Identification of non-model mammal species using the MinION DNA sequencer from Oxford Nanopore.

Background: The Neotropics harbors the largest species richness of the planet; however, even in well-studied groups, there are potentially hundreds of species that lack a formal description, and likewise, many already described taxa are difficult to identify using morphology. Specifically in small mammals, complex morphological diagnoses have been facilitated by the use of molecular data, particularly from mitochondrial sequences, to obtain accurate species identifications. Obtaining mitochondrial markers implies the use of PCR and specific primers, which are largely absent for non-model organisms. Oxford Nanopore Technologies (ONT) is a new alternative for sequencing the entire mitochondrial genome without the need for specific primers. Only a limited number of studies have employed exclusively ONT long-reads to assemble mitochondrial genomes, and few studies have yet evaluated the usefulness of such reads in multiple non-model organisms. Methods: We implemented fieldwork to collect small mammals, including rodents, bats, and marsupials, in five localities in the northern extreme of the Cordillera Central of Colombia. DNA samples were sequenced using the MinION device and Flongle flow cells. Shotgun-sequenced data was used to reconstruct the mitochondrial genome of all the samples. In parallel, using a customized computational pipeline, species-level identifications were obtained based on sequencing raw reads (Whole Genome Sequencing). ONT-based identifications were corroborated using traditional morphological characters and phylogenetic analyses. Results: A total of 24 individuals from 18 species were collected, morphologically identified, and deposited in the biological collection of Universidad EAFIT. Our different computational pipelines were able to reconstruct mitochondrial genomes from exclusively ONT reads. We obtained three new mitochondrial genomes and eight new molecular mitochondrial sequences for six species. Our species identification pipeline was able to obtain accurate species identifications for up to 75% of the individuals in as little as 5 s. Finally, our phylogenetic analyses corroborated the identifications from our automated species identification pipeline and revealed important contributions to the knowledge of the diversity of Neotropical small mammals. Discussion: This study was able to evaluate different pipelines to reconstruct mitochondrial genomes from non-model organisms, using exclusively ONT reads, benchmarking these protocols on a multi-species dataset. The proposed methodology can be applied by non-expert taxonomists and has the potential to be implemented in real-time, without the need to euthanize the organisms and under field conditions. Therefore, it stands as a relevant tool to help increase the available data for non-model organisms, and the rate at which researchers can characterize life specially in highly biodiverse places as the Neotropics.

Signals of positive selection in genomes of palearctic Myotis-bats coexisting with a fungal pathogen.

Disease can act as a driving force in shaping genetic makeup across populations, even species, if the impacts influence a particularly sensitive part of their life cycles. White-nose disease is caused by a fungal pathogen infecting bats during hibernation. The mycosis has caused massive population declines of susceptible species in North America, particularly in the genus Myotis. However, Myotis bats appear to tolerate infection in Eurasia, where the fungal pathogen has co-evolved with its bat hosts for an extended period of time. Therefore, with susceptible and tolerant populations, the fungal disease provides a unique opportunity to tease apart factors contributing to tolerance at a genomic level to and gain an understanding of the evolution of non-harmful in host-parasite interactions. To investigate if the fungal disease has caused adaptation on a genomic level in Eurasian bat species, we adopted both whole-genome sequencing approaches and a literature search to compile a set of 300 genes from which to investigate signals of positive selection in genomes of 11 Eurasian bats at the codon-level. Our results indicate significant positive selection in 38 genes, many of which have a marked role in responses to infection. Our findings suggest that white-nose syndrome may have applied a significant selective pressure on Eurasian Myotis-bats in the past, which can contribute their survival in co-existence with the pathogen. Our findings provide an insight on the selective pressure pathogens afflict on their hosts using methodology that can be adapted to other host-pathogen study systems.

Chromosome-level genome and population genomics of the intermediate horseshoe bat ( Rhinolophus affinis) reveal the molecular basis of virus tolerance in Rhinolophus and echolocation call frequency variation.

Horseshoe bats (genus Rhinolophus, family Rhinolophidae) represent an important group within chiropteran phylogeny due to their distinctive traits, including constant high-frequency echolocation, rapid karyotype evolution, and unique immune system. Advances in evolutionary biology, supported by high-quality reference genomes and comprehensive whole-genome data, have significantly enhanced our understanding of species origins, speciation mechanisms, adaptive evolutionary processes, and phenotypic diversity. However, genomic research and understanding of the evolutionary patterns of Rhinolophus are severely constrained by limited data, with only a single published genome of R. ferrumequinum currently available. In this study, we constructed a high-quality chromosome-level reference genome for the intermediate horseshoe bat ( R. affinis). Comparative genomic analyses revealed potential genetic characteristics associated with virus tolerance in Rhinolophidae. Notably, we observed expansions in several immune-related gene families and identified various genes functionally associated with the SARS-CoV-2 signaling pathway, DNA repair, and apoptosis, which displayed signs of rapid evolution. In addition, we observed an expansion of the major histocompatibility complex class II (MHC-II) region and a higher copy number of the HLA- DQB2 gene in horseshoe bats compared to other chiropteran species. Based on whole-genome resequencing and population genomic analyses, we identified multiple candidate loci (e.g., GLI3) associated with variations in echolocation call frequency across R. affinis subspecies. This research not only expands our understanding of the genetic characteristics of the Rhinolophus genus but also establishes a valuable foundation for future research.

The genome sequence of the endemic Mexican common mustached Bat, Pteronotus mexicanus. Miller, 1902 [Mormoopidae; Pteronotus].

We describe here the first characterization of the genome of the bat Pteronotus mexicanus, an endemic species of Mexico, as part of the Mexican Bat Genome Project which focuses on the characterization and assembly of the genomes of endemic bats in Mexico. The genome was assembled from a liver tissue sample of an adult male from Jalisco, Mexico provided by the Texas Tech University Museum tissue collection. The assembled genome size was 1.9 Gb. The assembly of the genome was fitted in a framework of 110,533 scaffolds and 1,659,535 contigs. The ecological importance of bats such as P. mexicanus, and their diverse ecological roles, underscores the value of having complete genomes in addressing information gaps and facing challenges regarding their function in ecosystems and their conservation.

Distinct Genes with Similar Functions Underlie Convergent Evolution in Myotis Bat Ecomorphs.

Convergence offers an opportunity to explore to what extent evolution can be predictable when genomic composition and environmental triggers are similar. Here, we present an emergent model system to study convergent evolution in nature in a mammalian group, the bat genus Myotis. Three foraging strategies-gleaning, trawling, and aerial hawking, each characterized by different sets of phenotypic features-have evolved independently multiple times in different biogeographic regions in isolation for millions of years. To investigate the genomic basis of convergence and explore the functional genomic changes linked to ecomorphological convergence, we sequenced and annotated 17 new genomes and screened 16,426 genes for positive selection and associations between relative evolutionary rates and foraging strategies across 30 bat species representing all Myotis ecomorphs across geographic regions as well as among sister groups. We identify genomic changes that describe both phylogenetic and ecomorphological trends. We infer that colonization of new environments may have first required changes in genes linked to hearing sensory perception, followed by changes linked to fecundity and development, metabolism of carbohydrates, and heme degradation. These changes may be linked to prey acquisition and digestion and match phylogenetic trends. Our findings also suggest that the repeated evolution of ecomorphs does not always involve changes in the same genes but rather in genes with the same molecular functions such as developmental and cellular processes.

Comparative genomics reveals convergent signals associated with the high metabolism and longevity in birds and bats.

Birds and bats have long lifespans relative to their body size compared with non-flying animals. However, the genomic basis associated with longer lifespan of flying species despite their higher metabolism was unclear. In this study, we hypothesized that genes involved in the regulation of metabolism and lifespan changed with the acquisition of flight and searched for genes that show specific evolutionary patterns in flying species. As a result, we identified several genes that show different evolutionary rates in bird and bat lineages. Genes in pathways involved in lifespan regulation were conserved in birds, while they evolved at an accelerated rate in bats. We also searched for genes in which convergent amino acid substitutions occurred in birds and bats and found such substitutions in genes involved in cancer, reactive oxygen species control and immunity. Our study revealed genomic changes associated with the acquisition of flight in birds and bats and suggested that multiple genes involved in the regulation of lifespan and metabolism support both high metabolism and longevity in flying species.

Complete mitochondrial genomes of three vulnerable cave bat species and their phylogenetic relationships within the order Chiroptera.

The IUCN Red List of Threatened Species contains 175 Brazilian bat species that are threatened by extinction in some degree. From this perspective, it is essential to expand the knowledge about the genetic diversity of vulnerable bats. Genomic sequencing can be useful to generate robust and informative genetic references, increasing resolution when analyzing relationships among populations, species, or higher taxonomic levels. In this study, we sequenced and characterized in detail the first complete mitochondrial genomes of Furipterus horrens, Lonchorhina aurita, and Natalus macrourus, and investigated their phylogenetic position based on amino acid sequences of protein-coding genes (PCGs). The mitogenomes of these species are 16,516, 16,697, and 16,668 bp in length, respectively, and each comprises 13 PCGs, 22 tRNA genes, two rRNA genes, and a putative control region (CR). In the three species, genes were arranged similarly to all other previously described bat mitogenomes, and nucleotide composition was also consistent with the reported range. The length and arrangement of rrnS and rrnL were also consistent with those of other bat species, showing a positive AT-skew and a negative GC-skew. Except for trnS1, for which we did not observe the DHU arm, all other tRNAs showed the cloverleaf secondary structure in the three species. In addition, the mitogenomes showed minor differences in start and stop codons, and in all PCGs, codons ending in adenine were more common compared to those ending in guanine. We found that PCGs of the three species use multiple codons to encode each amino acid, following the previously documented pattern. Furthermore, all PCGs are under purifying selection, with atp8 experiencing the most relaxed purifying selection. Considering the phylogenetic reconstruction, F. horrens was recovered as sister to Noctilio leporinus, L. aurita and Tonatia bidens shared a node within Phyllostomidae, and N. macrourus appeared as sister to Molossidae and Vespertilionidae.

Sugar assimilation underlying dietary evolution of Neotropical bats.

Dietary specializations in animals lead to adaptations in morphology, anatomy and physiology. Neotropical bats, with their high taxonomic and trophic diversity, offer a unique perspective on diet-driven evolutionary adaptations. Here we assess the metabolic response to different dietary sugars among wild-caught bats. We found that insectivorous bats had a pronounced metabolic response to trehalose, whereas bats with nectar and fruit-based diets showed significantly higher blood glucose levels in response to glucose and sucrose, reaching levels over 750 mg dl-1. The genomic analysis of 22 focal species and two outgroup species identified positive selection for the digestive enzyme trehalase in insect eaters, while sucrase-isomaltase showed selection in lineages with omnivorous and nectar diets. By examining anatomical and cellular features of the small intestine, we discovered that dietary sugar proportion strongly impacted numerous digestive traits, providing valuable insight into the physiological implications of molecular adaptations. Using hybridization chain reaction (HCR) RNA fluorescence in situ hybridization, we observed unusually high expression in the glucose transporter gene Slc2a2 in nectar bats, while fruit bats increased levels of Slc5a1 and Slc2a5. Overall, this study highlights the intricate interplay between molecular, morphological and physiological aspects of diet evolution, offering new insights into the mechanisms of dietary diversification and sugar assimilation in mammals.

Unraveling genomic features and phylogenomics through the analysis of three Mexican endemic Myotis genomes.

Background: Genomic resource development for non-model organisms is rapidly progressing, seeking to uncover molecular mechanisms and evolutionary adaptations enabling thriving in diverse environments. Limited genomic data for bat species hinder insights into their evolutionary processes, particularly within the diverse Myotis genus of the Vespertilionidae family. In Mexico, 15 Myotis species exist, with three-M. vivesi, M. findleyi, and M. planiceps-being endemic and of conservation concern. Methods: We obtained samples of Myotis vivesi, M. findleyi, and M. planiceps for genomic analysis. Each of three genomic DNA was extracted, sequenced, and assembled. The scaffolding was carried out utilizing the M. yumanensis genome via a genome-referenced approach within the ntJoin program. GapCloser was employed to fill gaps. Repeat elements were characterized, and gene prediction was done via ab initio and homology methods with MAKER pipeline. Functional annotation involved InterproScan, BLASTp, and KEGG. Non-coding RNAs were annotated with INFERNAL, and tRNAscan-SE. Orthologous genes were clustered using Orthofinder, and a phylogenomic tree was reconstructed using IQ-TREE. Results: We present genome assemblies of these endemic species using Illumina NovaSeq 6000, each exceeding 2.0 Gb, with over 90% representing single-copy genes according to BUSCO analyses. Transposable elements, including LINEs and SINEs, constitute over 30% of each genome. Helitrons, consistent with Vespertilionids, were identified. Values around 20,000 genes from each of the three assemblies were derived from gene annotation and their correlation with specific functions. Comparative analysis of orthologs among eight Myotis species revealed 20,820 groups, with 4,789 being single copy orthogroups. Non-coding RNA elements were annotated. Phylogenomic tree analysis supported evolutionary chiropterans' relationships. These resources contribute significantly to understanding gene evolution, diversification patterns, and aiding conservation efforts for these endangered bat species.

Activity of DNA Repair Systems in the Cells of Long-Lived Rodents and Bats.

Damages of various origin accumulated in the genomic DNA can lead to the breach of genome stability, and are considered to be one of the main factors involved in cellular senescence. DNA repair systems in mammalian cells ensure effective damage removal and repair of the genome structure, therefore, activity of these systems is expected to be correlated with high maximum lifespan observed in the long-lived mammals. This review discusses current results of the studies focused on determination of the DNA repair system activity and investigation of the properties of its key regulatory proteins in the cells of long-lived rodents and bats. Based on the works discussed in the review, it could be concluded that the long-lived rodents and bats in general demonstrate high efficiency in functioning and regulation of DNA repair systems. Nevertheless, a number of questions around the study of DNA repair in the cells of long-lived rodents and bats remain poorly understood, answers to which could open up new avenues for further research.