systematics molecular systematics of coptotermes (isoptera: … · 2020. 2. 17. · systematics...

SYSTEMATICS

Molecular Systematics of Coptotermes (Isoptera: Rhinotermitidae)From East Asia and Australia

BENG-KEOK YEAP, AHMAD SOFIMAN OTHMAN, AND CHOW-YANG LEE1

School of Biological Sciences, Universiti Sains Malaysia, 11800 Penang, Malaysia

Ann. Entomol. Soc. Am. 102(6): 1077Ð1090 (2009)

ABSTRACT Analyses of mitochondrial DNA sequences (12S, 16S, and COII) were conducted todetermine the phylogenetic relationships among the following 11 putative subterranean termites ofCoptotermes: Coptotermes cochlearus Xia & He, Coptotermes curvignathus Holmgren, Coptotermesdimorphus Xia & He, Coptotermes formosanus Shiraki, Coptotermes gestroi (Wasmann), Coptotermeskalshoveni Kemner, Coptotermes sepangensis Krishna, and Coptotermes travians (Haviland) from EastAsia, andCoptotermes acinaciformis Froggatt,Coptotermes frenchiHill, andCoptotermes lacteus (Frog-gatt) from Australia. Available sequences for these species and those of Coptotermes guangzhouensisPing from GenBank also were included in the analyses. Maximum parsimony and maximum likelihoodof the combined nucleotide matrices of the 12S, 16S, and COII genes resulted in two major clades withsix subclades: I (C. acinaciformis), II (C. lacteus andC. frenchi), III (C. curvignathus), IV (C. kalshoveni,C. sepangensis and C. travians), V (C. gestroi) and VI (C. formosanus, C. cochlearus, C. dimorphus andC. guangzhouensis).C. cochlearus andC.dimorphus are possibly junior synonyms ofC. formosanuswithnucleotide differences of up to 1.0%.

KEY WORDS subterranean termites, Coptotermes, phylogenetics, Asia, mitochondrial DNA.

Coptotermes is a genus of the family Rhinotermitidaethat is widely distributed in pantropical and subtrop-ical regions. There is a growing concern about theeconomic impact of subterranean termites, especiallythose from genus Coptotermes, on urban structures, inforestry, and in agricultural crops in most subtropicaland tropical countries of the world (Su and Scheffrahn2000). These termites represent the major pest speciesin the Americas, Asia, and Australia (Lo et al. 2006,Takematsu et al. 2006). In Malaysia, Coptotermes spp.cause �90% of all infestations in buildings and struc-tures (Lee 2002, 2007).

In addition, several invasive Coptotermes have beentransported from their native range in the Orient toother parts of the world (Takematsu et al. 2006). Thesesubterranean termites can extend themselves well be-yond their normal habitation range. For example,Cop-totermes gestroi (Wasmann) have established them-selves as serious structural pests in Florida, WestIndies, Mexico, Brazil, and Taiwan (Scheffrahn and Su2000, Kirton and Brown 2003, Tsai and Chen 2003,Ferraz and Mendez-Montiel 2004, Yeap et al. 2007, Liet al. 2009).

The international termite taxonomy is in severedecline, especially due to the irregular taxonomicpractice in China and Africa (Eggleton 1999). De-scription rates had risen enormously in China sincethe mid-1970s, but they decreased greatly in Africa

over the same period. In China, between 1946 and1996, 24 new species of Coptotermes were describedbased on morphological characteristics that over-lapped greatly with sympatric and allopatric speci-mens in China (Crosland 1995, Eggleton 1999). TwoCoptotermes spp. evaluated in this study, namely Cop-totermes cochlearus Xia & He and Coptotermes dimor-phus Xia & He were described by Xia and He (1986)in a controversial taxonomic compilation of Chinesetermites. Li (2000) synonymized several Coptotermesspp., but his efforts have been limited as most of theearlier species were included without further scruti-nization. The lack of robust morphological charactersand the limited number of available specimens, espe-cially the imago caste, have made identiÞcation oftermite at the species level difÞcult and unreliable(Tho 1992).

On the contrary, outside China, several synonymi-ties were described among the Coptotermes spp. Yeapet al. (2007) synonymized Coptotermes vastator Lightwith C. gestroi. Kirton and Brown (2003) reportedtaxonomic inconsistencies on the pest status of Cop-totermes havilandi Holmgren in different regionswithin its geographic range and concluded that it is ajunior synonym of C. gestroi. The authors also pro-posed Coptotermes javanicus Kemner to be a juniorsynonym to the latter species. Proper identiÞcation ofspecies is imperative for generating accurate a goodtermite taxonomy that provides the baseline for allcomparative biology. Moreover, accurate identiÞca-1 Corresponding author, e-mail: [email protected].

0013-8746/09/1077Ð1090$04.00/0 � 2009 Entomological Society of America

tion of species is needed for the effective managementof these insects in urban settings and for establishingan environmentally sound management strategy (Co-pren et al. 2005, Takematsu et al. 2006).

Many recent studies on Rhinotermitidae have dem-onstrated the potential of using DNA sequence anal-ysis in species identiÞcation, which would enable bet-ter understanding of phylogenies (Jenkins et al. 2000,2007; Austin et al. 2004; Lo et al. 2004; Ohkuma et al.2004; Takematsu et al. 2006). Mitochondrial geneshave been widely used as reliable genetic markers formolecular phylogenetic studies of termites. However,the taxa analyzed so far include few Coptotermes spp.

In this study, we used mitochondrial DNA(mtDNA) sequences (12S, 16S, and COII) to revisethe taxonomy and reveal the relationships among 11putative Coptotermes spp. from 29 populations col-lected from East Asia and Australia. We also includedCoptotermes sequences from GenBank into our phy-logenetic analysis. By integrating molecular system-atic data with morphometric analysis, we present herethe phylogenetic relationship between various Cop-totermes spp. from East Asia and Australia.

Materials and Methods

Sample Collection.Twenty-nine populations of the11 putative Coptotermes spp. were studied. Coptot-ermes samples were collected and preserved in 100%ethanol (Table 1). Preserved soldier specimens wereidentiÞed up to species level based on morphometric

characters using an Olympus SZ2-LGB stereomicro-scope connected to a computer-assisted imaging cam-era. The following eight morphological characteristicsof the soldier termites were measured: maximum headwidth, head width at base of mandibles, length of headfrom base of mandibles, maximum width of gula, min-imum width of gula, gula length, pronotum length, andpronotum width. These measurement data along withoriginal data from Yeap et al. (2007) were subjectedto analysis of variance (ANOVA), and means wereseparated by TukeyÕs honestly signiÞcant difference(HSD). The sequences for the outgroup species,Glo-bitermes sulfureus Haviland and Reticulitermes flavi-ceps Oshima, were procured from GenBank from thework of Yeap et al. (2007) and Li et al. (2009), re-spectively. Voucher specimens were preserved in100% ethanol and kept at the Vector Control ResearchUnit, School of Biological Sciences, Universiti SainsMalaysia, Penang, Malaysia. Published sequences ofCoptotermes spp. from GenBank (www.ncbi.nlm.nih-.gov) also were included in the phylogenetic analysis(Table 2).DNAExtraction.Total genomic DNA was extracted

from a single worker termite from the 29 populations.The preserved specimen was washed with distilledwater and laid ßat to dry on a piece of Þlter paper. Theintact termite was then frozen with liquid nitrogenand ground in a 1.5-ml tube. After grinding, 800 �l ofsterile STE buffer (50 mM sucrose, 25 mM Tris-HCl,pH 7.0, and 10 mM EDTA) was added. DNA wasextracted by incubation with proteinase K at 55�C for

Table 1. Information about the termite specimens collected and used in this study

Samplecodea

Species Collecting sitesGenBank accession no.

12S 16S COII

CG001TW C. gestroi Taiwan, Tainan 1 FJ235115 FJ376672 FJ384649CG002TW C. gestroi Taiwan, Chang Jung Christian University FJ235116 FJ376673 FJ384650CG003TW C. gestroi Taiwan, Tainan 2 FJ235117 FJ376674 FJ384651CF004JP C. formosanus Japan, Kagoshima, colony A FJ235103 FJ376661 FJ384640CF005JP C. formosanus Japan, Kagoshima, colony B FJ235104 FJ376662 FJ384641CF006JP C. formosanus Japan, Kagoshima, colony C FJ235105 FJ376663 FJ384642CF007JP C. formosanus Japan, Kagoshima, colony 1 FJ235106 FJ376664 FJ384643CF008JP C. formosanus Japan, Kagoshima, colony 3 FJ235107 FJ376665 FJ384644CF009JP C. formosanus Japan, Kagoshima, colony 5 FJ235108 FJ376666 FJ384645CF010JP C. formosanus Japan, Kagoshima, colony 6 FJ235109 FJ376667 FJ384646CF001TW C. formosanus Taiwan, Taichung 1 FJ235096 FJ376659 FJ384647CF002TW C. formosanus Taiwan, Taichung 2 FJ235097 FJ376660 FJ384648CF001CN C. formosanus China, Guangzhou, Guangdong Entomological Institute FJ235098 FJ376668 FJ384636CF002CN C. formosanus China, Guangzhou, Sun Yat-sen University, Pu Garden FJ235099 FJ376669 FJ384637CF003CN C. formosanus China, Guangzhou, AVON Co. FJ235100 FJ376670 FJ384638CF004CN C. formosanus China, Guangzhou, Guangdong Entomological Institute FJ235101 FJ376671 FJ384639CK001MY C. kalshoveni Malaysia, Penang, USM FJ235118 FJ376684 N.ACK002MY C. kalshoveni Malaysia, Penang, Pantai Keracut FJ235119 FJ376685 FJ384652CC001MY C. curvignathus Malaysia, Penang, USM FJ235110 FJ376686 FJ384634CC002MY C. curvignathus Malaysia, Penang FJ235111 FJ376680 FJ384635CC001SG C. curvignathus Singapore, Nim Road FJ235112 FJ376681 N.ACT001MY C. travians Malaysia, Perak, Teluk Intan FJ915823 FJ915824 N.ACS001MY C. sepangensis Malaysia, Perak, Bagan Datoh estate FJ235120 FJ376679 N.ACCO001CN C. cochlearus China FJ235113 FJ376682 N.ACD001CN C. dimorphus China FJ235114 FJ376683 N.ACFR001AU C. frenchi Australia, Canberra, ACT FJ235094 FJ376677 FJ384632CL001AU C. lacteus Australia, Canberra, ACT FJ235095 FJ376678 FJ384633CA001AU C. acinaciformis Australia, Darwin, NT FJ235092 FJ376675 FJ384630CA002AU C. acinaciformis Australia, GrifÞth, NSW FJ235093 FJ376676 FJ384631

a Sample code XX001YY: XX is species; 001 is sample vial number, and YY is country of collection.

1078 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 102, no. 6

Table 2. Published GenBank sequences used in this study

Species Collection sitesGenBank accession no.

References12S 16S COII

C. acinaciformis Australia: Darwin, Northern Territory AY536380 AY302701 UnpublishedC. acinaciformis Australia, GrifÞn, Canberra, New South Wales AY536381 AY302702 UnpublishedC. acinaciformis DQ441668 Inward et al. (2007)C. acinaciformis Australia: Darwin DQ367826 Lo et al. (2006)C. acinaciformis Australia: Townsville DQ367827 Lo et al. (2006)C. acinaciformis Australia: Sydney DQ367828 Lo et al. (2006)C. acinaciformis Australia: Olney State Forest, NSW DQ367829 Lo et al. (2006)C. acinaciformis Australia: Canberra DQ367831 Lo et al. (2006)C. acinaciformis Australia: Walpeup DQ367833 Lo et al. (2006)C. frenchi Australia: Canberra DQ367836 Lo et al. (2006)C. frenchi Australia: Melbourne DQ367837 Lo et al. (2006)C. lacteus Australia: Canberra, New South Wales AY536390 AY302710 UnpublishedC. lacteus Australia: Olney State Forest, NSW DQ367840 Lo et al. (2006)C. lacteus BYU IGC IS41 EU253886 Lo et al. (2006)C. curvignathus Malaysia AY683210 AJ854177 UnpublishedC. curvignathus Malaysia AJ854178 UnpublishedC. curvignathus Indonesia AY575853 UnpublishedC. formosanus Japan, Wakayama EF379978 EF379959 EF379941 Yeap et al. (2007)C. formosanus Japan, Wakayama EF379979 EF379960 EF379942 Yeap et al. (2007)C. formosanus Japan, Okayama EF379980 EF379961 EF379943 Yeap et al. (2007)C. formosanus USA, Hawaii, Oahu EF379976 EF379958 EF379940 Yeap et al. (2007)C. formosanus USA: GA AY536385 AY302706 UnpublishedC. formosanus USA: HI AY536386 UnpublishedC. formosanus USA: New Orleans, LA AY536387 AY302707 UnpublishedC. formosanus USA: Florida, Golden Beach AY168212 AY168225 Ye et al. (2004)C. formosanus China: Guangzhou AY558911 Scheffrahn et al. (2004)C. formosanus China: Guangzhou AB262474 Noda et al. (2007)C. formosanus Japan: Kagoshima, Amami AB262501 UnpublishedC. formosanus USA: Hawaii, Oahu AY453588 Austin et al. (2004)C. formosanus China FJ423459 UnpublishedC. formosanus Japan AB109529 Ohkuma et al. (2004)C. formosanus Japan DQ493744 Yashiro and Matsuura (2007)C. guangzhouensis Japan AB036673 UnpublishedC. gestroi Malaysia, Penang, USM EF379982 EF379963 EF379945 Yeap et al. (2007)C. gestroi Malaysia, Kuala Lumpur, Bangsar EF379987 EF379969 EF379951 Yeap et al. (2007)C. gestroi Malaysia, Johor, Muar EF379988 EF379970 EF379952 Yeap et al. (2007)C. gestroi Singapore, Serenity Terrace EF379983 EF379964 EF379946 Yeap et al. (2007)C. gestroi Singapore, Serangoon Avenue 3 EF379985 EF379967 EF379949 Yeap et al. (2007)C. gestroi Thailand, Bangkok 1 EF379977 EF379965 EF379947 Yeap et al. (2007)C. gestroi Thailand, Bangkok 2 EF379986 EF379968 EF379950 Yeap et al. (2007)C. gestroi Indonesia, Cibinong EF379981 EF379962 EF379944 Yeap et al. (2007)C. gestroi Indonesia, Bogor. EF379984 EF379966 EF379948 Yeap et al. (2007)C. gestroi Singapore: Sommerville Wak DQ004476 Jenkins et al. (2007)C. gestroi Singapore: Tampines DQ004477 DQ923419 Jenkins et al. (2007)C. gestroi Singapore: Sime Ave. DQ004478 Jenkins et al. (2007)C. gestroi Puerto Rico: Las Mareas DQ004485 DQ923418 Jenkins et al. (2007)C. gestroi Australia, Queensland, Hamilton DQ004487 Jenkins et al. (2007)C. gestroi Thailand, Bangkok, Royal Forest Department DQ004488 EF092290 Jenkins et al. (2007)C. gestroi USA: Cleveland, Ohio DQ004495 DQ923420 Jenkins et al. (2007)C. gestroi USA: Florida, Key West EF156760 EF092291 Jenkins et al. (2007)C. gestroi USA: Miama, FL AY558907 Scheffrahn et al. (2004)C. gestroi Turks and Caicos Islands: Grand Turk AY558906 Scheffrahn et al. (2004)C. gestroi Antigua and Barbuda AY558905 Scheffrahn et al. (2004)C. vastator USA, Hawaii, Oahu EF379990 EF379971 EF379953 Yeap et al. (2007)C. vastator Philippines, Laguna Philippines, Los Banos,

colony 1EF379989 EF379972 EF379954 Yeap et al. (2007)

C. vastator Philippines, Laguna Philippines, Los Banos,colony 2

EF379991 EF379973 EF379955 Yeap et al. (2007)

C. vastator Philippines, Laguna Philippines, Los Banos,colony 3

EF379992 EF379974 EF379956 Yeap et al. (2007)

C. vastator USA: Kalaeloa, Oahu Island, Honolulu, HI AY536391 AY302711 UnpublishedC. vastator Philippines: Manila AY536392 AY302712 UnpublishedC. vastator Philippines: Wedgewood, Laguna, Manila AY536393 AY302713 UnpublishedC. vastator Philippines: Manila AY536394 UnpublishedC. sepangensis Malaysia AJ854163 UnpublishedC. sepangensis Malaysia AJ854165 UnpublishedC. travians Malaysia AJ854169 UnpublishedC. travians Malaysia AJ854170 UnpublishedR. flaviceps Taiwan, Taitung County, Lanyu Township EU667782 EU627778 EU627780 Li et al. (2008)G. sulphurues Malaysia, Penang, USM EF379993 EF379975 EF379957 Yeap et al. (2007)

November 2009 YEAP ET AL.: MOLECULAR SYSTEMATICS OF Coptotermes 1079

30 min, followed by the addition of 10% SDS andincubation for 3 h. After a single extraction usingphenol/chloroform, total DNA was precipitated withethanol and then resuspended in 25 �l of distilledwater.Polymerase Chain Reaction (PCR) Amplificationand DNA Sequencing. PCR was conducted using theprimers shown in Table 3 for ampliÞcation of 12S, 16S,and COII mtDNA genes (Kambhampati and Smith1995, Miura et al. 1998). PCR ampliÞcation was per-formed in a standard 25- or 50-�l reaction volume with2 �l of total genomic DNA, 1 pmol of each primer, 1.5mM MgCl2, 2 mM dNTPs, and 5 U/�l TaqDNA poly-merase. AmpliÞcation was performed in a PTC-200Peltier Thermol Cycle (MJ Research, Watertown,MA) with a proÞle consisting of precycle denaturationat 94�C for 2 min, a postcycle extension at 72�C for 10min, and 35 cycles of a standard three-step PCR (51.3,53.1, and 58.2�C annealing). AmpliÞed DNA from in-dividual termites was puriÞed using a SpinClean gelextraction kit (Intron, Seongnam-Si, Gyeonggi-do,Korea). Samples were sent to Macrogen Inc. (Seoul,South Korea) for direct sequencing in both directions,which was conducted under BigDye terminator (Ap-plied Biosystems, Foster City, CA) cycling conditions.The reacted products were then puriÞed by usingethanol precipitation and ran for analysis using a DNAanalyzer (Automatic Sequencer 3730xl, Applied Bio-systems).Phylogenetic Analysis. BioEdit version 7.0.5 soft-

ware was used to edit individual electropherogramsand form contigs (Hall 1999). Multiple consensus se-quences were aligned using ClustalW (Thompson etal. 1994). Multiple alignment parameters for gap open-ing and extension penalties were 10 and 0.2, respec-tively. DNA sequences of other Coptotermes spp. ob-tained from GenBank were included in the alignmentsfor phylogenetic comparisons.

The distance matrix option of PAUP* 4.0b10 (Swof-ford 2002) was used to calculate genetic distance ac-cording to the Kimura 2-parameter model of sequenceevolution (Kimura 1980). Maximum parsimony (MP)analysis was performed with TBR branch swappingand 10 random taxon addition replicates under a heu-ristic search, saving no �100 equally parsimonioustrees per replicate. To estimate branch support on therecovered topology, nonparametric bootstrap valueswere assessed with 1,000 bootstrap pseudo-replicates(Felsenstein 1985).

Before the maximum likelihood (ML) analysis,Modeltest 3.7 was used to Þnd the optimal model ofDNA substitution (Posada and Crandall 1998). Ac-

cording to Posada and Buckley (2004), the Akaikeinformation criterion (AIC) (Akaike 1974) is moreadvantageous than the hierarchical likelihood ratiotest. Therefore, our phylogenetic reconstruction formaximum likelihood was based on the best-Þt model,which was selected by AIC. Heuristic ML searchesusing tree bisection-reconnection (TBR) branchswapping were performed in PAUP 4.0b10 (Swofford2002). ML nodal support was estimated using the non-parametric bootstrap (Felsenstein 1985) and was re-stricted to 1,000 pseudo-replicates.

Results and Discussion

Morphology. The soldier heads in the genus Cop-totermes are pear-shaped and rounded laterally. Milkyexudate from the fontanelle was produced on theanterior part of the head when the soldier termite wasdisturbed. No teeth were apparent on the mandibles.Most standard morphological characters used for theidentiÞcation of Coptotermes spp. have overlappingranges; thus, identiÞcation can be based only on sev-eral key characters. Mandibles of all of the Coptot-ermes spp. in this study seemed to be parallel, exceptfor those of Coptotermes curvignathus Holmgren,which strongly curved inwards. C. curvignathus wasthe largest species among all of the genera studied.Coptotermes kalshoveni Kemner is morphologicallysimilar to Coptotermes sepangensis Krishna, but it isconsiderably smaller and the head capsule is narrow atthe anterior end. Coptotermes formosanus Shiraki isreadily distinguished from C. gestroi; the former hastwo pairs of setae that project dorsal laterally from thebase of the fontanelle compared with only one pair ofsetae in the latter species. For the Australian Coptot-ermes, Coptotermes frenchi Hill is relatively small; thesoldierÕs head is circular behind and has short man-dibles.Coptotermes lacteusFroggatt is relatively largerand has a ßat head and long mandibles. CoptotermesacinaciformisFroggatt is generally larger than all otherAustralian Coptotermes spp., and the soldierÕs head inthis species is relatively long and has long mandibles.

Table 4 lists morphological measurements of thetermite soldiers. It is clearly shown that the morpho-logical characteristics of many Coptotermes spp. ex-amined in this study were overlapped. Even within thesame species, variations in the characters occur. Forexample, intraspecies comparisons of several popula-tions of C. formosanus and C. gestroi revealed signiÞ-cant differences (P � 0.05) in morphological charac-teristics between the populations (Table 4). C. gestroicollected from the natural sites (secondary rain for-ests) were found to be larger than those collectedfrom urban areas (data not shown). Dissimilarity insize between two populations of C. curvignathus fromMalaysia also has been observed in this study(CC001MY and CC002MY). Light (1929) and Kirtonand Brown (2003) both reported a continuous varia-tion in size and shape within a single Coptotermes spp.In addition, termite morphology is inßuenced by theage and state of the colony and by habitat factors(Scheffrahn et al. 2005). Grace et al. (1995) noted that

Table 3. Primers used for PCR and sequencing

Gene Name Sequence (5�Ð3�)

16S LR-J-13007 TTA CGC TGT TAT CCC TAALR-N-13398 CGC CTG TTT ATC AAA AAC AT

12S 12SF TAC TAT GTT ACG ACT TAT12SR AAA CTA GGA TTA GAT ACC C

COII C2F2 ATA CCT CGA CGW TAT TCA GATKN3785 GTT TAA GAG ACC AGT ACT TG


Tab

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(in

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(20

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that

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930

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0.44

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037

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213

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319

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b0.

511

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674

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UC.lacteus

101.

134

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567

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333

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249

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383

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689

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608

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600

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597

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377

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924

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380

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0.76

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585

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399

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025f

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worker size is correlated with colony population sizein aging colonies. Husseneder et al. (2008) reported anegative correlation between worker size and thenumber of reproductives in extended family coloniesof the Formosan subterranean termite. These obser-vations explain the variation we observed in soldierhead morphology in different populations of a givenspecies. They also illustrate that morphological iden-tiÞcation ofCoptotermes samples at the species level isdifÞcult and has limited reliability.DNA Sequence Results. DNA sequencing of 12S,

16S, and COII mtDNA amplicons from Coptotermesspp. revealed an average size of 420, 428, and 680 bp,respectively. The level of sequence variation based onthe Kimura two-parameter differed among the threegenes. Sequence divergence ranged from 0.0% be-tween C. formosanus and C. dimorphus to 6.5% be-tween C. sepangensis and C. curvignathus in the 12Sgene, from 0.73% between C. dimorphus and C. co-chlearus to 7.6% betweenC. formosanus andC. traviansin the 16S gene, and from 4.4% betweenC. lacteus andC. frenchi to 11.5% betweenC. gestroi andC. kalshoveniin the COII gene.Phylogenetic Analysis. The mtDNA sequences ofCoptotermes spp. obtained in this study together withsequences from GenBank were aligned by using G.sulfureus and R. flaviceps as the outgroup taxa. Thealigned DNA matrices are available at TreeBASE(http://www.treebase.org, submission SN4594). Be-fore the analysis, 31 characters, 29 characters, and 71characters were removed from the alignments of 12S,16S, and COII, respectively. The multiple sequencealignment for the 12S gene, including the outgrouptaxa, has 389 characters, of which 299 are constant and39 are parsimony-informative. For the 16S gene, thereare 399 characters, of which 297 are constant and61 are parsimony-informative. For the COII gene,there are 609 characters, of which 401 are constant and144 are parsimony-informative.

Maximum parsimony analysis of 12S, 16S, and COIInucleotide matrices resulted in a total of 53, 100, 100(initial MaxTrees setting � 100) equally most parsi-monious trees, respectively (Fig. 1: length � 257, con-sistency index [CI] � 0.805, retention index [RI] �0.878; Fig. 2: length � 219, CI � 0.932, RI � 0.939; Fig. 3:length � 372, CI � 0.616, RI � 0.838). The best modelfor the maximum likelihood analysis of the 12S genewas “GTR�I�G” with the following parameter set-tings: base � (0.4526, 0.2227, 0.1242, 0.2005), Nst � 6,Rmat � (553073.9375, 831360.3125, 286817.6250,98618.3984, 1777170.8750), rates � gamma shape �0.4675, and pinvar � 0.3652. For the 16S gene, theselected model was “HKY�I�G” with the followingparameter settings: base � (0.4426, 0.2410, 0.0834,0.2330), Nst � 2, TRatio � 3.2315, rates � gammashape � 0.2658, and pinvar � 0.4684. For the COIIgene, the “GTR�I�G” model was selected with thefollowing parameter settings: base � (0.3826, 0.2676,0.1382, 0.2115), Nst � 6, Rmat � (2.6028, 14.4212,2.1720, 0.5625, 45.7613), rates � gamma shape �1.1930, and pinvar � 0.5309. A single tree was recov-ered for each of the genes (�In L 1369.6742 for 12S,

�In L 1421.4773 for 16S, and �In L 2792.4729 for COII;trees not shown). The most parsimonious trees withtopology similar to that generated by the ML analysiswere chose (Figs. 1Ð3).

A comparison of the three bootstrap trees revealedno cases in which a grouping with �50% bootstrapsupport in one tree conßicted with a grouping with�50% bootstrap support in another tree. Based on theoverall low level of conßict between the three genes,the data were combined, and the most parsimonioustree was constructed. Two major clades within theCoptotermes genus with six subclades were apparent:I (C. acinaciformis), II (C. lacteus and C. frenchi), III(C. curvignathus), IV (C. kalshoveni,C. sepangensis andC. travians), V (C. gestroi) and VI (C. formosanus)(Fig. 4). Poor phylogenetic resolution (as indicated bya low bootstrap value) was found among most of thesesix subclades. However, the monophyletic Coptot-ermes clade demonstrated that genetic partitioning isstrong for the studied species. The continuous over-lapping nature of the data makes it difÞcult to code themorphological data in the molecular matrix (Table 4).Therefore, only a few distinct morphological charac-ters were mapped on the combined tree (Fig. 4).Australian Coptotermes.When other sequences ofC. acinaciformis from GenBank were added into theanalysis of the 12S, 16S, and COII genes, C. acinaci-formis from Darwin (CA001AU) and C. acinaciformisfrom GrifÞth (CA002AU) branched out as a sistergroup with high bootstrap values in the 16S and COIItrees (Figs. 2 and 3). The genetic diversity betweenthese two populations of C. acinaciformis was low forthe 12S gene (0.5%). However, for the 16S and COIIgenes, the genetic diversity between CA001AU andCA002AU was notably high (3.0% and 3.5%, respec-tively), despite the morphological similarity of the twopopulations. Samples from Darwin were more closelyrelated to the samples from Townsville (Fig. 3). Sam-ples from New South Wales, GrifÞth, Sydney, andCanberra were clustered into the same group, with theNew South Wales and GrifÞth samples being closelyrelated to each another. Previous cuticular hydrocar-bon and molecular studies suggested that C. acinaci-formiswas genetically diverse and may even consist ofseveral species (Brown et al. 2004, Lo et al. 2006).Besides nucleotide differences, termites of this speciesalso are biologically distinct. In the north (Darwin),they are mound builders, whereas in southern Aus-tralia (e.g., GrifÞth) they generally nest inside trees,stumps, poles, or Þlled-in verandahs where timber hasbeen buried. These habitat differences might explainwhy C. acinaciformis from Darwin and GrifÞth do notshare the same lineage, as shown by differences intheir 12S, 16S, and COII genes. Relative to the otherspecies,C. acinaciformis exhibited a close relationshipwith the other two Australian Coptotermes (C. frenchiand C. lacteus). C. lacteus formed a sister group withC. frenchi, with genetic diversity of 1.1, 1.3, and 4.4%for the 12S, 16S, and COII genes, respectively.Southeast Asian Coptotermes. C. curvignathus is

widely distributed in Southeast Asia and has causeddamage to wooden construction and tree plantations


(Roonwal 1970, Lee 2002, Takematsu et al. 2006). C.curvignathus from Malaysia and Singapore share iden-tical sequences across the 12S, 16S, and COII genes.The soldier measurements of oneC. curvignathus sam-ple (CC002MY) collected in Penang, Malaysia, wereconsiderably smaller compared with those from otherC. curvignathus samples. However after phylogeneticanalysis, CC002MY clustered together with the otherC. curvignathus (Clade III) (Fig. 4). This Þnding

clearly indicates that size variation affects the reliabil-ity of morphologically based taxonomy (Takematsu etal. 2006).

The phylogenetic trees constructed based on the12S, 16S, COII, and the combination of the three genesshowed that C. kalshoveni, C. sepangensis, and C. tra-vians are closely related species. These three speciesclustered under the same clade in all of the trees,although C. kalshoveni had a closer relationship with

Fig. 1. The most parsimonious tree obtained for the 12S gene using a heuristic search option in PAUP4.0b10 (Swofford2002). Bootstrap values for 1,000 replicates are listed above the branches supported at �50%.


C. sepangensis than with C. travians. C. sepangensisclosely resembles C. kalshoveni based on the soldiercaste (Kemner 1934); morphologically, they can only

be separated by the shape and the curvature of themandibles (Tho 1992). Strong quantitative support bybootstrap analysis also indicated thatC. kalshoveni and

Fig. 2. The most parsimonious tree obtained for the 16S gene using a heuristic search option in PAUP4.0b10 (Swofford2002). Bootstrap values for 1,000 replicates are listed above the branches supported at �50%.


C. sepangensis are closely related. This result is sup-ported by high degrees of similarity in the 12S (97%),16S (99%), and COII (99%) genes. Further assessmentof the morphological features of alate forms and typespecimens would be required to resolve the relation-ship between these two species. The phylogenetictrees inferred from the three mitochondrial genesdemonstrated that Australian Coptotermes spp. have acloser relationship withC. curvignathus, C. kalshoveni,

C. sepangensis, and C. travians than with C. gestroi.However, this relationship was not supported by highbootstrap values.C. gestroi. C. gestroi is believed to be native to

Southeast Asia but has spread to the Marquesas Islands(PaciÞc Ocean), Mauritius and Reunion (IndianOcean), the New World tropics (Brazil and Barba-dos), some islands of the West Indies, southern Mex-ico, the Southeastern United States (Scheffrahn and

Fig. 3. The most parsimonious tree obtained for the COII gene using a heuristic search option in PAUP4.0b10 (Swofford2002). Bootstrap values for 1,000 replicates are listed above the branches supported at �50%.


Su 2000, Ferraz and Mendez-Montiel 2004), and morerecently to Taiwan (Tsai and Chen 2003). The phy-logenetic tree generated from the three mitochondrialgenes revealed that the populations ofC. gestroi could

be divided into three geographical groups: group I:Taiwan, the Philippines, and Hawaii populations(node support of 86%); group II: Thailand, Malaysia,and Singapore populations (node support of 67%); and

Fig. 4. The most parsimonious tree obtained for combined genes using a heuristic search option in PAUP4.0b10 (Swofford2002) with morphological characters (bold number below branches). Bootstrap values for 1,000 replicates are listed abovethe branches supported at �50%. 1, pear-shaped head; 2, large size head; 3, small size head; 4, ßat head; 5, number of teethon mandibles; 6, long mandibles; 7, short mandibles; 8, mandibles strongly curve inward; 9, one pair of setae at fontanelle;and 10, two pairs of setae at fontanelle.


group III: Indonesia populations (node support of96%) (Fig. 4). These results agree with the report ofLi et al. (2009).

In this study, we found that the C. gestroi samplesfrom southern Taiwan were more closely related tothose from the Philippines and Hawaii than to thosefrom other geographic locations. Luzon Strait is thepassage between northern Luzon, the Philippines, andsouthern Taiwan. It is an important strait connectingthe China Sea on the west with the Philippine Seaon the east, and it extends 200 miles (320 km) betweenthe islands of Taiwan (north) and Luzon, Philippines(south) and is used for shipping and communication.Many ships from the Americas use this route to reachimportant East Asian ports. It is possible thatC. gestroiwas introduced to Taiwan via this maritime route.

Thailand, Malaysia, and Singapore share a well-de-veloped transportation infrastructure, including someworld-renowned port systems, railway network, andmodern highway system that accommodate intra- andintercountry travel and commerce (Jenkins et al.2007). Furthermore, a partnership between railwaysystems in Thailand and Malaysia allows minimuminspection of sealed railway containers. The numerousmodes of transportation, many characterized by min-imal inspection of cargo, may have enabled the easyspread of C. gestroi across the three countries. Thismay explain the close relationship between the C.gestroi samples collected from Thailand, Malaysia, andSingapore.

The two populations of C. gestroi from Indonesia(Cibinong and Bogor) branched out as a distinctgroup. This will certainly require more C. gestroi sam-ples from Sumatra, Borneo, and Sulawesi to furthersubstantiate current Þndings.

The high adaptability of C. gestroi allowed it to betransported and fortuitously introduced into manycountries.C.vastator(�C.gestroi; seeYeapet al. 2007)was recently found in Minami Torishima (Marcus Is-land), Japan (Morimoto and Ishii 2000). This was theÞrst record of C. gestroi in Japan. The island is locatedat 24� 18� N 153� 58� E, which shares a similar latitudewith Miami, FL in the United States (25� 79� N 80� 22�W) where activities of C. gestroi have been recorded.According to Bess (1970), in 1963 a number of C.vastator (�C. gestroi) imagos and soldiers were foundin several buildings in Honolulu, HI. This was theperiod during which Minami Torishima (Marcus Is-land) was occupied by the U.S. army (i.e., between1952 and 1968). Thus, material transportation con-ducted by the U.S. Army might be related to theexpansion of Coptotermes to various places within thePaciÞc Ocean (Morimoto and Ishii 2000).Chinese Coptotermes. C. formosanus is believed to

have originated from southern China and Taiwan be-cause of the higher level of genetic variability (Li etal. 2009). It ishighlyadapted to theurbanenvironmentand has been dispersed by the railway and by ships(Jenkins et al. 2002, Scheffrahn and Su 2005, Austin etal. 2008) through 10 southeastern states in the UnitedStates over the past 50 yr (Su 2003). It accounts for aconsiderable amount of the total termite damages (Su

2002). In our study, amongC. formosanus populations,samples form Taiwan, Japan, and China grouped to-gether (node support of 63%) and separated outthe Hawaii samples and one sample from China(CF001CN) (Fig. 4). This result agrees with data in Liet al. (2009). However, more samples ofC. formosanusfrom Hawaii and Florida are required to generate aclearer picture of their population structures.

In this study we only obtained sequences of the 12Sand 16S genes for C. dimorphus and C. cochlearus.Therefore, these two species could not be included inthe overall analysis of the combined genes. C. dimor-phus and C. cochlearus fell within the subclade of C.formosanus in the 12S tree (Fig. 1); in fact, theyshowed 100% similar toC. formosanus for the 12S gene.They branched out as a sister group to C. formosanusin the 16S tree (Fig. 2). As the 16S gene is a fasterevolving gene than the 12S gene (Ye et al. 2004), thismay explain why greater genetic diversity found be-tween these species when the 16S gene was used.

From the phylogenetic analysis, only two haplo-types were found in the 12S gene among 17 popula-tions ofC. formosanus from China, Taiwan, Japan, andHawaii (Table 5). These two haplotypes differ only bya single nucleotide. Both C. dimorphus and C. cochlea-rus differed from C. formosanus by only a single nu-cleotide in the 12S sequence. For the 16S gene, fourhaplotypes were found among the 17 populations ofC.formosanus (Table 6). These four haplotypes exhib-ited only one or two nucleotide differences amongthem. In total, C. dimorphus and C. cochlearus haveeight and ten nucleotides that differ from those of C.formosanus, respectively. Thus, the molecular datastrongly suggests that both C. dimorphus and C. co-chlearus are junior synonyms of C. formosanus. How-ever, because of the limited sample size was used inthis study, it is important to have more samples ex-amined, including their morphological characters, be-fore the current Þndings can be conÞrmed. In addi-

Table 5. Nucleotide and haplotype variations for 12S geneamong C. formosanus, C. dimorphus, and C. cochlearus

Species Haplotype 28 124 348

CF001JP F1 A Ð ACF002JP F1 * Ð *CF003JP F1 * Ð *CF004JP F1 * Ð *CF005JP F1 * Ð *CF006JP F1 * Ð *CF007JP F2 * Ð GCF008JP F1 * Ð *CF009JP F1 * Ð *CF010JP F1 * Ð *CF001CN F1 * Ð *CF002CN F1 * Ð *CF003CN F1 * Ð *CF004CN F1 * Ð *CF001TW F1 * Ð *CF002TW F1 * Ð *CF001HW F1 * Ð *CD001CN * A *CCO001CN G Ð *

Note: Ð refers to a gap, * refers to the same nucleotide as thenucleotide on the Þrst row.


tion, C. guangzhouensis sequence from the GenBankalso was grouped within the C. formosanus subclade(Fig. 2). A thorough molecular phylogenetic study ofCoptotermes spp. in China is urgently warranted.

Accurate identiÞcation and information about phy-logenetic diversity of Coptotermes are important forthe improvement of termite management strategies.With the Þnding of synonymous species, informationconcerning those species from different geographicalregions can now be pooled. Regulatory authoritiesmay now be able to accept efÞcacy assessments oftermite management strategies from any given regionif the targeted species is the same, as compared withthe past when they were thought to be different spe-cies. This will beneÞt all parties and will in the long runsave time and resources (Kirton 2005).

In summary, the following six subclades with poorrelationships among the Coptotermes spp. from EastAsia and Australia were revealed based on phylo-genetic analyses of three mitochondrial genes: (I)C. acinaciformis; (II) C. frenchi and C. lacteus; (III) C.curvignathus; (IV)C. kalshoveni, C. sepangensis, andC.travians; (V)C. gestroi; and (VI)C. formosanus. Basedon the genetic evidence, it is likely that C. dimorphusand C. cochlearus are possibly junior synonyms of C.formosanus; however, more samples would be re-quired to further substantiate this observation. Mor-phological characters are often ambiguous and theeight measurements on the soldier termites used inthis study overlapped for the 11 Coptotermes spp. ex-amined. These results suggest that the use of bothmolecular and morphological approaches is crucial inensuring accurate species identiÞcation for this genus.

Acknowledgments

We thank Michael Lenz (Commonwealth ScientiÞc andIndustrial Research Organization Entomology, Canberra,Australia) and six anonymous reviewers for comments on themanuscript drafts, Tomoki Sumino (Universiti Sains Malay-

sia) for translating some Japanese texts, and the followingindividuals who helped with the collection of the termitespecimens: Charunee Vongkaluang (Royal Forest Depart-ment, Bangkok, Thailand); Carlos Garcia (Forest ProductDepartment, Laguna, the Philippines); Julian Yates, III (Uni-versity of Hawaii, Honolulu, HI); Tsuyoshi Yoshimura(Kyoto University, Kyoto, Japan); Lim Kay Min (NLC Gen-eral Pest Control, Penang, Malaysia); Kean-Teik Koay(Ridpest Shah Alam, Selangor, Malaysia), Sulaeman Yusuf(LIPI, Bogor, Indonesia), Theo Evans (Commonwealth Sci-entiÞc and Industrial Research Organization Entomology,Canberra, Australia); John Ho (Singapore Pest ManagementAssociation, Singapore); Chun-Chun Tsai (Aletheia Univer-sity, Tainan, Taiwan); Nancy Lee (ChungHsi Chemical Co.,Taipei, Taiwan); Xie (Jia Fei Jie Pest Control Co., Tainan,Taiwan); and Junhong Zhong (Guangdong EntomologicalInstitute, Guangzhou, China). B.-K.Y. was supported by aPh.D. scholarship from Ministry of Science, Technology andInnovation, Malaysia. This study was supported under a Re-search University (RU) grant from Universiti Sains Malaysia.

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Received 22 April 2009; accepted 8 September 2009.


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