Diversification and introgression in four chromosomal taxa of the Pearson's horseshoe bat (Rhinolophus pearsoni) group.

Chromosomal variation among closely related taxa is common in both plants and animals, and can reduce rates of introgression as well as promote reproductive isolation and speciation. In mammals, studies relating introgression to chromosomal variation have tended to focus on a few model systems and typically characterized levels of introgression using small numbers of loci. Here we took a genome-wide approach to examine how introgression rates vary among four closely related horseshoe bats (Rhinolophus pearsoni group) that possess different diploid chromosome numbers (2n = 42, 44, 46, and 60) resulting from Robertsonian (Rb) changes (fissions/fusions). Using a sequence capture we obtained orthologous loci for thousands of nuclear loci, as well as mitogenomes, and performed phylogenetic and population genetic analyses. We found that the taxon with 2n = 60 was the first to diverge in this group, and that the relationships among the three other taxa (2n = 42, 44 and 46) showed discordance across our different analyses. Our results revealed signatures of multiple ancient introgression events between the four taxa, with evidence of mitonuclar discordance in phylogenetic trees and reticulation events in their evolutionary history. Despite this, we found no evidence of recent and/or ongoing introgression between taxa. Overall, our results indicate that the effects of Rb changes on the reduction of introgression are complicated and that these may contribute to reproductive isolation and speciation in concert with other factors (e.g. phenotypic and genic divergence).

Mitonuclear mismatch alters nuclear gene expression in naturally introgressed Rhinolophus bats.

BACKGROUND: Mitochondrial function involves the interplay between mitochondrial and nuclear genomes. Such mitonuclear interactions can be disrupted by the introgression of mitochondrial DNA between taxa or divergent populations. Previous studies of several model systems (e.g. Drosophila) indicate that the disruption of mitonuclear interactions, termed mitonuclear mismatch, can alter nuclear gene expression, yet few studies have focused on natural populations. RESULTS: Here we study a naturally introgressed population in the secondary contact zone of two subspecies of the intermediate horseshoe bat (Rhinolophus affinis), in which individuals possess either mitonuclear matched or mismatched genotypes. We generated transcriptome data for six tissue types from five mitonuclear matched and five mismatched individuals. Our results revealed strong tissue-specific effects of mitonuclear mismatch on nuclear gene expression with the largest effect seen in pectoral muscle. Moreover, consistent with the hypothesis that genes associated with the response to oxidative stress may be upregulated in mitonuclear mismatched individuals, we identified several such gene candidates, including DNASE1L3, GPx3 and HSPB6 in muscle, and ISG15 and IFI6 in heart. CONCLUSION: Our study reveals how mitonuclear mismatch arising from introgression in natural populations is likely to have fitness consequences. Underlying the processes that maintain mitonuclear discordance is a step forward to understand the role of mitonuclear interactions in population divergence and speciation.

Echolocation call frequency variation in horseshoe bats: molecular basis revealed by comparative transcriptomics.

Recently diverged taxa with contrasting phenotypes offer opportunities for unravelling the genetic basis of phenotypic variation in nature. Horseshoe bats are a speciose group that exhibit a derived form of high-duty cycle echolocation in which the inner ear is finely tuned to echoes of the narrowband call frequency. Here, by focusing on three recently diverged subspecies of the intermediate horseshoe bat (Rhinolophus affinis) that display divergent echolocation call frequencies, we aim to identify candidate loci putatively involved in hearing frequency variation. We used de novo transcriptome sequencing of two mainland taxa (himalayanus and macrurus) and one island taxon (hainanus) to compare expression profiles of thousands of genes. By comparing taxa with divergent call frequencies (around 15 kHz difference), we identified 252 differentially expressed genes, of which six have been shown to be involved in hearing or deafness in human/mouse. To obtain further validation of these results, we applied quantitative reverse transcription-PCR to the candidate gene FBXL15 and found a broad association between the level of expression and call frequency across taxa. The genes identified here represent strong candidate loci associated with hearing frequency variation in bats.

Genome-wide data reveal discordant mitonuclear introgression in the intermediate horseshoe bat (Rhinolophus affinis).

Closely related taxa often exhibit mitonuclear discordance attributed to introgression of mitochondrial DNA (mtDNA), yet few studies have considered the underlying causes of mtDNA introgression. Here we test for demographic versus adaptive processes as explanations for mtDNA introgression in three subspecies of the intermediate horseshoe bat (Rhinolophus affinis). We generated sequences of 1692 nuclear genes and 13 mitochondrial protein-coding genes for 48 individuals. Phylogenetic reconstructions based on 320 exon sequences and 2217 single nucleotide polymorphisms (SNPs) both revealed conflicts between the species tree and mtDNA tree. These results, together with geographic patterns of mitonuclear discordance, and shared identical or near-identical mtDNA sequences, suggest extensive introgression of mtDNA between the two parapatric mainland subspecies. Under demographic hypotheses, we would also expect to uncover traces of ncDNA introgression, however, population structure and gene flow analyses revealed little nuclear admixture. Furthermore, we found inconsistent estimates of the timing of population expansion and that of the most recent common ancestor for the clade containing introgressed haplotypes. Without a clear demographic explanation, we also examined whether introgression likely arises from adaptation. We found that five mtDNA genes contained fixed amino acid differences between introgressed and non-introgressed individuals, including putative positive selection found in one codon, although this did not show introgression. While our evidence for rejecting demographic hypotheses is arguably stronger than that for rejecting adaptation, we find no definitive support for either explanation. Future efforts will focus on larger-scale resequencing to decipher the underlying causes of discordant mitonuclear introgression in this system.

Gut transcriptomic changes during hibernation in the greater horseshoe bat (Rhinolophus ferrumequinum).

BACKGROUND: The gut is the major organ for nutrient absorption and immune response in the body of animals. Although effects of fasting on the gut functions have been extensively studied in model animals (e.g. mice), little is known about the response of the gut to fasting in a natural condition (e.g. hibernation). During hibernation, animals endure the long term of fasting and hypothermia. RESULTS: Here we generated the first gut transcriptome in a wild hibernating bat (Rhinolophus ferrumequinum). We identified 1614 differentially expressed genes (DEGs) during four physiological states (Torpor, Arousal, Winter Active and Summer Active). Gene co-expression network analysis assigns 926 DEGs into six modules associated with Torpor and Arousal. Our results reveal that in response to the stress of luminal nutrient deficiency during hibernation, the gut helps to reduce food intake by overexpressing genes (e.g. CCK and GPR17) that regulate the sensitivity to insulin and leptin. At the same time, the gut contributes energy supply by overexpressing genes that increase capacity for ketogenesis (HMGCS2) and selective autophagy (TEX264). Furthermore, we identified separate sets of multiple DEGs upregulated in Torpor and Arousal whose functions are involved in innate immunity. CONCLUSION: This is the first gut transcriptome of a hibernating mammal. Our study identified candidate genes associated with regulation of food intake and enhance of innate immunity in the gut during hibernation. By comparing with previous studies, we found that two DEGs (CPE and HSPA8) were also significantly elevated during torpor in liver and brain of R. ferrumequinum and several DEGs (e.g. TXNIP and PDK1/4) were commonly upregulated during torpor in multiple tissues of different mammals. Our results support that shared expression changes may underlie the hibernation phenotype by most mammals.

Resolving evolutionary relationships among six closely related taxa of the horseshoe bats (Rhinolophus) with targeted resequencing data.

Recently diverged taxa are often characterised by high rates of introgressive hybridization and incomplete lineage sorting, both of which can complicate phylogenetic reconstructions of species histories. Here we use a sequence capture approach to obtain genome-wide data to resolve the evolutionary relationships, and infer the extent and timescale of hybridization and introgression events, among six recently diverged taxa of the horseshoe bat species complexes Rhinolophus sinicus and R. thomasi. We show that two different methods of species tree reconstruction applied to a set of ~1500 nuclear loci all recover species trees with similar topologies, differing from the previous phylogeny based on two nuclear loci. By comparing the tree topology obtained from the nuclear loci with that inferred from the mitochondrial genome, we observed at least three cases of conflict, each of which likely results from past introgression. Of these, the occurrence of a highly similar mitogenome sequence shared by individuals of two taxa in a sympatric region points to very recent mtDNA introgression. The other cases are characterised by greater divergence and strong phylogeographic structure among putative introgressed individuals and their source populations, and thus likely reflect more ancient hybridization events. These results also suggest that two of the subspecies (R. s. septentrionalis and the undescribed taxon R. s. ssp) are likely to represent full species, warranting full taxonomic descriptions. This work adds a growing number of studies showing the potential problems of relying solely on mitochondrial sequences, or a limited number of loci, to infer phylogenetic relationships among recently diverged taxa.

Differential introgression among loci across a hybrid zone of the intermediate horseshoe bat (Rhinolophus affinis).

BACKGROUND: Hybrid zones formed by the secondary contact of divergent lineages represent natural laboratories for studying the genetic basis of speciation. Here we tested for patterns of differential introgression among three X-linked and 11 autosomal regions to identify candidate loci related to either reproductive isolation or adaptive introgression across a hybrid zone between two Chinese mainland subspecies of the intermediate horseshoe bat Rhinolophus affinis: R. a. himalayanus and R. a. macrurus. RESULTS: Our results support the previous suggestion that macrurus formed when a third subspecies (R. a. hainanus) recolonized the mainland from Hainan Island, and that himalayanus is the ancestral taxon. However, this overall evolutionary history was not reflected in all loci examined, with considerable locus-wise heterogeneity seen in gene tree topologies, levels of polymorphism, genetic differentiation and rates of introgression. Coalescent simulations suggested levels of lineage mixing seen at some nuclear loci might result from incomplete lineage sorting. Isolation with migration models supported evidence of gene flow across the hybrid zone at one intronic marker of the hearing gene Prestin. CONCLUSIONS: We suggest that phylogenetic discordance with respect to the species tree seen here is likely to arise via a combination of incomplete lineage sorting and a low incidence of introgression although we cannot rule out other explanations such as selection and recombination. Two X-linked loci and one autosomal locus were identified as candidate regions related to reproductive isolation across the hybrid zone. Our work highlights the importance of including multiple genomic regions in characterizing patterns of divergence and gene flow across a hybrid zone.

Introgression of mitochondrial DNA promoted by natural selection in the Japanese pipistrelle bat (Pipistrellus abramus).

Introgression of mitochondrial DNA (mtDNA) between closely related taxa can be promoted by either neutral processes or natural selection. Since mitochondrial gene-encoded proteins play critical roles in oxidative metabolism, mtDNA genes are commonly considered to experience strong selective constraint. However, metabolic requirements vary across climatic and ecological gradients, thus modifying potential selective pressures acting on mtDNA genes. Here we conducted tests to detect adaptive evolution occurring in two mtDNA genes (Cytb and ND5) in individuals of Japanese pipistrelle bat (Pipistrellus abramus) across the mainland of China and Hainan Island. Nuclear DNA markers identified two clades in both the mainland and Hainan Island populations, whereas each of these regions had a specific mtDNA clade. This cyto-nuclear discordance is most likely caused by introgression of the mtDNA by ruling out two other alternative scenarios (incomplete lineage sorting and sex-biased gene flow). Although population-based analyses revealed purifying selection acting on Cytb and neutrality in ND5, multiple nonsynonymous substitutions in both Cytb and ND5 were suggested to have been caused by positive selection by a divergence-based analysis. Our study supports the view that molecular adaptation can occur at genes under strong purifying selection if nonsynonymous substitutions cause radical changes in the physicochemical properties of amino acids.

Sex Differences in Cochlear Transcriptomes in Horseshoe Bats.

Sexual dimorphism of calls is common in animals, whereas studies on the molecular basis underlying this phenotypic variation are still scarce. In this study, we used comparative transcriptomics of cochlea to investigate the sex-related difference in gene expression and alternative splicing in four Rhinolophus taxa. Based on 31 cochlear transcriptomes, we performed differential gene expression (DGE) and alternative splicing (AS) analyses between the sexes in each taxon. Consistent with the degree of difference in the echolocation pulse frequency between the sexes across the four taxa, we identified the largest number of differentially expressed genes (DEGs) and alternatively spliced genes (ASGs) in R. sinicus. However, we also detected multiple DEGs and ASGs in taxa without sexual differences in echolocation pulse frequency, suggesting that these genes might be related to other parameters of echolocation pulse rather than the frequency component. Some DEGs and ASGs are related to hearing loss or deafness genes in human or mice and they can be considered to be candidates associated with the sexual differences of echolocation pulse in bats. We also detected more than the expected overlap of DEGs and ASGs in two taxa. Overall, our current study supports the important roles of both DGE and AS in generating or maintaining sexual differences in animals.

Complexin-1 enhances ultrasound neurotransmission in the mammalian auditory pathway.

Unlike megabats, which rely on well-developed vision, microbats use ultrasonic echolocation to navigate and locate prey. To study ultrasound perception, here we compared the auditory cortices of microbats and megabats by constructing reference genomes and single-nucleus atlases for four species. We found that parvalbumin (PV)+ neurons exhibited evident cross-species differences and could respond to ultrasound signals, whereas their silencing severely affected ultrasound perception in the mouse auditory cortex. Moreover, megabat PV+ neurons expressed low levels of complexins (CPLX1-CPLX4), which can facilitate neurotransmitter release, while microbat PV+ neurons highly expressed CPLX1, which improves neurotransmission efficiency. Further perturbation of Cplx1 in PV+ neurons impaired ultrasound perception in the mouse auditory cortex. In addition, CPLX1 functioned in other parts of the auditory pathway in microbats but not megabats and exhibited convergent evolution between echolocating microbats and whales. Altogether, we conclude that CPLX1 expression throughout the entire auditory pathway can enhance mammalian ultrasound neurotransmission.

Historical introgression and the persistence of ghost alleles in the intermediate horseshoe bat (Rhinolophus affinis).

Phylogenetic conflicts between genetic markers can help to disentangle complex histories of phylogeography and introgression among taxa. We previously proposed that the Chinese mainland subspecies of the intermediate horseshoe bat Rhinolophus affinis himalayanus colonized Hainan Island to form the subspecies R. a. hainanus. Subsequent recolonization of the mainland formed a third taxon, R. a macrurus, and a secondary contact zone with the ancestral himalayanus. To test for historical and recurrent genetic exchange between these mainland subspecies, we sampled populations of each from two parapatric zones and undertook analyses using one mtDNA marker, three nuclear genes and 14 microsatellites. Nuclear DNA, echolocation call frequencies and morphological data all recovered two taxa; however, a mtDNA phylogeny revealed two himalayanus clades, of which one clustered with macrurus, as well as some shared or related mtDNA haplotypes in eastern populations. Isolation-with-migration (IM) models suggested some mtDNA gene flow from macrurus to himalayanus. However, strong population structure in himalayanus raises the possibility that macrurus captured mtDNA from a coastal population of himalayanus that has since become rare or extinct. To reconcile these two sets of results, we suggest that the IM estimates might reflect historical mtDNA gene flow among populations of himalayanus, before mtDNA was subsequently captured by macrurus. Finally, microsatellite-based ABC analyses supported the island origin of macrurus; however, mtDNA-based ABC analyses suggest this taxon might have evolved on the mainland. Our findings highlight the importance of understanding population history and structure for interpreting hybridization and introgression events.

Sex differences in gene expression and alternative splicing in the Chinese horseshoe bat.

Sexually dimorphic traits are common in sexually reproducing organisms and can be encoded by differential gene regulation between males and females. Although alternative splicing is common mechanism in generating transcriptional diversity, its role in generating sex differences relative to differential gene expression is less clear. Here, we investigate the relative roles of differential gene expression and alternative splicing between male and female the horseshoe bat species, Rhinolophus sinicus. Horseshoe bats are an excellent model to study acoustic differences between sexes. Using RNA-seq analyses of two somatic tissues (brain and liver) from males and females of the same population, we identified 3,471 and 2,208 differentially expressed genes between the sexes (DEGs) in the brain and liver, respectively. DEGs were enriched with functional categories associated with physiological difference of the sexes (e.g.,gamete generation and energy production for reproduction in females). In addition, we also detected many differentially spliced genes between the sexes (DSGs, 2,231 and 1,027 in the brain and liver, respectively) which were mainly involved in regulation of RNA splicing and mRNA metabolic process. Interestingly, we found a significant enrichment of DEGs on the X chromosome, but not for DSGs. As for the extent of overlap between the two sets of genes, more than expected overlap of DEGs and DSGs was observed in the brain but not in the liver. This suggests that more complex tissues, such as the brain, may require the intricate and simultaneous interplay of both differential gene expression and splicing of genes to govern sex-specific functions. Overall, our results support that variation in gene expression and alternative splicing are important and complementary mechanisms governing sex differences.

Chromosome-level genome assembly of the Stoliczka's Asian trident bat (Aselliscus stoliczkanus).

Stoliczka's Asian trident bat (Aselliscus stoliczkanus) is a small-bodied species and very sensitive to climate change. Here, we presented a chromosome-level genome assembly of A. stoliczkanus by combining Illumina sequencing, Nanopore sequencing and high-throughput chromatin conformation capture (Hi-C) sequencing technology. The genome assembly was 2.18 Gb in size with 98.26% of the genome sequences anchored onto 14 autosomes and two sex chromosomes (X and Y). The quality of the genome assembly is very high with a contig and scaffold N50 of 72.98 and 162 Mb, respectively, Benchmarking Universal Single-Copy Orthologs (BUSCO) score of 96.6%, and the consensus quality value (QV) of 47.44. A total of 20,567 genes were predicted and 98.8% of these genes were functionally annotated. Syntenic blocks between A. stoliczkanus and Homo sapiens, together with previous comparative cytogenetic studies, provide valuable foundations for further comparative genomic and cytogenetic studies in mammals. The reference-quality genome of A. stoliczkanus contributes an important resource for conservative genomics and landscape genomics in predicting adaptation and vulnerability to climate change.

Impacts of seasonality on gene expression in the Chinese horseshoe bat.

Seasonality can cause changes in many environmental factors which potentially affects gene expression. Here, we used a bat species (Rhinolophus sinicus) from eastern China as a model to explore the molecular mechanisms of seasonal effects, in particular during phenological shifts in the spring and autumn. Based on the analysis of 45 RNA-seq samples, we found strong seasonal effects on gene expression, with a large number of genes identified as either specific or biased to each season. Weighted gene co-expression network analysis also identified multiple modules significantly associated with each season. These seasonal genes were further enriched into different functional categories. Consistent with effects of phenological shifts on bats, we found that genes related to promoting food intake were highly expressed in both autumn and spring. In addition, immunity genes were also highly expressed in both seasons although this seasonal immune response had tissue specificity in different seasons. In female bats, genes related to the delay of ovulation (e.g., NPPC, natriuretic peptide precursor type C) were highly expressed in October, while genes associated with the promotion of reproduction (e.g., DIO2, iodothyronine deiodinase 2) were biasedly expressed in April. Lastly, we found multiple known core clock genes in both October-biased and April-biased expressed genes, which may be involved in regulating the start and end of hibernation, respectively. Overall, together with studies in other land and aquatic animals, our work supports that seasonal gene expression variations may be a general evolutionary response to environmental changes in wild animals.

Characterization of microRNA and gene expression in the cochlea of an echolocating bat (Rhinolophus affinis).

MicroRNAs (miRNAs) are important post-transcriptional regulators of gene expression and play key roles in many biological processes, such as development and response to multiple stresses. However, little is known about their roles in generating novel phenotypes and phenotypic variation during the course of animal evolution. Here, we, for the first time, characterized the miRNAs of the cochlea in an echolocating bat (Rhinolophus affinis). We sampled eight individuals from two R. affinis subspecies with significant echolocation call frequency differences. We identified 365 miRNAs and 121 of them were novel. By searching sequences of these miRNAs precursors in multiple high-quality mammal genomes, we found one specific miRNA shared by all echolocating bats but not present in all other nonecholocating mammals. The targeted genes of this miRNA included several known hearing genes (e.g., KCNQ4 and GJB6). Together with the matched mRNA-seq data, we identified 1766 differentially expressed genes (DEGs) between the two subspecies and 555 of them were negatively regulated by differentially expressed miRNAs (DEMs). We found that almost half of known hearing genes in the list of all DEGs were regulated negatively by DEMs, suggesting an important role of miRNAs in call frequency variation of the two subspecies. These targeted DEGs included several important hearing genes (e.g., Piezo1, Piezo2, and CDH23) that have been shown to be important in ultrasonic hearing of echolocating mammals.

Extensive alternative splicing triggered by mitonuclear mismatch in naturally introgressed Rhinolophus bats.

Mitochondrial function needs strong interactions of mitochondrial and nuclear (mitonuclear) genomes, which can be disrupted by mitonuclear mismatch due to mitochondrial DNA (mtDNA) introgression between two formerly isolated populations or taxa. This mitonuclear disruption may cause severe cellular stress in mismatched individuals. Gene expression changes and alternative splicing (AS) are two important transcriptional regulations to respond to environmental or cellular stresses. We previously identified a naturally introgressed population in the intermediate horseshoe bat (Rhinolophus affinis). Individuals from this population belong to R. a. himalayanus and share almost identical nuclear genetic background; however, some of them had mtDNA from another subspecies (R. a. macrurus). With this unique natural system, we examined gene expression changes in six tissues between five mitonuclear mismatched and five matched individuals. A small number of differentially expressed genes (DEGs) were identified, and functional enrichment analysis revealed that most DEGs were related to immune response although some may be involved in response to oxidative stress. In contrast, we identified extensive AS events and alternatively spliced genes (ASGs) between mismatched and matched individuals. Functional enrichment analysis revealed that multiple ASGs were directly or indirectly associated with energy production in mitochondria which is vital for survival of organism. To our knowledge, this is the first study to examine the role of AS in responding to cellular stress caused by mitonuclear mismatch in natural populations. Our results suggest that AS may play a more important role than gene expression regulation in responding to severe environmental or cellular stresses.

The complete mitochondrial genome of Rhinolophus affinis himalayanus.

Here we generated the complete mitochondrial genome of one subspecies of R. affinis (R. affinis himalayanus) using next generation sequencing and Sanger sequencing. The length of the complete mitochondrial genome was 16,886 bp, containing 13 protein-coding genes, 22 tRNAs, 2 rRNAs, and a non-coding control region. A maximum-likelihood tree based on the 13 concatenated mitochondrial protein-coding genes of 16 Rhinolophus taxon and one outgroup Hipposideros armiger indicates that R. affinis shows a closer relationship with R. sinicus complex than with other taxa.

Historical male-mediated introgression in horseshoe bats revealed by multilocus DNA sequence data.

Instances of hybridization between mammalian taxa in the wild are rarely documented. To test for introgression between sibling species of horseshoe bat (Rhinolophus yunanensis and R. pearsoni) and two subspecies of the latter (R. p. pearsoni and R. p. chinensis), we sequenced two mtDNA and two ncDNA markers in individuals sampled from multiple localities within their overlapping ranges. The interspecific mtDNA gene tree corresponded to the expected taxonomic divisions, and coalescent-based analyses suggested divergence occurred around 4 MYA. However, these relationships strongly conflicted with those recovered from two independent nuclear gene trees, in which R. yunanensis clustered with R. p. pearsoni to the exclusion of R. p. chinensis. This geographically widespread discordance is best explained by large-scale historical introgression of ncDNA from R. yunanensis to R. pearsoni by male-mediated exchange in mixed species colonies during Pleistocene glacial periods, when ranges may have contracted and overlapped more than at present. Further species tree-gene tree conflicts were detected between R. p. pearsoni and R. p. chinensis, also indicating past and/or current introgression in their overlapping regions. However, here the patterns point to asymmetric mtDNA introgression without ncDNA introgression. Analyses of coalescence times indicate this exchange has occurred subsequent to the divergence of these subspecies from their common ancestor. Our work highlights the importance of using multiple data sets for reconstructing phylogeographic histories and resolving taxonomic relationships.

Transcriptome sequencing of cochleae from constant-frequency and frequency-modulated echolocating bats.

Echolocating bats are fascinating for their ability to 'see' the world in the darkness. Ultrahigh frequency hearing is essential for echolocation. In this study we collected cochlear tissues from constant-frequency (CF) bats (two subspecies of Rhinolophus affinis, Rhinolophidae) and frequency-modulated (FM) bats (Myotis ricketti, Vespertilionidae) and applied PacBio single-molecule real-time isoform sequencing (Iso-seq) technology to generate the full-length (FL) transcriptomes for the three taxa. In total of 10103, 9676 and 10504 non-redundant FL transcripts for R. a. hainanus, R. a. himalayanus and Myotis ricketti were obtained respectively. These data present a comprehensive list of transcripts involved in ultrahigh frequency hearing of echolocating bats including 26342 FL transcripts, 24833 of which are annotated by public databases. No further comparative analyses were performed on the current data in this study. This data can be reused to quantify gene or transcript expression, assess the level of alternative splicing, identify novel transcripts and improve genome annotation of bat species.