The genus Ilex belongs to the sole family and is the single genus within the order Aquifoliales, exhibiting significant phenotypic diversity. However, the genetic differences underlying these phenotypic variations have rarely been studied. In this study, collinearity analyses of three Ilex genomes, Ilex latifolia Thunb., Ilex polyneura (Hand.-Mazz.) S. Y. Hu, and Ilex asprella Champ. ex Benth., indicated a recent fusion event contributing to the reduction of chromosomes in I. asprella. Comparative genome analyses showed slight differences in gene annotation among the three species, implying a minimal disruption of genes following chromosomal fusion in I. asprella. Comprehensive annotation of transposable elements (TEs) revealed that TEs constitute a significant portion of the Ilex genomes, with LTR transposons being predominant. TEs exhibited an inverse relationship with gene density, potentially influencing gene regulation and chromosomal architecture. TE insertions were shown to affect the conformation and binding sites of key genes such as 7-deoxyloganetin glucosyltransferase and transmembrane kinase (TMK) genes, highlighting potential functional impacts. The structural variations caused by TE insertions suggest significant roles in the evolutionary dynamics, leading to either loss or gain of gene function. This study underscores the importance of TEs in shaping the genomic landscape and evolutionary trajectories of Ilex species.

MicroRNAs (miRNAs) are key post-transcriptional regulators of gene expression and play important roles in plant development and reproduction. Liriodendron chinense, a representative woody species of Magnoliaceae, produces a large quantity of pollen but exhibits low natural seed set. Despite numerous studies on factors influencing its low seed production, the molecular mechanisms underlying this reproductive limitation remain poorly understood. Here, we performed small RNA sequencing and transcriptome analyses across five tissues of L. chinense, including leaf, calyx, petal, pistil, and pollen. Genome-wide identification yielded 688 miRNAs, comprising both conserved and species-specific members. Expression-based clustering revealed that miRNAs are organized into distinct, tissue-associated modules rather than being uniformly expressed across organs. Among these, a pollen-enriched miRNA module was clearly separated from those associated with leaves and other floral tissues. By integrating sRNA-seq and RNA-seq data, we identified miRNA–target pairs displaying anticorrelated expression patterns, providing expression-level support for miRNA mediated regulation. In pollen, two complementary regulatory modes were observed: low-abundance miRNAs associated with highly expressed target genes, and highly expressed miRNAs associated with repressed targets. The predicted targets of pollen-associated miRNAs were enriched in biological processes central to pollen development, including signal transduction, polarity establishment, vesicle trafficking, and cell wall biogenesis. Overall, this study provides a comprehensive, tissue-resolved view of miRNA expression and pollen-associated miRNA–target relationships in L. chinense, offering candidate regulatory modules for future functional studies of pollen development.