Evidence for Long Isolation Among
Populations of a Pacific Cycad: Genetic
Diversity and Differentiation in Cycas
seemannii A.Br. (Cycadaceae)
G. Keppel, S.-W. Lee, and P. D. Hodgskiss
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 ⴝ 1.2, P ⴝ 21.3, Ho ⴝ 0.047, and He ⴝ 0.057)
and a high degree of differentiation among populations (FST ⴝ 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 ⴝ 0.165). The decline in abundance of C. seemannii,
coupled with the low level of genetic diversity, suggest that conservation strategies
are urgently needed.
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 441350, Republic of Korea ( Lee), and Institute of Forest
Genetics, USDA, Forest Service, Placerville, CA 95667
( Hodgskiss). 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. Address correspondence to Gunnar Keppel at the address
above or e-mail: [email protected]. Seok-Woo Lee can
be contacted at the Forestry Research Institute, P.O.
Box 24, Suwon 441-350, Republic of Korea, or e-mail:
[email protected].
2002 The American Genetic Association 93:133–139
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.
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 re-
133
Table 1. Locations of the five study populations of Cycas seemannii
a
Population code
Location
Longitude, latitude
EU
NB
NC
SG
VA
’Eua, Tonga
Nabou, Viti Levu, Fiji
Baie des Tortues, New Caledonia
Naduri, Vanua Levu, Fijia
Devils Point, Efate, Vanuatu
21⬚3S,
17⬚6S,
21⬚4S,
16⬚3S,
17⬚1S,
174⬚6W
177⬚2E
165⬚3E
179⬚1E
168⬚1E
Plants were collected from young, transplanted trees at D. Watling’s farm in the Sigatoka Valley.
mains 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.
Methods and Materials
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 (␳)
艐 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 (␳ 艐 55
plants/ha) on steep slopes densely cov-
Figure 1. Location of study sites.
134 The Journal of Heredity 2002:93(2)
ered 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 (␳ 艐 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 (␳ 艐 200 plants/ha) and the other
about 20 trees (␳ 艐 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.
Two hundred milligrams of foliar sample
was crushed to powder in 400 ␮l 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).
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
Table 2. Enzyme systems consistently resolved in this study
Enzyme
Locus abbreviation
EC no.
Buffer
Aspartate aminotransferase
Fluorescent esterase
Formaldehyde dehydrogenase
Fructose-biphosphate aldolase
Glucose-6-phosphate dehydrogenase
Glutamate dehydrogenase
Isocitrate dehydrogenase
Malate dehydrogenase
Phosphogluconate dehydrogenase
Phosphoglucomutase
Shikimate dehydrogenase
Triose-phosphate isomerase
UTP-glucose-1-phosphate uridyltransferase
AAT-1, -2
FEST-1
FDH-1
FBA-1
G6PDH-1
GTDH-1
IDH-1
MDH-1, -2, -3, -4
PGDH-1, -2
PGM-1
SKDH-1
TPI-1, -2
UGUT-1, -2
2.6.1.1
3.1.1.1.2.1.1
4.1.2.13
1.1.1.49
1.4.1.2
1.1.1.42
1.1.1.37
1.1.1.44
5.4.2.2
1.1.1.25
5.3.1.1
2.7.7.9
B
A
A
D
B
B
D
D
D
D
D
A
B
a
Nomenclature and abbreviations follow Murphy et al. (1996), based on IUBNC. EC no. ⫽ Enzyme Commission
number.
a
A, B, and D refer to buffers A (tris citrate/lithium borate), B (citrate/sodium borate), and a pH 8 version of buffer
D (morpholine citrate) of Conkle et al. (1982).
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
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, TPI1, 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
Table 3. Allele frequencies for 10 polymorphic loci in five populations of Cycas seemannii
Allele frequency
Locus
Allele
’Eua
Nabou
New
Caledonia
Naduri
Efate
FEST-1
1
2
3
1
2
3
1
2
3
4
1
2
1
2
1
2
1
2
1
2
3
1
2
1
2
0.000
0.000
1.000
1.000
0.000
0.000
0.966
0.034
0.000
0.000
0.621
0.379
0.167
0.833
1.000
0.000
0.393
0.607
1.000
0.000
0.000
1.000
0.000
0.950
0.050
0.063
0.938
0.000
1.000
0.000
0.000
0.958
0.042
0.000
0.000
1.000
0.000
1.000
0.000
1.000
0.000
1.000
0.000
1.000
0.000
0.000
1.000
0.000
1.000
0.000
1.000
0.000
0.000
0.328
0.500
0.172
0.000
0.000
0.931
0.069
1.000
0.000
1.000
0.000
1.000
0.000
1.000
0.000
0.154
0.846
0.000
1.000
0.000
1.000
0.000
0.000
0.000
1.000
0.946
0.054
0.000
0.611
0.389
0.000
0.000
1.000
0.000
0.857
0.143
0.911
0.089
1.000
0.000
0.865
0.000
0.135
0.857
0.143
1.000
0.000
1.000
0.000
0.000
0.483
0.417
0.100
0.900
0.000
0.100
0.000
1.000
0.000
0.950
0.050
0.883
0.117
1.000
0.000
0.533
0.467
0.000
1.000
0.000
1.000
0.000
IDH-1
MDH-2
MDH-4
PGDH-1
PGDH-2
PGM-1
SKDH-1
TPI-1
TPI-2
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.
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).
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.
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).
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.
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
Keppel et al • Genetic Diversity in Cycas seemannii 135
Table 4. Genetic diversity parameters in Cycas seemannii (standard errors in parentheses)
Population
x
’Eua
Nabou
New Caledonia
Naduri
Efate
Average
Total
29.5
24.4
28.5
27.5
30.0
28.0
140.0
A
(0.2)
(0.6)
(0.1)
(0.2)
(0.0)
(0.2)
(1.1)
1.3
1.1
1.2
1.3
1.3
1.2
1.8
AP
(0.1)
(0.1)
(0.1)
(0.1)
(0.1)
(0.1)
(0.2)
2.0
2.0
2.3
2.0
2.2
2.1
2.7
(0.2)
(0.2)
(0.2)
(0.2)
(0.2)
(0.2)
(0.3)
P
Ho
20.0
5.0
15.0
30.0
25.0
19.0
35.0
0.047
0.000
0.052
0.047
0.088
0.047
0.048
Table 5. Contingency chi-squared tests (with
degrees of freedom, df) and estimates of Wright’s
(1951) F statistics for the 10 polymorphic loci in
Cycas seemannii
He
(0.027)
(0.000)
(0.039)
(0.019)
(0.046)
(0.026)
(0.019)
0.071
0.010
0.051
0.074
0.079
0.057
0.138
(0.035)
(0.007)
(0.033)
(0.030)
(0.039)
(0.029)
(0.044)
x ⫽ mean sample size per locus, A ⫽ mean number of alleles per locus, AP ⫽ mean number of alleles per
polymorphic locus, P ⫽ percentage of loci polymorphic, Ho ⫽ mean observed heterozygosity, He ⫽ expected
heterozygosity (unbiased estimate; Nei 1978).
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
The genetic diversity of C. seemannii, an
outcrossing cycad with a discontinuous
distribution and mainly or entirely gravityand 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).
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).
136 The Journal of Heredity 2002:93(2)
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:
t ⫽ (1 ⫺ Fe)/(1 ⫹ Fe),
where Fe is the equilibrium fixation index
(Allard et al. 1968). Assuming that FIS represents the fixation index at equilibrium, t
would be 0.72. This value suggests that C.
seemannii is not a completely outcrossing
species, but may permit inbreeding to
some degree. However, because the t value is an indirect estimate, caution has to
be exercised in its interpretation ( Ledig et
al. 1997). If inbreeding was indeed significant, observed heterozygosity should be
lower than expected heterozygosity,
which is the case in three of the five populations studied. The positive overall FIS
value (0.165) and the mostly positive individual FIS values that differ significantly
Locus
␹2
df
FIS
FIT
FST
FEST-2
IDH-1
MDH-2
MDH-4
PGDH-1
PGDH-2
PGM-1
SKD-1
TPI-1
TPI-2
Mean
1000.00
263.55
1392.17
14.50
291.42
68.33
230.98
129.60
73.00
30.95
2480.628
3
3
6
1
1
1
1
3
1
1
21
1.000
⫺0.232
0.817
⫺0.025
0.499
⫺0.117
0.401
⫺0.120
0.125
⫺0.053
0.165
1.000
0.190
0.929
0.311
0.810
⫺0.043
0.732
0.478
0.228
⫺0.010
0.661
0.963
0.343
0.610
0.328
0.621
0.066
0.553
0.534
0.118
0.040
0.594
All chi-squared values are significant at P ⬍ .001.
from panmixia for all polymorphic loci
( Table 5) also indicate an excess of homozygotes within the populations. For a
better understanding of this issue, further
studies on the mating system of C. seemannii are needed.
In addition to inbreeding, the Wahlund
effect ( Hartl and Clark 1989) may provide
a partial explanation for the excess of homozygotes within populations. The justdescribed 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 winddisseminated 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
Figure 2. Graph showing the relation between genetic distance ( Nei 1972) and geographic distance for the
five populations of Cycas seemannii.
Figure 3. Wagner tree derived by rooting at the midpoint of greatest patristic distance (Cavalli-Sforza and Edwards 1967).
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 ap-
pears 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 mi-
Table 6. Genetic diversity and genetic differentiation in cycad species
Species
N
L
A
P
Ho
He
GST
Reference
Cycas pectinata
Cycas seemannii
Cycas siamensis
Macrozamia communis
Macrozamia heteromera
Macrozamia riedlei
Stangeria eriopus
Zamia pumila
Gymnosperms
Dicotyledons
11
5
13
5
—
15
2
2
—
—
17
20
17
18
16
14
16
15
—
—
1.82
1.80
1.48
1.61
1.30
2.43
—
1.75
2.35
1.79
58.5
50.0
58.9
50.0
58.0
93.0
62.5
16.7
70.9
44.8
0.066
0.048
0.114
—
—
0.263
—
0.047
—
—
0.076
0.138
0.134
0.045
0.060
0.274
—
0.041
0.173
0.136
0.387
0.418
0.291
0.270
0.100
0.092
—
—
0.068
0.273
Yang and Meerow 1996
Present study
Yang and Meerow 1996
Ellstrand et al. 1990
Sharma et al. 1999
Byrne and James 1991
McLellan and Ndamase 1995
Walters and Decker-Walters 1991
Hamrick and Godt 1989
Hamrick and Godt 1989
N ⫽ number of populations, L ⫽ number of loci, A ⫽ mean number of alleles per locus, P ⫽ percentage of
polymorphic loci, Ho ⫽ observed mean heterozygosity, He ⫽ expected mean heterozygosity, GST ⫽ relative degree
of gene diversity between populations, — ⫽ no data or not applicable.
gration 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 longterm survival of this particular population
doubtful.
We suggest the following guidelines for
the conservation of genetic resources of C.
seemannii:
1. 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.
2. 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
Keppel et al • Genetic Diversity in Cycas seemannii 137
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.
Godt MJW and Hamrick JL, 1998. Allozyme diversity in
the endangered pitcher plant Sarracenia rubra ssp. alabamensis (Sarraceniaceae) and its close relative S.
rubra ssp. rubra. Am J Bot 85:802–810.
3. 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.
Hamrick JL, Godt MJW, and Sherman-Broyles SL, 1992.
Factors influencing levels of genetic diversity in plant
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genetic comparison of forest and grassland Stangeria
eriopus. In: Proceedings of the Third International Conference of Cycad Biology ( Vorster P, ed). Stellenbosch,
South Africa: Cycad Society of South Africa; 255–261.
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Received December 12, 2000
Accepted December 31, 2001
Corresponding Editor: Brandon Gaut
Keppel et al • Genetic Diversity in Cycas seemannii 139