- The isolates, which were initially identified as three distinct species based on sequence variation in the 16S rRNA locus, could be distinguished as 41 different species based on genome-wide average nucleotide identity. - Of the 32 biosynthetic gene cluster (BGC) classes detected, non-ribosomal peptide synthetases, siderophores, and terpenes were present in all genomes. - Finally, we show that genome-wide recombination has partly contributed to the large genomic variation among strains of the same species.. - Many of the drugs used against infectious diseases and other medical disorders have been historically derived from molecules synthesized by environmental microbes, with the most notable belonging to the genus Streptomy- ces (phylum Actinobacteria) [1–3]. - The current, estimated number of known Strepto- myces species is approximately 650 [4], thus making it one of the largest genera in the bacterial domain. - Strep- tomyces are ubiquitous in the environment. - The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. - If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. - To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.. - The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.. - Full list of author information is available at the end of the article. - These isolates were selected from a culture collection compris- ing 632 isolates, which were initially identified using se- quence variation in the 16S rRNA locus [19]. - Herein, the feeding strategies of the bats include aerial hawking or gleaning of arthropods from different surfaces. - We initially used sequence variation in the 16S rRNA locus to delineate species boundaries. - 1a), with each cluster representing a distinct species based on the 97% sequence similarity threshold in the 16S rRNA gene (Fig. - Within each of the three clusters, sequence similar- ities between isolates ranged from . - to compare isolates, the majority of isolates within each of the three 16S rRNA-based clusters fell below the 95%. - ≥95% of the genomes (Additional file 4: Table S3 and Additional file 5: Fig. - Within each of the 16S rRNA- defined clusters, genomes exhibited ANI values ranging from and in clusters 1, 2 and 3, respectively (Fig. - Of the 32 BGC classes detected, we found non-ribosomal peptide synthetases (NRPS), siderophores, and terpenes present in all genomes. - The matrix shows genome-wide, average nucleotide identity (ANI) values calculated for every pair of genomes in the entire data set. - not identify any specific class of BGC that is unique to any of the three ANI clusters nor to any of the nine bat species.. - We found differences in the number and classes of BGCs, including hybrid BGCs (i.e., BGCs with genes that code for more than one type of scaffold-synthesizing enzymes [28, 31. - between strains of the same species (Fig. - However, we did not find any significant differences in the number of either accessory genes (Fig. - b Strain distribution of genes in the pangenome of each species. - Comparison of the d total number of genes, e accessory genes and f singleton genes in the pan-genome of each species. - Using the core genome alignment of each of the three species, we tested for evidence of recombination using the.. - The Splitstree analysis shows the reticulations in the phylogenetic relationships between strains of each species (Fig. - 4 A phylogenetic network of the core genome of Streptomyces species 20, 29 and 38 generated using SplitsTree4. - In that study, sequence variation in the 16S rRNA and five other housekeeping genes were used to characterize the phylogenetic diversity and rela- tionships of Streptomyces, with isolates representing 15 novel species that exhibited antifungal activity [19].. - For example, the presence on all genomes of sidero- phores, which are involved in the acquisition of ferric ions, may reflect the need for Streptomyces to cope in environments with limited iron supply [41]. - However, differences in the abundance of siderophores per gen- ome may be due to interactions with specific bacterial partners, as has been previously reported [42, 43]. - This is because cultivation fails to dis- cover novel strains that are recalcitrant to specific culti- vation methods, media and laboratory conditions commonly used because they do not precisely mimic the ecological niche of the bacterium in nature. - Hence, we recognize that the extent of genomic diversity we found in our dataset is likely an under-estimation of the true scale of Streptomyces diversity in bats, suggesting that additional species with unique genetic and phenotypic features remain to be discovered. - Overall, our study provides an initial genome-based as- sessment of the bat-associated Streptomyces diversity that will be critical to prioritizing those strains with the greatest ability to produce novel bioactive compounds, including those that can strongly inhibit P. - Collection and isolation of Streptomyces. - Immediately following swabbing of each bat, plates were inoculated and kept at 4 °C during transport and at 20 °C in the la- boratory during initial growth. - We used the program Roary [50] to characterize the core and accessory genes in the pan-genome of the 73 strains.. - We aligned the sequences of the 16S rRNA gene ex- tracted from the genomes using MAFFT [51] and counted all pairwise single nucleotide polymorphism (SNP) differences using snp-dists v0.6.3 (https://github.. - We also built a phylogenetic tree using the concatenated sequence alignments of the core genes using RAxML with the GTR model and four gamma categories. - Due to their high number, hybrid BGCs were split and counted as individual BGC classes in the genus tree (e.g., terpene-t1PKS BGC would count as both one ter- pene and one t1PKS). - Using the alignment of the concatenated core genes of each of the three species as input, we ran the Pairwise Homoplasy Index test for recombination with 100 per- mutations using PhiPack [56]. - The online version contains supplementary material available at https://doi.. - org/10.1186/s w.. - Accession numbers, metadata and genome characteristics of the 73 Streptomyces isolates. - 16S rRNA tree of Streptomyces isolates with bootstrap values.. - Pan-genome analysis of the 73 Streptomy- ces genomes estimated using Roary.. - Biosynthetic gene clusters (BGCs) of all genomes of Streptomyces estimated using antiSMASH.. - Pan-genome analyses of Streptomyces sp.. - We also thank the students in the Northup lab at the University of New Mexico for assisting with our bat netting efforts.. - Funding for the creation of the bat Actinobacteria culture collection in the Northup lab at UNM was provided by the Eppley Foundation, the National Park Service through the Colorado Plateau Cooperative Ecosystem Studies unit (CPCESU) for work at the El Malpais National Monument, Carlsbad Caverns National Monument, and Grand Canyon-Parashant National Monu- ment. - The funders had no role in the study design, data collection and interpretation, or the decision to. - 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