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1 Journal of Systematics and Evolution 47 (3): (2009) doi: /j x Morphological characteristics of leaf epidermis and size variation of leaf, flower and fruit in different ploidy levels in Buddleja macrostachya (Buddlejaceae) 1,2 Gao CHEN 1 Wei-Bang SUN 3 Hang SUN 1 (Kunming Botanical Garden, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming , China) 2 (Graduate School of Chinese Academy of Sciences, Beijing , China) 3 (Key Laboratory of Biodiversity and Biogeography, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming , China) Abstract Buddleja macrostachya (Buddlejaceae) is a widespread shrub native to the Sino-Himalayan mountains and beyond. It has been found to occur at two ploidy levels, hexaploid, 2n=6x=114 and dodecaploid, 2n=12x=228. To determine if morphological characters might be used as indicators of ploidy levels, we measured floral and fruit length, relative and absolute leaf size, trichome density on both leaf surfaces, and stomatal density and length in different populations of B. macrostachya. In general, flower and fruit length, absolute leaf size, and stomatal length increased with an increase at ploidy level (P<0.01), whereas adaxial cell and stomatal density decreased with an increase at ploidy level (P<0.01). We found no conspicuous differences in relative leaf size (P>0.05) in different populations. Other characters studied such as trichome type, cuticular membrane and ornamentation of stomata, cell and stomatal shape, and anticlinal wall pattern were quite constant in this species. Thus it appears that flower and fruit length, absolute leaf size, and stomatal frequency and length can be used to distinguish hexaploid from dodecaploid cytotypes either in the field or in herbarium specimens. Key words Buddleja macrostachya, dodecaploid, hexaploid, leaf epidermis, ploidy level, stomata. Many species of ferns and flowering plants exist as polyploid cytotypes; this is undoubtedly important in the evolutionary pathways of these taxa (Halverson et al., 2008). In fact, researchers require methods that are simple, reliable and rapid to separate plants from different ploidy levels in their work. Although chromosome counting is exact, it is time consuming. Therefore, attempts have been made to find other methods of ploidy determination. Measurements widely used for the identification of ploidy levels are pollen grain diameter (Pan, 1994), stomatal characters (Evans, 1955; Geok-Yong Tan & Dunn, 1973; Rajendra et al., 1978; Przywara et al., 1988; Sreenivasan et al., 1992; Mishra, 1997; Aryavand et al., 2003), the number of plastids or cell length in guard cells (Bingham, 1968) and DNA content of nuclei (Barker et al., 2001; Kawakami et al., 2007; Halverson et al., 2008). In a review of chromosome numbers in the Asiatic species of Buddleja, several species were determined to have polyploidy cytotypes (Chen et al., 2007). In this study, particular attention was given to Buddleja macrostachya because of the variation in morphology, reproductive system and ploidy level in isolated popula- Received: 1 September 2008 Accepted: 4 December 2008 Author for correspondence. hsun@mail.kib.ac.cn; Tel & Fax: tions. The high number of chromosomes (2n=6x=114 or 2n=12x=228) combined with their small size (less than 2 μm) and the low number of dividing cells that can be obtained from shoot or root tip squashes means a tedious and rather inaccurate procedure to identify the polyploidy level in different populations. The objective of this study was to determine if ploidy level differences in morphological characteristics of leaf epidermis and size variation of leaf, flower and fruit in B. macrostachya populations could be used as accurate, rapid and reliable procedures to identify the level of ploidy of fresh material. 1 Material and methods The details of the plant material (four hexaploid populations and two dodecaploid populations) used for the present study and the ploidy level of these materials are shown in Table 1. To measure the length of the flowers and fruits, 50 randomly selected flowers or fruits from 10 plants, five flowers or fruits per plant, were measured within one population using a metric ruler. Similarly, the length and width of the leaves was measured from 50 randomly selected leaves from 10 plants (five leaves per plant; leaf selections were based on leaf position and

2 232 Journal of Systematics and Evolution Vol. 47 No Table 1 Populations of Buddleja macrostachya with location and voucher data in this study Population Ploidy Altitude (m) Locality Latitude Longitude Voucher XM 6x 1730 Ximeng, Yunnan, China Sun 023 (KUN) BC 6x 1500 Binchuan, Yunnan, China Chen 044 (KUN) DH 6x 1840 Dehong, Yunnan, China Sun 021 (KUN) JS 6x 1340 Jianshui, Yunnan, China Chen 078 (KUN) SM 12x 1430 Simao, Yunnan, China Sun 029 (KUN) DL 12x 1920 Dali, Yunnan, China Chen 045 (KUN) Fig. 1. Comparison of absolute leaf size of hexaploids and dodecaploids of Buddleja macrostachya. The interval between leaf base and leaf apex of individual leaf was divided into 15 equal segments and the width of each segment was measured. Fifty randomly selected mature leaves from 10 plants were measured and the mean leaf width of each segment was calculated to simulate the ideal leaf size of B. macrostachya. a, hexaploid (2n=6x=114); b, dodecaploid (2n=12x=228). Bar = 18 mm. age, and all sampled leaves were homotypic and mature, from the base of the previous year s branches) were measured within one population using the same ruler. To determine absolute leaf size, the interval between the leaf base and leaf apex was divided into 15 equal segments, the width of each segment was measured, and 50 randomly selected mature leaves from 10 plants per population were used (Fig. 1). The relative leaf size of populations of B. macrostachya of different ploidy levels was calculated according to the following equation: R i =W i /W max, where R i is the ratio, W i is the width of each segment in an individual leaf (the leaf was divided into 15 segments), and W max is the maximum width of the leaf (Fig. 2). Samples of B. macrostachya were taken from fresh material planted in the Botanical Garden, Kunming Institute of Botany, Chinese Academy of Sciences (Kunming, China). The material for light microscopy was boiled in water before being macerated in Jeffrey s solution (Stace, 1965). The macerated leaf epidermis was stained with safranin alcohol (50%), then dehydrated in an ethanol series before being mounted in Canada balsam. To check the constancy of the epidermal structure, at least three slides were made from a single leaf and this was replicated three times with other leaves from the population. To determine trichome density, minute pieces of leaf tissue were attached to stubs for study with a scanning electron microscope. After gold sputtering, the specimens were examined and photographed under a Hitachi S-520 scanning electron Fig. 2. Comparison of relative leaf size of hexaploids and dodecaploids of Buddleja macrostachya. The interval between leaf base and leaf apex of individual leaf was divided into 15 equal segments, and the width of each segment and the maximum width of the leaf were measured. The relative leaf size was calculated according to the equation: R i =W i /W max,wherer i is the ratio, W i is the width of each segment in an individual leaf, and W max is the maximum width of the leaf. The average R i value of 50 randomly selected mature leaves from 10 plants were measured to simulate the ideal leaf shape of B. macrostachya. a, hexaploid (2n=6x=114); b, dodecaploid (2n=12x=228). microscope (Tokyo, Japan). Stomatal terminology is based on the classification proposed by Baranova (1972) and that for other characters is based on the classification of Wilkinson (1979). Trichome, cell and stomatal density per field (416 μm 312 μm) were determined based on the three slides made from a single leaf and replicated for three leaves per population. Measurements of stomatal

3 CHEN et al.: Morphology of Buddleja macrostachya 233 Table 2 Morphological characters of leaf epidermis and size variation of leaf, flower and fruit in different ploidy levels of Buddleja macrostachya Character B. macrostachya (hexaploid) B. macrostachya (dodecaploid) Significance Figure Length of single flower (mm) ( ) ( ) Not shown Length of fruit (mm) 8.74 ( ) ( ) Not shown Length of leaf (cm) 19.6 ( ) 27.6 ( ) Not shown Width of leaf (cm) 6.19 ( ) 9.54 ( ) Not shown Leaf width/leaf length 0.31 ( ) 0.34 ( ) Not shown Absolute leaf size NA NA Relative leaf size NA NA Trichome density of Ab (mm 2 ) 241 ( ) 144 ( ) Not shown Trichome density of Ad (mm 2 ) 72 (59 83) 67 (61 74) Not shown Cell density of Ad (mm 2 ) 638 ( ) 544 ( ) Not shown Stomatal density of Ab (mm 2 ) 440 ( ) 276 ( ) Not shown Stomatal length of Ab ( ) ( ) Not shown Trichome type Gl + Can Gl + Can NA 5, 8 Cell shape of Ab Irregular Irregular NA 10, 12 Cell shape of Ad Polygonal Polygonal NA 9, 11 Anticlinal cell wall pattern of Ab Sinuous Sinuous NA 10, 12 Anticlinal cell wall pattern of Ad Straight Straight NA 9, 11 Stomatal shape of Ab Elliptical Elliptical NA 4, 7 Ab, abaxial epidermis; Ad, adaxial epidermis; Can, candelabra hair; Gl, glandular hair; NA, not applicable. P<0.05 and P<0.01 mean significant at the 0.05 and 0.01 probability levels, respectively. Data of differences from statistical software SPSS version 13.0 (SPSS, Chicago, IL, USA). length were obtained using an Olympus light microscope (Tokyo, Japan) equipped with an ocular micrometer. The average size of the stomata was established on the basis of 25 measurements per sample. 2 Results and Discussion The length of the flower and fruit and the absolute leaf size show significant differences between the hexaploid and dodecaploid plants of B. macrostachya (Table 2, P<0.05). The mean length of a flower for the hexaploid is mm, with a range of mm, and for the dodecaploid is mm, with a range of mm. The mean length of a fruit for hexaploid is 8.74 mm, with a range of mm, and for the dodecaploid is mm, with a range of mm, for the dodecaploid. The absolute leaf size of the hexaploid and dodecaploid plants of B. macrostachya show very significant differences (P<0.01; Table 2 and Fig. 1). The differences indicate that some traits increase in size with an increase at ploidy level. The mean width : length ratio of the leaf revealed significant differences between hexaploids and dodecaploids (P<0.05); however, the relative leaf size of the hexaploids and dodecaploids of B. macrostachya were not significantly different (P=0.524; Table 2 and Fig. 2), indicating that the leaf width : length ratio is not an accurate means of determining ploidy level. Relative leaf size may be constant and can be used to judge the leaf shape within B. macrostachya (Fig. 2). Leaf epidermal characters are shown in Table 2 and Figs The epidermal cells of B. macrostachya are usually polygonal in the adaxial epidermis (Ad) and irregular in the abaxial epidermis (Ab), with the anticlinal cell walls sinuous on Ab and straight on Ad (Figs. 9 12). The cuticle on the adaxial epidermis forms conspicuous ridges in B. macrostachya (Figs. 3 8). Both leaf surfaces have the same glandular and candelabra trichomes. The stomatal apparatus of the Ad is absent and the stomata of the Ab are elliptic (Figs. 4, 7). The ornamentation of the outer stomatal ledge is smooth and the inner margin of the outer stomatal ledge is nearly smooth (Figs. 4, 7). The above features of leaf epidermis are constant in the two ploidy levels of B. macrostachya populations. To identify if morphological characters in different populations of B. macrostachya show significant differences, the characters of leaf epidermis and size variation of leaf, flower and fruit in six populations were compared according to different paired units (15 pairs, details in Table 3). When the significant differences were identified among populations, the significance between the hexaploid cytotype and the dodecaploid cytotype were compared in this study. The mean trichome density of Ad and Ab show different significance between hexaploid and dodecaploid populations (Table 3), which indicates that trichome density on epidermis might not be used to identify ploidy levels of B. macrostachya. Except for the trichome density on epidermis and the relative leaf size, the other morphological data revealed significant differences between hexaploid and dodecaploid populations (Table 3). Trichome density differed between the two ploidy levels. Furthermore, the abaxial leaf surface always showed more trichomes than the abaxial surface. The mean trichome density of the Ab for the hexaploid is 241/mm 2, with a range of , and for the dodecaploid is 144/mm 2, with a range of The

234 Journal of Systematics and Evolution Vol. 47 No. 3 2009 Figs. 3 8. Characteristics of epidermis of Buddleja macrostachya with a scanning electron microscope. Figs. 3, 6.

The ornamentation of outer stomatal ledge, the inner margin of outer stomatal ledge and the stomatal shape of B. macrostachya on the abaxial epidermis (hexaploid and dodecaploid, respectively); Figs.

4 234 Journal of Systematics and Evolution Vol. 47 No Figs Characteristics of epidermis of Buddleja macrostachya with a scanning electron microscope. Figs. 3, 6. The ridges of the cuticle on the adaxial epidermis (hexaploid and dodecaploid, respectively). Figs. 4, 7. The ornamentation of outer stomatal ledge, the inner margin of outer stomatal ledge and the stomatal shape of B. macrostachya on the abaxial epidermis (hexaploid and dodecaploid, respectively); Figs.5,8.Glandular and candelabra hairs on the abaxial epidermis (hexaploid and dodecaploid, respectively). Figs Characteristics of epidermal cells of Buddleja macrostachya using a light microscope. Figs. 9, 10. Adaxial and abaxial surface of hexaploid (2n=6x=114), respectively. Figs. 11, 12. Adaxial and abaxial surface of dodecaploid (2n=12x=228), respectively. Arrow: trichome base. Scale bars: 50 μm in Figs. 9, 11, and 20 μm in Figs. 10, 12. difference was significant (P<0.01; Table 2). However, the mean trichome density of the Ad for the hexaploid is 72/mm 2, with a range of 59 83, and for the dodecaploid is 67/mm 2, with a range of 61 74, showing no significant difference (P>0.05; Table 2). The mean cell density of the adaxial epidermis for the hexaploid is 638/mm 2, with a range of , and for the dodecaploid is 544/mm 2, with a range of There were significant differences in mean cell density on the adaxial leaf surface (P<0.01; Table 2). Because of the density of the glandular and candelabra hairs, and the crumpled and distorted epidermal cells (Figs. 10, 12), it was difficult to count the number of cells of the Ab, so the stomatal index and cell density of the Ab could not be calculated in this study. A positive relationship was found between ploidy level and stomatal length. On average, the hexaploid had a mean length of μm, significantly shorter (P<0.001) than the dodecaploid (Table 2) with a mean length of μm. It was noted that on examination

5 CHEN et al.: Morphology of Buddleja macrostachya 235 Table 3 Morphological characters of leaf epidermis and size variation of leaf, flower and fruit in different populations of Buddleja macrostachya Character Hex1 Hex1 Hex1 Hex2 Hex2 Hex3 Dod1 Hex1 Hex2 Hex3 Hex4 Hex1 Hex2 Hex3 Hex4 Hex2 Hex3 Hex4 Hex3 Hex4 Hex4 Dod2 Dod1 Dod1 Dod1 Dod1 Dod2 Dod2 Dod2 Dod2 Length of single flower (mm) Length of fruit (mm) Length of leaf (cm) Width of leaf (cm) Absolute leaf size Relative leaf size Trichome density of Ab (mm 2 ) Trichome density of Ad (mm 2 ) Cell density of Ad (mm 2 ) Stomatal density of Ab (mm 2 ) Stomatal length of Ab All plant populations sourced from Yunnan, China. Ab, abaxial epidermis; Ad, adaxial epidermis; Hex, hexaploid; Hex1, XM population (Ximeng locality); Hex2, BC population (Binchuan); Hex3, DH population (Dehong); Hex4, JS population (Jianshui); Dod, dodecaploid; Dod1, SM population (Simao); Dod2, DL population (Dali). P<0.05 and P<0.01 mean significant at these probability levels, respectively. P values of compared paired populations are shown. Data of differences from statistical software SPSS version 13.0 (SPSS, Chicago, IL, USA). of both the hexaploid and dodecaploid populations separately, there were no significant differences (P=0.847 for the hexaploids and P=0.793 for the dodecaploids) among the slides within each ploidy level. Thus there was only between-population, not within-population, variation in stomatal length for both ploidy levels, which indicates that stomatal length can be used to identify ploidy levels of B. macrostachya (Table 3). It was found that an inverse relationship exists between the frequency of stomata and the ploidy level. The hexaploid has a higher density of stomata per mm 2 than dodecaploid, and this difference is statistically significant in B. macrostachya populations (Table 2; P<0.01). Stomatal density varied from 413 to 482 stomata per mm 2 on the Ab of the hexaploid and from 247 to 302 stomata per mm 2 on the Ab of the dodecaploid. Similarly, on examination of both the hexaploid and dodecaploid populations separately, there were no significant differences in stomatal frequency for either ploidy level (P=0.596 for the hexaploid and P=0.703 for the dodecaploid) among the slides, which indicates that stomatal frequency also can be used as a criterion for identifying ploidy levels in B. macrostachya. The high chromosome number (2n=114 or 228) and their small size (less than 2 μm) makes it difficult to count chromosomes in B. macrostachya. Initial studies of the two cytotypes did not reveal significant differences in corolla length or leaf width (Chen et al., 2007); however, much larger samples of floral and fruit length and leaf dimensions and vestiture did reveal that these traits were significantly different in hexaploids and dodecaploids. We suspect that the dosage effect of DNA or C-values (amount of DNA in the unreplicated gametic nucleus) might impact on the epigenetic characters of B. macrostachya). Thus it appears that the macro (flower and fruit length, absolute leaf size) and micro (stomatal length and frequency) morphological characters might be used as indicators of ploidy levels of B. macrostachya both in the field and in herbarium specimens. Our recent work (Chen et al., 2007) indicated a great range of ploidy levels (from 2n=2x to 24x) in Buddleja, making it an ideal model for the investigation of polyploidization and speciation. B. macrostachya is a widely distributed species and presents a series of morphological variations and different ploidy levels in different populations. It is a good subject for investigating cytotypes, phytogeography and vicariance in the Sino-Himalayan region. An accurate, rapid and reliable method to identify the level of ploidy of fresh material of B. macrostachya in the field is just the beginning, and further field observations and molecular data are certainly needed.

6 236 Journal of Systematics and Evolution Vol. 47 No Acknowledgements The authors thank Professor Eliane Norman (Stetson University, DeLand, FL, USA) for her great help in editing the text. This study was supported by grants-in-aid from the National Natural Science Foundation of China (NSFC , ) and the Yunnan Natural Science Foundation (2008CC013). References Aryavand A, Ehdaie B, Tran B, Waines JG Stomatal frequency and size differentiate ploidy levels in Aegilops neglecta. Genetic Resources and Crop Evolution 50: Baranova M Systematic anatomy of the leaf epidermis in the Magnoliaceae and some related families. Taxon 21: Barker RE, Kilgore JA, Cook RL, Garay AE, Warnke SE Use of flow cytometry to determine ploidy level of ryegrass. Seed Science & Technology 29: Bingham ET Stomatal chloroplasts in alfalfa at four ploidy levels. Crop Science 8: Chen G, Sun H, Sun WB Ploidy variation in Buddleja L. (Buddlejaceae) in the Sino-Himalayan region and its biogeographical implications. Botanical Journal of the Linnean Society 154: Evans AM The production and identification of polyploids in red clover, white clover and lucerne. New Phytologist 54: Geok-Yong Tan, Dunn GM Relationship of stomatal length and frequency and pollen-grain diameter to ploidy level in Bromus inermis Leyss. Crop Science 13: Kawakami SM, Kato J, Serizawa S Ploidy chimeras induced in haploid sporophytes of Osmunda claytoniana and Osmunda japonica. Journal of Plant Research 120: Halverson K, Stephen B, Heard SB, John D, Nason JD, Stireman JO Origins, distribution, and local co-occurrence of polyploid cytotypes in Solidago altissima (Asteraceae). American Journal of Botany 95: Mishra MK Stomatal characteristics at different ploidy levels in Coffea L. Annals of Botany 80: Pan JT Phylogeny classification and geographic distribution of Rodgersia Gray. Acta Phytotaxonomica Sinica 92: Przywara L, Pandey KK, Sanders PM Length of stomata as an indicator of ploidy level in Actinidia deliciosa. New Zealand Journal of Botany 26: Rajendra BR, Mujeeb KA, Bates LS Relationships between 2x Hordeum sp., 2xSecale sp. and 2x, 4x,6x, Triticum sp. for stomatal frequency, size and distribution. Environmental and Experimental Botany 18: Stace CA Cuticular studies as an aid to plant taxonomy. Bulletin of the British Museum (Natural History) Botany 4: Sreenivasan MS, Prakash NS, Mishra MK Evaluation of some indirect ploidy indicators in Coffea L. Café Cacao Thé 36: Wilkinson HP The plant surface (mainly leaf). In: Metcalfe CR, Chalk L eds. Anatomy of the dicotyledons. 2nd ed. Oxford: Clarendon Press

Stomatal length and frequency as a measure of ploidy level in black wattle, Acacia mearnsii (de Wild)

Stomatal length and frequency as a measure of ploidy level in black wattle, Acacia mearnsii (de Wild) Blackwell Science, LtdOxford, UKBOJBotanical Journal of the Linnean Society0024-4074The Linnean Society of London, 2003 141 Original Article S. L. BECK ET AL. STOMATAL INDICATORS OF PLOIDY LEVEL IN BLACK