Evolutionary Dynamics Research Articles

Web Visualization for Spatiotemporal Genomic Epidemiology, Annotation, and Mutation Dynamics of Orthohantavirus hantanense Using Nextstrain.

The phylogeographic inference approach aims to connect genomic data with epidemiology to understand the spread and evolution of pathogens using visualization of spatiotemporal reconstructions. Orthohantavirus hantanense (HTNV), the causative agent of hemorrhagic fever with renal syndrome (HFRS), represents a significant global public health concern. Here, we introduce a localized Nextstrain platform for HTNV, offering a comprehensive resource for facilitating spatiotemporal genomic surveillance and the study of evolutionary dynamics of viral genomes. Nextstrain enables web-based visualization and simple sharing of graphic and numeric data through unique web addresses. The Nextstrain build for HTNV stands out for its user-friendly interface and is readily accessible online at https://nextstrain.org/community/KU-MV/Hantavirus. This study provides valuable insights into genomic surveillance, viral phylodynamics, and the evolutionary history of orthohantaviruses for the development of public health policies against endemic HFRS outbreaks.

Genomic Epidemiology of Erwinia amylovora Strains That Caused the Fire Blight Outbreak in Korea.

Fire blight, a devastating bacterial disease affecting rosaceous plants such as apples and pears, is caused by Erwinia amylovora. The disease, known for its rapid spread and destructive potential, can lead to severe symptoms and often result in the death of infected plants. In Korea, the observation of E. amylovora was first recorded in 2015, and subsequent dissemination has been noted across the peninsula. We previously determined the genomic characteristics of five Korean E. amylovora strains isolated in 2015, demonstrating a close phylogenetic relationship to a North American lineage. Based on these results, we have now sequenced a collection of 92 E. amylovora genomes from various fire blight cases in Korea spanning from 2016 to 2018. Through the population genomic approach, we explored the genetic diversity among the isolates. Our comprehensive genomic assessment facilitated the reconstruction of phylogenetic relationships, the correlation of genetic and spatial distances, and the inference of the most recent common ancestor of Korean E. amylovora. We conclude that E. amylovora was introduced to Korea several years before the first disease outbreak. Genomic insights obtained from our investigation will be invaluable for understanding the genetic characteristics, geographic spread and evolutionary dynamics of Korean E. amylovora.

Evolutionary Dynamics and Expression Divergence of the MADS-Box Gene Family During Recent Speciation of AA-Genome Oryza Species

To investigate the evolutionary trajectory during the recent speciation of AA-genome Oryza species, we conducted a comprehensive analysis of the MADS-box gene family across eight Oryza species. We identified 1093 MADS-box genes in total and systematically examined their evolutionary history, gene family expansion, and expression divergence. Our results revealed that extensive lineage-specific expansions occurred in AA-genome Oryza species, which were primarily generated by proximal and tandem duplications, with a particularly notable episode in Type-I genes. Despite the significant expansion, Type-I genes were generally expressed at low levels or not expressed across various organs. In contrast, the expansion of Type-II genes was primarily observed in the AG, AGL12, SOC1, GGM13, and MIKC* subfamilies, which exhibited high levels of expression in reproductive organs such as panicles and stigmas. Additionally, we found species-specific gene expression in the two out-crossing wild rice species, Oryza rufipogon and Oryza longistaminata. Notably, a unique MADS-box gene in O. longistaminata exhibited high expression levels in rhizomes and stems, which may be associated with the species’ distinctive rhizomatous growth habit.

Evolutionary Mechanism of Trust for Public–Private Partnership Projects with Public Participation

Trust plays a pivotal role in sustaining cooperative relationships and serves as the cornerstone of successful cooperation among participants in public–private partnership (PPP) projects. A decline in trust can result in unethical behavior, fractured relationships, and even project failure. Despite its critical importance, there is an insufficiency of research exploring the evolutionary dynamics of trust, particularly from the perspective of public participation. To address this, an evolutionary game model is constructed to analyze the evolution of behavioral strategies and identify their stable state from a dynamic perspective. The model examines the influence of trust levels and public participation on cooperation, revealing that trust asymmetry negatively impacts collaborative outcomes in PPP projects. There is an optimal trust level: trust positively influences cooperation up to a certain peak, beyond which excessive trust becomes detrimental and hinders cooperative outcomes. When trust levels are equal, the selection of behavioral strategies is influenced by the initial willingness of participants. A threshold exists for this initial willingness, above which cooperation is more likely to succeed. Additionally, public participation is found to significantly enhance cooperation. These conclusions advance the theoretical advancements of trust in PPP projects and offer valuable insights for policymakers and stakeholders in addressing trust-related challenges, thereby fostering sustainable collaboration.

Phylogenetic Analysis of Chandipura virus: Insights from a Preliminary Genomic Study

Chandipura virus (CHPV) is an arthropod-borne virus linked to encephalitis in humans, primarily in India. Its evolutionary dynamics and transmission pathways remain poorly understood due to limited genomic data. This study analyzed 23 publicly available CHPV genomes, including isolates from humans, sandflies, and a hedgehog, retrieved from GenBank. Phylogenetic analyses were conducted to explore host-specific and geographic evolutionary patterns. Phylogenetic analysis revealed distinct evolutionary lineages. Human-derived genomes collected in India between 2003 and 2024 formed a well-supported monophyletic clade, suggesting a unique evolutionary lineage. In contrast, sandfly-derived genomes exhibited diverse clustering patterns. Notably, Kenyan sandfly isolates from 2016–2017 were phylogenetically closer to human-derived sequences, suggesting possible shared evolutionary pressures. These findings provide preliminary insights into CHPV evolution and emphasize the need for enhanced genomic surveillance in both human and non-human populations. Expanding genomic data is essential to validate these observations and inform public health strategies.

Asymmetric Metabolic Adaptations Undermine Stability in Microbial Syntrophy

Abstract Syntrophic interaction, driven by metabolite exchange, is widespread within microbial communities. However, co-inoculation of most auxotrophic microorganisms often fails to establish a stable metabolite exchange relationship. Here, we engineered two auxotrophic Escherichia coli strains, each dependent on the other for essential amino acid production, to investigate the dynamics of syntrophic relationships. Through invasion-from-rare experiments, we observed the rapid formation of syntrophic consortia stabilized by frequency-dependent selection, converging to a 2:1 ratio of lysine-to-arginine auxotrophs. However, laboratory evolution over 25 days revealed that syntrophic interactions were evolutionarily unstable, with cocultures collapsing as ΔL cells dominated the population. Reduced fitness in cocultures was driven by the emergence of a “selfish” ΔL phenotype, characterized by decreased arginine production and exploitation of lysine produced by ΔA cells. Dynamic metabolic assays revealed that metabolite production and utilization patterns strongly influenced the fitness of each strain. ΔL cells displayed metabolic plasticity, adjusting lysine utilization in response to lysine availability, which enabled them to outcompete ΔA cells. In contrast, ΔA cells lacked similar plasticity, resulting in their negative selection. These findings demonstrate that asymmetric metabolic responses and the emergence of selfish phenotypes destabilize syntrophic relationships. Our work underscores the importance of balanced metabolic exchanges for developing sustainable synthetic microbial consortia and offers insights into the evolutionary dynamics of microbial cooperation.

Novel H16N3 avian influenza viruses isolated from migratory gulls in China in 2023

As a rare subtype of avian influenza virus, H16 viruses are predominant in gulls but rarely found in domestic birds. The low prevalence of H16 viruses has limited our understanding of their epidemiology and evolutionary dynamics. In this study, we isolated three novel H16N3 viruses from migratory gulls in East Asian–Australasian Flyway in eastern China in 2023, which are significantly different from previously identified isolates. To fully understand the epidemiology and genetics characteristics of the global H16 viruses, we compared the host divergence of several rare subtypes and determined that the H13 and H16 subtypes were predominantly pooled into different species of gulls by sharing their internal genes, whereas the waterfowl of Anatidae served as the primary natural reservoirs of the H8, H11, H12, H14, and H15 subtypes. Detailed phylogenetic analysis revealed the evolutionary divergence of globally circulating H16 viruses and their frequent gene reassortment. Furthermore, the gull origin H13 and H16 viruses collectively served as gene donors for the newly emerged highly pathogenic clade 2.3.4.4b H5N1 viruses because the H13/H16-like PA, NP, and NS genes have been introduced into circulating H5N1 viruses since May 2022 in Europe. To date, the H5N1 reassortants containing the H13/H16-like gene segments have been detected in wild and domestic birds and resulted in mammal and human infections. These results improve our knowledge of the ecology and genetics of H16 viruses and emphasize the need for surveillance to monitor the emergence of novel avian influenza viruses in migratory birds.

Exploring the Frontier of Genetic Diversity: Recent Advances in Pan-genomics

Pan-genomics has revolutionized our understanding of genetic diversity within species by providing a comprehensive view of both core and variable genes across multiple genomes. Recent advances in this field, driven by innovations in sequencing technologies, computational methods, and integrative approaches with other -omics fields, have unveiled new layers of complexity in genome architecture, gene regulation, and evolutionary dynamics. This mini-review explores key developments in pan-genomics, including the construction of graph-based pan-genomes, applications in crop improvement, integration with epigenomics, and the emerging role of machine learning. These advances underscore the potential of pan-genomics to drive future discoveries in genomics, with significant implications for agriculture, evolutionary biology, and personalized medicine.

OrthoBrowser: Gene Family Analysis and Visualization

Abstract Motivation The analysis of gene families across diverse species is pivotal in elucidating evolutionary dynamics and functional genomic landscapes. Typical analysis approaches often require significant computational expertise and user time. Results We introduce OrthoBrowser, a static site generator that will index and serve phylogeny, gene trees, multiple sequence alignments, and novel multiple synteny alignments. This greatly enhances the usability of tools like OrthoFinder by making the detailed results much more visually accessible. This interface can scale reasonably up to hundreds of genomes, allows a user to filter this large dataset to a subset of samples they are interested in at that particular moment in time, or “zoom in” to only a subtree of the orthogroup. The multiple synteny alignment method uses a progressive hierarchical alignment approach in the protein space using orthogroup membership to establish orthology. Orthobrowser makes it easy for users to identify, interact with, explore, and share key information about their gene families of interest. Availability and implementation OrthoBrowser is pip installable and is available under MIT license at: https://gitlab.com/salk-tm/orthobrowser. Complete example OrthoBrowser results are available at: https://orthobrowserexamples.netlify.app/.

Chromosome-level genome assembly and annotation of Japanese anchovy (Engraulis japonicus)

The Japanese anchovy (Engraulis japonicus), a finfish with the largest biomass of a single species in the Yellow and East China Seas, plays an important pivotal role in converting zooplanktons into high trophic fish in the food web. As a result, the fish is regard as a key species in its habiting ecosystem. However, the lack of genomic resources hampers our understanding of its genetic diversity and differentiation, as well as the evolutionary dynamics. Here, we firstly report a complex chromosome-level genome assembly of E. japonicus with a large size of 1.4 Gb, with features of high repetitive sequences (54.9%), high heterozygosity (2.3%) and a number of protein-coding genes (24,405). The genome sequence exhibited a remarkable degree of completeness, valued 94.07% of the complete BUSCO. This work firstly reported the genome sequence of E. japonicus, offering the crucial resources for further studies on the genetic diversity and adaptive evolution of this species.

DOP128 Spatial transcriptomic analysis reveals evolutionary dynamics in colitis-associated colorectal cancer

Abstract Background Colitis-associated colorectal cancer (CAC) is a severe complication of inflammatory bowel disease, particularly ulcerative colitis. Despite its clinical significance, the spatiotemporal dynamics of neoplastic progression in CAC remain poorly understood. This study aimed to elucidate the evolutionary trajectory of CAC using spatial transcriptomics. Methods We employed spatial transcriptomics to analyze four tissue samples from three patients with ulcerative colitis at various stages of neoplastic progression. Whole-slide spatial transcriptomics RNA sequencing was performed, enabling simultaneous analysis of gene expression and tissue architecture. Integrated spatial transcriptomic data revealed recurring epithelial programs along the progression of copy number alteration. Results Our analysis using copy number variation (CNV) score and Hematoxylin and Eosin (H&E) staining image revealed progressive changes in gene expression patterns as tissue transitioned from non-tumor to pre-tumor and finally to tumor states. This progression was quantified using beta coefficient values, allowing us to track gradual alterations in gene expression across the disease continuum. We identified a core set of 48 genes that exhibited consistent changes across all four analyzed samples. Among these, 40 genes showed a progressive decrease in expression, while eight genes, including CLDN1, E2F1, and RECQL4, demonstrated a gradual increase in expression as the tissue advanced towards a tumorous state. These upregulated genes pointed to potential oncogenic drivers or compensatory mechanisms activated during tumor development. Pathway analysis of the 48 genes revealed enrichment in key cellular processes, including p53 signaling, transcriptional regulation by E2F factors, and role of PKR in interferon induction and antiviral response. Upstream Regulator analysis revealed YAP1 and TGFB1 as the most significant regulators, highlighting the importance of transcriptional control and growth factor signaling in CAC development. Conclusion This study provides unprecedented insights into the spatiotemporal dynamics of CAC progression. By leveraging spatial transcriptomics, we have mapped the evolutionary trajectory of CAC at high resolution, revealing key molecular events and spatial features associated with disease progression. These findings not only enhance our understanding of CAC pathogenesis but also highlight potential targets for early intervention and personalized treatment strategies.

The origin and transmission of HIV-1 CRF80_0107 among two major first-tier cities in China

BackgroundCRF01_AE and CRF07_BC are the two most prevalent HIV-1 genotypes in China, and the co-circulation of these two genotypes has led to the continuous generation of CRF_0107 viruses in recent years. However, little is known about the origin and spread of CRF_0107 viruses thus far. This study focused on HIV-1 CRF80_0107, which we previously identified among the MSM population in Beijing and Hebei Province, to explore the demographic distribution, transmission links, and temporal-spatial evolutionary features of the HIV-1 CRF80_0107 strain in China.MethodsWith the partial pol region fragment of the HIV-1 CRF80_0107 subtype standard sequence as a reference, BLAST was used to search for highly similar sequences in the Los Alamos HIV Sequence Database, followed by preliminary subtype identification via COMET. Further phylogenetic and recombination breakpoint analyses were conducted to verify the subtypes and recombination patterns. We also performed a distance-based molecular network analysis to identify potential relationships among different HIV-positive individuals. In addition, spatiotemporal evolutionary dynamics analysis of the candidate CRF80_0107 sequences was performed via a Bayesian approach.ResultsA total of 36 partial pol gene sequences of HIV-1 CRF80_0107 were identified from 2009 to 2018 from 5 provinces in China. Phylogenetic and spatial-temporal dynamics analyses indicated that CRF80_0107 likely originated in Beijing around 2009 and spread to Guangdong Province around 2012. Population dynamics analysis revealed that CRF80_0107 experienced a significant increase in population size from 2009 to 2011 and then stabilized. The study also found that the number of cases in Guangdong Province was second only to that in Beijing and formed 2 relatively independent transmission clusters in the MSM population in Shenzhen, Guangdong Province.ConclusionsThe HIV-1 CRF80_0107 strain has spread to cities beyond its origin, particularly the MSM population in Shenzhen city, Guangdong Province, which is an area with a high incidence of HIV. This highlights the importance of continuous monitoring for the emergence and dynamic changes of novel HIV-1 recombinant viruses and the necessity of implementing effective preventive measures targeting specific populations in particular regions.

Geographic distance shapes morphological and molecular disparities in a common diatom species as revealed by geometric morphometry and metabarcoding

Abstract Diatoms, a highly diverse group, are sensitive indicators of aquatic environmental conditions, yet doubts persist regarding species distribution and distance-based factors influencing their populations. This study explores the relationship between geographical distance and the morphological and genetic differences among Achnanthidium minutissimum populations in freshwater ecosystems. Cell outlines were objectively captured by means of geometric morphometry, whereas rbcL marker was used for molecular approach. Our results indicate that the geographical factors significantly influence the morphological and molecular disparities among populations. Despite the absence of evident physical barriers, persistent spatial distance contributes to the observed differentiation among diatom populations. This emphasizes the crucial role of geographical isolation in shaping genetic divergence and morphological disparities, highlighting the need to consider spatial factors in understanding diatom diversity and distribution. Additionally, the study emphasizes the significance of molecular tools in revealing hidden genetic patterns and exploring evolutionary dynamics within diatom species.

Cross-species single-cell transcriptomics reveals neuronal similarities and heterogeneity in amniote pallium.

The amniote pallium, a vital component of the forebrain, exhibits considerable evolutionary divergence across species and mediates diverse functions, including sensory processing, memory formation, and learning. However, the relationships among pallial subregions in different species remain poorly characterized, particularly regarding the identification of homologous neurons and their transcriptional signatures. In this study, we utilized single-nucleus RNA sequencing to examine over 130 000 nuclei from the macaque ( Macaca fascicularis) neocortex, complemented by datasets from humans ( Homo sapiens), mice ( Mus musculus), zebra finches ( Taeniopygia guttata), turtles ( Chrysemys picta bellii), and lizards ( Pogona vitticeps), enabling comprehensive cross-species comparison. Results revealed transcriptomic conservation and species-specific distinctions within the amniote pallium. Notable similarities were observed among cell subtypes, particularly within PVALB + inhibitory neurons, which exhibited species-preferred subtypes. Furthermore, correlations between pallial subregions and several transcription factor candidates were identified, including RARB, DLX2, STAT6, NR3C1, and THRB, with potential regulatory roles in gene expression in mammalian pallial neurons compared to their avian and reptilian counterparts. These results highlight the conserved nature of inhibitory neurons, remarkable regional divergence of excitatory neurons, and species-specific gene expression and regulation in amniote pallial neurons. Collectively, these findings provide valuable insights into the evolutionary dynamics of the amniote pallium.

Rapid radiation of a plant lineage sheds light on the assembly of dry valley biomes.

Southwest China is characterized by high plateaus, large mountain systems, and deeply incised dry valleys formed by major rivers and their tributaries. Despite the considerable attention given to alpine plant radiations in this region, the timing and mode of diversification of the numerous dry valley plant lineages remain unknown. To address this knowledge gap, we investigated the macroevolution of Isodon (Lamiaceae), a lineage commonly distributed in the dry valleys in southwest China and wetter areas of Asia and Africa. We reconstructed a robust phylogeny encompassing nearly 90% of the approximately 140 extant Isodon species using transcriptome and genome-resequencing data. Our results suggest a rapid radiation of Isodon during the Pliocene that coincided with a habit shift from herbs to shrubs and a habitat shift from humid areas to dry valleys. The shrubby growth form likely acted as a preadaptation allowing for the movement of Isodon species into these dry valleys. Ecological analyses highlight drought-related factors as key drivers influencing the niche preferences of different growth forms and species richness of Isodon. The interplay between topography and the development of the East Asian monsoon since the middle Miocene likely contributed to the formation of the dry valley biome in southwest China. This study enhances our understanding of evolutionary dynamics and ecological drivers shaping the distinctive flora of southwest China and reveals the strategies employed by montane plants in response to climate change and dryland expansion, thus facilitating conservation efforts globally.

Vanadium-Dependent Haloperoxidase Gene Evolution in Brown Algae: Evidence for Horizontal Gene Transfer.

Compared with green plants, brown algae are characterized by their ability to accumulate iodine, contributing to their ecological adaptability in high-iodide coastal environments. Vanadium-dependent haloperoxidase (V-HPO) is the key enzyme for iodine synthesis. Despite its significance, the evolutionary origin of V-HPO genes remains underexplored. This study investigates the genomic and evolutionary dynamics of V-HPOs in brown algae, focusing on Laminariales species, particularly Saccharina japonica. Genomic analyses revealed the extensive expansion of the V-HPO gene family in brown algae, with 88 V-HPOs identified in S. japonica, surpassing the number in red algae. Phylogenetic analysis demonstrated distinct evolutionary divergence between brown and red algal V-HPOs, with the brown algal clade closely related to bacterial V-HPOs. These findings suggest horizontal gene transfer (HGT) played a key role in acquiring V-HPO genes, particularly from Acidobacteriota, a bacterial phylum known for genomic plasticity. Additionally, enriched active transposable elements were identified around V-HPO genomic clusters, highlighting their role in tandem gene duplications and rapid HGT processes. Expression profiling further revealed dynamic regulation of V-HPOs in response to environmental conditions. This study provides new insights into how HGT has driven kelp genomic adaptations and enhances understanding of marine ecological success and evolutionary processes.

Population genomics reveals strong impacts of genetic drift without purging and guides conservation of bull and giant kelp.

Kelp forests are declining in many parts of the northeast Pacific.1,2,3,4 In small populations, genetic drift can reduce adaptive variation and increase fixation of recessive deleterious alleles,5,6,7 but natural selection may purge harmful variants.8,9,10 To understand evolutionary dynamics and inform restoration strategies, we investigated genetic structure and the outcomes of genetic drift and purging by sequencing the genomes of 429 bull kelp (Nereocystis luetkeana) and 211 giant kelp (Macrocystis sp.) from the coastlines of British Columbia and Washington. We identified 6 to 7 geographically and genetically distinct clusters in each species. Low effective population size was associated with low genetic diversity and high inbreeding coefficients (including increased selfing rates), with extreme variation in these genetic health indices among bull kelp populations but more moderate variation in giant kelp. We found no evidence that natural selection is purging putative recessive deleterious alleles in either species. Instead, genetic drift has fixed many such alleles in small populations of bull kelp, leading us to predict (1) reduced within-population inbreeding depression in small populations, which may be associated with an observed shift toward increased selfing rate, and (2) hybrid vigor in crosses between small populations. Our genomic findings imply several strategies for optimal sourcing and crossing of populations for restoration and aquaculture, but these require experimental validation. Overall, our work reveals strong genetic structure and suggests that conservation strategies should consider the multiple health risks faced by small populations whose evolutionary dynamics are dominated by genetic drift.

Reconstructing NOD-like receptor alleles with high internal conservation in Podospora anserina using long-read sequencing.

NOD-like receptors (NLRs) are intracellular immune receptors that detect pathogen-associated cues and trigger defense mechanisms, including regulated cell death. In filamentous fungi, some NLRs mediate heterokaryon incompatibility, a self/non-self recognition process that prevents the vegetative fusion of genetically distinct individuals, reducing the risk of parasitism. The het-d and het-e NLRs in Podospora anserina are highly polymorphic incompatibility genes (het genes) whose products recognize different alleles of the het-c gene via a sensor domain composed of WD40 repeats. These repeats display unusually high sequence identity maintained by concerted evolution. However, some sites within individual repeats are hypervariable and under diversifying selection. Despite extensive genetic studies, inconsistencies in the reported WD40 domain sequence have hindered functional and evolutionary analyses. Here we demonstrate that the WD40 domain can be accurately reconstructed from long-read sequencing (Oxford Nanopore and PacBio) data, but not from Illumina-based assemblies. Functional alleles are usually formed by 11 highly conserved repeats, with different repeat combinations underlying the same phenotypic het-d and het-e incompatibility reactions. Protein structure models suggest that their WD40 domain folds into two 7-blade β-propellers composed of the highly conserved repeats, as well as three cryptic divergent repeats at the C-terminus. We additionally show that one particular het-e allele does not have an incompatibility reaction with common het-c alleles, despite being 11-repeats long. Our findings provide a robust foundation for future research into the molecular mechanisms and evolutionary dynamics of het NLRs, while also highlighting both the fragility and the flexibility of β-propellers as immune sensor domains.

Persistent, Private and Mobile genes: a model for gene dynamics in evolving pangenomes.

The pangenome of a species is the set of all genes carried by at least one member of the species. In bacteria, pangenomes can be much larger than the set of genes carried by a single organism. Many questions remain unanswered regarding the evolutionary forces shaping the patterns of presence/absence of genes in pangenomes of a given species. We introduce a new model for bacterial pangenome evolution along a species phylogeny that explicitly describes the timing of appearance of each gene in the species and accounts for three generic types of gene evolutionary dynamics: persistent genes that are present in the ancestral genome, private genes that are specific to a given clade, and mobile genes that are imported once into the gene pool and then undergo frequent horizontal gene transfers. We call this model the Persistent-Private-Mobile (PPM) model. We develop an algorithm fitting the PPM model and apply it to a dataset of 902 Salmonella enterica genomes. We show that the best fitting model is able to reproduce the global pattern of some multivariate statistics like the gene frequency spectrum and the parsimony vs. frequency plot. Moreover, the gene classification induced by the PPM model allows us to study the position of accessory genes on the chromosome depending on their category, as well as the gene functions that are most present in each category. This work paves the way for a mechanistic understanding of pangenome evolution, and the PPM model developed here could be used for dynamics-aware gene classification.

RNA Virus Discovery Sheds Light on the Virome of a Major Vineyard Pest, the European Grapevine Moth (Lobesia botrana).

The European grapevine moth (Lobesia botrana) poses a significant threat to vineyards worldwide, causing extensive economic losses. While its ecological interactions and control strategies have been well studied, its associated viral diversity remains unexplored. Here, we employ high-throughput sequencing data mining to comprehensively characterize the L. botrana virome, revealing novel and diverse RNA viruses. We characterized four new viral members belonging to distinct families, with evolutionary cues of cypoviruses (Reoviridae), sobemo-like viruses (Solemoviridae), phasmaviruses (Phasmaviridae), and carmotetraviruses (Carmotetraviridae). Phylogenetic analysis of the cypoviruses places them within the genus in affinity with other moth viruses. The bi-segmented and highly divergent sobemo-like virus showed a distinctive evolutionary trajectory of its encoding proteins at the periphery of recently reported invertebrate Sobelivirales. Notably, the presence of a novel phasmavirus, typically associated with mosquitoes, expands the known host range and diversity of this family to moths. Furthermore, the identification of a carmotetravirus branching in the same cluster as the Providence virus, a lepidopteran virus which replicates in plants, raises questions regarding the biological significance of this moth virus to the grapevine host. We further explored viral sequences in several publicly available transcriptomic datasets of the moth, indicating potential prevalence across distinct conditions. These results underscore the existence of a complex virome within L. botrana and lay the foundation for future studies investigating the ecological roles, evolutionary dynamics, and potential biocontrol applications of these viruses in the L. botrana-vineyard ecosystem.