The Journal of Heredity 2002:93(2)
© 2002 The American Genetic Association 93:133-139
Evidence for Long Isolation Among Populations of a Pacific Cycad: Genetic Diversity and Differentiation in Cycas seemannii A.Br. (Cycadaceae)
From the Biology Department, Room N224, School of Pure and Applied Sciences, University of the South Pacific, Suva, Fiji (Keppel), Korea Forest Research Institute, 44-3 Omokchun-dong, Kwonsun-ku, Suwon 441-350, Republic of Korea (Lee), and Institute of Forest Genetics, USDA, Forest Service, Placerville, CA 95667 (Hodgskiss).
Address correspondence to Gunnar Keppel at the address above or e-mail: keppel_g{at}usp.ac.fj. Seok-Woo Lee can be contacted at the Forestry Research Institute, P.O. Box 24, Suwon 441-350, Republic of Korea, or e-mail: swlee66{at}hotmail.com.
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Abstract |
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The genetic structure of Cycas seemannii A.Br. (Cycadaceae), sampled throughout its range in Vanuatu, New Caledonia, Fiji, and Tonga, was studied using starch-gel electrophoresis. Twenty enzyme loci in 13 enzyme systems were examined. Low genetic diversity within populations (A = 1.2, P = 21.3, Ho = 0.047, and He = 0.057) and a high degree of differentiation among populations (FST = 0.594) were found. This, together with low gene flow estimates, suggests genetic drift by isolation to have been most critical to the current genetic structure of the species. Inbreeding may occur to some extent (FIS = 0.165). The decline in abundance of C. seemannii, coupled with the low level of genetic diversity, suggest that conservation strategies are urgently needed.
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Introduction |
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Cycads are an ancient gymnosperm lineage that tends to be long lived, outcrossing (by wind and/or insect pollination), and generally have restricted ranges (Norstog and Nicholls 1997). The few isozyme studies on cycads show that genetic variation within populations is generally low, while differentiation among populations is generally high (Ellstrand et al. 1990; Walters and Decker-Walters 1991; Yang and Meerow 1996). However, high intrapopulation variation and low interpopulation differentiation were observed in Macrozamia riedlei (Byrne and James 1991).
Cycas seemannii A.Br. (subsection Rumphiae), in contrast to most other cycads, has a relatively wide distribution, being native to the islands of Vanuatu, New Caledonia, Fiji, and Tonga (Figure 1). This distribution is highly fragmented, with many populations being separated by hundreds of kilometers of ocean. Observations suggest this dioecious, arborescent cycad is slow growing (5–15 cm/year), mainly outcrossing by wind and having relatively inefficient seed dispersal (Keppel 2001). Probably about 100 or more populations of C. seemannii exist, the exact number being difficult to assess because of its scattered distribution on numerous small islands. Within the Fiji group, 44 populations and 4112 individuals have been counted (Keppel 2002). The species is predominantly found in coastal habitats and, consequently, most populations have been disturbed by human activities, especially agriculture. As a result, the number of populations of C. seemannii has decreased (Doyle 1998; Hill 1994; Keppel 1999, 2002), making the study of this species for conservation purposes desirable.
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Genetic diversity within populations is influenced by the geographic distribution, mating system, method of seed dispersal, and method of reproduction of a species (Hamrick et al. 1992). Species with restricted ranges and/or discontinuous distribution, selfing and/or mixed mating systems, and gravity- or wind-dispersed seeds often have low genetic diversity within populations as compared to species with widespread geographic ranges, outcrossing mating systems, and ingested or animal attached seed dispersal (Hamrick et al. 1979, 1992). Differentiation among populations was found to be greatest in autogamous monocarpic annuals that occur in the early stages of succession and have gravity-dispersed seeds (Loveless and Hamrick 1984). However, the above are generalized trends that explain only a small part of the total genetic variation observed, and the variation within populations and the differentiation between them are also the result of a species' unique evolutionary history (Hamrick and Godt 1996; Hamrick et al. 1992).
The objectives of this study were to identify and describe the amount and distribution of genetic variation in C. seemannii using isozyme markers and to compare the results to those from other cycad species. Starch-gel electrophoresis, which remains popular for investigating the genetic variability within plant populations (Godt and Hamrick 1998; Ledig et al. 1997, 1999; Lee et al. 1998), was used in this study. This information will be valuable in designing strategies to conserve the species.
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Methods and Materials |
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Five populations from New Caledonia, Vanuatu, Fiji (2), and Tonga were studied (Figure 1). The population from New Caledonia is located at the Baie des Tortues, a few kilometers southeast of Bourail. About 200 cycads (estimated density (
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150 plants/ha) grow on a limestone terrace on cliffs overhanging the ocean in a thicket dominated by Leucaena leucocephala, a serious weed on the island (Jaffré T, personal communication). Signs of human disturbance were evident in the form of pedestrian tracks through the Leucaena thicket.
In Vanuatu, the cycad population consists of hundreds of individuals ( 55 plants/ha) on steep slopes densely covered by lowland rainforest best described as "medium-stature forest heavily covered with liana" (Mueller-Dombois and Fosberg 1998). The population is in close proximity to a place known as Devils Point on the island of Efate.
A dense ( 350 plants/ha) stand of more than 1000 cycads was located on the southernmost tip of the island of 'Eua in Tonga. As in New Caledonia, the trees were growing on a limestone terrace above almost vertical cliffs, descending for about 100 m. They form a prominent component of the understory of the beach forest and grow on a thin to nonexistent soil layer with limestone outcrops. The vegetation is similar to the cliff vegetation and Hernandia-Terminalia coastal forest described by Drake et al. (1996).
In Fiji, a wild cycad population growing among a planted stand of commercial pine, Pinus caribaea, at the Nabou Pine Station on the leeward, drier, western side of Viti Levu was studied. The pine plantation is located on hilly terrain at about 210 m altitude, 5–10 km from the coast. Tree clusters consisting of 20–70 individuals are present on the plantation and are fragmented relics of a single, large population. Samples were obtained from two such fragments, one consisting of about 60 trees ( 200 plants/ha) and the other about 20 trees ( 100 plants/ha). These are on slopes with talasiga vegetation. In the botanical sense talasiga, a Fijian word pronounced "talasinga" and literally meaning "sunburnt land," refers to Fiji's pyrophytic grasslands and shrub lands, most of which resulted from burning of the original forest cover by man. The resulting soil erosion is thought to be responsible for the poor soils of the talasiga (Latham 1979, 1983; Southern 1986).
The final population sampled was from a forest near Naduri on the other major island of Fiji, Vanua Levu. Samples were obtained from individuals transplanted to the Sigatoka Valley on Viti Levu, Fiji, because the natural population is under threat through annual burning by man.
Leaf sections from at least 30 different individuals of C. seemannii were collected in each of the five populations (Table 1, Figure 1). The samples were wrapped in moist paper towels, packed in a plastic bag, and stored at 6°C at the University of the South Pacific, Suva, Fiji, before being sent by air to the Institute of Forest Genetics, Placerville, CA, in an insulated shipping container and stored at 4°C until needed.
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Two hundred milligrams of foliar sample was crushed to powder in 400 µl of extraction buffer (0.2 M phosphate buffer, pH 7.5, 10% PVP-40, 2% sodium ascorbate, 0.3% bovine albumin, 0.1% dithiothreitol; Hodgskiss 1999) and 10 ml of liquid nitrogen. The powder was immediately transferred, with a chilled spatula, into a 2 ml microtube and placed on dry ice. The 2 ml aliquots were then stored at -80°C until use.
Prior to electrophoresis, the enzyme extracts were thawed in slushy water ice, centrifuged, the supernatant absorbed onto wicks, and the wicks loaded into the starch gels. The procedures of starch-gel electrophoresis were similar to those described by Conkle et al. (1982) and Hodgskiss (1999). Twenty presumptive loci of 13 enzyme systems were consistently scored and used in the statistical analysis (Table 2).
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BIOSYS1 (Swofford and Selander 1989) was used to calculate the allele frequencies, the mean number of alleles per locus (A), alleles per polymorphic locus (AP), percentage of polymorphic loci (P; at the 95% level and for the entire population also at the 99% level), observed mean heterozygosity (Ho), expected heterozygosity (He) in Hardy–Weinberg equilibrium (Nei 1978), Wright's F statistics (FIS, FIT, and FST), and to construct a Wagner tree based on Edwards distances (Cavalli-Sforza and Edwards 1967). To test whether FIS differs significantly from panmixia (FIS = 0) at each polymorphic locus, a one-tailed contingency chi-squared test of Ho (FIS = 0) (Li and Horvitz 1953) was performed. POPGENE, version 1.21 (Yeh et al. 1997), was used to calculate the pairwise genetic distances (D) (Nei 1972) between the populations investigated. Genetic data analysis (Lewis and Zaykin 2001) software was used to calculate the coancestry coefficient (
) (Reynolds et al. 1983; Weir and Cockerham 1984). Gene flow was calculated from the FST estimates using Nm = (1/FST - 1)/4 (Wright 1951). GenePop (Raymond and Rousset 1995) was used to calculate gene flow by the rare allele method (Slatkin 1985). |
Results |
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Of the 20 loci, 10 (AAT-1, AAT-2, FDH-1, FBA-1, G6PDH-1, GTDH-1, MDH-1, MDH-3, UGUT-1, and UGUT-2) were invariant in all populations. Four loci (MDH-4, PGM-1, TPI-1, and TPI-2) were polymorphic in one population each. A locus was considered polymorphic when any variants were observed in any population. Four of the five populations had a total of seven private alleles (i.e., an allele found in only one population) at seven loci (see Table 3). The population on 'Eua had the largest number of private alleles.
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The percentage of polymorphic loci per population (P) ranged from 5 to 30% (average, Pp = 19%; pooled, Ps = 35% at the 95% level and 50% at the 99% level), the number of alleles per locus (A) ranged from 1.1 to 1.3 (Ap = 1.2, As = 1.8), the number of alleles per polymorphic locus (AP) ranged from 2.0 to 2.3 (APp = 2.1, APs = 2.7), the observed heterozygosity (Ho) ranged from 0.000 to 0.088 (Hop = 0.047, Hos = 0.048), and the expected heterozygosity (He, unbiased estimate) ranged from 0.010 to 0.079 (Hep = 0.057, Hes = 0.138) (Table 4).
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Observed heterozygosity was less than the expected heterozygosity in three of the five populations, which suggests some degree of inbreeding and/or Wahlund effect. Wright's FIS (0.165; Table 5), a measure of the deviation of the genotype proportions from the Hardy–Weinberg equilibrium at the population level, supports the occurrence of inbreeding and/or a Wahlund effect, as positive values suggest an excess of observed homozygotes (Wright 1965). Also, at all loci the FIS values showed significant (P = .001) deviations from panmixia (Table 5). The high value of Wright's FIT (0.661) indicates a homozygote excess at the species level, as well.
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Wright's F statistics (Table 5) partition genetic diversity into among and within population components. Diversity among populations, FST, was 0.594, which means that 40.6% of the observed variation resided within populations. In other words, most of the genetic variation observed in C. seemannii was due to interpopulational differentiation. Nei's (1972) genetic distance (mean value of D = 0.117) further indicated substantial differentiation among populations. The genetic distance between populations was strongly related to geographic distance (r2 = 0.741, P = .001; Figure 2).
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The Wagner tree (Figure 3) shows that the groups of Vanuatu/New Caledonia and those of Tonga/Fiji were first to split of the populations investigated. Within the Fiji/Tonga group, the population in Nabou, Viti Levu, Fiji is closest to the root.
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Indirect estimates of gene flow between populations were low. Wright's FST and Weir and Cockerham's
were 0.594 and 0.695, respectively, and the corresponding Nm values were 0.17 and 0.11 migrants per generation, respectively. Using Slatkin's method of private alleles, a slightly lower estimate of 0.07 migrants per generation was obtained. Irrespective of the exact value, rates of gene flow are either now or were in the recent past low enough to permit extensive differentiation between populations by random genetic drift (Wright 1969). |
Discussion |
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The genetic diversity of C. seemannii, an outcrossing cycad with a discontinuous distribution and mainly or entirely gravity- and ocean-dispersed seeds (Keppel 2001), was, at the species level [As = 1.8, Ps = 50.0 (at the 99% level), and Hes = 0.138], similar to that for plants in general (As = 1.97, Ps = 51.3, and Hes = 0.150) (Hamrick et al. 1992). However, at the population level (Ap = 1.2, Pp = 19.0, and Hep = 0.057) values were lower compared to plant species in general (Ap = 1.52, Pp = 34.6, and Hep = 0.113) and those of other tree species with similar ecological and biological traits (Hamrick et al. 1992). The values were similar to those reported for woody plants with endemic ranges (Ap = 1.48, Pp = 26.3, Hep = 0.056, and As = 1.82, Ps = 42.5, Hes = 0.078) (Hamrick et al. 1992) and slightly lower than those reported for other cycad species (Table 6). They were also lower than those reported for other gymnosperms and for dicotyledons (Table 6).
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More than 59% of the total genetic variation in C. seemannii was among populations (FST = 0.594; Table 4), which indicates high differentiation. This is also reflected in Nei's (1973) GST value (another estimate of Wright's FST) of 0.418, which is much higher than that for other woody plants with similar ecological traits (GST = 0.119 for trees with a tropical distribution; GST = 0.077 for species that outcross by wind pollination; GST = 0.131 for seeds dispersed by gravity) (Hamrick et al. 1992) and of woody plants in general. It is also higher compared to the GST values reported previously for other cycads (Table 6).
Therefore results of this study imply that while there is considerable genetic diversity in the species as a whole, this diversity is distributed over the various populations, each having comparatively little genetic variation. The genetic data also support the hypothesis that low intrapopulation variation and high interpopulation differentiation are biological and evolutionary characteristics of cycads (Yang and Meerow 1996).
Reduced levels of intrapopulation diversity may occur for several reasons, including reproductive mode, phylogenetic history, genetic drift, founder effects, or combinations of these and other factors. One possible explanation for the low amount of genetic variation observed in C. seemannii may be its mating system. Although the species appears to be wind pollinated (Keppel 1999, 2001), some degree of inbreeding may occur through consanguineous mating. The gravity-disseminated seeds of C. seemannii and the comparatively heavy pollen of cycads (Norstog and Nicholls 1997) may promote mating between individuals in close proximity within populations.
Fixation indices can be used to estimate the outcrossing rate (t) under the assumption that equilibrium has been reached:
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In addition to inbreeding, the Wahlund effect (Hartl and Clark 1989) may provide a partial explanation for the excess of homozygotes within populations. The just-described inbreeding scenario, together with the heavy pollen and gravity-disseminated seeds of C. seemannii, suggest that different family groups within a population may form, each characterized by a slightly different genetic composition. This phenomenon of "family structure" has been found in a number of conifers with wind-disseminated seeds (Furnier and Adams 1986; Knowles 1984). More detailed studies, sampling more individuals per population, and recording exact locality information for each individual of C. seemannii would be needed to test whether family structure exists in populations of this species.
Since C. seemannii usually occurs in rather small populations isolated on different Pacific islands, another plausible explanation for the lack of genetic variation may be genetic drift and/or the founder effect. Most populations are likely to have been founded by the chance arrival of a few seeds carrying a fraction of the genetic variation of their source population. It has been suggested that in small populations, such as that of a few founders, genetic drift may result in a reduction in genetic variation (Neigel 1996). Natural selection could also explain the genetic differentiation between populations (Baur and Schmid 1996). However, considering that all populations studied here are distributed in similar climatic conditions as well as similar edaphic conditions, especially for the populations on 'Eua and New Caledonia, drift by isolation is likely to be the more important factor.
The large number of private alleles with high frequencies indicates genetic drift and may also imply that this species is now in the process of speciation. However, to address this issue, further studies using molecular marker systems as well as more detailed morphometric studies are needed. The latter should focus on the morphology of the reproductive structures of C. seemannii and not only foliage, the morphology of which is not correlated with the genetic differences observed (Keppel 1999).
As no insect pollinator for the species has yet been identified, most populations are distant enough to make gene flow among them via their relatively heavy pollen unlikely. In addition, seed dispersal appears to occur mostly via gravity and ocean currents. Many populations are growing too distant from the coast and are geographically too far apart (Figure 2) for ocean dispersal to provide consistent gene flow. As a result, gene flow via pollen and seeds may be very restricted. This hypothesis is supported by the low estimates of Nm and the significant correlation between the genetic and geographic distances (r = 0.876). Theoretical studies have indicated that a relatively small amount of gene flow (Nm > 1) is sufficient to prevent population differentiation due to genetic drift (Wright 1965). Therefore drift by isolation and/or the founder effect may explain the high degree of population differentiation.
Of all the populations investigated, the one on Vanuatu had the greatest genetic variation. This may suggest that it is the oldest of all the populations investigated, but may also be due to the large population size. A possible scenario for the colonization sequence of this easternmost Cycas species would therefore be an initial colonization of the Vanuatu archipelago and later spread to other Pacific islands. The great abundance of C. seemannii on Vanuatu and, possibly, the Wagner tree (Figure 3) can also be seen to support this hypothesis. Hence the Wagner tree could be interpreted to be consistent with the theory of a relatively recent, stepwise migration from Australasia into the Pacific (Hill 1996). This theory of an eastward migration into the Pacific has also been proposed for other plant groups (Balgooy et al. 1996; Thorne 1963; Woodroffe 1987). However, neither ocean currents, which mainly flow from east to west between the island groups investigated (Wauthy 1986), nor human colonization in the opposite direction, seem to offer additional support, as the latter was a rapid process that occurred some 3000 years ago (Austin 1999; Cann and Lunn 1996; Diamond 1988; Kirch and Hunt 1988) and could not explain the high genetic differentiation observed.
Many of the cycad's natural populations, for example, that in Nabou on Viti Levu, Fiji (see Materials and Methods), have been much disturbed and decimated by human activities. Small populations rapidly lose genetic variability (Frankel and Soulé 1981) and this may explain why the lowest amount of genetic variability was found in the Nabou population (Table 4).
Considering the low level of genetic diversity in C. seemannii and its declining abundance, a strategy for the conservation of its genetic resources is urgently needed. The maintenance of genetic diversity is crucial to the survival of organisms, because it allows them to evolve and adapt to changing environmental conditions (Frankel and Soulé 1981; Franklin 1980; Lande 1988; Lynch 1996; Maxted et al. 1997). Bearing this in mind, the lower genetic diversity observed in the population in Nabou, likely to be the result of fragmentation and decimation of the original cycad population for the establishment of a pine plantation, makes the long-term survival of this particular population doubtful.
We suggest the following guidelines for the conservation of genetic resources of C. seemannii:
- Considering the high genetic differentiation among populations, preservation of any one population will not protect all the variation in the species. Therefore several populations throughout the entire range should be considered for conservation. If possible, all populations studied here should be conserved, as a minimum.
- Some populations, such as that in Nabou, have been seriously disturbed by anthropogenic activities. In such cases, seeds could be collected and seedlings grown and transplanted back to the disturbed sites. If seedlings survive and mature, this would help maintain an effective size, which is important, as indicated by the loss of genetic diversity in the Nabou population. For this approach to be effective, and to enhance natural regeneration, anthropogenic impacts on natural populations must be reduced. Fire and livestock grazing especially threaten young plants and seedlings.
- Ex situ conservation strategies, based on seed and germplasm collections, in botanical gardens or other institutions (i.e., field ex situ conservation) would be of practical value for the conservation of genetic diversity in C. seemannii, as in the ex situ population from Naduri, which is located on a farm in Sigatoka Valley.
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Acknowledgments |
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This work was funded by the University of the South Pacific and the French Embassy, Suva, Fiji, through the French Ministry of Foreign Affairs. The latter also financially supported the Master's studies of G. Keppel. We are very grateful for the assistance we received from these two organizations. M.F. Doyle had the idea for this project. We are indebted to various people and organizations that significantly contributed to its success: L'Institut Francaise de Recherche Scientifique pour le Development en Cooperation (IRD), Noumea, New Caledonia; the Department of Forestry of the Republic of Vanuatu, especially P. Ala and S. Chanel; the Institute of Forest Genetics, Placerville, CA; the Nabou Pine Station; and V. Tonga, D. Watling, P. F. Newell, and T. Jaffré. We also would like to acknowledge the helpful reviews by F. T. Ledig and S. A. Ghazanfar.
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Footnotes |
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Corresponding Editor: Brandon Gaut
Received December 12, 2000
Accepted December 31, 2001
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