red palm weevil

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Oriental Insects
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Phylogeny of red palm weevil (Rhynchophorus
ferrugineus) based on ITS1 and ITS2
Monther T. Sadder, Polana S. P. V. Vidyasagar, Saleh A. Aldosari, Mahmoud
M. Abdel-Azim & Abdullah A. Al-Doss
To cite this article: Monther T. Sadder, Polana S. P. V. Vidyasagar, Saleh A. Aldosari, Mahmoud
M. Abdel-Azim & Abdullah A. Al-Doss (2015): Phylogeny of red palm weevil (Rhynchophorus
ferrugineus) based on ITS1 and ITS2, Oriental Insects, DOI: 10.1080/00305316.2015.1081639
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2. Oriental Insects, 2015
http://dx.doi.org/10.1080/00305316.2015.1081639
Phylogeny of red palm weevil (Rhynchophorus
ferrugineus) based on ITS1 and ITS2
Monther T. Saddera,b
, Polana S. P. V. Vidyasagarc
, Saleh A. Aldosaric
,
Mahmoud M. Abdel-Azimc
and Abdullah A. Al-Dossb
a
Faculty of Agriculture, Department of Horticulture and Crop Science,The University of Jordan, Amman,
Jordan; b
Department of Plant Production, College of Food and Agricultural Sciences, King Saud
University, Riyadh, Saudi Arabia; c
Department of Plant Protection, College of Food and Agricultural
Sciences, Chair of Date Palm Research, King Saud University, Riyadh, Saudi Arabia
Introduction
Red palm weevil (RPW), Rhynchophorus ferrugineus (Olivier) (Curculionidae:
Coleoptera), was reported as a pest of coconut palm in India for more than one
hundred years ago (Lefroy 1906; Gosh 1912). Recently, it became the major
destructive insect of date palms (Abraham et al. 1998; Gómez and Ferry 1999).
RPW is polyphagous and attacks both commercial palms and several ornamental
palms as well. The invasive pest is very difficult to detect in early stages of infesta-
tion because of its cryptic nature (Nirula 1956). Several control methods have been
practiced to control PRW such as chemical control with pesticides, mechanical
control and cultural practices (Vidyasagar and Bhat 1991).
ABSTRACT
Red palm weevil (Rhynchophorus ferrugineus Olivier)
populations were collected from several regions in Saudi
Arabia. Insects were graded based on different patterns of
pronotum markings. The entire ITS1-5.8S-ITS2 region was
cloned and sequenced for both R. ferrugineus and its related
species R. vulneratus (Panzer). The novel ITS1 sequence
form Rhynchophorus was found to be unique in the current
Genbank database. Discrimination power of ITS1 region
was shown to be much higher than ITS2 region. Penetrance
of different pronotum markings varied from one region to
another. The pronotum-based clustering diverged from
that revealed by ribosomal sequence. Several Indels and
nucleotide substitutions were detected along the ITS1-5.8S-
ITS2 region between R. ferrugineus and R. vulneratus. The data
support a two-species classification rather than considering
them colour morphs of the same species.
© 2015 Taylor & Francis
KEYWORDS
5.8S; ITS1; ITS2;
Rhynchophorus ferrugineus;
R. vulneratus
ARTICLE HISTORY
Received 21 October 2013
Accepted 6 August 2015
CONTACT Monther T. Sadder sadderm@ju.edu.jo
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3. 2 M. T. Sadder et al.
The insect has been spreading steadily westward since the mid 1980s. It reached
the eastern region of Kingdom of Saudi Arabia (KSA) in 1987 (Al-Abdulmohsin
1987) and spread to many other areas in the country (Abraham et al. 1998). A total
of about 25 million date palms are in risk of infestation with RPW in KSA (Ministry
of Agriculture 2010). It was first recorded in northern United Arab Emirates (UAE)
in 1985. Afterwards, it has covered the entire country and neighbouring Oman
(El-Ezaby et al. 1998). In Iran, it was discovered in southern regions in 1990 (Faghih
1996) and in Egypt at the end of 1992 (Cox 1993). In 1994, it had been captured in
Spain (Barranco et al. 1996) and subsequently spread to other EU countries (Italy,
Portugal, France, Greece and Cyprus). RPW was found in Jordan in 1999 (Kehat
1999), in Japan in 2003 (Abe et al. 2009) and in USA in 2010 (USDA-APHIS-PPQ
2010). Therefore, it is considered a universal and key pest of all palm tree species
and new innovative methods should be utilised in combating this menace.
PCR-based DNA markers have been successfully used for estimating genetic
diversity and studying population genetics on a wide range of geographic scale.
These techniques vary in complicity, reliability and information generating
capacity. Among others, randomly amplified polymorphic DNA (RAPD) was
extensively used for genetic diversity studies in different insect species. It was
used to identify necrophagous insects (Benecke 1998), to distinguish species and
strains of rice weevil (Hidayat et al. 1996), gypsy moth (Garner and Slavicek
1996) and Indianmeal moth (Dowdy and MeGaughey 1996). RAPD was also
used to study introgression in budworm species (Deverno et al. 1998), genetic
structuring and gene flow among populations of boll weevils in South America
(Scataglini et al. 2000), gene flow among curculionid weevils (Scataglini et al. 2000;
Vandewoestijne and Baguette 2002; Ayres et al. 2003) and RPW genetic diversity
(Hallett et al. 2004; Gadelhak and Enan 2005). However, RAPD technique is not
suitable for phylogenetic analysis and has several drawbacks (Landry and Lapointe
1996; Bardakci 2001). A phylogeny can be achieved utilising sequence data, e.g.
cytochrome oxidase subunit 1 (COI) (El-Mergawy, Faure, et al. 2011) and internal
transcribed spacer (ITS) (El-Mergawy, Al Ajlan, et al. 2011).
Although the biology of RPW has been studied extensively, the population
genetics and biodiversity is still elusive. The aim of this study was to elucidate
the diversity of RPW from different countries based on pronotum markings and
ribosomal sequence.
Materials and methods
Insect samples
For collection and preservation of R. ferrugineus (Olivier) adult samples, 300 ml
glass jars were filled with 95% ethanol. Jars were securely packed in a spill-proof
box. Insects were collected under the jurisdiction of the Ministry of Agriculture
from seven different areas in Saudi Arabia (Table 1). From each location, fresh
and healthy adults (20 males and females each) were selected and preserved.
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4. Oriental Insects 3
Collection glass jars were labelled (governorate, location, sex, number and name
of collector or authorised person) and sent to the laboratory. In total, 280 insect
samples were procured and kept in refrigerator for further use.
Table 1. Palm weevil samples utilised in this study along with their origins.
*
Wattanapongsiri (1966).
# Country, region Host palm GPS coordinates Species Existence
(years)
1 KSA, Qatif Date palm 26°33′18.21″N R. ferrugineus ~25
49°59′10.45″E
2 KSA, Al Ahsa Date palm 25°23′5.07″N R. ferrugineus ~22
49°36′50.66″E
3 KSA, Kharj Date palm 24°13′0.88″N R. ferrugineus ~17
47°15′43.42″E
4 KSA, Diriyah Date palm 24°44′47.05″N R. ferrugineus ~18
46°33′25.85″E
5 KSA, Wadi Al Dawasir Date palm 20°28′4.79″N R. ferrugineus ~21
44°51′32.48″E
6 KSA, Mecca Date palm 21°25′6.11″N R. ferrugineus ~ 19
39°46′40.67″E
7 KSA, Najran Date palm 17°27′48.29″N R. ferrugineus ~18
44°6′10.41″E
8 UAE, Abu Dhabi Date palm NA R. ferrugineus ~24
9 Spain, Catalonia Canary Island date palm NA R. ferrugineus ~16
10 Italy, Sicily Canary Island date palm NA R. ferrugineus ~14
11 Indonesia Coconut NA R. vulneratus ~100*
Figure1. PronotummarkingsusedtocategoriseRhynchophorusferrugineuspopulationscollected
from KSA.
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5. 4 M. T. Sadder et al.
Adult insects were studies in a careful and non-destructive way to record
the dark colour markings on the pronotum. Depending on the number of dots
or marks present on the pronotum, nine categories were prepared (Figure 1).
Examined insects were grouped into these nine categories and their percentage in
the population was calculated. Data were subjected to hierarchical cluster analysis
based on Euclidean distance using SPSS software version 20.
Molecular analysis
A total of 20 insects, preserved in absolute ethanol, were used for the molecular
analysis (Table 1). Two R. ferrugineus insects (male and female) were used for
each KSA collection region. In addition, two R. ferrugineus samples were used
from UAE, a field sample and laboratory-reared one and two European samples
one from Italy and another from Spain. Furthermore, two R. vulneratus (Panzer)
insects (male and female) from Indonesia served as outgroup species.
Samples were washed thoroughly with water and ground with sand using mor-
tar and pestle. Total gDNA was isolated using Wizard genomic DNA purification
kit (Promega, USA) with some modifications. Each ground insect was incubated
overnight in rotary incubator at 55 °C in insect lysis buffer (500 μl nuclei lysis
solution, 120 μl EDTA (pH 8.0), 17.5 μl of 20 mg/ml proteinase K (Sigma, USA)).
Isolated DNA was quantified with gel electrophoresis and spectrophotometer.
Samples were diluted accordingly and stored at −20 °C.
DNA was amplified using thermocycler machine with heated lid (ABI, USA).
The PCR programme includes 3 min at 95 °C, 35 amplification cycles (94 °C for
30 s; 50 °C for 30 s; 72 °C for 1 min) and an extra extension cycle for 10 min at 72 °C.
PCR reactions were carried out in 20 μl volumes using DNA polymerase master
mix (Promega, USA), 0.4 μM primer (IDT-DNA, Belgium) and 10 ng of gDNA.
PCR primers were designed to amplify ribosomal DNA regions; a primer pair
for 12S ribosomal DNA (forward: 5′-TAGTAGTAGCTATGTTCTTG-3′; reverse:
5′-GAGTGTAAAGTTGTATTTCC-3′) and another pair for 18S(partial)-ITS1–
5.8S-ITS2–28S(partial) region (forward: 5′-AACCTGCGGAAGGATCATTA-3′;
reverse: 5′-AGTCTCACCTGCTCTGAGG-3′).
PCR amplicons were run using 1% agarose gel electrophoresis for 90 min at
100 volt in 1x TAE buffer (40 mM Tris-acetate, 1 mM EDTA, pH 8.0). Hyperladder
VI (Bioline, USA) was used as DNA marker. Gels were stained with SYBR Gold
(Life Technologies, USA) and bands were excised out with scalpel under safe blue
light. DNA was recovered from gel pieces using Wizard Gel and PCR Clean-Up kit
(Promega, USA). Fragments were cloned into pGEM vector (Promega, USA) and
transformed with heat shock into E. coli DH5α cells (Stratagene, USA). Competent
E. coli cells were chemically prepared with CaCl2
(Sambrook et al. 1989). Putative
colonies with recombinant plasmids were selected over LB plates containing ampi-
cillin, IPTG and X-Gal (Sambrook et al. 1989). Plasmids were isolated from over-
night cultures from selected clones using plasmid Miniprep kit (Promega, USA)
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6. Oriental Insects 5
and bi-directionally sequenced with Sanger method (ABI, USA). The sequences
were deposited in Genbank with accession numbers (KC954631 to KC954642).
ITS2 sequences (621 bp) for Rhynchophorus spp. and 5.8S ribosomal sequences
(163 bp)fromrelatedinsectswereretrievedfromGenbank(NCBI2013).Sequences
were aligned using ClustalW multiple alignment function available in BioEdit
(Hall 1999). Alignments were bootstrapped 100 times using SEQBOOT func-
tion available in Phylip (Felsenstein 1989) and subjected to maximum likelihood
method using the DNAML function (Felsenstein 1989; Felsenstein and Churchill
1996). The transition/transversion ratio was two and a global rearrangement was
selected, which causes each possible group to be removed and re-added after the
last species is added to the tree. Extended majority rule consensus tree was gener-
ated using CONSENSE function and plotted with TreeView software (Page 1996).
Results
Pronotum markings
The penetrance of pronotum markings in adult RPW varied for studied regions.
Insects without any marking or with one spot (marking 1) were recorded only
in Mecca with 16.67% and 3.33%, respectively. On the other hand, insects with
multiple spots (marking 9) were prominent in Wadi Al Dawasir region (30%).
Four different markings were recorded for insects from Qatif region with 22.22,
16.67, 22.22 and 38.89% for markings 3, 5, 6 and 8, respectively.
Pronotummarkings-basedclusteranalysisrevealedtwomajorclusters(Figure 2).
The first cluster grouped insects form Najran, Kharj, Mecca and Wadi Al Dawasir.
The second cluster grouped insects from Diriyah, Al Ahsa and Qatif regions.
Phylogeny of RPW
Repetitive 12S ribosomal DNA was used to screen isolated gDNA for PCR. 12S
ribosomal DNA was successfully amplified in all samples. Likewise, the second
designed primer pair covering the entire region spanning ITS1–5.8S-ITS2 was
Figure 2. Dendrogram clustering R. ferrugineus populations collected from KSA based on
pronotum markings.
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7. 6 M. T. Sadder et al.
utilised and successfully amplified in all samples. Insects with both males and
females showed identical sequences, therefore, just one sequence was used in the
phylogenetic analysis. A tree was constructed for R. ferrugineus samples from KSA,
UAE, Spain and Italy (Figure 3). The tree was based on 1325-bp long sequence
comprising the novel ITS1, 5.8S and ITS2 ribosomal regions. Outgroup species
R. vulneratus was clearly separated from R. ferrugineus samples. RPW samples were
clustered in three major clades. The first clade grouped KSA Al Ahsa (KC954632)
with international samples from Spain, Italy and UAE (two samples) (KC954640,
KC954641, KC954638 and KC954639, respectively). European samples were clus-
tered together in a subclade, while UAE sample formed another subclade. The
second clade encompassed two samples: KSA Mecca (KC954637) and KSA Najran
(KC954631). Both are located in the western part of the country. The third clade
grouped the remaining KSA samples, where a subclade grouped KSA Wadi Al
Dawasir (KC954633) and KSA Qatif (KC954634) and another subclade grouped
KSA Kharj (KC954635) and KSA Diriyah (KC954636). Three of them (Wadi Al
Dawasir, Kharj and Diriyah) are located in central part, while Qatif is located in
eastern part.
Published R. ferrugineus data (NCBI 2013) cover only ITS2 sequences. These
sequences were retrieved to construct another phylogenetic tree (Figure 4). The
tree showed a distinct separation for a sample from Japan (Accession numbers
HM043697). On the other hand, a major clade was resolved for KSA samples and
one sample from Majorda, India. Those KSA samples include four samples from
this study (KSA Diriyah, KSA Kharj, KSA Qatif and KSA Mecca) and two samples
published earlier (Accession numbers JX295850 and JX295851). The remaining
samples were weakly clustered in pairs along the tree.
Figure 3. Phylogeny of R. ferrugineus from KSA, UAE, Spain and Italy based on ITS1–5.8S-ITS2
sequence (1325 bp). R. vulneratus served as an outgroup species. Bootstrap values are shown at
branching points.
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8. Oriental Insects 7
Rhynchophorus spp
An additional phylogenetic investigation was applied to reveal the relationship
among Rhynchophorus spp. Available ITS2 sequences for related species R. phoe-
nicis, R. bilineatus and R. palmarum were retrieved from Genbank (NCBI 2013).
The phylogenetic tree clearly separated the outgroup species Sitophilus zeamais
(Figure 5). The tree revealed a consequent evolution of Rhynchophorus spp.,
where R. palmarum was branched out first, followed by R. phoenicis and then R.
­ferrugineus. On the other hand, the two species R. bilineatus and R. vulneratus
clustered in one clade with good bootstrap value (63%). Moreover, the novel ITS1
sequence supports the speciation of palm weevils into two species: R. ferrugineus
and R. ­vulneratus. The alignment of the consensus sequence between R. ferrugi-
neus and R. vulneratus resolved eleven Insertion–Deletion (Indel) polymorphisms
Figure 4. Phylogeny of R. ferrugineus from this study (Accession numbers KC955631–42) and
published data (NCBI 2013) based on ITS2 sequence (621 bp). Bootstrap values are shown at
branching points.
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9. 8 M. T. Sadder et al.
and sixteen nucleotide substitutions in the ITS1 region (Table 2), while one Indel
and eleven nucleotide substitutions were detected in ITS2 region (Table 2).
Complete 5.8S ribosomal sequence of the genus Rhynchophorus was further
utilised to get a prospective of palm weevils among related insects. The 5.8S
sequence of 19 different insects were retrieved and utilised to construct a phy-
logenetic tree with Rhynchophorus (Figure 6). The tree was resolved into three
major clades, while Cryptolaemus montrouzieri did not cluster with any clade.
First clade clustered the genera Campoletis, Anselmella, Mallada, Dasymutilla,
Anthonomus and Sitophilus. Second clade grouped the genera Bracon, Leiophron,
Tenebrio, Trichogramma and Solenopsis. And finally, Rhynchophorus was clustered
in a third clade with Altica litigata and Lysathia ludoviciana.
Figure 5. Phylogeny of Rhynchophorus species based on ITS2 sequence (621 bp). R. ferrugineus
(KC954636KSADiriyah)andR.vulneratus(KC954642)arefromthisstudy.R.phoenicis(HM043703),
R. bilineatus (HM043701) and R. palmarum (HM043699) are from Genbank (NCBI 2013). Sitophilus
zeamais (AF276518) served as an outgroup species. Bootstrap values are shown at branching
points.
Table 2. Indels and nucleotide substitutions detected in ITS1 and ITS2 regions sequenced from
both R. ferrugineus and R. vulneratus.
Base Region R. ferrugineus R. vulneratus Base Region R. ferrugineus R. vulneratus
30 ITS1 T 636 ITS1 A C
31 ITS1 T 672 ITS1 T
44 ITS1 T 673 ITS1 C
45 ITS1 A 674 ITS1 G
46 ITS1 T 690 ITS1 G C
47 ITS1 A 695 ITS1 G T
93 ITS1 T C 704 ITS1 G A
120 ITS1 G A 998 ITS2 C G
212 ITS1 A G 917 ITS2 T A
228 ITS1 G A 919 ITS2 T C
233 ITS1 T 942 ITS2 T C
234 ITS1 A 983 ITS2 A G
309 ITS1 G C 1032 ITS2 C T
318 ITS1 A T 1072 ITS2 A ∙
347 ITS1 C T 1081 ITS2 C T
369 ITS1 A G 1177 ITS2 T C
489 ITS1 C G 1200 ITS2 A G
507 ITS1 A C 1252 ITS2 G A
508 ITS1 C T 1264 ITS2 T A
516 ITS1 T G
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10. Oriental Insects 9
Discussion
Ministry of Agriculture in KSA has implemented various control programmes to
combat RPW. A pioneer IPM project for RPW was carried out in 1992 (Abraham
and Vidyasagar 1992). The project included pheromone trapping techniques and
pest management procedures. However, RPWs are still spreading in KSA and
worldwide. Our data revealed a detectable genetic diversity among investigated
samples. Such diversity could help the insect to escape management schemes and
combating plans.
Collected populations of R. ferrugineus demonstrated a wide spectrum of
pronotum markings (Figure 1). In a comprehensive survey, 24 different pat-
terns of pronotum markings were recorded for R. ferrugineus (Wattanapongsiri
1966), only two of which overlapped with ours (Figure 1). It is worth noting
that Wattanapongsiri’s survey (1966) was conducted when the insects were still
restricted to Southeast Asia (India, Philippines, Vietnam, Thailand, Taiwan and
Burma). Accordingly, it is important to understand the nature behind changes in
pronotum markings along the spread and distribution of the insect.
We investigated the potential use of these markings for diversity studies. The
pronotum markings-based tree (Figure 2) was compared with the phylogenetic
tree (Figure 3). Clustering data correlates for some populations but contradicts for
others. Similar conclusions were found in earlier reports utilising different genetic
data. Marvaldi et al. (2002) studied phylogeny of weevils based on combined 18S
rDNA and morphological data. They suggested that diversification in weevils
Figure 6. Phylogeny of Rhynchophorus (this study) and related insects based on 5.8S sequence
(163 bp). Accession numbers are followed by scientific names (NCBI 2013). Bootstrap values are
shown at branching points.
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11. 10 M. T. Sadder et al.
was accompanied by niche shifts in host–plant associations and larval habits.
Pronounced conservatism was evident in larval feeding habits, particularly in the
host tissue consumed. On the other hand, a genetic relationship between three
phenotypic different forms of RPW from one location of KSA Al Ahsa (Al-Hassa)
region was investigated using six RAPD primers (Al-Ayied et al. 2006). Although
weevils were collected from the same geographical region, it was suggested that
banding profile acquired from black and brown coloured morphs were genetically
closer compared to the brown spotted morphs. Intra genetic variation was lower in
black than brown and brown spotted morphs. The authors explained this genetic
variation by either mutated non-spotted/spotted morphs or a different weevil race.
Evaluation of genetic diversity based on morphological traits does not usually
provide accurate estimates of genetic differences as they are highly influenced by
environmental factors. This can also be influenced by genetic diversity between
insect species and even within populations of the same species (Apostol et al. 1993;
Stevens and Wall 1995). Nonetheless, some pronotum markings were found to
be more frequent than others in certain RPW populations. Therefore, it is highly
recommended to investigate both the penetrance and expressivity of pronotum
markings under controlled laboratory conditions. This can be achieved by utilising
planned mating systems for insects with various pronotum markings.
Limited genetic diversity studies have been published for the RPW (Salama and
Saker 2002; Gadelhak and Enan 2005; Al-Ayied et al. 2006). From Egypt, three
morphologically different forms of RPWs collected from infested date palm fields
were subjected to nine RAPD primers (Salama and Saker 2002). PCR amplification
products indicated that three different RAPD patterns align with three different
forms of weevils. They concluded that the detected genetic variations could be
assigned to the generation of possible mutants or they may belong to different
races. Seven other populations of the RPW insects collected from different loca-
tions from UAE were analysed using six RAPD 10-mers (Gadelhak and Enan
2005). A 51.4% of the generated bands were polymorphic. The populations were
clustered into three groups with genetic variability ranging from 38 to 94% and
good correlation between genetic and geographical distances among investigated
populations. However, RAPD markers generate non-specific amplifications, which
is a major drawback when applied for genetic diversity studies. DNA from RPW
gut microorganism (Khiyami and Alyamani 2008) can also be amplified. These
microorganisms vary from one isolate to another (Ibrahim et al. 2011) and from
one season to another (Jia et al. 2013).
Phylogenetic data of R. ferrugineus were limited to ITS2 region as this was
the only published sequence (El-Mergawy, Al Ajlan, et al. 2011; NCBI 2013).
El-Mergawy, Faure, et al. (2011) could not detect ITS2 polymorphism among
different RPW samples from thirteen international locations. The limited discrim-
ination power of ITS2 resulted in consolidation of some clades (Figure 4), which
could be resolved after incorporating ITS1 sequence (Figure 3). This scenario
emphasises the polymorphic features embedded in the novel ITS1 sequence for
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12. Oriental Insects 11
R. ferrugineus. This sequence is so unique that no single blastn hit could be
detected in the Genbank (NCBI 2013).
There are reports of about ten species of Rhynchophorus distributed in various
geographical regions of the world. Among them, R. ferrugineus and R. ­vulneratus
are sympatric in nature. Based on morphological and molecular studies, it was
suggested to consider them two morphs of one species (Hallett et al. 2004).
Our findings disagree with this classification for several reasonable facts. The
most comprehensive description of the genus Rhynchophorus was published by
Wattanapongsiri (1966), where several morphological features were described
to distinguish between the two species R. ferrugineus and R. vulneratus. On the
other hand, supporting data for considering them as synonyms (Hallett et al.
2004) was based on two molecular tools. The first was RAPD technique, which
cannot be utilised for phylogenetic analysis, while the second was COI, which
has some inherent limitations. In some cases, COI was found to have little reso-
lution at the species level (Derycke et al. 2010), while in other cases, the nuclear
copies of COI can lead to overestimation of the taxonomic diversity (Song et al.
2008). Nonetheless, our conclusion is supported by recent work based on COI
(Rugman-Jones et al. 2013). Moreover, detected Indels and nucleotide substi-
tutions based on both novel ITS1 and ITS2 sequences (Table 2) revealed an
undisputed evidence of molecular diversification between R. ferrugineus and
R. vulneratus. Finally, the phylogenetic analysis encountering available sequences
of related Rhynchophorus spp. revealed a striking genetic proximity between
R. vulneratus and R. bilineatus rather than between R. vulneratus and R. ferrug-
ineus (Figure 5).
Acknowledgements
The authors would like to thank the Ministry of Agriculture in KSA for facilitating the collec-
tion of insect samples and our collaborators for kindly supplying the International samples.
Disclosure statement
No potential conflict of interest was reported by the authors.
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