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Phylogenomic subsampling and upsampling for efficient evolutionary analyses of big data.

Long runtimes, high memory demands, and reliance on high-performance computing impede phylogenomic analyses. We review a scalable phylogenomic subsampling with upsampling (PSU) framework to address this challenge, which reduces runtime and memory requirements by orders of magnitude. In PSU, small subsamples of sites from a concatenated alignment are analyzed, which are expanded by upsampling before inference, and the resulting inferences are aggregated to obtain evolutionary estimates. PSU harnesses the fact that the computational cost of maximum likelihood analysis is strongly influenced by the number of distinct site patterns in the concatenated alignment, whereas statistical power depends primarily on the amount of evolutionary information represented by the total number of sites and substitutions. By reducing the former while restoring the latter through upsampling, PSU can approximate many full-alignment analyses at substantially lower computational cost. Analysis of simulated and empirical datasets shows that PSU can accurately estimate bootstrap support values, select the optimal substitution model, test evolutionary hypotheses, and infer branch lengths, divergence times, and associated uncertainty measures. PSU also provides distributions of inferred clade support across independent subsamples, enabling detection of conflicting phylogenetic signals that may remain hidden in conventional bootstrap analysis of concatenated alignments. Automated tuning of subsample size, the number of subsamples, and the number of upsampling replicates make PSU practical. We suggest that PSU is a general approach for scalable phylogenomic inference using a broad range of statistical methods. By enabling analyses of genome-scale alignments on commodity hardware, PSU broadens research access and reduces environmental and infrastructural costs of big-data phylogenomics.

Phylogeny

Phylogenomic subsampling and upsampling for efficient evolutionary analyses of big data.

Long runtimes, high memory demands, and reliance on high-performance computing impede phylogenomic analyses. We review a scalable phylogenomic subsampling with upsampling (PSU) framework, in which small subsamples of sites from a concatenated alignment are expanded by upsampling before inference, and the resulting analyses are then aggregated to obtain evolutionary estimates. PSU harnesses the fact that the computational cost of maximum likelihood analysis is strongly influenced by the number of distinct site patterns in the concatenated alignment, whereas statistical power depends primarily on the amount of evolutionary information represented by the total number of sites and substitutions. By reducing the former while restoring the latter through upsampling, PSU can approximate many full-data analyses at substantially lower computational cost. Analysis of simulated and empirical datasets shows that PSU can accurately estimate bootstrap support values, select the optimal substitution model, test evolutionary hypotheses, and infer branch lengths, divergence times, and associated uncertainty measures, while reducing runtime and memory requirements by orders of magnitude. PSU also provides distributions of inferred clade support across independent subsamples, enabling detection of conflicting phylogenetic signals that may remain hidden in conventional bootstrap analysis. Automated tuning of subsample size, the number of subsamples, and the number of upsampling replicates make PSU practical across diverse datasets. We suggest that PSU is a general strategy for scalable phylogenomic inference using a broad range of statistical methods. By enabling analyses of genome-scale alignments on commodity hardware, PSU broadens research access and reduces environmental and infrastructural costs of big-data phylogenomics.

confidence limits

Moraxella species isolated from blood cultures in Europe (MORAXEu): a multicentre study of epidemiology and antimicrobial susceptibility with complementary phylogenomic analysis of publicly available genomes.

INTRODUCTION: Moraxella species are fastidious Gram-negative bacteria capable of causing opportunistic infections, including bloodstream infections, especially in immunocompromised patients. Data on their epidemiology, antimicrobial susceptibility, and phylogenomics in Europe remains limited. METHODS: We conducted a multicentre, retrospective, observational study across 56 European hospital centres between January 1st 2020 and December 31st 2024. All Moraxella species isolated from blood cultures (BCs) were included. Species distribution and antimicrobial susceptibility profiles were analysed. We also performed a phylogenomic analysis of Moraxella genomes deposited in GenBank. RESULTS: A total of 709 Moraxella isolates were included. Moraxella osloensis (61.1%; n = 433/709) and Moraxella catarrhalis (20.4%; n = 145/709) were the most frequently identified species, followed by Moraxella nonliquefaciens (6.5%; n = 46/709) and Moraxella atlantae (4.8%; n = 34/709). Species distribution differed by age. M. catarrhalis was predominant in paediatric patients, whereas M. atlantae was more common in adults. Most isolates showed > 90% susceptibility to amoxicillin/clavulanate, cefotaxime, fluoroquinolones, and trimethoprim/sulfamethoxazole. Cefotaxime resistance in M. osloensis was more frequent in adults than in children (49% vs. 8%, p = 0.009). Phylogenomic analysis demonstrated the distinction of a core Moraxella group from the divergent Faucicola lineage, with M. osloensis and M. atlantae clustering within the latter. CONCLUSIONS: The epidemiology of Moraxella species from BCs in Europe showed age-group-specific differences in species distribution and generally favourable antimicrobial susceptibility patterns. Phylogenomic data corroborated recent taxonomic revisions, highlighting the need for improved diagnostics, harmonized nomenclature and sustained surveillance to inform management and stewardship of Moraxella bacteraemia.

Faucicola

Comparative phylogenomics and transcriptional regulatory networks of AQPs, HSPs, and LEA proteins in salt-stressed Portulaca oleracea.

Soil salinization severely threatens global food security, necessitating systematic investigations of halophytes like Portulaca oleracea to decode the molecular mechanisms of environmental resilience. Utilizing an integrated framework of deep learning-based genome annotation (58,817 predicted genes; 96.5% BUSCO completeness), multi-tissue RNA-Seq, phylogenomics, and gene regulatory network (GRN) inference, the synergistic orchestration of 78 aquaporins (AQPs), 525 heat shock proteins (HSPs), and 119 late embryogenesis abundant (LEA) proteins was elucidated. The active transcriptome, encompassing 39,065 expressed loci, revealed a systemic growth-defense trade-off. Tissues displayed distinct adaptive mechanisms: leaves modulated intracellular water balance via specialized AQPs, whereas adult roots maintained proteostasis through robust HSP20/HSP70 induction. Phylogenomic clustering across 154 species demonstrated that salinity tolerance constitutes an evolutionary mosaic, identifying 81 halophyte-exclusive orthogroups and 1129 species-specific clusters. Comparative topology across six independent GRNs (4.2M-5.3 M edges) unmasked a highly modular transcriptional reprogramming strategy governed by a core apparatus of 22 stress-exclusive regulators, with functional enrichment heavily prioritizing protein dimerization and chromatin remodeling. Theoretically, the distinct convergence of Trihelix transcription factors with guard cell differentiation pathways offers a candidate transcriptomic framework to explain the plant's characteristic C4-CAM photosynthetic plasticity under severe osmotic pressure. Practically, these evolutionary blueprints and specific master switches transcend single-gene transgenic limitations. Utilizing these root-sustained and stress-inducible targets under localized promoters provides a naturally optimized, network-level precision engineering roadmap to transfer robust, compartmentalized halotolerance to sensitive glycophytic crops.

Gene Regulatory Networks

Phylogenomics of Desulfuromonadia supports reclassification of Geobacter psychrophilus as Irobacter psychrophilus comb. nov. and proposal of Geosyntrophus gen. nov.

Genome-resolved phylogenomics reveals widespread misclassification of metal-reducing bacteria historically assigned to Geobacter based on 16S rRNA gene phylogeny, and highlights species that persist only as 16S rRNA entries without genomes for robust taxonomic resolution. Here, we resolve two such lineages by integrating whole-genome phylogeny with average amino acid identity (AAI) and percentage of conserved proteins (POCP) across 418 dereplicated genomes of Desulfuromonadia. We report a draft genome of the psychrophilic iron-reducing bacterium Geobacter psychrophilus (100% completeness). Phylogenomic analyses place both Geobacter psychrophilus and the GTDB placeholder genus g__JACRCG01 within the family 'Pseudopelobacteraceae', outside Geobacteraceae sensu stricto. Within this framework, G. psychrophilus forms a distinct, well-supported lineage separated from neighbouring genera by discontinuities in AAI and POCP, supporting its reclassification as Irobacter psychrophilus comb. nov. Additionally, we show that Geosyntrophus acetoxidans, a non-axenic syntrophic bacterium, forms a coherent genus with 51 other environmental genomes (placeholder genus g__JACRCG01), for which we propose the replacement name Geosyntrophus gen. nov. Comparative genome analysis revealed conserved family-level metabolic traits together with genus-specific differences in respiratory metabolism, while ANI-based clustering identified substantial species-level diversity within both proposed genera. Metagenome and 16S rRNA-gene survey data further show that Geosyntrophus and Irobacter occur in broadly similar aquatic and subsurface habitats spanning from the Arctic to the Antarctic. Together, these results resolve the taxonomy of two previously ambiguous Desulfuromonadales lineages and shed light on their environmental distribution.

AAI

Phylogenomics and female reproductive morphology reframe the classification of the Halymeniales (Rhodophyta).

The red algal order Halymeniales (Rhodophyta) exhibits remarkable morphological and taxonomic diversity but its higher-level relationships remain poorly resolved. Here, we present a comprehensive phylogenomic analysis based on newly generated plastid (170 protein-coding genes), mitochondrial (23 genes), and complete nuclear ribosomal cistron sequences from 56 taxa, complemented with an expanded rbcL dataset encompassing 334 sequences. Our results provide a robust phylogenomic framework for the Halymeniales, offering a taxonomic backbone for future systematic studies. The analyses consistently recover six early-diverging lineages (Acrodiscus, Isabbottia, Norrissia, Pachymenia, Zymurgia, and Tsengia) and two strongly supported larger clades (Halymenia s.l. and Grateloupia s.l.). While most small and recently described genera are monophyletic, several traditional genera (e.g., Halymenia, Cryptonemia, Grateloupia) are poly- or paraphyletic, requiring considerable taxonomic revision. At the family level, the data indicate that reinstatement of the Grateloupiaceae sensu Kim et al. (2021) would entail a revised circumscription of the Halymeniaceae and the recognition of at least five small families to accommodate the early-diverging lineages. Although such a revised classification would result in monophyletic families, it is not supported by morpho-anatomical characters. Instead, we propose a more stable two-family system, recognizing a broadly circumscribed Halymeniaceae that is sister to the Tsengiaceae. Female reproductive characters, particularly the structure of carpogonial and auxiliary cell ampullae, support this two-family system and further characterize many genus-level clades, although substantial convergence across lineages exists.

Phylogeny

Phylogenomics and evolution of the Lauraceae based on targeted capture data.

The family Lauraceae, a hyper-diverse magnoliid family comprising approximately 63 genera and over 3,000 species, plays a key ecological role in tropical and subtropical forests. Yet deep relationships among its nine tribes remain unresolved, likely due to limited sampling and complex evolutionary processes such as incomplete lineage sorting (ILS) and gene flow. To address these challenges, we generated datasets of 255 single-copy nuclear genes and chloroplast genomes using a newly designed Lauraceae-specific probe set, achieving the most comprehensive genus-level sampling (84%) to date. Phylogenomic analyses reconstructed a robust nuclear tree, which resolved the Neocinnamomeae as sister to the Caryodaphnopsideae and revealed pronounced gene tree conflict and pervasive cytonuclear discordance. To investigate the evolutionary processes underlying these patterns, comprehensive analyses were conducted. The results indicate that conflicting nuclear gene trees reflect the combined effects of ILS, gene tree estimation error, and gene flow, with ILS dominating across the core Lauraceae, whereas cytonuclear discordance is primarily driven by extensive gene flow. Diversification analyses further indicate that episodes of rapid lineage accumulation coincide with major gene flow events, suggesting a potential role of gene flow in the diversification of Lauraceae. Overall, this study provides a robust nuclear phylogenomic framework for Lauraceae and demonstrates that gene flow had profound effects on its evolutionary history, shedding light on the contribution of gene flow to the diversification of hyper-diverse tropical plant lineages.

Cytonuclear discordance

Beyond species trees: pervasive gene flow limits phylogenomic resolution in the diversification of Juniperus from the Qinghai-Tibet Plateau.

Understanding how lineages diversify despite persistent ancestral polymorphism and recurrent gene flow remains a central challenge in evolutionary biology. Juniperus distributed across the Qinghai-Tibet Plateau provide an ideal system for addressing this question because repeated geological uplift and climatic oscillations have likely promoted cycles of lineage divergence, range shifts, and secondary contact. Here, we combined approximately 1.08 million genome-wide SNPs from 164 individuals representing thirteen Juniperus lineages with phylogenomic datasets comprising 3,381 nuclear single-copy genes and nearly complete plastomes. We detected extensive phylogenomic discordance and cytonuclear incongruence across genomic datasets. Topology weighting, coalescent simulations, quartet-based tests, and analyses of gene flow and reticulation collectively support the interpretation that these patterns were shaped by the combined effects of prolonged incomplete lineage sorting and gene flow during lineage diversification. Ecological niche analyses further provide a spatial and climatic context in which environmentally similar lineages may have had greater opportunities for secondary contact during historical range shifts. Collectively, our results reveal that the evolutionary history of Qinghai-Tibet Plateau Juniperus is characterized by reticulate diversification rather than strictly bifurcating evolution, and demonstrate how genome-wide discordance can provide biological insights into the evolutionary processes underlying lineage diversification.

Gene Flow

Nuclear single-copy orthologous genes as phylogenomic markers for resolving the closely related firefly genera Pteroptyx, Medeopteryx, and Trisinuata (Coleoptera: Lampyridae: Luciolinae).

Fireflies (Lampyridae) are bioluminescent beetles with broad ecological roles across temperate and tropical ecosystems, occupying diverse habitats including forests, wetlands, grasslands, mangroves, and riverine systems. The subfamily Luciolinae is primarily distributed across Asia and the Indo-Pacific. Phylogenetic relationships among three closely related Luciolinae genera - Medeopteryx, Pteroptyx, and Trisinuata - remain unresolved using mitochondrial genome data alone. This study used nuclear genome data to resolve relationships among these genera and identify a lighter-weight nuclear marker panel for expanding taxon sampling. Draft genomes were reconstructed for fifteen firefly species, eight from the focal genera, and analyzed with five published firefly genomes. Using BUSCO and OrthoFinder, 1,011 nuclear single-copy orthologs (SCOs) were identified for phylogenomic inference. Discordance between concatenation- and coalescence-based phylogenies indicated incomplete lineage sorting (ILS). The coalescence-based phylogeny recoveredPteroptyxas monophyletic and sister to a (Medeopteryx,Trisinuata) clade, with Trisinuata nested within a non-monophyletic Medeopteryx; however, quartet support at the base of Pteroptyx, particularly at Pt. valida, was low.Filtering for compositional homogeneity, clock-likeness, and species-tree concordance yielded 103 SCOs with a significantly higher proportion of parsimony-informative sites than non-selected loci, retaining the backbone topology with higher gene concordance support at scored clades, while ILS-driven discordance at Pt. valida persists - confirming that the reduced panel retains phylogenetic resolving power for future taxon sampling. These findings demonstrate a practical framework for using nuclear SCOs to resolve close phylogenetic relationships within Luciolinae. Future work should expand taxon sampling - especially forTrisinuata - alongside long-read assemblies, for a more robust phylogenomic framework.

Fireflies

Phylogenomic evidence provides insights into the phylogeny and speciation patterns in the subgenus Caloscordum (Allium, Amaryllidaceae).

Phylogenomics with abundant informative sites offers a powerful means for elucidating complex diversification history. Here, we collected 22 samples from 18 populations representing all species of subgenus Caloscordum (Allium). Using transcriptome and whole-genome resequencing data, we generated 1755 low-copy nuclear genes and 81 plastid genes. By integrating morphological and phylogenomic evidence, we clarified the subgenus's complex evolutionary histories and speciation patterns. A total of 18 morphological characteristics were analysed, with a taxonomic framework established. Our analyses resolved robust species relationships despite detecting extensive phylogenetic discordances, which were attributed to incomplete lineage sorting (ILS) and hybridization. Specifically, our results suggest that A. inutile originated via rapid budding speciation from the widespread A. tubiflorum. This process likely coincided with mid-Pleistocene glacial-interglacial cycles and may have been reinforced by geographic isolation and ecological adaptation. In contrast, the sole tetraploid species, A. peikingense, was confirmed to be of hybrid origin, derived from A. neriniflorum and A. tubiflorum. This allopolyploidization event appears to have been facilitated by secondary contact between the parent species, which was likely associated with climatic oscillations within the 35° N-45° N arid belt. Overall, our findings elucidate the intricate speciation patterns within Caloscordum and highlight how the interplay of polyploidization, ecological isolation, and tectonic uplift-driven aridification has shaped plant diversity in East Asia.

Phylogeny

Cross-Kingdom Genomic Conservation of Putative Human Sleep-Related Genes: Phylogenomic Evidence From Chlamydomonas reinhardtii.

Sleep is a widespread and evolutionarily conserved process observed in diverse organisms, from jellyfish to mammals, hinting at its origin as a life-supporting mechanism over 500 million years ago. Although its fundamental purpose and mechanisms remain unclear, sleep's evolution and adaptive significance continue to be debated. This study explores the evolutionary origins of sleep using Chlamydomonas reinhardtii as a model organism, identifying 112 putative sleep-related genes across species and highlighting the evolutionary conservation of sleep-regulatory pathways. Additionally, discovering uncharacterized proteins with high sequence similarity and significant e-values suggests unexplored roles in sleep regulation, underscoring the potential of C. reinhardtii to reveal new insights into the molecular basis of sleep. This study provides a foundation for identifying previously unknown sleep-associated proteins, particularly within single-celled organisms, which may offer novel perspectives on the biological role of sleep. The study demonstrates that phylogenomic analysis of diverse model organisms can expand our understanding of the evolutionary trajectory of sleep and its fundamental function, paving the way for further research in sleep biology and its health implications. Overall, the fundamental functions of sleep observed in higher animal phyla originated from its primordial activities, demonstrating an evolutionary continuum wherein more specialized tasks were integrated with sleep's essential restorative properties.

Chlamydomonas reinhardtii

Phenotypic and phylogenomic characterization of Lactococcus garvieae isolates from rainbow trout (Oncorhynchus mykiss) in Türkiye.

Lactococcosis is an important bacterial disease of farmed fish and causes substantial economic losses in rainbow trout (Oncorhynchus mykiss) aquaculture. In this study, Lactococcus garvieae isolates recovered from rainbow trout farms in Türkiye were characterized using phenotypic, molecular, and phylogenomic methods. Among 32 presumptive Lactococcus isolates recovered from 127 dead rainbow trout, four were confirmed as L. garvieae and exhibited identical biochemical characteristics, Pulsed Field Gel Electrophoresis (PFGE) profiles, and broad growth tolerance across different pH, salinity, and temperature conditions. All isolates were presumptively classified as resistant to ciprofloxacin and florfenicol, while remaining susceptible to tetracycline and penicillin. Based on the AMR profiles, strain LG2, which exhibited the most susceptible antimicrobial profile among the isolates, was selected for whole-genome sequencing (WGS). WGS of the representative isolate LG2 generated a single 2,214,687-bp chromosomal contig with 38.5% GC content and 99.0% BUSCO completeness. In silico PCR assigned LG2 to serotype I, and the genome contained an intact capsule-associated cps/kps locus. The chromosomal lsa(D) determinant and an mdt(A)-like efflux-associated gene were detected, whereas no plasmid replicons or acquired quinolone or florfenicol resistance genes were identified, indicating discordance between the phenotypic and genomic AMR results. Taxonomic verification of 236 publicly available Lactococcus assemblies yielded 41 verified public L. garvieae genomes, which, together with LG2, formed a 42-genome within-species dataset. LG2 was most closely related to the Turkish isolate OS-37, sharing 99.96% ANI and differing by three core SNPs; both belonged to ST109, whereas the other Turkish isolates belonged to ST139. cgMLST identified a conserved genomic backbone, while pan-genome analysis identified 5,655 gene clusters and an open pan-genome characterized by a large cloud-gene fraction. These findings demonstrate the importance of species verification in Lactococcus population genomics and reveal substantial accessory-genome diversity within L. garvieae. The genomic features of LG2 provide a basis for future pathogenicity and immunogenicity studies, although experimental validation is required. Overall, these findings highlight the importance of local genomic surveillance for understanding L. garvieae population structure and provide a genomic framework for future region-specific vaccine research.

Animals

Phylogenomic and Clinical Perspectives of an East Asia-Specific Cluster of Mycobacterium massiliense.

BACKGROUND: Mycobacterium abscessus subspecies massiliense (MAM) can form genetically related clusters through continuous within-host adaptations. RESEARCH QUESTION: What is the epidemiologic and clinical significance of the sequence type 120 (ST120) strain of MAM, an East Asia-specific cluster? STUDY DESIGN AND METHODS: Isolates were obtained from patients with MAM pulmonary disease at Seoul National University Hospital between October 1, 2019, and December 31, 2023. These isolates were analyzed using multilocus sequence typing and colony morphotyping and were evaluated for glycopeptidolipid biosynthesis-related gene deletions. Whole-genome sequencing was performed for phylogenomic and pangenome analyses, incorporating MAM genome data from public databases. Finally, the clinical course and treatment outcomes of patients infected with ST120 were evaluated. RESULTS: Among the isolates obtained from 136 patients, 50 isolates (36.8%) were classified as ST120. All ST120 isolates exhibited a rough colony morphotype and harbored deletions in glycopeptidolipid biosynthesis-related genes. From 796 global strains, ST120 was identified exclusively in the isolates obtained from South Korea (51/137 isolates [37.1%]), Japan (3/53 [5.7%]), and Taiwan (2/52 [3.8%]). Genomic analysis revealed the stepwise genetic evolution of ST120, suggesting transmission from Taiwan to Japan and from Japan to South Korea. The ST120 strains exhibited genetic mutations associated with virulence and structural alterations within the ESX system. Consequently, patients with ST120 strain infections had a higher prevalence of cavitary disease (23/50 [46%]) than those infected with non-ST120 strains (19/86 [22.1%]; P = .004). Among those who initiated treatment, the proportion of microbiological cure was lower in patients with ST120 infection (6/15 [40.0%]) than in those with non-ST120 infection (24/34 [70.6%]; P = .043). INTERPRETATION: Our results show that ST120, a strain predominantly found in South Korea, is characterized by the extensive loss of glycopeptidolipid biosynthesis-related genes and is associated with increased disease severity and worse treatment outcomes.

Humans

Characterisation of the chloroplast genome of Macrotyloma species: comparative analysis and phylogenomic insights.

Macrotyloma is an underutilised legume genus within the tribe Phaseoleae (Fabaceae) that includes nutritionally and agronomically important crops such as horse gram (Macrotyloma uniflorum) and Kersting's groundnut (Macrotyloma geocarpum). Despite their importance, knowledge of the chloroplast (cp.) genome of this genus remains limited. In this study, we assembled and analysed the complete chloroplast genomes of three Macrotyloma species: M. uniflorum, M. geocarpum, and M. axillare. The chloroplast genomes were assembled into two isoforms that differ in the orientation of the small single-copy (SSC) region. Genome sizes ranged from 150,811 to 151,013 bp and exhibited the canonical quadripartite structure, comprising a pair of inverted repeats (IRa and IRb; 26,416-26,436 bp each), a large single-copy region (LSC; 80,229-80,446 bp), and a small single-copy region (SSC; 17,710-17,711 bp). Each genome encoded 110 unique genes, including 4 rRNA genes, 30 tRNA genes, and 76 protein-coding genes. All three species also possessed the ~ 50 kb inversion in the LSC region, a synapomorphy shared among a large clade within the Papilionoideae subfamily of Fabaceae. Although overall chloroplast genome structure and organisation were highly conserved among Macrotyloma species, gene-wise nucleotide diversity analysis identified seven relatively variable genes: rps18, rps15, ccsA, ndhA, ycf1, ycf4, and psaI. Phylogenomic analysis based on complete chloroplast genomes robustly resolved Macrotyloma as a monophyletic group within the Phaseolinae clade of the Papilionoideae subfamily. Within the genus, M. uniflorum and M. axillare formed a strongly supported sister pair, with M. geocarpum sister to this clade. Overall, this study provides valuable insights into chloroplast genome evolution in Macrotyloma and enhances understanding of its phylogenetic placement within Phaseoleae, offering genomic resources for future evolutionary, taxonomic, and conservation studies of this underutilised legume genus.

Genome, Chloroplast

Phylogenomics reveals persistent gene-tree discordance in the Chenopodium album aggregate.

BACKGROUND AND AIMS: Complex genomic histories shaped by hybridisation and polyploidy can influence traits related to plant defence, stress tolerance and toxicity, particularly in Amaranthaceae, which includes crops such as quinoa and spinach. Within this family, white goosefoot (Chenopodium album), a widespread agricultural weed and traditional food resource, belongs to a diploid-polyploid aggregate with extensive phylogenetic discordance. Clarifying its evolutionary history provides context for interpreting ecologically and agronomically relevant trait variation across the aggregate. Building on the established genome-lineage framework, we tested whether discordance persists when constituent genome-lineage components are represented separately and whether the remaining signal is compatible with reticulate evolution. METHODS: We analysed 2,298 conserved nuclear BUSCO families across 27 assembly-level terminals using tree- and network-based approaches. Genome-lineage-aware analyses used 2,156 families after separating polyploid Chenopodium into A-H components, with Dysphania ambrosioides as outgroup. HyDe tested site-pattern asymmetry under global false-discovery-rate correction. KEY RESULTS: Assembly-level analyses grouped the Danish C. album aggregate accession Ca6-1 with hexaploid C. album sensu stricto dcCheAlbu1.1, whereas relationships among surrounding Chenopodium taxa were less stable. Genome-lineage-aware analyses recovered the expected B-, C- and D-affinity relationships, but substantial gene-family heterogeneity persisted. Reticulate network models fitted the assembly-level data better than bifurcating models, although inferred patterns differed between methods. HyDe detected significant site-pattern asymmetry in a small subset of loci, with most retained signal shared between the focal assemblies. CONCLUSIONS: Gene-tree discordance persists in the C. album aggregate after genome-lineage separation. The established genome-lineage framework captures the dominant phylogenomic structure, while residual heterogeneity is compatible with both tree-like and reticulate processes without identifying direct progenitors or a unique hybridisation history. This framework supports future analyses of lineage-specific and trait-associated loci related to plant defence, food quality and toxicity in C. album and related Amaranthaceae.

Chenopodium album

Resolving the "Yucca queretaroensis problem": Phylogenomic analysis of Yucca reveals the identity of an enigmatic species and the origin of an obligate pollination mutualism.

PREMISE: The genus Yucca is a group of ~50 species of woody monocots endemic to the North American arid regions. Their obligate pollination mutualism with yucca moths is considered a "textbook example" of coevolution and is hypothesized to have promoted rapid diversification. However, testing this hypothesis has been difficult due to uncertainty about the placement of a rogue taxon, Yucca queretaroensis, a rare endemic of the Sierra Gorda region of central Mexico. Past work placed this species in different positions within the Agavoideae, producing starkly different age estimates for Yucca (25 to 4 million years). METHODS: We generated new sequence capture data for 353 nuclear genes and for all coding regions of the plastid genome from wild-collected plants and samples included in previous studies to provide a new phylogeny and new age estimate for Yucca. RESULTS: The data presented here suggest that Y. queretaroensis is closely related to other species of Yucca. A relaxed molecular clock analysis of the plastid genome produced an estimated age for the genus of approximately 6.8 million years. CONCLUSIONS: The results resolve a mystery that has bedeviled evolutionary biologists for decades and provide a surprisingly young estimate for the age of Yucca, suggesting rapid diversification. The past difficulties in identifying the correct placement of Y. queretaroensis appear to be the product of laboratory errors, mistakes in field identification, and frequent hybridization with co-distributed taxa. The "Yucca queretaroensis problem" reaffirms the essential role for traditional botanical tools in phylogenomics.

ASTRAL

Phylogenomics and museomics reveal five distinct species of tiger cats in South America.

The evolutionary history of elusive organisms can be characterized through genomic analyses, which have the power to reveal previously unknown taxa even in groups assumed to be well studied, such as cats. We have analyzed complete genomes of 38 individuals from the Neotropical cat genus Leopardus, including 26 individuals representing multiple evolutionary units of the contentious tiger cat (Leopardus tigrinus) species complex. Eight genomes were generated from museum specimens, which allowed the first genetic assessment of the type locality for L. tigrinus in the Guiana Shield. We found that this complex comprises five distinct species, including a novel cat species, discovered in the Bolivian Yungas and described in this study as L. tilcayo. The Peruvian Yungas unit of this complex also represents a distinct taxonomic entity, which we describe here as a novel subspecies, L. tigrinus antisuyo. Our phylogenomic analyses resolve the evolutionary relationships among the tiger cat geographic units, thus stabilizing their recalcitrant taxonomy and enabling adequate conservation assessment of these threatened felids. We also address other aspects of their evolution, including biogeography, past episodes of interspecies admixture, demographic history of each taxonomic unit, and temporal changes in genetic diversity. Altogether, our results clarify the evolutionary history of a complex radiation of wild cats, reveal novel taxa, and serve as a basis for conservation planning on behalf of these elusive wild cats.

Bolivian Yungas

FUSE-PhyloTree: linking functions and sequence conservation modules of a protein family through phylogenomic analysis.

SUMMARY: FUSE-PhyloTree is a phylogenomic analysis software for identifying local sequence conservation associated with the different functions of a multi-functional (e.g. paralogous or multi-domain) protein family. FUSE-PhyloTree introduces an original approach that combines advanced sequence analysis with phylogenetic methods. First, local sequence conservation modules within the family are identified using partial local multiple sequence alignment. Next, the evolution of the detected modules and known protein functions is inferred within the family's phylogenetic tree using three-level phylogenetic reconciliation and ancestral state reconstruction. As a result, FUSE-PhyloTree provides a gene tree annotated with both predicted sequence modules and ancestral gene functions, enabling the association of functions with specific sequence regions based on their co-emergence. AVAILABILITY AND IMPLEMENTATION: FUSE-PhyloTree is provided as Docker and Singularity images including all the required software tools. Images, source code, test data, and documentation are available at https://github.com/OcMalde/fuse-phylotree and https://zenodo.org/records/15855068.

Phylogeny