© 2016 The Japan Mendel Society Cytologia 81(2): 195–205

Cytological Study of Tetraploid Species of subgenus Yulania ()

Ya-Ling Wang1,2, Zi-Can He2, Erland Ejder3, Jian-Fen Yang2 and Shou-Zhou Zhang2*

1 Xi’an Botanical Garden of Province, Xi’an 710061, 2 Laboratory of Southern Subtropical Diversity, Fairylake Botanical Garden, Shenzhen & Chinese Academy of Sciences, Shenzhen 518004, China 3 Swedish Magnolia Group, Laholm, Sweden

Received June 29, 2015; accepted April 5, 2016

Summary Cytological studies were conducted on five tetraploid species plus two varieties of Magnolia subgenus Yulania and a hybrid between two of these species. Two different chromosome configurations in meiosis are observed in this article. The first group was typical for M. acuminata, M. liliiflora and their relatives which share several cytological characteristics including chromosome configurations and behavior in meiosis. The cytological evidence indicate autotetraploid origin for these species, despite the homologous chromosomes likely having changed their structures causing the formation of heteromorphic multivalents, chromosome bridges and fragments during meiosis. The second group of chromosome configuration was found in M. cylindrica and M. concinna. These species were characterized by very rare trivalents and tetravalents. These observations sug- gest these taxa as allotetraploids which may have been formed from hybridisation between hexaploid and diploid parents as deduced from trivalents observed at some rate. Multivalent formation and possible translocations and inversions of chromosomes in M. liliiflora influence its propagation by causing low fertility. M. liliiflora is prob- ably a comparatively new species with an unstable chromosome constitution and is growing only in small popu- lations at low elevation. It is vulnerable, and the species could already be extinct in nature. On the other hand, M. acuminata shows a more regular chromosome behavior and fewer abortion spores than M. liliiflora, and should be a more stable species with longer evolution time. Thus, the superior fertility of M. acuminata has enabled it to develop a stable and extensive distribution from NE to SE USA.

Key words Magnolia liliiflora Desr., Magnolia acuminata L., Magnolia cylindrica E. H. Wilson, Autotetraploid, Allotetraploid, Speciation.

Magnolia, with a basic chromosome number of 19, and Casey 1980). Further comparative analysis helps to is regarded as a paleopolyploid genus (Stebbins 1980). explore the way of formation and evolution mode of the Two groups of this genus, subgenus Magnolia sec- polyploidy. Very little work has been done to study the tion Theorhodon Spach and subgenus Yulania (Spach) chromosome behavior during meiosis of with Reichenbach, have polyploid series based on tetraploid the exception of a single study on the chromosome con- to hexaploid multiples of 19 chromosomes (Janaki figuration of M. denudata Desr. (Li and He 2003) and a Ammal 1952, Darlington and Wylie 1955, Chen et al. study on the genome differentiation between Michelia 1985). Law’s classification has been used in this study and subgenus Yulania based on hybrid chromosome con- (Law et al. 2004). figuration (Zhang et al. 2011). Polyploidization in the speciation of Magnolia con- Subgen. Yulania includes three taxonomically well- tinues to attract the attention of taxonomists. The chro- established tetraploid species of which two, M. liliiflora mosome structure among Magnolia species varies little Desr. and M. cylindrica E. H. Wilson, occur in China and the karyotypes of all the species remain constantly and one, M. acuminata L., in North America. The latter at type 2B (Stebbins 1971, Okada 1975, Biswas and includes two varieties, M. acuminata var. acuminata and Sharma 1984, Li et al. 1998). The chromosome behavior M. acuminata var. subcordata (Spach) Dandy (Law et al. during meiosis is usually examined to determine the 1996). numerical and structural features of chromosomes. The M. liliiflora was described by Desrousseaux (de La- configuration at metaphase I (MI) of meiosis is com- marck 1792) with Kaempfer’s flower plates of monly showing the homologous degree between the dif- in Japan used as the type. This species is frequently ferent sets of chromosomes in polyploid plants (Jackson cultivated in gardens all over the world, but has never been recorded in the wild up to now. Existing herbarium * Corresponding author, e-mail: [email protected] samples do not verify an origin in the wild. The native DOI: 10.1508/cytologia.81.195 range is suggested to be eastern China (Law et al. 1996). 196 Y. L. Wang et al. Cytologia 81(2)

It shows a notable variation in the number and size of M.×brooklynensis Kalmbacher, the hybrid between . The chromosome number is 2n=4x=76 (Whita- M. acuminata and M. liliiflora, exceeds M. liliiflora in ker 1933, Biswas and Sharma 1984, Li et al. 1998). M. fertility and hardiness. The cytology of the clone M. × liliiflora Desr. var. gracilis (Salisb.) Rehd. which may brooklynensis ‘Woodsman’ was studied in this investiga- have originated in cultivation, has been described (Re- tion in order to confirm the cytological relationship of its hder 1916) and the history in cultivation has been dis- parents. cussed by Treseder (1978). We agree with the treatment This article presents a comparison of the chromosome of Frodin and Govaerts (1996) and Xia et al. (2008) to behavior in pollen mother cells (PMCs) of these five spe- see M. liliiflora var. gracilis Rehd. as a synonym of M. cies, plus two varieties and one hybrid, all belonging to liliiflora. M. polytepala Law & R. Z. Zhou & R. J. Zhang Magnolia subgenus Yulania. The study aims to provide was described as a new species with 12–16 tepals from cytological evidence to unravel the origin of these tet- Mt. Wuyi of Fujian Province (Zhang et al. 2006). In our raploids and the relationship among them. In order to field explorations, we have found that M. polytepala has study the intra-species variation on chromosome level, true bush habit with slim twigs and no ascending trunks several individuals of different provenances have been even in individuals 20-years old. In contrast, older plants observed. of M. liliiflora can have one or several trunks. A hexa- ploid of subgenus Yulania, M. sprengerii Pamp. also has Materials and methods rich variation in number (Kang and Ejder 2011). The tepal number is often a capricious character in sub- Seven taxa of Magnolia subgenus Yulania were stud- genus Yulania as discussed in that reference. Xia et al. ied including M. liliiflora Desr. (four samples from dif- (2008) treat M. polytepala as a synonym of M. liliiflora. ferent sources), M. liliiflora var. gracilis (Salisb.) Rehd. In our study, both taxa above are treated as only differ- (one sample), M. polytepala Law et R. Z. Zhou et R. J. ent variations of M. liliifora from different distributions, Zhang (one sample), M. acuminata L. (two samples with but of horticultural interest. No studies about their chro- different morphological characters), M. acuminata var. mosomes have been reported so far. subcordata (Spach) Dandy (one sample), M. cylindrica Magnolia cylindrica E. H. Wilson is distributed in E. H. Wilson (four samples with different morphologi- southeast China at an altitude of 800–1900 m. The flow- cal characters from different sources), M. concinna Law ers have nine tepals, and the outer three are sepal-like et R. Z. Zhou (one sample)- and in addition a hybrid M. with trianglar shape. The chromosome number is 2n=76 acuminata×M. liliiflora (one sample) (Table 1). (Parris et al. 2010). There is no earlier cytological infor- The living material and voucher specimens are grown mation about meiosis. M. concinna Law & R. Z. Zhou respectively deposited at Wuhan Botanical Garden was described as a new species with 12 tepals from Mt. (HIB), Shenzhen Fairylake Botanical Garden (SZG), Wuyi of Fujian Province (Law et al. 2004). Our long and Xi’an Botanical Garden (XBG). The M. cylindrica, time observation of live plants, in cultivation as well as M. cylindrica ‘Pegasus’ and M. cylindrica ‘Krossa’ used in the wild in Fujian Province, shows that M. concinna is originated from Huangshan (Anhui Prov.) or Lushan similar to M. cylindrica in the shape of leaves and fruit, (Jiangxi Prov.) in China and have been cultivated in only that it has more tepals (9–12) than the latter. Since Western gardens for a long time as M. cylindrica. The the occasional appearance of specimens with additional specimens used are growing in the gardens of Dr. Flinck tepals is a general phenomenon in Magnolia, it should (FG) or Dr. Ejder (EG) in Sweden (Table 1). be submersed into M. cylindrica (Xia et al. 2008, Kang Preparation of somatic cell chromosomes: Leaf api- and Ejder 2011). We treat this taxon as an ecotype of M. cal meristem was pre-treated in a mixed solution of cylindrica in this study. 0.1% colchicine and 0.002 mol/L 8-hydroxyquinoline Magnolia acuminata is distributed in eastern North for 3 h before fixation in Carnoy’s fixative (glacial acetic America from New York state and southern Ontario, acid : ethanol=1 : 3) for 1 h before squashing. It was dyed south to the Florida panhandle and west to Arkansas and and then observed with an optical microscope. Louisiana. The only species in subgenus Yulania with Preparation for PCMs: Flower buds were taken when yellow tinted flowers, M. acuminata typically produces the PMCs were in the meiotic phase during early spring. blooms that are fairly variable in tepal size and color After removal of the bud bracts and tepals, the imma- (from green, yellow-green to yellow) while there is little ture anthers were fixed in Carnoy’s fixative for 8–24 h. or no pubescence on the young twigs and abaxial sides After cellulase and pectinase digestion of cell walls, a of leaves (Callaway 1994). Its variety, M. acuminata var. flame-drying protocol was followed to make chromo- subcordata (Spach) Dandy, which is restricted to SE US, some preparations (Zhu 1982). These chromosomes has more consistently yellow flowers while its young were stained by the Giemsa method. All meiotic stages twigs and leaf-backs are covered with dense pubescence. were studied and photographed using a Zeiss Axioplan 2 It is also a smaller , sometimes even -like, than imaging microscope equipped with an Axiocam digital the much larger-growing M. acuminata var. acuminata. camera. 2016 Meiosis of Tetraploid Magnolia 197

Table 1. Sources of materials used in this study.

Taxon Locality Voucher

M. liliiflora Desr. Wuhou Temple, Mianxian, Shaanxi SZG M. liliiflora Desr. *Wuhan Botanical Garden, Wuhan, HIB M. liliiflora Desr. *Mt. Micang, Hanzhong, Shaanxi XBG M. liliiflora Desr. Hangzhou Botanical Garden, Hangzhou, Zhejiang SZG M. liliiflora var. gracilis (Salisb.) Rehd. Hangzhou Botanical Garden, Hangzhou, Zhejiang SZG M. polytepala Law et R. Z. Zhou *Huangkeng, Fujian SZG M. cylindrica E. H. Wilson Xi’an Botanical Garden, Shaanxi (Huangshan origin) XBG M. cylindrica E. H. Wilson Flinck Garden, Sweden (Huangshan origin) FG M. cylindrica ‘Pegasus’ Ejder Garden, Sweden EG M. cylindrica ‘Krossa’ Flinck Garden, Sweden FG M. concinna Law et R. Z. Zhou Mt. Wuyi, Fujian XBG M. acuminata L. (‘Kenneth’s Delight’) Flinck Garden, Sweden (US origin) FG M. acuminata L. (‘North Gold’) Flinck Garden, Sweden (Wyoming Co, NY, USA) FG M. acuminata L. var. subcordata (Spach) Dandy (‘Miss Honeybee’) Flinck Garden, Sweden (US origin) FG M. acuminata×M. liliiflora (‘Woodsman’) Flinck Garden, Sweden FG

*Suspected to be wild clone.

Results pollen grains observed by random selection, 65 were empty, indicating that more than half of the grains were Magnolia liliiflora Desr. sterile. The abnormal chromosome behavior in meiosis The following report is mainly based on observation and huge proportion of infertile pollen is a consequence of the sample from Wuhou Temple of Mian County in of multivalent formation at MI and irregular chromo- Shaanxi Province. some segregation at AI and AII. The PMCs of the four specimens of different prove- The chromosome behavior of PMCs and the arranging nances (Table 1) show similar chromosome performance patterns of microspore tetrads of M. liliiflora from Hubei during meiosis. The chromatin in prophase I contracted Province was similar to those described above. Multiva- into chromonemata which became thicker and shorter as lents could be observed both in diakinesis and early MI. a result of contraction and pairing of homologous chro- Up to one ring hexavalent and five tetravalents (of which mosomes. Because of the great number and length of the 4 were hetero-tetravalents) could be observed in early chromonemata and the multivalent pairing, the chromo- MI (Fig. 1I). Chromosome bridges and fragments were nemata were entangled with each other and could hardly found in about 10% of the cells in AI and AII. be identified during pachytene (Fig. 1A) and in diplotene The PMCs of the sample from Mount Micang of (Fig. 1B). A number of multivalents can be observed in Shaanxi Province, suspected to be a wild individual, ex- every PMC in the diakinesis stage (Fig. 1D). Multiva- hibited the same features during meiosis, with more ring lents were observed in early MI, but most of them will bivalents and tetravalents dominating in diakinesis. Up gradually resolve into bivalents in late MI. Bivalents to one hetero-hexavalent or five tetravalents (of which dominated though ring or chain tetravalents and one het- four are hetero-type) were observed in early MI (Fig. ero-hexavalent also coexisted in early MI (Fig. 1C, E). 1J). In AI, chromosome bridges and fragments were Among the 23 PMCs studied, the average chromosome seen in a few PMCs. configuration was 2n=34.83II+1.39IV+0.13VI (Table The samples from Hangzhou Botanical Garden of 2). The normal division figure of anaphase I (AI) exhib- Zhejiang Province demonstrated the same features as the its clearly the chromosome number as n=2x=38. Among other three: five tetravalents were found at most in one 101 randomly selected PMCs in AI, 12.9% of the PMC (Fig. 1K). cells displayed abnormalities like chromosome bridges, chromosome fragmentation and different chromosome Magnolia liliiflora var. gracilis Rehd. number in two daughter cells (Fig. 1F) (Table 3). The The chromosome number was found to be 2n=4x=76. chromosomes of the two daughter cells in metaphase Bivalents dominated, accompanied by hetero-tetravalents II (MII) were lined on the equatorial plates which were and hexavalents in early MI (Fig. 1L). Four tetravalents either perpendicular or parallel to each other. The cells were found in one PMC at most (Fig. 2A). The chromo- developed into isobilateral, decussate, linear to T-shaped some configuration is 2n=34.58II+1.47IV+0.16VI. The arrays of tetrads. Some cells in MII had either lagging behavior of chromosomes at AI, MII and AII is normal. chromosomes or micronuclei. According to our study of the samples, the plant mor- The chromosome number in anaphase II (AII) is phology is within the variation range of M. liliiflora. n=2x=38 (Fig. 1G). 15.6% of 45 cells examined had Its chromosome configuration and performance during either chromosome fragments or micronuclei. Of the 127 meiosis were essentially the same as those of M. liliiflora. 198 Y. L. Wang et al. Cytologia 81(2)

Fig. 1. Meiosis of tetraploids of subgenus Yulania in China. A–K. Meiosis of M. liliiflora. A, Pachytene. B, Diplotene. C, MI (early stage), arrow showing one ring hexavalent. D, MI (early stage), thick arrow showing chain hexaploid and thin arrows showing five tetravalents. E, MI, thick arrow showing ring hexaploid and thin arrows showing three tetravalents. F, AI, arrow showing lagging choromosome. G, AII. H, pollen. I, MI of M. liliiflora from Hubei, thin arrow showing hexavalent and thick arrow showing multivalent. J, MI of M. liliiflora from Micang Shan, thick arrow showing hexavalent and thin arrows showing two tetravalents. K, MI of M. liliiflora from Hangzhou, arrows showing five tetravalents. L, MI of M. liliiflora var. gracilis, arrow showing hexavalent.

These facts support the view above that M. liliiflora var. same features concerning both the chromosome behav- gracilis should be a synonym of M. liliiflora and not ior in PMCs and the tetrad type were observed as in given varietal status. M. liliiflora and M. liliiflora var. gracilis. Bivalents pre- vailed, while four ring or chain tetravalents or one het- Magnolia polytepala Law et R. Z. Zhou ex. R .J. Zhang ero-hexavalent existed in some PMCs (Fig. 2B–D). The The chromosome number is found to be 2n=76. The chromosome configuration is 2n=0.04I+34.32II+0.04I 2016 Meiosis of Tetraploid Magnolia 199

Table 2. Mean frequency of chromosome pairing and range values at MI of the tetraploids of subgenus Yulania.

Number of No. cells Mean chromosome pairing per cell Material chromosomes observed Monovalent Bivalent Trivalent Tetravalent Hexavalent

M. liliiflora Desr. 2n=76 23 0 34.831 (28–38)2 0 1.39 (0–6) 0.13 (0–1) M. liliiflora var. gracilis Rehd. 2n=76 19 0 34.58 (30–38) 0 1.47 (0–4) 0.16 (0–1) M. polytepala Law et R. Z. Zhou 2n=76 22 0.04 (0–1) 34.31 (32–38) 0.04 (0–1) 1.40 (0–4) 0.27 (0–1) M. cylindrica E. H. Wilson 2n=76 44 2.00 (0–8) 35.5 (30–38) 0.60 (0–3) 0.30 (0–1) 0 M. concinna Law et R. Z. Zhou 2n=76 18 0 37.66 (38–36) 0 0.17 (0–1) 0 M. acuminata L. 2n=76 56 0 33.84 (28–38) 0 1.63 (0–5) 0.30 (0–2) M. acuminata L. var. subcordata 2n=76 66 0 34.04 (24–38) 0 1.77 (0–7) 0.14 (0–1) (Spach) Dandy

1Mean number of bivalents appearing in one cell. Same principle in the rest of the table. 2Number of bivalents appearing in one cell, ranging from 28–38. Same principle in the rest of the table.

Table 3. Mean frequency of abnormal phenomena in PMCs and pollen of the tetraploids of subgenus Yulania.

Material AI AII Tetrads Pollen

M. liliiflora Desr. 12.9%* (101**) 15.6% (45) Not count 51.0% (127) M. cylindrica E. H. Wilson 14.0% (114) 20.0% (61) Not count 6.0% (276) M. acuminata L. 5.0% (171) 10.1% (128) 2% (138) 13.0% (300) M. acuminata L. var. subcordata (Spach) Dandy 14.0% (105) Not count Not count 11.0% (300)

*Mean frequency of abnormal phenomena in PMCs observed. **The number of PMCs observed.

II+1.41IV+0.27VI (Table 2). The behavior of chromo- 138 pollen grains of M. acuminata are of apparent good somes at AI, MII and AII is basically normal. Its chro- quality compared to only 49% of 127 in M. liliiflora, mosome configuration and performance during meiosis which supports the better reproductive capability of M. are totally the same as those of M. liliiflora. These facts acuminata. support the view above not to recognize M. polytepala as a separate species. M. acuminata L. var. subcordata (Spach) Dandy The chromosome behavior is similar to M. acu- Magnolia acuminata L. minata. During prophase I, especially at diplotene M. acuminata ‘Kenneth’s Delight’ and M. acuminata and diakinesis, several tetravalents and one hexavalent ‘North Gold’ are used in this study (Table 1). They show could be observed (Fig. 3I). Up to seven tetravalents in the same chromosome behavior during meiosis. M. acu- one PMC existed during early MI of which 4 were of minata is a tetraploid, 2n=4x=76, in agreement with hetero-type (Fig. 3J). The chromosome configuration previous studies (Whitaker 1933). is 2n=34.04II+1.77IV+0.14VI based on 66 PMCs at The chromosome behavior in PMCs is similar to that MI (Table 2). 14% of 105 PMCs during AI had lagging of M. liliiflora. Tetravalents can be clearly observed in chromosomes, chromosome bridges, and fragments. the zygotene stage (Fig. 3A). In diakinesis, ring bivalents Similar abnormal phenomena were also found in AII. and tetravalents are frequently seen (Fig. 3B and C), but only bivalents are seen in later MI. Bivalents dominate Magnolia acuminata×M. liliiflora (M.×brooklynensis during MI, though one to four ring or chain tetravalents ‘Woodsman’) and one hetero-hexavalents were observed (Fig. 3D and The chromosome configuration is nearly the same as E). The chromosome configuration of 56 PMCs at MI that of its parents. And also similar to its parents, one was 2n=33.84II+1.63IV+0.30VI (Table 2). Five tetra- chain hexavalent (Fig. 3K) was found in addition to sev- valents at most could be observed within one PMC (Fig. eral tetravalents during diakinesis. At MI, seven tetrava- 3D). Interlocked bivalents were found, which might give lents at most were observed in one PMC, of which four rise to inversions or translocations and lead to the ap- were hetero-type (Fig. 3L). A chain hexavalent, possibly pearance of chromosome fragments in AI. involving translocation chromosomes, was also recorded The figure of AI and AII show that the chromosome in this hybrid. number is n=2x=38 (Fig. 3F and G). Lagging chromo- This means the four chromosome sets of the genomes somes can be observed in 5% of the 171 PMCs at AI and of M. liliiflora and M. acuminata have not only the same 10.1% of the 128 PMCs at AII (Fig. 3G). 128 cells were type of polyploid origin, but also homologous chromo- observed at AII, 10% of which possessed lagging chro- somes. But there are still some differences in the chro- mosomes or microkernels. M. acuminata had only two mosome structures such as more ring bivalents at diaki- kinds of tetrads: decussate and isobilateral. Also 98% of nesis in M. acuminata than in M. liliiflora. 200 Y. L. Wang et al. Cytologia 81(2)

Fig. 2. Meiosis of tetraploids of subgenus Yulania in China. A, MI of M. liliiflora var. gracilis, arrows showing four tetravalents. B, MI of M. polytepala, thin arrow showing hexavalent and thick arrow showing chain tetravalent. C, MI of M. polytepala, arrow showing hexavalent. D, MI of M. polytepala, arrows showing four tetravalents. E, Pachytene of M. cylindrica, thin arrow showing chromonema without synapsis. F, MI of M. cylindrica, showing thirty-eight rod bivalents. G, MI of M. cylindrica, thick arrow showing one tetravalent and thin arrows showing two trivalents. H, MI of M. cylindrica, showing more and more bivalents separating from the early to late metaphase. I, MI of M. concinna, showing thirty-eight rod bivalents. J, MI of M. concinna, arrow showing tetravalent. K, AI of M. concinna, showing lagging chromosome. L, AII of M. concinna, arrows showing lagging chromosomes or chromosome fragments.

Magnolia cylindrica E. H. Wilson was collected (Rehder and Wilson 1927). The chromosome behavior of four specimens of M. The chromosome number is 2n=4x=76 in a number cylindrica of different origins are almost the same. The of provenances and clones, in agreement with previous following results are mainly based on our observation studies (Parris et al. 2010). of the sample in Xi’an Botanical Garden, which is from Both the chromosome behavior and the tetrad type Mount Huangshan in Anhui, where the type specimen differ very much from those of M. liliiflora. The homolo- 2016 Meiosis of Tetraploid Magnolia 201

Fig. 3. Meiosis of tetraploids of subgenus Yulania in North America. A, Zygotene of M. acuminata, arrow showing mutivalent. B, Diakinesis of M. acuminata, arrows showing six tetravalents. C, MI of M. acuminata, thick arrow showing spoon-like hexavalent and thin arrow showing allotype tetravalent. D, MI of M. acuminata, arrows showing five tetravalents. E, MI of M. acuminata, thin arrow showing hexavalent and thick arrows showing two tetravalents. F, AI of M. acuminata, showing chromosome number. G, AII of M. acuminata, arrows showing some lagging chromosomes. H, Pollen of M. acuminata. I, MI of M. acuminata var. subcordata, arrow showing allotype hexavalent. J, MI of M. acuminata var. sub- cordata, arrows showing seven tetravalents. K, Diakinesis of M.×brooklynensis, thick arrow showing chain hexavalent and thin arrows showing two tetravalents. L, MI of M.×brooklynensis, arrows showing seven tetravalents. gous chromosomes are paired to bivalents, dispersing tene (Fig. 2E). Bivalents line up along the equatorial along the edge of PMCs. Chromonemata without syn- plate normally in the early phase of MI, while some of apsis were partly entangled in the middle of the PMCs. them separate early in late MI (Fig. 2H). Heteropyknotic This pattern indicates non-simultaneous chromosome chromosomes were observed in a few PMCs of MI, synapsis in some PMCs. It was observed that the ends of which leads to abortion microspores. A solitary ring some homologous chromosomes did not pair in pachy- bivalent and 37 rod bivalents are observed in PMCs of 202 Y. L. Wang et al. Cytologia 81(2)

MI. the chromosomes at MI appear as bivalents and the lim- The chromosome configuration showed more regular ited number of multivalents might reflect the incomplete pairing patterns than those of M.liliiflora and M. acu- cytological diploidization of these tetraploids, irrespec- minata: 2n=2.00I+35.50II+0.60III+0.30IV (Table 2). tive of their exact mode of origin. Though bivalents were the dominating type, one hetero- Based on all the results above, the origin mode of tetravalent could occasionally be observed, and no the species of the M. liliiflora group, are inferred as hexavalent ever existed (Fig. 2F, G). At the AI and AII likely autotetraploids, but the homologous chromosomes stages, abnormal behaviors, like lagging chromosomes, among the four chromosome sets in the tetraploids bridges and fragments, were observed in a rate of 14% have undergone obvious changes in their structures, in AI and 20% in AII (Table 3). Few abortion tetrads and as heteromorphic multivalents, chromosome bridges pollen grains were seen. and fragments appeared during meiosis. The hetero- or homo- hexavalents and tetravalents suggest these taxa Magnolia concinna Law et R. Z. Zhou as translocation heterozygotes. The chromosome bridges The chromosome number of the somatic cells was and fragments in AI point to the existence of inversion found to be 2n=76, and its chromosome configuration in chromosomes. meiosis is similar to M. cylindrica, 2n=37.66II+0.17IV The second kind of chromosome configuration was (Table 2). Though bivalents are still the dominating found in the taxa of the second group comprising M. type, tetravalents are occasionally observed (Fig. 2I and cylindrica and M. concinna (called the “M. cylindrica J). At AI and AII, lagging chromosomes existed in about group” below), whose cytology differs greatly from the 50% of the cells (Fig. 2K and L). These facts support our taxa of the first group described above. The latter group view above not to recognize M. concinna as a separate shows dominating bivalents with very rare tetravalents species but treat it as an ecotype of M. cylindrica. and trivalents; synapsis chromosomes pairing loosely in pachytene and MI are observed and a great quantity of Discussion lagging chromosomes in AI and AII. The chromosome configuration indicates that the two taxa are possibly al- Cytological characters of tetraploid species in subgenus lotetraploids, most likely hybrids between hexaploid and Yulania diploid parents, as deduced from trivalents observed at Two types of chromosome configurations during some rate, loosening synapsis chromosomes in pachy- meiosis were observed in the tetraploids species of tene and early separating chromosomes. Magnolia subgenus Yulania. The first type includes five The possible hybrid origin of these tetraploids may taxa, M. liliiflora, M. liliiflora var. gracilis, M. polyte- contribute to a faster process of cytological diploidiza- pala, M. acuminata and M. acuminata var. subcordata tion, and thus fewer multivalents in meiosis. Genetic (called the “M. liliiflora group” below). The cytological regulation of meiosis in tetraploids should also be con- characteristics can be summarized as follows: Bivalents sidered (Cifuentes et al. 2010). were found to dominate while some tetravalents and Since the current results are only based on cytology, hexavalents existed at early MI, but multivalents were the interpretation is as above until further evidence is seldom found in late MI. Except for seven tetravalents made available by the application of alternative methods found in one PMC of M. acuminata var. subcordata, all such as genomic in situ hybridization (GISH). the species have five tetravalents at most, part of which are asymmetrical hetero-tetravalents; the very similar Translocation heterozygotes chromosome configurations in meiosis show the stable Except for the M. cylindrica group, one or very nature in the chromosome behavior of this first group rarely two ring hexavalents were seen in the PMCs of of species; the existence of ring hexavalents reveals the the M.liliiflora group. More pairing chromosomes were possible homoeology among 19 chromosomes of the ge- observed than expected, indicating translocation be- nome originating possibly partly from similar genomes; tween non-homologous chromosomes. Nevertheless, no they are all translocation heterozygotes, perhaps since a homozygote plants were found in the study. This begs limited number of clones are investigated. the question whether the frequency of homozygotes is As a comparison, Magnolia kobus ‘Norman Gould’ is very low due to translocation heterozygotes being more an artificial autotetraploid created by colchicine treat- competitive. This argument is consistent with reported ment (Janaki Ammal 1952). The chromosome configura- evidence obtained from diploid Oenothera biennis L. tion at MI is 2n=5.30I+28.54II+2.06III+1.86IV. A few and tetraploid Haworthia reinwardtii var. chalumnensis tetravalents can be observed in diakinesis and early MI G. G. Smith. In the latter taxon, the frequency of translo- (Zhang, S. Z., unpublished results). Since homologous cations can exceed 99% of the individuals per population chromosomes are often given priority in PMCs, the rate (Brandham 1974, Levy and Levin 1975). These patterns of the formation of multivalent pairing will be reduced are rather similar to our observations on M. liliiflora. a lot in nature in comparison to theory. The majority of Moreover, we noticed that multivalents were observed 2016 Meiosis of Tetraploid Magnolia 203 in almost all PMC at diakinesis and early MI in the tet- and middle altitude which has made the human impact raploids investigated. These multivalents were kept sepa- even more pronounced than for several other Magno- rated and turned into normal bivalents in MI because lia species. Thus populations were rapidly destroyed of no chiasmata and no matching of the chromosomal and may have been extinct already in the wild before it termini. The lack of cross over hold the chromosomes reached a stabilized genome (Parisod et al. 2010). together in ring multivalents, which in turn is helpful M. liliiflora, which is perhaps the earliest ornamental in preserving the translocation structure of the chromo- plant in the Magnoliaceae (with the possible exception somes. It is reported that heterozygote individuals in of M. denudata), has been cultivated for more than 2500 Oenothera can have high genetical variation and pro- years (Law et al. 1990). During this time its structural duce apparent variations without hybridization (Brand- variations in the chromosomes have been preserved due ham 1974). This may explain the wide degree of poly- to substantial vegetative propagation, such as artificial morphism (i.e., growth habit, flower color and shape) in cutting, grafting and plant division. M. liliiflora should both M. liliiflora and M. acuminata. be seen as species of a late speciation and further evolu- tion with a complicated genome restructuring. Genomic Speciation of M. liliiflora stabilization has not been effective during this phase of Autopolyploidy can be caused by unreduced, diploid decline. The possibility of repeated polyploidisation in gamete production, which can be as high as 4% in some different populations must also be considered (Soltis and angiosperm species (Ballington and Galletta 1976, Ma- Soltis 2000). ceira et al. 1992). A good example is M. pseudokobus In the results from DNA sequencing studies, no exist- Abe & Akasawa, which is a spontaneous triploid cyto- ing diploids, such as M. kobus D.C. or M. biondii Pamp., type of wild Japanese origin (Ueda 1986). Also, a trip- are close to M. liliiflora. But it has a very close relation- loid Michelia was obtained through artificial pollination ship with M. denudata Desr. and M. cylindrica (Qiu et (Wang et al. 2006). The fusing of unreduced gametes al. 1995, Azuma et al. 1999, Kim et al. 2001, Wang et may be the origin of the tetraploid M. liliiflora. In addi- al. 2002, Nie et al. 2008), and it should be noted that tion to production of unreduced gametes, autopolyploids both M. denudata and M. liliiflora are polyploids. Maybe can also arise as a result of somatic chromosome dupli- they share the same extinct diploid parent from earlier cation. periods of evolution. A polyploid offspring could have Reduced fertility of nascent polyploids is mostly due found a niche in the competition with the diploid parent to meiotic irregularities. Complicated configurations, at first. But during colder climatic periods, the diploid caused by polyploidization and inversions or transloca- parent may not have been able to adapt to the cold as tions, prevent it from stabilizing. Translocation chromo- well as the polypoid individuals (=polyploid advan- somes also severely affect the fertility of microspores tage) and thus became extinct. Tetraploid and hexaploid of M. liliiflora. However, the absence of some chro- populations may show a higher survival probability and mosome segments can be compensated or substituted disperse in high altitude mountains (Parisod et al. 2010, by other relevant normal chromosomes in polyploids. te Beest et al. 2012). Similar conditions have been dis- Even though there were no more abnormal divisions cussed for M. sprengeri by Kang and Ejder (2011). observed at the microspore mother cell meiosis stage than in the case of M. accuminata, M. acuminata var. Relationship of M. liliiflora and M. acuminata subcordata and M. cylindrica, the most serious abortion Subgenus Yulania has a disjunct distribution in east- of M. liliiflora should happen at the mononuclear and ern Asia and North America. Studies of fossils, palynol- late binuclear pollen stages. Only 49% normal pollen ogy and geography show that its present geographical can be observed finally, much lower than for other spe- distribution was formed by the end of the Miocene cies (Table 3). The same phenomena were reported for (Hebda and Irving 2004). M. acuminata is distributed autotetraploid rice pollen (Dai et al. 2006). It has been disjunctively from other species of subgenus Yulania, inferred that an insufficiency of nutrition is the reason but have homologous chromosomes in its four sets of in addition to the abnormal division of chromosome at genomes and likely originated in the same way as M. meiosis (Peng et al. 2003, Wang and Zhang 2008). liliiflora, an autotetraploid. The chromosome configura- The great number of pistils and stamens is another tions in meiosis of these two species show a remarkable means of making up for the high proportion of infertile level of agreement in the nature of the chromosome pollen and ovules. M. liliiflora may still exist as small behavior. M. liliiflora and M. acuminata originated from and isolated populations in low altitude forests (600– different diploid progenitors or, alternatively, they could 800 m). But the impaired fertility would be expected to have emerged from the same precursor diploid parent, limit expansion of populations of M. liliiflora. In addi- but then their subsequent allopatric separation beginning tion, forest and consistent human dig- in the Oligocene (Azuma et al. 2011, Hebda and Irving ging of plants likely increased the threat of local extinc- 2004, Nie et al. 2008) would account for their contrast- tion. This species seems to be naturally restricted to low ing morphologies. 204 Y. L. Wang et al. Cytologia 81(2)

Although both seem to be autotetraploids, there are it is indeed striking to find that M. cylindrica shows a still some differences in the chromosome structures such discordance between nuclear and cytoplasmic data. The as more ring bivalents at diakinesis in M. acuminata most recent cpDNA-based molecular phylogeny shows than in M. liliiflora. The tectate-perforate pollen in the M. cylindrica as most closely related to M. denudata exine structure of M. acuminata (Fig. 3H) is also further and M. liliiflora (Azuma et al. 2011). The most recent in evolution than the tectate-imperforate pollen of M. corresponding phylogeny based on nuclear DNA (Nie liliiflora (Fig. 1H) (Walker 1974). M. acuminata has a et al. 2008) has M. cylindrica as most closely related better fertility which enables it to be widely distributed, to a M. sprengeri clone and rather far removed from M. from the NE to the SE of USA, although never abundant denudata. The diploids M. kobus, M. salicifolia and M. (Smith 1990). The fast growth makes it competitive with zenii are all closer to M. sprengeri than M. denudata is other , without the risk of getting out-competed like (maximum likelihood tree only available). The position M. liliiflora. Although autotetraploids have reproductive of M. cylindrica has not been commented on in these problems because of the abnormal meiosis, fertility can two investigations, but while these sequencings are not be improved after subsequent genome evolution (Parisod ideal for high resolution at species level, they may give et al. 2010). M. acuminata should have undergone a lon- some support to our suggested speciation process for M. ger evolution period and be more stable in chromosome cylindrica. It should be noted that M. denudata and M. structure than M. liliiflora. cylindrica have several overlapping populations in east- Compared to M. acuminata, the polyploidisation and ern China, such as in Zhejiang and Jiangxi Provinces. speciation of M. liliiflora may be a relatively recently The hexaploid parent could be M. denudata, and the event. Nie et al. (2008) put forward that M. acuminata diploid parent might have been extinct during evolution. is the basal group of subgenus Yulania and Michelia based on the nuclear sequence data. The present study Acknowledgements shows that M. acuminata is more closely related to M. liliiflora and other species of subgenus Yulania than it is This research was supported by Special Fund for to Michelia (Zhang et al. 2011), and this is supported by Forest Scientific Research in the Public Welfare of results of cpDNA studies (Qiu et al. 1995, Azuma et al. China (201504322), Science and Technology Project of 1999, Kim et al. 2001, Wang et al. 2002). Shaanxi Province Academy (2014k-04), and National Natural Science Foundation of China (30970180). Our Speciation of M. cylindrica thanks go to Prof. Bengt Bentzer and Prof. Waheeb Allopolyploids are likely to have more capacity Heneen for assistance in funding application and to the for novel evolution than autopolyploids (Madlung and latter and Richard Figlar for important comments on the Wendel 2013). In fact, large wild populations of M. cy- manuscript. Thanks to Dr. K. E. Flinck for generously lindrica can often be found in high altitude mountains supplying Magnolias buds. Thanks to Prof. Waheeb He- (900–1900 m) of eastern China. It is most likely a hybrid neen and Kerstin Brismar of the Swedish University of between hexaploid and diploid parents, deduced from Agricultural Sciences for assistance in the pollen exine trivalents observed in some rate. 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