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Genetic differentiation within and among island populations of the endangered plant Aster miyagii (Asteraceae), an endemic to the Ryukyu Islands¹

Biological Institute, Graduate School of Science, Tohoku University, Aoba, Sendai, Miyagi, 980-8578 Japan

Received for publication January 4, 2001. Accepted for publication May 24, 2001.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Genetic diversity was examined at 17 putative allozyme loci in 18 populations of the insular endemic plant Aster miyagii (Asteraceae). This species is geographically restricted to only three islands of the Ryukyu Islands and is on the federal list of threatened plants. Genetic differentiation within an island is small, suggesting that gene flow among populations on the same island is sufficiently large to prevent divergence. By contrast, genetic differentiation among islands is large, especially between Amamioshima Island and the other two islands, suggesting that gene flow between the islands is highly restricted. Two unique alleles are nearly fixed in populations on Amamioshima Island, which is the southernmost island of the three. Comparatively, genetic diversity is the smallest on Amamioshima Island. This genetic paucity on Amamioshima Island is probably a result of a population bottleneck at colonization or the small effective population size on this island. Genetic diversity at the species level of A. miyagii is larger than those of the species with a similar life history and of the congeneric widespread species, suggesting that the species has an old origin as an insular endemic species.

Key Words: allozyme • Aster miyagii • gene flow • genetic diversity • hierarchical population structure • insular endemic plant • Ryukyu Islands • threatened plant

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Genetic diversity in natural populations is a major concern of evolutionary biologists because the amount and distribution of genetic diversity are likely to affect the evolutionary potential of species and/or populations (Futuyma, 1986 ). In the last three decades, a vast amount of data on genetic diversity in natural plant populations has accumulated, and correlations between genetic diversity and various species attributes have been examined (Hamrick and Godt, 1989). Although such genetic data have been collected for a wide variety of wild plant species, data on tropical to subtropical and noncontinental species are relatively limited (Sheely and Meagher, 1996 ).

Insular endemic plants have garnered the attention of many evolutionary biologists (Carlquist, 1974 ; Stuessy and Ono, 1998 ) because the plants often have unique characteristics that differ from those of their continental relatives. In addition, remarkable adaptive radiation is often found in insular endemic taxa (Baldwin, 1997 ; Francisco-Ortega et al., 1997 ). These unique aspects of evolution in island species are considered to result from the geographic isolation and lack of competition on islands (Crawford, Witkus, and Stuessy, 1987 ). To examine the effect of geographic isolation on plant evolution on islands, population genetic analyses estimating gene diversity within a population and interpopulation gene flow are essential.

In insular endemic species distributed on islands, population distribution is discontinuous, because the sea acts as a barrier, isolating populations on one body of land from those on others. Accordingly, gene flow tends to occur more easily among populations on the same island than among those on different islands, leading to the hierarchical population genetic structure of insular endemic species. If gene flow among islands is highly restricted, populations on different islands are expected to take different evolutionary pathways. Thus, it is meaningful to examine genetic diversity among island populations of insular endemic species.

In addition, island species are considered to be prone to extinction. One cause of this is the genetic paucity in populations (Frankham, 1997 ). Island populations tend to have a lower level of genetic diversity than do continental populations (Frankham, 1997 ), although much genetic diversity has often been found in island populations (Weller, Sakai, and Straub, 1996 ; Francisco-Ortega et al., 2000 ). From the viewpoint of conservation biology, it is important to estimate genetic diversity in island endemic species.

The Ryukyu Islands are located in the south of the Japan Archipelago and consist of 140 subtropical islands. The range has a history of more than one period of landbridge connection in various combinations with adjacent land masses (Kizaki and Oshiro, 1980 ). In addition, the islands have been connected to each other in various combinations, and some islands were submerged after the Ryukyu Islands were separated from the adjacent continent (Kizaki and Oshiro, 1980 ). Approximately 150 plant taxa are endemic to the Ryukyu Islands; 7% of the flora is endemic (Hatushima, 1980 ). Many endemic taxa of the Ryukyu Islands are on the federal list of threatened plants in Japan (Environment Agency of Japan, 2000 ). To date only a few studies examined genetic diversity of plants endemic to the Ryukyu Islands (Maki, 1999 ; Chou, Chiang, and Chiang, 2000 ).

Aster miyagii Koidz. (Asteraceae) is a perennial herb occurring on rocky places near the seashore and is endemic to only three islands of the Ryukyu Islands: Amamioshima Island and the contiguous small islands, Tokunoshima Island, and Okinawajima Island (Fig. 1). This species is 20–30 cm tall and flowers from October to December. Heads are 25 mm in diameter, ray florets are bluish purple, and the pappus is white and 3.5–4 mm long. Although main pollinators of the species are unknown, probably small insects such as halictid bees serve as major pollinators (S. Matsumura, Tohoku University, personal communication). The chromosome number of the species is 2n = 18, indicating that the species is diploid. Aster miyagii is listed on the federal list of threatened plants in Japan as "vulnerable" to extinction (Environment Agency of Japan, 2000 ). In addition to the restricted distribution of the species, its habitats have often been destroyed by the construction of facilities for tourist resorts or harbors.

View larger version (18K): [in this window] [in a new window]   Fig. 1. Distribution of the populations examined of Aster miyagii. Population codes and number of samples are shown in Table 1

	MATERIALS AND METHODS

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Sampling populations
We sampled four, five, and nine populations of A. miyagii from Amamioshima Island, Tokunoshima Island, and Okinawajima Island, respectively (Fig. 1). On Amamioshima Island, A. miyagii is restrictively distributed only on the eastern side of the island (M. Tabata, personal communication). On Tokunoshima Island, A. miyagii is probably distributed widely on the western side of the island, but the island, for the most part, is not accessible on the western side due to a steep bluff. No population was found on the eastern side of the island. On Okinawajima Island, A. miyagii is distributed widely on the eastern side of the island, but only one population was found on the western side. In Amamioshima Island, only two other populations were known to exist except those examined in this study. Compared to the Amamioshima Island, some other populations seem to exist in Tokunoshima and Okinawajima Island, although access to those populations is often difficult.

Population codes and the number of samples are given in Fig. 1 and Table 1. A small rosette was collected individually from each of the populations, and these samples were transported in plastic bags to the nursery at Tohoku University and planted in plastic pots filled with sand. Before electrophoresis, a sample of mature leaves was taken from each individual plant and transported on ice to the laboratory.

Statistical analysis
Allele frequencies in each population of A. miyagii were calculated for the loci encoding the 12 enzyme systems. The following indices were used to quantify the amount of genetic diversity within each population examined: the proportion of polymorphic loci (p) at 95% criterion, the number of alleles per locus (A), and the expected heterozygosity (h). Genetic diversity parameters (p, A, and h) were also calculated at the species level. Following Hamrick and Godt (1990), we treated the loci polymorphic in at least one population as polymorphic at the species level.

Genetic differentiation among the populations was estimated by Nei's gene diversity statistics (Nei, 1973 ) for polymorphic loci. The gene diversity statistics were calculated for each island and for the overall population. The amount of gene flow among these populations was estimated as Nm = (1/G_ST – 1)/4 (Slatkin and Barton, 1989 ).

Wright's (1951) fixation index (F) was estimated at each polymorphic locus as F = 1 – n_o/n_e, where n_o is the observed number of heterozygotes and n_e is the number of heterozygotes expected from the Hardy-Weinberg equilibrium. For loci with more than two alleles, the frequencies of the less frequent alleles were combined into a single class. Chi-square values for each polymorphic locus in a population were calculated as NF², where N is the number of individuals per population (Li and Horvitz, 1953 ).

Genetic identity and standard genetic distance (Nei, 1972 ) for each pairwise comparison of all populations examined were calculated. A phenogram based on the standard genetic distance was obtained using the neighbor-joining method (Saitou and Nei, 1987 ) using PHYLIP version 3.5c (Felsenstein, 1993 ).

	RESULTS

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Genetic diversity within a population
A total of 17 putative loci were scored: Adh, Aat-1, Aat-2, Est-2, Est-4, Fdh, Gdh, G2dh, Gpi, Lap, 6pgdh–1, 6pgdh–2, Pgm-2, Tpi-1, Tpi-2, Sod-1, and Sod-2. In all populations, Aat-2, Sod-1, and Sod-2 were monomorphic, and other loci were polymorphic in at least one population.

Table 1 summarizes the resultant values of p, A, and h for each population and each island, and at the species level. At the population level, the mean values of p, A, and h were 0.199, 1.314, and 0.077, respectively. All three parameters indicated genetic diversity within populations was smaller in populations on Amamioshima Island (p = 0.074, A = 1.147, and h = 0.019) than on Tokunoshima Island (p = 0.247, A = 1.400, and h = 0.092) and Okinawajima Island (p = 0.268, A = 1.340, and h = 0.096).

Genetic diversity at the species level
The proportion of polymorphic loci (p = 0.824) at the species level was approximately four times that of the mean population level (p = 0.199), and the mean number of polymorphic loci and the gene diversity at the species level (A = 2.647 and h = 0.148) were approximately twice those at a population level (A = 1.314 and h = 0.077) (Table 1).

Population genetic structure
The fixation index (F) for each polymorphic locus is shown in Table 2. Approximately 20% of the values (14 of 67) deviated significantly from zero, suggesting that the genotype frequency at the loci departed from the Hardy-Weinberg equilibrium. The mean values of the fixation indices across the polymorphic loci for the populations ranged from –0.036 to 0.646, and most of the values (13 of 17) were <0.3, suggesting that outcrossing is predominant in most of the populations of A. miyagii.

View larger version (24K): [in this window] [in a new window]   Fig. 2. Allele frequency variation at Lap (A) and Est-2 (B) among the populations examined of Aster miyagii

View larger version (15K): [in this window] [in a new window]   Fig. 3. Phenogram for the populations examined of Aster miyagii using the neighbor-joining method (Saitou and Nei, 1987 ) on Nei's (1972) standard genetic distance (D)

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

In contrast to intrapopulation genetic diversity, genetic variation at the species level of A. miyagii was relatively large (p = 0.824, A = 2.647, and h = 0.148). For example, those values for long-lived herbaceous plants are p = 0.396, A = 1.42, and h = 0.205 (Hamrick and Godt, 1990) and for the widespread congener A. spathulifoius are p = 0.733, A = 1.73, and h = 0.200 (Maki and Morita, 1998 ). DeJoode and Wendel (1992) compiled data for island endemic plants and reported even lower values (p = 0.250 and A = 1.32).

The genetic paucity in island species is believed to be due to founder effects and limited gene flow from source (continental) populations (Frankham, 1997 ). These conditions exist for endemic species on an oceanic island with a relatively short geographical history. Such species probably evolved rapidly on the islands from a small number of founders because of geographic isolation and lack of competition on islands (Crawford, Witkus, and Stuessy, 1987 ). By contrast, the Ryukyu Islands have a longer geographical history than the islands that have been examined extensively for their biota, such as the Hawaiian Islands (Kizaki and Oshiro, 1980 ). In particular, the three islands examined in this study have not been submerged since the Oligocene. Thus, A. miyagii probably has an older origin than those of the other insular endemics examined previously and recovered genetic diversity from the founder effect at speciation. This is partly supported by the fact that A. miyagii is not distributed on other low-elevation nearby islands, which were submerged in the early Pleistocene. In addition, under some conditions in which interpopulation gene flow is limited, the total gene diversity is maintained at a higher level than that in the cases of higher migration rates (Varvio, Chakraborty, and Nei, 1986 ). This may partly explain the genetic diversity at the species level of A. miyagii.

Aster asa-grayi, endemic to the Ryukyu Islands and belonging to the same section Pseudocalimeris as A. miyagii, is also distributed on Okinawajima Island and Amamioshima Island. In particular, on Amamioshima Island, these species occur adjacently. However, because a hybrid between A. asa-grayi and A. miyagii has not been reported to date, it is unlikely that hybridization between these species could be a source of genetic variation of A. miyagii.

Genetic diversity on Amamioshima Island is notably lower than that on Tokunoshima Island and Okinawajima Island. This is probably because fewer populations of A. miyagii exist on Amamioshima Island. Excepting those examined in this study, few large populations of A. miyagii are known to exist on Amamioshima Island (M. Tabata, personal communication). In addition, the Amamioshima Island populations are isolated from the other two island populations (see below), and the genetic diversity is susceptible to genetic drift. Another possible reason for the low genetic diversity on Amamioshima Island is the founder effect at colonization. If Amamioshima Island populations were recently established from a small number of individuals colonized from the other two islands, the genetic diversity has not recovered since the founder event. Because the factors above are not mutually exclusive, they may influence the genetic diversity on Amamioshima Island simultaneously.

Genetic differentiation within and among islands
Genetic diversity among populations on the same island is relatively small on Amamioshima Island and Tokunoshima Island. On these islands, the numbers of migrants per generation (Nm) are larger than unity. By contrast, the genetic diversity among populations on Okinawajima Island is rather high, and gene flow per generation is <1. When Nm <1, the level of genetic diversity maintained within a population is more susceptible to genetic drift. Although the reason for high interpopulation genetic differentiation on Okinawajima Island is not obvious at present, the wide and discontinuous distribution of A. miyagii on the island may contribute to this.

At the level of interisland genetic diversity, genetic differentiation between Amamioshima Island and the other two islands is larger than that between Tokunoshima Island and Okinawajima Island. For example, two unique alleles are nearly fixed in two loci in Amamioshima Island populations (Fig. 2). This high genetic differentiation between Amamioshima Island and the other two islands results from highly restricted gene flow among them. This is probably because most of the populations on Okinawajima Island are located on the eastern side of the island, directly opposite those on Tokunoshima Island, which are located on the western side of the island (Fig. 1). By contrast, the populations on Amamioshima Island are located on the eastern side of the island; thus, these populations are likely isolated from the other two island populations.

In seed plants, gene flow occurs in two ways: via seed and via pollen. Because A. miyagii appears to outcross predominantly, these two pathways are both likely. Because seeds of A. miyagii can float on seawater, gene flow by seeds can occur by ocean current. However, migration by seed may be relatively rare because A. miyagii is not distributed on the islands located between these three islands. Although main pollinators to A. miyagii have not been identified, small insects such as halictid bees frequently visit A. miyagii flowers (S. Matsumura, Tohoku University, unpublished data). Such insects may move occasionally from one island to other, for example by flying on the winds of a tropical storm, and such an event seems to occur more easily between Okinawajima Island and Tokunoshima Island because these two island populations are directly opposite each other.

	FOOTNOTES

 
¹ The author thanks T. Denda, S. Horie, S. Matsumura, T. Nishino, M. Tabata, T. Yamashiro, J. Yokoyama, and M. Yokota for sampling materials; K. Saitou, K. Sato, Y. Uyama, A. Yamashiro, T. Yamashiro, and H. Yamaji for culture of plant materials; J. Yokoyama for statistical analysis; and two anonymous reviewers for comments. This study was partly supported by a grant-in-aid from the Japanese Ministry of Education, Science, Sports and Culture, the Global Environment Research Fund (F-1) from the Japan Environment Agency, and the grant from the Sumitomo Foundation.

² maki{at}mail.cc.tohoku.ac.jp

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This Article Abstract Full Text (PDF) Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via ISI Web of Science (12) Citing Articles via Google Scholar Google Scholar Articles by Maki, M. Search for Related Content PubMed Articles by Maki, M. Agricola Articles by Maki, M.