For: Journal of Economic Entomology (Household and Structural Insects)

Corresponding author:
Chow-Yang Lee
School of Biological Sciences
Universiti Sains Malaysia
11800 Penang, Malaysia.
Email: chowyang@usm.my

Molecular phylogenetics of the Asian subterranean termite,

Coptotermes gestroi (Wasmann) and the Philippines milk
termite, Coptotermes vastator Light (Isoptera: Rhinotermitidae)

BENG-KEOK YEAP1, AHMAD SOFIMAN OTHMAN1, VANNAJAN SANGHIRAN LEE2

1,3
AND CHOW-YANG LEE

1
School of Biological Sciences, Universiti Sains Malaysia, 11800 Penang, Malaysia.
2
Department of Chemistry, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand.

_________________
3
Corresponding author, email: chowyang@usm.my.
 Yeap et al. Molecular phylogenetics of C. gestroi and C. vastator

ABSTRACT The Asian subterranean termite, Coptotermes gestroi (Wasmann) and the

Philippine milk termite, Coptotermes vastator Light were compared using molecular

phylogenetic techniques. Partial sequences of the ribosomal RNA small subunit 12S,

ribosomal RNA large subunit 16S and mitochondrial COII were obtained from 9

populations of C. gestroi from South East Asia (Malaysia, Singapore, Thailand and

Indonesia) and 4 populations of C. vastator from the Philippines and Hawaii (USA). In

addition, 4 populations of Coptotermes formosanus Shiraki and Globitermes sulphureus

(Haviland) were used as the outgroups. Consensus sequences were obtained and aligned.

C. vastator and C. gestroi are synonymous, based on high sequence homology across the

12S, 16S and COII genes and phylogenetic analysis.. The interspecific pairwise

sequence divergence, based on uncorrected “p” distance between C. gestroi and C.

vastator, varied only up to 2.79 %. Other findings that further support the synonymity of

the two species are also discussed.

KEY WORDS Coptotermes gestroi, Coptotermes vastator, 12-S ribosomal DNA, 16-S

ribosomal DNA, COII mitochondrial DNA, synonymity.

Amongst the termite genera within Rhinotermitidae, Coptotermes is probably regarded as

the most economically important genus worldwide. Several species of Coptotermes

including the Formosan subterranean termite, Coptotermes formosanus Shiraki, and the

Asian subterranean termite, Coptotermes gestroi (Wasmann) have been known for their

destructive nature to buildings and structures in subtropical and tropical regions,

2
 Yeap et al. Molecular phylogenetics of C. gestroi and C. vastator

respectively. Su (2002) reported that C. formosanus accounted for a considerable

proportion of the total damage by termites worldwide.

In Malaysia, Thailand and Singapore, C. gestroi contributes more than 85% of the

total termite damages in buildings and structures in the urban area (Lee 2002; Lee et al.

2003). Geographic distribution of C. gestroi occurs from Assam through Burma and

Thailand to Malaysia and the Indonesian archipelago (Kirton & Brown 2003). It is

marked as the most destructive pest termite, damaging structural wood in the urban areas

in Southeast Asia (Kirton 2005). C. gestroi, reported as C. havilandi in Gay (1969), has

been brought into new geographical regions including parts of North and South America

and Caribbean by “hitch-hiking” in the cargo onboard ships and wooden components of

sailing vessels.

Despite C. gestroi being widely distributed in the tropical South East Asia,

Coptotermes vastator Light is the primary subterranean termite species of urban

environments in the Philippines. C. vastator is a serious structural pest that accounts for

>90% of the termite damages to timber and wooden structures. These damages cost

nearly USD 1 million to residential and commercial properties of the Mariana Islands and

between USD 8 and 10 million for the damages in and around Manila (Yudin 2002).

This species was unintentionally introduced to Hawaii in 1918 during a shipment of

banana stumps from Manila (Ehrhorn 1934; Gay 1969). C. vastator was found infesting

a single structure in Honolulu at 1963, and it was not discovered again in Hawaii until

1999. To date, C. vastator is recognized and known to cause major problems in the

islands of Hawaii, Guam and Saipan (Woodrow et al. 2001; Yudin 2002).

3
 Yeap et al. Molecular phylogenetics of C. gestroi and C. vastator

Both C. gestroi and C. vastator are very similar based on analysis of morphological

characteristics, and it was suspected that C. vastator is a junior synonym of C. gestroi

(Kirton & Brown 2003; Kirton 2005). However, no attempt has been executed so far to

address this issue from a molecular phylogenetic perspective. Molecular phylogenetic

analyses are able to reveal the relationship among populations and to differentiate species

regardless of the termite caste (Szalanski et al. 2003). Mitochondrial genes are known to

evolve more rapidly than nuclear genes and are therefore good markers to analyze

relatively close relationships, such as species relationship within a genus (Miura et al.

2000). In this study, we combined analysis of three mitochondrial genes (12S, 16S, and

COII) to determine the relationship between C. gestroi and C. vastator. Together with

morphological information, these phylogenetic analyses lead us to propose that C.

vastator and C. gestroi are synonymous.

Materials and Methods

Termite samples and Genbank sequences. Termite samples collected from Malaysia,

Thailand, Singapore, Indonesia, the Philippines, Japan and the United States (Table 1)

were preserved in absolute ethanol. The pictures of soldier heads and whole bodies for

each species were taken using an Olympus SZ2-LGB microscope attached with a digital

imaging camera. Morphometric measurements of mandible length, maximum head width,

and head length were determined for populations of the three species of Coptotermes (n =

15 – 20 soldiers per population). Published sequences from Genbank

(www.ncbi.nlm.nih.gov) on C. gestroi and C. vastator were also included in phylogenetic

analyses.

4
 Yeap et al. Molecular phylogenetics of C. gestroi and C. vastator

DNA Extraction. Absolute ethanol preserved specimens were washed with distilled

water and dried on a filter paper. Specimens were then placed in a 1.5 ml tube and

homogenized in STE buffer (50 mM sucrose, 25 mM 1M Tris-HCL, pH 7, and 10 mM

0.5M EDTA). DNA was extracted by incubating with Proteinase K at 55ºC for 30 min,

with 10% SDS for further 3 hours incubation. After a single extraction using

phenol/chloroform, total DNA was precipitated with ethanol and then resuspended in 25

l dH2O.

Data collection. Amplification of the 12S, 16S, and COII genes was achieved by

polymerase chain reaction (PCR) using primers shown in Table 2 (Liu & Beckenbach

1992; Simon et al. 1994; Kambhampati & Smith 1995; Miura et al. 1998; Hayashi et al.

2003). PCR amplification was performed in a standard 25- or 50-
l reaction volume

with 2
l of total genomic DNA, 1 pmol of each primer, 1.5 mM MgCl2, 2 mM dNTPs,

and 5U/
l Taq DNA polymerase. Amplification was accomplished in a MJ Research

PTC-200, Peltier Thermol Cycle, with a profile consisting of a precycle denaturation at

94ºC for 2 min, a postcycle extension at 72ºC for 10 min, and 35 cycles of a standard

three-step PCR (51.3, 53.1, and 58.2 ºC annealing). Reaction conditions for each primer

set were optimized with respect to MgCl2 concentration and annealing temperature. 2 μl

of each PCR product was visualized using a UV transilluminator on a 1.2% agarose gel

containing 0.5 mg/ml ethidium bromide. Double-stranded PCR products were purified

using a SpinClean Gel Extraction Kit (column). Amplified DNA was sent to Macrogen

Inc (Seoul, South Korea) for DNA sequencing. Sequencing was conducted under

BigDyeTM terminatior cycling conditions. The reacted products were then purified using

ethanol precipitation and ran using Automatic Sequencer 3730xl.

5
 Yeap et al. Molecular phylogenetics of C. gestroi and C. vastator

Nucleotide Data Analysis. BioEdit v7.0.5 software was used to edit individual

electropherograms and form contigs. Multiple consensus sequences were aligned using

CLUSTAL X. The alignment results were adjusted manually for obvious alignment

errors. The data were imported into PAUP4.0 (Swofford 2000) and analyzed to generate

distance based neighbour -joining (NJ) tree and character based parsimony tree. The NJ

tree was calculated using distances from nucleotide data using an uncorrected ‘p’ distance

procedure. Bootstrapping was carried out on trees obtained from the two analyses. A

bootstrap test was used to test the reliability of trees (Felsenstein 1985). Parsimony

analysis was carried out using the heuristic search. Under the heuristic search, RANDOM

addition sequence procedure was employed using 1000 replicates and TBR branch

swapping was carried out. Equally parsimonious trees obtained were then summarized

using strict consensus. Support for clades within a tree was determined by bootstrap

analysis (Felsenstein, 1985) using 1000 replicates of heuristic searches with randon

addition sequences.

Amino Acid Sequence Analysis. The selected amino acid sequences of 12S, 16S, and

COII of C. gestroi and C. vastator were translated from nucleotide using ExPASy

Proteomics tools* with the mitochondrial translation codes AGA GCG ACG, TTA CGC,

and ATG ACA ACA, respectively. Multiple sequence alignment was derived using the

CLUSTAL X program, and default parameters were applied (Thompson et al. 1997). The

aligned result was inspected and adjusted manually to minimize the number of gaps and

insertions.

* available at http://us.expasy.org/

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 Yeap et al. Molecular phylogenetics of C. gestroi and C. vastator

Results and Discussion

Morphology. The soldier termites were used for morphological studies (Figure 1). C.

formosanus was readily distinguished from C. vastator and C. gestroi with two pairs of

setae projecting dorso-laterally from the base of the fontanelle, compared with only a pair

of setae in the latter two species (Figure 2). It is difficult to distinguish C. vastator from

C. gestroi by the size and shape of its post mentum and head, as they are highly parallel.

As reported in Kirton and Brown (2003), there is a continuous variation of size and shape

in a single species. The morphology of termites can be influenced by the age and state of

the colony or by environmental conditions. Furthermore, additional variability in

coloration may be attributed to sample age and storage condition (Scheffrahn et al. 2005).

Therefore, the slight variation in the size range between C. vastator and C. gestroi is not

significant to classify them into two distinct species (Table 3).

Nucleotide analyses. DNA sequences of approximately 420, 428, and 680 basepairs

were obtained for the genes 12S, 16S, and COII respectively. For 12S, 16S, and COII

genes, 35 bp from the 5’ end of the amplicon was excluded in the analysis to facilitate

phylogenetic comparisons with existing GenBank DNA sequences. The average base

frequencies were A = 0.45, C = 0.22, G = 0.13, and T = 0.20 for the 12S gene, A = 0.43,

C = 0.25, G = 0.11, and T = 0.22 for the 16S gene, A = 0.40, C = 0.24, G = 0.13, and T =

0.23 in the COII gene.

The multiple alignment of 12S gene sequences, including the outgroup taxon has 365

characters, of which 320 are constant and 14 parsimony-informative. For the 16S gene,

there are 385 characters, of which 326 are constant and 20 parsimony-informative. There

7
 Yeap et al. Molecular phylogenetics of C. gestroi and C. vastator

are 947 characters, of which 404 are constant and 75 parsimony-informative in the

alignment for COII gene. When these three genes were combined, the multiple sequence

alignment had 1729 characters, of which 1466 are constant and 142 parsimony-

informative.

The interspecific pairwise sequence divergence based on uncorrected “p” distance

between C. gestroi and C. vastator, C. gestroi and C. formosanus, C. vastator and C.

formosanus, as well as the three Coptotermes species and the outgroup are shown in

Table 4. The highest interspecific pairwise sequence divergence between C. gestroi and

C. vastator was 2.79 %. This value was very similar to the divergence between the two

populations of the same species, C. gestroi from Cibinong (Indonesia) (CG001IN) and

Penang (Malaysia) (CG001MY) for the COII gene (2.36 %). This finding implies that

the genetic distance between both species was minimal.

Within the aligned nucleotides of the 12S gene, there are only 2 characters (at

nucleotide positions -61 and -170) in which C. vastator differs from that of C. gestroi.

However, at nucleotide -170, C. gestroi from Cibinong (CG001IN) and Bogor

(CG002IN) (both from Indonesia) has the same nucleotide (T) as C. vastator. This

showed that C. gestroi from Indonesia is extremely similar in its 12S DNA sequence with

C. vastator.

There are 3 characters (at nucleotide positions -39, -103, and -135) that were different

between C. gestroi and C. vastator across the 16S gene sequence. However, C. gestroi

from Indonesia shared the same nucleotide (C) with C. vastator at position 39.

Among the 5 characters (at nucleotide positions -174, -350, -490, -493, and -646) in

the COII gene of C. vastator that were different from C. gestroi, positions 174 and 490

8
 Yeap et al. Molecular phylogenetics of C. gestroi and C. vastator

(C and T, respectively) were similar between C. gestroi from Indonesia and C. vastator.

Across the 3 genes, the transition rate was 100 %. Substitutions involving C and T

occurred between C. gestroi and C. vastator. Comparable with the results in Ye et al.

(2004), COII is considered as the fastest evolving gene of those examined, while 12S is

the most conserved gene. In this study, high similarity in the sequences of the three genes

suggests that C. gestroi and C. vastator are likely synonymous in nature. Point

mutations were found in sequences across the three genes of C. gestroi from Indonesia

(CG001IN and CG002IN) and C. vastator from colony 3 (CV003PH) from the

Philippines, making these populations deviate from the other populations in the NJ tree.

Comparison of amino acid sequences. The amino acid sequences of 12S, 16S, and

COII between C. gestroi and C. vastator were also compared. The sequences were

deduced from the nucleotide using with the mitochondrial translation codes AGA GCG

ACG, TTA CGC, and ATG ACA ACA respectively. Comparison of the translated amino

acid sequences of 12S, 16S, and COII demonstrated that all C. gestroi are homologous

throughout the 3 genes whereas C. vastator samples differ in 16S and COII gene.

Variations between C. gestroi and C. vastator were observed as COII > 16S > 12S.

For the 12S gene, two nucleotide differences mentioned above corresponded to only

one amino acid difference at position 54 from tyrosine for C. gestroi to isoleucine for C.

vastator. There was no variation in the amino acid sequence for the nucleotide at position

61.

For the 16S gene, the difference of the two clades can be classified into 2 groups of C.

vastator. The first group is CV001HW, CV001PH, CV002PH which differ from C.

gestroi in 2 amino acid positions at 31 (T to I for C. vastator) and 42 (P for C. gestroi to

9
 Yeap et al. Molecular phylogenetics of C. gestroi and C. vastator

S for C. vastator) whereas the other group is CV003PH which differs less from C.

gestroi. Only one position difference at 49 from N for C. gestroi to S for C. vastator was

observed. This difference is not detected in the first group.

In the COII gene, all C. gestroi and C. vastator in the case of CV002PH and

CV003PH have identical amino acid residues. This shows the close relationship between

these two groups. There are three amino acid differences in the case of CV001PH at

amino acid position 57, 79, and 95. In summary, the highest variation in amino acid

sequences was found in C. vastator for the CV001PH sample, especially in the COII gene

with four amino acid residue differences. The other C. vastator sequences were similar to

C. gestroi which showed only a single amino acid difference in 12S and 16S and no

difference in COII gene.

Phylogenetic relationships inferred from 12S, 16S, and COII genes. Neighbour-

joining dendrograms based on genetic distance from each of the genes were generated to

examine relationships among C. vastator, C. gestroi and C. formosanus. Maximum-

parsimony (MP) and Maximum Likelihood (ML) analyses on the same data were

performed (trees not shown) to determine if there are conflicts in tree topology from

those obtained by NJ. Results obtained from MP and ML analyses showed the same

topology as the one revealed by the NJ tree.

Phylogenetic analyses of 12S, 16S and COII genes sequences with Globitermes

sulphureus (Haviland) as the outgroup, revealed two major clades within the Coptotermes

genus (Figure 3 – 5). One clade includes C. gestroi and C. vastator with strong bootstrap

support of 99%, 100%, and 100%, respectively for the 3 genes. Within the Coptotermes

clade, there are two sub-clades, one with various populations of C. gestroi and C.

10
 Yeap et al. Molecular phylogenetics of C. gestroi and C. vastator

vastator (the C. gestroi/vastator subclade). The second Coptotermes clade is composed

of C. formosanus from Japan and Hawaii. The same conclusion can be made when the

three genes were combined and analysed concurrently in a single tree (Fig 6).

In the 12S and COII genes, populations from Cibinong (CG001IN) and Bogor

(CG002IN), Indonesia were distinct from the other C. gestroi populations due to the point

mutations within C. gestroi at nucleotide positions -166 in the 12S gene and at four

positions in the COII gene (positions -45, -138, -420, and -466). Point mutations were

also found in C. vastator (colony 3) from Laguna, Philippines (CV003PH) which slightly

diverged from the other C. vastator populations (at nucleotide positions -253 in 12S gene,

-373 and -608 in COII gene) .

In 16S gene tree, there was a branch composed of C. gestroi from Indonesia

(CG002IN) and C. vastator from Philippines (CV003PH) although it was supported by

only 55 % of bootstrap value. This was due to both these individuals sharing unique

nucleotides at positions 103 and 135.

Geographical Distribution C. gestroi is a native tropical Asian species, but has

spread to the Southeastern Asian region via human activity. Zoogeographical regions

inhabited by this species include Nearctic, Neotropical, and Oriental regions. It was

collected in the Marquesas Islands (Pacific Ocean) in 1932, Mauritius and Reunion

(Indian Ocean) in 1936 and 1957, respectively. In the New World tropics, C. gestroi was

first reported in Brazil (earlier known as Coptotermes havilandi which was later

described as a junior synonym to C. gestroi [Kirton & Brown 2003]) in 1923 and in

Barbados in 1937. Later, the distribution of this species became endemic to Antigua and

Barbuda, Grand Cayman, Grand Turk, Jamaica (Montego Bay and Port Antonio), Little

11
 Yeap et al. Molecular phylogenetics of C. gestroi and C. vastator

Cayman, Montserrat, Nevis, Providenciales, Peurto Rico (San Juan), St. Kits, and the

United States. It has also been collected in southern Mexico (Scheffrahn and Su 2000).

On the other hand, Coptotermes vastator is a serious pest in Guam and the

Philippines. However, little information has been published on this species and its

distribution remains unclear. C. vastator was first found in Hawaii in 1963 but was

eliminated and not detected for the following 25 years. However, this species has recently

resurfaced to infest several places in Hawaii and it has become a serious concern to the

pest control industry (J.K. Grace, personal communication).

Implication of the findings to pest management industry. Kirton (2005) reviewed

the importance of accurate termite identification to the pest management industry. With

the recognition of a single pest species of Coptotermes in South East Asia, information

concerning C. gestroi and C.vastator species from different geographical regions can be

pooled. Industry will also be able to avoid duplicative testing of termite management

strategies for what was originally thought to be different pest species in different regions,

which will in the long run save time, money and resources (Kirton 2004).

In summary, morphometric and molecular phylogenetic analyses using three

mitochondrial genes in this study suggested that both C. gestroi and C. vastator are

synonymous. In particular, (1) the morphological characters of both C. gestroi and C.

vastator are highly parallel, (2) the genetic distance between C. gestroi and C. vastator

was very low (0.54 - 2.79 %), and (3) there was only a minimal nucleotide difference

between C. gestroi and C. vastator as compared to C. gestroi/formosanus and C.

vastator/formosanus. There are only a total of ten characters difference detected across

the three genes between C. gestroi and C. vastator. Moreover, out of the ten, only 6

12
 Yeap et al. Molecular phylogenetics of C. gestroi and C. vastator

characters are species specific. In contrast, C. gestroi/vastator differ from C. formosanus

at 166 nucleotide positions across the three genes. This seems to suggest that nucleotide

differences between C. gestroi and C. vastator might only be due to population variation.

We envisage that if a higher number of populations were to be analysed, the number of

species specific characters might be reduced. More studies using larger sample sizes and

more populations within the mentioned geographical ranges are currently under

investigation to further substantiate current findings.

Acknowlegdments

We thank M.E. Scharf (University of Florida, Gainesville) for reviewing the manuscript

draft, J.K. Grace for information on the current status of C. vastator infestation in

Hawaii, and the following individuals who had helped in the collection of the termite

specimens in this study: Charunee Vongkaluang (Royal Forest Department, Thailand),

Carlos Garcia (Forest Product Department, the Philippines), Julian Yates III (University

of Hawaii, Honolulu, USA), Tsuyoshi Yoshimura (Kyoto University), Kean-Teik Koay

(NLC General Pest Control, Petaling Jaya, Malaysia), Sulaeman Yusuf (Indonesian

Institute of Science) and John Ho (Singapore Pest Management Association). This study

constitutes a portion of the Ph.D. thesis of the senior author.

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mitochondrial COII gene sequences. Ann. Entomol. Soc. Am. 91: 515-523.

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Termitidae: Nasutitermitinae), with evidence from morphology, genetics, and

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Su, N.-Y. 2002. Novel technologies for subterranean termite control. Sociobiology 40(1):

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Received _____________________; accepted _________________________

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 Yeap et al. Molecular phylogenetics of C. gestroi and C. vastator

List of tables and figures

Table 1. Termite specimens and Genbank published sequences used in this study.
Table 2. Primers used for PCR and sequencing
Table 3. Measurements of termite soldiers
Table 4. Genetic distances (%) among C. gestroi, C. vastator, C. formosanus and G.
sulphureus (outgroup) across the 3 genes

Figure 1. Pictures of C. gestroi soldier (A), C. vastator soldier (B), and C. formosanus
soldier (C).

Figure 2. Lateral view of C. gestroi, C. vastator, and C. formosanus with focus on

fontanelle .

Figure 3. 12S gene neighbour-joining tree, created with PAUP4.0 (Swofford 2000).
Bootstrap values for 1000 replicates are listed above the branches supported at

50%. GenBank accession numbers represent the samples that are pooled from
the NCBI database.

Figure 4. 16S gene neighbour-joining tree, created with PAUP4.0 (Swofford 2000).
Bootstrap values for 1000 replicates are listed above the branches supported at

50%. GenBank accession numbers represent the samples that are pooled from
the NCBI database.

Figure 5. COII gene neighbour-joining tree, created with PAUP4.0 (Swofford 2000).
Bootstrap values for 1000 replicates are listed above the branches supported at

50%. GenBank accession numbers represent the samples that are pooled from
the NCBI database.

Figure 6. Neighbour-joining tree analyzing a combination of the three genes 12S, 16S
and COII concurrently. The tree was created with PAUP4.0 (Swofford 2000).
Bootstrap values for 1000 replicates are listed above the branches supported at

50%. GenBank accession numbers represent the samples that are pooled from
the NCBI database.

17
 Yeap et al. Molecular phylogenetics of C. gestroi and C. vastator

Table 1. Termite specimens and Genbank published sequences used in this study.

Sample code Species Collecting sites GenBank Accession No.

Samples from 12S 16S COII
this study
CG001MY C. gestroi Malaysia, Penang, USM.
CG004MY C. gestroi Malaysia, Kuala Lumpur,
Bangsar.
CG005MY* C. gestroi Malaysia, Muar.
CG001SG C. gestroi Singapore, Serenity Terr.
CG002SG C. gestroi Singapore, Serangoon.
CG001TH C. gestroi Thailand, Bangkok1.
CG002TH C. gestroi Thailand, Bangkok2.
CG001IN C. gestroi Cibinong, Indonesia.
CG002IN C. gestroi Bogor, Indonesia.
CF001JP C. formosanus Japan, Wakayama.
CF002JP C. formosanus Japan, Wakayama.
CF003JP C. formosanus Japan, Okayama.
CF001HW C.formosanus USA, Hawaii, Oahu.
CV001HW C. vastator USA, Hawaii, Oahu.
CV001PHI C. vastator Los Banos, Laguna
Philippines, colony1.
CV002PHI C. vastator Los Banos, Laguna
Philippines, colony2.
CV003PHI C. vastator Los Banos, Laguna
Philippines, colony3.
GS001MY G. sulphurues Malaysia, Penang, USM.

Samples from
other studies
C. gestroi Malaysia, Penang Island. AY536388
C. gestroi Thailand, Bangkok. AY302709
C. gestroi USA, Miama, Florida. AY558907

C. gestroi Turks and Caicos Islands: AY558906

Grand Turk.
C. gestroi Antigua and Barbuda. AY558905

C. vastator Philippines, Manila. AY536394

C. vastator Philippines, Wedgewood. AY536393
C. vastator Philippines, Manila. AY536392
C. vastator Philippines, Wedgewood. AY302713
C. vastator Philippines, Manila. AY302712
C. vastator USA, Honolulu, Hawaii. AY302711
* Dried sample

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 Yeap et al. Molecular phylogenetics of C. gestroi and C. vastator

Table 2. Primers used for PCR and sequencing

Gene Name Sequence (5’- 3’)
16S LR-J-13007 TTA CGC TGT TAT CCC TAA
LR-N-13398 CGC CTG TTT ATC AAA AAC AT

12S 12SF TAC TAT GTT ACG ACT TAT

12SR AAA CTA GGA TTA GAT ACC C

COII C2F2 ATA CCT CGA CGW TAT TCA GA

TKN3785 GTT TAA GAG ACC AGT ACT TG

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 Yeap et al. Molecular phylogenetics of C. gestroi and C. vastator

Table 3. Measurements of termite soldiers of three species of Coptotermes.

________________________________________________________________________
Species (n) Mean range (mm)
______________________________________________________
Max head width Head width at Head length to base
base of mandibles of mandibles
________________________________________________________________________
C. vastator (135) 2.35 (2.27 – 2.47) 1.29 (1.15 – 1.32) 2.93 (2.66 – 3.02)
C. gestroi (80) 2.33 (2.22 – 2.44) 1.22 (1.15 – 1.31) 2.62 (2.45 – 2.80)
C. formosanus (80) 2.51 (2.41 – 2.60) 1.31 (1.25 – 1.38) 3.07 (2.90 – 3.17)
________________________________________________________________________

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 Yeap et al. Molecular phylogenetics of C. gestroi and C. vastator

Table 4. Genetic distances (%) among C. gestroi, C. vastator, C. formosanus and G.

sulphureus (out group) across three genes.

C. gestroi
12S 16S COII
C. gestroi 0.00 0.00 0.00-2.36
C. vastator 0.55-0.82 0.79 0.64-2.79
C. formosanus 3.59-3.61 5.00 7.66-9.03

C. vastator
12S 16S COII
C. gestroi 0.54-0.82 0.79 0.64-2.79
C. vastator 0.00 0.00-1.05 0.43-0.64
C. formosanus 3.31-3.59 4.74-5.52 7.63-8.28

C. formosanus
12S 16S COII
C. gestroi 3.59-3.61 5.00 7.66-9.03
C. vastator 3.31-3.59 4.74-5.52 7.63-8.28
C. formosanus 0.00 0.00 0.11-0.32

G. sulphurues
12S 16S COII
C. gestroi 10.32-10.57 13.20-13.23 16.91-18.65
C. vastator 10.55 13.46-13.72 17.24-17.45
C. formosanus 11.35 12.45-12.70 15.93-16.14

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 Yeap et al. Molecular phylogenetics of C. gestroi and C. vastator

Figure 1. Pictures of C. gestroi soldier (A), C. vastator soldier (B), and C. formosanus
soldier (C).

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 Yeap et al. Molecular phylogenetics of C. gestroi and C. vastator

Figure 2. Lateral view of C. gestroi, C. vastator, and C. formosanus with focus on

fontanelle .

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 Yeap et al. Molecular phylogenetics of C. gestroi and C. vastator

Figure 3. 12S gene neighbour-joining tree, created with PAUP4.0 (Swofford 2000).
Bootstrap values for 1000 replicates are listed above the branches supported at

50%. GenBank accession numbers represent the samples that are pooled from
the NCBI database.

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 Yeap et al. Molecular phylogenetics of C. gestroi and C. vastator

Figure 4. 16S gene neighbour-joining tree, created with PAUP4.0 (Swofford 2000).
Bootstrap values for 1000 replicates are listed above the branches supported at

50%. GenBank accession numbers represent the samples that are pooled from
the NCBI database.

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 Yeap et al. Molecular phylogenetics of C. gestroi and C. vastator

Figure 5. COII gene neighbour-joining tree, created with PAUP4.0 (Swofford 2000).
Bootstrap values for 1000 replicates are listed above the branches supported at

50%. GenBank accession numbers represent the samples that are pooled from
the NCBI database.

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 Yeap et al. Molecular phylogenetics of C. gestroi and C. vastator

Figure 6. Neighbour-joining tree analyzing a combination of the three genes 12S, 16S
and COII concurrently. The tree was created with PAUP4.0 (Swofford 2000).
Bootstrap values for 1000 replicates are listed above the branches supported at

50%. GenBank accession numbers represent the samples that are pooled from
NCBI database.

27