Large-scale genome sequencing of giant pandas improves the understanding of population structure and future conservation initiatives.

The extinction risk of the giant panda has been demoted from "endangered" to "vulnerable" on the International Union for Conservation of Nature Red List, but its habitat is more fragmented than ever before, resulting in 33 isolated giant panda populations according to the fourth national survey released by the Chinese government. Further comprehensive investigations of the genetic background and in-depth assessments of the conservation status of wild populations are still necessary and urgently needed. Here, we sequenced the genomes of 612 giant pandas with an average depth of ~26× and generated a high-resolution map of genomic variation with more than 20 million variants covering wild individuals from six mountain ranges and captive representatives in China. We identified distinct genetic clusters within the Minshan population by performing a fine-grained genetic structure. The estimation of inbreeding and genetic load associated with historical population dynamics suggested that future conservation efforts should pay special attention to the Qinling and Liangshan populations. Releasing captive individuals with a genetic background similar to the recipient population appears to be an advantageous genetic rescue strategy for recovering the wild giant panda populations, as this approach introduces fewer deleterious mutations into the wild population than mating with differentiated lineages. These findings emphasize the superiority of large-scale population genomics to provide precise guidelines for future conservation of the giant panda.

Haplotype-resolved chromosome-scale genomes of the Asian and African Savannah Elephants.

The Proboscidea, which includes modern elephants, were once the largest terrestrial animals among extant species. They suffered mass extinction during the Ice Age. As a unique branch on the evolutionary tree, the Proboscidea are of great significance for the study of living animals. In this study, we generate chromosome-scale and haplotype-resolved genome assemblies for two extant Proboscidea species (Asian Elephant, Elephas maximus and African Savannah Elephant, Loxodonta africana) using Pacbio, Hi-C, and DNBSEQ technologies. The assembled genome sizes of the Asian and African Savannah Elephant are 3.38 Gb and 3.31 Gb, with scaffold N50 values of 130 Mb and 122 Mb, respectively. Using Hi-C technology ~97% of the scaffolds are anchored to 29 pseudochromosomes. Additionally, we identify ~9 Mb Y-linked sequences for each species. The high-quality genome assemblies in this study provide a valuable resource for future research on ecology, evolution, biology and conservation of Proboscidea species.

Single-nucleus transcriptome inventory of giant panda reveals cellular basis for fitness optimization under low metabolism.

BACKGROUND: Energy homeostasis is essential for the adaptation of animals to their environment and some wild animals keep low metabolism adaptive to their low-nutrient dietary supply. Giant panda is such a typical low-metabolic mammal exhibiting species specialization of extremely low daily energy expenditure. It has low levels of basal metabolic rate, thyroid hormone, and physical activities, whereas the cellular bases of its low metabolic adaptation remain rarely explored. RESULTS: In this study, we generate a single-nucleus transcriptome atlas of 21 organs/tissues from a female giant panda. We focused on the central metabolic organ (liver) and dissected cellular metabolic status by cross-species comparison. Adaptive expression mode (i.e., AMPK related) was prominently displayed in the hepatocyte of giant panda. In the highest energy-consuming organ, the heart, we found a possibly optimized utilization of fatty acid. Detailed cell subtype annotation of endothelial cells showed the uterine-specific deficiency of blood vascular subclasses, indicating a potential adaptation for a low reproductive energy expenditure. CONCLUSIONS: Our findings shed light on the possible cellular basis and transcriptomic regulatory clues for the low metabolism in giant pandas and helped to understand physiological adaptation response to nutrient stress.

Alternative telomere maintenance mechanism in Alligator sinensis provides insights into aging evolution.

Lifespan is a life-history trait that undergoes natural selection. Telomeres are hallmarks of aging, and shortening rate predicts species lifespan, making telomere maintenance mechanisms throughout different lifespans a worthy topic for study. Alligators are suitable for the exploration of anti-aging molecular mechanisms, because they exhibit low or even negligible mortality in adults and no significant telomere shortening. Telomerase reverse transcriptase (TERT) expression is absent in the adult Alligator sinensis, as in humans. Selection analyses on telomere maintenance genes indicated that ATM, FANCE, SAMHD1, HMBOX1, NAT10, and MAP3K4 experienced positive selection on A. sinensis. Repressed pleiotropic ATM kinase in A. sinensis suggests their fitness optimum shift. In ATM downstream, Alternative Lengthening of Telomeres (ALT)-related genes were clustered in a higher expression pattern in A. sinensis, which covers 10-15% of human cancers showing no telomerase activities. In summary, we demonstrated how telomere shortening, telomerase activities, and ALT contributed to anti-aging strategies.

Genomic investigation of the Chinese alligator reveals wild-extinct genetic diversity and genomic consequences of their continuous decline.

Critically endangered species are usually restricted to small and isolated populations. High inbreeding without gene flow among populations further aggravates their threatened condition and reduces the likelihood of their long-term survival. Chinese alligator (Alligator sinensis) is one of the most endangered crocodiles in the world and has experienced a continuous decline over the past c. 1 million years. In order to identify the genetic status of the remaining populations and aid conservation efforts, we assembled the first high-quality chromosome-level genome of Chinese alligator and explored the genomic characteristics of three extant breeding populations. Our analyses revealed the existence of at least three genetically distinct populations, comprising two breeding populations in China (Changxing and Xuancheng) and one breeding population in an American wildlife refuge. The American population does not belong to the last two populations of its native range (Xuancheng and Changxing), thus representing genetic diversity extinct in the wild and provides future opportunities for genetic rescue. Moreover, the effective population size of these three populations has been continuously declining over the past 20 ka. Consistent with this decline, the species shows extremely low genetic diversity, a large proportion of long runs of homozygous fragments, and mutational load across the genome. Finally, to provide genomic insights for future breeding management and conservation, we assessed the feasibility of mixing extant populations based on the likelihood of introducing new deleterious alleles and signatures of local adaptation. Overall, this study provides a valuable genomic resource and important genomic insights into the ecology, evolution, and conservation of critically endangered alligators.

Antibacterial Properties and Potential Mechanism of Serum from Chinese Alligator.

The Chinese alligator (Alligator sinensis) is an ancient reptile with strong immunity that lives in wetland environments. This study tested the antibacterial ability of Chinese alligator serum (CAS) against Klebsiella pneumoniae, Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa and analyzed the potential underlying mechanisms. Results showed that the CAS had a marked antibacterial effect on K. pneumoniae, E. coli, and P. aeruginosa, while S. aureus was only mildly affected. However, these effects disappeared when Protease K was added to the serum. The serum proteome analysis revealed that the antibacterial ability of CAS was produced by interactions among various proteins and that the complement proteins played a major antibacterial role. Therefore, we made relevant predictions about the structure and function of complement component 3. In addition, sequence alignment and phylogenetic analysis of complement component 3d (C3d) in four mammalian species and two alligator species showed that the amino acids that make up the acid pocket on the concave surface of alligator C3d are not identical to those in mammals. This study provided evidence that CAS elicits significant antibacterial effects against some pathogens and provides the basis for further development of novel antibacterial drugs.

Sexual Dimorphism of the Gut Microbiota in the Chinese Alligator and Its Convergence in the Wild Environment.

The gut microbiota forms a complex microecosystem in vertebrates and is affected by various factors. As a key intrinsic factor, sex has a persistent impact on the formation and development of gut microbiota. Few studies have analyzed sexual dimorphism of gut microbiota, particularly in wild animals. We used 16S rRNA gene sequencing to analyze the gut microbiota of juvenile and adult Chinese alligators, and untargeted metabolomics to study serum metabolomes of adult alligators. We observed significant sexual differences in the community diversity in juvenile, but not adult, alligators. In terms of taxonomic composition, the phylum Fusobacteriota and genus Cetobacterium were highly abundant in adult alligators, similar to those present in carnivorous fishes, whereas the gut microbiota composition in juvenile alligators resembled that in terrestrial reptiles, indicating that adults are affected by their wild aquatic environment and lack sex dimorphism in gut microbiota. The correlation analysis revealed that the gut microbiota of adults was also affected by cyanobacteria in the external environment, and this effect was sex-biased and mediated by sex hormones. Overall, this study reveals sexual differences in the gut microbiota of crocodilians and their convergence in the external environment, while also providing insights into host-microbiota interactions in wildlife.

Incubation determines favorable microbial communities in Chinese alligator nests.

Nest materials are a major heat source due to rotting promoted by microbial activity. Additionally, they are a potential microbial source given their direct contact with eggshells. Microbial dynamics during incubation have been studied in wild birds; however, similar studies in reptiles remain elusive. Here, the study characterized microbial communities in the nest materials of Chinese alligator (Alligator sinensis) using high-throughput sequencing of bacterial 16S rRNA genes and fungal internal transcribed spacer (ITS) region sequences. The results showed that significant changes in the diversity and structure of microbial communities according to different incubation periods. The diversity and richness of bacterial species increased significantly over time, but the relative abundance of the most dominant bacteria in pre-incubation period, including some pathogenic bacteria, declined after incubation. In contrast, fungal species diversity and richness decreased significantly with time. Additionally, nest material composition significantly influenced microbial community structure rather than species diversity and richness. Notably, the fungal community structure showed a stronger response than bacteria to nest material composition, which varied due to differences in plant litter composition. Our results demonstrate the significant response of microbial community diversity and structure to differences in incubation periods and nest material composition in reptiles. It is further emphasized that the importance of incubation period in the conservation of the Chinese alligator and could inform similar studies in other reptiles and birds.

Comparative Genomics Provides Insights into Adaptive Evolution in Tactile-Foraging Birds.

Tactile-foraging birds have evolved an enlarged principal sensory nucleus (PrV) but smaller brain regions related to the visual system, which reflects the difference in sensory dependence. The "trade-off" may exist between different senses in tactile foragers, as well as between corresponding sensory-processing areas in the brain. We explored the mechanism underlying the adaptive evolution of sensory systems in three tactile foragers (kiwi, mallard, and crested ibis). The results showed that olfaction-related genes in kiwi and mallard and hearing-related genes in crested ibis were expanded, indicating they may also have sensitive olfaction or hearing, respectively. However, some genes required for visual development were positively selected or had convergent amino acid substitutions in all three tactile branches, and it seems to show the possibility of visual degradation. In addition, we may provide a new visual-degradation candidate gene PDLIM1 who suffered dense convergent amino acid substitutions within the ZM domain. At last, two genes responsible for regulating the proliferation and differentiation of neuronal progenitor cells may play roles in determining the relative sizes of sensory areas in brain. This exploration offers insight into the relationship between specialized tactile-forging behavior and the evolution of sensory abilities and brain structures.

Sex-Biased miRNAs in the Gonads of Adult Chinese Alligator (Alligator sinensis) and Their Potential Roles in Sex Maintenance.

MicroRNA (miRNA) is a category of single-stranded non-coding small RNA (sRNA) that regulates gene expression by targeting mRNA. It plays a key role in the temperature-dependent sex determination of Chinese alligator (Alligator sinensis), a reptile whose sex is determined solely by the temperature during the incubation period and remains stable thereafter. However, the potential function of miRNAs in the gonads of adult Chinese alligators is still unclear. Here, we prepared and sequenced sRNA libraries of adult female and male alligator gonads, from breeding (in summer) and hibernating (in winter) animals. We obtained 130 conserved miRNAs and 683 novel miRNAs, which were assessed for sex bias in summer and winter; a total of 65 miRNAs that maintained sex bias in both seasons were identified. A regulatory network of sex-biased miRNAs and genes was constructed. Sex-biased miRNAs targeted multiple genes in the meiosis pathway of adult Chinese alligator oocytes and the antagonistic gonadal function maintenance pathway, such as MOS, MYT1, DMRT1, and GDF9. Our study emphasizes the function of miRNA in the epigenetic mechanisms of sex maintenance in crocodilians.

Sex-Biased Gene Expression and Evolution in the Cerebrum and Syrinx of Chinese Hwamei (Garrulax canorus).

It is common that males and females display sexual dimorphisms, which usually result from sex-biased gene expression. Chinese hwamei (Garrulax canorus) is a good model for studying sex-biased gene expression because the song between the sexes is quite different. In this study, we analyze cerebrum and syrinx sex-biased gene expression and evolution using the de novo assembled Chinese hwamei transcriptome. In both the cerebrum and syrinx, our study revealed that most female-biased genes were actively expressed in females only, while most male-biased genes were actively expressed in both sexes. In addition, both male- and female-biased genes were enriched on the putative Z chromosome, suggesting the existence of sexually antagonistic genes and the insufficient dosage compensation of the Z-linked genes. We also identified a 9 Mb sex linkage region on the putative 4A chromosome which enriched more than 20% of female-biased genes. Resultantly, male-biased genes in both tissues had significantly higher Ka/Ks and effective number of codons (ENCs) than unbiased genes, and this suggested that male-biased genes which exhibit accelerated divergence may have resulted from positive selection. Taken together, our results initially revealed the reasons for the differences in singing behavior between males and females of Chinese hwamei.

Gut microbiota of endangered crested ibis: Establishment, diversity, and association with reproductive output.

Gut microbiota is known to influence the host's health; an imbalance of the gut microbial community leads to various intestinal and non-intestinal diseases. Research on gut microbes of endangered birds is vital for their conservation. However, a thorough understanding of the gut microbiome composition present in crested ibises at different ages and its correlation with crested ibis reproductive capacity has remained elusive. Here, we used 16S rRNA gene sequencing to explore the fecal microbial structure of nestlings and adult birds, and the difference in gut microbiota between healthy and sterile crested ibises. We observed that (1) bacterial microbiota, alpha and beta diversity of one-day-old nestlings significantly distinguished from other nestlings; abundance of Proteobacteria decreased, while that of Fusobacteria increased with an increase in the age of the nestlings; (2) there was no significant difference in community composition among adult crested ibises aged one, two, three, and five years; (3) the abundance of Proteobacteria and alpha diversity indices were higher in sterile crested ibises than in healthy crested ibises; thus, Proteobacteria can act as a diagnostic biomarker of reproductive dysfunction in crested ibises. This study significantly contributes to the field of ecology and conservation, as it provides a platform for assessing the reproductive capacity of endangered crested ibises, based on the gut microbiota composition. Further studies may unravel additional factors influencing crested ibises' reproductive health, which will further help the management and control of the crested ibis population.

Genome-Wide DNA Methylation and Transcriptome Analyses Reveal Epigenetic and Genetic Mechanisms Underlying Sex Maintenance of Adult Chinese Alligator.

The sexes of Chinese alligators are determined during embryonic development and remain fixed thereafter. In this study, we investigated the genetic and epigenetic mechanisms underlying sex maintenance in Chinese alligators through RNA sequencing and bisulfite sequencing data analyses of the adult gonads. We identified the genes and pathways (e. g., DMRT1-SOX9-AMH pathway for males and oocyte meiotic maturation pathway for females) involved in male and female sex maintenance and gonadal development of adult Chinese alligators. In contrast to their expression patterns in the embryo, both DMRT1 and the steroid hormone biosynthesis related genes showed a male-biased expression in adult gonads. The overall DNA methylation density and level were higher in testes than in ovaries. Hypermethylation in the gene bodies enhanced the expression of male-biased genes (such as DMRT1-SOX9-AMH and steroid hormone biosynthesis related genes) in the testis, as opposed to the normalization of gene expression. Our results provide insights into the genetic and epigenetic mechanisms underlying sex maintenance in adult Chinese alligators, and are expected to contribute to the development of scientific programs for the successful conservation of this endangered species.

Cape Feather Coloration Signals Different Genotypes of the Most Polymorphic MHC Locus in Male Golden Pheasants (Chrysolophus pictus).

Ornamental feather coloration is usually a reflection of male quality and plays an important role during courtship, whereas the essence of male quality at the genetic level is not well understood. Major histocompatibility complex (MHC)-based mate choice has been observed in various vertebrates. Here, we investigated the relationship between the coloration of cape feathers and the MHC genotypes in golden pheasants (Chrysolophus pictus). We found that feather coloration differed sharply among different individuals (brightness: 1827.20 ± 759.43, chroma: 1241.90 ± 468.21, hue: 0.46 ± 0.06). Heterozygous individuals at the most polymorphic MHC locus (IA2) had brighter feathers than homozygous individuals (Z = -2.853, p = 0.004) and were more saturated in color (Z = -2.853, p = 0.004). However, feather coloration was not related to other MHC loci or to overall genetic heterozygosity (p > 0.050). Our study suggested that coloration of cape feathers might signal IA2 genotypes in golden pheasants.

Chromosome-scale genomes provide new insights into subspecies divergence and evolutionary characteristics of the giant panda.

Extant giant pandas are divided into Sichuan and Qinling subspecies. The giant panda has many species-specific characteristics, including comparatively small organs for body size, small genitalia of male individuals, and low reproduction. Here, we report the most contiguous, high-quality chromosome-level genomes of two extant giant panda subspecies to date, with the first genome assembly of the Qinling subspecies. Compared with the previously assembled giant panda genomes based on short reads, our two assembled genomes increased contiguity over 200-fold at the contig level. Additional sequencing of 25 individuals dated the divergence of the Sichuan and Qinling subspecies into two distinct clusters from 10,000 to 12,000 years ago. Comparative genomic analyses identified the loss of regulatory elements in the dachshund family transcription factor 2 (DACH2) gene and specific changes in the synaptotagmin 6 (SYT6) gene, which may be responsible for the reduced fertility of the giant panda. Positive selection analysis between the two subspecies indicated that the reproduction-associated IQ motif containing D (IQCD) gene may at least partly explain the different reproduction rates of the two subspecies. Furthermore, several genes in the Hippo pathway exhibited signs of rapid evolution with giant panda-specific variants and divergent regulatory elements, which may contribute to the reduced inner organ sizes of the giant panda.

Multi-omics analysis reveals that natural hibernation is crucial for oocyte maturation in the female Chinese alligator.

BACKGROUND: Hibernation in an appropriate environment not only is important for the survival of hibernators in winter, but also is crucial for breeding in the following season for many hibernating species. However, the genetic and epigenetic mechanism underlying this process remain unclear. In the current study, we performed an integrative multi-omics analysis of gonads collected from Chinese alligators that overwintered in wild cave and artificial warmroom to explore transcriptomic and epigenomic alternations in these organs. RESULTS: The data revealed that in the breeding season, female alligators were more strongly affected in terms of gene expression than males by non-hibernation because of overwintering in a warm room, especially for genes related to oocyte maturation, and this effect commenced in winter with the downregulation of STAR, which is the rate limiting factor of steroid biosynthesis. Further, miRNAs were found to play essential roles in this negative effect of overwintering in the warm room on hibernation. The upregulated miRNAs likely were responsible for the suppression of oocyte maturation in the breeding season. Finally, DNA methylome changes, especially hypomethylation, were found to play an important role in the alterations in ovarian function-related gene expression induced by non-hibernation. CONCLUSIONS: Our study revealed the crucial role of hibernation quality for oocyte maturation in the Chinese alligator and the underlying genetic and epigenetic mechanisms, and highlights the importance of habitat, and especially, the overwintering site, in the conservation of not only the Chinese alligator, but also other endangered hibernators.

Transcriptome Comparison Reveals Key Components of Nuptial Plumage Coloration in Crested Ibis.

Nuptial plumage coloration is critical in the mating choice of the crested ibis. This species has a characteristic nuptial plumage that develops from the application of a black sticky substance, secreted by a patch of skin in the throat and neck region. We aimed to identify the genes regulating its coloring, by comparing skin transcriptomes between ibises during the breeding and nonbreeding seasons. In breeding season skins, key eumelanin synthesis genes, TYR, DCT, and TYRP1 were upregulated. Tyrosine metabolism, which is closely related to melanin synthesis, was also upregulated, as were transporter proteins belonging to multiple SLC families, which might act during melanosome transportation to keratinocytes. These results indicate that eumelanin is likely an important component of the black substance. In addition, we observed upregulation in lipid metabolism in breeding season skins. We suggest that the lipids contribute to an oil base, which imbues the black substance with water insolubility and enhances its adhesion to feather surfaces. In nonbreeding season skins, we observed upregulation in cell adhesion molecules, which play critical roles in cell interactions. A number of molecules involved in innervation and angiogenesis were upregulated, indicating an ongoing expansion of nerves and blood vessels in sampled skins. Feather ß keratin, a basic component of avian feather filament, was also upregulated. These results are consistent with feather regeneration in the black skin of nonbreeding season ibises. Our results provide the first molecular evidence indicating that eumelanin is the key component of ibis coloration.

A Unique Energy-Saving Strategy during Hibernation Revealed by Multi-Omics Analysis in the Chinese Alligator.

Many ectotherms hibernate in face of the harsh winter conditions to improve their survival rate. However, the molecular mechanism underlying this process remains unclear. Here, we explored the hibernation mechanism of Chinese alligator using integrative multi-omics analysis. We revealed that (1) the thyroid hormone biosynthesis, nutrition absorption and metabolism, muscle contraction, urinary excretion and immunity function pathways are overall downregulated during hibernation; (2) the fat catabolism is completely suppressed, contrasting with the upregulation of hepatic fatty-acid-transporter CPT1A, suggesting a unique energy-saving strategy that differs from that in hibernating mammals; (3) the hibernation-related genes are not only directly regulated by DNA methylation but also controlled by methylation-dependent transcription networks. In addition, we identified and compared tissue-specific, species-specific, and conserved season-biased miRNAs, demonstrating complex post-transcriptional regulation during hibernation. Our study revealed the genetic and epigenetic mechanisms underlying hibernation in the Chinese alligator and provided molecular insights into the evolution of hibernation regulation.

Metagenomics Reveals Seasonal Functional Adaptation of the Gut Microbiome to Host Feeding and Fasting in the Chinese Alligator.

As a natural hibernator, the Chinese alligator (Alligator sinensis) is an ideal and intriguing model to investigate changes in microbial community structure and function caused by hibernation. In this study, we used 16S rRNA profiling and metagenomic analysis to compare the composition, diversity, and functional capacity in the gut microbiome of hibernating vs. active Chinese alligators. Our results show that gut microbial communities undergo seasonal restructuring in response to seasonal cycles of feeding and fasting in the Chinese alligator, but this animal harbors a core gut microbial community primarily dominated by Proteobacteria, Fusobacteria, Bacteroidetes, and Firmicutes across the gut regions. During hibernation, there is an increase in the abundance of bacterial taxa (e.g., the genus Bacteroides) that can degrade host mucin glycans, which allows adaptation to winter fasting. This is accompanied by the enrichment of mucin oligosaccharide-degrading enzyme and carbohydrate-active enzyme families. In contrast, during the active phase (feeding), active Chinese alligators exhibit a carnivore gut microbiome dominated by Fusobacteria, and there is an increase in the relative abundance of bacteria (e.g., Cetobacterium somerae) with known proteolytic and amino acids-fermentating functions that improve host protein-rich food digestion efficiency. In addition, seasonal variations in the expression of ß-defensins play a protective role in intestinal immunity. These findings provide insights into the functional adaptations of host-gut microbe symbioses to seasonal dietary shifts to maintain gut homeostasis and health, especially in extreme physiological states.

Genome Resequencing Reveals Congenital Causes of Embryo and Nestling Death in Crested Ibis (Nipponia nippon).

The crested ibis (Nipponia nippon) is endangered worldwide. Although a series of conservation measures have markedly increased the population size and distribution area of these birds, the high mortality of embryos and nestlings considerably decreases the survival potential of this bird species. High-throughput sequencing technology was utilized to compare whole genomes between ten samples from dead crested ibises (including six dead embryos and four dead nestlings aged 0-45 days) and 32 samples from living birds. The results indicated that the dead samples all shared the genetic background of a specific ancestral subpopulation. Furthermore, the dead individuals were less genetically diverse and suffered higher degrees of inbreeding compared with these measures in live birds. Several candidate genes (KLHL3, SETDB2, TNNT2, PKP1, AK1, and EXOSC3) associated with detrimental diseases were identified in the genomic regions that differed between the alive and dead samples, which are likely responsible for the death of embryos and nestlings. In addition, in these regions, we also found several genes involved in the protein catabolic process (UBE4A and LONP1), lipid metabolism (ACOT1), glycan biosynthesis and metabolism (HYAL1 and HYAL4), and the immune system (JAM2) that are likely to promote the normal development of embryos and nestlings. The aberrant conditions of these genes and biological processes may contribute to the death of embryos and nestlings. Our data identify congenital factors underlying the death of embryos and nestlings at the whole genome level, which may be useful toward informing more effective conservation efforts for this bird species.