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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 (r2 = 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.

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INTRODUCTION

Lycoris radiata (L'Hérit.) Herb. (Amaryllidaceae) is a perennial, bulbous plant distributed from southwestern China eastward to Japan and South Korea, and is one of the most widely distributed species in genus Lycoris (Hsu, Kurita, Yu, & Lin, ). Since 1920s when the chromosome number of this taxon was first counted (Nishiyama, ), much work has been done on the chromosome number and karyotype of this species, particularly on the Japanese islands and East China (Bose, ; Kurita, ; Liu, Zheng, Xia, & Zhou, ; Qin, Zhou, & Wang, ; Shao, Yang, Zhang, & Nie, ; Zhou et al., ; Zhou, Yu, Luo, Hu, & Bi, ). For a long time, this species had been thought to harbor only two ploidy levels, diploid (2n = 22) and triploid (2n = 33), until about a decade ago the tetraploid was firstly found (Zhou et al., ). 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., ; Shi, Qiu, Li, Wu, & Fu, ). 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, ; Kurita, ). In China, diploid, triploid, and tetraploid plants have been found (Liu et al., ; Zhou et al., ). 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?

MATERIALS AND METHODS 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 ). 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.

Origins of materials and the karyotypes

Pop. Localities (voucher) Coordinates Altitude (m) Number of bulbs examined Chromosome formula Ploidy
1 Laojiahe Village, Lu'an, Anhui; LR16001 31°29′N/115°24′E 455 22 2n = 22A 2x
2 Maotanchang Town, Lu'an, Anhui; LR16021 31°19′N/116°32′E 164 20 2n = 22A 2x
3 Hengdu Town, Chizhou, Anhui; LR16035 30°11′N/117°33′E 78 40 2n = 22A 2x
4 Bagongshan, Huainan, Anhui; 90409 32°37′N/116°47′E 103 20 2n = 22A 2x
5 Marenshan, Wuhu, Anhui; LR15002 30°58′N/118°09′E 150 18 2n = 22A 2x
6 Fenghuangshan, Tongling, Anhui; LR15001 30°51′N/118°01′E 180 30 2n = 22A 2x
7 Dashan Village, Chizhou, Anhui; LR14002 30°01′N/117°21′E 340 25 2n = 22A 2x
8 Qinyunshan, Huangshan, Anhui; LR13016 29°48′N/118°02′E 265 24 2n = 22A 2x
9 Wenquan Town, Anqing, Anhui; LR15003 30°56′N/116°17′E 628 6 2n = 22A 2x
10 Eshan Town, Wuhu, Anhui; 708251 31°06′N/118°17′E 95 14 2n = 22A 2x
11 Yashan, Wuhu, Anhui; LR12001 30°48′N/117°59′E 310 21 2n = 22A 2x
12 Nanquan Village, Tongling, Anhui; LR13003 30°25′N/117°16′E 38 30 2n = 22A 2x
13 Xiaotian Town, Lu'an, Anhui; LR13009 31°11′N/116°35′E 138 13 2n = 22A 2x
14 Daguling, Yi County, Huangshan, Anhui; LR11002 30°04′N/117°48′E 281 10 2n = 22A 2x
15 Xin'anjiang, Huangshan, Anhui; LR11003 29°39′N/118°11′E 132 5 2n = 22A 2x
16 Shitan Town, Qingyuan, Guangdong; LR16046 24°11′N/112°40′E 130 26 2n = 22A 2x
17 Fanjingshan Village, Tongren, Guizhou; LR15010 27°50′N/108°48′E 642 22 2n = 22A 2x
18 Wuchenhe Town, Xinyang, Henan; LR16034 31°45′N/114°47′E 66 41 2n = 22A 2x
19 Wufeng Town, Yichang, Hubei; LR16002 30°11′N/110°41′E 760 11 2n = 22A 2x
20 Rongmei Town, Enshi, Hubei; LR16028 29°49′N/109°54′E 929 15 2n = 22A 2x
21 Shadaogou Town, Enshi, Hubei; LR16030 29°41′N/109°36′E 628 22 2n = 22A 2x
22 Jiangyangping Town, Yichang, Hubei; LR16032 31°06′N/110°50′E 543 20 2n = 22A 2x
23 Beifeng Village, Yichang, Hubei; LR16007 31°06′N/110°49′E 544 40 2n = 22A 2x
24 Shaping Town, Xianning, Hubei; LR15005 29°22′N/113°50′E 128 26 2n = 22A 2x
25 Wudangshan, Shiyan, Hubei; LR16040 32°26′N/111°03′E 353 11 2n = 22A 2x
26 Maogou Town, Xiangxi, Hunan; LR15030 28°35′N/109°22′E 273 30 2n = 22A 2x
27 Miaoshi Town, Changde, Hunan; LR16018 29°28′N/111°12′E 145 10 2n = 22A 2x
28 Wulingyuan, Zhangjiajie, Hunan; LR15016 29°21′N/110°29′E 423 28 2n = 22A 2x
29 Biyunfeng, Yiyang, Hunan; LR15017 28°26′N/112°22′E 488 23 2n = 22A 2x
30 Hougushan, Chenzhou, Hunan; LR15020 25°52′N/113°17′E 150 26 2n = 22A 2x
31 Lujiawan Village, Huaihua, Hunan; LR16022 28°35′N/110°27′E 123 20 2n = 22A 2x
32 Jiemuxi Town, Huaihua, Hunan; LR16024 28°47′N/110°25′E 236 20 2n = 22A 2x
33 Daping Town, Zhangjiajie, Hunan; LR16026 29°33′N/110°04′E 502 40 2n = 22A 2x
34 Liangshuikou Town, Zhangjiajie, Hunan; LR16027 29°00′N/110°30′E 349 43 2n = 22A 2x
35 Yixing forest park, Wuxi, Jiangsu; LR13001 31°17′N/119°45′E 70 23 2n = 22A 2x
36 Shipai Town, Suzhou, Jiangsu; LR11004 31°33′N/121°01′E 9 16 2n = 22A 2x
37 Zhuangkou Town, Ganzhou, Jiangxi; LR15033 25°40′N/115°39′E 160 23 2n = 22A 2x
38 Yangxi Town, Ji'an, Jiangxi; LR15035 27°18′N/114°12′E 196 8 2n = 22A 2x
39 Tongtianyan, Ganzhou, Jiangxi; LR15022 25°55′N/114°54′E 134 28 2n = 22A; 2n = 20A+1m 2x
40 Nashan Village, Jinggangshan, Jiangxi; LR15023 26°43′N/114°16′E 238 30 2n = 22A 2x
41 Longhushan, Yingtan, Jiangxi; LR15025 28°05′N/116°58′E 120 28 2n = 22A 2x
42 Sishiba Town, Shangrao, Jiangxi; LR15026 28°12′N/118°02′E 169 25 2n = 22A 2x
43 Beishan, Jinhua, Zhejiang; LR15027 29°12′N/119°37′E 501 33 2n = 22A 2x
44 Yantou Town, Wenzhou, Zhejiang; LR15028 28°20′N/120°43′E 53 31 2n = 22A 2x
45 Chichengshan, Taizhou, Zhejiang; LR15029 29°10′N/121°01′E 240 33 2n = 22A 2x
46 Jiufengshan, Zhejiang; LR13012 29°00′N/119°22′E 601 16 2n = 22A 2x
47 Jingling Town, Shaoxing, Zhejiang; LR13014 29°22′N/120°47′E 238 18 2n = 22A 2x
48 Yangduan Village, Jiujiang, Jiangxi; LR15004 29°32′N/115°22′E 510 24 2n = 22A 2x
49 Longpan Town, Nanchong, Sichuan; LR17001 30°49′N/105°53′E 386 10 2n = 22A 2x
50 Dayang Town, Lishui, Zhejiang; LR15032 28°31′N/120°11′E 900 18 2n = 22A 2x
51 Xidian Town, Ningbo, Zhejiang; LR15037 29°26′N/121°25′E 35 6 2n = 22A 2x
52 Qingliangfeng Town, Hangzhou, Zhejiang; LR13013 30°06′N/118°54′E 222 19 2n = 22A 2x
53 Jingtingshan, Xuancheng, Anhui; LR11001 30°59′N/118°43′E 161 25 2n = 33A 3x
54 Langyashan, Chuzhou, Anhui; LR08002 32°16′N/118°16′E 211 36 2n = 33A 3x
55 Huangpushan, Chuzhou, Anhui; 803271 32°20′N/118°00′E 151 30 2n = 33A 3x
56 Sanqi Town, Lu'an, Anhui; LR13010 31°14′N/116°41′E 176 8 2n = 33A 3x
57 Meijie Town, Chizhou, Anhui; LR12003 30°27′N/117°34′E 120 18 2n = 33A 3x
58 Longshe Town, Pengshui, Chongqin; LR16005 27°07′N/108°11′E 932 6 2n = 33A 3x
59 Xiannvshan Town, Wulong, Chongqin; LR16006 29°24′N/107°47′E 812 36 2n = 33A 3x
60 Wenfeng Town, Wuxi, Chongqing; LR16031 31°25′N/109°10′E 1,061 1 2n = 33A 3x
61 Jinhan Town, Ningde, Fujian; LR16042 26°41′N/119°28′E 148 26 2n = 33A 3x
62 Maixieyan Village, Putian, Fujian; LR16043 25°32′N/118°48′E 654 15 2n = 31A + 1m + 1B 3x
63 Heping Town, Zhangzhou, Fujian; LR16044 23°56′N/117°10′E 665 26 2n = 33A 3x
64 Laizhou Town, Nanping, Fujian; LR08006 26°37′N/117°58′E 450 10 2n = 33A 3x
65 Tianbaoyuan Reserve, Yong'an, Fujian; LR08004 25°58′N/117°22′E 715 15 2n = 33A 3x
66 Heshui Village, Shaoguan, Guangdong; LR16019 24°53′N/113°56′E 240 30 2n = 33A 3x
67 Wangbian Village, Shaoguan, Guangdong; LR15021 25°04′N/113°19′E 130 21 2n = 33A 3x
68 Lvtian Town, Conghua, Guangdong; LR16045 23°48′N/113°55′E 224 30 2n = 33A 3x
69 Licheng Town, Guilin, Guangxi; LR15006 24°29′N/110°24′E 148 24 2n = 33A 3x
70 Rongjiang Town, Guilin, Guangxi; LR15007 25°41′N/110°19′E 293 39 2n = 33A 3x
71 Tangjia Village, Guilin, Guangxi; LR15008 25°19′N/110°19′E 156 5 2n = 31A+1m + 1B 3x
72 Pingxi Village, Qindongnan, Guizhou; LR16014 27°07′N/107°46′E 716 30 2n = 33A 3x
73 Zhaibao Village, Tongren, Guizhou; LR15011 27°46′N/108°45′E 436 11 2n = 33A 3x
74 Dongfeng Town, Guiyang, Guizhou; LR15012 26°38′N/106°49′E 1,015 5 2n = 33A 3x
75 Ali Village, Guiyang, Guizhou; LR15013 26°34′N/106°48′E 1,052 25 2n = 33A 3x
76 Chengguan Town, Pan County, Liupanshui, Guizhou; LR15014 25°47′N/104°40′E 1,625 8 2n = 31A + 1m + 1m′ 3x
77 Yaojiatun Village, Anshun, Guizhou; LR15015 26°12′N/105°54′E 1,399 27 2n = 33A 3x
78 Hongshan Town, Suizhou, Hubei; LR15034 31°36′N/112°55′E 210 21 2n = 33A 3x
79 Cihe Town, Xiangyang, Hubei; LR15036 32°01′N/111°48′E 166 24 2n = 33A 3x
80 Bajiaodongzu Town, Enshi, Hubei; LR16004 30°07′N/109°23′E 590 24 2n = 33A 3x
81 Moudao Town, Lichuan, Hubei; LR16020 30°29′N/108°39′E 1,032 25 2n = 33A 3x
82 Zhonglu Town, Enshi, Hubei; LR16041 30°08′N/108°45′E 1,088 31 2n = 33A 3x
83 Majiawan, Hefeng County, Enshi, Hubei; LR16029 29°52′N/110°00′E 583 1 2n = 33A 3x
84 Xiaojiacun Town, Yongzhou, Hunan; LR16015 26°21′N/112°00′E 103 20 2n = 33A 3x
85 Dankou Town, Shaoyang, Hunan; LR16013 26°25′N/110°13′E 400 25 2n = 33A 3x
86 Bozhou Town, Huaihua, Hunan; LR16017 27°23′N/109°17′E 325 24 2n = 33A 3x
87 Yidushui Town, Shaoyang, Hunan; LR15009 26°34′N/111°12′E 349 29 2n = 33A 3x
88 Qingwei Village, Loudi, Hunan; LR15018 27°51′N/110°58′E 626 12 2n = 33A 3x
89 Liuxin Village, Loudi, Hunan; LR15019 27°51′N/110°59′E 693 31 2n = 33A 3x
90 Longtan Village, Zhuzhou, Hunan; LR16008 26°09′N/113°45′E 480 20 2n = 33A 3x
91 Huaguoshan, Lianyungang, Jiangsu; LR13002 34°39′N/119°16′E 131 15 2n = 33A 3x
92 Laoshan, Nanjing, Jiangsu; LR14001 32°06′N/118°36′E 104 3 2n = 33A 3x
93 Yushan, Lianyungang, Jiangsu; LR13004 34°38′N/119°15′E 30.9 13 2n = 33A 3x
94 Wutong Town, Guilin, Guangxi; LR16016 25°22′N/110°03′E 180 28 2n = 33A 3x
95 Wanger Town, Shangrao, Jiangxi; LR15031 28°17′N/117°30′E 66 22 2n = 33A 3x
96 Chenshan, Shanghai; LR16010 31°04′N/121°10′E 25 38 2n = 33A 3x
97 Sheshan, Shanghai; LR16011 31°05′N/121°11′E 57 37 2n = 33A 3x
98 Longwangou, Lueyang County, Shannxi; LR13015 33°22′N/106°09′E 692 6 2n = 33A 3x
99 Liyushan, Ankang, Shannxi; LR16036 32°41′N/108°55′E 300 6 2n = 33A 3x
100 Taibai Village, Hanzhong, Shannxi; LR16039 33°00′N/106°47′E 554 36 2n = 33A 3x
101 Dahanshan, Hanzhong, Shannxi; LR16038 32°57′N/106°56′E 950 6 2n = 33A 3x
102 Taibaishan, Baoji, Shannxi; LR16039 34°05′N/107°42′E 1,136 6 2n = 33A 3x
103 Motan Town, Guangyuan, Sichuan; LR16003 32°10′N/106°03′E 868 24 2n = 33A 3x
104 Dafo Town, Leshan, Sichuan; LR10001 29°46′N/104°03′E 436 27 2n = 33A 3x
105 Changyuangou Village, Nanchong, Sichuan; LR16009 30°51′N/105°59′E 310 15 2n = 33A 3x
106 Dayun Village, Yibin, Sichuan; LR13005 28°22′N/104°46′E 597 30 2n = 33A 3x
107 Futou Village, Yibin, Sichuan; LR13006 28°20′N/104°53′E 447 31 2n = 33A 3x
108 Gaojian Village, Yibin, Sichuan; LR13007 28°17′N/104°59′E 604 30 2n = 33A 3x
109 Emeishan, Leshan, Sichuan; LR16033 29°36′N/103°23′E 880 6 2n = 33A 3x
110 Baishanzu Town, Lishui, Zhejiang; LR08005 27°43′N/119°12′E 1,074 5 2n = 33A 3x
111 Huilongchang Village, Nanchong, Sichuan; LR16012 30°53′N/105°59′E 387 6 2n = 22A; 2n = 33A 2x(1) + 3x(5)
112 Lianhua Village, Wuhu, Anhui; LR13011 31°03′N/117°32′E 223 13 2n = 22A; 2n = 33A 2x(11) + 3x(2)
113 Caishiji park, Ma'anshan, Anhui; LR08003 31°39′N/118°27′E 101 26 2n = 22A; 2n = 22A + 1B; 2n = 33A; 2n = 31A + 1m + 1m′ 2x(16) + 3x(10)
114 Tangxi Town, Chizhou, Anhui; LR12002 30°20′N/117°36′E 112 36 2n = 22A; 2n = 44A 2x(35) + 4x(1)

The growth form and seasonality of L. radiata are very characteristic, that is, the productive and reproductive phases are separate (Figure a,b). The diploids can produce seeds and have sexual and asexual reproduction, while the triploids can only propagate by clone, producing no seed.

View Image - Photograph of Lycoris radiata. (a) Plants in vegetative phase in habitat. (b) Plants in flowering in habitat

Photograph of Lycoris radiata. (a) Plants in vegetative phase in habitat. (b) Plants in flowering in habitat

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., ) 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 (), Kurita (), and Liu et al. (): 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 A-type 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., ), 29 populations (Kurita, ), and 7 populations (Chung, ) were from China, Japan, and South Korea, respectively. In total, 157 populations with exact ploidy data were mapped using ArcMap 10.0.

RESULTS The ploidy and chromosome number of Lycoris radiata

A total of 2,447 individuals, from 114 populations of Lycoris radiata (Table ), 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 ).

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 Figure a,i. The representative karyotype consists of twenty-two A-type chromosomes of which two are SAT chromosomes. The measured and calculated values of each chromosome of the representative karyotype of diploid are summarized in Table . The short-arm length of both SAT chromosomes is much the same, and their r value is 10.73 and 9.27, respectively. Different types of SAT chromosome combination are observed in the inter- and intra-population. One SAT chromosome is observed in one bulb from population 4 in Bagongshan of Huainan city (Figure b,j). As shown in Figure c,k four SAT chromosomes are found in one bulb from population 114. The four SATs, reported for the first time here, are the maximum number of SAT chromosome in this species known so far. Of 1,195 triploid bulbs, 1,166 bulbs are typical triploid consisting of 33 A-type chromosomes with 0–3 SAT chromosomes. A representative karyotype of triploid is shown in Figure d,l.

View Image - The metaphase chromosome morphology and karyotypes. (a, i) a metaphase chromosome from population 114. Asterisks indicate two SAT chromosomes. (b, j) a metaphase chromosome from population 4. Asterisk indicates one SAT chromosomes. (c, k) a metaphase chromosome from population 114. Asterisks indicate four SAT chromosomes. (d, l) a metaphase chromosome from population 77 showing a representative karyotype of triploid. (e, m) a metaphase chromosome from population 39. The arrow indicates one m-type chromosome. (f, n) a metaphase chromosome from population 76. Arrow indicate one m-type chromosome and one m′-type chromosome. (g, o) a metaphase chromosome from population 71. The arrow indicates one m-type chromosome. Asterisks indicate two SAT chromosomes and one B-type chromosome. (h, p) a metaphase chromosome from population 62. The arrow indicates one m-type chromosome. Asterisks indicate one SAT chromosomes and one B-type chromosome

The metaphase chromosome morphology and karyotypes. (a, i) a metaphase chromosome from population 114. Asterisks indicate two SAT chromosomes. (b, j) a metaphase chromosome from population 4. Asterisk indicates one SAT chromosomes. (c, k) a metaphase chromosome from population 114. Asterisks indicate four SAT chromosomes. (d, l) a metaphase chromosome from population 77 showing a representative karyotype of triploid. (e, m) a metaphase chromosome from population 39. The arrow indicates one m-type chromosome. (f, n) a metaphase chromosome from population 76. Arrow indicate one m-type chromosome and one m′-type chromosome. (g, o) a metaphase chromosome from population 71. The arrow indicates one m-type chromosome. Asterisks indicate two SAT chromosomes and one B-type chromosome. (h, p) a metaphase chromosome from population 62. The arrow indicates one m-type chromosome. Asterisks indicate one SAT chromosomes and one B-type chromosome

Measurements of somatic chromosomes in a representative karyotype of diploid

[Table omitted. See PDF]
Note:

Asterisk indicates SAT chromosome.

Abbreviations: LL, relative length of long arm; SL, relative length of short arm; TL, total relative length; SL + LL=TL.

Some aberrant chromosomes which should be generated from A-type chromosome are found including metacentrics (m), small metacentrics (m′), and B-type chromosome (Table ). The occurrence of Robertsonian fusion of A-type chromosomes is confirmed in some bulbs from five populations, for example, population 39, 62, 71, 76, and 113 26, 32, 40, 60, and 72. The karyotype of one bulb from population 39 in Tongtianyan mountain of Ganzhou city comprises two types of chromosome (A- and m-type chromosome), formulated as 2n = 21 = 20A + 1m (Figure e,m). Eight bulbs from population 76 in Pan County and one bulb from population 113 at Caishiji park are abnormal triploids and have 33 chromosomes with 31 A-type chromosomes, one metacentric chromosome (m-type chromosome) and a small metacentric chromosome (m′-type chromosome). The karyotype of them is formulated as 2n = 33 = 31A + 1m + 1m′ (Figure f,n). In addition, five bulbs from population 71 at Tangjia village of Guilin city and fifteen bulbs from population 62 at Maixie village of Putian city also have the m-type chromosome, and the karyotype of them is formulated as 2n = 33 = 31A + 1m + 1B (Figure g,h,o,p). Two and one SAT chromosomes are observed in the population 71 and 62, respectively. The arm ratio of m-type chromosome in the bulbs from population 71 and population 62 is 1.0 and 1.1, respectively. Small B-type chromosome is also observed in two bulbs from population 113, with the karyotype formulated as 2n = 22 = 22A + 1B. Both m′-type chromosome and B-type chromosome should originate from A-type chromosomes, but the former has at least twofold length than the latter.

The percentage of bulbs having aberrant chromosomes

Ploidy level Number of bulbs examined Number of bulbs with aberrant chromosomes
m m′ B
Diploid 1,224 1 0 2
Triploid 1,195 20 9 20
Tetraploid 1 0 0 0
Total number of bulbs 2,420 21 9 22
Distribution of different cytotypes in China

Diploid cytotypes are located primarily in the middle and east regions of China. Isolated diploid populations are found in southeast areas of China. The triploid cytotypes have a wider distribution, occupying nearly the whole distribution range of this species in China. Six provinces located on the periphery of the distribution area have no more than one diploid population (Figure ).

View Image - 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

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

In Figure , it is indicated that the triploids have a broader altitudinal range than the diploids. The triploids grow over a broad range of altitudes, from 25 to 1,625 m, with the average being 513.39 ± 382.53 m, while the diploids have a narrower altitudinal range of 9–929 m, with an average of 299.06 ± 226.76 m. The difference in mean altitude of localities between diploid and triploid is highly significant (p < 0.01; Figure a). Moreover, a significantly positive but low correlation was found between the ploidy and altitude (r2 = 0.103, p < 0.01; Figure b).

View Image - (a) Altitudes comparing ploidy levels. Means significantly different at p [less than] 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 (r2 = 0.103, p [less than] 0.01)

(a) Altitudes comparing ploidy levels. Means significantly different at p [less than] 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 (r2 = 0.103, p [less than] 0.01)

DISCUSSION The chromosome number and karyotype of Lycoris radiata

Several cytological studies on L. radiata have been made by various authors (Bose, ; Kurita, ; Nishiyama, ; Qin et al., ; Shao et al., ; Zhou et al., ). Recently, Liu et al. () 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, ; Liu et al., ; Zhou et al., ), further confirm that L. radiata is a species complex concluding diploid with 2n = 22, triploid with 2n = 33, and tetraploid with 2n = 44.

The common karyotype of L. radiata only consists of typical acrocentric chromosomes (A-type chromosome) with different types of SATs combination. To date, many abnormal karyotypes of this complex have been reported, such as 2n = 22 = 1m + 20A + 1B (Shao et al., ); 2n = 33 = 1m + 31A + 1B, 2n = 32 = 1m + 31A (Bose, ; Kurita, ); 2n = 21 = 1m + 10st + 9T + 1B (Zhou et al., ); 2n = 21 = 1m + 20st, 2n = 25 = 1m + 20st + 2t + 2T (Zhou et al., ), due to the various rearrangements of A-type chromosome. Through careful examination on the karyotype of 519 bulbs of L. radiata in Japan, Kurita () observed 14 bulbs with aberrant chromosomes, whereas no bulb with aberrant chromosomes was found by Liu et al. () based on the cytological study of 466 bulbs of this species in China. As reported in the previous studies, some rearranged chromosomes from A-type chromosome which are aberrant have been also found in this paper. The number of bulbs with aberrant chromosomes is small, only accounting for 1.32% of total examined bulbs (32/2,420), and the aberrants could be classified into three types and include metacentric chromosome (m), small metacentric chromosome (m′), and B-type chromosome. The diploid populations have less abnormal individuals with aberrant chromosomes than the triploid populations. Because the diploids have both sexual and asexual reproduction, and the individual with aberrant chromosomes producing no seeds might be gradually excluded from the population due to intraspecific competition, so less individuals with aberrant chromosomes were found from the diploid population than from the triploid population.

The phenomenon of Robertsonian fusion of A-type chromosome was found in some bulbs from five different populations. As confirmed in some previously published researches, the m-type chromosome in one diploid bulb from population 40 may be produced by the fusion of two A-type chromosomes. Concerning the origin of the triploid individuals carrying aberrant m-type chromosome, there are two alternative explanations. The one is that the m-type chromosome was generated from a diploid population including the bulb with m-type chromosome by hybridization between a normal haploid gamete and an unreduced diploid gamete; the other is that it started from a somatic cell of a bulb having only A-type chromosomes, in which m-type chromosome was formed by Robertsonian change, as it was observed in the diploid bulb of population 40. Which explanation is reasonable, hybridization or somatic cell mutation? Eleven abnormal triploid bulbs with m-type chromosome had been also found by Kurita (). He considered that the latter, somatic cell mutation, might be the actual occurrence, because only a very small proportion of examined bulbs carried the aberrant m-type chromosome. The authors agreed with Kurita’ opinion, considering that the m-type chromosome in aberrant diploid and triploid bulbs should be produced by Robertsonian fusion.

Although the karyotypes of L. radiata have some variability among inter-populations and even within intra-population to some extent, it was convinced that L. radiata have a relatively stable karyotype composed of 22/33/44 A-type chromosomes on the basis of large-scale sampling from China and Japan.

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., ). 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, ; Odee, Wilson, Omondi, Perry, & Cavers, ; Segraves, Tompson, Soltis, & Soltis, ; Steussy, Weiss-Schneeweiss, & Keil, ). In this study, we conducted an exhaustive survey of the chromosomal races in most populations within the natural range of L. radiata complex across the entire distribution regions in China.

Although whether or not polyploids have a broader niche breadth than diploids remains controversial (te Beest et al., ; Glennon, Ritchie, & Segraves, ; Martin & Husband, ), in numerous complex species documented by many authors (Lowry & Lester, ; McIntyre, ; Treieret al., ), the polyploids show wider geographic ranges and greater stress tolerances of extreme ecological conditions. In comparison with the diploids, the triploids have a significantly larger geographic range. The triploids distribute from the southwest of China eastward to the south of Korea and south of Japan, covering the whole geographic distribution ranges of the complex. In contrast, the diploids are primarily limited to the middle and east of China, with some diploid populations scattering in the southeast of China. Together with some previous reports (Kurita, ; Liu et al., ), 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.

A positive correlated relationship between polyploidy and elevation is fairly well supported (Brochmann et al., ; Soltis, ; Stebbins, ). 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, ), Chamerion angustifolium (Husband & Schemske, ), Centaurea jacea (Hardy, Vanderhoeven, Loose, & Meerts, ), and Isoetes spp. (Liu, Gituru, & Wang, ). 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 less-disturbed 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, ; Kurita, ; Nishiyama, ). 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., ; Kurita, ; Zhou et al., ). Because the tetraploid has only recently been discovered (Zhou et al., ; this paper), most researchers agree to the latter interpretation (Hayashi et al., ; Kurita, ). 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 () 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, ). By analyzing the nucleotide sequences of genomic DNA regions in 15 triploid strains and two diploid strains from Japan and China, Hayashi et al. () 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., ) and the monomorphism on all 24 allozyme loci in Korean L. radiata populations (Chung, ), 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, ; Hayashi et al., ; Kurita, ). 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.

ACKNOWLEDGMENTS

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).

CONFLICT OF INTEREST

None declared.

AUTHORS' CONTRIBUTIONS

Liu, K. and Zhou, S.B. designed the research. Liu, K. and Meng, W.Q. collected the samples. Liu, K., Wang, L.D., Meng, W.Q., and Zheng, L. generated and analyzed the data. Liu, K. and Zhou, S.B. wrote the manuscript.

DATA ACCESSIBILITY

The data supporting the conclusions of this manuscript can be found in the manuscript.

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