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Patterns of genetic variation and demography of Cycas taitungensis in Taiwan.

 I. Abstract
 II. Introduction
III. Materials and Methods
 IV. Results and Discussion
 A. Ecological Survey
 B. Population Structure
 C. Electrophoretic Analysis
 V. Conclusion
 VI. Acknowledgments
VII. Literature Cited

II. Introduction

Cycas taitungensis C. F. Shen, K. D. Hill, C. H. Tsou & C. J. Chen, the Taitung cycads, has long been misidentified as C. taiwaniana (Shen et al., 1994). The species is endemic to Taiwan and grows on steep, stony slopes in the two isolated nature reserves in altitudes between 300 and 900 m. The main population is located in the 300-hectare Taitung Cycads Nature Preserve in the Lu-Yeh River valley at 120[degrees]57'E, 22[degrees]52'N; the smaller one, in Coast Range Taitung Cycads Nature Preserve at 121[degrees]15'E, 23[degrees]05'N. The two sites are 40 km apart. The plant is a good model for studying the evolution, conservation and biogeography of plant biology (Walters & Decker-Waiters, 1991; Given, 1994; Norstog & Nicholls, 1997; De Laubenfels & Adema, 1998; Chiang et al., 1999). However, detailed population study of C. taitungensis has been neglected in the past, thereby reflecting poor ecology and genetic knowledge of the species. In addition, populations of C. taitungensis, as well as other species of Cycas in Asia, were strictly distributed and critically threatened due to overcollection and human-disturbed habitat loss (Jones, 1993; Given, 1994; Norstog & Nicholls, 1997).

A series of genetic studies of the cycads showed that a moderate level of genetic diversity occurs in some species (Norstog & Nicholls, 1997). For example, patterns of allozyme variation was found to be high within populations and low among populations in Macrozamia riedlei from southwestern Australia (Byrne & James, 1991) and Zamia pumila of the northern Bahamas (Walters & Decker-Waiters, 1991). On the other hand, Ellstrand et al. (1990) reported high genetic differentiation among the populations of M. communis in New South Wales, Australia. Keppel and Doyle (pers. comm.) analyzed the Cycas rumphii complex in the insular Southwest Pacific by isozyme electrophoresis and found that most of the species have low intrapopulational genetic variation.

To interpret heterogeneity of allelic frequencies within and among populations, it has been shown that the genetic variation can be influenced by mating system, seed-dispersal mechanism, succession status, and geographical range (Loveless & Hamrick, 1984; Hamrick et al., 1989). On the other hand, species with inbreeding, insect pollination, and local and/or small populations due to drift and reduced gene flow have higher levels of genetic differentiation among populations. The observation that the male cone of this species released fruit aromatic odors implies that the Taitung cycad is an insect-pollinated species, like other species of Cycas (Jones, 1993; Tang, 1987); i.e., C. rumphii (Niklas & Norstog, 1984), C. media (Ornduff, 1991), C. armstrongii (Ornduff, 1992), and C. revoluta (Vorster, 1995). Thus, the level of genetic differentiation between the microhabitat subpopulations that are on the opposite banks of the river within site may be high because C. taitungensis is a species with small population sizes, early successional status, long life span, insect pollination, and gravity seed dispersal and/or rodent dispersal within a population.

This study presents a preliminary ecological and genetic analysis of Cycas taitungensis occurring in the reserve areas of southeastern Taiwan. The purpose of the present study was to find out whether genetic data are correlated with those of the environment. We address first the different ecological situations among the subpopulations located on opposite slopes and then the level of genetic differentiation between subpopulations within a population in the reserve area as compared with those in the main large population and another small population.

III. Materials and Methods

The main population of Cycas taitungensis Shen, Hill, Tsou & Chen, along the Lu-Yeh River, is distributed on both north- and south-facing slopes in the Taitung Hong-Yeh Village Cycad Nature Reserve (CY). The small population of the Coast Range Natural Preserve (CC) is found on the east-facing slopes along the Coastal Mountain Range some 40 km from the main group (Fig. 1). The surrounding hillsides are dominated by Machilus-Castanopsis forest. The weather data, such as monthly precipitation, relative moisture, and air temperature for 1997-1999, were collected from the Taiwan Central Weather Bureau. The daily air temperature and solar radiation data at CY were collected from July 1997 to May 1999 and showed that radiation ranged from 0 to 50 MJ/[m.sup.2] per day. The highest air temperature, 27[degrees]C-34[degrees]C, occurred in the summer; the low air temperature, 17[degrees]C-25[degrees]C, occurred in the winter; and the mean air temperature was 20[degrees]C-29[degrees]C. Yearly precipitation was 2400 mm in 1998. The dry seasons was from May to October, with the most monthly precipitation, 1028 mm, falling in October; the rainy season was from November to April, with the least monthly precipitation, 19 mm, falling in January. Thus, the climate is a typical tropical heavy moist situation.


In the main population, CY, a pair of Cycas taitungensis subpopulations from both north-facing slopes (NCY) and south-facing slopes (SCY) were selected for ecological and genetic evaluation. Information on 12,250 individuals was provided by the Taiwan Forestry Bureau and reorganized. Data on individual height, diameter at breast height (DBH), leaf number, and sex of each plant were analyzed to determine the sex ratio, the growth form, and the distribution within and between populations. Two yearly field-study cycles were repeated monthly from October 1997 to May 1999. Further analyses were performed using Microsoft Excel.

Material on Taitung cycads was obtained during the spring and summer of 1997 and 1998. Young, healthy leaves of adult trees were collected. All sampled plants were greater than 10 cm DBH. The minimum physical distance between the individuals was approximately 10 m within CY. Forty samples from the subpopulations from both NCY and SCY were collected. Meanwhile, 31 samples from CC were collected for isozyme studies.

Leaf materials were stored at -70[degrees]C, ground to powder in liquid nitrogen, and assayed for 13 enzyme systems. Ten drops of extraction buffer (Mitton et al., 1979; Conkle et al., 1982) were stirred together with the powder, forming a paste that was immediately chilled on ice. As the samples thawed, the leaf extract was absorbed by paper wicks. Preparation of buffer systems and enzyme staining solutions followed standard protocols (Soltis et al., 1983; Wendel & Weeden, 1989; Murphy et al., 1996). Thirteen enzyme systems were used: asparate aminotransferase (AAT), creatine kinase (CK), esterse (EST), fructose-bisphosphatase (FBP), glucose--6-phosphate isomerase (GPI), glycerol-3-phosphate dehydrogenase (G3PDH), isocitric dehydrogenase (IDH), malate dehydrogenase (MDH), malate dehydrogenase ([NADP.sup.+]) (MDHP), peroxidase (PER), 6-phosphogluconate dehydrogenase (PGDH), phosphoglucomutase (PGM), and superoxide dismutase (SOD). To test the genetic structure within and between populations, Wright's F-statistics (1943a, 1943b, 1965) were calculated and then analyzed using BIOSYS-1. To compare genetic relationships across localities for the NCY and SCY subpopulations, Nei's genetic distance (Nei, 1973, 1977; Nei et al., 1978) and Nei and Chesser's (1983) diversity statistics for unequal sample sizes were used with BIOSYS-2 (Swofford & Selander, 1997).

IV. Results and Discussion


A statistical survey of the morphology of plants on northern (SCY) and southern (NCY) slopes of the Lu-Yeh River in the reserve area showed that the annual growth of plants was 5 cm. The maximum recorded height of 520 cm indicated that most plants studied are less than 100 years old.

Ratios of plant height to DBH and plant height to leaf were determined. A positive correlation between the two ratios was found in plants less than 1 m tall (n = 347 and 495, respectively). The correlation broke down as the diameter of the tree trunk and the number of leaves produced per year reached their maximum size.

The 1997-1999 CY survey showed that the average heights of female and male plants was 201.0 [+ or -] 63.7 cm (n = 104) and 245.2 [+ or -] 128.8 cm (n = 63), respectively, in the 177 coning individuals, with the sex ratio 1.7:1. The annual reproduction of female plants was highly variable, with seed numbers ranging from 80 to 400 per plant. The dry weight of individual seeds was 9.27 [+ or -] 1.39 g. The proportion of reproductive plants in 1998 was 41%.


To examine subpopulation variation within the main populations, we analyzed the patterns of leaf production of each subpopulation. Plants with a trunk diameter greater than 20 cm were selected for the calculation. Samples of 297 individuals from NCY and 149 individuals from SCY slopes were compared to estimate whether selection acts in aspect-defined microhabitats. The results indicated that leaf production and DBH were significantly different between subpopulations in NCY and SCY (p <0.05 and p <0.01, respectively). Meanwhile, we found that the numbers of middle-aged adults in SCY were significantly lower than were those in NCY and in the total population. For example, the number of young plants with heights of 20-70, 70-120, and 120-170 cm in SCY were significantly less were than those in NCY and in the total preserved area. This is probably caused by illegal depletion, because these sizes are easily removed from the reserve area. As a matter of fact, SCY is much easier to reach than is NCY, and excess human activities exert a strong impact on the population structure of the cycad in the SCY subpopulations.

Vegetative growth in both NCY and SCY was measured and compared for subpopulation differentiation using the Wilcoxon Two-sample test. We found that plant height in SCY (222.40 [+ or -] 90.14 cm) and NCY (169.11 [+ or -] 101.99 cm) was significantly different (p <0.001). Also, plant trunk DBH between SCY and NCY (24.18 [+ or -] 3.83 cm versus 21.73 [+ or -] 6.83 cm) and leaf-production numbers (22.77 [+ or -] 12.46 versus 20.17 [+ or -] 7.71) were significantly different (p <0.05). The results clearly demonstrate that subpopulations in different microhabitats on opposite slopes within the same site grew differently, thus displaying a completely different ecological appearance on the two riverbanks.


The 13 isozyme systems used in this study yielded 21 loci and 27 alleles. Among them, four loci (AAT, EST, MDH, and SOD) contained polymorphism. The remaining loci were found to be fixed in all populations. The average number of alleles per locus (A) for all population combined was 1.1, with a mean proportion of loci polymorphism of 10.7%. The mean observed heterozygosity ([H.sub.O]) and expected heterozygosity ([H.sub.E]) in all populations were 0.021 [+ or -] 0.009 and 0.039 [+ or -] 0.008, respectively.

The detected genetic variability ([H.sub.E]) of Cycas taitungensis was extremely low compared with those of the other cycad populations such as C. pectinata (0.076-0.078) and C. siamensis (0.036-0.162) (Yang & Meerow, 1996), Macrozamia communis (0.09), as well as with those of other gymnosperms (0.160) (Hamrick & Godt, 1989) and other endemic species (0.063).

Values of fixation indices ([F.sub.IS]) ranged from -0.049 at locus MDH-1 to 0.726 at AAT-1 with a mean of 0.432, suggesting that inbreeding or assortative mating had occurred within populations. The F-statistics provide additional evidence that little genetic differentiation among subpopulations exists ([F.sub.ST] = 0.051). An excess heterozygosity occurring at the SOD locus (p <0.05) implies that the genetic differentiation among the populations may have been partially skewed by the distribution of this locus.

The gene diversity (Nei, 1973) for all populations ([H.sub.T]) was calculated using total limiting variance as proposed by Wright (1978). Levels of [H.sub.T] varied from 0.058 to 0.498 (Table I). Meanwhile, the degree of genetic differentiation among the populations ([G.sub.ST]) was 0.032, meaning that nearly 3.2% of genetic variability occurs among the four population/subpopulations studied. Pairwise genetic distance between subpopulations and populations was estimated (Nei, 1978), and very little distance (0.001) was found between CY and CC populations. However, the genetic distance between opposite subpopulations (SCY versus NCY) within this site (three subpopulations in CY) was 0.02, twice that between populations.

The results based on genetic distance and F-statistic analyses clearly indicate that the level of genetic differentiation between subpopulations within 1 km in the reserve area is higher than those across populations that are 40 km apart. Low population differentiation revealed by G-statistic analysis correlated with low [G.sub.ST] values (0.032). A high gene flow between the two populations of Taiwan's cycads was also detected by allozyme analysis (Sun, 1998; Lin et al., in press), in which the values of [N.sub.M] and [F.sub.ST] were 7.1 and 0.034, were found. Low genetic differentiation between populations of cycads based on allozyme data, even though the [N.sub.M] estimated from different data sets did not match, suggests a distinct identity between the two extant populations. The geographical separation may have been caused by recent fragmentation events due to human disturbance in the past few hundred years. The possible renewed isolation caused a low degree of genetic differentiation via genetic drift.

V. Conclusion

Subpopulations of Cycas taitungensis located on opposite slopes within the reserve area along the Lu-Yeh River show genetic differentiation that reflects potential contributions mostly from natural selection. It is likely that environmental stress, caused by aspect-defined microhabitat variation, has had a major influence on patterns of genetic structure within populations and has thereby promoted genetic differentiation between adjacent subpopulations on opposite slopes of the same valley.

VI. Acknowledgments

This study was supported by a grant from the Council of Agriculture of Taiwan to S. Huang (87-2101-01). We would like to thank C. S. Wu and his colleagues at the Taitung Forestry District Office for their assistance in sampling.

VII. Literature Cited

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Chiang, Y. C., S. Huang, C. H. Chou & T. Y. Chiang. 1999. Organelle DNA phylogeography of Cycas taitungensis, a relic species in Taiwan. P. 341 in Abstracts: XVI International Botanical Congress, St. Louis, USA, August 1-7.

Conkle, M. T., P. D. Hodgskiss, L. B. Nunnally & S. C. Hunter. 1982. Starch gel electrophoresis of conifer seeds: A laboratory manual. U.S. Department of Agriculture, Forest Service, Pacific Southwest Forest and Range Experiment Station, Berkeley, CA.

De Laubenfels, D. & F. Adema. 1998. A taxonomic revision of the genera Cycad and Epicycas gen. Nov. (Cycadaceae). Blumea 43: 351-400.

Ellstrand, N., R. Ornduff & J. M. Clegg. 1990. Genetic structure of the Australian cycad, Macrozamia communis (Zamiaceae). Amer. J. Bot. 77: 677-81.

Given, D. R. 1994. Principles and practice of plant conservation. Chapman & Hall, New York

Hamrick, J. L. & M. J. W. Godt. 1989. Allozyme diversity in plant species. Pp. 46-55 in A. D. H. Brown, M. T. Clegg, A. L. Kahler & B. S. Wies (eds.), Plant population genetics, breeding, and genetic resources. Sinauer Associates, Sunderland, MA.

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Jones, D. 1993. Cycads of the world. Smithsonian Institution Press, Washington, DC.

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Shen, C. F., K. D. Hill, C. H. Tsou & C. J. Chen. 1994. Cycas taitungensis C. F. Shen, K. D. Hill, C. H. Tsou & C. J. Chen, sp. nov. (Cycadaceae), a new name for the widely known cycad species endemic in Taiwan. Bot. Bull. Acad. Sin. 35: 133-140.

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(1) Department of Biology National Taiwan Normal University Taipei, Taiwan 105

(2) Institute of Botany Academia Sinica Taipei, Taiwan 115
Table I
Analysis of F-statistics and gene diversity at four loci
among the four studied subpopulations of Cycas taitungensis


Locus [F.sub.IS] [F.sub.IT] [F.sub.ST]

Malate dehydrogenase (MDH) -0.049 -0.031 0.017
Asparate aminotransferase (AAT) 0.726 0.736 0.035
Superoxide dismutase (SOD) -0.081 0.030 0.103
Esterse (EST) 0.078 0.136 0.063
 Mean 0.432 0.460 0.051


Locus [H.sub.T] [H.sub.S]

Malate dehydrogenase (MDH) 0.058 0.059
Asparate aminotransferase (AAT) 0.498 0.496
Superoxide dismutase (SOD) 0.160 0.147
Esterse (EST) 0.122 0.205
 Mean 0.210 0.205

Locus [D.sub.ST] [G.sub.ST]

Malate dehydrogenase (MDH) 0.0001 0.010
Asparate aminotransferase (AAT) 0.002 0.004
Superoxide dismutase (SOD) 0.013 0.081
Esterse (EST) 0.004 0.033
 Mean 0.005 0.032
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Author:Huang, Shong; Hsieh, Hui-Ting; Fang, Kang; Chiang, Yu-Chung
Publication:The Botanical Review
Geographic Code:9TAIW
Date:Jan 1, 2004
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