Karyotype reconstruction provides new insights into polyploid evolution in grasses (Poaceae)

Karyotype reconstruction provides new insights into polyploid evolution in grasses (Poaceae) | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Karyotype reconstruction provides new insights into polyploid evolution in grasses (Poaceae) Longjiang Fan, Haijun Cheng, Xuefang Huang, Yujie Huang, Linfeng Jin, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8993757/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Chromosomal rearrangements following whole-genome duplication (WGD) shape the diversity of genome structures in Poaceae. An accurate karyotype construction based on high-quality genomes of extant species will facilitate a deeper understanding of the origin and evolution of Poaceae. Here, we determine ancestral karyotypes for the BOP and PACMAD clades (ABPK, n = 12) and further infer ancestral karyotypes for subfamilies and tribes with distinct chromosome structures. Evolutionary trajectories from ancestral karyotypes to modern genomes are reconstructed for 29 species across 7 subfamilies through the characterization of multiple cascading fusion events. Our results reveal that subfamily-specific fusions contribute to the divergence and establishment of distinct subfamilies, while shared fusions among closely related species within each subfamily reflect the stable inheritance of karyotypic architecture. Based on the comparative karyotype analysis, we elucidate that Cenchrus purpureus and C. fungigraminus share two diploid progenitors contributing to their respective subgenomes A and B, and that Paleotetraploid S. maritimus and S. alterniflorus share the first allotetraploid WGD (WGD1) but experience independent second WGD (WGD2) events. Our results highlight the importance of chromosome fusion in descending dysploidy after recurrent polyploidization and shed light on the polyploidization and karyotype evolution across the Poaceae family. Ancestral karyotype Chromosome fusion Whole-genome duplication Poaceae Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Poaceae, one of the most ecologically and economically significant plant lineages, ranks as the fifth-largest angiosperm family, encompassing approximately 12,000 species distributed among 12 subfamilies 1 – 4 . The family's diversity is structured around two major clades: BOP (Bambusoideae, Oryzoideae, and Pooideae) and PACMAD (Panicoideae, Arundinoideae, Chloridoideae, Micrairoideae, Aristidoideae, and Danthonioideae). Additionally, three early-diverging subfamilies (Anomochlooideae, Pharoideae, and Puelioideae) occupy successive sister positions to the main BOP-PACMAD lineages 4 – 6 . In general, the Poaceae family has undergone three rounds of WGD events ( σ , τ , and ρ ) 7–11 . While the first two events are shared with other monocots, the third ρ -WGD event is specific to Poaceae and is thought to have driven the family's extensive adaptive radiation. Following the ρ-WGD event, numerous additional species-specific polyploidization events have occurred in the Poaceae family, such as tetraploidization and hexaploidization in Echinochloa species 12 . Polyploidization plays an essential role in plant evolution by providing raw genetic material for functional innovation and adaptive diversification 13 . This process has been recognized as a major driver of speciation, contributing to reproductive isolation and facilitating rapid adaptation to environmental changes 14 , 15 . Autopolyploids are formed through genome duplication, while allopolyploids arise from hybridization between different species with independent evolutionary histories, both of which are accompanied by chromosome doubling and gamete fusion 16 . Among polyploid species, allopolyploids are predominant, accounting for nearly 90% of cases, whereas autopolyploids represent only a small proportion 16 , 17 . The initial evolutionary stages of recently formed allotetraploids can be traced, as exemplified by Tragopogon species 18 . However, long-term evolution and divergence of allopolyploid lineages, such as Cenchrus and Sporobolus species, are more difficult to infer due to mutations, genomic rearrangements, and differential retention or loss of genes across subgenomes 19 – 21 . Disentangling their evolutionary histories is possible through karyotype reconstruction using chromosome-level genomes, which provide phylogenetic markers based on conserved collinear gene blocks and shared chromosome fusions. Post-polyploidization genome instability triggers chromosome breakages, abnormal recombination, and extensive gene losses, providing evolutionary opportunities for plant diversification 22 , 23 . Chromosome number reduction typically occurs through chromosome fusions accompanied by structural rearrangements 24 . By performing comparative analyses of extant genomes, researchers have attempted to reconstruct the ancestral karyotype and elucidate evolutionary trajectories of chromosomes 25 – 30 . Traditional methods identify contiguous ancestral regions (CARs) by detecting collinear gene blocks. However, chromosomal rearrangements and short syntenic blocks create undefined gap regions, resulting in parameter-sensitive outputs that limit downstream analysis of chromosomal evolution 31 . To address these limitations, pairwise genomic comparisons were employed to identify shared chromosome-like syntenic blocks (CLSBs) as protochromosomes 32 . Evolutionary trajectories from ancient origins to extant species can be reconstructed by tracing three principal types of chromosomal rearrangements: nested chromosome fusion (NCF), end-to-end joining (EEJ), and reciprocal chromosome translocation (RCT) 33 . NCF occurs when one chromosome with free ends invades and integrates within the pericentromeric region of another chromosome 28 . EEJ involves a crossover event near the termini of two chromosomes, resulting in the formation of one large chromosome and a dispensable mini-chromosome 33 . Both NCF and EEJ reduce chromosome number by one. However, RCT exchanges arms between two chromosomes through interstitial crossover, producing two recombinant chromosomes without altering chromosome numbers 24 . Additionally, different types of chromosomal rearrangements generate distinct numbers of fusion breakpoints: EEJ produces one breakpoint, while RCT and NCF each produce two breakpoints 34 . Karyotype evolution research in Poaceae has historically focused on only a few crops like rice and sorghum, without a systematic cross-lineage perspective 35 , 36 . With the accumulation of chromosome-level genomic resources in Poaceae and advances in karyotyping methodologies, more comprehensive and accurate karyotype reconstruction will become increasingly achievable, further advancing our knowledge of grass genome evolution. In this work, we reconstruct multiple ancestral karyotypes characterized by distinct chromosome structures within the Poaceae family. We use available chromosome-level genome assemblies from extant Poaceae species and analyze their karyotype evolution by explicitly considering three types of chromosomal rearrangements (RCTs, EEJs, and NCFs), rather than merely counting the number of fission and fusion events. Additionally, we investigate the origin and evolution of allopolyploid species in two genera based on comparative karyotype analysis. Results Ancestral karyotype of BOP and PACMAD We established a comprehensive genomic resource encompassing 29 species spanning 7 Poaceae subfamilies for karyotype construction (Fig. 1 ; Supplementary Fig. 1 ). These species exhibited diverse chromosome numbers and ploidy levels, representing the karyotypic diversity of the Poaceae family. All genome assemblies achieved high completeness (BUSCO > 95%) and continuity (LAI > 10), providing a reliable foundation for accurate karyotype construction. The only exception was S. maritimus , which displayed a lower LAI value (7.4) due to its scaffold-level assembly. Nevertheless, its high BUSCO score (98.6%) ensured the reliability of gene-based karyotype analysis. We reconstructed the ancestral karyotype of BOP and PACMAD following the workflow of WGDI tool (details in Methods) 32 . Synteny comparisons between O. sativa and species from different subfamilies of the BOP clade revealed that each O. sativa chromosome exhibited CLSBs with corresponding chromosomes in BOP species ( Supplementary Fig. 2a ), supporting O. sativa chromosomes as protochromosomes of the BOP clade. Using the same approach, chromosomes from P. australis subgenome A were identified as protochromosomes of the PACMAD clade ( Supplementary Fig. 2b ). Notably, the karyotypes of O. sativa and P. australis subgenome A were highly similar. Therefore, we designated the 12 chromosomes of the O. sativa genome as protochromosomes of the ancestral karyotype of BOP and PACMAD, named ABPK1 to ABPK12 ( Supplementary Fig. 2c ). Karyotype evolution in BOP clade We systematically examined chromosome number variation and fusion events across the Poaceae family (Fig. 1 ). Within the BOP clade, the paleodiploid Zizania latifolia ( n = 17, Oryzoideae) provides a clear instance of chromosome number reduction from a post-WGD state ( n = 24) via seven EEJs and one RCT ( Supplementary Fig. 3 ). Raddia guianensis and Olyra latifolia (both Bambusoideae) shared a nested fusion (abbreviated as ABPK_10 + 12_NCF), indicating that their divergence postdated this fusion. Notably, R. guianensis represented the only documented case of chromosome fission in Poaceae (purple-dashed ellipse in Supplementary Fig. 4 ). As the most species-rich subfamily of Poaceae, Pooideae exhibits remarkable karyotypic variation. The ancestral karyotype of the Brachypodieae tribe (ABK, n = 10) was derived from ABPK through two NCFs (ABPK_8 + 10_NCF and ABPK_9 + 12_NCF), followed by one ABPK_(9 + 12_NCF)+11_RCT event ( Supplementary Fig. 5 ). The Brachypodium distachyon genome ( n = 5) was derived from the ABK genome through four NCFs (ABK_(2 + 6_NCF)+7_NCF, ABK_1 + 8_NCF, and ABK_3 + 4_NCF) and one EEJ event (ABK_5 + 10_EEJ), with the EEJ shared with Brachypodium sylvaticum ( n = 9) ( Supplementary Fig. 6 ). In the tetraploid Brachypodium hybridum genome ( n = 15), all five B. distachyon -specific chromosome fusions were identified in the 5-chromosome subgenome, whereas the 10-chromosome subgenome had the Brachypodium stacei genome structure without any rearrangement ( Supplementary Fig. 7 ). Based on the above results combined with the subgenome-level phylogenetic tree (Fig. 3 ), we concluded that B. distachyon and B. stacei were most likely the donors of the two B. hybridum subgenomes, respectively. This was consistent with previous conclusions 37 . The ancestral karyotype of the Poeae and Triticeae tribes (APTK, n = 7) was derived from ABPK through descending dysploidy involving four NCFs (ABPK_3 + 11_NCF, ABPK_4 + 7_NCF, ABPK_6 + 8_NCF, and ABPK_5 + 10_NCF) and one EEJ (ABPK_9 + 12_EEJ) ( Supplementary Fig. 8a, c ). Within the Poeae tribe, Alopecurus myosuroides evolved from APTK through one APTK_1 + 4_RCT ( Supplementary Fig. 9 ). Subsequently, the APTK genome was altered by a reciprocal translocation (APTK_1 + 6_RCT) towards the ancestral karyotype of Triticeae (ATK, n = 7) preceding the diversification of this tribe ( Supplementary Fig. 8b, c ). In relation to the Triticeae genome ( Supplementary Fig. 10a ), Aegilops umbellulata shared the ATK_4 + 5_RCT event with Triticum monococcum and had a reciprocal translocation (ATK_4 + 6_RCT) that arose independently from the similar translocation in Elymus sibiricus , as indicated by their different fusion breakpoint positions ( Supplementary Fig. 10b ). Karyotype evolution in PACMAD clade Within the PACMAD clade, although five subfamilies are recognized, chromosome-level genome assemblies for karyotype reconstruction are currently limited to three subfamilies: Panicoideae, Chloridoideae, and Arundinoideae (Fig. 2 ). Two NCF events (ABPK_3 + 10_NCF and ABPK_7 + 9_NCF) shaped the ancestral karyotype of Panicoideae subfamily (APEK, n = 10) from ABPK, and a subsequent NCF event (APEK_8 + 9_NCF) generated the ancestral karyotype of Paniceae tribe (APAK, n = 9) ( Supplementary Fig. 11 ). Miscanthus sinensis (Andropogoneae) underwent a nested fusion between chromosomes 4 and 7 (APEK_4 + 7_NCF), reducing its chromosome number to n = 19 ( Supplementary Fig. 12 ). In the Panicoideae tribe, Setaria italica and S. viridis retained the ancestral chromosome number ( n = 9) altered by one APAK_3 + 7_RCT ( Supplementary Fig. 13 ). Both subgenomes of the allotetraploid C. fungigraminus retained 7 chromosomes but differed in their evolutionary trajectories ( Supplementary Fig. 14a ). A shared APAK_3 + 6_NCF occurred prior to subgenome divergence ( Supplementary Fig. 14b ); subsequent APAK_4 + 9_RCT breakpoints differed between subgenomes, indicating independent fusion origins ( Supplementary Fig. 14c ). The ancestral karyotype of the Chloridoideae subfamily (ACK, n = 10) was shaped by two NCF events (ABPK_2 + 10_NCF and ABPK_6 + 9_NCF) ( Supplementary Fig. 15 ). Within the Cynodonteae tribe, the allotetraploid Cleistogenes songorica displayed asymmetric karyotype evolution: subgenome A retained the ancestral ACK structure without rearrangement, whereas subgenome B underwent two independent reciprocal translocations (ACK_1 + 5_RCT and ACK_3 + 7_RCT) ( Supplementary Fig. 16a ). The shared presence of ACK_3 + 10_NCF in both subgenomes of Cynodon dactylon and ACK_9 + 10_NCF in both subgenomes of Eleusine coracana indicated these fusion events occurred in diploid ancestors prior to the respective WGD events ( Supplementary Fig. 16b, c ). Within the Zoysieae tribe, pairwise karyotype comparisons of fused chromosomes between subgenomes (A and B) of S. maritimus and subgenomes (C and D) of S. alterniflorus revealed patterns of chromosomal rearrangement ( Supplementary Fig. 17 ). All four subgenomes shared four chromosome fusion events (ACK_4 + 9_NCF, ACK_6 + 8_NCF, ACK_5 + 10_NCF, and ACK_2 + 3_RCT), indicating these rearrangements predated the divergence of these subgenomes. In addition, three subgenomes (A-C) each underwent two further chromosomal rearrangements (ACK_4 + 6_NCF and ACK_1 + 10_NCF), reducing the chromosome number to n = 15. By contrast, subgenome D experienced a unique ACK_4 + 10_NCF event, bringing chromosome number down to n = 16. This distinct karyotypic pattern in subgenome D suggested that it diverged earlier from the other subgenomes, consistent with the K s results ( see the last section ). Moreover, the currently available tetraploid and hexaploid Arundinoideae species retained an ABPK-like karyotype (Fig. 2 ). Phylogenetic congruence of chromosome fusion distribution and uncertainty in the ancestral Poaceae karyotype To comprehensively illustrate the karyotype evolution across the Poaceae family, we systematically identified 57 distinct chromosome fusion events ( Supplementary Table 2 ) in representative species (excluding Pharus latifolius ), and constructed a subgenome-level phylogeny, with P. latifolius as the outgroup (Fig. 3 a). The resulting phylogeny resolved the evolutionary relationships of subgenomes across different subfamilies, which is consistent with previous studies 38 . The characterization of multiple cascading fusion events allowed us to construct the ABPK and other ancestral karyotypes, revealing different evolutionary trajectories from a common ancestor (Fig. 1 ). As shown in the heatmap, (i) chromosome fusions were not shared between subfamilies, providing evidence for independent karyotype evolution in each lineage, (ii) within each subfamily, species belonging to the same clade shared chromosome fusions, and the distribution of these shared fusions (marked by black rectangles) was highly congruent with the phylogenetic relationships inferred from single-copy orthologous genes (Fig. 3 a). Notably, several species retained the same karyotype as ABPK without undergoing any chromosome fusion events. Within Poaceae, Pharoideae serves as the sister lineage to the BOP and PACMAD clades. Karyotype comparisons between the P. latifolius genome, regarded as the ancestral karyotype of Pharoideae (APHK), and the ancestral ABPK genome revealed a reciprocal translocation (ABPK_1 + 7_RCT or APHK_1 + 7_RCT), but the evolutionary direction of this RCT event could not be determined (Fig. 3 b). Earlier studies suggested that the rice genome (referred to as ABPK) was the ancestral karyotype of Poaceae 36 . However, chromosome fusion analysis revealed two possible scenarios: either the ancestral karyotype resembled APHK, with APHK_1 + 7_RCT generating the ABPK genome configuration, or ABPK represented the ancestral karyotype state, with ABPK_1 + 7_RCT producing the APHK genome structure. To determine the directionality of the RCT event and thus infer the ancestral karyotype state, we extracted 100 genes flanking each side of the fusion breakpoints from RCT (ABPK_1 + 7_RCT or APHK_1 + 7_RCT) as individual segments and performed synteny analysis with closely related non-Poaceae outgroup species ( Ananas comosus and Juncus effusus ). In principle, when synteny blocks spanning the 200-gene segment are detected in outgroup genomes, the karyotype (ABPK or APHK) possessing the 200-gene segment is more closely related to the outgroup species and is considered to represent the ancestral karyotype of Poaceae 34 . However, we could not identify any syntenic blocks near the breakpoints ( Supplementary Fig. 18 ) owing to extensive chromosomal rearrangements during the Poaceae-specific ρ-WGD ( n = 7 to n = 12) 39 . Hence, the true ancestral Poaceae karyotype remains uncertain. The phylogenomic history of Cenchrus species The phylogeny of C. purpureus , C. fungigraminus , and C. americanus is currently undetermined. To investigate their evolutionary relationships, we conducted pairwise karyotype comparisons of (sub)genomes of three species. Although subgenomes A and B within each allotetraploid species showed significant karyotypic divergence ( Supplementary Fig. 19 ), subgenome A of both C. purpureus and C. fungigraminus exhibited highly conserved karyotypes largely consistent with genome of C. americanus (Fig. 4 a). Similarly, subgenome B of both C. purpureus and C. fungigraminus also exhibited high karyotypic concordance. We further performed whole-genome mapping analyses by aligning 150-mer reads derived from the diploid C. americanus genome against both C. purpureus and C. fungigraminus genomes. Subgenome A of C. purpureus and C. fungigraminus showed moderate coverage depths of approximately 45% and 50%, respectively ( Supplementary Fig. 20 ), suggesting that C. americanus was likely a close relative of the diploid progenitor that contributed subgenome A to both C. purpureus and C. fungigraminus . Following the same analytical approach, we aligned 150-mer reads from subgenomes A and B of C. purpureus against the C. fungigraminus genome, achieving coverage depths of approximately 80% and 85% for subgenomes A and B of C. fungigraminus , respectively (Fig. 4 d, e). The high level of sequence similarity between corresponding subgenomes strongly supported the hypothesis that C. purpureus and C. fungigraminus share two diploid progenitors for their respective A and B subgenomes. This interpretation is further validated by the subgenome tree (Fig. 3 a). We calculated the synonymous substitution rate ( K s) values for homeologous gene pairs between Cenchrus (sub)genomes. According to the K s peak, we determined that the two unknown diploid progenitors (diploids A and B) of the Cenchrus species diverged approximately 9.5 million years ago (mya). Subsequently, diploid A diverged from C. americanus around 4.5 mya (Fig. 4 b, c). The "bubble" peak in the transposable element (TE) divergence curve indicated that the allotetraploid speciation event occurred approximately 2.9 mya ( Supplementary Fig. 21 ). Furthermore, the divergence time between C. purpureus and C. fungigraminus was estimated at 0.6 mya. Allopolyploid evolution of Sporobolus species By using Eragrostis tef subgenome A as a reference and Chr3 as an example, we were able to readily identify four homeologous blocks in S. maritimus and S. alterniflorus , respectively. Based on the complementarity and completeness of these synteny blocks, we phased them into distinct sub-subgenomes (details in Methods; Fig. 5 a). To further test relationships among sub-subgenomes, we then extracted collinear genes from these synteny blocks and constructed gene trees, rooted on E. tef Chr3. Phylogenies constructed from 1243 collinear genes of sub-subgenomes with at least six collinear genes resulted in 1243 topologies, from which we obtained a consensus tree (Fig. 5 b). The coexisting sub-subgenomes within the genus Sporobolus formed two clusters (labeled S1 and S2) that fell into different clades, suggesting that the extant subgenomes (A-D) of Sporobolus species originated from an allotetraploid event. We further tested this hypothesis by phasing the Sporobolus subgenomes and constructing 10 phylogenetic trees using individual chromosomes from E. tef subgenome A as an outgroup, all of which consistently supported the allopolyploid origin ( Supplementary Figs. 22 and 23 ). We reconstructed karyotype schematics to trace the evolutionary history of two Sporobolus species (Fig. 5 d). Based on synchronous K s peaks at 0.22 (Fig. 5 c), the diploid ACK genome is estimated to have undergone WGD1 approximately 15 mya, yielding a shared tetraploid intermediate (n = 2x = 20). Subsequent descending dysploidy via chromosome fusions restructured the intermediate into two diploidized subgenomes with n = x = 16 and n = x = 15 chromosomes, respectively. These subgenomes then underwent lineage-specific WGD2, generating S. maritimus ( n = 2 x = 30) at approximately 0.5 mya and S. alterniflorus ( n = 2 x = 31) at approximately 0.9 mya ( Supplementary Fig. 24a, b ). Unlike the ancient shared WGD1, these recent WGD2 events occurred independently of each other. Chromosome-scale synteny analysis of selected homologous chromosomes and an ancient chromosome (marked by red triangles in Fig. 5 d) revealed distinct chromosomal syntenic scenarios (Fig. 5 e). Chromosomes with two distinct color blocks reflected their derivation from ancestral chromosome fusions. Notably, Chr31 of S. alterniflorus (Salt_31) displayed one-to-one syntenic correspondence with Chr6 of E. tef (Etef_6), demonstrating that Salt_31 represented an intact karyotype structure inherited from the ancestral chromosome, rather than a product of non-reciprocal translocation as previously proposed 21 . Discussion We have reconstructed ABPK ( n = 12) as the ancestral karyotype of the analyzed genomes of clades BOP and PACMAD. Our results show that modern genomes can be traced back to the ancestral karyotype through multiple cascading chromosomal rearrangements. Based on the order in which these chromosome fusions occur, we can determine the evolutionary trajectories of extant species. The Paniceae tribe provides a clear example: the ancestral karyotype ABPK underwent sequential chromosomal changes, first forming APEK, which then evolved into APAK, and subsequently gave rise to S. italica (Fig. 2 ) . This stepwise progression of fusion events reveals the evolutionary pathway more precisely than previous strategies 40 , 41 . By determining if genes on either side of a fusion breakpoint lie within a single synteny block, we could identify shared chromosomal rearrangements between species with significantly higher accuracy. Sometimes, seemingly identical fusion events from the same ancestral chromosome pairs show distinct breakpoint positions, such as APAK_4 + 9_RCT in the two subgenomes of C. fungigraminus and ATK_4 + 6_RCT in A. umbellulata versus E. sibiricus ( Supplementary Figs. 10 and 14) . Corresponding ancestral chromosomes as rearrangement hotspots experience multiple fusions, suggesting that certain chromosomal regions may be particularly prone to structural instability during genome evolution, similar fusions patterns have also been reported in the karyotype evolution of other plant species 34 . In this study, we identified only one chromosome fission event, consistent with a growing body of research suggesting that fusions, rather than fissions, are the primary drivers of karyotype changes 42 , 43 . The distribution of shared versus lineage-specific chromosome fusions provides a clear signature of karyotype evolution in Poaceae. The subfamily-specific fusions acted as key drivers of reproductive isolation, contributing to the divergence and establishment of distinct subfamilies, while shared fusions among closely related species reflect stable inheritance of karyotypic configurations (Fig. 3 a ) . Fusion events that predated divergence will be shared across descendant species, while those occurring post-divergence will be species-specific. For instance, in O. latifolia , ABPK_10 + 12_NCF occurred before divergence from Raddia guianensis , whereas ABPK_2 + 5_EEJ arose after divergence ( Supplementary Fig. 4) . Our karyotype reconstruction further proves its utility by providing crucial structural insights into evolutionary relationships of polyploid lineages. For Cenchrus species, pairwise karyotype comparisons between (sub)genomes, integrated with K s analysis and whole-genome sequence mapping, provide a high-resolution lens on their evolutionary history (Fig. 4 ). Our results clarify that C. americanus is a sister lineage to the diploid progenitor of subgenome A in the allopolyploids C. purpureus and C. fungigraminus , rather than the direct donor, consistent with previous conclusions 44 . We confirm that C. purpureus and C. fungigraminus share common diploid progenitors for their respective subgenomes A and B, resolving evolutionary relationships that remained ambiguous in prior studies 20 . We further reconstructed the karyotype of C. fungigraminus and characterized specific chromosome fusion events derived from the ancestral APAK genome ( Supplementary Fig. 14) , which significantly improved the accuracy of evolutionary trajectory inference relative to previous studies that only estimated the number of likely chromosomal rearrangements occurring during karyotype evolution 20 . Similarly, for Sporobolus species, through manual scaffold reordering ( Supplementary Fig. 25) , we generated a near-chromosome-level S. maritimus assembly and successfully partitioned the genomes of S. maritimus and S. alterniflorus into four subgenomes (A-D), each comprising two sub-subgenomes, confirming that both species are paleotetraploids rather than hexaploids as previously proposed 45 ( Supplementary Fig. 22) . The polyploid origin of Sporobolus species has remained contentious, recent work showed that distinguishing between autopolyploidy and allopolyploidy was not feasible due to the absence of extant diploid progenitors and tetraploid intermediates 21 . To address this challenge, we employed the E. tef subgenome A as a reference for partitioning homeologous chromosome blocks into their corresponding sub-subgenomes and constructed 10 phylogenetic trees using individual chromosomes of E. tef subgenome A as an outgroup, thereby establishing the initial allopolyploid origin of both Sporobolus species 45 ( Supplementary Fig. 23) . Karyotype evolution in Sporobolus species involves complex chromosomal rearrangements following polyploidization. Previous studies only characterized post-rediploidization karyotype changes and hypothesized the number of chromosome fusions in Sporobolus species, without considering the detailed karyotype changes from the ACK to Sporobolus species 21 . Here, we present a comprehensive karyotype reconstruction to trace the evolutionary trajectory of Sporobolus species. Contrary to the two kinds of karyotype evolution trajectories hypotheses proposed by Salmon et al. 21 , our findings revealed that the ACK genome underwent a shared WGD1 event followed by lineage-specific chromosomal rearrangements that generated distinct subgenomes (A-D). Subgenomes A and B subsequently underwent WGD2 to form S. maritimus , with karyotype evolution involving 12 chromosome fusions (including two RCTs) that reduced the chromosome complement from theoretical n = 40 (octoploid) to n = 30 (paleotetraploid) ( Supplementary Fig. 17 ). Similarly, WGD2 of subgenomes C and D produced S. alterniflorus, with 11 chromosome fusions (including two RCTs) decreasing the chromosome number from theoretical octoploid number ( n = 40) to the current paleotetraploid complement ( n = 31). The exact match between chromosome number reduction and chromosome fusion events robustly validates the accuracy of our karyotype constructions. Unlike previous reports 21 , our results indicate that WGD2 is not shared between S. maritimus and S. alterniflorus . By using the ACK genome as a reference, combined with chromosome karyotype reconstruction, we successfully disentangled the complex evolutionary relationships between two Sporobolus species. Identification of shared chromosome fusions between species provides an independent line of evidence that can corroborate and refine topology inferences, particularly when sequence-based phylogenetic methods are challenged by introgression, homoplasy, and incomplete lineage sorting over deep evolutionary timescales 46 . Nevertheless, the approach has inherent limitations that should be carefully considered. It is largely ineffective for phylogenetic inference when comparing genomes with conserved karyotypes, such as genomes from subfamily Arundinoideae 47 . Widespread gene losses in genomes can lead to the disappearance of gene collinearity, which may render the inference of chromosomal karyotypes extremely difficult. Additionally, karyotype reconstruction requires high-quality chromosome-level genome assemblies. Poor assembly quality can lead to the false-positive identification of chromosomal rearrangements and imprecise location of fusion breakpoints, resulting in misleading interpretations of evolutionary relationships between species. To date, the fate of diploid progenitors of subgenomes A and B in Cenchrus remains unclear—they may have gone extinct or may persist but lack chromosome-level genomic resources. Expanded taxonomic sampling across Cenchrus species will be necessary to resolve the outstanding question. Additionally, the structure of the most ancestral Poaceae karyotype remains unresolved. It may eventually be resolved as more chromosome-level genomes become available, especially those from early-diverging subfamilies. Methods Data collection We collected all available chromosome-level genomes in Poaceae, together with several phylogenetically related outgroup genomes from public databases 48 . Given the comprehensive characterization of Bambusoideae karyotypes in recent studies 49 , only two representative species from this subfamily were selected here. Following stringent criteria for assembly contiguity and completeness, we selected 29 representative genomes spanning the major Poaceae lineages (a list of genomes see Supplementary Table 1) for karyotype reconstruction and included additional species for comparative karyotype analysis. Phasing subgenomes with subphaser Subphaser is an automated pipeline based on subgenome-specific k -mers 50 . The assembled genome and homeologous chromosome relationships were input into SubPhaser (v 1.2.6) 50 to obtain subgenome phasing results. Potential inter-subgenomic translocations and homeologous exchanges between subgenomes were also detected. For S. maritimus , initial subgenome phasing revealed ambiguous assignments and large-scale apparent homeologous exchanges in several regions (marked by red dashed ellipses in Supplementary Fig. 25a, c ), likely reflecting scaffold ordering errors caused by high sequence similarity between homeologous chromosomes rather than genuine biological recombination events. Following manual scaffold repositioning ( Supplementary Table 3) , the optimized assembly achieved correct subgenome partitioning, with chromosomes 1–15 assigned to subgenome A and chromosomes 16–30 assigned to subgenome B ( Supplementary Fig. 25b, d ). The two subgenomes of S. alterniflorus were successfully phased in a default subphaser run, with chromosomes 1–15 assigned to subgenome C and chromosomes 16–31 assigned to subgenome D ( Supplementary Fig. 26a, b ). Ancestral karyotype reconstruction To reconstruct ancestral karyotypes of BOP and PACMAD clades, we performed pairwise genome comparisons using WGDI 32 . Within the BOP clade, we selected O. sativa ( n = 12) as the anchor genome because it has the highest haploid chromosome number within the clade and thus likely retains more ancestral karyotypic features than other BOP members. Three species from distinct subfamilies (Pooideae, Bambusoideae, and Oryzoideae) were then independently aligned against O. sativa using WGDI (with the '-d' parameter) to generate homologous dotplots. Similarly, for the PACMAD clade, we used P. australis subgenome A ( n = 12) as the anchor genome and performed alignments with three species representing Chloridoideae, Panicoideae, and Arundinoideae subfamilies, respectively. Given that fusion or fission of ancestral chromosomes occur gradually and randomly in the offspring lineages, intact protochromosomes are expected to be preserved in some descendants. Based on this reasoning, we identified shared CLSBs and extracted intact chromosomes as putative protochromosomes. Karyotypic evolution in Poaceae To reconstruct karyotype evolution in Poaceae, we employed a comparative genomics approach that inferred chromosomal changes from ancestral karyotypes to extant genomes. The WGDI workflow began with the identification of collinear gene pairs between species using the improved collinearity module ('-icl') followed by synteny block integration ('-bi'). Stringent filtering was then applied through the correspondence module ('-c') to eliminate syntenic regions potentially derived from earlier polyploidization events. Protochromosomes were mapped onto the chromosomes of the sample genomes via the karyotype mapping function ('-km'). Furthermore, the parameter '-sf' was employed to rapidly identify chromosome fusions, with shared fusions and their corresponding fusion breakpoints subsequently recorded. Fusion breakpoint coordinates were extracted using the '-fpd' parameter, which generated a comprehensive dataset recording fusion positions. Finally, we applied the '-fd' module to determine the occurrence of these breakpoints in other genomes. Phylogenomic analysis We reconstructed phylogenetic relationships using 43 subgenomes from 29 Poaceae species. Orthogroups were identified using OrthoFinder (v 2.5.4) 51 with subgenome protein sequences. We retained high-confidence orthogroups present in at least 35 subgenomes (> 80% taxonomic sampling), with a single gene copy per subgenome. Protein sequences within each orthogroup were aligned using MUSCLE (v 5.1) 52 , and the resulting multiple sequence alignments were concatenated into a supermatrix. Maximum-likelihood phylogenetic inference was performed using IQ-TREE (v 2.1.3) 53 under the best-fit substitution model selected by ModelFinder, with branch support assessed by 1,000 ultrafast bootstrap replicates. Divergence and merger time estimation We estimated the divergence time between subgenomes and subsequent merger time for allopolyploid Sporobolus species ( S. alterniflorus and S. maritimus ) and Cenchrus species ( C. americanus , C. fungigraminus , and C. purpureus ). We first aligned protein sequences among subgenomes using BLASTP (v 2.14.0) 54 with an E-value cutoff of 1e − 10 . DAGchainer (r02-06-2008) 55 was used to identify syntenic blocks with at least five homologous gene pairs within a region of 10 gene models based on best-hit blastp results. The K s values for syntenic homeologous gene pairs were calculated by K a K s_Calculator in NG mode. The K s distribution peaks were used to calculate the divergence time using the formula T = K s/2 r , where r is the nucleotide substitution rate ( r = 6.5 × 10 − 9 mutations × bp − 1 × generation − 1 ). TE divergence was assessed using PercDivs (percentage of substitutions in the matching region compared with the consensus), calculated by RepeatMasker (v 4.2.0) ( https://github.com/rmhubley/repeatmasker ). The TE sequence divergence between two subgenomes displaying a high degree of overlap suggested consistency in the TE evolutionary rate in the two subgenomes. The non-overlapping segregation region indicated the timeframe from diploid progenitor divergence to tetraploid genome formation. K -mer-based whole-genome mapping analysis To investigate evolutionary relationships between Cenchrus species, k -mer-based whole-genome mapping analysis was performed as previously described 12 . Query (sub)genome sequences were fragmented into 150-mers with a 5 bp step size, and these short reads were subsequently mapped to the reference genome using Bowtie2 (v 2.5.1) 56 . Coverage depth was calculated using SAMtools (v 1.23) 57 to quantify sequence similarity. Phasing sub-subgenomes with WGDI Subphaser toolkit 50 independently partitioned S. alterniflorus and S. maritimus into two subgenomes, each comprising two chromosome sets (termed sub-subgenomes). To resolve the evolutionary relationships among sub-subgenomes within the Sporobolus species, we used E. tef subgenome A as the reference for phasing of S. alterniflorus and S. maritimus . First, protein sequences from Sporobolus species were compared against those of the reference genome using DIAMOND (v 2.0.15) 58 . Collinear regions were identified and integrated using WGDI with the '-icl' and '-bi' parameters. Orthologous syntenic blocks derived from recent polyploidy events were retained using WGDI with the '-c' filtering option. The reference genome karyotype was then mapped onto Sporobolus chromosomes based on syntenic relationships using WGDI with the '-km' parameter. Sub-subgenomes were manually assigned based on complementarity and completeness of synteny blocks. Hierarchical gene lists (one sub-subgenome per column) were generated using WGDI with the '-pc' and '-a' parameters. For each chromosome of the reference genome, maximum likelihood gene trees were inferred from the merged hierarchical gene lists using built-in IQ-TREE with automatic selection of the best-fit substitution model (-m MFP) through the WGDI '-at' function. These gene trees were input to ASTRAL-Pro (v 1.19.3.6) 59 to construct the sub-subgenome phylogeny. The resulting phylogeny was visualized and evaluated using Newick utilities (v 1.6) 60 and Phytop (v 0.3) 61 . To ensure robust sub-subgenome classification, the analysis pipeline ('-pc', '-a', and '-at' options in WGDI, followed by ASTRAL-Pro) was iterated until topological consistency converged. Chromosome synteny visualization Chromosome-level synteny was analyzed across ten chromosomes from E. tef (Chr 6), S. alterniflorus (Chr 31, 3, 18, 2, and 17), and S. maritimus (Chr 3, 18, 2, and 17). Pairwise syntenic blocks between chromosomes were detected using JCVI (v 1.5.7) 62 , requiring ≥ 30 gene pairs per block with ≤ 20 intervening genes. Coordinates of anchor genes were extracted from BED files and converted to NGenomeSyn link format with strand directionality preserved. The resulting syntenic links were visualized using NGenomeSyn (v 1.43) 63 with customized color schemes for different collinearity relationships. Declarations Author countribution L.F., P.S., and D.W. led and coordinated the project. H.C. and L.J. collected the assemblies. X.H. and K.Y. performed quality control. H.C., S.Z., S.Z., R.Z., and Z.L. analyzed Poaceae genomes. H.C., X.H., and Y.Z. finished karyotype and phylogenetic analyses. H.C., P.S., D.W., Q.C., and Y.H. provided gramine quantification. L.F., P.S., D.W., H.C., and Y.H. discussed the results. L.F. and P.S. supervised all analyses. H.C. wrote the initial manuscript with input from all co-authors. All authors discussed the results and commented on the manuscript. Competing interests All authors declare no competing interests. Acknowledgements This work was supported by National Key Research and Development Program (2023YFD1400502). References Kellogg EA (2000) The grasses: A case study in macroevolution. Annu Rev Ecol Evol Syst 31:217–238 Kellogg EA (2001) Evolutionary history of the Grasses1. 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Bioinformatics 26:1669–1670 Shang H-Y et al (2025) Phytop: A tool for visualizing and recognizing signals of incomplete lineage sorting and hybridization using species trees output from ASTRAL. Hortic Res 12:uhae330 Tang H et al (2011) Screening synteny blocks in pairwise genome comparisons through integer programming. BMC Bioinf. 12 Gentili M, Martini L, Sponziello M, Becchetti L (2022) Biological random walks: Multi-omics integration for disease gene prioritization. Bioinformatics 38:4145–4152 Additional Declarations The authors declare no competing interests. Supplementary Files SupplementaryTables.xlsx SupplementaryFigures.docx Cite Share Download PDF Status: Posted 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. 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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-8993757","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":598476280,"identity":"9b631ce0-80ab-4d78-a1c8-bbb75a0d6629","order_by":0,"name":"Longjiang 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University","correspondingAuthor":false,"prefix":"","firstName":"Pengchuan","middleName":"","lastName":"Sun","suffix":""}],"badges":[],"createdAt":"2026-02-28 09:21:18","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-8993757/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8993757/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":103854770,"identity":"b0e59049-20a9-4e68-8003-bfade98f17e3","added_by":"auto","created_at":"2026-03-03 17:50:14","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":98572,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKaryotype reconstruction and evolution for a portion of the Poaceae family (including subfamily Pooideae, Oryzoideae, Bambusoideae from BOP clade and subfamily Pharoideae).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIdeograms are based on the published chromosome-level genome assemblies (\u003cstrong\u003eSupplementary Table 1\u003c/strong\u003e) and their karyotype reconstruction using the WGDI. ABPK: Ancestral karyotype of BOP and PACMAD clades, APHK: Ancestral Karyotype of Pharoideae subfamily, ABK: Ancestral Karyotype of Brachypodieae tribe, APTK: Ancestral Karyotype of Poeae and Triticeae tribes, ATK: Ancestral Karyotype of Triticeae tribe, RCT: reciprocal chromosome translocation, EEJ: end-to-end joining, NCF: Nested chromosome fusion. Arrows illustrate the evolutionary directions of the fusion events, while bidirectional arrows indicate uncertainty about the direction of evolution. Tetraploidization ismarked by a red circle. Triangle represents the PACMAD clade, and the reconstructed karyotype evolution is shown in Fig. 2.\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8993757/v1/aa712441498e560b25f546c2.jpg"},{"id":103854771,"identity":"6ac126d0-9e3f-4a16-9f86-9b3e7b8a9d0e","added_by":"auto","created_at":"2026-03-03 17:50:14","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":109709,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eReconstructed karyotype evolution for a portion of the Poaceae family (including subfamily Arundinoideae, Chloridoideae and Panicoideae from PACMAD clade).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAPEK: Ancestral Karyotype of Panicoideae subfamily, ACK: Ancestral Karyotype of Chloridoideae subfamily, APAK: Ancestral Karyotype of Panicoideae tribe. RCT: reciprocal chromosome translocation, EEJ: end-to-end joining, NCF: Nested chromosome fusion. Tetraploidization ismarked by a red circle; hexaploidization is marked by a red pentagram. See also legend for Fig. 1.\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8993757/v1/b9a3df947f8536f2a0ffc792.jpg"},{"id":104401290,"identity":"8eabb73f-b60c-460f-9d7d-11379c36e0e6","added_by":"auto","created_at":"2026-03-11 12:12:17","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":109832,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChromosome fusions in Poaceae during their karyotype evolution.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) Subgenome-level phylogenetic tree with \u003cem\u003eP. latifolius\u003c/em\u003e as an outgroup, accompanied by a heatmap illustrating the distribution of chromosome fusions across subgenomes from BOP and PACMAD clades. (b) The ancestral ABPK (identical to \u003cem\u003eO. sativa\u003c/em\u003e) genome and APHK (identical to \u003cem\u003eP. latifolius\u003c/em\u003e) genome are distinguished by a reciprocal translocation (ABPK_1+7_RCT or APHK_1+7_RCT). Bidirectional arrows indicate uncertain fusion direction. ABPK: Ancestral karyotype of BOP and PACMAD clades, APHK: Ancestral Karyotype of Pharoideae subfamily, ABK: Ancestral Karyotype of Brachypodieae tribe, APTK: Ancestral Karyotype of Poeae and Triticeae tribes, ATK: Ancestral Karyotype of Triticeae tribe, APEK: Ancestral Karyotype of Panicoideae subfamily, ACK: Ancestral Karyotype of Chloridoideae subfamily, APAK: Ancestral Karyotype of Paniceae tribe. RCT: reciprocal chromosome translocation, EEJ: end-to-end joining, NCF: Nested chromosome fusion.\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8993757/v1/6016ebc525ad038b39ffa4ad.jpg"},{"id":104401060,"identity":"a5e14189-5993-4649-987e-3fb7ebac704e","added_by":"auto","created_at":"2026-03-11 12:11:45","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":74127,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEvolutionary processes of the genomes in three \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eCenchrus\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e species.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) Pairwise karyotype comparisons among the (sub)genomes of \u003cem\u003eCenchrus\u003c/em\u003e species. (b) The synonymous substitution rate (\u003cem\u003eK\u003c/em\u003es) distribution of pairs of homeologous genes among the (sub)genomes from \u003cem\u003eC. purpureus\u003c/em\u003e (Cpur), \u003cem\u003eC. fungigraminus\u003c/em\u003e (Cfun), and \u003cem\u003eC. americanus\u003c/em\u003e (Came)\u003cem\u003e.\u003c/em\u003e (c) Brief diagram showing the evolution of \u003cem\u003eCenchrus\u003c/em\u003e species and estimation of polyploidization times. The grey dashed line represents two unknown ancestral diploid progenitors. Numbers beside each branch point show divergence or hybridization time. mya: million years ago. Coverage depth of two subgenomes in \u003cem\u003eC. fungigraminus\u003c/em\u003e by mapping 150-mer sequence from \u003cem\u003eC. purpureus\u003c/em\u003esubgenome A (d) or subgenome B (e).\u003c/p\u003e","description":"","filename":"Picture4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8993757/v1/92e60c2bc1da38d69f513644.jpg"},{"id":103854773,"identity":"6793a373-03d4-4949-aa58-3388d4f7aac3","added_by":"auto","created_at":"2026-03-03 17:50:14","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":74767,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEvolutionary relationships of the genomes between\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003etwo \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eSporobolus \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003especies.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) Identification of chromosome-like synteny blocks (CLSBs) using \u003cem\u003eEragrostis tef\u003c/em\u003e Chr3 as the reference against \u003cem\u003eS. maritimus \u003c/em\u003eand \u003cem\u003eS. alterniflorus\u003c/em\u003e. Letters within each oval indicate the sub-subgenome origin of the corresponding chromosome. (b) A phylogeny of the coexisting sub-subgenomes based on 1243 collinear genes from homologous blocks to \u003cem\u003eE. tef\u003c/em\u003e Chr3. Numbers above branches indicate concordance percentages between gene-based and chromosome-scale phylogenies; numbers below branches represent local posterior probabilities calculated by ASTRAL. Pie charts at nodes display the frequencies of the three primary gene tree topologies (q1, q2, and q3) as quantified by ASTRAL, reflecting discordance among gene trees. Branch lengths are proportional to substitutions per site (scale bar = 0.05). Species labels are color-coded to distinguish different accessions: \u003cem\u003eS. maritimus\u003c/em\u003e (A1, A2, B1, B2)\u003cem\u003e \u003c/em\u003eand\u003cem\u003eS. alterniflorus\u003c/em\u003e (C1, C2, D1, D2), with \u003cem\u003eE. tef\u003c/em\u003e subgenome A as the outgroup. (c) The synonymous substitution rate (\u003cem\u003eK\u003c/em\u003es) distribution of pairs of homeologous genes between subgenomes of \u003cem\u003eS. maritimus \u003c/em\u003eand\u003cem\u003e S. alterniflorus\u003c/em\u003e. (d) Karyotype reconstruction depicting the evolutionary trajectory from the Chloridoideae ancestor to extant \u003cem\u003eSporobolus\u003c/em\u003e species. Colored bars represent individual chromosomes, with each color denoting distinct ancestral chromosome segments. The red triangle highlights the chromosomes selected for downstream synteny analysis. (e) Synteny visualization of 10 selected chromosomes (red triangle in d) from \u003cem\u003eE. tef\u003c/em\u003e (Etef), \u003cem\u003eS. maritimus\u003c/em\u003e (Smar), and \u003cem\u003eS. alterniflorus\u003c/em\u003e (Salt).\u003c/p\u003e","description":"","filename":"Picture5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8993757/v1/69a093d36b4253de826933a7.jpg"},{"id":104407872,"identity":"847ea797-602b-449a-9d38-81115cd5b872","added_by":"auto","created_at":"2026-03-11 12:40:39","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2009927,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8993757/v1/d9568d72-2bc7-49f3-96ac-730db13305bf.pdf"},{"id":104400968,"identity":"a448b7ae-cde7-4165-84d6-dcbb8b599b34","added_by":"auto","created_at":"2026-03-11 12:11:35","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":24325,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTables.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8993757/v1/8b8c828a157a6e2663b9601c.xlsx"},{"id":103854775,"identity":"63e8628c-1b79-40fb-a655-2fa08574dfb3","added_by":"auto","created_at":"2026-03-03 17:50:15","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":96132212,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigures.docx","url":"https://assets-eu.researchsquare.com/files/rs-8993757/v1/7bb7bc1d90e189454e9eb71f.docx"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003eKaryotype reconstruction provides new insights into polyploid evolution in grasses (Poaceae)\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePoaceae, one of the most ecologically and economically significant plant lineages, ranks as the fifth-largest angiosperm family, encompassing approximately 12,000 species distributed among 12 subfamilies\u003csup\u003e\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. The family's diversity is structured around two major clades: BOP (Bambusoideae, Oryzoideae, and Pooideae) and PACMAD (Panicoideae, Arundinoideae, Chloridoideae, Micrairoideae, Aristidoideae, and Danthonioideae). Additionally, three early-diverging subfamilies (Anomochlooideae, Pharoideae, and Puelioideae) occupy successive sister positions to the main BOP-PACMAD lineages\u003csup\u003e\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn general, the Poaceae family has undergone three rounds of WGD events (\u003cem\u003eσ\u003c/em\u003e, \u003cem\u003eτ\u003c/em\u003e, and \u003cem\u003eρ\u003c/em\u003e)\u003csup\u003e7\u0026ndash;11\u003c/sup\u003e. While the first two events are shared with other monocots, the third \u003cem\u003eρ\u003c/em\u003e-WGD event is specific to Poaceae and is thought to have driven the family's extensive adaptive radiation. Following the ρ-WGD event, numerous additional species-specific polyploidization events have occurred in the Poaceae family, such as tetraploidization and hexaploidization in \u003cem\u003eEchinochloa\u003c/em\u003e species\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Polyploidization plays an essential role in plant evolution by providing raw genetic material for functional innovation and adaptive diversification\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. This process has been recognized as a major driver of speciation, contributing to reproductive isolation and facilitating rapid adaptation to environmental changes\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Autopolyploids are formed through genome duplication, while allopolyploids arise from hybridization between different species with independent evolutionary histories, both of which are accompanied by chromosome doubling and gamete fusion\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Among polyploid species, allopolyploids are predominant, accounting for nearly 90% of cases, whereas autopolyploids represent only a small proportion\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. The initial evolutionary stages of recently formed allotetraploids can be traced, as exemplified by \u003cem\u003eTragopogon\u003c/em\u003e species\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. However, long-term evolution and divergence of allopolyploid lineages, such as \u003cem\u003eCenchrus\u003c/em\u003e and \u003cem\u003eSporobolus\u003c/em\u003e species, are more difficult to infer due to mutations, genomic rearrangements, and differential retention or loss of genes across subgenomes\u003csup\u003e\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Disentangling their evolutionary histories is possible through karyotype reconstruction using chromosome-level genomes, which provide phylogenetic markers based on conserved collinear gene blocks and shared chromosome fusions.\u003c/p\u003e \u003cp\u003ePost-polyploidization genome instability triggers chromosome breakages, abnormal recombination, and extensive gene losses, providing evolutionary opportunities for plant diversification\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Chromosome number reduction typically occurs through chromosome fusions accompanied by structural rearrangements\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. By performing comparative analyses of extant genomes, researchers have attempted to reconstruct the ancestral karyotype and elucidate evolutionary trajectories of chromosomes\u003csup\u003e\u003cspan additionalcitationids=\"CR26 CR27 CR28 CR29\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Traditional methods identify contiguous ancestral regions (CARs) by detecting collinear gene blocks. However, chromosomal rearrangements and short syntenic blocks create undefined gap regions, resulting in parameter-sensitive outputs that limit downstream analysis of chromosomal evolution\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. To address these limitations, pairwise genomic comparisons were employed to identify shared chromosome-like syntenic blocks (CLSBs) as protochromosomes\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Evolutionary trajectories from ancient origins to extant species can be reconstructed by tracing three principal types of chromosomal rearrangements: nested chromosome fusion (NCF), end-to-end joining (EEJ), and reciprocal chromosome translocation (RCT)\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. NCF occurs when one chromosome with free ends invades and integrates within the pericentromeric region of another chromosome\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. EEJ involves a crossover event near the termini of two chromosomes, resulting in the formation of one large chromosome and a dispensable mini-chromosome\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Both NCF and EEJ reduce chromosome number by one. However, RCT exchanges arms between two chromosomes through interstitial crossover, producing two recombinant chromosomes without altering chromosome numbers\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Additionally, different types of chromosomal rearrangements generate distinct numbers of fusion breakpoints: EEJ produces one breakpoint, while RCT and NCF each produce two breakpoints\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eKaryotype evolution research in Poaceae has historically focused on only a few crops like rice and sorghum, without a systematic cross-lineage perspective\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. With the accumulation of chromosome-level genomic resources in Poaceae and advances in karyotyping methodologies, more comprehensive and accurate karyotype reconstruction will become increasingly achievable, further advancing our knowledge of grass genome evolution. In this work, we reconstruct multiple ancestral karyotypes characterized by distinct chromosome structures within the Poaceae family. We use available chromosome-level genome assemblies from extant Poaceae species and analyze their karyotype evolution by explicitly considering three types of chromosomal rearrangements (RCTs, EEJs, and NCFs), rather than merely counting the number of fission and fusion events. Additionally, we investigate the origin and evolution of allopolyploid species in two genera based on comparative karyotype analysis.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAncestral karyotype of BOP and PACMAD\u003c/h2\u003e \u003cp\u003eWe established a comprehensive genomic resource encompassing 29 species spanning 7 Poaceae subfamilies for karyotype construction (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e; \u003cb\u003eSupplementary Fig.\u0026nbsp;1\u003c/b\u003e). These species exhibited diverse chromosome numbers and ploidy levels, representing the karyotypic diversity of the Poaceae family. All genome assemblies achieved high completeness (BUSCO\u0026thinsp;\u0026gt;\u0026thinsp;95%) and continuity (LAI\u0026thinsp;\u0026gt;\u0026thinsp;10), providing a reliable foundation for accurate karyotype construction. The only exception was \u003cem\u003eS. maritimus\u003c/em\u003e, which displayed a lower LAI value (7.4) due to its scaffold-level assembly. Nevertheless, its high BUSCO score (98.6%) ensured the reliability of gene-based karyotype analysis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe reconstructed the ancestral karyotype of BOP and PACMAD following the workflow of WGDI tool (details in Methods)\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Synteny comparisons between \u003cem\u003eO. sativa\u003c/em\u003e and species from different subfamilies of the BOP clade revealed that each \u003cem\u003eO. sativa\u003c/em\u003e chromosome exhibited CLSBs with corresponding chromosomes in BOP species (\u003cb\u003eSupplementary Fig.\u0026nbsp;2a\u003c/b\u003e), supporting \u003cem\u003eO. sativa\u003c/em\u003e chromosomes as protochromosomes of the BOP clade. Using the same approach, chromosomes from \u003cem\u003eP. australis\u003c/em\u003e subgenome A were identified as protochromosomes of the PACMAD clade (\u003cb\u003eSupplementary Fig.\u0026nbsp;2b\u003c/b\u003e). Notably, the karyotypes of \u003cem\u003eO. sativa\u003c/em\u003e and \u003cem\u003eP. australis\u003c/em\u003e subgenome A were highly similar. Therefore, we designated the 12 chromosomes of the \u003cem\u003eO. sativa\u003c/em\u003e genome as protochromosomes of the ancestral karyotype of BOP and PACMAD, named ABPK1 to ABPK12 (\u003cb\u003eSupplementary Fig.\u0026nbsp;2c\u003c/b\u003e).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eKaryotype evolution in BOP clade\u003c/h3\u003e\n\u003cp\u003eWe systematically examined chromosome number variation and fusion events across the Poaceae family (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Within the BOP clade, the paleodiploid \u003cem\u003eZizania latifolia\u003c/em\u003e (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;17, Oryzoideae) provides a clear instance of chromosome number reduction from a post-WGD state (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;24) via seven EEJs and one RCT (\u003cb\u003eSupplementary Fig.\u0026nbsp;3\u003c/b\u003e). \u003cem\u003eRaddia guianensis\u003c/em\u003e and \u003cem\u003eOlyra latifolia\u003c/em\u003e (both Bambusoideae) shared a nested fusion (abbreviated as ABPK_10\u0026thinsp;+\u0026thinsp;12_NCF), indicating that their divergence postdated this fusion. Notably, \u003cem\u003eR. guianensis\u003c/em\u003e represented the only documented case of chromosome fission in Poaceae (purple-dashed ellipse in \u003cb\u003eSupplementary Fig.\u0026nbsp;4\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eAs the most species-rich subfamily of Poaceae, Pooideae exhibits remarkable karyotypic variation. The ancestral karyotype of the Brachypodieae tribe (ABK, \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;10) was derived from ABPK through two NCFs (ABPK_8\u0026thinsp;+\u0026thinsp;10_NCF and ABPK_9\u0026thinsp;+\u0026thinsp;12_NCF), followed by one ABPK_(9\u0026thinsp;+\u0026thinsp;12_NCF)+11_RCT event (\u003cb\u003eSupplementary Fig.\u0026nbsp;5\u003c/b\u003e). The \u003cem\u003eBrachypodium distachyon\u003c/em\u003e genome (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5) was derived from the ABK genome through four NCFs (ABK_(2\u0026thinsp;+\u0026thinsp;6_NCF)+7_NCF, ABK_1\u0026thinsp;+\u0026thinsp;8_NCF, and ABK_3\u0026thinsp;+\u0026thinsp;4_NCF) and one EEJ event (ABK_5\u0026thinsp;+\u0026thinsp;10_EEJ), with the EEJ shared with \u003cem\u003eBrachypodium sylvaticum\u003c/em\u003e (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;9) (\u003cb\u003eSupplementary Fig.\u0026nbsp;6\u003c/b\u003e). In the tetraploid \u003cem\u003eBrachypodium hybridum\u003c/em\u003e genome (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;15), all five \u003cem\u003eB. distachyon\u003c/em\u003e-specific chromosome fusions were identified in the 5-chromosome subgenome, whereas the 10-chromosome subgenome had the \u003cem\u003eBrachypodium stacei\u003c/em\u003e genome structure without any rearrangement (\u003cb\u003eSupplementary Fig.\u0026nbsp;7\u003c/b\u003e). Based on the above results combined with the subgenome-level phylogenetic tree (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003e), we concluded that \u003cem\u003eB. distachyon\u003c/em\u003e and \u003cem\u003eB. stacei\u003c/em\u003e were most likely the donors of the two \u003cem\u003eB. hybridum\u003c/em\u003e subgenomes, respectively. This was consistent with previous conclusions\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. The ancestral karyotype of the Poeae and Triticeae tribes (APTK, \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7) was derived from ABPK through descending dysploidy involving four NCFs (ABPK_3\u0026thinsp;+\u0026thinsp;11_NCF, ABPK_4\u0026thinsp;+\u0026thinsp;7_NCF, ABPK_6\u0026thinsp;+\u0026thinsp;8_NCF, and ABPK_5\u0026thinsp;+\u0026thinsp;10_NCF) and one EEJ (ABPK_9\u0026thinsp;+\u0026thinsp;12_EEJ) (\u003cb\u003eSupplementary Fig.\u0026nbsp;8a, c\u003c/b\u003e). Within the Poeae tribe, \u003cem\u003eAlopecurus myosuroides\u003c/em\u003e evolved from APTK through one APTK_1\u0026thinsp;+\u0026thinsp;4_RCT (\u003cb\u003eSupplementary Fig.\u0026nbsp;9\u003c/b\u003e). Subsequently, the APTK genome was altered by a reciprocal translocation (APTK_1\u0026thinsp;+\u0026thinsp;6_RCT) towards the ancestral karyotype of Triticeae (ATK, \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7) preceding the diversification of this tribe (\u003cb\u003eSupplementary Fig.\u0026nbsp;8b, c\u003c/b\u003e). In relation to the Triticeae genome (\u003cb\u003eSupplementary Fig.\u0026nbsp;10a\u003c/b\u003e), \u003cem\u003eAegilops umbellulata\u003c/em\u003e shared the ATK_4\u0026thinsp;+\u0026thinsp;5_RCT event with \u003cem\u003eTriticum monococcum\u003c/em\u003e and had a reciprocal translocation (ATK_4\u0026thinsp;+\u0026thinsp;6_RCT) that arose independently from the similar translocation in \u003cem\u003eElymus sibiricus\u003c/em\u003e, as indicated by their different fusion breakpoint positions (\u003cb\u003eSupplementary Fig.\u0026nbsp;10b\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eKaryotype evolution in PACMAD clade\u003c/h3\u003e\n\u003cp\u003eWithin the PACMAD clade, although five subfamilies are recognized, chromosome-level genome assemblies for karyotype reconstruction are currently limited to three subfamilies: Panicoideae, Chloridoideae, and Arundinoideae (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Two NCF events (ABPK_3\u0026thinsp;+\u0026thinsp;10_NCF and ABPK_7\u0026thinsp;+\u0026thinsp;9_NCF) shaped the ancestral karyotype of Panicoideae subfamily (APEK, \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;10) from ABPK, and a subsequent NCF event (APEK_8\u0026thinsp;+\u0026thinsp;9_NCF) generated the ancestral karyotype of Paniceae tribe (APAK, \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;9) (\u003cb\u003eSupplementary Fig.\u0026nbsp;11\u003c/b\u003e). \u003cem\u003eMiscanthus sinensis\u003c/em\u003e (Andropogoneae) underwent a nested fusion between chromosomes 4 and 7 (APEK_4\u0026thinsp;+\u0026thinsp;7_NCF), reducing its chromosome number to \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;19 (\u003cb\u003eSupplementary Fig.\u0026nbsp;12\u003c/b\u003e). In the Panicoideae tribe, \u003cem\u003eSetaria italica\u003c/em\u003e and \u003cem\u003eS. viridis\u003c/em\u003e retained the ancestral chromosome number (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;9) altered by one APAK_3\u0026thinsp;+\u0026thinsp;7_RCT (\u003cb\u003eSupplementary Fig.\u0026nbsp;13\u003c/b\u003e). Both subgenomes of the allotetraploid \u003cem\u003eC. fungigraminus\u003c/em\u003e retained 7 chromosomes but differed in their evolutionary trajectories (\u003cb\u003eSupplementary Fig.\u0026nbsp;14a\u003c/b\u003e). A shared APAK_3\u0026thinsp;+\u0026thinsp;6_NCF occurred prior to subgenome divergence (\u003cb\u003eSupplementary Fig.\u0026nbsp;14b\u003c/b\u003e); subsequent APAK_4\u0026thinsp;+\u0026thinsp;9_RCT breakpoints differed between subgenomes, indicating independent fusion origins (\u003cb\u003eSupplementary Fig.\u0026nbsp;14c\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe ancestral karyotype of the Chloridoideae subfamily (ACK, \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;10) was shaped by two NCF events (ABPK_2\u0026thinsp;+\u0026thinsp;10_NCF and ABPK_6\u0026thinsp;+\u0026thinsp;9_NCF) (\u003cb\u003eSupplementary Fig.\u0026nbsp;15\u003c/b\u003e). Within the Cynodonteae tribe, the allotetraploid \u003cem\u003eCleistogenes songorica\u003c/em\u003e displayed asymmetric karyotype evolution: subgenome A retained the ancestral ACK structure without rearrangement, whereas subgenome B underwent two independent reciprocal translocations (ACK_1\u0026thinsp;+\u0026thinsp;5_RCT and ACK_3\u0026thinsp;+\u0026thinsp;7_RCT) (\u003cb\u003eSupplementary Fig.\u0026nbsp;16a\u003c/b\u003e). The shared presence of ACK_3\u0026thinsp;+\u0026thinsp;10_NCF in both subgenomes of \u003cem\u003eCynodon dactylon\u003c/em\u003e and ACK_9\u0026thinsp;+\u0026thinsp;10_NCF in both subgenomes of \u003cem\u003eEleusine coracana\u003c/em\u003e indicated these fusion events occurred in diploid ancestors prior to the respective WGD events (\u003cb\u003eSupplementary Fig.\u0026nbsp;16b, c\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eWithin the Zoysieae tribe, pairwise karyotype comparisons of fused chromosomes between subgenomes (A and B) of \u003cem\u003eS. maritimus\u003c/em\u003e and subgenomes (C and D) of \u003cem\u003eS. alterniflorus\u003c/em\u003e revealed patterns of chromosomal rearrangement (\u003cb\u003eSupplementary Fig.\u0026nbsp;17\u003c/b\u003e). All four subgenomes shared four chromosome fusion events (ACK_4\u0026thinsp;+\u0026thinsp;9_NCF, ACK_6\u0026thinsp;+\u0026thinsp;8_NCF, ACK_5\u0026thinsp;+\u0026thinsp;10_NCF, and ACK_2\u0026thinsp;+\u0026thinsp;3_RCT), indicating these rearrangements predated the divergence of these subgenomes. In addition, three subgenomes (A-C) each underwent two further chromosomal rearrangements (ACK_4\u0026thinsp;+\u0026thinsp;6_NCF and ACK_1\u0026thinsp;+\u0026thinsp;10_NCF), reducing the chromosome number to \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;15. By contrast, subgenome D experienced a unique ACK_4\u0026thinsp;+\u0026thinsp;10_NCF event, bringing chromosome number down to \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;16. This distinct karyotypic pattern in subgenome D suggested that it diverged earlier from the other subgenomes, consistent with the \u003cem\u003eK\u003c/em\u003es results (\u003cb\u003esee the last section\u003c/b\u003e). Moreover, the currently available tetraploid and hexaploid Arundinoideae species retained an ABPK-like karyotype (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003ePhylogenetic congruence of chromosome fusion distribution and uncertainty in the ancestral Poaceae karyotype\u003c/h3\u003e\n\u003cp\u003eTo comprehensively illustrate the karyotype evolution across the Poaceae family, we systematically identified 57 distinct chromosome fusion events (\u003cb\u003eSupplementary Table\u0026nbsp;2\u003c/b\u003e) in representative species (excluding \u003cem\u003ePharus latifolius\u003c/em\u003e), and constructed a subgenome-level phylogeny, with \u003cem\u003eP. latifolius\u003c/em\u003e as the outgroup (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). The resulting phylogeny resolved the evolutionary relationships of subgenomes across different subfamilies, which is consistent with previous studies\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. The characterization of multiple cascading fusion events allowed us to construct the ABPK and other ancestral karyotypes, revealing different evolutionary trajectories from a common ancestor (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). As shown in the heatmap, (i) chromosome fusions were not shared between subfamilies, providing evidence for independent karyotype evolution in each lineage, (ii) within each subfamily, species belonging to the same clade shared chromosome fusions, and the distribution of these shared fusions (marked by black rectangles) was highly congruent with the phylogenetic relationships inferred from single-copy orthologous genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Notably, several species retained the same karyotype as ABPK without undergoing any chromosome fusion events.\u003c/p\u003e \u003cp\u003eWithin Poaceae, Pharoideae serves as the sister lineage to the BOP and PACMAD clades. Karyotype comparisons between the \u003cem\u003eP. latifolius\u003c/em\u003e genome, regarded as the ancestral karyotype of Pharoideae (APHK), and the ancestral ABPK genome revealed a reciprocal translocation (ABPK_1\u0026thinsp;+\u0026thinsp;7_RCT or APHK_1\u0026thinsp;+\u0026thinsp;7_RCT), but the evolutionary direction of this RCT event could not be determined (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). Earlier studies suggested that the rice genome (referred to as ABPK) was the ancestral karyotype of Poaceae\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. However, chromosome fusion analysis revealed two possible scenarios: either the ancestral karyotype resembled APHK, with APHK_1\u0026thinsp;+\u0026thinsp;7_RCT generating the ABPK genome configuration, or ABPK represented the ancestral karyotype state, with ABPK_1\u0026thinsp;+\u0026thinsp;7_RCT producing the APHK genome structure.\u003c/p\u003e \u003cp\u003eTo determine the directionality of the RCT event and thus infer the ancestral karyotype state, we extracted 100 genes flanking each side of the fusion breakpoints from RCT (ABPK_1\u0026thinsp;+\u0026thinsp;7_RCT or APHK_1\u0026thinsp;+\u0026thinsp;7_RCT) as individual segments and performed synteny analysis with closely related non-Poaceae outgroup species (\u003cem\u003eAnanas comosus\u003c/em\u003e and \u003cem\u003eJuncus effusus\u003c/em\u003e). In principle, when synteny blocks spanning the 200-gene segment are detected in outgroup genomes, the karyotype (ABPK or APHK) possessing the 200-gene segment is more closely related to the outgroup species and is considered to represent the ancestral karyotype of Poaceae\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. However, we could not identify any syntenic blocks near the breakpoints (\u003cb\u003eSupplementary Fig.\u0026nbsp;18\u003c/b\u003e) owing to extensive chromosomal rearrangements during the Poaceae-specific ρ-WGD (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7 to \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;12)\u003csup\u003e39\u003c/sup\u003e. Hence, the true ancestral Poaceae karyotype remains uncertain.\u003c/p\u003e\n\u003ch3\u003eThe phylogenomic history of Cenchrus species\u003c/h3\u003e\n\u003cp\u003eThe phylogeny of \u003cem\u003eC. purpureus\u003c/em\u003e, \u003cem\u003eC. fungigraminus\u003c/em\u003e, and \u003cem\u003eC. americanus\u003c/em\u003e is currently undetermined. To investigate their evolutionary relationships, we conducted pairwise karyotype comparisons of (sub)genomes of three species. Although subgenomes A and B within each allotetraploid species showed significant karyotypic divergence (\u003cb\u003eSupplementary Fig.\u0026nbsp;19\u003c/b\u003e), subgenome A of both \u003cem\u003eC. purpureus\u003c/em\u003e and \u003cem\u003eC. fungigraminus\u003c/em\u003e exhibited highly conserved karyotypes largely consistent with genome of \u003cem\u003eC. americanus\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). Similarly, subgenome B of both \u003cem\u003eC. purpureus\u003c/em\u003e and \u003cem\u003eC. fungigraminus\u003c/em\u003e also exhibited high karyotypic concordance.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe further performed whole-genome mapping analyses by aligning 150-mer reads derived from the diploid \u003cem\u003eC. americanus\u003c/em\u003e genome against both \u003cem\u003eC. purpureus\u003c/em\u003e and \u003cem\u003eC. fungigraminus\u003c/em\u003e genomes. Subgenome A of \u003cem\u003eC. purpureus\u003c/em\u003e and \u003cem\u003eC. fungigraminus\u003c/em\u003e showed moderate coverage depths of approximately 45% and 50%, respectively (\u003cb\u003eSupplementary Fig.\u0026nbsp;20\u003c/b\u003e), suggesting that \u003cem\u003eC. americanus\u003c/em\u003e was likely a close relative of the diploid progenitor that contributed subgenome A to both \u003cem\u003eC. purpureus\u003c/em\u003e and \u003cem\u003eC. fungigraminus\u003c/em\u003e. Following the same analytical approach, we aligned 150-mer reads from subgenomes A and B of \u003cem\u003eC. purpureus\u003c/em\u003e against the \u003cem\u003eC. fungigraminus\u003c/em\u003e genome, achieving coverage depths of approximately 80% and 85% for subgenomes A and B of \u003cem\u003eC. fungigraminus\u003c/em\u003e, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed, e). The high level of sequence similarity between corresponding subgenomes strongly supported the hypothesis that \u003cem\u003eC. purpureus\u003c/em\u003e and \u003cem\u003eC. fungigraminus\u003c/em\u003e share two diploid progenitors for their respective A and B subgenomes. This interpretation is further validated by the subgenome tree (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003eWe calculated the synonymous substitution rate (\u003cem\u003eK\u003c/em\u003es) values for homeologous gene pairs between \u003cem\u003eCenchrus\u003c/em\u003e (sub)genomes. According to the \u003cem\u003eK\u003c/em\u003es peak, we determined that the two unknown diploid progenitors (diploids A and B) of the \u003cem\u003eCenchrus\u003c/em\u003e species diverged approximately 9.5\u0026nbsp;million years ago (mya). Subsequently, diploid A diverged from \u003cem\u003eC. americanus\u003c/em\u003e around 4.5 mya (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb, c). The \"bubble\" peak in the transposable element (TE) divergence curve indicated that the allotetraploid speciation event occurred approximately 2.9 mya (\u003cb\u003eSupplementary Fig.\u0026nbsp;21\u003c/b\u003e). Furthermore, the divergence time between \u003cem\u003eC. purpureus\u003c/em\u003e and \u003cem\u003eC. fungigraminus\u003c/em\u003e was estimated at 0.6 mya.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAllopolyploid evolution of\u003c/b\u003e \u003cb\u003eSporobolus\u003c/b\u003e \u003cb\u003especies\u003c/b\u003e\u003c/p\u003e \u003cp\u003eBy using \u003cem\u003eEragrostis tef\u003c/em\u003e subgenome A as a reference and Chr3 as an example, we were able to readily identify four homeologous blocks in \u003cem\u003eS. maritimus\u003c/em\u003e and \u003cem\u003eS. alterniflorus\u003c/em\u003e, respectively. Based on the complementarity and completeness of these synteny blocks, we phased them into distinct sub-subgenomes (details in Methods; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). To further test relationships among sub-subgenomes, we then extracted collinear genes from these synteny blocks and constructed gene trees, rooted on \u003cem\u003eE. tef\u003c/em\u003e Chr3. Phylogenies constructed from 1243 collinear genes of sub-subgenomes with at least six collinear genes resulted in 1243 topologies, from which we obtained a consensus tree (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). The coexisting sub-subgenomes within the genus \u003cem\u003eSporobolus\u003c/em\u003e formed two clusters (labeled S1 and S2) that fell into different clades, suggesting that the extant subgenomes (A-D) of \u003cem\u003eSporobolus\u003c/em\u003e species originated from an allotetraploid event. We further tested this hypothesis by phasing the \u003cem\u003eSporobolus\u003c/em\u003e subgenomes and constructing 10 phylogenetic trees using individual chromosomes from \u003cem\u003eE. tef\u003c/em\u003e subgenome A as an outgroup, all of which consistently supported the allopolyploid origin (\u003cb\u003eSupplementary Figs.\u0026nbsp;22 and 23\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe reconstructed karyotype schematics to trace the evolutionary history of two \u003cem\u003eSporobolus\u003c/em\u003e species (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed). Based on synchronous \u003cem\u003eK\u003c/em\u003es peaks at 0.22 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec), the diploid ACK genome is estimated to have undergone WGD1 approximately 15 mya, yielding a shared tetraploid intermediate (n\u0026thinsp;=\u0026thinsp;2x\u0026thinsp;=\u0026thinsp;20). Subsequent descending dysploidy via chromosome fusions restructured the intermediate into two diploidized subgenomes with \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;\u003cem\u003ex\u003c/em\u003e = 16 and \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;\u003cem\u003ex\u003c/em\u003e = 15 chromosomes, respectively. These subgenomes then underwent lineage-specific WGD2, generating \u003cem\u003eS. maritimus\u003c/em\u003e (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2\u003cem\u003ex\u003c/em\u003e\u0026thinsp;=\u0026thinsp;30) at approximately 0.5 mya and \u003cem\u003eS. alterniflorus\u003c/em\u003e (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2\u003cem\u003ex\u003c/em\u003e\u0026thinsp;=\u0026thinsp;31) at approximately 0.9 mya (\u003cb\u003eSupplementary Fig.\u0026nbsp;24a, b\u003c/b\u003e). Unlike the ancient shared WGD1, these recent WGD2 events occurred independently of each other.\u003c/p\u003e \u003cp\u003eChromosome-scale synteny analysis of selected homologous chromosomes and an ancient chromosome (marked by red triangles in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed) revealed distinct chromosomal syntenic scenarios (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee). Chromosomes with two distinct color blocks reflected their derivation from ancestral chromosome fusions. Notably, Chr31 of \u003cem\u003eS. alterniflorus\u003c/em\u003e (Salt_31) displayed one-to-one syntenic correspondence with Chr6 of \u003cem\u003eE. tef\u003c/em\u003e (Etef_6), demonstrating that Salt_31 represented an intact karyotype structure inherited from the ancestral chromosome, rather than a product of non-reciprocal translocation as previously proposed\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eWe have reconstructed ABPK (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;12) as the ancestral karyotype of the analyzed genomes of clades BOP and PACMAD. Our results show that modern genomes can be traced back to the ancestral karyotype through multiple cascading chromosomal rearrangements. Based on the order in which these chromosome fusions occur, we can determine the evolutionary trajectories of extant species. The Paniceae tribe provides a clear example: the ancestral karyotype ABPK underwent sequential chromosomal changes, first forming APEK, which then evolved into APAK, and subsequently gave rise to \u003cem\u003eS. italica\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. This stepwise progression of fusion events reveals the evolutionary pathway more precisely than previous strategies\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e,\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eBy determining if genes on either side of a fusion breakpoint lie within a single synteny block, we could identify shared chromosomal rearrangements between species with significantly higher accuracy. Sometimes, seemingly identical fusion events from the same ancestral chromosome pairs show distinct breakpoint positions, such as APAK_4\u0026thinsp;+\u0026thinsp;9_RCT in the two subgenomes of \u003cem\u003eC. fungigraminus\u003c/em\u003e and ATK_4\u0026thinsp;+\u0026thinsp;6_RCT in \u003cem\u003eA. umbellulata\u003c/em\u003e versus \u003cem\u003eE. sibiricus\u003c/em\u003e (\u003cb\u003eSupplementary Figs.\u0026nbsp;10 and 14)\u003c/b\u003e. Corresponding ancestral chromosomes as rearrangement hotspots experience multiple fusions, suggesting that certain chromosomal regions may be particularly prone to structural instability during genome evolution, similar fusions patterns have also been reported in the karyotype evolution of other plant species\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn this study, we identified only one chromosome fission event, consistent with a growing body of research suggesting that fusions, rather than fissions, are the primary drivers of karyotype changes\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e,\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. The distribution of shared versus lineage-specific chromosome fusions provides a clear signature of karyotype evolution in Poaceae. The subfamily-specific fusions acted as key drivers of reproductive isolation, contributing to the divergence and establishment of distinct subfamilies, while shared fusions among closely related species reflect stable inheritance of karyotypic configurations (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003ea\u003cb\u003e)\u003c/b\u003e. Fusion events that predated divergence will be shared across descendant species, while those occurring post-divergence will be species-specific. For instance, in \u003cem\u003eO. latifolia\u003c/em\u003e, ABPK_10\u0026thinsp;+\u0026thinsp;12_NCF occurred before divergence from \u003cem\u003eRaddia guianensis\u003c/em\u003e, whereas ABPK_2\u0026thinsp;+\u0026thinsp;5_EEJ arose after divergence (\u003cb\u003eSupplementary Fig.\u0026nbsp;4)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eOur karyotype reconstruction further proves its utility by providing crucial structural insights into evolutionary relationships of polyploid lineages. For Cenchrus species, pairwise karyotype comparisons between (sub)genomes, integrated with \u003cem\u003eK\u003c/em\u003es analysis and whole-genome sequence mapping, provide a high-resolution lens on their evolutionary history (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Our results clarify that \u003cem\u003eC. americanus\u003c/em\u003e is a sister lineage to the diploid progenitor of subgenome A in the allopolyploids \u003cem\u003eC. purpureus\u003c/em\u003e and \u003cem\u003eC. fungigraminus\u003c/em\u003e, rather than the direct donor, consistent with previous conclusions\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. We confirm that \u003cem\u003eC. purpureus\u003c/em\u003e and \u003cem\u003eC. fungigraminus\u003c/em\u003e share common diploid progenitors for their respective subgenomes A and B, resolving evolutionary relationships that remained ambiguous in prior studies\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. We further reconstructed the karyotype of \u003cem\u003eC. fungigraminus\u003c/em\u003e and characterized specific chromosome fusion events derived from the ancestral APAK genome (\u003cb\u003eSupplementary Fig.\u0026nbsp;14)\u003c/b\u003e, which significantly improved the accuracy of evolutionary trajectory inference relative to previous studies that only estimated the number of likely chromosomal rearrangements occurring during karyotype evolution\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eSimilarly, for \u003cem\u003eSporobolus\u003c/em\u003e species, through manual scaffold reordering (\u003cb\u003eSupplementary Fig.\u0026nbsp;25)\u003c/b\u003e, we generated a near-chromosome-level \u003cem\u003eS. maritimus\u003c/em\u003e assembly and successfully partitioned the genomes of \u003cem\u003eS. maritimus\u003c/em\u003e and \u003cem\u003eS. alterniflorus\u003c/em\u003e into four subgenomes (A-D), each comprising two sub-subgenomes, confirming that both species are paleotetraploids rather than hexaploids as previously proposed\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e(\u003cb\u003eSupplementary Fig.\u0026nbsp;22)\u003c/b\u003e. The polyploid origin of \u003cem\u003eSporobolus\u003c/em\u003e species has remained contentious, recent work showed that distinguishing between autopolyploidy and allopolyploidy was not feasible due to the absence of extant diploid progenitors and tetraploid intermediates\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. To address this challenge, we employed the \u003cem\u003eE. tef\u003c/em\u003e subgenome A as a reference for partitioning homeologous chromosome blocks into their corresponding sub-subgenomes and constructed 10 phylogenetic trees using individual chromosomes of \u003cem\u003eE. tef\u003c/em\u003e subgenome A as an outgroup, thereby establishing the initial allopolyploid origin of both Sporobolus species\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e (\u003cb\u003eSupplementary Fig.\u0026nbsp;23)\u003c/b\u003e. Karyotype evolution in \u003cem\u003eSporobolus\u003c/em\u003e species involves complex chromosomal rearrangements following polyploidization. Previous studies only characterized post-rediploidization karyotype changes and hypothesized the number of chromosome fusions in Sporobolus species, without considering the detailed karyotype changes from the ACK to \u003cem\u003eSporobolus\u003c/em\u003e species\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Here, we present a comprehensive karyotype reconstruction to trace the evolutionary trajectory of \u003cem\u003eSporobolus\u003c/em\u003e species. Contrary to the two kinds of karyotype evolution trajectories hypotheses proposed by Salmon et al.\u003csup\u003e21\u003c/sup\u003e, our findings revealed that the ACK genome underwent a shared WGD1 event followed by lineage-specific chromosomal rearrangements that generated distinct subgenomes (A-D). Subgenomes A and B subsequently underwent WGD2 to form \u003cem\u003eS. maritimus\u003c/em\u003e, with karyotype evolution involving 12 chromosome fusions (including two RCTs) that reduced the chromosome complement from theoretical \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;40 (octoploid) to \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;30 (paleotetraploid) (\u003cb\u003eSupplementary Fig.\u0026nbsp;17\u003c/b\u003e). Similarly, WGD2 of subgenomes C and D produced S. alterniflorus, with 11 chromosome fusions (including two RCTs) decreasing the chromosome number from theoretical octoploid number (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;40) to the current paleotetraploid complement (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;31). The exact match between chromosome number reduction and chromosome fusion events robustly validates the accuracy of our karyotype constructions. Unlike previous reports\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e, our results indicate that WGD2 is not shared between \u003cem\u003eS. maritimus\u003c/em\u003e and \u003cem\u003eS. alterniflorus\u003c/em\u003e. By using the ACK genome as a reference, combined with chromosome karyotype reconstruction, we successfully disentangled the complex evolutionary relationships between two \u003cem\u003eSporobolus\u003c/em\u003e species.\u003c/p\u003e \u003cp\u003eIdentification of shared chromosome fusions between species provides an independent line of evidence that can corroborate and refine topology inferences, particularly when sequence-based phylogenetic methods are challenged by introgression, homoplasy, and incomplete lineage sorting over deep evolutionary timescales\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. Nevertheless, the approach has inherent limitations that should be carefully considered. It is largely ineffective for phylogenetic inference when comparing genomes with conserved karyotypes, such as genomes from subfamily Arundinoideae\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. Widespread gene losses in genomes can lead to the disappearance of gene collinearity, which may render the inference of chromosomal karyotypes extremely difficult. Additionally, karyotype reconstruction requires high-quality chromosome-level genome assemblies. Poor assembly quality can lead to the false-positive identification of chromosomal rearrangements and imprecise location of fusion breakpoints, resulting in misleading interpretations of evolutionary relationships between species.\u003c/p\u003e \u003cp\u003eTo date, the fate of diploid progenitors of subgenomes A and B in \u003cem\u003eCenchrus\u003c/em\u003e remains unclear\u0026mdash;they may have gone extinct or may persist but lack chromosome-level genomic resources. Expanded taxonomic sampling across \u003cem\u003eCenchrus\u003c/em\u003e species will be necessary to resolve the outstanding question. Additionally, the structure of the most ancestral Poaceae karyotype remains unresolved. It may eventually be resolved as more chromosome-level genomes become available, especially those from early-diverging subfamilies.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eData collection\u003c/h2\u003e \u003cp\u003eWe collected all available chromosome-level genomes in Poaceae, together with several phylogenetically related outgroup genomes from public databases\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. Given the comprehensive characterization of Bambusoideae karyotypes in recent studies\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e, only two representative species from this subfamily were selected here. Following stringent criteria for assembly contiguity and completeness, we selected 29 representative genomes spanning the major Poaceae lineages (a list of genomes see Supplementary Table\u0026nbsp;1) for karyotype reconstruction and included additional species for comparative karyotype analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003ePhasing subgenomes with subphaser\u003c/h2\u003e \u003cp\u003eSubphaser is an automated pipeline based on subgenome-specific \u003cem\u003ek\u003c/em\u003e-mers\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. The assembled genome and homeologous chromosome relationships were input into SubPhaser (v 1.2.6)\u003csup\u003e50\u003c/sup\u003e to obtain subgenome phasing results. Potential inter-subgenomic translocations and homeologous exchanges between subgenomes were also detected. For \u003cem\u003eS. maritimus\u003c/em\u003e, initial subgenome phasing revealed ambiguous assignments and large-scale apparent homeologous exchanges in several regions (marked by red dashed ellipses in \u003cb\u003eSupplementary Fig.\u0026nbsp;25a, c\u003c/b\u003e), likely reflecting scaffold ordering errors caused by high sequence similarity between homeologous chromosomes rather than genuine biological recombination events. Following manual scaffold repositioning (\u003cb\u003eSupplementary Table\u0026nbsp;3)\u003c/b\u003e, the optimized assembly achieved correct subgenome partitioning, with chromosomes 1\u0026ndash;15 assigned to subgenome A and chromosomes 16\u0026ndash;30 assigned to subgenome B (\u003cb\u003eSupplementary Fig.\u0026nbsp;25b, d\u003c/b\u003e). The two subgenomes of \u003cem\u003eS. alterniflorus\u003c/em\u003e were successfully phased in a default subphaser run, with chromosomes 1\u0026ndash;15 assigned to subgenome C and chromosomes 16\u0026ndash;31 assigned to subgenome D (\u003cb\u003eSupplementary Fig.\u0026nbsp;26a, b\u003c/b\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eAncestral karyotype reconstruction\u003c/h2\u003e \u003cp\u003eTo reconstruct ancestral karyotypes of BOP and PACMAD clades, we performed pairwise genome comparisons using WGDI\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Within the BOP clade, we selected \u003cem\u003eO. sativa\u003c/em\u003e (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;12) as the anchor genome because it has the highest haploid chromosome number within the clade and thus likely retains more ancestral karyotypic features than other BOP members. Three species from distinct subfamilies (Pooideae, Bambusoideae, and Oryzoideae) were then independently aligned against \u003cem\u003eO. sativa\u003c/em\u003e using WGDI (with the '-d' parameter) to generate homologous dotplots. Similarly, for the PACMAD clade, we used \u003cem\u003eP. australis\u003c/em\u003e subgenome A (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;12) as the anchor genome and performed alignments with three species representing Chloridoideae, Panicoideae, and Arundinoideae subfamilies, respectively. Given that fusion or fission of ancestral chromosomes occur gradually and randomly in the offspring lineages, intact protochromosomes are expected to be preserved in some descendants. Based on this reasoning, we identified shared CLSBs and extracted intact chromosomes as putative protochromosomes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eKaryotypic evolution in Poaceae\u003c/h2\u003e \u003cp\u003eTo reconstruct karyotype evolution in Poaceae, we employed a comparative genomics approach that inferred chromosomal changes from ancestral karyotypes to extant genomes. The WGDI workflow began with the identification of collinear gene pairs between species using the improved collinearity module ('-icl') followed by synteny block integration ('-bi'). Stringent filtering was then applied through the correspondence module ('-c') to eliminate syntenic regions potentially derived from earlier polyploidization events. Protochromosomes were mapped onto the chromosomes of the sample genomes via the karyotype mapping function ('-km'). Furthermore, the parameter '-sf' was employed to rapidly identify chromosome fusions, with shared fusions and their corresponding fusion breakpoints subsequently recorded. Fusion breakpoint coordinates were extracted using the '-fpd' parameter, which generated a comprehensive dataset recording fusion positions. Finally, we applied the '-fd' module to determine the occurrence of these breakpoints in other genomes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003ePhylogenomic analysis\u003c/h2\u003e \u003cp\u003eWe reconstructed phylogenetic relationships using 43 subgenomes from 29 Poaceae species. Orthogroups were identified using OrthoFinder (v 2.5.4)\u003csup\u003e51\u003c/sup\u003e with subgenome protein sequences. We retained high-confidence orthogroups present in at least 35 subgenomes (\u0026gt;\u0026thinsp;80% taxonomic sampling), with a single gene copy per subgenome. Protein sequences within each orthogroup were aligned using MUSCLE (v 5.1)\u003csup\u003e52\u003c/sup\u003e, and the resulting multiple sequence alignments were concatenated into a supermatrix. Maximum-likelihood phylogenetic inference was performed using IQ-TREE (v 2.1.3)\u003csup\u003e53\u003c/sup\u003e under the best-fit substitution model selected by ModelFinder, with branch support assessed by 1,000 ultrafast bootstrap replicates.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eDivergence and merger time estimation\u003c/h2\u003e \u003cp\u003eWe estimated the divergence time between subgenomes and subsequent merger time for allopolyploid \u003cem\u003eSporobolus\u003c/em\u003e species (\u003cem\u003eS. alterniflorus\u003c/em\u003e and \u003cem\u003eS. maritimus\u003c/em\u003e) and \u003cem\u003eCenchrus\u003c/em\u003e species (\u003cem\u003eC. americanus\u003c/em\u003e, \u003cem\u003eC. fungigraminus\u003c/em\u003e, and \u003cem\u003eC. purpureus\u003c/em\u003e). We first aligned protein sequences among subgenomes using BLASTP (v 2.14.0)\u003csup\u003e54\u003c/sup\u003e with an E-value cutoff of 1e\u003csup\u003e\u0026minus;\u0026thinsp;10\u003c/sup\u003e. DAGchainer (r02-06-2008)\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e was used to identify syntenic blocks with at least five homologous gene pairs within a region of 10 gene models based on best-hit blastp results. The \u003cem\u003eK\u003c/em\u003es values for syntenic homeologous gene pairs were calculated by \u003cem\u003eK\u003c/em\u003ea\u003cem\u003eK\u003c/em\u003es_Calculator in NG mode. The \u003cem\u003eK\u003c/em\u003es distribution peaks were used to calculate the divergence time using the formula \u003cem\u003eT\u003c/em\u003e\u0026thinsp;=\u0026thinsp;\u003cem\u003eK\u003c/em\u003es/2\u003cem\u003er\u003c/em\u003e, where \u003cem\u003er\u003c/em\u003e is the nucleotide substitution rate (\u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.5 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;9\u003c/sup\u003e mutations \u0026times; bp\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e \u0026times; generation\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). TE divergence was assessed using PercDivs (percentage of substitutions in the matching region compared with the consensus), calculated by RepeatMasker (v 4.2.0) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/rmhubley/repeatmasker\u003c/span\u003e\u003cspan address=\"https://github.com/rmhubley/repeatmasker\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The TE sequence divergence between two subgenomes displaying a high degree of overlap suggested consistency in the TE evolutionary rate in the two subgenomes. The non-overlapping segregation region indicated the timeframe from diploid progenitor divergence to tetraploid genome formation.\u003c/p\u003e \u003cp\u003e \u003cb\u003eK\u003c/b\u003e \u003cb\u003e-mer-based whole-genome mapping analysis\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo investigate evolutionary relationships between \u003cem\u003eCenchrus\u003c/em\u003e species, \u003cem\u003ek\u003c/em\u003e-mer-based whole-genome mapping analysis was performed as previously described\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Query (sub)genome sequences were fragmented into 150-mers with a 5 bp step size, and these short reads were subsequently mapped to the reference genome using Bowtie2 (v 2.5.1)\u003csup\u003e56\u003c/sup\u003e. Coverage depth was calculated using SAMtools (v 1.23)\u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e to quantify sequence similarity.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003ePhasing sub-subgenomes with WGDI\u003c/h2\u003e \u003cp\u003eSubphaser toolkit\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e independently partitioned \u003cem\u003eS. alterniflorus\u003c/em\u003e and \u003cem\u003eS. maritimus\u003c/em\u003e into two subgenomes, each comprising two chromosome sets (termed sub-subgenomes). To resolve the evolutionary relationships among sub-subgenomes within the \u003cem\u003eSporobolus\u003c/em\u003e species, we used \u003cem\u003eE. tef\u003c/em\u003e subgenome A as the reference for phasing of \u003cem\u003eS. alterniflorus\u003c/em\u003e and \u003cem\u003eS. maritimus\u003c/em\u003e. First, protein sequences from \u003cem\u003eSporobolus\u003c/em\u003e species were compared against those of the reference genome using DIAMOND (v 2.0.15)\u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. Collinear regions were identified and integrated using WGDI with the '-icl' and '-bi' parameters. Orthologous syntenic blocks derived from recent polyploidy events were retained using WGDI with the '-c' filtering option. The reference genome karyotype was then mapped onto \u003cem\u003eSporobolus\u003c/em\u003e chromosomes based on syntenic relationships using WGDI with the '-km' parameter. Sub-subgenomes were manually assigned based on complementarity and completeness of synteny blocks. Hierarchical gene lists (one sub-subgenome per column) were generated using WGDI with the '-pc' and '-a' parameters. For each chromosome of the reference genome, maximum likelihood gene trees were inferred from the merged hierarchical gene lists using built-in IQ-TREE with automatic selection of the best-fit substitution model (-m MFP) through the WGDI '-at' function. These gene trees were input to ASTRAL-Pro (v 1.19.3.6)\u003csup\u003e59\u003c/sup\u003e to construct the sub-subgenome phylogeny. The resulting phylogeny was visualized and evaluated using Newick utilities (v 1.6)\u003csup\u003e60\u003c/sup\u003e and Phytop (v 0.3)\u003csup\u003e61\u003c/sup\u003e. To ensure robust sub-subgenome classification, the analysis pipeline ('-pc', '-a', and '-at' options in WGDI, followed by ASTRAL-Pro) was iterated until topological consistency converged.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eChromosome synteny visualization\u003c/h2\u003e \u003cp\u003eChromosome-level synteny was analyzed across ten chromosomes from \u003cem\u003eE. tef\u003c/em\u003e (Chr 6), \u003cem\u003eS. alterniflorus\u003c/em\u003e (Chr 31, 3, 18, 2, and 17), and \u003cem\u003eS. maritimus\u003c/em\u003e (Chr 3, 18, 2, and 17). Pairwise syntenic blocks between chromosomes were detected using JCVI (v 1.5.7)\u003csup\u003e62\u003c/sup\u003e, requiring\u0026thinsp;\u0026ge;\u0026thinsp;30 gene pairs per block with \u0026le;\u0026thinsp;20 intervening genes. Coordinates of anchor genes were extracted from BED files and converted to NGenomeSyn link format with strand directionality preserved. The resulting syntenic links were visualized using NGenomeSyn (v 1.43)\u003csup\u003e\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e with customized color schemes for different collinearity relationships.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eAuthor countribution\u003c/h2\u003e \u003cp\u003eL.F., P.S., and D.W. led and coordinated the project. H.C. and L.J. collected the assemblies. X.H. and K.Y. performed quality control. H.C., S.Z., S.Z., R.Z., and Z.L. analyzed Poaceae genomes. H.C., X.H., and Y.Z. finished karyotype and phylogenetic analyses. H.C., P.S., D.W., Q.C., and Y.H. provided gramine quantification. L.F., P.S., D.W., H.C., and Y.H. discussed the results. L.F. and P.S. supervised all analyses. H.C. wrote the initial manuscript with input from all co-authors. All authors discussed the results and commented on the manuscript.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eAll authors declare no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eThis work was supported by National Key Research and Development Program (2023YFD1400502).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eKellogg EA (2000) The grasses: A case study in macroevolution. Annu Rev Ecol Evol Syst 31:217\u0026ndash;238\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKellogg EA (2001) Evolutionary history of the Grasses1. Plant Physiol 125:1198\u0026ndash;1205\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSoreng RJ et al (2015) A worldwide phylogenetic classification of the poaceae (gramineae). 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Bioinformatics 38:4145\u0026ndash;4152\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"National Key Research and Development Program","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Ancestral karyotype, Chromosome fusion, Whole-genome duplication, Poaceae","lastPublishedDoi":"10.21203/rs.3.rs-8993757/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8993757/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eChromosomal rearrangements following whole-genome duplication (WGD) shape the diversity of genome structures in Poaceae. An accurate karyotype construction based on high-quality genomes of extant species will facilitate a deeper understanding of the origin and evolution of Poaceae. Here, we determine ancestral karyotypes for the BOP and PACMAD clades (ABPK, \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;12) and further infer ancestral karyotypes for subfamilies and tribes with distinct chromosome structures. Evolutionary trajectories from ancestral karyotypes to modern genomes are reconstructed for 29 species across 7 subfamilies through the characterization of multiple cascading fusion events. Our results reveal that subfamily-specific fusions contribute to the divergence and establishment of distinct subfamilies, while shared fusions among closely related species within each subfamily reflect the stable inheritance of karyotypic architecture. Based on the comparative karyotype analysis, we elucidate that \u003cem\u003eCenchrus purpureus\u003c/em\u003e and \u003cem\u003eC. fungigraminus\u003c/em\u003e share two diploid progenitors contributing to their respective subgenomes A and B, and that Paleotetraploid \u003cem\u003eS. maritimus\u003c/em\u003e and \u003cem\u003eS. alterniflorus\u003c/em\u003e share the first allotetraploid WGD (WGD1) but experience independent second WGD (WGD2) events. Our results highlight the importance of chromosome fusion in descending dysploidy after recurrent polyploidization and shed light on the polyploidization and karyotype evolution across the Poaceae family.\u003c/p\u003e","manuscriptTitle":"Karyotype reconstruction provides new insights into polyploid evolution in grasses (Poaceae)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-03 17:49:53","doi":"10.21203/rs.3.rs-8993757/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8f54a6c4-16b1-45a2-9415-98e31713cd4c","owner":[],"postedDate":"March 3rd, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-03-03T17:49:53+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-03 17:49:53","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8993757","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8993757","identity":"rs-8993757","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}