Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 72(2): 3-7, 2019 Firenze University Press www.fupress.com/caryologiaCaryologia International Journal of Cytology, Cytosystematics and Cytogenetics ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.13128/caryologia-720 Citation: J.-X. Wang, Y.-J. Cao, Y.-C. Han, S.-. Zhou, K. Liu (2019) Karyo- type analysis of a natural Lycoris dou- ble-flowered hybrid. Caryologia 72(2): 3-7. doi: 10.13128/caryologia-720 Published: December 5, 2019 Copyright: © 2019 J.-X. Wang, Y.-J. Cao, Y.-C. Han, S.-. Zhou, K. Liu. This is an open access, peer-reviewed arti- cle published by Firenze University Press (http://www.fupress.com/caryo- logia) 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. Karyotype analysis of a natural Lycoris double- flowered hybrid Jin-Xia Wang1, Yuan-Jin Cao1, Yu-Chun Han1, Shou-Biao Zhou1,2, Kun Liu1,* 1 Anhui Provincial Key Laboratory of the Conservation and Exploitation of Biological Resources, College of Life Sciences, Anhui Normal University, Wuhu, Anhui, China 2 Anhui Provincial Engineering Laboratory of Water and Soil Pollution Control and Remediation, College of Environmental Science and Engineering, Anhui Normal Univer- sity, Wuhu, Anhui, China * Corresponding author: hudixiao@126.com Abstract. A putative natural double-flowered hybrid in Lycoris Herb. was found on Mt. Zhangjiajie in Hunan, China. The putative natural hybrid had a chromosome number of 2n = 18 and was karyotypically formulated as 2n = 4m + 6st + 5t + 3T. The karyo- type of the putative natural hybrid was classified as 2B type according to the degree of asymmetry and Stebbins’ criteria. According to the gross morphology, phenology and karyotype of the putative natural hybrid, it was suggested that this taxon was probably from the natural hybridization between L. aurea and L. radiata. Keywords. Karyotype, Lycoris Herb., natural hybrid. INTRODUCTION The genus Lycoris Herb., a small group of the family Amaryllidaceae, comprises approximately 20 species, distributed in warm temperate and sub- tropical zones of East Asia, with a few extending to northern Indochina and Nepal (Liu and Hsu 1989; Hsu et al. 1994; Shi et al. 2006). The Lycoris spe- cies are very popular bulb flowers characterized by their plentiful colors and beautiful and varied shapes (Hsu et al. 1994; Zhou et al. 2007). The hybridi- zation frequently happens in the genus Lycoris and causes a number of the presently established hybrid taxa, such as L. houdyshelii Traub, L. stramin- ea  Lindl., L. squamigera Maxim., L. incarnata Comes ex C. Sprenger and L. × hubeiensis K. Liu (Kurita 1987; Hsu et al. 1994; Shi et al. 2006; Meng et al. 2018). During our field investigations of the wild populations of Lyco- ris in Hunan Province, China, a mixed population of three taxa, namely L. aurea  (L’Her.) Herb., L. radiata (L’Her.) Herb., and a putative natural hybrid was found. The putative natural double-flowered hybrids attracted our more attention, which were discovered for the first time in Lycoris in the wild. To illustrate the origin of the natural double-flowered hybrids, some bulbs were successfully transplanted and cultivated in the experimental garden, together 4 Jin-Xia Wang et al. with their putative parents. The karyotypes of the three Lycoris taxa were analyzed in this study, indicating that the double-flowered taxon might be from the hybridiza- tion between L. aurea and L. radiata. MATERIALS AND METHODS The plants were collected from Mt. Zhangjiajie, Hunan, China (110º17’E, 29º19’N), and then transplant- ed to the experimental garden of Anhui Normal Uni- versity, Wuhu, China. The flowers of each taxon were shown in Fig. 1. For chromosome observation, actively growing root tips were pretreated in p-Dichlorobenzene solution at 4 °C for 5 h before they were fixed in Carnoy I (glacial acetic acid : absolute ethanol = 1:3) at 4 °C for 20 h. Then they were macerated in 1 mol L-1 hydrochlo- ric acid at 60 °C for 3 minutes, stained in Phenol-Fuch- sin for 20 h, and squashed in 45% acetic acid. The karyotype formula was based on the measure- ments of metaphase chromosomes taken from photo- graphs. For each taxon, measurements were taken from at least five well-spread metaphase cells in no fewer than three different individuals. For the description of karyotypes, the symbols had been adapted according to Levan et al. (1964): m for median-centromeric chro- mosome with arm ratio of 1.01-1.70; st for subterminal- centromeric chromosome with arm ratio of 3.01-7.00; t for terminal-centromeric chromosome with arm ratio of over 7.00; T for terminal-centromeric chromosome with no short arm. Karyotypes were classified on the basis of their degrees of asymmetry according to Stebbins (1971) and Li and Chen (1985). The intrachromosomal asym- metry index (A1) and interchromosomal asymmetry index (A2) were calculated using Romero Zarco’ equa- tions (1986). RESULTS 1. The putative natural Lycoris double-f lowered hybrid (Table 1, 2; Figure 2A, 2D) – The chromosomes were counted to be 2n = 18, consisting of 4 large medi- an-centromeric (m), 6 subterminal-centromeric (st), 5 terminal-centromeric (t) and 3 Terminal-centromeric (T). The karyotype was formulated as 2n = 4m + 6st + 5t + 3T. The average length of chromosome complement was 122.3 μm. The ratio of the length of the largest chro- mosome to that of the smallest was 3.39, and the pro- portion of chromosomes with arm ratio >2:1 was 77.8%. The karyotype was therefore of 2B type according to the degree of asymmetry and the chromosomes ranged from Fig 1. The flowers of the three taxa. A: the putative natural hybrid; B: L. aurea; C: L. radiata. 5Karyotype analysis of a natural Lycoris double-flowered hybrid 3.36~11.38 in relative length. The values of A1 and A2 were 0.72 and 0.50, respectively. 2. Lycoris aurea (Table 1, 3; Figure 2B, 2E) – The chromosome number was 2n = 14, consisting of 8 large median-centromeric (m) and 6 Terminal-centromeric (T). The karyotype formula was 2n = 8m + 6T. The aver- age length of chromosome complement was 138.6 μm. The ratio of the length of the largest chromosome to that of the smallest was 3.32, and the proportion of chromo- somes with arm ratio >2:1 was 42.9%. The karyotype type was 2B. The relative lengths of all chromosomes ranged from 3.49 to 11.59. The values of A1 and A2 were 0.47 and 0.44, respectively. Table 1. Karyotype characteristics of the studied Lycoris taxa. Species Karyotypic formula Stebbins’ type A1 A2 TCL (μm) Hybrid 2n = 4m + 6st + 5t + 3T 2B 0.72 0.50 122.3 L. aurea 2n = 8m + 6T 2B 0.47 0.44 138.6 L. radiata 2n = 12st + 10t 4A 0.86 0.10 111.2 A1: intrachromosomal asymmetry index, A2: interchromosomal asymmetry index, TCL: average length of total chromosome com- plement. Table 2. Measurements of somatic chromosomes of the putative natural hybrid. No. Relative length Arm ratio Type LL SL TL 1 5.71 5.67 11.38 1.01 m 2 5.81 5.38 11.19 1.08 m 3 6.11 4.65 10.76 1.31 m 4 4.39 4.09 8.48 1.07 m 5 4.63 0.37 5.00 12.51 t 6 4.63 0.33 4.96 14.03 t 7 3.97 0.65 4.62 6.11 st 8 4.53 0.00 4.53 ∞ T 9 4.49 0.00 4.49 ∞ T 10 3.90 0.46 4.36 8.48 t 11 4.34 0.00 4.34 ∞ T 12 3.47 0.54 4.01 6.43 st 13 3.61 0.31 3.92 11.65 t 14 3.39 0.49 3.88 6.92 st 15 3.22 0.46 3.68 7.00 st 16 2.99 0.60 3.59 4.98 st 17 2.99 0.46 3.45 6.50 st 18 3.03 0.33 3.36 9.18 t Note: LL, relative length of long arm; SL, relative length of short arm; TL, total relative length; LL + SL = TL. The same is below. Fig 2. The metaphase chromosome morphology and karyotypes of the putative natural hybrid, Lycoris aurea and L. radiata. A, D: the putative natural hybrid; B, E: L. aurea; C, F: L. radiata. Scale bar = 10 μm. Table 3. Measurements of somatic chromosomes of L. aurea. No. Relative length Arm ratio Type LL SL TL 1 5.81 5.76 11.57 1.01 m 2 5.56 5.38 10.94 1.03 m 3 5.52 5.34 10.86 1.03 m 4 5.47 4.93 10.40 1.11 m 5 4.75 4.13 8.88 1.15 m 6 4.39 4.26 8.65 1.03 m 7 4.13 3.90 8.03 1.06 m 8 3.99 3.41 7.40 1.17 m 9 4.93 0.00 4.93 ∞ T 10 3.81 0.00 3.81 ∞ T 11 3.77 0.00 3.77 ∞ T 12 3.68 0.00 3.68 ∞ T 13 3.59 0.00 3.59 ∞ T 14 3.49 0.00 3.49 ∞ T 6 Jin-Xia Wang et al. 3. Lycoris radiata (Table 1, 4; Figure 2C, 2F) – The chromosome number of this species was 2n = 22. It con- sisted of 12 subterminal-centromeric (st) and 10 termi- nal-centromeric (t). The karyotype was formulated as 2n = 12st + 10t. The average length of chromosome comple- ment was 111.2 μm. The ratio of the length of the larg- est chromosome to that of the smallest was 1.43, and the proportion of chromosomes with arm ratio >2:1 was 100%. The karyotype type was 4A. The relative lengths of all chromosomes ranged from 3.56 to 5.10. The values of A1 and A2 were 0.86 and 0.10, respectively. DISCUSSION The karyotype of the putative natural double-flow- ered hybrid is formulated as 2n = 18 = 4m + 6st + 5t + 3T. Lycoris aurea in this study has a chromosome num- ber of 2n = 14, in agreement with other reports (Liu and Hsu 1989; Hsu et al. 1994). L. radiata has a chromo- some number of 2n = 22, similar to some previous stud- ies (Shao et al. 1994; Zhou et al. 2007; Liu et al. 2016). Based on the values of the two indices, A1 and A2, L. radiata had the largest intrachromosomal asymmetry and the smallest interchromosomal asymmetry among the three taxa. Considering the sympatric distribution of L. aurea and L. radiata, it was supposed that this puta- tive natural hybrid might be a diploid between L. aurea, which produced the gamete having 4m + 3T, and L. radiata with a- reduced gamete having 6st + 5t. Accord- ing to our observation, the leaf of this putative natural hybrid emerged in September, the same as the putative parents. The sizes of leaf blade and bulb of this putative natural hybrid were intermediate between its two puta- tive parents. So far, some Lycoris species were confirmed to be hybrids by karyotype or molecular sequence anal- ysis, such as L. houdyshelii, L. straminea, and L. incar- nata (Hsu et al. 1994; Shi et al. 2006; Liu et al. 2011), and these hybrids had the similar flower characteris- tics with their putative parents. Here, a putative natural double-flowered hybrid with no pistil and stamen was discovered and its karyotype was described for the first time. Based on the karyotype analysis, further molecu- lar study was needed to uncover the origin of this natu- ral hybrid and distinguish the paternal donor from the maternal donor. Judging from the absence of seed, pistil and stamen, the putative natural hybrid could be sexually sterile. The Lycoris species all had considerable ornamental value. Compared with the normal Lycoris species, the double- flowered hybrid had more ornamental value. Because of the lack of seed, it was needed to propagate the double- flowered hybrid for the large application in landscaping in future by tissue culture and quick propagation technology. ACKNOWLEDGMENTS We thank the anonymous reviewers for helpful comments that improved the paper. FUNDING This work was financially funded by the National Natural Science Foundation of China (31400291) and Collaborative Innovation Center of Recovery and Recon- struction of Degraded Ecosystem in Wanjiang City Belt, Anhui Province; College of Life Sciences, Anhui Normal University. REFERENCES Hsu BS, Kurita S, Yu ZZ, Lin JZ. 1994. Synopsis of the genus Lycoris (Amaryllidaceae). SIDA. 16:301–331. Table 4. Measurements of somatic chromosomes of L. radiata. No. Relative length Arm ratio Type LL SL TL 1 4.60 0.75 5.35 6.13 st 2 4.43 0.67 5.10 6.61 st 3 4.38 0.70 5.08 6.26 st 4 4.25 0.70 4.95 6.07 st 5 4.63 0.27 4.90 17.15 t 6 4.23 0.62 4.85 6.82 st 7 4.25 0.57 4.82 7.46 t 8 4.05 0.67 4.72 6.05 st 9 4.18 0.52 4.70 8.04 t 10 3.81 0.85 4.66 4.48 st 11 4.08 0.55 4.63 7.42 t 12 4.01 0.60 4.61 6.68 st 13 4.08 0.51 4.59 8.00 t 14 3.91 0.67 4.58 5.84 st 15 4.11 0.38 4.49 10.81 t 16 3.73 0.55 4.28 6.78 st 17 3.81 0.45 4.26 8.47 t 18 3.86 0.30 4.16 12.87 t 19 3.38 0.55 3.93 6.15 st 20 3.41 0.52 3.93 6.56 st 21 3.38 0.47 3.85 7.19 t 22 3.21 0.35 3.56 9.17 t 7Karyotype analysis of a natural Lycoris double-flowered hybrid Kurita S. 1987. Variation and evolution on the karyotype of Lycoris, Amaryllidaceae II. Karyotype analysis of ten taxa among which seven are native to China. Cytologia. 52: 19–40. Levan L, Fredga K, Sandberg AA. 1964. Nomenclature for centromeric position on chromosomes. Hereditas. 52: 201–220. Li MX, Chen RY. 1985. A suggestion on the standardi- zation of karyotype analysis in plants. J Wuhan Bot Res. 3 (4): 297–302. Liu K, Zhou SB, Wang Y, Zhang D. 2011. A karyomor- phological study on two newly recorded taxa of Lyco- ris (Amaryllidaceae) in Anhui province, China. Cary- ologia. 64 (2): 158–163. Liu Y, Hsu BS. 1989. A study on karyotypes of the genus Lycoris. Acta Phyto Sin. 27 (4): 257–264. Liu YX, Zheng YH, Xia T, Zhou J. 2016. Karyotype stud- ies on Lycoris radiata populations from China. Genet Mol Res. 15 (1): 15017357. Meng WQ, Zheng L, Shao JW, Zhou SB, Liu K. 2018. A new natural allotriploid, Lycoris × hubeiensis hybr. nov. (Amaryllidaceae), identified by morphological, karyological and molecular data. Nord J Bot. 36 (6): e01780. Romero Zarco C. 1986. A new method for estimating karyotype asymmetry. Taxon 35 (3): 526–530. Shao JZ, Yang JG, Zhang DC, Nie LW. 1994. The discov- ery of diploid Lycoris radiata Herb. from Anhui. Acta Phyto Sin. 32 (6): 549–552. Shi SD, Qiu YX, Li EX, Wu L, Fu CX. 2006. Phylogenetic relationships and possible hybrid origin of Lycoris species (Amaryllidaceae) revealed by ITS sequences. Biochem Genet. 44: 198–206. Stebbins GL. 1971. Chromosomal evolution in higher plants. London: Edward Arnold Ltd. Zhou SB, Yu BQ, Luo Q, Hu JR, Bi D. 2007. Karyotypes of six populations of Lycoris radiata and discovery of the tetraploid. Acta Phyto Sin. 45 (4): 513–522. Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Volume 72, Issue 2 - 2019 Firenze University Press Karyotype analysis of a natural Lycoris double-flowered hybrid Jin-Xia Wang1, Yuan-Jin Cao1, Yu-Chun Han1, Shou-Biao Zhou1,2, Kun Liu1,* Insights on cytogenetic of the only strict African representative of genus Prunus (P. africana): first genome size assessment, heterochromatin and rDNA chromosome pattern Justine Germo Nzweundji1, Marie Florence Sandrine Ngo Ngwe2, Sonja Siljak-Yakovlev3,* Assessment of cytotoxicity and mutagenicity of insecticide Demond EC25 in Allium cepa and Ames Test Arzu Özkara Cytogenetic effects of Fulvic acid on Allium cepa L. root tip meristem cells Özlem Sultan Aslantürk Evaluation of the cytotoxic and genotoxic potential of some heavy metals by use of Allium test Ioan Sarac1, Elena Bonciu2,*, Monica Butnariu1, Irina Petrescu1, Emilian Madosa1 Fluorescence In Situ Hybridisation Study of Micronuclei in C3A Cells Following Exposure to ELF-Magnetic Fields Luc Verschaeve1,2,*, Roel Antonissen1, Ans Baeyens3, Anne Vral3, Annemarie Maes1 Phytochemical analysis and in vitro assessment of Polystichum setiferum extracts for their cytotoxic and antimicrobial activities Nicoleta Anca Şuţan1,*, Irina Fierăscu2, Radu Fierăscu2, Deliu Ionica1, Liliana Cristina Soare1 Telomeric heterochromatin and meiotic recombination in three species of Coleoptera (Dorcadion olympicum Ganglebauer, Stephanorrhina princeps Oberthür and Macraspis tristis Laporte) Anne-Marie Dutrillaux, Bernard Dutrillaux* A whole genome analysis of long-terminal-repeat retrotransposon transcription in leaves of Populus trichocarpa L. subjected to different stresses Alberto Vangelisti#, Gabriele Usai#, Flavia Mascagni#, Lucia Natali, Tommaso Giordani*, Andrea Cavallini Differences in C-band patterns between the Japanese house mice (Mus musculus) in Hokkaido and eastern Honshu Hikari Myoshu, Masahiro A. Iwasa* Karyotypic description and repetitive DNA chromosome mapping of Melipona interrupta Latreille, 1811 (Hymenoptera: Meliponini) Natália Martins Travenzoli1, Ingrid Cândido de Oliveira Barbosa2, Gislene Almeida Carvalho-Zilse2, Tânia Maria Fernandes Salomão3, Denilce Meneses Lopes1,*