MOLECULAR ECOLOGY AND EVOLUTION

Haplotype–Habitat Associations of Coptotermes gestroi (Termitoidae:Rhinotermitidae) From Mitochondrial DNA Genes

SHAWN CHENG,1,2 DINAIZ THINAGARAN,1 SEYEDEH ZEINAB MIRJALILI MOHANNA,3

AND NOR ANISAH MHD NOH3

Environ. Entomol. 43(4): 000Ð000 (2014); DOI: http://dx.doi.org/10.1603/EN13318

ABSTRACT Coptotermes gestroi (Wasmann) or the Asian subterranean termite is a serious structuralpest in urban settlements in Southeast Asia that has been introduced to other parts of the world throughhuman commerce. Although mitochondrial DNA markers were previously used to shed light on thedispersal history of the Asian subterranean termite, there were limited attempts to analyze or includepopulations of the termite found in thewild in SoutheastAsia. In this study,weanalyzed the 16S ribosomalRNA(16SrRNA)andcytochromecoxidase subunit1(cox1)genesofAsiansubterraneantermitecoloniesfound in mangrove swamps, beach forests, plantations, and buildings in semi-urban and urban areas todetermine the relationship between colonies found in the wild and the urban habitat, and to investigatethe possibility of different ecotypes of the termite in Peninsular Malaysia. Our Þndings show that the 16SrRNA haplotypes recovered from this study clustered into eastern, western, and southern populations ofthe termite,while the cox1haplotypeswere often speciÞc to an area or site. The 16S rRNAand cox1 genesorhaplotypesshowedthatthemostabundanthaplotypeoccupiedawiderangeofenvironmentsorhabitats.In addition, the cox1 tree showed evidence of historical biogeography where basal haplotypes inhabiteda wide range of habitats, while apical haplotypes were restricted to mangrove swamps and beach forests.Information on the haplotypeÐhabitat association of C. gestroi will enable the prediction of habitats thatmay harbor or be at risk of invasion in areas where they have been introduced.

KEY WORDS Coptotermes gestroi, Asian subterranean termite, mitochondrial DNA, 16S ribosomalRNA, cytochrome c oxidase subunit I

Termites are a serious threat to agricultural crops, forestplantations, and urban structures in Peninsular Malaysiawith subterranean termites from the genus Coptotermesresponsible for most of these infestations (Kirton andWong2001). Several species ofCoptotermes are found inMalaysia, namely, Coptotermes curvignathus Holmgren,Coptotermes gestroi (Wasmann), Coptotermes sepangen-sis Krishna, Coptotermes havilandi Holmgren, and Cop-totermes kalshoveni Kemner. C. curvignathus is the pri-mary pest of agriculture and forestry, where it is a pestof rubber, oil palm, andpine, all ofwhich are introducedplantation crops to the country. In the urban environ-ment, however,C. gestroi, C. curvignathus, andC. kalsho-venihave each been reported to infest homes and build-ings. A recent study, however, has shown that there hasbeen a rise in C. gestroi infestations in Peninsular Ma-laysia and Singapore and that it is now displacing otherCoptotermes as the main urban termite pest (Kirton andAzmi 2005).

C. gestroi or the Asian subterranean termite is dis-tributed from Burma through Thailand, PeninsularMalaysia, Borneo, and the Indonesian archipelago.

Like some of its relatives within the Coptotermes, theAsian subterranean termite has expanded its range intonew geographic areas over the past century with the aidof human dispersal via shipping and commerce. Popu-lations of this species are now found in Brazil, Hawaii,Mauritius, Ohio, Puerto Rico, the Reunion Islands, Tai-wan, Florida, the Marquesas Islands, Italy, and Australia(Light 1932, Moutia 1936, Araujo 1958, Su et al. 1997,Ghesini et al. 2001, Woodrow et al. 2001, Tsai and Chen2003, Jenkins et al. 2007, Yeap et al. 2009).

The accidental introduction of the Asian subterra-nean termite into Florida, Ohio, Puerto Rico, andAustralia was speculated to have Southeast Asian or-igins based on mitochondrial DNA (mtDNA) markeranalyses of population samples from these regions(Jenkins et al. 2007). The Asian subterranean termitewas thought to be primarily an urban-dwelling speciesthat has been transported across SoutheastAsia via thewell-developed transportation infrastructure in thisregion (Jenkins et al. 2007). An earlier study by Lee(2002) also suggested that most buildings and homesin Malaysia were prone to attack by the Asian subter-ranean termite because they were built over formerrubber and oil palm plantations that were thought tocontain large quantities of buried tree roots and plantdebris that harbors the termite.

1 Genetics Laboratory, Forest Research InstituteMalaysia (FRIM),52109 Kepong, Selangor, Malaysia.

2 Corresponding author, e-mail: shawn@frim.gov.my.3 Genetics Department, Institute of Biological Sciences, Universiti

Malaya, 50603, Kuala Lumpur, Malaysia.

0046-225X/14/0000Ð0000$04.00/0 � 2014 Entomological Society of America

In Florida, C. gestroi appears to be primarily foundin coastal areas (Scheffrahn and Su 2005), similar toanothermemberof thegenus,Coptotermes formosanusShiraki, which was introduced to Florida in 1980(Koehler 1980 in Scheffrahn and Su 2005). They oftencolonize boats and ships where they nest in cargo andwooden components of these sailing vessels (Uchimaand Grace 2003; Yeap et al. 2007, 2009), which wasmost likely their mode of arrival in Florida (Schef-frahn and Su 2005). In Peninsular Malaysia, Cop-totermes termites are rarely found in tropical rainfor-ests and are instead mainly present in mangrove andpeat swamps, and beach forests, which are typicallypoor in termite species richness(SalickandTho1984).

mtDNA is widely used in insect phylogenetic stud-ies. It is generally transmitted maternally; however, ininsects such as cicadas, honey bees, and fruit ßies, it istransmitted through males (Kondo et al. 1992, Meuseland Moritz 1993, Fontaine et al. 2007). As it is presentin high copy numbers, it can be easily isolated andampliÞed using universal mtDNA primers. mtDNAgene variants are also known as haplotypes because itis transmitted as a single unit (Neigel 1997). Here, weused mtDNA genes to determine the association be-tween mtDNA haplotypes and habitats that C. gestroiare found in, and the relationship between popula-tions found in the wild and urban areas. This wouldhelp shed light onwhetherpopulations found inurbanareas originated from the wild. Studies on the Asiansubterranean termitehave largelyneglectedanalyzingpopulations from thewild in SoutheastAsia, especiallyfrom mangrove swamps and beach forests where theyare particularly abundant. Data on the associationbetweenhaplotypes and habitats they inhabitwill alsobe useful for predicting habitats or environmentsdeemed vulnerable to its invasion, in areaswhere theyare an introduced pest.

Materials and Methods

Sample Collection. Asian subterranean termitesamples were collected from urban and natural hab-itats throughout Peninsular Malaysia between 2003and 2013. Sampling was carried out along the westernand southern coasts of the peninsula, while samplingon the eastern coast was limited to the states of Kelan-tan, Terengganu, and Pahang. The southeastern re-gion of Johore, however, was undersampled becauseof poor road access to the coastline. We sampled forC. gestroi in beach forests,mangroves, plantations, andfrom buildings in urban and semiurban areas.

We categorized buildings and homes in PeninsularMalaysia as occurring in urban or semiurban areasfollowing the deÞnitions of Kirton and Azmi (2005).Highly built-up areas with little to no vegetation werecategorized as “urban,” while less highly built-up areaswithin the vicinity of large tracts of trees or other veg-etation were characterized as “semiurban.” Coastal hab-itats such as beach forests and mangrove swamps weregenerally found on the eastern and western coasts ofPeninsular Malaysia, respectively. In beach forests, C.gestroi is normally found infestingCasuarina equisetifolia

L. trees internally, while in mangrove swamps it infestsdeadlogsandplantdebris lyingontheground. Inrubber,pine, and agroforestry plantations, C. gestroi also infestsdead trees and wood on the forest ßoor.

Whereverpossible, soldierswerecollected togetherwith workers and preserved in 75 and 100% undena-tured ethanol for morphological and molecular anal-ysis, respectively, and deposited at the Genetics Lab-oratory, Forest Research Institute Malaysia. Theidentities of the specimens were conÞrmed using thekeys provided by Tho (1991) and Kirton and Brown(2003).DNAwas extracted from the head, thorax, andall the legs of a single termite individual from eachvoucher sample or colony with Promega WizardGenomic DNA extraction kit (Promega Corp., Mad-ison, WI). The 16S ribosomal RNA (16S rRNA) genewas ampliÞed with 16 Sar (5�-CGCCTGTTTATCAAAAACAT-3�) and 16 Sbr (5�-CCGGTCTGAACTCAGATCACGT-3�; Palumbi 1996), while the cyto-chrome c oxidase subunit I (cox1) gene was ampliÞedas two separate but overlapping fragments using theprimers TL2341 (5�-CGAACGAATCCCACTATTTGT-3�) and TH2928 (5�-AATACTGCTCCTATAGATAG-3�); and TH2397 (5�-TTAGTAGTATTGTGATTGCTCC-3�) and TL1862 (5�-TACTTCGTATTCGGAGCTTGA-3�; Aanen et al. 2002).

Polymerase chain reaction(PCR)ampliÞcationwasperformed on a GeneAmp 9700 Thermal Cycler (Ap-plied Biosystem, Foster City, CA) in a reaction con-taining 2.0 �l of DNA template (5.0 ng/�l), 2.0 �ldNTPs (2.0 mM), 1.2 �l MgCl2 (25.0 mM), 2.0 �l 10�PCR Buffer, 0.6 �l of each primer (10.0 �M), 11.4 �ldouble-distilled water, and 0.2 �l GoTaq DNA poly-merase (Promega Corp., Madison, WI). PCR ampliÞ-cation was performed with the following parameters:initial denaturation at 94�C for 3 min, 36 cycles ofdenaturation at 94�C for 30 s, annealing at 47�C and48�C for 30 s for the 16S rRNA and cox1 primers,respectively, extension at 72�C for 1 min, and a Þnalextension at 72�C for 10 min. PCR products werepuriÞed with QIAquick PCR puriÞcation kit (Qiagen,Valencia, CA) and used in BigDye Terminator ver 3.1(Applied Biosystem) cycle sequencing reactions. Se-quencing was performed in both forward and reversedirections on an ABI3130xl Genetic Analyzer (Ap-plied Biosystems).

DNA sequences were edited and assembled withSeqScape 2.5 (Applied Biosystems) using the mtDNAgenome of C. formosanus (NCBI accession numberNC015800) as the reference sequence. We also in-cluded Asian subterranean termite gene sequencesavailable in GenBank, as well as sequences of its clos-est relatives, in the analysis (Table 1). Thedata setwasanalyzed for parsimony-informative characters usingPAUP* 4.0 (Swofford 2003). Then, mtDNA haplo-types were identiÞed with DNA Sequence Polymor-phism or DnaSP v5 (Librado and Rozas 2009) andhaplotype representatives were then used for maxi-mum-likelihood tree construction with RAxML onXSEDE (1.0) toolkit (Stamatakis 2006, Stamatakis etal. 2008) with the default parameter of 100 bootstrapreplicates, at theCyberinfrastructure forPhylogenetic

2 ENVIRONMENTAL ENTOMOLOGY Vol. 43, no. 4

Tab

le1

.D

etai

lsof

sam

ples

used

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udy,

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otyp

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enti

ties

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rent

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ter

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bers

(“—

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ple

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this

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August 2014 CHENG ET AL.: C. gestroi HAPLOTYPEÐHABITAT ASSOCIATIONS FROM mtDNA 3

Tab

le1

.C

onti

nued Loca

lity

Hab

itat

GenBan

kac

cess

ion

no.

16S

rRN

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ox1

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,C

hin

aU

rban

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8871

99(N

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Man

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(Mal

aysia)

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3210

Ð

Continued

on

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ing

pag

e

4 ENVIRONMENTAL ENTOMOLOGY Vol. 43, no. 4

Research Science Gateway Portal ver. 2.0 (www.phylo.org; Miller et al. 2010). Phylogenetic and min-imum-spanning network trees were reconstructed toinvestigate the relationships between C. gestroi pop-ulations found in PeninsularMalaysia and areaswherethey are an introduced species.

We also built minimum-spanning network treeswith TCS ver. 1.21 to examine haplotype relationshipsand to determine ancestral 16S rRNA and cox1 hap-lotypes (Clement et al. 2000,Castelloe andTempleton1994). We matched the haplotypes recovered fromour analyses to habitats they were found in such asbeach forests, mangrove swamps, plantations, and ur-ban, and semiurban habitats to identify the possibilityof different ecotypes of C. gestroi. Ecotypes are dis-tinct genotype or populations within a species thathave adapted to local environmental conditions butare still capable of interbreeding with individuals ofother ecotypes of the same species. We analyzed pop-ulation genetic structure in the Peninsular MalaysianC. gestroi samples according to the subregions (north-west, central west, southwest, northeast, and south)and the environments or habitats (urban, semiurban,plantation, mangrove, and beach forest) they oc-curred in. Genetic variation within and among sub-regions and habitats were determined through anal-yses of molecular variance (AMOVAs) of nucleotidediversity (�st) from the haplotype nucleotide se-quences with Arlequin ver. 3.1 (ExcofÞer et al. 2005).

Results

16S rRNA and cox1 Haplotype Distributions. Anal-ysis of the 16S rRNA gene recovered Þve haplotypes(Haplotypes 1Ð5) among the 50 Asian subterraneantermite colonies sampled in Peninsular Malaysia (Ta-ble 1; Fig. 1). Haplotype 1, which is the most frequenthaplotype in Peninsular Malaysia, occurred in all hab-itats sampled in this study. It was also the haplotypethat was introduced to North and Central America,Italy, and Australia (Table 1; Supp Fig. 1 [onlineonly]). In general, the 16S rRNA haplotypes fromPeninsular Malaysia clustered into eastern, western,and southern populations of the termite (Fig. 1).However, there was one instance where a haplotypethat was typically found on the west coast (Haplo-type 1) was also found in the east and southernregions of the peninsula. Similarly, Haplotype 2,which was generally found on the east coast, wasalso found on the west coast and the southern regionof Peninsular Malaysia (Fig. 1). There were morehaplotypes in the southern region (including Sin-gapore) comparedwith the rest of peninsulawith upto four haplotypes found here, one of which wasexclusive to the region (Fig. 1).

Twelve cox1 haplotypes (Haplotype AÐL) wereidentiÞed in Peninsular Malaysia (Fig. 2). Haplo-type B, which occurred in a wide range of habitats,was also the most widespread among the 12 cox1haplotypes; however, many of the haplotypes (e.g.,Haplotypes A, C, G, and J) were rather area-speciÞc(Fig. 2). C. gestroi colonies from urban and semi-

Tab

le1

.C

onti

nued Loca

lity

Hab

itat

GenBan

kac

cess

ion

no.

16S

rRN

AC

ox1

C.se

pange

nsis

Pah

ang,M

alay

sia

Man

gro

ve

KF85

3388

KF85

3390

Mal

aysia

ÐFJ3

7667

9Ð

C.ka

lshov

eni

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August 2014 CHENG ET AL.: C. gestroi HAPLOTYPEÐHABITAT ASSOCIATIONS FROM mtDNA 5

urban areas, however, were mainly composed ofHaplotypes D and E, although a single sample ofHaplotype E was also found in a plantation ecosys-tem in Perlis (Fig. 2).

16S and cox1 Maximum Likelihood Trees. The 16SrRNA likelihood tree recovered C. gestroi and Cop-totermes vastator Light, its junior synonym, as a singleclade with a bootstrap value of 58% (Fig. 3). Samplesof C. gestroi from Peninsular Malaysia, Singapore, thePhilippines, Taiwan, and populations of the speciesintroduced into North America and Hawaii formed awell-supported clade in the 16S rRNA tree, whilesamples from Turks and Caicos Islands and Antiguaand Barbuda, which clustered together, resolved sep-arately from this clade (Fig. 3). In the cox1 likelihoodtree, C. gestroi was also recovered as a single clade(Fig. 4). Two apically placed clades that were asso-ciatedwith speciÞchabitat typeswere seen in thecox1tree among the Peninsular Malaysian haplotypes(Haplotypes M and N were excluded, as the haplo-types belonged to populations introduced to China;Fig. 4).TheÞrst cladecomprisedHaplotypesA,C, and

K, which were found in beach forests, while the sec-ond clade, which consisted of Haplotypes G and I,were mainly associated with mangrove swamps (Fig.4). Both these clades had strong bootstrap support of�80%. Haplotypes B, D, E, J, and L, were recovered ata paraphyletic or basal position to the rest of the cox1haplotypes within the C. gestroi (Fig. 4). These hap-lotypes were associated with infestations in urban,semiurban areas, plantations, mangrove swamps, andbeach forests (Fig. 4).

Network Trees for the 16S rRNA and cox1 Genes.The aligned 16S rRNA data set was 571 nucleotides orcharacters in length, of which 553 were invariant and11 were parsimony-informative. The cox1 data setalignment was 1,102 characters in length, with 1,052invariant and 25 parsimony-informative characters.Figure 5 shows the minimum-spanning network treefor the 16S rRNA data set. Most of the 16S rRNAhaplotypes were separated by one to three mutations.Although Haplotype 1 was the most frequent or abun-dant haplotype, it was not the ancestral haplotype.Haplotype 2, which was mainly found along the east

PENANG

KEDAH

PERAKKELANTAN

TERENGGANU

JOHOR

PAHANG

SELANGOR

NEGERISEMBILAN

South China Sea

Straits of Malacca

2

2

2

2

2

22

2

22

1

1

1

1 1

11

11

1 1

1

11

1

2

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4

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220 50 100 km

12

PlantationMangrove swampBeach forest

Urban

Habitat

1

1

4

1 111

2

11

2 4 4

44

433

11

1

Semi-urban

11

Fig. 1. Distribution of 16S rRNA haplotypes (1Ð5) in Peninsular Malaysia. Each circle represents the haplotype of anindividual from a colony. (Online Þgure in color.)

6 ENVIRONMENTAL ENTOMOLOGY Vol. 43, no. 4

coast of Peninsular Malaysia, was recovered as theancestral haplotype by virtue of the haplotype havingthe highest root probability score and the most mu-tational derivatives among the 10 16S rRNA haplo-types (Figs. 1 and 5).

The cox1 minimum-spanning tree network, how-ever, recovered Haplotype B as the ancestral se-quence; this haplotype generally occurredon thewestcoastofPeninsularMalaysia(Figs. 2 and6).HaplotypeL was identiÞed as the most divergent of the cox1haplotypes as it had undergone �16 silent nucleotidesubstitutions (Fig. 6; Supp Data 1 [online only]). Thealigned amino acid sequences of the cox1 gene alsoshowed that there was little to no variation among C.gestroi amino acid sequences from the cox1 eventhough theirnucleotide sequenceswerequitevariable(Supp Data 1 [online only]).

PopulationGenetic StructureAnalysis.Pairwise16SrRNAnucleotidediversity values (�st) ofC. gestroipop-ulations in Peninsular Malaysia showed that most of thevariation (63.6%) was attributed to within-population

differences, while 36.4% of the genetic variation wasattributed to among-populations differences from theÞve different subregions (Table 2). When we assessedthe possibility of the presence of different “ecotypes” ofC. gestroi, that is, “urban,” “semiurban,” “mangrove,”“beach forest,” and “plantation,” most of the variationoccurred within the ecotypes (77.63%) as opposedto among the ecotypes (22.37%; Table 3). In the cox1data set, 27.3% of the total genetic variation wascaused by variation among populations from the Þvesubregions, while the remaining 72.7% was due towithin-population differences (Table 2). In com-parison, 19.78% of the total genetic variation wasdue to variation among the ecotypes with a majorityof the genetic variation (80.22%) occurring withinthe ecotypes (Table 3).

Discussion

Analysis of the 16S rRNA haplotype distributionshowed that the southern region of Peninsular Malaysia

D

1000 50 km

PENANG

KEDAH

PERAK KELANTANTERENGGANU

JOHOR

PAHANG

SELANGOR

NEGERISEMBILAN

South China Sea

Straits of Malacca

A

AA

B

BC

B

DDD D

EED D

B

IGB

J

B

B

F

B

K

H EB

DE

I

I

F

D DBLFB D

CC

B

PlantationMangrove swampBeach forest

Urban

Habitat

Semi-urban

Fig. 2. Distributionof cox1haplotypes (AÐL) inPeninsularMalaysia.Eachcircle represents thehaplotypeof an individualfrom a colony. (Online Þgure in color.)

August 2014 CHENG ET AL.: C. gestroi HAPLOTYPEÐHABITAT ASSOCIATIONS FROM mtDNA 7

had the highest haplotype diversity (Fig. 1). This couldbe because of the establishment or introduction of non-native haplotypes of C. gestroi transported via woodenpallets and timber features in ships to thepeninsula. This

is in view of Johore and Singapore being a commercialshipping and transportation hub in Southeast Asia. The16S rRNA haplotype distribution map also showed thateastern, western, and southern regions of the peninsula

C. sepangensis (TC1743)

C. sepangensis (FJ376679)

C. kalshoveni (FJ376684)

C. curvignathus (TC1688)

C. curvignathus (AY683210)

R. flavipes (AY702099)

N. castaneus (JN615267)

N. koshunensis (D89839)

Cryp. domesticus (D89835)

Cryp. brevis (FJ806145)

Haplotype 10

Haplotype 4

Haplotype 2

Haplotype 9 (Philippines & USA)

Haplotype 5

Haplotype 3

Haplotype 1Haplotype 8 (Antigua & Barbuda)

Haplotype 7 (Turks & Caicos Islands)

Haplotype 6 (Taiwan)

59

58

9689

51

96100

72

100

71

7

38

5

0

6

15

(Philippines)

Fig. 3. 16S rRNA maximum-likelihood tree with bootstrap values for 100 replicates shown at tree nodes. Haplotypes 1Ð8identiÞed as C. gestroi; Haplotypes 9 and 10 as C. vastator. Note: Cryptotermes abbreviated as Cryp. , Neotermes as N., andReticulitermes as R.

N M C K A I G H F E J B D L C. s

epan

gens

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thus

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100

100100

PlantationSemi-urban

MangroveBeach

Urban

+ + +

++

++ +

+++ +

++

+++

++

++ +

+

Fig. 4. Maximum-likelihood tree of 14 cox1 mtDNA haplotypes of C. gestroi (alphabets “A” to “N”) with their corre-sponding habitat associations superimposed on the tree. Haplotypes M and N originated from China; information on theirhabitat associations were from GenBank records.

8 ENVIRONMENTAL ENTOMOLOGY Vol. 43, no. 4

were each represented by distinct 16S rRNAhaplotypes(Fig. 1). However, the cox1 haplotype distributionshowed that many of the haplotypes were unique toparticular locations in Peninsular Malaysia (Fig. 2).Hence, examiningcox1genediversity and thehaplotypedistribution of the termite in countries or areas where itis native and where it is an introduced pest will provideamoredetailedandaccurateviewof thedispersal routesof the Asian subterranean termite.

The 16S rRNA and cox1 genes were both useful forreconstructing interspeciÞc relationships among Cop-totermes, but with the cox1 gene having the additionalproperty of being able to resolve intraspeciÞc rela-tionships among C. gestroi haplotypes (Figs. 3 and 4).The clades recovered by the cox1 gene also had theadvantageofhavinghigherbootstrapvaluescomparedwith theclades recoveredby the16S rRNAgene(Figs.3 and 4). This could be explained by the greater num-ber of informative sites in cox1 compared with the 16SrRNA. Cox1 plays an important role in the respiratoryelectron transport chain andhence inmetabolism, andhas a much faster evolution rate compared with the16S rRNA gene. In tenebrionids, the evolution rate ofcox1 is 3.54 versus 1.06% in the 16S rRNA (Papado-poulou et al. 2010).

In the 16S rRNA likelihood tree, Haplotypes 7 and8 from the Turks and Caicos and Antigua and Barbudaclustered separately from the remaining SoutheastAsian haplotypes (Fig. 3). This may be caused bygenetic drift, which is typical of founder populations(Page and Holmes 1998). Alternatively, Haplotype 7and 8 could originate from an unsampled region inSoutheastAsia. In thecox1 likelihood tree,HaplotypesM and N from China, which formed a clade, werefound to be closely related to populations from the

eastern and southern coasts of Peninsular Malaysia,indicating that it may have originated from these re-gions (Figs. 2 and 4).

The cox1 likelihood tree also showed evidence of ashift in the distribution of C. gestroi in Peninsular Ma-laysia, from being found in urban and semiurban areas,plantations, beach forests, and mangroves swamps to amorerestricteddistributionwhere theywereonly foundin mangrove swamps and beach forests in the api-cally placed haplotypes (Fig. 4). Haplotypes M andN from Yunnan and the island of Hainan in Chinaclustered with Haplotypes A, C, and K, which wereassociated with beach forests in Peninsular Malaysia(Fig. 4; Table 1). Although Haplotypes M and Nwere both found infesting urban and semiurbanareas in China, Haplotype N was found very close tothe coast and this was similar to the environmentinhabited by members of this clade in PeninsularMalaysia (Fig. 4). Information on haplotypeÐhabi-tat associations of C. gestroi from Peninsular Malay-sia would provide useful data for the developmentof models to predict the probability of areas at riskof invasion by C. gestroi (e.g., Li et al. 2013) or forpredicting habitats at risk of invasion by C. gestroi inareas where it has been introduced.

A slow invasion by the Termitidae, whose center ofdiversity lieoutsideSoutheastAsia, into the regionwaspreviously speculated to have led to the displacementof C. gestroi into low-productivity forests such as peatand mangrove swamps and beach forests as suggestedby Salick and Tho (1984). This possibly explains whythe apically placed C. gestroi colonies were associatedwithmangrove swampsandbeach forests, andwhythemore basal haplotypes could be found in a variety ofhabitats (Fig. 4). A recent example of Coptotermes

2

1

4

68

753 10

9

T-19-C T-44-C

A-331-T

G-13-A

A-355-G

A-220-G

T-14-C

G-325-C

G-83-T

T-1-A

T-117-C

C-85-T

C-144-T

A-143-G

(0.326)

(0.389)

(0.006)

(0.019)

(0.002)

(0.021)

(0.002)

(0.002)

(0.219) (0.012)

Fig. 5. 16S rRNA gene minimum-spanning network tree. (Note: Haplotype numbers in bold; nucleotide changes shownon branches; Þlled circles represent unsampled haplotypes; ancestral haplotype in rectangle; derived haplotypes in circles;Þgures in parenthesis indicate haplotype root probabilities).

August 2014 CHENG ET AL.: C. gestroi HAPLOTYPEÐHABITAT ASSOCIATIONS FROM mtDNA 9

displacement was when exotic rubber and pine treeplantations were introduced to Malaysia at the turn ofthe 20th century. Coptotermes gained dominance as aserious pest because of the reduction in termite di-versity caused by clear felling of the rainforest fol-lowed by the monoculture cultivation of rubber andpine trees in Malaysia. However, over time, as termitediversity recovered, Coptotermes became of lesser im-portance as pests (Tho 1974).

Thereweredifferences in the relationships of theC.gestroi samples suggested by the maximum-likelihoodtrees and theminimum-spanning tree of the 16S rRNAgene with reference to the identity of the ancestralhaplotype (Figs. 3 and 5). The likelihood tree recov-

ered Haplotypes 7 and 8 as the ancestral haplotypewhile the minimum-spanning 16S rRNA tree recov-ered Haplotype 2 as the ancestral haplotype by virtueof its higher root probability value compared with therest of the haplotypes (Figs. 3 and 5). The cox1 like-lihood tree recovered Haplotype L as the ancestralsequence while the minimum-spanning tree recov-ered Haplotype B as the ancestral haplotype (Figs. 4and 6). However, the ancestral haplotypes identiÞedfrom the 16S rRNA (Haplotype 2) and cox1 (Haplo-type B) minimum-spanning network trees were mostlikely the ancestral sequences of C. gestroi as colonieswith these haplotypes inhabited a wide range of en-vironments in Peninsular Malaysia.

Fig. 6. Cox1 gene minimum-spanning network tree. Conventions as in Fig. 5.

Table 2. AMOVA of 16S rRNA and cox1 (in parenthesis) mtDNA haplotypes of C. gestroi populations from five geographic subregionsin Peninsular Malaysia (northwest, central west, southwest, northeast, and south)

Source ofvariation

dfSum ofsquares

Variancecomponents

Variationpercentage (%)

Fixation index (�st) P value

Among subregions 4 (4) 10.471 (29.516) 0.18079 (0.73819) 36.40 (27.30) Ð 0.000* (0.000*)Within subregions 62 (37) 19.589 (72.723) 0.31595 (1.96547) 63.60 (72.70) Ð 0.000* (0.000*)Total 66 (41) 30.060 (102.238) 0.49674 (2.70367) Ð �st � 0.3640 (�st � 0.2730) 0.000 � 0.000* (0.000 � 0.000*)

* SigniÞcant at P 0.05.

10 ENVIRONMENTAL ENTOMOLOGY Vol. 43, no. 4

Differences in the haplotype relationships recoveredby maximum likelihood and network spanning could beexplained by the method or algorithm used to generatethe trees. Maximum-likelihood is commonly used to de-termine interspeciÞc relationships but may not be accu-rate in determining intraspeciÞc evolution. Maximum-likelihood methods have also been reported to result inphylogenetic trees or relationships that are resolvedoverconÞdently (Bandelt et al. 1995). This error stemsfrom its failure to incorporate the possibility of recom-bination events in phylogeny reconstruction (Clementet al. 2000). Network spanning, however, uses statisticalparsimony to calculate haplotype frequencies and esti-mates the outgroup that corresponds to the ancestralhaplotype (Castelloe and Templeton 1994). It groupssimilarities amonghaplotypes andgives empirical assess-ment of deviation from parsimony. Haplotypes are con-nected based on the least number of changes in nucle-otides, and nodes represent potential undiscoveredhaplotypes.

Anearlier studyby Jenkins et al. (2007) showed thatPeninsular Malaysian and Singaporean samples weresigniÞcantly differentiated from each other (�st �0.592); however, their Peninsular Malaysian sampleswere only from the island of Penang. The �st valuesobtained from the 16S rRNA and cox1 mtDNA genesin our study, however, showed more moderate lev-els of genetic differentiation among C. gestroi pop-ulations in Peninsular Malaysia at 0.364 and 0.273,respectively (see guidelines in Wright 1978). As oursampling of C. gestroi populations was more exten-sive than Jenkins et al. (2007), this would be a morereasonable measure of their genetic differentiation.In addition, our analyses indicated that variationamong populations of the termite was more likelyattributed to geographical differentiation ratherthan differences caused by habitat preferencesamong populations of C. gestroi in Peninsular Ma-laysia (Tables 2 and 3).

Thewell-developed transportation infrastructure inThailand,Malaysia, and Singaporewas previously pur-ported to have a role in the homogenization of hap-lotypes and the close relationship between samples ofC. gestroi in this region (Jenkins et al. 2007). A similartheorywas also put forward byYeap et al. (2011) frommicrosatellite analyses of populations from Penang,Kuala Lumpur, and Singapore, which showed nosigniÞcant isolation by distance between these pop-ulations. However, homogenization of haplotypesmay not be entirely because of C. gestroi beingtransported by rail and sea cargo through the var-

ious ports found in this region, as our study showsthat these haplotypes are already present in man-grove swamps and beach forests throughout coastalhabitats in Peninsular Malaysia, and possibly in Sin-gapore and Thailand too.

In conclusion, the 16S rRNA gene resolved into ÞvemtDNA haplotypes in Peninsular Malaysia and 10worldwide, while the cox1 sequences resolved into 12haplotypes in Peninsular Malaysia and 14 haplotypesworldwide. The 16S rRNA mtDNA haplotype distri-bution clustered the Asian subterranean termite intoeastern, western, and southern populations, while thecox1 haplotypes distribution showed that many of themtDNA haplotypes were area-speciÞc. The cox1 phy-logenetic tree also showed evidence of historical bio-geography in this primitive termite species; basalhaplotypes inhabited a wide range of habitats suchas semiurban and urban areas, plantations, man-groves, and beach forests while apically placed hap-lotypes were restricted to mangrove swamps andbeach forests only. The haplotypeÐhabitat associa-tion information from this study will enable theprediction of habitats or areas that may harbor C.gestroi colonies or at risk of C. gestroi invasion.Lastly, the cox1 gene would be more informativethan the 16S rRNA gene to infer a more detaileddispersal history of C. gestroi as it evolves at a muchfaster rate compared with the 16S rRNA, and todetermine other human-assisted dispersal routes ofthe Asian subterranean termite.

Acknowledgments

We thank the Ministry of Science, Technology and Inno-vation Malaysia (MOSTI) for funding this research, undergrant 06-03-10-SF0155 “Development of Strategies andTech-niques to Control the Asian subterranean termite Cop-totermes gestroi Wassmann (Rhinotermitidae) in Urban Set-tlements.” We also thank Ridpest Sdn. Bhd. Malaysia forproviding C. gestroi tissue samples for our research, andKaviarasu Munian for help on the analysis of molecular vari-ance. Finally, a special mention of thanks to Mohd. ShahFadir Ishakandmembersof theGeneticsLaboratory for theirassistance in both the Þeld and the laboratory, and MissAmira Nadaviana for editing the manuscript.

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Table 3. AMOVA of 16S rRNA and cox1 (in parenthesis) mtDNA haplotypes of C. gestroi populations from five different habitats(urban, semiurban, mangrove, beach forest, and plantation)

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Received 15 November 2013; accepted 29 April 2014.

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