Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 75(4): 93-101, 2022 Firenze University Press www.fupress.com/caryologia ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.36253/caryologia-1902 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: Peng Zhou, Jiao Li, Jing Huang, Fei Li, Qiang Zhang, Min Zhang (2022). Determination of genome size variation among varieties of Ilex cornuta (Aquifoliaceae) by fow cytom- etry. Caryologia 75(4): 93-101. doi: 10.36253/caryologia-1902 Received: November 11, 2022 Accepted: December 15, 2022 Published: April 28, 2023 Copyright: © 2022 Peng Zhou, Jiao Li, Jing Huang, Fei Li, Qiang Zhang, Min Zhang. This is an open access, peer- reviewed article published by Firenze University Press (http://www.fupress. com/caryologia) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All rel- evant data are within the paper and its Supporting Information files. Competing Interests: The Author(s) declare(s) no conflict of interest. ORCID QZ: 0000-0002-2025-9576 Determination of genome size variation among varieties of Ilex cornuta (Aquifoliaceae) by fow cytometry Peng Zhou1, Jiao Li2, Jing Huang1, Fei Li1, Qiang Zhang2,*, Min Zhang1,* 1 Jiangsu Academy of Forestry, Nanjing 211153, Jiangsu, China 2 Co‑Innovation Center for Sustainable Forestry in Southern China, Key Laboratory of State Forestry and Grassland Administration on Subtropical Forest Biodiversity Conser‑ vation, College of Biology and the Environment, Nanjing Forestry University, Nanjing 210037, Jiangsu, China *Corresponding author. E-mail: zhangqiang@njfu.edu.cn; E-mail: nmzhang@163.com Abstract. Ilex cornuta Lindl. & Paxton is a commercially important horticultural spe- cies worldwide, and extensive cultivation and hybridization have produced many varie- ties. Despite the considerable breeding, selection, widespread cultivation and domesti- cation, which may have a significant role in the composition of genomes, there are no other previous reports of intraspecific genome size variation in the different cultivars or hybrids of this species. In the present work, genome size of 12 varieties of I. cornuta was assessed and analyzed through high-resolution flow cytometry (FCM). Nuclear DNA was analyzed using nuclei isolated from young leaves, which used propidium iodide (PI) staining, with rice (Oryza sativa cv. Nipponbare) as internal reference. As a result, statistically significant differences in genome size were detected among all diploid I. cornuta varieties considered. The estimated genome size (2C value) of I. cornuta varieties ranged from 1.47 to 1.80 pg, with 1.22-fold variation and an average size of 1.65 pg. The domestication and interspecific hybridization induced variation of genome size in I. cornuta, and the genome size of hybrids exhibited a wider range of variation compared with that of cultivars. In summary, flow cytometry is a useful tool to analyze the genome size of I. cornuta. The first report of the genome sizes of varie- ties of this species would provide useful data for further research on I. cornuta, and enrich the C value database of Ilex L. What’s more, our findings could be the founda- tion in the future of I. cornuta genome sequencing and breeding programs. Keywords: Ilex cornuta, Genome size, DNA content, Flow cytometry, Hybrids, Culti- vars. INTRODUCTION The Ilex L. (holly) is the only living woody dioecious angiosperm genus, accounting for approximately 600 species with a broad distribution from tropics to temperate regions within the monogeneric family of Aquifoliace- ae (Loizeau et al. 2016). The Ilex species are prized for their glossy evergreen 94 Peng Zhou et al. foliage and abundant showy fruits that can bloom from autumn to early spring, when many other plants in the landscape are dormant (Yao et al. 2021). Ilex cornuta Lindl. & Paxton, one of very important native landscape woody materials and distributed only in eastern China and Korea (Hu 1949), has been utilized as a horticul- tural species because its leaves are distinctive rectangu- lar foliage (one or two spines per side) and its fruits are red berries (Park et al. 2019). I. cornuta, the most spe- ciose and commercially significant specie of the diverse genus Ilex, has a long and complex horticultural his- tory. Together with I. aquiforlium usually distributed in Europe, it is a typical species for Christmas tree inside home. In addition, it has been utilized as medical plant in China so that it contains several useful compounds (Zhang et al. 2012; Kang et al. 2014). Extensive cultiva- tion and hybridization have produced many varieties of I. cornuta, including commercially important horticul- tural species such as cultivated tea and iconic flowering shrubs (Hodges et al. 2001; Park et al. 2019). Genome size (C value/haploid nuclear DNA content) is an important attributes of living organisms, which is correlated with size of nucleus/cell, cellular process including DNA synthesis rate and ecological traits, etc. Genome size has fundamental significance in a wide range of applications including molecular biology, ecol- ogy, systematic biology, cytology, evolutionary biology and genomics (Jatt et al. 2019). Genome sizes of more than 7500 plant species have been estimated (Bennett and Leitch 2012), but the genome sizes of higher spe- cies are still poorly understood (Bennett and Leitch 2011; Doležel and Bartoš 2005). Flow cytometric analysis in plants has proved to be useful to determine DNA con- tent and ploidy level in different species and accessions (Sliwinska 2018; Pellicer et al. 2014; D’hondt et al. 2011). The genus Ilex is one of the largest plant genera, but of which only 6 species of genome size have been deter- mined (Bennett and Leitch 2012). As the most speciose member of this genus, I. cornuta has become widely cul- tivated throughout Asia, Europe and America. Despite the considerable breeding, selection, widespread cul- tivation and domestication of I. cornuta, which may have a significant role in the composition of genomes, except for I. cornuta (Zhang et al. 2013), there are no other previous reports of intraspecific genome size vari- ation in the different cultivars or hybrids of this species. Improved knowledge of genome size of key cultivars and complex hybrids would be a valuable resource for fur- ther breeding and improvement of I. cornuta. Therefore, in this study, the genome size of cultivars and hybrids of I. cornuta were identified and analyzed by FCM. The genome size variation of different I. cornuta varieties were explored to provide a basis for the development and utilization of I. cornuta germplasm resources. MATERIALS AND METHODS Plant materials The sampling site was located in the National Holly Germplasm Resources Repository of the Jiangsu Acad- emy of Forestry, which is located at Jiangning District, Nanjing City, Jiangsu Province, China. The tested mate- rials were 12 I. cornuta varieties in consideration of its commodity value; the age of trees is 5-7a, each was healthy and without pests and diseases. Diploid rice, Oryza sativa subsp. japonica cv. Nipponbare (1C = 389 Mb, GC = 43.6 %; International Rice Genome Sequence Project 2005), were used as an internal standard, which was provided by Nanjing Agricultural University and germinated in Petri dishes in the laboratory. Leaves were collected from 12 healthy I. cornuta varieties and rice. Flow cytometry analysis Experimental design Prior to FCM measurement, flow cytometer parame- ters were determined, based on external analyses of sam- ple and primary standard. Subsequently, internal FCM procedure was performed. Preparation of plant nuclei suspensions for f low cytometry (1) Sampling: 0.05 g of young leaves of I. cornuta and 0.05 g of young leaves of rice was collected, washed in distilled and deionized water successively to remove surface dirt, and dried on filter paper. (2) Dissociation: 1 mL of pre-cooled Tris dissociation solution was added to a pre- cooled culture dish, and the cut leaf tissues were immersed in this solu- tion and then chopped quickly with a razor blade. After chopping, 1 mL Tris dissociation solution was added, well mixed and allowed to stand for 1-3 min at 4 °C. (3) Filtration: The liquid mixture was drawn from the culture dish, filtered once through a 400 mesh mem- brane and placed into a centrifuge tube. The mix- ture was incubated at 4 °C for 5 min. (4) Centrifugation: The cell nuclear suspension was obtained by centrifugation at 4 °C at 1000 r/min for 5 min. (5) Dyeing: The supernatant was discarded. The nuclei were stained with propidium iodide (PI), and RNase 95Determination of genome size variation among varieties of Ilex cornuta (Aquifoliaceae) by fow cytometry was added to a final concentration of 50 μg/mL. The mixture was dyed at 4 °C for 5-10 min in the dark environment before being analyzed. Settings of flow cytometer and calculations Samples were run on a BD InfluxTM cell sorter (BD, Piscataway, NJ, USA) with an argon laser exciting at 488 nm. Pulse area was detected using 670 mean/30 band- width detector, as well as with side (SSC) and forward (FSC) scatters. Prior to analysis, the instrument was checked for linearity and the amplification was adjusted so that the peak corresponding to rice was positioned approximately at channel 10000. The voltage was main- tained at a constant high level throughout each experi- ment. Each plant was measured at least three times by the same operator to eliminate potential artefacts. If the difference among the three measurements exceeded 2%, the most deviating value was discarded and the sample was re-analyzed. Coefficient of variation values (CV) was used to evaluate the results. Nuclear genome size was calculated as a linear relation between the ratio of G0/G1 peak of the samples and the standard according to the following formula (Dolezel and Bartos 2005): Sam- ple genome size = [(sample G0/G1 peak mean)/(standard G0/G1 peak mean)] × standard genome size. Genome size data are presented in absolute terms in pg (1C and 2C value) and Mbp (1 pg DNA = 978 Mbp; Doležel 2003). Statistical analyses of genome size FCM detection results were edited and analyzed by BD FACS sortware1.0.0.650, and a flow histogram was obtained. Variance analysis was carried out using Excel 2003 and SPSS 13.0 with convective detection param- eters. A one-way ANOVA (analysis of variance) was used to compare genome sizes among individuals of the same varieties and among 12 sampled varieties respectively. Fisher’s least significant difference (LSD) test (P<0.05) was used for the multiple comparison. A t test was used to compare genome size values for four cultivars and eight hybrids to determine whether differences were significant between two groups. Genome size data were log10 transformed prior to analyses. RESULTS Optimization of flow cytometry for I. cornuta Based on recommendations from specialized FCM bibliography and small genome size values reported in Aquifoliaceae (Bennett and Leitch 2012), we chose rice as primary standard by internal standardization to lower the bias (Noirot et al. 2003; Lysák et al. 2000). The fluorescence intensity range of standard and sam- ple was determined by observing the position of peak in the flow cytometric histogram, when they were ana- lyzed separately on the machine. As shown in Figure 1 and Figure 2, the debris peak and nuclei peak were effectively separated, and the sample peak had good linearity, indicating that the nuclear dissociation solu- tion was suitable. The G0/G1 peak of rice was tuned to fluorescence channel 10000 (Figure 1b), and followed the same protocol, the G0/G1 peak of I. cornuta sample was positioned at channel number around 18000 (Figure 2b). When rice and I. cornuta being chopped simultane- ously, the SSC (Figure 3a) showed that the particles of the target species and the internal standard are clearly concentrated with good discrimination, and it was easy to distinguish two dominant G0/G1 peaks in histogram (Figure 3b). Nuclear DNA content was calculated as a linear relationship between the ratio of G0/G1 peaks of the sample and standard, indicating that the I. cornuta nuclei contained more DNA than rice nuclei. Therefore, in the flow cytometry histograms of mixed samples, the peak reflecting the nuclei isolated from rice should be positioned at the left side of the histogram, while the peak reflecting the nuclei isolated from I. cornuta sample should be positioned at the right side of the histogram (Figure 3b). Table 1. Variance analysis of FCM detection parameters of different varieties in I. cornuta. Index Variation source Sum of Squares Degree of freedom Mean Square F value Significance level 2C value Between varieties 0.390 11 0.035 17.631 0.000 Within varieties 0.058 29 0.002 Total variation 0.448 40 1C value Between varieties Within varieties 0.058 29 0.002 Total variation 0.448 40 96 Peng Zhou et al. FCM histograms of I. cornuta varieties The peak histograms of different classified I. cor‑ nuta varieties obtained with FCM were shown in Figure 4. Mean fluorescence values from 12 I. cornuta varie- ties showed G0/G1 nuclei peaks in a fluorescence range from 17240 to 23090. Flow cytometry analyses produced high-resolution histograms with CV values for the inter- nal standard and sample peaks varying between 2.77% and 5.10% (mean 4.15%) and between 1.82% and 5.15% (mean 3.95%), respectively, which suggested that the res- olutions of the high quality histograms were appropriate for genome size analysis (Table 2). Assessment of genome size of I. cornuta varieties The mean 2C value was determined for each varie- ties of I. cornuta by comparing the relative G0/G1 nuclei PI-fluorescence peak of rice (primary standard) to that of each I. cornuta sample. Analysis of variance (ANO- Figure 1. FCM detection analysis result for rice that was separately processed. (a) scatterplot on side scatter (SSC) versus PI fluores- cence with manually drawn polygon gate; (b) histogram of relative fluorescence intensity derived from nuclei isolated from rice only. Peak 4 represent G0/G1 nuclei of rice. Figure 2. FCM detection analysis result for I. cornuta sample that was separately processed. (a) scatterplot on side scatter (SSC) versus PI fluorescence with manually drawn polygon gate; (b) histogram of relative fluorescence intensity derived from nuclei isolated from I. cornuta only. Peak 4 represent G0/G1 nuclei of I. cornuta sample. Figure 3. FCM detection analysis result for mixed samples of rice and I. cornuta sample that were simultaneously processed (co- chopped). (a) scatterplot on side scatter (SSC) versus PI fluores- cence with manually drawn polygon gate, (b) histogram of relative fluorescence intensity derived from nuclei isolated from rice and I. cornuta processed simultaneously. Peak 4 represent G0/G1 leaf nuclei of rice, peak 5 represent G0/G1 nuclei of I. cornuta sample. Figure 4. FCM histograms obtained after analyses of propidium iodine-stained nuclei isolated I. cornuta varieties ’Burfordii’ (a), ’Dwarf Burford’ (b), ’Luteocarpa’ (c), ’’O’Spring’ (d), ’Emily Bruner’ (e), ’James Swan’ (f ), (g) ’Ilex dabieshanensis No.1’; ’Mary Nell’(h), ’Nellie R. Stevens’ (i) , ’Edward J. Stevens’ (j), ’Golden Nellie R Ste- vens’ (k) and ’China Girl’(l) with internal standard rice. Peak 4 rep- resent G0/G1 nuclei of rice, peak 5 represent G0/G1 nuclei of I. cor‑ nuta varieties. 97Determination of genome size variation among varieties of Ilex cornuta (Aquifoliaceae) by fow cytometry VA) of genome sizes variation in different varieties of I. cornuta was significant (Table 1). The 2C genome sizes (2C value) varied 1.22-fold among all I. cornuta varieties, ranging from 1.47±0.0217 to 1.80±0.0148 pg, thus with 1C genome size estimates for I. cornuta ranging from 717 to 881 Mb (0.733–0.901 pg) with an average of 805 Mb (0.823 pg). ‘Emily Bruner’ had the smallest genome size, whereas ’Nellie R. Stevens’ had the largest genome size (Table 2). Comparison of genome sizes between cultivars and hybrids The I. cornuta varieties measured were distinguished into two groups based on genetic origin, which were sig- nificantly different (P < 0.05). The first represents four I. cornuta-related cultivars, whereas the second includes eight interspecific hybrids developed based on I. cornuta. The 2C value of genome size was estimated between 1.61±0.0048 and 1.77±0.0263 pg for cultivar family and 1.47±0.0217 and 1.80±0.0148 pg for hybrids family (Table 2). Box and whisker plot showing differences in the 2C values assessed with different germplasm sources (Fig- ure 5). The median and average 2C value of genome sizes were 1.688 pg and 1.678 pg for cultivars and 1.634 pg and 1.627 pg for hybrids, respectively. The variation in genome size of hybrids (1.22-fold) was somewhat larger than that of cultivars (1.10-fold), possibly due to those hybrid nature. DISCUSSION Genome size estimation of I. cornuta In the present work, the genome sizes of 12 I. cor‑ nuta varieties were measured, according to their global commodity value in different countries. Results of flow cytometric analysis showed that the mean 2C genome size estimated of I. cornuta in this study ranged from 1.47 pg to 1.80 pg (1.63 pg on average), corresponding to the 1C genome size of 805 Mb or 0.823 pg, which indi- cated that genome size estimates of I. cornuta varieties were very small (1C≤1.4 pg; Leitch et al. 1998; Soltis et al. 2003) and considerable variations were found among all varieties. Therefore, caution should be taken when cultivars/genotypes are selected for genome sequencing and other genome-based studies. Nevertheless, these new data on the genome sizes of both cultivars and complex hybrids are larger than the previous result of I. cornuta (2C=1.31 pg; Zhang et al. 2013). The possible reasons for this difference might be due to the use of different origins of plant materials, reference plants, methods for lysates, staining proto- cols and instruments (Wang et al. 2019; Jatt et al. 2019; Kolano et al. 2012). Given the large variation in genome sizes of I. cornuta germplasm resources, it is essential to investigate a large number of varieties before a more accurate estimation of their average genome sizes can be achieved. Optimization of internal reference to estimate genome size accurately Due to its indirect nature, one of the important steps in FCM analysis was to choose the reference plant species used as an internal standard, which can reveal significant differences in DNA contents among the same cultivar or plant species (Ortega-Ortega et al. 2019; Jatt et al. 2019; Doležel et al. 2003). To be useful as a primary standard, a plant must have similar, but not identical, genome size to the analyzed plant, and the G0/G1 peaks of the standard should not overlap to the peaks of the sample and is located relatively at a distance from the samples that can help to decrease the errors in measur- ing DNA content (Doležel and Greilhuber 2010; Jatt et al. 2019). Ideally, the 2C peak of the target species should be located between the 2C and 4C peaks of the internal reference standard and the genome size of the target and internal standards should not differ more than four- fold (Suda and Leitch 2010). In addition, the standard must be easy to use, genetically stable, nuclei must be obtained in enough amounts for analysis (Ortega-Ortega Figure 5. Boxplot of genome sizes in cultivars and hybrids. The horizontal black line within each bar represents the median value of the genome size, while a black dot within the bar denotes the aver- age value of genome size. 98 Peng Zhou et al. et al. 2019). Rice has all these characteristics. Initially in this study, rice was selected as internal standard. The G0/ G1 peak of I. cornuta was about twice that of the diploid cultivated rice and they don’t overlap each other (Figure 3), which proved the rice was a advisable standard for I. cornuta flow cytometric analysis. Performance of flow cytometry for I. cornuta The CV has been considered an important FCM parameter, indicating the quality of nuclei suspensions (Favoreto et al. 2012). CV within 9% indicated that test results were relatively reliable (Georgiev et al. 2009); CV below 5% indicated the highest accuracy for FCM assess- ments in plants (Doležel and Bartoš 2005). In the pre- sent work, for ‘O Spring’ and ‘Golden Nellie R Stevens’, it was very difficult to obtain CV values at the level of below 5%, which mainly due to the high amount of autofluorescence and phenolics in the gold leaves ham- pering the dyeing and analysis of the nuclei (Choudhury et al. 2014). Except for these two varieties, the FCM pro- cedure used here provided fluorescence peaks of G0/G1 nuclei showing CV are all below 5%, which indicated that the extraction and staining procedure using Tris dissociation solution combined with a centrifugation step can result in the accepted histograms. Thus, the FCM procedure in this work is adequate for determina- tion of genome size for I. cornuta and can be applied in other FCM studies of Ilex. Intraspecific variations in genome size Statistical analysis can help assess the extent of genome size variation among varieties of related species or cultivars. In the case of I. cornuta it is important to determine the genetic variability between different cul- tivars (including genome size) since most of them are not biologically defined species, but rather the result of somatic mutations and artificial hybridization (Hodges et al. 2001). Thus, FCM analysis applied to estimate total nuclear DNA content in I. cornuta cultivars can help to identify those cultivars with higher possibilities of hav- Table 2. Genome size of the varieties of I. cornuta analyzed in this work. No Varieties Genome size CV (%) of samples CV (%) of standard 2C (pg) 1C (pg) Mean 1C (Mpb) MeanMean ± SD Min. Max. Significance* Ilex cornuta -related cultivars 1 ‘Burfordii’ 1.77±0.0263 1.72 1.81 ab 0.883 863 4.55 4.40 2 ‘Dwarf Burford’ 1.61±0.0048 1.60 1.62 def 0.805 787 3.50 2.77 3 ‘Luteocarpa’ 1.71±0.0351 1.63 1.81 bc 0.855 836 3.49 4.09 4 ‘O Spring’ 1.63±0.0282 1.57 1.67 de 0.813 794 5.15 4.94 Average 1.68 0.839 820 Interspecific hybrids Ilex (cornuta x latifolia) 5 ‘Emily Bruner’ 1.47±0.0217 1.42 1.53 g 0.733 717 4.56 4.81 6 ‘James Swan’ 1.55±0.0294 1.50 1.60 ef 0.774 757 4.85 4.90 Ilex 7 ‘dabieshanensis No.1’ 1.56±0.0355 1.50 1.62 ef 0.781 763 2.81 3.76 Ilex [(cornuta x pernyi) x latifolia] 8 ‘Mary Nell’ 1.71±0.0048 1.70 1.72 bc 0.856 837 4.57 4.97 Ilex (aquifoilium x cornuta) 9 ‘Nellie R. Stevens’ 1.80±0.0148 1.78 1.83 a 0.901 881 1.82 3.60 10 ‘Edward J. Stevens’ 1.54±0.0039 1.54 1.55 f 0.772 754 3.98 4.26 11 ‘Golden Nellie R Stevens’ 1.72±0.0146 1.69 1.76 bc 0.859 839 5.08 3.58 Ilex (rugosa x cornuta) 12 ‘China Girl’ 1.66±0.0093 1.64 1.68 cd 0.831 812 3.09 3.78 Average 1.63 0.813 795 Overall average 1.65±0.0165 1.42 1.83 0.823 805 3.95 4.15 99Determination of genome size variation among varieties of Ilex cornuta (Aquifoliaceae) by fow cytometry ing sexual compatibility and therefore hybridization via conventional breeding. Among Angiosperms, there is a great variation in genome sizes, ranging from 0.065 pg/1C of DNA in Genlisea margaretae Hutch. to 152.23 pg/1C in Paris japonica Franch (Kolano et al. 2012). Also, numerous studies revealed the existence of considerable varia- tion in genome size at the interspecific level, e.g. Coffea arabica (Ortega-Ortega et al. 2019; Noirot et al. 2003), Phoenix dactylifera (Jatt et al. 2019), Chenopodium qui‑ noa (Kolano et al. 2012), several Pisum species (Baranyi et al. 1996), three Saccharum species (Zhang et al. 2012), Agave tequilana (Palomino et al. 2003) and Arabidop‑ sis thaliana (Schmuths et al. 2004). Amongst the cul- tivars of I. cornuta there was a 1.16-fold range of varia- tion, and although some of this might be attributable to methodological variation, it is possible that not all can be explained in this way. Murray (2005) has suggested that intraspecific variation in C-value may be indicative of taxonomic heterogeneity and there is no doubt that I. cornuta is a highly variable species that exhibits a wide range of morphological variation. In general, these cultivars, resulting from spontane- ous mutations and being thus related, have remarkably similar genome sizes and a different DNA content rela- tive to their progenitors. This is the case of the I. cornuta cultivar that gave rise to ‘Burfordii’, ‘Dwarf Burford’, ‘Luteocarpa’ and ‘O’Spring’. In contrast, a wider range of variation in the genome size were exhibited among the hybrids, one possible explanation for which was that varieties of hybrid origin have undergone substantial genome size changes as compared with natural muta- tions (Ortega-Ortega et al. 2019). Other varieties have different genetic origin and yet possess similar DNA content, i.e., ‘Luteocarpa’ and ‘Golden Nellie R Stevens’. It is known that variations in genome size has been pri- marily attributed to fluctuation within highly repeti- tive DNA, variation in chromosome number, amplifica- tion/deletion of DNA sequences (Wang et al. 2017; Kolano et al. 2012; Sharma et al. 2019). Mechanisms underlying intraspecific and interspecific genome size variation in plants still remain controversial; thus, more research is fairly required in this regard (Ortega-Ortega et al. 2019; Huang et al. 2013). CONCLUSION In conclusion, genome size of 12 commercially important I. cornuta varieties was analyzed in pico- grams by flow cytometry technique for the first time. This study can fill a gap in the literature by providing information about the genome size of I. cornuta varie- ties and enrich the C value database of Ilex L. It also provides a valuable reference for other Ilex L. species to determine genome size by flow cytometry. This infor- mation can be helpful for I. cornuta breeding programs, give the paucity of genome size studies with FCM in dif- ferent important I. cornuta cultivars. Additionally, these results may be relevant for genomic analysis as well as for a better understanding of I. cornuta evolutionary relationships, diversification, hybridization, and poly- ploidy. ACKNOWLEDGMENTS Thanks go to Yanwei Zhou and Yiping Zou, who assisted with the material collection and Feng Lin for the technical support with FCM. AUTHOR CONTRIBUTIONS Conceptualization, P.Z. and M.Z.; formal analy- sis, Q.Z.; investigation, J.L. and P.Z.; data curation, J.H.; writing—original draft preparation, P.Z.; writing— review and editing, Q.Z. and M.Z.; visualization, F.L. and J.H.; funding acquisition, M.Z. and P.Z. All authors have read and agreed to the published version of the manuscript. 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