Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 78(2): 21-28, 2025 Firenze University Press https://riviste.fupress.net/index.php/caryologiaCaryologia International Journal of Cytology, Cytosystematics and Cytogenetics ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.36253/caryologia-3431 Citation: Kim, H.-r. & Heo, K. (2025). Kar- yotype analysis and chromosome evo- lution in Menyanthaceae using FISH. Caryologia 78(2): 21-28. doi: 10.36253/ caryologia-3431 Received: March 26, 2025 Accepted: July 17, 2025 Published: December 20, 2025 © 2025 Author(s). This is an open access, peer-reviewed article pub- lished by Firenze University Press (https://www.fupress.com) and distrib- uted, except where otherwise noted, under the terms of the CC BY 4.0 License for content and CC0 1.0 Uni- versal for metadata. 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 HRK: 0000-0002-1415-9675 KH: 0000-0003-3785-3974 Karyotype analysis and chromosome evolution in Menyanthaceae using FISH Hye-rin Kim, Kweon Heo* Department of Applied Plant Science, College of Agriculture and Life Sciences, Kangwon National University, Chuncheon 24341, Republic of Korea *Corresponding author. E-mail: laurus@kangwon.ac.kr Abstract. The Menyanthaceae, an aquatic plant family, is distinguished by extensive polyploidy and heterostyly. This study marks the first cytogenetic characterization of four Menyanthaceae species from Korea – Menyanthes trifoliata, Nymphoides peltata, N. indica, and N. coreana – employing fluorescence in situ hybridization (FISH) with 45S and 5S rDNA probes. All four species exhibit exclusively metacentric chromo- somes, with M. trifoliata and N. peltata being hexaploid (2n = 54), N. coreana tetra- ploid (2n = 36), and N. indica diploid (2n = 18). FISH mapping revealed between one to four 45S rDNA loci and one to three 5S rDNA loci per species, showing that rDNA site number does not correlate directly with ploidy level. The karyotypic data suggest a conserved base chromosome number (x = 9) and largely symmetrical karyotypes across these species. Notably, M. trifoliata presents fewer rDNA loci than expected for a hexaploid, indicating genomic rearrangements and rDNA locus loss through dip- loidization. These observations highlight an evolutionarily stable genome structure in M. trifoliata, despite its polyploid nature. This study elucidates the chromosomal organization and evolutionary dynamics of the Menyanthaceae, emphasizing the role of polyploidy and rDNA evolution in genome structuring. The findings enhance our understanding of plant cytogenetics in aquatic ecosystems and serve as a foundation for further comparative genomic and evolutionary studies in Menyanthaceae. Keywords: Menyanthaceae, polyploidy, karyotype analysis, Fluorescence in situ hybridization, rDNA loci. INTRODUCTION The Menyanthaceae family comprises perennial, floating-leaved aquatic plants prevalent across pantropical regions, including tropical America, Asia, and Australia. These plants are noted for heterostyly, a self-incompatible reproductive system that encourages outcrossing (Barrett 1992; Barrett and Shore 2008). The family predominantly exhibits distyly, though variants such as homostyly or other mating system alterations occur (Ornduff 1970, 1987, 1992). Reproduction is facilitated through both sexual (seed-based) and asex- ual (clonal propagation via root-derived turions) modes, promoting ecologi- cal resilience and adaptability in wetland habitats (Tippery et al. 2008, 2009). In the Korean Peninsula, Menyanthaceae is represented by Menyanthes tri- https://riviste.fupress.net/index.php/caryologia https://doi.org/10.36253/caryologia-3431 https://doi.org/10.36253/caryologia-3431 https://doi.org/10.36253/caryologia-3431 https://www.fupress.com https://creativecommons.org/licenses/by/4.0/legalcode https://creativecommons.org/publicdomain/zero/1.0/legalcode https://orcid.org/0000-0002-1415-9675 https://orcid.org/0000-0003-3785-3974 mailto:laurus@kangwon.ac.kr 22 Hye-rin Kim, Kweon Heo foliata L. and Nymphoides peltata (S. G. Gmel.). Kuntze, Nymphoides indica (L.) Kuntze, and Nymphoides coreana (H. Lév.) H. Hara, all crucial for biodiversity and eco- logical stability in aquatic environments (Ornduff 1970; Tippery et al. 2008, 2009; Watanabe 2022). Understanding chromosomal organization in Men- yanthaceae is crucial for unraveling their genetic and evolutionary dynamics, especially given their adapta- tion to aquatic environments. Cytogenetic studies have revealed substantial chromosomal variation within this family, including phenomena such as polyploidy, descending dysploidy, and chromosomal races, which contribute to speciation and environmental adaptation (Gillett 1968; Watanabe 2022; Leitch and Leitch 2013). In the genus Nymphoides (x = 9), detailed examinations reveal chromosome counts predominantly in diploid (2n = 18), tetraploid (2n = 36), and hexaploid (2n = 54) states, indicating dynamic karyotypic evolution (Ornduff 1970). Similarly, M. trifoliata shows a hexaploid chro- mosome count (2n = 54), contrasting with Nephrophyl- lidium crista-galli, which has been documented with 2n = 108, highlighting extensive polyploidization within the Menyanthaceae (Gillett 1968; Tippery et al. 2008). Fluorescence in situ hybridization (FISH) is a robust cytogenetic technique that facilitates the direct visualiza- tion of specific DNA sequences on chromosomes (Abbo et al. 1994). This method uses f luorescently labeled probes to map vital genomic elements, such as 45S and 5S rDNA loci, which act as molecular markers for assess- ing chromosomal polymorphisms and genetic diversity (Stebbins 1971; Stace 2000; Ilnicki 2014). Recent studies have underscored the role of rDNA loci in chromosomal evolution, particularly among plant species undergoing polyploidization and structural rearrangements (Weiss- Schneeweiss et al. 2013; Watanabe 2022). This study aims to investigate the chromosomal architecture of M. trifoliata using FISH to map repetitive DNA sequences, including 45S and 5S rDNA loci. By ana- lyzing the karyotype, we hope to elucidate the organiza- tion, composition, and evolutionary dynamics of the Men- yanthaceae genome. Comparative analyses with related taxa will provide deeper insights into chromosomal evo- lution and adaptation strategies in wetland environments, contributing to broader knowledge in plant cytogenetics, genome stability, and conservation biology. MATERIAL AND METHODS Root sample preparation for chromosome Plant materials were collected from natural popu- lations (Table 1). Roots were pre-treated with 2 mM 8-hydroxyquinoline solution for 4 hours at 12°C. They were subsequently fixed in Carnoy’s solution (3:1 etha- nol: acetic acid) for 24 hours and stored in 70% ethanol at 4°C until use. Chromosome spread preparation Somatic chromosome spreads were prepared using a modified version of the technique described by Kirov et al. (2014). After thorough washing with distilled water, the meristematic regions of the fixed root tips were excised and digested in an enzyme mix (2% cellulase, 1% pec- tolyase in 1× Citrate buffer) for 52 minutes at 37°C. The enzyme mix was removed, and 80 μL of Carnoy’s solu- tion was added; roots were suspended by vortexing, then centrifuged, and the pellet was resuspended in a 9:1 ace- tic acid–ethanol solution. Finally, the root suspension was dropped onto slides in a humid chamber to spread the chromosomes, and then the chromosomes were air-dried. Fluorescence in situ hybridization (FISH) The 45S rDNA was labeled with digoxigenin- 11-dUTP (Roche, Germany) via nick translation and detected with anti-digoxigenin FITC. The 5S rDNA was labeled with biotin-16-dUTP and detected with strepta- Table 1. Collection data of plant materials used in this study. Taxa Collection data Menyanthes trifoliata L. Korea, Gangwon-do, Goseong-gun, April 13, 2021, H. R. Kim and K. Heo s.n. (KWNU) Korea, Gangwon-do, Taebaek-si, May 25, 2021, H. R. Kim and K. Heo s.n. (KWNU) Nymphoides indica (L.) Kuntze Korea, Jeju-do, Jocheon-eup, August 8, 2023, K. Heo s.n. (KWNU) Korea, Jeju-do, Jocheon-eup, August 12, 2023, K. Heo s.n. (KWNU) Nymphoides coreana (H.Lév.) H.Hara Korea, Jeju-do, Seogwipo-si, July 30, 2023, H. R. Kim and J. S. Yang s.n (KWNU) Nymphoides peltata (S.G.Gmel.) Kuntze Korea, Gyeonggi-do, Yangpyeong-gun, September 11, 2022, H. R. Kim and K. Heo s.n. (KWNU) 23Karyotype analysis and chromosome evolution in Menyanthaceae using FISH vidin–avidin Cy3 (Table 2). Labeled DNA fragments ranging from 100 to 500 bp were used as probes. The hybridization mixture for FISH contained 50% forma- mide, 10% dextran sulfate, 2×SSC, and 500 ng/μL of each probe DNA, adjusted with distilled water to a total volume of 50 μL per slide. The mixture was denatured at 90°C for 10 minutes and immediately cooled on ice for 10 minutes. After applying the probe mixture, chro- mosome slides were denatured at 80°C for 3 minutes on a hotplate. The slides were then incubated in a humid chamber at 37°C for 18 hours to facilitate hybridization. Subsequently, the slides were treated with 2x SSC for 5 minutes at RT and 1x detection buffer for 10 minutes at RT. Biotin‐labelled 5S rDNA and digoxigenin labelled 45S rDNA were detected using Cy3-conjugated strepta- vidin and anti-digoxigenin-f luorescein isothiocyanate (FITC) at 37°C for 1 hour. Excess reagents were removed by washing three times in 1x detection buffer for 5 min- utes each. The slides were dehydrated in a series of etha- nol (70%, 90%, and 100%) for 3 minutes at RT, and air- dried. Then, the slides were counterstained with DAPI in VECTASHIELD. Chromosome spreads were examined using a phase-contrast fluorescence microscope (Axio Imager M2, Carl Zeiss, Germany). Chromosome length measurements and image acquisition were performed with ZEN software (Carl Zeiss). RESULTS This study provides the first cytogenetic charac- terization of four Menyanthaceae species using dou- ble-FISH. All species were found to possess exclusively metacentric chromosomes (Figs 1, 3). Chromosome counts confirm that M. trifoliata and N. peltata are hexa- ploid (2n = 54, Fig. 2A, B), N. coreana is tetraploid (2n = 36, Fig. 2C), and N. indica is diploid (2n = 18, Fig. 2D). Chromosome lengths varied from approximate- ly 1.0 μm to 3.7 μm across these four species (Table 3; Fig. 3). FISH mapping identified between one and four 45S rDNA loci and one to three 5S rDNA loci per spe- cies, indicative of some variation in rDNA copy number among the genomes (Fig. 2.). The precise chromosomal positions of the rDNA sig- nals were determined for each species. In M. trifoliata, green fluorescence signals corresponding to 45S rDNA were detected on chromosomes 3 and 15, while red fluo- rescence signals for 5S rDNA were observed on chromo- some 12. In N. peltata, 45S rDNA signals were detected on chromosomes 4 and 7, and 5S rDNA signals on chro- mosomes 3, 10, 12, 13, 23, and 27. N. coreana exhibited 45S rDNA signals on chromosome 3 and 5S rDNA sig- nals on chromosomes 1 and 4. N. indica displayed a 45S rDNA signal on chromosome 1 and a single 5S rDNA Table 2. Primers used in this study. Gene Type Sequence 5s rDNA Forward 5’-CGGTGCATTAATGCTGGTAT-3’ Reverse 5’-CCATCAGAACTCCGCAGTTA-3’ 45s rDNA Forward 5’-CGAAACCTGCAAGAGCA-3’ Reverse 5’-GTCTGATCTGGGGTCGCAA-3’ Figure 1. Somatic metaphase chromosomes of Menyanthaceae. (A, B) M. trifoliata (2n = 54), (C, D) N. peltata (2n = 54), (E, F) N. coreana (2n = 36), (G, H) N. indica (2n = 18). Scale bars 5 μm for A to H. 24 Hye-rin Kim, Kweon Heo signal, which exact position could not be determined (Table 3; Fig. 3). DISCUSSION Genetic evolutionary dynamics in Menyanthaceae The evolutionary trajectory of Menyanthaceae is characterized by extensive chromosomal variation and a prevalence of polyploidization, a key driver of specia- tion and morphological diversification. For example, the retention or breakdown of heterostyly can influence gene flow and mating patterns within Menyanthaceae popu- lations (Haddadchi 2013, 2015; Barrett and Shore 2008). Studies on Villarsia (Menyanthaceae) reveal distinct stig- ma morphology differences between distylous and non- heterostylous species, supporting the role of reproduc- tive adaptations in lineage diversification (Dulberger and Ornduff 2000). Cytogenetic studies have consistently revealed a base chromosome number of x = 9 across the family (Orn- duff 1970), with species exhibiting diploid (2n = 18), tetraploid (2n = 36), and hexaploid (2n = 54) karyotypes (Cook 1996). The genus Nymphoides exemplifies this pat- tern, wherein polyploidization appears to have enhanced ecological adaptability and geographic expansion (Soltis and Soltis 2016). Similar polyploidy-associated genomic modifications have been observed in other aquatic plant groups (Martel et al. 2004; Watanabe 2022). Polyploidy is a recurrent feature in angiosperm evolution, yet its interaction with descending dysploidy remains an active area of research (Kadereit 2007). At the molecular level, FISH has provided critical insights into chromosomal evolution, especially concerning the organization of ribosomal DNA (rDNA) loci. Studies have revealed substantial variation in both the number and chromosomal positioning of rDNA loci across Nym- phoides species, suggesting that post-polyploidization genomic reorganization is common (Rosato et al. 2015; Silvestri et al. 2015). Additionally, evidence of descend- ing dysploidy – where chromosome number is reduced following polyploidization – indicates that Menyan- thaceae species undergo structural karyotypic modifica- tions to stabilize their genomes (Semple and Watanabe 2023). This ongoing genomic reorganization underscores Figure 2. FISH signals in metaphase chromosomes of Menyan- thaceae species which were distributed across the chromosomes in (A) M. trifoliata, (B) N. peltata , (C) N. coreana , (D) N. indica. Localization of 5S rDNA (red), and 45S rDNA(green). The white arrow indicates the 45S signal, while the black arrow represents the 5S signal. Scale bars 5 μm for A to D. Figure 3. Ideogram of metaphase chromosomes of Menyanthaceae. (A) M. trifoliata, (B) N. peltata, (C) N. coreana and (D) N. indica. Red and green area indicated 5S and 45S rDNA loci. 25Karyotype analysis and chromosome evolution in Menyanthaceae using FISH the dynamic evolutionary landscape of the family, where a whole-genome duplication (WGD) is often followed by selective gene loss and structural rearrangements. Genomic status of Menyanthes trifoliata Despite cytogenetic confirmation that Menyanthes trifoliata is a hexaploid species with 2n = 54 chromo- somes (Peruzzi and Cesca 2004), its genetic behavior raises fundamental questions about whether it functions as a true hexaploid or has undergone extensive diploidi- zation. Unlike certain polyploid Nymphoides species displaying cytotype diversity, M. trifoliata has remained cytogenetically uniform across its widespread circumbo- real distribution. This stability suggests it may represent an ancient hexaploid lineage that has functionally revert- ed to a diploid-like state through genomic restructuring (Raabová et al. 2010). Several lines of evidence support the hypothesis that M. trifoliata has undergone diploidization. First, cytoge- netic analyses indicate predominantly bivalent chromo- some pairing during meiosis, characteristic typically associated with diploid-like inheritance (Mlinarec et al. 2012). Second, its rDNA organization deviates from what would be expected in a simple hexaploid genome, with fewer detectable rDNA loci than a direct tripling of the diploid number (Rosato et al. 2015). These patterns sug- gest that genomic streamlining has eliminated redun- dant rDNA arrays, favoring a more functionally efficient karyotype. Third, its consistent chromosome number across various geographic populations, with no evidence of aneuploidy or unstable cytotypes, further supports the notion of an evolutionarily stable genome structure Table 3. Chromosome analysis of Menyanthaceae species. Ch. No M. trifoliata N. peltata N. coreana N. indica CL (μm) AR (μm) Type CL (μm) AR (μm) Type CL (μm) AR (μm) Type CL (μm) AR (μm) Type (mean±SD) (mean±SD) (mean±SD) (mean±SD) (mean±SD) (mean±SD) (mean±SD) (mean±SD) 1 3.01±0.37 1.14±0.05 m 3.88±0.45 1.04±0.10 m 3.26±0.28 1.04±0.05 m* 3.71±0.30 1.14±0.06 m° 2 2.82±0.35 1.12±0.05 m 3.68±0.33 1.12±0.07 m 2.87±0.33 1.12±0.12 m 2.80±0.33 1.12±0.06 m 3 2.57±0.26 1.05±0.06 m* 3.06±0.38 1.11±0.05 m° 2.70±0.33 1.11±0.10 m° 2.75±0.22 1.05±0.04 m 4 2.42±0.22 1.08±0.08 m 3.31±0.31 1.03±0.13 m* 2.68±0.34 1.03±0.07 m* 2.61±0.25 1.08±0.07 m 5 2.34±0.22 1.09±0.04 m 3.20±0.36 1.07±0.06 m 2.56±0.38 1.07±0.05 m 2.52±0.14 1.09±0.04 m 6 2.27±0.18 1.08±0.06 m 3.17±0.57 1.07±0.10 m 2.52±0.39 1.07±0.13 m 2.47±0.28 1.08±0.05 m 7 2.21±0.17 1.09±0.09 m 3.13±0.28 1.01±0.05 m* 2.52±0.17 1.08±0.06 m 2.42±0.50 1.09±0.10 m 8 2.17±0.16 1.09±0.05 m 2.95±0.50 1.04±0.08 m 2.46±0.22 1.04±0.10 m 2.27±0.22 1.09±0.08 m 9 2.14±0.15 1.10±0.09 m 2.08±0.22 1.11±0.04 m 2.41±0.28 1.12±0.05 m 2.14±0.17 1.10±0.08 m 10 2.10±0.16 1.11±0.07 m 2.79±0.37 1.06±0.03 m° 2.37±0.60 1.06±0.08 m 11 2.04±0.16 1.08±0.07 m 2.75±0.19 1.09±0.04 m 2.35±0.54 1.09±0.04 m 12 1.99±0.14 1.08±0.04 m° 2.68±0.25 1.01±0.02 m° 2.31±0.31 1.01±0.03 m 13 1.96±0.14 1.07±0.09 m 2.64±0.23 1.06±0.05 m° 2.25±0.19 1.06±0.03 m 14 1.93±0.13 1.09±0.10 m 2.61±0.17 1.01±0.05 m 2.23±0.25 1.01±0.05 m 15 1.91±0.13 1.06±0.04 m* 2.58±0.15 1.05±0.06 m 2.22±0.25 1.07±0.02 m 16 1.88±0.14 1.09±0.12 m 2.56±0.60 1.14±0.06 m 2.22±0.19 1.14±0.05 m 17 1.85±0.13 1.10±0.03 m 2.50±0.33 1.06±0.05 m 2.21±0.36 1.06±0.07 m 18 1.83±0.12 1.09±0.02 m 2.45±0.16 1.00±0.03 m° 2.02±0.19 1.00±0.04 m 19 1.79±0.12 1.11±0.02 m 2.38±0.15 1.14±0.07 m 20 1.76±0.12 1.12±0.06 m 2.38±0.12 1.04±0.04 m 21 1.72±0.11 1.10±0.05 m 2.30±0.19 1.08±0.05 m 22 1.67±0.11 1.06±0.03 m 2.17±0.27 1.11±0.07 m° 23 1.63±0.12 1.10±0.09 m 2.11±0.22 1.07±0.10 m 24 1.59±0.10 1.12±0.06 m 2.09±0.23 1.04±0.05 m 25 1.52±0.10 1.11±0.10 m 2.06±0.14 1.12±0.03 m 26 1.46±0.09 1.09±0.05 m 1.97±0.20 1.08±0.10 m 27 1.37±0.07 1.10±0.04 m 1.86±0.24 1.10±0.13 m CL: Chromosome length, AR: Arm ratio, SD: Standard deviation, m: Metacentric chromosome, *: 45s rDNA, °:5s rDNA. 26 Hye-rin Kim, Kweon Heo (Soltis and Soltis 2016). Similar cases of diploidization have been observed in other polyploid plant taxa, includ- ing Nicotiana allopolyploids, where rDNA homogeniza- tion has played a role in genomic stabilization (Kovarik et al. 2008). The precise origins of M. trifoliata remain unre- solved. It may have arisen from autopolyploidy due to successive WGD events, or from allopolyploidy, in which hybridization between distinct ancestral genomes con- tributed to its karyotype (Watanabe 2022). Comparative genomic studies of M. trifoliata and closely related Nym- phoides species could offer deeper insight into whether its hexaploid genome originated from hybridization or from independent lineage expansion (Watanabe 2022). Advances in high-throughput sequencing technologies will be key in resolving this issue by enabling a com- prehensive analysis of genome duplication patterns and homeologous gene retention. Importantly, these genomic and cytogenetic observations can be interpreted within the framework of existing molecular phylogenies. Phylo- genetic analyses based on chloroplast DNA and nuclear ITS regions have consistently placed Menyanthes as sister to a clade of polyploid Nymphoides species (Tippery et al. 2008; Watanabe 2022). Our FISH-based findings sup- port this phylogenetic position by providing cytogenetic evidence that complements molecular data. Specifically, M. trifoliata exhibits diploid-like chromosomal behav- ior during meiosis and possesses fewer rDNA loci than would be expected under a strict hexaploid model. These features are consistent with a scenario in which a whole- genome duplication event occurred before the divergence of Menyanthes, followed by substantial genomic reorgan- ization and diploidization. Furthermore, the extensive chromosomal variation observed among Nymphoides species aligns with their high level of molecular diver- gence, suggesting that polyploidy and subsequent chro- mosomal restructuring have played a major role in driv- ing diversification within the genus. Thus, the cytoge- netic patterns revealed in this study provide a structural and evolutionary context that complements and reinforc- es existing phylogenetic hypotheses for Menyanthaceae. Discrepancy between FISH signals and ploidy level An intriguing anomaly in Menyanthaceae cytoge- netics is the absence of a direct correlation between the number of rDNA signals (as detected by FISH) and the ploidy level. Theoretically, a polyploid lineage derived from a diploid ancestor should display a proportional increase in rDNA loci. Yet, M. trifoliata exhibits fewer rDNA signals than expected for a strict hexaploid model (Fultz and Pikaard 2023). This discordance suggests that polyploid genomes undergo significant restructuring fol- lowing duplication, leading to selective retention, loss, or relocation of rDNA loci. Notably, similar observations have been made in certain polyploid Solanaceae, where fewer rDNA loci are present than expected for their ploidy level. One plausible explanation for this discrepancy is the selective loss of redundant rDNA loci. Polyploidi- zation often results in an initial surplus of rDNA cop- ies, but genome evolution may favor the retention of only the most functionally necessary loci, leading to the eventual elimination of extraneous rDNA sites (Mlin- arec et al. 2012). A targeted analysis using quantitative PCR or whole-genome sequencing could help determine whether the observed reduction in rDNA FISH signals corresponds to actual sequence loss. Another contrib- uting factor could be rDNA transposition and homog- enization. In some polyploids, rDNA loci are not static; instead, they may undergo concerted evolution, where a subset of rDNA sites expands while others diminish or relocate to different chromosomes (Rosato et al. 2015). This pattern is also observed in maize, where rDNA transposition significantly inf luences chromosomal architecture (Li and Arumuganathan 2001). Such pro- cesses may explain why M. trifoliata exhibits a lower- than-expected number of 45S and 5S rDNA loci despite its hexaploid genome structure. Employing FISH with additional chromosomal markers, such as probes for transposable elements, could reveal whether rDNA sites have been repositioned within the genome. Epigenetic modifications, particularly nucleolar dominance, fur- ther complicate the relationship between rDNA loci and ploidy. In allopolyploids and some autopolyploids, nucleolar dominance can result in the silencing of rDNA loci from one parental genome, leading to a functional reduction in active rDNA sites despite their genomic presence (Fultz and Pikaard 2023). If M. trifoliata exhib- its such a mechanism, certain rDNA loci may not be transcriptionally active, making them undetectable by FISH. RNA-seq analyses of rRNA transcription levels could elucidate whether epigenetic silencing contributes to the observed reduction in rDNA signals. The incon- sistency between FISH signal number and ploidy level underscores the complexity of genome evolution in poly- ploids. Rather than a straightforward duplication of all genetic elements, polyploid genomes undergo intricate modifications, including chromosomal rearrangements, rDNA loss, and epigenetic regulation. Future research integrating molecular cytogenetics, high-resolution sequencing, and transcriptomic analyses will be essential to fully characterize the evolutionary dynamics of M. tri- foliata and other polyploid Menyanthaceae taxa. 27Karyotype analysis and chromosome evolution in Menyanthaceae using FISH ACKNOWLEDGMENTS This study was supported by the Korea Research Foundation (KRF) with a grant under project number RS-2021-NR065781 (2021R1I1A2043432). REFERENCES Abbo, S., Miller, T. E., Reader, S. M., Dunford, R. P. and King, I. P. 1994. Detection of ribosomal DNA sites in lentil and chickpea by fluorescent in situ hybridiza- tion. – Genome 37: 713–716. Barrett, S. C. H. 1992. Heterostylous genetic polymor- phisms: Model systems for evolutionary analysis. – Am. Nat. 139: 421–435. Barrett, S. C. H. and Shore, J. S. 2008. New insights on heterostyly: Comparative biology, ecology and genet- ics. – Philos. Trans. R. Soc. B 363: 491–508. Cook, C. D. K. 1996. Aquatic Plant Book. 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