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BAD2matrix: Phylogenomic matrix concatenation, indel coding, and more

Common steps in phylogenomic matrix production include biological sequence concatenation, morphological data concatenation, insertion/deletion (indel) coding, gene content (presence/absence) coding, removing uninformative characters for parsimony analysis, recording with reduced amino acid alphabets, and occupancy filtering. Existing software does not accomplish these tasks on a phylogenomic scale using a single program. BAD2matrix is a Python script that performs the above-mentioned steps in phylogenomic matrix construction for DNA or amino acid sequences as well as morphological data. The script works in UNIX-like environments (e.g., LINUX, MacOS, Windows Subsystem for LINUX).

59 BASIC BIOLOGICAL SCIENCES↗

Adaptation strategies of giant viruses to low-temperature marine ecosystems

Abstract Microbes in marine ecosystems have evolved their gene content to thrive successfully in the cold. Although this process has been reasonably well studied in bacteria and selected eukaryotes, less is known about the impact of cold environments on the genomes of viruses that infect eukaryotes. Here, we analyzed cold adaptations in giant viruses (Nucleocytoviricota and Mirusviricota) from austral marine environments and compared them with their Arctic and temperate counterparts. We recovered giant virus metagenome-assembled genomes (98 Nucleocytoviricota and 12 Mirusviricota MAGs) from 61 newly sequenced metagenomes and metaviromes from sub-Antarctic Patagonian fjords and Antarctic seawater samples. When analyzing our data set alongside Antarctic and Arctic giant viruses MAGs already deposited in the Global Ocean Eukaryotic Viral database, we found that Antarctic and Arctic giant viruses predominantly inhabit sub-10°C environments, featuring a high proportion of unique phylotypes in each ecosystem. In contrast, giant viruses in Patagonian fjords were subject to broader temperature ranges and showed a lower degree of endemicity. However, despite differences in their distribution, giant viruses inhabiting low-temperature marine ecosystems evolved genomic cold-adaptation strategies that led to changes in genetic functions and amino acid frequencies that ultimately affect both gene content and protein structure. Such changes seem to be absent in their mesophilic counterparts. The uniqueness of these cold-adapted marine giant viruses may now be threatened by climate change, leading to a potential reduction in their biodiversity.

Environmental Sciences & Ecology↗

Genomic Characterization and Wetland Occurrence of a Novel Campylobacter Isolate from Canada Geese

Populations of resident, non-migratory Canada geese are rapidly increasing. Canada geese are known to transmit viral and bacterial diseases, posing a possible threat to human health. The most prevalent pathogens vectored by geese are Campylobacter species, yet the current understanding of the identity and virulence of these pathogens is limited. In our previous study, we observed a high prevalence of Campylobacter spp. in the Banklick Creek wetland—a constructed treatment wetland (CTW) located in northern KY (USA) used to understand sources of fecal contamination originating from humans and waterfowl frequenting the area. To identify the types of Campylobacter spp. found contaminating the CTW, we performed genetic analyses of Campylobacter 16s ribosomal RNA amplified from CTW water samples and collected fecal material from birds frequenting those areas. Our results showed a high occurrence of a Campylobacter canadensis-like clade from the sampling sites. Whole-genome sequence analyses of an isolate from Canada goose fecal material, called MG1, were used to confirm the identity of the CTW isolates. Further, we examined the phylogenomic position, virulence gene content, and antimicrobial resistance gene profile of MG1. Lastly, we developed an MG1-specific real-time PCR assay and confirmed the presence of MG1 in Canada goose fecal samples surrounding the CTW. Our findings reveal that the Canada goose-vectored Campylobacter sp. MG1 is a novel isolate compared to C. canadensis that possesses possible zoonotic potential, which may be of human health concern.

59 BASIC BIOLOGICAL SCIENCES↗

Novel Gloeobacterales spp. from Diverse Environments across the Globe

Photosynthetic Cyanobacteria and their descendants are the only known organisms capable of oxygenic photosynthesis. Their metabolism permanently changed the Earth’s surface and the evolutionary trajectory of life, but little is known about their evolutionary history. Genomes of the Gloeobacterales, an order of deeply divergent photosynthetic Cyanobacteria, may hold clues about the evolutionary process. However, there are only three published genomes within this order, and it is difficult to make broad inferences based on such little data. Here, I describe five species within the Gloeobacterales retrieved from publicly available databases and examine their photosynthetic gene content and the environments in which Gloeobacterales genomes and 16S rRNA gene sequences are found. The Gloeobacterales contain reduced photosystems and inhabit cold, wet-rock, and low-light environments. They are likely present in low abundances due to their low growth rate. Future searches for Gloeobacterales should target these environments, and samples should be deeply sequenced to capture the low-abundance taxa. Publicly available databases contain undescribed taxa within the Gloeobacterales. However, searching through all available data with current methods is computationally expensive. Therefore, new methods must be developed to search for these and other evolutionarily important taxa. Once identified, these novel photosynthetic Cyanobacteria will help illuminate the origin and evolution of oxygenic photosynthesis. Early branching photosynthetic Cyanobacteria such as the Gloeobacterales may provide clues into the evolutionary history of oxygenic photosynthesis, but there are few genomes or cultured taxa from this order. Five new metagenome-assembled genomes suggest that members of the Gloeobacterales all contain reduced photosystems and lack genes associated with thylakoids and circadian rhythms. Their distribution suggests that they may thrive in environments that are marginal for other species, including wet-rock and cold environments. These traits may aid in the discovery and cultivation of novel species in this clade.

59 BASIC BIOLOGICAL SCIENCES↗

Animal-associated jumbo phages as widespread and active modulators of gut microbiome ecology and metabolism

Huge phages are widespread in the biosphere, yet their prevalence and ecology in the human gut remain poorly characterized. Here, we report Jug (jumbo gut) phages with genomes of 360 to 402 kilobase pairs that comprise ~1.1% of the reads in human gut metagenomes, and are predicted to infect Bacteroides and/or Phocaeicola. Although three of the four major groups of Jug phages shared >90% genome-wide sequence identity, their large terminase subunits exhibited only 38 to 57% identity, suggesting horizontal acquisition from other phages. Over 1500 genomes of Jug phages were recovered from human and animal gut metagenomes, revealing their broad distribution, with largely shared gene content suggestive of frequent cross-animal-host transmission. Jug phages displayed high gene transcription activities, including the gene for a calcium-translocating P-type ATPase not detected previously in phages. These findings broaden our understanding of huge phages and highlight Jug phages as potential major players in gut microbiome ecology.

Chen, LinXing [University of Science and Technolog↗

Reekeekee- and roodoodooviruses, two different Microviridae clades constituted by the smallest DNA phages

Small circular single-stranded DNA viruses of the Microviridae family are both prevalent and diverse in all ecosystems. They usually harbor a genome between 4.3 and 6.3 kb, with a microvirus recently isolated from a marine Alphaproteobacteria being the smallest known genome of a DNA phage (4.248 kb). A subfamily, Amoyvirinae, has been proposed to classify this virus and other related small Alphaproteobacteria-infecting phages. Here, we report the discovery, in meta-omics data sets from various aquatic ecosystems, of sixteen complete microvirus genomes significantly smaller (2.991–3.692 kb) than known ones. Phylogenetic analysis reveals that these sixteen genomes represent two related, yet distinct and diverse, novel groups of microviruses—amoyviruses being their closest known relatives. We propose that these small microviruses are members of two tentatively named subfamilies Reekeekeevirinae and Roodoodoovirinae. As known microvirus genomes encode many overlapping and overprinted genes that are not identified by gene prediction software, we developed a new methodology to identify all genes based on protein conservation, amino acid composition, and selection pressure estimations. Surprisingly, only four to five genes could be identified per genome, with the number of overprinted genes lower than that in phiX174. These small genomes thus tend to have both a lower number of genes and a shorter length for each gene, leaving no place for variable gene regions that could harbor overprinted genes. Even more surprisingly, these two Microviridae groups had specific and different gene content, and major differences in their conserved protein sequences, highlighting that these two related groups of small genome microviruses use very different strategies to fulfill their lifecycle with such a small number of genes. The discovery of these genomes and the detailed prediction and annotation of their genome content expand our understanding of ssDNA phages in nature and are further evidence that these viruses have explored a wide range of possibilities during their long evolution.

59 BASIC BIOLOGICAL SCIENCES↗

Generation, Transfer, and Loss of Alternative Oxidase Paralogues in the Aspergillaceae Family

Alternative oxidase (Aox) is a terminal oxidase operating in branched electron transport. The activity correlates positively with overflow metabolisms in certain Aspergilli, converting intracellular glucose by the shortest possible path into organic acids, like citrate or itaconate. Aox is nearly ubiquitous in fungi, but aox gene multiplicity is rare. Nevertheless, within the family of the Aspergillaceae and among its various species of industrial relevance—Aspergillus niger, A. oryzae, A. terreus, Penicillium rubens—paralogous aox genes coexist. Paralogous genes generally arise from duplication and are inherited vertically. Here, we provide evidence of four independent duplication events along the lineage that resulted in aox paralogues (aoxB) in contemporary Aspergillus and Penicillium taxa. In some species, three aox genes are co-expressed. The origin of the A. niger paralogue is different than that of the A. terreus paralogue, but all paralogous clades ultimately arise from ubiquitous aoxA parent genes. We found different patterns of uncorrelated gene losses reflected in the Aspergillus pedigree, albeit the original aoxA orthologues persist everywhere and are never replaced. The loss of acquired paralogues co-determines the contemporary aox gene content of individual species. In Aspergillus calidoustus, the two more ancient paralogues have, in effect, been replaced by two aoxB genes of distinct origins.

59 BASIC BIOLOGICAL SCIENCES↗

A method for achieving complete microbial genomes and improving bins from metagenomics data

Metagenomics facilitates the study of the genetic information from uncultured microbes and complex microbial communities. Assembling complete microbial genomes ( i.e ., circular with no misassemblies) from metagenomics data is difficult because most samples have high organismal complexity and strain diversity. Less than 100 circularized bacterial and archaeal genomes have been assembled from metagenomics data despite the thousands of datasets that are available. Circularized genomes are important for (1) building a reference collection as scaffolds for future assemblies, (2) providing complete gene content of a genome, (3) confirming little or no contamination of a genome, (4) studying the genomic context and synteny of genes, and (5) linking protein coding genes to ribosomal RNA genes to aid metabolic inference in 16S rRNA gene sequencing studies. We developed a method to achieve circularized genomes using iterative assembly, binning, and read mapping. In addition, this method exposes potential misassemblies from k-mer based assemblies. We chose species of the Candidate Phyla Radiation (CPR) to focus our initial efforts because they have small genomes and are only known to have one ribosomal RNA operon. We present 34 circular CPR genomes, one circular Margulisbacteria genome, and two circular megaphage genomes from 19 public and published datasets. We demonstrate findings that would likely be difficult without circularizing genomes, including that ribosomal genes are likely not operonic in the majority of CPR, and that some CPR harbor diverged forms of RNase P RNA. Code and a tutorial for this method is available at https://github.com/lmlui/Jorg .

Lui, Lauren↗

A method for achieving complete microbial genomes and improving bins from metagenomics data

Metagenomics facilitates the study of the genetic information from uncultured microbes and complex microbial communities. Assembling complete genomes from metagenomics data is difficult because most samples have high organismal complexity and strain diversity. Some studies have attempted to extract complete bacterial, archaeal, and viral genomes and often focus on species with circular genomes so they can help confirm completeness with circularity. However, less than 100 circularized bacterial and archaeal genomes have been assembled and published from metagenomics data despite the thousands of datasets that are available. Circularized genomes are important for (1) building a reference collection as scaffolds for future assemblies, (2) providing complete gene content of a genome, (3) confirming little or no contamination of a genome, (4) studying the genomic context and synteny of genes, and (5) linking protein coding genes to ribosomal RNA genes to aid metabolic inference in 16S rRNA gene sequencing studies. We developed a semi-automated method called Jorg to help circularize small bacterial, archaeal, and viral genomes using iterative assembly, binning, and read mapping. In addition, this method exposes potential misassemblies from k-mer based assemblies. We chose species of the Candidate Phyla Radiation (CPR) to focus our initial efforts because they have small genomes and are only known to have one ribosomal RNA operon. In addition to 34 circular CPR genomes, we present one circular Margulisbacteria genome, one circular Chloroflexi genome, and two circular megaphage genomes from 19 public and published datasets. We demonstrate findings that would likely be difficult without circularizing genomes, including that ribosomal genes are likely not operonic in the majority of CPR, and that some CPR harbor diverged forms of RNase P RNA. Code and a tutorial for this method is available at https://github.com/lmlui/Jorg and is available on the DOE Systems Biology KnowledgeBase as a beta app.

59 BASIC BIOLOGICAL SCIENCES↗

Chromosome-level genome assemblies and genetic maps reveal heterochiasmy and macrosynteny in endangered Atlantic Acropora

Abstract Background Over their evolutionary history, corals have adapted to sea level rise and increasing ocean temperatures, however, it is unclear how quickly they may respond to rapid change. Genome structure and genetic diversity contained within may highlight their adaptive potential. Results We present chromosome-scale genome assemblies and linkage maps of the critically endangered Atlantic acroporids,Acropora palmataandA. cervicornis. Both assemblies and linkage maps were resolved into 14 chromosomes with their gene content and colinearity. Repeats and chromosome arrangements were largely preserved between the species. The family Acroporidae and the genusAcroporaexhibited many phylogenetically significant gene family expansions. Macrosynteny decreased with phylogenetic distance. Nevertheless, scleractinians shared six of the 21 cnidarian ancestral linkage groups as well as numerous fission and fusion events compared to other distantly related cnidarians. Genetic linkage maps were constructed from oneA. palmatafamily and 16A. cervicornisfamilies using a genotyping array. The consensus maps span 1,013.42 cM and 927.36 cM forA. palmataandA. cervicornis, respectively. Both species exhibited high genome-wide recombination rates (3.04 to 3.53 cM/Mb) and pronounced sex-based differences, known as heterochiasmy, with 2 to 2.5X higher recombination rates estimated in the female maps. Conclusions Together, the chromosome-scale assemblies and genetic maps we present here are the first detailed look at the genomic landscapes of the critically endangered Atlantic acroporids. These data sets revealed that adaptive capacity of Atlantic acroporids is not limited by their recombination rates. The sister species maintain macrosynteny with few genes with high sequence divergence that may act as reproductive barriers between them. In the AtlanticAcropora, hybridization between the two sister species yields an F1 hybrid with limited fertility despite the high levels of macrosynteny and gene colinearity of their genomes. Together, these resources now enable genome-wide association studies and discovery of quantitative trait loci, two tools that can aid in the conservation of these species.

Biotechnology & Applied Microbiology↗

Contrasting modes of macro and microsynteny evolution in a eukaryotic subphylum

Examination of the changes in order and arrangement of homologous genes is key for understanding the mechanisms of genome evolution in eukaryotes. Previous comparisons between eukaryotic genomes have revealed considerable conservation across species that diverged hundreds of millions of years ago (e.g., vertebrates, bilaterian animals, and filamentous fungi). However, understanding how genome organization evolves within and between eukaryotic major lineages remains underexplored. We analyzed high-quality genomes of 120 representative budding yeast species (subphylum Saccharomycotina) spanning ~400 million years of eukaryotic evolution to examine how their genome organization evolved and to compare it with the evolution of animal and plant genome organization. We, in this study, found that the decay of both macrosynteny (the conservation of homologous chromosomes) and microsynteny (the conservation of local gene content and order) was strongly associated with evolutionary divergence across budding yeast major clades. However, although macrosynteny decayed very fast, within ~100 million years, the microsynteny of many genes—especially genes in metabolic clusters (e.g., in the GAL gene cluster)—was much more deeply conserved both within major clades and across the subphylum. We further found that when genomes with similar evolutionary divergence times were compared, budding yeasts had lower macrosynteny conservation than animals and filamentous fungi but higher conservation than angiosperms. In contrast, budding yeasts had levels of microsynteny conservation on par with mammals, whereas angiosperms exhibited very low conservation. Our results provide new insight into the tempo and mode of the evolution of gene and genome organization across an entire eukaryotic subphylum.

59 BASIC BIOLOGICAL SCIENCES↗

Identification of mobile genetic elements with geNomad

Identifying and characterizing mobile genetic elements in sequencing data is essential for understanding their diversity, ecology, biotechnological applications and impact on public health. Here we introduce geNomad, a classification and annotation framework that combines information from gene content and a deep neural network to identify sequences of plasmids and viruses. geNomad uses a dataset of more than 200,000 marker protein profiles to provide functional gene annotation and taxonomic assignment of viral genomes. Using a conditional random field model, geNomad also detects proviruses integrated into host genomes with high precision. In benchmarks, geNomad achieved high classification performance for diverse plasmids and viruses (Matthews correlation coefficient of 77.8% and 95.3%, respectively), substantially outperforming other tools. Leveraging geNomad’s speed and scalability, we processed over 2.7 trillion base pairs of sequencing data, leading to the discovery of millions of viruses and plasmids that are available through the IMG/VR and IMG/PR databases. geNomad is available at https://portal.nersc.gov/genomad.

59 BASIC BIOLOGICAL SCIENCES↗

A large accessory genome and high recombination rates may influence global distribution and broad host range of the fungal plant pathogen Claviceps purpurea

Pangenome analyses are increasingly being utilized to study the evolution of eukaryotic organisms. While pangenomes can provide insight into polymorphic gene content, inferences about the ecological and adaptive potential of such organisms also need to be accompanied by additional supportive genomic analyses. In this study we constructed a pangenome of Claviceps purpurea from 24 genomes and examined the positive selection and recombination landscape of an economically important fungal organism for pharmacology and agricultural research. Together, these analyses revealed that C. purpurea has a relatively large accessory genome (~ 38%), high recombination rates (ρ = 0.044), and transposon mediated gene duplication. However, due to observations of relatively low transposable element (TE) content (8.8%) and a lack of variability in genome sizes, prolific TE expansion may be controlled by frequent recombination. We additionally identified that within the ergoline biosynthetic cluster the lpsA1 and lpsA2 were the result of a recombination event. However, the high recombination rates observed in C. purpurea may be influencing an overall trend of purifying selection across the genome. These results showcase the use of selection and recombination landscapes to identify mechanisms contributing to pangenome structure and primary factors influencing the evolution of an organism.

59 BASIC BIOLOGICAL SCIENCES↗

Warming is Associated With More Encoded Antimicrobial Resistance Genes and Transcriptions Within Five Drug Classes in Soil Bacteria: A Case Study and Synthesis

ABSTRACT The effect of warming on anti‐microbial resistance (AMR) genes in the environment has critical implications for public health but is little studied. We collected published soil bacterial genomes from the BV‐BRC database and tested the correlation between reported optimal growth temperature and the number of encoded AMR genes. Furthermore, we tested the relationship between temperature and AMR gene transcription in a natural ecosystem by analysing soil transcriptomes from a warming manipulation experiment in an Alaskan boreal forest. We hypothesised that there is a positive relationship between warming and AMR prevalence in gene content in bacterial genomes and transcriptomic sequences, and that this effect would vary by drug class. Regarding the bacterial genomes, we found a positive relationship between the fraction of encoded AMR genes and the reported optimal temperature of soil bacteria. The drug classes tetracycline and lincosamide/macrolide/streptogramin had the strongest positive relationship with reported optimal temperature. For the case study in a natural ecosystem, we found 61 significantly upregulated AMR gene‐associated transcripts spanning eight drug classes in warmed plots. In the Alaskan soil samples, we found that warming elicited the strongest positive effect on transcripts targeting lincosamide/streptogramin, beta‐lactam and phenicol/quinolone antibiotics. Overall, higher temperatures were linked to AMR gene prevalence.

Hacopian, Melanie T. [Department of Ecology and Ev↗

Genomic diversifications of five Gossypium allopolyploid species and their impact on cotton improvement

Abstract Polyploidy is an evolutionary innovation for many animals and all flowering plants, but its impact on selection and domestication remains elusive. Here we analyze genome evolution and diversification for all five allopolyploid cotton species, including economically important Upland and Pima cottons. Although these polyploid genomes are conserved in gene content and synteny, they have diversified by subgenomic transposon exchanges that equilibrate genome size, evolutionary rate heterogeneities and positive selection between homoeologs within and among lineages. These differential evolutionary trajectories are accompanied by gene-family diversification and homoeolog expression divergence among polyploid lineages. Selection and domestication drive parallel gene expression similarities in fibers of two cultivated cottons, involving coexpression networks and N 6 -methyladenosine RNA modifications. Furthermore, polyploidy induces recombination suppression, which correlates with altered epigenetic landscapes and can be overcome by wild introgression. These genomic insights will empower efforts to manipulate genetic recombination and modify epigenetic landscapes and target genes for crop improvement.

59 BASIC BIOLOGICAL SCIENCES↗

Defining the Environmental Adaptations of Genus Devosia: Insights into its Expansive Short Peptide Transport System and Positively Selected Genes

Devosia are well known for their dominance in soil habitats contaminated with various toxins and are best characterized for their bioremediation potential. In this study, we compared the genomes of 27 strains of Devosia with aim to understand their metabolic abilities. The analysis revealed their adaptive gene repertoire which was bared from 52% unique pan-gene content. A striking feature of all genomes was the abundance of oligo- and di-peptide permeases (oppABCDF and dppABCDF) with each genome harboring an average of 60.7±19.1 and 36.5±10.6 operon associated genes respectively. Apart from their primary role in nutrition, these permeases may help Devosia to sense environmental signals and in chemotaxis at stressed habitats. Through sequence similarity network analyses, we identified 29 Opp and 19 Dpp sequences that shared very little homology with any other sequence suggesting an expansive short peptidic transport system within Devosia. The substrate determining components of these permeases viz. OppA and DppA further displayed a large diversity that separated into 12 and 9 homologous clusters respectively in addition to large number of isolated nodes. We also dissected the genome scale positive evolution and found genes associated with growth (exopolyphosphatase, HesB_IscA_SufA family protein), detoxification (moeB, nifU-like domain protein, alpha/beta hydrolase), chemotaxis (cheB, luxR) and stress response (phoQ, uspA, luxR, sufE) were positively selected. The study highlights the genomic plasticity of the Devosia spp. for conferring adaptation, bioremediation and the potential to utilize a wide range of substrates. The widespread toxin-antitoxin loci and ‘open’ state of the pangenome provided evidence of plastic genomes and a much larger genetic repertoire of the genus which is yet uncovered.

59 BASIC BIOLOGICAL SCIENCES↗

A haplotype‐resolved reference genome of Quercus alba sheds light on the evolutionary history of oaks

Summary White oak ( Quercus alba ) is an abundant forest tree species across eastern North America that is ecologically, culturally, and economically important. We report the first haplotype‐resolved chromosome‐scale genome assembly of Q. alba and conduct comparative analyses of genome structure and gene content against other published Fagaceae genomes. We investigate the genetic diversity of this widespread species and the phylogenetic relationships among oaks using whole genome data. Despite strongly conserved chromosome synteny and genome size across Quercus , certain gene families have undergone rapid changes in size, including defense genes. Unbiased annotation of resistance (R) genes across oaks revealed that the overall number of R genes is similar across species – as are the chromosomal locations of R gene clusters – but, gene number within clusters is more labile. We found that Q. alba has high genetic diversity, much of which predates its divergence from other oaks and likely impacts divergence time estimations. Our phylogenetic results highlight widespread phylogenetic discordance across the genus. The white oak genome represents a major new resource for studying genome diversity and evolution in Quercus . Additionally, we show that unbiased gene annotation is key to accurately assessing R gene evolution in Quercus .

Larson, Drew A. [Department of Biology Indiana Uni↗

Permanent Draft Genome of Strain ESFC-1: Ecological Genomics of a Newly Discovered Lineage of Filamentous Diazotrophic Cyanobacteria

The nonheterocystous filamentous cyanobacterium, strain ESFC-1, is a recently described member of the order Oscillatoriales within the Cyanobacteria. ESFC-1 has been shown to be a major diazotroph in the intertidal microbial mat system at Elkhorn Slough, CA, USA. Based on phylogenetic analyses of the 16S RNA gene, ESFC-1 appears to belong to a unique, genus-level divergence; the draft genome sequence of this strain has now been determined. Here we report features of this genome as they relate to the ecological functions and capabilities of strain ESFC-1. The 5,632,035 bp genome sequence encodes 4914 protein-coding genes and 92 RNA genes. One striking feature of this cyanobacterium is the apparent lack of either uptake or bi-directional hydrogenases typically expected within a diazotroph. Additionally, a large genomic island is found that contains numerous low GC-content genes and genes related to extracellular polysaccharide production and cell wall synthesis and maintenance.

R Craig Everroad↗