Morphometric and Genetic Variation of Six Seed Sources of Azadirachta excelsa (Jack) Jacobs
Isozyme variability was examined in six seed sources
representing the Azadirachta excelsa on two provenance trials in
Batu Arang (Selangor, Malaysia) and Merchang (Terengganu, Malaysia). A
concomitant study of morphometric variation revealed a slight variation
in leaf morphology extending from quantitative to qualitative characteristics.
The existence of this small variation presented an ideal opportunity to
examine the genetic variation of these seed sources collected in Bukit
Lagong and Manong (West Coast of P. Malaysia), Pengkalan Arang and Pasir
Mas (East Coast of P. Malaysia), Semengoh (East Malaysia) and Narathiwat
(Thailand). Nineteen enzyme systems were used to determine the genetic
variation among seed sources using isozyme analysis. Allelic frequency
data indicated little differentiation between seed sources. The mean values
of observed heterozygosity (Ho) varied from 0.0229 (Pengkalan
Arang) to 0.0451 (Bukit Lagong) whereas the mean values expected heterozygosity
(He) varied from 0.0575 (Pengkalan Arang) to 0.0983 (Manong).
The percentages of proportion polymorphic loci were found to vary between
31.43% (Pengkalan Arang and Pasir Mas) and 42.86% (Bukit Lagong and Manong).
Genetic identities according to Nei ranged from 0.7727 to 0.9999. Despite
these high levels of genetic similarity, the populations appeared to be
highly inbred as indicated by positive mean of FIS and FIT
values with the mean values of 0.5643 and 0.8038.
Genetic studies have particularly been used to identify
superior species populations or provenances and played an important role
in the subsequent selection and breeding of the most desirable individuals
within these populations or provenances. Biochemical techniques could
provide an alternative approach for evaluating genetic diversity in tree
species. Genetic analysis of isozyme or also called isoenzyme electrophoresis
using starch gel has been extensively used over the past several decades
in investigations of the genetics of a large number organisms from fruit
flies and human to crop plants. With the fast technology advancements,
several other reliable markers have been introduced such as RAPD, RFLP,
AFLP and SSR. However, isozyme technique still stands by its own advantages
such as cheap, fast and not affected by environmental changes.
Isozyme analyses have not been widely used in tropical
tree species. The available studies were reported in Acacia auriculiformis
and A. crassicarpa (Moran et al., 1989a; Wickneswari and
Norwati, 1993), A. mangium (Moran et al., 1989b), A.
melanoxylon (Playford et al., 1993), Eucalyptus urophylla
and E. grandis (Martins-Corder and Lopes, 1997), Hevea brasiliensis
(Paiva et al., 1994a, b), Pterocarpus macrocarpus (Liengsiri
et al., 1995) and Tectona grandis (Kertadikara and Prat,
1995). Early works on Azadirachta excelsa were done by Norwati
et al. (1997).
A. excelsa is one of the indigenous potential
plantation species being selected to be investigated in this study in
order to gain more information especially about provenance variation to
ensure the suitability for a large scale plantation programmes in the
future. This study considered six seed sources (genotypes) of A. excelsa
in the assessment of its variation. The objective of this study is to
determine the variation of A. excelsa seed sources using morphological
and genetic markers.
MATERIALS AND METHODS
Seed sources and study sites: The study was done using six seed
sources of A. excelsa. The seeds originated from Bukit Lagong Forest Reserve (FRIM, Selangor), Pengkalan
Arang (Terengganu), Narathiwat (Thailand), Manong Forest Reserve (Perak),
Semengoh (Sarawak) and Pasir Mas (Kelantan). Table 1
shows all of seed origin and the location of the sources. The seeds were
sown and germinated immediately in seed tray and later the seedlings were
transplanted into polythene bags. They were about 3 months-old before
being transplanted to the field in Merchang (Terengganu) and Batu Arang
(Selangor) in year 2002.
|| Details of the six seed sources of A. excelsa
Evaluation of genetic variation: The selection of 30 trees from
each seed source in both provenance trials was done randomly and leaves
which were still attached to the twigs were preferred, in order to avoid
wilting. The leaf samples were kept in plastic bags and placed in a cool
container containing crushed ices. The samples were then ground immediately,
if possible or preserved fresh in refrigerator at -20 °C to avoid
the protein denaturation. An amount of 0.6 g of leaf was homogenised with
liquid nitrogen using mortar and pestle. Then 1200 μL of leaf extraction
buffer was added into the powdered form of leaf material with a ratio
of 1:2. This is to create a slurry homogenate. The sample was filled into
Eppendorf tube and labelled and later was kept in crushed ices before
centrifugation at -10 °C, 1000 rpm for 3 min. The clear filterate
was stored in a freezer at -70 °C.
Three types of gel and electrode buffers were used to
detect the various enzymes. These include Histidine (H), Lithium (L) and
Morpholine Citrate (MC). The mould frame was made of perspex, with internal
dimensions of 18.8x15.0x0.6 cm and a glass plate of equal area. Both the
frame and the glass plate were cleaned and dried properly by placing them
in the oven at 40 °C to prevent breakage when pouring the hot gel solution.
A 10.5% of potato hydrolysed starch (SIGMA-ALDRICH Inc.) was prepared
by adding 31.5 g of starch into 100 mL of cold buffer in a dry Buchner
flask. The remainder 200 mL of boiled buffer was then added into the flask
and the buffer was swirled thoroughly to create an even suspension. The
buffer was once again cooked together with the starch in the microwave
oven until it boiled or big bubbles started to form. When the starch was
fully cooked, the flask that contained the gel mixture was connected to
the suction pump for a degassing process. The starch gel solution
was then poured into the perspex mould and was left overnight at room
temperature for hardening.
||Enzymes assayed, their abbreviations and Enzymes Commission (EC)
|Source: Weeden (1989)
Each resulting homogenate was absorbed onto a filter
paper wick (i.e., 0.6x0.4 cm Whatman No. 3). This saturated wick and a
tracking marker were inserted into the sliced gel that was tightened up
by space bars before the electrophoretic run. The prepared gel (with wicks)
was placed between the 2 electrode tanks containing 500 mL of buffer.
The gel and the buffers were connected by two sets of bridge wicks (J-cloth
or sponge) that were placed with one end overlapping about 1.0 to 3.0
cm on the gel and the other end dipping into the buffer. The set-up was
covered with a thin polythene sheet to prevent evaporation of the buffer.
A bag or plastic container containing ice was placed on the polythene
sheet to avoid the drying up of gel during electrophoretic run due to
high voltage or current. The electrophoretic run was carried out in a
4 °C refrigerator with an appropriate electric voltage depending on
the buffer used. The wicks were taken out from the gel after 30 min of
After the electrophoretic run, the gel was removed from
the mould in the refrigerator and cut into a smaller size by discarding
only the sides. A mark was made to determine the origin of the sample
arrangement by slashing the side. Later, the gel was sliced into three
or four sheets with a thickness of approximately 2 mm per slice. The slices
were stained for 19 different enzyme systems. The different enzyme systems
used are as listed in Table 2. The stained gels were
incubated in an oven at 37 °C for 30 to 60 min or until coloured bands
developed. Then the gel was fixed using a fixing solution. Observation
on the pattern of the bands was made and a clear zymogram was photographed
whenever necessary. The banding of phenotypes was recorded in the prepared
data sheets and followed with the interpretation.
Data analysis: The soluble enzymatic proteins from the leaves
were assayed for each enzyme. The locus alleles were labeled as fast (F),
medium (M) and slow (S) according to the decreasing anodal mobility. Standard
measures of allozyme diversity within species and seed sources were calculated
as described in Hamrick and Godt (1989). These measures included the percentage
polymorphic loci (P), the mean number of alleles per locus (A), the effective
alleles per locus (Ae), observed (Ho) and expected (He)
heterozygosity, corrected for sample size (Nei, 1978). Other analyses
done on the isozyme data also included Shannon`s information index (I)
(Shannon and Weaver, 1949) and population genetic structure was measured
using F-Statistics and, fixation index (Wright, 1978), gene flow estimation
(Slatkin and Barton, 1989) and gene diversity among provenances (Nei,
1973). FIS compares the observed and expected levels of heterozygosity
within provenances. A positive FIS indicates a lower than expected
level of heterozygosity, possibly due to nonrandom mating (Wright, 1969).
FIT measures the heterozygosity of an individual relative to
the entire population. It is an indication of inbreeding due to nonrandom
mating and random genetic drift within the subpopulations. GST is
a measure of the level of variation that resides within, rather than between,
populations (Nei, 1973). Finally, the construction of a dendrogram was
made based on Nei`s genetic distance using UPGMA (Nei, 1978). The programme
is actually an adoption programme of NEIGHBOR and PHYLIP version 3.5c.
The data were analysed using POPGENE version 1.21. The banding patterns
were first encoded using Microsoft Excel for easier editing before being
transformed into a POPGENE data file. The formulas for calculating those
analyses are as follows:
The formula was used for allelic frequency are:
||= No. of homozygous for that allele
||= No. of heterozygotes for that allele
||= No. of individuals screened or examined
The observed heterozygosity (Ho) is simply
the proportion of all genotypes that was heterozygote and may be expressed
on a per-locus basis or averaged over all loci. Meanwhile, the expected
heterozygosity was estimated using formula:
He = (1 - Σ Xik2)
Where, Xik is the frequency of the kth
allele at a locus in the ith population.
The gene diversity in this populations is defined as
He = (1 - kΣ Xik2). The
average of He over loci is HS. The gene diversity
in the total population is:
Where, Xk = iΣ Xik/n
, n being the number of populations. Then HT-HS,
the diversity due to interpopulational gene differences, is denoted as
DST and the coefficient of gene differentiation is DST/HT
denoted as GST.
The genetic identity (GI) between the two population
or operational taxanomix unit (OTUs) was estimated according to Nei`s
coefficient using this formula:
Where, Xi and Yi = Frequencies
of the ith allele in the two OTUs or populations.
Morphological traits: A total of 40 trees were selected randomly
from each seed source in both provenance trials to determine the variability
of morphological characteristics when the trees were about two years old.
Morphological traits such as leaf shape, leaf margin, leaf base, leaf
angle and internode were also determined. The leaf angle was determined
using the triangulation method. The length between nodes (inter-node)
of two leaves (a) was measured. Then the length from the base of the node
to the centre length of the leaf (b) was measured. The distance from the
centre of the leaf to the base of the next leaf was then measured as (c).
The value of the distances (c) was obtained using the following formula:
The general analysis for morphological traits was done
according to one-way ANOVA. In addition, similarity coefficients based
on these morphological characteristics were estimated using the formula
||= No. of matches
||= No. of mismatches
Intra-population variation: Table 3 shows the
summary of genetic diversity from 35 loci scored including observed (Ho)
and expected (He) heterozygosities, Shannon`s information index
(I), effective number of alleles (Ae), proportion of polymorphic
loci and the average number of alleles per locus per seed source.
The values of the observed heterozygosity varied from
0.0000 to 0.3333 with mean values ranging from 0.0229 (Pengkalan Arang)
to 0.0451 (Bukit Lagong). Meanwhile, the values of expected heterozygosity
ranged from 0.0000 to 0.5161. The mean values of He were found
to vary from 0.0575 (Pengkalan Arang) to 0.0983 (Manong). Moreover, Shannon`s
information index showed the diversity within the seed source to vary
from 0.0000 to 0.8897 with mean values ranging from 0.1098 in the Pengkalan
Arang seed source to 0.1852 in the Manong seed source. The effective number
of alleles was found to range between 1.0000 and 2.0666 with mean values
found to be within 1.0808 to 1.1468.
Ten loci were found to be monomorphic for all seed sources
whereas the other 25 loci were polymorphic. The percentages of proportion
polymorphic loci were found to vary between 31.43% (Pengkalan Arang and
Pasir Mas) and 42.86% (Bukit Lagong and Manong). Meanwhile, the mean number
of alleles per locus ranged from 1.48 to 1.69.
Genetic structure: F-statistics revealed varying fixation indices
among the loci (Table 4). The estimates of FIS
revealed one locus (Pgi-3) with excess of heterozygotes, as revealed by
the negative mean fixation index value. The 34 remaining loci exhibited
heterozygote deficiencies. The FIS values calculated ranged
from -0.0265 at Pgi-3 to 1.0000 at Pgm-2 with the average of 0.5643. The
FIT values for all loci ranged from -0.0043 at Pgi-3 to 1.0000
at Pgm-2 with the mean value being 0.8038.
Genetic differentiation among seed sources as measured
by FST showed that 21.32% of the total genetic variation was
due to differences among seed sources; 78.68% of the isozyme variation
resided within seed sources. The FST values ranged from 0.0000
to 0.9681 among the polymorphic loci. The estimated gene flow (Nm) based
on the FST values was 2.9124.
The results obtained for genetic diversity in the seed
source, measured by Nei`s (1973) index, through the partition of total
genetic diversity (HT) in the components of diversity within
populations (HS) and between populations (DST) showed
relatively low values for total diversity (Table 5).
The average GST (coefficient of differentiation) value indicated
that approximately 29.84% of the observed variability was found among
seed sources, more than that estimated by FST.
Genetic distance: Nei`s genetic distance (Nei, 1978) and the UPGMA
dendrogram revealed low levels of genetic distance among seed sources.
Most of the seed sources produced high similarities to each other with
mean identity values ranging between 0.7727 and 0.9999 (Table
6). The most related seed sources were Pengkalan Arang and Pasir Mas
with a genetic identity of 0.9999 between them. There were two clusters
formed where the first cluster was made up of Pengkalan Arang, Pasir Mas,
Narathiwat and Semengoh seed sources while the second cluster consisted of Bukit Lagong and Manong seed sources
(Fig. 1). This clusteration is comparatively similar
to the ones based on morphological similarities with a few notable exceptions.
Four seed sources (Pengkalan Arang, Pasir Mas, Narathiwat and Semengoh)
were assigned to CLUSTER I on the basis of isozyme and morphological data.
|| Summary of genetic diversity for six seed sources of A. excelsa
|Ho: Heterozygosity observed, He:
Heterozygosity expected, I: Shannon`s information index, Ne: Effective
number of alleles
||F-statistics and gene flow for all loci
|FIS, FIT and FST: Inbreeding
coefficients, Nm: Gene flow
||Nei`s (1973) statistics of genetic diversity for 18 polymorphic
loci in six seed sources of A. excelsa
|1: Mean values do not include monomorphic
Morphological variability and similarity: Most seed sources have
the same pattern of qualitative characteristics for all leaf parameters
except for Narathiwat and Semengoh seed sources which had one extra pattern
in leaf margin (Table 7). Quantitative characteristics
of leaf angle and internode showed significant differences among
seed sources for both positions but no significant difference was detected
on other parameters (Table 8).
||Dendrogram of six seed sources of A. excelsa using Un-Weight
Pair Group Cluster Analysis of Identity Coefficients (Nei`s, 1972)
||Dendrogram of six seed sources of A. excelsa based on morphological
The mean morphological similarities for the qualitative
characteristics ranged from 0.7930 between Pasir Mas and Bukit Lagong
seed sources to 0.9952 between Semengoh and Bukit Lagong seed sources
(Table 9). The dendrogram for six seed sources of A.
excelsa based on these similarities is given in Fig. 2. Narathiwat-Pasir
Mas seed sources and Pengkalan Arang-Semengoh seed sources formed one
cluster while Bukit Lagong and Manong seed sources formed the other cluster.
||Nei`s (1972) Coefficients of genetic identity (above diagonal) and
genetic distance (below diagonal) among six seed sources of Azadirachta
|C: Seed source code
|| Leaf characteristics of six A. excelsa seed sources
||Analysis of variance for qualitative and quantitative traits of
|1Basal position, 2middle position,
*Significant at p < 0.05, ns: Not significant
Intra- and inter-provenance variation: Genetic variation within
populations is the basis for evolutionary change to occur. The amount
of such variation is dependent on the species level. According to Hopper
and Coates (1990), this is necessary to occur in order for plants to evolve
and adapt under different conditions and numerous environments that they
encounter during a single or many generations. There are five evolutionary
processes that affect the level of genetic variation such as random genetic
drift, selection, migration, mating and mutation. All these evolutionary
processes affect the levels and distribution of genetic variation and
the present state of the genetic resource is a result of their joint effects
The genetic variation among provenances is quantified
by measuring the mean heterozygosity, the percentage of polymorphic loci
and the number of alleles per locus. The range of expected heterozygosity
calculated in the present study was from 0.0575 (Pengkalan Arang) to 0.0983
(Manong) and found to be within the range recorded by Norwati et al.
(1997) on the same species but in different populations. These values
were also generally consistent with those reported on tropical species
by Hamrick and Loveless (1986).
The range of the proportion of polymorphic loci obtained
was from 31.43% (Pengkalan Arang and Pasir Mas) to 42.86% (Bukit Lagong
and Manong) and found to be within the range recorded on tropical species
by Hamrick and Loveless (1986) in general. This range was also found to
be similar to the ones reported by Norwati et al. (1997) on the
The range of the average number of allele per locus for
the six seed sources of A. excelsa obtained in the present study
was found to be within the range recorded on tropical species. This range
was also about similar than that recorded by Norwati et al. (1997)
but lower than those recorded on other popular tropical tree species for
plantation such as H. brasiliensis (Paiva et al., 1994a,
b), P. macrocarpus (Liengsiri et al., 1995) and T. grandis
(Kertadikara and Prat, 1995).
The mean average of expected heterozygosities was 0.077
and this value was found to be lower than those reported on tropical species
by Hamrick and Loveless (1986) in general. This value is comparable to
those reported by Mohamad et al. (1997) on the same species and
Wickneswari and Norwati (1993) on A. auriculiformis. The value
was again found to be lower than those reported on H. brasiliensis
(Paiva et al., 1994a, b), P. macrocarpus (Liengsiri et
al., 1995) and T. grandis (Kertadikara and Prat, 1995).
The mean value of the proportion of polymorphic loci
of six A. excelsa seed sources was 36.67. This value was found
to lower than those reported by Hamrick and Loveless (1986) on tropical
species. The value was comparatively similar to those reported by Norwati
et al. (1997) on the same species as well as A. auriculiformis
(Wickneswari and Norwati, 1993), A. mangium (Moran et al.
1989b) and S. macrophylla (Kanzaki et al., 1996).
Meanwhile, the value of the average mean number of alleles
per locus for six A. excelsa seed sources was found to be within
those reported by Hamrick and Loveless (1986) for tropical species in general. The mean value
obtained was again comparable to those reported by Norwati et al.
(1997) on the same species as well as A. auriculiformis (Wickneswari
and Norwati, 1993) and A. mangium (Moran et al., 1989b).
||Morphological identity (above diagonal) and morphological distance
(below diagonal) based on the morphological characteristics
|C: Seed source code
Species which are widespread, long-lived woody and primarily
outcrossed by wind or insect pollination have been reported to produce
high levels of genetic diversity (Brown, 1978; Loveless and Hamrick, 1984;
Hamrick and Godt, 1989; Hamrick et al., 1992). A. excelsa
is also expected to produce higher values of heterozygosities based on
mode of pollen and seed dispersal mechanisms. A. excelsa can be
classified as one of the entomophilus species pollinated by insects such
as bees and moth while the fruit is transported by bats and birds. In
contrast, lower level of heterozygosity was reported on all seed sources,
might be assumed that the seed sources consist of small population size.
This could be due to the species was confined to scattered distribution
along farm or plantation boundaries, roadsides or sparsely grown which
could lead to the reduction in population size. According to Kijkar and
Boontawee (1995), this species is thought to be an introduced species
in Peninsular Malaysia and not indigenous and may established from a very
restricted genetic base. Species with small population densities often
increased the possibility of genetic drift, which will reduce the genetic
variability as a result of bottlenecking and inbreeding (Moran and Hopper,
1987). This phenomena is well discussed by other researchers such as Hamrick
(1983), Chamberlain et al. (1996a, b) and Liengsiri et al.
Although the information gathered was inadequate to relate
concisely on the actual level of genetic variation especially on the number
of mother trees but the information gathered from Pengkalan Arang seed
source could be used to explain the lower level of genetic variation.
Lower values of heterozygosities would suggest that the seeds were collected
from a few mother trees. For instance, ground truthing at Pengkalan Arang
revealed that seed sources might originated from only two to three mother
trees separated about eight to 10 m away. Also, there is a possibility
that the seeds may be from the same maternal tree and thus leading to
genetic heterogeneity reduction and limited gene exchange.
In addition, seed sources from Bukit Lagong and Semengoh
were obtained from research plot which is an artificial stand where the
genetic base of the planted population is usually be rather narrow. This
postulation is supported by Dusan (1992) who found a lower value of mean
heterozygosity of 0.275 from artificial stand of Picea abies when
compared to the value (0.322) obtained from virgin forest. This is because
the artificial stand normally receives uniform treatment levels, which
explain for unnecessary increased for heterozygosity.
The percentage of proportion polymorphic loci among provenances
was 36.67%, which is lower than those reported for other tropical trees.
The low value of polymorphism suggests that the seed sources in the present
study had lesser opportunities to evolve. Normally, polymorphism is required
as part of the adaptive strategies of populations in a heterogenous environment
of the forest (Feret and Bergman, 1976). Consequently, a species has to
evolve to compete for optimum growth against other species for growth
resources such as space, light, nutrient and water. A concrete discussion
cannot be done further since information on the actual details of their
history, origin and the ecological background of source materials are
Genetic distance: Isozyme analysis in A. excelsa revealed
35 loci where 25 of them being polymorphic. One (Pgi-3) out of 25 polymorphic
loci showed significant excess of heterozygosity (Table 4). Meanwhile,
there was a general excess of homozygosity within seed sources, as revealed
by the positive mean fixation index value. According to Gregorius and
Namkoong (1983), a lack of heterozygosity can be observed in a population
because of restriction of gene flow within the whole provenance and of
increased of relatedness between neighbour individuals. Similarly, the
results noted in an isozyme analysis of population structure of Q.
rubra (Schwarzmann and Gerhold, 1991), in which observed heterozygosities
did not differ significantly from those expected under random mating and
in a population study of F. sylvatica (Cuguen et al., 1988;
Comps et al., 1990) in which FIS and FIT
values were generally positive indicating heterozygote deficit relative
to panmixia. Houston and Houston (1993) stated that the differences between
two species in population structure might reflect differences in their
reproductive strategies which result in differing mating patterns and
existing stand structures. The positive mean of FIT and FIS
values observed for this species implies an overall deficit of heterozygotes,
perhaps caused by assortative mating between near neighbours related descent.
Generally, FST values with average 0.213 obtained
in this study indicated moderate differentiation (Wright, 1978) between
the two provenances which approximately 78.7% of the observed variation
residing within seed source. This value was comparative to the one reported
on G. sepium (0.172) by Chamberlain et al. (1996a). However,
this value was higher than those reported on P. macrocarpus (0.121)
by Liengsiri et al. (1995) and other tropical trees (0.119) by
Hamrick et al. (1992). The value was also higher than those reported
on F. sylvatica (0.060) by Cuguen et al. (1988), Populus
tremuloides (0.030) by Jelinski and Cheliak (1992) as well as on European
beech (0.054) by Comps et al. (1990). This level of population
subdivision is high when compared with that of temperate tree species
and this supports the notion that there is greater population differentiation
in the tropics than in the temperate zone (Hamrick et al., 1992).
Possible reasons for higher levels of population differentiation in the
tropical species have been reviewed by Bawa (1983). They include lower
population densities, more widely scattered populations that reduce gene
flow and increase genetic drift and greater spatial variation in the natural
selection pressure. However, higher levels of population differentiation
in present study might also be due to bottleneck effects where the sources
of A. excelsa are mostly not from natural population (Nei, 1975).
Nei`s (1978) statistics of genetic diversity was also
calculated to analyse the distribution of allelic diversity. The average
GST value indicated that approximately 29.84% of the observed
variability was found among seed sources, more than the one estimated
by FST and was higher than the average value found in other
tropical tree species by Loveless (1992), i.e., GST = 10.9%.
This value was also found to be higher than those reported on F. sylvatica
(3.6%) by Comps et al. (1990), Pinus kesiya (3.9%), a wind-pollinated
tropical pine by Boyle et al. (1991), Q. macrocarpa (7.6%)
by Schnabel and Hamrick (1990) and Rugelia nudicaulis (1.5%) by
Godt and Hamrick (1995). This result once again showed that most of the
variation was found within seed sources.
The overall result corresponded to long-lived woody plants
which acquired relatively high genetic diversity within species but most
of the genetic diversity was found within populations and little existed
among populations. The distribution of allozyme variation among populations
is the product of interactions among several evolutionary factors. Of
primary importance are selection, effective population size and the ability
of the species to disperse pollen and seeds. On the other hand, species
with more pollen and seed movement should have less differentiation among
population than species with restricted gene flow (Hamrick, 1989). However,
low level of diversity among seed sources and the high level of diversity
within seed sources of A. excelsa in this study could be due to
the certain relatedness among the seed sources or land races of this species
whereby the seed sources might come from similar source in terms of ancestory
genetic constituent. The reason is simply that A. excelsa is not
usually found in the jungle or natural forests. It occurs mostly on farmland
or marginal lands through shifting cultivation (Kijkar and Boontawee,
1995) and therefore likely to be naturalised in this area.
Cluster analyses: The dendrograms produced by the UPGMA clustering
technique are presented in Fig. 1 and 2
for morphological and genetic values, respectively. The morphological
and genetic identity values were found to range between 0.7930 and 0.9952
and 0.7727 and 0.9999, respectively. Such high ranges revealed that they
were very much related both in terms of morphological and genetical aspects.
Two groups of clustering were formed using both techniques.
Both dendrograms showed seed source of Pengkalan Arang, Narathiwat, Semengoh
and Pasir Mas formed one cluster whereas Bukit Lagong and Manong seed
sources formed the other group. Such relatedness among the seed sources
are as expected based on their geographic distances among them. Narathiwat,
Pasir Mas and Pengkalan Arang seed sources were located at a closer latitudes
and longitudes. Similar clusteration based on latitudes, longitudes and
altitudes have been reported for Casuarina cunninghamiana (Moore
and Moran, 1989) and A. crassicarpa (Nor Aini et al., 2006).
Normally, as geographic distance between population is small or decreases,
then the genetic similarity will increase.
The pattern of such clusteration is also found to be
associated with historical factors. The Bukit Lagong seed source was once
thought to be originating from Perak while the source from Thailand was
believed to have some relationships with Borneon sources as a result from
the scientific expedition made earlier. This is because, (Roland Kueh,
unpublished) reported that a group of scientists from Thailand made an
expedition for seed collection of A. excelsa to Borneo Island
(i.e., Sabah and Sarawak). Perhaps, through such activity could possibly
caused such relatedness between Sarawak and Thailand.
The general pattern exhibited by the dendrogram based
on morphological similarity values is similar to that based upon genetic
identity values with a few notable exceptions. The uncertainty regarding
the phenetic-phylogenetic correspondence is due primarily to variation
caused by the confounding selective forces as a result of changes in environment.
The majority of allozyme loci are neutral with respect to natural selection
and their distribution is largely dependent on the stochastic events of
mutation, migration, drift and founder effect compared to morphological
characteristics. If so, UPGMA clustering of genetic identity values could
provide a less biased view of the evolutionary relationships of the species
than the clustering of morphological similarity values.
Genetic parameters were found to produce low levels
of genetic variability for all A. excelsa seed sources. The Manong
seed source was found to possess higher level of diversity followed by
Bukit Lagong, Semengoh, Narathiwat, Pasir Mas and Pengkalan Arang seed
sources. Moreover, low level of polymorphism might indicate that this
species has lesser opportunities to evolve in different environment pressures.
The overall result corresponded that most of the genetic
diversity found within seed sources with little existing among seed sources
where it is generalised the genetic diversity of woody plants. The founder
effect could help to explain such phenomenon for this species.
High values of morphological and genetic in cluster analyses
suggests that the close relatedness of the seed sources. This argument
is made based on the historical background and geographical distance among
those seed sources. Seed sources having similar latitudes and longitudes
were found to give smaller distance. Moreover, possible explanation on
the relatedness between groups was also due to the source of the land
We wish to express our deepest gratitude and thanks
to Associate Professor Dr. Kamis Awang for his invaluable suggestions
and comments. Our special thanks also extended to Mr. Salim Ahmad and
Mr. Zakaria Taha for their assistance and co-operation during sample and
information collection of the project. We would also like to thanks to
Forestry Department of Peninsular Malaysia for providing us the research
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