2017 Volume 86 Issue 3 Pages 379-388
Lilium auratum var. auratum Lindl. is distributed in the eastern part of Honshu, the main island of Japan. L. auratum var. platyphyllum Baker is endemic to the Izu archipelago, which consists of nine large islands located in south of Honshu’s Izu peninsula. Both varieties have been used as important parents of Oriental hybrid lily cultivars. They have large white flowers with yellow central stripes and colored spots on their tepals. L. auratum var. platyphyllum has larger flowers and wider leaves than L. auratum var. auratum. L. auratum var. platyphyllum has yellow spots, whereas L. auratum var. auratum has red or brown ones. Natural hybridization between these two taxa has been suggested on the basis of spot colors of populations in the Izu archipelago and the Izu peninsula. However, their genetic diversity and hybridity in nature have not been reported. We performed morphological analysis using 72 individuals of L. auratum var. auratum from seven populations and 72 individuals of L. auratum var. platyphyllum from six populations. We also performed simple sequence repeat (SSR) analysis using 102 individuals of L. auratum var. auratum from seven populations and 134 individuals of L. auratum var. platyphyllum from six populations. Both analyses revealed that L. auratum var. auratum and L. auratum var. platyphyllum are genetically different and that L. auratum var. platyphyllum has genetic diversity among populations in the archipelago.
Lilium auratum has two varieties: L. auratum var. auratum Lindl. and L. auratum var. platyphyllum Baker (syn. L. platyphyllum Makino). L. auratum var. auratum is distributed in the eastern part of Honshu (Fig. 1), the Japanese mainland (Shimizu, 1987). L. auratum var. platyphyllum is indigenous to the Izu archipelago, located in south of Honshu’s Izu peninsula. The natural distribution of L. auratum var. platyphyllum in five islands of the archipelago (Izu-Oshima, To-shima, Kouzu-shima, Miyake-jima, and Mikura-jima) was defined by Kikuchi and Kuramoto (2008).
Map of the 7 populations of L. auratum var. auratum and 6 populations of L. auratum var. platyphyllum used in this study. 1, Tateyama; 2, Zushi; 3, Miura; 4, Manazuru; 5, Matsuzaki; 6, Higashi-Izu; 7, Shimoda; 8, Izu-Oshima; 9, To-shima; 10, Kouzu-shima; 11, Miyake-jima; 12, Mikura-jima; 13, Aoga-shima. 1–7, L. auratum var. auratum; 8–13, L. auratum var. platyphyllum.
The Izu archipelago consists of nine large islands: Izu-Oshima, To-shima, Kouzu-shima, Nii-jima, Shikine-jima, Miyake-jima, Mikura-jima, Hachijyo-jima, and Aoga-shima, from north to south, and several small islands (Fig. 1). Izu-Oshima is the largest island and is approximately 20 km from the Izu peninsula. The altitudes of the highest points of the Izu archipelago range from 109 m (Shikine-jima) to 854 m (Hachjio-jima). The archipelago was formed as volcanic islands during the Pleistocene era and has never been connected to Honshu (Karig, 1975). The islands themselves have been separated by ocean since Quaternary glaciations 20000–80000 years ago when sea levels were 100–120 m lower (Gornitz, 1995). Eighteen plant species, 21 varieties, one form and two hybrids are endemic to the Izu archipelago (Ohba and Akiyama, 2002). Ohba and Iwatsuki (2006) indicated that the plant species of the Izu archipelago originated in the Honshu. The relationship of populations in this archipelago and Honshu has been investigated by the morphology in some plant species: for example, Campanula punctata and C. punctata var. microdantia (Inoue and Kawahara, 1990; Oiki et al., 2001), Hosta longipes (Yamada and Maki, 2014), Ligustrum obtusifolium, and L. ovalifolium (Yamada et al., 2014). These studies revealed morphological differentiation and diversity between the Izu archipelago and Honshu.
L. auratum var. auratum and L. auratum var. platyphyllum have racemose inflorescences and large white flowers with yellow central stripes, and colored spots on recurved tepals. L. auratum var. platyphyllum has larger flowers and wider leaves than those of L. auratum var. auratum. L. auratum var. auratum shows red or brown spots on tepals. In contrast, L. auratum var. platyphyllum has yellow spots on the tepals, although some individuals of this variety on Izu-Oshima have been reported to have red spots (Shimizu, 1971, 1987). Our preliminary investigation also found that some L. auratum var. platyphyllum individuals had red spots on their tepals (Yamamoto et al., 2014a). It has been proposed that L. auratum var. auratum and L. auratum var. platyphyllum were hybridized in Izu-Oshima by pollinators flying from the Izu peninsula (Shimizu, 1971), although Izu-Oshima is 20 km from the Izu peninsula by sea.
Several molecular approaches have been applied to the genus Lilium. Randomly amplified polymorphic DNA (RAPD) markers applied to L. japonicum showed genetic differences among populations (Haruki et al., 1998). However, the genome size of lilies is large and the C values of Lilium species are variable, ranging from 22 to 104 pg and averaging 56.3 pg (Peruzzi et al., 2009). Varshney et al. (2007) suggested that individual amplified fragment length polymorphism (AFLP) or RAPD fragments are complex in a large genome template, because the band number is large. Microsatellites or simple sequence repeat (SSR) markers are useful tools for genetic analysis because of their biparental inheritance and hypervariability. Some studies of Lilium employed SSR markers. Horning et al. (2003) developed six SSR markers in L. philadelphicum. Arzate-Fernández et al. (2005) applied inter simple sequence repeat (ISSR) markers in L. maculatum var. bukosaense. Kawase et al. (2010) developed three SSR markers in L. japonicum. Lee et al. (2011) developed 19 EST-SSR markers in L. regale using an expressed sequence tag (EST) database. Yuan et al. (2013) developed 118 EST-SSR markers in L. regale and related species, and applied them to Lilium cultivars. These EST-SSR markers were adopted for a wild population of L. auratum in our previous study (Yamamoto et al., 2014b).
Although L. auratum var. platyphyllum is an important genetic resource of Oriental hybrid lilies, there is little information about its genetic diversity. The wild population of L. auratum var. platyphyllum in the Izu archipelago has been decreasing and is listed in the red list of threatened species in Tokyo as a vulnerable plant (Ohba, 2011). The Aoga-shima population was once diminished by a volcanic eruption. The populations on Hachijyo-jima and Miyake-jima were once destroyed by excessive harvesting by humans for ornamental and edible use, and the Miyake-jima population was replanted from the Mikura-jima population.
Our preliminary studies using sequence related amplified polymorphism (SRAP) marker analysis and morphological observation suggested that L. auratum var. platyphyllum has genetic diversity in some populations of the Izu archipelago (Yamamoto et al., 2012). However, the precise genetic differences among populations in the Izu archipelago are still unclear, and the genetic relationship between L. auratum var. auratum and L. auratum var. platyphyllum have not yet been analyzed.
In the present study, we aimed to assess the genetic diversity of a wild population of L. auratum var. platyphyllum by morphological and SSR analysis. We also clarified the genetic relationship between L. auratum var. auratum and L. auratum var. platyphyllum.
Seventy-two individuals of L. auratum var. auratum from seven populations in coastal areas in Honshu near the Izu archipelago (Tateyama, 15; Zushi, 11; Miura, 12; Manazuru, 8; Matsuzaki, 8; Higashi-Izu, 3; Shimoda, 15) were investigated (Table 1; Fig. 1). Seventy-two individuals of L. auratum var. platyphyllum from six populations, representing six islands (Izu-Oshima, 26; To-shima, 12; Kouzu-shima, 12; Miyake-jima, 13; Mikura-jima, 3; Aoga-shima, 6) were investigated. In total, 144 individuals were used for morphological observation.
Population and the numbers of individuals used for morphological and SSR analysis.
Twenty-four characteristics were measured at flowering time: (1) flower diameter; (2) outer tepal length; (3) outer tepal width; (4) number of red spots on outer tepal; (5) number of yellow spots on outer tepal; (6) margin undulation of outer tepal; (7) recurving degree of outer tepal; (8) shape of outer tepal; (9) inner tepal length; (10) inner tepal width; (11) number of red spots on inner tepal; (12) number of yellow spots on inner tepal; (13) spot size of inner tepal; (14) margin undulation of inner tepal; (15) recurving degree of inner tepal; (16) shape of inner tepal; (17) stamen length; (18) stigma length; (19) stigma color; (20) coloration of stem anthocyanin; (21) distribution of stem anthocyanin; (22) leaf arrangement; (23) leaf length; and (24) leaf width. At first these morphological data were measured in four L. auratum var. auratum population (Tateyama, 15; Zushi, 11; Miura, 12; Manazuru, 8) and six L. auratum var. platyphyllum population (Izu-Oshima, 26; To-shima, 12; Kouzu-shima, 12; Miyake-jima, 13; Mikura-jima, 3; Aoga-shima, 6). To avoid misleading correlations, which can be affected by climate or annual changes, we selected 10 characteristics for further statistical analysis: (a) number of red spots on outer tepal; (b) number of red spots on inner tepal; (c) number of yellow spots on outer tepal; (d) number of yellow spots on inner tepal; (e) outer tepal length; (f) outer tepal width; (g) inner tepal length; (h) inner tepal width; (i) leaf length; and (j) leaf width.
We compared these 10 characteristics among populations and between two varieties using principal component analysis (PCA) using SPSS 20.0.
Plant materials for SSR analysisA total of 236 individuals were analyzed for SSR analysis. Samples of L. auratum var. auratum were collected from seven populations from coastal areas in Honshu near the Izu archipelago (Tateyama, 16; Zushi, 16; Miura, 16; Manazuru, 16; Matsuzaki 6; Higashi-Izu, 4; Shimoda, 18) (Table 1; Fig. 1). Samples of L. auratum var. platyphyllum were collected from six populations of the Izu archipelago (Izu-Oshima, 32; To-shima, 30; Kouzu-shima, 10; Miyake-jima, 13; Mikura-jima, 38; Aoga-shima, 11).
SSR analysisDNA was isolated from 20 mg of fresh leaf or stamens by the CTAB method of Doyle (1990). Eight EST-SSR markers (L20, L59, eL16, eL42, ivflmre252, ivflmre294, ivflmre330, and ivflmre850) were selected from Lee et al. (2011) and Yuan et al. (2013) (Table 2). PCR was performed in reaction mixtures with a final volume of 5 μL, which contained 0.4 μL of dNTP mix, 0.5 μL 10× reaction buffer, 0.3 μM of each pair of forward and reverse primers, 0.025 μL of Ex Taq polymerase (Takara, Tokyo, Japan), 20 ng of DNA and sterile distilled water. PCR amplification was performed using a T100 thermal cycler (BioRad, Hercules, CA, USA). For markers of L20, L59, eL16, and eL42, reaction cycles consisted of initial denaturation for 94°C for 2 min, followed by 35 cycles of 94°C for 45 s, 55°C for 45 s, 72°C for 1 min, and final extension for 5 min at 72°C (Lee et al., 2011). For markers of ivflmre252, ivflmre294, ivflmre330, and ivflmre850, a touchdown PCR amplification was performed; 94°C for 5 min, followed by 10 cycles of 94°C for 30 s, 63°C for 30 s (reducing by 0.5°C at each cycle), and 72°C for 45 s. Then, 25 cycles of 94°C for 30 s, 58°C for 30 s, and 72°C for 45 s, and final extension for 5 min at 72°C was carried out (Yuan et al., 2013). Forward primers of L20, L59, eL16, ivflmre330, and ivflmre850 were labeled with a fluorescent dye NED, and those of eL42, ivflmre252, and ivflmre294 were labeled with a fluorescent dye HEX (Applied Bio Systems, Carlsbad, CA, USA). PCR products were electrophoresed with an internal size standard (Gene scan-350; Applied Bio Systems) using a 3100 Genetic Analyzer (Applied Bio Systems). Genotypes were analyzed using Peak Scanner 1.0 (Applied Bio Systems).
Primer sequences of SSR markers (Lee et al., 2011; Yuan et al., 2013) used in this study.
The average number of alleles per locus (Na), observed heterozygosity (Ho), expected heterozygosity (He), Nei’s genetic diversity parameter (Nei) (Nei, 1987), and the analysis of molecular variance (AMOVA; Excoffier et al., 1992) were calculated with Arlequin version 3 (Excoffier and Lischer, 2010). A neighbor-joining (NJ) tree was generated based on Nei’s genetic distance (Nei et al., 1983) with 1000 bootstrap replicates using Poptree 2 (Takezaki et al., 2010). Principal coordinate analysis (PCoA) among populations was calculated with Genalex 6.5 (Peakall and Smouse, 2006, 2012). Bayesian clustering was calculated with Structure 2.3.4 (Falush et al., 2007; Pritchard et al., 2000). The numbers of distinct clusters (K) varied from one to 10. Ten iterations were run for each K, with a burn-in of 200000 and MCMC of 200000 iterations. The optimal K value was estimated by calculation of ΔK (Evanno et al., 2005).
Morphological variations of ten characteristics in 7 populations of L. auratum var. auratum in Honshu and 6 populations of L. auratum var. platyphyllum in the Izu archipelago are shown in Table 3. Honshu populations of L. auratum var. auratum had red spots only, whereas Izu archipelago populations of L. auratum var. platyphyllum had both red and yellow spots, except for the Aoga-shima population, which had only yellow spots. Numbers of red spots on outer and inner tepals were lower in populations from the Izu archipelago than in populations from Honshu. Numbers of yellow spots on the outer and inner tepals were significantly higher in populations of Aoga-shima and To-shima than in other populations from the Izu archipelago. Length of the inner tepal, width of the inner tepal, and length of the outer tepal, showed no significant differences among populations.
Variance and mean values of ten morphological characteristics of 7 populations of L. auratum var. auratum and 6 populations of L. auratum var. platyphyllum.
A comparison of mean values of 10 characteristics between L. auratum var. auratum and L. auratum var. platyphyllum is shown in Table 3. Tepal width, number of yellow spots on inner and outer tepals, and leaf length and width of L. auratum var. platyphyllum were significantly greater than those of L. auratum var. auratum. The number of red spots on outer and inner tepals was higher in L. auratum var. auratum than L. auratum var. platyphyllum. L. auratum var. platyphyllum had a large variation in the number of red spots because the Aoga-shima population had only yellow spots. Other characteristics showed no marked differences between the two varieties.
Eigenvalues of principal component 1 (PC1), PC2, and PC3 were 3.48, 2.87, and 1.16, respectively (Table 4). The contribution percentages of PC1, PC2, and PC3 were 34.78%, 28.66%, and 11.60%, respectively. The first three contributions cumulatively accounted for 75.04%. Eigenvalues for PC1 to PC3 are shown in Table 4. PC1 explained outer tepal length and width, number of yellow spots, length and width of the inner tepal, and leaf width. PC2 explained number of red spots. PC3 explained leaf length. A two-dimensional scatter diagram of the first and second components for ten morphological characteristics shows that L. auratum var. auratum and L. auratum var. platyphyllum are separate, but that individuals are not clearly clustered in populations within each variety (Fig. 2).
Factor loadings of ten morphological characteristics in the first three principal components (PCs) of the PCA.
Two-dimensional scatter diagram of the first and second components using ten morphological characteristics in 7 populations of L. auratum var. auratum and 6 populations of L. auratum var. platyphyllum.
The Na and He ranged from three (eL16, ivflmre850) to nine (ivflmre294) and from 0.24 (eL42) to 0.68 (ivflmre294), respectively (Table 2). The average Na, Ho, He, and Nei’s genetic diversity (Nei) values for the populations are shown in Table 5. Na ranged from 2.80 (Aoga-shima) to 4.86 (Matsuzaki, Shimoda). Ho ranged from 0.15 (Aoga-shima) to 0.63 (Zushi). He ranged from 0.43 (Aoga-shima) to 0.70 (Matsuzaki). Nei ranged from 0.27 (Aoga-shima) to 0.61 (Aoga-shima). AMOVA showed that variation within populations of L. auratum var. auratum and L. auratum var. platyphyllum accounted for 86% and 73%, respectively (Data not shown). AMOVA also indicated that the proportion of variation in populations was larger in L. auratum var. platyphyllum (27%) than L. auratum var. auratum (14%)
Genetic parameters of 7 populations of L. auratum var. auratum and 6 populations of L. auratum var. platyphyllum by SSR analysis.
According to the NJ tree, L. auratum var. auratum and L. auratum var. platyphyllum were clearly separated (Fig. 3). PCoA also clearly separated L. auratum var. auratum and L. auratum var. platyphyllum on the PC1 axis (Data not shown).
Neighbor-joining tree using Nei’s genetic distance of 7 populations of L. auratum var. auratum and 6 populations L. auratum var. platyphyllum by SSR analysis. Numbers on branches are bootstrap values based on 1000 replicates.
In the Bayesian cluster analysis, the highest ΔK was 2. When K = 2 (Fig. 4A), individuals of L. auratum var. auratum were assigned to cluster I and those of L. auratum var. platyphyllum were assigned to cluster II. When K = 3 (Fig. 4B), individuals of L. auratum var. auratum were assigned to clusters I. Most individuals of Izu-Oshima, To-shima, and Kouzu-shima were assigned to cluster II. Individuals of Miyake-jima, Mikura-jima, and Aoga-shima individuals were assigned to cluster III.
Bayesian cluster analysis of 7 populations of L. auratum var. auratum and 6 populations L. auratum var. platyphyllum by SSR analysis, estimated from data for K = 2 (A) and K = 3 (B). Population codes: 1, Tateyama; 2, Zushi; 3, Miura; 4, Manazuru; 5, Matsuzaki; 6, Higashi-Izu; 7, Shimoda; 8, Izu-Oshima; 9, To-shima; 10, Kouzu-shima; 11, Miyake-jima; 12, Mikura-jima; 13, Aoga-shima. 1–7, Lilium auratum var. auratum; 8–13, L. auratum var. platyphyllum.
L. auratum var. platyphyllum is an endemic variety in the Izu archipelago and has been used as genetic resource for lily breeding. Color and number of spots on tepals are important ornamental traits in lilies. From our morphological observations, L. auratum var. platyphyllum has fewer red spots on tepals than L. auratum var. auratum, and yellow spots are found only in this variety. Within the Izu archipelago, the Aoga-shima population had only yellow spots. Numbers of spots varied among population within the Izu archipelago. Numbers of yellow spots on outer tepals were markedly higher in populations from Aoga-shima and To-shima than in other populations. The To-shima population showed a large variation in spot characteristics, having both red and yellow spots.
In SSR analysis, the NJ tree showed features of the Aoga-shima population not present in other populations. These results show that the Aoga-shima population is genetically different from other populations in the Izu archipelago. A genetic difference of the Aoga-shima population was also suggested by our previous SRAP analysis (Yamamoto et al., 2012). This preliminary experiment showed that the Aoga-shima population is different from those of other islands. Aoga-shima is the southernmost island in the Izu archipelago, which is approximately 70 km from the nearest island Hachijo-jima. Four of 8 SSR loci (L20, L59, eL16, and eL42) were homozygous in the Aoga-shima population. These SSR loci were not homozygous in other L. auratum var. platyphyllum populations. Aoga-shima exploded in 1785 and most plant populations disappeared at that time. Our results indicate that L. auratum var. platyphyllum populations drastically decreased in number at one point in time, forming a bottleneck. The Miyake-jima population was replanted from the Mikura-jima population after disappearing following excessive harvesting. Our findings also suggested a similarity between the Miyake-jima and Mikura-jima populations by morphological and SSR analysis. There was no significant difference in terms of morphological characteristics between the populations of the two islands and results of the NJ tree showed their close relationship. The To-shima population had a higher number of yellow spots and wider leaves. The results of the NJ tree and structure analysis suggested that the To-shima population is close to the Izu-Oshima and Kouzu-shima populations. The reason for these differences in To-shima is unknown.
In our morphological and SSR analysis of L. auratum var. platyphyllum, there was a significant difference between the populations from Aoga-shima and those from the other islands of the Izu archipelago. The genetic structure of Prunus lannesiana var. speciosa on the mainland and in the Izu archipelago has been investigated by SSR and AFLP markers (Kato et al., 2011), who found highly significant genetic differentiation between Hachijyo-jima and other islands. Yamada and Maki (2012) investigated the genetic differentiation of Weigela coraeenisis between the Izu archipelago and the mainland using SSR markers. They also found that the Hachijyo-jima population formed a distinct cluster in Bayesian cluster analysis results. In our study, the results for the Aoga-shima population were similar to those of other studies for the Hachijyo-jima population.
The pollinator species of L. auratum var. auratum is a butterfly (Papilio bianor) in the daytime and a hawk moth (Meganonton analis) in the nighttime (Morinaga et al., 2009). There is no report regarding the pollinator species of L. auratum var. platyphyllum. The number of butterfly species in the Izu archipelago has decreased in comparison with that on the mainland. The number ranged from 34 to 112 in Tokyo towns (Nishitama Kontyu Dokokai, 2012). In contrast, 13 to 47 species were observed in the Izu archipelago. The number of Papilio species was eight in Tokyo and three to six in the Izu archipelago. Flowers adapted to nocturnal hawk moth pollination have whitish colors and strong aromas (Miyake, 2010). In our study, the number of red tepal spots was significantly decreased in populations of the Izu archipelago. This result indicates a change in pollinator species in the Izu archipelago. L. japonicum, a close relative of L. auratum, has pink flowers without spots. The pollinators of this species have been identified as hawk moths (Acosmeryx naga and Sphinx constricta) (Inagaki, 2003). These findings suggest that the main pollinator species of L. auratum var. platyphyllum, which has white flowers with a modest number of yellow spots, is the hawk moth. L. auratum var. platyphyllum has flower characteristics more adapted to the nocturnal hawk moth than to butterfly pollination. The number of Papilio species in Aoga-shima has been reported as three and for the other islands of the Izu archipelago as five to eight (Nishitama Kontyu Dokokai, 2012). We propose that because of the small number of butterfly species, hawk moths became the dominant pollinators and that only the yellow-spotted individuals adapted in Aoga-shima.
Genetic relationships between L. auratum var. platyphylum and nearby populations of L. auratum var. auratumShimizu (1971) reported finding populations with red spots on tepals in Izu-Oshima and suggested that hybridization had been brought about by pollinators flying from the Izu peninsula. Ogiwara (1962) found red-spotted individuals in Mikura-jima. In view of the distance of Mikura-jima from the Izu peninsula, Mikura-jima individuals were considered to belong to L. auratum var. platyphyllum. We have observed individuals with red spots in locations other than Izu-Oshima. However, there was a marked difference in spot number and color between the mainland and the Izu archipelago. The number of red spots was smaller in the Izu archipelago than in Honshu populations. SSR analysis also indicated genetic differences between Honshu and the Izu archipelago populations. Inoue and Kawahara (1990) distinguished a mainland group and an Izu archipelago group in Campanula punctate using morphological and allozyme analysis. The population of Izu-Oshima was similar to that of the mainland population in some morphological characteristics but was closely related to other Izu archipelago populations in their allozyme analysis. Our morphological and SSR findings were similar to these findings for Campanula species. In our study, L. auratum var. platyphyllum had red spots on tepals, a characteristic similar to that of L. auratum var. auratum. The SSR analysis showed a clear difference between the mainland and the Izu archipelago. These results suggest that L. auratum var. auratum and L. auratum var. platyphyllum populations have not been hybridized.
The large difference between mainland and Izu archipelago populations shown by SSR analysis is similar to that of Iwata et al. (2006). They investigated the genetic structure of Alunus sieboldiana, Miscanthus sinensis ssp. condensatus, and Polygonum cuspidatum var. terminalis populations in the Izu peninsula and the Izu archipelago. There was a significant correlation between genetic and geographical distance in M. sinensis ssp. condensatus and P. cuspidatum var. terminalis. Three spacer regions in chloroplast DNA showed that L. auratum var. auratum was close to L. rubrum and that L. auratum var. platyphyllum was close to L. japonicum (Nishikawa et al., 2002). These results also suggested a genetic difference between L. auratum var. auratum and L. auratum var. platyphyllum.
Our results indicate that L. auratum var. platyphyllum has high diversity in the Izu archipelago. This variety has been used for breeding cultivars. However, there have been not any studies on wild population diversity. The present study is the first to perform morphological and SSR analysis of L. auratum var. platyphyllum. These results provide basic information for this variety, the wild population is endemic to the Izu archipelago, and here we analyzed most of the populations of L. auratum var. platyphyllum.
We thank Mr. Toru Hishi and Mr. Kazunobu Kogi of Mikura island tourist information center and Mr. Mizuki Hara of Tokyo metropolitan Miyake office for collecting samples.
