Evolutionary dynamics of regulatory genes orchestrate secondary vascular innovation in Populus

Their work shows that 51% of regulatory genes in the Populus genome belong to the oldest phylostratum (PS1), tracing back to the dawn of cellular life, and that 65% of these ancestral genes expanded via the salicoid whole-genome duplication event. Genomic phylostratigraphy is a statistical genetic method. This method identifies the phylogenetic origin of genes by pinpointing the most distant branch on the tree of life where homologues are detected (Domazet-Lošo & Tautz, 2010). By assigning genes in six Populus genomes to 17 phylostrata (PS1, oldest: Cellular Organisms; PS17, youngest: Populus), Huang et al. observed that PS1 genes constitute > 51% of annotated genes across all species. This prevalence is markedly higher than in herbaceous plants, such as bamboo (c. 26%; Jin et al., 2021), suggesting a fundamental role for deeply conserved genes in woody plant evolution. Gene family clustering revealed significant expansion within PS1 families, with 77% of PS1 clusters containing multiple members, compared with only 3% in the youngest lineage-specific PS17 clusters. These expansive ancient families encode core cellular machinery (e.g. protein kinases, cytochrome P450s, and glycosyltransferases) and, crucially, key TF families, such as MYB, NAC, and WRKY, known master regulators of wood formation. This finding highlights how the expansion and diversification of ancient gene families underpin complex trait evolution. Gene duplication is a primary engine of evolutionary innovation. Huang et al.'s analysis shows that, on average, 81% of Populus genes exist as duplicates. The abundance of duplicate genes in plant genomes arises from ancient duplication events and the high retention of duplicate gene pairs (Panchy et al., 2016). The two main mechanisms generating duplicate genes are WGD and small-scale duplication (SSD). Genomic evolutionary analyses in Populus indicate that Salicaceae underwent a WGD event, the Salicoid WGD event, c. 58 million years ago, preceding the divergence to Populus and Salix (Shi et al., 2024). Huang et al. suggested that 68% of duplicated genes originated from this event, and within PS1, WGD accounted for 65% of gene expansion. WGD initially generates genetic redundancy; however, selection drives retained duplicates (ohnologues) along distinct evolutionary paths: nonfunctionalization (gene loss), neofunctionalization (one copy gains a new function), subfunctionalization (partitioning of ancestral functions), or dosage-balance conservation (Force et al., 1999; Panchy et al., 2016). Huang et al. provide compelling genomic evidence for biased retention and subsequent diversification. PS1 ohnologues have significantly longer coding sequences, more exons, and stronger signatures of purifying selection than younger genes or non-WGD PS1 genes. This suggests that WGD supplied the raw genetic material (expanded ancient gene families) while evolutionary constraints sculpted their structure and function, enabling functional innovation in vascular development. How did these ancient, WGD-expanded genes contribute to the evolution of the secondary vascular system? Huang et al. used phylotranscriptomics to analyse gene expression across cambium, xylem, and phloem in several Populus species. Although genes from younger phylostrata showed tissue-specific expression, PS1 ohnologues exhibited biased expression across the three vascular tissues, driving transcriptomic divergence. The Transcriptome Divergence Index (TDI) confirmed significant expression differences primarily driven by PS1 genes. Co-expression network analysis of P. tomentosa stem tissues identified 206 PS1-origin hub TFs. Among these, MYB, NAC, and WRKY TFs showed distinct evolutionary trajectories. Some ohnologues were dosage-constrained (retaining similar expression and likely ancestral co-regulation), whereas others were dosage-unconstrained. These unconstrained ohnologues evolved strong tissue specificity, particularly in the xylem, acting as hubs in tissue-specific modules. For example, dosage-unconstrained MYB and NAC TFs are highly enriched in xylem networks, consistent with their roles as master regulators of secondary cell wall biosynthesis (Zhong et al., 2019; Li et al., 2024). This suggests that WGD-derived ohnologues released from dosage constraints provided the substrate for evolving novel, tissue-specific regulatory circuits within the vascular GRN. Huang et al. demonstrate this principle through the functional characterization of PtoWRKY53. The gene PtoWRKY53, identified as a xylem-specific hub TF, forms an ohnologue pair with PtoWRKY41, generated by the salicoid WGD. In Arabidopsis, its homologue AtWRKY41 regulates cold stress responses, while PtoWRKY53 exhibits strong xylem specificity, highlighting their distinct functional divergence. Huang et al. proposed neofunctionalization as the underlying mechanism. PtoWRKY53 overexpression reduced plant height and stem diameter, while RNAi knockdown had the opposite effect. Microscopic observations revealed narrower xylem vessels, thinner fibre walls, and reduced cellulose content in overexpressors. Further analysis identified PtoCESA3 as a direct target of PtoWRKY53, which binds a W-box in the PtoCESA3 promoter to repress its transcription. The PtoWRKY53–PtoCESA3 regulatory module is conserved across Populus species (except P. euphratica) and present in Salix purpurea but absent in Arabidopsis. This suggests that the module originated within or before the divergence of the Salicaceae lineage. This exemplifies neofunctionalization: while PtoWRKY41 retained its ancestral stress-related function, PtoWRKY53 acquired a novel role in regulating cellulose deposition and xylem development by repressing a key biosynthetic gene (Fig. 1). It highlights how ancient TFs, generated by WGD, can be co-opted into new developmental programmes. Huang et al.'s work provides a foundational framework for understanding vascular evolution. Their findings reveal that the secondary vascular system is not solely driven by novel genes but relies on ancient genes (PS1) that were significantly expanded and diversified through the Salicoid WGD. This pattern aligns with broader evolutionary trends observed in other taxa, where WGDs facilitate rapid evolutionary innovation (Van de Peer et al., 2017). The study highlights the role of dosage-unconstrained ohnologues, particularly within key TF families, such as MYB, NAC, and WRKY, which evolved tissue-specific expression patterns. This tissue specificity enabled the rewiring of GRNs to support the development of xylem, cambium, and phloem. PtoWRKY53 exemplifies this pattern, evolving from a putative stress regulator to a xylem-specific repressor of wood formation. By integrating macroevolutionary patterns (e.g. phylostratigraphy and WGD) with microevolutionary processes (e.g. gene expression divergence and neofunctionalization) and developmental genetics (e.g. TF function and GRN architecture), the study offers a comprehensive understanding of vascular evolution. Populus, a fast-growing tree genus with significant economic and ecological value, has seen remarkable progress in genomics research. Recent advancements include the development of super-pangenomes (Shi et al., 2024) and telomere-to-telomere assemblies (Bi et al., 2025), alongside spatial and single-cell transcriptomics (Du et al., 2023; Tung et al., 2023). These tools provide unprecedented insights into the genetic basis of vascular evolution. Furthermore, large-scale genome-wide identification of copy number variations has established a statistical framework for linking structural genomic variations to quantitative traits, such as growth and wood properties (Wu & Adams, 2025). The strong predictive power of genomic selection across various traits (Crossa et al., 2025) underscores its potential for molecular breeding. These genomic resources have substantially enhanced our understanding of the molecular mechanisms underlying wood formation and have refined breeding approaches. Huang et al.'s work further emphasizes that the evolutionary success of wood is not only due to novel genes but also to the extensive repurposing and rewiring of ancient genetic toolkits, amplified by WGD. This integration of evolutionary insights with genomic tools paves the way for advancing molecular breeding and improving wood traits in Populus. This work was supported by the National Key Research and Development Program of China (2021YFD2200700), the National Natural Science Foundation of China (32401602), and the 111 Project (B16010). The New Phytologist Foundation remains neutral with regard to jurisdictional claims in maps and in any institutional affiliations.