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ABSTRACT The gut microbiome is a symbiotic microbial community associated with the host and plays multiple important roles in host physiology, nutrition, and health. A number of factors have been shown to influence the gut microbiome, among which diet is considered to be one of the most important; however, the relationship between diet composition and gut microbiota in wild mammals is still not well recognized. Herein, we characterized the gut microbiota of bats and examined the effects of diet, host taxa, body size, gender, elevation, and latitude on the gut microbiota. The cytochrome C oxidase subunit I (COI) gene and 16S rRNA gene amplicons were sequenced from the feces of eight insectivorous bat species in southern China, includingMiniopterus fuliginosus,Aselliscus stoliczkanus,Myotis laniger,Rhinolophus episcopus,Rhinolophus osgoodi,Rhinolophus ferrumequinum,Rhinolophus affinis,andRhinolophus pusillus. The results showed that the composition of gut microbiome and diet exhibited significant differences among bat species. Diet composition and gut microbiota were significantly correlated at the order, family, genus, and operational taxonomic unit levels, while certain insects had a marked effect on the gut microbiome at specific taxonomic levels. In addition, elevation, latitude, body weight of bats, and host species had significant effects on the gut microbiome, but phylosymbiosis between host phylogeny and gut microbiome was lacking. These findings clarify the relationship between gut microbiome and diet and contribute to improving our understanding of host ecology and the evolution of the gut microbiome in wild mammals. IMPORTANCEThe gut microbiome is critical for the adaptation of wildlife to the dynamic environment. Bats are the second-largest group of mammals with short intestinal tract, yet their gut microbiome is still poorly studied. Herein, we explored the relationships between gut microbiome and food composition, host taxa, body size, gender, elevation, and latitude. We found a significant association between diet composition and gut microbiome in insectivorous bats, with certain insect species having major impacts on gut microbiome. Factors like species taxa, body weight, elevation, and latitude also affected the gut microbiome, but we failed to detect phylosymbiosis between the host phylogeny and the gut microbiome. Overall, our study presents novel insights into how multiple factors shape the bat’s gut microbiome together and provides a study case on host-microbe interactions in wildlife. 

Differences in gene expression within tissues can lead to differences in tissue function. Understanding the transcriptome of a species helps elucidate the molecular mechanisms underlying phenotypic divergence. According to the presence or absence of a reference genome of for a studied species, transcriptome analyses can be divided into reference‑based and reference‑free methods, respectively. Presently, comparisons of complete transcriptome analysis results between those two methods are still rare. In this study, we compared the cochlear transcriptome analysis results of greater horseshoe bats (Rhinolophus ferrumequinum) from three lineages in China with different acoustic phenotypes using reference‑based and reference‑free methods to explore their differences in subsequent analysis. The results gained by reference-based results had lower false-positive rates and were more accurate because differentially expressed genes among the three populations obtained by this method had greater reliability and a higher annotation rate. Some phenotype-related enrichment terms, including those related to inorganic molecules and proton transmembrane channels, were also obtained only by the reference-based method. However, the reference‑based method might have the limitation of incomplete information acquisition. Thus, we believe that a combination of reference‑free and reference‑based methods is ideal for transcriptome analyses. The results of our study provided a reference for the selection of transcriptome analysis methods in the future. 

In the process of species differentiation and adaption, the relative influence of natural selection on gene expression variation often remains unclear (especially its impact on phenotypic divergence). In this study, we used differentially expressed genes from brain, cochlea, and liver samples collected from two species of bats to determine the gene expression variation forced by natural selection when comparing at the interspecific (Rhinolophus siamensis and R. episcopus episcopus) and the intraspecific (R. e. episcopus and R. episcopus spp.) levels. In both cases, gene expression variation was extensively adaptive (>66.0%) and mainly governed by directional selection, followed by stabilizing selection, and finally balancing selection. The expression variation related to acoustic signals (resting frequency, RF) and body size (forearm length, FA) was also widely governed by natural selection (>69.1%). Different functional patterns of RF- or FA-related adaptive expression variation were found between the two comparisons, which manifested as abundant immune-related regulations between subspecies (indicating a relationship between immune response and phenotypic adaption). Our study verifies the extensive adaptive expression variation between both species and subspecies and provides insight into the effects of natural selection on species differentiation and adaptation as well as phenotypic divergence at the expression level. 

Abstract Background In genus Rhinolophus , species in the Rhinolophus philippinensis and R. macrotis groups are unique because the horseshoe bats in these group have relatively low echolocation frequencies and flight speeds compared with other horseshoe bats with similar body size. The different characteristics among bat species suggest particular evolutionary processes may have occurred in this genus. To study the adaptive evidence in the mitochondrial genomes (mitogenomes) of rhinolophids, especially the mitogenomes of the species with low echolocation frequencies, we sequenced eight mitogenomes and used them for comparative studies of molecular phylogeny and adaptive evolution. Results Phylogenetic analysis using whole mitogenome sequences produced robust results and provided phylogenetic signals that were better than those obtained using single genes. The results supported the recent establishment of the separate macrotis group. The signals of adaptive evolution discovered in the Rhinolophus species were tested for some of the codons in two genes ( ND2 and ND6 ) that encode NADH dehydrogenases in oxidative phosphorylation system complex I. These genes have a background of widespread purifying selection. Signals of relaxed purifying selection and positive selection were found in ND2 and ND6 , respectively, based on codon models and physicochemical profiles of amino acid replacements. However, no pronounced overlap was found for non-synonymous sites in the mitogenomes of all the species with low echolocation frequencies. A signal of positive selection for ND5 was found in the branch-site model when R. philippinensis was set as the foreground branch. Conclusions The mitogenomes provided robust phylogenetic signals that were much more informative than the signals obtained using single mitochondrial genes. Two mitochondrial genes that encoding proteins in the oxidative phosphorylation system showed some evidence of adaptive evolution in genus Rhinolophus and the positive selection signals were tested for ND5 in R. philippinensis . These results indicate that mitochondrial protein-coding genes were targets of adaptive evolution during the evolution of Rhinolophus species, which might have contributed to a diverse range of acoustic adaptations in this genus. 

Disease outbreaks and pathogen introductions can have significant effects on host populations, and the ability of pathogens to persist in the environment can exacerbate disease impacts by fueling sustained transmission, seasonal epidemics, and repeated spillover events. While theory suggests that the presence of an environmental reservoir increases the risk of host declines and threat of extinction, the influence of reservoir dynamics on transmission and population impacts remains poorly described. Here we show that the extent of the environmental reservoir explains broad patterns of host infection and the severity of disease impacts of a virulent pathogen. We examined reservoir and host infection dynamics and the resulting impacts of Pseudogymnoascus destructans , the fungal pathogen that causes white-nose syndrome, in 39 species of bats at 101 sites across the globe. Lower levels of pathogen in the environment consistently corresponded to delayed infection of hosts, fewer and less severe infections, and reduced population impacts. In contrast, an extensive and persistent environmental reservoir led to early and widespread infections and severe population declines. These results suggest that continental differences in the persistence or decay of P. destructans in the environment altered infection patterns in bats and influenced whether host populations were stable or experienced severe declines from this disease. Quantifying the impact of the environmental reservoir on disease dynamics can provide specific targets for reducing pathogen levels in the environment to prevent or control future epidemics. 

Summary White‐nose syndrome, a disease that is caused by the psychrophilic fungusPseudogymnoascus destructans, has threatened several North America bat species with extinction. Recent studies have shown that East Asian bats are infected withP. destructansbut show greatly reduced infections. While several factors have been found to contribute to these reduced infections, the role of specific microbes in limitingP. destructansgrowth remains unexplored. We isolated three bacterial strains with the ability to inhibitP. destructans, namely,Pseudomonas yamanorumGZD14026,Pseudomonas brenneriXRD11711 andPseudomonas fragiGZD14479, from bats in China.Pseudomonas yamanorum, with the highest inhibition score, was selected to extract antifungal active substance. Combining mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy analyses, we identified the active compound inhibitingP. destructansas phenazine‐1‐carboxylic acid (PCA), and the minimal inhibitory concentration (MIC) was 50.12 μg ml⁻¹. Whole genome sequencing also revealed the existence of PCA biosynthesis gene clusters. Gas chromatography‐mass spectrometry (GC‐MS) analysis identified volatile organic compounds. The results indicated that 10 ppm octanoic acid, 100 ppm 3‐tert‐butyl‐4‐hydroxyanisole (isoprenol) and 100 ppm 3‐methyl‐3‐buten‐1‐ol (BHA) inhibited the growth ofP. destructans. These results support that bacteria may play a role in limiting the growth ofP. destructanson bats.