Kinetochore mutations and histone phosphorylation pattern changes accompany holo- and macro-monocentromere evolution

Domain: biology
Published: 2025-12-20
Editorial outcome: major_revision

Abstract
Centromeres are essential for kinetochore assembly and spindle attachment. While chromosomes of most species are monocentric with a single centromere, a minority exhibit holocentricity, with a centromere along the chromatid length. Sporadic emergence of holocentricity suggests multiple independent transitions. To explore this, we compare the centromere and (epi)genome organization of two sister genera with contrasting centromere types: Chamaelirium luteum with large macro-monocentromeres and Chionographis japonica with holocentromeres. Both exhibit chromosome-wide histone phosphorylation patterns distinct from typical monocentric species. Kinetochore analysis reveals similar chimeric Borealin in both species, with additional KNL2 loss and NSL1 chimerism in Cha. luteum. The broad-scale synteny between both genomes supports de novo holocentromere formation in Chi. japonica. Despite sharing features with both centromere types, macro-monocentromeres do not represent a direct link between mono- and holocentromeres. We propose a model for the divergent evolution involving kinetochore gene mutations, altered histone phosphorylation patterns, and centromeric satellite DNA amplification.

Introduction
Centromeres, the constricted regions of chromosomes that connect sister chromatids, are essential for chromosome segregation in eukaryotes. Most organisms harbor monocentric chromosomes, which are characterized by primary constrictions1, where the kinetochore assembles and spindle microtubules attach. A minority of species harbor an atypical centromeric organization known as holocentricity, in which centromeres are distributed over the entire chromatid length. Due to the telomere-to-telomere distribution of the holocentromere, sister chromatids cohere throughout their entire lengths and appear in mitotic chromosomes as two parallel structures, without a primary constriction2. Each holocentromere comprises multiple ‘centromere units’, which, depending on the species investigated, possess either centromere-specific or non-specific DNA on which a functional kinetochore protein complex is formed3. In contrast, monocentromeres are composed of a single centromere unit per chromosome, although also monocentrics with a few neighboring centromere units per chromosome have been observed, e.g., in Pisum and Lathyrus4,5 and the beetle Tribolium castaneum6, forming so-called ‘meta-polycentric’ chromosomes5.

The centromere-specific histone H3 (CENH3, also known as CENPA) specifies centromere identity in most species. It serves as a platform for kinetochore assembly, which includes the inner kinetochore constitutive centromere-associated network (CCAN) and the outer kinetochore KMN complex (KNL1c, MIS12c, and NDC80c)7. The precise balance between centromere-to-spindle-based tension and sister chromatid cohesion ensures accurate chromosome segregation8, monitored by the spindle assembly checkpoint (SAC), which verifies proper chromosome-spindle attachments before anaphase. Cell cycle-dependent histone phosphorylation is crucial for recruiting SAC proteins in this process9.

Histone H3 phosphorylation of the pericentromeric region is part of the histone modification network acting to control centromere function. These modifications can create a permissive environment for the correct assembly and function of centromeres9. In plants, with the onset of mitosis, only pericentromeres, where sister chromatids cohere, undergo histone H3 phosphorylation at serine 10 and 2810,11. In holocentrics, due to their distinct centromere arrangement, these epigenetic marks are dispersed along the whole length of condensed chromosomes. In monocentric species, meanwhile, both epi-marks are enriched adjacent to primary constrictions11. Histone H3 threonine 3 phosphorylation originates at pericentromeres in prophase and evenly distributes along chromosome arms at prometaphase in monocentric chromosomes12,13. In contrast, histone H2A threonine 120 (H2AT120ph; the threonine position refers to human H2A, and the corresponding positions differ between species) phosphorylation marks mirror the distribution of CENH3 in both centromere types14,15.

Since holocentric species appear sporadically within phylogenetic lineages that have monocentric chromosomes, it is believed that holocentric chromosomes evolved from monocentric ones. Such a one-way transition happened multiple times in various green algae, protozoans, invertebrates, and different plant families16. As a consequence of their independent evolution, holocentromeres are diverse in composition and organization (reviewed in refs. 3,17,18). However, the mechanisms underlying this mono-to-holocentromere transition are not yet understood. Analyzing closely related species with contrasting centromere types might illuminate this process. In the parasitic plant genus Cuscuta, the mono-to-holocentromere transition is associated with extensive changes in genes responsible for the structure and regulation of the kinetochore19. In insects such as Lepidoptera, the transition to holocentricity is related to the loss of CENH320, leading to a permissive chromatin state-based centromere identity21 and to CCAN-mediated kinetochore assembly in holocentric taxa22.

An independent transition to holocentricity occurred in the plant tribe Chionographideae, constituting the holocentric genus Chionographis and the monocentric, monotypic sister genus Chamaelirium, in the monocot family Melanthiaceae23–28 (Fig. 1A, Supplementary Fig. 1A, B). The two genera diverged about 23.5 million years ago (mya) and currently exhibit a disjunct distribution, with Chionographis found in East Asia and Chamaelirium in North America29. It has been suggested that the two genera be reclassified as parts of the merged genus Chamaelirium due to their otherwise considerable morphological similarities30.

Notably, the monocentric chromosome-typical primary constriction is absent in Chamaelirium luteum28. Instead, the species features unusually large heterochromatic centromeres that protrude poleward at metaphase (Supplementary Fig. 1A). It was assumed that the macro-monocentromeres of Cha. luteum might represent a precursor state from which holocentromeres in the sister genus Chionographis evolved28. In the holocentric Chi. japonica, each holocentromere is composed of merely 7 to 11, ~1.9 Mb-sized, minisatellite DNA-based, CENH3-positive centromere units that are arranged in a line along the poleward surface of each chromatid31 (Supplementary Fig. 1B). The centromere units of Chi. japonica are up to 200 times larger than those described for the repeat-based holocentromeres in Rhynchospora species32.

To gain insight into the evolution of atypical centromeres accompanying the mono-to-holocentromere transition, we resolved the genome and centromere organization of Cha. luteum. We compared the (epi)genome and kinetochore composition, as well as the genome synteny, taking advantage of the closely related species Chi. japonica, which possesses a contrasting centromere type and the same number of chromosomes (2n=24). Interestingly, the large macro-monocentromeres (up to 15 Mb) of Cha. luteum have characteristics in common with mono- and holocentromeres, but, in contrast to initial expectation, they do not constitute a direct link between mono- and holocentromeres. Instead, our comparative analysis of the kinetochore composition revealed a loss of the KNL2 gene and a chimeric origin of the NSL1 (a MIS12c component) gene in Cha. luteum and of the Borealin gene in Cha. luteum and Chi. japonica. The observed conservation of genome synteny between the two species suggests that the holocentromere formation occurred de novo in Chionographis. Additionally, comparison with representatives of the closely related monocentric tribe Heloniadeae revealed a distinct distribution pattern of the cell cycle-dependent histone H3 phosphorylation marks in the tribe Chionographideae. Notably, the distribution of these modifications along the entire length of the chromosomes does not distinguish between holo- and macro-monocentric species. Based on our findings, we discuss possible mechanisms driving the parallel transition from a typical monocentromere to either atypical macro-mono- or even holocentromeres.

Results
Cha. luteum chromosomes are monocentric despite the lack of a primary constriction
The primary constriction typical for monocentromeric chromosomes is absent in Cha. luteum. Instead, large heterochromatic regions protrude on metaphase chromosomes (Supplementary Fig. 1A). To determine whether the so-called macro-centromere28 acts as an active centromere, a Cha. luteum-specific CENH3 antibody was generated as a marker. For this purpose, we analyzed the Cha. luteum transcriptome and identified a single CENH3 gene. Its protein sequence phylogenetically grouped with that of the holocentric Chi. japonica (Supplementary Fig. 1D). As expected for CENH333, amino acid sequences differed most at the N-terminal tail (Supplementary Fig. 1E). The first twenty N-terminal amino acids were used to generate the anti-CENH3 antibody. Immunostaining of somatic metaphase cells revealed CENH3 signals decorating the protruding macro-monocentromeres (Fig. 1B), where multiple spindle microtubules attach (Fig. 1C, Supplementary Movie 1). During interphase, centromeres formed brightly DAPI-stained, almost equal-sized chromocenters (Fig. 1D). The number of CENH3-positive chromocenters (17–24, on average 20, counted in 30 nuclei) is less than or equal to the number of chromosomes, as in the case of other chromocenter-forming monocentric species, such as Arabidopsis thaliana34. A preferential distribution of chromocenters close to the nuclear double membrane was revealed (Fig. 1E). A similar nuclear membrane preference of centromeric chromocenters was also found in the holocentric Chi. japonica as well as in A. thaliana31. Our analysis of Cha. luteum demonstrates that the chromosome protrusion acts as a centromere and that a primary constriction is not a ubiquitous structural requirement for centromere function in a monocentric species.

Cha. luteum exhibit exceptionally large satellite-based monocentromeres
The genome size of Cha. luteum was determined as 887 Mb/1C using flow cytometry. To resolve the sequence and size of the Cha. luteum macro-monocentromeres, we assembled its genome using a combination of HiFi reads with Hi-C scaffolding, resulting in a ~745 Mb assembly representing ~84% of the Cha. luteum genome. The refined assembly retained 92.7% complete BUSCOs across 649 contigs (N50=2.1 Mb, L50=87) (Supplementary Table 1). Subsequent Hi-C scaffolding generated the top 22 scaffolds, each at least 10 Mb (10–68 Mb) used for downstream analysis (Supplementary Table 2, Supplementary Fig. 2A).

To determine whether the centromeres of Cha. luteum are composed of repetitive sequences, we first analyzed the repeat composition of its genome using RepeatExplorer and used the identified repeat clusters as references to assess their enrichment in CENH3-ChIP sequence data. Approximately 49% of the genome is composed of high-copy satellite repeats and transposable elements (Supplementary Table 3). Among these, the 60-bp-monomer satellite cluster CL1, named ‘Chama’, is the most abundant repeat (9.3%) (Fig. 2A). Its monomer sequence is rich in centromere-typical dyad symmetries, which may preferentially form a hairpin-loop structure (Fig. 2A). FISH demonstrated centromeric positioning of the satellite cluster (Fig. 2B), and its enrichment in the CENH3-precipitated fraction is further suggestive of a centromeric nature (Fig. 2C). Additionally, the enrichment of CENH3-ChIP reads in the Chama satellite arrays of the assembly (Fig. 2D), as well as the colocalization of CENH3-immuno and Chama-FISH signals in interphase chromocenters and heterochromatin regions of metaphase chromosomes (Fig. 2E), confirmed its centromeric localization. To determine the CENH3-positive fraction of the Chama arrays, we harnessed super-resolution microscopy. Colocalization of Chama FISH and CENH3 immunostaining interphase signals demonstrated that ~60 % of Chama arrays interact with CENH3-containing nucleosomes (Supplementary Table 4). Thus, a substantial fraction of the macro-monocentromeric regions forms the active centromere. In addition, the (AG/CT)n-containing CL51 and the GC-rich CL176 repeat cluster, both with very low genome abundance (<0.3%) and no sequence similarity to the Chama satellite, were also enriched in the CENH3-precipitated fraction (Fig. 2C).

Among the top 22 scaffolds of Cha. luteum, the eight CENH3-interacting Chama arrays range in size from 9.58 to 15.30 Mb, with an average size of 11.54 Mb (Supplementary Table 2). Unlike the frequently alternating orientation of the centromeric Chio satellite arrays observed in Chi. japonica31, in Cha. luteum the sequence arrays are highly homogeneous and consistently oriented, with higher-order repeat structures maintained across several hundred kilobases and in some cases extending over more than 1 Mb (Supplementary Fig. 2B–D). None of the identified Chama arrays was interspersed with retrotransposons. Our FISH analysis of naturally extended pachytene chromosomes, which feature knob-like centromeres, in line with our sequencing results, ruled out the possibility that the macro-monocentromeres of Cha. luteum are formed of multiple adjacent centromere units typical for ‘meta-polycentric’ chromosomes, as the number of Chama signals equaled the number of chromosome bivalents (Fig. 2F).

For a direct comparison of the centromere size with a species possessing similar chromosome dimensions, we performed immunolabeling of Sorghum bicolor centromeres with a S. bicolor-specific CENH3 antibody, resulting in CENH3 signals at primary constrictions (Fig. 2G)35. Despite comparable chromosome sizes (Cha. luteum (887 Mb/1C, n=12)≈73.9 Mb/chromosome, S. bicolor (789 Mb/1C, n=10)≈78.9 Mb/chromosome), Cha. luteum exhibits substantially larger centromeres.

The genomes of holocentric Chi. japonica and macro-monocentric Cha. luteum share broad-scale synteny, with the exception of their centromeres
Centromeres play a crucial role in shaping the genome architecture36. To investigate whether the evolution of the two centromere types from the sister genera was accompanied by genome shuffling, we analyzed syntenic orthologs of single-copy coding genes in the assembled scaffolds of Cha. luteum and the chromosome-level genome assembly of Chi. japonica. We identified 9,960 pairs of collinear genes, revealing large blocks and a high degree of genome conservation (Fig. 3A, Supplementary Fig. 3), although their genomes diverged 23.5 million years ago29. In particular, the chromosome-level arrangement of orthologs between chromosome 8 of Chi. japonica and scaffold 3 of Cha. luteum is identical (Fig. 3B). The order and orientation of all six non-centromeric intervals of Chi. japonica chromosome 8 were conserved in the corresponding chromosome-sized scaffold 3 of Cha. luteum. Besides chromosome-sized syntenic regions, 11 large-scale inversions and four inter-chromosomal translocations (scaffolds 2, 6, 7, and 13 of Cha. luteum) were found (Fig. 3A, Supplementary Fig. 3). Nevertheless, local gene synteny was mostly conserved. Notably, syntenic blocks were split by centromere units in Chi. japonica (Fig. 3B, Supplementary Fig. 3), indicating de novo origin of centromere units in this holocentric species. In addition, ATAC-seq revealed that accessible chromatin regions are evenly distributed across the chromosomes except in the centromeres of Cha. luteum (Fig. 3B). This suggests that the de novo holocentromere formation sites in Chi. japonica correspond to gene-rich, accessible chromatin regions in the syntenic Cha. luteum genome. According to the synteny of centromere flanking regions between the two genomes, the positions of the macro-monocentromeres in Cha. luteum (e.g., scaffold 3) and centromere units in Chi. japonica mostly do not correspond (Fig. 3B, Supplementary Fig. 3). Assuming that the centromere positions of Cha. luteum and of the ancestor of both species are conserved, the loss of monocentromeres was thus likely accompanied by de novo holocentromere formation in Chionographis.

To confirm the sequence-deduced conservation of syntenic chromosomal blocks and in silico-identified rearrangements between the two species, we designed oligo-FISH painting probes specific for the six non-centromeric intervals (NCIs) between the first and the eighth centromere unit of Chi. japonica chromosome 2 (Fig. 3C). FISH mapping on mitotic prometaphase and meiotic pachytene chromosomes of Chi. japonica confirmed the correctness of the sequence assembly and specificity of oligo-FISH probes (Fig. 3D, Supplementary Fig. 4A–C). In Cha. luteum, the signals of the six probes were located on three arms of two chromosome pairs (Fig. 3E, Supplementary Fig. 4A–C), corresponding to scaffolds 4 and 6, as predicted by our sequence analysis (Fig. 3A, E, Supplementary Fig. 4D). Additionally, we mapped the sequences of all 12 Chi. japonica-based, chromosome-wide oligopools to the top 22 scaffolds of Cha. luteum. This alternative in silico strategy verified the high chromosomal collinearity between the two genomes with contrasting centromere types as well as the absence of chromosome duplications (Supplementary Fig. 5A).

To determine whether this large-scale genome synteny is conserved beyond the tribe Chionographideae, we checked for a potential cross in situ hybridization in the closely related species of tribe Heloniadeae, which includes the genera Helonias, Heloniopsis, and Ypsilandra, and which diverged ~55 mya from tribe Chionographideae (Fig. 1A)29. However, the same oligo-FISH painting probes revealed no detectable signals on the chromosomes of Heloniopsis umbellata. Moreover, comparative repeat analysis revealed no shared high-copy repeats between H. umbellata and Cha. luteum or Chi. japonica (Supplementary Fig. 5B). Thus, despite their contrasting centromere types, the genomes of Cha. luteum and Chi. japonica are highly syntenic, yet divergent from those of Heloniadeae species.

Loss and alteration of kinetochore genes are associated with the transition to unconventional centromeres in Chionographideae
The protein composition of the kinetochore complex varies considerably across species with different centromere types18,19,37,38. To determine whether the evolution of holo- and macro-monocentromeres in different members of the Chionographideae was accompanied by changes of the kinetochore, we investigated the kinetochore protein composition of Chi. japonica, Cha. luteum, the related monocentric species Heloniopsis orientalis from tribe Heloniadeae (Fig. 1A, Supplementary Fig. 1C), and of Phoenix dactylifera, a phylogenetically distant monocot species for comparison. In total, we analyzed 29 structural and regulatory kinetochore proteins (Fig. 4, Supplementary Table 5).

In this in silico analysis, we identified homologs for all tested kinetochore proteins in the transcriptomes of Chi. japonica, H. orientalis, and P. dactylifera. In Cha. luteum, we found no sequence similar to the CENH3-loading protein KNL2 (Fig. 4A, B). A subsequent BLASTn search for the KNL2 gene in the genome assembly of this species using the complete gene sequence of Chi. japonica31, revealed only a short fragment at the 3’ end of the KNL2 gene, spanning only two of the seven exons; notably, the exons encoding the conserved SANTA domain and CENPC-k motif of KNL2 were lost (Supplementary Fig. 6A). This KNL2 fragment in Cha. luteum was found in the locus orthologous to the genomic region containing the intact KNL2 gene in Chi. japonica (Supplementary Fig. 6B), indicating that it is a remnant of a formerly intact KNL2 gene.

Comparative analysis of the identified kinetochore protein sequences revealed a remarkable N-terminal divergence in the MIS12c component NSL1 and the chromosomal passenger complex (CPC) module Borealin. Specifically, the N-terminus of NSL1 in Cha. luteum showed unique divergence (Fig. 5A, Supplementary Fig. 7A, B). In contrast, Borealin sequences were similar between Cha. luteum and Chi. japonica, but the two were distinct from the highly conserved sequences in H. orientalis and P. dactylifera (Fig. 5B, Supplementary Fig. 7C). Domain analysis revealed that the divergent N-terminus of NSL1 in Cha. luteum shares similarity with the BLOC-1 Related Complex Subunit 6 (BORCS6) protein (Supplementary Fig. 7B), while the N-terminus of Borealin in Cha. luteum and Chi. japonica showed similarity to ribosomal protein S17 (RPS17) (Supplementary Fig. 7C). This suggests a chimeric origin of the NSL1 gene in Cha. luteum and of the Borealin genes in both Cha. luteum and Chi. japonica, with the original N-terminus-coding regions replaced by sequences derived from BORCS6 and RPS17 genes, respectively (Fig. 5). By identifying the donors of the N-terminal sequences, we determined the acquired N-terminal lengths to be 104 aa for NSL1 (59% of the protein) (Supplementary Fig. 7B) and 50 aa for Borealin (20% of the protein) (Supplementary Fig. 7C). The chimeric nature of Borealin and NSL1 genes was further confirmed by transcriptional validation and genomic evidence (Supplementary Fig. 8).

NSL1 is a structural component of the MIS12 complex, which consists of NSL1, MIS12, DSN1, and NNF1, and interacts with the other two outer kinetochore complexes (NDC80 and KNL1) via NSL1 and DSN1, as well as with the inner kinetochore proteins via DSN139,40. Therefore, we wondered whether the observed change in NSL1 had an impact on kinetochore assembly in Cha. luteum. We took advantage of antibodies developed against the MIS12 and NDC80 proteins of Chi. japonica as well as a highly versatile antibody against KNL1 in plants31,41. All three antibodies specifically labeled holocentromeres in C. japonica and monocentromeres in Heloniadeae species31,41. It was anticipated that the antibodies would mark the centromeres of Cha. luteum if the kinetochore composition remained unchanged because the sequences of the KNL1, MIS12, and NDC80 antibody target domains are identical between Chi. japonica and Cha. luteum. We failed to detect signals for KNL1 and NDC80 in Cha. luteum, even though we were able to successfully detect MIS12 using well-established immunodetection procedures (Supplementary Fig. 7D). Therefore, we hypothesize that the alteration of NSL1 either prevents KMN complex formation or that these two proteins are present at concentrations below the sensitivity of our immunostaining assay. The presence of the intact MIS12c component DSN1 likely explains why the centromeric recruitment of MIS12 appears to be unaltered.

Macro-monocentric Cha. luteum shows holocentromere-typical cell cycle-dependent histone phosphorylation patterns
Phosphorylation of histone H3 serves to prime chromatin for faithful chromosome segregation9. Thus, we also analyzed the distribution of cell cycle-dependent, spindle assembly checkpoint-associated histone phosphorylation marks. Despite its monocentric chromosomes, anti-H3S10/S28/T3 phosphorylation signals were distributed throughout the mitotic metaphase chromosomes of Cha. luteum (Fig. 6A), as in holocentric Chi. japonica31 and other holocentric plants42. Surprisingly, H2AT120 phosphorylation, an otherwise conserved cell cycle-dependent centromeric mark15,43, was undetectable in both macro-monocentric Cha. luteum and holocentric Chi. japonica31.

To determine whether the holocentromere-like histone phosphorylation patterns evolved before the formation of the tribe Chionographideae, we examined the chromosomal distribution of the phosphorylated histone variants in the sister tribe Heloniadeae (Fig. 1A). Three species, Helonias bullata, Heloniopsis umbellata, and Ypsilandra thibetica, were selected as representatives of the corresponding genera. First, we confirmed the monocentricity of these three species by monitoring the distribution of the conserved outer kinetochore protein KNL141 (Fig. 6B, Supplementary Fig. 9). In these species, all analyzed histone H3 phosphorylation patterns were consistent with monocentricity, and H2AT120ph signals were detectable at centromeric regions (Fig. 6B, Supplementary Fig. 9).

To probe the large-scale organization of eu- and heterochromatin in Cha. luteum, we assayed the evolutionarily conserved histone marks histone H3K4me2 and H3K9me2. The euchromatin mark H3K4me2 showed uniform signals in metaphase chromosomes, except at centromeres, where the heterochromatin histone mark H3K9me2 was enriched (Fig. 6C). Similar labeling patterns were observed in interphase nuclei with H3K9me2 and H3K4me2 enriched in chromocenters and euchromatin, respectively (Fig. 6C). These cytological observations were confirmed at the genome-wide level by ChIP-seq, which showed that H3K9me2 is strongly enriched at (peri)centromeric regions (Fig. 2D) and markedly reduced in chromosome arms. In contrast, H3K4me2 is depleted at (peri)centromeric regions and enriched along the chromosome arms, closely resembling the general patterns observed in species with typical monocentromeres.

Thus, although Cha. luteum possesses a typical monocentric distribution of eu- and heterochromatin, it exhibits a cell cycle-dependent histone phosphorylation pattern remarkably similar to that of holocentric species, including Chi. japonica. This chromosome-wide phosphorylation pattern, unique to Chionographideae species and distinguishing them from Heloniadeae species with typical monocentromeres, likely evolved after the divergence of the two tribes.

Methods
Plant materials
Chamaelirium luteum (L.) A. Gray plants used in this study were provided by the Deutsche Homöopathie-Union (DHU), Germany, Chionographis japonica (Willd.) Maxim. plants were obtained from commercial nurseries in Japan, and the Helonias bullata L., Heloniopsis umbellata Baker, Heloniopsis orientalis var. breviscapa, and Ypsilandra thibetica Franch. species were purchased from British nurseries in the UK. The plants were grown at IPK Gatersleben (Germany) in a greenhouse: 16 h light (from 6 AM to 10 PM), day temperature 16°C, night temperature 12°C. Seeds of Sorghum bicolor BTx623 and Secale cereale L. inbred line Lo7 were obtained from the IPK Gene Bank (Gatersleben, Germany) and were germinated on wet filter papers to harvest roots and young leaves for experiments.

Genome size measurement
To isolate nuclei, approximately 0.5 cm2 of fresh leaf tissue of Cha. luteum was chopped together with equivalent amounts of leaf tissue of either of the two internal reference standards Glycine max (L.) Merr. convar. max var. max, cultivar ‘Cina 5202’ (Gatersleben genebank accession number: SOJA 392; 2.21 pg/2C) or Raphanus sativus L. convar. sativus, cultivar ‘Voran’ (Gatersleben genebank accession number: RA 34; 1.11 pg/2C), in a petri dish using the reagent kit ‘CyStain PI Absolute P’ (Sysmex-Partec) following the manufacturer’s instructions. The resulting nuclei suspension was filtered through a 50-μm CellTrics filter (Sysmex-Partec) and measured on a CyFlow Space flow cytometer (Sysmex-Partec, Germany). The gating strategy is shown in Supplementary Fig. 10. At least six independent measurements were performed for Cha. luteum. The absolute DNA content (pg/2C) was calculated based on the values of the G1 peak means and the corresponding genome size (Mbp/1C), according to ref. 72.

Short-read sequencing of DNA and RNA
Genomic DNA of Cha. luteum was extracted from leaf tissue using the DNeasy Plant Mini kit (Qiagen, Germany). Low-pass paired-end (2×150 bp) genome sequencing was performed using DNBSEQ system by BGI (China). Total RNAs from leaf, root, and fruit tissues of Cha. luteum were isolated using the SpectrumTM Plant total RNA kit (Sigma, USA, cat. no. STRN50). Library preparation (Illumina Stranded mRNA Prep Ligation Kit) and sequencing at IPK Gatersleben or Novogene (UK) (paired-end, 2×151 cycles, Illumina NovaSeq6000 system) involved standard protocols from the manufacturer (Illumina Inc., USA).

Isolation of HMW DNA, HiFi library preparation, and sequencing
For long-read PacBio sequencing, high-molecular weight (HMW) DNA of Cha. luteum was isolated from leaves of a single plant using the NucleoBond HMW DNA kit (Macherey Nagel, Germany). Quality was assessed using the FEMTO Pulse system (Agilent Technologies Inc., CA, USA). Quantification involved the Qubit device and the dsDNA High Sensitivity assay kit (Thermo Fisher Scientific, MA, USA). A HiFi library was prepared from 15 µg HMW DNA using the “SMRTbell prep Kit 3.0” according to the manufacturer’s protocol (Pacific Biosciences of California Inc., CA, USA). The initial DNA fragmentation was performed using the Megaruptor 3 device (Shearspeed: 29; Diagenode, Belgium). Finally, HiFi libraries were size-selected (narrow-size range: approximately 20 kb) using the SageELF system with a 0.75% Agarose Gel Cassette as described by the manufacturer (Sage Science Inc., MA, USA). Sequencing (HiFi CCS) was performed using the Pacific Biosciences Revio device (24 h movie time, 155 pM loading concentration, 2 h pre-extension time, diffusion loading, 100 min loading time, 22 kb mean insert length according to SMRT link raw data report, 55 Gb HiFi CCS yield) following standard manufacturer’s protocols (Pacific Biosciences of California Inc., Menlo Park, CA, USA) at IPK Gatersleben.

Chromosome conformation capture (Hi-C) sequencing
Hi-C sequencing libraries were generated from flowers of Cha. luteum as described previously73 using DpnII enzyme, and were sequenced (paired-end, 2×111 cycles) using the NovaSeq6000 device (Illumina Inc., USA) at IPK Gatersleben. A total of ~102 Gb paired-end reads were generated. After filtering, ~87 Gb Hi-C read pairs were used for scaffolding.

Genome assembly and Hi-C scaffolding
A total of ~53 Gb of PacBio HiFi reads (~59.5× coverage) of Cha. luteum were assembled into contigs using hifiasm (v0.19.3-r572; default)74. Contig statistics were calculated with Quast (v2.3)75, and gene content completeness was evaluated with Benchmarking Universal Single-Copy Orthologs (BUSCO) (v4.1.2; dataset: liliopsida_odb10, E-value=0.001)76. An initial 1.56-Gb primary assembly of Cha. luteum genome was constructed with a BUSCO completeness of 95.6% (Supplementary Table 1). The unexpectedly large assembly size and the high score of duplicated BUSCOs (68.0%) suggested the presence of notable heterozygosity in the primary contigs. To join the residual heterozygous contains, purge_dups (v 1.2.5; default)77 was used to identify and remove duplicated sequence segments in the primary assembly, resulting in a reduction of duplicated BUSCOs to 10.6% (Supplementary Table 1). The Arima Genomics mapping pipeline (https://github.com/ArimaGenomics/mapping_pipeline) was used to process the Hi-C data, including read mapping to the contigs, read filtering, read pairing, and PCR duplicate removal, and scaffolding was performed using YaHS (v1.2a.2; -e GATC --no-contig-ec)78. Hi-C contact maps and manual curation were accomplished by the bash scripts provided (https://github.com/c-zhou/yahs) and visualized using Juicebox (https://github.com/aidenlab/Juicebox).

Gene-based synteny analysis
The genome of Chi. japonica was re-annotated by mapping the clean RNA-seq data (EMBL ENA PRJEB58123) generated from our previous study31. For genome-directed transcriptome assembly, the RNA-seq datasets generated in this study from fruits (~7 Gb), leaves (~13 Gb), and roots (~13 Gb) of Cha. luteum (EMBL ENA PRJEB82608), were aligned to the assembled scaffolds using HISAT2 (v2.2.1, default parameters)79, and then processed to produce gene feature annotation with StringTie (v2.1.1, default parameters)80. Two sets of non-redundant transcripts from Chi. japonica and Cha. luteum were generated using gffread (v0.12.6)81. TransDecoder (v5.5.0)82 was used to annotate coding regions in transcripts. BRAKER3 pipeline was used to improve the annotation accuracy of protein-coding genes83.

To find the links of conserved single-copy proteins between the Chi. japonica and Cha. luteum genomes, a Python script was developed as follows. First, the translated protein sequences of Chi. japonica were aligned to those of Cha. luteum via blastp (v2.5.0, default). Second, alignments with an identity of <90% and an alignment length of <150 aa were treated as noisy alignments and were filtered out. Third, only when a sequence showed similarity to a single gene in the other genome were considered as conserved single-copy proteins. Finally, the genomic positions of the syntenic single-copy proteins were extracted from the gff3 files to create the links between the two genomes. NGenomeSyn (v1.41)84 was used to visualize the genome synteny.

Transcriptome-based identification of kinetochore protein genes
The clean RNA-seq datasets from various tissues of Cha. luteum were de novo assembled using Trinity 2.4.082,85 with default parameters. In addition, the genome-directed assembled transcriptome of Cha. luteum as described above was also applied for downstream analyses. Putative coding regions were first identified by TransDecoder (v5.5.0)82 with a minimum protein length of 100 aa as a threshold.

Trinity-made transcriptome assembly of Chi. japonica was obtained from our previous study31. The RNA-seq data and genome assembly used in the study were applied here for genome-directed transcriptome assembly using HISAT2 (v2.2.1, default)79 followed by StringTie (v2.1.1, default)80, as described above. De novo transcriptome assembly in H. orientalis was constructed using RNA-seq data downloaded from Sequence Read Archive (https://www.ncbi.nlm.nih.gov/sra; SRR28160651 and SRR28160654).

Protein sequence databases for these species were constructed by translating predicted open reading frames from de novo and genome-directed transcriptome assemblies generated using Trinity (all three species) and StringTie (Chi. japonica and Cha. luteum), respectively. The analysis was done using two types of sequence data; gene annotation models predicted in genome assemblies based on RNA-seq data and transcriptome assemblies.

Kinetochore protein sequences were first identified in P. dactylifera using BLASTp searches in the GenBank protein database, guided by sequences from previous studies19,37,86,87. These identified sequences were then used to search for homologs in the three Melanthiaceae species. The sequences of kinetochore proteins translated from de novo and genome-derived transcriptome assemblies matched well. Since the genome-derived assemblies were coupled with detailed information about the gene structures, we used these for further analyses. In a few cases, the gene structures had to be manually corrected by reassembling the RNA-seq reads assigned to specific loci and using the programs est2genome88 and genewise89 for the gene annotations.

Reverse transcription polymerase chain reaction (RT-PCR)
Total RNA was extracted from roots and young leaves of Cha. luteum using the Spectrum™ Plant Total RNA-Kit (Sigma-Aldrich). RNA samples were mixed and treated with DNase using the on-column DNA removal protocol (RNase-Free DNase I Kit, Norgen Biotek). cDNA was synthesized with RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific) using oligo(dT) primer. PCR amplification was performed using gene-specific primers (Clu_NSL1_F1: 5’-CGACGGGAAATGGGAGG-3’; Clu_NSL1_R1: 5’- CAAATAAAATTTCGAACAAGTCTGCT-3’; Clu_Borealin_F1: 5’-GGGTATTCCTCAGCTCAAAGA-3’; Clu_Borealin_F1: 5’-GCCTCCAAGTTATCGTCCTT-3’, Tm = 55°C) with GoTaq DNA Polymerase (Promega) following the manufacturer’s instructions. PCR products were size-verified on 1% agarose gels, and purified amplicons were Sanger sequenced by Eurofins (Germany).

Phylogenetic analysis
The CENH3 protein sequences of Cha. luteum and the other species derived from the NCBI GenBank (Supplementary Table 6) were aligned using the ClustalW algorithm implemented in MEGA X by default setting90,91. The maximum-likelihood tree was constructed via IQ-Tree web server (http://iqtree.cibiv.univie.ac.at/)92 and visualized using Interactive Tree Of Life (iTOL, http://itol.embl.de/)93,94.

Antibody production
The synthesized peptides of Cha. luteum CENH3 (ClCENH3: MAPTKKTKKTTENINNRPAL-C) were used for the immunization of rabbits to generate polyclonal antibodies. The peptide synthesis, immunization, and antibody purification were performed by LifeTein (www.lifetein.com, USA).

Indirect immunostaining
Mitotic chromosomes and interphase nuclei were prepared from root meristems. Roots were pretreated in ice-cold water overnight and fixed in 4% paraformaldehyde in Tris buffer (10 mM Tris, 10 mM EDTA, 100 mM NaCl, 0.1% Triton X-100, pH 7.5) for 5 min on ice under vacuum treatment, followed by another 25–30 min solely on ice. Root meristems were then chopped in lysis buffer LB01 (15 mM Tris, 2 mM Na2EDTA, 0.5 mM spermine, 80 mM KCl, 20 mM NaCl, 15 mM β-mercaptoethanol, and 0.1% (v/v) Triton X-100)95, the cell suspension was filtered through a 50-μm CellTrics filter (Sysmex-Partec) and subsequently centrifuged onto slides using a Cytospin 3 (Shandon, Germany) at 700 rpm (×55.32 g) for 5 min. The chromosome spreads were blocked in 3% BSA in 1× phosphate-buffered saline (PBS) at room temperature (RT) for 1 h and incubated with primary antibodies in 1% BSA/ 1× PBS at 4°C overnight. After three washes in 1× PBS at RT for 5 min each, secondary antibodies in 1% BSA/ 1× PBS were applied, followed by an incubation at 37°C for 1 h. After three washes, the slides were dehydrated in 70-90-100% ethanol series for 3 min each and counterstained with 10 µg/ml 4’,6-diamidino-2-phenylindoline (DAPI) in Vectashield antifade medium (Vector Laboratories, USA). For immunodetection of microtubules, root pretreatment with ice-cold water was omitted, and the Tris buffer and 1× PBS mentioned above were substituted by 1× MTSB buffer (50 mM PIPES, 5 mM MgSO4, and 5 mM EGTA, pH 7.2).

The primary antibodies used in this study included customized rabbit anti-Cha. luteum CENH3 (dilution 1:500), rabbit anti-Chi. japonica MIS12 (dilution 1:100)31, rabbit anti-Chi. japonica NDC80 (dilution 1:100)31, and rabbit anti-Cuscuta europaea KNL1 (dilution 1:500 or 1:1000)19, as well as the commercially available mouse anti-alpha-tubulin (Sigma-Aldrich, USA, cat. no. T9026-2, dilution 1:300), rabbit anti-histone H3K4me2 (abcam, UK, cat. no. ab7766, dilution 1:300), mouse anti-histone H3K9me2 (abcam, UK, cat. no. ab1220, dilution 1:300), mouse anti-histone H3S10ph (abcam, UK, cat. no. ab14955, dilution 1:1000), rat anti-histone H3S28ph (Sigma-Aldrich, USA, cat. no. H9908, dilution 1:1000), rabbit anti-H3T3ph (Sigma-Aldrich, USA, cat. no. 07-424, dilution 1:1000), and rabbit anti-H2AT120ph (Active Motif, USA, cat. no. 61196, dilution 1:500).

The anti-rabbit rhodamine (Jackson ImmunoResearch, USA, cat. no. 111-295-144, dilution 1:300), anti-rabbit Alexa488 (Jackson ImmunoResearch, USA, cat. no. 711-545-152, dilution 1:300), anti-mouse Alexa488 (Jackson ImmunoResearch, USA, cat. no. 715-546-151, dilution 1:300), and anti-rat Alexa488 (Jackson ImmunoResearch, USA, cat. no. 112-545-167, dilution 1:300) were used as secondary antibodies.

Repeatome analysis
The low-coverage genome skimming dataset of Cha. luteum was generated in this study, and those of Chi. japonica (ERR10639507, EMBL ENA, https://www.ebi.ac.uk/ena/)31 and H. umbellata (SRR15208642, NCBI SRA, https://www.ncbi.nlm.nih.gov/sra/)96 were publicly available. Genomic PE reads were assessed by FastQC97 implanted in the RepeatExplorer pipeline (https://repeatexplorer-elixir.cerit-sc.cz/galaxy/) and filtered by quality with 95% of bases equal to or above the cut-off value of 10. Qualified PE reads of Cha. luteum equivalent to 0.5× genome coverage were applied to analyze repetitive elements by a graph-based clustering method using RepeatExplorer98–100. The automatic annotation of repeat clusters was manually inspected and revised if necessary, followed by a recalculation of the genome proportion of each repeat type. The comparative clustering analysis was performed based on one million PE reads from each of the three species.

Chromatin immunoprecipitation (ChIP) sequencing
The CENH3-ChIP experiment was performed with minor modifications as described by Kuo et al.31. 0.65 g of Cha. luteum flower and 1.0 g of Secale cereale (inbred line Lo7) leaf tissue were ground with liquid nitrogen and homogenized separately in 10 ml nuclei isolation buffer (1 M sucrose, 5 mM KCl, 5 mM MgCl2, 60 mM HEPES pH 8.0, 5 mM EDTA, 0.6% Triton X-100, 0.4 mM PMSF, 1 µM pepstatin A, cOmplete protease inhibitor cocktail (Roche)). Nuclei fixation was performed in 1% PFA/nuclei isolation buffer at RT, 12 rpm for 10 min and terminated by adding glycine to a final concentration of 130 mM. The nuclei suspension was filtered through Miracloth (Millipore) twice and a 50-µm CellTrics filter (Sysmex) once and centrifuged at 4°C, 3,000 ×g for 10 min. The nuclei pellet was resuspended in 1 ml extraction buffer (0.25 M sucrose, 10 mM Tris-HCl pH 8.0, 10 mM MgCl2, 1% Triton X-100, 1 mM EDTA, 5 mM β-mercaptoethanol, 0.1 mM PMSF, 1 µM pepstatin A, cOmplete protease inhibitor cocktail), followed by centrifugation at 4°C, 12,000 g for 10 min. After removing the supernatant, nuclei were resuspended in 150 µl nuclei lysis buffer (20 mM Tris-HCl pH 8.0, 10 mM EDTA, 1% SDS, 0.1 mM PMSF, 1 µM pepstatin A, cOmplete protease inhibitor cocktail). Chromatins were sonicated for 14 cycles of 30 s ON, 30 s OFF at high power in a Bioruptor (Diagenode), followed by an addition of 100 µl ChIP dilution buffer (16.7 mM Tris-HCl pH 8.0, 167 mM NaCl, 1.1% Triton X-100, 1 mM EDTA, 20 mM Tris-HCl pH 8.0, cOmplete protease inhibitor cocktail), and continued sonication to a total of 31 cycles under the same setting. The sonicated samples were diluted 10 times with ChIP dilution buffer, centrifuged at 4°C, 13,000 g for 5 min, and the supernatant of each sample was transferred to new tubes. To dilute the high proportion of the putative Cha. luteum centromeric repeat, sonicated chromatin of Cha. luteum and S. cereale were mixed in a 1:3 ratio. The mixed chromatins were incubated with the ClCENH3 antibody (10 mg/ml) to a final 1:500 dilution at 4°C by shaking at 14 rpm for 12 h. DynabeadsTM Protein A (Invitrogen) in ChIP dilution buffer, corresponding to 0.1× volume of the chromatin solution, was added to the antibody-prebound chromatins and incubated at 4°C by shaking at 14 rpm for 1.5 h. The collected beads were then washed twice in low salt buffer (150 mM NaCl, 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0), followed by three washes in high salt buffer (500 mM NaCl, 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0), and another two washes in TE buffer at 4°C by shaking at 14 rpm for 5 min. The bead-bound chromatin was purified by using iPure kit v2 (Diagenode) following the manual and quantified using QubitTM dsDNA HS Assay kit (Invitrogen). ChIP-seq libraries were prepared by NEBNEXT® UltraTM II DNA Library Prep Kit for Illumina (New England Biolabs) and sequenced using NovaSeq6000 system (Illumina) by Novogene (UK) in the paired-end run (2×150 bp).

For histone mark ChIP, chromatin was isolated from 0.33 g of Cha. luteum flowers following the same nuclei isolation and sonication procedure described above. Antibodies H3K9me2 (ab1220, abcam) and H3K4me2 (ab7766, abcam) were added to a final dilution of 1:100. The subsequent immunoprecipitation, washing, DNA purification, and library preparation steps were performed as described for the CENH3-ChIP. The sequencing using DNBSEQ system was performed by BGI (China).

ChIP-seq data analysis
To evaluate the enrichment of repeats associated with CENH3-containing nucleosomes, single-end reads of CENH3-ChIP-seq and input-seq were quality filtered using the tool “Processing of FASTQ reads” (Galaxy Version 1.0.0.3), implemented in the Galaxy-based RepeatExplorer portal (https://repeatexplorer-elixir.cerit-sc.cz/galaxy/). ChIP-SeqMapper (Galaxy version 1.1.1.4) (Neumann et al.5) was used to map the ChIP- and input-seq reads on RepeatExplorer-derived contig sequences of repeat clusters. To analyze the size and position of Cha. luteum centromeres and histone modification patterns, the paired-end reads of ChIP- and input-seq were quality-filtered by Trimmomatic (Galaxy Version 0.39)101 and the resulting reads were mapped to the Cha. luteum genome assembly using Bowtie 2 (Galaxy Version 2.5.3)102 with default parameters. No filtering to remove multimapping reads was applied; therefore, all analyses include both uniquely and multimapping reads. Peak calling (--broad -887400000) was performed with MACS (v3.0.0)103. The deeptools bamCompare function (Galaxy Version 3.5.4)104 was used to generate the normalized ChIP-seq signal track as the average log2-ratio of ChIP over input read counts in genome-wide 1 kb windows. Visualization of chromosome regions with multiple tracks were plotted with pyGenomeTracks (Galaxy version 3.8)105.

ATAC sequencing and data analysis
Fresh leaves of Cha. luteum were finely chopped using a razor blade in 1 ml of nuclei isolation buffer (0.25 M sucrose, 10 mM Tris-HCl pH 8.0, 10 mM MgCl2, 1% Trion X-100, 5 mM β-mercaptoethanol) supplemented with 1× Halt™ Protease Inhibitor Cocktail (Thermo Scientific), and the slurry was filtered through a 50-μm cell strainer. The resulting nuclei were washed twice and resuspended with the same nuclei isolation buffer. An aliquot was analyzed using a flow cytometer for quality control and quantification. Based on the quantification, a volume containing approximately 75,000 nuclei was aliquoted, and the nuclei pellet was collected by centrifugation.

The nuclei pellet was resuspended in a transposition reaction mix containing Tagment DNA Enzyme (TDE1, Illumina, 20034197), 0.4× PBS, 0.01% digitonin, and 0.1% Tween-20, and incubated at 37°C for 30 min. Transposition products were purified using the MinElute PCR Purification Kit (QIAGEN, 28004). Libraries were amplified with the NEBNext® High-Fidelity 2× PCR Master Mix (NEB, M0541), and further purified using VAHTSTM DNA Clean Beads (Vazyme, N411). The final libraries were sequenced in paired-end mode (2× 151 cycles) on the Illumina NovaSeq6000 (Illumina Inc., USA).

Sequencing reads were adapter-trimmed using fastp (v0.20.0)106 and aligned to the reference genome with BWA-MEM (v0.7.17)107. SAMtools (v1.16.1)108 was used to remove duplicate and multi-mapping reads (-q30), followed by peak calling (-q0.01) with MACS (v3.0.0)103. For visualization, BAM files containing uniquely mapped reads were converted to BigWig format using deepTools bamCoverage (v3.5.1)104, with Reads Per Kilobase per Million mapped reads (RPKM) normalization.

Preparation of fluorescence in situ hybridization (FISH) probes
The consensus sequences of satellite repeats reconstructed by TAR-EAN (TAndem REpeat ANalyzer)109 were used to design fluorescence-modified oligonucleotides which were synthesized by Eurofins (Germany). The clone pAtT4110 was used as the probe to detect Arabidopsis-type telomeres. Plasmid DNA was labeled with ATTO 550-dUTP using Fluorescent Nick Translation Labeling kits (Jena Bioscience, Germany).

The chromosome 2-specific oligo painting probes were designed based on the genome assembly of Chi. japonica31 using the software Chorus2111. The predicted Chionographis-based oligos which matched the genome of Cha. luteum were all included in the synthesized myTags Immortal Libraries (Daicel Arbor Biosciences, USA) to improve the probe transferability to Cha. lutuem. Oligopools were labeled with fluorophores ATTO-594, Alexa 488, or Alexa 647 following the myTags Immortal Labeling Protocol (Daicel Arbor Biosciences, USA, https://arborbiosci.com/wp-content/uploads/2022/05/DaicelArborBio_myTags_Labeling_Protocol_v2-2.pdf).

Chromosome preparation and FISH
Mitotic chromosome spreads were prepared from root meristems using a dropping method31. Roots were pretreated in ice-cold water overnight, fixed in 3:1 (ethanol: glacial acetic acid) fixative at RT, overnight and kept in 70% ethanol at -20°C until use. Fixed roots were digested in an enzyme mixture (0.7% cellulose Onozuka R10 (Duchefa Biochemie, The Netherlands, cat. no. C8001), 0.7% Cellulase (Calbiochem, USA, cat. no. 219466), and 1.0% pectolyase (Sigma, USA, cat. no. 45-P3026)) in citric buffer (0.01 M sodium citrate dihydrate and 0.01 M citric acid) at 37°C for 30–40 min. Cell suspension in the 3:1 fixative was dropped onto slides on a hot plate at 55°C, and slides were further fixed in 3:1 fixative for 1 min, air-dried, and kept at 4°C for later use.

To prepare meiotic chromosomes, inflorescences of Chi. japonica and Cha. luteum were fixed as described above for roots. Anthers were digested at 37°C for 70-80 min in an enzyme mixture (0.23% cellulose Onozuka R10 (Duchefa Biochemie, The Netherlands, cat. no. C8001), 0.23% Cellulase (Calbiochem, USA, cat. no. 219466), 0.33% pectolyase (Sigma, USA, cat. no. 45-P3026), and 0.33% cytohelicase (Sigma, USA, cat. no. C8247)). Meiotic spreads were prepared by a typical squash method112. FISH mapping was performed as described in ref. 35. For oligo-FISH, the hybridization mixture containing 10% dextran sulfate, 50% formamide, 2× SSC, and 500–1000 ng of each labeled oligopool was used, and the hybridization at 37°C was extended to 36–48 h.

Microscopy and image analysis
Widefield fluorescence images were captured using an epifluorescence microscope BX61 (Olympus) equipped with a CCD camera (Orca ER, Hamamatsu, Japan) and pseudo-colored by the Adobe Photoshop 6.0 software. To analyze the chromatin and centromere ultrastructures at the super-resolution level, we applied spatial structured illumination microscopy (3D-SIM) using a 63×/1.40 Oil Plan-Apochromat objective of an Elyra 7 microscope system (Carl Zeiss GmbH, Germany). Image stacks were captured separately for each fluorochrome using 405, 488, and 561 nm laser lines for excitation and appropriate emission filters113. Maximum intensity projections from image stacks were calculated using the ZEN Black software (Carl Zeiss GmbH, Germany). Zoom-in sections were presented as single slices to indicate the subnuclear chromatin structures at the super-resolution level. 3D rendering to produce spatial animations was done based on 3D-SIM image stacks using the Imaris 9.7 (Bitplane, UK) software. The tool ‘Colocalization’ of the same software was applied to determine the CENH3 amount colocalizing to the Chama repeats based on voxel intensities. For it, the colocalization calculation threshold was automatically determined at P = 1.000. Based on this, the percentage of the CENH3 volume above the threshold colocalizing to the Chama volume was calculated per interphase nucleus.

Transmission electron microscopy (TEM)
For electron microscopic analysis, root tip cuttings were used for microwave-assisted fixation in 2.0% (v/v) glutaraldehyde and 2.0% (v/v) paraformaldehyde in 0.05 M cacodylate buffer (pH 7.3), dehydration with acetone, and embedding in Spurr’s resin. Ultra-thin sections (70 nm) were cut with a Leica microtome Ultracut S (Leica Microsystems, Wetzlar, Germany), and mounted on 70 mesh copper TEM grids. Prior to ultrastructure analysis at 120 kV in a Tecnai Sphera G2 transmission electron microscope (ThermoFisher Scientific, Eindhoven, Netherlands), sections were contrasted in a Leica EM AC 20 automatic contrasting device with homemade 2% uranyl acetate for 30 min, followed by a 90-second incubation in Leica Ultrastain 2 containing 3% Reynolds’ lead citrate.

Statistics and reproducibility
No statistical method was used to predetermine the sample size, no data were excluded from the analyses, the experiments were not randomized, and the investigators were not blinded to allocation during the experiments and during outcome assessment.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this very interesting study, Kuo and co-workers report a fundamentally different centromere/kinetochore organization from the monocentric versus holocentric organizations of known centromeres. By comparing the sequence organization of the holocentric flowering plant Chionographis japonica genome with that of its close relative, Chamaelirium luteum, they discovered a radically different “macro-monocentromere” sequence organization, which corresponded to the unique cytological appearance of C. luteum centromeres. These are dominated by extensive contiguous satellite sequence heterochromatin and lack a primary constriction. By comparison of the amino acid sequences of CENH3, they observed rapid evolution of the N-terminal tail, and a search for kinetochore proteins showed that Cha. luteum showed differences for some highly conserved components. Consideration of the functions of these kinetochore proteins led to a satisfying stepwise model for how this unique macro-monocentromere organization diverged from a monocentric common ancestor. They argue that holocentricity arose strictly in Chionographis, while Chamaelirium underwent a localized centromere expansion. This striking discovery will be of considerable interest to the broad readership of Nature Communications.\n\nThe authors state that the chimeric origin of NSL1 and Borealin, which evidently triggered the chain of events, is unprecedented, but I wonder if there are other examples for species that have been sequenced but which haven’t been studied cytologically with sufficient resolution to distinguish holocentricity from macro-monocentricity. For example, Lepidopterans are holocentric, exceptionally species rich, lack CENP-A/cenH3 and have small chromosomes, and perhaps there are clues in the sequences of their kinetochore proteins waiting to be discovered.\n\nMinor:\nPage10, line 296, Is the KNL2 gene in Chionographis known to be functional? It seems better to say it is intact.\nPage 7, lines 210-211 and Figure 2G: The text states that Chamaelinium has “the proportionally largest centromeres reported to date” and the figure displays the size ratio of centromere to chromosome as a percentage, with Chamaelinium centromeres encompassing 15.6 % of an average chromosome. The authors can consider whether to add Solenopsis invicta, in which the primary constriction and its CenSol satellite account for an average of 34% of the chromosome length [Huang et al 2016]. CenH3 localization has not been done, so it is possible that Solenopsis centromeres could be metapolycentric, like in Tribolium castaneum, where the distance between the most peripheral of the cenH3 dots accounts for 43% of the chromosome length [Grzan et al. 2020], or Pisum and Lathyrus, where the authors found the metapolycentric primary constriction encompasses a third of the chromosome.", "reviewer_2": "This manuscript by Kuo et al is valuable not only for its novel research results but also for its thoughtful summaries of widely different centromere and kinetochore structures, both at the chromosomal level and individual protein level. The figures are elegant, and few people can match the quality of their microscopy. In terms of novel results, the more important one here is Chamaelirium luteus, the sole member of the Chamaelirium genus and the most closely related species to the holocentric Chionographis genus, is monocentric but has unusual centromeres. They overlap large tandem arrays of a 60bp repeat, spanning up to 10 Mb total, and they do not form primary constrictions. Perhaps related, Chamaelirium and Chionographis have chromosomal distributions of histone phosphorylation in mitosis that is typical of holocentric species even though Chamaelirium is monocentric. Likewise, both have an unusual structural variant of the Borealin protein which is a core component of the chromosomal passenger complex. In addition, Chamaelirium but not Chionographis lacks the highly conserved protein KNL2, which normally functions in CENH3 loading to centromeres. These are important results that will be of interest to a large audience of scientists interested in centromeres, kinetochores, and chromosome biology.\n\nI have two major concerns, however, with the manuscript in its current state. First, the Kuo et al conclude based on CENH3 ChIP-seq that the size of the Chamaelirium centromeres match the sizes of the tandem repeat arrays. However, the data appears to suggest that the functional centromere defined by CENH3 occupies only a small segment of these large arrays. If this is true, the centromeres may in fact be well within the normal range of centromere sizes, and Chamaelirium is not the outlier that Kuo et al have concluded it is. Second, they draw conclusions about order of events and about causation without clearly explaining the underlying logic. Both concerns are explained in more detail below, as well as other comments and suggestions.\n\nSince the lack of a primary constriction in Chamaelirium luteus is one of the major results of the manuscript a few words on the significance of primary constrictions and how they form would be helpful.\n\nWhy are there only eight CENH3-interacting Chama arrays in the 22 scaffolds if there are 12 chromosomes?\n\nThe statement “The sequence arrays are highly homogeneous and consistent in their sequence orientation” needs further quantitative support. Supplementary Fig. 2 only shows an anecdotal example of a 5-kb region of a single Chama array.\n\nThe CENH3 ChIP-seq analysis included both multimapping and unique mapping reads, which will overestimate the size of the CENH3 occupied region if it overlaps with tandem repeats. For example, imagine that most of the CENH3 is concentrated in a single 1-Mb region of the array. When reads from those regions are mapped to the genome, they will be randomly distributed across the entire array.\n\nThe merely 2-fold enrichment of Chama for CENH3 suggests most of the Chama repeats are not bound to CENH3. This also suggests the actual centromere, as defined by CENH3 occupancy, is a small, possibly fluid, region within the large Chama array.\n\nRelated to the above two points, how complex is the sequence makeup of the centromeric chara arrays? Are they interspersed with retrotransposons? Could there be enough polymorphism in them that uniquely mapping reads could estimate the actual size of the centromeres within the larger arrays?\n\nThe statement “ATAC-seq revealed that the entire Cha. luteum chromosome, except for the centromere, exists in an open chromatin state” is misleading. ATAC-seq reveals nucleosome free regions. Even within genes there are peaks and valleys of ATAC-seq reads. What this data reveals is that accessible chromatin regions are evenly distributed across the chromosomes except in centromeres.\n\nWhile it is clear from these results that Chamaelirium centromeres are not syntenic with Chionographis, it is not clear whether Chamaelirium centromeres are syntenic with other monocentric relatives. Answering this question would increase the impact of this manuscript.\n\nThe logic behind the title of the paper and the evolution model expounded on figure 7 seems either severely lacking or needs to be communicated more clearly. It seems that every conclusion could be in the opposite direction. For example, could one also conclude from these data that constriction-free chromosomes arose first, which subsequently led to selection for a new Borealin variant? Or that formation of a holocentromere is what led to spread of novel repeats? Or that Chamaelirium monocentric centromeres reverted from Chionographis-like holocentromeres? Or that increased sister chromatid cohesion resulted in a need for stronger/more microtubule centromere attachments? Why do the data support the conclusions the authors make rather than these alternatives?\n\nFigure 2D: What are the units of the Y axis?\nFigure 3B. An X-axis scale would be helpful\nFigure 3C. “CjSat5” is not explained in the legend or main text. What is it and why is it included in the figure?", "reviewer_3": "This study provides a comprehensive comparison of two related species that have unusual centromere structures. The study was initiated to test if the unusually large centromeres of one species (Chamaeliurium luteum) might be a precursor to the evolution of holocentromeres in the other species (Chionographis japonica). The contributions here are mostly on the side of luteum since japonica had been the subject of a previous report. Notable observations are the large size of the satellite arrays in luteum, the fact that it has H3S10/S28 phosphorylation all along chromosome arms, that borealin is chimeric in both species, that NSL1 is chimeric in luteum, and that NDC80 and KNL1 are not observable by immunolocalization in luteum. The authors argue that changes in kinetochore proteins and phosphorylation led to changes in centromere structure.\nThe strengths of the study are the state of the art genome characterization and superb microscopy. A weakness is that the work is entirely comparative, that no firm conclusions can be made about cause and effect.\n\nMajor comments:\n1) Title. The word “preceded” should be removed from the title. An appropriate alternative would be “are correlated with”. It is just as likely that changes in centromere structure favored the changes in kinetochore proteins and phosphorylation. Only the borealin mutation can be said to have preceded the divergence of these species, and the proposed connection of borealin to changes to centromere structure involves long series of speculations that have not been tested in any way.\n2) Line 133-136. There is no evidence here that that H3 phosphorylation is “required but not sufficient” for holocentromere formation. This type of language is usually reserved for experiments where mutants are available or some type of overexpression is carried out. This study only involves staining and comparing.\n3) Lines 209-211. The language here should be softened. Centromere size is defined by the area occupied by CENH3. Because the satellite array is homogenized, it is not possible to know where CENH3 is in the array by ChIP-seq. The microscopy that supports this is excellent but does not have the resolution to show that CENH3 is continuous across the array.\n4) Lines 326-332 and 517-518. Neither KNL1 nor NDC80 could be detected by immunolocalization in luteum. This is perhaps the most surprising result in the study. If the KMN complex is absent that would suggest a completely different kinetochore structure. However, the as it is, the data are unconvincing. Indeed, both genes are present and expressed, so the data as shown suggest the KMN complex has a different form (or other proteins present) that change the accessibility of antibodies to their targets. If the authors want to suggest that KNL1 and NDC80 are absent, they need to do some western blots.\n5) Line 383. Change from “in short, the divergent..” to “in short, we propose that the divergent..”.\n6) The discussion can be improved. I found it to be rambling and overly speculative. In one section the authors suggest that KNL2 mutation is the most common cause of major shifts in centromere structure, yet in the next section, a case is made for borealin as the instigating event in this clade. If both are involved, then, taken together, the data suggest that there are multiple kinetochore alterations over long time frames, and seem more consistent with the view that centromeres changed first and the kinetochore mutations followed.", "reviewer_4": "This study focuses on a comparative analysis of two closely related species within the tribe Chionographideae that exhibit distinct centromere types. By integrating genomic, epigenomic, centromere structural, and kinetochore protein data, the research aims to elucidate the mechanisms underlying the transition from monocentric to holocentric chromosomes. The topic is novel, and the research framework is comprehensive, especially in its incorporation of multi-dimensional data across molecular, cytological, and structural levels, offering significant insights into evolutionary biology. However, concerns remain regarding the quality of the genome assemblies, the depth of functional annotation, and the completeness of the evidence supporting certain key conclusions. These aspects require further clarification or supplementation.\n\nA key concern is that the genome assembly used for Cha. luteum in this study is only 749 Mb, representing about 84% of the estimated genome size (~887 Mb), with a contig N50 of 2.1 Mb. This is relatively low given current standards in the era of HiFi and Hi-C sequencing, especially for analyzing highly repetitive regions such as centromeres or detecting gene family loss/fusion structural variations, where misinterpretation is likely. Improving the genome assembly quality and performing full-length transcriptome sequencing for Cha. luteum would enhance the reliability of protein structure annotation and help verify gene models.\n\nThe formation mechanism of the chimeric genes Borealin and NSL1 remains unclear, and the supporting evidence is insufficient. The authors suggest that the N-terminal regions of these two chimeric genes originate from RPS17 and BORCS6, respectively, based mainly on sequence homology analysis. However, the following critical evidence is lacking. No transcriptional validation of the fusion junctions (e.g., RT-PCR); No demonstration of adjacent structural changes on the chromosome (is this a true gene fusion or a misannotation?)\n\nThe conclusion that the holocentromeres in Chionographis arose de novo may be premature. The authors base this inference on the lack of synteny between the monocentromere position in Cha. luteum and the holocentromeric units in Chi. japonica, yet holocentromere formation is known to involve neocentromerization at multiple non-centromeric regions. The authors should consider conducting ancestral state reconstruction to infer whether the common ancestor of Cha. luteum and Chi. japonica was more likely monocentric or holocentric. Further searches for conserved unit positions and residual Chama-like centromeric sequences in Chi. japonica would also be valuable."}, "author_response": "Point-by-point response to reviewer’s comments\nReviewer #1 (Remarks to the Author)\nIn this very interesting study, Kuo and co-workers report a fundamentally different\ncentromere/kinetochore organization from the monocentric versus holocentric organizations of\nknown centromeres. By comparing the sequence organization of the holocentric flowering plant\nChionographis japonica genome with that of its close relative, Chamaelirium luteum, they\ndiscovered a radically different “macro-monocentromere” sequence organization, which\ncorresponded to the unique cytological appearance of C. luteum centromeres. These are\ndominated by extensive contiguous satellite sequence heterochromatin and lack a primary\nconstriction. By comparison of the amino acid sequences of CENH3, they observed rapid\nevolution of the N-terminal tail, and a search for kinetochore proteins showed that Cha. luteum\nshowed differences for some highly conserved components. Consideration of the functions of\nthese kinetochore proteins led to a satisfying stepwise model for how this unique macro-\nmonocentromere organization diverged from a monocentric common ancestor. They argue that\nholocentricity arose strictly in Chionographis, while Chamaelirium underwent a localized\ncentromere expansion. This striking discovery will be of considerable interest to the broad\nreadership of Nature Communications.\n\nThe authors state that the chimeric origin of NSL1 and Borealin, which evidently triggered the\nchain of events, is unprecedented, but I wonder if there are other examples for species that have\nbeen sequenced but which haven’t been studied cytologically with sufficient resolution to\ndistinguish holocentricity from macro-monocentricity. For example, Lepidopterans are holocentric,\nexceptionally species rich, lack CENP-A/cenH3 and have small chromosomes, and perhaps there\nare clues in the sequences of their kinetochore proteins waiting to be discovered.\nRESPONSE\nMany thanks for your insightful comment. Macro-monocentromere possessing species might\nlikely coexist in otherwise holocentric lineages. For example, a recent cytological finding in\nDrosera genus revealed that a macro-monocentromere-like structure in D. filiformis and\nholocentromeres in the related species D. rotundifolia (Hoshi and Terasaki 2025). However, no\ncomparative kinetochore analysis has been performed for this genus. Future analysis of\nkinetochore genes in Drosera species could tell whether chimeric kinetochore genes were formed\n1\nand would provide additional insights into the interplay between kinetochore genes and\ncentromere evolution.\nWe are not aware whether a similar situation exists in Lepidopterans. In moths and butterflies (in\ntotal ~160,000 species), only about 1.5 % of species have been karyotyped (De Vos et al. 2020),\nand only a small fraction have been analyzed in detail beyond simple chromosome counts. The\nkey will be a detailed analysis of different species using a combination of methods, from\nmicroscopy to gene identification.\nMinor:\nPage10, line 296, Is the KNL2 gene in Chionographis known to be functional? It seems better to\nsay it is intact.\nRESPONSE\nThanks for the suggestion. We agree and change the “functional” to “intact”.\nNow it reads (Page 10, Line 305-308): This KNL2 fragment in Cha. luteum was found in the locus\northologous to the genomic region containing the intact KNL2 gene in Chi. japonica\n(Supplementary Fig. 6B), indicating that it is a remnant of a formerly intact KNL2 gene.\nPage 7, lines 210-211 and Figure 2G: The text states that Chamaelinium has “the proportionally\nlargest centromeres reported to date” and the figure displays the size ratio of centromere to\nchromosome as a percentage, with Chamaelinium centromeres encompassing 15.6 % of an\naverage chromosome. The authors can consider whether to add Solenopsis invicta, in which the\nprimary constriction and its CenSol satellite account for an average of 34% of the chromosome\nlength [Huang et al 2016]. CenH3 localization has not been done, so it is possible that Solenopsis\ncentromeres could be metapolycentric, like in Tribolium castaneum, where the distance between\nthe most peripheral of the cenH3 dots accounts for 43% of the chromosome length [Grzan et al.\n2020], or Pisum and Lathyrus, where the authors found the metapolycentric primary constriction\nencompasses a third of the chromosome.\nThank you for pointing out additional cases of exceptionally large centromeric regions\nin Solenopsis invicta, Tribolium castaneum, Pisum sativum, and Lathyrus. We agree that these\nare highly relevant examples. We toned down our conclusion about the exceptional size of the\nactive centromere (interacting with CENH3-positive nucleosomes) in Cha. luteum. Therefore, we\nremoved Figure 2G and the corresponding Supplementary Table 4, but kept Figure 2H (now\nFigure 2G). This figure demonstrates directly the striking CENH3-size differences between Cha.\nluteus and a species (Sorghum bicolor) with similarly sized chromosomes.\nHowever, in our original Figure 2G, we restricted the comparison to the CENH3-ChIPseq-defined\ncentromeres in monocentric species. Thus, the metapolycentric species, which contain multiple\ncentromere units within a defined chromosomal region on each chromosome and species in which\ncentromere size estimates are based on cytological constrictions or satellite repeat abundance\nrather than direct CENH3 mapping, were not included. For example, in the well-studied\nmetapolycentric chromosome 6 of Pisum sativum, the primary constriction (CEN6) spans 81.6 Mb\n(Macas et al. 2023), yet the total size of CENH3-enriched domains within this region is only 4.6\nMb, leaving a large region devoid of CENH3. While larger primary constrictions or extended\ncentromeric regions have been described in some metapolycentric taxa (e.g., Pisum\nand Lathyrus) or in species where CENH3 localization has not yet been mapped\n(e.g., Solenopsis), our analysis was restricted to centromeres directly defined by CENH3-ChIP–\nseq in monocentric species.\nReviewer #2 (Remarks to the Author)\nThis manuscript by Kuo et al is valuable not only for its novel research results but also for its\nthoughtful summaries of widely different centromere and kinetochore structures, both at the\nchromosomal level and individual protein level. The figures are elegant, and few people can match\nthe quality of their microscopy. In terms of novel results, the more important one here is\nChamaelirium luteus, the sole member of the Chamaelirium genus and the most closely related\nspecies to the holocentric Chionographis genus, is monocentric but has unusual centromeres.\nThey overlap large tandem arrays of a 60bp repeat, spanning up to 10 Mb total, and they do not\nform primary constrictions. Perhaps related, Chamaelirium and Chionographis have\nchromosomal distributions of histone phosphorylation in mitosis that is typical of holocentric\n3\nspecies even though Chamaelirium is monocentric. Likewise, both have an unusual structural\nvariant of the Borealin protein which is a core component of the chromosomal passenger complex.\nIn addition, Chamaelirium but not Chionographis lacks the highly conserved protein KNL2, which\nnormally functions in CENH3 loading to centromeres. These are important results that will be of\ninterest to a large audience of scientists interested in centromeres, kinetochores, and\nchromosome biology.\nI have two major concerns, however, with the manuscript in its current state. First, the Kuo et al\nconclude based on CENH3 ChIP-seq that the size of the Chamaelirium centromeres match the\nsizes of the tandem repeat arrays. However, the data appears to suggest that the functional\ncentromere defined by CENH3 occupies only a small segment of these large arrays. If this is true,\nthe centromeres may in fact be well within the normal range of centromere sizes, and\nChamaelirium is not the outlier that Kuo et al have concluded it is.\nRESPONSE\nWe toned down our conclusion about the exceptional size of the active centromere (interacting\nwith CENH3-positive nucleosomes) in Cha. luteum. Therefore, we removed Figure 2G and the\ncorresponding Supplementary Table 4, but kept Figure 2H (now Figure 2G). This figure\ndemonstrates directly the striking CENH3-size differences between Cha. luteus and a species\n(Sorghum bicolor) with similarly sized chromosomes.\nSince our genome assembly of Cha. luteum is not complete; we quantified the Chama repeat\nproportion interacting with CENH3-containing nucleosomes by super-resolution microscopy. The\nnew analysis demonstrates that ~60 % of Chama arrays interact with CENH3-containing\nnucleosomes (new Supplementary Table 4). We added to the text (Page 7, Line 197-\n202): “To determine the CENH3-positive fraction of the Chama arrays, we harnessed super-\nresolution microscopy. Colocalization of Chama FISH and CENH3 immunostaining interphase\nsignals demonstrated that ~60 % of Chama arrays interact with CENH3-containing nucleosomes\n(new Supplementary Table 4). Thus, a substantial fraction of the macro-monocentromeric regions\nforms the active centromere.”\nexplaining the underlying logic. Both concerns are explained in more detail below, as well as other\ncomments and suggestions.\nRESPONSE\nWe rewrote the DISCUSSION and includied description of the proposed model.\nSince the lack of a primary constriction in Chamaelirium luteus is one of the major results of the\nmanuscript a few words on the significance of primary constrictions and how they form would be\nhelpful.\nRESPONSE\nThe absence of a primary constriction in Cha. luteus demonstrates that a chromosome\nconstriction is not a prerequisite for a functional monocentromere. Thus, the absence of a primary\nconstriction does not always mean that no monocentromere exists. We assume that a direct or\nindirect interplay between the formation of a primary constriction and a pericentromere-specific\nphosphorylation of histone H3S10/S28 exist. A number of years ago, we (Houben et al. 1999) and\nothers demonstrated that centromere inactivation correlates with the absence of pericentromeric\nhistone H3 phosphorylation marks. In Cha. luteum, due to the chromosome-wide distribution of\nthis cell cycle-dependent phosphorylation mark, no primary constriction is likely formed. We\nextended the DISCUSSION with (Page 19-20, Line 594-601): “The existence of constriction-free\ncentromeres in Cha. luteum suggests that the primary constriction, while typically distinguishing\nmonocentromeres, is not always necessary for centromere function. Consequently, the absence\nof this constriction does not necessarily indicate the absence of an active monocentromere. This\nmorphological exception may be explained by the species' immense centromere size and the\ndistribution of histone H3 phosphorylation marks along the entire length of the chromosomes; a\npattern otherwise characteristic of holocentromeres.\nWhy are there only eight CENH3-interacting Chama arrays in the 22 scaffolds if there are 12\nchromosomes?\nThank you very much for this question. The lower number of CENH3-interacting Chama arrays\nthan expected is caused by the challenges of assembling highly repetitive regions. Collapsing of\nsequence contigs occurs during genome assembly when repetitive DNA regions are incorrectly\nmerged into a single, shorter contig, losing the true number of repeats and creating an inaccurate\nrepresentation of the genome. This phenomenon is particularly prevalent with repetitive\nsequences, such as tandemly arrayed repeats, like the 60-bp-monomer Chama repeat which\nrepresents 9.3 % of the genome.\nOur approach to assembling the Cha. luteum genome largely mirrored our successful strategy for\nChi. japonica (Kuo et al. 2023a), where we generated a chromosome-scale reference genome\ndespite a high proportion of centromeric repeats. However, Cha. luteum presented challenges\nmuch earlier in the process, beginning with the initial step of high-quality DNA isolation. Both Cha.\nluteum and Chi. japonica leaf tissues are rich in secondary metabolites that severely compromise\nDNA yield and quality. To circumvent this, we aimed to propagate a single plant via tissue culture\nand isolate sufficient high-molecular-weight (HMW) DNA from the roots; unfortunately, this initial\nstep already proved difficult.\nHowever, unfortunately, Cha. luteum grows extremely slowly and was not suitable for tissue\nculture in our hands. Therefore, we isolated genomic DNA from leaves from a pool of plants to\nobtain sufficient material for PacBio long-read sequencing, generating 101.7 Gb of PacBio CCS\nreads (~114×). However, the initial assembly was highly fragmented (14,573 contigs, N50 of ~652\nkb). Moreover, the total assembled size was ~1.6 Gb, nearly twice the haploid genome size\nestimated by flow cytometry (1C = 887.4 Mb), which probably indicates an issue caused by\npooling the plant materials for sequencing.\nGenome assembly assessment of Cha. luteum\nSecond Assembly after\nFirst assembly\nassembly purge_dups\nNo. of contigs 14,573 4,087 649\nLargest contig 6,926,063 19,839,389 19,839,389\nTotal length 1,597,791,599 1,556,940,134 744,551,200\nN50 625,023 1,631,558 2,111,717\nN75 57,449 663,828 1,228,881\nL50 564 192 87\nGC (%) 41.55 40.51 41.13\nComplete BUSCOs - 2911 (90.0%) 2768 (85.5%)\nComplete and single-copy BUSCOs - 1167 (36.1%) 2477 (76.5%)\nComplete and duplicated BUSCOs - 1744 (53.9%) 291 (9.0%)\nFragmented BUSCO - 171 (5.3%) 215 (6.6%)\nMissing BUSCO - 154 (4.7%) 235 (7.9%)\nTotal BUSCO groups searched - 3236 3236\nTo overcome this, next, we extracted HMW DNA from the leaves of a single plant of Cha. luteum\nfor a second PacBio run (53 Gb, ~59.5× coverage). Applying the same assembly approach, the\nimproved assembly reduced contigs to 4,087 and increased the largest contig size (19.84 Mb),\nand raised the N50 to 1.63 Mb. Although we used Hifiasm (Cheng et al. 2021), a haplotype-\nresolved de novo assembler based on phased assembly graphs, the assembled genome size\n(~1.56 Gb) was nearly twice the 1C genome size of 887.4 Mb estimated by flow cytometry. This\nunexpectedly large assembly size, along with the high proportion of duplicated BUSCOs (68.0%),\nindicated substantial heterozygosity in the primary contigs. To address this, we applied\npurge_dups to remove redundant haplotypic sequences, which may also have included some\ncentromeric repeat arrays. The refined assembly contained 649 contigs (N50 = 2.1 Mb, L50 = 87)\nand retained 92.7% complete BUSCOs. Finally, Hi-C scaffolding generated the top 22 large\nscaffolds, each exceeding 10 Mb (10–68 Mb), used for downstream analyses. The limited plant\nmaterial from a single plant prevented us from performing Nanopore ultra-long sequencing to\nimprove the assembly further.\nImportantly, FISH mapping of the Chama repeat combined with CENH3 immunostaining revealed\ncolocalization signals on all 12 chromosomes of Cha. luteum. This demonstrates that each\nchromosome harbors a Chama-associated centromere repeat array, even though not all are\nrepresented in the assembly. Due to the large and highly repetitive nature of the centromeres, the\nhigh heterozygosity of this genome, we didn’t aim for a telomere-to-telomere level assembly in\nthis study. Instead, our focus was on identifying and characterizing centromere-associated\nrepeats, and we acknowledge that future improvements in sequencing and assembly strategies\nmay resolve all CENH3-interacting Chama arrays at the chromosomal level.\norientation” needs further quantitative support. Supplementary Fig. 2 only shows an anecdotal\nexample of a 5-kb region of a single Chama array.\nRESPONSE\nTo support the statement “the sequence arrays are highly homogeneous and consistent in their\nsequence orientation”, we generated scaffold-wide and megabase-scale dotplots by Gepard\n(Krumsiek et al. 2007), with the Chama array-containing scaffold Clu_scaffold_17 as an example.\nWe performed the analysis using two parameters: a less stringent word size of 20 (default = 10)\nto capture broader sequence similarity, and a strict word size of 60, matching the 60-bp Chama\nmonomer length, to detect individual repeat units precisely. We include the result in the\nSupplementary Fig. 2D. Dotplot analysis under the relaxed condition (word size 20) showed that\nChama arrays are highly homogeneous (Supplementary Fig. 2D, left). Under the strict condition,\nthe analysis revealed the presence of higher-order repeat structure (Supplementary Fig. 2D,\nmiddle). Furthermore, Chama arrays exhibit a consistent orientation spanning regions larger than\nseveral hundred kilobases. In summary, these large-scale dotplot analyses confirm that Chama\narrays are both highly homogeneous and consistently oriented, supporting the original statement.\nWe added to the text (Page 7, Line 210-214): Unlike the frequently alternating orientation of the\ncentromeric Chio satellite arrays observed in Chi. japonica 31, in Cha. luteum the sequence arrays\nare highly homogeneous and consistently oriented, with higher-order repeat structures\nmaintained across several hundred kilobases and extending in some cases over more than 1 Mb\n(Supplementary Fig. 2B–D).\nThe CENH3 ChIP-seq analysis included both multimapping and unique mapping reads, which will\noverestimate the size of the CENH3 occupied region if it overlaps with tandem repeats. For\nexample, imagine that most of the CENH3 is concentrated in a single 1-Mb region of the array.\nWhen reads from those regions are mapped to the genome, they will be randomly distributed\nacross the entire array.\nbound to CENH3. This also suggests the actual centromere, as defined by CENH3 occupancy,\nis a small, possibly fluid, region within the large Chama array.\nRelated to the above two points, how complex is the sequence makeup of the centromeric chara\narrays? Are they interspersed with retrotransposons? Could there be enough polymorphism in\nthem that uniquely mapping reads could estimate the actual size of the centromeres within the\nlarger arrays?\nRESPONSE\nWe agree with your concern. Analysis of the identified Chama repeat-containing centromere\narrays did not reveal intermingling retrotransposons. Further, using ChIPseq-Mapper, a genome\nassembly-independent CENH3-ChIPseq analysis tool did not identify retrotransposons. Besides\nthe Chama repeat, we identified two additional CENH3-enriched repeat clusters: the (AG/CT)n-\ncontaining CL51 and GC-rich CL176 repeat clusters (Fig. 2C), both with very low genome\nabundance (<0.3%) and no sequence similarity to the Chama satellite. Neither of these is a\nretrotransposon-annotated repetitive sequence.\nApart from the Chama satellite, the remaining repeat clusters in the Cha. luteum genome exhibits\na relatively low genomic proportion (<1.2%). Additionally, we performed FISH mapping of the two\nCRM LTR retrotransposons, CL66 and CL79, with genome proportions of 0.21 % and 0.18 %,\nrespectively. None of them showed centromere-specific signals. Thus, we did not identify a\nCENH3 nucleosome-interacting retrotransposon in Cha. luteum. A similar satellite-dominant\ncentromere composition was found in Chi. japonica (Kuo et al. 2023).\nTo determine the proportion of Chama array interacting with CENH3-containing nucleosomes,\nwe employed a ChIP/genome assembly-independent method. Using super-resolution microscopy of\nanti-CENH3/Chama-labelled nuclei, we found that ~60 % of Chama arrays interact with CENH3-\ncontaining nucleosomes (new Supplementary Table 4). We added to the text (Page 7, Line 197-\n202): “To determine the CENH3-positive fraction of the Chama arrays, we harnessed super-\nresolution microscopy. Colocalization of Chama FISH and CENH3 immunostaining interphase\nsignals demonstrated that ~60 % of Chama arrays interact with CENH3-containing nucleosomes\n(new Supplementary Table 4). Thus, a substantial fraction of the macro-monocentromeric regions\nforms the active centromere.”\ncentromere, exists in an open chromatin state” is misleading. ATAC-seq reveals nucleosome free\nregions. Even within genes there are peaks and valleys of ATAC-seq reads. What this data reveals\nis that accessible chromatin regions are evenly distributed across the chromosomes except in\ncentromeres.\nRESPONSE\nWe rephrased the sentence. Now it reads (Page 8, Line 248-250): “In addition, ATAC-seq revealed\nthat accessible chromatin regions are evenly distributed across the chromosomes except in the\ncentromeres of Cha. luteum (Fig. 3B).\nWhile it is clear from these results that Chamaelirium centromeres are not syntenic with\nChionographis, it is not clear whether Chamaelirium centromeres are syntenic with other\nmonocentric relatives. Answering this question would increase the impact of this manuscript.\nRESPONSE\nWe thank the reviewer for this suggestion. Unfortunately, current genomic resources do not allow\na direct test of centromere synteny with close relatives of Cha. luteum. The recent study by\nHenderson et al. (DOI: 10.21203/rs-7008504/v1) includes several monocot species, but none of\nthem are representative of Melanthiaceae or Liliales, and all diverged from Liliales over 100 million\nyears ago. By contrast, Chamaelirium and Chionographis diverged within Melanthiaceae only 20–\n40 million years ago. We also evaluated the recently published Melanthiaceae genomes by\nZeng et al. (Nature Plants, DOI: 10.1038/s41477-025-02060-3). But these provided no centromere-\nspecific annotation, and the data are insufficient for comparative analysis. Therefore, synteny with\ncloser monocentric relatives cannot be assessed with the available data.\nMoreover, the basic chromosome numbers (x) among the analyzed close monocentric relatives\nshow significant variation: x=17 in Heloniadeae species, x=8 in Veratrum, and x=5 in Paris\n(Pellicer et al. 2014). Since this differs substantially from the x=12 found in Chamaelirium and\nChionographis, such a high degree of karyotype divergence indicates that centromere synteny\nanalysis across these species would likely lack relevance or meaning.\"\neither severely lacking or needs to be communicated more clearly. It seems that every conclusion\ncould be in the opposite direction. For example, could one also conclude from these data that\nconstriction-free chromosomes arose first, which subsequently led to selection for a new Borealin\nvariant? Or that formation of a holocentromere is what led to spread of novel repeats? Or that\nChamaelirium monocentric centromeres reverted from Chionographis-like holocentromeres? Or\nthat increased sister chromatid cohesion resulted in a need for stronger/more microtubule\ncentromere attachments? Why do the data support the conclusions the authors make rather than\nthese alternatives?\nRESPONSE\nWe toned down the title, which now reads: “Kinetochore mutations and histone phosphorylation\npattern changes accompany holo- and macro-monocentromere evolution” and extended the\ndescription of the evolution model and adjusted Figure 7.\nQuestion: “could one also conclude from these data that constriction-free chromosomes arose\nfirst, which subsequently led to selection for a new Borealin variant?”\nRESPONSE\nWe agree with your suggestion. Now it reads (Page 14, Line 434-438): “The change in the\nBorealin gene might have acted as a molecular trigger, initiating the chromosome-wide distribution\nof histone phosphorylation and driving the evolution of primary constriction-free chromosomes in\nChionographideae. Conversely, constriction-free chromosomes may have arisen first, followed by\nthe selection for the new Borealin variant”.\nQuestion: “Or that formation of a holocentromere is what led to spread of novel repeats?”\nRESPONSE\nThe mechanism(s) that result(s) in the formation of a holocentromere are unknown. However, it\nis likely that partial similarity exists between de novo formation process of monocentromeres and\n11\nholocentromeres. However, in the case of a neo-monocentromere, the newly formed centromere\noften forms in the proximity of the “older” centromere. In contrast, the mono-to-holocentromere\ntransition process is accompanied by the formation of many de novo formed centromere units\nalong all chromosomes. Whether the formation of a metapolycentomere (e.g. Lathyrus, Pisum) is\ninvolved in this process as intermediate has been debated but not proven yet (Schubert et al.\n2020). In metapolycentric Lathyrus sativus, all centromere domains are composed of the same\nsatellite DNA family. However, in metapolycentric Pisum species closely related to Lathyrus, even\ncentromere units on the same chromosome are based on distinct repeats. Thus, it is not clear\nhow metapolycentromeres evolved. Thus, multiple conceivable scenarios, including the one\nmentioned by the reviewer.\nIt's plausible that the size of the centromere unit increased gradually in Chi. japonica. Under this\nmodel, a small-unit holocentromere first formed de novo (by an unknown mechanism). Over time,\nthese small units would have undergone massive expansion, reaching sizes of several\nmegabases (Mb) through repetitive DNA accumulation facilitated by mechanisms such as unequal\ncrossing over and gene conversion.\nQuestion: “Or that Chamaelirium monocentric centromeres reverted from Chionographis-like\nholocentromeres?”\nRESPONSE\nA de novo origin of macro-monocentromeres from holocentromeres is improbable for two main\nreasons. First, this process would necessitate multiple fissions of the holocentric chromosomes,\nwhich should result in a large, unobserved increase in the number of monocentric chromosomes.\nAlternatively, it could occur through the elimination of most holocentromere units from the genome,\npreceded or followed by changes in CENH3 localization. Such \"reversion scenario\" cannot be\ncompletely ruled out, but is unlikely because a mono-to-holo-to-mono transition is more complex\nthan a mono-to-holo transition. Second, there are no documented cases of an evolutionary\nreversion from a holocentric state back to monocentricity. We added (Page 14, Line 411-415): “ A\nde novo origin of macro-monocentromeres from holocentromeres is unlikely for two main reasons.\nFirst, such a process would require multiple fissions of holocentric chromosomes, resulting in a\n12\nlarge number of monocentric chromosomes, which is not observed. Second, there are no\ndocumented cases of a reversion from holocentricity to monocentricity.”\nQuestion: “Or that increased sister chromatid cohesion resulted in a need for stronger/more\nmicrotubule centromere attachments?”\nRESPONSE\nWe agree with this scenario. We have written (Page 20, Line 603-611): “Accurate chromosome\nsegregation relies on a delicate balance between sister chromatid cohesion and microtubule-\ngenerated pulling forces (Fig. 7B). Consequently, as an adaptation required for proper\nchromosome segregation, the expansion of centromeric regions in both Chamaelirium and\nChionographis probably increased microtubule attachment and pulling forces to offset the\nincreased sister chromatid cohesion brought on by the chromosome-wide distribution of\npericentromeric histone H3 phosphorylation. This sequence of events is more likely than the\nalternative scenario of independent histone H3 phosphorylation expansions in both genera,\nfollowed by centromere expansion.”\nFigure 2D: What are the units of the Y axis?\nRESPONSE\nThe Y axis of the ChIP-seq track is the average log2 ratio of IP/input reads in a genome-wide\n10 kb windows. The Y axis of the gene track is the coverage calculated as the number of reads\nper 1-kb bin. We included the units in Figure 2D.\nFigure 3B. An X-axis scale would be helpful\nRESPONSE\nWe included the X-axis in the image.\nthe figure?\nRESPONSE\nCjSat5, a satellite repeat identified in the Chi. japonica genome (Kuo et al. 2023b), was mapped\nonto chromosome 2. Our analysis demonstrated that high-copy CjSat5 sequences dominate the\ntwo distal ends of the chromosome. Because of this extreme enrichment, the design of oligo FISH-\npainting probes was restricted, and we could not extend them into these terminal regions. Now\nthe legend of Figure 3C reads: (Page 45, Line 1494-1497) \"Our mapping revealed that the ends\nof chromosome 2 are highly enriched with the CjSat5 satellite repeat, precluding the design of\noligo FISH-painting probes for those specific regions.\"\nReviewer #3 (Remarks to the Author)\nThis study provides a comprehensive comparison of two related species that have unusual\ncentromere structures. The study was initiated to test if the unusually large centromeres of one\nspecies (Chamaeliurium luteum) might be a precursor to the evolution of holocentromeres in the\nother species (Chionographis japonica). The contributions here are mostly on the side of luteum\nsince japonica had been the subject of a previous report. Notable observations are the large size\nof the satellite arrays in luteum, the fact that it has H3S10/S28 phosphorylation all along\nchromosome arms, that borealin is chimeric in both species, that NSL1 is chimeric in luteum,\nand that NDC80 and KNL1 are not observable by immunolocalization in luteum. The authors argue\nthat changes in kinetochore proteins and phosphorylation led to changes in centromere structure.\nThe strengths of the study are the state of the art genome characterization and superb microscopy.\nA weakness is that the work is entirely comparative, that no firm conclusions can be made about\ncause and effect.\nRESPONSE\nWe thank your thoughtful summary and highlighting both the strengths and limitations of our study.\nWe agree that our work is primarily comparative and does not allow us to draw firm conclusions\n14\nabout the direct cause and effect. This limitation stems largely from the uniqueness of the non-\nmodel plant materials, which constrain functional experimentation. We tested transgenic\napproaches, but we failed with this material so far. Nevertheless, we believe that the combination\nof genomic and cytological approaches provides an important foundation for future experimental\nwork. We hope our findings will serve as a framework for testing direct causal relationships in\nkinetochore and centromere evolution as new experimental tools become available and for\ninspiring studies in other independently evolved lineages.\nMajor comments:\n1) Title. The word “preceded” should be removed from the title. An appropriate alternative would\nbe “are correlated with”. It is just as likely that changes in centromere structure favored the\nchanges in kinetochore proteins and phosphorylation. Only the borealin mutation can be said to\nhave preceded the divergence of these species, and the proposed connection of borealin to\nchanges to centromere structure involves long series of speculations that have not been tested\nin any way.\nRESPONSE\nWe thank you for your suggestion. We agree that “preceded” in the title could imply a cause-effect\nrelationship, which has not been experimentally tested. We have revised the title to use\n“accompany”.\nNow the revised title reads: “Kinetochore mutations and histone phosphorylation pattern changes\naccompany holo- and macro-monocentromere evolution.”\n2) Line 133-136. There is no evidence here that H3 phosphorylation is “required but not sufficient”\nfor holocentromere formation. This type of language is usually reserved for experiments where\nmutants are available or some type of overexpression is carried out. This study only involves\nstaining and comparing.\nRESPONSE\nfunctional test that cannot be performed in both species. To avoid an overstatement, we have\nrevised the text.\nNow it reads: (Page 5, Line 133-140) “Additionally, comparison with representatives of the closely\nrelated monocentric tribe Heloniadeae revealed that a distinct distribution pattern of the cell cycle-\ndependent histone H3 phosphorylation marks in the Tribe Chionographideae. Notably, the\ndistribution of these modifications along the entire length of chromosomes does not distinguish\nbetween holo- and macro-monocentric species.”\n3) Lines 209-211. The language here should be softened. Centromere size is defined by the area\noccupied by CENH3. Because the satellite array is homogenized, it is not possible to know where\nCENH3 is in the array by ChIP-seq. The microscopy that supports this is excellent but does not\nhave the resolution to show that CENH3 is continuous across the array.\nRESPONSE\nWe toned down our conclusion about the exceptional size of the active centromere (interacting\nwith CENH3-positive nucleosomes) in Cha. luteum. Therefore, we removed Figure 2G, but kept\nFigure 2H (now Figure 2G). Figure 2G, demonstrates directly the striking CENH3-size differences\nbetween Cha. luteum and Sorghum bicolor, a species with similarly sized chromosomes.\nSince our genome assembly of Cha. luteum is not complete; we quantified the Chama repeat\nproportion interacting with CENH3-containing nucleosomes by super-resolution microscopy. The\nnew analysis demonstrates that ~60 % of Chama arrays interact with CENH3-containing\nnucleosomes (new Supplementary Table 4). We added to the text (Page 7, Line 197-202): “To\ndetermine the CENH3-positive fraction of the Chama arrays, we harnessed super-resolution\nmicroscopy. Colocalization of Chama FISH and CENH3 immunostaining interphase signals\ndemonstrated that ~60 % of Chama arrays interact with CENH3-containing nucleosomes (new\nSupplementary Table 4). Thus, a substantial fraction of the macro-monocentromeric regions\nforms the active centromere.”\n16\n4) Lines 326-332 and 517-518. Neither KNL1 nor NDC80 could be detected by\nimmunolocalization in luteum. This is perhaps the most surprising result in the study. If the KMN\ncomplex is absent that would suggest a completely different kinetochore structure. However, the\nas it is, the data are unconvincing. Indeed, both genes are present and expressed, so the data as\nshown suggest the KMN complex has a different form (or other proteins present) that change the\naccessibility of antibodies to their targets. If the authors want to suggest that KNL1 and NDC80\nare absent, they need to do some western blots.\nRESPONSE\nWe agree that the absence of detectable KNL1 and NDC80 signals in our immunolocalization\nexperiments does not prove that these proteins are absent. Both genes are indeed present in the\ngenome and expressed in transcriptome data. The lack of detectable signals most likely reflects\nantibody incompatibility, or conformational masking of epitopes in the kinetochore context, rather\nthan the absence of the proteins themselves.\nUnfortunately, due to the technical constraints, we are unable to perform Western blotting at this\ntime. To avoid overinterpretation, we have revised the text to state that KNL1 and NDC80 were\nnot detected by immunolocalization with the antibodies used in this study, and we refrain from\nconcluding that they are absent. We also discuss possible reasons for the lack of\nimmunodetection, as suggested by the reviewer. Now it reads (Page 18, Line 557-567): “NSL1,\nas a component of MIS12c, is one of the key structural kinetochore proteins. It is essential for\ninteraction with the other two complexes of the outer kinetochore, NDC80c and KNL1c. In\nDrosophila, NSL1 knockout mutants are lethal, underscoring its essential role for kinetochore\nfunction. The effects of the fusion of NSL1 with the N-terminal region of BORCS6 on the formation\nof KMN complex in centromeres of Cha. luteum remain unclear. While the lack of KNL1 and\nNDC80 immunosignals could indicate dramatic changes in kinetochore composition the results\ncould also be due to suggests antibody incompatibility or conformational masking of epitopes\nwithin the kinetochore contextor inaccessibility of antibody target domain. Thus, further\ninvestigation is needed to clarify the kinetochore composition and function in Cha. luteum.”\n5) Line 383. Change from “in short, the divergent..” to “in short, we propose that the divergent..”.\nThank you very much for the suggestion, we rewrote the Discussion. This sentence was removed.\n6) The discussion can be improved. I found it to be rambling and overly speculative. In one section\nthe authors suggest that KNL2 mutation is the most common cause of major shifts in centromere\nstructure, yet in the next section, a case is made for borealin as the instigating event in this clade.\nIf both are involved, then, taken together, the data suggest that there are multiple kinetochore\nalterations over long time frames, and seem more consistent with the view that centromeres\nchanged first and the kinetochore mutations followed.\nRESPONSE\nWe rewrote most of the DISCUSSION and considered the option that the centromeres change\nfirst and the kinetochore mutations follow.\nReviewer #4 (Remarks to the Author)\nThis study focuses on a comparative analysis of two closely related species within the tribe\nChionographideae that exhibit distinct centromere types. By integrating genomic, epigenomic,\ncentromere structural, and kinetochore protein data, the research aims to elucidate the\nmechanisms underlying the transition from monocentric to holocentric chromosomes. The topic\nis novel, and the research framework is comprehensive, especially in its incorporation of multi-\ndimensional data across molecular, cytological, and structural levels, offering significant insights\ninto evolutionary biology. However, concerns remain regarding the quality of the genome\nassemblies, the depth of functional annotation, and the completeness of the evidence supporting\ncertain key conclusions. These aspects require further clarification or supplementation.\nA key concern is that the genome assembly used for Cha. luteum in this study is only 749 Mb,\nrepresenting about 84% of the estimated genome size (~887 Mb), with a contig N50 of 2.1 Mb.\nThis is relatively low given current standards in the era of HiFi and Hi-C sequencing, especially\n18\nfor analyzing highly repetitive regions such as centromeres or detecting gene family loss/fusion\nstructural variations, where misinterpretation is likely. Improving the genome assembly quality\nand performing full-length transcriptome sequencing for Cha. luteum would enhance the reliability\nof protein structure annotation and help verify gene models.\nRESPONSE\nWe agree with your concern about the genome assembly of Cha. luteum. Unlike expected, we\nfaced several problems. Our approach to assembling the Cha. luteum genome largely mirrored\nour successful strategy for Chi. japonica (Kuo et al. 2023a), where we generated a chromosome-\nscale reference genome despite a high proportion of centromeric repeats. However, Cha. luteum\npresented challenges much earlier in the process, beginning with the initial step of high-quality\nDNA isolation. Both Cha. luteum and Chi. japonica leaf tissues are rich in secondary metabolites\nthat severely compromise DNA yield and quality. To circumvent this, we aimed to propagate a\nsingle plant via tissue culture and isolate sufficient high-molecular-weight (HMW) DNA from the\nroots; unfortunately, this initial step already proved difficult.\nHowever, unfortunately, Cha. luteum grows extremely slowly and was not suitable for tissue\nculture in our hands. Therefore, we isolated genomic DNA from leaves from a pool of plants to\nobtain sufficient material for PacBio long-read sequencing, generating 101.7 Gb of PacBio CCS\nreads (~114×). However, the initial assembly was highly fragmented (14,573 contigs, N50 of ~652\nkb). Moreover, the total assembled size was ~1.6 Gb, nearly twice the haploid genome size\nestimated by flow cytometry (1C = 887.4 Mb), which probably indicates an issue caused by\npooling the plant materials for sequencing.\nGenome assembly assessment of Cha. luteum\nSecond Assembly after\nFirst assembly\nassembly purge_dups\nNo. of contigs 14,573 4,087 649\nLargest contig 6,926,063 19,839,389 19,839,389\nTotal length 1,597,791,599 1,556,940,134 744,551,200\nN50 625,023 1,631,558 2,111,717\nN75 57,449 663,828 1,228,881\nL50 564 192 87\nL75 3163 564 205\nGC (%) 41.55 40.51 41.13\nComplete and single-copy BUSCOs - 1167 (36.1%) 2477 (76.5%)\nComplete and duplicated BUSCOs - 1744 (53.9%) 291 (9.0%)\nFragmented BUSCO - 171 (5.3%) 215 (6.6%)\nMissing BUSCO - 154 (4.7%) 235 (7.9%)\nTotal BUSCO groups searched - 3236 3236\nTo overcome this, next, we extracted HMW DNA from the leaves of a single plant of Cha. luteum\nfor a second PacBio run (53 Gb, ~59.5× coverage). Applying the same assembly approach, the\nimproved assembly reduced contigs to 4,087 and increased the largest contig size (19.84 Mb),\nand raised the N50 to 1.63 Mb. Although we used Hifiasm (Cheng et al. 2021), a haplotype-\nresolved de novo assembler based on phased assembly graphs, the assembled genome size\n(~1.56 Gb) was nearly twice the 1C genome size of 887.4 Mb estimated by flow cytometry. This\nunexpectedly large assembly size, along with the high proportion of duplicated BUSCOs (68.0%),\nindicated substantial heterozygosity in the primary contigs. To address this, we applied\npurge_dups to remove redundant haplotypic sequences, which may also have included some\ncentromeric repeat arrays. The refined assembly contained 649 contigs (N50 = 2.1 Mb, L50 = 87)\nand retained 92.7% complete BUSCOs. Finally, Hi-C scaffolding generated the top 22 large\nscaffolds, each exceeding 10 Mb (10–68 Mb), used for downstream analyses. The limited plant\nmaterial from a single plant prevented us from performing Nanopore ultra-long sequencing to\nimprove the assembly further.\nHowever, in our study, we did not intend to decipher the complete genome of Cha. luteum. We\naimed for insight into the evolution of atypical centromeres accompanying the mono- to\nholocentric transition. As demonstrated by our comparative chromosome painting experiment\n(Figure 3C–E), we confirmed by FISH the accuracy of the obtained genome analysis, because\nthe employed FISH probes were designed based on the determined sequences. Also, the\nobtained genome assembly in combination with ChIP-seq, immunostaining, and FISH was\nsufficient for the characterization of the macro-monocentromeres.\nsupporting evidence is insufficient. The authors suggest that the N-terminal regions of these two\nchimeric genes originate from RPS17 and BORCS6, respectively, based mainly on sequence\nhomology analysis. However, the following critical evidence is lacking. No transcriptional\nvalidation of the fusion junctions (e.g., RT-PCR); No demonstration of adjacent structural changes\non the chromosome (is this a true gene fusion or a misannotation?)\nRESPONSE\nWe understand your concern. We have now provided multiple lines of evidence to validate the\nchimeric nature of Borealin and NSL1 genes at both the genomic and transcriptional levels:\n1) Transcriptomic evidence: Transcripts corresponding to the two genes were also identified\nin a genome-independent transcriptome assembly (Trinity) derived from RNA-seq data,\nconfirming that these chimeric sequences are not assembly artefacts.\n2) Transcriptional validation: We performed RT-PCR for both chimeric Borealin and NSL1\ngenes, targeting the fusion junctions. The amplified products match the expected sizes,\nand Sanger sequencing of these products confirms the chimeric origin. The gel image and\nsequencing results are now provided in Supplementary Fig. 8A-B, and the corresponding\nmaterials and methods are included in the main text (Page 25-26, line 774-787).\n3) Genomic evidence: Chimeric Borealin (fused RPS17 and Borealin) and NSL1 (fused\nBORCS6 and NSL1) are present in single PacBio reads from both Cha. luteum and Chi.\njaponica. These reads span the entire gene structure, including exons and introns, of both\ngenes. We provide a new Supplementary Fig. 8C illustrating these PacBio reads and gene\nstructures.\nTaken together, these results provide strong evidence that Borealin and NSL1 are genuine\nchimeric genes rather than misannotations or assembly artefacts. Now it reads in the main text\n(Page 11, line 326-328): ”The chimeric nature of Borealin and NSL1 genes was further confirmed\nby transcriptional validation and genomic evidence (Supplementary Fig. 8).”\nThe conclusion that the holocentromeres in Chionographis arose de novo may be premature. The\nauthors base this inference on the lack of synteny between the monocentromere position in Cha.\nluteum and the holocentromeric units in Chi. japonica, yet holocentromere formation is known to\ninvolve neocentromerization at multiple non-centromeric regions. The authors should consider\n21\nconducting ancestral state reconstruction to infer whether the common ancestor of Cha. luteum\nand Chi. japonica was more likely monocentric or holocentric. Further searches for conserved unit\npositions and residual Chama-like centromeric sequences in Chi. japonica would also be valuable.\nRESPONSE\nOur conclusion regarding the de novo origin of holocentromeres in Chionographis is supported\nby the observed broad-scale synteny between its holocentric Chi. japonica and that of macro-\nmonocentric Cha. luteum, with the notable exception of their centromeres. We found a high\ndegree of genome conservation between both species, despite their divergence approximately\n23.5 million years ago. Specifically, syntenic blocks in Chi. japonica were found to be split by\ncentromere units, which we interpreted as an indication of the de novo origin of these centromere\nunits. The positions of the monocentromeres in Cha. luteum and the centromere units in Chi.\njaponica do not always correspond according to syntenic genes, suggesting that the loss of\nmonocentromeres in Chionographis was likely accompanied by de novo holocentromere\nformation. A de novo origin of macro-monocentromeres derived from holocentromeres is unlikely,\nbecause a de novo formation of monocentric chromosomes based on multiple fission events of a\nholocentric chromomosome would result in numerous monocentric chromosomes. However, the\nchromosome number of Cha. luteum is in the range of Chionographis species. In addition, it is\nvery likely that a common ancestor of Cha. luteum and Chi. japonica was monocentric because\nall known closely related species are monocentric (see Figure 1a, based on the phylogenetic trees\nof (Pellicer et al. 2014; Kim et al. 2019; Givnish et al. 2016). The tribes, holo/macro-monocentric\nChionographideae and monocentric Heloniadeae, appear to have originated in North America,\nat\n23.5 mya in the early Miocene and 27.3 mya in the late Oligocene, respectively, with the ancestors\nof Chionographis and monocentric Heloniopsis – monocentric Ypsilandra (China, Japan, and\nKorea) dispersing into East Asia. Thus, both tribes show an intercontinental disjunction between\nEast Asia and North America at the intergeneric level.\nOur findings are consistent with the reviewer's point that holocentromere formation is known to\ninvolve neocentromerization at multiple non-centromeric regions. Our study revealed that the de\nnovo holocentromere formation sites in Chi. japonica correspond to gene-rich, accessible\nchromatin regions in the syntenic Cha. luteum genome. This observation directly aligns with the\nconcept of neocentromerization occurring in regions that were previously non-centromeric and\ntranscriptionally active, rather than at pre-existing centromeric loci.\nrepeats between Heloniopsis umbellata and either Cha. luteum or Chi. japonica, we did identify a\nspecific conserved 9-base pair sequence (TTCGTACGA) that is shared between the Chama\nmonomer in Cha. luteum and the Chio monomer in Chi. japonica. Suggesting a potential ancient\nshared feature or functional significance.\nQuestion: “Further searches for conserved unit positions and residual Chama-like centromeric\nsequences in Chi. japonica would also be valuable.”\nRESPONSE\nSince we could not identify conserved unit position, we could not identifiy residual Chama-like\ncentromeric sequences in Chi. japonica. Also, after removing the shared 9-bp conserved motif\nbetween Chio and Chama monomers, the remaining Chama sequence showed no significant\nsimilarity to the genome of Chi. japonica in BLASTn (v2.5.0, default parameters). Given the\nabsence of conserved centromeric sequence features and extensive karyotype divergence\namong closely related taxa (e.g. Heloniadeae species with n=17, compared with n=12 in\nChamaelirium and Chionographis), an ancestral state reconstruction based solely on current\ngenomic and cytological data would not yield a reliable inference. Therefore, while we can’t\ndefinitely exlude alternative evolutionary scenarios, the available evidences best supports the\ninterpretation that holocentromeres in Chionographis arose de novo, rather than through\nmodification of ancestral monocentromeres.\nReferences\nCheng H, Concepcion GT, Feng X, Zhang H, Li H (2021) Haplotype-resolved de novo assembly\nusing phased assembly graphs with hifiasm. Nature methods 18 (2):170-175.\ndoi:10.1038/s41592-020-01056-5\nfission in Lepidoptera. Philosophical Transactions of the Royal Society B 375:20190539.\ndoi:https://doi.org/10.1098/rstb.2019.0539\nGivnish TJ, Zuluaga A, Marques I, Lam VKY, Gomez MS, Iles WJD, Ames M, Spalink D, Moeller\nJR, Briggs BG, Lyon SP, Stevenson DW, Zomlefer W, Graham SW (2016) Phylogenomics\nand historical biogeography of the monocot order Liliales: out of Australia and through\nAntarctica. Cladistics 32 (6):581-605. doi:10.1111/cla.12153\nHoshi Y, Terasaki K (2025) The AT-rich regions correspond to the localized centromeres in meiotic\nchromosomes of <i>Drosera filiformis</i> Raf. Cytologia 90 (2):101-108.\ndoi:10.1508/cytologia.90.101\nHouben A, Wako T, Furushima-Shimogawara R, Presting G, Kunzel G, Schubert I, Fukui K (1999)\nThe cell cycle dependent phosphorylation of histone H3 is correlated with the\ncondensation of plant mitotic chromosomes. Plant Journal 18 (6):675-679\nKim C, Kim SC, Kim JH (2019) Historical biogeography of Melanthiaceae: A case of out-of-North\nAmerica through the Bering land bridge. Front Plant Sci 10:396.\ndoi:10.3389/fpls.2019.00396\nKuo Y-T, Câmara AS, Schubert V, Neumann P, Macas J, Melzer M, Chen J, Fuchs J, Abel S,\nKlocke E, Huettel B, Himmelbach A, Demidov D, Dunemann F, Mascher M, Ishii T,\nMarques A, Houben A (2023) Holocentromeres can consist of merely a few megabase-\nsized satellite arrays. Nat Commun 14 (1):3502. doi:10.1038/s41467-023-38922-7\nMacas J, Avila Robledillo L, Kreplak J, Novak P, Koblizkova A, Vrbova I, Burstin J, Neumann P\n(2023) Assembly of the 81.6 Mb centromere of pea chromosome 6 elucidates the structure\nand evolution of metapolycentric chromosomes. PLoS Genet 19 (2):e1010633.\ndoi:10.1371/journal.pgen.1010633\nPellicer J, Kelly LJ, Leitch IJ, Zomlefer WB, Fay MF (2014) A universe of dwarfs and giants:\ngenome size and chromosome evolution in the monocot family Melanthiaceae. New\nPhytol 201 (4):1484-1497. doi:10.1111/nph.12617\nSchubert V, Neumann P, Marques A, Heckmann S, Macas J, Pedrosa-Harand A, Schubert I, Jang\nTS, Houben A (2020) Super-resolution microscopy reveals diversity of plant centromere\narchitecture. Int J Mol Sci 21 (10). doi:10.3390/ijms21103488\nReviewer #1 (Remarks to the Author):\nThe authors' responses to the reviewer comments are satisfactory. The re-written\nDiscussion is an improvement.\nLine 513: KNL should be KNL2\nRESPONSE\nWe have corrected KNL to KNL2.\nLines 547-552 and lines 554-560 have duplicated phrases that appear to be an editing\nerror.\nLines 548-549: Fix grammar: ”the results could also be due to suggests antibody\nincompatibility”.\nLine 550: “contextor” should be “context or”\nRESPONSE\nWe have corrected the errors and deleted the duplicated lines 554-560. Now it reads (line\n547-552): While the lack of KNL1 and NDC80 immunosignals could indicate dramatic\nchanges in kinetochore composition, the results could also arise from antibody\nincompatibility, conformational masking of epitopes within the kinetochore context, or\ninaccessibility of antibody target domain. Thus, further investigation is needed to clarify\nthe kinetochore composition and function in Cha. luteum.\nReviewer #2 (Remarks to the Author):\nMy comments have been addressed. I have three additional and very minor ones:\nThe title of Supplementary Table 4, \"Percentage of CENH3 amount colocalizing to Chama\nrepeats\" is incorrect. It should be something like “Percentage of Chama repeats\ncolocalizing with CENH3.”\nRESPONSE\nWe quantified the proportion of CENH3 signal volume that colocalizes with Chama\nrepeat signals. Accordingly, we have revised the title of Supplementary Table 4 to:\n“Percentage of CENH3 signal colocalizing with Chama repeats measured in 16 Cha.\nluteum root interphase nuclei”.\nDetailed methods describing how numbers in Supplementary Table 4 were generated are\nmissing.\nRESPONSE\nWe have added the methods in the Microscopy and image analysis of the Methods part.\nNow it reads (page 32, line 987-993): 3D rendering to produce spatial animations was\ndone based on 3D-SIM image stacks using the Imaris 9.7 (Bitplane, UK) software. The\ntool ‘Colocalization’ of the same software was applied to determine the CENH3 amount\ncolocalizing to the Chama repeats based on voxel intensities. For it, the colocalization\ncalculation threshold was automatically determined at P=1.000. Based on this, the\npercentage of the CENH3 volume above the threshold colocalizing to the Chama volume\nwas calculated per interphase nucleus.\nWhile it is implicit in the methods that multimapping ChIP-seq reads are included in the\nanalyses, it would be better to make this explicit since it a non-standard method. It would\nbe helpful to add a sentence such as this: “No filtering to remove multimapping reads was\napplied, so all analyses include both uniquely mapping.\"\nRESPONSE\nWe agree with this suggestion. We have added “No filtering to remove multimapping\nreads was applied; therefore, all analyses include both uniquely and multimapping reads.”\nin the ChIP-seq data analysis of Methods part (page 29, line 900-901).\nReviewer #3 (Remarks to the Author):\nThe manuscript is much improved and all of my comments have been addressed. The\ndiscussion is now more organized, cautious and balanced, as is warranted.\nThe authors may want to look at lines 547-558, where the wording is duplicated and\nsomewhat confusing.\nRESPONSE\nWe have corrected the errors and deleted the duplicated lines 554-560. Now it reads\n(page 18, line 547-551): While the lack of KNL1 and NDC80 immunosignals could indicate\ndramatic changes in kinetochore composition, the results could also arise from antibody\nincompatibility, conformational masking of epitopes within the kinetochore context, or\ninaccessibility of antibody target domain. Thus, further investigation is needed to clarify\nthe kinetochore composition and function in Cha. luteum.\nAlso, I could not locate the ChIP-seq or ATAC seq data at NCBI.\nRESPONSE\nWe apologize for the inconvenience. Now, the CENH3-ChIP-seq and ATAC-seq data\nare accessible through the links.\nCENH3-ChIP-seq (GEO accession GSE285103):\nAccess: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE285103\nATAC-seq (GEO accession GSE285102)\nAccess: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE285102\nThe newly included histone mark-ChIP-seq datasets have been submitted to the\neuropean GEO Annotare (accession number E-MTAB-16192) and are now in curation.\nReviewer #3 (Remarks on code availability):\nI am not capable of reviewing code.\nRESPONSE\nThe code used in this study was made publicly available, ensuring transparency and\nreproducibility.\nReviewer #4 (Remarks to the Author):\nThank you for your detailed response and for addressing my questions. I have no\nfurther comments.\nRESPONSE\nWe thank you for the positive feedback and for confirming that all your concerns have\nbeen addressed."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors' responses to the reviewer comments are satisfactory. The re-written Discussion is an improvement.\nLine 513: KNL should be KNL2\nLines 547-552 and lines 554-560 have duplicated phrases that appear to be an editing error.\nLines 548-549: Fix grammar: ”the results could also be due to suggests antibody incompatibility”.\nLine 550: “contextor” should be “context or”", "reviewer_2": "My comments have been addressed. I have three additional and very minor ones:\nThe title of Supplementary Table 4, \"Percentage of CENH3 amount colocalizing to Chama repeats\" is incorrect. It should be something like “Percentage of Chama repeats colocalizing with CENH3.”\nDetailed methods describing how numbers in Supplementary Table 4 were generated are missing.\nWhile it is implicit in the methods that multimapping ChIP-seq reads are included in the analyses, it would be better to make this explicit since it a non-standard method. It would be helpful to add a sentence such as this: “No filtering to remove multimapping reads was applied, so all analyses include both uniquely mapping.\"", "reviewer_3": "The manuscript is much improved and all of my comments have been addressed. The discussion is now more organized, cautious and balanced, as is warranted.\nThe authors may want to look at lines 547-558, where the wording is duplicated and somewhat confusing.\nAlso, I could not locate the ChIP-seq or ATAC seq data at NCBI.", "reviewer_4": "Thank you for your detailed response and for addressing my questions. I have no further comments."}, "author_response": ""}]

Decision letter