Decaploidy in Rosa praelucens Byhouwer (Rosaceae) Endemic to Zhongdian Plateau, Yunnan, China

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1 CARYOLOGIA Vol. 63, no. 2: , 2010 Decaploidy in Rosa praelucens Byhouwer (Rosaceae) Endemic to Zhongdian Plateau, Yunnan, China Hongying Jian, Hao Zhang, Kaixue Tang*, Shufa Li, Qigang Wang, Ting Zhang, Xianqin Qiu and Huijun Yan Key Laboratory for Flower Breeding of Yunnan Province, Flower Research Institute, Yunnan Academy of Agricultural Sciences, Taoyuan Village, Longtou Street, Kunming, Yunnan, , China. Abstract Genus Rosa (Rosaceae) has a base chromosome number of x = 7. Ploidy levels of wild species range from 2n = 2x = 14 to 2n = 8x = 56. In this paper, one decaploid, the highest naturally occurring ploidy never reported before in the genus was detected in the karyological study of an endemic wild species, R. praelucens Byhouwer to Zhongdian Plateau, Yunnan, China. Its karyotype formula was 2n = 10x = 70 = 2st + 6sm + 62m and the karyotype was 2B. Chromosome No. 6 was subterminal. Chromosome No. 15, No. 22 and No. 23 were submedian. According to its karyomorphology, R. praelucens derived not from auto polyploidization directly but from inter-specific hybridization. Polyploidization and inter-species hybridization both might have played important roles in the origin of R. praelucens associated with effect of high altitude environment. Further research on this species is warranted to provide helpful evidences to understand the differentiation and evolution of the Genus Rosa, and even clues to trace the process of speciation in Sino-Himalayan region. Key words: decaploid, endemic, hybridization, karyotype, polyploidy, Rosa praelucens Byhouwer. INTRODUCTION The genus Rosa contains about 200 species that are widely distributed throughout the Northern Hemisphere (KU and ROBERTSON 2003; WISSEMANN and RITZ 2005). It is among the first genera of garden flowers to attract the attention of cytologists because of the importance of cultivated roses as horticultural plants, in spite of the small size of its chromosomes (ROWLEY 1967). In order to clarify the genomic relationships, cytogeneticists and plant breeders have analyzed both somatic and meiotic metaphase chromosomes. Cytological reports in rose included chromosome counts, karyotypes and meiotic configuration frequencies by means of traditional squashing and pressing (LIU et al. 1985; MA et al. 1991, 1992; MA et al. 1997), C- banding (PRICE et al. 1981) and FISH (MA et al. *Corresponding author: kxtang@hotmail.com 1997; FERNANDEZ-ROMERO et al. 2001; AKASAKA et al. 2002; 2003). On the basis of both arm ratio and chromosome length the authors concluded that karyotypes were largely symmetric and that genomic uniformity existed across the genus. Genus Rosa exhibits a typical polyploidy series with a basic chromosome number of 7. According to the reported chromosome numbers, euploid predominated with range from 2n = 2x = 14 to 2n = 8x = 56 (DARLINGTON et al. 1955). The reported hexaploid R. sweginzowii Koehne was the known highest ploidy in China (TÄCKHOLM 1922; ROBERTS et al. 2009). R. praelucens Byhouwer, first nominated by BYHOUWER (1929), was collected from Zhongdian Plateau, Yunnan, China by Forrest in It has been included in Section Microphyllae, as it agreed in the following characters: styles free and short, stipules adnate and not laciniate, flowers solitary, receptaculum armed and depressed-globose (BYHOUWER 1929). Owing to its large and showy flower with diameter to 15 cm and pink flower color when full blooming, it arose much interest of horticulturist (LI et al. 2005; BAI et

2 DECAPLOIDY IN ROSA PRAELUCENS BYHOUWER (ROSACEAE) 163 al. 2008). However, it is endemic to Zhongdian Plateau, scattered in the highly exploited pastures and remains very few in number now according to our field survey. Zhongdian Plateau lies in the south-eastern edge of Himalaya range, with an average altitude of 3400 m. 14 species of the genus Rosa were recorded in this area (ZHOU et al. 2006). According to the published cytological data (Table 1), species in Section Pimpinellifoliae and Synstylae are all diploid with 14 chromosomes, while the chromosome of species in Section Cinnamomeae varies from diploid to hexaploid. During our comprehensive study on wild and cultivated Rose of Yunnan, China, the special karyology of R. praelucens was studied and reported here. MATERIALS AND METHODS The R. praelucens plants are grown in the rose germplasm base of Flower Research Institute, Yunnan Academy of Agricultural Sciences, China, which is located in the northern suburb of Kunming, Yunnan, with an altitude of 1900m. The plants were grafted from buds of wild R. praelucens plants, which were located at N 27 o and E 99 o , with an altitude of 3231m, Zhongdian, Yunnan. Shoot tips were collected from the plants showing vigorous growth, pretreated in a saturated dichlorobenzene solution for 2.5 hr at 23 C, then fixed with Carnoy (1:3 glacial acetic acid/absolute alcohol) at 4 C for 40 min. The fixed tips were hydrolyzed in 1N HCl at 60 C for 10 min, stained with carbol fuchsin for at least 30 min and squashed on glass slides for observation and photos were taken under a Nikon E800 microscope (NIKON, JAPAN). Observations were made on nuclei at the somatic mitotic interphase and metaphase, and measurements of chromosome arms were taken from at least ten well-spread metaphases of five shoot tips. The karyomorphological classification of the interphase and mitotic prophase chromosomes follows TANAKA (1971; 1977); the designation of the centromeric position as median (m), submedian (sm), and subterminal (st) was that of LEVAN et al. (1964). The symmetry of karyotype was classified according to STEBBINS (1971). AS.K% was calculated according to that of ARANO (1963). RESULTS The interphase nuclei and different cell mitosis stages of Rosa praelucens were shown in Fig 1. The interphase nucleus (Fig. 1A) belonged to the complex chromocenter type (TANAKA 1971; 1977) because they exhibited many darkly stained chromocenters with an irregularly protruding rough surface that gradually transformed into diffuse chromatin. The mitotic prophase chromosomes of this species (Fig. 1B), with very distinguishable heterochromatic and euchromatic segments and the heterochromatic segments distributing in the interstitial and proximal regions, were of interstitial type (TANAKA 1977). The metaphase separation (Fig. 1C, Fig. 2) of R.praelucens clearly showed that it had 70 chromosomes. The characteristics of the 35 pairs of chromosomes were shown in Fig. 3 and listed in Table 2. The karyotype formula of R. praelucens was 2n = 10x = 70 = 2st + 6sm + 62m. Chromosome No. 6 was very significantly subterminal (st), with an arm ratio of Chromosome No. 15, No. 22 and No. 23 were submedian chromosomes (sm) with the arm ratio of 1.75, 1.77 and 1.73, respectively. Except these 4 pairs, the others were all median chromosomes (m). The average arm ration of the whole genome is The AS. K% of this species was 58.53%. The ratio of relative length of longest chromosome to that of the shortest one was 2.14, and 2.86% Fig. 1 The interphase nuclei and different cell mitosis stages of Rosa praelucens. (A) Interphase nuclei. (B) Prophase. (C) Metaphase. (D) Anaphase. Bar=20µm.

3 164 JIAN, ZHANG, TANG, LI, WANG, ZHANG, QIU and YAN TABLE 1 Rosa species distributed in Zhongdian Plateau and the related cytological data. All are based on x=7. Species Section Reference 2n Ploidy Altitude Habitat R. omeiensis Rolfe Pimpinellifoliae TÄCKHOLM x Slopes, scrub and stream sides R. sericea Lindl. Pimpinellifoliae TÄCKHOLM x Sunny slopes R. mairei Lévl. Pimpinellifoliae No report Sunny slopes and scrubs R. sikangensis Yü et Ku Pimpinellifoliae No report Stream sides, road sides and scrubs R. graciliflora Rehd. et Wils. Pimpinellifoliae No report Scree Slopes, Scrubs R.soulieana Crép. Synstylae HURST x Picea forests and stream sides R.brunonii Lindl. Synstylae HURST x Forests and scrubs R. helenae Rehd. et Wils. Synstylae TÄCKHOLM x Stream sides, road sides and scrubs R. prattii Hemsl. Cinnamomeae DARLINGTON and WYLIE x Slopes, stream sides and scrubs R. willmottiae Hemsl. Cinnamomeae TÄCKHOLM x Slopes and scrubs R. moyesii Hemsl. et Wils. Cinnamomeae TÄCKHOLM x Dry slopes, pine forests and scrubs R. macrophylla Lindl. Cinnamomeae HURST , 28 2x, 4x Pine forest edges, scrubs and road sides R. sweginzowii Koehne Cinnamomeae TÄCKHOLM 1922; ROBERTS, GLADIS, BRUMME x Pine forest edges, scrubs and road sides R. praelucens Byhouwer Microphyllae No report Sunny slopes and scrubs chromosomes had an arm ratio of more than According to the classification of STEBBINS (1971), the karyotype of R. praelucens belonged to type 2B. DISCUSSION Polyploidy has long been recognized as a prominent force in evolutionary diversification and is one of the most important cytogenetic mechanisms in plant evolution and rapid speciation (STEBBINS 1971; GRANT 1981; LEVIN 2002). Alpine and arctic floras have been reported to have high frequencies of polyploidy (OHBA 1988; ABBOTT et al. 2003; BROCHMANN et al. 2004) and it had been known since Täckholm s work that species or forms in Rosa with the more arctic or alpine distribution were the higher polyploids (DARLINGTON 1942). Before, the highest chromosome number once found was in Rosa acicularis from Europe and Asia with 2n = 8x = 56(LEWIS 1959). Our report is the first of a naturally occurred decaploid rose species, Rosa praelucens, a rare species endemic to Zhongdian Plateau. Zhongdian Plateau, with an average altitude of 3400, the longitude from 99 o 23 to 100 o 3l E, latitude from 26 o 25 to 28 o 45 N, lies in the southeastern Himalayan range which is now a hot spot both for its biodiversity and for its role in chromosome evolution(boufford et al. 2000; NIE et al. 2005; CHEN et al. 2007). In this small area, rose species exhibit highly diverse ploidy level from 2x, 4x, 6x and now 10x, indicating that the extreme environment associating with the high altitude promotes the polyploidy in genus Rosa. LEVIN (2002) suggested the capacity of angiosperms forming new combinations via polyploidy may help to explain high incidence of endemism in a given region. The endemism of R. praelucens may be caused by its high ploidy level. It is well-known that interspecific hybridization is common in genus Rosa, even as a major evolutionary factor in the genus (WISSEMANN et al. 2007). According to the karyomorphology of R. praelucens,it was a significantly allopolyploid, although it still needs proof from meiosis data. Its karyotype was 2B, which was rather asymmetric compared to the other reported species in the genus, most of which was very symmetric (CRANE et al. 2003). So, it suggested that R. praelucens was a naturally occurred inter-specific descendent which was a somewhat advanced species in the genus as far as chromosome was concerned. Be-

4 DECAPLOIDY IN ROSA PRAELUCENS BYHOUWER (ROSACEAE) 165 Fig. 2 Metaphase chromosomes of Rosa praelucens. (a) Metaphase chromosomes. (b) Karyotype. Bar=20µm. Fig. 3 Karyotype of R. praelucens. Bar=5µm. cause aneuploids and the odd polyploids, except for special 5x Caninae, do not occur in the wild (ROWLEY 1967), the decaploidy of R. praelucens suggest that it may not have derived from autopolyploidization directly, but from inter-specific hybridization, during which the unreduced 2n gametes, which are common in Section Caninae (WERLEMARK et al. 1999), progenies of crossing between R. wichuraiana Crép. and R. rugosa Thunb (EL MOKADEM et al. 2002) and cultivated

5 166 JIAN, ZHANG, TANG, LI, WANG, ZHANG, QIU and YAN TABLE 2 The chromosomal characters of Rosa praelucens Byhouwer. No. Relative Length Arm ratio Centromeric index Type No. Relative Length Arm Ratio Centromeric index Type S L T S L T 1 1,74 2,04 3,78 1,17 46,03 m 19 1,26 1,58 2,84 1,25 44,37 m 2 1,47 2,27 3,74 1,58 39,3 m 20 1,26 1,56 2,82 1,24 44,68 m 3 1,61 1,99 3,6 1,24 44,72 m 21 1,15 1,63 2,78 1,41 41,37 m 4 1,52 1,99 3,51 1,31 43,3 m 22 1,15 1,58 2,73 1,37 42,12 m 5 1,5 1,83 3,33 1,22 45,05 m 23 0,98 1,74 2,72 1,77 36,08 sm 6 0,85 2,46 3,31 3,03 25,68 st 24 0,99 1,72 2,72 1,73 36,4 sm 7 1,36 1,85 3,21 1,37 42,37 m 25 1,12 1,59 2,71 1,42 41,33 m 8 1,34 1,85 3,19 1,38 42,01 m 26 1,03 1,63 2,66 1,58 38,72 m 9 1,29 1,86 3,16 1,44 40,82 m 27 1,06 1,61 2,67 1,53 39,7 m 10 1,5 1,66 3,16 1,11 47,47 m 28 0,98 1,63 2,61 1,66 37,55 m 11 1,2 1,91 3,11 1,59 38,59 m 29 1,09 1,39 2,48 1,3 43,95 m 12 1,22 1,8 3,02 1,48 40,4 m 30 0,99 1,39 2,38 1,4 41,6 m 13 1,23 1,74 2,97 1,41 41,41 m 31 0,98 1,37 2,35 1,4 41,7 m 14 1,26 1,66 2,92 1,31 43,15 m 32 0,96 1,23 2,19 1,28 43,84 m 15 1,06 1,85 2,91 1,75 36,43 sm 33 0,92 1,18 2,1 1,29 43,81 m 16 1,2 1,69 2,89 1,41 41,52 m 34 0,93 1,04 1,97 1,12 47,21 m 17 1,2 1,66 2,86 1,4 41,96 m 35 0,82 0,95 1,77 1,15 46,33 m 18 1,23 1,61 2,84 1,31 43,31 m Note: S, length of shorter arm; L, length of longer arm; T, total length roses (CRESPEL et al. 2006), might have played a very important role, just like in another genus of the same rose family, Fragaria, where also a naturally occurring decaploid was newly reported (HUMMER et al ). This unique decaploid of R. praelucens will provide helpful evidences in understanding the differentiation and evolution of Genus Rosa, and even clues to trace the process of speciation in Sino-Himalayan region. Unfortunately, many crucial issues on this species including its reproductive mechanism, meiotic process and phylogenetic relationships with other species were still lacking. Some conservation action is urgent because R. praelucens is endemic to Zhongdian Plateau and there remain very few numbers, threatened by destroyed habitat. Acknowledgements This study was supported by the Provincial Natural Science Foundation of Yunnan Province, China (Grant No. 2007C0003Z and 2008CD183). REFERENCES ABBOTT R.J. and BROCHMANN C., 2003 History and evolution of the arctic flora: in the footsteps of Eric Hulten. Molecular Ecology, 12: AKASAKA M., UEDA Y. and KOBA T., 2002 Karyotype analysis of wild rose species belonging to septets A by fluorescence in situ hybridization. Chromosome Science, 6: AKASAKA M., UEDA Y. and KOBA T., 2003 Karyotype analysis of wild rose species belonging to septets B, C, and D by Molecular cytogenetic method. Breeding Science, 53: ARANO H., 1963 Cytological studies in subfamily Carduoideae (Compositae) of Japan. Botanical Magazine, 76: BAI J.R. and ZHANG Q.X., 2008 Investigation on germplasm resources of Rosa L. in northwest Yunnan (in Chinese with English abstract). Journal of Anhui Agricultural Sciences, 36: BOUFFORD D.E., and VAN DIJK P.P., 2000 South-central China. In R.A. MIT- TERMEIER, N. MYERS and C.G. MIT- TERMEIER (Eds), Hotspots: earth s bio-

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