Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 77(2): 65-72, 2024 Firenze University Press www.fupress.com/caryologia ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.36253/caryologia-2404 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: Choi, B., & Jang, T.-S. (2024). Natural hybridization between Iris minutoaurea Makino and Iris odae- sanensis Y. N. Lee in Korea: evidence from cytological traits. Caryologia 77(2): 65-72. doi: 10.36253/caryologia-2404 Received: December 04, 2023 Accepted: September 19, 2024 Published: November 10, 2024 © 2024 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 BC: 0000-0001-9926-9656 T-SJ: 0000-0002-5527-1137 Natural hybridization between Iris minutoaurea Makino and Iris odaesanensis Y. N. Lee in Korea: evidence from cytological traits Bokyung Choi, Tae-Soo Jang* Department of Biological Science, Chungnam National University, Daejeon, Korea *Corresponding author. E-mail: jangts@cnu.ac.kr Abstract. Interspecific hybridization resulting in diploid-homoploid hybrids is rela- tively rare in natural populations. The Iris genus, which comprises numerous economi- cally important species, is a taxonomically complex group in which polyploidization and hybridization frequently contribute to taxon diversification and speciation in natu- ral populations. In Korea, populations of the diploid Iris odaesanensis (2n = 28) and Iris minutoaurea (2n = 22) come into contact with each other, leading to homoploid hybrids, as recently demonstrated by molecular phylogenetic and morphological sur- veys. In this study, we aimed to confirm whether the chromosome number, genome size, and pollen viability of the putative hybrids corresponded to the hybrid nature of their diploid parental species. As previously demonstrated, the hybrids exhibited inter- mediate tepal color traits between the parental taxa. Iris minutoaurea, I. odaesanensis, and the putative hybrids were consistently diploid (2n = 22, 28, and 25, respectively), confirming that homoploid hybridization mainly occurred in natural populations. The genome size of the putative hybrids (mean: 3.84 pg, range: 3.80–3.86 pg) was additive when compared with those of the parental diploid species (i.e., 3.72 pg in I. odaesan- ensis and 3.95 pg in I. minutoaurea). No fertile pollen grains were found in the puta- tive hybrids, which may have prevented the establishment of hybrid lineages and back- crosses with the parental species I. odaesanensis or I. minutoaurea. Together, these data confirm the existence of natural homoploid Iris hybrid populations in Korea and shed light on the dynamics of interspecific hybridization in the Iris genus. Keywords: chromosome number, genome size, homoploid hybrid, Iris, pollen abortion. INTRODUCTION Hybridization between closely related angiosperm taxa contributes to the diversification and speciation of natural populations (Soltis and Soltis 2009; Whitney et al. 2010; Yakimowski and Rieseberg 2014; Goulet et al. 2017; Kim et al. 2023). Natural hybridizations are collectively regarded as a cru- cial evolutionary mechanism, resulting in the most complex diversification of the Iridaceae family (Arnold et al. 1990; Young 1996; Makarevitch et al. 2003; Son et al. 2015; Samad et al. 2016; Niketić et al. 2018). A new hybrid lineage may form through allopolyploidy accompanied by the duplication http://www.fupress.com/caryologia https://doi.org/10.36253/caryologia-2404 https://doi.org/10.36253/caryologia-2404 https://www.fupress.com https://creativecommons.org/licenses/by/4.0/legalcode https://creativecommons.org/publicdomain/zero/1.0/legalcode https://orcid.org/0000-0001-9926-9656 https://orcid.org/0000-0002-5527-1137 mailto:jangts@cnu.ac.kr 66 Bokyung Choi, Tae-Soo Jang of chromosome sets or through homoploid hybrid spe- ciation between species of the same ploidy level (Soltis and Soltis 2009; Kadereit 2015; Schumer et al. 2014). To date, numerous homoploid hybrid species from natural populations have been recognized in many economically important plant families (Asteraceae–Lipman et al. 2013; Poaceae–Jiang et al. 2013; Amaryllidaceae–Smirnov et al. 2017; Lamiaceae–Arabaci et al. 2021; Iridaceae–Xiao et al. 2021; Isoëtaceae–Suissa et al. 2022). An increasing number of interspecific hybrids of the genus Iris have occasionally been reported, e.g., I. nelsonii, a natural hybrid between I. hexagona and I. fulva (Arnold 1993), I. verticolor, a hybrid between I. virginica, and I. setosa (Lim et al. 2007), and I. ×ampliflora, a hybrid between I. japonica and I. wattii (Xiao et al. 2021). However, little is known regarding the morphological, ecological, genet- ic, and cytological context between natural homoploid hybrid species (Yakimowski and Rieseberg 2014; Feliner et al. 2017). Iris L. ser. Chinense (Diels) Lawrence includes eight species that are mainly distributed in Korea, China, and Japan according to a recent taxonomic study (Wil- son 2020). Four Iris species (I. odaesanensis Y. N. Lee, I. minutoaurea Makino, I. koreana Nakai, and I. rossii Baker) are recognized in Korea (Lee 2003; Sim 2007; Choi et al. 2020a, 2022a, b, c). Iris odaesanensis is known to be an endemic species in Korea (Sim 2007). How- ever, some researchers have considered that the spe- cies are sub-endemic in Korea, with populations also being found in Jilin, China (Zhao et al. 2000; Son et al. 2015; Choi et al. 2020; Wilson 2020). However, whether the Iris population in China represents I. odaesanen- sis requires further investigation. Among the Korean Iris species within the Chinenses series, I. koreana was hypothesized to be of hybrid origin based on cytological data (i.e., chromosome number and genome size varia- tion; Choi et al. 2020a). Subsequent molecular cytoge- netic data substantiated I. koreana as a putative allopol- yploid species, with genomic contributions from two closely related species, I. minutoaurea and I. odaesanen- sis (Park et al. 2022). Interestingly, putative hybrid plants between I. minutoaurea and I. odaesanensis have been documented in Gasan, Korea, based on both molecu- lar analysis and morphological data, revealing interme- diate characteristics in tepal color and position of the ovary when compared with the parental species (Son et al. 2015; Yang et al. 2020). Despite increasing interest in these putative hybrid plants from natural populations, debate continues regarding whether these plants show conserved chromosome numbers, genome sizes, and karyotype features compared with their diploid parents, I. odaesanensis and I. minutoaurea. We recently investigated sympatric populations of I. odaesanensis (2n = 28) and I. minutoaurea (2n = 22) using molecular cytogenetic and complete plastome data (Choi et al. 2020a; Park et al. 2022). In those investiga- tions, we identified sympatric populations of putative hybrid individuals that appeared to be derived from two distinct genetic clusters corresponding to diploid I. odaesanensis and I. minutoaurea. To verify the plausible hybrid origin and relationship with I. odaesanensis and I. minutoaurea, we compared the chromosome numbers, genome size values, and pollen characters with those of the putative parental species using both light microscopy and scanning electron microscopy (SEM). In addition, we tested the pollen viability of the putative hybrid. MATERIALS AND METHODS Plant materials We selected two mixed-growing natural popula- tions of I. minutoaurea and I. odaesanensis and puta- tive hybrids between them for this study (Table 1; Fig- ure 1A–D). Living plants were transplanted at Chung- nam National University for cytological observations. We examined one to six different individuals of each species and the putative hybrids to check their chromo- some numbers and pollen viabilities and estimate their genome sizes (Table 1). All voucher specimens were deposited in the Herbarium of Chungnam National Uni- versity (CNUK, Daejeon, Korea). Chromosome numbers, karyotypes, and pollen viabilities of putative hybrid plants We determined chromosome numbers in root tip metaphasic plates using the standard Feulgen staining technique as described by Choi et al. (2020a). We placed pollen grains from two anthers per hybrid individual into aniline blue dye solution to distinguish between fertile and sterile pollen grains (Choi et al. 2020b; Kim et al. 2021). To make detailed palynological observa- tions, we dehydrated mature anthers from putative hybrid individuals in an ethanol series (70%, 90%, 95%, and 100%) at room temperature over 30 min. The dehy- drated anthers were immersed in liquid carbon diox- ide for critical point drying (Leica Microsystems, EM CPD300, Germany). All dried samples were mounted on aluminum stubs and coated using an ion-sputtering device (Hitachi, E-1010, Japan). The pollen grains in the dried anthers were gently removed using a needle and analyzed using SEM (Hitachi, S3000N, Japan) at 20 kV 67Natural hybridization between Iris minutoaurea and I. odaesanensis with a working distance of 10 mm, as described by Kim et al. (2021). Genome-size measurements The genome sizes of four I. minutoaurea individu- als and three putative hybrids were measured via flow cytometry, with Solanum pseudocapsicum L. (1C = 1.2946 pg; Temsch et al. 2010) serving as an internal standard. Genome-size estimations for Iris species were carried out as described by Choi et al. (2020a). Due to the variable genome-size data in our previous research, ranging from 3.70 pg/1C to 4.09 pg/1C with I. minutoaurea (Choi et al. 2020a), we included additional 1C values for the same species from another different natural population to clar- ify the range of genome-size data (Table 1). RESULTS Confirmation of homoploid hybridization based on the chromosomal number and genome size Despite floral color variations among the investi- gated taxa (e.g., I. odaesanensis in Figure 1A, I. minuto- aurea in Figure 1B, and their putative hybrids in Figure 1C–D), cytological analysis showed that I. minutoaurea, I. odaesanensis, and their putative hybrids were con- sistently diploid, with 2n = 22, 28, and 25, respectively (Figure 2), confirming that homoploid hybridization occurred in natural populations (Figures 1 –2). The kary- otypes in the putative hybrid plants could not be directly compared with their homologous pairs because of their odd chromosome numbers (2n = 25); thus, they were analyzed with reference to their parental karyotypes. The karyotypes of the hybrid plants were mostly com- posed of metacentric, sub-metacentric, and acrocentric chromosomes, regardless of their parental chromosomes (Figure 2B). Although satellites and nucleolar-organ- izer regions did not normally show clear localization in mitotic cells due to high chromosome condensation (Figure 2A–B), they were occasionally visible in hybrid plants (Figure 2C). Flow cytometric analyses yielded high-resolution histograms with distinct sample peaks and internal standards for all seven individuals investigated (Table 1). The results of our genome-size (1C) analysis are shown in Table 1, together with our previous data. The genome size of I. minutoaurea was higher (range: 3.85–4.09 pg) than that of I. odaesanensis (range: 3.67–3.76 pg). The Table 1. Plant material used for cytological analysis of I. odaesanensis, I. minutoaurea, and their putative hybrids in natural populations in Korea. Taxon; collection number Locality: global positioning satellite (GPS) coordinate Chromosome number Genome size 1C ± S.D. (pg) Reference I. odaesanensis* JCKC1932271 Gasan, Kyungsang 2n = 28 3.76 ± 0.021 Choi et al. 2020a JC041904 Gasan, Kyungsang 2n = 28 3.74 ± 0.042 Choi et al. 2020a JC041903 Gasan, Kyungsang 2n = 28 3.67 ± 0.015 Choi et al. 2020a JC041940 Gasan, Kyungsang 2n = 28 3.73 ± 0.018 Choi et al. 2020a Mean 3.72 I. minutoaurea Makino JC041913* Gasan, Kyungsang 2n = 22 4.09 ± 0.042 Choi et al. 2020a Gasan_8* Gasan, Kyungsang 2n = 22 3.92 ± 0.008 Choi et al. 2020a Che-01 Mt. Cheonma, Gyeonggi; N37°41’24’’, E127°24’36’’, 157 m 2n = 22 3.85 ± 0.009 This study Che-02 Mt. Cheonma, Gyeonggi; N37°41’24’’, E127°24’36’’, 157 m 2n = 22 3.94 ± 0.008 This study Go-1 Go-Nam, Gyeonggi; N37°40’22’’, E127°15’17’’, 157 m 2n = 22 3.93 ± 0.009 This study Hwa-3 Mt. Hwa-Ya, Gyeonggi; N36°56’25’’, E127°16’47’’, 162 m 2n = 22 4.02 ± 0.004 This study Mean 3.95 I. minutoaurea × I. odaesanensis* JC041927 Gasan, Kyungsang 2n = 25 3.86 ± 0.017 This study JC041906 Gasan, Kyungsang 2n = 25 3.85 ± 0.009 This study JC041924 Gasan, Kyungsang 2n = 25 3.80 ± 0.051 This study Mean 3.84 * The GPS coordinates, latitude, and longitude of the collection sites are not indicated for protection purposes, although the latter species has recently been excluded from the list of endangered Korean taxa. 68 Bokyung Choi, Tae-Soo Jang 1C mean value of the putative hybrids (mean: 3.84 pg, range: 3.80–3.86 pg) was additive with respect to the diploid parental species (i.e., 3.72 pg in I. odaesanensis and 3.95 pg in I. minutoaurea). Pollen fertility in the putative hybrids Mature anthers from all hybrid plants were removed, and all pollen grains were stained with Figure 1. Floral morphological variations in the studied Iris taxa. A. Iris odaesanensis: standards and styles were white, and falls were white with lower central half highlighted in yellow. B. Iris minutoaurea: standards and styles were yellow, and falls were yellow with its central lower half highlighted in dark purple. C–D. Putative hybrids between I. odaesanensis and I. minutoaurea. The putative hybrids showed tepal colour variations: with white standards and styles and yellowish falls with its lower central half highlighted in dark purple (C), yellow stand- ards and falls with their lower basal part highlighted in dark purple and yellow styles with its apical margin coloured in purple (D). Photo- graphs were taken by T.-S. Jang. 69Natural hybridization between Iris minutoaurea and I. odaesanensis aniline blue dye solution to assess the fertility of the putative hybrid plants (Figure 3A). However, despite staining, no fertile pollen grains were found, indicat- ing that most or all pollen grains were infertile (Fig- ure 3). SEM analysis performed on pollen samples from the putative hybrid individuals revealed that the pollen grains were monosulcate and mostly curled up or shriveled with folded aperture membranes (Fig- ure 3B–C). The exine ornamentation of the putative hybrids was reticulate with continuous muri (very rarely discontinuous). DISCUSSION Here, we provide cytological and palynological evi- dence for the presence of homoploid hybrids between I. minutoaurea and I. odaesanensis (I. ser. Chinense) in natural Korean populations, in agreement with previ- ous results based exclusively on molecular and mor- phological evidence (Son et al. 2015; Yang et al. 2020). Both diploid parental species differ in terms of morphol- ogy, chromosome number, genome size, and complete chloroplast genome composition (Sim 2007; Choi et al. 2020a; Park et al. 2022). Based on molecular phyloge- netic analyses of their matK and nrITS sequences, the Figure 2. Chromosome numbers and karyotypes of putative hybrids (2n = 2x = 25) of I. minutoaurea (2n = 2x = 22) and I. odaesanensis (2n = 2x = 28). A, C. Mitotic metaphase chromosomes with 2n = 25 (arrows indicate satellites of putative hybrids). B. Karyotype of a putative hybrid including a haploid chromosome of n = 11 (I. minutoaurea) and n = 14 (I. odaesanensis). Scale bars = 5 μm (n indicates a haploid chromosome number; karyotypes were analyzed in comparison to previous chromosome-counting data [Choi et al. 2020a]). Figure 3. Light and scanning electron microscopic photographs (LM and SEM) of sterile pollen grains in putative hybrids between Iris odaesanensis and I. minutoaurea. A. An unstained pollen grain (sterile pollen grains) in aniline blue dye solution. B, C. Sterile pollen grains observed by SEM. B. Stamens of putative hybrids produced sterile pollen grains. C. Enlargement of a sterile pollen grain by SEM. 70 Bokyung Choi, Tae-Soo Jang putative hybrids did not form a clearly separated mono- phyletic group when analyzed together with their paren- tal species (I. minutoaurea and I. odaesanensis; Son et al. 2015). The present chromosome number and genome- size data for the homoploid hybrids appeared to be more conclusive (Table 1). Thus, our results are consistent with the hypothesis that hybridization in the Iris genus of the Iridaceae family may have been important for taxon diversification and speciation (Arnold et al. 1990; Young 1996; Lim et al. 2007; Gao et al. 2021; Park et al. 2022). Natural homoploid hybrid speciation accompanied by reproductive isolation from putative parents has often been reported for the Iridaceae family (Arnold et al. 1990). The intermediate morphological variations of the Iris putative hybrid were not influenced by environmental factors. This lack of influence was attributable to a sympatric distribu- tion in their parental diploid species (Table 1). Instead, these variations seemed to correlate with cytological differ- ences, as frequently observed with other homoploid hybrid species (Peruzzi et al. 2012; Musiał et al. 2020; Xiao et al. 2021; Pellicer et al. 2022). Hybridization in plants can be affected by overlapping flowering periods in sympatric are- as (Paun et al. 2009; Abbott et al. 2010; Jiang et al. 2013; Kim et al. 2023) and is mainly documented for apomictic groups (Paun et al. 2006; Hörandl 2010; Hojsgaard and Hörandl 2019). Our results confirmed that homoploid hybrid species of Korean irises may have evolved partial intrinsic reproductive isolation from both diploid parents considering their different base chromosome numbers (x = 11 in I. minutoaurea and x = 14 in I. odaesanensis; Table 1). Such different base chromosome numbers and odd chromosome number could lead to infertility in individu- als and prevent the establishment of hybrid lineages and back-crosses with the parental species (Carnicero et al. 2023). However, only limited data are available regarding the emergence of Iris hybrids from natural populations, their potential for sexual and asexual reproduction (e.g., by rhizome disruptions), or morphological divergence from the parental taxa (Arnold et al. 1990; Niketić et al. 2018; Yang et al. 2020; Gao et al. 2021). Efforts to maintain homoploid hybrids from natu- ral populations are likely to fail because of their infer- tile offspring (Stebbins 1958; Yakimowski and Riese- berg 2014). Pollen abortion in putative hybrid Iris plants could be ref lected by impaired chromosomal pair- ing during meiosis between the two unequal parental genomes (x = 11 and 14). These hybrid plants are not amenable to sexual reproduction, suggesting that the hybrids probably represent a natural first-generation hybrid species, as has been reported for other species (Denduangboripant et al. 2007; Saito et al. 2007; Tseng and Hu 2014). Although such hybrid taxa may not even- tually produce well-differentiated hybrid species, they can facilitate the testing of key predictions from models of hybrid speciation, as observed with other plant groups (Barton 2001; Chen et al. 2022). Further investigation using molecular cytogenetic techniques and genomic in situ hybridization of the putative hybrid and its parental species will shed light on the speciation and diversifica- tion of Iris ser. Chinense. CONCLUSION In summary, our comprehensive data demonstrate the existence of populations of natural homoploid Iris hybrids. 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The role of chromosomal rearrangements, polyploidy, and genome size variation in the diversity and ecological distribution of Asparagus L. species: a landscape cytogenetics meta-analysis approach Paris Fouroutan1, Masoud Sheidai2,*, Fahimeh Koohdar3 First chromosome characterization and repetitive DNA of Barred Gliding Lizard, Draco taeniopterus Günther, 1861 (Draconinae: Agamidae: Squamata) Praween Supanuama, Sitthisak Jantaratb,*, Warakarn Khawporntipb, Thaintip Kraipromc, Somsak Buatipb, Sarun Jumrusthanasand, Sarawut Kaewsrid, Nattasuda Donbundite, Sukhonthip Ditcharoene, Weera Thongnetrf, Sumalee Phimphang, Alongklod Tanomtonge Cytotoxic assessment of aqueous extracts of Heliotropium keralense Sivar. & Manilal on Allium cepa root tip cells Athira. R. Mohan, Nisha Joseph* Chromosomal variations and genetic diversity in subpopulations of Senna alexandrina Mill. from Western Thar, India Sunita Arora, Monika Vyas, Meena Barupal* Application of Genomic In Situ Hybridization (GISH) and tandem repeat sequence amplification for identification of Erianthus – Saccharum introgression Valiya Purakkal Sobhakumari*, Krishnasamy Mohanraj Mapping of five classes of repetitive DNAs and microsatellite repeats in the genome of the Rainbow Shark, Epalzeorhynchos frenatum (Fowler, 1934) Kamika Sribenja1, Nuntaporn Getlekha2,* Natural hybridization between Iris minutoaurea Makino and Iris odaesanensis Y. N. Lee in Korea: evidence from cytological traits Bokyung Choi, Tae-Soo Jang*