Population genomic analysis provides evidence of the past success and future potential of South China tiger captive conservation.

BACKGROUND: Among six extant tiger subspecies, the South China tiger (Panthera tigris amoyensis) once was widely distributed but is now the rarest one and extinct in the wild. All living South China tigers are descendants of only two male and four female wild-caught tigers and they survive solely in zoos after 60 years of effective conservation efforts. Inbreeding depression and hybridization with other tiger subspecies were believed to have occurred within the small, captive South China tiger population. It is therefore urgently needed to examine the genomic landscape of existing genetic variation among the South China tigers. RESULTS: In this study, we assembled a high-quality chromosome-level genome using long-read sequences and re-sequenced 29 high-depth genomes of the South China tigers. By combining and comparing our data with the other 40 genomes of six tiger subspecies, we identified two significantly differentiated genomic lineages among the South China tigers, which harbored some rare genetic variants introgressed from other tiger subspecies and thus maintained a moderate genetic diversity. We noticed that the South China tiger had higher FROH values for longer runs of homozygosity (ROH > 1 Mb), an indication of recent inbreeding/founder events. We also observed that the South China tiger had the least frequent homozygous genotypes of both high- and moderate-impact deleterious mutations, and lower mutation loads than both Amur and Sumatran tigers. Altogether, our analyses indicated an effective genetic purging of deleterious mutations in homozygous states from the South China tiger, following its population contraction with a controlled increase in inbreeding based on its pedigree records. CONCLUSIONS: The identification of two unique founder/genomic lineages coupled with active genetic purging of deleterious mutations in homozygous states and the genomic resources generated in our study pave the way for a genomics-informed conservation, following the real-time monitoring and rational exchange of reproductive South China tigers among zoos.

Comparative analysis of the fecal microbiome and metabolomics of healthy versus captive South China tigers with mild diarrhea.

Diarrhea-predominant irritable bowel syndrome (IBS-D) is common among the captive South China tigers in zoos. 16S rRNA gene sequencing was performed to demonstrate the compositions and structures of the gastrointestinal microbiota of this species with IBS-D. Their healthy (F1) and mushy (F2) feces were allocated into two groups. A total of 21 and 31 fecal bacterial communities of major phyla and genera were detected, respectively. The F1 and F2 groups had five common microbiotas at the phylum level (Firmicutes, Proteobacteria, Fusobacteria, Actinobacteria, and Bacteroidetes). Among the five phyla, the abundance of Bacteroidetes in the F2 group was significantly lower than that in the F1 group. The diversity level of fecal microbiota within the mild-diarrhea stool was also significantly lower than that of the healthy counterpart. Thirty-two metabolites were correlated to four genus-level bacteria (Bacteroides, Pseudoclavibacter, Streptococcus, and Ruminococcaceae-UCG-005). Due to its normal role in protein degradation and metabolism, we hypothesized that the lower abundance of Bacteroides within the F2 group could be associated with the IBS-D symptoms. Therefore, this work implied that ameliorating the daily diet with a supplement of probiotics, such as Bacteroides, could improve the gut health of this species.

Sorting Out the Genetic Background of the Last Surviving South China Tigers.

The South China tiger (Panthera tigris amoyensis) is endemic to China and also the most critically endangered subspecies of living tigers. It is considered extinct in the wild and only about 150 individuals survive in captivity to date, whose genetic heritage, however, is ambiguous and controversial. Here, we conducted an explicit genetic assessment of 92 studbook-registered South China tigers from 14 captive facilities using a subspecies-diagnostic system in the context of comparison with other voucher specimens to evaluate the genetic ancestry and level of distinctiveness of the last surviving P. t. amoyensis. Three mtDNA haplotypes were identified from South China tigers sampled in this study, including a unique P. t. amoyensis AMO1 haplotype not found in other subspecies, a COR1 haplotype that is widespread in Indochinese tigers (P. t. corbetti), and an ALT haplotype that is characteristic of Amur tigers (P. t. altaica). Bayesian STRUCTURE analysis and parentage verification confirmed the verified subspecies ancestry (VSA) as the South China tiger in 74 individuals. Genetic introgression from other tigers was detected in 18 tigers, and subsequent exclusion of these and their offspring from the breeding program is recommended. Both STRUCTURE clustering and microsatellite-based phylogenetic analyses demonstrated a close genetic association of the VSA South China tigers to Indochinese tigers, an issue that could only be elucidated by analysis of historical South China tiger specimens with wild origin. Our results also indicated a moderate level of genetic diversity in the captive South China tiger population, suggesting a potential for genetic restoration.

Sequence Analysis of Mitochondrial Genome of Toxascaris leonina from a South China Tiger.

Toxascaris leonina is a common parasitic nematode of wild mammals and has significant impacts on the protection of rare wild animals. To analyze population genetic characteristics of T. leonina from South China tiger, its mitochondrial (mt) genome was sequenced. Its complete circular mt genome was 14,277 bp in length, including 12 protein-coding genes, 22 tRNA genes, 2 rRNA genes, and 2 non-coding regions. The nucleotide composition was biased toward A and T. The most common start codon and stop codon were TTG and TAG, and 4 genes ended with an incomplete stop codon. There were 13 intergenic regions ranging 1 to 10 bp in size. Phylogenetically, T. leonina from a South China tiger was close to canine T. leonina. This study reports for the first time a complete mt genome sequence of T. leonina from the South China tiger, and provides a scientific basis for studying the genetic diversity of nematodes between different hosts.

Insights for the Captive Management of South China Tigers Based on a Large-Scale Genetic Survey.

There is an urgent need to find a way to improve the genetic diversity of captive South China tiger (SCT, Panthera tigris amoyensis), the most critically endangered taxon of living tigers, facing inbreeding depression. The genomes showed that 13 hybrid SCTs from Meihuashan were divided into two groups; one group included three individuals who had a closer relationship with pureblood SCTs than another group. The three individuals shared more that 40% of their genome with pureblood SCTs and might be potential individuals for genetic rescuing in SCTs. A large-scale genetic survey based on 319 pureblood SCTs showed that the mean microsatellite inbreeding coefficient of pureblood SCTs decreased significantly from 0.1789 to 0.0600 (p = 0.000009) and the ratio of heterozygous loci increased significantly from 38.5% to 43.2% (p = 0.02) after one individual of the Chongqing line joined the Suzhou line and began to breed in the mid-1980s, which is a reason why the current SCTs keep a moderate level of microsatellite heterozygosity and nucleotide diversity. However, it is important to establish a back-up population based on the three individuals through introducing one pureblood SCT into the back-up population every year. The back-up population should be an important reserve in case the pureblood SCTs are in danger in the future.

Chromosome-scale genomes reveal genomic consequences of inbreeding in the South China tiger: A comparative study with the Amur tiger.

The South China tiger (Panthera tigris amoyensis, SCT) is the most critically endangered subspecies of tiger due to functional extinction in the wild. Inbreeding depression is observed among the captive population descended from six wild ancestors, resulting in high juvenile mortality and low reproduction. We assembled and characterized the first SCT genome and an improved Amur tiger (P. t. altaica, AT) genome named AmyTig1.0 and PanTig2.0. The two genomes are the most continuous and comprehensive among any tiger genomes yet reported at the chromosomal level. By using the two genomes and resequencing data of 15 SCT and 13 AT individuals, we investigated the genomic signature of inbreeding depression of the SCT. The results indicated that the effective population size of SCT experienced three phases of decline, ~5.0-1.0 thousand years ago, 100 years ago, and since captive breeding in 1963. We found 43 long runs of homozygosity fragments that were shared by all individuals in the SCT population and covered a total length of 20.63% in the SCT genome. We also detected a large proportion of identical-by-descent segments across the genome in the SCT population, especially on ChrB4. Deleterious nonsynonymous single nucleotide polymorphic sites and loss-of-function mutations were found across genomes with extensive potential influences, despite a proportion of these loads having been purged by inbreeding depression. Our research provides an invaluable resource for the formulation of genetic management policies for the South China tiger such as developing genome-based breeding and genetic rescue strategy.

Developmental stage variation in the gut microbiome of South China tigers.

South China tigers (Panthera tigris amoyensis, SC) are the most threatened tiger subspecies in the world. All the living SCs are captive in zoos or reserves and depend on artificial feeding. The composition of the gut microbiome plays an important role in sustaining the health of the host. A comprehensive understanding of the composition and development of the microbial community of SC is helpful to improve the feeding of captive SC. In this study, we collected 47 fecal samples, 37 of which were from SC of three developmental stages, 5 from adult Amur tigers (Am), and 5 from adult Bengal tigers (Bg), which were all housed in the same zoo. We investigated the diversity, richness, and composition of the bacterial microbiomes and we found that the gut microbiome of SC is strongly affected by host aging. The composition of the gut microbiome of juvenile SC experienced dramatic changes from 5 months old to 1 year old, and it showed much less difference when compared to the samples of 1 year old and the subadult. No significant differences were observed between the samples of subadult and the adult groups. The predominant phylum of 5-month-old SC is Fusobacteriota (33.99%) when the juvenile tigers were older than 5 months, and Firmicutes, but not Fusobacteriota, became the predominant phylum of bacteria in their gut. The gut microbiome of SC, Am, and Bg is possibly affected by their genetic variation; however, the core microbiome of these three subspecies is the same. Our data suggest that the gut microbiome of SC undergoes a developmental progression: a developmental phase (cub), a transitional phase (subadult), and a stable phase (adult). These results expand our understanding of the role of age in the development of the gut microbiome of SC.

New Evidence of Tiger Subspecies Differentiation and Environmental Adaptation: Comparison of the Whole Genomes of the Amur Tiger and the South China Tiger.

Panthera tigris is a top predator that maintains the integrity of forest ecosystems and is an integral part of biodiversity. No more than 400 Amur tigers (P. t. altaica) are left in the wild, whereas the South China tiger (P. t. amoyensis) is thought to be extinct in the wild, and molecular biology has been widely used in conservation and management. In this study, the genetic information of Amur tigers and South China tigers was studied by whole-genome sequencing (WGS). A total of 647 Gb of high-quality clean data was obtained. There were 6.3 million high-quality single-nucleotide polymorphisms (SNPs), among which most (66.3%) were located in intergenic regions, with an average of 31.72% located in coding sequences. There were 1.73 million insertion-deletions (InDels), among which there were 2438 InDels (0.10%) in the coding region, and 270 thousand copy number variations (CNVs). Significant genetic differences were found between the Amur tiger and the South China tiger based on a principal component analysis and phylogenetic tree. The linkage disequilibrium analysis showed that the linkage disequilibrium attenuation distance of the South China tiger and the Amur tiger was almost the same, whereas the r2 of the South China tiger was 0.6, and the r2 of the Amur tiger was 0.4. We identified functional genes and regulatory pathways related to reproduction, disease, predation, and metabolism and characterized functional genes related to survival in the wild, such as smell, vision, muscle, and predatory ability. The data also provide new evidence for the adaptation of Amur tigers to cold environments. PRKG1 is involved in temperature regulation in a cold climate. FOXO1 and TPM4 regulate body temperature to keep it constant. Our results can provide genetic support for precise interspecies conservation and management planning in the future.

Colonization and Development of the Fecal Microflora of South China Tiger Cubs (Panthera tigris amoyensis) by Sequencing of the 16S rRNA Gene.

Postnatal colonization and development of the gut microbiota is linked to health and growth. A comprehensive understanding of the postnatal compositional changes and development of the microbial community is helpful to understand the gut health and improve the survival rate of South China tiger cubs (Panthera tigris amoyensis). Fecal samples from three tiger cubs were collected on the day of birth in 2018 (June 17-21 [G0], July 18 [G1], July 31 [G2], and August 7 [G3]). The 16S rRNA genes of the fecal microflora were sequenced. Results showed that 38 phyla, 58 classes, 134 orders, 272 families, and 636 genera of bacteria from 3,059 operational taxonomic units were identified from 12 fecal samples. The diversity and abundance of species of group G0 were significantly higher (p < 0.05 or 0.01) than those of groups G2 and G3. The predominant phylum was Proteobacteria in groups G0 and G1 (38.85% and 48%, respectively) and Firmicutes in groups G2 and G3 (71.42% and 75.29%, respectively). At the phylum level, the abundance of Deinococcus-Thermus was significantly decreased in groups G1, G2, and G3 as compared to group G0 (p < 0.05), while that of Firmicutes was significantly increased in groups G2 and G3 (p < 0.05). At the genus level, the abundance of Faecalibacterium, Ralstonia, and unidentified Rickettsiales was significantly decreased in groups G1, G2, and G3 as compared with group G0 (p < 0.05), while that of Pseudomonas was significantly decreased in groups G2 and G3 (p < 0.05). The composition and structure of fecal microbiota of South China tiger cubs changed after birth.

Isolation, molecular characterization, and phylogenetic analysis of encephalomyocarditis virus from South China tigers in China.

Although encephalomyocarditis virus (EMCV) can infect many host species and cause myocarditis and sudden death in many species, little is known about EMCV infection in tigers. A virus was isolated from organs of dead South China tigers with sudden death in southern China. The production of cytopathic effect on BHK cells, and the results of PCR, electron microscopy (EM), and whole genome sequencing indicated that the pathogen was EMCV, the strain was named FJ13. Other pathogenic agents were excluded as possible pathogenic agents. Phylogenetic analyses of the whole genome, ORF (open reading frame) and CCR (capsid coding region) using the neighbour-joining method revealed that EMCV isolates cluster into two groups (group 1 and 2) with two sub-clusters within group 1 (group 1a and 1b), and FJ13 belongs to group 1a. Animal experiment showed that the isolated strain FJ13 could cause clinical symptoms and pathological changes. The results of this study indicated that FJ13 caused myocarditis of tigers and provided new epidemiologic data on EMCV in China.

Sequence Analysis of Mitochondrial Genome of Toxascaris leonina from a South China Tiger

Toxascaris leonina is a common parasitic nematode of wild mammals and has significant impacts on the protection of rare wild animals. To analyze population genetic characteristics of T. leonina from South China tiger, its mitochondrial (mt) genome was sequenced. Its complete circular mt genome was 14,277 bp in length, including 12 protein-coding genes, 22 tRNA genes, 2 rRNA genes, and 2 non-coding regions. The nucleotide composition was biased toward A and T. The most common start codon and stop codon were TTG and TAG, and 4 genes ended with an incomplete stop codon. There were 13 intergenic regions ranging 1 to 10 bp in size. Phylogenetically, T. leonina from a South China tiger was close to canine T. leonina. This study reports for the first time a complete mt genome sequence of T. leonina from the South China tiger, and provides a scientific basis for studying the genetic diversity of nematodes between different hosts.