INTRODUCTION

Quercus L. (Fagaceae.) is an evergreen or deciduous, shrub or tree. It is distinguished from other genus members in the Fagaceae for having a pendulous catkin as staminate inflorescence, which stiff, erect female inflorescence of 1-7 or more flowers individually subtended by a cupule, semi-spherical cupules, nuts circular in cross-section and one nut per cupule. About 450 species are widely distributed around temperate regions in the Northern hemisphere (Prantl, 1889; Camus, 1938-1954; Melchior, 1964; Hutchinson, 1967; Maleev, 1985; Mabberley, 1987; Kubitzki, 1993; Nixon, 1993; 1997; Huang et al., 1999).

The genus Quercus is divided into two subgenus on the basis of cupular scale shape; subgen. Quercus which is characterized by scales arranged spirally on the cupule, and subgen. Cyclobalanopsis (Oerst.) C. K. Schneid., which is characterized by united scales forming concentric rings on the cupule (Schneider, 1906; Camus, 1938-1954; Kubitzki, 1993; Nixon, 1993; Huang et al., 1999). Quercus subgen. Quercus includes about 350 species widely distributed from the subarctic zone to the tropical area of the Northern hemisphere including Asia, Europe, North America, North Africa, and while subgen. Cyclobalanopsis (Oerst.) C. K. Schneid. is finitely distributed in the warm regions of Southeast Asia and East Asia (Huang et al., 1999; Kubitzki, 1993; Nixon, 1993). In Korea, the genus Quercus constitutes twenty species; six species of subgen. Quercus and six species of subgen. Cyclobalanopsis. A variety of hybrid species have been reported resulting from the crossbreeding amongst the species of subgen. Quercus in Korea (Lee, 1980).

Quercus L. is one of the typical taxonomic groups where interspecific hybridization is often seen, and numerous hybrid species have been reported in the genus Quercus (MacDougal, 1907; Trelease, 1917; Nakai, 1926; Uyeki 1932; Camus, 1936-1954; Palmer, 1948; Lee, 1961b; 1961c; 1964; Hardin, 1975; Grant, 1981; Nixon, 1997). It is known that cross-pollination and wind-pollination are vastly seen in this genus (Hardin, 1975). Reproductive isolation amongst most of its species within the same section in Quercus rarely occur, hence the genus is known as a typical taxonomic group that form a local “syngameon” (Grant, 1981). In addition, hybridization between hybrid taxon and its parent species is observed in this genus (introgressive hyridization). Thus, there has been much confusion and difficulties in estimating specific delimitation, extent of variation in each taxon and in identifying hybrid taxa due to introgressive hybridization (Palmer, 1948: Lee, 1961a; 1961b; Hardin, 1975). It is understood that this phenomenon of vast hybridization within the genus of Quercus L. results from having the same chromosome number x=12, 2n=24, no reproductive barrier, same blooming period, and anemophilous flower (Lee, 1956; 1961b; Lee and Hashizume, 2004).

Research on Quercus L. in Korea began when the Western scholars like Skan (in Forbes and Hemsley, 1899), Palibin (1900), and Komarov (1903) started recording the distribution of Q. aliena Blume and Q. dentata Thunb. in Murray and several taxa of Quercus found in the Korean peninsula (Lee, 1961a). In Flora Koreana (Pars secunda), Nakai (1911) reported ten taxa belonging to Quercus which consists of eight species, two varieties, and two formas such as Q. aliena, Q. dentata, Q. glandulifera Blume (≡ Q. serrata Murray), Q. mongolica Fisch. ex Ledeb., etc. Later, he described several new species and changed the rank of some taxa in Quercus based on the morphological characters such as the leaf size and shape, tooth shape, number of lateral veins, distribution of trichome, and cupule scale (Nakai, 1915). These were compiled and reclassified into 20 taxa of Korean Quercus L. consisting of 10 species, 12 varieties, and 5 forms by Nakai (1917).

Quercus aliena and Q. serrata are morphologically differentiated; Q. aliena Blume shows clear differences in the size and shape of leaf, sizes and shape of tooth, distribution pattern (type and density) of trichomes found on the adaxial and abaxial leaf surfaces, size and shape of nut, size of cupule and cupule scale, twig diameter and its trichome distribution to those of Q. serrata Murray (Lee, 1961a; 1966; 1980; W. Lee, 1996; Y. Lee, 1996; Park et al., 2005; Park, 2009). Quercus aliena has bigger leaves and leaf teeth, and broader twigs in diameter than Q. serrata Murray. Also, two taxa show differences in the trichome distribution: Q. aliena has no trichomes on both upper leaf surface and on its twig but has dense fasciculate trichomes on the lower leaf surface, whereas Q. serrata Murray has solitary trichome on its upper leaf surface, and solitary and fasciculate trichomes on the lower leaf surface (Lee, 1961a; Park et al., 2005; Chang, 2007; Park, 2009). Additionally, Q. aliena, in comparison to Q. serrata, has broader and more hairy nuts, longer and broader cupule, and bigger cupule scales, showing clear distinctions in the characteristics of their reproductive organs (Lee, 1961a; Park et al., 2005; Park, 2009). And also, two species are positioned separately in the plot of principal component 1 and 2 in principal components analysis of numerical taxonomic research on the six species of Korean subgen. Quercus (Park et al., 2005).

These two species, however, belong to same section Prinus Loudon (Camus, 1938-1954), morphological intermediates resulting from interspecific hybridization between Q. aliena and Q. serrata were reported in earlier studies (Uyeki, 1934; Lee, 1961b; 1980). Blume (1851), a scholar from Netherlands who described numerous species based on the samples collected from Japan, close to Korea, described Q. aliena and Q. glandulifera (≡ Q. serrata) from Japan, and he also described an intermediate form between Q. aliena and Q. glandulifera as a morphologically different species naming Q. urticaefolia Blume in the same book. But, Lee (1961b; 1980) assumed Q. urticaefolia as one of the morpho-types of the hybrid taxon between Q. aliena and Q. serrata and hence used Q. × urticaefolia as its corrected name (Lee 1961b; 1980; Park et al. 2005; Park, 2009). Lee (1961b; 1980) reported diverse intermediates between the two species. Park et al. (2005) also reported that the putative hybrid individuals between Q. aliena and Q. serrata showed somewhat intermediate values between those of both presumed parent species in most of the morphological characteristics used in the numerical taxonomic analysis, and that they occupied an intermediate position between presumed parent species in the first two principal component axes in the principal components analysis.

Quercus forests vastly occupy 13.6 % (20.3 % of total forest area) of Korean territory, and the matter production of Quercus forest constitutes the starting point of the food chain and food web of the forest ecosystem. Therefore, Quercus is one of the very important woody genera in the ecology of Korea (Kim et al., 1981; Korea Forest Research Institute, 1988; Lee et al., 2006; Hong et al., 2010). Quercus aliena and Q. serrata are the main species found in mountains, and they coexist and are dispersed in regions with relatively low altitudes (Lee, 1961a; 1966; 1980; Chang, 2007; Korea Forest Service, 2012). In particular, Q. serrata is vastly dispersed in the lowlands of central Korea (Chung and Lee, 1965), and it is recognised as the dominant tree species and climax species in the forests of Southern region (Park, 1984; Kim and Kil, 2000; Sim and Han, 2003; Kim and Lee, 2006; Lee, 2007; Song, 2007; Lee and You, 2012; Park, 2014a; 2014b).

On the other hand, each of Q. aliena and Q. serrata have a wide spectrum of variation within species, and intermediates between the two species have overlapping characteristics which show various morphological variations caused by introgressive hybridization. Hence, there were high rate of misidentification in herbarium specimen labels (Park, personal observation).

This research aimed to understand identity and hybridization patterns of Q. aliena and Q. serrata, taxonomically and ecologically important species which are widely dispersed in Korea, through numerical taxonomic analysis (PCA) and phytochemical analysis of flavonoid compounds. Flavonoid compound, a secondary metabolite, which widely occurs in tracheophytes was found to be very useful in understanding the origins of hybridization as well as hybridization patterns in various taxa of tracheophytes. Generally, it has been reported that flavonoid profile features of hybrid taxa show features of the parent species (Alston and Turner, 1963; Levy and Levin, 1971; Wyatt and Hunt, 1991).

MATERIALS AND METHODS

1.Morphological Analysis

This research used samples collected across Korea between 1999 to 2007 from Mt. Jiri, Mt. Seorak, Mt. Byeonsan, Mt. Mani and Mt. Jeongjok in Ganghwa-do, Mt. Geum-san in Namhae-do, Mt. Gwanak, Mt. Samsung, Mt. Yumyeong, Is. Seokmo, Is. Naro-do, Geochang County, Sancheong County, Jangheung County, Haenam County, and Suwon City, etc. In addition, samples from SNU (herbarium of School of Biological Sciences, Seoul National University) and SNUA (herbarium in the arboretum of College of Agriculture and Life Sciences, Seoul National University) were used as research materials. The samples of individuals collected over the period of this research were placed at SNU as voucher specimens.

The above-mentioned samples were primarily classified into Q. aliena (ALI and ALI': ALI and ALI' are both typical Q. aliena but there is a slight difference in the density of trichomes on the lower leaf surface), Q. serrata (SER and SER': SER and SER' are both typical Q. serrata, but there are slight differences in the density of trichomes on the leaf surface and in number of ray of stellate trichome; SER has relatively lower density and ray number of stellate trichomes than SER’), and putative hybrids (AS: a hybrid-type close to Q. aliena, (AS): a hybrid-type, intermediate form equally distant from Q. aliena and Q. serrata, SA: a hybrid-type close to Q. serrata, S-A: an introgressed hybrid individuals very close to Q. serrata) by referring to protologue. And following morphological features and main characters of above classified types were analyzed and measured; leaf, twig, bud, nut (fruit), cupule, cupule scale, trichome density, and infructescence. The sixty-four characters (Figure 1, Table 1) which show morphological features of the two species were confirmed based on primary analysis. The characters of abovementioned samples, total 116 individuals, were measured: 39 Q. aliena individuals, 18 Q. serrata individuals, and 59 putative hybrids (8 AS individuals, 8 (AS) individuals, 27 SA individuals, 16 S-A individuals), and they were classified based on types and distribution patterns of trichome as well as the gross morphology. The samples used in the analysis were subject to having mature leaves and fruits where 64 morphological characters (Table 1) could be measured. They were selected in a way that geographical range of the country, altitude distribution, and ecological habitat of each taxa are well-reflected.

Each character was measured from organs in the same part in order to prevent any variations caused by measuring organs from different parts. The diameter of twigs (character 36), winter bud length and width (character 37-38), length of connate part of styles and divided part of styles (character 44-45), width and length of cupule scales (character 60-61) were measured using stereoscopic microscope in micrometers. The nut width (character 42), outer and inner diameter of cupules (character 53-54) were measured by using vernier caliper.

The research aimed to clarify the delimitation of morpho-types and variation pattern of diagnostic characters by conducting principal components analysis using the measurement data mentioned above. The principal components analysis was done using SAS program (SAS Institute, 1990: Release 6.11) for microcomputers by calculating correlation coefficient matrix from measurement value data matrix.

The distribution patterns of trichomes on the lower leaf surface were observed because distribution and type of trichomes are important diagnostic characters in classifying species of subgen. Quercus and in determining the presumed parent species of hybrids. The observation of trichome’s microstructure was conducted using the samples fixed in FAA. The samples were observed under a scanning electron microscope (Jeol Model JSM-6390LV) after dehydration in a graded series of ethanol concentration (Nyman, 1993) and gold-coating which was processed for three minutes with ion coater (Eiko Model IB-3) at 6 mA.

2.Flavonoid analysis

This research used samples of forty-two individuals collected across Korea between 1999 to 2007 from Mt.

Jiri, Mt. Seorak, Mt. Mani and Mt. Jeongjok in Ganghwado, Is. Naro-do, Sancheong County as research material. The samples of individuals collected over the period of this research were deposited at SNU as voucher specimens. The forty-two individuals were as follows: ten Q. aliena individuals (ALI or ALI'), three Q. serrata individuals (SER or SER'), four AS individuals, two (AS) individuals, twenty SA individuals, and three S-A individuals.

Four among them were utilized for full flavonoid analysis and the remaining individuals were examined for flavonoid composition by two-dimensional thin layer chromatography (2-D TLC) and high performance liquid chromatography (HPLC) described below (Figure 2).

Extraction and 2-D TLC: Flavonoids were extracted from air dried, ground leaf material (5-10 g) according to procedures described by Park (1987) and Mun and Park (1995). Preliminary analysis of the flavonoid extracts employed 2-D TLC. The concentrated extracts of each species were spotted on microcrystalline cellulose TLC plate (Merck, thickness 100 μm, 20 cm × 20 cm), and the chromatograms were developed in TBA (tertiary-butanol : acetic acid : water, 3 : 1 : 1, v/v/v) followed by 15% aqueous HOAc (Mabry et al., 1970). The flavonoid profiles were viewed under UV light, and colors as well as Rf values were recorded.

Purification of flavonoids: Bulk isolation and purification of the flavonoids employed one-dimensional paper chromatography (1-D PC) using Whatman 3MM paper followed by HPLC (Mun and Park, 1995). For HPLC, a Gilson Model 305-306/115UV dual pump system was used. A 50- to 100 μL sample of each concentrated flavonoid solution from the 1-D PC was injected into Waters semi-preparative lBondapak C18 column (7.8 mm × 30 cm), and it was eluted with 15-30 % acetonitrile in 2 % aqueous HOAc at a flow rate of 3 ml/min for 27 min cycle. Detection was accomplished at 254 nm with 0.5-1 a.u.f.s. sensitivity, and each flavonoid peak was collected for structural identification. Six to ten repeated runs were required to obtain sufficient amounts for complete structural identification. For relative retention, quercetin (acyl) 3-O-glucoside was used as an internal standard.

Identification of flavonoids: Purified flavonoids were identified by employing combinations of UV spectral analyses, acid and alkaline hydrolysis (Mabry et al., 1970; Markham, 1982), Rf values, retention times, and co-chromatography with standards using HPLC and/or TLC. Identification of sugars following the acid hydrolysis employed TLC using cellulose plates (Park, 1987; Mun and Park, 1995). Mild acid hydrolysis and H2O2 oxidation (Markham 1982) were also used for diglycosides. Mild alkaline hydrolysis (Markham 1982) was employed to detect the presence of acylated flavonoids. The flavonoid profile of each individuals were quantitatively and qualitatively analyzed by using the position of spots in 2-D TLC, spot intensity, retention time of HPLC, and the peak height.

RESULTS AND DISCUSSION

1.Morphological Analysis

Principal component analysis: The 64 characters (Table 1) that are related to main diagnostic characters such as size and shape of leaf, twig, winter bud, nuts, cupule, cupule scale and infructescence were used to conduct principal components analysis in order to investigate morphological variation patterns and to objectively understand the entity as well as to delimit Q. aliena, Q. serrata, and their hybrid morpho-types in Korea. The result of principal component analysis showed that first three principal components (PC 1, 2, 3) accounted for 57.8 % of the total variance, whereas subsequent components contributed less than 5.1 % each (Table 2). Principal component 1 accounted for 40.9 % of the total variance. It had relatively high loading for characters such as leaf length (character 1), characters related to leaf width (characters 2-8), distance from leaf apex to the widest point of leaf (character 9), ratio of leaf length to widest point of leaf (character 10), leaf apex width (character 20), petiole length (character 23), petiole diameter (character 24), size of the tooth and tooth apex angle (characters 28-32), twig diameter (character 36), length and width of winter bud (characters 37-38), nut width (character 42), ratio of nut length to nut width (character 43), diameter of nut scar (character 48), ratio of diameter of nut scar to nut width (character 55), diameter of pubescent region on nut and ratio of diameter of pubescent region on nut to nut width (characters 49, 56), height, outer and inner diameter of cupule (characters 52-54), length and width of cupule scale (characters 60-61) (Tables 1, 2). Principal component 2 accounted for 9.6 % of the total variance. It had relatively high loading for characters such as shape of the leaf (characters 11-18), widest point of the leaf (character 19), leaf base angle (character 25), nut length (character 41), and length of acorn (character 50) (Tables 1, 2). Principal component 3 accounted for 7.3 % of the total variance. It related to characters such as size and shape of the tooth (characters 28-31, 33-35), length of divided part of the style (characters 45, 47), number of styles (character 46), and cupule height (character 52) (Tables 1, 2).

Each individual was ordinated using principal component 1 and 2 as the axis, and the ordination plot showed the following classification: Q. aliena group Q. serrata, S-A, SA hybrid morpho-type group, and AS, (AS) hybrid morpho-type group (Figure 3).

Individuals of Quercus aliena were separated from the rest by the first component as they had high values of principal component 1 (Figure 3). This means that individuals of Q. aliena tend to have longer and wider leaves, higher ratio of leaf width at widest point to leaf length, wider leaf apex width, higher ratio of leaf apex length to apex width, longer petioles and wider petiole diameter, bigger teeth and obtuse or round tooth apex, thicker twig and longer and wider winter bud, wider nut width, lower ratio of nut width to nut length, wider diameter of nut scar, more pubescent nut surface, bigger cupule, greater difference between outer and inner diameter of cupule, longer and wider cupule scale than individuals of Q. serrata group and hybrid morpho-types [AS, (AS), SA, S-A] (Tables 1, 2).

Individuals of Q. serrata group and hybrid morpho-types between Q. aliena and Q. serrata [AS, (AS), SA, S-A] had relatively low value of first principal component and they were located to the left. The value of first principal component of hybrid morpho-types decreased in following order: AS, (AS), SA S-A (Fig. 3). Individuals of SA and S-A types were not separated from individuals of Q. serrata which means that gross morphology of SA and S-A types are somewhat similar to Q. serrata (Figure 3).

On the other hand, individuals of AS and (AS) types were located on the right side of Q. serrata. They occupied the position between Q. aliena and Q. serrata on the first principal component, but they were not completely separated from individuals of Q. serrata (Figure 3). It means that individuals of AS and (AS) types have intermediate forms between Q. aliena and Q. serrata, but they are slightly closer Q. serrata rather than Q. aliena in gross morphology.

Microstructure (trichomes): The distribution and type of trichomes have been recognized as important diagnostic characters in identifying different species of Quercus and in presuming the parental species of hybrid (Lee, 1958; 1961b; 1964; Hardin 1975; 1976; 1979; Nixon, 1997). Lee (1961a; 1961b) reported that different solitary trichomes, fasciculate and stellate trichomes are distributed in each four taxa of Korean subgen. Quercus sect. Prinus: Q. dentata, Q. mongolica, Q. aliena, and Q. serrata. In addition, he reported that there is glandular simple-uniseriate trichome (a type of solitary trichome) on the leaf of Q. aliena (Lee 1961a; 1961b).

The observation of trichomes with SEM showed that there are clear differences in the density and type of trichomes found on the abaxial leaf surface of Q. aliena, Q. serrata, and putative hybrids (Fig. 4). 8- to 12-rayed stellate trichomes were seen densely pubesent and short simple-uniseriate multicellular trichomes were seen on the abaxial leaf surface of Q. aliena (Figure 4A). 4- to (8)-rayed stellate trichomes, solitary, bending unicellular trichomes and short simple-uniseriate multicellular trichomes were seen moderately pubesent on the abaxial leaf surface of Q. serrata (Figure 4B).

On the other hand, (4)- to 8-rayed stellate trichomes were seen not densely but moderately pubesent and short simple-uniseriate multicellular trichome were seen moderately pubesent on the abaxial leaf surface of (AS), a putative hybrid type between Q. aliena and Q. serrata (Figure 4C). In comparison, the putative hybrids [AS, (AS)] between Q. aliena and Q. serrata, which showed slightly closer morphology to Q. aliena, tended to have fewer number of rays and lower density of stellate trichomes on the abaxial leaf surface than Q. aliena. In addition, they seldomly or did not at all had solitary, bending unicellular trichomes on the abaxial leaf surface (Figure 4C).

(4)- to 8-rayed stellate trichomes, solitary, bending or slightly erect unicellular trichomes and short simple-uniseriate multicellular trichomes were seen moderately pubesent on the abaxial leaf surface of SA which is a putative hybrid type between Q. aliena and Q. serrata (Figure 4D). In comparison, the putative hybrids [SA] between Q. aliena and Q. serrata, which showed closer morphology to Q. serrata, tended to have more number of rays and slightly higher density of stellate trichomes on the abaxial leaf surface than Q. serrata. Moreover, their solitary trichomes had tendency to becoming slightly erect unicellular trichomes on the abaxial leaf surface (Figure 4D).

Quercus aliena, Q. serrata, and putative hybrid morpho-types were well distinguished by trichome types and distribution pattern (density) on the abaxial leaf surface.

Putative hybrid individuals between taxa: Subgen. Quercus is one of the typical taxa where interspecific hybridization often occurs and various interspecific hybrids have been reported domestically and internationally (MacDougal, 1906; Trelease, 1917; Nakai, 1926; 1943; Uyeki, 1932; Palmer, 1948; Lee, 1961b; 1961c; 1964; Maze, 1968; Hardin, 1975; Jensen, 1977; Jensen et al., 1993; Nixon, 1997). Lee (1961b; 1961c) reported that all possible combinations of two-way and three-way crossings among the four species of section Prinus, Q. dentata, Q. mongolica, Q. aliena, and Q. serrata, in Korea have occurred, resulting in the distribution of double and/or triple hybrids domestically. The interspecific hybrid between Q. aliena and Q. serrata was recognized as Q. × urticaefolia Blume (Gal-jol-cham-na-mu) in Korea by Lee (1961b). The putative hybrids [AS, (AS), SA,] between Q. aliena and Q. serrata tended to have intermediate values of presumed parent species in most of the morphological characters used in numerical taxonomy in this research and they were positioned in the middle of two presumed parent species in principal component analysis as well (Figure 3).

Quercus aliena has dense 8- to 16-rayed stellate trichomes on the abaxial leaf surface but no hair on the adaxial leaf surface, and Q. serrata has mixture of 4- to 8-rayed stellate trichomes and solitary, bending unicellular trichomes on the abaxial leaf surface in Korea (Lee 1961a, b). On the other hand, Q. × urticaefolia has various trichome distribution patterns as follow: stellate trichome only type, mixture type of stellate trichomes and solitary (various degrees of density and ray number of stellate trichome, and erectness as well as density of solitary trichome) type, no hair type (sometimes trichome caducous) in Korea (Lee 1961b). The putative hybrids [AS, (AS), SA, S-A] analyzed in this research showed diverse distribution patterns of trichomes: AS and (AS) had lower density and fewer ray number of stellate trichomes than Q. aliena or had low density of stellate trichome only or no hairs on the abaxial leaf surface (Fig. 4C), on the other hand, SA and S-A had slightly higher density and more ray number of stellate trichomes than Q. serrata (Fig. 4D) (Some trichome distribution types are not shown).

The type and distribution pattern (density) are important characters in understanding the extent of hybridization and introgressive hybridization between Q. aliena and Q. serrata (Figure 4C).

2.Flavonoid Analysis

The flavonoid compounds identified from foliar extracts of 42 individuals representing Q. aliena, Q. serrata and putative hybrids are presented in Figure 5-6, and Table 3-4. The spectral properties of these compounds were not presented since no new flavonoids were reported.

Twenty-three flavonoid compounds were isolated and identified from Q. aliena, Q. serrata and putative hybrids (Table 3, 4). All of them were glycosylated derivatives of the flavonols kaempferol, quercetin, isorhamnetin, and myricetin. Among them, kaempferol 3-O-galactoside (compound 1), kaempferol 3-O-glucoside (compound 2), quercetin 3-O-galactoside (compound 10), quercetin 3-O-glucoside (compound 11), and isorhamnetin 3-O-glucoside (compound 18) were the main flavonoid constituents and were present in all individuals (Tables 3, 4). The sugars found in the flavonol O-glycosides include glucose, galactose, arabinose, rhamnose, and disaccharide, rhamnosylglucose, and arabinosylglucose (Figure 5, Table 3).

Three different forms of kaempferol 3-O-arabinoside (compound 3-5) and two different forms of quercetin 3-O-arabinoside (compounds 12-13) were detected from the individuals studied (Tables 3 and 4). Compounds 3, 4, 5, 12, 13 had Rf values and retention times identical to those of kaempferol 3-O-arabinoside I, II, III and kaempferol 3-Oarabinoside I and II, respectively, which were isolated previously from genus Fallopia, Q. mongolica, and Q. serrata (Kim et al. 2000a; 2000b; Park et al., 2011; Park, 2014a; 2014b). Arabinose is known to occur in both pyranose and furanose forms (Williams and Harborne 1994), and four different forms of quercetin 3-O-arabinoside, which differ in chemical properties including Rf values, were reported previously (Hattori 1962). Mild alkaline hydrolysis (Markham 1982) did not change the mobilities of these compounds in either the TBA or the 15 % HOAc solvent system or their retention times in HPLC. H2O2 oxidation (Markham 1982) of these compounds yielded arabinose. These results strongly suggest that those compounds have different forms of arabinose. However, the exact forms of arabinose attached to those compounds could not be determined.

Four acylated derivatives (compounds 9, 16-17, 23) were also isolated (Table 3, 4). Compound 9, 16, 17, 23, respectively yielded kaempferol 3-O-glucoside, quercetin 3-O-galactoside, quercetin 3-O-glucoside, and Myricetin 3-O-glucoside following mild alkaline hydrolysis (Markham 1982). However, the acyl groups of these compounds (9, 16, 17, 23) were not characterized. The occurrences of acylated flavonoid compounds have been previously reported from Q.

rubra L. and Q. mongolica (MacDougal and Parks, 1984; Park, 2014a; 2014b).

The result of flavonoid analysis showed that there is a clear difference in flavonoid profiles of Korean Q. aliena and Q. serrata and a minute variation within each two species (Table 4).

The flavonoid profiles of Q. aliena individuals showed a simple composition that consisted of five compounds, namely, kaempferol 3-O-galactoside (compound 1), kaempferol 3-O-glucoside (compound 2), quercetin 3-O-galactoside (compound 9), quercetin 3-O-glucoside (compounds 10), and isorhamnetin 3-O- glucoside (compounds 18) (Table 4). There were several individuals that had minor compounds in small quantities but these can be seen quantitavely insignificant or as qualitative intraspecific individual variation. All individuals of Q. aliena were found to have high concentrations of kaempferol 3-O glucoside, quercetin 3-O-galactoside, and quercetin 3-O-glucoside (compounds 2, 9 and 10) (Table 4).

On the other hand, the flavonoid profiles of Q. serrata individuals showed diverse composition which consisted of more than 20 compounds per individual. It contained not only the main compounds of Q. aliena (compounds 1, 2, 10, 11, 18), but also an abundance of rhamnosyl flavonol compounds (compounds 6, 14, and 22), making a clear distinction to the flavonoid profile of Q. aliena (Table 4). There were almost no variations within species except for minor quantitative and qualitative differences in compounds (compounds 4, 7 and 23) that exist in minute quantities in Q. serrata individuals.

The flavonoid profiles of putative hybrid individuals [AS, (AS), SA, S-A] between Q. aliena and Q. serrata showed proportional tendency towards having morphological similarities to the two putative parental species. In other words, the flavonoid profile of AS or (AS) putative hybrid which shared more morphological similarities to Q. aliena than SA or S-A tended to have less arabinosyl compounds (comp ounds 3-5, 12-13 and 21) or myricetin compounds (compounds 19-23) that are found in Q. serrata but not in Q. aliena. But, this tendency was not always observed in all putative hybrids. It is presumed that the compound compositions may not coincide with the morphological variation in putative hybrids between Q. aliena and Q. serrata where hybridization and introgression through bidirectional backcrossing take place in a highly complicated manner.

Conclusions are as follows: