Ancient allopolyploidy and specific subgenomic evolution drive adaptive radiation in poplars and willows

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However, detecting early allopolyploidy events in evolutionary history and understanding the specific subgenomic evolution that contributes to the origin of adaptive innovations for species radiation can be challenging. Here, we sequenced the genomes representing all three subfamilies of Salicaceae, a woody model clade, and collected epigenetic and transcriptomic samples. We revealed one shared ancient allopolyploidy event involving Populus, S alix and two sister genera, but followed by contrasted karyotypic and subgenomic evolution. The specific evolution drove the origin of unique photoperiod adaptation, flowering phenology and small, hairy seeds in the highly speciose Populus and Salix when compared with their species-depauperate sister genera. These adaptive traits may have ultimately led to the ecological adaptations and species radiation in both poplars and willows. Our findings underscore the previously overlooked role of ancient allopolyploidization and specific subgenomic evolution for fostering adaptive innovation and species diversification at deep nodes of the plant tree of life. One sentence summary: The specific subgenome evolution after ancient allopolyploidy drives the origin of unique adaptive traits that promote species radiation of the highly speciose Populus (poplars) and Salix (willows). Biological sciences/Evolution/Phylogenetics Biological sciences/Genetics/Genomics/Genome evolution Biological sciences/Plant sciences/Plant genetics/Polyploidy in plants Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Polyploidy, or whole-genome duplication (WGD), is common in flowering plants and has played a significant role in their evolution 1-3 . Initially, WGDs result in massive genetic redundancy that allows subfunctionalization of duplicated genes 4 and gives rise to the development of novel traits and adaptations 5-12 . One particularly consequential instance may have been the massive independent WGD events that took place near the Cretaceous-Paleocene (K-Pg) boundary around 60 million years ago (Ma). These events should have contributed to the survival of angiosperms during the mass extinction event at this stage 13-15 . Polyploids can arise through allopolyploidy or autopolyploidy, which involve the merging of genomes between distinct species or between plants within the same species 6 . However, identifying ancient allopolyploidy that have undergone rediploidization and distinguishing their subgenomes is challenging due to the possible extinction of parental lineages and often extensive chromosome rearrangements 6,16,17 . Therefore, despite reports suggesting that allopolyploids may have higher ecological adaptability and evolutionary potential relative to their progenitors and can lead to adaptive radiation 18-22 , the details of the contrasted subgenomic evolution of once ancient allopolyploids and their contributions to adaptive radiations with innovative traits, extensive species diversification and obvious niche shifts in higher taxonomic clades remains scarce 23 . The Salicaceae has been widely recognized as exemplary conducting diverse researches on woody species that encompasses molecular mechanisms, speciation, sex chromosomes, phenotypic variation, and ecosystem services 24 . This family comprises three subfamilies (Samydoideae, Scyphostegioideae, and Salicoideae) with approximately 56 genera 25 ( Supplementary Table 1 ). Speciation rates are extremely elevated in two genera of the Salicoideae, Populus and Salix 26 , which consist of ~100 and ~500 species as the well-known poplars and willows, respectively. Species in these two genera have successfully adapted to diverse environments across the Northern Hemisphere and play crucial roles in temperate and arctic forest ecosystems 24,27,28 . Traits including catkins, dehiscent capsules, hairy seeds, and early spring blooming possibly facilitated their rapid colonization of higher latitudes and an extensive adaptive radiation. In contrast, two other closely related monospecific and dispecific sister genera, Idesia and Itoa , are found in subtropical or tropical regions and have contrasting traits such as capsules or berry fruits, and glabrous seeds 29-32 ( Fig. 1a, data from: https://www.gbif.org/). Consequently, these genera have long been considered attractive model systems for studying adaptive innovation and species radiation in woody plants 33-35 . Previous studies reported that Populus and Salix underwent a 'salicoid' WGD event around the K-Pg boundary 35-37 , which was believed to be autopolyploidization due to the high similarity between subgenomes 38 . However, the detailed evolutionary history of this event and its impact on trait innovation and adaptive radiation across these genera remain poorly understood. Here, we selected species representing all three subfamilies and conducted comparative analysis to gain a better understanding of the origin and potential phenotypic effects of this WGD event (Supplementary Table 1) . Our study aimed to uncover the WGD-derived subgenomic evolution that led to the emergence of unique photoperiod adaptation, flowering phenology and small, hairy seeds in Populus and Salix when compared with the other species-depauperate genera, especially their sister genera Idesia and Itoa . The results revealed that these four genera originated from the common 'salicoid' WGD event, which is an allopolyploidy event involving the extinction of one parental lineage and subsequent divergent evolution of subgenomes. We further discovered that the dynamic gene retention following allopolyploidization, along with lineage-specific expression divergence between subgenomes may have facilitated innovative but contrasting phenotypic traits and ecological niches among these genera. Results Newly assembled genomes redefine the phylogenetic position of the 'salicoid' WGD event We combined Nanopore long reads, Illumina short reads, and high-throughput chromosome conformation capture reads ( Supplementary Table 2 ) to assemble eight chromosomal-level genomes of Salicaceae species from different genera, including seven species from the subfamily Salicoideae ( Dovyalis caffra , Scolopia chinensis , Xylosma longifolia , Flacourtia jangomas , Itoa orientalis , Idesia polycarpa , and Salix rehderiana ) and one species from the subfamily Samydoideae ( Caseria decandra ) (Fig. 1 b and Supplementary Table 1 ). These newly assembled genomes were anchored onto 9 to 21 pseudochromosomes and varied significantly in size, with I. polycarpa (1,214 Mb) and S. chinensis (274 Mb) having the largest and smallest genomes, respectively (Fig. 1 c and Supplementary Tables 2–4 ). As expected, their genome size is positively correlated with the content of repetitive sequence, primarily due to varying degrees of expansion of Gypsy and Copia transposable elements (TE) ( Supplementary Figs. 1 and 2 and Supplementary Table 5 ). Using 672 single-copy genes from 18 species identified by BUSCO and OrthoFinder methods, we reconstructed a high-resolution phylogenetic tree (Fig. 1 b), which matches a previous topology derived from complete plastomes 39 . The topology remained the same when STAG were used for all orthogroups containing multi-copy genes identified by OrthoFinder ( see Materials and Methods ). This tree confirmed that the Samydoideae and Salicoideae subfamilies were monophyletic. Within the Salicoideae, we identified two clades: one that consisted of taxa with n = 19–21 chromosomes (Clade I) and another with fewer chromosomes (n = 9–11; Clade II). The genera Itoa and Idesia were successive sisters to Populus and Salix in Clade I, while Dovyalis was sister to the remaining species in Clade II. Our estimates suggest that the Salicaceae diverged from its sister outgroup, Passiflora , around 93 Ma (95% confidence intervals: 86–101 Ma), and the Samydoideae and Salicoideae subfamilies diverged around 78 Ma (72–85 Ma). Clade I and Clade II of the Salicoideae subfamily diverged around 61 Ma (53–75 Ma) and extant genera within each clade began to diversify around 49 (47–51 Ma) and 36 Ma (22–52 Ma), respectively (Fig. 1 b). By comparing the chromosome and gene numbers of these genomes, we inferred that the 'salicoid' event may be restricted to Clade I species. To test this, we evaluated synonymous substitutions per synonymous site ( K S ) for paralogs in each genome ( Supplementary Fig. 3a ). We found that all these species experienced an ancient WGD (the core-eudicot-common γ event), but only those in Clade I underwent a second recent WGD. After correcting for unequal substitution rates among species, we confirmed that the K S peaks of the recent WGD were greater than those between orthologs within Clade I species, indicating it occurred before their diversification ( Supplementary Fig. 4 ). This was further supported by the well-preserved collinear relationships within Clade I species (2:2) and between Clade I and Clade II species (2:1) ( Supplementary Figs. 3b, 3c and 5 ). Taken together, these results consistently suggest that the 'salicoid' WGD event occurred on the ancestral branch of Clade I species. We next predicted the present distribution of the nine genera in the Salicaceae family using ecological niche modelling ( Supplementary Figs. 6 and 7 and Supplementary Tables 6 and 7 ). The results revealed that the four genera within Clade I, which underwent the 'salicoid' WGD, migrated to the Northern Hemisphere and exhibited niche differentiation, while the remaining lineages were primarily distributed in the Sounthern Hemisphere. Unlike Itoa and Idesia , which are restricted to subtropical regions, the genera Populus and Salix have expanded to higher latitudes, including temperate and polar regions, where they experienced extensive adaptive radiation. These findings suggest that the 'salicoid' WGD event may have played a critical role in driving ecological shifts and species diversification in their adaptability. Karyotypic evolution and chromosomal rearrangement Using genome collinearity among these species, we reconstructed the ancestral Salicaceae karyotype (ASK), which consists of 11 putative protochromosomes (Fig. 2 and Supplementary Figs. 8–10; see Materials and Methods ), rather than the previously suggested number of 10 35 . Drawing from the 'salicoid' WGD and genome rearrangement events, we further inferred their evolutionary relationships (Fig. 2 ), which align closely with the phylogenetic analyses (Fig. 1 b). Few fusion/fission events occurred in species from Clade II, which did not undergo the 'salicoid' WGD. Thus, in Clade II most protochromosomes were preserved intact, with the exception of at least four chromosomal rearrangements: i) protochromosome 8 underwent reciprocal translocation (RTA) with protochromosome 11 at the ancestral node of Clade II, ii) protochromosome 2 underwent end-to-end joining (EEJ) with protochromosome 3 to form chromosome 2 of D. caffra , iii) protochromosome 2 independently underwent reciprocal translocation (RTA) with protochromosome 4 to form the contemporary karyotype structure of S. chinensis , and iv) protochromosome 1 subsequently underwent an EEJ to form chromosome 1 of X. longifolia and F. jangomas (Fig. 2 and Supplementary Figs. 11a and 11b ). We inferred that the ancestral karyotype of Salicoideae Clade I species had a base number of 21 after the 'salicoid' WGD, achieved through an EEJ fusion between duplicated protochromosomes 5 and 9, with at least two subsequent inversions (Fig. 2 and Supplementary Fig. 11c ). Additionally, we also predicted multiple RTA events between several protochromosomes. Interestingly, these genome-wide reorganizations were fully preserved in I. polycarpa without other large fusion/fission events, indicating that their common ancestor already possessed a relatively stable karyotype during the rediploidization process. In contrast, our results indicated that I. orientalis underwent significant changes through multiple chromosomal rearrangements, resulting in a base chromosome number of 20. Ancestors of Populus and Salix also experienced a series of chromosome rearrangements, forming an ancient fused chromosome through successive nested chromosome fusions (NCF) of duplicated protochromosomes 3, 7 and 11 ( Supplementary Fig. 11d ). This new fused chromosome subsequently underwent two independent RTA events: one with protochromosome 1 in Populus to generate extant chromosomes 1 and 4, and a second with protochromosome 5 in Salix to generate extant chromosomes 4 and 16. This evolutionary trajectory resulted in a 19 chromosome karyotype in Populus and Salix (Fig. 2 ). The 'salicoid' WGD event is a cryptic allopolyploidization Combining the correspondence between ASK and extant chromosomes of each Salicaceae species, as well as phylogenetic analysis and subgenome division ( Supplementary Fig. 12; see Materials and Methods ), we found strong evidence to support that the ‘salicoid’ WGD is a cryptic allopolyploidization event. Specifically, according to the reconstructed protochromosomes (ASK1-11), we identified 1,634 full homologous gene groups (HGGs), among which all Clade I species retained paralogous genes from the 'salicoid' WGD, while other species retained the orthologous genes ( Supplementary Fig. 13 ). There were 77–265 full-HGGs across the 11 protochromosomes. Phylogenetic tree using the ASTRAL coalescent method showed that genes from Clade II were more closely related to one copy of 'salicoid' paralogs in Clade I species than the other copy (Fig. 3 a, topologies ‘q1’ and ‘q2’ in Fig. 3 b). This relationship was supported by 83.5–90.3% of full-HGGs on all 11 protochromosomes and 80.1–84.0% when considering partial-HGGs, where at least one species in Clade I retained 'salicoid' paralogs (Fig. 3 b and Supplementary Fig. 13 ). Consistently, we found that an RTA event between protochromosomes 8 and 11 that occurred in the ancestor of Clade II species was also present in only one chromosome in all Clade I species, further supporting their allopolyploid origin (Fig. 2 ). Based on their phylogenetic relationships with Clade II, we divided the genome of Clade I species into two subgenomes: the A subgenome consisted of chromosomes with genes closely related to Clade II, while the B subgenome was composed of the paralogous chromosomes from the 'salicoid' WGD (Fig. 3 c and Supplementary Fig. 14 ). As expected, K S distribution between Clade II species and A subgenome of Clade I species was generally smaller than that between Clade II and B subgenome, but both A and B subgenomes were similar in genetic distance to C. decandra ( Supplementary Fig. 15 ), a degree of divergence consistent with phylogenetic relationships under allopolyploidization. Phylogenetic trees reconstructed using the A and B subgenomes by coalescent and concatenated methods revealed the same topologies with high support ( Supplementary Fig. 16 ). These results indicate that the 'salicoid' event in Clade I species was an allopolyploidization event with one parent being the common ancestor of Clade II species. To identify the second possible parent of the 'salicoid' allopolyploidization, we assembled the draft genomes of four additional species from the subfamily Salicoideae (including Azara serrata , Prockia crucis , Banara guianensis and Homalium cochinchinense ) and one species from the subfamily Scyphostegioideae ( Dianyuea turbinata ). These draft genomes are fragmented but, based on gene prediction, cover most of the coding regions ( Supplementary Table 8 ). Therefore, we added gene sequences from these five more genera to the HGGs and constructed coalescent and concatenated trees ( Supplementary Fig. 17 ). In both trees, four of the new genera from Salicoideae clustered within Clade II, while D. turbinata was sister to both subgenomes, indicating that none of these species was direct donors of the B subgenome. Since these species represent all three subfamilies of Salicaceae 39 , 40 , w-e inferred that the parental donor of the B subgenome is likely extinct. Moreover, we inferred that the two parental lineages diverged for approximately 8 million years prior to the 'salicoid' WGD based on our estimates that the A and B subgenomes diverged about 68 Ma and Clade II diverged from the A subgenomes about 60 Ma (Fig. 3 a). Finally, based on K S values corrected for the 'salicoid' WGD, rates of molecular evolution were the slowest in I. polycarpa , followed by I. orientalis and Populus , while Salix evolved fastest (Fig. 3 d). Subgenome dominance and lineage-specific gene retention Subgenome evolution after polyploidization plays a crucial role in reducing gene redundancy and increasing subsequent potential for trait and adaptive innovation 12 , 41 . Our analysis of 21 Salicaceae genomes/subgenomes showed that A subgenome exhibited evolutionary dominance over B subgenome in Clade I species. First, we found that after the 'salicoid' event, the rate of gene retention was reduced significantly in Clade I species and was more lower in the B subgenome than in the A subgenome (Fig. 4 a). Second, B subgenome showed lower gene expression levels than A subgenome in all Clade I species (Fig. 4 b). This may be related to higher repeat content surrounding genes in the B subgenome ( Supplementary Fig. 18a ), although there was no significant difference in the expansion of TEs between subgenomes A and B overall ( Supplementary Fig. 18b ). Third, genes in the A subgenome had slightly but significantly higher chromatin accessibility levels ( Supplementary Fig. 18c ) and slightly lower methylation levels than those in the B subgenome ( Supplementary Figs. 18d and 18e ), suggesting that the differentiation between the A and B subgenomes is primarily reflected in sequence variation rather than in epigenetic landscapes. Fourth, protein evolution rate ( K a/ K S ) was significantly higher in the B subgenome, indicating relaxed purifying selection (Fig. 4 c). Finally, through an analysis of the contribution of subgenome-biased gene retention to lineage evolution, we found that genes specifically retained in the A subgenome of all Clade I species (n = 958), were enriched in functions such as 'chromosome segregation', 'double-strand break repair', 'meiotic cell cycle process', and 'spindle organization' (Fig. 4 d). In contrast, genes uniquely retained in the B subgenome (n = 558) did not exhibit enrichment in any specific functional categories. These findings suggest that the A subgenome may play a pivotal role in facilitating and coordinating chromosome recombination during the rediploidization process, while gene retention in the B subgenome appears to be more random and functionally diverse. Moreover, beyond subgenome dominance, gene retention may also exhibit lineage-specific dynamics over evolutionary time, which are closely linked to the diverse adaptation of different lineages to complex environments 9 . To explore this, we further analyzed gene retention at three key evolutionary nodes in Clade I (Fig. 4 e). We identified 1,865 WGD-derived gene pairs retained across all Clade I species (Node I). These genes are functionally associated with 'regulation of photoperiodism, flowering', 'inflorescence development', 'regulation of seed development', 'trichome morphogenesis', 'cold acclimation' and 'defense response' (Fig. 4 d). The retention of key genes related to photoperiod-flowering (such as FT , CIB1 and HUA2 ) 42 – 45 , cold acclimation and freezing tolerance ( MYB15 , ADF5 and GALS2 ) 46 – 48 , and trichome development ( GIS3 , ZFP6 and TOE1 ) 49 – 51 ( Supplementary Figs. 19a and 19b ) likely facilitated the divergence of Clade I species from their ancestral lineage by enabling adaptation to northern regions with variable photoperiods and temperatures, while promoting trait development. Consistent with this, more 402 pairs of genes with similar functions were specifically retained in Node II representing the deciduous lineages of Idesia , Populus and Salix that are distinct from the evergreen lineage Itoa (Fig. 4 d), including FD , NF-YB3, SPL4 , SPL5 and ELF9 52–55 , which are related to photeperiodic flowering (Fig. 4 e, Supplementary Figs. 19c and 19d ). Additionally, at Node III, representing the Populus and Salix lineages that are widely distributed in the Northern Hemisphere, we identified 129 uniquely retained gene pairs (Fig. 4 e). These genes are involved in specific functions like 'maintenance of floral organ identity', 'seed trichome differentiation', 'red or far-red light signaling pathway' and 'regulation of plant-type cell wall cellulose biosynthetic process' (Fig. 4 d), such as REV 56 related to the formation of inflorescence and floral meristem, and CSLD1 57 related to the development of root hairs and female gametophytes ( Supplementary Figs. 19e and 19f ). Overall, the dynamic patterns of gene retention in these lineages suggest that photoperiod adaptation, flowering, and traits development including inflorescence and seeds, have fostered the diversification of Clade I species, promoting their morphological innovation and environmental fitness. Lineage-specific subgenomic expressions contributed to adaptive innovation in multiple traits of Populus and Salix Expression divergence of gene pairs plays a pivotal role in driving adaptive radiation and evolutionary innovation after polyploidization 58 , 59 . To investigate the association between subgenome expression patterns and lineage diversification in the Salicaceae, we focused on gene pairs retained across all Clade I species. Transcriptome sequencing and cluster analysis of male and female flowers, as well as fruits at various developmental stages in Itoa , Idesia and Populus ( Supplementary Table 9 ), revealed that the expression biases between subgenomes A and B for gene pairs is largely consistent across tissues and developmental stages within the same species (Fig. 5 a). However, significant differences were observed between species. Specifically, among the 1,747 gene pairs examined, only 85 (C1) and 51 (C2) gene pairs showed the same expression bias towards subgenomes A and B, respectively, across the three lineages. In contrast, the vast majority (C4-C15, ~ 80%) of gene pairs showed clear differences in expression divergence between lineages (Fig. 5 a). Among the clusters with consistent expression biases, gene pairs in C1, with higher expression in the subgenome A (Fig. 5 a), were significantly enriched in functions related to reproductive transitions, seed maturation and stress responses ( Supplementary Table 10 ), including genes such as FT , HDA6, AP2 , BBX21 , GALS2 and transcription factors belonging to WRKY family 42 , 48 , 60 – 64 (Fig. 5 b). Conversely, gene pairs in C2, with higher expression in the subgenome B, were mainly associated with regulation of abscisic acid signaling and gene expression regulation ( Supplementary Table 10 ). These results suggest the significant role of the subgenome A in regulating reproductive and growth traits in these species. For example, the WGD-derived paralogs of flowering-related gene FT exemplified functional divergence: FT2 in subgenome A was highly expressed in our examined transcriptomes, while FT1 from subgenome B was rarely expressed (Fig. 5 b), supporting a role of FT2 in promoting flower and fruit development. Interestingly, previous studies on poplar have shown that FT1 is expressed in cold-exposed buds during winter, triggering the transition from vegetative meristems to the reproductive phase, while FT2 is primarily expressed during the growing season, regulating vegetative growth. These spatiotemporal shifts in expression are essential for the annual growth cycle of poplar 60 , 65 . To further explore the evolutionary insights into the functional divergence of this gene pair, we analyzed their expression patterns in leaves and buds (or shoot apex) from I . orientalis and I. polycarpa across late autumn to early spring (Fig. 5 c). The results showed that in leaves, both FT1 and FT2 were expressed during winter in I . orientalis , while only FT1 was expressed in I. polycarpa and poplar. In buds or shoot apex, FT1 was exclusively expressed in poplar, whereas FT2 was specifically expressed throughout winter in I . orientalis and only in late winter in I. polycarpa (Fig. 5 c). This unique functional divergence of FT genes in poplar is consistent with its phase change from vegetative to reproductive growth during the transition from active growth to winter dormancy, enabling earlier flowering and providing a reproductive advantage in spring. In contrast, the co-expression of FT1 and FT2 in I . orientalis likely correlates with its evergreen and non-dormant traits, while the expression patterns in I. polycarpa appear linked to the initiation of reproductive growth after spring warming. The lineage-specific divergence in FT expression have likely promote the variation in flowering phenology among these lineages. Furthermore, we found that the gene pairs with Populus -specific expression divergence (C4-C6 in Fig. 5 a) were significantly enriched in functions related to 'photoperiodism, flowering', 'regulation of circadian rhythm', flower development, 'regulation of seed development' and 'trichome morphogenesis' ( Supplementary Table 10 ). Among these, we identified several genes involved in the regulation of flower development, including FIL , PUB13 , BRM 66 – 68 , as well as ENO2 and XYL1 69,70 , which are associated with seed development and seed size determination. Additionally, we also identified ZFP6 and GIS3 , a pair of WGD-derived genes regulating trichome development 49 , 50 , which exhibited significantly higher expression levels in Populus compared to Idesia and Itoa (Fig. 5 b). Four Populus - Salix specific conserved noncoding elements (CNEs) were discovered in their promoter regions (Fig. 5 d). Reporter assays indicated that these CNEs possess potential enhancer activities, likely contributing to increased gene expression during seed trichome development in the Populus-Salix lineage (Fig. 5 e and Supplementary Table 11 ). In summary, these findings suggest that the lineage-specific divergence in subgenomic gene expression significantly contributed to the evolution of distinct flowering phenology and the development of small, hairy seeds in Populus and Salix . These traits likely played a critical role in enabling their rapid colonization and extensive adaptive radiation in high-latitude regions. Discussion Ancient allopolyploidization events are often obscured by large-scale subgenome reshuffling. In this study, we clearly demonstrate that the ancient polyploidization event, shared by the genera Populus and Salix , as well as their sister genera Idesia and Itoa in the Salicaceae, was caused by hybridization between two closely related lineages with a divergence time of approximately 8 million years. Due to the high degree of similarity between the two lineages 38 , this cryptic allopolyploidization event would have been difficult to detect without information from either parental lineage. Our analysis showed that one of the parental genomes is represented by the Clade II lineage of the Salicoideae subfamily, while the other parental lineage is now extinct. Based on this, we successfully separated the genomes of these polyploidized species into two distinct subgenomes. We further constructed the common ancestral karypotype and detected unique chromosomal fusions specific to Itoa , Idesia and Populus - Salix . The lineage-specific karyotypic evolution is well consistent with the phylogenomic tree and intact chromosomes are widespread and shared by three lineages ( Fig. 2 ) . These results also revealed that despite the 60 million years since allopolyploidization and subsequent species diversification, a considerable number of chromosomes have maintained relatively stable organization. Therefore, the Salicaceae, along with other lineages including Gossypium and related Malvaceae, Brassicaceae, and various subclades of Poaceae 71 – 73 , provides a potential model to investigate the long-term consequences of allopolyploidization and the subsequent evolution of subgenomes (Fig. 6 ) . This is in contrast to recent polyploids that have not undergone rediploidization or species diversification, or ancient polyploids with unknown parental lineages 74 , 75 . Our study shows that subgenomes derived from two parental lineages continue to exhibit evolutionary dominance even after a long time, regardless of the extent of chromosomal rearrangements in the respective descendant lineages. This dominance is typically manifested as biases in gene retention, variations in gene expression levels, and differences in evolutionary rates. Consistent with previous speculation on the role of paleopolyploidy in the adaptive evolution of angiosperms 15 , we found that genes associated with environmental responses, particularly those involved in photoperiod regulation, flowering cycles and cold adaptation, were preferentially retained following allopolyploidization. However, although we observed the same dominance of subgenome A over subgenome B, the retained genes and expression divergence exhibit significant variation between three lineages, Itoa , Idesia and Populus - Salix . Importantly, these lineage-specific evolutionary patterns align with their adaptive traits and ecological niches in the Northern Hemisphere, such as the distinct flowering phenology and highly-effective seed dispersal facilitated by hairs in the Populus - Salix lineage 27 , 28 , 32 , promoting their adaptive radiation at higher latitudes (Fig. 6 ) . In contrast, the other two lineages, Idesia and Itoa , which consist of only one and two species respectively, well adapt to tropical and subtropical environments with totally different gene retention and expressions, relying on animal-mediated pollination and animal- or wind-mediated fruit or seed dispersal. These findings suggest that allopolyploidization in Salicaceae conferred shared properties of evolutionary dominance to subgenomes, while contrasting gene retention and expression divergence provided significant potential for trait diversification and adaptive evolution across different descendant lineages. This supports the ancestral polyploidization as a driving force of key innovations and lineage diversification in angiosperms 10 . However, our results suggest not all allopolyploidy events will undoubtedly lead to the origin of the adaptive innovations and following species radiation as previously thought 18 – 22 . Overall, our study provides insights into different subgenome evolution after allopolyploidy underlying the diverse traits observed in the economically and ecologically significant Saliaceae, once woody model clade. This should prevail in multiple higher taxonomic clades of both plants and animals. In addition, our results further underscore the importance of distinguishing between autopolyploidization and allopolyploidization events as well as contrasted subgenomic evolution that foster adaptive innovation and species diversification at deep nodes of the tree of life. Materials and Methods Sample collection and genome sequencing Plant material of Casearia decandra , Dovyalis caffra , Scolopia chinensis , Xylosma longifolia and Flacourtia jangomas were collected in XiShuangBanNa Tropical Botanical Garden (Mengla, China), Itoa orientalis and Idesia polycarpa were collected in Chengdu, and Salix rehderiana is collected in Minya Konka of China, respectively. Fresh leaves were collected, and high-quality genomic DNA was extracted using the QIAGEN Genomic DNA extraction kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Three approaches were employed in DNA sequencing. First, genomic DNA was size-selected using the BluePippin system (sage Science), processed following the protocol of Ligation Sequencing Kit (LSK108), and sequenced using the Oxford Nanopore Technology sequencer. Sequencing was performed on the PromethION platform, and base calling was carried out using Guppy v3.2.8. Second, Paired-end libraries were constructed according to the manufacturer’s protocols and sequenced using the Illumina HiSeq 2500 System. Third, Hi-C (high-throughput chromatin conformation capture) libraries were prepared by chromatin extraction and digestion and DNA ligation, purification, fragmentation, and sequenced on an Illumina HiSeq 2500 76 . In addition, the dried leaves of Azara serrata , Prockia crucis , Banara guianensis and Homalium cochinchinense were used to extract DNA, construct Illumina paired-end libraries, and perform sequencing. For Dianyuea turbinata , The HiFi SMRTbell library was constructed using the SMRTbell Express Template Prep Kit v2.0 (Pacific Bioscience) and sequencing was carried out using the PacBio Sequel II platform (Berry Genomics, Beijing, China). Genome assembly and annotation Nanopore long reads were de novo assembled using the Nextdenovo v2.2.0 ( https://github.com/Nextomics/NextDenovo ). The initial assemblies were further corrected and polished using the program NextPolish v1.0 ( https://github.com/Nextomics/NextPolish ), by mapping the filtered Nanopore and Illumina reads to the genome using Minimap2 v2.17 77 and BWA v0.7.17 78 . Finally, contigs were clustered, ordered and anchored to the pseudochromosomes by LACHESIS 79 using validly mapped Hi-C reads. Illumina reads of A. serrata , P. crucis , B. guianensis and H. cochinchinense were assembled using Platanus v1.2.4 80 by implementing ‘assemble’, ‘scaffold’ and ‘gap_close’ program. PacBio HiFi reads were used to perform de novo genome assembly for D. turbinata with Hifiasm v0.14 81 . We utilized a combination of homolog-based and de novo approaches to annotate repetitive elements. RepeatMasker v.4.0.7 82 was firstly used to perform homolog prediction based on the Repbase database 83 . Next, RepeatModeler v.1.0.11 84 was used to perform de novo prediction of repeat sequence features and the results were then utilized by RepeatMasker v.4.0.7 82 to identify and classify repeat elements. Gene models were predicted based on de novo prediction, homologous identification and transcript data. In brief, Augustus v3.2.3 85 was used for de novo prediction of protein-coding genes. For homologous identification, we mapped the protein sequences of six published genomes ( P. trichocarpa 35 , P. pruinosa 86 , P. alba var. pyramidali 87 , S. suchowensis 36 , S. purpurea 88 , and Arabidopsis thaliana 89 ) onto the genomes using TBLASTN v2.6.0 90 and then used GENEWISE v2.4.1 91 to predict gene structures. RNA transcripts were used to predict gene models with PASA v2.3.3 92 . Finally, all of the predictions were integrated using EvidenceModeler v1.1.1 93 to generate consensus gene sets. Assembly and annotation completeness was assessed with BUSCO (Benchmarking Universal Single-Copy Orthologs) v3.0 94 . Phylogenetic analysis We conducted a phylogenomic analysis for eight newly sequenced species and three poplar ( P. trichocarpa 35 , P. alba var. pyramidalis 87 and P. euphratica 95 ) and two willow species ( S. chaenomeloides 96 and S. purpurea 97 ), using Passiflora edulia 98 , Passiflora organensis 99 , Arabidopsis thaliana 89 , Rosa chinensis 100 and Vitis vinifera 101 as outgroups. We constructed two phylogenetic datasets using different strategies: targeted identification of phylogenomic markers (BUSCO) and de novo inference (OrthoFinder). Conserved single-copy genes were identified by BUSCO analyses with the embryophyta_odb10 dataset (1,614 BUSCOs) 94 , resulting in 426 single-copy orthologs retained across 18 species. OrthoFinder v2.3.11 102 was used to de novo identify orthologous sequences shared among species with default parameters, resulting in 422 single-copy orthologs. To reconstruct a high-resolution phylogenetic tree, we merged the two datasets and obtained a total of 672 single-copy orthologs. For all orthologs, protein sequences were aligned using MAFFT v7.313 103 , and converted to codon alignments using PAL2NAL 104 . A maximum likelihood (ML) phylogenetic tree was then constructed using RAxML v8.2.11 105 with the GTR + gamma model and 1000 bootstrap replicates. Additionally, STAG from the OrthoFinder pipeline was also used to infer a species tree based on all orthogroups (including multi-copy genes) identified by OrthoFinder. The topologies of the phylogenetic trees constructed by the two methods were the same. The divergence times among species were estimated using the MCMCtree program 106 . Three constraints obtained from the TIMETREE database ( http://timetree.org/ ) were used for time calibrations: (1) the divergence between Vitis and Rosa (109–124 Mya), (2) the divergence between Arabidopsis and Populus (102–113 Mya), (3) the divergence between Populus and Salix (28–60 Mya). Analysis of WGD events We identified and localized WGD events in Salicaceae by combining intra- and inter-species synteny analysis and Ks distribution. First, we used the BLASTP v2.7.1 107 with a cutoff e-value of 1e-5 to align protein sequences within species and between species ( P. trichocarpa vs. I. polycarpa , P. trichocarpa vs. I. orientalis ). WGDI ( https://github.com/SunPengChuan/wgdi ) 108 with the ‘-icl’ parameter was used to identify the intergenomic synteny blocks between P. trichocarpa and others, as well as intragenomic synteny blocks within each species. The Ks between collinear genes was estimated by Nei-Gojobori approach in PAML with the parameter ‘-ks’ of WGDI, and the ‘-bk’ parameter was applied to generate a dot plot of collinear genes and Ks values, visualizing intra- and inter-species synteny. Additionally, the K S peaks were fitted using the ‘-pf’ parameter, and the density distribution curve of K S was displayed using the ‘-kf’ parameter. The location of the WGD event was identified based on the comparison of the Ks peaks between paralogs within species and orthologs between species. To address potential inaccuracies in detecting WGD events due to differing substitution rates among candidate species, we further applied KsRates v1.1.3 to bring all the distributions to a common Ks scale by compensating for the differences in synonymous substitution rates relative to the focal species, and the rate-adjusted mixed paralog-ortholog Ks distribution was then used to position adjusted WGD events. Ecological niche modelling The data collection for each genus was obtained from the Global Biodiversity Information Facility (GBIF: https://www.gbif.org/ ). To remove spatial autocorrelation and sampling bias, the obtained distribution data were subjected to 5 km spatial dilution using SDMtoolbox 109 , and the final distribution points ( Salix : 131,974, Populus : 82,944, Idesia : 1,183, Itoa : 113, Flacourtia : 2,905, Xylosma : 3,802, Scolopia : 3,689, Dovyalis : 2,039, Casearia : 14,416) were used for Maxent modeling analysis. Environmental layers for 19 bioclimatic variables at current time (1970–2000) were downloaded from the WorldClim v2.1 dataset ( http://www.worldclim.com/ ) at a spatial resolution of 10 arc minutes (see Supplementary Table 6 ). Pairwise correlations were examined for 19 variables within the distribution of each genus. Taxon distributions were reconstructed using variables with a pairwise Pearson correlation coefficient below 0.8 and the most ecological significance. Ecological niche modeling (ENM) was performed using Maxent 3.4.3 110 to simulate potentially suitable habitats under the current climate for each genus. The test output of the models was set at 30%. The accuracy of the model was assessed using the area under the curve (AUC) of the receiver operating characteristic (ROC) plot. AUC values above 0.7 were considered indicative of good model performance 111 . ArcGIS 10.8 was utilized for mapping the suitable distribution range. To examine niche differences, ENMtools 112 was employed to calculate niche overlap statistics Schoener's D 113 and Hellinger’s-based I 114 , with 100 pseudo-replicates. Values of D and I range from 0 (no ecological niche overlap) to 1 (identical ecological niches). Ancestral karyotype reconstruction Comparing gene collinearity between genomes can reflect karyotype changes, revealing the trajectory of the formation of existing chromosomes and inferring evolutionary relationships independently. During karyotype evolution, ancestral chromosomes (protochromosomes) may have fused or remained as independent chromosomes within existing genomes. We first applied the workflow by Sun et al. 71 , which identifies protochromosomes and reconstructs ancestral karyotypes by searching for independent chromosomes or chromosome-like homologous blocks shared across different lineages. A detailed example of the workflow is available at https://github.com/SunPengChuan/wgdi-example/blob/main/Karyotype_Evolution.md . Specifically, using the Casearia decandra genome as a reference, we aligned the remaining 12 genomes using WGDI 108 with the parameter ‘-d’. Synteny blocks shared between independent chromosomes were first searched in all genomes, and synteny blocks of independent chromosomes identified in at least three genera were assumed to represent Salicaceae protochromosomes. For example, protochromosome 6 (ASK6: homologous to Chr6 of Casearia ) of the ancestral Salicaceae karyotype is retained as an independent chromosome in Chr6 of Scolopia , Chr5 of Xylosma and Flacourtia , Chr20 of Itoa , Chr5 of Idesia , Chr13 of Populus and Salix ( Supplementary Figs. 8 and 9a ). Similarly, ASK1, ASK2, ASK4, ASK5, ASK7, ASK9, ASK10 and ASK11 are retained as independent chromosomes in at least three genera. Therefore, these independent chromosomes were extracted as protochromosomes. Next, all identified synteny blocks were removed from fused chromosomes in existing genomes, and the remaining parts were connected as a chromosomes for a new round of exploration. After removing ASK11 (Chr11 of Casearia ), Chr7 and Chr10 in Flacourtia were connected, corresponding to partial fragments of Chr3 and Chr8 in Casearia , which also remain intact in many other species and therefore were identified as ASK8 ( Supplementary Fig. 8 ). After further removing ASK8, Chr2 in Flacourtia corresponds to segments of Chr3 and Chr8 in Casearia , which are independent chromosomes in many other species, and therefore was identified as ASK3 ( Supplementary Fig. 8 ). Ultimately, each extant genome had no remaining genomic blocks, and a total of 11 putative protochromosomes were extracted, hypothesized to form the ancestral Salicaceae karyotype (ASK). Additionally, we also used the MLGO web service ( http://www.geneorder.org/ ) 115 to infer ancestral genomes based on information from synteny blocks between species and the phylogenetic tree constructed using single-copy orthologs, which also resulted in 11 ancestral chromosomes. Moreover, to obtain a more complete ancestral karyotype gene set, we expanded protochromosomes based on five species (Clade II and C. decandra ) that had not undergone the second WGD and retained relatively complete ancestral karyotypes ( Supplementary Fig. 9b ). Specifically,the genomes were aligned to the initial protochromosomes to identify syntenic blocks. If five or fewer gene clusters surrounded by collinear genes on the chromosomes of an existing species corresponded to two ordered ancestral genes on the protochromosomes, these intermediate genes were added between the two ancestral genes to extend the protochromosomes 116 . This process resulted in 11 putative protochromosomes containing as many genes as possible. The 13 extant genomes were aligned with the expanded protochromosomes, and WGDI with the parameter ‘-km’ was used to determine the karyotype composition from protochromosomes based on syntenic blocks, allowing the inference of chromosome fusion and evolutionary patterns ( Supplementary Fig. 10 ). Identification of allopolyploidization and subgenomes We followed the workflow in the Supplementary Fig. 12 to identify polyploidy types and split subgenomes. 1) We performed synteny analysis of all 13 Salicaceae species with the reconstructed protochromosomes, and performed K S calculation and information integration of collinearity fragments using the ‘-ks’ and ‘-bi’ programs of WGDI. Since the Clade I species experienced 'salicoid' WGD, we first split the two homologous subgenomes of P. alba var. pyramidalis corresponding to each protochromosomes according to the collinear fragment information, while the subgenomes of the remaining 7 Clade I species were split according to the collinear relationship with P. alba var. pyramidalis . The split information was added to the integrated collinear fragment information file, and the homologous gene list between the protochromosomes and each species was obtained through the "-a" program. Finally, the homologous genes of all species were merged to obtain the 1:2 homologous gene groups (HGGs) among CladeII/ C. decandra and CladeI. We then classified the HGGs (C1-C12) based on the number of gene copies in each species, with Full-HGGs and Partial-HGGs used for subsequent analysis ( Supplementary Fig. 13 ). 2) Gene trees were constructed for each Full and Partial-HGG using RAxML v8.2.11 105 with the two Passiflora species as outgroups. 3) The gene trees were utilized by ASTRAL v.5.6.2 117 to infer species trees with quartet scores and posterior probabilities for each protochromosome. The polyploidization type was inferred by counting the proportion of different topologies of the gene tree of protochromosome. In addition, GRAMPA v1.4.0, a topology-based gene-tree reconciliation algorithm, was also used to infer the mode of polyploidization, and the optimal tree with the lowest score was consistent with the topology of the ASTRAL tree. Next, according to the topology of the gene trees, the gene closer to Clade II is classified as the A subgenome, and the other paralogous gene belongs to the B subgenome. 4) Genes from different subgenomes were mapped onto the chromosomes of their respective species, and the genome was split into A and B subgenomes based on the gene locations. 5–6) The genes belonging to different subgenomes were concatenated, and the concatenated tree was constructed using RAxML v8.2.11 105 to further verify the polyploidization type. Divergence times were estimated using a phylogenetic tree constructed from full-HGGs and MCMCTree 106 in the PAML package, based on the divergence time between Populus and Salix . 7) Finally, yn00 function in PAML 106 was used to calculate the Ks values between the A subgenome and Clade II/Casearia, as well as between the B subgenome and Clade II/Casearia, thereby obtaining the divergence levels. Because the Clade I species share 'salicoid' WGD event, and genes from different subgenomes were identified in each species, we employed relative rate tests to estimate the evolutionary rate ( K S ) after the recent WGD event for each species. Since I. orientalis is at the base of the Clade I, we separately calculated the K S values of the remaining seven species after they diverged from I. orientalis based on the methods in previous studies 118 . Gene retention and repeat sequence content of subgenomes Based on homologous genomic data from 13 species, we compared the genomic and subgenomic characteristics of these species. To quantify the gene retention in the Clade II species, as well as A and B subgenomes of Clade I species, we first selected HGGs that retained C. decandra and then calculated the percentage of gene retention in these genomes, using non-overlapping windows of 100 genes along the protochromosomes. In addition, based on the results from RepeatMasker and RepeatModeler, we computed the repeat sequence content of genes and their surrounding 2k regions in all genomes. We further searched the genome of Clade I species using the LTRharvest 119 and LTRdigest 120 programs to de novo detect intact LTRs according to the pipeline in previous studies 121 . The 5′ and 3′ repeats of each LTR were aligned by MUSCLE v3.8.31 122 to estimate the substitution rate, and insertion times were finally estimated by assuming a mutation rate of 2.5×10 − 9 per year 123 . Sequencing and analysis of transcriptome data Total RNA was extracted from leaf, bud, shoot apex, flower and fruit tissues of Salicaceae species, respectively ( Supplementary Table 9 ). The extracted RNA was purified using poly-T oligo-attached magnetic beads. All transcriptome libraries were constructed using the Illumina TruSeq library Stranded mRNA Prep Kit and sequenced on an Illumina HiSeq 2000 platform. Quality-filtered reads were aligned to their own genomes using HISAT2 v2.1.0 124 , and then the expression levels (TPM) for each gene were calculated and normalized by StringTie v1.3.3b 125 . Transcriptome data were used in the following studies: 1)The expression levels in mature leaves of 13 species were used to assess differences within the intraspecific subgenomes. 2) Interspecific expression divergence between A and B subgenomes during flower and fruit development. This involved selecting retained duplicated gene pairs shared by all four genera and analyzing tissues from different developmental stages of three species ( I. orientalis , I. polycarpa , and P. deltoides ). 3) The dynamic expression changes of FT1 and FT2 were detected using the expression levels in leaves, buds and shoot apex of I. orientalis and I. polycarpa from different months. Sequencing and analysis of epigenetic data ATAC sample preparation from leaves was performed as described in a previous paper 126 . Vazyme TD501 manual was used to build the ATAC-seq library. For whole genome bisulfite sequencing (WGBS), genomic DNA was extracted from leaves with the DNeasy plant mini kit (Qiagen) and libraries were constructed following procedures described previously 127 . All libraries were sequenced on an Illumina HiSeq 2000 platform. Quality-filtered reads were aligned to the reference genome using Bowtie2 v2.4.1 128 (ATAC-seq) and Bismark v0.22.3 129 (WGBS), respectively. We extracted the 2k region upstream of each gene in 13 species and divided it into 20 bins. Subsequently, we counted the number of reads in each bin and normalized it to reads per bin per million mapped reads according to the RPKM method 130 . This was used to assess the chromatin accessibility of the genomes and subgenomes. Meanwhile, methylation levels of the gene body and the flanking 2k regions were determined by dividing the regions into 30 and 20 bins, respectively. Gene Ontology (GO) enrichment analysis Gene ontology (GO) enrichment analysis was performed on each group of genes using the enricher function in the "clusterProfiler" package 131 within R software. After p-value correction using the Benjamini-Hochberg method, terms with q-value < 0.05 were selected as the significant functions. Identification of conserved noncoding elements (CNEs) We applied AVID v2.1 132 to perform alignments of Salicaceae species and detect Populus - Salix specific CNEs using a 100 bp, 70% identity criterion. Finally, the alignments were visualized using VISTA 133 . Dual-luciferase assay The synthetic and cloned fragments of the four CNEs were fused with the 35S minimal promoter to drive the LUC expression as the reporters, and the 35S empty vector was used as a control. These constructs were transiently expressed in N. benthamiana leaves. After incubation in the dark for 2 days and the light for 1 day, the enzyme mixture was prepared according to the manufacturer's instructions in the Dual-Luciferase Reporter Assay System kit (Promega). Firefly ( LUC ) and Renilla (internal control, REN ) luciferase signals were detected using a multimode reader (Synergy H1; BioTek, Winooski, VT). All primers used are listed in Supplementary Table 11 . Declarations Data availability All raw sequence data, genome assembly and annotation information have been deposited in the National Genome Data Center (NGDC; https://bigd.big.ac.cn/bioproject) under BioProject accession number PRJCA022976. Acknowledgments This work was supported by the National Key Research and Development Program of China (2021YFD2201100, 2021YFD2200202 and 2016YFD0600101), National Natural Science Foundation of China (31922061, 32271828 and 32071732), Fundamental Research Funds for the Central Universities (2020SCUNL207, SCU2021D006 and 2020SCUNL103), and the US National Science Foundation (1542599). We thank Susanne S. Renner (Department of Biology, Washington University, Saint Louis, USA.) for insightful comments. We thank Yanping Su, Fuchuan Wu (Center for Gardening and Horticulture, Xishuangbanna Tropical Botanical Garden, Chinese Academy of Science), and Lei Zhang (College of Biological Science & Engineering, North Minzu University, Yinchuan, China) for providing plant samples. Author contributions TM, JQL and MO led the project. TM, JQL, MO, DYW and ZXX conceived and designed the research. DYW, MML, WLY, KC, JLZ, LXS, PCS, LX, YLL, YC, JXX, YBW, HH, TNL and JLL performed data analysis. DYW, MML, ZQL and ZXX collected samples. YZJ and QJH provided comments for improving the manuscript. DYW and TM drafted the manuscript. JQL and MO edited the manuscript. All authors approved the final manuscript. Competing interests The authors declare no competing interests. References Leebens-Mack, J.H. et al. One thousand plant transcriptomes and the phylogenomics of green plants. Nature 574 , 679-685 (2019). Jiao, Y. et al. Phylogenomic analysis reveals ancient genome duplications in seed plant and angiosperm history. Nature 473 , 97-100 (2011). Albert, V.A. et al. The Amborella genome and the evolution of flowering plants. Science 342 , 1241089 (2013). Force, A. et al. Preservation of duplicate genes by complementary, degenerative mutations. Genetics 151 , 1531-1545 (1999). Adams, K.L. & Wendel, J.F. Polyploidy and genome evolution in plants. Curr. Opin. Plant Biol. 8 , 135-141 (2005). Doyle, J.J. et al. Evolutionary genetics of genome merger and doubling in plants. Annu. Rev. Genet. 42 , 443-461 (2008). Hegarty, M.J. & Hiscock, S.J. Genomic clues to the evolutionary success of polyploid plants. Curr. Biol. 18 , R435-R444 (2008). Soltis, P.S., Liu, X., Marchant, D.B., Visger, C.J. & Soltis, D.E. Polyploidy and novelty: Gottlieb's legacy. Philos. Trans. R. Soc. Lond., B, Biol. Sci. 369 , 20130351 (2014). Soltis, P.S., Marchant, D.B., Van de Peer, Y. & Soltis, D.E. Polyploidy and genome evolution in plants. Curr. Opin. Genet. Dev. 35 , 119-125 (2015). Soltis, P.S. & Soltis, D.E. Ancient WGD events as drivers of key innovations in angiosperms. Curr. Opin. Plant Biol. 30 , 159-165 (2016). Van de Peer, Y., Maere, S. & Meyer, A. The evolutionary significance of ancient genome duplications. Nat. Rev. Genet. 10 , 725-732 (2009). Van de Peer, Y., Mizrachi, E. & Marchal, K. The evolutionary significance of polyploidy. Nat. Rev. Genet. 18 , 411-424 (2017). Fawcett, J.A., Maere, S. & Van De Peer, Y. Plants with double genomes might have had a better chance to survive the Cretaceous-Tertiary extinction event. Proc. Natl Acad. Sci. USA 106 , 5737-5742 (2009). Vanneste, K., Maere, S. & Van de Peer, Y. Tangled up in two: a burst of genome duplications at the end of the Cretaceous and the consequences for plant evolution. Philos. Trans. R. Soc. Lond., B, Biol. Sci. 369 , 20130353 (2014). Wu, S., Han, B. & Jiao, Y. Genetic contribution of paleopolyploidy to adaptive evolution in angiosperms. Mol. Plant 13 , 59-71 (2020). Schnable, J.C., Springer, N.M. & Freeling, M. Differentiation of the maize subgenomes by genome dominance and both ancient and ongoing gene loss. Proc. Natl Acad. Sci. USA 108 , 4069-4074 (2011). Wendel, J.F., Jackson, S.A., Meyers, B.C. & Wing, R.A. Evolution of plant genome architecture. Genome Biol. 17 , 1-14 (2016). Barker, M.S., Arrigo, N., Baniaga, A.E., Li, Z. & Levin, D.A. On the relative abundance of autopolyploids and allopolyploids. New Phytol. 210 , 391-398 (2016). Fowler, N.L. & Levin, D.A. Ecological constraints on the establishment of a novel polyploid in competition with its diploid progenitor. Am. Nat. 124 , 703-711 (1984). Ramsey, J. Polyploidy and ecological adaptation in wild yarrow. Proc. Natl Acad. Sci. USA 108 , 7096-7101 (2011). Parisod, C. & Broennimann, O. Towards unified hypotheses of the impact of polyploidy on ecological niches. New Phytol. 212 , 540-542 (2016). Luo, X. et al. Chasing ghosts: allopolyploid origin of Oxyria sinensis (Polygonaceae) from its only diploid congener and an unknown ancestor. Mol. Ecol. 26 , 3037-3049 (2017). Stull, G.W., Pham, K.K., Soltis, P.S. & Soltis, D.E. Deep reticulation: the long legacy of hybridization in vascular plant evolution. Plant J. 114 , 743-766 (2023). Stettler, R.F. Biology of Populus and its implications for management and conservation , (NRC Research Press, 1996). Chase, M.W. et al. When in doubt, put it in Flacourtiaceae: a molecular phylogenetic analysis based on plastid rbcL DNA sequences. Kew Bull. 57 , 141-181 (2002). Sanderson, B.J. et al. Phylogenomics reveals patterns of ancient hybridization and differential diversification that contribute to phylogenetic conflict in willows, poplars, and close relatives. Syst. Biol. 72 , 1120-1232 (2023). Argus, G.W. Infrageneric classification of Salix (Salicaceae) in the new world. Syst. Bot. Monogr. , 1-121 (1997). Tuo-Ya, D. Origin, divergence and geographical distribution of Salicaceae. Plant Divers. 17 , 1 (1995). Cronk, Q.C., Needham, I. & Rudall, P.J. Evolution of catkins: inflorescence morphology of selected Salicaceae in an evolutionary and developmental context. Front. Plant Sci. 6 , 1030 (2015). Lemke, D.E. A synopsis of Flacourtiaceae. Aliso 12 , 29-43 (1988). Zhang, Z.-S., Zeng, Q.-Y. & Liu, Y.-J. Frequent ploidy changes in Salicaceae indicates widespread sharing of the salicoid whole genome duplication by the relatives of Populus L. and Salix L. BMC Plant Biol. 21 , 1-17 (2021). Steyn, E., Smith, G. & Van Wyk, A. Functional and taxonomic significance of seed structure in Salix mucronata (Salicaceae). Bothalia 34 , 53-59 (2004). Cronk, Q. Plant eco‐devo: the potential of poplar as a model organism. New Phytol. 166 , 39-48 (2005). Jansson, S. & Douglas, C.J. Populus : a model system for plant biology. Annu. Rev. Plant Biol. 58 , 435-458 (2007). Tuskan, G.A. et al. The genome of black cottonwood, Populus trichocarp a (Torr. & Gray). Science 313 , 1596-1604 (2006). Dai, X. et al. The willow genome and divergent evolution from poplar after the common genome duplication. Cell Res. 24 , 1274-1277 (2014). Rodgers-Melnick, E. et al. Contrasting patterns of evolution following whole genome versus tandem duplication events in Populus . Genome Res. 22 , 95-105 (2012). Liu, Y. et al. Two highly similar poplar paleo-subgenomes suggest an autotetraploid ancestor of Salicaceae plants. Front. Plant Sci. 8 , 252555 (2017). Li, M. et al. Intergeneric relationships within the family Salicaceae sl based on plastid phylogenomics. Int. J. Mol. Sci. 20 , 3788 (2019). Shang, C., Liao, S., Guo, Y.J. & Zhang, Z.X. Dianyuea gen. nov.(Salicaceae: Scyphostegioideae) from southwestern China. Nord. J. Bot. 35 , 499-505 (2017). Cheng, F. et al. Gene retention, fractionation and subgenome differences in polyploid plants. Nat. Plants 4 , 258-268 (2018). Kardailsky, I. et al. Activation tagging of the floral inducer FT . Science 286 , 1962-1965 (1999). Kobayashi, Y., Kaya, H., Goto, K., Iwabuchi, M. & Araki, T. A pair of related genes with antagonistic roles in mediating flowering signals. Science 286 , 1960-1962 (1999). Liu, H. et al. Photoexcited CRY2 interacts with CIB1 to regulate transcription and floral initiation in Arabidopsis . science 322 , 1535-1539 (2008). Doyle, M.R. et al. HUA2 is required for the expression of floral repressors in Arabidopsis thaliana. Plant J. 41 , 376-385 (2005). Kim, S.H. et al. Phosphorylation of the transcriptional repressor MYB15 by mitogen-activated protein kinase 6 is required for freezing tolerance in Arabidopsis . Nucleic Acids Res. 45 , 6613-6627 (2017). Zhang, P. et al. Arabidopsis ADF5 acts as a downstream target gene of CBFs in response to low-temperature stress. Front. Cell Dev. Biol. 9 , 635533 (2021). Takahashi, D. et al. Structural changes in cell wall pectic polymers contribute to freezing tolerance induced by cold acclimation in plants. Curr. Biol. 34 , 958-968.e5 (2024). Sun, L. et al. GLABROUS INFLORESCENCE STEMS3 ( GIS3 ) regulates trichome initiation and development in Arabidopsis . New Phytol. 206 , 220-230 (2015). Zhou, Z. et al. Zinc Finger Protein 6 ( ZFP6 ) regulates trichome initiation by integrating gibberellin and cytokinin signaling in Arabidopsis thaliana . New Phytol. 198 , 699-708 (2013). Liu, Y., Yang, S., Khan, A.R. & Gan, Y. TOE1/TOE2 interacting with GIS to control trichome development in Arabidopsis . Int. J. Mol. Sci. 24 , 6698 (2023). Zhu, Y. et al. TERMINAL FLOWER 1-FD complex target genes and competition with FLOWERING LOCUS T. Nat. Commun. 11 , 5118 (2020). Kumimoto, R.W. et al. The Nuclear Factor Y subunits NF-YB2 and NF-YB3 play additive roles in the promotion of flowering by inductive long-day photoperiods in Arabidopsis . Planta 228 , 709-723 (2008). Jung, J.-H., Lee, H.-J., Ryu, J.Y. & Park, C.-M. SPL3/4/5 integrate developmental aging and photoperiodic signals into the FT-FD module in Arabidopsis flowering. Mol. Plant 9 , 1647-1659 (2016). Song, H.-R. et al. The RNA binding protein ELF9 directly reduces SUPPRESSOR OF OVEREXPRESSION OF CO1 transcript levels in Arabidopsis , possibly via nonsense-mediated mRNA decay. Plant Cell 21 , 1195-1211 (2009). Zhang, Z. et al. Convergence of the 26S proteasome and the REVOLUTA pathways in regulating inflorescence and floral meristem functions in Arabidopsis . J. Exp. Bot. 62 , 359-369 (2011). Yoo, C.-M., Quan, L. & Blancaflor, E.B. Divergence and redundancy in CSLD2 and CSLD3 function during Arabidopsis thaliana root hair and female gametophyte development. Front. Plant Sci. 3 , 111 (2012). Throude, M. et al. Structure and expression analysis of rice paleo duplications. Nucleic Acids Res. 37 , 1248-1259 (2009). Huminiecki, L. & Conant, G.C. Polyploidy and the evolution of complex traits. Int. J. Evol. Biol. 2012 , 292068 (2012). Hsu, C.-Y. et al. FLOWERING LOCUS T duplication coordinates reproductive and vegetative growth in perennial poplar. Proc. Natl Acad. Sci. USA 108 , 10756-10761 (2011). Yu, C.-W. et al. HISTONE DEACETYLASE6 interacts with FLOWERING LOCUS D and regulates flowering in Arabidopsis. Plant Physiol. 156 , 173-184 (2011). Bertran Garcia de Olalla, E. et al. Coordination of shoot apical meristem shape and identity by APETALA2 during floral transition in Arabidopsis. Nat. Commun. 15 , 6930 (2024). Zhao, X., Heng, Y., Wang, X., Deng, X.W. & Xu, D. A positive feedback loop of BBX11–BBX21–HY5 promotes photomorphogenic development in Arabidopsis . Plant Commun. 1 (2020). Li, P., Li, X. & Jiang, M. CRISPR/Cas9-mediated mutagenesis of WRKY3 and WRKY4 function decreases salt and Me-JA stress tolerance in Arabidopsis thaliana . Mol. Biol. Rep. 48 , 5821-5832 (2021). André, D. et al. FLOWERING LOCUS T paralogs control the annual growth cycle in Populus trees. Curr. Biol. 32 , 2988-2996. e4 (2022). Sawa, S., Ito, T., Shimura, Y. & Okada, K. FILAMENTOUS FLOWER controls the formation and development of Arabidopsis inflorescences and floral meristems. Plant Cell 11 , 69-86 (1999). Li, W. et al. The U-Box/ARM E3 ligase PUB13 regulates cell death, defense, and flowering time in Arabidopsis . Plant Physiol. 159 , 239-250 (2012). Zhang, C. et al. Gibberellin signaling modulates flowering via the DELLA–BRAHMA–NF-YC module in Arabidopsis. Plant Cell 35 , 3470-3484 (2023). Liu, Z. et al. ENO2 affects the seed size and weight by adjusting cytokinin content and forming ENO2-bZIP75 complex in Arabidopsis thaliana . Front. Plant Sci. 11 , 574316 (2020). Di Marzo, M. et al. Cell wall modifications by α-XYLOSIDASE1 are required for control of seed and fruit size in Arabidopsis. J. Exp. Bot. 73 , 1499-1515 (2021). Sun, P. et al. Subgenome-aware analyses reveal the genomic consequences of ancient allopolyploid hybridizations throughout the cotton family. Proc. Natl Acad. Sci. USA 121 , e2313921121 (2024). Cai, X. et al. Impacts of allopolyploidization and structural variation on intraspecific diversification in Brassica rapa . Genome Biol. 22 , 166 (2021). Marcussen, T. et al. Ancient hybridizations among the ancestral genomes of bread wheat. Science 345 , 1250092 (2014). Ramírez-González, R. et al. The transcriptional landscape of polyploid wheat. Science 361 , eaar6089 (2018). Wang, J. et al. A common whole-genome paleotetraploidization in Cucurbitales. Plant Physiol. 190 , 2430-2448 (2022). Zhang, L. et al. Bioinformatic analysis of chromatin organization and biased expression of duplicated genes between two poplars with a common whole-genome duplication. Hortic. Res. 8 , 62 (2021). Li, H. Minimap2: pairwise alignment for nucleotide sequences. Bioinformatics 34 , 3094-3100 (2018). Li, H. & Durbin, R. Fast and accurate short read alignment with Burrows–Wheeler transform. Bioinformatics 25 , 1754-1760 (2009). Burton, J.N. et al. Chromosome-scale scaffolding of de novo genome assemblies based on chromatin interactions. Nat. Biotechnol. 31 , 1119-1125 (2013). Kajitani, R. et al. Efficient de novo assembly of highly heterozygous genomes from whole-genome shotgun short reads. Genome Res. 24 , 1384-1395 (2014). Cheng, H., Concepcion, G.T., Feng, X., Zhang, H. & Li, H. Haplotype-resolved de novo assembly using phased assembly graphs with hifiasm. Nat. Methods 18 , 170-175 (2021). Chen, N. Using Repeat Masker to identify repetitive elements in genomic sequences. Curr. Protoc. Bioinf. 5 , 4.10. 1-4.10. 14 (2004). Bao, W., Kojima, K.K. & Kohany, O. Repbase Update, a database of repetitive elements in eukaryotic genomes. Mob. DNA 6 , 1-6 (2015). Price, A.L., Jones, N.C. & Pevzner, P.A. De novo identification of repeat families in large genomes. Bioinformatics 21 , i351-i358 (2005). Stanke, M. et al. AUGUSTUS: ab initio prediction of alternative transcripts. Nucleic Acids Res. 34 , W435-W439 (2006). Yang, W. et al. The draft genome sequence of a desert tree Populus pruinosa . Gigascience 6 , gix075 (2017). Ma, J. et al. Genome sequence and genetic transformation of a widely distributed and cultivated poplar. Plant Biotechnol. J. 17 , 451-460 (2019). Zhou, R. et al. Characterization of a large sex determination region in Salix purpurea L.(Salicaceae). Mol. Genet. Genomics 293 , 1437-1452 (2018). Iniative, A.G. Analysis of the genome sequence of the flowering plant Arabidopsis thaliana . Nature 408 , 796-815 (2000). Camacho, C. et al. BLAST+: architecture and applications. BMC Bioinformatics 10 , 1-9 (2009). Birney, E., Clamp, M. & Durbin, R. GeneWise and genomewise. Genome Res. 14 , 988-995 (2004). Campbell, M.A., Haas, B.J., Hamilton, J.P., Mount, S.M. & Buell, C.R. Comprehensive analysis of alternative splicing in rice and comparative analyses with Arabidopsis . BMC Genomics 7 , 1-17 (2006). Haas, B.J. et al. Automated eukaryotic gene structure annotation using EVidenceModeler and the Program to Assemble Spliced Alignments. Genome Biol. 9 , 1-22 (2008). Simão, F.A., Waterhouse, R.M., Ioannidis, P., Kriventseva, E.V. & Zdobnov, E.M. BUSCO: assessing genome assembly and annotation completeness with single-copy orthologs. Bioinformatics 31 , 3210-3212 (2015). Zhang, Z. et al. Improved genome assembly provides new insights into genome evolution in a desert poplar ( Populus euphratica ). Mol. Ecol. Resour. 20 , 781-794 (2020). Wang, D. et al. Repeated turnovers keep sex chromosomes young in willows. Genome Biol. 23 , 1-23 (2022). Zhou, R. et al. A willow sex chromosome reveals convergent evolution of complex palindromic repeats. Genome Biol. 21 , 1-19 (2020). Ma, D. et al. Chromosome‐level reference genome assembly provides insights into aroma biosynthesis in passion fruit ( Passiflora edulis ). Mol. Ecol. Resour. 21 , 955-968 (2021). Costa, Z.P. et al. A genome sequence resource for the genus Passiflora , the genome of the wild diploid species Passiflora organensis . Plant Genome 14 , e20117 (2021). Raymond, O. et al. The Rosa genome provides new insights into the domestication of modern roses. Nat. Genet. 50 , 772 - 777 (2018). Shi, X. et al. The complete reference genome for grapevine ( Vitis vinifera L.) genetics and breeding. Hortic. Res. 10 (2023). Emms, D.M. & Kelly, S. OrthoFinder: solving fundamental biases in whole genome comparisons dramatically improves orthogroup inference accuracy. Genome Biol. 16 , 1-14 (2015). Katoh, K., Misawa, K., Kuma, K.i. & Miyata, T. MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform. Nucleic Acids Res. 30 , 3059-3066 (2002). Suyama, M., Torrents, D. & Bork, P. PAL2NAL: robust conversion of protein sequence alignments into the corresponding codon alignments. Nucleic Acids Res. 34 , W609-W612 (2006). Stamatakis, A. RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics 30 , 1312-1313 (2014). Yang, Z. PAML 4: phylogenetic analysis by maximum likelihood. Mol. Biol. Evol. 24 , 1586-1591 (2007). Altschul, S.F., Gish, W., Miller, W., Myers, E.W. & Lipman, D.J. Basic local alignment search tool. J. Mol. Biol. 215 , 403-410 (1990). Sun, P. et al. WGDI: A user-friendly toolkit for evolutionary analyses of whole-genome duplications and ancestral karyotypes. Mol. Plant 15 , 1841-1851 (2022). Brown, J.L. SDM toolbox: a python‐based GIS toolkit for landscape genetic, biogeographic and species distribution model analyses. Methods Ecol. Evol. 5 , 694-700 (2014). Phillips, S.J., Anderson, R.P. & Schapire, R.E. Maximum entropy modeling of species geographic distributions. Ecol. Model. 190 , 231-259 (2006). Fielding, A.H. & Bell, J.F. A review of methods for the assessment of prediction errors in conservation presence/absence models. Environ. Conserv. 24 , 38-49 (1997). Warren, D.L. et al. ENMTools 1.0: An R package for comparative ecological biogeography. Ecography 44 , 504-511 (2021). Schoener, T.W. The Anolis lizards of Bimini: resource partitioning in a complex fauna. Ecology 49 , 704-726 (1968). Warren, D.L., Glor, R.E. & Turelli, M. Environmental niche equivalency versus conservatism: quantitative approaches to niche evolution. Evolution 62 , 2868-2883 (2008). Hu, F., Lin, Y. & Tang, J. MLGO: phylogeny reconstruction and ancestral inference from gene-order data. BMC Bioinformatics 15 , 1-6 (2014). Sun, P. et al. Early diversification and karyotype evolution of flowering plants. (2022). Zhang, C., Rabiee, M., Sayyari, E. & Mirarab, S. ASTRAL-III: polynomial time species tree reconstruction from partially resolved gene trees. BMC Bioinformatics 19 , 15-30 (2018). Ding, Y. et al. Genome structure-based Juglandaceae phylogenies contradict alignment-based phylogenies and substitution rates vary with DNA repair genes. Nat. Commun. 14 , 617 (2023). Ellinghaus, D., Kurtz, S. & Willhoeft, U. LTRharvest, an efficient and flexible software for de novo detection of LTR retrotransposons. BMC Bioinformatics 9 , 1-14 (2008). Steinbiss, S., Willhoeft, U., Gremme, G. & Kurtz, S. Fine-grained annotation and classification of de novo predicted LTR retrotransposons. Nucleic Acids Res. 37 , 7002-7013 (2009). Zhou, S. et al. A comprehensive annotation dataset of intact LTR retrotransposons of 300 plant genomes. Sci. Data 8 , 174 (2021). Edgar, R.C. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 32 , 1792-1797 (2004). Ingvarsson, P.K. Multilocus patterns of nucleotide polymorphism and the demographic history of Populus tremula . Genetics 180 , 329-340 (2008). Kim, D., Langmead, B. & Salzberg, S.L. HISAT: a fast spliced aligner with low memory requirements. Nat. Methods 12 , 357-360 (2015). Pertea, M. et al. StringTie enables improved reconstruction of a transcriptome from RNA-seq reads. Nat. Biotechnol. 33 , 290-295 (2015). Bajic, M., Maher, K.A. & Deal, R.B. Identification of open chromatin regions in plant genomes using ATAC-Seq. Methods Mol. Biol. , 183-201 (2018). Su, Y. et al. Single-base-resolution methylomes of Populus euphratica reveal the association between DNA methylation and salt stress. Tree Genet. Genom. 14 , 1-11 (2018). Langmead, B. & Salzberg, S.L. Fast gapped-read alignment with Bowtie 2. Nat. Methods 9 , 357-359 (2012). Krueger, F. & Andrews, S.R. Bismark: a flexible aligner and methylation caller for Bisulfite-Seq applications. Bioinformatics 27 , 1571-1572 (2011). Mortazavi, A., Williams, B.A., McCue, K., Schaeffer, L. & Wold, B. Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nat. Methods 5 , 621-628 (2008). Yu, G., Wang, L.-G., Han, Y. & He, Q.-Y. clusterProfiler: an R package for comparing biological themes among gene clusters. OMICS: J. Integrative Biol. 16 , 284-287 (2012). Bray, N., Dubchak, I. & Pachter, L. AVID: A global alignment program. Genome Res. 13 , 97-102 (2003). Frazer, K.A., Pachter, L., Poliakov, A., Rubin, E.M. & Dubchak, I. VISTA: computational tools for comparative genomics. Nucleic Acids Res. 32 , W273-W279 (2004). Additional Declarations There is NO Competing Interest. Supplementary Files NatureCommunicationsSupplementaryTables.xlsx Supplementary Tables 1-11 NatureCommunicationsSupplementaryFigures.pdf Supplementary Figures 1-19 111.pdf Reporting Summary Cite Share Download PDF Status: Published Journal Publication published 25 Jul, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5852798","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":406729925,"identity":"f27c8450-f832-4c62-8e5f-321741e01d44","order_by":0,"name":"Jianquan 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04:45:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5852798/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5852798/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41467-025-62178-y","type":"published","date":"2025-07-25T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":74915072,"identity":"800e476b-82e1-4b43-913a-3095e854b8dc","added_by":"auto","created_at":"2025-01-28 09:51:16","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":258811,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe distribution and phylogenetic relationships of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePopulus,\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e S\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ealix\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and the related genera in the Salicaceae.\u003c/strong\u003e \u003cstrong\u003ea,\u003c/strong\u003e The geographical distributions of the nine genera in Salicaceae in our study and illustrations depicting the morphological characteristics of the species. a-i correspond to the respective branches in Fig. 1b. \u003cstrong\u003eb, \u003c/strong\u003eThe phylogenetic relationships and divergence times of species in the Salicaceae, using \u003cem\u003ePassiflora edulia\u003c/em\u003e, \u003cem\u003ePassiflora organensis\u003c/em\u003e, \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, \u003cem\u003eRosa chinensis \u003c/em\u003eand \u003cem\u003eVitis vinifera \u003c/em\u003eas outgroups. Bootstrap support at each node are all 100. Arrows indicate calibration nodes. The 95% confidence intervals (CI) for divergence times are shown next to the nodes and represented by light blue error bars. The red star represents the 'salicoid' WGD event. \u003cstrong\u003ec,\u003c/strong\u003e The chromosome number, genome size (top), contig N50 (top), repetitive sequences content and types (bottom), and the number of genes (bottom) for each species on the evolutionary tree. The images on the right correspond to branches a-i in Fig. 1b, depicting their flowers and fruits.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5852798/v1/5e8ae2e184dbe270e87c7946.png"},{"id":74914921,"identity":"29f3bd92-7e25-484e-9f83-fcca91cd2f67","added_by":"auto","created_at":"2025-01-28 09:43:16","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":140393,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKaryotype evolution and evolutionary relationship of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePopulus,\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e S\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ealix\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and the related genera from the common ancestral karyotype.\u003c/strong\u003e The evolutionary relationships were constructed based on the shared polyploidy events and chromosome fusions with the shortest steps. The inferred ancestral karyotype contains 11 protochromosomes, marked with different colors. Leaf nodes show the modern karyotypes of species, and chromosomal changes leading to the formation of these modern karyotypes, including reciprocal translocations (RTA), end-to-end joining (EEJ), and nested chromosome fusions (NCF), are marked on the ancestral nodes of the phylogenetic tree. Among them, the ancestor of Clade II experienced a RTA, resulting in the karyotype of ancestor A, and subsequently hybridized with ancestor B of another branch (which is now extinct), leading to an allopolyploidization event. Chromosomes from ancestor B are distinguished by white asterisks in the center of the chromosomes. The dashed lines indicate unresolved relationships for karyotypes of existing species.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5852798/v1/917684e90281ddfb653186a2.png"},{"id":74914936,"identity":"6e735a06-c3f8-4cc4-862e-f16ab0de71af","added_by":"auto","created_at":"2025-01-28 09:43:17","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":208864,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIdentification of the allopolyploidization event for \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePopulus,\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e S\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ealix\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and two sister genera.\u003c/strong\u003e \u003cstrong\u003ea,\u003c/strong\u003e The evolutionary relationships and divergence time estimates of the two subgenomes of Clade I species relative to Clade II and \u003cem\u003eC. decandra\u003c/em\u003e. The subgenome closer to Clade II is designated as A, while the other subgenome is named B. The 95% confidence intervals (CI) for divergence times are shown next to the nodes and represented by light purple error bars. \u003cstrong\u003eb, \u003c/strong\u003eThe frequency of three topologies (q1-q3) around internal branches (marked with a star in Fig. 3a) of ASTRAL species trees for each protochromosome (ASK 1-11) in the datasets Full-HGGs and Full-/Partial-HGGs. \u003cstrong\u003ec,\u003c/strong\u003eThe collinearity between the two subgenomes of Clade I species (here \u003cem\u003eI. orientalis\u003c/em\u003e, \u003cem\u003eI. polycarpa\u003c/em\u003e, \u003cem\u003eP. trichocarpa\u003c/em\u003e and \u003cem\u003eS. purpurea\u003c/em\u003e), where the inner circle is the A subgenome and the outer circle is the B subgenome. The color of each chromosome indicates its ancestral origin. \u003cstrong\u003ed, \u003c/strong\u003eThe comparison of the corrected molecular evolutionary rates for Clade I species using the Mann-Whitney U test. **** p\u0026lt;0.0001. All species names are abbreviations. Ipo: \u003cem\u003eI. polycarpa\u003c/em\u003e, Ior: \u003cem\u003eI. orientalis\u003c/em\u003e, Pal: \u003cem\u003eP. alba\u003c/em\u003e var. \u003cem\u003epyramidalis\u003c/em\u003e, Peu: \u003cem\u003eP. euphratica\u003c/em\u003e, Ptr: \u003cem\u003eP. trichocarpa\u003c/em\u003e, Scha: \u003cem\u003eS. chaenomeloides\u003c/em\u003e, Spu: \u003cem\u003eS. purpurea\u003c/em\u003e, Sre: \u003cem\u003eS. rehderiana\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5852798/v1/58f61ae85104afb36b3dd71e.png"},{"id":74914929,"identity":"e6519bce-4923-4e50-8bfe-6e1d5b576b6a","added_by":"auto","created_at":"2025-01-28 09:43:16","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":237792,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSubgenome dominance and gene retention of the \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePopulus-\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eS\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ealix\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and two sister lineages. a, \u003c/strong\u003eThe percentage of gene retention in the Clade II species and two subgenomes of Clade I species.\u003cstrong\u003e b, \u003c/strong\u003eGene expression in the \u003cem\u003eC. decandra\u003c/em\u003e, Clade II, and two subgenomes of Clade I species. \u003cstrong\u003ec, \u003c/strong\u003eThe rates of protein evolution (\u003cem\u003eKa/Ks\u003c/em\u003e) of the A and B subgenome branches calculated using codeml. \u003cstrong\u003ea-c, \u003c/strong\u003eThe A and B subgenomes are represented in blue and green, respectively. Significant values of the Mann-Whitney U test for the two subgenomes are indicated by asterisks: *p \u0026lt; 0.05; **p \u0026lt; 0.01; ***p \u0026lt; 0.001, ****p\u0026lt;0.0001. All species names are the same as abbreviations in Fig. 3d. Cde: \u003cem\u003eC. decandra\u003c/em\u003e, Dca: \u003cem\u003eD. caffra\u003c/em\u003e, Sch:\u003cem\u003e S. chinensis\u003c/em\u003e, Xlo: \u003cem\u003eX. longifolia\u003c/em\u003e, Fja: \u003cem\u003eF. jangomas\u003c/em\u003e. \u003cstrong\u003ed, \u003c/strong\u003eEnriched GO terms (q value \u0026lt; 0.05) for genes specifically retained in the A subgenome of all Clade I species (A), as well as duplicated gene pairs commonly retained in four genera (Node I: \u003cem\u003eItoa\u003c/em\u003e, \u003cem\u003eIdesia\u003c/em\u003e, \u003cem\u003ePopulus\u003c/em\u003e and \u003cem\u003eSalix\u003c/em\u003e), three genera (Node II: \u003cem\u003eIdesia\u003c/em\u003e, \u003cem\u003ePopulus\u003c/em\u003e and \u003cem\u003eSalix\u003c/em\u003e), and two genera (Node III: \u003cem\u003ePopulus\u003c/em\u003e and \u003cem\u003eSalix\u003c/em\u003e), respectively. These three categories (Node I-III) correspond to the three ancestral nodes in Fig. 4e. \u003cstrong\u003ee, \u003c/strong\u003eThe number of duplicated gene pairs specifically retained in four genera (Node I), three genera (Node II), and two genera (Node III), and the microhomology visualization of gene \u003cem\u003eFD\u003c/em\u003e identified in the Node II. Only one copy of the duplicated gene was retained in the A subgenome of \u003cem\u003eItoa\u003c/em\u003e, while both copies were retained in \u003cem\u003eIdesia\u003c/em\u003e, \u003cem\u003ePopulus\u003c/em\u003e, and \u003cem\u003eSalix\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5852798/v1/2a6b5a77794017e1bddacddb.png"},{"id":74914931,"identity":"f387bf3a-b597-4770-bfd1-a57b6e51fbc5","added_by":"auto","created_at":"2025-01-28 09:43:17","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":387659,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLineage-specific differences in subgenome expression and regulatory patterns of retained duplicated gene pairs within the \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePopulus-\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eS\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ealix\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and two sister lineages.\u003c/strong\u003e \u003cstrong\u003ea, \u003c/strong\u003eCluster results of expression divergence of duplicated gene pairs between the A and B subgenomes during different developmental stages of female and male flowers and fruits in\u003cem\u003e I. orientalis\u003c/em\u003e (Ior),\u003cem\u003e I. polycarpa\u003c/em\u003e (Ipo) and \u003cem\u003eP. deltoides \u003c/em\u003e(Pde). The subgenome expression divergence was represented by the TPM ratio (A-B)/(A+B), where a ratio greater than 0 indicates expression dominance in the A subgenome, while a ratio less than 0 indicates expression dominance in the B subgenome. The heatmap results are shown on the left, and the mean and standard deviation of the TPM ratio for each sample are displayed on the right. \u003cstrong\u003eb,\u003c/strong\u003e The expression levels of duplicated gene pairs related to the development of adaptive traits in different tissues and developmental stages. These genes are distributed in four clusters, including C1, which is more highly expressed in the A subgenome across all species, and clusters (C4-C6) with lineage-specific expression divergence in \u003cem\u003ePopulus\u003c/em\u003e. The circle size and color are positively correlated with the expression levels. \u003cstrong\u003ec,\u003c/strong\u003e Expression of \u003cem\u003eFT1\u003c/em\u003e and \u003cem\u003eFT2\u003c/em\u003e in leaves and buds/shoot apex of \u003cem\u003eI. orientalis\u003c/em\u003e (Ior) \u003cem\u003eI. polycarpa\u003c/em\u003e (Ipo) and \u003cem\u003eP. deltoides \u003c/em\u003e(Pde) from late autumn to early spring. Relative fold change in expression levels of \u003cem\u003eFT1\u003c/em\u003e and \u003cem\u003eFT2\u003c/em\u003e relative to the lowest expression (TPM) within a tissue are shown. The results for\u003cem\u003e P. deltoides \u003c/em\u003e(Pde) are from a previous study\u003csup\u003e60\u003c/sup\u003e.\u003cstrong\u003e d, \u003c/strong\u003eVISTA sequence conservation plot of the \u003cem\u003ePopulus\u003c/em\u003e-\u003cem\u003eSalix\u003c/em\u003e specific CNE around \u003cem\u003eZFP6\u003c/em\u003e and \u003cem\u003eGIS3\u003c/em\u003e, using \u003cem\u003eS. rehderiana\u003c/em\u003e as a reference. The A and B subgenomes of Clade I species are represented in blue and green, respectively. All species names are the same as abbreviations in Fig. 4b. \u003cstrong\u003ee, \u003c/strong\u003eThe Dual-luciferase assay revealed four CNEs could significantly improve the expression level of luciferases and have potential enhancer activities. Significant values of the t test are indicated by asterisks: *p \u0026lt; 0.05; **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****p\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-5852798/v1/97c1b9824675027b38128e85.png"},{"id":74914944,"identity":"0c2684f4-d905-436b-b751-9fed17ffe6d2","added_by":"auto","created_at":"2025-01-28 09:43:17","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":111581,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEvolutionary model for allopolyploidization and the following specific subgenomic evolution drive the origin of adaptive traits in the \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePopulus-\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eS\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ealix\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e lineage compared with other related genera of Salicaceae with one or two species. \u003c/strong\u003eThe model shows three subfamilies of the Salicaceae, with an ancestral lineage (Ancestor A) in subfamily Salicoideae hybridized with another extinct ancestral lineage (Ancestor B). The resulting hybrid underwent rediploidization and gradually diverging into four lineages with subgenomic dominance. Duplicated genes underwent dynamic retention changes and subgenome expression divergence, contributing to the ecological adaptation and trait evolution of these lineages. Among these, the \u003cem\u003ePopulus\u003c/em\u003e-\u003cem\u003eSalix\u003c/em\u003e lineage developed innovative traits related to seeds and flowering, which facilitated its adaptive radiation.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-5852798/v1/a80209d88cc477a4aa71d922.png"},{"id":88506786,"identity":"4c173091-b150-44ad-b9ea-a35e82dc38be","added_by":"auto","created_at":"2025-08-07 07:35:08","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3565203,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5852798/v1/859f5f1b-f848-485a-b10f-dfea3beaa393.pdf"},{"id":74914918,"identity":"c628011f-b68d-4a13-9f94-8d6e75c826a1","added_by":"auto","created_at":"2025-01-28 09:43:16","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":78713,"visible":true,"origin":"","legend":"Supplementary Tables 1-11","description":"","filename":"NatureCommunicationsSupplementaryTables.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5852798/v1/36b8183ee774541219c57414.xlsx"},{"id":74914940,"identity":"a2639154-ebbc-46a3-a59c-6e78ca4778b5","added_by":"auto","created_at":"2025-01-28 09:43:17","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":16569916,"visible":true,"origin":"","legend":"Supplementary Figures 1-19","description":"","filename":"NatureCommunicationsSupplementaryFigures.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5852798/v1/e0ab02b1485cf3e4269a2090.pdf"},{"id":74914927,"identity":"b059756b-2215-452c-8598-58d7253b23a2","added_by":"auto","created_at":"2025-01-28 09:43:16","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":1995878,"visible":true,"origin":"","legend":"Reporting Summary","description":"","filename":"111.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5852798/v1/a2d73365c11d6c76a5b37bc0.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Ancient allopolyploidy and specific subgenomic evolution drive adaptive radiation in poplars and willows","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePolyploidy, or whole-genome duplication (WGD), is common in flowering plants and has played a significant role in their evolution\u003csup\u003e1-3\u003c/sup\u003e. Initially, WGDs result in massive genetic redundancy that allows subfunctionalization of duplicated genes\u003csup\u003e4\u003c/sup\u003e and gives rise to the development of novel traits and adaptations\u003csup\u003e5-12\u003c/sup\u003e. One particularly consequential instance may have been the massive independent WGD events that took place near the Cretaceous-Paleocene (K-Pg) boundary around 60 million years ago (Ma). These events should have contributed to the survival of angiosperms during the mass extinction event at this stage\u003csup\u003e13-15\u003c/sup\u003e. Polyploids can arise through allopolyploidy or autopolyploidy, which involve the merging of genomes between distinct species or between plants within the same species\u003csup\u003e6\u003c/sup\u003e. However, identifying ancient allopolyploidy that have undergone rediploidization and distinguishing their subgenomes is challenging due to the possible extinction of parental lineages and often extensive chromosome rearrangements\u003csup\u003e6,16,17\u003c/sup\u003e. Therefore, despite reports suggesting that allopolyploids may have higher ecological adaptability and evolutionary potential relative to their progenitors and can lead to adaptive radiation\u0026nbsp;\u003csup\u003e18-22\u003c/sup\u003e, the details of the contrasted subgenomic evolution of once ancient allopolyploids and their contributions to adaptive radiations with innovative traits, extensive species diversification and obvious niche shifts in higher taxonomic clades remains scarce\u003csup\u003e23\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe Salicaceae has been widely recognized as exemplary conducting diverse researches on woody species that encompasses molecular mechanisms, speciation, sex chromosomes, phenotypic variation, and ecosystem services\u003csup\u003e24\u003c/sup\u003e. This family comprises three subfamilies (Samydoideae, Scyphostegioideae, and Salicoideae) with approximately 56 genera\u003csup\u003e25\u003c/sup\u003e (\u003cstrong\u003eSupplementary\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;Table 1\u003c/strong\u003e). Speciation rates are extremely elevated in two genera of the Salicoideae,\u0026nbsp;\u003cem\u003ePopulus\u003c/em\u003e and\u0026nbsp;\u003cem\u003eSalix\u003c/em\u003e\u003csup\u003e26\u003c/sup\u003e, which\u0026nbsp;consist\u0026nbsp;of ~100 and ~500 species as the well-known poplars and willows, respectively. Species in these two genera have successfully adapted to diverse environments across the Northern Hemisphere and play crucial roles in temperate and arctic forest ecosystems\u003csup\u003e24,27,28\u003c/sup\u003e. Traits including catkins, dehiscent capsules, hairy seeds, and early spring blooming possibly facilitated their rapid colonization of higher latitudes and an extensive adaptive radiation. In contrast, two other closely related monospecific and dispecific sister genera,\u0026nbsp;\u003cem\u003eIdesia\u003c/em\u003e and\u0026nbsp;\u003cem\u003eItoa\u003c/em\u003e, are found in subtropical or tropical regions and have contrasting traits such as capsules or berry fruits, and glabrous seeds\u003csup\u003e29-32\u003c/sup\u003e (\u003cstrong\u003eFig. 1a,\u0026nbsp;\u003c/strong\u003edata from: https://www.gbif.org/). Consequently, these genera have long been considered attractive model systems for studying adaptive innovation and species radiation in woody plants\u003csup\u003e33-35\u003c/sup\u003e. Previous studies reported that\u0026nbsp;\u003cem\u003ePopulus\u003c/em\u003e and\u0026nbsp;\u003cem\u003eSalix\u003c/em\u003e underwent a 'salicoid' WGD event around the K-Pg boundary\u003csup\u003e35-37\u003c/sup\u003e, which was believed to be autopolyploidization due to the high similarity between subgenomes\u003csup\u003e38\u003c/sup\u003e. However, the detailed evolutionary history of this event and its impact on trait innovation and adaptive radiation across these genera remain poorly understood.\u003c/p\u003e\n\u003cp\u003eHere, we selected species representing all three subfamilies and conducted comparative analysis to gain a\u0026nbsp;better\u0026nbsp;understanding of the origin and potential phenotypic effects of this WGD event \u003cstrong\u003e(Supplementary\u003c/strong\u003e \u003cstrong\u003eTable 1)\u003c/strong\u003e. Our study aimed to uncover the WGD-derived subgenomic evolution that led to the emergence of\u0026nbsp;unique photoperiod adaptation, flowering\u0026nbsp;phenology\u0026nbsp;and small, hairy seeds\u0026nbsp;in\u0026nbsp;\u003cem\u003ePopulus\u003c/em\u003e and\u0026nbsp;\u003cem\u003eSalix\u003c/em\u003e when compared with the other\u0026nbsp;species-depauperate\u0026nbsp;genera, especially their sister genera\u0026nbsp;\u003cem\u003eIdesia\u003c/em\u003e and \u003cem\u003eItoa\u003c/em\u003e. The results revealed that these four genera originated from the common 'salicoid' WGD event, which is an\u0026nbsp;allopolyploidy event\u0026nbsp;involving the extinction of one parental lineage and subsequent divergent evolution of subgenomes. We further discovered that the dynamic gene retention following allopolyploidization, along with lineage-specific expression divergence between subgenomes may have facilitated innovative but contrasting phenotypic traits and ecological niches among these genera.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec2\" class=\"Section2\"\u003e \u003ch2\u003eNewly assembled genomes redefine the phylogenetic position of the 'salicoid' WGD event\u003c/h2\u003e \u003cp\u003eWe combined Nanopore long reads, Illumina short reads, and high-throughput chromosome conformation capture reads (\u003cb\u003eSupplementary Table\u0026nbsp;2\u003c/b\u003e) to assemble eight chromosomal-level genomes of Salicaceae species from different genera, including seven species from the subfamily Salicoideae (\u003cem\u003eDovyalis caffra\u003c/em\u003e, \u003cem\u003eScolopia chinensis\u003c/em\u003e, \u003cem\u003eXylosma longifolia\u003c/em\u003e, \u003cem\u003eFlacourtia jangomas\u003c/em\u003e, \u003cem\u003eItoa orientalis\u003c/em\u003e, \u003cem\u003eIdesia polycarpa\u003c/em\u003e, and \u003cem\u003eSalix rehderiana\u003c/em\u003e) and one species from the subfamily Samydoideae (\u003cem\u003eCaseria decandra\u003c/em\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb \u003cb\u003eand Supplementary Table\u0026nbsp;1\u003c/b\u003e). These newly assembled genomes were anchored onto 9 to 21 pseudochromosomes and varied significantly in size, with \u003cem\u003eI. polycarpa\u003c/em\u003e (1,214 Mb) and \u003cem\u003eS. chinensis\u003c/em\u003e (274 Mb) having the largest and smallest genomes, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec \u003cb\u003eand Supplementary Tables\u0026nbsp;2\u0026ndash;4\u003c/b\u003e). As expected, their genome size is positively correlated with the content of repetitive sequence, primarily due to varying degrees of expansion of \u003cem\u003eGypsy\u003c/em\u003e and \u003cem\u003eCopia\u003c/em\u003e transposable elements (TE) (\u003cb\u003eSupplementary Figs.\u0026nbsp;1 and 2 and Supplementary Table\u0026nbsp;5\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eUsing 672 single-copy genes from 18 species identified by BUSCO and OrthoFinder methods, we reconstructed a high-resolution phylogenetic tree (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb), which matches a previous topology derived from complete plastomes\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. The topology remained the same when STAG were used for all orthogroups containing multi-copy genes identified by OrthoFinder (\u003cb\u003esee Materials and Methods\u003c/b\u003e). This tree confirmed that the Samydoideae and Salicoideae subfamilies were monophyletic. Within the Salicoideae, we identified two clades: one that consisted of taxa with n\u0026thinsp;=\u0026thinsp;19\u0026ndash;21 chromosomes (Clade I) and another with fewer chromosomes (n\u0026thinsp;=\u0026thinsp;9\u0026ndash;11; Clade II). The genera \u003cem\u003eItoa\u003c/em\u003e and \u003cem\u003eIdesia\u003c/em\u003e were successive sisters to \u003cem\u003ePopulus\u003c/em\u003e and \u003cem\u003eSalix\u003c/em\u003e in Clade I, while \u003cem\u003eDovyalis\u003c/em\u003e was sister to the remaining species in Clade II. Our estimates suggest that the Salicaceae diverged from its sister outgroup, \u003cem\u003ePassiflora\u003c/em\u003e, around 93 Ma (95% confidence intervals: 86\u0026ndash;101 Ma), and the Samydoideae and Salicoideae subfamilies diverged around 78 Ma (72\u0026ndash;85 Ma). Clade I and Clade II of the Salicoideae subfamily diverged around 61 Ma (53\u0026ndash;75 Ma) and extant genera within each clade began to diversify around 49 (47\u0026ndash;51 Ma) and 36 Ma (22\u0026ndash;52 Ma), respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003eBy comparing the chromosome and gene numbers of these genomes, we inferred that the 'salicoid' event may be restricted to Clade I species. To test this, we evaluated synonymous substitutions per synonymous site (\u003cem\u003eK\u003c/em\u003e\u003csub\u003eS\u003c/sub\u003e) for paralogs in each genome (\u003cb\u003eSupplementary Fig.\u0026nbsp;3a\u003c/b\u003e). We found that all these species experienced an ancient WGD (the core-eudicot-common γ event), but only those in Clade I underwent a second recent WGD. After correcting for unequal substitution rates among species, we confirmed that the \u003cem\u003eK\u003c/em\u003e\u003csub\u003eS\u003c/sub\u003e peaks of the recent WGD were greater than those between orthologs within Clade I species, indicating it occurred before their diversification (\u003cb\u003eSupplementary Fig.\u0026nbsp;4\u003c/b\u003e). This was further supported by the well-preserved collinear relationships within Clade I species (2:2) and between Clade I and Clade II species (2:1) (\u003cb\u003eSupplementary Figs.\u0026nbsp;3b, 3c and 5\u003c/b\u003e). Taken together, these results consistently suggest that the 'salicoid' WGD event occurred on the ancestral branch of Clade I species.\u003c/p\u003e \u003cp\u003eWe next predicted the present distribution of the nine genera in the Salicaceae family using ecological niche modelling (\u003cb\u003eSupplementary Figs.\u0026nbsp;6 and 7 and Supplementary Tables\u0026nbsp;6 and 7\u003c/b\u003e). The results revealed that the four genera within Clade I, which underwent the 'salicoid' WGD, migrated to the Northern Hemisphere and exhibited niche differentiation, while the remaining lineages were primarily distributed in the Sounthern Hemisphere. Unlike \u003cem\u003eItoa\u003c/em\u003e and \u003cem\u003eIdesia\u003c/em\u003e, which are restricted to subtropical regions, the genera \u003cem\u003ePopulus\u003c/em\u003e and \u003cem\u003eSalix\u003c/em\u003e have expanded to higher latitudes, including temperate and polar regions, where they experienced extensive adaptive radiation. These findings suggest that the 'salicoid' WGD event may have played a critical role in driving ecological shifts and species diversification in their adaptability.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eKaryotypic evolution and chromosomal rearrangement\u003c/h2\u003e \u003cp\u003eUsing genome collinearity among these species, we reconstructed the ancestral Salicaceae karyotype (ASK), which consists of 11 putative protochromosomes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e \u003cb\u003eand Supplementary Figs.\u0026nbsp;8\u0026ndash;10; see Materials and Methods\u003c/b\u003e), rather than the previously suggested number of 10\u003csup\u003e35\u003c/sup\u003e. Drawing from the 'salicoid' WGD and genome rearrangement events, we further inferred their evolutionary relationships (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), which align closely with the phylogenetic analyses (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). Few fusion/fission events occurred in species from Clade II, which did not undergo the 'salicoid' WGD. Thus, in Clade II most protochromosomes were preserved intact, with the exception of at least four chromosomal rearrangements: i) protochromosome 8 underwent reciprocal translocation (RTA) with protochromosome 11 at the ancestral node of Clade II, ii) protochromosome 2 underwent end-to-end joining (EEJ) with protochromosome 3 to form chromosome 2 of \u003cem\u003eD. caffra\u003c/em\u003e, iii) protochromosome 2 independently underwent reciprocal translocation (RTA) with protochromosome 4 to form the contemporary karyotype structure of \u003cem\u003eS. chinensis\u003c/em\u003e, and iv) protochromosome 1 subsequently underwent an EEJ to form chromosome 1 of \u003cem\u003eX. longifolia\u003c/em\u003e and \u003cem\u003eF. jangomas\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e \u003cb\u003eand Supplementary Figs.\u0026nbsp;11a and 11b\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eWe inferred that the ancestral karyotype of Salicoideae Clade I species had a base number of 21 after the 'salicoid' WGD, achieved through an EEJ fusion between duplicated protochromosomes 5 and 9, with at least two subsequent inversions (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e \u003cb\u003eand Supplementary Fig.\u0026nbsp;11c\u003c/b\u003e). Additionally, we also predicted multiple RTA events between several protochromosomes. Interestingly, these genome-wide reorganizations were fully preserved in \u003cem\u003eI. polycarpa\u003c/em\u003e without other large fusion/fission events, indicating that their common ancestor already possessed a relatively stable karyotype during the rediploidization process. In contrast, our results indicated that \u003cem\u003eI. orientalis\u003c/em\u003e underwent significant changes through multiple chromosomal rearrangements, resulting in a base chromosome number of 20. Ancestors of \u003cem\u003ePopulus\u003c/em\u003e and \u003cem\u003eSalix\u003c/em\u003e also experienced a series of chromosome rearrangements, forming an ancient fused chromosome through successive nested chromosome fusions (NCF) of duplicated protochromosomes 3, 7 and 11 (\u003cb\u003eSupplementary Fig.\u0026nbsp;11d\u003c/b\u003e). This new fused chromosome subsequently underwent two independent RTA events: one with protochromosome 1 in \u003cem\u003ePopulus\u003c/em\u003e to generate extant chromosomes 1 and 4, and a second with protochromosome 5 in \u003cem\u003eSalix\u003c/em\u003e to generate extant chromosomes 4 and 16. This evolutionary trajectory resulted in a 19 chromosome karyotype in \u003cem\u003ePopulus\u003c/em\u003e and \u003cem\u003eSalix\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eThe 'salicoid' WGD event is a cryptic allopolyploidization\u003c/h3\u003e\n\u003cp\u003eCombining the correspondence between ASK and extant chromosomes of each Salicaceae species, as well as phylogenetic analysis and subgenome division (\u003cb\u003eSupplementary Fig.\u0026nbsp;12; see Materials and Methods\u003c/b\u003e), we found strong evidence to support that the \u0026lsquo;salicoid\u0026rsquo; WGD is a cryptic allopolyploidization event. Specifically, according to the reconstructed protochromosomes (ASK1-11), we identified 1,634 full homologous gene groups (HGGs), among which all Clade I species retained paralogous genes from the 'salicoid' WGD, while other species retained the orthologous genes (\u003cb\u003eSupplementary Fig.\u0026nbsp;13\u003c/b\u003e). There were 77\u0026ndash;265 full-HGGs across the 11 protochromosomes. Phylogenetic tree using the ASTRAL coalescent method showed that genes from Clade II were more closely related to one copy of 'salicoid' paralogs in Clade I species than the other copy (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, topologies \u0026lsquo;q1\u0026rsquo; and \u0026lsquo;q2\u0026rsquo; in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). This relationship was supported by 83.5\u0026ndash;90.3% of full-HGGs on all 11 protochromosomes and 80.1\u0026ndash;84.0% when considering partial-HGGs, where at least one species in Clade I retained 'salicoid' paralogs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb \u003cb\u003eand Supplementary Fig.\u0026nbsp;13\u003c/b\u003e). Consistently, we found that an RTA event between protochromosomes 8 and 11 that occurred in the ancestor of Clade II species was also present in only one chromosome in all Clade I species, further supporting their allopolyploid origin (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBased on their phylogenetic relationships with Clade II, we divided the genome of Clade I species into two subgenomes: the A subgenome consisted of chromosomes with genes closely related to Clade II, while the B subgenome was composed of the paralogous chromosomes from the 'salicoid' WGD (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec \u003cb\u003eand Supplementary Fig.\u0026nbsp;14\u003c/b\u003e). As expected, \u003cem\u003eK\u003c/em\u003e\u003csub\u003eS\u003c/sub\u003e distribution between Clade II species and A subgenome of Clade I species was generally smaller than that between Clade II and B subgenome, but both A and B subgenomes were similar in genetic distance to \u003cem\u003eC. decandra\u003c/em\u003e (\u003cb\u003eSupplementary Fig.\u0026nbsp;15\u003c/b\u003e), a degree of divergence consistent with phylogenetic relationships under allopolyploidization. Phylogenetic trees reconstructed using the A and B subgenomes by coalescent and concatenated methods revealed the same topologies with high support (\u003cb\u003eSupplementary Fig.\u0026nbsp;16\u003c/b\u003e). These results indicate that the 'salicoid' event in Clade I species was an allopolyploidization event with one parent being the common ancestor of Clade II species.\u003c/p\u003e \u003cp\u003eTo identify the second possible parent of the 'salicoid' allopolyploidization, we assembled the draft genomes of four additional species from the subfamily Salicoideae (including \u003cem\u003eAzara serrata\u003c/em\u003e, \u003cem\u003eProckia crucis\u003c/em\u003e, \u003cem\u003eBanara guianensis\u003c/em\u003e and \u003cem\u003eHomalium cochinchinense\u003c/em\u003e) and one species from the subfamily Scyphostegioideae (\u003cem\u003eDianyuea turbinata\u003c/em\u003e). These draft genomes are fragmented but, based on gene prediction, cover most of the coding regions (\u003cb\u003eSupplementary Table\u0026nbsp;8\u003c/b\u003e). Therefore, we added gene sequences from these five more genera to the HGGs and constructed coalescent and concatenated trees (\u003cb\u003eSupplementary Fig.\u0026nbsp;17\u003c/b\u003e). In both trees, four of the new genera from Salicoideae clustered within Clade II, while \u003cem\u003eD. turbinata\u003c/em\u003e was sister to both subgenomes, indicating that none of these species was direct donors of the B subgenome. Since these species represent all three subfamilies of Salicaceae\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e,\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e, w-e inferred that the parental donor of the B subgenome is likely extinct. Moreover, we inferred that the two parental lineages diverged for approximately 8\u0026nbsp;million years prior to the 'salicoid' WGD based on our estimates that the A and B subgenomes diverged about 68 Ma and Clade II diverged from the A subgenomes about 60 Ma (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Finally, based on \u003cem\u003eK\u003c/em\u003e\u003csub\u003eS\u003c/sub\u003e values corrected for the 'salicoid' WGD, rates of molecular evolution were the slowest in \u003cem\u003eI. polycarpa\u003c/em\u003e, followed by \u003cem\u003eI. orientalis\u003c/em\u003e and \u003cem\u003ePopulus\u003c/em\u003e, while \u003cem\u003eSalix\u003c/em\u003e evolved fastest (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eSubgenome dominance and lineage-specific gene retention\u003c/h3\u003e\n\u003cp\u003eSubgenome evolution after polyploidization plays a crucial role in reducing gene redundancy and increasing subsequent potential for trait and adaptive innovation\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. Our analysis of 21 Salicaceae genomes/subgenomes showed that A subgenome exhibited evolutionary dominance over B subgenome in Clade I species. First, we found that after the 'salicoid' event, the rate of gene retention was reduced significantly in Clade I species and was more lower in the B subgenome than in the A subgenome (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). Second, B subgenome showed lower gene expression levels than A subgenome in all Clade I species (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). This may be related to higher repeat content surrounding genes in the B subgenome (\u003cb\u003eSupplementary Fig.\u0026nbsp;18a\u003c/b\u003e), although there was no significant difference in the expansion of TEs between subgenomes A and B overall (\u003cb\u003eSupplementary Fig.\u0026nbsp;18b\u003c/b\u003e). Third, genes in the A subgenome had slightly but significantly higher chromatin accessibility levels (\u003cb\u003eSupplementary Fig.\u0026nbsp;18c\u003c/b\u003e) and slightly lower methylation levels than those in the B subgenome (\u003cb\u003eSupplementary Figs.\u0026nbsp;18d and 18e\u003c/b\u003e), suggesting that the differentiation between the A and B subgenomes is primarily reflected in sequence variation rather than in epigenetic landscapes. Fourth, protein evolution rate (\u003cem\u003eK\u003c/em\u003ea/\u003cem\u003eK\u003c/em\u003e\u003csub\u003eS\u003c/sub\u003e) was significantly higher in the B subgenome, indicating relaxed purifying selection (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). Finally, through an analysis of the contribution of subgenome-biased gene retention to lineage evolution, we found that genes specifically retained in the A subgenome of all Clade I species (n\u0026thinsp;=\u0026thinsp;958), were enriched in functions such as 'chromosome segregation', 'double-strand break repair', 'meiotic cell cycle process', and 'spindle organization' (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). In contrast, genes uniquely retained in the B subgenome (n\u0026thinsp;=\u0026thinsp;558) did not exhibit enrichment in any specific functional categories. These findings suggest that the A subgenome may play a pivotal role in facilitating and coordinating chromosome recombination during the rediploidization process, while gene retention in the B subgenome appears to be more random and functionally diverse.\u003c/p\u003e \u003cp\u003eMoreover, beyond subgenome dominance, gene retention may also exhibit lineage-specific dynamics over evolutionary time, which are closely linked to the diverse adaptation of different lineages to complex environments\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. To explore this, we further analyzed gene retention at three key evolutionary nodes in Clade I (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee). We identified 1,865 WGD-derived gene pairs retained across all Clade I species (Node I). These genes are functionally associated with 'regulation of photoperiodism, flowering', 'inflorescence development', 'regulation of seed development', 'trichome morphogenesis', 'cold acclimation' and 'defense response' (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). The retention of key genes related to photoperiod-flowering (such as \u003cem\u003eFT\u003c/em\u003e, \u003cem\u003eCIB1\u003c/em\u003e and \u003cem\u003eHUA2\u003c/em\u003e)\u003csup\u003e\u003cspan additionalcitationids=\"CR43 CR44\" citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e, cold acclimation and freezing tolerance (\u003cem\u003eMYB15\u003c/em\u003e, \u003cem\u003eADF5\u003c/em\u003e and \u003cem\u003eGALS2\u003c/em\u003e)\u003csup\u003e\u003cspan additionalcitationids=\"CR47\" citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e, and trichome development (\u003cem\u003eGIS3\u003c/em\u003e, \u003cem\u003eZFP6\u003c/em\u003e and \u003cem\u003eTOE1\u003c/em\u003e)\u003csup\u003e\u003cspan additionalcitationids=\"CR50\" citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e (\u003cb\u003eSupplementary Figs.\u0026nbsp;19a and 19b\u003c/b\u003e) likely facilitated the divergence of Clade I species from their ancestral lineage by enabling adaptation to northern regions with variable photoperiods and temperatures, while promoting trait development. Consistent with this, more 402 pairs of genes with similar functions were specifically retained in Node II representing the deciduous lineages of \u003cem\u003eIdesia\u003c/em\u003e, \u003cem\u003ePopulus\u003c/em\u003e and \u003cem\u003eSalix\u003c/em\u003e that are distinct from the evergreen lineage \u003cem\u003eItoa\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed), including \u003cem\u003eFD\u003c/em\u003e, \u003cem\u003eNF-YB3, SPL4\u003c/em\u003e, \u003cem\u003eSPL5\u003c/em\u003e and \u003cem\u003eELF9\u003c/em\u003e\u003csup\u003e52\u0026ndash;55\u003c/sup\u003e, which are related to photeperiodic flowering (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee, \u003cb\u003eSupplementary Figs.\u0026nbsp;19c and 19d\u003c/b\u003e). Additionally, at Node III, representing the \u003cem\u003ePopulus\u003c/em\u003e and \u003cem\u003eSalix\u003c/em\u003e lineages that are widely distributed in the Northern Hemisphere, we identified 129 uniquely retained gene pairs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee). These genes are involved in specific functions like 'maintenance of floral organ identity', 'seed trichome differentiation', 'red or far-red light signaling pathway' and 'regulation of plant-type cell wall cellulose biosynthetic process' (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed), such as \u003cem\u003eREV\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e related to the formation of inflorescence and floral meristem, and \u003cem\u003eCSLD1\u003c/em\u003e\u003csup\u003e57\u003c/sup\u003e related to the development of root hairs and female gametophytes (\u003cb\u003eSupplementary Figs.\u0026nbsp;19e and 19f\u003c/b\u003e). Overall, the dynamic patterns of gene retention in these lineages suggest that photoperiod adaptation, flowering, and traits development including inflorescence and seeds, have fostered the diversification of Clade I species, promoting their morphological innovation and environmental fitness.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eLineage-specific subgenomic expressions contributed to adaptive innovation in multiple traits of\u003c/b\u003e \u003cb\u003ePopulus\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eSalix\u003c/b\u003e\u003c/p\u003e \u003cp\u003eExpression divergence of gene pairs plays a pivotal role in driving adaptive radiation and evolutionary innovation after polyploidization\u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e,\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e. To investigate the association between subgenome expression patterns and lineage diversification in the Salicaceae, we focused on gene pairs retained across all Clade I species. Transcriptome sequencing and cluster analysis of male and female flowers, as well as fruits at various developmental stages in \u003cem\u003eItoa\u003c/em\u003e, \u003cem\u003eIdesia\u003c/em\u003e and \u003cem\u003ePopulus\u003c/em\u003e (\u003cb\u003eSupplementary Table\u0026nbsp;9\u003c/b\u003e), revealed that the expression biases between subgenomes A and B for gene pairs is largely consistent across tissues and developmental stages within the same species (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). However, significant differences were observed between species. Specifically, among the 1,747 gene pairs examined, only 85 (C1) and 51 (C2) gene pairs showed the same expression bias towards subgenomes A and B, respectively, across the three lineages. In contrast, the vast majority (C4-C15, ~\u0026thinsp;80%) of gene pairs showed clear differences in expression divergence between lineages (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003eAmong the clusters with consistent expression biases, gene pairs in C1, with higher expression in the subgenome A (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea), were significantly enriched in functions related to reproductive transitions, seed maturation and stress responses (\u003cb\u003eSupplementary Table\u0026nbsp;10\u003c/b\u003e), including genes such as \u003cem\u003eFT\u003c/em\u003e, \u003cem\u003eHDA6, AP2\u003c/em\u003e, \u003cem\u003eBBX21\u003c/em\u003e, \u003cem\u003eGALS2\u003c/em\u003e and transcription factors belonging to \u003cem\u003eWRKY\u003c/em\u003e family\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e,\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e,\u003cspan additionalcitationids=\"CR61 CR62 CR63\" citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). Conversely, gene pairs in C2, with higher expression in the subgenome B, were mainly associated with regulation of abscisic acid signaling and gene expression regulation (\u003cb\u003eSupplementary Table\u0026nbsp;10\u003c/b\u003e). These results suggest the significant role of the subgenome A in regulating reproductive and growth traits in these species. For example, the WGD-derived paralogs of flowering-related gene \u003cem\u003eFT\u003c/em\u003e exemplified functional divergence: \u003cem\u003eFT2\u003c/em\u003e in subgenome A was highly expressed in our examined transcriptomes, while \u003cem\u003eFT1\u003c/em\u003e from subgenome B was rarely expressed (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb), supporting a role of \u003cem\u003eFT2\u003c/em\u003e in promoting flower and fruit development. Interestingly, previous studies on poplar have shown that \u003cem\u003eFT1\u003c/em\u003e is expressed in cold-exposed buds during winter, triggering the transition from vegetative meristems to the reproductive phase, while \u003cem\u003eFT2\u003c/em\u003e is primarily expressed during the growing season, regulating vegetative growth. These spatiotemporal shifts in expression are essential for the annual growth cycle of poplar\u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e,\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e. To further explore the evolutionary insights into the functional divergence of this gene pair, we analyzed their expression patterns in leaves and buds (or shoot apex) from \u003cem\u003eI\u003c/em\u003e. \u003cem\u003eorientalis\u003c/em\u003e and \u003cem\u003eI. polycarpa\u003c/em\u003e across late autumn to early spring (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). The results showed that in leaves, both \u003cem\u003eFT1\u003c/em\u003e and \u003cem\u003eFT2\u003c/em\u003e were expressed during winter in \u003cem\u003eI\u003c/em\u003e. \u003cem\u003eorientalis\u003c/em\u003e, while only \u003cem\u003eFT1\u003c/em\u003e was expressed in \u003cem\u003eI. polycarpa\u003c/em\u003e and poplar. In buds or shoot apex, \u003cem\u003eFT1\u003c/em\u003e was exclusively expressed in poplar, whereas \u003cem\u003eFT2\u003c/em\u003e was specifically expressed throughout winter in \u003cem\u003eI\u003c/em\u003e. \u003cem\u003eorientalis\u003c/em\u003e and only in late winter in \u003cem\u003eI. polycarpa\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). This unique functional divergence of \u003cem\u003eFT\u003c/em\u003e genes in poplar is consistent with its phase change from vegetative to reproductive growth during the transition from active growth to winter dormancy, enabling earlier flowering and providing a reproductive advantage in spring. In contrast, the co-expression of \u003cem\u003eFT1\u003c/em\u003e and \u003cem\u003eFT2\u003c/em\u003e in \u003cem\u003eI\u003c/em\u003e. \u003cem\u003eorientalis\u003c/em\u003e likely correlates with its evergreen and non-dormant traits, while the expression patterns in \u003cem\u003eI. polycarpa\u003c/em\u003e appear linked to the initiation of reproductive growth after spring warming. The lineage-specific divergence in \u003cem\u003eFT\u003c/em\u003e expression have likely promote the variation in flowering phenology among these lineages.\u003c/p\u003e \u003cp\u003eFurthermore, we found that the gene pairs with \u003cem\u003ePopulus\u003c/em\u003e-specific expression divergence (C4-C6 in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea) were significantly enriched in functions related to 'photoperiodism, flowering', 'regulation of circadian rhythm', flower development, 'regulation of seed development' and 'trichome morphogenesis' (\u003cb\u003eSupplementary Table\u0026nbsp;10\u003c/b\u003e). Among these, we identified several genes involved in the regulation of flower development, including \u003cem\u003eFIL\u003c/em\u003e, \u003cem\u003ePUB13\u003c/em\u003e, \u003cem\u003eBRM\u003c/em\u003e\u003csup\u003e\u003cspan additionalcitationids=\"CR67\" citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u003c/sup\u003e, as well as \u003cem\u003eENO2\u003c/em\u003e and \u003cem\u003eXYL1\u003c/em\u003e\u003csup\u003e69,70\u003c/sup\u003e, which are associated with seed development and seed size determination. Additionally, we also identified \u003cem\u003eZFP6\u003c/em\u003e and \u003cem\u003eGIS3\u003c/em\u003e, a pair of WGD-derived genes regulating trichome development\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e,\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e, which exhibited significantly higher expression levels in \u003cem\u003ePopulus\u003c/em\u003e compared to \u003cem\u003eIdesia\u003c/em\u003e and \u003cem\u003eItoa\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). Four \u003cem\u003ePopulus\u003c/em\u003e-\u003cem\u003eSalix\u003c/em\u003e specific conserved noncoding elements (CNEs) were discovered in their promoter regions (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed). Reporter assays indicated that these CNEs possess potential enhancer activities, likely contributing to increased gene expression during seed trichome development in the \u003cem\u003ePopulus-Salix\u003c/em\u003e lineage (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee \u003cb\u003eand Supplementary Table\u0026nbsp;11\u003c/b\u003e). In summary, these findings suggest that the lineage-specific divergence in subgenomic gene expression significantly contributed to the evolution of distinct flowering phenology and the development of small, hairy seeds in \u003cem\u003ePopulus\u003c/em\u003e and \u003cem\u003eSalix\u003c/em\u003e. These traits likely played a critical role in enabling their rapid colonization and extensive adaptive radiation in high-latitude regions.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eAncient allopolyploidization events are often obscured by large-scale subgenome reshuffling. In this study, we clearly demonstrate that the ancient polyploidization event, shared by the genera \u003cem\u003ePopulus\u003c/em\u003e and \u003cem\u003eSalix\u003c/em\u003e, as well as their sister genera \u003cem\u003eIdesia\u003c/em\u003e and \u003cem\u003eItoa\u003c/em\u003e in the Salicaceae, was caused by hybridization between two closely related lineages with a divergence time of approximately 8\u0026nbsp;million years. Due to the high degree of similarity between the two lineages\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e, this cryptic allopolyploidization event would have been difficult to detect without information from either parental lineage. Our analysis showed that one of the parental genomes is represented by the Clade II lineage of the Salicoideae subfamily, while the other parental lineage is now extinct. Based on this, we successfully separated the genomes of these polyploidized species into two distinct subgenomes. We further constructed the common ancestral karypotype and detected unique chromosomal fusions specific to \u003cem\u003eItoa\u003c/em\u003e, \u003cem\u003eIdesia\u003c/em\u003e and \u003cem\u003ePopulus\u003c/em\u003e-\u003cem\u003eSalix\u003c/em\u003e. The lineage-specific karyotypic evolution is well consistent with the phylogenomic tree and intact chromosomes are widespread and shared by three lineages \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. These results also revealed that despite the 60\u0026nbsp;million years since allopolyploidization and subsequent species diversification, a considerable number of chromosomes have maintained relatively stable organization. Therefore, the Salicaceae, along with other lineages including \u003cem\u003eGossypium\u003c/em\u003e and related Malvaceae, Brassicaceae, and various subclades of Poaceae\u003csup\u003e\u003cspan additionalcitationids=\"CR72\" citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e\u003c/sup\u003e, provides a potential model to investigate the long-term consequences of allopolyploidization and the subsequent evolution of subgenomes (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. This is in contrast to recent polyploids that have not undergone rediploidization or species diversification, or ancient polyploids with unknown parental lineages\u003csup\u003e\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e,\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOur study shows that subgenomes derived from two parental lineages continue to exhibit evolutionary dominance even after a long time, regardless of the extent of chromosomal rearrangements in the respective descendant lineages. This dominance is typically manifested as biases in gene retention, variations in gene expression levels, and differences in evolutionary rates. Consistent with previous speculation on the role of paleopolyploidy in the adaptive evolution of angiosperms\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e, we found that genes associated with environmental responses, particularly those involved in photoperiod regulation, flowering cycles and cold adaptation, were preferentially retained following allopolyploidization. However, although we observed the same dominance of subgenome A over subgenome B, the retained genes and expression divergence exhibit significant variation between three lineages, \u003cem\u003eItoa\u003c/em\u003e, \u003cem\u003eIdesia\u003c/em\u003e and \u003cem\u003ePopulus\u003c/em\u003e-\u003cem\u003eSalix\u003c/em\u003e. Importantly, these lineage-specific evolutionary patterns align with their adaptive traits and ecological niches in the Northern Hemisphere, such as the distinct flowering phenology and highly-effective seed dispersal facilitated by hairs in the \u003cem\u003ePopulus\u003c/em\u003e-\u003cem\u003eSalix\u003c/em\u003e lineage \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e, promoting their adaptive radiation at higher latitudes (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. In contrast, the other two lineages, \u003cem\u003eIdesia\u003c/em\u003e and \u003cem\u003eItoa\u003c/em\u003e, which consist of only one and two species respectively, well adapt to tropical and subtropical environments with totally different gene retention and expressions, relying on animal-mediated pollination and animal- or wind-mediated fruit or seed dispersal. These findings suggest that allopolyploidization in Salicaceae conferred shared properties of evolutionary dominance to subgenomes, while contrasting gene retention and expression divergence provided significant potential for trait diversification and adaptive evolution across different descendant lineages. This supports the ancestral polyploidization as a driving force of key innovations and lineage diversification in angiosperms\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. However, our results suggest not all allopolyploidy events will undoubtedly lead to the origin of the adaptive innovations and following species radiation as previously thought\u003csup\u003e\u003cspan additionalcitationids=\"CR19 CR20 CR21\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Overall, our study provides insights into different subgenome evolution after allopolyploidy underlying the diverse traits observed in the economically and ecologically significant Saliaceae, once woody model clade. This should prevail in multiple higher taxonomic clades of both plants and animals. In addition, our results further underscore the importance of distinguishing between autopolyploidization and allopolyploidization events as well as contrasted subgenomic evolution that foster adaptive innovation and species diversification at deep nodes of the tree of life.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003eSample collection and genome sequencing\u003c/h2\u003e\n \u003cp\u003ePlant material of \u003cem\u003eCasearia decandra\u003c/em\u003e, \u003cem\u003eDovyalis caffra\u003c/em\u003e, \u003cem\u003eScolopia chinensis\u003c/em\u003e, \u003cem\u003eXylosma longifolia\u003c/em\u003e and \u003cem\u003eFlacourtia jangomas\u003c/em\u003e were collected in XiShuangBanNa Tropical Botanical Garden (Mengla, China), \u003cem\u003eItoa orientalis\u003c/em\u003e and \u003cem\u003eIdesia polycarpa\u003c/em\u003e were collected in Chengdu, and \u003cem\u003eSalix rehderiana\u003c/em\u003e is collected in Minya Konka of China, respectively. Fresh leaves were collected, and high-quality genomic DNA was extracted using the QIAGEN Genomic DNA extraction kit (Qiagen, Hilden, Germany) according to the manufacturer\u0026rsquo;s instructions. Three approaches were employed in DNA sequencing. First, genomic DNA was size-selected using the BluePippin system (sage Science), processed following the protocol of Ligation Sequencing Kit (LSK108), and sequenced using the Oxford Nanopore Technology sequencer. Sequencing was performed on the PromethION platform, and base calling was carried out using Guppy v3.2.8. Second, Paired-end libraries were constructed according to the manufacturer\u0026rsquo;s protocols and sequenced using the Illumina HiSeq 2500 System. Third, Hi-C (high-throughput chromatin conformation capture) libraries were prepared by chromatin extraction and digestion and DNA ligation, purification, fragmentation, and sequenced on an Illumina HiSeq 2500\u003csup\u003e76\u003c/sup\u003e. In addition, the dried leaves of \u003cem\u003eAzara serrata\u003c/em\u003e, \u003cem\u003eProckia crucis\u003c/em\u003e, \u003cem\u003eBanara guianensis\u003c/em\u003e and \u003cem\u003eHomalium cochinchinense\u003c/em\u003e were used to extract DNA, construct Illumina paired-end libraries, and perform sequencing. For \u003cem\u003eDianyuea turbinata\u003c/em\u003e, The HiFi SMRTbell library was constructed using the SMRTbell Express Template Prep Kit v2.0 (Pacific Bioscience) and sequencing was carried out using the PacBio Sequel II platform (Berry Genomics, Beijing, China).\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eGenome assembly and annotation\u003c/h3\u003e\n\u003cp\u003eNanopore long reads were \u003cem\u003ede novo\u003c/em\u003e assembled using the Nextdenovo v2.2.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/Nextomics/NextDenovo\u003c/span\u003e\u003c/span\u003e). The initial assemblies were further corrected and polished using the program NextPolish v1.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/Nextomics/NextPolish\u003c/span\u003e\u003c/span\u003e), by mapping the filtered Nanopore and Illumina reads to the genome using Minimap2 v2.17\u003csup\u003e77\u003c/sup\u003e and BWA v0.7.17\u003csup\u003e78\u003c/sup\u003e. Finally, contigs were clustered, ordered and anchored to the pseudochromosomes by LACHESIS\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e79\u003c/span\u003e\u003c/sup\u003e using validly mapped Hi-C reads. Illumina reads of \u003cem\u003eA. serrata\u003c/em\u003e, \u003cem\u003eP. crucis\u003c/em\u003e, \u003cem\u003eB. guianensis\u003c/em\u003e and \u003cem\u003eH. cochinchinense\u003c/em\u003e were assembled using Platanus v1.2.4\u003csup\u003e80\u003c/sup\u003e by implementing \u0026lsquo;assemble\u0026rsquo;, \u0026lsquo;scaffold\u0026rsquo; and \u0026lsquo;gap_close\u0026rsquo; program. PacBio HiFi reads were used to perform \u003cem\u003ede novo\u003c/em\u003e genome assembly for \u003cem\u003eD. turbinata\u003c/em\u003e with Hifiasm v0.14\u003csup\u003e81\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eWe utilized a combination of homolog-based and \u003cem\u003ede novo\u003c/em\u003e approaches to annotate repetitive elements. RepeatMasker v.4.0.7\u003csup\u003e82\u003c/sup\u003e was firstly used to perform homolog prediction based on the Repbase database\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e83\u003c/span\u003e\u003c/sup\u003e. Next, RepeatModeler v.1.0.11\u003csup\u003e84\u003c/sup\u003e was used to perform \u003cem\u003ede novo\u003c/em\u003e prediction of repeat sequence features and the results were then utilized by RepeatMasker v.4.0.7\u003csup\u003e82\u003c/sup\u003e to identify and classify repeat elements. Gene models were predicted based on \u003cem\u003ede novo\u003c/em\u003e prediction, homologous identification and transcript data. In brief, Augustus v3.2.3\u003csup\u003e85\u003c/sup\u003e was used for \u003cem\u003ede novo\u003c/em\u003e prediction of protein-coding genes. For homologous identification, we mapped the protein sequences of six published genomes (\u003cem\u003eP. trichocarpa\u003c/em\u003e\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e, \u003cem\u003eP. pruinosa\u003c/em\u003e\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e86\u003c/span\u003e\u003c/sup\u003e, \u003cem\u003eP. alba\u003c/em\u003e var. \u003cem\u003epyramidali\u003c/em\u003e \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e87\u003c/span\u003e\u003c/sup\u003e, \u003cem\u003eS. suchowensis\u003c/em\u003e\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e, \u003cem\u003eS. purpurea\u003c/em\u003e\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e88\u003c/span\u003e\u003c/sup\u003e, and \u003cem\u003eArabidopsis thaliana\u003c/em\u003e\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e89\u003c/span\u003e\u003c/sup\u003e) onto the genomes using TBLASTN v2.6.0\u003csup\u003e90\u003c/sup\u003e and then used GENEWISE v2.4.1\u003csup\u003e91\u003c/sup\u003e to predict gene structures. RNA transcripts were used to predict gene models with PASA v2.3.3\u003csup\u003e92\u003c/sup\u003e. Finally, all of the predictions were integrated using EvidenceModeler v1.1.1\u003csup\u003e93\u003c/sup\u003e to generate consensus gene sets. Assembly and annotation completeness was assessed with BUSCO (Benchmarking Universal Single-Copy Orthologs) v3.0\u003csup\u003e94\u003c/sup\u003e.\u003c/p\u003e\n\u003ch3\u003ePhylogenetic analysis\u003c/h3\u003e\n\u003cp\u003eWe conducted a phylogenomic analysis for eight newly sequenced species and three poplar (\u003cem\u003eP. trichocarpa\u003c/em\u003e\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e, \u003cem\u003eP. alba\u003c/em\u003e var. \u003cem\u003epyramidalis\u003c/em\u003e\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e87\u003c/span\u003e\u003c/sup\u003e and \u003cem\u003eP. euphratica\u003c/em\u003e\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e95\u003c/span\u003e\u003c/sup\u003e) and two willow species (\u003cem\u003eS. chaenomeloides\u003c/em\u003e\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e96\u003c/span\u003e\u003c/sup\u003e and \u003cem\u003eS. purpurea\u003c/em\u003e\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e97\u003c/span\u003e\u003c/sup\u003e), using \u003cem\u003ePassiflora edulia\u003c/em\u003e\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e98\u003c/span\u003e\u003c/sup\u003e, \u003cem\u003ePassiflora organensis\u003c/em\u003e\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e99\u003c/span\u003e\u003c/sup\u003e, \u003cem\u003eArabidopsis thaliana\u003c/em\u003e\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e89\u003c/span\u003e\u003c/sup\u003e, \u003cem\u003eRosa chinensis\u003c/em\u003e\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e100\u003c/span\u003e\u003c/sup\u003e \u003cem\u003eand Vitis vinifera\u003c/em\u003e\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e101\u003c/span\u003e\u003c/sup\u003e as outgroups. We constructed two phylogenetic datasets using different strategies: targeted identification of phylogenomic markers (BUSCO) and de novo inference (OrthoFinder). Conserved single-copy genes were identified by BUSCO analyses with the embryophyta_odb10 dataset (1,614 BUSCOs)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e94\u003c/span\u003e\u003c/sup\u003e, resulting in 426 single-copy orthologs retained across 18 species. OrthoFinder v2.3.11\u003csup\u003e102\u003c/sup\u003e was used to de novo identify orthologous sequences shared among species with default parameters, resulting in 422 single-copy orthologs. To reconstruct a high-resolution phylogenetic tree, we merged the two datasets and obtained a total of 672 single-copy orthologs. For all orthologs, protein sequences were aligned using MAFFT v7.313\u003csup\u003e103\u003c/sup\u003e, and converted to codon alignments using PAL2NAL\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e104\u003c/span\u003e\u003c/sup\u003e. A maximum likelihood (ML) phylogenetic tree was then constructed using RAxML v8.2.11\u003csup\u003e105\u003c/sup\u003e with the GTR\u0026thinsp;+\u0026thinsp;gamma model and 1000 bootstrap replicates. Additionally, STAG from the OrthoFinder pipeline was also used to infer a species tree based on all orthogroups (including multi-copy genes) identified by OrthoFinder. The topologies of the phylogenetic trees constructed by the two methods were the same.\u003c/p\u003e\n\u003cp\u003eThe divergence times among species were estimated using the MCMCtree program\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e106\u003c/span\u003e\u003c/sup\u003e. Three constraints obtained from the TIMETREE database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://timetree.org/\u003c/span\u003e\u003c/span\u003e) were used for time calibrations: (1) the divergence between \u003cem\u003eVitis\u003c/em\u003e and \u003cem\u003eRosa\u003c/em\u003e (109\u0026ndash;124 Mya), (2) the divergence between \u003cem\u003eArabidopsis\u003c/em\u003e and \u003cem\u003ePopulus\u003c/em\u003e (102\u0026ndash;113 Mya), (3) the divergence between \u003cem\u003ePopulus\u003c/em\u003e and \u003cem\u003eSalix\u003c/em\u003e (28\u0026ndash;60 Mya).\u003c/p\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003eAnalysis of WGD events\u003c/h2\u003e\n \u003cp\u003eWe identified and localized WGD events in Salicaceae by combining intra- and inter-species synteny analysis and \u003cem\u003eKs\u003c/em\u003e distribution. First, we used the BLASTP v2.7.1\u003csup\u003e107\u003c/sup\u003e with a cutoff e-value of 1e-5 to align protein sequences within species and between species (\u003cem\u003eP. trichocarpa\u003c/em\u003e vs. \u003cem\u003eI. polycarpa\u003c/em\u003e, \u003cem\u003eP. trichocarpa\u003c/em\u003e vs. \u003cem\u003eI. orientalis\u003c/em\u003e). WGDI (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/SunPengChuan/wgdi\u003c/span\u003e\u003c/span\u003e)\u003csup\u003e108\u003c/sup\u003e with the \u0026lsquo;-icl\u0026rsquo; parameter was used to identify the intergenomic synteny blocks between \u003cem\u003eP. trichocarpa\u003c/em\u003e and others, as well as intragenomic synteny blocks within each species. The \u003cem\u003eKs\u003c/em\u003e between collinear genes was estimated by Nei-Gojobori approach in PAML with the parameter \u0026lsquo;-ks\u0026rsquo; of WGDI, and the \u0026lsquo;-bk\u0026rsquo; parameter was applied to generate a dot plot of collinear genes and \u003cem\u003eKs\u003c/em\u003e values, visualizing intra- and inter-species synteny. Additionally, the \u003cem\u003eK\u003c/em\u003e\u003csub\u003eS\u003c/sub\u003e peaks were fitted using the \u0026lsquo;-pf\u0026rsquo; parameter, and the density distribution curve of \u003cem\u003eK\u003c/em\u003e\u003csub\u003eS\u003c/sub\u003e was displayed using the \u0026lsquo;-kf\u0026rsquo; parameter. The location of the WGD event was identified based on the comparison of the \u003cem\u003eKs\u003c/em\u003e peaks between paralogs within species and orthologs between species. To address potential inaccuracies in detecting WGD events due to differing substitution rates among candidate species, we further applied KsRates v1.1.3 to bring all the distributions to a common \u003cem\u003eKs\u003c/em\u003e scale by compensating for the differences in synonymous substitution rates relative to the focal species, and the rate-adjusted mixed paralog-ortholog \u003cem\u003eKs\u003c/em\u003e distribution was then used to position adjusted WGD events.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003eEcological niche modelling\u003c/h2\u003e\n \u003cp\u003eThe data collection for each genus was obtained from the Global Biodiversity Information Facility (GBIF: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.gbif.org/\u003c/span\u003e\u003c/span\u003e). To remove spatial autocorrelation and sampling bias, the obtained distribution data were subjected to 5 km spatial dilution using SDMtoolbox\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e109\u003c/span\u003e\u003c/sup\u003e, and the final distribution points (\u003cem\u003eSalix\u003c/em\u003e: 131,974, \u003cem\u003ePopulus\u003c/em\u003e: 82,944, \u003cem\u003eIdesia\u003c/em\u003e: 1,183, \u003cem\u003eItoa\u003c/em\u003e: 113, \u003cem\u003eFlacourtia\u003c/em\u003e: 2,905, \u003cem\u003eXylosma\u003c/em\u003e: 3,802, \u003cem\u003eScolopia\u003c/em\u003e: 3,689, \u003cem\u003eDovyalis\u003c/em\u003e: 2,039, \u003cem\u003eCasearia\u003c/em\u003e: 14,416) were used for Maxent modeling analysis. Environmental layers for 19 bioclimatic variables at current time (1970\u0026ndash;2000) were downloaded from the WorldClim v2.1 dataset (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.worldclim.com/\u003c/span\u003e\u003c/span\u003e) at a spatial resolution of 10 arc minutes (see \u003cstrong\u003eSupplementary Table\u0026nbsp;6\u003c/strong\u003e). Pairwise correlations were examined for 19 variables within the distribution of each genus. Taxon distributions were reconstructed using variables with a pairwise Pearson correlation coefficient below 0.8 and the most ecological significance. Ecological niche modeling (ENM) was performed using Maxent 3.4.3\u003csup\u003e110\u003c/sup\u003e to simulate potentially suitable habitats under the current climate for each genus. The test output of the models was set at 30%. The accuracy of the model was assessed using the area under the curve (AUC) of the receiver operating characteristic (ROC) plot. AUC values above 0.7 were considered indicative of good model performance\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e111\u003c/span\u003e\u003c/sup\u003e. ArcGIS 10.8 was utilized for mapping the suitable distribution range. To examine niche differences, ENMtools\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e112\u003c/span\u003e\u003c/sup\u003e was employed to calculate niche overlap statistics Schoener\u0026apos;s D\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e113\u003c/span\u003e\u003c/sup\u003eand Hellinger\u0026rsquo;s-based I\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e114\u003c/span\u003e\u003c/sup\u003e, with 100 pseudo-replicates. Values of D and I range from 0 (no ecological niche overlap) to 1 (identical ecological niches).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003eAncestral karyotype reconstruction\u003c/h2\u003e\n \u003cp\u003eComparing gene collinearity between genomes can reflect karyotype changes, revealing the trajectory of the formation of existing chromosomes and inferring evolutionary relationships independently. During karyotype evolution, ancestral chromosomes (protochromosomes) may have fused or remained as independent chromosomes within existing genomes. We first applied the workflow by Sun et al.\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e71\u003c/span\u003e\u003c/sup\u003e, which identifies protochromosomes and reconstructs ancestral karyotypes by searching for independent chromosomes or chromosome-like homologous blocks shared across different lineages. A detailed example of the workflow is available at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/SunPengChuan/wgdi-example/blob/main/Karyotype_Evolution.md\u003c/span\u003e\u003c/span\u003e.\u003c/p\u003e\n \u003cp\u003eSpecifically, using the \u003cem\u003eCasearia decandra\u003c/em\u003e genome as a reference, we aligned the remaining 12 genomes using WGDI\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e108\u003c/span\u003e\u003c/sup\u003e with the parameter \u0026lsquo;-d\u0026rsquo;. Synteny blocks shared between independent chromosomes were first searched in all genomes, and synteny blocks of independent chromosomes identified in at least three genera were assumed to represent Salicaceae protochromosomes. For example, protochromosome 6 (ASK6: homologous to Chr6 of \u003cem\u003eCasearia\u003c/em\u003e) of the ancestral Salicaceae karyotype is retained as an independent chromosome in Chr6 of \u003cem\u003eScolopia\u003c/em\u003e, Chr5 of \u003cem\u003eXylosma\u003c/em\u003e and \u003cem\u003eFlacourtia\u003c/em\u003e, Chr20 of \u003cem\u003eItoa\u003c/em\u003e, Chr5 of \u003cem\u003eIdesia\u003c/em\u003e, Chr13 of \u003cem\u003ePopulus\u003c/em\u003e and \u003cem\u003eSalix\u003c/em\u003e (\u003cstrong\u003eSupplementary Figs.\u0026nbsp;8 and 9a\u003c/strong\u003e). Similarly, ASK1, ASK2, ASK4, ASK5, ASK7, ASK9, ASK10 and ASK11 are retained as independent chromosomes in at least three genera. Therefore, these independent chromosomes were extracted as protochromosomes. Next, all identified synteny blocks were removed from fused chromosomes in existing genomes, and the remaining parts were connected as a chromosomes for a new round of exploration. After removing ASK11 (Chr11 of \u003cem\u003eCasearia\u003c/em\u003e), Chr7 and Chr10 in \u003cem\u003eFlacourtia\u003c/em\u003e were connected, corresponding to partial fragments of Chr3 and Chr8 in \u003cem\u003eCasearia\u003c/em\u003e, which also remain intact in many other species and therefore were identified as ASK8 (\u003cstrong\u003eSupplementary Fig.\u0026nbsp;8\u003c/strong\u003e). After further removing ASK8, Chr2 in \u003cem\u003eFlacourtia\u003c/em\u003e corresponds to segments of Chr3 and Chr8 in \u003cem\u003eCasearia\u003c/em\u003e, which are independent chromosomes in many other species, and therefore was identified as ASK3 (\u003cstrong\u003eSupplementary Fig.\u0026nbsp;8\u003c/strong\u003e). Ultimately, each extant genome had no remaining genomic blocks, and a total of 11 putative protochromosomes were extracted, hypothesized to form the ancestral Salicaceae karyotype (ASK). Additionally, we also used the MLGO web service (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.geneorder.org/\u003c/span\u003e\u003c/span\u003e)\u003csup\u003e115\u003c/sup\u003e to infer ancestral genomes based on information from synteny blocks between species and the phylogenetic tree constructed using single-copy orthologs, which also resulted in 11 ancestral chromosomes.\u003c/p\u003e\n \u003cp\u003eMoreover, to obtain a more complete ancestral karyotype gene set, we expanded protochromosomes based on five species (Clade II and \u003cem\u003eC. decandra\u003c/em\u003e) that had not undergone the second WGD and retained relatively complete ancestral karyotypes (\u003cstrong\u003eSupplementary Fig.\u0026nbsp;9b\u003c/strong\u003e). Specifically,the genomes were aligned to the initial protochromosomes to identify syntenic blocks. If five or fewer gene clusters surrounded by collinear genes on the chromosomes of an existing species corresponded to two ordered ancestral genes on the protochromosomes, these intermediate genes were added between the two ancestral genes to extend the protochromosomes\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e116\u003c/span\u003e\u003c/sup\u003e. This process resulted in 11 putative protochromosomes containing as many genes as possible. The 13 extant genomes were aligned with the expanded protochromosomes, and WGDI with the parameter \u0026lsquo;-km\u0026rsquo; was used to determine the karyotype composition from protochromosomes based on syntenic blocks, allowing the inference of chromosome fusion and evolutionary patterns (\u003cstrong\u003eSupplementary Fig.\u0026nbsp;10\u003c/strong\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003eIdentification of allopolyploidization and subgenomes\u003c/h2\u003e\n \u003cp\u003eWe followed the workflow in the \u003cstrong\u003eSupplementary Fig.\u0026nbsp;12\u003c/strong\u003e to identify polyploidy types and split subgenomes.\u003c/p\u003e\n \u003cp\u003e1) We performed synteny analysis of all 13 Salicaceae species with the reconstructed protochromosomes, and performed \u003cem\u003eK\u003c/em\u003e\u003csub\u003eS\u003c/sub\u003e calculation and information integration of collinearity fragments using the \u0026lsquo;-ks\u0026rsquo; and \u0026lsquo;-bi\u0026rsquo; programs of WGDI. Since the Clade I species experienced \u0026apos;salicoid\u0026apos; WGD, we first split the two homologous subgenomes of \u003cem\u003eP. alba\u003c/em\u003e var. \u003cem\u003epyramidalis\u003c/em\u003e corresponding to each protochromosomes according to the collinear fragment information, while the subgenomes of the remaining 7 Clade I species were split according to the collinear relationship with \u003cem\u003eP. alba\u003c/em\u003e var. \u003cem\u003epyramidalis\u003c/em\u003e. The split information was added to the integrated collinear fragment information file, and the homologous gene list between the protochromosomes and each species was obtained through the \u0026quot;-a\u0026quot; program. Finally, the homologous genes of all species were merged to obtain the 1:2 homologous gene groups (HGGs) among CladeII/\u003cem\u003eC. decandra\u003c/em\u003e and CladeI. We then classified the HGGs (C1-C12) based on the number of gene copies in each species, with Full-HGGs and Partial-HGGs used for subsequent analysis (\u003cstrong\u003eSupplementary Fig.\u0026nbsp;13\u003c/strong\u003e).\u003c/p\u003e\n \u003cp\u003e2) Gene trees were constructed for each Full and Partial-HGG using RAxML v8.2.11\u003csup\u003e105\u003c/sup\u003e with the two \u003cem\u003ePassiflora\u003c/em\u003e species as outgroups.\u003c/p\u003e\n \u003cp\u003e3) The gene trees were utilized by ASTRAL v.5.6.2\u003csup\u003e117\u003c/sup\u003e to infer species trees with quartet scores and posterior probabilities for each protochromosome. The polyploidization type was inferred by counting the proportion of different topologies of the gene tree of protochromosome. In addition, GRAMPA v1.4.0, a topology-based gene-tree reconciliation algorithm, was also used to infer the mode of polyploidization, and the optimal tree with the lowest score was consistent with the topology of the ASTRAL tree. Next, according to the topology of the gene trees, the gene closer to Clade II is classified as the A subgenome, and the other paralogous gene belongs to the B subgenome.\u003c/p\u003e\n \u003cp\u003e4) Genes from different subgenomes were mapped onto the chromosomes of their respective species, and the genome was split into A and B subgenomes based on the gene locations.\u003c/p\u003e\n \u003cp\u003e5\u0026ndash;6) The genes belonging to different subgenomes were concatenated, and the concatenated tree was constructed using RAxML v8.2.11\u003csup\u003e105\u003c/sup\u003e to further verify the polyploidization type. Divergence times were estimated using a phylogenetic tree constructed from full-HGGs and MCMCTree\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e106\u003c/span\u003e\u003c/sup\u003e in the PAML package, based on the divergence time between \u003cem\u003ePopulus\u003c/em\u003e and \u003cem\u003eSalix\u003c/em\u003e.\u003c/p\u003e\n \u003cp\u003e7) Finally, yn00 function in PAML\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e106\u003c/span\u003e\u003c/sup\u003e was used to calculate the \u003cem\u003eKs\u003c/em\u003e values between the A subgenome and Clade II/Casearia, as well as between the B subgenome and Clade II/Casearia, thereby obtaining the divergence levels.\u003c/p\u003e\n \u003cp\u003eBecause the Clade I species share \u0026apos;salicoid\u0026apos; WGD event, and genes from different subgenomes were identified in each species, we employed relative rate tests to estimate the evolutionary rate (\u003cem\u003eK\u003c/em\u003e\u003csub\u003eS\u003c/sub\u003e) after the recent WGD event for each species. Since \u003cem\u003eI. orientalis\u003c/em\u003e is at the base of the Clade I, we separately calculated the \u003cem\u003eK\u003c/em\u003e\u003csub\u003eS\u003c/sub\u003e values of the remaining seven species after they diverged from \u003cem\u003eI. orientalis\u003c/em\u003e based on the methods in previous studies\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e118\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003eGene retention and repeat sequence content of subgenomes\u003c/h2\u003e\n \u003cp\u003eBased on homologous genomic data from 13 species, we compared the genomic and subgenomic characteristics of these species. To quantify the gene retention in the Clade II species, as well as A and B subgenomes of Clade I species, we first selected HGGs that retained \u003cem\u003eC. decandra\u003c/em\u003e and then calculated the percentage of gene retention in these genomes, using non-overlapping windows of 100 genes along the protochromosomes. In addition, based on the results from RepeatMasker and RepeatModeler, we computed the repeat sequence content of genes and their surrounding 2k regions in all genomes. We further searched the genome of Clade I species using the LTRharvest\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e119\u003c/span\u003e\u003c/sup\u003e and LTRdigest\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e120\u003c/span\u003e\u003c/sup\u003e programs to \u003cem\u003ede novo\u003c/em\u003e detect intact LTRs according to the pipeline in previous studies\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e121\u003c/span\u003e\u003c/sup\u003e. The 5\u0026prime; and 3\u0026prime; repeats of each LTR were aligned by MUSCLE v3.8.31\u003csup\u003e122\u003c/sup\u003e to estimate the substitution rate, and insertion times were finally estimated by assuming a mutation rate of 2.5\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;9\u003c/sup\u003e per year\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e123\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n \u003ch2\u003eSequencing and analysis of transcriptome data\u003c/h2\u003e\n \u003cp\u003eTotal RNA was extracted from leaf, bud, shoot apex, flower and fruit tissues of Salicaceae species, respectively (\u003cstrong\u003eSupplementary Table\u0026nbsp;9\u003c/strong\u003e). The extracted RNA was purified using poly-T oligo-attached magnetic beads. All transcriptome libraries were constructed using the Illumina TruSeq library Stranded mRNA Prep Kit and sequenced on an Illumina HiSeq 2000 platform. Quality-filtered reads were aligned to their own genomes using HISAT2 v2.1.0\u003csup\u003e124\u003c/sup\u003e, and then the expression levels (TPM) for each gene were calculated and normalized by StringTie v1.3.3b\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e125\u003c/span\u003e\u003c/sup\u003e. Transcriptome data were used in the following studies: 1)The expression levels in mature leaves of 13 species were used to assess differences within the intraspecific subgenomes. 2) Interspecific expression divergence between A and B subgenomes during flower and fruit development. This involved selecting retained duplicated gene pairs shared by all four genera and analyzing tissues from different developmental stages of three species (\u003cem\u003eI. orientalis\u003c/em\u003e, \u003cem\u003eI. polycarpa\u003c/em\u003e, and \u003cem\u003eP. deltoides\u003c/em\u003e). 3) The dynamic expression changes of \u003cem\u003eFT1\u003c/em\u003e and \u003cem\u003eFT2\u003c/em\u003e were detected using the expression levels in leaves, buds and shoot apex of \u003cem\u003eI. orientalis\u003c/em\u003e and \u003cem\u003eI. polycarpa\u003c/em\u003e from different months.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n \u003ch2\u003eSequencing and analysis of epigenetic data\u003c/h2\u003e\n \u003cp\u003eATAC sample preparation from leaves was performed as described in a previous paper\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e126\u003c/span\u003e\u003c/sup\u003e. Vazyme TD501 manual was used to build the ATAC-seq library. For whole genome bisulfite sequencing (WGBS), genomic DNA was extracted from leaves with the DNeasy plant mini kit (Qiagen) and libraries were constructed following procedures described previously\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e127\u003c/span\u003e\u003c/sup\u003e. All libraries were sequenced on an Illumina HiSeq 2000 platform. Quality-filtered reads were aligned to the reference genome using Bowtie2 v2.4.1\u003csup\u003e128\u003c/sup\u003e (ATAC-seq) and Bismark v0.22.3\u003csup\u003e129\u003c/sup\u003e (WGBS), respectively. We extracted the 2k region upstream of each gene in 13 species and divided it into 20 bins. Subsequently, we counted the number of reads in each bin and normalized it to reads per bin per million mapped reads according to the RPKM method\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e130\u003c/span\u003e\u003c/sup\u003e. This was used to assess the chromatin accessibility of the genomes and subgenomes. Meanwhile, methylation levels of the gene body and the flanking 2k regions were determined by dividing the regions into 30 and 20 bins, respectively.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n \u003ch2\u003eGene Ontology (GO) enrichment analysis\u003c/h2\u003e\n \u003cp\u003eGene ontology (GO) enrichment analysis was performed on each group of genes using the enricher function in the \u0026quot;clusterProfiler\u0026quot; package\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e131\u003c/span\u003e\u003c/sup\u003e within R software. After p-value correction using the Benjamini-Hochberg method, terms with q-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were selected as the significant functions.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\n \u003ch2\u003eIdentification of conserved noncoding elements (CNEs)\u003c/h2\u003e\n \u003cp\u003eWe applied AVID v2.1\u003csup\u003e132\u003c/sup\u003e to perform alignments of Salicaceae species and detect \u003cem\u003ePopulus\u003c/em\u003e-\u003cem\u003eSalix\u003c/em\u003e specific CNEs using a 100 bp, 70% identity criterion. Finally, the alignments were visualized using VISTA\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e133\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\n \u003ch2\u003eDual-luciferase assay\u003c/h2\u003e\n \u003cp\u003eThe synthetic and cloned fragments of the four CNEs were fused with the 35S minimal promoter to drive the LUC expression as the reporters, and the 35S empty vector was used as a control. These constructs were transiently expressed in \u003cem\u003eN. benthamiana\u003c/em\u003e leaves. After incubation in the dark for 2 days and the light for 1 day, the enzyme mixture was prepared according to the manufacturer\u0026apos;s instructions in the Dual-Luciferase Reporter Assay System kit (Promega). \u003cem\u003eFirefly\u003c/em\u003e (\u003cem\u003eLUC\u003c/em\u003e) and \u003cem\u003eRenilla\u003c/em\u003e (internal control, \u003cem\u003eREN\u003c/em\u003e) luciferase signals were detected using a multimode reader (Synergy H1; BioTek, Winooski, VT). All primers used are listed in \u003cstrong\u003eSupplementary Table\u0026nbsp;11\u003c/strong\u003e.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll raw sequence data, genome assembly and annotation information have been deposited in the National Genome Data Center (NGDC; https://bigd.big.ac.cn/bioproject) under BioProject accession number PRJCA022976.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Key Research and Development Program of China (2021YFD2201100, 2021YFD2200202 and 2016YFD0600101), National Natural Science Foundation of China (31922061, 32271828 and 32071732), Fundamental Research Funds for the Central Universities (2020SCUNL207, SCU2021D006 and 2020SCUNL103), and the US National Science Foundation (1542599). We thank Susanne S. Renner (Department of Biology, Washington University, Saint Louis, USA.) for insightful comments. We thank Yanping Su, Fuchuan Wu (Center for Gardening and Horticulture, Xishuangbanna Tropical Botanical Garden, Chinese Academy of Science), and Lei Zhang (College of Biological Science \u0026amp; Engineering, North Minzu University, Yinchuan, China) for providing plant samples.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTM, JQL and MO led the project. TM, JQL, MO, DYW and ZXX conceived and designed the research. DYW, MML, WLY, KC, JLZ, LXS, PCS, LX, YLL, YC, JXX, YBW, HH, TNL and JLL performed data analysis. DYW, MML, ZQL and ZXX collected samples. YZJ and QJH provided comments for improving the manuscript. DYW and TM drafted the manuscript. JQL and MO edited the manuscript. All authors approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eLeebens-Mack, J.H. et al. One thousand plant transcriptomes and the phylogenomics of green plants. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e574\u003c/strong\u003e, 679-685 (2019).\u003c/li\u003e\n \u003cli\u003eJiao, Y. et al. Phylogenomic analysis reveals ancient genome duplications in seed plant and angiosperm history. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e473\u003c/strong\u003e, 97-100 (2011).\u003c/li\u003e\n \u003cli\u003eAlbert, V.A. et al. The \u003cem\u003eAmborella\u0026nbsp;\u003c/em\u003egenome and the evolution of flowering plants. \u003cem\u003eScience\u003c/em\u003e \u003cstrong\u003e342\u003c/strong\u003e, 1241089 (2013).\u003c/li\u003e\n \u003cli\u003eForce, A. et al. Preservation of duplicate genes by complementary, degenerative mutations. \u003cem\u003eGenetics\u003c/em\u003e \u003cstrong\u003e151\u003c/strong\u003e, 1531-1545 (1999).\u003c/li\u003e\n \u003cli\u003eAdams, K.L. \u0026amp; Wendel, J.F. Polyploidy and genome evolution in plants. \u003cem\u003eCurr. Opin. Plant Biol.\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, 135-141 (2005).\u003c/li\u003e\n \u003cli\u003eDoyle, J.J. et al. Evolutionary genetics of genome merger and doubling in plants. \u003cem\u003eAnnu. Rev. Genet.\u003c/em\u003e \u003cstrong\u003e42\u003c/strong\u003e, 443-461 (2008).\u003c/li\u003e\n \u003cli\u003eHegarty, M.J. \u0026amp; Hiscock, S.J. Genomic clues to the evolutionary success of polyploid plants. \u003cem\u003eCurr. Biol.\u003c/em\u003e \u003cstrong\u003e18\u003c/strong\u003e, R435-R444 (2008).\u003c/li\u003e\n \u003cli\u003eSoltis, P.S., Liu, X., Marchant, D.B., Visger, C.J. \u0026amp; Soltis, D.E. Polyploidy and novelty: Gottlieb\u0026apos;s legacy. \u003cem\u003ePhilos. Trans. R. Soc. Lond., B, Biol. Sci.\u003c/em\u003e \u003cstrong\u003e369\u003c/strong\u003e, 20130351 (2014).\u003c/li\u003e\n \u003cli\u003eSoltis, P.S., Marchant, D.B., Van de Peer, Y. \u0026amp; Soltis, D.E. Polyploidy and genome evolution in plants. \u003cem\u003eCurr. Opin. Genet. Dev.\u003c/em\u003e \u003cstrong\u003e35\u003c/strong\u003e, 119-125 (2015).\u003c/li\u003e\n \u003cli\u003eSoltis, P.S. \u0026amp; Soltis, D.E. Ancient WGD events as drivers of key innovations in angiosperms. \u003cem\u003eCurr. Opin. Plant Biol.\u003c/em\u003e \u003cstrong\u003e30\u003c/strong\u003e, 159-165 (2016).\u003c/li\u003e\n \u003cli\u003eVan de Peer, Y., Maere, S. \u0026amp; Meyer, A. The evolutionary significance of ancient genome duplications. \u003cem\u003eNat. Rev. Genet.\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, 725-732 (2009).\u003c/li\u003e\n \u003cli\u003eVan de Peer, Y., Mizrachi, E. \u0026amp; Marchal, K. The evolutionary significance of polyploidy. \u003cem\u003eNat. Rev. Genet.\u003c/em\u003e \u003cstrong\u003e18\u003c/strong\u003e, 411-424 (2017).\u003c/li\u003e\n \u003cli\u003eFawcett, J.A., Maere, S. \u0026amp; Van De Peer, Y. Plants with double genomes might have had a better chance to survive the Cretaceous-Tertiary extinction event. \u003cem\u003eProc. Natl Acad. Sci. USA\u003c/em\u003e \u003cstrong\u003e106\u003c/strong\u003e, 5737-5742 (2009).\u003c/li\u003e\n \u003cli\u003eVanneste, K., Maere, S. \u0026amp; Van de Peer, Y. Tangled up in two: a burst of genome duplications at the end of the Cretaceous and the consequences for plant evolution. \u003cem\u003ePhilos. Trans. R. Soc. Lond., B, Biol. Sci.\u003c/em\u003e \u003cstrong\u003e369\u003c/strong\u003e, 20130353 (2014).\u003c/li\u003e\n \u003cli\u003eWu, S., Han, B. \u0026amp; Jiao, Y. Genetic contribution of paleopolyploidy to adaptive evolution in angiosperms. \u003cem\u003eMol. Plant\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 59-71 (2020).\u003c/li\u003e\n \u003cli\u003eSchnable, J.C., Springer, N.M. \u0026amp; Freeling, M. Differentiation of the maize subgenomes by genome dominance and both ancient and ongoing gene loss. \u003cem\u003eProc. Natl Acad. Sci. USA\u003c/em\u003e \u003cstrong\u003e108\u003c/strong\u003e, 4069-4074 (2011).\u003c/li\u003e\n \u003cli\u003eWendel, J.F., Jackson, S.A., Meyers, B.C. \u0026amp; Wing, R.A. Evolution of plant genome architecture. \u003cem\u003eGenome Biol.\u003c/em\u003e \u003cstrong\u003e17\u003c/strong\u003e, 1-14 (2016).\u003c/li\u003e\n \u003cli\u003eBarker, M.S., Arrigo, N., Baniaga, A.E., Li, Z. \u0026amp; Levin, D.A. On the relative abundance of autopolyploids and allopolyploids. \u003cem\u003eNew Phytol.\u003c/em\u003e \u003cstrong\u003e210\u003c/strong\u003e, 391-398 (2016).\u003c/li\u003e\n \u003cli\u003eFowler, N.L. \u0026amp; Levin, D.A. Ecological constraints on the establishment of a novel polyploid in competition with its diploid progenitor. \u003cem\u003eAm. Nat.\u003c/em\u003e \u003cstrong\u003e124\u003c/strong\u003e, 703-711 (1984).\u003c/li\u003e\n \u003cli\u003eRamsey, J. Polyploidy and ecological adaptation in wild yarrow. \u003cem\u003eProc. Natl Acad. Sci. USA\u003c/em\u003e \u003cstrong\u003e108\u003c/strong\u003e, 7096-7101 (2011).\u003c/li\u003e\n \u003cli\u003eParisod, C. \u0026amp; Broennimann, O. Towards unified hypotheses of the impact of polyploidy on ecological niches. \u003cem\u003eNew Phytol.\u003c/em\u003e \u003cstrong\u003e212\u003c/strong\u003e, 540-542 (2016).\u003c/li\u003e\n \u003cli\u003eLuo, X. et al. Chasing ghosts: allopolyploid origin of \u003cem\u003eOxyria sinensis\u003c/em\u003e (Polygonaceae) from its only diploid congener and an unknown ancestor. \u003cem\u003eMol. Ecol.\u003c/em\u003e \u003cstrong\u003e26\u003c/strong\u003e, 3037-3049 (2017).\u003c/li\u003e\n \u003cli\u003eStull, G.W., Pham, K.K., Soltis, P.S. \u0026amp; Soltis, D.E. Deep reticulation: the long legacy of hybridization in vascular plant evolution. \u003cem\u003ePlant J.\u003c/em\u003e \u003cstrong\u003e114\u003c/strong\u003e, 743-766 (2023).\u003c/li\u003e\n \u003cli\u003eStettler, R.F. \u003cem\u003eBiology of Populus and its implications for management and conservation\u003c/em\u003e, (NRC Research Press, 1996).\u003c/li\u003e\n \u003cli\u003eChase, M.W. et al. When in doubt, put it in Flacourtiaceae: a molecular phylogenetic analysis based on plastid rbcL DNA sequences. \u003cem\u003eKew Bull.\u003c/em\u003e \u003cstrong\u003e57\u003c/strong\u003e, 141-181 (2002).\u003c/li\u003e\n \u003cli\u003eSanderson, B.J. et al. Phylogenomics reveals patterns of ancient hybridization and differential diversification that contribute to phylogenetic conflict in willows, poplars, and close relatives. \u003cem\u003eSyst. Biol.\u003c/em\u003e \u003cstrong\u003e72\u003c/strong\u003e, 1120-1232 (2023).\u003c/li\u003e\n \u003cli\u003eArgus, G.W. Infrageneric classification of \u003cem\u003eSalix\u003c/em\u003e (Salicaceae) in the new world. \u003cem\u003eSyst. Bot. Monogr.\u003c/em\u003e, 1-121 (1997).\u003c/li\u003e\n \u003cli\u003eTuo-Ya, D. Origin, divergence and geographical distribution of Salicaceae. \u003cem\u003ePlant Divers.\u003c/em\u003e \u003cstrong\u003e17\u003c/strong\u003e, 1 (1995).\u003c/li\u003e\n \u003cli\u003eCronk, Q.C., Needham, I. \u0026amp; Rudall, P.J. Evolution of catkins: inflorescence morphology of selected Salicaceae in an evolutionary and developmental context. \u003cem\u003eFront. Plant Sci.\u003c/em\u003e \u003cstrong\u003e6\u003c/strong\u003e, 1030 (2015).\u003c/li\u003e\n \u003cli\u003eLemke, D.E. A synopsis of Flacourtiaceae. \u003cem\u003eAliso\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, 29-43 (1988).\u003c/li\u003e\n \u003cli\u003eZhang, Z.-S., Zeng, Q.-Y. \u0026amp; Liu, Y.-J. Frequent ploidy changes in Salicaceae indicates widespread sharing of the salicoid whole genome duplication by the relatives of \u003cem\u003ePopulus\u0026nbsp;\u003c/em\u003eL. and \u003cem\u003eSalix\u0026nbsp;\u003c/em\u003eL. \u003cem\u003eBMC Plant Biol.\u003c/em\u003e \u003cstrong\u003e21\u003c/strong\u003e, 1-17 (2021).\u003c/li\u003e\n \u003cli\u003eSteyn, E., Smith, G. \u0026amp; Van Wyk, A. Functional and taxonomic significance of seed structure in \u003cem\u003eSalix mucronata\u0026nbsp;\u003c/em\u003e(Salicaceae). \u003cem\u003eBothalia\u003c/em\u003e \u003cstrong\u003e34\u003c/strong\u003e, 53-59 (2004).\u003c/li\u003e\n \u003cli\u003eCronk, Q. Plant eco‐devo: the potential of poplar as a model organism. \u003cem\u003eNew Phytol.\u003c/em\u003e \u003cstrong\u003e166\u003c/strong\u003e, 39-48 (2005).\u003c/li\u003e\n \u003cli\u003eJansson, S. \u0026amp; Douglas, C.J. \u003cem\u003ePopulus\u003c/em\u003e: a model system for plant biology. \u003cem\u003eAnnu. Rev. Plant Biol.\u003c/em\u003e \u003cstrong\u003e58\u003c/strong\u003e, 435-458 (2007).\u003c/li\u003e\n \u003cli\u003eTuskan, G.A. et al. The genome of black cottonwood, \u003cem\u003ePopulus trichocarp\u003c/em\u003ea (Torr. \u0026amp; Gray). \u003cem\u003eScience\u003c/em\u003e \u003cstrong\u003e313\u003c/strong\u003e, 1596-1604 (2006).\u003c/li\u003e\n \u003cli\u003eDai, X. et al. The willow genome and divergent evolution from poplar after the common genome duplication. \u003cem\u003eCell Res.\u003c/em\u003e \u003cstrong\u003e24\u003c/strong\u003e, 1274-1277 (2014).\u003c/li\u003e\n \u003cli\u003eRodgers-Melnick, E. et al. Contrasting patterns of evolution following whole genome versus tandem duplication events in \u003cem\u003ePopulus\u003c/em\u003e. \u003cem\u003eGenome Res.\u003c/em\u003e \u003cstrong\u003e22\u003c/strong\u003e, 95-105 (2012).\u003c/li\u003e\n \u003cli\u003eLiu, Y. et al. Two highly similar poplar paleo-subgenomes suggest an autotetraploid ancestor of Salicaceae plants. \u003cem\u003eFront. Plant Sci.\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, 252555 (2017).\u003c/li\u003e\n \u003cli\u003eLi, M. et al. Intergeneric relationships within the family Salicaceae sl based on plastid phylogenomics. \u003cem\u003eInt. J. Mol. Sci.\u003c/em\u003e \u003cstrong\u003e20\u003c/strong\u003e, 3788 (2019).\u003c/li\u003e\n \u003cli\u003eShang, C., Liao, S., Guo, Y.J. \u0026amp; Zhang, Z.X. \u003cem\u003eDianyuea\u003c/em\u003e gen. nov.(Salicaceae: Scyphostegioideae) from southwestern China. \u003cem\u003eNord. J. Bot.\u003c/em\u003e \u003cstrong\u003e35\u003c/strong\u003e, 499-505 (2017).\u003c/li\u003e\n \u003cli\u003eCheng, F. et al. Gene retention, fractionation and subgenome differences in polyploid plants. \u003cem\u003eNat. Plants\u003c/em\u003e \u003cstrong\u003e4\u003c/strong\u003e, 258-268 (2018).\u003c/li\u003e\n \u003cli\u003eKardailsky, I. et al. Activation tagging of the floral inducer \u003cem\u003eFT\u003c/em\u003e. \u003cem\u003eScience\u003c/em\u003e \u003cstrong\u003e286\u003c/strong\u003e, 1962-1965 (1999).\u003c/li\u003e\n \u003cli\u003eKobayashi, Y., Kaya, H., Goto, K., Iwabuchi, M. \u0026amp; Araki, T. A pair of related genes with antagonistic roles in mediating flowering signals. \u003cem\u003eScience\u003c/em\u003e \u003cstrong\u003e286\u003c/strong\u003e, 1960-1962 (1999).\u003c/li\u003e\n \u003cli\u003eLiu, H. et al. Photoexcited CRY2 interacts with CIB1 to regulate transcription and floral initiation in \u003cem\u003eArabidopsis\u003c/em\u003e. \u003cem\u003escience\u003c/em\u003e \u003cstrong\u003e322\u003c/strong\u003e, 1535-1539 (2008).\u003c/li\u003e\n \u003cli\u003eDoyle, M.R. et al. \u003cem\u003eHUA2\u003c/em\u003e is required for the expression of floral repressors in Arabidopsis thaliana. \u003cem\u003ePlant J.\u003c/em\u003e \u003cstrong\u003e41\u003c/strong\u003e, 376-385 (2005).\u003c/li\u003e\n \u003cli\u003eKim, S.H. et al. Phosphorylation of the transcriptional repressor MYB15 by mitogen-activated protein kinase 6 is required for freezing tolerance in \u003cem\u003eArabidopsis\u003c/em\u003e. \u003cem\u003eNucleic Acids Res.\u003c/em\u003e \u003cstrong\u003e45\u003c/strong\u003e, 6613-6627 (2017).\u003c/li\u003e\n \u003cli\u003eZhang, P. et al. \u003cem\u003eArabidopsis ADF5\u0026nbsp;\u003c/em\u003eacts as a downstream target gene of CBFs in response to low-temperature stress. \u003cem\u003eFront. Cell Dev. Biol.\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, 635533 (2021).\u003c/li\u003e\n \u003cli\u003eTakahashi, D. et al. Structural changes in cell wall pectic polymers contribute to freezing tolerance induced by cold acclimation in plants. \u003cem\u003eCurr. Biol.\u003c/em\u003e \u003cstrong\u003e34\u003c/strong\u003e, 958-968.e5 (2024).\u003c/li\u003e\n \u003cli\u003eSun, L. et al. \u003cem\u003eGLABROUS INFLORESCENCE STEMS3\u0026nbsp;\u003c/em\u003e(\u003cem\u003eGIS3\u003c/em\u003e) regulates trichome initiation and development in \u003cem\u003eArabidopsis\u003c/em\u003e. \u003cem\u003eNew Phytol.\u003c/em\u003e \u003cstrong\u003e206\u003c/strong\u003e, 220-230 (2015).\u003c/li\u003e\n \u003cli\u003eZhou, Z. et al. \u003cem\u003eZinc Finger Protein 6\u0026nbsp;\u003c/em\u003e(\u003cem\u003eZFP6\u003c/em\u003e) regulates trichome initiation by integrating gibberellin and cytokinin signaling in\u003cem\u003e\u0026nbsp;Arabidopsis thaliana\u003c/em\u003e. \u003cem\u003eNew Phytol.\u003c/em\u003e \u003cstrong\u003e198\u003c/strong\u003e, 699-708 (2013).\u003c/li\u003e\n \u003cli\u003eLiu, Y., Yang, S., Khan, A.R. \u0026amp; Gan, Y. TOE1/TOE2 interacting with GIS to control trichome development in \u003cem\u003eArabidopsis\u003c/em\u003e. \u003cem\u003eInt. J. Mol. Sci.\u003c/em\u003e \u003cstrong\u003e24\u003c/strong\u003e, 6698 (2023).\u003c/li\u003e\n \u003cli\u003eZhu, Y. et al. TERMINAL FLOWER 1-FD complex target genes and competition with FLOWERING LOCUS T. \u003cem\u003eNat. Commun.\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 5118 (2020).\u003c/li\u003e\n \u003cli\u003eKumimoto, R.W. et al. The Nuclear Factor Y subunits NF-YB2 and NF-YB3 play additive roles in the promotion of flowering by inductive long-day photoperiods in \u003cem\u003eArabidopsis\u003c/em\u003e. \u003cem\u003ePlanta\u003c/em\u003e \u003cstrong\u003e228\u003c/strong\u003e, 709-723 (2008).\u003c/li\u003e\n \u003cli\u003eJung, J.-H., Lee, H.-J., Ryu, J.Y. \u0026amp; Park, C.-M. SPL3/4/5 integrate developmental aging and photoperiodic signals into the FT-FD module in Arabidopsis flowering. \u003cem\u003eMol. Plant\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, 1647-1659 (2016).\u003c/li\u003e\n \u003cli\u003eSong, H.-R. et al. The RNA binding protein ELF9 directly reduces \u003cem\u003eSUPPRESSOR OF OVEREXPRESSION OF CO1\u003c/em\u003e transcript levels in \u003cem\u003eArabidopsis\u003c/em\u003e, possibly via nonsense-mediated mRNA decay. \u003cem\u003ePlant Cell\u003c/em\u003e \u003cstrong\u003e21\u003c/strong\u003e, 1195-1211 (2009).\u003c/li\u003e\n \u003cli\u003eZhang, Z. et al. Convergence of the 26S proteasome and the \u003cem\u003eREVOLUTA\u0026nbsp;\u003c/em\u003epathways in regulating inflorescence and floral meristem functions in \u003cem\u003eArabidopsis\u003c/em\u003e. \u003cem\u003eJ. Exp. Bot.\u003c/em\u003e \u003cstrong\u003e62\u003c/strong\u003e, 359-369 (2011).\u003c/li\u003e\n \u003cli\u003eYoo, C.-M., Quan, L. \u0026amp; Blancaflor, E.B. Divergence and redundancy in \u003cem\u003eCSLD2\u0026nbsp;\u003c/em\u003eand \u003cem\u003eCSLD3\u0026nbsp;\u003c/em\u003efunction during \u003cem\u003eArabidopsis thaliana\u0026nbsp;\u003c/em\u003eroot hair and female gametophyte development. \u003cem\u003eFront. Plant Sci.\u003c/em\u003e \u003cstrong\u003e3\u003c/strong\u003e, 111 (2012).\u003c/li\u003e\n \u003cli\u003eThroude, M. et al. Structure and expression analysis of rice paleo duplications. \u003cem\u003eNucleic Acids Res.\u003c/em\u003e \u003cstrong\u003e37\u003c/strong\u003e, 1248-1259 (2009).\u003c/li\u003e\n \u003cli\u003eHuminiecki, L. \u0026amp; Conant, G.C. Polyploidy and the evolution of complex traits. \u003cem\u003eInt. J. Evol. Biol.\u003c/em\u003e \u003cstrong\u003e2012\u003c/strong\u003e, 292068 (2012).\u003c/li\u003e\n \u003cli\u003eHsu, C.-Y. et al. \u003cem\u003eFLOWERING LOCUS T\u003c/em\u003e duplication coordinates reproductive and vegetative growth in perennial poplar. \u003cem\u003eProc. Natl Acad. Sci. USA\u003c/em\u003e \u003cstrong\u003e108\u003c/strong\u003e, 10756-10761 (2011).\u003c/li\u003e\n \u003cli\u003eYu, C.-W. et al. HISTONE DEACETYLASE6 interacts with FLOWERING LOCUS D and regulates flowering in Arabidopsis. \u003cem\u003ePlant Physiol.\u003c/em\u003e \u003cstrong\u003e156\u003c/strong\u003e, 173-184 (2011).\u003c/li\u003e\n \u003cli\u003eBertran Garcia de Olalla, E. et al. Coordination of shoot apical meristem shape and identity by APETALA2 during floral transition in Arabidopsis. \u003cem\u003eNat. Commun.\u003c/em\u003e \u003cstrong\u003e15\u003c/strong\u003e, 6930 (2024).\u003c/li\u003e\n \u003cli\u003eZhao, X., Heng, Y., Wang, X., Deng, X.W. \u0026amp; Xu, D. A positive feedback loop of BBX11\u0026ndash;BBX21\u0026ndash;HY5 promotes photomorphogenic development in \u003cem\u003eArabidopsis\u003c/em\u003e. \u003cem\u003ePlant Commun.\u003c/em\u003e \u003cstrong\u003e1\u003c/strong\u003e(2020).\u003c/li\u003e\n \u003cli\u003eLi, P., Li, X. \u0026amp; Jiang, M. CRISPR/Cas9-mediated mutagenesis of \u003cem\u003eWRKY3\u0026nbsp;\u003c/em\u003eand \u003cem\u003eWRKY4\u0026nbsp;\u003c/em\u003efunction decreases salt and Me-JA stress tolerance in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e. \u003cem\u003eMol. Biol. Rep.\u003c/em\u003e \u003cstrong\u003e48\u003c/strong\u003e, 5821-5832 (2021).\u003c/li\u003e\n \u003cli\u003eAndr\u0026eacute;, D. et al. \u003cem\u003eFLOWERING LOCUS T\u0026nbsp;\u003c/em\u003eparalogs control the annual growth cycle in \u003cem\u003ePopulus\u0026nbsp;\u003c/em\u003etrees. \u003cem\u003eCurr. Biol.\u003c/em\u003e \u003cstrong\u003e32\u003c/strong\u003e, 2988-2996. e4 (2022).\u003c/li\u003e\n \u003cli\u003eSawa, S., Ito, T., Shimura, Y. \u0026amp; Okada, K. \u003cem\u003eFILAMENTOUS FLOWER\u003c/em\u003e controls the formation and development of Arabidopsis inflorescences and floral meristems. \u003cem\u003ePlant Cell\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 69-86 (1999).\u003c/li\u003e\n \u003cli\u003eLi, W. et al. The U-Box/ARM E3 ligase PUB13 regulates cell death, defense, and flowering time in \u003cem\u003eArabidopsis\u003c/em\u003e. \u003cem\u003ePlant Physiol.\u003c/em\u003e \u003cstrong\u003e159\u003c/strong\u003e, 239-250 (2012).\u003c/li\u003e\n \u003cli\u003eZhang, C. et al. Gibberellin signaling modulates flowering via the DELLA\u0026ndash;BRAHMA\u0026ndash;NF-YC module in Arabidopsis. \u003cem\u003ePlant Cell\u003c/em\u003e \u003cstrong\u003e35\u003c/strong\u003e, 3470-3484 (2023).\u003c/li\u003e\n \u003cli\u003eLiu, Z. et al. \u003cem\u003eENO2\u0026nbsp;\u003c/em\u003eaffects the seed size and weight by adjusting cytokinin content and forming ENO2-bZIP75 complex in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e. \u003cem\u003eFront. Plant Sci.\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 574316 (2020).\u003c/li\u003e\n \u003cli\u003eDi Marzo, M. et al. Cell wall modifications by \u0026alpha;-XYLOSIDASE1 are required for control of seed and fruit size in Arabidopsis. \u003cem\u003eJ. Exp. Bot.\u003c/em\u003e \u003cstrong\u003e73\u003c/strong\u003e, 1499-1515 (2021).\u003c/li\u003e\n \u003cli\u003eSun, P. et al. Subgenome-aware analyses reveal the genomic consequences of ancient allopolyploid hybridizations throughout the cotton family. \u003cem\u003eProc. Natl Acad. Sci. USA\u003c/em\u003e \u003cstrong\u003e121\u003c/strong\u003e, e2313921121 (2024).\u003c/li\u003e\n \u003cli\u003eCai, X. et al. Impacts of allopolyploidization and structural variation on intraspecific diversification in\u003cem\u003e\u0026nbsp;Brassica rapa\u003c/em\u003e. \u003cem\u003eGenome Biol.\u003c/em\u003e \u003cstrong\u003e22\u003c/strong\u003e, 166 (2021).\u003c/li\u003e\n \u003cli\u003eMarcussen, T. et al. Ancient hybridizations among the ancestral genomes of bread wheat. \u003cem\u003eScience\u003c/em\u003e \u003cstrong\u003e345\u003c/strong\u003e, 1250092 (2014).\u003c/li\u003e\n \u003cli\u003eRam\u0026iacute;rez-Gonz\u0026aacute;lez, R. et al. The transcriptional landscape of polyploid wheat. \u003cem\u003eScience\u003c/em\u003e \u003cstrong\u003e361\u003c/strong\u003e, eaar6089 (2018).\u003c/li\u003e\n \u003cli\u003eWang, J. et al. A common whole-genome paleotetraploidization in Cucurbitales. \u003cem\u003ePlant Physiol.\u003c/em\u003e \u003cstrong\u003e190\u003c/strong\u003e, 2430-2448 (2022).\u003c/li\u003e\n \u003cli\u003eZhang, L. et al. Bioinformatic analysis of chromatin organization and biased expression of duplicated genes between two poplars with a common whole-genome duplication. \u003cem\u003eHortic. Res.\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, 62 (2021).\u003c/li\u003e\n \u003cli\u003eLi, H. Minimap2: pairwise alignment for nucleotide sequences. \u003cem\u003eBioinformatics\u003c/em\u003e \u003cstrong\u003e34\u003c/strong\u003e, 3094-3100 (2018).\u003c/li\u003e\n \u003cli\u003eLi, H. \u0026amp; Durbin, R. Fast and accurate short read alignment with Burrows\u0026ndash;Wheeler transform. \u003cem\u003eBioinformatics\u003c/em\u003e \u003cstrong\u003e25\u003c/strong\u003e, 1754-1760 (2009).\u003c/li\u003e\n \u003cli\u003eBurton, J.N. et al. Chromosome-scale scaffolding of de novo genome assemblies based on chromatin interactions. \u003cem\u003eNat. Biotechnol.\u003c/em\u003e \u003cstrong\u003e31\u003c/strong\u003e, 1119-1125 (2013).\u003c/li\u003e\n \u003cli\u003eKajitani, R. et al. Efficient de novo assembly of highly heterozygous genomes from whole-genome shotgun short reads. \u003cem\u003eGenome Res.\u003c/em\u003e \u003cstrong\u003e24\u003c/strong\u003e, 1384-1395 (2014).\u003c/li\u003e\n \u003cli\u003eCheng, H., Concepcion, G.T., Feng, X., Zhang, H. \u0026amp; Li, H. Haplotype-resolved de novo assembly using phased assembly graphs with hifiasm. \u003cem\u003eNat. Methods\u003c/em\u003e \u003cstrong\u003e18\u003c/strong\u003e, 170-175 (2021).\u003c/li\u003e\n \u003cli\u003eChen, N. Using Repeat Masker to identify repetitive elements in genomic sequences. \u003cem\u003eCurr. Protoc. Bioinf.\u003c/em\u003e \u003cstrong\u003e5\u003c/strong\u003e, 4.10. 1-4.10. 14 (2004).\u003c/li\u003e\n \u003cli\u003eBao, W., Kojima, K.K. \u0026amp; Kohany, O. Repbase Update, a database of repetitive elements in eukaryotic genomes. \u003cem\u003eMob. DNA\u003c/em\u003e \u003cstrong\u003e6\u003c/strong\u003e, 1-6 (2015).\u003c/li\u003e\n \u003cli\u003ePrice, A.L., Jones, N.C. \u0026amp; Pevzner, P.A. De novo identification of repeat families in large genomes. \u003cem\u003eBioinformatics\u003c/em\u003e \u003cstrong\u003e21\u003c/strong\u003e, i351-i358 (2005).\u003c/li\u003e\n \u003cli\u003eStanke, M. et al. AUGUSTUS: ab initio prediction of alternative transcripts. \u003cem\u003eNucleic Acids Res.\u003c/em\u003e \u003cstrong\u003e34\u003c/strong\u003e, W435-W439 (2006).\u003c/li\u003e\n \u003cli\u003eYang, W. et al. The draft genome sequence of a desert tree\u003cem\u003e\u0026nbsp;Populus pruinosa\u003c/em\u003e. \u003cem\u003eGigascience\u003c/em\u003e \u003cstrong\u003e6\u003c/strong\u003e, gix075 (2017).\u003c/li\u003e\n \u003cli\u003eMa, J. et al. Genome sequence and genetic transformation of a widely distributed and cultivated poplar. \u003cem\u003ePlant Biotechnol. J.\u003c/em\u003e \u003cstrong\u003e17\u003c/strong\u003e, 451-460 (2019).\u003c/li\u003e\n \u003cli\u003eZhou, R. et al. Characterization of a large sex determination region in\u003cem\u003e\u0026nbsp;Salix purpurea\u003c/em\u003e L.(Salicaceae). \u003cem\u003eMol. Genet. Genomics\u003c/em\u003e \u003cstrong\u003e293\u003c/strong\u003e, 1437-1452 (2018).\u003c/li\u003e\n \u003cli\u003eIniative, A.G. Analysis of the genome sequence of the flowering plant \u003cem\u003eArabidopsis thaliana\u003c/em\u003e. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e408\u003c/strong\u003e, 796-815 (2000).\u003c/li\u003e\n \u003cli\u003eCamacho, C. et al. BLAST+: architecture and applications. \u003cem\u003eBMC Bioinformatics\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, 1-9 (2009).\u003c/li\u003e\n \u003cli\u003eBirney, E., Clamp, M. \u0026amp; Durbin, R. GeneWise and genomewise. \u003cem\u003eGenome Res.\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, 988-995 (2004).\u003c/li\u003e\n \u003cli\u003eCampbell, M.A., Haas, B.J., Hamilton, J.P., Mount, S.M. \u0026amp; Buell, C.R. Comprehensive analysis of alternative splicing in rice and comparative analyses with \u003cem\u003eArabidopsis\u003c/em\u003e. \u003cem\u003eBMC Genomics\u003c/em\u003e \u003cstrong\u003e7\u003c/strong\u003e, 1-17 (2006).\u003c/li\u003e\n \u003cli\u003eHaas, B.J. et al. Automated eukaryotic gene structure annotation using EVidenceModeler and the Program to Assemble Spliced Alignments. \u003cem\u003eGenome Biol.\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, 1-22 (2008).\u003c/li\u003e\n \u003cli\u003eSim\u0026atilde;o, F.A., Waterhouse, R.M., Ioannidis, P., Kriventseva, E.V. \u0026amp; Zdobnov, E.M. BUSCO: assessing genome assembly and annotation completeness with single-copy orthologs. \u003cem\u003eBioinformatics\u003c/em\u003e \u003cstrong\u003e31\u003c/strong\u003e, 3210-3212 (2015).\u003c/li\u003e\n \u003cli\u003eZhang, Z. et al. Improved genome assembly provides new insights into genome evolution in a desert poplar (\u003cem\u003ePopulus euphratica\u003c/em\u003e). \u003cem\u003eMol. Ecol. Resour.\u003c/em\u003e \u003cstrong\u003e20\u003c/strong\u003e, 781-794 (2020).\u003c/li\u003e\n \u003cli\u003eWang, D. et al. Repeated turnovers keep sex chromosomes young in willows. \u003cem\u003eGenome Biol.\u003c/em\u003e \u003cstrong\u003e23\u003c/strong\u003e, 1-23 (2022).\u003c/li\u003e\n \u003cli\u003eZhou, R. et al. A willow sex chromosome reveals convergent evolution of complex palindromic repeats. \u003cem\u003eGenome Biol.\u003c/em\u003e \u003cstrong\u003e21\u003c/strong\u003e, 1-19 (2020).\u003c/li\u003e\n \u003cli\u003eMa, D. et al. Chromosome‐level reference genome assembly provides insights into aroma biosynthesis in passion fruit (\u003cem\u003ePassiflora edulis\u003c/em\u003e). \u003cem\u003eMol. Ecol. Resour.\u003c/em\u003e \u003cstrong\u003e21\u003c/strong\u003e, 955-968 (2021).\u003c/li\u003e\n \u003cli\u003eCosta, Z.P. et al. A genome sequence resource for the genus \u003cem\u003ePassiflora\u003c/em\u003e, the genome of the wild diploid species \u003cem\u003ePassiflora organensis\u003c/em\u003e. \u003cem\u003ePlant Genome\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, e20117 (2021).\u003c/li\u003e\n \u003cli\u003eRaymond, O. et al. The \u003cem\u003eRosa\u0026nbsp;\u003c/em\u003egenome provides new insights into the domestication of modern roses. \u003cem\u003eNat. Genet.\u003c/em\u003e \u003cstrong\u003e50\u003c/strong\u003e, 772 - 777 (2018).\u003c/li\u003e\n \u003cli\u003eShi, X. et al. The complete reference genome for grapevine (\u003cem\u003eVitis vinifera\u003c/em\u003e L.) genetics and breeding. \u003cem\u003eHortic. Res.\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e(2023).\u003c/li\u003e\n \u003cli\u003eEmms, D.M. \u0026amp; Kelly, S. OrthoFinder: solving fundamental biases in whole genome comparisons dramatically improves orthogroup inference accuracy. \u003cem\u003eGenome Biol.\u003c/em\u003e \u003cstrong\u003e16\u003c/strong\u003e, 1-14 (2015).\u003c/li\u003e\n \u003cli\u003eKatoh, K., Misawa, K., Kuma, K.i. \u0026amp; Miyata, T. MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform. \u003cem\u003eNucleic Acids Res.\u003c/em\u003e \u003cstrong\u003e30\u003c/strong\u003e, 3059-3066 (2002).\u003c/li\u003e\n \u003cli\u003eSuyama, M., Torrents, D. \u0026amp; Bork, P. PAL2NAL: robust conversion of protein sequence alignments into the corresponding codon alignments. \u003cem\u003eNucleic Acids Res.\u003c/em\u003e \u003cstrong\u003e34\u003c/strong\u003e, W609-W612 (2006).\u003c/li\u003e\n \u003cli\u003eStamatakis, A. RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. \u003cem\u003eBioinformatics\u003c/em\u003e \u003cstrong\u003e30\u003c/strong\u003e, 1312-1313 (2014).\u003c/li\u003e\n \u003cli\u003eYang, Z. PAML 4: phylogenetic analysis by maximum likelihood. \u003cem\u003eMol. Biol. Evol.\u003c/em\u003e \u003cstrong\u003e24\u003c/strong\u003e, 1586-1591 (2007).\u003c/li\u003e\n \u003cli\u003eAltschul, S.F., Gish, W., Miller, W., Myers, E.W. \u0026amp; Lipman, D.J. Basic local alignment search tool. \u003cem\u003eJ. Mol. Biol.\u003c/em\u003e \u003cstrong\u003e215\u003c/strong\u003e, 403-410 (1990).\u003c/li\u003e\n \u003cli\u003eSun, P. et al. WGDI: A user-friendly toolkit for evolutionary analyses of whole-genome duplications and ancestral karyotypes. \u003cem\u003eMol. Plant\u003c/em\u003e \u003cstrong\u003e15\u003c/strong\u003e, 1841-1851 (2022).\u003c/li\u003e\n \u003cli\u003eBrown, J.L. SDM toolbox: a python‐based GIS toolkit for landscape genetic, biogeographic and species distribution model analyses. \u003cem\u003eMethods Ecol. Evol.\u003c/em\u003e \u003cstrong\u003e5\u003c/strong\u003e, 694-700 (2014).\u003c/li\u003e\n \u003cli\u003ePhillips, S.J., Anderson, R.P. \u0026amp; Schapire, R.E. Maximum entropy modeling of species geographic distributions. \u003cem\u003eEcol. Model.\u003c/em\u003e \u003cstrong\u003e190\u003c/strong\u003e, 231-259 (2006).\u003c/li\u003e\n \u003cli\u003eFielding, A.H. \u0026amp; Bell, J.F. A review of methods for the assessment of prediction errors in conservation presence/absence models. \u003cem\u003eEnviron. Conserv.\u003c/em\u003e \u003cstrong\u003e24\u003c/strong\u003e, 38-49 (1997).\u003c/li\u003e\n \u003cli\u003eWarren, D.L. et al. ENMTools 1.0: An R package for comparative ecological biogeography. \u003cem\u003eEcography\u003c/em\u003e \u003cstrong\u003e44\u003c/strong\u003e, 504-511 (2021).\u003c/li\u003e\n \u003cli\u003eSchoener, T.W. The Anolis lizards of Bimini: resource partitioning in a complex fauna. \u003cem\u003eEcology\u003c/em\u003e \u003cstrong\u003e49\u003c/strong\u003e, 704-726 (1968).\u003c/li\u003e\n \u003cli\u003eWarren, D.L., Glor, R.E. \u0026amp; Turelli, M. Environmental niche equivalency versus conservatism: quantitative approaches to niche evolution. \u003cem\u003eEvolution\u003c/em\u003e \u003cstrong\u003e62\u003c/strong\u003e, 2868-2883 (2008).\u003c/li\u003e\n \u003cli\u003eHu, F., Lin, Y. \u0026amp; Tang, J. MLGO: phylogeny reconstruction and ancestral inference from gene-order data. \u003cem\u003eBMC Bioinformatics\u003c/em\u003e \u003cstrong\u003e15\u003c/strong\u003e, 1-6 (2014).\u003c/li\u003e\n \u003cli\u003eSun, P. et al. Early diversification and karyotype evolution of flowering plants. (2022).\u003c/li\u003e\n \u003cli\u003eZhang, C., Rabiee, M., Sayyari, E. \u0026amp; Mirarab, S. ASTRAL-III: polynomial time species tree reconstruction from partially resolved gene trees. \u003cem\u003eBMC Bioinformatics\u003c/em\u003e \u003cstrong\u003e19\u003c/strong\u003e, 15-30 (2018).\u003c/li\u003e\n \u003cli\u003eDing, Y. et al. Genome structure-based Juglandaceae phylogenies contradict alignment-based phylogenies and substitution rates vary with DNA repair genes. \u003cem\u003eNat. Commun.\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, 617 (2023).\u003c/li\u003e\n \u003cli\u003eEllinghaus, D., Kurtz, S. \u0026amp; Willhoeft, U. LTRharvest, an efficient and flexible software for de novo detection of LTR retrotransposons. \u003cem\u003eBMC Bioinformatics\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, 1-14 (2008).\u003c/li\u003e\n \u003cli\u003eSteinbiss, S., Willhoeft, U., Gremme, G. \u0026amp; Kurtz, S. Fine-grained annotation and classification of de novo predicted LTR retrotransposons. \u003cem\u003eNucleic Acids Res.\u003c/em\u003e \u003cstrong\u003e37\u003c/strong\u003e, 7002-7013 (2009).\u003c/li\u003e\n \u003cli\u003eZhou, S. et al. A comprehensive annotation dataset of intact LTR retrotransposons of 300 plant genomes. \u003cem\u003eSci. Data\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, 174 (2021).\u003c/li\u003e\n \u003cli\u003eEdgar, R.C. MUSCLE: multiple sequence alignment with high accuracy and high throughput. \u003cem\u003eNucleic Acids Res.\u003c/em\u003e \u003cstrong\u003e32\u003c/strong\u003e, 1792-1797 (2004).\u003c/li\u003e\n \u003cli\u003eIngvarsson, P.K. Multilocus patterns of nucleotide polymorphism and the demographic history of\u003cem\u003e\u0026nbsp;Populus tremula\u003c/em\u003e. \u003cem\u003eGenetics\u003c/em\u003e \u003cstrong\u003e180\u003c/strong\u003e, 329-340 (2008).\u003c/li\u003e\n \u003cli\u003eKim, D., Langmead, B. \u0026amp; Salzberg, S.L. HISAT: a fast spliced aligner with low memory requirements. \u003cem\u003eNat. Methods\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, 357-360 (2015).\u003c/li\u003e\n \u003cli\u003ePertea, M. et al. StringTie enables improved reconstruction of a transcriptome from RNA-seq reads. \u003cem\u003eNat. Biotechnol.\u003c/em\u003e \u003cstrong\u003e33\u003c/strong\u003e, 290-295 (2015).\u003c/li\u003e\n \u003cli\u003eBajic, M., Maher, K.A. \u0026amp; Deal, R.B. Identification of open chromatin regions in plant genomes using ATAC-Seq. \u003cem\u003eMethods Mol. Biol.\u003c/em\u003e, 183-201 (2018).\u003c/li\u003e\n \u003cli\u003eSu, Y. et al. Single-base-resolution methylomes of\u003cem\u003e\u0026nbsp;Populus euphratica\u0026nbsp;\u003c/em\u003ereveal the association between DNA methylation and salt stress. \u003cem\u003eTree Genet. Genom.\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, 1-11 (2018).\u003c/li\u003e\n \u003cli\u003eLangmead, B. \u0026amp; Salzberg, S.L. Fast gapped-read alignment with Bowtie 2. \u003cem\u003eNat. Methods\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, 357-359 (2012).\u003c/li\u003e\n \u003cli\u003eKrueger, F. \u0026amp; Andrews, S.R. Bismark: a flexible aligner and methylation caller for Bisulfite-Seq applications. \u003cem\u003eBioinformatics\u003c/em\u003e \u003cstrong\u003e27\u003c/strong\u003e, 1571-1572 (2011).\u003c/li\u003e\n \u003cli\u003eMortazavi, A., Williams, B.A., McCue, K., Schaeffer, L. \u0026amp; Wold, B. Mapping and quantifying mammalian transcriptomes by RNA-Seq. \u003cem\u003eNat. Methods\u003c/em\u003e \u003cstrong\u003e5\u003c/strong\u003e, 621-628 (2008).\u003c/li\u003e\n \u003cli\u003eYu, G., Wang, L.-G., Han, Y. \u0026amp; He, Q.-Y. clusterProfiler: an R package for comparing biological themes among gene clusters. \u003cem\u003eOMICS: J. Integrative Biol.\u003c/em\u003e \u003cstrong\u003e16\u003c/strong\u003e, 284-287 (2012).\u003c/li\u003e\n \u003cli\u003eBray, N., Dubchak, I. \u0026amp; Pachter, L. AVID: A global alignment program. \u003cem\u003eGenome Res.\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 97-102 (2003).\u003c/li\u003e\n \u003cli\u003eFrazer, K.A., Pachter, L., Poliakov, A., Rubin, E.M. \u0026amp; Dubchak, I. VISTA: computational tools for comparative genomics. \u003cem\u003eNucleic Acids Res.\u003c/em\u003e \u003cstrong\u003e32\u003c/strong\u003e, W273-W279 (2004).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-5852798/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5852798/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAllopolyploidy involves the fusion of genomes from different lineages through hybridization and chromosome doubling. However, detecting early allopolyploidy events in evolutionary history and understanding the specific subgenomic evolution that contributes to the origin of adaptive innovations for species radiation can be challenging. Here, we sequenced the genomes representing all three subfamilies of Salicaceae, a woody model clade, and collected epigenetic and transcriptomic samples. We revealed one shared ancient allopolyploidy event involving \u003cem\u003ePopulus,\u003c/em\u003eS\u003cem\u003ealix\u003c/em\u003e and two sister genera, but followed by contrasted karyotypic and subgenomic evolution. The specific evolution drove the origin of unique photoperiod adaptation, flowering phenology and small, hairy seeds in the highly speciose \u003cem\u003ePopulus\u003c/em\u003e and \u003cem\u003eSalix\u003c/em\u003ewhen compared with their species-depauperate sister genera. These adaptive traits may have ultimately led to the ecological adaptations and species radiation in both poplars and willows. Our findings underscore the previously overlooked role of ancient allopolyploidization and specific subgenomic evolution for fostering adaptive innovation and species diversification at deep nodes of the plant tree of life.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOne sentence summary: \u003c/strong\u003eThe specific subgenome evolution after ancient allopolyploidy drives the origin of unique adaptive traits that promote species radiation of the highly speciose \u003cem\u003ePopulus\u003c/em\u003e (poplars) and \u003cem\u003eSalix\u003c/em\u003e(willows).\u003c/p\u003e","manuscriptTitle":"Ancient allopolyploidy and specific subgenomic evolution drive adaptive radiation in poplars and willows","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-28 09:43:11","doi":"10.21203/rs.3.rs-5852798/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"81eeae76-9737-4775-894c-e64a4a6e7e56","owner":[],"postedDate":"January 28th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":43375987,"name":"Biological sciences/Evolution/Phylogenetics"},{"id":43375988,"name":"Biological sciences/Genetics/Genomics/Genome evolution"},{"id":43375989,"name":"Biological sciences/Plant sciences/Plant genetics/Polyploidy in plants"}],"tags":[],"updatedAt":"2025-08-07T07:27:50+00:00","versionOfRecord":{"articleIdentity":"rs-5852798","link":"https://doi.org/10.1038/s41467-025-62178-y","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2025-07-25 04:00:00","publishedOnDateReadable":"July 25th, 2025"},"versionCreatedAt":"2025-01-28 09:43:11","video":"","vorDoi":"10.1038/s41467-025-62178-y","vorDoiUrl":"https://doi.org/10.1038/s41467-025-62178-y","workflowStages":[]},"version":"v1","identity":"rs-5852798","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5852798","identity":"rs-5852798","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}