Genetic Variation of Vanda foetida J.J.Sm.; a Rare and Endemic Orchid in South Sumatra Based on RAPD Analysis

Abstract

Vanda foetida J.J.Sm., which is an endemic orchid species in Sumatra has taxonomically disappeared for over 100 years since it was described in 1906. The distribution of V. foetida was limited due to over-exploitation of this orchid and changes to land-use. The study of genetic variation of V. foetida in its natural habitat, Mount Dempo and Padiampe, South Sumatra was conducted based on Random Amplified Polymorphic DNA (RAPD) analysis for genetic variation using 8 primers (OPU 3, OPU 5, OPU 6, OPU 7, OPU 12, OPU 13, OPU 14 and OPU 16). Genetic variation in each population was detected by h value (Nei’s genetic diversity) which was 0.1999 and 0.1778 for Mount Dempo and Padiampe, respectively. This value was higher compared to those of other rare orchid species even though it only has a small population. V. foetida originated from two populations forming two main clusters in dendrogram with 67 % (0.67) degree of similarity. The dendrogram indicated that the two populations are connected, and assumed as one large population in the past and were separated from larger population by habitat fragmentation. This species was not genetically in danger and will be able to survive if its natural habitats are remained.

Full text

Journal of Tropical Biology and Conservation 12: 99–112, 2015 ISSN 1823-3902
Received 23 May 2015
Accepted 17 August 2015
Published 15 October 2015
Genetic Variation of Vanda foetida J.J.Sm.; a Rare and
Endemic Orchid in South Sumatra Based on RAPD Analysis
Ridesti Rindyastuti1*, Destario Metusala1, Dian Aruni Kumalawati2 , Budi Setiadi
Daryono2
1 Purwodadi Botanic Garden, Indonesian Institute of Sciences, Jl. Raya Surabaya-
Malang, Km. 65, Purwodadi, Pasuruan, East Java, Indonesia
2 Faculty of Biology, Gadjah Mada University, Jl. Teknika Selatan, Sekip Utara,
Yogyakarta, Indonesia
*Corresponding author: ride17@gmail.com
Abstract
Vanda foetida J.J.Sm., which is an endemic orchid species in Sumatra has
taxonomically disappeared for over 100 years since it was described in 1906. The
distribution of V. foetida was limited due to over-exploitation of this orchid and
changes to land-use. The study of genetic variation of V. foetida in its natural
habitat, Mount Dempo and Padiampe, South Sumatra was conducted based on
Random Amplified Polymorphic DNA (RAPD) analysis for genetic variation using 8
primers (OPU 3, OPU 5, OPU 6, OPU 7, OPU 12, OPU 13, OPU 14 and OPU 16).
Genetic variation in each population was detected by h value ( Nei’s genetic
diversity) which was 0.1999 and 0.1778 for Mount Dempo and Padiampe,
respectively. This value was higher compared to those of other rare orchid species
even though it only has a small population. V. foetida originated from two
populations forming two main clusters in dendrogram with 67 % (0.67) degree of
similarity. The dendrogram indicated that the two populations are connected, and
assumed as one large population in the past and were separated from larger
population by habitat fragmentation. This species was not genetically in danger
and will be able to survive if its natural habitats are remained.
Keywords: orchid, Vanda foetida, genetic variation, RAPD, conservation.
Introduction
Vanda foetida J.J.Sm., based on its first description in 1906, has a very unique
and white-purple flower. It was described as the mixing of V. dearei (sepal and
petal was widely opened) and V. tricolor (which has bilobed and midlobe
down-recurved labellum). The flower has an unpleasant smell, thus this orchid
has the species name “foetida”, which means unpleasant smell. Since
described in 1906, this species has not been seen in its habitat and the world’s
orchid collections (Comber, 2001; Hartini & Puspitaningtyas, 2005).
Research Article

100 Rindyastuti et al.
Furthermore, the holotype of V. foetida (BO) was only a dry herbarium of two
bunches of flowers and a leaf and wet collection of a leaf and two bunches of
flowers. The isotype (L) is only a bunch of dry flowers. Thus, it is assumed this
species was scientifically lost for over 100 years, being rare and endemic
(Metusala et al., 2010).
The population of V. foetida is very limited, and can only be found in Pagar
Alam, South Sumatra province. This species is distributed only in small,
fragmented and isolated populations in remnant tropical forests in South
Sumatra. The populations are fragmented and isolated by tea and coffee
plantations. Based on ecological research, the decreasing population was
accelerated by deforestation, habitat fragmentation and overexploitation. In
the Mount Dempo population, V. foetida was threatened by tea plantations,
while in Padiampe, this species is threatened by coffee plantations as these
estates are routinely regenerated about every 25 years. Based on interviews
with locals, orchids that grow in coffee trees are highly threatened as farmers
view epiphyte orchids as a parasite (Metusala et al., 2010).
Genetic structure is influenced by mating systems, effective population size,
mutation rates and gene flow among populations (Raymond et al., 2003).
Genetic variation appears in morphological characters of individuals such as
size, colour and shape of flower and leaf as the consequences of different
genetic structure among or within populations. The variations stem from
different structure of DNA and even protein produced by the individual.
Genetic variation is the response of the individual adapting in changed
conditions of the environment (Frankham et al., 2002). Environmental changes
become a factor that forced genetic variation to lead to adaptation
mechanisms for those changes. Nowadays, changes in the environment are
mostly caused by change in land use and deforestation. These have led to
decrease, fragmentation and smaller habitats for plant populations. Habitat
degradation and fragmentation yielded small patches of habitat which
automatically impair population size. This may lead to limitation of pollen,
reproduction connectivity and gene flow (Andrianoelina et al., 2009; Lander et
al., 2010; Vranckx et al., 2012).
In small populations, gene flow is considered to be lower due to geographic
isolation (Fischer et al., 2000; Parab & Krishnan, 2008). Geographic isolation
consequently increases inbreeding of individuals within a population and the
ability of reproduction then becomes very low. A species with restricted
distribution and very small population generally maintains lower level of

Genetic Variation of Vanda foetida Based on RAPD Analysis 101
genetic variation compared to common species (Chung, 2009; Li et al., 2006).
On the other hand, the high fitness of species and heterozigosity support the
occurrence of species in its habitat because it in turn supports the ability to
adapt to environmental changes and diseases (Allendorf & Luikart, 2007).
Thus, genetic variation maintenance was the main focus of plant conservation
because in the long term, it affects plant fitness (Fischer et al., 2000;
Frankham et al., 2002; Sun & Wong, 2001).
Genetic variation can be measured by either molecular or quantitative
methods. RAPD is a fingerprinting method using short, random, oligonucleotide
primers to search for variation in the entire genomic DNA. Hence, this
technique is easy, allows for rapid analysis and relatively low cost. It is
available to a large number of primers and requires a very small amount of
DNA for analysis (Goh et al., 2005; Kishor & Sharma, 2009; Kishor & Devi, 2009;
Shin et al. 2002; Ferreira et al., 2006). According to Li & Jin (2006), RAPD has
superiority in detecting variation of genome compared with allozyme and can
reveal greater genetic divergence between populations. This method is
appropriate for study of rare species and successfully showed the relationship
between population size and genetic variation.
Genetic variation among individuals and between two populations of V. foetida
in South Sumatera is the main object to be investigated, that it may reveal
adequate data of gene flow limitation between two populations, as the mating
system only occurs between individuals within the population (inbreeding). The
two populations of V. foetida from Dempo and Padiampe are separated by
many kilometers in Pagar Alam city (Figure 1). To obtain information about
Figure 1. Location of Vanda foetida population in Sumatra and details of two
separate populations in Pagar Alam, South Sumatra

102 Rindyastuti et al.
population of V. foetida for its conservation, we studied RAPD profiles of two
populations then addressed the questions, (1) Is there less genetic variation of
V. foetida among individuals within each population that reveals inbreeding
depression? (2) Are both populations still connected to each other?
Materials and Methods
Study of Species
The first rediscovery of V. foetida in its natural habitat occurred in 2008, by
identification of the plant and its flower. V. foetida was discovered growing at
unproductive rubber tree plantations. This orchid has been growing as an
epiphyte at old rubber trees, and this species was destroyed by people who
saw it as a weed. The rate of forest conversion and palm tree rejuvenation
became a serious threat for conservation of this orchid. A recent investigation
in 2010 rediscovered this species to be distributed only in small, fragmented
and isolated populations in a tropical forest in Pagar Alam, South Sumatra.
Only two populations of V. foetida were found in this area. In fragmented
forests, this species was suggested as having high occurrence of inbreeding.
Study site
The two population of V. foetida were found in Pagar Alam, South Sumatra
Province. Pagar Alam is bounded by Bengkulu Province to the south, the
district of Kota Agung to the east, the district of Jarai to the north and the
district of Tanjung Sakti to the west. These two populations are from the
population of Mount Dempo and Padiampe. The population of Mount Dempo is
located in Mount Dempo at coordinates from S4.02485 E103.15432 until
S4.02912 E103.18136 and altitude at 1186-2019 metres above sea level. Most of
the area in Mount Dempo was converted into tea plantations belonging to a
government company, and has remnants of several small patches of original
habitats near watersheds and rivers. The population of Padiampe is located in
Padiampe Mountain and located at coordinates from S4.15115 E103.21304 until
S4.16205 E103.24909 at 1290-1375 metres above sea level. Most areas in
Padiampe were converted into coffee plantations belonging to local people.
RAPD-PCR Analysis
Ten plants of each population were used in this research. DNA isolation was
successfully established using commercial DNA isolation kit Illustra Phytopure
RPN-8511. Orchid samples and its code are presented in Table 1. Young leaves
were cut, approximately 50-100 mg of specimen were used as the source of
DNA extraction. The procedure of isolation followed the modified Tepnel Life

Genetic Variation of Vanda foetida Based on RAPD Analysis 103
Sciences Company’s procedure. The extracted DNA concentration and purity
were quantitatively tested by GeneQuant (spectrophotometer) with wave
length of 260 and 280 nm. The DNA and primers were diluted to obtain the
appropriate and optimum concentration for PCR procedure, which was 25 ρM
for primers. The PCR-RAPD profile of samples were performed by PCR mix Go
Taq® Green (Promega) using 8 specific primers successfully used for Vanda
(Orchidaceae), i.e OPU-3, OPU-5, OPU-6, OPU-7, OPU-12, OPU-13, OPU-14 and
OPU-16 (Lim et al., 1998). Horizontal agarose gel electrophoresis apparatus
(BioRad electrophorator) was used to run DNA through gels. Visual profiles of
band in electrophoresis gels were printed and the absence and presence of
band in every locus from all orchid samples were scored.
Data Analysis
The percentages of polymorphic locus for each primer were counted in this
study. The percentages of polymorphic locus in each population were analyzed
using POPGEN 1.32 (Yeh et al., 1999). Genetic variation of each population (h-
value) was analyzed using the POPGENE version 1.32 programme (Yeh et al.,
1999) while genetic variation between populations was analyzed through the
Unweighted Pair-group Method Using Arithmetic Average (UPGMA) (Sneath &
Sokal, 1963). A pPhylogeny tree was constructed and its data was carried out
in NTSYSpc21. The binner data was used to construct scatter plots using the
GenAlEx version 6.1 programme to show molecularly position of all samples in
the population.
Table 1. Sample code of V. foetida
No.
Sample Code
Population
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
139
275
276
278
279
283
285
290
299
22.11
314
350
367
368
369
372
373
384
385
397
Dempo
Dempo
Dempo
Dempo
Dempo
Dempo
Dempo
Dempo
Dempo
Dempo
Padiampe
Padiampe
Padiampe
Padiampe
Padiampe
Padiampe
Padiampe
Padiampe
Padiampe
Padiampe

104 Rindyastuti et al.
Results and Discussion
DNA Amplification result
Amplification result of total genom in 20 samples of V. foetida using 8 primers
revealed that there were 117 DNA fragments from 150 – 3000 bp. Among these,
107 fragments were polymorphic (91.45 %) and only 10 fragments were
monomorphic (8.55 %). Bands of all orchid samples using OPU 12 primer is
shown in Figure 2.
Based on the percentage of polymorphic loci in each primer (Table 2), it shows
that in general, 8 primers can be used in RAPD method for V. foetida because
it produced a fairly high polymorphism (above 50 %). The percentage of
polymorphic loci of 100 % was generated through the OPU-3 primer, OPU-6,
OPU-7 and OPU-13. This indicated that four primers can amplify loci on the
genome of V. foetida more than other primers used in this study. Moreover,
1000
3000
500
100
Figure 2. Amplification result of V. foetida using OPU 12 primer. M = DNA marker
(Vivantis 100 bp), numbers are showing sample code
Tabel 2. Details of 10-mer random primers screened for this study and degree of
polymorphism and information content for 8 RAPD random primers along with their
sequences, applied to 20 samples of V. foetida
Primer
Nucleotide
Sequence (5’-3’)
Amplified
Locus
Monomorphic
Locus
Polymorphic
Locus
(%)
OPU 3
OPU 5
OPU 6
OPU 7
OPU 12
OPU 13
OPU 14
OPU 16
CTATGCCGAC
TTGGCGGCCT
ACCTTTGCGG
CCTGCTCATC
TCACCAGCCA
TGGGTCCCTC
CTGCGCTGGA
GGCTGGTTCC
16
13
15
17
14
12
14
16
0
1
0
0
1
0
1
7
16
12
15
17
13
12
13
9
100
92.31
100
100
92.86
100
92.86
56.25
Total
117
10
107
91.45%

Genetic Variation of Vanda foetida Based on RAPD Analysis 105
the primers can also amplify some specific fragments in the sample of V.
foetida, so that the primer was optimal for this species
Genetic variation
In this study, the analysis of polymorphic percentage was established. This
analysis aimed to identify the difference in polymorphism level in each
population of V. foetida which revealed genetic variation. In this study, 117
loci were successfully amplified from eight primers. The percentage of
polymorphic loci analysis results are presented in Table 3.
The number and percentage of polymorphic loci in each population showed the
presence of more than one allele in a locus. Based on the above data, it
showed that both populations have a fairly high polymorphism, above 70 %.
The existence of polymorphic loci led to the presence of genetic variations
(Chung, 2009; Li et al., 2002). The high level of polymorphic loci was also
reported in a study of the genetic relationship between species in
Phalaenopsis, which were between 26-54 polymorphic loci for each primer
(Niknejad et al., 2009).
Polymorphism in populations of V. foetida may occur, due to random mating in
the population. Vanda is compatible to the pollen of other orchid genus.
However, genus of Vanda evolved specifically to form the pollen and its
pollinator becomes very specific (Motes, 1997). This mechanism occurs
because of orchid pollen called polinaria. Polinaria causes pollen to stick well
on pollinators so that pollen can be carried over to another pistil of different
trees (Arditti, 1992; Cozzolino & Widmer, 2005). Furthermore, random mating
in a population of cross-pollinated plants will tend to maintain heterozygosity
in nature (Frankham et al., 2002).
In V. foetida, cross-pollination occurs between individuals in similar
population. This occurs because two populations of V. foetida (Mount Dempo
and Padiampe) were separated by an extensive urban area, Pagar Alam City.
With the considerable barrier between the two populations, pollination can
only occur between individuals within a population, without any cross-
pollination between the two populations. Therefore, there is no gene flow
Table 3. Percentage of polymorphic locus in each population
Population
Polimorphic Locus
Polimorphic Locus Percentage
Dempo
Padiampe
89
83
76.07 %
70.94 %

106 Rindyastuti et al.
between the two populations. If each population lost most of its members as a
result of habitat exploitation, both populations will experience inbreeding
depression due to reduced random mating events. Inbreeding is an event which
produces offspring of close related individuals. Inbreeding can decrease the
level of reproduction, survival, and power successor descent. This leads to
offspring which have high risk of death (Allendorf & Luikart, 2007). It could
threaten the survival of the population.
Genetic variation within and between populations
Genetic variation in each population was detected by h value (Nei’s genetic
diversity) which was 0.1999 and 0.1778 for Mount Dempo and Padiampe,
respectively (Table 4). According to Chung (2009), genetic variation within the
population of V. foetida was higher than those of some rare species originating
from South Korea, Gymnadenia cucullata, Gymnadenia camtschatica,
Amitostigma gracile, and Pogonia minor. H value of their population is only
0.036, 0.067, 0.009 and 0.014, respectively. This result showed that each
population of V. foetida still had high genetic diversity. The existence of
genetic variation showed that the population of V. foetida was not in danger of
extinction even though the population was small. The information of genetic
variation can be used to support V. foetida conservation, which mainly focuses
on increasing the number of individuals and also enriching genetic variation to
maintain the existence of V. foetida populations.
Based on genetic variation, it was known that genetic variation in the
population of Mount Dempo was higher than those of Padiampe. This happens
with regards to habitat conditions of both populations. At Mount Dempo, most
of the area which was originally a forest has been converted into tea
plantations. The high genetic variation in the population of Mount Dempo
indicated that initially this population was a huge and stable and genetic
variation was well maintained. Genetic variation of V. foetida in Mount Dempo
was maintained by the residents by growing this species in their garden
(Metusala et al., 2010). Padiampe population is a natural population located in
protected forest area. However, this protected forest has changed as a result
of coffee-growing by local communities. Then, some species of host plants for
Table 4. The value of h (Nei’s genetic diversity)
Population
h (gene diversity)
Dempo
Padiampe
0.1999 ± 0.1673
0.1778 ± 0.1714

Genetic Variation of Vanda foetida Based on RAPD Analysis 107
V. foetida were replaced by coffee trees. The alteration of host plants may
cause genetic changes that triggered the onset of genetic variation in the
Padiampe population. Genetic variation in both populations of V. foetida was a
result of cross-pollination that occurs between individuals within a population.
The diversity between populations of V. foetida was analyzed using NTSYSpc21
expressed by genetic similarity coefficient (Rohlf, 1997). Similarity coefficient
indicates the extent of genetic similarity among members of a population so
that it can be used to formulate individual clusters by using UPGMA. Individual
clusters show the diversity between populations (Singh, 1999). Analysis using
NTSYSpc21 formed dendrogram presented in Figure 3.
In addition, description of population composition based genetic analysis was
established using the GenAlEx 6.1 programme (Peakall & Smouse, 2005).
Scatter plot showed the distribution of the individuals in population of V.
foetida based on molecular character of RAPD analysis. Scatter plot is
presented in Figure 4. Based on RAPD analysis for genetic variation using the 8
primers, it showed that V. foetida which originated from two populations
formed two main clusters in dendrogram with 67 % (0.67) degree of similarity.
The two populations of V. foetida were closely related. This phenomenon was
also exhibited by the grouping of individuals from both populations into one
cluster in dendrogram with 74 % (0.74) degree of similarity. Dendrogram
(Figure 3) revealed that individuals formed two large clusters, namely A and B.
Figure 3. Dendrogram of V. foetida from two populations, population of Dempo (D) and
Padiampe (P). Individual samples of V. foetida were identified using numeric code as it
was collected in Purwodadi Botanic Garden, East Java, Indonesia. Numeric codes of
populations correspond to those in Table 1

108 Rindyastuti et al.
In cluster A, most individuals of V. foetida, from both populations of Mount
Dempo and Padiampe, clumped and mingle. These individuals are clustered at
the level of similarity 72 % (0.72). This is different from the concept of
similarity by Singh (1999) which states that individuals with morphological
similarity up to 85 % can be suggested as the same species. The level of
similarity in this study refers to the phenetic molecular basis character, so that
the level of similarity in one species can have different values from the
concept of morphological similarity.
Based on the dendrogram and scatter plot, it is known that there are some
individuals withlower levels of similarity compared to other individuals.
Individuals with code number 283 were contained in cluster A, and individuals
with sample code 397, 278, and 290 were contained in cluster B. Individuals
with sample code 397 came from Padiampe, while individuals 278, 290, and
283 were from the population of Mount Dempo. The individual of 283 were
grouped in clusters A at the level of similarity 70 % (0.70), while the individuals
of 397, 278, and 290 were in large clusters which have level of similarity 67 %
(0.67) with clusters A. The individual distribution on scatter plot in Figure 4
also showed that individuals with the sample code 283 seemed a bit detached
with another individual of cluster A, while individuals which come from cluster
B did not seem to be clustered with members of cluster A. This showed that
the two populations revealed genetic variation in the populations.
139
275
276
278
279
283
285 290
299
2211
314
350
367
368
369
372
373
384
385
397
Coord. 2
Coord. 1
Principal Coordinates
dempo
padiampe
Figure 4. Scatter plot of RAPD analysis of V. Foetida in two populations. Red dots
indicated individuals from population of Mount Dempo. Green dots indicated individuals
from population of Padiampe

Genetic Variation of Vanda foetida Based on RAPD Analysis 109
Individuals from population of Mount Dempo which are 276, 283, 290, 278 and
385 clustered with individuals in cluster B in the level of similarity 67 %. This
clustering showed that V. foetida population which was currently divided into
two populations are still connected and assumed as one large population in the
past. This large population was then separated by habitat fragmentation due
to the development of human urban civilization which became a barrier for
both populations. This was supported by the appearance of the estimated
population distribution of V. foetida on a scatter plot (Figure 4). From the
scatter plot, we know that most of individuals from Mount Dempo and
Padiampe populations are clustered. So, it was clear that the two populations
were in the beginning one large population.
Habitat fragmentation also occurred in populations of the ground orchid
Pterostylis gibbosa from Australia. Genetic variation in populations of P.
gibbosa is indicated by the percentage of polymorphic loci 69 % and
heterozygosity (h) 0.261 (Sharma et al., 1999). This orchid population showed
high genetic variation. Small population as the result of habitat fragmentation
still can have high genetic variation, yet it is needs to be sustainably
maintained for its conservation (Li & Ge, 2006). In populations of V. foetida,
the possibility of natural gene flow through cross-pollination between the two
populations is very low because both are constrained by geographic barriers.
But, the high genetic variation can compensate the negative impact of habitat
fragmentation which reveals the capability of adaptation to a changing
environment. This species can have stable population with huge population
size and high genetic diversity if the number of individuals increase and cross-
pollination between the two populations occurs. Ex-situ conservation strategy
should be established to increase population size and genetic diversity using
artificial cross-pollination between the two populations. Therefore,
introduction of its offspring into the natural habitat will able to maintain its
existence in nature (Chung, 2009; Young et al., 1996).
Conclusion
We can conclude that the two populations of V. foetida which are Mount
Dempo and Padiampe, Pagar Alam, South Sumatra have high genetic variation
even though this species only survives in small populations. This species is not
genetically in danger and will able to survive if its natural habitats are
remained. The two populations are genetically connected and are assumed as
one large population in the beginning.

110 Rindyastuti et al.
Acknowledgement
This research was supported by I-MHERE Research Grant 2010, Faculty of
Biology, Gadjah Mada University. The authors thank PIC of this grant and Dr.
Irawati from Bogor Botanic Garden-LIPI as a referee.
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