Taking a color photo: A homozygous 25-bp deletion in Bace2 may cause brown-and-white coat color in giant pandas.

Brown-and-white giant pandas (hereafter brown pandas) are distinct coat color mutants found exclusively in the Qinling Mountains, Shaanxi, China. However, its genetic mechanism has remained unclear since their discovery in 1985. Here, we identified the genetic basis for this coat color variation using a combination of field ecological data, population genomic data, and a CRISPR-Cas9 knockout mouse model. We de novo assembled a long-read-based giant panda genome and resequenced the genomes of 35 giant pandas, including two brown pandas and two family trios associated with a brown panda. We identified a homozygous 25-bp deletion in the first exon of Bace2, a gene encoding amyloid precursor protein cleaving enzyme, as the most likely genetic basis for brown-and-white coat color. This deletion was further validated using PCR and Sanger sequencing of another 192 black giant pandas and CRISPR-Cas9 edited knockout mice. Our investigation revealed that this mutation reduced the number and size of melanosomes of the hairs in knockout mice and possibly in the brown panda, further leading to the hypopigmentation. These findings provide unique insights into the genetic basis of coat color variation in wild animals.

Identifying priority protection areas of key food resources of the giant panda.

Animals that live in seasonal environments adjust their reproduction cycle to optimize seasonal forage quality. Giant pandas ( Ailuropoda melanoleuca) are seasonal altitudinal migrants that feed on bamboo shoots and leaves with different nutritional quality. However, the importance of bamboo shoots to giant pandas, especially small and isolated populations, is not fully appreciated. Here, we explored whether mating time of giant pandas is shaped by bamboo shoot phenology. We also assessed the intensity of ongoing bamboo shoot harvesting by local communities in 42 giant panda reserves based on questionnaire surveys. Varying intensity and protection levels of bamboo shoot harvesting were found. From these data, we developed a priority ranking scheme of protection areas for this key food resource. Our study showed that pandas time their mating behavior to coincide with bamboo shoot phenology due to the high nutritional demands associated with mating and pregnancy. We also found that bamboo shoots were not well protected in many places. Liangshan, Daxiangling, and Xiaoxiangling, containing the most isolated panda populations, were identified as the areas with the most urgent need of protection. Furthermore, equal attention should be paid to the QiongL-B population, as this region holds considerable potential to serve as a corridor between the Minshan and Qionglai populations. To address the challenges posed by bamboo shoot harvesting, we recommend establishing more practical bamboo shoot management policies, increasing public awareness of bamboo shoot protection, and providing alternative sources of income for local communities.

Stable seasonal migration patterns in giant pandas.

A critical function of animal movement is to maximize access to essential resources in temporally fluctuating and spatially heterogeneous environments. Seasonally mediated resource fluctuations may influence animal movements, enabling them to track changing resource distributions, resulting in annual migration patterns. The conservation-dependent giant panda ( Ailuropoda melanoleuca) displays seasonal movement patterns; however, the key factor driving these seasonal migration patterns remains poorly understood. Here, we used GPS tracking collars to monitor the movements of six giant pandas over a 12-year period across different elevations, and performed statistical analysis of seasonal migration directions, routes, habitat revisitation, home range overlap, first arrival events, and stability. Our results revealed a compelling pattern of seasonal migrations that facilitated the ability of the pandas to forage at the appropriate time and place to maximize nutritional intake. Our results indicated that pandas utilize spatial memory to locate reliable food resources, as evidenced by their annual return to the same or similar winter and summer home ranges and the consistently maintained percentage of home range overlap. These novel insights into giant panda foraging and movement ecology not only enhance our understanding of its ability to adapt to nutritionally poor dietary resources but also provide important information for the development of resource utilization-based protection and management strategies.

Community structure of the solitary giant pandas is maintained by indirect social connections.

BACKGROUND: Indirect interactions between individual solitary mammals, such as the giant panda, are often overlooked because of their nature, yet are important for maintaining the necessary sociality in solitary species. METHODS AND RESULTS: Here, we determined the genetic identity of all giant panda individuals in a local population and matched these identities with their associations to determine social network of this solitary animal. Total thirty-five giant panda individuals were found in our field survey, and we constructed genetic and social networks for thirty-three individuals who successfully obtained genetic, age and sex information. The results showed that sex had great impact on both social network and genetic network, and age may have the potential to influence the social network of the giant pandas. Adult males, mostly in the central of the social network, which appeared significantly larger network connections than adult females. Due to the female-biased dispersal pattern of wild giant pandas, male-male pairs showed higher relatedness than female-female ones and multi-generational patrilinear assemblages are expected in the study area. CONCLUSIONS: The relatedness of individuals has an influence on the formation of community social structure of giant pandas, and indirect interactions among solitary giant pandas potentially function to reduce competition for resources and inbreeding.

A single nucleotide mutation in the dual-oxidase 2 (DUOX2) gene causes some of the panda's unique metabolic phenotypes.

The giant panda (Ailuropoda melanoleuca) is an iconic bear native to China, famous for eating almost exclusively bamboo. This unusual dietary behavior for a carnivore is enabled by several key adaptations including low physical activity, reduced organ sizes and hypothyroidism leading to lowered energy expenditure. These adaptive phenotypes have been hypothesized to arise from a panda-unique single-nucleotide mutation in the dual-oxidase 2 (DUOX2) gene, involved in thyroid hormone synthesis. To test this hypothesis, we created genome-edited mice carrying the same point mutation as the panda and investigated its effect on metabolic phenotype. Homozygous mice were 27% smaller than heterozygous and wild-type ones, had 13% lower body mass-adjusted food intake, 55% decreased physical activity, lower mass of kidneys (11%) and brain (5%), lower serum thyroxine (T4: 36%), decreased absolute (12%) and mass-adjusted (5%) daily energy expenditure, and altered gut microbiota. Supplementation with T4 reversed the effects of the mutation. This work uses a state-of-the-art genome editing approach to demonstrate the link between a single-nucleotide mutation in a key endocrine-related gene and profound adaptive changes in the metabolic phenotype, with great importance in ecology and evolution.

Seasonal shift of the gut microbiome synchronizes host peripheral circadian rhythm for physiological adaptation to a low-fat diet in the giant panda.

Characteristics of the gut microbiome vary synchronously with changes in host diet. However, the underlying effects of these fluctuations remain unclear. Here, we performed fecal microbiota transplantation (FMT) of diet-specific feces from an endangered mammal (the giant panda) into a germ-free mouse model. We demonstrated that the butyrate-producing bacterium Clostridium butyricum was more abundant during shoot-eating season than during the leaf-eating season, congruent with the significant increase in host body mass. Following season-specific FMT, the microbiota of the mouse model resembled that of the donor, and mice transplanted with the microbiota from the shoot-eating season grew faster and stored more fat. Mechanistic investigations revealed that butyrate extended the upregulation of hepatic circadian gene Per2, subsequently increasing phospholipid biosynthesis. Validation experiments further confirmed this causal relationship. This study demonstrated that seasonal shifts in the gut microbiome affect growth performance, facilitating a deeper understanding of host-microbe interactions in wild mammals.

The giant panda is cryptic.

The giant panda (Ailuropoda melanoleuca) is an iconic mammal, but the function of its black-and-white coloration is mysterious. Using photographs of giant pandas taken in the wild and state-of-the-art image analysis, we confirm the counterintuitive hypothesis that their coloration provides camouflage in their natural environment. The black fur blends into dark shades and tree trunks, whereas white fur matches foliage and snow when present, and intermediate pelage tones match rocks and ground. At longer viewing distances giant pandas show high edge disruption that breaks up their outline, and up close they rely more on background matching. The results are consistent across acuity-corrected canine, feline, and human vision models. We also show quantitatively that the species animal-to-background colour matching falls within the range of other species that are widely recognised as cryptic. Thus, their coloration is an adaptation to provide background matching in the visual environment in which they live and simultaneously to afford distance-dependent disruptive coloration, the latter of which constitutes the first computational evidence of this form of protective coloration in mammals.

Geographic distributions shape the functional traits in a large mammalian family.

Traits of organisms are shaped by their living environments and also determined in part by their phylogenetic relationships. For example, phylogenetic relationships often affect the geographic distributions of animals and cause variation in their living environments, which usually play key roles in the life history and determine the functional traits of species. As an ancient family of mammals, bears widely distribute and have evolved some specific strategies for survival and reproduction during their long-term evolutionary histories. Many studies on the ecology of bears have been conducted in recent decades, but few have focused on the relationships between their geographic distributions and ecological adaptations. Here, using bears as a model system, we collected and reanalyzed data from the available literatures to explore how geographic distributions and phylogenetic relationships shape the functional traits of animals. We found a positive relationship between phylogenetic relatedness and geographic distributions, with bears distributed in adjacent areas applying more similar strategies to survive and reproduce: (a) Bears living at high latitudes consumed a higher proportion of vertebrates, which may provide more fat for adaptation to low temperatures, and (b) their reproduction rhythms follow fluctuations in seasonal forage availability and quality, in which bears reach mating status from March to May and give birth in approximately November or later.

Multi-omics reveals the positive leverage of plant secondary metabolites on the gut microbiota in a non-model mammal.

BACKGROUND: Flavonoids are important plant secondary metabolites (PSMs) that have been widely used for their health-promoting effects. However, little is known about overall flavonoid metabolism and the interactive effects between flavonoids and the gut microbiota. The flavonoid-rich bamboo and the giant panda provide an ideal system to bridge this gap. RESULTS: Here, integrating metabolomic and metagenomic approaches, and in vitro culture experiment, we identified 97 flavonoids in bamboo and most of them have not been identified previously; the utilization of more than 70% flavonoid monomers was attributed to gut microbiota; the variation of flavonoid in bamboo leaves and shoots shaped the seasonal microbial fluctuation. The greater the flavonoid content in the diet was, the lower microbial diversity and virulence factor, but the more cellulose-degrading species. CONCLUSIONS: Our study shows an unprecedented landscape of beneficial PSMs in a non-model mammal and reveals that PSMs remodel the gut microbiota conferring host adaptation to diet transition in an ecological context, providing a novel insight into host-microbe interaction. Video abstract.

Symbiotic bacteria mediate volatile chemical signal synthesis in a large solitary mammal species.

Mammalian chemosignals-or scent marks-are characterized by astounding chemical diversity, reflecting both complex biochemical pathways that produce them and rich information exchange with conspecifics. The microbiome of scent glands was thought to play prominent role in the chemical signal synthesis, with diverse microbiota metabolizing glandular products to produce odorants that may be used as chemosignals. Here, we use gas chromatography-mass spectrometry and metagenomic shotgun sequencing to explore this phenomenon in the anogenital gland secretions (AGS) of the giant panda (Ailuropoda melanoleuca). We find that this gland contains a diverse community of fermentative bacteria with enzymes that support metabolic pathways (e.g., lipid degradation) for the productions of volatile odorants specialized for chemical communication. We found quantitative and qualitative differences in the microbiota between AGS and digestive tract, a finding which was mirrored by differences among chemical compounds that could be used for olfactory communication. Volatile chemical compounds were more diverse and abundant in AGS than fecal samples, and our evidence suggests that metabolic pathways have been specialized for the synthesis of chemosignals for communication. The panda's microbiome is rich with genes coding for enzymes that participate in the fermentation pathways producing chemical compounds commonly deployed in mammalian chemosignals. These findings illuminate the poorly understood phenomena involved in the role of symbiotic bacteria in the production of chemosignals.

Diet drives convergent evolution of gut microbiomes in bamboo-eating species.

Gut microbiota plays a critical role in host physiology and health. The coevolution between the host and its gut microbes facilitates animal adaptation to its specific ecological niche. Multiple factors such as host diet and phylogeny modulate the structure and function of gut microbiota. However, the relative contribution of each factor in shaping the structure of gut microbiota remains unclear. The giant (Ailuropoda melanoleuca) and red (Ailurus styani) pandas belong to different families of order Carnivora. They have evolved as obligate bamboo-feeders and can be used as a model system for studying the gut microbiome convergent evolution. Here, we compare the structure and function of gut microbiota of the two pandas with their carnivorous relatives using 16S rRNA and metagenome sequencing. We found that both panda species share more similarities in their gut microbiota structure with each other than each species shares with its carnivorous relatives. This indicates that the specialized herbivorous diet rather than host phylogeny is the dominant driver of gut microbiome convergence within Arctoidea. Metagenomic analysis revealed that the symbiotic gut microbiota of both pandas possesses a high level of starch and sucrose metabolism and vitamin B12 biosynthesis. These findings suggest a diet-driven convergence of gut microbiomes and provide new insight into host-microbiota coevolution of these endangered species.

Genomic Signatures of Coevolution between Nonmodel Mammals and Parasitic Roundworms.

Antagonistic coevolution between host and parasite drives species evolution. However, most of the studies only focus on parasitism adaptation and do not explore the coevolution mechanisms from the perspective of both host and parasite. Here, through the de novo sequencing and assembly of the genomes of giant panda roundworm, red panda roundworm, and lion roundworm parasitic on tiger, we investigated the genomic mechanisms of coevolution between nonmodel mammals and their parasitic roundworms and those of roundworm parasitism in general. The genome-wide phylogeny revealed that these parasitic roundworms have not phylogenetically coevolved with their hosts. The CTSZ and prolyl 4-hydroxylase subunit beta (P4HB) immunoregulatory proteins played a central role in protein interaction between mammals and parasitic roundworms. The gene tree comparison identified that seven pairs of interactive proteins had consistent phylogenetic topology, suggesting their coevolution during host-parasite interaction. These coevolutionary proteins were particularly relevant to immune response. In addition, we found that the roundworms of both pandas exhibited higher proportions of metallopeptidase genes, and some positively selected genes were highly related to their larvae's fast development. Our findings provide novel insights into the genetic mechanisms of coevolution between nonmodel mammals and parasites and offer the valuable genomic resources for scientific ascariasis prevention in both pandas.

Why wild giant pandas frequently roll in horse manure.

Attraction to feces in wild mammalian species is extremely rare. Here we introduce the horse manure rolling (HMR) behavior of wild giant pandas (Ailuropoda melanoleuca). Pandas not only frequently sniffed and wallowed in fresh horse manure, but also actively rubbed the fecal matter all over their bodies. The frequency of HMR events was highly correlated with an ambient temperature lower than 15 °C. BCP/BCPO (beta-caryophyllene/caryophyllene oxide) in fresh horse manure was found to drive HMR behavior and attenuated the cold sensitivity of mice by directly targeting and inhibiting transient receptor potential melastatin 8 (TRPM8), an archetypical cold-activated ion channel of mammals. Therefore, horse manure containing BCP/BCPO likely bestows the wild giant pandas with cold tolerance at low ambient temperatures. Together, our study described an unusual behavior, identified BCP/BCPO as chemical inhibitors of TRPM8 ion channel, and provided a plausible chemistry-auxiliary mechanism, in which animals might actively seek and utilize potential chemical resources from their habitat for temperature acclimatization.

Seasonal dynamics of parasitism and stress physiology in wild giant pandas.

Many factors, including the inner status of the individuals and external environment, can influence the parasite infections and stress physiology in mammals. Here, we explored the influence of the sex, age, reproductive season and seasonal food availability on the parasitism and stress physiology in wild giant pandas (Ailuropoda melanoleuca) through nutrient and steroid hormone analysis and parasitic infection measurement. Diet composition had significant influences on the faecal cortisol levels and parasite load of wild giant pandas. The seasonal dynamic of the cortisol levels and parasite load in faeces co-vary with the seasonal nutrient intake levels of the pandas, which concurrently arrived the peaks at the wood bamboo shoot-eating period in May (parasite infection intensity, 41.47 ± 12.11 eggs/g of wet faeces; cortisol levels, 619.34 ± 70.55 ng/g dry faeces) that the nutrition intake by wild pandas was the highest (protein/fibre, 69.23 ± 9.93). Meanwhile, age class is also as an important factor to affect the parasite load and stress physiology of wild giant pandas. Cubs and sub-adults suffered more helminth burden and stress physiology than adults and old individuals. This is the first study to evaluate the inner and external factors influence on parasitism and stress physiology in wild giant pandas. The findings facilitate a better understanding of how environmental factors might influence the physiology, behaviour and health of pandas and other species and have implications for the conservation and management of the endangered species.

Impact of sympatric carnivores on den selection of wild giant pandas.

Interspecific killing is a primary reason for the low survival rates of some animal species. The giant panda ( Ailuropoda melanoleuca) is an altricial eutherian mammal and thus, in comparison to other infants, panda cubs are highly vulnerable, which may significantly influence the selection of breeding sites by females. Here, we used infrared camera traps to monitor giant panda dens for 5.5 years in Foping National Nature Reserve (FNNR) to determine how interspecific factors affect den selection by wild female pandas. Results indicated that Asian black bears ( Ursus thibetanus), yellow-throated martens ( Martes flavigula), leopard cats ( Prionailurus bengalensis), and masked palm civets ( Paguma larvata) visited the dens frequently, and the presence of these species negatively influenced den selection by female pandas. Interestingly, the presence of rodents and terrestrial birds appeared to indicate den safety, and female giant pandas were not averse and even preferred dens with a high abundance index of rodents and terrestrial birds. The den suitability index (DSI) was a reliable tool for evaluating whether dens were suitable for female giant pandas to give birth to and rear cubs, with preference for dens with high DSI values. This study increases our understanding of the den selection criteria of female giant pandas and the main threats to the survival of their cubs, thus providing important guidance for the conservation and management of this species.

Giant Pandas Are Macronutritional Carnivores.

Giant pandas are unusual in belonging to a primarily carnivorous clade and yet being extremely specialized herbivores that feed almost exclusively on highly fibrous bamboo [1]. Paradoxically, they appear inconsistently adapted to their plant diet, bearing a mix of herbivore and carnivore traits. Herbivore traits include a skull, jaw musculature, and dentition that are adapted for fibrous diets and a specialized "pseudo-thumb" used for handling bamboo [2, 3]. They have lost functional versions of the T1R1 gene codes for umami taste receptors, which are often associated with meat eating [3]. They also have an herbivore-like subcellular distribution of the metabolic enzyme alanine: glyoxylate aminotransferase [4]. But meanwhile, giant pandas have a digestive tract [5], digestive enzymes [6], and a gut microbiota composition that resemble those of carnivores and not of herbivores [6, 7]. We draw on recent developments in multi-dimensional niche theory [8] to examine this apparent paradox. We show that the pandas' diet clustered in a macronutrient space among carnivores and was distinct from that of herbivores. The similarity with carnivore diets applied not only to the ingested diet but also to the absorbed diet, with the absorbed macronutrient ratios similar to those of the ingested foods. Comparison of the macronutrient composition of pandas' milk with those of other species shows that the carnivore-like dietary macronutrient composition extends across the life cycle. These results cast new light on the seemingly incongruous constellation of dietary adaptations in pandas, suggesting that the transition from carnivorous and omnivorous ancestry to specialized herbivory might be less abrupt than it might otherwise appear.

Diet Evolution and Habitat Contraction of Giant Pandas via Stable Isotope Analysis.

The ancestral panda Ailurarctos lufengensis, excavated from the late Miocene, is thought to be carnivorous or omnivorous [1]. Today, giant pandas exclusively consume bamboo and have distinctive tooth, skull, and muscle characteristics adapted to a tough and fibrous bamboo diet during their long evolution [1, 2]. A special feature, the pseudo-thumb, has evolved to permit the precise and efficient grasping of bamboo [3, 4]. Unlike those of extant pandas, little is known about the diet and habitat preferences of extinct pandas. Prevailing studies suggest that the panda shifted to specialized bamboo feeding in the Pleistocene [5, 6]; however, this remains questionable. Pandas now survive in a fraction of their historical habitat [7], but no specific information has been reported. Stable isotope analyses can be used to understand diet- and habitat-related changes in animals [8]. Isotopic signals in bone collagen reflect dietary compositions of ancient human diets [9, 10] and dietary changes between historical and modern animal populations [11, 12]. Here, we conduct stable isotope analyses of bone and tooth samples from ancient and modern pandas and from sympatric fauna. We show that pandas have had a diet dominated by C3 resources over time and space and that trophic niches of ancient and modern pandas are distinctly different. The isotopic trophic and ecological niche widths of ancient pandas are approximately three times larger than those of modern pandas, suggesting that ancient pandas possibly had more complex diets and habitats than do their modern counterparts. Our findings provide insight into the dietary evolution and habitat contraction of pandas.

Seasonal and reproductive variation in chemical constituents of scent signals in wild giant pandas.

Seasonally reproducing animals show many behavioral and physiological changes during the mating period, including increased signaling for intrasexual competition and mate attraction. We collected 102 anogenital gland secretions (AGS) from marking trees in Foping Nature Reserve, and used gas chromatography mass spectrometry analyze these chemical composition. Of these marks, all but one were from males, confirmed with DNA analysis. We found that several chemical constituents, especially volatile compounds, is present only during the mating season and that the relative abundance of many compounds changed as a function of breeding season, whereas nonvolatile compounds were lower in the mating season. This seasonal variation in chemical composition of AGS most likely plays an important role in governing giant panda reproduction, including mate location, attraction, and male-male competition. The chemical properties of many of these putative chemosignals-such as volatility and longevity-are suggestive of these roles, and undoubtedly contribute to successful reproduction for this species with a characteristically sophisticated chemical communication system. We also found a number of important differences between the chemical constituents of AGS from wild pandas and those found in previous studies with captive pandas, suggesting that inappropriate chemosignal composition may contribute to poor reproductive success in captive breeding programs.

Conservation metagenomics: a new branch of conservation biology.

Multifaceted approaches are required to monitor wildlife populations and improve conservation efforts. In the last decade, increasing evidence suggests that metagenomic analysis offers valuable perspectives and tools for identifying microbial communities and functions. It has become clear that gut microbiome plays a critical role in health, nutrition, and physiology of wildlife, including numerous endangered animals in the wild and in captivity. In this review, we first introduce the human microbiome and metagenomics, highlighting the importance of microbiome for host fitness. Then, for the first time, we propose the concept of conservation metagenomics, an emerging subdiscipline of conservation biology, which aims to understand the roles of the microbiota in evolution and conservation of endangered animals. We define what conservation metagenomics is along with current approaches, main scientific issues and significant implications in the study of host evolution, physiology, nutrition, ecology and conservation. We also discuss future research directions of conservation metagenomics. Although there is still a long way to go, conservation metagenomics has already shown a significant potential for improving the conservation and management of wildlife.