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ISSN 2087-3940 (PRINT) | ISSN 2087
Jatropha curcas photo by A. Abdurrahman | Nus Biosci | vol. 3 | no. 1 | pp. 1‐58 | March 2011 |
ISSN 2087‐3940 (PRINT) | ISSN 2087‐3956 (ELECTRONIC)
| Nus Biosci | vol. 3 | no. 1 | pp. 1‐58 | March 2011 | ISSN 2087‐3940 (PRINT) | ISSN 2087‐3956 (ELECTRONIC) I S E A J o u r n a l o f B i o l o g i c a l S c i e n c e s FIRST PUBLISHED:
2009
ISSN:
2087-3940 (printed edition), 2087-3956 (electronic edition)
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(Sebelas Maret University, Surakarta), Ehsan Kamrani (Hormozgan University, IR Iran), Eko Handayanto (Brawijaya University,
Malang), Endang Sutariningsih (Gadjah Mada University, Yogyakarta), Faturochman (Gadjah Mada University, Yogyakarta), Iwan
Yahya (Sebelas Maret University, Surakarta), Jamaluddin (R.D. University, Jabalpur, India), Lien A. Sutasurya (Bandung Institute of
Technology, Bandung), Magdy Ibrahim El-Bana (Suez Canal University, Al-Arish, Egypt), Mahendra K. Rai (Amravati University,
India), Marsetyawan H.N. Ekandaru (Gadjah Mada University, Yogyakarta), Oemar Sri Hartanto (Sebelas Maret University, Surakarta),
R. Wasito (Gadjah Mada University, Yogyakarta), Rugayah (Indonesian Institute of Science, Cibinong-Bogor), Sameer A. Masoud
(Philadelphia University, Amman, Jordan), Supriyadi (Balitbiogen, Bogor), Sri Margana (Gadjah Mada University, Yogyakarta), Suranto
(Sebelas Maret University, Surakarta), Sutarno (Sebelas Maret University, Surakarta), Sutiman B. Sumitro (Brawijaya University,
Malang), Taufikurrahman (Bandung Institut of Technology, Bandung), Wayan T. Artama (Gadjah Mada University, Yogyakarta)
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ISSN: 2087-3940 (print)
ISSN: 2087-3956 (electronic)
Vol. 3, No. 1, Pp. 1-6
March 2011
Optimization of DNA extraction of physic nut (Jatropha curcas) by
selecting the appropriate leaf
EDI PRAYITNO, EINSTIVINA NURYANDANI♥
Open University, UPBJJ Semarang. Jl. Semarang-Kendal, Mangkang Wetan, Semarang 50156, Central Java, Indonesia, Tel. +62-24-8666044, Fax. +6224-8666045; ♥email: [email protected]
Manuscript received: 11 November 2010. Revision accepted: 24 February 2011 (stay empty)
Abstract. Prayitno E, Nuryandani E. 2011. Optimization of DNA extraction of physic nut (Jatropha curcas) by selecting the appropriate
leaf. Nusantara Bioscience 3: 1-6. Jatropha curcas L. has important roles as renewable source of bioenergy. The problem occurs on
difficult of DNA extraction for its molecular breeding programs. The objectives of this research were to study which leaf best as source
of DNA extraction. Four accession were used, namely J1 and J2 (Jawa Tengah), S1 (South Sumatra), and S2 (Bengkulu). First, third,
fifth, seventh, and yellow leaves for each accession were extracted using modification of Doyle and Doyle (1987) method. Visualization
and comparation with Lambda DNA, Spectrophotometer UV-Vis and cutting DNA with EcoRI enzyme were show quality and quantity
of DNA. The result showed that third leaves have sufficient quality and quantity as source of DNA. Third leaves DNA quantity for J1
(19.33 µg/mL), J2 (26.21 µg/mL), S1 (31.20 µg/mL), dan S2 (61.03 µg/mL), and quality for each accession were 1.9063 (J1), 2.0162
(J2), 2.0116 (S1), and 2.0856 (S2).
Key words: Jatropha curcas, DNA extraction, appropriate, leaf.
Abstrak. Prayitno E, Nuryandani E. 2011. Optimalisasi ekstraksi DNA jarak pagar (Jatropha curcas) melalui pemilihan daun yang
sesuai. Nusantara Bioscience. Nusantara Bioscience 3: 1-6. Jarak pagar (Jatropha curcas L.) mempunyai peran penting sebagai sumber
bahan bakar nabati. Usaha pemuliaan tanaman ini secara molekuler sering terkendala sulitnya ekstraksi DNA. Penelitian ini bertujuan
untuk mengetahui daun yang sesuai untuk digunakan sebagai sumber DNA. Penelitian ini dilakukan pada empat aksesi jarak pagar yaitu
J1 dan J2 (Jawa Tengah), S1 (Sumatera Selatan), dan S2 (Bengkulu). Ekstraksi dilakukan pada daun pertama, ketiga, kelima, ketujuh,
dan daun kuning dari setiap aksesi dengan metode Doyle and Doyle (1987) yang dimodifikasi. Kualitas dan kuantitas DNA hasil
ekstraksi diketahui melalui visualisasi dengan pembanding DNA lambda, spektrofotometer UV-Vis pada panjang gelombang 260/280,
dan pemotongan menggunakan enzim EcoRI. Hasil penelitian menunjukkan bahwa daun ketiga memadai untuk digunakan sebagai
sumber DNA. Kuantitas DNA daun ketiga J1 (19,33 µg/mL), J2 (26,21 µg/mL), S1 (31,20 µg/mL), dan S2 (61,03 µg/mL). Sedangkan
kemurniannya masing-masing yaitu 1,9063 (J1), 2,0162 (J2), 2,0116 (S1), dan 2,0856 (S2).
Kata kunci: Jatropha curcas, ekstraksi DNA, daun, sesuai.
INTRODUCTION
Increased economic growth and spur the growth of
population of high energy consumption. Energy source the
world today is still dominated by fossil fuel that cannot be
renewed (unrenewable). Various efforts have been made to
solve energy problems (Raharjo 2007). Fuel from the plant
has several advantages such as ease of storage and
environmentally friendly, therefore biofuels were given
priority for development. On January 25, 2006, the
President of Indonesia issued Presidential Regulation No.
5/2006 regarding the national energy policy and
Presidential Instruction No. 1/2006 concerning the
provision and use of biofuels as alternative fules. Then on
July 1, 2006, presidential and state officials conducting a
retreat in the village of Losari, Grabag subdistric,
Magelang district, and decided to develop a bioenergy or
biofuel as an alternative energy.
Biofuel can be divided into two major categories,
namely bioethanol and biodiesel. Bioethanol is ethanol
derived from fermentation of raw materials that contain
starch or sugar such as molasses and cassava. This fuel can
be used to replace regular gasoline (gasoline). Ethanol can
be used is alcohol-free pure water (anhydrous alcohol) and
levels of more than 99.5%, or called with a fuel grade
ethanol (FGE). Blend of premium and FGE is called
gasohol. In Indonesia, Pertamina give biopremium
trademark for the product. Biodiesel is a popular name for
FAME (fatty acid methyl ester), is a biofuel that is used to
power diesel engines as an alternative to diesel. This fuel
derived from vegetable oils are converted through chemical
and physical reactions, so that the nature of the chemical
has changed from its original nature. Currently, Pertamina
has issued such a product with trade name which is a
blending FAME biodiesel with regular diesel (petrosolar)
(Prihandana et al. 2007).
2
3 (1): 1-6, March 2011
Jatropha curcas is a native plant of Central America
(Fairless 2007) and has been naturalized in tropical and
subtropical regions, including Indonesia. This species is
drought resistant and is commonly planted as a garden
fence, but is also useful as an ornamental plant shrubs and
herbs. Oil from the seeds is useful for medicine,
insecticides, making soap and candles, as well as raw
material for biodiesel (Gubitz et al. 1999). The use of
castor oil as biodiesel ingredient is an ideal alternative,
because it is a renewable oil resources (renewable fuels)
and non-edible oil so it does not compete with human
consumption requirements, such as palm oil, corn,
soybeans and others (Dwimahyani 2005). In addition,
Jatropha also contains secondary metabolites which are
useful as protectant for plants and as an ingredient for
human
medicine
(Debnath
and
Bisen
2008)
Some of the obstacles encountered in developing castor oil,
among others, lack of information about varieties that have
beneficial properties such as high production, fast
multiplication, high oil yield in seeds, as well as resistance
to pests and diseases. This happens because so far the
Jatropha plant is only regarded as hedgerows that have low
economic value so that research and development of this
plant is rarely done. To overcome this, plant breeding has a
significant role.
Characterization of jatropha plant in Indonesia is
carried out simply and not be universal. Often, the mention
of Jatropha plant species is based solely on phenotypic
appearance or region of origin. Characterization using
morphological or phenotypic description has limitations
because it is very influenced by the environment. Different
morphological features can be caused by environmental
stress, whereas the same genotype, whereas the same
morphological features do not necessarily indicate that both
types of plants are closely related, because the outer shape
of a plant is the result of cooperation between the genotype
by environment (Joshi et al. 1999; Karsinah 1999 .)
Therefore, it is necessary to develop universal genetic
information. Molecular markers can provide information
universally because it is not influenced by the environment
(Azrai 2005), so that they can answer the problem in the
characterization of physic nut plants.
Jatropha curcas is one of the many plants that contain
latex, which is a true plant secondary metabolites. The
presence of secondary metabolites such as polyphenols,
tannins, and polysaccharides can inhibit the action of the
enzyme (Porebski 1997; Pirtilla et al. 2001). Isolation of
plant DNA at a distance often experienced problems due to
high levels of secondary metabolites in the form of
polysaccharides and polyphenols. According to Sharma et
al. (2002) the presence of metabolites in several crops
affect DNA isolation procedure, he was using a modified
CTAB to isolate DNA from plant tissue containing high
polysaccharide. In line with this Kiefer et al. (2000), Pirtilla
et al. (2001) and Sanchez-Hernandes, C. and J.C. GaytanOyarzun (2006), states that the extraction of DNA and
RNA from plants containing polysaccharides, polyphenols
as well as sap and difficult.
Proper techniques of DNA extraction is needed in the
plant breeding process to obtain DNA with a high quality
and quantity. To obtain pure DNA from plant sap,
generally carried out repeated purification and modification
of procedures (Kiefer et al. 2000), thus requiring additional
cost and effort. For that, you can use parts of plants that
contain little secondary metabolites. The content of
secondary metabolites in plant tissues fluctuate in line with
its development. Secondary metabolites may vary because
of differences in age and plant part (Cirak et al. 2007a, b,
2008; Achakzai et al. 2009). Therefore, to simplify the
DNA extraction process jatropha, have done research to
learn the parts of plants containing secondary metabolites
in small amounts and produce DNA with high quality and
quantity.
This research aims to study the jatropha plant leaves at
different levels of development that have the potential to
produce the best quality and quantity of DNA in the DNA
extraction process.
MATERIALS AND METHODS
Time and place of study
Research was conducted at the Open University UPBJJSemarang, Central Research Laboratory Tropical Fruit IPB,
Bogor, West Java, and Laboratory of Structure and
Function of Plant Diponegoro University in March to
November 2009.
Plant material
Jatropha plant materials used in this study are the three
accessions of jatropha plants originated from areas of
Klaten (Central Java) with the code J1 and J2, Palembang
(South Sumatra) with codes S1, and Bengkulu, with the
code S2.
Procedures
Isolation of DNA. About 0.5 g of leaves from the first,
third, fifth, seventh and yellow leaves from each sample
was crushed in porcelain bowls by adding 0.1 grams of
silica sand to be easily crushed. To prevent network
browning by oxidation, polivinilpolipirilidon (PVPP) as
much as 40 mg and added extraction buffer (2% CTAB,
100 mM Tris-HCl pH 8, 1.4 M NaCl, 20 mM EDTA) as
much as 1 mL is added into a cup containing the sample
which has added 1% merkaptoetanol. Samples that have
been incorporated into the fine volume of 1.5 mL
Eppendorf tube. Subsequently the mixture incubated at
65oC for 30 minutes while inverted, and then added 1 mL
solution of chloroform: isoamilalchohol (24:1 = v/v) and
divortek for 5 seconds. This solution was then separated
using a centrifuge with a speed of 11,000 rpm for 10
minutes at a room temperature. Supernatant was separated
from the pellet by putting it into a new Ependorf tube.
DNA in the supernatant was purified by adding 1 mL
solution of chloroform: isoamilalkohol (24:1 = v/v) and
disentrifuse at a speed of 11,000 rpm for 10 minutes at
room temperature. Supernatant was transferred into a tube
and added with 1 mL of cold isopropanol, shaken gently
until white threads arise, which is DNA. Subsequently
DNA was precipitated by incubation for 30 minutes at a
PRAYITNO & NURYANDANI – Optimization of DNA extraction of Jatropha curcas
present in yellow leaves, except on J1 where J1k (yellow)
has a thicker ribbon smears compared J15 and J17 (Figure
1).
In genomic DNA extracted from young leaves, which
are visible smear on the bottom of genomic DNA. Ribbon
smear is a molecule with varying weights that can be
derived from degraded DNA or other follow-up material
that is not known (Herison 2003). Smears indicated that the
isolated genomic DNA was not intact anymore, probably
dismembered during the extraction takes place (Sisharmini
et al. 2001). Genomic DNA damage can be caused by
degradation of secondary compounds that are released
when the cells were destroyed or damaged due to physical
handling. The decline is likely influenced by the smear of
secondary metabolites of plants and physical handling. In
this case the physical handling for each sample the same
can be said for using the same standard procedure,
therefore, the greatest influences that cause differences in
high and low smear is a secondary metabolite from the
leaves of plants (Milligan 1992).
In certain plants, plant metabolites will be seen visually
in the form of sap. Jatropha curcas is a plant sap, with pink
latex (de Padua et al. 1999) or nodes in the young gradually
turns cloudy/older if left in free air or dark brown when
taken from the older plants (Heyne 1987). Young leaves
contain more secondary metabolites than older leaves
(Badawi 2006; Mulyani 2006). Young leaves generally
contain secondary metabolites and enzymes that high
because it requires in the process of growth, development,
and division of cells’ leaf. In the development of plant
secondary metabolite concentrations will gradually decline
as the decline in leaf growth activity, and the leaves have
yellowed, the concentration of enzymes and secondary
metabolites in the leaves decreased significantly due to the
ongoing process of senesensi (Salisbury and Ross 1995).
At this stage the plant will attract substances and enzymes
that are still useful to the plant from old leaves for use in
the process of development of the younger plants, so the
possibility of plant secondary metabolites present in a very
low level so that the DNA is not much degraded by the
follow-up compound (Salisbury and Ross 1995; Herison
2003). Although the smear on the older leaves less and less,
but the quantity of genomic DNA was also decreased,
which lights up genomic DNA bands at the top of the wells
that are running low on older leaves.
temperature of -20ºC. Solution containing the DNA that
has been purified disentrifuse with speed 11 000 rpm for 10
minutes at room temperature and then the supernatant was
discarded. DNA precipitate was washed with 70% alcohol
and dried at room temperature. Further the DNA samples
that was obtained was dissolved in 100 mL TE buffer (10
mM Tris-HCl pH 7.5, 10 mM EDTA) and incubated at 37°
C for one hour and then mixed until uniform to further test
its quality.
Test the quality and quantity of DNA. The quantity
(concentration) and quality of DNA determined by UV-Vis
spectrophotometer at wavelength 260 and 280 nm.
Determination of the total DNA quantity was calculated
based on the value of absorbance at a wavelength of 260
nm. A at 260 = 1.0 equivalent amount of DNA is 50
ug/mL. λ DNA quality is considered good if the value of
A260/280 approaching 1.8 to 2. To determine the
concentration and quality of DNA, electrophoresis results
were soaked in a solution of 1% EtBr and then observed
under UV transluminator. The quantity of DNA is based on
the thickness of the electrophoresis results of DNA samples
are compared with the amount of lambda DNA of known
concentration, ie 250 ug/mL. This study also tested the
quality of DNA by cutting genomic DNA using EcoRI
enzyme are visualized by electrophoresis on agarose gel.
RESULTS AND DISCUSSION
Visualization of the extracted DNA
The success of the isolation and extraction process of
genomic DNA can be marked with resultant large DNA
(high molecular weight DNA), that is not degraded during
extraction and purification process, and can be cut by
restriction enzymes that has been used (Herison 2003).
Results of isolation and extraction of jatropha’s DNS
employed Doyle and Doyle method (1987) which has been
modified to produce the desired genomic DNA bands,
although relatively small quantity when compared to
lambda DNA. Genomic DNA was seen as a ribbon that
lights up at the top sinks electrophoresis results.
In general, smear on DNA extracted from young leaves
(code J11, J21, S11, S21) and concentrated look taller than
the smear on DNA extracted from the older leaves, then
gradually decreasing concentration smear on leaves more
old (leaves the third, fifth, and seventh), and smear the least
L
J11
J13
J15
J17
J1K
J21
J23
J25
J27
J2K
S11
3
S13
S15
S17
S1K
L
L
S21
S23
S25
S27
S2K
L
Figure 1. Visualization of the extracted DNA from four accessions of Jatropha curcas Klaten (J1, J2), Palembang (S1) and Bengkulu
(S2). L = LAMDA (ladder)
4
3 (1): 1-6, March 2011
The young leaves have a high cleavage activity. In the
division process, DNA replication will experience, so the
amount of DNA will double itself, thus DNA concentration
is relatively high in young leaves. On older leaves, the
division process could decrease, until finally stopped
altogether. On the leaves that have yellowed, in addition to
the absence of the division process, it also exacerbated the
death of cells that were old, so the amount of DNA was
also decreased dramatically (Salisbury and Ross 1995).
Test the quality and quantity of DNA with UV-Vis
spectrophotometer
The quantity (concentration) and quality of DNA
determined by UV-Vis spectrophotometer at wavelength
260 and 280 nm. Determination of the total DNA quantity
was calculated based on the value of absorbance at a
wavelength of 260 nm. The highest DNA purity can be
seen in the A260/280 ratio that produces the value of 1.8 to
2. According to Sambrook et al. (1989) DNA with a ratio
in the range of figures have met the requirements of purity
required in molecular analysis. Spectrophotometer results
show relatively good purity DNA that has yet to reach
100% purity in some accessions. The concentration and
purity of genomic DNA was analyzed using UV-Vis
spectrophotometer can be seen in Table 1.
Genomic DNA which has a purity of 100% contained in
the accession J1 was extracted from the third leaf with
value ratio of 1.9063. Genomic DNA from the first leaf
accession J1 has a value ratios approaching 100% purity
with ratio of 2.0131. While the three other leaves, that
leaves the fifth, seventh, and yellow leaves have a value
ratio of less than 1.8 respectively, 1.7417, 1.2578, and
1.2356. Results DNA extraction leaves first, third, and fifth
of the accession J2 has a value closer to purity ratio,
respectively 2.0697, 2.0162, 2.0914, while the seventh
leaves and yellow leaves have a ratio value that is still far
from
purity,
namely
1.5873
and
1,
1940.
On the accession of S1, almost all of the extracted DNA
purity approaching leaves, each leaf of the first, third, fifth,
and seventh ratio is 2.0768, 2.0116, 2.0792, 2.0225, while
the yellow leaves have value ratio far from the purity of
1.4434. DNA extracted first and third leaf from the
accession of S2 close to the purity of the value ratio of
2.0611 and 2.0856. While leaf fifth, seventh, and yellow
leaves have a ratio that is far from the purity of the
respective ratios 2.2187, 2.1782, and 1.5177.
Besides the purity of genomic DNA samples, another
consideration that must be considered is the quantity of
genomic DNA was generated from the DNA extraction
process. Readings A260 = 1 means the concentration of
DNA obtained at 50 ug/mL (Herison 2003). The
concentration of genomic DNA was extracted was
calculated by the formula: DNA concentration (ug/mL) =
A260 x dilution factor x 50 ug/mL.
DNA concentration resulting from the extraction
process represents the amount of DNA contained in the leaf
tissue used for the sample and treatment methods used in
each sample is the same. Table 1 below is the concentration
of DNA from samples of twenty leaves from four
accessions of jatropha plant that is used. From Table 1,
note the concentration ratio of genomic DNA from leaf
tissue of each first, third, fifth, seventh, and yellow leaves,
and comparison of genomic DNA concentration between
sections. In general, genomic DNA concentration
decreased with increasing age of leaves used as a sample.
Samples from the first leaf shows the quantity of
genomic DNA is much larger than the sample leaves the
third, fifth, seventh, and yellow leaves. Measurement of the
quantity of genomic DNA samples from accessions J1
genomic DNA in Klaten produces relatively little
compared to the accession of J2, S1, and S2, which is 27.69
ug/mL for the first leaf, 19.33 ug/mL for the third leaf, 3.68
tg/mL for the fifth leaf, 2.03 g/mL for the seventh leaf, and
4.51 ug/mL for yellow leaves. This is due to a smaller
sample size compared to other accessions due to spill some
of the samples by laboratory staff who worked on, so that
DNA samples that were tested got reduced. While the
accession J2, where accession was also derived from the
same home with the accession of J1, which was from
Klaten, Central Java, and comes from the same parent, the
quantity of genomic DNA generated greater than J1, which
is 62.06 ug/mL for the extraction of the first leaf, 26.21
ug/mL for the third leaf, 27.69 ug/mL for the fifth leaf,
5.37 g/mL for the seventh leaf, and 4.37 ug/mL for yellow
leaves.
The concentration of genomic DNA for S1 accession on
the first leaves produced 67.61 g/mL DNA, whereas the
third leaf, the concentration of genomic DNA was 31.20
ug/mL, on the fifth leaves of 46.71 ug/mL, on the seventh
leaf, 22, 90 ug/mL, and the yellow leaves of 7.59 g/mL.
Accession S2 on the first leaves produced 101.35 g/mL
genomic DNA, while the third leaf, the concentration of
genomic DNA was 61.03 ug/mL, on the fifth leaves of
44.18 ug/mL, leaves the seventh, 26.27 ug/mL , and the
yellow leaves of 5.37 g/mL. The Figure 1 shows the
concentration of the extracted genomic DNA of
Table 1. Test the quality (purity) and quantity (concentration) of DNA using UV-Vis spectrophotometer in four accessions of jatropha
from Klaten (J1, J2), Palembang (S1) dan Bengkulu (S2).
Leaves
First
Third
Fifth
Seventh
Yellow
J1
2.0131
1.9063
1.7417
1.2578
1.2356
DNA purity
J2
S1
2.0697
2.0768
2.0162
2.0116
2.0914
2.0792
1.5873
2.0225
1.1940
1.4434
S2
2.0611
2.0856
2.2187
2.1782
1.5177
J1
27.69
19.33
3.68
2.03
4.51
DNA concentration (µg/mL)
J2
S1
62.06
67.61
26.21
31.20
27.69
46.71
5.37
22.90
4.37
7.59
S2
101.35
61.03
44.18
26.27
5.37
PRAYITNO & NURYANDANI – Optimization of DNA extraction of Jatropha curcas
as secondary metabolites, carbohydrates, proteins, and
others, will hinder the work restriction enzymes.
Whether DNA can be cut with restriction enzymes is
visible from at least smear results of electrophoresis bands
after DNA cut with EcoRI enzyme (Herison 2003). EcoRI
produce DNA bands when smears were electrophoresed
because this restriction enzyme included in the frequent
cutter (Vos et al. 1995). The result of cutting with EcoRI
enzyme produces DNA fragments that appear as a smear
on some samples, but most other samples can not be cut by
this enzyme because of the high follow-up compounds that
inhibit enzymes work. Smear only be observed in J13 and
J15, while the other samples have not seen a clear smear as
a result of enzyme EcoRI. Visible is the presence of minor
compounds in the lower section sinks. possible follow-up
material that inhibits this enzyme EcoRI work so as not to
cut the genomic DNA tested jatropha.
The description above discussion shows that differences
in leaf tissue age used influence the extraction of genomic
DNA where the younger leaves will produce a quantity of
genomic DNA was higher but also accompanied by the
high follow-up material in the form of plant secondary
metabolites that inhibit the work in the field of molecular
further. Older leaves to produce the amount of genomic
DNA are relatively fewer compared to young leaves, but
the following secondary metabolites was also reduced in
number. This study shows that the third leaf is better used
as a source of genomic DNA since the DNA purity is better
than the other leaves, and the quantity produced enough
DNA to be used for further molecular analysis.
diminishing. This is related to the phase of leaf
development that has been outlined above.
Results spectrophotometer for quantity of genomic
DNA of the above shows that the largest quantity of
genomic DNA from four accessions were found in the
extraction of the first leaf. But considering the quality of
the resulting DNA, the highest purity approaching 100%
are found in the sample using the third leaf as a source of
genomic DNA, although in terms of quantity, the number is
lower than the samples originated from the first leaf.
Comparison of DNA extracted from five types of leaf
samples from accessions used in J1 and J2 from Klaten,
from the same parent tree can be seen in Table 1. From
Table 1 it can be seen that the DNA genome of the first and
second leaf (accession J1) and leaves the first, third, and
fifth (accession J2) approached the purity, but purity is
closest to the third leaf (accession J1 on the ratio of 1, 9063
(purity 100%) and the accession to the ratio of 2.0162 A2).
But in terms of quantity, J1 and J2 are not comparable
although originating from the same parent because of the
sample is not the same J1 J2 terms of number of samples
tested for spill samples by the laboratory.
Some researches indicate that generally young leaves
are used in DNA extraction because of the ease in getting
the DNA with a high quantity. Mansyah et al. (2003) who
conducted research on mangosteen states that extraction of
DNA from old leaves is more difficult when compared
with young leaves, so as to obtain DNA from old leaves
with a sufficient quantity is required special treatment,
namely with the addition of the extracted leaves up to 2 g
and DNA purification with the addition of RNase. While
Prana (2003) who perform DNA extraction on taro plants
also use the young leaves (in this case the leaf shoots) as
the source of DNA.
CONCLUSION
The third leaf physic nut plants suitable for use as a
source of DNA for molecular analysis of genomes, as in
quantity and quality sufficient to produce genomic DNA
for further molecular analysis such as PCR. Genomic DNA
extracted from the third leaf is generally close to 100%
purity and quantity of DNA produced is also large enough
to be used for further molecular analysis.
Test the quality of DNA by using the enzyme EcoRI
cuts
The purity of DNA can be seen from the absence of a
DNA sample can be cut by restriction enzyme such as
EcoR1 (Figure 2). If a DNA sample has high purity, this
DNA would be easy to cut by restriction enzymes. But if
this is still contain DNA samples follow-up materials such
M
J11
J13
J15
J17
J1k
J21
J23
J25
J27
J2K
5
S11
S13
S15
S17
S1K
S21
S23
S25
S27
S2k
M
Figure 2. Visualization of results by the enzyme EcoRI cuts at the four accessions of jatropha from Klaten (J1, J2), Palembang (S1) and
Bengkulu (S2).
6
3 (1): 1-6, March 2011
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ISSN: 2087-3940 (print)
ISSN: 2087-3956 (electronic)
Vol. 2, No. 1, Pp. 7-14
March 2010
Characterisation of taro (Colocasia esculenta) based on morphological and
isozymic patterns markers
TRIMANTO1,♥, SAJIDAN², SUGIYARTO²
¹ SMP Negeri 2 Gemolong, Sragen, Jl. Citro Sancakan No. 249, Sragen 57274, Central Java, Indonesia; Tel.: +92-0818754378
² Bioscience Program, School of Graduates, Sebelas Maret University, Surakarta 57126, Central Java, Indonesia
Manuscript received: 25 October 2009. Revision accepted: 15 February 2010.
Abstract. Trimanto, Sajidan, Sugiyarto. 2011. Characterization of taro (Colocasia esculenta) based on morphological and isozymic
patterns markers. Nusantara Bioscience: 7-14. The aims of this research were to find out: (i) the variety of Colocasia esculenta based
on the morphological characteristics; (ii) the variety of C. esculenta based on the isozymic banding pattern; and (iii) the correlation of
genetic distance based on the morphological characteristics and isozymic banding pattern. Survey research conducted in the
Karanganyar district, which include high, medium and low altitude. The sample was taken using random purposive sampling technique,
including 9 sampling points. The morphological data was elaborated descriptively and then made dendogram. The data on isozymic
banding pattern was analyzed quantitatively based on the presence or absence of bands appeared on the gel, and then made dendogram.
The correlation based on the morphological characteristics and isozymic banding pattern were analyzed based on the product-moment
correlation coefficient with goodness of fit criterion. The result showed : (i) in Karanganyar was founded 10 variety of C. esculenta; (ii)
morphological characteristics are not affected by altitude; (iii) isozymic banding pattern of peroxides forms 14 banding patterns, esterase
forms 11 banding patterns and shikimic dehydrogenase forms 15 banding patterns; (iv) the correlation of morphological data and the
isozymic banding pattern of peroxidase has good correlation (0.893542288) while esterase and shikimic dehydrogenase isozymes have
very good correlation (0.917557716 and 0.9121985446); (v) isozymic banding pattern of data supports the morphological character data.
Key words: taro, Colocasia esculenta, morphology, isozyme.
Abstrak. Trimanto, Sajidan, Sugiyarto. 2011. Karakterisasi talas (Colocasia esculenta) berdasarkan penanda morfologi dan pola pita
isozim. Nusantara Bioscience: 7-14. Tujuan penelitian ini adalah untuk mengetahui: (i) keragaman Colocasia esculenta berdasarkan
karakter morfologi; (ii) keragaman C. esculenta berdasarkan pola pita isozim, dan (iii) hubungan jarak genetik berdasarkan karakter
morfologi dan pola pita isozim. Survei penelitian dilakukan di Kabupaten Karanganyar, di ketinggian tinggi, sedang dan rendah. Sampel
diambil menggunakan teknik random sampling purposif, mencakup 9 titik cuplikan. Data morfologi diuraikan secara deskriptif dan
kemudian dibuat dendogram kekerabatan. Data pola pita isozim dianalisis secara kuantitatif berdasarkan ada atau tidaknya pita di gel,
kemudian dibuat dendogramnya. Korelasi berdasarkan karakter morfologi dan pola pita isozim dianalisis berdasarkan korelasi koefisien
momen-produk kriteria goodness of fit. Hasil penelitian menunjukkan: (i) di Karanganyar terdapat 10 varietas C. esculenta; (ii) karakter
morfologi tidak terpengaruh oleh ketinggian; (iii) peroksidase membentuk 14 pola pita isozim, esterse membentuk 11 pola pita dan
shikimate dehidrogenase membentuk 15 pola pita; (iv) data morfologi dengan isozim peroksidase memiliki korelasi yang baik
(0,893542288), sementara data morfologi dengan isozim esterse dan shikimate dehidrogenase memiliki korelasi yang sangat baik
(0,917557716 dan 0,9121985446); (v) data pola pita isozim mendukung data karakter morfologi.
Kata kunci: talas, Colocasia esculenta, morfologi, isozim
INTRODUCTION
The diversity of food crops in Indonesia can be
developed to overcome the food problem. Types of tubers
that can be utilized more optimally as a staple food rice
substitutes include cassava, sweet potato, taro, purse,
arrowroot and canna. These tubers have a lot of the preeminent, among them having a high content of
carbohydrates as energy sources (Liu et al. 2006), not
containing gluten (Rekha and Padmaja 2002), containing
angiotensin (Lee et al. 2003), antioxidative ( Nagai et al.
2006),which can be applied to various purposes (Aprianita
2009), and produce more energy per hectWEREthan rice
and wheat. Tubers can be grown on marginal areas
(Louwagie et al. 2006), where other plants cannot grow and
can be stored in the form of flour and starch (Aboubakar et
al. 2008).
Taro has a good variety of morphological characters
such as tubers, leaves and flowers as well as chemicals
such as flavor, aroma and others (Xu et al. 2001).
Characterization of taro plants now has started to be
developed through two approaches. The diversity among
the varieties can be distinguished based on morphological
and molecular markers. Diversity based on morphological
marker has a weakness, because the morphological
characteristics do not necessarily indicate genetic diversity.
Morphological diversity is influenced by the environment,
because every environment has different conditions, so the
plants do adapt to their home range.
8
2 (1): 7-14, March 2010
Molecular marker is an effective technique in genetic
analysis of a plant variety. Molecular markers have been
applied widely in plant breeding programs. Molecular
marker that is often used to distinguish plant diversity is a
marker of isozyme and DNA (Asains et al. 1995; Setyo
2001). Isozyme is a direct product of genes and relatively
free from environmental factors. Isozyme can be used as a
genetic trait to study and identify the diversity of
individuals or a cultivar. Isozymes were enzymes that have
active molecules and different chemical structure, but
catalyze the same chemical reaction. Different forms of an
enzyme molecule can be used as the basis of chemical
separation, by electrophoresis method will result in banding
patternsproduced by different distances (Purwanto et al. 2002).
Information about the genetic diversity of taro
(Colocasia esculenta L.) is needed for plant breeding and
improvement for the offsprings to obtain superior varieties.
Based on the background, the research was conducted on
taro plants in different areas in a region that had high
altitude, medium and low that included morphological
characters and isozyme banding patter pita on different
varieties of taro plants in Karanganyar, Central Java.
MATERIALS AND METHODS
The experiment was conducted in March 2009 to
August 2009. Taro plants (Colocasia esculenta L.) were
collected from Karanganyar District, Central Java
differentiated by differences in altitude, namely: (i) the
highlands (> 1000 m asl), (ii) plain medium (500-1000 m
asl), and (iii) Lowland (<500 m asl). Location of the study
covers nine districts in the district Karanganyar (Table 1).
Characterization of isozyme of taro plants were conducted
at the Faculty of Forestry Gadjah Mada University,
Yogyakarta, using three enzyme systems namely esterase
(EST), peroxidase (POD) and shikimate dehydrogenase
(ShDH).
Characterization of morphology
Characterization of morphology includes: range of
plants, plant’s height, stolon’s number, stolon’s length, leaf
shape of basalt, the dominant position of leaves, leaf edges,
leaf color, leaf blade edge color, pattern Petiole junction,
crossing the color, the color of the liquid at the tip of the
leaf blade, the main color of the leaves of bone, bone leaf
pattern, the ratio petiole length/leaf blade length, color
Petiole upper third, middle third Petiole color, lower third
Petiole color, color Petiole lines, color Petiole ring bottom,
bottom Petiole transverse incision, midrib length
ratio/display total Petiole, leaf midrib color, waxy coating
on leaves. Manifestation cormus, cormus length, branch
cormus, cormus shape, weight, cortex color, and the flesh
color the middle, the color of the meat fibers, cormus skin
surface, skin thickness cormus, cormus fiber levels, and
color shoots.
Isozyme analysis
The third leaf from top in was extracted with a mortal,
by adding a solution of extract buffer ± 1 mL. Once
crushed and homogenized, the sample was inserted into the
eppedorf, then played with the speed of 15,000 rpm for 20
minutes. Making the gel: Gel Poliacrilamide consists of
two parts, ie running a gel that lies at the bottom with a
concentration of 7.5% and spacer gel located on top of
running gel with a concentration of 3.75%. Electrophoresis:
electrophoresis tanks were filled with a solution of
electrode buffer tanks as high as ± 2 cm. Mounted on gel
electrophoresis, supernatant solution was filled into the
hole 5 mL samples using injection equipment (stepper).
Electrophoresis process carried out by an electric current ±
100 mA for 180-200 min. Staining performed after gel
electrophoresis, namely by putting that has been removed
from the glass electrophoresis into a plastic tray, then
soaked in dye solution of dye esterase (EST), peroxidase
(POD) and shikimate dehydrogenase (ShDH). Observations
gel performed after fixation with seeing a pattern emerging
bands, and copy it in the form zimogram.
Data analysis
Taro plant morphology data were described by
descriptive method that covers all the observed variables in
accordance with Kusumo et al. (2002). On isozyme data,
the tape that emerged was given a value of 1 while the ones
that did not arise given the value 0. Dendogram analysis
performed with the method of grouping Average Linkage
Cluster Method with DICE coefficient (Rohlf 2005). The
grouping was done by UPGMA (Unweigthed Pair Group
with arithmatic mean) is calculated by SHAN on NTSYS
program (Numerical Taxonomy and Multivariate Analysis
System) version of 2:02, while the dendogram analysis
using the statistical program Minitab 14 Average linkage
method with Euclidean distance measurement. The result
made dendogram based isozimnya relationship. Results
were analyzed by distance dendogram relationship more
than 60% similarity (Cahyarini 2004). The correlation
between genetic distance based on morphological
characteristics and genetic similarity based on isozyme
banding pattern was analyzed based on product-moment
correlation coefficient with the criteria of goodness of fit
based on the correlation according to Rohlf (2005).
RESULTS AND DISCUSSION
Characterization of morphology of C. esculenta
The results of characterization of taro plants performed
on three different plains in the district Karanganyar
ketinggianya obtained 11 variants showed that the plant C.
esculenta were scattered in several districts, namely:
Benthul, Lompongan, Laos, Mberek, Kladi, Plompong,
Sarangan, Kladitem, Jabon, Japan, and Linjik. In this study
18 samples taken taro with research sites with
environmental factors as listed in Table 1. The diversity
seen in the type of plant, leaf and cormus (bulb). The
characterization results show that there is a difference
between 11 variants of taro. Description of the morphology
of the leaf, midrib, and cormus (bulb) in each of the
varieties of taro were as Table 2.
TRIMANTO et al. – Characterization of taro based on morphological and isozyme markers
Tabel 1. Environmental conditions where the growth of taro in Karanganyar.
Locations/
Subdistricts
Lowland
Gondangrejo
Environmental factors
Altitude Temp. Type of
Rainfall CultiType of taro
Shade
(meter) (°C)
soil
(mm/y) vation
Benthul
Mberek
Kladi
Linjik
Lompongan
Plompong
150
150
98
98
320
95
29
29
29
29
30
29
Grumosol
Grumosol
Aluvial
Aluvial
Mediteran
Mediteran
√
-
1537
1537
1680
1680
2012
2012
√
√
√
√
Plain medium
Karangpandan Benthul
Lompongan
Sarangan
Matesih
Jabon
Laos
Linjik
650
600
650
700
750
700
28
28
28
28
27
28
Mediteran
Mediteran
Mediteran
Litosol
Litosol
Litosol
√
√
2818
2818
2818
2480
2480
2480
√
√
√
√
-
Plateau
Tawangmangu Kladitem
Benthul
Lompongan
Ngargoyoso
Laos
Jatiyoso
Sarangan
Jepang
1500
1700
1500
1000
1300
1200
23
22
23
26
26
26
Andosol
Andosol
Andosol
Andosol
Andosol
Andosol
√
√
√
-
3299
3299
3299
3182
3098
3098
√
√
√
√
√
Jaten
Karanganyar
Kebakkramat
In the dendrogram similarity coefficient of 60% was
used to analyze the phylogenetic relationship of the 18
samples found in different locations with 11 different
varieties. According Cahyarini (2004) said the similarity
distance away if less than 0.60 or 60%, so that separate
groups at a distance of less than 0.60 still has a close
resemblance. In this dendogram analysis, the number 1 or
100% indicates that the group members have a perfect
resemblance, while getting closer to the number 0 means
the similarity distance farther.
Benthul
Dendogram analysis results showed that the Benthul
taro of different height have the same morphological
characteristics and have a high relationship. This is evident
in the coefficient of 0.60 which was still in one group. But
there was a tendency that Benthul of different heights
showes different sizes, ranging from leaf size, plant height,
stem and tuber. Benthul is commonly grown as a crop
population between the rice fields and gardens, and
allowed to grow without special treatment. Environmental
factors such as temperature at any altitude, soil and
availability of different light and water, thought to cause
the size of the plants experience the difference. According
to Park et al. (1997) and Djukri (2006) each deal with
environmental stress of plants continues to do the
adaptation, including changes in morphological
characteristics and physiology.
Benthul that grows in the highlands appear higher with
habitus width, leaf midrib and stalk thin and big. This was
observed in taro grown in ketinggianya more than 1500 m
with high 22°C, and high rainfall reaches 2299 mm
/±humidity, low temperature year. According to Basri
9
(2002) plant growth is influenced by
environmental factors. Altitude above 1500
m cause gas and water vapor content
(humidity) and the number of clouds
blocking sunlight to the plants, so plants
were capturing light by raising levels of
chlorophyll and surface area. Taro plants
tend to have broad leaves because of the
availability of adequate water due to high
rainfall in the area still support the optimum
process in photosynthesis.±Low temperature
22°C
Benthul that grows in the lowlands tend
to have narrower leaves and smaller and
lighter bulbs. According to Menzel (1980)
the temperature is too high may cause leaves
to hinder the development of broad and
narrow leaf photosynthetic rate high as a
result reducing the weight of tuber. But
when the temperature is too low to reach less
than 10°C, the plant tissue can be damaged
and an interruption of growth so the plants
tend to be stunted.
Lompongan
Dendogram Lompongan relationship
found in three different heights showed only
the size difference. Broadly speaking taro
from the highlands, medium and low still have the same
morphological characteristics. Lompongan plants grow
wildly around the edge of rice fields and waterways.
Lompongan plants from the highlands have differences
with the lowlands, such as: green leaf color is more
concentrated, browner midrib color, and the size is larger.
Unlike Lompongan plants in the highlands that were often
found on the outskirts of the river with shade trees around
it, the ones in the lowlands were found in around the edges
of fields full of water. Environmental factors in the form of
light, temperature and humidity cause the plants to have
different adaptations. According to Taiz and Zeiger (1991),
leaf surface area increased because of the shade, and color
changes due to the increased levels of chlorophyll a and b.
In the circumstances shaded light spectrum that is
active in the process of photosynthesis (wavelength 400700 nm) get decreased. Plants will make adjustments to
streamline the capture of light energy that is by increasing
leaf area, plant height and chlorophyll a and b (Lambers et
al. 1998).
Altitude causes humidity, light, temperature, and
moisture content to vary. According to Fitter and Hay
(1998) environmental factors were related one another so
that the plant held a response to the environment. High
water levels in the soil cause leaf’s cell turgor to increase
which in turns causes leaf’s expansion. Reduced light
causes the leaves to add the proportion of mesophyll tissue.
Temperatures that were too high (> 40 ° C) cause defective
enzyme and respiration is rapid, so the plants have stunted
growth. The temperature is too low (<1°C) causes
decreased enzyme activity cause plant tissue damage and
death. The optimum temperature for photosynthesis is 20-30°C.
10
2 (1): 7-14, March 2010
Table 2. 18 samples of C. esculenta in Karanganyar district with
characteristics
Characteristics
Varieties
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
A. Type of plant
1. Rentang tanaman
1. Sempit
2. Sedang
3. Lebar
√
2. Tinggi tanaman
1. Kerdil (< 50 cm) 2. Sedang (< 50 cm) 3. Tinggi (< 50 cm) √
3. Jumlah stolon
1. 1-5 buah
2. 6- 10 buah
√
3. 11-20 buah
4. Panjang stolon
1. Pendek (<15 cm) √
2. Panjang (>15 cm) B. Cormus (umbi)
1. Manifestasi cormus
√
1. Ada
2. Tidak ada
2. Panjang Cormus
1. Pendek (± 8 cm)
2. Sedang (± 12 cm) √
3. Panjang (± 18 cm) 3. Cabang cormus
1. Bercabang
2. Tidak bercabang √
4. Bentuk cormus
√
1. Kerucut
2. Membulat
3. Silindris
4. Memanjang
5. Datar dan terbuka 5. Berat cormus
1. Ringan (± 0.5 kg) 2. Sedang (± 2 kg)
√
3. Berat (± 4 kg)
6. Warna korteks cormus
1. Putih
2. Kuning-orange
√
7. Warna daging tengah
1. Putih
2. Kuning
√
3. Orange
8. Warna serat daging
1. Putih
2. Kuning muda
3. Kuning-orange
√
4. Merah
9. Permukaan kulit cormu
1. Berserabut
2. Bersisik
3. Berserabut dan bersisik√
10. Ketebalan kulit
√
1. Tebal
2. Tipis
11. Tingkat serabut
1. Sedikit
√
2. Banyak
12. Warna tunas
1. Kuning hijau
2. Merah muda
√
3. Ungu
-
- - - - √√√√√ - - - - √ - - √√√√- - - - - √ √ √ √ - - - √
- - - - - - - - - - - - - - √ √ - - - - √√√- - - - - - √ - - √√√√- - - √√ √ √ √ √ - - - - - - - - - - - - - - - - - √ √ √
- - - - - - - √√ - - - √ - √ √ √
√√- - - - - - - √ √ √ - - - - - - √√√√√- - - - - - √ - - √√- - √√√√√ √ √ √ - √ √ - √
- - √√- - - - - - - - √ - - √ √√- - - √√√- - √ - √ √ √ - √
- - √√√- - - √ √ - √ - - - √ - √- - - √√- - - - - - - - √ √- - √√- - - - - - √ √ √ √ - - - √- - - - √√ √ √ - - - - - √
- - - - - √√- - - - - √ √ - - √√√√√- - √√ √ √ √ - - √ √ √
√
-
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-
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-
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-
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-
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-
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-
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- - - - - √√- - - - √ - - - √ - √√√√- - √√ - - - √ √ √ - √- - - - - - - - √ √ - - - - - √
- - - - - √√√√ - - - √ √ √ - √
√√√√√- - - - √ √ √ - - - √ - - √√√√√√√ √ √ - √ √ √ √ √
√√- - - - - - - - - - - - - - - - - - - - - - - - - √ - - - - √
√
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-
√
-
- - √√√- - - - √ √ - - - - √ - - - - - √√√√ - - √ - - √ - √
√√- - - - - - - - - - √ √ - - √√- - - - - √√ - - √ √ √ - - - - √√√√√- - √ √ - - - √ √ √
- - √√√√√- - - - - - - - √ √√- - - - - √√ √ √ √ √ √ √ - √
- - - - - √√- - √ √ - √ √ √ √ √√√√√- - √√ - - - - - - - √
- - - - - - - - - - - √ - - - - -
C. Daun
1. Posisi daun dominan
1. Mendatar
2. Mangkok
√
3. Tegak keatas
4. Tegak kebawah
2. Tepi daun
1. Penuh
2. Bergelombang
3. Berlekok-lekok
√
3. Warna Helai daun
1. Hijau
2. Hijau tua
√
3. Ungu
4. Warna tepi helai daun
1. Keputihan
2. Hijau
3. Merah muda
4. Ungu
√
5. Warna cairan ujung daun
1. Keputihan
2. Kuning
3. Merah muda
√
4. Merah tua
6. Warna utama tulang daun
1. Kuning
2. Hiaju
3. Merah muda
√
4. Ungu
7. Pola utama tulang daun
1. Bentuk Y
√
2. Bentuk Y meluas 8. Warna petiole
Sepertiga atas
1. Kuning
2. Hijau muda
√
3. Cokelat
4. Ungu
Sepwertiga bawah
1. Kuning
2. Hijau muda
3. Cokelat
√
4. Ungu
9. Warna garis petiole
1. Hijau
2. Ungu
√
10 Irisan melintang bawa
1. Terbuka
√
2. Tertutup
11. Warna cincin petiole
1. Putih
2. Kuning kehijauan √
3. Merah muda
4. Ungu
12. Warna pelepah daun
1. Keputihan
2. Hijau muda
3. Merah Keunguan √
13. Lapisan lilin
1. Tidak ada
2. Rendah
√
3. Sedang
4. Tinggi
-
√
-
√
-
√
-
√
-
√
-
√
-
√
-
√
√
√
√
√
√
-
√
-
√
√
√
-
- - - - - - - - - - - - √ √ - - - - √√√- - √√ - - √ - - √ √ √√- - - √√- - √ √ - - - - - √
- - √√√√√√√ √ √ - √ √ - - √√- - - - - - - - - - - - √ √ √
- - - - - - - - - - - √ - - - - √
√
-
√
-
√
-
√
-
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-
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-
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-
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-
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-
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-
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-
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- √ √ √
√
- - - - - - -
√
-
√
-
√
-
√
√
√
√
-
√
-
√
-
√
-
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-
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-
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-
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-
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√
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-
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-
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-
√√√√√√√- - √ √ - √ √ √ √ √
- - - - - - - √√ - - √ - - - - √
-
√
-
√
√
√
√
-
√
-
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-
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-
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-
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-
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√
-
√
-
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-
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-
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-
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-
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-
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-
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-
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-
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-
√
-
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√
-
√
-
√
-
- √
- √
- -
√
- - - - - √√- - √ √ - √ √ - √ √√√√√- - √√ - - √ - - √ - √
√√√√√- - √√ √ √ √ √ √ √ - √
- - - - - √√- - - - - - - - √ √
-
√
-
√
-
√
-
√
-
√
-
√
-
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-
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-
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-
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-
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-
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-
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-
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-
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-
- - - - - √√- - - - - - - - - - - - - - - - - - √ √ - √ √ √ √
√√√√√- - √√ - - √ - - - - √
√
-
√
-
√
-
√
-
√
-
√
-
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-
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-
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-
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-
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-
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-
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-
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-
√
√
-
√
TRIMANTO et al. – Characterization of taro based on morphological and isozyme markers
Note: 1. Benthul (plateau), 2. Benthul (plain medium), 3.Benthul
(lowlands), 4. Lompongan (plateau), 5.Lompongan (plain medium),
6. Lompongan (lowlands), 7. Laos (plateau), 8. Laos (plain
medium), 9. Linjik (plain medium), 10. Linjik (lowland), 11.
Sarangan (plateau), 12. Sarangan (plain medium), 13. Kladitem
(plateau), 14. Plompong (lowland), 15. Kladi (lowland), 16. jabon
(plain medium), 17. Mberek (lowland), 18. Japan (plateau).
Figure 1. Dendogram relationship 18 samples of C. esculenta
from three different heights based on morphological characters.
Description: No. 1-18 same as Table 2.
Characterization of isozyme taro
Peroxidase
Results with the dye peroxidase isozyme analysis,
shikimate dehydrogenase and esterase can be seen in
Figure 2. Peroxidase in 18 samples of C. esculenta tested to
form 14 different banding pattern. Banding pattern I with
migration distance (Rf) 0586, 0630, 0717, 0761 and 0804
being owned by the sample 1. Banding pattern II with Rf
0586, 0717, 0761 and 0804 were owned by the sample 2
and 3. Banding pattern III is owned by sample 5 with Rf
0630, 0739, 0782 and 0826. Banding pattern IV with Rf
0630, 0739, 0782 owned by samples 4 and 6. Banding
pattern V with Rf 0652, 0739, 0782 and 0874 were owned
by the sample 7 and 8. Rf banding pattern VI 0652, 0739,
0782 and 0869 were owned by the sample 9 and 10.
Banding pattern VII with Rf 0565, 0717 0739 and 0847
held by the sample 11. Banding pattern VIII with Rf 0565,
0717 0739 owned by sample 12. IX banding pattern with a
distance of 0630 and 0739 held by the sample 13. Banding
pattern X with Rf 0630 and 0739 held by the sample 14. XI
banding pattern with a distance of 0630, 0739, and 0804 is
owned by the sample 15. XII banding pattern with a
distance of 0630, 0739, and 0826 is owned by the sample
16. XIII banding pattern with a distance of 0630, 0717 and
0761 held by the sample 17. Banding pattern XIV with Rf
0607, 0652 and 0.761 were owned by the sample 18.
Shikimate dehydrogenase
Isozyme analysis results with dye shikimate
dehydrogenase (ShDH) on 18 samples of C. esculenta
tested to form 15 different banding pattern. Banding pattern
I with Rf 0523, 0568, and 0863 is owned by the sample 1.
Banding pattern II with Rf 0523, 0568, 0614 and 0863
were owned by the sample 2 and 3. Banding pattern III
with Rf 0523, 0568, 0614 and 0840 were owned by the
sample 4. Banding pattern IV with Rf 0500, 0523, 0568,
11
0614 and 0840 were owned by samples 5 and 6. Banding
pattern V with Rf 0568 and 0840 were owned by the
sample 7 and 8. Banding pattern VI with Rf 0523 and 0840
held by the sample 9. Banding pattern VII with Rf 0500,
0523 and 0840 were owned by the sample 10. Banding
pattern VIII with Rf 0523 and 0818 held by the sample 11.
Banding pattern IX with Rf 0500, 0523 and 0818 were held
by the sample 12. Banding pattern X with Rf 0416, 0432,
0523, 0795 owned by sample 13. Banding pattern of Rf
0500 XI, 0523, 0727 and 0750 were owned by the sample
14. XII banding pattern of Rf 0523, 0546, 0581 and 0818
held by the sample 15. Banding pattern XIII with Rf 0523,
0546, 0568 and 0795 held by the sample 16. Banding
pattern XIV with Rf 0500, 0546, and 0795 was owned by
the sample 17. Banding pattern XV with Rf 0546, 0568 and
0795 were held by the sample 18.
Esterase
Results with the dye esterase isozyme analysis on 18
samples of C. esculenta were tested forming 11 different
banding patterns. Banding pattern I with Rf the same but
having different shapes, and shown at Rf 0.22, 12:26 and
12:32 were owned by the sample 1, 2 and 3 (quantitative
and qualitative). Banding pattern II with Rf 0.20, 0:28, 0:32
and 0.36 were owned by the sample 4, 5 and 6 (quantitative
and qualitative). Banding pattern III with Rf 0:30, 0:34,
0:38, 0:40 was owned by the sample 7 and 8 (quantitative
and qualitative). Banding pattern IV with Rf 0.20, 0:30,
0:34, 0:38 and 0:44 is owned by the sample 9 and 10.
Banding pattern V with Rf 0.20, 12:26 and 12:38 were
owned by the sample 11 and 12 (quantitative and
qualitative). VI banding pattern was owned by the sample
13 with Rf 0.20, 0:28, 0:30, 0:46, 0:48. Banding pattern VII
owned by the sample 14 with Rf 0.20, 0.26, 0:30, 0:34.
VIII banding pattern VIII was owned by the sample 15
with Rf 0.20, 0.26, 0:30, 0:36. Banding pattern IX was
owned by the sample 16 with Rf 0.20, 0:22, 0:26, 0:32.
Banding pattern X was owned by the sample 17 with Rf
0.20, 0.24, 0:32 and banding pattern XI with Rf 0.20, 12:28
and 12:32 were owned by the sample 18.
Similarity on taro genetics based on isozyme markers
Genetic similarity between samples can be tested using
cluster analysis (group average analysis), which results in
the form dendogram or tree diagram. The end result is a
dendogram of relationship were tested by three different
enzymes (peroxidase, shikimat dehydroginase, and
esterase) (Figure 3).
Election peroxidase has advantages including: a broad
spectrum and has a very important role in the process of
plant physiology. This enzyme can be isolated and
scattered in the cell or plant tissue, especially in plant
tissues that had been developed (Butt 1980; Hartati 2001).
Shikimate dehydrogenase (ShDH) is an enzyme which
spread to most living things. Shikimate dehydrogenase
involved in oxidoreductase that catalyzes NADP +
shikimate into three main products dehydroshikimate +
NADPH+H+. At the plant, esterase is a hydrolytic enzyme
that functions to withhold simple esters in organic acids,
inorganic acids and phenols and alcohols have low
molecular weight and easily soluble.
12
2 (1): 7-14, March 2010
A
B
C
Figure 2. The variation of 18 isozyme banding pattern of sample C. esculenta from three different heights. Description: a. Banding
pattern of peroxidase, b. Shikimate dehydrogenase banding pattern, c. Esterase banding pattern. No. 1-18 same as Table 2.
A
B
C
Figure 3. Relationship dendogram 18 samples of C. esculenta from three different heights based on isozyme banding pattern. A.
peroxidase, B. shikimate dehydrogenase, c. Esterase. No. 1-18 same as Table 2.
Results dendogram relationship between the use of
peroxidase enzymes, shikimate dehydrogenase and esterase
showed generally taro of the same variety have the same
banding pattern, although from different locations
ketinggianya, so that enzymatically still have a high
relationship, since it is estimated the same parent. On a
different taro varieties tend to have a different banding
pattern. Formation of the group between the use of
esterase, peroxidase and shikimate dehydrogenase gave
different relationship relations, but in one variety is
generally joined in one group at a distance of more than
60% similarity, although originating from different
locations’ height.
Esterase formed seven groups which were of more than
60% similarity between one another, where there were taro
who joined another group. Jabon formed a group with
Plompong which were of 0.80 similarity. Kladi formed a
group with Plompong at a distance of 0.75 similarities.
Lompongan joined with Japan at a distance of 0.70
similarities. Laos and Linjik form one group at a distance
of 0.67 similarity. Even when they are different taro
varieties butwhen they form one group, they still have a
high genetic relationship.
Peroxidase also formed seven groups. In general, in a
variety of taro is still present in one group even though
planted in different locations altitude place. Peroxidase
formed a different group variation taro with esterase. In
peroxidase, Laos and Linjik in one group that were of 0.75
similarity. Plompong and Kladi form one group, but Jabon
joined at a distance of 0.70 similarity. Lompongan and
Kladitem form one group at a distance of 0.75 similarity.
Peroxidase added information that was not the presence of
new groups formed on the use of esterase.
Shikimate dehydrogenase provided the formation of
different groups of taro with esterase and peroxidase. In
shikimate dehydrogenase,formed three groups originating
from different varieties, but it formed one group that was of
more than 60% similarity. Lompongan and Benthul joined
at 0.65 similarity distance. Kladi joined Sarangan at a
distance of 0.62 similarity. Mberek and Japan formed one
group that was of 0.67 similarity.
The use of different enzymes gave results in different
groups, although there is formation of the same group with
TRIMANTO et al. – Characterization of taro based on morphological and isozyme markers
a different enzyme’s use. The use of different enzyme will
complement the formation of groups of different taro
varieties. The genetic pattern of bands that formed in the
use of enzymes is the expression of taro varieties in
question. With a specific enzyme that cannot afford some
taro that express ribbon patterns, but with other enzymes
can express ribbon patterns. So that more types of enzymes
used then it will complete the formation of groups on
varieties of taro.
Results dendogram through morphological markers and
isozyme banding pattern shows the difference. From the
morphological marker of the 11 varieties, obtained taro
formed 10 groups at a distance of 0.60 similarity. Different
taro varieties, most will form a separate group means
morphologically different taro varieties have different
morphological characteristics. Talas who formed a group
on the analysis of relationship is Kladi and Plompong.
When the isozyme was used, more groups were
formed, this means that between the different taro varieties
there is still a high relationship. If the different varieties of
taro belong to one group with a distance of close to 1 it is
possible that the similarity comes from the older of taro.
Environmental factors affect plant morphology, if the
environmental factor is more dominant than genetic factors,
the plant will experience a change in morphology (Suranto
1999, 2001). In the long term it is possible crop genetic
trait changes in her body. Plants that were stressed
environment would be possible to have mutations, so that
in the long term can happen speciation.
New types were also possible as a result of
hybridization, so having a close relationship with both of
the parent species. The property of taro which has a close
relationship is what can be used to search for a superior
taro through crossbreeding. Some taros found in
Karanganyar were a wild taro. Wild Taro and of likely no
benefit are possibly to have genetic traits that superior, so
that the hybridization process to obtain high yielding
varieties can be applied.
Generative breeding of taro is naturally difficult to
occur because the male and female flowersg et mature at
different times and a new flowering occurs after more than
6 months of age. Many plants are not considered going
through a flowering because the flowering process is too
long. Many cultivated plants are harvested before
adulthood, so many plants are difficult to perform in a
generative breeding.
Characterization of taro plants through morphological
marker is more easily done, by observing external nature,
taro plants can be assumed to have superior properties. But
genetic markers also play an important role because it is
more fundamental and is not influenced environment. Data
morphology and isozyme banding pattern on taro plants in
Karanganyar can be used in addition to the identification of
the food plant breeding efforts.
Characterization relations of morphology and isozyme
The correlation between genetic distance based on
morphological markers and similarity based on isozyme
banding pattern were analyzed based on product-moment
correlation coefficient with the criteria of goodness of fit
13
according to Rohlf (1993). Result of calculation correlation
between genetic distance based on morphological markers
and genetic similarity based on isozyme banding pattern
showed that between morphology and isozyme has a good
correlation and a very good (Table 4). Correlation between
morphological data and isozyme banding pattern of
peroxidase, esterase, and shikimate dehydrogenase,
respectively, also were on the value of 0.893542288,
0.917557716,
0.9121985446.
This
shows
the
characterization of taro based on morphological markers
consistent with isozyme banding pattern, so that the
isozyme data support the morphological data.
Diversity is difficult to observe the morphological
marker would be more accurate if you have the genetic
markers such as isozymes. Morphological characters that
were equipped with the character of isozyme banding
pattern adds accuracy of the data to identify plant diversity.
Isozyme has advantages because it requires little sample of
the plant, were not inhibited during plant dormancy, can be
used to perform characterization of the plant in very much.
Table 4. Relationships and morphological characterization
characterization results based on isozyme banding pattern
Characters that correlated
Level
Criteria
Morphology and POD
Morphology and EST
Morphology and ShDH
0.893542288
0.917557716
0.9121985446
good
very good
very good
The relationships of taro plants obtained from places of
different heights can be made into a dendogram between
morphology and marker pattern of the isozyme’s ribbon.
Dendogram based on morphological markers and isozyme
banding pattern of peroxidase, shikimate dehydrogenase,
and esterase showed that taro with the same type from a
different altitude did not show any difference at a distance
of 60% similarity. Of the eighteen samples were divided
into 10 groups. Each taro with the same type, although
located in different places still reflect the height of high
relationship. This proved that taro plants of the same type
belonged to a single group.
Figure 4. Dendogram relationship 18 samples of C. esculenta
from three different heights based on morphological markers and
isozyme banding pattern of peroxidase, esterase, and shikimate
dehydrogenase. Description: No. 1-18 same as Table 2.
14
2 (1): 7-14, March 2010
Taro varieties which become one group is based on
morphological markers and isozyme banding pattern,
where the isozyme banding pattern supports the
morphological data. This is evident in samples 1, 2, 3, ie
Bentul from three different height locations which join one
group. Other evidence were sample 4, 5 and 6, which were
from three different altitude sites that also formed one
group. This indicated that the isozyme data support the
morphological data, so as to identify the plant in addition to
morphological data, isozyme data is also needed to increase
the accuracy of the data. There were varieties of taro which
have a a close relationship that are Kladi and Plompong
that have a high relationship when viewed from the merger
with its isozyme morphological characteristics, both were
at the coefficient of 0.68. Allegedly the two taro plants
have elders who have a high kindship, because almost the
same its relation of morphology and isozyme almost the
same. From the characterization results obtained that has a
relationship Kladi and Plompong highest compared with
other varieties of taro. Taro with different varieties formed
their own groups at a distance of 60% similarity. This
means that at a distance of 60% of all varieties of taro had
different characters.
CONCLUSION
There is a diversity of morphological characters in 18
samples of taro plants (Colocasia esculenta L.) that grow in
Karanganyar. Taro is still in one variety that is at different
height diversity appears only on the size of the vegetative
plant. The results showed isozyme banding pattern of the
variability in isozyme banding pattern of peroxidase,
esterase and shikimate dehydrogenase in taro varieties
found in different locations. Characterization of taro based
on morphological markers is consistent with the
characterization based on isozymes. Isozyme data support
the morphological character data.
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Taiz L, Zeiger E. 1991. Plant physiology. Benyamin/Cumming. Tokyo.
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ISSN: 2087-3940 (print)
ISSN: 2087-3956 (electronic)
Vol. 3, No. 1, Pp. 15-22
March 2011
Study on floristic and plant species diversity in the Lebanon oak (Quercus
libani) site, Chenareh, Marivan, Kordestan Province, western Iran
HASSAN POURBABAEI♥, SHIVA ZANDI NAVGRAN
Determent of Forestry, Faculty of Natural Resources, University of Guilan, Somehsara, P.O.Box 1144, Tel.: +98-182-3220895, Fax.: +98-182-3223600,
♥
E-mail: [email protected]
Manuscript received: 28 Augustus 2010. Revision accepted: 4 October 2010.
Abstract. Pourbabaei H, Navgran SZ. 2011. Study on floristic and plant species diversity of the Lebanon oak site (Quercus libani) in
Chenareh, Marivan, Kordestan Province, western Iran. Nusantara Bioscience 3: 15-22. In order to study floristic and plant species
diversity, approximately 450 ha of oak forests were selected in Chenareh, Marivan of Kordestan province in western Iran. Inventory was
selectively carried out in 50 m elevation range in the different aspects. Vegetation was surveyed in the four layers including: tree (dbh
>5 cm), regeneration (dbh <5 cm), shrub and herb. Diversity and richness indices were used to analyze data in the different vegetation
layers. Results indicated that 82 plant species found in the studied site, comprise of 9 tree, 3 shrub and 70 herbaceous. The mean
diversities and richness measures were found to be the highest in southwestern and lowest in southeastern and northern aspects for the
tree layer. Whereas for the regeneration layer, the mean diversity measures were found the highest in northeastern (i.e., 1-D and H′) and
southern (i.e., N2 and N1) and lowest in southwestern (i.e., 1-D, H′ and N1) and southeastern (i.e., N2). The mean diversities were found
the highest in northern (i.e. N2 and H′) and northwestern (i.e. 1-D and N1) and lowest in northeastern aspect in the shrub layer. The mean
diversities were also found the highest in western and lowest in northeastern aspect in the herbaceous layer. Moreover, Mean richness
and diversity were found the highest in 1500 m asl and lowest in 1750 and 1800 m asl in the tree and shrub layers. Mean richness and
diversity were found the highest in 1500 m asl and lowest in 1750 m asl in the regeneration layer. Also, the mean diversities were found
the highest in elevation 1700 m asl and lowest in elevation 1800 m asl in the herbaceous layer.
Key words: floristic, plant diversity, aspect, elevation, western Iran.
Abstrak. Studi keanekaragaman floristic dan jenis tumbuhan pada situs Lebanon ek (Quercus libani) di Chenareh, Marivan, Provinsi
Kordestan, Iran barat. Nusantara Bioscience 3: 15-22. Dalam rangka meneliti floristik dan jenis tumbuhan, sekitar 450 ha hutan kayu
ek dipilih di Chenareh, Marivan, Provinsi Kordestan di Iran barat. Inventarisasi dilakukan dilakukan secara selektif pada kisaran elevasi
50 m dengan aspek yang berbeda-beda. Vegetasi disurvei dalam empat lapisan termasuk: pohon (dbh> 5 cm), regenerasi (dbh <5 cm),
semak dan herbal. Indeks keanekaragaman dan kekayaan digunakan untuk menganalisis data dalam lapisan vegetasi yang berbeda. Hasil
penelitian menunjukkan bahwa 82 jenis tumbuhan ditemukan di lokasi penelitian, terdiri dari 9 pohon, 3 semak dan 70 herba. Pada
pengukuran lapisan pohon mean keanekaragaman dan kekayaan ditemukan bahwa yang tertinggi di barat daya dan yang terendah di
tenggara dan utara. Pada pengukuran lapisan regenerasi mean keanekaragaman ditemukan yang tertinggi di timur laut (yaitu, 1-D dan H
') dan selatan (yaitu, N2 dan N1) dan yang terendah di barat daya (yaitu, 1-D, H' dan N1) dan tenggara (yaitu, N2). Pada lapisan semak
keanekaragaman ditemukan mean tertinggi di utara (N2 yaitu dan H ') dan barat laut (yaitu 1-D dan N1) dan terendah di timur laut. Pada
keanekaragaman lapisan herba ditemukan mean tertinggi di barat dan terendah di timur laut. Selain itu, mean kekayaan dan
keanekaragaman lapisan pohon dan semak ditemukan tertinggi pada ketinggian 1500 m dpl dan terendah pada 1750 dan 1800 m dpl.
Mean kekayaan dan keragaman lapisan regenerasi ditemukan tertinggi pada ketinggian 1500 m dpl dan terendah pada 1750 m dpl. Juga,
ditemukan mean keragaman lapisan herba tertinggi ditemukan pada ketinggian 1700 m dpl dan terendah pada 1800 m dpl.
Kata kunci: flora, keanekaragaman tanaman, aspek, ketinggian, Iran barat.
INTRODUCTION
The Zogros forests of western Iran extend from
Piranshahr city in western Azarbayejan province in the
Zagros and Bakhtiary mountains to around the Jahroum
and Fasa cities in the Fars province. These forests cover
approximately 5 million ha area, and because of dominancy
of species of oak genus, these forests are called as western
oak forests (Mohadjer 2005). The western oak forests have
remarkable significance in regard to ecological services
including water and soil conservation. Therefore, these
forests play an important role in preventing of soil erosion.
About 40% of surface waters of our country run from
Zogros mountains basin in which seven rivers exist with
fresh water. In the other hand, these forests produce by
products through their woody species, and also maintain
sustainable agriculture in the lowlands.
In general, forest communities in these forests from
lowland to highland as follows: Amygdaletum scopariae,
Pistacio-Amygdaletum, Quercetum persicae (Q.brantiae),
Juniperetum polycarpae. Juniperetum community has a
large extent from Khorasan to Azarbayejan, Zagros,
Bandar Abbas and Baluchestan (Sabeti 1994). In the
Pistacio-Amygdaletum community, Amygdalus species
16
3 (1): 15-22, March 2011
affect on the natural regeneration of Pistacia species, that
is, the seedlings of Pistacia species would be protected
under the spiny bushes of Amygdalus species.
The Zogros mountains are divided into two parts:
northern and southern. The northern Zagros is consisted of
the growing site of Quercus infectoria Oliv. and also
Q.libani Oliv. and Q.persica J. & Sp. (Q.brantii Lindl.)
species are found in this part. However, the southern
Zagros is included Q.persica site which it extended to Fars
province (i.e., 29º 5´ N). The northern Zagros is wetter and
cooler than the southern one. The dispersion areas of
Lebanon oak (Q.libani) are mostly restricted to central and
eastern mountains of Tavrous and Amanous of Anatolia in
Turkey, the mountains of northeastern of Iraq and
northwestern of Syria and western part of Iran (i.e.,
Kordestan province) (Browicz 1994). In addition, this
species is found over 1000 m asl elevation and the best
conditions range from 1200 to 1600 even to 1800 m asl to
growing it, and also this species is found higher than 2000
m asl elevation in Ahir dagi and Herakol dagi mountains in
southern Anatolia, Turkey. Western borderline of this
species is located in the Goniah province in Anatolia and
northern borderline restricted to latitude 40ºN in Erzincon
province in Turkey (Davis 1982). In flora of Iraq, the
distribution area of this species was cited in central regions
of Iraq forests, north of Syria, Palestinian, Turkey and Iran
on hillsides on the metamorphic and igneous rocks and on
loam soils, and elevation ranges from 1800 to 2000
(occasionally 2100) m asl (Townsend and Guest 1980) . In
Iran, the distribution of this species is restricted to
highlands of Sardasht in Kordestan and Euromiah
provinces, and horizontal distribution is from north of
Sardasht to south of Marivan in Kordestan province and
vertical distribution is from 1400 to 2150 m asl elevation
(Fattahi 1994). This species is situated in latitude from 25º
to 36º N and longitude from 45º to 46º E and is grown cold
humid, cold sub humid or humid climates. The pure type of
this species is found in highlands and the mixed one mostly
found associated with Q.infectoria and Q.brantii species.
This species is in relation to soil and climatic conditions.
The forests of this species are found as high and coppice
forms, and the species covers 106316 ha area in western
Iran of which 83844 is located in the Kordestsn province
(Fattahi 1994).
There are numerous studies in relation to floristic
composition all over the world (e.g., Andel 2001; Nebel et
al. 2001; Ipor et al. 2002; Blanckaert et al. 2004; WardellJohnson et al. 2004; Ramírez et al. 2007; Cayuela et al.
2008; Gole et al. 2008; Macía 2008; El-Ghanim et al. ,
2010; Figueroa et al. , 2011). In addition, plant species
diversity has been assessed in forest ecosystems in recent
decades (e.g., Brockway 1998; Pitkänen 1998; Khera et al.
2001; Ashton and Macintosh 2002; Aubert et al. 2003;
Nagaike 2003; Jobidon et al. 2004; Chiarucci and Bonini
2005; Pant and Samant 2007; Aparicio et al. 2008; Macía
2008; Pe´rez-Ramos 2008; Hayat et al. , 2010). Whereas
there is less studies about plant species diversity in Zagros
forest ecosystems (Mirzaei et al. 2008; Pourbabaei et al. ,
2010).
The aim of this study was to determine floristic
composition and plant species diversity in the Lebanon oak
site in Kordestan province of Iran.
MATERIALS AND METHODS
Study area
The study area is located in Marivan city of Kordestan
province in western Iran, and Chenareh is situated 25 km
from northwestern Marivan city (35° 29′ to 35° 45′ N
latitude, 46° 14′ to 46° 29′ W longitude). Mean annual
precipitation is 909.5 mm, ranging from 590.8 to 1422.2
mm (Figure 1).
Kordestan Chenareh forest Marivan City Figure 1. Study site maps Chenareh’s forests (blank circle) in Marivan District, Kordestan Province, IR Iran.
POURBABAEI & NAVGRAN – Plant species diversity of Chenareh forest
Mean annual temperature is 13.3º C, and the length of
dry season is 4 month (based on embrothermic curve) from
June to August. Type of climate is sub humid with cold
winters in the basis of Emberger’s formula (Department of
Forestry 2002). Edaphically, soils consist of developed
brown (calciferous and eutroph), deep and semi deep, and
young soils consisting of litho sol and colluvium which
often are less deepness and shallow. Quercus brantii
community are predominantly found on calcico brown
soils, and Q.libani community often found on eutroph
brown soils.
The research was conducted in 450 ha of Chenareh’s
forests where included Lebanon oak and altitude ranges
from 1500 to 1800 m asl These forests are located steep
areas, and slope is more than 50% in the most area. Main
aspects of these forests are northern and southern. These
forests have been under anthropogenic disturbances in the
past, therefore they are considered as manipulated forests
now.
Sampling
At first, oak site was quantified on the map with
1:50000 scale with surveying forests. Inventory was
selectively carried out in 50 m elevation range from 1500
m to 1800 m asl in the different aspects in the basis of
distribution of Lebanon oak population. Sampling plot area
was 1000 m2 in size and circular (Zobeiri 1994). In total,
42 sampling plots were made. At each plot, diameter at 1.3
m (DBH) of tree ≥ 5 cm was measured and identified (high
and coppice origin), and crown diameters (i.e., large and
small) of regeneration with DBH < 5 cm were measured.
For shrub species, the number of individuals were recorded
and identified. To collect herbaceous data, nested plot
sampling was performed at center the plot (MullerDombois 1974), and minimal area ranged from 32 to 1000
m2 in the basis of different altitudes. Cover percentage was
visually estimated, as accurately as possible, for each
herbaceous species in the nested plots, and type of species
was identified in the Herbarium of Faculty of Natural
Resources, University of Guilan.
Data analysis
Species richness (total number of species present) and
evenness (the manner in which abundance is distributed
among species) are the two principal components of
diversity. Species richness is frequently characterized by
the number of species present (S), Margalef species
richness (R1) and Menhinick species richness (R2) (Ludwig
and Reynolds 1988). In this study, Smith and Wilson’s
evenness index (Evar) was applied to calculate evenness
measures (Krebs 1999). Diversity indices combine species
richness and evenness components into a single numeric
value. The most commonly used indices of diversity,
Simpson (1-D) and Shannon-Wiener (H′) were used in this
study (Magurran 2004). Moreover, Hill’s N2 and
McArthur’s N1 were calculated in the basis of these indices
(Krebs 1999). Vegetation data were analyzed in four layers
(i.e., tree, regeneration, shrub and herb) using richness,
evenness and diversity indices. In tree layer, DBH was
converted to basal area (m2) for each individual tree and
summed for each species, and then substituted for the
17
number of individuals in the diversity formula.
Furthermore, crown cover area (m2) was computed for each
regeneration species and applied the formula. Data analyses
were performed using Ecological Methodology and SPSS
13.0 software (Krebs 1999; Kinnear 2001).
RESULTS AND DISCUSSION
Floristic composition
A total of 82 plant species were found in the studied
area, of which 12 woody species (9 trees, 3 shrubs) and 70
herbaceous species existed (Table 1) while 4 trees, 3
shrubs, one bush and 78 herbaceous species were identified
in Ilam forests of Zagros (Pourbabaei et al. 2010).
Therefore, it is concluded that tree richness is high in the
studied area. Also, it can be deduced from Table 1 that
Rosaceae and Fagaceae families play an important role in
among woody species. Moreover, Asteraceae and Poaceae
families were most abundant amongst herbaceous species.
In addition, results were revealed that the Asteraceae
family was dominant in Ilam forests of Zagros (Pourbabaei
et al. 2010).
The number of plant species was considerable in the
studied area when compare with northern Zagros
mountains since there is 165 woody species (tree and
shrub) in Zagros and 182 bush and herbaceous species only
in northern Zagros (Jazirehi and Rostaghi 2003). The
highest richness of woody species belong to Fagaceae and
Rosaceae and the highest richness of herbaceous species
belong to Asteraceae and Poaceae families in the studied
area, these results were confirmed in the Zagros zone
(Jazirehi and Rostaghi 2003).
Plant diversity based on different aspects
Plant species diversity of four growth layers was
obtained in terms of different aspects. The highest and
lowest population of Lebanon oak was found in eastern
(32%) and northwestern (24%) aspect, respectively. Figure
1 displays mean tree (high and coppice forms) diversity in
the basis of different aspects.
The mean diversities were highest in southwestern and
lowest in southeastern and northern aspects in the tree
layer. The ANOVA test indicated that there were no
significant differences amongst mean diversity measures in
the different aspects (P > 0.05). Figure 3. displays mean
tree richness, Margalef (R1) and Menhinick (R2) and
evenness measures in the different aspects.
The mean richness, R1 and R2 measures were highest in
southwestern, and lowest in southeastern and northern
aspects, respectively. The mean Evar was the highest in
southeastern and lowest in northeastern aspect. The
Kruskal-Wallis test showed that there were no significant
differences amongst mean richness values in the different
aspects. Whereas, the ANOVA test indicated that there
were significant differences amongst mean R1 and R2 in the
different aspects (P < 0.05), and Tukey test showed that
there was significant difference between southwestern and
southeastern aspects in view of mean R1. Also, there was
significant difference between southwestern and other
aspects except southern in view of mean R2.
18
3 (1): 15-22, March 2011
Table 1. Plant species list based on growth layers
Layer
Species
Tree
Acer monspessulanum L. (Aceraceae), Amygdalus communis L. (Rosaceae), Cerasus mahaleb L. (Rosaceae), Crataegus
pontica C.Koch. (Rosaceae), Pistacia atlantica (Anacardiaceae), Pyrus syriaca Boiss. (Rosaceae), Quercus brantii Lindl.
(Fagaceae), Q.infectoria Oliv. (Fagaceae), Q.libani Oliv. (Fagaceae).
Shrub
Cerasus microcarpa (C.A.Mey) Boiss. (Rosaceae), Cotoneaster nummularia Fisch & Mey. (Rosaceae), Lonicera
nummularifolia Jaub & Spach. (Caprifoliaceae).
Herbaceous
Acanthus dioscoridus L. (Acantaceae), Achillea filipendula L. (Asteraceae), A.millefolium L. (Asteraceae), Aegilops
triuncialis L. (Poaceae), A.triuncialis L. (Poaceae), Alopecurus myosuroides Ovcz. (Poaceae), Antemis tinctoria L.
(Asteraceae), Astragalus curvirstris Boiss. (Papilionaceae), A. michauxianus Boiss. (Papilionaceae), A. (tragacantha )
sp. (Papilionaceae), Aristolochia bottae Jaub & Spach. (Aristolochiaceae), Boissiera squarrosa Hochst. (Poaceae),
Bromus tectorum L. (Poaceae), Buchingera axillaris Boiss. (Cruciferae), Bunium elegans (Fenzl.) Freyn. (Umbelliferae),
Callipeltis cucularia Stev. (Rubiaceae), Centaurea virgata Lam. (Asteraceae), Cephalaria syriaca (L)Schrad.
(Dipsaceae), Chaerophyllum macropodum Boiss. (Umbelliferae), Cornilla varia L. (Papilionaceae), Dactylis glomerata
L. (Poaceae), Dianthus tabrizianus Adams. (Caryophylaceae), Echinops orientalis Trauth. (Asteraceae), E.ritrodes
Bunge. (Asteraceae), Eremopoa persica (Trin.) Roshev (Poaceae), Eryngium thyrsoides F.Delaroche. (Umbelliferae),
Euphorbia macroclada Boiss (Euphorbiaceae), Ferula orientalis L. (Umbelliferae), Fibijia macrocarpa Boiss.
(Cruciferae), Galium aparine L. (Rubiaceae), Grammosciadium platycarpum Boiss. (Umbelliferae), Gundelia
tournefortii L. (Asteraceae), Helianthemum ledifolium (L.) Miller. (Cistaceae), Heteranthelium piliferum (Banks &
Soland) (Poaceae), Hordeum bulbosum L. (Poaceae), Hypericum scabrum L. (Hypericaceae), Inula britanica L.
(Asteraceae), Lamium album L. (Labiatae), Marrubium vulgare L. (Labiatae), Mesostemma kotschyanum Wed.
(Caryophylaceae), Milium pedicellare Bornm. (Poaceae), Onopordon kurdicum Bornm& Beauv (Asteraceae), Onosma
elwendicum L. (Boraginaceae), O. microcarpa DC. (Boraginaceae), Phlomis olivieri Benth. (Labiatae), P.rigida Labill.
(Labiatae), Picnomon acarna L. (Asteraceae), Poa bulbosa L. (Poaceae), Potentila kurdica Boiss & Hohen. (Rosaceae),
Prangos ferulaceae L. (Umbelliferae), Rhaponticum insigne Boiss. (Asteraceae), Rhabdoscidium aucheri Boiss.
(Umbelliferae), Salvia bracteata Banks & Soland. (Labiatae), Sanguisorba minor Scop. (Rosaceae), Scabiosa
calocephala Boiss.(Dipsacaceae), S. leucactis Patzak. .(Dipsacaceae), Scutellaria pinnatifida A.Hamilt. (Labiatae),
Serratula grandifolia Boiss. (Asteraceae), Smyrnium aucheri Boiss. (Umbelliferae), Stachys inflata Benth. (Labiatae),
Taeniatherum crinitum (Schreb).Nevski (Poaceae), Teucrium polium L. (Labiatae), Trifolium campestre Schreb.
(Papilionaceae), T.pratens L. (Papilionaceae), Turginia latifolia L. (Umbelliferae), Valerianella dactylophylla Boiss &
Hohen. (Valerianaceae), Veronica kurdica Benth. (Scrophulariaceae), Vicia variabilis Freyn & Sint. (Papilionaceae),
Xeranthemum inaepertum Boiss. (Asteraceae), Zoegea leptaurea L. (Asteraceae).
The mean diversity measures were highest in
northeastern (i.e., 1-D and H′) and southern (i.e., N2 and
N1) and lowest in southwestern (i.e., 1-D, H′ and N1) and
southeastern (i.e., N2) in the regeneration layer (Figure 4).
The ANOVA test showed that there were significant
differences amongst mean 1-D measures in the different
aspects, but no significant differences amongst other
diversity indices. In addition, Tukey test showed that there
was significant difference between northeastern and
southwestern aspects.
The mean richness, R1 and R2 measures were highest in
northern, southern and lowest in southwestern and
southeastern aspects, and mean Evar was the highest in
southwestern and lowest in northern aspect (Figure 5).
There were significant differences amongst mean richness,
R1 and R2 measures. Tukey test showed that there was
significant difference between northern and southwestern
aspect in view of richness.
The mean diversities were highest in northern (i.e. N2
and H′) and northwestern (i.e. 1-D and N1) and lowest in
northeastern aspect in the shrub layer (Figure 6). The
ANOVA test showed that there were no significant differences
amongst mean diversities measures in the different aspects.
The mean richness and R1 measures were highest in
northern, while R2 was the highest in eastern aspect. The
mean richness was lowest in other aspects and also the
mean of R1 and R2 were found the lowest in northwestern
aspect, and the Evar were found the highest in northwestern
and lowest in northeastern (Figure 7). There were no
significant differences amongst mean richness, R1, R2 and
Evar measures in the different aspects.
The mean diversities were highest in western and
lowest in northeastern aspect in the herbaceous layer
(Figure 8). The ANOVA test showed that there were
significant differences amongst mean diversities measures
in the different aspects. The differences among means were
detected using Tukey’s test which are characterized by
different letters on the histogram of Figure 8.
The mean richness and Evar measures were highest in
western and lowest in northern aspect in the herbaceous
layer and there were significant differences amongst mean
measures in different aspects (Figure 9).
The Lebanon oak was found in all aspects, but it had
the most abundant in eastern and the least in northwestern
aspect since it requires plenty of sunlight in eastern aspect
(Maroufi 2000). This species is preferred northern and
eastern aspects and ecological needs of Q.libani is higher
than Q.infectoria and Q.brantii (Jazirehi and Rostaghi 2003).
The tree species diversity was found the highest in
southwestern and lowest in southeastern and northern
aspects, because richness and richness indices had the
highest and lowest values the mentioned aspects, and
evenness had the highest and lowest values in southeastern
and northeastern aspects, respectively.
POURBABAEI & NAVGRAN – Plant species diversity of Chenareh forest
4.5
1-D
4
N2
2.5
2
H'
3
Diversity indices
Diversity indices
3.5
19
N1
2.5
2
1.5
1-D
1.5
N2
H'
1
1
N1
0.5
0.5
0
0
1
2
3
4
5
6
7
1
8
2
3
4
Aspect code
Aspect code
Figure 1. Mean diversity measures and their standard errors based
on different aspects in the tree layer (1. northern, 2. northeastern,
3. northwestern, 4. eastern, 5. southern, 6. southwestern, 7.
southeastern, 8. western).
S
6
R2
5
S
3
R1
R1
Richness and evenness indices
Richness and evenness indices
Figure 6. Mean diversity measures and their standard errors based
on different aspects in the shrub layer.
Evar
4
3
2
1
2.5
R2
Evar
2
1.5
1
0.5
0
0
1
2
3
4
5
6
7
1
8
2
3
4
Aspect code
Aspect code
Figure 3. Mean richness, R1, R2 and Evar measures and their
standard errors based on different aspects in the tree layer.
Figure 7. Mean richness, R1, R2 and Evar measures and their
standard errors based on different aspects in the shrub layer.
1-D
14
2.5
12
Diversity indices
3
Diversity indices
cd
1-D
N2
H'
N1
3.5
2
1.5
cd
a
6
ab
a
4
a
2
abc
ab
a
1
0.5
abc
ab
abc
a
a
bc
ab
bcd
ab
abc
N1
abcd abc
abc
abc
8
abc
cd
cd
abc
cde
bcd
0
0
1
2
3
4
5
6
1
7
2
3
4
5
6
7
8
Aspect code
Aspect code
Figure 4. Mean diversity measures and their standard errors based
on different aspects in the regeneration layer.
Figure 8. Mean diversity measures and their standard errors based
on different aspects in the herbaceous layer (The same letters on
the histogram indicate that there are no significant differences
amongst mean values).
S
4.5
R1
4
R2
3.5
Evar
3
2.5
2
1.5
1
0.5
0
14
Richness and evenness indices
5
Richness and evenness indices
H'
bcd
bc
10
N2
d
S
cd
bc
Evar
abc
12
abc
abc
10
a
a
a
8
6
4
2
a
ab
bc
ab
ab
ab
bc
bc
0
1
2
3
4
5
6
7
Aspe ct cod
Figure 5. Mean richness, R1, R2 and Evar measures and their
standard errors based on different aspects in the regeneration
layer.
1
2
3
4
5
6
7
8
As pe ct code
Figure 9. Mean richness and Evar measures and their standard
errors based on different aspects in the herbaceous layer.
20
3 (1): 15-22, March 2011
3.5
1-D
3
N2
N1
2
1.5
1
Diversity indices
Diversity indices
2
H'
2.5
1-D
N2
H'
N1
2.5
1.5
1
0.5
0.5
0
0
1500
1550
1600
1650
1700
1750
1500
1800
1550
1600
1650
1700
Elevation (m a.s.l.)
Figure 10. Mean diversity measures and their standard errors
based on elevation classes in the tree layer.
Richness and evenness indices
R1
4.5
R2
4
Evar
3.5
3
2.5
2
1.5
1
0.5
0
1500
1550
1600
1650
1700
1750
S
3
R1
2.5
R2
Evar
2
1.5
1
0.5
0
1500
1800
1550
1600
1650
1700
1750
1800
Elevation (m a.s.l.)
Elevation (m a.s .l.)
1-D
N2
H'
N1
3.5
Diversity indices
3
2.5
2
1.5
1
Figure 15. Mean richness, R1, R2 and Evar measures and their
standard errors based on elevation classes in the shrub layer.
0.5
0
1500
1550
1600
1650
1700
1750
1-D
10
9
8
7
6
5
4
3
2
1
0
Diversity indices
Figure 11. Mean richness, R1, R2 and Evar measures and their
standard errors based on elevation classes in the tree layer.
N2
H'
N1
1500
1800
1550
1600
1650
1700
S
R1
R2
Evar
1800
Figure 16. Mean diversity measures and their standard errors
based on elevation classes in the herbaceous layer.
12
Richness and evenness indices
Richness and evenness indices
5
4.5
4
3.5
3
2.5
2
1.5
1
0.5
0
1750
Elevation (m a.s.l.)
Elevation (m a.s.l.)
Figure 12. Mean diversity measures and their standard errors
based on elevation classes in the regeneration layer.
1800
Figure 14. Mean diversity measures and their standard errors
based on elevation classes in the shrub layer
Richness and evenness indices
S
5
1750
Elevation (m a.s.l.)
S
10
Evar
8
6
4
2
0
1500
1550
1600
1650
1700
1750
1800
Elevation (m a.s.l.)
Figure 13. Mean richness, R1, R2 and Evar measures and their
standard errors based on elevation classes in the regeneration
layer.
1500
1550
1600
1650
1700
1750
1800
Elevation (m a.s .l.)
Figure 17. Mean richness and Evar measures and their standard
errors based on elevation classes in the herbaceous layer.
POURBABAEI & NAVGRAN – Plant species diversity of Chenareh forest
These results are to be confirmed with obtained results
from Zagros forests in Ilam (Mirzaei et al. 2008;
Pourbabaei et al. 2010). The Lebanon oak trees have been
overexploited in southwestern aspect and as a result,
population of other species such as Amygdalus communis
and Crataegus pontica have increased in this aspect and
also caused to increase tree species diversity.
The regeneration diversity of woody species was found
the highest in northeastern (i.e., 1-D and H´) and southern
(i.e., N1 and N2) and the lowest in southwestern (i.e., 1-D
and H´) and southeastern (i.e., N2). The highest value of
richness, R1 and R2 were found in northern and southern
aspects and the lowest in southwestern and southeastern
aspects. The highest value of evenness was found in
southwestern and the lowest in northern aspect.
The shrub diversity was found the highest in northern
(i.e., H´ and N2) and northwestern (i.e., 1-D and N1) and the
lowest in northeastern aspect. The highest value of richness
and R1 was found in northern and R2 in eastern aspect. The
lowest value of richness was found in other aspects, and R1
and R2 in northwestern aspect. The highest value of
evenness was found in northwestern and the lowest in
northeastern.
The herbaceous diversity was highest in western and
the lowest in northeastern aspect. The highest value of
richness and evenness were found in western and the
lowest were in northern aspect. The number of tree
individuals per hectare and its crown cover were low in
western aspect and as a result the herbaceous diversity was
the highest in this aspect. The population of tree species
was more in northeastern aspect and crown coverage was
60 to 80 percent and as a result the herbaceous diversity
was lower in this aspect.
Plant diversity based on elevation classes
The elevation distribution of Lebanon oak species stretch
from 1500 to 1800 m asl in the studied area. The highest
and lowest Lebanon oak population was found from 1600
to 1750 m asl (18%) and from 1500 to 1600 m asl (8%),
respectively. The mean diversities were found the highest
in elevation 1500 m asl and lowest in elevation 1800 m asl in
the tree layer (Figure 10). There were no significant
differences amongst mean diversity measures in the
different elevations (P > 0.05). These results are to be
confirmed with gained results of Zagros forests in Ilam
(Mizaei et al. 2008).
The mean richness, R1 and R2 measures were found the
highest and lowest in elevation 1500 and 1800 m asl,
respectively in the tree layer, while the highest and lowest
of mean Evar was found in elevation 1650 and 1600 m asl,
respectively (Figure 11). There were no significant differences
amongst mean these parameters in the different elevations.
The mean diversities were found the highest in
elevation 1500 m asl and lowest in elevation 1800 m asl in
the regeneration layer (Figure 12). The mean richness and
R1 measures were found the highest in elevation 1500 m
asl, and the highest value of R2 was in elevation 1700 m asl
and these parameters were lowest in elevation 1650 and
1800 m asl, respectively in the regeneration layer. The
highest and lowest of Evar were found in elevation 1700 and
1800 m asl, respectively (Figure 13). There were no
21
significant differences amongst mean diversity, richness
and evenness measures in elevation classes in the
regeneration layer.
The mean diversities were found the highest in
elevation 1500 m asl and lowest in elevation 1750 m asl in
the shrub layer (Figure 14). The mean richness and R1
measures were also found the highest in elevation 1500 m
asl, and the highest value of R2 was in elevation 1750 m asl
and these parameters were lowest in elevation 1600 m asl
in the shrub layer. The highest and lowest of Evar were
found in elevation 1600 and 1750 m asl, respectively
(Figure 15). There were no significant differences amongst
mean diversity, richness and evenness measures in
elevation classes in the shrub layer.
The mean diversities were found the highest in
elevation 1700 m asl and lowest in elevation 1800 m asl in
the herbaceous layer (Figure 16). The mean richness was
found the highest in elevation 1500 m asl, and lowest in
elevation 1800 m asl, and the highest and lowest of Evar
were found in elevation 1600 and 1800 m asl, respectively,
in the this layer (Figure 17). There were no significant
differences amongst mean diversity, richness and evenness
measures in elevation classes in the herbaceous layer.
The most population of Lebanon oak was found from
1600 to 1750 m asl elevation. Maroufi (2000) indicated that
this tree was distributed upper 1400 m asl elevation and it
formed pure stands in elevation from 1600 to 1700 m asl
The Quercus brantii, Q.infectoria and Q.libani species
were observed with each other in elevation from 1500 to
1600 m asl, and in elevation of 1600 to 1650 m asl
Q.infectoria and Q.libani species found with together, and
from 1650 to 1800 m asl just Q.libani was found
(Tabatabaei and Geisarani 1992).The Q.libani species is
distributed from 1500 to 2100 m asl and the best
elevational range of this species was characterized from
1600 to 1800 m asl (Jazirehi and Rostaghi 2003).
The herbaceous species of Vicia variabilis Fren & Sint.
has more population in sites where Q.libani population is
plentiful. With increasing elevation up to 1700 m asl,
V.variabilis population is also increased. The Q.libani
forms pure stands in higher elevations (1650 to 1800 m asl)
and population of Mesostemma kotschyanum is increased
in comparing with V.variabilis since ecological needs of
Mesostemma kotschyanum lower than is Vicia variabilis.
The herbaceous coverage is to be increased in western and
eastern aspects due to decreasing crown cover of oak
species, and Turginia latifolia L. species is formed the
most coverage percent since it has less ecological needs
and it also is a thorny species.
CONCLUSION
The Zogros are divided into two parts: northern and
southern. Northern Zagros is determined in the basis of
distribution of Quercus infectoria Oliv. and Q. libani Oliv.
Southern Zagros is also determined based on distribution of
Quercus brantii Lindl. The Lebanon oak was found in all
aspects, but it had the most population in eastern aspect and
also this species was preferred northern aspect due to high
22
3 (1): 15-22, March 2011
ecological needs. The most population of Lebanon oak was
found from 1600 to 1750 m asl elevation because of
suitable humidity and edaphically conditions. In fact,
elevational distribution of Lebanon oak is as spindle shape,
that is, population of this species is increasing when the
elevation is increasing and the population is decreasing in
higher elevation. The disturbance is approximately high in
elevation of 1500 m asl, as a result herbaceous and other
woody species have been dominated and Lebanon oak
decreased. Therefore, in order to rehabilitate the northern
Zagros it is recommended that plantation of Lebanon oak is
greatly conducted in the mentioned aspects and elevations.
Regarding that plant species diversity and richness are
considerable in studied area, it is better that this site is
considered as genetic reservoir.
ACKNOWLEDGEMENTS
We would like to thank to Hosein Maroufi who helped
us in identification of plant species specimens. Also, we
wish to acknowledge our field assistants that had helped us
during the data collection.
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ISSN: 2087-3940 (print)
ISSN: 2087-3956 (electronic)
Vol. 3, No. 1, Pp.: 23-27
March 2011
Evaluation structural diversity of Carpinus betulus stand in Golestan
Province, North of Iran
VAHAB SOHRABI1,♥, RAMIN RAHMANI1, SHAHROKH JABBARI2, HADI MOAYERI1
Faculty of Forestry, Gorgan University of Agricultural Science and Natural Resources. PO Box 386, Shahid Beheshti Street, Gorgan, Golestan, Islamic
Republic of Iran, Tel. +98 (171) 222 0028, Fax. +98 (171) 222 598, ♥Email: [email protected]
2
Super Council of Forests, Range, Watershade Management Organization, Islamic Republic of Iran.
Manuscript received: 19 February 2011 Revision accepted: 3 March 2011.
Abstract. Sohrabi V, Rahmani R, Jabbari S, Moayeri H. 2011. Evaluation structural diversity of Carpinus betulus stand in Golestan
Province, Northern Iran. Nusantara Bioscience 3: 23-27. In order to investigate structural diversity of Carpinus betulus type in Golestan
province 30 modified Whittaker plots by systematic random system were located. Per plot the characteristic of trees and shrubs species
(Species name, diameter and height of trees) are recorded. The heterogenity indices of Simpson, Shannon–Wiener, Simpson’s reciprocal
and number of equally common species were used for the quantitative data. Toward better understand from diversity condition in
horizontal and vertical composition of stand, the diameter divided in 10 cm classes and Method of Mohajer and the height divided in 10
m height classes and dominant height, then number of diversity of each class extracted by Ecological Methodology software V.7. The
results showed with increase of diameter and height classes, decrease species diversity. Also regeneration layers diversity has significant
difference with trees layers. Thus, the study of biodiversity changes in different diameter and height category cause ecologically precise
perspective in management of forest stands.
Key words: structure diversity, indices diversity, diameter and height classes.
Abstrak. Sohrabi V, Rahmani R, Jabbari S, Moayeri H. 2011. Evaluasi keragaman struktur tegakan Carpinus betulus di Provinsi
Golestan, Iran bagian utara. Nusantara Bioscience 3: 23-27. Dalam rangka untuk menyelidiki struktur keragaman tipe Carpinus betulus
di provinsi Golestan, 30 plot Whittaker yang telah dimodifikasi dibuat secara sistem random sistematis. Pada setiap plot, karakteristik
spesies pepohonan dan semak (nama spesies, diameter dan tinggi pohon) dicatat. Indeks heterogenitas dari beberapa macam indeks
Simpson, Shannon-Wiener, Simpson’s reciprocal dan jumlah spesies yang umum ditemukan digunakan untuk data kuantitatif. Untuk
lebih memahami kondisi keanekaragaman dalam tegakan horizontal dan vertikal, maka dikelompokkan ke dalam diameter dalam kelas
10 cm, metode Mohajer, tinggi dalam kelas 10 m, dan ketinggian yang dominan, kemudian jumlah keragaman setiap kelas ditentukan
dengan software Ecological Methodology v.7.0. Hasil penelitian menunjukkan bahwa peningkatan kelas diameter dan tinggi,
menyebabkan penurunan keragaman spesies. Keragaman lapisan regenerasi memiliki perbedaan signifikan dengan lapisan pohon. Studi
perubahan keanekaragaman hayati dengan kategori diameter dan tinggi yang berbeda memerlukan perspektif ekologis yang tepat dalam
pengelolaan tegakan hutan.
Kata kunci: keanekaragaman struktur, indeks diversitas, kelas diameter dan tinggi.
INTRODUCTION
Human knows the concept and the importance of
biodiversity from earlier century. Plato frequently point out
the diversity and believe that if there is more diversity in
the world, the world will be better (Beasapour 2000,
Ejtehadi et al. 2009). Today, the word of biodiversity
applies by various science experts, such as ecologists. The
convention of biodiversity of USA, describe biodiversity
as: there is a difference in all the life type all sources such
as marine, ground and ecological complex combination and
include the diversity within species, between species and
ecosystems (Markandya et al. 2008). One of the constant
keys of management of uneven age forest is the true
understanding about spatial structure of forest (Costanza et
al. 2007). Forest structure is the important feature in
management of forest ecosystems (Zenner and Hibbs
2000). Structural features are used to determine the species
neech heterogeneous experiment and plant dynamic time,
management of regeneration patterns and fragmentation
dynamic, description of microclimate diversity and
predicting the wood production (Youngblooda et al. 2004).
Management of forest stands performs by stands structure
control (age, size and tree density) and forest structure (size
and spatial order of tree) because the concept of forest
structure is more important than species combination
(Oheimb et al. 2005). The study of natural forests structure
defined the way of desired structure that the use of
appropriate silviculture operation and stimulation of natural
structure in under management stands considered as the
way to keep the biological diversity and forest dynamic and
stability (Markandya et al. 2003). The study of forest
24
3 (1): 23-27, March 2011
structure especially in virgin forests is very important and
gives us comprehensive information about the condition in
forest for programming. The diversity of a forest stand may
not be sufficiently described by tree species diversity alone.
Structural diversity, resulting from recruitment of trees of
different sizes into multilayered canopies, should also be
taken into account (Liang et al. 2007). This characteristic,
which can be approximated by the diversity of tree size,
affects the amount of light and precipitation received by
subordinate trees and understory plants (Anderson et al.
1969), and may thus influence the productivity of forest
ecosystems. Thus manipulating tree-size diversity is a
practical tool for forestmanagers who strive for greater
biodiversity and/or greater productivity (Varga et al.
2005).Varus studing done about in forest structure.
Ahani et al (2006) do the research about species
diversity of tree based on the diameter class in Acer sites in
Shafarud forests. So, rhombus plots in half hectare study in
forest according to Acer (34 plots). First the feature within
each plot, its slope, aspect, height from sea level and then
total diameter of trees up to more than 10 cm
measured.Biodiversity accounted in four diameter alasses
(10-30, 35-50, 55-80, 80-120 cm). The result showed that
the Shanon and N1 Mac Arthur indices in diameter class of
35-50 cm, have greatest amount, while the index of
Simpson and N2 hill shows the greatest amount in diameter
class of 10-30 cm. The purpose of this paper is the
evaluation of structural diversity in diameter and height
classes and their changing process with changing of
diameter classes and height category in Carpinus betulus
(Persian: Mamarz) type in Golestan province, IR Iran.
MATERIALS AND METHODS
The regions of study
Kohmian forestry plan is located in 98 wateshade
domain which is limited in north is village of Kohmian,
Fazel Abad, Khanduz Sadat and Marzbone, in south and
west to Naeem s forestry plan and in east to vatan forestry
plan. Its east longitude is 55-14-49 to 55-10-30 and its
north width is 37-65-15 to 37-00-00 degrees (Figure 1).
Golestan 6
4
5
3
2
1
Figure 1. Map of the site study in in Golestan Province, North of Iran. 1. Shastkalateh, 2. Tavir, 3. Kohmian, 4. Takht, 5. Loveh, 6.
Farsian.
SOHRABI et al. – Diversity of Carpinus betulus stand in Golestan, Iran
25
Table 1. Indices used in this paper (Ejtehadi et al. 2009)
Equation
1 − D = 1 − ∑ ( pi )
Index
2
Simpson
H ′ = ∑ ( Pi )( Log 2Pi )
Shannon–Wiener
1
Simpson’s
reciprocal
s
i =1
D
=
1
∑ pi
2
N1 = eH ′
Number of equally
common species
Description of equation
(1-D) = Simson’s index of diversity
p1 = proportion of individual species I in the community
H’ = information content of sample (bits/individual) = index of species diversity
s = number of species
p1 = proportion of total sample belonging to i-th species
1/D = Simson’s reciprocal index (= Hill’s N2)
p1 = proportion of individual species i in the community
H’ = information content of sample (bits/individual) = index of species diversity
s = number of species
p1 = proportion of total sample belonging to i-th species
Research method
This research is basee on sampling by systematic
random system and the center of plots in forest is
determined. To study and investigation, 30 modified
Whittaker plots in range of 850-950 m altitude from the sea
level in north aspect were located. In this 20x50 meter
frame, the characteristic of trees and shrubs species
(species name, diameter and height of trees) are recorded.
The heterogenity indices of Simpson, Shannon–Wiener,
Simpson’s reciprocal and number of equally common
species and evenness indices of Simpson, Camargo, SmithWilson and modified nee were used for the quantitative
data (Table 1). Then aforesaid characteristics saved as
information bank in Excell 2010. Then indices account by
Ecological Methodology software v.7.0 (Krebs 1999).
Analyze of data was done by analyze of variance
(ANOVA) and Duncan’s multiple range test (DMRT).
Diversity indices in 10 cm diameter classes
The under study diversity indices in this paper shows
the decrease in the diameter classes of 10cm with increase
of classes. The most diversity number is in diameter class
of 0-10 cm and the least diversity number is in diameter
class of 90-100 cm. other than Simpson diversity index that
shows the least diversity number in diameter class of more
than 100cm, the significant different is between diameter
classes in 1% level (Figure 2).
Diversity indices in diameter classes by method of
Mohajer
Diversity indices shows decrease process with the
increase of diameter classes but it increase again in last
class (dbh>80). The most diversity number is in the class of
0-10 cm and the least diversity number is in class of 60_80
cm. The diameter classas (20-30, 30-60, dbh>80) are not
significant different. The significant different is between
diameter classes in 1% level (Figure 3).
RESULTS AND DISCUSSION
Next of survey recorded number of 10 trees species
dependent of 8 families and 3 shrubs species dependent of
2 families that show notable statistics (Table 2).
Table 2. Composition of trees and shrubs species.
Scientific name
Quercus castanefolia
Carpinus betulus
Parrotia persica
Tilia begunda
Acer insigne
Ulmus glabra
Acer cappadocicum
Alnus glutinosa
Crataegus monogyna
Mespilus germanica
Prunus avium
Sorbus torminalis
Diospyros lotus
Family
Fagaceae
Betulaceae
Hamameliadaceae
Tiliaceae
Acearaceae
Ulmaceae
Acearaceae
Betulaceae
Rosaceae
Rosaceae
Rosaceae
Rosaceae
Ebenaceae
Trees/Shrubs
T
T
T
T
T
T
T
T
S
S
S
T
S
Diversity indices in 10m height classes
Diversity indices have orderly decrease process. The
most diversity number is in height class of 0-10m and the
least diversity number is in height class of 40-50m. , the
significant different is between height classes in 1% level
(Figure 4).
Diversity indices in dominant height of height classes
The most diversity number in all indices is for h<1/3hm
class and the least diversity number is for 1/3h<h>2/3hm
height class. Diversity indices, first, decrease then increase
in third class. First height class has significant difference
with other classes in level of 1% (Figure 5).
Discussion
Forest structure is the important feature in management
of forest ecosystems (Zenner and Hibbs 2000). The study
of natural forests structure defined the way of desired
structure that the use of appropriate silviculture operation
and stimulation of natural structure in under management
stands considered as the way to keep the biological
diversity and forest dynamic and stability (Markandya et al.
2003). Whereas, structure characterize the building
(vertical and horizontal), composition and diversity of
26
3 (1): 23-27, March 2011
Figure 2. The comparison of diversity indices in 10cm diameter
classes. A. Simpson, B. Simpson’s reciprocal, C. Shannon–
Wiener, D. Number of equally common species.
Figure 3. The comparison of diversity indices by method of
Mohajer (2005). A. Simpson, B. Simpson’s reciprocal, C.
Shannon–Wiener, D. Number of equally common species.
Figure 4. The comparison of diversity indices in 10m height
classes. A. Simpson, B. Simpson’s reciprocal, C. Shannon–
Wiener, D. Number of equally common species
Figure 5. The comparison of diversity indices in height classes by
dominant height. A. Simpson, B. Simpson’s reciprocal, C.
Shannon–Wiener, D. Number of equally common species.
forest stands. Forest stands have different structure in
various sections (linear and phenomenal) like a building.
For recognition, study and precise programming of forest
stands, its features need to consider according to different
sections. Various profiles (linear and phenomenal) could be
dividing for forest stands. The study of forest stand profile
especially in virgin forests is very important and gives us
comprehensive information about structure of these forests
(Mohajer 2005). For better understanding of the structure
of forest stand, we analyzed it according to the vertical and
horizontal structure. Species diversity of tree and shrub in
this type have significant different in low diameter and
height classes with up diameter and height classes classes.
Diameter and height classes below of 10 cm, account as 10
regeneration layer, so diversity of regeneration layer is
more than the diversity of tree layers (Pourbabaei et al.
2006; Sohrabi 2010). This is due to the decrease of canopy
of small saplings and it need low light than higher age
process in this classes. By the increase of diametrical and
height classes, the diversity decrease. It is obvious that the
structure diversity naturally in the virgin forest decrease
depend on site condition and with increase of stand age and
its move toward climax, because gradually increase of trees
age dominant species dominant against the under species.
Trees are the main elements in forest ecosystems that other
living thing life of this ecosystem depends on the life of
them. Therefore removing of the tree threatened the life of
the existent in this ecosystem. The main role of forest
engineer is the marketing of forest (Mohajer 2005). In this
step choosing of trees perform by considering of target
diameter from defined species and gradually the number of
trees in defined diameter decreased and so the repeating act
might remove some class of trees. It is threatened the
structure diversity and the species diversity. Trees diversity
in higher diametrical and altitudinal categories is part of the
lower diametrical category diversity. Any changes in above
level might change the ground cover. Tree dimension
diversity has an effect on the amount of light and raining
by small plant and trees (Anderson et al.1969). This has
influence on the produce of forest ecosystems.
SOHRABI et al. – Diversity of Carpinus betulus stand in Golestan, Iran
CONCUSION
The increasing of diameter and height classes, decrease
species diversity. Regeneration layers diversity has
significant difference with trees layers. Thus, the study of
biodiversity changes in different diameter and height
category cause ecologically precise perspective in
management of forest stands.
ACKNOWLEDGEMENTS
Therefore I express gratitude to any ones who is useful
in my life. By the way, I thank Ezazi to give us translation
of this paper.
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ISSN: 2087-3940 (print)
ISSN: 2087-3956 (electronic)
Vol. 3, No. 1, Pp.: 28-35
March 2011
Microanatomy alteration of gills and kidneys in freshwater mussel
(Anodonta woodiana) due to cadmium exposure
FUAD FITRIAWAN1,♥, SUTARNO², SUNARTO²
¹ Open University, UPBJJ Bandar Lampung. Jl. Soekarno-Hatta No. 108 B Rajabasa, Bandar Lampung 35144, Lampung, Indonesia. Tel.: +92-721704772. Fak.: +92-721-709026. E-mail: [email protected], [email protected]
² Bioscience Program, School of Graduates, Sebelas Maret University, Surakarta 57126, Central Java, Indonesia
Manuscript received: 15 December 2010. Revision accepted: 26 February 2011.
Abstract. Fitriawan F, Sutarno, Sunarto. 2011. Microanatomy alteration of gills and kidneys in freshwater mussel (Anodonta
woodiana) due to cadmium exposure. Nusantara Bioscience 3: 28-35. The purpose of this study were to determine the level of Cd
accumulation in the gills and kidneys, to khow the changes in microanatomic structure of A. woodiana after the various treatments of
heavy metals. Completely randomized design pattern of 5 x 3 as used in this laboratory experiment. The amount of exposure of heavy
metals Cd were (0 ppm, 0.5 ppm, 1 ppm, 5 ppm, 10 ppm), while the variation of length of exprosure time to Cd were (7 days, 14 days,
and 30 days). The parameters of Cd accumulation in the gills and kidney was analyzed by using AAS method, while abnormalities of
gills and kidney were detected by microanatomy structure. Data collected were then analyzed using the analysis of variance (ANOVA)
and continued with further test the DMRT. The results indicated that there is a significant effect in 475.3 > 0.000 and 60150.3 >0.000
with 5% significance level (P<0.05) of Cd treatment on gill and kidney microanatomy of A. woodiana. The changes in microanatomy
structure of those organs are including edema, hyperplasia, fusion of lamella, necrosis and atrophy.
Key words: gills, kidneys, Anodonta woodiana, cadmium.
Abstrak. Fitriawan F, Sutarno, Sunarto. 2011. Perubahan mikroanatomi pada insang dan ginjal kerang air tawar (Anodonta
woodiana) terhadap paparan kadmium. Nusantara Bioscience 3: 28-35. Tujuan penelitian ini untuk mengetahui tingkat akumulasi,
perubahan struktur mikroanatomi setelah perlakuan logam berat Cd pada insang dan ginjal A. woodiana. Jenis penelitian yang
digunakan yaitu eksperimental laboratorium dengan rancangan acak lengkap (5 x 3) berupa besarnya paparan Cd (0 ppm, 0,5 ppm, 1
ppm, 5 ppm, 10 ppm) dan waktu pemaparan Cd (setelah 7 hari, 14 hari, dan 30 hari). Parameter pengujian mencakup uji akumulasi Cd
pada insang dan ginjal dengan metode AAS, dan abnormalitas insang dan ginjal akibat akumulasi Cd dengan metode preparasi. Analisis
akumulasi Cd pada insang dan ginjal menggunakan analisis varian (ANAVA) dan dilanjutkan dengan uji lanjut jarak berganda Duncan
(DMRT). Hasil penelitian menunjukkan pengaruh pemberian beberapa perlakuan Cd terhadap kontrol insang dan ginjal A. woodiana
signifikan sebesar 475,3 > 0,000 dan 60150,3 > 0,000 dengan taraf signifikansi rata-rata 5% (P<0,05) yang ditandai dengan perubahan
struktur mikroanatomi pada insang berupa edema, hiperplasia, fusi lamella, nekrosis hingga atropi. Sedangkan pada ginjal berupa
edema, hiperplasia dan nekrosis pada tubulus, glomerulus, dan mineralisasi pada sel darah hingga mengalami pendarahan.
Kata kunci: insang, ginjal, Anodonta woodiana, kadmium.
INTRODUCTION
Cadmium (Cd) is one type of heavy metals that are
useful in several industries. For example in the textile
batteries industry and electroplating, as coloring matters in
ink. Cd also exisst naturally in foods even if only in small
amounts absorbed by the intestine (5-8%) (Palar 1994). But
on the other hand, a heavy metal can cause problems;
problems can occur more severe if waste management is
not done properly, so that it will have an impact on the
environment as a micro-pollutants (Soegianto et al. 2004).
Incoming cadmium in fresh water will be joined with a
metal ion cofactor so that the shape of Cd2+ causes the
toxicity of the water. Cd2+ ‘s toxicity levels in water depend
on salinity. The toxicity of Cd2+ in the water will rise if the
salinity is low.
To determine the level of pollution in a region we can
use a particular bioindicator organism typical of one that
can be used, namely A. woodiana. The advantages of this
animal is that they settle in one place, and have a slow
movement, so that if an environment is exposed to heavy
metal waste Cd then indirectly affects the lives of these
biota. Cd accumulation in an organism other than cause
exposure to the organ, it will also cause interference on
enzyme activity. The nature of the toxic metal is due to its
very effectiveness in binding itself withh the group of
sulfuhidril (SH) in the enzyme system of cells that form
bonds and metaloprotein metaloenzim so that enzyme
activity for cell life processes cannot take place (Connell
and Miller 1995).
Gills and kidneys are vital organs. Gills play a role in
the process of respiration, acid-base balance, ionic and
osmotic regulation because of the branchial epithelium
FITRIAWAN et al. – Effect of Cd on freshwater mussels A. woodiana
tissue which became the meeting place of active transport
between organisms and the environment (Soegianto et al.
2004). Renal function begins in the glomerular ultrafilter
that is formed from the plasma. Ultrafilter will enter the
Bowman's capsule and into the lumen of the tubule.
Filtering through the various segments of the tubules
causes changes in the volume and composition of fluid
filtration as a result of the process of reabsorption and
secretion along the tubules (Tresnati et al. 2007).
Glomerulus is composed of blood capillaries to function as
a selective filter from the blood mainly in the normal blood
screening (Takashima and Hibiya 1995). Following
through on glomerular filtration and being re-absorbed in
the tubular, it produces urine as a result of secretion in
normal circumstances (Tresnati et al. 2007).
The purpose of this study are: (i) to know the content of
Cd accumulation, (ii) changes in the microanatomy
structure, and (iii) in gill and kidney of A. woodiana after
treatment.
MATERIALS AND METHODS
Time and place
The research of the Cd treatment on A. woodiana
conducted at the Laboratory of Pharmacy and Food
Academy Analyst Sunan Giri, Roxburgh. Analysis of
heavy metal content by AAS method was carried out in sub
lab Chemistry Laboratory of Mathematics and Science
Center UNS Surakarta, while the preparation for the
analysis was conducted in the laboratory animal anatomy
Faculty of Veterinary Medicine, Gadjah Mada University
in Yogyakarta. The experiment was conducted in OctoberNovember 2009.
Materials
Freshwater mussels (A. woodiana) were obtained from
farms in the fishing village of the tourist site of Janti,
Polanharjo Subdistrict, Klaten District, Central Java.
Procedures
Freshwater bivalve A. woodiana was selected based
onthe maximum growth and uniform size. The shellfish
was acclimatized for 15 days, after which it was examined
by using the compound of Cd for 30 days with repeated 3
times at day 7, 14 and 30. Physical-chemical parameters
measured mencakuip pH, DO and water temperature where
the experiment. Cd content of the examination conducted
on the gill and kidney A. woodiana with AAS method.
Preparation of gill and kidney preparations performed with
haematoxylin-eosin (HE) method with treatment stages, ie
trimming, dehydration, embedding, cutting, stainning,
mounting and reading the results.
Data analysis
Environmental chemistry parameters (pH, DO,
temperature) were described with a descriptive method.
Effects of Cd exposure on the gill and kidney A. woodiana
were analyzed by ANOVA one-way significance level of
5% (P> 0.05), followed by a further test of significant
29
difference or Duncan's multiple range test (DMRT).
Abnormality in the microanatomy structure of gills and
kidneys of A. woodiana was directly observed and
described with a descriptive method.
RESULTS AND DISCUSSION
Water environmental parameters
Examination of physical and chemical parameters of
water quality used in this study include the degree of
acidity (pH), dissolved oxygen (DO), and water temperature.
Degree of acidity (pH)
The degree of acidity or pH is a value that shows the
activity of hydrogen ions in water. The pH of a water
reflects the balance between acid and base in these waters.
The pH range 1-14, pH 7 is the boundary halfway between
the acid and alkaline (neutral). The higher the pH of the
water, the greater the base nature will be, and the lower the
pH the more acidic the water. PH value is influenced by
several parameters, including biological activity,
temperature, oxygen content and the ions. From the
biological activity, CO2 gas is generated as a result of
respiration. This gas will form a buffer or buffer ions to
maintain the pH range in the waters in order to remain
stable (Erland 2007).
In this study, the pH is very important as water quality
parameters, for controlling the type and rate of speed of
reaction some materials in the water. In addition, A.
woodiana live at a certain pH interval, so that by knowing
the value of pH, it can be known whether or not the water
supports their lives. Based on Figure 1A, it is known that
the higher Cd concentration, the higher the value of the
range of pH waters. On day 7, pH values ranged from 7.34
to 8.44, on day 14 ranged from 7.37 to 8.40, and the dayto-30 range from 7.31 to 8.68. According to Erland (2007),
pH to function as an index of environmental conditions and
limiting factors, where each organism has a different
tolerance to pH maximum, minimum and optimal.
According to Erland (2007) pH value of water has a
special characteristic, the hydrogen ion concentration be
measured by the balance between acids and bases. Acidfree mineral acid and carbonic acid will lower pH value
(acid), while the carbonate (CO3), hydroxide (OH-) and
bicarbonate to raise pH (alkaline). Rochyatun et al. (2006)
states, that at a relatively high metal content will be
alkaline pH values (pH 7.40 to 8.59), where the metal is
difficult to dissolve and settle to the bottom of the water.
When in the treatment, pH values from 0.5 to 10 ppm, in
the study it ranged from 7.92 to 8.68, indicating water has
been polluted quite heavily, with the level of alkalinity in
excess of tolerance. According to Connell and Miller
(1995) increase in pH in the waters will be followed by
decreasing the solubility of heavy metals that tend to settle.
Deposition can occur in sediments and food; the food will
enter and accumulate in the body of A. woodiana. Given
the Cd is a non-essential metal that cannot be degraded so
that it will cause interference with the organs, such as the
gill and kidney.
30
3 (1): 28-35, March 2011
increasing the rate of respiration and dissolved CO2
increases, so the toxins more and more absorbed in the
body through the gills. The higher the level of aquatic
toxicity, the higher the rate of breathing will be (Budiono
2003).
Dissolved oxygen is essential for respiratory zoobentos
and other aquatic organisms (Odum 1993). In addition, the
solubility of oxygen is also affected by temperature, at high
temperature and low oxygen solubility at low temperatures
the high oxygen solubility. Each species of aquatic biota
have a range of different tolerances to the concentration of
dissolved oxygen in the water. Species with wide tolerance
range and wide distribution of species with narrow
tolerance range only live in certain places. Budiono (2003)
stated that the excessive presence of heavy metals in the
waters will affect the respiratory system of aquatic
organisms, causing low dissolved oxygen levels, which
disturb the life of aquatic organisms.
Water solubility of oxygen (DO)
Oxygen is one of the gases dissolved in natural waters
with varying levels are influenced by temperature, salinity,
water turbulence, and atmospheric pressure. Besides
necessary for the survival of aquatic organisms, oxygen is
also needed in the process of decomposition of organic
compounds. Sources of dissolved oxygen are mainly
derived from the diffusion of oxygen from the atmosphere.
This diffusion occurs directly on stagnant conditions
(silent), or because of agitation (water mass unrest) caused
by waves or wind.
Figure 1B shows that the higher concentration of Cd
treatment, then progressively decreasing levels of dissolved
oxygen (DO) in water. Ardi (2002) classified water quality
based on the DO into four types namely; not contaminated
(> 6.5 mg/L), lightly polluted (4.5 to 6.5 mg/L), being
contaminated (2.0 to 4, 4 mg/L) and heavily polluted (<2.0
mg/L). In this study, the DO in the first test of 10.10 ppm
decreased to 3.19 ppm, in the second test from 10.11 ppm
to 3.25 ppm, and the third repeat of 10.26 ppm to 3.76
ppm. From the above results the pollutuion can still be
considered moderate.
Decreased levels of oxygen in the water is inversely
proportional to the high Cd treatment in these waters.
Cadmium is an inorganic contaminants/minerals that can
accumulate in water or in food. In general, the Cd that
entered the waters will be Cd2+ which causes the toxicity
waters, and the presence of sediment in the diet will be
very easy to be consumed by aquatic biota, including A.
woodiana. Solubility of oxygen is essential for the
sustainability of aquatic life, oxygen is used as a tool of
biota metabolism so it can carry out their duties as
pendekomposisi and degrading organic materials in order
to more easily broken down by bacteria (Warlina 2004;
Ardi 2002). If an aquatic tecemar by a heavy metal
inorganic, then A. woodiana not able to decompose organic
materials, so that the decomposition process is highly
dependent aerobic bacteria that need oxygen is very high,
and can cause a deficit of oxygen in these waters.
According to Destiany (2007) with increasing
concentrations of heavy metals, the dissolved oxygen
content will decrease, and the CO2 will rose, due to low
oxygen levels that require aquatic biota such as A.
woodiana to pump water through their gills, thereby
(
9
7.5
8.44
8.22
8.018.09
8.3
7.92
7.31
8
27.4
27.2
27.5
10.1
10.26
10.11
10
7.96
7.37
7.34
8.68
8.568.61
8.44 8.4
26.9
27
7.57
7.27
6.89
5.68
6
4
6.146.02
5.164.92
5.17
3.76
3.25
3.19
nilai (derajad celc
8
)
12
n ilai DO (p p m
nilai pH
8.5
Temperature
Each of aquatic organisms has different tolerance limits
to changes in water temperature to the life and growth of
aquatic organisms. Therefore, the temperature is one factor
that physically is very important for aquatic organisms or
aquatic life. In general, the temperature directly affects the
aquatic biota of enzymatic reactions in the organism and
does not directly influence the structure of organs and the
spread of aquatic animals (Nontji 1984).
From Figure 1C, it is known that the temperature of
different water looks increasingly high Cd treatment at
each treatment, which ranged from 25.8 to 26.6°C on the
first test, and the second test ranged from 26.2 to 27°C, the
third test ranged from 26.8 to 27.4°C. This is influenced
metal accumulation in each treatment with the higher
concentration, thus causing the water temperature value is
also higher. It is inversely proportional to the solubility of
oxygen in water, ie at high temperature low oxygen
solubility, and low solubility of oxygen at high temperature
(Odum 1993).
The Relationship between the temperature rise of heavy
metal accumulation in the water is strong. Cd is an
inorganic non-essential metal that cannot be in the
degradation of benthos organisms and microorganism. The
presence of metal causes the metabolic rate of aquatic biota
26.6
26.5
27
26.926.9
26.8
26.6
26.5
26.2
26
25.925.9
25.8
26
25.5
7
2
25
6.5
7 hari
14 hari
30 hari
7 hari
0
7 hari
pemeriksaan
14 hari
14 hari
30 hari
30 hari
pemeriksaan
pemeriksaan
0 ppm
0 ppm 0.5 ppm 1 ppm 5 ppm 10 ppm
0 ppm 0.5 ppm 1 ppm 5 ppm 10 ppm
A
B
0.5 ppm
1 ppm
5 ppm
10 ppm
C
Figure 1. The physical-chemical parameters condition of waters in the experimental location after administration of Cd. A. the degree of
acidity (pH), B. DO, C. temperature.
FITRIAWAN et al. – Effect of Cd on freshwater mussels A. woodiana
increased in order to defend themselves, so that
automatically the oxygen demand is very much while on
the other hand, given the concentration of heavy metals
higher, thus increasing the concentration of heavy metals
that enter the more carbon dioxide (CO2) are released that
cause the oxygen content dwindling waters so that the
rising water temperature.
According to Connell and Miller (1995) the role of
water temperature is very important to help the body's
metabolism of aquatic animals. The increase in water
temperature can cause the immune system of aquatic biota
to decrease. So if a toxic Cd2+ enters the body of A.
woodiana the biota will be very difficult to retain yourself
from the poison.
Accumulation of Cd in the gills of A. woodiana
The result of the content of Cd in the gill and kidney A.
woodiana with AAS method are shown in Table 1. From
the results it is known that the increasing Cd treatment, the
increase of Cd accumulatd in the gill of A. woodiana. In the
control (0 ppm), Cd accumulation in the gills of A.
woodiana was 0.12 ppm, the accumulation in the control is
still below the maximum tolerance limit Cd accumulation
in organs, as specified by the FAO (1972) and MOH
(1989), namely a maximum accumulation of Cd in organs
of 1 ppm. It is also in accordance with preliminary studies
that have been made to the content of Cd in water samples,
with the result that the content of Cd in Janti aquaculture
that was still in the normal state that was 0.0028 ppm (IGR
No. 82/2001; EPA 1986).
After the examination after 7 days the average values
obtained Cd accumulation in gill A. woodiana in the
treatment of 0.5 ppm was 0.58 ppm, treatment of 1 ppm
was 0.87 ppm, 5 ppm was 1.00 ppm and 10 ppm was 2.15
ppm. Meanwhile, after the examination on day 14, obtained
an average value of accumulated Cd at 0.5 ppm treatment
was 0.78, treatment of 1 ppm 0.93 ppm, 5 ppm treatment at
1.24 ppm, and on treatment 10 ppm at 2.34 ppm. After the
examination on day-30, it was obtained an average value of
Cd accumulation in the treatment of 0.5 ppm 1.43 ppm, 1
ppm treatment at 1.01, 5 ppm treatment at 2.58, and the
treatment of 10 ppm 3.49 ppm.
Darmono (1995) states that the relationship between the
amount of metal absorption and metal content in water is
usually in proportion, the increase in metal content in the
network in accordance with the increase of metal content in
water. According Sunarto (2007) Cd will also experience
the process of biotransformation and bioaccumulation in
aquatic biota. Cadmium enters the body along the water or
food consumed, but water or food has been contaminated
by Cd. The amount of metal that accumulates in the gills
will continue to increase, even very likely continue to enter
through the accumulation of Cd digestive tract to the
kidney; in addition to increasing levels of pollutants in the
presence of Cd may also biomagnification process in the
water body. If the amount of Cd that enters the body and
has exceeded the threshold value, it will experience death
and even extinction.
Cd treatment against gill A. woodiana with a
concentration of 0 ppm, 0.5 ppm, 1 ppm, 5 ppm and 10
31
ppm for 7 days, 14 days and 30 days to yield significant
results (P <0.05). This is in accordance with the opinion
Darmono (1995) which states, that the relationship between
the amount of metal absorption and metal content in water
is usually in proportion, the increase in metal content in the
network in accordance with the increase of metal content in
water.
ANOVA test and Duncan's range test showed no
association of Cd accumulation in gill of A. woodiana with
the concentration of Cd (Table 1). The higher the
concentration of Cd is given, the higher the exposure to Cd
on the gills of A. woodiana will be. The most obvious
difference is indicated by the treatment concentration of 10
ppm, which is the highest concentration, thus giving an
average value of accumulated most Cd concentrations
higher than below it. As for the concentration of 0.5 ppm
and 1 ppm, obtained test results are less tangible difference,
possibly because the treatment they are not too much
difference compared with other treatments, so that the
results of cadmium exposure on A. woodiana not too
apparent.
Table 1. Treatment test results in the accumulated Cd
concentration of Cd in gill and kidney A. woodiana.
Treatment of Cd
Average Cd
Average Cd
concentration
accumulation in
accumulation in
(ppm)
gill (ppm)
kidneys (ppm)
0
0.12 a
0.018933 a
0.5
0.93 b
0.045200 b
1
0.94 b
0.042956 b
5
1.61 c
0.082844 c
10
2.66 d
0.660111 d
Note: Value having the same letter notation means that no effect
significantly different
ANOVA test and Duncan's range test that also shows a
relationship between the length of the treatments with the
level of Cd accumulation in the gill of A. woodiana (Table
2). The longer treatment time means the accumulation of
Cd in the gills. In Table 2, It can be seen that a 30-day
treatment gave the highest average of the Cd accumulation
on the gills of of A. woodiana. Then further test the
distance from Duncan to get the illustration relations and
exposure levels of Cd accumulation in the gill of A.
woodiana to the long treatment, where the old high Cd
treatment is given, the higher accumulation of Cd in the
gills of A. woodiana.
Table 2. Old test results the treatment of Cd accumulation in gill
and kidney A. woodiana
Average Cd
Average Cd
accumulation in gill
accumulation in
(ppm)
the kidney (ppm)
7
0.940313 a
0.048420 a
14
1.081420 a
0.063740 a
30
1.728753 b
0.397867 b
Note: Value having the same letter notation means that no effect
significantly different
Length of
treatment (days)
32
3 (1): 28-35, March 2011
Analysis of treatment outcome ofthe renal Cd of A.
woodiana
Test results on the Cd content of the kidney A.
woodiana after treatment is shown in Table 1. The average
value of the kidney of A. woodiana in the control group
amounted to 0.019 ppm (0.018933 ppm), the value
accumulated in the control was still below the maximum
tolerance limit Cd accumulation in organs, as specified by
the FAO (1972) and MOH (1989) that is equal to 1 ppm. It
is also in accordance with preliminary studies that have
been made to the content of Cd in the water, that the
content of Cd in the water of Janti aquaculture is still in the
normal state is 0.0028 ppm (IGR No. 82/2001; EPA 1986).
Later in the treatment of 0.5 ppm Cd in the kidneys
after examination, AAS average accumulation after 7 days
was at 0.020 ppm, after 14 days was at 0.029 ppm, after 30
days was at 0.086 ppm. Later in the treatment of 1 ppm
after 7 days accumulation of 0.030 ppm, 0.031 ppm after
14 days, and after 30 days at 0.066 ppm. Later in the
treatment of 5 ppm Cd accumulation after 7 days at 0.057
ppm, after 14 days at 0.085 ppm, and after 30 days at 0.107
ppm. And in the treatment of 10 ppm Cd accumulation in
the kidney after 7 days at 0.116 ppm, after 14 days at 0.150
ppm and after 30 days showed the exposure of 1.717 ppm.
From the above data, it is known that the higher the
concentration of Cd treatment A. woodiana, the higher the
value of exposure to cadmium in the kidneys of A.
woodiana. This is similar to what has been mentioned by
Sunarto (2007) that Cd will also experience the process of
biotransformation and bioaccumulation in aquatic biota.
Cadmium enters the body along the water or food
consumed, where water or food has been contaminated by
Cd. The amount of metal that accumulates in the gills will
continue to increase, even very likely continue to enter
through the accumulation of Cd digestive tract to the kidney.
Based on ANOVA statistical test, it is known that Cd
treatment on the kidney of A. woodiana with a
concentration of 0 ppm, 0.5 ppm, 1 ppm, 5 ppm and 10
ppm for 7 days, 14 days and 30 days, yield significant
results (P <0.05). This is in line with the significance
indicated on the gills of A. woodiana. Furthermore, Duncan
range test indicated that the higher the concentration of Cd
treatment, the higher the accumulation of Cd in the kidneys
of A. woodiana. Treatment of Cd concentration of 10 ppm
gave the highest cumulative impact with the most obvious
significance level, of the treatment concentration
underneath. Later the treatment of 0.5 ppm and 1 ppm did
not show significant differences, it might be that the range
of Cd concentrations that had been given is not far adrift,
so that the value of exposure and accumulation of Cd in the
kidneys was directly proportional to the treatment given.
Cd concentration of 10 ppm gave the most obvious
difference.
To know the effect of long exposure to the treatment of
kidney due to accumulation of Cd in A. woodiana, it was
also performed ANOVA test followed by a significantly
different test (Duncan), where the longer treatment time
means the higher the impact on exposure accumulation of
Cd in the kidneys of A. woodiana, where this long a period
of 30 days of treatment gave the greatest average rating of
accumulation.
According Destiany (2007) said that, the process of
accumulation of chemicals in living things is described as
follows: foods that accumulate heavy metals such as
cadmium, will be eaten by aquatic biota, including the
types of bivalves and will enter into the digestive tract.
From within the digestive (gastrointestinal) through the
walls will go to the circulatory fluid, then after the
circulatory fluid would most foodstuffs in metabolism and
partly met with several networks, so it will be in storage in
fat tissue. Then chemicals such as cadmium in the fluid
circulatory oxidized to Cd2+ that cause toxicity and will
accumulate in the liver, because the nature of the Cd is a
material non esesial, its presence in the liver cannot be
inactivated by the enzyme, so it continues to settle the
kidneys and and create sediment there.
Microanatomical changes in the gill of A. woodiana
after exposure to Cd
Accumulation of Cd has caused a variety of
physiological damage to the organs of A. woodiana,
because the nature of the toxicity of Cd accumulated in the
body has exceeded the maximum threshold of 1 ppm (FAO
1972), where the metal LC50 at 3 ppm occurs 48-72 hours
after treatment (Kraak et al. 1992). In this experiment,
some damaged organ/tissue shrinkage as siphon, foot, gill
and kidney. According to Palar (1994) Cd can damage
aquatic biota in the physiological system urinary system,
gill, kidney and blood circulation. Damage caused by Cd
contacts continuously through the cell membrane results in
the degeneration of the membrane. If Cd enters through the
gill, the gill will cause the deficiency so that the body's
metabolic function gets disrupted.
Result analysis of changes in cellular of the
microanatomical structure of the gill and kidney of A.
woodiana is shown in Table 3. From the data results of the
transverse slice preparations of the gill of A. woodiana after
accumulation of Cd, it is known that the symptoms of cell
damage on the gill was known at a concentration of 0.5
ppm with marked with edema in the lamella branchialis, so
that on day 14 and day 30 the hiperflasia was more visible
impact. The worst damage at the cellular level of gills
occured on thetreatment of 10 ppm, where the gills showed
symptoms of edema that was accompanied by hyperplasia,
and eventually the entire network of experienced fusion up
to each lamella having atrhopi (Figure 2).
In the normal gill at the concentrations of 0 ppm, with a
concentration of 0.1004 to 0.1321 ppm Cd accumulation
(Figure 2A), it can be seen all the parts of cells from
epithelial cells, basement membrane, Lacuna, the blood
cells until the cell pillar that were still in normal
circumstances. The accumulation of these metals has been
carried by each of biota samples from the sampling site that
is in the area of aquaculture of Janti. So to make a sample
without the accumulated metal is extremely difficult. In
addition, according to Rahman (2006) in general, heavy
metal content in a body of water is different form the one
with heavy metals that have been dissolved in the aquatic
sediments especially heavy metals in the organ. A heavy
metal when the waters would go down and settles to form
sedimentation, it will cause the organisms that eat at the
FITRIAWAN et al. – Effect of Cd on freshwater mussels A. woodiana
were also seen pillar cells began to separate from the
bottom of epithelial cells (middle lamella). When
experiencing edema,l Cd accumulation in gills occurred at
the accumulation of 0.5111 ppm.
Gills in Figure 2C has undergone thorough hyperplasia
and the fusion is taking place in two parts of the middle
lamella, with a marked by the epithelial cells started to
scarp, accompanied by the loss widened Lacuna red blood
cells and pillar cells apart. Laksman (2003) says that
hyperplasia is a process of formation of excessive tissue
due to the increase in cell volume. Hyperplasia caused by
excessive edema so that red blood cells out of kapilernya
and separated from the backers. In the event this hyperplasia
Cd accumulation began at 0.6829 ppm exposure level.
Condition of cells and gill tissue had fused to lamella
(Figure 2D), and began to show marked necrosis with
epithelial cells in each lamella started together with
epithelial cells on the other lamella, Lacuna also began to
rupture causing respiratory function failure which affects
the metabolism of A. woodiana. Secondary lamella fusion
caused by the swelling in the cells of the gills (edema). The
occurrence of secondary lamella fusion resulting in
impaired function of the secondary lamella in the case of
oxygen-making process and therefore contributes to the
death of A. woodiana (Susilowati 2005). At a concentration
of 5 ppm after 30 days A. woodiana experience death. In
this incident the gills accumulate heavy metals at a
concentration of 0.9280 ppm.
At that last stage of a gill would experience the highest
levels of damage, this damage can lead A. woodiana to
experience the death of the level of necrosis and atrophy.
Condition of cells and gill tissue necrosis and atrophy
experienced (Figure 2E), characterized by the merging of
each cell in lamella and lamella with bone loss starting
institutions. Atrophy is a reduction (shrinking) the size of a
cell, tissue, organ or body part (Harjono 1996). In this
study occurred atrophy in primary lamella. Atrophy occurs
due to experimental animals exposed to cadmium at high
concentrations and in a long exposure time. Cells in
primary lamella shrinkage (atrophy).
Laksman (2003) states that the necrosis is cell’s death
that occurrs due to hyperplasia and excessive fusion of
secondary lamella, so that the gill tissue is no longer intact
form or in other words necrosis occurs accompanied with
the death of a biota. In the event necrosis and atrophy of
the accumulated Cd in the gills of A. woodiana started at
2.1279 ppm exposure and atrophy starting at the level of
accumulation of 2.337 ppm.
bottom waters, such as A. woodiana (bivalves) will have a
great opportunity for exposure to heavy metals that have
been bound and form sediment.
Table 3. Changes in cellular structure mikroanatomi A. Gill
woodiana after exposure to heavy metals cadmium with HE
staining preparation.
Time of
Fusion
Concentration
Hypersurgery Edema
of Necrosis Atrophy
(ppm)
plasia
(days)
lamella
0
7
14
30
0,5
7
+
14
++
+
30
+++ ++
1
7
++
+
14
++
++
++
30
++++ +++
+++ 5
7
++++ +++
+
14
++++ ++++ +++ (dead)
30
++++ ++++ ++++ +++
10
7
++++ +++
+
14
++++ ++++ ++++ ++++ ++
(mati)
30
++++ ++++ ++++ ++++ +++
Note: -: no change in the microanatomical structure (0%); +: there
was a slight change in the microanatomical structure (1% -25%);
+ +: there are changes in the microanatomical structure (26% 50%); + + +: occurred many changes in the microanatomical
structure (51% -75%); + + + +: there are very many changes in
the microanatomical structure (76% -100%).
Gill cellular conditions experiencing edema (Figure
2B), visible basement membrane began to stretch out, the
field narrowing Lacuna cell deficiency causes gill function
and difficulty in breathing process, so that the metabolism
of the body began to fail. Edema is swelling of the cell or
excessive accumulation of fluid in body tissues (Laksman
2003). The presence of edema can cause fusion of
secondary lamella of the lamella. In this study the
occurrence of edema caused by the influx of Cd into the
gills of A. woodiana resultied in the cell cell irritating so
that the cell would swell.
The process of entry of Cd into the gills by Palar
(1994), together with other metal ions and the food that has
been accumulated Cd, and will form ions that can dissolve
in fat. Ions were able to penetrate the gill cell membrane,
so it can get into the gills, and then there will be a process
of loss of volume regulation in the cell. In this treatment
A
B
33
C
D
E
Figure 2. Structural changes in gill cells A. woodiana. Note: A. Tues normal gills, B. Tues gill edema, C. Tues gill hyperplasia, D. Tues
fusion gill lamella, E. Tues gill necrosis.
34
3 (1): 28-35, March 2011
Microanatomical changes in kidney of A. woodiana after
exposure to Cd
Changes in the microanatomical structure of the kidney
of A. woodiana after administration of Cd are shown in
Table 4. From this table, it is known that changes in
cellular structure mikroanatomi kidneys began to occur at a
concentration of 0.5 ppm for 7 days, edema of the tubules
begin to appear and be perfect edema at 30 days and began
to show more than 25% hyperplasia. Perfect hyperplasia is
shown at a concentration of 1 ppm after 14 days of
inspection, then the fusion epithelium of the kidney evenly
shown at concentrations of 5 ppm after 30 days of inspection.
Table 4. Changes in cellular structure of kidney mikroanatomi A.
woodiana after exposure to heavy metals cadmium with
haematoxylin-eosin staining preparation.
Time of
surgery
(Days)
Concentration
(ppm)
0
7
14
30
0.5
7
14
30
1
7
14
30
5
7
14
(dead)
30
10
7
14
(dead)
30
Note: same as Table 9.
Edema
Hyperplasia
+
+++
++++
+++
++++
++++
++++
++++
++++
++++
++++
++++
++
+
++++
++++
+++
++++
++++
+++
++++
++++
Fusion
of
Necrosis
lamella
++
+++
++
+++
+
++++
+++
+++
++++
+++
++++
++++
The kidney cells have shown complete necrosis at a
concentration of 10 ppm after 30 days, while the
concentration of 5 ppm to 10 ppm starting from day 14 and
day of the 30th state of A. woodiana have been many who
experienced the death (LC50), so the kidneys and gills
partially preserved in a freezer with a temperature of -4 ° C
for further examination. In Figure 2A, are shown in the
picture kidney that is still in normal circumstances from the
control A. woodiana.
Situation normal kidney cells and tissues in the control
A. woodiana or the treatment of 0 ppm (Figure 3A), visible
layer between cells in glomeruli and tubules and blood
cells are still visible above and below normal. The metal
accumulation in the kidney ranged from 0.0095 to 0.0242
A
B
ppm. Cd pollution levels, according to FAO (1972) still in
the normal category below the threshold of fishery water
quality (1 ppm), so it can be said that the content of Cd in
the kidneys of A. woodiana the control is still normal. State
accumulation is still at normal levels it may also occur due
to A. woodiana, located diantrara aduktor posterior kidney,
heart and pericardium (Suwignyo et al. 2005). Kidney position
located on the inside and is relatively protected from the
environment cause the accumulation of Cd is relatively
small when compared to the accumulation of Cd in the gills.
In Figure 3B, indicated changes in cell structure that
has undergone kidney mikroanatomi edema in all parts of
the tubules to glomeruli (indicated by black color), and
seems to bleed blood cells due to accumulated Cd logan
continuously. In clinical edema in kidney cells caused by
erasifikasi proteins in the renal tubules in the network, so
that the urine comes out containing excessive protein
(Anonymous 2008). In these conditions, the accumulation
of Cd to the kidney began to be exposed at a concentration
of 0.0200 ppm.
Then on further changes, where the higher Cd exposure
then suffered kidney cell hyperplasia (Figure 3C), which is
marked by the outbreak of the tubules, and the resulting
mixing of intra cell with extra fluid cell, and then also in
the glomerulus looks very black, because the glomerulus
has accumulated more Cd long, which will result in
epitelnya cells will rupture at any time. Then the blood
cells were also seen indicating blackish blood has been
contaminated with Cd. The range of Cd accumulation in
kidney condition hyperplasia began to occur on exposure of
0.0849 ppm.
Highest level of damage to the kidney, necrosis of
kidney cells have shown in Figure 3D, which has entered
the stage of renal cell necrosis seen any broken tubules,
glomeruli also broken so that mixed the cells with extra
fluid cells, and whole blood cells were blackened due to
Acute accumulation of Cd.
The content of Cd in kidney like this occured at the
exposure of 0.0786 ppm.
According to Atdjas (2008) accumulated Cd at the highest
level will cause some kidney disorder that is poisoning the
nephrons of the kidney (nephrotoxicity), proteinuria or
protein in the form contained in the urine, diabetes where
there is the content of glucose in the urine (glikosuria), and
aminoasidiuria or amino acid content in the urine
accompanied by a decline in kidney filtration rate glumerolus.
C
D
Figure 3. Structural changes in renal cell woodiana. Notes: A. Tues normal gills, B. Tues gill edema, C. Tues fusion gill lamella, D.
Tues gill necrosis
FITRIAWAN et al. – Effect of Cd on freshwater mussels A. woodiana
CONCLUSION
There is significant heavy metal accumulation of Cd in
each treatment against the gill and kidney A. woodiana as
evidenced by the Anova test data for 475.3> 60150.3 0.000
and> 0.000 with an average significance level of 5% (P
<0.05). There are structural changes in the kidneys marked
by microanatomical forms which were edema, hyperplasia,
fusion of lamella and necrosis, whereas in the kidney in proved
by the occurrence of edema, hyperplasia and necrosis of the
tubules, glomeruli and mineralization in the blood cells to
bleed.
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(Cd) on some species of crustacean on the beach Batakan and
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Sunarto 2007. Bioindicator of heavy metal pollutant cadmium (Cd) with
microanatomical structural analysis, the efficiency of gill function,
morphology and condition of shells of freshwater mussels (Anodonta
woodiana Lea). [Dissertation]. Airlangga University. Surabaya.
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Susilowati E. 2005. Acute effect of administration of cadmium to milkfish
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Tresnati J, Djawad MI, Bulqys AS. 2007. Kidney damage of ikan pari
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ISSN: 2087-3940 (print)
ISSN: 2087-3956 (electronic)
Vol. 3, No. 1, Pp. 36-43
March 2011
Site suitability to tourist use or management programs South Marsa
Alam, Red Sea, Egypt
1
MOHAMMED SHOKRY AHMED AMMAR1,♥, MOHAMMED HASSANEIN2,
HASHEM ABBAS MADKOUR1, AMRO ABD-ELHAMID ABD-ELGAWAD2
National Institute of Oceanography and Fisheries (NIOF), Suez, P.O. Box 182, Egypt. Tel. (Inst.) 0020 62 3360015. Fax. (Inst.) 0020 62 3360016. ♥
Email: [email protected]
2
Tourism Development Authority, Cairo, Egypt
Manuscript received: 3 February 2011. Revision accepted: 3 March 2011.
Abstract. Ammar MSA, Hassanein M, Madkour HA, Abd-Elgawad AE. 2011. Site suitability to tourist use or management programs
South Marsa Alam, Red Sea, Egypt. Nusantara Bioscience 3: 36-43. Twenty sites in the southern Egyptian Red Sea (Marsa Alam-Ras
Banas sector) were surveyed principally for sensitivity significance throughout the periode 2002-2003. Sensitivity of the study area was
derived from internationally known criteria, the key words of each criterion and a brief description of its use was described. The present
study assigned for the first time a numerical total environmental significance score that gives a full sensitivity significance evaluation for
any site to decide to select either for tourist use or management purposes. However, the results of the study still have the availability to
arrange sites with respect to one criterion or only two or many of the used criteria whichever needed. Sites selected for protection are
categorized as belonging to the following protected area categories: sites 7, 10 (category vi), site 18 (category ib), site 5 (category iv),
sites 16, 17 (category ii). Sites selected for tourist uses are suggested to be classified into 2 categories: first category sites (sites 1, 3, 8,
11, 13, 15) which are recommended as tourist use sites with management of the sensitive resources beside non consumptive recreational
activities like swimming, diving, boating, surfing, wind-surfing, jet skiing, bird watching, snorkelling, etc.; second category sites (sites
2, 4, 6, 9, 12, 14, 19, 20) which are recommended as tourist use sites with both non consumptive and managed consumptive recreational
activities like fishing.
Key words: sensitivity significance, selection criteria, tourist use, management programs, Marsa Alam, Red Sea, Egypt
Abstrak. Ammar MSA, Hassanein M, Abd-Elmegid AE. 2011. Kesesuaian untuk lokasi wisata atau program manajemen Marsa Alam
Selatan, Laut Merah, Mesir. Nusantara Bioscience 3: 36-43. Dua puluh situs di Laut Merah bagian selatan Mesir (sektor Marsa AlamRas Banas) disurvei terutama untuk signifikansi sensitivitas sepanjang periode 2002-2003. Sensitivitas suatu daerah penelitian
merupakan kriteria yang dikenal secara internasional, kata kunci setiap kriteria dan deskripsi singkat tentang penggunaannya dijelaskan.
Penelitian ini dilakukan untuk pertama kalinya berupa skor nilai total signifikansi lingkungan yang memberikan arti evaluasi sensitivitas
penuh untuk situs apapun untuk memutuskan memilih baik untuk tujuan wisata atau tujuan pengelolaan lainnya. Namun, hasil penelitian
ini masih memiliki ketersediaan untuk mengatur situs-situs yang berkaitan dengan satu kriteria, hanya dua atau banyak dari kriteria yang
digunakan mana yang diperlukan. Situs dipilih untuk perlindungan dikategorikan sebagai milik kategori kawasan lindung sebagai
berikut: situs 7, 10 (kategori vi), situs 18 (kategori ib), situs 5 (kategori iv), situs 16, 17 (kategori ii). Situs yang dipilih untuk keperluan
wisatawan disarankan harus diklasifikasikan menjadi dua kategori: situs kategori pertama (situs 1, 3, 8, 11, 13, 15) yang
direkomendasikan sebagai situs menggunakan wisata dengan manajemen sumber daya sensitif di samping kegiatan rekreasi non
konsumtif seperti berenang, menyelam, berperahu, berselancar, wind-surfing, jet ski, mengamati burung, snorkeling, dan lain-lain; situs
kategori kedua (situs 2, 4, 6, 9, 12, 14, 19, 20) yang direkomendasikan sebagai tempat wisata baik kegiatan non konsumtif atau kegiatan
rekreasi non konsumtif yang dikelola seperti memancing.
Kata kunci: signifikansi sensitivitas, kriteria seleksi, kegunaan wisata, program manajemen, Marsa Alam Selatan, Laut Merah
INTRODUCTION
South Marsa Alam’s diverse coastal and marine
environments are valuable community resource which may
be good sites providing recreation and pleasure for visitors
and tourists or scientific materials for scientists to do
monitoring and conservation programs. There is no getting
around the fact that tourism is huge, already categorized as
the world’s largest industry and will continue to be the
dominant developing force in the 21st century (Hill 1998).
As environmental conservation and protection is critically
important in some sites, sustainable tourism is critically
important as well since it may provide source of finance for
parks and conservation, serve as an economic justification
for park protection, offer local people economically sound
and sustainable alternatives to natural resource depletion or
destruction, promote conservation and build support with
commercial constituencies (Hawkins 1998).
Tourist uses includes a diversity of activities that take
place in both coastal zone and coastal waters (Watson et al.
2000), which involve the development of tourism
capacities (hotels, resorts, second homes, restaurants, etc.)
AMMAR et al. – Tourist and management of South Marsa Alam, Egypt
and support infrastructures (ports, marinas, fishing, diving
shops and other facilities). Coastal recreation activities
include two main types: consumptive and non-consumptive
ones: Activities such as fishing, shell fishing and shell
collection, etc. belong to the consumptive recreational uses
while the non consumptive activities include swimming,
diving, boating, surfing, wind-surfing, jet skiing, bird
watching, snorkelling, etc (Porter and Bright 2003). Tourist
uses is based on a unique resource combination at the
interface of land and sea offering amenities such as water,
beaches, scenic beauty, rich terrestrial and marine
biodiversity, diversified cultural and historic heritage,
healthy food and good infrastructure.
Management programs are the programs that are used
for preserving an area to provide lasting protection for part
or all of the natural marine environments therein. IUCN
(1994) defined the protected area as an area of land and/or
sea especially dedicated to the protection and maintenance
of biological diversity, and of natural and associated
cultural resources, and managed through legal or other
effective means. To help improve understanding and
promote awareness of protected area purposes, IUCN has
developed a six category system of protected areas
identified by their primary management objective (IUCN
1994) as follows:
I. Strict Nature Reserve/Wilderness Area: Protected area
managed mainly for science or wilderness protection.
Ia. Strict Nature Reserve: Protected area managed mainly
for science.
Ib. Wilderness Area: Protected area managed mainly for
wilderness protection.
II. National Park: Protected area managed mainly for
ecosystem protection and recreation.
III. Natural Monument: Protected area managed mainly for
conservation of specific natural features.
IV. Habitat/Species Management Area: Protected area
managed mainly for conservation through management
intervention.
V. Protected Landscape/Seascape: Protected area managed
mainly for landscape/seascape conservation and
recreation.
VI. Managed Resource Protected Area: Protected area
managed mainly for the sustainable use of natural
ecosystem
Carrying capacity is important to discuss on dealing
with coastal sustainable tourism. The term "carrying
capacity" is the number of organisms the resources of a
given area can support over a given time period (MPA
NEWS 2004). Adapted to tourism management, it has a
similar meaning: the number of people who can use a given
area without an unacceptable alteration in the physical
environment. Carrying capacity can differ from site to site.
Dixon et al. (1994), on analyzing coral cover, they
estimated that the diver carrying capacity threshold for the
Bonaire Marine Park is between 4000 and 6000 dives per
site per year. Surveying the percent of damaged coral
colonies in the Red Sea Ras Mohammed National Park,
Hawkins and Roberts (1997) suggest 5000 to 6000 dives
per site per year in the absence of a site specific data.
Sampling a suite of invertebrates (hard corals, soft corals,
37
sea fans, branching hydrocorals, and erect sponges),
Chadwick-Furman (1996) found the threshold for diving
sites in the US Virgin Islands to be only 500 dives per site
per year and attributed this significantly lower estimate to
the fragility of the various reef organisms in the study area.
However, effective diver education programs can allow
coral reef managers to increase carrying capacities (Medio
et al. 1997). Mooring buoys and the management of the
number of vessels using mooring buoys with respect to
time and location are other effective tools coral reef
managers use in reducing the anchor and diver damage to
coral reefs.
The purpose of this study is not to replace existing
criteria with a new set, but to use existing frameworks for
site selection to classify south Marsa Alam sites either for
tourist use or management programs in order to assign sites
either to EEAA (Egyptian Environmental Affairs Agency)
for management purposes or to TDA (Tourism
Development Authority) for tourist uses. It is also aimed to
develop a total numerical value of sensitivity significance
(by scoring and summing techniques) that can be used for
site selection (tourist use or management programs), then
using single criteria scoring for a particular management or
tourist use.
MATERIALS AND METHODS
Twenty sites in the southern Egyptian Red Sea
(between Marsa Alam and Ras Banas) were surveyed
principally for sensitivity significance. The survey was
conducted throughout the periode 2002-2003. The sites
were determined by fixing more or less equal distances
between them, however determining the position of sites
was done during the preliminary survey. The area of study
is shown in Figure 1.
The ecological survey was performed using Scuba
diving. For corals and other benthic fauna and flora, the
transect line method applied by Rogers et al. (1983) was
used by using a 30 m long tape for surveying the percent
cover. The intercepted lengths of every individual coral and
any other benthic organism or habitat were measured; these
lengths are then used to calculate the percent cover using
the formula:
% cover = (intercepted length/transect length) * 100
Three transects were used per depth zone and the
average was calculated for all transects.
For fishes, the stationary fish census applied by
Bohnsack and Bannerot (1986) was used by using a 50 m
long transect for the survey. Transects were laid parallel to
the shore at 4 m depth in the deep reefs or just above the
reef patch in case of the patchy reefs. The survey was
basically done at 4m depth since it is the area of maximum
fish abundance.
Sensitivity significance of the study area is derived
from internationally known criteria, however the key words
of each criterion and a brief description of its use can be
described as follow:
Diversity (Ratcliffe 1977; IEEM 2006): large numbers
of species, particularly when represented by large popu-
38
3 (1): 36-43, March 2011
lations are to be valued. A high species diversity is usually
also reflected by a high diversity of different communities
which show variation in environmental conditions.
Rarity (Tubbs and Blackwood 1971; Wittig et al. 1983;
Edwards-Jones et al. 2000): Applied to habitats or species
where areas are limited, population numbers low or the
habitat or species limited in distribution.
Fragility (Ratcliffe 1977; IEEM 2006): Habitats or
species vulnerable to disturbance and loss because of small
area, low population or reliance on a single key resource.
Ecological functions (IEEM 2006): Loss of ecological
function of the physical conditions can be measured by
calculating the area of vegetation that is removed or the
area of nearshore habitat that is covered by the pier
structure.
Typicalness (Fandiño 1996; Edwards-Jones et al.
2000): A measure of how well a site reflects all the habitats
that are expected to occur in that geographical region.The
more representative a site is of a region, the better.
Naturalness (Ratcliffe 1977; IEEM 2006): Habitats
largely unmodified by human activity (e.g. salt marsh,
blanket bog).
Scientific value (Wright 1977, Edwards-Jones et al.
2000): The degree of interest of a natural area in terms of
current or potential research. It may also be related to the
extent to which a site has been used for past research. Sites
with good histories (e.g., description of ecosystems’
dynamics in the past 50 years) are more valuable to science
because they enhance our understanding of ecology
Environmental
significance
(IEEM
2006):
Significance of the site to the environment where that
significance is global, natural or local
Scenic value (Ratcliffe 1977): The combination of
landforms and habitats is identified as having high scenic
value in the context of surrounding landscape
Size (Ratcliffe 1977; IEEM 2006): In general, nature
conservation value increases with size. Large sites in
general contain more species and larger populations of
animals and plants than small ones. Chance extinction of
species, either as a result of natural or man-made factors, is
reduced if a species is present in large numbers.
10, so 0.2% rare biota or habitats = an estimated score of 2
(0.2*10) and so on.
Fragility: Each 1% fragile habitats (nesting, feeding,
breeding), relative to the total cover, was given an optimal
score of 10, so each 0.3% fragile habitats = an estimated
score of 3 (0.3*10) and so on.
Ecological function: Each 6.66% vital ecological
function (vegetation or habitats not removed by physical
conditions) was assigned a score of 1 (6.66/6.66), thus a
vital ecological function of 26.64% will have an estimated
score of 26.64/6.66 = an estimated score of 4 and so on.
Typicalness: A site representing 80% of the number of
the characteristic ecosystems of a geographical area was
assigned a score of 10% (80/8), thus a site having 24%
characteristic ecosystems will have an estimated score of
24/8=3% and so on.
Naturalness: A 10% virgin area (with no human
caused alteration) was assigned a score of 1 (=10/10), thus
a 30% virgin area has an estimated score of 30/10=3 and a
virgin area of 50% has an estimated score of 50/10=5 and
so on.
Scientific value: A site used for scientific research for
the past 10 years was assigned a score of 1 (=10/10), thus a
site used for the past 30 years will have an estimated score
of 3 (=30/10), a site used for the past 50 years will have an
estimated score of 5 (=50/10) and so on.
Environmental significance: Global significance was
assigned a score of 3, each of national and local
significance was given a score of 1.
Scenic value: Scenic value of the landscape depends on
the value of the following dimensions: 1-visual dimension
2-geology 3-topography 4-soils 5-ecology 6-landscape
history
7-Anthropology
8-architecture
9-culture
associations 10-public places. A site that fulfil the scenic
value with respect to those 10 items was assigned a score
of 5 (=10/2), thus a site that fulfil 4 items will have an
estimated score of 4/2=2, a site that fulfil 2 items will have
an estimated score of 2/2=1 and so on.
Size: Each 5000m2 habitats was assigned a score of 1
(=5000/5000), so a size of 10000m2 will have an estimated
score of 2 (10000/5000) and so on.
Estimating sensitivity significance (developed by the
author)
An optimal sensitivity score (the optimal score) was
supposed for each criterion; this was the score at which the
site could be optimal. In addition, an estimated score was
assigned to each crierion depending on how much the site
meets the conditions of the optimal score, then all
sensitivity scores for each site were summed to get the total
sensitivity significance. Methods of how values have been
assigned to each site per each criterion is described as
follows (developed by the author) (Table 1).
Diversity: Diversity value of 1 (according to ShannonWiener 1948 formula) was assigned a sensitivity
significance score of 5, so .diverrsity value of 1.2 =
estimated score of 6 (1.2*5) and so on.
Rarity: Each 1% of rare biota, relative to the total
abundance, was assigned a sensitivity significance score of
List of sites and their positions
Site 1. Marsa Nakry: 24o55`35.476”N, 34o57`40.993”E
Site 2. between Marsa Nakry and Gabal Dorry:
24o54`36.428”N, 34o58`25.453”E
Site 3. 1 km south of Gabal Dorry: 24o47`33.942”N, 34o59`
14.139”E
Site 4. South Host Mark: 24o 47`33.942”N, 35o01`58.197”E
Site 5. Northern Sharmel Fokairy:
Transect 1: 24o45`16.192”N, 35o03`55.792”E
Transect 2: 24o45`22.126”N, 35o03`50.218” E
Site 6. Southern Sharmel Fokairy: 24o38`20”N, 35o04`51” E
Site 7. Sha’b North Ras Baghdadi:24o40`25``N,35 o05`38``E
Site 8. Northern Ras Baghdadi: 24o40`05.900”N,
35o05`52.625”E
Site 9. Southern Ras Baghdadi: 24o39`16.800”N,
35o05`54.200”E
Site 10. North Sharmel Loly: 24o36`50.460”N,
35o06`59.248”E
AMMAR et al. – Tourist and management of South Marsa Alam, Egypt
Site 11. Southern Sharmel Loly: 24o36`39.2666”N,
35o07`08.795”E
Site 12. North Hankourab: 24o34`49.624”N, 35o08`40.185”E
Site13. South Hankourab: 24o33`23.20”N, 35o09`02.405”E
Site 14. North Ummel Abas: 24o30`44.200”N,
35o08`16.927”E
Site 15. Middle Ummel Abas: 24o30`46.024”N,
35o08`16.300”E
Site 16. South Ummel Abas: 24o30`24.642”N,
35o08`31.717”E
Site 17. Wadi El-Mahara: 24o24`27.674”N, 35o13` 41.471” E
Site 18. a mangroove area: 24o16`32.400”N, 35o3`15.815”E
Site 19. South Hamata city: 24o 16` 32.400” N, 35o23`
15.815” E
Site 20: Lahmy; South El-Gharabawy: 24o12`09.494”N,
35o25`37.744”E
39
RESULTS AND DISCUSSION
Site priorities for management and protection
Dealing with the total assigned value of sensitivity
significance and considering sensitivity significance score
≥ 50 to be suitable for management purposes, the following
site priorities are suggested for management purposes: sites
10, 7, 18, 17, 5 and 16 having significant scores of 86, 77,
73, 61, 57 and 54 respectively. However, dealing with each
criterion separately, site 10 has first priority for managing
diversity and rarity; site 18 for fragility; sites 7, 10, 18 for
ecological functions, scientific value, and environmental
significance; sites 7, 10 for typicalness and size; site 10 for
naturalness; site 18 for scenic value. Moreover, if we used
many few of the used criteria, we'll have different site
priorities according to the criteria selected for comparison.
1
23
45
67
8
9
10
11
12
13
14
15
16
17
18
19
20
Figure 1. Location of the study area South Marsa Alam (from Marsa Alam to Ras Banas) on the Egyptian Red Sea.
40
3 (1): 36-43, March 2011
Also, choosing a higher number of criteria used for
comparison gives rise to shefting the priority into the site
that is appropriate to most of the used criteria.
Site priorities for tourist uses
Acoording to the total assigned sensitivity
significance and considering a sensitivity
significance score < 50 to be suitable for
touristic use, 16 sites were selected are
suggested for touristc uses. As many of these
sites have some sensitive resources, these sites
are suggested to be divided into two categories:
first category sites includes sites with some
sensitive resources and with 30 ≤ sensitivity
significance < 50, second category sites include
sites without sensitive resources and with
sensitivity significance < 30. First category sites
are sites 1, 3, 8, 11, 13, 15. Second category
sites are sites 2, 4, 6, 9, 12, 14, 19, 20. Sensitive
habitats for first category sites are rarity for site
1, diversity and rarity for site 3, fragility for site
8, diversity for site 11 and typicalness for site
13.
Table 1. Environmental sensitivity of each of the studied sites
Zone
Site 1
Site 2
Site 3
Site 4
Site 5
Site 6
Site 7
Site 8
Site description
Site 1 (Marsa Nakry) is characterized by a
95% degraded reef flat, 38% dead corals with
increase in the hydrocoral Millepora dichotoma
at 1-5m zone. However, one threatened manta
ray (Taenura lymma) was observed. Site 2 is
very poor and far from the shore with increased
algae, sands and dead corals. Site 3 has 45%
dead corals and one threatened stingray
(Taenura lymma). Site 4 is dominated with
algae while site 5 is characterized by two main
ecosystems: a coral reef ecosystem and a
seagrass ecosystem, 38.5% live corals, 66.5%
dead corals and one threatened threatened
Manta Ray (Himantura uarnak). Site 6 has the
shoreline heavily condensed with quite a lot
amount of plastic bags, glass and plastic bottles,
wood pieces, steel pieces, robes, old shoes,
small and big canes with a very poor marine
life. Site 7 has 65% live corals, 14% dead corals
and one endangered reptile, (the green turtle
Chelonia mydas. Site 8 has 20% live corals and
55% dead corals, site 9 has 46.5% live corals
and 50% dead corals while site 10 has 91% live
corals and 5% dead corals. Sites 11 and 12 have
a poor marine life except some algae and spots
of corals while site 13 is a clean sandy beach
with few small coral patches having 47% dead
corals and 35% live corals. Site 14 is mostly
sand with few patches of algae, sea grasses and
a well developed reef. Site 15 has a seagrass
patch, a small reef patch suffering from old
dynamite fishing and a lot of deep crab niches
on the shoreline while site 16 has a fringing
reef, a patch reef and a barrier reef with a
seagrass bed in between. Site 17 has a a
degraded reef flat, with 40% live corals, 30% dead corals
with juveniles of the threatened organ pipe coral Tubipora
musica attaching rocks, dead corals and rubble on the reef
crest, fishes were mostly of large sizes. while site 18 has
heavily condensed mangrove trees on both land and water
Site 9
Site 10
Site 11
Site 12
Site 13
Site 14
Site 15
Site 16
Site 17
Site 18
Site 19
Site 20
Div
(15)
1
5
1.2
6
1.6
8
0.4
2
1.2
6
1
5
2.4
12
1.4
7
1
5
3
15
1.4
7
0.6
3
1
5
0.6
3
1.2
6
1.6
8
1.4
7
2.6
13
1.6
8
0.8
4
Rar
(15)
1
10
0.2
2
1
10
0
0
0.9
9
0.2
2
1
10
0
0
0
0
1.4
14
0.4
4
0
0
0.6
6
0.1
1
0.3
3
0.3
3
1.3
13
0.4
4
0
0
0
0
Frag
(15)
0.3
3
0.2
2
0.3
3
0
0
1
10
0.2
2
0.9
9
0.9
9
0
0
1
10
0.4
4
0
0
0.5
5
0.3
3
0.9
9
1
10
0.9
9
1.3
13
0
0
0
0
EcFu
(15)
26.64
4
13.32
2
19.98
3
6.66
1
59.94
9
6.66
1
86.58
13
39.96
6
33.3
5
86.58
13
26.64
4
0
0
39.96
6
19.98
3
59.94
9
79.92
12
66.6
10
86.58
13
0
0
0
0
Typ
(10)
24
3
24
3
24
3
0
0
64
8
0
0
80
10
48
6
32
4
80
10
24
3
16
2
40
5
24
3
24
3
48
6
48
6
64
8
24
3
16
2
Nat
(10)
30
3
30
3
30
3
0
0
50
5
30
3
70
7
30
3
20
2
80
8
30
3
30
3
30
3
10
1
30
3
20
2
40
4
60
6
40
4
30
3
SciVa
(5)
30
3
20
2
20
2
10
1
30
3
20
2
40
4
30
3
10
1
40
4
30
3
20
2
20
2
10
1
20
2
30
3
30
3
40
4
40
4
20
2
EnSi SceVa Size
(5)
(5)
(5)
4
3
2
2
4
2
2
4
4
2
2
4
4
2
2
1
4
2
2
3
2
1
1
2
6
4
3
5
4
3
2
3
2
1
1
3
6
4
3
5
4
3
2
3
6
2
3
3
4
2
2
2
2
1
1
1
4
2
2
2
6
3
3
4
4
3
2
4
8
4
4
4
4
2
2
3
2
2
1
2
Tot
(100)
38
28
40
9
57
19
77
42
22
86
36
18
38
18
41
54
61
73
26
16
Note: Div = diversity, Rar = rarity, Frag = fragility, EcFu = Ecological function, Typ =
typicalness, Nat = naturalness, SciVa = scientific value, EnSi = environmental
significance, SceVa = scenic value, Tot = total. Site 2 = between Marsa Nakry and
Dorry. Values in parenthesis are the optimum score for each criterion. Diversity: upper
value in the table is the Shannon estimate of diversity, lower value is the estimated
score. Rarity: upper value in the table is the percent rare biota or habitats, lower value
is the estimated score. Fragility: upper value is the percent fragile habitats, lower value
is the estimated score. Ecological function: upper value is the percent non removed
vegetation or habitats, lower value is the estimated score. Typicalness: upper value is
the percent of characteristic ecosystems of a geographical area, lower value is the
estimated score. Naturalness: upper value is the percent of area of no human caused
alteration, lower value is the estimated score. Scientific value: upper value is the
number of past years the site has been used for scientific researches, lower value is the
estimated score. Environmental significance: values in the table are the estimated
scores. Scenic value: upper value is the number of items the site fulfil for scenic value,
lower value is the estimated score. Size: values in the table are the estimated scores.
AMMAR et al. – Tourist and management of South Marsa Alam, Egypt
beside having seagrasses (10% of the bottom cover). Site
19 is characterized by a dirty shoreline full of plastic bags,
robs, bottles and canes with a very poor reef while site 20
is a typical example of sandy beach having also a trace of
an old reef completely degraded and buried with sand.
Approaching a total mathematical sensitivity
significance score
Evaluation of sensitivity significance criteria in the
previous studies dealt just phonetically with each criterion
separately like for example Ratcliffe (1977), IEEM (2006)
for evaluation of diversity as high, medium or low, fragility
as reversible or irreversible, naturalness as virgin, semivirgin or altered, size as large, medium or small. Other
criteria were phonetically evaluated like Tubbs and
Blackwood for evaluation of rarity; IEEM (2006) for
ecological functions; Fandiño (1996) and Edwards-Jones et
al. (2000) for typicalness; Wright (1977) and EdwardsJones et al. (2000) for scientific value; IEEM (2006) for
environmental significance; Ratcliffe (1977) for scenic
value. Such phonetic evaluation can only deal with each
criterion separately making it difficult to compare several
sites for a group of criteria together, in turn making it
difficult to arrange those group of sites according to their
importance with respect to several criteria. The present
study solved that problem by assigning for the first time a
numerical score for each criterion (explained in the
material and methods section), then summing all
mathematical scores to give a total sensitivity significance
score. However, the study still has the availability to
arrange the sites with respect to one criterion or only two or
many of the used criteria whichever needed according to
the management purpose. Although Croom and Crosby
(1998) mentioned that scoring and summing techniques
was used to minimize the personal bias, he used scoring
and summing techniques with respect to only one separate
criterion e.g. rarity. Approaching a total sensitivity
significance score in the present study is important to select
a site that is much appropriate with most of the used
criteria. Salm and Clark (1984) and Ray and Legates
(1998) expected that extremely complicated scoring and
summing techniques may seem the most objective and
defensible way to choose a priority site. They further
related the reason of using a simple assessment system to
the fact that it is easier to use, requires fewer resources and
can be evaluated by a diverse group of individuals with
varying levels of expertise.
Site priorities for management purposes
Since priorities for site selection with respect to a single
criterion differ from those given on using another criterion
and from those given on using the total sensitivity
significance; it is important, after selecting sites for
management purposes, to use the appropriate criterion for
selecting the appropriate site for the appropriate
management. Parkes (1990) favoured the rating of
individual assets, but differed in how multiple values at a
site should be reconciled. He suggested that, where a site
has several assets of varying levels of biological
significance, the site rating should be based on the value of
41
the dominant asset at the site, or the majority of assets at
the site. Selection criteria can be used to order candidate
sites according to priority in the selection process (Nilsson
1998). However, the present study has been directed
mainly to solve the struggle between EEAA (Egyptian
Environmental Affairs Agency) and TDA (Tourist
Development Authority) for attaining as many sites as
possible to EEAA for management purposes or to TDA for
tourist uses. Therefore, it was important to think in
developing a numerical total environmental significance
score by which we can decide either to assign the site for
EEAA or for TDA. Latimer (2009) stated that the use of
precise numerical criteria, or indices for the evaluation of
size, diversity or rarity could provide a guideline reference
scale, he further mentioned that professional judgement is
also important. According to the purpose of the study and
considering a total sensitivity significance ≥50 to be
significant and appropriate for assigning the site for
management (protection) purposes, priorities of site
selection assigned for management purposes are site 10,
site 7, site 18, site 17, site 5 and site 16, other sites are
assigned for touristic uses.
Categorization, carrying capacity and management
objectives of sites selected for management purposes
Although sites 7 and 10 have high sensitivity
significance with respect to all criteria, they are
recommended as managed resource protected areas
(category VI) since they contain fishing communities and
fishing activities. It is important to sustain fishery resources
by restricting fishing activities seasonally or temporarily to
let the areas recover. Areas managed to sustain fisheries are
very rarely promoted to MPAs, but there are exceptions
like the fish habitat reserves in Australia. Site 18 having the
highest sensitivity significance with respect to fragility and
ecological functions, and being inhabited with mangrove
trees, is recommended as wilderness area (category Ib)
which is managed mainly for wilderness protection. Sites 7,
10 and 18 having fragile habitats should have a diver
carrying capacity threshold of 500 dives per site per year
according to Chadwick-Furman (1996). However, site 5
has considerable sensitivity significance with respect to
fragility and ecological functions, being inhabited with the
fragile seagrasses, it is recommended as habitat/species
management area (protected area, category IV). Similar to
sites 7, 10, 18; site 5 should have a diver carrying capacity
of 500 dives per site per year. Sites 16 and 17 though
having considerable sensitivity significance with respect to
diversity, rarity, fragility, ecological functions and
typicalness, they are recommended as national park
(protected areas, category II) since they have a significant
size which will increase their diver carrying capacity so as
to tolerate recreation. According to Dixon et al. (1994) in
Bonaire Marine Park and Hawkins and Roberts (1997) in
Ras Mohammed National Park, sites 16 and 17 should have
a diver carrying capacity of 4000-6000 dives per site per
year. A matrix of management objectives in the sites
assigned as protected areas are explained (Table 2)
according to IUCN (1994).
42
3 (1): 36-43, March 2011
Table 2. Management objectives of sites selected for management purposes
Sites 7, 10
Site 18
Site 5
Category VI
Category Ib
Category IV
Scientific research
3
3
2
Wilderness protection
2
1
3
Preservation of species and genetic diversity (biodiversity)
1
2
1
Maintenance of environmental services
1
1
1
Protection of specific natural / cultural features
3
–
3
Tourism and recreation
3
2
3
Education
3
–
2
Sustainable use of resources from natural ecosystems
1
3
2
Maintenance of cultural/traditional attributes
2
–
–
Note: 1 = Primary objective; 2 = Secondary objective; 3 = Potentially applicable objective; – = not applicable.
Management objective
Site priorities for tourist uses
Sites classified as first category sites (sites 1, 3, 8, 11,
13 and 15) are recommended as tourist use sites with
management of the sensitive resources and non
consumptive recreational activities like swimming, diving,
boating, surfing, wind-surfing, jet skiing, bird watching,
snorkelling, etc. Locations of recreational activities could
have a carrying capacity of up to 6000 dives per site per
year (Roberts 1997) while in the sensitive locations, it
should not exceed 500 dives per site per year (ChadwickFurman 1996). However, effective diver education
programs can allow coral reef managers to increase
carrying capacities (Medio et al. 1997), also mooring buoys
and the management of the number of vessels using
mooring buoys with respect to time and location are other
effective tools coral reef managers use in reducing the
anchor and diver damage to coral reefs. Management of
sensitive habitats in first category of tourist use sites
includes protection of rarity for sites 1, diversity and rarity
for site 3, fragility for site 8, diversity for site 11 and
typicalness for site 13. Second category sites (sites 2, 4, 6,
9, 12, 14, 19 and 20) are recommended as tourist use sites
with non consumptive and managed consumptive
recreational activities like fishing. Diver carrying capacity
of these sites could approach 6000 dives per site per year.
Site 4 having the lowest sensitivity significance and most
minimum values with respect to every sensitivity criterion
is suggested to allocate a part of it for building an artificial
reef to restore the damaged ones (Ammar 2009a).
Site description
Damaged reef flat in site 1 is due to the absence of reef
access points to deep water. Ammar (2009b) indicated the
importance of reef access points in his assessment of some
coral reef sites along the Gulf of Aqaba, Egypt. Increased
algae and sands in site 2 with increased dead corals agree
with Pearson (1981) and Nezali et al. (1998) that algae are
among the most important factors which can influence
coral recolonization. The high percentage cover of the
hydrocoral Millepora dichotoma at 1-5m depth in Marsa
Nakry as well as in other sites having that species, agrees
with the finding of Ammar (2004) that, Millepora sp. (a
hydrocoral) prefers high illumination and has a strong
skeletal density to tolerate strong waves. The relatively low
sensitivity significance in spite of the presence of the
Sites 16, 17
Category II
2
2
1
1
2
1
2
3
–
threatened species (the blue spotted stingray Taenura
lymma) in sites 1 and 3, indicates the importance of using a
particular criterion when dealing with a particular
management purpose. The green turtle Chelonia mydas
found in site 7 is categorized as a taxon having an
observed, estimated, inferred or suspected reduction of at
least 80% over the last 10 years or three generations,
whichever is the longer (IUCN 2002). The lower recorded
amount of dead corals in site 10 (Sharm El Loly) though it
is highly used by fishing boats, is due to the fact that these
boats anchor on the inlet terminal, away from the reef and
go to open water through the middle of the inlet. Reporting
juveniles of the vulnerable organ pipe coral Tubipora
musica in site 17 (Wadi El-Mahara) is the reason of
increased sensitivity significance with respect to rarity in
that site. Ammar (2005) categorized the organ pipe coral
Tubipora musica as vulnerable according to IUCN (2001),
as there is an estimated population size reduction of ≥ 50%
over the last 10 years, based on the index of abundance and
the decline in area of occupancy. Site 18 having a
mangrove ecosystem, a seagrass ecosystem and a coral reef
ecosystem integrating together helped to increase most of
the selection criteria, in turn increasing the overall
sensitivity significance. Broody (1998) stated that selection
criteria help to provide a rational basis for choosing among
potential sites.
CONCLUSIONS
The present study approached for the first time a
numerical total sensitivity significance score for each site
to select a site that is much appropriate with most of the
used criteria. This is important to classify a group of sites
to be suitable either for tourist use or management
purposes. Since priorities for site selection differ from one
sensitivity criterion to the other and from the total
sensitivity significance, it is important, after selecting a site
for management (using the total sensitivity significance), to
specify the appropriate criterion for deciding the
appropriate management purpose per site. Sites selected for
management (protection) purposes are categorized as
belonging to the following protected area categories: sites
7, 10 (category vi), site 18 (category ib), site 5 (category
iv), sites 16, 17 (category ii). Sites selected for tourist uses
are classified into 2 categories: 1- First category sites (sites
AMMAR et al. – Tourist and management of South Marsa Alam, Egypt
1, 3, 8, 11, 13, 15) which are recommended as tourist use
sites with management of the sensitive resources and non
consumptive recreational activities like swimming, diving,
boating, surfing, wind-surfing, jet skiing, bird watching,
snorkelling, etc. 2- Second category sites (sites 2, 4, 6, 9,
12, 14, 19, 20) which are recommended as tourist use sites
with non consumptive and managed consumptive
recreational activities like fishing.
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ISSN: 2087-3940 (print)
ISSN: 2087-3956 (electronic)
Vol. 3, No. 1, Pp.: 44-58
March 2011
Review: Natural products from Genus Selaginella (Selaginellaceae)
AHMAD DWI SETYAWAN♥
Department of Biology, Faculty of Mathematics and Natural Sciences, Sebelas Maret University, Surakarta 57126. Jl. Ir. Sutami 36A Surakarta 57126,
Tel./fax. +62-271-663375, email: [email protected]
Manuscript received: 28 Augustus 2010. Revision accepted: 4 October 2010.
Abstract. Setyawan AD. 2011. Natural products from Genus Selaginella (Selaginellaceae). Nusantara Bioscience 3: 44-58. Selaginella
is a potent medicinal-stuff, which contains diverse of natural products such as alkaloid, phenolic (flavonoid), and terpenoid. This species
is traditionally used to cure several diseases especially for wound, after childbirth, and menstrual disorder. Biflavonoid, a dimeric form
of flavonoids, is the most valuable natural products of Selaginella, which constituted at least 13 compounds, namely amentoflavone,
2',8''-biapigenin, delicaflavone, ginkgetin, heveaflavone, hinokiflavone, isocryptomerin, kayaflavone, ochnaflavone, podocarpusflavone
A, robustaflavone, sumaflavone, and taiwaniaflavone. Ecologically, plants use biflavonoid to response environmental condition such as
defense against pests, diseases, herbivory, and competitions; while human medically use biflavonoid especially for antioxidant, antiinflammatory, and anti carcinogenic. Selaginella also contains valuable disaccharide, namely trehalose that has long been known for
protecting from desiccation and allows surviving severe environmental stress. The compound has very prospects as molecular stabilizer
in the industries based bioresources.
Key words: natural products, biflavonoid, trehalose, Selaginella.
Abstrak. Setyawan AD. 2011. Bahan alam dari Genus Selaginella (Selaginellaceae). Nusantara Bioscience 3: 44-58. Selaginella adalah
bahan baku obat yang potensial, yang mengandung beragam metabolit sekunder seperti alkaloid, fenolik (flavonoid), dan terpenoid.
Spesies ini secara tradisional digunakan untuk menyembuhkan beberapa penyakit terutama untuk luka, nifas, dan gangguan haid.
Biflavonoid, suatu bentuk dimer dari flavonoid, adalah salah satu produk alam yang paling berharga dari Selaginella, yang meliputi
sekurang-kurangnya 13 senyawa, yaitu amentoflavone, 2',8''-biapigenin, delicaflavone, ginkgetin, heveaflavone, hinokiflavone,
isocryptomerin, kayaflavone, ochnaflavone, podocarpusflavone A, robustaflavone, sumaflavone, dan taiwaniaflavone. Secara ekologis,
tumbuhan menggunakan biflavonoid untuk merespon kondisi lingkungan seperti pertahanan terhadap hama, penyakit, herbivora, dan
kompetisi, sedangkan manusia menggunakan biflavonoid secara medis terutama untuk antioksidan, anti-inflamasi, dan anti
karsinogenik. Selaginella juga mengandung trehalosa suatu disakarida yang telah lama dikenal untuk melindungi dari pengeringan dan
memungkinkan bertahan terhadap tekanan lingkungan hidup yang keras. Senyawa ini sangat berpotensi sebagai stabilizer molekul
dalam industri berbasis sumberdaya hayati.
Kata kunci: produk alami, biflavonoid, trehalosa, Selaginella.
INTRODUCTION
Medicinal plant is plant containing substance which can
be used for the medication or become precursor of drug
synthesis (Sofowora 1982). Medicinal plant has been
source of human health since ancient time, whereas about
60-75% of world populations require plant for carrying
health (Farnsworth 1994; Joy et al. 1998; Harvey 2000).
Plants and microbes are the main source of natural products
(Hayashi et al. 1997; Armaka et al. 1999; Lin et al.
1999a,b; Basso et al. 2005), and consistently become main
source of the newest drugs (Harvey 2000). The drug
development from natural sources are based on the
bioassay-guided isolation of natural products, due to the
traditional uses of local plants (ethnobotanical and
ethanopharmacological applications) (Atta-ur-Rahman and
Choudhary 1999).
Traditional medication system by using plant medicines
has been developed during thousands of year especially by
Chinese (Wu-Hsing) and India (Ayurveda, Unani and
Siddha) (Peter 2004; Ahmad et al. 2006), while the most
advanced, widespread and oldest traditional medication
system in Nusantara or Malay Archipelago (Malesia) is
jamu which developed by Javanese. Jamu contains several
recipes that compiled by about 30 plant species. Relief at
Borobudur temple about making jamu indicates that jamu
has been widely recognized since early 9th century (Jansen
1993). This system has been documented for centuries in
many serat and primbon, Javanese literary (Soedibjo 1989,
1990; Sutarjadi 1990); and spreaded by trading, migration
and expansion of several kingdoms such as Mataram Hindu
(Sanjaya), Srivijaya (Saylendra) and Majapahit.
Selaginella Pal. Beauv. (Selaginellaceae Reichb.) has
been used as complementary and alternative medicines in
several traditional medication. This matter is traditionally
used to cure wound, after childbirth, menstrual disorder,
skin disease, headache, fever, infection of exhalation
channel, infection of urethra, cirrhosis, cancer, rheumatism,
bone fracture, etc. Part to be used is entire plant, though
only referred as leaves or herbs (Setyawan 2009; Setyawan
SETYAWAN – Natural products of Selaginella
and Darusman 2008). The usage can be conducted single or
combination, fresh or dried, direct eaten or boiled
(Dalimartha 1999; Wijayakusuma 2004). This plant has
sweet taste and gives warm effect on the body (Bensky et
al. 2004). The use of Selaginella as medicinal matter is
occurred in the entire world. The largest usage is conducted
by Chinese, especially for S. tamariscina, S. doederleinii,
S. moellendorffii, S. uncinata, and S. involvens (Lin et al.
1991; Chang et al. 2000; Wang and Wang 2001).
Unfortunately, Selaginella is rarely exploited in Nusantara.
Traditional jamu of Java use more cultivated spices and
rhizomes than wild herbs or grasses.
Plant medicinal properties are contributed by natural
products or secondary metabolites, such as phenolic
(flavonoid), alkaloid, terpenoid, as well as non protein
amino acid (Smith 1976). Natural products are chemical
compounds or substances produced by a living organism
and found in nature that usually has a biological activity for
use in pharmaceutical drug discovery and drug design
(Cutler and Cutler 2000). In this following discourse, the
authors studied diversity of natural products from
Selaginella, especially biflavonoid and trehalose
compounds; and biological activity of Selaginella’s
bifavonoid in modern medication.
NATURAL PRODUCTS DIVERSITY
Previous phytochemical studies on the constituents of
genus Selaginella leds to the discovery of many
compounds, including biflavonoids, the main secondary
metabolite of Selaginella (Sun et al. 1997; Silva et al. 1995;
Chen et al. 2005b; Lin et al. 1994; 2000). Biflavonoid has
also distributed to Selaginellales, Psilotales, and
Gymnosperms (Seigler 1998), several Bryophytes and
about 15 families of Angiosperms (DNP 1992). The other
compounds are including lignin (White and Towers 1967);
lignan (Lin et al. 1994), lignanoside (Lin et al. 1990; Zheng
et al. 2004, 2008b), alkaloid (Zheng et al. 2004; Lin et al.
1997), selaginellin (Zhang et al. 2007; Cheng et al. 2008),
glycosides (Man and Takahashi 2002; Zhu et al. 2008),
glucosides (Dai et al. 2006; Yuan et al. 2008), Cglycosylflavones (Richardson et al. 1989), etc. Selaginella
species of Java contains alkaloid, phenolic (flavonoid,
tannin, saponin), and terpenoid (triterpene, steroid)
(Chikmawati and Miftahudin. 2008; Chikmawati et al.
2008). Some species of Japan consist of a steroid type
namely ekdisteroid (Takemoto et al. 1967, Hikino et al.
1973; Yen et al. 1974). The diversity and content of other
compound is relatively lower than biflavonoid,
nevertheless they have also certain bioactivities.
Water extracts of S. tamariscina also has several natural
products such as ferulic acid, caffeic acid, vanillic acid,
syringic acid, umbelliferone (Bi et al. 2004b);
tamariscinoside A, tamariscinoside B, adenosine,
guanosine, arbutin (Bi el al. 2004a); tamariscinoside C,
tyrosine, D-mannitol, and shikimic acid (Zheng et al.
2004). The EtOH extract of the whole herbs of S.
tamariscina that fractionated by chloroform and ethyl
acetate contains selaginellin A and selaginellin B (Cheng et
al. 2008). The main constituen of S. tamariscina
45
subsequently is amentoflavone, robustaflavone, bilobetin,
hinokiflavone, isocryptomerin and an apigenin-diglucoside
(Yuan et al. 2008). S. tamariscina has also many sterols
that inhibit the growth of human leukemia HL-60 cells
indicating anti cancer property (Gao et al. 2007). The aerial
parts of S. pulvinata has steroid constituent (Zheng et al.
2007), and several Selaginella has also sterol (Chiu et al.
1988). Steroid compound namely ekdisteroid has been
found in Japanese species of S. deliculata, S. doederleinii,
S. moellendorffii, S. nipponica, S. involvens (= S.
pachystachys), S. stauntoniana (= S. pseudo-involvens), S.
remotifolia var. japonica, S. tamariscina, and S. uncinata
(Takemoto et al. 1967; Hikino et al. 1973; Yen et al. 1974).
Methanolic extract of S. lepidophylla contains 3methylenhydroxy-5-methoxy-2,4-dihydroxy
tetrahydrofurane, which can a slight inhibitory effect on the uterus
contraction (Perez et al. 1994). S. lepidophylla is also
reported contain volatile oils (Andrade-Cetto and Heinrich
2005). The acetone extract of S. sinensis contains
selaginellin A, an unusual flavonoid pigment (Zhang et al.
2007). S. sinensis has a glucoside, namely selaginoside
(Dai et al. 2006), a sesquilignan, namely sinensiol A (Wang
et al. 2007), secolignans, namely styraxlignolide D and
neolloydosin (Feng et al. 2009), and (+)-pinoresinol
(Umezawa 2003a,b). S. uncinata also has chromone
glycosides, namely uncinoside A and uncinoside B (Man
and Takahashi 2002), which shows antiviral activities
against RSV and PIV-3 (Ma et al. 2003). Ethanol extract
of S. uncinata also contains flavonoids that possessing a
benzoic acid substituent (Zheng et al. 2008a).
S. doederleinii contains several phenolic compounds
such as (+)-matairesinol, (-)-lirioresinol A, (-)-lirioresinol
B, (-)-nortracheloside (Lin et al. 1994), and (-)matairesinol, (+)-syringaresinol, (+)-wikstromol, (+)nortrachelogenin (Umezawa 2003a,b). The (-)-matairesinol
has inhibitory activity against cAMP and acts as an
insecticide synergist, while (+)-syringaresinol has cytotoxic
effect (Harborne et al. 1999). S. doederleinii also contains a
glycosidic hordenine (Markham et al. 1992), which
increases hypertension (Lin et al. 1991).
S. caulescens, S. involvens, and S. uncinata contain
about 0.2% silicon, higher than the most of other club
mosses and true ferns (Ma and Takahashi 2002), which
may improve plant tolerant to disease, drought, and metal
toxicities (Epstein 1999; Richmond and Sussman 2003; Ma
2004). S. labordei contains 4'-methylether robustaflavone,
robustaflavone, eriodictyol and amentoflavone (Tan et al
2009). S. apoda yields substantial amounts of 3-O-methylD-galactose (Popper et al. 2001). S. moellendorfii contains
several pyrrolidinoindoline alkaloids (Wang et al. 2009).
Other natural products, beside biflavonoid and trehalose,
also have several molecular properties that can increase
human health and have economical values; and need for
further observation.
Natural products of Selaginella can vary depend on
climate, location, and soil factors; as well as harvesting and
extraction procedure (Nahrstedt and Butterweck 1997); and
also plant species or variety, parts to be extracted and age.
The different species of Selaginella shows different HPLC
fingerprint characteristic. The samples of the similar
46
3 (1): 44-58, March 2011
species, collected in different period, different environment
or different locations shows certain difference in
fingerprints. However, it also generate main fingerprint
peaks, which can be used to evaluate and distinguish the
different species or infra species (Fan et al. 2007).
Phenol
BIFLAVONOID
Selaginella species have a large number of bioactive
compounds, the most important being biflavonoids (Silva
et al. 1995; Lin et al. 1999). Biflavonoids are naturally
occurring compounds that are ubiquitous in all vascular
plants and have many favorable biological and
pharmacological effects (Lee et al. 1996; Baureithel et al.
1997; Lobstein-Guth et al. 1998). One of flavonoid
structure that has high medicinal valuable is biflavonoid; a
dimeric form of flavonoid which formed by binding of two
flavone units or mixture between flavone and flavanon or
aurone (Geiger and Quinn 1976; DNP 1992; Ferreira et al.
2006).
Flavonoid (or flavanoid) is widespread plant natural
products (5-10%); its chemical structure and biological role
are very diverse (Macheix et al. 1990). This compound is
formed by shikimate and phenylpropanoid pathways
(Harborne 1989), with a few alternative biosynthesis
(Robards and Antolovich 1997). Flavonoid is derived from
phenols having basic structure of phenylbenzopiron
(tocopherol) (Middleton et al. 2000); distinguished by 15
carbon skeletons (C6-C3-C6) consisted of one oxygenated
ring and two aromatic rings (Figure 1). Substitution of
chemical group at flavonoid is generally hydroxylation,
methoxylation, methylation and glycosilation (Harborne
1980). Flavonoid is classified diversely; among them are
flavone, flavonone, isoflavone, flavanol, flavanon,
anthocyanin, and chalchone (Porter 1994; Ferreira and
Bekker 1996; Ferreira et al. 1999a,b). More than 6467
flavonoid compounds have been identified and amount of
new discovery is consistently increasing (Harborne and
Baxter 1999). This compound is playing important role in
determining color, favor, aroma, and quality of nutritional
food (Macheix et al. 1990). Flavonoid is mostly monomeric
form, but there is also dimer (biflavonoid), trimer, tetramer,
and polymer (Perruchon 2004).
Biflavonoid (or biflavonil, flavandiol) is a dimeric form
of flavonoid which formed by bonding of two flavone units
or mixture between flavone and flavanon or aurone (Geiger
and Quinn 1976; DNP 1992; Ferreira et al. 2006). Basic
structure of biflavonoid is 2,3-dihydroapigeninil-(I-3’,II3’)-apigenin (Figure 1.). This compound has interflavanil
C-C bond between carbon C-3’ at each flavone group.
There is also some biflavonoid with interflavanil C-O-C
bonding (Bennie et al. 2000, 2001, 2002; Ferreira et al.
2006). Locksley (1973) suggest generic term ‘biflavanoid’
to replace ‘biflavonil’ which is early used. Term
‘biflavanoid’ is assumed more accurate than ‘biflavonoid’
because indicating saturated in nature. Suffix ‘oid’
indicates homogeneous dimeric type, including biflavanon,
biflavon, biflavan, etc. However, term ‘biflavonoid’ is
more regularly used because articulated easier.
B
C
Flavonoid
Biflavonoid
Figure 1. Basic structure of phenol, flavanoid and biflavanoid.
Bicyclic ring system is named A and C rings, while unicyclic ring
is named B ring. The two unit of monomeric biflavonoid is
marked by Roman number I and II. Position number at each
monomer is started from containing oxygen atom ring, position of
C-9 and C-10 indicate unification of them (Rahman et al. 2007; ).
Biflavonoid is found at fruit, vegetable, and other part
of plant. This compound is originally found by Furukawa
in 1929 (Lin et al. 1997) from leaf extract of G. biloba in
form of yellow colored compound, later named ginkgetin
(I-4’, I-7-dimetoxy, II-4’, I-5, II-5, II-7-tetrahydroxy I-3’,
II-8 biflavone) (Baker and Simmonds 1940). Nowdays,
amount of biflavonoid which isolated and characterized
from nature continually increase (Oliveira et al. 2002;
Ariyasena et al. 2004; Chen et al. 2005a), but learning to
bioactivity is still limited. The most observed biflavonoid is
ginkgetin, isoginkgetin, amentoflavone, morelloflavone,
robustaflavone, hinokiflavone, and ochnaflavone. Those
compounds have similar basic structure, i.e. 5,7,4’trihydroxy flavonoid, but differing at nature and position of
flavonoid bond (Rahman et al. 2007).
Biflavonoid has several namenclaturing systems, such
as Loksley, IUPAC, and vernacular name. The first of two
systems is the most systematic, but the most used is
vernacular
name.
Locksley
(1973)
standardize
nomenclature and position number of biflavonil ring
skeleton. Every monomer unit is marked by Roman
numerals I and II that indicate bonding between monomer,
followed by Arabic numerals indicate that bonding
position. The two numeral from two monomer unit
compiled dimeric, than paired with hyphen to show
bonding position of two monomer. Number of substitution
group at monomer unit follow IUPAC system for flavone.
In Locksley system, amentoflavone named I-4’, II-4’, I-5,
II-5, I-7, II-7-hexahydroxy I-3’, II-8 biflavone, while
hinokiflavone which its flavone unit bonded with an
oxygen is named by II-4’, I-5, II-5, I-7, II-7-pentahydroxy
I-4’-O-II-6 biflavone. This system is intuitive, logical, and
depicts the chemical structure. In IUPAC, amentoflavone is
named by 8-5-(5,7-dihydroxy-4-oxo-4H-chromen-2-il)-2hydroxyphenyl-5,7-dihydroxy2-(4-hydroxy-phenyl)chromen-4-on, while hinokiflavone is 6-4-(5,7-dihydroxy4-oxo-4H-chromen-2-il)-phenoxy5,7-dihydroxy-2-(4-
SETYAWAN – Natural products of Selaginella
hydroxyphenyl)- chromen-4-on. Basic difference between
two systems is reference of structural skeleton. Locksley
use flavanoid structure, while IUPAC use chromen
structure that more complex (Rahman et al. 2007). The
above two nomenclature is rarely used because its
complication. Vernacular name that given by each inventor
is often used because simpler and easier, though it is not
systematic and does not depict chemical structure, such as
amentoflavone, hinokiflavone, ginkgetin, etc.
In vivo biosynthesis of flavonoid in nature is relatively
mysterious, but there are some approaches by in vitro to
explain biosynthesis. According to Rahman et al. (2007)
there are nine pathways of biflavonoid synthesis, namely:
(i) Ullmann coupling halogenated flavones; (ii) synthesis of
biflavones via 1,1’-biphenyls; (iii) metal catalyzed cross
coupling of flavones; (iv) Wessely-Moser rearrangements;
(v) phenol oxidative coupling of flavones; (vi) Ullmann
condensation with flavone salts; (vii) nucleophilic
substitution; (viii) dehydrogenation of biflavanones into
biflavones; and (ix) dehydrogenation of biflavone into
biflavanone.
In East Asia, biflavonoid is usually produced from leaf
of Ginkgo biloba which main constituent is ginkgetin
(Krauze-Baranowska and Wiart 2002; Dubber 2005). In
sub Sahara-Africa, it is especially produced from seed of
Garcinia cola which main constituent is kolaviron (Iwu
and Igboko 1982; Iwu 1985, 1999; Iwu et al. 1987, 1990;
Braide 1989, 1993; Han et al. 2006; Farombi et al. 2005;
Adaramoye and Medeiros 2009). The biflavanones are the
most dominant in the most Garcinia species (Waterman
and Hussain 1983), pericarp of Javanese mangosteen (G.
mangoestana) contains amentoflavone and other flavonoids
(ADS 2008, data not be shown). In Europe, biflavonoid is
commonly produced from herbs of Hypericum perforatum
which main constituent is amentoflavone (Berghofer and
Holzl 1987, 1989; Nahrstedt and Butterweck 1997; Borlis
et al. 1998; Tolonen 2003; Kraus 2005). Selaginella has
potent as source of biflavonoid, which can yield various
biflavonoid compounds depending on species. It has
cosmopolitanly distributed and able to cultivate almost all
the words depending on species.
DIVERSITY OF BIFLAVONOID
Selaginella is one of the potential medicinal plants as a
source biflavonoid in Nusantara, where 200 of the 700-750
species from the entire world are found (Setyawan 2008).
A total of 13 biflavonoid compounds have been isolated
from Selaginella, including amentoflavone (3',8”biapigenin), 2',8''-biapigenin, delicaflavone, ginkgetin,
heveaflavone, hinokiflavone, isocryptomerin, kayaflavone,
ochnaflavone, podocarpusflavone A, robustaflavone,
sumaflavone, and taiwaniaflavone (Figure 2). In Setyawan
and Darusman (2008) mentioned that the number is only 12
biflavonoid compounds. Some biflavonoid are easily found
at various species of Selaginella, but the other is only
found at certain species. Amentoflavone and ginkgetin is
biflavonoid compound of the most Selaginella, while
sumaflavone is only reported from S. tamariscina (Yang et
47
al. 2006; Lee et al. 2008) and delicaflavone is only reported
from S. delcatula (Andersen and Markham 2006). At least
11 species of Selaginella have been tested by
amentoflavone content (Sun et al. 2006). There are also
biflavonoid which is rarely found at Selaginella but it is
commonly found at other species. Preliminary study shows
that amentoflavone is found in high content (> 20%) at two
of about 35 species of Malesian Selaginella, namely S.
subalpina and S. involvens (ADS 2008, data not be shown).
In Selaginella, taiwaniaflavone is only reported from S.
tamariscina (Pokharel et al. 2006), while this is also found
at other plant such as Taiwania cryptomerioides (Kamil et
al. 1981).
Selaginella is generally extracted from whole plant,
though it is only conceived as herbs or leaves. Extraction
can be conducted by various solvent, i.e. polar, semi-polar
and non polar. For example: boiling in water, extraction by
using methanol, ethanol, buthanol, ethyl acetate,
chloroform, or extraction by using solvent mixture such as
alcohol-water, ethanol-ethyl acetate, and ethanolchloroform. Methanol and ethanol are the most solvent
used for biflavonoid extraction. Solvent types and
extraction procedure can influence obtaining chemical
structure and bioactivity of extract. Disease which is most
treated by Selaginella extract is cancer. Besides,
Selaginella extract also has many other usefulness, namely
antioxidant, anti-inflammatory, antimicrobial (virus,
bacterium, fungi, and protozoa), anti UV irradiation, anti
allergy, vasorelaxation, anti diabetes, blood pressure
stability, anti hemorrhagic, and antinociceptive.
Biflavonoid needs evaluation for its medical and nutritional
value (Harborne and Williams 2000). Selaginella contains
various biflavonoid with difference medical properties
(Table 2).
Amentoflavone. Amentoflavone, the most common
biflavonoid of Selaginella, has various biological and
pharmacological effects, including antioxidant (Mora et al.
1990; Cholbi et al. 1991; Shi et al. 2008), anti cancer (Silva
et al. 1995; Lee et al. 1996; Lin et al. 2000;
Guruvayoorappan and Kuttan 2007), anti-inflammatory
(Gambhir et al. 1978; Baureithel et al. 1997; Gil et al.
1997; Kim et al. 1998; Lin et al.. 2000; Woo et al.. 2005),
antimicrobial (Woo et al. 2005; Jung et al. 2007), antivirus
such as influenza (A, B), hepatitis (B), human
immunodeficiency virus (HIV-1), herpes (HSV-1, HSV-2),
herpes zoster (VZV), measles (Lin et al. 1998, 1999a,b,
2002; Flavin et al. 2001, 2002), and respiratory syncytial
virus (RSV) (Lin et al. 1999a,b; Ma et al. 2001),
vasorelaxation (Kang et al. 2004), anti-urcerogenic
(Gambhir et al. 1987), anti stomachic-ache (Kim et al.
1998), anti depressant (Baureithel et al. 1997), anxiolytic
(Cassels et al. 1998, 1999), analgesic (Silva et al. 2001),
and anti-angiogenesis agent (Lee et al. 2009c).
2',8''-biapigenin. 2',8''-biapigenin is an anticancer,
which inhibit transactivation of iNOS gene and
cyclooxigenase-2 (COX-2) through inactivate nuclear
factor-κB (NF-κB) and prevent translocation of p65 (Chen
et al. 2005b; Woo et al. 2006); and anti-inflammatory
(Grijalva et al. 2004; Woo et al. 2005 2006; Pokharel et al.
2006).
48
3 (1): 44-58, March 2011
OH
OH
HO
OH
O
HO
HO
OH
2’
8”
O
O
OH
HO
O
Delicaflavone
2’,8”-biapigenin
Amentoflavone
(3',8”-biapigenin)
OCH3
OH
OCH3
OH
CH3O
HO
CH3O
Hinokiflavon
CH3O
OH
O
Hinokiflavone
OH
Heveaflavone
Ginkgetin
OCH3
OCH3
CH3O
HO
CH3O
Ochnaflavone
Isocryptomerin
Kayaflavone
OCH3
HO
HO
Podocarpusflavone A
Sumaflavone
OH
OH
HO
OH
OH
Robustaflavone
HO
Taiwaniaflavone
Figure 2. Structure of biflavonoid from Selaginella, namely: amentoflavone, 2',8''-biapigenin, delicaflavone, ginkgetin, heveaflavone,
hinokiflavone, Isocryptomerin, kayaflavone, ochnaflavone, podocarpusflavone A, robustaflavone, sumaflavone, and taiwaniaflavone.
SETYAWAN – Natural products of Selaginella
Delicaflavone. Its bioactivity is not observed yet from
Selaginella.
Ginkgetin. This compound is the second most studied
biflavonoid of Selaginella beside amentoflavone. It has
several properties including antioxidant (Su et al. 2000;
Sah et al. 2005; Shi et al. 2008), anti-inflammatory
(Grijalva et al. 2004; Woo et al. 2005, 2006; Pokharel et al.
2006), anti viral such as herpes and cytomegalovirus
(Hayashi et al. 1992); anti protozoan such as Trypanosoma
cruzi (Weniger et al. 2006); anti cancer (Sun et al. 1997;
Kim and Park 2002; Yang et al. 2007), such as such as
ovarian adenocarcinoma (OVCAR-3), cervical carcinoma
(HeLa) and foreskin fibroblast (FS-5) (Su et al. 2000).
Ginkgetin is the strongest biflavonoid that inhibit cancer
(Kim and Park 2002). Besides, this matter increase activity
of neuroprotective against cytotoxic stress, and has potent
for curing neurodegenerative disease such as stroke and
Alzheimer (Kang et al. 2004; Han et al. 2006). Ginkgetin
can also replace caffeine in food-stuff and medicines
without generating addiction (Zhou 2002).
Heveaflavone. Heveaflavone has cytotoxic activity
against cancer cell of murine L 929 (Lin et al. 1994).
Hinokiflavone. Hinokiflavone has antioxidant, antiviral
and anti protozoan effect. This matter assists cell growth
and protect from free radical cased by hydrogen peroxide
(H2O2) (Sah et al. 2005). It also inhibit sialidase influenza
virus (Yamada et al. 2007; Miki et al. 2008); has high
resistance to HIV-1 by in vivo and to polymerase HIV-1
RTASE by in vitro (Lin et al. 1997). Lin et al. (1998,
1999a,b, 2002) and Flavin et al. (2001, 2002) is patenting
antiviral effect of hinokiflavone and others to influenza
virus (A, B), hepatitis (B), human immunodeficiency virus
(HIV-1), herpes (HSV-1, HSV-2), herpes zoster (VZV),
and measles. It has antiprotozoan activity by in
vitro against
Plasmodium
falciparum, Leishmania
donovani and Trypanosoma sp. (Kunert et al. 2008).
Isocryptomerin. Isocryptomerin has anti cancer
property as well as anti-inflammatory, immunosuppressant
and analgesic (Kang et al. 1998, 2001). It has cytotoxic
activity against various cancer cells (Silva et al. 1995),
including P-388 and HT-29 (Chen et al. 2005b). It has
antibacterial activity against Gram-positive and Gramnegative bacteria (Lee et al. 2009b); and also has antifungal
properties, which can depolarize fungal plasma membrane
of Candida albicans (Lee et al. 2009a).
Kayaflavone. Kayaflavone has moderately anti cancer
property (Sun et al. 1997; Yang et al. 2007) and
antioxidant, such as depleting H2O2 (Su et al. 2000).
Ochnaflavone. Ochnaflavone derivatives may have
antioxidant activity that inhibits expression of gene COX-2
at colon cancer cell (Chen et al. 2005b).
Podocarpusflavone A. It has moderately anti cancer
(Sun et al. 1997; Yang et al. 2007) and antioxidant
properties (Su et al. 2000; Shi et al. 2008).
Robustaflavone. Robustaflavone has anti cancer and
anti virus properties. This matter significantly cytotoxic to
various cancer cells (Silva et al. 1995) and significantly
inhibits tumor cell of Raji and Calu-1 (Lin et al. 2000),
cancer cell of P-388 and HT-29 (Chen et al. 2005b). It has
also antiviral properties, which indicates high resistance to
49
polymerase HIV-1 RTASE by in vitro (Lin et al. 1997) and
also influenza virus (A, B), hepatitis (B), human
immunodeficiency virus (HIV-1), herpes (HSV-1, HSV-2),
herpes zoster (VZV), and measles (Lin et al. 1998, 1999a,b
2002; Flavin et al. 2001, 2002).
Sumaflavone. Sumaflavone has anti-inflammatory
property that able to inhibit production of NO, by mean
blocking lipopolysaccharide formation that induces iNOS
gene expression (Yang et al. 2006). It can also significantly
inhibit ability of UV irradiation to induce matrix
metalloprotease-1 and -2 (MMP-1 and -2) activities at
fibroblast of primary human skin (Lee et al. 2008).
Taiwaniaflavone. It has anti-inflammatory, such as
induce iNOS and COX-2 at macrophage of RAW 264.7
(Pokharel, et al. 2006).
MOLECULAR BIOACTIVITIES
Selaginella is traditionally treated to cure several
disease depending on species, such as cancer or tumor
(uterus, nasopharyngeal, lung, etc), wound, after childbirth,
menstrual disorder, female reproduction disease, expulsion
of the placenta, tonic (for after childbirth, increase body
endurance, anti ageing, etc), pneumonia, respiratory
infection, exhalation channel infection, inflamed lung,
cough, tonsil inflammation, asthma, urethra infection,
bladder infection, kidney stone, cirrhosis, hepatitis, cystisis,
bone fracture, rheumatism, headache, fever, skin diseases,
eczema, depurative, vertigo, toothache, backache, blood
purify, blood coagulation, amenorrhea, hemorrhage
(resulting menstrual/obstetrical hemorrhage, stomachic,
pile or prolepses of the rectum), diarrhea, stomach-ache,
sedative, gastric ulcers, gastro-intestinal disorder, rectocele,
itches, ringworm, bacterial disease, bellyache, neutralize
poison caused by snakebite or sprained, bruise, paralysis,
fatigue, dyspepsia, spleen disease (diabetic mellitus),
emmenagogue, diuretic, and to refuse black magic
(Martinez 1961; Bouquet et al. 1971; Dixit and Bhatt 1974;
Ahmad and Raji 1992; Bourdy and Lee et al. 1992; Bourdy
and Walter 1992; Nasution 1993; Lin et al. 1994; Kambuou
1996; Caniago and Siebert 1998; Sequiera 1998;
Dalimartha 1999; Mathew et al. 1999; Abu-Shamah et al.
2000; van Andel 2000; Uluk et al. 2001; Harada et al.
2002; Man and Takahashi 2002; Warintek 2002; Winter
and Jansen 2003; ARCBC 2004; Batugal et al. 2004; de
Almeida-Agra and Dantas 2004; DeFilipps et al. 2004;
Wijayakusuma 2004; Mamedov 2005; Khare 2007; Pam.
2008; Setyawan and Darusman 2008) (Table 1). This plant
has sweet taste, and gives warm effect on the body (Bensky
et al. 2004).
Plants ecologically use biflavonoid to response
environmental condition such as defense against pests,
diseases, herbivory, and competitions; while human
medically use as antioxidant, anti-inflammatory, anti
cancer, anti allergy, antimicrobial, antifungal, antibacterial,
antivirus, antiprotozoan, protection to UV irradiation,
vasorelaxation (vasorelaxant), heart strengthener, anti
hypertension, anti blood coagulation, and influence enzyme
50
3 (1): 44-58, March 2011
metabolism (Havsteen 1983, 2002; Kandaswami and
Middleton 1993, 1994; Lale et al. 1996; Bisnack et al.
2001; Duarte et al. 2001; Kromhout 2001; Kang et al.
2004; Moltke et al. 2004; Arts and Hollman 2005; Martens
and Mithofer 2005; Yamaguchi et al. 2005). The
antioxidant, anti cancer and anti-inflammatory are the most
important bioactivities of this secondary metabolite.
Selaginella is known possess various molecular
bioactivities depending on species, but only a few species
has been detailed observe in the advanced research. Several
species that also distributed in Nusantara are observed,
such as S. tamariscina, S. doederleinii, S. involvens, S.
moellendorffii, S. uncinata, and S. willdenowii; while the
most distributed Selaginella in Nusantara namely S. plana
has not been investigated yet (Table 2).
S. tamariscina is the most powerful and the most useful
plant Selaginella in the world. This herb is widely used as
anti cancer, antioxidant and anti-inflammatory; and also
used as anti UV irradiation, anti allergy, vasorelaxation,
anti diabetic, immunosuppressant, analgesic, neuro
protectant, antibacteria, antifungal, and possess estrogenic
activity. As anti cancer, S. tamariscina can decrease
expression of MMP-2 and -9, urokinase plasminogen
activator, and inhibits growth of metastasis A549 cell and
Lewis lung carcinoma (LLC) (Yang et al. 2007); inhibits
proliferation of mesangial cell which activated by IL-1β
and IL-6 (Kuo et al. 1998); inhibits leukemia cancer cell of
HL-60 cell (Lee et al. 1999); induces expression of tumor
suppressor gene of p53 (Lee et al. 1996); degrades
leukemia cancer cell of U937 (Lee et al. 1996; Yang et al.
2007); reduces proliferation nucleus antigen cell from
stomach epithelium (Lee et al. 1999); chemopreventive for
gastric cancer (Lee et al. 1999); induces apoptosis of cancer
cell trough DNA fragmentation and nucleus clotting (Ahn
et al. 2006); and induces breast cancer apoptosis through
blockade of fatty acid synthesis (Lee et al. 2009c). This
property is mostly given by amentoflavone and
isocryptomerin (Kang et al. 1998, 2001; Lee et al. 2009c),
while ginkgetin is also acted as anti cancer to OVCAR-3
(Sun et al. 1997). As antioxidant, amentoflavone from S.
tamariscina inhibits production of NO, which inactivates
NF-κB, while sumaflavone blocks lipopolysaccharide
formation that induces iNOS gene expression (Yang et al.
2006).
As
anti-inflammatory,
amentoflavone,
taiwaniaflavone and ginkgetin from S. tamariscina inhibit
inflammation that induce iNOS and COX-2 at macrophage
RAW 264.7 which stimulated by lipopolysaccharide
(Grijalva et al. 2004; Woo et al. 2005; Pokharel et al.
2006). Amentoflavone inhibits activity of phospholipase
Cγ1 (Lee et al. 1996); phospholipase A-2 (PLA-2) and
COX-2 (Kim et al. 1998), while 2',8''-biapigenin inhibits
transactivation of iNOS gene and COX-2 through
inactivate NF-κB and prevent translocation of p65 (Woo et
al. 2006).
Amentoflavone from S. tamariscina inhibits fungi (Junk
et al. 2006), anti influenza and resist to HSV-1 and -2
(Rayne and Mazza 2007); hinokiflavone inhibits sialidase
influenza virus (Yamada et al. 2007; Miki et al. 2008) and
resists to HIV-1 (Lin et al. 1997); robustaflavone and
hinokiflavone resist to polymerase HIV-1 RTASE (Lin et
al. 1997); ginkgetin inhibits herpes and cytomegalovirus
(Hayashi et al. 1992), by degrading protein synthesis of
virus and depress gene transcription (Middleton et al.
2000). Isocryptomerin from S. tamariscina shows potent
antibacterial activity against Gram-positive and Gramnegative (Lee et al. 2009b). Amentoflavone from S.
tamariscina inhibits several pathogenic fungi (Woo et al.
2005; Jung et al. 2007). Isocryptomerin from S.
tamariscina can depolarize fungal plasma membrane of C.
albicans (Lee et al. 2009a).
S. tamariscina is effective ingredient to prevent and
cure acute brain degenerative disease, such as stroke and
dementia (Han et al. 2006). Capability to prevent brain
damage is especially given by amentoflavone (Kang et al.
1998). S. tamariscina can elastic vascular smooth muscle
through endothelium related to nitric oxide (NO) activity
(Yin et al. 2005). Amentoflavone from S. tamariscina
induces relaxation of phenylephrin which responsible to
aorta contraction (Kang et al. 2004; Yin et al. 2005). S.
tamariscina containing sumaflavone and amentoflavone
inhibit ability of UV irradiation to induce MMP-1 and -2 at
fibroblast (Lee et al. 2008). S. tamariscina reduces
histamine from peritoneal mast cell causing allergic
reaction (Dai et al. 2005). S. tamariscina decreases sugar
blood and lipid peroxide, and also increases insulin
concentration (Miao et al. 1996). Amentoflavone from S.
tamariscina inhibits activity of tyrosine phosphatase 1B to
maintain type-2 diabetic and obesity (Na et al. 2007).
S. articulate is treated as anti hemorrhagic. Water
extract of this matter can moderately neutralize
hemorrhagic effect and inhibits proteolysis of casein by
venom (Otero et al. 2000; Winter and Jansen 2003).
S. bryopteris acts as antioxidant, anti-inflammatory,
antiprotozoan, anti UV-irradiation and anti spasmodic.
Water extract of S. bryopteris increases endurance to
oxidative stress; and assists cell growth and protects from
free radical stress caused by H2O2 (Sah et al. 2005). S.
bryopteris is treated as anti-inflammatory and cures veneral
disease (Agarwal and Singh 1999). Amentoflavone and
hinokiflavone from S. bryopteris have antiprotozoan
activity
against P.
falciparum, L.
donovani
and
Trypanosoma sp (Kunert et al. 2008). Water extract of S.
bryopteris also significantly reduces potent cell dying
caused by UV irradiation (Sah et al. 2005), while ethanolic
extract can cure stomachic (Pandey et al. 1993).
S. delicatula acts as anti cancer and antioxidant. Water
extract of S. delicatula has antioxidant characteristic and
degrades blood cholesterol (Gayathri et al. 2005). Extract
of S. delicatula that contained by robustaflavone and
amentoflavone or its derivatives is cytotoxic against cancer
cell of P-388, HT-29 (Chen et al. 2005b), Raji, Calu-1,
lymphoma and leukemia (Lin et al. 2000)
S. doederleinii is usually treated as anti cancer, but also
acts as antiviral and anti-inflammatory. Water extract of S.
doederleinii has antimutagenic against both picrolonic
acid- and benzo[α]pyrene-induced mutation to cancer cell
(Lee and Lin 1988). Ethanolic extract of S. doederleinii
that is amentoflavone and heveaflavone has cytotoxic
activity against cancer cell of murine L 929 (Lin et al.
1994). Extract of S. doederleinii also has cytotoxic against
SETYAWAN – Natural products of Selaginella
the three human cancer cell lines, HCT, NCI-H358, and
K562 (Lee et al. 2008), and has anti mutagenic effect
against cholangiocarcinoma cancer, but my cause bone
marrow depression (Pan et al. 2001). Amentoflavone from
S. doederleinii has potent as antiviral and antiinflammatory agents (Lin et al. 2000). However, hordenine
that isolated from S. doederleinii increases hypertension
(Lin et al. 1991).
S. involvens has characteristics as antioxidant, antiinflammatory and anti bacteria. Extract of S. involvens can
inhibit production and effect of free radicals of NO and
expression of iNOS/IL-1β (Joo et al. 2007). Water extract
of S. involvens has significantly antioxidant effect to lipid
peroxides (EC50 = 2 ug/mL). This extract is non toxic and
degrades blood cholesterol (Gayathri et al. 2005). Water
extract of S. involvens kills the various Leptospira strains,
which causes infectious of leptospirosis diseases (Wang et
al. 1963). Extract of S. involvens depresses activity of
Propionibacterium acnes (> 100 ug/mL), which responses
to acne inflammation; although has no antibiotic property
(Joo et al. 2007). Beside, water extract of S. involvens may
have analgesic activity (ECMM 1997; Ko et al. 2007).
S. labordei indicates antioxidant, anti cancer, and anti
virus characteristics. S. labordei can inhibit activity of
xanthine oxidase (XOD) and lipoxygenase (LOX), and
absorb free radical (Chen et al. 2005b; Tan et al. 2009). It
also down-regulate COX-2 gene expression in human
colon adenocarcinoma CaCo-2 cells (Chen et al. 2005b).
Robustaflavone of S. labordei can inhibit hepatitis B virus
(Tan et al. 2009)
S. lepidophylla has hypoglycemic property (AndradeCetto and Heinrich 2005); while non-biflavonoid
compound from methanolic extract of S. lepidophylla, 3methylenhydroxy-5-methoxy-2,4-dihydroxy
tetrahydrofuran, has moderate resistance to uterus
contraction (Perez et al. 1994).
S. moellendorffii contains antioxidant and anti cancer
properties. Ethyl acetate extract of S. moellendorffii
contains amentoflavone, hinokiflavone, podocarpusflavone
A, and ginkgetin that has antioxidant properties (Shi et al.
2008). Ginkgetin that extracted by ethanol or ethyl acetate
from S. moellendorffii can inhibit cancer cell growth of
OVCAR-3, HeLa, and FS-5 (Sun et al. 1997; Su et al.
2000). It also act as anti-metastasis at lung cancer cell of
A549 and LLC (Yang et al. 2007); and apoptosis resulting
caspase activation by H2O2 (Su et al. 2000); while
amentoflavone and its derivatives, kayaflavone, and
podocarpusflavone A, have no this bioactivity (Sun et al.
1997).
S. pallescens has moderately antimicrobials and anti
spasmodic activities. S. pallescens contains an endophytic
Fusarium sp. that produce pentaketide anti fungal agent,
CR377 (Brady and Clardy 2000). Chloroform-methanolic
extract of S. pallescens can inhibit spontaneously
contraction of ileum muscle (Rojas et al. 1999).
S. rupestris contains amentoflavone which has
antispasmodic effect to ileum; and strengthening heart in
case of normodinamic and hypodinamic (Chakravarthy et
al. 1981)
51
S. sinensis contains amentoflavone which has antiviral
actifity against RSV (Ma et al. 2001)
S. uncinata has activity as anti virus but generated by
non biflavonoid compounds. S. uncinata has chromone
glycosides, namely uncinoside A and B (Man and
Takahashi 2002), which showed antiviral activities against
RSV and PIV-3 (Ma et al. 2003).
S. willdenowii contains isocryptomerin and derivatives
of amentoflavone and robustaflavone which significantly
cytotoxic against various cancer cell (Silva et al. 1995).
TREHALOSE
Trehalose is formed by α,α-1,1-glycosidic linkage of
two low energy hexose moieties (Paiva and Panek 1996;
Elbein et al 2003; Grennan 2007). This matter is a unique
simple sugar which non reactive, very stable, colorless,
odor-free, non-reducing disaccharide, and capable to
protect biomolecules against environmental stress
(Schiraldi et al. 2002). Therefore, this compound is a
natural product, although not as commonly secondary
metabolites of natural products. It works as osmoprotectant
during desiccation stress (Adams et al. 1990); such as
compatible solute in the stabilization of biological
structures under abiotic stress (Garg et al. 2002); serves as
a source of energy and carbon (Elbein et al 2003;
Schluepmann et al. 2003); serves as signaling molecule to
control certain metabolic pathways (Muller et al. 2001;
Elbein et al 2003; Avonce et al 2005); protects proteins and
cellular membranes from inactivation or denaturation
caused by harsh environmental stress, such as desiccation,
dehydration (drought), thermal heat, cold freezing,
oxidation, nutrient starvation, and salt (Avigad 1982;
Elbein et al. 2003; Wu et al. 2006). Trehalose acts as a
global protectant against abiotic stress (Jang et al. 2003).
This matter is proved to be an active stabilizer of enzymes,
proteins, biomasses, pharmaceutical preparations and even
organs for transplantation (Schiraldi et al. 2002), and very
prospects as molecular bio stabilizer in cosmetic, pharmacy
and food (Roser 1991; Kidd and Devorak 1994). These
multiple effects of trehalose on protein stability and folding
suggest promising applications (Singer and Lindquist
1998).
Trehalose has long been known for protecting certain
organisms from desiccation. The accumulation of the
disaccharide trehalose in anhydrobiotic organisms allows
them to survive severe environmental stress (Zentella et al.
1999). Trehalose also promotes survival under extreme
heat conditions, by enabling proteins to retain their native
conformation at elevated temperatures and suppressing the
aggregation of denatured proteins (Singer and Lindquist
1998). Desiccation can reduce the lipid component in
thylakoid membranes (Guschina et al. 2002). However, in
desiccation-tolerant plants, membrane integrity appears not
to be affected during drought-stress. S. lepidophylla retain
their structural organization as intact bilayers (Platt et al.
1994) and often referred as resurrection plant because able
to live on long drought and recovery through rehydration
52
3 (1): 44-58, March 2011
process (Crowe et al. 1992), even when the most water
body (99%) is evaporated (Schiraldi et al. 2002; van Dijck
et al. 2002). Another species, S. tamariscina, can also
remain alive in a desiccated state and resurrect when water
becomes available (Liu et al. 2008). The drought can
change fluorescence and pigmentation, but can not cause
dying (Casper et al. 1993).
Trehalose exists in a wide variety of organisms,
including bacteria, yeast, fungi, insects, invertebrates, and
lower and higher plants (Elbein 1974; Crowe et al. 1984;
Elbein et al. 2003), but rarely find in Angiosperms (Muller
et al. 1995) and does not find in mammals (Teramoto et al
2008), and it is not accumulated to detectable levels in the
most plants (Garg et al. 2002). This sugar plays important
roles in cryptobiosis of Selaginella and other organisms,
which revive with water from a state of suspended
animation induced by desiccation (Teramoto et al 2008).
Trehalose is the major sugar formed in photosynthesis of
Selaginella (White and Towers 1967). Some Selaginella
contains high concentration of trehalose, such as S.
lepidophylla (Adams et al. 1990; Mueller et al. 1995;
Zentella et al. 1995), S. sartorii (Iturriaga et al. 2000), S.
martensii (Roberts and Tovey 1969), S. densa, and S.
wallacei (White and Towers 1967). Trehalose can reach
10-15% of cell dry weight (Grba et al. 1975).
Trehalose is not merely chemical compounds that
responsible to resurrection ability of Selaginella. The
protective effect of trehalose is correlated with a trapping
of the protein in a harmonic potential, even at relatively
high temperature (Cordone et al. 1999). Deeba et al (2009)
suggest that S. bryopteris, one kind of resurrection plants,
has about 250 proteins that expressed in response to
dehydration and rehydration, and involved in transport,
targeting and degradation in the desiccated fronds. Harten
and Eickmeier (1986) suggest that several conservationed
enzymes are beneficial for rapid resumption of metabolic
activity of S. lepidophyla. Furthermore, Eickmeier (1979;
1982) suggests that both organelle- and cytoplasm-directed
protein syntheses are necessary for full photosynthetic
recovery during rehydration of S. lepidophyla.
FUTURE RESEARCH
Research on Selaginella is still widely challenging. In
the most elementary study of plant taxonomy, the high
morphological variation of Selaginella causes several
misidentification of this taxon. In ecology, global warming,
habitat fragmentation and degradation that affected on
sustainability of this resource need to be observed. In
physiology, changes of fluorescens and pigmentation
caused by environmental factor and age need to be
explained. In biochemistry, several natural products are not
exploited yet. One of non-biflavonoid compound from
Selaginella that needs to be further investigated is
trehalose. Molecular study is also required clarifying
certain identity and phylogenetics relationship.
In Indonesia, several authors often misidentify
Selaginella species, especially on popular article. This
matter is often identified as S. doederleinii, including
Javanese wild species. The most authors agree that S.
doederleinii is recognized as non native plant of Indonesia,
which natural distribution is India, Burma, Thailand, Laos,
Cambodia, Vietnam, Malaya, Chinese, Hong Kong,
Taiwan, and Japan (Huang 2006; USDA 2008). Java has no
species of S. doederleinii according to Alston (1935a) and
observation on Selaginella collection of Herbarium
Bogoriense, through several Kalimantan collection is
suspected and has morphological similarity to this species
(ADS 2007, data is not shown). This matter is possibility
caused by referring to Dalimartha (1999), which include S.
doederleinii in Indonesian plant medicines. Harada et al.
(2002) conduct similar misidentification, which cite S.
plana as one of plant medicine in Mount Halimun NP
(nowadays Mount Halimun-Salak NP), but the main picture
presented is S. willdenowii. Field survey indicate that S.
willdenowii is easily found in road side to Cikaniki
Research Station of Mount Halimun-Salak NP, at rice field,
shrubs land, primary and secondary forest, while S. plana is
easier to be found in countrifield at lower height (ADS
2008, personal observation).
Species misidentification impacts on drug properties,
because each species differ chemical constituent. Natural
products content of Selaginella highly vary depending on
species, although does not always congruent with
traditional medical recipes. Sundanese people of Mount
Halimun-Salak NP complementarily or substitutionally
uses several Selaginella for treatment of after childbirth
including S. ornata, S. willdenowii, S. involvens, and S.
intermedia, but for similar recipe Sundanese around Bogor
only uses S. plana (ADS 2008, personal observation).
Morphological diversity at infra specific level, and changes
on pigmentation caused by age, drought and other
environmental factors able to entangle identify base on
morphological characteristics. It needs identification base
on molecular characteristic, such as Korall et al. (1999) and
Korall and Kenrick (2002, 2004). Beside, taxonomy of
Malesian Selaginella needs to revise, because still based on
old literature namely Alderwereld van Rosenburgh
(1915a,b; 1916, 1917, 1918, 1920, 1922) and Alston (1934,
1935a,b; 1937, 1940). In a research brief about the
traditional utilization of Selaginella in Indonesia, Setyawan
(2009) collect at least 40 species of which half are
estimated to new species or new records.
Completely research on variability of biflavonoid
compounds of various Selaginella species with various
solvent have not conducted yet. This matter is only
conducted to certain species, compounds, and solvents.
Natural products of certain plant determine economical
value that required in industrial scale of modern pharmacy.
Species with various low content of natural products less
value than species with restricted high content, because
modern pharmacy exploits natural products at molecular
level. However, this matter is not always become
consideration in traditional medication, because it generally
uses simplicia that can be easily substituted by each others.
In phytochemistry and chemotaxonomy, high variety of
natural products can assist identification, though each has
no high content. However, a very low compound is not
SETYAWAN – Natural products of Selaginella
significantly important for identification, because often
influenced by environmental factors, not merely to genetic
factor. Bioactivity of each biflavonoid also requires to be
observed because nowadays only bioactivity of
amentoflavone and ginkgetin has been completely studied.
HPLC is potent method for analyzing of natural products of
Selaginella (Fan et al. 2007).
Besides, trehalose observation on Selaginella is still
restricted on a few species, and need to be conducted to
amount of other species caused by potent economic value
that can be generated. It can preliminary indicated by
species that incurling leaves in hot weather or drough
condition.
Biflavonoid study of Selaginella is still require attention
such as: (i) the importance of assuring species identity
caused by height morphological variety including by using
molecular method; (ii) the importance of extending
research coverage most of biflavonoid type, species, and
extraction method; and also (iii) the importance of
extending investigation on bioactivity, including nonbiflavonoid compound, which also have high economic
potent such as trehalose.
CONCLUSION
Selaginella is a potent medicinal matter, which mostly
contains phenolic (flavonoid), alkaloid, and terpenoid. This
matter is traditionally used to cure several diseases
especially for wound, after childbirth, and menstrual
disorder. Biflavonoid, a dimeric form of flavonoids, is one
of the most valuable natural products of Selaginella, which
constituted at least 13 compounds, namely amentoflavone,
2',8''-biapigenin, delicaflavone, ginkgetin, heveaflavone,
hinokiflavone, isocryptomerin, kayaflavone, ochnaflavone,
podocarpusflavone A, robustaflavone, sumaflavone, and
taiwaniaflavone. Human medically use biflavonoid
especially for antioxidant, anti-inflammatory, and anti
cancer. Selaginella also contains several natural products,
such as trehalose which valuable for bioindustry.
Selaginella research exhaustively needs to be conducted to
explore all natural products constituents and their
bioactivities.
ACKNOWLEDGEMENTS
I would like to thank Prof. Dr. Keon Wook Kang from
Chosun University, Gwangju, South Korea which delivered
a number of valuable articles on chemistry of biflavonoid. I
also thank Prof. Dr. Umesh R. Desai from Commonwealth
University, Virginia, USA for permitting to cite a very
interesting article on biosynthesis of biflavonoid. Grateful
thank to Prof. Dr. Raphael Ghogomu-Tih from University
of Yaoundé, Cameron for suggesting to write this article in
international language. Sincere thanks are expressed to the
late Dr. Muhammad Ahkam Soebroto from Research
Center for Biotechnology, Indonesian Institute of Science,
53
Cibinong-Bogor and anonymous peer reviewer of this
article for their criticism. I also would like to thank Prof.
Dr. Wasmen Manalu from SEAMEO-BIOTROP Bogor
which had invited me for training in writing scientific
articles.
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Zhou JH. 2002. Herbal caffeine replacement composition and food
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GUIDANCE FOR AUTHORS
NUSANTARA BIOSCIENCES, the ISEA Journal of Biological
Sciences publishes scientific articles, namely original full research and
review in all Biological Sciences, including: Agricultural Sciences,
Anthropology, Applied Biological Sciences, Biochemistry, Natural
Product Biochemistry, Biophysics and Computational Biology, Cell
Biology, Developmental Biology, Ecology, Environmental Sciences,
Evolution, Genetics, Immunology, Medical Sciences, Microbiology,
Neuroscience, Pharmacology, Physiology, Plant Biology, Population
Biology, Psychological and Cognitive Sciences, Sustainability Science,
and Systems Biology. Scientific feedback (short communication) is only
received for manuscript, which criticize published article before.
Manuscripts will be reviewed by managing editor, editorial board and
invited peer review according to their disciplines. The only articles written in
English (U.S. English) and Bahasa Indonesia are accepted for publication.
This journal periodically publishes in March, July and November. In order to
support reduction of global warming and forest degradation, editor prefers
receiving manuscripts via e-mail rather than in hard copy. Manuscript and its
communications can only be addressed to the managing editor; better to
forward to one of the editorial board member for accelerating evaluation. A
letter of statement expressing that the author (s) is responsible for the
original content of manuscript, the result of author(s)’s research and never
been published must be declared. Manuscript of original research should be
written in no more than 25 pages (including tables and figures), each page
contain 700-800 word, or proportional with article in this publication number.
Invited review articles will be accommodated. Avoid expressing idea with
complicated sentence and verbiage, and used efficient and effective sentence.
Manuscript is typed at one side of white paper of A4 (210x297 mm2)
size, in a single column, double space, 12-point Times New Roman font,
with 2 cm distance step aside in all side. Smaller letter size and space can be
applied in presenting table. Word processing program or additional software
can be used, however, it must be PC compatible and Microsoft Word based.
Names of sub-species until phylum should be written in italic, except for
italic sentence. Scientific name (genera, species, author), and cultivar or
strain should be mentioned completely at the first time mentioning it,
especially for taxonomic manuscripts. Name of genera can be shortened after
first mentioning, except generating confusion. Name of author can be
eliminated after first mentioning. For example, Rhizopus oryzae L. UICC
524, hereinafter can be written as R. oryzae UICC 524. Using trivial name
should be avoided, otherwise generating confusion. Mentioning of scientific
name completely can be repeated at Materials and Methods. Biochemical
and chemical nomenclature should follow the order of IUPAC-IUB, while
its translation to Indonesian-English refers to Glossarium Istilah AsingIndonesia (2006). For DNA sequence, it is better used Courier New font.
Symbols of standard chemical and abbreviation of chemistry name can
be applied for common and clear used, for example, completely written
butilic hydroxytoluene to be BHT hereinafter. Metric measurement use IS
denomination, usage other system should follow the value of equivalent with
the denomination of IS first mentioning. Abbreviation set of, like g, mg, mL,
etc. do not follow by dot. Minus index (m-2, L-1, h-1) suggested to be used,
except in things like “per-plant” or “per-plot”. Equation of mathematics
can be written separately. Number one to ten are expressed with words,
except if it relates to measurement, while values above them written in
number, except in early sentence. Fraction should be expressed in decimal.
In text, it should be used “%” rather than “gratuity”.
Title of article should be written in compact, clear, and informative
sentence preferably not more than 20 words (generally 135 characters
including spaces). Name of author(s) should be completely written. Running
title is about five words, refelcting the idea of the manuscript. Name and
institution address should be also completely written with street name and
number (location), zip code, telephone number, facsimile number, and e-mail
address. Manuscript written by a group, author for correspondence along
with address is required. First page of the manuscript is used for writing
above information.
Abstract should not be more than 250 words, written in English, on
page two of the manuscript. Keywords is about five words, covering
scientific and local name (if any), research theme, and special methods
which used. Introduction is about 400-600 words, covering background and
aims of the research. Materials and Methods should emphasize on the
procedures and data analysis. Results and Discussion should be written as a
series of connecting sentences, however, for manuscript with long discussion
should be divided into sub titles. Thorough discussion represents the causal
effect mainly explains for why and how the results of the research were
taken place, and do not only re-express the mentioned results in the form of
sentences. Conclusion should preferably be given at the end of the
discussion. Acknowledgments list and funding sources are expressed in a
brief. Dedications are rarely allowed.
Figures and Tables of maximum of three pages should be clearly
presented. Title of a picture is written down below the picture, while title of a
table is written in the above the table. Colored picture and photo can be
accepted if information in manuscript can lose without those images. Photos
and pictures are preferably presented in a digital file. JPEG format should be
sent in the final (accepted) article. Author could consign any picture or photo
for front cover, although it does not print in the manuscript. There is no
appendix, all data or data analysis are incorporated into Results and
Discussions. For broad data, it can be displayed in website as Supplement.
Citation in manuscript is written in “name and year” system; and is
arranged from oldest to newest and from A to Z. The sentence sourced from
many authors, should be structured based on the year of recently. In citing an
article written by two authors, both of them should be mentioned, however,
for three and more authors only the family (last) name of the first author is
mentioned followed by et al., for example: Saharjo and Nurhayati (2006) or
(Boonkerd 2003a, b, c; Sugiyarto 2004; El-Bana and Nijs 2005; Balagadde et
al. 2008; Webb et al. 2008). Extent citation as shown with word “cit” should
be avoided, and suggested to refer an original reference.
References. APA style in double space is used in the journal reference.
Only published or in-press papers and books may be cited in the reference
list. Unpublished abstracts of papers presented at meetings or references to
"data not shown" are not permitted. References should be cited in alphabetic
order. All authors should be named in the citation (unless there are more than
five). If there are more than five, list the first author's name followed by et al.
Include the full title for each cited article. Authors must translate foreign
language titles into English, with a notation of the original language (except
for Spanish, France, and Germany). For Indonesian manuscript, translation
of Indonesian title to English is not necessary. For correct abbreviations of
journal titles, refer to Chemical Abstracts Service Source Index (CASSI).
Provide inclusive volume, number, and page ranges for journal articles, but
not for book or book chapters.
Journal:
Saharjo BH, Nurhayati AD. 2006. Domination and composition structure
change at hemic peat natural regeneration following burning; a case
study in Pelalawan, Riau Province. Biodiversitas 7: 154-158.
Book:
Rai MK, Carpinella C. 2006. Naturally occurring bioactive compounds.
Elsevier, Amsterdam.
Chapter in book:
Webb CO, Cannon CH, Davies SJ. 2008. Ecological organization, biogeography,
and the phylogenetic structure of rainforest tree communities. In: Carson
W, Schnitzer S (eds) Tropical forest community ecology. WileyBlackwell, New York.
Abstract:
Assaeed AM. 2007. Seed production and dispersal of Rhazya stricta. 50th
annual symposium of the International Association for Vegetation
Science, Swansea, UK, 23-27 July 2007.
Proceeding:
Alikodra HS. 2000. Biodiversity for development of local autonomous
government. In: Setyawan AD, Sutarno (eds) Toward mount Lawu
national park; proceeding of national seminary and workshop on
biodiversity conservation to protect and save germplasm in Java island.
Sebelas Maret University, Surakarta, 17-20 July 2000. [Indonesia]
Thesis, Dissertation:
Sugiyarto. 2004. Soil macro-invertebrates diversity and inter-cropping plants
productivity in agroforestry system based on sengon. [Dissertation].
Brawijaya University, Malang. [Indonesia]
Information from internet:
Balagadde FK, Song H, Ozaki J, Collins CH, Barnet M, Arnold FH, Quake
SR, You L. 2008. A synthetic Escherichia coli predator-prey ecosystem.
Mol Syst Biol 4: 187. www.molecularsystemsbiology.com
Progress of manuscript. Notification of manuscript whether it is accepted
or refused will be notified in about three months since the manuscript
received. Manuscript is refused if the content does not in line with the
journal mission, low quality, inappropriate format, complicated language
style, dishonesty of research authenticity, or no answer of correspondence in
a certain period. Author or first authors at a group manuscript will get one
original copy of journal containing manuscript submitted not more than a
month after publication. Offprint or reprint is only available with special request.
NOTE: Author(s) agree to transfer copy right of published paper to
NUSANTARA BIOSCIENCES, the ISEA Journal of Biological Sciences.
Authors shall no longer be allowed to publish manuscript completely without
publisher permission. Authors or others allowed multiplying article in this
journal as long as not for commercial purposes. For the new invention,
authors suggested to manage its patent before publishing in this journal.
NOTIFICATION: All communications are strongly recommended to be undertaken through email.
| Nus Biosci | vol. 3 | no. 1 | pp. 1‐58 | March 2011 | ISSN 2087‐3940 (PRINT) | ISSN 2087‐3956 (ELECTRONIC) I S E A J o u r n a l o f B i o l o g i c a l S c i e n c e s
Optimization of DNA extraction of physic nut (Jatropha curcas) by selecting the appropriate leaf EDI PRAYITNO, EINSTIVINA NURYANDANI 1‐6 Characterisation of taro (Colocasia esculenta) based on morphological and isozymic patterns markers TRIMANTO, SAJIDAN, SUGIYARTO 7‐14 Study on floristic and plant species diversity of the Lebanon oak site (Quercus libani) in Chenareh, Marivan, Kordestan Province, western Iran HASSAN POURBABAEI, SHIVA ZANDI NAVGRAN 15‐22 Evaluation structural diversity of Carpinus betulus stand in Golestan Province, North of Iran VAHAB SOHRABI, RAMIN RAHMANI, SHAHROKH JABBARI, HADI MOAYERI 23‐27 Microanatomy alteration of gills and kidneys in freshwater mussel (Anodonta woodiana) due to cadmium exposure FUAD FITRIAWAN, SUTARNO, SUNARTO 28‐35 Site suitability to tourist use or management programs South Marsa Alam, Red Sea, Egypt MOHAMMED SHOKRY AHMED AMMAR, MOHAMMED HASSANEIN, HASHEM ABBAS MADKOUR, AMRO ABD‐ELHAMID ABD‐ELGAWAD 36‐43 Review: Natural products from Genus Selaginella (Selaginellaceae) AHMAD DWI SETYAWAN 44‐58 Published three times in one year PRINTED IN INDONESIA ISSN 2087‐3940 (print) ISSN 2087‐3956 (electronic)