GENE FLOW BETWEEN IMPATIENS RADICANS AND I. JAVENSIS (BALSAMINACEAE) IN GUNUNG PANGRANGO,

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1 American Journal of Botany 91(12): BRIEF COMMUNICATION GENE FLOW BETWEEN IMPATIENS RADICANS AND I. JAVENSIS (BALSAMINACEAE) IN GUNUNG PANGRANGO, CENTRAL JAVA, INDONESIA HIROKAZU TSUKAYA 2 National Institute for Basic Biology/Center for Integrated Bioscience, Myodaiji-cho, Okazaki , Japan; and School of Advanced Sciences, The Graduate University for Advanced Studies, Shonan Village, Hayama, Kanagawa , Japan This study reports the natural hybridization of two species of Impatiens in the mountain forests of Gunung Gede-Pangrango, in central Java, Indonesia. Impatiens radicans, which inhabits the highest altitude zone in the mountains of this park, and I. javensis, which inhabits a zone of lower altitude, can be easily distinguished by their morphological features. However, some populations that inhabit regions between the two zones have characteristics that appear to be combinations of traits of I. radicans and I. javensis. Sequence analysis of the 800-bp nuclear ITS region and 449-bp plastid trnl-f locus demonstrated that hybridization between the two species has occurred, and suggests gene flow between the species. Key words: trnl-f. Balsaminaceae; Impatiens javensis; Impatiens radicans; Indonesia; ITS; Java; natural hybridization; Southeast Asia; The mountain forests of the Gunung Gede-Pangrango National Park in central Java, Indonesia, are the habitat of three Impatiens species: Impatiens radicans Zoll. & Mor., which is endemic to west Java; Impatiens javensis Steud.; and the widespread Impatiens platypetala Lindl. (Mt. Gede Pangrango National Park, 1996). These species inhabit three different altitude zones in the Gunung Gede-Pangrango Park. Impatiens radicans, which has speckled petals and whorled, small, narrow leaves with sharply pointed serrations on the margins, is frequently found in mountain forests at altitudes greater than 2470 m a.s.l. Impatiens javensis, which is distinguished by a single reddish blotch in the center of the flower and oval leaves with a crenatus-serratus margin, is found at altitudes ranging from 2200 to 2400 m (Fig. 1). Impatiens platypetala, which is a common species in Southeast Asia, resides at altitudes of less than 2150 m, as well as in many other localities in Southeast Asia (e.g., in Sumatra; Grey-Wilson, 1989). During a botanical survey in Gunung Pangrango, in March 2003, I observed vigorous Impatiens individuals at an altitude of around 2420 m a.s.l. These plants had Impatiens radicans-like flower characteristics and narrow but large leaves with ciliate serrations (Fig. 1B and C). Both the morphological characteristics and the habitat of these individuals suggested that they might be hybrids between Impatiens radicans and Impatiens javensis. Although natural hybridization occurs widely in vascular plants and has an important role in their evolution (Arnold, 1997; Rieseberg and Carney, 1998), 1 Manuscript received 24 September 2003; revision accepted 30 July The authors thanks the Indonesian Institute of Science (LIPI) for kindly allowing the present study in the Gunung Gede-Pangrango National Park, Java (1122/SU/KS./2003), with permission from Departemen Dalam Negeri Republik Indonesia (#500.02/126-D.IV) and Professor Hiroshi Okada (Osaka City University, Osaka, Japan) for valuable discussions and kind assistance during the field trip. This study was supported by a grant from the SOKEN- DAI group research project of the Graduate University for Advanced Studies, and a Grant-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology, Japan. 2 Reprint requests: tsukaya@nibb.ac.jp hybridization in natural plant populations is not well understood, with the exception of several species from temperate zones (Cruzan and Arnold, 1993; Burke et al., 1998; Arnold, 2000; Johnston et al., 2001; Tsukaya et al., 2003). Natural hybridization in tropical Asian plants has been examined in relatively few studies (e.g., Okada, 1990). In the present study, I investigated the role of gene flow and natural hybridization in the establishment of biodiversity in tropical Asian islands. MATERIALS AND METHODS Plant specimens were collected from Gunung Pangrango (Mt. Pangorango) on 7 March 2003, with permission from the National Park Office of Gunung Gede-Pangrango and the Indonesian Institute of Science (LIPI). Herbarium specimens were deposited in the Herbarium Bogoriense (Bogor, Indonesia) and at the University of Tokyo (TI: Tokyo, Japan; Table 1). Before measuring the dimensions of leaves and flowers, fresh samples were dried in the manner of herbarium specimens. Data for each individual are represented as mean 1 SD. Genomic DNA was collected on FTA Cards (Whatman, Japan, Tokyo), as described previously (Tsukaya, 2003). Fresh samples of young leaves were squashed on the FTA Cards and then dried at room temperature in the presence of silica gel. The FTA Cards were washed with a buffer solution according to the manufacturer s protocol, and the DNA that was immobilized on the cards was used as a template for amplification using Pyrobest DNA polymerase (Takara Biomedicals, Tokyo, Japan) or the REDExtract-N-Amp Plant Polymerase Chain Reaction (PCR) kit (Sigma, St. Louis, Missouri, USA), according to the manufacturers protocols. The template for each PCR reaction was the DNA that was immobilized in a 1 2-mm square segment of an FTA Card. To amplify the ITS and trnl-f loci, PCR was performed in a 50- L volume almost until saturation, using the following protocol: incubation at 94 C for 30 s; 10 cycles of 94 C for 1.5 min, 45 C for 2 min, and 60 C for 3 min; followed by cycles of 94 C for 1 min, 45 C for 1 min, and 60 C for 3 min; with a final extension at 72 C for 15 min. The primers used to amplify the loci examined have been described elsewhere (Douzery et al., 1999; White et al., 1990; Tsukaya et al., 2003). DNA sequences were analyzed using the GENETYX Mac version 9.0 program (GE- NETYX, Tokyo, Japan).

2 2120 AMERICAN JOURNAL OF BOTANY [Vol. 91 Fig. 1. Differences in floral coloration, floral shape and leaf arrangement in the Impatiens species examined in this study. (A) Typical Impatiens javensis; (B) and (C) hybrid ; (D) and (E) typical Impatiens radicans. Photographs were taken of individuals in their native habitats in Gunung Pangrango, central Java, Indonesia, on 7 March RESULTS AND DISCUSSION As summarized in Table 1, the morphological characteristics of the putative hybrids of Impatiens javensis and Impatiens radicans living at around 2420 m a.s.l. were intermediate between those of the hypothetical parents. The leaves of the putative hybrid plants had dimensions between Fig. 2. Comparison of leaf arrangement and leaf shape of the Impatiens species examined in this study. A typical shoot (A to C) and part of the leaf margin (D to F) are shown for each strain. A and D, Impatiens javensis; B and E, hybrid ; C and F, Impatiens radicans. Bar 1 cm. Note that the morphological characteristics and dimensions of the putative hybrid are intermediate between those of Impatiens javensis and Impatiens radicans. those of the leaves of the two species (Table 1), and had spurs the length of those in Impatiens javensis. The morphology and floral coloration of the putative hybrids were very similar to those of Impatiens radicans (Fig. 1); the most prominent difference between the putative hybrids and Impatiens radicans was that of leaf dimensions (Table 1; Fig. 2). The leaf arrangement and leaf margin of the putative hybrids were similar to, but slightly different from, those of Impatiens radicans (Table 1; Fig. 2); they also had some characteristics of Impatiens javensis. The leaf arrangements of Impatiens radicans and the putative hybrids were both whorled, but the number of leaves per node in these plants differed (Fig. 2), with 3 7 in Impatiens radicans (6 2[1 SE]; 23 nodes examined) and 2 7 in the putative hybrid (3 1; 29 nodes examined). In contrast, the Impatiens javensis leaves were arranged oppositely, and the number of leaves per node was 2 1 (18 nodes examined). The shape of the leaf margin also differed among the three populations, and that of the putative hybrid had a shape that was intermediate between those of Impatiens radicans and Impatiens javensis (Fig. 2D F). Because these morphological characteristics strongly suggested that natural hybridization had occurred between Impatiens radicans and Impatiens javensis, I analyzed genomic sequences from these individuals. Sequences of a region of approximately 800 base pairs (bp) of the nuclear ITS locus and a region of 499 bp of the plastid trnl-f locus were obtained for six typical Impatiens javensis individuals, thirteen typical Impatiens radicans individuals, and eight putative hybrid individuals, which had been collected from their natural habitats

3 December 2004] TSUKAYA GENE FLOW BETWEEN IMPATIENS RADICANS AND I. JAVENSIS 2121 Fig. 3. Alignment of sequences of the ITS region of Impatiens species. Sequences from six individuals of Impatiens javensis (j-1 to j-6) and thirteen individuals of Impatiens radicans (r-1 to r-13) are shown. Note that eighteen sites in the 800-bp region indicate species specificity (indicated by the red letters a to s).

4 2122 AMERICAN JOURNAL OF BOTANY [Vol. 91 TABLE 1. Morphological characteristics of the Impatiens species examined in this study. Strain (specimen #) Impatiens javensis (#2316) hybrid (#2322) Impatiens radicans (#2317) Flower coloration A single red blotch on pale pink (white)* Dark speckles on pale pink (white) Dark speckles on pale pink (white) Leaf length ** (12 leaves from 4 individuals) (18 leaves from 6 individuals) * (21 leaves from 7 individuals) Leaf width Length of leaf petiole Leaf margin ** ** crenatusserratus* sharply serrated Arrangement of leaves Length of spur oppositus* (5 from 4 individuals) whorled (8 from 6 individuals) ** serrated* whorled ** (10 from 7 individuals) Note: All measurements were made on dried herbarium specimens collected from Gede-Pangrango National Park, West Java, Indonesia, on 6 7 March, 2003, by Hirokazu Tsukaya, Hiroshi Okada, Ryoko Imaichi, Hiroshi Ikeda, & Titien Ng. Praptosuwiryo. Star (*) indicates that the characteristic is different from that of the putative hybrid. Double star (**) indicates a significant difference from the data of the putative hybrid (P 0.05; Student s t test). in the Gede-Pangrango National Park (Table 2). These loci were also examined in one individual of Impatiens platypetala that inhabited the lower zone of the park; the data from this individual were clearly different from the above three populations (data not shown). Nineteen substitution sites in the ITS region distinguished the two haplotypes that represented Impatiens javensis and Impatiens radicans (Fig. 3). Within these sequences, seven of the eight putative hybrid individuals showed heterozygosity (shown in bold in Table 3) and appeared to have resulted from hybridization of the two haplotypes, the Impatiens javensis type and the Impatiens radicans type. Moreover, seven of the eight putative hybrid individuals had novel substitutions at these sites (indicated by underlines in Table 3). These variants did not appear to be sequences intermediate between the sequences from Impatiens javensis and Impatiens radicans, and seven of the nineteen speciesspecific sites in the sequences obtained from all of the putative hybrids were the same as those of Impatiens radicans. As the sequences from the putative hybrid individuals were mosaics of sequences from Impatiens radicans, sequences from the intermediate (heterozygote) plants, and novel substitutions, it is probable that hybridization occurred between the two species. As heterogeneity was rare at these sites in the putative hybrids, and these plants produced fertile seeds (data not shown), these individuals might not be F1 hybrids but instead the progeny of the original hybrids, possibly under the influence of backcrossing. Because all of the putative hybrid individuals had plastid trnl-f sequences of the Impatiens radicans type (Fig. 4), the maternal line might have been derived from Impatiens TABLE 2. ITS a trnl-f Accession numbers for sequences of the ITS and trnl-f loci. Locus Species Accession I. javensis I. radicans I. javensis I. radicans AB AB AB AB AB AB Note: a Polymorphisms among six individuals for I. javensis and thirteen individuals for I. radicans were assigned independent accession numbers. radicans and may have been back-crossed with Impatiens radicans. Both the species-specific haplotyping and the morphological characteristics strongly support the idea that the populations that inhabit the areas between the habitats of Impatiens javensis and Impatiens radicans are hybrids of the two species. Alternatively, the differentiation of Impatiens javensis and Impatiens radicans might have occurred only recently, and these plants might not yet represent different species, despite their obvious differences in morphology and flower color. To test this hypothesis, a more detailed analysis of the genomes of Impatiens species across Southeast Asia will be required. Natural hybridization of plants has been extensively studied in temperate zones, but only a few tropical plants have been fully analyzed for the occurrence of natural hybridization. One such study showed gene flow between two Monophyllaea species with segregated habitats in Southeast Asian forests (Okada, 1990). Future surveys of natural hybridization in tropical forests will reveal the richness of the biodiversity of tropical Asian islands, as well as suggest TABLE 3. Alignment of ITS sequences obtained from examined Impatiens individuals. Site* a b c d e f g h i j k l m n o p q r s Impatiens javensis T C C G T T G T G T C T C A C C C T T Impatiens radicans C A Y A C C T C A G T - T T G Y Y C C Putative hybrids #1 Y M Y R C C W Y A G Y - K T G C T C T #2 Y M Y A C C T C A G Y - K T G C T C G #3 C M T A C C K C A G Y - K T G C T C C #4 Y C Y R C Y G Y R G Y - G T G C T C T #5 Y M Y R C C K Y A G Y - K T G C T C T #6 C A T A C C T C A G T - T T G C T C C #7 C M T A C C W C A G Y - K T G C T C C #8 C M T A C C K C A G Y - K T G C T C C Note: Shown in Fig. 3 in red letter (number: bp site from the 5 end). Y, T or C; M, C or A; R, G or A; W, T or A; K, T or G. Heterozygote sites between sequences of Impatiens javensis type and of Impatiens radicans type and novel sites that differ from both sequences of Impatiens javensis-type and of Impatiens radicans-type are recognized.

5 December 2004] TSUKAYA GENE FLOW BETWEEN IMPATIENS RADICANS AND I. JAVENSIS 2123 Fig. 4. Alignment of sequences of the plastid trnl-f locus of Impatiens species. Note that there is only one polymorphism site in the 449-bp region shown. methods for conserving the biodiversity of Southeast Asian countries. LITERATURE CITED ARNOLD, M. L Natural hybridization and evolution. Oxford University Press, New York, New York. ARNOLD, M. L Anderson s paradigm: Louisiana Irises and the study of evolutionary phenomena. Molecular Ecology 9: BURKE, J. M., T. J. VOSS, AND M. L. ARNOLD Genetic interactions and natural selection in Louisiana iris hybrids. Evolution 52: CRUZAN, M. B., AND M. L. ARNOLD Ecological and genetic associations in an Iris hybrid zone. Evolution 47: DOUZERY, E. J. P., A. M. PRIDGEON, P. KORES, H. P. LINDER, H. KURZWEIL, AND M. W. CHASE Molecular phylogenetics of Diseae (Orchidaceae): a contribution from nuclear ribosomal ITS sequences. American Journal of Botany 86: GREY-WILSON, C A revision of Sumatran Impatiens. Kew Bulletin 44: JOHNSTON, J. A., R. A. WESSELINGH, A. C. BOUCK, L. A. DONOVAN, AND M. L. ARNOLD Intimately linked or hardly speaking? The relationship between genotype and environmental gradients in a Louisiana iris hybrid population. Molecular Ecology 10: MT. GEDE PANGRANGO NATIONAL PARK Mt. Gede Pangrango National Park: introduction book series, vol. 2, 106. Cipanas-Cianjur, Indonesia. OKADA, H A natural hybrid of Monophyllaea (Gesneriaceae) in the tropical rain forests of west Sumatra. Plant Systematics and Evolution 169: TSUKAYA, H A simple method for collecting DNA samples in the field. Newsletter of Himalayan Botany 32: TSUKAYA, H., T. FUKUDA, AND J. YOKOYAMA Hybridization and introgression between Callicarpa japonica and C. mollis (Verbenaceae) in central Japan, as inferred from nuclear and chloroplast DNA sequences. Molecular Ecology 12: WHITE, T. J., T. BRUNS, S. LEE, AND J. TAYLOR Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In M. Innis, D. Gelfand, J. Sninsky, and T. White [eds.], PCR protocols: a guide to methods and applications, Academic Press, San Diego, California, USA.