The coastal wetlands of the tropics and the subtropics of the world are characterized
by the presence of a unique group of plant species, the mangroves. Despite their
unique status as intertidal forests, hosting numerous faunal organisms and providing
essential functions and services to tropical and subtropical zones and their
populations, mangroves are one of the world's most threatened ecosystems (Triest,
2008). Mangrove forests all over the world are heavily exploited for wood
and fishpond operations, as well as other activities. The exploitation of mangroves
has resulted in the loss of genetic diversity in mangrove ecosystems (Maguire
et al., 2000).
Since, the mangrove ecosystems are seriously affected, the conservation and
suitable management of mangroves is a major priority in coastal areas of many
countries. To better design effective management strategies, it is important
to understand basic population parameters such as inbreeding, dispersal and
regional and local population parameters for key members of the mangrove community
(Souza et al., 2006).
Avicennia marina (Forsk.) Vierh, as a pioneer tree species of mangrove
forest ecosystems, is widely distributed from East Africa and Persian Gulf,
throughout Asia to China and Japan, to the Southwestern Pacific, New Zealand
and Australia (Giang et al., 2003). It can grow
and reproduce across a wide range of climatic, saline and tidal conditions.
The wide geographical and climatic distribution of A. marina indicates
that there is a large amount of genetic diversity available, which can be exploited
for conservation, breeding programs and afforestration schemes (Maguire
et al., 2002).
Several studies have been carried out on mangrove species in order to assess
genetic diversity using genetic markers such as Random Amplified Polymorphic
DNA (RAPD), Restriction Fragment Length Polymorphism (RFLP) (Balakrishma,
1995; Parani et al., 1997) and recently microsatellite
and Amplified Fragment Length Polymorphism (AFLP) (Maguire
et al., 2000, 2002; Dodd
et al., 2002; Giang et al., 2003).
Microsatellite markers are short tandem repeats of mono- to tetra-nucleotide
repeats, which are assumed to be randomly distributed in the nuclear genome.
Such sequence repeats are relatively abundant and have high mutation rates in
comparison to other markers, which make them useful for various types of population
studies (Lowe et al., 2004).
In this study, four populations of A. marina encompassing the coastal areas of Persian Gulf were studied using microsatellite markers. The aim of this study was to determine the genetic variation within and between A. marina populations in Iran.
MATERIALS AND METHODS
Sample Collection and DNA Isolation
A total of 44 individuals, representing four natural populations, were sampled
over the entire Iranian range of A. marina; namely Khamir, Qeshm, Tiab
and Jask located in 25° 41 to 27° 5 North latitude and 55° 28
to 57°48 East longitude (Fig. 1). Leaf materials from
each population were collected between June and July 2007. Total genomic DNA
was isolated from leaf tissue using a modified CTAB method (Maguire
et al., 1994).
Primer sequences specific for five microsatellite loci described by Maguire
et al. (2000) were used in this study (Table 1).
The Polymerase Chain Reaction (PCR) conditions were optimized for the five microsatellite
loci as necessary to produce scorable amplification products. The PCR was performed
in a 20 μL reaction volume containing 100 ng of template DNA, 10 pmol of
each primer, 400 μM each of the dNTPs, 1 U of Taq DNA polymerase (Cinnagen,
Iran), 1.5 mM MgCl2 and 1xPCR buffer. The temperature profile consisted
of 3 min initial denaturation at 94°C followed by 30 cycles of: 30 sec at
94°C, 45 sec at the annealing temperature (55°C) and 30 sec at 72°C,
ending with 5 min at 72°C. The PCR products were separated on 6% polyacrylamide
gels stained with silver nitrate.
The recorded microsatellite genotypes were used as input data for the GenAlex
software version 6 package (Peakall and Smouse, 2006)
in order to calculate allele and genotype frequencies, observed (Ho)
and expected (He) heterozygosities and to test for deviations from
Hardy-Weinberg Equilibrium (HWE). The phylogenetic relationship among the four
populations was estimated from Nei's standard genetic distance (D) and genetic
similarity index (I) (Nei, 1972). Genetic differentiation
between populations was also evaluated by the calculation of pairwise estimates
of Fst values and testing their significance using the FSTAT software
showing sampling locations of four populations of A. marina in
the Persian Gulf
five microsatellite loci used in this study
We also estimated an overall inbreeding coefficient (Fis; (Weir
and Cockerham, 1984)) for each population and locus, which can measure HWE
departures within a population. A UPGMA tree was constructed based on Nei's
genetic distance using TFPGA version 1.3 (Miller, 1997).
The genetic variability indices estimated for the four A. marina populations
are summarized in Table 2. All the five microsatellite loci
were polymorphic in all the populations examined and the levels of the polymorphism
varied depending on the locus. The only private allele (M81-4) at the population
level was observed in Jask. The average number of alleles per locus ranged from
4 in the Khamir population to 4.6 in the Qeshm and Tiab populations, showing
no significant difference among the four populations (p>0.05). The differences
between populations were not statistically significant (p>) for the average
observed heterozygosity (Ho).
of five microsatellite loci in four A. marina populations from
No. of alleles; Ho: Observed heterozygosity; He:
Expected heterozygosity; P: p-values of Chi-Square tests for Hardy-Weinberg
equilibrium; Fis: Fixation index. Statistically significant
values are marked with asterisks. *p<0.05, **p= 0.001
The average Ho ranged from 0.782 in the Tiab population to 0.960
in the Khamir population. The expected heterozygosity (He) was high,
ranging from 0.590 to 0.826.
Significant to highly significant deviations from Hardy-Weinberg expectations were observed in 10 out of 20 (five loci H four populations) cases (Table 3). Most of Fis values were negative and significantly different from zero, thus suggesting excess of heterozygosity.
The population differentiation (Fst) value between Tiab and Khamir populations was the highest (0.100) and significant among the population pair, while the Fst value between the Khamir and Qeshm populations (0.021) was the lowest and not significant (Table 3). The estimated gene flow (Nm) value between the Qeshm and Khamir population across all the studied loci was the highest, while the Nm value between Khamir and Tiab populations was the lowest (Table 4).
Genetic distance (D) and genetic similarity index (I) between any two populations are shown in Table 4. The genetic distance was the smallest (0.189) between the Qeshm and Khamir populations, whereas the largest distance (0.482) was between Khamir and Tiab populations. The UPGMA dendrogram constructed on the basis of the Nei's genetic distance showed the four populations allocated into two groups (Fig. 2), that is, one group including the Qeshm and Khamir populations and the other group including the Tiab and Jask populations.
Nm (above diagonal) and Fst values (below diagonal)
between pairs of A. marina populations across all loci
distance (D) (above diagonal) and genetic similarity (I) (below diagonal)
between pairs of A.marina populations
UPGMA dendrogram shows genetic distance between four populations allocated
into two groups
Using five microsatellite loci a total of 24 alleles were detected. The average
number of alleles per locus per population ranged from 4 to 4.6, showing the
same level of allelic diversity comparing to the values detected earlier for
A. marina sampled from the worldwide range (Maguire
et al., 2000).
The observed heterozygosity (Ho) detected over all loci, ranging
from 0.782 to 0.960 was comparable in the Iranian populations of A. marina.
This is much higher than the levels of heterozygosity described earlier, where
estimates of Ho ranged from 0.210 (Giang et
al., 2003) to 0.407 (Maguire et al., 2000).
Furthermore, A. marina populations in Iran were found to be generally
outcrossing with no inbreeding, which do not correspond to earlier data (Giang
et al., 2003; Maguire et al., 2000).
This is not unexpected as these populations may have not been subjected to repeated
bottleneck or founder effects in earlier times, due to episodes of glaciations
and transgressions. It has been reported that populations of A. marina
show reduced levels of polymorphism due to the constant use of foliage for fodder
and grazing as well as environmental pollution (Parani et
al., 1997). Regarding the higher levels of genetic heterozygosity in
our study, it could be inferred that the Iranian populations of A. marina
may have not been severely subjected to environmental impacts compared to other
populations of A. marina in the entire worldwide range.
Pairwise genetic differentiation (Fst) was used to assess genetic
differentiation, which is the acquisition of allele frequencies that differ
among populations (Daniel and Clark, 1997). The value
of Fst is a useful measure of genetic differentiation among populations
and different values mean different variation degrees. Significant population
differentiation was observed between Tiab and Khamir and also between Jask and
Khamir populations. In our study, the microsatellite analysis showed low genetic
differentiation among the populations (mean Fst = 0.044). However,
high values of genetic differentiation have been found in worldwide populations
of A. marina using microsatellite analysis (Fst = 0.410, Maguire
et al., 2000). The low value of Fst in A. marina
in Iran could be explained by the remarkable gene flow (Nm>1)
among populations. The ocean-borne propagules accompanied by insect pollination
may act as the efficient means of gene flow.
A dendrogram based on genetic distance (Fig. 2) showed two major clusters corresponding to the delineated geographical region. Our results indicate that geographical distance has caused genetic divergence among A. marina populations in Iran due to limitation of propagules dispersal.
The data generated in this study provide useful information on the genetic variation and differentiation in the Iranian populations of A. marina. Since, the Iranian mangrove forests showed higher genetic variation, suitable management strategies should be considered to avoid the loss of genetic diversity in the Iranian mangrove ecosystems.
The authors would like to thank by Department of Marine Environment, Iran for financial support of this study. We also express our sincere gratitude to Dr. Mohammad Ali Salari for his assistance.