Cytogeography and chromosomal variation of the endemic East Asian herb Lycoris radiata

Abstract Information on the spatial distribution of cytotypes and karyotype variation in plants is critical for studies of the origin and evolution of polyploid complexes. Here, the spatial distribution of cytological races and intraspecific variation in the karyotype of Lycoris radiata, an endemic species to East Asia, is investigated. Conventional karyotype analysis methods were used to determine ploidy level and karyotypical characteristics in 2,420 individuals from 114 populations of L. radiata nearly covering the whole distribution areas in China. Of 114 populations studied, 52 (45.61%), 58 (50.88%), and 4 (3.51%) are diploid, triploid, and mixoploid populations, respectively, with 1,224, 1,195, and 1 individuals being diploid, triploid, and tetraploid, respectively. The triploid possesses a much wider distribution range than the diploid, with the former almost occupying the entire range of this complex species in East Asia and the latter distributing in the middle and east regions of China. Triploids tend to occur at high altitudes, and the relationship between the ploidy and altitude is significantly positive but low (r 2 = 0.103, p < 0.01). About 98.6% of examined bulbs have a common karyotype consisting of 22 or 33 acrocentric (A) chromosomes. Some aberrant chromosomes which should be generated from A‐type chromosome have been found including metacentrics (m), small metacentrics (m′), and B‐type chromosome. The results can provide a fundamental cytogeographic data for further studies on the evolutionary origins and adaptive divergences of polyploids, especially the triploid, within L. radiata using molecular and/or ecological methods in the future.

firstly found (Zhou et al., 2007). This species exhibits great variation in karyotypes and chromosome number throughout its geographical range. The main chromosome numbers observed in this species complex are as follows: 2n = 21, 22, 32, 33, and 44 corresponding to the abnormal diploid, diploid, abnormal triploid, triploid, and tetraploid levels, respectively (Liu et al., 2016;Shi, Qiu, Li, Wu, & Fu, 2006). Cytogeographic patterns on the Japanese islands and South Korea have been depicted clearly, based on 58 and 11 populations of L. radiata, respectively, and only triploids were discovered in Japan and South Korea (Chung, 1999;Kurita, 1987). In China, diploid, triploid, and tetraploid plants have been found (Liu et al., 2016;Zhou et al., 2007). However, compared with researches on the populations in Japan, cytological investigations of the Chinese populations, especially those populations in southwest and southeast of China, are cursory to some extent.
As part of a broader investigation into the chromosomal variation and evolution of Lycoris species, our objective in this study was to examine the diversity and distribution of L. radiata cytotypes in China. Particularly, we addressed the following questions: (a) what are the frequencies of diploid, triploid, and tetraploid plants? (b) what are the geographical patterns of cytotype variation? (c) do polyploids have a wider distribution range than diploid?

| Plant materials
Lycoris radiata is mainly distributed in middle east, southwest, and southeast regions of China, preferring such place as riversides and the edges of farmlands, or growing under evergreen or deciduous broad-leaved forests. Two thousand four hundred and twenty individuals of L. radiata were collected from 114 populations during the past decade (from 2007 to 2017) nearly throughout its distribution range (Table 1). Bulbs collected were more than 3 m apart to avoid biasing sampling of the same clone due to extensive vegetative propagation in this species. All bulbs collected from the wild were cultivated in experiment garden. The corresponding voucher specimens were deposited in Anhui Normal University.
The growth form and seasonality of L. radiata are very characteristic, that is, the productive and reproductive phases are separate (Figure 1a,b). The diploids can produce seeds and have sexual and asexual reproduction, while the triploids can only propagate by clone, producing no seed.

| Karyotype analysis
All bulbs with the old roots cut were maintained in wet soil/tap water culture, and conventional karyotype analysis methods (Zhou et al., 2007) were used. The karyotype formula was based on the measurements of mitosis metaphase chromosomes taken from two or three well-spread metaphase cells. For the karyotype description and comparison, the simplified symbols were adapted according to Levan, Fredga, and Sandberg (1964), Kurita (1986), and Liu et al. (2012): m for large metacentric chromosome with arm ratio of 1.00-1.70; m′ for small metacentrics; st for subtelocentric chromosome with arm ratio of 3.01-7.00; t for acrocentric chromosome with arm ratio of 7.01-20.0; T for telocentric chromosome having mostly terminal centromere with dot-like short arm whose length is very short and with the arm ratio being more than 20.0; A-type chromosome includes both st-and t-type chromosome; SAT for Atype satellite chromosome; B for very small chromosome; r means arm ratio.

| Analysis of cytotype distribution
The relationship between altitude and ploidy of the sampled populations was tested using Pearson correlation analysis by SPSS v22.0. In order to exactly reveal the geographical distribution patterns of each cytotype of L. radiata complex in East Asia, we choose 43 previously published populations with precise chromosome number data and geographical location or longitude and latitude information, of which 7 populations (Liu et al., 2016), 29 populations (Kurita, 1987), and 7 populations (Chung, 1999) were from China, Japan, and South Korea, respectively. In total, 157 populations with exact ploidy data were mapped using ArcMap 10.0.

| The ploidy and chromosome number of Lycoris radiata
A total of 2,447 individuals, from 114 populations of Lycoris radiata (Table 1), were examined to determine the chromosome number and karyotype. Of 114 populations investigated, 52 (45.61%), 58 (50.88%), and 4 (3.51%) were diploid, triploid, and mixoploid populations, respectively. A total of 1,224 bulbs and 1,195 bulbs are diploid and triploid, respectively, with a few bulbs possessing abnormal chromosomes and B chromosomes, and only one bulb from population 114 is tetraploid having 2n = 4x = 44 (Table 1).

| Karyomorphology
The karyotypes of 1,221 bulbs are all composed of 22 A-type chromosomes, of which 0-4 are satellite chromosomes. One example of representative chromosome constitution of diploid bulb from population 114 at Tangxi Zhen of Chizhou city is shown in      ference in mean altitude of localities between diploid and triploid is highly significant (p < 0.01; Figure 4a). Moreover, a significantly positive but low correlation was found between the ploidy and altitude (r 2 = 0.103, p < 0.01; Figure 4b).

| The chromosome number and karyotype of Lycoris radiata
Several cytological studies on L. radiata have been made by various authors (Bose, 1963;Kurita, 1987;Nishiyama, 1928;Qin et al., 2004;Shao et al., 1994;Zhou et al., 2007). Recently, Liu et al. (2016) reported the somatic chromosome numbers of four hundred and sixty-six individuals from 25 populations of L. radiata in China, of which 10 were diploid (2n = 22) and 15 were triploid (2n = 33), and no tetraploid cytotype was found. In this study, 1,224 diploid individuals, 1,195 triploid individuals, and one tetraploid individual were detected. Despite the small proportion for the tetraploid, these new counts, together with data from the previous reports (Kurita, 1987;Liu et al., 2016;Zhou et al., 2007), further confirm that L. radiata is a species complex concluding diploid with 2n = 22, triploid with 2n = 33, and tetraploid with 2n = 44.

| The distribution patterns of cytotypes and origin of polyploids
Information on the geographical variation of cytotypes is critical for studies of origin and evolution of polyploids (Wu et al., 2016). A detailed investigation of the distribution of diploids and derivative polyploids can provide critical insights into the origins and establishment of new polyploids and cryptic speciation within a morphological species (Baack, 2004;Odee, Wilson, Omondi, Perry, & Cavers, 2015;Segraves, Tompson, Soltis, & Soltis, 1999;Steussy, Weiss-Schneeweiss, & Keil, 2004). In this study, we conducted an exhaus-   (Kurita, 1987;Liu et al., 2016), it can be concluded that the triploid shows a significantly wider distribution range than the diploid, with the former occupying almost the whole distribution range of this complex in East Asia.
F I G U R E 3 Distribution map of cytologically investigated and previously published populations of Lycoris radiata in East Asia. Shaded areas show the distribution range of diploid to our knowledge F I G U R E 4 (a) Altitudes comparing ploidy levels. Means significantly different at p < 0.01 are indicated by the different letter (two-tailed unpaired t test). Horizontal lines represent the median, and boxes, and whiskers, respectively, the interquartile range and the nonoutlier ranges. Black circles denote outliers. (b) Scatter plot of ploidy (diploid and triploid) versus altitude (meters). The linear relationship shows the significantly positive association between the ploidy and altitude (r 2 = 0.103, p < 0.01) A positive correlated relationship between polyploidy and elevation is fairly well supported (Brochmann et al., 2004;Soltis, 1984;Stebbins, 1984). However, in several other cases, the polyploids occupy the lower latitude localities, and a negative correlation of polyploidy with elevation was found, for example, Atriplex confertifolia (Stutz & Sanderson, 1983), Chamerion angustifolium (Husband & Schemske, 1998), Centaurea jacea (Hardy, Vanderhoeven, Loose, & Meerts, 2000), and Isoetes spp. (Liu, Gituru, & Wang, 2004). In general, the triploids tend to prefer such place as roadsides, riversides, and the edges of rice paddies or farmlands, and occupy the high altitude regions. Diploids are specific to undisturbed or lessdisturbed habitats, frequently growing under forests, in the lower latitude localities. Moreover, the triploids are expected to present a stronger tolerance to cold temperature, because the triploids show a higher relative distribution dominance than the diploids at higher latitudes. In regard to the north boundary of L. radiata distributed in East Asia, the latitude value of diploid and triploid is about 32.6° and 39.0°, respectively. However, this hypothesis needs to be empirically confirmed by further controlled experiments in common garden or greenhouse.
In addition, there is a complicated and perplexing question about the origin and distribution pattern of the polyploids, especially the triploid. The triploid was usually supposed to be an autotriploid (Hayashi, Saito, Mukai, Kurita, & Hori, 2005;Kurita, 1987;Nishiyama, 1928). About the origin of the triploid, there are two key hypotheses.
The first is that they are generated from the hybridization of diploid with tetraploid. The second is that they are derived from a crossing between a normal haploid gamete and a nonreduced diploid gamete (Hsu et al., 1994;Kurita, 1987;Zhou et al., 2007). Because the tetraploid has only recently been discovered (Zhou et al., 2007;this paper), most researchers agree to the latter interpretation (Hayashi et al., 2005;Kurita, 1987). To date, only two mixploid populations with the tetraploid cytotype are found, and no independent tetraploid population is detected. In view of the very few tetraploid individuals in natural populations, the authors also agree with the latter explanation.
However, based on an extensive cytogenetical study on the Japanese triploid populations, Kurita (1987) thought that L. radiata var. radiata is not a simple autotriploid. Namely, the triploid is structurally heterozygous at least in regard to the SAT chromosomes (Kurita, 1987). By analyzing the nucleotide sequences of genomic DNA regions in 15 triploid strains and two diploid strains from Japan and China, Hayashi et al. (2005) found some genetic variations between the Japanese and Chinese triploid strains, indicating that L. radiata var. radiata is not a typical autotriploid, supporting Kurita's notion. In our extensive field investigations, we found that the triploids in China also have different SAT chromosome combination, supporting their notion.
Another perplexing problem is why the triploids are distributed very commonly in Japan and South Korea where no diploid mother taxon can be found. The diploids are only distributed in China, and no diploid cytotype has been found so far in Japan and South Korea.
Based on the genetic constancy of Japanese triploids in both the nuclear and chloroplast DNA sequences (Hayashi et al., 2005) and the monomorphism on all 24 allozyme loci in Korean L. radiata populations (Chung, 1999), they thought that the sterile triploids in Japan and South Korea were introduced from China, that is, one and more triploid bulb were brought to Japan and South Korea firstly, and then via extensive asexual reproduction by the rapid formation of new bulbs the triploid spread throughout Japan (except Hokkaido) and South Korea, accompanying human activities to some extent, such as rice cultivation and movement of monks in temples (Chung, 1999;Hayashi et al., 2005;Kurita, 1987). Because the peripheral regions of China, including Yunnan, Guangxi, and South Guizhou possess only triploid populations, as Japan and South Korea do, it is conceivable that it may be the same reason responsible for the formation of the current distribution pattern of L. radiata complex cytotypes and nonexistence of the diploid population on the periphery of the distribution range.
With regard to the geographical patterns of different cytotypes of L. radiata complex, especially the triploids, there are two possible interpretations. The one is that the triploids are generated from the diploids located in the middle and east of China, then they spread to the surrounding areas where the triploids generally prefer more local environment; the other is that the parental diploid taxon which once had been distributed relatively widely in China, Japan, and South Korea was extinct in Japan, South Korea and many peripheral distribution areas of China for some unknown reasons, only leaving the triploid bulbs. In order to find the key to the questions of the origin, migration routes and distribution patterns of polyploids of L. radiata, some molecular and cytogenetic methods are needed in the future study.

ACK N OWLED G M ENTS
We thank the anonymous reviewers for their useful comments and suggestions. The authors thank Yuanjin Cao, Qingyang Zheng, Xiaofan Ye, Fang Liu, Dong Zhang, and Chengfeng Tang for helping with the sampling of Lycoris radiata populations. This study was financially supported by the National Natural Science Foundation of China (31400291).

CO N FLI C T O F I NTE R E S T
None declared.

AUTH O R S ' CO NTR I B UTI O N S
Liu, K. and Zhou, S.B. designed the research. Liu, K. and Meng, W.Q.

DATA ACCE SS I B I LIT Y
The data supporting the conclusions of this manuscript can be found in the manuscript.