Effect of locomotor preference on the evolution of mitochondrial genes in Bovidae.

Locomotor preferences and habitat types may drive animal evolution. In this study, we speculated that locomotor preference and habitat type may have diverse influences on Bovidae mitochondrial genes. We used selection pressure and statistical analysis to explore the evolution of mitochondrial DNA (mtDNA) protein-coding genes (PCGs) from diverse locomotor preferences and habitat types. Our study demonstrates that locomotor preference (energy demand) drives the evolution of Bovidae in mtDNA PCGs. The habitat types had no significant effect on the rate of evolution in Bovidae mitochondrial genes. Our study provides deep insight into the adaptation of Bovidae.

Mitochondrial Genomic Evidence of Selective Constraints in Small-Bodied Terrestrial Cetartiodactyla.

Body size may drive the molecular evolution of mitochondrial genes in response to changes in energy requirements across species of different sizes. In this study, we perform selection pressure analysis and phylogenetic independent contrasts (PIC) to investigate the association between molecular evolution of mitochondrial genome protein-coding genes (mtDNA PCGs) and body size in terrestrial Cetartiodactyla. Employing selection pressure analysis, we observe that the average non-synonymous/synonymous substitution rate ratio (ω) of mtDNA PCGs is significantly reduced in small-bodied species relative to their medium and large counterparts. PIC analysis further confirms that ω values are positively correlated with body size (R2 = 0.162, p = 0.0016). Our results suggest that mtDNA PCGs of small-bodied species experience much stronger purifying selection as they need to maintain a heightened metabolic rate. On the other hand, larger-bodied species may face less stringent selective pressures on their mtDNA PCGs, potentially due to reduced relative energy expenditure per unit mass. Furthermore, we identify several genes that undergo positive selection, possibly linked to species adaptation to specific environments. Therefore, despite purifying selection being the predominant force in the evolution of mtDNA PCGs, positive selection can also occur during the process of adaptive evolution.

Bacterial and fungal community structures in Hulun Lake are regulated by both stochastic processes and environmental factors.

Microorganisms are a crucial component of lake ecosystems and significant contributors to biogeochemical cycles. However, the understanding of how primary microorganism groups (e.g., bacteria and fungi) are distributed and constructed within different lake habitats is lacking. We investigated the bacterial and fungal communities of Hulun Lake using high-throughput sequencing techniques targeting 16S rRNA and Internal Transcribed Spacer 2 genes, including a range of ecological and statistical methodologies. Our findings reveal that environmental factors have high spatial and temporal variability. The composition and community structures vary significantly depending on differences in habitats. Variance partitioning analysis showed that environmental and geographical factors accounted for <20% of the community variation. Canonical correlation analysis showed that among the environmental factors, temperature, pH, and dissolved oxygen had strong control over microbial communities. However, the microbial communities (bacterial and fungal) were primarily controlled by the dispersal limitations of stochastic processes. This study offers fresh perspectives regarding the maintenance mechanism of bacterial and fungal biodiversity in lake ecosystems, especially regarding the responses of microbial communities under identical environmental stress.IMPORTANCELake ecosystems are an important part of the freshwater ecosystem. Lake microorganisms play an important role in material circulation and energy flow owing to their unique enzymatic and metabolic capacity. In this study, we observed that bacterial and fungal communities varied widely in the water and sediments of Hulun Lake. The primary factor affecting their formation was identified as dispersal limitation during stochastic processes. Environmental and geographical factors accounted for <20% of the variation in bacterial and fungal communities, with pH, temperature, and dissolved oxygen being important environmental factors. Our findings provide new insights into the responses of bacteria and fungi to the environment, shed light on the ecological processes of community building, and deepen our understanding of lake ecosystems. The results of this study provide a reference for lake management and conservation, particularly with respect to monitoring and understanding microbial communities in response to environmental changes.

Analysis of the Complete Mitochondrial Genome of Pteronura brasiliensis and Lontra canadensis.

P. brasiliensis and L. canadensis are two otter species, which successfully occupied semi-aquatic habitats and diverged from other Mustelidae. Herein, the full-length mitochondrial genome sequences were constructed for these two otter species for the first time. Comparative mitochondrial genome, selection pressure, and phylogenetic independent contrasts (PICs) analyses were conducted to determine the structure and evolutionary characteristics of their mitochondrial genomes. Phylogenetic analyses were also conducted to confirm these two otter species' phylogenetic position. The results demonstrated that the mitochondrial genome structure of P. brasiliensis and L. canadensis were consistent across Mustelidae. However, selection pressure analyses demonstrated that the evolutionary rates of mitochondrial genome protein-coding genes (PCGs) ND1, ND4, and ND4L were higher in otters than in terrestrial Mustelidae, whereas the evolutionary rates of ND2, ND6, and COX1 were lower in otters. Additionally, PIC analysis demonstrated that the evolutionary rates of ND2, ND4, and ND4L markedly correlated with a niche type. Phylogenetic analysis showed that P. brasiliensis is situated at the base of the evolutionary tree of otters, and then L. canadensis diverged from it. This study suggests a divergent evolutionary pattern of Mustelidae mitochondrial genome PCGs, prompting the otters' adaptation to semi-aquatic habitats.

Comparative Analyses of the Fecal Microbiome of Five Wild Black-Billed Capercaillie (Tetrao parvirostris) Flocks.

Black-billed capercaillie (Tetrao parvirostris) was listed as a first-class state-protected animal because it was endangered in China (Category I). This study is the first to examine the diversity and composition of T. parvirostris gut microbiome in the wild. We collected fecal samples from five black-billed capercaillie flock roosting sites (each 20 km apart) in one day. Thirty fecal samples were sequenced with 16S rRNA gene amplicons on the Illumina HiSeq platform. This study is the first to analyze the fecal microbiome composition and diversity of black-billed capercaillie in the wild. At the phylum level, Camplyobacterota, Bacillota, Cyanobacteria, Actinomycetota, and Bacteroidota were the most abundant in the fecal microbiome of black-billed capercaillie. At the genus level, unidentified Chloroplast, Escherichia-Shigella, Faecalitalea, Bifidobacterium, and Halomonas were the dominant genera. Based on alpha and beta diversity analyses, we found no significant differences in the fecal microbiome between five flocks of black-billed capercaillie. Protein families: genetic information processing; protein families: signaling and cellular processes, carbohydrate metabolism; protein families: metabolism and energy metabolism are the main predicted functions of the black-billed capercaillie gut microbiome through the PICRUSt2 method. This study reveals the composition and structure of the fecal microbiome of the black-billed capercaillie under wild survival conditions, and this study provides scientific data for the comprehensive conservation of the black-billed capercaillie.

Characteristics of Microbiota in Different Segments of the Digestive Tract of Lycodon rufozonatus.

The gastrointestinal tract of animals contains microbiota, forming a complex microecosystem. Gut microbes and their metabolites can regulate the development of host innate and adaptive immune systems. Animal immune systems maintain intestinal symbiotic microbiota homeostasis. However, relatively few studies have been published on reptiles, particularly snakes, and even fewer studies on different parts of the digestive tracts of these animals. Herein, we used 16S rRNA gene sequencing to investigate the microbial community composition and adaptability in the stomach and small and large intestines of Lycodon rufozonatus. Proteobacteria, Bacteroidetes, and Firmicutes were most abundant in the stomach; Fusobacteria in the small intestine; and Proteobacteria, Bacteroidetes, Fusobacteria, and Firmicutes in the large intestine. No dominant genus could be identified in the stomach; however, dominant genera were evident in the small and large intestines. The microbial diversity index was significantly higher in the stomach than in the small and large intestines. Moreover, the influence of the microbial community structure on function was clarified through function prediction. Collectively, the gut microbes in the different segments of the digestive tract revealed the unique features of the L. rufozonatus gut microbiome. Our results provide insights into the co-evolutionary relationship between reptile gut microbiota and their hosts.

Adaptive Evolution of the OAS Gene Family Provides New Insights into the Antiviral Ability of Laurasiatherian Mammals.

Many mammals risk damage from virus invasion due to frequent environmental changes. The oligoadenylate synthesis (OAS) gene family, which is an important component of the immune system, provides an essential response to the antiviral activities of interferons by regulating immune signal pathways. However, little is known about the evolutionary characteristics of OASs in Laurasiatherian mammals. Here, we examined the evolution of the OAS genes in 64 mammals to explore the accompanying molecular mechanisms of the antiviral ability of Laurasiatherian mammals living in different environments. We found that OAS2 and OAS3 were found to be pseudogenes in Odontoceti species. This may be related to the fact that they live in water. Some Antilopinae, Caprinae, and Cervidae species lacked the OASL gene, which may be related to their habitats being at higher altitudes. The OASs had a high number of positive selection sites in Cetartiodactyla, which drove the expression of strong antiviral ability. The OAS gene family evolved in Laurasiatherian mammals at different rates and was highly correlated with the species' antiviral ability. The gene evolution rate in Cetartiodactyla was significantly higher than that in the other orders. Compared to other species of the Carnivora family, the higher selection pressure on the OAS gene and the absence of positive selection sites in Canidae may be responsible for its weak resistance to rabies virus. The OAS gene family was relatively conserved during evolution. Conserved genes are able to provide better maintenance of gene function. The rate of gene evolution and the number of positively selected sites combine to influence the resistance of a species to viruses. The positive selection sites demonstrate the adaptive evolution of the OAS gene family to the environment. Adaptive evolution combined with conserved gene function improves resistance to viruses. Our findings offer insights into the molecular and functional evolution of the antiviral ability of Laurasian mammals.

Evolutionary Impacts of Pattern Recognition Receptor Genes on Carnivora Complex Habitat Stress Adaptation.

Many mammals develop specific immune responses owing to the changes in their ecological niche and diet that are essential for animal survival. However, pattern recognition receptors (PRRs) serve as the first line of defense in innate immunity and generate immune responses in the host. However, the evolutionary impacts on PRR genes in Carnivora are not well studied. Herein, we explored the evolution of 946 PRR gene sequences in 43 Carnivora species to elucidate the molecular mechanisms of carnivore adaptation to complex habitats. We found that the PRRs were relatively conserved, and different gene families showed different evolutionary patterns. PRRs were highly purified based on their overall roles in Carnivora species but interspersed with positive-selection patterns during evolution. Different niche types may have jointly driven the evolution of PRR genes. In particular, the selection pressure of toll-like receptor (TLR) 10 was relaxed in seven species with pseudogenes, which may have emerged during recent evolutionary events. We speculated that a "functional compensation" mechanism may exist for genes with overlapping functions in the TLR gene family. Additionally, TLR2, TLR4, NLRC5, and DECTIN1 were subject to positive selection in semi-aquatic species, and the adaptive evolution of these genes may have been related to the adaptation to semi-aquatic environments. In summary, our findings offer valuable insights into the molecular and functional evolution of PRR genes, which are important for immune adaptations in Carnivora.

Environmental factors and stochasticity affect the fungal community structures in the water and sediments of Hulun Lake, China.

Aquatic fungi form both morphologically and ecologically diverse communities. However, lake ecosystems are frequently overlooked as fungal habitats, despite the potentially important role of fungi in matter cycling and energy flow. Hulun Lake is a typical example of a seasonal glacial lake; however, previous studies have only focused on bacteria in this ecosystem. Therefore, in the current study, internal transcribed spacer ribosomal RNA (ITS rRNA) gene high-throughput sequencing was used to investigate the fungal communities in paired water and sediment samples from the Hulun Lake Basin in China. A significant difference was found between the fungal communities of the two sample types. Across all samples, we identified nine phyla, 30 classes, 78 orders, 177 families, and 307 genera. The dominant phyla in the lake were Ascomycota, Basidiomycota and Chytridiomycota. Our results show that both water and sediments have very high connectivity, are dominated by positive interactions, and have similar interaction patterns. The fungal community structures were found to be significantly affected by environmental factors (temperature, chemical oxygen demand, electrical conductivity, total phosphorus, and pH). In addition, the dispersal limitations of the fungi affected the structure of the fungal communities, and it was revealed that stochasticity is more important than deterministic mechanisms in influencing the structure and function of fungal communities. This study provides unique theoretical support for the study of seasonally frozen lake fungal communities and a scientific basis for the future management and protection of Hulun Lake.

Convergent evolution of the gut microbiome in marine carnivores.

The gut microbiome can help the host adapt to a variety of environments and is affected by many factors. Marine carnivores have unique habitats in extreme environments. The question of whether marine habitats surpass phylogeny to drive the convergent evolution of the gut microbiome in marine carnivores remains unanswered. In the present study, we compared the gut microbiomes of 16 species from different habitats. Principal component analysis (PCA) and principal coordinate analysis (PCoA) separated three groups according to their gut microbiomes: marine carnivores, terrestrial carnivores, and terrestrial herbivores. The alpha diversity and niche breadth of the gut microbiome of marine carnivores were lower than those of the gut microbiome of terrestrial carnivores and terrestrial herbivores. The gut microbiome of marine carnivores harbored many marine microbiotas, including those belonging to the phyla Planctomycetes, Cyanobacteria, and Proteobacteria, and the genus Peptoclostridium. Collectively, these results revealed that marine habitats drive the convergent evolution of the gut microbiome of marine carnivores. This study provides a new perspective on the adaptive evolution of marine carnivores.

Uncovering the Fecal Bacterial Communities of Sympatric Sika Deer (Cervus nippon) and Wapiti (Cervus canadensis).

Microbial symbiotic associations may be beneficial, neutral, or harmful to the host. Symbionts exploit the host space and nutrition or use hosts as carriers to spread to other environments. In order to investigate the fecal bacterial communities of wild sika deer (Cervus nippon) and wapiti (Cervus canadensis), this study aimed to sequence and explore the composition of, and similarity between, the fecal microbiota of sika deer and wapiti using high-throughput sequencing. The composition and relative abundance of fecal microbiota, alpha diversity, and differences in beta diversity between the two species were analyzed. We found that no pathogenic bacteria were present in large quantities in the hosts. The dominant bacterial phyla found in the two deer species were similar and included Firmicutes, Bacteroidetes, Proteobacteria, and Spirochaetes. Moreover, the deer also shared similar dominant genera, including the Rikenellaceae RC9 gut group, Ruminococcaceae_UCG-010, Ruminococcaceae_UCG-005, and Bacteroides. These results demonstrate that the sika deer and wapiti share a similar fecal microbiotal structure, probably due to their common diet and living environment, but there was some evidence of a difference at the species level. These analyses provide new insights into the health status of deer populations outside protected environments and offer a scientific framework for monitoring the health conditions of sika deer and wapiti.

High-Altitude Drives the Convergent Evolution of Alpha Diversity and Indicator Microbiota in the Gut Microbiomes of Ungulates.

Convergent evolution is an important sector of evolutionary biology. High-altitude environments are one of the extreme environments for animals, especially in the Qinghai Tibet Plateau, driving the inquiry of whether, under broader phylogeny, high-altitude factors drive the convergent evolution of Artiodactyla and Perissodactyla gut microbiomes. Therefore, we profiled the gut microbiome of Artiodactyla and Perissodactyla at high and low altitudes using 16S rRNA gene sequencing. According to cluster analyses, the gut microbiome compositions of high-altitude Artiodactyla and Perissodactyla were not grouped together and were far from those of low-altitude Artiodactyla and Perissodactyla. The Wilcoxon's test in high-altitude ungulates showed significantly higher Sobs and Shannon indices than in low-altitude ungulates. At the phylum level, Firmicutes and Patescibacteria were significantly enriched in the gut microbiomes of high-altitude ungulates, which also displayed a higher Firmicutes/Bacteroidetes value than low-altitude ungulates. At the family level, Ruminococcaceae, Christensenellaceae, and Saccharimonadaceae were significantly enriched in the gut microbiomes of high-altitude ungulates. Our results also indicated that the OH and FH groups shared two significantly enriched genera, Christensenellaceae_R_7_group and Candidatus_Saccharimonas. These findings indicated that a high altitude cannot surpass the order level to drive the convergent evolution of ungulate gut microbiome composition but can drive the convergent evolution of alpha diversity and indicator microbiota in the gut microbiome of ungulates. Overall, this study provides a novel perspective for understanding the adaptation of ungulates to high-altitude environments.

Adaptability and Evolution of Gobiidae: A Genetic Exploration.

The Gobiidae family occupy one of the most diverse habitat ranges of all fishes. One key reason for their successful colonization of different habitats is their ability to adapt to different energy demands. This energy requirement is related to the ability of mitochondria in cells to generate energy via oxidative phosphorylation (OXPHOS). Here, we assembled three complete mitochondrial genomes of Rhinogobius shennongensis, Rhinogobius wuyanlingensis, and Chaenogobius annularis. These mitogenomes are circular and include 13 protein-coding genes (PCGs), two rRNAs, 22 tRNAs, and one non-coding control region (CR). We used comparative mitochondrial DNA (mtDNA) genome and selection pressure analyses to explore the structure and evolutionary rates of Gobiidae mitogenomics in different environments. The CmC model showed that the ω ratios of all mtDNA PCGs were <1, and that the evolutionary rate of adenosine triphosphate 8 (atp8) was faster in Gobiidae than in other mitochondrial DNA PCGs. We also found evidence of positive selection for several sites of NADH dehydrogenase (nd) 6 and atp8 genes. Thus, divergent mechanisms appear to underlie the evolution of mtDNA PCGs, which might explain the ability of Gobiidae to adapt to diverse environments. Our study provides new insights on the adaptive evolution of Gobiidae mtDNA genome and molecular mechanisms of OXPHOS.

Comparative Analysis of the Gut Microbiota of Mongolian Gazelle (Procapra gutturosa) Under Fragmented Habitats.

The existence of man-made facilities such as pasture fences makes the grassland ecosystem fragmented and endangers the survival of local wild animals. The Mongolian gazelle is highly sensitive to hunting and habitat destruction, and is one of the most threatened artiodactyls in Eurasia. It provides a critical model to studying gut microbiota under fragmented habitats. Therefore, we applied metagenomics sequencing to analyze the gut microbiota communities and functions of Mongolian gazelle under fragmented habitats. The results demonstrated that there were no significant differences in gut microbial communities between the different groups at both the phylum and genus level. The functional analyses showed that the Mongolian gazelle in fragmented habitat had a stronger ability to degrade naphthalene, but their ability to absorb carbohydrates was weaker. This study provided fundamental information about the gut microbiota of Mongolian gazelle, and we recommend reducing habitat fragmentation to better protect the Mongolian gazelle.

Factors affecting seasonal variation of microbial community structure in Hulun Lake, China.

Microbial communities play an important role in water quality regulation and biogeochemical cycling in freshwater ecosystems. However, there is a lack of research on the seasonal variation in lake water microorganisms in cold environments. In this study, 16S rRNA gene high-throughput sequencing was used to explore the microbial community and its influencing factors in Hulun Lake water during different seasons. The results showed that Proteobacteria, Actinobacteria, and Bacteroidetes were the most important phyla in the microbial community of Hulun Lake, but they had significant seasonal differences in their distribution. In addition, significant seasonal differences were observed in the α diversity of microorganisms, with bacterial diversity being higher in winter than in summer. Changes in environmental variables were significantly correlated with changes in the microbial community, and the rapid changes in temperature, pH, and dissolved oxygen are potentially the major factors influencing seasonal bacterial diversity trends. The findings of the present study enhance our understanding of the microbial communities in alpine lake ecosystems and are of great significance for the management and protection of lake ecosystems.

Comparative Analyses of the Gut Microbiome of Two Fox Species, the Red Fox (Vulpes Vulpes) and Corsac Fox (Vulpes Corsac), that Occupy Different Ecological Niches.

The gut microbiome is integral for the host's living and environmental adaptation and crucially important for understanding host adaptive mechanisms. The red fox (Vulpes vulpes) dominates a wider ecological niche and more complicated habitat than that of the corsac fox (V. corsac). However, the adaptive mechanisms (in particular, the gut microbiome responsible for this kind of difference) are still unclear. Therefore, we investigated the gut microbiome of these two species in the Hulunbuir grassland, China, and evaluated their microbiome composition, function, and adaptive mechanisms. We profiled the gut microbiome and metabolism function of red and corsac foxes via 16S rRNA gene and metagenome sequencing. The foxes harbored species-specific microbiomes and functions that were related to ecological niche and habitat. The red fox had abundant Bacteroides, which leads to significant enrichment of metabolic pathways (K12373 and K21572) and enzymes related to chitin and carbohydrate degradation that may help the red fox adapt to a wider niche. The corsac fox harbored large proportions of Blautia, Terrisporobacter, and ATP-binding cassette (ABC) transporters (K01990, K02003, and K06147) that can help maintain corsac fox health, allowing it to live in harsh habitats. These results indicate that the gut microbiome of the red and corsac foxes may have different abilities which may provide these species with differing capabilities to adapt to different ecological niches and habitats, thus providing important microbiome data for understanding the mechanisms of host adaptation to different niches and habitats.

Evolutionary and dietary relationships of wild mammals based on the gut microbiome.

Gut microbiome influence the health and evolution of mammals and multiple factors modulate the structure and function of gut microbiome. However, the specific changes of the diets and phylogeny on the gut microbiome were unclear. Here, we compared the gut microbiome of 16 rare wild mammals. All data (>200G 16S rRNA gene sequences) were generated using a high-throughput sequencing platform. Firmicutes and Bacteroidetes were the most predominant phyla in all mammals. However, Proteobacteria was an additionally dominant phylum specifically detected in the microbiome of carnivores and omnivores. Moreover, the dominant phyla in canids were Firmicutes, Bacteroidetes, Proteobacteria, and Fusobacteria. Phylogenetic reconstructions based on the gut microbiome and mitochondrial genome of these mammals were similar. The impact of the host on the microbiome community composition was most evident when considering conspecific and congeneric relationships. Similarity clustering showed that the gut microbiome of herbivores was clustered together, and the other clade comprised both omnivores and carnivores. Collectively, these results revealed that phylogenetic relationships and diet have an important impact on the gut microbiome, and thus the gut microbiome community composition may reflect both the phylogenetic relationships and diets. This study provides valuable basic data to facilitate future efforts related to animal conservation and health.

High-altitude adaptation in vertebrates as revealed by mitochondrial genome analyses.

The high-altitude environment may drive vertebrate evolution in a certain way, and vertebrates living in different altitude environments might have different energy requirements. We hypothesized that the high-altitude environment might impose different influences on vertebrate mitochondrial genomes (mtDNA). We used selection pressure analyses and PIC (phylogenetic independent contrasts) analysis to detect the evolutionary rate of vertebrate mtDNA protein-coding genes (PCGs) from different altitudes. The results showed that the ratio of nonsynonymous/synonymous substitutions (dN/dS) in the mtDNA PCGs was significantly higher in high-altitude vertebrates than in low-altitude vertebrates. The seven rapidly evolving genes were shared by the high-altitude vertebrates, and only one positive selection gene (ND5 gene) was detected in the high-altitude vertebrates. Our results suggest the mtDNA evolutionary rate in high-altitude vertebrates was higher than in low-altitude vertebrates as their evolution requires more energy in a high-altitude environment. Our study demonstrates the high-altitude environment (low atmospheric O2 levels) drives vertebrate evolution in mtDNA PCGs.

Complete mitochondrial genome sequence and phylogenetic analysis of Arion ater (Stylommatophora: Arionidae).

Arion ater are a group of mollusks, almost all of which live in a dark and humid environment during their life cycle. In this study, the complete mitogenome of Arion ater was sequenced using next generation sequencing, analyzed and compared with other Stylommatophorans. The total length of the mitochondrial genome is 14,314 bp and consists of 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes, and 1 control region. The genome has a typical mitochondrial gene sequence of Arion. Phylogenetic analysis of the mitochondrial genomes of 27 species of Stylommatophora showed that the Arion ater, Arion rufus and Arion vulgaris forms a monophyletic group with other species.

Gut Microbiota in Dholes During Estrus.

The co-evolution of gut microbes and the host plays a vital role in the survival and reproduction of the host. The dhole (Cuon alpinus) has been listed as endangered species by the International Union for Conservation of Nature; therefore, conservation and effective breeding of dholes are essential. Effective estrus can promote reproduction. However, little is known about the relative contribution of estrus in shaping the structure and the functions of fecal microbiota. Here, we investigated the potential association between estrus and the fecal microbiota in dholes using shotgun metagenomic sequencing. We found that the estrus stages in dholes vary significantly in terms of gut bacterial composition and microbiome metabolism and function. Compared with that of non-estrus, adult dholes, the microbiome of estrus adult dholes had a significantly higher abundance of Bacillus faecalis and Veillonella, which play a key role in the synthesis of sex hormones and nucleic acids, energy production, and reproductive cell division. The insulin and energy metabolism-related pathways are significantly enhanced in the gut microbes and the related gluconeogenic enzymes are significantly enriched during estrus. These findings suggest that the structure and metagenome of the fecal microbiome during estrus have a significant effect in promoting estrus in dholes, thus providing a new perspective for dhole conservation.