Divergent Lineages Research Articles

Climatically Specialized Lineages of Batrachochytrium dendrobatidis, and its Likely Asian Origins.

Chytridiomycosis is a wildlife disease that has caused significant declines in amphibian populations and species extinctions worldwide. Asia, where the causal pathogens Batrachochytrium dendrobatidis (Bd) and B. salamndrivorans (Bsal) originated, has not witnessed mass die-offs. It is hypothesized that Asian amphibians may have evolved immunity to clinical Batrachochytrium infection, but this has not been explored in depth due to limited knowledge of endemic lineages and infection patterns. We investigated Bd's genetic diversity and infection patterns in south China's Guangxi region using the internal transcribed spacer (ITS) marker and nested PCR. Across the 17 forest sites studied (N = 1088 individuals; 1012 adults and 76 tadpoles), the overall prevalence of Bd infection was 4.74% in adult individuals and 5.26% in tadpoles. We found seven new haplotypes, four of which were closely related to the BdASIA-1 lineage from South Korea. The most prevalent haplotype (genetically similar to BdASIA-3) was found in 11 out of 15 infected species, including a salamander with non-lethal skin lesions. A generalized linear model of our environmental data indicates that Bd infection is correlated with mean temperature of the warmest quarter and elevation, with higher infection prevalence associated with lower temperature and relatively higher elevation in southern China. Our findings suggest significant undiscovered genetic diversity of Asian Bd lineages in this region. Longer-term studies are required to further investigate Bd diversity, prevalence, seasonality, and impact on native species and populations in Southern China and across the region of origin in Asia.

YHSeqY3000 panel captures all founding lineages in the Chinese paternal genomic diversity database

BackgroundThe advancements in second-/third-generation sequencing technologies, alongside computational innovations, have significantly enhanced our understanding of the genomic structure of Y-chromosomes and their unique phylogenetic characteristics. These researches, despite the challenges posed by the lack of population-scale genomic databases, have the potential to revolutionize our approach to high-resolution, population-specific Y-chromosome panels and databases for anthropological and forensic applications.ObjectivesThis study aimed to develop the highest-resolution Y-targeted sequencing panel, utilizing time-stamped, core phylogenetic informative mutations identified from high-coverage sequences in the YanHuang cohort. This panel is intended to provide a new tool for forensic complex pedigree search and paternal biogeographical ancestry inference, as well as explore the general patterns of the fine-scale paternal evolutionary history of ethnolinguistically diverse Chinese populations.ResultsThe sequencing performance of the East Asian-specific Y-chromosomal panel, including 2999-core SNP variants, was found to be robust and reliable. The YHSeqY3000 panel was designed to capture the genetic diversity of Chinese paternal lineages from 3500 years ago, identifying 408 terminal lineages in 2097 individuals across 41 genetically and geographically distinct populations. We identified a fine-scale paternal substructure that was correlating with ancient population migrations and expansions. New evidence was provided for extensive gene flow events between minority ethnic groups and Han Chinese people, based on the integrative Chinese Paternal Genomic Diversity Database.ConclusionsThis work successfully integrated Y-chromosome-related basic genomic science with forensic and anthropological translational applications, emphasizing the necessity of comprehensively characterizing Y-chromosome genomic diversity from genomically under-representative populations. This is particularly important in the second phase of our population-specific medical or anthropological genomic cohorts, where dense sampling strategies are employed.

DNA barcode and sequence related amplified polymorphism markers reveal phylogenetic relationship among Pinus species representing closed lineage divergence

DNA barcode and sequence related amplified polymorphism markers reveal phylogenetic relationship among Pinus species representing closed lineage divergence

Exploring Environmental Microfungal Diversity Through Serial Single Cell Screening.

Known for its remarkable diversity and ecological importance, the fungal kingdom remains largely unexplored. In fact, the number of unknown and undescribed fungi is predicted to exceed the number of known fungal species by far. Despite efforts to uncover these dark fungal taxa, we still face inherent sampling biases and methodological limitations. Here, we present a framework that combines taxonomic knowledge, molecular biology and data processing to explore the fungal biodiversity of enigmatic aquatic fungal lineages. Our work is based on serial screening of environmental fungal cells to approach unknown fungal taxa. Microscopic documentation is followed by DNA analysis of laser micro-dissected cells, coupled with a ribosomal operon barcoding step realised by long-read sequencing, followed by an optional whole genome sequencing step. We tested this approach on a range of aquatic fungal cells mostly belonging to the ecological group of aquatic hyphomycetes derived from environmental samples. From this initial screening, we were able to identify 60 potentially new fungal taxa in the target dataset. By extending this methodology to other fungal lineages associated with different habitats, we expect to increasingly characterise the molecular barcodes of dark fungal taxa in diverse environmental samples. This work offers a promising solution to the challenges posed by unknown and unculturable fungi and holds the potential to be applied to the diverse lineages of undescribed microeukaryotes.

Genomic Rewilding of Domestic Animals: The Role of Hybridization and Selection in Wolfdog Breeds.

Background/Objectives: The domestication of the grey wolf (Canis lupus) and subsequent creation of modern dog breeds have significantly shaped the genetic landscape of domestic canines. This study investigates the genomic effects of hybridization and breeding management practices in two hybrid wolfdog breeds: the Czechoslovakian Wolfdog (CSW) and the Saarloos Wolfdog (SAW). Methods: We analyzed the genomes of 46 CSWs and 20 SAWs, comparing them to 12 German Shepherds (GSHs) and 20 wolves (WLFs), which served as their ancestral populations approximately 70-90 years ago. Results: Our findings highlight that hybridization can increase genetic variability and mitigate the effects of inbreeding, as evidenced by the observed heterozygosity levels in both wolfdog breeds. However, the SAW genome revealed a higher coefficient of inbreeding and longer runs of homozygosity compared to the CSW, reflecting significant inbreeding during its development. Discriminant Analysis of Principal Components and fixation index analyses demonstrate that the CSW exhibits closer genetic proximity to the GSH than the SAW, likely due to differences in the numbers of GSHs used during their creation. Maximum likelihood clustering further confirmed clear genetic differentiation between these hybrid breeds and their respective ancestors, while shared ancestral polymorphism was detectable in all populations. Conclusions: These results highlight the role of controlled hybridization with captive-bred wolves and peculiar breeding strategies in shaping the genetic structure of wolfdog breeds. To ensure the long-term genetic health of these breeds, it is recommended to promote adequate and sustainable breeding practices to maintain genetic diversity, minimize inbreeding, and incorporate the careful selection of unrelated individuals from diverse lineages, while avoiding additional, uncontrolled crossings with wild wolves.

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.

Nr4a1 and Nr4a3 redundantly control clonal deletion and contribute to an anergy-like transcriptome in auto-reactive thymocytes to impose tolerance in mice

The Nr4a nuclear hormone receptors are transcriptionally upregulated in response to antigen recognition by the T cell receptor (TCR) in the thymus and are implicated in clonal deletion, but the mechanisms by which they operate are not clear. Moreover, their role in central tolerance is obscured by redundancy among the Nr4a family members and by their reported functions in Treg generation and maintenance. Here we take advantage of competitive bone marrow chimeras and the OT-II/RIPmOVA model to show that Nr4a1 and Nr4a3 are essential for the upregulation of Bcl2l11/BIM and thymic clonal deletion by self-antigen. Importantly, thymocytes lacking Nr4a1/3 acquire an anergy-like signature after escaping clonal deletion and Treg lineage diversion. We further show that the Nr4a family helps mediate a broad transcriptional program in self-reactive thymocytes that resembles anergy and may operate at the margins of canonical thymic tolerance mechanisms to restrain self-reactive T cells after thymic egress.

Cryo-EM structure and evolutionary history of the conjugation surface exclusion protein TraT

Conjugation plays a major role in dissemination of antimicrobial resistance genes. Following transfer of IncF-like plasmids, recipients become refractory to a second wave of conjugation with the same plasmid via entry (TraS) and surface (TraT) exclusion mechanisms. Here, we show that TraT from the pKpQIL and F plasmids (TraTpKpQIL and TraTF) exhibits plasmid surface exclusion specificity. The cryo-EM structures of TraTpKpQIL and TraTF reveal that they oligomerise into decameric champagne bottle cork-like structures, which are anchored to the outer membrane via a diacylglycerol and palmitic acid modified α-helical barrel domain. Unexpectedly, we identify chromosomal TraT homologues from multiple Gram-negative phyla which form numerous divergent lineages in a phylogenetic tree of TraT sequences. Plasmid-associated TraT sequences are found in multiple distinct lineages, including two separate clades incorporating TraT from Enterobacteriaceae IncF/F-like and Legionellaceae F-like plasmids. These findings suggest that different plasmid backbones have acquired and co-opted TraT on independent occasions.

Spatial transcriptomic characterization of a Carnegie stage 7 human embryo.

Gastrulation marks a pivotal stage in mammalian embryonic development, establishing the three germ layers and body axis through lineage diversification and morphogenetic movements. However, studying human gastrulating embryos is challenging due to limited access to early tissues. Here we show the use of spatial transcriptomics to analyse a fully intact Carnegie stage 7 human embryo at single-cell resolution, along with immunofluorescence validations in a second embryo. Employing 82 serial cryosections and Stereo-seq technology, we reconstructed a three-dimensional model of the embryo. Our findings reveal early specification of distinct mesoderm subtypes and the presence of the anterior visceral endoderm. Notably, primordial germ cells were located in the connecting stalk, and haematopoietic stem cell-independent haematopoiesis was observed in the yolk sac. This study advances our understanding of human gastrulation and provides a valuable dataset for future research in early human development.

Tip-to-base bark cross-sectional areas contribute to understanding the drivers of carbon allocation to bark and the functional roles of bark tissues.

Along their lengths, stems experience different functional demands. Because bark and wood traits are usually studied at single points on stems, it remains unclear how carbon allocation changes along tip-to-base trajectories across species. We examined bark vs wood allocation by measuring cross-sectional areas of outer and inner bark (OB and IB), IB regions (secondary phloem, cortex, and phelloderm), and wood from stem tips to bases of 35 woody angiosperm species of diverse phylogenetic lineages, climates, fire regimes, and bark morphologies. We examined how varied bark vs wood allocation was and how it was affected by precipitation, temperature, soil fertility, leaf habit, and fire regime. Allocation to phloem (relative to wood) varied little across species, whereas allocation to other tissues, strongly affected by the environment or shed in ontogeny, varied widely. Allocation to parenchyma-rich cortex and phloem was higher at drier sites, suggesting storage. Higher allocation to phloem and cortex also occurred on infertile soils, and to phloem in drought-deciduous vs cold-deciduous and evergreen species. Allocation to OB was highest at sites with frequent fires and decreased with fire frequency. Our approach contextualizes inferences from across-species studies, allows testing functional hypotheses, and contributes to disentangling the functional roles of poorly understood bark tissues.

An ALK Immunohistochemical Pitfall: ALK-Positive Histiocytosis Versus Angiomatoid Fibrous Histiocytoma.

The proliferation of recognized neoplasms related to related to rearrangements involving kinase genes, arising in diverse somatic tissue lineages, poses an increasing challenge in surgical pathology. For instance, ALK rearrangements have been observed in diverse neoplasms of epithelial, connective tissue, and hematolymphoid lineages, many of which are associated with overexpression of ALK by immunohistochemistry as a useful biomarker. An even higher order challenge and pitfall would be the scenario where a tumor without an ALK rearrangement nonetheless overexpresses the protein, thereby simulating an ALK-defined neoplasm. Herein, we present this exact scenario, where an angiomatoid fibrous histiocytoma, exhibiting the recently-described phenomenon of its overexpression of ALK, clinically, histologically, and immunophenotypically simulated ALK-positive histiocytosis.

Pan-phylum genomes of hornworts reveal conserved autosomes but dynamic accessory and sex chromosomes.

Hornworts, one of the three bryophyte phyla, show some of the deepest divergences in extant land plants, with some families separated by more than 300 million years. Previous hornwort genomes represented only one genus, limiting the ability to infer evolution within hornworts and their early land plant ancestors. Here we report ten new chromosome-scale genomes representing all hornwort families and most of the genera. We found that, despite the deep divergence, synteny was surprisingly conserved across all hornwort genomes, a pattern that might be related to the absence of whole-genome duplication. We further uncovered multiple accessory and putative sex chromosomes that are highly repetitive and CpG methylated. In contrast to autosomes, these chromosomes mostly lack syntenic relationships with one another and are evolutionarily labile. Notable gene retention and losses were identified, including those responsible for flavonoid biosynthesis, stomata patterning and phytohormone reception, which have implications in reconstructing the evolution of early land plants. Together, our pan-phylum genomes revealed an array of conserved and divergent genomic features in hornworts, highlighting the uniqueness of this deeply diverged lineage.

Phylogenomic resolution of lampreys reveals the recent evolution of an ancient vertebrate lineage.

Jawless vertebrates once dominated Palaeozoic waters, but just two lineages have persisted to the present day: lampreys and hagfishes. Living lampreys are a relatively small clade, with just over 50 species described, but knowledge of their evolutionary relationships has always been based on either a few mitochondrial genes or a small number of taxa. Biogeographers have noted the disjunct antitropical distribution of living lamprey families. Here, we present a comprehensive phylogenomic analysis of living and fossil lampreys, sampling 36 species with phylogenomic data and 46 in total with genetic data. We present new divergence time estimates based on comprehensive nuclear data and analysis of their diversification dynamics. Our analysis indicates a central role for extreme global warming during the Late Cretaceous Cenomanian-Turonian Boundary Event as a likely cause for the antitropical distribution of living lampreys, and a notable increase in lineage diversification in Northern Hemisphere lampreys during the Miocene corresponding with a period of global cooling.

Multimodal hierarchical classification of CITE-seq data delineates immune cell states across lineages and tissues.

Single-cell RNA sequencing (scRNA-seq) is invaluable for profiling cellular heterogeneity and transcriptional states, but transcriptomic profiles do not always delineate subsets defined by surface proteins. Cellular indexing of transcriptomes and epitopes (CITE-seq) enables simultaneous profiling of single-cell transcriptomes and surface proteomes; however, accurate cell-type annotation requires a classifier that integrates multimodal data. Here, we describe multimodal classifier hierarchy (MMoCHi), a marker-based approach for accurate cell-type classification across multiple single-cell modalities that does not rely on reference atlases. We benchmark MMoCHi using sorted T lymphocyte subsets and annotate a cross-tissue human immune cell dataset. MMoCHi outperforms leading transcriptome-based classifiers and multimodal unsupervised clustering in its ability to identify immune cell subsets that are not readily resolved and to reveal subset markers. MMoCHi is designed for adaptability and can integrate annotation of cell types and developmental states across diverse lineages, samples, or modalities.

The effect of curcumin, catechin and resveratrol on viability, proliferation and cytotoxicity of human umbilical cord Wharton's jelly derived mesenchymal stem cells.

Mesenchymal stem cells possess the capability to proliferate and differentiate into diverse lineages. Their beneficial properties have been explored widely to treat various disorders. Phytochemicals like curcumin, catechin and resveratrol have been evaluated for their medicinal values and have promising potential in treating numerous diseases. In this study, we have elucidated the in vitro survival, proliferative and cytotoxic effects of these phytochemicals at selected range of concentrations on human umbilical cord derived Wharton's jelly mesenchymal stem cells (WJ-MSCs). The human WJ-MSCs were extracted using explant culture method and characterized as per International Society for Cellular Therapy (ISCT) guidelines. To analyse the effect of different phytochemicals, the WJ-MSCs were treated with various concentrations ranging from 0.1 to 1000 µM and the viability, proliferative and toxicity effects were assayed using (3-(4,5-dimethylthioazolyl-2,5-diphenyltetrozolium bromide) MTT. Curcumin and catechin elicited no cytotoxic effect on WJ-MSCs after 48 hours of treatment between the concentrations ranging from 0.1 to 10 µM and the viability was maintained above 80 %. For both the phytochemicals, there was a significant decrease in the viability of WJ-MSCs after 50 µM. Resveratrol was well tolerated at higher doses till 100 µM with a viability above 90 % and cytotoxic effect was observed above 250 µM. Curcumin, catechin and resveratrol, affect the viability and proliferation of WJ-MSCs differently at varying concentrations. This data will be useful in deciding the dose of phytochemicals when employed concomitantly with stem cells to increase their efficiency.

Cophylogeny, narrow host breadth and local conditions drive highly specialized bird-haemosporidian associations in West-Central African sky islands.

The parasite island syndrome denotes shifts in parasite life histories on islands, which affect parasite diversity, prevalence and specificity. However, current evidence of parasite island syndromes mainly stems from oceanic islands, while sky islands (i.e. mountains isolated by surrounding low-elevation habitats) have received limited attention. To explore the parasite syndrome in Afrotropical sky islands, we examined haemosporidian blood parasites and their bird hosts in two Afromontane regions in Cameroon. Analysing more than 1300 bird blood samples from the Bamenda Highlands and Mount Cameroon, we found considerably reduced parasite lineage diversity and total prevalence in Mt Cameroon, but not in the Bamenda Highlands. We found highly specific parasite-host interactions at both sites and these associations showed significant phylogenetic congruence, suggesting that closely related parasites infect phylogenetically related hosts. These parasite-host associations tend to be shaped mainly by duplications, switches, losses and failures to diverge rather than through co-speciation events. Overall, the high specificity and parasite-host association differences at local scales largely agree with the limited insights from other sky islands. Yet the drivers of these interactions differ geographically, suggesting that unique dynamics of fragmentation and isolation of montane habitats can lead to similar patterns of host-parasite interactions that are shaped by different underlying drivers.

Reshaping the Battlefield: A Decade of Clonal Wars Among Staphylococcus aureus in China

BackgroundLong-term comprehensive studies on the genomic epidemiology of both methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) isolates are limited in China. Here, we aimed to assess the genomic epidemiological characteristics and population dynamics of S. aureus in China. MethodsWe performed whole-genome sequencing and resistance phenotyping on 3,848 S. aureus isolates from bloodstream infections across 72 hospitals in 22 provinces, from 2011 to 2020 in China. We explored the dynamic trends in the resistance/virulence genes and mobile genetic element profiles across lineages, and conducted time-scaled phylogenetic investigation for prevalent lineages. FindingsThe results revealed 315 different sequence types (STs) among all strains, 205 of which were novel. Significant shifts in MRSA population structure were observed, with ST59 replacing ST239 as the dominant lineage, exhibiting widespread inter-hospital transmission and increasing lineage diversity. In contrast, the composition of predominant MSSA lineages, ST188 (11.21%), ST7 (9.79%), ST22 (9.10%), ST5 (8.56%) and ST398 (7.91%), remained relatively stable over time, with the diversity among MSSA strains consistently preserved at the population level. Phylogenetic reconstruction showed that ST59, ST398, ST22 and ST188 MSSA could evolve into corresponding MRSA lineages through the acquisition of staphylococcal cassette chromosome mec (SCCmec) elements. Moreover, the distribution patterns of resistance and virulence genes closely correlated with different lineages, where the proportion of PVL+ isolates in MRSA is rising. Concurrently, changes in the MRSA population structure led to an overall decrease in the number of resistance and virulence genes, significantly increased antimicrobial sensitivity. InterpretationThe shifting genomic landscape of S. aureus in China underscores the need for tailored antimicrobial stewardship and enhances understanding of its epidemiological trends over the past decade.

Protists as mediators of complex microbial and viral associations.

Microbial eukaryotes (aka protists) are known for their important roles in nutrient cycling across different ecosystems. However, the composition and function of protist-associated microbiomes remains largely elusive. Here, we employ cultivation-independent single-cell isolation and genome-resolved metagenomics to provide detailed insights into underexplored microbiomes and viromes of over 100 currently uncultivable ciliates and amoebae isolated from diverse environments. Our findings reveal unique microbiome compositions and hint at an intricate network of complex interactions and associations with bacterial symbionts and viruses. We observed stark differences between ciliates and amoebae in terms of microbiome and virome compositions, highlighting the specificity of protist-microbe interactions. Over 115 of the recovered microbial genomes were affiliated with known endosymbionts of eukaryotes, including diverse members of the Holosporales, Rickettsiales, Legionellales, Chlamydiae, Dependentiae, and more than 250 were affiliated with possible host-associated bacteria of the phylum Patescibacteria. We also identified more than 80 giant viruses belonging to diverse viral lineages, of which some were actively expressing genes in single cell transcriptomes, suggesting a possible association with the sampled protists. We also revealed a wide range of other viruses that were predicted to infect eukaryotes or host-associated bacteria. Our results provide further evidence that protists serve as mediators of complex microbial and viral associations, playing a critical role in ecological networks. The frequent co-occurrence of giant viruses and diverse microbial symbionts in our samples suggests multipartite associations, particularly among amoebae. Our study provides a preliminary assessment of the microbial diversity associated with lesser-known protist lineages and paves the way for a deeper understanding of protist ecology and their roles in environmental and human health.

Potential Evolutionary Convergence in Trophic Adaptations of Two Booidean Snake Lineages as Evidenced by Skull Morphology.

Booidean snakes are a diverse and widespread lineage with an intriguing evolutionary and biogeographic history. By means of cranial morphology and osteology, this study investigates the evolutionary convergence in the Neotropical genera Boa and Corallus on the one hand and the Malagasy clade comprising Acrantophis and Sanzinia on the other. We hypothesize that the mostly arboreal Corallus and Sanzinia present larger jaws and longer teeth to keep hold of the prey and resist gravity and torsional forces acting on their skull while hanging from branches, while terrestrial genera such as Acrantophis show thinner jaws with shorter teeth because they can rely on the full length of their coils to immobilize and constrict the prey together with a substrate that supports the whole of their body. Overall, we highlight how booidean snakes can serve as intriguing subjects for the study of contingency, determinism, and opportunity in the evolution of distant lineages both phylogenetically and geographically. We also provide the first complete description of the skull of Boa constrictor.

Effects of Oregano Essential Oil and/or Yeast Cultures on the Rumen Microbiota of Crossbred Simmental Calves.

This study hypothesized that combining oregano essential oil (OEO) and yeast cultures (YCs) would modulate rumen microbiota to promote gastrointestinal homeostasis and function. Twenty-four newborn, healthy, disease-free, crossbred Simmental male calves (birth weight ≥ 35 kg) were assigned to one of four treatments based on birth data. Treatments were as follows: (1) Control (CON), calves fed calf starter without additives; (2) OEO, calves fed calf starter containing 60 mg/kg body weight (BW) of OEO per day; (3) YCs, calves fed calf starter containing 45 mg/kg BW of YC per day; and (4) MIX, calves fed calf starter with OEO (60 mg/kg, BW) and YC (45 mg/kg, BW) combination. The experimental period lasted 70 days. Rumen fluid was collected on the final day, and 16S rRNA sequencing was performed to assess alterations in rumen microbiota. Calves fed MIX exhibited significantly greater microbial richness, species diversity, and lineage diversity (p < 0.05) compared with calves in the other groups. MIX-fed calves also showed changes (p < 0.05) in the relative abundance of certain rumen species, identified as through LEfSe analysis (LDA > 4, p < 0.05). These biomarkers included f_Rikenellaceae, g_Rikenellaceae_RC9_gut_group, g_Erysipelotrichaceae_UCG-002, c_Saccharimonadia, o_Saccharimonadales, f_Saccharimonadaceae, and g_Candidatus_Saccharimonas. Pathways enriched (p < 0.05) in MIX-fed calves involved nucleotide metabolism, lipid metabolism, glycan biosynthesis and metabolism, amino acid metabolism, terpenoids and polyketides metabolism, antimicrobial drug resistance, xenobiotic biodegradation and metabolism, antineoplastic drug resistance, and excretory system pathways. In conclusion, this study demonstrates that the OEO and YC combination enhances rumen microbial community modulation in calves more effectively than OEO or YCs fed individually or with the control diet.