n = 320 individuals; 33 localities), (b) 19 whole mtDNA genomes, and (c) nuclear recombinase activating gene 2 (RAG2; n = 88 individuals; 19 localities). Phylogenetic analyses based on mtDNA datasets uncovered seven divergent mitochondrial clades (A–G), which likely originated in association with the uplifting of mountains during the Late Miocene, specific habitat requirements, barriers including mountains and drainages and lower dispersal ability. The distributions of clades were geographic partitioned and confined in neighboring regions. Furthermore, we discovered some mountains, rivers, and provinces harbored more than one clades. RAG2 analyses revealed no obvious geographic patterns among the five alleles detected. Our study depicts a relatively intact distribution map of A. davidianus clades in natural species range and provides important knowledge that can be used to improve monitoring programs and develop a conservation strategy for this critically endangered organism.     

Determining threatened species distributions in the face of limited data: Spatial conservation prioritization for the Chinese giant salamander (Andrias davidianus)

The purpose of this study was to determine whether limited occurrence data for highly threatened species can provide useful spatial information to inform conservation. The study was conducted across central and southern China. We developed a habitat suitability model for the Critically Endangered Chinese giant salamander (Andrias davidianus) based on one biotic and three abiotic parameters from single‐site locality records, which represent the only relevant environmental data available for this species. We then validated model quality by testing whether increased percentage of predicted suitable habitat at the county level correlated with independent data on giant salamander presence. We randomly selected 48 counties containing historical records which were distinct from, and independent of, the single‐site records used to develop the model, and 47 additional counties containing >50% predicted suitable habitat. We interviewed 2,812 respondents near potential giant salamander habitat across these counties and tested for differences in respondent giant salamander reports between counties selected using each method. Our model predicts that suitable giant salamander habitat is found widely across central and southern China, with counties containing ≥50% predicted suitable habitat distributed in 13 provinces. Counties with historical records contain significantly more predicted suitable habitat than counties without historical records. There are no statistical differences in any patterns of respondent giant salamander reports in surveyed counties selected from our model compared with the areas of known historical giant salamander distribution. A Chinese giant salamander habitat suitability model with strong predictive power can be derived from the restricted range of environmental variables associated with limited available presence‐only occurrence records, constituting a cost‐effective strategy to guide spatial allocation of conservation planning. Few reported sightings were recent, however, with most being over 20 years old, so that identification of areas of suitable habitat does not necessarily indicate continued survival of the species at these locations.     

Genetic diversity of Chinese giant salamander (Andrias davidianus) based on the novel microsatellite markers

Chinese giant salamander (Andrias davidianus) is a rare amphibian species in the world. Microsatellite markers are a promising tool for accurate estimation population structure and genetic diversity. In this paper, we isolated novel microsatellite marker for Chinese giant salamander using fast isolation by AFLP of sequences containing repeats (FIASCO) method. More than 50% sequences in 132 clones had repeat number over ten times with di or trinucleeotide repeat except of (GA)₁₂. Seventy pairs of primers were designed and eleven polymorphic microsatellite loci were characterized for wild and cultivated Chinese giant salamander populations. The allele number was from 3 to 9 in different loci. Polymorphism information content was from 0.544 to 0.702 in cultivated population. The results implied more alleles and PIC were in the wild population than cultivated population. The observed heterozygosities in two populations were higher than 0.553. The data analysis suggested that the cultivated population has lower genetic diversity than wild population, which it??s perhaps owing to inbreeding in artificial breeding. To our knowledge, it??s the first time to isolated microsatellite markers for Chinese giant salamander. The result indicated that the markers were suitable for the population genetic analysis of Chinese giant salamander.     

Future climate change will severely reduce habitat suitability of the Critically Endangered Chinese giant salamander

1. Being the largest extant amphibian in the world, the IUCN Critically Endangered Chinese giant salamander Andrias davidianus is a charismatic species with great international public interest. While threats such as commercial overexploitation and habitat degradation have been extensively documented to affect natural populations of A. davidianus, still no information is available about the species sensitivity to climate change.
2. Here, we develop an ensemble of species distribution models (SDMs) for A. davidianus and projected its habitat suitability under present-day and future climate change scenarios. We based our SDMs on bioclimatic and topographic predictors, and recent (2012–2018) field-collected occurrence data across the whole distribution range of the species.
3. The ensemble SDMs exhibited good predictive capacity and suggested that slope, maximum temperature of warmest month, precipitation of driest month, and isothermality are the most influential predictors in determining distribution patterns in this species. The projections of our models point to a pronounced impact of climate changes over A. davidianus, with more than two-thirds of its suitable range expected to be lost in all scenarios of future climates tested.
4. In concert with the numerous other threats that are affecting this species, climate change poses a serious hindrance to the long-term survival of A. davidianus. We emphasise the urgent need of undertaking strict measures to manage this species and safeguard the few remaining available suitable habitats. We suggest that adaptive management strategies including designation of new reserves should be considered to mitigate the impacts of climate change on A. davidianus.

     

Telocytes in ileum of the Chinese giant salamander: ultrastructural evidence

Telocytes (TCs) and their telopodes (Tps) have been found in various organs of many mammals, including in lower animals. However, knowledge of TCs in lower animals is still very limited. This study identified TCs and their Tps in the ileum of the Chinese giant salamander, Andrias davidianus (Amphibia: Caudata), by transmission electron microscopy. The TCs/Tps were found near epithelial cells, glandular cells and unmyelinated nerve fibres. Moreover, exosomes were also found to be present in between TCs/Tps and these cells.     

大鲵保护生物学及其研究进展

大鲵（Andrias davidianus)为我国特有的珍稀两栖动物,已列入国家二级保护动物名录,并列入CITES公约附录Ⅰ中。自20世纪50年代起,由于过度收购、非法捕杀和栖息地丧失等原因,大鲵种群数量下降极为严重,湖南、安徽等地的大鲵产量在20世纪50年代至70年代下降超过80％,分布区也极度萎缩,形成了12块岛屿状区域,目前分布于以我国中部山区的长江流域为主的17个省区。部分已建立的大鲵保护区经费短缺,人员不足,管理不力,状况不容乐观。非法捕捉和栖息地丧失仍是威胁大鲵生存的主要因素,保护管理力度还需进一步加强。     

Reduced genetic variation in the Japanese giant salamander, Andrias japonicus (Amphibia: Caudata)

The phylogenetic relationships among 46 samples from 27 populations of the Japanese giant salamander, Andriasjaponicus and its congener, A. davidianus from China was investigated, using 3664 bp sequences of the mitochondrial genes NADH1, NADH3, cyt b and CR, partial NADH6 and intervening genes. In phylogenetic trees constructed by MP, ML, and Bayesian methods, the family Cryptobranchidae and the genus Andrias both form monophyletic groups. Japanese A. japonicus and Chinese A. davidianus are sister taxa and can be regarded as separate species despite a small degree of genetic differentiation. Andriasjaponicus is divided into central and western clades, but the phylogenetic relationships within the latter clade are unresolved. As previously reported from allozyme analyses, A. japonicus exhibits little genetic differentiation, in strong contrast to salamanders of the genus Hynobius with which their distributions overlap. This reduced genetic variability in A. japonicus is attributable to a unique mating system of polygyny, delayed sexual maturity, notable longevity, life in a stable aquatic environment, and gigantism, as well as bottleneck effects following habitat fragmentation and extinction of local populations during Quaternary glaciations. The species is thus susceptible to extinction by potential environmental fluctuations, and requires extensive conservation measures.     

Inferring the roles of vicariance,climate and topography in population differentiation in Salamandra algira (Caudata,Salamandridae)

Integrating information from species occurrence data, environmental variables and molecular markers can provide valuable insights about the processes of population persistence and differentiation. In this study, we present the most comprehensive overview of the evolutionary history of the North African salamander Salamandra algira (Caudata, Salamandridae) to date, including analyses of climatic and topographical variables, and sequences of two mitochondrial and two nuclear DNA fragments, with a special focus on Algerian populations, under‐represented in previous studies. Coalescent‐based phylogenetic analyses of mtDNA data recover four well‐supported population groups corresponding to described subspecies, with a western clade including populations in north‐western Morocco (with two subclades corresponding to the subspecies tingitana and splendens), and an eastern clade including populations from north‐eastern Morocco (subspecies spelaea) and Algeria (subspecies algira). Inferred split times between major clades date back to the Miocene, with additional splits within each major clade in the Plio‐Pleistocene. Present climatic (aridity) and topographical factors account for geographical discontinuities across population groups and help identify potential areas of secondary contact between clades corresponding to the subspecies tingitana and splendens in the Rif mountains in Morocco. Niche analysis indicates the absence of phylogenetic signal in the use of environmental space in this species.     

Electrical discharges in Chinese salamander <Emphasis Type="Italic">Andrias davidianus</Emphasis>

In 2-year-old Chinese giant salamanders Andrias davidianus, occasional electric discharges with a characteristic pattern similar to the electric discharges of weakly electric catfish, Polypterus and Protopterus, were recorded for the first time. The discharges markedly differ in shape from the myograms accompanying abrupt movements of the salamander or exceeded them in amplitude by more than an order of magnitude. The discharges were recorded both in the autonomous experiment in the absence of experimenters and at a weak tactile stimulation.     

High genetic variation and phylogeographic relations among Palearctic fairy shrimp populations reflect persistence in multiple southern refugia during Pleistocene ice ages and postglacial colonisation

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Telocytes in pancreas of the Chinese giant salamander (Andrias davidianus)

Telocytes (TCs), novel interstitial cells, have been identified in various organs of many mammals. However, information about TCs of lower animals remains rare. Herein, pancreatic TCs of the Chinese giant salamanders (Andrias davidianus) were identified by CD34 immunohistochemistry (IHC) and transmission electron microscopy (TEM). The IHC micrographs revealed CD34⁺ TCs with long telopodes (Tps) that were located in the interstitium of the pancreas. CD34⁺ TCs/Tps were frequently observed between exocrine acinar cells and were close to blood vessels. The TEM micrographs also showed the existence of TCs in the interstitium of the pancreas. TCs had distinctive ultrastructural features, such as one to three very long and thin Tps with podoms and podomers, caveolae, dichotomous branching, neighbouring exosomes and vesicles. The Tps and exosomes were found in close proximity to exocrine acinar cells and α cells. It is suggested that TCs may play a role in the regeneration of acinar cells and α cells. In conclusion, our results demonstrated the presence of TCs in the pancreas of the Chinese giant salamander. This finding will assist us in a better understanding of TCs functions in the amphibian pancreas.     

Diversity among peripheral populations: genetic and evolutionary differentiation of Salamandra atra at the southern edge of the Alps

Separate populations at the edge of a species range are receiving great attention and have been shown to be often different from populations in the core area. However, it has rarely been tested whether neighboring peripheral populations are genetically and evolutionarily similar to each other, as expected for their geographical proximity and similar ecological conditions, or differ due to historical contingency. We investigated isolation and differentiation, within‐population genetic diversity and evolutionary relationships among multiple peripheral populations of a cold‐adapted terrestrial salamander, Salamandra atra, at the southern edge of the species core range. We carried out population genetic, phylogeographic, and phylogenetic analyses on various molecular markers (10 autosomal microsatellite loci, three mitochondrial loci with total length >2,100 bp, two protein‐coding nuclear genes) sampled from more than 100 individuals from 13 sites along the southern Prealps. We found at least seven isolated peripheral populations, all highly differentiated from the remaining populations and differentiated from each other at various levels. The within‐population genetic diversity was variable in the peripheral populations, but consistently lower than in the remaining populations. All peripheral populations along the southern Prealps belong to an ancient lineage that is also found in the Dinarides but did not contribute to the postglacial recolonization of the inner and northern Alps. All fully melanistic populations from the Orobian mountains to the southern Dinarides represent a single clade, to the exclusion of the two yellow‐patched populations inhabiting the Pasubio massif and the Sette Comuni plateau, which are distinguished as S. atra pasubiensis and S. atra aurorae, respectively. In conclusion, multiple populations of S. atra at the southern edge of the species core area have different levels of differentiation, different amount of within‐population genetic diversity, and different evolutionary origin. Therefore, they should be regarded as complementary conservation targets to preserve the overall genetic and evolutionary diversity of the species.     

Phylogeography and diversification of an Amazonian understorey hummingbird: paraphyly and evidence for widespread cryptic speciation in the Plio‐Pleistocene

Straight‐billed Hermit Phaethornis bourcieri inhabits the understorey of upland terra firme forest throughout most of the Amazon basin. Currently, two allopatric taxa regarded as subspecies are recognized: P. b. bourcieri and P. b. major. However, the validity, interspecific limits and evolutionary history of these taxa are not yet fully elucidated. We use molecular characters to propose a phylogenetic hypothesis for populations and taxa grouped under Phaethornis bourcieri. Our results showed that P. bourcieri is part of the ‘Ametrornis’ clade, along with Phaethornis philippii and Phaethornis koepckeae, and that the subspecies major is more closely related to the latter two species than to populations grouped under nominate bourcieri. Our phylogenetic hypotheses recovered three main reciprocally monophyletic clades under nominate bourcieri separated by the lower Negro River and the Branco River or the Branco–Negro interfluve (clades B and C) and the upper Amazon (Solimões) or lower Marañon/Ucayali Rivers (clades C and D). Based on multi‐locus phylogeographic and population genetics approaches, we show that P. b. major is best treated as a separate species, and that P. b. bourcieri probably includes more than one evolutionary species, whose limits remain uncertain. The diversification of the ‘Ametrornis’ clade (P. bourcieri, P. philippii and P. koepckeae) is centred in the Amazon and appears to be closely linked to the formation of the modern Amazon drainage during the Plio‐Pleistocene.     

Phylogeographic inference using Bayesian model comparison across a fragmented chorus frog species complex

Fragmented species complexes provide an interesting system for investigating biogeographic history and the present distribution of genetic variation. Recent advances in sequencing technology and statistical phylogeography enable the collection and rigorous analysis of large multilocus data sets, but designing studies that produce meaningful phylogeographic inferences remains challenging. We implemented a Bayesian model comparison approach to investigate previous biogeographic hypotheses while simultaneously inferring the presence of genetic structure in a chorus frog species complex. The Illinois chorus frog (Pseudacris illinoensis), originally described as a subspecies of the broadly distributed Strecker's chorus frog (Pseudacris streckeri), occurs in small, disjunct regions associated with scarce sand prairie habitats that have been impacted by human development. We used high‐throughput sequencing to develop and collect a multitiered genetic data set comprised of three different marker types (23 anonymous nuclear sequence loci, four mitochondrial genes and 14 microsatellite loci) designed to address questions across different evolutionary timescales. Phylogenetic analyses uncovered a deep divergence between populations in the Edwards Plateau of central Texas and all other P. streckeri/P. illinoensis populations, but suggest the disjunct distribution of P. illinoensis occurred more recently. Our best‐supported migration model is consistent with the hypothesis that central Texas represented a refugium from which populations expanded via multiple routes. This model also indicates that disjunct northern and southern regions of P. illinoensis should be considered genetically distinct management units. Our study provides an evolutionary context for future studies and conservation efforts in P. illinoensis and demonstrates the utility of model‐based approaches for phylogeographic inference.     

互花米草防治和麋鹿保护的系统耦合作用——基于自然的解决方案

互花米草(Spartina alterniflora)是典型的外来入侵物种,因其繁殖力和适应力强难以防治,直接威胁我国滨海滩涂湿地生物多样性,改变了该生态系统的结构和功能;麋鹿(Elaphurus davidianus)是我国I级保护动物,是野生动物迁地保护和回归自然的典范,但仍面临野生种群稳定性低、与保护区环境承载力矛盾尖锐的问题。麋鹿喜食互花米草,其牧食行为可有效调控互花米草的时空分布格局,提升滨海滩涂湿地生态系统功能和稳定性,并为其自身野化回归提供良好的环境。目前针对互花米草防治和麋鹿保护的研究较多,但少有二者耦合作用的探讨。尝试借助草地农业系统理论阐释互花米草防治和麋鹿保护(互花米草&麋鹿)耦合发展的范式和潜在机制,即寻求基于自然的克服系统相悖的解决方案。以期打破珍稀物种保护和入侵物种防控各自为战的范式,探索二者耦合发展思路和陆海农业实践。同时,探讨了利用无人机等技术搭建滨海滩涂湿地互花米草&麋鹿耦合系统的空-天-地一体化监测、分析和管理体系。     

Molecular and morphological assessment of Australia’s most endangered snake, <Emphasis Type="Italic">Hoplocephalus bungaroides</Emphasis>, reveals two evolutionarily significant units for conservation

The Broad-headed snake Hoplocephalus bungaroides is one of Australia’s most endangered vertebrates. Extant populations of H. bungaroides are restricted to several geographically isolated reserves to the north, west, and south of Sydney. We analysed mitochondrial DNA from 184 specimens drawn from across the geographic range of the Broad-headed snake. Phylogenetic analysis demonstrated that H. bungaroides comprises two divergent mitochondrial lineages with a “northern” clade comprising populations west and north of Sydney and a “southern” clade comprising animals in Morton National Park. The two clades differ by an uncorrected genetic distance of 1.7%, which implies a divergence dating to approximately 755,000–850,000 years ago. We complemented our molecular data set with a detailed analysis of morphological variation both between and within the genetic clades. The two H. bungaroides genetic clades are morphologically indistinguishable and show little sexual dimorphism. Our results demonstrate that the populations north and south of this biogeographic split function as two distinct populations with no recent gene flow. There is no reason for separate taxonomic recognition of these two clades, but they do represent distinct evolutionarily significant units (ESUs) that require separate conservation management. In addition, within the northern ESU, populations from Royal National Park, Blue Mountains National Park, Wollemi National Park, and the Sydney Water Catchment supply areas should be considered as separate management units to conserve both evolutionary and ecological processes.     

Purification,characterization and application of a novel antimicrobial peptide from Andrias davidianus blood

The Andrias davidianus has been known as a traditional Chinese medicine for a long time. Its blood is considered as a waste or by‐product of the meat production industry. Although there are reports on isolation of the antimicrobial peptides from different resources, there are no reports of their isolation from A. davidianus blood. In this work, an antimicrobial peptide, andricin B, was isolated from the blood of A. davidianus by an innovative method in which the magnetic liposome adsorption was combined with reversed‐phase high‐performance liquid chromatography. The structure, antimicrobial activity and safety of andricin B were further investigated. Amino acid sequence was determined by N‐terminal sequencing and found to be Gly‐Leu‐Thr‐Arg‐Leu‐Phe‐Ser‐Val‐Ile‐Lys. Circular dichroism (CD) spectra and prediction of three‐dimensional structure by bioinformatics software suggested the presence of a well‐defined random coil conformation. Andricin B was found to be active against all bacteria tested in this study as well as some fungi. The minimum inhibitory concentrations (MICs) were in the range 8–64 μg ml^?1. Moreover, the haemolytic testing also suggested that andricin B could be considered safe at the MICs. Finally, andricin B was shown to inhibit the growth of Staphylococcus aureus in the cooked meat of A. davidianus. This study shows that andricin B is a promising novel antimicrobial peptide that may provide further insights towards the development of new drugs.

Significance and Impact of the Study

This is the pioneer study on screening and isolation of antimicrobial peptide from the blood of Andrias davidianus. Here, we have developed a novel method by combining magnetic liposomes adsorption with reversed‐phase high‐performance liquid chromatography to purify and screen the antimicrobial peptides. From this screen, we identified a novel antimicrobial peptide which we name as andricin B. Andricin B is unique as it checks the growth of both Gram‐positive and Gram‐negative bacteria as well as few fungal species.     

Long‐distance dispersal or postglacial contraction? Insights into disjunction between Himalaya–Hengduan Mountains and Taiwan in a cold‐adapted herbaceous genus,Triplostegia

Current disjunct patterns can result from long‐distance dispersal or postglacial contraction. We herein investigate the evolutionary history of Triplostegia to elucidate the disjunction between the Himalaya–Hengduan Mountain region (HHM) and Taiwan (TW). Genetic structure of Triplostegia was investigated for 48 populations using sequences from five chloroplast loci and the ribosomal nuclear internal transcribed spacer. Divergence time estimation, ancestral area reconstruction, and species distribution modeling (SDM) were employed to examine the biogeographic history of Triplostegia. Substantial genetic differentiation among populations from southwestern China (SW), Central China (CC), and TW was detected. Triplostegia was inferred to have originated in SW, and diversification began during the late Miocene; CC was colonized in the mid‐Pliocene, and TW was finally colonized in the early Pleistocene. SDM suggested an expansion of climatically suitable areas during the Last Glacial Maximum and range contraction during the Last interglacial in Triplostegia. Disjunction between HHM and TW in Triplostegia is most likely the consequence of topographic isolation and postglacial contraction. The potential climatic suitability areas for Triplostegia by 2070s (2061–2080) are predicted to slightly shrink and move northward. With continued global warming and human‐induced deforestation, extinction risk may increase for the cold‐adapted species, and appropriate strategies should be employed for ecosystem conservation.