Population Genetic Structure of Acacia Brevispica from
East Africa
Joseph Harsh
Communicated by: Dr. Andrew Schnabel
Department of Biology
abstract
The genus Acacia consists of nearly 1,200 species of woody plants that play many key ecological roles as the
dominant woody vegetation in dry tropical forest-savannas worldwide. More than 100 of these species can
be found in the savannas of East Africa. Here we report allozyme variation in two populations of Acacia
brevispica from the Mplala Research Centre in Kenya. Of the 12 putative loci, 11 were polymorphic in at least
one population. Individuals exhibited high heterozygosity (27%) and dierences between populations were low
(1.3%). The latter result suggests high levels of gene movement between populations located within 10 km of
one another. This gene movement could result from long-distance pollinator movement or dispersal of seeds by
large herbivorous mammals.
Introduction
Acacia is the common name for plants of the genus Acacia in the bean family, Leguminosae. This genus consists
of approximately 1,100-1,200 species primarily of trees, but
also including some shrubs and climbers. Globally distributed, acacia species are located in Asia, Madagascar,
the Caribbean and Pacic islands, the Americas, and most
prominently in Australia and Africa in arid and semiarid
tropical zones. Those tropical regions that have long, dry
winters and short wet summers often support shrubby vegetation known as thorn scrub or savanna. Acacia trees constitute much of the woody vegetation in such plant communities (Ross 1981). The trees may grow to 30 meters
with a characteristic umbrella shape or they may be more
shrub-like with extensive basal branching of the stems. The
owers, usually yellow or white, grow in crowded, globose
heads or cylindrical spikes. Thorns are also another common
feature of acacias, which supposidly provide for protection
from herbivores, and, in some species, providing a symbiotic
home for colonies of ants (Janzen 1966; Stone and Willmer
1996).
Within the savanna woodland ecosystem of East Africa, acacias serve an essential ecological and economic role. Due to
a high protein content (15-20% of dry weight), acacia leaves
serve as an essential food source for wildlife, especially during the dry season (Berchem 1994). Because they comprise
much of the arboreal habitat, acacias are also critically important sources of nesting for native birds. Many pollinating
insects are also dependent on acacias, which provide a major
source of pollen and nectar when in ower. In East Africa,
acacias are thus a classic example of keystone species, organisms essential to the functioning of a community (Primack
1993). Acacias not only inuence the ecology of the community, but also the economy (Berchem 1994), with uses in
lumber production of a termite resistant wood, feeding of
livestock, and a wide variety of native uses.
We investigated genetic diversity within populations of Acacia brevispica from Kenya. This work was motivated by recent studies of Stone et al. (1996, 1998), who have been
studying the role of competition for pollination as a factor
structuring owering time in acacia communities. In their
studies, Stone et al. (1996, 1998) have examined the mechanisms reducing pollen movement between plants of dierent
species in a community of 10 acacia species. Because several
acacia species ower simultaneously and share pollinators,
interspecic pollen transfer is possible. The activity of these
common pollinators is structured through the day as a result of temporal dierences in the timing of pollen release
among acacia species - some species release pollen early in
the day, some release pollen later in the day. Pollinators
harvest available pollen and move from one species to the
next, following the daily sequence of pollen release. The
temporal structuring of pollen release is compatible with
patterns predicted to result from natural selection on owering time to reduce wasteful interspecic pollen transfer.
In addition, support for a competition-based explanation
comes from the observation that acacias without competitors do not have set peak times of pollen release during the
day.
The genetic study presented here was designed to complement ongoing ecological investigations. In particular, Stone
et al. (1998) questioned on what spatial scale acacia populations are divided into genetically identiable subunits,
because this determines the scale over which signicant owering time dierences could be maintained by natural selection. Genetic dierences between populations can only be
maintained if gene movement between populations is low.
If gene movement between populations is high, then the effects of natural selection are counteracted by the inux of
new genes. The results of the present study should therefore enable a greater understanding of pollination biology
in East African acacias and its relationship to genetic diversity within and between populations. Therefore, two specic
J. Harsh
40
questions were addressed in this study: How much genetic
variation is there within populations of A. brevispica ? How
large are the genetic dierences between populations of a
species?
Materials and Methods
Sample collections
Whole seedlings of A. brevispica were used in the genetic
analysis. Seed samples from two populations were collected
by Dr. A. Schnabel (Indiana University South Bend) and
Dr. G. Stone (University of Edinburgh) in June 1999 at
the Mpala Research Centre in Kenya. Leaf collections were
made from 5 sites (35 plants at each site) and pods were collected from two sites. Seeds were removed from pods and
sorted by pod and maternal tree. To aid in germination,
seeds were scaried briey (~15 min.) in sulfuric acid, and
rinsed prior to being planted in individual pots for germination. These plants remained in the IUSB greenhouse until
the seedlings had sucient tissue for analysis (2-3 weeks).
Enzyme extractions and starch gel
electrophoresis
Enzymes were extracted by grinding leaf tissue in 1-2 mL
of a phosphate extraction buer (0.1 M KH2 PO4 , pH 7.5,
5% PVP 40, and 0.1% mercaptoethanol) and absorbing the
enzyme/buer solution onto lter paper wicks (2 x 10 mm).
These wicks were then stored at -70 degrees Celsius until use. Horizontal electrophoresis was carried out using
11.5% starch gels. Electrophoresis is a method of separating molecules in a gel-like matrix based on dierences
in size, shape and electrical charge. Enzymes are encoded
by genes and therefore enzyme variation (present in several forms, called allozymes) is a direct reection of allelic variation. Starch gel electrophoresis of allozymes to
assess patterns and levels of genetic variation has been a
standard method widely used by evolutionary ecologists for
many years (Wendel and Weeden 1989; Weeden and Wendel 1989; Murphy et al. 1996). The allozyme analysis
of A.brevispica included acid phosphatase (ACP), aconitase (ACO), aspartate aminotransferase (AAT), alcohol dehydrogenase (ADH), adenylate kinase (ADK), diaphorase
(DIA), esterases (Est), glucose-6-phosphate dehydrogenase
(G6PDH), glutamate dehydrogenase (GDH), hexokinase (HK), isocitrate dehydrogenase (IDH), leucine aminopeptidase
(LAP), malate dehydrogenase (MDH), malic enzyme (ME),
peroxidase (PER), phosphoglucoisomerase (PGI), phosphoglucomutase (PGM), 6-phosphogluconate dehydrogenase (PGD), and shikimate dehydrogenase (SkDH). Several enzymatic buer systems were used to keep enzymatic systems
at a constant pH during electrophoresis (Table 1).
Data Analysis
Electrophoretic phenotypes were interpreted genetically in
light of previous acacia studies and known enzyme subunit
numbers (Wendel and Weeden 1989; Weeden and Wendel
1989; Joly et al. 1992). Genetic diversity within populations was estimated by the proportion of loci that were
polymorphic, the average number of alleles per locus, and
the average proportion of individuals that were heterozygous (heterozygosity) (Hamrick and Godt 1989). Chi-square
tests were used to determine whether genotypes were in
Hardy-Weinberg frequencies. Weir and Cockerham's (1984)
theta statistic was used as a measure of genetic dierentiation. Theta estimates the proportion of the total variation that is due to dierences between populations. Data
analysis was carried out using the program Tools for Population Genetic Analysis (TFPGA) written by M.P. Miller
(http://her.bio.nau.edu/~miller/tfpga.htm).
Electrode Buer
0.223M Tris
0.069M Citric acid
pH 7.2
Gel Buer
0.008M Tris
0.002M Citric acid
pH 7.2
0.100M NaOH
0.300 Boric acid
pH 8.6
0.0039M LiOH
0.263 Boric acid
pH 8.0
0.015M Tris
0.004M Citric acid
pH 7.8
0.042M Tris
0.007M Citric acid
0.004M LiOH
0.025M Boric acid
pH 7.6
0.009M L-Histidine
0.002 Citric acid
pH 5.7
0.065M 1-Histidine
free base,
0.015M to 0.016M
Citric acid,
pH 5.7
0.400M Citric acid
trisodium salt,
pH 7.0
Morpholine-Citrate
pH 6.1
0.005M Histidine-HCl
pH 7.0
Morpholine-Citrate
pH 6.1
Enzyme Tested
ACO, ACP
ADK, DIA
FDH, G6PDH
GDH, HK, IDH
LAP, MDH
ME, PER
PGI, 6-PGD
AAT, ACP
Est, DIA
PGE, PGM
AAT, ACP
EST, LAP
PGI
ACP, LAP
MDH, ME
PGM, SkDH
DIA, LAP
IDH, ME
ACO, ACP
ADH, DIA
FDH, GDH
IDH, LAP
MDH, ME
PGI, PGD
Table 1. Enzyme and electrophoretic buer systems surveyed
in the population study of A.brevispica. All electrode/gel buer
recipes come from Solstis et al (1983), except for the morpholinecitrate buer which was taken from Wendel and Weeden (1989).
Although most enzymes were tested on more than one buer, only
one system per enzyme was used for the nal electrophoretic runs.
These are indicated by asterisks. Not all enzymes listed were used
in the nal runs (runs which provided interpretable data).
Results
Of the 12 loci (coding genes) tested in both populations,
only pgm-1 showed no variation (Table 2). The number of
alleles per locus ranged from 1 to 6, with an average (+/SD) of 2.6 +/- 1.4 for both populations. Population 1 was
found to have relatively high heterozygosity ranging from 0
to 76% (Table 2). Population 2 also demonstrated substantial heterozygosity from 0 to 58%. The total average heterozygosity over all loci in both populations was estimated
to be 28%. For most loci, genotype frequencies conformed
to expectations of the Hardy-Weinberg theorem, suggesting
that the populations are nearly randomly mating. The average theta value across loci was 0.0127. The 95% condence
Population Genetic Structure of Acacia Brevispica from East Africa
Putative
Locus
Aat-1
Est-1
Fdh
Gdh
Idh
Mdh-1
Mdh-2
Mdh-3
Pgd-2
Pgi-1
Pgm-1
Pgm-2
Mean
SD
Alleles per
Locus
2
4
2
2
6
2
3
2
4
2
1
3
2.6
1.4
N
50
36
43
40
50
50
50
40
50
40
22
22
41
10
Pop. 1
H0
0.24
0.56
0.47
0.58
0.76
0.00
0.20
0.02
0.33
0.02
0.00
0.30
0.28
0.26
2
1.64
0.14
0.47
9.55*
0.02
0.13
0.01
10.57
0.01
1.50
N
45
40
44
36
45
45
45
42
45
42
18
14
38
11
Pop. 2
H0
0.20
0.70
0.57
0.39
0.58
0.02
0.04
0.05
0.51
0.00
0.00
0.43
0.29
0.27
2
1.00
0.07
10.20*
3.41
1.80
0.01
0.05
0.05
0.72
0.03
Table 2. Summary of allozyme diversity at 12 loci in two Acacia brevispica populations from the Mpala Research Centre in
Kenya. N= sample size, Ho = proportion of individuals that were
heterozygous. The 2 statistic indicates whether or not genotypes
were in Hardy-Weinberg frequencies (df = 1; * = P < 0.01).
interval based on 1,000 bootstrap replications over loci was
-0.005 to 0.0239, which indicated that there are no signicant dierences between these two populations for the loci
studied.
Discussion
Several ecological and life history characteristics of acacias
suggest clear answers to the questions addressed in this
study. For example, woody species often have high genetic
variability, as do species with broad geographic ranges like
A.brevispica (Hamrick and Godt 1989; Loveless 1992). Likewise, plant species that cannot self-fertilize, might be expected to have greater gene movement between populations
than those that are self-fertilizing, leading to less variability
between populations. All acacia species studied to date have
been shown to exhibit very little self-fertilization, and many
are thought to be self-incompatible (Kenrick and Knox 1989;
Muona et al. 1990).
Acacia species, therefore, generally possess traits that are
correlated with high variability within populations and small
genetic dierences between populations (Hamrick and Godt
1989). Few studies exist, however, that test these hypothesis. Moreover, most studies have been conducted on Australian species with limited research on the African species.
There is a wide range of levels of genetic diversity, and unexpectedly high levels of diversity between populations, in
the Australian species studied to date (Moran et al. 1989;
Wickneswaria and Norwati 1993; Playford et al.1993). The
one African species studied, A. albida , has a high genetic diversity and moderate dierences between populations (Joly
et al. 1992).
41
In summary, the genetic data for A. brevispica agree closely
with expectations based on its ecology and life history. Genetic diversity, as estimated by allozyme variation, is high
within populations. Genotype frequencies generally conform
to Hardy-Weinberg expectations, suggesting strongly that
the species, like other acacias, is highly outcrossed. Genetic dierences between the two populations, located approximately 10 km from one another, were not statistically
signicant, suggesting that gene movement between populations is high. Gene movement between populations could be
mediated through strong ying insect pollinators that carry
pollen between trees or through large herbivorous mammals
that move through these populations, feeding on fruits in
one population and defecaing seeds at a distant location.
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is a senior Biology major with a minor in Chemistry. He is scheduled to graduate in May 2000 and plans to
continue his education at MSU in the Plant Biology Graduate Program. He is also a member of the IUSB Mens
Basketball program, both as a player and as Assistant Couch. Joe began his research when he had the opportunity
to work with Dr. Andrew Schnabel, who served as his mentor. His research was supported by a SMART Summer
Fellowship.
Joe