Comparative analysis of organellar genomes between diploid and tetraploid Chrysanthemum indicum with its relatives

Chrysanthemum indicum, a species native to Eastern Asia is well known as one of the progenitor species of the cultivated Chrysanthemum which is grown for its ornamental and medicinal value. Previous genomic studies on Chrysanthemum have largely ignored the dynamics of plastid genome (plastome) and mitochondria genome (mitogenome) evolution when analyzing this plant lineage. In this study, we sequenced and assembled the plastomes and mitogenomes of diploid and tetraploid C. indicum as well as the morphologically divergent variety C. indicum var. aromaticum. We used published data from 27 species with both plastome and mitogenome complete sequences to explore differences in sequence evolution between the organellar genomes. The size and structure of organellar genome between diploid and tetraploid C. indicum were generally similar but the tetraploid C. indicum and C. indicum var. aromaticum were found to contain unique sequences in the mitogenomes which also contained previously undescribed open reading frames (ORFs). Across Chrysanthemum mitogenome structure varied greatly but sequences transferred from plastomes in to the mitogenomes were conserved. Finally, differences observed between mitogenome and plastome gene trees may be the result of the difference in the rate of sequence evolution between genes in these two genomes. In total the findings presented here greatly expand the resources for studying Chrysanthemum organellar genome evolution with possible applications to conservation, breeding, and gene banking in the future.


Background
Chrysanthemum indicum, a perennial herbaceous plant in the Asteraceae family, is widely used as a medicinal, ornamental, and food plant (Luo and Li, 2013).Previous studies have found that C. indicum possesses variable traits across the species range and also varies in ploidy level (Li et al., 2013;Klie et al., 2014;Qi et al., 2021), including diploid, tetraploid, andhexaploid (Si-Lan. et al., 1998;C. et al., 2004;El-Twab and Kondo, 2007;Li et al., 2013;Klie et al., 2014).For example C. indicum from coastal saline soils were noted to be dwarfed, while those found on Mount Lu (Jiangxi Province, China) were found to have a greater density of trichomes on their leaves than plants from the other regions (Luo and Li, 2013).Frequent gene flow has been reported between species in the Chrysanthemum genus (Si-Lan. et al., 1998;C. and S, 2002;C. et al., 2004;El-Twab and Kondo, 2007;Luo et al., 2016), as well as among different accessions within C. indicum (Li et al., 2013;Luo et al., 2016;Ma et al., 2020;Qi et al., 2021).In previous phylogenetic studies about Chrysanthemum genus, C. indicum were frequently found to be most closely related with C. zawadskii and C. nankingenese (C. et al., 2004).However, substantial gene flow has led to conflicting phylogenetic outcomes within the genus and among wild populations of C. indicum (C. et al., 2004;El-Twab and Kondo, 2007).Previous studies have suggested that tetraploid C. indicum originated from hybridization between diploid C. indicum and related species (Li et al., 2013), while other studies have found that some C. indicum polyploid populations were homoeologous polyploids (Li et al., 2014).Within C. indicum the variety C. indicum var.aromaticum was described to account for individuals that possess thicker leaf blades and glandular hairs (Luo and Li, 2013), but studies on this variety have also found that gene flow has occurred between it and other C. indicum lineages (Luo and Li, 2013;Yuan et al., 2022).Much of the research regarding C. indicum has been in its contribution to the Chinese cultivated Chrysanthemum which is an allohexaploid lineage used as a horticultural ornament (Si-Lan. et al., 1998;C. and S, 2002;C. et al., 2004;El-Twab and Kondo, 2007).The above-mentioned evidence for genetic admixture in Chrysanthemum has been derived from the nuclear loci with little known about how mitogenomes and plastomes have been sorted and evolved during episodes of admixture and increases in ploidy.
Sequence data from plant mitogenomes and plastomes has been widely used for plant phylogenetic research due to the presence of conserved nonrecombinant sequences that flank more rapidly evolving regions (Kelchner, 2002;Rogalski et al., 2015;Uncu et al., 2015).For example, the phylogeny of the three large families: Arecaceae, Moraceae, and Saxifragaceae, have been resolved with plastome data (Liu et al., 2020;Zhang et al., 2022;Yao et al., 2023).With the decreasing cost of high-throughput sequencing and the improvement of assembly techniques, an increasing number of studies are using complete organellar genomes instead of one or several loci to conduct phylogenetic and evolutionary studies in plants.Currently, 1852 plastomes (mostly from different varieties of the same species) and 62 mitogenomes of Asteraceae are available in GenBank.The smaller number of published mitogenomes is a result of the difficulty in assembling plant mitochondrial genomes (high frequency of rearrangements) using short read sequencing techniques (Uncu et al., 2015).For example, it is difficult to resolve complex mitogenome assembly results with different contig-repeat-contig combinations by using only short read sequencing data (Zhang et al., 2023).However, the issue of complete mitogenome assembly is gradually being addressed with the use of long-read sequencing methods.Although many organellar genomes have been published from the Asteraceae, most studies have been limited to phylogenetic research, without further exploring the evolutionary patterns within a species or how organellar genome evolution correlates with changes in nuclear genome ploidy (Xia et al., 2016;Kim et al., 2018;Won et al., 2018;Feng et al., 2020;Xia et al., 2021a;Xia et al., 2021b;Yu et al., 2021;Masuda et al., 2022).
In this study, we sequenced and assembled the complete mitogenomes and plastomes of diploid and tetraploid C. indicum, as well as C. indicum var.aromaticum, and compared them with those of 23 Asteraceae species for which public data was available for both organellar genomes.This is the first study to explore the evolutionary histories of varieties of C. indicum with different ploidy levels and phenotypic variations using organellar genomes as a basis for historical inference.We compared the differences of the sequence and structure of organellar genomes among different varieties of C. indicum, resolved phylogenetic relationships among different varieties of C. indicum, analyzed the diversity of simple sequence repeats (SSRs), and quantified the gene transfers from plastomes to mitogenomes.

Genome characteristics of Chrysanthemum organelle
By utilizing long PacBio HiFi reads, the mitogenomes of diploid (2x) and tetraploid (4x) C. indicum were assembled with published C. indicum mitogenome (NCBI accession number: MH716014.1)used to extract sequences from whole genome data.In addition the mitogenome of C. indicum var.aromaticum was also assembled with Illumina reads.The mitogenome of C. indicum (2x) was assembled into a single circular genome, and was used to resolve the mitogenome of C. indicum (4x) and C. indicum var.aromaticum with multiple linkage structures (Figure S1).The sizes of the three circular mitogenome were 192,408 bp with 45.51% GC content for C. indicum (2x), 193,563 bp with 45.43% GC content for C. indicum (4x), and 198,095 bp with 45.33% GC content for C. indicum var.aromaticum, similar with that in found in most Asteraceae species sequenced at present (Figure 1).All three of these newly assembled mitogenomes contain 51 unique genes, including 33 protein coding genes (PCGs) genes, three rRNA genes, and 16 tRNA, with trnS-GCU having two copies and trnM-CAU having three copies in C. indicum (2x) and C. indicum (4x), but five copies in C. indicum var.aromaticum (Table 1).Among the PCGs, eight genes contained introns, three of which (ccmFC, nad4 and rps3) contained one intron, and five others (cox2, nad1, nad2, nad5, and nad7) contained two or more introns.Among the three mitogenomes, all PCGs are single copy, except atp9 that has two copies in C. indicum var.aromaticum.
All three newly assembled chloroplast genomes maintain the classical dumbbell-shaped genome structure.The plastome sizes of the three samples were very similar at 151,033 bp for C. indicum (2x), 151,036 bp for C. indicum (4x) and 151,053 bp for C. indicum var.aromaticum with the same GC content at 37.47% for all these plastomes (Figure S2).All three of these newly assembled plastomes of Chrysanthemum contain 109 unique genes, including 80 PCGs, 25 tRNAs, and four rRNAs with the IR region containing six protein coding genes, eight tRNAs and four rRNAs, with 13 PCGs containing introns, and two of these (rps12 and ycf3) have more than one intron.Overall, compared to mitogenomes, these three plastomes differ only slightly in a few regions and are generally conserved in sequence similarity.

Differences between organelle genome sequences of Chrysanthemum
To better understand organellar genome evolution in Chrysanthemum, the three mitogenomes from Chrysanthemum indicum assembled in this study were used for genome-wide collinear comparisons with published mitogenomes of four Chrysanthemum species C. indicum (MH716014), C. boreale (NC_039757), C. makinoi (OU343227), and C. zawadskii (ON053202) (Figure 2).The four accessions from C. indicum have a more similar mitogenome structure in the genome-wide alignment results for sequence lengths greater than 1 Kb, except in C. indicum (MH716014) which did not have the 22.6 Kb and 23.7 Kb inversions found in the other three species (Figure 2).Between the four Chrysanthemum species mitogenomes highly divergent rearrangements were found pointing to a process of rapid structural turnover between species (Figure 2).Among the three newly assembled mitogenomes, C. indicum (4x) has a unique sequence of 2.8 Kb compared to C. indicum (2x), on which two ORFs encoding proteins of 496aa and 72aa in length were predicted.In C. indicum var.aromaticum two unique sequences of length 3.3 Kb and 5.6 Kb were found with ORFs encoding proteins of 307aa, 165aa, and 73aa in length predicted on the 3.3 Kb sequence.On the 5.6 Kb sequence ORFs encoding proteins 383 aa, 346 aa, 94 aa, 72 aa, and 71 aa in length, and two trnM-CAU were predicted (Table S2).Comparative sequence analysis of the plastomes corresponding to seven Chrysanthemum accessions showed that the plastome sequences were conserved within the genus (Figure S3).In addition, to understand the structural differences of mitogenomes within cultivated C. indicum, the three newly assembled mitogenomes were compared separately for intra-genomic collinearity, and it was found that these mitogenomes all contain few repetitive sequences, which explains the relatively small size of these mitogenomes.The C. indicum (2x) and C. indicum (4x) mitogenomes were more similar in intra-genomic collinearity patterns and showed similarity between their genome structure (Figure S4).In general, the mitogenomes showed large structural differences between species within Chrysanthemum and greater synteny within species while the plastomes were structurally conserved across all samples.
To further understand the differences between organellar genomes of Chrysanthemum repeat sequences were annotated and compared among seven Chrysanthemum accessions (Figure 3).The Chrysanthemum organellar genomes contained repeat types Complement (C), Forward (F), Palindromic (P), and Reverse (R) at different ratios, with repeats less than 50 bp being the most common (Figures 3A, B).In all accessions, the number of repeats of the F and P type were dominant in both genomes but in plastomes the repeat types were more evenly distributed especially among the shorter repeat sequences (Figures 3A, B).The abundance of F and P type repeats in the mitogenome is correlated with larger mitogenomes.To better understand the dynamics of repetitive sequences in the organelles of the Chrysanthemum plastome and mitogenome, SSR sequences of seven accessions were analyzed (Figures 3C, D).A total of 23, 24, 25, 23, 25, 39, and 3D), respectively.The number of SSRs in plastomes was greater than that in the corresponding mitogenomes (Figures 3C, D).The number of A/T SSRs in the plastomes and mitogenomes far exceeded the combined number of all other types of SSRs while the mitogenome of C. makinoi had a much higher number of A/T repeats than the other accessions (Figures 3C, D).The mitogenomes of all four accessions from C. indicum did not contain AG/CT SSRs, but C. indicum (4x) possessed a unique ACAGAT/ATCTGT SSR, and C. boreale possessed a unique AAGGCT/AGCCTT SSR (Figure 3C).In the plastomes the number and type of SSRs are more conserved, except for C. boreale and C. zawadskii that did not contain any C/G SSRs, all other accessions contained four C/G SSR loci (Figure 3D).

Intracellular gene transfer between organellar genomes
In plants, the exchange of genetic material between cellular compartments (nucleus, mitochondria, and chloroplasts) is often referred to as Intracellular Gene Transfer (IGT) (Bergthorsson et al., 2003;Aubin et al., 2021).Characterizing these IGT events is key to understanding the genetics and evolution of organellar genomes.We did this in Chrysanthemum, by quantifying the sequence transfers from plastomes to mitogenomes (Figure 4).Each accession had a total of 8-10 (from 5220-9176 bp in total length) fragments from a plastomic origin integrated into 8-12 (5212-9164 bp) locations in the mitogenome.The least number (8) of transferred sequences was in C. indicum (2x) and the most (10) in C. makinoi.The length of the transferred fragments ranged from the shortest at 79 bp to the longest of 2556 bp (Table 2; Table S1).Correlation analyses indicated that the number of plastome transferred sequences and the size of the corresponding mitogenomes were positively correlated, as the number and length of plastome transfer sequences was greater for accessions with large mitogenomes (Figure S5).The transferred sequences across the seven accessions were conserved in respect to both size and sequence of origin.Specifically all accessions contained seven completely transferred gene sequences (petL, petG, trnM-CAU, trnH-GUG, trnN-GUU, trnW-CCA, and trnP-UGG) and five partially transferred gene sequences (16S rRNA, rps11, ycf2, psaB, and psaA) (Table 2) S1).These results suggest that the plastomic fragments were transferred to the mitogenome predating speciation, and that shared transfers specifically between C. indicum var.aromaticum, and other Chrysanthemum species may be the result of past hybridization, incomplete lineage sorting, or retention of ancestral transfers lost in other C. indicum species.

Phylogenetic relationships
A maximum likelihood (ML) phylogenetic tree of 26 Asteraceae species (Platycodon grandifiorus was set as outgroup) with complete mitogenome and plastome data was constructed based on 80 plastome genes and 32 mitogenome genes, respectively (Figure 5).In our study, the phylogenetic topology at tribe level was generally consistent with previous research (Zhang et al., 2021).However, the internal phylogenetic relationships within the Anthemideae were somewhat different.On the mitogenome tree, C. boreale grouped with Artemisia giraldii, while on plastome tree, it grouped with Chrysanthemum.This may be due to the fact that the four mitogenome genes (atp9, mttb, nad2 and nad6) in C. boreale show greater similarity with that in A. giraldii, especially the atp9 gene which was identical between the two species.In a previous study, it was suggested that C. boreale may be an early diverging species within Chrysanthemum, and thus may have retained more ancestral characters (El-Twab and Kondo, 2007).From the phylogenetic tree all four genomes of C. indicum clustered together in both plastome and mitogenome analyses.The phylogenetic analysis also suggested that the maternal donor of the tetraploid C. indicum may be the diploid C. indicum, while C. indicum var.aromaticum appears to have undergone some degree of divergence from the other C. indicum.
With the set model parameters, we obtained two time tree result based on plastome estimation and mitogenome estimation (Figures S6, S7).From the results, the 95% intervals obtained for plastomes are more convergent, while those for mitogenomes are broader, which may be related to the mutation rate and the number of mutation sites in mitochondria (Figure S6).Here we take the plastome results as the basis.From the results, we learnt that the C. indicum was born at 1.43 Mya, and C. indicum var.aromaticum and C. indicum were separated at 0.81 Mya; while tetraploid C. indicum and diploid C. indicum were separated at 0.38 Mya (Figure S7).

Discussion
Interspecific diversity and intraspecific conservation of mitogenome structure and sequence in Chrysanthemum Plant mitogenomes have undergone tremendous structural changes during evolution, which have resulted in a near 200-fold variation in mitogenome size among plants (from ~66 Kb of Viscum scurruloideum to ~12 Mb of Larix sibirica) and frequent rearrangements with low gene density, in contrast to plant plastomes which are conserved in genome size from 115-165 Kb and containing a dumbbell-shaped genome structure (Putintseva et al., 2020;Wu et al., 2020).Owing to the release of a large amount of plant whole genome sequencing data, an increasing number of mitogenomes have been assembled and subjected to comparative studies, in which many mitogenomes have been found to exhibit a Synteny between Chrysanthemum mitogenomes.Mitogenomes from seven accessions with sequence rearrangements longer than 1 Kb between two pairs shown.
reticulated complex genomic structure.The diversity of many mitochondrial genomic structures is thought to the result of recombination mediated by repetitive sequences (Wu and Sloan, 2019).However, the three mitogenomes of C. indicum newly assembled in this study show a simple monocyclic structure, along with a low number and percentage (0.9%-2.2%) of repetitive sequences, which may account for the structure and relatively small size at just over 40 Kb longer than their respective plastomes.In a comparison among four accessions of C. indicum, it was found that the mitogenome structure and sequence within species is relatively conserved, in contrast to large differences observed among mitogenomes within cultivated sorghum (Zhang et al., 2023).Meanwhile, only a 2.8 Kb segment of the mitogenome differed between accessions of C. indicum of different ploidy levels, two ORFs encoding proteins 496aa and 72aa in length were predicted on this segment, and the functions of these two ORFs have yet to be investigated by mitochondrial gene editing techniques (Kazama et al., 2019).However, when comparing among four different species in Chrysanthemum, it was found that the mitogenomes differed not only in size, but also in structure, mainly in genome sequence order.In conclusion, the relationship between mitogenome sequence and structural variation

Conserved intracellular gene transfers between organellar genomes in Chrysanthemum mitogenomes
IGT occurs frequently and continuously during nucleoplasmic coevolution between different genomes in plant cells (Zhang et al., 2020).In this study, we assembled the mitogenomes and plastomes of three C. indicum accessions and examined the sequence transfers from plastome to mitogenome in combination with four published organellar genomes of species in Chrysanthemum.It was found that the number and the size of these transferred sequences within Chrysanthemum showed a positive correlation with the size of the mitogenome.The transferred genes were conserved among the accessions, and the size of the transferred fragments containing these genes was also generally conserved.All samples contained seven completely transferred gene sequences from the plastome (petL, petG, trnM-CAU, trnH-GUG, trnN-GUU, trnW-CCA, and trnP-UGG) and five partially transferred genes (16S rRNA, rps11, ycf2, psaB, and psaA).These results suggest that these sequence transfers occurred prior to the divergence of Chrysanthemum species and have been retained through time albeit in different locations of the genome given rapid turnover in structure from recombination.Interestingly, all four accessions of C. indicum have the same transferred gene, except for C. indicum var.aromaticum that has an additional 135 bp transferred sequence containing a partially transferred sequence of the 23S rRNA gene.This transferred fragment shared between C. indicum var.aromaticum and the other species in Chrysanthemum may suggest that this lineage is early diverging or that introgression or incomplete lineage sorting may have been involved in the evolution of this variety.

Phylogenetic relationships
We observed different topologies between phylogenetic analysis based on plastome and mitogenome sequences.Overall, the results obtained from chloroplasts are consistent with previous studies based on nuclear genes, while mitochondria tend to exhibit conflicting resolutions compared with nuclear gene results in adjacent clades (Zhang et al., 2021).This may be due to the slow mutation rate in sequences that have been observed in plant mitogenomes (Duff and Nickrent, 1999;Xi et al., 2013;Grewe et al., 2014).Additionally, the high frequency of RNA editing sites in mitochondrial genes imposes difficulties for mitochondrial gene tree construction (Drouin et al., 2008;Knoop, 2011).For example, in angiosperms, the number of editing sites in the mitogenome ranges from 200 to 500, while in the plastome, there are only 30 to 50 editing sites (Malek et al., 1996;Knoop, 2011;Zhang et al., 2021).Furthermore, the editing sites in the mitochondrial genome are not only evident in the coding region, but also in tRNA, introns, and 5' and 3' untranslated regions (Malek et al., 1996;Oldenkott et al., 2014).Our study supports the view that mitochondrial genes may not be appropriate for plant phylogenetic inferences at some levels of taxonomic organization et al., 1996;Drouin et al., 2008;Zhang et al., 2021).

Conclusions
The current research used highly accurate long-read HiFi data to assemble mitogenomes from diploid and tetraploid C. indicum and short read sequencing data for C. indicum var.aromaticum using assembly software GSAT.We compared the differences among mitogenomes from different species within Chrysanthemum and different accessions of C. indicum using the newly assembled and previously published organellar genomes, and found that between species large structural differences are present, but within C. indicum species the structure is more conserved between accessions of different ploids.Meanwhile the plastome is structurally conserved across Asteraceae.Transferred genes from plastome to mitogenome showed high levels of sequence conservation within the accessions of different ploids from C. indicum species.In addition, we resolved phylogenetic relationships using plastome and mitogenome CDSs of 27 Asteraceae accessions, with Platycodon grandiflorus as an outgroup and found conflicting resolutions between these gene sets potentially because of the extremely slow rates of evolution among mitogenome CDSs.
All gene pairs alignments were converted into PAML format using EasyCodeML (Yang, 2007;Gao et al., 2019).All the sequences pairs was merge in one file.Molecular divergence times were estimated by placing soft boundaries on the split nodes using records from the timetree database (http://timetree.org/).For divergence time markers we used two nodes, one for the divergence of the outgroup from the Asteraceae, set at 67.7-93.0Mya, and the other divergence time for the node represented by Arctium lappa from the node represented by Chrysanthemum indicum, set at 36.6-45.1 Mya.The MCMCtree module of PAML software was implemented to estimate the divergence time of each node (Dos Reis, 2022).Tracer was used to evaluate whether the results of MCMCtree have converged.The result of divergence time tree results are visualized using ggtree package (Xu et al., 2022).

FIGURE 1
FIGURE 1 Mitogenome structure from three C. indicum accessions in this study.* indicates gene containing intron (s).
. Interestingly, C. indicum var.aromaticum, C. boreale, and C. makinoi have partially transferred sequences of the 23S rRNA gene, but the lengths of these fragments vary from 135 bp in C. indicum var.aromaticum to 357 bp and 443 bp in C. makinoi, and 238 bp in C. boreale (Table

FIGURE 4
FIGURE 4 Schematic diagram of homologous sequences identified among the two organellar genomes of seven C. indicum accessions.Plastomes are on the left and corresponding mitogenomes are on the right.Lengths are given in Kb.

TABLE 1
Gene composition in the mitogenome of Chrysanthemum indicum.