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Search indexed bioRxiv preprints in genomics, neuroscience, cell biology and bioinformatics. Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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bioRxiv2025

Helicobacter pylori genome aggregation database reveals complex evolutionary forces shaping its genomic landscape and clinical impact in East Asia

We assembled HPgnomAD, a comprehensive global genome aggregation database of Helicobacter pylori (H. pylori) featuring 7,544 high-quality genomes from 278 populations, along with the first species-wide haplotype reference panel for genotype imputation. The panel provides high accuracy across diverse H. pylori populations, including those from low-coverage data, and is openly accessible with integrated analysis tools (https://www.hpgnomad.top/). Variant discovery revealed 1.82 million SNPs and 0.65 million InDels, with African strains exhibiting the most tremendous diversity and East Asian strains showing unexpectedly high novel variation. Fine-scale analysis of the hpEAsia lineage revealed six new sublineages shaped by altitude-, latitude-related divergence and region-specific admixture. Phylogenomic dating revealed two divergence waves in East Asia, paralleling Upper Paleolithic settlement and Neolithic human expansions, with highland-lowland separation at [~]18.8 kya, followed by the formation of complex geography-related population substructures. Genome-wide scans revealed adaptive loci related to metal acquisition, nitrogen metabolism, surface adhesion, and membrane transport, including altitude-associated highly differentiated variants linked to antibiotic resistance and gastric disease. By integrating genomic, evolutionary, environmental, and clinical data, HPgnomAD offers a framework for understanding H. pylori evolution, host-pathogen adaptation, and precision medicine worldwide.

evolutionary biology↗

bioRxiv2025

Water Stress Tolerance, Genomic Selection and Identification of Genomic Regions in a MAGIC Population of Eggplant

Horticultural crops are increasingly affected by water stress due to climate change, making the development of stress-tolerant varieties an urgent need. In this study, we evaluated a set of 184 Multi-parent Advanced Generation Inter-Cross (MAGIC) eggplant lines under water stress conditions, consisting of irrigation at 30% of field capacity. After 21 days of stress, we assessed traits related to growth, water content, plant pigments, and proline content. The MAGIC population displayed a high variability for water stress tolerance, as transgressive lines over parental values were found for most of the traits. Key traits associated with water stress tolerance were identified, including increased root growth and elevated proline, and flavonoid content. Genome-Wide Association Study (GWAS) analysis allowed identification of three genomic regions associated with total dry weight, water content and flavonoids, traits that contribute significantly to stress tolerance. Additionally, a linear genomic selection index was constructed based on total dry weight, dry weight increase during the stress period, root dry weight, water content, and proline content to identify water stress-tolerant and susceptible lines. The models predictive ability was calculated, and the value of the selection index was predicted for 141 unevaluated MAGIC lines, with prediction accuracy for the index traits ranging from 0.11 to 0.53. This study presents a comprehensive analysis, identifying critical traits, genomic regions and lines with genetic potential, providing valuable information for future breeding programmes to improve water stress tolerance in eggplant.

genetics↗

bioRxiv2025

Genome reorganisation and expansion shape 3D genome architecture and define a distinct regulatory landscape in coleoid cephalopods

How genomic changes translate into organismal novelties is often confounded by the multi-layered nature of genome architecture and the long evolutionary timescales over which molecular changes accumulate. Coleoid cephalopods (squid, cuttlefish, and octopus) provide a unique system to study these processes due to a large-scale chromosomal rearrangement in the coleoid ancestor that resulted in highly modified karyotypes, followed by lineage-specific fusions, translocations, and repeat expansions. How these events have shaped gene regulatory patterns underlying the evolution of coleoid innovations, including their large and elaborately structured nervous systems, novel organs, and complex behaviours, remains poorly understood. To address this, we integrate Micro-C, RNA-seq, and ATAC-seq across multiple coleoid species, developmental stages, and tissues. We find that while topological compartments are broadly conserved, hundreds of chromatin loops are species- and context-specific, with distinct regulation signatures and dynamic expression profiles. CRISPR-Cas9 knockout of a putative regulatory sequence within a conserved region demonstrates the role of loops in neural development and the prevalence of long-range, inter-compartmental interactions. We propose that differential evolutionary constraints across the coleoid 3D genome allow macroevolutionary processes to shape genome topology in distinct ways, facilitating the emergence of novel regulatory entanglements and ultimately contributing to the evolution and maintenance of complex traits in coleoids.

evolutionary biology↗

bioRxiv2025

Genomic analysis of Megalocytivirus genomes reveals widespread recombination

Megalocytiviruses are pathogens of global significance that can lead to substantial economic losses in aquaculture. Recombination among megalocytiviruses is typically assumed to be rare, although it has been relatively understudied. Here, we uncover widespread recombination within megalocytiviruses through detailed analyses of 63 Megalocytivirus genomes, including two which are newly sequenced and assembled. We also identify a number of genes which megalocytiviruses have likely obtained from outside the family Iridoviridae (iridovirids). These results have serious implications for the biosecurity management of megalocytiviruses, as they indicate that Megalocytivirus strains could be misclassified based on traditional approaches which target individual loci in the genome. We use this new knowledge of recombination to estimate updated phylogenetic trees of megalocytiviruses at the family-, genus-, and species-level. These trees show strong support for the designation of two novel species within the genus Megalocytivirus and highlight the difficulty of placing highly recombinant genomes in a single phylogenetic framework. We discuss the implications of our work for disease management, and the importance of genome-wide recombination detection and phylogenomic analysis in the classification and genetic characterisation of megalocytiviruses.

evolutionary biology↗

bioRxiv2018

A large-scale whole-genome sequencing analysis reveals highly specific genome editing by both Cas9 and Cpf1 nucleases in rice

Targeting specificity has been an essential issue for applying genome editing systems in functional genomics, precise medicine and plant breeding. Understanding the scope of off-target mutations in Cas9 or Cpf1-edited crops is critical for research and regulation. In plants, only limited studies had used whole-genome sequencing (WGS) to test off-target effects of Cas9. However, the cause of numerous discovered mutations is still controversial. Furthermore, WGS based off-target analysis of Cpf1 has not been reported in any higher organism to date. Here, we conducted a WGS analysis of 34 plants edited by Cas9 and 15 plants edited by Cpf1 in T0 and T1 generations along with 20 diverse control plants in rice, a major food crop with a genome size of ~380 Mb. The sequencing depth ranged from 45X to 105X with reads mapping rate above 96%. Our results clearly show that most mutations in edited plants were created by tissue culture process, which caused ~102 to 148 single nucleotide variations (SNVs) and ~32 to 83 insertions/deletions (indels) per plant. Among 12 Cas9 single guide RNAs (sgRNAs) and 3 Cpf1 CRISPR RNAs (crRNAs) assessed by WGS, only one Cas9 sgRNA resulted in off-target mutations in T0 lines at sites predicted by computer programs. Moreover, we cannot find evidence for bona fide off-target mutations due to continued expression of Cas9 or Cpf1 with guide RNAs in T1 generation. Taken together, our comprehensive and rigorous analysis of WGS big data across multiple sample types suggests both Cas9 and Cpf1 nucleases are very specific in generating targeted DNA modifications and off-targeting can be avoided by designing guide RNAs with high specificity.

molecular biology↗

bioRxiv2018

Functional genomics and programmed genome editing of omega-1 of the blood fluke Schistosoma mansoni

CRISPR/Cas9 based genome editing has yet been reported in parasitic or indeed any species of the phylum Platyhelminthes. We tested this approach by targeting omega-1 ({omega}1) of Schistosoma mansoni as a proof of principle. This secreted ribonuclease is crucial for Th2 priming and granuloma formation, providing informative immuno-pathological readouts for programmed genome editing. Schistosome eggs were either exposed to Cas9 complexed with a synthetic guide RNA (sgRNA) complementary to exon 6 of {omega}1 by electroporation or transduced with pseudotyped lentivirus encoding Cas9 and the sgRNA. Some eggs were also transduced with a single stranded oligodeoxynucleotide donor transgene that encoded six stop codons, flanked by 50 nt-long 5-and 3-microhomology arms matching the predicted Cas9-catalyzed double stranded break (DSB) within {omega}1. CRISPResso analysis of amplicons spanning the DSB revealed [~]4.5% of the reads were mutated by insertions, deletions and/or substitutions, with an efficiency for homology directed repair of 0.19% insertion of the donor transgene. Transcripts encoding {omega}1 were reduced >80% and lysates of {omega}1-edited eggs displayed diminished ribonuclease activity indicative that programmed editing mutated the {omega}1 gene. Whereas lysates of wild type eggs polarized Th2 cytokine responses including IL-4 and IL-5 in human macrophage/T cell co-cultures, diminished levels of the cytokines followed the exposure to lysates of {omega}1-mutated schistosome eggs. Following injection of schistosome eggs into the tail vein of mice, the volume of pulmonary granulomas surrounding {omega}1-mutated eggs was 18-fold smaller than wild type eggs. Programmed genome editing was active in schistosomes, Cas9-catalyzed chromosomal breakage was repaired by homology directed repair and/or non-homologous end joining, and mutation of {omega}1 impeded the capacity of schistosome eggs both to drive Th2 polarization and to provoke formation of pulmonary circumoval granulomas. Knock-out of {omega}1 and the impaired immunological phenotype showcase the novel application of programmed gene editing in and functional genomics for schistosomes.

molecular biology↗

bioRxiv2019

The genomic landscape of metastatic castration-resistant prostate cancers using whole genome sequencing reveals multiple distinct genotypes with potential clinical impact

Here we present whole-genome sequencing (WGS) analysis of fresh-frozen metastatic biopsies from 197 castration-resistant prostate cancer patients. Using hierarchical unsupervised clustering based on genomic aberrations only, we defined eight different clusters. We detected four distinct and potentially clinically relevant genotypes harboring unique genomic features, including: 1) Microsatellite Instability; 2) Homologous Recombination Deficiency (HRD) with enriched genomic deletions and BRCA2 aberrations; 3) tandem duplication phenotype associated with biallelic CDK12 mutations; and 4) a subgroup enriched for chromothripsis events. Our data suggest that classifying patients using WGS characteristics may improve classification of HRD patients. Moreover, we confirmed that important regulators of AR-mediated signaling are located in non-coding regions. Using ChIP sequencing data, we showed that the amplified AR and MYC promoter regions contain open chromatin and bind AR, suggesting a role in AR mediated biology. Thus, high-resolution WGS may be used to improve patient stratification.

cancer biology↗

bioRxiv2019

Genomes from Bacteria Associated with the Canine Oral Cavity: a Test Case for Automated Genome-Based Taxonomic Assignment

Taxonomy for bacterial isolates is commonly assigned via sequence analysis. However, the most common sequence-based approaches (e.g. 16S rRNA gene-based phylogeny or whole genome comparisons) are still labor intensive and subjective to varying degrees. Here we present a set of 33 bacterial genomes, isolated from the canine oral cavity. Taxonomy of these isolates was first assigned by PCR amplification of the 16S rRNA gene, Sanger sequencing, and taxonomy assignment using BLAST. After genome sequencing, taxonomy was revisited through a manual process using a combination of average nucleotide identity (ANI), concatenated marker gene phylogenies, and 16S rRNA gene phylogenies. This taxonomy was then compared to the automated taxonomic assignment given by the recently proposed Genome Taxonomy Database (GTDB). We found the results of all three methods to be similar (25 out of the 33 had matching genera), but the GTDB approach was less subjective, and required far less labor. The primary differences in the remaining taxonomic assignments related to proposed taxonomy changes by the GTDB team.

bioinformatics↗

bioRxiv2019

The user’s guide to comparative genomics with EnteroBase. Three case studies: micro-clades within Salmonella enterica serovar Agama, ancient and modern populations of Yersinia pestis, and core genomic diversity of all Escherichia

EnteroBase is an integrated software environment which supports the identification of global population structures within several bacterial genera that include pathogens. Here we provide an overview on how EnteroBase works, what it can do, and its future prospects. EnteroBase has currently assembled more than 300,000 genomes from Illumina short reads from Salmonella, Escherichia, Yersinia, Clostridiodes, Helicobacter, Vibrio, and Moraxella, and genotyped those assemblies by core genome Multilocus Sequence Typing (cgMLST). Hierarchical clustering of cgMLST sequence types allows mapping, a new bacterial strain to predefined population structures at multiple levels of resolution within a few hours after uploading its short reads. Case study 1 illustrates this process for local transmissions of Salmonella enterica serovar Agama between neighboring social groups of badgers and humans. EnteroBase also supports SNP calls from both genomic assemblies and after extraction from metagenomic sequences, as illustrated by case study 2 which summarizes the microevolution of Yersinia pestis over the last 5,000 years of pandemic plague. EnteroBase can also provide a global overview of the genomic diversity within an entire genus, as illustrated by case study 3 which presents a novel, global overview of the population structure of all of the species, subspecies and clades within Escherichia.

microbiology↗

bioRxiv2019

Genomic re-assessment of the transposable element landscape of the potato genome

Transposable elements (TEs) are DNA sequences with the ability to auto-replicate and move throughout the host genome. TEs are major drivers in stress response and genome evolution. Given their significance, the development of clear and efficient TE annotation pipelines has become essential for many species. The latest de novo TE discovery tools, along with available TEs from Repbase and sRNA-seq data, allowed us to perform a reliable potato TEs detection, classification and annotation through an open-source and freely available pipeline (https://github.com/DiegoZavallo/TE_Discovery). Using a variety of tools, approaches and rules, our pipeline revealed that ca. 16% of the potato genome can be clearly annotated as TEs. Additionally, we described the distribution of the different types of TEs across the genome, where LTRs and MITEs present a clear clustering pattern in pericentromeric and subtelomeric/telomeric regions respectively. Finally, we analyzed the insertion age and distribution of LTR retrotransposon families which display a distinct pattern between the two major superfamilies. While older Gypsy elements concentrated around heterochromatic regions, younger Copia elements located predominantly on euchromatic regions. Overall, we delivered not only a reliable, ready-to-use potato TE annotation files, but also all the necessary steps to perform de novo detection for other species. Key MessageWe provide a comprehensive and reliable potato TE landscape, based on a wide variety of identification tools and integrative approaches, producing clear and ready-to-use outputs for the scientific community.

plant biology↗

bioRxiv2019

Extraction of genome-wide reads distribution pattern through whole genome sequencing of cell-free DNA for tracking tissue-of-origin in cancer patients

Somatic mosaicism is widespread among tissues and could indicate distinct tissue origins of circulating cell-free DNA (cfDNA), DNA fragments released by lytic cells into the blood. By investigating the alignment patterns of whole genome sequencing reads with the genomic DNA of different tissues, we found that the read distributions formed type-specific patterns in some regions as a result of somatic mosaicism. We then utilized this information to construct a tissue-of-origin mapping model and evaluated its predictive performance on whole genome sequencing data from tissue and cfDNA samples. In total, 1,545 tissue samples associated with 13 cancer types were included, and identification of the tissue of origin achieved a specificity of 82% and a sensitivity of 80%. Furthermore, a total of 30 cfDNA samples from lung cancer and liver cancer patients and healthy controls were analyzed to predict their tissues of origin with a specificity of 87% and a sensitivity of 87%. Our results show that read distribution patterns from whole genome sequencing could be used to identify cfDNA tissues of origin with high accuracy, suggesting the potential application of our model to early cancer detection and diagnosis.

bioinformatics↗

bioRxiv2019

Uniform Genomic Data Analysis in the NCI Genomic Data Commons

The goal of the National Cancer Institute (NCI) Genomic Data Commons (GDC) is to provide the cancer research community with a data repository of uniformly processed genomic and associated clinical data that enables data sharing and collaborative analysis in the support of precision medicine. The initial GDC dataset include genomic, epigenomic, proteomic, clinical and other data from the NCI TCGA and TARGET programs. Data production for the GDC started in June, 2015 using an OpenStack-based private cloud. By June of 2016, the GDC had analyzed more than 50,000 raw sequencing data inputs, as well as multiple other data types. Using the latest human genome reference build GRCh38, the GDC generated a variety of data types from aligned reads to somatic mutations, gene expression, miRNA expression, DNA methylation status, and copy number variation. In this paper, we describe the pipelines and workflows used to process and harmonize the data in the GDC. The generated data, as well as the original input files from TCGA and TARGET, are available for download and exploratory analysis at the GDC Data Portal and Legacy Archive (https://gdc.cancer.gov/).

bioinformatics↗

bioRxiv2025

Reverse gyrase and 3D genome architecture suppress hyperthermophile genome instability arising from horizontal gene transfer

Reverse gyrase (Rgy), a distinctive topoisomerase conserved in all hyperthermophiles, has the unique ability to introduce positive DNA supercoils. It has long been hypothesized that Rgy overwinds genomic DNA to prevent its detrimental denaturation at high temperature. However, its role in vivo has remained unresolved for more than four decades. In the course of investigating how Rgy affects genome organization in the archaeon Thermococcus kodakarensis, we find that Rgy suppresses heat-induced clustering of AT-rich genes, most of which bear signatures of horizontal gene transfer. This function depends on the topoisomerase active site of Rgy, underscoring a critical role of its action on DNA topology. The clustering of AT-rich genes is accompanied by their aberrant recruitment of the single-stranded DNA-binding protein RPA, indicative of extensive DNA melting. Genetic analysis further provides evidence that RPA sequesters denatured loci and mitigates genome instability in the absence of Rgy. We propose that DNA topology and higher-order genome organization constitute a multilayered mechanism that stabilizes horizontally acquired genes at high temperature.

molecular biology↗

bioRxiv2025

Whole-Genome Population Genomics Reveals Lineage Structure and Adaptive Potential of Philaenus spumarius, the Principal Vector of Xylella fastidiosa in Europe

Philaenus spumarius (L.), the meadow spittlebug, is the principal European vector of Xylella fastidiosa. This xylem-feeding insect has a broad host range, ecological plasticity, and mobility, making it an efficient vector across diverse landscapes. Yet, major gaps remain in understanding its genetic diversity, migration patterns, and local adaptation, limiting effective control of X. fastidiosa outbreaks. To address these gaps, we worked with a global network of researchers and citizen scientists to assemble a geographically and ecologically diverse collection of P. spumarius samples. We generated a chromosome-level genome assembly for P. spumarius and high-quality assemblies for four related spittlebug species, providing a robust genomic framework for evolutionary and epidemiological studies. Resequencing 430 P. spumarius individuals from across the globe uncovered boreal, temperate, and semi-arid lineages shaped by geography and climate. The lineages vary in reproductive isolation and in the degree of mito-nuclear divergence. Iberian populations grouped with an individual identified as P. tesselatus contributing to regional genetic complexity. Genomic scans identified loci under selection in Apulian populations, including signatures in sulfotransferase (SULT) genes potentially linked to behavioural plasticity, host plant specialisation, or insecticide resistance in olive-growing regions. Migration analyses indicated limited long-distance dispersal but strong local connectivity, consistent with the rapid regional spread of X. fastidiosa. Together, these findings reveal the lineage structure and adaptive potential of P. spumarius, the key vector of X. fastidiosa in Europe. They underscore the importance of targeted surveillance of locally adapted populations and provide genomic tools for monitoring vector dynamics and mitigating emerging disease risks.

evolutionary biology↗

bioRxiv2026

Global genomic epidemiology of Candida auris: analysis of 12,644 whole genome sequences from 1997-2024

Candida auris is a critical priority fungal pathogen (World Health Organization) that has emerged into human populations pre-1996 from an unknown environmental reservoir. Genomic sequencing data has been used extensively in the last decade, leading to a largely unharnessed dataset with potential to unlock understanding of emerging fungal pathogen evolution. Here, we compiled publicly available Illumina paired-end whole genome sequences (WGS) for variant calling, supplemented by isolates from the worlds first four-clade single-facility outbreak in Algeria (n = 7), totaling 12,644 WGS. We describe the geographic, clinical, and temporal epidemiology of the outbreak across the globe between 1997 and 2024, indicating a conserved six-clade structure. Despite the evidence of earlier species diversity in low- and lower-middle income countries (LLMIC), the majority of WGS derived from high-income (12,035, 93.2%) and upper-middle-income countries (719, 5.57%), where cases are believed to be imported from endemic regions, with few from lower-middle-income countries (165, 1.28%) and none from lower-income countries. Copy number variation was present, including azole drug target ERG11, with seven isolates displaying ten to fifteen copies. Alarmingly, the standing variation of C. auris reveals emerging variation in hot-spots of the echinocandin drug target FKS1, which encodes a {beta}-1,3-glucan synthase. Eighteen emerging FKS1 hot-spot variants have not been detected or described in databases, and mostly occured in Northern America (57/61, 93.4%), where echinocandin monotherapy is standard treatment. Well-studied FKS1 variants known to cause resistance are significantly more common in clade I isolates derived from urine compared to blood, consistent with a role for the urinary niche as a low echinocandin concentration safe-haven for the development of resistance. The insights from this global genomic epidemiology survey of C. auris highlight sequencing inequality and detect ongoing genomic innovation in clinical settings, raising predictable and urgent concerns around the ongoing use of echinocandin monotherapy and potential emerging antifungal drug resistance-related genotypes in high-income settings.

microbiology↗

bioRxiv2026

Genomic correlates of hyperthermostability revisited: a large-scale validation across 1,963 microbial genomes

In 2003, Suhre and Claverie demonstrated that the difference between the fraction of charged amino acids and the fraction of polar uncharged amino acids in a proteome (the CvP-bias) was the single genomic feature that most strongly discriminates hyperthermophilic microorganisms from their mesophilic and thermophilic counterparts. The original analysis was based on 71 completely sequenced genomes available at the time. Here, using modern genome databases -- specifically the Bacterial and Viral Bioinformatics Resource Center (BV-BRC) for curated optimal growth temperature (OGT) metadata and the NCBI RefSeq FTP archive for sequence data -- the same analysis is repeated at approximately 28-fold larger scale, covering 1,963 bacterial and archaeal genomes (103 hyperthermophiles, 409 thermophiles, 1,451 mesophiles). The original finding is confirmed with high statistical confidence: CvP-bias is elevated in hyperthermophiles (mean 11.1 {+/-} 3.1%) relative to mesophiles (5.0 {+/-} 2.0%; ANOVA F = 496, p < 10-170), with a large effect size (Cohens d = 2.32) and area under the receiver operating characteristic curve of 0.94 for binary hyper/meso classification. Principal component analysis confirms that the first principal component, explaining 47% of variance, is loaded by CvP-bias as a major contributor, separating hyperthermophiles from other organisms. These results establish that the CvP-bias signal identified in 2003 is not an artifact of small sample size but a genuine, robust property of hyperthermophilic proteomes.

microbiology↗

bioRxiv2026

Genome-wide association study and genomic prediction of lucerne traits shaping living mulch performance

Lucerne is gaining interest as a living mulch in agroecological productions. However, its vigorous growth can lead to competition with cash crops for light and nutrients, necessitating new ideotypes. This study investigated the genetic basis of traits relevant to ideotype breeding: dormancy, spring regrowth, height, growth habit, leaflet size, stem diameter, and plant structure. Individuals from a diversity panel of 27 accessions and a synthetic population were phenotyped in a spaced plant nursery. Over 100,000 SNP markers were used for genotyping. Genome-wide association study (GWAS) and genomic prediction were conducted, considering population structure. Heritability estimates ranged from moderate to high in diversity panel (h{superscript 2} = 0.36-0.70) but were lower in synthetic population (h{superscript 2} = 0.17-0.33), reflecting reduced genetic variance. Trait correlations differed markedly between populations, indicating the possibility of recombining traits to create new ideotypes. GWAS identified a few QTL (r{superscript 2} up to 0.27) for leaflet size, height, growth habit, and plant structure, with candidate genes linked to growth, stress response, and signalling pathways. Genomic prediction was highly accurate in diversity panel, where broad genetic variation allowed reliable estimation of marker effects, with prediction accuracies exceeding 0.8 for heritable traits, including growth habit and leaflet size. In contrast, accuracies were low in synthetic population, reflecting its limited diversity and small size, whether training was based on the synthetic population itself or on the diversity panel. These results highlight the potential to recombine traits and develop lucerne ideotypes using molecular tools such as QTL detection and genomic prediction.

plant biology↗

bioRxiv2026

Whole-Genome Sequencing and Phylogenetic Analysis of Anolis adenovirus 2 Reveals Conserved Genome Organization and Gene-Specific Evolutionary Patterns

Adenoviruses, which infect vertebrates, have a rich history of evolution that includes both host switching and coevolution, particularly within Barthadenovirus, a genus that infects squamate reptiles, birds, and mammals. Potential host-switching events can be identified by comparing the evolutionary histories between viruses and their hosts; however, many Barthadenovirus phylogenies have been inferred based on a limited number of easily-amplifiable gene segments. Whole-genome sequencing novel strains of Barthadenovirus can provide greater phylogenetic confidence, and therefore more accurately identify host-switching when it occurs. Here, we present the whole-genome sequence, annotation, reconciled species tree, and molecular evolution analyses of two isolates of Anolis adenovirus 2, a member of Barthadenovirus. Our two isolates of Anolis adenovirus 2 are sister lineages with very high sequence similarity. Our results support existing hypotheses regarding the ancestral hosts of Barthadenovirus (squamate reptiles), and proposed host switching events within and between squamate reptiles and other vertebrate classes. We leverage our novel genome annotations to perform comparative synteny analyses, identifying a set of shared genes across Barthadenovirus whose gene order is largely conserved within the genus. Finally, our molecular evolution analyses highlight trends in evolutionary pressures on individual genes: Genes associated with viral replication and structure have experienced slower rates of evolution than those encoding proteins involved in host interaction. Our two sequenced isolates of Anolis adenovirus 2 add to an expanding number of Adenovirus genomic resources and facilitate future investigations into the patterns and processes shaping adenovirus diversification.

evolutionary biology↗