Cover Single.cdr


BIOTROPIA Vol. 27 No. 2, 2020: 179 - 188 DOI: 10.11598/btb.2020.27.2.1211

GENETIC VARIATION OF TEAK MISTLETOE ( Dendrophthoe
pentandra (L.) MIQ.) BASED ON RANDOM AMPLIFIED

POLYMORPHIC DNA (RAPD) MARKERS**

ZAINAL MUTTAQIN1', SRI WILARSO BUDI2, BASUKI WASIS2,
ISKANDAR ZULKARNAEN SIREGAR2 AND CORRYANTI3

1Faculty of Forestry, Universitas Nusa Bangsa, Bogor 16161, Indonesia
2T)epartment f Silviculture, Faculty of Forestry, IPB University, Bogor 16680, Indonesia

5Study Program of Environmental Sciences, Graduate Program, Institut Teknologi Yogyakarta, Yogyakarta 55171, Indonesia

Received 31 January 2019 / Accepted 21 May 2019

ABSTRACT

Mistletoes are hemiparasitic macroparasite plants which interfere with trees and other wild plants in nutrient
acquisition. As the plant has low leaf water potential, it draws water from teak wood tissues during the deciduous
stage of the teak host, thereby killing the twigs and eventually, the teak tree. Mistletoes are also a key player in
plant diversity. Therefore, the mistletoe population needs to be regulated not only as a parasite but also as a
keystone species affecting biodiversity. Knowledge scarcity on the status of mistletoes includes its genetic
variation. Hence, the purpose of this study is to analyze the level of genetic variation of teak mistletoe
( Dendrophthoe pentandra ) using RAPD marker. At Padangan teak Clonal Seed Orchard (CSO), it was randomly
collected leaf samples from three layers of the mistletoe’s crown (upper, middle, and below) were taken from five
host teak trees randomly selected from each of the sub-observation measure plots (OMP). Four OMP units
inside the observation sample plots (OSP) (n = 3, 50 x 50 m) at different levels of infestation (light, moderate
and heavy) were established. Analysis of the genetic variation and genetic distance of mistletoes hanging on the
different crown layers were conducted using RAPD markers. The leaf samples from the crown layers, UU (upper
crown and sub-section upper), UM (upper crown and sub-section middle), and UB (upper crown and sub-section
below), which include U (upper crown) had significantly greater genetic variation (He = 0.181 — 0.255) than those
from M layer (middle crown, He = 0.227) and the B layer (below crown, He = 0.114). Furthermore, the widest
genetic distance significantly occurred between the mistletoes of the UB and B crown layers (0.310), whereas the
nearest genetic distance significantly occurred between mistletoes of UU and UM layers in the upper crown
(0.038). Practical implications of the low genetic variation in this study include the control of mistletoe
D. pentandra infestation by means of restricting its population so that Perhutani State Owned Forestry Enterprise
can maintain the level of damage below the economic threshold.

Keywords: Dendrophthoe pentandra, genetic variation, mistletoe, RAPD, Teak

INTRODUCTION

Mistletoes are hemiparasitic macroparasite
plants which interfere in the nutrient acquisition
of cultivated plants including trees and other
wild plants. Based on their habitat, mistletoes
attach themselves to certain parts of a host plant,
such as branches, twigs, and occasionally stems.
Many research aspects about mistletoe

^Corresponding author, e-mail: znldeg@yahoo.com
**This paper was presented at the 3rd International
Conference on Tropical Biology 2018, 20-21 September
2018, Bogor, West Java, Indonesia

interaction with their hosts and bird dispersers
have been conducted in both plantation and
natural forest ecosystems. However, there is a
lack of information concerning the effect of
environmental variability of the canopy on the
fate of mistletoe seeds and seedlings growth
(Mellado & Zamora 2014b), include aspect of
genetic variation in this study and DNA barcode
characterization (Muttaqin et al. 2017).

Mistletoe infestations can decrease the
production of quality seeds in seed orchards and
in timber plantations. Alarmingly, with repeated
infestations, mistletoes can kill the host trees.

179



BIOTROPIA Vol. 27 No. 2, 2020

The initial process of growth and development
of mistletoes takes place when mistletoe seeds
spread on those parts of stem by the assistance
of birds as main agents (Mellado & Zamora
2014a). The seeds then germinate and develop
to form the haustorium organ which penetrates
into the xylem of a host (xylem tapping) to
absorb important nutrients such as water,
minerals, and components of sugar and amino
acids.

A study on the ecophysiology of teak and its
canopy hemiparasite Dendrophthoe falcata var.
pubescens revealed that mistletoes always maintain
lower leaf water potential compared to the host
teak. During the deciduous stage of the host
teak, the misdetoe is drawing most of the water
from the neighbouring wood tissue, thereby
causing death of the twigs, and finally the death
of the teak. The water use efficiency of the
mistletoe is lower compared to that of the teak.
Its photosynthetic performance concerning
electron transport ability and quantum-energy-

use efficiency are better in the mistletoe leaves.
Since K and Na are phloem mobile minerals,
these are highly concentrated in the misletoe
leaves. That is indicative of the absence of any
phloem connections between the host and the
mistletoe. The mistletoe can photosynthesis at
shade and exposed condition, showing its high
adaptability to the host (Kallarackal et al. 2003).
Moreover, misfietoe-host plants’ antagonistic
interactions, along with bird dispersers, may
form complex networks whose function and
structure can influence fragmentation at
different scales, e.g., molecular or population
levels (Arroyo et al. 2013).

In the Central Oregon, USA, pruning was
effective in controlling the severity of dwarf
misdetoes and in increasing the longevity of
Douglas-fir, even if not all the misteltoes were
removed because of the delayed mistletoe
intensification (Maffei et al. 2016). In Perhutani,
mistletoe control by silvicultural method was
also applied by planting ‘kersen’ (Muntingia
calabura), together with kesambi plant, dowet
(Sy^ygium cumini) and salam (Sy^ygiumpolyanthum),
as edge and filler plants. These plants, serving as
substitute hosts of teak, have fruits which are
foraged by the primary agent of mistletoe
dispersal, the cabai bird (Dicaeum sp). The mix
planting of teak for mistletoe control was also
conducted in Indonesia (Corryanti et al 2012).

Three mistletoe species were found at
Padangan teak clonal seed orchard (CSO), East
Jawa Province, Indonesia, namely Dendrophthoe
pentandra (Loranthaceae), Macrosolen tetragonus
(Loranthaceae), and Viscum articulatum
(Santalaceae) (Muttaqin et al. 2017). D. pentandra
was the most numerous and the most widely
distributed at the orchard. Those mistletoes
belong to indigenous plant groups that usually
grow on suitable host plants and spread to
tropical regions including Indonesia’s forested
areas.

The main goal of sustainable management at
Padangan orchard is to control mistletoe
infestation and develop immediate conservation
measures. It requires synergistic, not
antagonistic, support through the conservation
of genetic variation of teak mistletoes which can
be a valuable input and be correlated with the
result of assessing the intensity of mistletoe
infestation, including the True Mistletoe Rating
(TMR) modified 8-class rating requirement
(Muttaqin 2016). Sustainable management also
requires accurate data or information on the
level and range of mistletoes genetic variation
that validates the presence or scarcity of
mistletoe species exhibiting mistletoe main
characters and high level of adaptation to
environmental changes. The value of high
genetic variation would take effect towards
species ability to adapt on environmental
condition, and vice versa. So as, this study
hypothesis has two alternatives; if genetic
variation of D. pentandra is low then the control
of this mistletoe would be restricted to moderate
and heavy infestation. If the genetic variation is
moderate until high then the control of this
mistletoe would be conducted against all levels
of infestation from light, to moderate, to heavy.
Therefore, knowledge about the genetic
variation in mistletoes will improve the
sustainable management of teak seed orchards,
especially protection from pests and diseases,
like mistletoes. Also, it be constitute candidate
population for inclusion in future conservation
programmes for mistletoe of Dendrophthoe
pentandra that grow on teak stand in Perhutani
area, Indonesia.

For genetic resource conservation, the
genetic diversity and population structure of
mistletoe D. pentandra need immediate
investigation (Kim et al. 2017). Studies on the

180



Genetic variation of teak mistletoe ( Dendrophthoepentandra (L.) Miq.) — Muttaqin et at

genetic variation of mistletoes had applied some
markers such as RAPD, Microsatellite, AFLP
(Crichton et al. 2012; Yi et al. 2013; Amico et al.
2014; Kim et al. 2017) and the study on the
desert mistletoe Phoradendron californicum
(Santalaceae) had used isolation of 18
Microsatellite loci (Arroyo et al 2013). Despite
the ecological and medical importance of D.
pentandra, only few studies were conducted
(Poerba & Sunaryo 2006) and no studies have
evaluated the genetic diversity of its wild
populations in Indonesia. Therefore, this
research was conducted to analyze the level of
genetic variation of teak mistletoe D. pentandra
using the Random Amplified Polymorphic
DNA (RAPD) marker.

MATERIALS AND METHODS

Sampling Sites

The field data gathering and genetic material
sampling were conducted at the Padangan teak
CSO compartments or blocks, located at
111°34,57.3,? E and 07°12’56.1” S and also at
Bancer and Payaman Villages, Ngeraho District,
Bojonegoro Regency, East Java Province,
Indonesia (Fig. 1). The teak CSO area was
divided into eight compartments that are further
divided into blocks. A total of 132 blocks, at ± 5
ha each totalling to + 660 ha, were established.
Some 144 clones were planted repeatedly in the
blocks from 1983 until 1996 (Corryanti 2015).

The molecular analysis was carried out at the
Genetics and Forestry Molecular Laboratory,
Silviculture Department, Faculty of Forestry,
IPB University, Indonesia.

Collection of Samples
Leaf samples of D. pentandra were randomly

collected from five host teaks randomly selected
from the observation measure plots (OMP
subplots). Four OMPs were established at the
observation sample plots (OSP units) (n = 3, 50
x 50 m in size) depending on the level of
infestation (light, moderate and heavy)
(Muttaqin 2016), but one control OSP plot was
not included because there was no infestation or
no leaf samples of D. pentandra were found,
referring to modified EFF or TS/CRC990
(Drescher et al 2016) (Figs. 1, 2a, 2b). The
number and distribution of mistletoe leaf
samples were collected from the crown layers of
each host teak. The crown layers consisted of
UU (upper and sub-section upper), UM (upper
and sub-section middle), UB (upper and sub¬

section under), M (middle), and B layer (below).
The number of collected samples (n) for each
layer were UU (n = 45), UM (n = 57), UB (n =
31), M (n = 56), and B (n = 15) totalling to 2014
leaf samples of D. pentandra. That procedure
included the use of binoculars, digital cameras,
GPS of Garmin Oregon 550 and a map of
mistletoe infestation at Padangan teak CSO with
scale 1 : 18,000 covering the infestation from
years 2010-2014.

%

»

>2

i -

NAP Of
OBSERVATION AND MEASUREMENT

SAMPLE PLOTS M
TEAK CLONAL SEED ORCHARD (C$0) PADANGAN

A

A/ *>*
A/

MatermtMtr Hid forage pljntx lor Bvn

CmSERWIlOM SAMPLE PHIPOIKT5 :

•
L19W

MAP OF EAST JAVA PROVINCE

Figure 1 Location map of OSPs at the OMP in Padangan teak CSO (Muttaqin 2016)

181



BIOTROPIA Vol. 27 No. 2, 2020

OSP
1

m UMP

0
0

n
Q-

c L M
<a>

H

5 m

A B O D E F G H I J
1 .

20
30
47~

sT~

6~

i7~

80
9
~

ToT

A

C

- 50 m -
(b)

Figure 2 Field layout of the sampling units: (a) Position of the OMPs (n = 4) in the OSP units; (b) Position of the sub-

OMPs (n = 5) in an OMP unit (source: EFForTS/CRC990 2012, modified for this research)
Notes: C = control; L = light; M = medium; H = heavy.

Laboratory Procedures

DNA was extracted from leaf samples and
isolated using the modified CTAB (Cetyltrimetyl
ammonium bromide) method (Doyle 1991;
Aritonang el al 2007). DNA quality was carried
by PDA (Potato Dextrose Agar) electrophoresis
of 1% agarose gel at 100 volts using the buffer
TE 50 [xL, 3 [iL DNA and 2 [xL BJ (Blue Juice).

The product of electroforesis was given the
solution Gelred Tm nucleid acid and
photographed on UV transiluminator model
TFX-20.LM following Aritonang el al (2007).
Five random universal primers, namely; OPP-9,
OPP-15, OPP-19, OPBH-20, OPBH-16, and 10
primers used by Amico et al. (2014) for Tristeric
corymbosus (Loranthacea) of same family as
D. penlandra were used in this research (Table 1).

Table 1 Tested primers and base sequences for the PCR-RAPD analysis of D. pentandra

No. Primers Base sequences
(5’-3’)

1 OPP-9* GTGGTCCGCA
2 OPP-15* GGAAGCCAAC
3 OPP-19* GGGAAGGACA
4 OPBH-20* CACCGACATC
5 OPBH-16* CTGCGGGTTC
6 OPA-17 GACCGCTTGT
7 OPB-04 GGACTGGAGT
8 OPB-07 GGTGACGCAG
9 OPB-12 CCTTGACGCA

10 OPB-15 GGAGGGTGTT
11 OPA-04 AATCGGGCTG
12 OPB-18 CCACAGCAGT
13 OPB-20 GGACCCTTAC
14 OPF-11 TTGGTACCCC
15 OPL-12 GGGCGGTACT

Note: * — primer selected for this study (Kissinger 2013; Amico et al 2014).

182



Genetic variation of teak mistletoe ( Dendrophthoepentandra (L.) Miq.) — Muttaqin et at

Table 2 Amplification steps of the PCR-RAPD marker

Stages Temperature
(°c)

Time
(minutes) Cycle

Pre-denaturation 92 5 1
Denaturation 92 1

Annealing 32; 35 1 35a; 45b

Extension 73 1
Final Extension 73 10 1

Notes: aPCR machine at MJ Research PTC-100; b PCR machine at AB Applied Biosystem VeritiTM Thermal Cycler.

The primers were selected by temperature
optimization (annealing) with the PCR process
ranging from 32 °C, 33 °C, 34 °C, 35 °C, 36 °C,
to 37 °C. Through electrophoresis, the five
primers, i.e., OPP-9, OPP-15, OPP-19,
OPBH-20, OPBH-16 showed clear DNA
fragment bands at optimization temperature of
32 °C and 35 °C with 35 and 45 cycles (Tables 1
and 2). However, the other primers obtained
unclear DNA fragment bands. The primers used
were then diluted (5x, lOx) or as ratios 1:50,
1:100 in ddH20 to elucidate the crystals of
DNA bands by electrophoresis as a mixture of
materials in a microtube for the PCR-RAPD
process. Ingredients in the PCR-RAPD process
of lx reaction comprised of Psd H20 (2.0 pL),
Go Taq® Green Master Mix (6.0 pL), Primer
oligonucleic (2.0 pL), DNA template (2.0 pL).

The amplified PCR-RAPD marker was
identified by electrophoresis using 2% agarose
gel in 50 pL TE buffer, 3 pL DNA, and 2 pL BJ
(Blue Juice) for 45 min at 100 volts. The yields
of the electrophoresis were given a Gelred Tm
nucleic acid solution and were photographed on
the UV transilluminator TFX-20. LM model
(Nybom et al 2014; Aritonang et al. 2007) to
identify the clear bands.

The scoring used for locus marker was 1 if
having a band and 0 if having no band. Further
interpretation of each primer was carried out
using the software POPGENE version 1.31 and
NTSYSpc version 2.02. POPGENE was also
used to compute for other statistics (e.g. allele
frequency, gene diversity, genetic distance, F-

statistics, multilocus structure. Meanwhile,
NTSYS NTSYSpc was used in the cluster
analysis of qualitative molecular genetic data
(Rohlf 1998; Yeh et al. 1999; Aritonang et al.
2007).

RESULTS AND DISCUSSION

Genetic Variation

The 204 PCR amplified samples, using the
five RAPD markers (OPBH 16, OPBH 20, OPP
9, OPP 15, and OPP 19), produced 46 clear
polymorphic DNA bands (loci) with the base
length ranging from 50 to 1500 bp in size
(Fig. 3). Each primer produced a range of 8 to
11 loci (mean of 9.2 loci), and the polymorphic
loci percentage (PLP) ranged from 32.61 to
86.96% (Table 4). These results confirmed those
in the study conducted by Poerba and Sunaryo
(2006) on the same D. pentandra on 22 hosts,
excluding teak, at Eka Karya Botanical Garden,
Bali. Different from those used in this study,
their study used two primers consisting of OPA-

11 which produced 12 polymorphic bands (PLP
100%) and OPC-12 which produced 9 bands
with 8 polymorphic bands (PLP 88.89%) and 1
monomorphic band. Both primers have bands
ranging from 150 to 1700 bp in size. The same
primer for predicting the genetic variation of
Nepenthes gracilis Korth. in Kerangas Forest,
Indonesia, produced a band ranging from 150 to
1400 bp in size and PLP of > 82.75% (Kissinger
2013).

183



BIOTROPIA Vol. 27 No. 2, 2020

100 bp «

500 bp 4

1000 bp,

00 bp 500 bp I /

\ A"*•1000 bp 1100 bp
.

(a ) (b)
SO bp

300 bp

/

50 bp

50 bp

5 * - I P*

(c)

50 bp

500 bp

1100 bp

h750 bp

(e)

Figure 3 PCR amplification pattern with five markers: (a) OPBH 16, (b) OPBH 20, (c) OPP 9, (d) OPP 15, and (e)
OPP 19

Table 3 The primer sequence and number of polymorphic bands

No Primer Sequence Number of
polymorphic bands

Base length
(bp)

1 OPBH 16 5’CTGCGGGTTC ‘3 8 50-1500
2 OPBH 20 5’ CACCGACATC £3 10 100-1500
3 OPP 9 5’ GTGGTCCGCA c3 11 50-1500
4 OPP 15 5’ GGAAGCCAAC £3 8 100-1100
5 OPP 19 5’ GGGAAGGACA £3 9 50-1100

Total 46

Based on the five parameters of genetic
diversity (He) within population, the highest
value (0.255) was obtained by those mistletoes
in the UM crown layer and the lowest (0.114)
was obtained by those in the B crown layer
(Table 4). The higher the He, the higher is the
Shannon index (I) and the Ne (the sum of
effective alleles). In addition, genetic variation of
those in the UU, UM, and UB crown layers
which have the upper crown sections (He =
0.181 — 0.255) are greater than those in the M
layer (middle crown) (He = 0.227) and B layer
(below crown) (He = 0.114). This indicated that
the dispersal agent of mistletoe seeds, the birds
cabai jawa (Dicaeum trochilium) and other

specialist frugivores, preferred the upper and
middle crown sections than the layer below
(Muttaqin et al. 2016). The birds attached the
seeds on branches and twigs through their bird
droppings.

The He value of D. pentandra using the RAPD
marker was classified as rather low (0.194). This
value was lower than Tristeric corymbosus using
RAPD marker. Comparison with the different
marker, It was lower than Arcethobium spp. and
Aiscum album using AFLP marker, also it lower
than Ficus deltoidea using ISSR marker. However,
It be within the range of V. coloratum (He =
0.032-0.672) using Microsatellite marker
(Table 5).

184



Genetic variation of teak mistletoe (Dendrophthoepentandra (L.) Miq.) — Muttaqin et al.

Table 4 Genetic variation using RAPD analysis of mistletoe Dendrophthoe pentandra (L.) Miq. at the different crown layers
of infected host teak trees at Padangan teak CSO, Indonesia

No Layer3

(crown part)b N PLP
(%) Na Ne He I

1 UU 45 60.870 1.609 1.306 0.181 0.273
2 UM 57 86.960 1.870 1.429 0.255 0.391
3 UB 31 65.220 1.652 1.323 0.192 0.295
4 M 56 76.090 1.761 1.377 0.227 0.346
5 B 15 32.610 1.326 1.186 0.114 0.171

Sum 204 321.750 8.218 6.622 0.969 1.476
Average on type level 41 64.350 1.644 1.324 0.194 0.295
Standard deviation (SD) 18 20.430 0.204 0.091 0.054 0.083

Notes: Hrawan (2004), modified for this study: N = sum of samples; PLP = Polymorphic Locus Percentage; Na = sum
of observed samples alleles; Ne = sum of effective alleles; He = Heterozygosity expectation; I = Shannon
Index;

bUU = upper crown sub-section upper, UM = upper crown sub-section middle, UB = upper crown sub-section
under; M = middle crown, B = below crown.

Table 5 Comparison of genetic variation of mistletoes in this study and other studies using RAPD and other markers
Species He Marker Origin Source

Dendrophthoe pentandra 0.194 RAPD Indonesia, Padangan teak CSO This study
Tristerix corymbosus 0.365 RAPD Chile Amico et al. 2014
Malaysian mistletoe Fig 0.500 - 0.750 ISSR Malaysia Zimisuhara et al. 2015
(Ficus deltoideaJack)
Viscum album 0.820 AFLP Korea Yi et al. 2013
Arcethobium spp. Viscaceae 0.238 AFLP Western North America, Reif et al. 2015

Melampyrum sylvaticum 0.330 - 0.750 Microsatellite
Oregon, California

The United Kingdom Crichton et al. 2012
Phoradendron californicum 0.364 - 0.924 Microsatellite Mexico Arroyo et al. 2013
Viscum coloratum 0.032 - 0.672 Microsatellite Korea, Japan, China Kim et al. 2017

The 19 novel polymorphic microsatellite
markers for mistletoe Viscum coloratum have been
developed for its use as anti-cancer medicinal
plants and have become a novelty in the
molecular analysis of misdetoes (Kim et al.
2017). Fortunately, cross-species amplification
indicated that those markers can also be used for
molecular analysis associated with other species
coming from the same family (Santalaceae). That
result was also tested for cross-species
amplification in V. articulatum as a potential
medicinal plant.

The level of genetic variation of mistletoes
play an important role in their capability to
adapt to changes in environmental condition,
such as lack of water, nutrient, and their
regeneration. Low genetic variation implies a
high sensitivity to heterogeneous environment
that is detrimental to the growth and
development of mistletoes. D. pentandra has a
rather low genetic diversity (Table 4), therefore,
it is important to maintain the genetic variation
of D. pentandra in order to protect its existence
and prevent it from extinction, and this requires
effective conservation strategies. The massive
utilization of D. pentandra as a medicinal plant is

a cause of concern even as they are hanging on
host tree and crops.

Genetic diversity estimates among and within
populations of mistletoes species, will further
help in the development of effective strategies
for their conservation (Kim et al. 2017). The
knowledge gained from research using
Microsatellite markers was needed in designing
conservation management programs for M.
sylvaticum in the United Kingdom (Crichton et al.
2012). Such information will also be useful for
other related species of endangered non-weedy
hemiparasites, according to Mellado & Zamora
(2019), with similar traits and life history.

Genetic Distance among Mistletoes on
Different Canopy Layers

The largest genetic distance (0.131) was
obtained significantly between mistletoes
hanging on the UB and B layers which have
different crown sections while the shortest
genetic distance (0.038) was significantly
between those at the UU and UM layers which
were at the same upper crown sections
(Table 6).

185



BIOTROPIA Vol. 27 No. 2, 2020

Table 6 Genetic distances between mistletoes hanging on different crown layers of host teaks

Crown layer UM M B UB UU
UM ****
M 0.041 ****
B 0.119 0.100

UB 0.092 0.065 0.131 ****
UU 0.038 0.083 0.124 0.101

UM

UU

M

UB

B

0.04 0.06 0.08 0.10 0.12

Figure 4 UPGMA dendrogram of the genetic distances, based on RAPD marker, of D. pentandra misdetoes growing on
the different crown layers of teak
Notes: Crown layers: UU = upper crown sub-section upper, UM = upper crown sub-section middle, UB =

upper crown sub-section under; M = middle crown, B = below crown.

The genetic distance or relatedness among
mistletoes growing on different crown layers
(described by a cluster or a dendrogram) were
spread over a Eucledian distance consisting of
eight units (0.04 - 0.13) (Fig. 4). Two different
clades were identified in which the first clade
consisted of mistletoes from the UM, UU, M,
UB crown layers, while the second clade only
contained those mistletoes in the B crown layer
which was considered an outgroup. In this case,
the mistletoes in the B layer were more
genetically distant from other misdetoes in the
UU (0.124), UM (0.119), UB (0.131), M (0.100)
(Table 6). Hence, as predicted, the two clusters
were of different genetic structure.

Another cluster analysis of 22 D. pentandra
collected from several different locations had
separated the samples into two clusters; one
cluster consisted of 3 groups, namely; group 1 (6
collections), group 2 (13 collections), and group
3 (2 collections), whereas the other cluster, the
outgroup, consisted of only 1 collection (Poerba
& Sunaryo 2006). The Euclidean distance was
43 units (0.490 - 0.920) longer than this study
result (0.038 - 0.131). The different clustering is
due to the different genetic distant that D.
pentandra grow on teak and others as host tree

on different locations. While in this study, D.
pentandra grow only on teak of crown layers
differently.

CONCLUSION

Based on the RAPD marker, the genetic
variation, He, of D. pentandra mistletoe was rather
low (He = 0.114 - 0.255). The highest He (0.255)
was found among the mistletoes of the upper
middle layer of the canopy, UM layer, while the
lowest He (0.114) was among the mistletoes in
the layer below the crown (B). The genetic
distance in the five layers or sections of teak
crown inhabited by D. pentandra ranged from
0.038 to 0.131, and was clustered into two
clades. The first clade consisted of mistletoes in
the UM, UU, M, and UB layers, while the
second clade consisted of those mistletoes in the
B layer.

For Perhutani, the state-owned forestry
enterprise, this study results practically implied
the thorough removal of mistletoes by pruning
them from the entire sections of the crown, as
was done successfully on the dwarf mistletoes of
Douglas fir.

186



Genetic variation of teak mistletoe (Dendrophthoe pmtandra (L.) Miq.) — Muttaqin et al.

ACKNOWLEDGEMENTS

This study was supported by DIPA
SEAMEO BIOTROP 2015 (No. 045.17/PSRP/
SC/SPK-PNLT/111/2015), Indonesia. The
authors would like to also acknowledge and
express their gratitude to Mr. Suwarno, the
Head of Research and Development Center of
Perhutani, Indonesia and his technical staff for
their invaluable assistance during the field
sample collection.

REFERENCES

Amico GJ, Vidal-RusseU R, Aizen MA, Nickrent D. 2014.
Genetic diversity and population structure of the
misdetoe Tristerix corymbosus (Loranthaceae). Plant
Syst Evol 300:152-62.

Aritonang KV, Siregar IZ, Yunanto T. 2007. Manual of
genetic analysis of forest plant in Silviculture
laboratory. Bogor (ID): Faculty of Forestry, IPB
University.

Arroyo JM, Munguia-Vega A, Rodriguiz-Estrella R,
Bascompte J. 2013. Isolation of 18 microsatellite
loci in the desert mistletoe Phoradendron californicum
(Santalaceae) via 454 pyrosequencing. Appl Plant
Sci 1(12):1-4.

Corryanti, Purwanto, Suhudi M, Suryanaji, Mawardi K,
Hartanto B. 2012. Study of mix planting in teak for
controlling of mistletoe. Cepu (ID): Research and
Development Center Perum Perhutani.

Corryanti. 2015. Character of teak trees in Clonal Seed
Orchard. Cepu (ID): Research and Development
Center Perum Perhutani.

Crichton RJ, Squirrell J, Woodin SJ, Dalrymple SE,
Hollingsworth PM. 2012. Isolation of
microsatellite primers for Melampyrum sylvaticum
(Orobanchaceae), an endangered plant in The
United Kingdom. American J Bot:e457-9.

Doyle J. 1991. DNA protocols for plants. New York (US):
Bailey Hortorium 462 Mann Library, Cornell
University.

Drescher J, Rembold K, Allen K, Beckschafer P, Buchori
D, Clough Y, . .. Scheu S. 2016. Ecological and
socio-economic functions across tropical land use
systems after rainforest conversion [Abstract]. Phil
Trans R Soc B 371(1694)20150275.

Irawan B. 2004. Ironwood ( Eusideroxylon givageri Teijsm. &
Binn.) and its varieties in Jambi, Indonesia
[Dissertation]. Gottingen (DE): University of
Gottingen.

Kallarackal J, Somen CK, Rajesh N. 2003. Teak and its
canopy parasite Dendrophthoe - water relations and
ecophysiology. In: Bhat KM, Nair KKN, Bhat KV,

Muralidharan EM, Sharma JK, editors. Quality
Timber Products of Teak from Sustainable Forest
Management. Proceedings of the International
Conference, p. 188-97.

Kim BY, Park HS, Kim SN, Kim YD. 2017.
Development of microsatellite markers for Discum
coloratum (Santalaceae) and their application to wild
populations. Appl Plant Sci 5(1):1600102.

Kissinger. 2013. Bioprospection of heath forest: Analysis
of Nepenthes gracilis Korth as conservation stimulus
[Dissertation]. Retrieved from IPB University.

Maffei HM, Filip GM, Grulke NE, Oblinger BW,
Margolis EQ, Chadwick KL. 2016. Pruning high-

value Douglas-fir can reduce dwarf mistletoe
severity and increase longevity in Central Origon.
Forest Ecol Manag 379:11-9.

Mellado A, Zamora R. 2014a. Generalist birds govern the
seed dispersal of a parasitic plant with strong
recruitment constraints. Oecologia 176:139-47.

Mellado A, Zamora R. 2014b. Linking safe sites for
recruitment with host-canopy heterogeneity: The
case of a parasitic plant, Discum album subsp.
Austriacum (Viscaceae). Am J Bot 101(6):000-000.

Mellado A, Zamora R. 2019. Ecological consequences of
parasite host shifts under changing environments:
More than a change of partner. J Ecol, in press.

Muttaqin Z. 2016. Karakter biologi benalu pada jati di
Kebun Benih Klonal (KBK) Padangan, Perum
Perhutani [Character of biology of mistletoe on
teak in Clonal Seed Orchard (CSO), Padangan,
Perum Perhutani (State Owned Forestry
Enterprise)] [Dissertation]. Retrieved from IPB
University.

Muttaqin Z, Budi SW, Wasis B, Siregar IZ, Corryanti.
2016. Peranan burung sebagai agen penyebaran
benalu pada jati di Kebun Benih Klonal (KBK)
Padangan, Perum Perhutani [Role of bird as agent
of mistletoe spread in Padangan teak Clonal Seed
Orchard (CSO), Perum Perhutani (State Owned
Forestry Enterprise)]. Zoo Indonesia 25(2):90-106.

Muttaqin Z, Budi SW, Wasis B, Siregar IZ, Corryanti.
2017. DNA barcode characterization of mistletoe
infestation in teak clonal seed orchard (CSO) in
Padangan, East Java Province, Indonesia.
BIOTROPIA 24(2):140-52.

Nybom H, Weising K, Rotter B. 2014. DNA
fingerprinting in botany: Past, present, future.
Investig Genet 5(1):1-35.

Poerba YS, Sunaryo. 2006. Genetic variation study of
parasite Dendrophthoe pentandra (L.) Miq. based on
random amplified polymorphic DNA marker.
Bogor (ID): Technical Report. Scope Botany,
Center of Biology Research, LIPI.

Reif BP, Mathiasen RL, Kenaley SC, Allan GJ. 2015.
Genetic structure and morphological
differentiation of three Western North American

187



BIOTROPIA Vol. 27 No. 2, 2020

dwarf mistletoes (Arceuthobiunr. Viscaceae). SYST
BOT 40(l):191-207.

Rohlf FJ. 1998. NTSYS-pc version 2.0. Numerical
taxonomy and multivariate analysis system. New
York (US): Exeter Software, Setauket, New York.

Yeh FC, Yang R-C, Boyle T. 1999. POPGENE version
1.31. Microsoft Windows-based freeware for
population genetics analysis. Canada (CA):
Department of Renewable Resources, University
of Alberta, Edmonton, Alberta, Canada.

Yi JS, Kim CW, Lee JK, Meng FJ. 2013. Identification of
populations of Korean mistletoe (Viscum album L.
coloratum) from Gangwon-Do in Korea by AFLP.
Biotechnol & Biotechnol Eq 27(5):4091-7.

Zimisuhara B, Valdiani A, Shaharuddin NA,
Qamaruzzaman F, Maziah M. 2015. Structure and
principal components analyses reveal an
Intervarietal fusion in Malaysian misdetoe fig
( Ficus deltoidea Jack) populations. Int J Mol Sci
16:14369-94.

188